Chuangang Fu

Red and processed meat intake and risk of colorectal adenomas: A meta‐analysis of observational studies

Inconsistent results regarding the association between red and processed meat intake and the risk of colorectal adenoma (CRA), the precursor of colorectal cancer (CRC), have been reported. To provide a quantitative assessment of this association, we summarized the evidence from observational studies. Relevant studies were identified in MEDLINE and EMBASE until December 31, 2011. Summary relative risks (SRRs) with 95% confidence intervals (CIs) were pooled with a random‐effects model. Between‐study heterogeneity was assessed using the Cochrans Q and I2 statistics. A total of 21 studies (16 case–control studies and five cohort/nested case–control studies) were included in this meta‐analysis. The SRRs of CRA were 1.36 (95% CI = 1.17–1.58) for every 100 g/day increase in red meat intake, and 1.24 (95% CI = 1.12–1.36) for the highest versus the lowest level of red meat intake. Nonlinear dose‐response meta‐analysis indicated that CRA risk increased approximately linearly with increasing intake of red meat up to ∼ 90 g/day, where the curve reached its plateau. Subgrouped analyses revealed that the increased risk of CRA with intake of red meat was independent of geographic locations, design and confounders. The SRRs of CRA was 1.28 (95% CI = 1.03–1.60) for per 50 g/day increase in processed meat intake, and 1.17 (95% CI = 1.08–1.26) for the highest versus the lowest level of processed meat intake. Increased intake of red and processed meat is associated with significantly increased risk of CRA.

Dietary intake of vitamins A, C, and E and the risk of colorectal adenoma: a meta-analysis of observational studies.

To comprehensively summarize the association between dietary intake of vitamins A, C, and E and the risk of colorectal adenoma (CRA), the precursor of colorectal cancer, relevant studies were identified in MEDLINE and EMBASE up to 31 October 2012. Summary relative risks (SRRs) with 95% confidence intervals (CIs) were pooled with a random-effects model. Between-study heterogeneity was assessed using Cochran’s Q and I2 statistics. A total of 13 studies with 3832 CRA cases were included in this meta-analysis. On the basis of the highest versus lowest analysis, dietary intake of vitamin C reduced the risk of CRA by 22% (SRRs 0.78, 95% CIs: 0.62–0.98). Subgroup analyses showed that this relation was not modified by BMI, smoking status, and dietary energy intake. Further, dietary intake of &bgr;-carotene was also inversely associated with the risk of CRA. However, dietary intake of vitamins A and E was not associated with the risk of CRA in overall and subgroup analyses (vitamin A: SRRs 0.87, 95% CIs: 0.67–1.14; vitamin E: SRRs 0.87, 95% CIs: 0.69–1.10). Our results indicate that dietary intake of vitamin C and &bgr;-carotene, but not vitamins A and E, could reduce the risk of CRA. However, these associations seem to be limited. Further investigation using large samples and a rigorous methodology is warranted.

Gene expression profiling-derived immunohistochemistry signature with high prognostic value in colorectal carcinoma

Objective Gene expression profiling provides an opportunity to develop robust prognostic markers of colorectal carcinoma (CRC). However, the markers have not been applied for clinical decision making. We aimed to develop an immunohistochemistry signature using microarray data for predicting CRC prognosis. Design We evaluated 25 CRC gene signatures in independent microarray datasets with prognosis information and constructed a subnetwork using signatures with high concordance and repeatable prognostic values. Tumours were examined immunohistochemically for the expression of network-centric and the top overlapping molecules. Prognostic values were assessed in 682 patients from Shanghai, China (training cohort) and validated in 343 patients from Guangzhou, China (validation cohort). Median follow-up duration was 58 months. All p values are two-sided. Results Five signatures were selected to construct a subnetwork. The expression of GRB2, PTPN11, ITGB1 and POSTN in cancer cells, each significantly associated with disease-free survival, were selected to construct an immunohistochemistry signature. Patients were dichotomised into high-risk and low-risk subgroups with an optimal risk score (1.55). Compared with low-risk patients, high-risk patients had shorter disease-specific survival (DSS) in the training (HR=6.62; 95% CI 3.70 to 11.85) and validation cohorts (HR=3.53; 95% CI 2.13 to 5.84) in multivariate Cox analyses. The signature better predicted DSS than did tumour-node-metastasis staging in both cohorts. In those who received postoperative chemotherapy, high-risk score predicted shorter DSS in the training (HR=6.35; 95% CI 3.55 to 11.36) and validation cohorts (HR=5.56; 95% CI 2.25 to 13.71). Conclusions Our immunohistochemistry signature may be clinically practical for personalised prediction of CRC prognosis.

