Abstract
Background
Clear cell renal cell carcinoma (ccRCC) is a major worldwide health
problem due to its high prevalence and mortality rate. A disintegrin
and metalloproteinase 12 (ADAM12) is aberrantly expressed in various
cancers and plays an important role in tumor progression. However, its
explicit effect and molecular mechanism in ccRCC remain unclear.
Methods
We investigated the dysregulation of ADAM12 in ccRCC through public
databases and bioinformatics analyses. The expression of ADAM12 was
further verified in ccRCC tissues by RT-qPCR and immunohistochemistry
(IHC). The relationship between ADAM12 expression and
clinicopathological characteristics was analyzed statistically. The
effects of ADAM12 on the proliferation, migration and invasion of ccRCC
cells were examined by in vitro and in vivo experiments.
Results
ADAM12 was significantly upregulated in ccRCC tissues and associated
with poor prognosis in ccRCC patients. ADAM12 promoted ccRCC cell
proliferation, migration and invasion in vitro and the growth of
subcutaneous tumors in vivo. Knockdown of ADAM12 successfully
suppressed its oncogenic function. Mechanistically, its overexpression
induced epithelial-mesenchymal transition (EMT) by downregulating
E-cadherin and upregulating N-cadherin and Snail. Moreover, ADAM12
participated in the epidermal growth factor receptor (EGFR) pathway and
activated the downstream signal ERK1/2 by shedding the EGFR ligand,
thereby upregulating target genes including c-Myc, enhancing cell
survival and invasion ability, and promoting tumor progression,
metastasis and the induction of EMT.
Conclusions
High expression of ADAM12 induced EMT and promoted cell proliferation,
migration, and invasion by activating the EGFR/ERK signaling pathway in
ccRCC.
Supplementary Information
The online version contains supplementary material available at
10.1186/s12967-023-03913-1.
Keywords: ADAM12, EGFR Pathway, EMT, Tumor Progression, Clear cell
renal cell carcinoma
Background
Renal cell carcinoma (RCC) is a fatal genitourinary malignancy that
accounts for approximately 5% of cancer cases in adult men and 3% in
women [[33]1, [34]2]. It was estimated that there were about 431
thousand incidences and 179 thousand deaths globally in 2020 (Global
Cancer Observatory). RCC is a group of malignancies with diverse
histologic types, specific cytogenetic properties, and different
prognoses and therapeutic responses. It includes the most common
subtype, clear cell renal cell carcinoma (ccRCC), which accounts for
about 75%, papillary renal cell carcinoma (pRCC), chromophobe renal
cell carcinoma (chRCC), and other less common subtypes [[35]3].
Approximately 33 to 50% of patients have metastases by the time of
detection [[36]1], and nearly one-third of patients might experience
recurrence or metastasis after nephrectomy. Patients with metastatic
disease only have a median survival of 13 months [[37]4]. Although the
widespread application of imaging contributes to the early detection of
tumors, further exploration of the molecular mechanisms underlying RCC
development is urgently needed.
A disintegrin and metalloproteinases (ADAMs) are a multidomain and
multifunctional family of type I transmembrane proteins [[38]5] that
are vital for regulating cell adhesion and mediating proteolysis of a
variety of cell surface receptors and signal molecule extracellular
domains. Substrates for ADAMs include Notch receptor and ligand,
epidermal growth factor receptor (EGFR) ligand, interleukin-6 receptor
(IL-6R), tumor necrosis factor (TNF) and its receptor, E-cadherin and
CD44 [[39]6]. Owing to their abnormal expression or dysregulation,
ADAMs may lead to the initiation and progression of tumors [[40]6].
The human ADAM12 gene is located on chromosome 10q26.3 and can
alternatively splice to produce a long membrane-bound variant
(ADAM12-L) and a short-secreted variant (ADAM12-S) [[41]7]. As an
active metalloprotease, ADAM12 is primarily responsible for the
sheddases of EGF-like ligands, including heparin-binding EGF (HB-EGF),
EGF, and betacellulin (BTC), thereby activating the EGFR pathway
[[42]8, [43]9]. In addition, studies have suggested that the expression
of ADAM12 is increased in bladder [[44]10], colorectal [[45]11],
gastric [[46]12], lung [[47]13], and breast cancers [[48]6] and leads
to poor prognosis. In renal cancer, Gao et al. found an upregulation of
ADAM12 in ccRCC tissues and cells. Additionally, it was found to be
significantly correlated with gender, TNM stage and clinical grade of
patients [[49]14]. However, the function of ADAM12 in ccRCC and its
molecular mechanism have not been clarified. Here, we demonstrate the
oncogenic role of ADAM12 in ccRCC, both in vivo and in vitro. In
addition, we for the first time, demonstrate that ADAM12 can further
induce EMT and promote tumor progression in ccRCC through the EGFR/ERK
signaling pathway. Overall, the results of this study suggest that the
functions of ADAM12 in modulating EGFR/ERK signaling and EMT contribute
greatly to its role in ccRCC progression.
