Expression Of Bcl-2-associated Athanogene 3 Research Articles

Optimal combination of immune checkpoint and senescence molecule predicts adverse outcomes in patients with acute myeloid leukemia

Background High expression of immune checkpoints (ICs) and senescence molecules (SMs) contributes to T cell dysfunction, tumor escape, and progression, but systematic evaluation of them in co-expression patterns and prognosis in acute myeloid leukemia (AML) was lacking. Methods Three publicly available datasets (TCGA, Beat-AML, and GSE71014) were first used to explore the effect of IC and SM combinations on prognosis and the immune microenvironment in AML, and bone marrow samples from 68 AML patients from our clinical center (GZFPH) was further used to validate the findings. Results High expression of CD276, Bcl2-associated athanogene 3 (BAG3), and SRC was associated with poor overall survival (OS) of AML patients. CD276/BAG3/SRC combination, standard European Leukemia Net (ELN) risk stratification, age, and French-American-British (FAB) subtype were used to construct a nomogram model. Interestingly, the new risk stratification derived from the nomogram was better than the standard ELN risk stratification in predicting the prognosis for AML. A weighted combination of CD276 and BAG3/SRC positively corrected with TP53 mutation, p53 pathway, CD8+ T cells, activated memory CD4+ T cells, T-cell senescence score, and Tumor Immune Dysfunction and Exclusion (TIDE) score estimated by T-cell dysfunction. Conclusion High expression of ICs and SMs was associated with poor OS of AML patients. The co-expression patterns of CD276 and BAG3/SRC might be potential biomarkers for risk stratification and designing combinational immuno-targeted therapy in AML. Key Messages High expression of CD276, BAG3, and SRC was associated with poor overall survival of AML patients. The co-expression patterns of CD276 and BAG3/SRC might be potential biomarkers for risk stratification and designing combinational immuno-targeted therapy in AML.

Abstract 11262: Effect of Active Cancer/Cachexia on the Development of Cardiac Dysfunction in Mice

Introduction: Cancer is independently associated with the alteration of cardiac function prior to cardiotoxic chemotherapy (CCT) exposure. Similar to cancer associated cachexia (CAC), the elevation and the deleterious role of IL-6 in plasma was associated with a reduced cardiac function in heart failure (HF) patients subpopulation. Cancer cells manipulate BCL-2-associated athanogene 3 (BAG3)-HSP70-regulated pathways in tumor cells, which is a key regulator of protein turnover and contractility in cardiomyocyte. Hypothesis: Here, we aimed to characterize the progression of cardiac dysfunction and the expression of BAG3 and HSP70 in tumor-bearing mice. Methods: Colon-26 adenocarcinoma cells (C26; n=22) with/without shIL-6 (C26 shIL-6; n=22) were injected subcutaneously adult male BALB/c mice. Control mice were injected with PBS (n=13). Echocardiographic examinations and invasive hemodynamic measurements ( in vivo and ex vivo using isolated working hearts system) were performed at 10 (early) and 20 (late) days post injection, respectively. The expression of BAG3 and Hsp70 were determined by Western blot. Results: The tumor size was comparable between the cancer groups. However, only C26 group showed a significant loss of subcutaneous fat and skeletal muscle (p<0.05, respectively), confirming cachectic condition. Echocardiography results show a tendency to decline of ejection fraction at the early phase (p~0.08 vs Control), and turned significance lower at late stage (p<0.05 vs Control) in tumor-bearing mice. In line with that, invasive hemodynamic and isolated working heart measurements confirmed LV systolic and diastolic dysfunction (late stage, p<0.05 vs Control, respectively). Interestingly, heart rate and aortic flow were predominantly declined in cachectic animals (p<0.05 vs Control). Importantly, cardiac dysfunction was associated with a significant reduction in both BAG3 and Hsp70 in the myocardium independently of cachexia. Conclusions: Cancer rather than CAC is a main driver for the development of cardiac contractile dysfunction prior to CCT exposure. In addition, our data suggest that targeting BAG3-Hsp70 complex in the cardiomyocytes may provide a novel strategy to improve the cancer associated cardiac dysfunction.

