Targeting the crosstalk between estrogen receptors and membrane growth factor receptors in breast cancer treatment: Advances and opportunities.

Estrogens play a critical role in the initiation and progression of breast cancer. Estrogen receptor (ER)α, ERß, and G protein-coupled estrogen receptor are the primary receptors for estrogen in breast cancer. These receptors are mainly activated by binding with estrogens. The crosstalk between ERs and membrane growth factor receptors creates additional pathways that amplify the effects of their ligands and promote tumor growth. This crosstalk may cause endocrine therapy resistance in ERα-positive breast cancer. Furthermore, this may explain the resistance to anti-human epidermal growth factor receptor-2 (HER2) treatment in ERα-/HER2-positive breast cancer and chemotherapy resistance in triple-negative breast cancer. Accordingly, it is necessary to understand the complex crosstalk between ERs and growth factor receptors. In this review, we delineate the crosstalk between ERs and membrane growth factor receptors in breast cancer. Moreover, this review highlights the current progress in clinical treatment and discusses how pharmaceuticals target the crosstalk. Lastly, we discuss the current challenges and propose potential solutions regarding the implications of targeting crosstalk via pharmacological inhibition. Overall, the present review provides a landscape of the crosstalk between ERs and membrane growth factor receptors in breast cancer, along with valuable insights for future studies and clinical treatments using a chemotherapy-sparing regimen to improve patient quality of life.

Current progress and prospects for G protein-coupled estrogen receptor in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a biologically and clinically heterogeneous disease. The G protein-coupled estrogen receptor (GPER) plays a crucial role in mediating the effect of estrogen and estrogen-like compounds in TNBC cells. Compared with other subtypes, GPER has a higher expression in TNBC. The GPER mechanisms have been thoroughly characterized and analyzed in estrogen receptor α (ERα) positive breast cancer, but not in TNBC. Our previous work revealed that a higher expression of GPER mRNA indicates a better prognosis for ERα-positive breast cancer; however, its effects in TNBC differ. Whether GPER could serve as a predictive prognostic marker or therapeutic target for TNBC remains unclear. In this review, we provide a detailed introduction to the subcellular localization of GPER, the different effects of various ligands, and the interactions between GPER and closely associated factors in TNBC. We focused on the internal molecular mechanisms specific to TNBC and thoroughly explored the role of GPER in promoting tumor development. We also discussed the interaction of GPER with specific cytokines and chemokines, and the relationship between GPER and immune evasion. Additionally, we discussed the feasibility of using GPER as a therapeutic target in the context of existing studies. This comprehensive review highlights the effects of GPER on TNBC, providing a framework and directions for future research.

Endoscopic-Assisted Forehead Augmentation with Polyetheretherketone (PEEK) Patient-Specific Implant (PSI) for Aesthetic Considerations.

BACKGROUND: Forehead augmentation have become popular aesthetic procedures among Asians in recent years. However, the use of polyetheretherketone (PEEK) patient-specific implant (PSI) in the facial contouring surgery for aesthetic considerations is not well documented in the existing studies. The purpose of this study was to develop a novel method for forehead augmentation and assess the clinical outcomes and complications in patients who underwent forehead augmentation with PEEK PSI assisted by endoscopy. METHODS: The PEEK PSIs were fabricated using the virtual surgical planning (VSP) and the computer-aided manufacturing (CAM) for each patient, preoperatively. The implant pockets were dissected in the subperiosteal plane, and PEEK PSIs were placed in their designed position and fixed assisting by endoscopy via small incision within the hairline. All patients were asked to complete the FACE-Q questionnaire before and 6 months after the operation. Pre- and postoperative demographics, photographs, and other clinical data of patients were collected and analyzed. RESULTS: 11 patients underwent forehead augmentation were enrolled in this study. All procedures were completed successfully with the help of endoscope. The average patient age was 30.63 ± 2.54 years. The mean thickness and size of PEEK PSI were 4.44 ± 1.77 mm and 38.43 ± 22.66 cm2, respectively. The mean operative time was 83.00 ± 29.44 min, and the mean postoperative follow-up period was 11.00 ± 6.50 months. No implant exposure, extrusion or removal were reported. The FACE-Q scores of patients in satisfaction with the forehead increased from 47.64 ± 7.15 to 78.81 ± 6.35. CONCLUSIONS: PEEK PSIs can be prefabricated to achieve accurate remodeling of the frontal contour with good esthetic outcomes. The endoscope provides direct and magnified vision, which allow easy access to the supraorbital rim and lateral edge of the eyebrow arch and confirming the position of the implants without damaging nerves and vessels. Endoscopic-assisted forehead augmentation with PEEK PSI is safe and effective. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these evidence-based medicine ratings, please refer to the Table of contents or the online Instructions to Authors www.springer.com/00266 .

