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TIFF file for supplementary figure S2

Purpose: Abnormal activation of the NF-κB pathway induces a more aggressive phenotype of cutaneous melanoma. Understanding the mechanisms involved in melanoma NF-κB activation may identify novel targets for this pathway. KPC1, an E3 ubiquitin ligase, is a regulator of the NF-κB pathway. The objective of this study was to investigate the mechanisms regulating KPC1 expression and its clinical impact in melanoma.Experimental Design: The clinical impact of KPC1 expression and its epigenetic regulation were assessed in large cohorts of clinically well-annotated melanoma tissues (tissue microarrays; n = 137, JWCI cohort; n = 40) and The Cancer Genome Atlas database (TCGA cohort, n = 370). Using melanoma cell lines, we investigated the functional interactions between KPC1 and NF-κB, and the epigenetic regulations of KPC1, including DNA methylation and miRNA expression.Results: We verified that KPC1 suppresses melanoma proliferation by processing NF-κB1 p105 into p50, thereby modulating NF-κB target gene expression. Concordantly, KPC1 expression was downregulated in American Joint Committee on Cancer stage IV melanoma compared with early stages (stage I/II P = 0.013, stage III P = 0.004), and low KPC1 expression was significantly associated with poor overall survival in stage IV melanoma (n = 137; HR 1.810; P = 0.006). Furthermore, our data showed that high miR-155-5p expression, which is controlled by DNA methylation at its promoter region (TCGA; Pearson's r −0.455; P < 0.001), is significantly associated with KPC1 downregulation (JWCI; P = 0.028, TCGA; P = 0.003).Conclusions: This study revealed novel epigenetic regulation of KPC1 associated with NF-κB pathway activation, promoting metastatic melanoma progression. These findings suggest the potential utility of KPC1 and its epigenetic regulation as theranostic targets. Clin Cancer Res; 23(16); 4831–42. ©2017 AACR.

The file contains Supplementary Table S1 and figure legends for Supplementary Figure S1 to S4

This study aimed to elucidate the benefits and limitations of preoperative chemoradiotherapy (CRT) in rectal cancer treatment, specifically in T4b rectal cancer. This retrospective cohort study reviewed 1014 consecutive patients with clinical T3/4a/T4b adenocarcinomas of the lower rectum, who underwent total mesorectal excision at the Department of Surgical Oncology of the University of Tokyo Hospital and 22 referral institutions affiliated with the Japanese Study Group for Postoperative Follow-up of Colorectal Cancer. Patients were divided into two cohorts: cohort 1 comprised 298 consecutive patients who underwent CRT followed by radical surgery and cohort 2 comprised 716 consecutive patients who underwent curative surgery without preoperative therapy. We assessed the prognostic differences between the two cohorts, focusing particularly on T stages. In T3/4a patients, cohort 1 showed a significantly lower local recurrence rate than cohort 2 (4.8% vs. 9.4%, p=0.024), but not in T4b patients (23.5% vs. 16.0%, p=0.383). In contrast, no significant differences in survival were observed between T3/4a and T4b patients. T4b classification was found to be an independent predictive factor of local recurrence in cohort 1, but not in cohort 2. In T4b rectal cancer, preoperative CRT demonstrated a limited benefit for local control and survival. In cases of suspected T4b rectal tumors, additional therapies such as induction chemotherapy to conventional CRT may contribute to better outcomes.

Central venous port (CVP) is a widely used totally implantable venous access device. Recognition of risks associated with CVP-related complications is clinically important for safe, reliable, and long-term intravenous access. We therefore investigated factors associated with CVP infection and evulsion, including the device type. A total of 308 consecutive patients with initial CVP implantation between January 2011 and December 2017 were retrospectively reviewed, and the association of clinical features with CVP-related complications were analyzed. Intraoperative and postoperative complications occurred in 11 (3.6%) and 39 (12.7%) patients, respectively. The overall rate of CVP availability at six months was 91.4%. Malignancy and 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymer-coated catheter use were negatively associated with the incidence of CVP infections. Accordingly, malignancy and MPC polymer-coated catheter use were independent predictors for lower CVP evulsion rate (odds ratio, 0.23 and 0.18, respectively). Furthermore, both factors were significantly associated with longer CVP availability (hazard ratio, 0.24 and 0.27, respectively). This retrospective study identified factors associated with CVP-related complications and long-term CVP availability. Notably, MPC polymer-coated catheter use was significantly associated with a lower rate of CVP infection and longer CVP availability, suggesting the preventive effect of MPC coating on CVP infection.

Background/Aim: This study aimed to determine the effectiveness of surgical site infection (SSI) prevention approaches in rectal cancer surgery. Patients and Methods: A total of 1,408 patients who underwent elective rectal cancer surgery between 1995 and 2017 were reviewed. Patients were divided into three groups: control group (group A, n=245), SSI prevention intervention group (group B, n=516), and laparoscopic or robotic surgery group (group C, n=647). The groups were compared in terms of SSI and anastomotic leakage (AL) incidences, and risk factors for SSI were investigated. Results: The overall SSI and AL rates were 19.4% and 3.6%, respectively. These rates were significantly lower in Group C (9.3%, 1.7%), compared to Groups A (40.0%, 6.1%) and B (22.5%, 3.5%). Abdominoperineal resection, open surgery, operation time, intraoperative bleeding, lack of absorbable sutures, lack of mechanical bowel preparation, and lack of oral antibiotics were independently associated with SSI. Conclusion: SSI reduction after rectal cancer surgery was achieved through various intervention strategies.

