Diabetes and cancer: a consensus report.

Epidemiologic evidence suggests that cancer incidence is associated with diabetes as well as certain diabetes risk factors and treatments. This consensus statement of experts assembled jointly by the American Diabetes Association and the American Cancer Society reviews the state of science concerning 1) the association between diabetes and cancer incidence or prognosis; 2) risk factors common to both diabetes and cancer; 3) possible biologic links between diabetes and cancer risk; and 4) whether diabetes treatments influence the risk of cancer or cancer prognosis. In addition, key unanswered questions for future research are posed.

Risk factors for colorectal cancer in man induce aberrant crypt foci in rats: Preliminary findings.

Epidemiological studies have demonstrated clear associations between specific dietary and environmental risk factors and incidence of colorectal cancer, but the mechanisms responsible for these associations are not known. An animal model could facilitate such an understanding. Both genotoxic and nongenotoxic carcinogens induce aberrant crypt foci (ACF) in the colons of F344 rats. F344 rats were provided with diets that contained putative risk factors for CRC: low calcium and low vitamin D, high iron, high fructose, and decreased light (UV) exposure or a control diet for 14 wk. The rats were then assessed with biochemical measures and by topological examination for evidence of colon abnormalities. Circulating ionized calcium was decreased from 2.85 to 1.69 mmol/L, and ACF were increased from 0.7 to 13.6 lesions/colon (both P < 0.001). Rats exposed to the multiple environmental conditions associated with colon cancer, developed ACF similar to the heterogeneous or ill-defined ACF in the human colon. Heterogeneous ACF are the most frequently seen in humans and are also seen in rats shortly after exposure to the non-genotoxic colon carcinogen, dextransulfate sodium. The rodent model could be used to assess the pathways from diet and environment to colon cancer and to provide guidance for clinical studies.

Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity.

Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from (14)C-labeled acetate to those supplied exogenously as (14)C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2-3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells.

Inhibition of fatty acid synthase and Sp1 expression by 3,3'-diindolylmethane in human breast cancer cells.

The putative cancer preventive agent 3,3'-diindolylmethane (DIM) is formed in the acidic environment of the stomach following consumption of indole-3-carbinol (I3C), which is present in vegetables of the Brassica genus. We have recently shown that the transcription factor Sp1 is involved in the regulation of both proliferation and de novo lipogenesis in cancer cells. Here we show that DIM inhibits the proliferation of 3 human breast cancer cell lines, MCF-7, MDA-MB-231, and SKBr-3, and concomitantly inhibits the expression of Sp1 and fatty acid synthase (FAS). There were no DIM-related effects on the proliferation or expression of Sp1 or FAS in the nontumorigenic human breast epithelial cell line MCF-10A. These results suggest that inhibition of Sp1 and/or FAS expression could contribute to the anticancer properties of the dietary indoles.

Ezetimibe reverses the inhibitory effects of dietary cholesterol on mammary tumorigenesis in rats.

There are concerns regarding increased cancer incidence in patients treated with ezetimibe, an inhibitor of the absorption of dietary cholesterol. Here we tested the hypothesis that ezetimibe will accelerate mammary tumorigenesis in rats. The drug was administered at a dose of 1 ppm in an AIN-93G diet that contained 0.3% cholesterol. This experimental diet and control diets that contained either no additions or cholesterol or ezetimibe only, were fed to groups of 30 Sprague-Dawley rats 3 days after they were treated with 50 mg/kg methylnitrosourea (MNU). All rats were euthanized 22 weeks after MNU administration. Tumor multiplicity was significantly smaller in rats fed cholesterol than those fed no cholesterol (1.84 +/- 0.42 vs. 3.86 +/- 0.86 respectively, P < 0.05), but was significantly greater in the cholesterol/ezetimibe group than the group fed only cholesterol (3.48 +/- 0.59 vs. 1.84 +/- 0.42 respectively, P < 0.04). The average weight of tumors/rat was also significantly larger in the cholesterol/ezetimibe group than those fed cholesterol alone (5.67 +/- 1.15 vs. 2.56 +/- 0.71 respectively, P < 0.04). As expected, ezetimibe prevented the cholesterol raising effect of the dietary cholesterol. These results show that ezetimibe reverses the inhibitory effect of dietary cholesterol on the development of rat mammary tumors.

