Cutaneous Squamous Cell Carcinoma Tissues Research Articles

MicroRNA-124 plays an inhibitory role in cutaneous squamous cell carcinoma cells via targeting SNAI2, an immunotherapy determinant

PurposeMicroRNAs (miRs) play multiple roles during cutaneous squamous cell carcinoma (CSCC) progression. Previous studies suggest miR-124 could inhibit cancer development in CSCC. Methods: Obtained 63 pairs of CSCC and adjacent tissues for analysis. Cultured HaCaT and two CSCC cell lines (A431 and SCL-1) in DMEM (10 % FBS). Transfected cells using Lipofectamine 2000 with various miR-124 mimics, inhibitors, or Snail family transcriptional repressor 2 (SNAI2) expression plasmid. Performed a series of assays, including real-time quantitative PCR, Western blot, CCK8, wound healing, transwell, and luciferase reporter gene assay, to examine the effects of miR-124 on CSCC cells. Results: An evident downregulation of miR-124 in CSCC tissues, which was related to advanced disease stage and nodal metastasis. Overexpressing miR-124 could reduce the proliferation, migration, and invasion abilities of CSCC cells. It was verified that miR-124 targets the SNAI2 in CSCC cells. Moreover, ectopic expression of SNAI2 rescued the suppressive effects on CSCC cells induced by miR-124 overexpression. Furthermore, miR-124 increased cell sensitivity to cisplatin. Besides, SNAI2 is a critical factor in the immune-related aspects of CSCC and its modulation may influence the response to immunotherapy. Conclusion: We demonstrate that miR-124 inhibits CSCC progression through downregulating SNAI2, and thus it may be a molecular candidate for treating CSCC in the clinic.

Implication of Amyloid Precursor-like Protein 2 Expression in Cutaneous Squamous Cell Carcinoma Pathogenesis.

Regulatory functions of amyloid precursor-like protein 2 (APLP2) expression in intracellular trafficking of major histocompatibility complex class I (MHC-I) and biological behavior of tumor cells have been reported in various types of malignancies but not in cutaneous squamous cell carcinoma (CSCC). This study aimed to investigate the role of APLP2 expression in the pathogenesis of CSCC. The expression of APLP2 and a key modulator of cancer immune escape, MHC-I, were determined in CSCC tissue samples obtained from 141 patients using immunohistochemistry. The regulatory effects of APLP2 expression on the biological behavior and surface expression of MHC-I in CSCC cells were investigated by trypan blue assay, Matrigel invasion assay, and in vivo xenograft analysis. APLP2 immunoreactivity was high in 73 (51.8%) tissue samples from patients with CSCC and was significantly related to subcutaneous fat invasion and poor prognosis in our cohort. Moreover, proliferation of and invasion by CSCC cells were significantly reduced after APLP2 knockdown in CSCC cells both in vitro and in vivo. A significant association was found between APLP2 and membrane MHC-I expression in patients with CSCC. In vivo xenograft analysis showed that APLP2 knockdown increased membrane MHC-I expression in CSCC cells. APLP2 not only acts as an oncogene in CSCC progression but also as a possible modulator of cancer immune escape by influencing MHC-I expression on the cell surface. APLP2 may serve as a novel molecular biomarker and therapeutic target for patients with CSCC.

CircIFFO1 suppresses tumor growth and metastasis of cutaneous squamous cell carcinoma by targeting the miR-424-5p/NFIB axis.

Cutaneous squamous cell carcinoma (CSCC) is a severe malignancy derived from the skin. Circular RNAs (circRNAs) play an important role in the pathological process of many malignant tumors. Moreover, circIFFO1 is reported to be down-regulated in CSCC tissues compared with non-lesional skin tissues. This study aimed to explore the specific role and potential mechanism of circIFFO1 in CSCC progression. Cell proliferation ability was analyzed by 3-(4, 5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU) incorporation, and colony-formation assays. Cell cycle progression and apoptosis were detected by flow cytometry. Cell migration and invasion were examined by transwell assays. The interaction between microRNA-424-5p (miR-424-5p) and circIFFO1 or nuclear factor I/B (NFIB) was validated by dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation (RIP) assays. Xenograft tumor assay and immunohistochemistry (IHC) assay were employed to analyze the tumorigenesis in vivo. CircIFFO1 level was down-regulated in CSCC tissues and cell lines. CircIFFO1 overexpression suppressed the proliferation, migration, invasion, and promoted apoptosis of CSCC cells. CircIFFO1 acted as a molecular sponge for miR-424-5p. The anti-tumor effects mediated by circIFFO1 overexpression in CSCC cells could be reversed by miR-424-5p overexpression. miR-424-5p interacted with the 3' untranslated region (3'UTR) of Nuclear Factor I/B (NFIB). miR-424-5p knockdown suppressed the malignant behaviors of CSCC cells, and NFIB knockdown counteracted the anti-tumor effects of miR-424-5p absence in CSCC cells. Additionally, circIFFO1 overexpression restrained xenograft tumor growth in vivo. CircIFFO1 suppressed the malignant behaviors of CSCC by mediating the miR-424-5p/NFIB axis, which provided new insights into the pathogenesis of CSCC.

