Carboxyl Terminus Of Hsc70-interacting Protein Expression Research Articles

CHIP-mediated ubiquitin degradation of BCAT1 regulates glioma cell proliferation and temozolomide sensitivity

Glioma, a malignant and infiltrative neoplasm of the central nervous system, poses a significant threat due to its high mortality rates. Branched-chain amino acid transaminase 1 (BCAT1), a key enzyme in branched-chain amino acid (BCAA) catabolism, exhibits elevated expression in gliomas and correlates strongly with poor prognosis. Nonetheless, the regulatory mechanisms underlying this increased BCAT1 expression remains incompletely understood. In this study, we reveal that ubiquitination at Lys360 facilitates BCAT1 degradation, with low ubiquitination levels contributing to high BCAT1 expression in glioma cells. The Carboxyl terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase, interacts with BCAT1 via its coiled-coil (CC) domain, promoting its K48-linkage ubiquitin degradation through proteasomal pathway. Moreover, CHIP-mediated BCAT1 degradation induces metabolic reprogramming, and impedes glioma cell proliferation and tumor growth both in vitro and in vivo. Furthermore, a positive correlation is observed between low CHIP expression, elevated BCAT1 levels, and unfavorable prognosis among glioma patients. Additionally, we show that the CHIP/BCAT1 axis enhances glioma sensitivity to temozolomide by reducing glutathione (GSH) synthesis and increasing oxidative stress. These findings underscore the critical role of CHIP/BCAT1 axis in glioma cell proliferation and temozolomide sensitivity, highlighting its potential as a diagnostic marker and therapeutic target in glioma treatment.

E3 ubiquitin ligase CHIP interacts with transferrin receptor 1 for degradation and promotes cell proliferation through inhibiting ferroptosis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the major form of liver malignancy with high incidence and mortality. Identifying novel biomarkers and understanding regulatory mechanisms underlying the development and progression of HCC are critical for improving diagnosis, treatment and patient outcomes. Carboxyl terminus of Hsc-70-interacting protein (CHIP) is a well-described U-box-type E3 ubiquitin ligase which promotes the ubiquitination and degradation of numerous tumor-associated proteins. Recent studies have shown that CHIP can play as a tumor-suppressor gene or an oncogene in different kinds of malignancies. To date, the function and mechanism of CHIP in hepatocellular carcinoma remains largely unknown. Based on TCGA data, we found that compared with high CHIP expression, the overall survival of HCC patients with low expression of CHIP was better. In addition, CHIP overexpression markedly enhanced HCC cell proliferation and colony formation. Conversely, knockdown of CHIP restrained the proliferation and colony formation of HCC cells. Meanwhile, knockdown of CHIP decreased mitochondrial cristae or ruptured outer mitochondrial membrane, promoted the accumulation of Fe2+ and ferroptosis of HCC cells. Further research for the first time confirmed that CHIP interacts and degrades transferrin receptor 1 (TfR1) by ubiquitin-proteasome pathway, which leads to the inhibition of ferroptosis and promotes the proliferation of HCC cells. The analysis of proteomics data from CPTAC revealed a negative correlation between CHIP and TfR1 protein expression levels in HCC. These findings indicate that CHIP acts as a negative modulator of ferroptosis and functions as an oncogene in HCC.

Ubiquitin ligase CHIP regulates OTUD3 stability and suppresses tumour metastasis in lung cancer

Ovarian tumour domain-containing protein 3 (OTUD3), a key OTU (ovarian tumour protease) family deubiquitylase, plays context-dependent roles in cancers. It suppresses tumorigenesis in breast, colon, liver and cervical cancer through stabilizing PTEN (phosphatase and tension homologue deleted on chromosome 10) while promotes lung tumorigenesis through stabilizing GRP78 (The glucose-regulated protein 78 kDa). The regulation especially post-translational modification of OTUD3 remains unclear. Here, we report that the carboxyl terminus of Hsc70-interacting protein (CHIP) is a ubiquitin ligase for OTUD3. CHIP interacts with, polyubiquitylates OTUD3 and promotes OTUD3 degradation. Knockdown of CHIP stabilizes OTUD3 which leads to elevated GRP78 levels in lung cancer cells. CHIP-knockdown lung cancer cells exhibit increased invasion in OTUD3 and GRP78 dependent manner. Further study demonstrates that CHIP-knockdown lung cancer cells are more prone to metastasize to mice lung when injected intravenously or subcutaneously. Moreover, the expression of CHIP is low in human lung cancer tissues and inversely correlates with OTUD3 expression and GRP78 expression. Furthermore, we identified CHIP mutations in human lung cancers, which reduce CHIP catalytic activity. These findings demonstrate that CHIP is a negative regulator of OTUD3 and CHIP suppresses lung cancer metastasis through inhibiting OTUD3-GRP78 signaling axis.