Abstract 12697: Pharmacological Inhibition of BAG3-HSP70 With the Proposed Breast Cancer Therapeutic JG-98: In-vivo Mice Study

Introduction: The co-chaperone Bcl2-associated athanogene-3 (BAG3), along with its cofactor heat shock protein 70 (HSP70), is fundamental for protein quality control and cell survival in a healthy heart. However, elevated expression of BAG3 is also associated with metastasis in breast and other cancers, and small molecules such as JG-98 that disrupt BAG3-HSP70 binding have been shown to reduce cancer cell proliferation. We previously showed JG-98 is cardiotoxic in neonatal rat ventricular myocytes. Hypothesis: As BAG3-HSP70 is fundamental for autophagic protein turnover, JG-98 will reduce tumor size but have cardiotoxic effects. Methods: BT474 Resistant breast cancer cells were injected bilaterally into mammary fat pads of female athymic nude mice. When tumors reached 25 mm 2 , mice received a baseline echocardiography and were randomized to intraperitoneal injections of either PBS/DMSO vehicle or 3 mg/kg JG-98 twice weekly for 3 weeks (n = 4/group) or 6 weeks (n=10/group). Tumor growth was monitored using calipers and echocardiography was performed prior to sacrifice. Results: After 3 weeks, tumor volume increased by ~44% in Vehicle treated mice but decreased by 17% in JG-98 treated mice, effectively halting tumor growth. However, cardiac echocardiography did not reveal any functional or structural differences between the vehicle and JG-98 treated mice after either 3 or 6 weeks. JG-98 did result in dysregulation of BAG3 and its interactome in the LV. JG-98 slightly increased BAG3 levels in the LV at 3 weeks, but slightly decreased levels at 6 weeks. This agrees with data showing acute stress upregulates BAG3, but chronic stress downregulates it. Similarly, we found altered protein expression of the BAG3 binding partners HSBP8, HSPB5, and HSP70. Conclusions: JG-98 had no overt cardiotoxic effects, suggesting the adult heart may be resistant to disrupted BAG3-HSP70. However, we also observed dysregulated protein levels in the BAG3-mediated autophagy pathways that could precede dysfunction with more chronic treatment. Furthermore, mutations in BAG3 or altered expression levels could sensitize individuals to JG-98 treatment. Further work is necessary to understand the cardiac impact of cancer therapeutics that target BAG3.

Dissecting the progression of cardiac dysfunction in tumor-bearing mice

Abstract Funding Acknowledgements Type of funding sources: None. Background Cancer patients undergoing heart-related complications result in high incidences of mortality. Nevertheless, it is still not fully understood whether localized tumors affect heart function prior to the onset of cachexia, hence, making the heart more vulnerable for functional abnormalities in later stages of the disease. In addition to analyse heart function, we focus on the expression BCL-2–associated athanogene 3 (BAG3), a co-chaperone protein and Hsp70, which are highly expressed in tumor but decrease in cardiomyocytes (CM) in heart failure (HF). Methods Colon-26 adenocarcinoma cells (C26; n=22) with/without shIL-6 (C26 shIL-6; n=22) were injected subcutaneously into the right flank of 10-11 weeks old BALB/c male mice. Control mice were injected with vehicle (PBS; n=8). Cardiac function was assessed by echocardiography and invasive hemodynamic measurements 10 (early) and 20 (late) days after the injection, respectively. In addition, the expression of BAG3 and Hsp70 were determined by Western blot as well as the extend of cardiac fibrosis was determined by Masson-Goldner's trichrome staining. Results The tumor size was comparable between the two injected groups. However, only C26 group showed a significant loss of subcutaneous fat and skeletal muscle (p<0.05, respectively), suggesting cachexia. Heart weight normalized to tibia length was not changed in the injected groups as compared to controls (day 20). However, left ventricular ejection fraction (LVEF) showed a tendency to decline in the early phase (p~0.08) in both injected group and it reached significance at late stage (p<0.05). Invasive hemodynamic assessment also confirmed the contractile dysfunction, resulting in a decrease in LV systolic pressure and increase of LV end-diastolic pressure (p<0.05, respectively). Importantly, these functional changes in the heart in tumor-bearing mice were associated with a marked reduction in both BAG3 and Hsp70 in the myocardium. Furthermore, there was no sign of cardiac fibrosis in the injected groups. Discussion Our study shows for the first time that tumor rather than cancer cachexia plays a significant maladaptive role in the progression of cardiac dysfunction in a mouse model of C26 injection-induced cachexia. The progression of cardiac contractile dysfunction was associated with a decline in BAG3 and Hsp70 in tumor-bearing mice, suggesting changes of BAG3/Hsp 70 signalling may be a critical component as well as target.

