The Drosophila tumour suppressor Lgl and Vap33 activate the Hippo pathway through a dual mechanism.

The tumour suppressor, Lethal (2) giant larvae [Lgl; also known as L(2)gl], is an evolutionarily conserved protein that was discovered in the vinegar fly Drosophila, where its depletion results in tissue overgrowth and loss of cell polarity. Lgl links cell polarity and tissue growth through regulation of the Notch and the Hippo signalling pathways. Lgl regulates the Notch pathway by inhibiting V-ATPase activity via Vap33. How Lgl regulates the Hippo pathway was unclear. In this current study, we show that V-ATPase activity inhibits the Hippo pathway, whereas Vap33 acts to activate Hippo signalling. Vap33 physically and genetically interacts with the actin cytoskeletal regulators RtGEF (Pix) and Git, which also bind to the Hippo protein (Hpo) and are involved in the activation of the Hippo pathway. Additionally, we show that the ADP ribosylation factor Arf79F (Arf1), which is a Hpo interactor, is involved in the inhibition of the Hippo pathway. Altogether, our data suggest that Lgl acts via Vap33 to activate the Hippo pathway by a dual mechanism: (1) through interaction with RtGEF, Git and Arf79F, and (2) through interaction and inhibition of the V-ATPase, thereby controlling epithelial tissue growth.

Transcriptional repression of Myc underlies the tumour suppressor function of AGO1 in Drosophila.

Here, we report novel tumour suppressor activity for the Drosophila Argonaute family RNA-binding protein AGO1, a component of the miRNA-dependent RNA-induced silencing complex (RISC). The mechanism for growth inhibition does not, however, involve canonical roles as part of the RISC; rather, AGO1 controls cell and tissue growth by functioning as a direct transcriptional repressor of the master regulator of growth, Myc. AGO1 depletion in wing imaginal discs drives a significant increase in ribosome biogenesis, nucleolar expansion and cell growth in a manner dependent on Myc abundance. Moreover, increased Myc promoter activity and elevated Myc mRNA in AGO1-depleted animals requires RNA polymerase II transcription. Further support for transcriptional AGO1 functions is provided by physical interaction with the RNA polymerase II transcriptional machinery (chromatin remodelling factors and Mediator Complex), punctate nuclear localisation in euchromatic regions and overlap with Polycomb Group transcriptional silencing loci. Moreover, significant AGO1 enrichment is observed on the Myc promoter and AGO1 interacts with the Myc transcriptional activator Psi. Together, our data show that Drosophila AGO1 functions outside of the RISC to repress Myc transcription and inhibit developmental cell and tissue growth.This article has an associated 'The people behind the papers' interview.

Using Mouse and Drosophila Models to Investigate the Mechanistic Links between Diet, Obesity, Type II Diabetes, and Cancer.

Many of the links between diet and cancer are controversial and over simplified. To date, human epidemiological studies consistently reveal that patients who suffer diet-related obesity and/or type II diabetes have an increased risk of cancer, suffer more aggressive cancers, and respond poorly to current therapies. However, the underlying molecular mechanisms that increase cancer risk and decrease the response to cancer therapies in these patients remain largely unknown. Here, we review studies in mouse cancer models in which either dietary or genetic manipulation has been used to model obesity and/or type II diabetes. These studies demonstrate an emerging role for the conserved insulin and insulin-like growth factor signaling pathways as links between diet and cancer progression. However, these models are time consuming to develop and expensive to maintain. As the world faces an epidemic of obesity and type II diabetes we argue that the development of novel animal models is urgently required. We make the case for Drosophila as providing an unparalleled opportunity to combine dietary manipulation with models of human metabolic disease and cancer. Thus, combining diet and cancer models in Drosophila can rapidly and significantly advance our understanding of the conserved molecular mechanisms that link diet and diet-related metabolic disorders to poor cancer patient prognosis.

Lgl reduces endosomal vesicle acidification and Notch signaling by promoting the interaction between Vap33 and the V-ATPase complex.

