Determination of Region-Specific Roles of the M3 Muscarinic Acetylcholine Receptor in Gastrointestinal Motility.

BACKGROUND: The specific role of the M3 muscarinic acetylcholine receptor in gastrointestinal motility under physiological conditions is unclear, due to a lack of subtype-selective compounds. AIMS: The objective of this study was to determine the region-specific role of the M3 receptor in gastrointestinal motility. METHODS: We developed a novel positive allosteric modulator (PAM) for the M3 receptor, PAM-369. The effects of PAM-369 on the carbachol-induced contractile response of porcine esophageal smooth muscle and mouse colonic smooth muscle (ex vivo) and on the transit in mouse small intestine and rat colon (in vivo) were examined. RESULTS: PAM-369 selectively potentiated the M3 receptor under the stimulation of its orthosteric ligands without agonistic or antagonistic activity. Half-maximal effective concentrations of PAM activity for human, mouse, and rat M3 receptors were 0.253, 0.345, and 0.127 µM, respectively. PAM-369 enhanced carbachol-induced contraction in porcine esophageal smooth muscle and mouse colonic smooth muscle without causing any contractile responses by itself. The oral administration of 30 mg/kg PAM-369 increased the small intestinal transit in both normal motility and loperamide-induced intestinal dysmotility mice but had no effects on the colonic transit, although the M3 receptor mRNA expression is higher in the colon than in the small intestine. CONCLUSIONS: This study provided the first direct evidence that the M3 receptor has different region-specific roles in the motility function between the small intestine and colon in physiological and pathophysiological contexts. Selective PAMs designed for targeted subtypes of muscarinic receptors are useful for elucidating the subtype-specific function.

Chronic intestinal pseudo-obstruction due to al amyloidosis: a case report and literature review.

A 59-year-old woman presented to our hospital with a 6-month history of nausea, weight loss, and abdominal distension. Physical examination revealed abdominal distension without tenderness, and edema, numbness, and multiple peripheral neuropathy in the limbs. Blood test results showed anemia, hypoproteinemia, and hypoalbuminemia. Immunoelectrophoresis detected kappa-type Bence-Jones protein in both the serum and urine. Bone marrow examination did not reveal an increase of plasma cells. Computed tomography showed intestinal distension and retention of intestinal contents. No obstructive intestinal lesions were observed. Lower gastrointestinal endoscopy showed a decrease in the vascular visibility of the rectal mucosa. Histological findings showed amyloid deposition, which was positive for amyloid light-chain (AL) κ. Thus, she was diagnosed with chronic intestinal pseudo-obstruction (CIPO) due to gastrointestinal and neurological involvement of AL amyloidosis. Her abdominal symptoms were gradually improved by the insertion of an ileus tube and medication. Although we recommended chemotherapy for stopping her disease progression, she did not want to receive it. She died 1 year later because of her pneumonia. We should keep in mind that amyloidosis is an important cause of CIPO. Histopathological examination by endoscopic biopsy is required for exact diagnosis and appropriate treatment for CIPO due to amyloidosis.

MicroRNA-31 reflects IL-6 expression in cancer tissue and is related with poor prognosis in bile duct cancer.

Bile duct cancer is a highly aggressive malignancy wherein early diagnosis is difficult and few treatment options are available. MicroRNA-31 (miR-31) is reported to be related with survival in patients with gastrointestinal cancers; however, the regulatory mechanism of miR-31 and association between miR-31 expression and survival in patients with bile duct cancer cases have not been established. Thus, we evaluated miR-31 expression in bile duct cancer tissues and assessed its relationship with prognosis. Additionally, we examined the effects of several cytokines on miR-31 expression. The study included 81 samples of bile duct cancer tissues. MiR-31 expression in bile duct cancer cells was significantly higher than that in normal bile duct epithelial cells (P = 0.038). There were no significant associations between miR-31 expression and clinical or pathological characteristics, except for tumour size (P = 0.012). In Kaplan-Meier analysis, high miR-31 expression was significantly associated with shorter survival (log-rank test, P = 0.0082). In multivariate Cox regression analysis, high miR-31 expression was significantly associated with prognosis (P = 0.043), independent of clinical or pathological features. Interleukin-6 (IL-6) significantly promoted miR-31 expression and cell proliferation in a dose-dependent manner, and the inhibition of STAT-3 signalling significantly suppressed miR-31 expression and cell proliferation. In conclusion, high expression was significantly associated with poor prognosis in bile duct cancer patients. The IL-6-STAT-3 signalling regulated bile duct cancer cell proliferation and miR-31 expression. Our findings suggest that miR-31 may be a promising biomarker that reflects IL-6 expression in bile duct cancer tissues and predicts poor prognosis.

