Heterogeneous Nuclear Ribonuclear Protein Research Articles

Role of Heterogeneous Nuclear Ribonucleoproteins in the Cancer-Immune Landscape

Cancer remains the second leading cause of death, accounting for approximately 20% of all fatalities. Evolving cancer cells and a dysregulated immune system create complex tumor environments that fuel tumor growth, metastasis, and resistance. Over the past decades, significant progress in deciphering cancer cell behavior and recognizing the immune system as a hallmark of tumorigenesis has been achieved. However, the underlying mechanisms controlling the evolving cancer-immune landscape remain mostly unexplored. Heterogeneous nuclear ribonuclear proteins (hnRNP), a highly conserved family of RNA-binding proteins, have vital roles in critical cellular processes, including transcription, post-transcriptional modifications, and translation. Dysregulation of hnRNP is a critical contributor to cancer development and resistance. HnRNP contribute to the diversity of tumor and immune-associated aberrant proteomes by controlling alternative splicing and translation. They can also promote cancer-associated gene expression by regulating transcription factors, binding to DNA directly, or promoting chromatin remodeling. HnRNP are emerging as newly recognized mRNA readers. Here, we review the roles of hnRNP as regulators of the cancer-immune landscape. Dissecting the molecular functions of hnRNP will provide a better understanding of cancer-immune biology and will impact the development of new approaches to control and treat cancer.

Long Noncoding RNA lncRHL Regulates Hepatic VLDL Secretion by Modulating hnRNPU/BMAL1/MTTP Axis.

Dysregulation of hepatic VLDL secretion contributes to the pathogenesis of metabolic diseases, such as nonalcoholic fatty liver disease (NAFLD) and hyperlipidemia. Accumulating evidence has suggested that long noncoding RNAs (lncRNAs) had malfunctioning roles in the pathogenesis of NAFLD. However, the function of lncRNAs in controlling hepatic VLDL secretion remains largely unillustrated. Here, we identified a novel lncRNA, lncRNA regulator of hyperlipidemia (lncRHL), which was liver-enriched, downregulated on high-fat diet feeding, and inhibited by oleic acid treatment in primary hepatocytes. With genetic manipulation in mice and primary hepatocytes, depletion of lncRHL induces hepatic VLDL secretion accompanied by decreased hepatic lipid contents. Conversely, lncRHL restoration reduces VLDL secretion with increased lipid deposition in hepatocytes. Mechanistic analyses indicate that lncRHL binds directly to heterogeneous nuclear ribonuclear protein U (hnRNPU), and thereby enhances its stability, and that hnRNPU can transcriptional activate Bmal1, leading to inhibition of VLDL secretion in hepatocytes. lncRHL deficiency accelerates the protein degradation of hnRNPU and suppresses the transcription of Bmal1, which in turn activates VLDL secretion in hepatocytes. With results taken together, we conclude that lncRHL is a novel suppressor of hepatic VLDL secretion. Activating the lncRHL/hnRNPU/BMAL1/MTTP axis represents a potential strategy for the maintenance of intrahepatic and plasma lipid homeostasis.

A viral interferon regulatory factor degrades RNA-binding protein hnRNP Q1 to enhance aerobic glycolysis via recruiting E3 ubiquitin ligase KLHL3 and decaying GDPD1 mRNA.

Reprogramming of host metabolism is a common strategy of viral evasion of host cells, and is essential for successful viral infection and induction of cancer in the context cancer viruses. Kaposi's sarcoma (KS) is the most common AIDS-associated cancer caused by KS-associated herpesvirus (KSHV) infection. KSHV-encoded viral interferon regulatory factor 1 (vIRF1) regulates multiple signaling pathways and plays an important role in KSHV infection and oncogenesis. However, the role of vIRF1 in KSHV-induced metabolic reprogramming remains elusive. Here we show that vIRF1 increases glucose uptake, ATP production and lactate secretion by downregulating heterogeneous nuclear ribonuclear protein Q1 (hnRNP Q1). Mechanistically, vIRF1 upregulates and recruits E3 ubiquitin ligase Kelch-like 3 (KLHL3) to degrade hnRNP Q1 through a ubiquitin-proteasome pathway. Furthermore, hnRNP Q1 binds to and stabilizes the mRNA of glycerophosphodiester phosphodiesterase domain containing 1 (GDPD1). However, vIRF1 targets hnRNP Q1 for degradation, which destabilizes GDPD1 mRNA, resulting in induction of aerobic glycolysis. These results reveal a novel role of vIRF1 in KSHV metabolic reprogramming, and identifying a potential therapeutic target for KSHV infection and KSHV-induced cancers.

