Cutaneous ischemia-reperfusion injury is exacerbated by IL-36 receptor antagonist deficiency.

BACKGROUND: Loss-of-function homozygous or compound heterozygous mutations in IL36RN, which encodes interleukin-36 receptor antagonist (IL-36Ra), has been implicated in the pathogenesis of skin disorders. However, the pathogenic role of IL-36Ra in cutaneous ischemia-reperfusion (I/R) injury remains unclear. OBJECTIVES: We investigated the role of IL36Ra in cutaneous I/R injury. METHODS: We examined I/R injury in Il36rn-/- mice. The area of wounds, numbers of infiltrated cells, apoptotic cells and neutrophil extracellular trap (NET) formation were assessed. The expression levels of various genes were analysed using real-time RT-PCR. The expression of high mobility group box 1 (HMGB1), an endogenous toll-like receptor (TLR) 4 ligand, was confirmed using immunohistology, and serum HMGB1 levels were measured by ELISA. Cytokine production by stimulated cultured J774A.1 and HaCaT cells was examined. RESULTS: IL-36Ra deficiency resulted in significantly delayed wound healing and increased neutrophil and macrophage infiltration into the wound tissues. Il36rn-/- mice had increased mRNA expression levels of CXCL1, CXCL2, CCL4, TNF-α, TGF-ß, IL-1ß, IL-6 and IL-36γ relative to wild-type mice. Apoptosis was identified in keratinocytes by TUNEL assay. HMGB1 expression in the I/R site was decreased in both keratinocytes and adnexal cells, while serum HMGB1 levels were significantly elevated after reperfusion. The mRNA levels of various cytokines, including IL-1ß, were elevated in J774A.1 cells through TLR4 signalling by HMGB1 stimulation. In addition, HaCaT cells stimulated with IL-1ß showed significantly increased CXCL1, TNF-α, IL-6, IL-36ß and IL-36γ mRNA expression. Furthermore, NET formation was increased by IL-36Ra deficiency. Finally, either the blockade of TLR4 signalling by TAK-242 or inhibition of NET formation by Cl-amidine normalized exacerbated I/R injury in Il36rn-/- mice. CONCLUSIONS: This study indicated that IL-36Ra deficiency exacerbates cutaneous I/R injury due to excessive inflammatory cell recruitment, NET formation, and excessive cytokine and chemokine production via the TLR4 pathway by HMGB1 released from epidermal apoptotic cells.

Lactose drives Enterococcus expansion to promote graft-versus-host disease.

Disruption of intestinal microbial communities appears to underlie many human illnesses, but the mechanisms that promote this dysbiosis and its adverse consequences are poorly understood. In patients who received allogeneic hematopoietic cell transplantation (allo-HCT), we describe a high incidence of enterococcal expansion, which was associated with graft-versus-host disease (GVHD) and mortality. We found that Enterococcus also expands in the mouse gastrointestinal tract after allo-HCT and exacerbates disease severity in gnotobiotic models. Enterococcus growth is dependent on the disaccharide lactose, and dietary lactose depletion attenuates Enterococcus outgrowth and reduces the severity of GVHD in mice. Allo-HCT patients carrying lactose-nonabsorber genotypes showed compromised clearance of postantibiotic Enterococcus domination. We report lactose as a common nutrient that drives expansion of a commensal bacterium that exacerbates an intestinal and systemic inflammatory disease.

Effect of Donor Age on Graft Function and Pathologic Findings in Living Donor Transplantation.