PRDM14 directly interacts with heat shock proteins HSP90α and glucose-regulated protein 78.

PRDM14 is overexpressed in various cancers and can regulate cancer phenotype under certain conditions. Inhibiting PRDM14 expression in breast and pancreatic cancers has been reported to reduce cancer stem-like phenotypes, which are associated with aggressive tumor properties. Therefore, PRDM14 is considered a promising target for cancer therapy. To develop a pharmaceutical treatment, the mechanism and interacting partners of PRDM14 need to be clarified. Here, we identified the proteins interacting with PRDM14 in triple-negative breast cancer (TNBC) cells, which do not express the three most common types of receptor (estrogen receptors, progesterone receptors, and HER2). We obtained 13 candidates that were pulled down with PRDM14 in TNBC HCC1937 cells and identified them by mass spectrometry. Two candidates-glucose-regulated protein 78 (GRP78) and heat shock protein 90-α (HSP90α)-were confirmed in immunoprecipitation assay in two TNBC cell lines (HCC1937 and MDA-MB231). Surface plasmon resonance analysis using GST-PRDM14 showed that these two proteins directly interacted with PRDM14 and that the interactions required the C-terminal region of PRDM14, which includes zinc finger motifs. We also confirmed the interactions in living cells by NanoLuc luciferase-based bioluminescence resonance energy transfer (NanoBRET) assay. Moreover, HSP90 inhibitors (17DMAG and HSP990) significantly decreased breast cancer stem-like CD24-  CD44+ and side population (SP) cells in HCC1937 cells, but not in PRDM14 knockdown HCC1937 cells. The combination of the GRP78 inhibitor HA15 and PRDM14 knockdown significantly decreased cell proliferation and SP cell number in HCC1937 cells. These results suggest that HSP90α and GRP78 interact with PRDM14 and participate in cancer regulation.

EZH2 expression is a prognostic biomarker in patients with colorectal cancer treated with anti-EGFR therapeutics.

The polycomb group protein enhancer of zeste homolog 2 (EZH2) is a methyltransferase that suppresses microRNA-31 (miR-31) in various human malignancies including colorectal cancer. We recently suggested that miR-31 regulates the signaling pathway downstream of epidermal growth factor receptor (EGFR) in colorectal cancer. Therefore, we conducted this study for assessing the relationship between EZH2 expression and clinical outcomes in patients with colorectal cancer treated with anti-EGFR therapeutics. We immunohistochemically evaluated EZH2 expression and assessed miR-31 and gene mutations [KRAS (codon 61/146), NRAS (codon 12/13/61), and BRAF (codon 600)] in 109 patients with colorectal cancer harboring KRAS (codon 12/13) wild-type. We also evaluated the progression-free survival (PFS) and overall survival (OS). In the result, low EZH2 expression was significantly associated with shorter PFS (log-rank test: P = 0.023) and OS (P = 0.036) in patients with colorectal cancer. In the low-miR-31-expression group and the KRAS (codon 61/146), NRAS, and BRAF wild-type groups, a significantly shorter PFS (P = 0.022, P = 0.039, P = 0.021, and P = 0.036, respectively) was observed in the EZH2 low-expression groups than in the high-expression groups. In the multivariate analysis, low EZH2 expression was associated with a shorter PFS (P = 0.046), independent of the mutational status and miR-31. In conclusion, EZH2 expression was associated with survival in patients with colorectal cancer who were treated with anti-EGFR therapeutics. Moreover, low EZH2 expression was independently associated with shorter PFS in patients with cancer, suggesting that EZH2 expression is a useful additional prognostic biomarker for anti-EGFR therapy.

