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An in vivo RNAi mini-screen in Drosophila cancer models reveals novel potential Wnt targets in liver cancer.

INTRODUCTION

Wnt signaling is an evolutionary conserved pathway in various organisms from worms to mammals and plays an important role in several biological processes, such as development, differentiation, cellular proliferation, morphology, motility, and cell fate. Wnt proteins constitute a family of secreted cysteine-rich glycoproteins that exhibit distinct expression patterns in the embryo and adult organisms (1). In mammals, 12 distinct Wnt protein families exist that might induce at least four different pathways: canonical Wnt/[beta]-catenin/TCF, noncanonical Wnt/calcium, Wnt/planar cell polarity, and Wnt/G protein (2). However, alterations of the canonical Wnt/[beta]-catenin/TCF pathway are implicated in tumorigenesis.

If the Wnt/[beta]-catenin signaling pathway is not activated, cytoplasmic [beta]-catenin levels are maintained low through continuous proteasome-mediated degradation, which is controlled by a multiprotein complex containing glycogen synthase kinase 3[beta] (GSK-3[beta]), adenomatous polyposis coli, and Axin.

The activation of the Wnt/[beta]-catenin signaling pathway is initiated by binding of a Wnt ligand to Frizzled receptor and low-density lipoprotein receptor-related protein 5/6 co-receptor. In this case, Dishevelled inhibits the GSK-3[beta]-dependent phosphorylation of [beta]-catenin in response to the Wnt signal. Consequently, [beta]-catenin is dissociated from the destruction complex and starts to accumulate in the cytosol. The accumulated [beta]-catenin is then translocated into the nucleus, binds to the T cell factor (TCF)/lymphoid enhancer-binding factor family of transcription factors, and activates the expressions of several cell cycle and differentiation-related target genes, such as Axin, c-myc, and cyclin D1 (3).

Since [beta]-catenin mutations and activated Wnt signaling pathway are found to be closely related with hepatocellular carcinoma (HCC) development and progression, we had previously performed the overexpression of mutant [beta]-catenin in the HCC cell line human hepatoma 7 (Huh7) in order to mimic the active state of the Wnt/[beta]-catenin signaling in HCC. Huh7 is well-differentiated and has an inactive Wnt/[beta]-catenin pathway with no accumulation of endogenous [beta]-catenin in the nucleus. [beta]-Catenin levels in the cells were increased by transfection of this cell line with the constitutively active form of mutant [beta]-catenin (S33Y), which has a missense mutation of serine to tyrosine at codon 33 and is therefore insensitive to GSK-3[beta]-mediated phosphorylation and proteasomal degradation. Consequently, Huh7 cells transfected with mutant [beta]-catenin led more rapidly to larger tumors in nude mice than the cells transfected with a control plasmid (4).

In order to identify the novel transcriptional targets of the Wnt/[beta]-catenin signaling pathway, genome-wide transcriptomic profiling analyses were performed in hyperactive [beta]-catenin-expressing Huh7 cells by using the serial analysis of gene expression (SAGE) and microarray techniques. Finally, several putative Wnt/[beta]-catenin target genes were detected with the differential expression profile on [beta]-catenin induction in the HCC cell line.

More than 100 novel putative Wnt/[beta]-catenin targets were identified using SAGE and microarray. Among them, several genes were primarily selected for further examinations according to some parameters. First, the selected genes were mostly affected by [beta]-catenin induction (from +2 to -1) in Huh7 cells according to the results of SAGE and microarray approaches. Second, the selected genes were not associated with any specific cancer type so far. Finally, the genes were distinguished that have a homolog in Drosophila melanogaster. Thus, 15 putative Wnt/[beta]-catenin target genes were selected for further experimental investigations (Supplementary Table 1). These 15 putative Wnt/[beta]-catenin target genes are either novel genes that are not yet fully elucidated or genes that are partially defined but with no clear roles in cancer.

Mannosyl alpha-1,3-glycoprotein beta-1,2-N-acetyl-glucosaminyltransferase (MGAT1)

MGAT1 is a medial Golgi enzyme that catalyzes the first step in the conversion of oligomannose-type N-glycans into complex and hybrid N-glycans. Proteins on the cell surface that are N-glycosylated by MGAT1 are required for cell-cell interactions and for the binding of cytokines and other factors to the outer cell membrane. N-linked glycosylation is further important for the folding of some eukaryotic proteins (1).

