Expression Of Lipolysis-stimulated Lipoprotein Receptor Research Articles

Tumor cell-expressed lipolysis-stimulated lipoprotein receptor negatively regulates T-cell function.

Lipolysis-stimulated lipoprotein receptor (LSR) is known as a lipoprotein receptor. LSR is expressed in various solid tumors, including epithelial ovarian, gastric, and colon cancers. High LSR expression is significantly associated with poor prognosis, but its role in cancer has not been fully elucidated. LSR belongs to the Ig protein superfamily, which is conserved in B7 family. Here, we assessed LSR as a novel immune checkpoint molecule. We developed a novel anti-LSR antibody (#27-6 mF-18) that defects antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity. The #27-6 mF-18 cross-reacts with both human and mouse LSR. We found that LSR was expressed on 4T1 murine breast cancer cell line. The #27-6 mF-18 exhibited antitumor effects against the 4T1 syngeneic tumor model, a poor immunogenic model refractory to treatment with anti-PD-1 or anti-CTLA-4 antibodies. Compared with control antibody-treated mice, mice treated with #27-6 mF-18 showed significantly increased numbers of CD8+ T cells and a ratio of activated CD8+ T cells infiltrated in the tumor tissue. This antitumor effect was abrogated by CD8+ T-cell depletion through anti-CD8 antibody treatment, indicating that LSR negatively regulates tumor immunity by repressing CD8+ T cells. These findings show that LSR negatively regulates T-cell immune activity. LSR targeting could provide immune checkpoint inhibitors for cancer immunotherapy.

Lipolysis-Stimulated Lipoprotein Receptor Acts as Sensor to Regulate ApoE Release in Astrocytes

Astroglia play an important role, providing de novo synthesized cholesterol to neurons in the form of ApoE-lipidated particles; disruption of this process can increase the risk of Alzheimer’s disease. We recently reported that glia-specific suppression of the lipolysis-stimulated lipoprotein receptor (LSR) gene leads to Alzheimer’s disease-like memory deficits. Since LSR is an Apo-E lipoprotein receptor, our objective of this study was to determine the effect of LSR expression modulation on cholesterol and ApoE output in mouse astrocytes expressing human ApoE3. qPCR analysis showed that siRNA-mediated lsr knockdown significantly increased expression of the genes involved in cholesterol synthesis, secretion, and metabolism. Analysis of media and lipoprotein fractions showed increased cholesterol and lipidated ApoE output in HDL-like particles. Further, lsr expression could be upregulated when astrocytes were incubated 5 days in media containing high levels (two-fold) of lipoprotein, or after 8 h treatment with 1 µM LXR agonist T0901317 in lipoprotein-deficient media. In both conditions of increased lsr expression, the ApoE output was repressed or unchanged despite increased abca1 mRNA levels and cholesterol production. We conclude that LSR acts as a sensor of lipoprotein content in the medium and repressor of ApoE release, while ABCA1 drives cholesterol efflux, thereby potentially affecting cholesterol load, ApoE lipidation, and limiting cholesterol trafficking towards the neuron.

Anti-lipolysis-stimulated lipoprotein receptor monoclonal antibody as a novel therapeutic agent for endometrial cancer

BackgroundEndometrial cancer (EC) is a common gynecologic malignancy and patients with advanced and recurrent EC have a poor prognosis. Although chemotherapy is administered for those patients, the efficacy of current chemotherapy is limited. Therefore, it is necessary to develop novel therapeutic agents for EC. In this study, we focused on lipolysis-stimulated lipoprotein receptor (LSR), a membrane protein highly expressed in EC cells, and developed a chimeric chicken–mouse anti-LSR monoclonal antibody (mAb). This study investigated the antitumor effect of an anti-LSR mAb and the function of LSR in EC.MethodsWe examined the expression of LSR in 228 patients with EC using immunohistochemistry and divided them into two groups: high-LSR (n = 153) and low-LSR groups (n = 75). We developed a novel anti-LSR mAb and assessed its antitumor activity in an EC cell xenograft mouse model. Pathway enrichment analysis was performed using protein expression data of EC samples. LSR-knockdown EC cell lines (HEC1 and HEC116) were generated by transfected with small interfering RNA and used for assays in vitro.ResultsHigh expression of LSR was associated with poor overall survival (hazard ratio: 3.53, 95% confidence interval: 1.35–9.24, p = 0.01), advanced stage disease (p = 0.045), deep myometrial invasion (p = 0.045), and distant metastasis (p < 0.01). In EC with deep myometrial invasion, matrix metalloproteinase (MMP) 2 was highly expressed along with LSR. Anti-LSR mAb significantly inhibited the tumor growth in EC cell xenograft mouse model (tumor volume, 407.1 mm3versus 726.3 mm3, p = 0.019). Pathway enrichment analysis identified the mitogen-activated protein kinase (MAPK) pathway as a signaling pathway associated with LSR expression. Anti-LSR mAb suppressed the activity of MAPK in vivo. In vitro assays using EC cell lines demonstrated that LSR regulated cell proliferation, invasion, and migration through MAPK signaling, particularly MEK/ERK signaling and membrane-type 1 MMP (MT1-MMP) and MMP2. Moreover, ERK1/2-knockdown suppressed cell proliferation, invasion, migration, and the expression of MT1-MMP and MMP2.ConclusionsOur results suggest that LSR contributes to tumor growth, invasion, metastasis, and poor prognosis of EC through MAPK signaling. Anti-LSR mAb is a potential therapeutic agent for EC.

