Association between being breastfed in infancy and adult colorectal cancer risk among Japanese men and women.

It has been postulated that being breastfed in infancy affects not only health status in childhood but also disease risk in adulthood. To investigate the association of being breastfed with the risks of adult colorectal cancer and benign tumor, we conducted a case-control study including 1190 colorectal cancer and 1585 benign tumor cases and 5301 controls, admitted to a single hospital in Miyagi Prefecture, Japan, between 1997 and 2013. History of having been breastfed was assessed using a self-administered questionnaire, and odds ratios (ORs) were estimated using unconditional logistic regression. There was no association between being breastfed and colorectal cancer risk (breastfed versus formula-only fed, OR = 1.21; 95% CI 0.87-1.67). There was also no association with the risk of benign tumor (OR = 1.04). On the other hand, analyses stratified by sex and birth year found heterogeneous associations. Women born after 1950 who had been breastfed tended to have increased risks of colorectal cancer (OR = 1.58) and benign tumor (OR = 1.51) relative to those who had been formula-only fed, although not statistically significant. In men born after 1950, being breastfed was associated with a significantly decreased risk of benign tumor (OR = 0.57; 95% CI 0.33-0.98).

Associations of cigarette smoking, alcohol drinking and body mass index with survival after colorectal cancer diagnosis by anatomic subsite: a prospective patient cohort study in Japan.

BACKGROUND: Cigarette smoking, alcohol drinking and obesity are known to be risk factors for colorectal cancer. These factors may affect survival after diagnosis, but evidence has been inconsistent. We investigated subsite-specific associations between prediagnosis smoking, alcohol drinking and body mass index and survival in colorectal cancer. METHODS: Subjects were 1300 patients (colon 778; rectum 502; concurrent 20) with histologically confirmed colorectal cancer diagnosed during 1997-2013 at a single institution in Japan. Histories of smoking and alcohol drinking, height and prediagnosis weight were assessed using a self-administered questionnaire. Using Cox proportional hazards model, hazard ratios and 95% confidence intervals of mortality were estimated. RESULTS: During a median follow-up period of 6.7 years, 479 deaths were documented. Ever-smoking was associated with an increased risk of all-cause death among patients with colon cancer (hazard ratio: 1.47; 95% confidence interval: 1.07-2.02 compared with never-smoking). According to colon subsite, this increased risk was clear in patients with proximal colon cancer (hazard ratio: 2.09; 95% confidence interval: 1.28-3.40). There was no association between smoking and rectal cancer survival. Alcohol drinking was not associated with survival for either colon or rectal cancer. Among patients with rectal cancer, higher body mass index was associated with a lower risk of all-cause (Ptrend = 0.0006) and disease-specific death (Ptrend = 0.02). For colon cancer, lower body mass index tended to be associated with a higher risk of all-cause death (Ptrend = 0.05). CONCLUSIONS: The results indicate that lifestyles identified as risk factors for colorectal cancer may impact differently on patient survival according to anatomic subsite.

Tumor Microenvironment in Mixed Neuroendocrine Non-Neuroendocrine Neoplasms: Interaction between Tumors and Immune Cells, and Potential Effects of Neuroendocrine Differentiation on the Tumor Microenvironment.

The tumor microenvironment is considered to play a pivotal role in various human malignancies. Neuroendocrine and non-neuroendocrine neoplasms are considered to have different tumor microenvironments. However, owing to differences in the systemic and/or local immune statuses, tumor microenvironments in different patients may be difficult to compare. Mixed neuroendocrine non-neuroendocrine neoplasms (MiNENs), although rare, could be useful for exploring the effects of neuroendocrine differentiation on the tumor microenvironment, because both neuroendocrine and non-neuroendocrine components are present in the same tumor. Here, we examined 33 cases of histologically confirmed MiNENs and evaluated the influence of neuroendocrine differentiation on the tumor microenvironment by comparing tumor-infiltrating lymphocytes, tumor-associated macrophages, and other relevant factors in the two components the same tumor. The immunoreactivity of those examined above was evaluated quantitatively. The values of vasohibin-1-positive density (p < 0.0001) and immunoreactivity (p < 0.0001) (representing the neoangiogenesis status) were significantly higher in neuroendocrine as compared to non-neuroendocrine areas of the same tumors. In addition, the Foxp3/CD8 (p = 0.0717) and the PD-1/CD8 ratios (p = 0.0176) (representing tumor immunity suppression) tend to increase in neuroendocrine carcinomas. Immunoreactivity of CD163, a marker of M2-like macrophages, was also higher in the neuroendocrine areas. Our findings indicate that neuroendocrine and non-neuroendocrine tumors differ from each other with respect to the characteristics of both tumor cells and the tumor microenvironment.