Background Evaluating anorectal function using real-time tissue elastography (RTE) has not been reported. A previous study reported that in the internal anal sphincter (IAS) of surgical specimens of patients with rectal cancer who underwent abdominoperineal resection, there was an increased fibrosis trend in those who underwent pre-operative chemoradiotherapy (CRT) compared with non-CRT. We speculated that CRT might have induced sclerosis of the IAS because of fibrosis. Therefore, we aimed to establish a method of quantitating the degree of IAS hardness using RTE on endoanal ultrasonography. Methods RTE was performed with freehand manual compression under a defined pressure at the middle anal canal. Using the most compressive point in the strain graph, we traced the region of interest in the IAS. The strain histogram showed a frequency distribution of colours according to the degree of strain (numeric scan ranging from 0 to 255; smaller number indicated harder tissue). We defined the mean of the strain histogram as 'elasticity'. Ten patients with locally advanced rectal cancer who underwent pre-operative CRT were prospectively enrolled. We statistically evaluated the correlation between IAS elasticity and maximum resting pressure (MRP) values both at pre- and post-CRT. MRP was examined concurrently with the examination of IAS elasticity. Results Representativity of elasticity measurements was demonstrated. It revealed a trend: IAS elasticity had a moderate inverse correlation with MRP (r = 0.41, P = 0.07), regardless of whether measurements were made before or after CRT. Conclusion We established a completely novel method for the assessment of elasticity of the IAS, using RTE on endoanal ultrasonography.

Background/Aims: Restorative proctocolectomy (RPC) with ileal pouch-anal anastomosis and handsewn anastomosis for ulcerative colitis requires pulling down of the ileal pouch into the pelvis, which can be technically challenging. We examined risk factors for the pouch not reaching the anus.Methods: Clinical records of 62 consecutive patients who were scheduled to undergo RPC with handsewn anastomosis at the University of Tokyo Hospital during 1989–2019 were reviewed. Risk factors for non-reaching were analyzed in patients in whom hand sewing was abandoned for stapled anastomosis because of nonreaching. Risk factors for non-reaching in laparoscopic RPC were separately analyzed. Anatomical indicators obtained from presurgical computed tomography (CT) were also evaluated.Results: Thirty-seven of 62 cases underwent laparoscopic procedures. In 6 cases (9.7%), handsewn anastomosis was changed to stapled anastomosis because of non-reaching. Male sex and a laparoscopic approach were independent risk factors of non-reaching. Distance between the terminal of the superior mesenteric artery (SMA) ileal branch and the anus > 11 cm was a risk factor for non-reaching.Conclusions: Laparoscopic RPC with handsewn anastomosis may limit extension and induction of the ileal pouch into the anus. Preoperative CT measurement from the terminal SMA to the anus may be useful for predicting non-reaching.

FOLFOX therapy, a standard treatment for colorectal cancer (CRC), causes a rare, but serious adverse event, hyperammonemia. However, the risk factors of hyperammonemia remain unknown. We examined 74 patients who received mFOLFOX6 therapy with or without biologics for CRC between April 2013 and March 2018 in Yaizu City Hospital. Clinicopathological factors were retrospectively reviewed in association with hyperammonemia, and risk factors of hyperammonemia during mFOLFOX6 therapy were analyzed in 32 patients with the available data. Seven patients developed hyperammonemia, with onset exclusively on day 2 or 3 in the first cycle of therapy. They were treated with branched chain amino acid administration and hydration; however, one patient with stage G4 chronic kidney disease (CKD) died. By multivariate analysis, estimated glomerular filtration rate (eGFR)

Background: Systemic therapy can cause loss of skeletal muscle mass in colorectal cancer (CRC) patients in the neoadjuvant and palliative settings. However, it is unknown how the body composition is changed by chemotherapy rendering unresectable CRC to resectable disease or how it affects the prognosis. This study aimed at elucidating the effects of systemic therapy on skeletal muscles and survival in stage IV CRC patients who underwent conversion therapy. Methods: We reviewed 98 stage IV CRC patients who received systemic therapy in our hospital. According to the treatment setting, patients were divided into the conversion, neoadjuvant chemotherapy (NAC), and palliation groups. The cross-sectional area of skeletal muscles at the third lumbar level and changes in the skeletal muscle index (SMI), defined as the area divided by height squared, during systemic therapy were compared among patient groups. The effects of these parameters on prognosis were analyzed in the conversion group. Results: The mean SMI increased by 9.4% during systemic therapy in the conversion group ( n = 38), whereas it decreased by 5.9% in the NAC group ( n = 18) and 3.7% in the palliation group ( n = 42, p Conclusions: Stage IV CRC patients who underwent conversion to resection often had an increased SMI. On the other hand, a decrease in the SMI during systemic therapy was a negative prognostic factor in such patients.

