Abstract B121: A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation

Abstract

Abstract It is known that Bruton's tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel oncogenic isoform expressed in colon carcinomas. Standard procedures were used for cloning the full length p65BTK-encoding mRNA and raise anti-p65BTK specific polyclonal antibodies. Quantitative PCR, western blot, RNA immunoprecipitation, silencing experiments and fluorescence assay upon transfection with bi-cistronic vectors were used to demonstrate post-transcriptional regulation of p65BTK expression. Immunohystochemistry was employed to study p65BTK expression in colon cancer patients specimens. Soft agar assay and foci assay were carried out to assess p65BTK oncogenic properties. Cell growth, cell viability and colony assays were performed to study the effects of p65BTK inhibition (by a specific kinase inhibitor) on colon cancer cells biology. We found that p65BTK differs from the already known 77 kDa isoform for the lack of the N-terminal PH domain and is translated - through an IRES-dependent mechanism -from a transcript containing an alternative first exon in the 5'UTR. Moreover, p65BTK mRNA translation requires phosho-hnRNPK binding to its cognate sites (located in the alternative first exon) and is post-transcriptionally regulated, via hnRNPK, by the MAPK pathway. We demonstrate that p65BTK is endowed with strong transforming activity that depends on active ERK1/2 and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Finally we show that p65BTK inhibition affects growth and survival of colon cancer cells. In conclusion, our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approach. Citation Format: Emanuela Grassilli, Fabio Pisano, Annamaria Cialdella, Sara Bonomo, Carola Missaglia, Maria Grazia Cerrito, Laura Masiero, Leonarda Ianzano, Robert Narloch, Filomena D'Amato, Barbara Noli, Gian Luca Ferri, Biagio E. Leone, Giorgio Stanta, Serena Bonin, Kristian Helin, Roberto Giovannoni, Marialuisa Lavitrano. A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B121.