Inhibition of lncRNA PART1 Chemosensitizes Wild Type but Not KRAS Mutant NSCLC Cells

Abstract

Background: Lung cancer has the highest incidence among solid tumors in men and is the third most common cancer in women. Despite improved understanding of genomic and mutational landscape in non-small cell lung cancer (NSCLC), the five-year survival in these patients has remained stagnant at a dismal 15%. The first line of treatment commonly adapted for NSCLC patients with somatic mutation in EGFR is tyrosine kinase inhibitor gefitinib or erlotinib. EGFR mutant cells seem to be intrinsically sensitive to tyrosine kinase inhibitors; however, the remaining 20-30% patients are resistant to tyrosine kinase inhibitor.

Materials and methods: Here we show, using in vitro normal and NSCLS cell lines, that the lncRNA Prostate androgen-regulated transcript 1 (PART1) is expressed at higher levels in NSCLC cells compared to normal lung epithelial cell line, corroborating two earlier studies.

Results: We additionally show that these cells are resistant to erlotinib which is reversed in some, but not all, cell lines following suppression of PART1 expression. The differential response to erlotinib following siRNA-mediated knockdown of PART1 was found to be related to the mutational status of KRAS. Only in cells with wild-type KRAS suppression of PART1 sensitized them to erlotinib. Knockdown of mutant KRAS did not sensitize those cell lines to erlotinib. But knockdown of mutant KRAS along with suppression of PART1 sensitized the cells to treatment with erlotinib. The results from the study reveal a yet undefined and important role of lncRNA PART1 in defining sensitivity to erlotinib. This action is mediated by mutation status of KRAS.

Conclusion: Even though preliminary, our results indicate PART1 might be a potential candidate for targeted therapy or used as a predictor of chemosensitivity in patients with NSCLC.

Keywords: KRAS; PART1; chemosensitivity; erlotinib; lncRNA; non-small cell lung cancer.

Figure 1

Differential PART1 expression and resistance to erlotinib in NSCLC cells. (A) Relative expression of EGFR determined by qRT-PCR. Post-normalization to 18s rRNA expression, expression relative to BEAS-2B is shown. Error bars, SEM. Error bars, SD. *P<0.05. (B, C) Western blot (B) and IF (C) analysis of EGFR expression in the normal lung epithelial cell line BEAS-2B or the NSCLC cell line, H-2444, A549, NCI-H647, and NCI-H23. Scale bar, 35 µm. (D) The normal lung epithelial cell line BEAS-2B or the NSCLC cell line, H-2444, A549, NCI-H647, and NCI-H23 cells were treated with indicated doses of erlotinib for 3 days and cell viability was measured. Data is representative of three independent experiments, each done in triplicate. Error bars, SD. (E) Relative expression of lncRNA PART1 determined by qRT-PCR. Post-normalization to 18s rRNA expression, expression relative to BEAS-2B is shown. Error bars, SEM. Error bars, SD. *P<0.05.

Figure 2

Overexpression of PART1 makes BEAS-2B cells resistant to erlotinib. (A) Relative expression of lncRNA PART1 determined by qRT-PCR. Post-normalization to 18s rRNA expression, expression relative to mock transduction (BEAS-2B) or transfection (other cell lines). Error bars, SEM. Error bars, SD. *P<0.05. (B) Mock or PART1 transduced BEAS-2B cells were treated with indicated doses of erlotinib for 3 days and cell viability was measured. Data is representative of three independent experiments, each done in triplicate. Error bars, SD. *P<0.05.

Figure 3

Differential sensitivity of NSCLC cell lines to erlotinib following knockdown of PART1. Indicated cells were transfected with PART1 or negative control siRNA for 48 hours. Post-48 hours of transfection, cells (NCI-H2444 (A), NCI-H647 (B), A549 (C), and NCI-H23 (D)) were treated with indicated doses of erlotinib for 3 days and cell viability was measured. Data is representative of three independent experiments, each done in triplicate. Error bars, SD. *P<0.05.

Figure 4

NSCLC cells with mutant KRAS become sensitive to erlotinib following combined knockdown of PART1 and KRAS. (A) Relative expression of KRAS determined by qRT-PCR. Post-normalization to 18s rRNA expression, expression relative to respective controls are shown. Error bars, SEM. Error bars, SD. ^*P<0.05 versus mock-KRAS group; ^#P<0.05 versus mock-PART1 group. (B) Immunoblot analysis of KRAS in NCI-H2444 and NCI-H647 cells transduced with negative control or KRAS shRNA. Blots were probed with GAPDH to confirm loading across lanes. Shown are representative blots from three independent experiments. (C, D) KRAS knockdown NCI-H2444 (C) and NCI-H647 (D) cells were transiently transfected with negative control (mock) or PART1 siRNA. Post-48 hours of transfection, cells (NCI-H2444 (C), and NCI-H647 (D)) were treated with indicated doses of erlotinib for 3 days and cell viability was measured. Data is representative of three independent experiments, each done in triplicate. Error bars, SD. *P<0.05, ^#P<0.05.