Targeting Metabolic Adaptations in the Breast Cancer-Liver Metastatic Niche Using Dietary Approaches to Improve Endocrine Therapy Efficacy.

Estrogen receptor-positive (ER+) metastatic tumors contribute to nearly 70% of breast cancer-related deaths. Most patients with ER+ metastatic breast cancer (MBC) undergo treatment with the estrogen receptor antagonist fulvestrant as standard of care. Yet, among such patients, metastasis in liver is associated with reduced overall survival compared with other metastasis sites. The factors underlying the reduced responsiveness of liver metastases to ER-targeting agents remain unknown, impeding the development of more effective treatment approaches to improve outcomes for patients with ER+ liver metastases. We therefore evaluated site-specific changes in MBC cells and determined the mechanisms through which the liver metastatic niche specifically influences ER+ tumor metabolism and drug resistance. We characterized ER activity of MBC cells both in vitro, using a novel system of tissue-specific extracellular matrix hydrogels representing the stroma of ER+ tumor metastatic sites (liver, lung, and bone), and in vivo, in liver and lung metastasis mouse models. ER+ metastatic liver tumors and MBC cells grown in liver hydrogels displayed upregulated expression of glucose metabolism enzymes in response to fulvestrant. Furthermore, differential ERα activity, but not expression, was detected in liver hydrogels. In vivo, increased glucose metabolism led to increased glycogen deposition in liver metastatic tumors, while a fasting-mimicking diet increased efficacy of fulvestrant treatment to reduce the metastatic burden. Our findings identify a novel mechanism of endocrine resistance driven by the liver tumor microenvironment. IMPLICATIONS: These results may guide the development of dietary strategies to circumvent drug resistance in liver metastasis, with potential applicability in other metastatic diseases.

Combined Targeting of Estrogen Receptor Alpha and Exportin 1 in Metastatic Breast Cancers.

The majority of breast cancer specific deaths in women with estrogen receptor positive (ER+) tumors occur due to metastases that are resistant to therapy. There is a critical need for novel therapeutic approaches to achieve tumor regression and/or maintain therapy responsiveness in metastatic ER+ tumors. The objective of this study was to elucidate the role of metabolic pathways that undermine therapy efficacy in ER+ breast cancers. Our previous studies identified Exportin 1 (XPO1), a nuclear export protein, as an important player in endocrine resistance progression and showed that combining selinexor (SEL), an FDA-approved XPO1 antagonist, synergized with endocrine agents and provided sustained tumor regression. In the current study, using a combination of transcriptomics, metabolomics and metabolic flux experiments, we identified certain mitochondrial pathways to be upregulated during endocrine resistance. When endocrine resistant cells were treated with single agents in media conditions that mimic a nutrient deprived tumor microenvironment, their glutamine dependence for continuation of mitochondrial respiration increased. The effect of glutamine was dependent on conversion of the glutamine to glutamate, and generation of NAD+. PGC1α, a key regulator of metabolism, was the main driver of the rewired metabolic phenotype. Remodeling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and our findings reveal a critical role that ERα-XPO1 crosstalk plays in reducing cancer recurrences. Combining SEL with current therapies used in clinical management of ER+ metastatic breast cancer shows promise for treating and keeping these cancers responsive to therapies in already metastasized patients.

Lysosome Membrane Permeabilization and Disruption of the Molecular Target of Rapamycin (mTOR)-Lysosome Interaction Are Associated with the Inhibition of Lung Cancer Cell Proliferation by a Chloroquinoline Analog.

Lysosomes degrade cellular proteins and organelles and regulate cell signaling by providing a surface for the formation of critical protein complexes, notably molecular target of rapamycin (mTOR) complex 1 (mTORC1). Striking differences in the lysosomes of cancer versus normal cells suggest that they could be targets for drug development. Although the lysomotropic drugs chloroquine (CQ) and hydroxychloroquine (HCQ) have been widely investigated, studies have focused on their ability to inhibit autophagy. We synthesized a novel compound, called EAD1, which is structurally related to CQ but is a 14-fold more potent inhibitor of cell proliferation. Here we find that EAD1 causes rapid relocation, membrane permeabilization (LMP), and deacidification of lysosomes, and it induces apoptosis and irreversibly blocks proliferation of human lung cancer H460, H520, H1299, HCC827, and H1703 cells. EAD1 causes dissociation of mTOR from lysosomes and increases mTOR's perinuclear versus cytoplasmic localization, changes previously shown to inactivate mTORC1. The effect on mTOR was not seen with HCQ, even at >10-fold greater concentrations. Phosphorylation of a downstream target of mTORC1, ribosomal protein S6, was inhibited by EAD1. Although EAD1 also inhibited autophagy, it retained full antiproliferative activity in autophagy-deficient H1650 lung cancer cells, which have a biallelic deletion of Atg7, and in H460 Atg7-knockout cells. As Atg7 is critical for the canonical autophagy pathway, it is likely that inhibition of autophagy is not how EAD1 inhibits cell proliferation. Further studies are needed to determine the relationship of LMP to mTORC1 disruption and their relative contributions to drug-induced cell death. These studies support the lysosome as an underexplored target for new drug development.

