UbcH10 Expression Research Articles

Abstract 3075: A novel transcriptional role of spindle assembly checkpoint protein Cdc20 regulating the expression of mitotic ubiquitin carrier protein UbcH10

Abstract Progression through mitosis requires precisely timed ubiquitin-dependent degradation of specific substrates. UbcH10, an ubiquitin-conjugating enzyme, plays a crucial role with anaphase promoting complex/ cyclosome (APC/C) in progression of and exit from mitotis. Similarly, spindle assembly checkpoint (SAC) protein Cdc20 plays an important role in transition from metaphase to anaphase by activating APC/C. Cdc20 itself gets ubiquitinated by UbcH10 and thus activated to bind with APC/C. Any defect in chromosome alignment activates SAC by blocking Cdc20 and thus anaphase transition is arrested. Earlier our lab reported that Cdc20 overexpression led to defective SAC resulting in aneuploidy. This prompted us to examine the regulation of UbcH10 expression in Cdc20 upregulated condition. Initially, we observed a correlation in the expression of both Cdc20 and UbcH10 in different primary tumors and cancer cell lines. Next, a dose-dependent increase in endogenous UbcH10 levels was found upon ectopic expression of Cdc20. Alternatively, CDC20 knockdown by siRNA led to downregulation of endogenous UbcH10. Activation of UBCH10 promoter-luciferase activity by Cdc20 indicated the regulation at transcriptional level. ChIP assay showed the presence of Cdc20 on UBCH10 promoter leading to chromatin remodelling by interacting with CBP/p300. APC/C-CBP/p300 complex is reported to be involved in transcription regulation. Here, we established the presence of APC/C on the UBCH10 promoter using ChIP assay. Further from ChIP and Co-IP assay we established that Cdc20 regulates APC/C and CBP/p300 association and thus modulates APC/C-CBP/p300 mediated transcriptional regulation of UBCH10. Also, we found cell cycle specific regulation of expression of UbcH10 by Cdc20. Finally, Cyclin B1 degradation assay showed physiological relevance of Cdc20 mediated UbcH10 regulation on mitotic progression. In summary, our data showed a novel role of Cdc20 as a transcription regulator and it regulates UbcH10 expression in a cell cycle specific manner. The co-regulated expression of both these proteins maintains proper mitotic progression which otherwise may results in aneuploidy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3075. doi:10.1158/1538-7445.AM2011-3075

Compromised Spindle Assembly Checkpoint due to Altered Expression of Ubch10 and Cdc20 in Human Papillomavirus Type 16 E6- and E7-Expressing Keratinocytes

Cells expressing human papillomavirus type 16 (HPV-16) E6 and E7 proteins exhibit deregulation of G2/M genes, allowing bypass of DNA damage arrest signals. Normally, cells with DNA damage that override the G2 damage checkpoint would precociously enter mitosis and ultimately face mitotic catastrophe and apoptotic cell death. However, E6/E7-expressing cells (E6/E7 cells) have the ability to enter and exit mitosis in the presence of DNA damage and continue with the next round of the cell cycle. Little is known about the mechanism that allows these cells to gain entry into and exit from mitosis. Here, we show that in the presence of DNA damage, E6/E7 cells have elevated levels of cyclin B, which would allow entry into mitosis. Also, as required for exit from mitosis, cyclin B is degraded in these cells, permitting initiation of the next round of DNA synthesis and cell cycle progression. Proteasomal degradation of cyclin B by anaphase-promoting complex/cyclosome (APC/C) is, in part, due to elevated levels of the E2-conjugating enzyme, Ubch10, and the substrate recognition protein, Cdc20, of APC/C. Also, in E6/E7 cells with DNA damage, while Cdc20 is complexed with BubR1, indicating an active checkpoint, it is also present in complexes free of BubR1, presumably allowing APC/C activity and slippage through the checkpoint.

