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Background: Despite effective prevention and screening methods, the incidence and mortality rates associated with colorectal cancer (CRC) are still high. Insulin receptor substrate 1 (IRS-1), a signaling molecule involved in cell proliferation, survival and metabolic responses has been implicated in carcinogenic processes in various cellular and animal models. However, the role of IRS-1 in CRC biology and its value as a clinical CRC biomarker has not been well defined., Aim: To evaluate if and how IRS-1 expression and its associations with the apoptotic and proliferation tumor markers, Bax, Bcl-xL and Ki-67 are related to clinicopathological features in human CRC., Methods: The expression of IRS-1, Bax, Bcl-xL and Ki-67 proteins was assessed in tissue samples obtained from 127 patients with primary CRC using immunohistochemical methods. The assays were performed using specific antibodies against IRS-1, Bax, Bcl-xL, Ki-67. The associations between the expression of IRS-1, Bax, Bcl-xL, Ki-67 were analyzed in relation to clinicopathological parameters, i.e. , patient age, sex, primary localization of tumor, histopathological type, grading, staging and lymph node spread. Correlations between variables were examined by Spearman rank correlation test and Fisher exact test with a level of significance at P < 0.05., Results: Immunohistochemical analysis of 127 CRC tissue samples revealed weak cytoplasmatic staining for IRS-1 in 66 CRC sections and strong cytoplasmatic staining in 61 cases. IRS-1 expression at any level in primary CRC was associated with tumor grade (69% in moderately differentiated tumors, G2 vs 31% in poorly differentiated tumors, G3) and with histological type (81.9% in adenocarcinoma vs 18.1% in adenocarcinoma with mucosal component cases). Strong IRS-1 positivity was observed more frequently in adenocarcinoma cases (95.1%) and in moderately differentiated tumors (85.2%). We also found statistically significant correlations between expression of IRS-1 and both Bax and Bcl-xL in all CRC cases examined. The relationships between studied proteins were related to clinicopathological parameters of CRC. No significant correlation between the expression of IRS-1 and proliferation marker Ki-67, excluding early stage tumors, where the correlation was positive and on a high level ( P = 0.043, r = 0.723)., Conclusion: This study suggests that IRS-1 is co-expressed with both pro- and antiapoptotic markers and all these proteins are more prevalent in more differentiated CRC than in poorly differentiated CRC., Competing Interests: Conflict-of-interest statement: All the Authors have no conflict of interest related to the manuscript., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Experimental and clinical data suggest that pro-angiogenic, pro-inflammatory and mitogenic cytokine leptin can be implicated in ocular neovascularization and other eye pathologies. At least in part, leptin action appears to be mediated through functional interplay with vascular endothelial growth factor (VEGF). VEGF is a potent regulator of neoangiogenesis and vascular leakage with a proven role in conditions such as proliferative diabetic retinopathy, age-related macular degeneration and diabetic macular edema. Accordingly, drugs targeting VEGF are becoming mainstream treatments for these diseases. The crosstalk between leptin and VEGF has been noted in different tissues, but its involvement in the development of eye pathologies is unclear. Leptin is coexpressed with VEGF during ocular neovascularization and can potentiate VEGF synthesis and angiogenic function. However, whether or not VEGF regulates leptin expression or signaling has never been studied. Consequently, we addressed this aspect of leptin/VEGF crosstalk in ocular models, focusing on therapeutic exploration of underlying mechanisms. Here we show, for the first time, that in retinal (RF/6A) and corneal (BCE) endothelial cells, VEGF (100 ng/mL, 24 h) stimulated leptin mRNA synthesis by 70 and 30%, respectively, and protein expression by 56 and 28%, respectively. In parallel, VEGF induced RF/6A and BCE cell growth by 33 and 20%, respectively. In addition, VEGF upregulated chemotaxis and chemokinesis in retinal cells by ~40%. VEGF-dependent proliferation and migration were significantly reduced in the presence of the leptin receptor antagonist, Allo-aca, at 100-250 nmol/L concentrations. Furthermore, Allo-aca suppressed VEGF-dependent long-term (24 h), but not acute (15 min) stimulation of the Akt and ERK1/2 signaling pathways. The efficacy of Allo-aca was validated in the rat laser-induced choroidal neovascularization model where the compound (5 μg/eye) significantly reduced pathological vascularization with the efficacy similar to that of a standard treatment (anti-VEGF antibody, 1 μg/eye). Cumulatively, our results suggest that chronic exposure to VEGF upregulates leptin expression and function. As leptin can in turn activate VEGF, the increased abundance of both cytokines could amplify pro-angiogenic and pro-inflammatory environement in the eye. Thus, combined therapies targeting ObR and VEGF should be considered in the treatment of ocular diseases.

Adiponectin, an adipose tissue-excreted adipokine plays protective roles in metabolic and cardiovascular diseases and exerts anti-cancer activities, partially by interfering with leptin-induced signaling. Previously we identified the active site in the adiponectin protein, and generated both a nanomolar monomeric agonist of the adiponectin receptor (10-mer ADP355) and an antagonist (8-mer ADP400) to modulate various adiponectin receptor-mediated cellular functions. As physiologically circulating adiponectin forms multimeric complexes, we also generated an agonist dimer with improved biodistribution and in vitro efficacy. In the current report, we attempted to optimize the monomeric agonist structure. Neither extension of the peptide up to 14-mer analogs nor reinstallation of native residues in permissible positions enhanced significantly the activity profile. The only substitutions that resulted in 5-10-fold improved agonistic activity were the replacement of turn-forming Gly4 and Tyr7 residues with Pro and Hyp, respectively, yielding the more active native β-sheet structure. All peptides retained good stability in human serum exhibiting half-lives >2 h. The cellular efficacy and stability rankings among the peptides followed expected structure-activity relationship trends. To investigate whether simultaneous activation of adiponectin pathways and inhibition of leptin-induced signals can result in cytostatic and anti-oncogenic signal transduction processes, we developed a chimera of the leptin receptor antagonist peptide Allo-aca (placed to the N-terminus) and ADP355 (at the C-terminus). The in vitro anti-tumor activity and intracellular signaling of the chimera were dominated by the more active Allo-aca component. The ADP355 part, however, reversed unfavorable in vivo metabolic effects of the leptin receptor antagonist., (© 2015 Wiley Periodicals, Inc.)

