Cutaneous Squamous Cell Carcinoma Cells 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.

The Use of Low-Level Laser Therapy (LL LT) in the Symptomatic Treatment of Vulvar Cancer IV st.

Cutaneous squamous cell carcinoma (CSCC) is a malignant tumor originating from keratinocytes in the epidermis, which seriously affects people's life safety. Heat shock proteins (HSPs) are molecular chaperones that help maintain protein stability and are involved in tumor activities. However, there is currently no systematic study of HSPs in CSCC. In this study, we used qPCR and WB to explore the expression of 14 HSPs at mRNA and protein levels in human immortalized keratinocytes HACAT cells, human CSCC cell lines COLO-16 and SCL-1 cells, and collected human CSCC samples for histological verification. Eventually, we found that the expression levels of different HSPs in CSCC were different. HSPB6, HSPB8 and HSPB11 showed no dysregulation in CSCC. HSPB4, HSPB5, HSPB7, HSPB10 and HSP40 were significantly upregulated in CSCC cells, while HSPB1, HSPB9, HSP60, HSP70, HSP90 and HSP100 were significantly downregulated. In summary, we validated the expression of 14 common HSPs in CSCC, which may lay the foundation for the study of HSPs in CSCC.

Differential expression of heat shock proteins in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (CSCC) is a malignant tumor originating from keratinocytes in the epidermis, which seriously affects people's life safety. Heat shock proteins (HSPs) are molecular chaperones that help maintain protein stability and are involved in tumor activities. However, there is currently no systematic study of HSPs in CSCC. In this study, we used qPCR and WB to explore the expression of 14 HSPs at mRNA and protein levels in human immortalized keratinocytes HACAT cells, human CSCC cell lines COLO-16 and SCL-1 cells, and collected human CSCC samples for histological verification. Eventually, we found that the expression levels of different HSPs in CSCC were different. HSPB6, HSPB8 and HSPB11 showed no dysregulation in CSCC. HSPB4, HSPB5, HSPB7, HSPB10 and HSP40 were significantly upregulated in CSCC cells, while HSPB1, HSPB9, HSP60, HSP70, HSP90 and HSP100 were significantly downregulated. In summary, we validated the expression of 14 common HSPs in CSCC, which may lay the foundation for the study of HSPs in 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.

Global status of research on cutaneous squamous cell carcinoma and its programmed cell death: Bibliometric and visual analysis from 2012 to middle 2022.

This study was aimed to analyze the research status and development trend of cutaneous squamous cell carcinoma (CSCC), focusing on the field of programmed cell death of CSCC, and providing suggestions for the research of CSCC. The publications related to CSCC and CSCC programmed cell death were searched in the Web of Science Core Collection (WOSCC) database, and timespan was set from 2012 to middle 2022. Research trends, authors, major country collaborations, research institutions, representative journals, publishers, and keywords were analyzed with CiteSpace and VOSviewer. After screening, a total of 3656 publications on CSCC and 156 publications on CSCC cell programmed death were obtained. The number of published articles increased gradually with the years. The United States ranked first in terms of the number of published papers. Research in this field had focused on dermatology. Most of the institutions in both regions were from European and American countries. Harvard University was the most prolific institution. Wiley was the most prolific publisher. The popular keywords for programmed cell death in CSCC were cutaneous squamous cell carcinoma, diagnosis, pd 1, head, nivolumab and risk. Keywords in CSCC field were divided into 7 clusters: cutaneous squamous cell carcinoma, sentinel lymph node biopsy, skin cancer, B-Raf Proto-Oncogene, Serine/Threonine Kinase (BRAF) inhibitor and human Papillomaviruses, and P63 expression. Squamous cell carcinoma, cancer, head and expression were the most popular keywords. The popular keywords for programmed cell death in CSCC were cutaneous squamous cell carcinoma, diagnosis, pd 1, head, nivolumab and risk. This study analyzed the research status of cutaneous squamous cell carcinoma and programmed cell death from 2012 to middle 2022. Understanding research status and hotspots can help scholars, countries and policymakers to better understand the background and research frontier of CSCC, and guide further research directions.

Natural killer cells suppress human cutaneous squamous cell carcinoma cancer cell survival and tumor growth.

