Leptin pro-angiogenic signature in breast cancer is linked to IL-1 signalling

Background: Leptin and interleukin-1 (IL-1) upregulate vascular endothelial growth factor (VEGF), promote angiogenesis and are related to worse prognosis of breast cancer. However, it is unknown whether leptin regulates IL-1, and whether these effects are related to leptin-induction of VEGF/VEGFR2 in breast cancer. Methods: Several genetic and pharmacological approaches were used to determine the mechanisms involved in leptin regulation of IL-1 system (IL-1α, IL-1β, IL-1Ra and IL-1R tI) and the impact of IL-1 signalling on leptin-induced VEGF/VEGFR2 expression in mouse mammary cancer 4T1 cells (a model that resembles invasive and highly metastatic human breast cancer). Results: Leptin increased protein and mRNA levels of all components of the IL-1 system. IL-1 upregulation involved leptin activation of JAK2/STAT3, MAPK/ERK 1/2, PI-3K/AKT1, PKC, p38 and JNK. Leptin-induced phosphorylation of mTOR/4E-BP1 increased IL-1β and IL-1Ra expression, but downregulated IL-1α. Leptin upregulation of IL-1α promoter was linked to SP1 and NF-κB transcription factors. In addition, leptin receptor (Ob-Rb) was upregulated by leptin. Interestingly, leptin upregulation of VEGF/VEGFR2 was partially mediated by IL-1/IL-1R tI signalling. Conclusions: We show for the first time that leptin induces several signalling pathways to upregulate the translational and transcriptional expression of IL-1 system in breast cancer cells. Moreover, leptin upregulation of VEGF/VEGFR2 was impaired by IL-1 signalling blockade. These data suggest that leptin pro-angiogenic signature in breast cancer is linked to, or regulated, in part by IL-1 signalling.

