An Experimental Study on the Addition of Bacteria to Residual Anticancer Drugs: Evaluation of the Effect on Bacterial Growth.

Using anticancer drugs as examples, we examined the possibility of reusing residual drugs. The use of residual drugs is not widespread owing to concerns regarding bacterial contamination. We combined anticancer drugs and bacteria to investigate their effects on bacterial growth. The anticancer drugs carboplatin, paclitaxel, etoposide, irinotecan, methotrexate, and 5-fluorouracil (5-FU) were mixed with Staphylococcus aureus, Enterococcus faecalis, Serratia marcescens, and Escherichia coli. After a certain period, the bacteria were counted. Irinotecan showed no antibacterial activity, whereas 5-FU exhibited high antibacterial activity against the tested bacteria. The 5-FU also showed a minimum inhibitory concentration value in the range of 8-80 µg/mL, depending on the bacterial species. 5-FU dose-dependently inhibited S. aureus growth at more than 0.8 µg/mL. Because protein synthesis systems are reportedly antibiotic targets, we used a cell-free protein synthesis system to confirm the mechanism of the antibacterial activity of the anticancer agent. 5-FU and methotrexate had direct inhibitory effects on protein synthesis. It has been suggested that even if residual drugs are contaminated with bacteria, there will be no microbial growth, or the microbes will be killed by the drug. With careful monitoring, 5-FU can potentially be used for antimicrobial purposes.

Outcomes of regenerative treatment for over 200 patients with tympanic membrane perforation.

OBJECTIVE: To evaluate outcomes of a regenerative treatment (RT) for over 200 patients with tympanic membrane perforation (TMP). The RT-TMP method involves a gelatin sponge, basic fibroblast growth factor (bFGF) and fibrin glue. METHODS: The study population included 216 patients and 234 ears (male: female =100:116; age 1-93 years). All enrolled patients were treated with RT-TMP in which TMP edges were disrupted mechanically and a gelatin sponge immersed in bFGF was inserted into the perforation. Fibrin glue was then dripped over the sponge. Patient outcomes including TMP closure rates, change in hearing level, and complications were obtained from retrospective medical chart reviews. The TMP was examined three or more weeks after surgery. The treatment was repeated up to 4 times until complete TMP closure was achieved. RESULTS: After mechanical disruption, the perforation size was Grade I, ≤1/3 of entire TM area in 22 ears (9.4 %), Grade II, 1/3-2/3 of entire TM in 77 ears (32.9 %) and Grade III, ≥2/3 of entire TM area in 135 ears (57.7 %). The overall TMP closure rates were 97.0 % (227/234). Complete TMP closure was achieved in 68.8 % (161/234), 22.6 % (53/234), 4.7 % (11/234) and 0.9 % (2/234) of ears after 1, 2, 3 and 4 treatments, respectively. In 7 of 234 ears (3.0 %), the TMPs were not closed completely after 4 treatments. There was no correlation between TMP size after mechanical disruption and number of treatments required to achieve complete closure (Fisher's exact test p = 0.70). The mean air-conduction hearing threshold at low frequency improved from 57.3 ± 16.7 dB before treatment to 37.3 ± 16.0 dB (p < 0.0001) after closure of TMPs. For middle and high frequencies, the improvement was 49.0 ± 19.3 dB to 36.9 ± 17.9 dB (p < 0.0001) and 57.7 ± 22.9 dB to 49.2 ± 23.3 dB (p < 0.0001), respectively. The mean air-bone gaps also improved significantly, and were within 10 dB at 250 Hz, 500 Hz and 1 kHz, and 11 dB at 2 kHz. One or more complications occurred in 32 patients (32/216; 14.8 %). The most common complication was formation of an epithelial pearl (16 ears; 6.8 %), followed by severe TM retraction (9 ears; 3.8 %) and otitis media with effusion (6 ears; 2.6 %). There were no serious complications that caused deterioration of the patient's general condition. CONCLUSION: Our results showed that RT-TMP had high success rates for TMP closure and good hearing improvement and produced no severe complications that could affect general health status. This novel therapy is simple, safe and minimally invasive, and could help improve the quality of life in patients with TMP.