Materials and methods
ccRCC cell culture and tissue specimens
Human ccRCC cell lines (769-P, 786-O, Caki-1, Caki-2, ACHN) and the
human proximal tubular epithelial cell line HK-2 were acquired from the
American Type Culture Collection (ATCC). They were cultured at 37 °C
and 5% CO[2] in recommended culture media that contained 10% fetal
bovine serum and 100 U/mL penicillin/streptomycin. Tissue samples were
collected from patients diagnosed at the Department of Urology, Peking
University Shenzhen Hospital, China. In total, 30 pairs of ccRCC
tissues and adjacent normal kidney tissues were collected from ccRCC
patients confirmed by pathology and before receiving any treatment. All
experiments obtained ethical approval from the hospital’s Ethics
Committee, and patients were informed of their specimen content,
potential risks, and purposes of the study and signed written informed
consent.
Immunohistochemistry staining
Single spot tissue microarray (TMA) slides (150 cases of ccRCC, 30
cases of adjacent normal kidney tissues, HKidE180Su02, Shanghai Outdo
Biotech Company, Shanghai, China) were prepared. The ADAM12 rabbit
antibody (Proteintech, 1:3000) and the rabbit streptavidin–biotin
detection system (Beijing Zhongshan Golden Bridge Biotechnology,
Peking, China) were used for staining. According to the staining
intensity (0 (negative), 0.5 + , 1 + , 2 + , and 3 +) and the staining
positive rate (0–100%), two experienced pathologists evaluated the
results. Then, the total score (0–300%) was calculated as the product
of the staining intensity score and positive staining rate score. The
low expression group had a score less than or equal to the median
score, whereas the high expression group represented the opposite.
RNA isolation and RTq-PCR
TRIzol reagent (Takara, Japan) was used to extract the total RNA from
specimens. Then, for each sample, 1 µg of total RNA was reverse
transcribed by the PrimeScript^™ RT Reagent Kit with gDNA Eraser
(Takara, Japan) and amplified by qPCR in a LightCycler 480 (Roche, USA)
with the SYBR Premix Ex Taq^™ II Kit (Takara, Japan). GAPDH was
selected as the internal reference gene, and the relative expression
was calculated by the 2^−ΔΔCt method. Primer sequences were synthesized
by Sangon Biotech Company (Shanghai, China) and are listed below:
ADAM12-F: CGAGGGGTGAGCTTATGGAAC;
ADAM12-R: GCTTTCCCGTTGTAGTCGAATA;
GAPDH-F: CCACTCCTCCACCTTTGACG;
GAPDH-R: CTGGTGGTCCAGGGGTCTTA.
Western blot
Protein extraction and western blot assays were conducted as previously
described [[50]15]. The primary antibodies used in this study were
anti-ADAM12 (Proteintech, 1:1000), anti-E-cadherin, anti-N-cadherin,
anti-Vimentin, anti-Snail, anti-ERK1/2, anti-phosphorylated-ERK1/2 (all
from Cell Signaling Technology, 1:1000), anti-GAPDH (Abclonal, 1:7000),
anti-c-Myc (Abcam, 1:1000), anti-EGFR (Abcam, 1:5000), and
anti-phosphorylated-EGFR (Abcam, 1:3000). The secondary antibody used
here was HRP-linked anti-IgG (Cell Signaling Technology, 1:2000).
Viral infection
The human ADAM12 gene was amplified by PCR and cloned into a pHBLV-puro
lentivirus vector. The ADAM12-targeting short hairpin RNA (shRNA)
sequences were also cloned into pHBLV-puro, generated by HanBio
(Shanghai, China). The shRNA sequences were CCACTATCTGCAAGACGGTACTGAT
and ACCCATTCACCAGCCTCCATGAATT.
To establish a stable ccRCC cell line that could regulate the
expression of ADAM12, 3 × 10^4 cells per well were seeded in a 6-well
plate and infected with the lentiviruses. After 48–72 h, the expression
efficiency of GFP was preliminarily observed by fluorescence
microscopy, and stable infections were selected with puromycin at
4 μg/ml twice.