Divergent features of ERß isoforms in triple negative breast cancer: progress and implications for further research.

Estrogen receptor ß (ERß) was discovered more than 20 years ago. However, the extent and role of ERß expression in breast cancer remain controversial, especially in the context of triple-negative breast cancer (TNBC). ERß exists as multiple isoforms, and a series of studies has revealed an inconsistent role of ERß isoforms in TNBC. Our recent results demonstrated contrasting functions of ERß1 and ERß2/ß5 in TNBC. Additional research should be conducted to explore the functions of individual ERß isoforms and develop targeted drugs according to the relevant mechanisms. Consequently, a systematic review of ERß isoforms is necessary. In this review, we overview the structure of ERß isoforms and detail what is known about the function of ERß isoforms in normal mammary tissue and breast cancer. Moreover, this review highlights the divergent features of ERß isoforms in TNBC. This review also provides insights into the implications of targeting ERß isoforms for clinical treatment. In conclusion, this review provides a framework delineating the roles and mechanisms of different ERß isoforms in TNBC and sheds light on future directions for basic and clinical research.

Progress and prospects in research and clinical practice of hormone receptor-positive, HER-2-negative breast cancer with BRCA1/2 mutations.

Breast cancer (BC) is a heterogeneous disease that is the most common cancer in women worldwide. However, precise subtyping and corresponding treatments have improved patient outcomes. Hormone receptor (HR)-positive, human epidermal growth factor receptor type 2 (HER2)-negative (HR+/HER2-) BC with BRCA1 and/or BRCA2 mutations (BRCA1/2m) is a unique BC subset with dual drivers: homologous recombination deficiency and hormone receptor signaling. Wild-type BRCA1/2 suppresses estrogen receptor-mediated signaling. Loss-of-function mutations in BRCA1/2 release estrogen receptor suppression, leading to reduced sensitivity to endocrine therapy. Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) exert antitumor effects against this subtype and can be used in combination with endocrine therapy. Although PARPis have been evaluated in metastatic triple-negative breast cancer, their efficacy against HR+/HER2- BC has not been clearly established. The present review summarizes recent advances and prospects in the progress of the HR+/HER2-/BRCA1/2m subgroup. As such, this article provides theoretical guidance for future research and promotes the use of PARPis for the treatment of HR+/HER2-/BRCA1/2m BC.

Efficacy and safety of endocrine therapy after mastectomy in patients with hormone receptor positive breast ductal carcinoma in situ: Retrospective cohort study.

Background: More than half of Chinese patients with hormone receptor positive (HR+) ductal carcinoma in situ (DCIS) are treated with mastectomy, and usually subjected to postoperative endocrine therapy (ET). Given that long-term ET can cause severe adverse effects it is important to determine the beneficial effect and safety of post-mastectomy ET on the disease-free survival (DFS) and adverse events in patients with HR+ DCIS. Methods: To explore beneficial effect and safety of post-mastectomy ET in patients with HR+ DCIS, we performed a multicenter, population-based study. This retrospective study analyzed the DFS and adverse events in 1037 HR+ DCIS Chinese patients with or without post-mastectomy ET from eight breast centers between 2006 and 2016. The median follow-up time period was 86 months. Results: There were 791 DCIS patients receiving ET (ET group). Those patients were followed up for a median of 86 months (range, 60-177 months). There were 23 cases with tumor recurrence or distant metastasis. There were similar 5-year DFS rates and DFS between the ET and non-ET groups, even for those with high-risk factors. Conversely, 37.04% of patients suffered from adverse events after ET, which were significantly higher than those in the non-ET group. Conclusions: ET after mastectomy did not benefit patients with HR+ DCIS for their DFS, rather increased adverse events in those patients. Therefore, ET after mastectomy may not be recommended for patients with HR+ DCIS, even for those with high-risk factors, such as multifocal, microinvasive, and higher T stage. Funding: This study was supported by grants from Outstanding Scientific Fund of Shengjing Hospital (201803) and Outstanding Young Scholars of Liaoning Province (2019-YQ-10).

