The landscape of long non-coding RNA during cSCC progression.

Background: Cutaneous squamous cell carcinoma (cSCC) is a common cancer with high morbidity and poor prognosis for metastatic disease. Disease may progress from pre-malignant Actinic Keratosis (AK) to invasive and metastatic cSCC but perhaps is best characterised as a disease continuum progressing from a differentiated to a progenitor like state. The critical molecular mediators of this process remain poorly defined. Long non-coding RNAs (lncRNAs), a relatively unexplored class of RNA molecules over 200 nucleotides long, are likely to have important functional roles in cSCC.

Objectives: Here we wished to provide a comprehensive landscape of lncRNA expression during the cSCC continuum and identify potentially functional lncRNA drivers of disease progression.

Methods: We interrogated bulk RNA sequencing data from 110 patient samples, encompassing normal sun-exposed skin (n=26), Actinic Keratosis (n=14), primary cSCC (n=66), and metastases (n=4)1 to identify changes in lncRNA expression during disease progression. We developed a bioinformatics pipeline to infer lncRNA function based on co-expression patterns and generated a lncRNA signature score which we validated in head and neck squamous cell carcinoma (HNSC), and pancreatic adenocarcinoma (PAAD). We performed bulk RNA sequencing on 15 patient-derived cell lines and integrated this data to identify tumour cell specific lncRNAs and validated our findings in multiple other cSCC gene expression cohorts. Using in-vitro knockdown approaches we investigated the functional role of LINC00941.

Results: We characterised the landscape of lncRNAs during cSCC disease progression and revealed that lncRNA expression alone is sufficient to identify disease states and progression along the disease continuum. Correlation analysis revealed potentially functionally relevant lncRNAs and the processes that they may regulate. We developed a 267 lncRNA signature that correlates with a progenitor like state and predicts poor prognosis in HNSC and PAAD. Bulk RNA sequencing of patient derived cell lines (PDCLs) revealed tumour cell specific lncRNAs and knockdown of LINC00941 indicated that this is required for cell proliferation and colony formation in vitro.

Conclusions: Our findings provide a comprehensive description of lncRNA transcriptomic changes in cSCC and demonstrate their functional relevance as both biomarkers and drivers of disease progression in this and potentially other cancers.