Jianzhi Zhang

Expression of hiwi gene in human gastric cancer was associated with proliferation of cancer cells

Stem cell genetics research may be critical to our understanding of carcinogenesis, as both stem cells and cancer cells possess the ability to self‐renew. Recent discoveries have indicated that the piwi family of genes plays an essential role in stem cell self‐renewal in diverse organisms. The hiwi gene, the human homolog of the piwi family, participates in germ cell proliferation and its overexpression may cause the development of germ cell malignancy, but its expression and function in epithelial solid cancers have not been explored. In the present study, we investigated whether there was an association between hiwi expression and human gastric cancer and its potential mechanism. RT‐PCR findings demonstrated that hiwi was expressed in different gastric cancer cell lines. To identify the HIWI protein in gastric cancer, we developed a specific monoclonal antibody against HIWI and immunohistochemistry was performed on various gastric tissues. We found that the expression ratio of hiwi in normal gastric tissues, atrophic gastritis, intestinal metaplasia and gastric cancers was 10% (5/50), 36% (18/50), 36% (18/50) and 76% (38/50), respectively, which was consistent with precancerous development. Notably, the expression pattern of hiwi in gastric cancer tissues was similar to that of Ki67, which was used as a marker of proliferation. Moreover, the suppression of hiwi by antisense or RNAi inhibited the growth of gastric cancer cells and induced cell cycle arrest in G2/M phase. These results suggest that hiwi may be involved in the development of gastric cancer and is a potential target for cancer therapy.

MicroRNA let-7c inhibits migration and invasion of human non-small cell lung cancer by targeting ITGB3 and MAP4K3.

MicroRNAs play an important regulatory role in carcinogenesis and cancer metastasis. Different members of let-7 family have been reported to be decreased in human lung tumors. However, the effect of specific let-7 member on metastasis of NSCLC remains undefined. Our current study detected the expression of let-7 members in 94 cases of NSCLC and a significant association was noticed between low levels of let-7c expression and metastasis, venous invasion, advanced TNM stages and poor survival of NSCLC patients. Consistently, ectopic expression of let-7c in relatively highly metastatic cells remarkably suppressed their migration and invasion. Inhibition of let-7c in cells with relatively low metastatic potential promoted their motility and invasion. We then analyzed the potential targets of let-7c and found that ITGB3 and MAP4K3 were directly repressed by let-7c. Upon restoring the expression of ITGB3 and MAP4K3, the effects of let-7c on tumor metastasis were partially reversed, and more importantly, the expression levels of ITGB3 and MAP4K3 were inversely correlated with let-7c in 64 NSCLC tissues. Collectively, our results suggest that let-7c, by degrading ITGB3 and MAP4K3, prevents NSCLC metastasis.

p37 from Mycoplasma hyorhinis promotes cancer cell invasiveness and metastasis through activation of MMP-2 and followed by phosphorylation of EGFR

High Mycoplasma infection in gastric cancer tissues suggests a possible association between Mycoplasma infection and tumorigenesis. By using human gastric cancer cells AGS and mouse melanoma cells B16F10 stably expressing p37, the major immunogen of Mycoplasma hyorhinis, we found that p37 enhanced cell motility, migration, and invasion in vitro. With experimental metastasis model in C57BL/6 mice, p37 adenovirus-infected B16F10 cells formed more metastasis lesions in the lung. Furthermore, p37 promoted the phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase and the activity of matrix metalloproteinase-2 (MMP-2). Inhibitor of MMPs significantly blocked p37-induced EGFR but has little effect on extracellular signal-regulated kinase phosphorylation, whereas the p37-induced MMP-2 activation was only partially suppressed by inhibitor of MEK1/2 or by inhibitor of EGFR. However, all these inhibitors significantly reduced the p37-induced invasiveness of AGS cells. These results suggest that p37 may stimulate invasion by increasing the activity of MMP-2, thereby inducing EGFR phosphorylation and contributing to tumor metastasis on M. hyorhinis infection. p37 and its regulated molecules could be the potential targets for cancer therapy. [Mol Cancer Ther 2008;7(3):530–7]

