CUL4A Overexpression Research Articles

A novel tRNA-derived fragment tRF-3023b suppresses inflammation in RAW264.7 cells by targeting Cul4a through NF-κB signaling.

The role of transfer RNA (tRNA)-derived fragment (tRF) in various diseases has been established. However, the effect of tRF-3023b on inflammation remains unclear. Inflammation was imitated in RAW264.7 cells by adding Lipopolysaccharide (LPS). Cells were first divided into control, LPS, and LPS + Bulleyaconitine A (BLA) groups. The contents of TNF-α, IL-6, and MCP-1 were quantified using ELISA. The levels of cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), and the phosphorylation of nuclear factor-kappa B (NF-κB)-P65 (p-P65) were detected by Western blotting. RNA sequencing was utilized to find differentially expressed tRFs (DE-tRFs) among three groups. The levels of various tRFs were checked by quantitative real-time PCR (qRT-PCR). Cell cycle and apoptosis were checked by flow cytometry. Dluciferase reporter assay was applied to predict and confirm the interaction between tRF-3023b and Cullin 4A (Cul4a), subsequently RNA pull-down followed by mass spectrometry analysis were conducted. BLA treatment decreased the contents of TNF-α, IL-6, MCP-1, and the expression levels of COX2, iNOS, p-P65. We found 6 DE-tRFs in LPS + BLA group compared to LPS group, tRF-3023b was high expression in control and BLA groups, and the lowest in LPS group. Cul4a was a direct target of tRF-3023b. tRF-3023b mimic affected the cell cycle distribution, promoted cells apoptosis, and suppressed the TNF-α, IL-6, MCP-1, COX2, iNOS and p-P65. The suppression of Cul4a affected the cell cycle distribution, resulted in an increase of cell apoptosis while a decrease of TNF-α, IL-6, MCP-1, COX2, iNOS and p-P65. Furthermore, Cul4a overexpression reversed the effect of tRF-3023b mimic. Cul4a knockdown reversed the effect of tRF-3023b inhibitor. Our study positions tRF-3023b as a compelling candidate, through its interaction with Cul4a, the underlying mechanism on inflammation maybe related to NF-κB pathway. The study provides a basis for exploring new therapeutic strategies for inflammation.

Cul4a ublažava LPS-indukovanu akutnu insuficijenciju bubrega blokiranjem NF-kB signalnog puta u slučaju sepse

BackgroundAcute kidney injury (AKI) is a common disease that can develop into end-stage kidney disease. Sepsis is one of the main causes of AKI. Currently, there is no satisfactory way to treat septic AKI. Therefore, we have shown the protective function of Cul4a in septic AKI and its molecular mechanism.MethodsThe cellular and animal models of septic AKI were established by using lipopolysaccharide (LPS). Western blot (WB) was employed to analyze Cul4a expression. RT-qPCR was employed to test the expression of Cul4a, SOD1, SOD2, GPX1, CAT, IL-6, TNF-a, Bcl-2, IL1b, Bax and KIM-1 mRNA. ELISA was performed to detect the contents of inflammatory factors and LDH. CCK-8 was utilized to detect cell viability. Flow cytometry was utilized to analyze the apoptosis. DHE-ROS kit was used to detect the content of ROS.ResultsCul4a was down-regulated in cellular and animal models of septic AKI. Oxidative stress is obviously induced by LPS, as well as apoptosis and inflammation. However, these can be significantly inhibited by up-regulating Cul4a. Moreover, LPS induced the activation of the NF-kB pathway, which could also be inhibited by overexpression of Cul4a.ConclusionsCul4awas found to be a protective factor in septic AKI, which could inhibit LPS-induced oxidative stress, apoptosis and inflammation of HK-2 cells by inhibiting the NF-kB pathway.

LncRNA NEAT1 facilitates the progression of colorectal cancer via the KDM5A/Cul4A and Wnt signaling pathway.

