Jiequn Ma

Knockdown of Lymphoid Enhancer factor 1 Inhibits Colon Cancer Progression In Vitro and In Vivo

Expression of lymphoid enhancer factor 1 (LEF1) is frequently altered in different human cancers. This study aimed to assess LEF1 expression in colon cancer tissues and to explore changed phenotypes, gene expressions, and the possible mechanism after knocked down LEF1 expression in colon cancer cell lines. A total of 106 colon cancer and matched paratumorous normal tissues were used to assess LEF1 expression using immunohistochemistry and qRT-PCR. LEF1 lentivirus was used to knockdown LEF1 expression for the assessment of cell viability, cell cycle distribution, apoptosis, and gene expressions. The nude mouse xenograft assay was performed to detect the effects of LEF1 knockdown in vivo. The data showed that the levels of LEF1 mRNA and protein were significantly increased in human colon cancer tissues compared to the matched paratumorous normal tissues and were associated with infiltration depth, lymph node and distant metastases, advanced TNM (tumor-node-metastasis) stages, and shorter overall survival. Furthermore, LEF1 knockdown reduced tumor cell viability, invasion capacity, MMP2 and MMP-9 expression, but induced apoptosis. Nude mouse xenograft assay showed that LEF1 knockdown suppressed tumor formation and growth in vivo. In addition, the expression of Notch pathway-related proteins RBP-jκ and Hes1 was reduced in LEF1 knockdown cells. Taken together, LEF1 protein was overexpressed in colon cancer tissues and knockdown of LEF1 expression inhibited colon cancer growth in vitro and in vivo. These data suggest that targeting of LEF1 expression should be further evaluated for colon cancer prevention and therapy.

Loss of p57 expression and RhoA overexpression are associated with poor survival of patients with hepatocellular carcinoma

p57 and Ras homology A (RhoA) have been implicated in the growth and metastasis of several types of human cancers. This study aimed to detect their expression in hepatocellular carcinoma (HCC) tissue specimens and to determine a possible association with clinicopathological data and patient survival. A total of 80 HCC and corresponding distant normal tissue specimens were processed for immunohistochemical and qPCR analyses of p57 and RhoA expression. The data showed that expression of p57 mRNA and protein was reduced in HCC tissues when compared to that in distant non-cancer tissues (P<0.05), while expression of RhoA mRNA and protein was significantly higher in HCC tissue specimens when compared to that of the distant normal tissues. Loss of p57 expression was associated with HCC with higher α-fetoprotein (AFP) levels (>400 ng/ml; P=0.044), larger tumor size (>5 cm, P=0.004), poor tumor differentiation (P=0.020), advanced TNM stage (P=0.027), capsule invasion (P=0.018) and tumor thrombosis (P=0.008), whereas expression of RhoA protein was significantly associated with poor tumor differentiation (P=0.042), capsule invasion (P=0.022), and tumor thrombosis (P=0.002). Furthermore, there was a strong inverse relationship between p57 and RhoA expression in HCC tissues, indicating that loss of p57 expression may contribute to RhoA overexpression in HCC tissues. The median survival time of HCC patients with p57+ and p57- expression was 13.0 and 9.0 months, respectively, whereas the median survival time of HCC patients with RhoA+ and RhoA- was 9.0 and 15.0 months. Univariate analysis revealed that the levels of AFP, tumor size, TNM stage, histological grade, capsule invasion, tumor thrombosis, p57, RhoA and co-expression of p57 and RhoA were all significant prognostic indicators for overall survival of HCC patients. Multivariate analysis showed that tumor size, TNM stage, p57, RhoA and combined loss of p57 with RhoA were risk factors for poor survival of HCC patients. This study indicates that the abnormal expression of p57 and RhoA contributes to progression of HCC and poor survival of patients.

