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Summary: Background: The incidence of epidermal keratinocyte‐derived cutaneous squamous cell carcinoma (cSCC) is increasing worldwide. Objectives: To study the role of the complement classical pathway components C1q, C1r and C1s in the progression of cSCC. Methods: The mRNA levels of C1Q subunits and C1R and C1S in cSCC cell lines, normal human epidermal keratinocytes, cSCC tumours in vivo and normal skin were analysed with quantitative real‐time polymerase chain reaction. The production of C1r and C1s was determined with Western blotting. The expression of C1r and C1s in tissue samples in vivo was analysed with immunohistochemistry and further investigated in human cSCC xenografts by knocking down C1r and C1s. Results: Significantly elevated C1R and C1S mRNA levels and production of C1r and C1s were detected in cSCC cells, compared with normal human epidermal keratinocytes. The mRNA levels of C1R and C1S were markedly elevated in cSCC tumours in vivo compared with normal skin. Abundant expression of C1r and C1s by tumour cells was detected in invasive sporadic cSCCs and recessive dystrophic epidermolysis bullosa‐associated cSCCs, whereas the expression of C1r and C1s was lower in cSCC in situ, actinic keratosis and normal skin. Knockdown of C1r and C1s expression in cSCC cells inhibited activation of extracellular signal‐related kinase 1/2 and Akt, promoted apoptosis of cSCC cells and significantly suppressed growth and vascularization of human cSCC xenograft tumours in vivo. Conclusions: These results provide evidence for the role of tumour‐cell‐derived C1r and C1s in the progression of cSCC and identify them as biomarkers and putative therapeutic targets in cSCC. What's already known about this topic? The incidences of actinic keratosis, cutaneous squamous cell carcinoma (cSCC) in situ and invasive cSCC are increasing globally.Few specific biomarkers for progression of cSCC have been identified, and no biological markers are in clinical use to predict the aggressiveness of actinic keratosis, cSCC in situ and invasive cSCC. What does this study add? Our results provide novel evidence for the role of complement classical pathway components C1r and C1s in the progression of cSCC. What is the translational message? Our results identify complement classical pathway components C1r and C1s as biomarkers and putative therapeutic targets in cSCC. Linked Comment: Kopecki. Br J Dermatol 2020; 182:530–531. Plain language summary available online [ABSTRACT FROM AUTHOR]

