Conditional activation of Neu in the mammary epithelium of transgenic mice results in reversible pulmonary metastasis

AbstractTo determine the impact of tumor progression on the reversibility of Neu-induced tumorigenesis, we have used the tetracycline regulatory system to conditionally express activated Neu in the mammary epithelium of transgenic mice. When induced with doxycycline, bitransgenic MMTV-rtTA/TetO-NeuNT mice develop multiple invasive mammary carcinomas, essentially all of which regress to a clinically undetectable state following transgene deinduction. This demonstrates that Neu-initiated tumorigenesis is reversible. Strikingly, extensive lung metastases arising from Neu-induced mammary tumors also rapidly and fully regress following the abrogation of Neu expression. However, despite the near universal dependence of both primary tumors and metastases on Neu transgene expression, most animals bearing fully regressed Neu-induced tumors ultimately develop recurrent tumors that have progressed to a Neu-independent state

Investigation of the in vivo stress response to oncogenic mutation and tumor suppressor inactivation in mammary carcinogenesis

Breast cancer is believed to arise from the cumulative mutation of oncogenes and tumor suppressors within a small number of precursor cells within the mammary gland, yet the mechanism by which these mutations induce the transformation of normal mammary epithelial cells into malignant tumor cells remains undefined. Better definition of the functions mediated by genes mutated in breast cancer is essential to our understanding of the means by which mammary tumors are initiated. The two most commonly mutated genes in human cancers are the p53 and Ink4A/Arf loci. One process clearly regulated by both of these loci is the induction of cellular senescence. Moreover, several tumorigenic stimuli have been noted to induce senescence, among them activation of oncogenes such as RAS, and the inactivation of certain tumor suppressors such as BRCA2. The fact that senescence is activated by genetic alterations present in human cancers strongly suggests a tumor suppressive role for this process. For these reasons, the goals of this research are to investigate the role of the senescence in response to Ras activation and inactivation of the tumor suppressor Brca2. In this thesis we have determined the existence of a senescent-like growth arrest induced by RAS in vivo. Using a murine model for induction of oncogenic Ras in the mammary epithelium, we have found that low levels of oncogenic Ras induce moderate proliferation without growth arrest. In contrast, high levels of Ras activation lead to rapid proliferation followed by a strong growth arrest, in addition to increases in p53, p21, p16, and p19. This occurs in the absence of increased cell death. In addition, we have found that induction of Ras leads to a reversible inhibition of ductal elongation of the mammary epithelium. Moreover, deinduction of Ras in chronically induced glands leads to both increased apoptosis and resumption of proliferation of mammary epithelial cells, demonstrating that Ras-induced growth arrest is reversible. In our research we have also analyzed the murine Brca2 protein, which in murine models has been shown to trigger premature senescence when deleted. Despite overall low homology to human BRCA2, we have demonstrated that murine Brca2 interacts with Rad51 and Brca1 as has been shown for human BRCA2. Furthermore, we report that murine and human BRCA2 utilize the same domains in interaction with RAD51. We also show that murine Brca2 is capable of interaction with human BRCA1. Finally, we have demonstrated that murine and human BRCA2 truncation products differ in their nuclear localization properties, thus explaining why murine models harboring truncating mutations in Brca2 do not exhibit null phenotypes as is predicted for human BRCA2 truncation products

Disruption of neural progenitors along the ventricular and subventricular zones in periventricular heterotopia

Periventricular heterotopia (PH) is a disorder characterized by neuronal nodules, ectopically positioned along the lateral ventricles of the cerebral cortex. Mutations in either of two human genes, Filamin A (FLNA) or ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2), cause PH (Fox et al. in ‘Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia'. Neuron, 21, 1315–1325, 1998; Sheen et al. in ‘Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex'. Nat. Genet., 36, 69–76, 2004). Recent studies have shown that mutations in mitogen-activated protein kinase kinase kinase-4 (Mekk4), an indirect interactor with FlnA, also lead to periventricular nodule formation in mice (Sarkisian et al. in ‘MEKK4 signaling regulates filamin expression and neuronal migration'. Neuron, 52, 789–801, 2006). Here we show that neurons in post-mortem human PH brains migrated appropriately into the cortex, that periventricular nodules were primarily composed of later-born neurons, and that the neuroependyma was disrupted in all PH cases. As studied in the mouse, loss of FlnA or Big2 function in neural precursors impaired neuronal migration from the germinal zone, disrupted cell adhesion and compromised neuroepithelial integrity. Finally, the hydrocephalus with hop gait (hyh) mouse, which harbors a mutation in Napa [encoding N-ethylmaleimide-sensitive factor attachment protein alpha (α-SNAP)], also develops a progressive denudation of the neuroepithelium, leading to periventicular nodule formation. Previous studies have shown that Arfgef2 and Napa direct vesicle trafficking and fusion, whereas FlnA associates dynamically with the Golgi membranes during budding and trafficking of transport vesicles. Our current findings suggest that PH formation arises from a final common pathway involving disruption of vesicle trafficking, leading to impaired cell adhesion and loss of neuroependymal integrity