DamID-Seq analysis of cutaneous squamous cell carcinoma cells

In this study, we interrogated the changes in the fibronectin-induced adhesome in cancer cells using patient-derived malignant keratinocytes from cutaneous squamous cell carcinoma (cSCC). We found that the human enabled homologue, Mena (encoded by ENAH; also known as hMena), an actin regulatory protein of the Ena/VASP family, is upregulated in the adhesome of metastatic cSCC cells compared to equivalent cells from the primary tumour. Using a network analysis approach, we found that Mena clusters within an actin-binding/regulatory module that is upregulated in metastatic keratinocytes. Our proteomic analysis connected Mena to the LINC complex component nesprin-2, and we uncovered a novel role for Mena at the perinuclear region of metastatic keratinocytes. We found that Mena locates adjacent to, and interacts with the C-terminal spectrin repeats of, nesprin-2 at the nuclear membrane. Mena loss results in the reduction of nesprin-2 interactions with actin and lamin A/C, affecting nuclear morphology. Moreover, Mena loss results in the reduction of emerin tyrosine phosphorylation and affects the regulation of PTX3 (which encodes pentraxin-3, an immunomodulatory component of the complement system) via the recruitment of its putative enhancer region to the nuclear lamina DamID sequencing analysis of Dam-lamin B1 and untethered Dam in Met4 and Met4 Mena-/- cutaneous squamous cell carcinoma cells