Colorectal cancer (CRC) is a major cause of cancer‑related mortality. The aberrant expression of long non‑coding RNAs (lncRNAs) is implicated in the pathogenesis of CRC. The present study investigated the role of lncRNA nuclear paraspeckle assembly transcript1 (NEAT1) in CRC. lncRNA NEAT1 expression was detected in CRC tissues and cell lines. HCT116 cells were transfected with si‑NEAT1, and the malignant behavior of the cells was detected. The binding associations between NEAT1 and E2F1, as well as between E2F1 and KDM5A were verified. si‑NEAT1‑transfected cells were also transfected with si‑KDM5A. H3K4me3 methylation and cullin4A (Cul4A) expression in HCT116 cells were detected. The si‑NEAT1‑transfected cells were also transfected with pc‑Cul4A. Proteins related to the Wnt pathway were detected. A xenograft model of CRC using nude mice was established and the mice were injected with si‑NEAT1‑transfected HCT116 cells. lncRNA NEAT1 was found to be upregulated in CRC tissues and cells. NEAT1 silencing inhibited the malignant behaviors of the HCT116 cells. lncRNA NEAT1 inhibited KDM5A expression by binding to E2F1. The downregulation of KDM5A reversed the inhibitory effects of NEAT1 silencing on the malignant behavior of the cells. KDM5A inhibited Cul4A expression via the demethylation of H3K4me3. The overexpression of Cul4A promoted the malignant behavior of the si‑NEAT1‑transfected HCT116 cells. lncRNA NEAT1 activated the Wnt pathway via KDM5A/Cul4A. Invivo experiments confirmed the role of NEAT1 in CRC. On the whole, the present study demonstrates that lncRNA NEAT1 facilitates the progression of CRC via the KDM5A/Cul4A/Wnt axis.

Autophagy Plays a Role in the CUL4A-Related Poor Prognosis of Intrahepatic Cholangiocarcinoma.

CUL4A regulate the termination of autophagy in a physical process. However, the relationship between CUL4A and autophagy in cancer is unclear. We retrospectively investigated 99 intrahepatic cholangiocarcinoma (iCCA) cases. Whole sections were used for immunohistochemical analysis for p62, and LC3B expression. Q-score was defined as the sum of the labeling intensity and proportion. The cut-off point for immunoreactivity was set. CUL4A was overexpressed in cell lines and autophagy reflux was compared after manipulation. The iCCA cases with CUL4A overexpression had significantly higher prevalence of intact activated autophagy (42.4 vs. 15.2%; p = 0.003), which was significantly associated with advance tumor stage (34.1% vs. 15.4%; p = 0.032), less extensive necrosis (8.3 vs. 49.3%; p < 0.001), and shortened disease-free survival (mean, 19.6 vs. 65.5 months, p = 0.015). In vitro, iCCA cells with CUL4A overexpression significantly increased LC3II level as compared to the cells under basal condition. Although both cell types showed intact autophagy with increased LC3II expression after bafilomycin A1 treatment, the accumulation of LC3II was higher in CUL4A-overexpressing cells. CUL4A overexpression increased the proliferation of cells as compared with control cells. After treatment with bafilomycin A1, proliferation was inhibited in both cell types, but the effects were more prominent in the cells overexpressing CUL4A. CUL4A promotes autophagy, and exhibits significantly higher autophagic flux which affects the proliferation of iCCA cells; these effects correlated with advance tumor stage and poor prognosis. Thus, targeting autophagy may be potentially therapeutic in iCCA.

CUL4A, ERCC5, and ERCC1 as Predictive Factors for Trabectedin Efficacy in Advanced Soft Tissue Sarcomas (STS): A Spanish Group for Sarcoma Research (GEIS) Study

