Bacillaceae serine proteases and Streptomyces epsilon-poly-L-lysine synergistically inactivate Caliciviridae by inhibiting RNA genome release.

Human norovirus (HuNoV) is an enteric infectious pathogen belonging to the Caliciviridae family that causes occasional epidemics. Circulating alcohol-tolerant viral particles that are readily transmitted via food-borne routes significantly contribute to the global burden of HuNoV-induced gastroenteritis. Moreover, contact with enzymes secreted by other microorganisms in the environment can impact the infectivity of viruses. Hence, understanding the circulation dynamics of Caliciviridae is critical to mitigating epidemics. Accordingly, in this study, we screened whether environmentally abundant secretase components, particularly proteases, affect Caliciviridae infectivity. Results showed that combining Bacillaceae serine proteases with epsilon-poly-L-lysine (EPL) produced by Streptomyces-a natural antimicrobial-elicited anti-Caliciviridae properties, including against the epidemic HuNoV GII.4_Sydney_2012 strain. In vitro and in vivo biochemical and virological analyses revealed that EPL has two unique synergistic viral inactivation functions. First, it maintains an optimal pH to promote viral surface conformational changes to the protease-sensitive structure. Subsequently, it inhibits viral RNA genome release via partial protease digestion at the P2 and S domains in the VP1 capsid. This study provides new insights regarding the high-dimensional environmental interactions between bacteria and Caliciviridae, while promoting the development of protease-based anti-viral disinfectants.

Doublet chemotherapy, triplet chemotherapy, or doublet chemotherapy combined with radiotherapy as neoadjuvant treatment for locally advanced oesophageal cancer (JCOG1109 NExT): a randomised, controlled, open-label, phase 3 trial.

BACKGROUND: Neoadjuvant therapy is the standard treatment for patients with locally advanced oesophageal squamous cell carcinoma (OSCC). However, the prognosis remains poor and more intensive neoadjuvant treatment might be needed to improve patient outcomes. We therefore aimed to compare the efficacy and safety of neoadjuvant doublet chemotherapy, triplet chemotherapy, and doublet chemotherapy plus radiotherapy in patients with previously untreated locally advanced OSCC. METHODS: In this randomised, open-label, phase 3 trial, patients aged 20-75 years with previously untreated locally advanced OSCC and an Eastern Cooperative Oncology Group performance status of 0 or 1 were recruited from 44 centres across Japan. Patients were randomly assigned (1:1:1) centrally via a web-based system to receive neoadjuvant doublet chemotherapy (two courses of fluorouracil [800 mg/m2 per day intravenously on days 1-5] and cisplatin [80 mg/m2 per day on day 1] separated by an interval of 3 weeks [NeoCF]), triplet chemotherapy (three courses of fluorouracil [750 mg/m2 per day on days 1-5], cisplatin [70 mg/m2 per day on day 1], and docetaxel [70 mg/m2 per day on day 1] repeated every 3 weeks [NeoCF+D]), or doublet chemotherapy (two courses of fluorouracil [1000 mg/m2 per day on days 1-4] and cisplatin [75 mg/m2 per day on day 1] separated by an interval of 4 weeks) plus 41·4 Gy radiotherapy [NeoCF+RT]) followed by oesophagectomy with regional lymph node dissection. Randomisation was stratified by T stage and institution. Participants, investigators, and those assessing outcomes were not masked to group assignment. The primary endpoint was overall survival, analysed by intention to treat. Analysis of safety included all patients who received at least one course of chemotherapy, and analysis of surgical complications included those who also underwent surgery. This study is registered with the Japan Registry of Clinical Trials, jRCTs031180202, and the trial is complete. FINDINGS: A total of 601 patients (529 male individuals and 72 female individuals) were randomly assigned between Dec 5, 2012, and July 20, 2018, with 199 patients in the NeoCF group, 202 patients in the NeoCF+D group, and 200 patients in the NeoCF+RT group. Compared with the NeoCF group, during a median follow-up period of 50·7 months (IQR 23·8-70·7), the 3-year overall survival rate was significantly higher in the NeoCF+D group (72·1% [95% CI 65·4-77·8] vs 62·6% [55·5-68·9]; hazard ratio [HR] 0·68, 95% CI 0·50-0·92; p=0·006) but not in the NeoCF+RT group (68·3% [61·3-74·3]; HR 0·84, 0·63-1·12; p=0·12). Grade 3 or higher febrile neutropenia occurred in two (1%) of 193 patients in the NeoCF group, 32 (16%) of 196 patients in the NeoCF+D group, and nine (5%) of 191 patients in the NeoCF+RT group. Treatment-related adverse events leading to termination of neoadjuvant therapy were more common in the NeoCF+D group (18 [9%] of 202 participants) than in the NeoCF+RT group (12 [6%] of 200) and NeoCF group (eight [4%] of 199). There were three (2%) treatment-related deaths during neoadjuvant therapy in the NeoCF group, four (2%) deaths in the NeoCF+D group, and two (1%) deaths in the NeoCF+RT group. Grade 2 or higher postoperative pneumonia, anastomotic leak, and recurrent laryngeal nerve paralysis were reported in 19 (10%), 19 (10%), and 28 (15%) of 185 patients, respectively, in the NeoCF group; 18 (10%), 16 (9%), and 19 (10%) of 183 patients, respectively, in the NeoCF+D group; and 23 (13%), 23 (13%), and 17 (10%) of 178 patients, respectively, in the NeoCF+RT group. The in-hospital deaths following surgery included three deaths in the NeoCF group, two deaths in the NeoCF+D group, and one in the NeoCF+RT group. INTERPRETATION: Neoadjuvant triplet chemotherapy followed by oesophagectomy resulted in a statistically significant overall survival benefit compared with doublet chemotherapy and might be the new standard of care for locally advanced OSCC who are in good condition in Japan. Neoadjuvant doublet chemotherapy plus radiotherapy did not show significant improvement of survival compared with doublet chemotherapy. FUNDING: Japan Agency for Medical Research and Development and National Cancer Center Research and Development Fund.