Obesity is a significant risk factor for the development of different cancer types and has been associated with poorer response to oncotherapies and linked to earlier recurrence of the neoplastic disease. While molecular mechanisms of these associations are still under investigation, functional dysregulation of two major fat tissue-derived adipokines, leptin and adiponectin, appears to play an important role. Leptin is known to activate carcinogenic pathways, while adiponectin appears to exert antineoplastic activities and interfere with leptin-induced processes. Because excess body fat is associated with increased leptin expression and adiponectin downregulation, therapeutic rebalancing of these pathways may benefit cancer patients, especially the obese subpopulations. This review focuses on our novel leptin receptor antagonists and adiponectin receptor agonists designed for therapeutic modulation of obesity-associated pathways in cancer.

The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM-low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.

HIV protease inhibitors (PI) are fundamental to combination antiretroviral therapy, which has revolutionized HIV clinical care and produced significant reductions in HIV-associated morbidity and mortality. However, PI administration is frequently associated with severe metabolic impairment, including lipodystrophy, dyslipidemia, and insulin resistance; all of which can contribute to cardiovascular and neurologic co-morbidities. Experimental and epidemiological data support a potentially important role for the adipokine adiponectin in both metabolic and neurologic physiology. This study examined if ADP355, a novel, peptide-based adiponectin receptor agonist, could neutralize the detrimental effects of PI treatment in experimental animal models. Adult male C57BL/6 mice were subjected to a clinically relevant, 4-week regimen of lopinavir/ritonavir, with daily injections of ADP355 administered only during the final 2 weeks of PI exposure. Comprehensive metabolic, neurobehavioral, and biochemical analyses revealed that ADP355 administration partially reversed PI-induced loss of subcutaneous adipose tissue, attenuated PI-induced hyperinsulinemia, hypertriglyceridemia, and hypoadiponectinemia, and prevented PI-induced cognitive impairment and brain injury. Collectively, these data reinforce the link between metabolic co-morbidities and cognitive impairment and suggest that pharmacological reactivation of adiponectin pathways could remediate key aspects of PI-induced metabolic syndrome in clinical settings. Furthermore, therapeutic targeting of adiponectin receptors could show utility in reducing the prevalence and/or severity of HIV-associated neurocognitive disorders.

The leptin receptor antagonist peptide Allo-aca exhibits picomolar activities in various cellular systems and sub-mg/kg subcutaneous efficacies in animal models making it a prime drug candidate and target validation tool. Here we identified the biochemical basis for its remarkable in vivo activity. Allo-aca decomposed within 30 min in pooled human serum and was undetectable beyond the same time period from mouse plasma during pharmacokinetic measurements. The C max of 8.9 μg/mL at 5 min corresponds to approximately 22% injected peptide present in the circulation. The half-life was extended to over 2 h in bovine vitreous fluid and 10 h in human tears suggesting potential efficacy in ophthalmic diseases. The peptide retained picomolar anti-proliferation activity against a chronic myeloid leukemia cell line; addition of a C-terminal biotin label increased the IC50 value by approximately 200-fold. In surface plasmon resonance assays with the biotin-labeled peptide immobilized to a NeutrAvidin-coated chip, Allo-aca exhibited exceptionally tight binding to the binding domain of the human leptin receptor with ka = 5 × 10(5) M(-1) s(-1) and kdiss = 1.5 × 10(-4) s(-1) values. Peptides excel in terms of high activity and selectivity to their targets, and may activate or inactivate receptor functions considerably longer than molecular turnovers that take place in experimental animals.

Obesity is a recognized risk factor for breast cancer development and poorer response to therapy. Two major fat tissue-derived adipokines, leptin and adiponectin have been implicated in mammary carcinogenesis. Leptin appears to promote breast cancer progression through activation of mitogenic, antiapoptotic, and metastatic pathways, while adiponectin may restrict tumorigenic processes primarily by inhibiting cell metabolism. Furthermore, adiponectin is known to counteract detrimental leptin effects in breast cancer models. Thus, therapeutic inhibition of pro-neoplastic leptin pathways and reactivation of anti-neoplastic adiponectin signaling may benefit breast cancer patients, especially the obese subpopulation. This review focuses on current experimental strategies aiming at leptin and adiponectin pathways in breast cancer models. Novel leptin receptor antagonists and adiponectin receptor agonists as well as other compounds for therapeutic modulation of adipokine pathways are discussed in detail, including potential pharmacological advantages and limitations of these approaches.

Background: Emerging evidence suggests that angiogenic and pro-inflammatory cytokine leptin might be implicated in ocular neovascularization. However, the potential of inhibiting leptin function in ophthalmic cells has never been explored. Here we assessed mitogenic, angiogenic, and signaling leptin activities in retinal and corneal endothelial cells and examined the capability of a specific leptin receptor (ObR) antagonist, Allo-aca, to inhibit these functions., Methods and Results: The experiments were carried out in monkey retinal (RF/6A) and bovine corneal (BCE) endothelial cells. Leptin at 50-250 ng/mL stimulated the growth of both cell lines in a dose-dependent manner. The maximal mitogenic response (35±7 and 27±3% in RF6A and BCE cells, respectively) was noted at 24 h of 250 ng/mL leptin treatments. Leptin-dependent proliferation was reduced to base levels with 10 and 100 nM Allo-aca in BCE and RF6A cells, respectively. In both cell lines, leptin promoted angiogenic responses, with the maximal increase in tube formation (163±10 and 133±8% in RF6A and BCE cultures, respectively) observed under a 250 ng/mL leptin treatment for 3 h. Furthermore, in both cell lines 250 ng/mL leptin modulated the activity or expression of several signaling molecules involved in proliferation, inflammatory activity and angiogenesis, such as STAT3, Akt, and ERK1/2, COX2, and NFκB. In both cell lines, leptin-induced angiogenic and signaling responses were significantly inhibited with 100 nM Allo-aca. We also found that leptin increased its own mRNA and protein expression in both cell lines, and this autocrine effect was abolished by 100-250 nM Allo-aca., Conclusions: Our data provide new insights into the role of leptin in ocular endothelial cells and represent the first original report on targeting ObR in ophthalmic cell models.

