The Proto-oncogene PKCι regulates the alternative splicing of Bcl-x pre-mRNA

Abstract

Two splice variants derived from the Bcl-x gene via alternative 5' splice site selection (5'SS) are proapoptotic Bcl-x(s) and antiapoptotic Bcl-x(L). Previously, our laboratory showed that apoptotic signaling pathways regulated the alternative 5'SS selection via protein phosphatase-1 and de novo ceramide. In this study, we examined the elusive prosurvival signaling pathways that regulate the 5'SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broad-based approach by using a number of small-molecule inhibitors of various mitogenic/survival pathways, we found that only treatment of non-small cell lung cancer (NSCLC) cell lines with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (50 μmol/L) or the pan-protein kinase C (PKC) inhibitor Gö6983 (25 μmol/L) decreased the Bcl-x(L)/(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCι and PKCζ, had no effect on the Bcl-x(L)/(s) mRNA ratio. Additional studies showed that downregulation of the proto-oncogene, PKCι, in contrast to PKCζ, also resulted in a decrease in the Bcl-x(L)/(s) mRNA ratio. Furthermore, downregulation of PKCι correlated with a dramatic decrease in the expression of SAP155, an RNA trans-acting factor that regulates the 5'SS selection of Bcl-x pre-mRNA. Inhibition of the PI3K or atypical PKC pathway induced a dramatic loss of SAP155 complex formation at ceramide-responsive RNA cis-element 1. Finally, forced expression of Bcl-x(L) "rescued" the loss of cell survival induced by PKCι siRNA. In summary, the PI3K/PKCι regulates the alternative splicing of Bcl-x pre-mRNA with implications in the cell survival of NSCLC cells.

Figure 1. The Bcl-x(L)/(s) mRNA ratio is dysregulated in NSCLC tumors

A population of cDNAs from pathologist-verified lung adenocarcinomas, squamous cell carcinomas, and large cell carcinomas (Origene; Rockville, MD) underwent quantitative/competitive PCR for expression of Bcl-x splice variants. A) A representation of the RT-PCR analysis of matched normal and tumor samples used to determine the degree of Bcl-x(L)/(s) dysregulation. N = normal lung tissue and T = tumor tissue. B) Quantitative/competitive PCR analysis of Bcl-x splice variants demonstrate that 46% of NSCLC tumors present a moderately dysregulated Bcl-x(L)/(s) mRNA ratio (Bcl-x(L)/(s) ratio of 5.0–7.5) and 32% of NSCLC tumors present a highly dysregulated Bcl-x(L)/(s) mRNA ratio (Bcl-x(L)/(s) ratio >7.5) (N=41), as determined by densitometric analysis of PCR products. C) The lung NSCLC samples (N=41) utilized above and SCLC samples (N=3) were grouped according to tumor stage to depict the correlation between tumor stage and degree of Bcl-x(L)/(s) mRNA dysregulation.

Figure 2. The effect of PI₃ kinase inhibiton on the alternative splicing of Bcl-x pre-mRNA

Quantitative/competitive RT-PCR analysis of Bcl-x splice variants and the corresponding Bcl-x(L)/(s) mRNA ratios from A) A549s, B) H226s, and C) H292s treated with either structurally inactive LY303511 [50μM] or LY294002 [50μM]. D) Western immunoblot analysis of Bcl-x(L) expression from A549s, H226, H292s treated with LY303511 control or LY294002. E) Quantitative/competitive RT-PCR analysis of Bcl-x splice variants and the corresponding Bcl-x(L)/(s) mRNA ratios from A549s treated with 0.1% DMSO or the PI₃K inhibtor, HWT [10μM]. The ratio of Bcl-x(L) to Bcl-x(s) mRNA was determined by densitometric analysis of RT-PCR fragments (p<0.01, N=6). Data are expressed as means ± s.d. Data are representative of three separate determinations on two separate occasions.

