Ets Proteins Research Articles

Fli‑1 promotes metastasis by regulating MMP2 signaling in hepatocellular carcinoma.

Friend leukemia virus integration 1 (Fli‑1) is a newly identified ETS protein, and has critical roles in many malignancies. However, the physiological characters and potential mechanisms of Fli‑1 in hepatocellular carcinoma (HCC) progression remains unclear. In the present study, Fli‑1 was highly expressed in HCC samples and tumor cell lines. knockdown of Fli‑1 with small interfering (si)RNAs significantly reduced the colony formation and metastasis capacity of HCC cell lines invitro. Subsequent investigation identified that Fli‑1 functioned as an oncogene in HCC carcinogenesis and it exerted its promoting metastatic effect primarily by modulating the matrix metalloproteinase (MMP)2 signaling pathway. Collectively, these data provide a novel insight into the mechanism of Fli‑1/MMP2 signaling pathway in the pathogenesis of HCC, and Fli‑1 may serve as a novel therapeutic target for HCC.

MiR-129 inhibits cell proliferation and metastasis by targeting ETS1 via PI3K/AKT/mTOR pathway in prostate cancer

New evidence suggests that microRNAs (miRNAs) play an important role in regulating the development and progression of prostate cancer. However, their specific functions and mechanisms remained to be further explored. MiR-129 has been reported in gastric cancers, colon cancer and lung cancer. In this study, we disclosed a new tumor suppresser function of miR-129 in prostate cancer. The purpose of our study is to clarify the effects of miR-129 in cellular processes correlated with cancer development and progression of prostate cancer cell by regulating ETS1. MiR-129 and ETS1 expression in prostate cancer tissues, tumor adjacent tissues and cells were tested by quantitative real-time PCR. We validated the target relationship between miR-129 and ETS1 by dual luciferase reporter gene system. MTT, colony formation, tumorigenesis assays, flow cytometry, wound healing and transwell assays were used to analyze cell viability, proliferation, migration, and invasiveness in vivo and in vitro. The level of ETS1 protein expression was detected by western blot. Here we demonstrate that miR-129 have a relatively reduced expression in prostate cancer cell lines and tissues. Morever, the miR-129 inhibits the expression of ETS1 by binding its 3′-UTR. The overexpression of miR-129 can inhibit PC-3 cell viability, proliferation, migration and invasion through targeting ETS1 by PI3K/AKT/mTOR signaling pathway. These findings suggested that miR-129 could directly suppress ETS1, which might be one of potential mechanisms in inhibiting cell processes including viability, proliferation, migration and invasiveness of prostate cancercells and it provides new clues for us to understand the carcinogenesis of prostate cancer. In addition, it may help to develop a treatment approach for ETS1-activated prostate cancer.

ETS1, nucleolar and non-nucleolar TERT expression in nevus to melanoma progression.

TERT (telomerase reverse transcriptase) is the catalytic component of telomerase. TERT shows little expression in normal somatic cells but is commonly re-expressed in cancers, facilitating immortalization. Recently-discovered TERT promoter mutations create binding sites for ETS-family transcription factors to upregulate TERT. ETS1 is reported to be important for TERT upregulation in melanoma. However it is unclear when in melanoma progression TERT and ETS1 proteins are expressed. To elucidate this question, ETS1 and TERT immunohistochemistry were performed on a panel of benign (n=27) and dysplastic nevi (n=34), radial growth phase (n=29), vertical growth phase (n=25) and metastatic melanomas (n=27). Lesions were scored by percentage of positive cells. ETS1 was readily detectable in all lesions, but not in normal melanocytes. TERT was located in either the nucleolus, the nucleoplasm (non-nucleolar) or both. Non-nucleolar TERT increased in prevalence with progression, from 19% of benign nevi to 78% of metastases. It did not however correlate with cell proliferation (Ki-67 immunostaining), nor differ significantly in prevalence between primary melanomas with or without a TERT promoter mutation. These results demonstrate that ETS1 is expressed very early in melanoma progression, and interestingly only non-nucleolar TERT correlates clearly in prevalence with melanoma progression. It can be acquired at various stages and by mechanisms other than promoter mutations.

