| 1. |
- Das Mahapatra, Kunal, et al.
(author)
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A comprehensive analysis of coding and non-coding transcriptomic changes in cutaneous squamous cell carcinoma
- 2020
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Journal article (peer-reviewed)abstract
- Cutaneous Squamous Cell Carcinoma (cSCC) is the most common and fastest-increasing cancer with metastatic potential. Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are novel regulators of gene expression. To identify mRNAs, lncRNAs and circRNAs, which can be involved in cSCC, RNA-seq was performed on nine cSCCs and seven healthy skin samples. Representative transcripts were validated by NanoString nCounter assays using an extended cohort, which also included samples from pre-cancerous skin lesions (actinic keratosis). 5,352 protein-coding genes, 908 lncRNAs and 55 circular RNAs were identified to be differentially expressed in cSCC. Targets of 519 transcription factors were enriched among differentially expressed genes, 105 of which displayed altered level in cSCCs, including fundamental regulators of skin development (MYC, RELA, ETS1, TP63). Pathways related to cell cycle, apoptosis, inflammation and epidermal differentiation were enriched. In addition to known oncogenic lncRNAs (PVT1, LUCAT1, CASC9), a set of skin-specific lncRNAs were were identified to be dysregulated. A global downregulation of circRNAs was observed in cSCC, and novel skin-enriched circRNAs, circ_IFFO2 and circ_POF1B, were identified and validated. In conclusion, a reference set of coding and non-coding transcripts were identified in cSCC, which may become potential therapeutic targets or biomarkers.
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| 2. |
- Das Mahapatra, Kunal
(author)
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Understanding the role of non-coding RNAs in skin homeostasis and cancer
- 2020
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Doctoral thesis (other academic/artistic)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 growthinhibitory 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, selfrenewal, 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).
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| 3. |
- Li, Chen, et al.
(author)
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Long noncoding RNA plasmacytoma variant translocation 1 is overexpressed in cutaneous squamous cell carcinoma and exon 2 is critical for its oncogenicity
- 2023
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In: British Journal of Dermatology. - : Oxford University Press. - 0007-0963 .- 1365-2133. ; 190:3, s. 415-426
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Journal article (peer-reviewed)abstract
- BackgroundCutaneous squamous cell carcinoma (cSCC) is one of the most common and fastest increasing forms of cancer worldwide with metastatic potential. Long noncoding RNAs (lncRNAs) are a group of RNA molecules with essential regulatory functions in both physiological and pathological processes.ObjectivesTo investigate the function and mode of action of lncRNA plasmacytoma variant translocation 1 (PVT1) in cSCC.MethodsQuantitative reverse transcriptase polymerase chain reaction and single-molecule in situ hybridization were used to quantify the expression level of PVT1 in normal skin, premalignant skin lesions, actinic keratosis (AK) and primary and metastatic cSCCs. The function of PVT1 in cSCC was investigated both in vivo (tumour xenografts) and in vitro (competitive cell growth assay, 5-ethynyl-2′-deoxyuridine incorporation assay, colony formation assay and tumour spheroid formation assay) upon CRISPR-Cas9-mediated knockout of the entire PVT1 locus, the knockout of exon 2 of PVT1, and locked nucleic acid (LNA) gapmer-mediated PVT1 knockdown. RNA sequencing analysis was conducted to identify genes and processes regulated by PVT1.ResultsWe identified PVT1 as a lncRNA upregulated in cSCC in situ and cSCC, associated with the malignant phenotype of cSCC. We showed that the expression of PVT1 in cSCC was regulated by MYC. Both CRISPR-Cas9 deletion of the entire PVT1 locus and LNA gapmer-mediated knockdown of PVT1 transcript impaired the malignant behaviour of cSCC cells, suggesting that PVT1 is an oncogenic transcript in cSCC. Furthermore, knockout of PVT1 exon 2 inhibited cSCC tumour growth both in vivo and in vitro, demonstrating that exon 2 is a critical element for the oncogenic role of PVT1. Mechanistically, we showed that PVT1 was localized in the cell nucleus and its deletion resulted in cellular senescence, increased cyclin-dependent kinase inhibitor 1 (p21/CDKN1A) expression and cell cycle arrest.ConclusionsOur study revealed a previously unrecognized role for exon 2 of PVT1 in its oncogenic role and that PVT1 suppresses cellular senescence in cSCC. PVT1 may be a potential biomarker and therapeutic target in cSCC.