E3 ubiquitin ligase CHIP attenuates cellular proliferation and invasion abilities in triple-negative breast cancer cells.

Carboxyl terminus of Hsc-70-interacting protein (CHIP), as U-box-type ubiquitin ligase, connects the chaperone and proteasome systems and plays a pivotal role in maintaining protein homeostasis in the cytoplasm. CHIP induces the ubiquitination and degradation of diverse oncogenic substrate proteins and therefore involves in the progression of tumorigenesis. In this study, the CHIP expression was examined in different human breast cancer cell lines and a group of breast cancer tissues. We found, for the first time, that CHIP expression was correlated with the molecular subtyping of breast cancer. CHIP was least expressed in the base-like subtype of breast cancer, which are triple-negative breast cancer (TNBC) breast cancer predominantly. Accordingly, CHIP expression was evidently decreased in the TNBC MDA-MB-231 breast cancer cell line. Enforced induction of CHIP in the MDA-MB-231 cells exerted no obvious influences on cellular growth and cell cycle. The apoptotic and proliferation cells in hCHIP cells were both reduced compared to the ctrl cells. The mRNA and protein expressions of the anti-apoptotic Bcl-2 and Bcl-xL were markedly increased in the hCHIP cells compared to that of the ctrl cells. The expression of RelA was significantly reduced in the nuclear extract in hCHIP cells compared to that in the ctrl cells. The protein expressions of IKKβ were markedly decreased in the hCHIP cells compared to the ctrl cells. The reduced cellular proliferation was largely due to the attenuated IKKβ-p65/NF-κB activity. Meanwhile, the invasion ability but not the migration ability was diminished when CHIP was overexpressed in the MDA-MB-231 cells. The activity of MMP2 but not MMP9 was significantly decreased in the hCHIP cells compared to the ctrl cells. Taken together, these observations here provide functional evidence for CHIP behaved as a tumor suppressor in the TNBC breast cancer cells. CHIP influenced diverse biological aspects of the MDA-MB-231 breast cancer cells. Importantly, CHIP expression is a useful indicator of the molecular subtyping of breast cancer.

Inactivation of carboxyl terminus of Hsc70-interacting protein prevents hypoxia-induced pulmonary arterial smooth muscle cells proliferation by reducing intracellular Ca2+ concentration.

Carboxyl terminus of Hsc70-interacting protein (CHIP) is a 35-kDa cytoplasmic protein expressed in human striated muscle, brain, aortic smooth muscle, endothelial cells, and other tissues. Studies have confirmed that CHIP regulates cell growth, apoptosis, cell phenotype, metabolism, neurodegeneration, etc. However, whether CHIP is involved in pulmonary artery smooth muscle cell (PASMC) proliferation, a vital contributor to chronic hypoxia-induced pulmonary hypertension (CHPH), remains unknown. In this study, we first evaluated CHIP expression in the pulmonary arteries (PAs) of CHPH model rats. Subsequently, by silencing CHIP, we investigated the effect of CHIP on hypoxia-induced PASMC proliferation and the underlying mechanism. Our results showed that CHIP expression was upregulated in the PAs of CHPH model rats. Silencing CHIP significantly suppressed the hypoxia-triggered promotion of proliferation, [Ca2+]i, store-operated Ca2+ entry (SOCE), and some regulators of SOCE such as TRPC1 and TRPC6 in cultured PASMCs. These results indicate that CHIP likely contributes to hypoxia-induced PASMC proliferation by targeting the SOCE-[Ca2+]i pathway through the regulation of TRPC1 and TRPC6 in the PASMCs. In conclusion, the findings of the current study clarify the role of CHIP in hypoxia-induced PASMC proliferation.