The obesity hormone leptin has been implicated in breast cancer development. Breast cancer cells express the leptin receptor and are able to synthesize leptin in response to obesity-related stimuli. Furthermore, leptin is a positive regulator of vascular endothelial growth factor (VEGF) and high levels of both proteins are associated with worse prognosis in breast cancer patients. Peroxisome proliferator-activated receptor γ (PPARγ) ligands are therapeutic agents used in patient with Type 2 diabetes and obesity which have recently been studied for their potential anti-tumor effect. Here, we studied if these compounds, ciglitazone and GW1929, can affect the expression of leptin and VEGF in breast cancer cells. In MDA-MB-231 and MCF-7 breast cancer cells, treatment with submolar concentrations of ciglitazone and GW1929 elevated the expression of leptin and VEGF mRNA and protein, and increased cell viability and migration. These effects coincided with increased recruitment of PPARγ to the proximal leptin promoter and decreased association of a transcriptional factor Sp1 with this DNA region., (Copyright © 2012 Wiley Periodicals, Inc.)

The obesity hormone leptin has been implicated in the development and progression of different cancer types, and preclinical studies suggest that targeting leptin signaling could be a new therapeutic option for the treatment of cancer, especially in obese patients. To inhibit pro-neoplastic leptin activity, we developed leptin receptor (ObR) peptide antagonists capable of blocking leptin effects in vitro and in vivo. Our lead compound (Allo-aca), however, crosses the blood-brain-barrier (BBB), inducing undesirable orexigenic effects and consequent weight gain. Thus, redesigning Allo-aca to uncouple its central and peripheral activities should produce a superior compound for cancer treatment. The aim of this study was to generate novel Allo-aca analogs and test their biodistribution in vivo and anti-neoplastic activity in vitro in breast and colorectal cancer cells. Examination of several Allo-aca analogs resulted in the identification of the peptidomimetic, d-Ser, that distributed only in the periphery of experimental animals. d-Ser inhibited leptin-dependent-proliferation of ObR-positive breast and colorectal cancer cells in vitro at 1nM concentration without exhibiting any partial agonistic activity. d-Ser efficacy was demonstrated in monolayer and three-dimensional cultures, and its antiproliferative action was associated with the inhibition of several leptin-induced pathways, including JAK/STAT3, MAPK/ERK1/2 and PI3K/AKT, cyclin D1, and E-cadherin. In conclusion, d-Ser is the first leptin-based peptidomimetic featuring peripheral ObR antagonistic activity. The novel peptide may serve as a prototype to develop new therapeutics, particularly for the management of obesity-related cancers., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Metformin, a derivative of biguanide, is a first-line therapy for type 2 diabetes mellitus. Since the drug has been shown to significantly reduce the risk of various cancers and cancer mortality in diabetic patients, it is being considered as a potential anticancer therapeutic or preventive agent. In cellular models, metformin inhibits the growth of many types of cancer cells; however, its effects on glioblastoma multi-forme (GBM) are not well characterized. Here, we analyzed the effects of metformin on the growth and migration of LN18 and LN229 GBM cells cultured under basal conditions or exposed to leptin, a cytokine that has recently been implicated in GBM development. We found that 2-16 mM metformin reduced basal and leptin-stimulated growth of GBM cells in a dose-dependent manner. Furthermore, the drug significantly inhibited the migration of GBM cells. The action of metformin was mediated through the upregulation of its main signaling molecule, the adenosine monophosphate-activated protein kinase (AMPK), as well as through the downregulation of the signal transducer and activator of transcription 3 (STAT3) and the Akt/PKB serine/threonine protein kinase. In leptin-treated cells, the drug reversed the effects of the cytokine on the AMPK and STAT3 pathways, but modulated Akt activity in a cell-dependent manner. Our results suggest that metformin or similar AMPK-targeting agents with optimized blood-brain-barrier penetrability could be developed as potential treatments of GBM and could be used in conjunction with other target drugs such as leptin receptor antagonists.

Background: 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., Results: 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. 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., Conclusions: 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.

A potential link between obesity, circulating leptin levels and autoimmune disease symptoms suggests that targeting the leptin receptor (ObR) might be a viable novel strategy to combat rheumatoid arthritis (RA). However, studies in animal models and evaluation of clinical cases did not provide clear view on leptin's involvement in RA. To validate ObR as RA target, we used our peptide-based ObR agonists and antagonist in different in vitro and in vivo models of the disease. In human peripheral blood mononuclear cells, leptin and its agonist fragment, desI(2)-E1/Aca, moderately induced constitutive activation of a major proinflammatory transcription factor, NF-κB, while the ObR antagonist peptide Allo-aca inhibited the process. Leptin administration itself did not induce arthritis in rats, but worsened the clinical condition of mice given K/BxN serum transfer arthritis. Simultaneous administration of Allo-aca reduced leptin-dependent increase in disease severity by more than 50%, but the antagonist was ineffective when injected with a 3-day delay. In rats inflicted with mild adjuvant-induced arthritis, both leptin and Allo-aca reduced the extent of joint swelling and the number of arthritic joints. In a more aggressive disease stage, Allo-aca decreased the number of arthritic joints in a dose-dependent manner but did not affect other arthritis markers. In summary, leptin exerts diverse effects on RA depending on the experimental model. This might reflect the heterogeneous character of RA, which is differently impacted by leptin and is unmasked by ObR antagonism. Nevertheless, the results suggest that ObR antagonists might become useful therapeutics in leptin-sensitive early stages of RA., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Leptin is a pleiotropic hormone whose mitogenic and angiogenic activity has been implicated in the development and progression of several malignancies, including brain tumors. In human brain cancer, especially in glioblastoma multiforme (GBM), leptin and its receptor (ObR) are overexpressed relative to normal tissue. Until present, the potential of intratumoral leptin to exert proangiogenic effects on endothelial cells has not been addressed. Using in vitro models, we investigated if GBM can express leptin, if leptin can affect angiogenic and mitogenic potential of endothelial cells, and if its action can be inhibited with specific ObR antagonists. Leptin effects were compared with that induced by the best-characterized angiogenic regulator, VEGF., Results: We found that GBM cell lines LN18 and LN229 express leptin mRNA and LN18 cells secrete detectable amounts of leptin protein. Both lines also expressed and secreted VEGF. The conditioned medium (CM) of LN18 and LN 229 cultures as well as 200 ng/mL pure leptin or 50 ng/mL pure VEGF stimulated proliferation of human umbilical vein endothelial cells (HUVEC) at 24 h of treatment. Mitogenic effects of CM were ~2-fold greater than that of pure growth factors. Furthermore, CM treatment of HUVEC for 24 h increased tube formation by ~5.5-fold, while leptin increased tube formation by ~ 80% and VEGF by ~60% at 8 h. The mitogenic and angiogenic effects of both CM were blocked by Aca 1, a peptide ObR antagonist, and by SU1498, which inhibits the VEGF receptor. The best anti-angiogenic and cytostatic effects of Aca1 were obtained with 10 nM and 25 nM, respectively, while for SU1498, the best growth and angiogenic inhibition was observed at 5 μM. The combination of 5 μM SU1498 and Aca1 at 25 nM (growth inhibition) or at 10 nM (reduction of tube formation) produced superior effects compared with single agent treatments., Conclusions: Our data provide the first evidence that LN18 and LN 229 human GBM cells express leptin mRNA and might produce biologically active leptin, which can stimulate tube formation and enhance proliferation of endothelial cells. Furthermore, we demonstrate for the first time that a peptide ObR antagonist inhibits proangiogenic and growth effects of leptin on endothelial cells, and that the pharmacological potential of this compound might be combined with drugs targeting the VEGF pathway.

