V2 mRNA Research Articles

Alternative splicing of its 5′-UTR limits CD20 mRNA translation and enables resistance to CD20-directed immunotherapies

AbstractAberrant skipping of coding exons in CD19 and CD22 compromises the response to immunotherapy in B-cell malignancies. Here, we showed that the MS4A1 gene encoding human CD20 also produces several messenger RNA (mRNA) isoforms with distinct 5′ untranslated regions. Four variants (V1-4) were detected using RNA sequencing (RNA-seq) at distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma, only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform contained upstream open reading frames and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, whereas V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed chimeric antigen receptor T cells were able to kill both V3- and V1-expressing cells, but the bispecific T-cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on 4 postmosunetuzumab follicular lymphoma relapses and discovered that in 2 of them, the downregulation of CD20 was accompanied by a V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies.

Cloning and characterization of a novel functional organic anion transporting polypeptide 3A1 isoform highly expressed in the human brain and testis.

Organic anion transporting polypeptide 3A1 (OATP3A1, encoded by the SLCO3A1 gene) is a prostaglandin, oligopeptide, and steroid/thyroid hormone transporter with wide tissue distribution, expressed, e.g., in the human brain and testis. Although the physiological importance of OATP3A1 has not yet been clarified, based on its expression pattern, substrate recognition, and evolutionary conservation, OATP3A1 is a potential pharmacological target. Previously, two isoforms of OATP3A1, termed as V1 and V2, have been characterized. Here, we describe the cloning and functional characterization of a third isoform, OATP3A1_V3. The mRNA of isoform V3 is formed by alternative splicing and results in an OATP3A1 protein with an altered C-terminus compared to isoforms V1 and V2. Based on quantitative PCR, we demonstrate the widespread expression of SLCO3A1_V3 mRNA in human organs, with the highest expression in the brain and testis. By generation of an isoform V3-specific antibody and immunostaining, we show that the encoded protein is expressed in the human choroid plexus, neurons, and both germ and Sertoli cells of the testis. Moreover, we demonstrate that in contrast to isoform V1, OATP3A1_V3 localizes to the apical membrane of polarized MDCKII cells. Using HEK-293 cells engineered to overexpress OATP3A1_V3, we verify the protein’s functionality and identify dehydroepiandrosterone sulfate as a novel OATP3A1 substrate. Based on their distinct expression patterns but overlapping functions, OATP3A1 isoforms may contribute to transcellular (neuro)steroid transport in the central nervous system.

MP03-03 TARGETING V1/CHASE TO OVERCOME GEMCITABINE RESISTANCE

You have accessJournal of UrologyCME1 May 2022MP03-03 TARGETING V1/CHASE TO OVERCOME GEMCITABINE RESISTANCE Semih Sarcan, Daley S. Morera, Marie C. Hupe, Karina Aguilar, Anuj Sharma, Sung Alexander, Joseph McDaniels, Huabin Zhu, Martin Hennig, Martha Terris, Axel S. Merseburger, and Vinata B. Lokeshwar Semih SarcanSemih Sarcan More articles by this author , Daley S. MoreraDaley S. Morera More articles by this author , Marie C. HupeMarie C. Hupe More articles by this author , Karina AguilarKarina Aguilar More articles by this author , Anuj SharmaAnuj Sharma More articles by this author , Sung AlexanderSung Alexander More articles by this author , Joseph McDanielsJoseph McDaniels More articles by this author , Huabin ZhuHuabin Zhu More articles by this author , Martin HennigMartin Hennig More articles by this author , Martha TerrisMartha Terris More articles by this author , Axel S. MerseburgerAxel S. Merseburger More articles by this author , and Vinata B. LokeshwarVinata B. Lokeshwar More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002515.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Precision-based treatment approaches could improve the prognosis of patients with bladder cancer (BC). We recently discovered a first-in-class human Chondroitinase (Chase) that degrades chondroitin sulfate. This Chase is a splice variant of HYAL4 (V1) and promotes a malignant phenotype and chemoresistance in BC cells. We evaluated V1/Chase expression and functions in BC and inhibitors to overcome V1/Chase-mediated Gemcitabine (Gem) resistance. METHODS: Cohort 1: 86 bladder tissues (normal (NBL)=34; tumor (TBL)=52); cohort 2: 40 cystectomy specimens from MIBC patients who received adjuvant Gemcitabine plus cisplatin (G+C) treatment. Cohort 3: 144 urine specimens (BCA=42, non-BCa=102). Gene expression was measured by q-PCR in all cohorts and urine was assayed for Chase activity. V1/Chase was stably expressed or silenced in normal urothelial and three BC cell lines. Transfectants were analyzed for sensitivity to Gemcitabine and Cisplatin. The mechanism of Gem resistance was evaluated in preclinical in vitro and in vivo models. RESULTS: In cohort 1, V1 mRNA levels were an independent predictor of metastasis and death due to BC (P=0.0008). In cohort 2, high V1 levels significantly predicted with G+C treatment failure (P <0.0001). In cohort 3, Chase levels were 13 to 75-fold elevated in low and high-grade BC patients’ urine compared to non-BC cases (92.7% sensitivity; 88.2% specificity). V1-expressing urothelial and BC cells were resistant to Gem but not to Cisplatin. V1 expression increased Gem metabolism and subsequent efflux of an inactive metabolite dFdU. V1 increased CD44 shedding the conditioned media. This triggered intracellular activation of the JAK2/STAT3 pathway, resulting in the upregulation of cytidine deaminase (CDA). CDA induces Gem metabolism and dFdU efflux. JAK2, STAT3 and CDA inhibitors, (some FDA-approved), synergistically re- sensitized V1-expressing cells to Gem. V1-induced muscle invasive tumors that were metastatic. While Gem inhibited BC xenograft growth, V1-expressing tumors were resistant. Combination of Gem with a CDA inhibitor (tetrahydrouridine) abrogated V1 tumor growth with minimal toxicity. CONCLUSIONS: V1 is a functional biomarker that drives muscle-invasion, metastasis and Gem resistance in BC. Inhibitors of the JAK2/STAT3/CDA pathway overcome V1- mediated Gem resistance, suggesting a precision-based treatment approach to improve treatment response. Source of Funding: 1R01CA227277-01A1 (VBL); Department of Defence-PRCRP (W81XWH1810277 (CA170470; PRCRP) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e19 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Semih Sarcan More articles by this author Daley S. Morera More articles by this author Marie C. Hupe More articles by this author Karina Aguilar More articles by this author Anuj Sharma More articles by this author Sung Alexander More articles by this author Joseph McDaniels More articles by this author Huabin Zhu More articles by this author Martin Hennig More articles by this author Martha Terris More articles by this author Axel S. Merseburger More articles by this author Vinata B. Lokeshwar More articles by this author Expand All Advertisement PDF DownloadLoading ...

