Loss of Cell Adhesion Increases Tumorigenic Potential of Polarity Deficient Scribble Mutant Cells

Epithelial polarity genes are important for maintaining tissue architecture, and regulating growth. The Drosophila neoplastic tumor suppressor gene scribble (scrib) belongs to the basolateral polarity complex. Loss of scrib results in disruption of its growth regulatory functions, and downregulation or mislocalization of Scrib is correlated to tumor growth. Somatic scribble mutant cells (scrib-) surrounded by wild-type cells undergo apoptosis, which can be prevented by introduction of secondary mutations that provide a growth advantage. Using genetic tools in Drosophila, we analyzed the phenotypic effects of loss of scrib in different growth promoting backgrounds. We investigated if a central mechanism that regulates cell adhesion governs the growth and invasive potential of scrib mutant cells. Here we show that increased proliferation, and survival abilities of scrib- cells in different genetic backgrounds affect their differentiation, and intercellular adhesion. Further, loss of scrib is sufficient to cause reduced cell survival, activation of the JNK pathway and a mild reduction of cell adhesion. Our data show that for scrib cells to induce aggressive tumor growth characterized by loss of differentiation, cell adhesion, increased proliferation and invasion, cooperative interactions that derail signaling pathways play an essential role in the mechanisms leading to tumorigenesis. Thus, our study provides new insights on the effects of loss of scrib and the modification of these effects via cooperative interactions that enhance the overall tumorigenic potential of scrib deficient cells

Scribble Acts in the Drosophila Fat-Hippo Pathway to Regulate Warts Activity

Epithelial cells are the major cell-type for all organs in multicellular organisms. In order to achieve correct organ size, epithelial tissues need mechanisms that limit their proliferation, and protect tissues from damage caused by defective epithelial cells. Recently, the Hippo signaling pathway has emerged as a major mechanism that orchestrates epithelial development. Hippo signaling is required for cells to stop proliferation as in the absence of Hippo signaling tissues continue to proliferate and produce overgrown organs or tumors. Studies in Drosophila have led the way in providing a framework for how Hippo alters the pattern of gene transcription in target cells, leading to changes in cell proliferation, survival, and other behaviors. Scribble (Scrib) belongs to a class of neoplastic tumor suppressor genes that are required to establish apical-basal cell polarity. The disruption of apical-basal polarity leads to uncontrolled cell proliferation of epithelial cells. The interaction of apical basal polarity genes with the Hippo pathway has been an area of intense investigation. Loss of Scrib has been known to affect Hippo pathway targets, however, its functions in the Hippo pathway still remain largely unknown. We investigated the interactions of Scrib with the Hippo pathway. We present data suggesting that Drosophila Scrib acts downstream of the Fat (Ft) receptor, and requires Hippo signaling for its growth regulatory functions. We show that Ft requires Scrib to interact with Expanded (Ex) and Dachs (D), and for regulating Warts (Wts) levels and stability, thus placing Scrib in the Hippo pathway network

Intercellular Cooperation and Competition in Brain Cancers: Lessons From Drosophila and Human Studies

Glioblastoma (GBM) is a primary brain cancer with an extremely poor prognosis. GBM tumors contain heterogeneous cellular components, including a small subpopulation of tumor cells termed glioma stem cells (GSCs). GSCs are characterized as chemotherapy- and radiotherapy-resistant cells with prominent tumorigenic ability. Studies in Drosophila cancer models demonstrated that interclonal cooperation and signaling from apoptotic clones provokes aggressive growth of neighboring tumorigenic clones, via compensatory proliferation or apoptosis induced proliferation. Mechanistically, these aggressive tumors depend on activation of Jun-N-terminal kinase (upstream of c-JUN), and Drosophila Wnt (Wg) in the apoptotic clones. Consistent with these nonmammalian studies, data from several mammalian studies have shown that c-JUN and Wnt are hyperactivated in aggressive tumors (including GBM). However, it remains elusive whether compensatory proliferation is an evolutionarily conserved mechanism in cancers. In the present report, we summarize recent studies in Drosophila models and mammalian models (e.g., xenografts of human cancer cells into small animals) to elucidate the intercellular interactions between the apoptosis-prone cancer cells (e.g., non-GSCs) and the hyperproliferative cancer cells (e.g., GSCs). These evolving investigations will yield insights about molecular signaling interactions in the context of post-therapeutic phenotypic changes in human cancers. Furthermore, these studies are likely to revise our understanding of the genetic changes and post-therapeutic cell-cell interactions, which is a vital area of cancer biology with wide applications to many cancer types in humans

