NR0B2 re-educates myeloid immune cells to reduce regulatory T cell expansion and progression of breast and other solid tumors.

Although survival from breast cancer has dramatically increased, many will develop recurrent, metastatic disease. Unfortunately, survival for this stage of disease remains very low. Activating the immune system has incredible promise since it has the potential to be curative. However, immune checkpoint blockade (ICB) which works through T cells has been largely disappointing for metastatic breast cancer. One reason for this is a suppressive myeloid immune compartment that is unaffected by ICB. Cholesterol metabolism and proteins involved in cholesterol homeostasis play important regulatory roles in myeloid cells. Here, we demonstrate that NR0B2, a nuclear receptor involved in negative feedback of cholesterol metabolism, works in several myeloid cell types to impair subsequent expansion of regulatory T cells (Tregs); Tregs being a subset known to be highly immune suppressive and associated with poor therapeutic response. Within myeloid cells, NR0B2 serves to decrease many aspects of the inflammasome, ultimately resulting in decreased IL1ß; IL1ß driving Treg expansion. Importantly, mice lacking NR0B2 exhibit accelerated tumor growth. Thus, NR0B2 represents an important node in myeloid cells dictating ensuing Treg expansion and tumor growth, thereby representing a novel therapeutic target to re-educate these cells, having impact across different solid tumor types. Indeed, a paper co-published in this issue demonstrates the therapeutic utility of targeting NR0B2.

Development of NR0B2 as a therapeutic target for the re-education of tumor associated myeloid cells.

Immune checkpoint blockade (ICB) has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. A paper co-published in this issue describes how NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Here, we develop NR0B2 as a potential therapeutic target. NR0B2 in tumors is associated with improved survival for several cancer types including breast. Importantly, NR0B2 expression is also prognostic of ICB success. Within breast tumors, NR0B2 expression is inversely associated with FOXP3, a marker of Tregs. While a described agonist (DSHN) had some efficacy, it required high doses and long treatment times. Therefore, we designed and screened several derivatives. A methyl ester derivative (DSHN-OMe) emerged as superior in terms of (1) cellular uptake, (2) ability to regulate expected expression of genes, (3) suppression of Treg expansion using in vitro co-culture systems, and (4) efficacy against the growth of primary and metastatic tumors. This work identifies NR0B2 as a target to re-educate myeloid immune cells and a novel ligand with significant anti-tumor efficacy in preclinical models.

Cholesterol Metabolite 27-Hydroxycholesterol Enhances the Secretion of Cancer Promoting Extracellular Vesicles by a Mitochondrial ROS-Induced Impairment of Lysosomal Function.

Extracellular vesicles (EVs) serve as crucial mediators of cell-to-cell communication in normal physiology as well as in diseased states, and have been largely studied in regard to their role in cancer progression. However, the mechanisms by which their biogenesis and secretion are regulated by metabolic or endocrine factors remain unknown. Here, we delineate a mechanism by which EV secretion is regulated by a cholesterol metabolite, 27-Hydroxycholesterol (27HC), where treatment of myeloid immune cells (RAW 264.7 and J774A.1) with 27HC impairs lysosomal homeostasis, leading to shunting of multivesicular bodies (MVBs) away from lysosomal degradation, towards secretion as EVs. This impairment of lysosomal function is caused by mitochondrial dysfunction and subsequent increase in reactive oxygen species (ROS). Interestingly, cotreatment with a mitochondria-targeted antioxidant rescued the lysosomal impairment and attenuated the 27HC-mediated increase in EV secretion. Overall, our findings establish how a cholesterol metabolite regulates EV secretion and paves the way for the development of strategies to regulate cancer progression by controlling EV secretion.

Re-education of myeloid immune cells to reduce regulatory T cell expansion and impede breast cancer progression.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy but has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. We demonstrate that NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the NLRP3 inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Loss of NR0B2 increased mammary tumor growth and metastasis. Small molecule agonists, including one developed here, reduced Treg expansion, reduced metastatic growth and improved the efficacy of ICB. This work identifies NR0B2 as a target to re-educate myeloid immune cells providing proof-of-principle that this cholesterol-homeostasis axis may have utility in enhancing ICB.

