Ubiquitination of DNA Damage-Stalled RNAPII Promotes Transcription-Coupled Repair.

Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.

Human Rad52 Promotes XPG-Mediated R-loop Processing to Initiate Transcription-Associated Homologous Recombination Repair.

Given that genomic DNA exerts its function by being transcribed, it is critical for the maintenance of homeostasis that DNA damage, such as double-strand breaks (DSBs), within transcriptionally active regions undergoes accurate repair. However, it remains unclear how this is achieved. Here, we describe a mechanism for transcription-associated homologous recombination repair (TA-HRR) in human cells. The process is initiated by R-loops formed upon DSB induction. We identify Rad52, which is recruited to the DSB site in a DNA-RNA-hybrid-dependent manner, as playing pivotal roles in promoting XPG-mediated R-loop processing and initiating subsequent repair by HRR. Importantly, dysfunction of TA-HRR promotes DSB repair via non-homologous end joining, leading to a striking increase in genomic aberrations. Thus, our data suggest that the presence of R-loops around DSBs within transcriptionally active regions promotes accurate repair of DSBs via processing by Rad52 and XPG to protect genomic information in these critical regions from gene alterations.

RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.

RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype.

Preoperative Prediction of Long-Term Survival After Surgery in Patients with Resectable Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Although some clinical trials have demonstrated the benefits of neoadjuvant therapy for resectable pancreatic ductal adenocarcinoma (PDAC), its optimal candidate has not been clarified. This study aimed to detect predictive prognostic factors for resectable PDAC patients who underwent upfront surgery and identify patient cohorts with long-term survival without neoadjuvant therapy. PATIENTS AND METHODS: A total of 232 patients with resectable PDAC who underwent upfront surgery between January 2008 and December 2019 were evaluated. RESULTS: The median overall survival (OS) time and 5-year OS rate of resectable PDAC with upfront surgery was 31.5 months and 33.3%, respectively. Multivariate analyses identified tumor diameter in computed tomography (CT) ≤ 19 mm [hazard ratio (HR) 0.40, p < 0.001], span-1 within the normal range (HR 0.54, p = 0.023), prognostic nutritional index (PNI) ≥ 44.31 (HR 0.51, p < 0.001), and lymphocyte-to-monocyte ratio (LMR) ≥ 3.79 (HR 0.51, p < 0.001) as prognostic factors that influence favorable prognoses after upfront surgery. According to the prognostic prediction model based on these four factors, patients with four favorable prognostic factors had a better prognosis with a 5-year OS rate of 82.4% compared to others (p < 0.001). These patients had a high R0 resection rate and a low frequency of tumor recurrence after upfront surgery. CONCLUSIONS: We identified patients with long-term survival after upfront surgery by prognostic prediction model consisting of tumor diameter in CT, span-1, PNI, and LMR. Evaluation of anatomical, biological, nutritional, and inflammatory factors may be valuable to introduce an optimal treatment strategy for resectable PDAC.

Serum elastase-1 predicts malignancy in intraductal papillary mucinous neoplasm of the pancreas.

BACKGROUND: The indication for surgical resection of intraductal papillary mucinous neoplasms (IPMNs) is defined by imaging features, such as mural nodules. Although carbohydrate antigen (CA) 19-9 was selected as a parameter for worrisome features, no serum biomarkers were considered when deciding on surgical indications in the latest international consensus guideline. In this study, we assessed whether clinical factors, imaging findings, and serum biomarkers are useful in predicting malignant IPMNs. METHODS: A total of 234 resected IPMN cases in Chiba University Hospital from July 2005 to December 2021 were retrospectively analyzed. RESULTS: Among the 234 patients with resected IPMNs diagnosed by preoperative imaging, 117 were diagnosed with malignant pathologies (high-grade dysplasia and invasive IPMNs) according to the histological classification. In the multivariate analysis, cyst diameter ≥30 mm; p = 0.035), enhancing mural nodules on multidetector computed tomography (≥5 mm; p = 0.018), and high serum elastase-1 (≥230 ng/dl; p = 0.0007) were identified as independent malignant predictors, while CA19-9 was not. Furthermore, based on the receiver operator characteristic curve analyses, elastase-1 was superior to CA19-9 for predicting malignant IPMNs. Additionally, high serum elastase-1 levels (≥230 ng/dl; p = 0.0093) were identified as independent predictors of malignant IPMNs in patients without mural nodules on multidetector computed tomography (MDCT) in multivariate analysis. CONCLUSION: The serum elastase-1 level was found to be a potentially useful biomarker for predicting malignant IPMNs.

