Identification of the MRTFA/SRF pathway as a critical regulator of quiescence in cancer

Abstract

Chemoresistance is a major driver of cancer deaths. One understudied mechanism of chemoresistance is quiescence. We used single cell culture to identify, retrieve, and RNA-Seq profile primary quiescent ovarian cancer cells (qOvCa). We found that many qOvCa differentially expressed genes are transcriptional targets of the Myocardin Related Transcription Factor/Serum Response Factor (MRTF/SRF) pathway. We also found that genetic disruption of MRTF-SRF interaction, or an MRTF/SRF inhibitor (CCG257081) impact qOvCa gene expression and induce a quiescent state in cancer cells. Suggesting a broad role for this pathway in quiescence, CCG257081 treatment induced quiescence in breast, lung, colon, pancreatic and ovarian cancer cells. Furthermore, CCG081 (i) maintained a quiescent state in patient derived breast cancer organoids and, (ii) induced tumor growth arrest in ovarian cancer xenografts. Together, these data suggest that MRTF/SRF pathway is a critical regulator of quiescence in cancer and a possible therapeutic target.

Figure 1.. Identifying and single-cell-RNA-Seq profiling quiescent ovarian cancer stem cells identifies key quiescence genes and pathways.

A. Image displaying the single-cell microfluidic culture platform with photo-mechanical cell detachment and retrieval capacity used to identify and isolate quiescent ovarian cancer cells. B. Images of live cells after 5 days in culture, identifying (left) a single non-dividing/quiescent cell and (right) a well with multiple cells that derived from a single proliferating cell. C. Waterfall plot of differentially expressed gene in quiescent cells vs proliferating cells. D and E. GO analysis of the top down- and upregulated molecular pathways. F. STRING analysis to the significantly enriched downregulated genes.

Figure 2.. Downregulation of EIF4G1, MHY9, and NCL contributes to quiescence.

(A) Cell counts and (B) cell cycle summary of PT412 cells treated with EIF4G1, MHY9, or NCL siRNA compared to a scrambled control. C. FUCCI cell cycle analysis of HEY1 cells treated with scrambled control siRNA or EIF4G1, MHY9, or NCL siRNA. D. Quantification of total p27 expression of SiRNA knockdown compared to control. E. p27KIP1-mVenus and CDT1-mCherry-positive cells in G0, showing an increase in SiRNA knockdowns as compared to control. Each experiment was repeated, in triplicate, a minimum of three times. Results were compared with Student’s T-test, *p<0.05, **p<0.01.

Figure 3.. Chemical inhibition of MHY9 and NCL reduce cell proliferation in vitro and in vivo.

A-B. Normalized (i) cell counts and (ii) viability percentage in PT340 cells line treated with the indicated doses of oridonin or blebbistatin for 72 hours. Three μM cisplatin was used as a cell death viability control. C-D. Tumor growth curve and final tumor weights of PT340 tumor xenografts (n=10/treatment group). Black bars indicate treatment with 20mg/Kg oridonin, 2mg/Kg blebbistatin, or DMSO control daily. E-F. Representative images and quantification of Ki67 staining. In vitro assays were replicated at least three times. Tumor Ki67 was analyzed for 6 to 8 independent sections of 3 independent tumors for each treatment group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4.. The SRF/MRTF transcriptional inhibitor CCG257081 acts through NCL, MHY9 and EIF4G1 to induces a quiescent state in cancer cells.

A. Venn diagram showing overlap of differentially expressed genes of scRNAseq of primary quiescent cells and previously reported SRF targets . B. Relative mRNA expression for the indicated genes as assesses by qRT-PCR in PT412 cells treated with DMSO (control) or CCG257081 for 72-h. C. IncuCyte real time imaging analysis of cellular confluence and viability analysis of control and CCG257081 (15μM every 48 h for 4 days). D. Normalized cell counts of PT412 treated with CCG257081 or Vehicle control (DMSO). E-F. DAPI chromatin IF and measure of nuclear invariant homogeneity for PT340 cell line (E) treated with DMSO or CCG257081 or, (F) isolated CFSE labeled slow/non-dividing (Bright) or rapidly proliferating (Dim) cells. G. Normalized cell counts of colon (SW480, HT-29), breast (MCF7, MDA-MB-231), lung (H522, H2170), and pancreatic (PATU) cell lines, treated with DMSO or CCG257081. H-I. Summary and representative IF image of EdU staining in control and CCG257081-treated RFP-MDA-MB231. Except were otherwise indicated, CCG257081 treatment was 15μM every 48 hours for 72 hours. In vitro assays were replicated in triplicate at least three times and compared with an ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5.. CCG257081 acts through p27 to induce quiescence.

A-B p27 (p27kip1) Western blot and densitometry in control and CCG081-treated cells. C. p27 Relative mRNA expression in control and CCG081-treated PT340 cells. D-E. FUCCI cell cycle analysis of control and CCG081-treated HEY1 cells. F-G. FUCCI cell cycle analysis and quantification of p27-mVenus expression in HEY1 cells treated with scrambled control, MRTFA, SRF, or MRTFA+ SRF siRNA. H. Cell counts of PT340 cells treated with the indicated SiRNAs. I. Percent of cells in G0/G1 as measured by PI staining in Pt340 cells transfected with wild type (WT) MRTF-A, MRTF-A with a deletion of the SAP domain (ΔSAP), or MRTF-A with a deletion of the SRF binding domain (ΔSRF). J. Cell counts of PT340 cells treated with the indicated compounds alone or in combination with 15 μM of CCG257081. In all experiments, CCG257081 treatment was every 48 hours for 3 days. All assays were replicated, in triplicate, at least three times and compared with an ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6.. RNA sequencing of CCG257081-treated cells supports induction of quiescence.

A. Volcano plots of differential gene expression between control and CCG257081-treated cells. B. Gene Ontology (GO) analysis of the top involved pathways following CCG257081 treatment. C. Kyoto Encyclopedia of Genes and Genomes (KEGG) in involved in CCG257081-treated cells. D. FUCCI cell cycle analysis of HEY1 cells treated with either AXL (bemcetinib), Aurora Kinase-A (AURKA, alisertib), or polio-like kinase 1 (PLK1, onvansertib) inhibitors. E-G. Percent p27+ cells, normalized cell counts, and percent live cells in cells treated with the indicated inhibitors.

Figure 7.. CCG257081 growth arrest in tumor models.

A-D. Normalized cell counts, hematoxylin-eosin stain, and Ki67 IHC, with quantification from two triple negative breast cancer cell (TNBC) organoid cultures treated with DMSO (control) or CCG257081. E. Quantification of Ki67 stain from tumor MDA-231 cells expressing the indicated MRTF-A constructs. F-G. Tumor growth and overall survival curves for PT340 xenografts treated with 20 mg/Kg CCG257081 or vehicle (DMSO) control daily (black bars indicate treatment window). H. qRT-PCR-based relative mRNA expression for the indicated genes in control and CCG257081-treated tumors. I-J. Representative images and quantification of Ki67 staining in control and CCG257081 treated tumors, 6 to 8 independent sections of 3 independent tumors for each treatment group. *P<0.05, **P<0.01, ***P<0.001. HPF - High Power Field