Background/aim It remains unclear whether rectal cancers down-staged by preoperative chemoradiotherapy (CRT) have similar prognoses to those of the same stage without preoperative CRT. We compared prognoses of pT1 rectal cancer patients stratified by preoperative CRT. Patients and methods We retrieved data of patients with pathological T1 rectal cancer between 2003 and 2020. Patients were divided into the "ypT1 group" who received preoperative CRT following surgery and the "pT1 group" who underwent surgery alone. Factors associated with relapse-free survival (RFS) were investigated. Results Among 86 patients, ypT1 and pT1 groups comprised 18 and 68 patients, respectively. There was no significant difference in RFS between the groups (p=0.19). Tumor location within 5 cm from the anal verge was associated with recurrence (hazard ratio: 0.13, p=0.034). Conclusion The prognosis of patients with ypT1 rectal cancer was similar to that of patients with pT1. Low tumor location was a poor prognostic factor.

This study examined the role of the ubiquitin E3-ligase RNF123 in modulating downstream NF-&kappa, B1 targets in glioblastoma (GB) tumor progression. Our findings revealed an oncogenic pathway (miR-155-5p-RNF123-NF-&kappa, B1-p50-SerpinE1) that may represent a new therapeutic target pathway for GB patients with isocitrate dehydrogenase 1 and 2 (IDH) WT (wild type). Mechanistically, we demonstrated that RNF123 is downregulated in IDH WT GB patients and leads to the reduction of p50 levels. RNA-sequencing, reverse-phase protein arrays, and in vitro functional assays on IDH WT GB cell lines with RNF123 overexpression showed that SerpinE1 was a downstream target that is negatively regulated by RNF123. SERPINE1 knockdown reduced the proliferation and invasion of IDH WT GB cell lines. Both SerpinE1 and miR-155-5p overexpression negatively modulated RNF123 expression. In clinical translational analysis, RNF123, SerpinE1, and miR-155-5p were all associated with poor outcomes in GB patients. Multivariable analysis in IDH WT GB patients showed that concurrent low RNF123 and high SerpinE1 was an independent prognostic factor in predicting poor overall survival (p &lt, 0.001, hazard ratio (HR) = 2.93, 95% confidence interval (CI) 1.7&ndash, 5.05), and an increased risk of recurrence (p &lt, 0.001, relative risk (RR) = 3.56, 95% CI 1.61&ndash, 7.83).

The role of post-translational modifications (PTM) of the key epigenetic factor DNMT1 protein has not been well explored in cutaneous metastatic melanoma progression. The acetylated DNMT1 (ac-DNMT1) protein level was assessed using an anti-acetylated lysine antibody in a clinically annotated melanoma patient tumor specimen cohort. In this study, we showed that surgically resected tumors have significantly higher DNMT1 protein expression in metastatic melanoma (stage III metastasis n = 17, p = 0.0009, stage IV metastasis n = 164, p = 0.003) compared to normal organ tissues (n = 19). Additionally, reduced ac-DNMT1 protein levels were associated with melanoma progression. There was a significant inverse correlation between ac-DNMT1 and DNMT1 protein levels in stage IV metastatic melanoma (r = −0.18, p = 0.02, n = 164). Additionally, ac-DNMT1 protein levels were also significantly positively correlated with TIP60 (r = 0.6, p &lt, 0.0001) and USP7 (r = 0.74, p &lt, 0.0001) protein levels in stage IV metastatic melanoma (n = 164). Protein analysis in metastatic melanoma tumor tissues showed that with high ac-DNMT1 (p = 0.006, n = 59), or concurrent high ac-DNMT1 with low DNMT1 (p = 0.05, n = 27), or high TIP60 (p = 0.007, n = 41), or high USP7 (p = 0.01, n = 48) consistently showed better 4-year melanoma-specific survival (MSS). Multivariate Cox proportional hazard analysis showed that ac-DNMT1 level is a significant independent factor associated with MSS (HR, 0.994, 95% confidential interval (CI), 0.990–0.998, p = 0.002). These results demonstrated that low ac-DNMT1 levels may represent an important regulatory factor in controlling metastatic melanoma progression and a promising factor for stratifying aggressive stage IV metastasis.

The CDK4/6 pathway is frequently dysregulated in cutaneous melanoma. Recently, CDK4/6 inhibitors have shown promising clinical activity against several cancer types, including melanoma. Here, we show that microRNA-200a decreases CDK6 expression and thus reduces the response of CDK4/6 inhibitor in highly proliferative metastatic melanoma. Down-regulation of microRNA-200a expression in melanoma cells is associated with disease progression and a higher number of lymph node metastases. Furthermore, microRNA-200a expression is epigenetically modulated by both DNA methylation at the promoter region and chromatin accessibility of an upstream genomic region with enhancer activity. Mechanistically, overexpression of miR-200a in metastatic melanoma cells induces cell cycle arrest by targeting CDK6 and decreases the levels of phosphorylated-Rb1 and E2F-downstream targets, diminishing cell proliferation; these effects are recovered by CDK6 overexpression. Conversely, low microRNA-200a expression in metastatic melanoma cells results in higher levels of CDK6 and a more significant response to CDK4/6 inhibitors. We propose that microRNA-200a functions as a "cell cycle brake" that is lost during melanoma progression to metastasis and provides the ability to identify melanomas that are highly proliferative and more prompted to respond to CDK4/6 inhibitors.