The roles of tight junction protein cingulin in human endometrioid endometrial cancer.

The bicellular tight junction molecule cingulin (CGN) binds to microtubules in centrosomes. Furthermore, CGN contributes to the tricellular tight junction (tTJ) proteins lipolysis-stimulated lipoprotein receptor (LSR) and tricellulin (TRIC). CGN as well as LSR decreased during the malignancy of endometrioid endometrial cancer (EEC). Although tTJ protein LSR is involved in the malignancy of some cancers, including EEC, the role of CGN is unknown. In this study, we investigated the roles of CGN with tTJ proteins in human EEC cells by using the CGN-overexpressing EEC cell line Sawano. In 2D cultures, CGN was colocalized with LSR and TRIC at tTJ or at γ-tubulin-positive centrosomes. In immunoprecipitation with CGN antibodies, CGN directly bound to LSR, TRIC, and ß-tubulin. Knockdown of CGN by the siRNA decreased the epithelial barrier and enhanced cell proliferation, migration and invasion, as well as knockdown of LSR. In the Sawano cells cocultured with normal human endometrial stromal cells, knockdown of CGN decreased expression of LSR and TRIC via MAPK and AMPK pathways. In 2.5D cultures, knockdown of CGN induced the formation of abnormal cysts and increased the permeability of FD-4 to the lumen. In 2D and 2.5D cultures, treatment with ß-estradiol with or without EGF or TGF-ß decreased CGN expression and the epithelial permeability barrier and enhanced cell migration, and pretreatment with EW7197+AG1478, U0126 or an anti-IL-6 antibody prevented this. In conclusion, CGN, with tTJ proteins might suppress the malignancy of human EEC and its complex proteins are sensitive to estrogen and growth factors derived from stromal cells.

Serum growth differentiation factor 15 is a novel biomarker with high predictive capability for liver cancer occurrence in patients with MASLD regardless of liver fibrosis.

BACKGROUND AND AIMS: Although metabolic dysfunction-associated steatotic liver disease (MASLD) patients with a Fib-4 index >1.3 are recommended for fibrosis evaluation via elastography or biopsy, a more convenient method identifying high-risk populations requiring follow-up is needed. We explored the utility of serum levels of growth differentiation factor-15 (GDF15), a cell stress-responsive cytokine related to metabolic syndrome, for stratifying the risk of clinical events in MASLD patients. METHODS: Serum GDF15 levels were measured in 518 biopsy-performed MASLD patients, 216 MASLD patients for validation, and 361 health checkup recipients with MASLD. RESULTS: In the biopsy-MASLD cohort, multivariate analysis indicated that the serum GDF15 level was a risk factor for liver cancer, independent of the fibrosis stage or Fib-4 index. Using a GDF15 cutoff of 1.75 ng/mL based on the Youden index, high-GDF15 patients, regardless of fibrosis status, had a higher liver cancer incidence rate. While patients with a Fib-4 index <1.3 or low-GDF15 rarely developed liver cancer, high-GDF15 patients with a Fib-4 index >1.3 developed liver cancer and decompensated liver events at significantly higher rates and had poorer prognoses. In the validation cohort, high-GDF15 patients had significantly higher incidences of liver cancer and decompensated liver events and poorer prognoses than low-GDF15 patients, whether limited to high-Fib-4 patients. Among health checkup recipients with MASLD, 23.0% had a Fib-4 index >1.3, 2.7% had a Fib-4 index >1.3 and >1.75 ng/mL GDF15. CONCLUSIONS: Serum GDF15 is a biomarker for liver cancer with high predictive capability and is useful for identifying MASLD patients requiring regular surveillance.

