Journal: American Journal of Cancer Research

Article Title: Nuclear moonlighting of the secreted growth factor heregulin drives endocrine-resistant breast cancer independently of HER2/HER3 signaling

doi:

Figure Lengend Snippet: The isolated deficiency of nuclear heregulin-β2 but not of secreted heregulin-β2 to bind HER receptors abolishes heregulin-β2-driven endocrine resistance in vitro and in vivo. A. Estradiol (E2)-depleted cells were plated in soft agarose containing E2 (10-9 mol/L), tamoxifen (10-7 mol/L), fulvestrant (10-7 mol/L), their combinations, or ethanol (v/v) or DMSO (v/v) vehicle only for 7-10 days. Colony formation (≥ 60 μm) was assessed using a colony counter. Each experimental value represents the mean colony number (columns) ± S.D. (bars) from at least three separate experiments in which triplicate dishes were counted. B. Representative immunoblotting analyses showing the expression of P-HER/total HER, P-AKT/total AKT, P-MAPK/total MAPK, CYR61, ERα, and MTA1, in cells cultured in the absence and presence of serum. C. Shown are the mean tumor volumes (±SD) in MCF-7/pBABE, MCF-7/HRG, MCF-7/HRG∆NLS, MCF-7/HRG∆EGF, and MCF-7/HRG∆EGF + ∆NLS xenograft-bearing nude mice following treatment with estradiol, tamoxifen (TAM), fulvestrant (FVT), or their combinations for seven weeks (n = 10 animals/experimental group).

Article Snippet: Goat polyclonal anti-MTA1 antibody (45-094) was purchased from ProSci (Poway, CA).

Techniques: Isolation, In Vitro, In Vivo, Western Blot, Expressing, Cell Culture

Journal: American Journal of Cancer Research

Article Title: Nuclear moonlighting of the secreted growth factor heregulin drives endocrine-resistant breast cancer independently of HER2/HER3 signaling

doi:

Figure Lengend Snippet: Nuclear moonlighting of heregulin-β2 drives endocrine resistance in breast cancer: A working model. A. Isolated deficiency of nuclear HRG abolishes the HRG-driven endocrine resistance phenotype in vitro and in mouse xenograft models, while preserving its capacity to activate the HRG/HER/MAPK autocrine signaling axis. Conversely, isolated deficiency of secreted HRG to bind HER receptors does not impair endocrine resistance. B. Left. Nucleolin is a/the key driver of HRG nuclear shuttling in breast cancer cells. HRG interacts with nucleolin via a nuclear localization signal motif located at the N-terminal extracellular domain of HRG. Nucleolin interacts with HRG via aspartate/glutamate-rich acidic stretches located at the N-terminal domain of nucleolin. Right. MTA proteins (MTA1/2) are part of the chromatin and histone deacetylation complex (NuRD), which contains histone deacetylases (HDAC1/2) and functions in chromatin remodeling and histone deacetylation to drive biologically aggressive cancer phenotypes. MTA1 can directly interact with the ligand-binding domain of the ERα and nuclear HRG can stimulate the association of MTA1 with HDAC2 on the chromatin site of an ER-responsive element (ERE) in the promoter regions of estrogen responsive genes [32]. The repressive function of MTA1 protein on ERα is mediated via histone deacetylation by HDAC1/2, thereby suggesting that nuclear HRG might enhance/stabilize the potent co-repressor epigenetic function of NuRD during the trans-activation of ERα. The MTA1-containing NuRD complex can deacetylate also non-histone proteins such as the tumor-suppressor PTEN. In the presence of nuclear HRG, the MTA1/HDAC complex might be stabilized and localized in the nucleus where it can bind to acetylated PTEN. The MTA1/HDAC complex drives deacetylation (inactivation) of PTEN, which can diffuse into the cytoplasmic compartment but lacking a tumor suppressive function against phospho-active AKT. The isolated deficiency of nuclear HRG suffices to down-regulate MTA1 and disrupt MTA1/HDAC functionality, thereby increasing the acetylated (active) form of PTEN that can then inhibit AKT activity while preserving the HRG capacity to activate the HER/MAPK autocrine signaling axis. The ability of nuclear HRG to drive alterations in acetylation of both histone and non-histone proteins via regulation of the master co-regulatory molecule MTA-1 might explain how the nucleolin-driven nuclear moonlighting of HRG uncouples its well-known role as a driver of endocrine resistance from its canonical HER receptor-activating role in breast cancer cells.

