Knockdown Induced Cell Cycle Arrest Research Articles

Macrophage immigration inhibitory factor promotes cell proliferation and inhibits apoptosis of cervical adenocarcinoma.

As a multifunctional cytokine, macrophage migration inhibitory factor (MIF) is associated with inflammation and tumorigenesis; however, the role of MIF in cervical adenocarcinoma (ADC) is not fully understood. In this study, we aimed to examine the expression of MIF in ADC and explore the mechanism of MIF in ADC progression. MIF expression was positively related to ADC clinicopathological features of carcinoma diameter and lymph node metastasis. MIF knockdown induced cell cycle arrest of G1/S transition in ADC cells, upregulation of the expressions of p21 and p27, and downregulation of the expressions of Cdk4, CyclinD2, and CyclinE2. In MIF knockdown cells, the expressions of proapoptotic proteins of Bax, caspase-3, cleaved caspase-3, and cleaved-PARP were upregulated, and the expressions of antiapoptotic proteins of Bcl-2, pAkt, and p53 were downregulated. It indicated that MIF knockdown inhibited cell proliferation and induced apoptosis in ADC cells. MIF might be a novel molecular marker in diagnosis and therapy of ADC.

SiRNA-mediated knockdown against NUF2 suppresses pancreatic cancer proliferation in vitro and in vivo.

NUF2 (NUF2, Ndc80 kinetochore complex component) plays an important role in kinetochore-microtubule attachment. It has been reported that NUF2 is associated with multiple human cancers. However, the functional role of NUF2 in pancreatic cancer remains unclear. In this study, we found that NUF2 expression was stronger in tumour tissues than in normal pancreatic tissues, and its overexpression could be related to poor prognosis. Moreover, NUF2 was highly expressed in several human pancreatic cancer cell lines. We took advantage of lentivirus-mediated siRNA (small interfering RNA) to suppress NUF2 expression in PANC-1 and Sw1990 cell lines aiming to investigate the role of NUF2 in pancreatic cancer. NUF2 silencing by RANi (RNA interference) reduced the proliferation and colony formation ability of pancreatic cancer cells in vitro. Cell cycle analysis showed that NUF2 knockdown induced cell cycle arrest at G0/G1 phase via suppression of Cyclin B1, Cdc2 and Cdc25A. More importantly, NUF2 silencing was able to alleviate in vivo tumourigenesis in pancreatic cancer xenograft nude mice. Collectively, the present study indicates that the siRNA-mediated knockdown against NUF2 may be a promising therapeutic method for the treatment of pancreatic cancer.

Synthetic lethal interaction of combined CD26 and Bcl-xL inhibition is a powerful anticancer therapy against hepatocellular carcinoma.

CD26 is a membrane glycoprotein that has multiple functions, including dipeptidyl peptidase IV activity. CD26 expression varies in different tumor types, and its role in tumor growth in hepatocellular carcinoma (HCC) remains unclear. CD26 expression levels were examined in resected HCC and surrounding non-cancerous lesions. The effect of CD26 knockdown on the cellular proliferation of HepG2 or Huh7 cells, both of which highly express CD26, was studied in vitro. CD26 mRNA expression levels were significantly increased in HCC compared with their surrounding non-cancerous lesions. We confirmed that various HCC cell lines, especially HepG2 and Huh7 cells, showed high expression levels of CD26. siRNA-mediated knockdown of CD26 suppressed hepatoma cell growth in vitro. CD26 knockdown induced cell cycle arrest through the upregulation of Cip/Kip family proteins, p21 in HepG2 cells and p27 in Huh7 cells. CD26 knockdown did not affect apoptosis, but it increased expressions of the pro-apoptotic proteins Bim and Bak and the anti-apoptotic protein Bcl-xL, suggesting an addiction of CD26 knockdown cells to Bcl-xL for survival. We thus treated CD26 knockdown cells with ABT-737, a Bcl-xL/-2/-w inhibitor, and observed that the synthetic lethal interaction of combined Bcl-xL and CD26 inhibition induced significant apoptosis and impaired cellular viability. CD26 mRNA was overexpressed in HCC, and its inhibition suppressed cellular proliferation through cell cycle arrest. The combined use of CD26 knockdown with a Bcl-xL inhibitor further elicited substantial apoptosis and therefore may serve as a powerful anticancer combination therapy against HCC.