Diagnosis of Spontaneous Cerebrospinal Fluid Rhinorrhea Using Thin-Slice Computed Tomographic Images With Multiplanar Reconstruction and Three-Dimensional Virtual View Endoscopy.

Spontaneous cerebrospinal fluid (CSF) rhinorrhea represents an important clinical entity, which is associated with elevated intracranial pressure and is rarely treated successfully without surgical intervention. Here we report a case of spontaneous CSF rhinorrhea. The patient was a 54-year-old male, who presented with bacterial meningitis and was referred to the Department of Otorhinolaryngology for a detailed examination of the nose and sinuses. Reconstructed thin-slice computed tomography (CT) revealed multiple fistulae on the clivus. The defect was successfully repaired by transnasal endoscopic surgery, with the assistance of virtual endoscopic images, which were created by the surgical planning and navigation system from thin-slice CT images. This incremental improvement in the imaging technique helped with the diagnosis and surgical treatment of CSF rhinorrhea.

Predictive factors of acute sensorineural hearing loss in adult Japanese patients for clinical application by primary care doctors: a cross-sectional study.

BACKGROUND: Several methods are used for hearing loss screening; however, their benefits are uncertain. In this study, we aimed to determine the predictive factors of acute sensorineural hearing loss for clinical application by primary care doctors. METHODS: This retrospective, cross-sectional study included 365 patients with acute sensorineural hearing loss without prior therapy. The patients' clinical data, demographic information, and medical histories were obtained, and they were asked about comorbidities. In addition, we assessed lifestyle factors such as stress level, alcohol consumption, marital status, and socioeconomic level. Logistic regression analysis was performed to investigate the diagnostic predictive ability of the selected factors associated with acute sensorineural hearing loss. The hearing levels of all patients were evaluated using pure tone audiometry. RESULTS: We identified significant predictive factors for acute sensorineural hearing loss. The absence of hyperacusis was a predictive factor for sudden sensorineural hearing loss. Younger age, female sex, and marital status were predictive factors for acute low-tone hearing loss. High body mass index, high socioeconomic level, low alcohol consumption, high stress level, hyperacusis, and vertigo/dizziness were predictive factors for Ménière's disease. High body mass index and ear fullness were predictive factors for perilymph fistula. Low stress level was a predictive factor for acoustic tumours. CONCLUSIONS: Our findings can be used to distinguish between the types of acute sensorineural hearing loss. Symptoms, physical status, and lifestyle factors identified during this study are useful markers for predicting acute sensorineural hearing loss occurrence.

Classification of PD-L1 expression in various cancers and macrophages based on immunohistocytological analysis.

Programmed death (PD)-1/PD-ligand 1 (PD-L1) antibodies have shown an intense clinical effect in some patients with PD-L1+ tumors, and their applications have rapidly expanded to various cancer types with or without the application of new companion diagnostics (CDx) with a lower cutoff value and inclusion of macrophage evaluation. However, the pathological background explaining the difference in the cutoff value remains unknown. To address this, we evaluated tissue array samples from 231 patients with lung adenocarcinoma, 186 with lung squamous cell carcinoma, and 38 with renal cell carcinoma (RCC) who were not receiving PD-1/PD-L1 antibodies to investigate the relationship between PD-L1 expression on tumor cells and CD8+ T-cell infiltration in tumor tissues. PD-L1 expression in RCC was clearly lower than that in non-small-cell lung cancer (NSCLC) tissue, whereas CD8+ T-cell infiltration was low in all cancers. We next analyzed PD-L1 expression by interferon (α, ß, and γ) and LPS stimulation in both macrophages and 41 cancer cell lines derived from various organs and histological types. The PD-L1 expression patterns were classified into three types, which differed depending on each organ or tissue type. Interestingly, NSCLC cell lines showed highly diverse PD-L1 expression patterns compared with RCC cell lines. Conversely, PD-L1 expression was stronger and more prolonged in macrophages than in typical cell lines. Here, we revealed the diversity of the PD-L1 expression patterns in tumor cells and macrophages, demonstrating the pathological and cytological significance of the transition of cutoff values in PD-L1 CDx for PD-1/PD-L1 antibody administration.