BACKGROUND: The frequency of renal transplants from elderly living donors has increased because of a shortage of donors. However, the results of renal transplantation using aged kidney grafts have yet to be determined conclusively. METHODS: We evaluated 45 patients who underwent living donor kidney transplantation at our institution. The patients were categorized according to donor age at the time of the transplant: ≥ 60 years (elderly donor group, n = 21) and <60 years (young donor group, n = 24). We reviewed the renal function of the recipients and pathologic findings of the graft including interstitial fibrosis score, tubular atrophy score, tubular atrophy and interstitial fibrosis grades, and arteriosclerosis up to 2 years posttransplantation. RESULTS: Significant differences were observed in the preoperative creatinine clearance of the donor, prevalence of hypertension in the donor, and age of the recipient. Serum creatinine levels in the elderly donor group were significantly higher from 2 months to 1 year posttransplantation, and the estimated glomerular filtration rate was significantly lower from 7 days to 1 year posttransplantation. However, the decline in estimated glomerular filtration rate from 14 days to up to 2 years posttransplantation was similar in the 2 groups. There was no significant difference in the renal biopsy findings between the 2 groups except for arteriosclerosis 1 year posttransplantation. CONCLUSION: Kidney grafts from elderly living donors were not associated with a deterioration in renal function, and their pathologic findings were comparable with those of young donors for up to 2 years posttransplantation.

Phase II trial of combination treatment with paclitaxel, carboplatin and cetuximab (PCE) as first-line treatment in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck (CSPOR-HN02).

Background: The standard of care for first-line treatment of recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN) is combination treatment with platinum, 5-FU and cetuximab (PFE). However, this regimen requires hospitalization to ensure proper hydration and continuous infusion of 5-FU, and causes severe nausea and anorexia. We evaluated the efficacy and safety of paclitaxel, carboplatin and cetuximab (PCE) as first-line treatment in patients with R/M SCCHN. Patients and methods: Eligibility criteria included recurrent and/or metastatic, histologically proven SCC of the oropharynx, oral cavity, hypopharynx or larynx; PS 0-1; adequate organ function; no suitable local therapy for R/M SCCHN; and no prior systemic chemotherapy for R/M SCCHN. Chemotherapy consisted of paclitaxel 100 mg/m2 on days 1, 8; carboplatin area under the blood concentration-time curve 2.5 on days 1, 8, repeated every 3 weeks for up to 6 cycles; and cetuximab at an initial dose of 400 mg/m2, followed by 250 mg/m2 weekly until disease progression or unacceptable toxicities. Primary end point was overall response rate. Secondary end points were safety, treatment completion rate, progression-free survival, overall survival, and clinical benefit rate. Planned sample size was 45 patients. Results: Forty-seven subjects were accrued from July 2013 to October 2014. Of 45 evaluable, 40 were male; median age was 63 years; Eastern Cooperative Oncology Group Performance Status was 0/1 in 23/22 cases; site was the hypopharynx/oropharynx/oral cavity/larynx in 17/11/10/7 cases; and 36/9 cases were smokers/nonsmokers, respectively. Overall response rate, the primary end point, was 40%. Median overall survival was 14.7 months and progression-free survival was 5.2 months. Grade 3/4 adverse events included neutropenia (68%), skin reaction (15%), fatigue (9%) and febrile neutropenia (9%). A potentially treatment-related death occurred in one patient with intestinal pneumonia. Conclusions: The PCE regimen shows promising activity with acceptable toxicity in the outpatient clinic. Further studies are needed to compare PCE with PFE in this population. Registered clinical trial number: UMIN000010507.

Surfactant protein D inhibits activation of non-small cell lung cancer-associated mutant EGFR and affects clinical outcomes of patients.