Cancer stem cells in human gastrointestinal cancer.

Cancer stem cells (CSCs) are thought to be responsible for tumor initiation, drug and radiation resistance, invasive growth, metastasis, and tumor relapse, which are the main causes of cancer-related deaths. Gastrointestinal cancers are the most common malignancies and still the most frequent cause of cancer-related mortality worldwide. Because gastrointestinal CSCs are also thought to be resistant to conventional therapies, an effective and novel cancer treatment is imperative. The first reported CSCs in a gastrointestinal tumor were found in colorectal cancer in 2007. Subsequently, CSCs were reported in other gastrointestinal cancers, such as esophagus, stomach, liver, and pancreas. Specific phenotypes could be used to distinguish CSCs from non-CSCs. For example, gastrointestinal CSCs express unique surface markers, exist in a side-population fraction, show high aldehyde dehydrogenase-1 activity, form tumorspheres when cultured in non-adherent conditions, and demonstrate high tumorigenic potential in immunocompromised mice. The signal transduction pathways in gastrointestinal CSCs are similar to those involved in normal embryonic development. Moreover, CSCs are modified by the aberrant expression of several microRNAs. Thus, it is very difficult to target gastrointestinal CSCs. This review focuses on the current research on gastrointestinal CSCs and future strategies to abolish the gastrointestinal CSC phenotype.

The relationship between EZH2 expression and microRNA-31 in colorectal cancer and the role in evolution of the serrated pathway.

Polycomb group protein enhancer of zeste homolog 2 (EZH2) is a methyltransferase that correlates with the regulation of invasion and metastasis and is overexpressed in human cancers such as colorectal cancer. MicroRNA-31 (miR-31) plays an oncogenic role and is associated with BRAF mutation and poor prognosis in colorectal cancer. EZH2 is functionally considered to suppress miR-31 expression in human cancers; however, no study has reported its relationship with colon cancer. We therefore evaluated EZH2 expression using immunohistochemistry and assessed miR-31 and epigenetic alterations using 301 colorectal carcinomas and 207 premalignant lesions. Functional analysis was performed to identify the association between EZH2 and miR-31 using cancer cell lines. In the current study, negative, weak, moderate, and strong EZH2 expressions were observed in 15%, 19%, 25%, and 41% of colorectal cancers, respectively. EZH2 was inversely associated with miR-31 (P < 0.0001), independent of clinicopathological and molecular features. In a multivariate stage-stratified analysis, high EZH2 expression was related to favorable prognosis (P = 0.0022). Regarding premalignant lesions, negative EZH2 expression was frequently detected in sessile serrated adenomas/polyps (SSA/Ps) (76%; P < 0.0001) compared with hyperplastic polyps, traditional serrated adenomas, and non-serrated adenomas (25-36%). Functional analysis demonstrated that the knockdown of EZH2 increased miR-31 expression. In conclusion, an inverse association was identified between EZH2 and miR-31 in colorectal cancers. Our data also showed that upregulation of EZH2 expression may be rare in SSA/Ps. These results suggest that EZH2 suppresses miR-31 in colorectal cancer and may correlate with differentiation and evolution of serrated pathway.

Association of Fusobacterium nucleatum with immunity and molecular alterations in colorectal cancer.