Macrophage-derived extracellular vesicle-packaged WNTs rescue intestinal stem cells and enhance survival after radiation injury.

WNT/ß-catenin signalling is crucial for intestinal homoeostasis. The intestinal epithelium and stroma are the major source of WNT ligands but their origin and role in intestinal stem cell (ISC) and epithelial repair remains unknown. Macrophages are a major constituent of the intestinal stroma. Here, we analyse the role of macrophage-derived WNT in intestinal repair in mice by inhibiting their release using a macrophage-restricted ablation of Porcupine, a gene essential for WNT synthesis. Such Porcn-depleted mice have normal intestinal morphology but are hypersensitive to radiation injury in the intestine compared with wild-type (WT) littermates. Porcn-null mice are rescued from radiation lethality by treatment with WT but not Porcn-null bone marrow macrophage-conditioned medium (CM). Depletion of extracellular vesicles (EV) from the macrophage CM removes WNT function and its ability to rescue ISCs from radiation lethality. Therefore macrophage-derived EV-packaged WNTs are essential for regenerative response of intestine against radiation.

Amphiregulin Is a Critical Downstream Effector of Estrogen Signaling in ERα-Positive Breast Cancer.

Estrogen stimulation promotes epithelial cell proliferation in estrogen receptor (ERα)-positive breast cancer. Many ERα target genes have been enumerated, but the identities of the key effectors mediating the estrogen signal remain obscure. During mouse mammary gland development, the estrogen growth factor receptor (EGFR) ligand amphiregulin acts as an important stage-specific effector of estrogen signaling. In this study, we investigated the role of amphiregulin in breast cancer cell proliferation using human tissue samples and tumor xenografts in mice. Amphiregulin was enriched in ERα-positive human breast tumor cells and required for estrogen-dependent growth of MCF7 tumor xenografts. Furthermore, amphiregulin levels were suppressed in patients treated with endocrine therapy. Suppression of EGF receptor signaling appeared necessary for the therapeutic response in this setting. Our findings implicate amphiregulin as a critical mediator of the estrogen response in ERα-positive breast cancer, emphasizing the importance of EGF receptor signaling in breast tumor pathogenesis and therapeutic response.

Discovery of autophagy inhibitors with antiproliferative activity in lung and pancreatic cancer cells.

The autophagy inhibitors chloroquine (CQ) and hydroxychloroquine (HCQ) have single agent antiproliferative activity against human cancer cell lines; however, low potency may limit their antitumor efficacy clinically. We synthesized a series of chloroquine analogs that retained the 4-aminoquinoline subunit and incorporated different substituted triazoles into the target structure. These compounds were tested for growth inhibition against H460 and HCC827 human lung cancer and BxPC3 pancreatic cancer cells. The most potent compound, EAD1, had an IC50 of 5.8 µM in the BxPC3 cells and was approximately 8-fold more potent than CQ and HCQ. EAD1 inhibited autophagy, as judged by the cellular accumulation of the autophagy-related autophagosome proteins LC3-II and p62 and induced apoptosis. The increases in LC3-II levels by the analogues were highly correlated with their growth inhibitory IC50s, suggesting that autophagy blockade is closely linked to inhibition of cell proliferation. EAD1 is a viable lead compound for evaluation of the antitumor activity of autophagy inhibitors in vivo.

Independent anti-angiogenic capacities of coagulation factors X and Xa.

Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti-angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti-angiogenic properties but did not modify the anti-angiogenic effect of FX. Furthermore, only the anti-angiogenic activity of FXa is PAR-1dependent. Using in vivo models, we show that FX and FXa are anti-angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non-hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX.

Progesterone promotes focal adhesion formation and migration in breast cancer cells through induction of protease-activated receptor-1.