Metformin inhibits growth of human non‑small cell lung cancer cells via liver kinase B‑1‑independent activation of adenosine monophosphate‑activated protein kinase

Metformin, the most widely administered oral anti-diabetic therapeutic agent, exerts its glucose-lowering effect predominantly via liver kinase B1 (LKB1)-dependent activation of adenosine monophosphate-activated protein kinase (AMPK). Accumulating evidence has demonstrated that metformin possesses potential antitumor effects. However, whether the antitumor effect of metformin is via the LKB1/AMPK signaling pathway remains to be determined. In the current study, the effects of metformin on proliferation, cell cycle progression, and apoptosis of human non-small cell lung cancer (NSCLC) H460 (LKB1-null) and H1299 (LKB1-positive) cells were assessed, and the role of LKB1/AMPK signaling in the anti-growth effects of metformin were investigated. Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell cycle distribution and apoptosis were assessed by flow cytometry, and protein expression levels were measured by western blotting. Metformin inhibited proliferation, induced significant cell cycle arrest at the G0–G1 phase and increased apoptosis in NSCLC cells in a time- and concentration-dependent manner, regardless of the level of LKB1 protein expression. Furthermore, knockdown of LKB1 with short hairpin RNA (shRNA) did not affect the antiproliferative effect of metformin in the H1299 cells. Metformin stimulated AMPK phosphorylation and subsequently suppressed the phosphorylation of mammalian target of rapamycin and its downstream effector, 70-kDa ribosomal protein S6 kinase in the two cell lines. These effects were abrogated by silencing AMPK with small interfering RNA (siRNA). In addition, knockdown of AMPK with siRNA inhibited the effect of metformin on cell proliferation in the two cell lines. These results provide evidence that the growth inhibition of metformin in NSCLC cells is mediated by LKB1-independent activation of AMPK, indicating that metformin may be a potential therapeutic agent for the treatment of human NSCLC.

High Skp2/Low p57Kip2 Expression is Associated with Poor Prognosis in Human Breast Carcinoma

Downregulation of p57Kip2 is involved in tumor progression, and S-phase kinase-associated protein 2 (Skp2) is an E3 ligase that regulates a variety of cell cycle proteins. However, the prognostic value of p57Kip2 and its correlation with Skp2 in breast cancer have not been fully elucidated. Here we report our study on the expression of p57Kip2 and Skp2 in 102 breast cancer patients by immunohistochemistry, and analysis of clinicopathologic parameters in relation to patient prognosis. The expression of p57Kip2 was negatively associated with Skp2 expression in breast cancer (r = −0.26, P = 0.009). Kaplan–Meier analysis indicated that both high Skp2 and low p57Kip2 correlated with poor disease-free survival (DFS) (P < 0.05), and a group with the combination of high Skp2/low p57Kip2 demonstrated even worse DFS (log-rank = 21.118, P < 0.001). In addition, univariate analysis showed that Skp2, p57Kip2, histological grade, lymph node metastasis, and estrogen and progesterone receptors (ER and PR) were all associated with DFS, and multivariate analysis revealed that lymph node metastasis and Skp2 were independent prognostic biomarkers. The correlation between p57 and Skp2 was further demonstrated in multiple breast cancer cell lines and cell cycle phases. Half-life and immunoprecipitation (IP) experiments indicated that Skp2 directly interacts with p57Kip2 and promotes its degradation, rather than its mutant p57Kip2 (T310A). Overall, our findings demonstrate that Skp2 directly degrades p57Kip2, and an inverse correlation between these proteins (high skp2/low p57Kip2) is associated with poor prognosis in breast cancer. Thus, our results indicate a combined prognostic value of these markers in breast cancer diagnosis and treatment.

Reduced expression of liver kinase B1 and Beclin1 is associated with the poor survival of patients with non-small cell lung cancer