Hsa_circ_0005085 may suppress cutaneous squamous cell carcinoma growth and metastasis through targeting the miR-186-5p/LAMC1 axis.

Cutaneous squamous cell carcinoma (CSCC) is asevere malignancy derived from the skin. Mounting evidence suggests that circular RNAs (circRNAs) participate in diverse biological functions in human cancers, containing CSCC. However, the biological functions and underlyingmechanism of hsa_circ_0005085 in CSCC have not been clearly studied. Expression levels of hsa_circ_0005085, microRNA-186-5p (miR-186-5p), and Laminin subunit gamma 1 (LAMC1) were detected by reverse transcription-quantitative polymerase chain reaction. Cell counting kit-8 assay, colony formation assay, and 5-Ethynyl-2'-deoxyuridine assay were used to assess cell proliferation. Transwell assay was conducted to detect cell migration and invasion. Cell apoptosis was analyzed by flow cytometry. Protein expression of LAMC1, E-cadherin, Snail, and slug were assessed using western blot assay. Using bioinformatics software, the binding between miR-186-5p and hsa_circ_0005085 or LAMC1 was predicted, followed by verification using a dual-luciferase reporter and RNA-Immunoprecipitation. The mouse xenograft model was established to investigate the role of hsa_circ_0005085 in vivo. Hsa_circ_0005085 level was downregulated in CSCC tissues and cells. Overexpression of hsa_circ_0005085 inhibited cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and promoted cell apoptosis in CSCC. MiR-186-5p could restore the effect of hsa_circ_0005085 overexpression on CSCC cells, and the knockdown of LAMC1 reversed the regulation of the miR-186-5p inhibitor. In mechanism, hsa_circ_0005085 served as a sponge for miR-186-5p to regulate LAMC1 expression. Overexpression of hsa_circ_0005085 reduced growth of tumor via miR-186-5p/LAMC1 axis in vivo. In our study, hsa_circ_0005085 might inhibit CSCC development by targeting the miR-186-5p/LAMC1 axis, which might provide a promising therapeutic target for CSCC.

Circ_0068631 sponges miR-139-5p to promote the growth and metastasis of cutaneous squamous cell carcinoma by upregulating HOXB7.

Circular RNAs (circRNAs) are often dysregulated in cancers and closely related to cancer progression, including cutaneous squamous cell carcinoma (CSCC). However, the role and mechanism of circ_0068631 in CSCC progression have not been reported. The expression of circ_0068631, microRNA-139-5p (miR-139-5p), and homeobox B7 (HOXB7) was measured by real-time quantitative polymerase chain reaction (RT-qPCR). Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, and colony formation assay were used to measure cell proliferation. Cell apoptosis was assessed by flow cytometry. Cell migration was detected by transwell assay. The interaction between miR-139-5p and circ_0068631 or HOXB7 was confirmed by dual-luciferase reporter assay. A xenograft tumor model was established to confirm the function of circ_0068631 in vivo. Circ_0068631 was upregulated in CSCC tissues and cells, and its silencing could inhibit CSCC cell proliferation and metastasis while promoting apoptosis in vitro, as well as restrain CSCC tumor growth in vivo. Circ_0068631 acted as a sponge of miR-139-5p, and miR-139-5p inhibition reversed the repressive effect of circ_0068631 knockdown on CSCC cell progression. Furthermore, HOXB7 was a target of miR-139-5p, and miR-139-5p inhibited the malignant behaviors by downregulating HOXB7 expression in CSCC cells. Further, circ_0068631 sponged miR-139-5p to regulate HOXB7 expression. Circ_0068631 functioned as a novel oncogene in CSCC progression by regulating miR-139-5p/HOXB7 axis, suggesting that circ_0068631 may be a potential target for CSCC treatment. Circ_0068631 was overexpressed in CSCC tissues and cells. Circ_0068631 downregulation suppressed CSCC progression via miR-139-5p. Circ_0068631 regulated HOXB7 via sponging miR-139-5p.

Circ_0001402 knockdown suppresses the chemoresistance and development of DDP-resistant cutaneous squamous cell carcinoma cells by functioning as a ceRNA for miR-625-5p.

