Progression from actinic keratosis to cutaneous squamous cell carcinoma involves miR-204 silencing and a switch towards STAT3 activation
ABSTRACT: Squamous cell carcinoma is the second most common skin cancer and frequently progress from an intraepithelial actinic keratosis. The role of microRNAs during the progression from actinic keratosis to cutaneous squamous cell carcinoma (cSCC) remains to be elicited. By using an Agilent microRNA expression microarray we found the expression of miR-204 to be markedly downregulated in cSCC when compared to actinic keratoses. DNA methylation of the TRPM3 promoter region upstream of miR-204-5p was identified as one of the repressive mechanisms that accounts for miR-204 silencing in cSCC. Functional studies on HaCaT cells revealed that this microRNA downregulates the Signal Transducer and Activator of Transcription 3 (STAT3) pathway and favours the MAPK signaling pathway, likely acting through PTPN11, a tyrosine phosphatase that is a direct miR-204 target. We found that activated STAT3, as detected by pY705-STAT3 immunofluorescence, is retained in the membrane and cytoplasm compartment in AK, whereas cSCC displayed STAT3 in the nuclei. Taken together, our data indicates that MiR-204 may act as a “rheostat” that controls the signaling towards the MAPK pathway or the STAT3 pathway. Overall design: 4 samples were analyzed: HaCaT tansduced with control shRNA (n=2) and HaCaT transduced with shRNA against miR-204 (n=2)
INSTRUMENT(S): [HuGene-2_0-st] Affymetrix Human Gene 2.0 ST Array [transcript (gene) version]
Project description:Skin carcinogenesis is known to be a multi-step process with several stages along its malignant evolution. We hypothesized that transformation of normal epidermis to cutaneous squamous cell carcinoma (cSCC) is causally linked to alterations in miRNA expression. For this end we decided to evaluate their alterations in the pathologic states ending in cSCC. Total RNA was extracted from FFPE biopsies of five stages along the malignant evolution of keratinocytes towards cSCC: Normal epidermis, severe solar elastosis (SE), actinic keratosis (KIN1-2), advanced actinic keratosis, (KIN3) and well differentiated cSCC. Next generation small RNA sequencing was performed. We found that 18 miRNAs are over expressed and 28 miRNAs are under expressed in cSCC compared to normal epidermis. miR-424, miR-320, miR- 222 and miR-15a showed the highest fold change among the over expressed miRNAs. And miR-100, miR-101 and miR-497 showed the highest fold change among the under expressed miRNAs. Heat map of hierarchical clustering analysis of significantly changed miRNAs and principle component analysis disclosed that the most prominent change in miRNAs expression occurred in the switch from “early” stages; normal epidermis, solar elastosis and early actinic keratosis to the “late” stages of epidermal carcinogenesis; late actinic keratosis and cSCC. We found several miRNAs with "stage specific" alterations while others display a clear “gradual”, either progressive increase or decrease in expression along the malignant evolution of keratinocytes. The observed alterations focused in miRNAs involved in the regulation of AKT/mTOR or in those involved in epithelial to mesenchymal transition. We chose to concentrate on the evaluation of the molecular role of miR- 497. We found that it induces reversion of epithelial to mesenchymal transition. We proved that SERPINE-1 is its biochemical target. The present study allows us to further study the pathways which are regulated by miRNAs along the malignant evolution of keratinocytes towards cSCC. Overall design: Comparisons were made with regards to miRNA content between total RNA extracted from paraffin samples on the spectrum between normal skin and cSCC, and additional cell line RNA reference.
Project description:Analysis of the combined dataset of normal skin, actinic keratosis and cutaneous squamous cell carcinoma (cSCC) previously submitted in (GSE45216) at genomic/transciptomic level. We identified potential cSCC driver genes significantly mutated and upregulated in cSCC relative to normal skin. Overall design: RNA was extracted by laser capture microdissection from actinic keratosis (AK), normal Sun exposed skin (SE) and normal non-Sun exposed skin (NSE), for analysis using the Affymetrix HG U133 Plus 2.0 microarrays.
