A proteomic classifier panel for early screening of colorectal cancer: a case control study.

BACKGROUND: Diagnosis of colorectal cancer (CRC) during early stages can greatly improve patient outcome. Although technical advances in the field of genomics and proteomics have identified a number of candidate biomarkers for non-invasive screening and diagnosis, developing more sensitive and specific methods with improved cost-effectiveness and patient compliance has tremendous potential to help combat the disease. METHODS: We enrolled three cohorts of 479 subjects, including 226 CRC cases, 197 healthy controls, and 56 advanced precancerous lesions (APC). In the discovery cohort, we used quantitative mass spectrometry to measure the expression profile of plasma proteins and applied machine-learning to select candidate proteins. We then developed a targeted mass spectrometry assay to measure plasma concentrations of seven proteins and a logistic regression classifier to distinguish CRC from healthy subjects. The classifier was further validated using two independent cohorts. RESULTS: The seven-protein panel consisted of leucine rich alpha-2-glycoprotein 1 (LRG1), complement C9 (C9), insulin-like growth factor binding protein 2 (IGFBP2), carnosine dipeptidase 1 (CNDP1), inter-alpha-trypsin inhibitor heavy chain 3 (ITIH3), serpin family A member 1 (SERPINA1), and alpha-1-acid glycoprotein 1 (ORM1). The panel classified CRC and healthy subjects with high accuracy, since the area under curve (AUC) of the training and testing cohort reached 0.954 and 0.958. The AUC of the two independent validation cohorts was 0.905 and 0.909. In one validation cohort, the panel had an overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 89.9%, 81.8%, 89.2%, and 82.9%, respectively. In another blinded validation cohort, the panel classified CRC from healthy subjects with a sensitivity of 81.5%, specificity of 97.9%, and overall accuracy of 92.0%. Finally, the panel was able to detect APC with a sensitivity of 49%. CONCLUSIONS: This seven-protein classifier is a clear improvement compared to previously published blood-based protein biomarkers for detecting early-stage CRC, and is of translational potential to develop into a clinically useful assay.

The ubiquitin E3 ligase MDM2 induces chemoresistance in colorectal cancer by degradation of ING3.

Chemoresistance is an obstacle for colorectal cancer (CRC) treatment. This study investigates the role of the ubiquitin E3 ligase MDM2 in affecting cell growth and chemosensitivity in CRC cells by modifying the transcription factor inhibitor of growth protein 3 (ING3). The expression of MDM2 and ING3 in CRC tissues was predicted by bioinformatics analysis, followed by expression validation and their interaction in CRC HCT116 and LS180 cells. Ectopic overexpression or knockdown of MDM2/ING3 was performed to test their effect on proliferation and apotptosis as well as chemosensitivity of CRC cells. Finally, the effect of MDM2/ING3 expression on the in vivo tumorigenesis of CRC cells was examined through subcutaneous tumor xenograft experiment in nude mice. MDM2 promoted ubiquitin-proteasome pathway degradation of ING3 through ubiquitination and diminished its protein stability. Overexpression of MDM2 downregulated ING3 expression, which promoted CRC cell proliferation and inhibited the apoptosis. The enhancing role of MDM2 in tumorigenesis and resistance to chemotherapeutic drugs was also confirmed in vivo. Our findings highlight that MDM2 modifies the transcription factor ING3 by ubiquitination-proteasome pathway degradation, thus reducing ING3 protein stability, which finally promotes CRC cell growth and chemoresistance.

GLS1 promotes proliferation in hepatocellular carcinoma cells via AKT/GSK3ß/CyclinD1 pathway.

Glutamine metabolism is an important metabolic pathway for cancer cell survival, and there is a critical connection between tumor growth and glutamine metabolism. However, the role of GLS1 in hepatocellular carcinoma (HCC) progression remains to be elucidated. In this study, we reported that GLS1 expression was significantly increased in HCC tissues and correlated with serum AFP, tumor differentiation, lymphatic metastasis, TNM stage, and poorer patient outcome. We further showed that GLS1 promoted colony formation and cell proliferation of HCC cells. Furthermore, our data showed that GLS1 inhibitor compound 968 inhibited the proliferation of HCC cells in a dose-dependent manner. Importantly, we found that GLS1 overexpression increased p-AKT, p-GSK3ß and cyclinD1 expression, and had no influence on total AKT and GSK3ß protein level, indicating that GLS1 was involved in AKT/GSK3ß/CyclinD1 pathway. It is suggested that GLS1 promotes proliferation in HCC cells probably via AKT/GSK3ß/CyclinD1 pathway and may be a potential target for anti-hepatocellular carcinoma cancer.

MicroRNA-377-3p promotes cell proliferation and inhibits cell cycle arrest and cell apoptosis in hepatocellular carcinoma by affecting EGR1-mediated p53 activation.

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive malignant carcinoma with a high fatality rate. MicroRNAs (miRNAs) have been found to regulate the development of multiple cancers, including HCC. MATERIALS AND METHODS: Quantitative polymerase chain reaction (qPCR) were implemented to evaluate RNA level and western blot to detect protein level. Cell counting kit-8 (CCK-8), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), flow cytometry and in vivo assays were performed to evaluate the biological functions of RNAs on HCC cell proliferation, cell cycle and apoptosis. Luciferase reporter gene and chromatin immunoprecipitation (ChIP) assays were carried out to evaluate the underlying mechanisms. RESULTS: MiR-377-3p promotes cell proliferation and inhibits cell cycle arrest and cell apoptosis in HCC. MiR-377-3p downregulates transcription factor EGR1 expression to weaken the activation of p53. p53 inhibits CCNB1, CCNB2 and CHEK1 expressions and activates THBS1, IGFBP3 and TRIM22 expressions. p53 knockdown promotes the proliferation and inhibits the cell cycle arrest and apoptosis of HCC cells. CONCLUSION: Our study demonstrated the role and underlying mechanisms of miR-377-3p in HCC. MiR-377-3p facilitates the proliferation and suppresses the cell cycle arrest and apoptosis in HCC by affecting transcription factor EGR1-mediated p53 activation.

