The Mechanisms Of CHIP Regulates Angiogenesis In Human Gastric Cancer

Despite the improved prognosis of patients with gastric cancer (GC) resulting from early diagnosis, radical surgery, and the development of adjuvant therapy, GC remains the fourth most common cancer in the world and the second leading cause of cancer-related death worldwide. The high mortality rates in GC patients are mostly due to the metastatic disease.Metastasis is the result of several sequential steps of tumor development. Angiogenesis is a critical hallmark of malignancy and is induced surprisingly early during the multi-stage development of invasive cancers both in animal models and in humans. Although angiogenesis is an important aspect of tumor growth and metastasis and considered as an important predictor of overall survival in GC, little is known about the molecular events critical to the GC angiogenesis. Unraveling the factors driving this process is thus important also for future therapeutic interventions.The nuclear factor NF-κB is a critical transcription factor in various cancers that regulates genes associated with a variety of cellular functions, such as cell survival, proliferation, angiogenesis, and cancer metastasis. The p65(also called RelA) is a member of the NF-κB family, which is normally sequestered in the cytoplasm of non-stimulated cells by inhibitors of NF-κB (IκBs). Recently, a novel pathway of terminating NF-κB activation was revealed and showing that nuclear p65protein level is regulated by ubiquitin-proteasome-dependent degradation. Therefore, the molecules involved in controlling the aberrant activation of NF-κB signaling are attractive targets for therapeutic strategies against cancer.CHIP (carboxy terminus of Hsc70interacting protein), encoded by the STUB1gene, which functions as a E3ubiquitin ligase activity, link between the chaperone and proteasome systems. Substantial evidences indicate that CHIP functions as a tumor suppressor because it could induce ubiquitination and degradation of several oncogenic proteins. A recent study showed that CHIP overexpression inhibits metastatic potential and knockdown of CHIP increased the microvessel density in breast tumors. However, the possible function of CHIP in the development and progression of GC and the prognostic role in GC patients remains unclear, and the precise mechanisms of CHIP in angiogenesis have not yet been identified. In this study, the biological function and clinic significance of CHIP in GC was examined.Methods:The prognostic value of CHIP expression was evaluated using tissue microarray and immunohistochemical staining in two independent human GC cohorts. The role of CHIP on tumorigenicity and angiogenesis was determined in vitro and in vivo.Results:1. Reduced CHIP expression in GC versus non-cancer tissuesThe mRNA and protein levels of CHIP were found to be significantly lower in12of16(75%) and14of16(87.5%) gastrlic tumors compared with the paired normal gastric mucosa by real-time PCR and Western blot, respectively. Immunohisto-chemical staining of the gastric tissues showed that CHIP seemed to be predominantly localized in the cytoplasm of the GC cells, but presented in both the nucleus and cytoplasm of the normal cells. Moreover, expression of CHIP was significantly decreased in GC cells. These results were confirmed in TMA of GC patients, which showed the expression of CHIP was significantly decreased in64of74(86.5%) of GCs compared with the matched normal gastric tissues (P<0.001).2. Decreased CHIP expression correlates with clinicopathological characteristics and a shorter survival in GC patientsThe expression of CHIP in the cancerous tissues of training cohort significantly correlated with clinicopathological features, such as depth of invasion, lymph node metastasis, distant metastasis, TNM stage and histological type (P<0.05for all). Moreover, these findings was confirmed in the validation cohort of GC patients.In the training cohort, Kaplan-Meier analysis revealed that low CHIP expression was significantly correlated with poorer overall5-year survival of all GC patients (P <0.001, log-rank test). The MST of patients whose primary GC scored high for CHIP expression was more than60months, whereas low CHIP expression was correlated to a shortened MST of23months. This finding was confirmed in the validation cohort (P<0.001). Next, multivariate Cox regression analysis indicated that CHIP expression was an independent positive prognostic factor in both cohorts (HR=0.34,95%CI=0.15-0.76, training cohort; HR=0.21,95%CI=0.16-0.26, validation cohort).To further evaluate the predictive ability of CHIP expression, we conducted a time-dependent ROC analysis, which indicated that the combination of the clinical risk score (TNM stage, histological type and tumor diameter) and CHIP risk score contributed much more than either one alone in both cohorts. The AUC at year5was0.688(95%CI=0.550-0.826) and0.723(95%CI=0.675-0.770) for a clinical risk score, whereas it was significantly increased to0.760(95%CI=0.621-0.899) and0.842(95%CI=0.806-0.878) for the combination of the clinical risk score with CHIP risk score in the training cohort and validation cohort. 