TRIM22 induces cellular senescence by targeting PHLPP2 in hepatocellular carcinoma.

The ubiquitin-proteasome system is a vital protein degradation system that is involved in various cellular processes, such as cell cycle progression, apoptosis, and differentiation. Dysregulation of this system has been implicated in numerous diseases, including cancer, vascular disease, and neurodegenerative disorders. Induction of cellular senescence in hepatocellular carcinoma (HCC) is a potential anticancer strategy, but the precise role of the ubiquitin-proteasome system in cellular senescence remains unclear. In this study, we show that the E3 ubiquitin ligase, TRIM22, plays a critical role in the cellular senescence of HCC cells. TRIM22 expression is transcriptionally upregulated by p53 in HCC cells experiencing ionizing radiation (IR)-induced senescence. Overexpression of TRIM22 triggers cellular senescence by targeting the AKT phosphatase, PHLPP2. Mechanistically, the SPRY domain of TRIM22 directly associates with the C-terminal domain of PHLPP2, which contains phosphorylation sites that are subject to IKKß-mediated phosphorylation. The TRIM22-mediated PHLPP2 degradation leads to activation of AKT-p53-p21 signaling, ultimately resulting in cellular senescence. In both human HCC databases and patient specimens, the levels of TRIM22 and PHLPP2 show inverse correlations at the mRNA and protein levels. Collectively, our findings reveal that TRIM22 regulates cancer cell senescence by modulating the proteasomal degradation of PHLPP2 in HCC cells, suggesting that TRIM22 could potentially serve as a therapeutic target for treating cancer.

ICSBP-induced PD-L1 enhances osteosarcoma cell growth.

Background: Interferon (IFN) consensus sequence binding protein (ICSBP) is a transcription factor induced by IFN-γ. We previously reported that ICSBP expression promotes osteosarcoma progression by enhancing transforming growth factor-ß signaling. In cancer cells, programmed death-ligand 1 (PD-L1) contributes to immune escape and may also be involved in tumor progression. Because IFN-γ induces the expression of both ICSBP and PD-L1, we explored the association between ICSBP and PD-L1 expression in terms of osteosarcoma progression. Methods: Three osteosarcoma cell lines (Saos2, U2OS, and 143B) were employed. Gene expression was measured by qRT-PCR, and protein levels were assessed by immunoblotting. PD-L1 expression was evaluated in cells overexpressing ICSBP and in ICSBP knockdown cells. The effects of PD-L1 expression on cell growth were examined by MTS assays, Incucyte analysis, soft agar assays, and three-dimensional (3D) culture. Cell cycle and apoptosis were evaluated by FACS analysis of cells stained with propidium iodide (PI) and annexin V/PI, respectively. The antitumor effects of PD-L1 knockdown without or with doxorubicin treatment were evaluated in vivo in nude mice bearing ICSBP-overexpressing 143B cell xenograft. The clinical relevance of PD-L1 and ICSBP expression was evaluated immunohistochemically using a human osteosarcoma microarray and through analysis of publicly available data using Gene Expression Profiling Interactive Analysis2. Results: ICSBP overexpression upregulated PD-L1 expression in all three cell lines, whereas ICSBP knockdown decreased the PD-L1 expression. PD-L1 knockdown attenuated the cell growth and reduced colony-forming capacity in both soft agar assays and 3D culture. PD-L1 knockdown increased apoptosis and induced G2/M arrest, which was associated with decreased expression of survivin, cyclin-dependent kinase 4 (CDK4), cyclin E, and cyclin D1 expression and increased the expression of p27, phosphorylated Cdc2, and phosphorylated Wee1. PD-L1 knockdown decreased the growth of tumor xenografts and increased the doxorubicin sensitivity of ICSBP-overexpressing 143B cells both in vitro and in vivo. PD-L1 was expressed in human osteosarcoma tissues, and its expression was moderately correlated with that of ICSBP in osteosarcoma patients. Conclusion: ICSBP regulates PD-L1 expression in osteosarcoma cells, and PD-L1 knockdown combined with doxorubicin treatment could represent a strategy for controlling osteosarcoma expressing ICSBP.