Translationally controlled tumor protein 1 (TPT1)

The TPT1 gene product has been suggested to function as an antiapoptotic protein since the overexpression of the gene inhibits apoptosis, whereas its knockdown promotes this process (6). Gene knockout studies revealed that TPT1 deficient mice (7) and Drosophila (8) die early during embryogenesis, presumably due to unregulated apoptosis at a critical stage. These studies clearly indicate that TPT1 may play a critical role in the control of cell survival in vivo.

Calmodulin 3 (CALM3)

Calmodulin is a structurally conserved and functionally preserved protein that is encoded by the CALM3 gene. It serves as an intracellular calcium receptor and mediates the calcium regulation of cyclic nucleotide and glycogen metabolism, secretion, motility, and calcium transport (9).

TGF-[beta] inducible nuclear protein 1 (TINP1)

TINP1 was originally identified as one of the putative tumor suppressor genes involved in the pathogenesis of human cell leukemia with an upregulated expression on the stimulation with TGF-[beta] (10). However, in another study, the human TINP1 gene product was identified as a nucleolar protein acting as a cell growth promoting regulator in the cell cycle progression. The overexpression of TINP1 promoted cell growth in different cell lines by regulating the G1/S transition in the cell cycle, whereas its knockdown attenuated the cell growth and dramatically blocked the cell cycle in the G1/S transition (11).

Flap structure-specific endonuclease 1 (FEN1)

The FEN1 gene product is a structure-specific metallonuclease best known for its essential roles in the penultimate steps of Okazaki fragment maturation and long-patch base excision repair. The protein encoded by this gene removes 5' overhanging flaps in DNA repair and processes the 5' ends of Okazaki fragments in lagging strand during DNA synthesis. Furthermore, other studies indicate that FEN1 protein is a multifunctional nuclease that participates in distinct DNA metabolic pathways (12). Since the overexpression of this gene may confer a growth advantage to tumors, FEN1 has been also suggested as a potential cancer therapeutic target (13).

Histidine triad nucleotide-binding protein 1 (HINT1)

HINT1 is a member of the evolutionarily conserved family of histidine triad proteins. In previous studies, mice with deletions in the HINT1 gene are more prone to develop spontaneous hepatoma (14). Furthermore, an increased expression of HINT1 resulted in the growth suppression of several cell lines including lung and colon cancer cell lines, suggesting a potential role for this gene in tumori-genesis as a suppressor (15).

Acidic calponin 3 (CNN3)

Calponin, which is a protein encoded by the CNN3 gene, regulates actin cytoskeleton rearrangement, which is needed for the plasma trophoblast membranes to become fusion competent (16). Furthermore, the gene is found to be upregulated in several brain tumors, suggesting a possible role for this gene in tumorigenesis (17).

Differentially expressed in FDCP 8 homolog (DEF8) (mouse)

DEF8 is a novel gene whose role in the cellular system is not yet fully elucidated. A recent molecular analysis revealed that DEF8 is differentially expressed in primary hemopoietic tissues in mice (18).

Insulin-degrading enzyme (IDE)

Insulin-degrading enzyme (IDE) encodes a zinc metallopeptidase that degrades intracellular insulin, thereby terminating insulin activity as well as participating in intercellular peptide signaling by degrading diverse peptides, such as glucagon, amylin, bradykinin, and kallidin (19). Deficiencies in this protein's function are found to be associated with Alzheimer's disease (20) and type 2 diabetes mellitus (21).

Mortalin (heat shock 70 kDa protein 9)

Mortalin, a member of the heat shock protein 70 family, was first identified as a human mitochondrial heat shock protein, playing important roles in stress response and glucose regulation (22). Despite the role of mortalin in tumorigenesis is not fully elucidated, it is thought to exert its tumorigenic effects through various binding partners including p53 (23).

ADP-ribosylation factor 1 (ARF1)

The ARF1 protein is localized to the Golgi apparatus and has a central role in intra-Golgi transport. Furthermore, the ARF1 expression is downregulated in human leukemia cell line depending on vitamin D treatment (24). However, the role of this gene in tumorigenesis is still largely undefined.

