MINIREVIEW

Minireview: Obesity and Breast Cancer: A Tale of Inflammation and Dysregulated Metabolism Evan R. Simpson and Kristy A. Brown Prince Henry’s Institute of Medical Research, Monash Medical Centre, Clayton, Victoria 3168, Australia In addition to the spectrum of conditions known collectively as the Metabolic Syndrome, obesity is now recognized to be associated with increased risk of several cancers including colon, endometrial, and breast cancer. Obesity and carcinogenesis share 2 characteristics in common. On the one hand, they involve inflammatory pathways, and on the other hand, they involve dysregulated metabolism. In this review we focus on postmenopausal breast cancer and discuss the metabolic and cellular mechanisms whereby obesity and breast cancer are related. Because a majority of postmenopausal breast tumors are estrogen responsive, we include a discussion of the action of obesity-related factors on estrogen formation within the breast. (Molecular Endocrinology 27: 715–725, 2013)

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t has become a truism that we are facing a global “pandemic” of obesity, which currently affects hundreds of millions of women and men worldwide, not only in the developed world but in many developing countries also. Obesity is commonly associated with the metabolic syndrome, which is a spectrum of conditions including insulin resistance, type 2 diabetes, hyperlipidemia, hypertension, as well as increased risk of cardiovascular disease, stroke, and kidney failure. In general, there is a positive correlation between body mass index and the risk of type 2 diabetes, as has been shown in a number of epidemiological investigations. However, added to these pathologic abnormalities noted above, the metabolic syndrome is now recognized to be an established risk factor for a number of cancers (1). A conclusion from several studies (reviewed in 2) is that both obesity and type 2 diabetes increase the risk of postmenopausal breast cancer. The results concerning women with premenopausal breast cancer were less clear as positive as well as inverse relationships have been described in women from different ethnic groups. On the other hand, when employing waistto-hip ratio rather than body mass index, a positive correlation has been observed between elevated waist-to-hip ratio and risk of breast cancer in premenopausal women as well as postmenopausal women (reviewed in 3). Al-

though most postmenopausal breast cancers are estrogen receptor (ER) -positive, many premenopausal breast cancers are ER-negative or triple negative, that is to say they lack ER, PR, and HER2-neu (4). Obesity and carcinogenesis share 2 properties in common. On the one hand, they involve inflammatory pathways (5), and on the other, they are characterized by dysregulated metabolism (6). In this review we discuss the cellular and molecular mechanisms whereby obesity and cancer are linked, with particular reference to postmenopausal breast cancer. In this case it should be emphasized again that most postmenopausal breast cancers are ERpositive and that the ovaries cease to synthesize estrogens at the time of menopause. There is now widespread acceptance of the view that the increased risk of breast cancer in postmenopausal women is due to the production of estrogens by the adipose tissue (reviewed in 7). Estrogen synthesis in adipose tissue increases with obesity but also with aging and is associated with an increased expression of aromatase, the enzyme responsible for estrogen biosynthesis (8, 9). Thus the importance of estrogen formation in the adipose tissue and aromatase expression in the breast adipose, in particular, has been emphasized. Accordingly, in this review we devote space to discussing how inflammatory processes and dysregulated metabo-

ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2013 by The Endocrine Society Received January 15, 2013. Accepted March 14, 2013. First Published Online March 21, 2013

Abbreviations: AMPK, AMP-activated protein kinase; COX2, cyclooxygenase-2; CREB, CREbinding protein; CRTC2, CREB regulated transcription coactivator 2; EP, epinephrine; ER, estrogen receptor; HIF1␣, hypoxia-inducible factor 1-␣, LKB1, liver kinase B1; MCF, macrophage chemotactic factor; mTOR, mammalian target of rapamycin; mTORC, mammalian target of rapamycin complex 1; NAD, nicotinamide adenine dinucleotide; NF␬B, nuclear factor ␬B; NSAIDs, nonsteroidal anti-inflammatory drugs; OR, odds ratio; PGE2, prostaglandin E2; PI3K, phosphoinositide 3-kinase; TCA, trichloroacetic acid; TNF␣, tumour necrosis factor ␣.