Progesterone and progestins have been demonstrated to enhance breast cancer cell migration, although the mechanisms are still not fully understood. The protease-activated receptors (PARs) are a family of membrane receptors that are activated by serine proteases in the blood coagulation cascade. PAR1 (F2R) has been reported to be involved in cancer cell migration and overexpressed in breast cancer. We herein demonstrate that PAR1 mRNA and protein are upregulated by progesterone treatment of the breast cancer cell lines ZR-75 and T47D. This regulation is dependent on the progesterone receptor (PR) but does not require PR phosphorylation at serine 294 or the PR proline-rich region mPRO. The increase in PAR1 mRNA was transient, being present at 3  h and returning to basal levels at 18  h. The addition of a PAR1-activating peptide (aPAR1) to cells treated with progesterone resulted in an increase in focal adhesion (FA) formation as measured by the cellular levels of phosphorylated FA kinase. The combined but not individual treatment of progesterone and aPAR1 also markedly increased stress fiber formation and the migratory capacity of breast cancer cells. In agreement with in vitro findings, data mining from the Oncomine platform revealed that PAR1 expression was significantly upregulated in PR-positive breast tumors. Our observation that PAR1 expression and signal transduction are modulated by progesterone provides new insight into how the progestin component in hormone therapies increases the risk of breast cancer in postmenopausal women.

VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling.

Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2(+/-);Vegfr3(+/-) compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.

Progesterone utilizes distinct membrane pools of tissue factor to increase coagulation and invasion and these effects are inhibited by TFPI.

Tissue factor (TF) serving as the receptor for coagulation factor VII (FVII) initiates the extrinsic coagulation pathway. We previously demonstrated that progesterone increases TF, coagulation and invasion in breast cancer cell lines. Herein, we investigated if tissue factor pathway inhibitor (TFPI) could down-regulate progesterone-increased TF activity in these cells. Classically, TFPI redistributes TF-FVII-FX-TFPI in an inactive quaternary complex to membrane associated lipid raft regions. Herein, we demonstrate that TF increased by progesterone is localized to the heavy membrane fraction, despite progesterone-increased coagulation originating almost exclusively from lipid raft domains, where TF levels are extremely low. The progesterone increase in coagulation is not a rapid effect, but is progesterone receptor (PR) dependent and requires protein synthesis. Although a partial relocalization of TF occurs, TFPI does not require the redistribution to lipid rafts to inhibit coagulation or invasion. Inhibition by TFPI and anti-TF antibodies in lipid raft membrane fractions confirmed the dependence on TF for progesterone-mediated coagulation. Through the use of pathway inhibitors, we further demonstrate that the TF up-regulated by progesterone is not coupled to the progesterone increase in TF-mediated coagulation. However, the progesterone up-regulated TF protein may be involved in progesterone-mediated breast cancer cell invasion, which TFPI also inhibits.

2-Methoxyestradiol inhibits progesterone-dependent tissue factor expression and activity in breast cancer cells.

2-Methoxyestradiol (2ME) is an endogenous metabolite of 17ß-estradiol with antiangiogenic and antitumor properties, although its mechanisms of action remain unclear. Progestins in hormone replacement therapy increase the risk of breast cancer. Progesterone also enhances the procoagulant activity and invasive potential of progesterone receptor (PR)-positive breast cancer cell lines, an effect largely mediated by induction of tissue factor (TF), the cellular activator of the coagulation cascade. Here we show that 2ME abrogates the induction TF expression in progesterone-treated breast cancer cells via a mechanism that does not involve the estrogen receptor. Instead, we demonstrate that by selectively antagonizing ERK1/2 signaling in breast cancer cells, 2ME limits the transactivation potential of ligand-bound PR and inhibits the expression of endogenous progesterone targets, such as TF and signal transducer and activator of transcription 5. We further demonstrate that 2ME can alter the phosphorylation status of PR. Thus, 2ME prevents progesterone-dependent increase in breast cancer cell invasiveness and procoagulant activity by uncoupling PR from the ERK1/2 signal transduction pathway.

A semi-quantitative assay to screen for angiogenic compounds and compounds with angiogenic potential using the EA.hy926 endothelial cell line.

Angiogenesis, the development of new capillary vessels, has a host of clinical manifestations. The identification of agents that increase or decrease angiogenesis is of great pharmaceutical interest. Classically, in vitro angiogenesis utilizes human umbilical vein endothelial cells (HUVEC) grown in matrigel. This valid and simple method has the drawbacks that each cell population is distinct and the constraint of obtaining primary source material. Herein we utilize the established EA.hy926 endothelial cell line as our model for in vitro angiogenesis and present a novel formula to quantify endothelial cell remodeling to identify pro- and anti-angiogenic agents. Furthermore, our technique details the procedures to identify and quantify compounds that have the capacity to generate pro- or anti-angiogenic factors when given to non-endothelial cells, which we define herein as angiogenic potential. In conclusion, we propose a novel formula that we are confident accurately reflects the degree of in vitro angiogenesis allowing the quantification of prospective angiogenic compounds.