Cancer testis antigen PASD1 expression and immunogenicity in human colorectal cancer and polyps.

Colorectal cancer (CRC) is a malignant tumor arising from a human inner colon lining that may spread to other organs such as the liver and lungs. Per ARNT Sim domain containing 1 (PASD1) is a cancer-testis antigen expressed in cancers including CRC but not in normal tissues except for normal testes. This study aims to study PASD1 protein as a potential target for CRC immunotherapy. A total of 90 CRC and polyps tissue samples were investigated for PASD1 RNA and protein expression using a real-time polymerase chain reaction and immunohistochemical staining, respectively. Matched patients' peripheral blood mononuclear cells were pulsed with PASD1 peptides and measured for immunogenicity, cell cytotoxicity, and cytokine assays. The clinical data were collected and analyzed accordingly. Our results show that PASD1_v2 mRNA expression was highly expressed in CRC (46.0%) and polyps samples (33.3%). Both PASD1-1 and PASD1-2 proteins were expressed in 31.7% of CRC and 29.4% of polyps samples. Protein expression was weak to moderate positive in the cytoplasm and/or nucleus of the tissues. Immune responses towards CD4-specific PASD1 peptides were detected in 21.7% of CRC and 23.5% of polyps patients. The most immunogenic peptide was PASD1 (1) in CRC while PASD1 (3) in polyps. Cytotoxicity effects were detected up to 57.20% observed in CRC samples while IL-17A and IL-6 cytokines were highly expressed. The demographic data suggest that Chinese female patients more than 60 years old, diagnosed with late-stage rectosigmoid tumors may benefit from the PASD1 peptide immunotherapy approach. This is the first report describing CD4-positive T-helper response to the PASD1 positive CRC patients and its cytotoxicity.

Human Corneal Expression of SLC4A11, a Gene Mutated in Endothelial Corneal Dystrophies

Two blinding corneal dystrophies, pediatric-onset congenital hereditary endothelial dystrophy (CHED) and some cases of late-onset Fuchs endothelial corneal dystrophy (FECD), are caused by SLC4A11 mutations. Three N-terminal SLC4A11 variants: v1, v2 and v3 are expressed in humans. We set out to determine which of these transcripts and what translated products, are present in corneal endothelium as these would be most relevant for CHED and FECD studies. Reverse transcription PCR (RT-PCR) and quantitative RT-PCR revealed only v2 and v3 mRNA in human cornea, but v2 was most abundant. Immunoblots probed with variant-specific antibodies revealed that v2 protein is about four times more abundant than v3 in human corneal endothelium. Bioinformatics and protein analysis using variant-specific antibodies revealed that second methionine in the open reading frame (M36) acts as translation initiation site on SLC4A11 v2 in human cornea. The v2 variants starting at M1 (v2-M1) and M36 (v2-M36) were indistinguishable in their cell surface trafficking and transport function (water flux). Structural homology models of v2-M36 and v3 suggest structural differences but their significance remains unclear. A combination of bioinformatics, RNA quantification and isoform-specific antibodies allows us to conclude that SLC4A11 variant 2 with start site M36 is predominant in corneal endothelium.

