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Background: Diffuse large B-cell lymphoma (DLBCL) is the main pathological type of non-Hodgkin lymphoma (NHL). Chromobox (CBX) family proteins are classical components of polycomb group (PcG) complexes in many cancer types, resulting in accelerated carcinogenesis. Nevertheless, the prognostic, functional and expression significance of these CBX family members in DLBCL remain unclear and elusive., Methods: CBX transcriptional levels were confirmed using Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA) and Cancer Cell Line Encyclopedia (CCLE) databases. The protein levels of CBX family members were analysed using The Human Protein Atlas (HPA) database. Information on the PPI network, functional enrichment, drug sensitivity, prognostic value, miRNA network, protein structure, genetic alteration and immune cell infiltration were generated using the GeneMANIA, Metascape, GSCALite, GEPIA, PDB, cBioPortal, and TIMER databases, and the correlation of these factors with CBX expression levels in DLBCL was assessed., Results: CBX1/2/3/5/6/8 mRNA levels were significantly enhanced in DLBCL tissues compared to corresponding normal tissues. CBX1/3/4/5/8 protein expression levels were obviously increased, whereas CBX7 was obviously decreased. This difference might be attributed to miRNA regulation based on the miRNA network. Overall survival (OS) analysis showed that CBX levels were not correlated with prognosis in DLBCL patients, indicating that CBXs are not good biomarkers for DLBCL patients. Furthermore, functional enrichment analyses indicated that CBXs were closely related to DNA duplex unwinding, covalent chromatin modification, and histone lysine methylation. The levels of CBXs were also significantly associated with diverse immune cell infiltration in DLBCL., Conclusions: This study reveals that dysregulated CBXs are involved in DLBCL development and might represent potential therapeutic targets for DLBCL., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The chromobox (CBX) proteins CBX2, CBX4, CBX6, CBX7, and CBX8, also known as Polycomb (Pc) proteins, are canonical components of the Polycomb repressive complex 1 (PRC1). Abundant evidence indicates that abnormal expression of Pc proteins is associated with a variety of tumors, but their role in the pathogenesis of hepatocellular carcinoma (HCC) has not been fully elucidated. In the present study, we performed a case-control study to investigate the relationship between single nucleotide polymorphisms (SNPs) of CBX genes and HCC., Methods: Nine SNPs on CBX genes (rs7217395, rs2036316 of CBX2 ; rs3764374, rs1285251, rs2289728 of CBX4 ; rs7292074 of CBX6 ; and rs710190, rs139394, rs5750753 of CBX7 ) were screened and genotyped using MassARRAY technology in 334 HCC cases and 321 controls. The association between SNPs and their corresponding gene expressions was analyzed through bioinformatics methods using the Ensembl database and Blood eQTL browser online tools., Results: The results indicated that rs2289728 (G>A) of CBX4 ( P = 0.03, OR = 0.56, 95% CI: 0.33-0.94) and rs139394 (C>A) of CBX7 ( P = 0.02, OR = 0.55, 95% CI: 0.33-0.90) decreased the risk of HCC. Interaction between rs2036316 and HBsAg increased the risk of HCC ( P = 0.02, OR = 6.88, 95% CI: 5.20-9.11), whereas SNP-SNP interaction between rs710190 and rs139394 reduced the risk of HCC ( P = 0.03, OR = 0.33, 95% CI: 0.12-0.91). Gene expression analyses showed that the rs2289728 A allele and the rs139394 A allele significantly reduced CBX4 and CBX7 expression, respectively., Conclusion: Our findings suggest that CBX4 rs2289728 and CBX7 rs139394 are protective SNPs against HCC. The two SNPs may reduce the risk of HCC while suppressing the expression of CBX4 and CBX7 .

Aberrant chromobox (CBX) family protein expression has been reported in a variety of human malignancies. However, the role of CBX6 in hepatocellular carcinoma (HCC) progression and patient prognosis remains unknown. In this study, we found that CBX6 was frequently up-regulated in HCC clinical samples and HCC cell lines and that CBX6 expression was significantly correlated with larger tumor sizes (≥ 5 cm, p = 0.011) and multiple tumors (n ≥ 2, p = 0.018). Survival analyses indicated that patients with higher CBX6 expression levels had significantly shorter recurrence-free survival (RFS) and overall survival (OS) than patients with lower CBX6 expression levels, and multivariate analyses confirmed that increased CBX6 expression was an independent unfavorable prognostic factor for HCC patients. Functional study demonstrated that CBX6 profoundly promoted HCC cell growth both in vitro and in vivo, and mechanistic investigation revealed that the S100A9/NF-κB/MAPK pathway was essential for mediating CBX6 function. In conclusion, our results represent the first evidence that CBX6 contributes to tumor progression and indicate that the protein may serve as a novel prognostic biomarker for HCC and as a therapeutic target in the treatment of the disease.

