HDAC10 expression is associated with DNA mismatch repair gene and is a predictor of good prognosis in colon carcinoma
- Authors:
- Published online on: August 24, 2017 https://doi.org/10.3892/ol.2017.6818
- Pages: 4923-4929
Abstract
Introduction
Histone deacetylases (HDACs) was the name originally given to a family of proteins responsible for deacetylation of histone proteins, which were later shown to be also involved in the deacetylation of non-histone proteins (1,2). HDACs are divided into four classes based on structure: Class I including HDAC1, HDAC2, HDAC3 and HDAC8; class II, which is further divided into class IIa (HDAC4, HDAC5, HDAC7 and HDAC9) and class IIb (HDAC6 and HDAC10); class III comprising SIRT1 to SIRT7; and class IV, consisting of only HDAC11. While HDAC6 is a well-investigated class II HDAC, little is known about HDAC10.
HDAC10 has been reported to be involved in homologous recombination (3), melanogenesis (4), cells autophagy (5–7), cell cycle regulation (8), DNA mismatch repair (9) and cancer progression (10–16). While HDAC10 was reported to suppresses the proliferation and invasion of clear cell renal cell carcinoma (13), it was also demonstrated to promote cell proliferation via AKT phosphorylation in lung cancer (15). Based on these contradictory observations, we hypothesized that the function of HDAC10 in cancer is more complex and may be dependent on the type of tissue.
Colorectal carcinoma is among the five most commonly diagnosed cancers accounting for over 50% of the top five cancers all cases in China (17). Owing to the high risk of relapse and metastasis, the treatment for advanced colon cancer poses a significant challenge. Thus, it is crucial to discover new and competent therapeutic targets for colon cancer, thereby enabling the discovery of new diagnostic and therapeutic drugs.
To date, the expression of HDAC10, especially the prognostic role and its association with clinicopathological features in colon cancer has not been investigated. In this study we analyzed 100 colon cancer specimens in a tissue microarray (TMA) to assess HDAC10 expression and to evaluate the clinical significance of HDAC10 in colon cancer.
Patients and methods
Patients
A total of 100 colon cancer patients (54 male, 45 female and one missed gender information) aged between 24 and 90 were recruited in this study. All patients underwent surgery during July 2006 to May 2007 and received no prior extra therapy. Of these, 6 were cTNM stage I, 54 were cTNM stage II, 35 were cTNM stage III, and 3 were cTNM stage IV according to AJCC. Following surgery, a long-term follow-up was implemented for all patients up to July 2015. During the follow-up time, 61 patients died of colon carcinoma with a median overall survival time of 26 months. Detailed patient information is listed in Table I.
Immunohistochemistry
Colon adenocarcinoma TMA (HColA180Su09) containing 100 tumor tissues and 80 paired adjacent tissues was obtained from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China) for standardization. Deparaffinization of the TMA was performed by xylene and graded alcohol following incubation at high temperature for an hour. After antigen retrieval by EDTA and blocking with goat serum, the TMA was incubated with the primary anti-HDAC10 antibody (24913-1-AP) obtained from ProteinTech Group, Inc. (Chicago, IL, USA) at a dilution of 1:2,500 at 4°C overnight, and subsequently incubated with horse radish peroxidase (HRP) labeled secondary-antibody (K8000; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 30 min. Diaminobenzidine (DAB) and hematoxylin redyeing were performed for visualization. Three random fields having more than 100 cells were visually analyzed and scored by pathologists. The colon cancer patients were divided into three subgroups based on differences in HDAC10 expression as follows: 0–60%, low expression; 61–90%, median expression; 91–100%, high expression. The expression of mutL homolog 1 (MLH1) (1:5,000, sc-56160; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mutS homolog (MSH)2 (1:100, sc-56163; Santa Cruz Biotechnology, Inc.), MSH6 (1:5,000, 66172-1-Ig; ProteinTech Group, Inc.) and PMS2 (1:1,500, sc-618; Santa Cruz Biotechnology, Inc.) was also detected in these patients with the same protocol. The specificity of the anti-HDAC10 antibody used in the present study was validated by Wang et al in a previous study (18).
