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muPDF file - 102K, Supplementary Figure 2. Canonical pathways affected in tumor tissue. Metabolites that were altered in tumor tissue vs. normal mucosa were analyzed by IPA and significantly affected canonical pathways are shown.

PDF file - 48K, Representative EI fragmentation mass spectra for key metabolites. GC/MS EI fragmentation patterns for sarcosine (A) and 2-hydroxyglutarate (B).

PDF file - 68K, The identity and effect size of changes (AOM vs. saline-treated mice) based on log transformed data are shown for biochemicals whose changes are significant in feces and colorectum, plasma and colorectum and all 3 matrices, 7 weeks after the last injection.

Supplementary Figure 1 Legend from Effects of Tobacco Smoke on Gene Expression and Cellular Pathways in a Cellular Model of Oral Leukoplakia

Commentaries for This Article from The Effect of HIV and HPV Coinfection on Cervical COX-2 Expression and Systemic Prostaglandin E2 Levels

Perspective on this Article from Effects of Cigarette Smoke on the Human Oral Mucosal Transcriptome

Use of tobacco is responsible for ∼30% of all cancer-related deaths in the United States, including cancers of the upper aerodigestive tract. In the current study, 40 current and 40 age- and gender-matched never smokers underwent buccal biopsies to evaluate the effects of smoking on the transcriptome. Microarray analyses were carried out using Affymetrix HGU133 Plus 2 arrays. Smoking altered the expression of numerous genes: 32 genes showed increased expression and 9 genes showed reduced expression in the oral mucosa of smokers versus never smokers. Increases were found in genes involved in xenobiotic metabolism, oxidant stress, eicosanoid synthesis, nicotine signaling, and cell adhesion. Increased numbers of Langerhans cells were found in the oral mucosa of smokers. Interestingly, smoking caused greater induction of aldo-keto reductases, enzymes linked to polycyclic aromatic hydrocarbon–induced genotoxicity, in the oral mucosa of women than men. Striking similarities in expression changes were found in oral compared with the bronchial mucosa. The observed changes in gene expression were compared with known chemical signatures using the Connectivity Map database and suggested that geldanamycin, a heat shock protein 90 inhibitor, might be an antimimetic of tobacco smoke. Consistent with this prediction, geldanamycin caused dose-dependent suppression of tobacco smoke extract–mediated induction of CYP1A1 and CYP1B1 in vitro. Collectively, these results provide new insights into the carcinogenic effects of tobacco smoke, support the potential use of oral epithelium as a surrogate tissue in future lung cancer chemoprevention trials, and illustrate the potential of computational biology to identify chemopreventive agents. Cancer Prev Res; 3(3); 266–78

Supplementary Data from Obesity Is Associated with Inflammation and Elevated Aromatase Expression in the Mouse Mammary Gland

Supplementary Figure 1 from Effects of Tobacco Smoke on Gene Expression and Cellular Pathways in a Cellular Model of Oral Leukoplakia

Supplementary Data from Dietary Polyphenols Suppress Elevated Levels of Proinflammatory Mediators and Aromatase in the Mammary Gland of Obese Mice

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Perspective on this Article from NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase Regulates Levels of Bioactive Lipids in Non–Small Cell Lung Cancer

Perspective from Effects of Tobacco Smoke on Gene Expression and Cellular Pathways in a Cellular Model of Oral Leukoplakia

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Supplementary Methods, Tables 1-5 from Effects of Cigarette Smoke on the Human Oral Mucosal Transcriptome

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Effective diagnosis of inflammation and cancer by molecular imaging is challenging because of interference from nonselective accumulation of the contrast agents in normal tissues. Here, we report a series of novel fluorescence imaging agents that efficiently target cyclooxygenase-2 (COX-2), which is normally absent from cells, but is found at high levels in inflammatory lesions and in many premalignant and malignant tumors. After either i.p. or i.v. injection, these reagents become highly enriched in inflamed or tumor tissue compared with normal tissue and this accumulation provides sufficient signal for in vivo fluorescence imaging. Further, we show that only the intact parent compound is found in the region of interest. COX-2–specific delivery was unambiguously confirmed using animals bearing targeted deletions of COX-2 and by blocking the COX-2 active site with high-affinity inhibitors in both in vitro and in vivo models. Because of their high specificity, contrast, and detectability, these fluorocoxibs are ideal candidates for detection of inflammatory lesions or early-stage COX-2–expressing human cancers, such as those in the esophagus, oropharynx, and colon. Cancer Res; 70(9); 3618–27. ©2010 AACR.