Background: Variation in cancer risk among somatic tissues has been attributed to variations in the underlying rate of stem cell division. For a given tissue type, variable cancer risk between individuals is thought to be influenced by extrinsic factors which modulate this rate of stem cell division. To date, no molecular mitotic clock has been developed to approximate the number of stem cell divisions in a tissue of an individual and which is correlated with cancer risk., Results: Here, we integrate mathematical modeling with prior biological knowledge to construct a DNA methylation-based age-correlative model which approximates a mitotic clock in both normal and cancer tissue. By focusing on promoter CpG sites that localize to Polycomb group target genes that are unmethylated in 11 different fetal tissue types, we show that increases in DNA methylation at these sites defines a tick rate which correlates with the estimated rate of stem cell division in normal tissues. Using matched DNA methylation and RNA-seq data, we further show that it correlates with an expression-based mitotic index in cancer tissue. We demonstrate that this mitotic-like clock is universally accelerated in cancer, including pre-cancerous lesions, and that it is also accelerated in normal epithelial cells exposed to a major carcinogen., Conclusions: Unlike other epigenetic and mutational clocks or the telomere clock, the epigenetic clock proposed here provides a concrete example of a mitotic-like clock which is universally accelerated in cancer and precancerous lesions.

To maintain a particular cell fate, a unique set of genes should be expressed while another set is repressed. One way to repress gene expression is through Polycomb group (PcG) proteins that compact chromatin into a silent configuration. In addition to cell fate maintenance, PcG proteins also maintain normal cell physiology, for example cell cycle. In the absence of PcG, ectopic activation of the PcG-repressed genes leads to developmental defects and malignant tumors. Little is known about the molecular nature of ectopic gene expression; especially what differentiates expression of a given gene in the orthotopic tissue (orthotopic expression) and the ectopic expression of the same gene due to PcG mutations. Here we present that ectopic gene expression in PcG mutant cells specifically requires dBRWD3, a negative regulator of HIRA/Yemanuclein (YEM)-mediated histone variant H3.3 deposition. dBRWD3 mutations suppress both the ectopic gene expression and aberrant tissue overgrowth in PcG mutants through a YEM-dependent mechanism. Our findings identified dBRWD3 as a critical regulator that is uniquely required for ectopic gene expression and aberrant tissue overgrowth caused by PcG mutations., Competing Interests: The authors have declared that no competing interests exist.

Polycomb group response elements (PREs) in Drosophila are DNA-elements that recruit Polycomb proteins (PcG) to chromatin and regulate gene expression. PREs are easily recognizable in the Drosophila genome as strong peaks of PcG-protein binding over discrete DNA fragments; many small but statistically significant PcG peaks are also observed in PcG domains. Surprisingly, in vivo deletion of the four characterized strong PREs from the PcG regulated invected-engrailed (inv-en) gene complex did not disrupt the formation of the H3K27me3 domain and did not affect inv-en expression in embryos or larvae suggesting the presence of redundant PcG recruitment mechanism. Further, the 3D-structure of the inv-en domain was only minimally altered by the deletion of the strong PREs. A reporter construct containing a 7.5kb en fragment that contains three weak peaks but no large PcG peaks forms an H3K27me3 domain and is PcG-regulated. Our data suggests a model for the recruitment of PcG-complexes to Drosophila genes via interactions with multiple, weak PREs spread throughout an H3K27me3 domain.

