Mechanism of leptin expression in breast cancer cells: role of hypoxia-inducible factor-1alpha.

We reported previously that the obesity hormone leptin is overexpressed in breast cancer biopsies. Here, we investigated molecular mechanisms involved in this process, focusing on conditions that are associated with obesity, that is, hyperinsulinemia and induction of hypoxia. By using quantitative real-time PCR, immunofluorescent detection of proteins and enzyme-linked immunosorbent assays, we found that treatment of MCF-7 breast cancer cells with high doses of insulin or the hypoxia-mimetic agent CoCl2, or culturing the cells under hypoxic conditions significantly increased the expression of leptin mRNA and protein. Notably, the greatest leptin mRNA and protein expression were observed under combined hyperinsulinemia and hypoxia or hypoxia-mimetic treatments. Luciferase reporter assays suggested that increased leptin synthesis could be related to the activation of the leptin gene promoter. DNA affinity precipitation and chromatin immunoprecipitation experiments revealed that insulin, CoCl2 and/or hypoxia treatments augmented nuclear accumulation of hypoxia-inducible factor-1a (HIF-1a) and increased its interaction with several upstream leptin regulatory sequences, especially with the proximal promoter containing four hypoxia-response elements and three GC-rich regions. By using reverse chromatin precipitation, we determined that loading of HIF-1a on the proximal leptin promoter concurred with the recruitment of p300, the major HIF coactivator, suggesting that the HIF/p300 complex is involved in leptin transcription. The importance of HIF-1a in insulin- and CoCl2-activated leptin mRNA and protein expression was confirmed using RNA interferenc

Bergamot natural products eradicate cancer stem cells (CSCs) by targeting mevalonate, Rho-GDI-signalling and mitochondrial metabolism

Here, we show that a 2:1 mixture of Brutieridin and Melitidin, termed “BMF”, has a statin-like properties, which blocks the action of the rate-limiting enzyme for mevalonate biosynthesis, namely HMGR (3-hydroxy-3-methylglutaryl- CoA-reductase). Moreover, our results indicate that BMF functionally inhibits several key characteristics of CSCs. More specifically, BMF effectively i) reduced ALDH activity, ii) blocked mammosphere formation and iii) inhibited the activation of CSC-associated signalling pathways (STAT1/3, Notch and Wnt/ beta-catenin) targeting Rho-GDI-signalling. In addition, BMF metabolically inhibited mitochondrial respiration (OXPHOS) and fatty acid oxidation (FAO). Importantly, BMF did not show the same toxic side-effects in normal fibroblasts that were observed with statins. Lastly, we show that high expression of the mRNA species encoding HMGR is associated with poor clinical outcome in breast cancer patients, providing a potential companion diagnostic for BMF-directed personalized therapy

Relationships between hypoxia markers and the leptin system, estrogen receptors in human primary and metastatic breast cancer: effects of preoperative chemotherapy

<p>Abstract</p> <p>Background</p> <p>Tumor hypoxia is marked by enhanced expression of hypoxia-inducible factor-α (HIF-1α) and glucose transporter-1 (Glut-1). Hypoxic conditions have also been associated with overexpression of angiogenic factors, such as leptin. The aim of our study was to analyze the relationships between hypoxia markers HIF-1α, Glut-1, leptin, leptin receptor (ObR) and other breast cancer biomarkers in primary and metastatic breast cancer in patients treated or untreated with preoperative chemotherapy.</p> <p>Methods</p> <p>The expression of different biomarkers was examined by immunohistochemistry in 116 primary breast cancers and 65 lymph node metastases. Forty five of these samples were obtained form patients who received preoperative chemotherapy and 71 from untreated patients.</p> <p>Results</p> <p>In primary tumors without preoperative chemotherapy, HIF-1α and Glut-1 were positively correlated (p = 0.02, r = 0.437). HIF-1α in primary and metastatic tumors without preoperative therapy positively correlated with leptin (p < 0.0001, r = 0.532; p = 0.013, r = 0.533, respectively) and ObR (p = 0.002, r = 0.319; p = 0.083, r = 0.387, respectively). Hypoxia markers HIF-1α and Glut-1 were negatively associated with estrogen receptor alpha (ERα) and positively correlated with estrogen receptor beta (ERβ). In this group of tumors, a positive correlation between Glut-1 and proliferation marker Ki-67 (p = 0.017, r = 0.433) was noted. The associations between HIF-1α and Glut-1, HIF-1α and leptin, HIF-1α and ERα as well as Glut-1 and ERβ were lost following preoperative chemotherapy.</p> <p>Conclusions</p> <p>Intratumoral hypoxia in breast cancer is marked by coordinated expression of such markers as HIF-1α, Glut-1, leptin and ObR. The relationships among these proteins can be altered by preoperative chemotherapy.</p

