This research was presented at the 2012 American Society for Cell Biology conference in San Francisco.

Crosstalk of Wnt and HGF Signaling increases growth of Colon Cancer Initiating Cells

G Chen, S Sprowl, A Konstorum, A Newman, L Alonzo, M Moya, S George, CC Hughes, J Lowengrub, RA Edwards, ML Waterman

UC Irvine

Wnt signaling directs cell differentiation, embryonic development, and cell polarity. Signals are commonly defined as β-catenin-dependent (“canonical”) and β-catenin-independent (“non-canonical”), though both defy traditional categorization, with individual Wnt ligands capable of activating multiple pathways.

Over eighty percent of colorectal carcinomas have genetic mutations that stabilize β-catenin, leading to sustained levels of Wnt target gene expression. Despite being constitutively active, aberrant Wnt signaling is sensitive to modulation by crosstalk from other signals such as Hepatocyte Growth Factor (HGF) and autocrine Wnt ligands. These signals originate from multiple sources in the tumor microenvironment, including myofibroblasts and the cancer cells themselves.

HGF regulates cell growth and motility through the c-Met receptor tyrosine kinase and making it a key factor in the development and metastasis of many cancers. The effect of HGF crosstalk on the growth and progression of Wnt-driven colon tumors is currently unknown, as are the molecular mechanisms that mediate influences on β-catenin. We utilized colon cancer initiating cultures (CCICs) embedded in fibrin gels to address these unknowns.  CCICs are patient-derived cells with multipotent properties and recapitulate the histology of the parental tumor in xenograft. Single CCICs were seeded into fibrin and treated with a range of HGF concentrations.  A quantitative assessment of colony area was used as a measure of growth rate. We observed a non-linear dose-response of HGF on CCIC colony growth and cell scattering; low doses of HGF stimulated proliferation while high doses of HGF showed no change over control. Co-treatment of the CCICs with HGF and the Wnt secretion inhibitor IWP2 led to a synergistic increase in cell scattering, suggesting that autocrine secretion of Wnt(s) opposes HGF-directed cell scatter.  Colon myofibroblast-conditioned media exerted a greater effect on CCIC colony growth and scattering compared to HGF alone, suggesting that CMFs either secrete an optimal amount of HGF or other factors that synergize with HGF.

To assess how CMFs influence CCIC colony growth and Wnt signaling in a physiologically relevant environment, we used new 3D tumor microchamber devices seeded with endothelial progenitor cells, fibroblasts, and CCICs transduced with a lentiviral Wnt reporter driving destabilized GFP (TOPdGFP). Within the chamber, endothelial cells form a microvessel network which anastamoses with microfluidic channels to create an actively perfused vascular network. We observe that CCICs require CMFs for growth and survival and that GFP expression is heterogeneous – suggesting that microenvironmental signals drive complex patterns of Wnt activity. Currently, we are identifying the Wnt ligands that mediate the HGF/Wnt effects and defining the influence of HGF-secreting fibroblasts on the heterogenous levels of Wnt activity.