[A Case of Pulmonary Pleomorphic Carcinoma in a Hemodialysis Patient Treated with Pembrolizumab].

The patient was a 67-year-old male undergoing maintenance hemodialysis due to chronic renal failure caused by diabetic nephropathy. A left upper lobe resection was carried out for non-small cell lung cancer of the left upper lobe. It was histologically confirmed as pleomorphic carcinoma pT3N0M0, Stage ⅡB. He suffered a relapse with multiple metastases occurring in both lungs 3 months following surgery. The PD-L1 tumor proportion score(TPS)was 90%, indicating a high level of expression; 200mg of pembrolizumab was administered every 3 weeks on non-dialysis days. Two courses of administration achieved a partial response. A total of 17 courses were administered until discontinuation due to drug-induced lung injury.

Underwater endoscopic mucosal resection versus endoscopic submucosal dissection for 20-30 mm colorectal polyps.

BACKGROUND AND AIM: Endoscopic submucosal dissection (ESD) for large polyps provides a high en bloc resection rate, accurate pathological diagnosis, and low recurrence rate. However, ESD requires advanced techniques, and underwater endoscopic mucosal resection (UEMR) is an alternative. We investigated the efficacy and safety of UEMR for 20-30 mm colorectal lesions compared with ESD. METHODS: We retrospectively evaluated systematically collected data of patients who underwent UEMR or ESD for 20-30 mm sessile colorectal lesions. Outcome measures were the incidence of local recurrence, procedure time, en bloc resection rate, and incidence of adverse events. We performed propensity score matching and inverse probability weighting adjustment to control for possible confounders. RESULTS: We evaluated 125 patients undergoing UEMR and 306 patients undergoing ESD. Using propensity score matching, we analyzed 74 lesions in each group. UEMR had a shorter procedure time than ESD [6.7 min (95% confidence interval (CI), 5.3-8.1 min) vs 64.8 min (95% CI, 57.4-72.2 min), respectively]. Although the en bloc resection rate with UEMR was inferior to ESD [61% (95% CI, 49-72%) vs 99% (95% CI, 93-100%), respectively], there was no significant difference in the local recurrence rate between the procedures [0% (95% CI, 0-4.0%) in each group]. Inverse probability weighting adjustment revealed that neither ESD nor UEMR had a significant association with local recurrence. CONCLUSIONS: Underwater endoscopic mucosal resection for 20-30  mm colorectal lesions was comparable with ESD regarding long-term outcomes, with a shorter procedure time, despite the lower en bloc resection rate.

Magnifying endoscopy with crystal violet staining for immune checkpoint inhibitor-associated colitis.

BACKGROUND AND AIM: There exists no evidence on the relationship between endoscopic and histologic findings. Furthermore, even after multiple biopsy specimens were obtained, histologic examination usually fails to show the characteristic features of immune checkpoint inhibitor-associated colitis. In this study, we explored the endoscopic and histologic findings of immune checkpoint inhibitor-associated colitis. METHODS: Patients diagnosed with immune checkpoint inhibitor-associated colitis at our hospital between March 2018 and December 2018 were retrospectively assessed. The degree of mucosal inflammation was evaluated using endoscopic inflammation grade (inactive, mild, moderate, or severe disease) and further observed using magnifying endoscopy with crystal violet staining. Pit structures were classified into three types: regularly arranged pits with circular or elliptical shape (R type), irregularly arranged pits with inhomogeneous size and morphology (IR type), and pits with reduced density or pits that partially disappeared (AD type). RESULTS: Eleven patients (median age, 71 years; range, 44-83 years) were diagnosed with immune checkpoint inhibitor-associated colitis. All characteristic histologic findings, including crypt distortion, crypt abscesses, and apoptotic bodies, were observed at sites with moderate-to-severe endoscopic inflammation but not at sites with inactive-to-mild endoscopic inflammation. Characteristic histologic features were observed in 0%, 50%, and 100% of R-type, IR-type, and AD-type mucosa, respectively. CONCLUSIONS: We revealed the possible utility of endoscopic images for selecting suitable target sites for biopsy and showed that endoscopic findings could reduce the time lag associated with tissue diagnosis and sampling errors due to biopsy.

Long-term outcomes after endoscopic submucosal dissection for differentiated-type early gastric cancer that fulfilled expanded indication criteria: A prospective cohort study.

