Mechanisms by which obesity regulates inflammation and anti-tumor immunity in cancer.

Obesity is associated with an increased risk for 13 different cancers. The increased risk for cancer in obesity is mediated by obesity-associated changes in the immune system. Obesity has distinct effects on different types of inflammation that are tied to tumorigenesis. For example, obesity promotes chronic inflammation in adipose tissue that is tumor-promoting in peripheral tissues. Conversely, obesity inhibits acute inflammation that rejects tumors. Obesity therefore promotes cancer by differentially regulating chronic versus acute inflammation. Given that obesity is chronic, the initial inflammation in adipose tissue will lead to systemic inflammation that could induce compensatory anti-inflammatory reactions in peripheral tissues to suppress chronic inflammation. The overall effect of obesity in peripheral tissues is therefore dependent on the duration and severity of obesity. Adipose tissue is a complex tissue that is composed of many cell types in addition to adipocytes. Further, adipose tissue cellularity is different at different anatomical sites throughout the body. Consequently, the sensitivity of adipose tissue to obesity is dependent on the anatomical location of the adipose depot. For example, obesity induces more inflammation in visceral than subcutaneous adipose tissue. Based on these studies, the mechanisms by which obesity promotes tumorigenesis are multifactorial and immune cell type-specific. The objective of our paper is to discuss the cellular mechanisms by which obesity promotes tumorigenesis by regulating distinct types of inflammation in adipose tissue and the tumor microenvironment.

The effects of environmental aryl hydrocarbon receptor ligands on signaling and cell metabolism in cancer.

Dioxin and dioxin-like compounds are chlorinated organic pollutants formed during the manufacturing of other chemicals. Dioxins are ligands of the aryl hydrocarbon receptor (AHR), that induce AHR-mediated biochemical and toxic responses and are persistent in the environment. 2,3,7,8- tetrachlorodibenzo para dioxin (TCDD) is the prototypical AHR ligand and its effects represent dioxins. TCDD induces toxicity, immunosuppression and is a suspected tumor promoter. The role of TCDD in cancer however is debated and context-dependent. Environmental particulate matter, polycyclic aromatic hydrocarbons, perfluorooctane sulfonamide, endogenous AHR ligands, and cAMP signaling activate AHR through TCDD-independent pathways. The effect of activated AHR in cancer is context-dependent. The ability of FDA-approved drugs to modulate AHR activity has sparked interest in their repurposing for cancer therapy. TCDD by interfering with endogenous pathways, and overstimulating other endogenous pathways influences all stages of cancer. Herein we review signaling mechanisms that activate AHR and mechanisms by which activated AHR modulates signaling in cancer including affected metabolic pathways.

mTOR Regulation of N-Myc Downstream Regulated 1 (NDRG1) Phosphorylation in Clear Cell Renal Cell Carcinoma.

The mechanistic target of rapamycin (mTOR) kinase is a component of two signaling complexes that are known as mTOR complex 1 (mTORC1) and mTORC2. We sought to identify mTOR-phosphorylated proteins that are differently expressed in clinically resected clear cell renal cell carcinoma (ccRCC) relative to pair-matched normal renal tissue. Using a proteomic array, we found N-Myc Downstream Regulated 1 (NDRG1) showed the greatest increase (3.3-fold) in phosphorylation (on Thr346) in ccRCC. This was associated with an increase in total NDRG1. RICTOR is a required subunit in mTORC2, and its knockdown decreased total and phospho-NDRG1 (Thr346) but not NDRG1 mRNA. The dual mTORC1/2 inhibitor, Torin 2, significantly reduced (by ~100%) phospho-NDRG1 (Thr346). Rapamycin is a selective mTORC1 inhibitor that had no effect on the levels of total NDRG1 or phospho-NDRG1 (Thr346). The reduction in phospho-NDRG1 (Thr346) due to the inhibition of mTORC2 corresponded with a decrease in the percentage of live cells, which was correlated with an increase in apoptosis. Rapamycin had no effect on ccRCC cell viability. Collectively, these data show that mTORC2 mediates the phosphorylation of NDRG1 (Thr346) in ccRCC. We hypothesize that RICTOR and mTORC2-mediated phosphorylation of NDRG1 (Thr346) promotes the viability of ccRCC cells.

