p65BTK targeting restores the apoptotic response to chemotherapy of p53-null drug-resistant colon cancer cells

• Published in: European journal of cancer (1990) 2016-12, Vol.69, p.S140-S140
• Main Authors: Ianzano, L, Bonomo, S, Cialdella, A, Pisano, F, Cerrito, M.G, Carola, M, McLean, C, Romano, G, Giovannoni, R, Agostini, M, Nitti, D, Voest, E, Helin, K, Lavitrano, M, Grassilli, E
• Format: Article
• Language: English
• Subjects: Antibodies; Apoptosis; Assaying; Bevacizumab; Biocompatibility; Bruton's tyrosine kinase; Cancer; Caspase; Cell death; Chemotherapy; Colon; Colon cancer; Colorectal cancer; Cytotoxicity; Drug resistance; Enzyme-linked immunosorbent assay; Epidermal growth factor receptors; Hematology, Oncology and Palliative Medicine; Immunohistochemistry; Inhibition; Inhibitor drugs; Inhibitors; Molecular chains; Molecular modelling; Monoclonal antibodies; Organoids; p53 Protein; Polyclonal antibodies; Protein arrays; Protein-tyrosine kinase; Proteins; Stem cells; Targeted cancer therapy; Toxicity; Transcription; Tumor cell lines; Tyrosine; Xenografts; Xenotransplantation
• ISSN: 0959-8049; 1879-0852
• DOI: 10.1016/S0959-8049(16)33016-7

We recently identified p65BTK, a novel oncogenic isoform of Bruton tyrosine kinase abundantly expressed in colon cancer cell lines and tissues (Grassilli et al, Oncogene 2016) and showed that its inhibition affects growth and survival of colon cancer cells. The purpose of this study was to investigate whether its expression: (1) varies in colon cancers at different stage and grade; (2) plays a role in the response to chemotherapy and targeted therapy. Immunohistochemistry and ELISA assay using anti-p65BTK specific polyclonal antibodies raised in the lab were employed to study p65BTK expression in colon cancer patient specimens. Silencing experiments in drug-resistant cells and overexpression experiments in drug-sensitive cells were performed to study the role of p65BTK in the response to therapy. Cell viability/cell death assays were performed to study the effects of p65BTK silencing or inhibition (by different specific inhibitors) on the response to therapy in vitro. Xenograft experiments were carried out to assess the effect of p65BTK inhibition on the response to chemotherapy in vivo. Caspase assay, protein arrays and transcriptional profiling were used to define the molecular mechanisms in cells re-sensitized to chemotherapy upon p65BTK inhibition. We found that p65BTK expression significantly increases with the stage and the grade of colon carcinoma and correlates with cancer progression; in addition, p65BTK is strongly expressed in organoids and in cancer stem cells derived from colon cancer specimens. In vitro, p65BTK silencing and its inhibition by different specific inhibitors sensitize drug-resistant p53-null colon cells and patient-derived organoids to 5FU. At variance, blocking p65BTK does not restore the response of resistant cells to anti-EGFR receptor antibodies (panitumumab, cetuximab) and inhibitors (afatinib, poziotinib) or to bevacizumab. Conversely, p65BTK overexpression (but not overexpression of a kinase-dead mutant) protects p53-wt colon cancer cells from 5FU-induced cytotoxicity. Accordingly, p65BTK inhibition sensitizes drug-resistant p53-null colon cancer cell to 5FU through induction of apoptosis. Finally, in xenograft experiments we confirmed that the combination of 5FU with a BTK inhibitor (Ibrutinib) significantly reduced tumor volume in mice compared to the use of 5FU alone. In conclusion, our data indicate that p65BTK targeting restores the apoptotic response to chemotherapy of p53-null drug-resistant colon cancer cells and