Sp1 coordinately regulates de novo lipogenesis and proliferation in cancer cells.

Cancers express high levels of fatty acid synthase (FAS) from which they derive fatty acids for membrane biosynthesis to sustain cell proliferation. How cancer cells coordinate de novo lipogenesis and proliferation has not been investigated. Transcription factors Sp1, Sp3 and Sp4 are overexpressed in a variety of cancers and regulate gene expression by interacting with GC-rich Sp1 binding sites. Genes encoding FAS and cell cycle proteins such as CDC25A contain Sp1 binding sites in their promoters. We demonstrate by RNA interference that Sp1, Sp3 and Sp4 all play a role in regulating CDC25A expression and proliferation in human breast cancer cells. Only Sp1, however, also regulates FAS. Furthermore, mithramycin, which blocks Sp1 binding sites, decreased proliferation, inhibited CDC25A and FAS expression and reduced binding of Sp1 to the promoters of these genes as assessed by ChIP assays. Conversely, 17beta-estradiol (E(2)) increased proliferation and CDC25A and FAS expression along with increased binding of Sp1 to the promoters of the 2 genes. In addition, we showed that the expression of sterol regulatory element-binding protein-1c (SREBP-1c), the only transcription factor that has been shown to regulate genes of lipogenic enzymes in cancer cells, is also regulated by Sp1. Finally, we demonstrated that Sp1 plays a role in sustaining proliferation and FAS expression in colon as well as prostate cancer cells. Overall, these observations suggest that Sp1 coordinately regulates de novo lipogenesis and proliferation in cancer cells.

Interleukin-24 induces expression of beta4 integrin but suppresses anchorage-independent growth of rat mammary tumor cells by a mechanism that is independent of beta4.

Wistar-Furth rats develop multiple mammary adenocarcinomas following initiation with methylnitrosourea, whereas Copenhagen rats are resistant to the development of mammary tumors. We have previously isolated cell lines from tumors induced in resistant Copenhagen x Wistar-Furth F(1) rats by infusion of a retrovirus harboring v-Ha-ras directly into the main mammary ducts. Some of the cell lines were able to grow in soft agar, but a significant number did not display anchorage-independent growth. Here, we compared by microarray analysis genes that are differentially expressed in these cell lines. The expression of interleukin-24 (IL-24) and beta(4) integrin was highly correlated with the inability of cells to grow in soft agar. Ectopic expression of IL-24 in anchorage-independent cells inhibited their growth in monolayer culture, in soft agar, and in nude mice in vivo and inhibited their ability to migrate and invade in in vitro assays. Furthermore, growth suppression by IL-24 was associated with the transcriptional up-regulation of p27(Kip1) via the activation of Stat3. We showed, for the first time, that beta(4) integrin is a downstream target of IL-24. However, beta(4) does not play a direct role in regulating the proliferative capacity of rat mammary tumor cells. Our results show that IL-24 suppresses the growth of rat mammary carcinoma cells and may play a role in the resistance of Copenhagen rats to mammary carcinogenesis.

The 10t,12c isomer of conjugated linoleic acid inhibits fatty acid synthase expression and enzyme activity in human breast, colon, and prostate cancer cells.

