AP2M1 Supports TGF-ß Signals to Promote Collagen Expression by Inhibiting Caveolin Expression.

The extracellular matrix (ECM) is important for normal development and disease states, including inflammation and fibrosis. To understand the complex regulation of ECM, we performed a suppressor screening using Caenorhabditis elegans expressing the mutant ROL-6 collagen protein. One cuticle mutant has a mutation in dpy-23 that encodes the µ2 adaptin (AP2M1) of clathrin-associated protein complex II (AP-2). The subsequent suppressor screening for dpy-23 revealed the lon-2 mutation. LON-2 functions to regulate body size through negative regulation of the tumor growth factor-beta (TGF-ß) signaling pathway responsible for ECM production. RNA-seq analysis showed a dominant change in the expression of collagen genes and cuticle components. We noted an increase in the cav-1 gene encoding caveolin-1, which functions in clathrin-independent endocytosis. By knockdown of cav-1, the reduced TGF-ß signal was significantly restored in the dpy-23 mutant. In conclusion, the dpy-23 mutation upregulated cav-1 expression in the hypodermis, and increased CAV-1 resulted in a decrease of TßRI. Finally, the reduction of collagen expression including rol-6 by the reduced TGF-ß signal influenced the cuticle formation of the dpy-23 mutant. These findings could help us to understand the complex process of ECM regulation in organism development and disease conditions.

Rab8 and Rabin8-Mediated Tumor Formation by Hyperactivated EGFR Signaling via FGFR Signaling.

The epidermal growth factor receptor (EGFR) signaling is important for normal development, such as vulval development in Caenorhabditis elegans, and hyperactivation of the EGFR is often associated with cancer development. Our previous report demonstrated the multivulva (Muv) phenotype, a tumor model in C. elegans (jgIs25 strain) by engineering LET-23/EGFR with a TKI-resistant human EGFR T790-L858 mutant. Because Rab proteins regulate vesicle transport, which is important for receptor signaling, we screened the RNAi in the jgIs25 strain to find the Rabs critical for Muv formation. Herein, we show that rab-8 RNAi and the rab-8 (-/-) mutation effectively reduce Muv formation. We demonstrate that RABN-8, an ortholog of Rabin8, known as a GEF for Rab8, is also required for Muv formation by promoting the secretion of EGL-17/FGF from vulval precursor cells. In addition, FGFR inhibitors decreased Muv formation mediated by mutant EGFR. Our data suggest that Rab8 and Rabin8 mediate Muv formation through FGF secretion in the EGFR-TKI-resistant nematode model. Furthermore, FGFR-TKIs more effectively inhibit the growth of lung cancer cell lines in H1975 (EGFR T790M-L858R; EGFR-TKI-resistant) than H522 (wild-type EGFR) and H1650 (EGFR exon 19 deletion; EGFR-TKI-sensitive) cells, suggesting that FGFR-TKIs could be used to control cancers with EGFR-TKI-resistant mutations.

Pygenic Acid A (PA) Sensitizes Metastatic Breast Cancer Cells to Anoikis and Inhibits Metastasis In Vivo.

Metastasis is the main cause of cancer-related deaths. Anoikis is a type of apoptosis caused by cell detachment, and cancer cells become anoikis resistant such that they survive during circulation and can successfully metastasize. Therefore, sensitization of cancer cells to anoikis could prevent metastasis. Here, by screening for anoikis sensitizer using natural compounds, we found that pygenic acid A (PA), a natural compound from Prunella vulgaris, not only induced apoptosis but also sensitized the metastatic triple-negative breast cancer cell lines, MDA-MB-231 cells (human) and 4T1 cells (mouse), to anoikis. Apoptosis protein array and immunoblotting analysis revealed that PA downregulated the pro-survival proteins, including cIAP1, cIAP2, and survivin, leading to cell death of both attached and suspended cells. Interestingly, PA decreased the levels of proteins associated with anoikis resistance, including p21, cyclin D1, p-STAT3, and HO-1. Ectopic expression of active STAT3 attenuated PA-induced anoikis sensitivity. Although PA activated ER stress and autophagy, as determined by increases in the levels of characteristic markers, such as IRE1α, p-elF2α, LC3B I, and LC3B II, PA treatment resulted in p62 accumulation, which could be due to PA-induced defects in autophagy flux. PA also decreased metastatic characteristics, such as cell invasion, migration, wound closure, and 3D growth. Finally, lung metastasis of luciferase-labeled 4T1 cells decreased following PA treatment in a syngeneic mouse model when compared with the control. These data suggest that PA sensitizes metastatic breast cancer cells to anoikis via multiple pathways, such as inhibition of pro-survival pathways and activation of ER stress and autophagy, leading to the inhibition of metastasis. These findings suggest that sensitization to anoikis by PA could be used as a new therapeutic strategy to control the metastasis of breast cancer.