Statistical analysis
The difference in HDAC10 expression between colon adenocarcinoma and adjacent tissues was evaluated by paired t-test. Spearman's rank correlation coefficient and two-tailed test were performed to evaluate the correlation between HDAC10 expression and clinical parameters. Pearson analysis was performed to assess the association between HDAC10 and MLH1/MSH2/MSH6/PMS2 expression. Based on HDAC10 and clinical parameters, overall survival curves were drawn according to the Kaplan-Meier method and log-rank test. Subsequently, COX multivariate regression survival analysis was performed to determine the independent prognostic marker. All statistical analyses were conducted using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA), with P<0.05 being considered significant.
Results
Evaluation of HDAC10 mRNA expression in colon carcinoma
The HDAC10 mRNA expression level in colon adenocarcinoma was investigated in the Oncomine database. As depicted in Fig. 1, HDAC10 mRNA expression in colon adenocarcinoma tissues was found to be significantly upregulated both in the Kaiser colon statistics involving 41 colon carcinoma tissues and 5 normal tissues (fold change=1.103, P<0.05; Fig. 1A) as well as in the TCGA colorectal database containing 101 colon adenocarcinoma tissues and 19 normal colon tissues (fold change=1.656, P=1.07e-7; Fig. 1B).
HDAC10 expression is high in colon carcinoma
To investigate HDAC10 expression in colon adenocarcinoma, two-step immunohistochemistry was performed on the TMA. As shown in Fig. 2, constitutively high HDAC10 expression was visualized both in the cytoplasm and nucleus in most colon cancer tissues when compared with the adjacent normal tissues. Subsequently, HDAC10 expression in colon cancer was systemically investigated by statistical analysis with the adjacent normal tissue as a control (cytoplasm, 93.12±12.98 vs. 31.65±26.50%; Fig. 2C; nucleus, 84.16±19.23 vs. 68.64±19.00%; Fig. 2D). The results indicated significantly elevated expression of HDAC10 protein at the tissue level. Regardless of the location, HDAC10 expression did not show a significant correlation with the tumor tissue when compared with the adjacent normal tissue (Table I). Interestingly, HDAC10 expression in the nucleus was associated with its cytoplasmic expression, both in the tumor tissue as well as in the adjacent normal tissue (Table II).
Correlation between HDAC10 expression and clinical parameters
HDAC10 expression in colon adenocarcinoma tissues showed no significant correlation with any clinicopathological factors, apart from a link between cytoplasmic HDAC expression and gender (r=0.265, P<0.05). Intriguingly however, HDAC10 expression in para-carcinoma tissues was highly associated with clinicopathological factors. Cytoplasmic HDAC10 expression was found to be positively correlated with lymph node metastasis (N stage, r=0.256, P<0.05) and distant metastasis (M stage, r=0.331, P<0.05). Detailed results of the correlation analysis are listed in Table III.
Different prognostic role of HDAC10 in colon carcinoma and para-carcinoma
High cytoplasmic expression of HDAC10 in tumor tissues predicted good prognosis in colon cancer patients, with 0% survival in the population with low HDAC10 expression after 8 years of follow-up, in contrast with 29.4% for population with median expression and 43.0% for population with high expression (Fig. 3A). Conversely, cytoplasmic HDAC10 expression in para-carcinoma tissues was associated with poor outcome of patients (43.3 vs. 20.0 vs. 0%, P<0.001; Fig. 3B). Nuclear HDAC10 expression in the tumor tissues did not correlate with overall increased survival of colon cancer patients (28.6 vs. 40.8 vs. 40.5%, P>0.05; Fig. 3C), while high nuclear HDAC10 expression in the para-carcinoma tissues was correlated with increased survival rate (43.5 vs. 38.9 vs. 0%, P<0.001; Fig. 3D). Furthermore, regional lymph node metastasis (N stage, 51.7 vs. 25.9 vs. 9.1%, P=0.000), distant metastasis (M stage, 40.2 vs. 0.0%, P<0.001), tumor location (right vs. left, 31.1 vs. 52.6%, P<0.05) and clinical stage (cTNM, 66.7 vs. 50.0 vs. 22.9 vs. 0.0%, P<0.001) were all correlated with overall survival time. Subsequent multivariate analysis indicated that only cytoplasmic HDAC10 expression was an independent prognostic marker for colon cancer (Table IV).