Actinic keratosis and cutaneous squamous cell carcinoma in situ are precancerous forms of cutaneous squamous cell carcinoma. In this single-centre retrospective study, patients with histopathologically confirmed actinic keratosis (n = 121) or cutaneous squamous cell carcinoma in situ (n = 99) as their initial keratinocyte-derived lesion were compared and evaluated with regard to development of cutaneous squamous cell carcinoma during a 5-year observation period. Patients with severely dysplastic actinic keratosis or cutaneous squamous cell carcinoma in situ as their initial lesion developed cutaneous squamous cell carcinoma more rapidly than patients with actinic keratosis with mild or moderate dysplasia. With either actinic keratosis or cutaneous squamous cell carcinoma in situ as an initial lesion, advanced age, male sex, comorbidity with basal cell carcinoma, and immunosuppressive medication were associated with elevated risk of cutaneous squamous cell carcinoma development. Regarding solely patient with actinic keratosis as their initial lesion male sex, advanced age, immunosuppressive medication, location of the initial lesion, and degree of dysplasia were associated with the risk of cutaneous squamous cell carcinoma. Among patients with cutaneous squamous cell carcinoma in situ as their initial lesion, only aspirin usage was associated with increased risk of cutaneous squamous cell carcinoma. This study indicates that, among the vast and increasing population of patients with cutaneous squamous cell carcinoma precursors, male patients with immunosuppressive medication who develop basal cell carcinoma should be regarded as at heightened risk of cutaneous squamous cell carcinoma development and warrant closer surveillance.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer and the metastatic disease is associated with poor prognosis. Cancer-associated fibroblasts (CAFs) promote progression of cancer, but their role in cSCC is largely unknown. We examined the potential of CAF markers in the assessment of metastasis risk and prognosis of primary cSCC. We utilized multiplexed fluorescence immunohistochemistry for profiling CAF landscape in metastatic and non-metastatic primary human cSCCs, in metastases, and in premalignant epidermal lesions. Quantitative high-resolution image analysis was performed with two separate panels of antibodies for CAF markers and results were correlated with clinical and histopathological parameters including disease-specific mortality. Increased stromal expression of fibroblast activation protein (FAP), α-smooth muscle actin, and secreted protein acidic and rich in cysteine (SPARC) were associated with progression to invasive cSCC. Elevation of FAP and platelet-derived growth factor receptor-β (PDGFRβ) expression was associated with metastasis risk of primary cSCCs. High expression of PDGFRβ and periostin correlated with poor prognosis. Multimarker combination defined CAF subset, PDGFRα-/PDGFRβ+/FAP+, was associated with invasion and metastasis, and independently predicted poor disease-specific survival. These results identify high PDGFRβ expression alone and multimarker combination PDGFRα-/PDGFRβ+/FAP+ by CAFs as potential biomarkers for risk of metastasis and poor prognosis., (© 2024 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: Cutaneous squamous cell carcinoma (cSCC) is one of the most common and fastest increasing forms of cancer worldwide with metastatic potential. Long noncoding RNAs (lncRNAs) are a group of RNA molecules with essential regulatory functions in both physiological and pathological processes., Objectives: To investigate the function and mode of action of lncRNA plasmacytoma variant translocation 1 (PVT1) in cSCC., Methods: Quantitative reverse transcriptase polymerase chain reaction and single-molecule in situ hybridization were used to quantify the expression level of PVT1 in normal skin, premalignant skin lesions, actinic keratosis (AK) and primary and metastatic cSCCs. The function of PVT1 in cSCC was investigated both in vivo (tumour xenografts) and in vitro (competitive cell growth assay, 5-ethynyl-2'-deoxyuridine incorporation assay, colony formation assay and tumour spheroid formation assay) upon CRISPR-Cas9-mediated knockout of the entire PVT1 locus, the knockout of exon 2 of PVT1, and locked nucleic acid (LNA) gapmer-mediated PVT1 knockdown. RNA sequencing analysis was conducted to identify genes and processes regulated by PVT1., Results: We identified PVT1 as a lncRNA upregulated in cSCC in situ and cSCC, associated with the malignant phenotype of cSCC. We showed that the expression of PVT1 in cSCC was regulated by MYC. Both CRISPR-Cas9 deletion of the entire PVT1 locus and LNA gapmer-mediated knockdown of PVT1 transcript impaired the malignant behaviour of cSCC cells, suggesting that PVT1 is an oncogenic transcript in cSCC. Furthermore, knockout of PVT1 exon 2 inhibited cSCC tumour growth both in vivo and in vitro, demonstrating that exon 2 is a critical element for the oncogenic role of PVT1. Mechanistically, we showed that PVT1 was localized in the cell nucleus and its deletion resulted in cellular senescence, increased cyclin-dependent kinase inhibitor 1 (p21/CDKN1A) expression and cell cycle arrest., Conclusions: Our study revealed a previously unrecognized role for exon 2 of PVT1 in its oncogenic role and that PVT1 suppresses cellular senescence in cSCC. PVT1 may be a potential biomarker and therapeutic target in cSCC., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Association of Dermatologists.)

Long non-coding RNAs (lncRNAs) have emerged as important regulators of cancer progression. Super enhancers (SE) play a role in tumorigenesis and regulate the expression of specific lncRNAs. We examined the role of BRD3OS , also named LINC00094 , in cutaneous squamous cell carcinoma (cSCC). Elevated BRD3OS ( LINC00094 ) expression was detected in cSCC cells, and expression was downregulated by SE inhibitors THZ1 and JQ1 and via the MEK1/ERK1/2 pathway. Increased expression of BRD3OS ( LINC00094 ) was noted in tumor cells in cSCCs and their metastases compared to normal skin, actinic keratoses, and cSCCs in situ. Higher BRD3OS ( LINC00094 ) expression was noted in metastatic cSCCs than in non-metastatic cSCCs. RNA-seq analysis after BRD3OS ( LINC00094 ) knockdown revealed significantly regulated GO terms Cell-matrix adhesion , Basement membrane , Metalloendopeptidase activity , and KEGG pathway Extracellular matrix-receptor interaction. Among the top-regulated genes were MMP1 , MMP10 , and MMP13 . Knockdown of BRD3OS ( LINC00094 ) resulted in decreased production of MMP-1 and MMP-13 by cSCC cells, suppressed invasion of cSCC cells through collagen I, and growth of human cSCC xenografts in vivo. Based on these observations, BRD3OS ( LINC00094 ) was named SERLOC (super enhancer and ERK1/2-Regulated Long Intergenic non-protein coding transcript Overexpressed in Carcinomas). These results reveal the role of SERLOC in cSCC invasion and identify it as a potential therapeutic target in advanced cSCC.

