A Case Report of Germline Compound Heterozygous Mutations in the BRCA1 Gene of an Ovarian and Breast Cancer Patient.

The germline carrier of the BRCA1 pathogenic mutation has been well proven to confer an increased risk of breast and ovarian cancer. Despite BRCA1 biallelic pathogenic mutations being extremely rare, they have been reported to be embryonically lethal or to cause Fanconi anemia (FA). Here we describe a patient who was a 48-year-old female identified with biallelic pathogenic mutations of the BRCA1 gene, with no or very subtle FA-features. She was diagnosed with ovarian cancer and breast cancer at the ages of 43 and 44 and had a strong family history of breast and gynecological cancers.

Heat Shock Factor 1 Epigenetically Stimulates Glutaminase-1-Dependent mTOR Activation to Promote Colorectal Carcinogenesis.

Heat shock factor 1 (HSF1) generally exhibits its properties under stress conditions. In tumors, HSF1 has a pleiotropic feature in regulating growth, survival, and aggressiveness of cancer cells. In this study, we found HSF1 was increased in colorectal cancer (CRC) and had a positive correlation with shorter disease-free survival (DFS). Knockdown of HSF1 in CRC cells attenuated their growth while inhibiting mTOR activation and glutamine metabolism. HSF1 inhibited the expression of microRNA137 (MIR137), which targeted GLS1 (glutaminase 1), thus stimulating GLS1 protein expression to promote glutaminolysis and mTOR activation. HSF1 bound DNA methyltransferase DNMT3a and recruited it to the promoter of lncRNA MIR137 host gene (MIR137HG), suppressing the generation of primary MIR137. The chemical inhibitor of HSF1 also reduced cell growth, increased apoptosis, and impaired glutamine metabolism in vitro. Moreover, both chemical inhibition and genetic knockout of HSF1 succeeded in increasing MIR137 expression, reducing GLS1 expression, and alleviating colorectal tumorigenesis in azoxymethane (AOM)/dextran sulfate sodium (DSS) mice. In conclusion, HSF1 expression was increased and associated with poor prognosis in CRC. By recruiting DNMT3a to suppress the expression of MIR137 that targets GLS1 mRNA, HSF1 stimulated GLS1-dependent mTOR activation to promote colorectal carcinogenesis. Therefore, targeting HSF1 to attenuate glutaminolysis and mTOR activation could be a promising approach for CRC treatment.

Comprehensive spectrum of BRCA1 and BRCA2 deleterious mutations in breast cancer in Asian countries.

Approximately 5%-10% of breast cancers are due to genetic predisposition caused by germline mutations; the most commonly tested genes are BRCA1 and BRCA2 mutations. Some mutations are unique to one family and others are recurrent; the spectrum of BRCA1/BRCA2 mutations varies depending on the geographical origins, populations or ethnic groups. In this review, we compiled data from 11 participating Asian countries (Bangladesh, Mainland China, Hong Kong SAR, Indonesia, Japan, Korea, Malaysia, Philippines, Singapore, Thailand and Vietnam), and from ethnic Asians residing in Canada and the USA. We have additionally conducted a literature review to include other Asian countries mainly in Central and Western Asia. We present the current pathogenic mutation spectrum of BRCA1/BRCA2 genes in patients with breast cancer in various Asian populations. Understanding BRCA1/BRCA2 mutations in Asians will help provide better risk assessment and clinical management of breast cancer.

Germline RECQL mutations in high risk Chinese breast cancer patients.

