Dissecting the Molecular Mechanisms Involved in Keloid Pathogenesis and Response to Therapy
Son, Yuna (author)
Gunjan, Akash (professor directing dissertation)
Dennis, Jonathan Hancock (university representative)
Arbeitman, Michelle N. (Michelle Nina) (committee member)
Wang, Yanchang (committee member)
Stefanovic, Branko (committee member)
Florida State University (degree granting institution)
College of Medicine (degree granting college)
Department of Biomedical Sciences (degree granting department)
2021
text
doctoral thesis
Keloids are disfiguring, painful and itchy but benign fibrotic skin lesions characterized by excessive dermal fibroblast proliferation and collagen deposition. They occur in susceptible individuals due to abnormal wound healing. Keloids are understudied and predominantly affect dark-skinned individuals, suggesting that genetic and/or epigenetic factors contribute strongly to keloid disease. However, the molecular mechanisms involved in driving keloid formation in susceptible individuals are unclear. Furthermore, currently there are no standardized treatments available for keloids and they have very high recurrence rates upon surgical resection alone. Although ionizing radiation is emerging as one of the most effective post-surgical adjuvant therapies to prevent recurrence, there are no systematic studies on the range of effective radiation doses for keloid therapy. My studies have revealed that a single 3Gy dose of low energy (50kV) X-ray radiation inhibited the proliferation of keloid fibroblasts by ~80% due to a combination of apoptosis, necrosis and senescence. Further, a single 9Gy dose at 50kV completely blocked the outgrowth of fibroblasts from keloid explants. Collagen levels were reduced independently of the cell death triggered by radiation. This suggests that additional treatments which block excessive collagen production in keloids can be used synergistically with radiation in keloid management. More importantly, a single 8Gy dose of superficial 50kV radiation delivered an average of 34 days after keloid excision was effective in minimize recurrence following surgical excision, including in individuals resistant to steroids. Higher radiation energies, doses, or fractions may be unnecessary for keloid therapy and in fact could increase the potential for harm. Keloids are commonly treated using steroids, although the response in patients is highly variable. I developed minimally invasive and non-invasive methods for testing steroid response in patients prior to initiating steroid therapy. To better understand the molecular mechanisms that drive keloid formation and determine their response to therapy, I have used a combination of genomic sequencing-based approaches including RNA-sequencing, microRNA-sequencing, and Enzymatic Methyl-sequencing. Using these, I have identified several potential molecular pathways that contribute to keloid pathogenesis and their differential response to steroid therapy. I found that altered expression of specific miRNAs are likely to regulate their target genes such as IGF2 (hsa-miR-100-5p), PLOD2 and FN1 (hsa-miR-615-3p), and SERPINE1 (hsa-miR-10a-5p). I have confirmed the observed alterations in the expression of several dozens of genes from my transcriptomics data using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses, and many of these genes have been previously implicated in keloid or other fibrotic diseases. Furthermore, I have identified many differentially expressed genes between steroid sensitive and resistant keloid fibroblasts. These include the EYA4 and IGF1 genes that are downregulated, while the MCAM, WFDC1, IGFBP2, and RBM24 genes are upregulated in steroid resistant keloid fibroblasts. The expression levels of these genes can potentially be used as molecular biomarkers to screen for steroid resistance in patients. Finally, based on my genomic studies, I have identified new molecular targets for keloid therapy. Since IGF related pathways are reported to promote cell invasion and metastasis, blocking IGF2 can be a promising target to inhibit keloid progression. I have shown that inhibiting IGF2 using Chromeceptin significantly reduced keloid fibroblast proliferation in vitro. I have also shown that Tiplaxtinin, a SERPIN1 inhibitor, can selectively inhibit the proliferation of keloid fibroblasts over normal fibroblasts. I have confirmed that Minoxidil, an FDA approved over-the-counter topical drug for treating hair loss and a known PLOD2 inhibitor, also inhibits the proliferation of keloid fibroblasts. Since PLOD2 is known to promote collagen remodeling and induce cell migration, minoxidil may be helpful in reducing keloid severity and recurrence as a topical ointment. I have also tested the effect of different HAT and FDA approved HDAC inhibitors on keloid fibroblasts and both classes of inhibitors have shown the significant effects in blocking keloid fibroblast proliferation. These results suggest that keloid fibroblasts may need very precise levels of histone acetylation and altering the levels of this histone modification can result in keloid cell death. These data also suggests that further studies are required to assay additional histone modifications to fully understand the epigenetic contribution in keloid pathogenesis. Taken together, the studies described in this dissertation provide a wealth of genomic and cell biological data that greatly improve our understanding of keloid pathogenesis and response to steroid therapy at the molecular level. The studies also provide several molecular targets for keloid therapy using existing FDA approved and pre-clinical drugs, as well as potential biomarkers for determining keloid susceptibility and response to steroid therapy.
November 19, 2021.
A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Includes bibliographical references.
Akash Gunjan, Professor Directing Dissertation; Jonathan Dennis, University Representative; Michelle Arbeitman, Committee Member; Yanchang Wang, Committee Member; Branko Stefanovic, Committee Member.
Florida State University
2021_Fall_Son_fsu_0071E_16803