The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage.

The biological response to DNA double-strand breaks acts to preserve genome integrity. Individuals bearing inactivating mutations in components of this response exhibit clinical symptoms that include cellular radiosensitivity, immunodeficiency, and cancer predisposition. The archetype for such disorders is Ataxia-Telangiectasia caused by biallelic mutation in ATM, a central component of the DNA damage response. Here, we report that the ubiquitin ligase RNF168 is mutated in the RIDDLE syndrome, a recently discovered immunodeficiency and radiosensitivity disorder. We show that RNF168 is recruited to sites of DNA damage by binding to ubiquitylated histone H2A. RNF168 acts with UBC13 to amplify the RNF8-dependent histone ubiquitylation by targeting H2A-type histones and by promoting the formation of lysine 63-linked ubiquitin conjugates. These RNF168-dependent chromatin modifications orchestrate the accumulation of 53BP1 and BRCA1 to DNA lesions, and their loss is the likely cause of the cellular and developmental phenotypes associated with RIDDLE syndrome.

Initial laparoscopic liver resection is associated with reduced adhesions and transfusions at the time of salvage liver transplantation.

BACKGROUND: Laparoscopic liver resection (LLR) has been associated with improved patient recovery as well as reduced postoperative adhesions compared to open LR (OLR) and could therefore facilitate redo liver surgery. LLR prior to liver transplantation (LT) is increasingly performed, LT being saved for HCC recurrence. LT is still performed by open surgery due to vascular reconstructions and underlying liver chronic disease. We evaluated the impact of laparoscopic approach for LR prior to LT in terms of intraoperative transfusions, adhesions severity and outcome. METHODS: Data from all patients who underwent a LT after LR in two French high-volume tertiary centers were retrospectively reviewed. The impact of LLR on per operative and postoperative outcome was assessed using logistic regression. RESULTS: 170 patients were included, 43 patients in the LLR group and 127 in the OLR group. Preoperative characteristics were comparable between both groups, except LLR group included more patients with "very early-stage" BCLC tumors than OLR group (51% vs. 33%, p = 0.02) and less anatomical resections (54% vs. 75%, p = 0.015). During LT, the transfusion rate was significantly higher in the OLR group (71.4% vs 44.2%, p = 0.002) as well as the proportion of type III adhesions according to OLSG classification (p < 0.001). Multivariate analysis found LLR to be an independent protective factor for adhesions and transfusion (p = 0.001 and p = 0.03 respectively). CONCLUSION: Laparoscopic liver resection was associated with reduced postoperative adhesions and transfusion requirements during subsequent liver transplantation.

Clinical benefits of pulmonary resection for lung metastases from pancreatic cancer.

PURPOSE: Systemic chemotherapy is generally used for metastatic pancreatic cancer; however, pulmonary resection may be a treatment option for lung oligometastases from pancreatic cancer. The current study aimed to clarify the oncological outcomes and clinical benefits of pulmonary resection for lung metastases. METHODS: Of 510 patients who underwent pancreatic resection for pancreatic cancer, 44 patients with recurrence of isolated lung metastases and one patient with simultaneous lung metastases were evaluated. RESULTS: Of the 45 patients, 20 patients were selected as candidates for pulmonary resection based on clinical factors such as recurrence-free interval (RFI) from pancreatectomy to lung metastases, number of lung metastases, and serum CA19-9 level. The post-recurrent survival of patients with pulmonary resection was significantly better than that of patients without pulmonary resection. Fourteen of the 20 patients with pulmonary resection developed tumor recurrence with a median disease-free survival (DFS) of 15 months. Univariate analyses revealed that an RFI from pancreatectomy to lung metastases of ≥28 months was associated with better DFS after pulmonary resection. Of the 14 patients with an RFI of ≥28 months, pulmonary resection resulted in prolonged chemotherapy-free interval in 12 patients. Furthermore, repeat pulmonary resection for recurrent tumors after pulmonary resection led to further cancer-free interval in some cases. CONCLUSIONS: Although many patients had tumor recurrence after pulmonary resection, pulmonary resection for lung metastases from pancreatic cancer may provide prolonged cancer-free interval without the need for chemotherapy. Pulmonary resection should be performed for the patients with a long RFI from pancreatectomy to lung metastases.

[Intraoperative Blood Flow Assessment of Gastric Tube Using Indocyanine Green fluorography during Pancreaticoduodenectomy Following Esophagectomy-Case Reports].

