A phenotype of IGFBP-3 knockout mice revealed by dextran sulfate-induced colitis.

BACKGROUND AND AIM: Insulin-like growth factor-1 (IGF-1) bioactivity has been shown to be attenuated by insulin-like growth factor binding protein-3 (IGFBP-3), one of six IGF-binding proteins. While prior work revealed no major phenotype associated with IGFBP-3 knockout mice, we explored the possibility that a phenotype could be revealed under specific conditions of gastrointestinal stress. METHODS: The dextran sodium sulfate (DSS) murine model of ulcerative colitis was used for this study. RESULTS: Insulin-like growth factor binding protein-3 knockout mice had significantly reduced colitis on exposure to DSS as measured by lower levels of pro-inflammatory cytokines IL-6 (P < 0.0001), TNF-α (P = 0.0035), and IL-1ß (P = 0.0112), reduced weight loss (P < 0.0001), reduced myeloperoxidase activity (P = 0.0025), and maintenance of colorectal length (P < 0.05), all relative to wild-type mice exposed to DSS. IGFBP-3 knockout mice also exhibited increased colon epithelial cell proliferation (P < 0.0001) following DSS exposure. Semi-quantitative immunohistochemistry showed greater IGF-1 receptor activation in colon epithelial cells of IGFBP-3 knockout mice compared with control mice following DSS exposure. CONCLUSION: Our data demonstrate that IGFBP-3 influences severity of DSS-induced colitis. The observations suggest that in the absence of IGFBP-3, enhanced IGF bioactivity leads to increased epithelial proliferation and mucosal barrier repair, thereby lessening inflammation.

Germ line knockout of IGFBP-3 reveals influences of the gene on mammary gland neoplasia.

Insulin-like growth factor binding protein-3 (IGFBP-3) is an important carrier protein for insulin-like growth factors (IGFs) in the circulation. IGFBP-3 antagonizes the growth-promoting and anti-apoptotic activities of IGFs in experimental systems, but in certain contexts can increase IGF bioactivity, probably by increasing its half-life. The goal of this study was to investigate the role of IGFBP-3 in breast carcinogenesis and breast cancer metastasis. In the first part of the study, we exposed IGFBP-3 knockout and wild-type female mice to dimethylbenz[a]anthracene (DMBA) and followed them for appearance of primary tumors for up to 13 months. In the second part, mice of each genotype received an IV injection of 4T1 mammary carcinoma cells and then lung nodules were counted. Our results show that IGFBP-3 knockout mice developed breast tumors significantly earlier than the wild-type (13.9 ± 1.1 versus 22.5 ± 3.3 weeks, respectively, P = 0.0144), suggesting tumor suppression activity of IGFBP-3. In tumors of IGFBP-3 knockout mice, levels of phospho-AKT(Ser473) were increased compared to wild-type mice. The lung metastasis assay showed significantly more and larger lung nodules in IGFBP-3 knockout mice than in wild-type mice. While we observed increased levels of IGFBP-5 protein in the IGFBP-3 knockout mice, our findings suggest that this was not sufficient to completely compensate for the absence of IGFBP-3. Even though knockout of IGFBP-3 is associated with only a subtle phenotype under control conditions, our results reveal that loss of this gene has measurable effects on breast carcinogenesis and breast cancer metastasis.

Distinct perturbation of the translatome by the antidiabetic drug metformin.

Metformin has been reported to lower cancer incidence among type II diabetics. Metformin exhibits antiproliferative and antineoplastic effects associated with inhibition of mammalian target of rapamycin complex 1 (mTORC1), but the mechanisms are poorly understood. We provide a unique genome-wide analysis of translational targets of canonical mTOR inhibitors (rapamycin and PP242) compared with metformin, revealing that metformin controls gene expression at the level of mRNA translation to an extent comparable to that of canonical mTOR inhibitors. Importantly, metformin's antiproliferative activity can be explained by selective translational suppression of mRNAs encoding cell-cycle regulators via the mTORC1/eukaryotic translation initiation factor 4E-binding protein pathway. Thus, metformin selectively inhibits translation of mRNAs encoding proteins that promote neoplastic proliferation, which should facilitate studies on metformin and related biguanides in cancer prevention and treatment.