doi: 10.1210/me.2013-1011

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ized by infiltration of macrophages into the adipose depots in both subPromoter II Promoter I.1 Promoter I.4 HBR cutaneous and visceral sites (10). AATAAA ATG ATTAAA These macrophages typically form what are known as crownlike struc2a I.4 I.1 I.2 I.3 II III IV V VI VII VIII IX X tures around the lipid-filled adiadipose, Placenta ovary, pocytes of obese individuals and adipose (ERRϒ, HIF1α) bone 10 KB characteristically stain for CD68. (class I cytokines, (cAMP, Hypoxia is another characteristic of TNFα) FSH, PGE2) adipose tissue of obese individuals Figure 1. Diagram of the human CYP19A1 (aromatase) gene. The 9 coding exons numbered II and may be due in part to the physthrough X are shown in yellow, and the untranslated first exons are shown in red together with ical limitation of the blood vessels to associated promoters. Also indicated are stimulatory factors and coregulators associated with supply the large lipid-engorged adieach promoter. Abbreviations: ERR␥, estrogen related ␥; FSH, follicle stimulating hormone; HBR, heme binding region. pocytes (11). This is associated in turn with an increase in the expreslism affect aromatase expression in the breast. A schesion of hypoxia-inducible factor 1-␣ (HIF1␣) by these matic of the gene encoding human aromatase (CYP19A1) adipocytes, which increases the levels of monocyte cheis shown in Figure 1. motactic protein 1. This in turn stimulates the recruitment of macrophages to these sites. Saturated fatty acids produced by the lipid-engorged adipocytes can act to stimuInflammation and Adiposity late the activity of inflammasomes present in both macObesity is associated with inflammation in the adipose rophages and adipocytes (12), which gives rise to a tissue, which is classified as subclinical and is character- cascade featuring caspase 1, IL-1␤, and ending with nuclear factor ␬B (NF␬B). In addition, saturated fatty acids such as palmitate can increase the activity of toll-like receptors, such as toll-like receptor-4 (13), which can also stimulate the formation of NF␬B. This inflammatory cofactor together with the macrophage recruitment factors can bring about an increase in the expression of inflammatory mediators such as tumour necrosis factor ␣ (TNF␣), IL-6, and prostaglandin E2 (PGE2). These results are summarized in Figure 2. This figure also indicates that these factors can in turn stimulate the expression of aromatase in the adipose tissue, which is discussed in the next section. Promoter I.3

Inflammation and Breast Cancer

Figure 2. Inflammatory cascade associated with lipid-laden adipocytes characteristic of obesity. Adipocytes and macrophages synergize to increase the production of inflammatory mediators, including TNF␣, IL-6, and PGE2. These inflammatory factors then act on the adjacent stroma to increase aromatase expression and estrogen biosynthesis. Abbreviations: MCP-1, monocyte chemotactic protein 1; MMIF, macrophage migration inhibitory factor.

These inflammatory factors have been suggested to contribute to the increased risk of breast cancer progression and mortality. For example, the NF␬B signaling pathway has been shown to be important for growth of anti-estrogen-resistant breast cancer cells using an macrophage chemotactic factor (MCF)7-derived cell model (14). Cyclooxygenase-2 (COX2), responsible for the ratelimiting step in PGE2 biosynthesis, has also been associated with increased breast cancer risk and several studies have indicated that the use of nonsteroidal anti-inflammatory drugs (NSAIDs), inhibitors of COX2, can reduce the risk of breast cancer in preclinical studies (15). Upregulation of COX2 and resulting increased PGE2 syn-

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expression is regulated primarily by 2 promoters, the proximal promoters II/I.3 and a distal promoter, I.4. EP1 EP2 (Promoters II and I.3 are splice-variants of each other and for the purposes of this discussion can be conPKC sidered as one) (reviewed in 18). PKA Expression driven by promoter I.4 is regulated by TNF␣ via an NF␬Blinked mechanism. In the case of class 1 cytokines such as IL-6, expression of aromatase driven by promoter I.4 is via a JAK1/STAT3 pathway, with STAT3 binding to a GAS element upstream of the promoter. PGC1α LRH-1 On the other hand, PGE2 regulates CRTC2 aromatase via promoters II/I.3 (Figure 3). Two signaling pathways are CREB employed; one is via the EP2 receptor linked to adenylate cyclase and ATF2 Nucleus phosphorylation of CREB, which binds to 2 CREs in the aromatase promoter II. The second pathway PGC1α via the EP1 receptor is linked to ATF2 phospholipase C stimulation with LRH-1 CRTC CRTC the resulting increase in protein kiCREB CREB nase C activity. This in turn stimulates the expression of LRH-1, a moCRE CRE Aromatase nomeric orphan member of the Figure 3. Schematic of action of PGE2 to stimulate aromatase promoter II activity in human nuclear receptor superfamily, which breast adipose stromal cells. binds to a nuclear receptor half-site on the aromatase promoter II, thesis are recognized as a marker for the progression of downstream of the proximal CRE. This binding is absomany cancers including breast cancer (16). PGE2 activates 4 lutely required for aromatase promoter II activity (19). receptors, epinephrine (EP) 1– 4. EP2 and EP4 are coupled to Recent studies from Dannenberg’s group have indiadenylate cyclase and cAMP formation, whereas EP1 is cated that crownlike structures, namely macrophages surlinked to phospholipase C, DAG formation, and activation rounding the large lipid-filled adipocytes of obese individof protein kinase C. Downstream targets of PGE2 include uals, are present in the breasts of obese women (20). vascular endothelial growth factor (17) as well as the CRE- These are associated with increased expression of NF␬B binding protein (CREB), MAPK, Src, and Akt pathways, and their numbers correlate with an increase in the exand also HIF1␣. On the other hand, TNF␣ acts primarily pression and activity of aromatase in the adipose tissue of through the IKK␤/NF␬B and mammalian target of rapamy- the breasts of these women. Further studies from this cin (mTOR) pathways, whereas IL-6 and other class 1 cy- group have shown that the increase in aromatase exprestokines act via the gp130/Jak1/STAT3 pathway as well as sion is associated with an increase in activity of the proxthe Ras/MAPK pathway. imal promoters II/I.3 and in turn is correlated with an One way in which TNF␣, class 1 cytokines, and PGE2 increase in COX2 expression and PGE2 levels in the can increase the risk of breast cancer is by stimulating the breasts of these women (21). COX2 is also expressed in expression of aromatase in adipose tissue, particularly many breast carcinomas where it correlates with tumor that of the breast. Aromatase synthesizes estrogens in ad- size, high-grade HER2 positivity, and a worse disease-free ipose tissue from circulating androgens. It is expressed in interval. the fibroblasts or stromal cells, which surround the adiPrevious studies have examined the activity and expocytes and the mammary ducts. In these cells, aromatase pression of aromatase in the tissue of breast quadrants PGE2