Case 1: A 73-year-old male, who had an intraductal papillary mucinous adenocarcinoma or resectable pancreatic cancer at the uncinate process of the pancreas five years after subtotal esophagectomy for esophageal cancer, underwent pylorus preserving pancreaticoduodenectomy(PPPD). Case 2: A 68-year-old male, who also had a resectable pancreatic cancer at the uncinate process of the pancreas 3 years after subtotal esophagectomy for esophageal cancer, underwent PPPD following neoadjuvant chemotherapy. In both cases, right gastroepiploic artery and vein were preserved to maintain the perfusion of the gastric tube during surgery. Indocyanine Green(ICG)fluorography was performed just before duodenal-jejunal anastomosis, which visually showed the well-perfused gastric tube. Both patients had no necrosis of the gastric tube, nor gastrointestinal obstruction after surgery. Intraoperative ICG fluorography was useful to evaluate the blood flow of the remaining gastric tube visually during PPPD for post-esophagectomy patients.

[Conversion Surgery Following Systemic Chemotherapy for Advanced Gallbladder Cancer with Peritoneal Dissemination-A Case Report].

A 73-year-old female was diagnosed with gallbladder cancer, but the future liver remnant volume was deemed insufficient for curative resection. Consequently, transileocolic portal vein embolization was performed. During laparotomy, multiple nodules were palpable on the peritoneal surface of the pelvic floor. Subsequently, staging laparoscopy confirmed the pathological diagnosis of adenocarcinoma in the resected nodules, indicating peritoneal dissemination of gall bladder cancer. Due to this peritoneal dissemination, surgical resection was deemed inappropriate, and the patient was initiated on systemic chemotherapy consisting of gemcitabine and cisplatin. Following 22 courses of chemotherapy, contrast-enhanced computed tomography demonstrated no significant changes in the size of the primary tumor or its location relative to the main vessels, although a small metastatic lesion was identified in the gallbladder bed. At the second staging laparoscopy, any nodules suggesting peritoneal dissemination were observed. Based on these findings, we decided to perform curative resection. The surgical procedure involved right hepatectomy plus segment 4a resection, extrahepatic bile duct resection, and hepaticojejunostomy. Pathological examination revealed ypT3bN0M1(HEP), ypStage ⅣB, with the achievement of R0 resection. The patient survived with no recurrences for 40 months after surgery. These results suggest that aggressive therapeutic strategies, including conversion surgery following systemic chemotherapy, may be beneficial for patients initially deemed unresectable due to gallbladder cancer.

[Retrospective Study of Preoperative Serial Pancreatic Juice Aspiration Cytological Examination(SPACE)for Early Preoperative Detection of Pancreatic Cancer].

Serial pancreatic juice aspiration cytological examination(SPACE)has been reported as a reliable preoperative diagnostic method for early pancreatic cancer, when combined with imaging findings suspecting early pancreatic cancer. Among 259 patients with suspected pancreatic cancer who underwent pancreatic resection at our hospital, SPACE was preoperatively performed in 14 cases(5.4%). Of these 14 cases, final pathological diagnosis was pancreatic cancer in 12 patients (86%), including 5 patients with Stage ⅠA pancreatic cancer(35.7%), all of whom had a mass on preoperative CT or EUS. On the other hand, in the other 2 cases(14.3%), CT/EUS detected no mass but focal pancreatic parenchymal atrophy and main pancreatic duct stenosis which were the imaging findings suspecting very early pancreatic cancer such as cancer in situ. Although preoperative SPACE results of these 2 cases were class Ⅳ, final pathological results of resected specimen were low-grade PanIN in both cases. SPACE was considered useful for preoperative diagnosis of pancreatic cancer in our study, however further study is needed to examine its diagnostic accuracy for early pancreatic cancer which does not appear as a mass in any imaging modality.

Fine-tuning of DNA damage-dependent ubiquitination by OTUB2 supports the DNA repair pathway choice.

DNA double-strand breaks (DSBs) are deleterious lesions that lead to genetic mutations and cell death. Protein ubiquitination mediated by the E3 ubiquitin ligase RNF8 within the regions surrounding DSBs recruits DNA DSB response (DDR) factors and induces chromatin remodeling, which supports cell survival after DNA damage. Nevertheless, the impact of RNF8-mediated ubiquitination on DNA repair remains to be elucidated. Here, we report that depletion of the deubiquitinating enzyme OTUB2 enhances RNF8-mediated ubiquitination in an early phase of the DDR and promotes faster DSB repair but suppresses homologous recombination. The rapid ubiquitination results in accelerated accumulation of 53BP1 and RAP80 at DSBs, which in turn protects DSB ends from resection in OTUB2-depleted cells. Mechanistically, OTUB2 suppresses RNF8-mediated L3MBTL1 ubiquitination and Lys 63-linked ubiquitin chain formation in a deubiquitinating activity-dependent manner. Thus, OTUB2 fine-tunes the speed of DSB-induced ubiquitination so that the appropriate DNA repair pathway is chosen.

