Abstract

   Urokinase plasminogen activator receptor (PLAUR) has been implicated in
   a variety of physiological and pathological conditions. The
   multi-functionality of PLAUR is due to its capacity to interact with
   many co-receptors to regulate extracellular proteolysis and
   intracellular signaling. Recent reports are identifying novel functions
   of PLAUR which were not evident in the past; however, the molecular
   mechanisms of PLAUR signaling are not completely understood. Here, we
   have compared the transcriptomes of silencing control (sicon) and PLAUR
   silenced (PLAURsi) MDA-MB-231 breast cancer cells on treatment with
   radiation. We isolated RNA from the cells, synthesized cDNA and
   measured the gene expression changes by microarray. We identified 24
   downregulated and 53 upregulated genes, which were significantly
   (P-value < 0.005) affected by PLAUR silencing. Our analysis revealed
   415 canonical pathways and 743 causal disease networks affected on
   silencing PLAUR. Transcriptomic changes and predicted pathways
   supported and consolidated some of the earlier understanding in the
   context of PLAUR signaling; including our recent observations in DNA
   damage and repair process. In addition, we have identified several
   novel pathways where PLAUR is implicated.

   Keywords: urokinase receptor, breast cancer, pathway analysis,
   microarray

INTRODUCTION

   High-throughput technologies such as microarray, genome sequencing,
   mass-spectrometry, and genome-wide association studies have been very
   helpful in deciphering the differences between normal and cancer cells
   [[34]1]. Protein profiling has helped us to understand the regulation
   of a huge number of proteins and also in the prediction of regulatory
   pathway networks. Despite decades of research on cancer, we are still
   missing links to its understanding. We try here to merge the high
   throughput power of microarray analysis and pathway prediction programs
   for better visualization and understanding of gene interaction
   networks.

   Most of the existing therapies for cancer involve the use of DNA
   damaging drugs and agents like radiation, which induce DNA lesions
   interfering with DNA replication and transcription [[35]2]. Even in a
   normal cell, DNA is constantly at risk of damage due to various
   endogenous and exogenous factors; it has been predicted that oxidative
   stress inside a cell can damage the DNA 10,000 times per day [[36]3].
   To battle DNA damage, cells have evolved numerous sophisticated repair
   mechanisms for specific kinds of damage; these mechanisms interact and
   overlap to maintain genome integrity.

   The role of urokinase plasminogen activator receptor (PLAUR) in cancer
   has been extensively studied and as per our knowledge, there is no data
   available which documents changes in the transcriptome of PLAUR
   silenced (PLAURsi) cells. PLAUR is a GPI anchored extracellular
   receptor and localizes the serine protease activity of its ligand
   urokinase plasminogen activator (PLAU) on the cell membrane [[37]4].
   Binding of PLAU leads to the activation of plasmin and generates a
   proteolytic cascade involving matrix metalloproteases. Thus, the
   PLAU/PLAUR system is involved in the regulation and remodeling of the
   extracellular matrix [[38]5, [39]6]. PLAUR is a multifunctional
   receptor and can interact with a variety of co-receptors for
   intracellular signaling [[40]4].

   PLAUR has been shown to regulate the migration and proliferation of
   pancreatic and breast cancer cells [[41]7, [42]8]. PLAUR overexpression
   has been observed in many cancers and is often associated with poor
   survival and prognosis [[43]9, [44]10]. Previously it was thought that
   PLAUR promotes cancer progression by regulation of extracellular
   proteolysis on the cell surface, but recent studies show that PLAUR is
   involved in many intracellular mechanisms promoting cell survival. It
   has been observed that PLAUR overexpressing cells have persistent
   activation of MAPK kinases, pathways involving tyrosine kinase
   receptors and also G-protein coupled receptors [[45]4, [46]5, [47]10].
   In agreement with this we have previously shown that PLAUR regulates
   the ubiquitin proteasome system during DNA damage response and
   silencing PLAUR impairs DNA repair [[48]11]. We have also recently
   demonstrated the role of PLAUR in regulating the homologous
   recombination (HR) DNA repair pathway in MDA-MB-231 and HeLa cells
   [[49]12].

   In this study, we were interested in observing the transcriptomic
   changes occurring in irradiated PLAURsi cells. We have used
   high-throughput experimental data to predict novel molecular pathways
   and functions, inferred from already existing knowledge from biological
   databases. The predicted pathways are in accordance with known
   literature from in vitro experiments. Our results can have a
   substantial impact on the understanding of gene interacting networks.

RESULTS

   Microarray data was obtained from sicon and PLAURsi MDA-MB-231 cells, 4
   h after irradiation of 9 Gy. This data was processed by two different
   methods (Figure [50]1A). The first method involved filtering the genes
   using Qlucore Omics explorer with a P-value cutoff of 0.05; this
   resulted in around 370 upregulated and 347 downregulated genes, these
   genes were further processed by ingenuity pathway analysis (IPA)
   software to reveal gene interacting networks and functional pathways.
   The second method involved pre-processing the data using Limma
   Bioconductor package in R to identify differentially regulated genes
   using a cutoff P-value < 0.05. Functional enrichment analysis was
   performed using the Database for Annotation, Visualization and
   Integrated Discovery (DAVID) for Gene ontology (GO) enrichment analysis
   and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
   analysis.

Figure 1. Schematics of microarray analysis on PLAURsi cells.

   [51]Figure 1
   [52]Open in a new tab

   (A) Flowchart showing the overview of microarray analysis. MDA-MB-231
   Cells were transfected with siRNA to silence PLAUR and then irradiated
   for induction of DNA damage, 4 h later RNA was isolated and hybridized
   on an Agilent chip in Dual color mode. Raw data obtained after feature
   extraction was analyzed using Qlucore omics explorer, followed by
   Ingenuity pathway analysis software (Method 1). Raw data was also used
   as an input for the limma bioconductor package in R, the shortlisted
   genes where processed by using DAVID and KEGG databases (Method 2).
   Both the methods were used to perform pathway analysis and genes were
   validated by qPCR; (B) MDA-MB-231 cells silenced for PLAUR were
   irradiated at 9 Gy, after 4 h protein lysates were made and subjected
   to western blotting for the detection of PLAUR.

