Differential effects of metformin on breast cancer proliferation according to markers of insulin resistance and tumor subtype in a randomized presurgical trial

Treatment of diabetics with metformin is associated with decreased breast cancer risk in observational studies, but it remains unclear if this drug has clinical antineoplastic activity. In a recent presurgical trial, we found a heterogeneous effect of metformin on breast cancer proliferation (ki-67) depending upon insulin resistance (HOMA index). Here, we determined the associations of additional serum biomarkers of insulin resistance, tumor subtype, and drug concentration with ki-67 response to metformin. Two-hundred non-diabetic women were randomly allocated to metformin (850 mg/bid) or placebo for 4 weeks prior to breast cancer surgery. The ki-67 response to metformin was assessed comparing data obtained from baseline biopsy (ki-67 and tumor subtype) and serum markers (HOMA index, C-peptide, IGF-I, IGFBP-1, IGFBP-3, free IGF-I, hs-CRP, adiponectin) with the same measurements at definitive surgery. For patients with a blood sample taken within 24 h from last drug intake, metformin level was measured. Compared with placebo, metformin significantly decreased ki-67 in women with HOMA > 2.8, those in the lowest IGFBP-1 quintile, those in the highest IGFBP-3 quartile, those with low free IGF-I, those in the top hs-CRP tertile, and those with HER2-positive tumors. In women with HOMA index > 2.8, drug levels were positively correlated with the ki-67 decrease, whereas no trend was noted in women with HOMA < 2.8 (p-interaction = 0.07). At conventional antidiabetic doses, the effect of metformin on tumor ki-67 of non-diabetic breast cancer patients varies with host and tumor characteristics. These findings are relevant to design breast cancer prevention and treatment trials with metformin.     

二甲双胍在乳腺癌中抗肿瘤作用研究进展

临床前研究表明降糖药二甲双胍对乳腺癌有抗肿瘤作用,能降低糖尿病患者乳腺癌发病风险和死亡风险,也能提高乳腺癌患者新辅助化疗后病理完全缓解率。体内外实验表明二甲双胍有效抑制各种乳腺癌细胞和移植瘤,并和化疗药物、HER2靶向药物、新型抗肿瘤药物有良好协同作用。主要分子机制包括全身性下调胰岛素及相关信号通路、肿瘤细胞内激活LKBl／AMPK、抑制下游mTOR通路等。目前各国开展了多个临床试验评估--sp双胍在乳腺癌防治中的应用价值。     

Metformin: Taking away the candy for cancer?

Metformin is widely used in the treatment of diabetes mellitus type 2 where it reduces insulin resistance and diabetes-related morbidity and mortality. Population-based studies show that metformin treatment is associated with a dose-dependent reduction in cancer risk. The metformin treatment also increases complete pathological tumour response rates following neoadjuvant chemotherapy for breast cancer, suggesting a potential role as an anti-cancer drug. Diabetes mellitus type 2 is associated with insulin resistance, elevated insulin levels and an increased risk of cancer and cancer-related mortality. This increased risk may be explained by activation of the insulin- and insulin-like growth factor (IGF) signalling pathways and increased signalling through the oestrogen receptor. Reversal of these processes through reduction of insulin resistance by the oral anti-diabetic drug metformin is an attractive anti-cancer strategy. Metformin is an activator of AMP-activated protein kinase (AMPK) which inhibits protein synthesis and gluconeogenesis during cellular stress. The main downstream effect of AMPK activation is the inhibition of mammalian target of rapamycin (mTOR), a downstream effector of growth factor signalling. mTOR is frequently activated in malignant cells and is associated with resistance to anticancer drugs. Furthermore, metformin can induce cell cycle arrest and apoptosis and can reduce growth factor signalling. This review discusses the role of diabetes mellitus type 2 and insulin resistance in carcinogenesis, the preclinical rationale and potential mechanisms of metformin’s anti-cancer effect and the current and future clinical developments of metformin as a novel anti-cancer drug.     

