A2 Isoforms Research Articles

Abstract 2844: New breast cancer protein as a potential biomarker in liquid biopsies

Abstract The decline in breast cancer mortality over the past 2 decades is primarily the result of advances in early detection and treatment. However, the five year relative survival rate of metastatic breast cancer (MBC) patients is only 27%. This requires the need of new biomarkers for early detection of MBC. Tumors secrete microvesicles (MV) in large quantities to blood circulation in order to communicate with distant cells such as immune cells and prepare the metastatic niche. Our recent studies showed that MV from metastatic breast cancer cells contain a specific peptide from the a2 isoform vacuolar ATPase (a2NTD). This a2NTD is undetectable in mammary epithelial cells MV but expressed abundantly by MV produced by breast tumor cells. We have previously shown that the MV containing a2NTD modulate immune cells, promotes tumor growth, metastasis and positively correlates with the invasiveness of breast cancer cells. Identifying the role of a2NTD in regulating immune cells in liquid biopsy will provide a new biomarker for breast cancer. Treatment of whole blood from healthy volunteers with MDA MB-231 MV or recombinant a2NTD drives the neutrophil transition from High density neutrophils (HDN) to Low density neutrophils (LDN). Flow cytometrical analysis showed that LDN possess the PMN-MDSCs markers CD11b+ CD15+ CD14- CD33- HLA-DR-. In addition a2NTD treatment was associated with decreased internal staining of Arg-1 in neutrophils that suggests the release of Arg-1. This data suggest that a2NTD can reprogram neutrophils to exert immunosuppressive phenotype. Further characterization of surface markers as well as functional assays demonstrate the exact phenotype of neutrophils. Together studying the impact of a2NTD present in MV on immune alteration during metastasis can be potential predictors of the growth and metastasis of breast cancer. Citation Format: Safaa A. Ibrahim, Valerie riehl, Kenneth D. Beaman. New breast cancer protein as a potential biomarker in liquid biopsies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2844.

Surfactant-secreted phospholipase A2 interplay and respiratory outcome in preterm neonates.

Secreted phospholipase A2 hydrolyzes surfactant phospholipids and is crucial for the inflammatory cascade; preterm neonates are treated with exogenous surfactant, but the interaction between surfactant and phospholipase is unknown. We hypothesize that this interplay is complex and the enzyme plays a relevant role in neonates needing surfactant replacement. We aimed to: 1) identify phospholipases A2 isoforms expressed in preterm lung; 2) study the enzyme role on surfactant retreatment and function and the effect of exogenous surfactant on the enzyme system; and 3) verify whether phospholipase A2 is linked to respiratory outcomes. In bronchoalveolar lavages of preterm neonates, we measured enzyme activity (alone or with inhibitors), enzyme subtypes, surfactant protein-A, and inflammatory mediators. Surfactant function and phospholipid profile were also tested. Urea ratio was used to obtain epithelial lining fluid concentrations. Follow-up data were prospectively collected. Subtype-IIA is the main phospholipase isoform in preterm lung, although subtype-IB may be significantly expressed. Neonates needing surfactant retreatment have higher enzyme activity (P = 0.021) and inflammatory mediators (P always ≤ 0.001) and lower amounts of phospholipids (P always < 0.05). Enzyme activity was inversely correlated to surfactant adsorption (ρ = -0.6; P = 0.008; adjusted P = 0.009), total phospholipids (ρ = -0.475; P = 0.05), and phosphatidylcholine (ρ = -0.622; P = 0.017). Exogenous surfactant significantly reduced global phospholipase activity (P < 0.001) and subtype-IIA (P = 0.005) and increased dioleoylphosphatidylglycerol (P < 0.001) and surfactant adsorption (P < 0.001). Enzyme activity correlated with duration of ventilation (ρ = 0.679, P = 0.005; adjusted P = 0.04) and respiratory morbidity score at 12 mo postnatal age (τ-b = 0.349, P = 0.037; adjusted P = 0.043) but was not associated with mortality, bronchopulmonary dysplasia, or other long-term respiratory outcomes.