Drug treatment
Gefitinib (50 nM; Selleck, USA), an EGFR inhibitor, was dissolved in
dimethyl sulfoxide (DMSO; Sigma‒Aldrich, Germany) and added to the
corresponding medium. Meanwhile, the EGFR activator NSC228155 (100 μM,
Selleck, USA) was dissolved in DMSO and used for subsequent rescue
experiments. DMSO at 0.02% concentration was used as a control. Cells
were harvested for 24 h, and then the corresponding inhibitor or
activator was added for further western blot and functional assays.
CCK8 assay
ccRCC cells were inoculated into 96-well plates at a concentration of
3 × 10^3/ well with 100 µl culture medium. The plates were wrapped in
tin foil and incubated for 5–6 h until the cells adhered to the wall.
At 0 h, 24 h, 48 h and 72 h after transfection, 10 µl/well CCK-8
reagent was added under dark conditions. After 3 h of incubation, the
culture plate was placed into a microplate reader, and the absorbance
value (O.D. value) of each well was detected at 450 nm and then
recorded.
Colony formation assay
Five hundred cells were cultured in 6-well plates for 2 weeks. Then,
the cells were fixed with 4% paraformaldehyde for 30 min and stained
with 0.1% crystal violet solution for 30 min. After that, images of the
colonies were taken after two washes with PBS. Finally, the crystal
violet was eluted completely with 33.3% glacial acetic acid and
transferred to 96-well plates. The absorbance of each well was detected
with a microplate plate at 590 nm.
Wound healing assay
Cells stably infected with lentivirus were cultured in 6-well plates. A
linear scratch wound was created via a 10 µl pipette tip, and cell
debris was removed by washing with PBS. Subsequently, the cells were
supplemented with 2 ml of serum-free medium. The wound width was
recorded by an inverted microscope every 24 h intervals, and the images
were evaluated using ImageJ.
Transwell assay
Cell invasion was examined by Transwell assay, where 200 µl serum-free
medium containing 3 × 10^4 cells was added to the upper chamber of
Transwell (8 µm pore size, BD Biosciences, USA) coated with 50 µl
Matrigel (BD Biosciences, USA). The lower chamber contained 500 µl of
medium supplemented with 10% FBS. After 24 h of incubation at 37 °C,
noninvaded cells were removed. Invaded cells were fixed with 4%
paraformaldehyde, stained with 0.1% crystal violet, and examined under
a microscope. After eluting crystal violet with 33.3% glacial acetic
acid, the liquid was transferred to 96-well plates, where the
absorbance value was examined by a microplate reader.
RNA extraction and transcriptome sequencing
Total RNA was extracted as previously described [[51]16]. After the
assessment of RNA integrity, quality and quantity, the samples were
transferred to Sangon Biotechnology (Shanghai) Co., Ltd. for library
preparation and sequencing.
Bioinformatics analysis
The limma package in R software (R version 4.0) was applied to compare
gene expression profiles between shNC and shADAM12 cell samples, Vector
and OE-ADAM12 cell samples. The cut‐off criteria for identifying
differentially expressed genes (DEGs) were |log2-fold‐change (FC)|> 2
and adjusted P < 0.05. A Venn diagram was constructed to obtain the
overlapping DEGs by FunRich ([52]http://www.funrich.org) [[53]17]. The
RobustRankAggreg (RRA) R package was used to plot the distribution of
DEGs on the heatmap [[54]18]. DEG identification via Gene Ontology (GO)
and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
analyses was conducted on the DAVID database (version 6.8,
[55]http://david.ncifcrf.gov), and their potential functional relevance
was explored. Biological processes (BP), molecular functions (MF), and
cell components (CC) were assessed by GO enrichment analysis, while
pathways were enriched by KEGG pathway analysis. An adjusted P < 0.05
was regarded as statistically significant.
In vivo xenograft model
The animal experiment protocol was approved by the Animal Ethics
Committee of Shenzhen PKU-HKUST Medical Center. Twenty-four BALB/c nude
mice (4–6 weeks) were split into 4 groups: shNC, shADAM12, Vector and
OE-ADAM12. Transfected ACHN cells were resuspended in cold PBS buffer
mixed with an equal volume of Matrigel. A mixture containing 3.5 × 10^6
cells was subcutaneously injected into the axilla of each mouse. The
tumor volume was measured and calculated in 3-day intervals by the
following formula: tumor volume = (length × width^2)/2. All mice were
sacrificed one month after injection. The subcutaneous tumor specimens
of the four groups were collected, weighed and photographed, and
further immunohistochemistry staining experiments were performed.
