Bruton's tyrosine kinase is a potential therapeutic target in prostate cancer

Abstract

Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase that has mainly been studied in haematopoietic cells. We have investigated whether BTK is a potential therapeutic target in prostate cancer. We find that BTK is expressed in prostate cells, with the alternate BTK-C isoform predominantly expressed in prostate cancer cells and tumors. This isoform is transcribed from an alternative promoter and results in a protein with an amino-terminal extension. Prostate cancer cell lines and prostate tumors express more BTK-C transcript than the malignant NAMALWA B-cell line or human lymphomas. BTK protein expression is also observed in tumor tissue from prostate cancer patients. Down regulation of this protein with RNAi or inhibition with BTK-specific inhibitors, Ibrutinib, AVL-292 or CGI-1746 decrease cell survival and induce apoptosis in prostate cancer cells. Microarray results show that inhibiting BTK under these conditions increases expression of apoptosis related genes, while overexpression of BTK-C is associated with elevated expression of genes with functions related to cell adhesion, cytoskeletal structure and the extracellular matrix. These results are consistent with studies that show that BTK signaling is important for adhesion and migration of B cells and suggest that BTK-C may confer similar properties to prostate cancer cells. Since BTK-C is a survival factor for these cells, it represents both a potential biomarker and novel therapeutic target for prostate cancer.

Keywords: BTK inhibitors; apoptosis; biomarker; cell survival; prostate cancer.

Figure 1.

BTK expression in prostate cell lines and tumors. BTK-C message is more abundant than the BTK-A isoform in prostate cancer cells and tumors. cDNA prepared from RNA isolated from prostate cancer cell lines (A) and human prostate normal and tumor tissues. (B) was subjected to qPCR using primers specific for BTK-A and BTK-C isoforms. Expression of each isoform was normalized to an actin control in the respective qPCR reaction. The data represent relative mRNA levels of each BTK isoform as fold increase of the normal tissue sample with the lowest expression. Lymphoma samples are shown for comparison. BTK-C was the predominant isoform in only 3% of lymphomas but 40% of prostate tumors (6 out of 15 cases).

Figure 2.

The BTK-(C)isoform is expressed in prostate cancer cell lines and tumors. (A) Schematic representation showing the domains of BTK-A and predicted BTK-C protein. (B) Total lysate from LNCaP, DU145 and Namalwa B-cells subjected to immunoblot analysis blotted and probed with an anti-BTK antibody. (C) Immunohistochemical staining shows increased expression of BTK in malignant prostate cancer tissue compared to hyperplasia prostate tissue. (D) Immunohistochemical staining shows expression of BTK in tonsil tissue using a BTK antibody.

Figure 3.

BTK shRNA and BTK-(C)specific siRNAs knock down decrease cell survival in prostate cancer cell lines. LNCaP (A) and DU145 (B) were transfected with shRNA and co-transfected with GFP to mark transfected cells. Transfected cells were counted at 24h and 72h and the 72h to 24h ratio was calculated and expressed as % of the control. LNCaP (C) and DU145 (D) were transfected with BTK-C specific siRNA or non-targeting siRNA. Co-transfection with a GFP expressing plasmid marks transfected cells. Transfected cells were counted and the 96h to 24h ratio was calculated and expressed as % of the control. (E) DU145 and NAMALWA cells were transfected with BTK-C specific siRNA and control siRNA for 48h. The cell lysates were prepared for immunoblotting. GAPDH is a loading control; the results show BTK-C siRNA just decreases the BTK-C protein and not BTK-A protein in NAMALWA cells. LNCaP (F) and DU145 (G) were transfected with BTK-C specific siRNA or non-targeting siRNA as a control for 48h. Increased cleaved caspase-3 was detected compared with control. Apoptotic cells for each treatment were calculated as fold increase in Caspase-3 positive cells of control. Mean of triplicate assays ± SD. Student t-test, *p < 0.05.

Figure 4.

BTK inhibitors decrease cell survival in prostate cancer cell lines. LNCaP and DU145 were treated with Ibrutinib (A and B), AVL-292 (C and D) and CGI-1746 (E and F) at the indicated concentrations. Treated cells were counted after 72h of treatment and were presented as % of the control. Mean of triplicate assays ± SD. Student t-test, *p < 0.05.

Figure 5.

Inhibition of BTK results in increased apoptosis in prostate cancer cells. DU145 and LNCaP cells were incubated with indicated concentration of Ibrutinib (A and B) and AVL-292 (C and D), CGI (E and F) for 48h results in increased cleaved caspase-3 compared with control cells treated with DMSO. Representative images of apoptotic cells after Ibrutinib or DMSO treatment are shown (G). Apoptotic cells for each treatment were calculated as fold increase of Caspase-3 positive cells of control. Mean of triplicate assays ± SD. Student t-test, *p < 0.05.

Figure 6.

BTK phosphorylation is reduced after treatment with BTK inhibitors in prostate cancer cells. (A) LNCaP or (B) DU145 cells containing the stably integrated BTK-A-Flag, BTK-C-Flag or control vector were treated with Ibrutinib, AVL-292 and CGI-1746 at the indicated concentrations or DMSO as a control for 24h, then cells were lysed, subjected to western blot analysis and probed with the indicated antibodies.

Figure 7.

BTK inhibition by Ibrutinib leads to upregulation of apoptotic genes. (A) A heatmap representation of transcripts that display at least a 1.5-fold change in expression (B) Results of the Functional Annotation Clustering Analysis after GO-term enrichment of up-regulated and down-regulated genes using the DAVID Bioinformatics Resource. BP; Biological Process, CC; Cellular Component and MF; Molecular Function.

Figure 8.

Elevated BTK expression affects gene expression in prostate cancer cells. (A) A heatmap representation of transcripts that display at least a 1.fold5- change in expression (B) Venn diagram showing the number of genes those are upregulated or downregulated with overexpression of BTK-A or BTK-C in Du145 cells. (C) Results of the Functional Annotation Clustering Analysis after GO-term enrichment of up-regulated and down-regulated genes using the DAVID Bioinformatics Resource. BP; Biological Process, CC; Cellular Component and MF; Molecular Function.