Abstract 3510: A role for the Ewing’s sarcoma breakpoint protein EWS in ERG-induced prostate tumorigenesis

Abstract The TMPRSS2-ERG gene fusion occurs in one-half of prostate tumors and results in expression of ERG, an ETS family transcription factor that promotes prostate tumorigenesis. Another 5-10% of prostate tumors have similar rearrangements that result in over-expression of other ETS family members such as ETV1, ETV4, and ETV5. However, not all ETS family members are oncogenic, and normal prostate cells express at least 15 of these other ETS factors. Therefore, it is important to understand mechanisms that differentiate oncogenic ETS factors from non-oncogenic ETS factors. We have recently discovered that ERG, ETV1, ETV4, and ETV5, and no other ETS factor can directly bind the protein EWS. This is striking, as EWS is fused to various ETS factors in the chromosomal rearrangements that cause Ewing’s sarcoma. We demonstrate that fusion of EWS to any ETS protein is sufficient for that protein to function like ERG in prostate cells. Furthermore, the EWS-ERG interaction is required for ERG to activate an oncogenic gene expression program and for ERG-mediated tumorigenesis. Therefore, these findings reveal a conserved oncogenic mechanism for prostate cancer and Ewing’s sarcoma. In prostate cancer, aberrant over-expression of an ETS factor that naturally interacts with EWS, brings EWS to ETS-bound regions. In Ewing’s sarcoma this interaction is forced by the presence of an EWS/ETS fusion protein. Therefore, this model predicts that treatments that target EWS function would be effective in both types of cancer. Citation Format: Vivekananda Kedage, Nagarathinam Selvaraj, Taylor R. Nicholas, Justin A. Budka, Travis J. Jerde, Peter C. Hollenhorst. A role for the Ewing’s sarcoma breakpoint protein EWS in ERG-induced prostate tumorigenesis [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 3510. doi:10.1158/1538-7445.AM2017-3510

Functional dissection of breast cancer risk-associated TERT promoter variants.

The multi-cancer susceptibility locus at 5p15.33 includes TERT, encoding the telomerase catalytic subunit. Genome-wide association studies (GWAS) have identified six single nucleotide polymorphisms (SNPs) in the TERT promoter associated with decreased breast cancer risk, although the precise causal variants and their mechanisms of action have remained elusive. Luciferase reporter assays indicated that the protective haplotype reduced TERT promoter activity in human mammary epithelial and cancer cells in an estrogen-independent manner. Using single variant constructs, we identified rs3215401 and rs2853669 as likely functional variants. Silencing of MYC decreased TERT promoter activity but neither MYC nor ETS2 silencing conferred allele-specificity. In chromatin immunoprecipitation experiments, the ETS protein GABPA, but not ETS2 or ELF1, bound rs2853669 in an allele-specific manner in mammary epithelial cells. Investigation of open chromatin in mammoplasty samples suggested involvement of three additional variants, though not rs3215401 or rs2853669. Chromosome conformation capture revealed no interaction of the TERT promoter with regulatory elements in the locus, indicating limited local impact of candidate variants on the TERT promoter. Collectively, our functional studies of the TERT-CLPTM1L breast cancer susceptibility locus describe rs2853669 as a functional variant of this association signal among three other potentially causal variants and demonstrate the versatile mechanisms by which TERT promoter variants may affect breast cancer risk.

Distinct Roles for Interfacial Hydration in Site-Specific DNA Recognition by ETS-Family Transcription Factors.

The ETS family of transcription factors is a functionally heterogeneous group of gene regulators that share a structurally conserved, eponymous DNA-binding domain. Unlike other ETS homologues, such as Ets-1, DNA recognition by PU.1 is highly sensitive to its osmotic environment due to excess interfacial hydration in the complex. To investigate interfacial hydration in the two homologues, we mutated a conserved tyrosine residue, which is exclusively engaged in coordinating a well-defined water contact between the protein and DNA among ETS proteins, to phenylalanine. The loss of this water-mediated contact blunted the osmotic sensitivity of PU.1/DNA binding, but did not alter binding under normo-osmotic conditions, suggesting that PU.1 has evolved to maximize osmotic sensitivity. The homologous mutation in Ets-1, which was minimally sensitive to osmotic stress due to a sparsely hydrated interface, reduced DNA-binding affinity at normal osmolality but the complex became stabilized by osmotic stress. Molecular dynamics simulations of wildtype and mutant PU.1 and Ets-1 in their free and DNA-bound states, which recapitulated experimental features of the proteins, showed that abrogation of this tyrosine-mediated water contact perturbed the Ets-1/DNA complex not through disruption of interfacial hydration, but by inhibiting local dynamics induced specifically in the bound state. Thus, a configurationally identical water-mediated contact plays mechanistically distinct roles in mediating DNA recognition by structurally homologous ETS transcription factors.

