Lung cancer has been the most common
cancer worldwide for several decades. The outcomes of patients with locally advanced
lung cancer remain dismal, and only a minority of patients survive more than 5 years. However,
tumor therapeutic resistance mechanisms are poorly studied. Identification of therapeutic resistance pathways in
lung cancer in order to increase the sensitivity of lung
tumor cells to therapeutic agents is a crucial but challenging need. To identify novel genes that modulate the response to
platinum-based
therapy, we performed a genome-wide high-throughput
ribonucleic acid interference (RNAi) screen via transfection of human
lung cancer (PC9) cells with a viral
short hairpin RNA (
shRNA) library. We further validated a potential target via
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic survival assays on PC9 and A549 lung
tumor cells transfected with small interfering RNAs (siRNAs) to successfully downregulate
protein expression and then treated with increasing doses of
cisplatin or X-ray radiation. We determined
protein expression by immunohistochemistry (IHC) after
chemoradiotherapy and analyzed gene expression-based survival outcomes in two cohorts of human
non-small-cell lung cancer (NSCLC) patients. The screen identified several targets involved in epithelial-to-mesenchymal transition (EMT), including Smurf1, Smurf2, YAP1, and CEBPZ, and glycolytic pathway
proteins, including PFKFB3. Furthermore, we found that the small molecule
proteasome inhibitor bortezomib significantly downregulated Smurf2 in
lung cancer cells. The addition of
bortezomib in combination with
cisplatin and
radiation therapy in PC9 and A549 cells led to an increase in
deoxyribonucleic acid (
DNA) double-strand breaks with increased numbers of γ-H2AX-positive cells and upregulation of apoptosis. Finally, we found that Smurf2
protein expression was upregulated in situ
after treatment with
cisplatin and
radiation therapy in a relevant cohort of patients with stage III NSCLC. Additionally, Smurf2 gene expression was the strongest predictor of survival in patients with squamous NSCLC after
chemotherapy or
chemoradiotherapy. We successfully identified and validated Smurf2 as both a common modulator of resistance and an actionable target in
lung cancer. These results suggest the urgent need to investigate clinical Smurf2 inhibition via
bortezomib in combination with
cisplatin and radiation for patients with locally advanced NSCLC.