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 HE...

In addition to being less toxic and having a lower impact on a patient’s quality of life, evidence suggests that low-dose or metronomic chemotherapy modulates adaptive and innate antitumor immune responses. In this study, we examined whether the treatment of breast cancer cells and animals bearing breast cancer cell-derived tumors with low, nontoxic doses of chemotherapies promotes sensitivity to natural killer (NK) cells, based on target cell killing in vitro and the control of tumor growth and metastasis in vivo. We have developed a low-dose doxorubicin treatment protocol that sensitizes triple-negative breast cancer (TNBC) cells (MDA-MB-231, MDA-MB-468) to killing by NK cells. Specifically, low-dose doxorubicin treatment induced a “senescent-like” state in breast cancer cells concomitant with an impaired proliferative capacity, as shown by a decreased expression of Ki-67. Treatment of breast cancer cells also upregulated their expression of ligands for activatory NK cell receptors (e.g., MICA/B, ULBP1, ULBP2, ULBP3) and markedly increased their sensitivity to lysis by donor-derived, primary NK cells and the NK-92 cell line, as assessed using an in vitro flow cytometry-based assay. The cytotoxicity of donor-derived NK cells was markedly increased by prior stimulation with IL-2. The therapeutic potential of low-dose chemotherapy alone and in combination with adoptively transferred resting and activated NK cells was evaluated using immunodeficient NSG mice bearing MDA-MB-231/RFP/LUC-derived human TNBC xenografts. Although low-dose doxorubicin treatment alone reduced the growth of the primary tumors, as assessed using caliper measurements and in vivo imaging, the growth of the primary tumor was more markedly reduced following the adoptive transfer of healthy-donor derived NK cells (mean tumor volume: 70.56mm3 versus 228.8mm3 in control groups at day 49). Importantly, although treatment with low-dose doxorubicin alone also delayed the onset of metastasis by 7 days (day 49 in control animals, day 56 in treated animals), no signs of metastasis were observed in healthy-donor derived NK cell-treated animals at the time of culling on day 50. The expression of MICA/B was 8.5 times greater on tumor cells derived from mice that received the combination treatment (p=0.0001) and these cells were more sensitive to killing by NK-92 cells in vitro (3-fold increase at E:T 1:5). Furthermore, in vitro studies demonstrating that the treatment of MDA-MB-231 cells with low-dose doxorubicin reduces the number of CD44High/CD24−/low/EpCAM+ Cancer Initiating Cells (CICs) were confirmed by there being a significantly lower number of CICs in tumors derived from treated mice. These novel findings indicate that this approach has the capacity of preventing the primary tumor “fueling” the dissemination of aggressive, metastatic disease. Taken together, these studies demonstrate that the treatment of breast cancer with low, nontoxic doses of chemotherapies promotes their sensitivity to killing by NK cells in vitro and in vivo. We have shown that administering human NK cells to mice bearing human breast tumors that have been treated with low-dose doxorubicin reduces the growth and metastasis of tumors and eliminates the aggressive cells that “feed” this process. Ongoing studies are further interrogating the influence of this approach on breast cancer metastasis. In summary, this strategy offers a promising opportunity for more effectively treating patients with aggressive, triple-negative breast cancer. Citation Format: Sarra Idri, Graham Pawelec, Yvonne Barnett, Graham Pockley. Combining low-dose chemotherapy with an NK cell-based immunotherapy as a treatment for triple-negative breast cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A135.

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.