Liver kinase B1 (LKB1) regulates cell polarity and has tumor-suppressor functions, while Beclin1 regulates autophagy and is associated with tumorigenesis. The present study quantified the expression level of LKB1 and Beclin1 in non-small cell lung cancer (NSCLC) tissue specimens and examined the possible association with clinicopathological characteristics and the survival of patients. Quantitative real-time reverse transcriptase‑polymerase chain reaction (qRT-PCR), western blotting and immunohistochemistry were used to assess expression of LKB1 and Beclin1 in 142 NSCLC tissue samples. The data revealed that expression levels of both LKB1 and Beclin1 mRNA and protein were significantly reduced in the NSCLC tissues compared to levels in the matched surrounding normal lung tissues, and expression of LKB1 protein was associated with Beclin1 expression in the NSCLC tissues. Reduced expression of LKB1 protein was significantly associated with tumor histology (P<0.001) and poor differentiation (P<0.001), but was not associated with tumor lymph node metastasis, TNM stage or patient gender and age, while the reduced expression of Beclin1 protein was associated with tumor histology (P<0.05) and poor differentiation (P<0.05). Univariate analysis revealed that adenocarcinoma, lymph node metastasis, advanced TNM stage and reduced expression of LKB1 and Beclin1 proteins are associated with the survival of NSCLC patients. Multivariate analysis indicated that adenocarcinoma (P<0.05), lymph node metastasis (P<0.05), advanced TNM stage (P<0.001) and reduced expression of LKB1 (P<0.05) and Beclin1 (P<0.001) are all independent prognostic indicators for the survival of NSCLC patients.

Cyclooxygenase-2 inhibitor is a robust enhancer of anticancer agents against hepatocellular carcinoma multicellular spheroids

Purpose Celecoxib, an inhibitor of cyclooxygenase-2 (COX2), was investigated for enhancement of chemotherapeutic efficacy in cancer clinical trials. This study aimed to determine whether celecoxib combined with 5-fluorouracil or sorafenib or gefitinib is beneficial in HepG2 multicellular spheroids (MCSs), as well as elucidate the underlying mechanisms. Methods The human hepatocellular carcinoma cell line HepG2 MCSs were used as in vitro models to investigate the effects of celecoxib combined with 5-fluorouracil or sorafenib or gefitinib treatment on cell growth, apoptosis, and signaling pathway. Results MCSs showed resistance to drugs compared with monolayer cells. Celecoxib combined with 5-fluorouracil or sorafenib exhibited a synergistic action. Exposure to celecoxib (21.8 μmol/L) plus 5-fluorouracil (8.1 × 10−3 g/L) or sorafenib (4.4 μmol/L) increased apoptosis but exerted no effect on COX2, phosphorylated epidermal growth-factor receptor (p-EGFR) and phosphorylated (p)-AKT expression. Gefitinib (5 μmol/L), which exhibits no growth-inhibition activity as a single agent, increased the inhibitory effect of celecoxib. Gefitinib (5 μmol/L) plus celecoxib (21.8 μmol/L) increased apoptosis. COX2, p-EGFR, and p-AKT were inhibited. Conclusion Celecoxib combined with 5-fluorouracil or sorafenib or gefitinib may be superior to single-agent therapy in HepG2 MCSs. Our results provided molecular evidence to support celecoxib combination-treatment strategies for patients with human hepatocellular carcinoma. MCSs provided a good model to evaluate the interaction of anticancer drugs.

Expression and significance of the novel tumor-suppressor gene SMG-1 in hepatocellular carcinoma

Recent studies have demonstrated that SMG-1, a newly characterized member of the family of phosphatidylinositol 3-kinase-related protein kinases (PIKKs), is involved in tumorigenesis as a new tumor suppressor. However, its expression and significance in hepatocellular carcinoma (HCC) remain obscure. The present study investigated SMG-1 expression in HCC tissue specimens, aimed at defining the association with clinicopathological significance. Both immunohistochemistry and qRT-PCR were employed to analyze SMG-1 expression in 157 HCC and corresponding distant normal tissue specimens. The results revealed that expression of SMG-1 was significantly lower in the HCC tissue specimens than that in the distant normal tissues. Moreover, a lower expression level of SMG-1 was significantly correlated with serum α-fetoprotein level (P=0.001), poorly differentiated tumors (P=0.009) and more advanced TNM stage (P<0.001). Further study showed that SMG-1 expression was exactly associated with tumor differentiation and clinical stage in HCC. Kaplan-Meier analysis indicated that low SMG-1 expression was related to poor overall survival, and the prognostic impact of SMG-1 was further confirmed by stratified survival analysis. Importantly, multivariate analysis revealed that low SMG-1 expression was an independent prognostic marker for an unfavorable overall survival. We conclude that SMG-1 is downregulated in HCC and may represent a promising biomarker for predicting the prognosis of HCC, including the prognosis of early-stage patients.

