The dark side of hippo signaling: A cancer promoter role

The Hippo signaling pathway regulates organ size and tissue homeostasis. Given this role it is unsurprising that dysregulation of this pathway has implications for cancer progression. A convincing body of literature shows that the Hippo pathway serves a tumor suppressive function with its inactivation leading to massive overgrowth. However, additional studies have also shown that activation of Hippo signaling can promote tumor progression. It remains unknown how a single pathway can produce such diametrically opposed effects. This lack of knowledge is in part due to our inability to make meaningful comparisons from studies which have taken place in a variety of cell types, tissues, and organisms. Recently however, we have published 2 studies using the Drosophila wing disk to study the Hippo pathway and have found that Hippo pathway activation can promote cell migration and invasion while Hippo pathway inactivation leads to overgrowth. Thus we propose here that Drosophila can provide a research platform with which to begin addressing how the Hippo pathway can both enhance and suppress tumor progression due to published pro- and anti-tumor functionalities of the Hippo pathway in the same tissue.     

HP1a-mediated heterochromatin formation inhibits high dietary sugar-induced tumor progression

High dietary sugar (HDS) is a modern dietary concern that involves excessive consumption of carbohydrates and added sugars, and increases the risk of metabolic disorders and associated cancers. However, epigenetic mechanisms by which HDS induces tumor progression remain unclear. Here, we investigate the role of heterochromatin, an important yet poorly understood part of the epigenome, in HDS-induced tumor progression of Drosophila Ras/Src and Ras/scrib tumor systems. We found that increased heterochromatin formation with overexpression of heterochromatin protein 1a (HP1a), specifically in tumor cells, not only decreases HDS-induced tumor growth/burden but also drastically improves survival of Drosophila with HDS and Ras/Src or Ras/scrib tumors. Moreover, HDS reduces heterochromatin levels in tumor cells. Mechanistically, we demonstrated that increased heterochromatin formation decreases wingless (wg) and Hippo (Hpo) signaling, thereby promoting apoptosis, via inhibition of Yorkie (Yki) nuclear accumulation and upregulation of apoptotic genes, and reduces DNA damage in tumor cells under HDS. Taken together, our work identified a novel epigenetic mechanism by which HP1a-mediated heterochromatin formation suppresses HDS-induced tumor progression likely by decreasing wingless and Hippo signaling, increasing apoptosis, and maintaining genome stability. Our model explains that the molecular, cellular, and organismal aspects of HDS-aggravated tumor progression are dependent on heterochromatin formation, and highlights heterochromatin as a therapeutic target for cancers associated with HDS-induced metabolic disorders.Subject terms: Cancer metabolism, Chromatin structure     

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.     

β-Spectrin Regulates the Hippo Signaling Pathway and Modulates the Basal Actin Network

Emerging evidence suggests functional regulation of the Hippo pathway by the actin cytoskeleton, although the detailed molecular mechanism remains incomplete. In a genetic screen, we identified a requirement for β-Spectrin in the posterior follicle cells for the oocyte repolarization process during Drosophila mid-oogenesis. β-spectrin mutations lead to loss of Hippo signaling activity in the follicle cells. A similar reduction of Hippo signaling activity was observed after β-Spectrin knockdown in mammalian cells. We further demonstrated that β-spectrin mutations disrupt the basal actin network in follicle cells. The abnormal stress fiber-like actin structure on the basal side of follicle cells provides a likely link between the β-spectrin mutations and the loss of the Hippo signaling activity phenotype.     

Hippo Signaling Mediates Proliferation,Invasiveness, and Metastatic Potential of Clear Cell Renal Cell Carcinoma