FOXD1-ALDH1A3 signaling is a determinant for the self-renewal and tumorigenicity of mesenchymal glioma stem cells

Glioma stem-like cells (GSC) with tumor-initiating activity orchestrate the cellular hierarchy in glioblastoma and engender therapeutic resistance. Recent work has divided GSC into two subtypes with a mesenchymal (MES) GSC population as the more malignant subtype. In this study, we identify the FOXD1-ALDH1A3 signaling axis as a determinant of the MES GSC phenotype. The transcription factor FOXD1 is expressed predominantly in patient-derived cultures enriched with MES, but not with the proneural GSC subtype. shRNA-mediated attenuation of FOXD1 in MES GSC ablates their clonogenicity in vitro and in vivo. Mechanistically, FOXD1 regulates the transcriptional activity of ALDH1A3, an established functional marker for MES GSC. Indeed, the functional roles of FOXD1 and ALDH1A3 are likely evolutionally conserved, insofar as RNAi-mediated attenuation of their orthologous genes in Drosophila blocks formation of brain tumors engineered in that species. In clinical specimens of high-grade glioma, the levels of expression of both FOXD1 and ALDH1A3 are inversely correlated with patient prognosis. Finally, a novel small-molecule inhibitor of ALDH we developed, termed GA11, displays potent in vivo efficacy when administered systemically in a murine GSC-derived xenograft model of glioblastoma. Collectively, our findings define a FOXD1-ALDH1A3 pathway in controling the clonogenic and tumorigenic potential of MES GSC in glioblastoma tumors

Investigation of altered cell-cell interactions and signaling mechanisms in Drosophila tumor models

Tumors develop as a result of mutations in multiple genes which typically include tumor suppressor genes, or proto-oncogenes. These mutations promote abnormal proliferation and evasion of cell death resulting in tumor progression and metastasis. Solid tumors arise and grow amongst normal cells. Therefore, they are constantly in communication with their normal neighbors. The intercellular signaling interactions between emerging cancer cells and their neighbors have significant impact on tumor growth. We used scribble loss of function as a model to study altered intercellular interactions between mutant cells and their neighbors in Drosophila melanogaster. scribble⁻/⁻ cells form neoplastic tumors in a homotypic environment. In contrast, they are eliminated when surrounded by normal cells. However, their growth potential can be enhanced in the presence of secondary growth promoting mutations. We discovered that altering the genetic background ofscribble⁻/⁻ cells results in alterations in intercellular interactions that underlie their growth potential. We found that scribble⁻/⁻ has context dependent effects on proliferation, apoptosis, differentiation, adhesion and metastasis that is linked to tumor growth and progression. Amongst all the genetic combinations we studied only overexpression of Ras V¹² in scribble⁻/⁻ cells gives rise to neoplastic tumors. All other growth promoting modifiers enhanced the survival of scribble⁻/⁻ cells without causing neoplasia suggesting that additional growth promoting mutations are required to cause their aggressive growth. We discovered a novel tumor specific signaling network where Wingless acts as a major driver of aggressive growth upstream of Dronc to induce Jun N-terminal Kinase-Yorkie signal amplification positive feedback loop. The organization of this signaling hierarchy is further important to promote invasiveness of Ras V¹², scribble⁻/⁻ tumors. As a side project, we also tested if the mechanism of aggressive growth identified in the epithelial tumors was conserved in tumors of other cell type such as glioma. Using previously established glioma model generated by overexpression of Ras V¹² and Phosphatidylinositol-4,5-bisphosphate 3-kinase pathway, we discovered that at least 2 signaling proteins (Wingless and Yorkie) are induced in glioma and may underlie glioma pathogenesis. We further show that during gliomagenesis, over proliferation of glial cells occurs due to increase in number of progenitor cells. Lastly, we established that Forkhead Domain 59 mediated regulation of Aldehyde Dehydrogenase is an evolutionarily conserved regulatory mechanism required for aggressive glioma growth in flies. Forkhead Box D1 (vertebrate homolog of Forkhead Domain 59) mediated regulation of Aldehyde Dehydrogenase 1A3 was identified in Dr. I Nakano\u27s lab (our collaborator) as a novel mechanism via which Mesenchymal Glioma Stem Cells maintain their phenotype and participate in aggressive growth. Taken together, our research identified novel key signaling network that promotes tumorigenesis in Drosophila epithelial models by altering intercellular interactions. We further show that this signaling network may also be conserved in glioma model. Our results can be extrapolated to other tumor models providing helpful insights into designing novel therapeutic approaches

