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Intrinsically Disordered SRC-3/AIB1 Protein Undergoes Homeostatic Nuclear Extrusion by Nuclear Budding While Ectopic Expression Induces Nucleophagy

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Intrinsically Disordered SRC-3/AIB1 Protein Undergoes Homeostatic Nuclear Extrusion by Nuclear Budding While Ectopic Expression Induces Nucleophagy

Miguel A Cabrita et al. Cells.

Abstract

SRC-3/AIB1 (Amplified in Breast Cancer-1) is a nuclear receptor coactivator for the estrogen receptor in breast cancer cells. It is also an intrinsically disordered protein when not engaged with transcriptional binding partners and degraded upon transcriptional coactivation. Given the amplified expression of SRC-3 in breast cancers, the objective of this study was to determine how increasing SRC-3 protein levels are regulated in MCF-7 breast cancer cells. We found that endogenous SRC-3 was expelled from the nucleus in vesicle-like spheres under normal growth conditions suggesting that this form of nuclear exclusion of SRC-3 is a homeostatic mechanism for regulating nuclear SRC-3 protein. Only SRC-3 not associated with CREB-binding protein (CBP) was extruded from the nucleus. We found that overexpression in MCF-7 cells results in aneuploid senescence and cell death with frequent formation of nuclear aggregates which were consistently juxtaposed to perinuclear microtubules. Transfected SRC-3 was SUMOylated and caused redistribution of nuclear promyelocytic leukemia (PML) bodies and perturbation of the nuclear membrane lamin B1, hallmarks of nucleophagy. Increased SRC-3 protein-induced autophagy and resulted in SUMO-1 localization to the nuclear membrane and formation of protrusions variously containing SRC-3 and chromatin. Aspects of SRC-3 overexpression and toxicity were recapitulated following treatment with clinically relevant agents that stabilize SRC-3 in breast cancer cells. We conclude that amplified SRC-3 levels have major impacts on nuclear protein quality control pathways and may mark cancer cells for sensitivity to protein stabilizing therapeutics.