Effects of Vacuolar H+‐ATPase Inhibition and the a2 Isoform on Secretion and Activation of Cathepsin B in MDA‐MB231 Breast Cancer Cells

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are important for immune functionality, synaptic signaling, and other processes. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. We hypothesize that V‐ATPases create a low pH environment that promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells are involved in metastasis. Cathepsins cleave extracellular matrix proteins and activate other proteases, facilitating tumor invasion. We are interested in a connection between cell surface V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor reduced the activity of secreted cathepsin B, but did not cathepsin L. However, although there was a trend, difference in activity for secreted cathepsin B after V‐ATPase inhibition was not statistically significant. Our current data builds on that trend to show a significant difference in the activity of secreted cathepsin B. To investigate the role of V‐ATPases in the activation of cathepsins further we are using CRISPR/Cas9 to knockout the gene for the V‐ATPase a2 isoform of subunit “a”. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. Additionally, it was found that the a1 and a2 isoforms are not present on the cell surface, thus we hypothesize that the a2 isoform knockout will not affect the activity of secreted cathepsin B, but will affect the activity of cathepsin B in the cell interior. Separate abstracts submitted by our lab are focused on the a1, a3, and a4 isoform knockouts.Support or Funding InformationDenison University Anderson Summer Research Assistantship

Effects of Vacuolar H+‐ATPase Inhibition and the a4 Isoforms on Secretion and Activation of Cathepsin D in MDA‐MB231 Breast Cancer Cells

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are important for acid‐base balance in the blood and sperm maturation. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. V‐ATPases are hypothesized to create a localized low pH environment that promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells are involved in metastasis by cleaving extracellular matrix proteins and activating other proteases, which facilitates tumor invasion. We are interested in a connection between cell surface V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor showed a trend of reducing the activity of secreted cathepsin B, but not cathepsin L. However, that trend, was not statistically significant. Cathepsin D is another protease that is secreted by cancer cells including metastatic breast cancer. For this study we looked at the activity of cathepsin D following inhibition of V‐ATPases. Our preliminary data indicates that inhibition of V‐ATPases reduces the activity of secreted cathepsin D. To investigate the role of V‐ATPases in the activation of cathepsins further we are using CRISPR/Cas9 to knockout the gene for the a4 isoform of the V‐ATPase “a” subunit. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. We hypothesize that the a4 isoform knockout cells will have reduced activation of secreted cathepsin D while not effecting activation of cathepsin L. Separate abstracts submitted by our lab are focused on the a1, a2 and a3 isoform knockouts.Support or Funding InformationAnderson Summer Research Assistantship

Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166

While several clathrin-independent endocytic processes have been described so far, their biological relevance often remains elusive, especially in pathophysiological contexts such as cancer. In this study, we find that the tumor marker CD166/ALCAM (Activated Leukocyte Cell Adhesion Molecule) is a clathrin-independent cargo. We show that endophilin-A3—but neither A1 nor A2 isoforms—functionally associates with CD166-containing early endocytic carriers and physically interacts with the cargo. Our data further demonstrates that the three endophilin-A isoforms control the uptake of distinct subsets of cargoes. In addition, we provide strong evidence that the construction of endocytic sites from which CD166 is taken up in an endophilin-A3-dependent manner is driven by extracellular galectin-8. Taken together, our data reveal the existence of a previously uncharacterized clathrin-independent endocytic modality, that modulates the abundance of CD166 at the cell surface, and regulates adhesive and migratory properties of cancer cells.

Cancer-associated V-ATPase induces delayed apoptosis of protumorigenic neutrophils.

