Overexpression of Mcl-1L splice variant is associated with poor prognosis and chemoresistance in oral cancers

Abstract

Background: Altered expression of Mcl-1, an anti-apoptotic member of the Bcl-2 family, has been linked to the progression and outcome of a variety of malignancies. We have previously reported the overexpression of Mcl-1 protein in human oral cancers. The present study aimed to evaluate the clinicopathological significance of the expression of three known Mcl-1 isoforms in oral tumors and the effect of targeting Mcl-1L isoform on chemosensitivity of oral cancer cells.

Methods: The expression of Mcl-1 isoforms- Mcl-1L, Mcl-1S & Mcl-1ES was analyzed in 130 paired oral tumors and 9 oral cell lines using quantitative real-time PCR & protein by western blotting. The Mcl-1 mRNA levels were correlated with clinicopathological parameters and outcome of oral cancer patients. The effect of Mcl-1L shRNA or Obatoclax (a small molecule Mcl-1 inhibitor), in combination with Cisplatin on chemosensitivity of oral cancer cells was also assessed.

Results: Anti-apoptotic Mcl-1L was predominantly expressed, over low or undetectable pro-apoptotic Mcl-1S and Mcl-1ES isoforms. The Mcl-1L transcripts were significantly overexpressed in all cancer cell lines and in 64% oral tumors versus adjacent normals (P<0.02). In oral cancer patients, high Mcl-1L expression was significantly associated with node positivity (P = 0.021), advanced tumor size (P = 0.013) and poor overall survival (P = 0.002). Multivariate analysis indicated Mcl-1L to be an independent prognostic factor for oral cancers (P = 0.037). Mcl-1L shRNA knockdown or its inhibition by Obatoclax in combination with Cisplatin synergistically reduced viability and growth of oral cancer cells than either treatment alone.

Conclusion: Our studies suggest that overexpression of Mcl-1L is associated with poor prognosis and chemoresistance in oral cancers. Mcl-1L is an independent prognostic factor and a potential therapeutic target in oral cancers.

Figure 1. Expression of Mcl-1 isoforms in oral cell lines.

(a) Expression of Mcl-1L & Mcl-1S mRNA in oral cell lines; Mcl-1 ES levels were undetectable (* P≤0.03) (b) Mcl-1L protein expression in oral cell lines.

Figure 2. Correlation of Mcl-1L mRNA expression in oral normal versus tumor tissues.

(a) Expression of Mcl-1L mRNA in normal vs. oral tumors of different subsites (* P≤0.02); (b) Mcl-1L protein expression in adjacent normal versus tumors.

Figure 3. Survival analysis of oral cancer patients.

(a-c) Kaplan–Meier estimates of overall survival of oral cancer patients with low or high expression of Mcl-1 isoforms; (d) Multivariate analysis of oral cancer patients.

Figure 4. Down regulation of Mcl-1L expression in oral cancer cell lines.

(a & b) shRNA mediated down regulation of Mcl-1L mRNA & protein in AW8507, UPCI:SCC040 & SCCC29B oral cancer cells as compared to the control.

Figure 5. Estimation of Cisplatin IC50 for seven oral cancer cell lines by MTT assay (a) and the effect of Mcl-1L knockdown and/or Cisplatin on induction of cell death by PI staining (b) (* & ^δ P<0.05, vs. individual treatments).

Figure 6. Effect of combination of BH3 mimetic Obatoclax and Cisplatin on cell viability & growth.

(a) Confocal microscopic representative images of AW8507 cells post different treatments; (b) Assessment of percent cell viability of oral cancer cells post different treatments by trypan blue dye exclusion assay (* P<0.05, vs. individual treatments); (c) Analysis of proliferation of oral cancer cells after different treatments by MTT assay. (* P<0.05, vs. individual treatments).