Supplementary Figure 5. Schematic of FST CRISPR KO guides and in vivo experiment. A. Binding of the two commercially available FST CRISPR guides to FST to mediate KO. B. Mice were injected with SKOV3 cells expressing FST sgRNA #2. Mice were then treated 3 times with 10mg/kg Taxol intraperitoneal Paclitaxel at days 7, 14 and 21. C Dissection of control mice presenting with multiple metastases and an FST-KO mice which showed no residual disease. White arrows point to sites of disease.
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.