BAG3 protects chondrocytes against lumbar facet joint osteoarthritis by regulating autophagy and apoptosis.

Bcl2-associated athanogene3 (BAG3) protein, mainly induced by stressful stimuli, has been confirmed to participate in apoptosis and autophagy. In recent studies, BAG3 has gradually become a key molecule in tumors. However, the role of BAG3 in the progression of lumbar facet joint osteoarthritis (FJOA) and whether it can regulate chondrocyte apoptosis and autophagy are still unknown. In both human and FJOA rat models, we observed an upregulation of BAG3 and apoptosis and autophagy-related proteins compared with healthy tissues. Then, we established the chondrocytes injury model in vitro by using IL-1β to stimulate human SW1353 cells. Western blot analysis data showed significant expression of BAG3, apoptosis, and autophagy-related proteins in SW1353 cells. Finally, by knocking down and overexpressing BAG3, we discovered possible anti-apoptotic and autophagy-promoted effects of BAG3 in FJOA through various experimental methods. This study demonstrated that BAG3 actively participates in regulating chondrocyte apoptosis and autophagy in FJOA and may be a highly interesting targetfor pharmacological interventions.

Abstract 10321: Disrupting the Bag3-hsp70 Complex with the Proposed Cancer Therapeutic Jg-98 is Detrimental for Cardiomyocytes

The heat shock protein 70 (HSP70) co-chaperone Bcl-2-associated athanogene-3 (BAG3) is critical for cell viability through facilitating HSP70-dependent protein quality control (PQC). Unfortunately, the same vital functions of BAG3-HSP70 in healthy cells are known to promote cell survival and proliferation in various cancers. Recent promising studies found that disrupting BAG3-HSP70 with the small molecule JG-98 has potent anti-cancer effects in cultured cells and in vivo . However, it is unknown what impact this therapy has on other cell types such as cardiomyocytes, where BAG3-mediated PQC is important for function. Using neonatal rat ventricular myocytes (NRVMs), we assessed the impact of JG-98 on cardiomyocyte viability and found apoptosis increased, even at doses 10-100-fold lower than those used with cancer cells. Next, we measured autophagic flux using ammonium chloride to block lysosomal degradation and found flux was reduced with JG-98. To explore mechanism, we assessed expression of BAG3, HSP70, and three other proteins that assemble with the complex during autophagy (HSPB5, HSPB8, SYNPO2). We found BAG3, HSPB8, HSPB5, and SYNPO2 expression decreased at the protein and transcript level with JG-98, while HSP70 expression was not changed. Next, we tested whether the BAG3-HSP70 interaction was important for protein stability of the complex members by treating NRVMs with JG-98 in the presence of cycloheximide to block translation. JG-98 decreased the protein half-life of BAG3, HSPB8, and HSPB5, indicating that protein stability was reduced, while SYNPO2 and HSP70 half-lives were unaffected. As we previously showed BAG3-HSP70 was important for maintaining sarcomere PQC, we next assessed the impact of JG-98 on sarcomere structure. In agreement, we found here that JG-98 caused sarcomere structural disintegration, which was exacerbated with stress. Finally, the detrimental effects of JG-98 on cell viability and sarcomere structure could be partially ameliorated with autophagy induction by rapamycin. Taken together, our findings indicate the importance of BAG3-HSP70 in cardiomyocytes and caution against broadly targeting this complex therapeutically.

Pharmacological inhibition of BAG3-HSP70 with the proposed cancer therapeutic JG-98 is toxic for cardiomyocytes.

The co-chaperone Bcl2-associated athanogene-3 (BAG3) maintains cellular protein quality control through the regulation of heat shock protein 70 (HSP70). Cancer cells manipulate BAG3-HSP70-regulated pathways for tumor initiation and proliferation, which has led to the development of promising small molecule therapies, such as JG-98, whichinhibit the BAG3-HSP70 interaction and mitigate tumor growth. However, it is not known how these broad therapies impact cardiomyocytes, where the BAG3-HSP70 complex is a key regulator of protein turnover and contractility. Here, we show that JG-98 exposure is toxic in neonatal rat ventricular myocytes (NRVMs). Using immunofluorescence microscopy to assess cell death, we found that apoptosis increased in NRVMs treated with JG-98 doses as low as 10 nM. JG-98 treatment also reduced autophagy flux and altered expression of BAG3 and several binding partners involved in BAG3-dependent autophagy, including SYNPO2 and HSPB8. We next assessed protein half-life with disruption of the BAG3-HSP70 complex by treating with JG-98 in the presence of cycloheximide and found BAG3, HSPB5, and HSPB8 half-lives were reduced, indicating that complex formation with HSP70 is important for their stability. Next, we assessed sarcomere structure using super-resolution microscopy and found that disrupting the interaction with HSP70 leads to sarcomere structural disintegration. To determine whether the effects of JG-98 could be mitigated by pharmacological autophagy induction, we cotreated NRVMs with rapamycin, which partially reduced the extent of apoptosis and sarcomere disarray. Finally, we investigated whether the effects of JG-98 extended to skeletal myocytes using C2C12 myotubes and found again increased apoptosis and reduced autophagic flux. Together, our data suggest that nonspecific targeting of the BAG3-HSP70 complex to treat cancer may be detrimental for cardiac and skeletal myocytes.