BACKGROUND AND AIM: Endoscopic resection for early gastric cancer (EGC) is widely performed. However, there is still a paucity of strong evidence regarding long-term outcomes after endoscopic submucosal dissection (ESD) for the expanded indication criteria of the Japanese guidelines (ver. 2010). METHODS: Endoscopic submucosal dissection was performed in patients with EGC that met the expanded indication criteria: (i) cT1a, differentiated-type EGC of 2 to 5 cm, ulcer negative or (ii) cT1a, differentiated-type EGC of ≤3 cm, ulcer positive. Patients whose pathological examination fulfilled the curative resection criteria were then enrolled in this cohort study: negative vertical margin, negative lymphovascular invasion, and (i) pT1a, differentiated-type, and ulcer negative; (ii) pT1a, differentiated-type, ≤3 cm, and ulcer positive; or (iii) pT1b1 (<500-µm submucosal invasion), differentiated-type, and ≤3 cm. Patients with only a positive horizontal margin as a noncurative factor were included for follow-up. RESULTS: From September 2003 to February 2012, a total of 356 patients underwent ESD, and 214 were enrolled in the survival analysis. One hundred twenty patients (56%) had >2 cm in diameter and ulcer-negative lesions, and 94 (44%) had ≤3 cm and ulcer-positive lesions. The vital status at 5 years after ESD was confirmed in all (100%) patients. No local or metastatic recurrence was detected; however, 26 metachronous gastric cancers developed, and 1 patient died of metachronous gastric cancer. The 5-year disease-specific and overall survival rates were 99.5% (95% confidence interval [CI], 97.2%-100%) and 93.9% (95% CI, 89.8%-96.4%), respectively. CONCLUSION: ESD for EGC that fulfills the expanded criteria is feasible and shows favorable long-term outcomes.

Endoscopic detection and differentiation of esophageal lesions using a deep neural network.

BACKGROUND AND AIMS: Diagnosing esophageal squamous cell carcinoma (SCC) depends on individual physician expertise and may be subject to interobserver variability. Therefore, we developed a computerized image-analysis system to detect and differentiate esophageal SCC. METHODS: A total of 9591 nonmagnified endoscopy (non-ME) and 7844 ME images of pathologically confirmed superficial esophageal SCCs and 1692 non-ME and 3435 ME images from noncancerous lesions or normal esophagus were used as training image data. Validation was performed using 255 non-ME white-light images, 268 non-ME narrow-band images/blue-laser images, and 204 ME narrow-band images/blue-laser images from 135 patients. The same validation test data were diagnosed by 15 board-certified specialists (experienced endoscopists). RESULTS: Regarding diagnosis by non-ME with narrow-band imaging/blue-laser imaging, the sensitivity, specificity, and accuracy were 100%, 63%, and 77%, respectively, for the artificial intelligence (AI) system and 92%, 69%, and 78%, respectively, for the experienced endoscopists. Regarding diagnosis by non-ME with white-light imaging, the sensitivity, specificity, and accuracy were 90%, 76%, and 81%, respectively, for the AI system and 87%, 67%, and 75%, respectively, for the experienced endoscopists. Regarding diagnosis by ME, the sensitivity, specificity, and accuracy were 98%, 56%, and 77%, respectively, for the AI system and 83%, 70%, and 76%, respectively, for the experienced endoscopists. There was no significant difference in the diagnostic performance between the AI system and the experienced endoscopists. CONCLUSIONS: Our AI system showed high sensitivity for detecting SCC by non-ME and high accuracy for differentiating SCC from noncancerous lesions by ME.

Narrow band imaging under less-air condition improves the visibility of superficial esophageal squamous cell carcinoma.