   Western blotting of lysates from siRNA transfected cells confirmed that
   there was efficient silencing of PLAUR expression (Figure [53]1B). We
   had two parameters to assess, to see the effect of PLAUR silencing
   alone or effects of PLAUR silencing on induction of DNA damage; the 100
   most up- and down-regulated genes in these cases are shown in
   [54]Supplementary Tables 1-[55]4. We used Qlucore omics explorer to
   shortlist genes based on a P-value cutoff of 0.005, which resulted in
   53 upregulated and 24 downregulated genes; they have been represented
   as a heatmap (Figure [56]2A). Table [57]1 lists the genes in the
   heatmap along with their biological functions from UniProt [[58]13].

Figure 2. Differentially regulated genes and their functions which are
affected on silencing PLAUR.

   [59]Figure 2
   [60]Open in a new tab

   (A) Heat map depicting the differentially expressed genes with a
   P-value < 0.005; (B) Ingenuity Pathway Analysis of microarray data
   showing top 16 gene functions which are affected in PLAURsi cells on
   DNA damage.

Table 1. List of genes represented in the heatmap with known biological
functions.

   Gene Name p-value Fold change Biological Functions
   TTC3 2.89E-05 -0.84159 protein K48-linked ubiquitination,
   ubiquitin-dependent protein catabolic process
   LY6G6F 0.00058139 -0.66572 downstream signal transduction pathway
   SPTY2D1 0.00094336 -0.3819215 nucleosome assembly, regulation of
   chromatin assembly and transcription
   Down regulated BRCA2 0.00105166 -1.21529975 DNA damage response, DNA
   repair, double-strand break repair via homologous recombination
   WAPAL 0.00108458 -0.066805 cell division, regulation of chromosome
   segregation and chromatid cohesion
   PEX13 0.00139249 -0.414465 fatty acid alpha-oxidation, cell migration,
   mitophagy in response to mitochondrial depolarization
   MUTED 0.00140741 -0.1462995 vesicle-mediated transport
   [61]AK098147 0.00144621 -0.284735 unknown function
   PPP1R15B 0.00150601 -0.040375 peptidyl-serine dephosphorylation,
   endoplasmic reticulum stress-induced eIF2 alpha phosphorylation
   ENST00000390468 0.00231577 -0.0954755 unknown function
   LOC100270680 0.00235518 -1.437085 unknown function
   GOLGA6L6 0.00243931 -0.2633705 unknown function
   ENST00000326147 0.00248927 -0.8985405 unknown function
   A_33_P3232066 0.00266984 -0.0864295 unknown function
   DUX4 0.00284781 -0.43064 negative regulation of G0 to G1 transition,
   apoptotic process, transcription
   LOC100132733 0.00298165 -0.191127 unknown function
   COPB1 0.0031026 -0.19355765 intracellular protein transport, ER to
   Golgi vesicle-mediated transport
   ENST00000367860 0.00374651 -0.265195 unknown function
   RRM2B 0.00382213 -0.114055 DNA repair, mitochondrial DNA replication,
   deoxyribonucleotide biosynthetic process
   HELZ 0.00421811 -0.892889 cellular lipid metabolic process,
   transcription
   A_24_P704878 0.00422942 -1.1351695 unknown function
   ACP5 0.00453005 -0.46311 bone morphogenesis and resorption, osteoclast
   differentiation, positive regulation of cell migration
   PCBP3 0.00494525 -0.20857 mRNA metabolic process, nucleic acid binding
   ENST00000390426 0.00498692 -1.15069145 unknown function
   A_33_P3307192 6.56E-05 0.471136 unknown function
   CLEC1B 7.00E-05 0.17857 cell surface receptor signaling pathway,
   platelet activation and formation
   LOC644280 7.01E-05 0.40716 unknown function
   CCNT1 0.00019679 0.63888 regulation of cell cycle and transcription,
   regulation of cyclin-dependent protein serine/threonine kinase activity
   LOC100128126 0.00020899 0.932285 unknown function
   KRTCAP2 0.00026415 0.1043485 oligosaccharyltransferase activity
   Up regulated MYO7A 0.00026875 1.213465 actin filament-based movement,
   intracellular protein transport, lysosome organization
   IMP5 0.00042784 1.555375 protein transport, NLS-bearing protein import
   into nucleus
   RICH2 0.00043576 0.185575 exocytosis, regulation of small GTPase
   mediated signal transduction
   LOC100126447 0.00047818 0.840095 unknown function
   SCARNA7 0.00052182 0.57064 unknown function
   GPT 0.00073301 1.126995 cellular amino acid biosynthetic process
   ENST00000316698 0.00073633 0.559268 unknown function
   ZNF799 0.00089184 0.416855 regulation of transcription
   CACNA2D3 0.00094156 0.419785 cardiac conduction, regulation of calcium
   ion transport
   C19orf38 0.00108418 0.394503 unknown function
   ENST00000368941 0.00108575 1.406455 unknown function
   STAB2 0.00149055 0.913515 angiogenesis, cell adhesion, endocytosis
   TAP1 0.00151008 0.0403275 adaptive immune response, transmembrane
   transport, antigen processing and presentation
   AURKA 0.00169358 0.196458 cell division, DNA damage response,
   regulation of cytokinesis
   MYADML2 0.00175261 0.31679 unknown function
   C2orf81 0.00177281 0.1399515 unknown function
   ENST00000350501 0.00182808 0.20949 central nervous system development,
   positive regulation of GTPase activity, wound healing
   AGPAT1 0.00195538 0.078979 triglyceride biosynthetic process,
   phospholipid metabolic process
   ENST00000390376 0.00227851 0.255237 unknown function
   C1orf226 0.00241231 0.81491 unknown function
   GPSM1 0.00247166 0.5260285 cell differentiation, regulation of
   G-protein coupled receptor protein signaling pathway
   C10orf92 0.00256855 0.981 unknown function
   FAM90A10 0.00278555 1.31478 unknown function
   DOLK 0.0027918 0.0563 dolichyl diphosphate biosynthetic process
   A_33_P3314352 0.00286309 0.861685 unknown function
   KCNS1 0.00294728 1.180625 potassium ion transport, protein
   homooligomerization
   GDPD5 0.00304713 0.280104 lipid metabolic process, positive regulation
   of cell cycle, cell differentiation
   GRHPR 0.0031282 0.162362 dicarboxylic acid metabolic process,
   oxidation-reduction process
   SULT2A1 0.00321491 1.008145 3’-phosphoadenosine 5’-phosphosulfate
   metabolic process, sulfation
   PGLYRP3 0.003229 0.880755 defense response to Gram-positive bacterium,
   negative regulation of interferon-gamma production
   EXD1 0.0032326 0.206491 gene silencing by RNA, piRNA metabolic process,
   meiotic cell cycle
   SEPW1 0.00329521 0.1417625 unknown function
   FBXO48 0.00340037 0.4103129 unknown function
   ASPDH 0.00374399 0.570644 NAD biosynthetic process, NADP catabolic
   process
   FAM20C 0.00381368 0.2310795 biomineral tissue development, osteoclast
   maturation, positive regulation of bone mineralization
   FLJ37396 0.00381504 1.060099 unknown function
   SNAR-G1 0.00393914 0.64063 unknown function
   RGS9 0.00395577 0.3190388 dopamine receptor signaling pathway, nervous
   system development, response to estrogen
   DLEU2L 0.00437627 0.168915 unknown function
   VPS37A 0.00441433 0.1255275 autophagy, endosomal transport, protein
   transport
   ENST00000304813 0.00444994 1.0253 unknown function
   NOX5 0.00445637 0.8472815 angiogenesis, apoptotic process, cell
   proliferation, cytokine secretion
   TTLL12 0.00445823 0.117902 cellular protein modification process
   LOC100131686 0.00464655 0.2040485 unknown function
   LOC100128319 0.00469658 0.320885 unknown function
   ENST00000379256 0.00485664 0.4476945 unknown function
   CLDN19 0.00491595 0.173423 apical junction assembly,
   calcium-independent cell-cell adhesion, neuronal action potential
   propagation
   [62]Open in a new tab

