- Research article
- Open Access
An Rb1-dependent amplification loop between Ets1 and Zeb1 is evident in thymocyte differentiation and invasive lung adenocarcinoma
© Dean et al.; licensee BioMed Central. 2015
Received: 1 August 2014
Accepted: 26 February 2015
Published: 19 March 2015
Ras pathway mutation leads to induction and Erk phosphorylation and activation of the Ets1 transcription factor. Ets1 in turn induces cyclin E and cyclin dependent kinase (cdk) 2 to drive cell cycle progression. Ets1 also induces expression of the epithelial-mesenchymal transition (EMT) transcription factor Zeb1, and thereby links Ras mutation to EMT, which is thought to drive tumor invasion. Ras pathway mutations are detected by the Rb1 tumor suppression pathway, and mutation or inactivation of the Rb1 pathway is required for EMT.
We examined linkage between Rb1, Ets1 and Zeb1. We found that an Rb1-E2F complex binds the Ets1 promoter and constitutively limits Ets1 expression. But, Rb1 repression of Zeb1 provides the major impact of Rb1 on Ets1 expression. We link Rb1 repression of Zeb1 to induction of miR-200 family members, which in turn target Ets1 mRNA. These findings suggest that Ets1 and Zeb1 comprise an amplification loop that is dependent upon miR-200 and regulated by Rb1. Thus, induction of Ets1 when the Rb1 pathway is lost may contribute to deregulated cell cycle progression through Ets1 induction of cyclin E and cdk2. Consistent with such an amplification loop, we correlate expression of Ets1 and Zeb1 in mouse and human lung adenocarcinoma. In addition we demonstrate that Ets1 expression in thymocytes is also dependent upon Zeb1.
Taken together, our results provide evidence of an Rb1-dependent Ets1-Zeb1 amplification loop in thymocyte differentiation and tumor invasion.
c-Ets1 was identified as a proto-oncogene based on v-ets in the genome of the avian leukemia retrovirus E26, and is the founding member of the Ets family of transcription factors . Elevated Ets1 expression has been observed in many invasive and metastatic solid tumors  and Over-expression of Ets1 is sufficient for transformation of NIH3T3 fibroblasts . In adults, expression of Ets1 becomes restricted primarily to lymphoid tissues , and mutation of Ets1 in mice leads to defects in maturation of lymphocytes [5-7]. Ets1 interacts with Tlx to cause the critical maturation arrest in T cell acute lymphoblastic leukemia . Induction of Ets1 in solid tumors triggers neovascularization and the epithelial-mesenchymal transition (EMT) that drives tumor invasion [9,10]. Ras pathway signaling is critical for normal development, and constitutively activating Ras mutations in tumors short-circuit the pathway leading to growth factor-independent cell proliferation, neovascularization and EMT [11,12]. Ets1 is phosphorylated and activated by Erk phosphorylation when the Ras pathway is engaged [13-15], and this induction of Ets1 is a mediator of Ras-initiated EMT. Accordingly, a downstream target of Ets1 is the EMT transcription factor Zeb1 , which is required for maintaining epithelial vs. mesenchymal balance in vivo . When induced in response to Ras mutation, Zeb1 causes transition to an invasive mesenchymal phenotype . A key sensor of mutant Ras is the Rb1 family of cell cycle regulators, whose activation in response to Ras mutation represses Zeb1 and blocks EMT .
Recent studies have found that Ets is repressed by miR-200 family members . miR-200 also represses Zeb1, but in a double negative loop Zeb1 binds the promoters of miR-200 family members and represses their expression [20,21]. Such findings raised the possibility that Zeb1 might feedback to induce Ets1 via its repression of miR-200, and that Rb1 might also influence Ets1 expression via its regulation of Zeb1. Here, we examined potential linkage between Rb1, Ets1, and Zeb1. Although Rb1 can interact with genes in a cell cycle-dependent fashion to regulate proliferation, it is also found constitutively at other genes including pro-apoptotic factors and mutation or inactivate of Rb1 is required for induction of such genes . We found here that Rb1 is present constitutively at the Ets1 promoter and removal of an Rb1-E2F complex using a dominant negative-E2F led to induction of Ets1. Thus, Rb1 directly diminishes the level of Ets1 expression. We also provide evidence that Zeb1 induces Ets1, and we show that an additional and major effect of Rb1 on Ets1 expression is mediated through Rb repression of Zeb1. We link the effect of Zeb1 to its regulation of miR-200, which in turn target Ets1.
