USP12 facilitates gastric cancer progression via stabilizing YAP.

The dysregulation of Hippo signaling is a crucial factor driving the progression of gastric cancer, making the targeting of the Hippo pathway a promising therapeutic strategy. However, effective drugs targeting the Hippo/YAP axis remain unavailable. Thus, identifying potential therapeutic targets and mechanisms that inhibit the activity of the Hippo/YAP axis in gastric cancer is of paramount importance. The ubiquitination modification of the Hippo/YAP pathway plays a significant role in signaling transduction and cancer progression. In an effort to shed light on effective therapeutic targets, we conducted a screening using a deubiquitinase small interfering RNA library, leading to the identification of USP12 as an important deubiquitinase in the context of Hippo/YAP axis and the progression of gastric cancer. Our bioinformatic analysis further demonstrated a correlation between USP12 and poor survival, as well as a positive association with classical YAP target genes in gastric cancer samples. Notably, USP12 depletion was found to inhibit gastric cancer progression via the Hippo/YAP axis, whereas USP12 overexpression exhibited the opposite effect, promoting gastric cancer growth and enhancing YAP activity. Further studies through immuno-staining and immuno-precipitation assays indicated the nuclear localization of USP12 and its association with YAP to enhance YAP stability. Specifically, our findings revealed that USP12 could inhibit K48-linked poly-ubiquitination of YAP, predominantly at the K315 site. As a result, we have identified a novel regulatory mechanism involving USP12 and Hippo signaling in the progression of gastric cancer, with the potential for blockade of USP12 to materialize as a promising strategy for combating gastric cancer.

Regulation of the Hippo/YAP axis by CXCR7 in the tumorigenesis of gastric cancer.

BACKGROUND: The Hippo pathway is crucial in organ size control and tumorigenesis. Dysregulation of the Hippo/YAP axis is commonly observed in gastric cancer, while effective therapeutic targets for the Hippo/YAP axis are lacking. Identification of reliable drug targets and the underlying mechanisms that could inhibit the activity of the Hippo/YAP axis and gastric cancer progression is urgently needed. METHODS: We used several gastric cancer cell lines and xenograft models and performed immunoblotting, qPCR, and in vivo studies to investigate the function of CXCR7 in gastric cancer progression. RESULTS: In our current study, we demonstrate that the membrane receptor CXCR7 (C-X-C chemokine receptor 7) is an important modulator of the Hippo/YAP axis. The activation of CXCR7 could stimulate gastric cancer cell progression through the Hippo/YAP axis in vitro and in vivo, while pharmaceutical inhibition of CXCR7 via ACT-1004-1239 could block tumorigenesis in gastric cancer. Molecular studies revealed that the activation of CXCR7 could dephosphorylate YAP and facilitate YAP nuclear accumulation and transcriptional activation in gastric cancer. CXCR7 functions via G-protein Gαq/11 and Rho GTPase to activate YAP activity. Interestingly, ChIP assays showed that YAP could bind to the promoter region of CXCR7 and facilitate its gene transcription, which indicates that CXCR7 is both the upstream signalling and downstream target of the Hippo/YAP axis in gastric cancer. CONCLUSION: In general, we identified a novel positive feedback loop between CXCR7 and the Hippo/YAP axis, and blockade of CXCR7 could be a plausible strategy for gastric cancer.