Cytoplasmic G Protein-Coupled Estrogen Receptor 1 as a Prognostic Indicator of Breast Cancer: A Meta-Analysis.

Purpose: To determine whether G protein-coupled estrogen receptor 1 (GPER1) is a suitable biomarker to predict the treatment outcome of breast cancer (BC). Methods: A meta-analysis of the literature was performed to clarify the correlation between GPER1 protein expression and BC outcome. The relationship between GPER1 mRNA expression and survival was analyzed using Breast Cancer Gene-Expression Miner (bc-GenExMiner) v4.6 software. Results: Six studies involving 2697 patients were included in the meta-analysis. Four studies reported the correlation between GPER1 protein expression and relapse-free survival (RFS) and 4 others reported the impact of GPER1 protein expression on overall survival (OS). The results showed that high GPER1 protein expression was not associated with RFS (hazard ratio [HR] = 1.58; 95% confidence interval [CI] = 0.71-3.48; P = .26) or OS (HR = 1.18; 95% CI = 0.64-2.18; P = .60). Subgroup analysis suggested that nuclear expression of GPER1 was not associated with OS (HR = 0.91; 95% CI = 0.77-1.08; P = .30), but high expression of cytoplasmic GPER1 was significantly associated with longer OS (HR = 0.69; 95% CI = 0.55-0.86; P = .001). Furthermore, the association of GPER1 mRNA and OS of BC patients was analyzed using bc-GenExMiner v4.6. Two data sets involving 4016 patients were included in the analysis. The targeted prognostic analysis results showed that high mRNA expression of GPER1 was predictive of better OS in BC patients (HR = 0.71; 95% CI = 0.59-0.86; P = .0005), which was remarkably similar to the result of cytoplasmic GPER1. Further subgroup analysis demonstrated that high mRNA expression of GPER1 was predictive of better OS in estrogen receptor (ER)-positive, but not ER-negative or triple-negative BC patients. Conclusions: High mRNA and cytoplasmic protein expression of GPER1 were predictive of better OS of BC patients.

Estrogen Receptor Beta 1: A Potential Therapeutic Target for Female Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor alpha, progesterone receptor, and HER2. These receptors often serve as targets in breast cancer treatment. As a result, TNBCs are difficult to treat and have a high propensity to metastasize to distant organs. For these reasons, TNBCs are responsible for over 50% of all breast cancer mortalities while only accounting for 15% to 20% of breast cancer cases. However, estrogen receptor beta 1 (ERß1), an isoform of the ESR2 gene, has emerged as a potential therapeutic target in the treatment of TNBCs. Using an in vivo xenograft preclinical mouse model with human TNBC, we found that expression of ERß1 significantly reduced both primary tumor growth and metastasis. Moreover, TNBCs with elevated levels of ERß1 showed reduction in epithelial to mesenchymal transition markers and breast cancer stem cell markers, and increases in the expression of genes associated with inhibition of cancer cell invasiveness and metastasis, suggesting possible mechanisms underlying the antitumor activity of ERß1. Gene expression analysis by quantitative polymerase chain reaction and RNA-seq revealed that treatment with chloroindazole, an ERß-selective agonist ligand, often enhanced the suppressive activity of ERß1 in TNBCs in vivo or in TNBC cells in culture, suggesting the potential utility of ERß1 and ERß ligand in improving TNBC treatment. The findings enable understanding of the mechanisms by which ERß1 impedes TNBC growth, invasiveness, and metastasis and consideration of ways by which treatments involving ERß might improve TNBC patient outcome.

Dual-mechanism estrogen receptor inhibitors.

Efforts to improve estrogen receptor-α (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity.

Contrasting activities of estrogen receptor beta isoforms in triple negative breast cancer.

