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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.

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

Background: Cutaneous squamous cell carcinoma (cSCC) possesses metastatic potential and causes significant mortality. However, clinical assessment of metastasis risk is challenging. We approached this challenge by harnessing artificial intelligence (AI) algorithm to identify metastatic primary cSCCs. Methods: Residual neural network-architectures were trained and fine-tuned to identify metastatic cSCCs on tumor tiles extracted from clinician annotated, hematoxylin and eosin-stained whole slide images representing metastatic (n=45) and non-metastatic (n=59) primary cSCCs. Findings: Several models were trained and tested with best area under the receiver operating characteristic curve (AUROC) of 0·689 when all primary tumors (n=104) regardless of time to metastasis were included. Metastatic primary tumors were further divided into two groups that metastasize rapidly (≤180d) (n=22) or slowly (>180d) after primary tumor detection (n=23). The algorithm was able to predict whether primary tumor was non-metastatic or classified as rapidly metastatic with slide-level AUROC of 0·747. This prediction was superior to blind assessment by pathologist and did not appear to rely on classical clinicopathological features or staging systems. Furthermore, risk factor (RF) model taking into account prediction by AI, Clark’s level and tumor diameter provided higher AUROC (0·917) than other tested RF models and predicted high 5-year disease specific survival (DSS) for patients with cSCC with 0 or 1 RFs (100% and 95·7%) and poor 5-year DSS for patients with cSCCs with 2 or 3 RFs (41·7% and 40·0%). Interpretation: These results provide evidence, that AI recognizes unknown features associated with metastasis and provides added value to challenging clinical risk assessment of metastasis and prognosis of primary cSCC. Funding: Sigrid Juselius Foundation, Finnish Cancer Research Foundation, Jane and Aatos Erkko Foundation, Cancer Foundation of the Southwest Finland, Finnish Dermatological Society, The Maud Kuistila Memorial Foundation, and Turku University Hospital. JSK is a doctoral candidate in the Doctoral Program for Clinical Investigation of the University of Turku. Declaration of Interest: None to declare. Ethical Approval: The study was approved by The Ethics Committee of the Hospital District of Southwest Finland (187/2006) and Auria Biobank’s Scientific Steering Committee (AB15-9721).

Simple Summary The incidence of the most common metastatic skin malignancy, cutaneous squamous cell carcinoma (cSCC), is growing worldwide, and the prognosis of the metastatic disease is poor. Presently, there are no biomarkers or therapeutic targets for high-risk cSCCs. Recent studies have demonstrated the essential role of autocrine complement synthesis in the progression of cSCC. Here, we have evaluated the role of complement Factor D (FD), the rate-limiting enzyme of the alternative complement pathway, in cSCC development. The results identify FD as a novel biomarker and putative therapeutic target for cSCC and propose the small-molecule FD inhibitor Danicopan as a highly specific drug candidate in the therapy of advanced cSCC. It is expected that the discovery of complement-associated molecular markers for cSCC progression would improve diagnosis, classification, prognostication, and targeted therapy of cSCC and its precursors in the future. 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-gamma and interleukin-1 beta. 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.