Integrin-associated CD151 is a suppressor of prostate cancer progression

Abstract

Owing to the complexity of interacting molecular networks on the cell surface, integrin-associated tetraspanin CD151 remains controversial regarding its clinical importance and functional impact in prostate cancer. The current study evaluated dynamics and clinical importance of CD151 expression and its function in prostate cancer by IHC analysis of two independent patient cohorts (n=80, 181), bioinformatic interrogation of the TCGA database, and evaluation of gene knockdown effect at the cellular level. Our data showed that aside from high mRNA expression, CD151 was primarily localized to intercellular junctions at the plasma membrane in normal prostate glands or benign tissues, regardless of nature of antibodies used. By contrast, in primary tumors from patients with metastatic disease, CD151 was largely localized in the cytosol. Furthermore, the level of the cell-cell junction-linked CD151 was inversely associated with Gleason grade and tumor stage (P<0.001 for both). The portion of primary tumors expressing junctional CD151 was also three-fold less in the metastatic patient population than its counterpart (P<0.001). In line with these observations, CD151 and its associated α3β1 or α6β4 integrin inversely correlated with androgen receptor (AR) at the mRNA level (Spearman coefficient: -0.44, -0.48 and -0.42) in the TCGA cohort. Expression of these adhesion molecules also correlated with DNA methylation in their promoters (Spearman coefficient: -0.37, -0.71 and -0.82). Combined, these data suggest that CD151 and associated integrins are linked to tumor metastasis through AR and the epigenetic program. Meanwhile, CD151 knockdown in E-cadherin-positive tumor cells led to increased cell proliferation and induction of the epithelial-mesenchymal transition (EMT)-like phenotype. Given the strong RGD-binding integrin dependence of EMT-featured tumor cells, we examined focal adhesion kinase (FAK), their key signaling effector, in the above patient cohorts. In contrast to CD151, FAK exhibited positive correlation with tumor grade and stage as well as AR and p53 inactivation at either mRNA, protein or genomic level. Taken together, our results suggest that CD151 represses prostate cancer by antagonizing cell proliferation, EMT and the signaling of RGD-binding integrins. Since this anti-tumorigenic role is prone to the AR-mediated transcriptional and epigenetic regulation, CD151 and possibly α3β1 and α6β4 integrins are of potential biomarkers for metastatic prostate cancer.

Keywords: CD151; EMT; Prostate cancer; integrins; metastasis; tetraspanin.

Figure 1

Profiling CD151 expression in a commercial prostate cancer TMA patient cohort (N=80). Tissue was stained for CD-151 with the 11G5a monoclonal antibody (Abcam). A. Representative image of diverse CD151 staining patterns in primary tumors (100×). B. Subcellular localization of CD151 stratified by Gleason grade (a) and tumor stage (b). IHC imaging are representative of staining patterns and intensity by stage/grade. Percentages of diffused (filled bar) or cell-cell adhesion (hashed bar) were plotted against each of Gleason grades and tumor stages. Differences between groups were assessed using the Chi-Square analysis and p-values were indicated.

Figure 2

Profiling CD151 expression in a local prostate cancer TMA patient cohort (N=181). Tissue was stained with the NCL-CD151 monoclonal antibody. A. Representative image of diverse CD151 staining patterns in primary tumors (100×, 200× insert). B. Subcellular localization of CD151 stratified by Gleason grade and tumor stage. IHC imaging are representative of staining patterns and intensity by stage/grade. Percentages of diffused (filled bar) or cell-cell adhesion (hashed bar) were plotted against each of Gleason grades and tumor stages. Differences between groups were assessed using the Chi-Square analysis and p-values were indicated.

Figure 3

Differential expression of CD151 in primary tumors from patients with metastatic or non-metastatic disease. A, B. Representative images of primary tumors from patients with distant metastasis and organ-confined diseases are depicted for comparison. Primary tumors were subjected to IHC staining with 11G5a or CD151-NCL monoclonal antibodies. Differences between groups were assessed by Chi-Square analysis. p-values were indicated. Percentages of diffused (filled bar) or cell-cell adhesion (hashed bar) in the cohort were calculated and plotted for each of patient groups without disease recurrence or with node or distant metastasis.

Figure 4

A link between epigenetic and AR regulation and expression of CD151 and associated α3β1 and α6β4 integrins in prostate tumors at mRNA level. The TCGA cohort (n=333, Cell 2015) was interrogated regarding: A. (a) the association between CD151 mRNA expression and Gleason grade; (b) A genomic link between CDd151 and AR. (c) Plots of CD151 mRNA expression and state of methylation of its promoter region. (d) Volcano blot of the list of genes being co-overexpressed or underexpressed with CD151 at mRNA level. B-D. The association between mRNA expression of α3β1 and α6β4 integrins and E-cadherin and AR or state of methylation of its promoter region (a, b). The Spearman and Pearson correlation coefficients were shown.

Figure 5

Effect of CD151 downregulation on proliferation and the EMT-like phenotype in cultured prostate tumor cells. A, B. Tumor cells were subjected to CD151 known via siRNA oligos or shRNA, followed by analysis of cell proliferation and morphologies. Degree of CD151 knockdown was determined by immunoblotting. A. BPH line; B. 22RV1 line. C. Modulation of tumor cell sensitivity to Dasatinib. Tumor cells with or without stable knockdown of CD151 were treated with indicated inhibitors for 72 h, followed by analyses of cell viability by MTT assay and paired t-test analysis. *: p value <0.05; **: p value <0.01.

Figure 6

Reprehensive image of FAK staining in human prostate tumors. A. TMA from the local prostate cancer patient cohort was subjected to IHC analysis with an FAK-specific antibody. a-f. FAK staining in tumors with benign feature or varying in Gleason grade or stage. Scale: 100×, 200× insert. B. MTT analysis of CD151 knockdown on tumor cell sensitivity to FAK inhibitor (VS-6063) or chemotherapeutic agent (Docetaxel). BPH Tumor cells with or without stable knockdown of CD151 were treated with indicated agents for 72 h, followed by analyses of cell viability by MTT assay and paired t-test analysis. *: p value <0.05; **: p value <0.01. C. FAK deregulation at genomic and mRNA levels and association with oncogenic drivers in the TCGA prostate cancer patient cohort (Cell, 2015). a, b. Association between mRNA expression of FAK and gene copy number. CD151 mRNA expression and Gleason grade. c-e. Plots of FAK mRNA expression and tumor Gleason grade, AR and p53.