Targeted Suppression of Lipoprotein Receptor LSR in Astrocytes Leads to Olfactory and Memory Deficits in Mice.

Perturbations of cholesterol metabolism have been linked to neurodegenerative diseases. Glia–neuron crosstalk is essential to achieve a tight regulation of brain cholesterol trafficking. Adequate cholesterol supply from glia via apolipoprotein E-containing lipoproteins ensures neuronal development and function. The lipolysis-stimulated lipoprotein receptor (LSR), plays an important role in brain cholesterol homeostasis. Aged heterozygote Lsr+/− mice show altered brain cholesterol distribution and increased susceptibility to amyloid stress. Since LSR expression is higher in astroglia as compared to neurons, we sought to determine if astroglial LSR deficiency could lead to cognitive defects similar to those of Alzheimer’s disease (AD). Cre recombinase was activated in adult Glast-CreERT/lsrfl/fl mice by tamoxifen to induce astroglial Lsr deletion. Behavioral phenotyping of young and old astroglial Lsr KO animals revealed hyperactivity during the nocturnal period, deficits in olfactory function affecting social memory and causing possible apathy, as well as visual memory and short-term working memory problems, and deficits similar to those reported in neurodegenerative diseases, such as AD. Furthermore, GFAP staining revealed astroglial activation in the olfactory bulb. Therefore, astroglial LSR is important for working, spatial, and social memory related to sensory input, and represents a novel pathway for the study of brain aging and neurodegeneration.

Abstract 1989: LSR is a novel prognostic factor by regulating cell proliferation via MEK/ERK pathway in endometrial cancer:Analysis of signal transduction, bioinformatics, and in vitro/in vivo study

Abstract Introduction: Advanced endometrial cancer (EC) still has poor prognosis and requires the development of new therapeutic agents. In ovarian and gastric cancer, we revealed that high expression of lipolysis-stimulated lipoprotein receptor (LSR), a cell-surface membrane protein, was associated with poor prognosis and LSR could be a new therapeutic target. However, in EC, the clinical and cell biological role of LSR is still unclear. We aimed to investigate the functions of LSR in EC. Methods: We evaluated LSR expression by immunohistochemistry and analyzed overall survival (OS) and clinicopathological factors in 228 EC patients. To investigate the mechanism by which LSR affects the prognosis of EC patients, the pathway enrichment analysis and gene ontology analysis were conducted using the publicly available proteogenomic dataset of EC. Cell proliferation was analyzed by WST-8 assay using two LSR-knockdown cell lines (HEC1 and HEC116) developed by transfection of LSR-siRNA, and the activity of several signaling pathways were examined by Western blotting. Results: Patients were divided into two groups based on LSR expression; High (darkly stained cells in ≥25% of the area, n=153) and Low (darkly stained cells in &amp;lt;25% of the area, n=75) groups. 5-year OS rate in High group was significantly lower than Low group (hazard ratio: 3.53, 95% confidence interval: 1.35 - 9.24, p=0.01). Stage III-IV, deep myometrial invasion (≥75% of uterine myometrium), tumor involvement of adnexa or serosa, and distant metastasis were more frequently observed in High group than in Low group (p&amp;lt;0.05, respectively). The pathway analysis demonstrated that genes correlated with high LSR expression were enriched in MAPK signaling pathway. In gene ontology analysis, these genes were enriched in regulation of ERK1/2 and MAPK cascade. In vitro, LSR-knockdown suppressed cell proliferation (p&amp;lt;0.01) and phosphorylation of MEK1/2, ERK1/2, and p90RSK, suggesting that LSR promoted cell proliferation via MEK/ERK pathway. However, LSR did not regulate the other signaling pathways (SAPK/JNK, p38-MAPK, JAK/STAT3, and PI3K/AKT/mTOR pathway). Conclusion: LSR regulated cell proliferation via MEK/ERK pathway, and contributed poor prognosis in EC. LSR may be a new therapeutic target of advanced EC. Citation Format: Yoshikazu Nagase, Kosuke Hiramatsu, Satoshi Nakagawa, Shinya Matsuzaki, Toshihiro Kimura, Satoshi Serada, Yutaka Ueda, Tetsuji Naka, Tadashi Kimura. LSR is a novel prognostic factor by regulating cell proliferation via MEK/ERK pathway in endometrial cancer:Analysis of signal transduction, bioinformatics, and in vitro/in vivo study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1989.