3. CHIP suppresses GC cell growth and angiogenesisInvestigation on CHIP regulation of GC cell growth indicated that in soft agar colony-formation assay, there were50%fewer colonies formed in CHIPOE stable cells; and in agreement with this, knockdown of CHIP expression significantly increased the number of colonies compared to the control cells.To further study the functional role of CHIP in GC angiogenesis, HUVECs growth and tube formation in vitro were investigated. The growth of HUVECs in conditioned medium from CHIPOE BGC823cells was inhibited by48%; and in line with this, CHIP knockdown promoted the HUVECs growth by1.82-fold compared with the corresponding controls. The average number of complete tubular structures formed by HUVECs was significantly decreased in conditioned medium from CHIPOE BGC823cells, but increased in that from CHIP siRNA cells compared with corresponding controls.To further investigate the role of CHIP in tumorigenesis in vivo, stable CHIPOE and vector control BGC823cells were injected subcutaneously into nude mice and tumor growth was monitored. The data showed that tumor growth of BGC823cells was suppressed in the CHIP overexpression group.Microvessel density evaluated by CD31immunohistochemical staining showed a significant reduction in tumor tissues of CHIPOE group, compared with vector controls.4. Tumor angiogenesis and metastasis is inhibited by CHIP expression in GC cellsTo examine the roles of CHIP on invasive and metastatic potential of GC cells, we performed adhesion and invasion assays. In adhesion assay, we found that CHIPOE decreased cell attachment ability of BGC823cells on fibronectin and vitronectin-coated plates by41%and32%, respectively; and CHIP knockdown increased this ability by88%and79%, respectively, compared with the corresponding controls. We also investigated the invasiveness of these cells, and found BGC823and MGC803CHIP0E cells had significantly decreased ability to penetrate the matrigel-coated membrane by51%and48%, respectively; whereas silencing of CHIP in BGC823and MGC803cells increased the invasive ability by175%and90%, respectively, compared with the corresponding controls.5. CHIP inhibits NF-κB activity and the expression of its target pro-angiogenesis genesWe then investigated whether CHIP exerts inhibitory effect on GC cells angiogenesis through inhibiting NF-κB activity. It was shown that the NF-κB activity was increased following cotransfection with NF-KB-p65expression plasmid or exposure to NF-κB-p65activator (either TNF-a or LPS), whereas significantly reduced with or without these treatments in CHIP-overexpressing cells. In contrast, knockdown CHIP enhanced NF-κB activity. This effect was further abolished by knockdown of NF-κB-p65expression with a specific siRNA or suppression of NF-κB-p65activity with specific inhibitor (Bay-11).The EMSA indicated that NF-κB affinity was decreased in CHIP-overexpressing cells with or without TNF-α treatment, and increased in CHIP knockdown cells, when compared to the corresponding controls. Furthermore, we performed immuno-fluorescence to examine the effects of CHIP on p65localization. It was shown that myc-CHIP inhibited p65translocation into the nucleus and attenuated p65protein levels with or without TNF-a treatment.Moreover, our data revealed that CHIP overexpression decreased NF-κB-responsive genes, such as IL-852%; and CHIP knockdown elevated these mRNA levels by2.0, compared with corresponding controls..The ELISA was used to confirm alterations of secreted IL-8protein corresponding to CHIP modulation. We found that CHIP overexpression decreased IL-8protein in the medium from0.85ng/ml to0.52ng/ml. In CHIP knockdown cells IL-8protein increased from0.90ng/ml to1.75ng/ml in the medium. CHIP overexpression also decreased p65-induced IL-8mRNA and protein production, whereas silencing of p65blocked CHIP siRNA-induced IL-8mRNA and protein production.To confirm the role of IL-8in CHIP-regulated GC cell angiogenesis, we performed IL-8rescue and IL-8blocking assays. We found that the inhibited tubular structure formations in the conditioned medium from CHIP-overexpressing BGC823cells can be rescued by addition of0.4ng/ml recombinant IL-8. Application of sufficient IL-8antibody abrogated the elevated tube formation in that from CHIP-knockdown cells.6. CHIP promotes NF-λB ubiquitination for degradation by the proteasomeFurthermore, we investigated potential mechanisms of the NF-λB signalling pathway controlled by CHIP. NF-λB/p65and its phosphorylated (Serine-536) levels were decreased in CHIPOE cells, and increased in CHIP knockdown cells compared with corresponding control cells.When the cells were treated with CHX, the degradation of p65was promoted by overexpression of CHIP, whereas suppressed by CHIP knockdown. Additionally, a physical interaction between CHIP and p65was determined by co-immuno-precipitation (co-IP) in both endogenous and exogenous (myc-CHIP and Flag-p65) settings. Furthermore, co-IP experiments using cytoplasmic and nuclear fractions showed that Myc-CHIP interacted with Flag-p65protein only in the nuclear extracts, but not from the cytoplasmic fraction.It was also shown that loss of p65expression in CHIPOE cells was inhibited by pretreatment with the proteasome inhibitor MG132. Simultaneously, we found ubiquitinated p65was increased due to MG132inhibited its degradation, and CHIP overexpression further enhanced the ubiquitination of p65. These data indicated that CHIP functioned as an E3ligase to mediate the ubiquitination of p65for degradation by the proteasome.Conclusions:Decreased CHIP expression in GC resulted in increased angiogenesis and contributed to GC progression and poor prognosis. CHIP expression is a GC candidate clinical prognostic marker and a putative treatment target.