The human intestinal microbiome plays a major role in human health and diseases, including colorectal cancer. Colorectal carcinogenesis represents a heterogeneous process with a differing set of somatic molecular alterations, influenced by diet, environmental and microbial exposures, and host immunity. Fusobacterium species are part of the human oral and intestinal microbiota. Metagenomic analyses have shown an enrichment of Fusobacterium nucleatum (F. nucleatum) in colorectal carcinoma tissue. Using 511 colorectal carcinomas from Japanese patients, we assessed the presence of F. nucleatum. Our results showed that the frequency of F. nucleatum positivity in the Japanese colorectal cancer was 8.6% (44/511), which was lower than that in United States cohort studies (13%). Similar to the United States studies, F. nucleatum positivity in Japanese colorectal cancers was significantly associated with microsatellite instability (MSI)-high status. Regarding the immune response in colorectal cancer, high levels of infiltrating T-cell subsets (i.e., CD3+, CD8+, CD45RO+, and FOXP3+ cells) have been associated with better patient prognosis. There is also evidence to indicate that molecular features of colorectal cancer, especially MSI, influence T-cell-mediated adaptive immunity. Concerning the association between the gut microbiome and immunity, F. nucleatum has been shown to expand myeloid-derived immune cells, which inhibit T-cell proliferation and induce T-cell apoptosis in colorectal cancer. This finding indicates that F. nucleatum possesses immunosuppressive activities by inhibiting human T-cell responses. Certain microRNAs are induced during the macrophage inflammatory response and have the ability to regulate host-cell responses to pathogens. MicroRNA-21 increases the levels of IL-10 and prostaglandin E2, which suppress antitumor T-cell-mediated adaptive immunity through the inhibition of the antigen-presenting capacities of dendritic cells and T-cell proliferation in colorectal cancer cells. Thus, emerging evidence may provide insights for strategies to target microbiota, immune cells and tumor molecular alterations for colorectal cancer prevention and treatment. Further investigation is needed to clarify the association of Fusobacterium with T-cells and microRNA expressions in colorectal cancer.

Analysis of the molecular features of rectal carcinoid tumors to identify new biomarkers that predict biological malignancy.

Although gastrointestinal carcinoid tumors are relatively rare in the digestive tract, a quarter of them are present in the rectum. In the absence of specific tumor biomarkers, lymphatic or vascular invasion is generally used to predict the risk of lymph node metastasis. We, therefore, examined the genetic and epigenetic alterations potentially associated with lymphovascular invasion among 56 patients with rectal carcinoid tumors. We also conducted a microRNA (miRNA) array analysis. Our analysis failed to detect mutations in BRAF, KRAS, NRAS, or PIK3CA or any microsatellite instability (MSI); however, we did observe CpG island methylator phenotype (CIMP) positivity in 13% (7/56) of the carcinoid tumors. The CIMP-positive status was significantly correlated with lymphovascular invasion (P = 0.036). The array analysis revealed that microRNA-885 (miR-885)-5p was the most up-regulated miRNA in the carcinoid tumors with lymphovascular invasion compared with that in those without invasion. In addition, high miR-885-5p expression was independently associated with lymphovascular invasion (P = 0.0002). In conclusion, our findings suggest that miR-885-5p and CIMP status may be useful biomarkers for predicting biological malignancy in patients with rectal carcinoid tumors.

Association of Fusobacterium species in pancreatic cancer tissues with molecular features and prognosis.

Recently, bacterial infection causing periodontal disease has attracted considerable attention as a risk factor for pancreatic cancer. Fusobacterium species is an oral bacterial group of the human microbiome. Some evidence suggests that Fusobacterium species promote colorectal cancer development; however, no previous studies have reported the association between Fusobacterium species and pancreatic cancer. Therefore, we examined whether Fusobacterium species exist in pancreatic cancer tissue. Using a database of 283 patients with pancreatic ductal adenocarcinoma (PDAC), we tested cancer tissue specimens for Fusobacterium species. We also tested the specimens for KRAS, NRAS, BRAF and PIK3CA mutations and measured microRNA-21 and microRNA-31. In addition, we assessed epigenetic alterations, including CpG island methylator phenotype (CIMP). Our data showed an 8.8% detection rate of Fusobacterium species in pancreatic cancers; however, tumor Fusobacterium status was not associated with any clinical and molecular features. In contrast, in multivariate Cox regression analysis, compared with the Fusobacterium species-negative group, we observed significantly higher cancer-specific mortality rates in the positive group (p = 0.023). In conclusion, Fusobacterium species were detected in pancreatic cancer tissue. Tumor Fusobacterium species status is independently associated with a worse prognosis of pancreatic cancer, suggesting that Fusobacterium species may be a prognostic biomarker of pancreatic cancer.