Diagnosis of gastric cancer using decision tree classification of mass spectral data

Although gastric cancer is the second leading cause of cancer death worldwide, specific and sensitive biomarkers that can be used for its diagnosis are still unavailable. Attempting to improve on current approaches to the serological diagnosis of gastric cancer, we subjected serum samples from 245 individuals (including 127 gastric cancer patients, 100 age‐ and sex‐matched healthy individuals, nine benign gastric lesion patients and nine colorectal cancer patients) for analysis by surface‐enhanced laser desorption/ionization (SELDI) mass spectrometry. Peaks were detected with Ciphergen SELDI software version 3.1.1 and analyzed with Biomarker Patterns’ software 5.0. We developed a classifier for separating the gastric cancer groups from the healthy groups. Three protein masses with 1468, 3935 and 7560 m/z were selected as a potential ‘fingerprint’ for the detection of gastric cancer. It was able to distinguish the gastric cancer patients from the health volunteers with a sensitivity of 95.6% and a specificity of 92.0% in the training set. In the blinding set, it was capable of differentiating the gastric cancer samples from the others with a specificity of 88.0%, a sensitivity of 85.3%, and an accuracy of 86.4%. These values were all higher than those achieved in a parallel analysis by measuring serum carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19‐9 together. Therefore, the decision tree analysis of serum proteomic patterns has the potential to be used in gastric cancer diagnosis. (Cancer Sci 2007; 98: 37–43)

Suppression of tumor growth and metastasis by a VEGFR-1 antagonizing peptide identified from a phage display library

Although the VEGF‐Flk‐1‐pathway has been known as the major driving force of angiogenesis, new evidence has shown that VEGFR‐1/Flt‐1 plays important roles during the neovascularization under pathological conditions including tumor, atherosclerosis and arthritis. In search of Flt‐1 receptor antagonizing peptides, we screened a phage display 12‐mer‐peptide library with recombinant Flt‐1 protein. Seven candidate peptides were identified that specifically bound to VEGF receptor Flt‐1, of which peptide F56 (WHSDMEWWYLLG) almost abolished VEGF binding to receptor Flt‐1 in vitro. In vivo, F56 fused with DHFR (DHFR‐F56) inhibited angiogenesis in a CAM assay. Moreover, DHFR‐F56 significantly inhibited the growth of nodules of human gastric cancer cell line MGC‐803 in BALB/c nude mice. Histological analyses showed that necrosis of the implanted tumor was markedly enhanced following treatment with DHFR‐F56. In the severe combined immunodeficiency disease (SCID) mouse model for studying metastasis of the human breast cancer cell line BICR‐H1, synthetic peptide F56 significantly inhibited tumor growth and lung metastases. Taken together, our results have demonstrated that peptide F56, as a Flt‐1 receptor antagonist, fulfilled the antiangiogenic and antimetastatic effects by specifically interfering with the interaction between VEGF and receptor Flt‐1. Thus, short peptide F56 may have clinical potential in tumor therapy.