The objective of this study was to determine whether downregulation of fatty acid synthase (FAS) expression and/or inhibition of its activity by the two major CLA isomers, 10t,12c and 9c,11t CLA, could contribute to their inhibitory effect on the growth of human breast (MCF-7), colon (HT-29) and prostate (LNCaP) cancer cell lines. We first confirmed and extended the results of others showing that the inhibitory action of CLA on proliferation is dependent on the cell type as well as the structure of the isomer, the 10,12 isomer being a more potent inhibitor than the 9,11 isomer in the concentration range 25-100 microM. By Western analysis, we showed that 10,12 CLA downregulated FAS expression in all of the cell lines in a concentration-dependent manner, but the 9,11 isomer had no effect. Both isomers inhibited FAS enzyme activity, but 10,12 CLA was again more potent than the 9,11 isomer. Our results suggest that downregulation of FAS by 10,12 CLA, but not by the 9,11 isomer, as well as inhibition of FAS enzyme activity by both isomers, may contribute to growth inhibition of cancer cells but only at relatively high concentrations.

Fatty acid synthase is over-expressed in large aberrant crypt foci in rats treated with azoxymethane.

Fatty acid synthase (FAS) is over-expressed in many human cancers including colon. High levels of FAS expression have also been observed in a number of lesions that are precursors to invasive colorectal cancer. However, FAS expression in aberrant crypt foci (ACF), the earliest identifiable lesions in colon cancer development, has not been investigated. In this study, we treated Fisher rats with a single dose of the colon carcinogen azoxymethane then evaluated ACF 100 days later. We showed that large ACF (>or=4 crypts/focus) have a significantly higher level of immunohistochemical staining for FAS than either small ACF (

Elevated circulating adiponectin and elevated insulin sensitivity in adiponectin transgenic mice are not associated with reduced susceptibility to colon carcinogenesis.

Obesity, particularly visceral adiposity, is an established risk factor for colorectal cancer (CRC) and this is thought to result, at least in part, from insulin resistance and chronic hyperinsulinemia that may be mediated by adipokines. Serum levels of adiponectin, the most abundant protein secreted from adipocytes, are decreased in obesity and are inversely associated with insulin resistance and hyperinsulinemia. The objective of this study was to determine whether elevated circulating adiponectin plays a role in colon carcinogenesis using adiponectin transgenic (AdTg) mice that have 2-3-fold elevated circulating adiponectin but similar body weights as wildtype (WT) littermates used as controls. Eight-week old male and female AdTg and WT mice were treated with 4 weekly injections of the colon-specific carcinogen azoxymethane (AOM). One week following the last dose of AOM, all mice were placed on a high-fat diet and killed 24 weeks later, at 36 weeks of age, for the analysis of colon tumors. Glucose tolerance tests (GTT) were performed by injecting 2g/kg dextrose or 1.25-1.5 g/kg dextrose into all 12-week and 32-35-week-old mice respectively, and measuring blood from the tail vein 15, 30, 60 and 120 min following glucose administration. There were no significant differences in colon tumor incidence, number or size between AdTg and WT mice of either sex. AdTg mice of both sexes displayed resistance to diet-induced decreases in insulin sensitivity. Our results show that constitutively elevated levels of circulating adiponectin in AdTg mice do not confer protection against the development of colon tumors.

Colon carcinogenesis in liver-specific IGF-I-deficient (LID) mice.

Circulating insulin-like growth factor I (IGF-I) is associated with increased risk of colorectal cancer. It is not clear, however, whether IGF-1 plays a direct causative role in colon carcinogenesis or whether it mediates the known promoting effects of insulin. The objective of this study was to determine the role of IGF-1 in colon carcinogenesis using liver-specific IGF-I deficient (LID) mice that exhibit 70% reductions in circulating IGF-I. Female and male LID mice were treated with the colon-specific carcinogen azoxymethane to induce aberrant crypt foci (ACF) or colon tumors. Female LID mice developed significantly fewer ACF and had normal insulin levels compared to controls. Male LID mice, however, were hyperinsulinemic and exhibited no significant differences in ACF development compared to controls. In the tumor study, both male and female LID mice were hyperinsulinemic and had no significant differences in tumor incidence or multiplicity compared to their respective controls. There was a significant 25% reduction in tumor size, however, in both male and female LID mice compared to controls. These data suggest that IGF-I deficiency attenuates the promoting effect of insulin on colon carcinogenesis and that IGF-I is an independent promoter of the growth of established tumors. Our findings implicate both IGF-I and insulin as important promoters of colon cancer development.