Metformin-induced reductions in tumor growth involves modulation of the gut microbiome.

BACKGROUND/PURPOSE: Type 2 diabetes and obesity increase the risk of developing colorectal cancer. Metformin may reduce colorectal cancer but the mechanisms mediating this effect remain unclear. In mice and humans, a high-fat diet (HFD), obesity and metformin are known to alter the gut microbiome but whether this is important for influencing tumor growth is not known. METHODS: Mice with syngeneic MC38 colon adenocarcinomas were treated with metformin or feces obtained from control or metformin treated mice. RESULTS: We find that compared to chow-fed controls, tumor growth is increased when mice are fed a HFD and that this acceleration of tumor growth can be partially recapitulated through transfer of the fecal microbiome or in vitro treatment of cells with fecal filtrates from HFD-fed animals. Treatment of HFD-fed mice with orally ingested, but not intraperitoneally injected, metformin suppresses tumor growth and increases the expression of short-chain fatty acid (SCFA)-producing microbes Alistipes, Lachnospiraceae and Ruminococcaceae. The transfer of the gut microbiome from mice treated orally with metformin to drug naïve, conventionalized HFD-fed mice increases circulating propionate and butyrate, reduces tumor proliferation, and suppresses the expression of sterol response element binding protein (SREBP) gene targets in the tumor. CONCLUSION: These data indicate that in obese mice fed a HFD, metformin reduces tumor burden through changes in the gut microbiome.

Effects of castration on insulin levels and glucose tolerance in the mouse differ from those in man.

BACKGROUND: Plasma insulin concentration is increased in prostate cancer patients during androgen deprivation therapy (ADT) and hyperinsulinemia has been associated with aggressive prostate cancer behavior. To investigate the possible role of castration-induced hyperinsulinemia as a mechanism that may attenuate the beneficial effects of ADT in patients with prostate cancer, a murine model would be useful. We therefore investigated long-term metabolic effects of castration in several mouse models. METHODS: We studied the long-term influence of castration on energy intake, body weight, glucose tolerance, plasma-insulin, plasma insulin-like growth factor-1 (IGF-1), plasma adiponectin, and plasma leptin in C57BL/6, Swiss nu/nu, and CB17 scid mice receiving various diets. In each case, mice were randomized to have either bilateral orchiectomy or a sham operation. RESULTS: Energy intake, body weight, blood glucose levels in glucose tolerance test, plasma insulin, plasma IGF-1, and plasma leptin level in all had a trend to be decreased in castrated as compared to sham operated mice. Plasma adiponectin level was increased in the castrated mice. CONCLUSIONS: The effects of castration on glucose, insulin, and related markers in several mouse models studied does not coincide with clinical observations; further studies in this area will require clinical research and/or the use of alternate models such as the dog.

Metformin and rapamycin have distinct effects on the AKT pathway and proliferation in breast cancer cells.

Rapamycin and its analogues inhibit mTOR, which leads to decreased protein synthesis and decreased cancer cell proliferation in many experimental systems. Adenosine 5'- monophosphate-activated protein kinase (AMPK) activators such as metformin have similar actions, in keeping with the TSC2/1 pathway linking activation of AMPK to inhibition of mTOR. As mTOR inhibition by rapamycin is associated with attenuation of negative feedback to IRS-1, rapamycin is known to increase activation of AKT, which may reduce its anti-neoplastic activity. We observed that metformin exposure decreases AKT activation, an action opposite to that of rapamycin. We show that metformin (but not rapamycin) exposure leads to increased phosphorylation of IRS-1 at Ser(789), a site previously reported to inhibit downstream signaling and to be an AMPK substrate phosphorylated under conditions of cellular energy depletion. siRNA methods confirmed that reduction of AMPK levels attenuates both the IRS-1 Ser(789) phosphorylation and the inhibition of AKT activation associated with metformin exposure. Although both rapamycin and metformin inhibit mTOR (the former directly and the latter through AMPK signaling), our results demonstrate previously unrecognized differences between these agents. The data are consistent with the observation that maximal induction of apoptosis and inhibition of proliferation are greater for metformin than rapamycin.