Epithelial cell polarity is linked to the control of tissue growth and tumorigenesis. The tumor suppressor and cell polarity protein lethal-2-giant larvae (Lgl) promotes Hippo signaling and inhibits Notch signaling to restrict tissue growth in Drosophila melanogaster Notch signaling is greater in lgl mutant tissue than in wild-type tissue because of increased acidification of endosomal vesicles, which promotes the proteolytic processing and activation of Notch by γ-secretase. We showed that the increased Notch signaling and tissue growth defects of lgl mutant tissue depended on endosomal vesicle acidification mediated by the vacuolar adenosine triphosphatase (V-ATPase). Lgl promoted the activity of the V-ATPase by interacting with Vap33 (VAMP-associated protein of 33 kDa). Vap33 physically and genetically interacted with Lgl and V-ATPase subunits and repressed V-ATPase-mediated endosomal vesicle acidification and Notch signaling. Vap33 overexpression reduced the abundance of the V-ATPase component Vha44, whereas Lgl knockdown reduced the binding of Vap33 to the V-ATPase component Vha68-3. Our data indicate that Lgl promotes the binding of Vap33 to the V-ATPase, thus inhibiting V-ATPase-mediated endosomal vesicle acidification and thereby reducing γ-secretase activity, Notch signaling, and tissue growth. Our findings implicate the deregulation of Vap33 and V-ATPase activity in polarity-impaired epithelial cancers.

miR-9a mediates the role of Lethal giant larvae as an epithelial growth inhibitor in Drosophila.

Drosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, we hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, we found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling we identified a core set of ten miRNAs that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which we found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, we overexpressed it in the context of lgl knock-down by RNAi and found it able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling in lgl mutant tissues and human breast cancer cells identified thrombospondin (tsp) as a common factor altered in both fly and human breast cancer tumorigenesis models. Our work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates that miR-9a mediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression.

A Kinome RNAi Screen in Drosophila Identifies Novel Genes Interacting with Lgl, aPKC, and Crb Cell Polarity Genes in Epithelial Tissues.

In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila "cell polarity" eye and wing phenotypes. Initially, we identified kinase or phosphatase genes whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, Jun kinase (JNK), and Notch signaling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras, and lipid/Phospho-inositol-3-kinase (PI3K) signaling pathways. Additionally, we demonstrated that the "nutrient sensing" kinases Salt Inducible Kinase 2 and 3 (SIK2 and 3) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, our screen has revealed novel cell polarity-interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development.

Defective Hfp-dependent transcriptional repression of dMYC is fundamental to tissue overgrowth in Drosophila XPB models.

Nucleotide excision DNA repair (NER) pathway mutations cause neurodegenerative and progeroid disorders (xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD)), which are inexplicably associated with (XP) or without (CS/TTD) cancer. Moreover, cancer progression occurs in certain patients, but not others, with similar C-terminal mutations in the XPB helicase subunit of transcription and NER factor TFIIH. Mechanisms driving overproliferation and, therefore, cancer associated with XPB mutations are currently unknown. Here using Drosophila models, we provide evidence that C-terminally truncated Hay/XPB alleles enhance overgrowth dependent on reduced abundance of RNA recognition motif protein Hfp/FIR, which transcriptionally represses the MYC oncogene homologue, dMYC. The data demonstrate that dMYC repression and dMYC-dependent overgrowth in the Hfp hypomorph is further impaired in the C-terminal Hay/XPB mutant background. Thus, we predict defective transcriptional repression of MYC by the Hfp orthologue, FIR, might provide one mechanism for cancer progression in XP/CS.

Regulation of Notch signaling and endocytosis by the Lgl neoplastic tumor suppressor.

The evolutionarily conserved neoplastic tumor suppressor protein, Lethal (2) giant larvae (Lgl), plays roles in cell polarity and tissue growth via regulation of the Hippo pathway. In our recent study, we showed that in the developing Drosophila eye epithelium, depletion of Lgl leads to increased ligand-dependent Notch signaling. lgl mutant tissue also exhibits an accumulation of early endosomes, recycling endosomes, early-multivesicular body markers and acidic vesicles. We showed that elevated Notch signaling in lgl(-) tissue can be rescued by feeding larvae the vesicle de-acidifying drug chloroquine, revealing that Lgl attenuates Notch signaling by limiting vesicle acidification. Strikingly, chloroquine also rescued the lgl(-) overgrowth phenotype, suggesting that the Hippo pathway defects were also rescued. In this extraview, we provide additional data on the regulation of Notch signaling and endocytosis by Lgl, and discuss possible mechanisms by which Lgl depletion contributes to signaling pathway defects and tumorigenesis.

Lgl regulates Notch signaling via endocytosis, independently of the apical aPKC-Par6-Baz polarity complex.