A semi-quantitative assay to screen for angiogenic compounds and compounds with angiogenic potential using the EA. hy926 endothelial cell line

Angiogenesis, the development of new capillary vessels, has a host of clinical manifestations. The identification of agents that increase or decrease angiogenesis is of great pharmaceutical interest. Classically, in vitro angiogenesis utilizes human umbilical vein endothelial cells (HUVEC) grown in matrigel. This valid and simple method has the drawbacks that each cell population is distinct and the constraint of obtaining primary source material. Herein we utilize the established EA.hy926 endothelial cell line as our model for in vitro angiogenesis and present a novel formula to quantify endothelial cell remodeling to identify pro- and anti-angiogenic agents. Furthermore, our technique details the procedures to identify and quantify compounds that have the capacity to generate pro- or anti-angiogenic factors when given to non-endothelial cells, which we define herein as angiogenic potential. In conclusion, we propose a novel formula that we are confident accurately reflects the degree of in vitro angiogenesis allowing the quantification of prospective angiogenic compounds.

The action of ovarian hormones in cardiovascular disease.

The incidence of cardiovascular disease (CAD) differs between men and women, in part because of differences in risk factors and hormones. This sexual dimorphism means a lower incidence in atherosclerotic diseases in premenopausal women, which subsequently rises in postmenopausal women to eventually equal that of men. These observations point towards estrogen and progesterone playing a lifetime protective role against CAD in women. As exogenous estrogen and estrogen plus progesterone preparations produce significant reductions in low-density lipoprotein (LDL) cholesterol levels and significant increases in high-density lipoprotein (HDL) cholesterol, this should in theory lower the risk of CAD. However, results from oral contraceptive (OC) use and combined estrogen and progesterone hormone replacement therapy (HRT) have suggested that hormone replacement regimes do not provide cardiovascular protection. In fact, depending on the preparation and the presence or absence of genetic risk factors, an increased risk of cardiovascular diseases such as venous thrombosis, myocardial infarction (MI) and stroke have been observed. Interestingly, in the majority of studies the increase in risk was highest in the first year, after which an increase in risk was not observed, and in some studies a lower risk of CAD was evident after four or five years of exogenous hormone administration. While the debate continues about the merits of HRT, and several good reviews exist on the statistics of CAD in relation to exogenous hormones, we have decided to review the literature to piece together the physiological actions of estrogen and progesterone preparations on the individual mechanistic components leading to CAD; namely, the altered endothelium and the haemostatic balance between coagulation and fibrinolysis. We present possible mechanisms for how HRT and OCs protect against MI in the absence of cardiovascular risk factors but increase the incidence of MI in their presence. We also speculate on the roles played by hormones on the short- and long-term risks of cardiovascular disease.

The action of ovarian hormones in cardiovascular disease

The incidence of cardiovascular disease (CAD) differs between men and women, in part because of differences in risk factors and hormones. This sexual dimorphism means a lower incidence in atherosclerotic diseases in premenopausal women, which subsequently rises in postmenopausal women to eventually equal that of men. These observations point towards estrogen and progesterone playing a lifetime protective role against CAD in women. As exogenous estrogen and estrogen plus progesterone preparations produce significant reductions in low-density lipoprotein (LDL) cholesterol levels and significant increases in high-density lipoprotein (HDL) cholesterol, this should in theory lower the risk of CAD. However, results from oral contraceptive (OC) use and combined estrogen and progesterone hormone replacement therapy (HRT) have suggested that hormone replacement regimes do not provide cardiovascular protection. In fact, depending on the preparation and the presence or absence of genetic risk factors, an increased risk of cardiovascular diseases such as venous thrombosis, myocardial infarction (MI) and stroke have been observed. Interestingly, in the majority of studies the increase in risk was highest in the first year, after which an increase in risk was not observed, and in some studies a lower risk of CAD was evident after four or five years of exogenous hormone administration. While the debate continues about the merits of HRT, and several good reviews exist on the statistics of CAD in relation to exogenous hormones, we have decided to review the literature to piece together the physiological actions of estrogen and progesterone preparations on the individual mechanistic components leading to CAD; namely, the altered endothelium and the haemostatic balance between coagulation and fibrinolysis. We present possible mechanisms for how HRT and OCs protect against MI in the absence of cardiovascular risk factors but increase the incidence of MI in their presence. We also speculate on the roles played by hormones on the short- and long-term risks of cardiovascular disease.