Recent work has identified dysfunctional Hippo signaling to be involved in maintenance and progression of various human cancers, although data on clear cell renal cell carcinoma (ccRCC) have been limited. Here, we provide evidence implicating aberrant Hippo signaling in ccRCC proliferation, invasiveness, and metastatic potential. Nuclear overexpression of the Hippo target Yes-associated protein (YAP) was found in a subset of patients with ccRCC. Immunostaining was particularly prominent at the tumor margins and highlighted neoplastic cells invading the tumor-adjacent stroma. Short hairpin RNA-mediated knockdown of YAP significantly inhibited proliferation, migration, and anchorage-independent growth of ccRCC cells in soft agar and led to significantly reduced murine xenograft growth. Microarray analysis of YAP knockdown versus mock-transduced ccRCC cells revealed down-regulation of endothelin 1, endothelin 2, cysteine-rich, angiogenic inducer, 61 (CYR61), and c-Myc in ccRCC cells as well as up-regulation of the cell adhesion molecule cadherin 6. Signaling pathway impact analysis revealed activation of the p53 signaling and cell cycle pathways as well as inhibition of mitogen-activated protein kinase signaling on YAP down-regulation. Our data suggest CYR61 and c-Myc as well as signaling through the endothelin axis as bona fide downstream effectors of YAP and establish aberrant Hippo signaling as a potential therapeutic target in ccRCC.     

Par-1 Regulates Tissue Growth by Influencing Hippo Phosphorylation Status and Hippo-Salvador Association

The evolutionarily conserved Hippo (Hpo) signaling pathway plays a pivotal role in organ size control by balancing cell proliferation and cell death. Here, we reported the identification of Par-1 as a regulator of the Hpo signaling pathway using a gain-of-function EP screen in Drosophila melanogaster. Overexpression of Par-1 elevated Yorkie activity, resulting in increased Hpo target gene expression and tissue overgrowth, while loss of Par-1 diminished Hpo target gene expression and reduced organ size. We demonstrated that par-1 functioned downstream of fat and expanded and upstream of hpo and salvador (sav). In addition, we also found that Par-1 physically interacted with Hpo and Sav and regulated the phosphorylation of Hpo at Ser30 to restrict its activity. Par-1 also inhibited the association of Hpo and Sav, resulting in Sav dephosphorylation and destabilization. Furthermore, we provided evidence that Par-1-induced Hpo regulation is conserved in mammalian cells. Taken together, our findings identified Par-1 as a novel component of the Hpo signaling network.     

In silico analysis of DND1 and its co-expressed genes in human cancers

Dead-End (DND1) is an RNA-binding protein involved in translational regulation. Defects in DND1 gene causes germ cell tumors and sterility in rodents. Experimental studies with human somatic cancer cells indicate that DND1 has anti-proliferative and pro-apoptotic function in some while oncogenic function in other cells. We examined The Cancer Genome Atlas data for gene alterations and gene expression changes in DND1 in a variety of human cancers. We found that DND1 is amplified, deleted or mutated in multiple human cancers. In different cancers, DND1 alteration correlates with increased diagnosis age of patients, shift in tumor spectrum or change of tumor sites and in some cases is significantly associated with worse survival for cancer patients. For 15 cancers, we retrieved expression data of thousands of genes that co-expressed with DND1. We found that these cancers contain different percentage of genes that are positively or negatively co-expressed with DND1. Ingenuity Pathway Analysis was performed to explore the biological implications of these genes. More than 10 canonical pathways were identified and each cancer type exhibits unique pathway profiles. Comparison analysis across all 15 cancer types showed that some cancers exhibit strikingly similar profiles of DND1-correlated signaling pathway activation or suppression. Our data reinforce the notion that the biological role of DND1 is cell-type specific and suggest that DND1 may play opposing role by exerting anti-proliferative effects in some cancer cells while being pro-proliferative in others. Our study provides valuable insights to direct experimental investigations of DND1 function in somatic cancers.     

The tumor-suppressor gene fat controls tissue growth upstream of expanded in the hippo signaling pathway

BACKGROUND: The tight control of cell proliferation and cell death is essential to normal tissue development, and the loss of this control is a hallmark of cancers. Cell growth and cell death are coordinately regulated during development by the Hippo signaling pathway. The Hippo pathway consists of the Ste20 family kinase Hippo, the WW adaptor protein Salvador, and the NDR kinase Warts. Loss of Hippo signaling in Drosophila leads to enhanced cell proliferation and decreased apoptosis, resulting in massive tissue overgrowth through increased expression of targets such as Cyclin E and Diap1. The cytoskeletal proteins Merlin and Expanded colocalize at apical junctions and function redundantly upstream of Hippo. It is not clear how they regulate growth or how they are localized to apical junctions. RESULTS: We find that another Drosophila tumor-suppressor gene, the atypical cadherin fat, regulates both cell proliferation and cell death in developing imaginal discs. Loss of fat leads to increased Cyclin E and Diap1 expression, phenocopying loss of Hippo signaling. Ft can regulate Hippo phosphorylation, a measure of its activation, in tissue culture. Importantly, fat is needed for normal localization of Expanded at apical junctions in vivo. Genetic-epistasis experiments place fat with expanded in the Hippo pathway. CONCLUSIONS: Together, these data suggest that Fat functions as a cell-surface receptor for the Expanded branch of the conserved Hippo growth control pathway.     

