NOVEL EPIGENETIC THERAPY FOR HYPERTHERMAL INTRAPERITONEAL CHEMOTHERAPY ENABLES GENE REMODELING OF THE TUMOR MICROENVIRONMENT
Olivia K. Coburn-Flynn*1, Margaret V. Butchy1, Gennaro Calendo2, Robert Emery1, Vincent Verchio1, Kristen Knapp1, Zachary Ladd3, Ping Zhang1, Francis Spitz1, Jaroslav Jelinek2, Young K. Hong1
1Surgery, Cooper University Health Care, Camden, NJ; 2Coriell Institute for Medical Research, Camden, NJ; 3Rowan University Cooper Medical School, Camden, NJ
Introduction:
Cytoreductive surgery followed by HIPEC is a treatment option for peritoneal carcinomatosis for colorectal cancer; however, there is a lack of consensus on the benefit and type of chemotherapy agent utilized. We propose a novel epigenetic agent Mithramycin A (MA), as an alternative treatment to Mitomycin C as a cytotoxic agent with enhanced capability to remodel the tumor genetic landscape.
Methods:
Utilizing two colon cancer cell lines, HT-29 and CaCO2, we performed cell proliferation assays after treatment with MA (750nM) or MC (4.48uM) incubated at 37° or 42° Celsius for 90 min. RNA Sequencing using triplicate samples of the HT-29 cell line was performed. Resultant data were organized into 50 hallmark gene sets, normalized enrichment scores, log fold changes, and adjusted p-values. The top 6 hallmark gene sets with the greatest change in expression measured by adjusted p-value were cross-referenced against the Catalog of Somatic Mutations in Cancer (COSMIC) list of 733 known cancer-associated genes and the Tumor Suppressor Gene Database (TSGD) list of 535 known tumor suppressor genes in colon cancer.
Results:
Treatment of CaCo2 and HT-29 at 42° Celsius demonstrated comparable cell viability (CaCo2: MA 48.9% vs. MC 40.9%, p=0.23) (HT-29: MA 38.1% vs. MC 25.1%, p=0.18). RNA sequencing results showed MA treatment had dramatic global effects on gene expression, with 3,523 (26.1%) genes upregulated and 4,128 (30.6%) downregulated compared to MC, which had only 369 (2.7%) upregulated and 947 (7.0%) downregulated. Compared to the COSMIC data set, the top six hallmark pathways contained 57 unique gene expressions versus control with statistical significance. Two genes whose upregulation has been previously implicated in colon cancer progression saw significant downregulation when treated with MA versus control: MYB (Log fold D= -3.45, adj p-value= 3.11x10-5) and MYH9 (Log fold D= -3.96, adj p-value= 4.65x10-7). Top six hallmark pathways were compared to the TSGD database and 21 genes with a significant positive log fold change were found, including CDO1 (log fold D= 5.07, adj p-value=2.01x10-4), GPX3 (log fold D=5.44, adj p-value 5.34x10-7), DNAJB4 (log fold D= 2.43, adj p-value= 5.44x10-4), CDKN1C (log fold D= 1.97, adj p-value= 0.002) CDKN1A (log fold D=2.28, adj p-value= 1.88x10-5) and CAV1 (log fold D= 3.24, adj p-value= 7.66x10-4).
Conclusion:
While Mithramycin A shows comparable efficacy in vitro against colon cancer cell lines to Mitomycin C, MA genetically remodels tumorigenesis by epigenetic modulation. Further investigation of this novel agent is warranted as a potential agent to reduce tumor recurrence by upregulation of previously repressed tumor suppressor genes.
Fig1: Volcano plot of relative expression of individual genes when treated with mithramycin A (left) or mitomycin C (right)
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