PP6 deficiency in mice with KRAS mutation and Trp53 loss promotes early death by PDAC with cachexia-like features.

To examine effects of PP6 gene (Ppp6c) deficiency on pancreatic tumor development, we developed pancreas-specific, tamoxifen-inducible Cre-mediated KP (KRAS(G12D) plus Trp53-deficient) mice (cKP mice) and crossed them with Ppp6cflox / flox mice. cKP mice with the homozygous Ppp6c deletion developed pancreatic tumors, became emaciated and required euthanasia within 150 days of mutation induction, phenotypes that were not seen in heterozygous or wild-type (WT) mice. At 30 days, a comparative analysis of genes commonly altered in homozygous versus WT Ppp6c cKP mice revealed enhanced activation of Erk and NFκB pathways in homozygotes. By 80 days, the number and size of tumors and number of precancerous lesions had significantly increased in the pancreas of Ppp6c homozygous relative to heterozygous or WT cKP mice. Ppp6c-/- tumors were pathologically diagnosed as pancreatic ductal adenocarcinoma (PDAC) undergoing the epithelial-mesenchymal transition (EMT), and cancer cells had invaded surrounding tissues in three out of six cases. Transcriptome and metabolome analyses indicated an enhanced cancer-specific glycolytic metabolism in Ppp6c-deficient cKP mice and the increased expression of inflammatory cytokines. Individual Ppp6c-/- cKP mice showed weight loss, decreased skeletal muscle and adipose tissue, and increased circulating tumor necrosis factor (TNF)-α and IL-6 levels, suggestive of systemic inflammation. Overall, Ppp6c deficiency in the presence of K-ras mutations and Trp53 gene deficiency promoted pancreatic tumorigenesis with generalized cachexia and early death. This study provided the first evidence that Ppp6c suppresses mouse pancreatic carcinogenesis and supports the use of Ppp6c-deficient cKP mice as a model for developing treatments for cachexia associated with pancreatic cancer.

Ppp6c deficiency accelerates K-rasG12D -induced tongue carcinogenesis.

BACKGROUND: Effective treatments for cancer harboring mutant RAS are lacking. In Drosophila, it was reported that PP6 suppresses tumorigenicity of mutant RAS. However, the information how PP6 regulates oncogenic RAS in mammals is limited. METHODS: We examined the effects of PP6 gene (Ppp6c) deficiency on tongue tumor development in K (K-rasG12D)- and KP (K-rasG12D + Trp53-deficient)-inducible mice. RESULTS: Mice of K and KP genotypes developed squamous cell carcinoma in situ in the tongue approximately 2 weeks after the induction of Ppp6c deficiency and was euthanized due to 20% loss of body weight. Transcriptome analysis revealed significantly different gene expressions between tissues of Ppp6c-deficient tongues and those of Ppp6c wild type, while Trp53 deficiency had a relatively smaller effect. We then analyzed genes commonly altered by Ppp6c deficiency, with or without Trp53 deficiency, and identified a group concentrated in KEGG database pathways defined as 'Pathways in Cancer' and 'Cytokine-cytokine receptor interaction'. We then evaluated signals downstream of oncogenic RAS and those regulated by PP6 substrates and found that in the presence of K-rasG12D, Ppp6c deletion enhanced the activation of the ERK-ELK1-FOS, AKT-4EBP1, and AKT-FOXO-CyclinD1 axes. Ppp6c deletion combined with K-rasG12D also enhanced DNA double-strand break (DSB) accumulation and activated NFκB signaling, upregulating IL-1ß, COX2, and TNF.

