cSCC Cells Research Articles

MiR-106b-5p Promotes Malignant Behaviors of Cervical Squamous Cell Carcinoma Cells by Targeting TIMP2.

The objective of this study was to investigate modulatory mechanism of miR-106b-5p and tissue inhibitor of metalloproteinases 2 (TIMP2) on cervical squamous cell carcinoma cells. Differentially expressed genes in CSCC were analyzed via bioinformatics analysis. The targeting impact of miR-106b-5p on TIMP2 was validated through dual-luciferase assay and RNA immunoprecipitation assay. MiR-106b-5p level and TIMP2 mRNA level were assessed via qRT-PCR. TIMP2 protein level was measured via western blot. Malignant behaviors of CSCC cells were evaluated by functional experiments. The EMT and apoptosis-related proteins were determined via western blot. MiR-106b-5p was noticeably elevated in CSCC cells. Its downstream target was TIMP2. MiR-106b-5p and TIMP2 levels were inversely correlated. MiR-106b-5p overexpression fostered malignant phenotypes of CSCC cells, and vice versus. TIMP2 overexpression weakened the promotive impact of forced expression of miR-106b-5p on CSCC cell growth. EMT was facilitated by forced expression of miR-106b-5p. MiR-106b-5p regulates the progression of CSCC cells via targeting TIMP2, which may provide novel value for development of therapeutic targets for CSCC.

Anti-Malignant Effect of Tensile Loading to Adherens Junctions in Cutaneous Squamous Cell Carcinoma Cells.

Actomyosin contractility regulates various cellular processes including proliferation and differentiation while dysregulation of actomyosin activity contributes to cancer development and progression. Previously, we have reported that actomyosin-generated tension at adherens junctions is required for cell density-dependent inhibition of proliferation of normal skin keratinocytes. However, it remains unclear how actomyosin contractility affects the hyperproliferation ability of cutaneous squamous cell carcinoma (cSCC) cells. In this study, we find that actomyosin activity is impaired in cSCC cells both in vitro and in vivo. External application of tensile loads to adherens junctions by sustained mechanical stretch attenuates the proliferation of cSCC cells, which depends on intact adherens junctions. Forced activation of actomyosin of cSCC cells also inhibits their proliferation in a cell-cell contact-dependent manner. Furthermore, the cell cycle arrest induced by tensile loading to adherens junctions is accompanied by epidermal differentiation in cSCC cells. Our results show that the degree of malignant properties of cSCC cells can be reduced by applying tensile loads to adherens junctions, which implies that the mechanical status of adherens junctions may serve as a novel therapeutic target for cSCC.

TGFβ1 Secreted by Cancer-Associated Fibroblasts as an Inductor of Resistance to Photodynamic Therapy in Squamous Cell Carcinoma Cells.

Simple SummaryPhotodynamic therapy (PDT) is used for the treatment of in situ cutaneous squamous cell carcinoma (cSCC), the second most common form of skin cancer, as well as for its precancerous form, actinic keratosis. However, relapses after the treatment can occur. Transforming growth factor β1 (TGFβ1) produced by cancer-associated fibroblasts (CAFs) in the tumor microenvironment has been pointed as a key player in the development of cSCC resistance to other therapies, such as chemotherapy. Here, we demonstrate that TGFβ1 produced by CAFs isolated from patients with cSCC can drive resistance to PDT in SCC cells. This finding opens up novel possibilities for strategy optimization in the field of cSCC resistance to PDT and highlights CAF-derived TGFβ1 as a potential target to improve the efficacy of PDT.As an important component of tumor microenvironment, cancer-associated fibroblasts (CAFs) have lately gained prominence owing to their crucial role in the resistance to therapies. Photodynamic therapy (PDT) stands out as a successful therapeutic strategy to treat cutaneous squamous cell carcinoma. In this study, we demonstrate that the transforming growth factor β1 (TGFβ1) cytokine secreted by CAFs isolated from patients with SCC can drive resistance to PDT in epithelial SCC cells. To this end, CAFs obtained from patients with in situ cSCC were firstly characterized based on the expression levels of paramount markers as well as the levels of TGFβ1 secreted to the extracellular environment. On a step forward, two established human cSCC cell lines (A431 and SCC13) were pre-treated with conditioned medium obtained from the selected CAF cultures. The CAF-derived conditioned medium effectively induced resistance to PDT in A431 cells through a reduction in the cell proliferation rate. This resistance effect was recapitulated by treating with recombinant TGFβ1 and abolished by using the SB525334 TGFβ1 receptor inhibitor, providing robust evidence of the role of TGFβ1 secreted by CAFs in the development of resistance to PDT in this cell line. Conversely, higher levels of recombinant TGFβ1 were needed to reduce cell proliferation in SCC13 cells, and no induction of resistance to PDT was observed in this cell line in response to CAF-derived conditioned medium. Interestingly, we probed that the comparatively higher intrinsic resistance to PDT of SCC13 cells was mediated by the elevated levels of TGFβ1 secreted by this cell line. Our results point at this feature as a promising biomarker to predict both the suitability of PDT and the chances to optimize the treatment by targeting CAF-derived TGFβ1 in the road to a more personalized treatment of particular cSCC tumors.