Statistics
GraphPad Prism 9 was utilized for statistical analysis, and data were
presented as mean ± standard deviation (SD) with at least 3 repeats.
Independent Student’s t test and one-way ANOVA were used for intergroup
comparisons. Pearson’s chi-squared test or Fisher’s exact test was used
to compare clinicopathological characteristics. Kaplan‒Meier survival
analysis was performed for patient prognosis. P < 0.05 was considered
statistically significant.
Results
ADAM12 was upregulated in ccRCC and correlated with poor prognosis
To investigate the potential role of ADAM12 in ccRCC, we compared the
expression profiles of ADAM12 between ccRCC tissues and adjacent normal
kidney tissues with The Cancer Genome Atlas (TCGA) database and Gene
Expression Omnibus (GEO) database. TCGA database revealed that ADAM12
was significantly upregulated in ccRCC tissues and was positively
correlated with T stage and tumor stage in ccRCC patients (p < 0.001)
(Fig. [56]1A–C). And its high expression indicated a poor prognosis for
ccRCC patients (Fig. [57]1D). By analyzing the clinicopathological
characteristics and clinical outcomes, we discovered that the
dysregulation of ADAM12 in ccRCC was associated with age, gender, local
invasion (T stage), lymph node involvement (N stage), and distant
metastasis (M stage) (Table [58]1). Moreover, [59]GSE53757 and
[60]GSE73731 data sets showed a significantly higher expression of
ADAM12 in ccRCC tissues tightly associated with tumor stage and grade
(p < 0.05) (Fig. [61]1E–G). Furthermore, we assessed ADAM12 expression
in 30 pairs of ccRCC and adjacent normal kidney tissues and discovered
that it was significantly upregulated in cancer tissues (Fig. 1H–I).
These results were further confirmed by immunohistochemistry staining
(p = 0.006) (Fig. [62]1J, Table [63]2). In addition, both the
transcription and translation levels of ADAM12 detected in ccRCC cell
lines suggested that ADAM12 had significantly higher expression in
ccRCC cells than in the HK-2 cell line (Fig. [64]1K–L).
Fig. 1.
[65]Fig. 1
[66]Open in a new tab
ADAM12 was upregulated in ccRCC tissues and cell lines. A The
expression of ADAM12 in ccRCC tissues was significantly higher than
that in adjacent normal kidney tissues according to TCGA. B, C ADAM12
expression was positively associated with T stage and tumor stage in
ccRCC patients. D The expression of ADAM12 was negatively correlated
with overall survival in ccRCC patients. E [67]GSE53757 data set showed
a significantly higher expression of ADAM12 in ccRCC tissues. F, G
[68]GSE73731 data set revealed that the expression of ADAM12 in ccRCC
was associated with tumor stage and grade. H, I The relative expression
of ADAM12 in 30 pairs of ccRCC tissues and adjacent normal kidney
tissues. Statistical analysis revealed that ADAM12 was dramatically
upregulated in 30 ccRCC tissues compared to normal tissues. J IHC
assays showed ADAM12 protein expression in different tumor stages of
ccRCC tissues and adjacent normal kidney tissues. Scale bar = 50 μm. K,
L The expression of ADAM12 in ccRCC cell lines and HK-2 cells at both
the transcriptional and translational levels. *P < 0.05, **P < 0.01,
***P < 0.001
Table 1.
Correlation between ADAM12 expression and clinicopathological
characters of patients with clear cell renal cell carcinoma (TCGA)
Clinico- pathological variables No. of cases ADAM12 expression χ^2 p
value
Low High
All cases 535 267 268
Gender Male 157 192 9.723 0.0018**
Female 110 76
Age
[MATH: < :MATH]
60 109 139 6.557 0.0104*
[MATH: ≥ :MATH]
60 158 129
Pathologic T T1 147 128 9.143 0.0274*
T2 39 31
T3 79 100
T4 2 9
Pathologic N N0 124 116 9.297 0.0096**
N1 2 14
NX 141 138
Pathologic M M0 209 215 14.24 0.0008***
M1 32 46
MX 25 6
TNM stage I 144 125 4.695 0.1955
II 32 26
III 56 67
IV 35 47
Survival
[MATH: < :MATH]
5 181 200 3.612 0.0574
[MATH: ≥ :MATH]
5 85 65
[69]Open in a new tab
^*P < .05
^**P < .01
^***P < .001 was considered significant (Chi-square test or Fisher’s
exact test)
Table 2.