GPER mediates the angiocrine actions induced by IGF1 through the HIF-1α/VEGF pathway in the breast tumor microenvironment

The G protein estrogen receptor GPER/GPR30 mediates estrogen action in breast cancer cells as well as in breast cancer-associated fibroblasts (CAFs), which are key components of microenvironment driving tumor progression. GPER is a transcriptional target of hypoxia inducible factor 1 alpha (HIF-1α) and activates VEGF expression and angiogenesis in hypoxic breast tumor microenvironment. Furthermore, IGF1/IGF1R signaling, which has angiogenic effects, has been shown to activate GPER in breast cancer cells. We analyzed gene expression data from published studies representing almost 5000 breast cancer patients to investigate whether GPER and IGF1 signaling establish an angiocrine gene signature in breast cancer patients. Next, we used GPER-positive but estrogen receptor (ER)-negative primary CAF cells derived from patient breast tumours and SKBR3 breast cancer cells to investigate the role of GPER in the regulation of VEGF expression and angiogenesis triggered by IGF1. We performed gene expression and promoter studies, western blotting and immunofluorescence analysis, gene silencing strategies and endothelial tube formation assays to evaluate the involvement of the HIF-1α/GPER/VEGF signaling in the biological responses to IGF1. We first determined that GPER is co-expressed with IGF1R and with the vessel marker CD34 in human breast tumors (n = 4972). Next, we determined that IGF1/IGF1R signaling engages the ERK1/2 and AKT transduction pathways to induce the expression of HIF-1α and its targets GPER and VEGF. We found that a functional cooperation between HIF-1α and GPER is essential for the transcriptional activation of VEGF induced by IGF1. Finally, using conditioned medium from CAFs and SKBR3 cells stimulated with IGF1, we established that HIF-1α and GPER are both required for VEGF-induced human vascular endothelial cell tube formation. These findings shed new light on the essential role played by GPER in IGF1/IGF1R signaling that induces breast tumor angiogenesis. Targeting the multifaceted interactions between cancer cells and tumor microenvironment involving both GPCRs and growth factor receptors has potential in future combination anticancer therapies

Design and development of a peptide-based adiponectin receptor agonist for cancer treatment

<p>Abstract</p> <p>Background</p> <p>Adiponectin, a fat tissue-derived adipokine, exhibits beneficial effects against insulin resistance, cardiovascular disease, inflammatory conditions, and cancer. Circulating adiponectin levels are decreased in obese individuals, and this feature correlates with increased risk of developing several metabolic, immunological and neoplastic diseases. Thus, pharmacological replacement of adiponectin might prove clinically beneficial, especially for the obese patient population. At present, adiponectin-based therapeutics are not available, partly due to yet unclear structure/function relationships of the cytokine and difficulties in converting the full size adiponectin protein into a viable drug.</p> <p>Results</p> <p>We aimed to generate adiponectin-based short peptide that can mimic adiponectin action and be suitable for preclinical and clinical development as a cancer therapeutic. Using a panel of 66 overlapping 10 amino acid-long peptides covering the entire adiponectin globular domain (residues 105-254), we identified the 149-166 region as the adiponectin active site. Three-dimensional modeling of the active site and functional screening of additional 330 peptide analogs covering this region resulted in the development of a lead peptidomimetic, ADP 355 (H-DAsn-Ile-Pro-Nva-Leu-Tyr-DSer-Phe-Ala-DSer-NH₂). In several adiponectin receptor-positive cancer cell lines, ADP 355 restricted proliferation in a dose-dependent manner at 100 nM-10 μM concentrations (exceeding the effects of 50 ng/mL globular adiponectin). Furthermore, ADP 355 modulated several key signaling pathways (AMPK, Akt, STAT3, ERK1/2) in an adiponectin-like manner. siRNA knockdown experiments suggested that ADP 355 effects can be transmitted through both adiponectin receptors, with a greater contribution of AdipoR1. <it>In vivo</it>, intraperitoneal administration of 1 mg/kg/day ADP 355 for 28 days suppressed the growth of orthotopic human breast cancer xenografts by ~31%. The peptide displayed excellent stability (at least 30 min) in mouse blood or serum and did not induce gross toxic effects at 5-50 mg/kg bolus doses in normal CBA/J mice.</p> <p>Conclusions</p> <p>ADP 355 is a first-in-class adiponectin receptor agonist. Its biological activity, superior stability in biological fluids as well as acceptable toxicity profile indicate that the peptidomimetic represents a true lead compound for pharmaceutical development to replace low adiponectin levels in cancer and other malignancies.</p

