Xiaohang Zhao

A Key Role for Early Growth Response-1 and Nuclear Factor-κB in Mediating and Maintaining GRO/CXCR2 Proliferative Signaling in Esophageal Cancer

Although early growth response-1 (EGR-1) has been shown as a key transcription factor in controlling cell growth, proliferation, differentiation, and angiogenesis, its role in the development of esophageal cancer is poorly understood despite the high frequency of this disease in many parts of the world. Here, immunohistochemistry showed that EGR-1 is overexpressed in 80% of esophageal tumor tissues examined. Furthermore, EGR-1 is constitutively expressed in all esophageal cancer cell lines analyzed. Esophageal squamous carcinoma WHCO1 cells stably transfected with EGR-1 short hairpin RNA displayed a 55% reduction in EGR-1 protein levels, 50% reduction in cell proliferation, a 50% reduction in cyclin-dependent kinase 4 levels, and a 2-fold induction in p27Kip1 levels associated with a G2-M cell cycle arrest. EGR-1 knockdown also caused a marked induction in IκBα expression, an effect also observed in GROβ RNA interference-expressing WHCO1 cells, because EGR-1 lies downstream of GRO/CXCR2 signaling. Furthermore, p65 mRNA levels were also reduced in cells treated with either short hairpin RNA EGR-1 or small interfering RNA EGR-1. Immunohistochemical analysis indicated that p65 is elevated in 78% (n = 61) of esophageal tumor sections analyzed. Moreover, nuclear factor-κB inhibition with either sodium salicylate or p65 RNA interference led to a significant reduction in GROα and GROβ expression. These results indicate that EGR-1 and nuclear factor-κB mediate GRO/CXCR2 proliferative signaling in esophageal cancer and may represent potential target molecules for therapeutic intervention.(Mol Cancer Res 2009;7(5):755–64)

Detection of apoptotic circulating tumor cells in advanced pancreatic cancer following 5-fluorouracil chemotherapy.

The objective of the present study was to evaluate CA19-9 and CK8/18 expression patterns in pancreatic cancer cell lines induced by 5-fluorouracil (5-Fu), and in circulating tumor cells (CTCs) in peripheral blood of patients previously untreated with advanced pancreatic cancer. Furthermore, the goal was to test the relationship of dynamic of CTCs with the effects of the first cycle of chemotherapy. To accomplish this study, CD45 antibody coated beads were used to discard white blood cells in peripheral blood. This was done in combination with CA19-9-Alexa488 and CK8/18-Alexa594 immunofluorescence staining to identify CTCs in circulation from 41 advanced pancreatic cancer patients, before and after chemotherapy. The PL45 pancreatic cancer cell line was incubated with 20 µmmol/L 5-Fu for 12 and 24 hours to induce apoptosis, and the expression patterns of CA19-9 and CK8/18 were measured, and the extent of apoptosis was evaluated. Subsequently, apoptotic cells and CTCs were measured. Of 41 patients with stage III and Ⅳ pancreatic cancer, 80.5% were detected with more than two CTCs in 7.5mL peripheral blood before any therapy and the median number of CTCs was 16.8±16.0 (0-59). After 7 days by the first cycle of 5-Fu chemotherapy, only 29.3% (12/41) of these patients were detected more than two CTCs in 7.5mL peripheral blood and the median number of CTCs was 3.8±7.8 (0-40) (P=0.000). And no CTCs were detected in 20 healthy donors from 7.5mL peripheral blood (P=0.000). Apoptotic CTCs were detected after advanced pancreatic patients were administered the first cycle of chemotherapy. Punctate, granular, and bubble-like morphologies with CA19-9 and CK8/18 staining were found and may reflect apoptosis in pancreatic cancer cells and CTCs. Apoptotic CTCs may indicate the efficacy of chemotherapy in pancreatic cancer patients. However, further studies are required to follow up these findings.