Utility of G protein-coupled receptor 35 expression for predicting outcome in colon cancer

The utility of mRNA and protein determinations of G protein-coupled receptor 35, that is, GPR35a (GPR35 V1) and GPR35b (GPR35 V2/3), as indicators of outcome for colon cancer patients after curative surgery was investigated. Expression levels of V1 and V2/3 GPR35, carcinoembryonic antigen and CXCL17 mRNAs were assessed in primary tumours and regional lymph nodes of 121 colon cancer patients (stage I-IV), colon cancer cell lines and control colon epithelial cells using real-time quantitative reverse transcriptase-polymerase chain reaction. Expression of G protein-coupled receptor 35 was investigated by two-colour immunohistochemistry and immunomorphometry. GPR35 V2/3 mRNA, but not V1 mRNA, was expressed in colon cancer cell lines, primary colon tumours and control colon epithelial cells. Haematoxylin and eosin positive (H&E(+)), but not H&E(-), lymph nodes expressed high levels of GPR35 V2/3 mRNA (P<0.0001). GPR35b and carcinoembryonic antigen proteins were simultaneously expressed in many colon cancer tumour cells. Kaplan-Meier and hazard ratio analysis revealed that patients with lymph nodes expressing high levels of GPR35 V2/3 mRNA and, in particular, in the group of patients with lymph nodes also expressing carcinoembryonic antigen mRNA, had a short disease-free survival time, 67 months versus 122 months at 12-year follow-up (difference: 55 months, P = 0.001; hazard ratio: 3.6, P = 0.002). In conclusion, high level expression of G protein-coupled receptor 35 V2/3 mRNA in regional lymph nodes of colon cancer patients is a sign of poor prognosis.

Functional Interaction between SEPT9_v1 and EGFR in Triple‐Negative and Inflammatory Breast Cancer

The poor prognosis for patients with inflammatory breast cancer (IBC) emphasizes the need to better understand the molecular signature of this disease with the goal of developing effective targeted therapeutics. Importantly, 20–40% of IBC cases are triple‐negative breast cancers (TNBC) in which targeted hormone therapy is not effective. Interestingly, approximately 1/2 of cases of IBC overexpress the epidermal growth factor receptor (EGFR), a member of the ErbB family of receptors involve in cell proliferation, migration, and cytoskeleton remodeling. Recently, SEPT9, a member of a large family of cytoskeletal proteins important in diverse biological processes, was found to interact with EGFR. Dysregulation of SEPT9 isoforms have been associated in different cancer types, including breast cancer. To date, no studies have looked at the potential pro‐oncogenic role of SEPT9 isoforms in IBC or TNBC. We will examine the functional relationship between SEPT9_v1, cytoskeleton remodeling and EGFR signaling in the IBC and TNBCs cell lines. Our hypothesis is that SEPT9_v1 contributes to oncogenesis by regulating the activity of ErbB receptors in IBC and/or TNBC. To test our hypothesis, generation of stable cell lines with knockdown or overexpression of SEPT9_v1 using lentiviral and delivering vectors will be developed. Establishment of 2D and 3D cultures of the cell lines will be performed to analyze if the acquisition of pro‐oncogenic phenotypes. To assess these phenotypes, we will use cell proliferation assays using alamar blue to determine the viability of cells, in vitro transwell migration, invasion assays and fluorescent microscopy to determine if there is aberrant morphology or cytoskeleton remodeling. We will determine the specific effect of SEPT9_v1 overexpression and knockdown to the activation of the EGFR signaling. Stability of ErbB receptors upon SEPT9_v1 overexpression or knockdown will be also measured by protein stability assays and by looking directly in the plasma membrane of cells with fluorescent microscopy. Downstream activation of the EGFR pathway will be assessed by Western blot (WB) analysis of activated effector proteins. Co‐immunoprecipitation analysis will be performed to test if there is a specific interaction between EGFR and SEPT9_v1. Our preliminary data shows that approximately 50% of breast cancer cell lines overexpress SEPT9_v1 mRNA when compared to a panel of human mammary epithelial cell lines. Also, by WB analysis we determined that SUM149 expresses two isoforms, SEPT9_v1 and SEPT9_v3. This work will provide new insights into the functional relationship between SEPT9_v1 and EGFR and their contribution to the aggressive metastatic phenotype in IBC. Furthermore, it will help us to identify new potential therapeutic targets for the better management of this disease.Support or Funding InformationThis project is supported by UPR Fondos Institucionales para la Investigación (FIPI to E.Peterson ) and MARC program (5T34GM008721‐38 to A. Agosto)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Empagliflozin Contributes to Polyuria via Regulation of Sodium Transporters and Water Channels in Diabetic Rat Kidneys.

