DEPTOR Expression Negatively Correlates with mTORC1 Activity and Tumor Progression in Colorectal Cancer

The mammalian target of rapamycin (mTOR) signaling pathway is upregulated in the pathogenesis of many cancers, including colorectal cancer (CRC). DEPTOR is an mTOR inhibitor whose expression is negatively regulated by mTOR. However, the role of DEPTOR in the development of CRC is not known. The aim of this study was to investigate the expression of DEPTOR and mTORC1 activity (P-S6) in a subset of CRC patients and determine their relation to tumor differentiation, invasion, nodal metastasis and disease-free survival. Here, Immunohistochemical expression of P-S6 (S235/236) and DEPTOR were evaluated in 1.5 mm tumor cores from 90 CRC patients and in 90 samples of adjacent normal mucosa by tissue microarray. The expression of P-S6 (S235/236) was upregulated in CRC, with the positive rate of P-S6 (S235/236) in CRC (63.3%) significantly higher than that in control tissues (36.7%, 30%) ( p <0.05). P-S6 (S235/236) also correlated with high tumor histologic grade ( p =0.002), and positive nodal metastasis ( p =0.002). In contrast, the expression level of DEPTOR was correlated with low tumor histological grade ( p =0.006), and negative nodal metastasis ( p =0.001). Interestingly, P-S6 (S235/236) expression showed a significant negative association with the expression of DEPTOR in CRC ( p =0.011, R= -0.279). However, upregulation of P-S6 (S235/236) ( p =0.693) and downregulation of DEPTOR ( p =0.331) in CRC were not significantly associated with overall survival. Thus, we conclude that expression of DEPTOR negatively correlates with mTORC1 activity and tumor progression in CRC. DEPTOR is a potential marker for prognostic evaluation and a target for the treatment of CRC.


Introduction
Colorectal cancer (CRC) is the third most common malignancy and represents 4% of all tumors (Ostendorff et al., 2013).Although 70% of CRC cases undergo curative surgery, 50% of surgically cured patients will have an advanced local recurrence or metastases.CRC is a common cancer with a poor prognosis and significant mortality worldwide (Huh et al., 2009).The extent of local invasion and tumor metastasis are important factors in determining disease outcome.Distant metastases increase treatment failure, and patients are then subjected to palliative treatment (Kunimura et al., 2009).To predict disease outcome and improve therapeutic interventions, continuous efforts are needed to identify more prognostic markers.
The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that integrates signals from growth factors, nutrients, and stresses to regulate multiple processes, including

DEPTOR Expression Negatively Correlates with mTORC1 Activity and Tumor Progression in Colorectal Cancer
Er-Yong Lai 1& , Zhen-Guo Chen 2& , Xuan Zhou 2 , Xiao-Rong Fan 2 , Hua Wang 2 , Ping-Lin Lai 2 , Yong-Chun Su 2 , Bai-Yu Zhang 3 , Xiao-Chun Bai 2 *, Yun-Feng Li 1 * mRNA translation, cell cycle progression, autophagy, and cell survival (Sarbassov et al., 2005;Jacobs et al., 2013;Sarkar., 2013).It is increasingly apparent that deregulation of the mTOR pathway occurs in common diseases, including cancer, emphasizing the importance of identifying and understanding the function of the components of the mTOR signaling network (Guertin et al., 2007).The activation of p70 S6K1 and S6 by mTOR enhances the translation of mRNAs that bear a 50 terminal oligopyrimidine tract, which encodes proteins related to the translational apparatus such as ribosomal proteins, elongation factors eEF1A, eEF2 and the poly A-binding protein (Bjornsti et al., 2004;Mueller et al., 2012).The phosphorylation status of p70S6K (T389) and S6 (S235/236) is commonly used as a marker of mTOR activity and for pharmacodynamic monitoring of mTOR inhibition (Mueller et al., 2012;Sun et al., 2013).Moreover, scientists have identified DEPTOR, also called DEPDC6, as an mTOR-interacting protein whose expression is negatively regulated by mTORC1 and mTORC2.Loss of DEPTOR activates S6K1, Akt, and SGK1; promotes cell growth and survival; and activates mTORC1 and mTORC2 kinase activities.DEPTOR overexpression suppresses S6K1, but by relieving feedback inhibition from mTORC1 to PI3K signaling, activates Akt (Zhao et al., 2011;Wang et al., 2012;Meng et al., 2013).Consistent with many human cancers with activated mTORC1 and mTORC2 pathways, DEPTOR has low expression in most cancers (Peterson et al., 2009).mTOR has been proved to be one of the most important targets in cancer therapy, and its inhibitors have exhibited encouraging effects in animal experiments and clinical trials (Bjornsti et al., 2004;Baldo et al., 2008).However, the expression of DEPTOR and mTORC1 activity in CRC tissue and their relationship to the prognosis of CRC patients is not known.
In this study, we examined the expression of mTOR-related proteins such as P-S6 (S235/236) and DEPTOR in a subset of primary CRC patients using immunoreactive scores and determined their relation to tumor differentiation, invasion, nodal metastasis, clinicopathologic features and prognosis.From these results, we attempted to evaluate the role of DEPTOR and the mTOR pathway in the development of CRC and its prognosis.

