The FOXC2 Transcription Factor Promotes Melanoma Outgrowth and Regulates Expression of Genes Associated With Drug Resistance and Interferon Responsiveness

Abstract

Background/Aim: The FOXC2 transcription factor promotes the progression of several cancer types, but has not been investigated in the context of melanoma cells. To study FOXC2's influence on melanoma progression, we generated a FOXC2-deficient murine melanoma cell line and evaluated The Cancer Genome Atlas (TCGA) patient datasets. Materials and Methods: We compared tumor growth kinetics and RNA-seq/qRT-PCR gene expression profiles from wild-type versus FOXC2-deficient murine melanomas. We also performed Kaplan-Meier survival analysis of TCGA data to assess the influence of FOXC2 gene expression on melanoma patients' response to chemotherapy and immunotherapy. Results: FOXC2 promotes melanoma progression and regulates the expression of genes associated with multiple oncogenic pathways, including the oxidative stress response, xenobiotic metabolism, and interferon responsiveness. FOXC2 expression in melanoma correlates negatively with patient response to chemotherapy and immunotherapy. Conclusion: FOXC2 drives a tumor-promoting gene expression program in melanoma and is a prognostic indicator of patient response to multiple cancer therapies. Melanoma, a highly aggressive form of cancer arising from pigment-producing melanocytes, is responsible for the majority of skin cancer-related mortality, accounting for~6 0,000 annual deaths worldwide (1). Importantly, the incidence of melanoma has risen substantially over the last 40 years (2), a trend that is expected to continue to at least 2031 (3). Although surgical removal of primary lesions is typically successful in the treatment of early-stage disease, many melanoma patients are not diagnosed until later stages of metastatic disease in which surgery is either not possible or largely ineffective. Unfortunately, malignant melanoma is highly resistant to radiation and chemotherapy, and the only FDA-approved chemotherapeutic for the treatment of melanoma, dacarbazine (DTIC), has a minimal impact on patient survival (4). While advances in targeted therapy and immunotherapy have improved the prognosis for melanoma in recent years, there are still patients who do not respond to these regimens, and relapse of therapy-resistant tumors remains an ongoing challenge in many patients who do achieve clinical benefit (5, 6). Therefore, in order to improve the clinical outcome of melanoma patients going forward, it is necessary to gain additional insight into factors that promote melanoma progression and resistance to these therapeutic modalities. FOXC2 is a member of the forkhead box family of transcription factors that control a variety of cellular processes in embryonic and adult tissues. In addition to its normal regulation of development, growth, and metabolism in various tissue types, FOXC2 has recently emerged as a driver of several hallmarks of cancer progression as well. Within vascular endothelial cells, FOXC2 promotes expression of multiple genes that enhance angiogenesis (7-9). FOXC2 can also become overexpressed or dysregulated in tumor cells themselves, where it is asso...

RNA-seq Analysis of Wild-Type vs. FOXC2-Deficient Melanoma Cells Reveals a Role for the FOXC2 Transcription Factor in the Regulation of Multiple Oncogenic Pathways

“…Our recent study highlighted several differentially expressed genes upregulated in B16-F1 that are associated with GO Terms related to the cellular response to xenobiotics and oxidative stress. We also noted in that study the downregulation in B16-F1 of several genes associated with GO Terms related to IFN responsiveness (16). Our current KEGG Pathway analysis supports these findings, with significant enrichment of B16-F1 upregulated genes in pathways related to xenobiotic metabolism and glutathione metabolism as well as significant enrichment of B16-F1 downregulated genes in pathways related to viruses and Nod-like receptor signaling, all of which include several genes involved in cellular responses to IFN.…”

“…Another previously unappreciated function of FOXC2 revealed by our data is its negative regulation of genes associated with IFN signaling, a finding that is particularly intriguing in light of recent studies demonstrating that both type I IFN and IFNγ signaling pathways within tumor cells are critical to the efficacy of cancer immunotherapies (34)(35)(36)(37). Indeed, our recent analysis of melanoma patient TCGA data showed that FOXC2 expression correlates negatively with progression-free survival (PFS) of patients treated with the CTLA-4 immune checkpoint inhibitor ipilimumab (16). Though the mechanism by which FOXC2 might promote resistance to checkpoint blockade therapy remains to be elucidated, it is interesting that in our murine model FOXC2 negatively regulated the expression of several IFN signaling pathway components, including the Ddx58 gene encoding RIG-I and the Stat1/Stat2/Stat3 and Irf7/Irf9 transcription factor genes.…”

“…While previous studies have focused on FOXC2 in the context of cancers originating from epithelial tissues, we recently demonstrated that FOXC2 is also a key contributor to the progression of melanoma (16). Using CRISPR-Cas9 gene editing technology, we engineered a variant of the B16-F1 murine melanoma cell line that carries a bi-allelic disruption in the Foxc2 gene and that does not express FOXC2 protein, and we reported that this B16-F1 FOXC2 variant grows out with slower kinetics as a subcutaneous tumor than its parental counterpart.…”

“…B16-F1 murine melanoma cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and grown in RPMI-1640 medium supplemented with 2 mM Lglutamine, 2 g/l glucose, and 2 g/l sodium bicarbonate (Thermo Fisher Scientific, Waltham, MA, USA), as well as 10% fetal bovine serum (Premium Select, Atlanta Biologicals, Norcross, GA, USA). B16-F1 FOXC2 cells were generated as described (16) and maintained in the same growth medium as the parental cell line. All cultures were grown at 37 • C in a 5% CO 2 incubator and passaged at 80-90% confluence.…”

“…We recently reported that expression of the FOXC2 gene in melanoma biopsies is an unfavorable prognostic indicator of patient survival following treatment with either chemotherapy or immunotherapy (16). In that study, we also described a novel CRISPR-Cas9 gene-edited variant of the murine B16-F1 melanoma that we engineered to lack the FOXC2 transcription factor (B16-F1 FOXC2).…”