Besides lowering glucose, empagliflozin, a selective sodium-glucose cotransporter-2 (SGLT2) inhibitor, have been known to provide cardiovascular and renal protection due to effects on diuresis and natriuresis. However, the natriuretic effect of SGLT2 inhibitors has been reported to be transient, and long-term data related to diuretic change are sparse. This study was performed to assess the renal effects of a 12-week treatment with empagliflozin (3 mg/kg) in diabetic OLETF rats by comparing it with other antihyperglycemic agents including lixisenatide (10 μg/kg), a glucagon-like peptide receptor-1 agonist, and voglibose (0.6 mg/kg), an α-glucosidase inhibitor. At 12 weeks of treatment, empagliflozin-treated diabetic rats produced still high urine volume and glycosuria, and showed significantly higher electrolyte-free water clearance than lixisenatide or voglibose-treated diabetic rats without significant change of serum sodium level and fractional excretion of sodium. In empagliflozin-treated rats, renal expression of Na+-Cl- cotransporter was unaltered, and expressions of Na+/H+ exchanger isoform 3, Na+-K+-2Cl- cotransporter, and epithelial Na+ channel were decreased compared with control diabetic rats. Empagliflozin increased an expression of aquaporin (AQP)7 but did not affect AQP3 and AQP1 protein expressions in diabetic kidneys. Despite the increased expression in vasopressin V2 receptor, protein and mRNA levels of AQP2 in empagliflozin-treated diabetic kidneys were significantly decreased compared to control diabetic kidneys. In addition, empagliflozin resulted in the increased phosphorylation of AQP2 at S261 through the increased cyclin-dependent kinases 1 and 5 and protein phosphatase 2B. These results suggest that empagliflozin may contribute in part to polyuria via its regulation of sodium channels and AQP2 in diabetic kidneys.

Abstract 3445: Functional interaction between SEPT9_v1 and EGFR in triple-negative and inflammatory breast cancer

Abstract The poor prognosis for patients with inflammatory breast cancer (IBC) emphasizes the need to better understand the molecular signature of this disease with the goal of developing effective targeted therapeutics. Importantly, 20-40% of IBC cases are triple-negative breast cancers (TNBC) in which targeted hormone therapy is not effective. Interestingly, approximately 1/2 of cases of IBC overexpress the epidermal growth factor receptor (EGFR), a member of the ErbB family of receptors involve in cell proliferation, migration, and cytoskeleton remodeling. Recently, SEPT9, a member of a large family of cytoskeletal proteins important in diverse biological processes, was found to interact with EGFR. Dysregulation of SEPT9 isoforms have been associated in different cancer types, including breast cancer. To date, no studies have looked at the potential pro-oncogenic role of SEPT9 isoforms in IBC or TNBC. We will examine the functional relationship between SEPT9_v1, cytoskeleton remodeling and EGFR signaling in the IBC cell lines SUM149, SUM190 and TNBCs. Our hypothesis is that SEPT9_v1 contributes to oncogenesis by regulating the activity of ErbB receptors in IBC and/or TNBC. To test our hypothesis, generation of stable cell lines with knockdown or overexpression of SEPT9_v1 using lentiviral and delivering vectors will be developed. Establishment of 2D and 3D cultures of the cell lines will be performed to analyze if the acquisition of pro-oncogenic phenotypes and the effects on cytoskeleton dynamics are SEPT9_v1 and/or EGFR dependent. To assess these phenotypes, we will use cell proliferation assays using alamar blue to determine the viability of cells over time, in vitro transwell migration, invasion assays and fluorescent microscopy to determine if there is aberrant morphology or cytoskeleton remodeling. We will determine the specific effect of SEPT9_v1 overexpression and knockdown to the activation of the EGFR signaling. Stability of ErbB receptors upon SEPT9_v1 overexpression or knockdown will be also measured by protein stability assays and by looking directly in the plasma membrane of cells with fluorescent microscopy. Downstream activation of the EGFR pathway will be assessed by Western blot analysis of activated effector proteins. Co-immunoprecipitation analysis will be performed to test if there is a specific interaction between EGFR and SEPT9_v1. Our preliminary data shows that approximately 50% of breast cancer cell lines overexpress SEPT9_v1 mRNA when compared to a panel of human mammary epithelial cell lines. Also, by WB analysis we determined that SUM149 expresses two isoforms, SEPT9_v1 and SEPT9_v3. This work will provide new insights into the functional relationship between SEPT9_v1 and EGFR and their contribution to the aggressive metastatic phenotype in IBC. Furthermore, it will help us to identify new potential therapeutic targets for the better management of this disease. Citation Format: Amanda Agosto, Jose G. Cirino, Kimberleve Rolón, Gabriel E. Figueroa, Gabriela Martinez, Katixa Montañez-Negrón, Keishla Rodríguez-Mártir, Xavier Bittman, Esther A. Peterson. Functional interaction between SEPT9_v1 and EGFR in triple-negative and inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3445.