Recently, RECQL was reported as a new breast cancer susceptibility gene. RECQL belongs to the RECQ DNA helicase family which unwinds double strand DNA and involved in the DNA replication stress response, telomere maintenance and DNA repair. RECQL deficient mice cells are prone to spontaneous chromosomal instability and aneuploidy, suggesting a tumor-suppressive role of RECQL in cancer. In this study, RECQL gene mutation screening was performed on 1110 breast cancer patients who were negative for BRCA1, BRCA2, TP53 and PTEN gene mutations and recruited from March 2007 to June 2015 in the Hong Kong Hereditary and High Risk Breast Cancer Program. Four different RECQL pathogenic mutations were identified in six of the 1110 (0.54 %) tested breast cancer patients. The identified mutations include one frame-shift deletion (c.974_977delAAGA), two splicing site mutations (c.394+1G>A, c.867+1G>T) and one nonsense mutation (c.796C>T, p.Gln266Ter). Two of the mutations (c.867+1G>T and p.Gln266Ter) were seen in more than one patients. This study provides the basis for existing of pathogenic RECQL mutations in Southern Chinese breast cancer patients. The significance of rare variants in RECQL gene in the estimation of breast cancer risk warranted further investigation in larger cohort of patients and in other ethnic groups.

miR-199a-5p confers tumor-suppressive role in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) remains a poor prognostic factor for breast cancer since no effective targeted therapy is readily available. Our previous studies confirmed miR-199a-5p is a TNBC-specific circulating biomarker, however, its functional roles in breast cancer is largely unknown. Thus, we investigated the functional implication of miR-199a-5p in TNBC and its potential underlying mechanisms. METHODS: MTT assay was performed to investigate the cell proliferation after transient transfection of miR-199a-5p in MDA-MB-231 cell line, followed by cell cycle analysis. Transwell invasion assay and wound healing assay were used to study the invasion and migration ability respectively. To further investigate the stemness-related characteristics of miR-199a-5p in breast cancer cells, single-cell clonogenic assay and aldehyde dehydrogenase (ALDH) assay were performed. 32 normal and 100 breast cancer patients' plasma were recruited to identify the potential circulating markers by qPCR. RESULTS: Cell proliferation assay revealed significant inhibition after miR-199a-5p ectopic expression (p < 0.0001), as a result of decreased S phase (p = 0.0284), increased G0/G1 phase (p = 0.0260) and apoptosis (p = 0.0374). Invasiveness (p = 0.0005) and wound healing ability were also decreased upon miR-199a-5p overexpression. It significantly altered EMT-related genes expression, namely CDH1, ZEB1 and TWIST. Single-cell clonogenic assay showed decreased colonies in miR-199a-5p (p = 0.0182). Significant downregulation (p = 0.0088) and inhibited activity (p = 0.0390) of ALDH was observed in miR-199a-5p. ALDH1A3, which is the dominant isoform of ALDH, is significantly upregulated in breast cancer plasma especially in TNBC (p = 0.0248). PIK3CD was identified as a potential downstream target of miR-199a-5p. CONCLUSIONS: Taken together, we unraveled, for the first time, the tumor-suppressive role of miR-199a-5p in TNBC, which attributed to EMT and cancer stemness properties, providing a novel therapeutic options towards this aggressive disease.

A new paradigm of genetic testing for hereditary breast/ovarian cancers.

INTRODUCTION: Genetic risk factors and family history play an important role in breast cancer development. This review aimed to summarise the current genetic testing approach to hereditary breast/ovarian cancer. METHODS: A systematic literature review was performed by searching the PubMed database. Publications available online until January 2015 that addressed issues related to hereditary breast/ovarian cancer genetic counselling/testing were selected. The search terms used were "familial breast/ovarian cancer", "susceptibility genes", "genetic counselling", and "genetic testing". The data extracted for this review were analysed by the authors, with a focus on genetic testing for hereditary breast/ovarian cancer. RESULTS: Although a greater proportion of inherited breast/ovarian cancers are due to the BRCA1 and BRCA2 mutations, a number of new genes have emerged as susceptibility candidates, including rare germline mutations in high penetrance genes, such as TP53 and PTEN, and more frequent mutations in moderate/low penetrance genes, such as PALB2, CHEK2 and ATM. Multi-gene testing, if used appropriately, is generally a more cost- and time-effective method than single-gene testing, and may increase the number of patients who can be offered personal surveillance, risk-reduction options, and testing of high-risk family members. CONCLUSIONS: Recent advances in molecular genetics testing have identified a number of susceptibility genes related to hereditary breast and/or ovarian cancers other than BRCA1 and BRCA2. The introduction of multi-gene testing for hereditary cancer has revolutionised the clinical management of high-risk patients and their families. Individuals with hereditary breast/ovarian cancer will benefit from genetic counselling/testing.