USP1 modulates hepatocellular carcinoma progression via the Hippo/TAZ axis.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies worldwide. The Hippo signaling pathway has emerged as a significant suppressive pathway for hepatocellular carcinogenesis. The core components of the Hippo pathway constitute a kinase cascade, which inhibits the functional activation of YAP/TAZ. Interestingly, the overactivation of YAP/TAZ is commonly observed in hepatocellular carcinoma, although the inhibitory kinase cascade of the Hippo pathway is still functional. Recent studies have indicated that the ubiquitin‒proteasome system also plays important roles in modulating Hippo signaling activity. Our DUB (deubiquitinase) siRNA screen showed that USP1 is a critical regulator of Hippo signaling activity. Analysis of TCGA data demonstrated that USP1 expression is elevated in HCC and associated with poor survival in HCC patients. RNA sequencing analysis revealed that USP1 depletion affects Hippo signaling activity in HCC cell lines. Mechanistic assays revealed that USP1 is required for Hippo/TAZ axis activity and HCC progression. USP1 interacted with the WW domain of TAZ, which subsequently enhanced TAZ stability by suppressing K11-linked polyubiquitination of TAZ. Our study identifies a novel mechanism linking USP1 and TAZ in regulating the Hippo pathway and one possible therapeutic target for HCC.

Ubiquitous mitochondrial creatine kinase promotes the progression of gastric cancer through a JNK-MAPK/JUN/HK2 axis regulated glycolysis.

BACKGROUND: Ubiquitous mitochondrial creatine kinase (uMtCK) transfers high-energy phosphates from mitochondrially generated ATP to creatine to generate phosphocreatine. uMtCK overexpression has been reported in several malignant tumors, however, the clinical significance and impact of uMtCK in gastric cancer (GC) has not been comprehensively studied. METHODS: We first examined uMtCK expression in GC by quantitative real-time PCR and western blot assays. Then the clinicopathological significance of aberrant uMtCK expression was determined by immunohistochemical staining in a GC tissue microarray. Kaplan-Meier analysis was used for survival analysis. The biological functions of uMtCK in GC cells were explored by wound-healing, transwell assays and glucose metabolism assays in vitro as well as a liver metastasis model by spleen injection in nude mice in vivo. RESULTS: We verified that the expression of uMtCK was substantially elevated in GC tissues, significantly associating with a poorer prognosis in GC patients, especially for those with advanced stage. In univariate and multivariate analyses, uMtCK expression emerged as an independent prognostic factor for both disease-free survival and overall survival. Functionally, we demonstrated that uMtCK promoted glycolysis in GC cells and facilitated their migration, invasion and liver metastasis in vitro and in vivo. Mechanistically, uMtCK enhanced GC progression in a HK2-dependent glycolysis via acting the JNK-MAPK/JUN signaling pathway. CONCLUSIONS: uMtCK could serve as a novel independent prognostic biomarker as well as potential therapeutic target for GC patients, particularly for GC patients with an advanced UICC stage and tumor recurrence.

USP36 facilitates esophageal squamous carcinoma progression via stabilizing YAP.

Esophageal squamous carcinoma (ESCC) is the major subtype of esophageal cancer in China, accounting for 90% of cases. Recent studies revealed that abnormalities in the Hippo/YAP axis are pervasive in ESCC and are recognized as the important driver of ESCC progression. Since the activity of Hippo signaling is controlled by phosphorylation cascade, it is a mystery why the major effector YAP is still over-activated when the cascade is inhibited. Several studies suggested that in addition to phosphorylation, other protein modifications such as ubiquitination also play important roles in manipulating Hippo/YAP signaling activity. Since YAP protein stability is controlled via an appropriate balance between E3 ubiquitin ligases and deubiquitinases, we performed deubiquitinase siRNA screening and identified USP36 as a deubiquitinase significantly related to Hippo/YAP signaling activity and ESCC progression. USP36 expression was elevated in ESCC samples and correlated with poor differentiation. USP36 expression was correlated with YAP protein levels in ESCC samples. Molecular studies demonstrated that USP36 associated with the YAP protein and enhanced YAP protein stability by blocking the K48-linked polyubiquitination of YAP. In conclusion, our study revealed a novel deubiquitinase in regulating Hippo signaling in ESCC, which could be an encouraging drug target for Hippo-driven ESCC.

OTUB1 suppresses Hippo signaling via modulating YAP protein in gastric cancer.