PGE2

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obtained at the time of mastectomy because of the presence of a tumor. These studies found that aromatase expression (22) and activity (23) were highest in the quadrant of the breast that contained the tumor, and that there was a gradient of aromatase expression that was highest in the tumor and in the tumor-bearing quadrant and decreased with increased distance from the tumor. This suggested that the tumor produced a factor or factors that stimulated aromatase expression in the surrounding adipose tissue and in particular the cancer-associated fibroblasts. When this aromatase expression was examined, it was found that the increase was due primarily to an increase in expression from promoters II/I.3, strongly suggesting that it was PGE2 produced by the tumor that was largely responsible for this increase in expression (24). Taking these data together, the results are indicative that inflammatory mediators and, in particular, PGE2 produced both by the adipose tissue in the breasts of obese women and by breast tumors can drive aromatase expression locally in the breast (Figure 2). This in turn leads to the local production of estrogens within the breast that stimulate the proliferation of the tumorous breast epithelium due to a positive feed-on mechanism resulting from epithelial-mesenchymal interactions.

Dysregulated Metabolism and Carcinogenesis The concept of dysregulated metabolism and carcinogenesis was first enunciated by the great German biochemist/cell biologist Otto Warburg over 80 years ago. Otto Warburg received the Nobel Prize in 1931 for the discovery of cytochrome oxidase but he also showed that tumor cells exhibit high rates of aerobic glycolysis, a phenomenon that became known as the Warburg effect. Warburg then stated that “this was the cause of cancer” (25). This concept lay fallow for decades because people lost interest in metabolism following the development of molecular biology and recombinant DNA technology and it is only in the last 10 to 15 years or so that interest in metabolism has revived with the development of techniques to study its regulation. Therefore, interest in the Warburg effect has also revived and certainly it correlates with proliferative capacity and is the basis for positron emission tomography scanning. However it has been generally assumed that these changes in metabolism follow the changes in gene expression associated with cellular proliferation. However, there is increasing evidence that the reverse is also true, thus at least partially vindicating Warburg. For example, oncogenes such as HIF1␣ and Myc are potent direct stim-

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ulators of glycolysis at multiple steps, including glucose uptake, hexokinase, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase (26). In addition, HIF1␣ stimulates pyruvate dehydrogenase kinase, which is inhibitory of pyruvate dehydrogenase and thus inhibits the entry of pyruvate into the mitochondria and the formation of acetyl CoA. Thus these oncogenes recapitulate many aspects of the Warburg effect. Furthermore, the downstream signaling pathway of growth factors that activate mTOR, such as insulin, IGF-1, and epidermal growth factor, also regulates metabolism in the direction favoring cell proliferation. Thus mammalian target of rapamycin complex 1 (mTORC1), via posttranscriptional mechanisms, increases the levels of HIF1␣ (27), which as we have seen, results in a stimulation of glucose uptake and glycolysis. However, it also stimulates the processing and activation of SREBP1 and SREBP2, which lead to stimulation of lipid and sterol biosynthesis, respectively (28). Interestingly these factors also stimulate the oxidative limb of the pentose phosphate pathway, which is required for the production of reduced nicotinamide adenine dinucleotide (NAD) phosphate to drive lipid and sterol biosynthesis (Figure 4). These are examples of factors with oncogenic activity that are directly involved in dysregulated metabolism. Other factors do the opposite, namely, those which have tumor-suppressing activity. Examples of these are p53 and AMP-activated protein kinase (AMPK), and indeed p53 is inhibitory of glycolysis at multiple steps, including the uptake of glucose via glucose transporter, GLUT1 and GLUT4; it stimulates TIGAR, which is inhibitory of phosphofructokinase-2. Thus it lowers the levels of fructose-2,6-bisphosphate and so inhibits phosphofructokinase-1 and thus glycolysis. p53 also inhibits phosphoglucomutase. On the other hand, it stimulates the expression of SCO2 (stimulator of cytochrome oxidase 2), which increases the expression of cytochrome oxidase in the mitochondria and thus stimulates oxidative Growth Factor RTK