Glioma Stem Cells: Markers,Hallmarks and Therapeutic Targeting by Metformin

Malignant gliomas are among the deadliest primary brain tumors. Despite multimodal therapy and advances in chemotherapy, imaging, surgical and radiation techniques, these tumors remain virtually incurable. Glioma stem cells may be responsible for resistance to traditional therapies and tumor recurrence. Therefore, elimination of glioma stem cells may be crucial for achieving therapeutic efficacy. Metformin, a small molecule drug widely used in the therapy of type 2 diabetes, has shown significant anti-tumor effects in patients with breast cancer and prostate cancer. Recent preclinical data suggest that metformin also has therapeutic effects against glioma. Here we review the markers and hallmarks of glioma stem cells, and the molecular mechanisms involved in therapeutic targeting of glioma stem cells by metformin.     

Targeting metabolism in breast cancer: How far we can go?

Adjuvant therapies for breast cancer have achieved great success in recent years and early breast cancer is now a curable or chronic disease. Targeted therapies, including endocrine therapy and human epidermal growth factor receptor-2 targeted therapy, marked a new era of breast cancer treatment. However, except for chemotherapy, an efficient drug treatment to improve the overall survival of breast cancer patients is still lacking for triple negative breast cancer. Furthermore, a certain proportion of breast cancer patients present with resistance to drug therapy, making it much more difficult to control the deterioration of the disease. Recently, altered energy metabolism has become one of the hallmarks of cancer, including breast cancer, and it may be linked to drug resistance. Targeting cellular metabolism is becoming a promising strategy to overcome drug resistance in cancer therapy. This review discusses metabolic reprogramming in breast cancer and the possible complex mechanism of modulation. We also summarize the recent advances in metabolic therapy targeted glycolysis, glutaminolysis and fatty acids synthesis in breast cancer.     

Metformin suppresses breast cancer growth via inhibition of cyclooxygenase-2

Pre-clinical and on-going trials have indicated the advantage of using metformin as an anticancer drug alone or in combination with other chemotherapeutics for the treatment of patients with breast cancer. However, the mechanisms by which metformin attenuates tumorigenesis remain to be further elucidated. The present study investigated the anticancer effects of metformin in breast cancer and identified potential molecular targets of metformin using western blotting and immunohistochemical analysis. Metformin significantly decreased tumor cell proliferation in vitro and suppressed tumor growth in vivo. Moreover, it induced the activation of AMP-induced protein kinase and suppression of phosphorylated-eukaryotic translation initiation factor 4E-binding protein 1 (p-4E-BP1), a downstream effector of the mTOR signaling pathway, and decreased cyclin D1 levels in in vitro and in vivo experimental models. Additionally, metformin inhibited cyclooxygenase (COX)-2 expression. Clinically, high expression levels of COX-2 and p-4E-BP1 in tissues of patients with breast cancer were significantly associated with enhanced lymphatic metastasis and distant metastasis. Thus, the current data suggested that metformin may have potential value as a synergistic therapy targeting both the COX-2 and mTOR signaling pathways.     

选择性雌激素受体下调剂氟维司群耐药机制的研究进展

氟维司群（fulvestrant）是一种选择性雌激素受体下调剂,能特异性结合雌激素受体（estrogen receptor,ER）,抑制其下游靶基因的转录并促进ER蛋白的快速降解。随着氟维司群在乳腺癌内分泌治疗中的广泛应用,耐药现象逐步突显。探索氟维司群的耐药机制、靶向机制中的核心分子是突破治疗瓶颈、改善预后的关键。本文将就氟维司群的耐药机制及其联合用药等新治疗方案的研究进展进行综述。      

Dichloroacetate enhances apoptotic cell death via oxidative damage and attenuates lactate production in metformin-treated breast cancer cells