Health-related quality-of-life analysis from KEYNOTE-590: pembrolizumab plus chemotherapy versus chemotherapy for advanced esophageal cancer.

BACKGROUND: In the KEYNOTE-590 study, first-line pembrolizumab plus chemotherapy provided statistically significant improvement in overall survival, progression-free survival, and objective response rate compared with chemotherapy, with a manageable safety profile in patients with advanced esophageal cancer. Prespecified health-related quality-of-life (HRQoL) outcomes are reported. MATERIALS AND METHODS: Change from baseline to week 18 in the EORTC Quality of Life Questionnaire Core 30 (QLQ-C30) global health status/QoL (GHS/QoL) and QLQ-Esophageal cancer module (OES18) dysphagia, pain, and reflux scales were evaluated. RESULTS: The HRQoL analysis included 730 patients who received treatment and completed ≥1 HRQoL assessment. Least squares mean (LSM) change from baseline to week 18 was similar between treatment groups for QLQ-C30 GHS/QoL and physical functioning and QLQ-OES18 reflux scales. The QLQ-OES18 dysphagia (LSM difference, -5.54; 95% CI, -10.93 to -0.16) and pain (LSM difference, -2.94; 95% CI, -5.86 to -0.02) scales favored pembrolizumab plus chemotherapy over placebo plus chemotherapy. Median time to confirmed deterioration (TTD) was similar between treatment groups for QLQ-C30 GHS/QoL and physical functioning and QLQ-OES18 dysphagia and reflux scales. Compared with chemotherapy, pembrolizumab plus chemotherapy prolonged median TTD, as seen on the QLQ-OES18 pain scale (HR, 0.69; 95% CI, 0.51 to 0.95). CONCLUSION: The use of pembrolizumab plus chemotherapy maintained HRQoL at week 18 relative to baseline and was comparable with placebo plus chemotherapy. These HRQoL results together with published reports of efficacy, support the use of pembrolizumab plus chemotherapy as first-line therapy for advanced/metastatic esophageal cancer. CLINICALTRIALS.GOV ID: NCT03189719.

Definitive chemoradiotherapy induces T-cell-inflamed tumor microenvironment in unresectable locally advanced esophageal squamous cell carcinoma.

BACKGROUND: Chemoradiotherapy (CRT) modulates the tumor immune microenvironment of multiple cancer types, including esophageal cancer, which potentially induces both immunogenicity and immunosuppression by upregulating the presentation of tumor-specific antigens and immune checkpoint molecules in tumors, respectively. The prognostic effects of immune modification by CRT in esophageal squamous cell carcinoma (ESCC) remain controversial because of the lack of detailed immunological analyses using paired clinical specimens before and after CRT. We aimed to clarify the immunological changes in the tumor microenvironment caused by CRT and elucidate the predictive importance of clinical response and prognosis and the rationale for the necessity of subsequent programmed cell death protein 1 (PD-1) inhibitor treatment. METHODS: In this study, we performed a comprehensive immunological analysis of paired biopsy specimens using multiplex immunohistochemistry before and after CRT in patients with unresectable locally advanced ESCC. RESULTS: CRT significantly increased the intra-tumoral infiltration and PD-1 expression of CD8+ T cells and conventional CD4+ T cells but decreased those of regulatory T cells and the accumulation of tumor-associated macrophages. Multivariate analysis of tumor-infiltrating T-cell phenotypes revealed that the density of PD-1+CD8+ T cells in the tumor after CRT could predict a confirmed complete response and favorable survival. CONCLUSIONS: This study showed that CRT improved the immunological characteristics of unresectable locally advanced ESCC and identified the density of PD-1+CD8+ T cells as a predictive factor for prognosis. This finding supports the rationale for the necessity of subsequent PD-1 inhibitor treatment.

Accuracy of the Majority Voting Method with Multiple IOL Power Formulae.