Unlabelled: ANRIL, a long noncoding RNA (lncRNA), has recently been reported to have a direct role in recruiting polycomb repressive complexes PRC2 and PRC1 to regulate the expression of the p15/CDKN2B-p16/CDKN2A-p14/ARF gene cluster. Expression analysis of ANRIL, EZH2, SUZ12, EED, JARID2, CBX7, BMI1, p16, p15, and p14/ARF genes was evaluated in a large cohort of invasive breast carcinomas (IBC, n = 456) by qRT-PCR and immunohistochemistry (IHC) was performed on CBX7, EZH2, p14, p15, p16, H3K27me3, and H3K27ac. We observed significant overexpression in IBCs of ANRIL (19.7%) and EZH2 (77.0%) and an underexpression of CBX7 (39.7%). Correlations were identified between these genes, their expression patterns, and several classical clinical and pathologic parameters, molecular subtypes, and patient outcomes, as well as with proliferation, epithelial-mesenchymal transition, and breast cancer stem cell markers. Multivariate analysis revealed that combined EZH2/CBX7 status is an independent prognostic factor (P = 0.001). In addition, several miRNAs negatively associated with CBX7 underexpression and EZH2 overexpression. These data demonstrate a complex pattern of interactions between lncRNA ANRIL, several miRNAs, PRC2/PRC1 subunits, and p15/CDKN2B-p16/CDKN2A-p14/ARF locus and suggest that their expression should be considered together to evaluate antitumoral drugs, in particular the BET bromodomain inhibitors., Implications: This study suggests that the global pattern of expression rather than expression of individual family members should be taken into account when defining functionality of repressive Polycomb complexes and therapeutic targeting potential. Mol Cancer Res; 14(7); 623-33. ©2016 AACR., (©2016 American Association for Cancer Research.)

Polycomb group (PcG) proteins belong to a family of epigenetic modifiers and play a key role in dynamic control of their target genes. Several reports have found that aberrations in PcG-microRNA (miRNA) interplay in various cancer types often associated with poor clinical prognosis. This review discusses important PcG-miRNA molecular networks which act as critical interfaces between chromatin remodeling, and transcriptional and post-transcriptional regulation of their target genes in cancer. Moreover, here are discussed several compounds influencing the activity of PcG proteins entered in clinical arena for the treatment of solid tumors, multiple myeloma and B lymphomas, thus highlighting the therapeutic potential of targeting this protein family., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The Drosophila melanogaster Piwi protein regulates both niche and intrinsic mechanisms to maintain germline stem cells, but its underlying mechanism remains unclear. Here we report that Piwi interacts with Polycomb group complexes PRC1 and PRC2 in niche and germline cells to regulate ovarian germline stem cells and oogenesis. Piwi physically interacts with the PRC2 subunits Su(z)12 and Esc in the ovary and in vitro. Chromatin coimmunoprecipitation of Piwi, the PRC2 enzymatic subunit E(z), histone H3 trimethylated at lysine 27 (H3K27me3) and RNA polymerase II in wild-type and piwi mutant ovaries demonstrates that Piwi binds a conserved DNA motif at ∼ 72 genomic sites and inhibits PRC2 binding to many non-Piwi-binding genomic targets and H3K27 trimethylation. Moreover, Piwi influences RNA polymerase II activities in Drosophila ovaries, likely via inhibiting PRC2. We hypothesize that Piwi negatively regulates PRC2 binding by sequestering PRC2 in the nucleoplasm, thus reducing PRC2 binding to many targets and influencing transcription during oogenesis.

Polycomb repressive complexes (PRCs) play crucial roles in transcriptional repression and developmental regulation in both plants and animals. In plants, depletion of different members of PRCs causes both overlapping and unique phenotypic defects. However, the underlying molecular mechanism determining the target specificity and functional diversity is not sufficiently characterized. Here, we quantitatively compared changes of tri-methylation at H3K27 in Arabidopsis mutants deprived of various key PRC components. We show that CURLY LEAF (CLF), a major catalytic subunit of PRC2, coordinates with different members of PRC1 in suppression of distinct plant developmental programs. We found that expression of flower development genes is repressed in seedlings preferentially via non-redundant role of CLF, which specifically associated with LIKE HETEROCHROMATIN PROTEIN1 (LHP1). In contrast, expression of embryo development genes is repressed by PRC1-catalytic core subunits AtBMI1 and AtRING1 in common with PRC2-catalytic enzymes CLF or SWINGER (SWN). This context-dependent role of CLF corresponds well with the change in H3K27me3 profiles, and is remarkably associated with differential co-occupancy of binding motifs of transcription factors (TFs), including MADS box and ABA-related factors. We propose that different combinations of PRC members distinctively regulate different developmental programs, and their target specificity is modulated by specific TFs.

