Ruben Rene Gonzalez

Leptin Signaling Promotes the Growth of Mammary Tumors and Increases the Expression of Vascular Endothelial Growth Factor (VEGF) and Its Receptor Type Two (VEGF-R2)

To gain insight into the mechanism(s) by which leptin contributes to mammary tumor (MT) development we investigated the effects of leptin, kinase inhibitors, and/or leptin receptor antagonists (LPrA2) on 4T1 mouse mammary cancer cells in vitro and LPrA2 on 4T1-MT development in vivo. Leptin increases the expression of vascular endothelial growth factor (VEGF), its receptor (VEGF-R2), and cyclin D1 through phosphoinositide 3-kinase, Janus kinase 2/signal transducer and activator of transcription 3, and/or extracellular signal-activated kinase 1/2 signaling pathways. In contrast to leptin-induced levels of cyclin D1 the changes in VEGF or VEGF-R2 were more dependent on specific signaling pathways. Incubation of 4T1 cells with anti-VEGF-R2 antibody increased leptin-mediated VEGF expression suggesting an autocrine/paracrine loop. Pretreatment of syngeneic mice with LPrA2 prior to inoculation with 4T1 cells delayed the development and slowed the growth of MT (up to 90%) compared with controls. Serum VEGF levels and VEGF/VEGF-R2 expression in MT were significantly lower in mice treated with LPrA2. Interestingly, LPrA2-induced effects were more pronounced in vivo than in vitro suggesting paracrine actions in stromal, endothelial, and/or inflammatory cells that may impact the growth of MT. Although all the mechanism(s) by which leptin contributes to tumor development are unknown, it appears leptin stimulates an increase in cell numbers, and the expression of VEGF/VEGF-R2. Together, these results provide further evidence suggesting leptin is a MT growth-promoting factor. The inhibition of leptin signaling could serve as a potential adjuvant therapy for treatment of breast cancer and/or provide a new target for the designing strategies to prevent MT development.

Leptin-Signaling Inhibition Results in Efficient Anti-Tumor Activity in Estrogen Receptor Positive or Negative Breast Cancer

IntroductionWe have shown previously that treatment with pegylated leptin peptide receptor antagonist 2 (PEG-LPrA2) reduced the expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor type 2 (VEGFR2) and growth of 4T1-breast cancer (BC) in syngeneic mice. In this investigation, PEG-LPrA2 was used to evaluate whether the inhibition of leptin signaling has differential impact on the expression of pro-angiogenic and pro-proliferative molecules and growth of human estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) BC xenografts hosted by immunodeficient mice.MethodsTo test the contribution of leptin signaling to BC growth and expression of leptin-targeted molecules, PEG-LPrA2 treatment was applied to severe immunodeficient mice hosting established ER+ (MCF-7 cells; ovariectomized/supplemented with estradiol) and ER- (MDA-MB231 cells) BC xenografts. To further assess leptin and PEG-LPrA2 effects on ER+ and ER- BC, the expression of VEGF and VEGFR2 (protein and mRNA) was investigated in cell cultures.ResultsPEG-LPrA2 more effectively reduced the growth of ER+ (>40-fold) than ER- BC (twofold) and expression of pro-angiogenic (VEGF/VEGFR2, leptin/leptin receptor OB-R, and IL-1 receptor type I) and pro-proliferative molecules (proliferating cell nuclear antigen and cyclin D1) in ER+ than in ER- BC. Mouse tumor stroma in ER+ BC expressed high levels of VEGF and leptin that was induced by leptin signaling. Leptin upregulated the transcriptional expression of VEGF/VEGFR2 in MCF-7 and MDA-MB231 cells.ConclusionsThese results suggest that leptin signaling plays an important role in the growth of both ER+ and ER- BC that is associated with the leptin regulation of pro-angiogenic and pro-proliferative molecules. These data provide support for the potential use of leptin-signaling inhibition as a novel treatment for ER+ and ER- BC.

A peptide derived from the human leptin molecule is a potent inhibitor of the leptin receptor function in rabbit endometrial cells

In this article we show that rabbit endometrial cells express leptin receptor and that human leptin triggers phosphorylation of signal transducer and activator of transcription 3 and up-regulates the expression of interleukin-1 receptor type I as was previously found in human endometrial cells. Interestingly, leptin also upregulates the secretion of leukemia inhibitory factor and expression of its receptor by rabbit endometrial cells. Analysis of a structural model of the leptin-leptin receptor complex suggested that helices I and III of the human leptin structure were likely sites of interaction with the cytokine binding domain of leptin receptor. Accordingly, we synthesized a peptide (LPA-2) comprising helix III (residues 70–95) and investigated its ability to inhibit leptin receptor function. The effects of LPA-2 were assayed in rabbit endometrial cells, and an antileptin receptor antibody and a scrambled version of LPA-2 were used as positive and negative controls, respectively. LPA-2 binds specifically and with high affinity (Ki ∼ 0.6×10−10M) to leptin receptor and is a potent inhibitor of its functions in rabbit endometrial cells. Because leukemia inhibitory factor and interleukin-1 have been implicated in embryo implantation, our results raise the possibility that the LPA-2-induced inhibition of leptin receptor may be exploited to study the actions of leptin in endometrium and in other tissues under conditions characterized by abnormal leptin production.

