Colorectal cancer liver metastatic growth depends on PAD4-driven citrullination of the extracellular matrix

Abstract

Citrullination of proteins, a post-translational conversion of arginine residues to citrulline, is recognized in rheumatoid arthritis, but largely undocumented in cancer. Here we show that citrullination of the extracellular matrix by cancer cell derived peptidylarginine deiminase 4 (PAD4) is essential for the growth of liver metastases from colorectal cancer (CRC). Using proteomics, we demonstrate that liver metastases exhibit higher levels of citrullination and PAD4 than unaffected liver, primary CRC or adjacent colonic mucosa. Functional significance for citrullination in metastatic growth is evident in murine models where inhibition of citrullination substantially reduces liver metastatic burden. Additionally, citrullination of a key matrix component collagen type I promotes greater adhesion and decreased migration of CRC cells along with increased expression of characteristic epithelial markers, suggesting a role for citrullination in promoting mesenchymal-to-epithelial transition and liver metastasis. Overall, our study reveals the potential for PAD4-dependant citrullination to drive the progression of CRC liver metastasis.

Citrullination in Cancer

“…In keeping with these findings, GSK3b was reported to induce EMT (58) and control several transcription factors related to cancer progression (59). Additionally, in a mouse model of hepatic metastases, pharmacological inhibition of PADs abated liver metastatic growth and enhanced the expression of mesenchymal markers, whereas the reverse process, mesenchymal-to-epithelial transition (MET) was suggested to be of importance in initiating metastatic colony formation (28). These studies collectively suggest that PAD4/citrullination axis might be implicated in acquisition of prometastatic phenotype by tumors through regulating EMT.…”

“…Approximately 40% of cells in malignant lymphomas also expressed PAD4, indicating that expression of this protein is associated with cancer development from all embryological lineages. Benign tumors and nontumor inflamed tissues did not express PAD4 (37), whereas metastasis exhibited much higher PAD4 levels compared with corresponding primary tumors (28), implicating citrullination in the progression from benign neoplasm to invasive malignancy. Taken together, these data indicate that PAD4 is not uncommonly overexpressed in tumors and thus may represent a biomarker or a putative therapeutic target for cancer treatment.…”

“…26,27). Citrullination of the extracellular matrix (ECM) also exists; for example, ECM deposition of PAD4 along with extensive ECM protein citrullination were observed in colorectal cancer liver metastasis with exosomal route of PAD4 delivery being proposed as a mechanism (28). Others, however, speculated that extracellular citrullination can be potentiated by the leak of PADs during cell death (29).…”

Protein citrullination was introduced into animals by horizontal gene transfer from cyanobacteria

“…The bestestablished role of citrullination is in innate immunity, through mediating the release of neutrophil extracellular traps (NETs) 12 but a plethora of studies have shown that PADIs also regulate gene expression, chromatin compaction, nerve myelination, skin homeostasis and the establishment of ground state pluripotency 8,13,14 . Notably, deregulation of PADIs is strongly implicated in the aetiology of a host of pathologies including autoimmunity (rheumatoid arthritis, ulcerative colitis, psoriasis and type I diabetes), neurodegeneration (multiple sclerosis, Alzheimer's and prion diseases) and metastatic cancer 13,[15][16][17][18] . In the case of rheumatoid arthritis, autoantibodies against citrullinated endogenous proteins (Anti-Citrullinated Protein Antibodies, ACPAs) serve as diagnostic and prognostic markers as they precede the onset of symptoms by several years and correlate with disease severity and response to treatment 19 .…”

Citrullination regulates wound responses and tissue regeneration in zebrafish

“…Binding of the neutrophil chemokine, Cxcl8, to its receptors, Cxcr1 and Cxcr2, regulates neutrophil directional and reverse migration (Powell et al, 2017); interestingly, citrullination of Cxcl8 alters its binding to its receptors (Proost et al, 2008). Alternatively, citrullination of ECM components affects cell migration (Shelef et al, 2012; Sipilä et al, 2014; Yuzhalin et al, 2018), and could potentially regulate inflammation by altering the wound ECM.…”