A translational study was designed to analyze the expression of nucleotide excision repair (NER) and homologous recombination (HR) genes as potential predictive biomarkers for trabectedin in soft-tissue sarcoma (STS). This study is part of a randomized phase II trial comparing trabectedin plus doxorubicin versus doxorubicin in advanced STS. Gene expression levels were evaluated by qRT-PCR, while CUL4A protein levels were quantified by immunohistochemistry. Expression levels were correlated with patients’ progression-free survival (PFS) and overall survival (OS). Gene expression was also evaluated in cell lines and correlated with trabectedin sensitivity. In doxorubicin arm and in the whole series, which includes samples from both arms, no significant differences in terms of PFS were observed amongst the analyzed genes. In the group treated with trabectedin plus doxorubicin, the median of PFS was significantly longer in cases with CUL4A, ERCC1, or ERCC5 overexpression, while BRCA1 expression did not correlated with PFS. Gene expression had no prognostic influence in OS. CUL4A protein levels correlated with worse PFS in doxorubicin arm and in the whole series. In cell lines, only overexpression of ERCC1 was significantly correlated with trabectedin sensitivity. In conclusion, CUL4A, ERCC5, and mainly ERCC1 acted as predictive factors for trabectedin efficacy in advanced STS.

Cul4A Modulates Invasion and Metastasis of Lung Cancer Through Regulation of ANXA10.

Cullin 4A (Cul4A) is overexpressed in a number of cancers and has been established as an oncogene. This study aimed to elucidate the role of Cul4A in lung cancer invasion and metastasis. We observed that Cul4A was overexpressed in non-small cell lung cancer (NSCLC) tissues and the overexpression of Cul4A was associated with poor prognosis after surgical resection and it also decreased the expression of the tumor suppressor protein annexin A10 (ANXA10). The knockdown of Cul4A was associated with the upregulation of ANXA10, and the forced expression of Cul4A was associated with the downregulation of ANXA10 in lung cancer cells. Further studies showed that the knockdown of Cul4A inhibited the invasion and metastasis of lung cancer cells, which was reversed by the further knockdown of ANXA10. In addition, the knockdown of Cul4A inhibited lung tumor metastasis in mouse tail vein injection xenograft models. Notably, Cul4A regulated the degradation of ANXA10 through its interaction with ANXA10 and ubiquitination in lung cancer cells. Our findings suggest that Cul4A is a prognostic marker in NSCLC patients, and Cul4A plays important roles in lung cancer invasion and metastasis through the regulation of the ANXA10 tumor suppressor.

Abstract 4733: Dysregulation of CUL4A and CUL4B ubiquitin ligases in lung cancer

Abstract Background: The Cullin-RING ubiquitin ligases 4 (CRL4) is implicated in controlling cell cycle, DNA damage repair and checkpoint response based on studies employing cell lines and mouse models. CRL4 proteins, including CUL4A and CUL4B are often highly accumulated in human malignancies. Elevated CRL4 attenuates DNA damage repair and increases genome instability that is believed to facilitate tumorigenesis. However, this has yet to be evaluated in human cancer patients. Methods: 352 lung cancer and 62 benign lung specimens of Asian origin were constructed into tissue microarrays (TMAs) of four distinct lung cancer subtypes. Expression of CUL4A, CUL4B and their substrates was detected by immunohistochemistry (IHC), and analyzed statistically for their prognostic value and association with DNA damage response and genomic instability. Results: Both CUL4A and CUL4B are overexpressed in the majority of lung carcinomas (PCUL4A&amp;lt;0.001, PCUL4B&amp;lt;0.001), and significantly associated with tumor size (PCUL4A&amp;lt;0.001, PCUL4B=0.002), lymphatic invasion (PCUL4A=0.004, PCUL4B&amp;lt;0.001), metastasis (PCUL4A=0.019, PCUL4B=0.006), and advanced TNM stage (PCUL4A&amp;lt;0.001, PCUL4B&amp;lt;0.001), which parallels gene amplification and abnormal activation of the canonical Wnt signaling. Moreover, overexpression of CUL4A, but not CUL4B, is significantly associated with tobacco smoking (P=0.01), and inversely correlated with XPC and p21, both of which are substrates of CUL4A (PCUL4A=0.019, PCUL4B=0.006). Higher levels of CUL4A or CUL4B are significantly associated with patients’ OS (PCUL4A&amp;lt;0.001, PCUL4B&amp;lt;0.001) and PFS (PCUL4A&amp;lt;0.001, PCUL4B=0.001). Conclusion: Our findings revealed that CUL4A and CUL4B are differentially associated with etiological factors for pulmonary malignancies and are independent prognostic markers for survival of distinct lung cancer subtypes. Citation Format: Hui Li, Lei Jia, Fan Yan, Pengbo Zhou. Dysregulation of CUL4A and CUL4B ubiquitin ligases in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4733. doi:10.1158/1538-7445.AM2017-4733