Validation of Bmi1 as a therapeutic target of hepatocellular carcinoma in mice.

Bmi1 is a member of the polycomb group family of proteins, and it drives the carcinogenesis of various cancers and governs the self-renewal of multiple types of stem cells. Our previous studies have revealed that Bmi1 acts as an oncogene in hepatic carcinogenesis in an INK4a/ARF locus independent manner. However, whether Bmi1 can be used as a potential target for hepatocellular carcinoma treatment has not been fully confirmed yet. Here, we show that perturbation of Bmi1 expression by using short hairpin RNA can inhibit the tumorigenicity and tumor growth of hepatocellular carcinoma cells both in vitro and in vivo. Importantly, Bmi1 knockdown can block the tumor growth, both in the initiating stages and the fast growing stages. Cellular biology analysis revealed that Bmi1 knockdown induces cell cycle arrest and apoptosis. Our findings verify Bmi1 as a qualified treatment target for hepatocellular carcinoma (HCC) and support Bmi1 targeting treatment with chemotherapeutic agents.

Multidrug resistance protein 4/ ATP binding cassette transporter 4: a new potential therapeutic target for acute myeloid leukemia.

Less than a third of adults patients with acute myeloid leukemia (AML) are cured by current treatments, emphasizing the need for new approaches to therapy. We previously demonstrated that besides playing a role in drug-resistant leukemia cell lines, multidrug resistance protein 4 (MRP4/ABCC4) regulates leukemia cell proliferation and differentiation through the endogenous MRP4/ABCC4 substrate, cAMP. Here, we studied the role of MRP4/ABCC4 in tumor progression in a mouse xenograft model and in leukemic stem cells (LSCs) differentiation. We found a decrease in the mitotic index and an increase in the apoptotic index associated with the inhibition of tumor growth when mice were treated with rolipram (PDE4 inhibitor) and/or probenecid (MRPs inhibitor). Genetic silencing and pharmacologic inhibition of MRP4 reduced tumor growth. Furthermore, MRP4 knockdown induced cell cycle arrest and apoptosis in vivo. Interestingly, when LSC population was isolated, we observed that increased cAMP levels and MRP4/ABCC4 blockade resulted in LSCs differentiation. Taken together, our findings show that MRP4/ABCC4 has a relevant role in tumor growth and apoptosis and in the eradication of LSCs, providing the basis for a novel promising target in AML therapy.

Knockdown of CUG-binding protein 1 induces apoptosis of human laryngeal cancer cells.

To investigate the role of CUG-binding protein 1 (CUGBP1) in human laryngeal cancer, we employed lentivirus-mediated short hairpin RNA (shRNA) to knockdown CUGBP1 expression in Hep-2 cells. Depletion of CUGBP1 remarkably inhibited the proliferation of Hep-2 cells. CUGBP1 knockdown induced cell cycle arrest in S phase, especially in the sub-G1 phase, representing apoptotic cells. Knockdown of CUGBP1 in Hep-2 cells markedly increased the expression of LIP and cleavage of PARP, which could contribute to apoptosis. Thus CUGBP1 has a critical role in modulating cell growth and apoptosis, and serves as a potential therapeutic target in laryngeal cancer.

ERBB3 knockdown induces cell cycle arrest and activation of Bak and Bax-dependent apoptosis in colon cancer cells.