miR-320b suppresses cell proliferation by targeting c-Myc in human colorectal cancer cells

BackgroundMicroRNAs (miRNAs) are small noncoding RNAs that potentially play a critical role in tumorigenesis. Mounting evidence indicates that one specific miRNA: miR-320b is down regulated in numerous human cancers, including colorectal cancer (CRC); making the hypothesis that miR-320b may play a key role in tumorigenesis plausible. However, its role in carcinogenesis remains poorly defined. The goal of this study is to better clarify the role of miR-320b in tumor growth of CRC.MethodsQuantitative reverse-transcription polymerase chain reaction (qRT-PCR) was conducted to detect the expression of miR-320b in CRC tissues and 5 CRC cell lines. The effect of miR-320b on cell proliferation was analyzed in vitro and in vivo. Furthermore, a luciferase reporter assay was performed to measure the target effects of miR-320b. Lastly, the messenger RNA (mRNA) and protein levels of the gene c-MYC were measured in CRC cell lines and tissues by qRT-PCR, and confirmed via Western blot and Immunohistochemical (IHC) staining.ResultsThe results presented here showed that miR-320b expression was down regulated in both CRC tissues and cells. Overexpression of miR-320b in CRC cells was statistically correlated with a decrease of cell growth in vitro and in vivo, while c-MYC was identified as a target gene of miR-320b in CRC. Furthermore, it was found that up-regulation of c-Myc can attenuate the effects induced by miR-320b.ConclusionsOur identification of c-MYC as a target gene of miR-320b provides new insights into the pathophysiology of CRC proliferation, and identifies miR-320b as a novel therapeutic target for the treatment of CRC.

HMGA1 Interacts with β-Catenin to Positively Regulate Wnt/β-Catenin Signaling in Colorectal Cancer Cells

The high mobility group A1 (HMGA1) protein plays an important role in numerous biological processes, such as embryogenesis, cell proliferation, differentiation, apoptosis and carcinogenesis. Wnt/β-catenin signaling pathway plays a key role in development and cancer. Although previous reports have shown HMGA1 protein level can be induced by Wnt/β-catenin signaling pathway, however, the specific mechanism of HMGA1 on regulating Wnt/β-catenin signaling remains unclear. Here, we reported that HMGA1 interacted with β-catenin by using coimmunoprecipitation approach with exogenous and endogenous protein samples. HMGA1 positively regulated Wnt/β-catenin signaling, as determined by that HMGA1 increased the TOP-FLASH activity in a dose-dependent manner and β-catenin downstream target gene expression. Moreover, HMGA1 induced proliferation of colorectal cancer cells. Mechanistically, HMGA1 increased the β-catenin–TCF4 complex formation. Importantly, there was a correlation between HMGA1 and β-catenin expression in human colorectal cancer tissues. In summary, HMGA1 positively regulates Wnt/β-catenin signaling through interacting with β-catenin, which leads to increase the β-catenin–TCF4 complex formation. This suggests that targeting HMGA1 may be a useful therapeutic option in clinical application.

Tumor cells positive and negative for the common cancer stem cell markers are capable of initiating tumor growth and generating both progenies.

The cancer stem cell (CSC) model depicts that tumors are hierarchically organized and maintained by CSCs lying at the apex. CSCs have been “identified” in a variety of tumors through the tumor-forming assay, in which tumor cells distinguished by a certain cell surface marker (known as a CSC marker) were separately transplanted into immunodeficient mice. In such assays, tumor cells positive but not negative for the CSC marker (hereby defined as CSC+ and CSC− cells, respectively) have the ability of tumor-forming and generating both progenies. However, here we show that CSC+ and CSC− cells exhibit similar proliferation in the native states. Using a cell tracing method, we demonstrate that CSC− cells exhibit similar tumorigenesis and proliferation as CSC+ cells when they were co-transplanted into immunodeficient mice. Through serial single-cell derived subline construction, we further demonstrated that CSC+ and CSC− cells from CSC marker expressing tumors could invariably generate both progenies, and their characteristics are maintained among different generations irrespective of the origins (CSC+-derived or CSC−-derived). These findings demonstrate that tumorigenic cells cannot be distinguished by common CSC markers alone and we propose that cautions should be taken when using these markers independently to identify cancer stem cells due to the phenotypic plasticity of tumor cells.