Rationale Hormone therapies, particularly those targeting estrogen and its receptors, are a key treatment modality for patients with estrogen receptor (ER)-positive breast or ovarian cancer. Some gastric cancers (GCs) express ERs, and preclinical studies suggest the potential of estrogen-targeting hormone therapy on GC; however, the clinical relevance of this hormone therapy on GC treatment has not been well elucidated. Patient concerns An 80-year-old female was admitted to our department with hypogastric pain and vomiting. Computed tomography demonstrated small bowel obstruction, and laparotomy after bowel decompression revealed peritoneal dissemination consisting of a poorly-differentiated adenocarcinoma. Intestinal bypass between the ileum and transverse colon was performed. Diagnoses The tumor was ER- and mammaglobin-positive, indicating that it originated from a breast cancer. Diagnostic imaging revealed no evidence of breast cancer; however, right axillary ER- and mammaglobin-positive lymphadenopathy was found. Interventions The patient received hormone therapy using letrozole based on a clinical diagnosis of occult breast cancer with peritoneal dissemination and right axillary lymph node metastasis. Outcomes The patient remained disease free until 37 months but deceased at 53 months from the onset of disease. An autopsy revealed no tumor cells in the right breast tissue; however, there was a massive invasion of cancer cells in the stomach. Lessons A patient with ER positive GC with peritoneal dissemination and right axillary lymph node metastasis presented remarkable response to letrozole. The long-term survival obtained using letrozole for a patient with GC with distant metastasis suggests the potential of estrogen targeting hormone therapies for GC.

A mouse model of epilepsy was generated by inducing status epilepticus (SE) for either 1.5 or 4.5h with pilocarpine to study anxiety-related behaviors, changes in the electroencephalogram of the cerebral cortex and hippocampus, and expression of hippocampal proteins. The viability and rate of success of SE induction were high in C57BL/6N mice but not in C57BL/6J mice. C57BL/6N mice were immotile during the first 2days after SE; however, by the third day, most mice were recovered and exhibited strong anxiety-related behaviors in response to the light/dark preference test and open field test. There was a striking difference in the temporal appearance of anxiety-related behavior between the two SE durations: 1.5h SE mice exhibited strong anxiety-related behavior 3days after SE that gradually attenuated over the next few weeks, whereas 4.5h SE mice exhibited strong anxiety-related behavior 3days after SE that persisted even at nearly 1year after SE. Mice receiving both SE durations exhibited generalized seizures (GS) after SE; however, there was a marked difference in the timing and duration of GS appearance. Mice in the 4.5h SE group exhibited spontaneous GS from 4days to at least 96days after SE. In contrast, mice in the 1.5h SE group exhibited GS only within the first several days after SE; however, epileptic spike clusters continuously appeared in the cerebral cortex and hippocampus for up to twelve days after SE. Among the hippocampal proteins tested, only brain derived-neurotrophic factor (BDNF) exhibited altered expression in parallel with anxiety-related behavior. These results showed the possibility that BDNF expression in the hippocampus might cause anxiety-related behavior in adulthood.

The use of intraoperative colonoscopy has increased alongside progress in the development of colonoscopy-associated devices and techniques, including the colonoscope itself. In the present review, we focus on four circumstances in which intraoperative colonoscopy is beneficial to colorectal surgery: (i) intraoperative determination of a tumor's location; (ii) observation of the proximal colon in cases of obstructive colorectal cancer; (iii) confirmation of the integrity of anastomosis; and (iv) novel surgical techniques that combine laparoscopic and endoscopic surgery. In light of the findings of our review, a combination of colonoscopy and surgery-especially laparoscopic surgery-is expected to facilitate the optimal handling of a variety of colorectal tumors, ranging from benign cases to advanced and obstructive cases.

Background Primary mucosal melanoma (MM) is a rare subtype of melanoma that arises from melanocytes in the mucosa. MM has not been well profiled for mutations and its etiology is not well understood, rendering current treatment strategies unsuccessful. Hence, we investigated mutational landscape for MM to understand its etiology and to clarify mutations that are potentially relevant for MM treatment. Methods Forty one MM and 48 cutaneous melanoma (CM) tissues were profiled for mutations using targeted deep next-generation sequencing (NGS) for 89 cancer-related genes. A total of 997 mutations within exons were analyzed for their mutational spectrum and prevalence of mutation, and 685 non-synonymous variants were investigated to identify mutations in individual genes and pathways. PD-L1 expression from 21 MM and 18 CM were assessed by immunohistochemistry. Results Mutational spectrum analysis revealed a lower frequency of UV-induced DNA damage in MM than in CM (p = 0.001), while tobacco exposure was indicated as a potential etiologic factor for MM. In accordance with low UV damage signatures, MM demonstrated an overall lower number of mutations compared to CM (6.5 mutations/Mb vs 14.8 mutations/Mb, p = 0.001), and less PD-L1 expression (p = 0.003). Compared to CM, which showed frequent mutations in known driver genes (BRAF 50.0%, NRAS 29.2%), MM displayed lower mutation frequencies (BRAF; 12.2%, p