Anti-Tumor Effects of Sodium Meta-Arsenite in Glioblastoma Cells with Higher Akt Activities.

Glioblastoma is a type of aggressive brain tumor that grows very fast and evades surrounding normal brain, lead to treatment failure. Glioblastomas are associated with Akt activation due to somatic alterations in PI3 kinase/Akt pathway and/or PTEN tumor suppressor. Sodium meta-arsenite, KML001 is an orally bioavailable, water-soluble, and trivalent arsenical and it shows antitumoral effects in several solid tumor cells via inhibiting oncogenic signaling, including Akt and MAPK. Here, we evaluated the effect of sodium meta-arsenite, KML001, on the growth of human glioblastoma cell lines with different PTEN expression status and Akt activation, including PTEN-deficient cells (U87-MG and U251) and PTEN-positive cells (LN229). The growth-inhibitory effect of KML001 was stronger in U87-MG and U251 cells, which exhibited higher Akt activity than LN229 cells. KML001 deactivated Akt and decreased its protein levels via proteasomal degradation in U87-MG cells. KML001 upregulated mutant PTEN levels via inhibition of its proteasomal degradation. KML001 inhibited cell growth more effectively in active Akt-overexpressing LN229 cells than in mock-expressing LN229 cells. Consistent with these results, KML001 sensitized PTEN-deficient cells more strongly to growth inhibition than it did PTEN-positive cells in prostate and breast cancer cell lines. Finally, we illustrated in vivo anti-tumor effects of KML001 using an intracranial xenograft mouse model. These results suggest that KML001 could be an effective chemotherapeutic drug for the treatment of glioblastoma cancer patients with higher Akt activity and PTEN loss.

Anti-cancer effect of doxorubicin is mediated by downregulation of HMG-Co A reductase via inhibition of EGFR/Src pathway.

Doxorubicin is a widely used DNA damage-inducing anti-cancer drug. However, its use is limited by its dose-dependent side effects, such as cardiac toxicity. Cholesterol-lowering statin drugs increase the efficacy of some anti-cancer drugs. Cholesterol is important for cell growth and a critical component of lipid rafts, which are plasma membrane microdomains important for cell signaling. 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMG-CR) is a critical enzyme in cholesterol synthesis. Here, we show that doxorubicin downregulated HMG-CR protein levels and thus reduced levels of cholesterol and lipid rafts. Cholesterol addition attenuated doxorubicin-induced cell death, and cholesterol depletion enhanced it. Reduction of HMG-CR activity by simvastatin, a statin that acts as an HMG-CR inhibitor, or by siRNA-mediated HMG-CR knockdown enhanced doxorubicin cytotoxicity. Doxorubicin-induced HMG-CR downregulation was associated with inactivation of the EGFR-Src pathway. Furthermore, a high-cholesterol-diet attenuated the anti-cancer activity of doxorubicin in a tumor xenograft mouse model. In a multivulva model of Caenorhabditis elegans expressing an active-EGFR mutant, doxorubicin decreased hyperplasia more efficiently in the absence than in the presence of cholesterol. These data indicate that EGFR/Src/HMG-CR is a new pathway mediating doxorubicin-induced cell death and that cholesterol control could be combined with doxorubicin treatment to enhance efficacy and thus reduce side effects.