Development of aberrant crypt foci in the colons of ob/ob and db/db mice: evidence that leptin is not a promoter.

Leptin is elevated in obesity and has been suggested to increase the risk of colorectal cancer (CRC), although the evidence is conflicting. The objective of this study was to compare the susceptibility to colon carcinogenesis of db/db mice that have highly elevated circulating leptin and leptin-deficient ob/ob mice, both of which are obese. Seven-week-old male ob/ob, db/db, and WT mice received 4 weekly i.p. injections of 5 mg/kg azoxymethane (AOM) and were killed 14 wk later for the analysis of putative preneoplastic aberrant crypt foci (ACF). There were no differences in ACF number or multiplicity between ob/ob and db/db mice. Leptin has been shown to induce CYP2E1, the main enzyme that activates AOM, but we observed no differences in hepatic CYP2E1 activity or colonic CYP2E1 protein levels between ob/ob and db/db mice. We also induced ACF with 2 oral doses 3 d apart of 30 mg/kg methylnitrosourea (MNU), a direct-acting carcinogen. There were no differences in ACF number or multiplicity between the two groups of obese animals 5 wk following the last dose of MNU. The colonic mucosa of db/db mice expressed significantly lower mRNA levels of ObRa, the predominant short form of the leptin receptor, compared to ob/ob mice, and following i.p. injection with 1 mg/kg recombinant mouse leptin, exhibited significantly reduced p44/42 pMAPK compared to saline-treated controls. These results show that ObRa is functionally active in the colons of db/db mice. We conclude that leptin does not play a significant role in ACF development.

Adiponectin is a negative regulator of bone mineral and bone strength in growing mice.

Obesity is associated with increased bone mineral density (BMD) but the mechanism for this is unclear. Serum levels of the adipokine adiponectin are inversely correlated with obesity, but results from studies on its relationship to bone mass are conflicting. The objective of this study was to compare bone mineral content (BMC), BMD and biomechanical strength properties of femur and lumbar vertebrae in 8- and 16-week old adiponectin transgenic mice (AdTg). These mice exhibit significantly elevated circulating adiponectin but have similar body weights compared to wild-type (WT) littermates that were used as controls. Female AdTg mice displayed significantly lower femur BMC at 8 and 16 weeks of age and femur neck peak load was significantly lower in 8-week old AdTg mice of both genders compared to controls. The peak load from compression testing of an individual lumbar vertebra was significantly lower in female AdTg mice compared to WT at 8 weeks, and this difference persisted at 16 weeks of age. In addition, lumbar vertebrae BMC was significantly lower in 16-week old male AdTg mice compared to WT although vertebra peak load was not different. Serum adiponectin levels were inversely correlated with femur BMC. In summary, elevated circulating adiponectin inhibits the acquisition of bone mass in growing mice and results in decreased biomechanical measures of functional strength that are surrogate measures of susceptibility to fractures. These results support a role for circulating adiponectin as a metabolic link that can explain, at least in part, the positive relationship between obesity and both bone mass and reduced susceptibility to fractures.

Dehydroalanine and lysinoalanine in thermolyzed casein do not promote colon cancer in the rat.