Cutaneous squamous cell carcinoma (CSCC) is the most common metastatic skin cancer. Circular RNAs (circRNAs) are differentially expressed in CSCC and can sequester and sponge microRNAs. GSE74758 shows that hsa_circ_0001402 (circ_0001402) is the most overexpressed circRNA in CSCC. Expression of circ_0001402, microRNA(miR)-625-5p and karyopherin subunit alpha 4 (KPNA4) was detected by quantitative real-time polymerase chain reaction and/or Western blot. Colon formation, flow cytometry, Transwell assays and xenograft tumour model confirmed the development of CSCC cells. The competing endogenous RNA (ceRNA) interaction was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Circ_0001402 was significantly upregulated in CSCC tissues and cells, and higher expression of circ_0001402 was found in DDP-resistant samples. Functionally, circ_0001402 knockdown induced apoptosis and inhibited half maximal inhibitory concentration of DDP, colony formation, migration and invasion of DDP-resistant CSCC cells, accompanied with the depressed multi-drug resistance-1 (MDR1) and MDR-related protein-1, while miR-625-5p inhibitor could counteract these effects. Mechanically, circ_0001402 mediated the expression regulation of KPNA4 via functioning as a ceRNA for miR-625-5p. KPNA4 re-expression could abate the functions of miR-625-5p. Furthermore, circ_0001402 knockdown could hinder tumour growth of DDP-resistant CSCC. Circ_0001402 knockdown can suppress the development and chemoresistance of DDP-resistant CSCC cells at least partly through targeting miR-625-5p/KPNA4 axis.

The Role of NFATC4 Gene in Human Cutaneous Squamous Cell Carcinoma.

Nuclear factor of activated T-cells, cytoplasmic 4 (NFATC4) has been implicated in keratinocyte development and several types of cancer. A well-defined role for NFATC4 in cutaneous squamous cell carcinoma (CSCC) has not yet been established. In this study, NFATC4 gene function in CSCC development was examined. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to measure the mRNA expression of NFATC4 in CSCC tissues and controls. A431 and Colo16 cell proliferation, invasion, and apoptosis were measured by CCK-8 assay, transwell invasion, and flow cytometry, respectively, after an NFATC4 expression lentivirus infection. Animal models were applied to validate the function of the NFATC4 gene. (1) CSCC tissues showed a significant decrease in NFATC4 expression compared to controls. (2) Overexpression of NFATc4 suppresses A431 and Colo16 cell proliferation and invasion but promotes cell apoptosis. (3) Mouse models overexpressing NFATC4 showed reduced tumourigenesis. It was suggested that NFATC4 might be a tumour suppressor gene in CSCC.

LINC00460 Promotes Cutaneous Squamous Cell Carcinoma Progression Through Stabilizing ELAVL1 Protein

Long intergenic noncoding ribonucleic acid (lncRNA) 460 is reportedly associated with carcinogenesis and progression in various types of cancer. However, the mechanisms underlying its action in cutaneous squamous cell carcinoma (CSCC) remain unclear. LINC00460 mRNA expression was analysed using data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Cell growth, migration, and invasion were evaluated using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), transwell migration and invasion assays after inducing LINC00460 knockdown. A xenograft tumour model was used to determine the effects of LINC00460 on tumour growth and metastasis in vivo. To examine the interaction between LINC00460 and ELAVL1, RNA pulldown and RNA immunoprecipitation assays were performed. LINC00460 was found to be significantly upregulated in CSCC tissues and cell lines. Functionally, LINC00460 knockdown inhibited cell proliferation, migration, and invasion in vitro. Consistent with this, when LINC00460 expression decreased, CSCC tumorigenesis and metastasis in vivo were inhibited. Mechanistically, LINC00460 binds to embryonic lethal abnormal vision like RNA binding protein 1 (ELAVL1) and enhances its stability by inhibiting the β-transducin repeats-containing protein (β-TrCP)-mediated ubiquitination of ELAVL1. Moreover, the effect of LINC00460 silencing on the proliferation, migration, and invasion of CSCC cells could be reversed by overexpressing ELAVL1. Our findings demonstrated that LINC00460 plays a critical role in regulating ELAVL1 function. This highlights the potential targets for the clinical diagnosis and treatment of CSCC.

Knockdown of circRNA circEBF1 inhibits cutaneous squamous cell carcinoma progression by regulating the miR-1247-5p/CSF3 axis.