CUL4A overexpression as an independent adverse prognosticator in intrahepatic cholangiocarcinoma

BackgroundCUL4A has been known for its oncogenic properties in various human cancers. However, its role in intrahepatic cholangiocarcinoma (iCCA) has not been explored.MethodsWe retrospectively investigated 105 iCCA cases from a single medical institution. Tissue microarrays were used for immunohistochemical analysis of CUL4A expression. CUL4A expression vectors were introduced in cell lines. Cell migration and invasion assays were used to compare the mobility potential of iCCA cells under basal conditions and after manipulation. Then we evaluated the effects of CUL4A on the cell growth by proliferation assay, and further checked the susceptibility to cisplatin in iCCA cells with or without CUL4A overexpression.ResultsCUL4A overexpression was detected in 34 cases (32.4%). Patients with CUL4A-overexpressing tumors exhibited shortened disease-free survival (mean, 27.7 versus 90.4 months; P = 0.011). In the multivariate analysis model, CUL4A overexpression was shown to be an independent unfavorable predictor for disease-free survival (P = 0.045). Moreover, stably transfected CUL4A-overexpressing iCCA cell lines displayed an increased mobility potential and enhanced cell growth without impact on susceptibility to cisplatin.ConclusionsOur data demonstrate that overexpression of CUL4A plays an oncogenic role in iCCA and adversely affects disease-free survival. Thus, it may prove to be a powerful prognostic factor and a potential therapeutic target.

Dysregulation of CUL4A and CUL4B Ubiquitin Ligases in Lung Cancer

The Cullin-RING ubiquitin ligase 4 (CRL4) is implicated in controlling cell cycle, DNA damage repair, and checkpoint response based on studies employing cell lines and mouse models. CRL4 proteins, including CUL4A and CUL4B, are often highly accumulated in human malignancies. Elevated CRL4 attenuates DNA damage repair and increases genome instability that is believed to facilitate tumorigenesis. However, this has yet to be evaluated in human patients with cancer. In our study, 352 lung cancer and 62 normal lung specimens of Asian origin were constructed into tissue microarrays of four distinct lung cancer subtypes. Expression of CUL4A, CUL4B, and their substrates was detected by immunohistochemistry and analyzed statistically for their prognostic value and association with DNA damage response and genomic instability. Our results show that both CUL4A and CUL4B are overexpressed in the majority of lung carcinomas (PCUL4A <0.001 and PCUL4B <0.001) and significantly associated with tumor size (PCUL4A <0.001 and PCUL4B = 0.002), lymphatic invasion (PCUL4A = 0.004 and PCUL4B <0.001), metastasis (PCUL4A = 0.019 and PCUL4B = 0.006), and advanced TNM stage (PCUL4A <0.001 and PCUL4B <0.001), which parallels gene amplification and abnormal activation of the canonical WNT signaling. Moreover, overexpression of CUL4A, but not CUL4B, is significantly associated with tobacco smoking (p = 0.01) and is inversely correlated with XPC and P21, both of which are substrates of CUL4A (PCUL4A = 0.019 and PCUL4B = 0.006). Higher levels of CUL4A or CUL4B are significantly associated with the overall survival of patients (PCUL4A <0.001 and PCUL4B <0.001) and progression-free survival (PCUL4A <0.001 and PCUL4B = 0.001). Our findings revealed that CUL4A and CUL4B are differentially associated with etiologic factors for pulmonary malignancies and are independent prognostic markers for the survival of distinct lung cancer subtypes.

CUL4A promotes proliferation and metastasis of colorectal cancer cells by regulating H3K4 trimethylation in epithelial-mesenchymal transition.

