Retinol-binding Protein-1 Research Articles

ELOA promotes tumor growth and metastasis by activating RBP1 in gastric cancer.

Elongin A (ELOA), our previous work revealed, serves as a novel tumor suppressor in colorectal cancer. However, the function and mechanism of ELOA in other cancer types, including gastric cancer (GC), remain to be elucidated. The expression of ELOA was measured by quantitative reverse transcription-polymerase chain reaction and western blot. The effects of ELOA on GC growth and metastasis were assessed through a series of in-vitro and in-vivo assays. Furthermore, the potential mechanism of ELOA was revealed by RNA sequencing, dual luciferase reporter assay, chromatin immunoprecipitation, and rescue experiments in GC. We uncovered increased expression of ELOA in GC tissues compared with paired normal tissues via bioinformatic analyses and our sample detection. Enhanced ELOA expression in GC tissues was obviously correlated with poor tumor differentiation, lymph node metastasis, advanced tumor stage, and a poor prognosis. A series of functional experiments showed that ELOA promoted the proliferation and metastasis of GC. Mechanistically, we revealed that the decreased levels of miR-490-3p caused the upregulation of ELOA in GC. Both RNA-seq and ChIP assays revealed that ELOA transcriptionally activated retinol-binding protein 1 (RBP1) by binding to its promotor. Furthermore, specific knockdown of RBP1 reduced the tumor-promoting ability of ELOA in GC cells. In summary, our findings demonstrate that ELOA exerts oncogenic properties by activating RBP1 expression, providing the basis for a promising therapeutic target in GC.

TMIC-72. ALL-TRANS RETINOIC ACID INDUCES DURABLE TUMOR IMMUNITY IN IDH MUTANT GLIOMAS AND CIRCUMVENTS REPRESSION OF CRBP1-RETINOIC ACID AXIS

Abstract Diffuse gliomas are fatal, immunologically cold tumors characterized by mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) genes. IDH mutations have been shown to result in global genomic hypermethylation and drastically altered transcriptomic landscape. We and others have shown that IDH mutation is direct responsible for the immunological cold landscape observed in IDH mutated glioma. Using a combination of animal models and patient-derived glioma stem cells (GSCs), we identify retinoic acid signaling as a primary regulator of innate and adaptive immune activation in IDHm gliomas. Using the Sleeping Beauty (SB) orthotopic murine model of IDH glioma, which faithfully recapitulates the TME seen in patients, we show that intracellular accumulation of oncometabolite 2-HG leads to hypermethylation and transcriptional repression of Retinol Binding Protein-1 (RBP1), which encode CRBP1, a key chaperone protein required for intracellular all-trans-retinoic-acid biosynthesis in glial cells. Restoration retinoic acid signaling through the addition of all-trans retinoic acid (ATRA) resulted in spontaneous tumor regression and prolonged survival. ATRA administration is further correlated with increased CD8, NK, M1 macrophages and decreased Treg and MDSC populations within the TME. Single-cell sequencing of the TME shows increased type I interferon signaling, myeloid cell polarization, T cell activation, and TCR diversity. Using knockdown and overexpression techniques in patient-derived GSCs, we show that CRBP1 activity is critical for NK mediated immune surveillance and cytotoxicity through regulation of NKG2D ligand expression and immune cell migration through differential regulation of CXCL12 and CCL2. In summary, epigenetic silencing of RBP1 by 2-HG induced hypermethylation leads to a global immune dysregulation resulting in an immunologically cold landscape. Restoration of retinoic acid signaling is a promising immunotherapeutic approach for the treatment of IDHm gliomas.

Urinary Proteomics Analysis of Active Vitiligo Patients: Biomarkers for Steroid Treatment Efficacy Prediction and Monitoring.