Application of artificial intelligence using a convolutional neural network for detecting cholesteatoma in endoscopic enhanced images.

OBJECTIVE: We examined whether artificial intelligence (AI) used with the novel digital image enhancement system modalities (CLARA+CHROMA, SPECTRA A, and SPECTRA B) could distinguish the cholesteatoma matrix, cholesteatoma debris, and normal middle ear mucosa, and observe the middle ear cavity during middle ear cholesteatoma surgery. METHODS: A convolutional neural network (CNN) was trained with a set of images chosen by an otologist. To evaluate the diagnostic accuracy of the constructed CNN, an independent test data set of middle ear images was collected from 14 consecutive patients with 26 cholesteatoma matrix lesions, who underwent transcanal endoscopic ear surgery at a single hospital from August 2018 to September 2019. The final test data set included 58 total images, with 1‒5 images from each modality for each case. RESULTS: The CNN required only 10 s to analyze more than 58 test images. Using SPECTRA A and SPECTRA B, the CNN correctly diagnosed 15 and 15 of 26 cholesteatoma matrix lesions, with a sensitivity of 34.6% and 42.3%, and with a specificity of 81.3% and 87.5%, respectively. CONCLUSION: Our preliminary study revealed that AI and novel imaging modalities are potentially useful tools for identifying and visualizing the cholesteatoma matrix during endoscopic ear surgery. The diagnostic ability of the CNN is not yet appropriate for implementation in daily clinical practice, based on our study findings. However, in the future, these techniques and AI tools could help to reduce the burden on surgeons and will facilitate telemedicine in remote and rural areas, as well as in developing countries where the number of surgeons is limited.

Hearing Outcomes and Complications of Cochlear Implantation in Elderly Patients over 75 Years of Age.

OBJECTIVE: Populations are aging in many countries, and the proportion of elderly people with severe to profound hearing loss is increasing in parallel with the increasing average life span. The objective of this study was to investigate the outcomes of cochlear implant (CI) surgery in elderly patients compared to those in younger patients. METHODS: The outcomes of CI surgery were retrospectively investigated for 81 adults (32 men and 49 women) who underwent CI surgery at our hospital. They were divided according to age at the time of implantation into the younger group (<75 years of age; n = 49) or elderly group (≥75 years of age; n = 32). RESULTS: The mean sentence recognition score on the CI-2004 Japanese open-set test battery (±standard deviation) was 82.9% ± 24.1 in the younger group and 81.9% ± 23.2 in the elderly group, with no significant difference between the groups (Mann-Whitney U test). The incidence of major complications that required surgical treatment was not significantly different between the groups (4.1% vs. 6.2%, respectively). Thus, there were no severe complications that could affect general health status in either group. Three patients in each group died for reasons unrelated to CI surgery during follow-up. The proportion of patients who were alive and continued to use the CI five years after surgery was 92.8% and 91.5%, respectively. CONCLUSION: Our results show good speech recognition and a low incidence of major complications in elderly patients. This comprehensive report on the outcomes of CI surgery in elderly patients will be helpful to the elderly with severe to profound hearing loss when deciding whether to undergo CI surgery.

Tim4 recognizes carbon nanotubes and mediates phagocytosis leading to granuloma formation.

Macrophage recognition and phagocytosis of crystals is critical for the associated fibrosis and cancer. Of note, multi-walled carbon nanotubes (MWCNTs), the highly representative products of nanotechnology, induce macrophage NLRP3 inflammasome activation and cause asbestosis-like pathogenesis. However, it remains largely unknown how macrophages efficiently recognize MWCNTs on their cell surfaces. Here, we identify by a targeted screening of phagocyte receptors the phosphatidylserine receptors T cell immunoglobulin mucin 4 (Tim4) and Tim1 as the pattern-recognition receptors for carbon crystals. Docking simulation studies reveal spatiotemporally stable interfaces between aromatic residues in the extracellular IgV domain of Tim4 and one-dimensional carbon crystals. Further, CRISPR-Cas9-mediated deletion of Tim4 and Tim1 reveals that Tim4, but not Tim1, critically contributes to the recognition of MWCNTs by peritoneal macrophages and to granuloma development in a mouse model of direct mesothelium exposure to MWCNTs. These results suggest that Tim4 recognizes MWCNTs through aromatic interactions and mediates phagocytosis leading to granulomas.

