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Adaption of Osteosarcoma Tumor Cells to ATG7 Knockout and Autophagy Deficiency is Dependent on Parental Cell Line and does not Sensitize Cells to Doxorubicin

2022; Elsevier BV; Volume: 174; Linguagem: Inglês

10.1016/s0959-8049(22)01084-x

1879-0852

Deborah Gustafson, Lindsey Viola, Kellon S Banks, Sandra Witta, Sunetra Das, Dawn L. Duval, K. Van Eaton,

Cancer-related gene regulation

Background: Autophagy inhibitors such as hydroxychloroquine (HCQ) are an active area of research in cancer therapy due to the role autophagy plays in tumor cell survival during physiologic and pharmacologic stress. Recent studies have suggested that tumor cell survival following ATG7 knockout is dependent on NRF2 expression leading to stress responses including macropinocytosis. Characterization of tumor cell adaption to autophagy inactivation is a critical step in understanding how these cells may become resistant to clinical use of autophagy inhibitors. Recently, we have focused on the role of autophagy in the survival and drug response of osteosarcoma. Canine osteosarcoma (cOSA) is recognized as being similar to human osteosarcoma in terms of molecular, pathological and clinical features and to serve as a surrogate for the rarer human form in dogs that develop the disease. Materials and Methods: Validated and dual RFP/GFP labelled cOSA cell lines (Abrams, D17, Gracie, OSA8) were transfected with Cas9 and guide RNAs targeting GFP and ATG7 and resulting RFP only cells screened for loss of ATG7 and functional autophagy by Western Blotting for ATG7 and LC3I/LC3II conversion. Three ATG7-deficient clones were identified for each cell line for subsequent studies. Measures of tumor cell proliferation and death in response to growth conditions and drug treatment were done by live cell imaging of RFP and YOYO-1 labelled cells using an INCUCYTE ZOOMÒ. Lysosomal volume and macropinocytosis were determined using LysoTrackerÒ and uptake of Dextran-FITC, respectively. RNA was isolated from select ATG7-deficient clones for each cell line and RNASeq analysis performed. Results: Three ATG7-deficient clones were identified for each cell line. Response to low serum conditions varied across the deficient cell lines in a parental-dependent manner as did response to doxorubicin. Changes in lysosomal volume and upregulation of macropinocytosis were observed in some, but not all clones and these observed phenotypic changes were consistent with changes in lysosomal and macropinocytosis related gene expression in the RNASeq data. Interestingly, the ATG7-deficient clones maintained a similar sensitivity to HCQ and LysoV as the parental lines suggesting a disconnect between functional autophagy and sensitivity to drugs that inhibit autophagy by inhibiting lysosomal fusion. Conclusions: The results from these studies show that ATG7-deficient cOSA cells have drug sensitivities similar to those of the parental line from which they were derived for both HCQ, LysoV and doxorubicin. ATG7-deficiency also leads to changes in lysosomal volume and macropinocytosis activity that are varied across cell lines as well as changes in gene expression. These results will help in understanding tumor cell response to autophagy inhibition and how cell of origin can influence these responses. No conflict of interest.