Interconnected Gene Networks Underpin the Clinical Overlap of HNRNPH1-Related and Rubinstein-Taybi Intellectual Disability Syndromes.

Interconnected Gene Networks Underpin the Clinical Overlap of HNRNPH1-Related and Rubinstein-Taybi Intellectual Disability Syndromes.

Glioma pathogenesis-related protein 1 performs dual functions in tumor cells.

Glioma pathogenesis-related protein 1 (GLIPR1) was identified as an oncoprotein in some cancer types including gliomas, breast cancers, melanoma cancers, and Wilms tumors, but as a tumor suppressor in some other types of cancers, such as prostate cancers, lung cancers, bladder cancers, and thyroid cancers. In gliomas, GLIPR1 promotes the migration and invasion of glioma cells by interaction with the actin polymerization regulator Neural Wiskott-Aldrich syndrome protein (N-WASP) and then abolishes the negative effects of Heterogeneous nuclear ribonuclear protein K (hnRNPK). In prostate cancers, high levels of GLIPR1 induce apoptosis and destruction of oncoproteins. In lung cancers, overexpression of GLIPR1 inhibits the growth of lung cancer cells partially through inhibiting the V-ErbB avian erythroblastic leukemia viral oncogene homolog3 (ErbB3) pathway. However, the mechanisms that GLIPR1 performs its function in other tumors still remain unclear. The tumor suppressing role of GLIPR1 has been explored to the cancer treatment. The adenoviral vector-mediated Glipr1 (AdGlipr1) gene therapy and the GLIPR1-transmembrane domain deleted (GLIPR1-ΔTM) protein therapy both showed antitumor activities and stimulated immune response in prostate cancers. Whether GLPIR1 can be used to treat other tumors is an important topic to be explored. Among which, whether GLPIR1 can be used to treat lung cancer by atomizing inhalation is the key topic we care about. If it does, this therapy has a wide application prospect and is a great progression in lung cancer treatment.

A TNFR2-hnRNPK Axis Promotes Primary Liver Cancer Development via Activation of YAP Signaling in Hepatic Progenitor Cells.

Most primary liver cancer (PLC) cases progress mainly due to underlying chronic liver inflammation, yet the underlying mechanisms of inflammation-mediated PLC remain unclear. Here we uncover a TNF receptor II (TNFR2)-hnRNPK-YAP signaling axis in hepatic progenitor cells (HPC) essential for PLC development. TNFR2, but not TNF receptor I (TNFR1), was required for TNFα-induced activation of YAP during malignant transformation of HPCs and liver tumorigenesis. Mechanistically, heterogeneous nuclear ribonuclear protein K (hnRNPK) acted downstream of TNFα-TNFR2 signaling to directly interact with and stabilize YAP on target gene promoters genome-wide, therefore coregulating the expression of YAP target genes. Single-cell RNA sequencing confirmed the association of TNFR2-hnRNPK with YAP expression and the pathologic importance of HPC. Accordingly, expressions of TNFR2, hnRNPK, and YAP were all upregulated in PLC tissues and were strongly associated with poor prognosis of PLC including patient survival. Collectively, this study clarifies the differential roles of TNFRs in HPC-mediated tumorigenesis, uncovering a TNFR2-hnRNPK-centered mechanistic link between the TNFα-mediated inflammatory milieu and YAP activation in HPCs during PLC development. SIGNIFICANCE: This work defines how hnRNPK links TNFα signaling and Hippo pathway transcription coactivator YAP in hepatic progenitor cells during primary liver tumorigenesis.