C1r Upregulates Production of Matrix Metalloproteinase-13 and Promotes Invasion of Cutaneous Squamous Cell Carcinoma

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. Previous studies have shown the role of the complement system in cSCC progression. In this study, we have investigated the mechanistic role of serine proteinase C1r, a component of the classical pathway of the complement system, in cSCC. Knockout of C1r in cSCC cells using CRISPR/Cas9 resulted in a significant decrease in their proliferation, migration, and invasion through collagen type I compared with that of wild-type cSCC cells. Knockout of C1r suppressed the growth and vascularization of cSCC xenograft tumors and promoted apoptosis of tumor cells invivo. mRNA-sequencing analysis after C1r knockdown revealed significantly regulated Gene Ontology terms cell-matrix adhesion, extracellular matrix component, basement membrane, and metalloendopeptidase activity and Kyoto Encyclopedia of Genes and Genomes pathway extracellular matrix‒receptor interaction. Among the significantly regulated genes were invasion-associated matrix metalloproteinases (MMPs) MMP1, MMP13, MMP10, and MMP12. Knockout of C1r resulted in decreased production of MMP-1, MMP-13, MMP-10, and MMP-12 by cSCC cells in culture. Knockout of C1r inhibited the expression of MMP-13 by tumor cells, suppressed invasion, and reduced the amount of degraded collagen invivo in xenografts. These results provide evidence for the role of C1r in promoting the invasion of cSCC cells by increasing MMP production.

Kinectin1 depletion promotes EGFR degradation via the ubiquitin-proteosome system in cutaneous squamous cell carcinoma

Depletion of kinectin1 (KTN1) provides a potential strategy for inhibiting tumorigenesis of cutaneous squamous cell carcinoma (cSCC) via reduction of epidermal growth factor receptor (EGFR) protein levels. Yet, the underlying mechanisms of KTN1 remain obscure. In this study, we demonstrate that KTN1 knockdown induces EGFR degradation in cSCC cells by promoting the ubiquitin-proteasome system, and that this effect is tumor cell-specific. KTN1 knockdown increases the expression of CCDC40, PSMA1, and ADRM1 to mediate tumor suppressor functions in vivo and in vitro. Mechanistically, c-Myc directly binds to the promoter region of CCDC40 to trigger the CCDC40-ADRM1-UCH37 axis and promote EGFR deubiquitination. Furthermore, KTN1 depletion accelerates EGFR degradation by strengthening the competitive interaction between PSMA1 and ADRM1 to inhibit KTN1/ADRM1 interaction at residues Met1-Ala252. These results are supported by studies in mouse xenografts and human patient samples. Collectively, our findings provide novel mechanistic insight into KTN1 regulation of EGFR degradation in cSCC.

280 Complement Factor D is a biomarker for progression of cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) is the most prevalent metastatic skin cancer, and its incidence is increasing on a global scale. Previous studies have demonstrated the role of complement system in cSCC progression. Here, we have investigated the role of complement factor D (CFD), an activator of alternative pathway of complement, in development of cSCC. RT-qPCR analysis of cSCC cell lines (n=8) and normal human epidermal keratinocytes (NHEKs) (n=6), demonstrated significant up-regulation of CFD mRNA expression in cSCC cells compared to NHEKs.

Crucial Role of Reactive Oxygen Species (ROS) for the Proapoptotic Effects of Indirubin Derivatives in Cutaneous SCC Cells.

