Peijun Liu

The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2-independent mechanism.

DNA damage induced by the carcinogen benzo[a]pyrene dihydrodiol epoxide (BPDE) induces a Chk1-dependent S-phase checkpoint. Here, we have investigated the molecular basis of BPDE-induced S-phase arrest. Chk1-dependent inhibition of DNA synthesis in BPDE-treated cells occurred without detectable changes in Cdc25A levels, Cdk2 activity, or Cdc7/Dbf4 interaction. Overexpression studies showed that Cdc25A, cyclin A/Cdk2, and Cdc7/Dbf4 were not rate-limiting for DNA synthesis when the BPDE-induced S-phase checkpoint was active. To investigate other potential targets of the S-phase checkpoint, we tested the effects of BPDE on the chromatin association of DNA replication factors. The levels of chromatin-associated Cdc45 (but not soluble Cdc45) were reduced concomitantly with BPDE-induced Chk1 activation and inhibition of DNA synthesis. The chromatin association of Mcm7, Mcm10, and proliferating cell nuclear antigen was unaffected by BPDE treatment. However, the association between Mcm7 and Cdc45 in the chromatin fraction was inhibited in BPDE-treated cells. Chromatin immunoprecipitation analyses demonstrated reduced association of Cdc45 with the β-globin origin of replication in BPDE-treated cells. The inhibitory effects of BPDE on DNA synthesis, Cdc45/Mcm7 associations, and interactions between Cdc45 and the β-globin locus were abrogated by the Chk1 inhibitor UCN-01. Taken together, our results show that the association between Cdc45 and Mcm7 at origins of replication is negatively regulated by Chk1 in a Cdk2-independent manner. Therefore, Cdc45 is likely to be an important target of the Chk1-mediated S-phase checkpoint.

The novel SPARC family member SMOC-2 potentiates angiogenic growth factor activity

SMOC-2 is a novel member of the SPARC family of matricellular proteins. The purpose of this study was to determine whether SMOC-2 can modulate angiogenic growth factor activity and angiogenesis. SMOC-2 was localized in the extracellular periphery of cultured human umbilical vein endothelial cells (HUVECs). Ectopically expressed SMOC-2 was also secreted into the tissue culture medium. In microarray profiling experiments, a recombinant SMOC-2 adenovirus induced the expression of transcripts required for cell cycle progression in HUVECs. Consistent with a growth-stimulatory role for SMOC-2, its overexpression stimulated DNA synthesis in a dose-dependent manner. Overexpressed SMOC-2 also synergized with vascular endothelial growth factor or with basic fibroblast growth factor to stimulate DNA synthesis. Ectopically expressed SMOC-2 stimulated formation of network-like structures as determined by in vitro matrigel angiogenesis assays. Fetal calf serum enhanced the stimulatory effect of overexpressed SMOC-2 in this assay. Conversely, small interference RNA directed toward SMOC-2 inhibited network formation and proliferation. The angiogenic activity of SMOC-2 was also examined in experimental mice by subdermal implantation of Matrigel® plugs containing SMOC-2 adenovirus. SMOC-2 adenovirus induced a 3-fold increase in the number of cells invading Matrigel® plugs when compared with a control adenoviral vector. Basic fibroblast growth factor and SMOC-2 elicited a synergistic effect on cell invasion. Taken together, our results demonstrate that SMOC-2 is a novel angiogenic factor that potentiates angiogenic effects of growth factors.

TAZ induces growth factor-independent proliferation through activation of EGFR ligand amphiregulin

The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. We previously showed that the pivotal effector of this pathway, YAP, is amplified in tumors and promotes epithelial-to-mesenchymal transition (EMT) and malignant transformation. Here, we report that overexpression of TAZ, a paralog of YAP, in human mammary epithelial cells promotes EMT and, in particular, some invasive structures in 3D cultures. TAZ also leads to cell migration and anchorage-independent growth in soft agar. Furthermore, we identified amphiregulin (AREG), an epidermal growth factor receptor (EGFR) ligand, as a target of TAZ. We show that AREG functions in a non-cell-autonomous manner to mediate EGF-independent growth and malignant behavior of mammary epithelial cells. In addition, ablation of TEAD binding completely abolishes the TAZ-induced phenotype. Last, analysis of breast cancer patient samples reveals a positive correlation between TAZ and AREG in vivo. In summary, TAZ-dependent secretion of AREG indicates that activation of the EGFR signaling is an important non-cell-autonomous effector of the Hippo pathway, and TAZ as well as its targets may play significant roles in breast tumorigenesis and metastasis.