Purpose: This study aimed to evaluate the efficacy of a majority decision algorithm that integrates intraoperative aberrometry (IA) and two intraocular lens (IOL) frequency formulas. The primary objective was to compare the accuracy of three formulas (IA; Sanders, Retzlaff, and Kraff/Theoretical (SRK/T); and Barrett Universal II (BUII)), in achieving emmetropia in eyes implanted with TFNT lenses (Alcon). Patients and Methods: A total of 145 eyes of 145 patients were included in the evaluation. Preoperative data were obtained from IOLMaster 700, while intraoperative data were collected from ORA SYSTEMTM. Visual acuity ≥0.8 at the 3-month post-surgery mark was confirmed. We assessed refractive prediction error (RPE), which is the difference between predicted refraction (PR) and postoperative subjective refraction. This evaluation aimed to identify the optimal IOL power with the implemented algorithm. Results: Among the 145 eyes evaluated, 55.9%, 78.7%, and 97.2% achieved postoperative subjective refraction within ±0.13 Diopters (D), ±0.25 D, and ±0.50 D, respectively. The percentages of eyes within ±0.25 D of PR varied by formula type, with values of 57%, 57%, and 54% for IA, BUII, and SRK/T, respectively. For eyes with short to medium axial length (AL<26.00 mm), the percentages within ±0.25 D of RPE were 52%, 58%, and 58% for IA, SRK/T, and BUII, respectively. In contrast, for eyes with long axial length (≥26.00 mm) the percentages were 68%, 52%, and 45% for IA, BUII, and SRK/T, respectively. Conclusion: The proposed majority decision algorithm incorporating IA and two IOL frequency formulas was effective in reducing postoperative refractive error. IA was particularly beneficial for eyes with long axial length. These findings suggest the algorithm has potential to optimize IOL power selection to improve quality of life of patients and clinical practice outcomes.

First-line pembrolizumab plus chemotherapy for advanced/metastatic esophageal cancer: 1-year extended follow-up in the Japanese subgroup of the phase 3 KEYNOTE-590 study.

BACKGROUND: First-line pembrolizumab plus chemotherapy (pembrolizumab-chemotherapy) demonstrated improved efficacy and a manageable safety profile versus placebo plus chemotherapy (placebo-chemotherapy) in the subgroup analysis of Japanese patients with advanced/metastatic esophageal cancer in KEYNOTE-590 at a median follow-up of 24.4 months. Longer-term data from the Japanese subgroup analysis of KEYNOTE-590 are reported. METHODS: Patients were randomly assigned 1:1 to pembrolizumab 200 mg or placebo every 3 weeks for ≤ 35 cycles plus chemotherapy (cisplatin 80 mg/m2 and 5-fluorouracil 800 mg/m2/day). Endpoints included overall survival (OS) and progression-free survival (PFS; investigator-assessed per RECIST v1.1; dual primary) and safety (secondary). Early tumor shrinkage (ETS) and depth of response (DpR) were assessed post hoc. RESULTS: Overall, 141 patients were enrolled in Japan. As of July 9, 2021, median follow-up was 36.6 months (range, 29.8-45.7). Pembrolizumab-chemotherapy showed a trend toward favorable OS (hazard ratio [HR], 0.70; 95% confidence interval [CI] 0.47-1.03) and PFS (0.57; 0.39-0.83) versus placebo-chemotherapy. In the pembrolizumab-chemotherapy group, patients with ETS ≥ 20% (55/74; 74.3%) versus < 20% (19/74; 25.7%) had favorable OS (HR, 0.23; 95% CI 0.12-0.42) and PFS (0.24; 0.13-0.43). Patients with DpR ≥ 60% (31/74; 41.9%) versus < 60% (43/74; 58.1%) had favorable OS (HR, 0.37; 95% CI 0.20-0.68) and PFS (0.24; 0.13-0.43). Grade 3-5 treatment-related adverse events occurred in 55/74 patients (74.3%) with pembrolizumab-chemotherapy and 41/67 patients (61.2%) with placebo-chemotherapy. CONCLUSIONS: With longer-term follow-up of Japanese patients with advanced/metastatic esophageal cancer, efficacy continued to favor pembrolizumab-chemotherapy compared with placebo-chemotherapy, with no new safety signals observed. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT03189719.

Therapeutic Aqueous Humor Concentrations of Latanoprost Attained in Rats by Administration in a Very-High-Molecular-Weight Hyaluronic Acid Eye Drop.