Triple-negative breast cancers, which represent 10-20% of all mammary tumours, are characterised by the aggressive phenotype, are often found in younger women and have been associated with poor prognosis. Obesity increases the risk for triple-negative breast cancer development. Because triple-negative breast cancer patients are unresponsive to current targeted therapies and other treatment options are only partially effective, new pharmacological modalities are urgently needed. Here we examined if the leptin (obesity hormone) receptor is a viable target for the treatment of this cancer subtype. In human triple-negative breast cancer tissues, the leptin receptor was expressed in 92% (64/69) and leptin in 86% (59/69) of cases. In a model triple-negative breast cancer cell line MDA-MB-231, the leptin receptor antagonist peptide Allo-aca inhibited leptin-induced proliferation at 50 pM concentration. In an MDA-MB-231 orthotopic mouse xenograft model, Allo-aca administered subcutaneously significantly extended the average survival time from 15.4 days (untreated controls) to 24 and 28.1 days at 0.1 and 1mg/kg/day doses, respectively. In parallel, conventional treatment with 1mg/kg/day intraperitoneal cisplatin prolonged the average survival time to 18.6 days, while administration of 20mg/kg/day oral Tamoxifen (negative control) had no significant survival effects relative to controls. In normal CD-1 mice, Allo-aca produced no systemic toxicity up to the highest studied subcutaneous bolus dose of 50mg/kg, while, as expected, it induced a modest 6-10% body weight increase. Our results indicate that leptin receptor antagonists could become attractive options for triple-negative breast cancer treatment, especially in the obese patient population., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Leptin, a multifunctional hormone, controls various processes in both the central nervous system and in peripheral tissues. Because of the presence of multiple leptin/receptor (ObR) interaction sites and diverse leptin activities, the literature lacks truly monofunctional leptin protein derivatives or fragments. To date, selective ObR antagonists have not been reported. We developed short, pharmacologically advantageous peptide analogs of ObR-binding site III of leptin that acted as selective ObR inhibitors without any partial agonistic activity. These reduced leptin-dependent growth and signaling in cancer cell lines at picomolar and low nanomolar concentrations. In immunocompromised mice the peptides suppressed the growth of rapidly proliferating orthotopic human breast cancer xenografts by 50% when administered either intraperitoneally (i.p.) or subcutaneously (s.c.) for 38 days at a 0.1 mg/kg/day dose. The peptides were distributed to the brain, and when added to growing C57BL/6 normal mice i.p., s.c., or orally, the lead antagonist accelerated normal weight increase without producing any toxic effects. Weight gain increases could not be observed after 10-12 days of treatment indicating that the mice became resistant to the central nervous system activity of leptin antagonists. However, in normal growing rats the intranasal administration at 0.1 mg/kg/day for 20 days resulted in a 2% net total body weight gain without signs of resistance induction. In addition to the potential of these peptides in drug development against primary and metastatic tumors and cachexia, our data confirm that resistance to leptin resides at the blood-brain barrier.

Leptin, a pluripotent adipokine, has been discovered as a hormone controlling energy balance in hypothalamic neuroendocrine centers. In addition, recent studies provided ample evidence that leptin can be produced by cells other than adipocytes, and that the hormone can regulate many physiological processes other than energy balance and appetite. In this context, it is not surprising that both leptin excess as well as leptin insufficiency have been implicated in various pathologies. Consequently, despite initially disappointing results with recombinant leptin as the drug for obesity management, new leptin receptor modifiers have been developed and emerged as potential treatment modalities for numerous metabolic, immunological and neoplastic diseases. The major focus of this paper is a systematic review of current experimental leptin-based therapies, including pharmacological advantages and limitations of each prodrug category.

The incidence of colon cancer has increased in developed countries, possibly due to sedentary lifestyle and high caloric diet. Experimental and epidemiological evidence suggests a link between colon cancer development and adipose tissue-derived circulating hormones. Leptin, a pluripotent cytokine secreted by adipocytes, is a key regulator of appetite and energy balance acting in the brain. On the other hand, leptin also controls many physiological and pathological processes in peripheral organs. Recent studies in colon cancer cell lines and human tumors suggested that leptin and its receptor (ObR) are implicated in colon carcinogenesis, and may serve as new biomarkers and pharmacological targets. Here, we explored, for the first time, whether leptin can affect the biology of colorectal tumor stem cells (CTSCs). We found that our previously established and characterized CTSC clones express ObR and respond to leptin with cell proliferation, activation of the extracellular signal-related kinase (ERK)1/2 and AKT signaling pathways, enhanced growth in soft agar, and improved sphere formation associated with E-cadherin overexpression. Moreover, leptin counteracted cytotoxic effects of 5-fluorouracil, a common colon cancer therapeutic agent. These results suggest that obesity and increased leptin levels might promote colorectal cancer by increasing growth and survival of CTSCs.