Cutaneous squamous cell carcinoma (CSCC) is one of the causes of cancer-related death worldwide. Circular RNAs (circRNAs) play a vital role in the pathological process of many malignant tumors. This study aimed to explore the specific role and potential mechanism of circRNA EBF transcription factor 1 (circEBF1) in CSCC. The levels of circEBF1, microRNA-1247-5p (miR-1247-5p) and colony stimulating factor 3 (CSF3) were determined by quantitative real-time PCR and Western blot. Cell proliferation was assessed by colony formation assay. Cell migration, invasion and apoptosis were evaluated by Transwell, wound healing, flow cytometry and Western blot assays. Cellular glycolysis, including glucose consumption, lactate production, and ATP level, was detected by commercial kits. The binding relationship between miR-1247-5p and circEBF1 or CSF3 was verified by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay. Xenograft experiment was conducted to analyze tumor growth in vivo. circEBF1 and CSF3 were up-regulated, while miR-1247-5p was down-regulated in CSCC tissues and cells. Interference of circEBF1 hindered the proliferation, migration, invasion, and glycolysis of CSCC cells and promoted apoptosis. In addition, circEBF1 sponged miR-1247-5p to regulate CSCC cell development. Also, miR-1247-5p suppressed CSCC cell progression by inhibiting CSF3. Moreover, depletion of circEBF1 blocked CSCC tumor growth in vivo. Down-regulation of circEBF1 impeded CSCC progression by regulating the miR-1247-5p/CSF3 pathway, suggesting that circEBF1 might be a promising therapeutic target for CSCC.

From Bowen disease to cutaneous squamous cell carcinoma: eight markers were verified from transcriptomic and proteomic analyses

BackgroundBowen's disease is a cutaneous squamous cell carcinoma (CSCC) in situ. If left untreated, BD may progress to invasive CSCC. CSCC is one of the most common cutaneous carcinoma in the elderly and the advanced, metastasis CSCC usually have a poor outcomes. However, the mechanisms of invasion and metastasis from Bowen’s disease to CSCC is complicated and still unclear.ObjectivesThe aim of this study was to explore the biomarkers and molecular alterations in Bowen’s disease development process via analyzing the proteomics changes in tissues of CSCC, Bowen disease and healthy skin.MethodsA total of 7 individuals with CSCC (5 for proteomics study and 2 for validation), 7 individuals with Bowen disease (5 for proteomics study and 2 for validation) and 7 healthy controls (5 for proteomics study and 2 for validation) presented to the Department of Dermatology, Yijishan Hospital, the First Affiliated Hospital of Wannan Medical College between January 2021 and December 2021 were enrolled. The proteomics analysis was performed to screen differentially expressed proteins/gens (DEPs/DEGs) in the lesions of CSCC, Bowen disease and healthy skin tissues. The transcriptomic data (GSE32628) of CSCC was selected and downloaded from the GEO database. The common DEGs in our proteomics results and GSE32628 between CSCC and healthy skin tissues were selected. And then, the common DEGs which significantly up or down-regulated between CSCC and Bowen disease in our proteomics results were further screened to identify using Western blot methods in the validation group. CSCC A431 cells were transfected with SERPINB1 small interfering RNA (si-SERPINB1) or small interfering RNA negative control (si-NC). To explore the effect of SERPINB1 silencing on migration and invasion ability of A431 cells.ResultsA total of 501 proteins were differentially expressed between the CSCC and healthy skin tissues, with 332 up-regulated and 169 down-regulated at least 1.5-fold with a P value < 0.05. These DEPs involved multiple biological functions such as protein binding process, immune, inflammation, ribosome, protein digestion and absorption, ECM-receptor interaction, focal adhesion, PI3K-Akt signaling pathway and others. A total of 20 common DEGs (COL3A1, LUM, TNC, COL1A1, ALDH3A2, FSCN1, SERPINB4, SERPINB1, CD36, COL4A1, CSTB, GPX3, S100A7, ACTN1, SERPINB3, S100A8, RAB31, STAT1, SPRR1B, S100A9) between CSCC and healthy skin tissues in GSE32628 and our proteomics results were found. Besides, the proteins of TNC, FSCN1, SERPINB1, ACTN1 and RAB31 in CSCC were significantly up-regulated, while COL3A1, COL1A1 and CD36 were significantly down-regulated relative to Bowen disease in proteomics results. These proteins were mainly involved in multiple pathways, including Focal adhesion, ECM-receptor interaction, Human papillomavirus infection, PI3K-Akt signaling pathway, PPAR signaling pathway, AMPK signaling pathway and others. These eight proteins were selected for further validation. According to the Western blotting analysis, when compared with the Bowen disease and healthy skin tissues, we found that the relative expression levels of TNC, FSCN1, SERPINB1, ACTN1 and RAB31 in the CSCC were significantly increased, while COL1A1 and CD36 were significantly decreased, and the differences were statistically significant (P < 0.05). Furthermore, the relative expression levels of TNC, FSCN1, SERPINB1 in the Bowen disease were also significantly increased, while the COL3A1 were also significantly decreased relative to the healthy control. SERPINB1 siRNA inhibited the expression of SERPINB1 at mRNA and protein levels in the A431 cells. After interfering with the expression of SERPINB1, the migration and invasion ability in the A431 cells were significantly decreased (P < 0.05).ConclusionsThis study highlights that eight proteins, TNC, FSCN1, SERPINB1, ACTN1, RAB31, COL3A1, COL1A1, CD36, were significantly associated with the mechanisms of invasion and metastasis in Bowen’s disease.

