| This thesis describes an investigation of the consequences of hyperactivation of colony-stimulating factor receptor (CSF-1R), a potential contributor to breast tumorigenesis, in MCF-10A human mammary epithelial cells. Elevated CSF-1R expression in breast tumor epithelia correlates with invasiveness; however, the mechanisms whereby CSF-1R may contribute to pathogenesis are incompletely understood [1]. To characterize the biological activities of CSF-1R, I utilized basement membrane cultures in which MCF-10A cells assemble into hollow spherical structures reminiscent of in vivo lobuloaveolar acini. Autocrine stimulation of CSF-1R by co-overexpression of CSF-1 induced a striking disruption of acinar architecture, culminating in the dispersion of individual cells from acini. This perturbation was associated with redistribution of E-cadherin from the plasma membrane to intracellular puncta, emphasizing the importance of intercellular adhesion to maintaining an organized epithelium and indicating the significance of E-cadherin trafficking to its regulation. Cytoplasmic accumulation of E-cadherin was dependent upon Src activity downstream of CSF-1R, as mutation of the Src docking site on the receptor specifically prevented its relocalization. To discover relevant Src targets, I employed both candidate and unbiased strategies. Rap1 and Nf2, both known to stabilize adhesion, antagonized the CSF-1R phenotype, suggesting that these factors influence a target shared by the receptor. Furthermore, using phosphotyrosine immunoprecipitation and mass spectrometry, we identified ACAP2 and Rin1 as novel Src substrates that may regulate E-cadherin trafficking.; Finally, we sequenced the CSF-1R locus in breast tumor-associated tissue samples to determine whether mutation of CSF-1R might contribute to pathogenesis. Several missense variants were identified, and MCF-10A acinar culture and a panel of biochemical assays were employed to assess whether five of these changes could affect receptor activity. However, none of these polymorphisms resulted in altered cell behavior or acinar architecture. While this limited analysis does not discount a role for such variants in tumorigenesis, these findings suggest that autocrine stimulation may be the most common mode of CSF-1R activation in epithelial tumors. Collectively, these studies identify critical downstream modulators of the CSF-1R phenotype, and provide substantial motivation for the investigation of CSF-1R as a therapeutic target in breast cancer. |