Gastric cancer is one of the most lethal human malignancies in the world. Although great efforts are put in developing novel therapeutic targets, the effective targeting drugs are still limited. Recent studies reveal the abnormality of Hippo/YAP axis play critical role in the oncogenic process of gastric cancer. It is of great importance to demonstrate the regulation of Hippo signaling activity and YAP protein turnover in gastric cancer. Besides, the phosphorylation cascade on YAP function, which has been thoroughly investigated, the ubiquitination of YAP is also important in Hippo signaling status. Here, We utilized the DUB (Deubiquitinase) siRNA library to identify critical DUB for Hippo signaling. We discovered OTUB1 as a critical factor to facilitate gastric cancer cell stemness and progression, which deubiquitinated and stabilized YAP protein. The clinical data analysis implicated OTUB1 was higher expressed in gastric cancer, which correlated with YAP activity and poor survival. OUTB1 interacted with YAP protein via its OTU domain (Ovarian tumor domain) and deubiquitinated YAP at several lysine sites (K90, K280, K343, K494 and K497), which subsequently inhibited YAP degradation. Our study revealed a novel deubiquitinase of Hippo/YAP axis and one possible therapeutic target for YAP-driven gastric cancer.

DUB1 suppresses Hippo signaling by modulating TAZ protein expression in gastric cancer.

BACKGROUND: The Hippo pathway functions as a tumor suppressor pathway in human cancers, while dysfunction of the Hippo pathway is frequently observed in malignancies. Although YAP/TAZ activity is tightly controlled by the phosphorylation cascade of the MST-LATS-YAP/TAZ axis, it is still unclear why the YAP/TAZ proteins are activated in human cancers despite Hippo pathway activation. Recent studies have suggested that in addition to phosphorylation, several other posttranslational modifications, including ubiquitination, also play critical roles in modulating TAZ function. METHODS: We used several gastric cancer cell lines and performed western blot analysis, real-time PCR, immunoprecipitation assays, and in vitro ubiquitination assays and established a xenograft mouse model. RESULTS: Here, by screening a DUB (deubiquitinase) siRNA library, we discovered that DUB1 functions as a critical modulator that facilitates gastric cancer stemness and progression by deubiquitinating and activating the TAZ protein. We also found that DUB1 expression was elevated in gastric cancer and that elevated DUB1 expression correlated with TAZ activation and poor survival. DUB1 associates with the TAZ protein and deubiquitinates TAZ at several lysine residues, which subsequently stabilizes TAZ and facilitates its function. CONCLUSIONS: Our study revealed a novel deubiquitinase in the Hippo/TAZ axis and identified one possible therapeutic target for Hippo-driven gastric cancer.

UHRF1 modulates breast cancer cell growth via estrogen signaling.

The ubiquitination process, which involves that binding of an ubiquitin protein to certain substrates, regulates several human biological processes and human cancers. Several studies report that the abnormal expression of quite a few E3 ubiquitin ligases could play critical role in carcinogenic process and cancer progression. In our current study, we identify UHRF1 (Ubiquitin Like with PHD And Ring Finger Domain 1) is an important regulator for breast cancer growth. UHRF1 depletion significantly decreases breast cancer growth in vitro and in vivo. Clinical data analysis reveals that UHRF1 is dramatically elevated in breast cancer, compared to normal breast tissue. UHRF1 correlates with poor survival in luminal type of breast cancer patients, but not in ER-negative groups. The molecular biological studies show that UHRF1 localizes in the nuclear and interact with ERα via its SRA domain, which subsequently inhibits K48-linked ubiquitination of ERα and enhances ERα stability. Our study provides a novel function of UHRF1 in regulation estrogen signaling in breast cancer and a promising target for breast cancer therapeutics.

Totally laparoscopic gastrectomy with natural orifice (vagina) specimen extraction in gastric cancer: Introduction of a new technique.