The nuclear receptor TLX (NR2E1) inhibits growth and progression of triple- negative breast cancer.

Development of targeted therapies will be a critical step towards reducing the mortality associated with triple-negative breast cancer (TNBC). To achieve this, we searched for targets that met three criteria: (1) pharmacologically targetable, (2) expressed in TNBC, and (3) expression is prognostic in TNBC patients. Since nuclear receptors have a well-defined ligand-binding domain and are thus highly amenable to small-molecule intervention, we focused on this class of protein. Our analysis identified TLX (NR2E1) as a candidate. Specifically, elevated tumoral TLX expression was associated with prolonged recurrence-free survival and overall survival for breast cancer patients with either estrogen receptor alpha (ERα)-negative or basal-like tumors. Using two TNBC cell lines, we found that stable overexpression of TLX impairs in vitro proliferation. RNA-Seq analysis revealed that TLX reduced the expression of genes implicated in epithelial-mesenchymal transition (EMT), a cellular program known to drive metastatic progression. Indeed, TLX overexpression significantly decreased cell migration and invasion, and robustly decreased the metastatic capacity of TNBC cells in murine models. We identify SERPINB2 as a likely mediator of these effects. Taken together, our work indicates that TLX impedes the progression of TNBC. Several ligands have been shown to regulate the transcriptional activity of TLX, providing a framework for the future development of this receptor for therapeutic intervention.

The Liver X Receptor Is Selectively Modulated to Differentially Alter Female Mammary Metastasis-associated Myeloid Cells.

Dysregulation of cholesterol homeostasis is associated with many diseases such as cardiovascular disease and cancer. Liver X receptors (LXRs) are major upstream regulators of cholesterol homeostasis and are activated by endogenous cholesterol metabolites such as 27-hydroxycholesterol (27HC). LXRs and various LXR ligands such as 27HC have been described to influence several extra-hepatic biological systems. However, disparate reports of LXR function have emerged, especially with respect to immunology and cancer biology. This would suggest that, similar to steroid nuclear receptors, the LXRs can be selectively modulated by different ligands. Here, we use RNA-sequencing of macrophages and single-cell RNA-sequencing of immune cells from metastasis-bearing murine lungs to provide evidence that LXR satisfies the 2 principles of selective nuclear receptor modulation: (1) different LXR ligands result in overlapping but distinct gene expression profiles within the same cell type, and (2) the same LXR ligands differentially regulate gene expression in a highly context-specific manner, depending on the cell or tissue type. The concept that the LXRs can be selectively modulated provides the foundation for developing precision pharmacology LXR ligands that are tailored to promote those activities that are desirable (proimmune), but at the same time minimizing harmful side effects (such as elevated triglyceride levels).

Extracellular Vesicles-The Next Frontier in Endocrinology.

Extracellular vesicles (EVs), including exosomes, are emerging as important carriers of signals in normal and pathological physiology. As EVs are a long-range communication or signaling modality-just like hormones are-the field of endocrinology is uniquely poised to offer insight into their functional biology and regulation. EVs are membrane-bound particles secreted by many different cell types and can have local or systemic effects, being transported in body fluids. They express transmembrane proteins, some of which are shared between EVs and some being specific to the tissue of origin, that can interact with target cells directly (much like hormones can). They also contain cargo within them that includes DNA, RNA, miRNA, and various metabolites. They can fuse with target cells to empty their cargo and alter their target cell physiology in this way also. Similar to the endocrine system, the EV system is likely to be under homeostatic control, making the regulation of their biogenesis and secretion important aspects to study. In this review, we briefly highlight select examples of how EVs are implicated in normal physiology and disease states. We also discuss what is known about their biogenesis and regulation of secretion. We hope that this paper inspires the endocrinology field to use our collective expertise to explore these new multimodal "hormones."

The Cholesterol Metabolite 27HC Increases Secretion of Extracellular Vesicles Which Promote Breast Cancer Progression.