UbcH10 expression on thyroid fine‐needle aspirates

Thyroid fine-needle aspiration (FNA) samples belonging to the follicular neoplasm/suspicious for malignancy classes are controversial. The authors identified UbcH10 as a marker useful in the diagnosis of several neoplasms, including thyroid cancer. Here, analysis of UbcH10 expression by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry was applied to FNAs. A series of 84 follicular neoplasm/suspicious for malignancy FNAs with histological follow-up (30 malignant) was prospectively collected. UbcH10 immunostaining was performed on cell blocks and compared with that of the proliferation marker Ki-67. At the mRNA level, UbcH10 was compared with CCND2 and PCSK2 expression, these latter being the best performing components of the previously reported 3-gene assay; to determine the diagnostic accuracy, the area under the curve (AUC) of the receiver operating characteristic (ROC) curve for each gene individually and in combination was evaluated. UbcH10 and Ki-67 shared a similar pattern; although UbcH10 expression was higher in malignant than in benign lesions (P < .001), staining was sporadic, and the cutoff value derived by the ROC analysis was too low (1.25%) for routine application. Conversely, UbcH10 expression assessment by quantitative RT-PCR was effective. UbcH10 mRNA levels associated with malignant histology were significantly higher than those associated with benign histology (P = .02). The AUC was 0.74 for UbcH10, 0.81 for CCDN2, 0.62 for PCSK2, and 0.84 for UbcH10 and CCND2 combination. UbcH10 quantitative RT-PCR analysis, rather than immunohistochemistry, is useful to increase the detection of malignancy in thyroid FNAs. UbcH10 may be added as a panel component in quantitative RT-PCR-based assays.

Association of clinicopathological features with UbcH10 expression in colorectal cancer

UbcH10 is the cancer-related E2 ubiquitin-conjugating enzyme, and its overexpression has been demonstrated in a variety of malignancies. The aim of this study is to investigate the association of UbcH10 gene expression with the carcinogenesis and tumor progression of colorectal cancer. The expression levels of UbcH10 in human malignant colorectal carcinoma tissues and their adjacent normal tissues were examined using real-time quantitative RT-PCR and immunohistochemical analysis. The correlations of UbcH10 expression to the clinicalpathologic characteristics of the colorectal cancer were analyzed. Cell proliferation and Matrigel invasion assays were performed in HT-29 cells transfected with UbcH10 expression plasmid pcDNA3.1-UbcH10, UbcH10 RNA interference vector pUbcH10-RNAi as well as their control vectors. Our study demonstrated that the expression of UbcH10 in colorectal carcinoma tissues was significantly higher than that in non-cancerous tissues (P < 0.01), and the UbcH10 overexpression was related to the degree of tumor differentiation and lymph node metastasis of colorectal cancer patients (P < 0.05). In vitro, the overexpression of UbcH10 promoted cell proliferation and tumor invasiveness, but the downregulation of UbcH10 expression significantly reduced the growth rate and the invasiveness activity of tumor cell line. Our study suggests that the overexpression of UbcH10 gene plays a critical role in the carcinogenesis and tumor progression of colorectal cancer. It may be a new marker in diagnosis and prognosis of colorectal cancer, and the inhibition of UbcH10 may be a therapeutic potential for the treatment of colorectal cancer.

Knockdown of ubiquitin-conjugating enzyme E2C/UbcH10 expression by RNA interference inhibits glioma cell proliferation and enhances cell apoptosis in vitro

To address the role of ubiquitin-conjugating enzyme, E2C/UbcH10, in astrocytic carcinogenesis. Expression pattern of UbcH10 in U251 glioma cells was evaluated by immunohistochemistry and western blot. RNA interference was employed to downregulate UbcH10 expression in U251 cell line. The effect of UbcH10 silencing on cell proliferation was assessed by MTT assay and cell cycle analysis. Cell apoptosis was determined by flow cytometry, TUNEL staining and western blot. Levels of UbcH10 protein were significantly upregulated in U251 cells compared with normal brain tissues. Marked immunoreactivity for UbcH10 was demonstrated in the cytoplasm of U251 glioma cells, especially in the mitotic cells. The growth rate of U251 cells was significantly inhibited by depletion of UbcH10 by short interference RNA. Further, UbcH10 RNAi induced apoptosis through induction of Bax and p53, downregulation of Bcl-2 and G2/M arrest of the cell cycle. These data imply that knocking-down UbcH10 protein expression may represent a potential therapeutic option for glioma.

