Joana Liu Donaher

Twist, a Master Regulator of Morphogenesis, Plays an Essential Role in Tumor Metastasis

Metastasis is a multistep process during which cancer cells disseminate from the site of primary tumors and establish secondary tumors in distant organs. In a search for key regulators of metastasis in a murine breast tumor model, we have found that the transcription factor Twist, a master regulator of embryonic morphogenesis, plays an essential role in metastasis. Suppression of Twist expression in highly metastatic mammary carcinoma cells specifically inhibits their ability to metastasize from the mammary gland to the lung. Ectopic expression of Twist results in loss of E-cadherin-mediated cell-cell adhesion, activation of mesenchymal markers, and induction of cell motility, suggesting that Twist contributes to metastasis by promoting an epithelial-mesenchymal transition (EMT). In human breast cancers, high level of Twist expression is correlated with invasive lobular carcinoma, a highly infiltrating tumor type associated with loss of E-cadherin expression. These results establish a mechanistic link between Twist, EMT, and tumor metastasis.

Cyclin D1 provides a link between development and oncogenesis in the retina and breast

Mice lacking cyclin D1 have been generated by gene targeting in embryonic stem cells. Cyclin D1-deficient animals develop to term but show reduced body size, reduced viability, and symptoms of neurological impairment. Their retinas display a striking reduction in cell number due to proliferative failure during embryonic development. In situ hybridization studies of normal mouse embryos revealed an extremely high level of cyclin D1 in the retina, suggesting a special dependence of this tissue on cyclin D1. In adult mutant females, the breast epithelial compartment fails to undergo the massive proliferative changes associated with pregnancy despite normal levels of ovarian steroid hormones. Thus, steroid-induced proliferation of mammary epithelium during pregnancy may be driven through cyclin D1.

Growth Inhibitory and Tumor- Suppressive Functions of p53 Depend on its Repression of CD44 Expression

The p53 tumor suppressor is a key mediator of cellular responses to various stresses. Here, we show that under conditions of basal physiologic and cell-culture stress, p53 inhibits expression of the CD44 cell-surface molecule via binding to a noncanonical p53-binding sequence in the CD44 promoter. This interaction enables an untransformed cell to respond to stress-induced, p53-dependent cytostatic and apoptotic signals that would otherwise be blocked by the actions of CD44. In the absence of p53 function, the resulting derepressed CD44 expression is essential for the growth and tumor-initiating ability of highly tumorigenic mammary epithelial cells. In both tumorigenic and nontumorigenic cells, CD44s expression is positively regulated by p63, a paralogue of p53. Our data indicate that CD44 is a key tumor-promoting agent in transformed tumor cells lacking p53 function. They also suggest that the derepression of CD44 resulting from inactivation of p53 can potentially aid the survival of immortalized, premalignant cells.

LACTB is a tumour suppressor that modulates lipid metabolism and cell state

Post-mitotic, differentiated cells exhibit a variety of characteristics that contrast with those of actively growing neoplastic cells, such as the expression of cell-cycle inhibitors and differentiation factors. We hypothesized that the gene expression profiles of these differentiated cells could reveal the identities of genes that may function as tumour suppressors. Here we show, using in vitro and in vivo studies in mice and humans, that the mitochondrial protein LACTB potently inhibits the proliferation of breast cancer cells. Its mechanism of action involves alteration of mitochondrial lipid metabolism and differentiation of breast cancer cells. This is achieved, at least in part, through reduction of the levels of mitochondrial phosphatidylserine decarboxylase, which is involved in the synthesis of mitochondrial phosphatidylethanolamine. These observations uncover a novel mitochondrial tumour suppressor and demonstrate a connection between mitochondrial lipid metabolism and the differentiation program of breast cancer cells, thereby revealing a previously undescribed mechanism of tumour suppression.

Integrin-β4 identifies cancer stem cell-enriched populations of partially mesenchymal carcinoma cells