Leptin regulation of proangiogenic molecules in benign and cancerous endometrial cells.

Several proangiogenic/proinflammatory factors involved in endometrial cancer are regulated by leptin, but the signaling mechanisms responsible for these leptin‐induced actions are largely unknown. Here, we report that in benign (primary and HES) and cancerous‐endometrial epithelial cells (EEC) (An3Ca, SK‐UT2 and Ishikawa), leptin in a dose‐dependent manner regulates vascular endothelial growth factor, (VEGF); interleukin‐1 beta, (IL‐1β); leukemia inhibitory factor, (LIF) and their respective receptors, VEGFR2, IL‐1R tI and LIFR. Remarkably, leptin induces a greater increase in VEGF/VEGFR2 and LIF levels in cancer than in benign cells. However, IL‐1β was only increased by leptin in benign primary‐EEC. Cancer‐EEC expressed higher levels of leptin receptor (full‐length OB‐Rb and short isoforms) in contrast to benign primary‐EEC. Leptin‐mediated activation of JAK2 (janus kinase 2) was upstream to the activation of PI‐3K (phosphatidylinositol‐3 kinase) and/or MAPK (mitogen‐activated protein kinase) signaling pathways. Leptin induction of cytokines/receptors generally involved JAK2 and MAPK activation, but PI‐3K phosphorylation was required for leptin increase of LIF, IL‐1/IL‐1R tI. Leptin‐mediated activation of mTOR (mammalian target of Rapamycin), mainly linked to MAPK, played a central role in leptin regulation of all cytokines and receptors. These results suggest that leptins effects are cell‐specific and could confer a proliferative or cell survival advantage or possibly promote endometrial thickness. Leptins effects on proangiogenic molecules were more evident in malignant versus benign cells and may imply that there is an underlying shift in leptin‐induced cell signaling pathways in endometrial cancer cells.

Effects of leptin, interleukin-1α, interleukin-6, and transforming growth factor-β on markers of trophoblast invasive phenotype

Phenotypic changes of integrin and metalloproteinase secretion of the invasive human cytotrophoblast are regulated by cytokines and growth factors, but how this occurs is not completely understood. We used 24h cytotrophoblast cultures from first trimester pregnancies to investigate the effects of leptin and cytokines on the expression of the α2, α5, and α6 integrin subunits and on the activity of metalloproteinase-2 (gelatinase A) and metalloproteinase-9 (gelatinase B). The α2 subunit was marginally upregulated by leptin and interleukin-1α (IL-1α). All compounds tested upregulated, in some degree, the α5 expression. The α6 integrin subunit was massively upregulated, by leptin, interleukins, and transforming growth factor-β. None of the factors tested affected metalloproteinase-2 activity, but the activity of metalloproteinase-9 was upregulated by leptin and IL-1α. In conclusion, leptin and IL-1α actively induce some of the changes that cytotrophoblasts undergo to achieve a more invasive phenotype. A novel role for leptin is proposed during early pregnancy: leptin might be an autocrine/paracrine regulator of cytotrophoblast invasiveness during implantation and placentation.

Leptin upregulates β3-integrin expression and interleukin-1β upregulates leptin and leptin receptor expression in human endometrial epithelial cell cultures

Human endometrium and endometrial epithelial cells (EECs) either cultured alone or cocultured with human embryos express leptin and leptin receptor. This study compares the effect of leptin with that of interleukin-1β (IL-1β) on the expression of β3-EEC integrin, a marker of endometrial receptivity. Both cytokines increased the expression of β3-EEC at concentrations in the range of 0.06–3 nM; however, leptin exhibited a significantly greater effect than IL-1β. We also determined the regulatory effects of IL-1β on leptin secretion and on the expression of leptin and leptin receptor at the protein level in both EEC and endometrial stromal cell (ESC) cultures. In EEC cultures, IL-1β upregulated secretion of leptin and expression of both leptin and leptin receptors. No effect of IL-1β was found in the ESC cultures. However, leptin exhibited marginal upregulation of leptin receptor. The upregulation of β3-integrin and leptin/leptin receptor expression by IL-1β in EEC cultures indicates that both cytokines may be implicated in embryonic-maternal cross-talk during the early phase of human implantation. Our present data also raise the possibility that leptin is an endometrial molecular effector of IL-1β action on β3-integrin upregulation. Thus, a new role for leptin in human reproduction as an autocrine/paracrine regulator of endometrial receptivity is proposed.