Faculty Bibliography

BACKGROUNDS:Tamoxifen is typically used to treat patients with estrogen receptor alpha (ERα)-positive breast cancer. However, 30% of these patients gain acquired resistance to tamoxifen during or after tamoxifen treatment. As a Ras modulator, Nogo-B receptor (NgBR) is required for tumorigenesis through the signaling crosstalk with epidermal growth factor (EGF) receptor (EGFR)-mediated pathways. NgBR is highly expressed in many types of cancer cells and regulates the sensitivity of hepatocellular carcinoma to chemotherapy. In this study, we found the expression of NgBR is increased in tamoxifen-resistant ERα-positive breast cancer cells. METHODS:Tamoxifen-resistant ERα-positive MCF-7 and T47D breast cancer cell lines were established by culturing with gradually increased concentration of 4-hydroxytamoxifen (4-OHT). The effects of NgBR on tamoxifen resistance was determined by depleting NgBR in these cell lines using previously validated small interfering RNA (siRNA). The effects of 4-OHT on cell viability and apoptosis were determined using well-accepted methods such as clonogenic survival assay and Annexin V/propidium iodide staining. The alteration of EGF-stimulated signaling and gene expression was determined by western blot analysis and real-time PCR, respectively. RESULTS:NgBR knockdown with siRNA attenuates EGF-induced phosphorylation of ERα and restores the sensitivity to tamoxifen in ERα-positive breast cancer cells. Mechanistically, our data demonstrated that NgBR knockdown increases the protein levels of p53 and decreases survivin, which is an apoptosis inhibitor. CONCLUSIONS:These results suggested that NgBR is a potential therapeutic target for increasing the sensitivity of ERα-positive breast cancer to tamoxifen.

Nogo-B receptor (NgBR) plays fundamental roles in regulating angiogenesis, vascular development, and the epithelial-mesenchymal transition (EMT) of cancer cells. However, the therapeutic effect of NgBR blockade on tumor vasculature and malignancy is unknown, investigations on which requires an adequate delivery system for small interfering RNA against NgBR (NgBR siRNA). Here a surface charge switchable polymeric nanoparticle that was sensitive to the slightly acidic tumor microenvironment was developed for steady delivery of NgBR siRNA to tumor tissues. The nanoformulation was constructed by conjugating 2, 3-dimethylmaleic anhydride (DMMA) molecules to the surface amines of micelles formed by cationic co-polymer poly(lactic-co-glycolic acid)₂-poly(ethylenimine) and subsequent absorption of NgBR siRNAs. The nanoparticles remained negatively charged in physiological condition and smartly converted to positive surface charge due to tumor-acidity-activated shedding of DMMA. The charge conversion facilitated cellular uptake of siRNAs and in turn efficiently depleted the expression of NgBR in tumor-bearing tissues. Silencing of NgBR suppressed endothelial cell migration and tubule formation, and reverted the EMT process of breast cancer cells. Delivery of the nanoformulation to mice bearing orthotopic breast carcinoma showed no effect on tumor growth, but led to remarkable decrease of distant metastasis by normalizing tumor vessels and suppressing the EMT of breast cancer cells. This study demonstrated that NgBR is a promising therapeutic target in abnormal tumor vasculature and aggressive cancer cells, and the tumor-responsive nanoparticle with the feature of charge transformation offers great potential for tumor-specific delivery of gene therapeutics.

Intrinsic or acquired chemoresistance is a hurdle in oncology. Only 7%-16% of estrogen receptor α (ERα) positive breast cancer cases achieve a pathological complete response (pCR) after neo-adjuvant chemotherapy. Nogo-B receptor (NgBR) is a cell surface receptor that binds farnesylated Ras and promotes Ras translocation to the plasma membrane. Here, we demonstrate NgBR as a potential therapeutic target for ERα positive breast cancer patients to attenuate paclitaxel resistance. NgBR knockdown enhanced paclitaxel-induced cell apoptosis by modulating expression of p53 and survivin in ERα positive breast cancer cells via NgBR-mediated PI3K/Akt and MAPK/ERK signaling pathways. NgBR knockdown attenuated either 17β-estradiol or epidermal growth factor stimulated phosphorylation of ERα at Serine 118 residue. The ChIP-PCR assay further demonstrated that NgBR knockdown decreased ERα binding to the estrogen response element (ERE) of the ERα target gene and increased the binding of p53 to the promoter region of survivin to attenuate survivin transcription. In summary, our data suggest that NgBR expression is essential to promoting ERα positive breast cancer cell resistance to paclitaxel. Findings from this study implicate a novel therapeutic target for treating ERα positive breast cancer in neo-adjuvant/adjuvant chemotherapy.