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 (CoCl(2) exposure). Using immunoblotting, ELISA, and quantitative real-time PCR, we demonstrated that miR-20b decreased VEGF protein levels at 4 and 24 h following CoCl(2) 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 CoCl(2) treatment was associated with reduced levels of nuclear HIF-1 alpha subunit and STAT3. Chromatin immunoprecipitation (ChIP) assays revealed that HIF-1 alpha, but not STAT3, was recruited to the VEGF promoter following the 4 h of CoCl(2) 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 CoCl(2)-mediated HIF-1 alpha 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., ((c) 2010 Wiley-Liss, Inc.)

Although leptin and its receptor (ObR) have emerged as important cancer biomarkers, the role of the leptin system in brain tumor development remains unknown. We screened 87 human brain tumor biopsies using immunohistochemistry and detected leptin and ObR in 55.2% and 60.9% cases, respectively. In contrast, leptin and ObR were absent in 14 samples of normal brain tissue. The presence of leptin correlated with ObR with overall concordance 80.5%. The leptin/ObR system was highly expressed in glioblastomas and anaplastic astrocytomas, while lower expression of both markers was noted in low-grade astrocytomas and gangliogliomas. The association between leptin/ObR and the degree of tumor malignancy was highly significant (P < 0.001). Using double immunofluorescence of glioblastoma tissues, we found co-expression of leptin with ObR and with the proliferation marker Ki-67 in 87% and 64% of cells, respectively. The leptin/ObR-positive tissues also expressed activated forms of STAT3 and Akt. In line with this finding, ObR-positive glioblastoma cells responded to leptin with cell growth and induction of the STAT3 and Akt pathways as well as inactivation of the cell cycle suppressor Rb. In summary, our data demonstrate that the leptin/ObR system is expressed in malignant brain tumors and might be involved in tumor progression.

Both leptin and vascular endothelial growth factor (VEGF) are growth and angiogenic cytokines that are upregulated in different types of cancer and have been implicated in neoplastic progression. Here we investigated the molecular mechanism by which leptin and VEGF expression are regulated in colon cancer by epidermal growth factor (EGF). In colon cancer cell line HT-29, EGF induced the binding of signal transducer and activator transcription 3 (STAT3) to STAT3 consensus motifs within the VEGF and leptin promoters and stimulated leptin and VEGF mRNA and protein synthesis. All these EGF effects were significantly blocked when HT-29 cells were treated with an inhibitor of the phosphoinositide 3-kinase (PI3K) pathway, LY294002, or with small interfering RNA (siRNA) targeting STAT3. Thus, our study identified the EGF/PI3K/STAT3 signaling as an essential pathway regulating VEGF and leptin expression in EGF-responsive colon cancer cells. This suggests that STAT3 pathways might constitute attractive pharmaceutical targets in colon cancer patients where anti-EGF receptor drugs are ineffective., (Copyright 2009 Wiley-Liss, Inc.)

Leptin is overexpressed in human breast tumors and is produced by breast cancer cells in response to obesity-related stimuli. The leptin promoter polymorphism Lep-2548G/A can be associated with increased leptin secretion by adipocytes and elevated cancer risk. However, molecular mechanisms underlying the link between Lep-2548G/A and breast cancer have never been addressed. Lep-2548G/A is proximal to a binding site for the transcriptional factor Sp1. Furthermore nucleolin, a transcriptional repressor, can bind Sp1 or its consensus site. Consequently, we focused on the impact of Lep-2548G/A on Sp1- and nucleolin-dependent leptin transcription in breast cancer cells. The Lep-2548G/A was identified in a homozygous conformation in BT-474 and SK-BR-3 breast cancer cells, in a heterozygous conformation in MDA-MB-231 cells, and a wild-type Lep-2548G/G sequence was present in MCF-7 and ZR-75-1 cells. The occurrence of Lep-2548A/A and Lep-2548G/A coincided with high and intermediate leptin mRNA expression, respectively, while cells containing Lep-2548G/G expressed low leptin mRNA levels. We demonstrated that the existence of Lep-2548G/A improved efficient recruitment of Sp1 to DNA under insulin treatment, while Sp1 loading on DNA containing Lep-2548G/G was not insulin-dependent. In contrast, nucleolin binding to Lep-2548G/A was downregulated in response to insulin, while it was not regulated on Lep-2548G/G. The presence of Lep-2548G/A was studied in breast cancer epithelial cells by IHC and LCM. Interestingly, all 14 tumors expressing high leptin levels contained Lep-2548A/A. In conclusion, the occurrence of Lep-2548G/A can enhance leptin expression in breast cancer cells via Sp1- and nucleolin-dependent mechanisms and possibly contribute to intratumoral leptin overexpression., (2009 UICC.)

Breast cancer development and progression is regulated by growth factors and steroid hormones. Although the majority of human breast cancers expresses androgen receptor (AR), the role of androgens in breast tumorigenesis remains largely unexplored. Here we demonstrate that an AR ligand, 5-alpha-dihydrotestosterone (DHT), inhibits MCF-7 breast cancer cell growth induced by insulin like growth factor 1 (IGF-I). Our results show that DHT induces association of AR with IRS-1, the major IGF-1 receptor signaling molecule. The AR/IRS-1 complex translocates to the nucleus and is recruited to gene promoters containing androgen responsive elements causing an increase of AR transcriptional activity. Moreover, IRS-1 knockdown suggests that IRS-1/AR interaction decreases the ubiquitin/proteasome dependent degradation of AR, increasing its stability. Taken together, these data indicate that nuclear IRS-1 is a novel AR regulator required to sustain AR activity and demonstrate, for the first time in breast cancer cells, the existence of a functional interplay between the IGF system and AR. This interplay may represent the molecular basis of mechanisms through which androgens exert their inhibitory role on the proliferation of breast cancer cells.