Metformin attenuates the stimulatory effect of a high-energy diet on in vivo LLC1 carcinoma growth.

We investigated the effects of metformin on the growth of lewis lung LLC1 carcinoma in C57BL/6J mice provided with either a control diet or a high-energy diet, previously reported to lead to weight gain and systemic insulin resistance with hyperinsulinemia. Forty-eight male mice were randomized into four groups: control diet, control diet+metformin, high-energy diet, or high-energy diet+metformin. Following 8 weeks on the experimental diets, selected groups received metformin in their drinking water. Three weeks following the start of metformin treatment, mice were injected with 0.5x10(6) LLC1 cells and tumor growth was measured for 17 days. By day 17, tumors of mice on the high-energy diet were nearly twice the volume of those of mice on the control diet. This effect of diet on tumor growth was significantly attenuated by metformin, but metformin had no effect on tumor growth of the mice on the control diet. Metformin attenuated the increased insulin receptor activation associated with the high-energy diet and also led to increased phosphorylation of AMP kinase, two actions that would be expected to decrease neoplastic proliferation. These experimental results are consistent with prior hypothesis-generating epidemiological studies that suggest that metformin may reduce cancer risk and improve cancer prognosis. Finally, these results contribute to the rationale for evaluation of the anti-neoplastic activity of metformin in hyperinsulinemic cancer patients.

Translational and HIF-1α-Dependent Metabolic Reprogramming Underpin Metabolic Plasticity and Responses to Kinase Inhibitors and Biguanides.

There is increasing interest in therapeutically exploiting metabolic differences between normal and cancer cells. We show that kinase inhibitors (KIs) and biguanides synergistically and selectively target a variety of cancer cells. Synthesis of non-essential amino acids (NEAAs) aspartate, asparagine, and serine, as well as glutamine metabolism, are major determinants of the efficacy of KI/biguanide combinations. The mTORC1/4E-BP axis regulates aspartate, asparagine, and serine synthesis by modulating mRNA translation, while ablation of 4E-BP1/2 substantially decreases sensitivity of breast cancer and melanoma cells to KI/biguanide combinations. Efficacy of the KI/biguanide combinations is also determined by HIF-1α-dependent perturbations in glutamine metabolism, which were observed in VHL-deficient renal cancer cells. This suggests that cancer cells display metabolic plasticity by engaging non-redundant adaptive mechanisms, which allows them to survive therapeutic insults that target cancer metabolism.

Silibinin suppresses in vivo growth of human prostate carcinoma PC-3 tumor xenograft.

Chemoprevention is an upcoming approach to control cancer including prostate cancer (PCa). Here, we studied the efficacy and associated mechanisms of a chemopreventive agent silibinin against ectopically growing and established advanced human prostate carcinoma PC-3 tumor xenografts in athymic nude mice. Dietary silibinin (0.5%, w/w) did not show any adverse health effect in mice. In first protocol, silibinin started 1 week prior to xenograft implantation and continued for 60 additional days, whereas in the second protocol, silibinin treatment was started after 25 days of established tumors for 4, 8 and 16 days. Silibinin inhibited tumor growth rate in both protocols showing up to 35% (P = 0.010) and 18-56% (P = 0.002 to <0.001) decrease in tumor volume per mouse and 27% (P < 0.01) and 44% (P = 0.014) decrease in tumor weight per mouse, respectively. In first protocol, silibinin decreased (P < 0.001) tumor cell proliferation and microvessel density but increased (P < 0.001) apoptosis. An increase in insulin-like growth factor-binding protein-3 (IGFBP-3) expression with a concomitant decrease in vascular endothelial growth factor (VEGF) expression was noted. Silibinin strongly increased phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), Cip1/p21 and Kip1/p27 (cyclin-dependent kinase inhibitors) levels but moderately decreased Bcl-2 and survivin levels. In established tumors, similar biomarkers and molecular changes were observed due to silibinin corresponding to its antitumor efficacy. These findings identified in vivo antitumor efficacy of silibinin against PC-3 human PCa in both intervention protocols accompanied with its anti-proliferative, pro-apoptotic and anti-angiogenic activities. At molecular level, silibinin increased IGFBP-3, Cip1/p21, Kip1/p27 levels and ERK1/2 activation and decreased Bcl-2, survivin and VEGF levels in tumors.

