Lauren F Watt1,2, Nikita Panicker1,2, Abdul Mannan1,2, Ben Copeland1,2,3, Richard G S Kahl1,2, Matthew D Dun1,2, Barbara Young4,5, Severine Roselli1,2, Nicole M Verrills6,7. 1. Faculty of Health and Medicine, School of Biomedical Sciences and Pharmacy, and Priority Research Centre for Cancer Research, Innovation and Translation, University of Newcastle, Life Sciences Building, Callaghan, NSW, 2308, Australia. 2. Cancer Research Program, Hunter Medical Research Institute, New Lambton, NSW, Australia. 3. Beckman Research Institute, City of Hope, Duarte, CA, 91010, USA. 4. Department Anatomical Pathology, Hunter Area Pathology Service (HAPS), John Hunter Hospital, New Lambton Heights, NSW, Australia. 5. Faculty of Health and Medicine, School of Medicine and Public Health, University of Newcastle, Callaghan, NSW, Australia. 6. Faculty of Health and Medicine, School of Biomedical Sciences and Pharmacy, and Priority Research Centre for Cancer Research, Innovation and Translation, University of Newcastle, Life Sciences Building, Callaghan, NSW, 2308, Australia. nikki.verrills@newcastle.edu.au. 7. Cancer Research Program, Hunter Medical Research Institute, New Lambton, NSW, Australia. nikki.verrills@newcastle.edu.au.
Abstract
PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
PURPOSE: 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. METHODS:PP2A subunit expression was examined by immunohistochemistry in humanbreast 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. RESULTS: Expression of PP2A-Aα, PP2A-Bα and PP2A-B'α subunits was significantly lower in primary humanbreast 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. CONCLUSION: 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.
Entities:
Keywords:
3D culture; Breast cancer; MCF10A; PP2A; Phosphatase; Tumour suppressor
Authors: Ziran Zhao; Alison Kurimchak; Anna S Nikonova; Felicity Feiser; Jason S Wasserman; Holly Fowle; Tinsa Varughese; Megan Connors; Katherine Johnson; Petr Makhov; Cecilia Lindskog; Vladimir M Kolenko; Erica A Golemis; James S Duncan; Xavier Graña Journal: Oncogenesis Date: 2019-12-10 Impact factor: 7.485