Nogo-B receptor (NgBR) is a specific receptor of Nogo-B that regulates vascular remodeling and angiogenesis. Previously, we found that NgBR promotes the membrane translocation and activation of Ras in breast cancer cells and enhances the chemoresistance of hepatocellular carcinoma cells to 5-fluorouracil. However, the role of NgBR in lung cancer has not yet been elucidated. In the present study, we found that NgBR knockdown inhibited epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) cells in vitro and metastasis of NSCLC cells in vivo. In contrast, NgBR overexpression promoted EMT in and lung metastasis of NSCLC cells. At the molecular level, NgBR modulated the expression of EMT-related proteins and enhanced the protein expression of Snail1, a crucial transcription factor that represses epithelial cell protein marker E-cadherin. Moreover, we found that NgBR overexpression promoted the membrane localization of Ras and activation of downstream MEK/ERK signaling pathway and that NgBR knockdown by using a specific shRNA inversely affected the expression of EMT-related proteins in NSCLC cells. Thus, our results provide novel insights on the regulatory role of NgBR in the metastasis of NSCLC that should be investigated further for developing a therapeutic strategy for treating patients with NSCLC.

Low chemosensitivity considerably restricts the therapeutic efficacy of gemcitabine (GEM) in pancreatic cancer treatment. Using immunohistochemical evaluation, we investigated that decreased expression of human equilibrative nucleoside transporter-1 (hENT1, which is the major GEM transporter across cell membranes) and increased expression of ribonucleotide reductase subunit 2 (RRM2, which decreases the cytotoxicity of GEM) was associated with low GEM chemosensitivity. To solve these problems, we employed a nanomedicine-based formulation of cationic liposomes for co-delivery of GEM along with siRNA targeting RRM2. Due to the specific endocytic uptake mechanism of nanocarriers and gene-silencing effect of RRM2 siRNA, this nanomedicine formulation significantly increased GEM chemosensitivity in tumor models of genetically engineered Panc1 cells with low hENT1 or high RRM2 expression. Moreover, in a series of patient-derived cancer cells, we demonstrated that the therapeutic benefits of the nanomedicine formulations were associated with the expression levels of hENT1 and RRM2. In summary, we found that the essential factors of GEM chemosensitivity were the expression levels of hENT1 and RRM2, and synthesized nanoformulations can overcome these problems. This unique design of nanomedicine not only provides a universal platform to enhance chemosensitivity but also contributes to the precision design and personalized treatment in nanomedicine.

Deficiency of hepatic Nogo-B receptor (NgBR) expression activates liver X receptor α (LXRα) in an adenosine monophosphate-activated protein kinase α (AMPKα)-dependent manner, thereby inducing severe hepatic lipid accumulation and hypertriglyceridemia. Statins have been demonstrated non-cholesterol lowering effects including anti-nonalcoholic fatty liver disease (NAFLD). Herein, we investigated if the anti-NAFLD function of statins depends on activation of NgBR expression. In vivo, atorvastatin protected apoE deficient or NgBR floxed, but not hepatic NgBR deficient mice, against Western diet (WD)-increased triglyceride levels in liver and serum. In vitro, statins reduced lipid accumulation in nonsilencing small hairpin RNA-transfected (shNSi), but not in NgBR small hairpin RNA-transfected (shNgBRi) HepG2 cells. Inhibition of cellular lipid accumulation by atorvastatin is related to activation of AMPKα, and inactivation of LXRα and lipogenic genes. Statin also inhibited expression of oxysterol producing enzymes. Associated with changes of hepatic lipid levels by WD or atorvastatin, NgBR expression was inversely regulated. At cellular levels, statins increased NgBR mRNA and protein expression, and NgBR protein stability. In contrast to reduced cellular cholesterol levels by statin or β-cyclodextrin, increased cellular cholesterol levels decreased NgBR expression suggesting cholesterol or its synthesis intermediates inhibit NgBR expression. Indeed, mevalonate, geranylgeraniol or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate or farnesol, blocked atorvastatin-induced NgBR expression. Furthermore, we determined that induction of hepatic NgBR expression by atorvastatin mainly depended on inactivation of extracellular signal-regulated kinases 1/2 (ERK1/2) and protein kinase B (Akt). Taken together, our study demonstrates that statins inhibit NAFLD mainly through activation of NgBR expression.

