Optimization of adiponectin-derived peptides for inhibition of cancer cell growth and signaling.

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 ß-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.

The designer leptin antagonist peptide Allo-aca compensates for short serum half-life with very tight binding to the receptor.

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.

Leptin and adiponectin: emerging therapeutic targets in breast cancer.

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.

Effects of PPARγ agonists on the expression of leptin and vascular endothelial growth factor in breast cancer cells.

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.

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

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.

Glioblastoma-derived leptin induces tube formation and growth of endothelial cells: comparison with VEGF effects.

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.

miR-20b modulates VEGF expression by targeting HIF-1 alpha 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 (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.

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

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

Insulin receptor substrate 1 may play divergent roles in human colorectal cancer development and progression.

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.

Development of a pharmacologically improved peptide agonist of the leptin receptor.

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.

Estrogen receptor beta-mediated nuclear interaction between IRS-1 and Rad51 inhibits homologous recombination directed DNA repair in medulloblastoma.

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.

Expression of angiogenic regulators, VEGF and leptin, is regulated by the EGF/PI3K/STAT3 pathway in colorectal cancer cells.

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.

Functional analysis of the -2548G/A leptin gene polymorphism in breast cancer cells.

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.

Insulin receptor substrate 1 modulates the transcriptional activity and the stability of androgen receptor in breast cancer cells.

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.

Is BRCA1-5083del19, identified in breast cancer patients of Sicilian origin, a Calabrian founder mutation?

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/HER2 crosstalk in breast cancer: in vitro study and preliminary in vivo analysis.

BACKGROUND: Obesity in postmenopausal women is associated with increased breast cancer risk, development of more aggressive tumors and resistance to certain anti-breast cancer treatments. Some of these effects might be mediated by obesity hormone leptin, acting independently or modulating other signaling pathways. Here we focused on the link between leptin and HER2. We tested if HER2 and the leptin receptor (ObR) can be coexpressed in breast cancer cell models, whether these two receptors can physically interact, and whether leptin can transactivate HER2. Next, we studied if leptin/ObR can coexist with HER2 in breast cancer tissues, and if presence of these two systems correlates with specific clinicopathological features. METHODS: Expression of ObR, HER2, phospho-HER2 was assessed by immunoblotting. Physical interactions between ObR and HER2 were probed by immunoprecipitation and fluorescent immunostaining. Expression of leptin and ObR in breast cancer tissues was detected by immunohistochemistry (IHC). Associations among markers studied by IHC were evaluated using Fisher's exact test for count data. RESULTS: HER2 and ObR were coexpressed in all studied breast cancer cell lines. In MCF-7 cells, HER2 physically interacted with ObR and leptin treatment increased HER2 phosphorylation on Tyr 1248. In 59 breast cancers, the presence of leptin was correlated with ObR (the overall association was about 93%). This result was confirmed both in HER2-positive and in HER2-negative subgroups. The expression of leptin or ObR was numerically more frequent in larger (> 10 mm) tumors. CONCLUSION: Coexpression of HER2 and the leptin/ObR system might contribute to enhanced HER2 activity and reduced sensitivity to anti-HER2 treatments.

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.

Overexpression of the obesity hormone leptin in human colorectal cancer.

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.

Expression of nuclear insulin receptor substrate 1 in breast cancer.

BACKGROUND: Insulin receptor substrate 1 (IRS-1), a cytoplasmic protein transmitting signals from the insulin and insulin-like growth factor 1 receptors, has been implicated in breast cancer. Previously, it was reported that IRS-1 can be translocated to the nucleus and modulate oestrogen receptor alpha (ERalpha) activity in vitro. However, the expression of nuclear IRS-1 in breast cancer biopsy specimens has never been examined. AIMS: To assess whether nuclear IRS-1 is present in breast cancer and non-cancer mammary epithelium, and whether it correlates with other markers, especially ERalpha. Parallel studies were carried out for the expression of cytoplasmatic IRS-1. METHODS: IRS-1 and ERalpha expression was assessed by immunohistochemical analysis. Data were evaluated using Pearson's correlation, linear regression and receiver operating characteristic analysis. RESULTS: Median nuclear IRS-1 expression was found to be low in normal mammary epithelial cells (1.6%) and high in benign tumours (20.5%), ductal grade 2 carcinoma (11.0%) and lobular carcinoma (approximately 30%). Median ERalpha expression in normal epithelium, benign tumours, ductal cancer grade 2 and 3, and lobular cancer grade 2 and 3 were 10.5, 20.5, 65.0, 0.0, 80 and 15%, respectively. Nuclear IRS-1 and ERalpha positively correlated in ductal cancer (p<0.001) and benign tumours (p<0.01), but were not associated in lobular cancer and normal mammary epithelium. In ductal carcinoma, both nuclear IRS-1 and ERalpha negatively correlated with tumour grade, size, mitotic index and lymph node involvement. Cytoplasmic IRS-1 was expressed in all specimens and positively correlated with ERalpha in ductal cancer. CONCLUSIONS: A positive association between nuclear IRS-1 and ERalpha is a characteristic for ductal breast cancer and marks a more differentiated, non-metastatic phenotype.