Supplementary Methods, Figures 1-6 from Selective Visualization of Cyclooxygenase-2 in Inflammation and Cancer by Targeted Fluorescent Imaging Agents

Supplementary Figure 1 from Cyclooxygenase-2 Transcription Is Regulated by Human Papillomavirus 16 E6 and E7 Oncoproteins: Evidence of a Corepressor/Coactivator Exchange

Supplementary Figure 2 from Cyclooxygenase-2 Transcription Is Regulated by Human Papillomavirus 16 E6 and E7 Oncoproteins: Evidence of a Corepressor/Coactivator Exchange

Different mechanisms contribute to the development of sporadic, hereditary and colitis-associated colorectal cancer. Inhibitor of DNA binding/differentiation (Id) proteins act as dominant-negative antagonists of basic helix-loop-helix transcription factors. Id1 is a promising target for cancer therapy, but little is known about its role in the development of colon cancer. We used immunohistochemistry to demonstrate that Id1 is overexpressed in human colorectal adenomas and carcinomas, whether sporadic or syndromic. Furthermore, elevated Id1 levels were found in dysplasia and colon cancer arising in patients with inflammatory bowel disease. Because levels of PGE2 are also elevated in both colitis and colorectal neoplasia, we determined whether PGE2 could induce Id1. PGE2 via EP4 stimulated protein kinase A activity resulting in enhanced pCREB-mediated Id1 transcription in human colonocytes. To determine the role of Id1 in carcinogenesis, two mouse models were used. Consistent with the findings in humans, Id1 was overexpressed in tumors arising in both Apc(Min) (/+) mice, a model of familial adenomatous polyposis, and in experimental colitis-associated colorectal neoplasia. Id1 deficiency led to significant decrease in the number of intestinal tumors in Apc(Min) (/+) mice and prolonged survival. In contrast, Id1 deficiency did not affect the number or size of tumors in the model of colitis-associated colorectal neoplasia, likely due to exacerbation of colitis associated with Id1 loss. Collectively, these results suggest that Id1 plays a role in gastrointestinal carcinogenesis. Our findings also highlight the need for different strategies to reduce the risk of colitis-associated colorectal cancer compared with sporadic or hereditary colorectal cancer.

Elevated circulating estrogen levels are associated with increased risk of breast cancer in obese postmenopausal women. Following menopause, the biosynthesis of estrogens through CYP19 (aromatase)-mediated metabolism of androgen precursors occurs primarily in adipose tissue, and the resulting estrogens are then secreted into the systemic circulation. The potential links between obesity, inflammation, and aromatase expression are unknown. In both dietary and genetic models of obesity, we observed necrotic adipocytes surrounded by macrophages forming crown-like structures (CLS) in the mammary glands and visceral fat. The presence of CLS was associated with activation of NF-κB and increased levels of proinflammatory mediators (TNF-α, IL-1β, Cox-2), which were paralleled by elevated levels of aromatase expression and activity in the mammary gland and visceral fat of obese mice. Analyses of the stromal-vascular and adipocyte fractions of the mammary gland suggested that macrophage-derived proinflammatory mediators induced aromatase and estrogen-dependent gene expression (PR, pS2) in adipocytes. Saturated fatty acids, which have been linked to obesity-related inflammation, stimulated NF-κB activity in macrophages leading to increased levels of TNF-α, IL-1β, and Cox-2, each of which contributed to the induction of aromatase in preadipocytes. The discovery of the obesity → inflammation → aromatase axis in the mammary gland and visceral fat and its association with CLS may provide insight into mechanisms underlying the increased risk of hormone receptor-positive breast cancer in obese postmenopausal women, the reduced efficacy of aromatase inhibitors in the treatment of breast cancer in these women, and their generally worse outcomes. The presence of CLS may be a biomarker of increased breast cancer risk or poor prognosis.

