Maciej J. Zelazowski

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

BackgroundThe 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.MethodsGene 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.ResultsWWOX 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.ConclusionsWe 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.

Age-Dependent Alterations in Meiotic Recombination Cause Chromosome Segregation Errors in Spermatocytes

Faithful chromosome segregation in meiosis requires crossover (CO) recombination, which is regulated to ensure at least one CO per homolog pair. We investigate the failure to ensure COs in juvenile male mice. By monitoring recombination genome-wide using cytological assays and at hotspots using molecular assays, we show that juvenile mouse spermatocytes have fewer COs relative to adults. Analysis of recombination in the absence of MLH3 provides evidence for greater utilization in juveniles of pathways involving structure-selective nucleases and alternative complexes, which can act upon precursors to generate noncrossovers (NCOs) at the expense of COs. We propose that some designated CO sites fail to mature efficiently in juveniles owing to inappropriate activity of these alternative repair pathways, leading to chromosome mis-segregation. We also find lower MutLγ focus density in juvenile human spermatocytes, suggesting that weaker CO maturation efficiency may explain why younger men have a higher risk of fathering children with Down syndrome.

X marks the spot: PRDM9 rescues hybrid sterility by finding hidden treasure in the genome

Three recent reports explore how PRDM9 binds to meiotic hotspots within the genome and provide compelling evidence that hotspot erosion leads to speciation.

Abstract 5183: Loss of WWOX induces ANGPTL4 and ROS production in breast cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Expression of the WW-domain containing oxidoreductase (WWOX) gene is lost in many human malignancies including breast cancer. It has been suggested that WWOX is potentially acting as a tumor suppressor in breast cancer. In order to understand the effects of loss of WWOX expression we used an shRNA-mediated approach to silence expression of this gene in normal human breast cells (MCF10F). Microarray analysis identified 671 commonly deregulated probes between two WWOX-silenced lines generated from shRNAs targeting two independent regions of the WWOX transcript. We found that genes involved in cell cycle/proliferation and DNA damage were significantly enriched in the list of up-regulated genes while genes involved in oxidation-reduction and wound healing were enriched in the list of down-regulated genes. ANGPTL4 was found to be one of the most significantly up-regulated genes in WWOX-silenced cells. ANGPTL4 is known to be an important player in lipid metabolism and energy balance. Importantly recent findings have demonstrated ANGPTL4 to also play key roles in breast cancer progression and metastasis. We validated the upregulation of ANGPTL4 observed in the microarray at the mRNA level by qPCR and the protein level by ELISA. We also show that this upregulation can be reversed by reestablishing WWOX expression in previously silenced cells suggesting an inverse correlation between WWOX and ANGPTL4. Supporting these findings, meta-analysis of data from three independent breast cancer gene expression studies (n= 819 cases) demonstrated a significant inverse correlation between WWOX and ANGPTL4 expression. WWOXlow/ANGPTL4high tumors were enriched in triple-negative breast cancer and basal-like tumors. ANGPTL4 signaling is known to upregulate production of reactive oxygen species (ROS) in metastatic breast cancer cells. Interestingly, we found that silencing WWOX in human breast cells or MEFs results in a significant increase in NADPH oxidase-dependent ROS production. Since ROS is a well-known inducer of DNA damage we hypothesized that WWOX silenced cells could have higher levels of DNA damage. In tune with this hypothesis, further analysis of our gene expression data set revealed significant upregulation of multiple genes involved in DNA repair including BRCA1, RAD51 and FANCA in WWOX-silenced MCF10 cells. In additional studies, we determined that WWOX KO MEFs display significantly higher levels of DNA double-strand breaks when compared with WT counterparts as determined by colabeling with 53BP1 and phospho-H2AX antibodies. In summary, we show that loss of the putative tumor suppressor WWOX results in increased levels of the breast metastasis associated gene ANGPTL4 in breast cells and is associated with increased levels of ROS. Citation Format: Brent W. Ferguson, Xinsheng Gao, Maciej Zelazowski, Sabine Lange, Martin C. Abba, Richard D. Wood, C. Marcelo Aldaz. Loss of WWOX induces ANGPTL4 and ROS production in breast cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5183. doi:10.1158/1538-7445.AM2013-5183

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

DNA polymerase ν (pol ν), encoded by the POLN 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. POLN is very weakly expressed in most tissues, with the highest relative expression in testis. We constructed multiple mouse models for Poln disruption and detected no anatomic abnormalities, alterations in lifespan, or changed causes of mortality. Mice with inactive Poln 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 Poln in mouse embryonic fibroblasts did not alter cellular sensitivity to mitomycin C, cisplatin, or aldehydes. Depletion of POLN 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.