Association of Fusobacterium nucleatum with clinical and molecular features in colorectal serrated pathway.

Human gut microbiota is being increasingly recognized as a player in colorectal cancers (CRCs). Evidence suggests that Fusobacterium nucleatum (F. nucleatum) may contribute to disease progression and is associated with CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) in CRCs; however, to date, there are no reports about the relationship between F. nucleatum and molecular features in the early stage of colorectal tumorigenesis. Therefore, we investigated the presence of F. nucleatum in premalignant colorectal lesions. In total, 465 premalignant lesions (343 serrated lesions and 122 non-serrated adenomas) and 511 CRCs were studied. We determined the presence of F. nucleatum and analyzed its association with molecular features including CIMP, MSI and microRNA-31 status. F. nucleatum was detected in 24% of hyperplastic polyps, 35% of sessile serrated adenomas (SSAs), 30% of traditional serrated adenomas (TSAs) and 33% of non-serrated adenomas. F. nucleatum was more frequently detected in CIMP-high premalignant lesions than in CIMP-low/zero lesions (p = 0.0023). In SSAs, F. nucleatum positivity increased gradually from sigmoid colon to cecum (p = 0.042). F. nucleatum positivity was significantly higher in CRCs (56%) than in premalignant lesions of any histological type (p < 0.0001). In conclusion, F. nucleatum was identified in premalignant colorectal lesions regardless of histopathology but was more frequently associated with CIMP-high lesions. Moreover, F. nucleatum positivity increased according to histological grade, suggesting that it may contribute to the progression of colorectal neoplasia. Our data also indicate that F. nucleatum positivity in SSAs may support the "colorectal continuum" concept.

Clinicopathological and molecular characteristics of serrated lesions in Japanese elderly patients.

BACKGROUND: The population in Japan is aging more rapidly than in any other country. However, no studies have determined the characteristics of the large population of elderly patients with colorectal tumors. Therefore, we examined the clinicopathological and molecular features of these tumors in elderly patients. METHODS: In total, 1,627 colorectal tumors (393 serrated lesions, 277 non-serrated adenomas and 957 colorectal cancers) were acquired from patients. Tumor specimens were analyzed for BRAF and KRAS mutations, CpG island methylator phenotype-specific promoters (CACNA1G, CDKN2A, IGF2 and RUNX3), IGFBP7, MGMT, MLH1 and RASSF2 methylation, microsatellite instability (MSI) and microRNA- 31 (miR-31). RESULTS: The frequency of elderly patients (aged ≥75 years) with sessile serrated adenomas (SSAs) with cytological dysplasia was higher than that of those with other serrated lesions and non-serrated adenomas (p < 0.0001). In elderly patients, all SSAs were located in the proximal colon (particularly the cecum to ascending colon). High miR-31 expression, MLH1 methylation and MSI-high status were more frequently detected in SSAs from elderly patients than in those from non-elderly patients. In contrast, no significant differences were found between older age of onset and high-grade dysplasia for traditional serrated adenomas or non-serrated adenomas in any of these molecular alterations. CONCLUSION: In elderly patients, all SSAs were located in the proximal colon. Furthermore, cytological dysplasia and molecular alterations were more frequently detected in elderly patients with SSAs than in non-elderly patients. Thus, careful colonoscopic examinations of the proximal colon are necessary for elderly patients because SSAs in those patients may exhibit malignant potential.

Association of MicroRNA-31-5p with Clinical Efficacy of Anti-EGFR Therapy in Patients with Metastatic Colorectal Cancer.