Efficient drugs are needed for countering the worldwide high incidence of cutaneous squamous cell carcinoma (cSCC) and actinic keratosis. Indirubin derivatives represent promising candidates, but their effects in cSCC cells have not been reported before. Here, we investigated the efficacy of three indirubin derivatives (DKP-071, -073 and -184) in four cSCC cell lines. High efficacy was seen in SCL-I, SCL-II, SCC-12 and SCC-13, resulting in up to 80% loss of cell proliferation, 60% loss of cell viability and 30% induced apoptosis (10 µM). Apoptosis was further enhanced in combinations with TNF-related apoptosis-inducing ligand (TRAIL). Induction of reactive oxygen species (ROS) appeared as critical for these effects. Thus, antioxidative pretreatment completely abolished apoptosis as well as restored cell proliferation and viability. Concerning the pathways, complete activation of caspases cascades (caspases-3, -4, -6, -7, -8 and -9), loss of mitochondrial membrane potential, activation of proapoptotic PKCδ (protein kinase C delta), inhibition of STAT3 (signal transducer and activator of transcription 3), downregulation of antiapoptotic XIAP (X-linked inhibitor of apoptosis protein) and survivin as well as upregulation of the proapoptotic Bcl-2 protein Puma and the cell cycle inhibitor p21 were obtained. Importantly, all activation steps were prevented by antioxidants, thus proving ROS as a master regulator of indirubins’ antitumor effects. ROS induction presently develops as an important issue in anticancer therapy.

Sirolimus diminishes the expression of GRO-α (CXCL-1) /CXCR2 axis in human keratinocytes and cutaneous squamous cell carcinoma cells

BackgroundOrgan transplant recipients show a high incidence for the formation of cutaneous squamous cell carcinoma (cSCC), while sirolimus appears to reduce the risk. GRO-α is a chemokine, which is overexpressed in many tumor entities and associated with malignant transformation. However, little is known about the expression and function of GRO-α in human cSCC. ObjectiveOur aim was to investigate the relevance of the GRO-α (CXCL-1)/ CXCR2 axis in human cSCC and the potential impact of sirolimus. MethodsWe analyzed the GRO-α expression in human keratinocytes, different cSCC cell lines as well as cSCC tissue and investigated its effect on cell proliferation and migration. Additionally, we incubated cells with sirolimus and measured the expression of GRO-α and its receptor CXCR2. ResultsWe showed that both constitutive as well as induced GRO-α expression is higher in in cSCC cell lines compared to keratinocytes and that GRO-α protein is detectable in human cSCC tissue. By GRO-α exposure and shRNA knock down, we identified GRO-α as a driving factor in proliferation and migration. Moreover, in a dermis equivalent GRO-α knocked down cSCC cell lines displayed a reduced capacity in tumor nest formation. Incubation with sirolimus significantly inhibited GRO-α expression in keratinocytes as well as tumor cell lines. Moreover, sirolimus decreased the expression of the corresponding receptor CXCR2. ConclusionTaken together, our results suggest that the GRO-α/CXCR2 axis plays a role in human keratinocyte carcinogenesis and might represent a molecular mechanism for the preventive effect of mTOR inhibitors in cSCC development.

25686 Variability in the dosing of PD1 inhibitors used for treatment of nonmelanoma skin cancers

Nonmelanoma skin cancer (NMSC) represents the most common cancer diagnosed in the United States. These tumors are frequently treated by surgical modalities. Patients with advanced disease require a different approach to therapy. Programmed cell death protein 1 (PD1) inhibitors are an emerging immunotherapeutic option for the treatment of a variety of tumors including advanced NMSC. Cemiplimab is a PD-1 inhibitor approved for treatment of locally advanced or metastatic cutaneous squamous cell carcinoma (cSCC) not amenable to curative surgery or radiation. Pembrolizumab and nivolumab are PD1 inhibitors used as off-label therapies for advanced cSCC or basal cell carcinoma (BCC). We conducted a PubMed literature search up to August 8, 2020 to include studies on the use of PD-1 inhibitors in patients with advanced NMSC. Thirty-seven articles reported a total of 432 unique cases of advanced cSCC or BCC treated with PD1 inhibitors. The most common dosing schedules for pembrolizumab (2 mg/kg q3 weeks), nivolumab (3 mg/kg q2 weeks), or cemiplimab (3 mg/kg q2 weeks) were identified. A total of 10 different dosing schedules were identified. Outcomes of these patients ranged between progressive disease to achievement of a complete clinical response. All 3 PD1 inhibitors showed adequate safety profiles with few reported adverse events that were grade 3 or above. The use of cemiplimab, pembrolizumab, and nivolumab for advanced NMSC has shown promising results. However, the literature shows a wide range of dosing regimens for these therapies. Further studies are needed to guide clinicians toward the optimal dosage and the ideal anti-PD1 agent for their patients.