Figure 3. Inhibition of PKCι decreases the Bcl-x(L)/(s) mRNA ratio in A549 cells

Quantitative/competitive RT-PCR analysis of Bcl-x splice variants and the corresponding Bcl-x(L)/(s) mRNA ratios from A549s treated with either A) 0.1% DMSO or Gö6983 [10μM], B) PKCι siRNA, or C) PKCζ siRNA. The ratio of Bcl-x(L) to Bcl-x(s) mRNA was determined by densitometric analysis of RT-PCR fragments (p<0.01, N=6). Data are expressed as means ± s.d. B, C) Western immunoblot analysis of PKCι and PKCζ knockdown. Data are representative of three separate determinations on two separate occasions. D) Quantitative/competitive RT-PCR analysis of Bcl-x splice variants and the corresponding Bcl-x(L)/(s) mRNA ratios from A549s treated with either siCon, siPKCι, DMSO, or LY294002 (LY) as indicated. The ratio of Bcl-x(L) to Bcl-x(s) mRNA was determined by densitometric analysis of RT-PCR fragments (p<0.01, N=6). Data are expressed as means ± s.d.

Figure 4. Inhibition of the PI₃K/PKCι pathway decreases the levels of SAP155 protein expression in A549 cells

A) Quantitative/competitive RT-PCR analysis of Bcl-x splice variants and the corresponding Bcl-x(L)/(s) mRNA ratios from A549s treated with either control siRNA or SAP155 siRNA. The ratio of Bcl-x(L) to Bcl-x(s) mRNA was determined by densitometric analysis of RT-PCR fragments (p<0.01, N=6). Data are expressed as means ± s.d. Western immunoblot analysis of SAP155 knockdown. B) Total proteins from A549s treated with LY303511 control [50μM], LY294002 [50μM], 0.1% DMSO control, Gö6983 [10μM], Gö6976 [10μM], control siRNA, PKCι siRNA, or PKCι siRNA were subjected to western blot analysis to determine expression levels of SAP155 and β-actin. Quantiative real-time PCR analysis of SAP155 mRNA in A549s following treatment with C) LY303511 control or LY294002 and D) control siRNA or PKCι siRNA. Data are expressed as quantity of SAP155 mRNA over quantity of 18s rRNA. The columns represent the mean of two independent experiments ± S.E. E) Total proteins from A549s transfected with either control siRNA or PKCι siRNA were subjected to western blot analysis to determine expression levels of hnRNP K, Sam68, hnRNP L, hnRNP F/H and β-actin.

Figure 5. Inhibition of the PI₃K/PKCι pathway decreases the formation of the SAP155:CRCE 1 complex in A549 cells

The effect of LY294002 and Gö6983 on the formation of the SAP155:CRCE 1 complex was examined. Specifically, nuclear extracts were prepared from A549 cells after treatment with LY294002 (A) and Gö6983 (B) and subjected to EMSA binding conditions with labeled CRCE 1 as previously described. After a 25 min reaction equilibration, samples were then subjected to electrophoretic separation using a 5% TBE-polyacrylamide (37.5:1 acrylamide/bis-acrylamide). The position of the specific SAP155:CRCE 1 complex is indicated by the arrows. Data are representative of N=3 reproduced on two separate occasions.

Figure 6. Bcl-x(L) is required for PKCι to increase the clonogenic survival of NSCLC cells

A) A549 cells were transfected with the indicated siRNAs. Twenty-four hours post-transfection, cells were infected with control or Bcl-x(L) adenovirus. Twenty-four hours post-infection, cells were plated as single cells (125 cells/well) in 6-well dishes. Cells were then allowed to form colonies for 12 days after which colonies were fixed, stained with crystal violet, and counted. Total protein lysates were isolated from cells following treatment with the indicated siRNAs and adenoviruses and subjected to western immunoblot analysis to determine PKCι and Bcl-x(L) expression B) Representative photographs of stained tissue culture plates following treatment with the indicated siRNAs and adenoviruses. Data are expressed as mean ±SE and are representative of six separate determinations on two separate occasions.