Mingzhu Huang

Identification of stem-like cells and clinical significance of candidate stem cell markers in gastric cancer.

The existence of gastric cancer stem cells (CSCs) has not been definitively proven and specific cell surface markers for identifying gastric CSCs have largely not been identified. Our research aimed to isolate potential gastric CSCs and clarify their clinical significance, while defining markers for GCSC identification and verification. Here, we report that spheroid cells possess stem cell-like properties, and overexpress certain stem cell markers. CD133 or CD44-positive cells also exhibit properties of CSCs. The expression of Oct4, Sox2, Gli1, CD44, CD133, p-AKT, and p-ERK was significantly higher in metastatic lesions compared to that in primary lesions. Elevated expression of some of these proteins was correlated with a more aggressive phenotype and poorer prognosis, including Oct4, Sox2, Gli1, CD44, and p-ERK. Multivariate Cox proportional hazards model analysis showed that only CD44 is an independent factor. Knockdown of CD44 down-regulated the stem cell-like properties, which was accompanied by the down-regulation of p-ERK and Oct4. Oct4 overexpression could reverse the decreased CSCs properties induced by CD44 knockdown. Taken together, our research revealed that spheroid cell culture, and CD133 or CD44-labeled FACS methods can be used to isolate gastric CSCs. Some CSC markers have clinical significance in predicting the prognosis. CD44 is an independent prognostic factor and maintains the properties of CSCs in CD44-p-ERK-Oct4 positive feedback loop.

COX-2-765G>C Polymorphism Increases the Risk of Cancer: A Meta-Analysis

Background Chronic inflammation has been regarded as an important mechanism in carcinogenesis. Inflammation-associated genetic variants have been highly associated with cancer risk. Polymorphisms in the gene cyclooxygenase-2 (COX-2), a pro-inflammation factor, have been suggested to alter the risk of multiple tumors, but the findings of various studies are not consistent. Methods A literature search through February 2013 was performed using PubMed, EMBASE, and CNKI databases. We used odds ratios (ORs) with confidence intervals (CIs) of 95% to assess the strength of the association between the COX-2-765G>C polymorphism and cancer risk in a random-effect model. We also assessed heterogeneity and publication bias. Results In total, 65 articles with 29,487 cancer cases and 39,212 non-cancer controls were included in this meta-analysis. The pooled OR (95% CIs) in the co-dominant model (GC vs. GG) was 1.11 (1.02–1.22), and in the dominant model ((CC+GC) vs. GG), the pooled OR was 1.12 (1.02–1.23). In the subgroup analysis, stratified by cancer type and race, significant associations were found between the-765 C allele and higher risk for gastric cancer, leukemia, pancreatic cancer, and cancer in the Asian population. Conclusion In summary, the COX-2-765 C allele was related to increased cancer susceptibility, especially gastric cancer and cancer in the Asian population.

Bmi-1 regulates stem cell-like properties of gastric cancer cells via modulating miRNAs

BackgroundB cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) plays an important role in regulating stemness in some kinds of cancer. However, the mechanisms remain unclear. This study was to investigate whether and how Bmi-1 regulates stemness of gastric cancer.MethodsWe firstly explored the role of Bmi-1 in regulating stem cell-like features in gastric cancer. Secondly, we screened out its downstream miRNAs and clarified whether these miRNAs are involved in the regulation of stemness. Finally, we investigated the mechanisms how Bmi-1 regulates miRNAs.ResultsBmi-1 positively regulates stem cell-like properties of gastric cancer and upregulates miR-21 and miR-34a. There was a positive correlation between Bmi-1 and miR-21 expression in gastric cancer tissues. MiR-21 mediated the function of Bmi-1 in regulating stem cell-like properties, while miR-34a negatively regulates stem cell-like characteristics via downregulating Bmi-1. Bmi-1 binds to PTEN promoter and directly inhibits PTEN and thereafter activates AKT. Bmi-1 also regulates p53 and PTEN via miR-21. Bmi-1 activated NF-kB via AKT and enhanced the binding of NF-kB to the promoter of miR-21 and miR-34a and increased their expression.ConclusionsBmi-1 positively regulates stem cell-like properties via upregulating miR-21, and miR-34a negatively regulates stem cell-like characteristics by negative feedback regulation of Bmi-1 in gastric cancer. Bmi-1 upregulates miR-21 and miR-34a by activating AKT-NF-kB pathway.