The hedgehog pathway inhibitor cyclopamine increases levels of p27, and decreases both expression of IGF-II and activation of Akt in PC-3 prostate cancer cells.

The hedgehog signalling inhibitor cyclopamine has been shown to induce growth inhibition and cell cycle arrest in prostate cancer cell lines, but the mechanism of action has not been clearly defined, and observations between laboratories have not always been consistent. We first observed that albumin can protect PC-3 prostate cancer cells from cyclopamine-induced growth inhibition, suggesting that cyclopamine binds to albumin, and that only free cyclopamine is active. We then conducted a phospho-site protein kinase screen to elucidate the mechanism of cyclopamine-induced growth inhibition. Treatment of PC-3 cells with 5 or 10 microM cyclopamine for 72h resulted in a decrease in cell viability of approximately 50% and approximately 75%, respectively. A phospho-site protein kinase screen showed that cyclopamine decreased levels of phospho-Thr(187)-p27 by 71%. This phospho-site on p27 positively regulates its ubiquitin degradation; therefore a decrease in phospho-Thr(187)-p27 should correlate with increased levels of p27. Consistent with this hypothesis, treatment of PC-3 cells with cyclopamine resulted in a approximately 3-fold increase in p27 protein levels. Cdk-2 phosphorylates Thr(187)-p27, and immunoblotting demonstrated that cyclopamine treatment of PC-3 cells reduces the expression of cdk-2. Furthermore, cyclopamine decreased the levels of phosphorylated (activated) Akt, which is known to increase p27 degradation via Skp-2-induced ubiquitination. The mechanism by which cyclopamine decreases phosphorylated Akt is currently under investigation, but it may involve our observed cyclopamine-induced reduction in IRS-1 and IGF-II expression. These results demonstrate novel molecular correlates of cyclopamine-induced growth inhibition of prostate cancer cells.

A germ line mutation that delays prostate cancer progression and prolongs survival in a murine prostate cancer model.

Circulating insulin-like growth factor-I (IGF-I) levels have been shown to be related to risk of prostate cancer in epidemiologic studies. While specific genetic loci responsible for interindividual variation in circulating IGF-I levels in normal men have not been identified, candidate genes include those involved in the growth hormone (GH)-IGF-I axis such as the hypothalamic factors GH releasing hormone (GHRH) and somatostatin and their receptors. To investigate the role of the GH-IGF-I axis on in vivo prostate carcinogenesis and neoplastic progression, we generated mice genetically predisposed to prostate cancer (the TRAMP model) to be homozygous for lit, a mutation that inactivates the GHRH receptor (GHRH-R) and reduces circulating levels of GH and IGF-I. The lit mutation significantly reduced the percentage of the prostate gland showing neoplastic changes at 35 weeks of age (P=0.0005) and was also associated with improved survival (P<0.01). These data provide an example of a germ line mutation that reduces risk in an experimental prostate carcinogenesis model. The results suggest that prostate carcinogenesis and progression may be influenced by germ line variation of genes encoding signalling molecules in the GH-IGF-I axis.

Serine deprivation enhances antineoplastic activity of biguanides.

Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism.

Treatment with insulin analog X10 and IGF-1 increases growth of colon cancer allografts.