Ppp6c haploinsufficiency accelerates UV-induced BRAF(V600E)-initiated melanomagenesis.

According to TCGA database, mutations in PPP6C (encoding phosphatase PP6) are found in c. 10% of tumors from melanoma patients, in which they coexist with BRAF and NRAS mutations. To assess PP6 function in melanoma carcinogenesis, we generated mice in which we could specifically induce BRAF(V600E) expression and delete Ppp6c in melanocytes. In these mice, melanoma susceptibility following UVB irradiation exhibited the following pattern: Ppp6c semi-deficient (heterozygous) > Ppp6c wild-type > Ppp6c-deficient (homozygous) tumor types. Next-generation sequencing of Ppp6c heterozygous and wild-type melanoma tumors revealed that all harbored Trp53 mutations. However, Ppp6c heterozygous tumors showed a higher Signature 1 (mitotic/mitotic clock) mutation index compared with Ppp6c wild-type tumors, suggesting increased cell division. Analysis of cell lines derived from either Ppp6c heterozygous or wild-type melanoma tissues showed that both formed tumors in nude mice, but Ppp6c heterozygous tumors grew faster compared with those from the wild-type line. Ppp6c knockdown via siRNA in the Ppp6c heterozygous line promoted the accumulation of genomic damage and enhanced apoptosis relative to siRNA controls. We conclude that in the presence of BRAF(V600E) expression and UV-induced Trp53 mutation, Ppp6c haploinsufficiency promotes tumorigenesis.

BEX2 suppresses mitochondrial activity and is required for dormant cancer stem cell maintenance in intrahepatic cholangiocarcinoma.

Cancer stem cells (CSCs) define a subpopulation of cancer cells that are resistant to therapy. However, little is known of how CSC characteristics are regulated. We previously showed that dormant cancer stem cells are enriched with a CD274low fraction of cholangiocarcinoma cells. Here we found that BEX2 was highly expressed in CD274low cells, and that BEX2 knockdown decreased the tumorigenicity and G0 phase of cholangiocarcinoma cells. BEX2 was found to be expressed predominantly in G0 phase and starvation induced the USF2 transcriptional factor, which induced BEX2 transcription. Comprehensive screening of BEX2 binding proteins identified E3 ubiquitin ligase complex proteins, FEM1B and CUL2, and a mitochondrial protein TUFM, and further demonstrated that knockdown of BEX2 or TUFM increased mitochondria-related oxygen consumption and decreased tumorigenicity in cholangiocarcinoma cells. These results suggest that BEX2 is essential for maintaining dormant cancer stem cells through the suppression of mitochondrial activity in cholangiocarcinoma.

Associations of Japanese food intake with survival of stomach and colorectal cancer: A prospective patient cohort study.

Dietary factors may affect the prognosis of digestive tract cancer, but evidence has been sparse. We investigated the association between pretreatment intake of 6 Japanese foods (including soy food, miso [soybean paste] soup and seaweed) and the risk of death among patients with histologically confirmed major digestive tract cancers (stomach, 1931; colon, 793; rectum, 510) diagnosed during 1997-2013 at a single institution in Japan. Pretreatment dietary intake was assessed using a food frequency questionnaire, and the patients were followed until December 2016. The Cox proportional hazards model was used to estimate hazard ratios (HR) and 95% confidence intervals (CI). Among the patients with stomach cancer, frequent intake of soy food was inversely associated with the risk of all-cause (Ptrend for four frequency groups = 0.01; HR = 0.72, 95% CI: 0.50-1.04 for highest vs lowest group) and stomach cancer (Ptrend  = 0.03; HR = 0.63, 95% CI: 0.40-0.99) death. A similar inverse association was also found for intake of miso soup. In contrast, frequent seaweed intake was inversely associated with the risk of all-cause death among the patients with colon cancer (Ptrend  = 0.03). Rectal cancer patients who had frequently consumed seaweed tended to have a lower risk of rectal cancer death (Ptrend  = 0.02). These findings indicate that pretreatment intake of Japanese foods such as soybean products and seaweed may have favorable effects on patient survival of stomach and colorectal cancer, although this needs to be confirmed by further research.