TopBP1 deficiency impairs V(D)J recombination during lymphocyte development.

TopBP1 was initially identified as a topoisomerase II-ß-binding protein and it plays roles in DNA replication and repair. We found that TopBP1 is expressed at high levels in lymphoid tissues and is essential for early lymphocyte development. Specific abrogation of TopBP1 expression resulted in transitional blocks during early lymphocyte development. These defects were, in major part, due to aberrant V(D)J rearrangements in pro-B cells, double-negative and double-positive thymocytes. We also show that TopBP1 was located at sites of V(D)J rearrangement. In TopBP1-deficient cells, γ-H2AX foci were found to be increased. In addition, greater amount of γ-H2AX product was precipitated from the regions where TopBP1 was localized than from controls, indicating that TopBP1 deficiency results in inefficient DNA double-strand break repair. The developmental defects were rescued by introducing functional TCR αß transgenes. Our data demonstrate a novel role for TopBP1 as a crucial factor in V(D)J rearrangement during the development of B, T and iNKT cells.

Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis.

Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function.

Novel involvement of leukotriene B4 receptor 2 through ERK activation by PP2A down-regulation in leukotriene B4-induced keratin phosphorylation and reorganization of pancreatic cancer cells.

Perinuclear reorganization via phosphorylation of specific serine residues in keratin is involved in the deformability of metastatic cancer cells. The level of leukotriene B4 is high in pancreatic cancers. However, the roles of LTB4 and its cognate receptors in keratin reorganization of pancreatic cancers are not known. LTB4 dose-dependently induced phosphorylation and reorganization of Keratin 8 (K8) and these processes were reversed by LY255283 (BLT2 antagonist). BLT2 agonists such as Comp A and 15(S)-HETE also induced phosphorylation of serine 431 in K8. Moreover, Comp A-induced K8 phosphorylation and reorganization were blocked by LY255283. Gene silencing of BLT2 suppressed Comp A-induced K8 phosphorylation and reorganization in PANC-1 cells. Over-expression of BLT2 promoted K8 phosphorylation. Comp A promoted the migration of PANC-1 cells in a dose-dependent manner, and LY255283 blocked Comp A-induced migration, respectively. PD98059 (ERK inhibitor) suppressed Comp A-induced phosphorylation of serine 431 and reorganization of K8. Gene silencing of BLT2 suppressed the expression of pERK, and over-expression of BLT2 increased the expression of pERK even without Comp A. Comp A induced the expression of active ERK (pERK) and BLT2. These inductions were blocked by PD98059. Comp A decreased PP2A expression and hindered the binding of PP2A to the K8, leading to the activation of ERK. PD98059 suppressed the Comp A-induced migration of PANC-1 cells and BLT2 over-expression-induced migration of PANC-1 cells. Overall, these results suggest that BLT2 is involved in LTB(4)-induced phosphorylation and reorganization through ERK activation by PP2A downregulation, leading to increased migration of PANC-1 cells.

A genetic screen for aldicarb resistance of Caenorhabditiselegans dauer larvae uncovers 2 alleles of dach-1, a cytochrome P450 gene.

Animals exhibit phenotypic plasticity through the interaction of genes with the environment, and little is known about the genetic factors that change synaptic function at different developmental stages. Here, we investigated the genetic determinants of how animal's sensitivity to drugs that alter synaptic activity is regulated at a specific developmental stage using the free-living nematode Caenorhabditis elegans. C. elegans enters the stress-resistant dauer larval stage under harsh conditions. Although dauer is known to have reduced permeability and increased resistance to most known exogenous chemicals, we discovered that dauer is hypersensitive to a cholinesterase inhibitor, aldicarb. To investigate genes regulating dauer-specific acetylcholine transduction, we first screened for aldicarb-resistant mutations in dauer and then performed a secondary screen to rule out aldicarb-resistant mutations that also affect adults. We isolated 2 different mutations of a single gene called cyp-34A4 or dach-1 encoding a cytochrome P450. In the nondauer stages, dach-1 is mainly expressed in the intestine, but its expression is robustly increased in the epidermis of dauers. By tissue-specific rescue experiments, we found that dach-1 modulates aldicarb sensitivity in a cell nonautonomous manner. In addition, dach-1 plays pleiotropic functions in dauers by regulating quiescence and surviving heat shock and hyperosmolar stress. Our study reveals novel functions of the cytochrome P450 in synaptic and physiological changes during the developmental plasticity.