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 in vivo. 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 in vivo in xenografts. These results provide evidence for the role of C1r in promoting the invasion of cSCC cells by increasing MMP production., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Most cases of keratinocyte cancer can be treated effectively with surgery. However, survival is reduced in patients with advanced disease. This retrospective cohort study evaluated overall survival of patients with invasive keratinocyte cancers, and high-risk features for progression of the disease and mortality in Finnish patients in a real-world setting. A total of 43,143 patients with keratinocyte cancer types of basal cell carcinoma and 10,380 with cutaneous squamous cell carcinoma were identified nationwide. More detailed patient records were available for a subset of patients (basal cell carcinoma n = 5,020 and cutaneous squamous cell carcinoma n = 1,482) from a regional database. Fifty percent of patients with advanced cutaneous squamous cell carcinoma died approximately 4.5 years after diagnosis. Multivariable models suggested that risk factors for keratinocyte cancer progression were male sex, presence of comorbidities, immunosuppression, and pre-cancerous lesions, while risk factors for disease-specific mortality were advanced disease stage with immunosuppression, other malignancies, and consecutive surgical excisions. These results suggest that identifying patient and tumour factors associated with poor disease outcome could be important when determining appropriate treatment and follow-up; however, further studies are necessary.

Cutaneous squamous cell carcinoma (cSCC) is the most prevalent metastatic skin cancer. Previous studies have demonstrated the autocrine role of complement components in cSCC progression. We have investigated factor D (FD), the key enzyme of the alternative complement pathway, in the development of cSCC. RT-qPCR analysis of cSCC cell lines and normal human epidermal keratinocytes (NHEKs) demonstrated significant up-regulation of FD mRNA in cSCC cells compared to NHEKs. Western blot analysis also showed more abundant FD production by cSCC cell lines. Significantly higher FD mRNA levels were noted in cSCC tumors than in normal skin. Strong tumor cell-associated FD immunolabeling was detected in the invasive margin of human cSCC xenografts. More intense tumor cell-specific immunostaining for FD was seen in the tumor edge in primary and metastatic cSCCs, in metastases, and in recessive dystrophic epidermolysis bullosa-associated cSCCs, compared with cSCC in situ, actinic keratosis and normal skin. FD production by cSCC cells was dependent on p38 mitogen-activated protein kinase activity, and it was induced by interferon-γ and interleukin-1β. Blocking FD activity by Danicopan inhibited activation of extracellular signal-regulated kinase 1/2 and attenuated proliferation of cSCC cells. These results identify FD as a novel putative biomarker and therapeutic target for cSCC progression.

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., (© 2021 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

Skin cancers are the most common types of cancer worldwide, and their incidence is increasing. Melanoma, basal cell carcinoma (BCC), and cutaneous squamous cell carcinoma (cSCC) are the three major types of skin cancer. Melanoma originates from melanocytes, whereas BCC and cSCC originate from epidermal keratinocytes and are therefore called keratinocyte carcinomas. Chronic exposure to ultraviolet radiation (UVR) is a common risk factor for skin cancers, but they differ with respect to oncogenic mutational profiles and alterations in cellular signaling pathways. cSCC is the most common metastatic skin cancer, and it is associated with poor prognosis in the advanced stage. An important early event in cSCC development is mutation of the TP53 gene and inactivation of the tumor suppressor function of the tumor protein 53 gene (TP53) in epidermal keratinocytes, which then leads to accumulation of additional oncogenic mutations. Additional genomic and proteomic alterations are required for the progression of premalignant lesion, actinic keratosis, to invasive and metastatic cSCC. Recently, the role of p53 in the invasion of cSCC has also been elucidated. In this review, the role of p53 in the progression of cSCC and as potential new therapeutic target for cSCC will be discussed.

