Eva Surmacz

Leptin and cancer

The prevalence of obesity has markedly increased over the past two decades, especially in the industrialized countries. While the impact of excess body weight on the development of cardiac disease and diabetes has been well documented, the link between obesity and carcinogenesis is just being recognized. This review will focus on the link between leptin, a cytokine that is elevated in obese individuals, and cancer development. First, we briefly discuss the biological functions of leptin and its signaling pathways. Then, we summarize the effects of leptin on different cancer types in experimental cellular and animal models. Next, we analyze epidemiological data on the relationship between obesity and the presence of cancer or cancer risk in patients. Finally, leptin as a target for cancer treatment and prevention will be discussed. J. Cell. Physiol. 207: 12–22, 2006.

Increased expression of leptin and the leptin receptor as a marker of breast cancer progression : Possible role of obesity-related stimuli

Purpose: Recent in vitro studies suggested that the autocrine leptin loop might contribute to breast cancer development by enhancing cell growth and survival. To evaluate whether the leptin system could become a target in breast cancer therapy, we examined the expression of leptin and its receptor (ObR) in primary and metastatic breast cancer and noncancer mammary epithelium. We also studied whether the expression of leptin/ObR in breast cancer can be induced by obesity-related stimuli, such as elevated levels of insulin, insulin-like growth factor-I (IGF-I), estradiol, or hypoxic conditions. Experimental Design: The expression of leptin and ObR was examined by immunohistochemistry in 148 primary breast cancers and 66 breast cancer metastases as well as in 90 benign mammary lesions. The effects of insulin, IGF-I, estradiol, and hypoxia on leptin and ObR mRNA expression were assessed by reverse transcription-PCR in MCF-7 and MDA-MB-231 breast cancer cell lines. Results: Leptin and ObR were significantly overexpressed in primary and metastatic breast cancer relative to noncancer tissues. In primary tumors, leptin positively correlated with ObR, and both biomarkers were most abundant in G3 tumors. The expression of leptin mRNA was enhanced by insulin and hypoxia in MCF-7 and MDA-MB-231 cells, whereas IGF-I and estradiol stimulated leptin mRNA only in MCF-7 cells. ObR mRNA was induced by insulin, IGF-I, and estradiol in MCF-7 cells and by insulin and hypoxia in MDA-MB-231 cells. Conclusions: Leptin and ObR are overexpressed in breast cancer, possibly due to hypoxia and/or overexposure of cells to insulin, IGF-I, and/or estradiol.

Function of the IGF-I receptor in breast cancer.

The insulin-like growth factor-I receptor (IGF-IR)3 is a transmembrane tyrosine kinaseregulating various biological processes such as proliferation, survival, transformation, differentiation,cell-cell and cell-substrate interactions. Different signaling pathways may underlie thesepleiotropic effects. The specific pathways engaged depend on the number of activated IGF-IRs,availability of intracellular signal transducers, the action of negative regulators, and is influencedby extracellular modulators. Experimental and clinical data implicate the IGF-IR in breastcancer etiology. There is strong evidence linking hyperactivation of the IGF-IR with theearly stages of breast cancer. In primary breast tumors, the IGF-IR is overexpressed andhyperphosphorylated, which correlates with radio-resistance and tumor recurrence. In vitro,the IGF-IR is often required for mitogenesis and transformation, and its overexpression oractivation counteract effects of various pro-apoptotic treatments. In hormone-responsive breastcancer cells, IGF-IR function is strongly linked with estrogen receptor (ER) action. TheIGF-IR and the ER are co-expressed in breast tumors. Moreover, estrogens stimulate the expressionof the IGF-IR and its major signaling substrate IRS-1, while antiestrogens downregulateIGF-IR signaling, mainly by decreasing IRS-1 expression and function. On the other hand,overexpression of IRS-1 promotes estrogen-independence for growth and transformation. InER-negative breast cancer cells, usually displaying a more aggressive phenotype, the levelsof the IGF-IR and IRS-1 are often low and IGF is not mitogenic, yet the IGF-IR is stillrequired for metastatic spread. Consequently, IGF-IR function in the late stages of breastcancer remains one of the most important questions to be addressed before rationalanti-IGF-IR therapies are developed.

miR-20b modulates VEGF expression by targeting HIF-1α and STAT3 in MCF-7 breast cancer cells†