Predictors of a difficult Pringle maneuver in laparoscopic liver resection and evaluation of alternative procedures to assist bleeding control.

PURPOSE: To evaluate the predictors of a difficult Pringle maneuver (PM) in laparoscopic liver resection (LLR) and to assess alternative procedures to PM. METHODS: Data from patients undergoing LLR between 2013 and 2020 were reviewed retrospectively. Univariate and multivariate analyses were performed and the outcomes of patients who underwent PM or alternative procedures were compared. RESULTS: Among 106 patients who underwent LLR, PM could not be performed in 18 (17.0%) because of abdominal adhesions in 14 (77.8%) and/or collateral flow around the hepatoduodenal ligament in 5 (27.8%). Multivariate analysis revealed that Child-Pugh classification B (p = 0.034) and previous liver resection (p < 0.001) were independently associated with difficulty in performing PM in LLR. We evaluated pre-coagulation of liver tissue using microwave tissue coagulators, saline irrigation monopolar, clamping of the hepatoduodenal ligament using an intestinal clip, and hand-assisted laparoscopic surgery as alternatives procedures to PM. There were no significant differences in blood loss (p = 0.391) or transfusion (p = 0.518) between the PM and alternative procedures. CONCLUSIONS: Child-Pugh classification B and previous liver resection were identified as predictors of a difficult PM in LLR. The alternative procedures were found to be effective.

Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair.

CRISPR/Cas9, which generates DNA double-strand breaks (DSBs) at target loci, is a powerful tool for editing genomes when codelivered with a donor DNA template. However, DSBs, which are the most deleterious type of DNA damage, often result in unintended nucleotide insertions/deletions (indels) via mutagenic nonhomologous end joining. We developed a strategy for precise gene editing that does not generate DSBs. We show that a combination of single nicks in the target gene and donor plasmid (SNGD) using Cas9D10A nickase promotes efficient nucleotide substitution by gene editing. Nicking the target gene alone did not facilitate efficient gene editing. However, an additional nick in the donor plasmid backbone markedly improved the gene-editing efficiency. SNGD-mediated gene editing led to a markedly lower indel frequency than that by the DSB-mediated approach. We also show that SNGD promotes gene editing at endogenous loci in human cells. Mechanistically, SNGD-mediated gene editing requires long-sequence homology between the target gene and repair template, but does not require CtIP, RAD51, or RAD52. Thus, it is considered that noncanonical homology-directed repair regulates the SNGD-mediated gene editing. In summary, SNGD promotes precise and efficient gene editing and may be a promising strategy for the development of a novel gene therapy approach.

Early- and long-term outcomes of liver transplantation with rescue allocation grafts.

In France, liver grafts which have been refused by at least five centers are proposed as rescue allocation (RA). The aim of this study is to clarify the feasibility and safety of RA grafts in liver transplantation (LT). Short- and long-term outcomes of patients who received RA grafts (RA group) were compared with those of patients who received standard allocation (SA) grafts (SA group). From a total of 1635 patients, 102 patients received RA grafts. Before matching, the RA group was characterized primarily by less severe liver disease, but the quality of graft was worse. After matching recipients' characteristics of 102 patients who used RA grafts with 306 patients who used SA grafts, recipients' characteristics were well balanced (1:3 matching). Although the rate of primary dysfunction was significantly higher in the RA group, there is no significant difference in the occurrence of major complications, length of hospitalization, and mortality between two groups. Graft survival (GS) and overall survival (OS) in the RA group were not significantly different from the SA group (GS; HR = 1.03 P = .89, OS; HR = 1.03 P = .90). In the French allocation system, the feasibility and safety of RA grafts might be comparable to SA grafts for carefully selected patients.

BRCA1 ensures genome integrity by eliminating estrogen-induced pathological topoisomerase II-DNA complexes.