Increasing evidence suggests that CUL4A, a ubiquitin ligase, is involved in the promotion of cancer malignancy and correlated with worse clinical prognosis in several kinds of human cancers. Although its effect and mechanism on the progression of colorectal cancer (CRC) remain unknown. Our clinical data show that CUL4A protein is overexpressed, positively associated with lymph nodes status, differentiation degree, tumor size, and poor prognosis in 80 CRC patients. CUL4A overexpression promotes cell proliferation and colony formation of CRC cells. Knockdown of CUL4A inhibits cell proliferation and migration. CUL4A can significantly promote the in vitro migration of CRC cells via induction of the epithelial–mesenchymal transition process. And the modulation of CUL4A expression altered the level of H3K4 trimethylation at the E-cadherin, N-cadherin, and vimentin gene promoters, which in turn transcriptionally regulated their expression. Moreover, knockdown of CUL4A also decreased the tumor volume and tumor weight in vivo. Together, our results reveal that CUL4A plays as an oncogene in CRC and may become a potential therapeutic target in the treatment of colorectal cancer.

CUL4A facilitates hepatocarcinogenesis by promoting cell cycle progression and epithelial-mesenchymal transition

CUL4A, a member of the CULLIN family, functions as a scaffold protein for an E3 ubiquitin ligase. It was reported that the CUL4A gene showed amplification in some human primary hepatocellular carcinomas (HCC). However, the exact role of CUL4A in HCC remains unknown. Here, we aimed to investigate the expression and function of CUL4A in HCC development. Through immunohistochemistry study, we showed increased CUL4A expression in HCC tissues. Statistical analysis disclosed an inverse correlation between CUL4A expression and tumor differentiation grade, and patient survival, but a positive correlation with hepatocyte proliferation as well as lymphatic and venous invasion. CUL4A expression in HCC tissues was associated with HBeAg status in patients and upregulated by HBV in HCC cell lines. Further functional assay showed that CUL4A overexpression significantly promoted growth of H22 tumor homografts in BALB/c mice. Consistently, CUL4A knockdown inhibited the proliferation of established HCC cells, accompanied by S-phase reduction and Cyclin A and Cyclin B1 repression. Furthermore, CUL4A siRNA ameliorated the motility of HCC cell lines with altered expression of epithelial-mesenchymal transition (EMT)-associated molecules. Taken together, our findings indicate that CUL4A plays a pivotal role in HCC progression and may serve as a potential marker for clinical diagnosis and target for therapy.

Cul4A overexpression associated with Gli1 expression in malignant pleural mesothelioma.

Malignant pleural mesothelioma (mesothelioma) is a highly aggressive cancer without an effective treatment. Cul4A, a scaffold protein that recruits substrates for degradation, is amplified in several human cancers, including mesothelioma. We have recently shown that Cul4A plays an oncogenic role in vitro and in a mouse model. In this study, we analysed clinical mesothelioma tumours and found moderate to strong expression of Cul4A in 70.9% (51/72) of these tumours, as shown by immunohistochemistry. In 72.2% mesothelioma tumours with increased Cul4A copy number identified by fluorescence in situ hybridization analysis, Cul4A protein expression was moderate to strong. Similarly, Cul4A was overexpressed and Cul4A copy number was increased in human mesothelioma cell lines. Because Gli1 is highly expressed in human mesothelioma cells, we compared Cul4A and Gli1 expression in mesothelioma tumours and found their expression associated (P < 0.05, chi-square). In mesothelioma cell lines, inhibiting Cul4A by siRNA decreased Gli1 expression, suggesting that Gli1 expression is, at least in part, regulated by Cul4A in mesothelioma cells. Our results suggest a linkage between Cul4A and Gli1 expression in human mesothelioma.

PR-Set7 is Degraded in a Conditional Cul4A Transgenic Mouse Model of Lung Cancer.