Differential expression of ADAM12 in cancer and adjacent tissues
Expression of ADAM12 No. of cases Tissue type Chi-square p value
Adjacent tissues Cancer
All cases 175
Low 77 20 57 7.55 0.006**
High 98 10 88
[70]Open in a new tab
^**P < .01 was considered significant
ADAM12 promoted the proliferation of ccRCC cells in vitro
To explore the biological function of ADAM12 in ccRCC, ccRCC cell lines
with stable ADAM12 knockdown or overexpression were established. The
infection efficiencies were verified by RT‐qPCR and western blot
experiments. Figure [71]2A, B confirmed the successful knockdown of
ADAM12 in CAKI-2 and 786-O cells after infection with shADAM12
lentivirus at both the transcriptional and translational levels. In
contrast, ADAM12 was markedly overexpressed in ACHN and 786-O cells
when infected with OE-ADAM12 lentivirus (Fig. [72]2C, D). The CCK8
results revealed that downregulation of ADAM12 caused a remarkable
inhibition in the viability of CAKI-2 and 786-O cells (Fig. [73]2E),
whereas upregulation of ADAM12 had the opposite effect on proliferation
(Fig. [74]2F). The colony formation assay suggested that its knockdown
notably attenuated the colony formation capacity of CAKI-2 and 786-O
cells. Conversely, its overexpression facilitated colony formation of
ACHN and 786-O cells (Fig. [75]2G, H). These results confirmed that
ADAM12 promoted the proliferation of ccRCC cells.
Fig. 2.
[76]Fig. 2
[77]Open in a new tab
ADAM12 promoted the proliferation of ccRCC cells. A, B ADAM12 was
significantly downregulated in CAKI-2 and 786-O cells at both the
transcriptional and translational levels after infection with shADAM12
lentivirus. C, D ADAM12 was markedly overexpressed in ACHN and 786-O
cells at both the transcriptional and translational levels after
infection with OE-ADAM12 lentivirus. E–F CCK-8 assays showed the
proliferation capacity of ccRCC cells infected with the indicated
lentivirus. G, H Colony formation assay revealed the colony number of
ccRCC cells infected with the indicated lentivirus
ADAM12 facilitated migration and invasion and induced EMT in ccRCC cells
Wound healing assays and transwell assays were conducted to investigate
the effects of ADAM12 on the motility and invasiveness of ccRCC cells.
The wound healing assay showed that silencing ADAM12 markedly inhibited
the migration of CAKI-2 and 786-O cells, whereas restoring ADAM12
rescued the migration ability of ACHN and 786-O cells (Fig. [78]3A, B).
Moreover, the transwell assay revealed that the invasive capacity of
ccRCC cells was inhibited or strengthened after depletion or
restoration of ADAM12 in ccRCC cells, respectively (Fig. [79]3C, D). To
further examine whether EMT could be mediated by ADAM12, the expression
of EMT transcription factors and related markers were analyzed by
western blotting. In CAKI-2 and 786-O cells, after silencing ADAM12,
the expression of E-cadherin was remarkably elevated, and the
expression of N-cadherin and Snail were significantly reduced, while
Vimentin was unaffected (Fig. [80]3E). We then overexpressed ADAM12 to
explore whether it could reverse these changes. The results revealed
that E-cadherin expression was downregulated, while N-cadherin and
Snail expression were notably upregulated, leaving Vimentin intact
(Fig. [81]3F). These changes further validated our results in the
ADAM12 knockdown experiments and illustrated the role of ADAM12 in EMT
induction.
Fig. 3.
