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

Abstract

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.

Main

Ovarian carcinomas have a great impact in human pathology, in fact ovarian cancer is the leading cause of death from gynecological neoplasias and the fifth most common cancer among women worldwide (Ozols et al., 2004; Jemal et al., 2005).

Research aimed to determine the specific genes involved in the development of ovarian cancers would help to understand how normal ovarian epithelial cells escape regulation of proliferation, apoptosis and senescence. It has been already (Welcsh and King, 2001) determined that approximately 10% of ovarian cancers arises in women who have inherited mutations in cancer-susceptibility genes such as BRCA1, BRCA2 and other DNA repair genes. Conversely, the vast majority of ovarian cancers are sporadic, presumably resulting from the accumulation of genetic damage over lifetime. Several genes involved in ovarian carcinogenesis have been identified, most notably the p53 tumor suppressor (Feki and Irminger-Finger, 2004).

Recently in our laboratory, by a microarray gene expression profiling, we found a gene, UbcH10, that was upregulated in thyroid anaplastic carcinoma samples vs the normal thyroid tissues, suggesting a correlation with the malignant progression (Pallante et al., 2005). A high expression of UbcH10 has also been found in carcinomas of different anatomic origin (Wagner et al., 2004).

The UbcH10 gene belongs to the E2 gene family and codes for a protein of 19.6 kDa that is involved in the ubiquitin-dependent proteolysis. In this pathway, ubiquitin-conjugating enzyme (E2), together with ubiquitin ligase (E3), transfers ubiquitin to specific substrate proteins (Hershko and Ciechanover, 1998; Joazeiro and Weissman, 2000).

The aim of our work was to investigate whether the UbcH10 expression might be a new useful indicator for the diagnosis and prognosis of ovarian cancer.

Therefore, we evaluated the expression of UbcH10 by reverse transcription–polymerase chain reaction (RT–PCR) and Western blot in ovarian carcinoma cell lines in comparison with the normal ovarian tissue. All of the carcinoma cell lines showed a high UbcH10 expression, which was barely detectable in the normal tissue (Figure 1a and b).

Figure 1
figure1

UbcH10 expression in human ovarian carcinoma cell lines and tissues. (a) UbcH10 gene expression analysis by RT–PCR in human ovarian carcinoma cell lines (SKOV-3, IGROV, OVCAR-3, OVCAR-4, OVCAR-5 and OVCAR-8; Masciullo et al., 2003) vs normal tissue. β-Actin gene expression was evaluated as control to normalize the amount of the used RNAs. (b) UbcH10 protein expression analysis by Western blot in the same human ovarian carcinoma cell lines. Blot against α-tubulin has been performed as control for equal protein loading. (c) RT–PCR analysis of UbcH10 expression in human ovarian tumor samples (thirty ovarian carcinoma samples were collected) vs their normal ovarian counterparts. β-Actin expression shows the same amount of RNAs used. NO, normal ovarian tissue. (d) Western blot analysis of UbcH10 protein expression in a panel of ovarian neoplasias. The level of α-tubulin has been used as loading control. NO, normal ovarian tissue. PCR reactions, primers and conditions to amplify UbcH10 and β-actin are as reported elsewhere (Pallante et al., 2005). Protein extraction was performed according to standard procedure as reported by Pierantoni et al. (2005).

The same analyses, performed on ovarian carcinoma tissues, showed a different expression in the different histotypes analysed. In fact, as shown in Figure 1c and d, UbcH10 is expressed at low levels in clear cell carcinomas, endometrioid carcinomas and serous cystadenocarcinomas, whereas the undifferentiated carcinomas express high level of UbcH10.

Immunohistochemistry essentially confirms the data obtained by RT–PCR and Western blot analyses. Whereas benign ovary lacked UbcH10 expression (Figure 2f), the different histotypes of ovarian carcinomas showed certain variability in the range of neoplastic cells expressing UbcH10 (Figure 2a–e). However, there was a clear relationship between the loss of tumor differentiation and the gain of UbcH10 reactivity. This was evident both when examining tumor type and tumor grade: the average levels of UbcH10 expression were low in tumors belonging to the differentiated histotypes, as in serous (16.6% mean, range: 12.3–19.8%), clear cell (7.1% mean, range: 1.3–10.0%) and endometrioid (9.2% mean, range: 8.3–10.8%) carcinomas. Similarly the only case examined of the mucinous histotype showed a low level of expression (5%). On the contrary, higher levels (33.6% mean, range: 22.9–42.1%) of UbcH10 expression were observed in the undifferentiated histotype of ovarian cancer. All seven cases of undifferentiated ovarian carcinomas had levels of UbcH10 expression higher than the median value (P=0.015).

