Yijian Zhang

Fibronectin promotes cell proliferation and invasion through mTOR signaling pathway activation in gallbladder cancer

Fibronectin (FN), a heterodimeric glycoprotein overexpressed in several types of tumors, has been implicated in cancer progression via the activation of integrin-mediated pro-oncogenic pathways. The FN level in human bile fluid is dramatically increased in malignant biliary diseases; however, FN expression and its biological functions in gallbladder cancer (GBC) remain unknown. In this study, we found that FN was overexpressed in GBC tissues and was associated with a worse prognosis in GBC patients. In vitro experimental studies indicated that exogenous FN significantly enhanced cell proliferation, invasion and active MMP-9 secretion in human GBC cell lines (GBC-SD and NOZ). Moreover, the key kinases of the mTOR signaling pathway, including FAK, Akt, mTOR and 4E-BP1, were markedly activated in a time-dependent manner in FN-treated GBC-SD and NOZ cells. The IHC statistical analyses validated that FN expression was positively correlated with the phosphorylation levels of the 4E-BP1 protein in GBC tissues. Furthermore, rapamycin, a specific inhibitor of mTOR, almost completely blocked FN-induced phosphorylation of 4E-BP1 and also partially abrogated the stimulatory effects of FN on GBC cell proliferation and invasion. In vivo, FN treatment significantly promoted the proliferation and metastasis of GBC cells and markedly activated Akt/mTOR/4E-BP1 signaling cascade. These findings demonstrate that FN may play a critical role in the modulation of cell proliferation and invasion via mTOR signaling pathway activation during GBC progression.

20(S)-ginsenoside Rg3 promotes senescence and apoptosis in gallbladder cancer cells via the p53 pathway.

Gallbladder cancer (GBC), the most frequent malignancy of the biliary tract, is associated with high mortality and extremely poor prognosis. 20(S)-ginsenoside Rg3 (20(S)-Rg3) is a steroidal saponin with high pharmacological activity. However, the anticancer effect of 20(S)-Rg3 in human GBC has not yet been determined. In this study, we primarily found that 20(S)-Rg3 exposure suppressed the survival of both NOZ and GBC-SD cell lines in a concentration-dependent manner. Moreover, induction of cellular senescence and G0/G1 arrest by 20(S)-Rg3 were accompanied by a large accumulation of p53 and p21 as a result of murine double minute 2 (MDM2) inhibition. 20(S)-Rg3 also caused a remarkable increase in apoptosis via the activation of the mitochondrial-mediated intrinsic caspase pathway. Furthermore, intraperitoneal injection of 20(S)-Rg3 (20 or 40 mg/kg) for 3 weeks markedly inhibited the growth of xenografts in nude mice. Our results demonstrated that 20(S)-Rg3 potently inhibited growth and survival of GBC cells both in vitro and in vivo. 20(S)-Rg3 attenuated GBC growth probably via activation of the p53 pathway, and subsequent induction of cellular senescence and mitochondrial-dependent apoptosis. Therefore, 20(S)-Rg3 may be a potential chemotherapeutic agent for GBC therapy.

MicroRNA-29c-5p suppresses gallbladder carcinoma progression by directly targeting CPEB4 and inhibiting the MAPK pathway.

Gallbladder cancer (GBC) is a leading cause of cancer-related deaths worldwide, and its prognosis remains poor, with a 5-year survival rate of ~5%. Given the crucial role of microRNAs (miRNAs) in cancer metastasis, we aimed to analyze the expression and function of the metastasis-associated miRNA miR-29c-5p in GBC.We validated that expression of miR-29c-5p was significantly downregulated in GBC and was closely associated with lymph node metastasis, overall survival and disease-free survival in 40 GBC patients who were followed clinically. Ectopic overexpression of miR-29c-5p dramatically repressed proliferation, metastasis, and colony formation and induced apoptosis in vitro, and it suppressed tumorigenicity in vivo through the MAPK pathway. Cytoplasmic polyadenylation element binding protein 4 (CPEB4) was identified as a critical effector target of miR-29c-5p. Enforced expression of miR-29c-5p significantly inhibited the expression of CPEB4, and restoration of CPEB4 expression reversed the inhibitory effects of miR-29c-5p on GBC cell proliferation and metastasis. Transforming growth factor-β (TGF-β) upregulated CPEB4 by downregulating miR-29c-5p, leading to MAPK pathway activation. In conclusion, the TGF-β/miR-29c-5p/CPEB4 axis has a pivotal role in the pathogenesis and poor prognosis of GBC, suggesting that miR-29c-5p is a tumor-suppressive miRNA that may serve as potential prognostic biomarker or therapeutic target for GBC.

