Complement system in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) consists 20% of keratinocytederived non-melanoma skin cancers (NMSC), the incidence of which is increasing globally. cSCC is the most common metastatic skin cancer and it causes approximately 20% of skin cancer-related deaths. At present, there are no molecular markers for predicting which cSCC lesions are aggressive or metastasize rapidly. UV radiation is the most important risk factor for cSCC. During the development of cSCC, normal epidermal keratinocytes are transformed and form actinic keratosis (AK), which progresses to cSCC in situ (cSCCIS, Bowen’s disease) and finally to invasive and metastatic cSCC. Inflammatory factors and cells are a part of cancer microenvironment and cSCC can develop in the chronically irritated skin or in the context of chronic inflammation. The complement system is a central part of innate immunity and it regulates normal immunological and inflammatory processes. In this study, the role of complement system components and inhibitors were studied in the progression of cSCC in culture and in vivo. Elevated expression of complement factor H (CFH), complement factor I (CFI), complement component C3 and complement factor B (CFB) was noted in cSCC cells in culture. The analysis with immunohistochemistry (IHC) revealed that the expression of CFH, CFI, C3 and CFB was specifically noted in tumor cells in vivo. The staining intensity of CFH, CFI, C3 and CFB was also stronger in invasive cSCC than in AK or cSCCIS samples. The knockdown of CFH, CFI and CFB with specific siRNAs decreased cSCC cell viability and migration, whereas the knockdown of C3 reduced only cSCC cell migration. Moreover, the knockdown of CFI, C3 and CFB inhibited growth of cSCC xenograft tumors established in SCID mice in vivo. In these tumors, CFI, C3 and CFB knockdown decreased the number of proliferating cells. Moreover, the knockdown of CFI increased local inflammation and complement activation. This study provides evidence for the roles of CFH, CFI, C3 and CFB in the tumor progression indicating these as molecular biomarkers and putative therapeutic targets of cSCC.Komplementtijärjestelmä ihon okasolusyövässä Keratinosyyttiperäiset ei-melanoottiset syövät lisääntyvät maailmanlaajuisesti ja näistä syövistä 20% on ihon okasolusyöpää. Ihon okasolusyöpä on yleisimmin metastasoiva ihosyöpä ja se aiheuttaa 20% ihosyöpäkuolemista. Tällä hetkellä ihon okasolusyövän aggressiivisuutta tai nopeaa metastasointia ennustavia merkkitekijöitä ei tunneta. UV-säteily on tärkein riskitekijä ihon okasolusyövän kehittymiselle. Normaalit ihon keratinosyytit voivat muuttua ja muodostaa aktiinisia keratooseja, mitkä etenevät in situ muotoiseksi okasolusyöväksi (Bowenin tauti) ja lopulta ihon okasolusyöväksi. Tulehdussolut ja –tekijät kuuluvat syöpien mikroympäristöön. Myös ihon okasolusyöpä voi kehittyä kroonisesti ärsyyntyneelle tai tulehtuneelle ihoalueelle. Komplementtijärjestelmä on tärkeä osa ihmisen synnynnäistä immuniteettia ja se säätelee tulehduksellisia tapahtumasarjoja. Tässä työssä tutkittiin komplementtijärjestelmän komponenttien ja inhibiittoreiden osuutta ihon okasolusyövän kehittymisessä soluviljelmässä ja in vivo. Komplementin tekijä H:ta (CFH), komplementin tekijä I:tä (CFI), komplementin komponenttia C3:a ja komplementin tekjä B:tä (CFB) havaittiin kohonneina pitoisuuksina soluviljelmässä ihon okasolusyöpäsoluissa. Immunohistokemiallisessa analyysissä paljastui, että erityisesti kasvainsolut ilmensivät CFH:ta, CFI:tä, C3:a ja CFB:tä in vivo ja värjäysintensiteetti oli vahvempi okasolusyöpäsoluissa kuin aktiinisessa keratoosissa tai Bowenin taudissa. Kun spesifistä siRNA:ta käytettiin hiljentämään CFH:n, CFI:n ja CFB:n ilmaantuvuus, solujen elinkykyisyys ja liikkuminen väheni. C3 geenin hiljentäminen vähensi solujen liikkumista. Lisäksi CFI-, C3- ja CFB-geenin hiljentäminen vähensi okasolusyövän kasvua in vivo SCID-hiirissä kasvatetuissa ksenograftikasvaimissa. CFI-, C3- ja CFB-geenin hiljentäminen vähensi solujen jakautumista. CFI-geenin hiljentäminen lisäsi lisäksi paikallista tulehdusta ja komplementin aktivaatiota näissä kasvaimissa. Tässä tutkimuksessa osoitetaan CFH:n, CFI:n, C3:n ja CFB:n rooli okasolusyöpäkasvaimen kehittymisessä. CFH, CFI, C3 tai CFB voisivat myös toimia merkkitekijöinä ja hoitokohteina ihon okasolusyövälle.Siirretty Doriast