Multidrug resistance-associated protein 3 confers resistance to chemoradiotherapy for rectal cancer by regulating reactive oxygen species and caspase-3-dependent apoptotic pathway

This study aimed to clarify the role of multidrug resistance-associated protein 3 (MRP3) in resistance to neoadjuvant chemoradiotherapy and long-term prognosis of advanced rectal cancer. Immunohistochemistry was used to measure MRP3 expression in biopsy specimens of 144 stage II-III rectal cancer patients who received preoperative chemoradiotherapy. The effect of MRP3 expression on short-term pathological response and postoperative long-term prognosis were assessed using the Cox proportional hazards model. Short interfering RNAs targeting MRP3 were synthesized and used to transfect human colorectal carcinoma cell lines. The effect of MRP3 down-regulation on cell proliferation and apoptosis in response to 5-fluorouracil and/or irradiation were examined in vitro and in xenograft mouse models, respectively. The content of intracellular reactive oxygen species and the activity of caspase-3-dependent apoptotic pathway in response to irradiation were further evaluated. High expression (immunoreactive score > 6) of MRP3 significantly predicted poor pathological response to chemoradiotherapy (tumor regression grade ≤ 2 vs. ≥3, p = 0.002) in univariate analysis and unfavorable long-term prognosis (5-year overall survival: HR = 1.612, 95% CI, 1.094-2.375, p = 0.016; 5-year disease-free survival: HR = 1.513, 95% CI, 1.041-2.200, p = 0.030) in multivariate Cox analysis. MRP3 down-regulation significantly increased 5-fluorouracil or irradiation-induced cell apoptosis and attenuated tumor growth following irradiation in animal models. MRP3 inhibition significantly reduced intracellular reactive oxygen species exporting from cells following irradiation, and increased expression of cleaved poly ADP-ribose polymerase and caspase-3. Aberrant expression of MRP3 in rectal cancer confers chemo-radioresistance. MRP3 might be a predictive factor and an attractive target in treating advanced rectal cancer.

Nuclear orphan receptor NR4A2 confers chemoresistance and predicts unfavorable prognosis of colorectal carcinoma patients who received postoperative chemotherapy

BACKGROUND NR4A2, an orphan nuclear receptor essential in neuron generation, has been recently linked to inflammatory and metabolic pathways of colorectal carcinoma (CRC). However, the effects of NR4A2 on chemo-resistance and postoperative prognosis of CRC remain unknown. METHODS NR4A2 was transfected into CRC cells to investigate its effects on chemo-resistance to 5-fluorouracil and oxaliplatin and chemotherapeutics-induced apoptosis. We also investigated prostaglandin E2 (PGE2)-induced NR4A2 expression and its effect on chemo-resistance. Tissue microarrays including 51 adenoma, 14 familial adenomatous polyposis with CRC, 17 stage IV CRC with adjacent mucosa and 682 stage I-III CRC specimens were examined immunohistochemically for NR4A2 expression. Median follow-up time for stage I-III CRC patients was 53 months. RESULTS Ectopic expression of NR4A2 increased the chemo-resistance, and attenuated the chemotherapeutics-induced apoptosis. Transient treatment of PGE2 significantly up-regulated NR4A2 expression via protein kinase A pathway and increased the chemo-resistance. NR4A2 expression in epithelials consecutively increased from adenoma, adjacent mucosa to CRC (P(trend)<0.001). In multivariate Cox regression analyses, high NR4A2 expression in cancer nuclei (immunoreactive score ≥ 4) significantly predicted a shorter disease-specific survival (DSS) of CRC patients (hazard ratio [HR]=1.88, P=0.024). High NR4A2 expression specifically predicted a shorter DSS of colon cancer patients (dichotomisation, HR=2.55, log-rank test P=0.011), especially for those who received postoperative 5-fluorouracil/leucovorin plus oxaliplatin (FOLFOX) chemotherapy (3-score range, HR=1.86, log-rank test P=0.020). CONCLUSION High expression of NR4A2 in CRC cells confers chemo-resistance, attenuates chemotherapeutics-induced apoptosis, and predicts unfavorable prognosis of colon cancer patients, especially for those who received postoperative chemotherapy. NR4A2 may be prognostic and predictive for colon cancer.