ERBB3 is an emerging target for cancer therapy among the EGFR family. Contrary to resistance against EGFR and ERBB2 targeting, the genetic inhibition of ERBB3 results in anti-tumorigenic in HCT116 colon cancer cells harboring constitutively active KRAS and PIK3CA mutations. Still, the anti-tumorigenic molecular mechanism has not been defined. We demonstrated in this study that ERBB3 knockdown resulted in cell cycle arrest and activation of Bak and Bax-dependent apoptosis. Apoptosis was irrelevant to the majority of BH3-only pro-apoptotic proteins and correlated with the transcriptional upregulation of Bak and p53-dependent Bax translocation. Treatment with LY294002, a PI3K inhibitor, resulted in cell cycle arrest without apoptosis and a concomitant down-regulation of cap-dependent translation by the suppression of the PI3K/AKT/mTOR pathway. However, the inhibition of cap-dependent translation by ERBB3 knockdown occurred without altering the PI3K/AKT/mTOR pathway. In addition, ERBB3 knockdown-induced cell cycle arrest was observed in most colon cancer cells but was accompanied by apoptosis in p53 wild-type cells. These results indicate that ERBB3 is a potential target for EGFR- and ERBB2-resistant colon cancer therapy.

HOXB7 mRNA is overexpressed in pancreatic ductal adenocarcinomas and its knockdown induces cell cycle arrest and apoptosis

BackgroundHuman homeobox genes encode nuclear proteins that act as transcription factors involved in the control of differentiation and proliferation. Currently, the role of these genes in development and tumor progression has been extensively studied. Recently, increased expression of HOXB7 homeobox gene (HOXB7) in pancreatic ductal adenocarcinomas (PDAC) was shown to correlate with an invasive phenotype, lymph node metastasis and worse survival outcomes, but no influence on cell proliferation or viability was detected. In the present study, the effects arising from the knockdown of HOXB7 in PDAC cell lines was investigated.MethodsReal time quantitative PCR (qRT-PCR) (Taqman) was employed to assess HOXB7 mRNA expression in 29 PDAC, 6 metastatic tissues, 24 peritumoral tissues and two PDAC cell lines. siRNA was used to knockdown HOXB7 mRNA in the cell lines and its consequences on apoptosis rate and cell proliferation were measured by flow cytometry and MTT assay respectively.ResultsOverexpression of HOXB7 mRNA was observed in the tumoral tissues and in the cell lines MIA PaCa-2 and Capan-1. HOXB7 knockdown elicited (1) an increase in the expression of the pro-apoptotic proteins BAX and BAD in both cell lines; (2) a decrease in the expression of the anti-apoptotic protein BCL-2 and in cyclin D1 and an increase in the number of apoptotic cells in the MIA PaCa-2 cell line; (3) accumulation of cell in sub-G1 phase in both cell lines; (4) the modulation of several biological processes, especially in MIA PaCa-2, such as proteasomal ubiquitin-dependent catabolic process and cell cycle.ConclusionThe present study confirms the overexpression of HOXB7 mRNA expression in PDAC and demonstrates that decreasing its protein level by siRNA could significantly increase apoptosis and modulate several biological processes. HOXB7 might be a promising target for future therapies.

Expression and prognostic significance of Livin in gastric cancer

Livin is one of the most important members of the inhibitor of apoptosis protein family. It is overexpressed in several types of tumors and may have prognostic significance. The present study investigated the biological role of Livin in the oncogenic behavior of gastric cancer cells, the expression of Livin in gastric cancer tissue and the relationship of its expression with various clinicopathological parameters and patient survival. Small interfering RNA blocked Livin gene expression in AGS and SNU638 human gastric cancer cell lines. The expression of Livin was investigated in gastric cancer tissues by RT-PCR, western blotting and immunohistochemistry. The associations with various clinicopathological parameters and survival were analyzed. Livin knockdown inhibited tumor cell migration, invasion and proliferation in AGS and SNU638 cells. Livin knockdown induced apoptosis by activating caspase-3, caspase-7 and PARP. Livin knockdown induced cell cycle arrest by a decrease in cyclin D1, cyclin-dependent kinase 4 and 6 and an increase in expression of p21 and p27. The ERK1/2 and JNK signaling pathways were inhibited by Livin knockdown. Livin expression was upregulated in gastric cancer tissues at the mRNA and protein levels. However, no significant correlation was found between Livin expression and various clinicopathological parameters including survival. In conclusion, Livin expression may be important in the alteration of invasive and oncogenic phenotypes of gastric cancer cells. The prognostic relevance of Livin remains unclear.

