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- Material and methods
We previously demonstrated that leptin, an adipose-derived hormone, induces cell proliferation in a model of preneoplastic (IMCE (ApcMin/+), but not normal (YAMC (Apc+/+), colon epithelial cells by inducing autocrine IL-6 production and trans-IL-6 signaling. Low serum adiponectin is associated with colon, prostate and breast cancer. Adiponectin is secreted by white adipose tissue; the levels of adiponectin in the blood decrease as body mass index (and leptin) increases. In our study, we tested whether murine recombinant globular adiponectin (gArcp30) could modulate leptin-induced cell proliferation, autocrine IL-6 production, trans-IL-6 signaling and other leptin-induced cell signaling events previously observed in IMCE cells but not YAMC cells. Under serum-free conditions, adiponectin (1 μg/ml) inhibited leptin-induced autocrine IL-6 production, soluble IL-6 receptor shedding, trans-IL-6 signaling and subsequent STAT3 phosphorylation in IMCE cells. Adiponectin inhibited leptin-induced cell proliferation in the IMCE cells and this inhibition was associated with IκB-α phosphorylation, IκB-α degradation and decreased NF-κB p65 DNA activation and binding. These data indicate that adiponectin acts on preneoplastic colon epithelial cells to regulate cell growth via 2 distinct pathways inhibiting leptin-induced NF-κB-dependent autocrine IL-6 production and trans-IL-6 signaling. We hypothesize that adiponectin may be an important regulator of colon epithelial cell homeostasis by linking the observed reduced risk for cancer in populations with high serum adiponectin concentrations to specific mechanisms of cell number homeostasis in a model of preneoplastic colon epithelial cells. These data may have broad implications for diet and lifestyle strategies for the prevention and treatment of obesity-associated cancers. © 2008 Wiley-Liss, Inc.
White adipose tissue secretes hormones and cytokines (also referred to as adipokines) that may play a role in the development of the systemic inflammatory state that is associated with obesity and subsequent cancer risk.1 Adipokines, such as leptin and adiponectin, and cytokines like interleukin-6 (IL-6) may play a direct role in a variety of inflammatory conditions both in vivo and in vitro.2 While these hormones/cytokines are critical to normal cell functioning at homeostatic concentrations, when levels are altered from normal (as in obesity) they may become pathologic. There is strong biological plausibility that an imbalance in these systemic mediators is causally related to obesity-associated cancers like colorectal cancer. However, the mechanisms underlying the obesity-cancer link are not understood.
Inflammation is associated with epithelial cell transformation and the process of carcinogenesis.3, 4 Truncating mutations in the adenomatous polyposis coli (Apc) gene are initiating events in colorectal carcinogenesis; a majority of adenomas in inherited and sporadic forms of colorectal cancers have mutations in Apc.5 In inflammation-associated colorectal cancer, such as cases associated with inflammatory bowel disease, nongenetic stimuli also encourage the survival and proliferation of initiated cells.3 We propose that an imbalance in adipokines, particularly those produced by visceral adipose tissue, may trigger inflammatory stimuli for preneoplastic cell proliferation and survival.