[82]Fig. 3
[83]Open in a new tab
ADAM12 facilitated migration and invasion and induced EMT in ccRCC
cells. A, B Wound healing assay showed the migration capacity of ccRCC
cells infected with the indicated lentivirus. C, D Transwell assay
revealed that the invasive capacity of ccRCC cells was obviously
inhibited or strengthened after depletion or restoration of ADAM12 in
ccRCC cells, respectively. E The effect of ADAM12 knockdown on EMT
marker expression was assessed by western blotting. F The effect of
ADAM12 overexpression on EMT marker expression in ACHN and CAKI-2 cells
ADAM12 enhanced the EGFR/ERK signaling pathway in ccRCC
To reveal the mechanism by which ADAM12 mediated the progression and
EMT in ccRCC, we performed transcriptome sequencing and bioinformatics
analysis. Volcano plots were generated for all DEGs by comparing shNC
with shADAM12 cell samples and Vector with OE-ADAM12 cell samples
(Fig. [84]4A). Notably, Venn software revealed that 17 overlapping DEGs
were screened out (Additional file [85]1: Fig. S1, Additional file
[86]1: Table S1). Through the RRA R package, the distribution of 17
overlapping DEGs was plotted on the heatmap (Fig. [87]4B). Then, GO and
KEGG pathway enrichment analyses were conducted to explore the
potential functional relevance among DEGs. The GO results suggested
that DEGs were mainly involved in biological adhesion, multicellular
organismal process, behavior and developmental process in BP; membrane
part, extracellular matrix and region, and synapse in CC; and receptor
regulator activity and transporter activity in MF (Fig. [88]4C). In
addition, KEGG pathway enrichment analysis indicated that the Ras
signaling pathway might be involved in ccRCC cells after ADAM12
stimulation (Fig. [89]4D). Moreover, previous studies reported that
ADAM12-mediated shedding of EGFR ligands resulted in inducing the
phosphorylation of EGFR and activating subsequent downstream signaling,
including the Ras-MEK-ERK/MAPK, PI3K/AKT and JAK/STAT pathways [[90]19,
[91]20]. To further determine whether the EGFR/ERK pathway could be
activated by ADAM12 in ccRCC cells, we conducted western blotting. The
results revealed that silencing ADAM12 suppressed the phosphorylation
levels of EGFR and ERK1/2 and downregulated c-Myc expression. However,
overexpression of ADAM12 markedly promoted c-Myc expression and the
phosphorylation of EGFR and ERK1/2 (Fig. [92]4E, F). These changes
validated the role of ADAM12 in the EGFR/ERK signaling pathway.
Fig. 4.
[93]Fig. 4
[94]Open in a new tab
ADAM12 enhanced the EGFR/ERK signaling pathway in ccRCC. A Volcano
plots of the distribution of DEGs by comparing shNC with shADAM12 cell
samples and Vector with OE-ADAM12 cell samples. B The distribution of
DEGs was plotted on the heatmap. C, D The biological functions of the
integrated DEGs were explored by GO and KEGG enrichment analysis. E, F
Western blot revealed the expression of EGFR/ERK and c-Myc proteins
upon the knockdown and overexpression of ADAM12
Silencing ADAM12 abolished the positive action of the EGFR activator on the
proliferation, metastasis and EMT process of ccRCC cells
To examine whether its effect on EGFR/ERK signaling affected the
progression and metastasis of ccRCC, we treated the cells with
NSC228155, a specific EGFR agonist. The CCK8 assay revealed that the
activation of EGFR dramatically elevated the viability of CAKI-2 and
786-O cells, and silencing ADAM12 counteracted this effect
(Fig. [95]5A). Colony formation assays suggested that activating EGFR
led to a remarkable increase in the colony number, while the knockdown
of ADAM12 narrowed the change (Fig. [96]5B). Both wound healing and
transwell assays indicated that the capacities of migration and
invasion were improved after treatment with NSC228155. In addition, the
depletion of ADAM12 reversed the promoting effect of NSC228155 on cell
migration and invasion (Fig. [97]5C, D). Protein level analysis showed
that the phosphorylation levels of EGFR and ERK1/2 rose significantly
under NSC228155 treatment in CAKI-2 and 786-O cells. E-cadherin
expression was markedly decreased, while c-Myc, N-cadherin and Snail
expression were increased (Fig. [98]5E, F). However, this regulatory
effect could be abolished by silencing ADAM12. Collectively, the EGFR
phosphorylation activator reversed the inhibitory action of ADAM12 on
proliferation, migration, invasion and EMT in ccRCC.
Fig. 5.
[99]Fig. 5
[100]Open in a new tab
Silencing ADAM12 abolished the positive action of the EGFR activator on
the proliferation, metastasis and EMT process of ccRCC cells. A CCK8
assay revealed the viability of CAKI-2 and 786-O cells treated with
shADAM12 lentivirus and EGFR activator NSC228155. B Colony formation
assays showed the colony number of CAKI-2 and 786-O cells treated with
shADAM12 lentivirus and NSC228155. C The wound healing assay showed the
migration capacity of CAKI-2 and 786-O cells treated with shADAM12
lentivirus and NSC228155. D Transwell assay indicated the invasion
capacity of CAKI-2 and 786-O cells treated with shADAM12 lentivirus and
NSC228155. E–F Western blot assay revealed the expression of the
indicated proteins in CAKI-2 and 786-O cells treated with shADAM12
lentivirus and NSC228155
EGFR blocking antagonized the effects of ADAM12 in ccRCC
To better clarify how ADAM12-induced proliferation, migration, invasion
and EMT were regulated via the EGFR/ERK signaling pathway, ccRCC cells
were treated with gefitinib, an EGFR phosphorylation inhibitor. Cell
proliferation assays indicated that suppressing EGFR phosphorylation
significantly reduced cell viability and cell growth in ACHN and 786-O
cells. Overexpression of ADAM12 eliminated these inhibitory effects
(Fig. [101]6A, B). Wound healing and transwell assays showed that
gefitinib treatment dramatically decreased the number of migrated and
invaded cells, while restoration of ADAM12 antagonized EGFR inhibition
(Fig. [102]6C, D). In addition, western blot analysis showed that
gefitinib significantly reduced the phosphorylation levels of EGFR and
ERK1/2 and the expression levels of c-Myc, N-cadherin and Snail but
increased E-cadherin expression (Fig. [103]6E, F). However, this
inhibitory effect could be reversed by overexpression of ADAM12. These
findings supported that the effects of ADAM12 on various ccRCC cell
activities were achieved by phosphorylating EGFR.