MicroRNAs (miRNAs) are small non‐coding RNAs that regulate the expression of different genes, including genes involved in cancer progression. A functional link between hypoxia, a key feature of the tumor microenvironment, and miRNA expression has been documented. We investigated whether and how miR‐20b can regulate the expression of vascular endothelial growth factor (VEGF) in MCF‐7 breast cancer cells under normoxic and hypoxia‐mimicking conditions (CoCl2 exposure). Using immunoblotting, ELISA, and quantitative real‐time PCR, we demonstrated that miR‐20b decreased VEGF protein levels at 4 and 24 h following CoCl2 treatment, and VEGF mRNA at 4 h of treatment. In addition, miR‐20b reduced VEGF protein expression in untreated cells. Next, we investigated the molecular mechanism by which pre‐miR‐20b can affect VEGF transcription, focusing on hypoxia inducible factor 1 (HIF‐1) and signal transducer and activator of transcription 3 (STAT3), transcriptional inducers of VEGF and putative targets of miR‐20b. Downregulation of VEGF mRNA by miR‐20b under a 4 h of CoCl2 treatment was associated with reduced levels of nuclear HIF‐1α subunit and STAT3. Chromatin immunoprecipitation (ChIP) assays revealed that HIF‐1α, but not STAT3, was recruited to the VEGF promoter following the 4 h of CoCl2 treatment. This effect was inhibited by transfection of cells with pre‐miR‐20b. In addition, using siRNA knockdown, we demonstrated that the presence of STAT3 is necessary for CoCl2‐mediated HIF‐1α nuclear accumulation and recruitment on VEGF promoter. In summary, this report demonstrates, for the first time, that the VEGF expression in breast cancer cells is mediated by HIF‐1 and STAT3 in a miR‐20b‐dependent manner. J. Cell. Physiol. 224:242–249, 2010

Growth factor receptors as therapeutic targets: strategies to inhibit the insulin-like growth factor I receptor

Neoplastic transformation is often related to abnormal activation of growth factor receptors and their signaling pathways. The concept of targeting specific tumorigenic receptors and/or signaling molecules has been validated by the development and successful clinical application of drugs acting against the epidermal growth factor receptor 2 (HER2/neu, Erb2), the epidermal growth factor receptor 1 (EGFR, HER1), the Brc-Abl kinase, the platelet-derived growth factor receptor, and c-kit. This review will focus on the next promising therapeutic target, the insulin-like growth factor I receptor (IGF-IR). IGF-IR has been implicated in a number of neoplastic diseases, including several common carcinomas. From a pharmaceutical standpoint, of particular importance is that IGF-IR appears to be required for many transforming agents (genetic, viral, chemical) to act, but is not obligatory for the function of normal adult cells. The tumorigenic potential of IGF-IR is mediated through its antiapoptotic and transforming signaling, and in some cases through induction of prometastatic pathways. Preclinical studies demonstrated that downregulation of IGF-IR reversed the neoplastic phenotype and sensitized cells to antitumor treatments. The strategies to block IGF-IR function employed anti-IGF-IR antibodies, small-molecule inhibitors of the IGF-IR tyrosine kinase, antisense oligodeoxynucleotides and antisense RNA, small inhibitory RNA, triple helix, dominant-negative mutants, and various compounds reducing ligand availability. The experience with these strategies combined with the knowledge gained with current anti-growth factor receptor drugs should streamline the development of anti-IGF-IR therapeutics.

Leptin Interferes with the Effects of the Antiestrogen ICI 182,780 in MCF-7 Breast Cancer Cells

Purpose: Obesity is a risk factor for breast cancer development in postmenopausal women and correlates with shorter disease-free and overall survival in breast cancer patients, regardless of menopausal status. Adipose tissue is a major source of leptin, a cytokine regulating energy balance and controlling different processes in peripheral tissues, including breast cancer cell growth. Here, we investigated whether leptin can counteract antitumorigenic activities of the antiestrogen ICI 182,780 in breast cancer cells. Experimental Design: Mitogenic response to leptin and the effects of leptin on ICI 182,780-dependent growth inhibition were studied in MCF-7 estrogen receptor α-positive breast cancer cells. The expression of leptin receptor and the activation of signaling pathways were studied by Western immunoblotting. The interference of leptin with ICI 182,780-induced estrogen receptor α degradation was probed by Western immunoblotting, fluorescence microscopy, and pulse-chase experiments. Leptin effects on estrogen receptor α–dependent transcription in the presence and absence of ICI 182,780 were studied by luciferase reporter assays and chromatin immunoprecipitation. Results: MCF-7 cells were found to express the leptin receptor and respond to leptin with cell growth and activation the signal transducers and activators of transcription 3, extracellular signal-regulated kinase-1/2, and Akt/GSK3/pRb pathways. The exposure of cells to 10 nmol/L ICI 182,780 blocked cell proliferation, induced rapid estrogen receptor α degradation, inhibited nuclear estrogen receptor α expression, and reduced estrogen receptor α–dependent transcription from estrogen response element–containing promoters. All of these effects of ICI 182,780 were significantly attenuated by simultaneous treatment of cells with 100 ng/mL leptin. Conclusions: Leptin interferes with the effects of ICI 182,780 on estrogen receptor α in breast cancer cells. Thus, high leptin levels in obese breast cancer patients might contribute to the development of antiestrogen resistance.