Obesity, a pandemic in the United States, is associated with more than 100 000 incidents of cancer in the United States every year, particularly cancers of the breast, colon and endometrium. Obese breast cancer patients have increased mortality compared with non-obese (Whiteman et al, 2005). Obesity negatively impacts the survival of breast cancer patients regardless of menopausal status, as it has been positively associated with increased risk of recurrence and increased proportion of breast cancer irresponsive to oestrogens (Daling et al, 2001). The obese gene ligand (leptin), a 16-kDa cytokine, is mainly produced by adipose tissue. Higher levels of leptin are found in female, postmenopausal women and obese individuals. Leptin is a pro-angiogenic, pro-inflammatory and mitogenic factor, the actions of which are reinforced through crosstalk with cytokines/growth factors (Caldefie-Chézet et al, 2005;Gonzalez et al, 2006;Rene Gonzalez et al, 2009;Jarde et al, 2010).
Breast carcinoma cells express higher levels of leptin and its receptor, Ob-R, than normal mammary cells (Miyoshi et al, 2006), and a significant correlation between leptin/Ob-R levels with metastasis and lower survival of breast cancer patients has been found. Moreover, studies on leptin (ob/ob) and Ob-R (db/db) mutant mice have provided compelling data supporting a role for leptin in breast cancer development. These obese mice with deficiency in leptin signalling show a significantly lower incidence of mammary tumours than their lean littermates. MMTV/TGF-a mice have a proclivity to develop mammary tumours, but when crossed with leptin/Ob-R-deficient mice, there is a reduced incidence of mammary tumours in their progeny (Cleary et al, 2003(Cleary et al, , 2004. Furthermore, our published data strongly suggest that leptin is an important factor for breast cancer development. We have shown that the inhibition of leptin signalling in vitro and in vivo by our innovative leptin peptide receptor antagonists (PEG-LPrA) significantly decreased the levels of vascular endothelial growth factor (VEGF) and its receptor type 2 (VEGFR2) before hypoxia is manifested  in breast cancer and stroma cells, while simultaneously reducing establishment and growth of tumours in syngeneic , xenograft (Rene Gonzalez et al, 2009) and 7,12-dimethylbenz(a)anthracene-diet-induced obesity (unpublished) mouse models of breast cancer.
One critical event for tumour growth and metastasis success is the growth of a new network of blood vessels that can be promoted by several cytokines derived from immune, endothelial, epithelial and stromal cells. Leukaemia inhibitory factor, VEGF, interleukin-1 (IL-1) and leptin are known angiogenic factors expressed by mammary cancer cells (Apte et al, 2006a, b). Aberrant inflammatory response over normal immunity facilitates the development of neoplasias. Increased levels of IL-1 are found in breast cancer (Miller et al, 2000). IL-1 family, one of the major pro-inflammatory cytokines, is represented by two ligands: IL-1a and IL-1b, an antagonist: IL-1 receptor antagonist (IL-1Ra) and two receptors: IL-1R tI (type I receptor) and IL-1R tII (type II receptor) (Boraschi et al, 1996). IL-1R tI is an 80-kDa protein, which has an intracytoplasmic domain of about 215 amino acids. IL-1R tI, is the only receptor responsible for transmitting signalling upon IL-1 binding. In contrast, IL-1R tII, a 60-kDa protein with a short intracytoplasmic domain (29 amino acids) serves as a decoy target that reduces the levels of IL-1. IL-1Ra is an inhibitor protein that can bind to IL-1R tI or IL-1R tII with similar affinity without triggering cell signalling effects. Therefore, IL-1Ra represents a natural inhibitor of IL-1 signalling (Apte et al, 2006a, b).
IL-1 is a known inducer of VEGF expression in different tissues and has been described as a factor in cancer development (Carmi et al, 2009;Valdivia-Silva et al, 2009 including VEGF, produced by tumour cells and other cell types in the tumour microenvironment. In turn macrophages and tumour cells secrete IL-1 that contributes to tumour progression by facilitating angiogenesis, matrix remodelling, invasion and metastasis (Chen et al, 2009). Strikingly, the inhibition of IL-1 signalling by exogenous IL-1Ra negatively impacted tumour angiogenesis in nude mice (Voronov et al, 2003). Although, all the mechanism(s) by which leptin contributes to tumour progression are unknown, our published data suggest that specific leptin signalling increase cancer-cell proliferation and the expression of VEGF/VEGFR2 in breast Rene Gonzalez et al, 2009;Gonzalez-Perez et al, 2010;Guo et al, 2010) and endometrial cancer (Carino et al, 2008), and in endometriotic lesions (Styer et al, 2008). The associated expression of leptin and IL-1 has been described in several pathological situations. In endometrial cancer cells leptin induces the expression of IL-1 system (Carino et al, 2008). Leptin and IL-1 signalling can activate NF-kB and increase the levels of VEGF and bcl-2 that could be linked to breast cancer progression (Caldefie-Chézet et al, 2005). Therefore, leptin and IL-1 may have synergistic functions in breast cancer progression. Moreover, their relationships might be more evident in obese (showing higher leptin levels) than in lean individuals.
Despite that both leptin (Caldefie-Chézet et al, 2005;Gonzalez et al, 2006;Rene Gonzalez et al, 2009;Gonzalez-Perez et al, 2010;Guo et al, 2010) and IL-1 systems (Kumar et al, 2003;Voronov et al, 2003) seem to have important roles in tumour angiogenesis and growth no published data is available on the potential relationships between leptin and IL-1 signalling in breast cancer. We hypothesise that the leptin-induced progression of breast cancer could involve the regulation of IL-1 system expression and activation of NF-kB and/ or SP1. Moreover, leptin-induced IL-1 could be related to leptin upregulation of essential pro-angiogenic factors in breast cancer: VEGF/VEGFR2. To test this hypothesis inhibitors of leptin-induced kinases, RNA knockdown for transcription factors and luciferase reporter for IL-1 gene promoter were used in mouse mammary 4T1 cells, a model that closely resembles human invasive/metastasic stage IV breast cancer Gonzalez-Perez et al, 2010). Present data suggest that leptin upregulates the IL-1 system at the transcriptional and translational levels. Remarkably, leptin-induced increase in VEGF and VEGFR2 levels were abrogated by the blockade of IL-1 signalling. Therefore, leptin pro-angiogenic actions in breast cancer may be linked to, or regulated in part by IL-1 signalling.