High constitutive activity of a broad panel of housekeeping and tissue-specific cis-regulatory elements depends on a subset of ETS proteins.

Enhancers and promoters that control the transcriptional output of terminally differentiated cells include cell type-specific and broadly active housekeeping elements. Whether the high constitutive activity of these two groups of cis-regulatory elements relies on entirely distinct or instead also on shared regulators is unknown. By dissecting the cis-regulatory repertoire of macrophages, we found that the ELF subfamily of ETS proteins selectively bound within 60 base pairs (bp) from the transcription start sites of highly active housekeeping genes. ELFs also bound constitutively active, but not poised, macrophage-specific enhancers and promoters. The role of ELFs in promoting high-level constitutive transcription was suggested by multiple evidence: ELF sites enabled robust transcriptional activation by endogenous and minimal synthetic promoters, ELF recruitment was stabilized by the transcriptional machinery, and ELF proteins mediated recruitment of transcriptional and chromatin regulators to core promoters. These data suggest that the co-optation of a limited number of highly active transcription factors represents a broadly adopted strategy to equip both cell type-specific and housekeeping cis-regulatory elements with the ability to efficiently promote transcription.

TK216, a Novel, Small Molecule Inhibitor of the ETS-Family of Transcription Factors, Displays Anti-Tumor Activity in AML and DLBCL

The ETS family of transcription factors is critical for development, differentiation, proliferation, and plays an important role in apoptosis and tissue remodeling. Transcriptional consequences of ETS protein deregulation by overexpression, gene fusion, and modulation by RAS/MAPK and PI3K signaling are linked to alterations in normal cell functions, and lead to increased proliferation, sustained angiogenesis, invasion, and metastasis. Overexpressed ETS proteins and ETS family fusion proteins have been reported in acute myeloid leukemia (AML) and diffuse large B cell lymphoma (DLBCL). In DLBCL, the 11q24.3 region has been identified as a recurrent lesion and a contributor to the pathogenesis of disease, leading to the deregulation of ETS family members, ETS1 and FLI1. Additionally, in AML, the overexpression and translocations of ERG, an ETS family member, has been shown to be associated with poor prognosis in complex or normal karyotypes.TK216 is a first in class, small molecule that directly binds EWS-FLI1 inhibiting the biological activity of ETS-family transcription factor oncoproteins and is currently under clinical investigation in patients with Ewing sarcoma (NCT02657005). The EWS1-FLI1 is a fusion protein that has been shown to be the driver of Ewing Sarcoma (ES). In preclinical potency models, TK216 blocked the binding between EWS-FLI1 and RNA helicase A, showed a significant transcriptional decrease in COS7 cells transfected with a EWS-FLI1 responsive promoter (EC50 < 100 nM), and inhibited the proliferation of A4573 cells (EWS-FLI1 expressing Ewing sarcoma cell line) at nanomolar concentrations (EC50 < 200 nM). Here we show that TK216 has anti-proliferative effects, causes cell cycle arrest, and induces apoptosis in a panel of AML and DLBCL cell lines with deregulated ETS family members. We report an upregulation of FLI1 and/or ERG ETS family members in 5 of 5 myeloid cell lines evaluated (HL60, Kasumi-1, ML-2, MOLM-13, and MOLM-16). Treatment with TK216 showed a decrease in cellular viability and induced dose-dependentapoptosis of cells at 48 hours. Similarly, in a panel of DLBLC cell lines (TMD8, HBL1, U2932, DOHH2, WSUDLCL2 and OCI-Ly18), TK216 treatment resulted in a decrease in cellular proliferation and an increase in apoptosis. In vivo efficacy studies in xenograft models of DLBCL are underway; the preliminary anti-tumor activity that is being seen is encouraging and consistent with our in vitro findings.We believe that our findings provide compelling evidence of the utility and potential efficacy of TK216 in the treatment of AML and DLBCL by targeting the aberrant expression and translocations in the ETS-family of transcription factors, which contribute to the pathogenesis of the disease. DisclosuresJessen:Oncternal: Employment. Otuski:Oncternal: Employment, Equity Ownership. Breitmeyer:Oncternal: Employment, Equity Ownership. Freddo:Oncternal: Employment, Equity Ownership. Toretsky:Oncternal: Research Funding. Lannutti:Oncternal: Employment, Equity Ownership.