Conditional Ror1 knockout reveals crucial involvement in lung adenocarcinoma development and identifies novel HIF-1α regulator.

We previously reported that ROR1 is a crucial downstream gene for the TTF-1/NKX2-1 lineage-survival oncogene in lung adenocarcinoma, while others have found altered expression of ROR1 in multiple cancer types. Accumulated evidence therefore indicates ROR1 as an attractive molecular target, though it has yet to be determined whether targeting Ror1 can inhibit tumor development and growth in vivo. To this end, genetically engineered mice carrying homozygously floxed Ror1 alleles and an SP-C promoter-driven human mutant EGFR transgene were generated. Ror1 ablation resulted in marked retardation of tumor development and progression in association with reduced malignant characteristics and significantly better survival. Interestingly, gene set enrichment analysis identified a hypoxia-induced gene set (HALLMARK_HYPOXIA) as most significantly downregulated by Ror1 ablation in vivo, which led to findings showing that ROR1 knockdown diminished HIF-1α expression under normoxia and clearly hampered HIF-1α induction in response to hypoxia in human lung adenocarcinoma cell lines. The present results directly demonstrate the importance of Ror1 for in vivo development and progression of lung adenocarcinoma, and also identify Ror1 as a novel regulator of Hif-1α. Thus, a future study aimed at the development of a novel therapeutic targeting ROR1 for treatment of solid tumors such as seen in lung cancer, which are frequently accompanied with a hypoxic tumor microenvironment, is warranted.

Inhibition of heat shock protein 90 destabilizes receptor tyrosine kinase ROR1 in lung adenocarcinoma.

We have previously identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a direct transcriptional target of TTF-1/NKX2-1, a lineage-survival oncogene in lung adenocarcinoma. ROR1 sustains prosurvival signaling from multiple receptor tyrosine kinases including epidermal growth factor receptor, MET, and insulin-like growth factor 1 receptor in part by maintaining the caveolae structure as a scaffold protein of cavin-1 and caveolin-1. In this study, a high throughput screening of the natural product library containing 2560 compounds was undertaken using a cell-based FluoPPI assay detecting ROR1-cavin-1 interaction. As a result, geldanamycin (GA), a known inhibitor of heat shock protein 90 (HSP90), was identified as a potential inhibitor of ROR1. Geldanamycin, as well as two GA derivatives tested in the clinic, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), decreased ROR1 protein expression. We found that ROR1 physically interacted with HSP90α, but not with other HSP90 paralogs, HSP90ß or GRP94. Geldanamycin in turn destabilized and degraded ROR1 protein in a dose- and time-dependent manner through the ubiquitin/proteasome pathway, resulting in a significant suppression of cell proliferation in lung adenocarcinoma cell lines, for which the kinase domain of ROR1, but not its kinase activity or N-glycosylation, was required. Our findings indicate that HSP90 is required to sustain expression of ROR1 crucial for lung adenosarcoma survival, suggesting that inhibition of HSP90 could be a promising therapeutic strategy in ROR1-positive lung adenocarcinoma.

ROR1-CAVIN3 interaction required for caveolae-dependent endocytosis and pro-survival signaling in lung adenocarcinoma.