HMGB1-downregulated angulin-1/LSR induces epithelial barrier disruption via claudin-2 and cellular metabolism via AMPK in airway epithelial Calu-3 cells

A non-histone chromatin-associated protein, high mobility group box 1 (HMGB1), which impairs the airway epithelial barrier, is involved in the induction of airway inflammation in patients with allergy, asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). Tricellular tight junctions (tTJs) form at the convergence of bicellular tight junctions (bTJs). Angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) is a novel molecule present at tricellular contacts and contributes to the epithelial barrier and cellular metabolism. Adenosine monophosphate-activated protein kinase (AMPK) is a central metabolic regulator and has a reciprocal association with TJs. In the present study, to examine how HMGB1 contributes to airway epithelial barrier disruption and the cellular metabolism indicated as mitochondrial respiration, bronchial epithelial Calu-3 cells were transfected with siRNAs of angulin-1/LSR or treated with HMGB1 and the relationship between HMGB1 and angulin-1/LSR was investigated. Knockdown of angulin-1/LSR upregulated the expression of the tight junction molecule claudin-2, AMPK activity, and mitochondrial respiration, and downregulated the epithelial barrier. Treatment with HMGB1 downregulated angulin-1/LSR expression and the epithelial barrier, and upregulated claudin-2 expression, AMPK activity and mitochondrial respiration. Treatment with EW-7197, a transforming growth factor-β (TGF-β) type I receptor kinase inhibitor, prevented all the effects of HMGB1 in Calu-3 cells. HMGB1-downregulated angulin-1/LSR induced epithelial barrier disruption via claudin-2 and cellular metabolism via AMPK in airway epithelial Calu-3 cells. The effects of HMGB1 contribute to TGF-β signaling and EW-7197 shows potential for use in therapy for HMGB1-induced airway inflammation.

ASPP2 suppression promotes malignancy via LSR and YAP in human endometrial cancer.

Apoptosis-stimulating p53 protein 2 (ASPP2) is an apoptosis inducer that acts via binding with p53 and epithelial polarity molecule PAR3. Lipolysis-stimulated lipoprotein receptor (LSR) is an important molecule at tricellular contacts, and loss of LSR promotes cell migration and invasion via Yes-associated protein (YAP) in human endometrial cancer cells. In the present study, to find how ASPP2 suppression promotes malignancy in human endometrial cancer, we investigated its mechanisms including the relationship with LSR. In endometriosis and endometrial cancers (G1 and G2), ASPP2 was observed as well as PAR3 and LSR in the subapical region. ASPP2 decreased in G3 endometrial cancer compared to G1. In human endometrial cancer cell line Sawano, ASPP2 was colocalized with LSR and tricellulin at tricellular contacts and binding to PAR3, LSR, and tricellulin in the confluent state. ASPP2 suppression promoted cell migration and invasion, decreased LSR expression, and induced expression of phosphorylated YAP, claudin-1, -4, and -7 as effectively as the loss of LSR. Knockdown of YAP prevented the upregulation of pYAP, cell migration and invasion induced by the ASPP2 suppression. Treatment with a specific antibody against ASPP2 downregulated ASPP2 and LSR, affected F-actin at tricellular contacts, upregulated expression of pYAP and claudin-1, and induced cell migration and invasion via YAP. In normal human endometrial epithelial cells, ASPP2 was in part colocalized with LSR at tricellular contacts and knockdown of ASPP2 or LSR induced expression of claudin-1 and claudin-4. ASPP2 suppression promoted cell invasion and migration via LSR and YAP in human endometrial cancer cells.

Tricellular tight junction protein LSR/angulin-1 contributes to the epithelial barrier and malignancy in human pancreatic cancer cell line.

Lipolysis-stimulated lipoprotein receptor (LSR)/angulin-1 is a crucial molecule of tricellular contacts in the epithelial barrier of normal cells and the malignancy of cancer cells. To investigate whether LSR/angulin-1 affects the epithelial barrier and malignancy in human pancreatic cancer, human pancreatic cancer cell line HPAC was used. Treatment with EGF or TGF-β increased the expression of LSR, but not tricellulin (TRIC), and induced the localization of LSR and TRIC to bicellular tight junctions from tricellular tight junctions. TGF-β receptor type-1 inhibitor EW-7197 prevented changes of the distribution and the barrier function of LSR by TGF-β. Knockdown of LSR increased cell migration, invasion, proliferation and EGF ligand amphiregulin expression and decreased the epithelial barrier. Treatment with amphiregulin induced cell migration and invasion and knockdown of amphiregulin prevented the increases of cell migration, invasion and proliferation caused by knockdown of LSR. Treatment with LSR ligand peptide angubindin-1 decreased the epithelial barrier and the expression of LSR, but not TRIC, and increased cell invasion. Knockdown of TRIC decreased cell migration and the epithelial barrier. In immunohistochemical analysis of human pancreatic cancer tissues, LSR and TRIC were found to be localized at the cell membranes of normal pancreatic ducts and well-differentiated pancreatic ductal adenocarcinomas (PDAC), whereas in poorly differentiated PDAC, LSR was weakly detected in the cytoplasm. Amphiregulin was highly expressed in the cytoplasm of well- and poorly differentiated PDAC. In pancreatic cancer, LSR contributes to the epithelial barrier and malignancy via growth factors and may be a potential targeting molecule in the therapy.