Prognostic significance and molecular mechanism of ATP-binding cassette subfamily C member 4 in resistance to neoadjuvant radiotherapy of locally advanced rectal carcinoma.

Background Mechanism of radioresistance in rectal carcinoma remains largely unknown. We aimed to evaluate the predictive role of ATP-binding cassette subfamily C member 4 (ABCC4) in locally advanced rectal carcinoma and explore possible molecular mechanisms by which ABCC4 confers the resistance to neoadjuvant radiotherapy. Methods The expression of ABCC4 and P53 mutant in biopsy tissue specimens from 121 locally advanced rectal carcinoma patients was examined using immunohistochemistry. The factors contributing to 3-year overall survival and disease-free survival were evaluated using the Kaplan-Meier method and Cox proportional hazard model. Lentivirus-mediated small hairpin RNA was applied to inhibit ABCC4 expression in colorectal carcinoma cell line RKO, and investigate the radiosensitivity in xenograft model. Intracellular cyclic adenosine monophosphate concentration and cell cycle distribution following irradiation were detected. Results High expression of ABCC4 and p53 mutant in pretreated tumors, poor pathological response, and high final tumor staging were significant factors independently predicted an unfavorable prognosis of locally advanced rectal carcinoma patients after neoadjuvant radiotherapy. Down-regulation of ABCC4 expression significantly enhanced irradiation-induced suppression of tumor growth in xenograft model. Furthermore, down-regulation of ABCC4 expression enhanced intracellular cyclic adenosine monophosphate production and noticeable deficiency of G1-S phase checkpoint in cell cycle following irradiation. Conclusions Our study suggests that ABCC4 serves as a novel predictive biomarker that is responsible for the radioresistance and predicts a poor prognosis for locally advanced rectal carcinoma after neoadjuvant radiotherapy.

Abstract C217: Prognostic significance and molecular mechanism of ATP-Binding Cassette subfamily C member 4 in resistance to neoadjuvant radiotherapy of locally advanced rectal carcinoma.

Mechanism of radioresistance in rectal carcinoma remains largely unknown. The aim of this study is to verify the predictive value of ATP-Binding Cassette subfamily C member 4 (ABCC4) in locally advanced rectal carcinoma and explore possible molecular mechanisms by which ABCC4 confers the resistance to neoadjuvant radiotherapy. Methods: Expression of ABCC4 protein in pretreated biopsy tissue specimens from locally advanced rectal carcinoma patients was examined using immunohistochemistry. The 3-year overall survival and disease-free survival were used to determine long-term prognosis, and significant clinicopathological factors were identified by univariate log-rank test and multivariate Cox regression analysis. Lentivirus-mediated small hairpin RNA inhibited ABCC4 expression in colorectal carcinoma cell lines, and altered radiosensitivity was confirmed by xenograft model in vivo. Intracellular cyclic adenosine monophosphate concentration and cell cycle distribution following irradiation were further detected. Results: High expression of ABCC4 in pretreated tumors, p53 mutant type, high serum CEA and positive lymph node predicted an unfavorable postoperative prognosis for locally advanced rectal carcinoma patients after neoadjuvant radiotherapy in univariate analysis. Multivariate Cox regression analysis further demonstrated that high expression of ABCC4 and p53 mutant type were significant factors independently predicted an unfavorable long-term prognosis. Downregulation of ABCC4 expression significantly enhanced irradiation-induced suppression of tumor growth in xenograft model in vivo. Furthermore, inhibition of ABCC4 expression induced intracellular cyclic adenosine monophosphate accumulation and a noticeable deficiency of G1-S phase checkpoint in cell cycle following irradiation. Conclusions: Our study suggests ABCC4 as a novel predictive biomarker in predicting radioresistance and prognosis for locally advanced rectal carcinoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C217. Citation Format: Zhiqi Yu, Chang Zhang, Rui Chai, Yan Du, Xianhua Gao, Junjie Xing, Guangwen Cao, Chuangang Fu. Prognostic significance and molecular mechanism of ATP-Binding Cassette subfamily C member 4 in resistance to neoadjuvant radiotherapy of locally advanced rectal carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C217.