Cholesterol has been implicated in the clinical progression of breast cancer, a disease that continues to be the most commonly diagnosed cancer in women. Previous work has identified the cholesterol metabolite 27-hydroxycholesterol (27HC) as a major mediator of the effects of cholesterol on breast tumor growth and progression. 27HC can act as an estrogen receptor (ER) modulator to promote the growth of ERα+ tumors, and as a liver X receptor (LXR) ligand in myeloid immune cells to establish an immune-suppressive program. In fact, the metastatic properties of 27HC require the presence of myeloid cells with neutrophils (polymorphonuclear neutrophils; PMNs) being essential for the increase in lung metastasis in murine models. In an effort to further elucidate the mechanisms by which 27HC alters breast cancer progression, we made the striking finding that 27HC promoted the secretion of extracellular vesicles (EVs), a diverse assortment of membrane bound particles that includes exosomes. The resulting EVs had a size distribution that was skewed slightly larger than EVs generated by treating cells with vehicle. The increase in EV secretion and size was consistent across 3 different subtypes: primary murine PMNs, RAW264.7 monocytic cells, and 4T1 murine mammary cancer cells. Label-free analysis of 27HC-EVs indicated that they had a different metabolite composition to those from vehicle-treated cells. Importantly, 27HC-EVs from primary PMNs promoted tumor growth and metastasis in 2 different syngeneic models, demonstrating the potential role of 27HC-induced EVs in the progression of breast cancer. EVs from PMNs were taken up by cancer cells, macrophages, and PMNs, but not T cells. Since EVs did not alter proliferation of cancer cells, it is likely that their protumor effects are mediated through interactions with myeloid cells. Interestingly, RNA-seq analysis of tumors from 27HC-EV-treated mice do not display significantly altered transcriptomes, suggesting that the effects of 27HC-EVs occur early on in tumor establishment and growth. Future work will be required to elucidate the mechanisms by which 27HC increases EV secretion, and how these EVs promote breast cancer progression. Collectively, however, our data indicate that EV secretion and content can be regulated by a cholesterol metabolite, which may have detrimental effects in terms of disease progression, important findings given the prevalence of both breast cancer and hypercholesterolemia.

Arsenic-induced immunomodulatory effects disorient the survival-death interface by stabilizing the Hsp90/Beclin1 interaction.

Ground water arsenic contamination is a global menace. Since arsenic may affect the immune system, leading to immunesuppression, we investigated the effects of acute arsenic exposure on the thymus and spleen using Swiss albino mice, exposed to 5 ppm, 15 ppm and 300 ppm of sodium arsenite for 7 d. Effects on cytokine balance and cell survivability were subsequently analyzed. Our data showed that arsenic treatment induced debilitating alterations in the tissue architecture of thymus and spleen. A dose-dependent decrease in the ratio of CD4+-CD8+ T-cells was observed along with a pro-inflammatory response and redox imbalance. In addition, pioneering evidences established the ability of arsenic to induce an up regulation of Hsp90, eventually resulting in stabilization of its client protein Beclin-1, an important autophagy-initiating factor. This association initiated the autophagic process, confirmed by co-immunoprecipitation assay, acridine orange staining and Western blot, indicating the effort of cells trying to survive at lower doses. However, increased arsenic assault led to apoptotic cell death in the lymphoid organs, possibly by increased ROS generation. There are several instances of autophagy and apoptosis taking place either simultaneously or sequentially due to oxidative stress. Since arsenic is a potent environmental stress factor, exposure to arsenic led to a dose-dependent increase in both autophagy and apoptosis in the thymus and spleen, and cell death could therefore possibly be induced by autophagy. Therefore, exposure to arsenic leads to serious effects on the immune physiology in mice, which may further have dire consequences on the health of exposed animals.

Impairment of ultrastructure and cytoskeleton during progression of cardiac hypertrophy to heart failure.

