Chiung-Nien Chen

Gene Expression Profile Predicts Patient Survival of Gastric Cancer After Surgical Resection

PURPOSE This study was conducted to characterize gene expression profile of survival in patients with surgically curable gastric cancer by using an in-house membrane microarray and developing a survival prediction model. MATERIALS AND METHODS Data of cDNA microarrays were obtained from 18 pairs of cancerous and noncancerous gastric tissues. Nine patients who survived > 30 months were identified as good survival, and the other nine, who survived < 12 months, were identified as poor survival. Supervised analysis was performed to identify a gene expression profile by good and poor survival. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to confirm the microarray data in 10 patients with sufficient RNA. Using these 10 patients and another 10 patients selected randomly from 40 newly enrolled patients as the training group, the RT-PCR status of the confirmed genes was used for predicting good versus poor survival. Finally, the prediction model was tested in the remaining 30 newly enrolled gastric cancer patients. RESULTS A survival prediction model consisting of three genes (CD36, SLAM, PIM-1) was developed. This model could correctly predict poor or good survival in 23 (76.7%) of 30 newly enrolled patients, and yielded a specificity of 80% and a sensitivity of 73.3%. The survival rate of the patients predicted to have good survival was significantly higher than that of those predicted to have poor survival in the test group as a whole (N = 30; P = .00531) and in stage III patients (n = 16; P = .04467). CONCLUSION The semiquantitative RT-PCR gene expression profiling of three genes extracted from microarray study can accurately predict surgery-related outcome in gastric cancer patients.

CTGF enhances the motility of breast cancer cells via an integrin-αvβ3–ERK1/2-dependent S100A4-upregulated pathway

Connective tissue growth factor (CTGF) expression is elevated in advanced stages of breast cancer, but the regulatory role of CTGF in invasive breast cancer cell phenotypes is unclear. Presently, overexpression of CTGF in MCF-7 cells (MCF-7/CTGF cells) enhanced cellular migratory ability and spindle-like morphological alterations, as evidenced by actin polymerization and focal-adhesion-complex aggregation. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) impaired cellular migration and promoted a change to an epithelial-like morphology. A neutralizing antibody against integrin αvβ3 significantly attenuated CTGF-mediated ERK1/2 activation and cellular migration, indicating that the integrin-αvβ3–ERK1/2 signaling pathway is crucial in mediating CTGF function. Moreover, the cDNA microarray analysis revealed CTGF-mediated regulation of the prometastatic gene S100A4. Transfection of MCF-7/CTGF cells with AS-S100A4 reversed the CTGF-induced cellular migratory ability, whereas overexpression of S100A4 in MDA231/AS cells restored their high migratory ability. Genetic and pharmacological manipulations suggested that the CTGF-mediated S100A4 upregulation was dependent on ERK1/2 activation, with expression levels of CTGF and S100A4 being closely correlated with human breast tumors. We conclude that CTGF plays a crucial role in migratory/invasive processes in human breast cancer by a mechanism involving activation of the integrin-αvβ3–ERK1/2–S100A4 pathway.

miR-107 promotes tumor progression by targeting the let-7 microRNA in mice and humans

MicroRNAs (miRNAs) influence many biological processes, including cancer. They do so by posttranscriptionally repressing target mRNAs to which they have sequence complementarity. Although it has been postulated that miRNAs can regulate other miRNAs, this has never been shown experimentally to our knowledge. Here, we demonstrate that miR-107 negatively regulates the tumor suppressor miRNA let-7 via a direct interaction. miR-107 was found to be highly expressed in malignant tissue from patients with advanced breast cancer, and its expression was inversely correlated with let-7 expression in tumors and in cancer cell lines. Ectopic expression of miR-107 in human cancer cell lines led to destabilization of mature let-7, increased expression of let-7 targets, and increased malignant phenotypes. In contrast, depletion of endogenous miR-107 dramatically increased the stability of mature let-7 and led to downregulation of let-7 targets. Accordingly, miR-107 expression increased the tumorigenic and metastatic potential of a human breast cancer cell line in mice via inhibition of let-7 and upregulation of let-7 targets. By mutating individual sites within miR-107 and let-7, we found that miR-107 directly interacts with let-7 and that the internal loop of the let-7/miR-107 duplex is critical for repression of let-7 expression. Altogether, we have identified an oncogenic role for miR-107 and provide evidence of a transregulational interaction among miRNAs in human cancer development.