Methylated septin 9 gene for noninvasive diagnosis and therapy monitoring of breast cancer

Background: Blood-based biomarkers specific for breast cancer is urgently demanded to facilitate the screening and reduce breast cancer-related deaths. This study evaluated the potential of methylated septin 9 gene ( mSEPT9 ) as a biomarker for breast cancer. Methods: A total of 86 breast biopsy samples were collected from 59 patients with breast cancer and from 27 patients with benign breast tumor. Two hundred and seven plasma samples were collected from 80 patients with breast cancer, 27 patients with benign breast tumor patients, and 100 healthy volunteers. mSEPT9 was detected using the Epi proColon 2.0 CE test. Additionally, 40 tissue cases of the tumor samples were analysed by real time PCR for SEPT9_v2 mRNA expression and by immunohistochemical staining for SEPT9 protein expression. Serum CEA, CK19 and CA15-3 concentrations were analyzed using an Electrochemiluminescence Immunoassay Kit. Results: The sensitivity of plasma mSEPT9 for detecting breast cancer was 26.3% with a specificity of 97.6%. The plasma mSEPT9 -positive rate was higher in patients with later stage lymph node metastasis (P=0.018), intravascular tumor thrombus (P=0.035), ki67-positive (P=0.033) and p53-positive (P=0.044). Despite its limitation in sensitivity, conversion of plasma mSEPT9 to negative status in mSEPT9 -positive breast cancer patients following cancer treatment was correlated with favorable responses, while persistent positivity was associated with poor responses. mSEPT9 performed comparably with CK19 (P>0.05) and better than CEA (P=0.002) and CA15-3 (P mSEPT9 was detected in 83.1% (49/59) of the breast cancer tissues, whereas 8.0% (2/25) of the benign breast tumor tissues were mSEPT9 positive (P mSEPT9 in breast cancer tissue was associated with tumor size of >2 cm (P=0.026) and with invasion of adipose tissue (P=0.015). Conclusions: Our results indicate that mSEPT9 test has the potential to become a valuable tool in the diagnosis and treatment monitoring of breast cancer for Chinese patients.

Immunohistochemical and Molecular Investigations Show Alteration in the Inflammatory Profile of Multiple System Atrophy Brain.

Multiple system atrophy (MSA) is an adult-onset neurodegenerative disease characterized by aggregation of α-synuclein in oligodendrocytes to form glial cytoplasmic inclusions. According to the distribution of neurodegeneration, MSA is subtyped as striatonigral degeneration (SND), olivopontocerebellar atrophy (OPCA), or as combination of these 2 (mixed MSA). In the current study, we aimed to investigate regional microglial populations and gene expression in the 3 different MSA subtypes. Microscopy with microglial marker Iba-1 combined with either proinflammatory marker CD68 or anti-inflammatory marker Arginase-1 was analyzed in control, SND, and OPCA cases (n = 5) using paraffin embedded sections. Western immunoblotting and cytokine array were used to determine protein expression in MSA and control brain regions. Gene expression was investigated using the NanoString nCounter Human Inflammation panel v2 mRNA Expression Assay. Analysis of neuropathological subtypes of MSA demonstrated a significant increase in microglia in the substantia nigra of OPCA cases. There was no difference in the microglial activation state in any region. Cytokine expression in MSA was comparable with controls. Decreased expression of CX3CL1 precursor protein and significantly greater CX3CR1 protein was found in MSA. NanoString analysis revealed the >2-fold greater expression of ARG1, MASP1, NOX4, PTGDR2, and C6 in MSA.

Abstract 1396: Role of epigenetic reader and alternative mRNA splicing variant MBD2 variant 2 in triple-negative breast cancer

