[This corrects the article on p. 67 in vol. 3, PMID: 27790618.].

1130 Background: Obesity and HER2 overexpression are associated with poor prognosis in breast cancer (BC). Recent studies suggest the existence of functional crosstalk between leptin, a hormone correlated with adiposity, and HER2. Specifically, stimulation with leptin can transactivate HER2 and activate its downstream signaling, while chronic exposure to leptin increases HER2 stability in presence or absence of Herceptin. In our study we evaluated if excess adipocity (associated with leptin overexpression) can impact the clinical outcome in HER2-positive patients treated with Herceptin. Methods: We analyzed retrospectively 155 patients with HER2-positive BC treated with Herceptin for early stage or metastatic disease in the period 2003-2008. The patients were grouped into normal, overweight, and obese categories according to Body Mass Index (BMI) following WHO guidelines. Overall survival (OS) and time to progression (TTP) were calculated by the Kaplan-Meier method and comparisons were made using the log-rank test. Results: 103 patients were treated with adjuvant Herceptin. 12.5% of the population relapsed; of these, 31% and 23% were overweight and obese, respectively. Inrelapsed patients, OS and TTP decreased with increased BMI: the median OS for normal, overweight and obese patients was 40, 36.5, and 31 months, respectively (p=ns); median TTP was 11, 8, and 5 months, respectively (p=0.0351). 52 patients received Herceptin for metastatic disease in association with chemotherapy. In this population, 33% and 15% patients were overweight and obese, respectively; median OS was 65 months and TTP 10.5 months. Decreased OS and TTP were associated with increased BMI: median OS for normal weight, overweight and obese patients was 67, 54, and 39 months, respectively (p=0.0010); median TTP was 12, 7.5, and 7 months, respectively (p=0.0442). The negative impact of increasing BMI was confirmed in subgroups differentiated by hormone receptor and menopausal status. Conclusions: The results suggest that obesity is not only a risk factor and an indicator of poor prognosis in BC patients, but also a negative predictive parameter in HER2-positive subjects. Our in vivo data validate in vitro observation that leptin can impair response to Herceptin in BC cells. No significant financial relationships to disclose.

Aim: To design, manufacture and test a second generation leptin receptor (ObR) agonist glycopeptide derivative. The major drawback to current experimental therapies involving leptin protein is the appearance of treatment resistance. Our novel peptidomimetic was tested for efficacy and lack of resistance induction in rodent models of obesity and appetite reduction. Methods: The glycopeptide containing two additional non‐proteinogenic amino acids was synthesized by standard solid‐phase methods. Normal mice were fed with peanuts until their blood laboratory data and liver histology showed typical signs of obesity but not diabetes. The mice were treated with the peptidomimetic at 0.02, 0.1 or 0.5 mg/kg/day intraperitoneally side‐by‐side with 0.1 mg/kg/day leptin for 11 days. After termination of the assay, the blood cholesterol and glucose amounts were measured, the liver fat content was visualized and quantified and the remaining mice returned to normal diet and were allowed to mate. In parallel experiments normal rats were treated intranasally with the glycopeptide at 0.1 mg/kg/day for 10 days. Results: The 12‐residue glycosylated leptin‐based peptidomimetic E1/6‐amino‐hexanoic acid (Aca) was designed to target a principal leptin/ObR‐binding interface. E1/Aca induced leptin effects in ObR‐positive cell lines at picomolar concentrations and readily crossed the blood‐brain barrier (BBB) following intraperitoneal administration. The peptide initiated typical leptin‐dependent signal transduction pathways both in the presence and absence of leptin protein. The peptide also reduced weight gain in mice fed with high‐fat peanut diet in a dose‐dependent manner. Obese mice receiving peptide E1/Aca at a 0.5 mg/kg/day dose lost weight, corresponding to a net 6.5% total body weight loss, while similar mice treated with leptin protein did not. Upon cessation of the weight loss treatment, several obesity‐related pathologies (i.e. abnormal metabolic profile and liver histology as well as infertility) normalized in peptide‐, but not leptin‐treated, mice. Peptide E1/Aca added intranasally to growing normal rats decelerated normal weight gain corresponding to a net 6.8% net total body weight loss with statistical significance. Conclusions: No resistance induction to peptide E1/Aca or toxicity in either obese or healthy rodents was observed, indicating the potential for widespread utility of the peptidomimetic in the treatment of leptin‐deficiency disorders. We provide additional proof for the hypothesis that difficulties in current leptin therapies reside at the BBB penetration stage, and we document that by either glycosylation or intranasal peptide administration we can overcome this limitation.

BRCA1 and BRCA2 germline mutations contribute to a significant number of familial and hereditary breast and/or ovarian cancers. The proportion of high-risk families with breast and/or ovarian cancer cases due to mutations in these tumor suppressor genes varies widely among populations. In some population, a wide spectrum of different mutations in both genes are present, whereas in other groups specific mutations in BRCA1 and BRCA2 have been reported with high frequency. Most of these mutations are prevalent in restricted populations as consequence of a founder effect. The comparison of haplotypes between families with the same mutation can distinguish whether high-frequency alleles derive from an older or more recent single mutational event or whether they have arisen independently more than once. Here, we review some of the most well-known and significant examples of founder mutations in BRCA genes found in European and non-European populations. In conclusion, the identification of the ethnic group of families undergoing genetic counseling enables the geneticist and oncologist to make more specific choices, leading to simplify the clinical approach to genetic testing carried out on members of high-risk families. Futhermore, the high frequency of founder mutations, allowing to analyze a large number of cases, might provide accurate information regarding their penetrance.

BACKGROUND:Insulin receptor substrate 1 (IRS-1) is a signaling molecule that exerts a key role in mediating cross talk between estrogen receptor alpha (ERalpha) and insulin-like growth factor 1 (IGF-1) in breast cancer cells. Previously, we demonstrated that a fraction of IRS-1 binds ERalpha, translocates to the nucleus, and modulates ERalpha-dependent transcription at estrogen response elements (ERE). Here, we studied structure-function relationships of the ERalpha:IRS-1 complex under IGF-1 and/or estradiol (E2) stimulation.MATERIALS AND METHODS:ERalpha and IRS-1 deletion mutants were used to analyze structural and functional ERalpha/IRS-1 interactions. IRS-1 binding to ERE and IRS-1 role in ERalpha-dependent ERE transcription was examined by chromatin immunoprecipitation and gene reporter analysis, respectively. The requirement for IRS-1 in ERalpha function was tested with RNAi technology.RESULTS:Nuclear translocation of IRS-1 was induced by E2, IGF-1, and a combination of both stimuli. ERalpha/IRS-1 binding was direct and involved the activation function-1 (AF-1)/DNA binding domain (DBD) region of ERalpha and two discrete regions of IRS-1 (the N-terminal pleckstrin homology domain and a region within the C-terminus). IRS-1 knock down abrogated IGF-1-dependent transcriptional activity of unliganded ERalpha, but induced the activity of liganded ERalpha.CONCLUSIONS:ERalpha/IRS-1 interactions are direct and involve the ERalpha AF-1/DBD domain and IRS-1 domains mapping within N- and C-terminus. IRS-1 may act as a repressor of liganded ERalpha and coactivator of unliganded ERalpha.

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-1α (HIF-1α) 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-1α 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-1α in insulin- and CoCl2-activated leptin mRNA and protein expression was confirmed using RNA interference.