Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer and its incidence is increasing worldwide. Previous studies have demonstrated the role of complement system in cSCC progression. In this study we have investigated in detail the mechanistic role of serine protease C1r, a component of the C1 complex of the classical pathway of complement system. CRISPR/Cas9 technology was used to generate C1r negative cSCC cell clones. Knockout of C1r was confirmed by sequencing the PCR product obtained by amplification of genomic DNA and by Western blot analysis of the conditioned media of the cell clones. Pooled C1r knockout cSCC cell clones showed significantly decreased proliferation, migration and invasion through collagen type I, as compared to wild type cSCC cells. Knockout of C1r also significantly suppressed growth of human cSCC xenografts in vivo. RNA-Seq analysis of cSCC cells after C1r knockdown with specific siRNA revealed significantly regulated GO terms Cell-matrix adhesion, Extracellular matrix component, Basement membrane and Metallopeptidase activity. Among the significantly downregulated genes were matrix metalloproteinase (MMP) genes MMP1, MMP13 and MMP10. Downregulation of MMP1, MMP13 and MMP10 mRNA levels after C1r knockdown was confirmed by qRT-PCR. Decreased levels of MMP-1 (collagenase-1), MMP-13 (collagenase-3) and MMP-10 (stromelysin-2) were detected in the conditioned media of cSCC cells by Western blotting after C1r knockdown. These results provide new evidence for the role of C1r in regulating invasion of cSCC cells by increasing production of invasion-associated proteinases MMP-1, MMP-13, and MMP-10.

Complement component C1r upregulates the expression of matrix metalloproteinase-1, -13 and -10 and promotes invasion of cutaneous squamous carcinoma cells