Knockout of ATG5 leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2

Abstract

Autophagy can either promote or inhibit cell death in different cellular contexts. In this study, we investigated the role of autophagy in ATG5 knockout (KO) cell line established using CRISPR/ Cas9 system. In ATG5 KO cells, RT-PCR and immunoblot of LC3 confirmed the functional gene knockout. We found that knockout of ATG5 significantly increased proliferation of NIH 3T3 cells. In particular, autophagy deficiency enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and a soft agar colony formation assay. We also found that ATG5 KO cells had a greater migration ability as compared to wild-type (WT) cells. Moreover, ATG5 KO cells were more resistant to treatment with a Src family tyrosine kinase inhibitor (PP2) than WT cells were. Cyto-ID Green autophagy assay revealed that PP2 failed to induce autophagy in ATG5 KO cells. PP2 treatment decreased the percentage of cells in the S and G 2 /M phases among WT cells but had no effect on cell cycle distribution of ATG5 KO cells, which showed a high percentage of cells in the S and G 2 /M phases. Additionally, the proportion of apoptotic cells significantly decreased after treatment of ATG5 KO cells with PP2 in comparison with WT cells. We found that expression levels of p53 were much higher in ATG5 KO cells. The ATG5 KO seems to lead to compensatory upregulation of the p53 protein because of a decreased apoptosis rate. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to PP2.Autophagy is a crucial adaptive response that functions as a primary route of degradation for long-lived proteins and cytoplasmic organelles during normal cellular homeostasis (Mizushima & Komatsu, 2011). Nonetheless, there are conflicting data on the involvement of autophagy in the regulation of cell death or survival. In fact, autophagy can either promote or inhibit cell death in different cellular contexts (Maycotte & Thorburn, 2011; White et al., 2011). Our previous studies also showed that autophagy can serve either pro-survival or -death functions (Ahn, Lee, Ahn, & Lee, 2014;Kim & Lee, 2014;Kim, Han, & Lee, 2016). We recently reported a cytoprotective role of autophagy against BH3 mimetic gossypol in melanoma cells and glioma cells (Kim & Lee, 2014;Kim et al., 2016). Conversely, we found that an inhibitor (UAI-201) of oncogenic protein BRAF induces cell cycle arrest and autophagy in BRAF-mutant glioma cells (Ahn et al., 2014). In particular, deletion of ATG5 in mice results in the development of a benign tumor in the liver (Takamura et al., 2011), suggesting that autophagy has tumor-suppressive effects.Src family kinases (SFKs) are kept in an inactive state but can be switched to an active state by abnormal events such as a mutation (Okada, 2012). Active forms of SFKs are central mediators in multiple signaling pathways that are important in oncogenesis (Kim, Song, & Haura, 2009). Thus, Src inhibition has been proposed recently as a strategy for cancer therapy (Kim et al., 2...

Application of CRISPR/Cas9 gene editing technique in the study of cancer treatment

“…The use of CRISPR/Cas9‐mediated ATG5 knockout can significantly promote cell proliferation of the mouse embryonic fibroblasts cell line NIH3T3 and enhance the cell susceptibility to transformation. Compared with wild‐type cells, ATG5 KO cells had stronger migration ability and significantly increased resistance to the Src tyrosine kinase inhibitor PP2 . The results showed that autophagy deficiency can induce malignant cell transformation and resistance to PP2.…”

“…56,88,94,97,100,113,[141][142][143][144][145][146][147][148][149][150][151][152][153][154][155] Compared with wild-type cells, ATG5 KO cells had stronger migration ability and significantly increased resistance to the Src tyrosine kinase inhibitor PP2. 156 The results showed that autophagy deficiency can induce malignant cell transformation and resistance to PP2.…”

Autophagy inhibition in 3T3-L1 adipocytes breaks the crosstalk with tumor cells by suppression of adipokine production

“…In contrast, other evidence indicates that the predominant role of autophagy in cancer cells is to maintain tumor cell survival (Degenhardt et al 2006). Our previous studies also showed that autophagy could play a dual role in either pro-cell survival or pro-cell death in response to anticancer drugs (Hwang et al 2018).…”

“…Autophagy was measured by the conversion of LC3-I to LC3-II by immunoblot analysis as previously (Hwang et al 2018). Detection was achieved using the Bio-Rad ChemiDoc XRS+ instrument (Hercules, CA, USA), and the data were visualized using the Bio-Rad Image Lab software version 5.2.1 (Bio-Rad Laboratories).…”

“…In vitro cell migration assay An in vitro cell migration assay was carried out using a 24-well transwell culture system as described previously (Hwang et al 2018). For quantitation, the crystal violet dye retained on the filters was acid extracted, and cell migration was measured by reading the absorbance at 550 nm.…”