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

Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control

Abstract p38 mitogen-activated protein kinases (MAPKs) participate in autophagic signaling; and previous reports suggest that pyridinyl imidazole p38 MAPK inhibitors, including SB203580 and SB202190, induce cell death in some cancer cell-types through unrestrained autophagy. Subsequent studies, however, have suggested that the associated cytoplasmic vacuolation resulted from off-target inhibition of an unidentified enzyme. Herein, we report that SB203580-induced vacuolation is rapid, reversible, and relies on the class III phosphatidylinositol 3-kinase (PIK3C3) complex and the production of phosphatidylinositol 3-phosphate [PI(3)P] but not on autophagy per se. Rather, vacuolation resulted from the accumulation of Rab7 on late endosome and lysosome (LEL) membranes, combined with an osmotic imbalance that triggered severe swelling in these organelles. Inhibition of PIKfyve, the lipid kinase that converts PI(3)P to PI(3,5)P2 on LEL membranes, produced a similar phenotype in cells; therefore, we performed in vitro kinase assays and discovered that both SB203580 and SB202190 directly inhibited recombinant PIKfyve. Cancer cells treated with either drug likewise displayed significant reductions in the endogenous levels of PI(3,5)P2. Despite these results, SB203580-induced vacuolation was not entirely due to off-target inhibition of PIKfyve, as a drug-resistant p38α mutant suppressed vacuolation; and combined genetic deletion of both p38α and p38β dramatically sensitized cells to established PIKfyve inhibitors, including YM201636 and apilimod. The rate of vacuole dissolution (i.e., LEL fission), following the removal of apilimod, was also significantly reduced in cells treated with BIRB-796, a structurally unrelated p38 MAPK inhibitor. Thus, our studies indicate that pyridinyl imidazole p38 MAPK inhibitors induce cytoplasmic vacuolation through the combined inhibition of both PIKfyve and p38 MAPKs, and more generally, that p38 MAPKs act epistatically to PIKfyve, most likely to promote LEL fission

Nanog1 in NTERA-2 and recombinant NanogP8 from somatic cancer cells adopt multiple protein conformations and migrate at multiple M.W species.

Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8, which is ~99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/NanogP8 is ∼35 kD, both have been reported to migrate, on Western blotting (WB), at apparent molecular masses of 29-80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore, detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies, immunoprecipitation, mass spectrometry, and studies using recombinant proteins, here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ~22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8) proteins made in bacteria using cDNAs from multiple cancer cells also migrate, on denaturing SDS-PAGE, at ~28 kD to 180 kD. Interestingly, different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins, which can spontaneously form high M.W protein species. Finally, we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether, the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells

Nanog proteins in NTERA-2 human EC cells.^*

<p>*Presented are the results of various experiments using the 8 anti-Nanog Abs. The Biolegend Ab is the only Ab that does not recognize the 42 kD band as the major protein band and does not recognize the 35 kD band. Presented molecular masses are all estimated based on their migrations on SDS-PAGE.</p

WB analysis of endogenous Nanog1 protein species in NTERA-2 cells.

<p>(<b>A</b>) Schematic of the human Nanog protein and 8 anti-Nanog Abs used in this study. Shown in parentheses are epitopes of individual Abs. ND, N-terminus domain; HD, homeodomain; CD1 and CD2, C-terminus domain 1 and 2; WR, tryptophan-rich domain. The asterisk in ND indicates the Leu61 residue recognized by the eBioscience mAb (arrow) mapped from our present studies. (B–H) WB analysis in NTERA-2 NE (N, two different batches) or cytosol (C) using 8 anti-Nanog Abs. Individual Ab is indicated at the bottom and M.W on the left. Black arrowhead, the predicted 35 kD Nanog protein; red arrowhead, the main 42 kD band; green arrows, additional bands (especially after longer exposures).</p

Examples of Nanog antibodies and their recognized protein bands.

<p>*This table lists the information for the 8 antibodies used in our current study, together with several others commercial and home-made antibodies. Shown in parenthesis are catalog numbers.</p><p><b>Abbreviations</b>: EC, embryonal carcinoma; EG, embryonic germ cells; hESCs, human embryonic stem cells; hNanog,</p><p>human Nanog; mESCs, mouse embryonic stem cells; mAb, monoclonal antibody; mNanog, mouse Nanog protein;</p><p>pAb, polyclonal antibody; Rb, rabbit; rhNanog, recombinant human Nanog protein; SC, Santa Cruz.</p

IP and ID studies with 5 anti-Nanog antibodies in NTERA-2 and cancer cells.

<p>(<b>A</b>) The NE of NTERA-2 and various cancer cells was used in IP with the Kamiya anti-Nanog Rb pAb followed by WB with the eBioscience mAb. Lanes 1-6 were regular WB with either cytosol (C) or NE. Red arrowhead, the 42 kD Nanog band; IgH, IgG heavy chain (∼53 kD). Note that the prominent 42 kD protein band was detected on WB (lane 4) and immunoprecipitated down (lane 9) <i>only</i> in NTERA-2 NE. (<b>B</b>) The NTERA-2 NE or Du145 cytosol (cyto) or NE was used in IP with the CS anti-Nanog rabbit mAb followed by WB with the R&D goat pAb. Lanes 1-3 were regular WB. Red arrowhead, the 42 kD Nanog band; IgH, IgG heavy chain. Note that the 42 kD Nanog protein was detected on WB (lane 1) and immunoprecipitated down (lane 9) only in NTERA-2 NE. (<b>C</b>) The NE of NTERA-2 and Du145 cells was used in IP with the SC anti-Nanog rabbit pAb H155 followed by WB with eBioscience mAb. Lanes 1–4 were regular WB using two independent preparations of Du145 or NTERA-2 NE. Red arrowhead, the 42-kD band; black arrowhead, the 35-kD Nanog band; IgH, IgG heavy chain. Note that the 42-kD protein band was detected on WB (lane 3 and 4) and immunoprecipitated down (lane 8) <i>only</i> in NTERA-2 NE. The right-pointing bracket indicates the cluster of Nanog protein bands below the dominant 42 kD band. (<b>D</b>) The NE of NTERA-2 cells and MCF7 cells (two independent preparations) was used in IP with the R&D anti-Nanog goat pAb (goat IgG used as the control) followed by WB with the eBioscience mAb. Red arrowhead, the 42 kD Nanog band; IgH, IgG heavy chain. Note that the 42-kD protein band was detected on WB (lane 4; input) and immunoprecipitated down (lane 3) only in NTERA-2 NE. (<b>E–F</b>) Nanog protein ID by MALDI-TOF/TOF in NTERA-2 NE following IP using the R&D goat pAb. Shown are SYPRO Ruby gel image (E; NTRD1-4 refer to the 4 gel slices cut out for protein elution) and the Nanog peptides recovered from each gel slice (F).</p

Reactivity of rNanogP8 and rNanog proteins towards 8 anti-Nanog Abs.

<p>WB analysis using 8 anti-Nanog Abs (A-H). Cell types from which the initial cDNAs were cloned are indicated on top. Individual Abs are indicated on the right and M.W on the left. For some Abs, both a long (LE) and short (SE) exposures were shown. N: non-induced; I: induced by IPTG (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090615#s2" target="_blank">Methods</a>). The red arrowheads in each panel indicate the 42 kD major Nanog protein and green arrows point to minor upper bands. In panel F, the two arrows point to the ∼48/54 kD doublets recognized by the BioLegend Rb pAb.</p