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transcriptional target of the lineage-survival oncogene NKX2-1/TTF-1 in lung adenocarcinomas. In addition to its kinase-dependent role, ROR1 functions as a scaffold protein to facilitate interaction between caveolin-1 (CAV1) and CAVIN1, and consequently maintains caveolae formation, which in turn sustains pro-survival signaling toward AKT from multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET (proto-oncogene, receptor tyrosine kinase), and IGF-IR (insulin-like growth factor receptor 1). Therefore, ROR1 is an attractive target for overcoming EGFR-TKI resistance due to various mechanisms such as EGFR T790M double mutation and bypass signaling from other RTKs. Here, we report that ROR1 possesses a novel scaffold function indispensable for efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site distinct from sites for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore, evidence of a mechanistic link between ROR1-CAVIN3 interaction and consequential caveolae trafficking, which was found to utilize a binding site distinct from those for ROR1 interactions with CAV1 and CAVIN1, with RTK-mediated pro-survival signaling towards AKT in early endosomes in lung adenocarcinoma cells was also obtained. The present findings warrant future study to enable development of novel therapeutic strategies for inhibiting the multifaceted scaffold functions of ROR1 in order to reduce the intolerable death toll from this devastating cancer.

Clinical outcomes of end-flexible-rigidscopic transoral surgery (E-TOS) in patients with T1-selected T3 pharyngeal and supraglottic cancers.

BACKGROUND: Endoscopic laryngo-pharyngeal surgery (ELPS), which is a transoral minimally invasive surgery using a gastrointestinal endoscope and a curved laryngopharyngeal retractor, is effective to treat primary lesions of superficial laryngopharyngeal cancers. To extend concepts of ELPS to invasive laryngopharyngeal cancers, we developed end-flexible-rigidscopic transoral surgery (E-TOS) from ELPS by changing a gastrointestinal endoscope to a flexible-tip rigid endoscope. AIMS/OBJECTIVES: To retrospectively evaluate oncological outcomes and laryngopharyngeal functional preservation of E-TOS in patients with T1-selected T3 laryngopharyngeal cancers. MATERIAL AND METHODS: In 47 patients T1-selected T3 pharyngeal and supraglottic cancers were resected by E-TOS using the flexible-tip rigid endoscope and curved instruments. Negative resection margin was histopathologically evaluated. The survival, preservation of larynx, and disease control rates were estimated using Kaplan-Meier method. RESULTS: Curative resection was achieved in 94% of patients. No patient complained prolonged swallowing dysfunction or hoarseness after E-TOS. Postoperative bleeding, stenosis of the pharynx and esophageal entrance, and local recurrence was observed each in one patient. The 3-year overall survival, disease-specific survival, laryngeal preservation, local control, and locoregional control rates were, 86%, 93%, 100%, 98%, and 79%, respectively. CONCLUSION: E-TOS is an effective minimally invasive surgery for T1-selected T3 pharyngeal and supraglottic cancers with preserving laryngeal function.

ROR1 sustains caveolae and survival signalling as a scaffold of cavin-1 and caveolin-1.

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) sustains prosurvival signalling directly downstream of the lineage-survival oncogene NKX2-1/TTF-1 in lung adenocarcinoma. Here we report an unanticipated function of this receptor tyrosine kinase (RTK) as a scaffold of cavin-1 and caveolin-1 (CAV1), two essential structural components of caveolae. This kinase-independent function of ROR1 facilitates the interactions of cavin-1 and CAV1 at the plasma membrane, thereby preventing the lysosomal degradation of CAV1. Caveolae structures and prosurvival signalling towards AKT through multiple RTKs are consequently sustained. These findings provide mechanistic insight into how ROR1 inhibition can overcome EGFR-tyrosine kinase inhibitor (TKI) resistance due to bypass signalling via diverse RTKs such as MET and IGF-IR, which is currently a major clinical obstacle. Considering its onco-embryonic expression, inhibition of the scaffold function of ROR1 in patients with lung adenocarcinoma is an attractive approach for improved treatment of this devastating cancer.

Receptor tyrosine kinase-like orphan receptor 1, a target of NKX2-1/TTF-1 lineage-survival oncogene, inhibits apoptosis signal-regulating kinase 1-mediated pro-apoptotic signaling in lung adenocarcinoma.