Cutaneous squamous cell carcinoma (CSCC), which develops in response to ultraviolet irradiation exposure, is among the most common cancers. CSCC lesions can be removed by surgical excision, but 4.5% of these cancers reappear as aggressive and therapy-resistant tumors. CSCC tumors display a high mutation burden, and tumor frequency is dramatically increased in immune-suppressed patients, indicating a vital role for the immune system in controlling cancer development. Natural killer cells (NK cells) play a key role in cancer immune surveillance, and recent studies suggest that NK cells from healthy donors can be expanded from peripheral blood for use in therapy. In the present study, we test the ability of ex vivo expanded human NK cells to suppress the CSCC cell cancer phenotype and reduce tumor growth. We expanded human NK cells from multiple healthy donors, in the presence of IL-2, and tested their ability to suppress the CSCC cell cancer phenotype. NK cell treatment produced a dose-dependent reduction in SCC-13 and HaCaT cell spheroid growth and matrigel invasion and induced SCC-13 and HaCaT cell apoptosis as evidenced by increased procaspase 9, procaspase 3, and PARP cleavage. Moreover, two important CSCC cell pro-cancer signaling pathways, YAP1/TAZ/TEAD and MEK1/2-ERK1/2, were markedly reduced. Furthermore, tail-vein injection of NK cells markedly suppressed the growth of SCC-13 xenograft tumors in NSG mice, which was also associated with a reduction in YAP1 and MEK1/2-P levels and enhanced apoptosis. These findings show that NK cell treatment suppresses CSCC cell spheroid formation, invasion, viability, and tumor growth, suggesting NK cell treatment may be a candidate therapy 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.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 transfection of guide RNA targeting on MMP9 as anti-cancer therapy in human cutaneous squamous cell carcinoma cell line A431.

Cutaneous squamous cell carcinoma (SCC) is the second most common form of skin malignancy, representing around 20% of all skin cancers. It is the main cause of death due to non-melanoma skin cancer every year. Metastatic cutaneous SCC is associated with poor prognosis in patients and warrants a more effective and specific approach such as disruption of genes associated with cancer metastasis. Matrix metalloproteinases (MMPs) are enzymes involved in cancer progression and are regarded as major oncotargets. Among others, MMP9 plays critical roles in tumour progression, angiogenesis, and invasion of cutaneous SCC. We aimed to determine whether the MMP9 gene is a suitable gene target for anti-cancer therapy for cutaneous SCC. We performed clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 transfection of guide RNA (gRNA) targeting the MMP9 gene into human cutaneous SCC cell line A431. Following CRISPR transfection treatment, the viability (p < 0.01) and migratory activities (p < 0.0001) of in vitro cutaneous SCC cells were found to be reduced significantly. The use of quantitative polymerase chain reaction (qPCR) also revealed downregulation of the mRNA expression levels of cancer-promoting genes TGF-β, FGF, PI3K, VEGF-A, and vimentin. Direct inhibition of the MMP9 gene was shown to decrease survivability and metastasis of cutaneous SCC cell line A431. Our findings provided direct evidence that MMP9 is important in the viability, proliferation, and metastasis of cutaneous SCC cells. It serves as a positive foundation for future CRISPR-based targeted anti-cancer therapies in treating skin cancer and other forms of malignancies that involve MMPs as the key determinants.

Pitavastatin Induces Apoptosis of Cutaneous Squamous Cell Carcinoma Cells through Geranylgeranyl Pyrophosphate-Dependent c-Jun N-Terminal Kinase Activation.

Pitavastatin is a cholesterol-lowering drug and is widely used clinically. In addition to this effect, pitavastatin has shown the potential to induce apoptosis in cutaneous squamous cell carcinoma (SCC) cells. The purpose of this study is to investigate the effects and possible action mechanisms of pitavastatin. SCC cells (SCC12 and SCC13 cells) were treated with pitavastatin, and induction of apoptosis was confirmed by Western blot. To examine whether pitavastatin-induced apoptosis is related to a decrease in the amount of intermediate mediators in the cholesterol synthesis pathway, the changes in pitavastatin-induced apoptosis after supplementation with mevalonate, squalene, geranylgeranyl pyrophosphate (GGPP) and dolichol were investigated. Pitavastatin dose-dependently induced apoptosis of cutaneous SCC cells, but the viability of normal keratinocytes was not affected by pitavastatin at the same concentrations. In supplementation experiments, pitavastatin-induced apoptosis was inhibited by the addition of mevalonate or downstream metabolite GGPP. As a result of examining the effect on intracellular signaling, pitavastatin decreased Yes1 associated transcriptional regulator and Ras homolog family member A and increased Rac family small GTPase 1 and c-Jun N-terminal kinase (JNK) activity. All these effects of pitavastatin on signaling molecules were restored when supplemented with either mevalonate or GGPP. Furthermore, pitavastatin-induced apoptosis of cutaneous SCC cells was inhibited by a JNK inhibitor. These results suggest that pitavastatin induces apoptosis of cutaneous SCC cells through GGPP-dependent JNK activation.

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.