Cofilin 1 (CFL1)

The CFL1 gene product is a widely distributed intracellular actin-modulating protein that binds and depolymerizes filamentous F-actin and inhibits the polymerization of monomeric G-actin in a pH-dependent manner. It is involved in the translocation of actin-cofilin complex from the cytoplasm to the nucleus (25). In a recent study, knockdown of CFL1 in zebrafish interferes with the epibolic movement of the deep cell layer but not in the enveloping layer, and the defect can be specifically rescued by the overexpression of CFL1, suggesting an effective role for this gene in adhesion and cell movements (26).

Protein tyrosine phosphatase, receptor type, F (PTPRF)

The protein encoded by this gene is a member of the PTP family, which are signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation (27). PTPRF was shown to function in the regulation of epithelial cell-cell contacts at the adherens junctions (28). An increased expression level of this protein was found in the insulin-responsive tissue of obese, insulin-resistant individuals and may contribute to the pathogenesis of insulin resistance (29). However, an association between this gene and cancer has not yet been identified.

RAP1B (member of RAS oncogene family)

Rap proteins are small GTPases that belong to the Ras family. Genetic analysis of Rap1b function in the lower eukaryotes is critical for development, as its loss-of-function mutations are lethal in Drosophila (30). There is increasing evidence that Rap signaling is involved in the regulation of multiple cellular processes including cell differentiation and adhesion (31).

ARHGAP1 (Rho GTPase activating protein 1)

The ARHGAP1 gene product is a member of the Rho GTPase family known to regulate multiple eukaryotic cell functions including actin cytoskeleton reorganization, polarity establishment, and cell growth. A recent study revealed that the ARHGAP1 protein plays an important role in regulating mammalian cell genomic stability. The ARHGAP1 knockout primary cells show reduction of DNA damage repair ability, elevation of genomic abnormalities, and induction of multiple cell cycle inhibitors including p53, suggesting an activity for this gene in genome maintenance and cell cycle regulation (32).

MATERIALS AND METHODS

Drosophila stocks

ey-GAL4, GS88A8, UAS-Dl/CyO (eyeful), and ey-GAL4 fly strains were used. UAS-Dl/CyO (sensitized) flies were all kindly provided by Prof. Bassem Hassan from the University of Leuven in Belgium. (33) The following were obtained from the Vienna Drosophila RNAi Center: UAS-RNAi-Mgat1 (stock no.: v103609), UAS-RNAi-Tctp (stock no.: v26632), UAS-RNAi-Cam (stock no.: v102004), UAS-RNAi-Ip259 (stock no.: v110697), UAS-RNAi-Fen1 (stock no.: v108738), UAS-RNAi-HINT1 (stock no.: v110597), UAS-RNAi-Mp20 (stock no.: v40554), UAS-RNAi-DEF8 (stock no.: v108938), UAS-RNAi-Ide (stock no.: v101317), UAS-RNAi-Hsc70-5 (stock no.: v106236), UAS-RNAi-Arf79f (stock no.: v103572), UAS-RNAi-YL-1 (stock no.: v107951), UAS-RNAi-Liprin-alpha (stock no.: v106588), UAS-RNAi-Roughened (stock no.: v9055), UAS-RNAi-RhoGAP68F (stock no.: v107775), UAS-RNAi-Axin (stock no.: v7748), and UAS-RNAi-white (stock no.: v40657) (Table 1). All flies were raised in a fly incubator at 25 [degrees]C on a standard fly food.

Examination of tumorigenesis and metastasis

Eyeful and sensitized flies with desired genes' RNAi downregulations were anesthetized with C[O.sub.2] and analyzed under a stereo microscope. For each gene's down-regulation, 180 flies were analyzed in each of the eyeful and sensitized backgrounds. Tumor formations in the eye tissue and metastasis formations in the entire body were analyzed. Each eye of the flies was scored separately, and the eyes were considered with tumors when the eye tissue showed at least one folding. Metastasis formations were detected as amorphous red-pigmented cells outside of the eye field.

Statistical analysis

The results of the analyzed putative target genes' down-regulations were normalized to the results of the negative control white gene's downregulation. Student's t-test was used to determine the significance of the differences between the obtained results. A p value <0.05 compared with the control group was considered as significantly different.

Ethics committee approval is not required because our study does not involve any human or animal tissues.