Obesity and type 2 diabetes are associated with an increased risk for development of certain forms of cancer, including colon cancer. The publication of highly controversial epidemiological studies in 2009 raised the possibility that use of the insulin analog glargine increases this risk further. However, it is not clear how mitogenic effects of insulin and insulin analogs measured in vitro correlate with tumor growth-promoting effects in vivo. The aim of this study was to examine possible growth-promoting effects of native human insulin, insulin X10 and IGF-1, which are considered positive controls in vitro, in a short-term animal model of an obesity- and diabetes-relevant cancer. We characterized insulin and IGF-1 receptor expression and the response to treatment with insulin, X10 and IGF-1 in the murine colon cancer cell line (MC38 cells) in vitro and in vivo. Furthermore, we examined pharmacokinetics and pharmacodynamics and monitored growth of MC38 cell allografts in mice with diet-induced obesity treated with human insulin, X10 and IGF-1. Treatment with X10 and IGF-1 significantly increased growth of MC38 cell allografts in mice with diet-induced obesity and we can therefore conclude that supra-pharmacological doses of the insulin analog X10, which is super-mitogenic in vitro and increased the incidence of mammary tumors in female rats in a 12-month toxicity study, also increase growth of tumor allografts in a short-term animal model.

Metformin decreases glucose oxidation and increases the dependency of prostate cancer cells on reductive glutamine metabolism.

Metformin inhibits cancer cell proliferation, and epidemiology studies suggest an association with increased survival in patients with cancer taking metformin; however, the mechanism by which metformin improves cancer outcomes remains controversial. To explore how metformin might directly affect cancer cells, we analyzed how metformin altered the metabolism of prostate cancer cells and tumors. We found that metformin decreased glucose oxidation and increased dependency on reductive glutamine metabolism in both cancer cell lines and in a mouse model of prostate cancer. Inhibition of glutamine anaplerosis in the presence of metformin further attenuated proliferation, whereas increasing glutamine metabolism rescued the proliferative defect induced by metformin. These data suggest that interfering with glutamine may synergize with metformin to improve outcomes in patients with prostate cancer.

Metformin improves healthspan and lifespan in mice.

Metformin is a drug commonly prescribed to treat patients with type 2 diabetes. Here we show that long-term treatment with metformin (0.1% w/w in diet) starting at middle age extends healthspan and lifespan in male mice, while a higher dose (1% w/w) was toxic. Treatment with metformin mimics some of the benefits of calorie restriction, such as improved physical performance, increased insulin sensitivity, and reduced low-density lipoprotein and cholesterol levels without a decrease in caloric intake. At a molecular level, metformin increases AMP-activated protein kinase activity and increases antioxidant protection, resulting in reductions in both oxidative damage accumulation and chronic inflammation. Our results indicate that these actions may contribute to the beneficial effects of metformin on healthspan and lifespan. These findings are in agreement with current epidemiological data and raise the possibility of metformin-based interventions to promote healthy aging.

Carbon source and myc expression influence the antiproliferative actions of metformin.

Epidemiologic and experimental data have led to increased interest in possible roles of biguanides in cancer prevention and/or treatment. Prior studies suggest that the primary action of metformin is inhibition of oxidative phosphorylation, resulting in reduced mitochondrial ATP production and activation of AMPK. In vitro, this may lead to AMPK-dependent growth inhibition if AMPK and its effector pathways are intact or to an energetic crisis if these are defective. We now show that the effect of exposure of several transformed cell lines to metformin varies with carbon source: in the presence of glutamine and absence of glucose, a 75% decrease in cellular ATP and an 80% decrease in cell number is typical; in contrast, when glucose is present, metformin exposure leads to increased glycolysis, with only a modest reduction in ATP level and cell number. Overexpression of myc was associated with sensitization to the antiproliferative effects of metformin, consistent with myc involvement in "glutamine addiction". Our results reveal previously unrecognized factors that influence metformin sensitivity and suggest that metformin-induced increase in glycolysis attenuates the antiproliferative effects of the compound.

Stimulation of MC38 tumor growth by insulin analog X10 involves the serine synthesis pathway.