A Hybrid Epithelial to Mesenchymal Transition in Ex Vivo Cutaneous Squamous Cell Carcinoma Tissues.

While most cases of cutaneous squamous cell carcinoma (cSCC) are benign, invasive cSCC is associated with higher mortality and is often more difficult to treat. As such, understanding the factors that influence the progression of cSCC are important. Aggressive cancers metastasize through a series of evolutionary changes, collectively called the epithelial-to-mesenchymal transition (EMT). During EMT, epithelial cells transition to a highly mobile mesenchymal cell type with metastatic capacities. While changes in expression of TGF-β, ZEB1, SNAI1, MMPs, vimentin, and E-cadherin are hallmarks of an EMT process occurring within cancer cells, including cSCC cells, EMT within tissues is not an “all or none” process. Using patient-derived cSCC and adjacent normal tissues, we show that cells within individual cSCC tumors are undergoing a hybrid EMT process, where there is variation in expression of EMT markers by cells within a tumor mass that may be facilitating invasion. Interestingly, cells along the outer edges of a tumor mass exhibit a more mesenchymal phenotype, with reduced E-cadherin, β-catenin, and cytokeratin expression and increased vimentin expression. Conversely, cells in the center of a tumor mass retain a higher expression of the epithelial markers E-cadherin and cytokeratin and little to no expression of vimentin, a mesenchymal marker. We also detected inverse expression changes in the miR-200 family and the EMT-associated transcription factors ZEB1 and SNAI1, suggesting that cSCC EMT dynamics are regulated in a miRNA-dependent manner. These novel findings in cSCC tumors provide evidence of phenotypic plasticity of the EMT process occurring within patient tissues, and extend the characterization of a hybrid EMT program occurring within a tumor mass. This hybrid EMT program may be promoting both survival and invasiveness of the tumors. A better understanding of this hybrid EMT process may influence therapeutic strategies in more invasive disease.

LncRNA NEAT1 Targets miR-342-3p/CUL4B to Inhibit the Proliferation of Cutaneous Squamous Cell Carcinoma Cells.

Objective This study investigated whether lncRNA NEAT1 could inhibit the proliferation of cutaneous squamous cell carcinoma (CSCC) cells by targeting miR-342-3p/CUL4B, thereby affecting the occurrence and development of CSCC. Methods Fluorescence quantitative PCR was used to detect the expression of lncRNA NEAT1 and miR-42-3p in skin squamous cell carcinoma and adjacent tissues. Bioinformatics software and luciferase reporter gene assay were used to analyze the association of lncRNA NEAT1 and miR-342-3p. The effect of overexpression or knockdown of miR-342-3p on the proliferation of CSCC cells was examined by MTT and colony formation assays. Western blotting was used to detect the proteins of the miR-342-3p/CUL4B signaling axis. Results The lncRNA NEAT1 is abnormally overexpressed in CSCC tissues and cell lines. The expression of lncRNA NEAT1 and miR-342-3p in CSCC was negatively correlated. Bioinformatics prediction analysis revealed that lncRNA NEAT1 regulates the expression of miR-342-3p. The results of MTT and plate colony formation experiments showed that the transfection of miR-342-3p mimics significantly inhibited the proliferation and plate colony formation of CSCC cells, while the transfection of miR-342-3p inhibitor significantly promoted the proliferation and plate colony-forming ability of CSCC cells. Western blot results showed that lncRNA NEAT1 affected CSCC cell proliferation through miR-342-3p/CUL4B/PI3K-Akt signaling pathway. Conclusion The expression of lncRNA NEAT1 and miR-342-3p in CSCC tissues was negatively correlated. This study is the first to demonstrate that the lncRNA NEAT1, as a ceRNA, affects the proliferation of skin squamous cell carcinoma cells through the miR-342-3p/CUL4B/PI3K-Akt signaling pathway. Therefore, lncRNA NEAT1 could be a biological marker or target for CSCC diagnosis or treatment.

Abstract LB022: A hybrid epithelial to mesenchymal transition in ex-vivo cutaneous squamous cell carcinoma tissues