Tyrosine kinase inhibitor (TKI)-sensitive and TKI-resistant mutations of epidermal growth factor receptor (EGFR) are associated with lung adenocarcinoma. EGFR mutants were previously shown to exhibit ligand-independent activation. We have previously demonstrated that pulmonary surfactant protein D (SP-D, SFTPD) suppressed wild-type EGFR signaling by blocking ligand binding to EGFR. We herein demonstrate that SFTPD downregulates ligand-independent signaling in cells harboring EGFR mutations such as TKI-sensitive exon 19 deletion (Ex19del) and L858R mutation as well as TKI-resistant T790M mutation, subsequently suppressing cellular growth and motility. Lectin blotting and ligand blotting in lung cancer cell lines suggested that EGFR mutants express oligomannose-type N-glycans and interact with SFTPD directly. Cross-linking assay indicated that SFTPD inhibits ligand-independent dimerization of EGFR mutants. We also demonstrated that SFTPD reduced dimerization-independent phosphorylation of Ex19del and T790M EGFR mutants using point mutations that disrupted the asymmetric dimer interface. It was confirmed that SFTPD augmented the viability-suppressing effects of EGFR-TKIs. Furthermore, retrospective analysis of 121 patients with lung adenocarcinoma to examine associations between serum SFTPD levels and clinical outcome indicated that in TKI-treated patients with lung cancer harboring EGFR mutations, including Ex19del or L858R, high serum SFTPD levels correlated with a lower number of distant metastases and prolonged overall survival and progression-free survival. These findings suggest that SFTPD downregulates both TKI-sensitive and -resistant EGFR mutant signaling, and SFTPD level is correlated with clinical outcome. These findings illustrate the use of serum SFTPD level as a potential marker to estimate the efficacy of EGFR-TKIs.

Efficacy and safety of rebamipide liquid for chemoradiotherapy-induced oral mucositis in patients with head and neck cancer: a multicenter, randomized, double-blind, placebo-controlled, parallel-group phase II study.

BACKGROUND: Recent preclinical and phase I studies have reported that rebamipide decreased the severity of chemoradiotherapy-induced oral mucositis in patients with oral cancer. This placebo-controlled randomized phase II study assessed the clinical benefit of rebamipide in reducing the incidence of severe chemoradiotherapy-induced oral mucositis in patients with head and neck cancer (HNC). METHODS: Patients aged 20-75 years with HNC who were scheduled to receive chemoradiotherapy were enrolled. Patients were randomized to receive rebamipide 2% liquid, rebamipide 4% liquid, or placebo. The primary endpoint was the incidence of grade ≥ 3 oral mucositis determined by clinical examination and assessed by central review according to the Common Terminology Criteria of Adverse Events version 3.0. Secondary endpoints were the time to onset of grade ≥ 3 oral mucositis and the incidence of functional impairment (grade ≥ 3) based on the evaluation by the Oral Mucositis Evaluation Committee. RESULTS: From April 2014 to August 2015, 97 patients with HNC were enrolled, of whom 94 received treatment. The incidence of grade ≥ 3 oral mucositis was 29% and 25% in the rebamipide 2% and 4% groups, respectively, compared with 39% in the placebo group. The proportion of patients who did not develop grade ≥ 3 oral mucositis by day 50 of treatment was 57.9% in the placebo group, whereas the proportion was 68.0% in the rebamipide 2% group and 71.3% in the rebamipide 4% group. The incidences of adverse events potentially related to the study drug were 16%, 26%, and 13% in the placebo, rebamipide 2%, and rebamipide 4% groups, respectively. There was no significant difference in treatment compliance among the groups. CONCLUSIONS: The present phase II study suggests that mouth washing with rebamipide may be effective and safe for patients with HNC receiving chemoradiotherapy, and 4% liquid is the optimal dose of rebamipide. TRIAL REGISTRATION: ClinicalTrials.gov under the identifier NCT02085460 (the date of trial registration: March 11, 2014).

Outcomes of fulvestrant therapy among japanese women with advanced breast cancer: a retrospective multicenter cohort study (JBCRG-C06; Safari).