BACKGROUND: The current virtual chromoendoscopy equipment cannot completely detect superficial squamous cell carcinoma (SCC) in the esophagus, despite its development in the recent years. Thus, in this study, we aimed to elucidate the appropriate air volume during endoscopic observation to improve the visibility of esophageal SCC. METHODS: This retrospective study included a total of 101 flat type esophageal SCCs identified between April 2017 and January 2019 at the Department of Gastrointestinal Oncology, Osaka International Cancer Institute. Video images of narrow band imaging (NBI) under both less-air and standard-air conditions were recorded digitally. Videos were evaluated by five endoscopists. Relative visibility between less-air and standard-air conditions of the brownish area, brownish color change of the epithelium, and dilated intrapapillary capillary loop (IPCL) were graded as 5 (definitely better under less-air condition) to 1 (definitely worse under less-air condition), with 3 indicating average visibility (equivalent to standard-air observation). RESULTS: The mean (standard deviation) visibility score of the brownish area, brownish color change of the epithelium, and dilated IPCLs under less-air condition were 3.94 (0.58), 3.73 (0.57), and 4.13 (0.60), respectively, which were significantly better than that under standard-air condition (p < 0.0001). Esophageal SCC evaluated as ≥ 4 in the mean visibility score of the brownish area, brownish color change of the epithelium, and dilated IPCLs accounted for 50% (51/101 lesions), 34% (34/101 lesions), and 67% (68/101 lesions), respectively. CONCLUSIONS: The present results suggested that NBI with less air might improve the visibility of flat type esophageal SCC compared with NBI with standard air. Less-air NBI observation may facilitate the detection of flat type esophageal SCC. TRIAL REGISTRATION: The present study is a non-intervention trial.

Does cold snare polypectomy completely resect the mucosal layer? A prospective single-center observational trial.

BACKGROUND AND AIM: The true incidence of incomplete muscularis mucosa resection with cold snare polypectomy (CSP) is unknown. We examined the incidence of incomplete muscularis mucosa resection both with and without cold snare defect protrusion (CSDP). METHODS: We prospectively enrolled patients undergoing polypectomy for 4 to 9mm nonpedunculated polyps. We evaluated the presence of CSDP immediately following CSP and biopsied the CSDP or the center of the mucosal defect without CSDP. The presence of the muscularis mucosa and any residual polyp in the biopsies was evaluated histologically. The primary outcome was the incidence of incomplete mucosal layer resection defined as the presence of muscularis mucosa or residual polyp in the biopsies. RESULTS: From August 2017 to October 2018, 188 patients were screened, and 357 polyps were included. CSDP was detected in 122/355 (34%) evaluated mucosal defects. Excluding five lesions requiring hemostasis immediately following CSP, 352 mucosal defects were biopsied. After excluding 102 biopsies containing normal mucosa, we evaluated 250 biopsies. The overall incidence of incomplete mucosal layer resection was 63% (159/250), 76% (68/90) with CSDP and 57% (91/159) without CSDP (P < 0.01). Both univariate and multivariate analyses showed that size (≥ 6 mm), resection time (≥ 5 s), and serrated lesions were risk factors for CSDP. CONCLUSIONS: Cold snare defect protrusion (CSDP), which was present with 36%, was a good indicator for incomplete mucosal layer resection. Even in nonCSDP polypectomies, 57% of the mucosal layer was not removed completely. Thus, CSP should be used for intra-epithelial lesions only, and careful pretreatment evaluation is recommended.

Feasibility of underwater endoscopic mucosal resection and management of residues for superficial non-ampullary duodenal epithelial neoplasms.

BACKGROUND AND AIM: Underwater endoscopic mucosal resection (UEMR) is effective for superficial non-ampullary duodenal epithelial neoplasms (SNADEN). However, the incidence of residual lesion after UEMR, especially for large lesions (≥20 mm), and their prognosis remain unclear. We aimed to assess the incidence of residual lesions and further outcomes after UEMR for SNADEN. METHODS: We carried out a retrospective study at a tertiary cancer institute. Candidates for the study were systematically retrieved from an endoscopic and pathological database from January 2013 to April 2018. RESULTS: A total of 162 SNADEN resected with UEMR were analyzed. Median (range) procedure time was 5 (1-70) min. En bloc resection rates for large lesions (≥20 mm) and small lesions (<20 mm) were 14% and 79%, respectively. Intraprocedural bleeding occurred in one (0.6%) case, but no intraprocedural perforation occurred during the study. Delayed bleeding occurred in two (1.2%) cases and delayed perforation occurred in one (0.6%) case. A total of 157 (97%) lesions were followed up by at least one endoscopic examination. Of these lesions, residual lesions were recognized in seven cases (5%). Additional UEMR was carried out in five lesions and underwater cold snare polypectomy in one lesion. One lesion was observed without additional treatment. After salvage intervention, no cases experienced further residual lesions. CONCLUSION: Although UEMR for SNADEN can be relevant when other efficacious procedures are unavailable, careful follow up for residual lesions is required especially after piecemeal resection for large lesions.

Impact of age at diagnosis of head and neck cancer on incidence of metachronous cancer.