PURPOSE: Triple negative breast cancer (TNBC), an aggressive subtype of breast cancer, lacks the three major receptors for predicting outcome or targeting therapy. Hence, our aim was to evaluate the potential of estrogen receptor beta (ERß) as a possible endocrine therapy target in TNBC. METHODS: The expression and prognostic effect of ERß isoforms were analyzed using TCGA breast tumor data, and the expression of ERß isoform mRNA and protein in TNBC cell lines was assayed. Endogenous ERß2 and ERß5 were knocked down with siRNA, and ERß2, ERß5, and ERß1 were upregulated using a doxycycline-inducible lentiviral system. Cell proliferation, migration and invasion, and specific gene expressions were evaluated. RESULTS: ERß2 and ERß5 were the predominant endogenous forms of ERß in TNBC tumors and cell lines. High ERß2 predicted worse clinical outcome. Knockdown of endogenous ERß2/ERß5 in cell lines suppressed proliferation, migration and invasion, and downregulated proto-oncogene survivin expression. ERß2/ERß5 upregulation did the reverse, increasing survivin and these cell activities. ERß1 was barely detectable in TNBC cell lines, but its upregulation reduced survivin, increased tumor suppressor expression (E-cadherin and cystatins), and suppressed proliferation, migration and invasion in both ligand-independent and dependent manners, suggesting the possible translational benefit of ERß ligands. CONCLUSIONS: ERß2/ERß5 and ERß1 exhibit sharply contrasting activities in TNBC cells. Our findings imply that delineating the absolute amounts and relative ratios of the different ERß isoforms might have prognostic and therapeutic relevance, and could enable better selection of optimal approaches for treatment of this often aggressive form of breast cancer.

Association of two polymorphisms Asp299Gly and Thr399Ile in Toll-like receptor 4 with rheumatoid arthritis: A meta-analysis.

Our meta-analysis aims to evaluate the association of Asp299Gly and Thr399Ile with rheumatoid arthritis (RA) susceptibility and severity. By manually searching 3 electronic databases (PubMed, Embase and Web of Science), relevant articles were collected. After checking eligibility for every study, this meta-analysis on eligible studies was performed under 5 genetic models: (1) allelic contrast; (2) heterozygous model; (3) homozygous model; (4) dominant model; (5) recessive model. In Spanish populations, a significantly decreased RA risk was identified in allelic comparison (odds ratio [OR] = 0.73, 95% CI 0.55 ~ 0.96) and dominant model (OR = 0.74, 95% CI 0.56 ~ 0.99) of Asp299Gly polymorphism. A trend of reduced risk was also observed under the heterozygous model (OR = 0.77, 95% CI 0.58 ~ 1.03). As for Thr399Ile, it might also have a protective effect on Spanish populations in allelic comparison (OR = 0.71, 95% CI 0.44 ~ 1.15). In contrast, for both Asp299Gly and Thr399Ile, a higher risk of RA was detected in Chinese Han populations. The frequency of both Asp299Gly and Thr399Ile increased in rheumatoid factor (RF)-positive subjects in Chinese patients (Asp299Gly, RF+:RF- = 0.165:0.145; Thr399Ile, RF+:RF- = 0.170:0.161) and decreased in Spanish patients (Asp299Gly, RF+:RF- = 0.060:0.073; Thr399Ile, RF+:RF- = 0.046:0.056), but not to a statistically significant extent. Our meta-analysis suggested that both Asp299Gly and Thr399Ile might have a protective effect on Spanish populations, but the 2 polymorphisms could act as a susceptible factor in Chinese Han populations. To confirm our results, further investigation concerning the functional impacts of Asp299Gly and Thr399Ile are still needed.

The predictive ability of plasma ESR1 mutations for the efficacy of endocrine therapy in hormone-receptor-positive advanced breast cancer.