Lipolysis-stimulated lipoprotein receptor overexpression is a novel predictor of poor clinical prognosis and a potential therapeutic target in gastric cancer.

The prognosis of patients with advanced gastric cancer (GC) remains poor despite the recent advances in molecular targeted therapies, and the search for biomarkers that can predict prognosis and additional new agents with acceptable toxicity profiles are needed. Lipolysis-stimulated lipoprotein receptor (LSR) is a lipoprotein receptor that binds to triglyceride-rich lipoproteins and related to some malignancies. Herein, we examined the association between LSR expression and the prognosis of patients with GC, and investigated the antitumor effect of a previously developed anti-human LSR monoclonal antibody (#1–25). We first performed immunohistochemical analysis of LSR protein expression in GC and normal tissues, and then examined its association with the prognosis of 110 patients with GC. LSR was overexpressed in most of primary GC and metastatic tumors, but not in normal tissues. Patients with strong LSR expression (N = 80, 72.7%) had significantly poorer overall survival (OS) than those with weak expression (P = 0.017). Multivariate analysis identified strong LSR (as well as pT) as independent and significant prognostic factors for OS. Next, we demonstrated that very low density lipoprotein (VLDL) treatment increases cell proliferation in LSR-expressing GC cell lines in vitro; LSR inhibition using #1–25 inhibited VLDL-induced proliferation by suppressing JAK/STAT and PI3K signaling. In vivo, we demonstrated a marked antitumor effect of #1–25 in 2 distinct GC cell line xenograft mice models. Our findings suggest that LSR plays a key functional role in GC development, and that this antigen can be therapeutically targeted to improve GC treatment.

Guanylate binding protein-1-mediated epithelial barrier in human salivary gland duct epithelium

Guanylate-binding protein-1 (GBP-1) is an interferon-inducible large GTPase involved in the epithelial barrier at tight junctions. To investigate the role of GBP-1 in the epithelial barrier, primary human salivary gland duct epithelial cells were treated with the the proinflammatory cytokines IFNγ, IL-1β, TNFα and the growth factor TGF-β. Treatment with IFNγ, IL-1β, or TNFα markedly enhanced GBP-1 and the epithelial barrier function, and induced not only CLDN-7 but also the tricellular tight junction molecule lipolysis-stimulated lipoprotein receptor (LSR). Knockdown of GBP-1 by its siRNA induced endocytosis of tight junction molecules, and prevented the increases of CLDN-7 and LSR with the upregulation of the epithelial barrier function induced by treatment with IFNγ or TNFα. Treatment with a PKCα inhibitor induced expression of GBP-1, CLDN-7 and LSR and enhanced the epithelial barrier function. In almost intact salivary gland ducts from patients with IgG4-related disease (IgG4-RD) indicated significant infiltration of IgG-positive plasma cells, expression of GBP-1, CLDN-7 and LSR was increased. These findings indicated that GBP-1 might play a crucial role in barrier function of normal human salivary gland duct epithelium and perform a preventive role in the duct epithelium of IgG4-RD disease.

LSR Antibody Therapy Inhibits Ovarian Epithelial Tumor Growth by Inhibiting Lipid Uptake.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy, but it still lacks effective treatment options. In this study, we utilized proteomic technology to identify lipolysis-stimulated lipoprotein receptor (LSR) as a new tumor antigen of EOC. Immunohistochemical analysis of EOC tissues in conjunction with survival analysis of EOC patients showed that high expression of LSR is associated with poor prognosis. High LSR expression also occurred in tumor metastases including to the lymph node and omentum. To evaluate the possible benefits of blocking this antigen in EOC, we raised a new monoclonal antibody (mAb) to human LSR (hLSR). In mouse xenograft models of hLSR+ EOC (cell lines or patient-derived tumors), we found that administration of anti-hLSR mAb inhibited tumor growth in a manner independent of both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Mechanistic investigations showed that hLSR expression increased incorporation of very-low-density lipoprotein (VLDL) into EOC cells and that anti-hLSR mAb inhibited lipid uptake in vitro and in vivo Moreover, VLDL promoted cell proliferation in hLSR-positive EOC cells in vitro, and this effect was inhibited by anti-hLSR mAb. While the anti-hLSR mAb studied cross reacted with the mouse antigen, we observed no adverse effects on normal organs and lipid metabolism in murine hosts. Our findings suggest that hLSR plays a key functional role in EOC development and that this antigen can be therapeutically targeted by specific mAb to improve EOC treatment.Significance: These findings offer preclinical evidence of the therapeutic efficacy of a novel targeted antibody therapy against deadly epithelial ovarian cancers. Cancer Res; 78(2); 516-27. ©2017 AACR.

