Autoantibodies against the cell surface-associated chaperone GRP78 stimulate tumor growth via tissue factor.

Tumor cells display on their surface several molecular chaperones that normally reside in the endoplasmic reticulum. Because this display is unique to cancer cells, these chaperones are attractive targets for drug development. Previous epitope-mapping of autoantibodies (AutoAbs) from prostate cancer patients identified the 78-kDa glucose-regulated protein (GRP78) as one such target. Although we previously showed that anti-GRP78 AutoAbs increase tissue factor (TF) procoagulant activity on the surface of tumor cells, the direct effect of TF activation on tumor growth was not examined. In this study, we explore the interplay between the AutoAbs against cell surface-associated GRP78, TF expression/activity, and prostate cancer progression. First, we show that tumor GRP78 expression correlates with disease stage and that anti-GRP78 AutoAb levels parallel prostate-specific antigen concentrations in patient-derived serum samples. Second, we demonstrate that these anti-GRP78 AutoAbs target cell-surface GRP78, activating the unfolded protein response and inducing tumor cell proliferation through a TF-dependent mechanism, a specific effect reversed by neutralization or immunodepletion of the AutoAb pool. Finally, these AutoAbs enhance tumor growth in mice bearing human prostate cancer xenografts, and heparin derivatives specifically abrogate this effect by blocking AutoAb binding to cell-surface GRP78 and decreasing TF expression/activity. Together, these results establish a molecular mechanism in which AutoAbs against cell-surface GRP78 drive TF-mediated tumor progression in an experimental model of prostate cancer. Heparin derivatives counteract this mechanism and, as such, represent potentially appealing compounds to be evaluated in well-designed translational clinical trials.

Nuclear transportation of exogenous epidermal growth factor receptor and androgen receptor via extracellular vesicles.

Epidermal growth factor receptor (EGFR) plays a central role in the progression of several human malignancies. Although EGFR is a membrane receptor, it undergoes nuclear translocation, where it has a distinct signalling pathway. Herein, we report a novel mechanism by which cancer cells can directly transport EGFR to the nucleus of other cells via extracellular vesicles (EVs). The transported receptor is active and stimulates the nuclear EGFR pathways. Interestingly, the translocation of EGFR via EVs occurs independently of the nuclear localisation sequence that is required for nuclear translocation of endogenous EGFR. Also, we found that the mutant receptor EGFRvIII could be transported to the nucleus of other cells via EVs. To assess the role of EVs in the regulation of an actual nuclear receptor, we studied the regulation of androgen receptor (AR). We found that full-length AR and mutant variant ARv7 are secreted in EVs derived from prostate cancer cell lines and could be transported to the nucleus of AR-null cells. The EV-derived AR was able to bind the androgen-responsive promoter region of prostate specific antigen, and recruit RNA Pol II, an indication of active transcription. The nuclear-translocated AR via EVs enhanced the proliferation of acceptor cells in the absence of androgen. Finally, we provide evidence that nuclear localisation of AR could occur in vivo via orthotopically-injected EVs in male SCID mice prostate glands. To our knowledge, this is the first study showing the nuclear translocation of nuclear receptors via EVs, which significantly extends the role of EVs as paracrine transcriptional regulators.

Regulation of the tumor suppressor PTEN through exosomes: a diagnostic potential for prostate cancer.

PTEN is a potent tumor-suppressor protein. Aggressive and metastatic prostate cancer (PC) is associated with a reduction or loss of PTEN expression. PTEN reduction often occurs without gene mutations, and its downregulation is not fully understood. Herein, we show that PTEN is incorporated in the cargo of exosomes derived from cancer cells. PTEN is not detected in exosomes derived from normal, noncancerous cells. We found that PTEN can be transferred to other cells through exosomes. In cells that have a reduction or complete loss of PTEN expression, the transferred PTEN is competent to confer tumor-suppression activity to acceptor cells. In PC patients, we show that PTEN is incorporated in the cargo of exosomes that circulate in their blood. Interestingly, normal subjects have no PTEN expression in their blood exosomes. Further, we found that the prostate-specific antigen (PSA) is incorporated in PC patients' and normal subjects' blood exosomes. These data suggest that exosomal PTEN can compensate for PTEN loss in PTEN deficient cells, and may have diagnostic value for prostate cancer.