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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. [ABSTRACT FROM AUTHOR]

Summary: Background: Cutaneous squamous cell carcinoma (cSCC) is a common cancer that invades the dermis through the basement membrane. The role of the basement membrane in poorly differentiated cSCC is not well understood. Objectives: To study the effect that loss of the laminin subunit alpha‐3 (α3) chain from the tumour microenvironment has on tumour invasion and inflammatory cell recruitment. Methods: We examined the role of the basement membrane proteins laminin subunits α3, β3 and γ2 in SCC invasion and inflammatory cell recruitment using immunohistochemistry, short hairpin RNA knockdown, RNA‐Seq, mouse xenograft models and patient tumour samples. Results: Analysis of SCC tumours and cell lines using antibodies specific to laminin chains α3, β3 and γ2 identified a link between poorly differentiated SCC and reduced expression of laminin α3 but not the other laminin subunits investigated. Knockdown of laminin α3 increased tumour invasion both in vitro and in vivo. Western blot and immunohistochemical staining identified increased phosphorylated myosin light chain with loss of laminin α3. Inhibition of ROCK (rho‐associated protein kinase) but not Rac1 significantly reduced the invasive potential of laminin α3 knockdown cells. Knockdown of laminin subunits α3 and γ2 increased monocyte recruitment to the tumour microenvironment. However, only the loss of laminin α3 correlated with increased tumour‐associated macrophages both in xenografted tumours and in patient tumour samples. Conclusions: These data provide evidence that loss of the laminin α3 chain in cSCC has an effect on both the epithelial and immune components of cSCC, resulting in an aggressive tumour microenvironment. What is already known about this topic?The tumour microenvironment, including the extracellular matrix, plays an important role in modulating tumour behaviour and inflammatory infiltration.Data on laminin expression in poorly differentiated squamous cell carcinomas (cSCCs) are limited. What does this study add?Poorly differentiated cSCCs have significantly reduced laminin α3 expression.Loss of laminin α3 increases tumour invasion, amoeboid migration and ROCK (rho‐associated protein kinase) signalling.Expression of laminin α3 in patient tumours is inversely correlated with macrophage recruitment.Our results provide novel evidence for the role of laminin α3 in the recruitment of tumour‐associated macrophages in SCC. What is the translational message?There is a potential prognostic value of laminin α3 expression in predicting cSCC metastasis.There is potential for targeting ROCK activity in cSCCs lacking laminin α3 to block tumour invasion.The role played by the laminin α3 chain in macrophage polarization into tumour‐associated macrophages presents additional targets for immunotherapy or immunoregulation in cSCC. Linked Comment: Leb‐Reichl et al. Br J Dermatol 2021; 184:799. [ABSTRACT FROM AUTHOR]

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]