Samples and tissue microarray
The tissue microarray was obtained from Shanghai Outdo Biotech Company and contained 90 samples of CRC and 90 samples of adjacent normal mucosal tissue.The 90 patients who were diagnosed with CRC underwent surgical procedures such as right hemicolectomy, left hemicolectomy, sigmoidectomy, anterior resection, or abdominoperineal resection and had no prior history of neo-adjuvant chemotherapy.Samples from these patients were included in this study.All cases were reviewed and classified according to the criteria of the National Comprehensive Cancer Network (NCCN) classification.Pathologic staging was reviewed based on the Tumor Node Metastasis (TNM) staging system of the American Joint Committee on Cancer (AJCC 7th Edition) (Edge et al., 2010).The patients were grouped according to age, gender, tumor location, TNM stage, tumor invasion, presence of lymph node metastasis, presence of distant metastasis, and histologic grade.The mean follow-up period was 5.3-6.1 years (operation time from 2006.7 to 2007.5, the last follow-up time was 2012.8).The median age of the patients was 70 years (range: 24 to 90 years).Representative portions of tumor and normal mucosa were marked for tissue microarray construction.One 1.5 mm core of tumor per case and one 1.5 mm core of representative normal mucosa were sampled for tissue microarray.The tumor tissue was cut into 4 μm slices for immunohistochemical staining.All information was provided by Shanghai Outdo Biotech Company.Table 1 shows the clinicopathologic characteristics of this subset of patients.

Statistical analysis
All statistical analyses were performed using the SPSS 17.0 statistical software.Statistical significance was determined at p<0.05, and tests were two sided.Differences between two groups of cases for one variable were tested using the Mann-Whitney test.The Kruskal-Wallis test was used to test the association between three groups of cases for one independent variable.Differences in expression between normal mucosa and tumor were tested using the Wilcoxon signed-rank test.Correlations between expression of P-S6 and DEPTOR were analyzed

Expression of P-S6 (S235/236) and DEPTOR, and Correlations with Clinicopathologic Characteristics
The expression of P-S6 (S235/236) and DEPTOR in normal colorectal mucosa and in 90 patients with CRC was observed using the cytoplasm staining pattern, respectively.In CRC, the expression of DEPTOR in both cytoplasm and brush border was less with reduced staining compared with P-S6.The positive expression of P-S6 and DEPTOR in CRC was 57 (63.3%) and 53 (58.9%), respectively.The sampled normal mucosa showed weak expression with cytoplasm staining (Figure 1).
A high level of P-S6 protein expression was observed in tumor samples (63.3% of cases) compared with adjacent normal mucosa (36.7% of cases) (p<0.001).However, the expression of DEPTOR in tumor samples and adjacent normal mucosa was not significantly different (p=0.093)(Table 2).
The distribution of positive immunoexpression in CRC showed that DEPTOR and P-S6 were not significantly related to clinicopathologic features such as gender, age, tumor location, tumor size, invasion, depth of primary tumor and status at end point (p>0.05).However, the altered expression of P-S6 was correlated with high tumor histologic grade (p=0.002) and positive nodal metastasis (p=0.002).In contrast, the altered expression of DEPTOR was correlated with low tumor histologic grade (p=0.006) and negative nodal metastasis (p=0.001)(Table 3).

Correlation between P-S6 and DEPTOR expression.
The expression of P-S6 showed a significant negative    4).

Survival analysis
A Cox proportional hazard model was used in the univariate analysis of overall survival (Table 5).Of the analyzed clinicopathologic features, older age (≥70 years) (p=0.025),high TNM stage (p<0.001),left-sided tumor (p=0.047),large tumor size (p=0.034),presence of lymph node metastasis (p<0.01), and poorly differentiated histologic grade (stages III and IV) (p<0.01) were significantly associated with shorter overall survival.However, the overex-pression of P-S6 (p=0.693) and low expression of DEPTOR (p=0.331) were not associated with overall survival.In addition, gender (p=0.749) was not associated with overall survival.
The factors considered in multivariate analysis were age, tumor location, tumor size, histologic grade, nodal metastasis and the expression of P-S6 and DEPTOR (Table 6).In this analysis, age, tumor size, histologic grade, and nodal metastasis were independent prognostic factors significantly associated with overall survival.