Vitiligo is a common acquired skin disorder caused by immune-mediated destruction of epidermal melanocytes. Systemic glucocorticoids (GCs) have been used to prevent the progression of active vitiligo, with 8.2–56.2% of patients insensitive to this therapy. Currently, there is a lack of biomarkers that can accurately predict and evaluate treatment responses. The goal of this study was to identify candidate urinary protein biomarkers to predict the efficacy of GCs treatment in active vitiligo patients and monitor the disease. Fifty-eight non-segmental vitiligo patients were enrolled, and 116 urine samples were collected before and after GCs treatment. Patients were classified into a treatment-effective group (n = 42) and a treatment-resistant group (n = 16). Each group was divided equally into age- and sex-matched experimental and validation groups, and proteomic analyses were performed. Differentially expressed proteins were identified, and Ingenuity Pathway Analysis was conducted for the functional annotation of these proteins. Receiver operating characteristic curves were used to evaluate the diagnostic value. A total of 245 and 341 differentially expressed proteins between the treatment-resistant and treatment-effective groups were found before and after GCs treatment, respectively. Bioinformatic analysis revealed that the urinary proteome reflected the efficacy of GCs in active vitiligo patients. Eighty and fifty-four candidate biomarkers for treatment response prediction and treatment response evaluation were validated, respectively. By ELISA analysis, retinol binding protein-1 and torsin 1A interacting protein 1 were validated to have the potential to predict the efficacy of GCs with AUC value of 1 and 0.875, respectively. Retinol binding protein-1, torsin 1A interacting protein 1 and protein disulfide-isomerase A4 were validated to have the potential to reflect positive treatment effect to GCs treatment in active vitiligo with AUC value of 0.861, 1 and 0.868, respectively. This report is the first to identify urine biomarkers for GCs treatment efficacy prediction in vitiligo patients. These findings might contribute to the application of GCs in treating active vitiligo patients.

Retinol binding protein 1-dependent activation of NF- κB signaling enhances the malignancy of non-glioblastomatous diffuse gliomas.

Nonglioblastomatous diffuse glioma (non‐GDG) is a heterogeneous neuroepithelial tumor that exhibits a varied survival range from 4 to 13 years based on the diverse subtypes. Recent studies demonstrated novel molecular markers can predict prognosis for non‐GDG patients; however, these findings as well as pathological classification strategies show obvious limitations on malignant transition due to the heterogeneity among non‐GDGs. Therefore, developing reliable prognostic biomarkers and therapeutic targets have become an urgent need for precisely distinguishing non‐GDG subtypes, illuminating the underlying mechanism. Nuclear factor κβ (NF‐κB) has been proved to be a significant nuclear transcriptional regulator with specific DNA‐binding sequences to participate in multiple pathophysiological processes. However, the underlying mechanism of NF‐κB activation still needs to be further investigated. Herein, our results indicated retinol‐binding protein 1 (RBP1) was significantly upregulated in the IDHWT and 1p19qNon co‐del non‐GDG subtypes and enriched RBP1 expression was markedly correlated with more severe outcomes. Additionally, malignant signatures of the non‐GDG cells including proliferation, migration, invasion, and self‐renewal were significantly suppressed by lentiviral knockdown of RBP1. To further explore the underlying molecular mechanism, bioinformatics analysis was performed using databases, and the results demonstrated RBP1 was strongly correlated with tumor necrosis factor α (TNFα)–NF‐κB signaling. Moreover, exogenous silencing of RBP1 reduced phosphorylation of IkB‐kinase α (IKKα) and thus decreased NF‐κB expression via decreasing the degradation of the IκBα protein. Altogether, these data suggested RBP1‐dependent activation of NF‐κB signaling promoted malignancy of non‐GDG, indicating that RBP1 could be a reliable prognostic biomarker and potential therapeutic target for non‐GDG.

Abstract 2538: Retinol binding protein-1 and E6-E7 HR-HPV mRNA expression in normal cervical tissue and high grade squamous intraepithelial lesions