Keywords: PML; intrinsically disordered protein; microtubules; nuclear protein quality control; nucleophagy; protein aggregation; transcriptional coactivator.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
SRC-3 undergoes nuclear extrusion dependent on association with CBP. (A) MCF-7 cells cultured as described in Methods were subjected to IF for SRC-3. DNA was visualized with DAPI. Arrows indicate nuclear SRC-3 protrusions (63× magnification). (B) MCF-7 cells transfected with SRC-3 (tSRC-3) in the absence (endogenous only- eCBP)(panel i) or presence of cotransfected (tCBP) CBP (panel ii). In panel (i) Z-stack images show SRC-3 aggregates overlapping with diffuse CBP (red arrows). CBP not associated with SRC-3 is indicated by the green arrow. In panel (ii) some aggregates merge as CBP and SRC-3 foci (white arrows), while others near the nuclear periphery contain only SRC-3 (red arrows) (100× magnification).
Figure 2
Figure 2
Establishment of SRC-3-overexpressing MCF-7 cells. (A) Immunoblot analysis showing SRC-3 protein expression in MCF-7 cells transfected with pCMX-RAC3 (SRC-3) or pcDNA3 and selected for puromycin resistance. SRC-3 stable cells were categorized according to the relative size of the average cell within a colony prior to expansion. Examples of clones that survived at least one passage are shown: control pcDNA3 cells (1,2,3), small-sized SRC-3 transfected clones (S1,2,5) and medium-sized SRC-3 clones (M1,2,4). Large cell clones did not survive passage. (B) Immunoblot of original clones showing SRC-3 expression relative to empty vector-transfected control. (C) Flow analysis of DNA content derived from the indicated clones. (D) DNA content graphs derived from the analysis of several clones. Bars are SD of means from: pcDNA control (n = 4), SRC-3 small (n = 15), SRC-3 medium (n = 4). (E) Phase-contrast images of control pcDNA3- and pCMX-RAC3- (SRC-3) transfected MCF-7 cells. Panels are (i) Control clones, (ii) small SRC-3-overexpressing clones, (iii and iv) medium SRC-3-overexpressing clones which were enlarged and flat with abundant cytoplasm. (F) Cell lysates harvested after infection at the indicated times after infection with Ad-LacZ or Ad-SRC-3 were probed with antibodies to SRC-3, P-Chk2, Chk2 (denoted by arrowhead), p21 and actin. (G) MCF-7 cells infected with the Ad-RFP and Ad-SRC-3 viruses were cultured for 72 h and assayed for senescence-associated (SA) β-galactosidase activity. (H) Immunoblot for SRC-3, cyclin E and PARP-1 in MCF-7 cell lysates 72 h post-transfection with empty vector (EV), wtSRC-3 or the stable mutant SRC-3(S102A). Note that although highly expressed relative to wtSRC-3 and to the gel loading control, S102A does not induce transcription of cyclin E. Actin was used as a protein loading control.
Figure 3
Figure 3
Overexpressed SRC-3 rapidly forms nuclear aggregates. (A) GFP imaging of MCF-7 cells 72 h post-transfection with wtSRC-3, three different SRC-3-GFP phospho-mutants, and SRC-3 deleted for the polyQ region (residues 1230–1300). 63× magnification (B) Still images from video-microscopy of aggregation of YFP-SRC-3 in transfected cells. MCF-7 cells were transfected with YFP-SRC-3 and microscopy was performed 24 h later on an Axiovert 200M inverted fluorescent microscope (Carl Zeiss, Toronto, ON, Canada) for a total of 24 using Axiovision 4.8 acquisition software (Carl Zeiss). Images were acquired using a 10× objective (EC Plan-Neofluar) with a side-mounted AxiocamHRm camera (Carl Zeiss). YFP was excited using the Colibri LED illumination system (LEDmodule 505nm, Carl Zeiss) and detected using the 46HEYFP filter (Carl Zeiss). Exposure times were 1 ms (brightfield/phase contrast) and 100ms (YFP) at 10 min intervals for 24 h and compiled into video files using Axiovision 4.8 software (Carl Zeiss). 20 min intervals are shown. Cells circled in blue showed continuous accumulation of SRC-3 while cells circled in orange appeared to resolve aggregates.
Figure 4
Figure 4
SRC-3 overexpression induces autophagy but does not affect the proteasome. (A) MCF-7 cells stably transfected with a nuclear-targeted GFP (NLS-GFPu) were mock-infected, or infected with Ad-SRC-3 or Ad-Lac-Z. Cells harvested 72 h after infection with or without 10 μM MG132 for the final 3 h of culture. Immunoblots were probed with anti-GFP and anti-SRC-3. Anti-actin reactivity was used as a control for protein loading on anti-GFP-probed blots. E-cadherin or vinculin were used as loading controls for blots probed with anti-SRC-3. (B) Cells infected as in A were seeded on coverslips and IF was performed after 72 h. GFP (green), anti-SRC-3/CY5 (red) and DAPI fluorescence are shown (63× magnification). (C) Graph depicting average of uGFP pixels/nuclear area within nuclei of MCF-7 cells described as above in A. Bars are S.E.M. (D) Immunoblots to detect the activation of the unfolded-protein response in LCC9 (L) (a tamoxifen-resistant MCF-7 derivative [30]) and MCF-7 (M) cells uninfected or infected with adenoviral (Ad)-LacZ or Ad-SRC-3. Actin reactivity serves as a protein loading control. (E) MCF-7 cells stably expressing LC3-EGFP were infected with Ad-SRC-3 or Ad-RFP. Anti-SRC-3 and GFP-LC-3 were detected by epifluorescence microscopy (63× magnification). (F) MCF-7 cells were infected with Ad-Lac Z or Ad-SRC-3 and treated with a vehicle or 20 μM HCQ for 48 h. Lysates were immunoblotted for SRC-3, LC3 or actin, (LE denotes long exposure).