Cell culture
The mouse mammary tumour cell line 4T1 (CRL-2539; American Type Culture Collection) was cultured on uncoated flat-bottomed plastic plates (cell densities of 1.0, 2.0 or 4.0 Â 10 5 cells per well for 24-, 12-or 6-well plates as described elsewhere . Semi-confluent cells were starved for 24 h in basal medium (RPMI-1640 without fetal bovine serum) and treated with different compounds. In all experiments triplicate wells, tubes and reactions were run and repeated at least three times with different cell preparations. Leptin dose -response and time -course effects 4T1 cells were starved as described above and incubated for 24 h with medium containing leptin (0, 0.6, 1.2 and 6.25 nM, equivalent to 0, 10, 20 and 100 ng ml À1 ). Cell lysates and culture supernatants were used to assess leptin dose -response effects on the levels of IL y c in P D 9 8 0 5 9 P D 9 8 0 5 9 A G 4 9 0 A G 4 9 0 S B 2 0 3 5 8 0 S B 2 0 3 5 8 0 S P 6 0 0 1 2 5 S P 6 0 0 1 2 5 G ö 6 9 7 6 G ö 6 9 7 6 W o r t m a n n in

Western blot analysis
Following cytokine and antibody treatment, cellular lysates were prepared for western blot as described elsewhere . b-actin was used as control.  Flow cytometry assay 4T1 cells were incubated in basal medium for 24 h (to synchronise cells) and further incubated with 1.2 nM leptin for additional 24 h. Cells were re-dispersed, permeabilized and incubated with antibodies for NH2 (all isoforms) and C-terminal (long-isoform) Ob-R domains as described elsewhere (Gonzalez et al, 2003). Goat anti-b-actin antibodies were used as positive control. Cells were analysed by fluorescence-activated cell sorting (BD FACScan, Becton Dickinson, Franklin Lakes, NJ, USA) and, data were analyzed using BD FACSDiva (Becton Dickinson) and FlowJo (TreeStar, Ashland, OR, USA) software.

Immunocytochemistry
To assess leptin effects on IL-1 system expression 4T1 cells (5 Â 10 5 cells per chamber) were cultured in immunocytochemistry-treated glass slides (BD Falcon, Belford, MA, USA) and incubated with 1.2 nM leptin and antibodies for IL-1a, IL-1b and IL-1R tI (Johnston et al, 2008). Negative controls have omitted the primary antibody.

RNA knockdown
Semi-confluent 4T1 cells were cotransfected with SP1 siRNA oligonucleotide, NF-kB1 shRNA and pGL3-IL-1a plasmids, and treated with 1.2 nM leptin for 24 h. Luciferase activities were determined as described above.
Blockade of IL-1R tI 4T1 cells were incubated with 1.2 nM leptin and anti-mouse IL-1R tI antibody or non-specific species-matched IgG2b (0.1 mg ml À1 ). mRNA levels and VEGF protein were determined by ELISA and real-time RT -PCR, respectively. The VEGF and VEGFR2 proteins in cell lysates were analyzed by western blot.

Data analysis
Student's t-test was used for data analysis. Data are presented as mean±s.e.m. Values for Po0.05 were considered statistically significant. The model included the main effects of treatments and replicates.

Leptin dose -response induction of IL-1 system
Leptin induced the expression of all IL-1 system components at protein and mRNA levels ( Figure 1). Leptin-mediated increase in IL-1 protein levels was initially determined by immunohistochemistry ( Figure 1A). Leptin (1.2 nM) increased protein levels of IL-1a, IL-1b and IL-1Ra as determined by ELISA. Leptin effects show bell-shaped dose -response patterns. Leptin upregulation of IL-1 protein levels were found significant at 1.2 nM ( Figure 1B -D). Moreover, leptin upregulation of IL-1 mRNA showed significant changes at lower concentration, that is, 0.6 nM ( Figure 1G -J). Western blot analysis showed that leptin at all doses tested increased the levels of IL-1a, IL-1b and IL-1R tI ( Figure 1E and F).

Leptin time -course regulation of IL-1 system
Protein levels of all components of IL-1 system in 4T1 cells were increased after 24 h of incubation with leptin (Figure 2A -E).

Leptin effects on the regulation of IL-1a promoter
Incubation of cells with leptin increased more than 50% the activity of IL-1a promoter compared with control (Po0.05) ( Figure 5A). The RNA silencing of SP1 and NF-kB negatively affected leptin-mediated induction of IL-1a reporter activity ( Figure 5B).

Leptin upregulates its receptor in 4T1 cells
4T1 cells express Ob-R in basal conditions. Leptin induced a similar increase of Ob-R total (22%; Figure 6B) and Ob-Rb (26%; Figure 6C). These results suggest that leptin mainly increases the expression of the full-functional Ob-Rb.

