Mammary Epithelial Cell Subtype-Specific Analysis of Ras Pathway Activation
Open Access
- Author:
- Plichta, Kristin Ann
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 06, 2010
- Committee Members:
- Edward Joseph Gunther, Dissertation Advisor/Co-Advisor
Edward Joseph Gunther, Committee Chair/Co-Chair
Andrea Manni, Committee Member
Christopher Alan Siedlecki, Committee Member
Lisa M Shantz, Committee Member - Keywords:
- Ras
mammary organoids
breast cancer
transgenic mice
mammary epithelial cells - Abstract:
- Breast cancers can be divided into subtypes based in part on how closely the tumor cells resemble mammary epithelial cell (MEC) subtypes resident in normal breast tissue. Though breast cancer subtypes differ in their aggressiveness and their response to treatment, the mechanisms by which distinct subtypes arise remain unknown. Traditionally, attempts to explain the varied clinical behavior of breast cancers rely on defining the genetic lesions harbored within individual tumors. However, recent evidence suggests that distinct breast cancer subtypes may arise from distinct MEC lineages, suggesting that some biological features of a given breast cancer subtype may be attributable to its antecedent “cell of origin”. Elucidating whether MEC lineage impacts the biology of descendant breast cancers presents a formidable challenge. By the time a tumor is clinically detectable, breast cancers have undergone clonal evolution that precludes a reliable retrospective determination of the cell of origin. As such, prospective analyses may be required to determine whether distinct MEC subtypes yield distinct cancer subtypes. We hypothesized that distinct MEC compartments yield different phenotypes in response to an identical oncogenic stimulus. To test this possibility, we generated mouse models that permit doxycycline-dependent expression of transgenes in an MEC compartment-restricted manner and developed strategies to monitor transgene-mediated phenotypes in real-time. As a first step toward studying malignant transformation of distinct MEC subtypes in mice, we tested experimental strategies designed to permit restriction of transgene expression to distinct MEC compartments in vitro. Transgenic mammary epithelium was partially disaggregated and propagated in 3D culture as mammary organoids, preserving the bilayered arrangement of the basal and luminal MEC compartments. Pairing a tet operator-driven H2B-eGFP reporter transgene with either a basal or luminal MEC transactivator enabled MEC compartment-restricted reporter gene expression. Notably, nuclear fluorescence resulting from H2B-eGFP expression enabled visualization of cellular dynamics within discrete MEC compartments. Through extended, multiparameter live cell imaging of organoids, time-lapse movies were generated that enabled visualization of MEC migration as well as tracking and quantitation of mitotic and apoptotic events. Next, we adapted this system to co-express the H2B-eGFP reporter together with a tet-operator-regulated oncogenic H-RASG12V allele. Whether expressed in a basal or luminal MEC-restricted manner, H-RASG12V expression reproducibly triggered aberrant organoid growth, reflected in measureable changes in organoid size and shape. Increases in organoid size were attributable to H-RASG12V-mediated increases in cell proliferation and decreases in cell death. In addition, H-RASG12V expression in either MEC compartment drove MECs to both traverse normal compartment boundaries and adopt modes of cellular migration distinct from those encountered in the setting of physiologic Ras signaling. Surprisingly, though H-RASG12V expression in each MEC compartment triggered similar perturbations in cell fate, inhibitor studies uncovered marked differences in the downstream signaling pathways critical for Ras-mediated overgrowth of basal versus luminal MECs. Most dramatically, inhibition of either the phosphoinositide 3-kinase (Pi3k) or the mammalian target of rapamycin (mTor) pathways completely suppressed growth perturbations due to the basal, but not the luminal expression of H-RASG12V. To extend these findings, future studies should examine whether MEC-specific dependencies on Ras pathway effectors are maintained during tumor progression. If so, then cell of origin may emerge as a powerful predictor of the drug sensitivity profile of descendant breast cancers. Finally, to further dissect cellular mechanisms of Ras-mediated MEC overgrowth, we developed novel methods for generating and analyzing chimeric mammary organoids engineered such that only discrete subsets of basal or luminal MECs express H-RASG12V. Overgrowth of H-RASG12V-expressing MEC subsets recapitulated those changes in organoid size and morphology triggered by broader, compartment-wide H-RASG12V expression. Aberrant growth was largely driven by expansion of H-RASG12V-expressing MEC clones. However, H-RASG12V-expressing MECs also impacted neighboring wild-type MECs by inducing them to form cellular protrusions and adopt aberrant modes of migration. These finding identify novel, cell non-autonomous effects of H-RASG12V-expression.