Women having BRCA1 germ-line mutations develop cancer in breast and ovary, estrogen-regulated tissues, with high penetrance. Binding of estrogens to the estrogen receptor (ER) transiently induces DNA double-strand breaks (DSBs) by topoisomerase II (TOP2) and controls gene transcription. TOP2 resolves catenated DNA by transiently generating DSBs, TOP2-cleavage complexes (TOP2ccs), where TOP2 covalently binds to 5' ends of DSBs. TOP2 frequently fails to complete its catalysis, leading to formation of pathological TOP2ccs. We have previously shown that the endonucleolytic activity of MRE11 plays a key role in removing 5' TOP2 adducts in G1 phase. We show here that BRCA1 promotes MRE11-mediated removal of TOP2 adducts in G1 phase. We disrupted the BRCA1 gene in 53BP1-deficient ER-positive breast cancer and B cells. The loss of BRCA1 caused marked increases of pathological TOP2ccs in G1 phase following exposure to etoposide, which generates pathological TOP2ccs. We conclude that BRCA1 promotes the removal of TOP2 adducts from DSB ends for subsequent nonhomologous end joining. BRCA1-deficient cells showed a decrease in etoposide-induced MRE11 foci in G1 phase, suggesting that BRCA1 repairs pathological TOP2ccs by promoting the recruitment of MRE11 to TOP2cc sites. BRCA1 depletion also leads to the increase of unrepaired DSBs upon estrogen treatment both in vitro in G1-arrested breast cancer cells and in vivo in epithelial cells of mouse mammary glands. BRCA1 thus plays a critical role in removing pathological TOP2ccs induced by estrogens as well as etoposide. We propose that BRCA1 suppresses tumorigenesis by removing estrogen-induced pathological TOP2ccs throughout the cell cycle.

[Laparoscopic Local Gastrectomy for Gastric Schwannoma and Extramurally Developed Gastric GIST].

Among gastric submucosal tumors, neurogenic tumors are considered to be rare diseases. We experienced a case of laparoscopic local gastrectomy of gastric schwannoma coexisting with extramurally developed gastric GIST found accidentally during surgery. A 61-year-old man was pointed out a gastric submucosal tumor with a diameter of 15 mm in a medical checkup. Endoscopic ultrasound-guided fine needle aspiration(EUS-FNA)was performed, and immunostaining showed that c-kit(-), CD34(-), S-100(+), SMA(-), MIB-1<2%. Diagnosis was gastric schwannoma. We performed laparoscopic local gastrectomy. During the surgery another extramural nodule was accidentally found with a diameter of 8 mm at the anterior wall of the gastric body near lesser curvature. Immunostaining showed c-kit(+), CD34(+)and was diagnosed GIST. Because a gastric schwannoma coexisting with GIST is a rare case, we decided to report it by adding discussion with some literatures.

Roles of Pin1 as a Key Molecule for EMT Induction by Activation of STAT3 and NF-κB in Human Gallbladder Cancer.

BACKGROUND: Despite developments in multidisciplinary treatment, the prognosis for advanced gallbladder cancer (GBC) still is poor because of its rapid progression. Epithelial-mesenchymal transition (EMT) plays a central role in promoting tumor invasion and metastasis in malignancies thorough signal transducer and activator of transcription-3 (STAT3) and nuclear factor κB (NF-κB) activation. Whereas Pin1 mediates STAT3 and NF-κB activation, the involvement of Pin1 in GBC progression is unclear. METHODS: Factors regulating Pin1-related STAT3 and NF-κB activation were evaluated using surgical specimens collected from 76 GBC patients, GBC cells, and orthotopic GBC xenograft mice. RESULTS: In the patients with GBC, high Pin1 expression in GBC was associated with aggressive tumor invasion and increased tumor metastasis, and was an independent factor for a poor prognosis. Pin1 expression was correlated with phosphorylation of STAT3(Ser727) and NF-κB-p65(Ser276), thereby activating STAT3 and NF-κB in GBC. Pin1-mediated STAT3 and NF-κB activation induced EMT in GBC. When Pin1 knockdown was performed in GBC cells, the phosphorylation of STAT3(Ser727) and NF-κB-p65(Ser276) was inhibited, and STAT3 and NF-κB activation was suppressed. Inactivation of STAT3 and NF-κB in Pin1-depleted cells decreased snail and zeb-2 expression, thereby reducing the rate of mesenchymal-like cells, suggesting that EMT was inhibited in GBC cells. PiB, a Pin1-specific inhibitor, inhibited EMT and reduced tumor cell invasion by inactivating STAT3 and NF-κB in vitro. Moreover, PiB treatment inhibited lymph node metastasis and intrahepatic metastasis in orthotopic GBC xenograft tumor in vivo. CONCLUSIONS: Pin1 accelerates GBC invasion and metastasis by activating STAT3 and NF-κB. Therefore, Pin1 inhibition by PiB is an excellent therapy for GBC by safely inhibiting its metastasis.