ALKBH3 Contributes to Survival and Angiogenesis of Human Urothelial Carcinoma Cells through NADPH Oxidase and Tweak/Fn14/VEGF Signals

The role and function of a novel human AlkB homologue, ALKBH3, in human urothelial carcinoma development were examined. Biologic roles of ALKBH3 were examined by gene silencing analysis using in vitro and in vivo siRNA transfection. Immunohistochemical analyses of ALKBH3 and the related molecules using human bladder cancer samples were conducted to estimate the association with clinicopathologic or prognostic parameters. ALKBH3 knockdown induced cell cycle arrest at the G1 phase through downregulation of NAD(P)H oxidase-2 (NOX-2)-mediated generation of reactive oxygen species (ROS). ALKBH3 knockdown reduced VEGF expression by reducing expression of tumor necrosis factor-like weak inducer of apoptosis (Tweak) and its receptor, fibroblast growth factor-inducible 14 (Fn14). Silencing of ALKBH3 or Tweak significantly suppressed invasion and angiogenesis of urothelial carcinoma in vivo as assessed both by a chorioallantoic membrane assay and in an orthotopic mouse model. Interestingly, not only urothelial carcinoma cells but also vascular endothelial cells within cancer foci expressed Fn14, which was strongly reduced by ALKBH3 and Tweak knockdown in vivo, suggesting that ALKBH3-dependent expression of Tweak stabilizes Fn14. Immunohistochemical examination showed high expression of ALKBH3, Tweak, and Fn14 in urothelial carcinoma, especially in high-grade, superficially, and deeply invasive carcinomas; moreover, Fn14-positive vessel counts within cancer foci were increased in invasive phenotypes. ALKBH3 contributes to development of urothelial carcinomas by accelerating their survival, angiogenesis, and invasion through NOX-2-ROS and Tweak/Fn14-VEGF signals.

The histone methyltransferase Wolf–Hirschhorn syndrome candidate 1‐like 1 (WHSC1L1) is involved in human carcinogenesis

Histone lysine methylation plays a fundamental role in chromatin organization. Although a set of histone methyltransferases have been identified and biochemically characterized, the pathological roles of their dysfunction in human cancers are still not well understood. In this study, we demonstrate important roles of WHSC1L1 in human carcinogenesis. Expression levels of WHSC1L1 transcript were significantly elevated in various human cancers including bladder carcinoma. Immunohistochemical analysis of bladder, lung, and liver cancers confirmed overexpression of WHSC1L1. WHSC1L1-specific small interfering RNAs significantly knocked down its expression and resulted in suppression of proliferation of bladder and lung cancer cell lines. WHSC1L1 knockdown induced cell cycle arrest at the G(2)/M phase followed by multinucleation of cancer cells. Expression profile analysis using Affymetrix GeneChip(®) showed that WHSC1L1 affected the expression of a number of genes including CCNG1 and NEK7, which are known to play crucial roles in the cell cycle progression at mitosis. As WHSC1L1 expression is significantly low in various normal tissues including vital organs, WHSC1L1 could be a good candidate molecule for development of novel treatment for various types of cancer.

