Mechanisms of Mitotic Spindle Orientation by Plexin-B2

Cells show certain asymmetries in morphology and molecular organization, a characteristic that is known as cell polarity. Polarity is generally regulated by protein complexes and Rho GTPases like Cdc42, Rho or Rac. Epithelial cells are polarized along the apicobasal axis, and this apicobasal polarity influences many cellular processes, including cell division. Polarized mitosis in epithelia is controlled by the orientation of the mitotic spindle. This is crucial in epithelial cells during development for a correct tissue morphogenesis, but also in the adult for maintenance of tissue homeostasis or damage repair. The orientation of the spindle is controlled by a protein complex that includes NuMA, which mediates pulling from the spindle poles and LGN, which links NuMA to the correct regions of the cell cortex. Semaphorin-Plexin signaling is a cell-cell communication pathway involved in many tissues and processes mainly through regulation of the cytoskeleton and adhesion. It has been shown that Plexin-B2 regulates mitotic spindle orientation in kidney epithelial cells and that this regulation is relevant for kidney morphogenesis and repair. However, the molecular mechanisms through which Plexin-B2 controls spindle orientation are still largely unclear. In this work, I demonstrate that Plexin-B2 localizes to cell-cell contacts in the kidney epithelium and in epithelial cell lines in 2D and 3D. Furthermore, I show that Plexin-B2 remains polarized during mitosis. I add that the basolateral localization of Plexin-B2 in the kidney is independent of its ligands, but depends on its intracellular juxtamembrane domain in 3D cultures. Furthermore, I show that this region contains a unique basolateral targeting motif present in all murine class B plexins and conserved in human Plexin-B2. Using CRISPR-Cas genome editing, I confirm that the deletion of Plexin-B2 impairs correct lumen formation in epithelial cell cysts. Additionally, I show that the lack of Plexin-B2 does not influence growth of these cells, but increases the proportion of cells in the S and G2/M phases of the cell cycle and aneuploidy. Importantly, I demonstrate that the deletion of Plexin-B2 does not have an effect on the normal localization of the spindle regulators LGN and NuMA in 3D cell cultures. Therefore, the role of the polarized expression of Plexin-B2 in its control of mitotic spindle orientation and its possible connection with mitotic spindle regulators need to be further investigate

Pals1 functions as a tumor suppressor regulating cell polarity, Hippo signaling and cancer progression

Epithelial cell polarity is of vital importance for the organization and function of epithelial tissues and is primarily maintained by three protein complexes, the Crumbs complex, the Par complex and the Scribble complex. Most of the polarity proteins within these complexes are highly conserved and play pivotal roles in embryonic development, cell-cell adhesion and cell migration. Recent studies have demonstrated that deregulation of epithelial polarity is a hallmark of tumor progression, in particular during the metastatic process. This study was conducted to examine the role of the tight junction protein Pals1 in the maintenance of cell polarity, and cancer. RNA interference and CRISPR/Cas9 gene deletion approaches were used to downregulate or deplete Pals1 in MDCKII, HCT116 and DLD1 cell lines. Reduction in Pals1 resulted in atypical expression levels of polarity proteins and defects in Hippo pathway regulation. Moreover, Pals1 loss caused E-cadherin reduction and enhanced cell migration. Pals1 deficient cells exhibited typical markers, inferring epithelial-to-mesenchymal transition. Further in vivo xenograft experiments revealed a function of Pals1 in cancer progression, as tumors derived from Pals1-deficient cells showed increased growth and more extensive liver and lung metastases. Taken together, these findings support a close link between epithelial cell polarity and tumorigenesis, and suggest the existence of a novel Pals1-mediated mechanism of tumor suppression. Thus, the pathophysiological consequences of Pals1 alteration must be investigated further, and used to develop new therapeutic strategies against this devastating disease

The Hippo pathway in immunity and cancer

The Hippo pathway integrates numerous stimuli into intracellular signaling that informs the cell of its structural features (actin cytoskeleton, polarity, cell shape), location, and surroundings (cell-cell contacts, growth factors, extracellular matrix), instructing cellular survival, proliferation, differentiation and fate. Dysregulated Hippo signaling leads to cell fate and developmental defects, and promotes tumorigenesis. We investigated the role of Hippo pathway transcriptional regulators YAP and TAZ (YAP/TAZ) in cancer metabolic reprograming and showed that they regulate the expression of glutamine transaminases that promote glutamine dependence in breast cancer cells. In breast cancer patients, YAP/TAZ activity positively correlates with transaminase expression, identifying transamination as a prospective clinical target in breast cancers driven by aberrant YAP/TAZ function. Critical regulators of cancer progression in the tumor microenvironment (TME) are T cells, whose activation and differentiation relies on signals regulated by the Hippo pathway in other contexts. We studied the effect of T cell specific YAP deficiency in anti-tumor T cell mediated immunity, which led us in identifying YAP as a suppressor of T cell activation and function. YAP deficiency enhanced CD4+ T cell differentiation to polarized subtypes. In in vivo mouse tumor models, YAP deficiency reduces tumor growth and augments the ability of CD8+ T cells to infiltrate tumors. Tumor infiltrating lymphocyte RNA-Seq identified YAP as a global suppressor of T cell responses in the TME and as a key negative regulator of T cell tumor infiltration and patient survival in many human cancers. Given these newly discovered effects of YAP in T cell biology, we further studied how YAP/TAZ deficiency affects the hallmark of adaptive immunity, T cell memory, during bacterial and viral infection and found that YAP/TAZ inhibit memory T cell generation. Lastly, we investigated the role of Hippo pathway kinases and YAP/TAZ inhibitors LATS1 and LATS2 in T cells and found that in contrast to YAP/TAZ they are indispensable for normal thymocyte development and function. Through this work, we define novel and critical roles of Hippo pathway signaling in T cell development and function, as well as cancer metabolic reprograming and propose T cell specific YAP inhibition for improving immune mediated cancer treatment.2021-01-30T00:00:00