Abstract Background: Gene expression differences between cervical healthy tissue and premalignant lesions could contribute to identify potential biomarkers for each step of this tumor progression. In a previous cDNA microarray analysis, we identified the gene Retinol Binding Protein 1 (RBP1), differentially expressed between normal cervix and a high grade squamous intraepithelial lesion (HSIL) with HPV-18 transforming infection. RBP1 codifies a retinol-binding protein implied in differentiation of epithelia and may play a role on inhibition of E2F-dependent gene expression and suppress cell growth. Design: The expression of RBP1 mRNA was evaluated using RNAscope chromogenic in situ hybridization (CISH) in a tissue microarray constructed from specimens (conization and hysterotomy) obtained from 169 patients, with mean age of 35.37 years (SD ±10.4). Stained sections from donor blocks were reviewed by two pathologist, who chose representative areas of normal cervix, low grade squamous intraepithelial lesion (LSIL), and HSIL. RNAscope 2.5HD Red RBP1 assay, and RNAscope 2.5HD Red HPV-HR18 , that detect E6/E7 mRNA for eighteen different high-risk (HR) HPV genotypes, were performed, the stainings were visually scored by two pathologist from 0 to 4 in a semi-quantitative analysis, based on the average number of red dots per cell. The mean score values for each tissue group were compared using GraphPad Prism® 7.0. A p< 0.05 was considered statistically significant. Results: The in situ expression of RBP1 mRNA was determined in a cervical TMA including, normal cervix: 66, LSIL: 74, and HSIL: 130, tissue cores. There was not a statistically significant difference (p=0.965) between the RBP1 mRNA expression score in the squamous epithelium from normal cervix (0.42, 95% CI:0.23-0.61), and LSIL (0.51, 95% CI:0.28-0.74). But, its expression was higher in HSIL (1.30, 95% CI:1.09-1.50), with a very significant difference compared to squamous epithelium from normal cervix (p<0.0001). Mean HPV E6/E7 mRNA expression in HSIL was significantly higher than mean HPV E6/E7 mRNA expression in LSIL (p<0.0001). There was positive-moderate correlation between RBP1 mRNA expression and E6/E7 HR-HPV mRNA expression (Spearman r =0.55). Conclusions: The in situ mRNA expression of RBP1 is similar in LSIL and squamous epithelium lining normal cervix, but the significantly different expression between healthy cervical tissue and HSIL, considered as the real premalignant cervical lesion, confirms our previous findings. There is a moderate correlation with the expression of E6/E7 mRNA for eighteen different HR-HPV. We could hypothesized that this overexpression represent a failed mechanism of regulation induced by the HR-HPV infection in cervical carcinogenesis. Citation Format: Ines Benedetti, Reinhard Rodríguez, Lia Barrios. Retinol binding protein-1 and E6-E7 HR-HPV mRNA expression in normal cervical tissue and high grade squamous intraepithelial lesions [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 2538.

Cellular retinol binding protein 1 transfection reduces proliferation and AKT-related gene expression in H460 non-small lung cancer cells

In recent years, new treatments with novel action mechanisms have been explored for advanced non-small cell lung cancer (NSCLC). Retinoids promote cancer cell differentiation and death and their trafficking and action is mediated from specific cytoplasmic and nuclear receptors, respectively. The purpose of this study was to investigate the effect of Cellular retinol binding protein-1 (CRBP-1) transfection in H460 human NSCLC cell line, normally not expressing CRBP-1. H460 cells were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBP-1 gene. For proliferation and apoptosis studies, cells were treated with different concentrations of all-trans Retinoic Acid (atRA) and retinol. AKT-related gene expression was analyzed by using western blot and Signosis array and results analysed by one-way analysis of variance (ANOVA) or by t-student test. CRBP-1+ showed reduced proliferation and viability in basal condition and after atRA treatment when compared to empty-transfected H460 cells. Reduced proliferation in CRBP-1+ H460 cells associated to the down-regulation of pAKT/pERK/pEGFR-related genes. In particular, gene array documented the down-regulation of AKT and Stat-3-related genes, including M-Tor, Akt1, Akt2, Akt3, Foxo1, p27, Jun. Restoration of CRBP-1 expression in H460 cells reduced proliferation and viability in both basal condition and after atRA treatment, likely by down-regulating AKT-related gene level. Further studies are needed to better clarify how those CRBP-1-related intracellular pathways contribute to counteract NSCLC progression in order to suggest a potential tool to improve efficacy of retinoid anti lung cancer adjuvant therapy.

Vitamin D receptor and cellular retinol-binding protein-1 immunohistochemical expression in normal, hyperplastic and neoplastic endometrium: Possible diagnostic and therapeutic implications

BackgroundWe conducted this study to assess the effect of VDR and CRBP-1 immunohistochemical expression on the endometrium and to explore their role in endometrial cancer carcinogenesis. MethodsThis study comprised two hundred paraffin-embedded endometrial tissue samples diagnosed as 42 and 63 proliferative and secretory endometrium respectively, 45 endometrial hyperplasias with atypia and 50 endometrial carcinomas (25 low-grade and 25 high-grade endometrial carcinomas). The immunohistochemical method was done to determine the expression of VDR and CRBP-1. ResultsVDR was strongly expressed in 8 (17.8%) cases with endometrial hyperplasia, 15 (60%) cases with low-grade endometrial carcinoma, and 22 (88%) cases with high-grade endometrial carcinoma. While CRPB1 overexpression was noted in cases with proliferative endometrium, secretory endometrium and endometrial hyperplasia with atypia, 37 (88.1%), 56 (88.9%) and 3 (6.7%) cases respectively and all malignant cases showed negative expression. ConclusionsIncreased VDR expression and reduced CRBP-1 expression are associated with malignant features of the endometrium with a significant statistical difference of immunoreactivity between groups of normal endometrium, hyperplastic changes & carcinoma. Our data suggested that increased VDR expression is partly associated with endometrial cancers through a premalignant phase. Also, increased VDR and reduced CRBP-1 expression are associated with the progression of endometrial carcinoma with higher grades.