ABCC4 copy number variation is associated with susceptibility to esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is the eighth most common cause of cancer-related death worldwide. However, previous genome-wide single nucleotide polymorphism association analyses have not explained the high heritability associated with ESCC. In this study, we performed genome-wide copy number variation (CNV) analysis on 128 discordant sibling pairs to identify novel genes that contribute to ESCC susceptibility. A total of 57 774 individual CNVs were identified, and an interactive network of common CNV-associated genes was constructed, which showed that several ABC transporter genes contain CNVs in ESCC patients. Independent validation of a CNV at 13q32.1 in 1048 northern Chinese Han subjects demonstrated that the amplification of ABCC4 significantly correlated with ESCC risk [odds ratio: 3.36 (1.65-7.93), P = 0.0013]. Immunohistochemistry staining suggested that high copy numbers correlated with increased protein levels. High expression of ABCC4 was an independent poor prognostic factor for ESCC [relative risk: 1.73 (1.10-2.73), P = 0.0181]. The CNV region showed strong enhancer activity. Furthermore, inhibition of ABCC4 protein in ESCC cells decreased cell proliferation and motility via the inhibition of COX-2, PGE2 receptors and c-Myc expression; AKT, extracellular signal-regulated kinase and cAMP response element-binding protein phosphorylation; and β-catenin nuclear translocation in ESCC cells. In conclusion, the CNV at 13q32.1 is associated with ESCC susceptibility, and a gene within this locus, ABCC4, activates the oncogenic pathways in ESCC and thus facilitates cancer cell development and progression. A direct genetic contribution of ESCC risk through CNV common variants was determined in this study, and ABCC4 might therefore have predictive and therapeutic potential for ESCC.

Annexin A2 is a discriminative serological candidate in early hepatocellular carcinoma

To date, the useful markers of hepatocellular carcinoma (HCC) remains incompletely developed. Here, we show that annexin A2 complement alpha-fetoprotein (AFP), a widely used liver cancer marker, in the serologically surveillance and early detection of HCC. First, differentially expressed proteins in HCC were identified using a subcellular proteomic approach. Annexin A2 was then selected for further verification. It was found to be overexpressed in HCC tissues (60.7%, 136/224). Using a self-estabished sandwich enzyme-linked immunosorbent assay, we found that annexin A2 significantly increased in the sera of HCC (n = 175, median, 24.75ng/µl) compared with the healthy (n = 49, median, 16.69ng/µl), benign tumors (n = 19, median, 19.92ng/µl), hepatitis (n = 23, median, 6.48ng/µl) and cirrhosis (n = 51, median, 7.39ng/µl) controls and other malignant tumors (n = 87). Importantly, raised concentrations of annexin A2 were observed in 83.2% (79/95) of early stage (median, 24.32ng/µl) and 78.4% (58/74) of AFP-negative (median, 24.09ng/µl) patients. Annexin A2 alone had a better area under the receiver-operating characteristic curve (AUC = 0.79, 95% confidence interval: 0.73–0.85) in comparison with AFP (AUC = 0.73, 95% confidence interval: 0.66–0.80) in detecting of early stage HCC. Combining both markers notably improved the diagnostic efficiency of early HCC with an achieved sensitivity of 87.4%. Additionally, the expression characteristics of annexin A2 during hepatocarcinogenesis were detected in p21-HBx gene knockin transgenic mice model. The results showed that annexin A2 expression was substantially elevated in HCC-bearing mice, in accordance with the finding in human samples. In conclusion, annexin A2 may be an independent serological candidate for hepatitis B virus–related HCC, especially in the early stage cases with normal serum AFP.

Expression profiling reveals dysregulation of cellular cytoskeletal genes in HBx–induced hepatocarcinogenesis

The molecular mechanisms underlying hepatitis B virus encoded HBx protein -mediated tumorigenesis are not fully understood. In order to gain a better view of the effects of HBx on transcriptional regulation and hepatocarcinogenesis, the expression profiles of liver and tumor tissues from 6 and 18 month-old p21-HBx transgenic and control mice were monitored using oligo microarrays. Data analysis demonstrated that 42 genes were de-regulated in both 6 and 18 month-old HBx transgenic mouse tissues. Gene ontology assisted analysis classified these genes into functionally related clusters that encode proteins related to metabolism, signal transduction, transcription regulation and stress responses. Among them, cytoskeletal genes, including microtubule genes tubulin-β2 (Tubb2), tubulin-β3 (Tubb3) and tubulin-β6 (Tubb6) , intermediate filament genes periplakin, keratin 8 (K8) and keratin 18 (K18) and actin-γ1 (Actg1) , were closely clustered and up-regulated in liver tissues. These results were validated by semi-quantitative RT-PCR in both mouse and human HCC tissues. The up-regulation of K8 and K18 was only detected in p21-HBx but not p21-HBsAg liver tissues, suggesting that the global change in the expression of cellular cytoskeletal genes was correlated with the expression of HBx transgene. These findings propose for the first time that systemic dysregulation of cellular cytoskeletal genes is involved in HBx–induced hepatocarcinogenesis.