RESULTS

In vivo RNAi screening in the eyeful and sensitized Dro-sophila cancer models

In order to identify the possible roles of the selected 15 putative Wnt/[beta]-catenin targets in tumorigenesis, an in vivo RNAi mini-screen has been performed in the eyeful and sensitized Drosophila cancer models. The Drosophila homologs of the selected genes were knockdowned using the RNAi system. First, the suitable transgenic UAS-RNAi fly lines of the genes of interest (Table 1) were crossed with the eyeful flies containing the ey-GAL4 constructs (Figure 1a) to enable an eye tissue-specific gene downregulation in the eyeful background. In the resulting progeny, flies with the downregulated target genes were selected, and the alterations in the existing tumor and metastasis formation prevalences on the knockdowns were examined.

Second, the candidate genes were knockdowned in the sensitized Drosophila cancer model in order to further investigate the potential of the same genes to induce novel tumor and/or metastasis formations. For this purpose, the transgenic RNAi fly lines were crossed with the sensitized flies bearing the ey-GAL4 drivers (Figure 1b). Thus, an eye tissue-specific knockdown of the candidate genes was achieved in the sensitized flies. In the resulting progeny, tumor and metastasis formation prevalences were examined.

In these mini-screens, three independent crosses were set up for each gene's downregulation, and from each cross, 180 flies were analyzed with the desired gene knockdown. In both of the eyeful and sensitized backgrounds, the white gene was used as a negative control that has a function in the formation of developing eye color but no effect on tumorigenesis. On the other hand, the Axin gene that is involved in the degradation of [beta]-catenin as a negative regulator of the Wnt/[beta]-catenin signaling pathway and has a tumor suppressor activity was used as a positive control.

Analysis of tumor and metastasis formation prevalences in the eyeful model

The tumor formation frequencies were examined on the downregulations of the target genes in the eyeful background. For this purpose, a total of 180 eyeful flies were analyzed for any tumor formation in the eye tissue.

Figure 2 shows several examples of the examined eyes of flies. During the analysis, each eye of the flies was scored separately. Figure 2a shows the eyeful flies with no active gene knockdown presented as a regular, single-layered, red-pigmented and round-shaped eye tissue. In comparison to eyeful flies, the downregulation of the negative control white gene resulted in lighter eye color formation (Figure 2b) since this gene is involved in the formation of developing eye color in Drosophila. When the eye tissue exhibited an excessive overgrowth and generated at least one outgrowing folding (Figure 2c, d) or additional red-pigmented eye tissue (Figure 2e, f) on the knockdowns, the eyes were accepted as tumorous. As long as the eye tissue was located on the fly head, it was considered as a tumor and not as metastasis.

The statistical analysis showed that the downregulation of the negative control white gene in the eyeful flies induced tumor formation in 42% of the examined eyes (data not shown here, presented as normalized data in Figure 3a). The knockdown of the positive control Axin gene in the eyeful flies resulted in increased frequency of eye tumors compared with the negative control white gene (Figure 3a). This result confirmed the known tumor suppressor activity of Axin in the eyeful cancer model. The knockdown of 11 tested genes (Mgat1, YL-1, HINT1, RhoGAP68f, DEF8, Liprin-alpha, Ide, Mp20, Roughened, Cam, and Tctp) resulted in significantly increased tumor formations ranging between 1.5- and 2.3-fold (Figure 3a), indicating a potential tumor suppressor activity for each of these examined genes. On the other hand, the downregulation of the other three genes (Hsc70-5, Arf79f, and Ip259) showed significantly decreased tumor formation prevalences (Figure 3a) in comparison to the negative control white gene, suggesting a putative oncogenic activity for these genes.

In addition to tumor formation prevalences, the metastasis formation frequencies have been also analyzed in the same 180 eyeful flies in a further step. Figure 3b shows the metastasis formation rates in these flies. Metastasis could be seen as amorphous red-pigmented cells outside of the eye field and formed in the dorsal part, ventral part, or neck of the flies.

The downregulation of the negative control white gene in the eyeful flies showed metastasis in 3% of the examined flies (data not shown here), whereas that of the positive control gene Axin strongly enhanced metastasis formations when downregulated (Figure 3b).

