Expression screening of 17q12-21 amplicon reveals GRB7 as an ERBB2-dependent oncogene

Gene amplification is a major genetic alteration in human cancers. Amplicons, amplified genomic regions, are believed to contain "driver" genes responsible for tumorigenesis. However, the significance of co-amplified genes has not been extensively studied. We have established an integrated analysis system of amplicons using retrovirus-mediated gene transfer coupled with a human full-length cDNA set. Applying this system to 17q12-21 amplicon observed in breast cancer, we identified GRB7 as a context-dependent oncogene, which modulates the ERBB2 signaling pathway through enhanced phosphorylation of ERBB2 and Akt. Our work provides an insight into the biological significance of gene amplification in human cancers.     

Combining Anti-ERBB3 Antibodies Specific for Domain I and Domain III Enhances the Anti-Tumor Activity over the Individual Monoclonal Antibodies

Background

Inappropriate signaling through the epidermal growth factor receptor family (EGFR1/ERBB1, ERBB2/HER2, ERBB3/HER3, and ERBB4/HER4) of receptor tyrosine kinases leads to unregulated activation of multiple downstream signaling pathways that are linked to cancer formation and progression. In particular, ERBB3 plays a critical role in linking ERBB signaling to the phosphoinositide 3-kinase and Akt signaling pathway and increased levels of ERBB3-dependent signaling is also increasingly recognized as a mechanism for acquired resistance to ERBB-targeted therapies.

Methods

We had previously reported the isolation of a panel of anti-ERBB3 single-chain Fv antibodies through use of phage-display technology. In the current study scFv specific for domain I (F4) and domain III (A5) were converted into human IgG1 formats and analyzed for efficacy.

Results

Treatment of cells with an oligoclonal mixture of the A5/F4 IgGs appeared more effective at blocking both ligand-induced and ligand-independent signaling through ERBB3 than either single IgG alone. This correlated with improved ability to inhibit the cell growth both as a single agent and in combination with other ERBB-targeted therapies. Treatment of NCI-N87 tumor xenografts with the A5/F4 oligoclonal led to a statistically significant decrease in tumor growth rate that was further enhanced in combination with trastuzumab.

Conclusion

These results suggest that an oligoclonal antibody mixture may be a more effective approach to downregulate ERBB3-dependent signaling.     

LncRNA SNHG17 promotes gastric cancer progression by epigenetically silencing of p15 and p57

Long noncoding RNAs (lncRNA) are attractive biomarkers and therapeutic targets because of their disease- and stage-restricted expression. Small nucleolar RNA host gene 17 (SNHG17) belongs to a large family of noncoding genes hosting small RNAs, with its expression pattern and biological function not clarified in gastric cancer (GC). Thus, we conducted this study to investigate the functional significance and the underlying mechanisms of SNHG17 in GC progression. Our results showed that SNHG17 expression was upregulated in GC tissues and cells, and its high expression was significantly correlated with increased invasion depth, lymphatic metastasis, and advanced TNM stage. The expression of plasma SNHG17 was also found upregulated in patients with GC compared with healthy controls, with a moderate accuracy for diagnosis of GC (area under the receiver operating characteristic curve = 0.748; 95% CI, 0.666–0.830). Gain- and loss-of-function of SNHG17 revealed that SNHG17 promoted GC cell proliferation, cell cycle progression, invasion, and migration and inhibited apoptosis. Mechanistic investigations showed that SNHG17 was associated with polycomb repressive complex 2 and that this association was required for epigenetic repression of cyclin-dependent protein kinase inhibitors, including p15 and p57, thus contributing to the regulation of GC cell cycle and proliferation. Furthermore, rescue experiments indicated that SNHG17 functioned as an oncogene via activating enhancer of zeste homolog 2 in GC cells. Our study provides a new perspective for SNHG17 acting as a noncoding oncogene in GC tumorigenesis, and it may serve as a novel early diagnostic marker and potential target for the treatment of GC.     