The temporal change in concentration of a novel medicine, Latanoprost (LP), was evaluated in the aqueous humor of rats (6-8-week-old Jcl:Wister rats) when delivered in a very-high-molecular-weight hyaluronic acid (vHiHA) eye drop. Animals were randomly assigned to three treatment groups (LP + vHiHA (LPvHiHA), commercial LP (cLP), and diluted LP (dLP)) and after instilling the eye drops, the aqueous humor (AH) was collected at 0.5, 1, 2, 4, and 6 h to measure the LP concentration using an enzyme-linked immunosorbent assay (ELISA). Although the LP concentration in the LPvHiHA eye drop formulation was 3.57 times lower than in the commercial eye drops used (cLP), the LP concentration in the AH following LPvHiHA administration reached a value close to that of cLP. The cLP was diluted to the same concentration of LP as in the LPvHiHA eye drops for the dLP group, but the LP concentration in the AH of these animals was lower than that of the LPvHiHA rats at all time points. The higher LP concentration in the AH of the LPvHiHA rats suggests that vHiHA may aid the transport of LP across the ocular surface epithelium.

Functional Studies of Deafness-Associated Pendrin and Prestin Variants.

Pendrin and prestin are evolutionary-conserved membrane proteins that are essential for normal hearing. Dysfunction of these proteins results in hearing loss in humans, and numerous deafness-associated pendrin and prestin variants have been identified in patients. However, the pathogenic impacts of many of these variants are ambiguous. Here, we report results from our ongoing efforts to experimentally characterize pendrin and prestin variants using in vitro functional assays. With previously established fluorometric anion transport assays, we determined that many of the pendrin variants identified on transmembrane (TM) 10, which contains the essential anion binding site, and on the neighboring TM9 within the core domain resulted in impaired anion transport activity. We also determined the range of functional impairment in three deafness-associated prestin variants by measuring nonlinear capacitance (NLC), a proxy for motor function. Using the results from our functional analyses, we also evaluated the performance of AlphaMissense (AM), a computational tool for predicting the pathogenicity of missense variants. AM prediction scores correlated well with our experimental results; however, some variants were misclassified, underscoring the necessity of experimentally assessing the effects of variants. Together, our experimental efforts provide invaluable information regarding the pathogenicity of deafness-associated pendrin and prestin variants.

Serotonergic regulation of cortical neurovascular coupling and hemodynamics upon awakening from sleep in mice.

Neurovascular coupling (NVC) is the functional hyperemia of the brain responding to local neuronal activity. It is mediated by astrocytes and affected by subcortical ascending pathways in the cortex that convey information, such as sensory stimuli and the animal condition. Here, we investigate the influence of the raphe serotonergic system, a subcortical ascending arousal system in animals, on the modulation of cortical NVC and cerebral blood flow (CBF). Raphe serotonergic neurons were optogenically activated for 30 s, which immediately awakened the mice from non-rapid eye movement sleep. This caused a biphasic cortical hemodynamic change: a transient increase for a few seconds immediately after photostimulation onset, followed by a large progressive decrease during the stimulation period. Serotonergic neuron activation increased intracellular Ca2+ levels in cortical pyramidal neurons and astrocytes, demonstrating its effect on the NVC components. Pharmacological inhibition of cortical neuronal firing activity and astrocyte metabolic activity had small hypovolemic effects on serotonin-induced biphasic CBF changes, while blocking 5-HT1B receptors expressed primarily in cerebral vasculature attenuated the decreasing CBF phase. This suggests that serotonergic neuron activation leading to animal awakening could allow the NVC to exert a hyperemic function during a biphasic CBF response, with a predominant decrease in the cortex.

Whole-transcriptome sequencing in advanced gastric or gastroesophageal cancer: A deep dive into its clinical potential.

Advanced gastric and gastroesophageal junction cancers (GC/GEJCs) harbor diverse molecular signatures, highlighting the need for intricate evaluations to identify potential therapeutic targets. Although whole-transcriptome sequencing (WTS) has emerged as a useful tool for understanding these molecular intricacies, its clinical implications have yet to be fully elucidated. This study evaluated the correlation between immunohistochemistry (IHC) and WTS, compared their clinical significance, and identified potential therapeutic targets undetectable through IHC alone. We enrolled 140 patients with advanced GC/GEJC and assessed them using IHC for six pivotal biomarkers: claudin-18 (CLDN18), human epidermal growth factor receptor 2 (HER2), multiple receptor tyrosine kinases (RTKs), and programmed death ligand 1 (PD-L1). Concurrently, WTS was employed as part of the analyses in MONSTAR-SCREEN-2, a multicenter multiomics study. IHC analysis revealed 16.4% HER2, 39.3% CLDN18 (2+/3 + ≥75%), and 15.8% PD-L1 (combined positive score ≥ 10) positivity, among other molecular markers. Significant correlations were observed between IHC and WTS for all six pivotal biomarkers. Among nineteen HER2 IHC-positive patients treated with anti-HER2 therapeutics, ERBB2 status in WTS was significantly associated with progression-free survival (ERBB2-high vs. -low: median 9.0 vs. 5.6 months, log-rank p = 0.046). IHC-based molecular profiling revealed significantly high expression of CLDN18 in RTK-negative patients, with 78.4% positive for either CLDN18 or PD-L1. Additionally, WTS revealed elevated expression of pivotal biomarkers in patients displaying negative targetable biomarkers via IHC. Our findings highlighted the significant correlation between IHC and WTS, reinforcing the clinical utility of WTS. A subset with IHC-negative but WTS-positive status may benefit from specific biomarker-targeted therapies.

