Abstract 431: FOXD1 promotes dedifferentiation and targeted therapy resistance in melanoma by regulating the expression of connective tissue growth factor
Authors: Sun Qian , Novak Daniel , Hüser Laura , Poelchen Juliane , Wu Huizi , Granados Karol , Federico Aniello Federico , Liu Ke , Steinfass Tamara , Vierthaler Marlene , Umansky Viktor , Utikal Jochen 
Published: April 2023

Abstract
Metastatic melanoma is one of the most aggressive skin cancers and is associated with poor prognosis. BRAF and MEK inhibitors are used to treat patients with BRAFV600E-mutated advanced melanoma. However, the development of resistances to these treatments compromises therapeutic success. Our lab previously demonstrated that forkhead box D1 (FOXD1) plays a critical role in melanoma migration and invasion. Here, we found that FOXD1 was highly expressed in melanoma cells. Immunohistochemical assessment of 105 samples from patients with metastatic melanoma revealed that high FOXD1 expression in tumors was associated with poor survival and correlated with low MITF, SOX10 and high AXL expression. Upregulation of FOXD1 expression enhanced the resistance of melanoma to vemurafenib (BRAF inhibitor) or combinatorial treatment with vemurafenib and cobimetinib (MEK inhibitor). On the other hand, loss of FOXD1 increased the sensitivity of naïve melanoma cells towards vemurafenib or combinatorial treatment with vemurafenib and cobimetinib. Furthermore, high FOXD1 expression levels were found in BRAF inhibitor (BRAFi)-resistant cells. Downregulation of FOXD1 resulted in a resensitization of BRAFi-resistant cells to vemurafenib. By using microarray analysis, connective tissue growth factor (CTGF) was found to be one of the most downregulated genes in FOXD1 knockdown (KD) cells while its expression was highly increased upon FOXD1 overexpression. Thus, CTGF was identified as a downstream factor of FOXD1. In addition, in vitro expression analysis and evaluation of clinical samples demonstrated that CTGF and FOXD1 expression were positively correlated. By using a CHIP assay and a dual reporter luciferase assay, we discovered that FOXD1 could regulate the expression of CTGF by directly binding to the CTGF promoter. This result was confirmed with RT-PCR and western blot. In addition, we found that the protein level of CTGF was highly increased in BRAFi-resistant cells. Similar to FOXD1 knockdown, the knockdown of CTGF resensitized BRAFi-resistant cells to vemurafenib. FOXD1 KD cells treated with recombinant CTGF protein were less sensitive towards vemurafenib compared to untreated FOXD1 KD cells. Based on these findings, we conclude that the transcription factor FOXD1 could promote dedifferentiation and targeted therapy-resistance in melanoma cells by regulating the expression of CTGF. FOXD1 might be a promising new diagnostical marker and a therapeutic target of targeted therapy resistant melanoma.
Citation Format: Qian Sun, Daniel Novak, Laura Hüser, Juliane Poelchen, Huizi Wu, Karol Granados, Aniello Federico Federico, Ke Liu, Tamara Steinfass, Marlene Vierthaler, Viktor Umansky, Jochen Utikal. FOXD1 promotes dedifferentiation and targeted therapy resistance in melanoma by regulating the expression of connective tissue growth factor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 431.