Project description:Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer type and arises from keratinocytes. Most cSCC progress from a UV-induced precancerous lesion termed actinic keratosis (AK). Despite various efforts to characterize these lesions molecularly, the etiology of AK and its progression to cSCC remain only partially understood. Here we have used Infinium MethylationEPIC BeadChips to interrogate the DNA methylation status of about 850.000 CpGs in epidermal preparations from healthy skin, AK and cSCC. Importantly, we found that the premalignant AK samples displayed classical features of cancer methylomes and were highly similar to cSCC methylomes. Further analysis identified typical features of stem cell methylomes, such as a reduced DNA methylation age, non-CpG methylation and stem cell-related keratin and enhancer methylation patterns. Interestingly, this signature was detected only in one half of the AK and cSCC samples, while the other half showed methylation patterns that were more closely related to the control epidermis. These findings suggest the existence of two distinct subclasses of AK and cSCC that originate from distinct keratinocyte differentiation stages.
Project description:Cutaneous squamous cell carcinoma (cSCC) is one of the most common malignancies in fair skinned populations worldwide and its incidence is increasing. Despite previous observations of multiple genetic abnormalities in cSCC, the oncogenic process remains elusive. The purpose of this study was to investigate the transcriptomes of cSCC and actinic keratoses (AK), to elucidate key differences between precursor AK lesions and invasive carcinoma. This study identified 196 genes that are differentially expressed between AK and cSCC, with enrichment for processes including epidermal differentiation, cell migration, cell cycle regulation and metabolism. Gene set enrichment analysis highlighted a key role for the MAPK pathway in the transition from AK to cSCC. Furthermore, the differentiation status of the tumor influenced the gene expression profile, which may have implications for drug sensitivity and response in clinical trials. These data indicate that progression to cSCC is associated with a complex pattern of molecular changes. We have identified relevant pathways involved in this process, in particular that the MAPK pathway may be pivotal to the transition from AK and may represent a potential therapeutic target. RNA was extracted by laser capture microdissection from 10 AK and 30 cSCC, for analysis using the Affymetrix HG U133 Plus 2.0 microarrays. The cSCC samples included a range of histological diagnoses (well differentiated through to poorly differentiatied) from both immunocompetent and immunosuppressed patients.
Project description:Keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, and its incidence is increasing globally. Chronic inflammation has been recognized as a risk factor for cSCC and inflammation is a typical feature of the progression of actinic keratosis lesions to invasive and metastatic cSCC. Inflammasomes are important components of the innate immune response involved in onset of inflammation. Inflammasome component AIM2 serves as a sensor for cytoplasmic double-strand DNA, and this way plays a key role in response to bacterial and viral colonization. Activation of inflammasome by cytoplasmic DNA in epidermal keratinocytes can promote the initiation of inflammation in autoimmune and autoinflammatory skin diseases. Whole transcriptome analysis of cSCC cells (n=8) and normal human epidermal keratinocytes (NHEKs, n=4) and oligonucleotide array-based expression analysis of cSCC cells (n=8) and normal human epidermal keratinocytes (NHEKs, n=5) revealed overexpression of AIM2 in cSCC cells (GSE66412 and GSE66368, respectively). We wanted to futher study the RNA expression profile of AIM2 knockdown cSCC cells. Overall design: Total RNAs from negative control and AIM2 siRNA transfected cSCC cell lines (n=3) were extracted. The samples were sequenced using Illumina sequencing.
Project description:BACKGROUND: The boundaries between actinic keratosis (AK), Bowen's disease (BD), and cutaneous squamous cell carcinoma (cSCC) are sometimes not clear. Large-scale proteomic profiling studies of these lesions are also non-existent. OBJECTIVE: To evaluate proteomic changes between normal epidermis, AK, BD and cSCC that could support a molecular classification and improve our understanding of disease progression. METHODS: Microdissected formalin-fixed paraffin embedded samples of normal epidermis (n = 4, pooled), AK (n = 10), BD (n = 10) and cSCC (n = 10) were analyzed by mass spectrometry. Following normalization and multiple testing adjustments, differential abundance analysis was performed using Linear Models for Microarray data. Proteins were filtered for significance (adjusted p-value ≤ 0.05) and fold change of at least ±1.5. Comparative bioinformatics analysis was performed using Ingenuity Pathway Analysis (IPA) software. Proteomic findings were subsequently substantiated using immunohistochemistry. RESULTS: 2073 unique proteins were identified. cSCC had the highest number of differentially abundant proteins (63 proteins) followed by BD (58 proteins) and AK (46 proteins). Six proteins (APOA1, ALB, SERPINA1, HLA-B, HP and TXNDC5) were differentially abundant in cSCC compared to AK. Immunohistochemical analysis corroborated changes in MIF, RPL37A and TXNDC5. IPA analysis predicted that cell proliferation, angiogenesis and inflammatory reactions were significantly activated in cSCC compared to BD and AK. Cell death and DNA damage were predicted to be inhibited in BD. CONCLUSION: Our study supports the concept that AK and BD are precursors of cSCC. The identification of proteome changes indicates disruption of repair, pro-apoptotic, and tumor promoting pathways. Our findings will help select targets for classification and treatment.
