Vasioukhin Lab Research

Cell polarity and cell adhesion in mammalian development and cancer.

Individual cells in all metazoans need to communicate with each other in order to coordinate their behavior to ensure survival of the entire organism. During embryonic development, non-differentiated totipotent cells divide asymmetrically and generate daughters, which are destined to become the myriads of different cell types that will stop proliferation, differentiate and generate organs of predetermined shape and size. This process of asymmetric cell division is repeated in all organs and tissues of adult organisms during maintenance by their respective stem cell populations. The overall general aim of our research is to understand the mechanisms responsible for orchestrating cellular behavior that help individual cells to work together to produce and maintain normal mammalian organism. Our laboratory is pursuing research in two major directions:

1.   We study the cell polarity mechanisms responsible for asymmetric cell division that help to ensure both the maintenance of pluripotent stem cell population and normal cell differentiation. We believe that the failure of these mechanisms is ultimately responsible for cancer.

2.   We are trying to understand how cells use cell adhesion structures as biosensors of their microenvironment and translate information obtained by these structures into critical decisions concerning cell proliferation, differentiation and programmed cell death.

Specifically, we are working on the following projects:

1. The mechanisms and role of Lethal giant larvae (Lgl) in regulation of asymmetric cell division during self-renewal and differentiation of mammalian stem and progenitor cells. Neural progenitor cells in the developing mammalian brain are highly polarized and organized as pseudostratified epithelium. These cells divide asymmetrically in a stem-cell fashion to replenish themselves and to generate daughters that differentiate into major brain cell types. Our laboratory uses neural progenitors as a model to study the mechanisms governing polarity and asymmetric cell division necessary for proper self-renewal and differentiation. We began these studies with the analysis of the function of the mammalian homolog of the Drosophila tumor-suppressor protein Lgl. Drosophila Lgl is necessary for asymmetric cell division of neuroblasts and Lgl mutants display abnormal increase in self-renewal and decrease in differentiation of neuroblasts, accumulation of dividing cells and cancer. We hypothesized that mammalian orthologs of Drosophila Lgl may also be involved in regulation of asymmetric cell division. To determine the role and significance of mammalian Lgl genes, we generated straight, and later, conditional Lgl1 knockout mice (1). We found that mammalian Lgl1 plays a critical role in regulation of asymmetric cell division, self-renewal and differentiation. The Lgl1-/- progenitors failed to withdraw from the cell cycle and continued to divide, leading to massive brain dysplasia resembling human primitive neuroectodermal tumors. We found that the important function of Lgl1 was asymmetric mitotic localization of the cell fate determinant Numb. Numb is a negative regulator of the Notch pathway. Loss of Lgl1 caused deregulation of Notch signaling and subsequent failures of proper cell fate determination and cell cycle withdrawal. We are now studying the molecular mechanisms responsible for Lgl function in asymmetric localization of cell fate determinants.

2. Role of the Dlg family of proteins in the maintenance of cell polarity and intercellular adhesion. Dlg belongs to a superfamily of membrane-associated guanylate kinase (MAGUK) proteins. Drosophila Dlg1 functions in the same pathway as Lgl to maintain cell polarity and regulate asymmetric cell division. Like Lgl, Drosophila Dlg1 is a tumor-suppressor protein. Our laboratory is studying the role of a distinct member of the Dlg family of proteins, Dlg5. We found that Dlg5 is a unique member of the family conserved throughout the animal kingdom. Although Dlg5 is present in all multicellular organisms, no Dlg5-mutant organism has ever been generated and analyzed.  Therefore, the function of the protein was unknown. We found that Dlg5 is ubiquitously expressed, and the Dlg5 protein localizes to cytoplasm and adherens junctions. To determine the role of Dlg5, we generated Dlg5-/- mice (2). We found that Dlg5-/- mice developed hydrocephalus and renal cysts. Our analysis of the knockout mice demonstrated that disorganization and disruption of adhesion and cell polarity were responsible for the brain and kidney defects. We analyzed the molecular mechanisms responsible for this phenotype and found that Dlg5 functions in plasma membrane delivery of cell adhesion proteins cadherins. Dlg5 links cadherin-containing transport vesicles with the t-SNARE membrane targeting complex. These findings uncovered a novel gene causally involved in hydrocephalus and renal cysts and revealed that targeted membrane delivery of cadherin-catenin adhesion complexes is critical for cell polarity and epithelial tube maintenance in live mammalian organism.

