SIRT1 Expression Is Associated with Good Prognosis in Colorectal Cancer

Abstract

Silent mating type information regulation 2 homolog 1 (SIRT1), an NAD+-dependent deacetylase, might act as a tumor promoter by inhibiting p53, but may also as a tumor suppressor by inhibiting several oncogenes such as β-catenin and survivin. Deleted in breast cancer 1 (DBC1) is known as a negative regulator of SIRT1. Immunohistochemical expressions of SIRT1, DBC1, β-catenin, surviving, and p53 were evaluated using 2 mm tumor cores from 349 colorectal cancer patients for tissue microarray. Overexpression of SIRT1, DBC1, survivin, and p53 was seen in 235 (67%), 183 (52%), 193 (55%), and 190 (54%) patients, respectively. Altered expression of β-catenin was identified in 246 (70%) patients. On univariate analysis, overexpression of SIRT1 (p=0.029) and altered expression of β-catenin (p=0.008) were significantly associated with longer overall survival. Expression of SIRT1 was significantly related to DBC1 (p=0.001), β-catenin (p = 0.001), and survivin (p=0.002), but not with p53. On multivariate analysis, age, tumor stage, differentiation, and expression of SIRT1 were independent prognostic factors significantly associated with overall survival. SIRT1 overexpression is a good prognostic factor for colorectal cancer, and SIRT1 may interact with β-catenin and survivin rather than p53. [PUBLICATION ABSTRACT]

Headnote

Background: Silent mating type information regulation 2 homolog 1 (SIRT1), an NAD+-dependent deacetylase, might act as a tumor promoter by inhibiting p53, but may also as a tumor suppressor by inhibiting several oncogenes such as β-catenin and survivin. Deleted in breast cancer 1 (DBC1) is known as a negative regulator of SIRT1. Methods: Immunohistochemical expressions of SIRT1, DBC1, β-catenin, surviving, and p53 were evaluated using 2 mm tumor cores from 349 colorectal cancer patients for tissue microarray. Results: Overexpression of SIRT1, DBC1, survivin, and p53 was seen in 235 (67%), 183 (52%), 193 (55%), and 190 (54%) patients, respectively. Altered expression of β-catenin was identified in 246 (70%) patients. On univariate analysis, overexpression of SIRT1 (p=0.029) and altered expression of β-catenin (p=0.008) were significantly associated with longer overall survival. Expression of SIRT1 was significantly related to DBC1 (p=0.001), β-catenin (p = 0.001), and survivin (p=0.002), but not with p53. On multivariate analysis, age, tumor stage, differentiation, and expression of SIRT1 were independent prognostic factors significantly associated with overall survival. Conclusions: SIRT1 overexpression is a good prognostic factor for colorectal cancer, and SIRT1 may interact with β-catenin and survivin rather than p53.

Keywords: Colon; Adenocarcinoma; SIRT1 ; DBC1 ; Betacafenin

Silent mating type information regulation 2 homolog 1 (SIRT1) is one of the mammalian homologue of silent informa- tion regulator 2 (Sir2) which is a nicotinamide adenine dinucle- otide (NAD+)-dependent histone deacetylase that belongs to the class III histone deacetlylases.1 SIRT1 deacetylates not only histones but also many non-histone proteins which are involved in adaptation to calorie restriction, cell growth, apoptosis, cell senescence, and tumorigenesis.2 Recent reports have revealed that SIRT1 expression is increased in various human malignant tumors such as colon and breast cancer.3,4 However, the role of SIRT1 in malignant tumors is controversial. SIRT1 might act as a tumor promoter by inhibiting tumor suppressor genes such as p53,5 but SIRT1 might also act as a tumor suppressor by re- pressing several oncogenes or oncoproteins such as β-catenin and survivin.6-7 Thus, SIRT1 can serve as a tumor promoter or tumor suppressor, depending on the oncogenic pathways spe- cific to particular tumors.

Colorectal cancer is one of the most common malignancies in the world. However, there are few studies revealing the clinical relevance of the expression of SIRT1 and related markers in colorectal cancer with human tissue.0,9 Although recent studies have revealed that SIRT1 plays an important role in colorectal tumorigenesis, the exact role of SIRT1 in colorectal tumorigen- esis is still controversial.610 Some studies showed conflicting re- sults regarding the relationship between SIRT1 and clinical fea- tures in several tumors depending on which proteins interacted with SIRT1.11,12

