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Background Chloride channel accessory 1 (CLCA1) belongs to the calcium-sensitive chloride conductance protein family, which is mainly expressed in the colon, small intestine and appendix. This study was conducted to investigate the functions and mechanisms of CLCA1 in colorectal cancer (CRC). Methods The CLCA1 protein expression level in CRC patients was evaluated by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and western blotting analysis. Using CRISPR/Cas9 technology, CLCA1-upregulated (CLCA1-ACT) and CLCA1-knockout cells (CLCA1-KO), as well as their respective negative controls (CLCA1-ACT-NC and CLCA1-KO-NC), were constructed from the SW620 cell line. Cell growth and metastatic ability were assessed both in vitro and in vivo. The association of CLCA1 with epithelial-mesenchymal transition (EMT) and other signaling pathways was determined by western blotting assays. Results The expression level of CLCA1 in CRC tissues was significantly decreased compared with that in adjacent normal tissue (P

Purpose: Secreted protein acidic and rich in cysteines-like 1 (SPARCL1) is an extracellular matrix glycoprotein with malignancy-suppressing potential. The hypothesis that SPARCL1 reduces cancer invasiveness and predicts better survival in colorectal cancers (CRC) was investigated. Experimental Design: Stable SPARCL1 transfectants, RKO-SPARCL1, and corresponding vector control were constructed and implanted into nude mice to generate a mouse xenograft model of liver metastasis. Also, a retrospective outcome study was conducted on the COH set (222 CRCs) and ZJU set (412 CRCs). The protein expression level of SPARCL1 was determined by immunohistochemistry. The Kaplan–Meier and Cox analyses were used for survival analysis. The association of SPARCL1 with mesenchymal–epithelial transition (MET) was examined by reverse transcription PCR (RT-PCR) and Western blot analysis. Results: The ectopic expression of SPARCL1 significantly reduced the potential for anchorage-independent growth, migration, invasion and induced cell differentiation in RKO and SW620 cells. In mouse xenograft model, the expression of SPARCL1 significantly reduced the liver metastasis (P < 0.01). The patient-based studies revealed that the expression of SPARCL1 was related to better differentiation (P < 0.01), less lymph node involvement [OR, 0.67; 95% confidence interval (CI), 0.45–1.00], and less distant metastasis (OR, 0.38; 95% CI, 0.18–0.79). The Kaplan–Meier and Cox analysis showed that the expression of SPARCL1 was associated with better overall survival (log-rank: P < 0.01; HR, 0.57; 95% CI, 0.39–0.84). Transfection of SPARCL1 induced MET of colon cancer cells. Conclusion: SPARCL1 functions as a tumor suppressor promoting differentiation possibly via MET, which inhibits the aggressiveness of CRCs. Clin Cancer Res; 18(19); 5438–48. ©2012 AACR.

Objective: To characterize the expression of ST13 protein in human tissues for investigation of the function of colorectal cancer related gene ST13.Methods: ST13 ORF was cloned and over-expressed in E.coli. The recombinant ST13 protein was purified by affinity chromatography. ST13 monoclonal antibodies were generated and affinity purified with the recombinant protein. Immunoblot and immunohistochemical staining were employed to analyze ST13 protein expression in human tissues. Results:The expression and purification of the recombinant ST13 protein were confirmed by SDS-PAGE. The protein yield reached about 2.5 mg/L of induced bacterial culture with a purity of 91.3%. Three strains of hybridoma were obtained with antibody titers from 104 to 105 in ascites fluids and with high specificity for ST13 protein. Immunoblot showed that the apparent Mr of ST13 protein in SW480 cells and human tissues estimated by SDS-PAGE mobility was approximately 50000, which was about 10000 larger than the 41324 calculated, but the glycosylation of the protein was excluded. Computer modeling revealed the protein to be a hydrophilic molecule. Immunohistochemical staining showed that ST13 protein was evenly distributed in cytoplasm and expressed in colon, stomach, liver, and other epithelial cells. Differences in the staining intensity of the protein were observed between normal and cancer tissues as well as among different normal or carcinoma tissues. Conclusion: ST13 protein is a cytoplasmic molecule with an apparent Mr of 50000. The protein is expressed in colorectal and other epithelial tissues. The expression level of the protein is down-regulated in colorectal cancer and varies among different normal and/or carcinoma tissues. Comparison of cDNA sequences and protein characteristics indicates that ST13 protein and hsp70-interacting protein (Hip) are same proteins, raising the possibility that ST13 protein is involved in the development of colorectal cancer through Hsp70 molecular chaperone machinery.

