Microenvironment‑driven resistance to B‑Raf inhibition in a melanoma patient is accompanied by broad changes of gene methylation and expression in distal fibroblasts

Abstract

The incidence of malignant melanoma is rapidly increasing and current medicine is offering only limited options for treatment of the advanced disease. For B-Raf mutated melanomas, treatment with mutation-specific drug inhibitors may be used. Unfortunately, tumors frequently acquire resistance to the treatment. Tumor microenvironment, namely cancer-associated fibroblasts, largely influence this acquired resistance. In the present study, fibroblasts were isolated from a patient suffering from acrolentiginous melanoma (Breslow, 4.0 mm; Clark, IV; B-Raf V600E mutated). The present study focused on the expression of structural and functional markers of fibroblast activation in melanoma-associated fibroblasts (MAFs; isolated prior to therapy initiation) as well as in autologous control fibroblasts (ACFs) of the same patient isolated during B-Raf inhibitor therapy, yet before clinical progression of the disease. Analysis of gene transcription was also performed, as well as DNA methylation status analysis at the genomic scale of both isolates. MAFs were positive for smooth muscle actin (SMA), which is a marker of myofibroblasts and the hallmark of cancer stoma. Surprisingly, ACF isolated from the distant uninvolved skin of the same patient also exhibited strong SMA expression. A similar phenotype was also observed in control dermal fibroblasts (CDFs; from different donors) exclusively following stimulation by transforming growth factor (TGF)-β1. Immunohistochemistry confirmed that melanoma cells potently produce TGF-β1. Significant differences were also identified in gene transcription and in DNA methylation status at the genomic scale. Upregulation of SMA was observed in ACF cells at the protein and transcriptional levels. The present results support recent experimental findings that tumor microenvironment is driving resistance to B-Raf inhibition in patients with melanoma. Such an activated microenvironment may be viable for the growth of circulating melanoma cells.

Dissecting Mechanisms of Melanoma Resistance to BRAF and MEK Inhibitors Revealed Genetic and Non-Genetic Patient- and Drug-Specific Alterations and Remarkable Phenotypic Plasticity

“…Analysis of whole-exome sequencing data revealed only a few mutations previously identified as associated with acquired resistance to targeted drugs. It is in line with the current view that in parallel to genetic alterations, melanoma cell-autonomous mechanisms of resistance can rely on complex transcriptomic adaptations supported by epigenetic regulations, including miRNA-mediated mechanisms, and extended by the interplay between tumor and stromal cells that involves cell-cell interactions, paracrine stimulation and extracellular vesicles to create a resistance-permissive niche [39][40][41][42][43][44][45][46]. Mechanisms of resistance primarily directed to support proliferation and survival of cancer cells implicate a plethora of transcriptional regulators, adaptive responses, and feedback loops that extensively affect melanoma cell phenotype enabling the expansion of distinct cell subpopulations in the presence of drug(s) [27,[47][48][49][50][51][52].…”

Melanoma in the Eyes of Mechanobiology

“…Some signaling pathways are more involved in tumor development, whereas other signals are more crucial in the metastization process. The description of biochemical pathways signaling is beyond of this review, however the readers can find more information in different sources (Dissanayake et al, 2007;Straussman et al, 2012;Moriceau et al, 2015;Stark et al, 2015;Wang et al, 2016;Ahmed and Haass, 2018;Kodet et al, 2018). Melanoma can be pigmented or not pigmented.…”

The Head and Neck Squamous Cell Carcinoma Microenvironment as a Potential Target for Cancer Therapy

“…For example, CAF isolated from melanoma or basal cell carcinoma influence the phenotype and migratory activity of cells of both breast cancer and glioblastoma [26,27]. However, the in vitro experimental setting used in the cited studies is not fully supported by evidence based on tumour tissue sample analyses, and therefore the results need further validation [28,29,30]. Recently, the epigenetic mechanisms controlling the CAF function within the tumour niche have been identified [30,31].…”