Histone deacetylase inhibition prevents cell death induced by loss of tricellular tight junction proteins in temperature-sensitive mouse cochlear cells.

Tricellular tight junctions (tTJs) are specialized structures that occur where the corners of three cells meet to seal adjacent intercellular space. The molecular components of tTJs include tricellulin (TRIC) and lipolysis-stimulated lipoprotein receptor (LSR) which recruits TRIC, are required for normal hearing. Although loss of TRIC causes hearing loss with degeneration of cochlear cells, the detailed mechanisms remains unclear. In the present study, by using temperature-sensitive mouse cochlear cells, US/VOT-E36 cell line, we investigated the changes of TRIC and LSR during cochlear cell differentiation and the effects of histone deacetylase (HDAC) inhibitors against cell degeneration induced by loss of TRIC and LSR. During cell differentiation induced by the temperature change, expression of TRIC and LSR were clearly induced. Treatment with metformin enhanced expression TRIC and LSR via AMPK during cell differentiation. Loss of TRIC and LSR by the siRNAs induced cell death in differentiated cells. Treatment with HDAC inhibitors trichostatin A and HDAC6 inhibitor prevented the cell death induced by loss of TRIC and LSR. Collectively, these findings suggest that both tTJ proteins TRIC and LSR have crucial roles for the differentiated cochlear cell survival, and that HDAC inhibitors may be potential therapeutic agents to prevent hearing loss.

Abstract 4410: LSR promotes ovarian cancer cell growth following the activation of β-oxidation and its antibody inhibits lipid catabolism

Abstract Ovarian cancer is the most lethal gynecologic malignancy; thus developing new treatment options is urgently required. Molecular targeted therapies for cancers, which are generally more tolerable than widely used cytotoxic agents, have shown highly specific inhibition of target molecules. In this study, we aimed to identify a new ovarian cancer antigen and to develop a novel monoclonal antibody (mAb). Furthermore we evaluated its preclinical efficacy and analyzed the function of its antigen in ovarian cancer.To identify a new ovarian cancer antigen, cell surface membrane proteins of normal ovarian epithelial and ovarian cancer cell lines were analyzed by iTRAQ-based proteomic technology. We identified lipolysis-stimulated lipoprotein receptor (LSR) as the new therapeutic target for ovarian cancer. By the immunohistochemical analysis, significant poor prognosis was observed in high-LSR expression patients with ovarian cancer compared to patients with low-LSR expression by survival assay (p &amp;lt; 0.05). Our newly developed anti-LSR mAb showed significant inhibition of tumor growth in vivo against xenograft model of LSR-positive ovarian cancer cell line and patient derived LSR-positive ovarian cancer tissue (p &amp;lt; 0.05). In LSR-positive ovarian cancer cells, high number and large lipid droplets were observed compared to LSR-negative cells and anti-LSR mAb decreased these droplets. Moreover addition of VLDL to LSR-positive ovarian cancer cells significantly promoted the cell proliferation (p &amp;lt; 0.05) and anti-LSR mAb inhibited that in vitro (p &amp;lt; 0.05). Supporting these data, addition of VLDL to LSR-positive ovarian cancer cells significantly promoted β-oxidation-mediated lipid catabolism (p &amp;lt; 0.05) and anti-LSR mAb also inhibited that (p &amp;lt; 0.05). In addition, this anti-LSR mAb which cross-reacted with mouse LSR did not show any cytotoxicity on normal organs and lipid metabolism in mice. In summary, high expression of LSR in ovarian cancer was the poor prognostic factor. Our newly developed anti-LSR mAb showed significant tumor growth inhibition against not only LSR-positive ovarian cancer cell line but also patient derived LSR-positive ovarian cancer tissue. In LSR-positive ovarian cancer cells, high number and large lipid droplets were observed and LSR promoted cell proliferation following β-oxidation-mediated lipid catabolism. Anti-LSR mAb inhibited these processes. Our preclinical data demonstrated that targeting LSR by mAb is a promising therapy for patients with LSR-positive ovarian cancer. Citation Format: Kosuke Hiramatsu, Satoshi Serada, Takayuki Enomoto, Takuhei Yokoyama, Yusuke Takahashi, Minoru Fujimoto, Kiyoshi Yoshino, Tadashi Kimura, Tetsuji Naka. LSR promotes ovarian cancer cell growth following the activation of β-oxidation and its antibody inhibits lipid catabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4410. doi:10.1158/1538-7445.AM2017-4410

Abstract 4998: Lipolysis-stimulated lipoprotein receptor (LSR) can be a novel therapeutic target of ovarian cancer