Transla
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PI3K Akt

4E-BP1

mTORC1 p70S6K SREBP1/2 Processing

Glucose Uptake & Glycolysis Pentose Phosphate Pathway (oxida
ve) Lipid/Sterol Biosynthesis

Figure 4. Action of growth factor signaling cascade to stimulate glycolysis and lipid and sterol biosynthesis. Abbreviations: PI3K, phosphoinositide 3-kinase; RTK, receptor tyrosine kinase; SREBP, sterol regulatory element-binding protein.

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(31), the primary action of the growth factors is to stimulate glycolysis at multiple steps and also to renucleodes Nucleic acids RTK direct mitochondrial metabolism PPP PI3K such that instead of the intermediNADPH Glycolysis ates of the TCA cycle being oxidized Akt to carbon dioxide, they and also inAmino acids 3-PG lipids termediates of glycolysis are used to Protein synth. generate metabolites that serve as inmTOR termediates for the biosynthesis of pyruvate Acetyl CoA proteins, purines, pyrimidines, and PDH lipids. Thus, for example, 3-phosProteins Acetyl CoA phoglycerate can be converted to serine and hence on to glycine and Citrate ACL CS cysteine; Myc stimulates the uptake Asp OAA OAA by the cell of glutamine, an imporMalate Malate Glu α-KG GLS Myc tant fuel metabolite, and its converglutamine sion in the mitochondrion to glutaRTK mate by the enzyme glutaminase. Glutamate in turn can be converted Growth factor glutamine in the mitochondria to ␣-ketoglutaFigure 5. Metabolism is a direct, and not an indirect, response to growth factor action; rate and on to oxaloacetate and asdiagram of role of growth factors to reprogram metabolism to provide intermediates for the partate. Oxaloacetate together with synthesis of proteins, lipids, and nucleic acids, rather than oxidation to CO2. Abbreviations: Asp,aspartate; ␣-KG, ␣-ketoglutarate; CS, citrate synthase; GLS, glutaminase; OAA, acetyl CoA derived from pyruvate oxaloacetate; 3-PG, 3-phosphoglycerate; NADPH, reduced NAD phosphate; PDH, phosphate via the action of citrate synthase dehydrogenase. forms citrate, which can exit the mitochondria and be converted via metabolism via the mitochondrial respiratory chain (29). ATP citrate lyase to acetyl CoA in the cytoplasm and In the case of AMPK, the liver kinase B1 (LKB1)/AMPK hence on to fatty acids. ATP citrate lyase itself is stimupathway is now recognized to be a master regulator of lated by Akt, an important intermediate downstream of energy homeostasis. AMPK in general stimulates path- growth factor signaling. ways that are involved in the generation of energy, such as In his original formulation, Warburg envisaged that glucose uptake, glycolysis, fatty acid oxidation, and mi- the increase in glycolysis observed in tumor cells was a tochondrial biogenesis, and is inhibitory of pathways that consequence of unspecified mitochondrial damage. require energy, such as fatty acid and cholesterol biosyn- Therefore, in this aspect he was incorrect because what in thesis, gluconeogenesis, mTOR activation, and protein fact is taking place is that the mitochondria are reprobiosynthesis (30). grammed. Therefore, instead of oxidizing TCA cycle inThus it appears that metabolism is a direct and not termediates to carbon dioxide, these intermediates are simply an indirect response to growth factor action. In used to provide precursors for the synthesis of proteins, what has been called the traditional demand model (31), purines, pyrimidines, and lipids, all of which are essential the growth factor action is envisioned primarily to be to for cell proliferation. Thus the supply of these key precurstimulate the transcription of genes involved in cell pro- sors comes under the control of metabolism and not the liferation and their translation. This results in a decrease other way around (31). in the ATP to ADP ratio, which leads in turn to an inHowever there is yet another even more direct way crease in glycolysis and oxidative metabolism via the tri- whereby metabolism can regulate the expression of genes, chloroacetic acid (TCA) cycle and mitochondrial respira- as was discussed in a recent review by Sassone-Corsi and tory chain, to generate sufficient ATP to maintain the his group (32). What they point out is that each cell conincreased rate of cell proliferation. In this case, most of the tains 2 meters of DNA, that histone H3 tails play a critical carbon from glucose and other fuel metabolites ends up as role in gene regulation, and that there are approximately carbon dioxide as a consequence of TCA cycle and mito- 3 ⫻ 109 of these per cell. The activity of these tails is in chondrial respiratory chain activity. In the new so-called turn regulated by epigenetic modifications, mainly acetysupply-based model of growth factor action (Figure 5) lation, methylation, and phosphorylation. The imporGrowth factor