The plant homeobox domain (PHD) proteins are widespread in eukaryotes, and play important roles in regulating chromatin and transcription. Comprehensive analyses of PHD-finger proteins have been performed in animals, but few plant PHD-finger proteins involved in growth and development have been characterized functionally. In this study, we conducted a genome-wide survey of PHD-finger proteins in Populus trichocarpa by describing the phylogenetic relationship, gene structure, and chromosomal location and microarray analyses of each predicted PHD-finger family member. We identified 73 PHD-finger genes (PtPHD1-73) and classified them into eleven subfamilies (A-K) by phylogenetic analysis. Seventy-two of the 73 genes were unevenly distributed on all 19 chromosomes, with seven segmental duplication events. Analysis of the Ka (non-synonymous substitution rate)/Ks (synonymous substitution rate) ratios suggested that the duplicated genes of the PHD-finger family mainly underwent purifying selection with restrictive functional divergence after the duplication events. Expression profiles analysis indicated that 67 PHD-finger genes were differentially expressed in various tissues. Quantitative real-time RT-PCR (qRT-PCR) analyses of nine selected PtPHD genes under high salinity, drought and cold stresses were also performed to explore their stress-related expression patterns. The results of this study provide a thorough overview of poplar PHD-finger proteins and will be valuable for further functional research of poplar PHD-finger genes to unravel their biological roles., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: SIRT1 is likely to play a role in the extension in healthspan induced by dietary restriction. Actions of SIRT1 are pleiotropic, and effects on healthspan may include effects on DNA methylation. Polycomb group protein target genes (PCGTs) are suppressed by epigenetic mechanisms in stem cells, partly through the actions of the polycomb repressive complexes (PRCs), and have been shown previously to correspond with loci particularly susceptible to age-related changes in DNA methylation. We hypothesised that SIRT1 would affect DNA methylation particularly at PCGTs. To map the sites in the genome where SIRT1 affects DNA methylation, we altered SIRT1 expression in human intestinal (Caco-2) and vascular endothelial (HuVEC) cells by transient transfection with an expression construct or with siRNA. DNA was enriched for the methylated fraction then sequenced (HuVEC) or hybridised to a human promoter microarray (Caco-2)., Results: The profile of genes where SIRT1 manipulation affected DNA methylation was enriched for PCGTs in both cell lines, thus supporting our hypothesis. SIRT1 knockdown affected the mRNA for none of seven PRC components nor for DNMT1 or DNMT3b. We thus find no evidence that SIRT1 affects DNA methylation at PCGTs by affecting the expression of these gene transcripts. EZH2, a component of PRC2 that can affect DNA methylation through association with DNA methyltransferases (DNMTs), did not co-immunoprecipitate with SIRT1, and SIRT1 knockdown did not affect the expression of EZH2 protein. Thus, it is unlikely that the effects of SIRT1 on DNA methylation at PCGTs are mediated through direct intermolecular association with EZH2 or through effects in its expression., Conclusions: SIRT1 affects DNA methylation across the genome, but particularly at PCGTs. Although the mechanism through which SIRT1 has these effects is yet to be uncovered, this action is likely to contribute to extended healthspan, for example under conditions of dietary restriction.

Our recent investigations showed that polycomb chromobox 4 (Cbx4) promotes angiogenesis and metastasis of hepatocellular carcinoma (HCC) through its sumoylating action on hypoxia-inducible factor-1α protein. Here, we attempt to identify the prognostic significances of Cbx4 by a retrospective analyses in 727 cases of HCC patients with and without postoperative transarterial chemoembolization (TACE) or transarterial embolization (TAE). Binary logistic regression tests indicated that Cbx4 is correlated with histological grading, tumor-node-metastasis stage, microvessel density, distant metastasis and hematogenous metastasis of HCC. By univariate and multivariate analyses, we show that Cbx4 is an independent prognostic factor of HCC, and both TAE and TACE treatments have no effects on the overall survival in HCC patients with low Cbx4 expression. More intriguingly, TACE prolongs, while TAE shortens, the overall survival of HCC patients with high Cbx4 expression, indicating that Cbx4 is a good biomarker on decision-making to perform postoperative TACE in HCC patients. Moreover, Cbx4 overexpression enhances while Cbx4 silencing antagonizes doxorubicin-induced cell death of HCC cell lines. In conclusion, Cbx4 is an independent prognostic factor for HCC patients, and the patients with high Cbx4 expression should receive postoperative TACE treatment to improve their survival.

