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

Abstract

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.

Figure 1

p65BTK, a novel isoform of Bruton’s tyrosine kinase, is widely expressed in colon carcinoma cell lines and tissues. (a, b) BTK expression in colon cancer cell lines (a) or patients’ biopsy (b) lysates. Western blots probed with a commercial BTK antibody (Santa Cruz, sc-1696). (c) Western blot showing that in colon carcinoma cells (HCT116) BTK has a lower molecular weight than in lymphoid leukaemia (Nalm-6). (d) Western blot of BTK expression in HCT116 cells after silencing with BTK-specific siRNA (exons 5+8). (e) Western blot of BTK expression in HCT116 cells upon silencing using exon 1b (B1–3)-targeting siRNAs. (f) BTK gene and mRNAs encoding p77BTK and p65BTK. ATG1 and ATG2: start codons, black/white boxes: translated/untranslated exons. Exon 1a and exon 1b are indicated. (g) BTK expression in 293T cells transiently transfected with empty vector (empty) and plasmids encoding p77BTK or p65BTK coding sequence (p77CDS, p65CDS), p77BTK CDS or p65BTK CDS full lengths (p77FL, p65FL). (h) Western blot of p65BTK expression in 293T cells transiently transfected with p65FL plasmid followed by silencing with exon1b-specific siRNAs. (i) p65 and p77 BTK protein organization: PH domain. BH, BTK homology region; PPR, PolyProline region; TH, Tec homology domain; *phosphoinositide binding site.

Figure 2

hnRNPK and active ERKs post-transcriptionally regulate p65BTK expression. (a) In vitro translation assay performed with the following plasmids: empty vector (empty); p65FL (wt), p65_msATG1, p65_nsATG1, p65_nsATG2, p77_5′UTR or p77_msATG1. +cnt indicates the positive control included in the commercial kit used for the reaction. (b) p65BTK mRNA expression in matched samples of tumoural and peritumoural colon tissue from CRC patients (same patients as in Figure 1b). mRNA was quantified by Taqman assay and expression levels normalized to phosphoglycerate kinase. (c) Western blot of 293T cells transfected with empty vector (empty) or the following plasmids: p65FL, p65_5′UTRΔK1, p65_5′UTRΔK2, p65_5′UTRΔK3, p65_5′UTRΔK4. Deletion of all four binding sites allowed p65BTK overexpression most likely by rendering the transcript as it would be a CDS. (d) Western blot of p65BTK levels in colon cancer cell lines after siRNA-mediated depletion of hnRNPK (K). Transfection with siRNAs targeting luciferase (luc) was used as a control. On the right, the percentage of hnRNPK and p65BTK protein expression of each sample as calculated and normalized to actin by ImageJ program (http://imagej.nih.gov/ij/). (e, top) Anti-hnRNPK and anti-phospho-hnRNPK western blots after RNA immunoprecipitation using anti-hnRNPK and isotype-matched control (Ig mouse) antibodies. (e, bottom) Real-time PCR of p65BTK mRNA recovered by RIP in hnRNPK and IgG immunoprecipitates. (f) Western blot of p65BTK expression and hnRNPK-Ser284 phosphorylation following ERK1/2 inhibition with the MEK1/2 inhibitor CI-1040 (10 μM). Levels of total and phospho-ERKs are also shown. Cell lysates were obtained 24 h after CI-1040 addition but for HCT116p53KO cells, where p65BTK reduction is most prominent, at 16 h. On the right, the percentage of p-hnRNPK and p65BTK protein expression of each sample was calculated and normalized to actin by ImageJ program.