PURPOSE: The predictive ability of plasma ESR1 mutations for outcomes among patients with advanced breast cancer undergoing endocrine therapy (ET) remains disputable. We performed a comprehensive meta-analysis of published studies to clarify the impact of plasma ESR1 mutations on clinical outcomes for patients after subsequent ET. MATERIALS AND METHODS: An electronic search was performed to identify eligible studies. Studies analyzing progression-free survival (PFS) and/or overall survival (OS) according to plasma ESR1 mutation status after subsequent ET were included. HRs were calculated using a fixed- or random-effects model according to heterogeneity. Pooled HRs and 95% CIs were used to estimate the effects. RESULTS: Six studies including 705 patients with advanced breast cancer met the inclusion criteria. The impact of plasma ESR1 mutations on PFS and OS after subsequent ET was reported in six studies (seven groups) and two studies, respectively. Meta-analysis results showed that the pooled HR for ESR1 mutations was 1.70 (95% CI, 1.05-2.74; P=0.03) for OS, which was statistically significant for predicting poor survival, and 1.56 (95% CI, 1.13-2.14; P=0.006) for PFS; however, Begg's and Egger's test results identified the presence of bias. The trim-and-fill method was used, and after incorporation of the imputed studies, the HR was 1.16 (95% CI, 0.88-1.53, P=0.30) for PFS, which indicates that plasma ESR1 mutation had no effect on PFS after subsequent ET. Subgroup analysis suggested that plasma ESR1 mutations were correlated with shorter PFS (HR, 1.98; 95% CI, 1.12-3.51; P=0.02) in patients subsequently treated with aromatase inhibitors (AIs), whereas no association with PFS was observed for patients subsequently treated with non-AI ET (HR, 1.08; 95% CI, 0.85-1.38; P=0.54) or fulvestrant (HR, 1.03; 95% CI, 0.79-1.34; P=0.83). CONCLUSION: The current meta-analysis demonstrates that plasma ESR1 mutation status is not a predictor of ET efficacy for all drugs without distinction in patients with hormone-receptor-positive advanced breast cancer. ESR1 mutation predicted a poor response to AIs, whereas it was not predictive of non-AI ET efficacy, especially for fulvestrant.

Prognostic significance of c-Met in breast cancer: a meta-analysis of 6010 cases.

BACKGROUND: The prognostic value of c-Met in breast cancer remains controversial. A meta-analysis of the impact of c-Met in breast cancer was performed by searching published data. METHODS: Published studies analyzing overall survival (OS) or relapse free survival (RFS) according to c-Met expression were searched. The principal outcome measures were hazard ratios (HRs) for RFS or OS according to c-Met expression. Combined HRs were calculated using fixed- or random- effects models according to the heterogeneity. RESULTS: Twenty-one studies involving 6,010 patients met our selection criteria. The impact of c-Met on RFS and OS was investigated in 12 and 17 studies, respectively. The meta-analysis results showed that c-Met overexpression significantly predicted poor RFS and OS in unselected breast cancer. Subgroup analysis indicated that c-Met overexpression was correlated with poor RFS and OS in Western patients, but was not associated with RFS or OS in Asian patients. C-Met was associated with poor OS in lymph node negative breast cancer and with poor RFS in hormone-receptor positive and triple negative breast cancer, but was not associated with prognosis in human epidermal growth factor receptor (HER)-2 positive breast cancer. CONCLUSIONS: C-Met overexpression is an adverse prognostic marker in breast cancer, except among Asian and HER-2 positive patients. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1869780799156041.

Tamoxifen with ovarian function suppression versus tamoxifen alone as an adjuvant treatment for premenopausal breast cancer: a meta-analysis of published randomized controlled trials.

BACKGROUND: Ovarian function suppression (OFS) significantly downregulates the concentration of plasma estrogens. However, it is unclear whether it offers any survival benefits if combined with adjuvant tamoxifen treatment in premenopausal women. This meta-analysis was designed to assess data from previous studies involving adjuvant tamoxifen treatment plus OFS in premenopausal breast cancer. METHODS: Electronic literature databases (PubMed, Embase, the Web of Science, and the Cochrane Library) were searched for relevant randomized controlled trials published prior to February 1, 2015. Only randomized controlled trials that compared tamoxifen alone with tamoxifen plus OFS for premenopausal women with breast cancer were selected. The evaluated endpoints were disease-free survival and overall survival. RESULTS: Four randomized controlled trials comprising 6,279 patients (OFS combination, n=3,133; tamoxifen alone, n=3,146) were included in the meta-analysis. There was no significant improvement in disease-free survival or overall survival with addition of OFS in either the whole population or the hormone receptor-positive subgroup. The risk of distant recurrence was not reduced with the addition of OFS in the whole population. A subgroup analysis showed that addition of OFS significantly improved overall survival in patients who were administered chemotherapy. CONCLUSION: Based on the available studies, concurrent administration of OFS and adjuvant tamoxifen treatment for premenopausal women with breast cancer has no effect on prolonging disease-free survival and overall survival, excluding patients who were administered chemotherapy. It should not be widely recommended, except perhaps for women who were hormone-receptor positive and who were also administered adjuvant chemotherapy.

The impact of IGF-1R expression on the outcomes of patients with breast cancer: a meta-analysis.