glucose

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tance of epigenetic signals has been emphasized by the recent large-scale ENCODE study (33). The enzymes involved in these modifications, namely the markers, require as cosubstrates, acetyl CoA, S-adenosylmethionine, and ATP, respectively. These of course require metabolism for their formation and the epigenetic demand is such that it appears to be regulated by the metabolic activity of the cell. Thus gene regulation itself and not just the supply of precursors for proteins, lipids, and DNA would appear to come under the control of metabolism.

Dysregulated Metabolism and Breast Cancer As indicated above, obesity must be considered the most common state of dysregulated metabolism in the human population. In addition to the role of inflammation in obesity-linked cancers, which has already been discussed in the context of breast cancer, examples of dysregulated metabolism in obesity include insulin resistance, increased synthesis of leptin by the adipose tissue (34), and decreased synthesis of adiponectin (35). Insulin as well as IGF-1 have both been associated with stimulation of growth and increased risk of a number of cancers. In particular, hyperinsulinemia, caused by insulin resistance in the liver, skeletal muscle, and adipose tissue, often predates the diagnosis of type 2 diabetes and has been shown to link obesity and type 2 diabetes to cancer (reviewed in 36). Higher levels of IGF-1 have also been correlated with elevated risk of cancer. In general, the mitogenic actions of insulin and IGF-1 are mediated by the activation of Ras and the MAPK pathway. However, both insulin and IGF-1 also signal via pathways involving phosphoinositide 3-kinase (PI3K), Akt, and mTORC1. mTORC1 activates the p70S6 kinase and inhibits 4EBP1, in both cases leading to a stimulation of protein synthesis and in turn to increased cellular proliferation (37). Although leptin is generally viewed as signaling via a JAK-STAT pathway, it also signals through the PI3K, Akt, and mTORC1 pathway, leading to activation of p70S6 kinase. Recently it has been shown that p70S6 kinase phosphorylates AMPK on serine 491 of the ␣-2 subunit. This is an inhibitory site and so signaling through this pathway leads to inhibition of AMPK activity. Furthermore it was shown that leptin inhibits the activity of AMPK in the hypothalamus via phosphorylation of this site by p70S6 kinase (38). AMPK is generally considered to be antitumorigenic and its role to inhibit lipid and cholesterol biosynthesis is well recognized (reviewed in 39). However, it also inhibits the activity of mTORC1. The tumor suppressor proteins

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TSC1 and TSC2, which are components of the mTOR complex, are known substrates of AMPK and many sporadic cancers are associated with mutations in TSC1 and TSC2, including those of breast, prostate, endometrium, colon, and lung (40). By inhibiting mTORC1 in this fashion, AMPK is inhibitory of protein synthesis. However, AMPK also plays an important role in the regulation of the tumor suppressor p53 whereby activation of AMPK leads to the upregulation of p53 and its phosphorylation at serine 15 (41). The apoptotic actions of p53 are well known but, in addition, p53 activates the cell-cycle inhibitors p21Sip and p27Kip and thus has an inhibitory effect on the cell cycle (42). Thus AMPK can inhibit cell proliferation at multiple sites. In addition, the emerging role of p53 as an inhibitor of aerobic glycolysis and stimulator of mitochondrial respiration provides an additional mechanism whereby AMPK regulates metabolism. As stated above, leptin via its action to stimulate mTORC1 and thus p70S6 kinase would be expected to inhibit the activity of AMPK; this has been shown in the case of the hypothalamus and in breast adipose stromal cells (38). On the other hand, adiponectin, which although synthesized in the adipose tissue, is reduced in obesity, has been shown to activate AMPK in a number of cell types, including hepatocytes, myocytes, preadipocytes, and adipocytes, and it is known to inhibit the proliferation and metastases of breast cancer cells. In the ER-positive MCF7 and T47D cells, adiponectin treatment leads to an increase in AMPK activity and inhibition of p70S6 kinase, which is dependent on LKB1 (43). This has also been shown in MDA-MB-231 breast cancer cells. Moreover the adiponectin receptor peptide agonist ADP355 has been shown to increase AMPK activity and inhibit the growth of orthotopic human breast cancer xenografts (44). Thus, although there are a few reports to the contrary, most studies suggest an antiproliferative role for AMPK in breast cancer. As indicated above, most postmenopausal breast cancers are estrogen-dependent and evidence suggests that it is the estrogen produced locally within the breast that is responsible for the increased proliferation of cancer cells. Recently AMPK has been shown to be a negative regulator of aromatase, the enzyme responsible for the biosynthesis of estrogens in human breast adipose stromal cells (45, 46). Results demonstrate that AMPK prevents nuclear translocation of the CREB coactivator CREB regulated transcription coactivator 2 (CRTC2) in these cells. This was shown initially by Montminy’s group in terms of inhibition of PEPCK gene expression in the liver (47). This is due to phosphorylation of CRTC2 by AMPK, resulting in its sequestration in the cytoplasm by 14-3-3. However a similar result has been shown in human breast

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tissue of obese women, resulting in an increase in the expression of aromatase.