MicroRNAs are differentially deregulated in mammary malignant phyllodes tumour.

AIMS: MicroRNAs (miRs) have been shown to play important roles in tumour progression. Their expression pattern can be useful for cancer classification. However, little is known about miRs in mammary phyllodes tumours (PT). METHODS AND RESULTS: In this study, polymerase chain reaction (PCR)-based miR profiling was performed in a small PT cohort to identify deregulated miRs in malignant PT. The purported roles and targets of these miRs were further validated. Unsupervised clustering of miR expression profiling segregated PT into different grades, implicating the miR profile in PT classification. Among the deregulated miRs, miR-21, miR-335 and miR-155 were validated to be higher in malignant than in lower-grade PT in the independent cohort by quantitative PCR (qPCR) (P ≤ 0.032). Their expression correlated with some of the malignant histological features, including high stromal cellularity, nuclear pleomorphism and mitosis. Subsequent analysis of their downstream proteins, namely PTEN for miR-21/miR-155 and Rb for miR-335, also showed an independent significant negative association between miR and protein expression. CONCLUSIONS: Differential expression of miRs in PT could be useful in diagnosis and grading of PT. Their deregulated expression, together with the altered downstream targets, implicated their active involvement in PT malignant transformation.

MicroRNA-143 is downregulated in breast cancer and regulates DNA methyltransferases 3A in breast cancer cells.

MicroRNAs (miRNAs) are small non-protein-coding RNAs that regulate expression of a wide variety of genes including those involved in cancer development. Here, we investigate the role of miR-143 in breast cancer. In this study, we showed that miR-143 was frequently downregulated in 80% of breast carcinoma tissues compared to their adjacent noncancerous tissues. Ectopic expression of miR-143 inhibited proliferation and soft agar colony formation of breast cancer cells and also downregulated DNA methyltransferase 3A (DNMT3A) expression on both mRNA and protein levels. Restoration of miR-143 expression in breast cancer cells reduces PTEN hypermethylation and increases TNFRSF10C methylation. DNMT3A was demonstrated to be a direct target of miR-143 by luciferase reporter assay. Furthermore, miR-143 expression was observed to be inversely correlated with DNMT3A mRNA and protein expression in breast cancer tissues. Our findings suggest that miR-143 regulates DNMT3A in breast cancer cells. These findings elucidated a tumor-suppressive role of miR-143 in epigenetic aberration of breast cancer, providing a potential development of miRNA-based treatment for breast cancer.

NF-κB targets miR-16 and miR-21 in gastric cancer: involvement of prostaglandin E receptors.

Cigarette smoke is one of the risk factors for gastric cancer and nicotine has been reported to promote tumor growth. Deregulation of microRNA (miRNA) and cyclooxygenase-2 (COX-2) expressions are hallmarks of many cancers including gastric cancer. Here, we used an miRNA array platform covering a panel of 95 human miRNAs to examine the expression profile in nicotine-treated gastric cancer cells. We found that miR-16 and miR-21 were upregulated upon nicotine stimulation, transfection with anti-miR-16 or anti-miR-21 significantly abrogated cell proliferation. In contrast, ectopic miR-16 or miR-21 expression exhibited a similar stimulatory effect on cell proliferation as nicotine. Nicotine-mediated IkappaBα degradation and nuclear factor-kappa B (NF-κB) translocation dose-dependently. Knockdown of NF-κB by short interfering RNA (siRNA) or specific inhibitor (Bay-11-7085) markedly suppressed nicotine-induced cell proliferation and upregulation of miR-16 and miR-21. Interestingly, NF-κB-binding sites were located in both miR-16 and miR-21 gene transcriptional elements and we showed that nicotine enhanced the binding of NF-κB to the promoters of miR-16 and miR-21. Furthermore, activation of COX-2/prostaglandin E2 (PGE2) signaling in response to nicotine was mediated by the action of prostaglandin E receptors (EP2 and EP4). EP2 or EP4 siRNA or antagonists impaired the nicotine-mediated NF-κB activity, upregulation of miR-16 and miR-21 and cell proliferation. Taken together, these results suggest that miR-16 and miR-21 are directly regulated by the transcription factor NF-κB and yet nicotine-promoted cell proliferation is mediated via EP2/4 receptors. Perhaps this study may shed light on the development of anticancer drugs to improve the chemosensitivity in smokers.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone promoted gastric cancer growth through prostaglandin E receptor (EP2 and EP4) in vivo and in vitro.