We previously identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a transcriptional target of the NKX2-1/TTF-1 lineage-survival oncogene in lung adenocarcinoma. ROR1 consequently sustains a favorable balance between pro-survival phosphatidylinositol 3-kinase-protein kinase B and pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1)-p38MAPK signaling. In contrast to recent advances in understanding how ROR1 sustains pro-survival signaling, the mechanism of ROR1 repression of pro-apoptotic signaling remains rather elusive. In the present study, we investigated the underlying mechanism of ROR1-mediated inhibition of the ASK1-p38MAPK signaling pathway. Growth inhibition mediated by siROR1 was partially but significantly alleviated by ASK1 co-knockdown in lung adenocarcinoma cell lines. Also, ASK1 phosphorylation at Thr845, which reflects its activated state, was clearly inhibited by ROR1 overexpression in both steady state and oxidative stress-elicited conditions in MSTO-211H cells. In addition, we found that ROR1 was physically associated with ASK1 at the C-terminal serine threonine-rich domain of ROR1. Furthermore, ROR1 kinase activity was shown to be required to repress the ASK1-p38 axis and oxidative stress-induced cell death. The present findings thus support our notion that ROR1 sustains lung adenocarcinoma survival, at least in part, through direct physical interaction with ASK1 and consequential repression of the pro-apoptotic ASK1-p38 axis in a ROR1 kinase activity-dependent manner.

Lung adenocarcinoma subtypes definable by lung development-related miRNA expression profiles in association with clinicopathologic features.

Accumulation of genetic and epigenetic changes alters regulation of a web of interconnected genes including microRNAs (miRNAs), which confer hallmark capabilities and characteristic cancer features. In this study, the miRNA and messenger RNA expression profiles of 126 non-small cell lung cancer specimens were analyzed, with special attention given to the diversity of lung adenocarcinomas. Of those, 76 adenocarcinomas were classified into two major subtypes, developing lung-like and adult lung-like, based on their distinctive miRNA expression profiles resembling those of either developing or adult lungs, respectively. A systems biology-based approach using a Bayesian network and non-parametric regression was employed to estimate the gene regulatory circuitry functioning in patient tumors in order to identify subnetworks enriched for genes with differential expression between the two major subtypes. miR-30d and miR-195, identified as hub genes in such subnetworks, had lower levels of expression in the developing lung-like subtype, whereas introduction of miR-30d or miR-195 into the lung cancer cell lines evoked shifts of messenger RNA expression profiles toward the adult lung-like subtype. Conversely, the influence of miR-30d and miR-195 was significantly different between the developing lung-like and adult lung-like subtypes in our analysis of the patient data set. In addition, RRM2, a child gene of the miR-30d-centered subnetwork, was found to be a direct target of miR-30d. Together, our findings reveal the existence of two miRNA expression profile-defined lung adenocarcinoma subtypes with distinctive clinicopathologic features and also suggest the usefulness of a systems biology-based approach to gain insight into the altered regulatory circuitry involved in cancer development.

NKX2-1/TTF-1: an enigmatic oncogene that functions as a double-edged sword for cancer cell survival and progression.

Emerging evidence indicates that NKX2-1, a homeobox-containing transcription factor also known as TTF-1, plays a role as a "lineage-survival" oncogene in lung adenocarcinomas. In T cell acute lymphoblastic leukemia, gene rearrangements lead to aberrant expression of NKX2-1/TTF-1. Despite accumulating evidence supporting its oncogenic role, it has become apparent that NKX2-1/TTF-1 expression also has biological and clinical functions in the opposite direction that act against tumor progression. Herein, we review recent findings showing these enigmatic double-edged characteristics, with special attention given to the roles of NKX2-1/TTF-1 in lung development and carcinogenesis.

MYBPH inhibits NM IIA assembly via direct interaction with NMHC IIA and reduces cell motility.