Exosomes derived from HCC cells with different invasion characteristics mediated EMT through TGF-ß/Smad signaling pathway.

BACKGROUND: Exosomes are nano-sized biological vesicles released by many kinds of cells, which play an important role in tumor metastasis through transporting cytokines, RNAs and proteins. However, the molecular mechanisms of exosomes in hepatocellular carcinoma (HCC) metastasis are not completely understood. MATERIALS AND METHODS: Exosomes derived from hepatoma cell lines with different invasion characteristics. Exosome characteristics, cell migration and invasion, and effects on major signal transduction pathways deregulated in cancer cells were analyzed by transmission electron microscopy (TEM), wound-healing assay, Trans-well invasion assay and Western blot-based assays, respectively. Moreover, exosomes effects on tumor metastasis in vivo were investigated by subcutaneous transplantation tumor model of athymic nude mice. RESULTS: Exosomes derived from hepatoma cells can promote the migration and invasion of recipient cells, induce the decrease of E-cadherin expression, increase the expression of Vimentin and promote epithelial-mesenchymal transition (EMT) in cells. Moreover, highly invasive hepatoma-cells-derived exosomes effects are stronger than low-invasive hepatoma cells and normal liver cells exosomes. Mechanistic studies showed that the biological alterations in recipient HCC cells treated with MHCC97H and MHCC97L-derived exosomes were caused by inducing EMT via TGF-ß/Smad signaling pathway. In vivo experiments also suggest that highly invasive hepatoma cells derived exosomes are more likely to promote lung metastasis of HCC in nude mice. CONCLUSION: Our results reveal the important role of tumor-derived exosomes in the migration and invasion of recipient cells and exosomes may be the novel therapeutic and prognostic targets for HCC patients.

MicroRNA-150 suppresses the growth and malignant behavior of papillary thyroid carcinoma cells via downregulation of MUC4.

Previous studies have revealed that microRNA (miR)-150 can act as an oncomiR or a tumor suppressor in numerous types of hematological malignancy and solid tumor. However, the function of miR-150 in papillary thyroid carcinoma (PTC) remains elusive. The present study aimed to investigate the function of miR-150 in PTC and its underlying molecular mechanism. The expression of miR-150 was identified to be significantly downregulated, whereas that of mucin (MUC)4 was significantly upregulated in PTC tissues and cell lines compared with corresponding controls. Further experiments demonstrated that MUC4 is a direct target of miR-150. PTC cell proliferation and capacity for migration and invasion decreased following miR-150 overexpression. It was also demonstrated that miR-150-mediated MUC4 downregulation was associated with an accompanying decrease in human epidermal growth factor receptor 2, as well as its phosphorylated form, resulting in suppressed activation of downstream signaling. In conclusion, the present study demonstrated that miR-150 may serve a key function in suppressing the malignant growth and aggressive behavior of PTC cells through the downregulation of MUC4. These findings may provide a novel approach for diagnostic and therapeutic strategies for PTC.

[Correlation analysis between the numbers of G-MDSCs and M-MDSCs in peripheral blood decrease and therapeutic effect in patients with colorectal cancer].

Objective To detect the frequencies of myeloid-derived suppressor cells (MDSCs) in the peripheral blood of patients with colorectal cancer (CRC) in an attempt to investigate the relationship between the change of MDSC frequencies and clinicopathologic characteristics of CRC patients. Methods The frequencies of granulocytic MDSCs (G-MDSCs) and monocytic MDSCs (M-MDSCs) in the peripheral blood of 82 CRC patients and 30 healthy volunteers were detected by flow cytometry. ANOVA and t-test were used to explore the relationship between the frequencies of MDSCs in the peripheral blood and the TNM stages, lymph node metastasis, tumor sites, histological grade as well as common therapeutic measures of CRC patients. Correlation analysis was performed to evaluate the correlation between the frequencies of G-MDSCs and M-MDSCs. Results Compared with the 30 healthy volunteers, the frequencies of G-MDSCs and M-MDSCs in the peripheral blood of the 82 CRC patients were statistically higher. The frequencies of G-MDSCs and M-MDSCs in the peripheral blood from TNF stage III and IV CRC patients were obviously higher than those from stage I and II patients, and CRC patients with lymph node metastasis had remarkably higher frequencies of G-MDSCs and M-MDSCs than the patients without lymph node metastasis. There were no statistical differences in the frequencies of G-MDSCs and M-MDSCs in the peripheral blood between CRC patients with different tumor sites as well as histological grades. Radical resection and effective adjuvant chemotherapy significantly decreased the frequencies of G-MDSCs and M-MDSCs in the peripheral blood of CRC patients. No statistical correlation was found between the frequencies of circulating G-MDSCs and M-MDSCs in CRC patients. Conclusion G-MDSC and M-MDSC frequencies in the peripheral blood of CRC patients significantly increased, and were closely associated with TNM stages and lymph node metastasis. Radical resection and effective adjuvant chemotherapy significantly reduced the frequencies of circulating G-MDSCs sand M-MDSCs of CRC patients.