Lymphatic type of adrenal cysts is most common; however, this type of endothelial cyst is quite rare in excessively large adrenal cysts. A 37-year-old Japanese woman was admitted to our institution with distension of her left flank and the upper quadrant of her abdomen. Abdominal ultrasonography revealed a cystic lesion with a homogenous anechoic texture, and measuring 21 cm in diameter. Computed tomography and magnetic resonance imaging displayed a giant cystic lesion adjacent to the liver, pancreas, kidney, and spleen. The origin of the cyst was not identified. We were not able to make a preoperative diagnosis; therefore, the patient underwent resection of the mass by open laparotomy for therapeutic diagnosis. Intraoperatively, the mass was identified to be cystic and adhered to the left adrenal gland. It was filled with more than 2000 mL of serous brown-red fluid. The content of the cyst contained no atypical cells on cytological examination. The wall of the cyst was composed of a lining of a single layer of lymphatic vessel–derived cells, and the cyst was pathologically classified as a true cyst. No abdominal symptoms were observed and a postoperative radiological work-up showed no evidence of recurrence during a 6-year follow-up period. We describe a case of a patient with a giant lymphatic cyst of the adrenal gland. The preset data suggest that surgeons should decide treatment strategy for large adrenal cysts in consideration of hormonal function, degree of size, and possibility of malignancy.

Purpose: Abnormal activation of the NF-κB pathway induces a more aggressive phenotype of cutaneous melanoma. Understanding the mechanisms involved in melanoma NF-κB activation may identify novel targets for this pathway. KPC1, an E3 ubiquitin ligase, is a regulator of the NF-κB pathway. The objective of this study was to investigate the mechanisms regulating KPC1 expression and its clinical impact in melanoma. Experimental Design: The clinical impact of KPC1 expression and its epigenetic regulation were assessed in large cohorts of clinically well-annotated melanoma tissues (tissue microarrays; n = 137, JWCI cohort; n = 40) and The Cancer Genome Atlas database (TCGA cohort, n = 370). Using melanoma cell lines, we investigated the functional interactions between KPC1 and NF-κB, and the epigenetic regulations of KPC1, including DNA methylation and miRNA expression. Results: We verified that KPC1 suppresses melanoma proliferation by processing NF-κB1 p105 into p50, thereby modulating NF-κB target gene expression. Concordantly, KPC1 expression was downregulated in American Joint Committee on Cancer stage IV melanoma compared with early stages (stage I/II P = 0.013, stage III P = 0.004), and low KPC1 expression was significantly associated with poor overall survival in stage IV melanoma (n = 137; HR 1.810; P = 0.006). Furthermore, our data showed that high miR-155-5p expression, which is controlled by DNA methylation at its promoter region (TCGA; Pearson's r −0.455; P < 0.001), is significantly associated with KPC1 downregulation (JWCI; P = 0.028, TCGA; P = 0.003). Conclusions: This study revealed novel epigenetic regulation of KPC1 associated with NF-κB pathway activation, promoting metastatic melanoma progression. These findings suggest the potential utility of KPC1 and its epigenetic regulation as theranostic targets. Clin Cancer Res; 23(16); 4831–42. ©2017 AACR.

Introduction Although several reports have documented the increased incidence of right-sided colorectal cancer (CRC) in the elderly, especially in women, the gender-specific, age-related changes in the characteristics of CRCs, especially related to the cancer localization, have not been fully investigated. This study evaluated the age-related changes in the clinicopathologic features of CRCs, according to the gender. Materials and Methods A total of 1059 consecutive patients with CRCs who were admitted to the authors' surgical department between February 2005 and June 2012 were retrospectively reviewed. The patients were divided into male (n = 632) and female (n = 427) groups and then according to the age group, and the correlation between the age group and the other clinicopathologic features was analyzed by univariate and multivariate analysis. Results The number of concomitant adenomas found was significantly increased along with increasing age in men, and the presence of concomitant adenoma was the only independent age-related factor of male CRC in the multivariate analysis (P = .0044). In contrast, in women, the location of the CRC progressively shifted to the right side (proximal colon) with increasing age, and the presence of right-sided CRC was the only independent factor of female CRC in the multivariate analysis (P Conclusion There was a significant gender-specific difference in the age-related changes in the characteristics of CRC. Increasing the number of concomitant adenomas and the shift of CRC localization to the proximal colon were the gender-specific characteristics of male and female CRC, respectively.

Background: Ectodysplasin (Eda) signaling is essential for the morphogenesis of several ectodermal appendages. Results: Here, we report a medaka mutant, all-fin less (afl), which has a nonsense mutation in its eda gene. The adult afl fish displayed various abnormalities of its dermal skeleton, such as short and twisted fin rays, missing and abnormally shaped scales and teeth, and skull deformation. Focusing on the developing fin rays in the caudal region of afl larvae, we found that the fin rays did not elongate; although the initial formation of fin rays proceeded normally. Additionally, eda expression was lost, and the expression pattern of edar, the gene for the receptor of Eda, was different from wild-type one. In vivo imaging of the double-transgenic medaka expressing enhanced green fluorescent protein under control of the edar promoter and DsRed under control of the osterix promoter revealed that edar expression preceded that of osterix and that the edar-expressing cells migrated in the direction of fin ray elongation, indicating that the Eda/Edar signaling event precedes osteoblast differentiation. Conclusions: Our findings provide evidence that Eda signaling accompanied with the binding of Eda to Edar are essential for fin ray formation guided by cell migration. Developmental Dynamics 243:765–777, 2014. © 2014 Wiley Periodicals, Inc.