miR-101 targeting ZFX suppresses tumor proliferation and metastasis by regulating the MAPK/Erk and Smad pathways in gallbladder carcinoma

Gallbladder cancer (GBC), the most common malignancy of the bile duct, is highly aggressive and has an extremely poor prognosis, which is a result of early metastasis. As it is regulated being at multiple levels, the metastatic cascade in GBC is complex. Recent evidence suggests that microRNAs (miRNAs) are involved in cancer metastasis and are promising therapeutic targets. In this study, miR-101 was significantly downregulated in tumor tissues, particularly in metastatic tissues. In GBC patients, low miR-101 expression was correlated with tumor size, tumor invasion, lymph node metastasis, TNM stage, and poor survival. Moreover, miR-101 was an independent prognostic marker for GBC. Additionally, miR-101 inhibited GBC cell proliferation, migration, invasion, and TGF-β-induced epithelial-mesenchymal transition (EMT) in vitro and in vivo. Mechanistically, the gene encoding the zinc finger protein X-linked (ZFX) was identified as a direct target of miR-101. More importantly, miR-101 significantly reduced activation of the MAPK/Erk and Smad signaling pathways, resulting in inhibition of TGF-β-mediated induction of EMT. Altogether, our findings demonstrate a novel mechanism by which miR-101 attenuates the EMT and metastasis in GBC cells and suggest that miR-101 can serve as a potential biomarker and therapeutic target for GBC management.

miR-122 inhibits cancer cell malignancy by targeting PKM2 in gallbladder carcinoma

Gallbladder cancer (GBC) is one of the lethal diseases of digestive system. Increasing evidence prompt that microRNAs (miRs) might provide a novel therapeutical target for malignant disease. The antitumor effect of miR-122 to GBC is worth to be investigated. miR-122 expression level in GBC tissue sample and cell lines were assayed by qRT-PCR. miR-122 mimics were transfected for upregulation of miR-122 expression. Cell function was assayed by CCK8, flow cytometry, wound healing assay, migration assay, and invasion assay. The target genes of miR-122 were predicated by TargetScan online program and verified by western blot and luciferase report gene assay. miR-122 was decreased in GBC tissue and cell lines. The exogenous introduction of miR-122 exhibits multiple antitumor effect in GBC cell proliferation, invasion, and metastasis. Further studies revealed that the PKM2 was a regulative target of miR-122 in GBC cell. miR-122 also inhibits TGF-β-induced epithelium mesenchymal transformation of GBC cell by downregulating PKM2 expression. These findings suggest that miR-122 plays an important role in tumorigenesis of GBC through interfering PKM2, highlighting its usefulness as a potential therapeutic agent in GBC.

A novel PI3K/AKT signaling axis mediates Nectin-4-induced gallbladder cancer cell proliferation, metastasis and tumor growth

Nectin-4 is a Ca(2+)-independent immunoglobulin-like cell adhesion molecule which has diverse functions in cell-cell adhesion via homophilic and heterophilic interactions. Cell-cell adhesive processes are central to cell polarization, differentiation, proliferation, survival and movement. Here we report that Nectin-4 is substantially overexpressed in gallbladder cancer (GBC), the most common biliary tract malignancy with a high risk of local tumor spread and invasion. Further, Nectin-4 high expression in GBC patients was associated with pathologic T stage and lymph node metastasis status, and the expression level of the downstream target Rac1 and poor prognoses were also correlated with Nectin-4. Ectopic expression of Nectin-4 promoted GBC cell growth, motility and tumor growth in a mouse model. The depletion of Nectin-4 inhibited GBC cell proliferation and migration both in cell culture and in mice. Our data suggest that activation of the PI3K/AKT pathway was involved in the oncogenic function of Nectin-4 to activate Rac1 in GBC. Inhibition of PI3K/AKT with LY294002 and/or Rac1 with NSC23766 impaired Nectin-4-mediated GBC cell proliferation and motility. We hypothesize that Nectin-4 is critical for GBC progression via PI3K/AKT pathway activation of Rac1. Nectin-4 may be a novel prognostic factor and therapeutic target in GBC patients.

Zinc finger X-chromosomal protein (ZFX) is a significant prognostic indicator and promotes cellular malignant potential in gallbladder cancer

The Zinc finger X-chromosomal protein (ZFX), a novel member of the Krueppel C2H2-type zinc finger protein family, has been implicated in multiple human cancers. However, the clinical significance of ZFX expression in gallbladder cancer (GBC) remains largely unknown. In this study, we focused on the clinical significance, biological function and mechanism of ZFX in GBC, and found that ZFX protein overexpression was frequently detected in GBC tissues. The expression of ZFX was significantly correlated with histological grade, perineural invasion, and margin status and lead to a significantly poorer prognosis in GBC patients(P <0.001). Furthermore, knockdown of ZFX result in significant inhibition of proliferation, migration, invasion and cause cell cycle arrest in GBC-SD cells, while over-expression of ZFX in NOZ shows the opposite results. Activation of PI3K/AKT pathway maybe the potential mechanism behind these effects. In conclusion, ZFX may serve as a oncogene and could be used as a potential prognostic marker and genetic treatment target for GBC patients.