Abstract Background: Among all breast cancer patients, those diagnosed with the triple-negative breast cancer (TNBC) subtype have the worst prognosis, in part attributable to the fact that TNBC lacks targets for effective molecularly-targeted therapies. The concept that EGFR inhibitor drugs could be used as a targeted treatment against TNBC has been put forth because roughly 50% of TNBC express high levels of EGFR. However clinical trials targeting EGFR did not significantly improve patient outcomes. Our recent work offers a potential explanation as to why EGFR inhibitors failed and supports an innovative therapeutic approach combining an EGFR tyrosine kinase inhibitor with the novel antioxidant biologic, CAT-SKL. Our data indicate that inhibition of cancer stem cell-like cells depends on antioxidant-induced downregulation of an alternative mRNA splice variant of the methyl-CpG binding domain 2 gene, MBD2_v2. Objective and Methods: The purpose of the present study was to evaluate the importance of MBD2_v2 in TNBC and better understand how CAT-SKL regulates its expression. Using TNBC cell lines we investigated the effects of MBD2_v2 expression on tumor initiation capacity. Furthermore we studied MBD2_v2 mRNA levels in an in vivo tumor xenograph mouse model of TNBC and in human TNBC samples using an existing database. Then by RNA-seq analysis we assessed how CAT-SKL regulates MBD2_v2 expression in TNBC cells through differential gene expression. Results: The results support that MBD2_v2 expression is regulated by redox signaling in TNBC cells, which is tightly linked to TNBC cell tumor initiation capacity. Furthermore, MBD2_v2 was upregulated in tumors harvested from overweight mice, which also displayed increased tumor take rate, suggesting that a pro-inflammatory tumor microenvironment could play a role in promoting MBD2_v2 expression. In addition, RNA-seq analysis identified an overrepresentation of mRNA splicing factors with expression downregulated by CAT-SKL treatment of TNBC cells. From this gene set two splicing co-factors are highlighted: SRRM1 and CPSF3. These co-factors are critical for splicing factor function, 3’ end formation and polyadenylation; and both have been linked via protein-protein interaction to a relevant exonic splicing enhancer site in exon 3 of the MBD2 _v2 transcript. Finally, according to the Kaplan Meier Plotter database high MBD2_v2 levels in BC were associated with shorter relapse free survival. Conclusion: Our investigation sheds light on the relevance of MBD2_v2 expression in TNBC, and how a select antioxidant regulates its expression. The application of which holds promise of a novel, targeted therapeutic modality for TNBC, the subtype with the worst prognosis and highest need for treatment options. Citation Format: Emily A. Girsch, Bin Bao, Cristina Mitrea, Gregory Dyson, Julie Boerner, Lisa Polin, Stan R. Terlecky, Aliccia Bollig-Fischer. Role of epigenetic reader and alternative mRNA splicing variant MBD2 variant 2 in triple-negative breast cancer [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 1396. doi:10.1158/1538-7445.AM2017-1396

Effects of 17β-estradiol on leptin signaling in anterior pituitary of ovariectomized rats.

Leptin is secreted predominantly by adipocytes and exerts its role mainly by interaction with the long form of leptin receptor (LEPR_V2). It has been identified that LEPR_V2 is widely distributed in various tissues, including the anterior pituitary. Cross-talk between leptin and estrogens has been indentified. Estrogen is known to modulate the tissue-specific expression of LEPR_V2 and leptin in ovariectomized (OVX) rats, a model of postmenopausal condition. Our previous data showed that 17β-estradiol (E2) up-regulated the expression of LEPR_V2 protein and mRNA in rat dorsal root ganglion (DRG) in an estrogen receptor alpha (ERα)-dependent manner. But it is still unclear whether estrogen can regulate leptin signalling in the pituitary of OVX rats. In the present study, we found that ovariectomy decreased the expressions of LEPR_V2. Administration of E2 increased the expressions of LEPR_V2 in a dose-dependent manner. In addition, E2 improved LEPR_V2, STAT3, and SOCS3 protein levels in OVX rats. The effects of exogenous E2 were attenuated by ICI 182,780, a specific estrogen receptors antagonist. However, E2 did not change the Lepr_v1, a type of short form of leptin receptor (LEPR), or leptin mRNA levels. Thus, E2 plays a crucial role in regulating pituitary sensitivity to leptin in OVX rats. Our findings implied that exogenous E2 had potential roles in modification of the function of pituitary in postmenopausal women.

Methylation of the SEPT9_v2 promoter as a novel marker for the detection of circulating tumor DNA in breast cancer patients.

The aim of the present study was to evaluate the promoter methylation status of SEPT9_v2 in breast cancer and to detect this methylated gene in circulating tumor DNA (ctDNA) in plasma. Bisulfite sequencing was performed with a next generation sequencer. Methylation of the SEPT9_v2 promoter was found in 67%(8/12) of breast cancer cell lines and 53% (10/19) of breast tumor tissue, but not in normal breast tissue (0/19). A clear inverse correlation was observed between the expression of SEPT9_v2 mRNA and the methylation index (MI) both in cell lines and breast cancer tissues. The MI of SEPT9_v2 was significantly higher in non-basal subtype of breast cancer (13.0%, n=84) than in basal subtype (3.0%, n=23) (P<0.0001). Methylated SEPT9_v2 ctDNA in plasma was detected in 11% (9/82) of primary breast cancer patients and 52% (26/50) of metastatic breast cancer patients, but not in the healthy controls (0/51). These results indicate that SEPT9_v2 promoter hypermethylation, which silences the expression of SEPT9_v2 mRNA, is observed in a significant proportion of breast tumors, and that methylated SEPT9_v2 may serve as a novel tumor marker for breast cancer.

