PRL3-zumab, a first-in-class humanized antibody for cancer therapy.

Novel, tumor-specific drugs are urgently needed for a breakthrough in cancer therapy. Herein, we generated a first-in-class humanized antibody (PRL3-zumab) against PRL-3, an intracellular tumor-associated phosphatase upregulated in multiple human cancers, for unconventional cancer immunotherapies. We focused on gastric cancer (GC), wherein elevated PRL-3 mRNA levels significantly correlated with shortened overall survival of GC patients. PRL-3 protein was overexpressed in 85% of fresh-frozen clinical gastric tumor samples examined but not in patient-matched normal gastric tissues. Using human GC cell lines, we demonstrated that PRL3-zumab specifically blocked PRL-3+, but not PRL-3-, orthotopic gastric tumors. In this setting, PRL3-zumab had better therapeutic efficacy as a monotherapy, rather than simultaneous combination with 5-fluorouracil or 5-fluorouracil alone. PRL3-zumab could also prevent PRL-3+ tumor recurrence. Mechanistically, we found that intracellular PRL-3 antigens could be externalized to become "extracellular oncotargets" that serve as bait for PRL3-zumab binding to potentially bridge and recruit immunocytes into tumor microenvironments for killing effects on cancer cells. In summary, our results document a comprehensive cancer therapeutic approach to specific antibody-targeted therapy against the PRL-3 oncotarget as a case study for developing antibodies against other intracellular targets in drug discovery.

PRL-3 activates mTORC1 in Cancer Progression.

PRL-3, a metastasis-associated phosphatase, is known to exert its oncogenic functions through activation of PI3K/Akt, which is a key regulator of the rapamycin-sensitive mTOR complex 1 (mTORC1), but a coherent link between PRL-3 and activation of mTOR has not yet been formally demonstrated. We report a positive correlation between PRL-3 expression and mTOR phospho-activation in clinical tumour samples and mouse models of cancer and demonstrate that PRL-3 increased downstream signalling to the mTOR substrates, p70S6K and 4E-BP1, by increasing PI3K/Akt-mediated activation of Rheb-GTP via TSC2 suppression. We also show that PRL-3 increases mTOR translocation to lysosomes via increased mTOR binding affinity to Rag GTPases in an Akt-independent manner, demonstrating a previously undescribed mechanism of action for PRL-3. PRL-3 also enhanced matrix metalloproteinase-2 secretion and cellular invasiveness via activation of mTOR, attributes which were sensitive to rapamycin treatment. The downstream effects of PRL-3 were maintained even under conditions of environmental stress, suggesting that PRL-3 provides a strategic survival advantage to tumour cells via its effects on mTOR.

Metastasis-associated PRL-3 induces EGFR activation and addiction in cancer cells.

Metastasis-associated phosphatase of regenerating liver-3 (PRL-3) has pleiotropic effects in driving cancer progression, yet the signaling mechanisms of PRL-3 are still not fully understood. Here, we provide evidence for PRL-3-induced hyperactivation of EGFR and its downstream signaling cascades in multiple human cancer cell lines. Mechanistically, PRL-3-induced activation of EGFR was attributed primarily to transcriptional downregulation of protein tyrosine phosphatase 1B (PTP1B), an inhibitory phosphatase for EGFR. Functionally, PRL-3-induced hyperactivation of EGFR correlated with increased cell growth, promigratory characteristics, and tumorigenicity. Moreover, PRL-3 induced cellular addiction to EGFR signaling, as evidenced by the pronounced reversion of these oncogenic attributes upon EGFR-specific inhibition. Of clinical significance, we verified elevated PRL-3 expression as a predictive marker for favorable therapeutic response in a heterogeneous colorectal cancer (CRC) patient cohort treated with the clinically approved anti-EGFR antibody cetuximab. The identification of PRL-3-driven EGFR hyperactivation and consequential addiction to EGFR signaling opens new avenues for inhibiting PRL-3-driven cancer progression. We propose that elevated PRL-3 expression is an important clinical predictive biomarker for favorable anti-EGFR cancer therapy.

PRL-3 suppresses c-Fos and integrin α2 expression in ovarian cancer cells.

BACKGROUND: Phosphatase of regenerating liver-3 (PRL-3), a protein tyrosine phosphatase, is highly expressed in multiple human cancers and strongly implicated in tumor progression and cancer metastasis. However, the mechanisms by which PRL-3 promotes cancer cell migration, invasion, and metastasis are not very well understood. In this study, we investigated the contribution and molecular mechanisms of PRL-3 in ovarian cancer progression. METHODS: PRL-3 protein expression was detected on ovarian cancer tissue microarrays using immunohistochemistry. Stable PRL-3 depleted cell lines were generated using short hairpin RNA (shRNA) constructs. The migration and invasion potential of these cells were analyzed using Transwell and Matrigel assays, respectively. Immunoblotting and immunofluorescence were used to detect protein levels and distribution in PRL-3-ablated cells and the control cells. Cell morphology was observed with hematoxylin-eosin staining and transmission electron microscopy. Finally, PRL-3-ablated and control cells were injected into nude mice for xenograft tumorigenicity assays. RESULTS: Elevated PRL-3 expression was detected in 19% (26 out of 135) of human ovarian cancer patient samples, but not in normal ovary tissues (0 out of 14). Stable depletion of PRL-3 in A2780 ovarian cancer cells resulted in decreased migration ability and invasion activity compared with control parental A2780 cells. In addition, PRL-3-ablated cells also exhibited flattened morphology and extended lamellipodia. To address the possible molecular basis for the altered phenotypes associated with PRL-3 down-regulation, we assessed the expression profiles of various proteins involved in cell-matrix adhesion. Depletion of PRL-3 dramatically enhanced both RNA and protein levels of the cell surface receptor integrin α2, but not its heterologous binding partner integrin ß1. Inhibition of PRL-3 also correlated with elevated expression and phosphorylation of paxillin. A pronounced increase in the expression and activation of c-fos, a transcriptional activator of integrin α2, was observed in these PRL-3 knock-down cells. Moreover, forced expression of EGFP-PRL-3 resulted in the suppression of both integrin α2 and c-fos expression in A2780 cells. Significantly, using a xenograft tumor model, we observed a greatly reduced tumorigenicity of A2780 PRL-3 knock-down cells in vivo. CONCLUSIONS: These results suggest that PRL-3 plays a critical role in ovarian cancer tumorigenicity and maintaining the malignant phenotype. PRL-3 may inhibit c-fos transcriptional regulation of integrin α2 signaling. Our results strongly support a role for PRL-3 as a promising therapeutic target and potential early biomarker in ovarian cancer progression.