BACKGROUND: Gene mutations in the pathway downstream of epidermal growth factor receptor (EGFR) are considered to induce resistance to anti-EGFR therapy in colorectal cancer (CRC). We recently reported that microRNA-31 (miR-31)-5p may regulate BRAF activation and play a role in the signaling pathway downstream of EGFR in CRC. Therefore, we hypothesized that miR-31-5p can be a useful biomarker for anti-EGFR therapy in CRC. METHODS: We evaluated miR-31-5p expression and gene mutations [KRAS (codon 61 or 146), NRAS (codon 12, 13, or 61), and BRAF (V600E)] in the EGFR downstream pathway in 102 CRC patients harboring KRAS (codon 12 or 13) wild-type who were treated with anti-EGFR therapeutics. Progression-free survival (PFS) and overall survival (OS) were evaluated. RESULTS: KRAS (codon 61 or 146), NRAS, and BRAF mutations were detected in 6.9, 6.9, and 5.9 % patients, respectively. Compared with CRCs with at least one mutation (n = 20), significantly better PFS (P = 0.0003) but insignificantly better OS were observed in CRCs harboring all wild-type genes (KRAS, NRAS, and BRAF). High miR-31-5p expression was identified in 11 % (n = 11) patients and was significantly associated with shorter PFS (P = 0.003). In CRCs carrying all wild-type genes, high miR-31-5p was associated with shorter PFS (P = 0.027). CONCLUSIONS: High miR-31-5p expression was associated with shorter PFS in patients with CRC treated with anti-EGFR therapeutics. Moreover, in CRCs carrying all wild-type genes, high miR-31-5p was associated with shorter PFS, suggesting that it may be a useful and additional prognostic biomarker for anti-EGFR therapy.

MicroRNA-31 expression in colorectal serrated pathway progression.

MicroRNAs have been increasingly recognized as useful biomarkers for colorectal cancers (CRC). We have recently observed that microRNA-31 (miR-31) expression is associated with BRAF mutation and prognosis in CRC. Moreover, high miR-31 expression is frequently detected in sessile serrated adenomas compared with hyperplastic polyps (HPs). These results suggest that miR-31 may contribute to the progression of serrated lesions. At a follow-up colonoscopy, we observed the case of a 75-year-old man with a 7-mm flat-elevated lesion in the cecum and diagnosed the lesion as an early invasive carcinoma with serrated features. Tissue specimens were obtained from the representative areas to compare the molecular alterations in the carcinoma component with those in the HP component. Higher miR-31 expression was observed in the carcinoma component (57-fold increase) and the HP component (8-fold increase) compared with the paired normal mucosa, suggesting that miR-31 may be one of the key molecules in serrated pathway progression.

IGF2 differentially methylated region hypomethylation in relation to pathological and molecular features of serrated lesions.

AIM: To investigate insulin-like growth factor 2 (IGF2) differentially methylated region (DMR)0 hypomethylation in relation to clinicopathological and molecular features in colorectal serrated lesions. METHODS: To accurately analyze the association between the histological types and molecular features of each type of serrated lesion, we consecutively collected 1386 formalin-fixed paraffin-embedded tissue specimens that comprised all histological types [hyperplastic polyps (HPs, n = 121), sessile serrated adenomas (SSAs, n = 132), traditional serrated adenomas (TSAs, n = 111), non-serrated adenomas (n = 195), and colorectal cancers (CRCs, n = 827)]. We evaluated the methylation levels of IGF2 DMR0 and long interspersed nucleotide element-1 (LINE-1) in HPs (n = 115), SSAs (n = 120), SSAs with cytological dysplasia (n = 10), TSAs (n = 91), TSAs with high-grade dysplasia (HGD) (n = 15), non-serrated adenomas (n = 80), non-serrated adenomas with HGD (n = 105), and CRCs (n = 794). For the accurate quantification of the relative methylation levels (scale 0%-100%) of IGF2 DMR0 and LINE-1, we used bisulfite pyrosequencing method. Tumor specimens were analyzed for microsatellite instability, KRAS (codons 12 and 13), BRAF (V600E), and PIK3CA (exons 9 and 20) mutations; MLH1 and MGMT methylation; and IGF2 expression by immunohistochemistry. RESULTS: The distribution of the IGF2 DMR0 methylation level in 351 serrated lesions and 185 non-serrated adenomas (with or without HGD) was as follows: mean 61.7, median 62.5, SD 18.0, range 5.0-99.0, interquartile range 49.5-74.4. The IGF2 DMR0 methylation level was divided into quartiles (Q1 ≥ 74.5, Q2 62.6-74.4, Q3 49.6-62.5, Q4 ≤ 49.5) for further analysis. With regard to the histological type, the IGF2 DMR0 methylation levels of SSAs (mean ± SD, 73.1 ± 12.3) were significantly higher than those of HPs (61.9 ± 20.5), TSAs (61.6 ± 19.6), and non-serrated adenomas (59.0 ± 15.8) (P < 0.0001). The IGF2 DMR0 methylation level was inversely correlated with the IGF2 expression level (r = -0.21, P = 0.0051). IGF2 DMR0 hypomethylation was less frequently detected in SSAs compared with HPs, TSAs, and non-serrated adenomas (P < 0.0001). Multivariate logistic regression analysis also showed that IGF2 DMR0 hypomethylation was inversely associated with SSAs (P < 0.0001). The methylation levels of IGF2 DMR0 and LINE-1 in TSAs with HGD (50.2 ± 18.7 and 55.7 ± 5.4, respectively) were significantly lower than those in TSAs (61.6 ± 19.6 and 58.8 ± 4.7, respectively) (IGF2 DMR0, P = 0.038; LINE-1, P = 0.024). CONCLUSION: IGF2 DMR0 hypomethylation may be an infrequent epigenetic alteration in the SSA pathway. Hypomethylation of IGF2 DMR0 and LINE-1 may play a role in TSA pathway progression.