Figure 2
figure2

Immunostaining pattern of UbcH10 expression in ovarian cancer. In all different histotypes of ovarian carcinoma, there was a clear relationship between loss of tumor differentiation and the gain of UbcH10 reactivity. Whereas in differentiated histotypes, as in mucinous (original magnification, 25 ×) (a), serous (original magnification, 25 ×) (b), clear cell (original magnification, 25 ×) (c) and endometrioid (original magnification, 25 ×) (d) carcinomas, there was variability in the levels of UbcH10 expression, all cases of undifferentiated ovarian carcinomas (original magnification, 25 ×) (e) had levels of UbcH10 expression higher than the median value. No immunoreactivity occurred in normal ovarian tissue (original magnification, 25 ×) (f). UbcH10 immunoreactivity has been evaluated in two TMAs (constructed at the Department of Histopathology, S Chiara Hospital, Trento, Italy), as described (Aldovini et al., 2006). TMAs included 60 carcinomas arising from the ovarian surface epithelium, each represented by three different 0.6 mm cores. Pathological data (tumor type, grade and stage), IHC profile (p53, RB245 and ER status) and clinical follow-up data (relapse-free and overall survival rates) were available for analysis. Tumors were typed according to the WHO criteria (Tavassoli and Devilee, 2003), including serous (n=38), mucinous (n=1), clear cell (n=4), endometrioid (n=6) and undifferentiated (n=7) carcinomas; four cases were excluded for technical reason. Staging was assessed in accordance with the International Federation of Gynecology and Obstetrics (FIGO) (Benedet et al., 2000). Grading informations were derived from the original diagnostic reports. As the tumors composing the TMAs were diagnosed in different years, special care was taken to adopt the same grading system and to grade consistently all cases, by using the same criteria. Consequently, the same four-tier classification system (Vacher-Lavenu et al., 1993), employed in the 1990s and used to diagnose the earlier cases included in the TMAs (with a longer follow-up) was consistently used also to score the cases more recently diagnosed. Thus, tumors were graded into well differentiated (G1), moderately well differentiated without nuclear atypia (G2), moderately well differentiated with nuclear atypia (G3) and poorly differentiated or undifferentiated (G4) (Vacher-Lavenu et al., 1993). Further details on TMAs generation are reported elsewhere (Aldovini et al., 2006). Complete follow-up data were available for all patients from the Departments of Gynecological Surgery and Medical Oncology of the S Chiara Hospital of Trento. Follow-up time was based on patient date of death or the last information in the medical records. As control carried out to assess UbcH10 expression in benign ovary, five samples of normal ovary were also processed. Special care was taken to evaluate UbcH10 expression only on adequately stained TMA slides. Immunostaining was processed by routine methods (Troncone et al., 2003). UbcH10 expression was evaluated by two pathologists in a double-blind fashion and any discordance was resolved after consultation at the double-headed microscope. Values relative to each tumor sample, present in triplicate, were derived by combining the percentage of any single cores. To analyse tissue microarrays, we employed maximum, minimum and mean as pooling methods for the replicates (Liu et al., 2004) and statistical analysis was carried out. We dichotomized biomarker expression against its median values, so as to ease biological interpretation. Finally, to account for the heterogeneity of protein expression across tumor tissues, cases with only one core section with valid staining were excluded by the statistical analysis. This resulted in 56 cases for ovarian data set. Statistical analysis was performed with R statistical package (R Development Core Team, 2004) and SPSS ver. 11.5 for Windows. (g) Correlation between ovarian tumor grade and UbcH10 expression. The correlation of UbcH10 expression vs tumor grade is well established as low levels of UbcH10 expression were recorded in the G1 well-differentiated tumors whereas there was a significant relationship between increasing cytological grading of malignancy and protein expression (P=0.002 Kruskall–Wallis test). Comparisons between UbcH10 expression and clinical–pathological data (histotype, grading, T, N), as well as with other biomarkers (p53, RB245, ER) were performed with Wilcoxon's signed rank test or χ2 or Kruskall–Wallis test on numerical expression data and with Fisher's exact test on categorized data.

Similarly, UbcH10 expression was also related to the tumor grade, as significant differences were recorded among the different grades (P=0.002 Kruskall–Wallis test). In fact, as shown in Figure 2g, low levels of UbcH10 expression were recorded in the G1 well-differentiated tumors (0.8%), whereas there was a significant relationship between increasing cytological grading of malignancy and protein expression. In G2 and in G3 carcinomas, the cells expressing UbcH10 were, respectively, 12.3 and 22%, whereas G4 high-grade tumors showed staining in more than a third of neoplastic cells (34.7%). The correlation with tumor grade is further established if we compare G1 and G2 vs G3 and G4 grades and UbcH10 expression (P-value=0.023 Fisher's exact test) (data not shown).