Fig. 6.
[104]Fig. 6
[105]Open in a new tab
EGFR blocking antagonized the effects of ADAM12 in ccRCC. A CCK-8 assay
revealed the viability of ACHN and 786-O cells treated with OE-ADAM12
lentivirus and the EGFR inhibitor gefitinib. B Colony formation assays
showed the colony number of ACHN and 786-O cells treated with OE-ADAM12
lentivirus and gefitinib. C The wound healing assay showed the
migration capacity of ACHN and 786-O cells treated with OE-ADAM12
lentivirus and gefitinib. D Transwell assay indicated the invasion
capacity of ACHN and 786-O cells treated with OE-ADAM12 lentivirus and
gefitinib. E, F Western blot assay revealed the expression of the
indicated proteins in ACHN and 786-O cells treated with OE-ADAM12
lentivirus and gefitinib
ADAM12 promoted the growth of ccRCC cells in vivo
Finally, an in vivo model was established to gain more information on
the role of ADAM12 in the growth of ccRCC. ACHN cells stably infected
with shADAM12 or OE-ADAM12 were injected subcutaneously into the axilla
of nude mice (n = 6 for each group). In the xenograft tumor model,
ADAM12 knockdown significantly repressed the volume and weight of the
tumors, whereas ADAM12 overexpression had the opposite effect
(Fig. [106]7A, B). Moreover, the IHC assay demonstrated that the levels
of ADAM12, c-Myc, N-cadherin and Snail drastically decreased after
silencing ADAM12, whereas E-cadherin markedly increased, suggesting
inhibition of tumor growth and the EMT process (Fig. [107]7C). The
opposite effects occurred after overexpressing ADAM12 (Fig. [108]7D).
Fig. 7.
[109]Fig. 7
[110]Open in a new tab
ADAM12 promoted the growth of ccRCC cells in vivo. A, B ACHN cells
stably infected with shADAM12 or OE-ADAM12 were injected subcutaneously
into the axilla of nude mice (n = 6 for each group) to create a
xenograft tumor model. The tumor growth curves were documented
according to the measurement of tumor volume every 3 days, and the
tumor weight was measured. C, D Representative immunohistochemistry
images of xenograft tumor tissues for HE, ADAM12, c-Myc, E-cadherin,
N-cadherin and Snail staining
Discussion
ADAM12, which belongs to the ADAM family, has unique characteristics,
including extracellular metalloproteinase, cell-binding functions, and
intracellular signal transduction capabilities [[111]21]. It is worth
noting that ADAM12, a secreted protein that can be detected in both
blood and body fluids, is a good potential tumor biomarker. Previous
evidence supported the oncogenic role of ADAM12 in multiple types of
tumors. It promoted cell proliferation in glioblastoma through the
stimulation of ectodomain shedding of proHB-EGF [[112]22]. Moreover,
ADAM12 significantly upregulated pro-angiogenic factors and increased
endothelial cell recruitment in a STAT3-dependent manner to promote
tumor angiogenesis in breast cancer [[113]23]. Although Gao et al.
predicted that ADAM12 might be a potential prognostic factor in ccRCC
by bioinformatics analysis [[114]14], both in vitro and in vivo
experimental validations were lacking. This study could supplement this
blank topic and elucidate the mechanism of ADAM12 in the development
and progression of ccRCC.
In this study, we utilized the TCGA and GEO databases combined with our
clinical specimens to evaluate the expression of ADAM12 in ccRCC and
its correlation with clinicopathological factors and clinical outcomes.
