The cancer gene WWOX behaves as an inhibitor of SMAD3 transcriptional activity via direct binding

Background: The WW domain containing protein WWOX has been postulated to behave as a tumor suppressor in breast and other cancers. Expression of this protein is lost in over 70% of ER negative tumors. This prompted us to investigate the phenotypic and gene expression effects of loss of WWOX expression in breast cells. Methods: Gene expression microarrays and standard in vitro assays were performed on stably silenced WWOX (shRNA) normal breast cells. Bioinformatic analyses were used to identify gene networks and transcriptional regulators affected by WWOX silencing. Co-immunoprecipitations and GST-pulldowns were used to demonstrate a direct interaction between WWOX and SMAD3. Reporter assays, ChIP, confocal microscopy and in silico analyses were employed to determine the effect of WWOX silencing on TGFβ-signaling. Results: WWOX silencing affected cell proliferation, motility, attachment and deregulated expression of genes involved in cell cycle, motility and DNA damage. Interestingly, we detected an enrichment of targets activated by the SMAD3 transcription factor, including significant upregulation of ANGPTL4, FST, PTHLH and SERPINE1 transcripts. Importantly, we demonstrate that the WWOX protein physically interacts with SMAD3 via WW domain 1. Furthermore, WWOX expression dramatically decreases SMAD3 occupancy at the ANGPTL4 and SERPINE1 promoters and significantly quenches activation of a TGFβ responsive reporter. Additionally, WWOX expression leads to redistribution of SMAD3 from the nuclear to the cytoplasmic compartment. Since the TGFβ target ANGPTL4 plays a key role in lung metastasis development, we performed a meta-analysis of ANGPTL4 expression relative to WWOX in microarray datasets from breast carcinomas. We observed a significant inverse correlation between WWOX and ANGPTL4. Furthermore, the WWOXlo/ANGPTL4hi cluster of breast tumors is enriched in triple-negative and basal-like sub-types. Tumors with this gene expression signature could represent candidates for anti-TGFβ targeted therapies. Conclusions: We show for the first time that WWOX modulates SMAD3 signaling in breast cells via direct WW-domain mediated binding and potential cytoplasmic sequestration of SMAD3 protein. Since loss of WWOX expression increases with breast cancer progression and it behaves as an inhibitor of SMAD3 transcriptional activity these observations may help explain, at least in part, the paradoxical pro-tumorigenic effects of TGFβ signaling in advanced breast cancer.Fil: Ferguson, Brent W.. University of Texas; Estados UnidosFil: Gao, Xinsheng. University of Texas; Estados UnidosFil: Zelazowski, Maciej J.. University of Texas; Estados UnidosFil: Lee, Jaeho. University of Texas; Estados UnidosFil: Jeter, Collene R.. University of Texas; Estados UnidosFil: Abba, Martín Carlos. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aldaz, Claudio Marcelo. University of Texas; Estados Unido

Wwox Deletion in Mouse B Cells Leads to Genomic Instability, Neoplastic Transformation, and Monoclonal Gammopathies

WWOX (WW domain containing oxidoreductase) expression loss is common in various cancers and characteristic of poor prognosis. Deletions, translocations, and loss of expression affecting the WWOX gene are a common feature of various B cell neoplasms such as certain B cell lymphomas and multiple myeloma. However, the role of this common abnormality in B cell tumor initiation and/or progression has not been defined. In this study, we conditionally deleted Wwox early in B cell development by means of breeding Cd19-Cre transgenic mice crossed to Wwox floxed mice (Cd19 Wwox KO). We observed a significant reduced survival in Cd19 Wwox KO mice and the development of B cell neoplasms including B cell lymphomas, plasma cell neoplasias characterized by increased numbers of CD138+ populations as well as monoclonal gammopathies detected by serum protein electrophoresis. To investigate whether Wwox loss could play a role in genomic instability, we analyzed DNA repair functions during immunoglobulin class switch joining between DNA segments in antibody genes. While class switch recombination (CSR) was only slightly impaired, Wwox deficiency resulted in a dramatic shift of double strand break (DSB) repair from normal classical-NHEJ toward the microhomology-mediated alternative-NHEJ pathway, a pathway associated with chromosome translocations and genome instability. Consistent with this, Wwox deficiency resulted in a marked increase of spontaneous translocations during CSR. This work defines for the first time a role for Wwox for maintaining B cell genome stability during a process that can promote neoplastic transformation and monoclonal gammopathies.Facultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada

Wwox seletion in mouse B cells leads to genomic instability, neoplastic transformation, and monoclonal gammopathies

WWOX (WW domain containing oxidoreductase) expression loss is common in various cancers and characteristic of poor prognosis. Deletions, translocations, and loss of expression affecting the WWOX gene are a common feature of various B cell neoplasms such as certain B cell lymphomas and multiple myeloma. However, the role of this common abnormality in B cell tumor initiation and/or progression has not been defined. In this study, we conditionally deleted Wwox early in B cell development by means of breeding Cd19-Cre transgenic mice crossed to Wwox floxed mice (Cd19 Wwox KO). We observed a significant reduced survival in Cd19 Wwox KO mice and the development of B cell neoplasms including B cell lymphomas, plasma cell neoplasias characterized by increased numbers of CD138+ populations as well as monoclonal gammopathies detected by serum protein electrophoresis. To investigate whether Wwox loss could play a role in genomic instability, we analyzed DNA repair functions during immunoglobulin class switch joining between DNA segments in antibody genes. While class switch recombination (CSR) was only slightly impaired, Wwox deficiency resulted in a dramatic shift of double strand break (DSB) repair from normal classical-NHEJ toward the microhomology-mediated alternative-NHEJ pathway, a pathway associated with chromosome translocations and genome instability. Consistent with this, Wwox deficiency resulted in a marked increase of spontaneous translocations during CSR. This work defines for the first time a role for Wwox for maintaining B cell genome stability during a process that can promote neoplastic transformation and monoclonal gammopathies.Fil: McBride, Kevin M.. University of Texas Health Science Center at Houston. University of Texas Md Anderson Cancer Center; Estados UnidosFil: Kil, Hyunsuk. University of Texas Health Science Center at Houston. University of Texas Md Anderson Cancer Center; Estados UnidosFil: Mu, Yunxiang. University of Texas Health Science Center at Houston. University of Texas Md Anderson Cancer Center; Estados UnidosFil: Plummer, Joshua B.. University of Texas Health Science Center at Houston. University of Texas Md Anderson Cancer Center; Estados UnidosFil: Lee, Jaeho. University of Texas Health Science Center at Houston. University of Texas Md Anderson Cancer Center; Estados UnidosFil: Zelazowski, Maciej J.. University of Texas Health Science Center at Houston. University of Texas Md Anderson Cancer Center; Estados UnidosFil: Sebastian, Manu. University of Texas Health Science Center at Houston. University of Texas Md Anderson Cancer Center; Estados UnidosFil: Abba, Martín Carlos. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Aldaz, Claudio Marcelo. University of Texas Health Science Center at Houston. University of Texas Md Anderson Cancer Center; Estados Unido

Analysis of DNA polymerase ν function in meiotic recombination, immunoglobulin class-switching, and DNA damage tolerance