PURPOSE: This retrospective study evaluated the effect of clinical background and treatment line on time to treatment failure (TTF) in advanced/metastatic breast cancer (AMBC) patients receiving F500 in Japan (UMIN 000015168). METHODS: Patients who commenced F500 treatment were registered at 16 sites in Japan. Correlations between baseline clinicopathological factors, treatment line, and TTF were investigated by Kaplan-Meier analysis. TTF data were analyzed using univariate analysis and multivariate analysis with a Cox proportional hazards model. RESULTS: Data for 1072 patients were available; 1031 patients (96.2%) were evaluable for efficacy. F500 was administered as first-line treatment in 2.0%, second-line in 22.7%, third-line in 26.7%, and ≥fourth-line in 48.6% patients. Median TTF was 5.4 months. Multivariate analysis found that earlier F500 use (first and second vs. third vs. ≥fourth line; hazard ratio (HR) = 0.80, 95% confidence interval (CI) 0.74-0.86; P < 0.001), longer period from AMBC diagnosis to F500 use (≥3 vs. <3 years; HR 0.60, 95% CI 0.51-0.70; P < 0.001), and no prior palliative chemotherapy administered for unresectable or metastatic breast cancer (no vs. yes; HR 0.69, 95% CI 0.60-0.80; P < 0.001) were associated with significantly longer TTF. Among 691 patients, where information on histologic/nuclear grade was available, a low grade was also associated with a longer TTF, but this finding was not maintained among patients with recurrent breast cancer (N = 558). Among women with recurrent breast cancer, a longer DFI between a patient's initial breast cancer diagnosis and their recurrence was associated with a longer TTF on F500 therapy. CONCLUSIONS: Our study showed that treatment period of F500 was longer when used in earlier-line treatment. For patients on F500, TTF was also longer for patients who had not received prior palliative chemotherapy and for those who had a longer period from their AMBC diagnosis to F500 use.

A phase I/II trial of intrabone marrow cord blood transplantation and comparison of the hematological recovery with the Japanese nationwide database.

Intrabone marrow cord blood transplantation (IB-CBT) was proposed as a promising treatment modality to improve hematological recovery. However, clinical advantages of IB-CBT over conventional IV CBT have been unclear. We conducted a prospective single-center trial of IB-CBT to evaluate its safety and superiority in terms of hematological recovery. Fifteen adults with hematological malignancies were enrolled. A thawed and unwashed single cord blood unit was injected into the bilateral superior-posterior iliac crests under local anesthesia. Engraftments of neutrophils and platelets were achieved in 13 cases, with medians of 17 and 45 days, respectively. For the control, we extracted data from the Japanese nationwide database and compared the hematological recovery of contemporaneously transplanted 1135 CBT cases. Multivariate analysis revealed that IB-CBT enhanced platelet recovery (hazard ratio, 2.13; P=0.007), but neutrophil recovery did not differ significantly (hazard ratio, 1.70; P=0.19). Better donor chimerism was seen in the bone marrow of the ilium than of the sternum on day 14, suggesting that the local hematopoiesis at the injected site was established earlier than that at the remote bone marrow site. Collectively, IB-CBT was well tolerated and may enhance local engraftment, which promotes prompter platelet recovery than does IV-CBT.

Long-term follow-up of composite bladder augmentation incorporating stomach in a multi-institutional cohort of patients with cloacal exstrophy.

INTRODUCTION: Composite bladder augmentation, incorporating gastric and bowel segments, has the theoretical advantage of metabolic neutrality while potentially avoiding the morbidities of gastrocystoplasty, such as hematuria-dysuria syndrome. The most common indication for this operation is a paucity of bowel, such as in cloacal exstrophy. Despite several early descriptive studies of this technique, there are no reports, to date, of long-term follow-up in this population. OBJECTIVE: To describe the outcomes of composite bladder augmentation utilizing stomach in a cohort of cloacal exstrophy patients. MATERIALS AND METHODS: A retrospective review of cloacal exstrophy patients who underwent composite bladder augmentation from 1984 to 2006 at two institutions was performed. The incidence of mortality and morbidities related to augmentation was evaluated. RESULTS: Eleven patients with cloacal exstrophy underwent composite bladder augmentation. Median age at initial augmentation was 6.4 years (interquartile range (IQR) 4.4-9.1). Median follow-up was 13.2 years (IQR 11.2-24.6). The Summary table describes the types of composite bladder augmentations. Of the three patients with pre-operative metabolic acidosis, two improved with composite bladder augmentation and one developed metabolic alkalosis. Three developed hematuria-dysuria syndrome: one improved with staged ileocystoplasty, and two had persistent symptoms successfully treated with H2 receptor blockers. Two of 11 developed symptomatic bladder stones. There were no reported bladder perforations, bladder malignancies, conversions to incontinent urinary diversions, or deaths. CONCLUSION: With long-term follow-up, very few patients developed metabolic acidosis/alkalosis after composite bladder augmentation. The composite bladder augmentation will continue to be used in patients with cloacal exstrophy, in order to minimize the impact on the pre-existing short gut in these patients.