BACKGROUND: Metachronous cancer in patients with head and neck cancer (HNC) is common and is associated with a poor prognosis. We aimed to evaluate the incidence of metachronous cancer at different sites according to age at diagnosis of index HNC. METHODS: We collected data on 2011 patients with oral cancer, oropharynx cancer, hypopharyngeal cancer, and laryngeal cancer as index cancers using the Osaka International Cancer Institute Cancer Registry database between 2005 and 2016. Among these, we analyzed 1953 patients after excluding 5 patients who were not followed-up and 53 patients with simultaneous multiple index cancers. We evaluated the cumulative incidence of metachronous cancer in the esophagus, lung, and other sites according to age at diagnosis of the index HNC using the Kaplan-Meier method. Multivariate logistic regression analysis was performed to identify factors that influenced the incidence of metachronous cancers following HNC. RESULTS: The cumulative incidence of metachronous esophageal cancer in young patients (< 65 years) was significantly higher than that in old patients (≥ 65 years) (12.1% vs 8.5% at 5 years, and 16.5% vs 11.2% at 10 years; p = 0.015). On the other hand, the cumulative incidence of the other cancers in young patients was significantly lower than that in old patients (7.8% vs 12.2% at 5 years, and 13.9% vs 15.3% at 10 years; p = 0.017). The cumulative incidence of lung cancer was not significance according to age at diagnosis of the index HNC. In the multivariate analysis, histological type (squamous cell carcinoma) and lesion location (hypopharynx and larynx) were independently associated with metachronous cancers. Moreover, age at diagnosis of the index HNC (< 65 years), histological type (squamous cell carcinoma) and lesion location (hypopharynx) were significant predictors of metachronous esophageal cancer incidence and lesion location (hypopharynx) was a significant predictor of metachronous lung cancer incidence. CONCLUSION: Risk stratification of metachronous cancers with age and other predictors may help to properly manage patients with HNC. TRIAL REGISTRATION: The present study is a non-intervention trial.

Classification for invasion depth of esophageal squamous cell carcinoma using a deep neural network compared with experienced endoscopists.

BACKGROUND AND AIMS: Cancer invasion depth is a critical factor affecting the choice of treatment in patients with superficial squamous cell carcinoma (SCC). However, the diagnosis of invasion depth is currently subjective and liable to interobserver variability. METHODS: We developed a deep learning-based artificial intelligence (AI) system based on Single Shot MultiBox Detector architecture for the assessment of superficial esophageal SCC. We obtained endoscopic images from patients with superficial esophageal SCC at our facility between December 2005 and December 2016. RESULTS: After excluding poor-quality images, 8660 non-magnified endoscopic (non-ME) and 5678 ME images from 804 superficial esophageal SCCs with pathologic proof of cancer invasion depth were used as the training dataset, and 405 non-ME images and 509 ME images from 155 patients were selected for the validation set. Our system showed a sensitivity of 90.1%, specificity of 95.8%, positive predictive value of 99.2%, negative predictive value of 63.9%, and an accuracy of 91.0% for differentiating pathologic mucosal and submucosal microinvasive (SM1) cancers from submucosal deep invasive (SM2/3) cancers. Cancer invasion depth was diagnosed by 16 experienced endoscopists using the same validation set, with an overall sensitivity of 89.8%, specificity of 88.3%, positive predictive value of 97.9%, negative predictive value of 65.5%, and an accuracy of 89.6%. CONCLUSIONS: This newly developed AI system showed favorable performance for diagnosing invasion depth in patients with superficial esophageal SCC, with comparable performance to experienced endoscopists.

Pulley Traction-Assisted Colonic Endoscopic Submucosal Dissection: A Retrospective Case Series.

BACKGROUND AND STUDY AIMS: We have reported the usefulness of traction-assisted colorectal endoscopic submucosal dissection (TAC-ESD) to overcome the technical difficulty of colorectal ESD. However, the direction of traction is toward the anal side only. We retrospectively evaluate the safety and efficacy of "pulley" TAC-ESD. PATIENTS AND METHODS: We retrospectively analyzed patients diagnosed with colorectal polyps and treated by "pulley" TAC-ESD at Osaka International Cancer Institute from December 2017 to June 2018. RESULTS: During the study period, 79 patients with 81 lesions were treated by ESD, and 54 of these patients were treated by traction-assisted ESD. Among them, 7 underwent "pulley" traction-assisted ESD (6 men, 1 woman; age, 48-69 years), resulting in en bloc resection with no complications. This afforded good visibility of the submucosal layer in 6 patients, but it was not effective in the remaining patient with the muscle-retracting sign. CONCLUSION: The "pulley" method affords good visibility of the submucosal layer by changing the traction direction in cases that are difficult to manage by conventional traction-assisted ESD.