LPCAT1 Promotes Cutaneous Squamous Cell Carcinoma via EGFR-Mediated Protein Kinase B/p38MAPK Signaling Pathways

Cutaneous squamous cell carcinoma (cSCC) is the second most common form of skin cancer. LPCAT1, a lysophosphatidylcholine acyltransferase, takes a center stage in membrane lipid remodeling. LPCAT1 is elevated in several cancers and contributes to cancer development. However, its role and molecular mechanisms in cSCC remain to be elucidated. In this study, we found that LPCAT1 was upregulated in cSCC tissues and in cell lines. Invitro, loss-of-function and gain-of-function experiments demonstrated that LPCAT1 facilitated cSCC cell proliferation, protected cells against apoptosis, accelerated epithelial‒mesenchymal transition, and enhanced cell metastasis. Mechanistically, LPCAT1 regulated EGFR signaling. The oncogenic effect of LPCAT1 was mediated by EGFR/protein kinase B and EGFR/p38MAPK pathways in cSCC. Using the xenograft mouse model, we consolidated the results mentioned earlier. In conclusion, LPCAT1 contributed to cSCC progression through EGFR-mediated protein kinase B and p38MAPK signaling pathways. LPCAT1 may serve as a target for therapeutic intervention in cSCC.

Modified 5-aminolevulinic acid photodynamic therapy (M-PDT) inhibits cutaneous squamous cell carcinoma cell proliferation via targeting PP2A/PP5-mediated MAPK signaling pathway

We previously demonstrated that M-PDT is painless and effective in precancerous skin diseases treatment. However, whether M-PDT is effective in cSCC and the underlying inhibitory mechanism remains enigmatic. We aims to unveil the effect of M-PDT on cSCC cell proliferation and the regulatory effect of M-PDT on MAPK signaling. The proliferation and migration of cSCC cells were revealed by CCK8 assay, tumor sphere formation assay and scratch assay respectively. The expression of MAPKs was examined by western blot. The activity of PP2A and PP5 was regulated by inhibitor and recombinant adenoviruses. Here, we show that M-PDT inhibits cSCC cell proliferation by activating p-JNK, p-p38 and inhibiting p-Erk1/2, as well as activation of PP2A and inactivation of PP5. Furthermore, pharmacological inhibition of PP2A conferred resistance to M-PDT's suppression on p-Erk1/2 and attenuated inhibitory effects of M-PDT on cell proliferation whereas overexpression of wild-type PP2A showed the contrary results. Pharmacological inhibition of PP5 potentiated M-PDT's elevation on p-JNK and strengthened inhibitory effects of M-PDT on cell proliferation whereas overexpression of wild-type PP5 exhibited the contrary results. Our findings indicate that M-PDT inhibits cSCC cell proliferation via targeting PP2A/PP5-mediated MAPK signaling pathway.

Ceramide kinase-like protein promotes cutaneous squamous cell carcinoma.

Linked Article:Meyer et al. Br J Dermatol 2021; 185:147-152. Ceramide kinase-like protein (CERKL) was originally described in retinal tissue in the eye. It has been shown to protect cells from oxidative stress, which is a process that kills cells; and mutations (faults) in CERKL cause an inherited disease called retinitis pigmentosa that leads to loss of vision. We set out to investigate CERKL in normal skin and in a type of skin cancer called cutaneous squamous cell carcinoma (cSCC). CERKL levels were low in normal skin but significantly increased in cSCC and also in actinic keratoses (areas of sun-damaged skin that sometimes can develop into skin cancer). Because of their fast metabolism, cancer cells have high levels of oxidative stress. CERKL appears to enable these cSCC cells to survive, because when we inactivated CERKL, the cSCC cells died. In conclusion, these findings suggest that CERKL may be important in disease progression in cSCC. Since CERKL is not expressed in normal skin cells, further investigation of CERKL could lead to novel strategies for prevention and treatment of cSCC.