Postnatal development of BAG3 expression in mouse cerebral cortex and hippocampus

The decreased efficiency of autophagic processing in the central nervous system during aging may be a contributing factor in neurodegenerative diseases. BAG3 (Bcl2 associated athanogene 3) is a major member of the BAG family of co-molecular chaperones that mediate selective macroautophagy. Therefore, we analyzed the expression and distribution of BAG3 in the brain at postnatal 0day (P0), P15, 1-, 2-, 9-, 12-, and 18month-old C57BL/6 mice, thus covering almost all ages. Except for a significant steep drop in mRNA and protein levels in the cortex and hippocampus soon after birth, there were minimal differences in the expression and distribution of BAG3 among P15, M1, M2, M9, and M12 mice; however, at 18months, BAG3 expression was significantly higher. Immunohistochemical analyses showed that BAG3 is mainly located in the neuronal cytoplasm and processes in C57BL/6 the cerebral cortex and hippocampus from P0 to M18 postnatal development. These findings indicate that BAG3 might be stable in young and middle-aged mice, but unstable in aged mice.

HSC70-JNK-BAG3 complex is critical for cardiomyocyte protection of BAG3 through its PXXP and BAG structural domains.

Notwithstanding previous studies have proved the anti-apoptotic effect of Bcl-2 associated athanogene3 (BAG3) in myocardium, the structural domains PXXP and BAG responsible for its protection are not reformed. Since BAG3 in cardiomyocytes is a new target for inhibiting apoptosis induced by hypoxia/reoxygenation (H/R) stress, we demonstrated that over-expression of BAG3 reduced the injury induced by H/R in either neonatal or adult rat cardiomyocytes (NRCMs and ARCMs, respectively) and PXXP and BAG domains play an important role in cellular protection in H/R stress. Apoptosis in cardiomyocytes induced by hypoxia-reperfusion was evaluated with propidium iodide (PI) staining, cleaved caspase-3, and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining in cultured NRCMS. Either increasing expression of BAG3 or its mutants was performed to manipulate the level of BAG3. Co-immunoprecipitation (Co-IP) was used to demonstrate the complex that BAG3 is binding to HSC70 and JNK. PXXP and BAG domains of BAG3 played an essential role in BAG3 attenuating cardiomyocytes apoptosis induced by H/R through the JNK signalling pathway. The cellular protection of BAG3 with its structural domain PXXP or BAG is associated with the binding with HSC70 and JNK. These results showed that the protective effect of BAG3 on apoptosis induced by H/R stress is closely related to its structural domains PXXP and BAG. The mechanism may provide a new therapeutic strategy for the patients suffering from ischemic cardiomyopathy and may be a critical role of its PXXP and BAG3 domains.

Knockdown of Bcl-2-Associated Athanogene-3 Can Enhance the Efficacy of BGJ398 via Suppressing Migration and Inducing Apoptosis in Gastric Cancer.