The RBP1\u2013CKAP4 axis activates oncogenic autophagy and promotes cancer progression in oral squamous cell carcinoma

Retinol-binding protein 1 (RBP1) is involved in several physiological functions, including the regulation of the metabolism and retinol transport. Studies have shown that it plays an important role in the pathogenesis of several types of cancer. However, the role of RBP1 and its correlation with autophagy in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. In this study, RBP1 was identified as the most significantly upregulated DEPs with a >2-fold change in OSCC samples when compared to normal tissues through iTRAQ-based proteomics analysis coupled with 2D LC–MS/MS. RBP1 overexpression was significantly associated with malignant phenotypes (differentiation, TNM stage, and lymphatic metastasis) of OSCC. In vitro experiments demonstrated that RBP1 was significantly increased in OSCC tissues and cell lines compared with control group. RBP1 overexpression promoted cell growth, migration, and invasion of OSCC cells. Silencing of RBP1 suppressed tumor formation in xenografted mice. We further demonstrated that the RBP1–CKAP4 axis was a critical regulator of the autophagic machinery in OSCC, inactivation of autophagy rescued the RBP1–CKAP4-mediated malignant biological behaviors of OSCC cells. Overall, a mechanistic link was provided by RBP1–CKAP4 between primary oncogenic features and the induction of autophagy, which may provide a potential therapeutic target that warrants further investigation for treatment of OSCC.

Protein-protein interaction of the putative magnetoreceptor cryptochrome 4 expressed in the avian retina

Migratory birds can sense the Earth’s magnetic field and use it for orientation over thousands of kilometres. A light-dependent radical-pair mechanism associated with the visual system is currently discussed as the underlying mechanism of the magnetic compass sense. The blue light receptor cryptochrome 4 (Cry4) is considered as the most likely primary sensory protein that detects the geomagnetic field. Since the protein interaction partners of Cry4 are completely unknown at present, here, we aim to identify potential candidate interaction partners of Cry4 in the avian retina. We used the yeast-two-hybrid system to screen avian cDNA libraries for possible interaction partners of Cry4 in the European robin. The UAS-GAL yeast two hybrid system was applied to confirm a group of candidate Cry4 interaction partners. Six proteins were found to be particularly promising candidates for interacting with European robin Cry4. The identified genes code for guanine nucleotide-binding protein G(t) subunit alpha-2 (GNAT2), long-wavelength-sensitive opsin (LWS, also called iodopsin), guanine nucleotide-binding protein subunit gamma 10 (GNG10), potassium voltage-gated channel subfamily V member 2 (KCNV2), retinol binding protein 1 (RBP1) and retinal G protein-coupled receptor (RGR). All genes are known to be expressed in vertebrate retinae of different species. We conclude by discussing putative signalling pathways that could connect cryptochrome 4 to one or more of these 6 candidates.

Combined proteomics/miRNomics of dendritic cell immunotherapy-treated glioblastoma patients as a screening for survival-associated factors

Glioblastoma is the most prevalent and aggressive brain cancer. With a median overall survival of ~15–20 months under standard therapy, novel treatment approaches are desperately needed. A recent phase II clinical trial with a personalized immunotherapy based on tumor lysate-charged dendritic cell (DC) vaccination, however, failed to prolong survival. Here, we investigated tumor tissue from trial patients to explore glioblastoma survival-related factors. We followed an innovative approach of combining mass spectrometry-based quantitative proteomics (n = 36) with microRNA sequencing plus RT-qPCR (n = 38). Protein quantification identified, e.g., huntingtin interacting protein 1 (HIP1), retinol-binding protein 1 (RBP1), ferritin heavy chain (FTH1) and focal adhesion kinase 2 (FAK2) as factor candidates correlated with a dismal prognosis. MicroRNA analysis identified miR-216b, miR-216a, miR-708 and let-7i as molecules potentially associated with favorable tissue characteristics as they were enriched in patients with a comparably longer survival. To illustrate the utility of integrated miRNomics and proteomics findings, focal adhesion was studied further as one example for a pathway of potential general interest.Taken together, we here mapped possible drivers of glioblastoma outcome under immunotherapy in one of the largest DC vaccination tissue analysis cohorts so far—demonstrating usefulness and feasibility of combined proteomics/miRNomics approaches. Future research should investigate agents that sensitize glioblastoma to (immuno)therapy—potentially building on insights generated here.

