RASSF1A Enhances Chemosensitivity of NSCLC Cells Through Activating Autophagy by Regulating MAP1S to Inactivate Keap1-Nrf2 Pathway

Abstract

Objective

Cisplatin (DDP) is an effective first-line therapy for non-small cell lung cancer (NSCLC) treatment; however, it can cause resistance and thus pose an obstacle to the efficacy of chemotherapy in NSCLC. This study aims to detect the effect of RASSF1A on DDP resistance of NSCLC and the underlying mechanism.

Methods

The expression levels of RASSF1A and microtubule-associated protein 1S (MAP1S) were investigated by qRT-PCR and Western blot and their interaction was testified by co-immunoprecipitation (Co-IP) analysis. The IC₅₀ value of DDP on A549 and A549/DDP cells (DDP-resistant cells) was measured. A549/DDP cells were transfected with pCDNA3.1-RASSF1A, pCDNA3.1-MAP1S, or si-RASSF1A, followed by treated with DDP. Cell counting kit-8 (CCK-8) and 5-ethynyl-2ʹ-deoxyuridine (EDU) were employed to measure cell survival rate. Western blot was applied to test the levels of autophagy-associated proteins p62, LC3II, and LC3I. Immunofluorescence staining was used to detect the green fluorescent protein (GFP)-LC3 puncta to evaluate the level of autophagy. Finally, a xenograft model in nude mice using A549/DDP cells was developed.

Results

RASSF1A and MAP1S were lowly expressed and positively correlated in NSCLC tissues. We observed that RASSF1A and MAP1S overexpression significantly enhanced DDP-induced effects in A549 and A549/DDP cells, including decreased cell viability, as well as increased autophagy levels. Besides, investigations into the mechanism between RASSF1A and MAP1S disclosed that RASSF1A could regulate MAP1S to inactivate the Keap1-Nrf2 pathway, thus activating autophagy to enhance chemosensitivity. Moreover, consistent results were confirmed in vivo experiments.

Conclusion

RASSF1A increases chemosensitivity in NSCLC by facilitating autophagy via MAP1S-mediated Keap1-Nrf2 pathway.

Keywords:

• RASSF1A
• MAP1S
• chemosensitivity
• autophagy
• Keap1-Nrf2
• A549/DDP

Acknowledgment

Thanks for all the contributors and participants.

Disclosure

The authors declare that they have no competing interests.

Additional information

Funding

This research was funded by the grants from Heilongjiang Postdoctoral Foundation (Grant No. LBH-Z18158);  Reseach Fund of Postdoctor in Heilongjiang Province (LBH-Q15094), Health and Family Planning Commission Research Foundation of Heilongjiang Province (Grant No. 2016-091) and Natural Science Foundation of Heilongjiang Province (Grant No. H2016005).