Matrix metalloproteinases in keratinocyte carcinomas

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-beta 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

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.Peer reviewe

The role of p53 in progression of cutaneous squamous cell carcinoma

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. </p

C1r Upregulates Production of Matrix Metalloproteinase-13 and Promotes Invasion of Cutaneous 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 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.Peer reviewe

Tumour-cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma

Summary Background Incidence of epidermal keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is increasing worldwide. Objectives To study the role of complement classical pathway components C1q, C1r and C1s in the progression of cSCC. Methods The mRNA levels of C1Q subunits, C1R and C1S in cSCC cell lines, normal human epidermal keratinocytes (NHEKs), cSCC tumors in vivo and normal skin were analyzed with quantitative RT-PCR. The production of C1r and C1s was determined with Western blotting. The expression of C1r and C1s in tissue samples in vivo was analyzed 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 to normal human epidermal keratinocytes. The mRNA levels of C1R and C1S were markedly elevated in cSCC tumors in vivo compared to normal skin. Abundant expression of C1r and C1s by tumor 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 ERK1/2 and Akt, promoted apoptosis of cSCC cells and significantly suppressed growth and vascularization of human cSCC xenograft tumors in vivo. Conclusions These results provide evidence for the role of tumor cell-derived C1r and C1s in the progression of cSCC and identify them as biomarkers and putative therapeutic targets in cSCC. This article is protected by copyright. All rights reserved.Peer reviewe

Risk Factors and Prognosis for Metastatic Cutaneous Squamous Cell Carcinoma: A Cohort Study

Cutaneous squamous cell carcinoma (cSCC) has me-tastatic potential. The aims of this study were to identify the risk factors for metastasis of primary cSCC and for poor prognosis in metastatic cSCC. Retrospective primary tumour cohorts of metastatic cSCCs (n = 85) and non-metastatic cSCCs (n = 218) were analysed. The mean annual rate of metastasis for primary cSCCs was 2.28%. In 49.4% of patients with metastatic cSCC, metastasis was detected within 6 months of diagnosis of the primary cSCC. There was no prior history of cSCC in 84.7% of metastatic cSCCs. Risk factors for metastasis included Clark's level 5, tumour diameter 20-29.9 mm, age at diagnosis = 3 nodal metastases and extranodal extension of metastasis. These results characterise new risk factors for metastatic cSCC

Complement Factor D Is a Novel Biomarker and Putative Therapeutic Target in Cutaneous Squamous Cell Carcinoma

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

Super Enhancer-Regulated LINC00094 (SERLOC) Upregulates the Expression of MMP-1 and MMP-13 and Promotes Invasion of Cutaneous Squamous Cell Carcinoma

Simple Summary Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, and its incidence is increasing worldwide. The prognosis of the metastatic disease is poor, and there are no established biomarkers for the assessment of metastasis risk or specific therapeutic targets for advanced or metastatic cSCCs. The role of long non-coding RNAs (lncRNAs) in the progression of cSCC has recently been emphasized. Super enhancers (SE) have been shown to play a role in tumorigenesis and regulate the expression of specific lncRNAs. In this study, we evaluated the role of SE-regulated BRD3OS (lncRNA LINC00094) in the progression of cSCC. Based on the results, we named this lncRNA SERLOC. The results identify SERLOC as a biomarker for invasion and metastasis of cSCC and as a putative therapeutic target in advanced cSCC. 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.</p