Taken together, our results provide evidence of an amplification loop consisting of Ets1 and Zeb1, which is mediated by miR-200 and regulated by Rb1. We also show that Zeb1 and Ets1 are expressed together at the invasive edge of K-Ras-initiated mouse lung adenocarcinomas, and there is a significant correlation between expression of Ets1 and Zeb1 in human lung adenocarcinoma. Like Ets1, Zeb1 is important for thymocyte differentiation, and Zeb1(−/−) mice show a reduction in thymocytes [5,23-27]. Importantly, we demonstrate that mutation of Zeb1 eliminates Ets1 expression in thymocytes, demonstrating dependence of Ets1 expression on Zeb1 in thymocytes, and thus potentially linking the Zeb1 phenotype in T cell differentiation to a lack of Ets1 expression.
Cells and cell culture
Rb family triple knockout (TKO) mouse embryo fibroblasts and control wild-type fibroblasts have been described and were a kind gift from T. Jacks and J. Sage . Three independent TKO and wild-type isolates were used with similar results. Mouse Zeb1 wild type and mutant fibroblasts were isolated from crosses of mice heterozygous for Zeb1 and genotyped as described . The human osteosarcoma U2OS cells expressing IPTG-inducible p16INK4a were described previously , as were the U2OS cells expressing both IPTG-inducible p16INK4a and DN-E2F-mER . U2OS cells were cultured with 1 mM IPTG in the medium for either one or three days to induce p16INK4a, or with 100 nM Tamoxifen (OHT) for one day to induce mER-DB-E2F expression. For combined treatments, cells were treated with IPTG for one day, and then OHT was added along with the IPTG for an additional day. All the above fibroblast cells were cultured in DMEM medium with 10% fetal bovine serum (FBS) and antibiotics at 10% CO2 and 37°C.
Zeb1 shRNA construct and lentivirus preparation
Mouse embryos and adult lung tumor tissues were fixed with 10% formalin, paraffin-embedded, and sectioned at 5 μm. Primary antibodies for Zeb1 (kind gift from Dr. Douglas Darling), CD3 (kind gift from Dr. Qingxian Lu), E-cadherin (Santa Cruz Technology), Ets1 (Santa Cruz Biotechnology) and the secondary antibodies are detailed in Additional file 1: Table S1. The slides were mounted with coverslips using anti-fade medium Permount with DAPI (Fisher) and viewed under a Zeiss fluorescent microscope.
Housing and handling of all mice was in accordance with procedures approved by the University of Louisville Institutional Animal Care and Use Committee (IACUC). K-RasLA1 mice  in a C57BL6 background were obtained from Jackson Laboratory. PCR genotyping was as described previously .
Human lung adenocarcinoma microarray analysis
No human tissue was utilized in this study. The processed microarray data were used from previously published work (the NCBI database GSE_1969163) that contains 59 samples of human lung adenocarcinomas sequenced for mutations in K-RAS and EGFR and patient matched control lung tissue . The expression levels of Zeb1 and Est1 mRNAs were selected for calculating Pearson correlation score.
Chromatin immunoprecipitation (ChIP) assay
ChIP assays were based on the UpState protocol (http://www.tc.umn.edu/~muell002/Laboratory/protocols/X-ChIP.htm) using formaldehyde to crosslink genomic DNA of wild-type MEF cells. The chromatin was sheared to an average length of 300–500 bp. Monoclonal antibodies for Rb (Santa Cruz sc-50), E2F1 (Santa Cruz sc-193), E2F4 (Santa Cruz sc-866), and polyclonal antiserum for Zeb1 (a kind gift from Dr. Douglas Darling) were used for immunoprecipitation. Equal amounts of anti-IgG or pre-immune serum were used as controls. The sequence of primers for Ets1 promoter and the expected size of the PCR products are show in Additional file 2: Table S2. ChIP PCR reactions were similar to those described below for real-time PCR, but with additional 1% BSA and 1% DMSO, and the PCR programs usually had a higher annealing temperature (e.g. 60 – 68°C) and longer extension time (e.g. 1 minute).
Protein extraction and electrophoretic mobility shift assay (EMSA)
The cultured adherent wild-type MEF cells were scraped off and lysed in the lysis buffer (50 mM Tris/HCl, pH7.4, 100 mM NaCl, 0.5% Triton X-100, 0.5% NP-40) on ice for 20 minutes, followed by 10-minute centrifuge at 13,000 g, the supernatant was thereafter collected as non-denatured protein extract immediately for the gel shift assay. Based on the sequence alignment of both human and mouse Ets1 promoters, an aligned 24-bp short sequence (5′-TCCATAATTTGCCACTGATAGAT-3′) with a putative E2F site (TTTGCCAC) is selected for synthesis of the double-strand oligos with and without artificial mutation of the E2F site (the mutant oligo sequence: 5′-TCCATAACCTAGCTAGATAGAT-3′). Twenty μg of the above crude protein lysate was mixed with 10 pmol of each of the double-strand oligos in a 10-μl protein-DNA binding buffer (5 mM Tris/HCl pH7.4, 100 mM KCl, 100 μM dithiothrreitol, 100 μM EDTA), and incubated at RT for 40 minutes. The binding reactions without protein lysate or oligos were served as background controls. All the binding reaction samples were separated on a 6% native polyacrylamide gel in 0.5× TBE buffer at constant 200 V and RT for 20 minutes. The finished gel was stained with 10,000× diluted SYBR green (Molecular Probe) in 0.5× TBE buffer at RT for 20 minutes, and then visualized under UV light.