ETS1 Expression Is Upregulated in Chronic Myeloid Leukemia (CML) and the ETS1 Transcriptional Program Correlates with Disease Progression Towards Blast Crisis

Chronic myeloid leukemia is a clonal myeloproliferative neoplasm characterized by the occurrence of the Ph1 chromosome in a primitive hematopoietic stem cell with a major amplification of myeloid compartment. Despite the major progress obtained by the use of tyrosine kinase inhibitors (TKI), resistances to these drugs occur with progression towards blast crisis (BC) during which there is an increase of BCR-ABL expression. To evaluate the potential targets of BCR-ABL in this context, a gene profiling analysis of the UT7-11 cell line overexpressing BCR-ABL fusion protein was performed using a whole transcriptome microarray. One of the highly upregulated genes was ETS1 with a fold change of +35.86. ETS1 proto-oncogene, the founder member of the ETS-domain family of transcription factors such as TEL and FLI1, is the human homolog of the avian erythroblastosis virus E26. They play a crucial role in stem cell biology, tumorigenesis and ETS1 has been shown to be involved in the regulation of granulocytic differentiation. Its role in CML pathophysiology has not been studied so far. We first showed by Western blots that ETS1 protein was highly increased in UT7-11 cells as compared to parental UT7 cells. Using a DOX-Inducible BCR-ABL system, we have shown that ETS1 expression was downregulated upon inhibition of BCR-ABL expression. ETS1 expression was a tyrosine kinase dependent event as its expression was reduced in the presence of TKI. To determine if ETS1 expression is upregulated in primary leukemic cells, we have analyzed leukemic cells of CML patients at diagnosis (n= 40) as compared to healthy controls (n= 30) showing an increase of ETS1 mRNA expression in primary CML cells in a highly significant manner (p < 0.0007). We then analyzed ETS1 targets using chip-sequencing in K562 cells, performed on HG19 human genome allowing to predict 3209 proximal promoters (-3000 upstream pb, + 50pb around Transcription Starting Sites) which represents a promoter enrichment of +33.68 (p-value=4.9E-324) as compared to the distribution of peaks in the whole genome. Chromosomes 19, 16, 17, 11, 12 and 1 were found over-represented with ETS1 bound events in Chip-sequencing. Integration of BCR-ABL transcriptome with ETS1 promoters identified by Chip-Sequencing led to the identification of 130 ETS1-targets activated by BCR-ABL, allowing reclassification of BCR-ABL transfected samples with transcriptome data by unsupervised classification. ETS1 transcriptional program regulated by BCR-ABL analysis performed on CD34+ from CML patients during progression of the disease (GSE47927) allowed discrimination of the different states of the progression (from the chronic phase to accelerated phase and BC p-value=1.63E-33). This analysis was also found significant by Gene Set Enrichment Analysis ETS1_BC specific geneset (NES=+1.52, p-value<0.0001). Among this ETS1 program specific of BC, we identified 2 targets that are discriminant for CD34+ cells of CML patients without cytogenetic response under Imatinib therapy. These two genes were Dynamin 3 (DNM3) and LIMS1/ PINCH1. Dynamin 3 is a member of the motor proteins implicated in cell motility and cytokinesis and it is implicated in megakaryocyte development whereas LIMS1 / PINCH1 is essentially involved in cell migration and adhesion. In order to validate ETS1 and its target expressions in a novel cohort of CML patients, we have analyzed the expression of DNM3 and PINCH1 in whole blood samples in CML patients ( n= 60 ) as compared to healthy controls (n= 32). We have found that, similar to ETS1, DNM3 and LIMS1 / PINCH1 mRNA were highly upregulated in primary CML and this increase was highly significant. In this new cohort of patients, DNM3 transcript levels were inversely correlated to WBC count (r=-0.32, p-value=0.011) and especially in male gender (r=-0.43, p-value=0.012). The expression of LIMS1 was inversely correlated with the age at diagnosis and the major molecular response at 12 months. Thus, our results show for the first time the major upregulation of ETS1 transcriptional program in CML. ETS1 transcriptional program has been found to correlate with the gene expression pattern involved in the progression of the disease in the transcriptomic analysis of CML CD34+ cells. The novel druggable targets that we identified in the BCR-ABL-activated transcriptional program is currently under investigation to determine their use in patients with resistance to targeted therapies. DisclosuresNo relevant conflicts of interest to declare.