In medulloblastomas, which are highly malignant cerebellar tumors of the childhood genotoxic treatments such as cisplatin or gamma-irradiation are frequently associated with DNA damage, which often associates with unfaithful DNA repair, selection of new adaptations and possibly tumor recurrences. Therefore, better understanding of molecular mechanisms which control DNA repair fidelity upon DNA damage is a critical task. Here we demonstrate for the first time that estrogen receptor beta (ERbeta) can contribute to the development of genomic instability in medulloblastomas. Specifically, ERbeta was found highly expressed and active in mouse and human medulloblastoma cell lines. Nuclear ERbeta was also present in human medulloblastoma clinical samples. Expression of ERbeta coincided with nuclear translocation of insulin receptor substrate 1 (IRS-1), which was previously reported to interfere with the faithful component of DNA repair when translocated to the nucleus. We demonstrated that ERbeta and IRS-1 bind each other, and the interaction involves C-terminal domain of IRS-1 (aa 931-1233). Following cisplatin-induced DNA damage, nuclear IRS-1 localized at the sites of damaged DNA, and interacted with Rad51--an enzymatic component of homologous recombination directed DNA repair (HRR). In medulloblastoma cells, engineered to express HRR-DNA reporter plasmid, ER antagonist, ICI 182,780, or IRS mutant (931-1233) significantly increased DNA repair fidelity. These data strongly suggest that both molecular and pharmacological interventions are capable of preventing ERbeta-mediated IRS-1 nuclear translocation, which in turn improves DNA repair fidelity and possibly counteracts accumulation of malignant mutations in actively growing medulloblastomas.

Various studies have been published in Italy regarding the different BRCA1 mutations, but only the BRCA1-5083del19 mutation is recurrent and specific to individuals of Italian descent with a founder effect on the Calabrian population. In our previous study, BRCA1-5083del19 mutation carriers were found in four index cases of 106 Sicilian patients selected for familial and/or hereditary breast/ovarian cancers. The high frequency rate of this mutation identified in the Sicilian population led us to perform haplotype analysis in all family carriers. Five highly polymorphic microsatellite markers were used (D17S1320, D17S932, D17S1323, D17S1326, D17S1325) to establish whether or not all these families had a common ancestor. This analysis showed that all mutation carriers of these families had a common allele. None of the non-carriers of the mutation or of the 50 healthy Sicilian controls showed this haplotype. This allelotype analysis highlighted the presence of a common allele (ancestor), thus suggesting the presence of a founder effect in the Sicilian population. Our results are in contrast with other studies but only the allelotype analysis of all the BRCA1-5083del19 mutation carriers of two neighboring regions of the south of Italy (Calabria and Sicily) will make it possible to identify the real ancestor of this mutation.

Leptin, a hormone produced by adipose tissue, regulates energy balance in the hypothalamus and is involved in fertility, immune response and carcinogenesis. The existence of disorders related to leptin deficit and leptin overabundance calls for the development of drugs activating or inhibiting the leptin receptor (ObR). We synthesized four proposed receptor-binding leptin fragments (sites I, IIa and IIb, III), their reportedly antagonist analogs, and a peptide chimera composed of the two discontinuous site II arms. To assess the pharmacological utility of leptin fragments, we studied the peptides' ability to stimulate the growth of ObR-positive and ObR-negative cells. The combined site II construct and site III derivatives selectively reversed leptin-induced growth of ObR-positive cells at mid-nanomolar concentrations. However, these peptides appeared to be partial agonists/antagonists as they activated cell growth in the absence of exogenous leptin. A designer site III analog, featuring non-natural amino acids at terminal positions to decrease proteolysis and a blood-brain barrier (BBB) penetration-enhancing carbohydrate moiety, proved to be full agonist to ObR, i.e., stimulated proliferation of different ObR-positive but not ObR-negative cells in the presence or absence of leptin. This glycopeptide bound to isolated ObR on solid-phase assays and activated ERK-1/2 signaling in ObR-positive MCF-7 cells at 100-500 nM concentrations. The glycopeptide was stable in mouse serum, readily crossed endothelial/astrocyte cell layers in a cellular BBB model, and was distributed into the brain of Balb/c mice after intraperitoneal administration. These characteristics suggest a potential pharmaceutical utility of the designer site III glycopeptide in leptin-deficient diseases.

Pathologic conditions associated with hyperinsulinemia, such as obesity, metabolic syndrome, and diabetes, seem to increase the risk of breast cancer. Here, we studied molecular mechanisms by which insulin activates the expression of leptin, an obesity hormone that has been shown to promote breast cancer progression in an autocrine or paracrine way. Using MDA-MB-231 breast cancer cells, we found that (a) insulin stimulated leptin mRNA and protein expression, which was associated with increased activation of the leptin gene promoter; (b) insulin increased nuclear accumulation of transcription factors hypoxia inducible factor (HIF)-1alpha and Sp1 and their loading on the leptin promoter; (c) small interfering RNA (siRNA)-mediated knockdown of either HIF-1alpha or Sp1 significantly down-regulated insulin-induced leptin mRNA and protein expression; further inhibition of leptin expression was observed under the combined HIF-1alpha and Sp1 siRNA treatment; (d) inhibition of extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol-3-OH kinase (PI-3K) pathways significantly, albeit partially, decreased insulin-dependent leptin mRNA and protein expression, which coincided with reduced association of HIF-1alpha and/or Sp1 with specific leptin promoter regions; and (e) inhibition of ERK1/2 reduced recruitment of both HIF-1alpha and Sp1 to the leptin promoter, whereas down-regulation of PI-3K influenced only HIF-1alpha binding. In summary, our data suggest that hyperinsulinemia could induce breast cancer progression through leptin-dependent mechanisms. In MDA-MB-231 cells, this process requires Sp1- and HIF-1alpha-mediated leptin gene transcription and is partially regulated by the PI-3K and ERK1/2 pathways.

Purpose: To evaluate the contribution of germline BRCA1 mutations in the incidence of hereditary and familial Breast Cancer (BC) and/or Ovarian Cancer (OC) in patients from Southern Italy (in the region of Sicily) and to identify a possible association between the higher frequency of BRCA1 mutations and a specific familial profile., Experimental Design: A consecutive series of 650 patients with BC and/or OC diagnosed between 1999 and 2005 were recruited from the Southern Italian region of Sicily, after interview at the "Regional Reference Centre for the Characterization and Genetic Screening of Hereditary Tumors" at the University of Palermo. Genetic counselling allowed us to recruit a total of 106 unrelated families affected with breast and/or ovarian cancer screened for mutations occurring in the whole BRCA1 gene by automatic direct sequencing., Results: Germline BRCA1 mutations were found in 17 of 106 (16%) Sicilian families. The HBOC profile had a major frequency (66%) of mutations (P < 0.01). A total of 28 sequence variants was identified. Seven of these were pathogenic, 5 unknown biological variant (UV) and 16 polymorphisms. We also identified a pathological mutation (4843delC) as a possible Sicilian founder mutation., Conclusions: The present study is the first BRCA1 disease-associated mutations analysis in Southern Italian families. The early age of onset of such tumors and the association with the HBOC familial profile could be two valid screening factors for the identification of BRCA1 mutation carriers. Finally, we identified a BRCA1 mutation with a possible founder effect.