Low-Dose Thalidomide Treatment for Advanced Hepatocellular Carcinoma

Objective: To analyze the efficacy of oral thalidomide in the treatment of advanced hepatocellular carcinoma (HCC). Methods: Sixty-eight patients with unresectable and nonembolizable HCC were consecutively enrolled in a compassionate treatment program of oral thalidomide. Tumor response and treatment-related toxicity were prospectively followed. Thalidomide was given at a starting dose of 200 mg per day. The dose was gradually escalated in 100-mg steps up to 600 mg per day if no limiting toxicities developed. Results: Sixty-three patients were evaluable for response. One complete and 3 partial responses, defined by World Health Organization criteria, were seen, with a response rate of 6.3% (95% CI 0–12.5). The duration of response was 50+, 24.6, 11.6+ and 8.7+ weeks, respectively. All 4 responders had a dramatic decrease in α-fetoprotein (α-FP) levels. Another 6 of the 42 patients with elevated α-FP levels before treatment had a more than 50% decrease in their α-FP levels after thalidomide treatment. Totally 10 patients had an objective response to thalidomide. The median overall survival for all of the 68 patients was 18.7 weeks (95% CI 11.8– 25.6) with a 1-year survival rate of 27.6%. The median overall survival of the 10 patients with an objective response to thalidomide was 62.4 weeks (95% CI 31.2–93.6 weeks). All responders responded at a dose equal to or less than 300 mg per day. Toxicities of thalidomide were generally manageable, and only 16, 6, and 0 patients developed grade 2, 3, and 4 toxicities, respectively. Conclusion: Low-dose thalidomide is safe and induces unequivocal tumor response in a minority of patients with advanced HCC.

Lysophospholipids increase IL‐8 and MCP‐1 expressions in human umbilical cord vein endothelial cells through an IL‐1‐dependent mechanism

Lysophosphatidic acid (LPA) and sphingosine 1‐phosphate (S1P) are both low‐molecular‐weight lysophospholipid (LPL) ligands which are recognized by the Edg family of G protein‐coupled receptors (GPCRs). In endothelial cells, these two ligands activate Edg receptors resulting in cell proliferation and cell migration. Interleukin‐8 (IL‐8) is a C‐X‐C chemokine and acts as a chemoattractant of neutrophils, whereas monocyte chemoattractant protein‐1 (MCP‐1) is a C‐C chemokine and functions mainly as a chemoattractant of monocytes/macrophages. Both factors are secreted from endothelial cells and have been implicated in the processes leading to atherosclerosis. We examined the effects of LPLs on the expression of IL‐8 and MCP‐1, key regulators of leukocyte recruitment in human umbilical cord vein endothelial cells (HUVECs). Work illustrated in this article showed that LPA and S1P enhanced IL‐8 and MCP‐1 mRNA expressions, and protein secretions in dose‐ and time‐dependent fashions. Maximal mRNA expression appeared at 16 hr post‐ligand treatment. Using prior treatments with chemical inhibitors, LPLs enhanced IL‐8 and MCP‐1 expressions through a Gi‐, Rho‐, and NFκB‐dependent mechanism. In a chemotaxis assay system, LPL treatments of endothelial cells enhanced monocyte recruitment through upregulating IL‐8 and MCP‐1 protein secretions. Pre‐incubation with AF12198, an IL‐1 receptor antagonist or IL‐1 functional blocking antibody both suppressed the enhanced effects elicited by LPLs of IL‐8 and MCP‐1 mRNA expressions in HUVECs. These results suggest that LPLs released by activated platelets might enhance the IL‐8‐ and MCP‐1‐dependent chemoattraction of monocytes toward the endothelium through an IL‐1‐dependent mechanism, which may play an important role in facilitating wound‐healing and inflammation processes. J. Cell. Biochem. 99: 1216–1232, 2006.

