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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.

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.

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.

Background Recently genome-based studies revealed that the abnormality of Hippo signaling is pervasive in TNBC and played important role in cancer progression. RING finger protein 31 (RNF31) comes to RING family E3 ubiquitin ligase. Our previously published studies have revealed RNF31 is elevated in ER positive breast cancer via activating estrogen signaling and suppressing P53 pathway. Methods We used several TNBC cell lines and xenograft models and performed immuno-blots, QPCR, in vivo studies to investigate the function of RNF31 in TNBC progression. Result Here, we demonstrate that RNF31 plays tumor suppressive function in triple negative breast cancer (TNBC). RNF31 depletion increased TNBC cell proliferation and migration in vitro and in vitro. RNF31 depletion in TNBC coupled with global genomic expression profiling indicated Hippo signaling could be the potential target for RNF31 to exert its function. Further data showed that RNF31 depletion could increase the level of YAP protein, and Hippo signaling target genes expression in several TNBC cell lines, while clinical data illustrated that RNF31 expression correlated with longer relapse-free survival in TNBC patients and reversely correlated with YAP protein level. The molecular biology assays implicated that RNF31 could associate with YAP protein, facilitate YAP poly-ubiquitination and degradation at YAP K76 sites. Interestingly, RNF31 could also repress PDL1 expression and sensitive TNBC immunotherapy via inhibiting Hippo/YAP/PDL1 axis. Conclusions Our study revealed the multi-faced function of RNF31 in different subtypes of breast malignancies, while activation RNF31 could be a plausible strategy for TNBC therapeutics.

Primary liver cancer is the sixth most frequently diagnosed cancer worldwide and the third leading cause of cancer-related death. The majority of the primary liver cancer cases are hepatocellular carcinoma and intrahepatic cholangiocarcinoma. Worldwide, there is an increasing incidence of primary liver cancer cases due to multiple risk factors ranging from parasites and viruses to metabolic diseases and lifestyles. Often, patients are diagnosed at advanced stages, depriving them of surgical curability benefits. Moreover, the efficacy of the available chemotherapeutics is limited in advanced stages. Furthermore, tumor metastases and recurrence make primary liver cancer management exceptionally challenging. Thus, exploring the molecular mechanisms for the development and progression of primary liver cancer is critical in improving diagnostic, treatment, prognostication, and surveillance modalities. These mechanisms facilitate the discovery of specific targets that are critical for novel and more efficient treatments. Consequently, the Hippo signaling pathway executing a pivotal role in organogenesis, hemostasis, and regeneration of tissues, regulates liver cells proliferation, and apoptosis. Cell polarity or adhesion molecules and cellular metabolic status are some of the biological activators of the pathway. Thus, understanding the mechanisms exhibited by the Hippo pathway is critical to the development of novel targeted therapies. This study reviews the advances in identifying therapeutic targets and prognostic markers of the Hippo pathway for primary liver cancer in the past six years.

Chemotherapy is widely used in a variety of solid tumors, such as lung cancer, gastric cancer and breast cancer. The genotoxic drugs, such as cisplatin, suppress cancer progression either by inhibition cell proliferation or facilitating apoptosis. However, the chemotherapy resistance remains an urgent challenge in cancer therapy, especially in advanced stages. Several studies showed that the activation of pro-survival pathways, such as PI3K-AKT, participated in mediating chemotherapy resistance. The insights into the molecular mechanisms for underlying chemotherapy resistance are of great importance to improve cancer patient survival in advanced stages. The HOIP protein belongs to the RING family E3 ubiquitin ligases and modulates several atypical ubiquitination processes in cellular signaling. Previous studies showed that HOIP might be an important effector in modulating cancer cell death under genotoxic drugs. Here, we report that HOIP associates with PTEN and facilitates PTEN degradation in cancer cells. Depletion of HOIP causes cell cycle arrest and apoptosis, which effects could be rescued by PTEN silencing. Besides, the survival data from public available database show that HOIP expression correlates with poor survival in several types of chemotherapy-treated cancer patients. In conclusion, our study establishes a novel mechanism by which HOIP modulates PTEN stability and facilitates chemotherapy resistance in malignancies.