The unique metabolism of breast cancer cells provides interest in exploiting this phenomenon therapeutically. Metformin, a promising breast cancer therapeutic, targets complex I of the electron transport chain leading to an accumulation of reactive oxygen species (ROS) that eventually lead to cell death. Inhibition of complex I leads to lactate production, a metabolic byproduct already highly produced by reprogrammed cancer cells and associated with a poor prognosis. While metformin remains a promising cancer therapeutic, we sought a complementary agent to increase apoptotic promoting effects of metformin while attenuating lactate production possibly leading to greatly improved efficacy. Dichloroacetate (DCA) is a well-established drug used in the treatment of lactic acidosis which functions through inhibition of pyruvate dehydrogenase kinase (PDK) promoting mitochondrial metabolism. Our purpose was to examine the synergy and mechanisms by which these two drugs kill breast cancer cells. Cell lines were subjected to the indicated treatments and analyzed for cell death and various aspects of metabolism. Cell death and ROS production were analyzed using flow cytometry, Western blot analysis, and cell counting methods. Images of cells were taken with phase contrast microscopy or confocal microscopy. Metabolism of cells was analyzed using the Seahorse XF24 analyzer, lactate assays, and pH analysis. We show that when DCA and metformin are used in combination, synergistic induction of apoptosis of breast cancer cells occurs. Metformin-induced oxidative damage is enhanced by DCA through PDK1 inhibition which also diminishes metformin promoted lactate production. We demonstrate that DCA and metformin combine to synergistically induce caspase-dependent apoptosis involving oxidative damage with simultaneous attenuation of metformin promoted lactate production. Innovative combinations such as metformin and DCA show promise in expanding breast cancer therapies.     

乳腺癌内分泌治疗耐药的研究进展

乳腺癌内分泌治疗作为乳腺癌综合治疗的方法之一,具有不可替代的重要性.然而,内分泌治疗的疗效因其耐药性受到限制.文章旨在对乳腺癌内分泌治疗耐药的机制及逆转耐药的治疗进展进行综述,为临床内分泌治疗提供新的方向.     

MicroRNA在乳腺癌内分泌耐药中的研究新进展

内分泌治疗可显著改善雌激素受体阳性的乳腺癌患者的无病生存期和总生存期,但内分泌耐药是导致治疗失败的重要原因。微小RNA（microRNA,miRNA）是肿瘤研究领域中的热点,新近研究证实miRNA的表达变化是乳腺癌耐药机制之一。miRNA通过上调药物外排转运、抗凋亡蛋白、调节多药耐药信号传导网络等方式促成上皮 间质转化并形成肿瘤干细胞。miRNA亦可能通过调节雌激素受体α表达、受体酪氨酸激酶信号传导、细胞生存信号及细胞凋亡等途径引发耐药。miRNA可能成为激素受体阳性乳腺癌的预后因子、内分泌治疗疗效评估的预测因子以及新的靶点。     

Metformin increases survival in hormone receptor-positive,HER2-positive breast cancer patients with diabetes

Introduction

Metformin use has recently been observed to decrease both the rate and mortality of breast cancer. Our study was aim to determine whether metformin use is associated with survival in diabetic breast cancer patients by breast cancer subtype and systemic treatment.

Methods

Data from the Asan Medical Center Breast Cancer Database from 1997 to 2007 were analyzed. The study cohort comprised 6,967 nondiabetic patients, 202 diabetic patients treated with metformin, and 184 diabetic patients that did not receive metformin. Patients who were divided into three groups by diabetes status and metformin use were also divided into four subgroups by hormone receptor and HER2-neu status.

Results

In Kaplan-Meier analysis, the metformin group had a significantly better overall and cancer specific survival outcome compared with non metformin diabetic group (P <0.005 for both). There was no difference in survival between the nondiabetic and metformin groups. In multivariate analysis, Compared with metformin group, patients who did not receive metformin tended to have a higher risk of metastasis with HR 5.37 (95 % CI, 1.88 to 15.28) and breast cancer death with HR 6.51 (95 % CI, 1.88 to 15.28) on the hormone receptor-positive and HER2-negative breast cancer. The significant survival benefit of metformin observed in diabetic patients who received chemotherapy and endocrine therapy (HR for disease free survival 2.14; 95 % CI 1.14 to 4.04) was not seen in diabetic patients who did not receive these treatments.