Substituted Syndecan-2-Derived Mimetic Peptides Show Improved Antitumor Activity over the Parent Syndecan-2-Derived Peptide.

We previously showed that a synthetic peptide (S2-P) corresponding to a portion of the human syndecan-2 (SDC2) sequence can bind to the pro-domain of matrix metalloproteinase-7 (MMP-7) to inhibit colon cancer activities. Since S2-P had a relatively weak binding affinity for the MMP-7 pro-domain, we herein modified the amino acid sequence of S2-P to improve the anticancer potential. On the basis of the interaction structure of S2-P and MMP-7, four peptides were generated by replacing amino acids near Tyr 51, which is critical for the interaction. The SDC2-mimetic peptides harboring an Ala-to-Asp substitution at the C-terminal side of Tyr 51 (S2-D) or with an Ala-to-Phe substitution at the N-terminal side of Tyr 51 and an Ala-to-Asp substitution at the C-terminal side of Tyr 51 (S2-FE) showed improved interaction affinities for the MMP-7 pro-domain. Compared to S2-P, S2-FE was better able to inhibit the SDC2-MMP-7 interaction, the cell surface localization of MMP-7, the gelatin degradation activity of MMP-7, and the cancer activities (cell migration, invasion, and colony-forming activity) of human HCT116 colon cancer cells in vitro. In vivo, S2-FE inhibited the primary tumor growth and lung metastasis of CT26 mouse colon cancer cells in a xenograft mouse model. Together, these data suggest that S2-FE could be useful therapeutic anticancer peptides for colon cancer.

Shed syndecan-2 enhances colon cancer progression by increasing cooperative angiogenesis in the tumor microenvironment.

Although shed syndecan-2 potentiated the tumorigenic activities of colon cancer cells, how shed syndecan-2 increases this tumorigenic potential remains unclear. Using an orthotopic mouse model of colon cancer, we show that shed syndecan-2 increases colon cancer progression by cooperatively promoting angiogenesis. Co-administration with a synthetic peptide of shed syndecan-2 (S2LQ) enhanced the survival and tumor engraftment of luciferase-expressing CT26 colon adenocarcinoma cells orthotopically implanted into the cecum of BALB/c mice. Intravenous injection of S2LQ further enhanced the growth of orthotopic tumors in the cecum, with increases in the tissue infiltration of macrophages and the formation of blood vessels, mainly in peripheral layers of the tumor facing the stroma. Furthermore, S2LQ stabilized HIF1α and enhanced the VEGF expression in human colon cancer cell lines, and increased the migration of RAW 264.7 murine macrophage cells and bone marrow-derived macrophages. Finally, S2LQ increased the tube formation of vascular endothelial cells in vitro. Together, these data demonstrate that shed syndecan-2 enhances tumorigenic activity by increasing the crosstalk of cancer cells with tumor-associated macrophages and endothelial cells to enhance angiogenesis for colon cancer progression in the tumor microenvironment.

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.

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.

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.

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.

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.

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.

Histologic Response and Toxicity following Interval-Compressed Four-Drug Therapy Given Preoperatively in Children and Young Adults with Osteosarcoma: A Retrospective Study.

OBJECTIVES: The histologic response to chemotherapy is an important prognostic factor in osteosarcoma. Thus, we attempted to develop an effective neoadjuvant regimen to achieve an improvement in histologic response. METHODS: Twenty-nine patients with a high-grade osteosarcoma received 2 courses of neoadjuvant chemotherapy non-randomly with either the MAP regimen (methotrexate 12 g/m2, cisplatin 120 mg/m2, and doxorubicin 75 mg/m2) or MAPI regimen (MAP plus ifosfamide 9 g/m2). We applied interval compression to MAPI by shortening the preoperative period to be aligned with that of MAP. Adjuvant chemotherapy was tailored according to the necrosis rate of resected tumor specimens. Necrosis rate, toxicity, and survival outcome were compared retrospectively between the 2 groups. RESULTS: The median interval between the beginning of neoadjuvant chemotherapy and surgery was 97.0 days in the MAPI group (17 patients) and 90.5 days in the MAP group (12 patients; p = 0.19). The good histologic response (>90% of necrosis) was observed in 71% of MAPI and in 42% of MAP (p = 0.12). Major toxicities of grade 3 or worse were not different between the 2 groups. The probability of 5-year progression-free survival and overall survival of the MAPI group were 74 and 83%, and those in the MAP group were 50 and 75%, showing no difference. CONCLUSIONS: Interval-compressed MAPI therapy given in a similar duration of the preoperative phase to that of conventional MAP therapy showed a marginal trend toward a better histologic response without a significant increase in major toxicities. Regarding the proportion of good histologic response, 71% is one of the highest values ever reported in the literature. The results warrant further testing in a prospective way in a larger cohort.