Prostaglandin E (EP) receptor is positively related with COX-2, which is involved in cancer biology. A mechanistic study on how 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) promotes gastric carcinogenesis is lacking. Recently, we found that nicotine promoted tumor growth through upregulation of the COX-2/prostaglandin E(2) pathway. This extended our study on the involvement of EP receptors in gastric carcinogenesis. Both in vitro and in vivo studies showed that NNK promoted cancer cell growth with concomitant EP2 and EP4 upregulation. We found that NNK stimulated vascular endothelial growth factor (VEGF) and angiogenesis, but suppressed apoptosis by increasing Bcl2 and decreasing caspase-3 expressions. Both EP2 and EP4 siRNA significantly impaired these tumorigenic actions of NNK in xenograft tumor. Cell cycle analysis showed that NNK increased S phase entry with increased cyclin D1 and the associated cyclin-dependent kinase 4/6, and downregulation of p21 and p27. The p38 phosphorylation was EP2/4-dependent, and SB203580 (p38 inhibitor) suppressed NNK-induced prostaglandin E(2) , VEGF, and cell proliferation. Antagonists of EP2 or EP4 abolished the elevated VEGF and VEGF receptor-2. These data strongly indicate that EP2/4 are important for NNK-promoted gastric carcinogenesis, thus providing a framework for future evaluation of EP antagonist(s) as anticancer drugs for smokers.

Germline PALB2 Mutation in High-Risk Chinese Breast and/or Ovarian Cancer Patients.

The prevalence of the PALB2 mutation in breast cancer varies across different ethnic groups; hence, it is of intense interest to evaluate the cancer risk and clinical association of the PALB2 mutation in Chinese breast and/or ovarian cancer patients. We performed sequencing with a 6-gene test panel (BRCA1, BRCA2, TP53, PTEN, PALB2, and CDH1) to identify the prevalence of the PALB2 germline mutation among 2631 patients with breast and/or ovarian cancer. In this cohort, 39 mutations were identified with 24 types of mutation variants, where the majority of the mutations were frame-shift mutations and resulted in early termination. We also identified seven novel PALB2 mutations. Most of the PALB2 mutation carriers had breast cancer (36, 92.3%) and were more likely to have family history of breast cancer (19, 48.7%). The majority of the breast tumors were invasive ductal carcinoma (NOS type) (34, 81.0%) and hormonal positive (ER: 32, 84.2%; PR: 23, 60.5%). Pathogenic mutations of PALB2 were found in 39 probands with a mutation frequency of 1.6% and 1% in breast cancer and ovarian cancer patients, respectively. PALB2 mutation carriers were more likely have hormonal positive tumors and were likely to have familial aggregation of breast cancer.

Competing Risk Analyses of Medullary Carcinoma of Breast in Comparison to Infiltrating Ductal Carcinoma.

The aim of current study was to use competing risk model to assess whether medullary carcinoma of the breast (MCB) has a better prognosis than invasive ductal carcinomas of breast cancer (IDC), and to build a competing risk nomogram for predicting the risk of death of MCB. We involved 3,580 MCB patients and 319,566 IDC patients from Surveillance, Epidemiology, and End Results (SEER) database. IDC was found to have a worse BCSS than MCB (Hazard ratio (HR) > 1, p < 0.001). The 5-year cumulative incidences of death (CID) was higher in IDC than MCB (p < 0.001). Larger tumor size, increasing number of positive lymph nodes and unmarried status were found to worsen the BCSS of MCB (HR > 1, p < 0.001). We found no association between ER, PR, radiotherapy or chemotherapy and MCB prognosis (p > 0.05). After a penalized variable selection process, the SH model-based nomogram showed moderate accuracy of prediction by internal validation of discrimination and calibration with 1,000 bootstraps. In summary, MCB patients had a better prognosis than IDC patients. Interestingly, unmarried status in addition to expected risk factors such as larger tumor size and increasing number of positive lymph nodes were found to worsen the BCSS of MCB. We also established a competing risk nomogram as an easy-to-use tool for prognostic estimation of MCB patients.