The CD200-CD200R Axis Promotes Squamous Cell Carcinoma Metastasis via Regulation of Cathepsin K.

The CD200-CD200R immunoregulatory signaling axis plays an etiologic role in the survival and spread of numerous cancers, primarily through suppression of antitumor immune surveillance. Our previous work outlined a prometastatic role for the CD200-CD200R axis in cutaneous squamous cell carcinoma (cSCC) that is independent of direct T-cell suppression but modulates the function of infiltrating myeloid cells. To identify effectors of the CD200-CD200R axis important for cSCC metastasis, we conducted RNA sequencing profiling of infiltrating CD11B+Cd200R+ cells isolated from CD200+ versus CD200-null cSCCs and identified the cysteine protease cathepsin K (Ctsk) to be highly upregulated in CD200+ cSCCs. CD11B+Cd200R+ cells expressed phenotypic markers associated with myeloid-derived suppressor cell-like cells and tumor-associated macrophages and were the primary source of Ctsk expression in cSCC. A Cd200R+ myeloid cell-cSCC coculture system showed that induction of Ctsk was dependent on engagement of the CD200-CD200R axis, indicating that Ctsk is a target gene of this pathway in the cSCC tumor microenvironment. Inhibition of Ctsk, but not matrix metalloproteinases, significantly blocked cSCC cell migration in vitro. Finally, targeted CD200 disruption in tumor cells and Ctsk pharmacologic inhibition significantly reduced cSCC metastasis in vivo. Collectively, these findings support the conclusion that CD200 stimulates cSCC invasion and metastasis via induction of Ctsk in CD200R+ infiltrating myeloid cells. SIGNIFICANCE: These findings highlight the relationship between CD200-CD200R and cathepsin K in cutaneous squamous cell carcinoma metastasis and suggest that either of these components may serve as a viable therapeutic target in this disease.

Hsa_circ_0008234 facilitates proliferation of cutaneous squamous cell carcinoma through targeting miR-127-5p to regulate ADCY7.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin malignant tumor with 25-50% of 5-year survival. There exist urgent needs for the identification of novel biomarkers for the diagnostic and therapeutic strategies of cSCC. The differentially expressed circRNAs in cSCC tissues and non-lesional skin tissues were obtained through analyzing the circular RNAs (circRNAs) microarray dataset GSE74758. The expression pattern of the indicated circRNAs in cSCC tissues was confirmed by qRT-PCR. FISH analysis was used to detect the location of hsa_circ_0008234 in cells. RIP experiment was used to detect the interaction between hsa_circ_0008234 and miR-127-5p. CCK-8 analysis and colony formation assay were used to detect the proliferation of cSCC cells. qRT-PCR and western blot were adopted to detect the expression of ACDY7. Three differential expressed circRNAs were obtained from the microarray data (GSE74758), and hsa_circ_0008234 was confirmed to be highly expressed in cSCC tissues by qRT-PCR. Hsa_circ_0008234 was mainly located in cytoplasm and stable in cSCC cells. RIP experiment revealed that hsa_circ_0008234 directly interacts with miR-127-5p in cSCC cells. Hsa_circ_0008234 increased the cell viability and colony formation of cSCC cells through acting as the sponge of miR-127-5p. MiR-127-5p inhibited the expression of ADCY7 in cSCC cells through binding the 3'UTR of ADCY7. Hsa_circ_0008234 was positively associated with ADCY7 expression in cSCC tissues. Hsa_circ_0008234 facilitates the proliferation of cSCC through targeting miR-127-5p to regulate ADCY7 expression and has the potential to be a novel therapeutic target for cSCC.