MicroRNA-31 expression in relation to BRAF mutation, CpG island methylation and colorectal continuum in serrated lesions.

The CpG island methylator phenotype (CIMP) is a distinct form of epigenomic instability. Many CIMP-high colorectal cancers (CRCs) with BRAF mutation are considered to arise from serrated pathway. We recently reported that microRNA-31 (miR-31) is associated with BRAF mutation in colorectal tumors. Emerging new approaches have revealed gradual changes in BRAF mutation and CIMP-high throughout the colorectum in CRCs. Here, we attempted to identify a possible association between miR-31 and epigenetic features in serrated pathway, and hypothesized that miR-31 supports the "colorectal continuum" concept. We evaluated miR-31 expression, BRAF mutation and epigenetic features including CIMP status in 381 serrated lesions and 222 non-serrated adenomas and examined associations between them and tumor location (rectum; sigmoid, descending, transverse and ascending colon and cecum). A significant association was observed between high miR-31 expression and CIMP-high status in serrated lesions with BRAF mutation (p = 0.0001). In contrast, miR-31 was slightly but insignificantly associated with CIMP status in the cases with wild-type BRAF. miR-31 expression in sessile serrated adenomas (SSAs) with cytological dysplasia was higher than that in SSAs, whereas, no significant difference was observed between traditional serrated adenomas (TSAs) and TSAs with high-grade dysplasia. The frequency of miR-31, BRAF mutation CIMP-high and MLH1 methylation increased gradually from the rectum to cecum in serrated lesions. In conclusion, miR-31 expression was associated with CIMP-high status in serrated lesions with BRAF mutation. Our data also suggested that miR-31 plays an important role in SSA evolution and may be a molecule supporting the colorectal continuum.

HER2 expression and PI3K-Akt pathway alterations in gastric cancer.

