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. 2013 Jul 8;24(1):30-44.
doi: 10.1016/j.ccr.2013.05.007. Epub 2013 Jun 13.

Par-4 downregulation promotes breast cancer recurrence by preventing multinucleation following targeted therapy

James V Alvarez  1 , Tien-Chi Pan, Jason Ruth, Yi Feng, Alice Zhou, Dhruv Pant, Joshua S Grimley, Thomas J Wandless, Angela Demichele; I-SPY 1 TRIAL Investigators; Lewis A Chodosh
Affiliations

Par-4 downregulation promotes breast cancer recurrence by preventing multinucleation following targeted therapy

James V Alvarez et al. Cancer Cell. .

Abstract

Most deaths from breast cancer result from tumor recurrence, but mechanisms underlying tumor relapse are largely unknown. We now report that Par-4 is downregulated during tumor recurrence and that Par-4 downregulation is necessary and sufficient to promote recurrence. Tumor cells with low Par-4 expression survive therapy by evading a program of Par-4-dependent multinucleation and apoptosis that is otherwise engaged following treatment. Low Par-4 expression is associated with poor response to neoadjuvant chemotherapy and an increased risk of relapse in patients with breast cancer, and Par-4 is downregulated in residual tumor cells that survive neoadjuvant chemotherapy. Our findings identify Par-4-induced multinucleation as a mechanism of cell death in oncogene-addicted cells and establish Par-4 as a negative regulator of breast cancer recurrence.

