The Roles of Cancer-Associated Fibroblasts in Colorectal Carcinogenesis

Abstract

Cancer-associated fibroblasts (CAF), key constituents of the tumour microenvironment, either promote or restrain tumour growth. Attempts to therapeutically target CAFs have been hampered by our rudimentary understanding of the functions and origins of these heterogeneous cells. In this thesis, I first addressed the functional heterogeneity of CAFs involving bone morphogenetic proteins (BMPs), a key growth factor in CRC progression. Using human CRC RNA expression data, I identified Gremlin 1 (Grem1) and immunoglobulin superfamily containing leucine-rich repeat (Islr) as CAF-specific genes involved in BMP signalling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signalling, respectively. GREM1 and ISLR marked ACTA2high and ACTA2low colorectal CAFs, respectively. Grem1 and Islr expression were differentially regulated by transforming growth factor-β (TGF-β) and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In CRC patients, high GREM1 and ISLR expression were associated with poor and favourable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signalling and improved mouse survival in our preclinical model of hepatic metastasis. Stromal BMP signalling predicts and modifies CRC progression and survival, and can be therapeutically targeted by novel AAV-directed gene delivery to the liver. Next, I examined the origins and contributions of colorectal CAFs. Using five different fatemapping models with BrdU dosing, this study revealed that half of ACTA2+ CAFs emerge through proliferation in a mouse model of CRC. Intestinal pericryptal Leptin receptor (Lepr)+ cells were the major origins of the proliferating CAFs. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker we identified using RNA-sequencing. High MCAM expression induced by TGF-β was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved mouse survival through decreased tumour-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor-ĸB-IL34/CCL8 signaling that promotes macrophage chemotaxis. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC. These data indicate that targeting these CAF subpopulations could be novel potential therapeutic strategies to inhibit CRC progression.