Differences in Clinical Course of Intraprocedural and Delayed Perforation Caused by Endoscopic Submucosal Dissection for Colorectal Neoplasms: A Retrospective Study.

BACKGROUND: Although the use of endoscopic submucosal dissection (ESD) as a minimally invasive treatment for large superficial colorectal neoplasms is increasing, colorectal ESD remains technically challenging. As perforation in the colorectum is generally considered to be associated with a higher risk of complications, the aim of this study was to investigate the characteristics of perforation caused by colorectal ESD. METHODS: This retrospective study included 635 lesions treated with colorectal ESD, between February 2011 and December 2015, in a tertiary cancer center. We evaluated and compared the clinical course and short-term outcomes of the intraprocedural perforation group with those of the delayed perforation and no perforation groups. RESULTS: Perforation occurred in 45 (7.1%) of the 635 cases. Thirty-six cases were intraprocedural perforation (5.7%), all of which were successfully closed with endoclips during the procedure. Nine cases of delayed perforation occurred (1.4%). No emergency surgery was performed in the intraprocedural perforation group; however, 5 of 9 cases underwent emergency surgery in the delayed perforation group (56%, p < 0.0001). There were statistically significant differences between the intraprocedural and delayed perforation groups with regard to the hospitalization period, fasting period, abdominal pain scale, peak white blood cell (WBC) count, and peak C-reactive protein (CRP), and between the intraprocedural and no perforation groups with regard to the location of the lesion, hospitalization period, fasting period, abdominal pain scale, peak WBC, peak CRP, and en bloc resection rate. CONCLUSIONS: While intraprocedural perforation due to colorectal ESD can be managed conservatively, delayed perforation can lead to serious adverse events.

Endoscopic findings corresponding to multiple Lugol-voiding lesions in the esophageal background mucosa.

BACKGROUND AND AIM: Multiple Lugol-voiding lesions (LVLs) on Lugol chromoendoscopy can predict the development of metachronous multiple cancers in the esophagus and the head and neck regions. However, Lugol chromoendoscopy sometimes causes adverse events such as chest pain and discomfort. We therefore investigated the endoscopic findings on narrow band imaging (NBI) or blue laser imaging (BLI) that correspond to the presence of multiple LVLs in patients with esophageal squamous cell carcinoma. METHODS: First, we investigated the NBI/BLI findings corresponding to individual small LVLs (one-to-one correspondence). Second, we investigated the association between the grade of multiple LVLs and the five endoscopic findings, including multiple foci of dilated vessels (MDV), multiple small brownish areas without microvascular irregularity, and a nonuniform color tone. RESULTS: One-to-one correspondence of endoscopic findings was analyzed in 106 small LVLs. The main findings matched with small LVLs were a focus of dilated vessels (44 lesions), a small brownish area (17 lesions), and a small brownish area with a focus of dilated vessels (19 lesions). The relationship between multiple LVLs and each finding assessed by NBI/BLI was assessed in 155 patients. Multivariate logistic regression indicated that the presence of MDV was the only finding independently associated with multiple LVLs (P < 0.01). CONCLUSIONS: The presence of MDV in the noncancerous background esophageal mucosa was significantly associated with multiple LVLs. This pilot study demonstrates that MDV has the potential to be a new risk factor for the development of metachronous multiple esophageal squamous cell carcinoma.

Pharyngeal observation via transoral endoscopy using a lip cover-type mouthpiece.

BACKGROUND AND AIM: Simultaneous and metachronous head and neck cancers are likely to develop in patients with current or previous esophageal cancer. The Valsalva maneuver facilitates the visualization of the hypopharyngeal area during endoscopic observation, but it requires transnasal endoscopy. We developed a novel Valsalva maneuver using transoral endoscopy with a lip cover-type mouthpiece. METHODS: Between March 2018 and July 2018, 107 patients with current or previous esophageal cancer who were scheduled to undergo upper gastrointestinal endoscopy were included in our pilot study. We performed the Valsalva maneuver using our newly developed mouthpiece and transoral endoscopy in the patients and evaluated the hypopharyngeal region, including the postcricoid area and the posterior wall of the hypopharynx. The outcome measure was procedural success rate, which was graded as "complete," "incomplete," and "none." RESULTS: Observation of the hypopharyngeal area was "complete" in 81 patients (76%) using the Valsalva maneuver. However, in 25 patients and in 1 patient, observation was "incomplete" and "none," respectively. Seven lesions were newly detected in seven patients during the study period. CONCLUSIONS: The Valsalva maneuver using transoral endoscopy with a lip cover-type mouthpiece is feasible for the visualization of the postcricoid area and the posterior wall of the hypopharynx. Particularly, this technique would contribute to early detection of head and neck cancers.

