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Abstract
Keloids are a disorder of pathologic wound healing caused by fibroblast hyperproliferation and excess collagen deposition following skin injury which have an increased incidence in Black and Hispanic populations. Keloidal scars may lead to an impaired quality of life with significant physical and emotional impacts associated with psychosocial and functional distress. There is currently no universally accepted treatment for keloids; however, corticosteroids, surgery, and radiotherapy are commonly used options. Unfortunately, the prognosis may not be satisfactory and some of those treatment approaches may lead to serious complications. Therefore, there is a need to find an alternative method aiming to improve the treatment outcomes for keloid patients.
Recent studies have shown that epigenetic modifications including DNA methylation, histone acetylation and non-coding RNAs all play major roles in keloid pathogenesis. Our lab has developed an epigenetic pathway small molecule inhibitor that can inhibit the CoREST repressor complex by targeting both HDAC1/2 and LSD1, corin, and has shown significant effects of corin in inhibiting melanoma tumor growth. The current literature suggests that epigenetic changes are critical for the formation of keloids and that the process of keloidal scar development is analogous to the changes seen in cancer development. We have therefore hypothesized that corin may have bioactivity in the prevention and/or treatment of keloidal scars. We further expect that the study of epigenetic changes in keloidal scarring may reveal significant biomolecular mechanisms shared by keloids and cancer which should prove to be useful in the diagnosis, prevention, and treatment of both diseases.
In this study, we hypothesize that the CoREST epigenetic repressor complex is directly involved in the development and progression of keloidal scarring. We therefore evaluate the role of epigenetic regulation in the development and progression of keloid fibroblasts by assessing proliferation, migration, invasion, and transcriptomic changes through RNA sequencing analysis, using an in-vitro model system as well as keloid patient-derived fibroblasts.
Human keloid fibroblast cells (ATCC CRL-1762), normal human fibroblasts cells (ATCC PCS-201-012), patient keloid 1 (PK1), patient keloid 2 (PK2), patient keloid 3 (PK3), patient keloid 4 (PK4) or patient normal fibroblasts 4 (PNF4) were treated with the following epigenetic drugs: corin, MS-275 (HDACi), LSD1-GSK (LSD1i) & A-485 (p300i) along with DMSO as a control. Proliferation assays were performed in cells treated with the epigenetic drugs for 72 hours (n = 2–3). Overall, corin and HDACi were found to demonstrate the greatest growth inhibition in all cells evaluated with significantly less growth inhibition being noted with LSD1 inhibition. The EP300 HAT inhibitor, A-485, failed to demonstrate any significant growth effects in ATCC cell lines, we therefore decided not to pursue further studies with this compound in patient-derived keloid or normal fibroblasts. These data overall demonstrated that both the dual-acting CoREST inhibitor, corin, and the HDAC inhibitor, MS-275, reduced fibroblast proliferation more than the LSD1 inhibitor in both normal and keloid fibroblasts as well as patient-derived keloid-associated primary fibroblasts.
Next, we evaluated the effect of epigenetic small molecule inhibitors on fibroblast migration after 24 hours of treatment by performing scratch wound migration assays (n = 3–4). The epigenetic drugs did not demonstrate a significant reduction in cellular migration in ATCC PCS-201-012 (normal fibroblasts), ATCC CRL-1762 (keloid fibroblasts), patient keloid 1 (PK1), or patient keloid 2 (PK2) fibroblasts. However, corin significantly reduced cellular migration by 30.76% in patient keloid 3 (PK3), by 27.79% in patient keloid 4 (PK4), and by 29.94% in patient normal fibroblasts 4 (PNF4) compared to DMSO (p < 0.05) (Figure 12). This data suggests that epigenetic inhibitors may have variable effects on keloid-associated fibroblast migration.
In order to evaluate the effects of epigenetic inhibitors on keloid-associated fibroblast invasion, we performed a 24-hour Boyden chamber transwell assay (n = 3). Overall, we found that keloid-associated fibroblast invasion was significantly reduced in the setting of all epigenetic drugs tested, with corin showing the greatest effect on cellular invasion. We found that 1 ?M of corin reduced invasion by 65% in CRL-1762 cells (p < 0.05), by 49% in PK1 (p < 0.001), by 70.4% in PK2 (p < 0.0001), by 63.4% in PK3 (p < 0.05), by 45.8% in PK4, and by 60% in PNF4. This data suggests that corin is the most effective inhibitor of keloid-associated fibroblast invasion across keloid cell lines (CRL-1762) and primary cells (PK1, PK2, PK3, PK4 and PNF4).
Lastly, we performed RNA sequencing analysis (RNA-seq) on ATCC cell lines and patient-derived primary cells treated for 24 hours with 1 ?m corin or DMSO as a control, in order to identify potential molecular mechanisms of corin effects in keloid fibroblasts. Not surprisingly, we found that corin treatment upregulated a significant number of genes in all cells tested. Gene set enrichment analysis (GSEA) of patient-derived keloid fibroblasts treated with corin compared to the same fibroblasts treated with DMSO as a control showed many significantly upregulated and downregulated (padj<1E-6) gene ontology (GO) pathways. The Kyoto Encyclopedia of Genes and Genomes (KEGG) cell adhesion molecules pathway was one of the significantly upregulated (padj<2E-3) enriched pathways, and the E2F target pathway was one of the more significantly downregulated (padj<1E-6) GO pathways. These data demonstrate that 24-hour treatment with corin significantly alters the transcriptome in patient keloid fibroblasts. Corin affected many pathways associated with neural differentiation as well as cell cycle, apoptosis, and adhesion pathways in keloid-associated fibroblasts, which is expected to have great implications for the mechanism of action of this compound in keloidal scarring.
Altogether, these results demonstrate a significant role for epigenetic regulation of cell growth and invasion properties of keloid-associated fibroblasts and support further studies of the use of epigenetic inhibitors in the prevention and/or treatment of keloidal scarring