Prognosis of patients with residual pathological disease after neoadjuvant docetaxel, cisplatin, and 5-fluorouracil therapy and surgery for esophageal squamous cell carcinoma: a retrospective cohort study.

Background: Docetaxel, cisplatin, and 5-fluorouracil (DCF) combination chemotherapy has been established as one of the standard neoadjuvant therapies for locally advanced esophageal squamous cell carcinoma (ESCC). However, little is known about prognostic factors in patients with residual pathological disease after neoadjuvant DCF followed by surgery for locally advanced ESCC who are candidates for adjuvant nivolumab. Objectives: This study aimed to investigate prognostic factors in patients with residual pathological disease after neoadjuvant DCF chemotherapy followed by surgery for locally advanced ESCC. Design: This was a retrospective cohort study. Methods: This retrospective cohort study included patients who received neoadjuvant DCF followed by surgery for locally advanced ESCC between June 2014 and January 2020 at the National Cancer Center Hospital East. Results: Among a total of 210 patients, 45 patients (21.4%) achieved a pathological complete response. The 3-year disease-free survival (DFS) rate was significantly lower in patients with residual pathological disease than in those with a pathological complete response [53.5% versus 74.5%; hazard ratio (HR): 2.09, 95% confidence interval (CI): 1.16-3.77, p = 0.01]. In patients with residual pathological disease (n = 165), multivariate analysis revealed that pathological node positivity (HR: 3.59, 95% CI: 1.92-6.71, p < 0.01), supraclavicular lymph node metastasis (HR: 2.15, 95% CI: 1.19-3.90, p = 0.01), and lymphovascular invasion (HR: 1.90, 95% CI: 1.14-3.17, p = 0.02) were significantly associated with poor DFS. Conclusion: In this largest-to-date cohort study, patients with residual pathological disease after neoadjuvant DCF followed by surgery for locally advanced ESCC had a poor prognosis. In these patients, pathological node positivity, including supraclavicular lymph node metastasis, and lymphovascular invasion were considered significant prognostic factors.

The Roles and Regulatory Mechanisms of Tight Junction Protein Cingulin and Transcription Factor Forkhead Box Protein O1 in Human Lung Adenocarcinoma A549 Cells and Normal Lung Epithelial Cells.

Tight junction (TJ) protein cingulin (CGN) and transcription factor forkhead box protein O1 (FOXO1) contribute to the development of various cancers. Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for some cancers. HDAC inhibitors affect the expression of both CGN and FOXO1. However, the roles and regulatory mechanisms of CGN and FOXO1 are unknown in non-small cell lung cancer (NSCLC) and normal human lung epithelial (HLE) cells. In the present study, to investigate the effects of CGN and FOXO1 on the malignancy of NSCLC, we used A549 cells as human lung adenocarcinoma and primary human lung epithelial (HLE) cells as normal lung tissues and performed the knockdown of CGN and FOXO1 by siRNAs. Furthermore, to investigate the detailed mechanisms in the antitumor effects of HDAC inhibitors for NSCLC via CGN and FOXO1, A549 cells and HLE cells were treated with the HDAC inhibitors trichostatin A (TSA) and Quisinostat (JNJ-2648158). In A549 cells, the knockdown of CGN increased bicellular TJ protein claudin-2 (CLDN-2) via mitogen-activated protein kinase/adenosine monophosphate-activated protein kinase (MAPK/AMPK) pathways and induced cell migration, while the knockdown of FOXO1 increased claudin-4 (CLDN-4), decreased CGN, and induced cell proliferation. The knockdown of CGN and FOXO1 induced cell metabolism in A549 cells. TSA and Quisinostat increased CGN and tricellular TJ protein angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) in A549. In normal HLE cells, the knockdown of CGN and FOXO1 increased CLDN-4, while HDAC inhibitors increased CGN and CLDN-4. In conclusion, the knockdown of CGN via FOXO1 contributes to the malignancy of NSCLC. Both HDAC inhibitors, TSA and Quisinostat, may have potential for use in therapy for lung adenocarcinoma via changes in the expression of CGN and FOXO1.

Functional studies of deafness-associated pendrin and prestin variants.