PGE2

PKA

LEPTIN

AMPK

LKB1

ADIPONECTIN METFORMIN

Compounds That Target Breast Cancer Therapeutically via Inflammatory and Metabolic Pathways

AMPK

The fact that inflammatory mediators as well as factors involved in CRTC2 dysregulated metabolism are inCREB volved in breast cancer proliferation, invasion, and metastasis has Nucleus led to a growing number of studies involving the use of these agents to CRTC CRTC determine their therapeutic use in CREB CREB breast cancer prevention and treatCRE CRE ment. In terms of endocrine therapy Aromatase for breast cancer, this field is curFigure 6. Action of PGE2 and leptin to stimulate, and adiponectin and metformin to inhibit, rently dominated by the use of aroaromatase promoter II expression in human breast adipose stromal cells. Abbreviations: CRE, matase inhibitors, which are genercAMP response element; CRTC, CREB regulated transcription coactivator; LKB, liver kinase B; PKA, protein kinase A. ally proven to be superior to tamoxifen in both neoadjuvant and adjuvant settings. The 3 compounds adipose stromal cells (Figure 6). CRTC2, a potent inducer in clinical use are letrozole, arimidex, and exemestane. of aromatase, is sequestered in the cytoplasm in situations where AMPK is activated (45). This appears to be pre- However there are a number of contraindications associdominantly due to an increase in expression of LKB1 as ated with the use of these compounds, such as bone loss, seen in cells stimulated by adiponectin, resulting in an joint pain or arthralgia, hot flashes, and possibly cogniincreased phosphorylation of AMPK at threonine 172, tive defects, as some studies indicate. The reason for this is that these compounds inhibit the catalytic activity of which results in activation of AMPK. On the other hand, the inflammatory mediator PGE2 aromatase and hence inhibit its activity globally, not just causes a downregulation of LKB1 expression and a de- in the breast but in other body sites where estrogens have crease in phosphorylation of AMPK at threonine 172. In important roles to play such as bone, brain, and the caraddition, PGE2 via PKA causes an increase in phosphor- diovascular system. Ideally, one would like to target the ylation of AMPK at the inhibitory serine 485/491 sites on breast specifically and leave other body sites unaffected. the ␣-1/2 subunits. The net effect of this inhibition of This is possible to do postmenopausally in the case of AMPK is to permit the translocation of CRTC2 to the aromatase because the aromatase promoters II/I.3 appear nucleus where it activates CREB and hence aromatase via to be employed specifically in the breast in postmenopromoter II. Similarly, leptin also causes a decrease in pausal women in whom the ovaries no longer synthesize LKB1 expression and a decrease in the phosphorylation estrogens, and bone and brain employ different aromaof AMPK at T172 (Figure 6) (45). However, the fact that tase promoters. leptin stimulates p70S6 kinase in the hypothalamus resulting in an increased phosphorylation of AMPK at the Factors that target inflammation As indicated above, COX2 is believed to be a key facinhibitory serine 491 site (38) suggests that this might also pertain in the human breast adipose stromal cells, al- tor during tumor initiation in tissues subject to chronic though this is still to be established. Thus yet another way inflammation (48). Furthermore COX2 expression in huwhereby obesity leads to an increase in breast cancer risk man breast cancer is correlated with reduced survival, is likely due to the increased formation of leptin and the increased tumor size, high tumor grade, Her-2 overexdecreased formation of adiponectin in the breast adipose pression, as well as metastases to lymph nodes and other 14-3-3