Strikingly, the knockdowns of nine genes (Mgat1, YL-1, HINT1, RhoGAP68f, Liprin-alpha, Ide, Mp20, Cam, and Tctp), which showed increased tumor formation prevalences, also resulted in significantly increased metastasis formation frequencies ranging between 2- and 7-fold (Figure 3b) in the eyeful flies, further supporting the potential tumor suppressor activity of these genes.

On the other hand, the downregulations of Hsc70-5 and Ip259, which showed decreased tumor formation prevalences, were able to totally suppress the metastasis formation in the eyeful background (Figure 3b), indicating a potential oncogenic function for these two genes.

Analysis of tumor and metastasis formation prevalences in the sensitized model

Analyzing the effects of the target genes on the existing cancerous background in the eyeful flies, the next potential of the same genes to induce any tumor and/or metastasis was questioned in the sensitized model. For this purpose, 180 sensitized flies with the specific gene knockdowns were analyzed for tumorigenic and metastatic eye tissues. Figure 4a shows the tumor formation prevalences in the analyzed sensitized flies. Knockdown of the positive control gene Axin strongly triggered tumor formation in the sensitized flies, confirming its tumor suppressor function. The downregulation of 11 candidate genes (Mgat1, YL-1, HINT1, RhoGAP68f, DEF8, Liprin-alpha, Ide, Mp20, Roughened, Cam, and Tctp), which increased tumor frequencies in the eyeful background, was able to induce tumor formation in the sensitized cancer model as well (Figure 4a). These data confirm the results obtained in the eyeful background and strikingly demonstrate a big evidence for a potential tumor suppressor function for each of these examined genes.

In contrast, the knockdown of the additional two candidate genes (Hsc70-5 and Ip259), which resulted in decreased tumor and metastasis formations in the eyeful background, suppressed the tumor formation in the sensitized flies (Figure 4a), indicating an oncogenic function for these genes and supporting the results of the eyeful background. Furthermore, the downregulation of the same two genes resulted in very small or no-eye pheno-type in 46%-58% of the fly eyes in the sensitized background (data not shown here), suggesting an effective role for each of these genes in development.

Analysis of metastasis formation prevalences in the sensitized model

After the examination of tumor formation prevalences on the downregulation of the target genes in the sensitized flies, metastasis formation frequencies were investigated in the same 180 sensitized flies in an additional step.

Figure 4b shows the metastasis formation prevalences in the analyzed sensitized flies. The positive control Axin gene was not able to induce metastasis formation in the sensitized background when downregulated, which could be a false-negative result due to the limited numbers of the examined flies. On the other hand, the downregulation of YL-1 and RhoGAP68f, which significantly enhanced tumor and metastasis formations in the eyeful background and induced tumor formation in the sensitized model, was able to trigger metastasis in the sensitized background as well. In contrast to this, Hsc70-5 and Ip259 knockdowns suppressed metastasis formations in the sensitized flies consistent with the results obtained in the eyeful model (Figure 4b).

DISCUSSION

The canonical Wnt/[beta]-catenin signaling is an evolutionary conserved pathway that is involved in various events during embryonic development, such as axis formation, cellular proliferation, differentiation, and morphogenesis. In addition to its role in development, the Wnt/[beta]-catenin pathway has the potential to initiate tumor formation when it is aberrantly activated. Molecular studies have revealed that activating mutations in the Wnt/[beta]-catenin signaling pathway are responsible for approximately 90% of colorectal cancer and somewhat less frequently in other cancer types, such as HCC. Those characteristics of the Wnt/[beta]-catenin signaling pathway make the pathway itself and its targets important key subjects for cancer studies.

In order to identify novel transcriptional targets of the Wnt/[beta]-catenin pathway, a microarray and SAGE screen was conducted in our laboratory. Consequently, a number of genes were differentially regulated on the accumulation of mutant [beta]-catenin in the human HCC cell line Huh7, by means of mimicking the active state of the Wnt/[beta]-catenin pathway. These genes are considered as novel candidates of the canonical Wnt/[beta]-catenin signaling pathway.

Since [beta]-catenin mutations and an activated Wnt signaling pathway have been found to be closely related with tumorigenesis, the significantly and differentially regulated putative target genes of the canonical Wnt/[beta]-catenin signaling pathway may play effective roles in cancer. The aim of the present study was to identify the possible effects of the selected 15 candidate genes on tumor and metastasis formations and characterize their potential roles in cancer. For this purpose, an in vivo RNAi mini-screen was performed in two Drosophila eye cancer models: eyeful and sensitized. In these cancer models, the Drosophila homologs of the selected 15 genes were perturbed, and the effects of the altered gene expressions on tumor formation and metastasis were examined.