Topical arginase inhibition decreases growth of cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinomas (cSCC) are among the most commonly diagnosed malignancies, causing significant morbidity and mortality. Tumor-associated macrophage (TAM) expression of arginase is implicated in tumor progression, and therapeutic use of arginase inhibitors has been studied in various cancers. However, investigating potential cSCC immunotherapies including arginase inhibition in pre-clinical models is hampered by the lack of appropriate tumor models in immunocompetent mice. PDV is a cSCC cell line derived from chemical carcinogenesis of mouse keratinocytes. PDVC57 cells were derived from a PDV tumor in C57BL/6 (B6) mice. Unlike PDV, PDVC57 tumors grow consistently in B6 mice, and have increased TAMs, decreased dendritic and T cell intra-tumor infiltration. Arginase inhibition in cSCC tumors using Nω-hydroxy-nor-arginine (nor-NOHA) reduced tumor growth in B6 mice but not immunodeficient Rag1-deficient mice. nor-NOHA administration increased dendritic and T cell tumor-infiltration and PD-1 expression. The combination of nor-NOHA and anti-PD-1 therapy with nivolumab enhanced anti-PD-1 therapeutic efficacy. This study demonstrates the therapeutic potential of transcutaneous arginase inhibition in cSCC. A competent immune microenvironment is required for tumor growth inhibition using this arginase inhibitor. Synergistic co-inhibition of tumor growth in these results, supports further examination of transcutaneous arginase inhibition as a therapeutic modality for cSCC.

Downregulation of ARMC8 promotes tumorigenesis through activating Wnt/β-catenin pathway and EMT in cutaneous squamous cell carcinomas

BackgroundAberrant expression of Armadillo repeat containing 8 (ARMC8) plays crucial roles in tumor growth and metastasis of various cancers. The specific role of ARMC8 in cutaneous squamous cell carcinoma (cSCC) is yet to be elucidated. ObjectiveThe present study aimed to investigate the molecular mechanisms of ARMC8 and epithelial-mesenchymal transition (EMT) in cSCC development and provide translational insights for future therapeutics. MethodscSCC tumor specimens were used to determine the ARMC8 by immunohistochemistry. Three cSCC cell lines including HSC-1, HSC-5 and A431 as well as BALB/C mouse tumor model was utilized to study the potential mechanisms in tumorigenesis. ResultsOur data identified ARMC8 as a direct downstream target of miR-664. We found that ARMC8 was remarkably low expression in cSCC patient specimens and cSCC cell lines. Knockdown of ARMC8 promotes tumorigenic behaviors such as increased cell proliferation, migration and invasion capacities in vitro and enhanced tumorigenicity in xenograft mouse model. Whereas ARMC8 over-expression inhibits tumorigenesis in cSCC. Together, it revealed ARMC8 functions as a tumor suppressor via restraining Wnt/β-catenin pathway and epithelial–mesenchymal transition in cSCC. ConclusionOur data verifies that aberrant expression of ARMC8 plays a vital role in carcinogenesis of cSCC. And overexpression of ARMC8 will facilitate future development of cSCC therapeutic interventions.

Complement factor I upregulates expression of matrix metalloproteinase-13 and -2 and promotes invasion of cutaneous squamous carcinoma cells.

The incidence of cutaneous squamous cell carcinoma (cSCC) is increasing globally. Here, we have studied the functional role of complement factor I (CFI) in the progression of cSCC. CFI was knocked down in cSCC cells, and RNA-seq analysis was performed. Significant downregulation of genes in IPA biofunction categories Proliferation of cells and Growth of malignant tumor, in Gene Ontology (GO) terms Metallopeptidase activity and Extracellular matrix component, as well as Reactome Degradation of extracellular matrix was detected after CFI knockdown. Further analysis of the latter three networks, revealed downregulation of several genes coding for invasion-associated matrix metalloproteinases (MMPs) after CFI knockdown. The downregulation of MMP-13 and MMP-2 was confirmed at mRNA, protein and tissue levels by qRT-qPCR, Western blot and immunohistochemistry, respectively. Knockdown of CFI decreased the invasion of cSCC cells through type I collagen. Overexpression of CFI in cSCC cells resulted in enhanced production of MMP-13 and MMP-2 and increased invasion through type I collagen and Matrigel, and in increased ERK1/2 activation and cell proliferation. Altogether, these findings identify a novel mechanism of action of CFI in upregulation of MMP-13 and MMP-2 expression and cSCC invasion. These results identify CFI as a prospective molecular marker for invasion and metastasis of cSCC.

Clinically relevant aberrant Filip1l DNA methylation detected in a murine model of cutaneous squamous cell carcinoma.