CRTC2

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organs. Moreover COX2 is overexpressed in roughly 50% of breast cancer specimens inclusive of ductal carcinoma in situ and invasive carcinomas. As a consequence, several epidemiological studies including prospective, case-control studies as well as meta-analyses have sought to determine the efficacy of NSAIDs in terms of breast cancer prevention and treatment. The results from these studies have proven to be somewhat inconclusive (49). Although most studies have found a small benefit from the use of aspirin, results on the use of ibuprofen have been mixed with some studies showing as much as a 40% reduction in breast cancer risk (50) and others showing no benefit. At least one study was able to obtain data in a case-controlled study of the use of selective COX2 inhibitors for 2 years or more, namely, celecoxib and rofecoxib, before these compounds were withdrawn from the market (51). In this study 2 years’ use of aspirin led to a benefit odds ratio (OR) of 0.5, whereas similar use of ibuprofen led to an OR of 0.4. The results obtained for the use of COX2-specific inhibitors such as rofecoxib led to a multivariate OR of 0.3. On the other hand, use of acetaminophen, which has little effect on COX2 activity, showed no benefit. Several in vitro studies have also been published examining the effect of COX2 inhibitors (reviewed in 51) as well as inhibitors of the PGE2 receptors on both the proliferation of breast cancer cells and the adipose stromal cells. In addition, one study used a syngeneic mouse breast cancer model of spontaneous lymphatic metastases (52). In general, these studies, whether they used as endpoints cancer cell migration and invasiveness or else aromatase expression (53), have found that mixed COX1- and COX2- or COX2-specific inhibitors inhibited proliferation, migration, and invasiveness as well as aromatase expression. Inhibitors of the 4 PGE2 receptors (54) have also been examined and in general inhibitors of the EP2 and EP4 receptors that are linked to adenylate cyclase were effective, whereas those targeting the EP3 receptor were less effective or ineffective (52, 55). Therefore, although the in vitro data are strongly suggestive of the efficacy of NSAIDs in terms of therapeutic benefit, the clinical data remain somewhat inconclusive. Perhaps this will remain the case until such time as specific COX2 inhibitors are developed that have no potentially lifethreatening contraindications. Factors that target dysregulated metabolism As mentioned above, AMPK is now generally recognized to be a master regulator of energy homeostasis. For example, it phosphorylates and thus inhibits acetyl CoA carboxylase 1 as well as HMG-CoA reductase, thus inhibiting both lipid and cholesterol biosynthesis. It also

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inhibits mTORC1 and thus inhibits angiogenesis, cell growth, and metabolism. However it also plays an important role in the regulation of the tumor suppressor p53 by leading to its upregulation as well as phosphorylation on serine 15. This in turn leads to inhibition of the cell cycle via the release of the sequestered CDK inhibitors p27Kip1 and p21Sip1. Thus AMPK acts to inhibit cellular proliferation by inhibition of protein, lipid, and cholesterol biosynthesis as well as by inhibition of the cell cycle (reviewed in 56). Consequently then, there is much interest in the development of factors that stimulate AMPK, not only in the context of its antidiabetic effects but also in the context of its potential ability to suppress cancer proliferation. The only drug to stimulate AMPK that is currently in clinical use is the antidiabetic drug metformin and most if not all of the actions of metformin are believed to be mediated by stimulation of AMPK (reviewed in 57). However metformin does not act directly on AMPK and the mechanism of this stimulation is not entirely clear. One major action seems to be the inhibition of complex 1 of the mitochondrial respiratory chain (58). This results in an increase in the ratio of AMP-ADP/ATP, which would bring about an activation of AMPK. This is due to binding of AMP or ADP to the ␥-subunit of AMPK, which causes a conformational change, allowing phosphorylation of the ␣-subunit at T172 by LKB1 or in some cases by CaMKK␤ (59, 60). Because of reported decreases in cancer incidence in diabetic patients treated with metformin, a number of studies have been undertaken or are being undertaken to determine whether this commonly prescribed drug may be useful for the treatment of a number of cancers, including those of the breast, colon, and endometrium. For example, in a preoperative window of opportunity randomized trial, nondiabetic women with operable invasive breast cancer were given metformin for 2 weeks (61). This resulted in a significant decrease in the proliferation marker Ki67, although insulin levels remained stable. Moreover, the treatment was associated with, for example, changes in TNF␣ signaling as well as the cell-cycle inhibitors. Genome-wide studies in a number of breast cancer cells have also identified that metformin regulates pathways involved in cell proliferation, for example, inhibitory effects on ribosomal proteins and mitosis-related gene family members (62). Metformin has also been shown to inhibit the proliferation of several cancer cells in culture including those of breast, prostate, ovary, colon, and pancreas (63– 66). The effect of metformin on specific breast cancer subtypes has also been explored. The expression of LKB1 is required for these actions of metformin because it has been shown that LKB1-deficient