Antitumor activity and inhibitory effects on cancer stem cell-like properties of Adeno-associated virus (AAV) -mediated Bmi-1 interference driven by Bmi-1 promoter for gastric cancer

Bmi-1 is aberrantly activated in various cancers and plays a vital role in maintaining the self-renewal of stem cells. Our previous research revealed that Bmi-1 was overexpressed in gastric cancer (GC) and its overexpression was an independent negative prognostic factor, suggesting it can be a therapeutic target. The main purpose of this investigation was to explore the antitumor activity of Bmi-1 interference driven by its own promoter (Ad-Bmi-1i) for GC. In this study, we used adenoviral vector to deliver Bmi-1 shRNA driven by its own promoter to treat GC. Our results revealed that Ad-Bmi-1i could selectively silence Bmi-1 in GC cells which overexpress Bmi-1 and suppress the malignant phenotypes and stem-like properties of GC cells in vitro and in vivo. Moreover, direct injection of Ad-Bmi-1i into xenografts suppressed tumor growth and destroyed cancer cells in vivo. Ad-Bmi-1i inhibited the proliferation of GC cells mainly via inducing senescence in vitro, but it suppressed tumor through inducing senescence and apoptosis, and inhibiting angiogenesis in vivo. Bmi-1 knockdown by Ad-Bmi-1i downregulated VEGF via inhibiting AKT activity. These results suggest that Ad-Bmi-1i not only inhibits tumor growth and stem cell-like phenotype by inducing cellular senescence directly, but also has an indirect anti-tumor activity by anti-angiogenesis effects via regulating PTEN/AKT/VEGF pathway. Transfer of gene interference guided by its own promoter by an adeno-associated virus (AAV) vector might be a potent antitumor approach for cancer therapy.

Maintenance treatment of Uracil and Tegafur (UFT) in responders following first-line fluorouracil-based chemotherapy in metastatic gastric cancer: a randomized phase II study

Background Maintenance therapy proves to be effective in advanced lung and breast cancer after initial chemotherapy. The purpose of this phase II study was to evaluate the efficacy and safety of Uracil and Tegafur (UFT) maintenance in metastatic gastric cancer patients following the first-line fluorouracil-based chemotherapy. Methods Metastatic gastric cancer patients with stable disease or a better response after the completion of first-line chemotherapy were randomized to oral UFT (360mg/m2 × 2 weeks) every 3 weeks until disease progression/intolerable toxicity or to observation (OBS). The primary endpoint was progression-free survival (PFS); the secondary endpoints were overall survival (OS) and safety. Results The trial was closed after the interim analysis of the 58 enrolled (120 planned) patients. Median PFS was not improved in the UFT group compared with the OBS group (3.2 months versus 3.6 months, P = 0.752), as well as the median OS (14.2 months for both, P = 0.983). However, subgroup analysis showed that low baseline hemoglobin (< 120 g/L) was associated with poorer PFS with maintenance therapy (P = 0.032), while the normal hemoglobin patients benefit from the UFT treatment (P = 0.008). Grade 3 to 4 toxicities in the UFT group were anemia (3.4%), thrombocytopenia (3.4%) and diarrhea (6.9%). Conclusions This trial did not show superiority of UFT maintenance in non-selected patients responding to fluorouracil-based first-line chemotherapy. The normal hemoglobin level at baseline is a predictive biomarker for favorable patient subsets from the maintenance treatment.

Mel-18 negatively regulates stem cell-like properties through downregulation of miR-21 in gastric cancer

Mel-18, a polycomb group protein, has been reported to act as a tumor suppressor and be down-regulated in several human cancers including gastric cancer. It was also found that Mel-18 negatively regulates self-renewal of hematopoietic stem cells and breast cancer stem cells (CSCs). This study aimed to clarify its role in gastric CSCs and explore the mechanisms. We found that low-expression of Mel-18 was correlated with poor prognosis and negatively correlated with overexpression of stem cell markers Oct4, Sox2, and Gli1 in 101 gastric cancer tissues. Mel-18 was down-regulated in cultured spheroid cells, which possess CSCs, and overexpression of Mel-18 inhibits cells sphere-forming ability and tumor growth in vivo. Besides, Mel-18 was lower-expressed in ovary metastatic lesions compared with that in primary lesions of gastric cancer, and Mel-18 overexpression inhibited the migration ability of gastric cancer cells. Interestingly, overexpression of Mel-18 resulted in down-regulation of miR-21 in gastric cancer cells and the expression of Mel-18 was negatively correlated with the expression of miR-21 in gastric cancer tissues. Furthermore, miR-21 overexpression partially restored sphere-forming ability, migration potential and chemo-resistance in Mel-18 overexpressing gastric cancer cells. These results suggests Mel-18 negatively regulates stem cell-like properties through downregulation of miR-21 in gastric cancer cells.

CBX7 regulates stem cell-like properties of gastric cancer cells via p16 and AKT-NF-κB-miR-21 pathways