Replication licensing promotes cyclin D1 expression and G1 progression in untransformed human cells

Defects in DNA replication are implicated as early and causal events in malignancy. However, the immediate effects of impaired DNA replication licensing on cell cycle progression of non-malignant human cells are unknown. Therefore, we have investigated the acute effects of Mcm7 ablation using synchronized cultures of untransformed Human Dermal Fibroblasts (HDF). Mcm7 ablation elicited a G1 delay associated with impaired activation of CDK4 and CDK2 and reduced Rb phosphorylation. The cell cycle delay of Mcm7-ablated cells was not associated with a DNA damage response. However, levels of cyclin D1 mRNA were specifically reduced and binding of RNA Polymerase II to the CYCD1 promoter was decreased in Mcm7-depleted cells. Similar to Mcm7-deficiency, Mcm2- or Cdc6-depletion led to impaired cyclin D expression. Ectopic over-expression of Cdc6 in quiescent cells promoted cyclin D1 expression, CDK4 activation and G1 progression. Therefore timely and efficient expression of cyclin D1 during G1 phase requires replication licensing. Reconstitution of cyclin D1 expression was insufficient to correct the G1 delay of Mcm7-depleted cells, indicating that additional cell cycle events during G1are dependent on replication licensing. However, ectopic expression of the HPV-E7 oncoprotein, and the resulting bypass of the requirement for cyclin D1-Rb signaling enabled Mcm7-depleted cells to enter S-phase. HPV-E7-induced S-phase entry of Mcm7-depleted cells led to a DNA damage response, a hallmark of pre-malignancy. Taken together, our results suggest the existence of a replication licensing restriction point that couples pre-RC assembly with G1 progression in normal cells to minimize replication stress, DNA damage and tumorigenesis.

Loss of LKB1 disrupts breast epithelial cell polarity and promotes breast cancer metastasis and invasion

BackgroundLKB1, also known as STK11, is a master kinase that serves as an energy metabolic sensor and is involved in cell polarity regulation. Recent studies have indicated that LKB1 is related to breast tumorigenesis and breast cancer progression. However, little work has been done on the roles of LKB1 in cell polarity and epithelial-mesenchymal transition in breast cancer. In this study, we tried to prove that loss of LKB1 disrupts breast epithelial cell polarity and causes tumor metastasis and invasion.MethodsThe relationships of LKB1 expression to clinic-pathological parameters and epithelial markers E-cadherin and high-molecular-weight -cytokeratin (HMW-CK) were investigated in 80 clinical breast cancer tissue samples and their paired normal control breast tissue samples by using immunohistochemistry. Then, the LKB1 expressions in metastatic and non-metastatic breast cancer cell lines were compared. The roles of LKB1 in cell polarity and epithelial-mesenchymal transition in breast cancer were determined by using immunofluorescence, western blot assay, and cell migration and invasive assays. Finally, the non-transformed human breast cell line MCF-10A was cultured in three dimensions to further reveal the role of LKB1 in breast epithelial cell polarity maintenance.ResultsHistopathological analysis showed that LKB1 expression level was significantly negatively correlated with breast cancer TNM stage, and positively correlated with ER/PR status and expression levels of E-cadherin and HMW-CK. Immunofluorescence staining showed that LKB1 was co-localized with E-cadherin at adheren junctions. In vitro analysis revealed that loss of LKB1 expression enhanced migration, invasion and the acquisition of mesenchymal phenotype, while LKB1 overexpression in MDA-MB-435 s cells, which have a low basal level of LKB1 expression, promoted the acquisition of epithelial phenotype. Finally, it was found for the first time that endogenous LKB1 knockdown resulted in abnormal cell polarity in acini formed by non-transformed breast epithelial cells grown in 3D culture.ConclusionOur data indicated that low expression of LKB1 was significantly associated with established markers of unfavorable breast cancer prognosis, such as loss of ER/PR, E-cadherin and HMW-CK. Knockdown of endogenous LKB1 gave rise to dysregulation of cell polarity and invasive phenotype of breast cancer cells.

