Mcl-1 mediates TWEAK/Fn14-induced non-small cell lung cancer survival and therapeutic response

Abstract

Insensitivity to standard clinical interventions, including chemotherapy, radiotherapy, and tyrosine kinase inhibitor (TKI) treatment, remains a substantial hindrance towards improving the prognosis of patients with non-small cell lung cancer (NSCLC). The molecular mechanism of therapeutic resistance remains poorly understood. The TNF-like weak inducer of apoptosis (TWEAK)-FGF-inducible 14 (TNFRSF12A/Fn14) signaling axis is known to promote cancer cell survival via NF-κB activation and the upregulation of prosurvival Bcl-2 family members. Here, a role was determined for TWEAK-Fn14 prosurvival signaling in NSCLC through the upregulation of myeloid cell leukemia sequence 1 (MCL1/Mcl-1). Mcl-1 expression significantly correlated with Fn14 expression, advanced NSCLC tumor stage, and poor patient prognosis in human primary NSCLC tumors. TWEAK stimulation of NSCLC cells induced NF-κB-dependent Mcl-1 protein expression and conferred Mcl-1-dependent chemo- and radioresistance. Depletion of Mcl-1 via siRNA or pharmacologic inhibition of Mcl-1, using EU-5148, sensitized TWEAK-treated NSCLC cells to cisplatin- or radiation-mediated inhibition of cell survival. Moreover, EU-5148 inhibited cell survival across a panel of NSCLC cell lines. In contrast, inhibition of Bcl-2/Bcl-xL function had minimal effect on suppressing TWEAK-induced cell survival. Collectively, these results position TWEAK-Fn14 signaling through Mcl-1 as a significant mechanism for NSCLC tumor cell survival and open new therapeutic avenues to abrogate the high mortality rate seen in NSCLC.

Implications: The TWEAK-Fn14 signaling axis enhances lung cancer cell survival and therapeutic resistance through Mcl-1, positioning both TWEAK-Fn14 and Mcl-1 as therapeutic opportunities in lung cancer.

Figure 1. Mcl-1 expression in human NSCLC specimens correlates with Fn14 expression

(A) Mcl-1 and Fn14 staining on representative samples from the same patient with lung adenocarcinoma (5× objective, Aperio GL Scanner). Tumor-cell specific Fn14 and Mcl-1 staining in each of the tumor punches was scored by a board-certified pathologist; a score of zero indicates staining level equal to adjacent non-tumor cells. A non-zero score indicates increased staining (1= minimum, 2= moderate, 3= strong positive). (B) A total of 290 samples were scored for Mcl-1 and Fn14 expression and the correlation between the two stains was analyzed using Kendall’s tau test.

Figure 2. TWEAK induces Mcl-1 in NSCLC cell lines in an NF-κB-dependent manner

Total cell lysates were prepared from serum-reduced (A) H1975 and (B) H2073 cell lines treated with TWEAK for the indicated times and immunoblotted with the indicated antibodies: Mcl-1, Bcl-xL, and phosphorylated-p65 (Ser536). Tubulin was used as a loading control. (C) Serum-reduced H2073 cells transfected ± IkBα mutant were treated with TWEAK for 24 hours. Cells were harvested, total cell lysates were prepared and immunoblotted with the indicated antibodies to both Mcl-1 and phospho-p65. All blots were run in duplicate and tubulin was used as a loading control.

Figure 3. TWEAK-induced NSCLC cell survival is dependent on Mcl-1 expression

H1975 (A) and H2073 (B) cells were transfected with luciferase (siCont) or siRNAs targeting Mcl-1. Total lysates were collected 72 hours post-transfection and immunoblotted for Mcl-1 and alpha-tubulin. H1975 (C and E) and H2073 (D and F) cells transfected with control or siRNA constructs targeting Mcl-1 were exposed to 1 µM cisplatin for 24 hours (C and D) or 2Gy ionizing radiation (E and F) ± pre-incubation with TWEAK (100 ng/mL). Cells were sparsely seeded into 6-well dishes and allowed to grow for 7 days prior to staining with crystal violet and colony counting. A colony was defined as containing at least 50 cells. Bars represent average of three independent wells ± standard error with the non-treated (first bar) set to 1. * represents a p value < 0.05 by ANOVA with Bonferroni posttest.

Figure 4. Depletion of Mcl-1 abrogates TWEAK-induced protection from cell death induced by DNA damage

H1975 cells were transfected with either siRNA targeting luciferase (control) or Mcl-1. Cells were exposed to 5uM cisplatin (A) or 8Gy radiation (B) for 0, 4 or 24 hours ± pre-incubation with TWEAK (100 ng/mL). Total cell lysates were prepared and immunblotted for cleaved-PARP (cPARP) and GAPDH as a loading control. All blots were run in duplicate.

Figure 5. Pharmacologic inhibition of Mcl-1 inhibits NSCLC cell growth

(A) H1975 cells were grown in the presence or absence of TWEAK (100 ng/mL) and EU-5148 (10µM). Cells were lysed and immunoprecipitated with anti-Bak antibodies. Protein expression Mcl-1, Bcl-xL and Bak after immunoprecipitation were resolved by immunoblot analysis. (B) Cell viability of DHL10, Bcl-2 1863 and Mcl-1 1780 cells was assessed by PrestoBlue assay. Cells were exposed to the indicated concentrations of EU-5148 in DMSO for 48 hours. Cell killing curves and EC-50 values were generated from triplicate runs in GraphPad Prism 5. (C) A panel of NSCLC cell lines was exposed to vehicle or 10 µM EU-5148 for 48 hours. Cell growth was assessed by Cell-Titer Glo assay. Bars represent the average of two wells with the untreated set to 100%.

Figure 6. Pharmacologic inhibition of Mcl-1 abrogates TWEAK-mediated cell survival

H1975 (A and B) cells were pre-incubated with TWEAK (100 ng/mL), (A) EU-5148 (10 µM), (B) ABT-737 (10 µM) or both drug and TWEAK prior to exposure to 2Gy ionizing radiation. Cells were sparsely seeded (125 cells) into 6-well dishes and allowed to grow for 7 days prior to staining with crystal violet and colony counting. A colony was defined as containing at least 50 cells. Bars represent average of three independent wells ± standard error with the non-treated (first bar) set to 1. * represents a p value < 0.05 by ANOVA with Bonferroni posttest.