BackgroundCutaneous squamous cell carcinomas (cSCC) are among the most common and highly mutated human malignancies. Understanding the impact of DNA methylation in cSCC may provide avenues for new therapeutic strategies.MethodsWe used reduced-representation bisulfite sequencing for DNA methylation analysis of murine cSCC. Differential methylation was assessed at the CpG level using limma. Next, we compared with human cSCC Infinium HumanMethylation BeadArray data. Genes were considered to be of major relevance when they featured at least one significantly differentially methylated CpGs (RRBS) / probes (Infinium) with at least a 30% difference between tumour vs. control in both a murine gene and its human orthologue. The human EPIC Infinium data were used to distinguish two cSCC subtypes, stem-cell-like and keratinocyte-like tumours.FindingsWe found increased average methylation in mouse cSCC (by 12.8%, p = 0.0011) as well as in stem-cell like (by 3.1%, p=0.002), but not keratinocyte-like (0.2%, p = 0.98), human cSCC. Comparison of differentially methylated genes revealed striking similarities between human and mouse cSCC. Locus specific methylation changes in mouse cSCC often occurred in regions of potential regulatory function, including enhancers and promoters. A key differentially methylated region was located in a potential enhancer of the tumour suppressor gene Filip1l and its expression was reduced in mouse tumours. Moreover, the FILIP1L locus showed hypermethylation in human cSCC and lower expression in human cSCC cell lines.InterpretationDeregulation of DNA methylation is an important feature of murine and human cSCC that likely contributes to silencing of tumour suppressor genes, as shown for Filip1l.FundingBritish Skin Foundation, Cancer Research UK

050 Loss of retinoic acid receptor-related receptor alpha (Rorα) promotes the progression of UV-induced cSCC

Cutaneous squamous cell carcinoma (cSCC) is prevalent in the world, accounting a huge part in non-melanoma skin cancer. Most cSCCs are associated with a distinct pre-cancerous lesion, the actinic keratosis (AK). However, the progression trajectory from normal skinto AK and cSCC has not fully demonstrated yet. To identify genes involved in this progression trajectory and possible threptic targets for cSCC, here we constructed a UV-induced cSCC mouse model covering the progression from normal skinto AK to cSCC, which mimicked the solar UV radiation perfectly using the solar-like ratio of UVA and UVB, firstly. Then, transcriptome analysis and a series of bioinformatics analysesand cell experiments proved that Rorα is a key transcript factor during cSCC progression.Rorα could down-regulate the expressions of S100a9 and Sprr2f in cSCC cells, which can inhibit theproliferation and migration in cSCC cells, but not the normal keratinocyte. Finally, further animal experiment confirmed the inhibitory effect of cSCC growth by Rorα in vivo.Our findings showed that Rorα would serve as a potential novel target for cSCC, which will facilitate the treatment of cSCC in the future.

487 Topical MEK inhibition as precision targeted chemoprevention

Cutaneous squamous cell carcinoma (cuSCC) comprises at least 20% of all non-melanoma skin cancers. Effective chemoprevention for cuSCC is lacking and high-risk cuSCC causes significant surgical morbidity. Previously, we conducted RNA-seq analysis of human cuSCC, which identified ETS2, a transcription factor in the ERK pathway, as an upstream regulator of cuSCC development. Consistent with this, our computational drug repositioning screen predicted the FDA-approved MEK1, 2 inhibitor, selumetinib, as a therapeutic for cuSCC based on its ability to reverse transcriptional signatures associated with cuSCC development. As a regulator of ERK activity, we reasoned MEK would be a viable chemopreventative target. Although systemic MEK inhibition suppresses tumor formation, systemic MEKi administration causes significant adverse effects. Here, we report the development of a topically formulated, metabolically labile MEK inhibitor, NFX-179, designed to potently and selectively suppress the ERK pathway in the skin prior to rapid degradation in the circulation. In our UV-induced model of cuSCC, topical application of NFX-179 gel reduced the formation of new cuSCCs by an average of 60% at doses of 0.1% and greater at 28 days. No systemic or skin toxicities were observed. Furthermore, we conducted a split-mouse randomized controlled study in 5 mice. NFX-179 0.5% gel was applied to half of the back and vehicle was applied to the other half of each of the UV-irradiated mice. Near complete suppression of cuSCC was observed only in the drug-treated area, demonstrating the targeted and local effect of the intervention. NFX-179 inhibits the growth of human cSCC cell lines in a dose-dependent manner and topical NFX-179 application penetrates human skin and inhibits ERK signaling in human cuSCC explants. Our data provide compelling evidence that topical application of NFX-179 is a safe, effective strategy for SCC chemoprevention in high risk individuals.