   For pathway prediction analysis we included more number of genes and
   processed it via IPA software, Figure [63]2B displays the top 16 gene
   functions which are affected in PLAURsi cells on DNA damage. The
   complete list of molecules from our microarray, which can be mapped
   into these gene functions are shown in [64]Supplementary Table 5. The
   top gene function affected on silencing PLAUR is predicted to be
   Cellular Movement and the predicted gene network is shown in Figure
   [65]3A. The role of PLAUR in cellular migration is well documented by
   numerous reports.

Figure 3. Gene networks affected on silencing PLAUR.

   [66]Figure 3
   [67]Open in a new tab

   (A) and (B) Gene interacting networks generated using IPA showing the
   interacting genes and biological processes, cell migration (A) and
   synthesis of DNA (B).

   The gene functions ‘DNA replication, recombination and repair’ and the
   related ‘Nucleic acid metabolism’ are novel functions of PLAUR which
   have been investigated by our group. We have demonstrated that PLAUR is
   essential for DNA damage-induced nuclear import of PSMD6 subunit of 26S
   proteasome and activation of proteasome activity [[68]11]. Furthermore,
   we have recently demonstrated that PLAUR is essential for HR repair via
   activation of CHK-1 and nuclear import of RAD51 protein [[69]12].
   Microarray data confirms the involvement of PLAUR in the regulation of
   DNA metabolism and repair. The gene interacting networks in Figure
   [70]3B and [71]4A displays the complexity involved in regulation of DNA
   repair by PLAUR. Many of the partners in the network are known to
   influence the process of DNA synthesis and repair. Regulation of some
   of these molecules such as STATs, MAPKs and PTEN by PLAUR system is
   well documented. Whereas, the interrelation of PLAUR with other members
   of the network awaits further investigation. Differential
   transcriptional profile of PLAURsi cells suggests impaired DNA repair,
   and this corresponds to our previous observations [[72]11, [73]12].
   Accordingly, PLAURsi cells demonstrate higher background level of
   oxidative DNA damage and have more persistent DNA damage after
   irradiation and 6 h of repair (Figure [74]4B).

Figure 4. Silencing PLAUR affects proteins involved in DNA metabolism,
synthesis and repair.

   [75]Figure 4
   [76]Open in a new tab

   (A) Gene interacting networks generated using IPA showing the
   interacting genes and biological processes involving metabolism and
   incorporation of nucleotides; (B) MDA-MB-231 cells silenced for PLAUR
   were irradiated at 9 Gy, after 6 h cells were fixed and stained for
   8-OHdG antibody. DraQ5 was used as nuclear stain. Scale Bar 20 μm.

   Further we performed real time PCR to validate some of the important
   target genes in our microarray. We selected a set of genes among the
   most regulated genes which were related to the regulation of cell cycle
   and DNA repair. RT-PCR was performed on irradiated MDA-MB-231 cells
   using verified KiCqStart primer pairs as described in the Materials and
   Methods. The RT-PCR data were largely in agreement with the results
   obtained from our microarray experiment (Figure [77]5A and [78]5B).
   Thus, the expression of RRM2B, HNRNPU, Dux4, BRCA2, and WAPAL in
   irradiated PLAURsi cells was significantly lower than in irradiated
   sicon cells. Whereas the expression of SYNCRYP, AURKA, WISP1, WDR33,
   DDX31, GANA, TRIM11, CCNT1, and TTC3 in irradiated PLAURsi cells was
   higher than in irradiated sicon cells. Change in expression of some
   selected genes (RRM2B, HNRNPU, and Dux4) have been also verified by
   western blotting (Figure [79]6A). These data were also in agreement
   with the microarray data.

Figure 5. PLAUR regulates mRNA expression of DNA damage related genes.

   [80]Figure 5
   [81]Open in a new tab

   MDA-MB-231 cells silenced for PLAUR were treated as for microarray
   experiment; then RNA was isolated, and SYBR-green RT-PCR for DNA
   damage-related genes was performed using predesigned KiCqStart^® SYBR^®
   Green primers sets (Sigma Aldrich) as described in the Material and
   Methods. mRNA expression of irradiated sicon and PLAURsi cells are
   shown as mean±s.d. (A) Genes downregulated in irradiated PLAURsi cells
   in comparison to sicon cells. (B) Genes upregulated in irradiated
   PLAURsi cells in comparison to sicon cells.