Alternative splicing of DENND1A, a PCOS candidate gene, generates variant 2

Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by hyperandrogenism and metabolic disorders. The excess androgens may be of both ovarian and adrenal origin. PCOS has a strong genetic component, and genome-wide association studies have identified several candidate genes, notably DENND1A, which encodes connecdenn 1, involved in trafficking of endosomes. DENND1A encodes two principal variants, V1 (1009 amino acids) and V2 (559 amino acids). The androgen-producing ovarian theca cells of PCOS women over-express V2. Knockdown of V2 in these cells reduces androgen production, and overexpression of V2 in normal theca cells confers upon them a PCOS phenotype of increased androgen synthesis. We report that human adrenal NCI-H295A cells express V1 and V2 mRNA and that the V2 isoform is produced by exonization of sequences in intron 20, which generates a unique exon 20A, encoding the C-terminus of V2. As in human theca cells from normal women, forced expression of V2 in NCI-H295A cells resulted in increased abundance of CYP17A1 and CYP11A1 mRNAs. We also found genetic variation in the intronic region 330 bp upstream from exon 20A, which could have the potential to drive the selective expression of V2. There was no clear association with these variants with PCOS when we analyzed genomc DNA from normal women and women with PCOS. Using minigene expression vectors in NCI-H295A cells, this variable region did not consistently favor splicing of the V2 transcript. These findings suggest increased V2 expression in PCOS theca cells is not the result of genomic sequence variation in intron 20.

Abstract 2068: Cyclooxygenase-1 gene expression contributes to multiple oncogenic pathways in high grade serous ovarian cancer

Abstract Ovarian cancer is the most lethal gynecologic malignancy. Most women diagnosed with high grade serous ovarian cancer present with advanced disease characterized by widespread peritoneal carcinomatosis. Recent epidemiological results indicate that cyclooxygenase (COX) inhibitors aspirin and other non-steroidal anti-inflammatory drugs are a promising preventive strategy to reduce the risk of developing ovarian cancer. COX-1 and COX-2 catalyze the rate-limiting step in the biosynthesis of prostaglandins and thromboxanes, which contribute to tumorigenesis by promoting proliferation, escape from apoptosis, and angiogenesis. Emerging evidence from our group and others suggests that COX-1 is an important molecular target in ovarian cancer, distinguishing ovarian cancer from most other solid tumors, where COX-2 is the dominant isoform. However, the precise roles of COX-1 and COX-2 in human ovarian cancer remain controversial. Therefore, the aim of this study was to gain additional insight into COX-1 gene expression and function in human ovarian cancer. First, we extracted RNA-seq V2 mRNA expression data for COX-1 and COX-2 in the PANCAN12 tumor panel of The Cancer Genome Atlas. COX-1 mRNA levels were significantly higher than those of COX-2 in high grade serous ovarian tumors, and higher than COX-1 expression in 11 other solid tumor types. We confirmed these findings in a tissue microarray generated from 209 ovarian cancer patients. COX-1 protein was highly expressed in a large subset of serous tumors, with significantly lower COX-2 expression present. In contrast, there was markedly lower COX-1 expression in endometrioid, mucinous and clear cell ovarian tumors compared to COX-2. Second, stable COX-1 knockdown in serous-representative OVCAR-3 cells significantly reduced cell viability compared to control cells. Pathway analysis from RNA-seq data revealed that COX-1 knockdown induced a coordinated transcriptional response consistent with reduced cell proliferation (eg. reduced cyclin D1 and increased p21 expression), cell migration/invasion (eg. reduced TGF-β signaling and expression of MMPs), angiogenesis (eg. reduced VEGF expression), epithelial-mesenchymal transition (eg. increased E-cadherin expression and reduced BMP signaling), and NF-κB signaling. Gene expression changes were validated by western blot. In SKOV-3 ovarian cancer cells stably overexpressing COX-1, opposite effects on cell viability and on these genes/pathways were observed. Our findings confirm that COX-1 is over-expressed in serous ovarian cancer, and likely plays a key role in tumor progression. Exploiting high COX-1 expression in ovarian cancer with COX-1-targeted molecules has the potential to improve the treatment and detection of this deadly disease. Citation Format: Andrew J. Wilson, Brian D. Lehmann, Jeanette Saskowski, MD Jashim Uddin, Cristina Daniel, Brenda C. Crews, Qi Liu, Shilin Zhao, Deok-Soo Son, Jennifer A. Pietenpol, Lawrence J. Marnett, Dineo Khabele. Cyclooxygenase-1 gene expression contributes to multiple oncogenic pathways in high grade serous ovarian cancer. [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 2068. doi:10.1158/1538-7445.AM2015-2068

Modulation of the UGT2B7 Enzyme Activity by C-Terminally Truncated Proteins Derived from Alternative Splicing