Helicobacter pylori promotes invasion and metastasis of gastric cancer cells through activation of AP-1 and up-regulation of CACUL1

Infection with Helicobacter pylori is important in the development and progression of gastric cancer. However, the mechanisms that regulate this activation in gastric tumors remain elusive. CACUL1 has been cloned and identified as a novel gene that is expressed in many types of cancer and is involved in cell cycle regulation and tumor growth. The current study aimed to examine the expression of CACUL1 in gastric cancer samples and analyze its correlation with H. pylori infection. We found that CACUL1 was highly expressed in gastric cancer tissues and negatively correlated with gastric cancer differentiation and TNM stage. In addition, CACUL1 expression was high in H. pylori-infected tissues compared with H. pylori non-infected tissue. We found that H. pylori could up-regulate CACUL1 expression through activating protein 1. The up-regulation of CACUL1 expression could promote matrix metalloproteinase 9 and Slug expression to increase invasion and metastasis of tumor cells. These results suggested that H. pylori-triggered CACUL1 production occurred in an activating protein 1-dependent manner and regulated matrix metalloproteinase 9 and Slug expression to affect the invasion and metastasis of tumor cells. Therefore, CACUL1 is a potential therapeutic target for the treatment of aggressive gastric cancer.

Pleiotrophin as a potential biomarker in breast cancer patients

BACKGROUND Pleiotrophin (PTN), a multifunctional growth factor, is up-regulated in many tumors. PTN is reported to play an important role in the regulation of several cellular processes. The objective of this study is to evaluate the clinical significance of PTN as a tumor marker in breast cancer (BC). METHODS Serum PTN levels were detected in 105 BC patients and 40 healthy volunteers using ELISA. In addition, PTN expression was examined in 80 BC tissues in a nested case-control study by immunohistochemistry. RESULTS Serum PTN levels were elevated in BC patients compared to healthy controls. Area under receiver operating characteristic (ROC) curve was 0.878 (95% CI: 0.824-0.932). The sensitivity of serum PTN was superior to CEA and CA15-3. High serum PTN levels were associated with TNM stage, histology grade, and distant metastasis. Moreover, serum PTN levels decreased significantly after surgical treatment. In BC tissues, PTN expression was significantly higher in BC tissues relative to paired paracancerous tissues. Tissue PTN expression proved to be a prognostic factor for breast cancer according to multivariable logistic regression analysis. CONCLUSION PTN could be considered as a potential biomarker for the presence of breast cancer.

Cytoplasmic liver kinase B1 promotes the growth of human lung adenocarcinoma by enhancing autophagy

Liver kinase B1 (LKB1) as a tumor suppression gene that is associated with various kinds of cancers, including lung cancer. In this study, we found that the effect of LKB1 on tumor growth was dependent on its subcellular expression in A549 and HCC827 cells. Full‐length LKB1 decreased the proliferation and clonogenicity of A549‐LKB1 and HCC827‐LKB1 cells, but increased their apoptosis. Opposite effects were observed in A549‐LKB1s and HCC827‐LKB1S cells that overexpressed truncated LKB1 without the nuclear localization sequence. The truncated cytoplasmic LKB1 enhanced the growth of implanted tumors in vivo. The truncated cytoplasmic LKB1 promoted autophagy, which was independent of AMP‐activated protein kinase and mTOR signaling in A549 and HCC827 cells. Further characterization indicated that higher levels of cytoplasmic LKB1 expression were associated with advanced TNM stage and reduced overall survival (OS) in 190 patients with adenocarcinoma. In contrast, high nuclear expression of LKB1 is associated with early TNM stage and longer OS. The high level of cytoplasmic LKB1 expression was an independent risk factor for poor overall survival in patients with adenocarcinoma. Together, our results revealed that cytoplasmic LKB1 promotes the growth of lung adenocarcinoma and could be a prognostic marker for lung adenocarcinoma.