The anti-HER2 antibody trastuzumab has led to an era of personalized therapy in gastric cancer (GC). As a result, HER2 expression has become a major concern in GC. HER2 overexpression is seen in 7-34% of GC cases. Trastuzumab is an antibody that targets the HER2 extracellular domain and induces antibody-dependent cellular cytotoxicity and inhibition of the HER2 downstream signals. Mechanisms of resistance to trastuzumab have been reported in breast cancer. There are various mechanisms underlying trastuzumab resistance, such as alterations of HER2 structure or surroundings, dysregulation of HER2 downstream signal effectors and interaction of HER2 with other membrane receptors. The PI3K-Akt pathway is one of the main downstream signaling pathways of HER2. It is well known that PIK3CA mutations and phosphate and tensin homolog (PTEN) inactivation cause over-activation of the downstream signal without an upstream signal activation. Frequencies of PIK3CA mutations and PTEN inactivation have been reported to be 4-25 and 16-77%, respectively. However, little is known about the association between HER2 expression and PI3K-Akt pathway alterations in GC. We have found that HER2 over-expression was significantly correlated with pAkt expression in GC tissues. Furthermore, pAkt expression was correlated with poor prognosis. These results suggest that the PI3K-Akt pathway plays an important role in HER2-positive GC. Moreover, PIK3CA mutations and/or PTEN inactivation might affect the effectiveness of HER2-targeting therapy. Hence, it is necessary to clarify not only HER2 alterations but also PI3K-Akt pathway alterations for HER2-targeting therapy in GC. This review will introduce recent investigations and consider the current status of HER2-targeted therapy for treatment of GC.

Association of microRNA-31 with BRAF mutation, colorectal cancer survival and serrated pathway.

BRAF is an important gene in colorectal cancers (CRCs) that is associated with molecular characterization and resistance to targeted therapy. Although microRNAs (miRNAs) are useful biomarkers of various cancers, the association between miRNA and BRAF in CRCs is undefined. Therefore, this study was conducted to identify a relationship between specific miRNA molecules and BRAF mutation in CRCs and serrated lesions. miRNA array was used for the measurement of 760 miRNAs in 29 CRCs. To assess the identified miRNAs, quantitative reverse transcription-PCR was performed on 721 CRCs, 381 serrated lesions and 251 non-serrated adenomas. Moreover, proliferation and invasion assays were conducted using cell lines. miRNA array analysis revealed that microRNA-31 (miR-31)-5p was the most up-regulated miRNA in CRCs with mutated BRAF (V600E) compared with CRCs possessing wild-type BRAF (including cases with KRAS mutation). High miR-31 expression was associated with BRAF and KRAS mutations and proximal location (P < 0.0001). High miR-31 expression was related to cancer-specific mortality [multivariate hazard ratio = 2.06, 95% confidence interval: 1.36-3.09, P = 0.0008]. Functional analysis demonstrated that miR-31 inhibitor decreased cell invasion and proliferation. With regard to serrated lesions, high miR-31 expression was less frequently detected in hyperplastic polyps compared with other serrated lesions. In conclusion, associations were identified between miR-31, BRAF and prognosis in CRC. Transfection of miR-31 inhibitor had an antitumour effect. Thus, miR-31 may be a promising diagnostic biomarker and therapeutic target in colon cancers. Moreover, high miR-31 expression in serrated lesions suggested that miR-31 may be a key molecule in serrated pathway.

Irinotecan as the key chemotherapeutic agent in second-line treatment of metastatic gastric cancer after failure of first-line S-1 or S-1/CDDP therapy.

BACKGROUND: S-1, an oral fluoropyrimidine, is one of the standard chemotherapeutic agents for the treatment of metastatic gastric cancer(MGC). However, the most effective second-line regimen after failure of treatment with first-line agents such as S-1 is yet to be determined. The aim of this study was to investigate the various second-line chemotherapy regimens in MGC patients. METHODS: We retrospectively studied patients with MGC who received second-line treatment after failure of the first-line S-1 or S-1/cisplatin treatment. The overall survival times with each second-line regimen were determined using the Kaplan-Meier method, and the effect on overall survival was analyzed using Cox regression analysis. RESULTS: The median survival time for all patients was 14. 2 months(95% confidence interval(CI): 12. 88-15. 43 months)with a 1-year survival rate of 60. 4%. Kaplan-Meier analysis revealed that the second-line regimens containing irinotecan significantly improved the median survival time as compared to regimens without irinotecan(median survival time: 16. 5 and 13. 8 months, respectively). Cox regression analysis showed that irinotecan-containing regimens were associated with improved overall survival(hazard ratio: 0. 165; 95% CI: 0. 041-0. 665). CONCLUSION: The use of irinotecan-containing regimens as second-line chemotherapy after failure of first-line S-1 therapy may be beneficial for MGC patients.