Significance It is widely appreciated that carcinoma cells exhibiting certain mesenchymal traits are enriched for cancer stem cells (CSCs) and can give rise to tumors with aggressive features. Whereas it has been proposed that mesenchymal carcinoma cell populations are internally heterogeneous, the field has made little progress in resolving the specific subtypes of mesenchymal carcinoma cells that pose the greatest risk for patients. We demonstrate the utility of integrin-β4 (ITGB4) in segregating these cells into distinct subpopulations with differing tumor-initiating abilities and pathological behaviors. In addition, we identified mechanistic links between ZEB1 (zinc finger E-box binding homeobox 1) and TAp63α (tumor protein 63 isoform 1) as regulators of ITGB4 expression and demonstrate that ITGB4 can be used as a marker to determine which patients are more likely to relapse after treatment. Neoplastic cells within individual carcinomas often exhibit considerable phenotypic heterogeneity in their epithelial versus mesenchymal-like cell states. Because carcinoma cells with mesenchymal features are often more resistant to therapy and may serve as a source of relapse, we sought to determine whether such cells could be further stratified into functionally distinct subtypes. Indeed, we find that a basal epithelial marker, integrin-β4 (ITGB4), can be used to enable stratification of mesenchymal-like triple-negative breast cancer (TNBC) cells that differ from one another in their relative tumorigenic abilities. Notably, we demonstrate that ITGB4+ cancer stem cell (CSC)-enriched mesenchymal cells reside in an intermediate epithelial/mesenchymal phenotypic state. Among patients with TNBC who received chemotherapy, elevated ITGB4 expression was associated with a worse 5-year probability of relapse-free survival. Mechanistically, we find that the ZEB1 (zinc finger E-box binding homeobox 1) transcription factor activity in highly mesenchymal SUM159 TNBC cells can repress expression of the epithelial transcription factor TAp63α (tumor protein 63 isoform 1), a protein that promotes ITGB4 expression. In addition, we demonstrate that ZEB1 and ITGB4 are important in modulating the histopathological phenotypes of tumors derived from mesenchymal TNBC cells. Hence, mesenchymal carcinoma cell populations are internally heterogeneous, and ITGB4 is a mechanistically driven prognostic biomarker that can be used to identify the more aggressive subtypes of mesenchymal carcinoma cells in TNBC. The ability to rapidly isolate and mechanistically interrogate the CSC-enriched, partially mesenchymal carcinoma cells should further enable identification of novel therapeutic opportunities to improve the prognosis for high-risk patients with TNBC.

Abstract B48: Spatiotemporal regulation of epithelial-mesenchymal transition is essential for carcinoma metastasis

During metastasis, epithelial tumor cells dissociate from each other, invade surrounding extracellular matrix, disseminate into the systemic circulation, and then establish secondary lesions in distant sites. A developmental program termed Epithelial-Mesenchymal Transition (EMT) has been implicated in giving rise to the dissemination of single carcinoma cells. During EMT, stationary epithelial cells undergo a series of cellular and molecular changes to convert into motile mesenchymal cells. However, the involvement of EMT in tumor metastasis is still controversial primarily due to the lack of a mesenchymal phenotype in human carcinoma distant metastases. In this study, we established a transgenic mouse model that specifically expresses the EMT-inducing transcription factor Twist1 on the basal layer of the skin in an inducible fashion. Using a chemical carcinogenesis skin tumor model, we report here that induction of Twist1 is sufficient to promote carcinoma cells to undergo EMT, invade through basement membrane, intravasate into blood circulation, and extravasate into distant organs. More importantly, we found that in distant sites, turning off Twist1 to allow reversion of EMT is essential for tumor cells to proliferate and form epithelial macrometastases. In contrast, continuous expression of Twist1 in disseminated tumor cells inhibits metastasis formation in distant organs. Consistent with these results, we found that expression of Twist1 is significantly higher in primary human carcinomas compared to their corresponding distant metastases. These results indicate that carcinoma cells undergo EMT to disseminate; once reaching distant site, they need to revert to an epithelial identity to form macrometastases. Our study demonstrates in vivo the requirement of “reversible EMT” in tumor metastasis and may resolve the controversy on the importance of EMT in carcinoma metastasis. EMT features are frequently observed in many types of primary human carcinoma, but not their corresponding metastases. Our findings indicate that reversible EMT likely represents a key driving force in human carcinoma metastasis. Delayed onset of metastasis following primary tumor removal is thought to be due to resurrection of latent carcinoma cells in distant organs. Our study raises the possibility that tumor dormancy could be due to the inability of disseminated tumor cells to revert EMT and proliferate. The dynamic involvement of EMT in metastasis cautions that therapies inhibiting EMT could be counterproductive in preventing distant metastases when patients already present circulating tumor cells. Instead, blocking EMT reversion may prevent dormant tumor cells from establishing macrometastases. Citation Format: Jeff H. Tsai, Joana Liu Donaher, Danielle A. Murphy, Sandra Chau, Jing Yang. Spatiotemporal regulation of epithelial-mesenchymal transition is essential for carcinoma metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B48.