In the eyeful cancer model, the concurrent overexpression of the Notch ligand Delta and two epigenetic silencers lola and pipsqueak resulted in tumor formation and metastasis in flies. When knockdowned in this background, the nine analyzed putative Wnt/[beta]-catenin target genes (Mgat1, YL-1, HINT1, RhoGAP68f, Liprin-alpha, Ide, Mp20, Cam, and Tctp) further enhanced the tumor and metastasis rates. In contrast to this, Hsc70-5 and Ip259 knockdowns totally suppressed the existing tumor and metastasis and resulted in smaller or no-eye phenotypes.

Further, the effects of the downregulations of the same genes were examined in the sensitized cancer model by ignoring the overexpression of lola and pipsqueak from the tumor-inducing background of eyeful flies. In this background, further supporting the results in the eyeful background, the knockdowns of nine candidate genes (Mgat1, YL-1, HINT1, RhoGAP68f, Liprin-alpha, Ide, Mp20, Cam, and Tctp) induced tumorigenesis, whereas those of two of them (YL-1 and RhoGAP68f) were also able to promote metastasis formation when downregulated. On the other hand, decreased levels of Hsc70-5 and Ip259 did not trigger any tumor or metastasis formation and resulted again in smaller or no-eye phenotypes, providing clues about a possible role for these genes in developmental pathways and linking differentiation and tumorigenesis in Drosophila. Overall, the results obtained from the eyeful and sensitized backgrounds are consistent with each other and suggest a tumor suppressor function for the analyzed nine candidate genes and an oncogenic or a developmental function for the other two genes for the first time.

However, the exact functions of the examined genes in cell proliferation and apoptosis mechanisms in the cells are still unclear. The examination of the effects of these genes on cell proliferation and apoptosis would be interesting by analyzing the levels of proliferation markers or apoptosis markers in the eye tissues of eyeful, sensitized, and wild-type flies on the downregulation of these candidate genes. These data may provide clues about the potential roles of these genes in tumorigenesis by identifying their effects on cell proliferation or apoptosis. Furthermore, these genes were identified as novel potential Wnt/[beta]-catenin target genes since they were found to be differentially expressed on [beta]-catenin induction, mimicking overactivated Wnt signaling. Therefore, in addition to their potential tumorigenic functions, the identification of their interaction partners and putative roles in the Wnt/[beta]-catenin signaling pathway is crucial in order to enlighten the molecular mechanisms and signaling networks in which they are involved in. Thus, a possible cross-talk between the Wnt/[beta]-catenin signaling pathway and other signaling cascades via these putative target genes might also be elucidated eventually.

In the present study, we identified novel potential Wnt/[beta]-catenin target genes with possible tumor suppressor or oncogenic functions that may be considered as novel subjects for future cancer studies. By elucidating the possible roles of these genes in tumorigenesis and metastasis formations and clarifying the molecular mechanisms behind their activities, these genes may be identified as novel targets for diagnostic and therapeutic processes of liver cancer as well as several other cancer types.

Ethics Committee Approval: Ethics committee approval is not required because our study does not involve any human or animal tissues.

Informed Consent: N/A.

Peer-review: Externally peer-reviewed.

Acknowledgements: The authors would like to thank Bassem Hassan and Duygu Esen Ozel for sharing their expertise and laboratory facilities, and Naseem Muhammed for proofreading of the manuscript.

Author Contributions: Concept - N.B.I.; Design - N.B.I., I.E.; Supervision - N.B.I.; Resources - N.B.I.; Materials - N.B.I., B.H.; Data Collection and/or Processing - I.A., I.E., N.B.I.; Analysis and/or Interpretation - I.A., I.E., N.B.I.; Literature Search - I.A., I.E.; Writing Manuscript - I.A., I.E., N.B.I.; Critical Reviews - N.M.

Conflict of Interest: The authors have no conflict of interest to declare.

Financial Disclosure: The authors declared that this study was supported by Bogazici University Research Projects (Project Number: 6734).