RNA extraction and real-time PCR
Total cellular RNA was extracted using TRIzol solution (Invitrogen, Carlsbad, California). Using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/), primer sets were designed to generate 150–250 base pair PCR products that bridge two separate exons. Primer sequence, melting temperature (Tm), and PCR product sizes are listed in Additional file 2: Table S2. First-strand complementary DNA (cDNA) synthesis was carried out in 20-μl reactions containing 1-2 μg of total RNA, 500 ng random hexamers, 10 mM dithiothreitol, 500 μM dNTP mix, 40 U RNaseOUT™ ribonuclease inhibitor, and 200 U M-MLV reverse transcriptase, at 37°C for 1 h according to the manufacturer’s protocol (Invitrogen, Carlsbad, California). Real-time quantitative PCR was performed in 25-μl reaction volumes containing 0.25-μl aliquots of cDNA, gene-specific primer pairs, and SYBR Green I fluorescent dye (Molecular Probes, Eugene, Oregon), in an Mx3000P Real-Time PCR System (Stratagene, Cedar Creek, Texas), according to the manufacturer’s instructions. The PCR cycle parameters were set at 95°C for 20 sec, 60°C for 30 sec, and 72°C for 30 sec, for a total of not more than 45 cycles. The fluorescent intensity of SYBR green was monitored at the end of each extension step; relative amounts of the target cDNA was estimated by the threshold cycle (Ct) number, and normalized to two internal control genes, β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Three independent samples were analyzed for each condition and/or cell type, and each sample was compared in at least 3 independent RT-PCR amplifications.
The quantitative RT-PCR to detect miRNAs is as described . Briefly, polyadenylation of at least 5 μg of the total RNA was completed by poly(A) polymerase kit (PAP, Ambion) in a 20 μl of reaction volume according to manufacturer’s instruction. The polyadenylated RNA was thereafter directly utilized for cDNA preparation using a reverse transcription kit (M-MLV reverse transcriptase, Invitrogen) and an adaptor primer (5′-GCGAGCACAGAATTAATACGACTCACTATAGG(T) 12VN*-3′) in a 40 μl of reaction volume. Real-time quantitative PCR was performed using a universal primer (5′-GCGAGCACAGAATTAATACGAC-3′) and a miRNA-specific primer (Additional file 2: Table S2) as described above.
The Rb1 family represses Ets1 expression
E2F1-Rb1 binds the Ets1 promoter and constitutively repressed the gene
Zeb1 repression of the miR-200 family is linked to induction of Ets1
Rb1 family repression of Zeb1 is a major component of inhibition of Ets1 by Rb1
Even though an Rb1-E2F complex binds to the Ets1 promoter to reduce its expression, it is of note that Rb1 also represses Zeb1 , leading us to ask whether Rb1 repression of Zeb1 was also contributing to the Rb1-dependent downregulation of Ets1. As we demonstrated previously , mutation of Rb1 family members led to induction of Zeb1 and this induction of Zeb1 was accompanied by repression of miR-200 (Figure 4C) and, as shown above, Ets1 (Figure 1). Knockdown of Zeb1 in the TKO MEFs using shRNA lenvirirus, as described previously , eliminated much of the induction of Ets1 (Figure 4D), suggesting that induction of Zeb1 and repression of miR-200 as Rb1 family members are mutated is contributing significantly to the upregulation of Ets1. These results suggest that Rb1-E2F binds the Ets1 promoter to limit its level of expression, but it is induction of Zeb1 and in turn repression of miR-200 when the Rb1 family activity is lost that is responsible for most of the induction of Ets1.
Ets1 and Zeb1 are expressed at the invasive front of K-Ras initiated mouse lung adenocarcinoma
Correlation between Ets1 and Zeb1 in human lung adenocarcinomas
Mutation of Zeb1 eliminates Ets1 expression in differentiating thymocytes
Our results provide evidence of an Rb1-dependent Ets1-Zeb1 amplification loop that is important in both cancer and in normal development of the thymus.
We thank Dr T. Jacks and Dr J. Sage for the gift of TKO and wild-type littermate control MEFs, Dr G. Leone for Rb heterozygous and null cells, Dr. D. Darling for Zeb1 antiserum, and Dr. Qingxian Lu for CD3 antibody. The studies were supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences (P20GM103453) to Y.L.
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