PURPOSE: The value of insulin-like growth factor 1 receptor (IGF-1R) for predicting survival of patients with breast cancer remains controversial. The purpose of this study was to perform a meta-analysis of the published data to attempt to clarify the impact of IGF-1R. METHODS: Studies published between January 1, 1990 and October 1, 2014 were identified using an electronic search to aggregate the available survival results. Studies were included if they reported detecting IGF-1R expression in the primary breast cancer and analyzed patient survival data according to IGF-1R status. The principal outcome measures were hazard ratios (HRs) for survival of IGF-1R-positive patients. Combined HRs and 95% confidence intervals (CIs) were estimated using fixed- or random-effects models according to between-study heterogeneity. RESULTS: Ten studies, involving 5,406 patients, satisfied our inclusion criteria. Data from five studies provided the impact of IGF-1R on overall survival (OS), three studies the impact on breast cancer-specific survival (BCSS), and seven studies the impact on disease-free survival (DFS). The results of meta-analysis showed that for DFS, membranous IGF-1R positivity was not a significant predictor. The combined HR for OS/BCSS was 0.63 (95% CI: 0.42-0.95, P=0.03), indicating that membranous IGF-1R positivity was a significant predictor of better survival. IGF-1R cytoplasmic positivity was significantly associated with longer DFS and OS/BCSS (combined HR: 0.56, 95% CI: 0.35-0.89, P=0.01; combined HR: 0.55, 95% CI: 0.35-0.85, P=0.008, respectively). The results of subgroup analysis suggested that membranous IGF-1R positivity in hormone-receptor-positive breast cancer was correlated with favorable DFS (combined HR: 0.61, 95% CI: 0.41-0.92, P=0.02) and OS/BCSS (combined HR: 0.73, 95% CI: 0.57-0.93, P=0.01). Membranous IGF-1R positivity in triple-negative breast cancer predicted worse DFS (combined HR: 1.86, 95% CI: 1.03-3.34, P=0.04). Membranous IGF-1R positivity in Her-2-positive or ER (estrogen receptor)-negative breast cancer was not found to be a significant prognostic indicator. CONCLUSION: The results of this meta-analysis suggest that IGF-1R expression has different prognostic values for patients with breast cancers of different molecular subtypes. It was a favorable prognostic indicator in unselected breast cancers and hormone-receptor-positive cancers, but indicated poor survival in triple-negative breast cancers.

Bufalin enhances TRAIL-induced apoptosis by redistributing death receptors in lipid rafts in breast cancer cells.

Studies have shown that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in cancer cells. However, breast cancer cells are generally resistant to TRAIL. In the present study, we explored the effect of bufalin on TRAIL-induced breast cancer cell apoptosis. The results showed that bufalin enhanced TRAIL-induced apoptosis in MCF-7 and MDA-MB-231 breast cancer cells by activating the extrinsic apoptotic pathway. Bufalin also promoted the clustering of death receptor 4 (DR4) and DR5 in aggregated lipid rafts. The cholesterol-sequestering agent methyl-ß-cyclodextrin reversed the DR4 and DR5 clustering and reduced bufalin+TRAIL-induced apoptosis. Overall, these results indicate that bufalin enhanced TRAIL-induced apoptosis in breast cancer cells by the partial redistribution of DRs in lipid rafts.

Down-regulation of Cbl-b by bufalin results in up-regulation of DR4/DR5 and sensitization of TRAIL-induced apoptosis in breast cancer cells.

PURPOSE: TNF-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic agent that preferentially induces apoptosis in cancer cells. However, breast cancer cells are generally resistant to TRAIL. Bufalin is a major active ingredient of the traditional Chinese medicine ChanSu. The present study aimed to assess the synergistic effect of bufalin and TRAIL and elucidate the underlying mechanisms in breast cancer cells. METHODS: Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. The expression of proteins was assayed by flow cytometry and/or Western blotting. Transfection studies were used to determine the involvement of DR4, DR5 and Cbl-b in the synergistic effect of bufalin and TRAIL. RESULTS: MCF-7 and MDA-MB-231 cells were resistant to TRAIL. Both cell lines were dramatically sensitized to TRAIL-induced apoptosis by bufalin. Further experiments indicated that bufalin up-regulated DR4 and DR5, activated ERK, JNK and p38 MAPK and down-regulated Cbl-b. Blocking the up-regulation of DR4 and DR5 by siRNA rendered cells less sensitive to apoptosis induced by the combination of bufalin and TRAIL. Inhibition of the activation of ERK, JNK and p38 MAPK by specific inhibitors attenuated DR4 and DR5 up-regulation. Moreover, down-regulation of Cbl-b by shRNA led to stronger activation of ERK, JNK and p38 MAPK, more up-regulation of DR4 and DR5, and a stronger synergistic effect of bufalin and TRAIL. CONCLUSIONS: Bufalin enhanced TRAIL-induced apoptosis by up-regulating the expression of DR4 and DR5. Bufalin-induced down-regulation of Cbl-b contributed to the up-regulation of DR4 and DR5, which might be partially mediated by the activation of ERK, JNK and p38 MAPK.