FAM188B Downregulation Sensitizes Lung Cancer Cells to Anoikis via EGFR Downregulation and Inhibits Tumor Metastasis In Vivo.

Anoikis is a type of apoptosis induced by cell detachment from the extracellular matrix (ECM), which removes mislocalized cells. Acquisition of anoikis resistance is critical for cancer cells to survive during circulation and, thus, metastasize at a secondary site. Although the sensitization of cancer cells to anoikis is a potential strategy to prevent metastasis, the mechanism underlying anoikis resistance is not well defined. Although family with sequence similarity 188 member B (FAM188B) is predicted as a new deubiquitinase (DUB) member, its biological function has not been fully studied. In this study, we demonstrated that FAM188B knockdown sensitized anoikis of lung cancer cell lines expressing WT-EGFR (A549 and H1299) or TKI-resistant EGFR mutant T790M/L858R (H1975). FAM188B knockdown using si-FAM188B inhibited the growth of all three human lung cancer cell lines cultured in both attachment and suspension conditions. FAM188B knockdown resulted in EGFR downregulation and thus decreased its activity. FAM188B knockdown decreased the activities of several oncogenic proteins downstream of EGFR that are involved in anoikis resistance, including pAkt, pSrc, and pSTAT3, with little changes to their protein levels. Intriguingly, si-FAM188B treatment increased EGFR mRNA levels but decreased its protein levels, which was reversed by treatment with the proteasomal inhibitor MG132, indicating that FAM188B regulates EGFR levels via the proteasomal pathway. In addition, cells transfected with si-FAM188B had decreased expression of FOXM1, an oncogenic transcription factor involved in cell growth and survival. Moreover, FAM188B downregulation reduced metastatic characteristics, such as cell adhesion, invasion, and migration, as well as growth in 3D culture conditions. Finally, tail vein injection of si-FAM188B-treated A549 cells resulted in a decrease in lung metastasis and an increase in mice survival in vivo. Taken together, these findings indicate that FAM188B plays an important role in anoikis resistance and metastatic characteristics by maintaining the levels of various oncogenic proteins and/or their activity, leading to tumor malignancy. Our study suggests FAM188B as a potential target for controlling tumor malignancy.

Fibronectin regulates anoikis resistance via cell aggregate formation.

The loss of cell-matrix interactions induces apoptosis, known as anoikis. For successful distant metastasis, circulating tumor cells (CTCs) that have lost matrix attachment need to acquire anoikis resistance in order to survive. Cell aggregate formation confers anoikis resistance, and CTC clusters are more highly metastatic compared to single cells; however, the molecular mechanisms underlying this aggregation are not well understood. In this study, we demonstrated that cell detachment increased cell aggregation and upregulated fibronectin (FN) levels in lung and breast cancer cells, but not in their normal counterparts. FN knockdown decreased cell aggregation and increased anoikis. In addition, cell detachment induced cell-cell adhesion proteins, including E-cadherin, desmoglein-2, desmocollin-2/3, and plakoglobin. Interestingly, FN knockdown decreased the levels of desmoglein-2, desmocollin-2/3, and plakoglobin, but not E-cadherin, suggesting the involvement of desmosomal junction in cell aggregation. Accordingly, knockdown of desmoglein-2, desmocollin-2, or plakoglobin reduced cell aggregation and increased cell sensitivity to anoikis. Previously, we reported that NADPH oxidase 4 (Nox4) upregulation is important for anoikis resistance. Nox4 inhibition by siRNA or apocynin decreased cell aggregation and increased anoikis with the downregulation of FN, and, consequently, decreased desmoglein-2, desmocollin-2/3, or plakoglobin. The coexpression of Nox4 and FN was found to be significant in lung and breast cancer patients, based on cBioPortal data. In vivo mouse lung metastasis model showed that FN knockdown suppressed lung metastasis and thus enhanced survival. FN staining of micro tissue array revealed that FN expression was positive for human lung cancer (61%) and breast cancer (58%) patients. Furthermore, the expression levels of FN, desmoglein-2, desmocollin-2, and plakoglobin were significantly correlated with the poor survival of lung and breast cancer patients, as per the Kaplan-Meier plotter analysis. Altogether, our data suggest that FN upregulation and enhanced desmosomal interactions are critical for cell aggregation and anoikis resistance upon cell detachment.