Germline Mutation in 1338 BRCA-Negative Chinese Hereditary Breast and/or Ovarian Cancer Patients: Clinical Testing with a Multigene Test Panel.

Differences in the mutation spectrum across ethnicities suggest the importance of identifying genes in addition to common high penetrant genes to estimate the associated breast cancer risk in China. A total of 1338 high-risk breast cancer patients who tested negative for germline BRCA1, BRCA2, TP53, and PTEN mutations between 2007 and 2017 were selected from the Hong Kong Hereditary Breast Cancer Family Registry. Patient samples were subjected to next-generation DNA sequencing using a multigene panel (Color Genomics). All detected pathogenic variants were validated by bidirectional DNA sequencing. The sequencing data were coanalyzed by a bioinformatics pipeline developed in-house. Sixty-one pathogenic variants (4.6%) were identified in this cohort in 11 cancer predisposition genes. Most carriers (77.1%) had early onset of breast cancer (age <45 years), 32.8% had family members with breast cancer, and 11.5% had triple-negative breast cancer. The most common mutated genes were PALB2 (1.4%), RAD51D (0.8%), and ATM (0.8%). A total of 612 variants of unknown significance were identified in 494 patients, and 87.4% of the variants of unknown significance were missense mutations. Pathogenic variants in cancer predisposition genes beyond BRCA1, BRCA2, TP53, and PTEN were detected in an additional 4.6% of patients using the multigene panel. PALB2 (1.4%) and RAD51D (0.8%) were the most commonly mutated genes in patients who tested mutation negative by a four-gene panel.

Impaired autophagic degradation of lncRNA ARHGAP5-AS1 promotes chemoresistance in gastric cancer.

Chemoresistance remains the uppermost disincentive for cancer treatment on account of many genetic and epigenetic alterations. Long non-coding RNAs (lncRNAs) are emerging players in promoting cancer initiation and progression. However, the regulation and function in chemoresistance are largely unknown. Herein, we identified ARHGAP5-AS1 as a lncRNA upregulated in chemoresistant gastric cancer cells and its knockdown reversed chemoresistance. Meanwhile, high ARHGAP5-AS1 expression was associated with poor prognosis of gastric cancer patients. Intriguingly, its abundance is affected by autophagy and SQSTM1 is responsible for transporting ARHGAP5-AS1 to autophagosomes. Inhibition of autophagy in chemoresistant cells, thus, resulted in the upregulation of ARHGAP5-AS1. In turn, it activated the transcription of ARHGAP5 in the nucleus by directly interacting with ARHGAP5 promoter. Interestingly, ARHGAP5-AS1 also stabilized ARHGAP5 mRNA in the cytoplasm by recruiting METTL3 to stimulate m6A modification of ARHGAP5 mRNA. As a result, ARHGAP5 was upregulated to promote chemoresistance and its upregulation was also associated with poor prognosis in gastric cancer. In summary, impaired autophagic degradation of lncRNA ARHGAP5-AS1 in chemoresistant cancer cells promoted chemoresistance. It can activate the transcription of ARHGAP5 in the nucleus and stimulate m6A modification of ARHGAP5 mRNA to stabilize ARHGAP5 mRNA in the cytoplasm by recruiting METTL3. Therefore, targeting ARHGAP5-AS1/ARHGAP5 axis might be a promising strategy to overcome chemoresistance in gastric cancer.

Breast and ovarian cancer penetrance of BRCA1/2 mutations among Hong Kong women.