Abstract Objective:Ovarian cancer is the most lethal gynecological malignancy. Five-year survival of advanced ovarian cancer patients remains less than 50% and the mortality rate has not changed in recent years. This study is aimed to screen for novel ovarian cancer therapeutic targets using quantitative proteomic approach and to develop antibody-based medicine targeting novel ovarian cancer antigens. Method:Exhaustive quantitative proteome analysis focused on cell surface membrane proteins were performed by isobaric tags for relative and absolute quantitation(iTRAQ) using a normal ovarian surface epithelial cell line and 7 ovarian cancer cell lines. By this approach, ovarian cancer-specific membrane proteins were identified. We confirmed the expression of lipolysis-stimulated lipoprotein receptor (LSR) using immunohistochemical staining, western blotting method, and flow cytometry. By modified MTT assay, cell growth inhibition was performed in LSR-knock down cells compared with control cells. Cell adhesion assay was also performed in vitro. Anti-LSR monoclonal antibody (anti-LSR mAb) was generated to evaluate the efficacy of the LSR targeted antibody therapy in vivo. For subcutaneous xenograft experiments, ovarian cancer cell line (RMG-1) was injected subcutaneously into the CB17/SCID mice. Mice were then randomly divided into two groups and administrated intraperitoneally anti-LSR mAb or control IgG antibody twice a week for 6 times. In addition, luciferase transfected ovarian cancer cell line (A2780-luc) was implanted intraperitoneally into the CB17/SCID mice. Mice were administrated anti-LSR mAb or control IgG antibody intraperitoneally twice a week for 5 times. Result:By iTRAQ analysis, we identified 1685 proteins and one of the ovarian cancer candidates protein, LSR was identified. We confirmed the expression of LSR in 8 out of 11 ovarian cancer cell lines and all of ovarian cancer clinical specimens. We performed proliferation assay using siRNA against ovarian cancer cell lines. When expression of LSR was suppressed, a tumor growth was significantly inhibited at day 4(p&amp;lt;0.01). In adhesion assay, we observed significant inhibition of cell adhesion in LSR knockdown cells. These results suggested that LSR is related to cell proliferation and adhesion. A significant anti-tumour effect with 78% was observed in the anti-LSR mAb antibody administrated group compared with control IgG antibody administrated group after 25 days (p &amp;lt;0.001). Furthermore, in intraperitoneal xenograft model, anti-LSR antibody administration group significantly decreased signal intensities compared with the control antibody administration group at day 22 after implantation. Conclusion:We showed that the LSR is related to cell proliferation and adhesion in vitro in ovarian cancer cells and proved the antitumor effect of anti-LSR mAb. These results suggested that LSR can be a new therapeutic target of ovarian cancer. Citation Format: Satoko Matsuzaki, Kosuke Hiramatsu, Satoshi Serada, Satoshi nakagawa, Shinya Matsuzaki, Yutaka Ueda, Minoru Fujimoto, Kiyoshi Yoshino, Tadashi Kimura, Tetsuji Naka. Lipolysis-stimulated lipoprotein receptor (LSR) can be a novel therapeutic target of ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4998.

Human apolipoprotein E allele and docosahexaenoic acid intake modulate peripheral cholesterol homeostasis in mice

Carrying at least one apolipoprotein E ε4 allele (E4+) is the main genetic risk factor for Alzheimer's disease (AD). Epidemiological studies support that consuming fatty fish rich in docosahexaenoic acid (DHA; 22:6ω3) is protective against development of AD. However, this protective effect seems not to hold in E4+. The involvement of APOE genotype on the relationship between DHA intake and cognitive decline could be mediated through cholesterol. Many studies show a link between cholesterol metabolism and AD progression. In this study, we investigated whether cholesterol metabolism is improved in E3+ and E4+ mice consuming a diet rich in DHA. Plasma cholesterol was 36% lower in E4+ mice compared to E3+ mice fed the control diet (P=.02), and in the liver, there was a significant genotype effect where cholesterol levels were 18% lower in E4+ mice than E3+ mice. The low-density lipoprotein receptor was overexpressed in the liver of E4+ mice. Plasma cholesterol levels were 33% lower after the DHA diet (P=.02) in E3+ mice only, and there was a significant diet effect where cholesterol level was 67% lower in the liver of mice fed DHA. Mice fed the DHA diet also had 62% less lipolysis stimulated lipoprotein receptor expression in the liver compared to mice fed the control diet (P<.0001), but there was no genotype effect. These findings suggest that plasma and liver cholesterol homeostasis and the receptors regulating uptake of cholesterol in the liver are modulated differently and independently by APOE allele and DHA intake.