Transcription Factor Ets-2 Acts as a Preinduction Repressor of Interleukin-2 (IL-2) Transcription in Naive T Helper Lymphocytes

IL-2 is the first cytokine produced when naive T helper (Th) cells are activated and differentiate into dividing pre-Th0 proliferating precursors. IL-2 expression is blocked in naive, but not activated or memory, Th cells by the transcription factor Ets-2 that binds to the antigen receptor response element (ARRE)-2 of the proximal IL-2 promoter. Ets-2 acts as an independent preinduction repressor in naive Th cells and does not interact physically with the transcription factor NFAT (nuclear factor of activated T-cells) that binds to the ARRE-2 in activated Th cells. In naive Th cells, Ets-2 mRNA expression, Ets-2 protein levels, and Ets-2 binding to ARRE-2 decrease upon cell activation followed by the concomitant expression of IL-2. Cyclosporine A stabilizes Ets-2 mRNA and protein when the cells are activated. Ets-2 silences directly constitutive or induced IL-2 expression through the ARRE-2. Conversely, Ets-2 silencing allows for constitutive IL-2 expression in unstimulated cells. Ets-2 binding to ARRE-2 in chromatin is stronger in naive compared with activated or memory Th cells; in the latter, Ets-2 participates in a change of the IL-2 promoter architecture, possibly to facilitate a quick response when the cells re-encounter antigen. We propose that Ets-2 expression and protein binding to the ARRE-2 of the IL-2 promoter are part of a strictly regulated process that results in a physiological transition of naive Th cells to Th0 cells upon antigenic stimulation. Malfunction of such a repression mechanism at the molecular level could lead to a disturbance of later events in Th cell plasticity, leading to autoimmune diseases or other pathological conditions.

Signatures of prostate-derived Ets factor (PDEF) in cancer.

The Ets proteins are a family of transcription factors characterized by an evolutionarily conserved DNA-binding domain and have diverse biological functions including tumor suppressor as well as tumor promoter functions. They are regulated via a complex and diverse number of mechanisms and control key cellular processes. Prostate-derived Ets transcription factor (PDEF), a unique member of the ETS family, is present in tissues with high epithelial content are hormone-regulated, such as prostate, breast, salivary glands, ovaries, colon, airways, and stomach tissues. PDEF (prostate-derived Ets factor) is also referred to as SPDEF (SAM pointed domain containing Ets transcription factor), PSE (mouse homolog), or hPSE (human PSE) in the literature and is the sole member of the PDEF ETS sub-family. The role of PDEF in cancer development is still not fully elucidated though. The present article focuses on the key findings about the PDEF's biological functions, interacting proteins, and its target genes. There is a strong urge to focus on the clinical studies in larger cohort, which elucidate the regulation of PDEF and its target genes, to determine the potential of PDEF as biomarker. Based on the studies discussed in the present article, one can anticipate that PDEF offers a great potential for developing therapeutics against cancer.

Statins affect ETS1-overexpressing triple-negative breast cancer cells by restoring DUSP4 deficiency.

We investigated the molecular mechanisms underlying statin-induced growth suppression of triple-negative breast cancer (TNBC) that overexpress the transcription factor ets proto-oncogene 1(ets-1) and downregulate dual specific protein phosphatase 4(dusp4) expression. We examined the gene expression of BC cell lines using the nCounter expression assay, MTT viability assay, cell proliferation assay and Western blot to evaluate the effects of simvastatin. Finally, we performed cell viability testing in TNBC cell line-transfected DUSP4. We demonstrated that ETS1 mRNA and protein were overexpressed in TNBC cells compared with other BC cell lines (P = <0.001) and DUSP4 mRNA was downregulated (P = <0.001). MTT viability assay showed that simvastatin had significant antitumor activity (P = 0.002 in 0.1 μM). In addition, simvastatin could restore dusp4 deficiency and suppress ets-1 expression in TNBC. Lastly, we found that si-DUSP4 RNA transfection overcame the antitumor activity of statins. MAPK pathway inhibitor, U0126 and PI3KCA inhibitor LY294002 also decreased levels of ets-1, phosphor-ERK and phosphor-AKT on Western blot assay. Accordingly, our study indicates that simvastatin potentially affects the activity of transcriptional factors such as ets-1 and dusp4 through the MAPK pathway. In conclusion, statins might be potential candidates for TNBC therapy reducing ets-1 expression via overexpression of dusp4.