Analysis of <i>eys</i> <sup>+/-</sup>; <i>lrp5</i> <sup>+/-</sup> Zebrafish Reveals LRP5 as a Strong Candidate for the Receptor Of All- <i>Trans</i> Retinol in the Visual Cycle

Vision is essential for vertebrates including humans. Sustained vision is accomplished by the retinoid metabolism, ‘visual cycle’, where all-trans retinol (atROL) is incorporated into the retinal pigment epithelium (RPE) from photoreceptors presumably through decade-long missing receptor(s). Here, we show that LDL-related receptor-5 (LRP5) protein is linked to retinol binding protein 1 (RBP1), the transporter of atROL in the visual cycle, by generating and analyzing the digenic eyes shut homolog+/-; lrp5+/- zebrafish, the same form of gene defects emerged from a human case report of a candidate inherited retinal degeneration. Global gene expression analysis followed by genetic study clarified that rbp1 played a role downstream of lrp5. RBP1 protein was colocalized with LRP5 protein at microvilli of RPE cells. Furthermore, RBP1 directly bound to C-terminal intracellular region of LRP5 in vitro. Collectively, these results strongly suggest that LRP5 is a potent candidate of the receptor of atROL in the visual cycle.

STRA6 Expression Serves as a Prognostic Biomarker of Gastric Cancer.

Despite advances in our understanding on the pathogenesis of gastric cancer (GC), patients face a poor prognosis. To improve clinical outcomes, effective approaches to diagnosis and treatment employing new diagnostic biomarkers are required to achieve early detection and predict recurrence and prognosis. Transcriptome analysis was conducted using surgically resected gastric tissues from four patients with metastatic GC. A total of 228 pairs of primary GC tissues and corresponding normal adjacent tissues were subjected to mRNA expression analysis. To validate our findings, we accessed an integrated microarray dataset and RNA sequencing data of GC cell lines. We identified stimulated by retinoic acid 6 (STRA6) as a differentially overexpressed gene, which encodes a transmembrane protein that mediates the cellular uptake of retinol. To investigate how STRA6 contributes to the malignant phenotype of GC cells, we mined public datasets and found the mRNA encoding retinol binding protein 1 (RBP1), which is associated with retinoid metabolism, was co-expressed with STRA6. Furthermore, STRA6 mRNA levels were significantly higher in GC tissues compared to the corresponding noncancerous adjacent tissues of 228 surgically resected gastric tissue samples. Moreover, patients with high levels of STRA6 mRNA experienced significantly shorter disease-free survival and overall survival. Multivariate analysis revealed that high levels of STRA6 served as a significant risk factor. Patients with high levels of STRA6 mRNA experienced significantly worse clinical outcomes, indicating that STRA6 may serve as a diagnostic and prognostic biomarker of GC.

Retinal proteomic evaluation of rats following streptozotocin‑injection using shotgun proteomics.

It is important to elucidate how retinal stimulation leads to retinal protection and dysfunction. The current study aimed to identify factors that are up‑ and downregulated in the retinas of streptozotocin (STZ)‑induced diabetic rats with acute retinal dysfunction. Retinal function was measured and changes in protein expressions were determined using electroretinograms (ERGs) and liquid chromatography/mass spectroscopy‑based shotgun proteomics, respectively. The results revealed that the plasma glucose levels of STZ rats were markedly higher when compared with normal rats. Furthermore, levels of a‑waves, b‑waves and oscillatory potential amplitudes on ERGs in STZ rats were decreased compared with healthy animals. With use of shotgun proteomics, 391proteins were identified in the retinas of normal rats and 541proteins were found in the retinas of STZ rats. Of the 560proteins identified in rat retinas, 372 (66.4%) were present in both normal and STZ rats. Of these, 19 (3.39%) were unique to normal rats and 169 (30.1%) were unique to STZ rats. Gene Ontology analysis was performed on the candidate proteins that were differentially regulated in the retinas of STZ rats and focused on those classified as 'protein binding', which serve important roles in retinal neurodegeneration. The results revealed an excessive expression of retinol‑binding protein1 (RBP1) and a negative expression of rod outer segment membrane protein1 (Rom-1) in the retinas of STZ rats. Therefore, retinal function may be decreased with STZ‑induced injury, and expressions of Rom‑1 and RBP1 may be altered in the retinas of STZ rats.

