Heterogeneity in Mammary Cancer: Using Mouse Models to Investigate Tumor Subtype Origins and Interclonal Interactions
Open Access
- Author:
- Cleary, Allison Shea
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 14, 2014
- Committee Members:
- Edward Joseph Gunther, Dissertation Advisor/Co-Advisor
Edward Joseph Gunther, Committee Chair/Co-Chair
David Feith, Committee Member
Leslie Joan Parent, Committee Member
Todd Schell, Committee Member
Raghu Sinha, Committee Member - Keywords:
- tumor heterogeneity
interclonal cooperation
breast cancer
mammary gland
transgenic mice
3D organoid culture
FACS - Abstract:
- Breast cancer is a heterogeneous disease on two levels. First, breast cancers display inter-tumor heterogeneity evidenced by the diversity of clinically and molecularly defined tumor subtypes that differ with respect to disease progression and drug sensitivity. Second, individual breast cancers display remarkable intra-tumor heterogeneity evidenced by the diversity of component tumor cell subtypes co-residing within each cancer that differ with respect to cell morphology, proliferation rate, metastatic potential, drug sensitivity, and capacity for tumor reconstitution. The cellular mechanisms that generate and maintain breast cancer heterogeneity, both at the level of tumor subtype and tumor cell subtype, remain poorly understood. Using mouse models of human breast cancer, this dissertation examines whether and how breast cancer heterogeneity derives from the diverse and highly interactive cell subtypes that comprise the normal mammary gland, breast cancer’s tissue-of-origin. In one set of experiments directed at uncovering the origin of tumor subtypes, we examined why transgenic mouse models of breast cancer nearly always yield a hormone receptor (HR)-negative mammary cancer subtype. In the mammary gland, mature ducts consist of basal and luminal mammary epithelial cell (MEC) subtypes. The luminal epithelial compartment can be further subdivided into hormone receptor (HR)-positive and HR-negative subsets. While human breast cancers frequently express HRs and depend on ovarian hormones for growth, transgenic mouse models of breast cancers show an unexplained bias toward HR-negative disease. Since the majority of mouse breast cancer models use the mouse mammary tumor virus long terminal repeat (MMTV-LTR) as a mammary-specific promoter element, we examined whether MMTV targets transgene expression to a specific MEC compartment. Using the MMTV-LTR to drive expression of a nuclear H2BGFP reporter transgene, we observed nuclear labeling restricted to HR-negative cells within the luminal compartment. Combining this labeling strategy with MMTV-directed expression of the Neu oncogene, we found Neu transgene expression was similarly enriched within HR-negative luminal MECs. Further, Neu-initiated neoplasias were comprised entirely of HR-negative cells from the carcinoma-in-situ stage onward. Thus, MMTV-driven Neu expression targets HR-negative luminal cells, culminating in HR-negative tumors. We propose that the HR-negative phenotype of many mouse breast cancer models can be explained by MMTV-driven transgene expression in HR-negative MECs. In another set of experiments, we sought to study interactions between tumor cell subtypes. To do this, we developed a novel experimental platform for culturing chimeric mammary organoids which permits analysis of both the cell-autonomous and non-autonomous effects of oncogene expression. By combining primary MECs from two different transgenic donors, chimeric mammary organoids were assembled consisting of intermingled populations of genetically distinct donor MECs that could each be tracked over time. We tested our system using transgenic mouse models engineered to inducibly express either an activated HRas allele or oncogenic Wnt1 in specific MECs. As expected, HRas-expressing cells expanded in number, which is consistent with a predominantly cell autonomous role for oncogenic HRas. By contrast, Wnt1-expressing cells did not expand in number. Instead, luminal expression of Wnt1 produced a dramatic and selective expansion of the basal epithelial cell compartment, as captured by live cell imaging. Thus, secreted Wnt1 primarily drove MEC overgrowth by acting in a paracrine rather than autocrine manner. Overall, chimeric organoid analysis can be used as a sensitive and effective tool for studying complex cell-cell interactions in the context of both normal and transformed mammary epithelium. In a final set of experiments directed at explaining how diverse tumor cell subtypes are maintained within mammary cancers, we examined the functional relationship between distinct tumor cell clones. Recent studies highlight the phenotypic and genetic diversity present locally within individual breast tumors, but whether this heterogeneity is a cause or a consequence of tumor progression remains unclear. Here, we used mouse models of breast cancer to demonstrate for the first time that interclonal cooperation can be essential for tumor maintenance. Aberrant expression of the secreted signaling molecule Wnt1 generates mixed-lineage mammary tumors composed of basal and luminal tumor cell subtypes, which purportedly derive from a bipotent malignant progenitor cell residing atop a tumor cell hierarchy. Using somatic HRas mutations as clonal markers, we showed that some Wnt tumors indeed conformed to a hierarchical configuration, but others unexpectedly harbored genetically distinct basal HRas mutant (HRasmut) and luminal HRas wild-type (HRaswt) subclones. Both subclones were required for efficient tumor propagation, which strictly depended on luminally-produced Wnt1. When biclonal tumors were challenged with Wnt withdrawal to simulate targeted therapy, analysis of tumor regression and relapse revealed that basal subclones recruited heterologous Wnt-producing cells to restore tumor growth. Alternatively, in the absence of a substitute Wnt source, the original subclones often evolved to rescue Wnt pathway activation and drive relapse, either by restoring cooperation or by switching to a defector strategy. Uncovering similar modes of interclonal cooperation in human cancers may inform efforts aimed at eradicating tumor cell communities.