Gastric cancer (GC) is one of the most common malignancies of the digestive tract worldwide, and cancer cell resistance against anticancer drugs remains a major challenge for GC treatment. Nvp-BGJ398 (BGJ398) is considered as a common drug for cancer treatment; however, Bcl-2-associated athanogene-3 (BAG3) plays an important role in drug resistance. To investigate the function of BAG3 on the sensitivity of GC cells to BGJ398. The expression of BAG3 in GC cells and GC resistance cells was examined by qRT-PCR and western blot. The resistance to BGJ398 was detected by viability assay, and a half-maximal inhibitory concentration (IC50) was calculated. The cell migration and apoptosis were determined by wound-healing assay and flow cytometry assay. BAG3 was highly expressed in drug-resistant cells Fu97R and Snu16R. BAG3 was also associated with sensitivity of Snu16 cells to BGJ398, promoting migration but inhibiting apoptosis. However, knockdown of heat shock transcription factor 1 (HSF1) suppressed BAG3 expression and lowered the sensitivity to BGJ398 in Snu16R cells. Knockdown of BAG3 inhibited tumor growth and cell apoptosis but induced cell apoptosis and amplified the sensitivity to BGJ398 in Snu16R cells, followed by enhancing BGJ398-induced antitumor function in a Snu16R-derived xenograft mouse model. The mechanism of resistance to BGJ398 in GC is mediated by BAG3/HSF1, and combined treatment with shBAG3 could improve the efficacy of BGJ398 in GC. Thus, BAG3-targeted therapy improves the antitumor efficacy of BGJ398, which might provide a novel therapeutic strategy for GC.

Association of GRP78, HIF-1α and BAG3 Expression with the Severity of Chronic Lymphocytic Leukemia.

Parallel with the progression of Chronic Lymphocytic Leukemia (CLL), the levels of 78KDa Glucose-Regulated Protein (GRP78) and Hypoxia-Inducible Factor 1 alpha (HIF-1α) are increased as they may activate the induction of anti-apoptotic proteins such as BCL2 Associated Athanogene 3 (BAG3). Previous studies have indicated that there is a positive correlation among GRP78, HIF-1α and BAG3. This study aimed to evaluate the effect of metabolic factors involved in invasive CLL on apoptotic factors. A case-control study was conducted on 77 patients diagnosed with CLL along with 100 healthy individuals. Cell blood count was performed for all participants. According to Binet's classification, CLL patients were divided into different groups. B cells were isolated from the peripheral blood of CLL patients by binding to anti-CD19 beads. The expression of BAG3, GRP78 and HIF-1α genes was analyzed using the RT-PCR method. To confirm the results of RT-PCR, western blot analysis was carried out. The results showed that there was a strong association among the expression of BAG3, GRP78 and HIF-1α. The stage of CLL in patients was highly correlated with the expression rate of each gene (p<0.001). Accordingly, the western blot analysis indicated that the concentrations of GRP78 and HIF-1α were significantly higher than the expression of BAG3, considering the stage of CLL. It was shown that increased expression of GRP78 and HIF-1α could result in the elevation of BAG3, as well as the disease progression. Therefore, the role of these metabolic factors might be more pronounced compared with the anti-apoptotic agents to monitor disease progression in CLL patients.

SAT-632 Involvement of NF-κB-p65 in BAG3 Regulation After Stress Stimuli

We previously identified the limb salvage QTL1 (LSQ-1) on mouse chromosome 7 as a locus that offered protection against ischemic injury following induction of hind limb ischemia (HLI) a model of experimental peripheral arterial disease (PAD) in mice. To better understand the role of the LSQ-1 locus in post ischemic adaptation we characterized several genes within this locus and identified a number of genes that were important in tissue adaptation to ischemia, including BCL2-associated athanogene 3 (BAG3). BAG3 is an anti-apoptotic protein that plays an important role in cell survival through the regulation of autophagy. BAG3 expression is induced in the gastrocnemius muscles of mice after hind limb ischemia but how ischemia regulates BAG3 expression is poorly understood. Additionally, the activation of NF-κB transcription factor is essential for cell survival after stress stimuli. We hypothesized that BAG3 upregulation following stress stimuli is regulated by NF-κB. We determined whether NF-κB is involved in BAG3-mediated survival of primary human skeletal muscle cells (HSMC) during ischemia and diabetic conditions. Within 6 hours of treatment, ischemia induced BAG3 mRNA (no ischemia vs ischemia: 1.0 ±0.09 vs 1.41±0.02; p<0.01) and protein expression (BAG3/total protein, no ischemia vs ischemia 1.0±0.01 vs 1.38±0.06; p<0.01). Knockdown of BAG3 expression by shRNA induced early cell damage in HMSC under ischemic conditions as measured by HMGB1 expression (HMGB1/total protein; control plasmid vs shRNA, 1.0±0.09 vs 1.71±0.04; p<0.01). Knockdown of p65 subunit of the NF-κB by shRNA significantly diminished BAG3 mRNA expression after ischemia (control plasmid vs shRNA: 2.11±0.18 vs 1.48±0.05; p<0.05). Moreover, treatment of HSMC with 750 uM palmitic acid (PA) and 100nM insulin for 3 days to mimic diabetic conditions significantly increased the expression of BAG3 mRNA (control vs PAL+Ins, 1.0±0.14 vs 2.27±0.08; p<0.01) and protein (BAG3/total protein, control vs PAL+Ins, 1.0±0.03 vs 1.39±0.11, p<0.01). Knocking down p65 attenuated these increase in BAG3 mRNA (PAL+Ins vs shRNA+PAL+Ins, 2.27±0.08 vs 1.56±0.02 p<0.01) and protein (BAG3/total protein; PAL+Ins vs shRNA+PAL+Ins, 1.39±0.11 vs 0.99±0.1; p<0.05) Thus, 1) BAG3 expression is important in cell survival under ischemic conditions, and 2) NF-kB plays a key role in upregulating the expression of BAG3 under diabetic and ischemic conditions.