UNC119 is a binding partner of tumor suppressor Ras-association domain family 6 and induces apoptosis and cell cycle arrest by MDM2 and p53.

Ras-association domain family 6 (RASSF6) is a tumor suppressor that interacts with MDM2 and stabilizes p53. Caenorhabditis elegans unc-119 encodes a protein that is required for normal development of the nervous system. Humans have 2 unc-119 homologues, UNC119 and UNC119B. We have identified UNC119 as a RASSF6-interacting protein. UNC119 promotes the interaction between RASSF6 and MDM2 and stabilizes p53. Thus, UNC119 induces apoptosis by RASSF6 and p53. UNC119 depletion impairs DNA repair after DNA damage and results in polyploid cell generation. These findings support that UNC119 is a regulator of the RASSF6-MDM2-p53 axis and functions as a tumor suppressor.

c-Jun N-terminal phosphorylation antagonises recruitment of the Mbd3/NuRD repressor complex.

AP-1 (activator protein 1) activity is strongly induced in response to numerous signals, including growth factors, cytokines and extracellular stresses. The proto-oncoprotein c-Jun belongs to the AP-1 group of transcription factors and it is a crucial regulator of intestinal progenitor proliferation and tumorigenesis. An important mechanism of AP-1 stimulation is phosphorylation of c-Jun by the Jun amino-terminal kinases (JNKs). N-terminal phosphorylation of the c-Jun transactivation domain increases target gene transcription, but a molecular explanation was elusive. Here we show that unphosphorylated, but not N-terminally phosphorylated c-Jun, interacts with Mbd3 and thereby recruits the nucleosome remodelling and histone deacetylation (NuRD) repressor complex. Mbd3 depletion in colon cancer cells increased histone acetylation at AP-1-dependent promoters, which resulted in increased target gene expression. The intestinal stem cell marker lgr5 was identified as a novel target gene controlled by c-Jun/Mbd3. Gut-specific conditional deletion of mbd3 (mbd3(ΔG/ΔG) mice) stimulated c-Jun activity and increased progenitor cell proliferation. In response to inflammation, mdb3 deficiency resulted in colonic hyperproliferation and mbd3(ΔG/ΔG) mice showed markedly increased susceptibility to colitis-induced tumorigenesis. Notably, concomitant inactivation of a single allele of c-jun reverted physiological and pathological hyperproliferation, as well as the increased tumorigenesis in mbd3(ΔG/ΔG) mice. Thus the transactivation domain of c-Jun recruits Mbd3/NuRD to AP-1 target genes to mediate gene repression, and this repression is relieved by JNK-mediated c-Jun N-terminal phosphorylation.

Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.

Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in cell proliferation and differentiation. It is phosphorylated by large tumor suppressor kinases, the core kinases of the tumor-suppressive Hippo pathway. Phosphorylation induces the cytoplasmic accumulation of TAZ and its degradation. In human cancers, the deregulation of the Hippo pathway and gene amplification enhance TAZ activity. TAZ interacts with TEA domain family members (TEAD), and upregulates genes implicated in epithelial-mesenchymal transition. It also confers stemness to cancer cells. Thus, TAZ activation provides cancer cells with malignant properties and worsens the clinical prognosis. Therefore, TAZ attracts attention as a therapeutic target in cancer therapy. We applied 18 606 small chemical compounds to human osteosarcoma U2OS cells expressing GFP-fused TAZ (GFP-TAZ), monitored the subcellular localization of GFP-TAZ, and selected 33 compounds that shifted GFP-TAZ to the cytoplasm. Unexpectedly, only a limited number of compounds suppressed TAZ-mediated enhancement of TEAD-responsive reporter activity. Moreover, the compounds that weakened TEAD reporter activity did not necessarily decrease the unphosphorylated TAZ. In this study, we focused on three compounds that decreased both TEAD reporter activity and unphosphorylated TAZ, and treated several human cancer cells with these compounds. One compound did not show a remarkable effect, whereas the other two compounds compromised the cell viability in certain cancer cells. In conclusion, the GFP-TAZ-based assay can be used as the first screening for compounds that inhibit TAZ and show anticancer properties. To develop anticancer drugs, we need additional assays to select the compounds.