BACKGROUND: The Drosophila melanogaster junctional neoplastic tumor suppressor, Lethal-2-giant larvae (Lgl), is a regulator of apicobasal cell polarity and tissue growth. We have previously shown in the developing Drosophila eye epithelium that, without affecting cell polarity, depletion of Lgl results in ectopic cell proliferation and blockage of developmental cell death due to deregulation of the Hippo signaling pathway. RESULTS: Here, we show that Notch signaling is increased in lgl-depleted eye tissue, independently of Lgl's function in apicobasal cell polarity. The upregulation of Notch signaling is ligand dependent and correlates with accumulation of cleaved Notch. Concomitant with higher cleaved Notch levels in lgl- tissue, early endosomes (Avalanche [Avl+]), recycling endosomes (Rab11+), early multivesicular bodies (Hrs+), and acidified vesicles, but not late endosomal markers (Car+ and Rab7+), accumulate. Colocalization studies revealed that Lgl associates with early to late endosomes and lysosomes. Upregulation of Notch signaling in lgl- tissue requires dynamin- and Rab5-mediated endocytosis and vesicle acidification but is independent of Hrs/Stam or Rab11 activity. Furthermore, Lgl regulates Notch signaling independently of the aPKC-Par6-Baz apical polarity complex. CONCLUSIONS: Altogether, our data show that Lgl regulates endocytosis to restrict vesicle acidification and prevent ectopic ligand-dependent Notch signaling. This Lgl function is independent of the aPKC-Par6-Baz polarity complex and uncovers a novel attenuation mechanism of ligand-activated Notch signaling during Drosophila eye development.

lgl Regulates the Hippo Pathway Independently of Fat/Dachs, Kibra/Expanded/Merlin and dRASSF/dSTRIPAK.

In both Drosophila and mammalian systems, the Hippo (Hpo) signalling pathway controls tissue growth by inhibiting cell proliferation and promoting apoptosis. The core pathway consists of a protein kinase Hpo (MST1/2 in mammals) that is regulated by a number of upstream inputs including Drosophila Ras Association Factor, dRASSF. We have previously shown in the developing Drosophila eye epithelium that loss of the apico-basal cell polarity regulator lethal-(2)-giant-larvae (lgl), and the concomitant increase in aPKC activity, results in ectopic proliferation and suppression of developmental cell death by blocking Hpo pathway signalling. Here, we further explore how Lgl/aPKC interacts with the Hpo pathway. Deregulation of the Hpo pathway by Lgl depletion is associated with the mislocalization of Hpo and dRASSF. We demonstrate that Lgl/aPKC regulate the Hpo pathway independently of upstream inputs from Fat/Dachs and the Kibra/Expanded/Merlin complex. We show depletion of Lgl also results in accumulation and mislocalization of components of the dSTRIPAK complex, a major phosphatase complex that directly binds to dRASSF and represses Hpo activity. However, depleting dSTRIPAK components, or removal of dRASSF did not rescue the lgl-/- or aPKC overexpression phenotypes. Thus, Lgl/aPKC regulate Hpo activity by a novel mechanism, independently of dRASSF and dSTRIPAK. Surprisingly, removal of dRASSF in tissue with increased aPKC activity results in mild tissue overgrowth, indicating that in this context dRASSF acts as a tumor suppressor. This effect was independent of the Hpo and Ras Mitogen Activated Protein Kinase (MAPK) pathways, suggesting that dRASSF regulates a novel pathway to control tissue growth.

Lgl, the SWH pathway and tumorigenesis: It's a matter of context & competition!

Loss of function of the neoplastic tumors suppressors, lgl, scrib and dlg or overexpression of the apical polarity components, Crumbs and atypical protein kinase C (aPKC), are associated with polarity loss and tissue overgrowth, however, the mechanism behind these effects is poorly understood. In our recent study, we showed that Lgl, aPKC and Crumbs mediate their effects on proliferation and survival via the Salvador/Warts/Hippo (SWH) tumor suppressor pathway. Loss of lgl can lead to substantial overgrowth, however the lgl mutant phenotype can be quite variable and the amount of overgrowth of the mutant tissue, its survival and ultimate fate is strongly determined by context and competition. In this extra-view we present a more detailed description of the lgl mutant phenotype and highlight the phenotypic differences between lgl and SWH pathway mutant phenotypes. In addition, we explore the role for the Jun kinase (JNK) pathway in the development of the lgl mutant phenotype.

Lgl/aPKC and Crb regulate the Salvador/Warts/Hippo pathway.