Tumors and neutrophils undergo an unexpected interaction, in which products released by tumor cells interact to support neutrophils that in turn support cancer growth, angiogenesis, and metastasis. A key protein that is highly expressed by cancer cells in tumors is the a2 isoform V‐ATPase (a2V). A peptide from a2V (a2NTD) is secreted specifically by cancer cells, but not normal cells, into the tumor microenvironment. This peptide reprograms neutrophils to promote angiogenesis, cancer cell invasiveness, and neutrophil recruitment. Here, we provide evidence that cancer‐associated a2V regulates the life span of protumorigenic neutrophils by influencing the intrinsic pathway of apoptosis. Immunohistochemical analysis of human cancer tissue sections collected from four different organs shows that levels of a2NTD and neutrophil counts are increased in cancer compared with normal tissues. Significant increases in neutrophil counts were present in both poorly and moderately differentiated tumors. In addition, there is a positive correlation between the number of neutrophils and a2NTD expression. Human neutrophils treated with recombinant a2NTD show significantly delayed apoptosis, and such prolonged survival was dependent on NF‐κB activation and ROS generation. Induction of antiapoptotic protein expression (Bcl‐xL and Bcl‐2A1) and decreased expression of proapoptotic proteins (Bax, Apaf‐1, caspase‐3, caspase‐6, and caspase‐7) were a hallmark of these treated neutrophils. Autocrine secretion of prosurvival cytokines of TNF‐α and IL‐8 by treated neutrophils prolongs their survival. Our findings highlight the important role of cancer‐associated a2V in regulating protumorigenic innate immunity, identifying a2V as a potential important target for cancer therapy.

Intracellular Ca2+-dependent formation of N-acyl-phosphatidylethanolamines by human cytosolic phospholipase A2ε

N-Acyl-phosphatidylethanolamines (NAPEs) are known to be precursors of bioactive N-acylethanolamines (NAEs), including the endocannabinoid arachidonoylethanolamide (anandamide) and anti-inflammatory palmitoylethanolamide. In mammals, NAPEs are produced by N-acyltransferases, which transfer an acyl chain from the sn-1 position of glycerophospholipid to the amino group of phosphatidylethanolamine (PE). Recently, the ɛ isoform of cytosolic phospholipase A2 (cPLA2ɛ) was found to be Ca2+-dependent N-acyltransferase. However, it was poorly understood which types of phospholipids serve as substrates in living cells. In the present study, we established a human embryonic kidney 293 cell line, in which doxycycline potently induces human cPLA2ɛ, and used these cells to analyze endogenous substrates and products of cPLA2ɛ with liquid chromatography-tandem mass spectrometry. When treated with doxycycline and Ca2+ ionophore, the cells produced various species of diacyl- and alkenylacyl-types of NAPEs as well as NAEs in large quantities. Moreover, the levels of diacyl- and alkenylacyl-types of PEs and diacyl-phosphatidylcholines (PCs) decreased, while those of lysophosphatidylethanolamines and lysophosphatidylcholines increased. These results suggested that cPLA2ɛ Ca2+-dependently produces NAPEs by utilizing endogenous diacyl- and alkenylacyl-types of PEs as acyl acceptors and diacyl-type PCs and diacyl-type PEs as acyl donors.

Conditional Deletion of the V-ATPase a2-Subunit Disrupts Intrathymic T Cell Development.

Proper orchestration of T lymphocyte development is critical, as T cells underlie nearly all responses of the adaptive immune system. Developing thymocytes differentiate in response to environmental cues carried from cell surface receptors to the nucleus, shaping a distinct transcriptional program that defines their developmental outcome. Our recent work has identified a previously undescribed role for the vacuolar ATPase (V-ATPase) in facilitating the development of murine thymocytes progressing toward the CD4+ and CD8+ αβ T cell lineages. Vav1Cre recombinase-mediated deletion of the a2 isoform of the V-ATPase (a2V) in mouse hematopoietic cells leads to a specific and profound loss of peripheral CD4+ and CD8+ αβ T cells. Utilizing T cell-restricted LckCre and CD4Cre strains, we further traced this deficiency to the thymus and found that a2V plays a cell-intrinsic role throughout intrathymic development. Loss of a2V manifests as a partial obstruction in the double negative stage of T cell development, and later, a near complete failure of positive selection. These data deepen our understanding of the biological mechanisms that orchestrate T cell development and lend credence to the recent focus on V-ATPase as a potential chemotherapeutic target to combat proliferative potential in T cell lymphoblastic leukemias and autoimmune disease.