Annexin A2 Silencing Induces G2 Arrest of Non-small Cell Lung Cancer Cells through p53-dependent and -independent Mechanisms

Annexin A2 (ANXA2) overexpression is required for cancer cell proliferation; however, the molecular mechanisms underlying ANXA2-mediated regulation of the cell cycle are still unknown. ANXA2 is highly expressed in non-small cell lung cancer (NSCLC) and is positively correlated with a poor prognosis. NSCLC A549 cells lacking ANXA2 exhibited defects in tumor growth in vivo and in cell proliferation in vitro without cytotoxicity. ANXA2 knockdown induced cell cycle arrest at G(2) phase. Unexpectedly, ANXA2 silencing increased the expression of p53 and its downstream genes, which resulted in p53-dependent and -independent G(2) arrest. Aberrant JNK inactivation, which was observed in ANXA2-deficient cells, inhibited cell proliferation following G(2) arrest. A lack of ANXA2 caused a loss of JNK-regulated c-Jun expression, resulting in an increase in p53 transcription. These results demonstrate a novel role for ANXA2 in NSCLC cell proliferation by facilitating the cell cycle partly through the regulation of p53 via JNK/c-Jun.

A Novel Senescence-Evasion Mechanism Involving Grap2 and Cyclin D Interacting Protein Inactivation by Ras Associated with Diabetes in Cancer Cells under Doxorubicin Treatment

Ras associated with diabetes (Rad) is a Ras-related GTPase that promotes cell growth by accelerating cell cycle transitions. Rad knockdown induced cell cycle arrest and premature senescence without additional cellular stress in multiple cancer cell lines, indicating that Rad expression might be critical for the cell cycle in these cells. To investigate the precise function of Rad in this process, we used human Rad as bait in a yeast two-hybrid screening system and sought Rad-interacting proteins. We identified the Grap2 and cyclin D interacting protein (GCIP)/DIP1/CCNDBP1/HHM, a cell cycle-inhibitory molecule, as a binding partner of Rad. Further analyses revealed that Rad binds directly to GCIP in vitro and coimmunoprecipitates with GCIP from cell lysates. Rad translocates GCIP from the nucleus to the cytoplasm, thereby inhibiting the tumor suppressor activity of GCIP, which occurs in the nucleus. Furthermore, in the presence of Rad, GCIP loses its ability to reduce retinoblastoma phosphorylation and inhibit cyclin D1 activity. The function of Rad in transformation is also evidenced by increased telomerase activity and colony formation according to Rad expression level. In vivo tumorigenesis analyses revealed that tumors derived from Rad knockdown cells were significantly smaller than those from control cells (P = 0.0131) and the preestablished tumors are reduced in size after the injection of siRad (P = 0.0064). Therefore, we propose for the first time that Rad may promote carcinogenesis at least in part by inhibiting GCIP-mediated tumor suppression.

PS208. Cytostatic Gene Therapy: RNAi-Mediated Survivin Knockdown Induces Cell Cycle Arrest, Polyploidy, and Reduced Migration of Vascular Smooth Muscle Cells (VS MC)

Survivin (SVV) has been implicated in the development of neointimal hyperplasia. We investigated the effects of RNA-interference (RNAi) mediated SVV knockdown on VS MC phenotype. SVV knockdown was achieved by either siRNA or lentiviral transduction of shRNA in primary cultured VS MC from human saphenous vein. Proliferation, cell cycle kinetics, apoptosis and migration were assessed. RNAi reduced SVV gene expression by qPCR (75%, p < 0.01), without loss of cell viability. RNAi treatment increased p53 levels, whereas total cellular SVV levels were variable. Subcellular fractionation revealed the majority of SVV protein in VS MC is mitochondrial. SVV RNAi treatment blocked proliferation in response to serum and PDGF-AB (p < 0.05, Fig 1). Cell cycle analysis revealed no evidence of G1/S block, but an increased G2/M fraction (27% SVV siRNA vs 17% control siRNA, p < 0.05) consistent with a mitotic defect. In a Transwell assay, SVV knockdown reduced migration to PDGF-AB (74% vs controls, p < 0.01), and actin-phalloidin staining revealed disorganized actin filaments and polygonal cell shape. Apoptosis was not induced by SVV RNAi, and sensitivity to apoptotic agonists (staurosporine, cytokines) was unchanged. RNAi targeting SVV in VS MC leads to cell cycle arrest at G2/M and impaired chemotaxis. Regulation of mitosis and apoptosis in VS MC appears to involve differentially regulated pools of SVV. Treatment of VS MC with SVV RNAi might limit the response to vascular injury without destabilizing the vessel wall.