REFERENCES

(1.) Cadigan KM, Nusse R. Wnt signaling: a common theme in animal development. Genes Dev 1997; 11: 3286-305. [CrossRef]

(2.) Wodarz A, Nusse R. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol 1998; 14: 59-88. [CrossRef]

(3.) Behrens J, von Kries JP, Kuhl M, et al. Functional interaction of [beta]-catenin with the transcription factor LEF-1. Nature 1996; 382: 638-42. [CrossRef]

(4.) Kavak E, Najafov A, Ozturk N, et al. Analysis of the Wnt/B-catenin/TCF4 pathway using SAGE, genome-wide microarray and promoter analysis: Identification of BRI3 and HSF2 as novel targets. Cell Signal 2010; 22: 1523-35. [CrossRef]

(5.) Yen CL, Stone SJ, Cases S, et al. Identification of a gene encoding MGAT1, a monoacylglycerol acyltransferase. Proc Natl Acad Sci 2002; 99: 8512-7. [CrossRef]

(6.) Diraison F, Hayward K, Sanders KL, et al. Translationally controlled tumour protein (TCTP) is a novel glucose-regulated protein that is important for survival of pancreatic beta cells. Diabetologia 2011; 54: 368-79. [CrossRef]

(7.) Chen SH, Wu PS, Chou CH, et al. A knockout mouse approach reveals that TCTP functions as an essential factor for cell proliferation and survival in a tissue- or cell type-specific manner. Mol. Biol. Cell 2007; 18: 2525-32. [CrossRef]

(8.) Hsu YC, Chern JJ, Cai Y, et al. Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase. Nature 2007; 445: 785-8. [CrossRef]

(9.) Zhang M, Yuan T. Molecular mechanisms of calmodulin's functional versatility. Biochem. Cell Biol 1998; 76: 313-23. [CrossRef]

(10.) Wu X, Ivanova G, Merup M, et al. Molecular analysis of the human chromosome 5q13.3 region in patients with hairy cell leukemia and identification of tumor suppressor gene candidates. Genomics 1999; 60: 161-71. [CrossRef]

(11.) Zhang H, Ma X, Shi T, et al. NSA2, a novel nucleolus protein regulates cell proliferation and cell cycle. Biochem Biophys Res Commun 2010; 391: 651-8. [CrossRef]

(12.) Liu Y, Kao HI, Bambara RA. Flap endonuclease 1: A central component of DNA metabolism. Annu Rev Biochem 2004; 73: 589-615. [CrossRef]

(13.) Zheng L, Jia J, Finger LD, et al. Functional regulation of FEN1 nuclease and its link to cancer. Nucleic Acids Res 2011; 39: 781-94. [CrossRef]

(14.) Li H, Zhang Y, Su T, et al. Hint1 is a haplo-insufficient tumor suppressor in mice. Oncogene 2006; 25: 713-21. [CrossRef]

(15.) Wang L, Li H, Zhang Y, et al. Restoration of HINT1 expression inhibits growth and AP-1 transcription factor activity in HepG2 human hepatoma cells. Cancer Res 2006; 66: 1003-13.

(16.) Shibukawa Y, Yamazaki N, Kumasawa K, et al. Calponin 3 regulates actin cytoskeleton rearrangement in trophoblastic cell fusion. Mol Biol Cell 2010; 21: 3973-84. [CrossRef]

(17.) Najafov A, Seker T, Even I, et al. MENA is a transcriptional target of the Wnt/beta-catenin pathway. PLoS One 2012; 7: e37013. [CrossRef]

(18.) Ronquist GK, Larsson A, Roquist G, et al. Prostasomal DNA characterization and transfer into human sperm. Mol Reprod Dev 2011; 78: 467-76. [CrossRef]

(19.) Authier F, Bergeron JJ, Ou WJ, et al. Degradation of the cleaved leader peptide of thiolase by a peroxisomal proteinase. Proc Natl Acad Sci 1995; U. S. A. 92: 3859-63.