BAG3 expression correlates with the grade of dysplasia in squamous intraepithelial lesions of the uterine cervix.

Bcl-2-associated athanogene 3 (BAG3) is a protein involved in apoptosis and stress response, which is overexpressed in invasive cervical cancer. However, nothing is known about BAG3 expression in precancerous lesions of the uterine cervix. We aimed to evaluate the expression of BAG3 in cervical intraepithelial neoplasia/squamous intraepithelial lesions (CIN/SIL). Forty patients (16 CIN1/L-SIL, 11 CIN2/H-SIL and 13 CIN3/H-SIL) were assessed by immunohistochemistry for BAG3. The intensity of BAG3 expression was categorized as null, minimal, weak, moderate or strong. The association of BAG2 intensity of expression with the grade of dysplasia was assessed using Chi-square test (significant P value <0.05). In all normal controls, BAG3 expression was negative. In L-SIL specimens, BAG3 expression was confined to the basal third of the epithelium, with an intensity minimal in nine cases (56.3%), weak in six (37.5%) and strong in one (6.3%). In H-SIL specimens, BAG3 expression involved also the two upper thirds of the epithelium, with an intensity moderate in 13 cases (54.2%; 8 CIN2 and 5 CIN3) and strong in 11 cases (45.8%; 3 CIN2 and 8 CIN3). The distribution of BAG3 expression correlated perfectly with the grade of dysplasia (P=0.0); a moderate/strong expression of BAG3 was significantly associated with H-SIL (P<0.0001), with no significant difference between CIN2 and CIN3 (P=0.1228). In CIN/SIL, both distribution and intensity of BAG3 expression correlate directly with the grade of dysplasia, supporting the involvement of BAG3 in all phases of cervical carcinogenesis and its possible diagnostic and prognostic role in cervical premalignant lesions.

Role of Bcl2-associated Athanogene 3 in Turnover of Gap Junction Protein, Connexin 43, in Neonatal Cardiomyocytes

Any pathological stress that impairs expression, turnover and phosphorylation of connexin 43 (Cx43), one of the major proteins of gap junctions, can adversely impact myocardial cell behavior, thus leading to the development of cardiac arrhythmias and heart failure. Our results in primary neonatal rat ventricular cardiomyocytes (NRVCs) show that impairment of the autophagy-lysosome pathway dysregulates degradation of Cx43, either by inhibiting lysosomal activity or suppressing the level of Bcl2-associated athanogene 3 (BAG3), a stress-induced pleiotropic protein that is involved in protein quality control (PQC) via the autophagy pathway. Inhibition of lysosomal activity leads to the accumulation of Cx43 aggregates and suppression of BAG3 significantly diminished turnover of Cx43. In addition, knock-down of BAG3 reduced the levels of Cx43 by dysregulating Cx43 protein stability. Under stress conditions, expression of BAG3 affected the state of Cx43 phosphorylation and its degradation. Furthermore, we found that BAG3 co-localized with the cytoskeleton protein, α-Tubulin, and depolymerization of α-Tubulin led to the intracellular accumulation of Cx43. These observations ascribe a novel function for BAG3 that involves control of Cx43 turnover under normal and stress conditions and potentially for optimizing communication of cardiac muscle cells through gap junctions.

FAM111B, a direct target of p53, promotes the malignant process of lung adenocarcinoma.