Background and objectiveMaintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer. PR-Set7 (also known as Set8) is a cell cycle regulated enzyme that catalyses monomethylation of histone 4 at Lys20 (H4K20me1) to promote chromosome condensation and prevent DNA damage. Recent studies show that CRL4CDT2-mediated ubiquitylation of PR-Set7 leads to its degradation during S phase and afer DNA damage. Tis might occur to ensure appropriate changes in chromosome structure during the cell cycle or to preserve genome integrity afer DNA damage.MethodsWe developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A. We have therefore used a mouse model to demonstrate for the frst time that Cul4A is oncogenic in vivo. With this model, staining of PR-Set7 in the preneoplastic and tumor lesions in AdenoCre-induced mouse lungs was performed. Meanwhile we identifed higher protein level changes of γ-tubulin and pericentrin by IHC.ResultsTe level of PR-Set7 down-regulated in the preneoplastic and adenocarcinomous lesions following over-expression of Cul4A. We also identifed higher levels of the proteins pericentrin and γ-tubulin in Cul4A mouse lungs induced by AdenoCre.ConclusionPR-Set7 is a direct target of Cul4A for degradation and involved in the formation of lung tumors in the conditional Cul4A transgenic mouse model.

Analysis of lung tumor initiation and progression in transgenic mice for Cre-inducible overexpression of Cul4A gene.

BackgroundLung cancer is the leading cause of morbidity and death worldwide. Although the available lung cancer animal models have been informative and further propel our understanding of human lung cancer, they still do not fully recapitulate the complexities of human lung cancer. The pathogenesis of lung cancer remains highly elusive because of its aggressive biologic nature and considerable heterogeneity, compared to other cancers. The association of Cul4A amplification with aggressive tumor growth and poor prognosis has been suggested. Our previous study suggested that Cul4A is oncogenic in vitro, but its oncogenic role in vivo has not been studied.MethodsViral delivery approaches have been used extensively to model cancer in mouse models. In our experiments, we used Cre-recombinase induced overexpression of the Cul4A gene in transgenic mice to study the role of Cul4A on lung tumor initiation and progression and have developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A.ResultsHere we show that the use of a recombinant adenovirus expressing Cre-recombinase (“AdenoCre”) to induce Cul4A overexpression in the lungs of mice allows controls of the timing and multiplicity of tumor initiation. Following our mouse models, we are able to study the potential role of Cul4A in the development and progression in pulmonary adenocarcinoma as well.ConclusionOur findings indicate that Cul4A is oncogenic in vivo, and this mouse model is a tool in understanding the mechanisms of Cul4A in human cancers and for testing experimental therapies targeting Cul4A.

CUL4A overexpression enhances lung tumor growth and sensitizes lung cancer cells to erlotinib via transcriptional regulation of EGFR.

BackgroundCUL4A has been proposed as oncogene in several types of human cancer, but its clinical significance and functional role in human non-small cell lung cancer (NSCLC) remain unclear.MethodsExpression level of CUL4A was examined by RT-PCR and Western blot. Forced expression of CUL4A was mediated by retroviruses, and CUL4A silencing by shRNAs expressing lentiviruses. Growth capacity of lung cancer cells was measured by MTT in vitro and tumorigenesis in vivo, respectively.ResultsWe found that CUL4A was highly expressed in human lung cancer tissues and lung cancer cell lines, and this elevated expression positively correlated with disease progression and prognosis. Overexpression of CUL4A in human lung cancer cell lines increased cell proliferation, inhibited apoptosis, and subsequently conferred resistance to chemotherapy. On other hand, silencing CUL4A expression in NSCLC cells reduced proliferation, promoted apoptosis and resulted in tumor growth inhibition in cancer xenograft model. Mechanistically, we revealed CUL4A regulated EGFR transcriptional expression and activation, and subsequently activated AKT. Targeted inhibition of EGFR activity blocked these CUL4A induced oncogenic activities.ConclusionsOur results highlight the significance of CUL4A in NSCLC and suggest that CUL4A could be a promising therapy target and a potential biomarker for prognosis and EGFR target therapy in NSCLC patients.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-252) contains supplementary material, which is available to authorized users.

