The functional activity of E-cadherin controls tumor cell metastasis at multiple steps.

E-cadherin is a tumor suppressor protein, and the loss of its expression in association with the epithelial mesenchymal transition (EMT) occurs frequently during tumor metastasis. However, many metastases continue to express E-cadherin, and a full EMT is not always necessary for metastasis; also, positive roles for E-cadherin expression in metastasis have been reported. We hypothesize instead that changes in the functional activity of E-cadherin expressed on tumor cells in response to environmental factors is an important determinant of the ability of the tumor cells to metastasize. We find that E-cadherin expression persists in metastatic lung nodules and circulating tumor cells (CTCs) in two mouse models of mammary cancer: genetically modified MMTV-PyMT mice and orthotopically grafted 4T1 tumor cells. Importantly, monoclonal antibodies that bind to and activate E-cadherin at the cell surface reduce lung metastasis from endogenous genetically driven tumors and from tumor cell grafts. E-cadherin activation inhibits metastasis at multiple stages, including the accumulation of CTCs from the primary tumor and the extravasation of tumor cells from the vasculature. These activating mAbs increase cell adhesion and reduce cell invasion and migration in both cell culture and three-dimensional spheroids grown from primary tumors. Moreover, activating mAbs increased the frequency of apoptotic cells without affecting proliferation. Although the growth of the primary tumors was unaffected by activating mAbs, CTCs and tumor cells in metastatic nodules exhibited increased apoptosis. Thus, the functional state of E-cadherin is an important determinant of metastatic potential beyond whether the gene is expressed.

RORα switches transcriptional mode of ERRγ that results in transcriptional repression of CYP2E1 under ethanol-exposure.

Increased cytochrome P450 2E1 (CYP2E1) expression is the main cause of oxidative stress, which exacerbates alcoholic liver diseases (ALDs). Estrogen-related receptor gamma (ERRγ) induces CYP2E1 expression and contributes to enhancing alcohol-induced liver injury. Retinoic acid-related orphan receptor alpha (RORα) has antioxidative functions; however, potential cross-talk between ERRγ and RORα in the regulation of CYP2E1 has not been studied. We report that RORα suppressed ERRγ-mediated CYP2E1 expression. A physical interaction of RORα with ERRγ at the ERRγ-response element in the CYP2E1 promoter was critical in this suppression. At this site, coregulator recruitment of ERRγ was switched from coactivator p300 to the nuclear receptor corepressor 1 in the presence of RORα. Cross-talk between ERRγ and RORα was demonstrated in vivo, in that administration of JC1-40, a RORα activator, significantly decreased both CYP2E1 expression and the signs of liver injury in ethanol-fed mice, and this was accompanied by coregulator switching. Thus, this non-classical RORα pathway switched the transcriptional mode of ERRγ, leading to repression of alcohol-induced CYP2E1 expression, and this finding may provide a new therapeutic strategy against ALDs.

Anoctamin 1 (TMEM16A) is essential for testosterone-induced prostate hyperplasia.

Benign prostatic hyperplasia (BPH) is characterized by an enlargement of the prostate, causing lower urinary tract symptoms in elderly men worldwide. However, the molecular mechanism underlying the pathogenesis of BPH is unclear. Anoctamin1 (ANO1) encodes a Ca(2+)-activated chloride channel (CaCC) that mediates various physiological functions. Here, we demonstrate that it is essential for testosterone-induced BPH. ANO1 was highly amplified in dihydrotestosterone (DHT)-treated prostate epithelial cells, whereas the selective knockdown of ANO1 inhibited DHT-induced cell proliferation. Three androgen-response elements were found in the ANO1 promoter region, which is relevant for the DHT-dependent induction of ANO1. Administration of the ANO1 blocker or Ano1 small interfering RNA, inhibited prostate enlargement and reduced histological abnormalities in vivo. We therefore concluded that ANO1 is essential for the development of prostate hyperplasia and is a potential target for the treatment of BPH.

22-S-Hydroxycholesterol protects against ethanol-induced liver injury by blocking the auto/paracrine activation of MCP-1 mediated by LXRα.