Discussion
The occurrence and development of CRC is the outcome of the combined action of many steps, including genetic expression of oncogenes and tumor suppressor genes in cells with an imbalance in apoptosis caused by excessive proliferation, which is one of the most important mechanisms.The importance of the PI3K/Akt/mTOR signaling network in CRC etiology has been noted recently (Martelli et al., 2011).Inhibitors of PI3K/Akt/mTOR signaling have been suggested as potential therapeutic preparations in CRC (Pandurangan., 2013).However, the way in which mTOR signaling pathway-related proteins influence the prognosis of CRC is still unclear.The present study examined the immunohistochemical expression of P-S6 (S235/236) and DEPTOR in human CRC and their relationships with clinicopathologic factors and prognostic significance.
As mentioned above, P-S6 (S235/236) is the downstream signal/target of mTORC1 (Hay et al., 2004).Direct targets of mTORC1 include ribosomal protein S6 kinase 1 (S6K1) which regulate protein translation (Fasolo et al., 2012).In addition, P-S6 (S235/236) is the downstream signal/target of S6K1.S6K1 and S6K2 have been shown to be upregulated in breast cancer (Lyzogubov et al., 2005), and in other cancers.Our study also revealed that expression of P-S6 (S235/236) was significantly higher in CRC tissue than in normal colorectal mucosa (p<0.01).When greatly overexpressed, DEPTOR inhibits mTORC1 (Peterson et al., 2009;Bruneau et al., 2013;Meng et al., 2013;Zhang et al., 2013).In this study, DEPTOR was equally expressed in CRC and adjacent   Previous studies reported that the overexpression of mTOR and P-mTOR may play an important role in colorectal carcinogenesis in relation to the degree of differentiation, invasiveness and metastasis (Wang et al., 2011;Nelson et al., 2012).Our study examined the expression of P-S6 (S235/236), DEPTOR and their relationship to clinicopathologic characteristics.The altered expression of P-S6 (S235/236) was correlated with high tumor histological grade and positive nodal metastasis.In contrast, the altered expression of DEPTOR was correlated with low tumor histological grade and negative nodal metastasis (Table 3).Therefore, these findings indicated that activation of the mTORC1 pathway plays an important role in colorectal carcinogenesis in relation to the degree of differentiation, invasiveness and metastasis.
A recent study revealed that mTOR overexpression was related to poor prognosis in patients with CRC (Iwaya et al., 2012;Mueller et al., 2012;Cai et al., 2014).It was recently demonstrated that mTOR inhibitors have anti-tumor effects (Bijnsdorp et al., 2011;Finn., 2012;Migliardi et al., 2012).However, our study failed to determine the relationship between the expression of P-S6 (S235/236), DEPTOR and overall survival in CRC.Older age (≥70 years), high TNM stage, left-sided tumor, large tumor size, presence of lymph node metastasis, and poorly differentiated histologic grade (stages III and IV) were significantly associated with shorter overall survival.
Recently, many more cancer-related genes or proteins were considered as prognostic markers for CRC, naming activating transcription factor 1 (ATF1) (Huang et al., 2012), Wip1 (Li et al., 2013), CEA andCA 19-9 (Sisik et al., 2013), POK erythroid myeloid ontogenic factor (Pokemon) (Zhao et al., 2013) and nucleophosmin (NPM) (Yang et al., 2014).All these conclusions, of course including ours, were based on the immunohistochemical analysis of the tumor tissues of CRC patients.However, it was uncertain that the aberrant protein expression of the genes above was the cause or the effect for the carcinogenesis of CRC, and the associated cellular and molecular mechanisms were also not clear.Thus, subsequent experiments were designed to further explore the roles of deptor in a mouse CRC model induced by azoxymethane/dextran sodium sulfate (AOM/DSS) in combination with Cre-loxp system (Car1-cre, deptorloxp).
In summary, our results demonstrate that P-S6 (S235/236) was greatly activated in CRC tissues, and was positively correlated with high tumor histological grade and positive nodal metastasis.In contrast, DEPTOR exhibited the opposite expression and action pattern, which confirmed its inhibitory effect on mTOR signaling and potential tumor suppressor role in CRC.Together, our findings suggest that expression of DEPTOR negatively correlates with mTORC1 activity and tumor progression in CRC.DEPTOR is a potential marker for prognostic evaluation and a target for the treatment of CRC.

Table 1 . Clinicopathologic Characteristics of 90 Cases of Colorectal Cancer
.doi.org/10.7314/APJCP.2014.15.11.4589DEPTORExpression NegativelyCorrelates with mTORC1 Activity and Tumor Progression in Colorectal Cancer by Pearson's χ 2 tests.Overall survival was calculated as the time from the date of surgery to the date of death or final contact.Univariate and multivariate analyses of overall survival were performed using the Cox proportional hazard model.

Table 2 . Scale of
p-S6 and DEPTOR Immunoexpression*Wilcoxon signed-rank test was used to test the difference expression between normal mucosa and tumor

Table 3 . Distribution of p-S6 and DEPTOR and Positive Immunoexpression in Relation to Clinicopathological Characteristics
The distribution is presented as percentage (%), *The Kruskal-Wallis test; other test: The Mann-Whitney test correlation with the expression of DEPTOR in CRC (p=0.011,R= -0.279) (Table

Table 5 . Clincopathologic Characteristics and their Overall Survival by Univariate Cox Proportional Hazards Regression Analysis
*HR: hazard ratio; CI: confidence interval

Table 4 . Expression Status and Correlation of
Correlation between p-S6 and DEPTOR was analyzed by Pearson's χ2 tests.The p value between p-S6 and DEPTOR is 0.011, R=-0.279 *