Ginsenoside Rp1, A Ginsenoside Derivative, Augments Anti-Cancer Effects of Actinomycin D via Downregulation of an AKT-SIRT1 Pathway.

Novel strategies for overcoming multidrug resistance are urgently needed to improve chemotherapy success and reduce side effects. Ginsenosides, the main active components of Panax ginseng, display anti-cancer properties and reverse drug resistance; however, the biological pathways mediating this phenomenon remain incompletely understood. This study aimed to evaluate the anti-cancer effects of ginsenoside Rp1, actinomycin D (ActD), and their co-administration in drug-resistant cells and murine xenograft model of colon cancer, and explore the underlying mechanisms. ActD increased expression and activity of SIRT1 in drug-resistant LS513 colon cancer, OVCAR8-DXR ovarian cancer, and A549-DXR lung cancer cells, but not in ActD-sensitive SW620 colon cancer cells. Inhibition of SIRT1, either pharmacologically, with EX527 or through siRNA, stimulated p53 acetylation and apoptosis in LS513 cells when treated with ActD. ActD also increased AKT activation in drug-resistant cells. Inhibition of AKT abrogated ActD-induced upregulation of SIRT1, suggesting that the AKT-SIRT1 pathway is important in ActD resistance. Rp1 inhibited both ActD-induced AKT activation and SIRT1 upregulation and re-sensitized the cells to ActD. Synergistic antitumor effects of Rp1 with ActD were also observed in vivo. Our results suggest that combining Rp1 with chemotherapeutic agents could circumvent drug resistance and improve treatment efficacy.

Isoharringtonine Induces Apoptosis of Non-Small Cell Lung Cancer Cells in Tumorspheroids via the Intrinsic Pathway.

Lung cancer is the major cause of cancer-associated death worldwide, and development of new therapeutic drugs is needed to improve treatment outcomes. Three-dimensional (3D) tumorspheroids offer many advantages over conventional two-dimensional cell cultures due to the similarities to in vivo tumors. We found that isoharringtonine, a natural product purified from Cephalotaxus koreana Nakai, significantly inhibited the growth of tumorspheroids with NCI-H460 cells in a dose-dependent manner and induced apoptotic cell death in our 3D cell culture system. On the other hand, A549 tumorspheroids displayed low sensitivity to isoharringtonine-induced apoptosis. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor known to regulate proliferation and apoptosis of cancer cells. We observed that knockdown of NR4A1 dramatically increased isoharringtonine-induced cancer cell death in A549 tumorspheroids by activating the intrinsic apoptosis pathway. Furthermore, treatment with combined isoharringtonine and iNR4A1 significantly inhibited multivulva formation in a Caenorhabditis elegans model and tumor development in a xenograft mouse model. Taken together, our data suggest that isoharringtonine is a potential natural product for treatment of non-small cell lung cancers, and inhibition of NR4A1 sensitizes cancer cells to anti-cancer treatment.

Integrin-mediated adhesions in regulation of cellular senescence.