Deleted in breast cancer 1 (DBC1) was found to be homozy- gously deleted in human chromosome 8p21 in breast cancer.1'' DBC1 enhances p53-mediated apoptosis through specific inhi- bition of deacetylase activity of SIRT.14 Recent studies have demonstrated that DBC1 expression is associated with poor prognosis in gastric and breast carcinoma.1215 A previous study revealed that, in colorectal cancer, DBC1 overexpression is re- lated to poor overall survival and poor prognostic indicators such as high tumor node metastasis (TNM) stage, poor histo- logic grade and presence of lymph node metastasis.16

β-catenin is a key regulator of Wnt signaling and is also known as an important regulator in cancer development, β-catenin is considered to play various roles in cadherin-medi- ated cell-to-cell adhesion, Wnt signaling transduction, and car- cinogenesis.17 The altered expression of β-catenin and its rela- tion to tumorigenesis have been investigated in several studies. One study reported that SIRT1 promotes cytoplasmic localiza- tion of β-catenin in colon cancer.6 Another study reported that altered expression of β-catenin was independently associated with poor prognosis in colon cancer.ls The diverse functions of β-catenin in tumorigenesis are thought to be associated with its altered expression. SIRT1 may be an important regulator of β-catenin in colorectal tumorigenesis.

Survivin, a member of the chromosomal passenger protein complex, is a small molecular weight protein that is an inhibi- tor of apoptosis, and is overexpressed in various cancers.19 SIRT1 is thought to inhibit survivin expression by deacety- lation of histone H3 within nucleosomes at the survivin pro- moter.7 Previous studies have reported that survivin expression is impaired by loss of p53 function, which is often observed in cancer cells.20

In this study, we examined the expression of SIRT1 and re- lated proteins such as DBC1, β-catenin, survivin, and p53, and their relationship to clinicopathologic features and prognosis. Through these results, we attempted to reveal the role of SIRT1 in colorectal cancer patients and their prognosis, as well as which proteins interact with SIRT1.

MATERIALS AND METHODS

Patients and samples

Three hundred forty nine patients who were diagnosed with colorectal cancer at the Korea University Guro Hospital from January 2002 to December 2009, who had surgical procedure such as right hemicolectomy, left hemicolectomy, sigmoidecto- my, anterior resection, and abdominoperineal resection and had no prior history of neo-adjuvant chemotherapy were included in this study. Clinicopathologic data such as sex, age, and dis- tant metastasis were collected from medical records. All of the cases were reviewed and classified according to the criteria of the World Health Organization (WHO) classification.21 Patho- logic staging was reviewed based on the TNM staging system of the American Joint Committee on Cancer.22 The patients were grouped according to age, sex, location, TNM stage, tu- mor invasion, presence of lymph node metastasis, presence of distant metastasis, and histologic grade. The mean follow-up period was 55.3 months. The median age was 63 years (range, 27 to 88 years). This study obtained approval from the institu- tional review board of Korea University Guro Hospital (KUG- GR-2011-015).

Tissue preparation and immunohistochemical staining

Hematoxylin and eosin-stained glass slides of selected patients were reviewed for construction of tissue microarray (TMA). Representative portions of tumor and normal mucosa were marked for TMA construction. One 2.0 mm core of tumor per case and one 2.0 mm core of representatively sampled normal mucosa were arrayed. TMA blocks were cut into 4 pm slices for immunohistochemical staining. A standard streptavidin-biotin peroxidase complex method was used. After deparaffinization and rehydration, slides were heated in a microwave oven for 15 minutes in 10 mM citrate buffer (pH 6.0) and treated with 3% hydrogen peroxide for 20 minutes. We used the Bond-maX au- tostainer (Leica, Wetzlar, Germany). The following antibodies were used: SIRT1 (1:50, clone H-300, Santa Cruz Biotechnolo- gy, Santa Cruz, CA, USA), DBC1 (1:100, polyclonal, Abeam, Cambridge, MA, USA), β-catenin (1:500, clone β-catenin-1, Dako, Carpintería, CA, USA), survivin (1:400, polyclonal, No- vus, Littleton, MA, USA), and p53 (1:500, clone DO-7, Novo- castra, Newcastle upon Tyne, UK). Each case was evaluated by estimating the percentages and intensity of tumor cells show- ing a nuclear staining pattern. Immunostaning for SIRT1 was considered positive if 30% or more of the tumor cells were stained with an antibody, referring to other previous studies us- ing the same antibody.1215 Immunostaining for DBC1 was con- sidered negative if more than 30% of tumor cells showed loss of expression, because DBC1 was expressed in all of the normal mucosa with nuclear staining pattern and at least moderate in- tensity; the cutoff value of 30% was chosen because DBC1 is known as negative regulator of SIRT1, so it was adjusted to the cutoff value of SIRT1. Survivin was also expressed in normal colonic mucosa with moderate intensity and is known to be in- hibited by SIRT1. In the same manner, immunostaining for survivin was considered negative if more than 30% of tumor cells showed loss of expression. Immunostaining for β-catenin was considered positive if the tumor cells showed cytoplasmic and/or nuclear expression, so called altered expression. Immu- nostaining for p53 was considered positive if more than 10% of tumor cells were stained with the antibody, according to previ- ous guidelines.23