The RBP1-CKAP4 Axis Activates Oncogenic Autophagy and Promotes Cancer Progression in Oral Squamous Cell Carcinoma

Background: Retinol-binding protein 1 (RBP1) is involved in several physiological functions including the regulation of the metabolism and retinol transport. RBP1 has been implicated as an important element in the development of several types of cancer. However, the roles of RBP1 and its correlation with autophagy in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. Methods: In this study, RBP1 was identified as the most significant up-regulated DEPs with fold change > 2 in OSCC samples versus normal tissues through iTRAQ-based proteomics analysis coupled with 2D LC-MS/MS and was significantly associated with malignant phenotypes (differentiation, TNM stage, lymphatic metastasis) of OSCC. Immunohistochemistry was used to measure protein expression in tissues. Cell proliferation was evaluated by clonogenic and MTT assay. Cell cycle was analyzed by flow cytometer. Cell migration and invasion potentials were assessed by wound healing assay and transwell assay as well as confirmed by in vivo xenograft mice model. QRT-PCR, western blot and immunofluorescence were performed to detect the expression of ATG5, Beclin 1 and LC3B. Transmission electron microscopy was utilized to identify autophagsomes. Immunofluorescent staining and coimmunoprecipitation were used to determine the relationship between RBP1 and CKAP4. Findings: In vitro experiments demonstrated that RBP1 overexpression promoted cell proliferation, migration and invasion as well as accelerated G1 phase progression. Silencing of RBP1 suppressed tumor formation in xenografted mice. We further showed that RBP1-CKAP4 axis was a critical regulator of the autophagic machinery, a process essential for maintaining metabolic homeostasis in OSCC. Interpretation: We discovered that RBP1 could mediate autophagy through the CKAP4 axis and regulate the proliferation, migration and invasion of OSCC cells. Therefore, we propose that RBP1 induces autophagy to promote cancer by regulating CKAP4. Funding Statement: This work was supported by the National Science Foundation of China (81502354, 81472520, 81600880), Qingdao/China Postdoctoral Science Foundation (2017M622145, 2017M612217), Source Innovation Planning of Qingdao (17-1-1-42-jch, 17-1-1-45-jch, 18-2-2-77-jch) and Natural Science Foundation of Shaanxi/Shandong Province (2015JM8396, 2016JQ8028, 2017WS215, ZR2018BH021). Declaration of Interests: The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article. Ethics Approval Statement: Ethical approval was ratified by the local Ethics Committee, and all patients provided written informed consent for the utilization of tissue samples in research. All animal experiments were approved by the Animal Care and Use Committee of the Affiliated Hospital of Qingdao University.

Cellular retinol-binding protein-1 expression increases with increasing clinical severity of alopecia areata.

Alopecia areata (AA) is multifactorial disease mostly autoimmune affecting anagen hair follicles. Many researchers hypothesize that adequate retinoic acid (RA) levels are important for proper hair follicle behavior. Previous animal studies revealed increase in RA synthesis proteins and decrease in RA degradation proteins in AA patients when compared with controls. To evaluate cellular retinol-binding protein-1 expression in lesional skin of alopecia areata in comparison with controls, in an attempt to know its role in the pathogenesis of alopecia areata . Immunohistochemical expression of cellular retinol-binding protein-1 CRBP1 was evaluated in skin biopsies taken from lesions of alopecia areata in 30 patients and 10 normal biopsy specimens taken from skin of healthy controls (HC) who were within the same age and sex. CRBP1 expression was significantly increased in lesional alopecia areata skin in comparison with normal skin of controls (P<0.001*). Significant positive correlation was found between expression of CRBP-1 and percentage of hair loss in the scalp (SALT score; r=0.840, P=<0.001). These results may enhance the idea of the possible role of CRBP1 in the pathogenesis of AA, and ensuring the importance of its level in AA treatment.

CRBP-1 over-expression is associated with poor prognosis in tongue squamous cell carcinoma