Prospective evaluation of laparoscopy-assisted colectomy versus laparotomy with resection for management of complex polyps of the sigmoid colon

Laparoscopy-assisted colectomy istechnically feasible, but objective evidence of its benefits remainsscarce. This study was done to evaluate the outcomes and operativestress of laparoscopy-assisted colectomy versus the traditional openmethod in the management of sigmoid complex polyps that cannot besafely or adequately removed by colonofibroscopy. Between January 1997and December 1999, a total of 42 patients were equally randomizedto the laparoscopy group and the laparotomy group by the blockedrandomization method. Three patients randomized to the laparoscopygroup did not complete the trial; therefore 18 patients treated bylaparoscopy-assisted sigmoidectomy and the other 21 treated by the openmethod were prospectively evaluated. These two groups of patients werewell matched in age, gender, symptoms, tumor location, localizationmethod, tumor size, morphology, histopathology, and the accuracy of theclinical diagnosis. Two standardized surgical strategies, thelateral-to-medial and medial-to-lateral dissection sequences, wereperformed in 14 and 4 patients of the laparoscopy group, respectively,according to whether their tumors were located above or below 20 cmabove the anal verge. After evaluating the surgical outcomes, we foundthat the laparoscopy group was significantly better than the laparotomygroup in regard to parameters that included severity of postoperativepain, wound size, postoperative complication rate, and the duration ofpostoperative ileus, hospitalization, and disability. There was nosignificant difference in the operating times for these two groups.However, the costs of the laparoscopy group were significantly higher.To evaluate the surgical stress, we measured the serum C-reactiveprotein (CRP) level, erythrocyte sedimentation rate (ESR), totallymphocyte count, and CD4+/CD8+ ratio 24 hoursbefore and after surgery. We found that the postoperative serum CRPlevel and the ESR were significantly less elevated and the totallymphocyte counts and CD4+/CD8+ ratio weresignificantly less depressed in the laparoscopy group than in thelaparotomy group. We thus concluded that laparoscopy-assistedsigmoidectomy can be safely performed with shorter convalescence andless operative stress but at a higher cost. We strongly recommended theuse of this technique in the management of sigmoid complex polyps ifthe patients economic status permits.

Integrative network analysis reveals active microRNAs and their functions in gastric cancer.

BackgroundMicroRNAs (miRNAs) are a class of endogenous, small and highly conserved noncoding RNAs that control gene expression either by degradation of target mRNAs or by inhibition of protein translation. They play important roles in cancer progression. A single miRNA can provoke a chain reaction and further affect protein interaction network (PIN). Therefore, we developed a novel integrative approach to identify the functional roles and the regulated PIN of oncomirs.ResultsWe integrated the expression profiles of miRNA and mRNA with the human PIN to reveal miRNA-regulated PIN in specific biological conditions. The potential functions of miRNAs were determined by functional enrichment analysis and the activities of miRNA-regulated PINs were evaluated by the co-expression of protein-protein interactions (PPIs). The function of a specific miRNA, miR-148a, was further examined by clinical data analysis and cell-based experiments. We uncovered several miRNA-regulated networks which were enriched with functions related to cancer progression. One miRNA, miR-148a, was identified and its function is to decrease tumor proliferation and metastasis through its regulated PIN. Furthermore, we found that miR-148a could reduce the invasiveness, migratory and adhesive activities of gastric tumor cells. Most importantly, elevated miR-148a level in gastric cancer tissues was strongly correlated with distant metastasis, organ and peritoneal invasion and reduced survival rate.ConclusionsThis study provides a novel method to identify active oncomirs and their potential functions in gastric cancer progression. The present data suggest that miR-148a could be a potential prognostic biomarker of gastric cancer and function as a tumor suppressor through repressing the activity of its regulated PIN.

Using ultrasound Nakagami imaging to assess liver fibrosis in rats

This study explored the feasibility of using the ultrasound Nakagami image to assess the degree of liver fibrosis in rats. The rat has been widely used as a model in investigations of liver fibrosis. Ultrasound grayscale imaging makes it possible to observe fibrotic rat livers in real time. Statistical analysis of the envelopes of signals backscattered from rat livers may provide useful clues about the degree of liver fibrosis. The Nakagami-model-based image has been shown to be useful for characterizing scatterers in tissues by reflecting the echo statistics, and hence the Nakagami image may serve as a functional imaging tool for quantifying rat liver fibrosis. To validate this idea, fibrosis was induced in each rat liver (n=21) by an intraperitoneal injection of 0.5% dimethylnitrosamine. Livers were excised from rats for in vitro ultrasound scanning using a single-element transducer. The backscattered-signal envelopes of the acquired raw ultrasound signals were used for Nakagami imaging. The Metavir score determined by a pathologist was used to histologically quantify the degree of liver fibrosis. It was found that the Nakagami image could be used to distinguish different degrees of liver fibrosis in rats, since the average Nakagami parameter increased from 0.55 to 0.83 as the fibrosis score increased from 0 (i.e., normal) to 4. This correlation may be due to liver fibrosis in rats involving an increase in the concentration of local scatterers and the appearance of the periodic structures or clustering of scatterers that would change the backscattering statistics. The current findings indicate that the ultrasound Nakagami image has great potential as a functional imaging tool to complement the use of the conventional B-scan in animal studies of liver fibrosis.