Synaptosomal-associated protein 25 (SNAP-25) plays an essential role in exocytotic neurotransmitter release as a t-SNARE protein. SNAP-25 is phosphorylated at Ser 187 in a protein kinase C (PKC)-dependent manner, but the mechanism for dephosphorylation has yet to be clarified. We investigated SNAP-25 dephosphorylation by comparing it to growth associated protein 43 (GAP-43), another PKC-dependent presynaptic phosphoprotein, in crude mouse brain synaptosome preparations. Phosphorylation levels for both SNAP-25 and GAP-43 increased significantly after treatment with PKC activator phorbol 12, 13-dibutyrate (PDB), and ionomycin treatment induced a striking reduction in a time-dependent manner. This dephosphorylation occurred only in the presence of extracellular Ca 2+ , indicating involvement of a Ca 2+ -dependent phosphatase. Ca 2+ -dependent dephosphorylation was not suppressed by calcineurin/PP2B inhibitors such as FK506 and cyclosporine A. SNAP-25 dephosphorylation, however, was suppressed by calyculin A, a non-selective inhibitor of PP1 and PP2A, and okadaic acid selective for PP2A, but not by tautomycin selective for PP1. In contrast, none of these inhibitors suppressed GAP-43 dephosphorylation. PDB-induced SNAP-25 phosphorylation was enhanced by okadaic acid in a concentration-dependent manner. These results suggest that PP2A participates in SNAP-25 dephosphorylation through Ca 2+ -dependent and Ca 2+ -independent mechanisms but is not involved in GAP-43 dephosphorylation.

Gas sensor material, which shows a resistivity change in response to change in concentration of volatile organochlorine compounds, is first developed based on a percolation principle. A composite of organic montmorillonite and carbon shows a reversible resistivity change in response to the concentration change of chloroform, dichloromethane, trichloroethylene, tetrachloroethylene, and tetrachloromethane, because the resistivity of the composite is closely dependent on the volume fraction of the insulating phase and the volume of the insulating phase is increased by incorporation of the organochlorine molecules. The response time is substantially improved by fabricating thick-film composites using classified organic montmorillonite.

Purpose: Abnormal activation of the NF-κB pathway promotes a more aggressive phenotype of cutaneous malignant melanoma. Understanding the mechanisms regulating the NF-κB pathway in melanoma is of critical importance. KPC1 (RNF123) is an E3 ubiquitin ligase that leads to proteasomal processing of precursor NF-κB1 p105 into mature p50, one of the most important steps in the NF-κB pathway regulation. We demonstrated novel epigenetic mechanisms affecting KPC1 expression that lead to an abnormal activation of the NF-κB pathway, which was significant during tumor progression in melanoma patients. Experimental Design: Initially using melanoma cell lines, we investigated the functional interactions between KPC1 and NF-κB, and the epigenetic regulations of KPC1, including microRNA targeted interaction and DNA methylation. The clinical impact of KPC1 expression and these epigenetic regulations were further assessed in large cohorts of clinically well-annotated melanoma tissues (tissue micro-arrays; n=137, JWCI cohort; n=40) and melanoma TCGA database cohort (n=370). High-throughput RNA sequencing, reverse-phase protein array and human methylation 450k platform were utilized for comprehensive analyses. Results: Initially using metastatic melanoma cell lines, we verified that KPC1 promotes processing of NF-κB1 p105 into p50, thereby modulates NF-κB-target gene expression and suppresses melanoma cell proliferation. Concordantly in melanoma tissue, KPC1 expression was down-regulated in AJCC stage IV melanoma compared to early stages (JWCI cohort stage I/II p=0.013, stage III p=0.004), whereby low KPC1 expression was significantly associated with poor overall survival in stage IV melanoma (tissue micro-arrays, n=137, Hazard Ratio 1.810, p=0.006). Furthermore, epigenetic mechanisms regulating KPC1, particularly miR-155-5p and DNA methylation level at its promoter region, was shown to be significant in melanoma lines. This regulatory mechanism was validated in clinical melanoma tissues. High miR-155-5p expression, which is negatively regulated by its promoter DNA methylation level (melanoma TCGA database cohort; Pearson's r -0.455, p Conclusions: We identified miR-155-5p, which is epigenetically controlled by its promoter methylation, has a regulatory role on KPC1 expression. These interactions promote to down-regulation of KPC1 and abnormal NF-κB pathway activation, leading to highly proliferative melanoma cells and poor clinical outcomes. These findings suggest utility of KPC1 expression level for stratification of stage IV melanoma patients, and the importance of the miR-155-5p-KPC1-NF-κB-axis in controlling melanoma proliferation. Citation Format: Yuuki Iida, Aaron Ciechanover, Diego M. Marzese, Keisuke Hata, Matias Bustos, Shigeshi Ono, Jinhua Wang, Matthew P. Salomon, Kevin Tran, Yelena Kravtsova-Ivantsiv, Gordon B. Mills, Michael A. Davies, Dave S.B. Hoon. Epigenetic regulation of KPC1 ubiquitin ligase has a regulatory role on the NF-κB pathway in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3541.