Impact of IL-1R tI blockade on leptin-induction of VEGF/VEGFR2
Leptin induced almost two-fold and three-fold increase in the levels of VEGF mRNA ( Figure 7A) and protein ( Figure 7C), respectively. Similarly, leptin induced three-fold and two-fold VEGFR2 mRNA ( Figure 7B) and protein ( Figure 7D), respectively. The addition of IgG control antibodies did not alter leptin-mediated effects on VEGF ( Figure 7A and C) or VEGFR2 ( Figure 7B and D). The blockade of IL-1R tI function significantly impaired leptin's effects on VEGF and VEGFR2 (see Figure 7 A -D).

DISCUSSION
Leptin regulates inflammatory cytokines, including IL-1, in diverse tissues and pathological conditions (Chala et al, 2006;Carino et al, 2008;Johnston et al, 2008). Nevertheless, published studies on the relationships between leptin and IL-1 in breast cancer are scarce (Miller et al, 2000). To address whether leptin could regulate IL-1 system in breast cancer, a mouse mammary cancer cell line 4T1 that closely resembles human invasive breast cancer was used. Data from the present investigation show for the first time that leptin upregulates the translational and transcriptional expression of all IL-1 system components. Leptin activates several canonical and non-canonical signalling pathways in 4T1 cells mainly leading to increased levels of two transcription factors, SP1 and NF-kB that were involved in IL-1 gene regulation. In addition, leptin-induced activation of PI-3K signalling pathway was related to increased levels of pmTOR, p70S6K1 and p4E-BP. Leptin upregulation of VEGF/VEGFR2 was partially dependant of IL-1 signalling ( Figure 8). Moreover, leptin upregulated its own receptor, Ob-R, that could reinforce leptin actions on the expression of IL-1 system. These data strongly suggest that leptin pro-inflammatory and pro-angiogenic effects are closely connected. Immunosuppression provoked by excessive inflammatory response could lead to increased tumourigenesis and tumour-cell invasion (Voronov et al, 2003). Several studies suggest that the IL-1 family of cytokines may be important in regulating protumourigenic activities within the breast cancer microenvironment (Miller et al, 2000;Pantschenko et al, 2003;Singer et al, 2003). IL-1b activates the NF-kB pathway and induces both cell migration and proliferation (Wang et al, 2005;Lewis et al, 2006;Streicher et al, 2007). Increased IL-1a expression correlated with the expression of prometastatic (IL-6 and IL-8) and anti-apoptotic genes (TRAF-1 and cIAP-2) (Bhat-Nakshatri et al, 1998;Newton et al, 1999;Patel et al, 2000) and is associated with invasive and metastatic cancer leading to poor prognosis (Miller et al, 2000). Moreover, increased levels of IL-1, due to functional polymorphism of IL-1a gene, enhance hepatocellular carcino susceptibility (Gao et al, 2009). Data from IL-1 KO mice strongly suggest that IL-1 is a crucial factor in determining the balance between immunity and inflammation in tumours (Voronov et al, 2010).
Tumour growth stimulation by IL-1 system appears to depend on cancer cell -stromal cell interactions. Elevated production and levels of IL-1 system components are found in invasive breast cancer (Apte and Voronov, 2002). However, no studies have been performed on the leptin regulation of IL-1 in breast cancer. Furthermore, it is a lack of information on whether high levels of leptin in obese patients parallel the levels of IL-1 in breast cancer. Indeed, one of the factors secreted by tumour and adipose cells is leptin, a small and pleiotropic cytokine, which could be involved in the regulation of tumour-related inflammatory response.
Accumulating evidence suggest that leptin is an important player in breast cancer growth and tumour angiogenesis (Vona-Davis and Rose, 2009;Ray and Cleary, 2010). Elevated levels of leptin/Ob-R in breast cancer are commonly linked to more invasive tumours and worse prognosis (Cleveland et al, 2010). Both leptin and Ob-R are over-expressed in cancer tissue relative to non-cancer epithelium (Ishikawa et al, 2004). Therefore, our present results open the possibilities to further explore these hypotheses.
Present results show that leptin induced the expression of the Ob-Rb with full-signalling capabilities. Therefore, it is possible that high levels of leptin found in the tumour microenvironment, derived either from tumour or adipose cells, can upregulate the expression of Ob-R by tumour cells leading to the increase of tumour growth (Otvos et al, 2008) and expression of proangiogenic and inflammatory factors (Guagnano et al, 2003). In addition, leptin activates several genes involved in cell proliferation by upregulation of CDK2 and cyclin D1 levels (Dieudonne et al, 2002;Okumura et al, 2002).
Leptin at concentrations similar to those found in the serum of normal-weight individuals (i.e., 10-20 ng ml À1 ) induced expression of IL-1 system. Therefore, it is anticipated that higher concentrations of leptin as those found in serum from obese individuals could impact on the expression of IL-1 in breast cancer. Leptin-induced expression of IL-1R tI involved several leptin-induced signalling pathways. However, JAK2 and ERK1/2 were mainly involved in the upregulation of IL-1 ligands and antagonist. These findings are in agreement with leptin's ability to upregulate Ob-Rb in 4T1 cells. Ob-Rb is the only leptin receptor that can signal through pSTAT3 (Frühbeck, 2006). In addition, leptin-induced activation of PKC and JNK signalling pathways increased IL-1Ra levels. Therefore, it could be expected that biological processes leading to reduction of PKC (i.e., by activated ras oncogene) (Weyman et al, 1988) or JNK (i.e., by NF-kB pathway) (Beevers et al, 2006;De Smaele et al, 2001) could downregulate IL-1Ra and increase the actions of IL-1 agonists. Present findings show that leptin-activated SP1 and NF-kB were involved in the regulation of IL-1a promoter Leptin-mediated activation of mTOR, a pathway deregulated in many cancers (Beevers et al, 2006;Knight et al, 2006), was involved in the regulation of IL-1 system. Indeed, leptin activation of pAKT1/ mTOR/p4E-BP1 increased the levels of IL-1b, IL-1Ra and IL-1R tI in 4T1 cells. Present data suggest that IL-1a promoter is under regulatory effects of leptin through activated SP1 and NF-kB. It is known that SP1 activation augments the angiogenic and metastatic capacity of tumour cells through overexpression of multiple downstream genes, including VEGF (Shi et al, 2001). Currently, how leptin activates SP1 in breast cancer cells is unknown.
Because both leptin and IL-1 are inflammatory and proangiogeneic factors that upregulate VEGF, we hypothesised that the association between IL-1 and leptin could be a critical event for tumour angiogenesis. Consequently, the blockade of IL-1R tI partially abrogated leptin-mediated increase of both VEGF and VEGFR2 protein and mRNA. These data strongly suggest that leptin pro-angiogenic signature in breast cancer could partially be mediated by IL-1 signalling. These cytokines could actively crosstalk in breast cancer eliciting pro-inflammatory and proangiogenic effects that contribute to cancer growth.

