Supplementary Figure 4. The effect of ATF2 siRNA on gene expression and PT340 FST induced chemoresistance. A. Schematic detailing the transwell experiments. CellTrace Violet cells were sorted for rapidly dividing (Dim) or quiescent cells (Bright) which were then plated in transwell chambers either as Dim:Dim or Bright:Dim. Cells were untreated or treated with taxol alone or in combination with IgG or anti-FST antibody. Cell counts were performed on the dim cells and data was expressed as fold chnage. B. Relative ATF2 gene expression of PT412 and PT340 treated with scrambled siRNA or two independent ATF2 siRNAs (ATF2 siRNA #1 and #2). C. Viable cell number of PT340 cells treated with control scrambled siRNA or knocked down with ATF2 siRNA and treated with cisplatin +/- FST. *P<0.05, ns = not significant.
ARTICLE ABSTRACT
We recently reported that the transcription factor NFATC4, in response to chemotherapy, drives cellular quiescence to increase ovarian cancer chemoresistance. The goal of this work was to better understand the mechanisms of NFATC4-driven ovarian cancer chemoresistance.
We used RNA sequencing to identify NFATC4-mediated differential gene expression. CRISPR-Cas9 and FST (follistatin)-neutralizing antibodies were used to assess impact of loss of FST function on cell proliferation and chemoresistance. ELISA was used to quantify FST induction in patient samples and in vitro in response to chemotherapy.
We found that NFATC4 upregulates FST mRNA and protein expression predominantly in quiescent cells and FST is further upregulated following chemotherapy treatment. FST acts in at least a paracrine manner to induce a p-ATF2–dependent quiescent phenotype and chemoresistance in non-quiescent cells. Consistent with this, CRISPR knockout (KO) of FST in ovarian cancer cells or antibody-mediated neutralization of FST sensitizes ovarian cancer cells to chemotherapy treatment. Similarly, CRISPR KO of FST in tumors increased chemotherapy-mediated tumor eradication in an otherwise chemotherapy-resistant tumor model. Suggesting a role for FST in chemoresistance in patients, FST protein in the abdominal fluid of patients with ovarian cancer significantly increases within 24 hours of chemotherapy exposure. FST levels decline to baseline levels in patients no longer receiving chemotherapy with no evidence of disease. Furthermore, elevated FST expression in patient tumors is correlated with poor progression-free, post–progression-free, and overall survival.
FST is a novel therapeutic target to improve ovarian cancer response to chemotherapy and potentially reduce recurrence rates.