Efficacy of oral administration of cystine and theanine in colorectal cancer patients undergoing capecitabine-based adjuvant chemotherapy after surgery: a multi-institutional, randomized, double-blinded, placebo-controlled, phase II trial (JORTC-CAM03).

PURPOSE: Capecitabine-based adjuvant chemotherapy for colorectal cancer patients often causes adverse events (AEs), such as diarrhea, stomatitis, anorexia, and hand-foot syndrome (HFS). Cystine and theanine were reported to attenuate some chemotherapy-associated AEs, and hence are also expected to attenuate capecitabine-induced AEs. Therefore, we aimed to investigate the safety and efficacy of cystine/theanine treatment in colorectal cancer patients undergoing capecitabine-based adjuvant chemotherapy after surgery. METHODS: A total of 100 colorectal cancer patients treated with capecitabine as an adjuvant chemotherapy after surgery were randomly allocated into the cystine/theanine group (n = 52) or the placebo group (n = 48). The primary endpoint was incidence rate of diarrhea of grade 1 or higher in accordance with the Common Terminology Criteria for AEs (CTCAE) v.4.0, Japanese Clinical Oncology Group (JCOG) version. The secondary endpoints included incidence rates of other AEs (CTCAE v.4.0-JCOG), as well as the incidence rate of HFS according to the HFS grading scale. RESULTS: There were no significant differences in capecitabine-induced AEs between the two groups. However, the incidence rate of diarrhea of grade 1 or higher tended to be lower in the cystine/theanine group than the placebo group (18.4% vs. 28.9%, p = 0.169) as well as the incidence rate of HFS of grade 1 or higher (CTCAE v.4.0-JCOG or HFS grading scale) (67.4% vs. 77.8%, p = 0.185, 67.3% vs. 80.0%, p = 0.124, respectively). CONCLUSION: This trial demonstrated that cystine/theanine treatment of colorectal cancer patients undergoing capecitabine-based adjuvant chemotherapy after surgery is safe and has the tendency to reduce the incidence rate of diarrhea or HFS. TRIAL REGISTRATION: UMIN000024784.

Loss of protein phosphatase 6 in mouse keratinocytes enhances K-rasG12D -driven tumor promotion.

Here, we address the function of protein phosphatase 6 (PP6) loss on K-ras-initiated tumorigenesis in keratinocytes. To do so, we developed tamoxifen-inducible double mutant (K-rasG12D -expressing and Ppp6c-deficient) mice in which K-rasG12D expression is driven by the cytokeratin 14 (K14) promoter. Doubly-mutant mice showed early onset tumor formation in lips, nipples, external genitalia, anus and palms, and had to be killed by 3 weeks after induction by tamoxifen, while comparably-treated K-rasG12D -expressing mice did not. H&E-staining of lip tumors before euthanasia revealed that all were papillomas, some containing focal squamous cell carcinomas. Immunohistochemical analysis of lips of doubly-mutant vs K-rasG12D mice revealed that cell proliferation and cell size increased approximately 2-fold relative to K-rasG12D -expressing mutants, and epidermal thickness of lip tissue greatly increased relative to that seen in K-rasG12D -only mice. Moreover, AKT phosphorylation increased in K-rasG12D -expressing/Ppp6c-deficient cells, as did phosphorylation of the downstream effectors 4EBP1, S6 and GSK3, suggesting that protein synthesis and survival signals are enhanced in lip tissues of doubly-mutant mice. Finally, increased numbers of K14-positive cells were present in the suprabasal layer of doubly-mutant mice, indicating abnormal keratinocyte differentiation, and γH2AX-positive cells accumulated, indicating perturbed DNA repair. Taken together, Ppp6c deficiency enhances K-rasG12D -dependent tumor promotion.

Associations of cigarette smoking and alcohol drinking with stomach cancer survival: A prospective patient cohort study in Japan.