CONCLUSIONS
Aberrant levels of inflammatory cytokines are a hallmark of breast cancer. Present study further supports the idea that leptin has an important role in breast cancer by increasing the expression of pro-angiogenic and pro-inflammatory factors. Leptin upregulation of IL-1 system could further enhance leptin's actions in breast cancer. Moreover, leptin and IL-1 could have synergistic functions in breast cancer progression promoting VEGF/VEGFR2 expression Rene Gonzalez et al, 2009; Figure 8 Signalling mechanisms for leptin regulation of IL-1 system in 4T1 cells. Leptin upregulated IL-1 system at transcriptional and translational levels. Leptin canonical signalling pathways (JAK2/STAT3, MAPK and PI-3K/ AKT1) were mainly involved in the upregulation of IL-1 system. Leptin activation of PI-3K/AKT1 was related to the phosphorylation of mTOR and its downstream target, 4E-BP1, which is probably linked to upregulation of IL-1b and IL-1Ra. In addition, mTOR negatively regulated IL-1a. Leptin upregulation of IL-1a promoter involved the activation of SP1 and NF-kB. Leptin-induced non-canonical signalling pathways (PKC, p38 and JNK) differentially impacted on the expression of some components of IL-1 system. Leptin-induced upregulation of VEGF/VEGFR2 system was partially mediated by IL-1/IL-1R tI signalling.
et al, 2010) and tumour macrophage recruitment (Cao et al, 2001) that could indirectly augment leptin-mediated angiogenic effects and breast cancer growth. In addition, VEGF signalling transduction is also required to IL-1 induction (Mills et al, 2008). Therefore, understanding of how these various factors cooperate to promote tumour progression could lead to the development of more effective combination therapies to fight breast cancer.