Downregulation of CHIP promotes ovarian cancer metastasis by inducing Snail-mediated epithelial-mesenchymal transition.

The epithelial–mesenchymal transition (EMT) plays a pivotal role in the conversion of early‐stage tumors into invasive malignancies. The transcription factor Snail, an extremely unstable protein whose subcellular levels are regulated by many E3 ubiquitin ligases, promotes EMT as well as associated pathological characteristics including migration, invasion, and metastasis. Through yeast two‐hybrid screening, we identified the carboxyl terminus of Hsc70‐interacting protein (CHIP) as a novel Snail ubiquitin ligase that interacts with Snail to induce ubiquitin‐mediated proteasomal degradation. Inhibition of CHIP expression increases Snail protein levels, induces EMT, and enhances in vitro migration and invasion as well as in vivo metastasis of ovarian cancer cells. In turn, Snail depletion abrogates all phenomena induced by CHIP depletion. Finally, Snail and CHIP expression is inversely correlated in ovarian tumor tissues. These findings establish the CHIP–Snail axis as a post‐translational mechanism of EMT and cancer metastasis regulation.

Decreased levels of UBE2Q1 and CHIP in the placentas of infection related preterm birth

Objective: To explore the role of ubiquitin proteasome system in the pathogenesis of infection related preterm birth. Materials and Methods: Thirteen women with spontaneous preterm delivery and 13 controls were included in present case-controlled study. The mRNA and protein levels of Ub-conjugating enzyme E2Q1 (UBE2Q1) and Carboxyl-terminus of Hsc70 interacting protein (CHIP) in the placenta were measured by immunohistochemistry, real-time RT-PCR, and Western blotting methods. Statistical significance (p t-test. Results: The mRNA levels of UBE2Q1 (0.48±0.05 vs. 0.67±0.07, p = 0.047) and CHIP (1.59±0.23 vs. 5.62±1.00, p = 0.002) in the placentas of preterm delivery were significant lower than those of term delivery. The protein levels of UBE2Q1 (0.64±0.09 vs. 1.49±0.22, p vs. 1.33±0.23, p = 0.001) in the placentas of preterm delivery were also significant lower than those of term delivery. Conclusions: Decreased mRNA and protein expression of UBE2Q1 and CHIP were both found in the syncytiotrophoblast and cytotrophoblast of placenta in preterm birth patients, which was speculated to play a role in the pathogenesis of infection related preterm birth.

CHIP functions as an oncogene by promoting colorectal cancer metastasis via activation of MAPK and AKT signaling and suppression of E-cadherin

BackgroundThe carboxyl terminus of Hsc70-interacting protein (CHIP) is an E3 ubiquitin ligase that plays a controversial role in different cancers, either as a tumor suppressor or a tumor promoter. To date, the exact function and underlying mechanism of CHIP in colorectal cancer (CRC) is not yet clear. Here we aimed to determine whether CHIP could affect the biological behaviors of CRC cells and its underlying mechanisms.MethodsStably transfected CHIP overexpression and depletion DLD-1 and HT-29 cells were established using Lipofectamine 2000. Cell growth was monitored by x-Celligence system. Cell proliferation was detected using CCK-8 and Brdu proliferation assay. Cell apoptosis and cell cycle were detected by flow cytometry analysis. Cell migration and invasion abilities were monitored by x-Celligence system, wound healing assay and transwell assay. In vivo intraperitoneal metastasis assay was performed to investigate the influence of CHIP on the tumor metastasis of CRC cells in nude mice. The expression of ERK, AKT, NF-кB signaling subunits and EMT related proteins were detected by Western blotting. The influence and function of CHIP on the protein expression of CRC cells were also elucidated by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. CRC microarray tissue was analyzed to investigate the CHIP expression and its clinical significance.ResultsCHIP depletion inhibited cell growth, migration and invasion potential of CRC cells, accompanied by downregulation of MAPK and AKT signaling activities and upregulation of E-cadherin. CHIP overexpression dramatically enhanced the migration and invasion abilities, due to the upregulation of MAPK and AKT signaling and downregulation of E-cadherin. The proteomic analysis confirmed that E-cadherin was decreased in CHIP-overexpressing CRC cells. Furthermore, clinical tissue data revealed that CHIP expression was upregulated in CRC samples and was significantly correlated with poor survival of CRC patients. Mechanically, CHIP probably activated the MAPK and AKT signaling, which inactivated GSK-3β. The GSK-3β inactivation, in turn, upregulated Slug and led to E-cadherin downregulation and EMT initiation.ConclusionsOur finding suggested that CHIP functions as an oncogene in the migration and metastasis of CRC, and is a potential unfavorable independent predictive biomarker for CRC. CHIP activates the AKT pathway to promote EMT and metastasis in CRC through the CHIP-MAPK/AKT-GSK-3β-Slug-E-cadherin pathways.