Behavioural phenotyping of thunder mice with a hypomorphic mutation of heterogeneous nuclear ribonuclear protein L-like (hnRNPLL) and reduced T cell function

Heterogeneous nuclear ribonuclear protein l-like (hnRNPLL) is an RNA binding protein that regulates alternative splicing of mRNA and is abundantly expressed in memory T lymphocytes of the immune system and in the brain. A hypomorphic allele of the gene encoding hnRNPLL (Hnrpllthunder) selectively reduces T cell accumulation in lymphoid tissues, but little is known about its effects in the brain. Therefore, we exposed Hnrpllthunder mice to a test battery with relevance for a range of psychiatric illnesses. Thunder mice showed enhanced immobility in the tail-suspension test for depression-related behaviours, impaired short-term spatial memory in the Y-maze and reduced avoidance learning in the active avoidance test. Thus, in addition to its reported effects on immune function, the hnRNPLL mutation in thunder mice selectively affected aspects of behaviour.

FUS Recognizes G Quadruplex Structures Within Neuronal mRNAs.

Fused in sarcoma (FUS), identified as the heterogeneous nuclear ribonuclear protein P2, is expressed in neuronal and non-neuronal tissue, and among other functions, has been implicated in messenger RNA (mRNA) transport and possibly local translation regulation. Although FUS is mainly localized to the nucleus, in the neurons FUS has also been shown to localize to the post-synaptic density, as well as to the pre-synapse. Additionally, the FUS deletion in cultured hippocampal cells results in abnormal spine and dendrite morphology. Thus, FUS may play a role in synaptic function regulation, mRNA localization, and local translation. Many dendritic mRNAs have been shown to form G quadruplex structures in their 3′-untranslated region (3′-UTR). Since FUS contains three arginine-glycine-glycine (RGG) boxes, an RNA binding domain shown to bind with high affinity and specificity to RNA G quadruplex structures, in this study we hypothesized that FUS recognizes these structural elements in its neuronal mRNA targets. Two neuronal mRNAs found in the pre- and post-synapse are the post-synaptic density protein 95 (PSD-95) and Shank1 mRNAs, which encode for proteins involved in synaptic plasticity, maintenance, and function. These mRNAs have been shown to form 3′-UTR G quadruplex structures and were also enriched in FUS hydrogels. In this study, we used native gel electrophoresis and steady-state fluorescence spectroscopy to demonstrate specific nanomolar binding of the FUS C-terminal RGG box and of full-length FUS to the RNA G quadruplex structures formed in the 3′-UTR of PSD-95 and Shank1a mRNAs. These results point toward a novel mechanism by which FUS targets neuronal mRNA and given that these PSD-95 and Shank1 3′-UTR G quadruplex structures are also targeted by the fragile X mental retardation protein (FMRP), they raise the possibility that FUS and FMRP might work together to regulate the translation of these neuronal mRNA targets.

Dysfunctional RNA binding proteins and stress granules in multiple sclerosis

Dysfunction of the RNA binding protein (RBP) heterogeneous nuclear ribonuclear protein A1 (hnRNP A1) has been shown to contribute to the pathogenesis of neurodegenerative diseases, but its involvement in multiple sclerosis (MS) is largely unknown. In a neuronal cell line, interferon-γ caused hnRNP A1 nucleocytoplasmic mislocalization; colocalization of hnRNP A1 in stress granules (SGs); and inhibition of translation. Neurons in the brain of a MS patient showed pathogenic RBP dysfunction, including nuclear depletion of hnRNP A1, its mislocalization to the cytoplasm, and its colocalization in SGs. These data indicate a role for dysfunctional hnRNP A1 in the pathogenesis of MS.

Autoantibodies to heterogeneous nuclear ribonuclear protein A1 (hnRNPA1) cause altered ‘ribostasis’ and neurodegeneration; the legacy of HAM/TSP as a model of progressive multiple sclerosis

Several years following its discovery in 1980, infection with human T-lymphotropic virus type 1 (HTLV-1) was shown to cause HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease biologically similar to progressive forms of multiple sclerosis (MS). In this manuscript, we review some of the clinical, pathological, and immunological similarities between HAM/TSP and MS with an emphasis on how autoantibodies to an RNA binding protein, heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), might contribute to neurodegeneration in immune mediated diseases of the central nervous system.