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| 4. |
- Lohcharoenkal, Warangkana, et al.
(author)
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Genome-Wide Screen for MicroRNAs Reveals a Role for miR-203 in Melanoma Metastasis.
- 2018
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In: Journal of Investigative Dermatology. - : Elsevier BV. - 0022-202X .- 1523-1747. ; 138:4, s. 882-892
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Journal article (peer-reviewed)abstract
- Melanoma is one of the deadliest human cancers with limited therapeutic options. MicroRNAs are a class of short noncoding RNAs regulating gene expression at the post-transcriptional level. To identify important miRNAs in melanoma, we compared the miRNome of primary and metastatic melanomas in The Cancer Genome Atlas dataset and found lower miR-203 abundance in metastatic melanoma. Lower level of miR-203 was associated with poor overall survival in metastatic disease. We found that the methylation levels of several CpGs in the MIR203 promoter negatively correlated with miR-203 expression and that treatment with the demethylating agent 5-aza-2-deoxycytidine induced miR-203 expression, which was associated with demethylation of the promoter CpGs, in melanoma cell lines. In vitro, there was a decreased expression of miR-203 in melanoma cell lines in comparison with primary melanocytes. Ectopic overexpression of miR-203 suppressed cell motility, colony formation, and sphere formation as well as the angiogenesis-inducing capacity of melanoma cells. In vivo, miR-203 inhibited xenograft tumor growth and reduced lymph node and lung metastasis. SLUG was shown as a target of miR-203, and knockdown of SLUG recapitulated the effects of miR-203, whereas its restoration was able to reverse the miR-203-mediated suppression of cell motility. These results establish a role for miR-203 as a tumor suppressor in melanoma which suppresses both early and late steps of metastasis. Hence, restoration of miR-203 has therapeutic potential in melanoma.
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| 5. |
- Lohcharoenkal, Warangkana, et al.
(author)
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MicroRNA-203 Inversely Correlates with Differentiation Grade, Targets c-MYC, and Functions as a Tumor Suppressor in cSCC.
- 2016
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In: Journal of Investigative Dermatology. - : Elsevier BV. - 0022-202X .- 1523-1747. ; 136:12, s. 2485-2494
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Journal article (peer-reviewed)abstract
- Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer and a leading cause of cancer mortality among solid organ transplant recipients. MicroRNAs (miR) are short RNAs that regulate gene expression and cellular functions. Here, we show a negative correlation between miR-203 expression and the differentiation grade of cSCC. Functionally, miR-203 suppressed cell proliferation, cell motility, and the angiogenesis-inducing capacity of cSCC cells in vitro and reduced xenograft tumor volume and angiogenesis in vivo. Transcriptomic analysis of cSCC cells with ectopic overexpression of miR-203 showed dramatic changes in gene networks related to cell cycle and proliferation. Transcription factor enrichment analysis identified c-MYC as a hub of miR-203-induced transcriptomic changes in squamous cell carcinoma. We identified c-MYC as a direct target of miR-203. Overexpression of c-MYC in rescue experiments reversed miR-203-induced growth arrest in cSCC, which highlights the importance of c-MYC within the miR-203-regulated gene network. Together, miR-203 acts as a tumor suppressor in cSCC, and its low expression can be a marker for poorly differentiated tumors. Restoration of miR-203 expression may provide a therapeutic benefit, particularly in poorly differentiated cSCC.
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| 6. |
- Lohcharoenkal, Warangkana, et al.