Figure 6. Level of PLAUR expression regulates expression of DNA damage
related proteins and efficiency of DNA repair.

   [82]Figure 6
   [83]Open in a new tab

   (A) MDA-MB-231 cells silenced for PLAUR were treated as for microarray
   experiment; then western blotting was performed. (B) Quantification of
   the western blotting data from (A) taken from three independent
   experiments. (C) MDA-MB-231 cells infected with control or
   PLAUR-overexpression lentivirus were treated as for the microarray
   experiment; then comet assay was performed after 4 h of repair. (D)
   MDA-MB-231 cells infected with control or PLAUR-overexpression
   lentivirus were treated as above; then western blotting was performed.
   (E) 26S proteasome activity was measured in control and PLAURsi
   MDA-MB-231 cells 6 h after irradiation.

   In our earlier publication, we reported that downregulation of PLAUR
   expression delays DNA repair in MDA-MB-231 cells and human primary
   smooth muscle cells [[84]11]. Accordingly, PLAUR overexpression in HEK
   cells results in increased efficiency of DNA repair [[85]12]. To
   directly link our data to DNA repair, we overexpressed PLAUR in
   MDA-MB-231 cells and performed comet assay to assess DNA repair. As
   shown in Figure [86]6C, overexpression of PLAUR in MDA-MB-231 indeed
   improves repair of radiation-induced DNA damage. Further, we tested the
   expression of some selected proteins from the microarray data. Also,
   basal level of expression of RRM2B, HNRNPU, and Dux4 were increased in
   PLAUR-overexpressing cells offering an explanation for improved repair
   of DNA damage (Figure [87]6D). However, when analyzing protein
   expression in PLAURsi cells, one should keep in mind the dependence of
   proteasome activity on PLAUR expression (Figure [88]6E). Thus, PLAURsi
   cells fail to upregulate 26S proteasome activity after DNA damage by
   irradiation.

   Further, we wanted to see if these transcriptional effects of PLAURsi
   can be observed in non-transformed cells. We have shown previously that
   DNA repair is delayed in PLAURsi human primary smooth muscle cells
   suggesting that this effect is not cancer cells-specific [[89]11].
   Tubular cells are primarily sensitive to nephrotoxic anticancer drugs
   and we used HK-2 kidney tubular epithelial cells in our experiments.
   First, we performed comet assay to verify impairment of DNA repair in
   PLAURsi tubular epithelial cells. As shown in Figure [90]7A, indeed
   repair of DNA damage was significantly delayed in PLAURsi HK-2 cells.
   Next, we analyzed expression of some selected genes identified in the
   microarray study. We observed that several genes, specifically RRM2B,
   HNRNPU, Dux4, SYNCRYP, and WISP-1 are regulated in a similar fashion in
   tubular epithelial cells as in breast cancer cells (Figure [91]7B)
   whereas remaining genes have not shown similar regulation (data not
   shown).

Figure 7.

   [92]Figure 7
   [93]Open in a new tab

   (A) HK-2 cells were nucleofected to downregulate PLAUR expression.
   Then, cells were treated with doxorubicin, and comet assay was
   performed. (B) HK-2 cells silenced for PLAUR were irradiated with 9 Gy.
   After 4 h RNA was isolated and RT-PCR for DNA damage-related genes was
   performed using predesigned KiCqStart^® SYBR^® Green primers sets
   (Sigma Aldrich) as described in the Material and Methods. mRNA
   expression of irradiated sicon and PLAURsi cells are shown as mean±s.d.

DISCUSSION

   The primary function of PLAUR is to localize the proteolytic activity
   of PLAU on the extracellular surface and aid in the degradation of
   extracellular matrix, thereby promoting cell migration [[94]6]. It has
   been demonstrated that antibodies which block the binding of PLAU to
   PLAUR affect the migration of endothelial and breast cancer cells
   [[95]8, [96]14]. In addition, catalytically inactive PLAU also promotes
   cell migration via binding to PLAUR and initiation of intracellular
   signaling mediated by integrins; along with other PLAUR co-receptors in
   cancer cells, neutrophils, endothelial and smooth muscle cells. PLAUR
   signaling towards cell movement is mediated by activation of focal
   adhesion kinase (FAK), myosin light chain kinase (MLCK) and small
   GTPases of Rho Family [[97]5, [98]15]. Cell adhesion is also regulated
   by PLAUR system [[99]16]. PLAUR is highly expressed in majority of
   cancers and high PLAUR expression correlates with poor patient survival
   and bad prognosis [[100]17–[101]19]. Genetic knockdown of PLAUR has
   demonstrated strong anti-tumor activity. However, despite this
   promising data, therapeutic targeting of PLAUR has not been
   conceivable. Recently, progress was achieved in targeting PLAUR by
   monoclonal antibody and small molecule inhibitors (reviewed in
   [[102]20]). An antibody that showed beneficial anti-cancer effects in
   preclinical studies was shown to interfere with interaction of PLAUR
   and integrins [[103]21, [104]22]. These inhibitory approaches however,
   work by preventing metastatic dissemination and cannot affect survival
   of tumor cells.

   Recent reports shed light on PLAU/PLAUR roles that are not directly
   related to cell migration; thus, PLAUR affects
   epithelial-to-mesenchymal transition [[105]23], stemness properties of
   cancer cells [[106]24], metabolic state of cancer cells [[107]25].
   However, to the best of our knowledge, our work is the first to address
   transcriptional effects of PLAUR.

   Our recent studies describe a new role of PLAUR in facilitating repair
   of damaged DNA. Our data suggest that high expression of PLAUR is
   associated with more efficient DNA repair after chemo- or
   radio-therapy. Specifically, we found homologous DNA DSB repair pathway
   to be affected by PLAUR [[108]12]. These data offer a new explanation
   for poor prognosis of cancer patients with high PLAUR expression. To
   further decipher transcriptional effects of PLAUR in cancer cells, we
   have performed a microarray study. Pathway analysis highlighted many
   already well-described roles of PLAUR, such as inflammation
   [[109]26–[110]30], cardiovascular events [[111]31–[112]33] and liver
   function [[113]34–[114]36]. Our microarray data has also highlighted a
   specific function of PLAUR in the kidney that was also documented in
   the literature (reviewed in [[115]37, [116]38]).

