Resminostat in EGFR-mutated lung cancer

Drug-tolerant cells are mediators of acquired resistance. BIM-intron2 deletion polymorphism (BIM-del) is one of the mechanisms underlying the resistance to epidermal growth factor tyrosine kinase inhibitor (EGFR-TKI)-mediated apoptosis that induces drug tolerance. Here, we investigated whether resminostat, a histone deacetylase inhibitor, circumvents BIM-del-associated apoptosis resistance. The human EGFR-mutated non-small cell lung cancer (NSCLC) cell line PC-9 and its homozygous BIM-del-positive variant (PC-9 BIMi2-/-), established by editing with zinc finger nuclease, were used. In comparison with PC-9 cells, PC-9 BIMi2-/- cells were less sensitive to apoptosis mediated by EGFR-TKIs such as gefitinib and osimertinib. The combined use of resminostat and an EGFR-TKI preferentially induced the expression of the pro-apoptotic BIM transcript containing exon 4 rather than that containing exon 3, increased the level of pro-apoptotic BIM protein (BIMEL), and stimulated apoptosis in vitro. In a subcutaneous tumor model derived from PC-9 BIMi2-/- cells, gefitinib monotherapy decreased tumor size but retained residual lesions, indicative of the presence of tolerant cells in tumors. The combined use of resminostat and gefitinib increased BIMEL protein level and induced apoptosis, subsequently leading to the remarkable shrinkage of tumor. These findings suggest the potential of resminostat to circumvent tolerance to EGFR-TKIs associated with BIM deletion polymorphism

Role of vinculin in paracellular barrier for ions

The paracellular barrier function of tight junctions (TJs) in epithelial cell sheets is robustly maintained against mechanical fluctuations, by molecular mechanisms that are poorly understood. Vinculin is an adaptor of a mechanosensory complex at the adherens junction. Here, we generated vinculin KO Eph4 epithelial cells and analyzed their confluent cell-sheet properties. We found that vinculin is dispensable for the basic TJ structural integrity and the paracellular barrier function for larger solutes. However, vinculin is indispensable for the paracellular barrier function for ions. In addition, TJs stochastically showed dynamically distorted patterns in vinculin KO cell sheets. These KO phenotypes were rescued by transfecting full-length vinculin and by relaxing the actomyosin tension with blebbistatin, a myosin II ATPase activity inhibitor. Our findings indicate that vinculin resists mechanical fluctuations to maintain the TJ paracellular barrier function for ions in epithelial cell sheets

Podoplanin promotes progression of MPM

Malignant pleural mesothelioma (MPM) is characterized by dissemination and aggressive growth in the thoracic cavity. Podoplanin (PDPN) is an established diagnostic marker for MPM, but the function of PDPN in MPM is not fully understood. The purpose of this study was to determine the pathogenetic function of PDPN in MPM. Forty-seven of 52 tumors (90%) from Japanese patients with MPM and 3/6 (50%) MPM cell lines tested positive for PDPN. Knocking down PDPN in PDPN-high expressing MPM cells resulted in decreased cell motility. In contrast, overexpression of PDPN in PDPN-low expressing MPM cells enhanced cell motility. PDPN stimulated motility was mediated by activation of the RhoA/ROCK pathway. Moreover, knocking down PDPN with short hairpin (sh) RNA in PDPN-high expressing MPM cells resulted in decreased development of a thoracic tumor in mice with severe combined immune deficiency (SCID). In sharp contrast, transfection of PDPN in PDPN-low expressing MPM cells resulted in an increase in the number of Ki-67-positive proliferating tumor cells and it promoted progression of a thoracic tumor in SCID mice. Interestingly, PDPN promoted focus formation in vitro, and a low level of E-cadherin expression and YAP1 activation was observed in PDPN-high MPM tumors. These findings indicate that PDPN is a diagnostic marker as well as a pathogenetic regulator that promotes MPM progression by increasing cell motility and inducing focus formation. Therefore, PDPN might be a pathogenetic determinant of MPM dissemination and aggressive growth and may thus be an ideal therapeutic target