(author)
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MiR-130a Acts as a Tumor Suppressor MicroRNA in Cutaneous Squamous Cell Carcinoma and Regulates the Activity of the BMP/SMAD Pathway by Suppressing ACVR1
- 2021
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In: Journal of Investigative Dermatology. - : Elsevier. - 0022-202X .- 1523-1747. ; 141:8, s. 1922-1931
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Journal article (peer-reviewed)abstract
- Cutaneous Squamous Cell Carcinoma (cSCC) is a malignant neoplasm of the skin resulting from the accumulation of somatic mutations due to solar radiation. It is one of the fastest increasing malignancies and it represents a particular problem among immunosuppressed individuals. MicroRNAs (miRNAs) are short non-coding RNAs that regulate the expression of protein-coding genes at the posttranscriptional level. Here we identify miR-130a to be downregulated in cSCC compared with healthy skin and with precancerous lesions (actinic keratosis) and demonstrate that it is regulated at the transcriptional level by HRAS and MAPK-signaling. We report that miR-130a suppresses the growth of cSCC xenografts in mice. We demonstrate that overexpression of miR-130a suppresses long-term capacity of growth, cell motility and invasion ability in human cSCC cell lines. Mechanistically, miR-130a directly targets Activin A receptor, type I (ACVR1/ALK2) and changes in miR-130a levels result in the diminished activity of BMP/SMAD1 pathway via ACVR1. These data reveal a link between activated MAPK-signaling and decreased expression of miR-130a, which acts as a tumor suppressor miRNA in cSCC and contributes to a better understanding of molecular processes in malignant transformation of epidermal keratinocytes.
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| 7. |
- Pasquali, Lorenzo, et al.
(author)
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The Keratinocyte Transcriptome in Psoriasis : Pathways Related to Immune Responses, Cell Cycle and Keratinization.
- 2019
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In: Acta Dermato-Venereologica. - : Medical Journals Sweden AB. - 0001-5555 .- 1651-2057. ; 99:2, s. 196-205
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Journal article (peer-reviewed)abstract
- Psoriasis is a common immune-mediated disease resulting from altered cross-talk between keratinocytes and immune cells. Previous transcriptomic studies have identified thousands of deregulated genes in psoriasis skin; however, the transcriptomic changes confined to the epidermal compartment remained poorly characterized. The aim of this study was to characterize the transcriptomic landscape of psoriatic keratinocytes, using sorted CD45neg epidermal cells. Genes with functions in innate immunity, type I interferon response, cell cycle and keratinization were enriched among deregulated genes in psoriatic keratinocytes. Gene set enrichment analysis indicated the dominance of interleukin (IL)-22/IL-17A signatures in the epidermal psoriasis-signature. A set of deregulated genes overlapped with psoriasis-associated genetic regions, suggesting that genetic variations affecting gene expression in keratinocytes contribute to susceptibility to psoriasis. Several psoriasis-susceptibility genes, which were previously believed to be expressed preferentially or exclusively in immune cells, were identified as having altered expression in psoriatic keratinocytes. These results highlight the role of keratinocytes in the pathogenesis of psoriasis, and indicate that both genetic factors and an inflammatory microenvironment contribute to epidermal alterations in psoriasis.
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| 8. |
- Sun, Chengxi, et al.
(author)
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MicroRNA-23b Plays a Tumor-Suppressive Role in Cutaneous Squamous Cell Carcinoma and Targets Ras-Related Protein RRAS2
- 2023
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In: Journal of Investigative Dermatology. - : Elsevier. - 0022-202X .- 1523-1747. ; 143:12, s. 2386-2396
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Journal article (peer-reviewed)abstract
- Cutaneous squamous cell carcinoma (cSCC) is one of the most common types of cancer with metastatic potential. MicroRNAs regulate gene expression at the post-transcriptional level. In this study, we report that miR23b is downregulated in cSCCs and in actinic keratosis and that its expression is regulated by the MAPK signaling pathway. We show that miR-23b suppresses the expression of a gene network associated with key oncogenic pathways and that the miR-23b-gene signature is enriched in human cSCCs. miR-23b decreased the expression of FGF2 both at mRNA and protein levels and impaired the angiogenesis-inducing ability of cSCC cells. miR23b overexpression suppressed the capacity of cSCC cells to form colonies and spheroids, whereas the CRISPR/Cas9-mediated deletion of MIR23B resulted in increased colony and tumor sphere formation in vitro. In accordance with this, miR-23b-overexpressing cSCC cells formed significantly smaller tumors upon injection into immunocompromised mice with decreased cell proliferation and angiogenesis. Mechanistically, we verify RRAS2 as a direct target of miR-23b in cSCC. We show that RRAS2 is overexpressed in cSCC and that interference with its expression impairs angiogenesis and colony and tumorsphere formation. Taken together, our results suggest that miR-23b acts in a tumor-suppressive manner in cSCC, and its expression is decreased during squamous carcinogenesis.
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