Ubiquitin ligase CHIP functions as an oncogene and activates the AKT signaling pathway in prostate cancer.

Carboxyl terminus of Hsc-70-interacting protein (CHIP) is an E3 ubiquitin ligase that induces the ubiquitination and degradation of numerous tumor-associated proteins and serves as a suppressor or promoter in tumor progression. To date, the molecular mechanism of CHIP in prostate cancer remains unknown. Therefore, the present study investigated the biological function of CHIP in prostate cancer cells and obtained evidence that CHIP expression is upregulated in prostate cancer tissues. The CHIP vector was introduced into DU145 cancer cells and the cell biological behaviour was examined through a series of experiments, including cell growth, cell apoptosis and migration and invasion assays. The results indicated that the overexpression of CHIP in DU145 prostatic cancer cells promoted cell proliferation through activation of the protein kinaseB(AKT) signaling pathway, which subsequently increased cyclinD1 protein levels and decreased p21 and p27 protein levels. The overexpression of CHIP significantly increased the migration and invasion of the DU145 cells, which is possible due to activation of the AKT signaling pathway and upregulation of vimentin. The expression level of CHIP was observed to be increased in human prostate cancer tissues compared with the adjacent normal tissue. Furthermore, the CHIP expression level exhibited a positively association with the Gleason score of the patents. These findings indicate that CHIP functions as an oncogene in prostate cancer.

Anisomycin prevents OGD-induced necroptosis by regulating the E3 ligase CHIP

Necroptosis is an essential pathophysiological process in cerebral ischemia-related diseases. Therefore, targeting necroptosis may prevent cell death and provide a much-needed therapy. Ansiomycin is an inhibitor of protein synthesis which can also activate c-Jun N-terminal kinases. The present study demonstrated that anisomycin attenuated necroptosis by upregulating CHIP (carboxyl terminus of Hsc70-interacting protein) leading to the reduced levels of receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3) proteins in two in vitro models of cerebral ischemia. Further exploration in this research revealed that losing neither the co-chaperone nor the ubiquitin E3 ligase function of CHIP could abolish its ability to reduce necroptosis. Collectively, this study identifies a novel means of preventing necroptosis in two in vitro models of cerebral ischemia injury through activating the expression of CHIP, and it may provide a potential target for the further study of the disease.

CHIP/Stub1 regulates the Warburg effect by promoting degradation of PKM2 in ovarian carcinoma.

Tumor cells preferentially adopt aerobic glycolysis for their energy supply, a phenomenon known as the Warburg effect. It remains a matter of debate as to how the Warburg effect is regulated during tumor progression. Here, we show that CHIP (carboxyl terminus of Hsc70-interacting protein), a U-box E3 ligase, suppresses tumor progression in ovarian carcinomas by inhibiting aerobic glycolysis. While CHIP is downregulated in ovarian carcinoma, induced expression of CHIP results in significant inhibition of the tumor growth examined by in vitro and in vivo experiments. Reciprocally, depletion of CHIP leads to promotion of tumor growth. By a SiLAD proteomics analysis, we identified pyruvate kinase isoenzyme M2 (PKM2), a critical regulator of glycolysis in tumors, as a target that CHIP mediated for degradation. Accordingly, we show that CHIP regulates PKM2 protein stability and thereafter the energy metabolic processes. Depletion or knockout of CHIP increased the glycolytic products in both tumor and mouse embryonic fibroblast cells. Simultaneously, we observed that CHIP expression inversely correlated with PKM2 levels in human ovarian carcinomas. This study reveals a mechanism that the Warburg effect is regulated by CHIP through its function as an E3 ligase, which mediates the degradation of PKM2 during tumor progression. Our findings shed new light into understanding of ovarian carcinomas and may provide a new therapeutic strategy for ovarian cancer.