Article Snippet: Goat polyclonal anti-MTA1 antibody (45-094) was purchased from ProSci (Poway, CA).

Techniques: Isolation, In Vitro, Preserving, Ligand Binding Assay, Activation Assay, Activity Assay

Journal: Biology of Reproduction

Article Title: Metastasis Tumor Antigen 2 (MTA2) Is Involved in Proper Imprinted Expression of H19 and Peg3 During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.110.086397

Figure Lengend Snippet: Mta RNA and protein expression patterns in oocyte and preimplantation embryos. A) Temporal pattern of expression of Mta1, Mta2, and Mta3. The experiment was conducted twice, and the data are expressed as mean ± range and are expressed relative to the value obtained for full-grown oocytes. The amount of Mta RNA was normalized to Gfp mRNA that was added as an external control prior to RNA isolation. B) Immunoblot analysis of MTA1, MTA2, and MTA3 expression. One hundred oocytes/embryos were loaded per lane, and beta-tubulin (TUBB) was used as a loading control. The experiment was conducted three times, and similar results were obtained in each case; a representative experiment is shown. C) Immunocytochemical analysis of MTA1, MTA2, and MTA3 expression. All samples for a given MTA were processed for immunocytochemistry together, and all images were taken at the same laser power, thereby enabling direct comparison of signal intensities. The experiment was conducted three times, and at least 25 oocytes/embryos were analyzed for each sample. Shown are representative examples. INC, incompetent oocyte; GV, fully grown oocyte; MII, metaphase II; 1C, one-cell embryo; 2C, two-cell embryo; 4C, four-cell embryo; 8C, eight-cell embryo; BL, blastocyst. Original magnification x80.

Article Snippet: The qRT-PCR analysis was performed with the ABI Taqman Assay-on-demand probe/primer sets for Mta1 (Mm00475337_m1), Mta2 (Mm00488671_m1), and Mta3 (Mm00475365_m1).

Techniques: Expressing, Isolation, Western Blot, Immunocytochemistry

Journal: Biology of Reproduction

Article Title: Metastasis Tumor Antigen 2 (MTA2) Is Involved in Proper Imprinted Expression of H19 and Peg3 During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.110.086397

Figure Lengend Snippet: Effects of RNAi-mediated knockdown of Mta2 on Mta1, Mta2, and Mta3 expression, and localization and amount of MTA1, MTA2, and MTA3. A) One-cell embryos were injected with either Gfp dsRNA (control) or Mta2 dsRNA and then cultured for 24, 48, or 72 h, at which time the relative abundance of Mta1, Mta2, and Mta3 transcripts was assayed by qRT-PCR and expressed relative to controls. The experiment was performed three times, and the data are expressed as mean ± SEM. B) The experiment was performed as described in A, and the samples were processed for immunocytochemical detection of MTA 1, 2, or 3 at the indicated times. The experiment was conducted three times, and at least 25 oocytes/embryos were analyzed for each sample. Original magnification x80. C) The experiment was performed as described in A, and the relative amount of MTA1 and MTA2 was determined by immunoblot analysis after 72 h of culture. The experiment was performed twice, and similar results were obtained. One hundred embryos were used for immunoblot analysis.

Article Snippet: The qRT-PCR analysis was performed with the ABI Taqman Assay-on-demand probe/primer sets for Mta1 (Mm00475337_m1), Mta2 (Mm00488671_m1), and Mta3 (Mm00475365_m1).

Techniques: Expressing, Injection, Cell Culture, Quantitative RT-PCR, Western Blot