Bioinformatic and functional data link integrin-mediated cell adhesion to cellular senescence; however, the significance of and molecular mechanisms behind these connections are unknown. We now report that the focal adhesion-localized ßPAK-interacting exchange factor (ßPIX)-G protein-coupled receptor kinase interacting protein (GIT) complex controls cellular senescence in vitro and in vivo. ßPIX and GIT levels decline with age. ßPIX knockdown induces cellular senescence, which was prevented by reexpression. Loss of ßPIX induced calpain cleavage of the endocytic adapter amphiphysin 1 to suppress clathrin-mediated endocytosis (CME); direct competition of GIT1/2 for the calpain-binding site on paxillin mediates this effect. Decreased CME and thus integrin endocytosis induced abnormal integrin signaling, with elevated reactive oxygen species production. Blocking integrin signaling inhibited senescence in human fibroblasts and mouse lungs in vivo. These results reveal a central role for integrin signaling in cellular senescence, potentially identifying a new therapeutic direction.

A forward genetic approach for analyzing the mechanism of resistance to the anti-cancer drug, 5-fluorouracil, using Caenorhabditis elegans.

Pyrimidine antagonists including 5-Fluorouracil (5-FU) have been used in chemotherapy for cancer patients for over 40 years. 5-FU, especially, is a mainstay treatment for colorectal cancer. It is a pro-drug that is converted to the active drug via the nucleic acid biosynthetic pathway. The metabolites of 5-FU inhibit normal RNA and DNA function, and induce apoptosis of cancer cells. One of the major obstacles to successful chemotherapy is the resistance of cancer cells to anti-cancer drugs. Therefore, it is important to elucidate resistance mechanisms to improve the efficacy of chemotherapy. We have used C. elegans as a model system to investigate the mechanism of resistance to 5-FU, which induces germ cell death and inhibits larval development in C. elegans. We screened 5-FU resistant mutants no longer arrested as larvae by 5-FU. We obtained 18 mutants out of 72,000 F1 individuals screened, and mapped them into three complementation groups. We propose that C. elegans could be a useful model system for studying mechanisms of resistance to anti-cancer drugs.

Thymidylate synthase and dihydropyrimidine dehydrogenase levels are associated with response to 5-fluorouracil in Caenorhabditis elegans.

5-Fluorouracil (5-FU), a pyrimidine antagonist, has a long history in cancer treatment. The targeted pyrimidine biosynthesis pathway includes dihydropyrimidine dehydrogenase (DPD), which converts 5-FU to an inactive metabolite, and thymidylate synthase (TS), which is a major target of 5-FU. Using Caenorhabditis elegans as a model system to study the functional and resistance mechanisms of anti-cancer drugs, we examined these two genes in order to determine the extent of molecular conservation between C. elegans and humans. Overexpression of the worm DPD and TS homologs (DPYD-1 and Y110A7A.4, respectively) suppressed germ cell death following 5-FU exposure. In addition, DPYD-1 depletion by RNAi resulted in 5-FU sensitivity, while treatment with Y110A7A.4 RNAi and 5-FU resulted in similar patterns of embryonic death. Thus, the pathway of 5-FU function appears to be highly conserved between C. elegans and humans at the molecular level.

The unfolded protein response regulates glutamate receptor export from the endoplasmic reticulum.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors mediate the majority of excitatory signaling in the CNS, and the functional properties and subcellular fate of these receptors depend on receptor subunit composition. Subunit assembly is thought to occur in the endoplasmic reticulum (ER), although we are just beginning to understand the underlying mechanism. Here we examine the trafficking of Caenorhabditis elegans glutamate receptors through the ER. Our data indicate that neurons require signaling by the unfolded protein response (UPR) to move GLR-1, GLR-2, and GLR-5 subunits out of the ER and through the secretory pathway. In contrast, other neuronal transmembrane proteins do not require UPR signaling for ER exit. The requirement for the UPR pathway is cell type and age dependent: impairment for receptor trafficking increases as animals age and does not occur in all neurons. Expression of XBP-1, a component of the UPR pathway, is elevated in neurons during development. Our results suggest that UPR signaling is a critical step in neural function that is needed for glutamate receptor assembly and secretion.

Oncogenic role of rab escort protein 1 through EGFR and STAT3 pathway.