Erratum

HistopathologyVolume 54, Issue 7 p. 918-918 Free Access Erratum This article corrects the following: UbcH10 expression in human lymphomas Giancarlo Troncone, Eliana Guerriero, Pierloronzo Pallante, Maria Teresa Berlingieri, Angelo Ferraro, Luigi Del Vecchio, Marisa Gorrese, Elisabetta Mariotti, Antonino Iaccarino, Emiliano A Palmieri, Pio Zeppa, Lucio Palombini, Alfredo Fusco, Volume 54Issue 6Histopathology pages: 731-740 First Published online: April 24, 2009 First published: 01 June 2009 https://doi.org/10.1111/j.1365-2559.2009.03317.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL In Troncone et al.,1 a typographical error appeared in an author’s name. The third author’s name should have appeared as Pierlorenzo Pallante. We apologize for these errors. Reference 1 Troncone G, Guerriero E, Pallante P et al. UbcH10 expression in human lymphomas. Histopathology 2009; 54; 731– 740. Volume54, Issue7June 2009Pages 918-918 ReferencesRelatedInformation

UbcH10 expression in human lymphomas

The UbcH10 ubiquitin-conjugating enzyme plays a key role in regulating mitosis completion. We have previously reported that UbcH10 overexpression is associated with aggressive thyroid, ovarian and breast carcinomas. The aim of this study was to investigate UbcH10 expression in human lymphomas. Cell lines and tissue samples of Hodgkin's lymphoma (HL) and of non-Hodgkin's lymphoma (NHL) were screened for UbcH10 expression at transcriptional and translational levels. UbcH10 expression was related to the grade of malignancy. In fact, it was low in indolent tumours and high in a variety of HL and NHL cell lines and in aggressive lymphomas. It was highest in Burkitt's lymphoma, as shown by quantitative real-time polymerase chain reaction and by tissue microarray immunohistochemistry. Flow cytometry of cell lines confirmed that UbcH10 expression is cell-cycle dependent, steadily increasing in S phase, peaking in G(2)/M phase and dramatically decreasing in G(0)/G(1) phases. We also showed that UbcH10 plays a relevant role in lymphoid cell proliferation, since blocking of its synthesis by RNA interference inhibited cell growth. Taken together, these results indicate that UbcH10 is a novel lymphoid proliferation marker encompassing the cell cycle window associated with exit from mitosis. Its overexpression in aggressive lymphomas suggests that UbcH10 could be a therapeutic target in this setting.

Overexpression of UbcH10 alternates the cell cycle profile and accelerate the tumor proliferation in colon cancer

BackgroundUbcH10 participates in proper metaphase to anaphase transition, and abrogation of UbcH10 results in the premature separation of sister chromatids. To assess the potential role of UbcH10 in colon cancer progression, we analyzed the clinicopathological relevance of UbcH10 in colon cancer.MethodsWe firstly screened the expression profile of UbcH10 in various types of cancer tissues as well as cell lines. Thereafter, using the colon cancer cells line, we manipulated the expression of UbcH10 and evaluated the cell cycle profile and cellular proliferations. Furthermore, the clinicopathological significance of UbcH10 was immunohistologically evaluated in patients with colon cancer. Statistical analysis was performed using the student's t-test and Chi-square test.ResultsUsing the colon cancer cells, depletion of UbcH10 resulted in suppression of cellular growth whereas overexpression of UbcH10 promoted the cellular growth and oncogenic cellular growth. Mitotic population was markedly alternated by the manipulation of UbcH10 expression. Immunohistochemical analysis indicated that UbcH10 was significantly higher in colon cancer tissue compared with normal colon epithelia. Furthermore, the clinicopathological evaluation revealed that UbcH10 was associated with high-grade histological tumors.ConclusionThe results show the clinicopathological significance of UbcH10 in the progression of colon cancer. Thus UbcH10 may act as a novel biomarker in patients with colon cancer.