Recent evidence suggests that type II diabetes is associated with increased risk and/or aggressive behavior of several cancers, including those arising from the colon. Concerns have been raised that endogenous hyperinsulinemia and/or exogenous insulin and insulin analogs might stimulate proliferation of neoplastic cells. However, the mechanisms underlying possible growth-promoting effects of insulin and insulin analogs in cancer cells in vivo, such as changes in gene expression, are incompletely described. We observed that administration of the insulin analog X10 significantly increased tumor growth and proliferation in a murine colon cancer model (MC38 cell allografts). Insulin and X10 altered gene expression in MC38 tumors in a similar fashion, but X10 was more potent in terms of the number of genes influenced and the magnitude of changes in gene expression. Many of the affected genes were annotated to metabolism, nutrient uptake, and protein synthesis. Strikingly, expression of genes encoding enzymes in the serine synthesis pathway, recently shown to be critical for neoplastic proliferation, was increased following treatment with insulin and X10. Using stable isotopic tracers and mass spectrometry, we confirmed that insulin and X10 increased glucose contribution to serine synthesis in MC38 cells. The data demonstrate that the tumor growth-promoting effects of insulin and X10 are associated with changes in expression of genes involved in cellular energy metabolism and reveal previously unrecognized effects of insulin and X10 on serine synthesis.

Metformin abolishes increased tumor (18)F-2-fluoro-2-deoxy-D-glucose uptake associated with a high energy diet.

Insulin regulates glucose uptake by normal tissues. Although there is evidence that certain cancers are growth-stimulated by insulin, the possibility that insulin influences tumor glucose uptake as assessed by ( 18) F-2-Fluoro-2-Deoxy-d-Glucose Positron Emission Tomography (FDG-PET) has not been studied in detail. We present a model of diet-induced hyperinsulinemia associated with increased insulin receptor activation in neoplastic tissue and with increased tumor FDG-PET image intensity. Metformin abolished the diet-induced increases in serum insulin level, tumor insulin receptor activation and tumor FDG uptake associated with the high energy diet but had no effect on these measurements in mice on a control diet. These findings provide the first functional imaging correlate of the well-known adverse effect of caloric excess on cancer outcome. They demonstrate that, for a subset of neoplasms, diet and insulin are variables that affect tumor FDG uptake and have implications for design of clinical trials of metformin as an antineoplastic agent.

IGF1/insulin receptor kinase inhibition by BMS-536924 is better tolerated than alloxan-induced hypoinsulinemia and more effective than metformin in the treatment of experimental insulin-responsive breast cancer.

Epidemiologic and experimental evidence suggest that a subset of breast cancer is insulin responsive, but it is unclear whether safe and effective therapies that target the insulin receptor (IR), which is homologous to oncogenes of the tyrosine kinase class, can be developed. We demonstrate that both pharmacologic inhibition of IR family tyrosine kinase activity and insulin deficiency have anti-neoplastic activity in a model of insulin-responsive breast cancer. Unexpectedly, in contrast to insulin deficiency, pharmacologic IR family inhibition does not lead to significant hyperglycemia and is well tolerated. We show that pharmacokinetic factors explain the tolerability of receptor inhibition relative to insulin deficiency, as the small molecule receptor kinase inhibitor BMS-536924 does not accumulate in muscle at levels sufficient to block insulin-stimulated glucose uptake. Metformin, which lowers insulin levels only in settings of hyperinsulinemia, had minimal activity in this normoinsulinemic model. These findings highlight the importance of tissue-specific drug accumulation as a determinant of efficacy and toxicity of tyrosine kinase inhibitors and suggest that therapeutic targeting of the IR family for cancer treatment is practical.

Loss of function of PTEN alters the relationship between glucose concentration and cell proliferation, increases glycolysis, and sensitizes cells to 2-deoxyglucose.

PTEN loss of function enhances proliferation, but effects on cellular energy metabolism are less well characterized. We used an inducible PTEN expression vector in a PTEN-null glioma cell line to examine this issue. While proliferation of PTEN-positive cells was insensitive to increases in glucose concentration beyond 2.5mM, PTEN-null cells significantly increased proliferation with increasing glucose concentration across the normal physiologic range to approximately 10mM, coinciding with a shift to glycolysis and "glucose addiction". This demonstrates that the impact of loss of function of PTEN is modified by glucose concentration, and may be relevant to epidemiologic results linking hyperglycemia to cancer risk and cancer mortality.