RUNX1 and FOXO3a dictates IGF1R expression driving the early stages of chemoresistance development in Ovarian Cancer Cells

Introduction

Chemoresistance has been a major hurdle in management of Epithelial Ovarian Cancer (EOC). Therefore, it is crucial to delineate molecular events responsible for initiation of chemoresistance. Using isogenic chemoresistant models of EOC cells, we recently reported augmented expression of IGF1R during onset of chemoresistance which subsequently decreased as cells progressed to highly resistant state. In this study we aim to comprehend the regulators of IGF1R during chemoresistance development.

Materials and methods

A2780, OAW42 EOC cells and their respective Cisplatin-Paclitaxel resistant models were used to asses IGF1R promoter reporter (IGF1R proximal promoter driving Firefly luciferase2-tdTomato fusion protein) activity and Chromatin immunoprecipitation (ChIP) assay. IGF1R-promoter binding transcription factor (TF) array was performed to identify putative TFs binding to IGF1R promoter. CBFβ-Runt-related transcription factor 1 (RUNX1) inhibitor (Ro5-3335) and CBFβ (RUNX1 binding protein) knockdown strategies were used for RUNX1 inhibition.

Results and discussion

TF array identified RUNX1 as a novel regulator for IGF1R promoter. Ro5-3335 treatment led to dose dependent decrease in IGF1R promoter-reporter activity which was more profound in A2780 and OAW42 early resistant (A2780^Cis-Pac-ER and OAW42^Cis-Pac-ER) cells than sensitive (A2780^S and OAW42^S) and late resistant (A2780^Cis-Pac-LR and OAW42^Cis-Pac-LR) cells. Ro5-3335 treatment also reduced IGF1R transcript levels in A2780^Cis-Pac-ER cells by 40% as compared to 11% and 8% decrease in A2780^S and A2780^Cis-Pac-LR cells. Bioinformatics analysis revealed that binding sites of RUNX1 and FOXO3a, a known IGF1R regulator are in close proximity on IGF1R promoter. Mutagenesis in FOXO3a binding sites on IGF1R promoter led to significant reduction of its promoter activity in A2780^Cis-Pac-ER and OAW42^Cis-Pac-ER cells which intriguingly further diminished after RUNX1 inhibition indicating a co-operative role of RUNX1 and FOXO3a in IGF1R regulation only at onset of resistance. Nuclear FOXO3a and phosphor-s-413 FOXO3a (activated form) levels were found to be elevated in early resistant cells than the respective sensitive and late resistant cells. ChIP studies showed enhanced binding of FOXO3a to IGF1R promoter in A2780^Cis-Pac-ER cells compared to A2780^S and A2780^Cis-Pac-LR cells. Inhibition of RUNX1 activity decreased FOXO3a binding to IGF1R promoter only in A2780^Cis-Pac-ER cells. RUNX1 inhibition reversed the chemoresistance development in A2780^Cis-Pac-ER cells and A2780^Cis-Pac-LR cells and significantly affected the clonogenic potential. In vivo studies to monitor effect of RUNX1 inhibition on chemoresistance are ongoing.

Conclusion

We identify FOXO3a and RUNX1 as key players involved in early onset of drug resistance by upregulating IGF1R levels and potential targets for blocking initial stages of drug resistance development.