A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells.

The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1-10 and GFP11 cells). GFP1-10 and GFP11 cells do not exhibit GFP signals until they are fused. The signal intensity correlates with the expression of myogenic markers and myofusion. Myogenesis-promoting reagents, such as insulin-like growth factor-1 (IGF1) and ß-guanidinopropionic acid (GPA), enhance the signals, whereas the poly-caspase inhibitor, z-VAD-FMK, suppresses it. GFP signals are observed when myotubes formed by GFP1-10 cells are fused with single nuclear GFP11 cells, and enhanced by IGF1, GPA, and IBS008738, a recently-reported myogenesis-promoting reagent. Fusion between myotubes formed by GFP1-10 and GFP11 cells is associated with the appearance of GFP signals. IGF1 and GPA augment these signals, whereas NSC23766, Rac inhibitor, decreases them. The conditioned medium of cancer cells suppresses GFP signals during myogenesis and reduces the width of GFP-positive myotubes after differentiation. Thus the novel split GFP-based assay will provide the useful method for the study of myogenesis, myofusion, and atrophy.

The RASSF6 tumor suppressor protein regulates apoptosis and the cell cycle via MDM2 protein and p53 protein.

Ras association domain family (RASSF) 6 is a member of the C-terminal RASSF proteins such as RASSF1A and RASSF3. RASSF6 is involved in apoptosis in various cells under miscellaneous conditions, but it remains to be clarified how RASSF6 exerts tumor-suppressive roles. We reported previously that RASSF3 facilitates the degradation of MDM2, a major E3 ligase of p53, and stabilizes p53 to function as a tumor suppressor. In this study, we demonstrate that RASSF6 overexpression induces G1/S arrest in p53-positive cells. Its depletion prevents UV- and VP-16-induced apoptosis and G1/S arrest in HCT116 and U2OS cells. RASSF6-induced apoptosis partially depends on p53. RASSF6 binds MDM2 and facilitates its ubiquitination. RASSF6 depletion blocks the increase of p53 in response to UV exposure and up-regulation of p53 target genes. RASSF6 depletion delays DNA repair in UV- and VP-16-treated cells and increases polyploid cells after VP-16 treatment. These findings indicate that RASSF6 stabilizes p53, regulates apoptosis and the cell cycle, and functions as a tumor suppressor. Together with the previous reports regarding RASSF1A and RASSF3, the stabilization of p53 may be the common function of the C-terminal RASSF proteins.

Characterization of RSF-1, the Caenorhabditis elegans homolog of the Ras-association domain family protein 1.

Mammals have 10 RASSF proteins, which are characterized by the Ras-association (RA) domain. Among them, RASSF1 to RASSF6 have the Salvador/RASSF/Hippo (SARAH) domain and form the subclass C-terminal RASSF proteins. Drosophila genome has a single C-terminal RASSF, dRASSF. All these RASSF proteins are related to the tumor suppressive Hippo pathway, and are considered to function as tumor suppressors. Caenorhabditis elegans T24F1.3 encodes a protein with the RA and the SARAH domains. The amino acid sequences are 40% and 55% similar to those of RASSF1 in the RA and the SARAH domains, respectively. We have characterized T24F1.3 gene product and named it RSF-1 as RASSF1 homolog. RSF-1 is widely expressed in epithelial cells. About 14% rsf-1 mutants exhibit defects in embryonal morphogenesis and the actin disorganization. The combinatorial synthetic lethal analysis demonstrates that the lethality is enhanced to more than 80% in rsf-1 mutants with the WASP-family verprolin homologous protein complex-related gene depletions and corroborates the implication of RSF-1 in the regulation of actin cytoskeleton. In rsf-1 mutants, the structures of muscle actin are preserved, but the swimming ability is impaired. Although we could not detect the direct physical interaction of LET-60 with RSF-1, rsf-1 mutants suppress the multivulva phenotype of the active let-60 mutants, suggesting that rsf-1 genetically interacts with the Ras signaling.