Understanding the role of non-coding RNAs in skin homeostasis and cancer

Abstract

The epidermis is a stratified epithelium with continuous self-renewing capacity. As the outermost layer of our body, it provides a protective barrier against external trauma, produces pigmentation, and keeps the skin hydrated. Keratinocytes are the primary constituent cells type within the epidermis. A fine balance is maintained between keratinocyte cell proliferation and differentiation to sustain a functional epidermis. The interplay between multiple signaling pathways, transcription factors, epigenetic modulators, and non-coding RNAs is the key to maintaining this balance. Disrupted epidermal homeostasis can cause various diseases, including cancer. Cutaneous squamous cell carcinoma (cSCC) is one such keratinocyte-derived cancer that begins with the accumulation of somatic mutations and genetic abnormalities. The pigment-producing melanocytes within the epidermis can undergo oncogenic transformation due to numerous genetic and environmental factors, and give rise to malignant melanoma. In this thesis, we have explored the role of non-coding RNAs in epidermal homeostasis and the development of skin cancers.

Paper I: In this study, we investigated the role of miR-203 in cSCC, and found that its expression was negatively correlated with the differentiation grade of the tumors. Functionally, miR-203 inhibited cell cycle progression, self-renewability, motility and proangiogenic-activity of cSCC cells in vitro, and reduced xenograft tumor growth and angiogenesis in vivo. We identified c-MYC as a potential upstream regulator of the transcriptomic changes caused by miR-203 overexpression, and subsequently demonstrated that c-MYC is a direct target of miR203 in cSCC. In line with these findings, overexpression of c-Myc rescued the growth-inhibitory effect of miR-203 in cSCC cell lines.

Paper II: In this study, we analyzed the small RNA-seq data from the skin cutaneous melanoma (TCGA SKCM-cohort) and found that miR-203 is the most downregulated miRNA in metastatic melanoma. Moreover, high miR-203 abundance seems to confer longer overall survival to patients with metastatic melanoma. Methylome data from patient samples, together with results from in vitro experiments, suggested that promoter hypermethylation could suppress miR-203 expression in metastatic tumors. Functionally, miR-203 acted as a tumor suppressor by inhibiting cancer/metastatic hallmarks such as cell migration, invasion, self-renewal, and angiogenesis. SLUG, an essential regulator of epithelial-mesenchymal transition, was found to be a direct target of miR-203. In vivo, miR-203 effectively suppressed melanoma metastasis to the inguinal lymph nodes and the lungs.

Paper III: In this study, we investigated the changes in the coding and non-coding landscape in cSCC using RNA-seq. We identified a large number of differentially expressed coding transcripts linear lncRNAs and circRNAs. Representative transcripts from each group were validated using an extended cohort. We found that several transcription factors regulating skin development and cSCC oncogenesis were altered at mRNA level. In addition to various lncRNAs with potential oncogenic function, we identified a set of skin-specific lncRNAs, which were mostly downregulated in cSCC. We observed a global downregulation of circRNA abundance in cSCC. Apart from previously annotated circRNAs, novel skin-enriched circRNAs were also identified and validated.

Paper IV: In this study, we characterized a skin-specific lncRNA located at the Epidermal Differentiation Complex (EDC) on human chromosome 1. It is highly induced during the late stages of keratinocyte differentiation and localized to the granular layer of the human epidermis. We showed that transcription factor YY1 suppresses its expression in the progenitor keratinocytes. CRISPR-mediated activation of this lncRNA locus led to an increased expression of late differentiation marker genes. In contrast, loss-of-function experiment in a 3D organotypic skin model resulted in impaired terminal differentiation program and formation of thinner cornified envelope. Due to its functional requirement in late differentiation, we have renamed this lncRNA as ELDAR (Epidermal Late Differentiation Associated RNA).