T-box transcription factor 2 mediates antitumor immune response in cutaneous squamous cell carcinoma by regulating the expression of programmed death ligand 1.

Cutaneous squamous cell carcinoma (CSCC) is the second largest nonmelanoma skin cancer in humans; effective treatment options for metastatic CSCC are still in short. In this study, we aimed to explore the function of T-box transcription factor 2 (TBX2) in CSCC. The expression level of TBX2 was determined in CSCC samples and cell lines. Programmed death ligand 1 (PD-L1) expression was also analyzed in human CSCC samples. Furthermore, SCC13 cells were transfected with TBX2-DN (loss of function) or normal TBX2 to check its role in regulating PD-L1. The expression level of TBX2 was positively correlated with the stage of CSCC. CSCC tumor cell lines have significantly higher expression levels of TBX2 than normal skin cell lines, and SCC13 cells showed the highest expression. PD-L1 expressions were upregulated during the progression of CSCC, and positively correlated with TBX2. Furthermore, PD-L1 expression increased in SCC13 cells overexpressing TBX2. However, TBX2 did not regulate the activation of IFNγ signal, but mediated the expression of interferon regulatory factor 1 (IRF1) and PD-L1 in both SCC13 and PDV cells. TBX2 could mediate antitumor immune response in CSCC by regulating the expression of PD-L1 through IRF1. It might be a prognostic marker in CSCC and synergistic target for PD-1 immunotherapy.

Alkannin exerts antitumor properties in cutaneous squamous cell carcinoma by inducing apoptosis and shifting the M1/M2 polarization of tumor-associated macrophages by upregulating PTEN.

Cutaneous squamous cell carcinoma (CSCC) is a common cancer in humans and is the second major type of skin cancer that causes death in humans. In this article, we investigated the effects of alkannin on CSCC progression. We revealed that alkannin curbed CSCC cell viability in a dose-dependent manner and accelerated CSCC cell apoptosis. In addition, alkannin expedited macrophage M1 polarization while curbing M2 polarization. Moreover, alkannin elevated phosphatase and tensin homolog (PTEN) abundance in CSCC cells. The results of bioinformatics analysis revealed that alkannin might modulate CSCC via PTEN. Downregulation of PTEN reversed the effects of alkannin on apoptosis of CSCC cells and M1/M2 polarization of macrophages. Alkannin reduced CSCC tumor growth in a mouse xenograft model. In conclusion, alkannin curbed the advancement of CSCC by expediting apoptosis and facilitating M1 polarization of macrophages by upregulating PTEN. These data may offer a therapeutic approach against CSCC.

Sulforaphane inhibits CD44v6/YAP1/TEAD signaling to suppress the cancer phenotype.

Sulforaphane (SFN) is a promising cancer prevention and treatment agent that strongly suppresses the cutaneous squamous cell carcinoma (CSCC) cell cancer phenotype. We previously showed that yes-associated protein 1 (YAP1)/TEAD signaling is a key procancer stimulator of the aggressive CSCC cell cancer phenotype. However, SFN-responsive upstream regulators of YAP1/TEAD signaling are not well characterized and so there is a pressing need to identify these factors. We show that CD44v6 knockdown reduces YAP1/TEAD-dependent transcription and target gene expression, and that this is associated with reduced spheroid formation, invasion and migration. CD44v6 knockout cell lines also display reduced YAP1/TEAD activity and target gene expression and attenuated spheroid formation, invasion, migration and tumor formation. An important finding is that SFN treatment suppresses CD44v6 level leading to a reduction in YAP1/TEAD signaling and marker gene expression. Sox2 level and epithelial-mesenchymal transition (EMT) are also reduced. Forced expression of constitutive active YAP1 in CD44v6 knockdown cells partially restores the aggressive cancer phenotype. These important findings suggest that CD44v6 drives YAP1/TEAD signaling to enhance the CSCC cell cancer phenotype and that SFN treatment reduces CD44v6 level/function which, in turn, reduces YAP1/TEAD signaling leading to reduced stemness, EMT and tumor growth.

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.

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.

LB887 Direct effects of zinc in proliferation and migration of human squamous cell carcinoma cell lines in vitro

Zinc is a trace element that requires for more than 2000 transcription factors. Thus, zinc could have functions in development, differentiation, and cell growth of many cell types including cancer cells. Prior studies reported that zinc induced apoptosis in cancer cells, such as glioma, bladder, prostate, breast, and melanoma. Topical application of high concentration zinc containing Mohs paste is effective to shrink skin cancers including squamous cell carcinoma (SCC), breast cancer. However, the evidences of direct effect of zinc in growth and cell migration of cutaneous SCC cells are scarce.

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.