Chronic ethanol consumption causes hepatic steatosis and inflammation, which are associated with liver hypoxia. Monocyte chemoattractant protein-1 (MCP-1) is a hypoxia response factor that determines recruitment and activation of monocytes to the site of tissue injury. The level of MCP-1 is elevated in the serum and liver of patients with alcoholic liver disease (ALD); however, the molecular details regarding the regulation of MCP-1 expression are not yet understood completely. Here, we show the role of liver X receptor α (LXRα) in the regulation of MCP-1 expression during the development of ethanol-induced fatty liver injury, using an antagonist, 22-S-hydroxycholesterol (22-S-HC). First, administration of 22-S-HC attenuated the signs of liver injury with decreased levels of MCP-1 and its receptor CCR2 in ethanol-fed mice. Second, hypoxic conditions or treatment with the LXRα agonist GW3965 significantly induced the expression of MCP-1, which was completely blocked by treatment with 22-S-HC or infection by shLXRα lentivirus in the primary hepatocytes. Third, over-expression of LXRα or GW3965 treatment increased MCP-1 promoter activity by increasing the binding of hypoxia-inducible factor-1α to the hypoxia response elements, together with LXRα. Finally, treatment with recombinant MCP-1 increased the level of expression of LXRα and LXRα-dependent lipid droplet accumulation in both hepatocytes and Kupffer cells. These data show that LXRα and its ligand-induced up-regulation of MCP-1 and MCP-1-induced LXRα-dependent lipogenesis play a key role in the autocrine and paracrine activation of MCP-1 in the pathogenesis of alcoholic fatty liver disease, and that this activation may provide a promising new target for ALD therapy.

SIGN-R1 and complement factors are involved in the systemic clearance of radiation-induced apoptotic cells in whole-body irradiated mice.

Although SIGN-R1-mediated complement activation pathway has been shown to enhance the systemic clearance of apoptotic cells, the role of SIGN-R1 in the clearance of radiation-induced apoptotic cells has not been characterized and was investigated in this study. Our data indicated that whole-body γ-irradiation of mice increased caspase-3(+) apoptotic lymphocyte numbers in secondary lymphoid organs. Following γ-irradiation, SIGN-R1 and complements (C4 and C3) were simultaneously increased only in the mice spleen tissue among the assessed tissues. In particular, C3 was exclusively activated in the spleen. The delayed clearance of apoptotic cells was markedly prevalent in the spleen and liver of SIGN-R1 KO mice, followed by a significant increase of CD11b(+) cells. These results indicate that SIGN-R1 and complement factors play an important role in the systemic clearance of radiation-induced apoptotic innate immune cells to maintain tissue homeostasis after γ-irradiation.

Retinoic acid receptor-related orphan receptor α-induced activation of adenosine monophosphate-activated protein kinase results in attenuation of hepatic steatosis.

UNLABELLED: There is increasing evidence that the retinoic acid receptor-related orphan receptor α (RORα) plays an important role in the regulation of metabolic pathways, particularly of fatty acid and cholesterol metabolism; however, the role of RORα in the regulation of hepatic lipogenesis has not been studied. Here, we report that RORα attenuates hepatic steatosis, probably via activation of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) and repression of the liver X receptor α (LXRα). First, RORα and its activator, cholesterol sulfate (CS), induced phosphorylation of AMPK, which was accompanied by the activation of serine-threonine kinase liver kinase B1 (LKB1). Second, the activation of RORα, either by transient transfection or CS treatment, decreased the TO901317-induced transcriptional expression of LXRα and its downstream target genes, such as the sterol regulatory element binding protein-1 (SREBP-1) and fatty acid synthase. RORα interacted physically with LXRα and inhibited the LXRα response element in the promoter of LXRα, indicating that RORα interrupts the autoregulatory activation loop of LXRα. Third, infection with adenovirus encoding RORα suppressed the lipid accumulation that had been induced by a free-fatty-acid mixture in cultured cells. Furthermore, we observed that the level of expression of the RORα protein was decreased in the liver of mice that were fed a high-fat diet. Restoration of RORα via tail-vein injection of adenovirus (Ad)-RORα decreased the high-fat-diet-induced hepatic steatosis. Finally, we synthesized thiourea derivatives that activated RORα, thereby inducing activation of AMPK and repression of LXRα. These compounds decreased hepatic triglyceride levels and lipid droplets in the high-fat-diet-fed mice. CONCLUSION: We found that RORα induced activation of AMPK and inhibition of the lipogenic function of LXRα, which may be key phenomena that provide the beneficial effects of RORα against hepatic steatosis.

Positive cross-talk between hypoxia inducible factor-1α and liver X receptor α induces formation of triglyceride-loaded foam cells.