The putative endogenous AHR ligand ITE reduces JAG1 and associated NOTCH1 signaling in triple negative breast cancer cells.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor. Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. TNBC expresses AHR and AHR ligands have anti-cancer activity in TNBC. The aggressiveness of TNBC is due in part to JAG1-NOTCH1 signaling. ITE is a putative endogenous AHR ligand. We show that ITE reduces the expression of JAG1 the amount of Notch 1 intracellular domain (NICD1) and the phosphorylation of STAT3 (at tyrosine 705) in TNBC MDA-MB-231 cells. The STAT3 inhibitor STATTIC also reduced JAG1. STAT3, thus, mediates regulation of JAG1 in MDA-MB-231 cells. Reducing the expression of JAG1 with short interfering RNA decreases the growth, migration and invasiveness of MDA-MB-231 cells. JAG1, therefore, has cellular effects in MDA-MB-231 cells under basal conditions. We consequently evaluated if exposing cells to greater amounts of JAG1 would counteract ITE cellular effects in MDA-MB-231 cells. The results show that JAG1 does not counteract the cellular effects of ITE. JAG1, thus, has no effect on growth or invasiveness in MDA-MB-231 cells treated with ITE. JAG1, therefore, has context dependent roles in MDA-MB-231 cells (basal versus ITE treatment). The results also show that other pathways, not inhibition of the JAG1-NOTCH1 pathway, are important for mediating the growth and invasive inhibitory effect of ITE on MDA-MB-231 cells.

The Regulation and Function of the L-Type Amino Acid Transporter 1 (LAT1) in Cancer.

The progression of cancer is associated with increases in amino acid uptake by cancer cells. Upon their entry into cells through specific transporters, exogenous amino acids are used to synthesize proteins, nucleic acids and lipids and to generate ATP. The essential amino acid leucine is also important for maintaining cancer-associated signaling pathways. By upregulating amino acid transporters, cancer cells gain greater access to exogenous amino acids to support chronic proliferation, maintain metabolic pathways, and to enhance certain signal transduction pathways. Suppressing cancer growth by targeting amino acid transporters will require an in-depth understanding of how cancer cells acquire amino acids, in particular, the transporters involved and which cancer pathways are most sensitive to amino acid deprivation. L-Type Amino Acid Transporter 1 (LAT1) mediates the uptake of essential amino acids and its expression is upregulated during the progression of several cancers. We will review the upstream regulators of LAT1 and the downstream effects caused by the overexpression of LAT1 in cancer cells.

Aryl hydrocarbon receptor (AHR) regulation of L-Type Amino Acid Transporter 1 (LAT-1) expression in MCF-7 and MDA-MB-231 breast cancer cells.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is regulated by environmental toxicants that function as AHR agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). L-Type Amino Acid Transporter 1 (LAT1) is a leucine transporter that is overexpressed in cancer. The regulation of LAT1 by AHR in MCF-7 and MDA-MB-231 breast cancer cells (BCCs) was investigated in this report. Ingenuity pathway analysis (IPA) revealed a significant association between TCDD-regulated genes (TRGs) and molecular transport. Overlapping the TCDD-RNA-Seq dataset obtained in this study with a published TCDD-ChIP-seq dataset identified LAT1 as a primary target of AHR-dependent TCDD induction. Short interfering RNA (siRNA)-directed knockdown of AHR confirmed that TCDD-stimulated increases in LAT1 mRNA and protein required AHR expression. TCDD-stimulated increases in LAT1 mRNA were also inhibited by the AHR antagonist CH-223191. Upregulation of LAT1 by TCDD coincided with increases in leucine uptake by MCF-7 cells in response to TCDD. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays revealed increases in AHR, AHR nuclear translocator (ARNT) and p300 binding and histone H3 acetylation at an AHR binding site in the LAT1 gene in response to TCDD. In MCF-7 and MDA-MB-231 cells, endogenous levels of LAT1 mRNA and protein were reduced in response to knockdown of AHR expression. Knockdown experiments demonstrated that proliferation of MCF-7 and MDA-MB-231 cells is dependent on both LAT1 and AHR. Collectively, these findings confirm the dependence of cancer cells on leucine uptake and establish a mechanism for extrinsic and intrinsic regulation of LAT1 by AHR.

Insulin/Insulin-like growth factors in cancer: new roles for the aryl hydrocarbon receptor, tumor resistance mechanisms, and new blocking strategies.

The insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor (IR) are receptor tyrosine kinases that are expressed in cancer cells. The results of different studies indicate that tumor proliferation and survival is dependent on the IGF1R and IR, and that their inhibition leads to reductions in proliferation and increases in cell death. Molecular targeting therapies that have been used in solid tumors include anti-IGF1R antibodies, anti-IGF1/IGF2 antibodies, and small molecule inhibitors that suppress IGF1R and IR kinase activity. New advances in the molecular basis of anti-IGF1R blocking antibodies reveal they are biased agonists and promote the binding of IGF1 to integrin ß3 receptors in some cancer cells. Our recent reports indicate that pharmacological aryl hydrocarbon receptor (AHR) ligands inhibit breast cancer cell responses to IGFs, suggesting that targeting AHR may have benefit in cancers whose proliferation and survival are dependent on insulin/IGF signaling. Novel aspects of IGF1R/IR in cancer, such as biased agonism, integrin ß3 signaling, AHR, and new therapeutic targeting strategies will be discussed.

Endogenous aryl hydrocarbon receptor promotes basal and inducible expression of tumor necrosis factor target genes in MCF-7 cancer cells.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that upon activation by the toxicant 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) stimulates gene expression and toxicity. AHR is also important for normal mouse physiology and may play a role in cancer progression in the absence of environmental toxicants. The objective of this report was to identify AHR-dependent genes (ADGs) whose expression is regulated by AHR in the absence of toxicants. RNA-Seq analysis revealed that AHR regulated the expression of over 600 genes at an FDR<10% in MCF-7 breast cancer cells upon knockdown with short interfering RNA. Pathway analysis revealed that a significant number of ADGs were components of TCDD and tumor necrosis factor (TNF) pathways. We also demonstrated that siRNA knockdown of AHR modulated TNF induction of MNSOD and cytotoxicity in MCF-7 cells. Collectively, the major new findings of this report are: (1) endogenous AHR promotes the expression of xenobiotic metabolizing enzymes even in the absence of toxicants and drugs, (2) AHR by modulating the basal expression of a large fraction of TNF target genes may prime them for TNF stimulation and (3) AHR is required for TNF induction of MNSOD and the cellular response to cytotoxicity in MCF-7 cells. This latter result provides a potentially new role for AHR in MCF-7 cancer progression as a mediator of TNF and antioxidant responses.

Insulin like growth factor 2 regulation of aryl hydrocarbon receptor in MCF-7 breast cancer cells.

Insulin like growth factor (IGF)-1 and IGF-2 stimulate normal growth, development and breast cancer cell proliferation. Cyclin D1 (CCND1) promotes cell cycle by inhibiting retinoblastoma protein (RB1). The aryl hydrocarbon receptor (AHR) is a major xenobiotic receptor that also regulates cell cycle. The purpose of this study was to investigate whether IGF-2 promotes MCF-7 breast cancer proliferation by inducing AHR. Western blot and quantitative real time PCR (Q-PCR) analysis revealed that IGF-2 induced an approximately 2-fold increase (P<.001) in the expression of AHR and CCND1. Chromatin immunoprecipitation (ChIP), followed by Q-PCR indicated that IGF-2 promoted (P<.001) a 7-fold increase in AHR binding on the CCND1 promoter. AHR knockdown significantly (P<.001) inhibited IGF-2 stimulated increases in CCND1 mRNA and protein. AHR knockdown cells were less (P<.001) responsive to the proliferative effects of IGF-2 than control cells. Collectively, our findings have revealed a new regulatory mechanism by which IGF-2 induction of AHR promotes the expression of CCND1 and the proliferation of MCF-7 cells. This previously uncharacterized pathway could be important for the proliferation of IGF responsive cancer cells that also express AHR.

Aryl hydrocarbon receptor ligands inhibit igf-ii and adipokine stimulated breast cancer cell proliferation.

Obesity increases human cancer risk and the risk for cancer recurrence. Adipocytes secrete paracrine factors termed adipokines that stimulate signaling in cancer cells that induce proliferation. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that plays roles in tumorigenesis, is regulated by exogenous lipophilic chemicals, and has been explored as a therapeutic target for cancer therapy. Whether exogenous AHR ligands modulate adipokine stimulated breast cancer cell proliferation has not been investigated. We provide evidence that adipocytes secrete insulin-like growth factor 2 (IGF-2) at levels that stimulate the proliferation of human estrogen receptor (ER) positive breast cancer cells. Using highly specific AHR ligands and AHR short interfering RNA (AHR-siRNA), we show that specific ligand-activated AHR inhibits adipocyte secretome and IGF-2-stimulated breast cancer cell proliferation. We also report that a highly specific AHR agonist significantly (P < 0.05) inhibits the expression of E2F1, CCND1 (known as Cyclin D1), MYB, SRC, JAK2, and JUND in breast cancer cells. Collectively, these data suggest that drugs that target the AHR may be useful for treating cancer in human obesity.