Abstract While most cases of cutaneous squamous cell carcinoma (cSCC) are benign, invasive cSCC is associated with higher mortality and is often more difficult to treat. As such, understanding the factors that influence the progression of cSCC are important. Aggressive cancers metastasize through a series of evolutionary changes, collectively called the epithelial-to-mesenchymal transition (EMT). During EMT, epithelial cells transition to a highly mobile mesenchymal cell type with metastatic capacities. While changes in expression of TGF-β, vimentin, and E-cadherin are hallmarks, EMT is not an “all or none” process. Using patient-derived cSCC and adjacent normal tissues (ANT), we observed a significant increase in mRNA expression of TGF-β (p&amp;lt;0.05), TGF-βR2 (p&amp;lt;0.001), and E-cadherin (p&amp;lt;0.005) in cSCC tissues by qPCR. Immunoblots performed on homogenized tissues yielded significant and parallel increases in protein expression of TGF-β (p&amp;lt;0.05) and E-cadherin (p&amp;lt;0.05). We also observed increased expression of the EMT-associated transcription factors Zeb1 and Snai1 in cSCC tissue sections using immunohistochemistry. Interestingly, we observed a significant increase in expression of vimentin (p&amp;lt;0.01) by immunoblot and differential expression of four oncogenic and EMT-associated miRNAs; miR-31 expression was significantly higher in cSCC tissues (p&amp;lt;0.001), while we observed a significant decrease in expression of miR-34a (p&amp;lt;0.05), miR-200b, and miR-200c in cSCC tissues. Examination of ANT and cSCC tissue sections using immunofluorescence and immunohistochemistry showed differential, and reverse, expression of E-cadherin and vimentin among cells within an individual tumor. Collectively, our data support a hypothesis that cSCC tumors are undergoing a hybrid EMT program, where there is variation in expression of EMT markers by cells within a tumor mass that may be facilitating invasion. This hybrid EMT program may be promoting both survival and invasiveness of the tumors. A better understanding of this hybrid EMT process may influence therapeutic strategies in more invasive disease. Citation Format: Christopher S. Pulford, Chandana K. Uppalapati, McKale R. Montgomery, Elizabeth E. Hull, Kathryn J. Leyva. A hybrid epithelial to mesenchymal transition in ex-vivo cutaneous squamous cell carcinoma tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB022.

UVB-induced SFRP1 methylation potentiates skin damage by promoting cell apoptosis and DNA damage.

In response to the accumulation of genetic mutations and cellular changes, ultraviolet radiation B (UVB) skin lesions undergo dysplasia and transform into a cutaneous squamous cell carcinoma (CSCC). Consistent with our previous findings that secreted frizzled-related protein 1 (SFRP1), a member of the SFRP gene family, was downregulated in human CSCC tissue samples, we found a significant downregulation of SFRP1 in HaCaT, A431, and SCL-1 cells after UVB irradiation. DNA methyltransferase 1 (DNMT1) was significantly increased in CSCC tissues as well as UVB-exposed A431 and SCL-1 cells. Bisulfite genomic sequencing analysis showed that the downregulation of SFRP1 was mainly due to methylation of the SFRP1 promoter, as indicated by increased methylation rate of SFRP1 after UVB irradiation in HaCaT cells. Moreover, demethylation treatment with 5-aza'-deoxycytidine (5-AzaC) increased SFRP1 expression and reduced the methylation rate of SFRP1 in HaCaT cells. Flow cytometry analyses demonstrated that 5-AzaC treatment or overexpression of SFRP1 ameliorated UVB-induced apoptosis, while knockdown of SFRP1 promoted UVB-induced apoptosis in HaCaT cells. In addition, a comet assay confirmed that 5-AzaC treatment reduced DNA damage following UVB irradiation, while knockdown of SFRP1 enhanced DNA damage following UVB irradiation. In conclusion, our study identified DNA methylation of SFRP1 as a key mediator in the UVB-induced apoptosis of keratinocytes. These findings indicate that reinforcing SFRP1 defences by 5-AzaC may help prevent UVB-induced skin damage.

FABP7 inhibits proliferation and invasion abilities of cutaneous squamous cell carcinoma cells via the Notch signaling pathway.

Cutaneous squamous cell carcinoma (CSCC) is one of the most common non-melanoma skin cancers worldwide. Fatty acid-binding protein 7 (FABP7) has been reported to be involved in the occurrence, development, metastasis and prognosis of various tumors. In addition, downregulated FABP7 expression was demonstrated in cutaneous malignant melanoma in a previous study. Therefore, we speculated that FABP7 may be a biomarker for CSCC diagnosis. The aim of the present study was to determine the molecular mechanism underlying the effects of FABP7 in CSCC, which may provide a new diagnostic biomarker or treatment target for CSCC. Reverse transcription-PCR, western blotting and immunohistochemistry assays were performed to detect the expression levels of FABP7 in CSCC tissues and cells. Overexpression of FABP7 was achieved in A431 and colo-16 cell lines by transfection with an overexpression vector (oeFABP7). Cell proliferation, colony formation, migration and invasion were detected by Cell Counting Kit-8, crystal violet, scratch and Transwell assays, respectively. Following FABP7 overexpression, western blotting was used to determine the expression levels of proliferation-, invasion- and Notch pathway-associated proteins, including Snail, N-cadherin, Twist, matrix metalloproteinase (MMP)-2, MMP-7, Notch 1 and Notch 3. In addition a CSCC model in nude mice was constructed. Immunohistochemistry was used to determine the expression levels of FABP7, Ki67, Notch 1 and Notch 3. It was demonstrated that FABP7 expression levels were significantly reduced in human CSCC tissues and cells compared with normal samples. Overexpression of FABP7 inhibited the proliferation, invasion and migration abilities of A431 and colo-16 cells compared with those in the negative control group. In addition, transfection with oeFABP7 reduced the expression levels of proliferation-, invasion- and Notch pathway-associated proteins compared with those in the negative control group. Overexpression of FABP7 also reduced the growth of CSCC tumors in vivo and inhibited the expression of Ki67, Notch 1 and Notch 3. Therefore, the results of the present study suggested that FABP7 may inhibit the proliferation and invasion of CSCC cells via the Notch signaling pathway.