Medulloblastoma sensitivity to 17-allylamino-17-demethoxygeldanamycin requires MEK/ERKM

ERBB2 increases the sensitivity of breast cancer cells to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). This has been attributed to the disruption of ERBB3/ERBB2 heterodimers that maintain a crucial cell survival signal via phosphatidylinositol 3-kinase/AKT. ERBB2 confers a poor clinical outcome in medulloblastoma, the most common malignant pediatric brain tumor. Here, we show that medulloblastoma cell sensitivity to 17-AAG is directly related to ERBB2 expression level. Furthermore, overexpression of exogenous ERBB2 in these cells induces spontaneous homodimerization, further enhancing cell sensitivity to 17-AAG. In contrast to breast cancer cells, this increased sensitivity to 17-AAG does not result from cell dependence on AKT1 activity. Rather, we show that 17-AAG generates a dose- and time-dependent increase in MEK/ERK signaling that is required for the drug to inhibit the proliferation of medulloblastoma cells and that ERBB2 sensitizes medulloblastoma cells to 17-AAG by up-regulating basal MEK/ERK signaling. We further show that down-regulation of MEK1 activity markedly reduces the sensitivity of medulloblastoma, breast, and ovarian cancer cells to 17-AAG, whereas expression of a constitutively active MEK1 potentiates the activity of 17-AAG against these cells. Therefore, intact MEK/ERK signaling may be required for optimal 17AAG activity against a variety of tumor cell types. These data identify a new mechanism by which 17-AAG inhibits the proliferation of cancer cells. Defining the precise mode of action of these agents within specific tumor cell types will be crucial if this class of drugs is to be efficiently developed in the clinic.     

The ErbB2-targeting antibody trastuzumab and the small-molecule SRC inhibitor saracatinib synergistically inhibit ErbB2-overexpressing gastric cancer

The anti-ErbB2 antibody trastuzumab has shown significant clinical benefits in ErbB2-overexpressing breast and gastric cancer, but resistance to the drug is common. Here, we investigated the antitumor activity of the combination of trastuzumab and the SRC inhibitor saracatinib in ErbB2-overexpressing trastuzumab-resistant gastric cancer. The ErbB2-overexpressing human gastric cancer cell line NCI-N87 was treated with trastuzumab to obtain the trastuzumab-resistant cell line NCI-N87R. The NCI-N87R cell line showed a marked increase in SRC activity and ErbB signaling compared with the NCI-N87 cell line. Our data demonstrated that trastuzumab plus saracatinib was much more potent than either agent alone in reducing the phosphorylation of ErbB3 and AKT in both NCI-N87 and NCI-N87R gastric cancer cell lines. Trastuzumab and saracatinib synergistically inhibited the in vitro growth of NCI-N87 and NCI-N87R cell lines. Further data showed that combination therapy of trastuzumab with saracatinib resulted in a significant benefit over either agent alone in both NCI-N87 and NCI-N87R xenograft models, suggesting its potential use for treating ErbB2-overexpressing gastric cancer.     

Biological Activities of Fractions from Methanolic Extract of <Emphasis Type="Italic">Picrasma quassioides</Emphasis>

Antioxidant, antidiabetes, and anticancer activities of fractions (water, hexane, EtOAc, and BuOH) from methanolic (MeOH) extract of Picrasma quassioides were evaluated in vitro. Among the four fractions, BuOH fraction showed cytotoxic activity on the human stomach carcinoma cells (NCI-N87) without cytotoxic effect on the human normal kidney cells (293) by MTT assay. Considering these results, we adapted BuOH fraction of MeOH extract to the assessment of apoptosis by flow cytometry and mRNA expression levels of established apoptotic-related genes on NCI-N87 cells. The experiments showed that BuOH fraction could induce apoptosis on NCI-N87 cells significantly. Take together these results, P. quassioides showed that it could be used for potential pharmaceutical products.     

BST2 promotes cell proliferation,migration and induces NF-κB activation in gastric cancer

Objectives

To investigate the functional roles of bone marrow stromal cell antigen 2 (BST2) in gastric cancer (GC) cells and its implications in the development of GC patients.

Results

BST2 was frequently overexpressed in GC tissues compared with the adjacent non-tumorous tissues, and high BST2 expression was correlated with tumor stage and lymphatic metastasis. Furthermore, in vitro experiments demonstrated that knockdown of BST2 by siRNA inhibited cell proliferation, induced apoptosis and repressed cell motility in GC cells. In addition, the pro-tumor function of BST2 in GC was mediated partly through the NF-κB signaling.

Conclusion

BST2 possesses the oncogenic potential in GC by regulating the proliferation, apoptosis, and migratory ability of GC cells, thereby BST2 could be a potential therapeutic target for the treatment of GC.

     

Phenylhexyl isothiocyanate has dual function as histone deacetylase inhibitor and hypomethylating agent and can inhibit myeloma cell growth by targeting critical pathways

Background

Amplification of the ERBB2 (Her-2/neu) oncogene, which occurs in approximately 25% of breast carcinomas, is a known negative prognostic factor. Available data indicate that a variable number of nearby genes on chromosome 17q may be co-amplified or deleted, forming a continuous amplicon of variable size. In approximately 25% of these patients, the amplicon extends to the gene for topoisomerase II alpha (TOP2A), a target for anthracyclines. We sought to understand the significance of these associated genomic changes for breast cancer prognosis and predicting response to therapy.