Project description:SAGE libraries from cultured, differentiated keratinocytes and human epidermis, both normal and affected by actinic keratosis Keywords = Keratinocyte, Epidermis, Homo sapiens, Actinic Keratosis, TNF alpha
Project description:The goal of the study is to compare the transcriptomic profile of patient-derived skin samples, Actinic Keratosis, Intraepidermal Carcinoma and Squamous Cell Carcinoma samples using next generation RNA Sequencing. We aim to identify perturbed pathways and similarities between the 4 conditions. Please note that detailed patients' information, such as gender and age, was withheld from this submission to protect the patients' identity.
Project description:normal skin (no), actinic keratosis (ak), and squamous cell carcinoma (scc) of the skin were examined:; BACKGROUND: Carcinogenesis is a multi-step process indicated by several genes up- or down-regulated during tumor progression. This study examined and identified differentially expressed genes in cutaneous squamous cell carcinoma (SCC). RESULTS: Three different biopsies of 5 immunosuppressed organ-transplanted recipients each normal skin (all were pooled), actinic keratosis (AK) (two were pooled), and invasive SCC and additionally 5 normal skin tissues from immunocompetent patients were analyzed. Thus, total RNA of 15 specimens were used for hybridization with Affymetrix HG-U133A microarray technology containing 22,283 genes. Data analyses were performed by prediction analysis of microarrays using nearest shrunken centroids with the threshold 3.5 and ANOVA analysis was independently performed in order to identify differentially expressed genes (p < 0.05). Verification of 13 up- or down-regulated genes was performed by quantitative real-time reverse transcription (RT)-PCR and genes were additionally confirmed by sequencing. Broad coherent patterns in normal skin vs. AK and SCC were observed for 118 genes. CONCLUSION: The majority of identified differentially expressed genes in cutaneous SCC were previously not described.
Project description:Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation and suppressed apoptosis. Downregulation of the BMPR2 gene along with activation of the transcription factor NFAT have been implicated in the maintenance of pro-proliferative and anti-apoptotic stages of cells. Since an increasing number of microRNAs have been implicated in the regulation of genes specifically important for cell proliferation and apoptosis, we hypothesized that microRNAs might be associated with these cellular features in the etiology of PAH. We demonstrate that downregulation of one such microRNA (miR-204) in human PAH-PASMC promotes the activation of an Src/STAT3/NFAT axis that increases PAH-PASMC proliferation and their resistance to apoptosis. Stimulation experiments using the pro-PAH factors (PDGF, endothelin-1 and angiotensin II) and time course analysis in experimental PAH show that STAT3 activation leads to miR-204 downregulation, thereby activating an Src-dependent positive feedback loop sustaining STAT3 and activating NFAT. More importantly, restoring miR-204 expression decreases proliferation and resistance to apoptosis in human and in an experimental PAH model. Taken together, our study uncovers a new STAT3-miR-204-Src/STAT3/NFAT axis that links the STAT3-dependent downregulation of BMPR2 with the NFAT-mediated pro-proliferative and anti-apoptotic phenotype observed in PAH. Our data point toward a novel potential strategy for treating patients with PAH. Comparative expression profiling of PAH versus healthy patients to evaluate the modulated genes in the disease. Following the demonstration of the downregulation of miR-204 in PAH we want to investigate the effect of the inhibition (using antagomir) of miR-204 expression in PASMC cells.