Neuronal protein synuclein γ predicts poor clinical outcome in breast cancer

Synuclein γ (SNCG), previously identified as a breast cancer‐specific gene (BCSG1), is highly expressed in breast carcinomas but not in normal epithelium. SNCG regulates many pathways in growth and progression of breast cancer. To determine if SNCG is a biomarker for clinical prognosis of breast cancer, we generated a panel of murine monoclonal antibodies (mAbs) against human SNCG and correlated SNCG protein expression in 358 clinical breast cancer specimens with clinical outcome. A panel of 14 mAbs was characterized by ELISA, immunoprecipitation (IP), Western blot, immunocytochemistry and immunohistochemistry. SNCG protein expression was determined in 438 clinical breast specimens by immunohistochemical analysis using mAb 5C5. Expression of SNCG was strongly correlated with the stage, lymph node involvement, metastasis, tumor size and Her‐2 status, but its expression was not associated with ER and PR expression status. While 71.4% of advanced breast cancers were positive for SNCG expression, only 26.8% of Stage I/II breast cancers were positive for SNCG expression and 5.2% of benign hyperplasia expressed SNCG. SNCG protein was not detectable in normal tissue adjacent to breast cancer. After a median follow‐up of 64 months, patients with an SNCG‐positive tumor had a significantly shorter disease‐free survival and overall survival and a high probability of death compared no expression of SNCG. Multivariate analysis demonstrated that SNCG was a strong independent prognostic variable. SNCG is a new unfavorable prognostic marker for breast cancer progression and a potential target for breast cancer treatment.

Generation of novel monoclonal antibodies and their application for detecting ARD1 expression in colorectal cancer

Arrest defective 1 (ARD1) is an acetyltransferase involved in cell cycle control in yeast. ARD1 interacts with human N-acetyltransferase (NATH) to form a functional N-terminal acetyltransferase complex. Recently it had been linked with proliferation and apoptosis in mammalian cells, but its function in cancer development remains unclear. To evaluate significance of ARD1 expression in human colorectal cancer, we generated a panel of monoclonal antibodies (mAbs) with high specificity and sensitivity against ARD1. All of the 10 different clones could be used in ELISA and Western blot, and clone 10C12, 13G2, and 4D10 can interact with ARD1 in eukaryotic cells by immunoprecipitation (IP). Clones of 14D4 and 10C12 were strongly reacted to ARD1 in immunocytochemistry (ICH) and immunohistochemistry (IHC). ARD1 expression was evaluated in human colorectal cancer and colitis tissues by immunohistochemical analysis with mAb 14D4. Forty-one were ARD1-positive in 50 colorectal cancer tissues and only 12 were weak positive in the 50 matched normal tissues (P < 0.001). Moreover, ARD1 expression was not detectable in 20 cases of colitis tissue (P < 0.001). Furthermore, all of the six human colorectal cancer cell lines we examined were also ARD1-positive at mRNA and protein levels. Taken together, the novel mAbs against ARD1 we generated could be good tools for both basic and clinical studies, and ARD1 could be a potential biomarker in colorectal cancer.

Upregulation of programmed cell death ligand 1 promotes resistance response in non‐small‐cell lung cancer patients treated with neo‐adjuvant chemotherapy

To assess the association of the programmed cell death ligand 1 (PD‐L1) with cisplatin‐based neo‐adjuvant chemotherapy (NAC) response, we investigated the level of PD‐L1 and found increased PD‐L1 expression in chemo‐resistant tumors compared with chemo‐sensitive tumors according to RNA‐Seq analysis. In a cohort of 92 patients with NAC, the positive staining of PD‐L1 was correlated with TNM stage, lower sensitive‐response rates and shorter overall survival rates. In another 30 paired tumor specimens pre‐ and post‐chemotherapy, the patients with high PD‐L1 expression post‐chemotherapy had a worse outcome and higher stable disease rate. CD8+ tumor‐infiltrating lymphocytes were found to be related to chemosensitive response and better prognosis and negative PD‐L1 expression. Furthermore, in two patient‐derived xenograft models and cell lines A549 and PC‐9, cisplatin upregulated PD‐L1 expression, and the enhancement of PD‐L1 in cancer cell lines was in a drug dose‐dependent manner. Moreover, the depletion of PD‐L1 significantly reduced cisplatin resistance. When phosphatidylinositol 3‐kinase/protein kinase B signaling was inhibited by corresponding inhibitors, PD‐L1 expression was downregulated and apoptosis was upregulated in the cisplatin‐treated cancer cells. These results suggest that the upregulation of PD‐L1 promotes a resistance response in lung cancer cells that might be through activation of the phosphatidylinositol 3‐kinase/protein kinase B pathway and suppression of tumor‐infiltrating lymphocytes. The high expression of PD‐L1 after NAC could be an indication of therapeutic resistance and poor prognosis in patients with non‐small‐cell lung cancer.