Background: Leptin is an adipocyte-derived neurohormone, high levels of which are found in obese individuals. Leptin controls energy expenditure, acting in the brain, and regulates different processes in peripheral organs. Recent studies have suggested that leptin may be involved in cancer development and progression., Aims: To analyse leptin expression in human colorectal cancer as well as in colorectal mucosa and colorectal adenomas., Methods: Leptin expression was assessed by immunohistochemistry in 166 colorectal cancers, 101 samples of colorectal mucosa and 41 adenomas. Leptin concentration in colorectal cancer was correlated with selected clinicopathological features., Results: Immunoreactivity for leptin was observed in 51.2% (85/166) of primary colorectal cancers. In adenomas leptin expression was observed in 14.6% (6/41) of studied cases. In normal mucosa, leptin was present at low levels, except in tumour bordering areas where its concentration appeared to reflect levels in the adjacent cancer tissue. Leptin expression in colorectal cancer significantly correlated with tumour G2 grade (p = 0.002) as well as with histological type (adenocarcinoma) of tumours (p = 0.044)., Conclusions: Results indicate that leptin is overexpressed in human colorectal cancer, which suggests that the hormone might contribute to colorectal cancer development and progression.

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.

Background: The obesity hormone, leptin, has been found to play a role in development and proliferation of normal and malignant tissues. Leptin activity is mediated through the leptin receptor (ObR) that is often expressed in different human cancer cells. Previously, we found that the expression of leptin and ObR can be stimulated by hypoxia-mimetic agents. The aim of this study was to analyze the abundance of and relationships among leptin, ObR and hypoxia-inducible factor-1alpha (HIF-1alpha, transcriptional regulator) in human colorectal cancer., Materials and Methods: We investigated the expression of leptin, ObR and HIF-1alpha in colorectal cancer specimens from 135 patients who underwent curative resection., Results: Immunoreactivity for leptin, ObR and HIF-1alpha protein was observed in 69 of 135 (51.1%), 129 of 135 (95.5%) and 88 of 135 (65.2%) of colorectal cancers, respectively. Statistically significant positive correlations were noted between leptin and HIF-1alpha (P = 0.005, r = 0.243), ObR and HIF-1alpha (P < 0.001, r = 0.325) as well as leptin and ObR (P < 0.001, r = 0.426) in the group of all patients as well as in various subgroups depending on clinicopathological features., Conclusions: The results indicate that the leptin system is overexpressed in human colorectal cancer and this overexpression appears to be associated with the abundance of HIF-1alpha.

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.

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.

Breast cancer development and progression is regulated by crosstalk between steroid hormones (SHs) (e.g., estrogens, progestins and androgens) and growth factors such as insulin-like growth factors (IGFs), insulin, epidermal growth factors (EGFs), transforming growth factors, and vascular endothelial growth factor. The biological effects of SHs are mediated by the nuclear receptors acting as transcriptional activators. Steroid hormone receptors (SRs), in addition to being induced by their own ligands, are also regulated by cellular kinases activated by growth factors. Growth factors are known to influence the expression and activity of SRs as well as regulate the action of various SR transcriptional co-factors. In turn, the expression of growth factor receptors, their ligands, and signaling molecules is often controlled by SHs. This review will focus on crosstalk between the IGF-I system and several SRs implicated in breast cancer.

The putative role of TP53 and p16(INK4A) tumor suppressor genes and Ras oncogenes in the development and progression of salivary gland neoplasias was studied in 28 cases of pleomorphic adenomas (PA), 4 cases of cystic adenocarcinomas, and 1 case of carcinoma ex-PA. Genetic and epigenetic alterations in the above genes were analyzed by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP) and sequencing and by Methylation Specific-PCR (MS-PCR). Mutations in TP53 were found in 14% (4/28) of PAs and in 60% (3/5) of carcinomas. Mutations in H-Ras and K-Ras were identified in 4% (1/28) and 7% (2/28) of PAs, respectively. Only 20% (1/5) of carcinomas screened displayed mutations in K-Ras. p16(INK4A) promoter hypermethylation was found in 14% (4/28) of PAs and 100% (5/5) carcinomas. All genetic and epigenetic alterations were detected exclusively in the epithelial and transitional tumor components, and were absent in the mesenchymal parts. Our analysis suggests that TP53 mutations and p16(INK4A) promoter methylation, but not alterations in the H-Ras and K-Ras genes, might be involved in the malignant progression of PA into carcinoma., (Copyright 2006 Wiley-Liss, Inc.)

Estrogens exhibit important biological functions and influence several pathological processes of hormone-dependent diseases. The biological actions of estrogens require their interaction with two estrogen receptors (ER-alpha and ER-beta), which are ligand-dependent transcription factors. ER-alpha and ER-beta exhibit distinct tissue expression patterns as well as show different patterns of gene regulation. In addition, it has been suggested that ER-beta works as a counter partner of ER-alpha through inhibition of the transactivating functions of ER-alpha. For instance, ER-beta seems to play a different role in breast tumorigenesis than ER-alpha, as ER-beta decreased expression in breast cancer has been correlated with bad prognosis. Biological activities of ER-alpha and ER-beta could be controlled by a number of interacting proteins such as activators/inhibitors, ligand binding and kinases. We have previously reported that pRb2/p130, retinoblastoma related protein, could be involved in the silencing of ER-alpha gene during breast tumorigenesis. Here, we report that ER-beta and pRb2/p130 proteins co-immunoprecipitate in both nucleus and cytoplasm of MCF-7 breast cancer cells. Our hypothesis is that the interaction of pRb2/130 with ER-beta may have a functional significance in regulating ER-beta activity.