Methods and patients

Archival tissue samples from 63 breast cancer patients with ERBB2 amplification, stages 0–IV, were previously analyzed with FISH probes for genes located near ERBB2. In the present study, the clinical outcome data were determined for all patients presenting at stages I–III for whom adequate clinical follow up was available.

Results

Four amplicon patterns (Classes) were identified. These were significantly associated with the clinical outcome, specifically, recurrence of breast cancer. The Amplicon class IV with deleted TOP2A had 67% (6/9) cases with recurrence, whereas the other three classes combined had only 12% (3/25) cases (p-value = 0.004) at the time of last follow-up. TOP2A deletion was also significantly associated with time to recurrence (p-value = 0.0002). After adjusting for age in Cox regression analysis, the association between TOP2A deletion and time to recurrence remains strongly significant (p-value = 0.002) whereas the association with survival is marginally significant (p-value = 0.06).

Conclusion

TOP2A deletion is associated with poor prognosis in ERBB2-amplified breast carcinomas. Clarification of the mechanism of this association will require additional study.     

Targeting Cadherin-17 Inactivates Ras/Raf/MEK/ERK Signaling and Inhibits Cell Proliferation in Gastric Cancer

Cadherin-17 (CDH17), one member of 7D-cadherin superfamily, was overexpressed in gastric cancer (GC) and was associated with poor survival, tumor recurrence, metastasis, and advanced tumor stage. So far the cellular function and signaling mechanism of CDH17 in GC remains unclear. In this study, we showed that over 66% of GC cell lines (20/30) were CDH17 positive. Tissue microarray (TMA) assay showed that 73.6% Chinese GC tissues (159/216) were CDH17 positive, while 37% respective adjacent normal tissues were CDH17 positive. Knockdown of CDH17 inhibited cell proliferation, migration, adhesion and colony formation, and also induced a cell cycle arrest and apoptosis in AGS human GC cells. On the other side, overexpression of CDH17 facilitated MGC-803 GC tumor growth in nude mice. Antibody array and Western blotting assay demonstrated that knockdown of CDH17 in AGS cells down-regulated integrin β series proteins, further inactivated the Ras/Raf/MEK/ERK pathway and led to p53 and p21 accumulation, which resulted in proliferation inhibition, cell-cycle arrest and apoptosis induction. Collectively, our data firstly demonstrate the capacity of CDH17 to regulate the activity of Ras/Raf/MEK/ERK pathway for cell proliferation in GC, and suggest that CDH17 can serve as an attractive therapeutic target for future research.     

miR-195通过靶向调控趋化因子5促进胃癌细胞增殖、转移及侵袭的实验研究

目的:研究miR-195通过靶向调控趋化因子5促进胃癌细胞增殖、转移及侵袭的分子机制。方法:选取MGC803及NCI-N87细胞,根据转染不同分为:miR-NC组(空质粒),miR-195-mimics组(模拟序列)。实时荧光定量PCR法检测miR-195表达;MTT检测细胞增殖能力;Transwell侵袭实验检测细胞侵袭力;细胞划痕实验检测细胞转移能力;流式细胞术检测细胞凋亡情况;Western blot检测chemokine 5表达水平;Spearman相关分析miR-195及chemokine 5相关性。荧光素酶实验验证miR-195与chemokine 5的靶向关系。结果:miR-195-mimics组细胞miR-195水平高于miR-NC组(P0.05);miR-195 mimics组第1、2、3、4 d细胞活力低于miR-NC组(P0.05);miR-195 mimics组G1细胞高于miR-NC组,G2期、S期细胞低于miR-NC组,G2/S期细胞比值低于miR-NC组(P0.05);miR-195 mimics组划痕距离大于miR-NC组(P0.05);miR-195 mimics组细胞侵袭数低于miR-NC组(P0.05);miR-195-mimics组细chemokine 5蛋白含量低于miR-NC组(P0.05);miR-195 m RNA水平与chemokine 5蛋白含量负相关(r=-0.398,P=0.00);miR-195可直接靶向chemokine 5。结论:miR-195可通过靶向chemokine 5促进胃癌MGC803及NCI-N87细胞的增殖、转移及侵袭。     

NRG1 / ERBB3 signaling in melanocyte development and melanoma: inhibition of differentiation and promotion of proliferation