Figure 3

hnRNPK post-transcriptionally regulates p65BTK expression via IRES-dependent translation of exon 1b-containing mRNA. (a) Anti-hnRNPK antibodies immunoprecipitate a complex containing hnRNPK, eIF4G2 (top) and p65BTK mRNA (bottom) from HCT116p53KO lysates. (b) Fluorescence of HeLa cells transfected with a bicistronic vector encoding RFP under the control of CMV promoter and GFP not preceded by a regulatory region (first row) or under the control of p65BTK 5′UTR (second to fourth row) and left untreated (second row) or treated with Rapamycin 200 nM (third row) or Cymarin 100 nM (fourth row) for 36 h. DAPI was used to stain nuclei. (c) Time-dependent variation of p65BTK expression after treatment of colon cancer cells with 200 nM Rapamycin (left) and 200 nM Cymarin (right). Fold variation of p65BTK protein expression of each sample was calculated and normalized to actin by ImageJ program. (d) HeLa cells were transfected with the same bicistronic reporter as in (b) and luc-targeted siRNAs (second row) or hnRNPK-targeted siRNAs (third row). DAPI was used to stain nuclei.

Figure 4

p65BTK is a novel oncogenic protein acting downstream of RAS/MAPK pathway and is overexpressed in colon cancers. (a) NIH3T3 cells transfected with empty vector or plasmids encoding p65BTK, p77 or mutated H-RAS (H-RASV12). p65BTK expression was assessed by p65BTK-specific polyclonal antibody BN49, whereas p77BTK was probed with a monoclonal antibody against the N-term of BTK (BD). (b) Phase contrast images of NIH3T3 transfected with empty vector or plasmids expressing p77BTK, p65BTK, H-RASV12; × 40 magnification. To note, p77BTK-transfected NIH3T3 maintain the same appearance of the empty vector-transfected untransformed fibroblasts, whereas p65BTK-transfected NIH3T3 are similar to H-RASV12-transformed fibroblasts. (c) In soft agar assay, p65BTK-transfected NIH3T3 fibroblasts showed a colony-forming activity higher than H-RASV12-transfected ones (× 10 magnification). Right: number of colonies (mean of three separate wells). (d) Focus assay of NIH3T3 cells transfected with empty vector, H-RASV12, p65BTK or p77BTK expression plasmids, grown in the absence or presence of BTK (Ibrutinib), RAS (FTI-277) or MEK1/2 (CI-1040) inhibitors; parallel samples of p65BTK-transfected cells were treated for 16 days with CI1040 or treated for 10 days with CI1040 followed by 6 days without drug; (× 10 magnification). (e) Immunohistochemical detection of p65BTK, hnRNPK and p-ERK-1/2 in formalin-fixed, paraffin-embedded specimens (× 40 magnification); tumour samples (T) showing predominant cytoplasmic hnRNPK expression and moderate to strong p-ERK-1/2 levels expressed the highest amounts of p65BTK, whereas low expression of p65BTK was detectable in peritumoural (PT) samples, in which hnRNPK was exclusively or predominantly nuclear and p-ERK-1/2 levels were very low. (f, g) Overexpression of p65BTK in patients with stage II colon cancer. Tissue microarray (TMA) analysis of p65BTK expression was performed in tumoural/peritumoural pairs of specimens from a cohort of 83 patients and results were grouped by comparing the expression in tumoural vs peritumoural tissues (f) and by the intensity of the staining in the tumour tissue (g).

Figure 5

p65BTK inhibition affects growth and survival of colon cancer cells. (a) Time course showing Ibrutinib dose response (0, 0.01, 0.1, 1, 10, 20 μM Ibru) of colon carcinoma cell lines characterized by different genetic background; cell proliferation was determined every 24 h by MTT assay on cells incubated with Ibrutinib at the indicated concentrations; error bars show s.e.m.; data are the average of 3–5 independent experiments. Ibrutinib at 10 and 20 μ M significatively decreases cell growth in all cell lines *10 vs 0 μ M Ibru P<0.05; **20 vs 0 μ M Ibru: P<0.05. (b) Clonogenicity was assessed by seeding cells at low density and incubating them with the indicated doses of Ibrutinib for 10–12 days, at the end of which colonies were stained by crystal violet. (c) Cell viability was assessed after 72 h of treatment with the indicated concentration of Ibrutinib; crystal violet assay was performed to quantify viable cells; data are presented as fold change of the initial cell number obtained from 3 independent experiments; error bars show s.e.m. *10 vs 0 μ M Ibru: P<0.05; **20 vs 0 μ M Ibru: P<0.05; ***30 vs 0 μ M Ibru: P< 0.05.

Figure 6

Proposed model of p65BTK regulation.