Upregulation of transforming growth factor-beta type I receptor by interferon consensus sequence-binding protein in osteosarcoma cells.

Transforming growth factor-beta (TGF-ß) is a known tumor suppressor, which also exerts a tumor promoting activity at an advanced stage of cancer. Previously, we reported that expression of interferon consensus sequence-binding protein (ICSBP), also known as interferon regulatory factor-8, is positively correlated with TGF-ß type I receptor (TGF-ß RI) expression in osteosarcoma patient tissues. In this study, we demonstrated that ICSBP upregulated TGF-ß RI and induced epithelial-to-mesenchymal transition-like phenomena in human osteosarcoma cell lines. As determined by soft agar growth of osteosarcoma cells and xenografted mouse models, ICSBP increased tumorigenicity, which was reversed by ICSBP knock-down or a TGF-ß RI inhibitor. To test whether ICSBP directly regulates the promoter activity of TGF-ß RI, we performed a TGF-ß RI promoter assay, an electro mobility shift assay, and a chromatin immunoprecipitation assay. We observed that TGF-ß RI promoter was activated in ICSBP-overexpressing osteosarcoma cells. Exploiting serial deletions and mutations of the TGF-ß RI promoter, we found a putative ICSBP-binding site at nucleotides -216/-211 (GGXXTC) in the TGF-ß RI promoter. Our data suggest that ICSBP upregulates TGF-ß RI expression by binding to this site, causing ICSBP-mediated tumor progression in osteosarcoma cells. In addition, we found a positive correlation between ICSBP and TGF-ß RI expression in several types of tumors using the cBioportal database. SUMMARY: We demonstrated that interferon consensus sequence-binding protein upregulates transforming growth factor-beta type I receptor (TGF-ß RI) expression by binding to nucleotides -216/-211 (GGXXTC) in the TGF-ß RI promoter, which resulted in increased tumorigenicity and tumor progression in human osteosarcoma cells.

Suppressor of Variegation 3-9 Homolog 2, a Novel Binding Protein of Translationally Controlled Tumor Protein, Regulates Cancer Cell Proliferation.

Suppressor of Variegation 3-9 Homolog 2 (SUV39H2) methylates the lysine 9 residue of histone H3 and induces heterochromatin formation, resulting in transcriptional repression or silencing of target genes. SUV39H1 and SUV39H2 have a role in embryonic development, and SUV39H1 was shown to suppress cell cycle progression associated with Rb. However, the function of human SUV39H2 has not been extensively studied. We observed that forced expression of SUV39H2 decreased cell proliferation by inducing G1 cell cycle arrest. In addition, SUV39H2 was degraded through the ubiquitin-proteasomal pathway. Using yeast two-hybrid screening to address the degradation mechanism and function of SUV39H2, we identified translationally controlled tumor protein (TCTP) as an SUV39H2-interacting molecule. Mapping of the interacting regions indicated that the N-terminal 60 amino acids (aa) of full-length SUV39H2 and the C-terminus of TCTP (120-172 aa) were critical for binding. The interaction of SUV39H2 and TCTP was further confirmed by co-immunoprecipitation and immunofluorescence staining for colocalization. Moreover, depletion of TCTP by RNAi led to up-regulation of SUV39H2 protein, while TCTP overexpression reduced SUV39H2 protein level. The half-life of SUV39H2 protein was significantly extended upon TCTP depletion. These results clearly indicate that TCTP negatively regulates the expression of SUV39H2 post-translationally. Furthermore, SUV39H2 induced apoptotic cell death in TCTP-knockdown cells. Taken together, we identified SUV39H2, as a novel target protein of TCTP and demonstrated that SUV39H2 regulates cell proliferation of lung cancer cells.