Obesity is a risk factor for the development of hormone receptor-positive breast cancer in post-menopausal women. Estrogen synthesis is catalyzed by aromatase. Recently, we identified an obesity-inflammation-aromatase axis in mouse models and women. In mouse models of obesity, inflammatory foci characterized by crown-like structures (CLS) consisting of dead adipocytes encircled by macrophages were found in the mammary gland (MG). CLS of the breast were found in most overweight and obese women. CLS were associated with adipocyte hypertrophy, activation of NF-κB, elevated levels of proinflammatory mediators and aromatase, and increased expression of the progesterone receptor (PR). Collectively, these findings provide a plausible explanation for the link between obesity, chronic inflammation, and post-menopausal breast cancer. Here we investigated whether caloric restriction (CR) reversed the inflammatory state and related molecular changes in the MG of obese mice. Obese ovariectomized C57BL/6J mice were subjected to 30% CR for 7 or 14 weeks. Findings in CR mice were compared with results in mice fed a high fat diet ad libitum or with control mice fed a low fat diet. CR was associated with more than a 75% decrease in mammary CLS/cm2. Reduced histological inflammation following CR was associated with decreased adipocyte diameter and MCP-1 levels, reduced NF-κB binding activity, and normalization of levels of proinflammatory mediators, aromatase and PR. In summary, obesity-related inflammation of the MG and elevated aromatase and PR levels were reversed with CR. Our results provide a rationale for determining whether weight loss can reverse breast inflammation associated with obesity in women.

Cyclooxygenase-2 (COX-2) and Id-1 are overexpressed in a variety of human malignancies. Recently, each of these genes was found to play a role in mediating breast cancer metastasis to the lungs, but their potential interdependence was not evaluated. Hence, the main objective of the current study was to determine whether COX-2-derived prostaglandin (PGE(2)) activated Id-1 transcription, leading in turn to increased invasiveness of mammary epithelial cells. In MDA-MB-231 cells, treatment with PGE(2) induced Id-1, an effect that was mimicked by an EP(4) agonist. PGE(2) via EP(4) activated the epidermal growth factor receptor (EGFR) --ERK1/2 pathway, which led to increased expression of Egr-1. PGE(2) stimulated EGFR signaling by inducing the release of amphiregulin, an EGFR ligand. The ability of PGE(2) to activate Id-1 transcription was mediated by enhanced binding of Egr-1 to the Id-1 promoter. Silencing of COX-2 or pharmacological inhibition of COX-2 led to reduced PGE(2) production, decreased Id-1 expression, and reduced migration of cells through extracellular matrix. A similar decrease in cell migration was found when Id-1 was silenced. The interrelationship between COX-2, PGE(2), Id-1, and cell invasiveness was also compared in nontumorigenic SCp2 and tumorigenic SCg6 mammary epithelial cells. Consistent with the findings in MDA-MB-231 cells, COX-2-derived PGE(2) induced Id-1, leading in turn to increased cell invasiveness. Taken together, these results suggest that PGE(2) via EP(4) activated the EGFR --ERK1/2 --Egr-1 pathway, leading to increased Id-1 transcription and cell invasion. These findings provide new insights into the relationship between COX-2 and Id-1 and their potential role in metastasis.

Obesity is associated with an increased incidence of high-grade prostate cancer and poor prognosis for prostate cancer patients. Recently, we showed that obesity-related periprostatic white adipose tissue (WAT) inflammation, characterized by crown-like structures (CLS) consisting of dead or dying adipocytes surrounded by macrophages, was associated with high-grade prostate cancer. It is possible, therefore, that agents that suppress periprostatic WAT inflammation will alter the development or progression of prostate cancer. Pioglitazone, a ligand of PPARγ, is used to treat diabetes and possesses anti-inflammatory properties. Here, our main objectives were to determine whether pioglitazone inhibited obesity-related periprostatic WAT inflammation in mice and then to elucidate the underlying mechanism. Treatment with pioglitazone reduced the density of CLS in periprostatic fat and suppressed levels of TNFα, TGFβ, and the chemokine monocyte chemoattractant protein-1 (MCP-1). Importantly, the ability of pioglitazone to suppress periprostatic WAT inflammation was abrogated in MCP-1 knockout mice. Pioglitazone caused dose-dependent induction of both adiponectin, an anti-inflammatory adipokine, and its receptor AdipoR2 in cultured 3T3-L1 cells and in periprostatic WAT of obese mice. Pioglitazone blocked TNFα-mediated induction of MCP-1 in 3T3-L1 cells, an effect that was attenuated when either adiponectin or AdipoR2 were silenced. Taken together, pioglitazone-mediated induction of adiponectin suppressed the elevation in MCP-1 levels, thereby attenuating obesity-related periprostatic WAT inflammation. These findings strengthen the rationale for future efforts to determine whether targeting the PPARγ–adiponectin–MCP-1 axis will decrease periprostatic adipose inflammation and thereby reduce the risk of high-grade prostate cancer or improve outcomes for men with prostate cancer. Cancer Prev Res; 11(4); 215–26. ©2017 AACR.

