Study on the molecular mechanism of Rac3 on regulating autophagy in human lung cancer cells.

PURPOSE: Rac3 plays an important role in regulating tumorigenesis. Autophagy plays a vital role in tumorigenesis and tumor progression. The relationship between the two remains unclear. The objective of the present study was to determine the specific molecular mechanism of intracellular Rac3 in regulating autophagy and reveal the relationship between tumor cell autophagy and apoptosis.

METHODS: A laser confocal microscope was used to photograph the accumulated EGFP-MAP1LC3 spots for investigating the relationship between Rac3 and autophagy at the cellular level. Immunoblotting was also used to investigate the relationship between Rac3 and autophagy. The autophagy flux arising from inhibition of Rac3 was detected with autophagy inhibitors and ATG5 and ATG7 siRNA interference experiments. ATF4 and DDIT4 siRNA interference and overexpression experiments were conducted to investigate the relationship between endoplasmic reticulum stress, the MTOR signaling pathway, and autophagy arising from inhibition of Rac3. Co-immunoprecipitation experiments were performed to investigate the interaction between Rac3 and proteins related to endoplasmic reticulum stress. Co-immunoprecipitation was performed to investigate the structural domains between Rac3 and HSPA5.

RESULTS: The expression of ATF4 and DDIT4 was upregulated, which inhibited the MTOR signaling pathway and induced autophagy of human non-small cell lung cancer cells after Rac3 siRNA was introduced. The degree of acetylation of the substrate, HSPA5, increased and the endoplasmic reticulum stress response was activated after Rac3 was inhibited.

CONCLUSION: In conclusion, the degree of acetylation of HSPA5 increased and it was dissociated from the receptor, EIF2AK3, on the endoplasmic reticulum membrane, thus causing the endoplasmic reticulum stress response. Endoplasmic reticulum stress activated the expression of the ATF4 protein, upregulated the level of DDIT4, inhibited the MTOR signaling pathway, and caused cellular autophagy.