Exploring roles of LASP1 in tumorigenesis in Human Papillomavirus positive cervical cancers

Persistent infection with high-risk human papillomaviruses is the main cause of a wide range of human cancers, including the majority of cervical cancer and an increasing number of oral cancers. LIM and SH3 protein (LASP1) is overexpressed in HPV positive cancer cell lines and tissues and its high expression correlates with a poor prognosis. LASP1 plays a pivotal role in promoting the proliferation of HPV+ cancer cells and has been demonstrated to be regulated by the tumour suppressor miR-203. Nevertheless, the molecular mechanism underlying LASP1 upregulation and its oncogenic functions remain incompletely understood. In this study, we firstly cloned the LASP1 promoter into the luciferase gene reporter system. The luciferase reporter assays showed the LASP1 promoter can be specifically activated by high-risk HPV E7 oncoproteins. To identify potential transcription factors that involved in the regulation of LASP1 promoter activation, we reanalysed published chromatin immunoprecipitation (ChIP) sequencing data obtained from HeLa cells and found that E2F1 is significantly enriched at the LASP1 promoter. Notably, E2F1 has been shown to be specifically activated by HPV E7. We have also demonstrated that the luciferase gene driven by the LASP1 promoter can be activated by E2F1 overexpression and suppressed by pRB overexpression, indicating that LASP1 is regulated by HPV E7 via the pRB/E2F1 signalling pathway. Furthermore, we have demonstrated the critical role of LASP1 in the migration of HPV+ cervical cancer cells. Wound healing assay indicated LASP1 overexpression enhances the migration ability of cervical cancer cells, while LASP1 knockdown inhibits cell migration, suggesting LASP1 may be involved in the Epithelial-Mesenchymal Transition (EMT). To uncover the links between LASP1 and EMT marker genes, we performed an integrative analysis, combining published ChIP-seq data and protein-protein interaction data from the IntAct database, and identified several potential binding partners for LASP1, including STAT3 and INTS11. Notably, STAT3 has been demonstrated to be essential in EMT by binding to promoters of EMT marker genes. Therefore, it is highly likely that LASP1 participates in the regulation of EMT via its interaction with STAT3. This interaction was further validated by the co-immunoprecipitation assay and its orthogonal techniques such as GST pull-down and PLA assay. Additionally, ChIP-qPCR assays further confirmed the significant enrichment of LASP1 and STAT3 at the promoters of SNAI1 and SNAI2. This work provides the first evidence of the regulation of LASP1 expression by HPV E7 through the pRB/E2F1 signalling pathway, and also the interplay between LASP1 and STAT3 at the promoters of EMT marker genes, which provides more clues for understanding of the general mechanism of HPV related cancer progression.