C/EBPß Is a Transcriptional Regulator of Wee1 at the G2/M Phase of the Cell Cycle.

The CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor that regulates cellular proliferation, differentiation, apoptosis and tumorigenesis. Although the pro-oncogenic roles of C/EBPß have been implicated in various human cancers, how it contributes to tumorigenesis or tumor progression has not been determined. Immunohistochemistry with human non-small cell lung cancer (NSCLC) tissues revealed that higher levels of C/EBPß protein were expressed compared to normal lung tissues. Knockdown of C/EBPß by siRNA reduced the proliferative capacity of NSCLC cells by delaying the G2/M transition in the cell cycle. In C/EBPß-knockdown cells, a prolonged increase in phosphorylation of cyclin dependent kinase 1 at tyrosine 15 (Y15-pCDK1) was displayed with simultaneously increased Wee1 and decreased Cdc25B expression. Chromatin immunoprecipitation (ChIP) analysis showed that C/EBPß bound to distal promoter regions of WEE1 and repressed WEE1 transcription through its interaction with histone deacetylase 2. Treatment of C/EBPß-knockdown cells with a Wee1 inhibitor induced a decrease in Y15-pCDK1 and recovered cells from G2/M arrest. In the xenograft tumors, the depletion of C/EBPß significantly reduced tumor growth. Taken together, these results indicate that Wee1 is a novel transcription target of C/EBPß that is required for the G2/M phase of cell cycle progression, ultimately regulating proliferation of NSCLC cells.

FAM188B enhances cell survival via interaction with USP7.

We have previously reported that FAM188B showed significant differential exon usage in cancers (NCBI GEO GSE30727), but the expression and function of FAM188B is not well characterized. In the present study, we explored the functions of FAM188B by a knockdown strategy, using siRNAs specific for FAM188B in colon cancer cell lines. FAM188B is a novel gene that encodes a protein that is evolutionarily conserved among mammals. Its mRNA has been found to be highly expressed in most solid tumors, including colorectal cancer. FAM188B knockdown induced cell growth inhibition due to an increase in apoptosis in colon cancer cell lines. Interestingly, siFAM188B treatment induced the upregulation and activation of p53, and consequently increased p53-regulated pro-apoptotic proteins, PUMA and BAX. Proteomic analysis of FAM188B immunocomplexes revealed p53 and USP7 as putative FAM188B-interacting proteins. Deletion of the putative USP7-binding motif in FAM188B reduced complex formation of FAM188B with USP7. It is noteworthy that FAM188B knockdown resulted in a decrease in overall ubiquitination in the p53 immunocomplexes, as well as p53 ubiquitination, because USP7 is involved in p53 deubiquitination. FAM188B knockdown inhibited both colony formation and anchorage-independent growth in vitro. In addition, FAM188B knockdown by siRNA reduced tumor growth in xenografted mice, with an increase in p53 proteins. Taken together, our data suggest that FAM188B is a putative oncogene that functions via interaction with USP7. Therefore, control of FAM188B could be a possible target to inhibit tumor growth.

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.

Regulation of anoikis resistance by NADPH oxidase 4 and epidermal growth factor receptor.