Cigarette smoking and alcohol drinking may affect the prognosis of stomach cancer, but evidence has been inconsistent. We investigated the associations between pretreatment smoking and alcohol drinking and the risk of all-cause and stomach cancer death among 1,576 patients with histologically confirmed stomach cancer diagnosed during 1997-2010 at a single hospital in Japan. Histories of smoking and alcohol drinking were assessed using a self-administered questionnaire. The patients were followed until December 31, 2013. The Cox proportional hazards model was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). During 9,625.1 person-years, 670 all-cause and 419 stomach cancer deaths were documented. Among the patients overall, ever-drinking was significantly associated with an increased risk of all-cause death (HR: 1.25; 95% CI: 1.03-1.51), but not stomach cancer death. Positive linear associations with the frequency of drinking (ptrend = 0.02) and the amount of alcohol consumed per day (ptrend = 0.03) were observed for the risk of all-cause death. Ever-smoking was not related to either the risk of all-cause or stomach cancer death. Conversely, among the patients who underwent curative resection, a significant positive association was found between ever-smoking and the risk of stomach cancer death (HR: 2.44; 95% CI: 1.17-5.08). A positive association was also found for earlier age at start of smoking (ptrend = 0.0046). Pretreatment smoking and alcohol drinking have significant effects on stomach cancer survival. Lifestyle adjustments throughout life may improve survival.

Pyruvate kinase type M2 contributes to the development of pancreatic ductal adenocarcinoma by regulating the production of metabolites and reactive oxygen species.

The majority of cancer cells maintain a high glycolytic activity and an increased lactate production, even in a well oxygenated environment. This phenomenon is known as the Warburg effect. Previous studies have revealed that various types of cancer selectively express the pyruvate kinase M2 isoform (PKM2), and that PKM2 plays a pivotal role in the Warburg effect. Although elevated PKM2 levels have been observed in pancreatic cancer and other types of cancer, little is known about the biological function of PKM2. In this study, in order to examine the expression and role of PKM2 in pancreatic ductal adenocarcinoma (PDAC), we knocked down PKM2 in PDAC cells by introducing small interfering and short hairpin RNAs, and examined the gene expression profiles in the cells by microarray analysis. We analyzed the energy-producing pathways in the cells by XFe Extracellular Flux Analyzers, and detected intracellular metabolites by capillary electrophoresis time-of-flight mass spectrometry. We found that the RNAi-mediated knockdown of PKM2 diminished the proliferative, migratory and tumorigenic ability of the PDAC cell-lines. PKM2 knockdown also resulted in lower glycolytic activities and decreased levels of some intracellular metabolites, such as pyruvate and polyamine; however, it led to elevated levels of reactive oxygen species. Microarray analysis revealed the functional association between PKM2 and the expression of genes that drive the cell cycle. On the whole, the findings of this study demonstrate that PKM2 plays an important role in metabolic activities, as well as in the malignancy of PDAC cells.

Drug delivery of oral anti-cancer fluoropyrimidine agents.

INTRODUCTION: Sixty years since its introduction, 5-FU still forms the core of chemotherapy regimens for many types of malignancies. 5-FU is a time-dependent drug but is rapidly degraded in plasma by dihydropyrimidine dehydrogenase (DPD). Although originally developed in an intravenous form, 5-FU oral prodrugs were developed with the goal of improving efficacy and minimizing toxicity as well as to capitalize on the advantages of oral drug administration. The inactive 5-FU prodrug is gradually converted into the active form in the systemic circulation. UFT, S-1, and capecitabine are oral 5-FU prodrugs currently in clinical use. However, the efficacy of 5-FU can be further improved by its combination with DPD inhibitors and biochemical modulators, such as uracil and leucovorin, in addition to modifying administration schedules. Areas covered: We focused on the drug delivery of oral 5-FU prodrugs, their pharmacokinetics, and the development of DPD inhibitors. Since oral 5-FU prodrugs have been formulated into combination drugs, we also discussed the regulatory approval of combination drugs. Expert opinion: Many regimens that include intravenously administered 5-FU can be replaced by oral 5-FU prodrugs. Patients would benefit from development of combination 5-FU oral prodrug formulations and its associated path through the combination drug regulatory approval process.

