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CIB1 depletion impairs cell survival and tumor growth in triple-negative breast cancer

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Abstract

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with generally poor prognosis and no available targeted therapies, highlighting a critical unmet need to identify and characterize novel therapeutic targets. We previously demonstrated that CIB1 is necessary for cancer cell survival and proliferation via regulation of two oncogenic signaling pathways, RAF–MEK–ERK and PI3K–AKT. Because these pathways are often upregulated in TNBC, we hypothesized that CIB1 may play a broader role in TNBC cell survival and tumor growth. Methods utilized include inducible RNAi depletion of CIB1 in vitro and in vivo, immunoblotting, clonogenic assay, flow cytometry, RNA-sequencing, bioinformatics analysis, and Kaplan–Meier survival analysis. CIB1 depletion resulted in significant cell death in 8 of 11 TNBC cell lines tested. Analysis of components related to PI3K–AKT and RAF–MEK–ERK signaling revealed that elevated AKT activation status and low PTEN expression were key predictors of sensitivity to CIB1 depletion. Furthermore, CIB1 knockdown caused dramatic shrinkage of MDA-MB-468 xenograft tumors in vivo. RNA sequence analysis also showed that CIB1 depletion in TNBC cells activates gene programs associated with decreased proliferation and increased cell death. CIB1 expression levels per se did not predict TNBC susceptibility to CIB1 depletion, and CIB1 mRNA expression levels did not associate with TNBC patient survival. Our data are consistent with the emerging concept of non-oncogene addiction, where a large subset of TNBCs depend on CIB1 for cell survival and tumor growth, independent of CIB1 expression levels. Our data establish CIB1 as a novel therapeutic target for TNBC.

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Acknowledgments

We thank Paul Truex, Dinesh Srinivasan, Thomas Freeman, and Thomas Stewart for helpful discussions. We also thank Charlene Santos and Mark Ross for assistance with mouse xenograft experiments, and Amy Perou for managing RNAseq processing and data collection. This work was supported by NHLBI 1R01HL092544 and NC TraCS 4DR11410 (LV Parise), AHA 13PRE16470024 (JL Black), the Triple Negative Breast Cancer Foundation and NCI Breast SPORE program (P50-CA58223-09A1) (JC Harrell and CM Perou), and NCBC 2013-MRG-1110 (CD Jones).

Conflict of interest

JL Black, TM Leisner, and LV Parise are co-founders of Reveris Therapeutics, LLC. CM Perou is an equity stock holder, consultant, and member of the board of directors of BioClassifier LLC and GeneCentric Diagnostics. The other authors declare no potential conflicts of interest.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of North Carolina, 120 Mason Farm Rd Ste 3010, Chapel Hill, NC, 27599, USA

    Justin L. Black, Tina M. Leisner, Melissa J. Fellmeth & Leslie V. Parise

  2. Department of Pathology, Virginia Commonwealth University, Richmond, VA, USA

    J. Chuck Harrell

  3. Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA

    Samuel D. George, Nicole M. Baker, Channing J. Der, Charles M. Perou & Leslie V. Parise

  4. Department of Mathematics and Computer Science, Clark University, Worcester, MA, USA

    Dominik Reinhold

  5. Carolina Center for Genomic Sciences, University of North Carolina, Chapel Hill, NC, USA

    Dominik Reinhold & Corbin D. Jones

  6. Department of Biology, University of North Carolina, Chapel Hill, NC, USA

    Corbin D. Jones

  7. Department of Pharmacology, University of North Carolina, Chapel Hill, NC, USA

    Nicole M. Baker & Channing J. Der

  8. Department of Genetics, University of North Carolina, Chapel Hill, NC, USA

    Charles M. Perou

  9. Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC, USA

    Charles M. Perou

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  1. Justin L. Black
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10549_2015_3458_MOESM1_ESM.tif