The adipocyte-derived hormone leptin, which plays a crucial role in regulating energy balance, is elevated in obese individuals.1 It is hypothesized that leptin may act locally within the gastrointestinal tract to influence intestinal functions, such as nutrient absorption.6 However, it is unclear whether elevated systemic leptin levels may have pathophysiologic implications for colon epithelial cells and the development of colon cancer, or if it may indirectly mediate the process via a wide variety of other cell types located in the gut. High serum leptin is associated with increased risk of several cancers including, colon,7 prostate8 and breast.7 In addition, leptin levels are positively correlated the likelihood of developing larger and riskier tumors.7, 9
In vitro data are much more consistent regarding the effects of leptin on cell fate. In tumor cell lines, leptin treatment induces cell proliferation in colon,10–12 breast,13, 14 gastric,14 prostate14, 15 and ovarian cancer.16 On the basis of these data, it is likely that leptin has cancer cell stage-specific and tissue-specific actions that ultimately result in a growth promoting effect on neoplastic cells. We previously demonstrated that leptin, a systemic mediator of inflammation associated with obesity, may act on the colon epithelial mucosal microenvironment to promote the survival and proliferation in a model of preneoplastic colon epithelial cells (IMCE (ApcMin/+)) but not normal colon epithelial cells (YAMC (Apc+/+)).17 The difference in leptin-induced cell proliferation we observed is likely due to a trans-IL-6 signaling mechanism activated in IMCE but not YAMC cells.18
Adiponectin is an adipocyte-derived hormone with diverse biological functions including, stimulation of glucose utilization, fatty-acid oxidation and inhibition of gluconeogenesis.19 Serum levels of adiponectin exceed 10 μg/ml, representing ∼0.01% of serum protein, in normal weight individuals.20 Adiponectin self-associates in serum into at least 3 oligomeric complexes, including trimer, hexamer and octadecamer (“high molecular weight;” HMW) forms.21 HMW forms of adiponectin predominate in serum of healthy human subjects; humans with diabetes and metabolic syndrome have a low percentage of HMW adiponectin relative to total adiponectin.22 However, as obesity increases, adiponectin levels decrease and this decrease is thought to be associated with an increased risk of diabetes and cardiovascular disease, implying an early role for adiponectin in disease development.23
To date, 2 specific adiponectin receptors have been identified. The receptors were identified primarily on muscle cells (AdipoR1) and liver cells (AdipoR2).24 AdipoR1 and AdipoR2 serve as receptors for adiponectin in vitro, and their reduction in obesity is correlated with reduced adiponectin sensitivity. Simultaneous disruption of both AdipoR1 and R2 abolished adiponectin binding and actions which resulted in increased tissue triglyceride content, inflammation and oxidative stress leading to insulin resistance and marked glucose intolerance.25
A role for adiponectin receptors in colon cancer progression has not been identified. However, low serum adiponectin is associated with several cancers, including colon,26 breast7 and prostate cancers.27 The mechanism by which low adiponectin may be involved in cancer risk/progression is not understood. It is hypothesized that high serum adiponectin may impart some protective/preventative effect against chronic diseases like cancer.2 Findings from recent in vitro studies suggest that adiponectin may control cancer cell growth. Specifically, globular adiponectin induced cell growth arrest and even apoptosis in MDA-MB-231 breast cancer cells28 as well as antiproliferative and apoptotic responses in human MCF7 breast cancer cells.29
In our study, we tested whether murine recombinant adiponectin could block our previously observed cell proliferation and cell signaling activity induced by leptin. We utilized a model system of conditionally immortalized colon epithelial cell lines to dissect these early events. These cell lines, YAMC (Apc+/+) cells and IMCE (ApcMin/+) cells, respectively display phenotypes consistent with normal and preneoplastic colon epithelial cells observed in human colon epithelial carcinogenesis.30
- Top of page
- Material and methods
Our laboratory has established that leptin-induced signaling pathways, NF-κB and trans-IL-6 pathways, are up regulated in preneoplastic but not normal colon epithelial cells resulting in preneoplastic cell proliferation. In metastatic tumor cells and in mouse models of colitis adiponectin blocked cell proliferation and decreased tumor burden respectively.37–39 These phenotypic observations led us to explore a possible preventative role for adiponectin on our previously observed leptin-induced cell proliferation. In the present study our primary goal was to determine whether adiponectin could inhibit the preneoplastic (IMCE (ApcMin/+) cells) colon epithelial cell proliferation induced by leptin as we previously observed.
While not physiologic based on the fact that adiponectin or leptin do not exist in vivo alone but rather coexist in serum, we wanted to take advantage of our reductionist model system to identify adiponectin-specific phenotypic effects. Initially, we treated both the normal YAMC (Apc+/+) and preneoplastic IMCE (ApcMin/+) cells with adiponectin across a range of concentrations (0.01–25.0 μg/ml) to determine the concentration-dependent effects of adiponectin alone on basal cell proliferation. In serum-free conditions, adiponectin treatment alone slightly blocked YAMC (Apc+/+) and IMCE (ApcMin/+) cell proliferation at the highest concentrations of 10 and 25 μg/ml (Fig. 1a). Co-treatment of IMCE cells with leptin and adiponectin together inhibited leptin-induced cell proliferation (Fig. 1b) and increased caspase activation (Fig. 1c). These data are consistent with the hypothesis that adiponectin desensitizes the cells to proliferative signals evidenced by decreased cell numbers across adiponectin treatment alone or in combination with leptin. In addition, cotreatment of IMCE cells with leptin and adiponectin activated caspases consistent with the phenotypic observation in Figure 1b. It is interesting, while not physiologic, that treatment of IMCE cells with 1 μg/ml adiponectin alone decreased caspase activity (Fig. 1c) with no observed change in cell number (1A). Note that 1 μg/ml adiponectin treatment alone led to decreased NF-κB activation, increased total cytoplasmic IκB-α and decreased phospho- IκB-α (Fig. 5). These observations provide indirect but consistent evidence for IκK activation resulting in inhibition of NF-κB and may explain part of the decreased cell proliferation because of adiponectin treatment alone.
