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 isoform abundantly expressed in colon carcinoma cell lines and tumour tissue samples. p65BTK protein is expressed, through heterogeneous nuclear ribonucleoprotein K (hnRNPK)-dependent and internal ribosome entry site-driven translation, from a transcript containing an alternative first exon in the 5′-untranslated region, and is post-transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. 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.

Purpose: Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy.Experimental Design: By using a kinase-directed short hairpin RNA library and HCT116p53KO drugresistant colon carcinoma cells, glycogen synthase kinase 3 beta (GSK3B) was identified as a target whose silencing bypasses drug resistance due to loss of p53. p53-null colon cancer cell lines with different sets of mutations were used to validate the role of GSK3B in sustaining resistance and to characterize cell death mechanisms triggered by chemotherapy when GSK3B is silenced. In vivo xenograft studies were conducted to confirm resensitization of drug-resistant cells to chemotherapy upon GSK3 inhibition. Colon cancer samples from a cohort of 50 chemotherapy-treated stage II patients were analyzed for active GSK3B expression.Results: Downregulation of GSK3B in various drug-resistant p53-null colon cancer cell lines abolished cell viability and colony growth after drug addition without affecting cell proliferation or cell cycle in untreated cells. Cell death of 5-fluorouracil (5FU)-treated p53-null GSK3B-silenced colon carcinoma cells occurred via PARP1-dependent and AIF-mediated but RIP1-independent necroptosis. In vivo studies showed that drug-resistant xenograft tumor mass was significantly reduced only when 5FU was given after GSK3B inhibition. Tissue microarray analysis of colon carcinoma samples from 5FU-treated patients revealed that GSK3B is significantly more activated in drug-resistant versus responsive patients.Conclusions: Targeting GSK3B, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant tumors.

Purpose of the study. GSK3A and GSK3B isoforms perform similar functions but they are not completely redundant, depending on the cell type, biological process and differentiative status (Rayasam GV et al, Br J Pharmacol, 2009). We recently demonstrated that GSK3B inhibition enables necroptosis in response to chemotherapy in drug-resistant colon carcinoma cells (Grassilli E et al, Clin Cancer Res, 2013). Here we investigated whether GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis. Materials and Methods. Cell lines stably interfered were obtained by retroviral infection and selection with the appropriate antibiotic. siRNA transfection was performed using Lipofectamine 2000 (Invitrogen). Cell viability was assessed by Trypan blue staining. Cell proliferation was evaluated by CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega). Cell cycle was analysed by flow cytometry upon PI staining using Modfit Cell Cycle Analysis (Becton Dickinson). Reporter activity was analysed by Dual-Glo Luciferase Assay (Promega). IF staining was performed on 4% paraformaldehyde-fixed cells. Active caspase-3/7 was quantified by the Caspase-Glo3/7 Assay System (Promega). Western blots were performed on lysates obtained by RIPA buffer. Results and Discussion. GSK3A stable silencing in HCT116p53KO cells did not alter cell proliferation as assessed by growth curve, cell cycle distribution and β-catenin activation assays. GSK3A depletion abolished colony formation after 5FU treatment and re-sensitized drug-resistant HCT116p53KO cells to 5FU-induced cell death. Blocking GSK3A did not interfere with DNA damage sensing upon 5FU treatment, as demonstrated by H2AX foci formation, but impaired the DNA repair response by affecting RPA70 foci formation. GSK3A silencing allowed a cell death response to DNA-damaging drugs in absence of p53: 5FU-treated, GSK3A-depleted HCT116p53KO cells underwent caspase-independent PARP1- and tBid-dependent cell death, which was unaffected by RIP1 inhibition and was characterized by PARP-dependent nuclear re-localization of AIF. Our data demostrate that GSK3A, like GSK3B, contributes to cell survival upon DNA-damaging chemotherapy by suppressing RIP-independent necroptosis and pinpoint that inhibition of one isoform is sufficient to bypass drug resistance of p53-null cells. Being GSK3A and GSK3B redundant in the response to DNA-damaging drugs, it is likely that, when used together with anticancer drugs, GSK3 inhibitors should be particularly effective in overcoming drug resistance even at low doses, since inhibition of only one isoform, or rather partial inhibition of overall cellular GSK3 activity, is enough to re-sensitize drug-resistant cells. We therefore propose that GSK3 inhibition in combination with DNA-damaging drugs would be an appealing strategy to induce necroptosis in p53-null drug-resistant tumor. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B113. Citation Format: Emanuela Grassilli, Elena Federzoni, Robert Narloch, Leonarda Ianzano, Kristian Helin, Marialuisa Lavitrano. GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B113.

Evasion from chemotherapy-induced apoptosis strongly contributes to drug resistance and tumor relapse. In particular, p53 loss renders colon carcinoma cells resistant to 5-fluorouracil-induced apoptosis. We have used the 5-fluorouracil-resistant HCT116p53KO colon carcinoma cell line and a kinase-directed retroviral shRNA library to identify kinases whose inactivation reverts resistance to drug-induced apoptosis. In this way we have identified 29 kinases whose downregulation abolished colony growth after 5-fluorouracil treatment. Silencing of these kinases in other drug-resistant p53-null colon cell lines also abolished cellular growth, suggesting that the effect is independent of the genetic background. We focused on GSK-3 since by tissue microarray we found that GSK-3 is overexpressed/activated in tumoral biopsies of infiltrating and invasive colon carcinomas. We demonstrated that GSK-3 silencing in different colon carcinoma cell lines reverts resistance not only to 5-fluorouracil, but also to oxaliplatin and the 2 drugs used in combination. Interestingly, we found that 5-fluorouracil-treated p53-null GSK-3-depleted colon carcinoma cells undergo caspase-independent but PARP-dependent and tBid- and AIF-mediated cell death. Therefore, we propose that GSK-3 is an excellent potential target for the development of anti-cancer therapy. Our results suggest that inhibitors of GSK-3 can be used in combination with chemotherapy to kill cancer cells unable to trigger caspase-dependent apoptosis.

<p>Supplementary Table 2 - PDF file 70K, Cohort of patients characterization</p>