Thermolysis of proteins produces xenobiotic amino-acids such as the potentially toxic lysinoalanine, and the alkylating agent, dehydroalanine, which have been considered possible health hazards. We observed that thermolyzed casein promoted aberrant crypt foci (ACF) and colon cancer growth in rats initiated with azoxymethane and speculated that promotion might be due to the formation of these compounds. To test this notion we first measured the concentration of the modified amino acids as a function of thermolysis time. The concentration of dehydroalanine in the casein paralleled the degree of promotion, that of lysinoalanine did not. We then tested diets containing foods with high levels of dehydroalanine (thermolyzed sodium-caseinate, cooked Swiss cheese) for their effect on ACF promotion. They decreased the number and/or size of ACF significantly, indicating that dehydroalanine did not promote, but protected rats against colon carcinogenesis. These results do not support the notion that lysinoalanine or dehydroalanine are a hazard with respect to colon carcinogenicity.

Farnesol decreases serum triglycerides in rats: identification of mechanisms including up-regulation of PPARalpha and down-regulation of fatty acid synthase in hepatocytes.

Obesity is associated with impaired fatty acid (FA) oxidation and increased de novo hepatic lipogenesis that may contribute to the development of hypertriglyceridemia, an important risk factor for the development of cardiovascular disease. Strategies to improve hepatocyte FA metabolism, including dietary interventions, are therefore important for the prevention of obesity-associated co-morbidities. Farnesol is consumed in the diet as a component of plant products. In the present study, we administered farnesol orally to rats for seven days and found significantly reduced serum triglyceride concentrations compared with controls. Potential mechanisms underlying the hypotriglyceridemic effect of farnesol were investigated using clone-9 cultured rat hepatocytes. Farnesol significantly upregulated expression of peroxisome proliferator-activated receptor alpha (PPARalpha) and the PPARalpha-regulated genes fatty acyl-CoA oxidase and carnitine palmitoyl transferase 1a, suggesting that increased hepatic FA oxidation may contribute to serum triglyceride lowering in rats. Farnesol did not change SREBP-1c mRNA levels, but significantly down-regulated fatty acid synthase (FAS) mRNA and protein levels and activity, indicating that attenuated lipogenesis may also contribute to hypotriglyceridemic effects of farnesol in vivo. Rescue experiments revealed that down-regulation of FAS by farnesol was not related to activation of PPARalpha, but rather was caused by a 9-cis retinoic acid mediated mechanism that involved down-regulation of retinoid X receptor beta. Diets rich in plant products are associated with a lower risk of cardiovascular disease. Our findings suggest that farnesol may contribute to this protective effect by lowering serum TG levels.

Celecoxib decreases fatty acid synthase expression via down-regulation of c-Jun N-terminal kinase-1.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are inhibitors of cyclooxygenase (COX). Our previous observations that celecoxib, a COX-2-specific inhibitor, not only inhibits rat mammary carcinogenesis, but also decreases fat deposition in rats fed a high-fat diet, prompted us to determine whether celecoxib affects lipid metabolism. At 57 days of age, two groups of 10 female Sprague Dawley rats were pair-fed a high-fat diet with or without 1500 ppm celecoxib for 15 weeks. Compared with controls, celecoxib-treated rats had 44.4% less hepatic triglycerides and 22.6% less intra-abdominal adipose tissue mass. In the liver and adipose tissue of several genes involved in fat metabolism and mobilization that we measured, only fatty acid synthase (FAS) was significantly down-regulated by celecoxib treatment. There were no differences in the level of prostaglandin E(2) in these tissues, indicating that celecoxib decreases fat accumulation by down-regulating FAS through a COX-2-independent mechanism. Among the potential molecular targets by which celecoxib may regulate FAS expression, only c-Jun N-terminal kinase-1 (JNK1) was significantly down-regulated. Furthermore, a known inhibitor of JNK suppressed FAS expression in rat hepatocytes. Our observations suggest that celecoxib suppresses FAS expression and decreases fat accumulation by down-regulating JNK1.

Bone abnormalities in adolescent leptin-deficient mice.