A key goal of developmental biology is to understand the mechanisms that coordinate organ growth. It has long been recognized that the genes that control apico-basal cell polarity also regulate tissue growth. How loss of cell polarity contributes to tissue overgrowth has been the subject of much speculation. Do loss-of-function mutations in cell polarity regulators result in secondary effects that globally deregulate cell proliferation, or do these genes specifically control growth pathways? Three recent papers have shown that the apico-basal polarity determinants Lgl/aPKC and Crb regulate tissue growth independently of their roles in cell polarity and coordinately regulate cell proliferation and cell death via the Salvador/Warts/Hippo (SWH) pathway. Lgl/aPKC are required for the correct localization of Hippo (Hpo)/Ras associated factor (RASSF), while Crb regulates the levels and localization of Expanded (Ex), indicating that cell polarity determinants modify SWH pathway activity by distinct mechanisms. Here, we review the key data that support these conclusions, highlight remaining questions and speculate on the underlying mechanisms by which the cell polarity complexes interact with the SWH pathway. Understanding the interactions between cell polarity regulators and the SWH pathway will improve our knowledge of how epithelial organization and tissue growth are coordinated during development and perturbed in disease states such as cancer.

Geminin and Brahma act antagonistically to regulate EGFR-Ras-MAPK signaling in Drosophila.

Geminin was identified in Xenopus as a dual function protein involved in the regulation of DNA replication and neural differentiation. In Xenopus, Geminin acts to antagonize the Brahma (Brm) chromatin-remodeling protein, Brg1, during neural differentiation. Here, we investigate the interaction of Geminin with the Brm complex during Drosophila development. We demonstrate that Drosophila Geminin (Gem) interacts antagonistically with the Brm-BAP complex during wing development. Moreover, we show in vivo during wing development and biochemically that Brm acts to promote EGFR-Ras-MAPK signaling, as indicated by its effects on pERK levels, while Gem opposes this. Furthermore, gem and brm alleles modulate the wing phenotype of a Raf gain-of-function mutant and the eye phenotype of a EGFR gain-of-function mutant. Western analysis revealed that Gem over-expression in a background compromised for Brm function reduces Mek (MAPKK/Sor) protein levels, consistent with the decrease in ERK activation observed. Taken together, our results show that Gem and Brm act antagonistically to modulate the EGFR-Ras-MAPK signaling pathway, by affecting Mek levels during Drosophila development.

Lgl, aPKC, and Crumbs regulate the Salvador/Warts/Hippo pathway through two distinct mechanisms.

BACKGROUND: The Drosophila neoplastic tumor suppressor Lethal (2) giant larvae (Lgl) controls apicobasal cell polarity and proliferation. We have previously shown that lgl(-) clones in the developing eye exhibit ectopic proliferation and suppress apoptosis without affecting apicobasal cell polarity. Ectopic expression of the apical polarity regulators atypical protein kinase C (aPKC) and Crumbs also leads to increased cell proliferation and/or survival. Here we investigate how these cell polarity regulators control proliferation and survival. RESULTS: We report that depletion of lgl in eye epithelial tissue, where polarity is maintained, results in upregulation of targets of the Salvador/Warts/Hippo (SWH) tumor suppressor pathway. Consistent with this, the SWH pathway transcriptional coactivator Yorkie is hyperactivated in Lgl-deficient tissue and is rate limiting for lgl(-) phenotypes. Overexpression of the apical polarity regulators Crumbs or aPKC also leads to ectopic expression of SWH pathway targets without affecting polarity. We show that Lgl depletion or aPKC overexpression results in comislocalization of Hippo and Ras-associated domain family protein (RASSF), consistent with RASSF's ability to block Hippo activation by Salvador. In contrast, Crumbs overexpression leads to mislocalization of Expanded away from the apical cortex, which is predicted to deregulate the pathway. CONCLUSIONS: Collectively, our data reveal that the cell polarity regulators Lgl, aPKC, and Crumbs regulate the SWH pathway by two distinct pathways: Lgl acts antagonistically to aPKC to regulate Hippo and RASSF localization, whereas Crumbs regulates Expanded localization. Thus, our study implicates Lgl, aPKC, and Crumbs as regulators of tissue growth via the SWH pathway.

Laboratory diagnostic aspects of drug resistant tuberculosis.

Multi-drug resistant strains of Mycobacterium tuberculosis (MDR-TB) are present world wide, and in many areas constitute a serious threat to the efficacy of TB control programs. The most effective strategies to limit further spread of MDR-TB are rapid detection of drug resistance followed by prompt and effective therapy. Routine laboratory diagnosis of drug resistance in TB requires a viable, pure culture of M. tuberculosis. Use of liquid media has decreased the turn around time for susceptibility test results however, because of the slow growth of M. tuberculosis these assays can still take 10 to 14 days. Alternatively, an increased understanding of the molecular basis for resistance to the antituberculosis drugs can greatly contribute to further decreasing turn around time. Based on this information, more precise and rapid molecular testing can be developed and lead to more appropriate and timely treatment regimens. In this review, we discuss methods for, and problems encountered in, performing TB drug susceptibility assays. Descriptions of routine protocols will be followed by recent developments in molecular detection of drug resistance.