Loss of CHIP Expression Perturbs Glucose Homeostasis and Leads to Type II Diabetes Through Defects in Microtubule Polymerization and Glucose Transporter Localization

Glucose homeostasis requires insulin signaling throughout the body to maintain physiological blood glucose levels. The loss of insulin sensitivity, or insulin resistance, is associated with a myriad of diseases including Type II diabetes, atherosclerosis, abdominal obesity, and hypertension. Cellular protein quality control pathways involving the chaperone and ubiquitin proteasome system have been associated with the pathophysiology of insulin resistance, but the molecular details are incompletely understood. We and others have previously demonstrated a role for the co‐chaperone and E3‐ubiquitin ligase CHIP (Carboxyl terminus of HSC70‐Interacting Protein) in the regulation of several metabolism‐associated proteins including AMPK, SirT6, AKT, SGK‐1 and PTEN. We demonstrate that mice genetically deficient in CHIP (gene locus Stub1) exhibit a striking Type II diabetes‐like phenotype, including impaired glucose tolerance, decreased insulin sensitivity, and decreased insulin‐stimulated glucose uptake in skeletal muscle in comparison to wild‐type animals. Interestingly, CHIP‐depleted C2C12 skeletal myoblasts exhibited a profound defect in the translocation of the glucose transporter Glut4 to the plasma membrane after glucose/insulin stimulation. The impairment of Glut4 translocation was accompanied by a decrease in both tubulin polymerization and phosphorylation of stathmin (Ser16), a microtubule‐regulating protein required for polymerization‐dependent protein trafficking. PIP3, required for microtubule reorganization, was sustained in wild‐type C2C12 cells following stimulation whereas CHIP‐depleted C2C12s exhibited only transient increases in PIP3 that diminished 30 min after stimulation. Moreover, CHIP‐depleted C2C12s exhibited a concomitant reduction in the levels of phosphorylated AKT (Thr308), a signaling event that requires PIP3‐dependent translocation of AKT to the plasma membrane. The sustained PIP3 levels in wildtype C2C12s correlated with decreasing PTEN levels, a negative regulator of PIP3, at a time when CHIP and PTEN co‐immunoprecipitate. Conversely, a decrease in PTEN is not observed in CHIP‐depleted cells. Together, these data describe a novel role for CHIP in an insulin induced phosphorylation signaling cascade regulating microtubule polymerization necessary for glucose transporter translocation, thereby promoting glucose homeostasis and sensitivity to insulin.

Carboxyl Terminus of HSC70-interacting Protein (CHIP) Down-regulates NF-κB-inducing Kinase (NIK) and Suppresses NIK-induced Liver Injury

Ser/Thr kinase NIK (NF-κB-inducing kinase) mediates the activation of the noncanonical NF-κB2 pathway, and it plays an important role in regulating immune cell development and liver homeostasis. NIK levels are extremely low in quiescent cells due to ubiquitin/proteasome-mediated degradation, and cytokines stimulate NIK activation through increasing NIK stability; however, regulation of NIK stability is not fully understood. Here we identified CHIP (carboxyl terminus of HSC70-interacting protein) as a new negative regulator of NIK. CHIP contains three N-terminal tetratricopeptide repeats (TPRs), a middle dimerization domain, and a C-terminal U-box. The U-box domain contains ubiquitin E3 ligase activity that promotes ubiquitination of CHIP-bound partners. We observed that CHIP bound to NIK via its TPR domain. In both HEK293 and primary hepatocytes, overexpression of CHIP markedly decreased NIK levels at least in part through increasing ubiquitination and degradation of NIK. Accordingly, CHIP suppressed NIK-induced activation of the noncanonical NF-κB2 pathway. CHIP also bound to TRAF3, and CHIP and TRAF3 acted coordinately to efficiently promote NIK degradation. The TPR but not the U-box domain was required for CHIP to promote NIK degradation. In mice, hepatocyte-specific overexpression of NIK resulted in liver inflammation and injury, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. Our data suggest that CHIP/TRAF3/NIK interactions recruit NIK to E3 ligase complexes for ubiquitination and degradation, thus maintaining NIK at low levels. Defects in CHIP regulation of NIK may result in aberrant NIK activation in the liver, contributing to live injury, inflammation, and disease.