Purpose: Lung adenocarcinoma (LUAD) is a main subtype of lung cancer, which is the leading cause of cancer-related deaths. The five-year survival rates of lung cancer patients are still comparatively low. Therefore, potential therapeutic targets are urgently needed to improve the survival of lung cancer patients. In this study, we identified FAM111B as an oncogene and potential therapeutic target for LUAD.Methods: The TCGA database and tissue microarray analysis were used to compare the expression of FAM111B in tumor tissue and normal tissues and evaluate the relationship between FAM111B expression and clinical survival. FAM111B was knocked down and overexpressed to observe whether FAM111B could affect the proliferation, migration, cell cycle, and apoptosis of LUAD cells in vivo and in vitro.Results: FAM111B was highly expressed in tumor tissues compared with normal tissues (P<0.01). LUAD patients with hyper-expression of FAM111B had a lower recurrence-free survival (P<0.01) and shorter overall survival (P<0.01). Knocking down FAM111B inhibited cell proliferation, migration and invasion in vitro and tumor growth in vivo. Silencing FAM111B could arrest LUAD cells at G2/M phase and increase apoptosis. Overexpression of FAM111B promoted the growth of lung cancer cells. FAM111B was identified as a direct target of p53 in existing researches by chip-seq analysis. Bioinformatics analysis predicted that FAM111B could directly bind to BAG3 (BCL2 associated athanogene 3). When FAM111B was down-regulated, both expression of BAG 3 and BCL2 were significantly reduced, whereas decreasing the expression of BAG3 had no effect on FAM111B.Conclusions: Our study indicated that FAM111B might be an oncogene and potential therapeutic target in LUAD which could be involved in the regulation of tumor cells by p53 signaling pathway and play an important role in the process of cell cycle and apoptosis by influencing the expression of BAG3 and BCL2.

Bcl-2-associated athanogene 3(BAG3) is associated with tumor cell proliferation, migration, invasion and chemoresistance in colorectal cancer

BackgroundCRC is one of the most common malignancies worldwide, and its molecular mechanisms remain unclear. Elevated levels of BAG3 have been reported in various tumors. The present study aimed to explore the expression and function of BAG3 in CRC.MethodsBAG3 protein expression was evaluated in 90 CRC specimens using immunohistochemistry in tissue microarrays, and the correlation between BAG3 expression and the clinicopathological features were assessed. In HCT116 cells BAG3 overexpression cell models were constructed, and CRISPR/Cas9 was used for BAG3 knockout. Western blotting and quantitative real-time PCR were used to determine BAG3 expression in HCT-116 Cells. Cell proliferation, migration and invasion were analyzed by cell counting, colony formation assay, EdU cell proliferation assay, RTCA growth curve assays, wound-healing migration assay and transwell invasion assay. The influence of BAG3 expression level on chemoresistance in HCT-116 cells was examined. Gene expression microarray and IPA analyses were employed to explore signaling pathways associated with the control of BAG3.ResultsUsing immunohistochemistry, this study found that BAG3 was markedly upregulated in colorectal cancer tissues and that BAG3 levels were significantly associated with tumor size and gender. BAG3 overexpression promoted HCT-116 cell growth, migration and invasion in vitro. In contrast, BAG3 knockout inhibited HCT-116 cell growth, migration and invasion. HCT-116 cells with high expression of BAG3 had higher cell viability and lower apoptosis rate than control cells after treatment with 5-FU, while the BAG3 knockout group demonstrated the opposite effects. So BAG3 expression level was associated with chemoresistance to 5-FU in HCT-116 cells. Gene expression microarrays and bioinformatics analyses of HCT-116 cells with BAG3 knockout demonstrated the involvement of BAG3 in signaling pathways associated with the control of cell proliferation, migration, invasion and chemoresistance in CRC.ConclusionsIn conclusion, this study provided evidence that BAG3 has a relevant role in CRC biology, and defined potential molecular pathways and networks. So BAG3 may be considered as a potential therapeutic target for anti-tumor therapy in colorectal cancer.