OBJECTIVE:The reduced adiponectin levels are associated with atherosclerosis. Adiponectin exerts its functions by activating adiponectin receptor (AdipoR). Proprotein convertase subtilisin kexin type 9 (PCSK9) degrades LDLR protein (low-density lipoprotein receptor) to increase serum LDL-cholesterol levels. PCSK9 expression can be regulated by PPARγ (peroxisome proliferator-activated receptor γ) or SREBP2 (sterol regulatory element-binding protein 2). The effects of AdipoR agonists on PCSK9 and LDLR expression, serum lipid profiles, and atherosclerosis remain unknown. APPROACH AND RESULTS:mice with amelioration of lipid profiles. CONCLUSIONS:mice.

Nogo-B receptor (NgBR) was identified as a specific receptor for binding Nogo-B and is essential for the stability of Niemann-Pick type C2 protein (NPC2) and NPC2-dependent cholesterol trafficking. Here, we report that NgBR expression levels decrease in the fatty liver and that NgBR plays previously unrecognized roles in regulating hepatic lipogenesis through NPC2-independent pathways. To further elucidate the pathophysiological role of NgBR in mammals, we generated NgBR liver-specific knockout mice and investigated the roles of NgBR in hepatic lipid homeostasis. The results showed that NgBR knockout in mouse liver did not decrease NPC2 levels or increase NPC2-dependent intracellular cholesterol levels. However, NgBR deficiency still resulted in remarkable cellular lipid accumulation that was associated with increased free fatty acids and triglycerides in hepatocytes in vitro and in mouse livers in vivo. Mechanistically, NgBR deficiency specifically promotes the nuclear translocation of the liver X receptor alpha (LXRα) and increases the expression of LXRα-targeted lipogenic genes. LXRα knockout attenuates the accumulation of free fatty acids and triglycerides caused by NgBR deficiency. In addition, we elucidated the mechanisms by which NgBR bridges the adenosine monophosphate-activated protein kinase alpha signaling pathway with LXRα nuclear translocation and LXRα-mediated lipogenesis.

Expression of ATP-binding cassette transporter G1 (ABCG1), a molecule facilitating cholesterol efflux to HDL, is activated by liver X receptor (LXR). In this study, we investigated if inhibition of ERK1/2 can activate macrophage ABCG1 expression and functions. MEK1/2 inhibitors, PD98059 and U0126, increased ABCG1 mRNA and protein expression, and activated the natural ABCG1 promoter but not the promoter with the LXR responsive element (LXRE) deletion. Inhibition of ABCG1 expression by ABCG1 siRNA did enhance the formation of macrophage/foam cells and it attenuated the inhibitory effect of MEK1/2 inhibitors on foam cell formation. MEK1/2 inhibitors activated macrophage cholesterol efflux to HDL in vitro, and they enhanced reverse cholesterol transport (RCT) in vivo. ApoE deficient (apoE(-/-)) mice receiving U0126 treatment had reduced sinus lesions in the aortic root which was associated with activated macrophage ABCG1 expression in the lesion areas. MEK1/2 inhibitors coordinated the RXR agonist, but not the LXR agonist, to induce ABCG1 expression. Furthermore, induction of ABCG1 expression by MEK1/2 inhibitors was associated with activation of SIRT1, a positive regulator of LXR activity, and inactivation of SULT2B1 and RIP140, two negative regulators of LXR activity. Taken together, our study suggests that MEK1/2 inhibitors activate macrophage ABCG1 expression/RCT, and inhibit foam cell formation and lesion development by multiple mechanisms, supporting the concept that ERK1/2 inhibition is anti-atherogenic.

Nogo-B receptor (NgBR), a type I single transmembrane domain receptor is the specific receptor for Nogo-B. Our previous work demonstrated that NgBR is highly expressed in breast cancer cells, where it promotes epithelial mesenchymal transition (EMT), an important step in metastasis. Here, we show that both in vitro and in vivo increased expression of NgBR contributes to the increased chemoresistance of Bel7402/5FU cells, a stable 5-FU (5-Fluorouracil) resistant cell line related Bel7402 cells. NgBR knockdown abrogates S-phase arrest in Bel7402/5FU cells, which correlates with a reduction in G1/S phase checkpoint proteins p53 and p21. In addition, NgBR suppresses p53 protein levels through activation of the PI3K/Akt/MDM2 pathway, which promotes p53 degradation via the ubiquitin proteasome pathway and thus increases the resistance of human hepatocellular cancer cells to 5-FU. Furthermore, we found that NgBR expression is associated with a poor prognosis of human hepatocellular carcinoma (HCC) patients. These results suggest that targeting NgBR in combination with chemotherapeutic drugs, such as 5-FU, could improve the efficacy of current anticancer treatments.