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MDA-MB-231 cells were unaffected by metformin treatment. On the other hand, in LKB1 expressing breast cancer cells, including MCF7 cells, metformin treatment led to the activation of AMPK and inhibition of the cell cycle via the release of the inhibitors p27 and p21 (64). Moreover, metformin was shown to cause a 30% decrease in global protein synthesis attributed to a decrease in translation initiation as a consequence of mTOR inhibition (67). Metformin also reduces insulin and IGF-1 signaling, suggesting that it may also inhibit tumor growth via these mechanisms (68). Thus, in an orthotopic model of ERnegative breast cancer, treatment with metformin caused a decrease in systemic IGF-1 and inhibition of tumor cell proliferation (66). Consistent with the action of AMPK to inhibit aromatase expression in human breast adipose stromal cells mentioned above, metformin also significantly inhibited the expression of aromatase in these cells at micromolar concentrations similar to the concentrations present in the blood of women treated with metformin for type II diabetes (69); this inhibition was associated with increased expression of LKB1 and phosphorylation of AMPK at T172 and with a decreased nuclear translocation of CRTC2 (Figure 6) (45, 46). The effects of metformin on aromatase expression were also shown to be promoter-specific, namely use of promoter II (70), implying that treatment may inhibit estrogen production specifically within the breast and thereby prevent side effects associated with current endocrine therapy. Because of these beneficial actions of metformin mediated by AMPK, albeit indirectly, there is considerable interest in developing specific activators of AMPK. However this turns out to be complicated because AMPK is a heterotrimeric enzyme composed of ␣, ␤, and ␥ subunits and multiple isoforms of each subunit have been identified, each of which is encoded by a distinct gene and these are expressed differentially in a tissue-specific fashion. Nevertheless, Lee et al (71) described that the AMPK activator OSU53 significantly inhibited the viability and clonogenic growth of triple negative breast cancer cells in vitro and in vivo, leaving the nonmalignant MCF10A cells unaffected. It also caused an almost 50% decrease in the growth of MDA-MB-231 cells in tumor-bearing mice. AICAR is a commonly used AMPK activator in in vitro studies, which is dependent on its conversion to ZMP, which is an AMP analog. Nevertheless this compound is unsuitable for clinical use. Another compound of potential interest is resveratrol, a polyphenol found in red wine, which has been reported to be a calorie restriction mimetic with potential antiaging and anti-diabetogenic properties in mouse models of diet-induced obesity (72) and in obese humans with

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impaired glucose tolerance (73), but not in nonobese women with normal glucose tolerance (74). The action of resveratrol has been thought primarily to be mediated by activation of SIRT1; however, recently evidence has been presented that its primary action is to activate AMPK. This results from the inhibition of cyclic AMP degrading phosphodiesterases, leading to elevated cyclic AMP levels. This results in the activation of EPAC1, a cyclic AMP effector protein, which in turn activates the CAMKK␤/ AMPK pathway (75). AMPK is known to increase the cellular concentrations of NAD⫹ due to an increase in nicotinamide phosphoribosyltransferase activity and NAD⫹ in turn is an activator of SIRT1, thus apparently explaining the ability of resveratrol to activate SIRT1. Interestingly in this context, resveratrol has been shown to inhibit proliferation of breast cancer cells in an AMPKdependent manner and independent of hormone receptor status (76) and also inhibits aromatase activity in breast cancer cells (77).

Conclusions Interest in metabolism has revived dramatically in the last decade or so with the development of techniques to study its regulation and with the realization that dysregulated metabolism is a key player on center stage in carcinogenesis. Thus there is hardly a current issue of journals such as Cancer Cell and Nature Reviews Cancer that does not feature some aspect of the relationship between these 2 processes. Likewise, inflammation has also emerged as a leading role in cancer biology. Obesity provides a direct link between inflammation and dysregulated metabolism and, not surprisingly therefore, has an emergent role in the etiology of numerous cancers. Although many factors play a part in this linkage, the role of obesity in postmenopausal breast cancer must also be seen in the context that estrogen plays a dominant role in driving this disease. It would seem plausible therefore that obesity should also play a role in the regulation of estrogen biosynthesis in adipose tissue, and this indeed is the case, metabolically in terms of the interplay between leptin and adiponectin and also because of the role of inflammatory mediators as stimulators of aromatase expression, especially PGE2, produced both in the adipose itself and in the tumor. Furthermore, evidence is emerging that these factors play a role in endometrial cancer, which is also estrogen-dependent and linked to obesity. As interest in obesity and carcinogenesis gains momentum, it is likely that we are seeing only the tip of the iceberg in terms of new knowledge and new facets of this deadly connection.

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Simpson and Brown

Inflammation and Obesity in Breast Cancer

Acknowledgments Address all correspondence and requests for reprints to: Evan R. Simpson, PhD, Prince Henry’s Institute of Medical Research– Monash Medical Centre, Block E, Level 4246 Clayton Road, PO Box 5152, Clayton, Victoria–3168, Australia. E-mail: [email protected]. This work was funded by National Health and Medical Research Council (NHMRC, Australia) Project Grant GNT1005735 (to K.A.B. and E.R.S.), the Victorian Government, through the Victorian Cancer Agency funding of the Victorian Breast Cancer Research Consortium (to E.R.S. and K.A.B.), and by the Victorian Government Operational Infrastructure Support Program. E.R.S. is supported by an NHMRC (Australia) Senior Principal Research Fellowship GNT0550900. K.A.B. is supported by an NHMRC (Australia) Career Development Award GNT1007714. Disclosure Summary: The authors have nothing to disclose.

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