High efficacy of third generation EGFR inhibitor AZD9291 in a leptomeningeal carcinomatosis model with EGFR-mutant lung cancer cells

Leptomeningeal carcinomatosis (LMC) remarkably decreases the quality of life of EGFR-mutant lung cancer patients. In contrast to the lesions outside the central nervous system (CNS), molecular mechanisms of EGFR tyrosine kinase inhibitor (TKI) resistance in CNS lesions including LMC are largely unknown. In this study, we established an in vivo imaging model for LMC with EGFR mutant lung cancer cell lines harboring an exon 19 deletion in EGFR and evaluated the effect of first generation EGFR-TKIs, erlotinib, second generation afatinib, and third generation AZD9291. In PC-9/ffluc model, erlotinib treatment slowed the development of LMC. Importantly, treatment with afatinib or AZD9291 apparently delayed the development of LMC. Moreover, treatment with a higher dose of AZD9291, also associated with inhibited phosphorylation of EGFR downstream molecule S6, regressed LMC refractory to the aforementioned EGFR-TKI treatments. These observations suggest that the third generation EGFR-TKI AZD9291 may be an effective treatment for first or second generation EGFR-TKI resistant LMC caused by EGFR-mutant lung cancer

Phase separation of an actin nucleator by junctional microtubules regulates epithelial function

Liquid-liquid phase separation (LLPS) is involved in various dynamic biological phenomena. In epithelial cells, dynamic regulation of junctional actin filaments tethered to the apical junctional complex (AJC) is critical for maintaining internal homeostasis against external perturbations; however, the role of LLPS in this process remains unknown. Here, after identifying a multifunctional actin nucleator, cordon bleu (Cobl), as an AJC-enriched microtubule-associated protein, we conducted comprehensive in vitro and in vivo analyses. We found that apical microtubules promoted LLPS of Cobl at the AJC, and Cobl actin assembly activity increased upon LLPS. Thus, microtubules spatiotemporally regulated junctional actin assembly for epithelial morphogenesis and paracellular barriers. Collectively, these findings established that LLPS of the actin nucleator Cobl mediated dynamic microtubule-actin cross-talk in junctions, which fine-tuned the epithelial barrier

Podoplanin promotes progression of malignant pleural mesothelioma by regulating motility and focus formation

Malignant pleural mesothelioma (MPM) is characterized by dissemination and aggressive growth in the thoracic cavity. Podoplanin (PDPN) is an established diagnostic marker for MPM, but the function of PDPN in MPM is not fully understood. The purpose of this study was to determine the pathogenetic function of PDPN in MPM. Forty-seven of 52 tumors (90%) from Japanese patients with MPM and 3/6 (50%) MPM cell lines tested positive for PDPN. Knocking down PDPN in PDPN-high expressing MPM cells resulted in decreased cell motility. In contrast, overexpression of PDPN in PDPN-low expressing MPM cells enhanced cell motility. PDPN stimulated motility was mediated by activation of the RhoA/ROCK pathway. Moreover, knocking down PDPN with short hairpin (sh) RNA in PDPN-high expressing MPM cells resulted in decreased development of a thoracic tumor in mice with severe combined immune deficiency (SCID). In sharp contrast, transfection of PDPN in PDPN-low expressing MPM cells resulted in an increase in the number of Ki-67-positive proliferating tumor cells and it promoted progression of a thoracic tumor in SCID mice. Interestingly, PDPN promoted focus formation in vitro, and a low level of E-cadherin expression and YAP1 activation was observed in PDPN-high MPM tumors. These findings indicate that PDPN is a diagnostic marker as well as a pathogenetic regulator that promotes MPM progression by increasing cell motility and inducing focus formation. Therefore, PDPN might be a pathogenetic determinant of MPM dissemination and aggressive growth and may thus be an ideal therapeutic target. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association