As far as the overall survival is concerned, there is a significant relationship with UbcH10 expression (P-value=0.044 – log-rank test), as shown in Figure 3, only if we consider the mean as pooling method. Regarding the other pooling methods, as well as for relapse-free survival, no statistical significant correlation was found.

Figure 3
figure3

Kaplan–Meier survival analysis with log-rank test was carried out both for overall and relapse-free survival. Fifty-six patients were included in the survival analysis as clinical information was entirely available. The picture shows the results for overall survival analysis. The median age at surgery is 61 years (range 34–84) and the median follow-up time is 62 months (range 0–121 months). A P-value less than 0.05 was considered statistically significant for each analysis. Cases showing higher levels of UbcH10 expression (>=median) were associated with a lower rate of overall survival.

We asked whether UbcH10 overexpression had a role in the process of ovarian carcinogenesis by evaluating the growth rate of one ovarian carcinoma cell line, in which the synthesis of UbcH10 protein was suppressed by RNA interference. The SKOV-3 cell line was treated with siRNA duplexes targeting to the UbcH10 mRNA. After transfection, we observed an efficient knock down of the UbcH10 protein levels at 48 h after treatment (Figure 4a). The cell growth analysis, in the presence or absence of the UbcH10 siRNA duplexes, revealed that the block of the UbcH10 protein synthesis significantly inhibits ovarian carcinoma cell growth. In fact, as shown in Figure 4b, a significant reduction in cell growth rate was observed in SKOV-3 cell line treated with UbcH10 siRNA in comparison with the untreated cells or those treated with the control scrambled siRNA.

Figure 4
figure4

The block of UbcH10 protein synthesis by RNA interference inhibits the proliferation of ovarian carcinoma cells. (a) Inhibition of UbcH10 protein expression by RNAi in SKOV-3 cell line evaluated by Western blot analysis. At 48 h after siRNA transfection, total cell lysates were prepared and normalized for protein concentration. The expression of α-tubulin was used to control equal protein loading (30 μg). (b) SKOV-3 cell line growth curve after siUbcH10 treatment. SKOV-3 cells were transfected with siUbcH10 duplexes (siUbcH10) and the relative number of viable cells was determined by MTT assay. Cells transfected with a scrambled duplex (siScrambled) and untransfected cells (Untransfected) were used as negative controls. Absorbance was read at 570 nm and the data are the mean of triplicates.

These results indicate a role of UbcH10 in neoplastic ovarian cell proliferation, for this reason we propose the UbcH10 expression as a possible tool to be used in the diagnosis and prognosis of ovarian carcinomas. Abundant expression of UbcH10 was detected in primary ovarian tumors compared with benign ovarian tissues and significantly correlated to tumor grade (P=0.0008) and undifferentiated histotype (P=0.015). These results are consistent with our previous published data (Pallante et al., 2005) and further support the involvement of UbcH10 in the differentiation process of several human epithelial tissues (Wagner et al., 2004).

UbcH10 is also a negative predictor of clinical outcome in our series, further suggesting its ability to confer a more aggressive phenotype to tumor cells. Studies are ongoing to assess this hypothesis on a larger series of patients with a longer follow-up.

Functional studies also demonstrate that the suppression of the UbcH10 expression by RNA interference reduced the growth of one ovarian carcinoma cell line indicating a role of UbcH10 overexpression in ovarian carcinogenesis, in particular, in influencing the hyperproliferative status of the most malignant cells.

In conclusion, all these data taken together suggest the possibility to use this gene as a marker for the diagnosis and prognosis of these neoplastic diseases.

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Acknowledgements

This work was supported by grants from the Associazione Italiana Ricerca sul Cancro (AIRC), Progetto Strategico Oncologia Consiglio Nazionale delle Ricerche, the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MIUR), and ‘Piani di Potenziamento della Rete Scientifica e Tecnologica’ CLUSTER C-04, the Programma Italia-USA sulla Terapia dei Tumori coordinated by Professor Cesare Peschle and ‘Ministero della Salute’. This work was supported from NOGEC-Naples Oncogenomic Center. We thank the Associazione Partenopea per le Ricerche Oncologiche (APRO) for its support.

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Berlingieri, M., Pallante, P., Guida, M. et al. UbcH10 expression may be a useful tool in the prognosis of ovarian carcinomas. Oncogene 26, 2136–2140 (2007). https://doi.org/10.1038/sj.onc.1210010

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Keywords

  • UbcH10
  • ovarian
  • carcinomas
  • immunohistochemistry

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