Neuregulin (NRG) signaling through the receptor tyrosine kinase, ERBB3, is required for embryonic development, and dysregulated signaling has been associated with cancer progression. Here, we show that NRG1/ERBB3 signaling inhibits melanocyte (MC) maturation and promotes undifferentiated, migratory and proliferative cellular characteristics. Embryonic analyses demonstrated that initial MC specification and distribution were not dependent on ERBB3 signaling. However NRG1/ERBB3 signaling was both necessary and sufficient to inhibit differentiation of later stages of MC development in culture. Analysis of tissue arrays of human melanoma samples suggests that ERBB3 signaling may also contribute to metastatic progression of melanoma as ERBB3 was phosphorylated in primary tumors compared with nevi or metastatic lesions. Neuregulin 1‐treated MCs demonstrated increased proliferation and invasion and altered morphology concomitant with decreased levels of differentiation genes, increased levels of proliferation genes and altered levels of melanoma progression and metastases genes. ERBB3 activation in primary melanomas suggests that NRG1/ERBB3 signaling may contribute to the progression of melanoma from benign nevi to malignancies. We propose that targeting ERBB3 activation and downstream genes identified in this study may provide novel therapeutic interventions for malignant melanoma.     

WWP2 regulates proliferation of gastric cancer cells in a PTEN-dependent manner

WW domain containing E3 Ub-protein ligase 2 (WWP2) plays an important role in tumor progression as an E3 ligase of PTEN. Here, we investigated the role of WWP2 in gastric cancer (GC). We found that WWP2 is overexpressed in GC tissues, which is closely related to poor prognosis of GC patients. Using a WWP2-shRNA lentivirus expressing system, we established WWP2 stable-knockdown GC cell lines and found that knockdown of WWP2 inhibits the proliferation of GC cells both in vitro and in vivo. Also, WWP2 silencing induced the up-regulation of PTEN protein level and down-regulation of AKT phosphorylation level. We further investigated the role of PTEN in this regulating process by performing rescue assay and found that PTEN is essential for WWP2-mediated regulation of GC cells proliferation. Taken together, our results demonstrated that WWP2 promotes proliferation of GC cells by downregulating PTEN, which may provide new therapeutic targets for GC.     

Association analysis of ERBB2 amplicon genetic polymorphisms and STARD3 expression with risk of gastric cancer in the Chinese population

The purpose of this study was to investigate whether risk of gastric cancer (GC) was associated with single nucleotide polymorphisms (SNPs) in a gene cluster on the chromosome 17q12-q21 (ERBB2 amplicon) in the Chinese Han population. We detected twenty-six SNPs in this gene cluster containing steroidogenic acute regulatory-related lipid transfer domain containing 3 (STARD3), protein phosphatase 1 regulatory subunit 1B (PPP1R1B/DARPP32), titin-cap (TCAP), per1-like domain containing 1(PERLD1/CAB2), human epidermal growth factor receptor-2 (ERBB2/HER2), zinc-finger protein subfamily 1A 3 (ZNFN1A3/IKZF3) and DNA topoisomerase 2-alpha (TOP2A) genes in 311 patients with GC and in 425 controls by Sequenom. We found no associations between genetic variations and GC risk. However, haplotype analysis implied that the haplotype CCCT of STARD3 (rs9972882, rs881844, rs11869286 and rs1877031) conferred a protective effect on the susceptibility to GC (P = 0.043, odds ratio [OR] = 0.805, 95% confidence intervals [95% CI] = 0.643–0.992). The STARD3 rs1877031 TC genotype endued histogenesis of gastric mucinous adenocarcinoma and signet-ring cell carcinoma (P = 0.021, OR = 2.882, 95% CI = 1.173–7.084). We examined the expression of STARD3 in 243 tumor tissues out of the 311 GC patients and 20 adjacent normal gastric tissues using immumohistochemical (IHC) analysis and tissue microarrays (TMA). The expression of STARD3 was observed in the gastric parietal cells and in gastric tumor tissues and significantly correlated with gender (P = 0.004), alcohol drinking (P < 0.001), tumor location (P = 0.007), histological type (P = 0.005) and differentiation (P = 0.023) in GC. We concluded that the combined effect of haplotype CCCT of STARD3 might affect GC susceptibility. STARD3 expression might be related to the tumorigenesis of GC in the Chinese population.     