Abstract 2881: Deubiquitinase Usp9x controls tumorigenicity through regulation of the Ets-1 transcription factor in melanoma

Abstract Transcription factors are frequently deregulated in cancer cells and may be good therapeutic targets, but few successful targeting strategies have been reported. Deubiquitinases (DUBs) are specialized enzymes that regulate the ubiquitin (Ub) content on many proteins and DUB expression and activity are elevated in a number of cancers where they can act to alter tumor suppressor and/or oncoprotein levels. We previously described Usp9x activity and expression in melanoma; here we sought to investigate its role in primary melanoma and metastatic disease. Usp9x was upregulated in tumor cells compared to normal melanocytes and Usp9x expression and activity were found to be essential for 3D growth and melanoma tumor expansion in vivo. We defined the Usp9x ubiquitinated protein landscape and demonstrate that Usp9x regulates Ets-1, a cancer-promoting transcription factor. Usp9x binds, deubiquitinates and thereby stabilizes Ets-1 protein, and primary tissue and tumor analysis demonstrated elevated and coincident Usp9x/Ets-1 protein expression in melanoma compared to normal skin or benign nevi. Usp9x knockdown or Usp9x inhibition with small molecule G9 reduced Ets-1 protein levels and blocked tumor growth in vitro and in vivo. Conversely, Usp9x overexpression in melanoma cells increased Ets-1 protein levels and enhanced 3D tumor growth in vitro and in vivo, which were all reversible by treatment with G9. We conclude that Usp9x is essential for Ets-1 protein stability and may be therapeutically exploited with small molecule Usp9x inhibitors to reduce Ets-1-dependent gene expression and tumorigenicity. Citation Format: Harish Potu, Luke F. Peterson Peterson, Malathi Kandarpa Kandarpa, Anupama Pal Pal, Hanshi Sun Sun, Paul W. Harms Harms, Peter C. Hollenhorst Hollenhorst, Ugur Eskiocak Eskiocak, Moshe Talpaz Talpaz, Nicholas J. Donato Donato. Deubiquitinase Usp9x controls tumorigenicity through regulation of the Ets-1 transcription factor in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2881.

Abstract 4223: Novel strategy for diagnosis of solid tumors by visualization of fusion transcripts

Abstract The discovery of chromosomal translocations as one major cause of cancers has led to the introduction of numerous diagnostic techniques to detect these chromosomal abnormalities. Chromosomal banding, DNA fluorescence in situ hybridization (DNA FISH), and RT PCR are all techniques used for the diagnosis of cancers. Despite the advancement these techniques have brought to the field, they are primarily optimized for the detection of blood cancers and are not very efficient for the detection of solid tumors which are very heterogeneous. One such cancer, Ewing's sarcoma (ES), is a prevalent pediatric, bone and soft tissue tumor that is caused by a chromosomal translocation between Ewing's sarcoma breakpoint region 1 (EWSR1) on chromosome 22 and various members of the ETS protein family. The most common fusion partner, in 85% of cases, is Friend leukemia integration 1 (FLI1) on chromosome 11 which leads to the formation of the fused EWS/FLI1 gene that codes for an aberrant transcription factor. However, even within this single translocation there are multiple breakpoint-fusion scenarios that can occur which results in heterogeneity and increases the chances of a faulty diagnosis with standard techniques. Considering the diversity that exists within the molecular abnormalities that result in Ewing's sarcoma and other solid tumors, there is a need for an alternative and more robust method of detection. The technique of single molecule fluorescence in situ hybridization (smFISH) has been modified by our group to target the EWS/FLI1 transcript that results from the chromosomal fusion; this new technique is referred to as “Fusion FISH”. In Fusion FISH, two differently labeled probe sets are designed to target each half of the EWS/FLI1 fusion transcript. A co-localization of the two probe sets indicates the presence of the EWS/FLI1 translocation and a positive test for Ewing's sarcoma. Preliminary data has established the success of Fusion FISH in detecting both type 1 and type 2 translocations in several Ewing's sarcoma cell lines. The fusion transcript is expressed at similar levels in all cell lines and clearly distinct from the signal of wild type EWS and FLI1 mRNA. Additional studies will examine this new technique in other solid cancers, patient tumor samples, and for validation of additional relevant mRNA biomarkers. Citation Format: Fatu Badiane Markey, Mona Batish. Novel strategy for diagnosis of solid tumors by visualization of fusion transcripts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4223.