OBJECTIVE: Atherosclerosis is a chronic and progressive inflammatory disease of the arteries that is characterized by subendothelial accumulation of lipid-rich macrophages, called foam cells. We sought to identify the molecular details of cross-talk between liver X receptor α (LXRα) and hypoxia-inducible factor 1α (HIF-1α) for the formation of triglyceride-rich foam cells under hypoxic conditions. METHODS AND RESULTS: We first observed that expression of LXRα and its target lipogenic genes was time-dependently induced in human primary macrophages and RAW 264.7 cells under hypoxia. Similarly, TO901317, an activator of LXRα, enhanced the expression level and the transcriptional activity of HIF-1α. Second, we demonstrated that LXRα increased HIF-1α protein stability through a physical interaction between the ligand binding domain of LXRα and the oxygen-dependent degradation domain of HIF-1α. Third, we found that the activation of HIF-1α or LXRα synergistically induced triglyceride accumulation in macrophages. Finally, we showed that LXRα and HIF-1α were codistributed in the macrophages of atherosclerotic lesions of patients. CONCLUSIONS: These results suggest that the positive feed-forward regulation of transcriptional activity and protein stability of LXRα and HIF-1α has an important impact in foam cell formation.

Liver X receptor mediates hepatitis B virus X protein-induced lipogenesis in hepatitis B virus-associated hepatocellular carcinoma.

UNLABELLED: Although hepatitis B virus X protein (HBx) has been implicated in abnormal lipid metabolism in hepatitis B virus (HBV)-associated hepatic steatosis, its underlying molecular mechanism remains unclear. Liver X receptor (LXR) plays an important role in regulating the expression of genes involved in hepatic lipogenesis. Here we demonstrate that LXRalpha and LXRbeta mediate HBV-associated hepatic steatosis. We have found that HBx induces the expression of LXR and its lipogenic target genes, such as sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and peroxisome proliferator-activated receptor, and this is accompanied by the accumulation of lipid droplets. RNA interference with LXR expression decreases the amount of lipid droplets as well as the expression of the lipogenic genes, and this indicates that HBx-induced lipogenesis is LXR-dependent. LXRalpha and HBx colocalize in the nucleus and are physically associated. HBx induces the transactivation function of LXRalpha by recruiting CREB binding protein to the promoter of the target gene. Furthermore, we have observed that expression of LXR is increased in the livers of HBx-transgenic mice. Finally, there is a significant increase in the expression of LXRbeta (P = 0.036), SREBP-1c (P = 0.008), FAS, and stearoyl-coenyzme A desaturase-1 (P = 0.001) in hepatocellular carcinoma (HCC) in comparison with adjacent nontumorous nodules in human HBV-associated HCC specimens. CONCLUSION: Our results suggest a novel association between HBx and LXR that may represent an important mechanism explaining HBx-induced hepatic lipogenesis during HBV-associated hepatic carcinogenesis.

Transcriptional activation of HIF-1 by RORalpha and its role in hypoxia signaling.

OBJECTIVE: Hypoxia-inducible factor 1alpha (HIF-1alpha) is primarily involved in the adapting of cells to changes in oxygen levels, which is essential for normal vascular function. Recently, physiological roles for retinoic acid-related orphan receptor alpha (RORalpha) have been implicated in cardiovascular diseases such as atherosclerosis. In this study, we have investigated the potential roles of RORalpha in the hypoxia signaling pathway in connection with activation of HIF-1alpha. METHODS AND RESULTS: Under hypoxic conditions, expression of RORalpha was induced. When RORalpha was introduced exogenously, protein level as well as transcriptional activity of HIF-1alpha was enhanced. Putative ligands of RORalpha, such as melatonin and cholesterol sulfate, induced transcriptional activity for HIF-1alpha, which was abolished by RNA interference against RORalpha. RORalpha was physically associated with HIF-1alpha through DNA binding domain, which was required to the RORalpha-induced stabilization and transcriptional activation of HIF-1alpha. Finally, either infection with adenovirus encoding RORalpha or treatment with ROR ligands enhanced the formation of capillary tubes by human umbilical vascular endothelial cells. CONCLUSIONS: Our results provide a new insight for the function of RORalpha in amplification of hypoxia signaling and suggest a potential application of RORalpha ligands for the therapy of hypoxia-associated vascular diseases.

Identification of formaldehyde-responsive genes by suppression subtractive hybridization.