Actomyosin filament assembly is a critical step in tumor cell migration. We previously found that myosin binding protein H (MYBPH) is directly transactivated by the TTF-1 lineage-survival oncogene in lung adenocarcinomas and inhibits phosphorylation of the myosin regulatory light chain (RLC) of non-muscle myosin IIA (NM IIA) via direct interaction with Rho kinase 1 (ROCK1). Here, we report that MYBPH also directly interacts with an additional molecule, non-muscle myosin heavy chain IIA (NMHC IIA), which was found to occur between MYBPH and the rod portion of NMHC IIA. MYBPH inhibited NMHC IIA assembly and reduced cell motility. Conversely, siMYBPH-induced increased motility was partially, yet significantly, suppressed by blebbistatin, a non-muscle myosin II inhibitor, while more profound effects were attained by combined treatment with siROCK1 and blebbistatin. Electron microscopy observations showed well-ordered paracrystals of NMHC IIA reflecting an assembled state, which were significantly less frequently observed in the presence of MYBPH. Furthermore, an in vitro sedimentation assay showed that a greater amount of NMHC IIA was in an unassembled state in the presence of MYBPH. Interestingly, treatment with a ROCK inhibitor that impairs transition of NM IIA from an assembly-incompetent to assembly-competent state reduced the interaction between MYBPH and NMHC IIA, suggesting that MYBPH has higher affinity to assembly-competent NM IIA. These results suggest that MYBPH inhibits RLC and NMHC IIA, independent components of NM IIA, and negatively regulates actomyosin organization at 2 distinct steps, resulting in firm inhibition of NM IIA assembly.

NKX2-1/TITF1/TTF-1-Induced ROR1 is required to sustain EGFR survival signaling in lung adenocarcinoma.

We and others previously identified NKX2-1, also known as TITF1 and TTF-1, as a lineage-survival oncogene in lung adenocarcinomas. Here we show that NKX2-1 induces the expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1), which in turn sustains a favorable balance between prosurvival PI3K-AKT and pro-apoptotic p38 signaling, in part through ROR1 kinase-dependent c-Src activation, as well as kinase activity-independent sustainment of the EGFR-ERBB3 association, ERBB3 phosphorylation, and consequential PI3K activation. Notably, ROR1 knockdown effectively inhibited lung adenocarcinoma cell lines, irrespective of their EGFR status, including those with resistance to the EGFR tyrosine kinase inhibitor gefitinib. Our findings thus identify ROR1 as an "Achilles' heel" in lung adenocarcinoma, warranting future development of therapeutic strategies for this devastating cancer.

MYBPH, a transcriptional target of TTF-1, inhibits ROCK1, and reduces cell motility and metastasis.

Cell migration driven by actomyosin filament assembly is a critical step in tumour invasion and metastasis. Herein, we report identification of myosin binding protein H (MYBPH) as a transcriptional target of TTF-1 (also known as NKX2-1 and TITF1), a master regulator of lung development that also plays a role as a lineage-survival oncogene in lung adenocarcinoma development. MYBPH inhibited assembly competence-conferring phosphorylation of the myosin regulatory light chain (RLC) as well as activating phosphorylation of LIM domain kinase (LIMK), unexpectedly through its direct physical interaction with Rho kinase 1 (ROCK1) rather than with RLC. Consequently, MYBPH inhibited ROCK1 and negatively regulated actomyosin organization, which in turn reduced single cell motility and increased collective cell migration, resulting in decreased cancer invasion and metastasis. Finally, we also show that MYBPH is epigenetically inactivated by promoter DNA methylation in a fraction of TTF-1-positive lung adenocarcinomas, which appears to be in accordance with its deleterious functions in lung adenocarcinoma invasion and metastasis, as well as with the paradoxical association of TTF-1 expression with favourable prognosis in lung adenocarcinoma patients.

Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis.

Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its significance has remained unknown. Here we demonstrated a direct interaction between Plk1 and vimentin-Ser55 phosphorylated by Cdk1, an event that led to Plk1 activation and further vimentin phosphorylation. Plk1 phosphorylated vimentin at approximately 1 mol phosphate/mol substrate, which partly inhibited its filament forming ability, in vitro. Plk1 induced the phosphorylation of vimentin-Ser82, which was elevated from metaphase and maintained until the end of mitosis. This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion. Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B. Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.