A chemical sensor based on the percolation principle is newly developed by fabricating a composite comprising of a conducting phase and an insulating phase whose volume increases by the incorporation of a desired substance. We find a composite of montmorillonite with carbon shows a remarkable resistivity increase in a humid atmosphere, because the volume of the montmorillonite phase increases by the incorporation of water. The resistivity increases in response to stepwise water vapor increases like a humidity sensor. The response time is substantially improved by fabricating a composite thick film using classified fine montmorillonite particles.

A robust method for preparing (3S)-3-alkyl-3-phenyl-1,4-dioxane-2,5-diones was developed using an improved cyclocondensation reaction between (S)-α-alkylmandelic acids and 2-bromocarbonyl halides. Subtle differences in the reaction conditions, including separate additions of triethylamine, significantly increased the yield compared with Schollkopf's original method.

The function of phosphatidylserine-specific phospholipase A1 (PS-PLA1), a phospholipase that acts specifically on phosphatidylserine and produces lysophosphatidylserine, a lysophospholipid mediator, has not been fully elucidated. We evaluated the role of PS-PLA1 in oncogenesis and metastasis of colorectal cancer (CRC).Specimens from 85 patients with CRC were immunostained with a monoclonal antibody against PS-PLA1. The correlation between PS-PLA1 expression and the clinicopathological variables was analyzed.Tumor depth and hematogenous metastasis independently positively correlated with PS-PLA1 expression. High PS-PLA1 expression was associated with shorter disease-free survival, although it was not an independent predictive factor.PS-PLA1 expression in CRC is associated with tumor invasion and metastasis.

Lysophosphatidylserine (lysoPS) is a type of lysophospholipid mediator, which is involved in allergic conditions and tumor progression. We investigated the physiological function of lysoPS on colorectal cancer (CRC) cell lines, as well as the involved receptor and signaling pathways.Expression of lysoPS receptors on six cell lines was examined by reverse transcription-polymerase chain reaction (RT-PCR). The physiological functions of lysoPS were investigated, and experiments using small interfering RNA (siRNA) or inhibitors of the signaling pathways were conducted.Among the three lysoPS receptors, GPR34 was highly expressed on all cell lines. LysoPS stimulated the chemotactic migratory ability. Wortmannin inhibited the migratory ability, as well as the GPR34 knock-down, strongly suggestive of the involvement of this receptor in the PI3K/Akt pathway.The involved receptor and pathways in the migratory ability in response to lysoPS was demonstrated, which opens premises for targeting as a new strategy for prevention and treatment of colorectal cancer.

Ectodysplasin (Eda) signaling is essential for the morphogenesis of several ectodermal appendages.Here, we report a medaka mutant, all-fin less (afl), which has a nonsense mutation in its eda gene. The adult afl fish displayed various abnormalities of its dermal skeleton, such as short and twisted fin rays, missing and abnormally shaped scales and teeth, and skull deformation. Focusing on the developing fin rays in the caudal region of afl larvae, we found that the fin rays did not elongate; although the initial formation of fin rays proceeded normally. Additionally, eda expression was lost, and the expression pattern of edar, the gene for the receptor of Eda, was different from wild-type one. In vivo imaging of the double-transgenic medaka expressing enhanced green fluorescent protein under control of the edar promoter and DsRed under control of the osterix promoter revealed that edar expression preceded that of osterix and that the edar-expressing cells migrated in the direction of fin ray elongation, indicating that the Eda/Edar signaling event precedes osteoblast differentiation.Our findings provide evidence that Eda signaling accompanied with the binding of Eda to Edar are essential for fin ray formation guided by cell migration.

Temsirolimus (TEM) is a novel, water-soluble mammalian target of rapamycin (mTOR) inhibitor that has shown activity against a wide range of cancers in preclinical models, but its efficacy against colorectal cancer (CRC) has not been fully explored. We evaluated the antitumor effect of TEM in CRC cell lines (CaR-1, HT-29, Colon26) in vitro and in vivo. In vitro, cell growth inhibition was assessed using a MTS assay. Apoptosis induction and cell cycle effects were measured using flow cytometry. Modulation of mTOR signaling was measured using immunoblotting. Antitumor activity as a single agent was evaluated in a mouse subcutaneous tumor model of CRC. The effects of adding chloroquine, an autophagy inhibitor, to TEM were evaluated in vitro and in vivo. In vitro, TEM was effective in inhibiting the growth of two CRC cell lines with highly activated AKT, possibly through the induction of G1 cell cycle arrest via a reduction in cyclin D1 expression, whereas TEM reduced HIF-1α and VEGF in all three cell lines. In a mouse subcutaneous tumor model, TEM inhibited the growth of tumors in all cell lines, not only through direct growth inhibition but also via an anti-angiogenic effect. We also explored the effects of adding chloroquine, an autophagy inhibitor, to TEM. Chloroquine significantly potentiated the antitumor activity of TEM in vitro and in vivo. Moreover, the combination therapy triggered enhanced apoptosis, which corresponded to an increased Bax/Bcl-2 ratio. Based on these data, we propose TEM with or without chloroquine as a new treatment option for CRC.

