Patrick O. Humbert

Control of tumourigenesis by the Scribble/Dlg/Lgl polarity module

The neoplastic tumour suppressors, Scribble, Dlg and Lgl, originally discovered in the vinegar fly Drosophila melanogaster, are currently being actively studied for their potential role in mammalian tumourigenesis. In Drosophila, these tumour suppressors function in a common genetic pathway to regulate apicobasal cell polarity and also play important roles in the control of cell proliferation, survival, differentiation and in cell migration/invasion. The precise mechanism by which Scribble, Dlg and Lgl function is not clear; however, they have been implicated in the regulation of signalling pathways, vesicle trafficking and in the Myosin II–actin cytoskeleton. We review the evidence for the involvement of Scribble, Dlg, and Lgl in cancer, and how the various functions ascribed to these tumour suppressors in Drosophila and mammalian systems may impact on the process of tumourigenesis.

E2F4 Is Essential for Normal Erythrocyte Maturation and Neonatal Viability

The retinoblastoma protein (pRB) plays a key role in the control of normal development and proliferation through the regulation of the E2F transcription factors. We generated a mutant mouse model to assess the in vivo role of the predominant E2F family member, E2F4. Remarkably, loss of E2F4 had no detectable effect on either cell cycle arrest or proliferation. However, E2F4 was essential for normal development. E2f4-/- mice died of an increased susceptibility to opportunistic infections that appeared to result from craniofacial defects. They also displayed a variety of erythroid abnormalities that arose from a cell autonomous defect in late stage maturation. This suggests that E2F4 makes a major contribution to the control of erythrocyte development by the pRB tumor suppressor.

The tumour-suppressor Scribble dictates cell polarity during directed epithelial migration: regulation of Rho GTPase recruitment to the leading edge

Altered expression of human Scribble is associated with invasive epithelial cancers, however, its role in tumour development remains unclear. Mutations in Drosophila Scribble result in loss of polarity, overproliferation and 3D-tumourous overgrowth of epithelial cells. Using complementation studies in Drosophila we recently demonstrated that expression of human Scribble can also regulate polarity and restrict tissue overgrowth. Here, we have undertaken a detailed study of human Scribble function in the polarized mammary cell line, MCF10A. We show that although Scribble does not seem to be required for apical-basal polarity or proliferation control in MCF10A cells, Scribble is essential for the control of polarity associated with directed epithelial cell migration. Scribble-depleted MCF10A cells show defective in vitro wound closure and chemotactic movement. The cells at the wound edge fail to polarize, show reduced lamellipodia formation and impaired recruitment of Cdc42 and Rac1 to the leading edge. Furthermore, we show that this function is relevant in vivo as Scribble mutant mice show defective epidermal wound healing. This data identifies an essential role for mammalian Scribble in the regulation of the polarity specifically involved in directed epithelial migration.

Multiscale Modeling of Form and Function

Topobiology posits that morphogenesis is driven by differential adhesive interactions among heterogeneous cell populations. This paradigm has been revised to include force-dependent molecular switches, cell and tissue tension, and reciprocal interactions with the microenvironment. It is now appreciated that tissue development is executed through conserved decision-making modules that operate on multiple length scales from the molecular and subcellular level through to the cell and tissue level and that these regulatory mechanisms specify cell and tissue fate by modifying the context of cellular signaling and gene expression. Here, we discuss the origin of these decision-making modules and illustrate how emergent properties of adhesion-directed multicellular structures sculpt the tissue, promote its functionality, and maintain its homeostasis through spatial segregation and organization of anchored proteins and secreted factors and through emergent properties of tissues, including tension fields and energy optimization.

Loss of human Scribble cooperates with H-Ras to promote cell invasion through deregulation of MAPK signalling

Activating mutations in genes of the Ras-mitogen-activated protein kinase (MAPK) pathway occur in approximately 30% of all human cancers; however, mutation of Ras alone is rarely sufficient to induce tumour development. Scribble is a polarity regulator recently isolated from a Drosophila screen for events that cooperate with Ras mutation to promote tumour progression and cell invasion. In mammals, Scribble regulates directed cell migration and wound healing in vivo; however, no role has been identified for mammalian Scribble in oncogenic transformation. Here we show that in human epithelial cells expressing oncogenic Ras or Raf, loss of Scribble promotes invasion of cells through extracellular matrix in an organotypic culture system. Further, we show that the mechanism by which this occurs is in the regulation of MAPK signalling by Scribble. The suppression of MAPK signalling is a highly conserved function of Scribble as it also prevents Raf-mediated defects in Drosophila wing development. Our data identify Scribble as an important mediator of MAPK signalling and provide a molecular basis for the observation that Scribble expression is decreased in many invasive human cancers.