Adiponectin, independently and in the presence leptin, blocked cell proliferation of preneoplastic colon epithelial cells. We hypothesized this effect of adiponectin on leptin-induced cell proliferation could be explained by blocking NF-κB activation or trans-IL-6 signaling. Previously, we showed that leptin induced phosphorylation of p38 and p42/44 and increased NF-κB nuclear translocation and DNA binding.17 Evidence suggests that both p38 and p42/44 MAPKs are involved in NF-κB transactivation.40, 41 MAPKs can mediate the IκK complex and lead to IκB-α freeing bound p65 NF-κB from the cytoplasm and allowing nuclear translocation and DNA binding.42 In addition, MAPKs can directly phosphorylate p65 NF-κB resulting in transactivation and increased DNA binding potential.42 Indeed, in our model system adiponectin cotreatment blocked phosphorylation of p38 and p42/44. Consistent with this observation, cotreatment with adiponectin blocked leptin-induced IκB-α phosphorylation, IκB-α degradation and subsequent NF-κB DNA activation and binding. The inhibitory action of adiponectin on NF-κB activation is consistent with previously reported effects of HMW adiponectin. In vitro, adiponectin blocks proliferation in MCF-7 breast cancer cells,29 MDA-MB-231 breast cancer cells28 and prostate cancer cells.43 Adiponectin also decreases lipopolysaccharide-induced IL-8 production by intestinal epithelial cells.44 The growth inhibitory effects of adiponectin could be due to the ability of adiponectin to selectively bind to several mitogenic growth factors45 and the ability of adiponectin to activate caspases and induce apoptosis.46 Moreover, adiponectin may inhibit NF-kB activation,47 consistent with our findings (Fig. 5).
Previously we showed that leptin activated 2 independent pathways resulting in cell proliferation of preneoplastic colon epithelial cells. After characterizing the effect on NF-kB activation, we wanted to determine whether adiponectin also blocked leptin-induced IL-6 production and trans-IL-6 signaling as previously described. Given that the IL-6 promoter contains a putative NF-κB binding site, it was possible that this upstream action of adiponectin in blocking NF-κB nuclear activation and translocation might be responsible for inhibition of the trans-IL-6 signaling pathway activation in IMCE cells.
Here we show that adiponectin cotreatment blocks leptin-induced IL-6 production and shedding of the IL-6R by IMCE (ApcMin/+) cells (Fig. 3). Blocking IL-6 production resulted in decreased proliferative signal for the IMCE cells. Importantly, another novel observation was that adiponectin cotreatment with IL-6 and leptin blocked shedding of the sIL-6 receptor and increased the shedding of the sgp130 receptor. It is thought that the homeostatic signaling of IL-6 is through the membrane bound IL-6R whereas the proinflammatory process is likely regulated by IL-6 trans-signaling.48 We show that adiponectin can block the trans-IL-6 signaling mechanism induced by leptin. Given these observations, we hypothesized that NF-κB was likely not the only pathway being attenuated by adiponectin. Therefore, we cotreated the cells with IL-6 and adiponectin to identify whether adiponectin could block IL-6 signaling independent of NF-κB. The increase in IL-6 induced by adiponectin alone may constitute a homeostatic signal. Adiponectin treatment alone increased both soluble IL-6R and gp130 not altering the overall balance of the receptors. These data support the hypothesis that IL-6 production may be important for epithelial cell homeostasis.49
Adiponectin blocked key signaling molecules involved in IL-6 signaling (Figs. 4 and 6). Adiponectin cotreatment was able to inhibit IL-6-induced STAT3 phosphorylation, nuclear translocation and activity (Fig. 6). Further we show that the IMCE cell proliferation induced by IL-6 was dependent on STAT3. Chemical inhibition of STAT3 phosphorylation and nuclear translocation was associated with inhibition of IL-6-induced cell proliferation (Fig. 6). Therefore, we concluded that adiponectin could also block IL-6-induced cell proliferation via inhibition of STAT3 activation.