Enhanced expression of the M2 isoform of pyruvate kinase is involved in gastric cancer development by regulating cancer-specific metabolism.

Recent studies have indicated that increased expression of the M2 isoform of pyruvate kinase (PKM2) is involved in glycolysis and tumor development. However, little is known about the role of PKM2 in gastric cancer (GC). Therefore, we examined the expression and function of PKM2 in human GC. We evaluated PKM1 and PKM2 expression by quantitative RT-PCR in gastric tissues from 10 patients who underwent gastric endoscopic submucosal dissection, 80 patients who underwent gastrectomy, and seven healthy volunteers, and analyzed the correlation with clinicopathological variables. To assess the function of PKM2, we generated PKM2-knockdown GC cells, and investigated the phenotypic changes. Furthermore, we examined the induction of PKM2 expression by cytotoxin-associated gene A (CagA), a pathogenic factor of Helicobacter pylori, using CagA-inducible GC cells. We found that PKM2 was predominantly expressed not only in GC lesions but also in the normal gastric regions of GC patients and in the gastric mucosa of healthy volunteers. The PKM2 expression was significantly higher in carcinoma compared to non-cancerous tissue and was associated with venous invasion. Knockdown of PKM2 in GC cells caused significant decreases in cellular proliferation, migration, anchorage-independent growth, and sphere formation in vitro, and in tumor growth and liver metastasis in vivo. The serine concentration-dependent cell proliferation was also inhibited by PKM2 silencing. Furthermore, we found that PKM2 expression was upregulated by CagA by way of the Erk pathway. These results suggested that enhanced PKM2 expression plays a pivotal role in the carcinogenesis and development of GC in part by regulating cancer-specific metabolism.

NDRG2, suppressed expression associates with poor prognosis in pancreatic cancer, is hypermethylated in the second promoter in human gastrointestinal cancers.

Although N-myc downstream regulated gene 2 (NDRG2) is frequently downregulated in various cancers and is considered to be a candidate tumor suppressor gene, molecular mechanisms of the expressional suppression that lead to cancers are largely unknown. Recent studies indicated that epigenetic suppression of NDRG2 involved carcinogenesis and progression in several tumor types, and we demonstrated positive association with NDRG2 suppression and poor prognosis in pancreatic cancer. In this study, we analyzed mRNA and protein expressions of NDRG2 in 26 cancer cell lines (20 colorectal and 6 gastric cancers) and found that many cell lines showed variously reduced NDRG2 expressions. Furthermore, NDRG2 expressions were significantly reduced in primary resected cancer tissues compared to corresponding normal tissues immunohistochemically (19 of 20 colorectal and 14 of 17 gastric cancers). Treatment with 5-Aza-2' deoxycytidine predominantly upregulated NDRG2 expressions in NDRG2 low-expressing cell lines. Bisulfite sequencing analyses and methylation specific PCR revealed that methylation status at one of the two promoters (around exon 2) correlated well with the suppressed expression, and this is the major promoter in colorectal and gastric cancer cell lines. Our present results suggest that hypermethylation in promoter around exon 2 is functioning as essential factors of NDRG2 silencing in gastrointestinal cancers.

Ex vivo model of non-small cell lung cancer using mouse lung epithelial cells.

Lung cancer is the most common cause of cancer mortality, however, efficient methods to culture, expand and transform lung epithelial (LE) cells have not been established. In the present study, an efficient ex vivo method was applied to recapitulate lung carcinogenesis using mouse LE cells. A Matrigel-assisted three-dimensional culture was used to isolate and selectively expand LE cells from mouse lungs. Purified LE cells were passaged and expanded for at least 2 to 3 months while maintaining epidermal growth factor-dependence. LE cells were also easily transformed by genetic manipulations using retroviral vectors. A SV40 large-T antigen, suppressing p53 and pRB, plus an activated oncogene, such as KrasG12V or EGFRex19del, were required to transform LE cells. Transformed cells formed tumors resembling non-small cell lung cancer (NSCLC) in allograft models and exhibited strong oncogene addiction. This experimental system provided a unique model system to study lung tumorigenesis and develop novel therapeutics against NSCLC.

