Differential expression of αVβ3 and αVβ6 integrins in prostate cancer progression

Abstract

Neuroendocrine prostate cancer (NEPrCa) arises de novo or after accumulation of genomic alterations in pre-existing adenocarcinoma tumors in response to androgen deprivation therapies. We have provided evidence that small extracellular vesicles released by PrCa cells and containing the αVβ3 integrin promote neuroendocrine differentiation of PrCa in vivo and in vitro. Here, we examined αVβ3 integrin expression in three murine models carrying a deletion of PTEN (SKO), PTEN and RB1 (DKO), or PTEN, RB1 and TRP53 (TKO) genes in the prostatic epithelium; of these three models, the DKO and TKO tumors develop NEPrCa with a gene signature comparable to those of human NEPrCa. Immunostaining analysis of SKO, DKO and TKO tumors shows that αVβ3 integrin expression is increased in DKO and TKO primary tumors and metastatic lesions, but absent in SKO primary tumors. On the other hand, SKO tumors show higher levels of a different αV integrin, αVβ6, as compared to DKO and TKO tumors. These results are confirmed by RNA-sequencing analysis. Moreover, TRAMP mice, which carry NEPrCa and adenocarcinoma of the prostate, also have increased levels of αVβ3 in their NEPrCa primary tumors. In contrast, the αVβ6 integrin is only detectable in the adenocarcinoma areas. Finally, analysis of 42 LuCaP patient-derived xenografts and primary adenocarcinoma samples shows a positive correlation between αVβ3, but not αVβ6, and the neuronal marker synaptophysin; it also demonstrates that αVβ3 is absent in prostatic adenocarcinomas. In summary, we demonstrate that αVβ3 integrin is upregulated in NEPrCa primary and metastatic lesions; in contrast, the αVβ6 integrin is confined to adenocarcinoma of the prostate. Our findings suggest that the αVβ3 integrin, but not αVβ6, may promote a shift in lineage plasticity towards a NE phenotype and might serve as an informative biomarker for the early detection of NE differentiation in prostate cancer.

Fig 1. αVβ3 integrin is selectively upregulated in the primary tumors of mice carrying neuroendocrine prostate cancer.

Immunostaining of the αVβ3 integrin (top panels), αVβ6 integrin (middle panels), and SYP (bottom panels) in prostate tumors from murine models with genetic knockdown of PTEN (SKO; n = 5), PTEN and RB1 (DKO; n = 5), and PTEN, RB1, and TRP53 (TKO; n = 5) in the prostatic epithelium. The bar at the bottom right corner of each panel represents 50 μm. First column: SKO; second column: DKO; third column: TKO.

Fig 2. αVβ3 integrin is selectively upregulated in lung metastases of mice carrying neuroendocrine prostate cancer.

Immunostaining of the αVβ3 integrin (top panels), αVβ6 integrin (middle panels), and SYP (bottom panels) in the lung metastases from murine models with genetic knockdown of PTEN and RB1 (DKO; n = 5) and PTEN, RB1, and TRP53 (TKO; n = 5) in the prostatic epithelium. The bar at the bottom right corner of each panel represents 20 μm. First column: DKO; second column: TKO.

Fig 3. Selective upregulation of αVβ3 integrin in the TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) mice.

IHC staining of αVβ3 (first row), αVβ6 (second row), and chromogranin A (CgA, third row) of prostate tumors from TRAMP mice (n = 24). Of the 24 samples analyzed, 13 show only a NE phenotype, 11 show only ADPrCa lesions, and 5 show both characteristics. IgG was used as negative control (last row). The bar at the bottom right corner of each panel represents 50 μm. Left column, ADPrCa; right column, NEPrCa.

Fig 4. Increased expression of the αVβ3 integrin, but not αVβ6, correlates with the neuroendocrine marker SYP in LuCaP PDXs and human prostate tumor samples.

Immunohistochemical analysis of 42 LuCaP PDX models. (A) representative IHC staining for αVβ3 (left) or αVβ6 (right) integrin of SYP positive (top row) or SYP-negative (bottom row) LuCaP PDX models is shown. The bar at the bottom right corner of each panel represents 20 μm. (B) Heat map of the signature score for SYP, αVβ3 or αVβ6 integrin of each LuCaP is shown. Raw data are reported in the S1 Table. (C) Immunostaining analysis of αVβ3 and αVβ6 integrins and SYP primary tumors from ADPrCa patients. The bar at the bottom right corner of each panel represents 20 μm.

Fig 5. Downregulation of androgen receptor does not increase αVβ3 or αVβ6 integrin expression.

Immunoblotting analysis of C4-2B and LNCaP cell lysates after AR downregulation by siRNA to AR. (A) Expression levels of αVβ3 integrin in C4-2B and LNCaP cells after AR downregulation. Immunoblotting was performed under reducing conditions. (B) Expression levels of αVβ6 integrin in C4-2B and LNCaP cells after AR downregulation. Immunoblotting was performed under non-reducing conditions. Actin or TSG101 serves as loading controls. NS, non-silencing.

Fig 6. Schematic representation of the findings described in this study.

SKO (PB-Cre4 PTEN^loxP/loxP) cancer cells do not metastasize and express low levels of αVβ3 integrin and high levels of αVβ6 integrin. On the other hand, DKO and TKO tumors (PB-Cre4 PTEN^loxP/loxP RB1^loxP/loxP and PB-Cre4 PTEN^loxP/loxP RB1^loxP/loxP TRP53^loxP/loxP respectively) express high levels of αVβ3 integrin and low levels of αVβ6 integrin. These αVβ3 positive tumors acquire metastatic behavior and expression of NE markers.