The ubiquitin ligase CHIP inactivates NF-κB signaling and impairs the ability of migration and invasion in gastric cancer cells.

Ubiquitin modification of proteins influences cellular processes related to carcinogenesis. The carboxyl terminus of Hsc-70-interacting protein (CHIP), as U-box-type ubiquitin ligase, induces ubiquitination and proteasome-mediated degradation of its substrate proteins. In this study, the role of CHIP in diverse aspects of gastric cancer cells was investigated. CHIP overexpression in the AGS gastric cancer cells caused impaired tumor growth. CHIP overexpression significantly inhibited the migration and invasion of the AGS cells. Moreover, we found that not only RelA/p65 but also RelB, the NF-κB subunits, was negatively regulated by CHIP, likely owing to the TRAF2 reduction. Downregulated target genes of NF-κB subunits, including MMP-2 and -9, integrin β-1 and Bcl-2 were involved in these processes. We also showed that the expression level of CHIP was frequently decreased in gastric cancer tissues and the low level of CHIP expression might be an indicator of an unfavorable prognosis. Taken together, these observations provide functional evidence for CHIP behaviors as a tumor suppressor in gastric cancer.

Ubiquitin ligase CHIP suppresses cancer stem cell properties in a population of breast cancer cells

Cancer stem cells (CSCs) have several distinctive characteristics, including high metastatic potential, tumor-initiating potential, and properties that resemble normal stem cells such as self-renewal, differentiation, and drug efflux. Because of these characteristics, CSC is regarded to be responsible for cancer progression and patient prognosis. In our previous study, we showed that a ubiquitin E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) suppressed breast cancer malignancy. Moreover, a recent clinical study reported that CHIP expression levels were associated with favorable prognostic parameters of patients with breast cancer. Here we show that CHIP suppresses CSC properties in a population of breast cancer cells. CHIP depletion resulted in an increased proportion of CSCs among breast cancers when using several assays to assess CSC properties. From our results, we propose that inhibition of CSC properties may be one of the functions of CHIP as a suppressor of cancer progression.

Abstract P2-11-18: Immunohistochemical expression of ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein) as a significant prognostic marker in postmenopausal invasive breast cancer patients

Abstract Background: CHIP is a U box type ubiquitin ligase that induces ubiquitination and degradation of its target proteins, which include several oncogenic proteins. We clarified that CHIP suppresses tumor growth and metastasis in human breast cancer by inhibiting oncogenic pathways (Nat Cell Biol 2009;11:312-9). In this study, we investigated the relationship between immunohistochemical CHIP expression and several biomarkers, and evaluated the correlation between CHIP expression and prognosis of patients with invasive breast cancer. Patients and Methods: In this study, 272 consecutive patients with invasive carcinoma of no special type with invasion >5 mm, treated in 2000-2001, were enrolled. We performed immunohistochemical staining with antibodies against CHIP, ER, PgR, and HER2. Cytoplasmic expression patterns of CHIP were scored as 0 (no stain), 1 (weak stain), 2 (moderate stain), and 3 (strong stain), and divided into high CHIP (score 3) and low CHIP (score 2, 1, 0) groups. We investigated the relationship between CHIP expression and ER, PgR, HER2, histological grade (HG) and lymph node status (pN). We also evaluated correlation between CHIP expression and prognosis (RFS) of patients. Results: CHIP expression was as follows: score 0, 49 patients (18.0%); score 1, 91 (33.5%); score 2, 83 (30.5%); score 3, 49 (18.0%). High CHIP correlated significantly with positive ER (p<0.001), positive PgR (P<0.001), negative HER2 (p = 0.02), and low HG (p = 0.01). RFS was relatively better in the high CHIP group (49 patients) than the low CHIP group (223 patients) (p = 0.09). In addition, in postmenopausal patients, RFS was significantly better in the high CHIP group (23 patients) than the low CHIP group (133 patients) (p = 0.04); this was not so in premenopausal cases (p = 0.68). Furthermore, RFS was significantly better in CHIP score 3 and ER positive cases (42 patients) than in CHIP score 0 and ER negative cases (34 patients) (p = 0.03). Conclusions: CHIP expression correlated with status of hormone receptors and HER2 and might be a potent prognostic factor in invasive breast cancer patients, especially postmenopausal cases. Immunohistochemical examination of CHIP is thought to be useful for evaluating prognosis of breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-18.

