CRISPR/Cas9-mediated ATG7 knockout results in enhanced osteosarcoma chemosensitivity

Masters thesis


Pears, A. 2022. CRISPR/Cas9-mediated ATG7 knockout results in enhanced osteosarcoma chemosensitivity. Masters thesis Middlesex University Natural Sciences
TypeMasters thesis
TitleCRISPR/Cas9-mediated ATG7 knockout results in enhanced osteosarcoma chemosensitivity
AuthorsPears, A.
Abstract

Due to the development of chemotherapy, the survival rates in Osteosarcoma (OS) have improved immensely in patients with the localised disease. Despite this, long-term survival rates for OS patients facing metastatic or recurrent disease remains unchanged. One of the main reasons for this is resistance to anti-cancer therapies, which has become a major obstacle to improving OS treatment. Chemoresistance in OS appears to be mediated by a myriad of mechanisms, and increasing evidence implicates autophagy, a ‘self-degradation’ pathway as one of the mechanisms involved.
The aim of the present investigation was to demonstrate that autophagy has a key role in OS progression and to further elucidate the effect of autophagy modulation to circumvent resistance mechanisms to re-sensitise OS cells to common chemotherapy drugs.
In the current study, CRISPR/Cas9 was used to knockout (KO) ATG7, a key gene in the autophagy process, in HOS-143B (highly metastatic/aggressive) osteosarcoma cells. Successful knockout of ATG7, and the autophagy process, was validated by qPCR and cell viability assays. Furthermore, a colony-forming assay was used to examine the effect of ATG7 KO on cell survival following treatment with Doxorubicin or Cisplatin. In vitro metastatic potential following chemotherapy treatment was investigated through assessment of cell migration in monolayer and by the formation of migratory bodies from monolayer cultures.
Gene expression of ATG7 was significantly reduced in CRISPR/Cas9 edited HOS-143B cells, with a 14.5-fold decrease compared to control cultures (p<0.01) Clonogenicity assays revealed a 1.4-fold reduction in clone formation in ATG7 KO cells compared to controls (p<0.05), and no colonies were observed in KO cells treated with Dox or Cis (15.6-fold and 4-fold reduction versus drug-treated controls, respectively; p<0.05). No difference in migration was observed in ATG7 KO cells compared to controls, however, Dox- and Cis- treated KO cells displayed a 93.7% and 97.2% decrease in migration, respectively (p<0.05). Interestingly, formation of migratory bodies was reduced by 48.9% in ATG7 KO cells compared to HOS-143B controls (p<0.05) and was comparable to the reduction of migratory bodies observed in MG63 cells (77.0%; p<0.05), which have low metastatic potential.
The findings presented here show strong evidence to suggest that in advanced tumours autophagy is employed to aid cells in their progression and survival. Upon chemotherapy introduction, autophagy is upregulated to overcome stresses induced by anti-cancer drugs and facilitates tumour cells survival, progression and ultimately makes chemotherapy agents untenable. We have shown that the KO of ATG7 in osteosarcoma cells results in increased sensitivity to chemotherapy. Further to this, the relationship between autophagy and metastasis has been displayed showing that inhibition of autophagy can indeed result in lesser metastatic characteristics in previously higher metastatic cells. This is seminal in the eventual development into cancer therapy as those with the metastatic disease see the worst survival rate.
Despite this data indicating the importance of autophagy in chemotherapeutic efficacy, targeting this pathway in vivo poses many questions. Not least the mechanism of action, potential patient stratification and tumour targeting. Nevertheless, this work highlights the importance of this cell survival pathway in the susceptibility of osteosarcoma to currently used chemotherapeutic agents.

Sustainable Development Goals3 Good health and well-being
Middlesex University ThemeHealth & Wellbeing
Department nameNatural Sciences
Institution nameMiddlesex University
Publication dates
Print18 Jan 2023
Publication process dates
Deposited18 Jan 2023
Accepted07 Sep 2022
Output statusPublished
LanguageEnglish
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