Enhanced proliferative potential of hematopoietic cells expressing degradation-resistant c-Myb mutants

The c-myb gene encodes a transcription factor required for proliferation, differentiation, and survival of hematopoietic cells. Expression of c-Myb is often increased in hematological malignancies, but the underlying mechanisms are poorly understood. We show here that c-Myb has a longer half-life ( at least 2-fold) in BCR/ABL-expressing than in normal hematopoietic cells. Such enhanced stability was dependent on a phosphatidylinositol 3-kinase ( PI-3K)/Akt/GSKIII beta pathway( s) as indicated by the suppression of c-Myb expression upon treatment with PI-3K inhibitors or co-expression with dominant negative Akt or constitutively active GSKIII beta. Moreover, inhibition of GSKIII beta by LiCl enhanced cMyb expression in parental 32Dcl3 cells. Compared with wild type c-Myb, three mutants ( Delta( 358 - 452), Delta( 389 - 418), and L389A/L396A c-Myb) of the leucine zipper domain had increased stability. However, only expression of Delta( 358 - 452) was not affected by inhibition of the PI-3K/Akt pathway and was not enhanced by a proteasome inhibitor, suggesting that leucine zipper-dependent and - independent mechanisms are involved in the regulation of c-Myb stability. Indeed, Delta( 389 - 418) carrying four lysine-to-alanine substitutions ( Delta( 389 - 418) K387A/K428A/ K442A/K445A) was as stable as Delta( 358 - 452) c-Myb. Compared with full-length c-Myb, constitutive expression of Delta( 358 - 452) and Delta( 389 - 418) c-Myb in Lin-Sca-1(+) mouse marrow cells increased cytokine-dependent primary and secondary colony formation. In K562 cells, expression of Delta( 358 - 452), Delta( 389 - 418), and L389A/L396A c-Myb led to enhanced proliferation after STI571 treatment. Thus, enhanced stability of c-Myb by activation of PI-3K-dependent pathway( s) might contribute to the higher proliferative potential of BCR/ABL-expressing and, perhaps, other leukemic cells

Insulin-like growth factor 1 differentially regulates estrogen receptor-dependent transcription at estrogen response element and AP-1 sites in breast cancer cells

Cross-talk between insulin-like growth factor 1 (IGF-1) and estrogen receptor alpha (ER) regulates gene expression in breast cancer cells, but the underlying mechanisms remain unclear. Here, we studied how 17-beta-estradiol (E2) and IGF-1 affect ER transcriptional machinery in MCF-7 cells. E2 treatment stimulated ER loading on the estrogen response element (ERE) in the pS2 promoter and on the AP-1 motif in the cyclin D1 promoter. On ERE, similar amounts of liganded ER were found at 1-24-h time points, whereas on AP-1, ER binding fluctuated over time. At 1 h, liganded ER was recruited to ERE together with histone acetyltransferases SRC-1 and p300, ubiquitin ligase E6-AP, histone methyltransferase Carm1 (Carm), and polymerase (pol) II. This coincided with increased histone H3 acetylation and up-regulation of pS2 mRNA levels. At the same time, E2 moderately increased cyclin D1 expression, which was associated with the recruitment of liganded ER, SRC-1, p300, ubiquitin ligase E6-AP (E6L), Mdm2, and pol II, but not other regulatory proteins, to AP-1. In contrast, at 1 h, IGF-1 increased the recruitment of the ER.SRC-1.p300.E6L.Mdm2.Carm.pol II complex on AP-1, but not on ERE, and induced cyclin D1, but not pS2, mRNA expression. Notably, ER knockdown reduced the association of ER, E6L, Mdm2, Carm, and pol II with AP-1 and resulted in down-regulation of cyclin D1 expression. IGF-1 potentiated the effects of E2 on ERE but not to AP-1 and increased E2-dependent pS2, but not cyclin D1, mRNA expression. In conclusion, E2 and IGF-1 differentially regulate ER transcription at ERE and AP-1 sites