Obesity (body mass index (BMI)⩾30 kg m−2) is associated with an increased risk of estrogen-dependent breast cancer after menopause. Levels of aromatase, the rate-limiting enzyme in estrogen biosynthesis, are elevated in breast tissue of obese women. Recently, the regulation of aromatase by the p53-hypoxia-inducible factor-1α (HIF1α)/pyruvate kinase M2 (PKM2) axis was characterized in adipose stromal cells (ASCs) of women with Li–Fraumeni Syndrome, a hereditary cancer syndrome that predisposes to estrogen-dependent breast cancer. The current study aimed to determine whether stimulation of aromatase by obesity-associated adipokine leptin involves the regulation of the p53-HIF1α/PKM2 axis. Human breast ASCs were used to characterize the p53-HIF1α/PKM2-aromatase axis in response to leptin. The effect of pharmacological or genetic modulation of protein kinase C (PKC), mitogen-activated protein kinase (MAPK), p53, Aha1, Hsp90, HIF1α and PKM2 on aromatase promoter activity, expression and enzyme activity was examined. Semiquantitative immunofluorescence and confocal imaging were used to assess ASC-specific protein expression in formalin-fixed paraffin-embedded tissue sections of breast of women and mammary tissue of mice following a low-fat (LF) or high-fat (HF) diet for 17 weeks. Leptin-mediated induction of aromatase was dependent on PKC/MAPK signaling and the suppression of p53. This, in turn, was associated with an increase in Aha1 protein expression, activation of Hsp90 and the stabilization of HIF1α and PKM2, known stimulators of aromatase expression. Consistent with these findings, ASC-specific immunoreactivity for p53 was inversely associated with BMI in breast tissue, while HIF1α, PKM2 and aromatase were positively correlated with BMI. In mice, HF feeding was associated with significantly lower p53 ASC-specific immunoreactivity compared with LF feeding, while immunoreactivity for HIF1α, PKM2 and aromatase were significantly higher. Overall, findings demonstrate a novel mechanism for the obesity-associated increase in aromatase in ASCs of the breast and support the study of lifestyle interventions, including weight management, which may reduce breast cancer risk via effects on this pathway.

Obesity is associated with breast white adipose tissue (WAT) inflammation, elevated levels of the estrogen biosynthetic enzyme, aromatase, and systemic changes that have been linked to the pathogenesis of breast cancer. Here, we determined whether metabolic obesity, including changes in breast biology and systemic effects, occurs in a subset of women with normal body mass index (BMI). Breast WAT and fasting blood were collected from 72 women with normal BMI ( See related article by Berger, p. 223–25.

Inositol-requiring enzyme 1[α] (IRE1[α])–X-box binding protein spliced (XBP1) signaling maintains endoplasmic reticulum (ER) homeostasis while controlling immunometabolic processes. Yet, the physiological consequences of IRE1a–XBP1 activation in leukocytes remain unexplored. We found that induction of prostaglandin-endoperoxide synthase 2 (Ptgs2/Cox-2) and prostaglandin E synthase (Ptges/mPGES-1) was compromised in IRE1a-deficient myeloid cells undergoing ER stress or stimulated through pattern recognition receptors. Inducible biosynthesis of prostaglandins, including the pro-algesic mediator prostaglandin E2 (PGE2), was decreased in myeloid cells that lack IRE1a or XBP1 but not other ER stress sensors. Functional XBP1 transactivated the human PTGS2 and PTGES genes to enable optimal PGE2 production. Mice that lack IRE1α–XBP1 in leukocytes, or that were treated with IRE1a inhibitors, demonstrated reduced pain behaviors in PGE2-dependent models of pain. Thus, IRE1a–XBP1 is a mediator of prostaglandin biosynthesis and a potential target to control pain.