BackgroundTongue squamous cell carcinoma (TSCC) is one of the most common malignancies of oral squamous cell carcinomas. Cellular retinol binding protein-1 (CRBP-1) as a carrier protein transports retinol from the liver storage site to peripheral tissue. Up-regulated expression of CRBP-1 is associated with some tumor types such as prostate cancer, breast cancer and ovarian cancer as reported, but its role in TSCC remains uncertain.MethodsIn this study, an integrated bioinformatics analysis based on the multiple cancer microarray data sets available from Oncomine database was conducted to view the differential expression of CRBP-1 between TSCC and the adjacent non-tumorous tissues. Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting (WB) and immunohistochemical (IHC) assays were performed to investigate CRBP-1 expression in 101 paraffin-embeded TSCC tissues and 48 pairs of freshly frozen tissues. Kaplan-Meier curve and univariate and multivariate Cox-regression analysis were used to estimate the association between CRBP-1 expression and patients’ prognosis. Then western blotting, MTT, transwell migration and invasion assays were performed in TSCC cell lines to investigate the effects of CRBP-1 on cellular proliferation and invasion.ResultsCompared with the matched adjacent non-tumorous tissues, the expression of CRBP-1 was significantly up-regulated in TSCC tissues, which correlated with the differentiation state (P = 0.003), N classification (P = 0.048), the clinical stage (P = 0.048) and death (P = 0.001). The Kaplan-Meier curve showed that TSCC patients with higher CRBP-1 expression levels had lower overall survival rates than those with lower CRBP-1 expression levels. A univariate and multivariate analysis demonstrated that CRBP-1 was an independent prognostic factor (P < 0.05). Furthermore, we knocked down CRBP-1 expression and observed that TSCC cell proliferation and invasion in vitro were significantly blocked, as determined by MTT and transwell assays.ConclusionsUp-regulated expression of CRBP-1 is associated with poor prognosis in TSCC, so it might potentially serve as an additional prognostic marker, and the inhibition of CRBP-1 might provide new therapeutic approaches for TSCC.

Characterization of voiding function and structural bladder changes in a rat model of neurogenic underactive bladder disease.

To create an animal model for neurogenic underactive bladder disease (UAB) and identify markers to describe secondary myogenic changes in the bladder wall. Male rats underwent either bilateral pelvic nerve injury or sham surgery. Four weeks after surgery functional evaluation was performed and tissue was harvested. Functional evaluation consisted of analysis of voiding pattern, 24-h urine collection in a metabolic cage, in vivo cystometry and in-vitro contractile function assessment. PCR and immunohistochemical localization of different smooth muscle cell and extracellular matrix markers was performed on bladder strips. After pelvic nerve injury, dry bladder weight increased and voiding contractions were absent, resulting in overflow incontinence. In-vitro contractile response to carbachol was decreased. This was paired with an upregulation of synthetic smooth muscle cell (SMC) markers mRNA expression such as retinol binding protein 1 (RBP1), myosin 10 (MYH10) and osteopontin (OPN), and a downregulation of contractile SMC marker smoothelin (SMTL). The SMTL/OPN mRNA ratio was 50 times higher in sham bladders compared to PNI bladders. The loss of in-vivo and in-vitro contractile function following pelvic nerve transection is characterized by a switch from a contractile to synthetic SMC phenotype, which is best characterized by the ratio SMTL/OPN mRNA expression. Modulating this phenotypical switch is a potential target for the development of UAB therapy. We suggest for the first time a set of markers that may be useful to evaluate therapeutic strategies on improvements in bladder wall structure.

Abstract 5574: Retinoic acid as a therapeutic adjuvant in low-grade gliomas

Abstract Introduction: Malignant gliomas are incurable brain tumors and the most common primary brain tumors in adults. Mutations in isocitrate dehydrogenase (IDH) are enriched in a subset of malignant low-grade gliomas (MLGGs) that arise as lower grade neoplasms and steadily progress to more aggressive and universally fatal glioblastomas (GBMs) usually within 5-10 years after initial diagnosis. Gliomas with IDH1/2 mutations are characterized by hypermethylation of multiple genes, including immune genes, thereby rendering them highly immune evasive. Retinol binding protein 1 (RBP1) gene, that encodes a protein involved in retinoic acid (RA) generation, is significantly hyper-methylated in IDH mutant cells. Since RA is instrumental in the induction of multiple immune genes including immune-activating ligands such as NKG2D ligands (NKG2D-L), we hypothesized that treatment of IDH mutant cells with RA will increase the expression of NKG2D-L on the tumor cells, leading to increased recognition and lysis by immune cells. In addition, we hypothesized that RBP1 methylation was crucial to development of the IDH mutant immune-evasive phenotype. Methods: Primary glioma cells (IDH WT and mutant) were treated with 1μM RA for 48 hours. The cells were assessed for changes in expression of NKG2D-L and other type-1 immune response genes. Effect of RA treatment on NK killing and immune cell chemotaxis were evaluated. We also assessed the impact of RBP1 knock-down in IDH WT cells. Finally, we evaluated the efficacy of RA as a therapeutic modality for low-grade gliomas in a murine model. Results: RA-treated IDH mutant gliomas had a higher expression of NKG2D-L and a higher sensitivity to killing by NK cells, when compared with untreated IDH mutant cells. RA treatment of IDH mutant cells also induced increased chemotaxis of NKs and CD8+ T cells in a CCL2-dependant manner. In addition, RA demonstrated superior anti-tumor efficacy in vivo, compared with controls. Finally, we demonstrated that RBP1 knock-down in IDH WT cells was sufficient to replicate the immune evasive phenotype observed in IDH mutant cells. Conclusions: RA treatment reverses the immune evasion in IDH mutant cells primarily by inducing the upregulation of NKG2D-L and by mediating increased NK and CD8+ T cell chemotaxis via CCL2 induction. Additionally, we established that RBP1 gene silencing is crucial for the development of the immune evasive IDH mutant phenotype. Note: This abstract was not presented at the meeting. Citation Format: Aparna Rao, Xiaoran Zhang, Jason Kim, Alexander Pomerantz, Youngmi Kim, Eric Holland, Nduka Amankulor. Retinoic acid as a therapeutic adjuvant in low-grade gliomas [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 5574. doi:10.1158/1538-7445.AM2017-5574