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Figures

Figure 1
Figure 1. Par-4 is down-regulated in recurrent mammary tumors
A. qRT-PCR analysis and B–D. Western analysis showing Par-4 expression in primary and recurrent HER2/neu, MYC, and Wnt1; p53+/− tumors. E. Quantification of Par-4 protein levels, normalized to tubulin. F. IF analysis of Par-4 in primary and recurrent HER2/neu tumors. Scale bar = 50 μm. Error bars denote mean +/− SEM. * p<.05, ** p<.01, *** p<.001. See also Figure S1.
Figure 2
Figure 2. Decreased Par-4 expression predicts poor response to neoadjuvant chemotherapy and is selected for following treatment
A. 5-year RFS for breast cancer patients in the I-SPY1 neoadjuvant trial with Hazards Ratio, 95% confidence intervals, and adjusted p-value. B. Par-4 expression in primary human breast cancers prior to treatment as a function of residual cancer burden following neoadjuvant therapy. C, D. Par-4 expression in matched pre-treatment breast cancer biopsies and residual tumor following neoadjuvant therapy at surgery. Error bars denote mean +/− SEM. * p<.05, ** p<.01. See also Figure S2 and Tables S1–5.
Figure 3
Figure 3. Par-4 down-regulation is necessary and sufficient for tumor recurrence
A. Western analysis showing Par-4 expression in control and Par-4 knockdown primary HER2/neu tumor cells. B, C. RFS for mice harboring control tumors or tumors expressing one of two shRNAs targeting Par-4. D. Western analysis showing Par-4 expression in four control (MLP) recurrent orthotopic tumors. E. RFS for mice harboring control tumors or tumors expressing a cDNA encoding Par-4. F. Western analysis showing endogenous and ectopic Par-4 expression in primary and recurrent tumors. See also Figure S3.
Figure 4
Figure 4. Par-4 is up-regulated following oncogene inhibition
A. Western analysis of Par-4 following HER2/neu down-regulation in primary tumor cells. B. Quantification of Par-4 levels from A. normalized to tubulin. C. IF analysis of Par-4 in primary tumors (on dox; HER2/neu-expressing) and tumors 3 or 7 days following HER2/neu down-regulation. Scale bar = 50 μm. D. Western analysis of Par-4 in HER2-amplified human breast cancer cells treated with 100 nM lapatinib. E. Quantification of Par-4 levels from D normalized to tubulin. Error bars denote mean +/− SEM. * p<.05, ** p<.01, *** p<.001. See also Figure S4.
Figure 5
Figure 5. Par-4 down-regulation promotes survival following oncogene withdrawal
A. Fluorescent micrographs from an in vivo competition assay showing primary tumors and residual lesions formed from injecting a 1:1 mixture of GFP- and mCherry-labeled control cells (MLP + MLP-Red) or a 1:1 mixture of GFP-labeled Par-4 knockdown cells and mCherry-labeled control cells (Par-4.891 + MLP Red and Par-4.1011 + MLP-Red). Scale bar = 50 μm. B. Quantification of the relative proportion of GFP-positive and mCherry-positive cells in primary tumors or residual lesions. C. Control (MLP) or Par-4 knockdown (Par-4.891 or Par-4.1011) cells labeled with GFP were mixed in a 1:1 ratio with control cells labeled with mCherry (MLP-Red) and the relative proportion of green to red cells was measured by flow cytometry following HER2/neu down-regulation in vitro. D. Percentage increase in non-viable cells following HER2/neu down-regulation in primary tumor cells expressing a control vector (MLP) or one of two shRNAs targeting Par-4. E. Clonogenic survival of cells of the indicated genotype grown in the presence of HER2/neu (+dox) or in the absence of HER2/neu (-dox) for 3 weeks. Dox was added back to a subset of plates for 1 week to re-induce HER2/neu expression (-dox → +dox). F. Quantification of the surviving colonies in E. Error bars denote mean +/− SEM. * p<.05, ** p<.01, *** p<.001. See also Figure S5.
Figure 6
Figure 6. Re-expressing Par-4 in recurrent tumor cells induces multinucleation accompanied by p53 activation, growth arrest, and apoptosis
A. Cell viability was measured following retroviral transduction of Par-4 into primary or recurrentcells. B. Western analysis showing Par-4 expression following Shld-1 treatment of recurrent tumor cells expressing L106P-par4 constructs. C. Growth curves of vehicle or Shld-1 treated control cells (L106P-YFP or Luc-L106P) or Par-4 inducible cells (L106P-Par-4). D. Recurrent tumor cells were treated with Shld-1 for 24 hr and visualized by light microscopy or fluorescence microscopy following immunostaining for Par-4. E. Quantification of multinucleated cells following 24 hr Shld-1 treatment. F. IF staining for p53 in multinucleated cells following Shld-1 treatment. G. IF analysis of BrdU incorporation in multinucleated and control cells following Shld-1 treatment. H. Quantification of percentage of BrdU-positive cells corresponding to the experiment shown in G. I. IF staining for cleaved caspase-3 in multinucleated cells following Shld-1 treatment or retroviral transduction of Par-4 in recurrent cells. All scale bars = 50 μm. Error bars denote mean +/− SEM. * p<.05, ** p<.01, *** p<.001. See also Figure S6.
Figure 7
Figure 7. Par-4 causes cytokinesis failure through ZIPK-mediated MLC2 phosphorylation
A. Stills from live-cell imaging of recurrent tumor cells expressing H2B-mCherry and L106P-Par-4 untreated (top row) or treated with Shld-1 (bottom row). Scale bar = 15 μm. B. Western analysis showing p-MLC2 levels following retroviral transduction of Par-4 into recurrent tumor cells. C. IF analysis showing p- MLC2 following Shld-1 treatment of recurrent cells expressing L106P-Par-4. Scale bar = 10 μm. D. qRT-PCR showing ZIPK expression following transfection of recurrent cells with a control pool or an siRNA pool targeting ZIPK. E. Western analysis of p-MLC2 levels in ZIPK knockdown cells 24 hours after Par-4 expression. F. Quantification of the percentage of multinucleated cells 24 hours after Par-4 expression in control and ZIPK knockdown cells. Error bars denote mean +/− SEM. * p<.05. See also Movies S1 and 2.
Figure 8
Figure 8. Oncogene inhibition leads to Par-4 dependent multinucleation
A. Photomicrographs of cells following HER2/neu down-regulation in primary MTB/TAN tumor cells. Scale bar = 50 μm. B. Quantification of percentage of multinucleated cells corresponding to the experiment shown in A. C. Percentage of multinucleated control or Par-4 knockdown MTB/TAN cells following HER2/neu down-regulation. D. Percentage of control or Par-4 knockdown BT-474 cells with polyploid DNA content following lapatinib treatment as measured by flow cytometry. E. Model for the role of Par-4 down-regulation in tumor recurrence. Oncogene inhibition or chemotherapy results in Par-4-dependent multinucleation, which in turn leads to p53 activation, growth arrest, and apoptosis. Together these cellular processes contribute to tumor regression, such that cells surviving tumor regression exhibit spontaneous Par-4down-regulation. These cells ultimately give rise to recurrent tumors with low Par-4 expression. Error bars denote mean +/− SEM. * p<.05, ** p<.01. See also Figure S7.
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Comment in

  • Par-4 prevents breast cancer recurrence.
    Hebbar N, Shrestha-Bhattarai T, Rangnekar VM. Hebbar N, et al. Breast Cancer Res. 2013;15(5):314. doi: 10.1186/bcr3562. Breast Cancer Res. 2013. PMID: 24164776 Free PMC article.

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