Abstract Background Insulin like growth factor binding proteins modulate the mitogenic and pro survival effects of IGF. Elevated expression of IGFBP2 is associated with progression of tumors that include prostate, ovarian, glioma among others. Though implicated in the progression of breast cancer, the molecular mechanisms involved in IGFBP2 actions are not well defined. This study investigates the molecular targets and biological pathways targeted by IGFBP2 in breast cancer. Methods Transcriptome analysis of breast tumor cells (BT474) with stable knockdown of IGFBP2 and breast tumors having differential expression of IGFBP2 by immunohistochemistry was performed using microarray. Differential gene expression was established using R-Bioconductor package. For validation, gene expression was determined by qPCR. Inhibitors of IGF1R and integrin pathway were utilized to study the mechanism of regulation of β-catenin. Immunohistochemical and immunocytochemical staining was performed on breast tumors and experimental cells, respectively for β-catenin and IGFBP2 expression. Results Knockdown of IGFBP2 resulted in differential expression of 2067 up regulated and 2002 down regulated genes in breast cancer cells. Down regulated genes principally belong to cell cycle, DNA replication, repair, p53 signaling, oxidative phosphorylation, Wnt signaling. Whole genome expression analysis of breast tumors with or without IGFBP2 expression indicated changes in genes belonging to Focal adhesion, Map kinase and Wnt signaling pathways. Interestingly, IGFBP2 knockdown clones showed reduced expression of β- catenin compared to control cells which was restored upon IGFBP2 re-expression. The regulation of β-catenin by IGFBP2 was found to be IGF1R and integrin pathway dependent. Furthermore, IGFBP2 and β-catenin are co-ordinately overexpressed in breast tumors and correlate with lymph node metastasis. Conclusion This study highlights regulation of β-catenin by IGFBP2 in breast cancer cells and most importantly, combined expression of IGFBP2 and β-catenin is associated with lymph node metastasis of breast tumors. Keywords: IGFBP2, Breast cancer, Wnt signaling, β-catenin, Integrin Background The Insulin like Growth Factor binding proteins (IGFBP) are a family of six proteins that bind with high affinity to Insulin like growth factors (IGF-I and IGF-II), prolong their half-life in circulation and thereby regulate IGF actions. Insulin like growth factor binding protein 2 (IGFBP2) is the second most abundant IGFBP in circulation and in a context dependent manner it can either inhibit or potentiate the actions of IGF [[38]1], thereby modulating the prosurvival and/or mitogenic effects of IGF. Elevated expression of IGFBP2 has been observed in multiple malignancies, including Glioblastoma multiforme [[39]2-[40]4], ovarian [[41]5,[42]6], pancreatic [[43]7], gastric [[44]8], prostate [[45]9], colon [[46]10], breast [[47]11,[48]12], leukemia [[49]13] and thyroid cancer [[50]14]. In addition, increased expression of IGFBP2 has been correlated with poor prognosis in prostate, glioblastoma and colon cancers [[51]15-[52]18]. It has been reported that IGFBP2 inhibits the IGF dependent proliferation of normal cells while in tumor cells, it promotes proliferation in an IGF1R dependent or independent manner [[53]19,[54]20]. Pro proliferative action of IGFBP2 has been reported in prostate, ovarian and colon cancer cells and non-transformed rat osteoblasts [[55]19,[56]21-[57]24]. IGFBP2 expression has also been shown to enhance migration and invasion in glioma, ovarian and bladder cancer cells [[58]3,[59]25-[60]27]. Recent studies in glioma implicate IGFBP2 in the activation of PI3K Akt pathway [[61]28], integrin/ILK/NF-B network which drives glioma progression in mice [[62]29] and binding to integrin α5 [[63]30] that brings about increased migration and invasion. In breast cancer, IGFBP2 over expression has been shown to confer drug resistance [[64]11] and increased expression has been reported to correlate with lymph node metastasis In T1 breast carcinomas [[65]31]. However, mechanisms that govern IGFBP2 actions in breast cancers are poorly understood. In the present study, to elucidate the cellular pathways influenced by IGFBP2 in breast cancer, gene expression profiling of IGFBP2 knockdown breast cancer cells was compared with the expression profile of IGFBP2 positive breast tumors. Our results highlight regulation of cell cycle and Wnt signaling pathways by IGFBP2. Most significantly, our data shows for the first time that the concomitant over expression of IGFBP2 and β-catenin in breast cancer is associated with increased incidence of lymph node metastasis. Results IGFBP2 perturbation by shRNA alters gene expression profile in breast cancer cells In view of the pro-tumorigenic actions of IGFBP2 reported in several cancers including breast tumors, we decided to delineate the molecular mechanism of IGFBP2 actions in breast cancers. Initially, stable sub lines of breast tumor cell line BT474 with knockdown of IGFBP2 were generated. Among several clones, two of the clones (C5 and C12) that showed considerable knock down of IGFBP2 (Figure [66]1a) were selected for further studies. Transcriptome analysis of the IGFBP2 knock down cells using Agilent whole human genome 4x44K arrays was performed against control cells (vector transfected). Data analysis revealed significant regulation of 4069 probes in both the clones compared to control cells. Among these, 2067 probes showed up regulation while 2002 probes showed down regulation (Additional file [67]1: Table S1). Hierarchical cluster revealed similar expression pattern of regulated genes in both the clones (Figure [68]1b). The list of top 25 up and down regulated genes is shown in Table [69]1. The differentially regulated genes were subjected to pathway enrichment analysis using GSEA (Table [70]2). This analysis revealed enrichment of down regulated genes belonging to cell cycle, DNA replication, repair, p53 signaling, oxidative phosphorylation, Wnt signaling, etc. qPCR analysis of some genes validated differential expression seen in microarray data (Figure [71]1c). Over expression of IGFBP2 in the knockdown cells resulted in up regulation of IGF1R, IGF2, TOP2A, p53, CCND1 and FOXM1 genes which were down regulated upon IGFBP2 knockdown (Additional file [72]2: Figure S1) suggesting the specificity of the regulation of these genes by IGFBP2. Hence, perturbation of IGFBP2 results in differential expression of several genes and pathways. Figure 1. [73]Figure 1 [74]Open in a new tab IGFBP2 regulated genes in BT474 breast cancer cells. a) Western blot analysis of IGFBP2 in the supernatant of IGFBP2 knockdown clones C5 and C12 and control cells. Lower panel is ponceau stained membrane shown as loading control. b) Hierarchical cluster of differentially expressed genes in IGFBP2 knockdown BT474 cells versus control cells. Differentially regulated genes were clustered using MeV software. The dendrogram on the left shows different clusters of genes segregated according to the pattern of regulation. Red and green indicate high and low expression of genes respectively. Black indicates no regulation. c) Validation of selected genes by qPCR. Bar graphs of differentially regulated genes in IGFBP2 Knockdown BT474 clones versus control cells. The graphs represent the fold change over control after normalization with the expression of RPL35a. Table 1. List of top 50 differentially regulated genes (p < 0.05) in IGFBP2 knockdown clones Probe Gene symbol Accession no. LOG2 ratios IGFBP2 (shRNA/scrambled) __________________________________________________________________ C5 C12 A_23_P70007 __________________________________________________________________ HMMR __________________________________________________________________ [75]NM_012484 __________________________________________________________________ −6.1994 __________________________________________________________________ −6.3340 __________________________________________________________________ A_23_P115872 __________________________________________________________________ CEP55 __________________________________________________________________ [76]NM_018131 __________________________________________________________________ −5.5493 __________________________________________________________________ −5.7555 __________________________________________________________________ A_23_P155815 __________________________________________________________________ HCAP-G __________________________________________________________________ [77]NM_022346 __________________________________________________________________ −5.9516 __________________________________________________________________ −5.6434 __________________________________________________________________ A_23_P49878 __________________________________________________________________ FAM64A __________________________________________________________________ [78]NM_019013 __________________________________________________________________ −5.3231 __________________________________________________________________ −5.5775 __________________________________________________________________ A_23_P401 __________________________________________________________________ CENPF __________________________________________________________________ [79]NM_016343 __________________________________________________________________ −5.6859 __________________________________________________________________ −5.5456 __________________________________________________________________ A_24_P297539 __________________________________________________________________ UBE2C __________________________________________________________________ [80]NM_181803 __________________________________________________________________ −6.0374 __________________________________________________________________ −5.5064 __________________________________________________________________ A_23_P52017 __________________________________________________________________ ASPM __________________________________________________________________ [81]NM_018136 __________________________________________________________________ −5.6626 __________________________________________________________________ −5.4146 __________________________________________________________________ A_23_P118815 __________________________________________________________________ BIRC5 __________________________________________________________________ [82]NM_001012271 __________________________________________________________________ −5.7127 __________________________________________________________________ −5.3579 __________________________________________________________________ A_23_P51085 __________________________________________________________________ SPBC25 __________________________________________________________________ [83]NM_020675 __________________________________________________________________ −5.3333 __________________________________________________________________ −5.2314 __________________________________________________________________ A_32_P62997 __________________________________________________________________ PBK __________________________________________________________________ [84]NM_018492 __________________________________________________________________ −5.3592 __________________________________________________________________ −5.1651 __________________________________________________________________ A_23_P379614 __________________________________________________________________ OIP5 __________________________________________________________________ [85]NM_007280 __________________________________________________________________ −5.4136 __________________________________________________________________ −5.0805 __________________________________________________________________ A_23_P138507 __________________________________________________________________ CDC2 __________________________________________________________________ [86]NM_001786 __________________________________________________________________ −5.3918 __________________________________________________________________ −4.9418 __________________________________________________________________ A_23_P74349 __________________________________________________________________ CDCA1 __________________________________________________________________ [87]NM_145697 __________________________________________________________________ −5.0628 __________________________________________________________________ −4.7854 __________________________________________________________________ A_23_P375 __________________________________________________________________ CDCA8 __________________________________________________________________ [88]NM_018101 __________________________________________________________________ −4.9988 __________________________________________________________________ −4.7055 __________________________________________________________________ A_23_P385861 __________________________________________________________________ CDCA2 __________________________________________________________________ [89]NM_152562 __________________________________________________________________ −4.8413 __________________________________________________________________ −4.6523 __________________________________________________________________ A_23_P107421 __________________________________________________________________ TK1 __________________________________________________________________ [90]NM_003258 __________________________________________________________________ −5.1628 __________________________________________________________________ −4.6138 __________________________________________________________________ A_23_P50108 __________________________________________________________________ KNTC2 __________________________________________________________________ [91]NM_006101 __________________________________________________________________ −4.5643 __________________________________________________________________ −4.6060 __________________________________________________________________ A_23_P124417 __________________________________________________________________ BUB1 __________________________________________________________________ [92]NM_004336 __________________________________________________________________ −4.8189 __________________________________________________________________ −4.5874 __________________________________________________________________ A_23_P88331 __________________________________________________________________ DLG7 __________________________________________________________________ [93]NM_014750 __________________________________________________________________ −4.8666 __________________________________________________________________ −4.5641 __________________________________________________________________ A_23_P118834 __________________________________________________________________ TOP2A __________________________________________________________________ [94]NM_001067 __________________________________________________________________ −4.9585 __________________________________________________________________ −4.5030 __________________________________________________________________ A_24_P234196 __________________________________________________________________ RRM2 __________________________________________________________________ [95]NM_001034 __________________________________________________________________ −4.6703 __________________________________________________________________ −4.4466 __________________________________________________________________ A_23_P65757 __________________________________________________________________ CCNB2 __________________________________________________________________ [96]NM_004701 __________________________________________________________________ −4.6828 __________________________________________________________________ −4.4265 __________________________________________________________________ A_23_P119943 __________________________________________________________________ IGFBP2 __________________________________________________________________ [97]NM_000597 __________________________________________________________________ −2.4765 __________________________________________________________________ −4.4074 __________________________________________________________________ A_23_P88731 __________________________________________________________________ RAD51 __________________________________________________________________ [98]NM_002875 __________________________________________________________________ −4.9625 __________________________________________________________________ −4.3833 __________________________________________________________________ A_23_P133123 __________________________________________________________________ MND1 __________________________________________________________________ [99]NM_032117 __________________________________________________________________ −4.6389 __________________________________________________________________ −4.3051 __________________________________________________________________ A_23_P31407 __________________________________________________________________ AGR2 __________________________________________________________________ [100]NM_006408 __________________________________________________________________ 4.8060 __________________________________________________________________ 4.2255 __________________________________________________________________ A_23_P106194 __________________________________________________________________ FOS __________________________________________________________________ [101]NM_005252 __________________________________________________________________ 2.1989 __________________________________________________________________ 4.2063 __________________________________________________________________ A_23_P429998 __________________________________________________________________ FOSB __________________________________________________________________ [102]NM_006732 __________________________________________________________________ 1.5980 __________________________________________________________________ 3.8822 __________________________________________________________________ A_23_P113952 __________________________________________________________________ [103]AY227436 __________________________________________________________________ [104]AY227436 __________________________________________________________________ 3.4910 __________________________________________________________________ 3.8165 __________________________________________________________________ A_23_P500000 __________________________________________________________________ SCEL __________________________________________________________________ [105]NM_144777 __________________________________________________________________ 3.6586 __________________________________________________________________ 3.2728 __________________________________________________________________ A_23_P169437 __________________________________________________________________ LCN2 __________________________________________________________________ [106]NM_005564 __________________________________________________________________ 2.5385 __________________________________________________________________ 3.2159 __________________________________________________________________ A_23_P380754 __________________________________________________________________ PRSS1 __________________________________________________________________ [107]NM_002769 __________________________________________________________________ 2.3278 __________________________________________________________________ 3.1664 __________________________________________________________________ A_23_P29773 __________________________________________________________________ LAMP3 __________________________________________________________________ [108]NM_014398 __________________________________________________________________ 4.2963 __________________________________________________________________ 3.1041 __________________________________________________________________ A_23_P5983 __________________________________________________________________ PLTP __________________________________________________________________ [109]NM_006227 __________________________________________________________________ 1.3270 __________________________________________________________________ 2.8955 __________________________________________________________________ A_23_P310274 __________________________________________________________________ PRSS2 __________________________________________________________________ [110]NM_002770 __________________________________________________________________ 2.0382 __________________________________________________________________ 2.8886 __________________________________________________________________ A_23_P71170 __________________________________________________________________ TRPV6 __________________________________________________________________ [111]NM_018646 __________________________________________________________________ 2.6719 __________________________________________________________________ 2.7426 __________________________________________________________________ A_23_P98121 __________________________________________________________________ FXYD4 __________________________________________________________________ [112]NM_173160 __________________________________________________________________ 3.0264 __________________________________________________________________ 2.6979 __________________________________________________________________ A_23_P88095 __________________________________________________________________ TBC1D4 __________________________________________________________________ [113]NM_014832 __________________________________________________________________ 0.9759 __________________________________________________________________ 2.6576 __________________________________________________________________ A_32_P230828 __________________________________________________________________ GAS5 __________________________________________________________________ [114]NR_002578 __________________________________________________________________ 1.9990 __________________________________________________________________ 2.6280 __________________________________________________________________ A_23_P369343 __________________________________________________________________ KLK8 __________________________________________________________________ [115]NM_144505 __________________________________________________________________ 2.0683 __________________________________________________________________ 2.5972 __________________________________________________________________ A_23_P18684 __________________________________________________________________ CLGN __________________________________________________________________ [116]NM_004362 __________________________________________________________________ 2.4448 __________________________________________________________________ 2.5689 __________________________________________________________________ A_32_P37592 __________________________________________________________________ SCARNA17 __________________________________________________________________ [117]NR_003003 __________________________________________________________________ 1.7724 __________________________________________________________________ 2.5574 __________________________________________________________________ A_23_P22735 __________________________________________________________________ BEX2 __________________________________________________________________ [118]NM_032621 __________________________________________________________________ 3.3099 __________________________________________________________________ 2.5138 __________________________________________________________________ A_24_P921446 __________________________________________________________________ EMP1 __________________________________________________________________ [119]BC017854 __________________________________________________________________ 1.1796 __________________________________________________________________ 2.4896 __________________________________________________________________ A_23_P121926 __________________________________________________________________ SEPP1 __________________________________________________________________ [120]NM_005410 __________________________________________________________________ 2.7851 __________________________________________________________________ 2.4722 __________________________________________________________________ A_23_P34915 __________________________________________________________________ ATF3 __________________________________________________________________ [121]NM_004024 __________________________________________________________________ 3.5213 __________________________________________________________________ 2.4288 __________________________________________________________________ A_23_P105803 __________________________________________________________________ FGF9 __________________________________________________________________ [122]NM_002010 __________________________________________________________________ 0.9259 __________________________________________________________________ 2.3751 __________________________________________________________________ A_24_P327886 __________________________________________________________________ TCEA3 __________________________________________________________________ [123]NM_003196 __________________________________________________________________ 2.1369 __________________________________________________________________ 2.3522 __________________________________________________________________ A_23_P391344 __________________________________________________________________ RASGEF1A __________________________________________________________________ [124]NM_145313 __________________________________________________________________ 3.5570 __________________________________________________________________ 2.3442 __________________________________________________________________ A_24_P18190 HSPA5 [125]NM_005347 2.8335 2.3293 [126]Open in a new tab Table 2. GSEA summary of pathways associated with genes down regulated upon IGFBP2 knockdown NAME SIZE NOM p-val FDR q-val FWER p-val KEGG_CELL_CYCLE __________________________________________________________________ 49 __________________________________________________________________ 0 __________________________________________________________________ 0 __________________________________________________________________ 0 __________________________________________________________________ KEGG_DNA_REPLICATION __________________________________________________________________ 28 __________________________________________________________________ 0 __________________________________________________________________ 0 __________________________________________________________________ 0 __________________________________________________________________ KEGG_NUCLEOTIDE_EXCISION_REPAIR __________________________________________________________________ 15 __________________________________________________________________ 0 __________________________________________________________________ 0 __________________________________________________________________ 0 __________________________________________________________________ KEGG_MISMATCH_REPAIR __________________________________________________________________ 15 __________________________________________________________________ 0 __________________________________________________________________ 0 __________________________________________________________________ 0 __________________________________________________________________ KEGG_PATHOGENIC_ESCHERICHIA_COLI_INFECTION __________________________________________________________________ 15 __________________________________________________________________ 0 __________________________________________________________________ 2.19E-04 __________________________________________________________________ 0.001 __________________________________________________________________ KEGG_HOMOLOGOUS_RECOMBINATION __________________________________________________________________ 15 __________________________________________________________________ 0 __________________________________________________________________ 1.83E-04 __________________________________________________________________ 0.001 __________________________________________________________________ KEGG_PYRIMIDINE_METABOLISM __________________________________________________________________ 21 __________________________________________________________________ 0 __________________________________________________________________ 1.56E-04 __________________________________________________________________ 0.001 __________________________________________________________________ KEGG_OXIDATIVE_PHOSPHORYLATION __________________________________________________________________ 19 __________________________________________________________________ 0 __________________________________________________________________ 0.00635 __________________________________________________________________ 0.039 __________________________________________________________________ KEGG_PARKINSONS_DISEASE __________________________________________________________________ 21 __________________________________________________________________ 0 __________________________________________________________________ 0.01229 __________________________________________________________________ 0.082 __________________________________________________________________ KEGG_PROGESTERONE_MEDIATED_OOCYTE_MATURATION __________________________________________________________________ 17 __________________________________________________________________ 0.006186 __________________________________________________________________ 0.013514 __________________________________________________________________ 0.098 __________________________________________________________________ KEGG_SPLICEOSOME __________________________________________________________________ 32 __________________________________________________________________ 0.002012 __________________________________________________________________ 0.01617 __________________________________________________________________ 0.128 __________________________________________________________________ KEGG_OOCYTE_MEIOSIS __________________________________________________________________ 30 __________________________________________________________________ 0 __________________________________________________________________ 0.02404 __________________________________________________________________ 0.199 __________________________________________________________________ KEGG_P53_SIGNALING_PATHWAY __________________________________________________________________ 26 __________________________________________________________________ 0 __________________________________________________________________ 0.027815 __________________________________________________________________ 0.247 __________________________________________________________________ KEGG_HUNTINGTONS_DISEASE __________________________________________________________________ 30 __________________________________________________________________ 0.004098 __________________________________________________________________ 0.032137 __________________________________________________________________ 0.3 __________________________________________________________________ KEGG_WNT_SIGNALING_PATHWAY __________________________________________________________________ 21 __________________________________________________________________ 0.045726 __________________________________________________________________ 0.138961 __________________________________________________________________ 0.804 __________________________________________________________________ KEGG_PURINE_METABOLISM __________________________________________________________________ 26 __________________________________________________________________ 0.047431 __________________________________________________________________ 0.150692 __________________________________________________________________ 0.844 __________________________________________________________________ KEGG_GAP_JUNCTION __________________________________________________________________ 17 __________________________________________________________________ 0.15251 __________________________________________________________________ 0.155502 __________________________________________________________________ 0.877 __________________________________________________________________ KEGG_TGF_BETA_SIGNALING_PATHWAY __________________________________________________________________ 15 __________________________________________________________________ 0.393214 __________________________________________________________________ 0.318836 __________________________________________________________________ 0.995 __________________________________________________________________ KEGG_SMALL_CELL_LUNG_CANCER __________________________________________________________________ 15 __________________________________________________________________ 0.55144 __________________________________________________________________ 0.400925 __________________________________________________________________ 0.999 __________________________________________________________________ KEGG_REGULATION_OF_ACTIN_CYTOSKELETON __________________________________________________________________ 25 __________________________________________________________________ 0.517578 __________________________________________________________________ 0.687552 __________________________________________________________________ 1 __________________________________________________________________ KEGG_TIGHT_JUNCTION __________________________________________________________________ 16 __________________________________________________________________ 0.787018 __________________________________________________________________ 0.687305 __________________________________________________________________ 1 __________________________________________________________________ KEGG_PATHWAYS_IN_CANCER __________________________________________________________________ 52 __________________________________________________________________ 0.229814 __________________________________________________________________ 0.731651 __________________________________________________________________ 1 __________________________________________________________________ KEGG_ALZHEIMERS_DISEASE 38 0.433663 0.706543 1 [127]Open in a new tab NOM P nominal P value. Differential expression of genes between tumors staining positive or negative for IGFBP2 In order to determine, whether expression of IGFBP2 regulated genes as revealed by IGFBP2 perturbation is also altered in tumors, we studied the gene expression patterns in tumors based on IGFBP2 expression. We selected 12 IGFBP2 positive and 7 IGFBP2 negative tumor RNAs for microarray expression analysis using Agilent whole human genome 4x44K arrays. Comparison of gene expression profiles between IGFBP2 positive and negative tumors revealed 3460 probes as significantly differentially regulated. Among them, 1635 probes were up regulated and 1825 probes were found to be down regulated in IGFBP2 positive tumors compared to IGFBP2 negative tumors (Additional file [128]3: Table S2). List of top 25 up or down regulated genes are shown in Table [129]3. To identify enriched pathways associated with differentially expressed genes, Gene set enrichment analysis (GSEA) was carried out. The genes up regulated in IGFBP2 positive tumor samples showed significant enrichment in Focal adhesion, MAPK signaling pathway, apoptosis, Chemokine signaling, cytokine-cytokine receptor interaction and ECM receptor interaction and Wnt signaling pathway (Table [130]4). Hierarchical cluster (Euclidean distance method) of log2 transformed differentially expressed genes between IGFBP2 positive and negative tumors revealed two major clusters consisting of predominantly either IGFBP2 positive or negative tumors. However, in one cluster, there is a sub cluster representing exclusively IGFBP2 positive tumors (Figure [131]2a). Microarray results were validated on few genes by qPCR. As shown in Figure [132]2b, qPCR revealed that CCND1(Cyclin D1), CDC42, GATA 3, SYT13 and SFRP2 and TMEM49 as up regulated in IGFBP2 positive tumors while IGFBP2, NR4A2 and SFRP2 were down regulated in IGFBP2 negative tumors. In addition, since Wnt pathway genes were significantly regulated in IGFBP2 knock down cells, we studied the expression of Wnt target genes in IGFBP2 positive and negative breast tumors. The Wnt target genes CCND1, SFRP2 (Figure [133]2b) MCAM, SP5 and IGF1 (Additional file [134]4: Figure S2) were found to be differentially expressed between IGFBP2 positive and negative tumors. Taken together, the data from the IGFBP2 knockdown cells and IGFBP2 positive breast tumors suggest a positive correlation of IGFBP2 with pro-tumorigenic pathways including Wnt pathway in breast cancer. Table 3. List of top 50 differentially regulated genes (p < 0.05) in IGFBP2 positive versus IGFBP2 negative tumors Probe Gene name Accession no. Fold change IGFBP2 +/ IGFBP2- A_23_P161940 __________________________________________________________________ SCGB2A2 __________________________________________________________________ [135]NM_002411 __________________________________________________________________ 4.2566 __________________________________________________________________ A_23_P8702 __________________________________________________________________ PIP __________________________________________________________________ [136]NM_002652 __________________________________________________________________ 2.8894 __________________________________________________________________ A_24_P137501 __________________________________________________________________ SFRP2 __________________________________________________________________ [137]NM_003013 __________________________________________________________________ 2.4919 __________________________________________________________________ A_23_P312300 __________________________________________________________________ SCGB2A1 __________________________________________________________________ [138]NM_002407 __________________________________________________________________ 2.4797 __________________________________________________________________ A_24_P347431 __________________________________________________________________ FOXA1 __________________________________________________________________ [139]NM_004496 __________________________________________________________________ 2.4462 __________________________________________________________________ A_23_P372234 __________________________________________________________________ CA12 __________________________________________________________________ [140]NM_001218 __________________________________________________________________ 2.3044 __________________________________________________________________ A_23_P393099 __________________________________________________________________ TFF3 __________________________________________________________________ [141]NM_003226 __________________________________________________________________ 2.2590 __________________________________________________________________ A_23_P215328 __________________________________________________________________ SFRP4 __________________________________________________________________ [142]NM_003014 __________________________________________________________________ 2.2567 __________________________________________________________________ A_23_P213745 __________________________________________________________________ CXCL14 __________________________________________________________________ [143]NM_004887 __________________________________________________________________ 2.1824 __________________________________________________________________ A_23_P413641 __________________________________________________________________ PREX1 __________________________________________________________________ [144]NM_020820 __________________________________________________________________ 2.1719 __________________________________________________________________ A_23_P99063 __________________________________________________________________ LUM __________________________________________________________________ [145]NM_002345 __________________________________________________________________ 2.1563 __________________________________________________________________ A_23_P75056 __________________________________________________________________ GATA3 __________________________________________________________________ [146]NM_001002295 __________________________________________________________________ 2.0939 __________________________________________________________________ A_24_P322771 __________________________________________________________________ TFF1 __________________________________________________________________ [147]NM_003225 __________________________________________________________________ 2.0888 __________________________________________________________________ A_23_P119943 __________________________________________________________________ IGFBP2 __________________________________________________________________ [148]NM_000597 __________________________________________________________________ 2.0290 __________________________________________________________________ A_23_P161659 __________________________________________________________________ SYT13 __________________________________________________________________ [149]NM_020826 __________________________________________________________________ 1.9984 __________________________________________________________________ A_23_P329768 __________________________________________________________________ GREB1 __________________________________________________________________ [150]NM_014668 __________________________________________________________________ 1.9528 __________________________________________________________________ A_23_P105212 __________________________________________________________________ THRSP __________________________________________________________________ [151]NM_003251 __________________________________________________________________ 1.9163 __________________________________________________________________ A_23_P95594 __________________________________________________________________ NAT1 __________________________________________________________________ [152]NM_000662 __________________________________________________________________ 1.9107 __________________________________________________________________ A_24_P264943 __________________________________________________________________ COMP __________________________________________________________________ [153]NM_000095 __________________________________________________________________ 1.8625 __________________________________________________________________ A_23_P89431 __________________________________________________________________ CCL2 __________________________________________________________________ [154]NM_002982 __________________________________________________________________ 1.8472 __________________________________________________________________ A_32_P133072 __________________________________________________________________ SPON1 __________________________________________________________________ [155]NM_006108 __________________________________________________________________ 1.8374 __________________________________________________________________ A_23_P33196 __________________________________________________________________ COL5A2 __________________________________________________________________ [156]NM_000393 __________________________________________________________________ 1.8357 __________________________________________________________________ A_23_P2920 __________________________________________________________________ SERPINA3 __________________________________________________________________ [157]NM_001085 __________________________________________________________________ 1.8217 __________________________________________________________________ A_23_P22970 __________________________________________________________________ PIK3R3 __________________________________________________________________ [158]NM_003629 __________________________________________________________________ 1.7963 __________________________________________________________________ A_23_P165778 __________________________________________________________________ MLPH __________________________________________________________________ [159]NM_024101 __________________________________________________________________ 1.7915 __________________________________________________________________ A_32_P184464 __________________________________________________________________ ROPN1 __________________________________________________________________ [160]NM_017578 __________________________________________________________________ −3.5588 __________________________________________________________________ A_23_P328545 __________________________________________________________________ GABRP __________________________________________________________________ [161]NM_014211 __________________________________________________________________ −2.9846 __________________________________________________________________ A_23_P66137 __________________________________________________________________ SOX8 __________________________________________________________________ [162]NM_014587 __________________________________________________________________ −2.7293 __________________________________________________________________ A_24_P417407 __________________________________________________________________ ROPN1B __________________________________________________________________ [163]NM_001012337 __________________________________________________________________ −2.5309 __________________________________________________________________ A_23_P53176 __________________________________________________________________ FOLR1 __________________________________________________________________ [164]NM_016725 __________________________________________________________________ −2.3657 __________________________________________________________________ A_23_P56197 __________________________________________________________________ CRLF1 __________________________________________________________________ [165]NM_004750 __________________________________________________________________ −2.2530 __________________________________________________________________ A_23_P369343 __________________________________________________________________ KLK8 __________________________________________________________________ [166]NM_144505 __________________________________________________________________ −2.2248 __________________________________________________________________ A_24_P236251 __________________________________________________________________ DLK1 __________________________________________________________________ [167]NM_003836 __________________________________________________________________ −2.1531 __________________________________________________________________ A_23_P78248 __________________________________________________________________ KRT23 __________________________________________________________________ [168]NM_015515 __________________________________________________________________ −2.1320 __________________________________________________________________ A_23_P47484 __________________________________________________________________ GLYATL2 __________________________________________________________________ [169]NM_145016 __________________________________________________________________ −2.1208 __________________________________________________________________ A_23_P10127 __________________________________________________________________ SFRP1 __________________________________________________________________ [170]NM_003012 __________________________________________________________________ −2.0505 __________________________________________________________________ A_23_P40108 __________________________________________________________________ COL9A3 __________________________________________________________________ [171]NM_001853 __________________________________________________________________ −2.0500 __________________________________________________________________ A_23_P47616 __________________________________________________________________ FOLH1 __________________________________________________________________ [172]NM_004476 __________________________________________________________________ −1.8461 __________________________________________________________________ A_23_P50815 __________________________________________________________________ TTYH1 __________________________________________________________________ [173]NM_020659 __________________________________________________________________ −1.7817 __________________________________________________________________ A_23_P137173 __________________________________________________________________ TMSL8 __________________________________________________________________ [174]NM_021992 __________________________________________________________________ −1.7054 __________________________________________________________________ A_23_P110234 __________________________________________________________________ CSN1S1 __________________________________________________________________ [175]NM_001890 __________________________________________________________________ −1.6859 __________________________________________________________________ A_23_P216448 __________________________________________________________________ NFIB __________________________________________________________________ [176]NM_005596 __________________________________________________________________ −1.6243 __________________________________________________________________ A_23_P78980 __________________________________________________________________ B3GNT3 __________________________________________________________________ [177]NM_014256 __________________________________________________________________ −1.5946 __________________________________________________________________ A_24_P924484 __________________________________________________________________ [178]K03200 __________________________________________________________________ [179]K03200 __________________________________________________________________ −1.5836 __________________________________________________________________ A_32_P157391 __________________________________________________________________ PSMAL __________________________________________________________________ [180]NM_153696 __________________________________________________________________ −1.4833 __________________________________________________________________ A_23_P110837 __________________________________________________________________ IRX4 __________________________________________________________________ [181]NM_016358 __________________________________________________________________ −1.4820 __________________________________________________________________ A_23_P43157 __________________________________________________________________ MYBL1 __________________________________________________________________ [182]X66087 __________________________________________________________________ −1.4800 __________________________________________________________________ A_23_P59960 __________________________________________________________________ CRISPLD1 __________________________________________________________________ [183]NM_031461 __________________________________________________________________ −1.4630 __________________________________________________________________ A_23_P422212 __________________________________________________________________ SLC35F3 __________________________________________________________________ [184]NM_173508 __________________________________________________________________ −1.4618 __________________________________________________________________ A_23_P37205 NDRG2 [185]NM_201535 −1.4531 [186]Open in a new tab Table 4. GSEA summary of pathways associated with genes up regulated in IGFBP2 positive tumors NAME SIZE NOM p-val FDR q-val FWER p-val KEGG_MAPK_SIGNALING_PATHWAY __________________________________________________________________ 43 __________________________________________________________________ 0 __________________________________________________________________ 0 __________________________________________________________________ 0 __________________________________________________________________ KEGG_LEISHMANIA_INFECTION __________________________________________________________________ 20 __________________________________________________________________ 0 __________________________________________________________________ 0 __________________________________________________________________ 0 __________________________________________________________________ KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION __________________________________________________________________ 35 __________________________________________________________________ 0 __________________________________________________________________ 0.007717 __________________________________________________________________ 0.013 __________________________________________________________________ KEGG_SYSTEMIC_LUPUS_ERYTHEMATOSUS __________________________________________________________________ 19 __________________________________________________________________ 0.001976 __________________________________________________________________ 0.006687 __________________________________________________________________ 0.015 __________________________________________________________________ KEGG_T_CELL_RECEPTOR_SIGNALING_PATHWAY __________________________________________________________________ 19 __________________________________________________________________ 0.00211 __________________________________________________________________ 0.016915 __________________________________________________________________ 0.045 __________________________________________________________________ KEGG_FOCAL_ADHESION __________________________________________________________________ 30 __________________________________________________________________ 0.007619 __________________________________________________________________ 0.025984 __________________________________________________________________ 0.084 __________________________________________________________________ KEGG_COMPLEMENT_AND_COAGULATION_CASCADES __________________________________________________________________ 16 __________________________________________________________________ 0.015968 __________________________________________________________________ 0.023016 __________________________________________________________________ 0.087 __________________________________________________________________ KEGG_ECM_RECEPTOR_INTERACTION __________________________________________________________________ 15 __________________________________________________________________ 0.002053 __________________________________________________________________ 0.022288 __________________________________________________________________ 0.096 __________________________________________________________________ KEGG_CHEMOKINE_SIGNALING_PATHWAY __________________________________________________________________ 32 __________________________________________________________________ 0.008403 __________________________________________________________________ 0.020502 __________________________________________________________________ 0.1 __________________________________________________________________ KEGG_APOPTOSIS __________________________________________________________________ 20 __________________________________________________________________ 0.008584 __________________________________________________________________ 0.018452 __________________________________________________________________ 0.1 __________________________________________________________________ KEGG_VIRAL_MYOCARDITIS __________________________________________________________________ 17 __________________________________________________________________ 0.005906 __________________________________________________________________ 0.017723 __________________________________________________________________ 0.105 __________________________________________________________________ KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY __________________________________________________________________ 27 __________________________________________________________________ 0.028902 __________________________________________________________________ 0.037213 __________________________________________________________________ 0.228 __________________________________________________________________ KEGG_GNRH_SIGNALING_PATHWAY __________________________________________________________________ 19 __________________________________________________________________ 0.044444 __________________________________________________________________ 0.113272 __________________________________________________________________ 0.563 __________________________________________________________________ KEGG_CELL_ADHESION_MOLECULES_CAMS __________________________________________________________________ 26 __________________________________________________________________ 0.095808 __________________________________________________________________ 0.195608 __________________________________________________________________ 0.805 __________________________________________________________________ KEGG_ALZHEIMERS_DISEASE __________________________________________________________________ 24 __________________________________________________________________ 0.087302 __________________________________________________________________ 0.183226 __________________________________________________________________ 0.805 __________________________________________________________________ KEGG_PATHWAYS_IN_CANCER __________________________________________________________________ 48 __________________________________________________________________ 0.098 __________________________________________________________________ 0.185527 __________________________________________________________________ 0.831 __________________________________________________________________ KEGG_AXON_GUIDANCE __________________________________________________________________ 17 __________________________________________________________________ 0.11553 __________________________________________________________________ 0.176978 __________________________________________________________________ 0.834 __________________________________________________________________ KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION __________________________________________________________________ 24 __________________________________________________________________ 0.136538 __________________________________________________________________ 0.210733 __________________________________________________________________ 0.893 __________________________________________________________________ KEGG_DILATED_CARDIOMYOPATHY __________________________________________________________________ 15 __________________________________________________________________ 0.165339 __________________________________________________________________ 0.237117 __________________________________________________________________ 0.925 __________________________________________________________________ KEGG_NEUROTROPHIN_SIGNALING_PATHWAY __________________________________________________________________ 19 __________________________________________________________________ 0.168932 __________________________________________________________________ 0.240447 __________________________________________________________________ 0.932 __________________________________________________________________ KEGG_MELANOGENESIS __________________________________________________________________ 15 __________________________________________________________________ 0.252033 __________________________________________________________________ 0.367091 __________________________________________________________________ 0.99 __________________________________________________________________ KEGG_LYSOSOME __________________________________________________________________ 18 __________________________________________________________________ 0.329389 __________________________________________________________________ 0.436596 __________________________________________________________________ 0.996 __________________________________________________________________ KEGG_CALCIUM_SIGNALING_PATHWAY __________________________________________________________________ 19 __________________________________________________________________ 0.367886 __________________________________________________________________ 0.427407 __________________________________________________________________ 0.996 __________________________________________________________________ KEGG_WNT_SIGNALING_PATHWAY __________________________________________________________________ 24 __________________________________________________________________ 0.361616 __________________________________________________________________ 0.414545 __________________________________________________________________ 0.996 __________________________________________________________________ KEGG_ENDOCYTOSIS __________________________________________________________________ 20 __________________________________________________________________ 0.365503 __________________________________________________________________ 0.423908 __________________________________________________________________ 0.998 __________________________________________________________________ KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION __________________________________________________________________ 15 __________________________________________________________________ 0.419958 __________________________________________________________________ 0.454951 __________________________________________________________________ 0.999 __________________________________________________________________ KEGG_HUNTINGTONS_DISEASE __________________________________________________________________ 18 __________________________________________________________________ 0.568465 __________________________________________________________________ 0.60777 __________________________________________________________________ 1 __________________________________________________________________ KEGG_REGULATION_OF_ACTIN_CYTOSKELETON __________________________________________________________________ 29 __________________________________________________________________ 0.74269 __________________________________________________________________ 0.769736 __________________________________________________________________ 1 __________________________________________________________________ KEGG_PURINE_METABOLISM 20 0.933054 0.931545 1 [187]Open in a new tab NOM P nominal P value. Figure 2. [188]Figure 2 [189]Open in a new tab Differential expression of genes in IGFBP2 positive and IGFBP2 negative tumor samples compared to control tissues. a) Genes were clustered using MeV software. The dendrogram on the left shows different clusters of genes segregated according to the pattern of regulation. Red and green indicate high and low expression of genes respectively. Black indicates no regulation. b) Validation of selected genes. Scatter plots of differentially regulated genes in IGFBP2 positive and IGFBP2 negative tumors compared to the expression in normal tissues. Log 2-transformed gene expression ratios obtained from real-time quantitative PCR analysis normalized to TBP expression are plotted. Each dot represents a data derived from one sample. Common genes differentially expressed in breast tumors and cell lines based on IGFBP2 expression In the previous experiments, we identified genes differentially expressed in breast tumors and breast cancer cells lines based on IGFBP2 expression. In order to identify the genes commonly regulated by IGFBP2 in cell lines and tumors, we compared the gene expression profiles of IGFBP2 positive versus negative tumors and IGFBP2 knockdown breast cancer cells. 654 probes were found to be common among IGFBP2 regulated genes in tumors and cell line. Among these 412 probes were down regulated in IGFBP2 positive tumors and up regulated upon IGFBP2 knockdown while 242 probes were up regulated in IGFBP2 positive tumors and down regulated upon IGFBP2 knockdown (Additional file [190]5: Table S3). Some genes that are differentially regulated in both are shown in Table [191]5. Genes such as FBLN1, ID1, FN1, LMO2, DCK, TLR4 which have important roles in tumor progression were up regulated in IGFBP2 positive tumors and were decreased upon IGFBP2 knockdown in breast cancer cells whereas genes such as SRPRB, POPDC3, ARHGEF4, KCNN4, BC11A which have negative role in tumorigenesis were down regulated in IGFBP2 positive tumors and were up regulated in IGFBP2 negative cells (p < 0.05). These results indicate that these genes or the pathways associated with these genes could be truly regulated by IGFBP2 in breast cancer. Some of these genes/pathways may have a role in IGFBP2 mediated tumor progression. Table 5. List of top 50 common genes differentially regulated between IGFBP2 positive tumors and IGFBP2 knockdown clones Probe Gene symbol Accession no. A_23_P119943 __________________________________________________________________ IGFBP2 __________________________________________________________________ [192]NM_000597 __________________________________________________________________ A_23_P118392 __________________________________________________________________ RASD1 __________________________________________________________________ [193]NM_016084 __________________________________________________________________ A_23_P211631 __________________________________________________________________ FBLN1 __________________________________________________________________ [194]NM_006486 __________________________________________________________________ A_23_P53126 __________________________________________________________________ LMO2 __________________________________________________________________ [195]NM_005574 __________________________________________________________________ A_23_P22433 __________________________________________________________________ RP2 __________________________________________________________________ [196]NM_006915 __________________________________________________________________ A_23_P252306 __________________________________________________________________ ID1 __________________________________________________________________ [197]NM_002165 __________________________________________________________________ A_24_P119745 __________________________________________________________________ FN1 __________________________________________________________________ [198]NM_212482 __________________________________________________________________ A_23_P70307 __________________________________________________________________ SMOC2 __________________________________________________________________ [199]NM_022138 __________________________________________________________________ A_23_P170986 __________________________________________________________________ TMCC1 __________________________________________________________________ [200]NM_001017395 __________________________________________________________________ A_32_P140139 __________________________________________________________________ F13A1 __________________________________________________________________ [201]NM_000129 __________________________________________________________________ A_23_P204850 __________________________________________________________________ RB1 __________________________________________________________________ [202]NM_000321 __________________________________________________________________ A_32_P216004 __________________________________________________________________ KCTD9 __________________________________________________________________ [203]AF130091 __________________________________________________________________ A_23_P83939 __________________________________________________________________ SYAP1 __________________________________________________________________ [204]NM_032796 __________________________________________________________________ A_23_P259438 __________________________________________________________________ DCK __________________________________________________________________ [205]NM_000788 __________________________________________________________________ A_32_P28284 __________________________________________________________________ TPM4 __________________________________________________________________ [206]NM_003290 __________________________________________________________________ A_23_P42257 __________________________________________________________________ IER3 __________________________________________________________________ [207]NM_003897 __________________________________________________________________ A_32_P152348 __________________________________________________________________ HIST1H2BD __________________________________________________________________ [208]BQ683489 __________________________________________________________________ A_32_P66881 __________________________________________________________________ TLR4 __________________________________________________________________ [209]NM_138554 __________________________________________________________________ A_32_P89709 __________________________________________________________________ TPM1 __________________________________________________________________ [210]NM_001018004 __________________________________________________________________ A_23_P380848 __________________________________________________________________ TXNL5 __________________________________________________________________ [211]NM_032731 __________________________________________________________________ A_24_P106297 __________________________________________________________________ AMACR __________________________________________________________________ [212]NM_014324 __________________________________________________________________ A_23_P401055 __________________________________________________________________ SOX2 __________________________________________________________________ [213]NM_003106 __________________________________________________________________ A_23_P89799 __________________________________________________________________ ACAA2 __________________________________________________________________ [214]NM_006111 __________________________________________________________________ A_23_P500799 __________________________________________________________________ CASP6 __________________________________________________________________ [215]NM_001226 __________________________________________________________________ A_23_P134237 __________________________________________________________________ RARRES2 __________________________________________________________________ [216]NM_002889 __________________________________________________________________ A_23_P113952 __________________________________________________________________ [217]AY227436 __________________________________________________________________ [218]AY227436 __________________________________________________________________ A_23_P88095 __________________________________________________________________ TBC1D4 __________________________________________________________________ [219]NM_014832 __________________________________________________________________ A_23_P369343 __________________________________________________________________ KLK8 __________________________________________________________________ [220]NM_144505 __________________________________________________________________ A_23_P371039 __________________________________________________________________ NTSR1 __________________________________________________________________ [221]NM_002531 __________________________________________________________________ A_24_P844917 __________________________________________________________________ [222]AF222023 __________________________________________________________________ [223]AF222023 __________________________________________________________________ A_23_P90601 __________________________________________________________________ STEAP3 __________________________________________________________________ [224]NM_182915 __________________________________________________________________ A_24_P57047 __________________________________________________________________ DLL3 __________________________________________________________________ [225]NM_203486 __________________________________________________________________ A_23_P363426 __________________________________________________________________ SRP46 __________________________________________________________________ [226]NM_032102 __________________________________________________________________ A_23_P204751 __________________________________________________________________ ACCN2 __________________________________________________________________ [227]NM_020039 __________________________________________________________________ A_23_P11262 __________________________________________________________________ F8A1 __________________________________________________________________ [228]NM_012151 __________________________________________________________________ A_23_P376591 __________________________________________________________________ CLYBL __________________________________________________________________ [229]NM_206808 __________________________________________________________________ A_23_P80773 __________________________________________________________________ SRPRB __________________________________________________________________ [230]NM_021203 __________________________________________________________________ A_23_P253221 __________________________________________________________________ ARHGEF4 __________________________________________________________________ [231]NM_032995 __________________________________________________________________ A_24_P163237 __________________________________________________________________ STOX2 __________________________________________________________________ [232]NM_020225 __________________________________________________________________ A_23_P8240 __________________________________________________________________ FAM50B __________________________________________________________________ [233]NM_012135 __________________________________________________________________ A_23_P67529 __________________________________________________________________ KCNN4 __________________________________________________________________ [234]NM_002250 __________________________________________________________________ A_24_P411186 __________________________________________________________________ BCL11A __________________________________________________________________ [235]NM_022893 __________________________________________________________________ A_23_P358597 __________________________________________________________________ POPDC3 __________________________________________________________________ [236]NM_022361 __________________________________________________________________ A_23_P78518 __________________________________________________________________ CEACAM19 __________________________________________________________________ [237]AK128234 __________________________________________________________________ A_23_P91702 __________________________________________________________________ EIF3S7 __________________________________________________________________ [238]NM_003753 __________________________________________________________________ A_23_P415558 __________________________________________________________________ ZNF212 __________________________________________________________________ [239]NM_012256 __________________________________________________________________ A_24_P414269 __________________________________________________________________ ALG3 __________________________________________________________________ [240]NM_005787 __________________________________________________________________ A_23_P130764 __________________________________________________________________ KCNJ14 __________________________________________________________________ [241]NM_170720 __________________________________________________________________ A_24_P152635 __________________________________________________________________ TXNDC14 __________________________________________________________________ [242]NM_015959 __________________________________________________________________ A_23_P82478 PUS7 [243]NM_019042 [244]Open in a new tab KEGG pathway analysis of common differentially regulated genes between IGFBP2 perturbed cells and IGFBP2 positive tumors revealed that the regulated genes belong to Glioma, Oxidative Phosphorylation, Apoptosis, Pathways in cancer and ErbB signaling pathway (Additional file [245]6: Table S4).Taken together, these data indicate that tumors with IGFBP2 expression phenotype are associated with distinct changes in expression of genes associated with the regulation of cell proliferation and tumorigenicity. β-catenin expression is regulated by IGFBP2 in breast cancer cells Since the GSEA analysis of differentially expressed genes in both tumors and knockdown cells revealed significant regulation of Wnt signaling pathway, we decided to examine if IGFBP2 regulates Wnt pathway. As β-catenin is an effector of Wnt pathway we determined β-catenin expression in IGFBP2 knockdown cells. As shown in Figure [246]3, knockdown of IGFBP2 in BT474 breast cancer cells substantially decreased the expression of β-catenin in both the clones C5 and C12, suggesting a direct regulation of β-catenin by IGFBP2. In good correlation, when IGFBP2 expression is restored in the knockdown cells, β-catenin expression is also restored (Figure [247]4). These results collectively demonstrate regulation of β-catenin expression by IGFBP2. Figure 3. [248]Figure 3 [249]Open in a new tab β-catenin and IGFBP2 expression in IGFBP2 knockdown and control cells. IGFBP2 knockdown clones and control cells were plated on coverslips and allowed to grow. 24 h after plating, cells were fixed, permeabilized, and stained for β-catenin and IGFBP2 expression. Expression of β-catenin and IGFBP2 is shown in green and blue, respectively. Nucleus was stained using propidium iodide (PI) as shown in red. Original magnification was 63×. Figure 4. [250]Figure 4 [251]Open in a new tab Regulation of β-catenin by IGFBP2 is IGF1R and FAK dependent. IGFBP2 knockdown clone C12 was transfected with IGFBP2 and 36 h. after transfection cells were fixed and analyzed for β-catenin, and IGFBP2. For inhibitor experiments, 24 h. after transfection cells were treated with IGF1R or FAK inhibitor for 12 h. Cells were fixed and analyzed for β-catenin and IGFBP2 expression. Expression of β-catenin and IGFBP2 is shown in green and blue, respectively. Nucleus was stained using propidium iodide (PI) as shown in red. Original magnification was 63×. V, Vector control; OE, Over expression; Inh, inhibitor. It has been known that some of the IGFBP2 actions are mediated in part by the activation of IGF1 receptor and also through integrin receptors [[252]20]. Hence, in order to identify the intermediates of IGFBP2 regulation of β-catenin, we studied the effect of IGF1R inhibitor (PPP, 10 μM) and Focal Adhesion Kinase inhibitor (PP2, 10 μM) on the regulation of β-catenin by IGFBP2. As described above, over expression of IGFBP2 in the knockdown clones increased β-catenin expression and in the presence of IGF1R inhibitor or FAK inhibitor, IGFBP2 induced β-catenin expression was abolished (Figure [253]4). Similar results were obtained using MDA-MB-231 cells which lack endogenous IGFBP2 expression (Additional file [254]7: Figure S3). These results suggest that IGFBP2 regulates β-catenin expression in an IGF1R and integrin dependent manner. IGFBP2 and β-catenin staining together correlates with the lymph node metastasis in human breast cancer Since the previous results showed an increase in β-catenin expression upon IGFBP2 over expression, we sought to examine the correlation of β-catenin and IGFBP2 staining in human breast cancer tissues. Towards this we performed IHC on 38 grade III Invasive Ductal Carcinoma tissues for β-catenin and IGFBP2 expression. A representative staining pattern of IGFBP2 and β-catenin expression is depicted in