Ubiquitin ligase CHIP induces TRAF2 proteasomal degradation and NF-κB inactivation to regulate breast cancer cell invasion

Transcriptional factor nuclear factor-kappaB (NF-κB) plays a crucial role in human breast cancer cell invasion and metastasis. The carboxyl terminus of Hsc70-interacting protein (CHIP) is a U-box-type ubiquitin ligase that induces ubiquitination and proteasomal degradation of its substrate proteins. In this study, we investigated the role of CHIP in the NF-κB pathway in the invasion of MDA-MB-231 cells, a highly aggressive breast cancer cell line. We showed that overexpression of CHIP significantly inhibits the invasion of the MDA-MB-231 cells. The overexpression of CHIP suppressed expression of urokinase plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) in MDA-MB-231 cells. Moreover, CHIP strongly inhibited the nuclear localization and the transcriptional activity of NF-κB. The activation of the IkappaB kinase complex (IKK) was also blocked by CHIP overexpression. Importantly, CHIP overexpression resulted in a significant decrease in the level of TNF receptor-associated factor 2 (TRAF2), an upstream key player in the NF-κB pathway. However, the level of TRAF2 was restored after treatment with a proteasome inhibitor, MG-132. Moreover, CHIP overexpression promoted the ubiquitination of TRAF2. We also found cell invasion significantly decreased in cells transfected with TRAF2 small interfering RNA (siRNA). In contrast, when CHIP expression was suppressed by siRNA in poorly invasive MCF-7 cells, cell invasion significantly increased in conjunction with enhanced NF-κB activation and TRAF2 levels. Taken together, these results suggest that CHIP regulates NF-κB-mediated cell invasion via the down-regulation of TRAF2.

Prognostic implications of carboxyl-terminus of Hsc70 interacting protein and lysyl-oxidase expression in human breast cancer

Background:Ubiquitin modification of proteins influences cellular processes relevant to carcinogenesis. CHIP (carboxyl-terminus of Hsc70-interacting protein) is a chaperone-dependent E3 ubiquitin ligase, regulating the stability of heat shock protein 90 (HSP90) interacting proteins. CHIP is implicated in the modulation of estrogen receptor (ESR1) and Her-2/neu (ERBB2) stability. LOX (lysyl-oxidase) serves intracellular roles and catalyses the cross-linking of extracellular matrix (ECM) collagens and elastin. LOX expression is altered in human malignancies and their peri-tumoral stroma. However, paradoxical roles are reported. In this study, the level of mRNA expression of CHIP and LOX were assessed in normal and malignant breast tissue and correlated with clinico-pathological parameters.Materials and Methods:Breast cancer (BC) tissues (n = 127) and normal tissues (n = 33) underwent RNA extraction and reverse transcription; transcript levels were determined using real-time quantitative PCR and normalized against CK-19. Transcript levels were analyzed against TNM stage, nodal involvement, tumor grade and clinical outcome over a ten-year follow-up period.Results:CHIP expression decreased with increasing Nottingham Prognostic Index (NPI): NPI-1 vs. NPI-3 (12.2 vs. 0.2, P = 0.0264), NPI-2 vs. NPI-3 (3 vs. 0.2, P = 0.0275). CHIP expression decreased with increasing TNM stage: TNM-1 vs. TNM-2 (12 vs. 0, P = 0.0639), TNM-1 vs. TNM-2-4 (12 vs. 0, P = 0.0434). Lower transcript levels were associated with increasing tumor grade: grade 1 vs. grade 3 (17.7 vs. 0.3, P = 0.0266), grade 2 vs. grade 3 (5 vs. 0.3, P = 0.0454). The overall survival (OS) for tumors classified as ‘low-level expression’, was poorer than those with ‘high-level expression’ (118.1 vs. 152.3 months, P = 0.039). LOX expression decreased with increasing NPI: NPI-1 vs. NPI-2 (3 vs. 0, P = 0.0301) and TNM stage: TNM-1 = 3854639, TNM-2 = 908900, TNM-3 = 329, TNM-4 = 1.232 (P = NS).Conclusion:CHIP expression is associated with favorable prognostic parameters, including tumor grade, TNM stage and NPI. CHIP expression predicts OS. LOX expression is associated with improved NPI. In addition to their prognostic utility, mechanistic insights into tumor suppressor function may offer potential therapeutic strategies.