Epidermal Wound Repair Is Regulated by the Planar Cell Polarity Signaling Pathway

The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. Here, we show that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, Celsr1, PTK7, and Scrb1, and the transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects, and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3(-)(/-) mice, we identified RhoGEF19, a homolog of a RhoA activator involved in PCP signaling in Xenopus, as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerization, cellular polarity, and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling and broadly implicate this pathway in epidermal repair.

SCRIB expression is deregulated in human prostate cancer, and its deficiency in mice promotes prostate neoplasia

Loss of cellular polarity is a hallmark of epithelial cancers, raising the possibility that regulators of polarity have a role in suppressing tumorigenesis. The Scribble complex is one of at least three interacting protein complexes that have a critical role in establishing and maintaining epithelial polarity. In human colorectal, breast, and endometrial cancers, expression of the Scribble complex member SCRIB is often mislocalized and deregulated. Here, we report that Scrib is indispensable for prostate homeostasis in mice. Scrib heterozygosity initiated prostate hyperplasia, while targeted biallelic Scrib loss predisposed mice to prostate intraepithelial neoplasia. Mechanistically, Scrib was shown to negatively regulate the MAPK cascade to suppress tumorigenesis. Further analysis revealed that prostate-specific loss of Scrib in mice combined with expression of an oncogenic Kras mutation promoted the progression of prostate cancer that recapitulated the human disease. The clinical significance of the work in mice was highlighted by our observation that SCRIB deregulation strongly correlated with poor survival in human prostate cancer. These data suggest that the polarity network could provide a new avenue for therapeutic intervention.

Polarity Regulators and the Control of Epithelial Architecture, Cell Migration, and Tumorigenesis

A large body of work on Drosophila melanogaster has identified and characterized a number of key polarity regulators, many of which are required for the regulation of multiple other processes including proliferation, migration, invasion, and tumorigenesis. Humans possess either single or multiple homologues of each of the Drosophila polarity proteins, and in most cases, these are highly conserved between species, implying an important and conserved function for each of the polarity complexes. Recent studies in cultured mammalian epithelial cells have shed some light on the requirement for the polarity complexes in the regulation of epithelial cell function, including an unexpected link to the regulation of directed cell migration. However, many questions still remain regarding the molecular mechanisms of polarity regulation and whether disruption of polarity protein function is an important step in the development of human cancers. Here we will review what is currently understood about the regulation of cell polarity, migration, and invasion and the level of functional conservation between Drosophila and mammalian tissues. Particular reference will be made as to how the Scribble and Par polarity complexes may be involved in the regulation of apical-basal polarity, migration, and tumorigenesis.

HScrib is a functional homologue of the Drosophila tumour suppressor Scribble

Scribble (scrib), discs large (dlg) and lethal giant larvae (lgl) encode proteins that regulate cell polarity and have been identified as neoplastic tumour suppressor genes in Drosophila melanogaster. Here, we have used the Drosophila model system to provide the first functional evidence that human Scribble (hScrib) can act as a tumour suppressor. We show that hScrib protein displays highly polarized localization in mammalian epithelial cells and colocalizes with mammalian Dlg, similar to D. melanogaster Scribble (DmScrib) distribution in Drosophila epithelium. Furthermore, hScrib can rescue the polarity and tumorous overgrowth defects of scrib mutant Drosophila. hScrib therefore can act as an effective tumour suppressor in vivo, regulating both apical–basal polarity and cellular proliferation in a manner similar to that of DmScrib in Drosophila. These data demonstrate that hScrib is a functional homologue of DmScrib and therefore predict an important role for hScrib in the suppression of mammalian tumorigenesis.

Asymmetric Cell Division of T Cells upon Antigen Presentation Uses Multiple Conserved Mechanisms

Asymmetric cell division is a potential means by which cell fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric division of T cells is paramount for determining the role of this process in the generation of effector and memory T cell subsets. In other cell types, asymmetric cell division is regulated by conserved polarity protein complexes that control the localization of cell fate determinants and spindle orientation during division. We have developed a tractable, in vitro model of naive CD8+ T cells undergoing initial division while attached to dendritic cells during Ag presentation to investigate whether similar mechanisms might regulate asymmetric division of T cells. Using this system, we show that direct interactions with APCs provide the cue for polarization of T cells. Interestingly, the immunological synapse disseminates before division even though the T cells retain contact with the APC. The cue from the APC is translated into polarization of cell fate determinants via the polarity network of the Par3 and Scribble complexes, and orientation of the mitotic spindle during division is orchestrated by the partner of inscuteable/G protein complex. These findings suggest that T cells have selectively adapted a number of evolutionarily conserved mechanisms to generate diversity through asymmetric cell division.