Obesity increases the risk of hormone receptor–positive breast cancer in postmenopausal women. Levels of aromatase, the rate-limiting enzyme in estrogen biosynthesis, are increased in the breast tissue of obese women. Both prostaglandin E(2) (PGE(2)) and hypoxia-inducible factor 1α (HIF-1α) contribute to the induction of aromatase in adipose stromal cells (ASCs). Sirtuin 1 (SIRT1) binds, deacetylates, and thereby inactivates HIF-1α. Here, we sought to determine whether SIRT1 also plays a role in regulating aromatase expression. We demonstrate that reduced SIRT1 levels are associated with elevated levels of acetyl–HIF-1α, HIF-1α, and aromatase in breast tissue of obese compared with lean women. To determine whether these changes were functionally linked, ASCs were utilized. In ASCs, treatment with PGE(2), which is increased in obese individuals, down-regulated SIRT1 levels, leading to elevated acetyl–HIF-1α and HIF-1α levels and enhanced aromatase gene transcription. Chemical SIRT1 activators (SIRT1720 and resveratrol) suppressed the PGE(2)-mediated induction of acetyl–HIF-1α, HIF-1α, and aromatase. Silencing of p300/CBP-associated factor (PCAF), which acetylates HIF-1α, blocked PGE(2)-mediated increases in acetyl–HIF-1α, HIF-1α, and aromatase. SIRT1 overexpression or PCAF silencing inhibited the interaction between HIF-1α and p300, a coactivator of aromatase expression, and suppressed p300 binding to the aromatase promoter. PGE(2) acted via prostaglandin E2 receptor 2 (EP(2)) and EP(4) to induce activating transcription factor 3 (ATF3), a repressive transcription factor, which bound to a CREB site within the SIRT1 promoter and reduced SIRT1 levels. These findings suggest that reduced SIRT1-mediated deacetylation of HIF-1α contributes to the elevated levels of aromatase in breast tissues of obese women.

Li-Fraumeni syndrome (LFS) patients harbor germ line mutations in the TP53 gene and are at increased risk of hormone receptor-positive breast cancers. Recently, elevated levels of aromatase, the rate-limiting enzyme for estrogen biosynthesis, were found in the breast tissue of LFS patients. Although p53 down-regulates aromatase expression, the underlying mechanisms are incompletely understood. In the present study, we found that LFS stromal cells expressed higher levels of Hsp90 ATPase activity and aromatase compared with wild-type stromal cells. Inhibition of Hsp90 ATPase suppressed aromatase expression. Silencing Aha1 (activator of Hsp90 ATPase 1), a co-chaperone of Hsp90 required for its ATPase activity, led to both inhibition of Hsp90 ATPase activity and reduced aromatase expression. In comparison with wild-type stromal cells, increased levels of the Hsp90 client proteins, HIF-1α, and PKM2 were found in LFS stromal cells. A complex comprised of HIF-1α and PKM2 was recruited to the aromatase promoter II in LFS stromal cells. Silencing either HIF-1α or PKM2 suppressed aromatase expression in LFS stromal cells. CP-31398, a p53 rescue compound, suppressed levels of Aha1, Hsp90 ATPase activity, levels of PKM2 and HIF-1α, and aromatase expression in LFS stromal cells. Consistent with these in vitro findings, levels of Hsp90 ATPase activity, Aha1, HIF-1α, PKM2, and aromatase were increased in the mammary glands of p53 null versus wild-type mice. PKM2 and HIF-1α were shown to co-localize in the nucleus of stromal cells of LFS breast tissue. Taken together, our results show that the Aha1-Hsp90-PKM2/HIF-1α axis mediates the induction of aromatase in LFS.

Li-Fraumeni syndrome (LFS) patients harbor germ line mutations in the TP53 gene and are at increased risk of hormone receptor-positive breast cancers. Recently, elevated levels of aromatase, the rate-limiting enzyme for estrogen biosynthesis, were found in the breast tissue of LFS patients. Although p53 down-regulates aromatase expression, the underlying mechanisms are incompletely understood. In the present study, we found that LFS stromal cells expressed higher levels of Hsp90 ATPase activity and aromatase compared with wild-type stromal cells. Inhibition of Hsp90 ATPase suppressed aromatase expression. Silencing Aha1 (activator of Hsp90 ATPase 1), a co-chaperone of Hsp90 required for its ATPase activity, led to both inhibition of Hsp90 ATPase activity and reduced aromatase expression. In comparison with wild-type stromal cells, increased levels of the Hsp90 client proteins, HIF-1α, and PKM2 were found in LFS stromal cells. A complex comprised of HIF-1α and PKM2 was recruited to the aromatase promoter II in LFS stromal cells. Silencing either HIF-1α or PKM2 suppressed aromatase expression in LFS stromal cells. CP-31398, a p53 rescue compound, suppressed levels of Aha1, Hsp90 ATPase activity, levels of PKM2 and HIF-1α, and aromatase expression in LFS stromal cells. Consistent with these in vitro findings, levels of Hsp90 ATPase activity, Aha1, HIF-1α, PKM2, and aromatase were increased in the mammary glands of p53 null versus wild-type mice. PKM2 and HIF-1α were shown to co-localize in the nucleus of stromal cells of LFS breast tissue. Taken together, our results show that the Aha1-Hsp90-PKM2/HIF-1α axis mediates the induction of aromatase in LFS.