SELEX aptamer used as a probe to detect circulating tumor cells in peripheral blood of pancreatic cancer patients.

Many studies have shown that the quantity and dynamics of circulating tumor cells (CTCs) in peripheral blood of patients afflicted with solid tumours have great relevance in therapeutic efficacy and prognosis. Different methods based on various strategies have been developed to isolate and identify CTCs, but their efficacy needs to be improved because of the rarity and complexity of CTCs. This study was designed to examine the possibility of using a SELEX aptamer (BC-15) as a probe to identify rare CTCs out of background nucleated cells. Aptamer BC-15 was selected from a random oligonucleotide library screened against human breast cancer tissue. Fluorescence staining showed that BC-15 had a high affinity for nuclei of human cancer cell lines of various origins as well as CTCs isolated from pancreatic cancer patients, whereas its binding capacity for non-tumor breast epithelial cells and leukocytes was almost undetectable. BC-15+/CD45- cells in cancer patient blood were also found to be cytokeratins 18-positive and aneuploid by immunofluorescence staining and fluorescent in situ hybridization, respectively. Finally, the aptamer method was compared with the well-established anti-cytokeratin method using 15 pancreatic cancer patient blood samples, and enumeration indicated no difference between these two methods. Our study establishes a novel way to identify CTCs by using a synthetic aptamer probe. This new approach is comparable with the anti-cytokeratin-based CTC identification method.

Role of Smac in Determining the Chemotherapeutic Response of Esophageal Squamous Cell Carcinoma

Purpose: Second mitochondria-derived activator of caspase (Smac) regulates chemotherapy-induced apoptosis. Smac mimetics have been tested in clinical trials as chemosensitizers. We determined the role of Smac in modulating the chemosensitivity of esophageal squamous cell carcinoma (ESCC). Experimental Design: Smac expression was evaluated in tissues from ESCC patients with differential chemotherapeutic responses. The effects of Smac knockdown and Smac mimetics on the chemosensitivity of ESCC cells and the molecular mechanisms by which Smac and Smac mimetics modulate chemosensitivity were determined. The therapeutic responses of ESCC cells with different Smac statuses were compared using xenograft models. Results: We found that Smac was significantly downregulated in most ESCC samples (36.8%, 25/68, P = 0.001), and Smac expression differed significantly (P < 0.05) between chemosensitive and chemoresistant tumors. The associations of tested factors and their responses were examined using logistic regression analysis. In ESCC cells treated with cisplatin, a common chemotherapeutic drug, Smac and cytochrome c were released from mitochondria, and caspase-3 and caspase-9 were activated. Knockdown of Smac abrogated cisplatin-induced apoptosis, mitochondrial dysfunction, cytochrome c release, and caspase activation. Smac deficiency also reduced the effect of cisplatin on long-term cell viability, and led to cisplatin resistance in xenograft tumors in vivo. LBW242, a small molecule Smac mimetic, enhanced cisplatin-induced apoptosis and caspase activation and restored cisplatin sensitivity in Smac-deficient cells. Conclusion: Our data suggested that downregulation of Smac may be a chemoresistance mechanism in ESCC. Combinations of Smac mimetics with chemotherapeutic agents may have therapeutic benefits for the treatment of esophageal cancer. Clin Cancer Res; 17(16); 5412–22. ©2011 AACR.