Background Breast cancer is the most common cancer in women worldwide. More than 70% of breast cancers are estrogen receptor (ER) alpha positive. Compared with ER alpha-negative breast cancer, which is more aggressive and has a shorter survival time, ER alpha-positive breast cancer could benefit from endocrine therapy. Selective estrogen receptor modulators, such as tamoxifen, are widely used in endocrine therapy. Approximately half of ER alpha-positive breast cancer patients will eventually develop endocrine resistance, making it a major clinical challenge in therapy. Thus, decoding the throughput of estrogen signaling, including the control of ER alpha expression and stability, is critical for the improvement of breast cancer therapeutics. Methods TRIM3 and ER alpha protein expression levels were measured by western blotting, while the mRNA levels of ER alpha target genes were measured by RT–PCR. A CCK-8 assay was used to measure cell viability. RNA sequencing data were analyzed by Ingenuity Pathway Analysis. Identification of ER alpha signaling activity was accomplished with luciferase assays, RT–PCR and western blotting. Protein stability assays and ubiquitin assays were used to detect ER alpha protein degradation. Ubiquitin-based immunoprecipitation assays were used to detect the specific ubiquitination modification on the ER alpha protein. Results In our current study, we found that TRIM3, an E3 ligase, can promote ER alpha signaling activity and breast cancer progression. TRIM3 depletion inhibits breast cancer cell proliferation and migration, while unbiased RNA sequencing data indicated that TRIM3 is required for the activity of estrogen signaling on the -genome-wide scale. The immunoprecipitation assays indicated that TRIM3 associates with ER alpha and promotes its stability, possibly by inducing K63-linked polyubiquitination of ER alpha. In conclusion, our data implicate a nongenomic mechanism by which TRIM3 stabilizes the ER alpha protein to control ER alpha target gene expression linked to breast cancer progression. Conclusion Our study provides a novel posttranslational mechanism in estrogen signaling. Modulation of TRIM3 expression or function could be an interesting approach for breast cancer treatment. Graphical abstract

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.

Breast cancer is one of the most common malignancies worldwide, while the luminal types (ERα positive) accounts for two third of all breast cancer cases. Although ERα positive breast cancer could be effective controlled by endocrine therapy, most of the patients will develop endocrine resistance, which becomes a headache clinical issue for breast cancer field. Endocrine resistance could be caused by multiple pathway disorders, the dys-regulation of ERα signaling might be a critical factor, which makes it urgent and important to reveal the potential molecular mechanism of ERα signaling. In our current study, we identified a new deubiquitination enzyme USP1 through screening the whole DUB (Deubiquitinases) siRNA library. The expression of USP1 is elevated in human breast cancer compared with normal mammary tissues. Importantly, USP1 expression levels are specially correlated with poor survival in ERα positive patients. USP1 depletion inhibited breast cancer cell progression and ERα signaling activity. Immuno-precipitation assays indicate that USP1 associates with ERα and promotes its stability possibly via inhibiting ERα K48-linked poly-ubiquitination. In conclusion, our data implicate a non-genomic mechanism by USP1 via stabilizing ERα protein controls ERα target gene expression linked to breast cancer progression.

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.

The tumor suppressor P53 plays critical role in preventing cancer. P53 is rarely mutated and remains functional in luminal-type breast cancer(1). According to current knowledge, wild-type P53 function is tightly controlled by posttranslational modifications, such as ubiquitination. Several ubiquitin ligases have been shown to regulate P53 ubiquitination and protein stability. Here, we report that RNF187, a RING family ubiquitin ligase, facilitates breast cancer growth and inhibits apoptosis by modulating P53 signaling. RNF187 expression was elevated in breast cancer and correlated with breast cancer survival only in the P53 wild-type groups. Bioinformatic analysis showed that the expression of RNF187 was negatively correlated with the expression of P53 target genes, such as IGFBP3 and FAS, in breast cancer. RNF187 depletion inhibited breast cancer growth and facilitated cell death. RNA sequencing analysis indicated that RNF187 could be an important modulator of P53 signaling. Further experiments showed that RNF187 interacts with P53 and promotes its degradation by facilitating its polyubiquitination in breast cancer cells. Interestingly, the in vitro ubiquitin assay showed that RNF187 can directly ubiquitinate P53 in a manner independent of MDM2. These findings reveal a novel direct P53 regulator and a potential therapeutic target for breast cancer.

Breast cancer is one of the most commonly diagnosed malignancies worldwide, while the triple negative breast cancer (TNBC) is the most aggressive and virulent subtype in breast cancers. Compared with luminal type breast cancers, which could be well controlled by endocrine treatment, TNBC is worse in prognosis and lack of effective targeted therapy. Thus, it would be interesting and meaningful to identify novel therapeutic targets for TNBC treatments. Recent genomic data showed the activation of Hippo/YAP signaling in TNBC, indicating its critical roles in TNBC carcinogenesis and cancer progression. Hippo/YAP signaling could subject to several kinds of protein modifications, including ubiquitination and phosphorylation. Quite a few studies have demonstrated these modifications, which controlled YAP protein stability and turnover, played critical role in Hippo signaling activation In our current study, we identified ZNF213 as a negative modifier for Hippo/YAP axis. ZNF213 depletion promoted TNBC cell migration and invasion, which could be rescued by further YAP silencing. ZNF213 knocking down facilitated YAP protein stability and Hippo target gene expression, including CTGF and CYR61. Further mechanism studies demonstrated that ZNF213 associated with YAP and facilitated YAP K48‐linked poly‐ubiquitination at several YAP lysine sites (K252, K254, K321 and K497). Besides, the clinical data showed that ZNF213 negatively correlated with YAP protein level and Hippo target gene expression in TNBC samples. ZNF213 expression correlated with good prognosis in TNBC patients. Our data provided novel insights in YAP proteolytic regulation and TNBC progression., The triple negative breast cancer (TNBC) is the most aggressive and virulent subtype in breast cancers.In our current study, we identified ZNF213 as a negative modifier for Hippo/YAP axis in TNBC. Our data provided novel insights in YAP proteolytic regulation and TNBC progression.