Abstract 1861: The role and regulation of MTA1-ETS2 axis in prostate cancer

Abstract Overexpression of the chromatin modifier protein, metastasis associated 1 (MTA1) in prostate cancer contributes to tumor aggressiveness, but the molecular mechanism involved has not been fully elucidated. MTA1 ChIP-Seq analysis in prostate tissues from the prostate-specific Pten heterozygous mice, which exhibit elevated levels of MTA1 revealed MTA1 recruitment onto the ETS2 promoter. Loss of function studies with MTA1 in various prostate cancer cell lines exhibited reduced expression for ETS2 at both mRNA and protein levels. We also observed increased expression of ETS2 at both mRNA and protein levels in prostate tissues from the prostate-specific MTA1 transgenic mice. Meta-analysis of prostate cancer samples in the GEO database exhibited a positive correlation between MTA1 and ETS2 levels. These results suggested that MTA1 functions as a coactivator for regulating ETS2 expression in prostate cancer. We found direct interaction between MTA1 and ETS2 in cells expressing ectopic MTA1 and ETS2. Interestingly, the prostate-specific Pten heterozygous mice, when fed with diet supplemented with pterostilbene, a natural pharmacological inhibitor of MTA1, showed a decrease in ETS2 mRNA and protein levels, perhaps due to decreased recruitment of MTA1 onto the ETS2 promoter. These results suggest that MTA1 functions as a coactivator for regulating ETS2 expression in prostate cancer at the transcriptional level and co-operates with ETS2 to promote prostate cancer progression. At the same time, pterostilbene may become a novel chemopreventive and therapeutic candidate for clinical development in prostate cancer due to its ability to target the MTA1-ETS2 axis. Citation Format: Avinash Kumar, Swati Dhar, Nasir Butt, Agnes Rimando, Luis Martinez, Anait Levenson. The role and regulation of MTA1-ETS2 axis in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1861.

Differential sensitivity to methylated DNA by ETS-family transcription factors is intrinsically encoded in their DNA-binding domains.

Transactivation by the ETS family of transcription factors, whose members share structurally conserved DNA-binding domains, is variably sensitive to methylation of their target genes. The mechanism by which DNA methylation controls ETS proteins remains poorly understood. Uncertainly also pervades the effects of hemi-methylated DNA, which occurs following DNA replication and in response to hypomethylating agents, on site recognition by ETS proteins. To address these questions, we measured the affinities of two sequence-divergent ETS homologs, PU.1 and Ets-1, to DNA sites harboring a hemi- and fully methylated CpG dinucleotide. While the two proteins bound unmethylated DNA with indistinguishable affinity, their affinities to methylated DNA are markedly heterogeneous and exhibit major energetic coupling between the two CpG methylcytosines. Analysis of simulated DNA and existing co-crystal structures revealed that hemi-methylation induced non-local backbone and groove geometries that were not conserved in the fully methylated state. Indirect readout of these perturbations was differentially achieved by the two ETS homologs, with the distinctive interfacial hydration in PU.1/DNA binding moderating the inhibitory effects of DNA methylation on binding. This data established a biophysical basis for the pioneering properties associated with PU.1, which robustly bound fully methylated DNA, but not Ets-1, which was substantially inhibited.

Bombyx mori E26 transformation-specific 2 (BmEts2), an Ets family protein, represses Bombyx mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in the silkworm Bombyx mori.

E26 transformation-specific (Ets) family transcription factors are known to play roles in various biological phenomena, including immunity, in vertebrates. However, the mechanisms by which Ets proteins contribute to immunity in invertebrates remain poorly understood. In this study, we identified a cDNA encoding BmEts2, which is a putative orthologue of Drosophila Yan and human translocation-ets-leukemia/Ets-variant gene 6, from the silkworm Bombyx mori. Expression of the BmEts2 gene was significantly increased in the fat bodies of silkworm larvae in response to injection with Escherichia coli and Staphylococcus aureus. BmEts2 overexpression dramatically repressed B. mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in silkworm cells. Conversely, gene knockdown of BmEts2 significantly enhanced BmRels activity. In addition, two κB sites located on the 5' upstream region of cecropin B1 were found to be involved in the repression of BmRels-mediated promoter activation. Protein-competition analysis further demonstrated that BmEts2 competitively inhibited binding of BmRels to κB sites. Overall, BmEts2 acts as a repressor of BmRels-mediated transactivation of antimicrobial protein genes by inhibiting the binding of BmRels to κB sites.