<div><p>DNA polymerase ν (pol ν), encoded by the <i>POLN</i> gene, is an A-family DNA polymerase in vertebrates and some other animal lineages. Here we report an in-depth analysis of pol ν–defective mice and human cells. <i>POLN</i> is very weakly expressed in most tissues, with the highest relative expression in testis. We constructed multiple mouse models for <i>Poln</i> disruption and detected no anatomic abnormalities, alterations in lifespan, or changed causes of mortality. Mice with inactive <i>Poln</i> are fertile and have normal testis morphology. However, pol ν–disrupted mice have a modestly reduced crossover frequency at a meiotic recombination hot spot harboring insertion/deletion polymorphisms. These polymorphisms are suggested to generate a looped-out primer and a hairpin structure during recombination, substrates on which pol ν can operate. Pol ν-defective mice had no alteration in DNA end-joining during immunoglobulin class-switching, in contrast to animals defective in the related DNA polymerase θ (pol θ). We examined the response to DNA crosslinking agents, as purified pol ν has some ability to bypass major groove peptide adducts and residues of DNA crosslink repair. Inactivation of <i>Poln</i> in mouse embryonic fibroblasts did not alter cellular sensitivity to mitomycin C, cisplatin, or aldehydes. Depletion of <i>POLN</i> from human cells with shRNA or siRNA did not change cellular sensitivity to mitomycin C or alter the frequency of mitomycin C-induced radial chromosomes. Our results suggest a function of pol ν in meiotic homologous recombination in processing specific substrates. The restricted and more recent evolutionary appearance of pol ν (in comparison to pol θ) supports such a specialized role.</p></div

Tumor multiplicity of <i>Poln</i>^ΔEx2/ΔEx2, <i>Poln</i>^+/ΔEx2, and <i>Poln</i>^+/+ mice at end of life.

<p>Tumor multiplicity of <i>Poln</i>^ΔEx2/ΔEx2, <i>Poln</i>^+/ΔEx2, and <i>Poln</i>^+/+ mice at end of life.</p

The inactivation of pol ν does not sensitize Tag immortalized mouse embryonic fibroblasts to DNA damaging agents.

<p>Cells were exposed to indicated doses of mitomycin C for 48 hr (A), cisplatin for 48 hr (B), bleomycin for 48 hr (C), hydroxyurea for 48 hr (D), formaldehyde for 96 hr (E) and acetaldehyde for 72 hr (F). Viability was determined by measuring ATP content as described in Materials and Methods. <i>Poln</i>^+/+ MEFs (black), <i>Poln</i>^+/ΔEx2 MEFs (grey) and <i>Poln</i>^ΔEx2/ΔEx2 MEFs (blue). The mean is shown for three independently derived MEFs for each genotype, separately plated in triplicate and treated, with SD indicated by error bars.</p

Depletion of pol ν from human cells does not change cellular sensitivity to mitomycin C or alter the frequency of mitomycin C-induced radial chromosomes.

<p>(A) Upper panel: immunoblot showing efficacy of siRNA-mediated knockdown of POLN (siN), FANCA (siA), and FANCD2 (siD2) in 293FT cells and 293T-REx doxycycline inducible POLN cells. siC served as a negative control and Tubulin as loading control. Polyclonal anti-pol ν antibody (PA434) recognized overexpressed pol ν [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006818#pgen.1006818.ref003" target="_blank">3</a>] but not endogenous pol ν. (B) Cells were exposed to the indicated doses of mitomycin C for 48 hr. Viability was determined by measuring ATP content as described in Materials and Methods. The mean of three separately plated and treated plates is shown, with SD indicated by error bars. (C) Mitomycin C -induced radial chromosomes in the cells shown in A. The cells were exposed to 40 ng/mL mitomycin C for 48 hr. 100 metaphases per sample were analyzed. (D) Arrows indicate radial chromosomes in <i>POLN</i>-depleted 293FT cells exposed to mitomycin C. Scale bar: 10μm.</p

Incidence of non-neoplastic findings in <i>Poln</i>^ΔEx2/ΔEx2, <i>Poln</i>^+/ΔEx2, and <i>Poln</i>^+/+ mice at end of life.

<p>Incidence of non-neoplastic findings in <i>Poln</i>^ΔEx2/ΔEx2, <i>Poln</i>^+/ΔEx2, and <i>Poln</i>^+/+ mice at end of life.</p