(20.) Kurochkin IV, Goto S. Alzheimer's [beta]-amyloid peptide specifically interacts with and is degraded by insulin degrading enzyme. FEBS Lett 1994; 345: 33-7. [CrossRef]

(21.) Duckworth WC, Bennett RG, Hamel FG. Insulin degradation: progress and potential. Endocr Rev 1998; 19: 608-24. [CrossRef]

(22.) Qu M, Zhou Z, Xu S, et al. Mortalin overexpression attenuates beta-amyloid-induced neurotoxicity in SH-SY5Y cells. Brain Res 2011; 1368: 336-45. [CrossRef]

(23.) Lu WJ, Lee NP, Kaul SC, et al. Mortalin-p53 interaction in cancer cells is stress dependent and constitutes a selective target for cancer therapy. Cell Death Differ 2011; 18: 1046-56. [CrossRef]

(24.) Savli H. Vitamin D dependent down regulation of Arf1 gene in human leukemia cell line HL-60 of medical sciences. Turkish J Med Sci 2003; 33: 255-7.

(25.) Chai X, Forster E, Zhao S, et al. Reelin stabilizes the actin cytoskeleton of neuronal processes by inducing n-cofilin phosphorylation at serine3. J Neurosci 2009; 29: 288-99. [CrossRef]

(26.) Lin CW, Yen ST, Chang HT, et al. Loss of cofilin 1 disturbs actin dynamics, adhesion between enveloping and deep cell layers and cell movements during gastrulation in zebrafish. PLoS One 2010; 5: e15331. [CrossRef]

(27.) Chernoff J. Protein tyrosine phosphatases as negative regulators of mitogenic signaling. J Cell Physiol 1999; 180: 173-81. [CrossRef]

(28.) Harder K, Saw J, Miki N, et al. Coexisting amplifications of the chromosome 1p32 genes (PTPRF and MYCL1) encoding protein tyrosine phosphatase LAR and L-myc in a small cell lung cancer line. Genomics 1995; 27: 552-3. [CrossRef]

(29.) Menzaghi C, Paroni G, De Bonis C, et al. The protein tyrosine phosphatase receptor type f (PTPRF) locus is associated with coronary artery disease in type 2 diabetes. J Intern Med 2008; 263: 653-4. [CrossRef]

(30.) Hariharan LK, Carthew RW, Rubin GM. The Drosophila roughened mutation: activation of a rap homolog disrupts eye development and interferes with cell determination. Cell 1991; 67: 717-22. [CrossRef]

(31.) Malchinkhuu E, Sato K, Maehama T, et al. Role of Rap1B and tumor suppressor PTEN in the negative regulation of lysophosphatidic acid-induced migration by isoproterenol in glioma cells. Mol Biol Cell 2009; 20: 5156-65. [CrossRef]

(32.) Wang L, Yang L, Debidda M, et al. Cdc42 GTPase-activating protein deficiency promotes genomic instability and premature aging-like phenotypes. Proc Natl Acad Sci 2007; 104: 1248-53. [CrossRef]

(33.) Bossuyt W, Geest ND, Aerts S, et al. The atonal proneural transcription factor links differentiation and tumor formation in Drosophila. PloS Biol 2009; 7: e1000040. [CrossRef]
Supplementary Table 1. Human and the corresponding Drosophila gene names

Human     Drosophila

MGAT 1    Mgat1
TPT1      Tctp
CALM3     Cam
TINP1     Ip259
FEN1      Fen1
HINT1     HINT1
CNN3      Hsc70-3
DEF8      DEF8
IDE       Ide
Mortalin  Hsc70-5
ARF1      Arf79f
CFL1      YL-1
PTPRF     Liprin-alpha
RAP1B     Roughened
ARHGAP1   RhoGAP68f


Ipek Even (1) [iD], Izzet Akiva (1) [iD], Necla Birgul Iyison (1,2) [iD]

(1) Department of Molecular Biology and Genetics, Bo c azici University, Istanbul, Turkey

(2) Center for Life Sciences and Technologies, Bogazii University, Istanbul, Turkey

Cite this article as: Even I, Akiva I, Birgul Iyison N. An in vivo RNAi mini-screen in Drosophila cancer models reveals novel potential Wnt targets in liver cancer. Turk J Gastroenterol 2019; 30(2): 198-207.

Corresponding Author: Necla Birgul Iyison; birgul@boun.edu.tr

Received: March 28, 2018 Accepted: August 8, 2018 Available online date: December 7, 2018

DOI: 10.5152/tjg.2018.18241
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Author:Even, Ipek; Akiva, Izzet; Iyison, Necla Birgul
Publication:The Turkish Journal of Gastroenterology
Article Type:Report
Date:Feb 1, 2019
Words:5248
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