Background Breast cancer ranks No. 1 in women cancer incidence, while triple negative breast cancer (TNBC) is the most aggressive and the worst prognostic subtype in all breast cancer subtypes. Compared with estrogen receptor alpha positive breast cancer, which could be well controlled by endocrine therapy, TNBC is lack of mature molecular targets for medical therapy. Thus, it is urgent and necessary to discovery the carcinogenic mechanism and potential therapeutic targets for TNBC. Recent studies reveal that Hippo/YAP signaling is an important mediator for TNBC progression. Our current study investigates the role of RING finger protein RNF181 in modulation Hippo/YAP signaling. Methods YAP and RN181 protein level were measured by western blot, while the Hippo classical target genes were measured by real-time PCR. WST1 assay were used to measure cell proliferation, the trans-well and wound healing were used to measure the cell migration and invasion capacity. Protein stability and ubiquitin assay were used to detect the YAP protein ubiquitin and stability. The immuno-precipitation assays were used to detect the protein interactions. Immuno-staining was used to detect the protein localization of YAP and RNF181, while the ubiquitin-based immuno-precipitation assays were used to detect the specific ubiquitination manner of YAP. Results Our current study identified a novel modulator-RNF181 as a positive mediator for Hippo/YAP signaling activation in TNBC. RNF181 depletion significantly inhibited TNBC cell migration, invasion and proliferation, which effect could be rescued by YAP overexpression. RNF181 depletion decreased YAP protein level and Hippo signaling target genes, such as CTGF and CYR61, in TNBC cell lines. Immuno-precipitation assay showed that RNF181 interact with YAP and promoted YAP stability by inhibition K48-linked poly-ubiquitination of YAP in TNBC cells. Besides, public available data showed that RNF181 is elevated in breast cancer and related to poor prognosis in TNBC patients. Conclusion Our study provides evidence to establish a non-proteolytic mechanism in modulating Hippo signaling in breast cancer. RNF181 could be an interesting marker for triple negative breast cancer prognostics and therapeutics.

Objective: To investigate the expression of glioma-associated oncogene homolog 1(Gli-1) in colon cancer and its association with clinicopathological parameters and postoperative liver metastasis. Methods: Expression of Gli-1 was detected by immunohistochemistry in paraffin-embedded specimens of 96 cases of colon cancer. Relationship between Gli-1 expression and clinicopathological parameters, postoperative liver metastasis were analyzed. Results: Gli-1 protein expression was significantly increased in colon cancer tissues compared to normal colon tissues (P = 0.037). Gli-1 expression in colon tissues was increased in patients with lymph node metastases (P = 0.022) and higher T stages (P = 0.030). Postoperative live metastasis-free survival period was significantly longer in low Gli-1 expression group than that of high Gli-1 expression group (48.22±10.03 months vs 20.46±6.32 months, P=0.001). Multivariate analysis showed that Gli-1 expression level is an independent prognostic factor for postoperative live metastasis-free survival. Conclusion: Colon cancer is associated with an upregulation of Gli-1 protein expression in colon tissues. In patients with colon cancer, Gli-1 expression level is closely related to lymph node metastases, T stages and postoperative live metastasis-free survival periods, indicative of a possible role of Gli-1 expression in colon cancer progression.

Aim: We investigated the association of WNT inhibitory factor-1 (WIF-1) gene methylation with the pathogenesis of multiple human tumors, using a meta-analysis based approach. Materials and Methods: Electronic databases and manual search was additionally employed to retrieve relevant published literature. The cohort studies relating to tumor and WIF-1 were screened based on predefined selection criteria, and all extracted data from the selected studies were analyzed through STATA software. Results: Sixteen studies were finally enrolled in our study involved 1112 tumor samples and 612 adjacent normal samples. The study result showed that WIF-1 gene methylations in tumor tissues were significantly higher compared with adjacent/normal tissues. The result of subgroup analysis on ethnicity revealed that in the Caucasians, Asians, and Africans, the methylation status of WIF-1 gene in tumor tissues was higher than adjacent/normal tissues. Further subgroup analysis on disease types revealed that WIF-1 gene methylation status is a widespread phenomenon that is, observed in tumor tissues of patients with multiple human tumors compared with that in adjacent/normal tissues. Interestingly, there was no significant difference in WIF-1 gene methylation between tumor tissues among patients with lung cancer, gastric cancer, astrocytoma, and adjacent/normal tissues, indicating the WIF-1 gene methylation not a general nonspecific phenomenon. Conclusion: WIF-1 gene methylation in tumor tissues was significantly more frequent as compared to that in adjacent normal tissues, indicating that WIF-1 gene methylation may be an important event in the pathogenesis of multiple human tumors.