Apolipoprotein A2 isoform and CA19-9 as biomarkers for early detection of pancreatic cancer - A prospective evaluation by the EPIC study

Apolipoprotein A2 isoform and CA19-9 as biomarkers for early detection of pancreatic cancer - A prospective evaluation by the EPIC study

Conditional deletion of the V-ATPase a2-subunit disrupts intrathymic T cell development

Abstract Proper orchestration of T lymphocyte development is critical, as T cells underlie nearly all responses of the adaptive immune system. Developing thymocytes differentiate in response to environmental cues carried from cell surface receptors to the nucleus, shaping a distinct transcriptional program that defines their developmental outcome. Our recent work has identified a previously undescribed role for the vacuolar ATPase (V-ATPase) in facilitating the development of murine thymocytes progressing toward the CD4+ and CD8+ αβ T cell lineages. Vav-Cre recombinase-mediated deletion of the a2 isoform of the V-ATPase (a2V) in mouse hematopoietic cells leads to a specific and profound loss of peripheral CD4+ and CD8+ αβ T cells. Utilizing T cell-restricted Lck- and CD4-Cre strains, we further traced this deficiency to the thymus and found that a2V plays a cell-intrinsic role throughout intrathymic development. Loss of a2V manifests as a partial obstruction in the double negative stage of T cell development, and later, a near complete failure of positive selection. These data deepen our understanding of the biological mechanisms that orchestrate T cell development, and lend credence to the recent focus on V-ATPase as a potential chemotherapeutic target to combat proliferative potential in T cell lymphoblastic leukemias and autoimmune disease.

CDNA and gene structures of two phospholipase A2 isoforms, acidic PLA2 PA4 AND PLA2 PA3A/PA3B/PA5, in Nemopilema nomurai jellyfish venom

cDNA and gene structures of two phospholipase A2 isoforms, acidic PLA2 PA4 AND PLA2 PA3A/PA3B/PA5, in Nemopilema nomurai jellyfish venom

Potential of plasma apolipoprotein A2 isoforms as a pancreatic cancer screening

Potential of plasma apolipoprotein A2 isoforms as a pancreatic cancer screening

1002: Expression of the a2 isoform of vacuolar ATPase influences outcome only in female-female twin pregnancies

1002: Expression of the a2 isoform of vacuolar ATPase influences outcome only in female-female twin pregnancies

Role of Annexin A2 isoform 2 on the aggregative growth of dermal papillae cells.

The dermal papilla is a major component of hair, which signals the follicular epithelial cells to prolong the hair growth process. Human Annexin A2 was preliminarily identified by two-dimensional gel electrophoresis (2-DE), MALDI-TOF-MS and database searching. The aim of the present study was to explore the role of Annexin A2 in the aggregative growth of dermal papillae cells (DPC). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were adopted to detect the expression of Annexin A2. And siRNA technique was used to suppress the expression of Annexin A2. Construction of over-expression vector was used to up-regulate the expression of Annexin A2. Cell Counting Kit 8 (CCK-8) and proliferating cell nuclear antigen (PCNA) were taken to detect the proliferation of DPC. The expression of Annexin A2 mRNA was up-regulated in passage 3 DPC compared with passage 10 DPC by RT-PCR. In line with the results at the mRNA level, Western blot analysis revealed that Annexin A2 isoform 2 was up-regulated significantly in passage 3 DPC compared with passage 10 DPC. The Annexin A2 isoform 2 siRNA was synthesized and transfected into passage 3 DPC. RT-PCR data showed the mRNA expression of Annexin A2 isoform 2 was suppressed in passage 3 DPC. Western blot results showed the expression level of Annexin A2 isoform 2 and PCNA were suppressed in passage 3 DPC. CCK-8 results showed that the proliferation of passage 3 DPC was suppressed (P < 0.05). Recombinant plasmid PLJM-Annexin A2 isoform 2-expression vector were constructed and were transfected into passage 10 DPC. RT-PCR data showed the mRNA expression of Annexin A2 isoform 2 was up-regulated in passage 10 DPC. Western blot results showed the expression level of annexin A2 isoform 2 and PCNA were up-regulated in passage 10 DPC. CCK-8 assay showed the proliferation of DPC was stimulated compared with control group (*P < 0.05). Our study proved that Annexin A2 isoform 2 may participate in regulating the proliferation of DPC and may be related to aggregative growth of dermal papilla cells. Therefore, our study suggests that Annexin A2 may be linked to hair follicle growth cycle.