Figure [255]5. It was observed that 27 out of 38 tumors stained positive for IGFBP2. There was a positive correlation between IGFBP2 and β-catenin expression with 26 out of 27 IGFBP2 positive tumor samples also staining positive for β-catenin (Table [256]6). Tissues with β-catenin expression exhibited a heterogeneous mixture of membranous and cytosolic β-catenin accumulation. In addition, more lymph node metastasis was observed in patients positive for both IGFBP2 and β-catenin proteins (18/24, 75%) compared with patients with low levels of both proteins (1/24, 4%) (p = 0.0006). Figure 5. [257]Figure 5 [258]Open in a new tab β-catenin and IGFBP2 expression in breast cancer tissues. Representative micrographs showing β-catenin staining in cancer tissues. a β-catenin staining of a section of IDC 3 breast tumors showing cytoplasmic and membrane staining. b represents breast cancer tissue section stained for IGFBP2 showing predominantly cytoplasmic staining. Table 6. IGFBP2 and β-catenin expression in breast cancer tissues S. No. Case No. IGFBP2 β-catenin Nodal status 1 __________________________________________________________________ 160 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 2 __________________________________________________________________ 159 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ - __________________________________________________________________ 3 __________________________________________________________________ 157 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ - __________________________________________________________________ 4 __________________________________________________________________ 171 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 5 __________________________________________________________________ 150 __________________________________________________________________ + __________________________________________________________________ - __________________________________________________________________ + __________________________________________________________________ 6 __________________________________________________________________ 151 __________________________________________________________________ - __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 7 __________________________________________________________________ 180 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ - __________________________________________________________________ 8 __________________________________________________________________ 176 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 9 __________________________________________________________________ 174 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 10 __________________________________________________________________ 168 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 11 __________________________________________________________________ 165 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 12 __________________________________________________________________ 162 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ - __________________________________________________________________ 13 __________________________________________________________________ 149 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 14 __________________________________________________________________ 148 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 15 __________________________________________________________________ 143 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 16 __________________________________________________________________ 142 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 17 __________________________________________________________________ 140 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 18 __________________________________________________________________ 138 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ - __________________________________________________________________ 19 __________________________________________________________________ 137 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ - __________________________________________________________________ 20 __________________________________________________________________ 134 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 21 __________________________________________________________________ 133 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ - __________________________________________________________________ 22 __________________________________________________________________ 132 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ - __________________________________________________________________ 23 __________________________________________________________________ 130 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 24 __________________________________________________________________ 70 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 25 __________________________________________________________________ 94 __________________________________________________________________ - __________________________________________________________________ - __________________________________________________________________ - __________________________________________________________________ 26 __________________________________________________________________ 87 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 27 __________________________________________________________________ 49 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 28 __________________________________________________________________ 63 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 29 __________________________________________________________________ 117 __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 30 __________________________________________________________________ 108 __________________________________________________________________ - __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 31 __________________________________________________________________ 96 __________________________________________________________________ - __________________________________________________________________ - __________________________________________________________________ - __________________________________________________________________ 32 __________________________________________________________________ 85 __________________________________________________________________ - __________________________________________________________________ + __________________________________________________________________ - __________________________________________________________________ 33 __________________________________________________________________ 77 __________________________________________________________________ - __________________________________________________________________ - __________________________________________________________________ + __________________________________________________________________ 34 __________________________________________________________________ 48 __________________________________________________________________ - __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 35 __________________________________________________________________ 57 __________________________________________________________________ - __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 36 __________________________________________________________________ 78 __________________________________________________________________ - __________________________________________________________________ + __________________________________________________________________ + __________________________________________________________________ 37 __________________________________________________________________ 30 __________________________________________________________________ - __________________________________________________________________ - __________________________________________________________________ - __________________________________________________________________ 38 31 - - - [259]Open in a new tab No significant association of combined expression of IGFBP2 and β-catenin was observed with ER, PR, Her2 or triple negative receptor status of breast tumors. Discussion Enhanced expression of IGFBP2 is associated with a large number of malignant cancers that include tumors of breast, ovarian, glioma and prostate. Primarily known for its growth inhibitory actions in physiological context, IGFBP2 has now been shown to promote growth and tumorigenesis in numerous cancer cells such as glioma, prostate and colon cancers [[260]15-[261]18]. To gain further insights into the role of IGFBP2 in breast cancer, we have attempted to identify the molecular players in IGFBP2 associated tumorigenesis in breast cancer. To elucidate the molecular targets of IGFBP2, we perturbed IGFBP2 expression by shRNA and the differential gene expression was determined using whole genome microarrays. IGFBP2 knockdown resulted in significant changes in the expression of genes associated with cellular proliferation and tumorigenicity. The down regulated genes were found to be associated with several pathways, notably Cell cycle, p53 and Wnt pathways as revealed by GSEA. Comparison of our data with a previous microarray study of IGFBP2 regulated genes in glioma cells [[262]29] revealed an overlap of about 22% genes with wild type IGFBP2 over expressing cells and 23% genes with RGE mutant IGFBP2 over expressing cells. Pathway comparisons revealed Cell cycle, p53 signaling, oxidative phosphorylation, nucleotide metabolism and Wnt signaling pathway to be common among the two data sets (Additional file [263]8: Figure S4). To further validate these results in breast cancer tissues, we performed whole genome expression analysis in 19 breast tumors which were categorized as IGFBP2 positive or negative based on immunohistochemical staining pattern. Compared to IGFBP2 negative tumors, IGFBP2 positive tumors showed increased expression of genes belonging to MAPK signaling, Focal adhesion and Wnt signaling. IGFBP2 correlation with proliferation has been studied extensively in several tumor cells including in breast cancer cells. The effect of IGFBP2 on proliferation has been shown to be context dependent. In prostate, ovarian, nephroblastoma cells, it has a pro proliferative action [[264]19,[265]21-[266]24]. In contrast IGFBP2 has an antiproliferative effect on HEK, Hs578T [[267]32,[268]33]. Our data on the regulation of different pathways such as MAPK, Cell cycle, Focal adhesion and Wnt corroborate the reported functional significance of IGFBP2 with respect to its pro proliferative and tumor promoting roles in breast cancer cells. One of the important and novel findings from this study is the regulation of Wnt signaling pathway genes by IGFBP2. So far, only IGFBP4 has been reported to activate Wnt signaling pathway in renal cell carcinoma [[269]34]. Activation of canonical Wnt signaling promotes tumorigenesis by regulating cell survival, proliferation and invasion of many cancers [[270]35]. In numerous tumors cytoplasmic and/or nuclear accumulation of β-catenin has been shown to be a strong indicator of aberrant Wnt pathway activation. Elevated cytosolic and nuclear accumulation of β-catenin has been associated with a variety of malignancies and inversely correlated with patient survival [[271]36-[272]39], Wnt activation leads to stabilization and translocation of β-catenin from cytoplasm to the nucleus where it associates with T-cell factor (TCF)/lymphocyte enhancer transcription (LEF) factors to activate target genes that are involved in cell survival, proliferation, and invasion [[273]40,[274]41]. In order to establish Wnt pathway activation by IGFBP2, we examined the canonical Wnt signaling target, β-catenin in IGFBP2 knockdown breast cancer cells. Compared to Vector transfected cells, IGFBP2 knockdown cells showed remarkably decreased levels of β-catenin. When IGFBP2 was re expressed in the knockdown cells, as expected there was substantial increase in β-catenin levels indicating that IGFBP2 regulates β-catenin. Interestingly, inhibition of IGF1R or integrin signaling resulted in the loss of β-catenin regulation by IGFBP2. These data suggest that IGFBP2 acts through IGF1R and integrin pathways in the regulation of β-catenin. Although the mechanisms are not clear, recently Uzoh et al. demonstrated an increased proliferation of prostate cancer cells by IGFBP2 in an IGF1R dependent manner [[275]20]. It is also known that IGF independent actions of IGFBP2 are mediated by the activation of integrin signaling through RGD motif present in the C-terminal region of IGFBP2 protein [[276]30]. Role of integrin receptors in pro-tumorigenic action of tumor cells is well established [[277]42,[278]43]. Hence, it is conceivable that activation of integrin signaling by IGFBP2 leading to FAK phosphorylation may be an important step in the activation of IGF1R by IGFBP2. In congruence with this, it has been reported that activated FAK phosphorylates and stabilizes IGF1R in mouse embryonic fibroblast [[279]44]. Very recently, IGFBP2 in association with IGF1 was found to activate IGF1R in endothelial cells [[280]45]. Taken together, regulation of Wnt pathway by IGFBP2 involves FAK and IGF1R in breast carcinogenesis. However, the mechanism (s) by which FAK and IGF1R signaling converge on the regulation of Wnt pathway by IGFBP2 needs further investigations. Another important finding from our data is the correlation of IGFBP2 