Circular RNA circFADS2 inhibits the progression of cutaneous squamous cell carcinoma by regulating miR-766-3p/HOXA9 axis.

Mounting evidence indicates that circular RNAs (circRNAs) play vital roles in human diseases, especially in cancers. However, the biological functions and underlying mechanism of circRNA fatty acid desaturase 2 (circFADS2) in cutaneous squamous cell carcinoma (CSCC) have not been reported. The expression levels of circFADS2, microRNA-766-3p (miR-766-3p) and homeobox A9 (HOXA9) were determined by quantitative real-time PCR (qRT-PCR). Flow cytometry analysis was used to determine cell cycle distribution. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) and colony formation assays. Wound healing and transwell assays were used to assess cell migration and invasion abilities. Glycolysis was examined via the measurement of extracellular acidification rate (ECAR). All protein levels were detected by western blot assay. The interaction between miR-766-3p and circFADS2 or HOXA9 was predicted by bioinformatics software and confirmed by dual-luciferase reporter, RNA pull-down, and RNA Immunoprecipitation (RIP) assays. The mouse xenograft model was established to investigate the role of circFADS2 in vivo. CircFADS2 was downregulated in CSCC tissues and cells. CircFADS2 overexpression inhibited CSCC cell proliferation, metastasis and glycolysis. Moreover, miR-766-3p was able to directly bind to circFADS2, and circFADS2 played an anti-cancer role in CSCC by downregulating miR-766-3p. In addition, HOXA9 was a direct target of miR-766-3p, and miR-766-3p inhibition suppressed CSCC cell proliferation, metastasis and glycolysis by upregulating HOXA9. Furthermore, circFADS2 acted as a sponge of miR-766-3p to regulate HOXA9 expression. Besides, circFADS2 suppressed tumor growth in vivo. CircFADS2 suppressed CSCC progression by regulating miR-766-3p/HOXA9 axis, which might provide a promising therapeutic target for CSCC.

Downregulation of lncRNA NEAT1 inhibits the proliferation of human cutaneous squamous cell carcinoma in vivo and in vitro.

BackgroundThe incidence of cutaneous squamous cell carcinoma (CSCC), a malignant tumor that threatens human life, is increasing every year, and yet its pathogenesis is still unclear. This study found that long noncoding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) was abnormally expressed in CSCC. However, the biochemical mechanisms of lncRNA NEAT1 in carcinogenesis and the development of cancer remain unclear.MethodsFluorescence quantitative polymerase chain reaction (qPCR) was conducted to determine lncRNA NEAT1 expression in CSCC and paracarcinoma tissues and investigate the correlation between NEAT1 levels and patients’ clinicopathological features. The invasion, proliferation, and migration of CSCC cells were measured using colony formation, Cell Counting Kit-8, and Transwell assays. Western blot assay was conducted to test whether NEAT1 knockdown affected invasion and migration-related proteins. In addition, a nude mouse subcutaneous tumorigenesis experiment was performed to determine whether the knockdown of NEAT1 affected the proliferation ability of CSCC cells.ResultsChanges in lncRNA NEAT1 expression in CSCC tissues were correlated with the degree of lymph node metastasis and the tumor, regional lymph nodes, and distant metastasis (TNM) grade of patients. The downregulation of NEAT1 lncRNA significantly impeded cell invasion, proliferation, and migration in CSCC. Through lncRNA NEAT1 knockdown, significant reductions in metalloproteinase-2, metalloproteinase-9, N-cadherin, and vimentin expression were observed, and the level of E-cadherin increased. In vivo experiments in nude mice revealed that knockdown of lncRNA NEAT1 greatly inhibited cell proliferation in CSCC.ConclusionsIn CSCC tissues, NEAT1 lncRNA was expressed at high levels and correlated with lymph node metastasis and TNM stage. The knockdown of NEAT1 lncRNA could significantly impede CSCC proliferation, metastasis, and invasion. Additionally, by measuring the expression level of lncRNA NEAT1, we may be able to detect the clinical and pathological characteristics of CSCC.

Research on Correlations of miR-196a Expression with Progression and Prognosis of Cutaneous Squamous Cell Carcinoma.

