Expression of the αVß3 integrin affects prostate cancer sEV cargo and density and promotes sEV pro-tumorigenic activity in vivo through a GPI-anchored receptor, NgR2.

It is known that small extracellular vesicles (sEVs) are released from cancer cells and contribute to cancer progression via crosstalk with recipient cells. We have previously reported that sEVs expressing the αVß3 integrin, a protein upregulated in aggressive neuroendocrine prostate cancer (NEPrCa), contribute to neuroendocrine differentiation (NED) in recipient cells. Here, we examine the impact of αVß3 expression on sEV protein content, density and function. sEVs used in this study were isolated by iodixanol density gradients and characterized by nanoparticle tracking analysis, immunoblotting and single vesicle analysis. Our proteomic profile of sEVs containing αVß3 shows downregulation of typical effectors involved in apoptosis and necrosis and an upregulation of tumour cell survival factors compared to control sEVs. We also show that the expression of αVß3 in sEVs causes a distinct reposition of EV markers (Alix, CD81, CD9) to a low-density sEV subpopulation. This low-density reposition is independent of extracellular matrix (ECM) protein interactions with sEVs. This sEV subset contains αVß3 and an αVß3 downstream effector, NgR2, a novel marker for NEPrCa. We show that sEVs containing αVß3 are loaded with higher amounts of NgR2 as compared to sEVs that do not express αVß3. Mechanistically, we demonstrate that sEVs containing NgR2 do not affect the sEV marker profile, but when injected in vivo intratumorally, they promote tumour growth and induce NED. We show that sEVs expressing NgR2 increase the activation of focal adhesion kinase (FAK), a known promoter of cancer cell proliferation, in recipient cells. We also show that NgR2 mimics the effect of sEVs containing αVß3 since it displays increased growth of NgR2 transfectants in vivo, as compared to control cells. Overall, our results describe the changes that occur in cargo, density and functions of cancer cell-derived sEVs containing the αVß3 integrin and its effector, NgR2, without affecting the sEV tetraspanin profiles.

Neuroendocrine gene subsets are uniquely dysregulated in prostate adenocarcinoma.

Prostate cancer has heterogeneous growth patterns, and its prognosis is the poorest when it progresses to a neuroendocrine phenotype. Using bioinformatic analysis, we evaluated RNA expression of neuroendocrine genes in a panel of five different cancer types: prostate adenocarcinoma, breast cancer, kidney chromophobe, kidney renal clear cell carcinoma and kidney renal papillary cell carcinoma. Our results show that specific neuroendocrine genes are significantly dysregulated in these tumors, suggesting that they play an active role in cancer progression. Among others, synaptophysin (SYP), a conventional neuroendocrine marker, is upregulated in prostate adenocarcinoma (PRAD) and breast cancer (BRCA). Our analysis shows that SYP is enriched in small extracellular vesicles (sEVs) derived from plasma of PRAD patients, but it is absent in sEVs derived from plasma of healthy donors. Similarly, classical sEV markers are enriched in sEVs derived from plasma of prostate cancer patients, but weakly detectable in sEVs derived from plasma of healthy donors. Overall, our results pave the way to explore new strategies to diagnose these diseases based on the neuroendocrine gene expression in patient tumors or plasma sEVs.

Targeting the αVß3/NgR2 pathway in neuroendocrine prostate cancer.

Highly aggressive, metastatic, neuroendocrine prostate cancer, which typically develops from prostate cancer cells acquiring resistance to androgen deprivation therapy, is associated with limited treatment options and hence poor prognosis. We have previously demonstrated that the αVß3 integrin is over-expressed in neuroendocrine prostate cancer. We now show that LM609, a monoclonal antibody that specifically targets the human αVß3 integrin, hinders the growth of neuroendocrine prostate cancer patient-derived xenografts in vivo. Our group has recently identified a novel αVß3 integrin binding partner, NgR2, responsible for regulating the expression of neuroendocrine markers and for inducing neuroendocrine differentiation in prostate cancer cells. Through in vitro functional assays, we here demonstrate that NgR2 is crucial in promoting cell adhesion to αVß3 ligands. Moreover, we describe for the first time co-fractionation of αVß3 integrin and NgR2 in small extracellular vesicles derived from metastatic prostate cancer patients' plasma. These prostate cancer patient-derived small extracellular vesicles have a functional impact on human monocytes, increasing their adhesion to fibronectin. The monocytes incubated with small extracellular vesicles do not show an associated change in conventional polarization marker expression and appear to be in an early stage that may be defined as "adhesion competent". Overall, these findings allow us to better understand integrin-directed signaling and cell-cell communication during cancer progression. Furthermore, our results pave the way for new diagnostic and therapeutic perspectives for patients affected by neuroendocrine prostate cancer.

STEAP1-4 (Six-Transmembrane Epithelial Antigen of the Prostate 1-4) and Their Clinical Implications for Prostate Cancer.

Six-Transmembrane Epithelial Antigen of the Prostate 1-4 (STEAP1-4) compose a family of metalloproteinases involved in iron and copper homeostasis and other cellular processes. Thus far, five homologs are known: STEAP1, STEAP1B, STEAP2, STEAP3, and STEAP4. In prostate cancer, STEAP1, STEAP2, and STEAP4 are overexpressed, while STEAP3 expression is downregulated. Although the metalloreductase activities of STEAP1-4 are well documented, their other biological functions are not. Furthermore, the properties and expression levels of STEAP heterotrimers, homotrimers, heterodimers, and homodimers are not well understood. Nevertheless, studies over the last few decades have provided sufficient impetus to investigate STEAP1-4 as potential biomarkers and therapeutic targets for prostate cancer. In particular, STEAP1 is the target of many emerging immunotherapies. Herein, we give an overview of the structure, physiology, and pathophysiology of STEAP1-4 to provide context for past and current efforts to translate STEAP1-4 into the clinic.