Clinicopathological relevance of UbcH10 in breast cancer

Abrogated mitotic progression is a common hallmark of cancer. UbcH10, one of the components of the ubiquitin/proteasome pathway, plays a pivotal role in the regulation of mitotic progression. Abnormal UbcH10 activity is reported in certain types of human cancers; its overexpression is occasionally encountered in cancerous tissue compared with adjacent normal tissue. Current studies have suggested the critical role of UbcH10 in the spindle assembly checkpoint and the subsequent accurate separation of sister chromatids, which is orchestrated by a series of molecular interactions governed by the complex and diverse cell cycle machinery. To validate the potential role of UbcH10 in cell proliferation in cancer, we have analyzed the clinicopathological relevance of UbcH10 in progression of breast cancer using a combinatorial approach of human tumor arrays and biochemical analyses. Our results show that the percentage of tested samples which stained positive for UbcH10 in breast cancer tissues is significantly higher compared to the adjacent nonmalignant tissue. Furthermore, results from the clinicopathological analysis have revealed that elevated expression of UbcH10 is associated with higher histological grade tumors. In addition, depletion of UbcH10 by RNA interference in breast cancer cells resulted in decreased cellular proliferation, while overexpression of UbcH10 significantly enhanced cellular growth in breast cancer. Our results suggest a pathological correlation between UbcH10 and cell proliferation in breast cancer. Thus, aberrant UbcH10 activity may induce the dysfunction of proper cell cycle progression and result in the aggressive behavior of tumor cells in patients with breast cancer.

Analysis of UbcH10 expression represents a useful tool for the diagnosis and therapy of astrocytic tumors

Previous studies suggest the expression of UbcH10 gene, that codes for a protein belonging to the ubiquitin-conjugating enzyme family, as a valid indicator of the proliferative and aggressive status of tumors of different origin. Therefore, to look for possible tools to be used as diagnostic markers in astrocytic neoplasias, we investigated UbcH10 expression in normal brain, gliosis and low-grade and high-grade astrocytic tumors by immunohistochemistry. UbcH10 expression was observed in low-grade astrocytoma and in glioblastoma. Our data indicate a clear correlation between UbcH10 expression and the histological grade of the astrocytic tumors. Moreover, the analysis of UbcH10 expression allows the differentiation between gliotic and malignant tissues. Finally, since proteasome inhibitors have recently been considered as possible drugs in the chemotherapy of various tumors, our results would suggest new perspectives for the treatment of brain malignancies based on the suppression of the UbcH10 function.

Expression of ubiquitin-conjugating enzyme E2C/UbcH10 in astrocytic tumors

UbcH10 is one of the key regulators of cell cycle progression through the mitotic spindle assembly checkpoint pathway. Recently, aberrantly high UbcH10 expression has been demonstrated in a variety of malignancies. However, its role in astrocytic carcinogenesis is not well defined. This study investigated the splice pattern of the UbcH10 gene and its expression status in astrocytomas of different grades. Consequently, UbcH10 splice variant 1 (GenBank accession nos. NM_007019) was detected in astrocytomas and normal brain tissues by RT-PCR and sequence analysis. Expression levels of UbcH10 mRNA were elevated in high- versus low-grade astrocytomas (64.33 ± 60.98 vs 8.36 ± 8.15, respectively; p = 0.000) or normal controls (64.33 ± 60.98 vs 1.00 ± 1.57, respectively; p = 0.000), as determined by quantitative real time PCR analysis. Similarly, immunohistochemistry study showed increased UbcH10 labelling index in high-grade astrocytomas versus low-grade tumors (10.53 ± 5.79% vs 4.23 ± 2.85%, respectively; p = 0.000) or normal controls (10.53 ± 5.79% vs 0.0 ± 0.0%, respectively; p = 0.000) and, a positive correlation between UbcH10 immunoreactivity and Ki-67 immunostaining was also noted (Spearman r = 0.63, p < 0.001). These data suggest that overexpression of UbcH10 may serve as one important molecular mechanism that underlies the astrocytic carcinogenesis.

UbcH10 is overexpressed in malignant breast carcinomas

Our group has recently demonstrated the overexpression of the UbcH10 gene in undifferentiated thyroid carcinomas. Subsequently, a clear correlation between UbcH10 overexpression and a reduced survival in ovarian carcinoma patients has been described indicating UbcH10 as a valid prognostic marker in this neoplastic disease. Here we have extended the analysis of the UbcH10 expression to neoplastic breast diseases. We demonstrated, by tissue micro-arrays immunohistochemical studies, a significant difference ( p = 0.0001) in the mean percentage of UbcH10 stained cells between benign (0.22%) and malignant (11.01%) neoplastic lesions. High UbcH10 expression was associated with intense Ki-67 staining ( p = 0.015) and ErbB2 positivity ( p = 0.092). The suppression of the ErbB2 expression in breast carcinoma cell lines induces a reduction of UbcH10 level. Consistently, the inhibition of breast carcinoma cell growth was achieved following the block of UbcH10 protein synthesis by RNA interference. Therefore, these results suggest the perspective of a therapy of aggressive breast carcinomas based on the suppression of the UbcH10 function.