The data suggested that ADAM12 was highly expressed in most ccRCC
tissues, mainly in the cytoplasm. Its expression was significantly
correlated with gender, age, T stage, N stage and M stage. In addition,
Kaplan‒Meier analysis showed that high expression of ADAM12 indicated
poorer overall survival. Both our in vitro and in vivo assays
demonstrated the oncogenic role of ADAM12 in ccRCC. We found that
abnormally high ADAM12 expression promoted tumor growth and metastasis
by enhancing cell proliferation, migration, and invasion. The positive
effect of ADAM12 on tumor growth was further confirmed in a
subcutaneous xenograft tumor mouse model. Nevertheless, ADAM12
knockdown significantly inhibited cell proliferation, clonogenicity,
cell migration and invasion. Therefore, we hypothesized that ADAM12
might potentially be a therapeutic target for ccRCC treatment.
It was well-known that EMT mediated the response to various signaling
factors, including embryonic development, fibrosis, wound healing,
cancer metastasis, and proliferation [[115]24]. EMT was typically
activated in malignant tumors and acted as the initiation of tumor
invasion and metastasis, triggering the separation of cancer cells from
primary cancer and subsequently spreading to distant sites [[116]25].
In this study, we observed that overexpression of ADAM12 induced the
specific EMT transcription factor Snail. This could later reduce the
expression of the epithelial marker E-cadherin and enhance the
expression of the mesenchymal marker N-cadherin. Nevertheless,
silencing ADAM12 had the opposite effects on EMT. These changes in all
biomarkers indicated that ADAM12-mediated invasion and metastasis of
ccRCC were correlated with the induction of EMT.
Previous studies reported that ADAM12 might mediate the release of
soluble EGFR ligands, thereby activating EGFR [[117]26, [118]27].
Notably, the phosphorylation of EGFR activated diverse intracellular
processes, such as accelerating cell proliferation and invasion,
suppressing apoptosis and stimulating tumor angiogenesis [[119]28].
Moreover, EGFR was an upstream regulator of the Ras signaling pathway
[[120]29], and its activation was a major trigger for the progression
of most carcinomas [[121]30]. Since EMT could be modulated by multiple
signaling pathways, including PTEN/Akt [[122]31], Wnt/β-Catenin
[[123]32], PI3K/Akt [[124]33], and MAPK [[125]34], we first speculated
whether ADAM12 induced EMT progression through the EGFR/RAS signaling
pathway in ccRCC.
To validate these predictions, we conducted biological experiments. We
found that ADAM12 overexpression markedly promoted EGFR and ERK1/2
phosphorylation and increased c-Myc expression. As an important target
downstream of the ERK/MAPK pathway, a high level of c-Myc expression
was tightly associated with cell proliferation and the induction of EMT
in various carcinomas [[126]35–[127]38]. Cho et al. demonstrated that
overexpression of c-Myc inactivated ERK-dependent GSK-3β and activated
Snail, which could induce EMT in mammary epithelial cells [[128]37]. In
our study, we inhibited EGFR with gefitinib and discovered decreased
phosphorylation levels of EGFR and ERK1/2. The transcription factors
c-Myc and Snail and the mesenchymal marker N-cadherin were
downregulated, whereas the epithelial marker E-cadherin was
upregulated. These results suggested that inhibiting the EGFR/ERK
pathway affected cell proliferation and EMT. However, overexpression of
ADAM12 partially eliminated the effects, which was consistent with the
evidence that ADAM12 was an upstream regulator of the EGFR/ERK pathway.
These results proved that the oncogenic function of ADAM12 in cell
proliferation and EMT was dependent on the activation of EGFR/ERK
pathway. However, its two distinct isoforms in the regulation of
EGFR/ERK signaling pathway in human cancers were unknown. The specific
isoform of ADAM12 may provide greater help for the precisely targeted
therapy of ccRCC in the future.
Conclusion
Taking together, ADAM12 acts to facilitate the EGFR/ERK signaling
pathway and enhance the function to activate c-Myc and EMT-related
molecules such as E-cadherin, N-cadherin and Snail, leading to the
induction of EMT and the promotion of ccRCC growth and metastasis.
However, it remains unclear whether ADAM12-L or ADAM12-S plays a more
vital role in ccRCC progression. All of the above will be further
investigated in our future study.
Supplementary Information
[129]12967_2023_3913_MOESM1_ESM.docx^ (80.5KB, docx)
Additional file 1. Table S1. The relative expression of 17 overlapping
DEGs via comparison between Vector and OE-ADAM12 group. Figure S1. Venn
diagram representing the number of overlapping DEGs.
Acknowledgements