Inhibiting cell migration and cell invasion by silencing the transcription factor ETS-1 in human bladder cancer.

As one of the members of the ETS gene family, the transcription factor v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS-1) plays key role in the regulation of physiological processes in normal cells and tumors. In this study, we aimed to investigate the relationship between the transcription factor ETS-1 and malignant phenotypes of bladder cancer. We demonstrated that ETS-1 was up-regulated in human bladder cancer tissue compared to paired normal bladder tissue. In order to evaluate the functional role of ETS-1 in human bladder cancer, vectors expressing ETS-1 shRNA and ETS-1 protein were constructed in vitro and transfected into the human bladder cancer T24 and 5637 cells. Our results showed that the transcription factor ETS-1 could promote cell migration and cell invasion in human bladder cancer, without affecting cell proliferation and apoptosis. In conclusion, ETS-1 plays oncogenic roles through inducing cell migration and invasion in human bladder cancer, and it can be used as a therapeutic target for treating human bladder cancer.

Molecular Self-Titration as a Mechanism of Gene Regulation

In systems biology, molecular titration is a mechanism for introducing sensitivity of genetic networks to their regulatory proteins. Since most eukaryotic transcription factors lack chemical control by low-molecular-weight hormones or metabolites, transcription factors have evolved various strategies, such as cooperative DNA binding and sequestering partner proteins, to generate sensitivity in the expression of the genes that they regulate. The ETS superfamily of transcription factors, which are broadly distributed in the animal kingdom, regulate target genes in an active monomeric state. On the one hand, many ETS members, such as the master transcription factor PU.1, are involved in critical cell-fate decisions and signaling pathways that require highly sensitive output responses, yet no coherent mechanism exists for generating the required sensitivity. On the other hand, dimerization of ETS proteins in both DNA-bound and free states via their DNA-binding domain has been reported, but its purpose and biophysical nature remain obscure. We propose that dimerization serves as a sensitivity-generating mechanism by molecular self-titration: self-association sequesters free ETS proteins in inactive dimers, while dimerization in the DNA-bound state inhibits transactivation by the active protein/DNA complex. With the positive auto-regulation of the PU.1 (Spi1) gene generating self-amplification of PU.1, homodimerization would provide the minimal negative feedback needed to prevent explosion and maintain stable dynamics of PU.1 target genes in the absence of additional interactions. We are defining the thermodynamic and structural parameters of the PU.1 homodimer in the free and DNA-bound states. The data show that dimerization involves previously unexplored molecular surfaces that are distinct from the “orthosteric” protein/DNA contact interface and offer insight into how molecular self-titration would participate in the dynamics of PU.1-dependent gene expression.

The Ets protein Pointed prevents both premature differentiation and dedifferentiation of Drosophila intermediate neural progenitors.

Intermediate neural progenitors (INPs) need to avoid both dedifferentiation and differentiation during neurogenesis, but the underlying mechanisms are not well understood. In Drosophila, the Ets protein Pointed P1 (PntP1) is required to generate INPs from type II neuroblasts. Here, we investigated how PntP1 promotes INP generation. By generating pntP1-specific mutants and using RNAi knockdown, we show that the loss of PntP1 leads to both an increase in type II neuroblast number and the elimination of INPs. The elimination of INPs results from the premature differentiation of INPs due to ectopic Prospero expression in newly generated immature INPs (imINPs), whereas the increase in type II neuroblasts results from the dedifferentiation of imINPs due to loss of Earmuff at later stages of imINP development. Furthermore, reducing Buttonhead enhances the loss of INPs in pntP1 mutants, suggesting that PntP1 and Buttonhead act cooperatively to prevent premature INP differentiation. Our results demonstrate that PntP1 prevents both the premature differentiation and the dedifferentiation of INPs by regulating the expression of distinct target genes at different stages of imINP development.