Proteomic Profiling for Identification of Novel Biomarkers Differentially Expressed in Human Ovaries from Polycystic Ovary Syndrome Patients.

ObjectivesTo identify differential protein expression pattern associated with polycystic ovary syndrome (PCOS).MethodsTwenty women were recruited for the study, ten with PCOS as a test group and ten without PCOS as a control group. Differential in-gel electrophoresis (DIGE) analysis and mass spectroscopy were employed to identify proteins that were differentially expressed between the PCOS and normal ovaries. The differentially expressed proteins were further validated by western blot (WB) and immunohistochemistry (IHC).ResultsDIGE analysis revealed eighteen differentially expressed proteins in the PCOS ovaries of which thirteen were upregulated, and five downregulated. WB and IHC confirmed the differential expression of membrane-associated progesterone receptor component 1 (PGRMC1), retinol-binding protein 1 (RBP1), heat shock protein 90B1, calmodulin 1, annexin A6, and tropomyosin 2. Also, WB analysis revealed significantly (P<0.05) higher expression of PGRMC1 and RBP1 in PCOS ovaries as compared to the normal ovaries. The differential expression of the proteins was also validated by IHC.ConclusionsThe present study identified novel differentially expressed proteins in the ovarian tissues of women with PCOS that can serve as potential biomarkers for the diagnosis and development of novel therapeutics for the treatment of PCOS using molecular interventions.

High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis.

Adenocarcinoma, the most common non-small cell lung cancer is a leading cause of death worldwide, with a low overall survival (OS) despite increasing attempts to achieve an early diagnosis and accomplish surgical and multimodality treatment strategies. Cellular retinol binding protein-1 (CRBP-1) regulates retinol bioavailability and cell differentiation, but its role in lung cancerogenesis remains uncertain. CRBP-1 expression, clinical outcome and other prognostic factors were investigated in 167 lung adenocarcinoma patients. CRBP-1 expression was evaluated by immunohistochemistry of tissue microarray sections, gene copy number analysis and tumor methylation specific PCR. Effects of CRBP-1 expression on proliferation/apoptosis gene array, protein and transcripts were investigated in transfected A549 lung adenocarcinoma cells. CRBP-1(High) expression was observed in 62.3% of adenocarcinomas and correlated with increased tumor grade and reduced OS as an independent prognostic factor. CRBP-1 gene copy gain also associated with tumor CRBP-1(High) status and dedifferentiation. CRBP-1-transfected (CRBP-1(+)) A549 grew more than CRBP-1(-) A549 cells. At >1μM concentrations, all trans-retinoic acid and retinol reduced viability more in CRBP-1(+) than in CRBP-1(-) A549 cells. CRBP-1(+) A549 cells showed up-regulated RARα/ RXRα and proliferative and transcriptional genes including pAkt, pEGFR, pErk1/2, creb1 and c-jun, whereas RARβ and p53 were strongly down-regulated; pAkt/pErk/ pEGFR inhibitors counteracted proliferative advantage and increased RARα/RXRα, c-jun and CD44 expression in CRBP-1(+) A549 cells. CRBP-1(High) expression in lung adenocarcinoma correlated with increased tumor grade and reduced OS, likely through increased Akt/Erk/EGFR-mediated cell proliferation and differentiation. CRBP-1(High) expression can be considered an additional marker of poor prognosis in lung adenocarcinoma patients.