Rab escort protein-1 (REP1) is linked to choroideremia (CHM), an X-linked degenerative disorder caused by mutations of the gene encoding REP1 (CHM). REP1 mutant zebrafish showed excessive cell death throughout the body, including the eyes, indicating that REP1 is critical for cell survival, a hallmark of cancer. In the present study, we found that REP1 is overexpressed in human tumor tissues from cervical, lung, and colorectal cancer patients, whereas it is expressed at relatively low levels in the normal tissue counterparts. REP1 expression was also elevated in A549 lung cancer cells and HT-29 colon cancer cells compared with BEAS-2B normal lung and CCD-18Co normal colon epithelial cells, respectively. Interestingly, short interfering RNA (siRNA)-mediated REP1 knockdown-induced growth inhibition of cancer cell lines via downregulation of EGFR and inactivation of STAT3, but had a negligible effect on normal cell lines. Moreover, overexpression of REP1 in BEAS-2B cells enhanced cell growth and anchorage-independent colony formation with little increase in EGFR level and STAT3 activation. Furthermore, REP1 knockdown effectively reduced tumor growth in a mouse xenograft model via EGFR downregulation and STAT3 inactivation in vivo. These data suggest that REP1 plays an oncogenic role, driving tumorigenicity via EGFR and STAT3 signaling, and is a potential therapeutic target to control cancers.

Development of zebrafish medulloblastoma-like PNET model by TALEN-mediated somatic gene inactivation.

Genetically engineered animal tumor models have traditionally been generated by the gain of single or multiple oncogenes or the loss of tumor suppressor genes; however, the development of live animal models has been difficult given that cancer phenotypes are generally induced by somatic mutation rather than by germline genetic inactivation. In this study, we developed somatically mutated tumor models using TALEN-mediated somatic gene inactivation of cdkn2a/b or rb1 tumor suppressor genes in zebrafish. One-cell stage injection of cdkn2a/b-TALEN mRNA resulted in malignant peripheral nerve sheath tumors with high frequency (about 39%) and early onset (about 35 weeks of age) in F0 tp53e7/e7 mutant zebrafish. Injection of rb1-TALEN mRNA also led to the formation of brain tumors at high frequency (58%, 31 weeks of age) in F0 tp53e7/e7 mutant zebrafish. Analysis of each tumor induced by somatic inactivation showed that the targeted genes had bi-allelic mutations. Tumors induced by rb1 somatic inactivation were characterized as medulloblastoma-like primitive neuroectodermal tumors based on incidence location, histopathological features, and immunohistochemical tests. In addition, 3' mRNA Quanti-Seq analysis showed differential activation of genes involved in cell cycle, DNA replication, and protein synthesis; especially, genes involved in neuronal development were up-regulated.

REP1 inhibits FOXO3-mediated apoptosis to promote cancer cell survival.

Rab escort protein 1 (REP1) is a component of Rab geranyl-geranyl transferase 2 complex. Mutations in REP1 cause a disease called choroideremia (CHM), which is an X-linked eye disease. Although it is postulated that REP1 has functions in cell survival or death of various tissues in addition to the eye, how REP1 functions in normal and cancer cells remains to be elucidated. Here, we demonstrated that REP1 is required for the survival of intestinal cells in addition to eyes or a variety of cells in zebrafish, and also has important roles in tumorigenesis. Notably, REP1 is highly expressed in colon cancer tissues and cell lines, and silencing of REP1 sensitizes colon cancer cells to serum starvation- and 5-FU-induced apoptosis. In an effort to elucidate the molecular mechanisms underlying REP1-mediated cell survival under those stress conditions, we identified FOXO3 as a binding partner of REP1 using a yeast two-hybrid (Y2H) assay system, and we demonstrated that REP1 blocked the nuclear trans-localization of FOXO3 through physically interacting with FOXO3, thereby suppressing FOXO3-mediated apoptosis. Importantly, the inhibition of REP1 combined with 5-FU treatment could lead to significant retarded tumor growth in a xenograft tumor model of human cancer cells. Thus, our results suggest that REP1 could be a new therapeutic target in combination treatment for colon cancer patients.