Statistical analysis

All statistical analyses were performed using IBM SPSS ver. 20 (IBM, Armonk, NY, USA). The end point of interest was overall survival. The endpoint of follow-up was the date of final contact or the date of death through July 2011. Correlations between immunohistochemical expression and the clinicopath- ological characteristics were analyzed by Pearson's tests. Overall survival was calculated as the time from the date of the surgery to the date of death or final contact. Univariate and multivariate analyses for overall survival were performed using the Cox proportional hazard model. In all statistical analyses, p< 0.05 was considered statistically significant.

RESULTS

Expression of SIRT1 and related proteins and correlations with clinicopathologic characteristics

The expression of SIRT1 and DBC1 in colorectal cancer was observed in 235 (67%) and 183 (52%) patients with nuclear staining pattern, respectively. All of the sampled normal muco- sa showed weak SIRT1 expression with nuclear staining. DBC1 was expressed in all of the normal mucosa with moderate inten- sity (Fig. 1). SIRT1 and DBC1 were not significantly related to clinicopathological features such as age, sex, location, stage, in- vasion depth of tumor, lymph node metastasis, distant metasta- sis, or histologic grade (Table 1).

All of the sampled normal mucosa showed membranous ex- pression of β-catenin with weak or moderate intensity. The nu- clear and/or cytoplasmic expression of β-catenin was observed in 246 (70%) patients (Fig. 1). The altered expression of β- catenin was correlated with left-located tumor (p= 0.001), ab- sence of distant metastasis (p= 0.027), and non-poorly differen- tiation (p= 0.026) (Table 1).

Survivin was expressed in all of the normal mucosa with moderate or strong intensity with nuclear staining pattern. The loss of expression of survivin was observed in 156 (45%) pa- tients (Fig. 1). The loss of nuclear expression of survivin was correlated with higher T stage (p = 0.004) (Table 1). Survivin also showed cytoplasmic expression in 70 (20%) patients. Cyto- plasmic expression of survivin was not related to overall survival or clinicopathological features such as age, sex, location, stage, invasion depth of tumor, lymph node metastasis, distant metas- tasis, or histologic grade.

Expression of p53 was observed in 190 (54%) patients. Tu- mor cells were stained with nuclear staining pattern of strong intensity. Normal sampled mucosa showed no expression of p53 (Fig. 1). Expression of p53 was positively correlated with TNM stage (p = 0.018) and distant metastasis (p = 0.001) (Table 1).

Expression of SIRT1 and related proteins with their correlation

The expression of SIRT1 was significantly related to the ex- pression of DBC1 (p< 0.001), β-catenin (p= 0.001), and sur- vivin (p= 0.002). The expression of DBC1 was significantly re- lated to the expression of β-catenin (p = 0.006) and survivin (p < 0.001). β-catenin was not related to DBC1 or survivin. p53 was not related to any other proteins evaluated in this study. Expres- sion of SIRT1 and related proteins and their correlations are summarized in Table 2.

Survival analysis

For univariate analysis of overall survival, a Cox proportional hazard model was used (Table 3). Among analyzed clinicopath- ologic features, old age (>65) (p = 0.005), high TNM stage (stages III and IV) (p< 0.001), left-side tumor (p= 0.034), inva- sion of more than the muscularis propria (p = 0.024), presence of lymph node metastasis (p= 0.001), presence of distant metas- tasis (p< 0.001), and poorly differentiated histologic grade (p = 0.001) were significantly associated with shorter overall surviv- al. The overexpression of SIRT1 and altered expression of β-catenin showed significant association with better overall sur- vival (p= 0.029 and p= 0.008, respectively). However, the ex- pression of DBC1 (p= 0.221), survivin (p= 0.537), and p53 (p = 0.218) was not associated with overall survival. Sex (p = 0.101) was also not associated with overall survival.

Multivariate analysis was performed (Table 4). The factors considered in the analysis were age, tumor location, tumor stage, histologic grade, and the expression of SIRT1 and β- catenin. On multivariate analysis, age, tumor stage, histologic grade, and SIRT1 expression were independent prognostic fac- tors significantly associated with overall survival.