The IAP Protein Family, SMAC Mimetics and Cancer Treatment.

Since the acquired resistance of cells to apoptosis is one of the major hallmarks of cancer, the endogenous inhibitors of apoptosis can be regarded as promising targets in the design of anticancer therapeutics. In addition to their antiapoptotic activity, inhibitor of apoptosis proteins (IAPs) are able to regulate numerous other cell functions, including proliferation, differentiation, and migration, as well as proinflammatory and immune responses. Study of the IAP family as target molecules in targeted therapies has recently focused on SMAC mimetics as synthetic IAP antagonists that have been under development as promising therapeutics. To overview the background of IAP proteins and to focus on the development of SMAC mimetics, the present review first looks at the mechanisms of IAP proteins' antiapoptotic activities and those for controlling those activities; then the SMAC mimetics, including birinapant, LCL161, and DEBIO1143/AT-406, and their clinical trials are introduced. To further clarify the processes to exert the efficacies of SMAC mimetics, it is necessary to determine therapeutic biomarkers that predict and assess them, which may include caspases and factors in the TNFα pathway.

Enhanced expression of semaphorin 3E is involved in the gastric cancer development.

Semaphorins and their receptors are abnormally expressed in various cancers, but little is known about the expression and function of semaphorin 3E (SEMA3E) and its receptor, plexin D1 (PLXND1), in gastric cancer development or metastasis. We evaluated SEMA3E and PLXND1 expression by quantitative RT-PCR in gastric tissues from 62 patients who underwent gastrectomy and analyzed the correlation between their expression and clinicopathological variables. To assess the function of SEMA3E, we generated human gastric cancer cell lines with suppressed or increased SEMA3E expression. The expression level of SEMA3E, but not PLXND1, was correlated with lymph node involvement and metastatic progression in gastric cancer. A significant association was observed between a high level of SEMA3E expression and poor differentiation or poor survival in the intestinal type of gastric cancer. SEMA3E knockdown in gastric cancer cells attenuated cell proliferation and metastatic ability in vitro and in vivo. Moreover, SEMA3E caused cell proliferation and anchorage-independent cell growth in the intestinal type of gastric cancer. These results suggested that SEMA3E is likely to be involved in the development of gastric cancer and might also be a therapeutic target for its treatment.

SERPINI1 regulates epithelial-mesenchymal transition in an orthotopic implantation model of colorectal cancer.

An increasingly accepted concept is that the progression of colorectal cancer is accompanied by epithelial-mesenchymal transition (EMT). In our study, in order to characterize the properties of EMT in 16 colorectal cancer cell lines, the cells were first orthotopically implanted into nude mice, and the tumors in vivo, as well as cells cultured in vitro, were immunostained for EMT markers. The immunostaining revealed that seven of the cells had an epithelial phenotype with a high expression of E-cadherin, whereas other cells showed opposite patterns, such as a high expression of vimentin (CX-1, COLO205, CloneA, HCT116, and SW48). Among the cells expressing vimentin, some expressed vimentin in the orthotopic tumors but not in the cultured cells (SW480, SW620, and COLO320). We evaluated these findings in combination with microarray analyses, and selected five genes: CHST11, SERPINI1, AGR2, FBP1, and FOXA1. Next, we downregulated the expression of SERPINI1 with siRNA in the cells, the results of which showed reverse-EMT changes at the protein level and in the cellular morphology. Along with immunohistochemical analyses, we confirmed the effect of the intracellular and secreted SERPINI1 protein of SW620 cells, which supported the importance of SERPINI1 in EMT. The development of therapeutic strategies targeting EMT is ongoing, including methods targeting the transforming growth factor-ß signaling pathway as well as the Wnt pathway. SERPINI1 is an important regulator of EMT. Our findings help to elucidate the signaling pathways of EMT, hopefully clarifying therapeutic pathways as well.