RESUMEN
BACKGROUND: Current treatments for castrate (hormone)-resistant prostate cancer (CRPC) remain limited and are not curative, with a median survival from diagnosis of 23 months. The PAP-specific Sipuleucel-T vaccine, which was approved by the FDA in 2010, increases the Overall Survival (OS) by 4 months, but is extremely expensive. We have previously shown that a 15 amino accid (AA) PAP sequence-derived peptide could induce strong immune responses and delay the growth of murine TRAMP-C1 prostate tumors. We have now substituted one amino acid and elongated the sequence to include epitopes predicted to bind to several additional HLA haplotypes. Herein, we present the immunological properties of this 42mer-mutated PAP-derived sequence (MutPAP42mer). METHODS: The presence of PAP-135-143 epitope-specific CD8+ T cells in the blood of patients with prostate cancer (PCa) was assessed by flow cytometry using Dextramer™ technology. HHDII/DR1 transgenic mice were immunized with mutated and non-mutated PAP-derived 42mer peptides in the presence of CAF®09 or CpG ODN1826 (TLR-9 agonist) adjuvants. Vaccine-induced immune responses were measured by assessing the proportion and functionality of splenic PAP-specific T cells in vitro. RESULTS: PAP-135-143 epitope-specific CD8+ T cells were detected in the blood of patients with PCa and stimulation of PBMCs from patients with PCa with mutPAP42mer enhanced their capacity to kill human LNCaP PCa target cells expressing PAP. The MutPAP42mer peptide was significantly more immunogenic in HHDII/DR1 mice than the wild type sequence, and immunogenicity was further enhanced when combined with the CAF®09 adjuvant. The vaccine induced secretory (IFNγ and TNFα) and cytotoxic CD8+ T cells and effector memory splenic T cells. CONCLUSIONS: The periphery of patients with PCa exhibits immune responsiveness to the MutPAP42mer peptide and immunization of mice induces/expands T cell-driven, wild-type PAP immunity, and therefore, has the potential to drive protective anti-tumor immunity in patients with PCa.
RESUMEN
BACKGROUND: Metformin, a biguanide, is one of the most commonly prescribed treatments for type 2 diabetes and has recently been recommended as a potential drug candidate for advanced cancer therapy. Although Metformin has antiproliferative and proapoptotic effects on breast cancer, the heterogenous nature of this disease affects the response to metformin leading to the activation of pro-invasive signalling pathways that are mediated by the focal adhesion kinase PYK2 in pure HER2 phenotype breast cancer. METHODS: The effect of metformin on different breast cancer cell lines, representing the molecular heterogenicity of the disease was investigated using in vitro proliferation and apoptosis assays. The activation of PYK2 by metformin in pure HER2 phenotype (HER2+/ER-/PR-) cell lines was investigated by microarrays, quantitative real time PCR and immunoblotting. Cell migration and invasion PYK2-mediated and in response to metformin were determined by wound healing and invasion assays using HER2+/ER-/PR- PYK2 knockdown cell lines. Proteomic analyses were used to determine the role of PYK2 in HER2+/ER-/PR- proliferative, migratory and invasive cellular pathways and in response to metformin. The association between PYK2 expression and HER2+/ER-/PR- patients' cancer-specific survival was investigated using bioinformatic analysis of PYK2 expression from patient gene expression profiles generated by the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) study. The effect of PYK2 and metformin on tumour initiation and invasion of HER2+/ER-/PR- breast cancer stem-like cells was performed using the in vitro stem cell proliferation and invasion assays. RESULTS: Our study showed for the first time that pure HER2 breast cancer cells are more resistant to metformin treatment when compared with the other breast cancer phenotypes. This drug resistance was associated with the activation of PTK2B/PYK2, a well-known mediator of signalling pathways involved in cell proliferation, migration and invasion. The role of PYK2 in promoting invasion of metformin resistant HER2 breast cancer cells was confirmed through investigating the effect of PYK2 knockdown and metformin on cell invasion and by proteomic analysis of associated cellular pathways. We also reveal a correlation between high level of expression of PYK2 and reduced survival in pure HER2 breast cancer patients. Moreover, we also report a role of PYK2 in tumour initiation and invasion-mediated by pure HER2 breast cancer stem-like cells. This was further confirmed by demonstrating a correlation between reduced survival in pure HER2 breast cancer patients and expression of PYK2 and the stem cell marker CD44. CONCLUSIONS: We provide evidence of a PYK2-driven pro-invasive potential of metformin in pure HER2 cancer therapy and propose that metformin-based therapy should consider the molecular heterogeneity of breast cancer to prevent complications associated with cancer chemoresistance, invasion and recurrence in treated patients.