The role of intracellular anionic phospholipids in the production of N-acyl-phosphatidylethanolamines by cytosolic phospholipase A2ɛ.

N-Acyl-phosphatidylethanolamines (NAPEs) represent a class of glycerophospholipids and serve as the precursors of bioactive N-acylethanolamines, including arachidonoylethanolamide (anandamide), palmitoylethanolamide and oleoylethanolamide. NAPEs are produced in mammals by N-acyltransferases, the enzymes which transfer an acyl chain of glycerophospholipids to the amino group of phosphatidylethanolamine. Recently, the ɛ isoform of cytosolic phospholipase A2 (cPLA2ɛ, also called PLA2G4E) was identified as Ca2+-dependent N-acyltransferase. We showed that the activity is remarkably stimulated by phosphatidylserine (PS) in vitro. In the present study, we investigated whether or not endogenous PS regulates the function of cPLA2ɛ in living cells. When PS synthesis was suppressed by the knockdown of PS synthases in cPLA2ɛ-expressing cells, the cPLA2ɛ level and its N-acyltransferase activity were significantly reduced. Mutagenesis studies revealed that all of C2, lipase and polybasic domains of cPLA2ɛ were required for its proper localization as well as the enzyme activity. Liposome-based assays showed that several anionic glycerophospholipids, including PS, phosphatidic acid and phosphatidylinositol 4,5-bisphosphate, enhance the Ca2+-dependent binding of purified cPLA2ɛ to liposome membrane and stimulate its N-acyltransferase activity. Altogether, these results suggested that endogenous PS and other anionic phospholipids affect the localization and enzyme activity of cPLA2ɛ.

Sex‐specific influence of the vacuolar adenosine triphosphatase a2 isoform on outcome in twin pregnancies

The influence of fetal sex on immune responses in multifetal pregnancies remains incompletely elucidated. The a2 isoform of vacuolar adenosine triphosphatase (a2V) is expressed on the cell membrane of maternal lymphoid cells and contributes to down-regulation of pro-inflammatory immune responses during gestation. The association between fetal sex and a2V expression on peripheral blood mononuclear cells (PBMCs) from mothers with twin gestations was assessed. Patients in this prospective study were 93 women with twin pregnancies in their mid-second or early third trimester-27 with two male, 30 with two female and 36 with one male and one female fetus. PBMCs were isolated and a2V was measured by ELISA in cell lysates. Demographic and clinical data were subsequently obtained and correlations between a2V and fetal sex, birthweight and pregnancy outcome were assessed by the Mann-Whitney and Spearman rank correlation tests. The mean a2V level was highest when both fetuses were male (2.0ng/mL) and lowest when both were female (1.5ng/mL; P=0.0184). Only when both fetuses were female did the a2V concentration negatively correlate with birthweight of the 1st (P=0.0011) and 2nd (P=0.0044) born fetus and with gestational age at delivery (P=0.0018). There were no associations between a2V and these outcomes in male only or mixed twin pregnancies. We conclude that the a2V-mediated regulation of maternal immunity during twin pregnancies is influenced by fetal sex.

Development of sesbania mosaic virus nanoparticles for imaging.