PurposeThis study aimed to investigate the correlation between miR-196a expression and the progression and prognosis of cutaneous squamous cell carcinoma (CSCC).Patients and MethodsTissue samples and corresponding paracancerous tissue samples from 117 patients with CSCC were collected. The qRT-PCR analysis was used to detect the expression levels of miR-196a. Kaplan–Meier curve and Cox regression analyses were used to analyze the relationship between miR-196a expression and patients’ prognosis. The CCK-8 and transwell assays were used to explore the effects of miR-196a on the abilities of cell proliferation, migration, or invasion.ResultsmiR-196a expression was significantly up-regulated in CSCC tissues or cell lines, compared with adjacent normal tissues or cell lines, respectively. High expression of miR-196a was associated with positive lymph node metastasis, high TNM stages, and a lower five-year survival rate. The expression level of miR-196a was up-regulated and the proliferation, migration or invasion ability of cells were significantly increased accordingly.ConclusionmiR-196a is highly expressed in CSCC, thus affecting the occurrence and development of CSCC. More importantly, miR-196a was shown to have potential as a prognostic marker for CSCC.

Circular RNA circ-LARP1B contributes to cutaneous squamous cell carcinoma progression by targeting microRNA-515-5p/TPX2 microtubule nucleation factor axis

ABSTRACT Circular RNAs (circRNAs) have shown pivotal regulatory roles in tumorigenesis and progression. Our purpose was to analyze the role of circRNA La ribonucleoprotein 1B (circ-LARP1B; hsa_circ_0070934) in cutaneous squamous cell carcinoma (CSCC) progression and its associated mechanism. Cell viability, colony formation ability, migration, and invasion were analyzed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay, colony formation assay, wound healing assay, and transwell invasion assay. Flow cytometry was performed to analyze cell apoptosis and cell cycle progression. Cell glycolytic metabolism was analyzed using Glucose Uptake Colorimetric Assay kit, Lactate Assay Kit II, and ATP colorimetric Assay kit. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the interaction between microRNA-515-5p (miR-515-5p) and circ-LARP1B or TPX2 microtubule nucleation factor (TPX2). Circ-LARP1B expression was up-regulated in CSCC tissues and cell lines. Circ-LARP1B knockdown suppressed cell viability, colony formation ability, migration, invasion, cell cycle progression, and glycolysis and triggered cell apoptosis in CSCC cells. miR-515-5p was a direct target of circ-LARP1B in CSCC cells, and circ-LARP1B silencing-mediated anti-tumor effects were largely counteracted by miR-515-5p knockdown. miR-515-5p directly interacted with the 3ʹ untranslated region (3ʹUTR) of TPX2. TPX2 overexpression largely overturned miR-515-5p-mediated anti-tumor effects in CSCC cells. Circ-LARP1B could up-regulate TPX2 expression by sponging miR-515-5p in CSCC cells. Circ-LARP1B knockdown suppressed tumor growth in vivo. In conclusion, circ-LARP1B contributed to CSCC progression by targeting miR-515-5p/TPX2 axis. The circ-LARP1B/miR-515-5p/TPX2 axis might provide novel therapeutic targets for CSCC patients.

Gene Expression Studies in Formalin-Fixed Paraffin-Embedded Samples of Cutaneous Cancer: The Need for Reference Genes.

Formalin-fixed paraffin-embedded (FFPE) tumour samples may provide crucial data regarding biomarkers for neoplasm progression. Analysis of gene expression is frequently used for this purpose. Therefore, mRNA expression needs to be normalized through comparison to reference genes. In this study, we establish which of the usually reported reference genes is the most reliable one in cutaneous malignant melanoma (MM) and cutaneous squamous cell carcinoma (CSCC). ACTB, TFRC, HPRT1 and TBP expression was quantified in 123 FFPE samples (74 MM and 49 CSCC biopsies) using qPCR. Expression stability was analysed by NormFinder and Bestkeeper softwares, and the direct comparison method between means and SD. The in-silico analysis with BestKeeper indicated that HPRT1 was more stable than ACTB and TFRC in MM (1.85 vs. 2.15) and CSCC tissues (2.09 vs. 2.33). The best option to NormFinder was ACTB gene (0.56) in MM and TFRC (0.26) in CSCC. The direct comparison method showed lower SD means of ACTB expression in MM (1.17) and TFRC expression in CSCC samples (1.00). When analysing the combination of two reference genes for improving stability, NormFinder indicated HPRT1 and ACTB to be the best for MM samples, and HPRT1 and TFRC genes for CSCC. In conclusion, HPRT1 and ACTB genes in combination are the most appropriate choice for normalization in gene expression studies in MM FFPE tissue, while the combination of HPRT1 and TFRC genes are the best option in analysing CSCC FFPE samples. These may be used consistently in forthcoming studies on gene expression in both tumours.