This mechanism is consistent with in vivo studies implicating a role for IL-6 and trans-IL-6 signaling in cancer. IL-6 is an important mediator of inflammatory bowel diseases which are a risk factor for colorectal cancer.50 Recent studies show that serum IL-6 levels were increased in patients with colon carcinoma and correlated with tumor size.51, 52 IL-6 also promotes cell proliferation in vitro in colon cancer cells.53 Persistent STAT3 activation in colon cancer is associated with enhanced cell proliferation and tumor growth.54 The use of STAT3 inhibitors to treat cancer is actively being pursued.55 Results from recent studies suggest that selective targeting of IL-6 trans-signaling may represent a viable strategy for treating cancers dependent on these signaling pathways.56–58 Future experiments will include the addition of recombinant sIL-6R and sgp130 to test the specific interactions between these mediators and leptin- and IL-6-induced IMCE cell growth.
We cannot neglect that the form of adiponectin is increasingly being shown to dictate specific signaling pathway activation. While a globular form (glycosylated homotrimer) of adiponectin was used in our studies, as well as many reported here, we cannot rule out the protein homotrimers associate in culture into oligomeric complexes. It was beyond the scope of this work to determine the distribution of adiponectin oligomers present and the receptors through which their effects are mediated. However, the inhibitory effect of adiponectin in NF-κB activation on these cells is consistent with previously reported effects of HMW adiponectin on NF-κB activation. Globular adiponectin induced NF-κB activation in several cell types while full length adiponectin inhibited activation.59, 60 Our data indicate that adiponectin treatment alone did not activate NF-κB consistent with the HMW adiponectin effect. Future research plans include characterizing the distributions of the forms of adiponectin found when used in culture.
The full length form of adiponectin predominates in serum and globular may be responsible for the proinflammatory effects sometimes observed with adiponectin treatment.61, 62 However, in breast cancer patients, modeling HMW instead of total adiponectin did not offer any additional predictive value of cancer risk.63In vivo, low total serum adiponectin is associated with several cancers including colon,26 prostate,9 breast,7 endometrial64 and gastric cancer.65 Data from the breast cancer study cited above suggests that low serum adiponectin levels and high serum leptin levels are associated with an increased risk for breast cancer.7 In addition, serum adiponectin levels were negatively associated with histologic grade and disease stage.7, 9 In a murine adiponectin KO model, adiponectin was protective against DSS- and TNBS-induced murine colitis.44 Paradoxically, Fayad et al.66 report, in an independently generated adiponectin KO mouse strain, that adiponectin deficiency protects mice from chemically induced colonic inflammation. There are several possible explanation for these differences. The authors attribute the observed disparate outcomes in these 2 adiponectin KO models to differences in the genetic background of mouse strains employed. Other important differences exist in the mouse model used by Fayed et al. These investigators reported the detection of adiponectin mRNA in normal and inflamed colonic tissue in addition to adipose tissue, a finding not observed by Nishihara. According to Nishihara, 7 days of 2.5% DSS treatment induces severe inflammation in both normal and adiponectin KO mice (Nishihara, personal communication). Overall, the preponderance of published data, in vivo and in vitro, supports an antiinflammatory role for adiponectin in inflammation-associated events in cancer.
Our in vitro data shows that adiponectin acts in a mechanistically distinct manner on models of normal and preneoplastic colon epithelial cells. These data indicate that adiponectin acts on preneoplastic colon epithelial cells to regulate cell growth via 2 distinct pathways inhibiting leptin-induced NF-κB-dependent autocrine IL-6 production and trans-IL-6 signaling. As such, adiponectin may be an important stage-dependent regulator of colon epithelial cell homeostasis. In summary, our data provide the first mechanistic evidence that adiponectin may abrogate the proliferative effect of leptin in a model of preneoplastic colon epithelial cells. These findings, if confirmed in relevant human model systems, enhances the biologic plausibility that adiponectin may act at an early stage of carcinogenesis to block leptin-induced colon epithelial cell proliferation.