A pre-operative predictive score for the outcome of eccentric rotational acetabular osteotomy in the treatment of acetabular dysplasia and early osteoarthritis of the hip in adults.

AIMS: The influence of identifiable pre-operative factors on the outcome of eccentric rotational acetabular osteotomy (ERAO) is unknown. We aimed to determine the factors that might influence the outcome, in order to develop a scoring system for predicting the prognosis for patients undergoing this procedure. PATIENTS AND METHODS: We reviewed 700 consecutive ERAOs in 54 men and 646 women with symptomatic acetabular dysplasia or early onset osteoarthritis (OA) of the hip, which were undertaken between September 1989 and March 2013. The patients' pre-operative background, clinical and radiological findings were examined retrospectively. Multivariate Cox regression analysis was performed using the time from the day of surgery to a conversion to total hip arthroplasty (THA) as an endpoint. A risk score was calculated to predict the prognosis for conversion to THA, and its predictive capacity was investigated. RESULTS: The congruity of the hip, age, the pre-operative minimum width of the joint space and range of abduction were identified as factors predicting conversion to THA. For three groups of patients (scoring 0 to 5, 6 to 7, and 8 to 9 points), the Kaplan-Meier event-free rates of survival at 15 years post-operatively for conversion to THA were 99.6%, 85.2% and 67.3%, respectively. CONCLUSION: These four pre-operative factors are easily measured and predict the prognosis for patients following ERAO. They may be used for decision making when offering surgical treatment to patients with acetabular dysplasia and early onset osteoarthritis. Cite this article: Bone Joint J 2016;98-B:1326-32.

Effects of neoadjuvant chemotherapy with or without zoledronic acid on pathological response: A meta-analysis of randomised trials.

PURPOSE: The addition of bisphosphonates to adjuvant therapy improves survival in postmenopausal breast cancer (BC) patients. We report a meta-analysis of four randomised trials of neoadjuvant chemotherapy (CT) +/- zoledronic acid (ZA) in stage II/III BC to investigate the potential for enhancing the pathological response. METHODS: Individual patient data from four prospective randomised clinical trials reporting the effect of the addition of ZA on the pathological response after neoadjuvant CT were pooled. Primary outcomes were pathological complete response in the breast (pCRb) and in the breast and lymph nodes (pCR). Trial-level and individual patient data meta-analyses were done. Predefined subgroup-analyses were performed for postmenopausal women and patients with triple-negative BC. RESULTS: pCRb and pCR data were available in 735 and 552 patients respectively. In the total study population ZA addition to neoadjuvant CT did not increase pCRb or pCR rates. However, in postmenopausal patients, the addition of ZA resulted in a significant, near doubling of the pCRb rate (10.8% for CT only versus 17.7% with CT+ZA; odds ratio [OR] 2.14, 95% confidence interval [CI] 1.01-4.55) and a non-significant benefit of the pCR rate (7.8% for CT only versus 14.6% with CT+ZA; OR 2.62, 95% CI 0.90-7.62). In patients with triple-negative BC a trend was observed favouring CT+ZA. CONCLUSION: This meta-analysis shows no impact from the addition of ZA to neoadjuvant CT on pCR. However, as has been seen in the adjuvant setting, the addition of ZA to neoadjuvant CT may augment the effects of CT in postmenopausal patients with BC.

Lymph node density predicts lung metastases in oral squamous cell carcinoma.