NSUN2 modified by SUMO-2/3 promotes gastric cancer progression and regulates mRNA m5C methylation

The 5-methylcytosine (m5C) RNA methyltransferase NSUN2 is involved in the regulation of cell proliferation and metastasis formation and is upregulated in multiple cancers. However, the biological significance of NSUN2 in gastric cancer (GC) and the modification of NSUN2 itself have not been fully investigated. Here, we analyzed the expression level of NSUN2 in tissue microarrays containing 403 GC tissues by immunohistochemistry. NSUN2 was upregulated in GC, and that it was a predictor of poor prognosis. NSUN2 promotes the proliferation, migration, and invasion of GC cells in vitro. We also demonstrated that small ubiquitin-like modifier (SUMO)-2/3 interacts directly with NSUN2 by stabilizing it and mediating its nuclear transport. This facilitates the carcinogenic activity of NSUN2. Furthermore, m5C bisulfite sequencing (Bis-seq) in NSUN2-deficient GC cells showed that m5C-methylated genes are involved in multiple cancer-related signaling pathways. PIK3R1 and PCYT1A may be the target genes that participate in GC progression. Our findings revealed a novel mechanism by which NSUN2 functions in GC progression. This may provide new treatment options for GC patients.Subject terms: Gastric cancer, Post-translational modifications     

LncRNA LINC00470 promotes the degradation of PTEN mRNA to facilitate malignant behavior in gastric cancer cells

Gastric cancer (GC) is the fourth most frequent malignancy worldwide. Recently, long noncoding RNA (lncRNA) LINC00470 has been demonstrated to play an oncogenic role in human cancer. However, the clinical significance and functional role of LINC00470 in the progression of GC is largely unknown. In this study, our findings showed that LINC00470 was significantly upregulated in GC tissues and cell lines, and correlated with distant metastasis, TNM stage and poor prognosis. Overexpression and knockdown experiments revealed its oncogenic functions on GC cell proliferation, migration and invasion. Mechanistically, LINC00470 associated with PTEN mRNA and suppressed its stability through interaction with the N⁶-methyladenosine (m⁶A) writer METTL3. We also showed that LINC00470-METTL3-mediated PTEN mRNA degradation relied on the m⁶A reader protein YTHDF2-dependent pathway. Taken together, LINC00470 might serve as a therapeutic target for GC patients.     

Sigma ligands inhibit the growth of small cell lung cancer cells

The effects of sigma ligands on small cell lung cancer (SCLC) cells were investigated. 125I-N-(2-(piperidino)ethyl)-2-iodobenazmide (2-IBP) bound with high affinity to SCLC cell line NCI-H209 and NCI-N417. Specific 125I-2-IBP binding was inhibited with high affinity by ifendipine, haloperidol, (2-piperidinyl-aminoethyl)-4-iodobenzamide (IPAB) and 1,3-ditolylguanidine (DTG) with IC50 values of 3, 10, 15 and 90 nM respectively. In vitro, 10 microM 2-IBP, haloperidol or IPAB inhibited NCI-N417 proliferation using a MTT or clonogenic assay. In vivo, 4 mg/kg IPAB or 2-IBP inhibited NCI-N417 xenograft proliferation. 125I-2-IBP localized to the SCLC tumors after subcutaneous injection. These results suggest that sigma ligands may be utilized to localize and inhibit the proliferation of SCLC tumors.     

人EGFR显性负性突变体对胃癌细胞体内成瘤及淋巴结转移的影响

为了研究人表皮生长因子显性负性突变体(dominant negative mutant of EGFR, dnEGFR)对胃癌细胞体内成瘤及淋巴结转移的影响,用目的质粒pEGFPN1 dnEGFR,空质粒载体pEGFP N1分别转染胃癌细胞NCI-N87.筛选出稳定转染株,实验共分3组：NCI-N87细胞未转染组（untreated NCI-N87,UN组）,NCI-N87细胞pEGFP-N1转染组（EN组）;NCI-N87细胞pEGFPN1-dnEGFR转染组（DN组）.将3组细胞接种于裸鼠右后足垫,6周后测量移植瘤大小及对应腹股沟转移淋巴结数目,HE染色验证,real-time PCR和Western 印迹检测3组细胞中AKT1、MAPK3 mRNA和蛋白的表达改变.结果发现,DN组移植瘤较UN、EN组明显缩小（P<0.05）,且右腹股沟转移淋巴结数目较UN、EN组减少（P<0.05）.DN组细胞中,AKT1、MAPK3 mRNA和蛋白水平较UN、EN组降低（P<0.05）.提示pEGFPN1-dnEGFR可抑制裸鼠体内胃癌细胞成瘤及淋巴结转移,AKT1及MAPK3信号通路可能参与其中.