   Further, we selected DNA damage and repair-related genes from the set
   of most regulated genes and verified their expression by RT-PCR. A set
   of genes including RRM2B, HNRNPU, BRCA2, and Dux4 were confirmed to be
   downregulated in PLAURsi cells in comparison to sicon MDA-MB-231 cells.
   RRM2B, a p53-dependent ribonucleotide reductase subunit B, is a stress
   response protein upregulated by oxidative stress and DNA damage
   [[117]39]. Mutations of RRM2B are associated with mitochondrial DNA
   depletion. Accordingly, mouse embryonic fibroblasts isolated from RRM2B
   -/- mice showed depletion of mitochondrial DNA [[118]40]. RRM2B
   protects cell from the oxidative stress by various mechanisms. Thus, it
   has intrinsic catalase activity [[119]41]. In addition, anti-oxidative
   function of RRM2B is mediated via direct interaction with
   Pyrroline-5-carboxylate reductases 1 and 2 (PYCR1, PYCR2) [[120]42].
   This complex is essential to maintain mitochondrial integrity and
   regulates metabolic shift in cancer cells towards cell proliferation
   [[121]42]. A recent report highlighted its essential role for the
   reduction of ribonucleotides to deoxyribonucleotides, under hypoxic
   conditions [[122]43]. Thus, RRM2B provides cancer cells with the
   ability to replicate DNA and therefore avoid accumulation of DNA damage
   in hypoxia. RRM2B -/- mice develop severe glomerular damage and kidney
   insufficiency and die by the age of 8 weeks, due to dNTP pool depletion
   and p53 activation in the kidney [[123]44, [124]45]. Hence, decreased
   RRM2B expression in PLAURsi cells can negatively regulate survival of
   cancer cells and provide a strong link of PLAUR to kidney damage
   pathways.

   HNRNPU, also called scaffold attachment factor A (SAF-A) is a member of
   the heterogeneous ribonucleoprotein family involved in a multiplicity
   of cellular processes such as mRNA turnover, transport, splicing,
   transcription, protein translation and mitosis [[125]46, [126]47].
   During DNA damage, HNRNPU is rapidly phosphorylated by DNA-PK and this
   is an essential mechanism for temporal regulation of various DNA repair
   pathways [[127]48]. Thus, HNRNPU phosphorylation leads to temporal
   delay of base exchange repair (BER) pathway via displacement of DNA
   glucosidases thus allowing DNA-PK-mediated NHEJ to take place first
   [[128]49]. Further, it is recognized that HNRNPU works as a part of
   anti-DNA:RNA hybrids mechanism [[129]50, [130]51] to arrest
   transcription in the proximity of DNA breaks. Since PLAUR silenced
   cells show low HNRNPU expression after DNA damage, one could expect it
   to result in accumulation of DNA damage in the absence of PLAUR via
   different mechanisms.

   Double homeobox 4 (DUX4) transcription factor is a main player in
   development of facioscapulohumeral (FSHD) dystrophy, one of the most
   common muscular dystrophies. It induces oxidative damage and
   constitutive DNA damage in FSHD myoblasts [[131]52]. In mammals it is
   expressed in the testis and epigenetically repressed in most of the
   tissues [[132]53]. DUX4 activity was recently detected in acute
   lymphoblastic leukemia [[133]54]. Further, in a recently characterized
   subset of high-grade sarcoma, so called CDS sarcoma a CIC-DUX4 gene
   fusion was detected [[134]55]. in vitro, CIC-DUX4 exhibited transformed
   potential in fibroblasts through modification of a cells transcriptome
   [[135]55]. Furthermore, DUX4 regulates cell cycle arrest through
   upregulation of p21 expression [[136]56].

   BRCA2 or the breast cancer susceptibility gene 2 is a tumor suppressor,
   which when mutated increases the risk of breast cancer [[137]57]. BRCA2
   plays a very important role in the error-free HR repair pathway; it is
   responsible for loading the recombinase RAD51 onto damaged DNA, where
   it aids in the search for homologous sequences. We have recently
   demonstrated that silencing PLAUR affects phosphorylation of RAD51 and
   impairs HR pathway in cancer cells [[138]12]. Downregulation of BRCA2
   after DNA damage can affect RAD51 function and impair the HR pathway
   [[139]58]. WAPAL is a subunit of the protein complex cohesin, which
   mediates sister chromatid cohesion during cell division. Cohesin is
   required for HR mediated DNA double strand break repair (reviewed in
   [[140]59]). WAPAL is responsible for the dissociation of the cohesin
   complex [[141]60], and hence may affect DNA repair. It is also known
   that cohesin recruitment occurs during late G2 phase to repair DNA
   strand breaks [[142]61]. Therefore, WAPAL downregulation in PLAURsi
   cells can also contribute to the impaired DNA repair. TTC3 is an E3
   ubiquitin ligase which mediates degradation of phosphorylated AKT
   [[143]62]. The AKT pathway regulates cell cycle progression by
   regulating purine nucleotide synthesis and affects the G1/S transition
   [[144]63]. Furthermore, recent reports demonstrated that radiation
   caused nuclear import of activated AKT and its association with DNA-PK
   at DSB that potentiated DNA repair [[145]64]. Also, inhibition of AKT
   pathway is shown to sensitize cancer cells to DNA damaging agents and
   radiation [[146]65, [147]66]. CDK9/cyclin T1 complex is a key regulator
   of RNA polymerase II activity [[148]67] and provides for expression of
   multiple anti-apoptotic genes. They are mainly recruited to particular
   promoter sequences and regulate transcription of specific genes
   [[149]68]. AURKA is known to deregulate DDR and is overexpressed in
   tumors with genomic instability [[150]69]. DDX31 is an RNA binding
   protein which is not well studied. Downregulation of DDX31 results in
   p53 stabilization and apoptosis [[151]70], therefore high expression
   can lead to genomic instability. TRIM11 (tripartite motif-containing
   protein 11), is an E3 ubiquitin ligase and high expression of TRIM11
   correlates with malignant glioma cells. Knockdown of TRIM11 inhibits
   proliferation and migration of glioblastoma cells; it's also necessary
   for the activation of EGFR and MAPK pathways [[152]71].