Frequent epigenetic inactivation of KIBRA,an upstream member of the Salvador/Warts/Hippo (SWH) tumor suppressor network,is associated with specific genetic event in B-cell acute lymphocytic leukemia

The WW-domain containing protein KIBRA has recently been identified as a new member of the Salvador/Warts/Hippo (SWH) pathway in Drosophila and is shown to act as a tumor suppressor gene in Drosophila. This pathway is conserved in humans and members of the pathway have been shown to act as tumor suppressor genes in mammalian systems. We determined the methylation status of the 5′ CpG island associated with the KIBRA gene in human cancers. In a large panel of cancer cell lines representing common epithelial cancers KIBRA was unmethylated. But in pediatric acute lymphocytic leukemia (ALL) cell lines KIBRA showed frequent hypermethylation and silencing of gene expression, which could be reversed by treatment with 5-aza-2′-deoxycytidine. In ALL patient samples KIBRA was methylated in 70% B-ALL but was methylated in <20% T-ALL leukemia (p = 0.0019). In B-ALL KIBRA methylation was associated with ETV6/RUNX1 [t(12;21) (p13;q22)] chromosomal translocation (p = 0.0082) phenotype, suggesting that KIBRA may play an important role in t(12;21) leukemogenesis. In ALL paired samples at diagnosis and remission KIBRA methylation was seen in diagnostic but not in any of the remission samples accompanied by loss of KIBRA expression in disease state compared to patients in remission. Hence KIBRA methylation occurs frequently in B-cell acute lymphocytic leukemia but not in epithelial cancers and is linked to specific genetic event in B-ALL.Key words: KIBRA, methylation, ALL, SWH pathway, ETV6/RUNX1 translocation     

Molecular insights of Hippo signaling in the chick developing lung

Hippo signaling pathway and its effector YAP have been recognized as an essential growth regulator during embryonic development. Hippo has been studied in different contexts; nevertheless, its role during chick lung branching morphogenesis remains unknown. Therefore, this work aims to determine Hippo role during early pulmonary organogenesis in the avian animal model. The current study describes the spatial distribution of Hippo signaling members in the embryonic chick lung by in situ hybridization. Overall, their expression is comparable to their mammalian counterparts. Moreover, the expression levels of phosphorylated-YAP (pYAP) and total YAP revealed that Hippo signaling is active in the embryonic chick lung. Furthermore, the presence of pYAP in the cytoplasm demonstrated that the Hippo machinery distribution is maintained in this tissue. In vitro studies were performed to assess the role of the Hippo signaling pathway in lung branching. Lung explants treated with a YAP/TEAD complex inhibitor (verteporfin) displayed a significant reduction in lung size and branching and decreased expression of ctgf (Hippo target gene) compared to the control. This approach also revealed that Hippo seems to modulate the expression of key molecular players involved in lung branching morphogenesis (sox2, sox9, axin2, and gli1). Conversely, when treated with dobutamine, an upstream regulator that promotes YAP phosphorylation, explant morphology was not severely affected. Overall, our data indicate that Hippo machinery is present and active in the early stages of avian pulmonary branching and that YAP is likely involved in the regulation of lung growth.     

Drosophila Casein Kinase 2 (CK2) Promotes Warts Protein to Suppress Yorkie Protein Activity for Growth Control

Drosophila Hippo signaling regulates Wts activity to phosphorylate and inhibit Yki in order to control tissue growth. CK2 is widely expressed and involved in a variety of signaling pathways. In this study we report that Drosophila CK2 promotes Wts activity to phosphorylate and inhibit Yki activity, which is independent of Hpo-induced Wts promotion. In vivo, CK2 overexpression suppresses hpo mutant-induced expanded (Ex) up-regulation and overgrowth phenotype, whereas it cannot affect wts mutant. Consistent with this, knockdown of CK2 up-regulates Hpo pathway target expression. We also found that Drosophila CK2 is essential for tissue growth as a cell death inhibitor as knockdown of CK2 in the developing disc induces severe growth defects as well as caspase3 signals. Taken together, our results uncover a dual role of CK2; although its major role is promoting cell survive, it may potentially be a growth inhibitor as well.     