MicroRNA-195 (miR-195) plays important roles in tumor metastasis and angiogenesis, yet its function and mechanism of action in hepatocellular carcinoma (HCC) remain to be elucidated. In this study, we aimed to confirm whether chromobox homolog 4 (CBX4) is a direct target gene of miR-195 and determine the functions of miR-195 through the CBX4 pathway. miR-195 expression was slightly lower in the HCC tissues compared with that in the adjacent normal tissues. In addition, western blotting and qRT-RCR results showed that both CBX4 mRNA and protein levels were downregulated upon miR-195 overexpression. Luciferase reporter assays revealed that CBX4 is a direct target gene of miR-195. Furthermore, overexpression of CBX4 significantly restored the proliferative, invasive and migratory capacities of the HepG2 cells. Finally, in vivo experiments confirmed that high expression of CBX4 in HepG2 cells promoted tumor growth. In conclusion, our study demonstrated that miR-195 acts as a tumor suppressor by directly targeting CBX4 in HCC. This finding suggests a potential novel strategy for therapeutic interventions of this disease.

Background: Polycomb group proteins form multicomponent complexes that are important for establishing lineage-specific patterns of gene expression. Mammalian cells encode multiple permutations of the prototypic Polycomb repressive complex 1 (PRC1) with little evidence for functional specialization. An aim of this study is to determine whether the multiple orthologs that are co-expressed in human fibroblasts act on different target genes and whether their genomic location changes during cellular senescence., Results: Deep sequencing of chromatin immunoprecipitated with antibodies against CBX6, CBX7, CBX8, RING1 and RING2 reveals that the orthologs co-localize at multiple sites. PCR-based validation at representative loci suggests that a further six PRC1 proteins have similar binding patterns. Importantly, sequential chromatin immunoprecipitation with antibodies against different orthologs implies that multiple variants of PRC1 associate with the same DNA. At many loci, the binding profiles have a distinctive architecture that is preserved in two different types of fibroblast. Conversely, there are several hundred loci at which PRC1 binding is cell type-specific and, contrary to expectations, the presence of PRC1 does not necessarily equate with transcriptional silencing. Interestingly, the PRC1 binding profiles are preserved in senescent cells despite changes in gene expression., Conclusions: The multiple permutations of PRC1 in human fibroblasts congregate at common rather than specific sites in the genome and with overlapping but distinctive binding profiles in different fibroblasts. The data imply that the effects of PRC1 complexes on gene expression are more subtle than simply repressing the loci at which they bind.

Mesoderm and embryonic axis formation in vertebrates is mediated by maternal and zygotic factors that activate the expression of target genes. Transcriptional derepression plays an important role in the regulation of expression in different contexts; however, its involvement and possible mechanism in mesoderm and embryonic axis formation are largely unknown. Here we demonstrate that XDSCR6, a Xenopus homologue of human Down syndrome critical region protein 6 (DSCR6, or RIPPLY3), regulates mesoderm and embryonic axis formation through derepression of polycomb group (PcG) proteins. Xdscr6 maternal mRNA is enriched in the endoderm of the early gastrula and potently triggers the formation of dorsal mesoderm and neural tissues in ectoderm explants; it also dorsalises ventral mesoderm during gastrulation and induces a secondary embryonic axis. A WRPW motif, which is present in all DSCR6 homologues, is necessary and sufficient for the dorsal mesoderm- and axis-inducing activity. Knockdown of Xdscr6 inhibits dorsal mesoderm gene expression and results in head deficiency. We further show that XDSCR6 physically interacts with PcG proteins through the WRPW motif, preventing the formation of PcG bodies and antagonising their repressor activity in embryonic axis formation. By chromatin immunoprecipitation, we demonstrate that XDSCR6 releases PcG proteins from chromatin and allows dorsal mesoderm gene transcription. Our studies suggest that XDSCR6 might function to sequester PcG proteins and identify a novel derepression mechanism implicated in embryonic induction and axis formation.