Pendrin and prestin are evolutionary conserved membrane proteins that are essential for normal hearing. Pendrin is an anion transporter required for normal development and maintenance of ion homeostasis in the inner ear, while prestin is a voltage-dependent motor responsible for cochlear amplification essential for high sensitivity and frequency selectivity of mammalian hearing. Dysfunction of these proteins result in hearing loss in humans, and numerous deafness-associated pendrin and prestin variants have been identified in patients. However, the pathogenic impacts of many of these variants are ambiguous. Here we report results from our ongoing efforts in experimentally characterizing pendrin and prestin variants using in vitro functional assays, providing invaluable information regarding their pathogenicity.

Multiomic molecular characterization of the response to combination immunotherapy in MSS/pMMR metastatic colorectal cancer.

BACKGROUND: Immune checkpoint inhibitor (ICI) combinations represent an emerging treatment strategies in cancer. However, their efficacy in microsatellite stable (MSS) or mismatch repair-proficient (pMMR) colorectal cancer (CRC) is variable. Here, a multiomic characterization was performed to identify predictive biomarkers associated with patient response to ICI combinations in MSS/pMMR CRC for the further development of ICI combinations. METHODS: Whole-exome sequencing, RNA sequencing, and multiplex fluorescence immunohistochemistry of tumors from patients with MSS/pMMR CRC, who received regorafenib plus nivolumab (REGONIVO) or TAS-116 plus nivolumab (TASNIVO) in clinical trials were conducted. Twenty-two and 23 patients without prior ICI from the REGONIVO and TASNIVO trials were included in this study. A biomarker analysis was performed using samples from each of these studies. RESULTS: The epithelial-mesenchymal transition pathway and genes related to cancer-associated fibroblasts were upregulated in the REGONIVO responder group, and the G2M checkpoint pathway was upregulated in the TASNIVO responder group. The MYC pathway was upregulated in the REGONIVO non-responder group. Consensus molecular subtype 4 was significantly associated with response (p=0.035) and longer progression-free survival (p=0.006) in the REGONIVO trial. CD8+ T cells, regulatory T cells, and M2 macrophages density was significantly higher in the REGONIVO trial responders than in non-responders. Mutations in the POLE gene and patient response were significantly associated in the TASNIVO trial; however, the frequencies of other mutations or tumor mutational burden were not significantly different between responders and non-responders in either trial. CONCLUSIONS: We identified molecular features associated with the response to the REGONIVO and TASNIVO, particularly those related to tumor microenvironmental factors. These findings are likely to contribute to the development of biomarkers to predict treatment efficacy for MSS/pMMR CRC and future immunotherapy combinations for treatment.

Gap junction beta-4 accelerates cell cycle progression and metastasis through MET-AKT activation in pancreatic cancer.

Despite continuing advances in the development of effective new therapies, including immunotherapies, the prognosis of pancreatic cancer remains extremely poor. Gap junction proteins have become attractive targets for potential cancer therapy. However, the role of gap junction beta-4 (GJB4) protein remains unexplored in pancreatic cancer. Through bioinformatic analyses we discovered pancreatic cancer tissues showed higher levels of GJB4 transcripts compared to normal pancreatic tissues and this had a negative effect on overall survival in patients that had pancreatic cancer. The high expression of nuclear GJB4 was identified as a negative prognostic factor in such patients. Knockdown of GJB4 in cultured pancreatic cancer cells resulted in G0/G1 arrest followed by decreased cell proliferation and suppression of metastatic potential. The overexpression of GJB4 accelerated cell proliferation, migration, and invasion in a SUIT-2 cell line, whereas MET inhibitor canceled the acceleration. GJB4 suppression with siRNA significantly inhibited tumor growth in a mouse xenograft model. Mechanistically, suppression of GJB4 inhibited MET-AKT activities. Such data suggest that targeting the GJB4-MET axis could represent a promising new therapeutic strategy for pancreatic cancer.

The interplay between the epithelial permeability barrier, cell migration and mitochondrial metabolism of growth factors and their inhibitors in a human endometrial carcinoma cell line.