Supplementary Figure S1. CIB1 depletion reduces cell proliferation in a panel of TNBC cell lines. A) A panel of 11 TNBC cells lines were transduced with either control (SCR) or two separate CIB1 shRNA targeting sequences (CIB1 shRNA1 or shRNA2). SCR was normalized to 1.0 (dotted line) and graph represents relative mean fold change in total cell count ± SEM for each CIB1 shRNA treated sample (n ≥ 3). B Average differentiation score of sensitive and insensitive cell lines from TNBC cell line panel. Cell lines sensitive to CIB1 depletion trend toward a lower differentiation score than insensitive cell lines (P = .0695). Supplementary material 1 (TIFF 3402 kb)

10549_2015_3458_MOESM2_ESM.tif

Supplementary Figure S2. Effect of CIB1 depletion on cell death in non-TNBC cell lines. A) A panel of three non-TNBC cell lines were transduced with either control or CIB1 shRNA. Graph represents average percent cell death ± SEM for each sample (n = 3). ZR-75-1 had significantly higher cell death upon CIB1 depletion. B) Relative levels of pAKT, AKT, pERK, ERK, CIB1, and Rac (loading control) in CIB1-depleted and control samples. CIB1 depletion results in reduction in pAKT and pERK in ZR-75-1 cells, corresponding to increased cell death. Supplementary material 2 (TIFF 2213 kb)

10549_2015_3458_MOESM3_ESM.tif

Supplementary Figure S3. CIB1 depletion decreases TNBC cell proliferation and increases cell death in vitro and in vivo. A) CIB1 depletion decreases MDA-468 proliferation and viability. Graphs represent mean total cell number ± SEM (Upper panel) and mean percentage of dead cells ± SEM (Lower panel) at each time point (n = 3). *P < .001; **P < .01. B) Measurement of cell surface phosphatidylserine (Annexin V binding) and cell permeability to 7-AAD. A higher percentage of MDA-468-CIB1shRNA+Dox cells are positive for both Annexin V and 7-AAD, indicating a higher percentage of dead cells. C-D) Relative levels of pAKT, AKT, pERK, ERK, CIB1, γH2AX, and Rac (loading control) in CIB1-depleted and control samples in vitro (C) and in vivo (D). MDA-468-CIB1shRNA+Dox exhibit complete depletion of CIB1, and decreased pERK and pAKT in vitro (C) and in vivo (D), and increased γH2Ax in vitro (C). E) Representative TUNEL-stained xenograft tumor sections show that more of cells from CIB1-depleted tumors are dead or dying (dark brown punctate dots) compared to control tumors. Supplementary material 3 (TIFF 3848 kb)

10549_2015_3458_MOESM4_ESM.tif

Supplementary Figure S4. Expression of constitutively active PAK1 partially rescues CIB1 depletion-induced cell death. A) caPAK1 was overexpressed in MDA-468 cells, followed by treatment with either control or CIB1 shRNA. caPAK1 expression partially rescued cells from CIB1-depletion induced cell death. Graph represents average percent dead cells ± SEM (n = 2). B) Relative levels of PAK1, pAKT, AKT, pERK, ERK, CIB1, and GAPDH (loading control) in control or CIB1 shRNA treated cells ± caPAK1 overexpression. Supplementary material 4 (TIFF 2345 kb)

10549_2015_3458_MOESM5_ESM.tif

Supplementary Figure S5. CIB1 expression is not prognostic for TNBC patient survival. A) CIB1 expression in a population of human ER- tumors does not correlate with probability of relapse-free survival by Kaplan–Meyer survival analysis (n = 256). B) Same as in (A) but focusing on subset of TNBC within the ER- tumors (n = 110). Supplementary material 5 (TIFF 2288 kb)

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Black, J.L., Harrell, J.C., Leisner, T.M. et al. CIB1 depletion impairs cell survival and tumor growth in triple-negative breast cancer. Breast Cancer Res Treat 152, 337–346 (2015). https://doi.org/10.1007/s10549-015-3458-4

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