3 Tesla MRI detects accelerated hippocampal volume reduction in postmenopausal women

Purpose To clarify age-related structural changes specific to hippocampal volume by hierarchizing according to age, gender, and menopausal status. Many studies report the neuroprotective effects of estrogen and age-related brain volume changes; however, there are no studies regarding age-related change specific to hippocampal volume in terms of age, gender, and menopausal status. Materials and Methods T1-weighted MR images were obtained in 412 healthy adults divided into eight groups according to age and gender, to analyze brain volume change focusing on hippocampal volume. Results Voxel-based morphometry (VBM) revealed significantly smaller gray matter volume in the hippocampus bilaterally in females aged in their fifties (51 of 59 females were at menopause) compared with females in their forties (3 of 46 females were at menopause). No significant difference was found, however, between female groups in their fifties versus sixties, or sixties versus seventies; or between male groups in their forties versus fifties, fifties versus sixties, or sixties versus seventies. In addition, VBM revealed significant hippocampal volume reduction bilaterally in all postmenopausal women compared with all premenopausal women. Conclusion The results of the current study suggest that the menopause may be associated with hippocampal volume reduction. © 2010 Wiley-Liss, Inc.Thesis of Goto,Masami / 後藤 政実 博士学位論文(金沢大学 / 大学院医薬保健学総合研究科

Effects of low-intensity pulsed ultrasound on osteoclasts: Analysis with goldfish scales as a model of bone

The effects of low-intensity pulsed ultrasound (LIPUS) on osteoclastogenesis were examined using fish scales that had both osteoclasts and osteoblasts. The binding of the receptor activator of NF-κB ligand (RANKL) in osteoblasts to the receptor activator of NF-κB (RANK) in osteoclasts induced osteoclastogenesis. Therefore, we focused on RANK/RANKL signaling. After 6 h of incubation following LIPUS treatment, mRNA expression of RANKL increased significantly. Resulting from the increased RANKL mRNA level, the expression of transcription-regulating factors significantly increased after 6 h of incubation, and then the mRNA expression of functional genes was significantly up-regulated after 12 h of incubation. However, the mRNA expression of osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, also significantly increased after 6 h of incubation and tended to further increase after 12 h of incubation. At 24 h of incubation, osteoclastic functional genes’ mRNA expression decreased to the level of the control. Furthermore, we performed an in vivo experiment with goldfish. Two weeks after daily LIPUS exposure, osteoclastic marker enzymes tended to decrease while osteoblastic marker enzymes were activated. The regeneration rate of the LIPUS-treated scales was significantly higher than that of the control scales. Thus, LIPUS moderately activates osteoclasts and induces bone formation. © 2017 Biomedical Research Foundation. All rights reserved

Transient IGF-1R inhibition combined with osimertinib eradicates AXL-low expressing EGFR mutated lung cancer

Drug tolerance is the basis for acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) including osimertinib, through mechanisms that still remain unclear. Here, we show that while AXL-low expressing EGFR mutated lung cancer (EGFRmut-LC) cells are more sensitive to osimertinib than AXL-high expressing EGFRmut-LC cells, a small population emerge osimertinib tolerance. The tolerance is mediated by the increased expression and phosphorylation of insulin-like growth factor-1 receptor (IGF-1R), caused by the induction of its transcription factor FOXA1. IGF-1R maintains association with EGFR and adaptor proteins, including Gab1 and IRS1, in the presence of osimertinib and restores the survival signal. In AXL-low-expressing EGFRmut-LC cell-derived xenograft and patient-derived xenograft models, transient IGF-1R inhibition combined with continuous osimertinib treatment could eradicate tumors and prevent regrowth even after the cessation of osimertinib. These results indicate that optimal inhibition of tolerant signals combined with osimertinib may dramatically improve the outcome of EGFRmut-LC