It is known that there are abnormalities of tight junction functions, cell migration and mitochondrial metabolism in human endometriosis and endometrial carcinoma. In this study, we investigated the effects of growth factors and their inhibitors on the epithelial permeability barrier, cell migration and mitochondrial metabolism in 2D and 2.5D cultures of human endometrioid endometrial carcinoma Sawano cells. We also investigated the changes of bicellular and tricellular tight junction molecules and ciliogenesis induced by these inhibitors. The growth factors TGF-ß and EGF affected the epithelial permeability barrier, cell migration and expression of bicellular and tricellular tight junction molecules in 2D and 2.5D cultures of Sawano cells. EW-7197 (a TGF-ß receptor inhibitor), AG1478 (an EGFR inhibitor) and SP600125 (a JNK inhibitor) affected the epithelial permeability barrier, cell migration and mitochondrial metabolism and prevented the changes induced by TGF-ß and EGF in 2D and 2.5D cultures. EW-7197 and AG1478 induced ciliogenesis in 2.5D cultures. In conclusion, TGF-ß and EGF promoted the malignancy of endometrial cancer via interplay among the epithelial permeability barrier, cell migration and mitochondrial metabolism. EW-7197 and AG1478 may be useful as novel therapeutic treatments options for endometrial cancer.

Accuracy of intraocular lens power formulas in eyes with keratoconus: Multi-center study in Japan.

PURPOSE: To assess the accuracy of intraocular lens (IOL) power formulas, namely, SRK/T, Haigis, Barrett Universal II, Barrett True-K for keratoconus, Kane formula, and Kane formula for keratoconus, for cataract with keratoconus in Japanese eyes. SETTING: Five surgical sites in Japan. DESIGN: A retrospective case series. METHODS: Eyes with keratoconus undergoing cataract surgery were included. Postoperative refraction was compared with the prediction by the formulas. Visual acuity, manifest spherical equivalent, prediction error (PE), and mean absolute errors (MAEs) were determined 1 month postoperatively. The PE within 0.50 diopter (D), 1.00 D, and 2.00 D were compared between IOL formulas. Subgroup analysis based on the steepest keratometry (stage 1, ≤ 48 D; stage 2, > 48 D and ≤ 53 D; and stage 3, > 53 D) was performed. The relationship between PE and preoperative biometric data were assessed. RESULTS: Fifty eyes were included. The MAE of the Barrett True-K for keratoconus, Kane keratoconus, and Kane formulas were significantly lower than that of Haigis. A statistically significant difference in the prediction accuracy within ± 0.50 D was found between Kane keratoconus and Haigis. The prediction accuracy of the Barrett True-K for keratoconus, SRK/T, and Kane within ± 1.00 D was statistically significant compared with that of Haigis. In stage 3, the Barrett True-K for keratoconus had a significantly lower MAE than SRK/T and Haigis. CONCLUSION: Keratoconus-specific formulas were more accurate than existing formulas in Japanese eyes. The Barrett True-K formula for keratoconus had higher prediction accuracy in severe keratoconus.

Second-line tislelizumab versus chemotherapy in Japanese patients with advanced or metastatic esophageal squamous cell carcinoma: subgroup analysis from RATIONALE-302.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) has a poor prognosis, with limited second-line systemic therapy options, and represents an increasing disease burden in Japan. In the phase 3 RATIONALE-302 study, the anti-programmed cell death protein 1 antibody, tislelizumab, significantly improved overall survival (OS) versus chemotherapy as second-line treatment for advanced/metastatic ESCC. Here, we report the Japanese patient subgroup results. METHODS: Patients with advanced/metastatic ESCC, with disease progression during/after first-line systemic therapy were randomized 1:1 to open-label tislelizumab 200 mg every 3 weeks or investigator's choice of chemotherapy (paclitaxel/docetaxel). Efficacy and safety were assessed in all randomized Japanese patients. RESULTS: The Japanese subgroup comprised 50 patients (n = 25 per arm). Tislelizumab improved OS versus chemotherapy (median: 9.8 vs. 7.6 months; HR 0.59; 95% CI 0.31, 1.12). Among patients with programmed death-ligand 1 score ≥ 10%, median OS was 12.5 months with tislelizumab (n = 10) versus 2.9 months with chemotherapy (n = 6) (HR 0.31; 95% CI 0.09, 1.03). Tislelizumab improved progression-free survival versus chemotherapy (median: 3.6 vs. 1.7 months, respectively; HR 0.50; 95% CI 0.27, 0.95). Objective response rate was greater with tislelizumab (32.0%) versus chemotherapy (20.0%), and responses were more durable (median duration of response: 8.8 vs. 2.6 months, respectively). Fewer patients experienced ≥ grade 3 treatment-related adverse events with tislelizumab (24.0%) versus chemotherapy (47.8%). Tislelizumab demonstrated an improvement in health-related quality of life versus chemotherapy. CONCLUSIONS: As second-line therapy for advanced/metastatic ESCC, tislelizumab improved OS versus chemotherapy, with a favorable safety profile, in the Japanese patient subgroup, consistent with the overall population. CLINICAL TRIAL REGISTRY: ClinicalTrials.gov: NCT03430843.