Ob/ob and db/db mice have different aberrations in leptin signaling that both lead to abnormalities in bone mineral density (BMD), and bone histological and histomorphometric outcomes. A few studies have directly compared bone metabolism in ob/ob and db/db mice, and biomechanical strength properties that are surrogate measures of fracture risk, have not been extensively studied. This study compared bone mineral content (BMC), BMD and biomechanical strength properties of femurs and lumbar vertebrae among 10 week old male ob/ob, db/db and C57Bl/6 wildtype (WT) mice. Femurs and lumbar vertebrae were specifically studied to determine if trabecular and cortical bone are regulated by leptin in a similar manner in ob/ob and db/db mice. Femurs of ob/ob and db/db mice had lower BMC, BMD and biomechanical strength properties, including peak load, compared to WT mice. In contrast, lumbar vertebrae BMC and BMD did not differ among genotypes, nor did the peak load from compression testing of an individual lumbar vertebra differ among groups. These findings suggest that leptin deficiency in adolescent male mice first results in changes in femurs, a representative long bone, and alterations in lumbar vertebrae may occur later in life.

The interplay between cell signalling and the mevalonate pathway in cancer.

The mevalonate (MVA) pathway is an essential metabolic pathway that uses acetyl-CoA to produce sterols and isoprenoids that are integral to tumour growth and progression. In recent years, many oncogenic signalling pathways have been shown to increase the activity and/or the expression of MVA pathway enzymes. This Review summarizes recent advances and discusses unique opportunities for immediately targeting this metabolic vulnerability in cancer with agents that have been approved for other therapeutic uses, such as the statin family of drugs, to improve outcomes for cancer patients.

Cyclin D1 is necessary but not sufficient for anchorage-independent growth of rat mammary tumor cells and is associated with resistance of the Copenhagen rat to mammary carcinogenesis.

To identify genes associated with the resistance of Copenhagen (Cop) rats to mammary carcinogenesis, we infused a retrovirus harboring v-Ha-ras directly into the main mammary ducts of resistant F1 rats from a cross between Cop and susceptible Wistar Furth (WF) rats. Adenocarcinomas formed in approximately 50% of infused glands. Cell lines derived from these tumors were clonal, but did not share a common viral integration site, suggesting that a high level of v-Ha-ras expression was able to overcome resistance in the F1 rats. Some of the cell lines were able to grow in soft agar, but a significant number did not display anchorage-independent growth. These growth characteristics were independent of v-Ha-ras expression levels. The ability to grow in soft agar was associated with the size of tumors induced by injecting the cells into nude mice, and showed a striking positive association with the expression of cyclin D1. Furthermore, while resistance to anchorage-independent growth was fully overcome by transfection of cyclin D1 in some clones, in the others the effect was partial. A similar pattern of cyclin D1 upregulation and growth in soft agar was also observed when the cells were transfected with an active form of beta-catenin. Hybrid cells from the somatic fusion of an anchorage-dependent to an anchorage-independent clone did not grow in soft agar. These results suggest that while a high expression level of cyclin D1 is necessary for anchorage-independent growth in all clones, it is not sufficient for full growth capacity in soft agar, raising the possibility that the loss of a tumor suppressor gene in the cell lines is required to fully confer anchorage-independent growth. Our anchorage-dependent and -independent rat mammary tumor-derived cell lines may recapitulate the resistance and susceptibility of Cop and WF rats, respectively, to mammary carcinogenesis that could facilitate the identification of breast cancer susceptibility genes.

Statins and cancer development.

There is epidemiologic evidence that the hydrophilic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor pravastatin increases the incidence of some extrahepatic cancers, although this finding has been attributed to chance. We hypothesize that pravastatin is able to promote the development of cancer by causing an induction of HMG-CoA reductase and, hence, mevalonate synthesis in extrahepatic tissues. We have shown that mevalonate, the product of HMG-CoA reductase, promotes the growth of breast cancer cells. Because there is no uptake of pravastatin by most extrahepatic cells, this statin will be unable to mitigate the increase in mevalonate synthesis in extrahepatic tissues that accompanies the decrease in circulating cholesterol caused by its inhibition of hepatic HMG-CoA reductase.