Extensive cross-regulation of post-transcriptional regulatory networks in Drosophila.

In eukaryotic cells, RNAs exist as ribonucleoprotein particles (RNPs). Despite the importance of these complexes in many biological processes, including splicing, polyadenylation, stability, transportation, localization, and translation, their compositions are largely unknown. We affinity-purified 20 distinct RNA-binding proteins (RBPs) from cultured Drosophila melanogaster cells under native conditions and identified both the RNA and protein compositions of these RNP complexes. We identified “high occupancy target” (HOT) RNAs that interact with the majority of the RBPs we surveyed. HOT RNAs encode components of the nonsense-mediated decay and splicing machinery, as well as RNA-binding and translation initiation proteins. The RNP complexes contain proteins and mRNAs involved in RNA binding and post-transcriptional regulation. Genes with the capacity to produce hundreds of mRNA isoforms, ultracomplex genes, interact extensively with heterogeneous nuclear ribonuclear proteins (hnRNPs). Our data are consistent with a model in which subsets of RNPs include mRNA and protein products from the same gene, indicating the widespread existence of auto-regulatory RNPs. From the simultaneous acquisition and integrative analysis of protein and RNA constituents of RNPs, we identify extensive cross-regulatory and hierarchical interactions in post-transcriptional control.

Identification of Salmonella Typhimurium Deubiquitinase SseL Substrates by Immunoaffinity Enrichment and Quantitative Proteomic Analysis.

Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here, we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification, and global quantitative proteomic analysis. As a model system to identify substrates, we used a virulence-related deubiquitinase, SseL, secreted by Salmonella enterica serovar Typhimurium into host cells. Using this approach, two SseL substrates were identified in the RAW 264.7 murine macrophage-like cell line, S100A6 and heterogeneous nuclear ribonuclear protein K, in addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. This method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.

Novel somatic single nucleotide variants within the RNA binding protein hnRNP A1 in multiple sclerosis patients.

Some somatic single nucleotide variants (SNVs) are thought to be pathogenic, leading to neurological disease. We hypothesized that heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), an autoantigen associated with multiple sclerosis (MS) would contain SNVs. MS patients develop antibodies to hnRNP A1 (293-304), an epitope within the M9 domain (AA (268-305)) of hnRNP A1. M9 is hnRNP A1's nucleocytoplasmic transport domain, which binds transportin-1 (TPNO-1) and allows for hnRNP A1's transport into and out of the nucleus. Genomic DNA sequencing of M9 revealed nine novel SNVs that resulted in an amino acid substitution in MS patients that were not present in controls. SNVs occurred within the TPNO-1 binding domain (hnRNP A1 (268-289)) and the MS IgG epitope (hnRNP A1 (293-304)), within M9. In contrast to the nuclear localization of wild type (WT) hnRNP A1, mutant hnRNP A1 mis-localized to the cytoplasm, co-localized with stress granules and caused cellular apoptosis. Whilst WT hnRNP A1 bound TPNO-1, mutant hnRNP A1 showed reduced TPNO-1 binding. These data suggest SNVs in hnRNP A1 might contribute to pathogenesis of MS.

Novel somatic single nucleotide variants within the RNA binding protein hnRNP A1 in multiple sclerosis patients