Lung tumourigenesis in a conditional Cul4A transgenic mouse model

Cullin4A (Cul4A) is a scaffold protein that assembles cullin-RING ubiquitin ligase (E3) complexes and regulates many cellular events, including cell survival, development, growth and cell cycle control. Our previous study suggested that Cul4A is oncogenic in vitro, but its oncogenic role in vivo has not been studied. Here, we used a Cul4A transgenic mouse model to study the potential oncogenic role of Cul4A in lung tumour development. After Cul4A over-expression was induced in the lungs for 32 weeks, atypical epithelial cells were observed. After 40 weeks, lung tumours were visible and were characterized as grade I or II adenocarcinomas. Immunohistochemistry (IHC) revealed decreased levels of Cul4A-associated proteins p21(CIP1) and tumour suppressor p19(ARF) in the lung tumours, suggesting that Cul4A regulated their expression in these tumours. Increased levels of p27(KIP1) and p16(INK4a) were also detected in these tumours. Moreover, the protein level of DNA replication licensing factor CDT1 was decreased. Genomic instability in the lung tumours was further analysed by the results from pericentrin protein expression and array comparative genomic hybridization analysis. Furthermore, knocking down Cul4A expression in lung cancer H2170 cells increased their sensitivity to the chemotherapy drug cisplatin in vitro, suggesting that Cul4A over-expression is associated with cisplatin resistance in the cancer cells. Our findings indicate that Cul4A is oncogenic in vivo, and this Cul4A mouse model is a tool in understanding the mechanisms of Cul4A in human cancers and for testing experimental therapies targeting Cul4A.

CUL4A Induces Epithelial–Mesenchymal Transition and Promotes Cancer Metastasis by Regulating ZEB1 Expression

The ubiquitin ligase CUL4A has been implicated in tumorigenesis, but its contributions to progression and metastasis have not been evaluated. Here, we show that CUL4A is elevated in breast cancer as well as in ovarian, gastric, and colorectal tumors in which its expression level correlates positively with distant metastasis. CUL4A overexpression in normal or malignant human mammary epithelial cells increased their neoplastic properties in vitro and in vivo, markedly increasing epithelial-mesenchymal transition (EMT) and the metastatic capacity of malignant cells. In contrast, silencing CUL4A in aggressive breast cancer cells inhibited these processes. Mechanistically, we found that CUL4A modulated histone H3K4me3 at the promoter of the EMT regulatory gene ZEB1 in a manner associated with its transcription. ZEB1 silencing blocked CUL4A-driven proliferation, EMT, tumorigenesis, and metastasis. Furthermore, in human breast cancers, ZEB1 expression correlated positively with CUL4A expression and distant metastasis. Taken together, our findings reveal a pivotal role of CUL4A in regulating the metastatic behavior of breast cancer cells.

Involvement of CUL4A in Regulation of Multidrug Resistance to P-gp Substrate Drugs in Breast Cancer Cells

CUL4A encodes a core component of a cullin-based E3 ubiquitin ligase complex that regulates many critical processes such as cell cycle progression, DNA replication, DNA repair and chromatin remodeling by targeting a variety of proteins for ubiquitination and degradation. In the research described in this report we aimed to clarify whether CUL4A participates in multiple drug resistance (MDR) in breast cancer cells. We first transfected vectors carrying CUL4A and specific shCUL4A into breast cancer cells and corresponding Adr cells respectively. Using reverse transcription polymerase chain reactions and western blots, we found that overexpression of CUL4A in MCF7 and MDA-MB-468 cells up-regulated MDR1/P-gp expression on both the transcription and protein levels, which conferred multidrug resistance to P-gp substrate drugs, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. On the other hand, silencing CUL4A in MCF7/Adr and MDA-MB-468/Adr cells led to the opposite effect. Moreover, ERK1/2 in CUL4A-overexpressing cells was highly activated and after treatment with PD98059, an ERK1/2-specific inhibitor, CUL4A-induced expression of MDR1/P-gp was decreased significantly. Lastly, immunohistochemistry in breast cancer tissues showed that P-gp expression had a positive correlation with the expression of CUL4A and ERK1/2. Thus, these results implied that CUL4A and ERK1/2 participated in multi-drug resistance in breast cancer through regulation of MDR1/P-gp expression.