Conclusion

Patients receiving metformin treatment when breast cancer diagnosis show a better prognosis only if they have hormone receptor-positive, HER2-positive tumors. Metformin treatment might provide a survival benefit when added to systemic therapy in diabetic patients.     

晚期雌激素受体阳性乳腺癌的治疗进展

针对晚期雌激素受体阳性乳腺癌虽然主要使用内分泌、靶向等治疗方法，但内分泌治疗的耐药问题在临床上较常见。随着对CDK4/6、PI3K-Akt1-mTOR通路等治疗靶点和耐药机制的研究，晚期雌激素受体阳性乳腺癌的靶向治疗已成为研究热点，利用基因靶向治疗等也可能成为新的突破点，同时在如何针对不同患者选择最佳治疗方案、最佳治疗时间等问题上仍存在挑战。本文将就晚期雌激素受体阳性乳腺癌的治疗进展进行综述。      

Metformin Use Is Associated With Better Survival of Breast Cancer Patients With Diabetes: A Meta‐Analysis

Background.

Diabetic patients with breast cancer receiving metformin and neoadjuvant chemotherapy have a higher pathologic complete response rate than do diabetic patients not receiving metformin, but findings on salvage treatment have been inconsistent. We performed a meta-analysis to assess the effect of adding metformin to standard therapy on the prognosis of breast cancer patients with diabetes.

Methods.

We searched PubMed, Embase, Web of Science (Thomson Scientific), China Knowledge Resource Integrated Database, VIP journal integration platform, and Chinese BioMedical Literature Database from inception to January 10, 2015, without language restrictions, including references related to metformin, breast cancer, and prognosis. We performed the meta-analysis using a random-effects model, with hazard ratios (HRs) and 95% confidence intervals (95% CIs) as effect measures.

Results.

A total of 11 studies consisting of 5,464 breast cancer patients with diabetes were included, comprising 2,760 patients who had received metformin and 2,704 patients who had not. The meta-analysis showed that metformin was associated with better overall survival times (HR: 0.53; 95% CI: 0.39-0.71) and cancer-specific survival times (HR: 0.89; 95% CI: 0.79-1.00). Subgroup analysis revealed that metformin improved the overall survival by 65% after adjusting for hormone receptor expression (HR: 0.35; 95% CI: 0.15–0.84). Taking metformin after the diagnosis of breast cancer was still associated with prolonged overall survival.

Conclusion.

The use of metformin in standard cancer therapy might improve both overall and cancer-specific survivals of diabetic patients with breast cancer.

Implications for Practice:

Diabetic patients with breast cancer receiving metformin and neoadjuvant chemotherapy have a higher pathologic complete response rate than diabetic patients not receiving metformin, but findings on salvage treatment have been inconsistent. The meta-analysis showed that metformin was associated with better overall survival times and cancer-specific survival times. Subgroup analysis revealed that metformin improved the overall survival by 65% after adjusting for hormone receptor expression. Taking metformin after the diagnosis of breast cancer was still associated with prolonged overall survival. The findings of this study highlight the potential usage of metformin in diabetic patients with breast cancer.     

Metformin decreases the dose of chemotherapy for prolonging tumor remission in mouse xenografts involving multiple cancer cell types

Metformin, the first-line drug for treating diabetes, selectively kills the chemotherapy resistant subpopulation of cancer stem cells (CSC) in genetically distinct types of breast cancer cell lines. In mouse xenografts, injection of metformin and the chemotherapeutic drug doxorubicin near the tumor is more effective than either drug alone in blocking tumor growth and preventing relapse. Here, we show that metformin is equally effective when given orally together with paclitaxel, carboplatin, and doxorubicin, indicating that metformin works together with a variety of standard chemotherapeutic agents. In addition, metformin has comparable effects on tumor regression and preventing relapse when combined with a four-fold reduced dose of doxorubicin that is not effective as a monotherapy. Finally, the combination of metformin and doxorubicin prevents relapse in xenografts generated with prostate and lung cancer cell lines. These observations provide further evidence for the CSC hypothesis for cancer relapse, an experimental rationale for using metformin as part of combinatorial therapy in a variety of clinical settings, and for reducing the chemotherapy dose in cancer patients.     