Hypothesis: Bioactive glass (BG) S53P4 reduces the viability of epidermal keratinocyte-derived immortalized cell line, HaCaT in sufficient concentrations in vitro., Background: Although used in mastoid obliteration surgery, there is no data available on whether BG S53P4 granules have an inhibitory or excitatory effect on keratinocytes, found in normal skin and ear cholesteatoma in vivo., Methods: HaCaT cell cultures were incubated with a direct BG S53P4 granule contact. Microscopic evaluation of the cultures was performed and interleukin-6 (IL-6) and -8 (IL-8) concentrations were measured from the medium samples. In addition, BG granules were incubated in two cell culture media for 6 days and the pure media were used in confluent HaCaT cultures preceding cell viability assay. Finally, a scratch assay test was performed to reveal the possible BG effect on HaCaT cultures., Results: Eight to ten cell thick layers of dead HaCaT cells were noticed after a 2-day BG granule contact. With a BG concentration of 2.5%, IL-6 and IL-8 concentrations were smaller compared with the control group without BG after 2 days' incubation. Overall, HaCaT cell viability decreased when BG was incubated in keratinocyte growth medium, but did not change in Dulbecco's modified Eagle's medium. In a scratch assay test, cell regrowth in the scratch area was notable in cultures without BG., Conclusions: BG S53P4 seems to have an inhibitory effect on HaCaT cell growth. Although further studies are needed, this observation seems advantageous for cholesteatoma treatment., Competing Interests: The authors disclose no conflicts of interest., (Copyright © 2021, Otology & Neurotology, Inc.)

The incidence of cutaneous keratinocyte-derived cancers is increasing globally. Basal cell carcinoma (BCC) is the most common malignancy worldwide, and cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. BCC can be classified into subtypes based on the histology, and these subtypes are classified further into low- and high-risk tumors. There is an increasing need to identify new therapeutic strategies for the treatment of unresectable and metastatic cSCC, and for aggressive BCC variants such as infiltrating, basosquamous or morpheaform BCCs. The most important risk factor for BCC and cSCC is solar UV radiation, which causes genetic and epigenetic alterations in keratinocytes. Similar gene mutations are noted already in sun-exposed normal skin emphasizing the role of the alterations in the tumor microenvironment in the progression of cSCC. Early events in cSCC progression are alterations in the composition of basement membrane and dermal extracellular matrix induced by influx of microbes, inflammatory cells and activated stromal fibroblasts. Activated fibroblasts promote inflammation and produce growth factors and proteolytic enzymes, including matrix metalloproteinases (MMPs). Transforming growth factor-β produced by tumor cells and fibroblasts induces the expression of MMPs by cSCC cells and promotes their invasion. Fibroblast-derived keratinocyte growth factor suppresses the malignant phenotype of cSCC cells by inhibiting the expression of several MMPs. These findings emphasize the importance of interplay of tumor and stromal cells in the progression of cSCC and BCC and suggest tumor microenvironment as a therapeutic target in cSCC and aggressive subtypes of BCC., (© 2020 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. The molecular basis of cSCC progression to invasive and metastatic disease is still incompletely understood. Here, we show that fibroblasts and transforming growth factor-β (TGF-β) signaling promote laminin-332 synthesis in cancer cells in an activated H-Ras-dependent manner, which in turn promotes cancer cell invasion. Immunohistochemical analysis of sporadic UV-induced invasive human cSCCs (n = 208) revealed prominent cSCC cell specific immunostaining for laminin-332 γ2 chain, located in the majority of cases (90%, n = 173) in the invasive edge of the tumors. To mimic the progression of cSCC we established 3D spheroid cocultures using primary skin fibroblasts and HaCaT/ras-HaCaT human keratinocytes. Our results indicate that in 3D spheroids, unlike in monolayer cultures, TGF-β upregulates laminin-332 production, but only in cells that harbour oncogenic H-Ras. Accumulation of laminin-332 was prevented by both H-Ras knock down and inhibition of TGF-β signaling by SB431542 or RAdKD-ALK5 kinase-defective adenovirus. Furthermore, fibroblasts accelerated the invasion of ras-HaCaT cells through collagen I gels in a Ras/TGF-β signaling dependent manner. In conclusion, we demonstrate the presence of laminin-332 in the invasive front of cSCC tumors and report a new Ras/TGF-β-dependent mechanism that promotes laminin-332 accumulation and cancer cell invasion., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Epidermal keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer with high mortality rates in the advanced stage. Chronic inflammation is a recognized risk factor for cSCC progression and the complement system, as a part of innate immunity, belongs to the microenvironment of tumors. The complement system is a double-edged sword in cancer, since complement activation is involved in anti-tumor cytotoxicity and immune responses, but it also promotes cancer progression directly and indirectly. Recently, the role of several complement components and inhibitors in the regulation of progression of cSCC has been shown. In this review, we will discuss the role of complement system components and inhibitors as biomarkers and potential new targets for therapeutic intervention in cSCC.