Cyclooxygenase-2 (COX-2) is considered to be a target for anticancer therapy. Histone deacetylase (HDAC) inhibitors exhibit antitumor activity, but the mechanisms of action are incompletely understood. We investigated whether HDAC inhibitors blocked AP-1-mediated activation of COX-2 transcription. Trichostatin A and suberoylanilide hydroxamic acid, two structurally related inhibitors of HDAC activity, blocked AP-1-mediated induction of COX-2 expression and prostaglandin E2 biosynthesis. Chromatin immunoprecipitation assays indicated that HDAC inhibitors suppressed c-Jun binding to the COX-2 promoter and thereby blocked transcription. The observed reduction in binding reflected reduced levels of c-Jun. HDAC inhibitors suppressed the induction of c-jun transcription by blocking the recruitment of the preinitiation complex (RNA polymerase II and TFIIB) to the c-jun promoter. HDAC3 but not HDAC1 or HDAC2 was required for AP-1-mediated stimulation of c-jun expression. Because HDAC inhibitors suppressed the induction of c-jun gene expression, resulting in reduced COX-2 transcription, it was important to determine whether other known AP-1 target genes were also modulated. Cyclin D1 and collagenase-1 are AP-1-dependent genes that have been implicated in carcinogenesis. HDAC inhibitors suppressed the induction of both cyclin D1 and collagenase-1 transcription by inhibiting the binding of c-Jun to the respective promoters. Taken together, these results suggest that HDAC inhibitors block the induction of c-jun transcription by inhibiting the recruitment of the preinitiation complex to the c-jun promoter. This led, in turn, to reduced expression of several activator protein-1-dependent genes (COX-2, cyclin D1, collagenase-1). These findings provide new insights into the mechanisms underlying the antitumor activity of HDAC inhibitors.

The p53 tumor suppressor gene encodes a homotetrameric transcription factor which is activated in response to a variety of cellular stressors, including DNA damage and oncogene activation. p53 mutations occur in50% of human cancers. Although p53 has been shown to regulate Wnt signaling, the underlying mechanisms are not well understood. Here we show that silencing p53 in colon cancer cells led to increased expression of Aha1, a co-chaperone of Hsp90. Heat shock factor-1 was important for mediating the changes in Aha1 levels. Increased Aha1 levels were associated with enhanced interactions with Hsp90, resulting in increased Hsp90 ATPase activity. Moreover, increased Hsp90 ATPase activity resulted in increased phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β), leading to enhanced expression of Wnt target genes. Significantly, levels of Aha1, Hsp90 ATPase activity, Akt, and GSK3β phosphorylation and expression of Wnt target genes were increased in the colons of p53-null as compared with p53 wild type mice. Using p53 heterozygous mutant epithelial cells from Li-Fraumeni syndrome patients, we show that a monoallelic mutation of p53 was sufficient to activate the Aha1/Hsp90 ATPase axis leading to stimulation of Wnt signaling and increased expression of Wnt target genes. Pharmacologic intervention with CP-31398, a p53 rescue agent, inhibited recruitment of Aha1 to Hsp90 and suppressed Wnt-mediated gene expression in colon cancer cells. Taken together, this study provides new insights into the mechanism by which p53 regulates Wnt signaling and raises the intriguing possibility that p53 status may affect the efficacy of anticancer therapies targeting Hsp90 ATPase.