Studies at the morphological and molecular level have found that transgenic (Tg) mice that overexpress myotrophin in the heart develop hypertrophy at the early age of 4 weeks; this condition worsens to heart failure (HF) at approximately 36 weeks. However, how the sustained effects of alteration in cytoarchitecture of the contractile machinery lead to malfunction of the normal heart remains unclear. Our data have shown that at 4 weeks, the cytoarchitecture observed in left ventricular (LV) tissue samples of Tg mice is similar to that of wild-type (WT) mice. However, as the disease progresses, cardiomyocytes show deterioration in some mitochondrial as well as myofibril features, evidenced by swelling of mitochondria, misalignment of myofibril structure, and blurring as well as breakage of Z-lines. At 36 weeks of age, Tg mice (the group in transition from hypertrophy to HF) show significant degenerative changes in cardiomyocytes, including swelling of mitochondria, disruption of the nuclear membrane, and absence of myofibril structure. Besides these, formation of myelin bodies was also observed, a feature typically found in human hearts with HF. Changes in Z-line architecture were further confirmed by alteration in the gene expression profile of desmin and tubulin, the two main cytoskeletal proteins. We thus conclude that Tg mice overexpressing myotrophin show no visible changes in the initiation phase (4 weeks); however, as the disease progresses, alterations in the cytoskeleton are found during the transition phase from hypertrophy to HF (36 weeks onward). Our data suggest that treatment for prevention/reversal of hypertrophy should start at the early stage of hypertrophy to prevent its transition to HF.

Silencing the myotrophin gene by RNA interference leads to the regression of cardiac hypertrophy.

Myotrophin-induced activation of NF-kappaB has been shown to be associated with cardiac hypertrophy (CH) that progresses to heart failure (HF). In the present study, we examined the cause-and-effect relationship between myotrophin and NF-kappaB activation using small hairpin RNA (shRNA) against myotrophin both in vitro (using neonatal rat myocytes) and in vivo [using myotrophin transgenic (Myo-Tg) mice, which overexpress myotrophin in the heart, develop CH, and gradually progress to HF]. Among several lentiviral vectors expressing myotrophin shRNAs, L-sh-109 showed the best silencing effect at both the mRNA (155.3 +/- 5.9 vs. 32.5 +/- 5.5, P < 0.001) and protein levels associated with a significant reduction of atrial natriuretic factor (ANF) and NF-kappaB. In vivo, when L-sh-109 was delivered directly into the hearts of 10-wk-old Myo-Tg mice, we observed a significant regression of cardiac mass (8.0 vs. 5.7 mg/g, P < 0.001) and myotrophin gene expression (54.5% over untreated Myo-Tg mice, P < 0.001) associated with a reduction in ANF and NF-kappaB signaling components. Our data suggest that using RNA interference to silence the myotrophin gene prevents NF-kappaB activation, associated with an attenuation of CH. This strategy could be an excellent therapeutic means for the treatment of CH and HF.

Prevention of cardiac hypertrophy and heart failure by silencing of NF-kappaB.

Activation of the nuclear factor (NF)-kappaB signaling pathway may be associated with the development of cardiac hypertrophy and its transition to heart failure (HF). The transgenic Myo-Tg mouse develops hypertrophy and HF as a result of overexpression of myotrophin in the heart associated with an elevated level of NF-kappaB activity. Using this mouse model and an NF-kappaB-targeted gene array, we first determined the components of NF-kappaB signaling cascade and the NF-kappaB-linked genes that are expressed during the progression to cardiac hypertrophy and HF. Second, we explored the effects of inhibition of NF-kappaB signaling events by using a gene knockdown approach: RNA interference through delivery of a short hairpin RNA against NF-kappaB p65 using a lentiviral vector (L-sh-p65). When the short hairpin RNA was delivered directly into the hearts of 10-week-old Myo-Tg mice, there was a significant regression of cardiac hypertrophy, associated with a significant reduction in NF-kappaB activation and atrial natriuretic factor expression. Our data suggest, for the first time, that inhibition of NF-kappaB using direct gene delivery of sh-p65 RNA results in regression of cardiac hypertrophy. These data validate NF-kappaB as a therapeutic target to prevent hypertrophy/HF.

Combination therapy with indolylquinoline derivative and sodium antimony gluconate cures established visceral leishmaniasis in hamsters.

2-(2"-Dichloroacetamidobenzyl)-3-(3'-indolylquinoline), designated indolylquinoline derivative A, reduced the splenic and the liver parasite burdens by >93.0% in Leishmania donovani-infected hamsters, whereas sodium antimony gluconate (SAG) reduced the burdens approximately 80.0%. Complete clearance of parasitemia from the livers and spleens was noticed when infected animals received indolylquinoline derivative A plus SAG, suggesting that indolylquinoline derivative A has potential as a new agent for sole or conjunctive therapy for leishmaniasis.