Some somatic single nucleotide variants (SNVs) are thought to be pathogenic, leading to neurological disease. We hypothesized that heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), an autoantigen associated with multiple sclerosis (MS) would contain SNVs. MS patients develop antibodies to hnRNP A1 293-304, an epitope within the M9 domain (AA 268-305) of hnRNP A1. M9 is hnRNP A1’s nucleocytoplasmic transport domain, which binds transportin-1 (TPNO-1) and allows for hnRNP A1’s transport into and out of the nucleus. Genomic DNA sequencing of M9 revealed nine novel SNVs that resulted in an amino acid substitution in MS patients that were not present in controls. SNVs occurred within the TPNO-1 binding domain (hnRNP A1 268-289) and the MS IgG epitope (hnRNP A1 293-304), within M9. In contrast to the nuclear localization of wild type (WT) hnRNP A1, mutant hnRNP A1 mis-localized to the cytoplasm, co-localized with stress granules and caused cellular apoptosis. Whilst WT hnRNP A1 bound TPNO-1, mutant hnRNP A1 showed reduced TPNO-1 binding. These data suggest SNVs in hnRNP A1 might contribute to pathogenesis of MS.

Novel somatic single nucleotide variants within the RNA binding protein hnRNP A1 in multiple sclerosis patients

Some somatic single nucleotide variants (SNVs) are thought to be pathogenic, leading to neurological disease. We hypothesized that heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), an autoantigen associated with multiple sclerosis (MS) would contain SNVs. MS patients develop antibodies to hnRNP A1 (293-304), an epitope within the M9 domain (AA (268-305)) of hnRNP A1. M9 is hnRNP A1's nucleocytoplasmic transport domain, which binds transportin-1 (TPNO-1) and allows for hnRNP A1's transport into and out of the nucleus. Genomic DNA sequencing of M9 revealed nine novel SNVs that resulted in an amino acid substitution in MS patients that were not present in controls. SNVs occurred within the TPNO-1 binding domain (hnRNP A1 (268-289)) and the MS IgG epitope (hnRNP A1 (293-304)), within M9. In contrast to the nuclear localization of wild type (WT) hnRNP A1, mutant hnRNP A1 mis-localized to the cytoplasm, co-localized with stress granules and caused cellular apoptosis. Whilst WT hnRNP A1 bound TPNO-1, mutant hnRNP A1 showed reduced TPNO-1 binding. These data suggest SNVs in hnRNP A1 might contribute to pathogenesis of MS.

Potential Role of Bacterial Infection in Autoimmune Diseases: A New Aspect of Molecular Mimicry

Molecular mimicry is an attractive mechanism for triggering autoimmunity. In this review, we explore the potential role of evolutionary conserved bacterial proteins in the production of autoantibodies with focus on granulomatosis with polyangiitis (GPA) and rheumatoid arthritis (RA). Seven autoantigens characterized in GPA and RA were BLASTed against a bacterial protein database. Of the seven autoantigens, proteinase 3, type II collagen, binding immunoglobulin protein, glucose-6-phosphate isomerase, α-enolase, and heterogeneous nuclear ribonuclear protein have well-conserved bacterial orthologs. Importantly, those bacterial orthologs are also found in human-associated bacteria. The wide distribution of the highly conserved stress proteins or enzymes among the members of the normal flora and common infectious microorganisms raises a new question on how cross-reactive autoantibodies are not produced during the immune response to these bacteria in most healthy people. Understanding the mechanisms that deselect auto-reactive B cell clones during the germinal center reaction to homologous foreign antigens may provide a novel strategy to treat autoimmune diseases.

A Human Proteome Microarray Identifies that the Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) Recognizes the 5′ Terminal Sequence of the Hepatitis C Virus RNA

Stem-loop I (SL1) located in the 5' untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.

Global Profiling of Alternative Splicing Events and Gene Expression Regulated by hnRNPH/F

In this study, we have investigated the global impact of heterogeneous nuclear Ribonuclear Protein (hnRNP) H/F-mediated regulation of splicing events and gene expression in oligodendrocytes. We have performed a genome-wide transcriptomic analysis at the gene and exon levels in Oli-neu cells treated with siRNA that targets hnRNPH/F compared to untreated cells using Affymetrix Exon Array. Gene expression levels and regulated exons were identified with the GenoSplice EASANA algorithm. Bioinformatics analyses were performed to determine the structural properties of G tracts that correlate with the function of hnRNPH/F as enhancers vs. repressors of exon inclusion. Different types of alternatively spliced events are regulated by hnRNPH/F. Intronic G tracts density, length and proximity to the 5′ splice site correlate with the hnRNPH/F enhancer function. Additionally, 6% of genes are differently expressed upon knock down of hnRNPH/F. Genes that regulate the transition of oligodendrocyte progenitor cells to oligodendrocytes are differentially expressed in hnRNPH/F depleted Oli-neu cells, resulting in a decrease of negative regulators and an increase of differentiation-inducing regulators. The changes were confirmed in developing oligodendrocytes in vivo. This is the first genome wide analysis of splicing events and gene expression regulated by hnRNPH/F in oligodendrocytes and the first report that hnRNPH/F regulate genes that are involved in the transition from oligodendrocyte progenitor cells to oligodendrocytes.