Radical excision by surgery is the main treatment method for gastric cancer and as the surgery develops, the laparoscopic treatment effect on gastric cancer is gradually being verified. The totally laparoscopic gastrectomy (TLG) with natural orifice specimen extraction surgery (NOSES) for gastric cancer has attracted people's attention by avoiding abdominal incision and further reducing surgical injury and provides ideas for the further development of minimally invasive surgical treatment on the basis of laparoscopy. Surgical technique of TLG with natural orifice (vagina) specimen extraction is detailed in the text. We have employed NOSES in 4 cases of TLG in the past year. The visual analogue scale score was low, and all patients had no complications during and after the operation. No recurrence or metastasis was found in the short-term follow-up. TLG with NOSES is feasible and has many advantages such as aesthetics, light post-operative pain.

The ubiquitin ligase RNF181 stabilizes ERα and modulates breast cancer progression.

ERα positive breast cancer accounts for 70% of breast malignancies. Compared with ERα negative types, ERα positive breast cancer could be effective controlled by endocrine therapy. However, more than half of the patients will develop endocrine resistance, making it an important clinical issue for breast cancer therapy. Endocrine resistance might be caused by multiple alternations, including the components of ERα signaling, during tumor progression. Thus, it is urgent and necessary to uncover the molecular mechanisms that controls ERα expression and stability to improve breast cancer therapeutics. In our current study, we identifies that the ubiquitin ligase RNF181 stabilizes ERα and facilitates breast cancer progression. The expression of RNF181 is correlated with ERα level in human breast tumors and relates to poor survival in endocrine-treated patients. RNF181 depletion inhibits breast cancer progression in vivo and in vitro, reduces ERα protein level and its target gene expression, such as PS2 and GREB1. Unbiased RNA sequencing analysis indicates RNF181 is necessary for ERα signature gene expression in whole genomic level. Immuno-precipitation assays indicate that RNF181 associates with ERα and promotes its stability possibly via inducing ERα K63-linked poly-ubiquitination. In conclusion, our data implicate a non-genomic mechanism by RNF181 via stabilizing ERα protein controls ERα target gene expression linked to breast cancer progression.

Initial experience of single-incision plus one port left-side approach totally laparoscopic distal gastrectomy with uncut Roux-en-Y reconstruction.

BACKGROUND: Single incision plus one port left-side approach (SILS+1/L) totally laparoscopic distal gastrectomy (TLDG) is an emerging technique for the treatment of gastric cancer. Reduced port laparoscopic gastrectomy has a number of potential advantages for patients compared with conventional laparoscopic gastrectomy: relieving postoperative pain, shortening hospital stay and offering a better cosmetic outcome. Nevertheless, there are no previous reports on the use of SILS+1/L TLDG with uncut Roux-en-Y (uncut R-Y) reconstruction. AIM: To investigate the initial feasibility of SILS+1/L TLDG with uncut Roux-en-Y digestive tract reconstruction (uncut R-Y reconstruction) to treat distal gastric cancer. METHODS: A total of 21 patients who underwent SILS+1/L TLDG with uncut R-Y reconstruction for gastric cancer were enrolled. All patients were treated at The Second Hospital of Shandong University. Reconstructions were performed intracorporeally with 60 mm endoscopic linear stapler and 45 mm no-knife stapler. The clinicopathological characteristics, surgical details, postoperative short-term outcomes, postoperative follow-up upper gastrointestinal radiography findings and endoscopy results were analyzed retrospectively. RESULTS: All SILS+1/L operations were performed by SILS+1/L TLDG successfully. The patient population included 13 men and 8 women with a mean age of 48.2 years (ranged from 40 years to 70 years) and median body mass index of 22.8 kg/m2. There were no conversions to open laparotomy, and no other port was placed. The mean operation time was 146 min (ranged 130-180 min), and the estimated mean blood loss was 54 mL (ranged 20-110 mL). The mean duration to flatus and discharge was 2.3 (ranged 1-3.5) and 7.3 (ranged 6-9) d, respectively. The mean number of retrieved lymph nodes was 42 (ranged 30-47). Two patients experienced mild postoperative complications, including surgical site infection (wound at the navel incision) and mild postoperative pancreatic fistula (grade A). Follow-up upper gastrointestinal radiography and endoscopy were carried out at 3 mo postoperatively. No patients experienced moderate or severe food stasis, alkaline gastritis or bile reflux during the follow-up period. No recanalization of the biliopancreatic limb was found. CONCLUSION: SILS+1/L TLDG with uncut R-Y reconstruction could be safely performed as a reduced port surgery.