Abstract 508: Synergistic effect of β-catenin knockdown and radiation combination toward AMC-HN9 head and neck cancer cells is associated with down-regulation of Ku expression.

Abstract Based on our previous report showing that up-regulation of Ku70/Ku80 in AMC-HN9 (HN9) cells is required for cell-cycle re-entry and cell proliferation following irradiation, further study to investigate the mechanism by which β-catenin knockdown sensitizes the cells to irradiation have been conducted. β-Catenin, as a multifunctional protein involved in cadherin-mediated cell-cell adhesion and Wnt signaling transduction, have been well known to regulates cell growth and survival following radiation in various types of cancer cells. However, the molecular mechanisms by which β-catenin affects radiation sensitivity are still not fully understood, which led us to explore, with regard to regulation of Ku70/Ku80 expression, the molecular mechanism of the synergistic effects of β-catenin silencing and radiation combination in radio-resistant head and neck cancer cells. β-Catenin silencing using small interfering RNA(siRNA) down-regulated β-catenin expression up to 72 h both in membranous and nuclear fraction of HN9 cells. β-Catenin knockdown induced cell cycle arrest on G1 phase and thus decreased cell proliferation, but didn't cause any DNA damage response and cell death. Whereas expression of Ku70/Ku80 was up-regulated in HN9 cell following irradiation (4Gy), in the cells treated with combination of radiation and β-catenin siRNA, Ku70/Ku80 expression were dramatically decreased. In addition, when β-catenin silenced cells were exposed to single dose of radiation, irradiation-induced cell death was much more increased from 3.1% to 13% and accompanied with caspase-3 activation and PARP cleavage. Taken together, these results suggest that inhibition of Ku expression through β-catenin silencing is associated with its radio-sentitizing effect in HN9 cells and further studies to clarify the pathway linking β-catenin signaling and regulation of Ku70/Ku80 expression are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 508.

Syntaxin 6, a Regulator of the Protein Trafficking Machinery and a Target of the p53 Family, Is Required for Cell Adhesion and Survival

The p53 family consists of p53, p63, and p73. It has been well characterized that all of the p53 family proteins are transcription factors and capable of regulating cell cycle and apoptosis. To determine whether the p53 family exerts tumor suppression by other mechanisms, we set to identify novel p53 family target genes. Here, we found that the gene encoding STX6 (syntaxin 6), a vesicle transporter protein, is directly regulated by each of the p53 family proteins. In addition, STX6 can be induced by DNA damage and Mdm2 inhibitor Nutlin-3 in a p53-dependent manner. To examine how STX6 mediates the activity of the p53 family, STX6 is inducibly overexpressed or knocked down in various cell lines. We found that overexpression of STX6 alone has limited effect on cell proliferation. In contrast, we found that knockdown of STX6 inhibits cell proliferation and survival. We also found that knockdown of STX6 leads to cell cycle arrest and apoptosis. Interestingly, we found that p53 is necessary for STX6 knockdown-induced cell cycle arrest and apoptosis. Furthermore, we found that STX6 is necessary for proper expression of focal adhesion kinase and integrin alpha5 adhesion receptor. Consistent with this observation, STX6 knockdown inhibits cell adhesion. Together, we postulate that STX6 is an effector and a modulator of the p53 family in the regulation of cell adhesion and survival.