Dephosphorylation of HuR Protein during Alphavirus Infection Is Associated with HuR Relocalization to the Cytoplasm

We have demonstrated previously that the cellular HuR protein binds U-rich elements in the 3' untranslated region (UTR) of Sindbis virus RNA and relocalizes from the nucleus to the cytoplasm upon Sindbis virus infection in 293T cells. In this study, we show that two alphaviruses, Ross River virus and Chikungunya virus, lack the conserved high-affinity U-rich HuR binding element in their 3' UTRs but still maintain the ability to interact with HuR with nanomolar affinities through alternative binding elements. The relocalization of HuR protein occurs during Sindbis infection of multiple mammalian cell types as well as during infections with three other alphaviruses. Interestingly, the relocalization of HuR is not a general cellular reaction to viral infection, as HuR protein remained largely nuclear during infections with dengue and measles virus. Relocalization of HuR in a Sindbis infection required viral gene expression, was independent of the presence of a high-affinity U-rich HuR binding site in the 3' UTR of the virus, and was associated with an alteration in the phosphorylation state of HuR. Sindbis virus-induced HuR relocalization was mechanistically distinct from the movement of HuR observed during a cellular stress response, as there was no accumulation of caspase-mediated HuR cleavage products. Collectively, these data indicate that virus-induced HuR relocalization to the cytoplasm is specific to alphavirus infections and is associated with distinct posttranslational modifications of this RNA-binding protein.

The lectin Helix pomatia agglutinin recognizes O-GlcNAc containing glycoproteins in human breast cancer

There has been considerable interest in understanding the epitopes that bind the lectin Helix pomatia agglutinin (HPA) in breast cancer as the lectin has been shown to identify glycosylation changes associated with the development of metastatic disease. HPA has previously been shown to recognize aberrant O-linked α-N-acetylgalactosamine (GalNAcα)/mucin glycosylation in cancer, including exposed Tn epitopes. However, recent glycan-array analysis reported that diverse epitopes are also recognized by the lectin, e.g. consortium for functional glycomics (CFG) data: GalNAcα1,3Gal; β-GalNAc; GlcNAcβ1,4Gal. The intriguing observations from the CFG array led to this study, in which HPA-binding epitopes were localized and characterized in an in vitro model of breast cancer metastasis. HMT3522 (benign disease), BT474 (primary cancer) and T47D/MCF7 (metastatic cancer) cells were assessed in confocal microscopy-based co-localization studies and a glycoproteomic analysis based on 2-dimensional electrophoresis (2DE), western blotting and mass spectrometry was adopted. HPA binding correlated with levels of integrin α6, transcription factors heterogeneous nuclear ribonuclear protein (HnRNP) H1, HnRNP D-like, HnRNP A2/B1 as well as heat shock protein 27 (Hsp27), glial fibrillary acidic protein and enolase 1 (ENO1). These glycoproteins were non-detectable in the non-metastatic breast cancer cell lines. The recognition of HnRNPs, Hsp27 and ENO1 by HPA correlated with O-GlcNAcylation of these proteins. Integrin α6 was the most abundant HPA glycoprotein in the breast cancer cells with a metastatic phenotype; this concurred with previous findings in colorectal cancer. This is the first report in which HPA has been shown to bind O-GlcNAcylated transcription factors. This class of proteins represents a new means by which HPA differentiates cancer cells with an aggressive metastatic phenotype.