The association between lymph node density and survival free of lung metastases in oral squamous cell carcinoma (SCC), has not been investigated so far to our knowledge. Lymph node density ≧ 0.07 has been reported by a multicentre international study to be a significant predictor of shorter survival in patients with oral SCC who have invaded nodes. We investigated whether a lymph node density of ≧ 0.07 correlates with shorter overall survival, survival free of distant metastases, and survival free of lung metastases, in patients with oral SCC and invaded lymph nodes. Thirty-five patients with histologically-confirmed invaded lymph nodes werestudied. Their density was calculated as the ratio of the number of invaded lymph nodes:total number of nodes. A density of ≧ 0.07 correlated significantly with shorter overall survival (p<0.02), survival free of distant metastases (p<0.01), and survival free of lung metastases (p<0.01) on log rank testing. On testing by Cox's proportional hazards model of multivariate survival analysis with adjustment for the pathological stage (pstage IV/pstage III), and invaded surgical margins or extracapsular spread, or both, we found that lymph node density ≧ 0.07 was associated with significantly shorter survival (p<0.02). We conclude that lymph node density predicts lung metastases in patients with oral SCC.

Hes1 suppresses acute myeloid leukemia development through FLT3 repression.

In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix-loop-helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL-AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL-AF9-expressing immortalized and leukemia cells with a Hes1- or RBPJ-null background. MLL-AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL-AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL-AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression profiles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.

Superovulatory response in Japanese Black cows receiving a single subcutaneous porcine follicle-stimulating hormone treatment or six intramuscular treatments over three days.

To reduce labor for superovulation treatment by twice-daily intramuscular (im) administration of FSH for more than 3 to 4 days, we investigated the superovulatory responses of Japanese Black cows to porcine FSH (pFSH) used as a single subcutaneous (sc) administration at two different doses in two different volumes of saline. In experiment 1, 20 Armour units (AU) of pFSH dissolved in either 10 mL (treatment A; n = 14) or 50 mL (treatment B; n = 14) of saline was administered subcutaneously in the neck region. In experiment 2, 30 AU of pFSH dissolved in either 10 mL (treatment C; n = 15) or 50 mL (treatment D; n = 15) of saline was administered subcutaneously in the neck region. The control animals in experiment 1 (n = 14) and experiment 2 (n = 15) received 20 AU of pFSH administered intramuscularly twice daily in decreasing doses for more than 3 days. In experiment 1, mean (±SEM) numbers of CL (15.4 ± 2.5, 18.1 ± 3.4, and 17.2 ± 2.6), total number of ova and embryos (12.9 ± 1.4, 15.9 ± 3.5, and 16.2 ± 2.8), and transferable embryos (7.5 ± 2.0, 10.4 ± 2.8, and 8.0 ± 2.1) did not differ among treatments A, B, and control. In experiment 2, mean (±SEM) numbers of CL (20.5 ± 4.3, 20.4 ± 2.7, and 20.1 ± 3.4), total number of ova and embryos (21.7 ± 4.2, 17.3 ± 3.4, and 16.5 ± 3.2), and transferable embryos (8.1 ± 1.6, 9.3 ± 2.2, and 9.5 ± 1.9) did not differ among treatments C, D, and control. Although there were no differences in serum pFSH concentrations among the three treatments at each of the time points in experiment 1, in experiment 2, the serum pFSH concentration at 6 and 8 hours after pFSH administration in treatment C (3.1 ± 0.8, 2.7 ± 0.5 ng/mL, mean ± SEM) was significantly greater (P < 0.05) than in the control (0.7 ± 0.1, 1.1 ± 0.2 ng/mL). At 10 hours after administration, the pFSH concentration had decreased and there were no differences among the three treatments at subsequent time points. These results suggest that increasing the volume of saline or the dose of pFSH does not affect the absorption pattern of pFSH administered as a single sc administration. In conclusions, single sc administration of pFSH at a dose of 20 or 30 AU dissolved in 10 or 50 mL of saline is able to induce a superovulatory response comparable with that obtained by twice-daily im administration in Japanese Black cows.

LIM-domain protein AJUBA suppresses malignant mesothelioma cell proliferation via Hippo signaling cascade.

