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.

GnRH regulates expression of LHB via transcriptional regulation of early growth response 1 (EGR1), an immediate early gene that encodes a zinc-finger DNA-binding protein. EGR1 interacts functionally with the orphan nuclear receptor steroidogenic factor 1 (SF1) and pituitary homeobox 1, a member of the paired-like homeodomain family. The functional synergism of this tripartite interaction defines the maximal level of LHB transcription that can occur in response to GnRH. Results presented herein provide new evidence that the interaction between SF1 and EGR1 also requires beta-catenin, a transcriptional coactivator and member of the canonical Wnt signaling pathway. For instance, targeted reduction of beta-catenin attenuates activity of a GnRH-primed LHB promoter. Additional gene reporter assays indicate that overexpression of beta-catenin, or its targeted reduction by small interfering RNA, modulates activity of both SF1 and EGR1 as well as their functional interaction. beta-Catenin coimmunoprecipitates with SF1. Moreover, an SF1 mutant that lacks a beta-catenin binding domain has compromised transcriptional activity and fails to interact synergistically with EGR1. Finally, GnRH promotes beta-catenin colocalization with SF1 and EGR1 on the endogenous mouse Lhb promoter-regulatory region. Taken together, these data suggest that beta-catenin binds to SF1 and that this interaction is required for subsequent functional interaction with EGR1. Thus, these data identify beta-catenin as a new and required member of the basal transcriptional complex that allows the LHB promoter to achieve maximal activity in response to GnRH.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) are widespread environmental pollutants. TCDD is well known for its adverse effects on female reproduction when administered acutely to immature or adult rats. It is also known that fetal/neonatal exposure to this compound alters reproductive parameters. It is unknown whether exposure to PCDF causes similar adverse effects in offspring. The objectives of the study were to investigate the effects of in utero and lactational (IUL) exposure to TCDD and PCDF on subsequent growth, estrous cycles, and ovulation. Additionally a gonadotropin-primed immature rat model was used to investigate possible direct effects on the ovary after IUL exposure to TCDD (2.5 microg/kg) by evaluating 1) ovarian morphometrics and 2) serum estradiol concentrations. Body weights were reduced in animals with IUL exposure to TCDD and PCDF relative to those in controls at 10 days of age (P < 0.05 for each), and this difference was maintained until termination of the experiment at 125-165 days of age (P < 0.05). Exposure to TCDD or PCDF also disrupted regular estrous cycles and inhibited ovulation rate. On Day 23 (before eCG stimulation), ovaries from animals exposed to TCDD contained the same number of primordial, primary, secondary, preantral, and antral follicles as ovaries from control animals. On Day 25 (48 h after eCG stimulation), ovaries from TCDD-exposed rats had significantly fewer large preovulatory follicles when compared with ovaries from controls. The numbers of smaller follicles (both antral and small antral) were not different. Serum estradiol was significantly lower in TCDD-exposed animals 48 h after eCG stimulation.

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.

GnRH binds its G-coupled protein receptor, GnRHR, on pituitary gonadotropes and stimulates transcription of Cga, Lhb, and Fshb. These three genes encode two heterodimeric glycoprotein hormones, LH and FSH, that act as gonadotropins by regulating gametogenesis and steroidogenesis in both the testes and ovary. GnRH also regulates transcription of Gnrhr. Thus, regulated expression of Cga, Lhb, Fshb, and Gnrhr provides a genomic signature unique to functional gonadotropes. Steadily increasing evidence now indicates that GnRH regulates transcription of its four signature genes indirectly through a hierarchical transcriptional network that includes distinct subclasses of DNA-binding proteins that comprise the immediate early gene (IEG) family. These IEGs, in turn, confer hormonal responsiveness to the four signature genes. Although the IEGs confer responsiveness to GnRH, they cannot act alone. Instead, additional DNA-binding proteins, including the orphan nuclear receptor steroidogenic factor 1, act permissively to allow the four signature genes to respond to GnRH-induced changes in IEG levels. Emerging new findings now indicate that beta-catenin, a transcriptional coactivator and member of the canonical WNT signaling pathway, also plays an essential role in transducing the GnRH signal by interacting with multiple DNA-binding proteins in gonadotropes. Herein we propose that these interactions with beta-catenin define a multicomponent transcriptional network required for regulated expression of the four signature genes of the gonadotrope, Cga, Lhb, Fshb, and Gnrhr.

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 was 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.

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 β3 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 β3 signaling, AHR, and new therapeutic targeting strategies will be discussed.

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.