The enzyme UGT2B7 is one of the most active UDP-glucuronosyltransferases (UGTs) involved in drug metabolism and in maintaining homeostasis of endogenous compounds. We recently reported the existence of 22 UGT2B7 mRNAs, two with a classic 5' region but alternative 3' ends namely UGT2B7_v5 (containing a novel terminal exon 6b) and _v7 (exon 5 excluded) that encode enzymatically inactive isoforms 2 and 4 (i2 and i4), respectively. The v1 mRNA encoding the UGT2B7 enzyme (renamed isoform 1 or i1) is coexpressed with the splice variants v5 and v7 in human liver, kidney, and small intestine and the hepatic cell lines HepG2 and C3A. The presence of alternate v5 and v7 transcripts in isolated polysomes from these hepatic cells further supports endogenous protein translation. Cellular fractionation of clonal HEK293 cell lines overexpressing UGT2B7 isoforms demonstrates that i1, i2, and i4 proteins colocalize in the microsomal/Golgi fraction, whereas i2 and i4 can also be found in the cytosol; a finding sustained by immunofluorescence experiments using tagged proteins. By modifying splice variant abundance in overexpression in HEK293 and HepG2 cells as well as RNA interference experiments in HepG2 and C3A cells, we observe drug glucuronidation phenotypes compatible with variant-mediated repression of UGT2B7 activity without consequent alteration of the apparent enzyme affinity (K(m)). Finally, coimmunoprecipitation experiments support a direct protein-protein interaction of i2 and i4 proteins with the functional UGT2B7 enzyme as a potential causative mechanism. These findings point toward a novel autoregulatory mechanism of the UGT2B7 glucuronidation pathway by naturally occurring alternative i2 and i4 proteins.

Quaking, an RNA-Binding Protein, Is a Critical Regulator of Vascular Smooth Muscle Cell Phenotype

RNA-binding proteins are critical post-transcriptional regulators of RNA and can influence pre-mRNA splicing, RNA localization, and stability. The RNA-binding protein Quaking (QKI) is essential for embryonic blood vessel development. However, the role of QKI in the adult vasculature, and in particular in vascular smooth muscle cells (VSMCs), is currently unknown. We sought to determine the role of QKI in regulating adult VSMC function and plasticity. We identified that QKI is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Quaking viable mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering noncontractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome, where it interacts with the myocardin pre-mRNA and regulates the splicing of alternative exon 2a. This post-transcriptional event impacts the Myocd_v3/Myocd_v1 mRNA balance and can be modulated by mutating the quaking response element in exon 2a of myocardin. Furthermore, we identified that arterial damage triggers myocardin alternative splicing and is tightly coupled with changes in the expression levels of distinct QKI isoforms. We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.

Abstract 532: The RNA-binding Protein Quaking Critically Regulates Vascular Smooth Muscle Cell Phenotype

In response to vascular injury, smooth muscle cells (VSMC) adopt a proliferative, synthetic hypocontractile phenotype. This phenotype switch is deemed instrumental in vascular remodeling in both health and disease. Here, we detail a decisive role for the RNA-binding protein Quaking (QKI) in regulating VSMC plasticity. We identified that the RNA-binding protein Quaking (QKI) is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Qk v mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering non-contractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome in proliferative VSMCs, where it interacts with and impacts myocardin (pre)-mRNA metabolism by mediating myocardin exon 2a exclusion. As such, in vitro and in vivo experiments indicate that the modulation of QKI expression directly influences the myocardin_v3 / myocardin_v1 mRNA balance, which could play a role in shifting the Myocardin-induced transcriptional coactivation profile following arterial damage. We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.

A Flanking Gene Problem Leads to the Discovery of a Gprc5b Splice Variant Predominantly Expressed in C57Bl/6J Mouse Brain and in Maturing Neurons

BackgroundGprc5b, a retinoic acid-inducible orphan G protein–coupled receptor (GPCR), is a member of the group C metabotropic glutamate receptor family proteins possibly involved in non-canonical Wnt signaling. Many GPCR transcripts are alternatively spliced, which diversifies this class of proteins in their cell- and tissue-specific signaling, regulatory and/or pharmacological properties. We previously generated p97FE65 isoform-specific knockout mice that showed learning/memory deficits. In this study, we further characterized the 97FE65 null mice using cDNA microarray and RT-PCR analyses.Methodology/Principal FindingsWe discovered a novel brain-specific C-terminal splice variant of Gprc5b, Gprc5b_v2, which was differentially expressed in p97FE65 wild type and null mouse brains. The null mice were generated in 129/Sv ES cells, and backcrossed to C57Bl/6J for ten generations. We found that expression of Gprc5b_v2 mRNA in the brains of p97FE65 null mice was dramatically down-regulated (more than 20 fold) compared to their wild type littermates. However, expression profiles of Gprc5b variants and SNP analysis surrounding the FE65 locus suggest that the down-regulation is unlikely due to the altered FE65 function, but rather is caused by gene retention from the 129/Sv ES cells. Consistently, in contrast to ubiquitously expressed Gprc5b_v1, Gprc5b_v2 was predominantly expressed in the brain tissues of C57Bl/6J mice. The alternative splicing of the 3′ terminal exon also altered the protein coding sequences, giving rise to the characteristic C-termini. Levels of Gprc5b_v2 mRNA were increased during neuronal maturation, paralleling the expression of synaptic proteins. Overexpression of both Gprc5b variants stimulated neurite-like outgrowth in a neuroblastoma cell line.Conclusions/SignificanceOur results suggest that Gprc5b-v2 may play a role during brain maturation and in matured brain, possibly through the regulation of neuronal morphology and protein-protein interaction. This study also highlights the fact that unexpected gene retention following repeated backcrosses can lead to important biological consequences.