乳腺癌芳香化酶抑制剂耐药的研究进展

乳腺癌是女性常见的恶性肿瘤,约70％的患者为雌激素受体(estrogen receptor,ER)和(或)孕激素受体(progesterone receptor,PR)阳性,内分泌治疗是激素受体(hormone receptor,HR)阳性乳腺癌的主要治疗方式之一.近几十年来,内分泌治疗药物不断发展并应用于临床,乳腺癌...     

Therapeutic metformin/AMPK activation promotes the angiogenic phenotype in the ERα negative MDA-MB-435 breast cancer model

Metformin, a first line treatment for type 2 diabetes, has been implicated as a potential anti-neoplastic agent for breast cancers as well as other cancers. Metformin is known to work in part through the activation of AMP-dependent kinase (AMPK). AMPK is a key regulator of cellular energy homeostasis, especially under stress conditions where biosynthetic pathways are blocked by the phosphorylation of downstream AMPK substrates. Stimulation of AMPK by metformin resulted in a significant repression of cell proliferation and active MAPK1/2 in both estrogen receptor α (ERα) negative (MDA-MB-231, MDA-MB-435) and positive (MCF-7, T47D) human breast cancer cell lines. However, when ERα negative MDA-MB-435 cells were treated with metformin, they demonstrated increased expression of vascular endothelial growth factor (VEGF) in an AMPK dependent manner; while the ERα positive MCF-7 cells did not. Systemic therapy with metformin was tested for efficacy in an orthotopic model of ERα negative breast cancer performed in athymic nude mice. Surprisingly, metformin therapy significantly improved tumorigenic progression as compared to untreated controls. The metformin-treated group showed increased VEGF expression, intratumoral microvascular density and reduced necrosis. Metformin treatment was sufficient, however, to reduce systemic IGF-1 and the proliferation rate of tumor cells in vascularized regions. The data presented here suggests that, although metformin significantly represses breast cancer cell growth in vitro, the efficacy with respect to its therapeutic application for ERα negative breast cancer lesions in vivo may result in promotion of the angiogenic phenotype and increased tumorigenic progression. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Metformin-induced preferential killing of breast cancer initiating CD44+CD24-/low cells is sufficient to overcome primary resistance to trastuzumab in HER2+ human breast cancer xenografts

Trastuzumab-refractory breast cancer stem cells (CSCs) could also explain the high rate of primary resistance to single-agent trastuzumab in HER2 gene-amplified breast cancer patients. The identification of agents with strong selective toxicity for trastuzumab-resistant breast CSCs may have tremendous relevance for how HER2+ breast cancer patients should be treated. Using the human breast cancer cell line JIMT-1, which was established from the pleural metastasis of a patient who was clinically resistant to trastuzumab ab initio, we examined whether preferential killing of the putative CD44+CD24-/low breast CSC population might be sufficient to overcome primary resistance to trastuzumab in vivo. Because recent studies have shown that the anti-diabetic biguanide metformin can exert antitumor effects by targeted killing of CSC-like cells, we explored whether metformin's ability to preferentially kill breast cancer initiating CD44+CD24-/low cells may have the potential to sensitize JIMT-1 xenograft mouse models to trastuzumab. Upon isolation for breast cancer initiating CD44+CD24-/low cells by employing magnetic activated cell sorting, we observed the kinetics of metformin-induced killing drastically varied among CSC and non-CSC subpopulations. Metformin's cell killing effect increased dramatically by more than 10-fold in CD44+CD24-/low breast CSC cells compared to non-CD44+CD24-/low immunophenotypes. While seven-weeks treatment length with trastuzumab likewise failed to reduce tumor growth of JIMT-1 xenografts, systemic treatment with metformin as single agent resulted in a significant two-fold reduction in tumor volume. When trastuzumab was combined with concurrent metformin, tumor volume decreased sharply by more than four-fold. Given that metformin-induced preferential killing of breast cancer initiating CD44+CD24-/low subpopulations is sufficient to overcome in vivo primary resistance to trastuzumab, the incorporation of metformin into trastuzumab-based regimens may provide a valuable strategy for treatment of HER2+ breast cancer patients.     