Lysophosphatidic acid upregulates vascular endothelial growth factor-C and tube formation in human endothelial cells through LPA1/3, COX-2, and NF-κB activation- and EGFR transactivation-dependent mechanisms

Lysophosphatidic acid (LPA) is a lipid bioactive mediator which binds to G-protein-coupled receptors and activates a variety of cellular functions. LPA modulates multiple behaviors in endothelial cells, including cell proliferation and migration, capillary-like tube formation in vitro, activation of proteases, interactions with leukocytes, and expressions of inflammation-related genes, thereby regulating vessel formation. LPA has been reported to modulate the angiogenesis process. However, the role of LPA in the lymphangiogenesis process has not been studied. In this study, we showed that LPA upregulated vascular endothelial growth factor-C (VEGF-C) mRNA expression in human umbilical vein endothelial cells (HUVECs) and subsequent endothelial cell tube formation in vitro and in vivo. These enhancement effects were LPA(1)- and LPA(3)-dependent and required cyclooxygenase-2 (COX-2), endothelial growth factor receptor (EGFR) transactivation and activation of nuclear factor kappaB (NF-kappaB). Moreover, LPA induced the protein expressions of the lymphatic markers, Prox-1, LYVE-1, and podoplanin, in HUVECs, and these enhancement effects were dependent on LPA(1) and LPA(3) activation and EGFR transactivation. Our results demonstrated that LPA might regulate VEGF-C and lymphatic marker expression in endothelial cells, which contributes to endothelial cell tube formation in vitro and in vivo, thus facilitating endothelial cell participation in the lymphangiogenesis process. This study clarifies the signaling mechanism of LPA-regulated VEGF-C expression and lymphatic marker expressions in endothelial cells, which suggest that LPA may be a suitable target for generating therapeutics against lymphangiogenesis and tumor metastasis.

The Interactions Between GPR30 and the Major Biomarkers in Infiltrating Ductal Carcinoma of the Breast in an Asian Population

OBJECTIVE G-protein-coupled receptor 30 (GPR30) has been reported to be a novel estrogen receptor alpha (ERalpha) in vitro. Therefore, the interactions among GPR30, ERalpha, progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2/neu), and their prognostic utilities in the infiltrating ductal carcinoma (IDC) of the breast were evaluated. MATERIALS AND METHODS Messenger RNA (mRNA) levels of GPR30, ERalpha, PR and HER-2/neu in the tumor samples of 118 Taiwanese IDC patients and 27 non-tumor mammary tissues were measured via quantitative polymerase chain reaction analyses. The correlations of GPR30 mRNA levels with clinical parameters, i.e. tumor/non-tumor, ERalpha, PR, HER-2/neu, age, lymph node metastasis, lymph-vascular invasion, grade, stage and patient survival, were assessed by using appropriate statistical analyses. RESULTS GPR30 expression was observed to be lower in IDC (p < 0.001) than in non-tumor mammary tissues. Importantly, GPR30 mRNA level was positively correlated with that of ERalpha (p = 0.001) and PR (p = 0.001) but not correlated with that of HER-2/neu when they were analyzed as continuous variables. However, lower GPR30 was noticed in tumors with HER-2/neu protein overexpression. GPR30 expression was not correlated with age, lymph node metastasis, lymph-vascular invasion, grade and stage in IDC. GPR30 expression was not an independent prognostic factor for patient survival. CONCLUSION GPR30 expression is downregulated in IDC. GPR30 is preferentially co-expressed with ER and/or PR but is lowly expressed in HER-2/neu(+) tumors. The correlation of GPR30 expression with clinical parameters, including patient survival, was not evident in this cohort.