BACKGROUND: Normal cells are sensitive to anoikis, which is a cell detachment-induced apoptosis. However, cancer cells acquire anoikis resistance that is essential for successful metastasis. This study aimed to demonstrate the function and potential mechanism of NADPH oxidase 4 (NOX4) and EGFR activation in regulating anoikis resistance in lung cancer. METHODS: Cells were cultured either in the attached or suspended condition. Cell viability was measured by cell counting and live and dead cell staining. Expression levels of NOX4 and EGFR were measured by PCR and immunoblotting. Reactive oxygen species (ROS) levels were measured by flow cytometry. Effects of NOX4 overexpression or NOX4 knockdown by si-NOX4 on anoikis sensitivity were explored. Levels of NOX4 and EGFR in lung cancer tissues were evaluated by IHC staining. RESULTS: NOX4 was upregulated but EGFR decreased in suspended cells compared with attached cells. Accordingly, ROS levels were increased in suspended cells, resulting in the activation of Src and EGFR. NOX4 knockdown decreased activation of Src and EGFR, and thus sensitised cells to anoikis. NOX4 overexpression increased EGFR levels and attenuated anoikis. NOX4 expression is upregulated and is positively correlated with EGFR levels in the lung cancer patient tissues. CONCLUSIONS: NOX4 upregulation confers anoikis resistance by ROS-mediated activation of EGFR and Src, and by maintaining EGFR levels, which is critical for cell survival.

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.

An in vivo C. elegans model system for screening EGFR-inhibiting anti-cancer drugs.

The epidermal growth factor receptor (EGFR) is a well-established target for cancer treatment. EGFR tyrosine kinase (TK) inhibitors, such as gefinitib and erlotinib, have been developed as anti-cancer drugs. Although non-small cell lung carcinoma with an activating EGFR mutation, L858R, responds well to gefinitib and erlotinib, tumors with a doubly mutated EGFR, T790M-L858R, acquire resistance to these drugs. The C. elegans EGFR homolog LET-23 and its downstream signaling pathway have been studied extensively to provide insight into regulatory mechanisms conserved from C. elegans to humans. To develop an in vivo screening system for potential cancer drugs targeting specific EGFR mutants, we expressed three LET-23 chimeras in which the TK domain was replaced with either the human wild-type TK domain (LET-23::hEGFR-TK), a TK domain with the L858R mutation (LET-23::hEGFR-TK[L858R]), or a TK domain with the T790M-L858R mutations (LET-23::hEGFR-TK[T790M-L858R]) in C. elegans vulval cells using the let-23 promoter. The wild-type hEGFR-TK chimeric protein rescued the let-23 mutant phenotype, and the activating mutant hEGFR-TK chimeras induced a multivulva (Muv) phenotype in a wild-type C. elegans background. The anti-cancer drugs gefitinib and erlotinib suppressed the Muv phenotype in LET-23::hEGFR-TK[L858R]-expressing transgenic animals, but not in LET-23::hEGFR-TK[T790M-L858R] transgenic animals. As a pilot screen, 8,960 small chemicals were tested for Muv suppression, and AG1478 (an EGFR-TK inhibitor) and U0126 (a MEK inhibitor) were identified as potential inhibitors of EGFR-mediated biological function. In conclusion, transgenic C. elegans expressing chimeric LET-23::hEGFR-TK proteins are a model system that can be used in mutation-specific screens for new anti-cancer drugs.

Anoikis resistance: an essential prerequisite for tumor metastasis.

Metastasis is a multistep process including dissociation of cancer cells from primary sites, survival in the vascular system, and proliferation in distant target organs. As a barrier to metastasis, cells normally undergo an apoptotic process known as "anoikis," a form of cell death due to loss of contact with the extracellular matrix or neighboring cells. Cancer cells acquire anoikis resistance to survive after detachment from the primary sites and travel through the circulatory and lymphatic systems to disseminate throughout the body. Because recent technological advances enable us to detect rare circulating tumor cells, which are anoikis resistant, currently, anoikis resistance becomes a hot topic in cancer research. Detailed molecular and functional analyses of anoikis resistant cells may provide insight into the biology of cancer metastasis and identify novel therapeutic targets for prevention of cancer dissemination. This paper comprehensively describes recent investigations of the molecular and cellular mechanisms underlying anoikis and anoikis resistance in relation to intrinsic and extrinsic death signaling, epithelial-mesenchymal transition, growth factor receptors, energy metabolism, reactive oxygen species, membrane microdomains, and lipid rafts.