Functional Significance of Type 1 Insulin-like Growth Factor-mediated Nuclear Translocation of the Insulin Receptor Substrate-1 and β-Catenin

Previous work has shown that the transcriptional regulator β-catenin can translocate to the nuclei when cells are stimulated with the type 1 insulin-like growth factor (IGF-1). We show by immunocoprecipitation and by confocal microscopy that β-catenin binds to and co-localizes with the insulin receptor substrate-1 (IRS-1), a docking protein for both the insulin and the IGF-1 receptors. IRS-1 is required for IGF-1-mediated nuclear translocation of β-catenin, resulting in the activation of the β-catenin target genes. IGF-1-mediated nuclear translocation of β-catenin is facilitated by the nuclear translocation of IRS-1. Both IRS-1 and β-catenin are recruited to the cyclin D1 promoter, an established target for β-catenin, but only IRS-1 is recruited to the ribosomal DNA (rDNA) promoter. UBF proteins (known to interact with both IRS-1 and β-catenin) are also detectable in the cyclin D1 and rDNA promoters. These results indicate that IRS-1 (activated by the IGF-1 receptor) is one of several proteins that regulate the subcellular localization and activity of β-catenin. The ability of IRS-1 to localize to both RNA polymerase II (with β-catenin) and RNA polymerase I-regulated promoters suggest an explanation for the effect of IRS-1 on both cell growth in size and cell proliferation. This possibility is supported by the demonstration that enforced nuclear localization of IRS-1 causes nuclear translocation of β-catenin and transformation of normal mouse embryo fibroblasts (colony formation in soft agar).

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

BackgroundAdiponectin, 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.ResultsWe 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. In vivo, 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.ConclusionsADP 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.

Obesity hormone leptin: a new target in breast cancer?

Leptin is a multifunctional hormone produced mainly by the adipose tissue and involved in the regulation of food intake and energy balance. In addition, leptin can stimulate mitogenic and angiogenic processes in peripheral organs. Because leptin levels are elevated in obese individuals and excess body weight has been shown to increase breast cancer risk in postmenopausal women, attempts have been made to evaluate whether leptin can promote breast cancer. Data obtained in cell and animal models and analyses of human breast cancer biopsies indeed suggest such an involvement. Furthermore, a recent report clearly shows that targeting leptin signaling may reduce mammary carcinogenesis. Thus, leptin should become a new attractive target in breast cancer.

Nuclear insulin receptor substrate 1 interacts with estrogen receptor α at ERE promoters

Insulin receptor substrate 1 (IRS-1) is a major signaling molecule activated by the insulin and insulin-like growth factor I receptors. Recent data obtained in different cell models suggested that in addition to its conventional role as a cytoplasmic signal transducer, IRS-1 has a function in the nuclear compartment. However, the role of nuclear IRS-1 in breast cancer has never been addressed. Here we report that in estrogen receptor α (ERα)-positive MCF-7 cells, (1) a fraction of IRS-1 was translocated to the nucleus upon 17-β-estradiol (E2) treatment; (2) E2-dependent nuclear translocation of IRS-1 was blocked with the antiestrogen ICI 182,780; (3) nuclear IRS-1 colocalized and co-precipitated with ERα; (4) the IRS-1:ERα complex was recruited to the E2-sensitive pS2 gene promoter. Notably, IRS-1 interaction with the pS2 promoter did not occur in ERα-negative MDA-MB-231 cells, but was observed in MDA-MB-231 cells retransfected with ERα. Transcription reporter assays with E2-sensitive promoters suggested that the presence of IRS-1 inhibits ERα activity at estrogen-responsive element-containing DNA. In summary, our data suggested that nuclear IRS-1 interacts with ERα and that this interaction might influence ERα transcriptional activity.