UbcH10 expression may be a useful tool in the prognosis of ovarian carcinomas

The UbcH10 gene codes for a protein that belongs to the ubiquitin-conjugating enzyme family. Previous studies of our group suggest UbcH10 expression as a valid indicator of the proliferative and aggressive status of thyroid carcinomas. Therefore, to better understand the process of ovarian carcinogenesis, and to look for possible tools to be used as prognostic markers in these neoplasias, we decided to extend the analysis of the UbcH10 expression to the ovarian neoplastic disease. We found that the UbcH10 gene was upregulated in some ovarian carcinoma cell lines analysed. Then, immunohistochemical studies demonstrate that UbcH10 expression significantly correlates with the tumor grade and the undifferentiated histotype of the ovarian carcinomas. Furthermore, a significant relationship between UbcH10 expression and overall survival was observed. Finally, the block of UbcH10 protein synthesis by RNA interference inhibited the growth of ovarian carcinoma cell lines, suggesting a role of UbcH10 overexpression in ovarian carcinogenesis. Therefore, all these data taken together suggest the possibility to use UbcH10 detection as a marker for the diagnosis and prognosis of these neoplastic diseases and open the perspective of a therapy of some ovarian carcinomas based on the suppression of the UbcH10 synthesis and/or function.

UbcH10 overexpression may represent a marker of anaplastic thyroid carcinomas

The hybridisation of an Affymetrix HG_U95Av2 oligonucleotide array with RNAs extracted from six human thyroid carcinoma cell lines and a normal human thyroid primary cell culture led us to the identification of the UbcH10 gene that was upregulated by 150-fold in all of the carcinoma cell lines in comparison to the primary culture cells of human normal thyroid origin. Immunohistochemical studies performed on paraffin-embedded tissue sections showed abundant UbcH10 levels in thyroid anaplastic carcinoma samples, whereas no detectable UbcH10 expression was observed in normal thyroid tissues, in adenomas and goiters. Papillary and follicular carcinomas were only weakly positive. These results were further confirmed by RT–PCR and Western blot analyses. The block of UbcH10 protein synthesis induced by RNA interference significantly reduced the growth rate of thyroid carcinoma cell lines. Taken together, these results would indicate that UbcH10 overexpression is involved in thyroid cell proliferation, and may represent a marker of thyroid anaplastic carcinomas.

Overexpression, genomic amplification and therapeutic potential of inhibiting the UbcH10 ubiquitin conjugase in human carcinomas of diverse anatomic origin.

Gene expression profiling of anatomically diverse carcinomas and their corresponding normal tissues was used to identify genes with cancer-associated expression. We show here that the ubiquitin conjugase, UbcH10, is significantly overexpressed in many different types of cancers and is associated with the degree of tumor differentiation in carcinomas of the breast, lung, ovary and bladder, as well as in glioblastomas. We also show that UbcH10 overexpression in gastro-esophageal, and probably other carcinomas may be a direct consequence of chromosomal amplification at the UbcH10 locus, 20q13.1, a region known to be amplified in diverse tumors. To evaluate whether inhibition of UbcH10 function may be therapeutically relevant in cancer, we used small interfering RNAs (siRNAs) to silence UbcH10 transcription selectively. Diminution of UbcH10 expression significantly inhibited both tumor and normal cell proliferation without inducing cell death. However, when combined with agonists of the DR5/TRAIL receptor, siRNAs directed against the UbcH10 transcript dramatically enhanced killing of cancer cells, but not of proliferating primary human epithelial cells or fibroblasts. Together, these data demonstrate that UbcH10 plays an important role in tumor development and that its inhibition in combination with agonists of the TRAIL receptor may provide an enhanced therapeutic index.