Germline mutations in BRCA1 and BRCA2 (BRCA1/2) are associated with increased risk of breast and ovarian cancer. The penetrance of breast and ovarian cancer in BRCA1/2 mutation carriers has been well characterized in Caucasian but not in Asian. Two studies have investigated the breast cancer risk in Asian women with BRCA1/2 mutations, and no published estimates are available for ovarian cancer. Therefore, we estimated the age-specific cumulative risk of BRCA1/2-associated breast and ovarian cancer in Chinese women. From Jan 2007 to Nov 2015, the Hong Kong Hereditary Breast Cancer Family Registry identified 1635 families with hereditary breast-ovarian cancer. Among probands in these families, 66 had BRCA1 mutations, 84 had BRCA2 mutations, and 1,485 tested negative for BRCA1/2 mutations. Using the female first-degree relatives of these probands, we estimated the risk of breast and ovarian cancer using a modified marginal likelihood approach. Estimates of breast cancer penetrance by age 70 were 53.7% (95% CI 34.5-71.6%) for BRCA1 mutation carriers and 48.3% (95% CI 31.8-68.5%) for BRCA2. The estimated risk of ovarian cancer by age 70 was 21.5% and 7.3% for Chinese women carrying BRCA1 or BRCA2 mutation respectively. A meta-analysis of available studies in Asian women revealed pooled estimates of breast cancer risk by age 70 of 44.8% (95% CI 33-57.2%) and 40.7% (95% CI 31.3-50.9%) for BRCA1 and BRCA2 mutation carriers respectively. These data suggest that BRCA1/2-associated breast cancer risk for Chinese women is similar to that for Caucasian women, although BRCA1/2-associated ovarian cancer risks are lower for Chinese women.

Rapid detection of BRCA1/2 recurrent mutations in Chinese breast and ovarian cancer patients with multiplex SNaPshot genotyping panels.

BRCA1/2 mutations are significant risk factors for hereditary breast and ovarian cancer (HBOC), its mutation frequency in HBOC of Chinese ethnicity is around 9%, in which nearly half are recurrent mutations. In Hong Kong and China, genetic testing and counseling are not as common as in the West. To reduce the barrier of testing, a multiplex SNaPshot genotyping panel that targeted 25 Chinese BRCA1/2 mutation hotspots was developed, and its feasibility was evaluated in a local cohort of 441 breast and 155 ovarian cancer patients. For those who tested negative, they were then subjected to full-gene testing with next-generation sequencing (NGS). BRCA mutation prevalence in this cohort was 8.05% and the yield of the recurrent panel was 3.52%, identifying over 40% of the mutation carriers. Moreover, from 79 Chinese breast cancer cases recruited overseas, 2 recurrent mutations and one novel BRCA2 mutation were detected by the panel and NGS respectively. The developed genotyping panel showed to be an easy-to-perform and more affordable testing tool that can provide important contributions to improve the healthcare of Chinese women with cancer as well as family members that harbor high risk mutations for HBOC.

Probiotic Lactobacillus rhamnosus GG enhances gastric ulcer healing in rats.

Probiotics are widely used as functional foods which have been advocated for the maintenance of gastrointestinal microflora equilibrium and treatment of gastrointestinal disorders. However, studying the role of probiotics in peptic ulcer disease is limited. The aim of the present study is to investigate the effect of a probiotic strain Lactobacillus rhamnosus GG on gastric ulcer and to elucidate the mechanisms involved. Gastric kissing ulcers were induced in rats by acetic acid (60% v/v). L. rhamnosus GG was given intragastrically at 10(8) cfu/day or 10(9) cfu/day for three consecutive days after ulcer induction. L. rhamnosus GG successfully colonized in the gastric mucosa especially at the ulcer margin. It also significantly and dose-dependently reduced gastric ulcer area. Cell apoptosis to cell proliferation ratio was strongly decreased and accompanied by significant up-regulation of ornithine decarboxylase (ODC) and B-cell lymphoma 2 (Bcl-2) protein expression at the ulcer margin. Angiogenesis was also significantly stimulated together with the induction of vascular endothelial growth factor (VEGF) expression. Furthermore, L. rhamnosus GG up-regulated the phosphorylation level of epidermal growth factor receptor (EGF receptor) without altering the total EGF receptor expression. These findings suggested that L. rhamnosus GG enhanced gastric ulcer healing via the attenuation of cell apoptosis to cell proliferation ratio and increase in angiogenesis. Regulators of these processes such as ODC, Bcl-2, VEGF and EGF receptor are likely to be involved in the healing action of L. rhamnosus GG for gastric ulcer.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone from cigarette smoke stimulates colon cancer growth via beta-adrenoceptors.

