Functional importance of PP2A regulatory subunit loss in breast cancer
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Protein phosphatase 2A (PP2A) is a family of serine/threonine phosphatases that regulate multiple cellular signalling pathways involved in proliferation, survival and apoptosis. PP2A inhibition occurs in many cancers and is considered a tumour suppressor. Deletion/downregulation of PP2A genes has been observed in breast tumours, but the functional role of PP2A subunit loss in breast cancer has not been investigated.
PP2A subunit expression was examined by immunohistochemistry in human breast tumours, and by qPCR and immunoblotting in breast cancer cell lines. PP2A subunits were inhibited by shRNA, and mutant PP2A genes overexpressed, in MCF10A and MCF7 cells, and growth and signalling in standard and three-dimensional cultures were assessed.
Expression of PP2A-Aα, PP2A-Bα and PP2A-B′α subunits was significantly lower in primary human breast tumours and lymph node metastases, compared to normal mammary tissue. PP2A-Aα and the regulatory subunits PP2A-Bα, -Bδ and -B′γ were also reduced in breast cancer cell lines compared to normal mammary epithelial cells. Functionally, shRNA-mediated knockdown of PP2A-Bα, -B′α and -B′γ, but not PP2A-Aα, induced hyper-proliferation and large multilobular acini in MCF10A 3D cultures, characterised by activation of ERK. Expression of a breast cancer-associated PP2A-A mutant, PP2A-Aα-E64G, which inhibits binding of regulatory subunits to the PP2A core, induced a similar hyper-proliferative phenotype. Knockdown of PP2A-Bα also induced hyper-proliferation in MCF7 breast cancer cells.
These results suggest that loss of specific PP2A regulatory subunits is functionally important in breast tumourigenesis, and support strategies to enhance PP2A activity as a therapeutic approach in breast cancer.
KeywordsBreast cancer PP2A MCF10A Tumour suppressor 3D culture Phosphatase
We thank Prof Stefan Strack for PP2A constructs, Prof Roger Daly for MCF10A cells, and Ms Helen Carpenter for technical assistance.
This work was supported by grants from the Cure Cancer Australia Foundation, the Pink Frangipani Ball, the Cancer Council NSW and the Hunter Medical Research Institute. LW was supported by an Australian Postgraduate Award, a University of Newcastle VC Award, and a Cancer Institute NSW Research Scholar Award. MDD and NMV were supported by Fellowships from the Cancer Institute NSW.
Compliance with ethical standards
Conflict of interest
the authors have no conflicts to declare.
- 3.Karakosta A, Golias C, Charalabopoulos A, Peschos D, Batistatou A, Charalabopoulos K (2005) Genetic models of human cancer as a multistep process. Paradigm models of colorectal cancer, breast cancer, and chronic myelogenous and acute lymphoblastic leukaemia. J Exp Clin Cancer Res. 24(4):505–514PubMedGoogle Scholar
- 11.Smith AM, Roberts KG, Verrills NM (2011) Ser/Thr phosphatases: the new frontier for Myeloid Leukemia therapy? In: Koschmieder S, Krug U (eds) Myeloid Leukemia—basic mechanisms of leukemogenesis. Intech, Croatia, pp 123–148Google Scholar
- 12.Roberts KG, Smith AM, McDougall F, Carpenter H, Horan M, Neviani P, Powell JA, Thomas D, Guthridge MA, Perrotti D, Sim AT, Ashman LK, Verrills NM (2010) Essential requirement for PP2A inhibition by the oncogenic receptor c-KIT suggests PP2A reactivation as a strategy to treat c-KIT+ cancers. Cancer Res 70(13):5438–5447CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Smith AM, Dun MD, Lee EM, Harrison C, Kahl R, Flanagan H, Panicker N, Mashkani B, Don AS, Morris J, Toop H, Lock RB, Powell JA, Thomas D, Guthridge MA, Moore A et al (2016) Activation of protein phosphatase 2A in FLT3+ acute myeloid leukemia cells enhances the cytotoxicity of FLT3 tyrosine kinase inhibitors. Oncotarget 7(30):47465–47478CrossRefPubMedPubMedCentralGoogle Scholar
- 15.Rincon R, Cristobal I, Zazo S, Arpi O, Menendez S, Manso R, Lluch A, Eroles P, Rovira A, Albanell J, Garcia-Foncillas J, Madoz-Gurpide J, Rojo F (2015) PP2A inhibition determines poor outcome and doxorubicin resistance in early breast cancer and its activation shows promising therapeutic effects. Oncotarget 6(6):4299–4314CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Calin GA, di Iasio MG, Caprini E, Vorechovsky I, Natali PG, Sozzi G, Croce CM, Barbanti-Brodano G, Russo G, Negrini M (2000) Low frequency of alterations of the alpha (PPP2R1A) and beta (PPP2R1B) isoforms of the subunit A of the serine-threonine phosphatase 2A in human neoplasms. Oncogene 19(9):1191–1195CrossRefPubMedGoogle Scholar
- 22.Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, Speed D, Lynch AG, Samarajiwa S, Yuan Y, Graf S, Ha G, Haffari G, Bashashati A, Russell R, McKinney S et al (2012) The genomic and transcriptomic architecture of 2000 breast tumours reveals novel subgroups. Nature 486(7403):346–352PubMedPubMedCentralGoogle Scholar
- 38.Yu XX, Du X, Moreno CS, Green RE, Ogris E, Feng Q, Chou L, McQuoid MJ, Pallas DC (2001) Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen. Mol Biol Cell 12(1):185–199CrossRefPubMedPubMedCentralGoogle Scholar
- 39.Ripple MO, Kalmadi S, Eastman A (2005) Inhibition of either phosphatidylinositol 3-kinase/Akt or the mitogen/extracellular-regulated kinase, MEK/ERK, signaling pathways suppress growth of breast cancer cell lines, but MEK/ERK signaling is critical for cell survival. Breast Cancer Res Treat 93(2):177–188CrossRefPubMedGoogle Scholar