Carboxyl terminus of heat-shock cognate 70-interacting protein degrades tau regardless its phosphorylation status without affecting the spatial memory of the rats

Accumulation of the hyperphosphorylated tau is a hallmark pathological event for the formation of neurofibrillary tangles in Alzheimer disease (AD). Until now, there is no effective way to antagonize this accumulation. Recently, it has been reported that carboxyl terminus of Hsc70-interacting protein (CHIP) is the specific tau E3 ligase and it mediates tau degradation in vitro. However, the nature of CHIP on in vivo tau degradation is not fully understood. Here, we demonstrated that hippocampal transfection of CHIP plasmid in rats could reach a time-dependent elevated expression of CHIP mRNA and protein in 24 h. Concomitantly, expression of exogenous CHIP could promote the degradation of the hyperphosphorylated tau induced by overactivation of glycogen synthase kinase-3 (GSK-3) or inhibition of protein phosphatase-2A (PP-2A) in the rat brains and N2A cells. CHIP also degraded tau in normal rats regardless the phosphorylation status of tau proteins. Though CHIP overexpression was not able to improve significantly the spatial memory retention deficits induced by overactivation of GSK-3, it did not cause significant damage to the memory function in normal rats. These results suggest that CHIP may degrade tau both in physiological and pathological conditions without affecting the behavior of the rats, implying that overexpression of CHIP may antagonize tau accumulation in the AD brains.

CHIP Controls the Sensitivity of Transforming Growth Factor-β Signaling by Modulating the Basal Level of Smad3 through Ubiquitin-mediated Degradation

Transforming growth factor-beta (TGF-beta) signaling is critical in a variety of biological processes such as cell proliferation, differentiation, and apoptosis. TGF-beta signaling is mediated by a group of proteins including TGF-beta receptors and Smads. It is known that different cells can exhibit different sensitivities to TGF-beta. Several molecular mechanisms, such as the differential expression of the receptor levels, have been suggested as contributing to these differences. Here, we report evidence for a novel mechanism of regulating TGF-beta sensitivity that depends on the role of CHIP (carboxyl terminus of Hsc70-interacting protein) in regulating the basal level of Smad3 via the ubiquitin-dependent degradation pathway. First, using a luciferase assay we found that overexpression of CHIP inhibited TGF-beta signaling, whereas silencing CHIP expression by small interfering RNAs led to increased TGF-beta signaling sensitivity. Second, based on the results of cell proliferation assays and JunB expression, we found that TGF-beta signaling could be abolished by stably overexpressing CHIP. Third, in those cell lines with stably expressed CHIP, we observed that the Smad3 protein level was dramatically decreased. Finally, we demonstrated that CHIP served as a U-box dependent E3 ligase that can directly mediate ubiquitination and degradation of Smad3 and that this action of CHIP was independent of TGF-beta signaling. Taken together, these findings suggest that CHIP can modulate the sensitivity of the TGF-beta signaling by controlling the basal level of Smad3 through ubiquitin-mediated degradation.