Obesity is a risk factor for the development of hormone receptor (HR)–positive breast cancer in postmenopausal women. Obesity causes subclinical inflammation in white adipose tissue (WAT), characterized by macrophages surrounding dead or dying adipocytes forming crown-like structures (CLS). Estrogen synthesis is catalyzed by aromatase. Previously, we demonstrated CLS and elevated levels of proinflammatory mediators and aromatase in the mammary glands of obese mice and breast tissue of obese women. Here, we tested the hypothesis that supplemental estrogen could prevent or reverse WAT inflammation (WATi) and related molecular changes in the mammary gland. C57BL/6J mice were ovariectomized (OVX) to simulate the postmenopausal state. Supplementation with 17β-estradiol (E2) protected against high fat diet (HFD)-induced weight gain and mammary glands WATi. Expression of proinflammatory mediators (Cox-2, TNFα, IL1β) and aromatase were also reduced in the mammary glands of mice that received supplemental E2. Next, to determine whether E2 supplementation can reverse WATi, obese OVX mice were treated with E2 or placebo and then continued on HFD. E2 supplementation induced weight loss, reversed mammary gland inflammation, and downregulated expression of proinflammatory mediators and aromatase. Finally, we determined whether the protective effects of E2 were mediated by estrogen receptor-α (ERα). Knocking out ERα in ovary intact mice fed a HFD led to weight gain, WATi and elevated levels of proinflammatory mediators and aromatase mimicking the effects of OVX. Taken together, our findings indicate that estrogen via ERα protects against weight gain, WATi and associated increases in proinflammatory mediators and aromatase in the mammary gland. Cancer Prev Res; 8(8); 751–9. ©2015 AACR.

Different mechanisms contribute to the development of sporadic, hereditary and colitis-associated colorectal cancer. Inhibitor of DNA binding/differentiation (Id) proteins act as dominant-negative antagonists of basic helix–loop–helix transcription factors. Id1 is a promising target for cancer therapy, but little is known about its role in the development of colon cancer. We used immunohistochemistry to demonstrate that Id1 is overexpressed in human colorectal adenomas and carcinomas, whether sporadic or syndromic. Furthermore, elevated Id1 levels were found in dysplasia and colon cancer arising in patients with inflammatory bowel disease. Because levels of PGE2 are also elevated in both colitis and colorectal neoplasia, we determined whether PGE2 could induce Id1. PGE2 via EP4 stimulated protein kinase A activity resulting in enhanced pCREB-mediated Id1 transcription in human colonocytes. To determine the role of Id1 in carcinogenesis, two mouse models were used. Consistent with the findings in humans, Id1 was overexpressed in tumors arising in both ApcMin/+ mice, a model of familial adenomatous polyposis, and in experimental colitis-associated colorectal neoplasia. Id1 deficiency led to significant decrease in the number of intestinal tumors in ApcMin/+ mice and prolonged survival. In contrast, Id1 deficiency did not affect the number or size of tumors in the model of colitis-associated colorectal neoplasia, likely due to exacerbation of colitis associated with Id1 loss. Collectively, these results suggest that Id1 plays a role in gastrointestinal carcinogenesis. Our findings also highlight the need for different strategies to reduce the risk of colitis-associated colorectal cancer compared with sporadic or hereditary colorectal cancer. Cancer Prev Res; 8(4); 303–11. ©2015 AACR.

Summary LGR5 and BMI1 mark intestinal stem cells in crypt base columnar cells and +4 position cells, respectively, but characterization of functional markers in these cell populations is limited. ID1 maintains the stem cell potential of embryonic, neural, and long-term repopulating hematopoietic stem cells. Here, we show in both human and mouse intestine that ID1 is expressed in cycling columnar cells, +4 position cells, and transit-amplifying cells in the crypt. Lineage tracing revealed ID1+ cells to be self-renewing, multipotent stem/progenitor cells that are responsible for the long-term renewal of the intestinal epithelium. Single ID1+ cells can generate long-lived organoids resembling mature intestinal epithelium. Complete knockout of Id1 or selective deletion of Id1 in intestinal epithelium or in LGR5+ stem cells sensitizes mice to chemical-induced colon injury. These experiments identify ID1 as a marker for intestinal stem/progenitor cells and demonstrate a role for ID1 in maintaining the potential for repair in response to colonic injury., Highlights • ID1 is expressed in mouse and human intestinal and colonic stem and progenitor cells • ID1+ cells are long-lived and multipotent • Deletion of Id1 in stem and progenitor cells sensitizes mice to colon injury, In this article, Benezra, Dannenberg, and colleagues show that the dominant-negative transcription factor ID1 marks the stem and progenitor cells of the gut and is required in the colon for an optimal response to injury in a mouse model of colitis.