3. Role of adherens junctions in monitoring and regulating cell accumulation during development, normal tissue homeostasis and cancer. Cells in the developing embryo can monitor their rates of accumulation and adjust their proliferation accordingly to produce organs of predetermined size. The mechanisms responsible for this function remain unknown. We hypothesized that cell-cell adhesion structures can provide cells with information about their immediate cellular environment and their location within the organ that can be translated into important decisions concerning cell proliferation, differentiation or death. Our laboratory studies the adherens junction protein αE-catenin. To determine the role and significance of αE-catenin in mammalian neural progenitor cells, we generated central nervous system (CNS)–specific αE-catenin-/- mice (4). We found that these mice developed massive brain dysplasia and hyperplasia, and had effectively doubled the normal number of brain cells at birth. Mutant neural progenitor cells lose polarity, hyperproliferate and disperse throughout the developing brain forming tumor-like masses characteristic of human brain tumors. We analyzed the molecular mechanisms responsible for hyperplasia and found that activation of the hedgehog signaling was responsible for abnormal accumulation of cells in αE-catenin-/- brains (3). Remarkably, we found that αE-catenin had no effect on the b-catenin signaling pathway (4). We concluded that cortical progenitors use cell-density-dependent cell-cell adhesion structures to control the developmental hedgehog pathway to ensure the proper mammalian brain size. We used these findings to formulate a “crowd control” hypothesis that proposes that an increase in cell density, sensed by the expansion of the adherens junctions, negatively regulates the hedgehog pathway, and this connection provides a negative feedback loop that controls the rates of cell accumulation in the developing mammalian brain (5). We are now studying the mechanisms connecting αE-catenin to the regulation of the hedgehog signaling.

4. Role of cell adhesion in prostate cancer progression. Extracellular and cell-surface proteases play an important role in the regulation of cell adhesion, and deregulation of these enzymes may be critical for tumor dissemination. Hepsin is a cell-surface serine protease markedly overexpressed in prostate cancer. To determine the significance of hepsin overexpression in prostate epithelia, we generated and analyzed transgenic mice expressing hepsin in prostate epithelia. We found that overexpression of hepsin in prostate epithelium in vivo causes disorganization of the basement membrane and promotes prostate cancer progression and metastasis (6). We concluded that overexpression of hepsin plays an important causal role in prostate cancer progression and metastasis, and hepsin represents a therapeutic target for the treatment of prostate cancer.

In a parallel study we investigated the significance of a recurrent chromosomal rearrangement event in human prostate cancer that results in overexpression of ETS family transcription factor ERG. We found that ERG overexpression results in initiation of prostate cancer. (7). We found that overexpression of ERG causes activation of cell-surface proteolytic system and this may be responsible for its function in prostate cancer.

1. Klezovitch O, Fernandez TE, Tapscott SJ, Vasioukhin V. Loss of cell polarity causes severe brain dysplasia in Lgl1 knockout mice. Genes Dev. 2004 Mar 1;18(5):559-71.
      Cover picture. Highlighted by Faculty of 1000.

2. Nechiporuk T, Fernandez T, Vasioukhin V. Failure of epithelial tube maintenance causes hydrocephalus and renal cysts in Dlg5-/- mice. Dev Cell, 2007. Sep;13(3):338-50, 2007.
     Cover picture. Highlighted by Faculty of 1000.

3. Lien W-H, Klezovitch O, Fernandez T, Delrow J, Vasioukhin V. E-catenin controls cerebral cortical size by regulating the hedgehog signaling pathway. Science. 2006 311(5767):1609-12.
     Highlighted in Science Perspectives, Sci. STKE Editor’s choice, Cell, J Cell Biol and Faculty of 1000.

4. Lien WH, Klezovitch ON, Null M, Vasioukhin V. Endogenous aE-catenin is not a significant regulator of  b-catenin transcriptional activity in the developing mammalian brain. J Cell Sci. 2008, Apr 8. Epub.

5. Lien W-H, Klezovitch O, Vasioukhin V. Cadherins and associated proteins in development. Curr Opin Cell Biol. 2006 18(5).

6. Klezovitch O, Chevillet J, Mirosevich J, Roberts RL, Matusik RJ, Vasioukhin V. Hepsin promotes prostate cancer progression and metastasis. Cancer Cell. 2004 Aug;6(2):185-95.
     Cover picture.

7. Klezovitch O, Risk M, Coleman I, Lucas J, Null M, True L, Nelson PS, Vasioukhin V.  A causal role for ERG in neoplastic transformation of prostate epithelium. PNAS USA. 2008, Feb 12;105(6):2105-10.