Wnt Signaling in Stem Cell Maintenance and Differentiation in the Drosophila Germarium

Wnt signaling is a conserved regulator of stem cell behaviors, and the Drosophila germarium has been an important model tissue for the study of stem cell maintenance, differentiation, and proliferation. Here we review Wnt signaling in the germarium, which houses two distinct types of ovarian stem cells: the anteriorly located germline stem cells (GSCs), which give rise to oocytes; and the mid-posteriorly located follicle stem cells (FSCs), which give rise to the somatic follicle cells that cover a developing oocyte. The maintenance and proliferation of GSCs and FSCs is regulated by the stem cell niches, whereas differentiation of the germline is regulated by the differentiation niche. Four distinct Wnt ligands are localized in the germarium, and we focus review on how these Wnt ligands and Wnt signaling affects maintenance and differentiation of both germline and follicle stem cells in their respective niches

Armadillo expression is disrupted at the AJs during neoplastic growth.

<p>Arm expression and localization (red, and greyscale in A-D) in eye discs containing GFP positive <i>eyFLP</i> MARCM clones of the genotype (A) Wild-type, (B) <i>scrib</i>^-, (C) <i>Ras</i>^<i>V12</i>, and (D) <i>Ras</i>^<i>V12</i>,<i>scrib</i>^- clones (GFP, green) is shown. The yellow arrows in panels A-D highlight mutant cells in different genotypes.</p

Models depicting changes in <i>scrib</i> mutant cells in different genetic backgrounds.

<p>The image shows changes in signaling pathways, and cell adhesion in <i>scrib</i> mutant cells, and changes to these signals in <i>scrib</i> cells combined with different growth modifiers.</p

Effect of loss of <i>scrib</i> on JNK/MMP1 signaling.

<p>Panels show eye imaginal discs stained for MMP1 (red, greyscale in A-F) from the following genotypes: (A) Wild-type, (B) <i>scrib</i>^-, (C) <i>scrib</i>^-<i>/M</i>, (D) <i>p35+scrib</i>^-, (E) <i>scrib</i>^-,<i>wts</i>^-, and (F) <i>Ras</i>^<i>V12</i>,<i>scrib</i>^-. Clones in A-B and D-F are GFP positive, and clones in C are GFP-negative, and are marked by yellow arrows.</p

Alterations in Fas2 expression due to loss of <i>scrib</i>.

<p>Eye imaginal discs stained with antibody against <i>Drosophila</i> Fas2 (red, and greyscale in A-F) are shown in somatic clones of the genotypes (A) Wild-type (B) <i>scrib</i>^- (C) <i>scrib</i>^-<i>/M</i>, (D) <i>p35+scrib</i>^-, (E) <i>scrib</i>^-,<i>wts</i>^-, and (F) <i>Ras</i>^<i>V12</i>,<i>scrib</i>^-. The effect on Fas2 expression, and localization was assayed in clones that were present posterior to the MF where Fas2 is endogenously expressed. The yellow arrows in panels B-F point to the clones depicting changes in Fas2 expression.</p