BRD4 Short Isoform Interacts with RRP1B, SIPA1 and Components of the LINC Complex at the Inner Face of the Nuclear Membrane

<div><p>Recent studies suggest that BET inhibitors are effective anti-cancer therapeutics. Here we show that BET inhibitors are effective against murine primary mammary tumors, but not pulmonary metastases. <i>BRD4</i>, a target of BET inhibitors, encodes two isoforms with opposite effects on tumor progression. To gain insights into why BET inhibition was ineffective against metastases the pro-metastatic short isoform of BRD4 was characterized using mass spectrometry and cellular fractionation. Our data show that the pro-metastatic short isoform interacts with the LINC complex and the metastasis-associated proteins RRP1B and SIPA1 at the inner face of the nuclear membrane. Furthermore, histone binding arrays revealed that the short isoform has a broader acetylated histone binding pattern relative to the long isoform. These differential biochemical and nuclear localization properties revealed in our study provide novel insights into the opposing roles of BRD4 isoforms in metastatic breast cancer progression.</p> </div

BRD4-SF interacts with SIPA1.

<p>(A) Interaction of epitope-tagged BRD4-SF and SIPA1 by reciprocal Co-IP in HEK293 cells using antibodies against the epitope tags, followed by western blot analysis. (B) Co-immunofluorescence of epitope-tagged SIPA1 (red) and SUN2 (green) in HeLa cells. Blue; DAPI: double-stranded DNA. (C) BiFC analysis of SIPA1 and SUN2 in HeLa cells followed by confocal microscopy. Green: fluorescence complementation, blue; DAPI: double-stranded DNA. (D) Immunofluorescence of a SIPA1 construct that has an SV40 nuclear localization signal (green) in HeLa cells followed by confocal microscopy. (E) Western blot analysis performed on the cytoplasmic fraction of HeLa cells isolated using Lamond lab cellular fractionation protocol after high speed centrifugation at 100,000 x g. Supernatant: cytosolic extract, pellet: membrane-enriched extract. (F) Immunohistochemical staining of breast cancer TMAs from the Human Protein Atlas for SIPA1 protein. All confocal microscopy pictures were taken at 63x magnification.</p

BRD4-SF interacts with RRP1B.

<p>(A) Interaction of epitope-tagged BRD4-SF and RRP1B by reciprocal Co-IP in HEK293 cells using antibodies against the epitope tags, followed by western blot analysis. (B, C) BiFC analysis of BRD4-SF and RRP1B in HeLa cells followed by confocal microscopy. Green: fluorescence complementation, blue; DAPI: double-stranded DNA. (D) Co-immunofluorescence of GFP-BRD4-SF (green) and mCherry-RRP1B (red) in HeLa cells followed by confocal microscopy. Blue; DAPI: double-stranded DNA. All pictures were taken at 63x magnification using confocal microscopy.</p

BRD4 isoforms have different binding affinities to histones.

<p>(A) MODified Histone Peptide Array of whole cell lysate from HEK293 cells transiently transfected with FLAG-tagged BRD4-LF, FLAG-tagged BRD4-SF or V5-tagged point mutations of both isoforms. The arrays were visualized using ECL plus western blot detecting reagent. (B) The specificity factor calculated as the ratio of average intensity of all spots containing the mark divided by average intensity of all spots not containing the mark. The graph shows the 10 modifications with the greatest specificity factors.</p

I-BET151 reduces primary tumor but not metastasis.

<p>(A) Layout of I-BET151 mouse experiment. FVB/NJ female mice were injected orthotopically into the mammary fat pad with Mvt1 or 6DT1 cells and 10 days post cell-injection, mice were treated daily with I-BET151 (30 mg/kg) or vehicle for 17 days. Mice were euthanized 27 days after cell injection. (B) Primary tumor weight of FVB/NJ female mice injected orthotopically into the mammary fat pad with Mvt1 or 6DT1 cell lines then treated with I-BET151 or vehicle. Weight assessed 27 days post-cell injection. (C) Quantification of pulmonary metastasis nodules after sectioning and H&E staining of lungs of mice injected orthotopically with cells then treated with I-BET151 or vehicle. (D) Normalization of pulmonary metastasis nodules count from H&E staining by primary tumor mass. Means ± SD, n=10 per group (n=9 for I-BET151-treated Mvt1-injected mice). <i>P</i> value calculated using Mann-Whitney test.</p

Epitope-tagged BRD4-SF localizes at nuclear membrane and interacts with SUN2.

<p>(A) Cellular fractionation of HeLa cells stably expressing myc-tagged BRD4-SF using Lamond lab protocol followed by western blot analysis. Control cells were transduced with an empty vector. (B) Confocal microscopy showing co-immunofluorescence of HeLa cells ectopically expressing myc-tagged BRD4-SF (green) after co-staining with the nuclear envelope marker lamin B1 (LMNB1; red) and double-stranded DNA (DAPI; blue). (C) Immunoprecipitation of HeLa cells stably expressing myc-tagged BRD4-SF using modified formaldehyde cross-linking protocol and anti-myc antibody for immunoprecipitation followed by western blot analysis. (D) BiFC analysis of BRD4-SF and SUN2 in HeLa cells followed by confocal microscopy. Green: fluorescence complementation, blue; DAPI: double-stranded DNA. (E) Immunoprecipitation from nuclear membrane-enriched fraction of HeLa cells ectopically expressing myc-tagged BRD4-SF using anti-myc antibody for immunoprecipitation followed by western blot analysis. All pictures were taken at 63x magnification using confocal microscopy.</p

BRD4-SF interacts with RRP1B at the nuclear periphery.

<p>(A) BiFC analysis of RRP1B and SUN2 in HeLa cells followed by confocal microscopy. Green: fluorescence complementation, blue; DAPI: double-stranded DNA. Pictures were taken at 63x magnification using confocal microscopy. (B) Immunohistochemical staining of breast cancer TMAs from the Human Protein Atlas for RRP1B expression.</p

BRD4-SF localizes at the nuclear membrane.

<p>(A, B) Cellular fractionation experiments of HeLa cells using either Lamond lab protocol, protocol 1 (A) or Gardner lab protocol, protocol 2 (B) followed by western blot analysis. </p

MOESM10 of Assessing histone demethylase inhibitors in cells: lessons learned

Additional file 10. Methods and chemical synthesis