BackgroundChromobox protein homolog 7 (CBX7), a member of the polycomb group (PcG) family of proteins, is involved in the regulation of cell proliferation and cancer progression. PcG family members, such as BMI, Mel-18, and EZH2, are integral constituents of the polycomb repressive complexes (PRCs) and have been known to regulate cancer stem cell (CSC) phenotype. However, the role of other PRCs’ constituents such as CBX7 in the regulation of CSC phenotype remains largely elusive. This study was to investigate the role of CBX7 in regulating stem cell-like properties of gastric cancer and the underlying mechanisms.MethodsFirstly, the role of CBX7 in regulating stem cell-like properties of gastric cancer was investigated using sphere formation, Western blot, and xenograft tumor assays. Next, RNA interference and ectopic CBX7 expression were employed to determine the impact of CBX7 on the expression of CSC marker proteins and CSC characteristics. The expression of CBX7, its downstream targets, and stem cell markers were analyzed in gastric stem cell spheres, common cancer cells, and gastric cancer tissues. Finally, the pathways by which CBX7 regulates stem cell-like properties of gastric cancer were explored.ResultsWe found that CBX7, a constituent of the polycomb repressive complex 1 (PRC1), plays an important role in maintaining stem cell-like characteristics of gastric cancer cells via the activation of AKT pathway and the downregulation of p16. Spearman rank correlation analysis showed positive correlations among the expression of CBX7 and phospho-AKT (pAKT), stem cell markers OCT-4, and CD133 in gastric cancer tissues. In addition, CBX7 was found to upregulate microRNA-21 (miR-21) via the activation of AKT-NF-κB pathway, and miR-21 contributes to CBX7-mediated CSC characteristics.ConclusionsCBX7 positively regulates stem cell-like characteristics of gastric cancer cells by inhibiting p16 and activating AKT-NF-κB-miR-21 pathway.

The prognostic value of age in non-metastatic gastric cancer after gastrectomy: a retrospective study in the U.S. and China

Purpose: We explored the influence of age on clinicopathologic features and survival of patients with M0 gastric cancer (GC). Methods: 16856 GC patients from Surveillance, Epidemiology and End Results (SEER) database and 1037 GC patients from Chinese multiple centers were enrolled in the U.S. and Chinese cohort, respectively. 50-year-old was treated as cutoff age. Propensity score method was used to carry out a 1:1 paired match. Results: In the U.S. cohort, we found that younger patients presented poor tumor behavior. However, in spite of worse outcome in stage I~IV cohort, young group showed better 3-year survival in M0 patients, especially for those who underwent a total gastrectomy. In a matched analysis, a better prognosis was still observed in younger group. The prognostic value of age was also validated in M0 GC patients with gastrectomy in Chinese cohort. Conclusions: In spite of the worse outcome in survival curve of stage I~IV GC cohort, young patients with gastrectomy presented favorable survival in M0 subgroup. It is also applicable in China. Early diagnosis and treatment should be taken seriously in young GC patients since they often possess poorer characteristics but benefited more from gastrectomy.

Influence of SLCO1B1 in gastric cancer patients treated with EOF chemotherapy

Cytochrome-P450 enzymes, ATP-binding cassette transporters, and solute carriers mediate drug metabolism as metabolic enzymes and membrane transporters, respectively. The present study investigated whether single nucleotide polymorphisms (SNPs) in genes encoding these proteins were predictive or prognostic factors in patients with metastatic gastric cancer (MGC) undergoing chemotherapy. A retrospective study of 108 MGC patients who received epirubicin, oxaliplatin, and 5-fluorouracil (EOF) as first-line treatment was performed. A total of 13 SNPs were genotyped, including SLCO1B1 (rs4149056), SLC2A9 (rs16890979, rs6449213, rs734553), ABCG2 (rs2231142), CYP2C9 (rs1057910, rs1799853), CYP2C19 (rs72552267, rs28399504, rs56337013, rs41291556) and CYP1A2 (rs12720461, rs56107638). The associations between these genotypes and disease-control rate (DCR), progression-free survival (PFS) and overall survival (OS) were analyzed. Patients with SLCO1B1 rs4149056 TT genotype had a significantly shorter OS compared with those with a C allele (CC + CT; 312 vs. 565 days, P=0.039). Multivariate analysis revealed that the rs4149056 TT homozygous genotype was an independent prognostic factor for shorter OS (hazard ratio: 2.565, 95% confidence interval: 1.215–5.415, P=0.014). However, no significant associations between SLCO1B1 rs4149056 and PFS were observed, between the other 12 SNPs and PFS or OS, or between any of the 13 SNPs and DCR. In conclusion, SLCO1B1 rs4149056 TT may be an independent predictor of survival in patients with MCG treated with EOF chemotherapy.

BRAF and EGFR inhibitors synergize to increase cytotoxic effects and decrease stem cell capacities in BRAF(V600E)-mutant colorectal cancer cells

Mutations in the oncogene BRAF(V600E) are found in ~10% of colorectal cancers (CRCs) and are associated with poor prognosis. However, BRAF(V600E) has a limited response to the small-molecule drug, vemurafenib, a BRAF inhibitor, and BRAF inhibition is thought to cause a feedback activation of EGFR signaling that supports continued proliferation. In this study, we explored the effect of combined use of dabrafenib, a BRAF inhibitor, and cetuximab, an EGFR inhibitor, on BRAF(V600E)-mutant CRC stem cells and its possible mechanisms. Through cell viability analysis, flow cytometry, sphere forming, and western blot analysis, we found that the dabrafenib can synergize with cetuximab to reduce cell viability, induce enhanced apoptotic rates and cell cycle arrest in BRAF(V600E)-mutant HT-29 cells and inhibits stem cell capacities. Further, western blot analysis revealed that PTEN/Src/c-Myc pathway is possibly involved in the synergism between dabrafenib and cetuximab. Overall, our study shows that the combination of dabrafenib and cetuximab results in increased antitumor activity and decreased stem cell capacities in BRAF(V600E)-mutant CRC cells.