   Hence, the deregulation of a set of DNA damage and repair
   pathway-associated genes in PLAURsi cells can have a consequence on the
   downstream DDR signaling, ultimately leading to impairment of DNA
   repair processes. The top canonical pathways that are affected in
   PLAURsi cells on irradiation are listed in Table [153]2. The top toxic
   functions associated with deficiency of PLAUR on DNA damage are shown
   in Table [154]3.

Table 2. List of the genes in most significantly affected canonical pathways.

   Ingenuity Canonical Pathways -log(p-value) Ratio Molecules
   Corticotropin Releasing Hormone Signaling 3.91E00 1.23E-01 PRKACB,
   MAPK1, BDNF, NOS3, MAPK11, RAP1A, BRAF, CALM1 (includes others), PRKCH,
   MEF2C, PTGS2, NPR2, ADCY7
   IL-22 Signaling 2.83E00 2.08E-01 MAPK1, IL10RB, IL22RA1, STAT3, MAPK11
   Coagulation System 2.09E00 1.43E-01 PLAUR, F13A1, TFPI, SERPINE1, F2
   Leukocyte Extravasation Signaling 1.96E00 7.25E-02 MAPK1, MMP3,
   ARHGAP4, MAPK11, RAP1A, MMP24, ARHGAP5, WIPF1, NCF2, PRKCH, VCL, ACTG2,
   MMP1, CTNND1
   RhoA Signaling 1.92E00 8.33E-02 ARHGAP5, PLXNA1, MPRIP, CFL2, LPAR1,
   MYLK2, MYL5, ARHGAP4, PIP5KL1, ACTG2
   CMP-N-acetylneuraminate Biosynthesis I (Eukaryotes) 1.91E00 4E-01 NAGK,
   NANP
   cAMP-mediated signaling 1.9E00 6.94E-02 PRKACB, MAPK1, CAMK1D, STAT3,
   RAP1A, AKAP11, PDE1C, BRAF, CALM1 (includes others), GPER1, LPAR1,
   DUSP1, PDE4D, PKIA, ADCY7
   Cellular Effects of Sildenafil (Viagra) 1.82E00 8.06E-02 PRKACB, CALM1
   (includes others), MPRIP, MYL5, MYH3, PDE4D, ACTG2, NOS3, ADCY7, PDE1C
   Protein Kinase A Signaling 1.79E00 5.96E-02 PRKACB, PTPRE, MAPK1,
   YWHAE, PTPN2, MYLK2, MYL5, NOS3, RAP1A, PTEN, AKAP11, PDE1C, BRAF,
   CALM1 (includes others), PTPRU, PTPRJ, DUSP1, PDE4D, PRKCH, PTGS2,
   ADCY7, EBI3
   UVC-Induced MAPK Signaling 1.76E00 1.19E-01 BRAF, MAPK1, PRKCH, SMPD3,
   MAPK11
   Extrinsic Prothrombin Activation Pathway 1.73E00 1.88E-01 F13A1, TFPI,
   F2
   Fatty Acid α-oxidation 1.73E00 1.88E-01 PTGS2, ALDH3A1, ALDH7A1
   [155]Open in a new tab

Table 3. Top toxic functions and diseases implicated on DNA damage in PLAUR
silenced cells.

   Category p-value Molecules
   Nephrosis 6.13E-04-3.63E-02 CEP290, CEP83, PAX8, NPHP1, NPHP3
   Kidney Failure 2.12E-03-5.4E-01 ADM, DACT3, XDH, AGA, TRPV1, NOS3, F2,
   MR1, AR, EDN1, DUSP1, CD274, PDE4D, PTGS2, CYR61, TFPI, SERPINE1, NPR2,
   TNS1
   Renal Inflammation 2.81E-03-3.97E-01 TRAF3, XDH, DGAT2, TRPV1, NOS3,
   FAS, FAN1, IFIH1, HRH1, PTGS2, TLR3, SERPINE1, TNS1
   Renal Nephritis 2.81E-03-3.97E-01 TRAF3, XDH, DGAT2, TRPV1, NOS3, FAS,
   FAN1, IFIH1, HRH1, PTGS2, TLR3, SERPINE1, TNS1
   Glutathione Depletion In Liver 1.29E-02-3.59E-01 GSTT2/GSTT2B, XDH,
   CYP1B1, PTEN
   Renal Damage 1.57E-02-6.18E-01 ADM, EDN1, DUSP1, PLAUR, TLR3, NOS3,
   SERPINE1, FAS
   Renal Tubule Injury 1.57E-02-1.57E-02 EDN1, PLAUR, SERPINE1
   Liver Damage 1.79E-02-5.57E-01 ADM, STAT3, NOS3, INHBA, FAS, F2, PTEN,
   EDN1, CD40, DUSP1, HLA-DRA, IFNLR1, CD274, PTGS2, TLR3, SERPINE1,
   ALDH3A1, EBI3
   Liver Inflammation/Hepatitis 1.79E-02-6.04E-01 TRAF3, PLAUR, STAT3,
   FAS, PTEN, PTPRJ, IL10RB, IFNLR1, CD274, PDE4D, TLR3, CYR61, TFPI,
   SERPINE1, CASP7
   Cardiac Inflammation 1.92E-02-1.92E-02 TRAF3, TLR3, NOS3, SERPINE1,
   CSF1R
   Liver Hemorrhaging 1.99E-02-3.63E-02 ADM, DGCR8, PTGS2, FAS
   Cardiac Hypertrophy 2.02E-02-6.04E-01 ADM, MAPK1, MYLK2, STAT3, NOS3,
   INHBA, PTEN, BRAF, ANKRD1, FHL2, EDN1, DUSP1, APLN, MEF2C, PTGS2,
   ACTG2, SERPINE1, NPR2, MMP1
   Glomerular Injury 2.02E-02-5.4E-01 ADM, MAPK1, DACT3, XDH, AGA, TRPV1,
   TRPC6, NOS3, RAP1A, EDN1, B4GALT1, DUSP1, PTGS2, SERPINE1, TNS1
   Renal Proliferation 2.17E-02-4.03E-01 FAT4, SAT1, PLAUR, STAT3, NOS3,
   ZBTB5, CSF1R, EDN1, DUSP1, APLN, CALCR, HAS2, PTGS2
   Cardiac Necrosis/Cell Death 2.23E-02-2.71E-01 ADM, MAPK1, XDH, STAT3,
   NOS3, MAPK11, INHBA, FAS, PTEN, NRG1, EDN1, APLN, NAMPT, CYR61
   [156]Open in a new tab