Abstract 4382: Anti-human LSR monoclonal antibody inhibits tumor growth of ovarian cancer directly

Abstract Ovarian cancer is the most lethal gynecologic malignancy; thus developing new treatment options is urgently required. Molecular targeted therapies for cancers, which are generally more tolerable than widely used cytotoxic agents, have shown highly specific inhibition of target molecules. We previously identified bone marrow stromal antigen 2 (BST2) as an endometrial cancer antigen using iTRAQ-based quantitative proteomic technology focused on cell surface membrane proteins, and also demonstrated the usefulness of an anti-BST2 monoclonal antibody (mAb) for endometrial cancer. In this study, we aimed to identify a new ovarian cancer antigen. We also aimed to develop a novel monoclonal antibody (mAb) and evaluate its preclinical efficacy against ovarian cancer. To identify a new ovarian cancer antigen, cell surface membrane proteins of normal ovarian epithelial and ovarian cancer cell lines were analyzed by iTRAQ-based proteomic technology. As the new therapeutic target for ovarian cancer, we identified lipolysis-stimulated lipoprotein receptor (LSR) which had one of the largest significant differences in protein level between normal ovarian epithelial and ovarian cancer cell lines. Immunohistochemical analysis showed that the overall survival of ovarian serous carcinoma patients with high LSR expression was significantly shorter than those with low LSR (p = 0.0293). We newly developed anti-LSR mAb and investigated its preclinical efficacy. Anti-LSR mAb showed significant in vivo inhibition of tumor growth against a xenograft model of hLSR-positive ovarian cancer in an antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) independent manner (p = 0.0001). And anti-LSR mAb also induced G0/G1 cell cycle arrest by regulation of MEK and p44/42 MAPK activities and expression levels of cell cycle related proteins in vitro. Furthermore, anti-hLSR mAb, which crossreacts with mouse LSR, had little detectable toxicity in mice. In summary, high expression of LSR in ovarian cancer was the poor prognostic factor. Our newly developed anti-LSR mAb showed significant tumor growth inhibition in ADCC and CDC independent manner in vivo. Anti-human LSR mAb also inhibited LSR function and showed direct tumor growth inhibition inducing G0/G1 cell cycle arrest in vitro. Our preclinical data demonstrated that targeting LSR by mAb is a promising therapy for patients with LSR-positive ovarian cancer. Citation Format: Kosuke Hiramatsu, Satoshi Serada, Takayuki Enomoto, Satoshi Nakagawa, Akiko Morimoto, Minoru Fujimoto, Takuhei Yokoyama, Yusuke Takahashi, Yutaka Ueda, Kiyoshi Yoshino, Eiichi Morii, Tadashi Kimura, Tetsuji Naka. Anti-human LSR monoclonal antibody inhibits tumor growth of ovarian cancer directly. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4382. doi:10.1158/1538-7445.AM2015-4382

Abstract 1987: The role of lipolysis-stimulated lipoprotein receptor in directing breast cancer cell behavior and subtype

Abstract The claudin-low molecular subtype of breast cancer is of particular interest for clinically the majority of these tumors are poor prognosis, triple negative, invasive ductal carcinomas. Claudin-low tumors are characterized by low gene expression of cell junction and adhesion proteins, and de-regulation of tight junction proteins is often been implicated in tumorigenesis. Herein, we sought to define the role of tricellular tight junction (tTJ) proteins in breast cancer and cancer cell behavior with a focus on lipolysis-stimulated lipoprotein receptor (LSR). LSR was expressed in epithelium, endothelium, adipose and stromal cells within the healthy breast tissue, as well as in tumor epithelium. LSR expression was significantly correlated with invasive ductal carcinomas compared to invasive lobular carcinomas, and ERα positive tumors and breast cancer cell lines. Consistent with the role of LSR in tTJs, LSR levels were significantly reduced in claudin-low breast cancer cell lines. Functional studies illustrated that that re-introduction of LSR into a claudin-low cell line suppressed the EMT phenotype and reduced individual cell migration. However, our data suggest that LSR may direct collective cell migration. Moreover, re-introduction of LSR in claudin-low breast cancer cell lines reestablished a family of TJ expression, thereby reverting claudin-low lines to other molecular subtypes. Overexpression of LSR enhanced proliferation and survival in anchorage independent conditions, suggesting that reestablishment of tight junctions is not sufficient to inhibit aggressive cell behaviors in vitro. Collectively, these data highlight an unanticipated role for the tTJ protein, LSR in directing aggressive breast cancer behavior. Citation Format: Jodie M. Fleming, Denise K. Reaves, Katerina D. Fagan-Solis, Karen Dunphy, Shannon D. Oliver. The role of lipolysis-stimulated lipoprotein receptor in directing breast cancer cell behavior and subtype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1987. doi:10.1158/1538-7445.AM2014-1987

Challenging the roles of CD44 and lipolysis stimulated lipoprotein receptor in conveying Clostridium perfringens iota toxin cytotoxicity in breast cancer.