Malignant mesothelioma (MM) is one of the most aggressive neoplasms usually associated with asbestos exposure and is highly refractory to current therapeutic modalities. MMs show frequent activation of a transcriptional coactivator Yes-associated protein (YAP), which is attributed to the neurofibromatosis type 2 (NF2)-Hippo pathway dysfunction, leading to deregulated cell proliferation and acquisition of a malignant phenotype. However, the whole mechanism of disordered YAP activation in MMs has not yet been well clarified. In the present study, we investigated various components of the NF2-Hippo pathway, and eventually found that MM cells frequently showed downregulation of LIM-domain protein AJUBA, a binding partner of large tumor suppressor type 2 (LATS2), which is one of the last-step kinases of the NF2-Hippo pathway. Although loss of AJUBA expression was independent of the alteration status of other Hippo pathway components, MM cell lines with AJUBA inactivation showed a more dephosphorylated (activated) level of YAP. Immunohistochemical analysis showed frequent downregulation of AJUBA in primary MMs, which was associated with YAP constitutive activation. We found that AJUBA transduction into MM cells significantly suppressed promoter activities of YAP-target genes, and the suppression of YAP activity by AJUBA was remarkably canceled by knockdown of LATS2. In connection with these results, transduction of AJUBA-expressing lentivirus significantly inhibited the proliferation and anchorage-independent growth of the MM cells that harbored ordinary LATS family expression. Taken together, our findings indicate that AJUBA negatively regulates YAP activity through the LATS family, and inactivation of AJUBA is a novel key mechanism in MM cell proliferation.

Surfactant protein D suppresses lung cancer progression by downregulation of epidermal growth factor signaling.

Surfactant protein D (SP-D) is a member of the collectin family that has an important role in maintaining pulmonary homeostasis. In this study, we demonstrated that SP-D inhibited the proliferation, migration and invasion of A549 human lung adenocarcinoma cells. We found that SP-D suppressed epidermal growth factor (EGF) signaling in A549 cells, H441 human lung adenocarcinoma cells and human EGF receptor (EGFR) stable expression CHO-K1 cells. A binding study using (125)I-EGF demonstrated that SP-D downregulated the binding of EGF to EGFR. A ligand blot indicated that SP-D bound to EGFR, and a lectin blot suggested that EGFR in A549 cells had both high-mannose type and complex type N-glycans. We purified the recombinant extracellular domain of EGFR (soluble EGFR=soluble EGFR (sEGFR)), and demonstrated that SP-D directly bound to sEGFR in a Ca(2+)-dependent manner. The binding of SP-D to sEGFR was suppressed by EDTA, mannose or N-glycopeptidase F treatment. Mass spectrometric analysis indicated that N-glycans in domain III of EGFR were of a high-mannose type. These data suggest that SP-D reduces EGF binding to EGFR through the interaction between the carbohydrate recognition domain of SP-D and N-glycans of EGFR, and downregulates EGF signaling. Our finding suggests the novel type of regulation system of EGF signaling involving lectin-to-carbohydrate interaction and downregulation of ligand binding.

Clinical applications of analysis of plasma circulating complete hydatidiform mole pregnancy-associated miRNAs in gestational trophoblastic neoplasia: a preliminary investigation.

The aim of this study was to investigate the clinical application of plasma complete hydatidiform mole pregnancy-associated microRNAs (CHM-miRNAs: hsa-miR-520b, hsa-miR-520f and hsa-miR-520c-3p). We measured plasma CHM-miRNA concentration by real-time quantitative reverse transcriptase polymerase chain reaction in two cases of CHM resulting in gestational trophoblastic neoplasia later. As progress of treatments in both cases, the plasma concentrations of CHM-miRNAs showed a decreasing tendency similar to the pattern for serum hCG concentration, but exhibited a transient increasing tendency after each course of chemotherapy, suggesting that the plasma CHM-miRNAs could be an additional follow-up marker for malignant changes of CHM.