MAPK signaling mediates sinomenine hydrochloride-induced human breast cancer cell death via both reactive oxygen species-dependent and -independent pathways: an in vitro and in vivo study

Sinomenine, the main alkaloid extracted from the medicinal plant Sinomenium acutum, is known for its anti-inflammatory effects. Recent studies have suggested its anti-cancer effect in synovial sarcoma, lung cancer and hepatic cancer. However, the underlying molecular mechanism for its anti-cancer effect still remains unclear. This study investigated the anti-tumor activity of sinomenine hydrochloride (SH), a hydrochloride form of sinomenine, in human breast cancer cells in vitro and in vivo. We found that SH potently inhibited cell viability of a broad panel of breast cancer cell lines. Two representative breast cancer cell lines, namely ER(−)/PR(−) MDA-MB-231 and ER(+)/PR(+) MCF-7, were used for further investigation. The results showed that SH induced G1/S cell cycle arrest, caused apoptosis and induced ATM/Chk2- and ATR/Chk1-mediated DNA-damage response in MDA-MB-231 and MCF-7. The anti-cancer effect of SH was regulated by increased expression levels of p-ERK, p-JNK and p-38 MAPK. Further studies showed that SH resulted in an increase in reactive oxygen species (ROS) and inhibition of ROS by N-acetyl-L-cysteine (NAC) almost blocked SH-induced DNA damage but only mitigated SH-induced MAPK expression changes, suggesting that both ROS-dependent and -independent pathways were involved in MAPK-mediated SH-induced breast cancer cell death. The in vivo study demonstrated that SH effectively inhibited tumor growth without showing significant toxicity. In conclusion, SH induced breast cancer cell death through ROS-dependent and -independent pathways with an upregulation of MAPKs, indicating that SH may be a potential anti-tumor drug for breast cancer treatment.

Superiority of Minimally Invasive Oesophagectomy in Reducing In-Hospital Mortality of Patients with Resectable Oesophageal Cancer: A Meta-Analysis

Background Compared with open oesophagectomy (OE), minimally invasive oesophagectomy (MIO) proves to have benefits in reducing the risk of pulmonary complications for patients with resectable oesophageal cancer. However, it is unknown whether MIO has superiority in reducing the occurrence of in-hospital mortality (IHM). Objective The objective of this meta-analysis was to explore the effect of MIO vs. OE on the occurrence of in-hospital mortality (IHM). Data Sources Sources such as Medline (through December 31, 2014), Embase (through December 31, 2014), Wiley Online Library (through December 31, 2014), and the Cochrane Library (through December 31, 2014) were searched. Study Selection Data of randomized and non-randomized clinical trials related to MIO versus OE were included. Interventions Eligible studies were those that reported patients who underwent MIO procedure. The control group included patients undergoing conventional OE. Study Appraisal and Synthesis Methods Fixed or random -effects models were used to calculate summary odds ratios (ORs) or relative risks (RRs) for quantification of associations. Heterogeneity among studies was evaluated by using Cochran’s Q and I2 statistics. Results A total of 48 studies involving 14,311 cases of resectable oesophageal cancer were included in the meta-analysis. Compared to patients undergoing OE, patients undergoing MIO had statistically reduced occurrence of IHM (OR=0.69, 95%CI =0.55 -0.86). Patients undergoing MIO also had significantly reduced incidence of pulmonary complications (PCs) (RR=0.73, 95%CI = 0.63-0.86), pulmonary embolism (PE) (OR=0.71, 95%CI= 0.51-0.99) and arrhythmia (OR=0.79, 95%CI = 0.68-0.92). Non-significant reductions were observed among the included studies in the occurrence of anastomotic leak (AL) (OR=0.93, 95%CI =0.78-1.11), or Gastric Tip Necrosis (GTN) (OR=0.89, 95%CI =0.54-1.49). Limitation Most of the included studies were non-randomized case-control studies, with a diversity of study designs, demographics of participants and surgical intervention. Conclusions Minimally invasive oesophagectomy (MIO) has superiority over open oesophagectomy (OE) in terms of the occurrence of in-hospital mortality (IHM) and should be the first-choice surgical procedure in esophageal surgery.