Context Most estrogen-dependent breast cancers occur after menopause, despite low levels of circulating estrogens. Breast expression of the estrogen-biosynthetic enzyme, aromatase, is proposed to drive breast cancer development after menopause. However, the effects of menopause on breast aromatase expression are unknown. Objective To determine the effect of menopause on breast aromatase expression in relation to body mass index (BMI), white adipose tissue inflammation (WATi), and systemic markers of metabolic dysfunction. Design, setting, and participants Cross-sectional study of 102 premenopausal (age 27 to 56) and 59 postmenopausal (age 45 to 74) women who underwent mastectomy for breast cancer treatment/prevention. Outcome Breast tissue was assessed for the presence of crown-like structures and the expression and activity of aromatase. Systemic markers examined include interleukin (IL)-6, insulin, glucose, leptin, adiponectin, high-sensitivity C-reactive protein (hsCRP), cholesterol, and triglycerides. Multivariable analysis was performed for aromatase messenger RNA (mRNA) in relation to BMI, WATi, and blood markers. Results Postmenopausal women had higher BMI and more breast WATi than premenopausal women. Fasting levels of IL-6, glucose, leptin, hsCRP, and homeostatic model assessment 2 insulin resistance score were higher in the postmenopausal group. BMI was positively correlated with aromatase mRNA in both pre- and postmenopausal women. Aromatase levels were higher in breast tissue of postmenopausal women, with levels being higher in inflamed vs noninflamed, independent of BMI. Adipocyte diameter and levels of leptin, hsCRP, adiponectin, and high-density lipoprotein cholesterol were more strongly correlated with aromatase in postmenopausal than premenopausal women. Conclusions Elevated aromatase in the setting of adipose dysfunction provides a possible mechanism for the higher incidence of hormone-dependent breast cancer in obese women after menopause.

The p53 tumor suppressor gene encodes a homotetrameric transcription factor which is activated in response to a variety of cellular stressors, including DNA damage and oncogene activation. p53 mutations occur in >50% of human cancers. Although p53 has been shown to regulate Wnt signaling, the underlying mechanisms are not well understood. Here we show that silencing p53 in colon cancer cells led to increased expression of Aha1, a co-chaperone of Hsp90. Heat shock factor-1 was important for mediating the changes in Aha1 levels. Increased Aha1 levels were associated with enhanced interactions with Hsp90, resulting in increased Hsp90 ATPase activity. Moreover, increased Hsp90 ATPase activity resulted in increased phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β), leading to enhanced expression of Wnt target genes. Significantly, levels of Aha1, Hsp90 ATPase activity, Akt, and GSK3β phosphorylation and expression of Wnt target genes were increased in the colons of p53-null as compared with p53 wild type mice. Using p53 heterozygous mutant epithelial cells from Li-Fraumeni syndrome patients, we show that a monoallelic mutation of p53 was sufficient to activate the Aha1/Hsp90 ATPase axis leading to stimulation of Wnt signaling and increased expression of Wnt target genes. Pharmacologic intervention with CP-31398, a p53 rescue agent, inhibited recruitment of Aha1 to Hsp90 and suppressed Wnt-mediated gene expression in colon cancer cells. Taken together, this study provides new insights into the mechanism by which p53 regulates Wnt signaling and raises the intriguing possibility that p53 status may affect the efficacy of anticancer therapies targeting Hsp90 ATPase.

The increasing rate of obesity worldwide is predicted to be associated with a surge in diseases. Notably, obesity has been linked to approximately 20% of cancer cases in the United States; obesity is associated with both increased risk and worse outcomes after diagnosis. Altered levels of circulating factors are strongly implicated, including insulin, insulin-like growth factor 1, leptin, adiponectin, and interleukin-6 (IL-6). In addition, increasing attention has focused on the consequences of local adipose inflammation. Inflammatory foci characterized by crown-like structures consisting of dead adipocytes encircled by macrophages occur in white adipose depots, including the breast tissue, of most overweight and obese women. Saturated fatty acids, released as a consequence of obesity-associated lipolysis, induce macrophage activation via Toll-like receptor 4, thereby stimulating NF-κB signaling. This, in turn, activates transcription of proinflammatory genes including COX-2, IL-6, IL-1β, and TNFα. Elevated levels of proinflammatory mediators cause both local and systemic effects. Of particular relevance with regard to breast cancer is increased transcription of the CYP19 gene encoding aromatase, the rate-limiting enzyme for estrogen synthesis. Notably, this obesity–inflammation–aromatase axis provides a plausible explanation for increased rates of postmenopausal, hormone receptor–positive breast cancer associated with obesity and hence may offer targets for interventions to attenuate risk or improve prognosis. Potential approaches include weight reduction, exercise, and suppression of obesity-driven signaling pathways using pharmaceutical or dietary agents. A key future goal is to identify biomarkers that accurately report adipose inflammation, both for identification of at-risk individuals and to assess the efficacy of interventions. Clin Cancer Res; 19(22); 6074–83. ©2013 AACR.