The centrosomal component CEP161 of Dictyostelium discoideum interacts with the Hippo signaling pathway

CEP161 is a novel component of the Dictyostelium discoideum centrosome which was identified as binding partner of the pericentriolar component CP250. Here we show that the amino acids 1-763 of the 1381 amino acids CEP161 are sufficient for CP250 binding, centrosomal targeting and centrosome association. Analysis of AX2 cells over-expressing truncated and full length CEP161 proteins revealed defects in growth and development. By immunoprecipitation experiments we identified the Hippo related kinase SvkA (Hrk-svk) as binding partner for CEP161. Both proteins colocalize at the centrosome. In in vitro kinase assays the N-terminal domain of CEP161 (residues 1-763) inhibited the kinase activity of Hrk-svk. A comparison of D. discoideum Hippo kinase mutants with mutants overexpressing CEP161 polypeptides revealed similar defects. We propose that the centrosomal component CEP161 is a novel player in the Hippo signaling pathway and affects various cellular properties through this interaction.     

Cross-species identification of PIP5K1-, splicing- and ubiquitin-related pathways as potential targets for RB1-deficient cells

The RB1 tumor suppressor is recurrently mutated in a variety of cancers including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. Finding new synthetic lethal (SL) interactions with RB1 could lead to new approaches to treating cancers with inactivated RB1. We identified 95 SL partners of RB1 based on a Drosophila screen for genetic modifiers of the eye phenotype caused by defects in the RB1 ortholog, Rbf1. We validated 38 mammalian orthologs of Rbf1 modifiers as RB1 SL partners in human cancer cell lines with defective RB1 alleles. We further show that for many of the RB1 SL genes validated in human cancer cell lines, low activity of the SL gene in human tumors, when concurrent with low levels of RB1 was associated with improved patient survival. We investigated higher order combinatorial gene interactions by creating a novel Drosophila cancer model with co-occurring Rbf1, Pten and Ras mutations, and found that targeting RB1 SL genes in this background suppressed the dramatic tumor growth and rescued fly survival whilst having minimal effects on wild-type cells. Finally, we found that drugs targeting the identified RB1 interacting genes/pathways, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol also elicit SL in human cancer cell lines. In summary, we identified several high confidence, evolutionarily conserved, novel targets for RB1-deficient cells that may be further adapted for the treatment of human cancer.     

DNA methylation profiling distinguishes histological subtypes of renal cell carcinoma

Renal cell carcinoma (RCC) accounts for around 3% of cancers in the UK, and both incidence and mortality are increasing with the aging population. RCC can be divided into several subtypes: conventional RCC (the most common, comprising 75% of all cases), papillary RCC (15%) and chromophobe RCC (5%). Renal oncocytoma is a benign tumor and accounts for 5% of RCC. Cancer and epigenetics are closely associated, with DNA hypermethylation being widely accepted as a feature of many cancers. In this study the DNA methylation profiles of chromophobe RCC and renal oncocytomas were investigated by utilizing the Infinium HumanMethylation450 BeadChips. Cancer-specific hypermethylation was identified in 9.4% and 5.2% of loci in chromophobe RCC and renal oncocytoma samples, respectively, while the majority of the genome was hypomethylated. Thirty (hypermethylated) and 41 (hypomethylated) genes were identified as differentially methylated between chromophobe RCC and renal oncocytomas (p < 0.05). Pathway analysis identified some of the differentially hypermethylated genes to be involved in Wnt (EN2), MAPK (CACNG7) and TGFβ (AMH) signaling, Hippo pathway (NPHP4), and cell death and apoptosis (SPG20, NKX6-2, PAX3 and BAG2). In addition, we analyzed ccRCC and papillary RCC data available from The Cancer Genome Atlas portal to identify differentially methylated loci in chromophobe RCC and renal oncocytoma in relation to the other histological subtypes, providing insight into the pathology of RCC subtypes and classification of renal tumors.