The capsids of viruses have a high degree of symmetry. Therefore, virus nanoparticles (VNPs) can be programmed to display many imaging agents precisely. Plant VNPs are biocompatible, biodegradable and non-infectious to mammals. We have carried out bioconjugation of sesbania mosaic virus (SeMV), a well characterized plant virus, with fluorophores using reactive lysine-N-hydroxysuccinimide ester and cysteine-maleimide chemistries. Monitoring of cellular internalization of labelled SeMV nanoparticles (NPs) by confocal microscopy and flow cytometry showed that the particles have a natural preference for entry into MDA-MB-231 (breast cancer) cells, although they could also enter various other cell lines. The fluorescence of SeMV NPs labelled via the cysteines with Cy5.5 dye was found to be more stable and was detectable with greater sensitivity than that of particles labelled via the lysines with Alexa Fluor. Live-cell imaging using SeMV internally labelled with Cy5.5 showed that it could bind to MDA-MB-231 cells in less than 5 minutes and enter the cells within 15 minutes. The particles undergo endolysosomal degradation by 6 h as evidenced by their co-localization with LAMP-1. Far-western blot analysis with a HeLa cell membrane protein fraction showed that SeMV interacts with 54-, 35- and 33-kDa proteins, which were identified by mass spectrometry as vimentin, voltage-dependent anion-selective channel protein (VDAC1), and annexin A2 isoform 2 (ANXA2), respectively, suggesting that the particles may bind and enter the cell through these proteins. The results presented here demonstrate that the SeMV NPs provide a new platform technology that could be used to develop in vivo imaging and targeted drug delivery agents for cancer diagnosis and therapy.

746P - Usefulness of the first screening using apolipoprotein A2 isoforms as the enrichment strategy for pancreatic cancer and its risk diseases

746P - Usefulness of the first screening using apolipoprotein A2 isoforms as the enrichment strategy for pancreatic cancer and its risk diseases

132P - Carbohydrate antigen 19-9 and apolipoprotein A2 isoform as early detection biomarkers for pancreatic cancer: A prospective evaluation by the EPIC study

132P - Carbohydrate antigen 19-9 and apolipoprotein A2 isoform as early detection biomarkers for pancreatic cancer: A prospective evaluation by the EPIC study

Dystonin-A3 upregulation is responsible for maintenance of tubulin acetylation in a less severe dystonia musculorum mouse model for hereditary sensory and autonomic neuropathy type VI.

Hereditary sensory and autonomic neuropathy type VI (HSAN-VI) is a recessive human disease that arises from mutations in the dystonin gene (DST; also known as Bullous pemphigoid antigen 1 gene). A milder form of HSAN-VI was recently described, resulting from loss of a single dystonin isoform (DST-A2). Similarly, mutations in the mouse dystonin gene (Dst) result in severe sensory neuropathy, dystonia musculorum (Dstdt). Two Dstdt alleles, Dstdt-Tg4 and Dstdt-27J, differ in the severity of disease. The less severe Dstdt-Tg4 mice have disrupted expression of Dst-A1 and -A2 isoforms, while the more severe Dstdt-27J allele affects Dst-A1, -A2 and -A3 isoforms. As dystonin is a cytoskeletal-linker protein, we evaluated microtubule network integrity within sensory neurons from Dstdt-Tg4 and Dstdt-27J mice. There is a significant reduction in tubulin acetylation in Dstdt-27J indicative of microtubule instability and severe microtubule disorganization within sensory axons. However, Dstdt-Tg4 mice have no change in tubulin acetylation, and microtubule organization was only mildly impaired. Thus, microtubule instability is not central to initiation of Dstdt pathogenesis, though it may contribute to disease severity. Maintenance of microtubule stability in Dstdt-Tg4 dorsal root ganglia could be attributed to an upregulation in Dst-A3 expression as a compensation for the absence of Dst-A1 and -A2 in Dstdt-Tg4 sensory neurons. Indeed, knockdown of Dst-A3 in these neurons resulted in a decrease in tubulin acetylation. These findings shed light on the possible compensatory role of dystonin isoforms within HSAN-VI, which might explain the heterogeneity in symptoms within the reported forms of the disease.