Insulin receptor substrate 1 (IRS-1) is a major downstream signaling protein for insulin and insulin-like growth factor I (IGF-I) receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In breast cancer, IRS-1 overexpression has been associated with tumor development, hormone-independence and antiestrogen-resistance. In part, these effects are related to potentiation of IRS-1/PI-3K/Akt signaling. In estrogen sensitive breast cancer cell lines, tamoxifen treatment reduces IRS-1 expression and function; consequently, inhibiting IRS-1/PI-3K signaling. We tested whether anti-IRS1 siRNA could inhibit growth and survival of estrogen-sensitive MCF-7 breast cancer cells, when used alone or in combination with TAM. Our results indicated: (a) out of four tested anti-IRS1 siRNAs, two siRNAs reduced IRS-1 protein by approximately three-fold in both growing and IGF-I-stimulated cells without affecting a closely related protein, IRS-2; (b) these effects paralleled IRS1 mRNA downregulation by approximately three-fold, measured by quantitative real time-polymerase chain reaction; (c) action of anti-IRS1 siRNAs induced the apoptotic response, observed by altered mitochondrial membrane potential coupled with downregulation of NF-kappaB target Bcl-xL and reduced cell viability; (d) anti-IRS1 siRNA treatment enhanced the cytotoxic effects of TAM by approximately 20%. In summary, anti-IRS1 RNAi strategy could become a potent tool to induce breast cancer cell death, especially if combined with standard TAM therapy., (Copyright 2006 Wiley-Liss, Inc.)

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.

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.

To prospectively evaluate the prognostic significance of TP53, H-, K-, and N-Ras mutations, DNA-ploidy and S-phase fraction (SPF) in patients affected by locally advanced laryngeal squamous cell carcinoma (LSCC). Eight-one patients (median follow-up was 71 months) who underwent resective surgery for primary operable locally advanced LSCC were analyzed. Tumor DNA was screened for mutational analysis by PCR/SSCP and sequencing. DNA-ploidy and SPF were performed by flow cytometric analyses. Thirty-six patients (44%) had, at least, a mutation in the TP53 gene. Of them, 22% (8/36) had double mutations and 3% (1/36) had triple mutations. In total, 46 TP53 mutations were observed. The majority (41%) of these occur in exon 5 (19/46), while the mutations in exons 6, 7, and 8 were represented in 14, 7, and 6 patients, respectively (31%, 15%, and 16%). Five LSCC patients (6%) showed a mutation in H-Ras gene. Sixty-three percent of the cases (51/81) were DNA aneuploidy, 14% of these (7/51) were multiclonal. Thirty-nine patients (48%) had an high SPF value. At Univariate analysis, the DNA aneuploidy, high SPF (>15.1%), TP53 mutations and, in particular, the mutations that occur in exons 5 and 8 were significantly related to quicker disease relapse and short OS. At Multivariate analysis, the major significant predictors for both disease relapse and death were high SPF and any TP53 mutations. While histological grade G3 was an independent factor only for relapse. In conclusions, any TP53 mutations and high SPF are important biological indicators to predict the outcome of LSCC patients., (Copyright 2005 Wiley-Liss, Inc.)

Previous work has shown that the transcriptional regulator beta-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 beta-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 beta-catenin, resulting in the activation of the beta-catenin target genes. IGF-1-mediated nuclear translocation of beta-catenin is facilitated by the nuclear translocation of IRS-1. Both IRS-1 and beta-catenin are recruited to the cyclin D1 promoter, an established target for beta-catenin, but only IRS-1 is recruited to the ribosomal DNA (rDNA) promoter. UBF proteins (known to interact with both IRS-1 and beta-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 beta-catenin. The ability of IRS-1 to localize to both RNA polymerase II (with beta-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 beta-catenin and transformation of normal mouse embryo fibroblasts (colony formation in soft agar).

Insulin receptor substrate-1 (IRS-1) mediates signaling from the insulin-like growth factor type-I receptor. We found that all-trans retinoic acid (RA) decreases IRS-1 protein levels in MCF-7, T47-D, and ZR75.1 breast cancer cells, which are growth arrested by RA, but not in the RA-resistant MDA-MB-231 and MDA-MB-468 cells. Based on prior reports of ubiquitin-mediated degradation of IRS-1, we investigated the ubiquitination of IRS-1 in RA-treated breast cancer cells. Two proteasome inhibitors, MG-132 and lactacystin, blocked the RA-mediated degradation of IRS-1, and RA increased ubiquitination of IRS-1 in the RA-sensitive breast cancer cells. In addition, we found that RA increases serine phosphorylation of IRS-1. To elucidate the signaling pathway responsible for this phosphorylation event, pharmacologic inhibitors were used. Two PKC inhibitors, but not a MAPK inhibitor, blocked the RA-induced degradation and serine phosphorylation of IRS-1. We demonstrate that RA activates PKC-delta in the sensitive, but not in the resistant cells, with a time course that is consistent with the RA-induced decrease of IRS-1. We also show that: (1) RA-activated PKC-delta phosphorylates IRS-1 in vitro, (2) PKC-delta and IRS-1 interact in RA-treated cells, and (3) mutation of three PKC-delta serine sites in IRS-1 to alanines results in no RA-induced in vitro phosphorylation of IRS-1. Together, these results indicate that RA regulates IRS-1 levels by the ubiquitin-proteasome pathway, involving a PKC-sensitive mechanism.

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 alpha-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 alpha degradation was probed by Western immunoblotting, fluorescence microscopy, and pulse-chase experiments. Leptin effects on estrogen receptor alpha-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 alpha degradation, inhibited nuclear estrogen receptor alpha expression, and reduced estrogen receptor alpha-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 alpha in breast cancer cells. Thus, high leptin levels in obese breast cancer patients might contribute to the development of antiestrogen resistance.

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 alpha (ERalpha)-positive MCF-7 cells, (1) a fraction of IRS-1 was translocated to the nucleus upon 17-beta-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 ERalpha; (4) the IRS-1:ERalpha complex was recruited to the E2-sensitive pS2 gene promoter. Notably, IRS-1 interaction with the pS2 promoter did not occur in ERalpha-negative MDA-MB-231 cells, but was observed in MDA-MB-231 cells retransfected with ERalpha. Transcription reporter assays with E2-sensitive promoters suggested that the presence of IRS-1 inhibits ERalpha activity at estrogen-responsive element-containing DNA. In summary, our data suggested that nuclear IRS-1 interacts with ERalpha and that this interaction might influence ERalpha transcriptional activity.