BAG3 promotes tumour cell proliferation by regulating EGFR signal transduction pathways in triple negative breast cancer

Triple-negative breast cancer (TNBC), is a heterogeneous disease characterised by absence of expression of the estrogen receptor (ER), progesterone receptor (PR) and lack of amplification of human epidermal growth factor receptor 2 (HER2). TNBC patients can exhibit poor prognosis and high recurrence stages despite early response to chemotherapy treatment. In this study, we identified a pro-survival signalling protein BCL2- associated athanogene 3 (BAG3) to be highly expressed in a subset of TNBC cell lines and tumour tissues. High mRNA expression of BAG3 in TNBC patient cohorts significantly associated with a lower recurrence free survival. The epidermal growth factor receptor (EGFR) is amplified in TNBC and EGFR signalling dynamics impinge on cancer cell survival and disease recurrence. We found a correlation between BAG3 and EGFR expression in TNBC cell lines and determined that BAG3 can regulate tumour cell proliferation, migration and invasion in EGFR expressing TNBC cells lines. We identified an interaction between BAG3 and components of the EGFR signalling networks using mass spectrometry. Furthermore, BAG3 contributed to regulation of proliferation in TNBC cell lines by reducing the activation of components of the PI3K/AKT and FAK/Src signalling subnetworks. Finally, we found that combined targeting of BAG3 and EGFR was more effective than inhibition of EGFR with Cetuximab alone in TNBC cell lines. This study demonstrates a role for BAG3 in regulation of distinct EGFR modules and highlights the potential of BAG3 as a therapeutic target in TNBC.

The role of heat shock proteins and co-chaperones in heart failure.

The ongoing contractile and metabolic demands of the heart require a tight control over protein quality control, including the maintenance of protein folding, turnover and synthesis. In heart disease, increases in mechanical and oxidative stresses, post-translational modifications (e.g., phosphorylation), for example, decrease protein stability to favour misfolding in myocardial infarction, heart failure or ageing. These misfolded proteins are toxic to cardiomyocytes, directly contributing to the common accumulation found in human heart failure. One of the critical class of proteins involved in protecting the heart against these threats are molecular chaperones, including the heat shock protein70 (HSP70), HSP90 and co-chaperones CHIP (carboxy terminus of Hsp70-interacting protein, encoded by the Stub1 gene) and BAG-3 (BCL2-associated athanogene 3). Here, we review their emerging roles in the maintenance of cardiomyocytes in human and experimental models of heart failure, including their roles in facilitating the removal of misfolded and degraded proteins, inhibiting apoptosis and maintaining the structural integrity of the sarcomere and regulation of nuclear receptors. Furthermore, we discuss emerging evidence of increased expression of extracellular HSP70, HSP90 and BAG-3 in heart failure, with complementary independent roles from intracellular functions with important therapeutic and diagnostic considerations. While our understanding of these major HSPs in heart failure is incomplete, there is a clear potential role for therapeutic modulation of HSPs in heart failure with important contextual considerations to counteract the imbalance of protein damage and endogenous protein quality control systems.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'.

Silencing of BAG3 inhibits the epithelial-mesenchymal transition in human cervical cancer.

Bcl2-associated athanogene 3 (BAG3) has been reported to be involved in aggressive progression of many tumors. In the present study, we examined the expression of BAG3 in human cervical cancer (CC) tissues and investigated the role of BAG3 in SiHa and HeLa cell growth, migration, and invasion. Here, we found that most of CC tissues highly expressed the protein and mRNA of BAG3, while their expression was obviously lower in paired normal tissues (all p<0.001). BAG3 expression was associated with FIGO stage and metastasis (all p<0.05). In-vitro analysis demonstrated that BAG3 siRNAs inhibited SiHa and HeLa cell growth, invasion and migration. Mechanically, BAG3 siRNAs inhibited the expression of EMT-regulating markers, involving MMP2, Slug and N-cadherin, and increased the expression of E-cadherin. In a xenograft nude model, BAG3 siRNAs inhibited tumor growth and the expression of EMT biomarkers. In conclusion, BAG3 is involved in the EMT process, including cell growth, invasion and migration in the development of CC. Thus, BAG3 target might be recommended as a novel therapeutic approach.

Expression of Anti-apoptotic Protein BAG3 in Human Sebaceous Gland Carcinoma of the Eyelid.

Bcl-2-associated athanogene 3 (BAG3), a co-chaperone of heat shock protein 70 (HSP70), has been shown to play a role in anti-apoptosis of various malignant tumors. In this study, the expression of BAG3 was examined in human sebaceous gland carcinoma of the eyelid. The expression of BAG3 was evaluated by immunohistochemistry of surgical samples from 5 patients with sebaceous gland carcinoma in the eyelid. BAG3 was positive diffusely in the cytoplasm in all patients. The average positive rate of BAG3 was 73.0±26.0% in tumor cells of all patients. BAG3 was highly expressed in sebaceous gland carcinoma of the eyelid. BAG3 may play an important role in the pathogenesis and progression of sebaceous gland carcinoma of the eyelid.