Protective effects of tumor necrosis factor-α blockade by adalimumab on articular cartilage and subchondral bone in a rat model of osteoarthritis.

We aimed to investigate the effects of an anti-tumor necrosis factor-α antibody (ATNF) on cartilage and subchondral bone in a rat model of osteoarthritis. Twenty-four rats were randomly divided into three groups: sham-operated group (n=8); anterior cruciate ligament transection (ACLT)+normal saline (NS) group (n=8); and ACLT+ATNF group (n=8). The rats in the ACLT+ATNF group received subcutaneous injections of ATNF (20 μg/kg) for 12 weeks, while those in the ACLT+NS group received NS at the same dose for 12 weeks. All rats were euthanized at 12 weeks after surgery and specimens from the affected knees were harvested. Hematoxylin and eosin staining, Massons trichrome staining, and Mankin score assessment were carried out to evaluate the cartilage status and cartilage matrix degradation. Matrix metalloproteinase (MMP)-13 immunohistochemistry was performed to assess the cartilage molecular metabolism. Bone histomorphometry was used to observe the subchondral trabecular microstructure. Compared with the rats in the ACLT+NS group, histological and Mankin score analyses showed that ATNF treatment reduced the severity of the cartilage lesions and led to a lower Mankin score. Immunohistochemical and histomorphometric analyses revealed that ATNF treatment reduced the ACLT-induced destruction of the subchondral trabecular microstructure, and decreased MMP-13 expression. ATNF treatment may delay degradation of the extracellular matrix via a decrease in MMP-13 expression. ATNF treatment probably protects articular cartilage by improving the structure of the subchondral bone and reducing the degradation of the cartilage matrix.

Up-regulation of PKM2 promote malignancy and related to adverse prognostic risk factor in human gallbladder cancer

Recently, pyruvate kinase M2 (PKM2) has been implicated in the progression of certain cancers and might play pivotal roles in the formation of malignancy. However, the role of PKM2 in gallbladder cancer had not been well investigated. This study analyzed associations between PKM2 expression status with various clinical and pathologic parameters in a large cohort of gallbladder cancer (GBC) patients from a long term follow up results. The expression level of pyruvate kinase isotypes in GBC tissues and their adjacent normal gallbladder tissues were estimated by qRT-PCR and Western blot. PKM2 mRNA level were significantly high in gallbladder cancer tissues than in adjacent noncancerous tissues (P < 0.001). High expression of the PKM2 was detected in 55.71% paraffin-embedded GBC tissue. The high PKM2 expression was independently associated with poorer overall survival in patients with GBC (median survival 11.9 vs 30.1 months; hazard ratio 2.79; 95% CI = 1.18 to 6.55; P = 0.02). These findings indicated elevated expression of PKM2 is a prognostic factor for poor GBC clinical outcomes, implied involving of PKM2 in GBC progression.

LASP-1 induces proliferation, metastasis and cell cycle arrest at the G2/M phase in gallbladder cancer by down-regulating S100P via the PI3K/AKT pathway.

LASP-1 is an actin-binding protein that regulates cytoskeletal dynamics and cell migration. LASP-1 was previously identified in a cDNA library from metastatic breast cancer samples. This protein has since been detected in multiple human cancers, including liver cancer, gastric cancer and pancreatic cancer. S100P is a small calcium-binding protein in the S100 protein family that regulates cellular, physiological and pathological processes in various cancers. However, the clinical significance of LASP-1 and S100P expression in gallbladder cancer (GBC) is not yet clear. In our study, we focused on the clinical significance, biological function and mechanism of LASP-1 in gallbladder cancer and detected LASP-1 and S100P overexpression in GBC tissues. The expression of LASP-1 was significantly correlated with poor prognosis in GBC patients (P < 0.05). Furthermore, down-regulation of LASP-1 expression resulted in the obvious inhibition of proliferation and migration and caused cell cycle arrest by down-regulating S100P via the PI3K/AKT pathway; in mice, tumor volume was significantly decreased. In conclusion, LASP-1 may act as an oncogene to regulate the expression of S100P to influence cellular functions in GBC. LASP-1 could serve as a genetic treatment target in GBC patients.