DISCUSSION

The present study examined the immunohistochemical ex- pressions of SIRT1, DBC1, β-catenin, survivin, and p53 in hu- man colorectal cancer and their relationships with clinicopatho- logic factors and prognostic significance. Only a few studies that have examined expression of SIRT1 and its relationship to prognosis in colorectal cancer.s i> Our study is the first to exam- ine expression of SIRT1 and its relationship to expression of other related markers, DBC1, β-catenin, survivin, and p53 in colorectal cancer with human tissue.

As mentioned above, SIRT1 might act as a tumor promoter by inhibiting tumor suppressor genes and might also act as a tumor suppressor by repressing several oncogenes.5"7 A number of recent studies have revealed that SIRT1 expression was in- creased in many human malignant tumors.3'4 However, the role of SIRT1 in human malignant tumors is controversial. Some previous studies have reported that SIRT1 overexpression was associated with shorter overall survival or poor prognostic indi- cators in breast and gastric carcinoma.1215 In contrast to these studies, one study reported that SIRT1 expression was gradually decreased as tumor progression and was associated with better overall survival in colorectal adenocarcinoma.8 Our study also revealed that expression of SIRT1 was significantly related to better overall survival. Taken together with these reports, our results suggest that SIRT1 expression is related to better prog- nosis in colorectal cancer and that SIRT1 acts differently de- pending on the specific organ or type of tumor involved.

Our study examined the expression of DBC1 and its relation- ship to prognosis in colorectal cancer. A recent study revealed that DBC1 overexpression was related to poor prognosis in colorectal cancer.16 However, our study failed to determine the relationship between the expression of DBC 1 and prognosis in colorectal cancer. DBC 1 is known as negative regulator of SIRT 1.14 The present study found that the expressions of SIRT1 and DBC1 were positively correlated with each other (p< 0.001). We could surmise that this result might be due to a close func- tional relationship between SIRT1 and DBC1 in carcinogenesis. However, further functional study will be needed to investigate the relationship between SIRT1 and DBC1.

β-catenin is a key regulator of Wnt signaling and is also known as an important regulator in cancer development.17 The adenomatous polyposis coli (APC) protein negatively regulates the Wnt signaling pathway.24 The APC gene mutation results in nuclear accumulation of β-catenin which becomes phosphor- ylated and is eventually degraded via the APC-dependent ubiq- uitin-proteosome pathway.25 In our study, normal colonic muco- sa showed membranous staining of β-catenin. The altered ex- pression of β-catenin was negatively correlated with the presence of distant metastasis and poorly differentiated histologic grade. The altered expression of β-catenin was related to better overall survival. This is a conflicting result to previous study which re- ported that altered expression of β-catenin was independently associated with poor prognosis in colon cancer.ls Therefore, the relationship between β-catenin expression and prognosis re- mains controversial, and further studies on Wnt/β-catenin and target gene activity and protein expression are needed.

Survivin is a protein that is an inhibitor of apoptosis, and is overexpressed in various cancers.19 In our study, normal colonic mucosa showed expression of survivin with nuclear staining pattern. Some studies have reported that altered expression of survivin was related to poor prognosis in several tumors.26,27 Another study reported that cytoplasmic expression of survivin was associated with a poor prognosis, but nuclear overexpres- sion was related to a better prognosis.20 Our study also showed that the loss of nuclear expression of survivin was related to ad- vanced invasion depth. Cytoplasmic expression of survivin was not associated with prognostic indicators; however, loss of nu- clear expression of survivin may be related to poor prognosis in colorectal cancer.

p53 is well known as a key regulator of cell cycle progression and apoptosis. SIRT1 is considered to inhibit p53-mediated apoptosis through deacetylation of p53.5 However, there was no relationship between the expression of SIRT1 and p53 in our study and a previous study.11

Overexpression of SIRT1 was related to better overall surviv- al. If SIRT1 predominantly acts like a tumor suppressor, it might have less effect on p53 in colorectal cancer. The finding that SIRT1 expression was correlated with altered expression of β-catenin and survivin which are considered to be oncoproteins, supports the hypothesis that SIRT1 predominantly acts as a tu- mor suppressor and might have less effect on p53 in colorectal cancer. Through multivariate analysis, we revealed that SIRT1 was an independent prognostic indicator for overall survival but β-catenin was not. This may also be due to the strong correla- tion between SIRT1 and β-catenin expression. However, to en- sure our hypothesis, further functional study is needed.

In summary, expression of SIRT1 is associated with better overall survival in colorectal cancer patients. Although the exact role of SIRT1 in colorectal cancer is not clear, nor whether it is a tumor suppressor or promoter, the results of our study suggest that the expression of SIRT1 is related to better prognosis and predominantly acts like tumor suppressor in colorectal cancer patients.

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

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