Purpose: This study aims to address the hypothesis that the high-mobility group A2 (HMGA2), an oncofetal protein, relates to survivability and serves as a prognostic biomarker for colorectal cancer (CRC). Experimental Design: This is a retroprospective multiple center study. The HMGA2 expression level was determined by performing immunohistochemistry on surgical tissue samples of 89 CRCs from a training set and 191 CRCs from a validation set. The Kaplan–Meier analysis and COX proportional hazard model were employed to analyze the survivability. Results: Multivariate logistic analysis indicated that the expression of HMGA2 significantly correlates with distant metastasis in training set (odds ratio, OR = 3.53, 95% CI: 1.37–9.70) and validation set (OR = 6.38, 95% CI: 1.47–43.95). Survival analysis revealed that the overexpression of HMGA2 is significantly associated with poor survival of CRC patients (P < 0.05). The adjusted HRs for overall survival were 2.38 (95% CI: 1.30–4.34) and 2.14 (95% CI: 1.21–3.79) in training and validation sets, respectively. Further investigation revealed that HMGA2 delays the clearance of γ-H2AX in HCT-116 and SW480 cells post γ-irradiation, which supports our finding that CRC patients with HMAG2-positive staining in primary tumors had augmented the efficacy of adjuvant radiotherapy (HR = 0.18, 95% CI: 0.04–0.63). Conclusion: Overexpression of HMGA2 is associated with metastasis and unequivocally occurred in parallel with reduced survival rates of patients with CRC. Therefore, HMGA2 may potentially serve as a biomarker for predicting aggressive CRC with poor survivability and as an indicator for better response of radiotherapy. Clin Cancer Res; 17(8); 2570–80. ©2011 AACR.

To study the effect of interleukin-18 gene transfection on the tumorigenesis of breast cancer cell line Bacp37, human breast cancer cell line Bcap37 were transfected with Lipofectamine and selected by G418. The biological expression of rhIL-18 was tested by RT-PCR and ELISA method; nude mice were injected with Bcap37 cell with or without the hIL-18 gene. The hIL-18 cDNA was successfully integrated into Bcap37 cell; 126.3+/-4.5 pg hIL-18 secreted by one million transduced cells in 24 hours. Nude mice injected with IL-18 gene engineered Bcap37 cell had no tumor growth. These findings indicated that human breast cancer cells were successfully modified by the gene of IL-18 cytokine; the IL-18 gene engineered Bcap37 cells secreted hIL-18 and lost their tumorigenicity. The Bcap37 cells transduced with IL-18 gene may be used as breast cancer vaccine.

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer and triple negative breast cancer (TNBC) are two subtypes with poor outcome of all breast carcinomas. However, among all the patients in these two subtypes some still yielded relatively better prognosis. Ribonucleotide reductase (RR) small subunits M2 was associated with cancer cell invasiveness and metastasis, which prognoses poor survival in many cancers. Here we investigated whether RRM2 could serve as a prognostic biomarker for breast cancers, especially HER2- positive and TNBC subgroups. One hundred seventy five assessable breast cancers (40 cases were HER2-positive or TNBC) were collected from affiliated hospital of Zhejiang University (ZJU set); and six published microarray data sets (total 1154 cases, 63 cases were identified as HER2- positive or TNBC) with pathoclinical and follow- up information were employed for validation. In the ZJU set, the protein level of RRM2 was determined by IHC. It was indicated that RRM2 was positively correlated with cell proliferative marker Ki-67 (trends P=0.018) and stem/progenitor cell marker CD44+/CD24-/low (P=0.044). Multivariate COX model revealed that RRM2 was an independent prognostic biomarker for both OS (Hazard Ratio, HR=2.79, 95%Cl 1.21-6.75) and PFS (Hazard Ratio, HR=1.82, 95%Cl 1.02-3.26) for all the breast cancers. Moreover, in HER2-positive or TNBC breast cancers, RRM2-high was significantly associated with poor survival (P=0.025). Consequently, none of the patients with RRM2-low died of breast cancer in HER2-positive or TNBC subgroups. Furthermore, above findings were validated in the published data sets. The mRNA level of RRM2 evaluated significantly in highly-invasive subtypes including Luminal B, HER2-positive and TNBC in comparison with Luminal A and unclassified subgroups (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 706. doi:1538-7445.AM2012-706