乳腺癌芳香化酶抑制剂耐药分子机制研究进展

芳香化酶抑制剂（ aromatase inhibitors,AIs）在绝经后雌激素依赖性乳腺癌的治疗中取得了令人鼓舞的疗效。大型的临床试验ATAC和BIG 1-98均证明第三代芳香化酶抑制剂作为绝经后乳腺癌的辅助治疗可明显提高患者的无病生存期,疗效优于他莫昔芬。随着AIs在临床广泛应用及用药时间的延长,AIs耐药也成了临床医生不可回避的问题,至今国内尚没有对AIs耐药分子机制的详细报道,本文通过对国内外一些实验室和临床研究结果的综述,拟概述乳腺癌AIs耐药分子机制的研究进展。     

Insulin priming effect on estradiol-induced breast cancer metabolism and growth

Diabetes is a risk factor for breast cancer development and is associated with poor prognosis for breast cancer patients. However, the molecular and biochemical mechanisms underlying the association between diabetes and breast cancer have not been fully elucidated. Here, we investigated estradiol response in MCF-7 breast cancer cells with or without chronic exposure to insulin. We found that insulin priming is necessary and specific for estradiol-induced cancer cell growth, and induces anaplerotic shunting of glucose into macromolecule biosynthesis in the estradiol treated cells. Treatment with ERK or Akt specific inhibitors, U0126 or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, respectively, suppressed estradiol-induced growth. Interestingly, molecular analysis revealed that estradiol treatment markedly increases expression of cyclin A and B, and decreases p21 and p27 in the insulin-primed cells. In addition, estradiol treatment activated metabolic genes in pentose phosphate (PPP) and serine biosynthesis pathways in the insulin-primed cells while insulin priming decreased metabolic gene expression associated with glucose catabolism in the breast cancer cells. Finally, we found that anti-diabetic drug metformin and AMPK ligand AICAR, but not thiazolidinediones (TZDs), specifically suppress the estradiol-induced cellular growth in the insulin-primed cells. These findings suggest that estrogen receptor (ER) activation under chronic hyperinsulinemic condition increases breast cancer growth through the modulation of cell cycle and apoptotic factors and nutrient metabolism, and further provide a mechanistic evidence for the clinical benefit of metformin use for ER-positive breast cancer patients with diabetes.     

Low-dose estrogen therapy to reverse acquired antihormonal resistance in the treatment of breast cancer

Estrogen is a potent stimulus for growth in its target organs: the uterus, vagina, and some estrogen receptor-positive breast cancers. However, estrogen is also able to control menopausal symptoms and maintain bone density in postmenopausal women. Until recently, there was also believed to be a link between estrogen and the prevention of cardiovascular disease. For these reasons, hormone replacement therapy (HRT) with an orally active estrogen and progesterone has been used routinely for more than 50 years to maintain physiologic homeostasis after menopause. Not surprisingly, HRT increases the risk of developing breast cancer. The link between estrogen and breast cancer growth served as the incentive to develop long-term tamoxifen therapy and, subsequently, the aromatase inhibitors (AIs) as successful "anti-estrogenic" treatments. Unfortunately, the consequence of exhaustive therapy is drug resistance. Laboratory studies have defined the evolution of tumor drug resistance to tamoxifen, raloxifene (used for breast and osteoporosis chemoprevention), and the AIs. Remarkably, the long-term exposure of breast cancers to antihormonal therapy also exposes a vulnerability that is being exploited in the clinic. Years of antihormonal therapy alters the cellular response mechanism to estrogen. Normally, estrogen is classified as a survival signal in breast cancer, but in sensitive antihormone-resistant cells, estrogen induces apoptosis. When resistant cells are killed, antihormonal therapy is once again effective. This new targeted approach to the treatment of metastatic breast cancer could open the door to novel approaches to treatment with drug combinations.