Receptor Interactive Protein Kinase 3 Promotes Cisplatin-Triggered Necrosis in Apoptosis-Resistant Esophageal Squamous Cell Carcinoma Cells

Cisplatin-based chemotherapy is currently the standard treatment for locally advanced esophageal cancer. Cisplatin has been shown to induce both apoptosis and necrosis in cancer cells, but the mechanism by which programmed necrosis is induced remains unknown. In this study, we provide evidence that cisplatin induces necrotic cell death in apoptosis-resistant esophageal cancer cells. This cell death is dependent on RIPK3 and on necrosome formation via autocrine production of TNFα. More importantly, we demonstrate that RIPK3 is necessary for cisplatin-induced killing of esophageal cancer cells because inhibition of RIPK1 activity by necrostatin or knockdown of RIPK3 significantly attenuates necrosis and leads to cisplatin resistance. Moreover, microarray analysis confirmed an anti-apoptotic molecular expression pattern in esophageal cancer cells in response to cisplatin. Taken together, our data indicate that RIPK3 and autocrine production of TNFα contribute to cisplatin sensitivity by initiating necrosis when the apoptotic pathway is suppressed or absent in esophageal cancer cells. These data provide new insight into the molecular mechanisms underlying cisplatin-induced necrosis and suggest that RIPK3 is a potential marker for predicting cisplatin sensitivity in apoptosis-resistant and advanced esophageal cancer.

Expression and phosphorylation of stathmin correlate with cell migration in esophageal squamous cell carcinoma

Microtubules play extensive roles in cellular processes, including cell motility. Stathmin is an important protein which destabilizes microtubules. The essential function of stathmin is closely associated with its phosphorylation status. Stathmin is overexpressed in many human cancers and has a significant relationship with clinical characteristics such as grade, tumor size and prognosis. We demonstrated that stathmin was overexpressed in ESCC tissues using both 2-DE and immunohistochemistry analysis. In addition, overexpression of stathmin was significantly correlated with histological grade in ESCC. However, no correlation was found with age, gender and lymph node metastasis. Knockdown of stathmin with siRNA impaired cell migration in KYSE30 and KYSE410 cells. When EC0156 cells were treated with paclitaxel, stathmin was stably phosphorylated and migration was impaired. These observations suggest that stathmin may have a more important function in ESCC development and migration. The present study provides further understanding of the importance of stathmin in ESCC therapy or diagnosis.

A Prolyl-Hydroxylase Inhibitor, Ethyl-3,4-Dihydroxybenzoate, Induces Cell Autophagy and Apoptosis in Esophageal Squamous Cell Carcinoma Cells via Up-Regulation of BNIP3 and N-myc Downstream-Regulated Gene-1

The protocatechuic acid ethyl ester ethyl-3,4-dihydroxybenzoate is an antioxidant found in the testa of peanut seeds. Previous studies have shown that ethyl-3,4-dihydroxybenzoate can effectively reduce breast cancer cell metastasis by inhibiting prolyl-hydroxylase. In this study, we investigated the cytotoxic effect of ethyl-3,4-dihydroxybenzoate on esophageal squamous cell carcinoma cells in vitro and identified key regulators of ethyl-3,4-dihydroxybenzoate-induced esophageal cancer cell death through transcription expression profiling. Using flow cytometry analysis, we found that ethyl-3,4-dihydroxybenzoate induced S phase accumulation, a loss in mitochondrial membrane permeabilization, and caspase-dependent apoptosis. Moreover, an expression profile analysis identified 46 up- and 9 down-regulated genes in esophageal cancer KYSE 170 cells treated with ethyl-3,4-dihydroxybenzoate. These differentially expressed genes are involved in several signaling pathways associated with cell cycle regulation and cellular metabolism. Consistent with the expression profile results, the transcriptional and protein expression levels of candidate genes NDRG1, BNIP3, AKR1C1, CCNG2 and VEGFA were found to be significantly increased in treated KYSE 170 cells by reverse-transcription PCR and western blot analysis. We also found that protein levels of hypoxia-inducible factor-1α, BNIP3, Beclin and NDRG1 were increased and that enriched expression of BNIP3 and Beclin caused autophagy mediated by microtubule-associated protein 1 light chain 3 in the treated cells. Autophagy and apoptosis were activated together in esophageal cancer cells after exposed to ethyl-3,4-dihydroxybenzoate. Furthermore, knock-down of NDRG1 expression by siRNA significantly attenuated apoptosis in the cancer cells, implying that NDRG1 may be required for ethyl-3,4-dihydroxybenzoate-induced apoptosis. Together, these results suggest that the cytotoxic effects of ethyl-3,4-dihydroxybenzoate were mediated by the up-regulation of NDRG1, BNIP3, Beclin and hypoxia-inducible factor-1α, initiating BNIP3 and Beclin mediated autophagy at an early stage and ultimately resulting in esophageal cancer cell apoptosis.