Comparative Analysis of Clinicopathologic Features, Treatment, and Survival of Asian Women With a Breast Cancer Diagnosis Residing in the United States

It has been established that disparities by ethnicity in the rates of breast cancer diagnoses and disease‐specific survival (DSS) exist in the United States. However, few studies have assessed differences specifically between Asians and other ethnic groups or among Asian subgroups.

Suppression of tumor angiogenesis by metformin treatment via a mechanism linked to targeting of HER2/HIF-1α/VEGF secretion axis

Anti-angiogenesis is currently considered as one of the major antitumor strategies for its protective effects against tumor emergency and later progression. The anti-diabetic drug metformin has been demonstrated to significantly inhibit tumor angiogenesis based on recent studies. However, the mechanism underlying this anti-angiogenic effect still remains an enigma. In this study, we investigated metformin-induced inhibitory effect on tumor angiogenesis in vitro and in vivo. Metformin pretreatment significantly suppressed tumor paracrine signaling-induced angiogenic promotion even in the presence of heregulin (HRG)-β1 (a co-activator of HER2) pretreatment of HER2+ tumor cells. Similar to that of AG825, a specific inhibitor of HER2 phosphorylation, metformin treatment decreased both total and phosphorylation (Tyr 1221/1222) levels of HER2 protein and significantly reduced microvessel density and the amount of Fitc-conjugated Dextran leaking outside the vessel. Furthermore, our results of VEGF-neutralizing and -rescuing tests showed that metformin markedly abrogated HER2 signaling-induced tumor angiogenesis by inhibiting VEGF secretion. Inhibition of HIF-1α signaling by using RNAi or YC-1, a specific inhibitor of HIF-1α synthesis, both completely diminished mRNA level of VEGF and greatly inhibited endothelial cell proliferation promoted by HER2+ tumor cell-conditioned medium in both the absence and presence of HRG-β1 pretreatment. Importantly, metformin treatment decreased the number of HIF-1α nucleus positive cells in 4T1 tumors, accompanied by decreased microvessel density. Our data thus provides novel insight into the mechanism underlying the metformin-induced inhibition of tumor angiogenesis and indicates possibilities of HIF-1α-VEGF signaling axis in mediating HER2-induced tumor angiogenesis.

Vascular Normalization Induced by Sinomenine Hydrochloride Results in Suppressed Mammary Tumor Growth and Metastasis

Solid tumor vasculature is characterized by structural and functional abnormality and results in a hostile tumor microenvironment that mediates several deleterious aspects of tumor behavior. Sinomenine is an alkaloid extracted from the Chinese medicinal plant, Sinomenium acutum, which has been utilized to treat rheumatism in China for over 2000 years. Though sinomenine has been demonstrated to mediate a wide range of pharmacological actions, few studies have focused on its effect on tumor vasculature. We showed here that intraperitoneally administration of 100u2005mg/kg sinomenine hydrochloride (SH, the hydrochloride chemical form of sinomenine) in two orthotopic mouse breast cancer models for 14 days, delayed mammary tumor growth and decreased metastasis by inducing vascular maturity and enhancing tumor perfusion, while improving chemotherapy and tumor immunity. The effects of SH on tumor vessels were caused in part by its capability to restore the balance between pro-angiogenic factor (bFGF) and anti-angiogenic factor (PF4). However 200u2005mg/kg SH didnt exhibit the similar inhibitory effect on tumor progression due to the immunosuppressive microenvironment caused by excessive vessel pruning, G-CSF upregulation, and GM-CSF downregulation. Altogether, our findings suggest that SH induced vasculature normalization contributes to its anti-tumor and anti-metastasis effect on breast cancer at certain dosage.