Formaldehyde is frequently used in indoor household and occupational environments. Inhalation of formaldehyde invokes an inflammatory response, including a variety of allergic signs and symptoms. Therefore, formaldehyde has been considered as the most prevalent cause of sick building syndrome, which has become a major social problem, especially in developing urban areas. Further formaldehyde is classified as a genotoxicant in the respiratory tract of rats and humans. To better understand the molecular mechanisms involved in formaldehyde intoxication, we sought differentially regulated genes by formaldehyde exposure to Hs 680.Tr human trachea cells, using polymerase chain reaction (PCR)-based suppression subtractive hybridization. We identified 27 different formaldehyde-inducible genes, including those coding for the major histocompatibility complex, class IA, calcyclin, glutathione S-transferase pi, mouse double minute 2 (MDM2), platelet-derived growth factor receptor alpha, and which are known to be associated with cell proliferation and differentiation, immunity and inflammation, and detoxification. Induction of these genes by formaldehyde treatment was confirmed by reverse transcription PCR and western blot analysis. Further, the expression of calcyclin, glutathione S-transferase pi, PDGFRA and MDM2 were significantly induced in the tracheal epithelium of Sprague Dawley rats after formaldehyde inhalation. Our results suggest that the elevated levels of these genes may be associated with the formaldehyde-induced toxicity, and that they deserve evaluation as potential biomarkers for formaldehyde intoxication.

E-cadherin in contact inhibition and cancer.

E-cadherin is a key component of the adherens junctions that are integral in cell adhesion and maintaining epithelial phenotype of cells. Homophilic E-cadherin binding between cells is important in mediating contact inhibition of proliferation when cells reach confluence. Loss of E-cadherin expression results in loss of contact inhibition and is associated with increased cell motility and advanced stages of cancer. In this review we discuss the role of E-cadherin and its downstream signaling in regulation of contact inhibition and the development and progression of cancer.

NR1D1 enhances oxidative DNA damage by inhibiting PARP1 activity.

Cancer cells exhibit an elevated intracellular level of reactive oxygen species (ROS) because of their accelerated metabolism, mitochondrial dysfunction, and antioxidant deficit. The oxidative stress in cancer cells may provide clinical benefits, which can be associated with a better response to anticancer therapies. Therefore, identifying the regulatory pathway of oxidative stress in cancer cells is important in the development of therapeutic targets that enhance sensitivity to ROS-generating anticancer therapies. Here, we report that nuclear receptor subfamily 1, group D, member 1 (NR1D1; Rev-erbα) inhibited DNA repair of ROS-induced DNA damage in breast cancer cells. NR1D1 interacted with poly(ADP-ribose) polymerase 1 (PARP1) and subsequently inhibited catalytic activity of PARP1. NR1D1 enhanced accumulation of DNA damage, which increased sensitivity of breast cancer cells to oxidative stress. Our findings suggest that NR1D1 could be a therapeutic target for breast cancer treatment, especially in those patients treated with ROS-inducing chemotherapeutic agents.

The trisaccharide raffinose modulates epidermal differentiation through activation of liver X receptor.

The epidermal barrier function requires optimal keratinocyte differentiation and epidermal lipid synthesis. Liver X receptor (LXR) α and ß, are important transcriptional regulators of the epidermal gene expression. Here, we show that raffinose, a ubiquitously present trisaccharide in plants, activated the transcriptional activity of LXRα/ß, which led to the induction of genes required for keratinocyte differentiation such as involucrin and filaggrin, and genes involved in lipid metabolism and transport including SCD1 and ABCA1 in both HaCaT and normal human epidermal keratinocytes. Raffinose induced the expression of JunD and Fra1, and their DNA binding in the AP1 motif in the promoters of involucrin and loricrin. Interestingly, LXR bound the AP1 motif upon raffinose treatment, and conversely, JunD and Fra1 bound the LXR response element in promoters of LXR target genes, which indicates the presence of a postive cross-talk between LXR and AP1 in the regualtion of these genes. Finally, the effect of raffinose in epidermal barrier function was confirmed by applying raffinose in an ointment formulation to the skin of hairless mice. These findings suggest that raffinose could be examined as an ingredient in functional cosmetics and therapeutic agents for the treatment of cutaneous disorders associated with abnormal epidermal barrier function.

NR1D1 Recruitment to Sites of DNA Damage Inhibits Repair and Is Associated with Chemosensitivity of Breast Cancer.

DNA repair capacity is critical for survival of cancer cells upon therapeutic DNA damage and thus is an important determinant of susceptibility to chemotherapy in cancer patients. In this study, we identified a novel function of nuclear receptor NR1D1 in DNA repair, which enhanced chemosensitivity in breast cancer cells. NR1D1 inhibited both nonhomologous end joining and homologous recombination double-strand breaks repair, and delayed the clearance of γH2AX DNA repair foci that formed after treatment of doxorubicin. PARylation of NR1D1 by PARP1 drove its recruitment to damaged DNA lesions. Deletion of the ligand binding domain of NR1D1 that interacted with PARP1, or treatment of 6-(5H)-phenanthridinone, an inhibitor of PARP1, suppressed the recruitment of NR1D1 to DNA damaged sites, indicating PARylation as a critical step for the NR1D1 recruitment. NR1D1 inhibited recruitment of the components of DNA damage response complex such as SIRT6, pNBS1, and BRCA1 to DNA lesions. Downregulation of NR1D1 in MCF7 cells resulted in resistance to doxorubicin, both in vitro and in vivo Analysis of four public patient data sets indicated that NR1D1 expression correlates positively with clinical outcome in breast cancer patients who received chemotherapy. Our findings suggest that NR1D1 and its ligands provide therapeutic options that could enhance the outcomes of chemotherapy in breast cancer patients. Cancer Res; 77(9); 2453-63. ©2017 AACR.