BackgroundTranslational exploration of bacterial toxins has come to the forefront of research given their potential as a chemotherapeutic tool. Studies in select tissues have demonstrated that Clostridium perfringens iota toxin binds to CD44 and lipolysis stimulated lipoprotein receptor (LSR) cell-surface proteins. We recently demonstrated that LSR expression correlates with estrogen receptor positive breast cancers and that LSR signaling directs aggressive, tumor-initiating cell behaviors. Herein, we identify the mechanisms of iota toxin cytotoxicity in a tissue-specific, breast cancer model with the ultimate goal of laying the foundation for using iota toxin as a targeted breast cancer therapy.MethodsIn vitro model systems were used to determine the cytotoxic effect of iota toxin on breast cancer intrinsic subtypes. The use of overexpression and knockdown technologies confirmed the roles of LSR and CD44 in regulating iota toxin endocytosis and induction of cell death. Lastly, cytotoxicity assays were used to demonstrate the effect of iota toxin on a validated set of tamoxifen resistant breast cancer cell lines.ResultsTreatment of 14 breast cancer cell lines revealed that LSR+/CD44- lines were highly sensitive, LSR+/CD44+ lines were slightly sensitive, and LSR-/CD44+ lines were resistant to iota cytotoxicity. Reduction in LSR expression resulted in a significant decrease in toxin sensitivity; however, overexpression of CD44 conveyed toxin resistance. CD44 overexpression was correlated with decreased toxin-stimulated lysosome formation and decreased cytosolic levels of iota toxin. These findings indicated that expression of CD44 drives iota toxin resistance through inhibition of endocytosis in breast cancer cells, a role not previously defined for CD44. Moreover, tamoxifen-resistant breast cancer cells exhibited robust expression of LSR and were highly sensitive to iota-induced cytotoxicity.ConclusionsCollectively, these data are the first to show that iota toxin has the potential to be an effective, targeted therapy for breast cancer.

The Role of Lipolysis Stimulated Lipoprotein Receptor in Breast Cancer and Directing Breast Cancer Cell Behavior

The claudin-low molecular subtype of breast cancer is of particular interest for clinically the majority of these tumors are poor prognosis, triple negative, invasive ductal carcinomas. Claudin-low tumors are characterized by cancer stem cell-like features and low expression of cell junction and adhesion proteins. Herein, we sought to define the role of lipolysis stimulated lipoprotein receptor (LSR) in breast cancer and cancer cell behavior as LSR was recently correlated with tumor-initiating features. We show that LSR was expressed in epithelium, endothelium, and stromal cells within the healthy breast tissue, as well as in tumor epithelium. In primary breast tumor bioposies, LSR expression was significantly correlated with invasive ductal carcinomas compared to invasive lobular carcinomas, as well as ERα positive tumors and breast cancer cell lines. LSR levels were significantly reduced in claudin-low breast cancer cell lines and functional studies illustrated that re-introduction of LSR into a claudin-low cell line suppressed the EMT phenotype and reduced individual cell migration. However, our data suggest that LSR may promote collective cell migration. Re-introduction of LSR in claudin-low breast cancer cell lines reestablished tight junction protein expression and correlated with transepithelial electrical resistance, thereby reverting claudin-low lines to other intrinsic molecular subtypes. Moreover, overexpression of LSR altered gene expression of pathways involved in transformation and tumorigenesis as well as enhanced proliferation and survival in anchorage independent conditions, highlighting that reestablishment of LSR signaling promotes aggressive/tumor initiating cell behaviors. Collectively, these data highlight a direct role for LSR in driving aggressive breast cancer behavior.

Spontaneously diabetic Ins2(+/Akita):apoE-deficient mice exhibit exaggerated hypercholesterolemia and atherosclerosis.

Type 1 diabetes (T1D) increases the risk of adverse coronary events. Among risk factors, dyslipidemia due to altered hepatic lipoprotein metabolism plays a central role in diabetic atherosclerosis. Nevertheless, the likely alterations in plasma lipid/lipoprotein profile remain unclear, especially in the context of spontaneously developed T1D and atherosclerosis. To address this question, we generated Ins2(+/Akita):apoE(-/-) mouse by cross-breeding Ins2(+/Akita) mouse (which has Ins2 gene mutation, causing pancreatic β-cell apoptosis and insulin deficiency) with apoE(-/-) mouse. Ins2(+/Akita):apoE(-/-) mice developed T1D spontaneously at 4-5 wk of age. At 25 wk of age and while on a standard chow diet, diabetic Ins2(+/Akita):apoE(-/-) mice exhibited an approximately threefold increase in atherosclerotic plaque in association with an approximatelty twofold increase in plasma non-HDL cholesterol, predominantly in the LDL fraction, compared with nondiabetic controls. To determine factors contributing to the exaggerated hypercholesterolemia, we assessed hepatic VLDL secretion and triglyceride content, expression of hepatic lipoprotein receptors, and plasma apolipoprotein composition. Diabetic Ins2(+/Akita):apoE(-/-) mice exhibited diminished VLDL secretion by ~50%, which was accompanied by blunted Akt phosphorylation in response to insulin infusion and decreased triglyceride content in the liver. Although the expression of hepatic LDL receptor was not affected, there was a significant reduction in the expression of lipolysis-stimulated lipoprotein receptor (LSR) by ~28%. Moreover, there was a marked decrease in plasma apoB-100 with a significant increase in apoB-48 and apoC-III levels. In conclusion, exaggerated hypercholesterolemia and atherosclerosis in spontaneously diabetic Ins2(+/Akita):apoE(-/-) mice may be attributable to impaired lipoprotein clearance in the setting of diminished expression of LSR and altered apolipoprotein composition of lipoproteins.