MiR-19-3p Induces Tumor Cell Apoptosis via Targeting FAS in Rectal Cancer Cells.

MicroRNAs are reported as a vital important factor in cancer cell initiation and progression processes. MicroRNA-19-3p has drawn the attention of many researchers in recent years because of its wide expression and its key role in serious kinds of tumor cells. However, the detailed mechanism of microRNA-19a-3p in these tumors is still poorly understood. So, in the present study, we aimed to explore the biological function and potential molecular mechanism of microRNA-19a-3p in different cancer cells. We first detect the relative level of miR-19a-3p in cancer cell lines and tumor tissues compared to normal cells and tissues. Results indicated the messenger RNA expression of microRNA-19a-3p existing in an aberrant low level in cancer cells and tissues. The overexpression of microRNA-19a-3p significantly reduced the cell proliferation, migration, and invasion ability in HCT116 cells. In addition to this, increased microRNA-19a-3p could induce cell apoptosis via promoting reactive oxygen species (ROS) accumulation, whereas inhibition of microRNA-19a-3p exhibited an opposite effect. Moreover, we predicated the target genes and the binding sites of microRNA-19a-3p and confirmed FAS as the targeting of microRNA-19a-3p through luciferase activity assay. Taken together, these results indicated that microRNA-19a-3p overexpression inhibited HCT116 cell proliferation, migration and invasion, induced cell apoptosis, and ROS accumulation via FAS targeting effect. It was conceivable that microRNA-19a-3p might serve as a potential molecular target for breast and liver cancer treatment.

Application value of enhanced recovery after surgery for total laparoscopic uncut Roux-en-Y gastrojejunostomy after distal gastrectomy.

AIM: To evaluate the safety and feasibility of enhanced recovery after surgery (ERAS) for total laparoscopic uncut Roux-en-Y gastrojejunostomy after distal gastrectomy. METHODS: The clinical data of 42 patients who were divided into an ERAS group (n = 20) and a control group (n = 22) were collected. The observed indicators included operation conditions, postoperative clinical indexes, and postoperative serum stress indexes. Measurement data following a normal distribution are presented as mean ± SD and were analyzed by t-test. Count data were analyzed by χ2 test. RESULTS: The operative time, volume of intraoperative blood loss, and number of patients with conversion to open surgery were not significantly different between the two groups. Postoperative clinical indexes, including the time to initial anal exhaust, time to initial liquid diet intake, time to out-of-bed activity, and duration of hospital stay of patients without complications, were significantly different between the two groups (t = 2.045, 8.685, 2.580, and 4.650, respectively, P < 0.05 for all). However, the time to initial defecation, time to abdominal drainage-tube removal, and the early postoperative complications were not significantly different between the two groups. Regarding postoperative complications, on the first and third days after the operation, the white blood cell count (WBC) and C reactive protein (CRP) and interleukin-6 (IL-6) levels in the ERAS group were significantly lower than those in the control group. CONCLUSION: The perioperative ERAS program for total laparoscopic uncut Roux-en-Y gastrojejunostomy after distal gastrectomy is safe and effective and should be popularized. Additionally, this program can also reduce the duration of hospital stay and improve the degree of comfort and satisfaction of patients.