[Expressions of c-Cbl, Cbl-b and EGFR and its role of prognosis in NSCLC].

BACKGROUND AND OBJECTIVE: Epidermal growth factor receptor (EGFR) is closely correlated with the progression of lung cancer. Its activity is modulated by Casitas B-lineage lymphoma (Cbl) family. The aim of this study is to investigate the expression and clinical relevance of c-Cbl, Cbl-b and EGFR in non-small cell lung cancer (NSCLC). METHODS: Expressions of c-Cbl, Cbl-b and EGFR protein were detected with tissue microarrays and immunohistochemistry technique in 94 cases of NSCLC. The correlations between the expression of the three proteins and clinicopathological parameters were analyzed. RESULTS: The positive expression rates of EGFR, c-Cbl and Cbl-b were 60.6% (57/94), 30.9% (29/94) and 84.0% (79/94), respectively. The expression of EGFR, c-Cbl and Cbl-b was not associated with age, pathological type, TNM stage, lymph node metastasis, and smoking history. c-Cbl and Cbl-b status was not significantly correlated with overall survival. Subgroup analyses showed that c-Cbl-positive patients had longer survival than c-Cbl-negative patients in EGFR-positive group (P=0.014). CONCLUSION: Detection of c-Cbl protein levels might contribute to the prognosis evaluation of EGFR-positive NSCLC.

Ubiquitin ligase c-Cbl is involved in tamoxifen-induced apoptosis of MCF-7 cells by downregulating the survival signals.

BACKGROUND: Tamoxifen (TAM) is a nonsteroidal antiestrogen that has been widely used in the treatment of breast cancer through its anti-estrogen activity. Recent studies show that TAM is cytotoxic to both estrogen receptor (ER)-positive and ER-negative cells via the induction of apoptosis. However, the molecular mechanisms of this effect are not well understood. In the present study, we investigated the roles of c-Src, ERK, AKT and c-Cbl ubiquitin ligases during TAM-induced apoptosis of MCF-7 cells. MATERIAL AND METHODS: MCF-7 cell proliferation and apoptosis were measured by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and flow cytometry, respectively. c-Cbl expression, and the activity of c-Src, ERK, AKT were assayed by Western blotting. Overexpression of the wild and the dominant-negative type of c-Cbl (70Z/Cbl) were achieved by transient transfection of plasmids encoding c-Cbl and 70Z/Cbl, respectively, and were confirmed by Western blotting. Statistical analysis was performed using the t-test, and a p-value <0.05 was considered to be statistically significant. RESULTS: A high concentration of TAM (25 µM) induced a time-dependent apoptosis of MCF-7 cells. ERK1/2 and AKT were activated during TAM-induced apoptosis. The ERK1/2 inhibitor PD98059, the PI3K/Akt inhibitor LY294002, and the c-Src inhibitor PP2 all enhanced TAM action. Moreover, the ubiquitin ligase c-Cbl was up-regulated during this process. Over-expression of c-Cbl significantly enhanced the apoptosis-inducing effects of TAM, while 70Z/Cbl suppressed the apoptosis-inducing effects of TAM. Further investigation revealed that, overexpression of c-Cbl significantly downregulated the c-Src protein levels and TAM-induced AKT activity. But 70Z/Cbl significantly upregulated TAM-induced ERK and AKT activity. CONCLUSIONS: This study demonstrates that c-Src, ERK, and AKT played a protective role during TAM-induced apoptosis, and that c-Cbl sensitized MCF-7 cells to TAM by modulating the expression of c-Src, and TAM-induced ERK and AKT activity.