HDAC10 may be associated with DNA mismatch repair
The implication of HDAC10 in DNA repair pathway via interaction with DNA mismatch repair gene MSH2 in HeLa cells prompted us to explore the possibility that HDAC10 is involved in the progression of colon cancer via its interaction with the DNA mismatch repair genes (9). Four major DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 were investigated by immunohistochemistry. Pearson analysis was performed to evaluate the their association with HDAC10. The results, listed in Table V, predicted that cytoplasmic HDAC10 expression in the tumor tissue was not associated with any of the four DNA mismatch repair genes. Instead it showed negative association with MLH1 (r=−0.244, P<0.05), MSH2 (r=−0.410, P<0.001) and MSH6 (r=−0.240, P<0.05) in the para-carcinoma tissues. Similarly, nuclear HDAC10 expression was negatively correlated with MLH1 expression (r=−0.288, P<0.05).
Table V.Association between HDAC10 expression and expression of DNA mismatch repair genes in colon cancer. |
Discussion
To the best of our knowledge, our findings highlight for the first time, the clinical significance of HDAC10 in colon cancer. HDAC10 was demonstrated to be a tumor suppresser in some types of cancer, including clear cell renal cell carcinoma (13), cervical cancer (19), gastric cancer (12,20), and ovarian cancer (16). The function of HDAC10 in lung cancer is a matter of debate (10,15). High HDAC10 expression was associated with good prognosis in non-small cell lung cancer (10) but has been lately demonstrated to promote lung cancer proliferation (15).
Class II HDACs have been reported to be able to shuttle between the nucleus and cytoplasm. In this study, we found similar behavior of HDAC10 expression in colon cancer tissues. Yang et al reported that HDAC10 is mainly expressed in the cytoplasm of lung cancer cells but is mainly located in the nucleus of normal lung cells, and suggested different functions of cytoplasmic and nuclear HDAC10 in lung cancer progression (15). The association of cytoplasmic and nuclear HDAC10 expression and clinicophathological and prognostic effect is inconsistent even in colon cancer. Moreover, in tumor tissues HDAC10 acted as a tumor suppressor, but showed quite different effect in adjacent tissues, suggesting that HDAC10 might act as a tumor suppressor in colon cancer tissues but may also function as a tumor promoter by promoting tumor metastasis to regional lymph node or to distant tissues.
A growing number of studies point to quite different prognosis of patients between right-sided and left-sided colon cancers (21). The probability of relapse of colon carcinoma in different sides is dependent on different molecular pathways (22). Our observation is consistent with the previous studies that survival of patients with right-sided colon cancer is less than those with left-sided colon cancer.
In summary, our findings suggest that HDAC10 expression in tumor tissues is associated with good prognosis of colon cancers but predicted poor prognostic outcomes in para-carcinoma tissues, probably owing to regulation of the DNA mismatch repair pathway. Further studies by altering the HDAC10 expression in colon cancer cells and normal colon cells to investigate the potential functionin invasion and metastasis is needed to test our notion that HDAC10 has different roles in tumor and para-carcinoma tissues.
Acknowledgements
The present study was supported by Foundation for Key Project of Natural Science Research Education Department of Anhui Province (KJ2016A726 and KJ2017A249).
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