Cigarette smoking is a risk factor for colorectal cancer. It is suggested that 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, mediates the carcinogenic action of cigarette smoking by promoting cancer growth. In the present study, the proliferative response of a cultured colon cancer cell line HT-29 to NNK was determined. It was found that NNK dose-dependently stimulated HT-29 cell proliferation. In this regard, the stimulatory action of NNK was abolished by atenolol and ICI 118,551, a beta1- and beta2-selective antagonist, respectively. In addition, cell growth was stimulated by the nonselective adrenergic agonist, noradrenaline, and more effectively by the beta-selective agonist, isoproterenol. The second message cyclic AMP level for beta-adrenoceptor activation was elevated by isoproterenol and NNK treatment. These agents also up-regulated cyclooxygenase-2 expression, cytosolic phospholipase A2 expression, and prostaglandin E2 release. Beta2-adrenoceptor blockade with ICI 118,551, in contrast, significantly decreased cyclooxygenase-2 expression, cytosolic phospholipase A2 expression and prostaglandin E2 release induced by NNK and isoproterenol. To conclude, it is proposed that NNK stimulates HT-29 cell proliferation through beta-adrenoceptors, preferentially beta2 receptors. Activation of the beta-adrenoceptors, and the consequent cyclic AMP elevation coupled with the downstream arachidonic acid pathway, is perhaps an important mechanistic cascade in the promotion of colon cancer growth. These findings partly elucidate the carcinogenic actions of cigarette smoke and shed new light on the novel modulatory role of beta-adrenoceptors in the development of colon cancer.

Nicotine induces cyclooxygenase-2 and vascular endothelial growth factor receptor-2 in association with tumor-associated invasion and angiogenesis in gastric cancer.

Blockade of angiogenesis is a promising strategy to suppress tumor growth, invasion, and metastasis. Vascular endothelial growth factor (VEGF), which binds to tyrosine kinase receptors [VEGF receptors (VEGFR) 1 and 2], is the mediator of angiogenesis and mitogen for endothelial cells. Cyclooxygenase-2 (COX-2) plays an important role in the promoting action of nicotine on gastric cancer growth. However, the action of nicotine and the relationship between COX-2 and VEGF/VEGFR system in tumorigenesis remain undefined. In this study, the effects of nicotine in tumor angiogenesis, invasiveness, and metastasis were studied with sponge implantation and Matrigel membrane models. Nicotine (200 microg/mL) stimulated gastric cancer cell proliferation, which was blocked by SC-236 (a highly selective COX-2 inhibitor) and CBO-P11 (a VEGFR inhibitor). This was associated with decreased VEGF levels as well as VEGFR-2 but not VEGFR-1 expression. Topical injection of nicotine enhanced tumor-associated vascularization, with a concomitant increase in VEGF levels in sponge implants. Again, application of SC-236 (2 mg/kg) and CBO-P11 (0.4 mg/kg) partially attenuated vascularization by approximately 30%. Furthermore, nicotine enhanced tumor cell invasion through the Matrigel membrane by 4-fold and promoted migration of human umbilical vein endothelial cells in a cocultured system with gastric cancer cells. The activity of matrix metalloproteinases 2 and 9 and protein expressions of plasminogen activators (urokinase-type plasminogen activator and its receptor), which are the indicators of invasion and migration processes, were increased by nicotine but blocked by COX-2 and VEGFR inhibitors. Taken together, our results reveal that the promoting action of nicotine on angiogenesis, tumor invasion, and metastasis is COX-2/VEGF/VEGFR dependent.