   Gene ontology analysis using DAVID depicts the enrichment of genes in
   cellular functions as shown in [157]Supplementary Table 6. The
   functions include cellular organization, response to cytokines, tissue
   remodeling, heart development, WNT signaling, adhesion and more; the
   results also show that nucleobase, nucleoside and nucleotide catabolic
   processes are affected. These functions directly relate to our results
   from IPA (Figure [158]3B and [159]4A) which shows that PLAURsi cells
   have defects in DNA synthesis, which can impact the cells ability to
   replicate DNA and resolve DNA damage.

   It is an interesting issue, how a plasma membrane receptor like PLAUR
   can affect DNA repair events. Our recent mechanistic studies suggested
   that PLAUR is essential for bystander effect signaling and senses
   damage-associated molecule(s) released by the irradiated cells
   [[160]12]. This assumption can be supported by the reports that known
   PLAUR-signaling network proteins like STAT3 and MAPK promote DNA repair
   [[161]72–[162]74]. The microarray data presented here confirms our
   previous observations and identifies several additional potential
   molecular mechanisms of PLAUR-related DNA metabolism and repair. In
   addition, many of the predicted biological processes and molecular
   functions are confirmed by mechanistic studies from our group and
   others. Furthermore, data from IPA were in agreement with results from
   DAVID and KEGG, strengthening our data. Our study aids in finding new
   roles for a cell surface receptor and in developing new diagnostic and
   therapeutic approaches.

   As shown in Table [163]3, PLAUR is strongly connected to various
   mechanisms of kidney pathology as well as in inflammation, toxicity of
   liver and cardiac tissues. Some of those mechanisms are already known
   whereas others await further investigations [[164]26, [165]37]. Based
   on our data, one can assume PLAUR is involved in resolving DNA damage
   caused by endogenous cues, inflammation [[166]75], chemotherapy or
   radiation bystander effects thus affecting cells fate and survival.

   Our microarray data and pathway analysis confirmed the connection of
   PLAUR to several reported cell mechanisms, including our recent
   observations linking PLAUR to DNA repair. This data awaits further
   investigation aiming at the deciphering the underlying mechanisms and
   the development of new therapeutics.

MATERIALS AND METHODS

Cell culture

   Cell line MDA-MB-231 was purchased from ATCC. Cells were tested and
   authenticated by morphology and western blotting for specific markers
   in our laboratory. MDA-MB-231 was cultured in DMEM (Lonza) supplemented
   with 10% FBS and pen strep. Cell lines were tested for mycoplasma
   contamination every six months.

   DNA damage was induced by irradiating the cells using a GammaCell 2000,
   having Caesium-137 as the radioactive source. The radiation dose was
   decided based on our previous publication, where we studied the
   regulation of the DNA damage response and repair pathways by the
   urokinase receptor [[167]12]. The kinetics of the formation and
   resolution of p-H2AX foci (which indicates sites of DNA damage) gave us
   a clear idea of the extent of DNA damage induced by our treatment. We
   tested different doses of radiation and performed kinetics of some of
   the important DNA repair proteins, and selected the dose of 9 Gy -
   which sufficiently elicited the DNA damage response and expression of
   important DNA repair proteins around the timepoint of 4 h. The effects
   arising due to radiation induced damage have been extensively studied.
   Ionising radiation results in numerous kinds of DNA lesions which are
   chemically caused to the generation of free oxygen species. It has been
   shown by Cadet et al [[168]76] that ionising radiation creates
   clustered damage sites on the DNA, corresponding to around 850
   pyrimidine lesions, 450 purine lesions, 1000 single-strand breaks (SSB)
   and 20-40 double-strand breaks (DSB)/cell/Gy.

   HK-2 tubular epithelial cell line was purchased from ATCC and cultured
   as recommended by the supplier.

Transfection and viral infection

   Scrambled control or PLAUR siRNA were obtained from Santa Cruz and
   transfected using Amaxa Nucleofector™ (Lonza) as previously described.
   Media was changed after 24 h and cells were used for the experiments 48
   h after transfection.

   Lentiviruses having a VSV-G envelope were produced using HEK-293T
   cells. Viral titer was determined by LV Lentiviral Titer kit (MoBiTec)
   and viruses were used at a multiplicity of infection (MOI) of 1 - 5
   using polybrene at a concentration of 2 μg/ml. PLAURsi and Flag-PLAUR
   overexpression viruses were described earlier [[169]11, [170]12].

Western blotting

   Western blotting was performed as previously described [[171]12].
   Briefly, cells were lysed in RIPA buffer containing protease inhibitors
   and subjected to sonication. Lysates were then centrifuged at 10,000
   rpm for 10 min, the supernatant was collected and protein was estimated
   by Bradford Reagent (BioRAD). Around 50 μg of protein was boiled with
   SDS loading buffer and run on a polyacrylamide gel. Proteins were
   transferred to a PVDF membrane, blocked with 3% BSA and incubated with
   Primary antibodies. Secondary antibodies conjugated to HRP were used to
   detect the proteins. The following antibody were used: PLAUR (#3937)
   from American Diagnostics. Tubulin (#2128) from Cell Signaling
   Technology, RRM2B (ab8105) from Abcam, HNRNP U (3G6) from Santa Cruz
   Biotechnology; Dux4 (NBP-49552SS) and TTC3 (NBP1-84293) from Novus
   Biologicals; WISP1 (AF1627) from R&D Systems; OHdG (bs-1278R) was from
   Bioss.