over expression with elevated β-catenin levels in breast tumors. In humans, breast tumors frequently exhibit elevated levels of IGFBP2 [[281]12] and β-catenin, with higher expression levels of β-catenin correlating with a decreased patient survival [[282]39]. In mice, over expression of an activated β-catenin leads to the development of mammary hyperplasia and adenocarcinomas [[283]46]. These studies coupled with our data suggest that regulation of β-catenin could be an important step for the pro-tumorigenic actions of IGFBP2. Most significantly, when both IGFBP2 and β-catenin expression was correlated with the lymph node status of breast cancers, we found a significant association of IGFBP2 and β-catenin staining with increased lymph node metastasis in comparison with tumors which did not show staining for either protein. Interestingly, in a previous report, expression of IGFBP2 and IGFBP5 were correlated with increased lymph node metastasis in T1 breast carcinoma. However our data shows a significant positive correlation of IGFBP2 and β-catenin in lymph node metastasis. Hence, evaluation of IGFBP2, IGFBP5 along with β-catenin may provide a stronger predictive value for the prognosis of breast cancer. Conclusion This study highlights the pathways and genes regulated by IGFBP2 in breast cancer. Most importantly, this study reports regulation of β-catenin by IGFBP2 and their association in the lymph node metastasis. These findings are highly relevant in the prediction of breast cancer progression. Methods All the tissues for this study were collected after obtaining written informed consent from the patients. This study and the protocols were approved by the Institutional Ethics Committee of Kidwai Memorial Institute of Oncology, where the patients were treated. Cell culture and transfection BT474, a breast cancer cell-line was cultured in DMEM (Sigma-Aldrich, USA) with 10% foetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin, 2.5 μg/ml fungizone (Invitrogen Life Sciences, USA). All the cells were maintained at 37°C in a humid atmosphere with 5% CO[2]. Transfections were performed using Lipofectamine 2000 (Invitrogen) based on the manufacturer’s instructions. In brief, breast cancer cells were transfected with IGFBP2 shRNA expression vector (Origene, Cat no. TR316590) or empty vector (Origene, Cat no. TR20003) and 48 hrs after transfection puromycin (1 μg/ml, Calbiochem) was added to the growth medium. Selection medium was replaced every 2–3 days until individual clones could be identified. After 3 weeks of selection, fourteen puromycin resistant clones of BT474 cells were isolated and expanded in the selective medium. Two clones (C5 and C12) which showed significant down regulation of IGFBP2 expression were selected for further experiments Reversion of IGFBP2 expression in IGFBP2 knockdown cells was achieved by transfecting IGFBP2 cDNA sub cloned into pcDNA3.1 vector (Invitrogen). Pathway inhibitor treatments were performed using IGF1R inhibitor (PPP, 10 μM, Calbiochem Cat. No. 407247) and Focal Adhesion Kinase inhibitor (PP2, 10 μM, Calbiochem Cat. No. 529573). Immunoblot analysis For immunoblot analysis, cells were grown in growth medium till they achieved 50-70% confluency, washed with serum free DMEM and cultured in serum free medium for another 48 h. The spent medium was collected, concentrated using centrifugal filter units (Millipore, Amicon ultra-3 k) and equal amounts of protein as determined by the Bio-Rad DC protein assay (Bio-Rad, USA) were separated on 12.5-15% polyacrylamide gel and electrophoretically transferred onto PVDF membranes (Immobilin P, Millipore). Membranes were pre-incubated for 1 h with 5% non-fat dry milk (Fluka, Sigma-Aldrich) in Tris buffered-saline containing 0.1% Tween 20 (TBST) and then were incubated overnight with primary antibody. (IGFBP2 C-18: sc-6001, Santa Cruz Biotechnology, Inc, CA). Membranes were washed thrice for 15 min in TBST at room temperature, incubated with appropriate horseradish-peroxidase conjugated IgG (Sigma-Aldrich) at a dilution of 1:2000 for 1 h at room temperature and the complex detected using Super Signal West Femto chemiluminescence (Pierce, Thermo Scientific), as per the manufacturer’s instructions. RNA extraction and gene expression profiling Total RNA from frozen tumor tissues and tumor cells was extracted using the TRI reagent (Sigma-Aldrich) according to the manufacturer’s protocol. The concentration of RNA was estimated by measuring the absorbance at 260 nm (Nano Drop ND-1000 spectrophotometer) and integrity was verified on a denaturing 1% MOPS-formaldehyde agarose gel followed by ethidium bromide staining. For expression profiling, microarray experiments using whole genome human arrays (4×44K, Agilent) were used. The microarray hybridizations were performed as described before [[284]47]. Microarray analysis was performed by R-Bioconductor (limma package) using subtract method for background correction [[285]48]. Loess normalization was applied for dye bias and Quantile normalization was applied for spatial variation [[286]49]. Linear model and empirical Bayes methods (limma) was used for assessing differentially regulated genes [[287]50]. Benjamini Hochberg correction was applied for P value correction. Hierarchical cluster was done by Mev4.1 using Euclidean distance metric. The data was clustered by averaged linkage [[288]51]. Adjusted p value cut-off was used as 0.05 for differentially regulated genes. Gene expression data are deposited into GEO (Clone arrays: [289]GSE40682, Breast cancer tissue arrays: [290]GSE40206). Real-time qPCR assay For RT-PCR, cDNA was synthesised from total RNA using the cDNA Archive kit (Applied Bio systems, USA). cDNA equivalent to 10 ng of total RNA was used for all the PCR reactions using Dynamo SYBR green mix (Finnzymes, Finland) in ABI Prism 7900HT sequence detection system (Applied Bio systems, USA). The sequences of the primers are shown in Additional file [291]9: Table S5. The analysis has been done using SDS 2.1 software (Applied Bio systems, USA). For normalization of RT-PCR data, ribosomal protein L35a (RPL 35a) and TATA Binding Protein (TBP) were used for cells and tissues, respectively. Immunoflourescence Cells were grown on sterile cover-slips till they were about 50% confluent. The growth medium was discarded; cells were washed twice with chilled DPBS and were fixed in ice cold methanol for 10 minutes at −20°C. The fixed cells were then washed with DPBS thrice. For blocking non-specific binding of the antibodies, the cells were incubated with 1% BSA in PBS for 60 min followed by overnight incubation with protein specific antibodies (β-catenin, 1:50; IGFBP2, 1:25) in a humidified chamber at 4°C. After the overnight incubation, the cells were washed thrice with PBS and incubated with the secondary antibody, 1:1500 dilution of alexa flur 488 (anti-rabbit, for β-catenin) and alexa flur 633 (anti-goat for IGFBP2) (Molecular probes, Invitrogen, USA) in PBS for 1 hour in dark. All steps thereafter were performed in the dark. After 1 h, the cells were again washed thrice with PBS and counterstained with 33 μg/ml Propidium Iodide for 5 minutes and mounted in anti‒fade solution on clean slides. The stained cells were visualized using a confocal microscope (LSM 510 Meta, Carl-Zeiss) and were photographed. Tissue samples and immunohistochemistry For histology, sections of breast tumor tissues were obtained from blocks archived in the Department of Pathology at the Kidwai Memorial Institute of Oncology (KMIO). The status of estrogen receptor (ER), progesterone receptor (PR), Her2/neu, and pathological data like tumor grade, size and lymph node status were obtained from the pathology records of the respective patients. Tissue sections (5 μm) from the paraffin embedded tumor specimens were collected on silane-coated slides and immunohistochemistry for IGFBP2 and β-catenin was performed on 38 samples. Antigen retrieval was done by heat treatment of the deparaffinised sections in Citrate buffer (10 mM; pH 6.0). After the initial processing steps, sections were incubated overnight with respective primary antibodies - IGFBP2 (C-18: sc-6001, Santa Cruz Biotechnology, Inc, CA) and β-catenin (C 2206, Sigma-Aldrich), at 4°C. This was followed by incubation with the linked streptavidin- biotinylated secondary antibody (Universal LSAB, DAKO, Denmark) for IGFBP2 and with supersensitive non-biotin horseradish peroxidase detection system (QD440-XAK, Biogenex) for β-catenin antibodies. 3, 3’-Diaminobenzidine (Sigma-Aldrich) was used as the chromogenic substrate. Evaluation of immunohistochemistry The scoring method used for IGFBP2 and β-catenin expression was based on semi quantitative scoring method as described before [[292]52] where both intensity and percentage of cells with positive staining were counted and a combined score was given. The combined score was arrived by the multiplication product of both the scores. The scores are, (1) percentage of cells: no staining = 0; 10% or less of cells stained = 1; 11–50% of cells stained = 2; and 50% or more of cells stained =3; (2) intensity: no staining = 0, weak staining = 1, moderate staining = 2, and strong staining = 3. Thus, the combined scores ranged from 0–9. Only scores from 4–9 were considered positive for staining. Statistical analysis Statistical significance for all experimental analyses (except microarray) was determined by Student’s t-test or one-way analysis of variance GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). For correlation analysis Fisher’s exact test was utilized. Competing interests The authors declare that they have no competing interests. Authors’ contributions PS planned, executed, interpreted experiments, manuscript writing; VRP, SP and AB executed some experiments; NK did the microarray analysis; MVK and GM contributed in the planning and recruitment of patients and samples, clinical evaluation; PK, planned the experiments; manuscript writing and resources. All authors read and approved the final manuscript. Supplementary Material Additional file 1 List of common probe between IGFBP2 Knockdown clones C5 and C12. [293]Click here for file^ (1.2MB, xls) Additional file 2: Figure S1 Gene expression changes in IGFBP2 knockdown cells upon IGFBP2 over expression. Cells were plated and 24 h later transfected with pcDNA3.1-IGFBP2 and /or pcDNA3.1 vector. 48 h post transfection, RNA was extracted and gene expression was analyzed by Semi quantitative RT-PCR analysis. Representative ethidium bromide gel shows the expression of genes regulated upon forced expression of IGFBP2 in a) clone C12 and b) clone C5. Expression values were quantitated and the graph (right) represents fold change over control after normalization with the expression of RPL35A. [294]Click here for file^ (429KB, ppt) Additional file 3 List of Differentially expressed probes between IGFBP2 positive and negative breast tumors. [295]Click here for file^ (940.5KB, xls) Additional file 4: Figure S2 Validation of Wnt target genes in IGFBP2 positive and IGFBP2 negative tumors. Scatter plots of differentially regulated genes in tumor tissues compared to the expression in normal tissues. Log 2-transformed gene expression ratios obtained from real-time quantitative PCR analysis normalized to TBP expression are plotted. Each dot represents data derived from one sample. [296]Click here for file^ (277.5KB, ppt) Additional file 5 List of differentially regulated probes common between IGFBP2 knockdown clones and IGFBP2 positive/negative tumors. [297]Click here for file^ (292.5KB, xls) Additional file 6 List of pathways associated with differentially regulated genes common between IGFBP2 knockdown clones and IGFBP2 positive/negative tumors. [298]Click here for file^ (28KB, xls) Additional file 7: Figure S3 Regulation of β-catenin by IGFBP2 is IGF1R and FAK dependent. MDA-MB-231 cells were transfected with IGFBP2 and 36 h. after transfection cells were fixed and analyzed for β-catenin and IGFBP2 protein. For inhibitor treatements, 24 h. after transfection cells were treated with IGF1R or FAK inhibitor for 12 h. Cells were fixed and analyzed for β-catenin and IGFBP2 expression. Expression of β-catenin and IGFBP2 is shown in green and blue, respectively. Nucleus was stained using propidium iodide (PI) as shown in red Original magnification was 63×. V, Vector control; OE, Over expression; Inh, inhibitor. [299]Click here for file^ (290.5KB, ppt) Additional file 8: Figure S4 Comparison of IGFBP2 regulated genes in knock down cells with other available data sets. a) Venn diagram showing genes common between IGFBP2 over expressing glioma cells (GEO accession no. [300]GSE35467) and IGFBP2 knock down breast cancer cells (Additional file [301]1: Table S1). b) Venn diagram showing genes common between RGE mutant IGFBP2 over expressing glioma cells (GEO accession no. [302]GSE35467) and IGFBP2 knock down breast cancer cells (Table S1). OV, over expression; KD, knockdown; RGE, RGE mutant IGFBP2. [303]Click here for file^ (269.5KB, ppt) Additional file 9 List of primer sequences used for qPCR. [304]Click here for file^ (29.5KB, xls) Contributor Information Priyanka Sehgal, Email: priay@mrdg.iisc.ernet.in. Neeraj Kumar, Email: neerajrana14@gmail.com. Varuvar Rajesh Praveen Kumar, Email: praveenjsr@mrdg.iisc.ernet.in. Shilpa Patil, Email: shilpa_sp@mrdg.iisc.ernet.in. Animesh Bhattacharya, Email: animesh.bhattacharya@gmail.com. Manavalan Vijaya Kumar, Email: mvijai2002@yahoo.com. Geetashree Mukherjee, Email: mukherjeegeeta@yahoo.co.in. Paturu Kondaiah, Email: paturu@mrdg.iisc.ernet.in. Acknowledgements