Differential regulation of estrogen receptor α expression in breast cancer cells by metastasis-associated protein 1.

Metastasis-associated protein 1 (MTA1) is a component of the nucleosome remodeling and histone deacetylase (HDAC) complex, which plays an important role in progression of breast cancer. Although MTA1 is known as a repressor of the transactivation function of estrogen receptor α (ERα), its involvement in the epigenetic control of transcription of the ERα gene ESR1 has not been studied. Here, we show that silencing of MTA1 reduced the level of expression of ERα in ERα-positive cells but increased it in ERα-negative cells. In both MCF7 and MDA-MB-231, MTA1 was recruited to the region +146 to +461 bp downstream of the transcription start site of ESR1 (ERpro315). Proteomics analysis of the MTA1 complex that was pulled down by an oligonucleotide encoding ERpro315 revealed that the transcription factor AP-2γ (TFAP2C) and the IFN-γ-inducible protein 16 (IFI16) were components of the complex. Interestingly, in MCF7, TFAP2C activated the reporter encoding ERpro315 and the level of ERα mRNA. By contrast, in MDA-MB-231, IFI16 repressed the promoter activity and silencing of MTA1 increased expression of ERα. Importantly, class II HDACs are involved in the MTA1-mediated differential regulation of ERα. Finally, an MDA-MB-231-derived cell line that stably expressed shIFI16 or shMTA1 was more susceptible to tamoxifen-induced growth inhibition in in vitro and in vivo experiments. Taken together, our findings suggest that the MTA1-TFAP2C or the MTA1-IFI16 complex may contribute to the epigenetic regulation of ESR1 expression in breast cancer and may determine the chemosensitivity of tumors to tamoxifen therapy in patients with breast cancer.

Identification of daidzein as a ligand of retinoic acid receptor that suppresses expression of matrix metalloproteinase-9 in HaCaT cells.

Retinoids have been used as therapeutics for diverse skin diseases, but their side effects limit clinical usage. Here, we report that extracts of two soybeans, Glycine max and Rhynchosia nulubilis, and their ethyl acetate fractions increased the transcriptional activity of retinoic acid receptors (RARs), and that daidzin and genistin were the major constituents of the active fractions. Daidzin and its aglycone, daidzein, induced transcriptional activity of RAR and RARγ. FRET analysis demonstrated that daidzein, but not daidzin, bound both RAR and RARγ with EC50 values of 28µM and 40µM, respectively. Daidzein increased expression of mRNA of RARγ through direct binding of RAR and recruitment of p300 to the RARγ2 promoter. Further, mRNA and gelatinolytic activity of matrix metalloproteinase-9 were decreased by daidzein in HaCaT cells. Together, these results indicate that daidzein functions as a ligand of RAR that could be a candidate therapeutic for skin diseases.

LXRalpha enhances lipid synthesis in SZ95 sebocytes.

Differentiation of sebocytes is strongly associated with enhanced lipid synthesis and accumulation in the cells. Liver X receptors (LXRs) are members of the nuclear receptor superfamily, which play a critical role in cholesterol homeostasis and lipid metabolism. We examined whether LXRalpha regulated lipid synthesis in the immortalized human sebaceous gland cell line SZ95. When the SZ95 sebocytes were treated with the ligand of LXR such as TO901317 or 22(R)-hydroxycholesterol, lipid droplets were accumulated in the majority of cells when examined by Oil Red O staining. The expression of the known LXR targets, such as fatty acid synthase and sterol regulatory-binding protein-1, was induced by TO901317. TO901317 treatment increased expression of LXRalpha but not that of LXRbeta. Transfection of antisense LXRalpha significantly decreased TO901317-induced target gene expression and lipid droplet accumulation, suggesting a major role of LXRalpha in differentiation of sebocytes. Further, TO901317 decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase that was induced by lipopolysaccharide treatment. Together, these results indicate that important roles of LXRalpha in differentiation and inflammatory signaling in sebaceous glands. Thus, we suggest that LXR ligands could provide a new class of therapeutic agents for sebaceous gland-associated disorders such as seborrhea and acne.