Blockade of Notch3 inhibits the stem-like property and is associated with ALDH1A1 and CD44 via autophagy in non-small lung cancer

Acquired resistance to standard chemotherapy causes treatment failure in patients with local advanced and advanced non-small lung cancer (NSCLC). Cancer stem cells (CSCs) are a small subpopulation within cancer that is thought to be resistant to conventional chemotherapy. The Notch pathway is one of the most intensively studied for putative therapeutic targets of CSCs in solid tumors. In our study, suppression of Notch3 decreased colony and sphere formation of stem-like property in lung cancer cells. In addition, Notch3 expression was demonstrated to be upregulated in the patients with chemoresistance and related to poor prognosis of NSCLC patients. Our results also showed that CSC markers ALDH1A1 and CD44 were highly expressed in NSCLC patients with chemoresistance and these two markers were positively correlated with Notch3 expression in lung cancer specimens from TCGA database. Furthermore, the lung cancer cells with drug resistance were shown to be associated with activation of autophagy. All the data support a crucial role of Notch3 in the increase of stem-like property in NSCLC cells that might be associated with upregulation of ALDH1A1 and CD44 and activation of autophagy.

A Retrospective Study of Stage I to IIIa Lung Adenocarcinoma After Resection: What Is the Optimal Adjuvant Modality for Patients With an EGFR Mutation?

UNLABELLED We retrospectively reviewed a total of 257 stage I to IIIa lung adenocarcinoma after resection, tested them for the epidermal growth factor receptor (EGFR) mutation, and analyzed the effect of perioperative treatment on survival. The results showed that in patients with an EGFR mutation, adjuvant EGFR-tyrosine kinase inhibitor monotherapy after complete resection significantly prolongs disease-free survival compared with adjuvant chemotherapy and/or radiotherapy. BACKGROUND Adjuvant cisplatin-based chemotherapy improves non-small-cell lung cancer (NSCLC) 5-year survival rates after resection. However, adjuvant epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) efficacy and the optimal adjuvant treatment are unclear. PATIENTS AND METHODS The clinical records of patients tested for EGFR mutation after complete NSCLC resection were reviewed and tested for significance; EGFR mutation and adjuvant therapy effects on survival were assessed using univariate and Cox regression analyses. RESULTS We enrolled 257 patients (stage I, 126; stage II-IIIa, 131); 138 had EGFR mutation. EGFR mutation status was unrelated to recurrence (hazard ratio [HR], 0.83; 95% confidence interval [CI], 0.572-1.204; P = .326) or death (HR, 0.679; 95% CI, 0.406-1.136; P = .14). Thirty-one patients with EGFR mutation received adjuvant EGFR-TKIs; most (87.1%) received EGFR-TKI monotherapy. Patients who received adjuvant EGFR-TKIs had longer disease-free survival (DFS) than those who did not (P = .033) or received conventional adjuvant chemotherapy (P = .038). Adjuvant EGFR-TKIs did not affect overall survival (OS; P = .258), although the recipients had better 3-year OS (92.5% vs. 81%). Eight patients who received adjuvant EGFR-TKI developed disease recurrence, which occurred in 7 patients during adjuvant treatment. In the adjuvant EGFR-TKI group patients with a primary tumor EGFR mutation, EGFR mutation in the corresponding metastatic lymph nodes did not affect DFS, but patients who received EGFR-TKI after recurrence had longer progression-free survival (P = .087). CONCLUSION In patients with an EGFR mutation, adjuvant EGFR-TKI monotherapy after complete resection significantly prolongs DFS compared with adjuvant chemotherapy and/or radiotherapy.