Abstract 91: The CUL4A ubiquitin ligase, a putative target gene at the 13q34 amplicon and its role in breast cancer pathogenesis &amp; progression

Abstract Breast cancer is a complex and heterogeneous disease and one of the most frequent causes of cancer deaths in developed countries. DNA copy number alterations occur frequently in breast tumours. Regions of DNA amplification are of major interest since often contain oncogenes whose increased expression confers tumour cells a selective advantage and contributes to the carcinogenesis process. In our laboratory prior analysis of familiar and sporadic breast tumours by using array-CGH identified an amplification event at the 13q34 region with an overall frequency of 4.5% that increased to 8.1% in BRCA1-associated tumours. Comprehensive characterization of the amplicon allowed us to define CUL4A as one of the most likely putative oncogenes. CUL4A is an E3 Ubiquitin Ligase which is amplified and/or overexpressed in primary breast tumours and involved in functions such as chromatin regulation, cell cycle regulation or DNA repair through ubiquitylation of NER pathway members. Its deregulation could have implications for oncogenic transformation and treatment response to DNA-damaging agents. One of such compounds is Trabectedin a marine-derived drug that presents antitumor activity in sarcomas and ovarian cancer. To elucidate the possible implication of CUL4A in the initial steps of the tumorigenic process we conducted the stable up-regulation of CUL4A in human mammary epithelial cells (184B5), and in NIH3T3 mouse-derived cell line by lentiviral infection. We also stably silenced CUL4A in the human breast cancer cell lines MDA-MB-157 and HCC1937 that present amplification and/or overexpression of CUL4A. With these modified cell lines we performed functional assays in different in vitro systems such as viability experiments, colony formation both in plastic and in soft agar and 3-D cultures in Matrigel. In order to measure sensitivity to Trabectedin we used silenced CUL4A breast cancer cell lines to perform clonogenic assays. Our preliminary results in CUL4A overexpression in 184B5 model system showed increase in proliferation and colony formation ability. On the other hand, CUL4A silencing in breast cancer cell lines resulted in diminished proliferation and decreased colony-forming abilities in both anchorage dependent and independent conditions. Concerning DNA-repair implication, it seems that high levels of CUL4A could be a good predictor of Trabectedin response. Our results indicate that CUL4A would play a role in promoting oncogenesis and/or in modulating breast cancer progression. Further evaluation of CUL4A amplification/overexpression impact on the maintenance of genome integrity will help elucidate its full potential for therapeutic intervention in breast and other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 91. doi:1538-7445.AM2012-91

Abstract 210: Cul4A is an oncogene in malignant pleural mesothelioma

Abstract Cullin 4A (Cul4A) is important in cell survival, development, growth and the cell cycle, but its role in mesothelioma has not been studied. For the first time, we identified amplification of the Cul4A gene in four of five mesothelioma cell lines. Consistent with increased Cul4A gene copy number, we found that Cul4A protein was overexpressed in mesothelioma cells as well. Cul4A protein was also overexpressed in 64% of primary malignant pleural mesothelioma (MPM) tumours. Furthermore, knockdown of Cul4A with shRNA in mesothelioma cells resulted in up-regulation of p21 and p27 tumour suppressor proteins in a p53-independent manner in H290, H28 and MS-1 mesothelioma cell lines. Knockdown of Cul4A also resulted in G0/G1 cell cycle arrest and decreased colony formation in H290, H28 and MS-1 mesothelioma cell lines. Moreover, G0/G1 cell cycle arrest was partially reversed by siRNA down-regulation of p21 and/or p27 in Cul4A knockdown H290 cell line. In the contrary, overexpression of Cul4A resulted in down-regulation of p21 and p27 proteins and increased colony formation in H28 mesothelioma cell line. Both p21 and p27 showed faster degradation rates in Cul4A overexpressed H28 cell line and slower degradation rates in Cul4A knockdown H28 cell line. Our study indicates that Cul4A amplification and overexpression play an oncogenic role in the pathogenesis of mesothelioma. Thus, Cul4A may be a potential therapeutic target for malignant pleural mesothelioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 210. doi:1538-7445.AM2012-210