Synaptosomal-associated protein of 25 kDa (SNAP-25), a t-SNARE protein, plays a crucial role in neurotransmitter release by exocytosis. Protein kinase C phosphorylates SNAP-25 at Ser(187), however the physiological significance of this phosphorylation event in brain function remains unclear. In the present study, we found that SNAP-25 phosphorylation increased rapidly in the mouse brain following cold-water restraint stress. Both basal and stress-induced phosphorylation of SNAP-25 were high in stress-related brain regions, including the cerebral cortex, hippocampus, and amygdala, and the extent of phosphorylation increased with increasing amounts of stress. Intravenous administration of adrenaline increased SNAP-25 phosphorylation, although stress-induced phosphorylation was still observed in adrenalectomized mice. These results indicate that SNAP-25 phosphorylation is regulated in a stress-dependent manner through both central and peripheral mechanisms.

Background: Although the physiological functions of various phospholipases, such as lysophospholipase D (Autotaxin), has been extensively investigated, those of Phosphatidylserine-Specific Phospholipase A1 (PSPLA1), a kind of phospholipase involved in allergic reactions, has not been fully elucidated yet. Here, we evaluated the role of PSPLA1 in oncogenesis and tumor metastasis of colorectal cancer. Materials and Methods: Surgical specimens of 85 consecutive patients with colorectal cancer who received surgery in our department between January 2005 and December 2005 were immunostained with a monoclonal antibody to PSPLA1. The correlation between PSPLA1 expression and the clinico-pathological variables was analyzed by univariate and multivariate analyses. The impact of PSPLA1 expression on the prognosis of the patients was assessed, in comparison with the other clinico-pathological variables. Results: PSPLA1 expression significantly correlated with tumor size, tumor depth and hematogenous metastasis in univariate analysis, and tumor depth and hematogenous metastasis were independent variables in multivariate analysis. The disease-free survival (DFS) of patients with high-PSPLA1 expression was significantly shorter compared to those with low expression. The presence of hematogenous metastasis, however, was the only independent factor associated with poor DFS. Conclusion: PSPLA1 expression in colorectal cancer was associated with tumor invasion and metastasis. Citation Format: Yuuki Iida, Eiji Sunami, Soichiro Ishihara, Hironori Yamaguchi, Nelson H. Tsuno, Joji Kitayama, Toshiaki Watanabe. Phosphatidylserine-specific phospholipase A1 (PSPLA1) expression in colorectal cancer correlates with tumor invasion and hematogenous metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 31. doi:10.1158/1538-7445.AM2014-31

// Katsuya Ohta 1 , Hiromitsu Hoshino 1 , Jinhua Wang 1 , Shigeshi Ono 1 , Yuuki Iida 1 , Keisuke Hata 1 , Sharon K. Huang 1 , Steven Colquhoun 2 and Dave S. B. Hoon 1 1 Department of Molecular Oncology, John Wayne Cancer Institute at Providence Saint John’s Health Center, Santa Monica, CA, USA 2 Liver Disease and Transplant Center, Cedars-Sinai Medical Center, Beverly Hills, CA, USA Correspondence: Dave S. B. Hoon, email: // Keywords : miR-93, hepatocellular carcinoma, drug-sensitivity, sorafenib and tivantinib Received : September 09, 2014 Accepted : December 23, 2014 Published : December 26, 2014 Abstract To assess the role of microRNAs (miR) in hepatocellular carcinoma (HCC), we performed comprehensive microRNA expression profiling using HCC cell lines and identified miR-93 as a novel target associated with HCC. We further verified miR-93 expression levels in advanced HCC tumors (n=47) by a direct PCR assay and found that elevated miR-93 expression level is significantly correlated with poor prognosis. Elevated miR-93 expression significantly stimulated in vitro cell proliferation, migration and invasion, and additionally inhibited apoptosis. We confirmed that miR-93 directly bound with the 3’ untranslated regions of the tumor-suppressor genes PTEN and CDKN1A , respectively,and inhibited their expression. As a result of this inhibition, the c-Met/PI3K/Akt pathway activity was enhanced. IHC analysis of HCC tumors showed significant correlation between c-Met protein expression levels and miR-93 expression levels. Knockdown of c-Met inhibited the activation of the c-Met/PI3K/Akt pathway regardless of hepatocyte growth factor (HGF) treatment, and furthermore reduced the expression of miR-93 in these HCC cells. miR-93 also rendered cells to be more sensitive to sorafenib and tivantinib treatment. We concluded that miR-93 stimulated cell proliferation, migration, and invasion through the oncogenic c-Met/PI3K/Akt pathway and also inhibited apoptosis by directly inhibiting PTEN and CDKN1A expression in human HCC.