The polycomb group proteins have recently captured the attention of cancer biologists. enhancer of zeste homologue 2 (EZH2) and B lymphoma Mo-MLV insertion region 1 homolog (BMI-1) are the best-characterized polycomb group proteins; their deregulation contributes to the development of many malignancies including gastric cancers. H3 trimethylation at lysine 27 and DNA methylase DNA methyltransferase 3B proteins are associated with the recruitment of polycomb group proteins. Overexpression of polycomb group proteins is associated with poor prognoses in some types of cancers but with favorable prognoses in others. In the present study, we investigated the expression of the polycomb group proteins EZH2 and BMI-1 and the associated proteins H3 trimethylation at lysine 27 and DNA methyltransferase 3B in advanced gastric cancers. Based on immunohistochemical detection, we evaluated the clinical relevance of these proteins in 178 cases of advanced gastric cancers that were managed with radical surgery and adjuvant systemic chemotherapy. BMI-1, enhancer of zeste homologue 2, H3 trimethylation at lysine 27, and DNA methyltransferase 3B proteins were overexpressed in the nuclei of gastric carcinoma compared with adjacent nonneoplastic gastric parenchyma. The high-level expression of BMI-1, enhancer of zeste homologue 2, H3 trimethylation at lysine 27, and DNA methyltransferase 3B proteins were frequently noted in advanced gastric cancer tissues (70.8%, 92.1%, 58.4%, and 64.6% of cases, respectively) and well intercorrelated in expression (P < .05). The expression level of BMI-1, enhancer of zeste homologue 2, and DNA methyltransferase 3B showed correlation with sex, gross type, and histologic type of the tumor among clinicopathologic variables. In terms of patient survival, low-level expression of enhancer of zeste homologue 2 was associated with cancer-related death (P = .018) and shorter overall survival (P = .005). Low-level expression of enhancer of zeste homologue 2 may represent a negative prognostic marker (P = .005) and indicate high risk in patients with advanced gastric cancer after surgery and adjuvant chemotherapy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a nucleocytoplasmic shuttling protein that is a key player in the p53-triggered DNA damage response, acting as a cofactor for p53 in response to DNA damage. hnRNP K is a substrate of the ubiquitin E3 ligase MDM2 and, upon DNA damage, is de-ubiquitylated. In sharp contrast with the role and consequences of the other post-translational modifications, nothing is known about the role of SUMO conjugation to hnRNP K in p53 transcriptional co-activation. In the present work, we show that hnRNP K is modified by SUMO in lysine 422 within its KH3 domain, and sumoylation is regulated by the E3 ligase Pc2/CBX4. Most interestingly, DNA damage stimulates hnRNP K sumoylation through Pc2 E3 activity, and this modification is required for p53 transcriptional activation. Abrogation of hnRNP K sumoylation leads to an aberrant regulation of the p53 target gene p21. Our findings link the DNA damage-induced Pc2 activation to the p53 transcriptional co-activation through hnRNP K sumoylation.

A major function of long non-coding RNAs (lncRNAs) is regulating gene expression through changes in chromatin state. Experimental evidence suggests that in cancer, they can influence Polycomb Repressive Complexes (PRC) to retarget to an occupancy pattern resembling that of the embryonic state. We have previously demonstrated that the expression level of lncRNA in the HOX locus, including HOTAIR, is a predictor of breast cancer metastasis. In this current project, RNA in situ hybridization of probes to three different lncRNAs (HOTAIR, ncHoxA1, and ncHoxD4), as well a immunohistochemical staining of EZH2, is undertaken in formalin-fixed paraffin-embedded breast cancer tissues in a high throughput tissue microarray format to correlate expression with clinicopathologic features. Though overall EZH2 and HOTAIR expression levels were highly correlated, the subset of cases with strong HOTAIR expression correlated with ER and PR positivity, while the subset of cases with strong EZH2 expression correlated with an increased proliferation rate, ER and PR negativity, HER2 underexpression, and triple negativity. Co-expression of HOTAIR and EZH2 trended with a worse outcome. In matched primary and metastatic cancers, both HOTAIR and EZH2 had increased expression in the metastatic carcinomas. This is the first study to show that RNA in situ hybridization of formalin fixed paraffin-embedded clinical material can be used to measure levels of long non-coding RNAs. This approach offers a method to make observations on lncRNAs that may influence the cancer epigenome in a tissue-based technique.