Comet assay

   Comet assay was performed as described [[172]77] with some
   modifications. Briefly, cells were stimulated with γ-radiation and
   allowed to repair DNA for 4 hrs. Cells were further trypsinised and
   counted. Approximately 10,000 cells were mixed with 1% low melting
   agarose and spread on normal agarose pre-coated glass slides. Agarose
   was allowed to solidify for 30 min at 4 °C, slides were incubated in
   lysis buffer (1% Triton X-100 in 10mM Tris, 100 mM EDTA) for 4 h, at 4
   °C. The slides were incubated in alkaline running buffer (10N NaOH, 200
   mM EDTA, pH>13) for 20 min, before performing electrophoresis for 20
   min at 300 mA, 25 V. After electrophoresis the slides were 2×5 min
   incubated with neutralization buffer (0.4M Tris, pH-7.5), placed in
   cold 100% ethanol for 5 min and dried overnight at 4 °C. Staining was
   performed with vista green dye (Cell Biolabs) and comets were observed
   under a fluorescence microscope. Analysis of comet tails was performed
   by CASP (CaspLab) Software. 50-100 cells were used for quantification
   of comet tails.

Immunofluorescence

   Immunofluorescence was performed on cells grown overnight on coverslips
   and then treated with γ-radiation; they were then fixed with 2%
   formaldehyde at the required time points, permeabilized with 0.1%
   Triton X-100 and blocked with 3% BSA in PBS at 4 °C overnight. Cells
   were labeled with primary and corresponding Alexa Fluor^® 488- or Alexa
   Fluor^® 594-conjugated secondary antibody (Invitrogen) for 1 h at room
   temperature. DRAQ5™ (Biostatus) was used for nuclear staining. Cells
   were then mounted with Aqua-Poly-Mount mounting medium (Polysciences)
   and analyzed on a Leica TCS-SP2 AOBS confocal microscope. Antibody for
   8-OHdG (bs 1278R) was from Bioss.

Proteasome activity assay

   Total proteasome activity in cell lysates was measured using the 20S
   proteasome assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) as
   described by the manufacturer. In brief, cells were treated with
   γ-radiation. The cell lysates were incubated with 10 μM substrate
   (SUC-LLVY-AMC) for 1 h at 37 °C, the fluorescence was read using a
   Magellan GENIOUS (Tecan, Männedorf, Switzerland) at 360 nm (excitation)
   and 480 nm (emission). The enzymatic activity was normalized to the
   protein concentration. The results are reported as mean ± SD.

Microarray and data analysis

   sicon and PLAURsi MDA-MB-231 cells were treated with radiation of 9 Gy.
   RNA was prepared after 4 h, by Qiagen RNeasy kit; a technical replicate
   was included. Microarray data was then generated by the Research Core
   Unit Transcriptomics (RCUT) of the Hannover Medical School. Briefly,
   RNA quality was assessed by RNA 6000 Nano Kit assay (Agilent
   Bioanalyzer 2100). RNA samples from sicon and PLAURsi cells were
   hybridized on an Agilent chip (Whole Human Genome Oligo Microarray v2,
   4×44K) in Dual color mode according to recommended protocols in the
   ‘Dual-Color Microarray-Based Gene Expression Analysis Protocol V5.7’.
   Slides were scanned on the Agilent Micro Array Scanner G2565CA (pixel
   resolution 5 μm, bit depth 20). Data extraction was performed with the
   ‘Feature Extraction Software V10.7.3.1’. Extracted data was further
   processed using Omics Explorer software v3.0 (Qlucore) and the genes
   were filtered using P-value to generate a heat map. The filtered genes
   were used to perform pathway analysis using IPA (Ingenuity Pathway
   Analysis) software; functional and causal networks were generated using
   the information available in the Ingenuity Knowledge Base.

   Since the methods used for data analysis are likely to influence the
   final observation, primary data files obtained after ‘Feature
   Extraction’ were further analyzed independently by another method. The
   datasets were subjected to noise reduction, normalization, and
   differential gene expression analysis. Limma Bioconductor package in R
   was used to perform data pre-processing and differential gene
   expression analysis of the microarray dataset (Agilent Dye-swap)
   [[173]78]. The background noise was eliminated (using normexp method
   with an offset value=16) and the spot intensity values of both PLAURsi
   and sicon were converted to a log2 scale. The arrays were then
   normalized using Loess normalization method; this was followed by the
   Empirical Bayes method to identify differentially expressed genes
   between PLAURsi and sicon. The differentially expressed genes
   identified were screened using a cutoff P-value < 0.05. The probe IDs
   were converted into gene names based on the platform Whole Human Genome
   Oligo Microarray v2 (4×44K).

   Functional enrichment analysis was performed by submitting the
   differentially expressed genes as input to DAVID for GO enrichment
   analysis and KEGG pathway enrichment analysis [[174]79]. The data was
   screened with the cutoff P-Value < 0.05 in order to identify genes
   involved in functional pathways.

   Microarray data are available in the ArrayExpress database
   ([175]www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-5298.

Quantitative RT-PCR analysis

   Total RNA was isolated from MDA-MB-231 cells, 4 h after irradiation of
   9Gy using RNeasy miniprep kit (Qiagen). Real-time quantitative RT-PCR
   was performed on a LightCycler^® 480 Real-Time PCR System. SYBR Green
   RT-PCR was performed using Applied Biosystems master mix. Predesigned
   KiCqStart^® SYBR^® Green primers were purchased from Sigma.
   Oligonucleotide sequence is given in the [176]Supplementary Table 7.

SUPPLEMENTARY MATERIALS FIGURES AND TABLES

   [177]oncotarget-08-101572-s001.pdf^ (1,007.1KB, pdf)
   [178]oncotarget-08-101572-s002.xlsx^ (13.1KB, xlsx)
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   [180]oncotarget-08-101572-s004.xlsx^ (12.7KB, xlsx)
   [181]oncotarget-08-101572-s005.xlsx^ (12.7KB, xlsx)
   [182]oncotarget-08-101572-s006.xlsx^ (14.5KB, xlsx)
   [183]oncotarget-08-101572-s007.xlsx^ (14.6KB, xlsx)

Acknowledgments