Figure 5
Figure 5
SRC-3 aggregates are associated with microtubules, redistribute PML and SUMO and compromise nuclear membrane integrity. (A) 3D deconvoluted model constructed as described in Materials and Methods using Z-stacks acquired with confocal microscopy from SRC-3-transfected cells and immunostained for SRC-3 (red) and β-tubulin (green) and imaged. Blue is DAPI (100× magnification). (B) Immunofluorescence of β-tubulin (green) and SRC-3 (red) showing regions of increased perinuclear tubulin density in SRC-3 overexpressing cells. Nuclear protrusion of SRC-3 and DNA (blue) are indicated with arrows (63× magnification). (C) SRC-3 expression interferes with mitotic microtubule dynamics. MCF-7 cells were infected with Ad-LacZ or Ad-SRC-3 on coverslips and 48 h later were treated with taxol for 18hrs and IF performed to detect mitotic microtubules and SRC-3 (63× magnification). (D) Cells treated in C were enumerated for the presence of mitotic figures. Bars are S.D., ** p < 0.01. (E) Z-stack images of cells transfected with SRC-3-YFP and immunostained for lamin B1 (red) and tubulin (green) immunofluorescence showing nuclear membrane distortion, and degradation associated with cytoplasmic SRC-3 aggregates (100× magnification). (F) IF of lamin B1 in SRC-3-GFP-transfected cells showing cells with discontinuous nuclear membranes, lamin B1 fragments, and distortions of nuclear shape.
Figure 6
Figure 6
Effects of increased SRC-3 expression on PML: (A) IF of PML and SRC-3 in SRC-3 transfected cells shows ring-like aggregates of SRC-3 adjacent to PML bodies. (B) Dispersion of PML bodies (circled nuclei in panels i and ii’) in MCF-7 cells containing varying levels of homogenously distributed SRC-3. Nuclear membrane accumulation into caps of PML was observed in a subset of SRC-3 transfected cells (arrow in panels ii and ii’) (63× magnification). (C) Diffuse accumulations of PML form near the nuclear membrane. Cytoplasmic SRC-3(red open arrow) and PML (green open arrow). (D) Immunoblot of PML 96hrs following SRC-3 transfection. “V” denotes empty vector control. Vinculin was used as a protein loading control.
Figure 7
Figure 7
Overexpressed SRC-3 is SUMOylated and disrupts the SUMO pathway. (A) Immunoprecipitation of protein from control (pcDNA3) or SRC-3 transfected MCF-7 cells using anti-SRC-3 or non-immune IgG was immunoblotted with anti-SUMO and anti-SRC-3. (B) SRC-3 immunoblot of input cell lysates in A as indicated. (C) MCF-7 cells were transfected with SRC-3-GFP then IF performed for UBC9. A representative cell expressing transfected SRC-3 (SRC-3-GFP+) is shown (63× magnification). (D) Graph depicting mean of UBC9 pixels/nuclear area within cells transfected with SRC-3-GFP (n = 12 GFPpos cells and n = 20 GFPneg cells as shown) or transfected with pcDNA3 (n = 25 cells) ± S.E. * p < 0.05 (paired t-test). (E) Examples of SUMO IF in SRC-3 overexpressing MCF-7 cells. Panel (i): SUMO-1 puncta in the nucleus and associated with an SRC-3/chromatin nuclear protrusion. Panel (ii): Nuclear membrane-associated SUMO-1 with loss of nuclear puncta in an SRC-3 overexpressing cell. Panel (iii): MCF-7 cell with SUMO-1 aggregates and SUMO-1 distribution at the nuclear membrane. Note the absence of detectable nucleoli. Images on the right of each panel represent inverted and posturized (IP) DAPI channel images (Photoshop CS6) to visualize extranuclear DNA. (F) RanGAP1 IF (red) in MCF-7 cells transfected with SRC-3 (green) shows redistribution of ranGAP1 at the nuclear periphery. DNA is stained with DAPI (blue) (E and F, 63× magnification).
Figure 8
Figure 8
Pharmacological protein stabilization results in strong increases in cytoplasmic SRC-3 and correlates with growth inhibition. (A) Immunoblot of SRC-3 after a 48hr treatment with vehicle, HCQ, B or HCQ+B. Vinculin immunoreactivity was used is a protein loading control. (B) MCF-7 cells were treated as indicated for 96 h and viability assessed by Alamar blue assay. Cells were assayed in quadruplicate. Bars are mean ± S.D. ** p < 0.01, *** p < 0.01 (One-way ANOVA). (C) IF of SRC-3 in MCF-7 cells treated with vehicle or B or HCQ+B for 48hrs. Panels (i, ii, iii) merged DAPI (blue)/anti-SRC-3 (red) pseudocolored IF, panels (i’,ii’,iii’) anti-SRC-3 epifluorescence only (identical exposure images inverted in Photoshop CS6). Panel iii’’ represents DAPI only (grayscale) enlargement of the boxed area in (iii). Arrows point to extranuclear chromatin. All images 63× magnification. (D) Immunoblot for SRC-3 in MCF-7 lysates from cells treated for the indicated times in 5 μM tamoxifen. Vinculin was used as a protein loading control. (E), IF for SRC-3 in cells treated with vehicle or 5 μM Tam for 3 h. Cells with SRC-3 nuclear protrusions are boxed. Cells with reduced nucleoli are indicated with closed arrows. (F), Nucleolar regions were enumerated from DAPI-stained images in cultures treated as in (E). Bars are S.E.M. ** p < 0.01. (G), IF to detect PML protein in MCF-7 cells treated as in (E). (H) Enumeration of discrete PML bodies in cells treated as in (E). Bars are S.E.M. *** p < 0.001(paired t-test).

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