Biomarker-driven therapeutic groups sensitive to PP2A activators in cancer patients

Abstract

Deregulation of the feedback mechanism of protein phosphatase 2 A (PP2A) is implicated in tumorigenesis directly through reduced expression of functional catalytic subunit of PP2A (PP2A-C) or through overexpression of its inhibitors a4, SET, SETBP1 and CIP2A. PP2A controls proliferation, growth and apoptosis, attenuating various signalling pathways. The cBIO Portal for Cancer Genomics (cBIOPortal) shows that the PP2A is deregulated in 63% of Triple Negative Breast Cancer (TNBC) patients. TNBC patients derive little benefit from traditional target-specific therapies due to lack of the favourable prognostic markers ER, PR and HER2. In the breast cancer cell line models, sensitivity to a PP2A activator (FTY720) was observed solely in TNBC cell lines. The significant overexpression of CIP2A in the sensitive cell line MDAMB231, exemplifies PP2A activity regulation. Results showed that PP2A core complex and regulatory subunit expression at transcript and protein level could not directly predict FTY720 sensitivity, hence the need for PP2A activity biomarkers. A candidate set of PP2A activity biomarkers was derived from in silica analysis of The Cancer Genome Atlas (TCGA) expression data from the invasive breast cancer cohort. PP2A activity biomarkers were differentially expressed in cases with potential PP2A deregulation, as defined by low expression of PP2A core subunits and overexpression of PP2A regulators. A biological link to PP2A was established through pathway analysis of a short-list of candidate genes, identifying 5 potential biomarkers. The expression of PP2A core components and regulators, breast cancer signature genes and the 5 biomarker candidates were assessed in a Maltese cohort of breast cancer patients (N = 90) using a specific technique for RNA analysis in formalin fixed paraffin embedded (FFPE) tissue (Quantigene v2.0). lmmunohistochemical analysis of PP2A regulators and PP2A phospho-targets, pAKT and pS6K, was carried out on the same local breast cancer patients and on a cohort of 302 breast cancer cases on tissue microarrays (TMAs) provided by our collaborators at Leeds University. The multi-level approach provided evidence for various mechanisms for PP2A activity regulation: isoform formation of the catalytic subunit a. isoform of PP2A (PPP2CA}; post translational modifications of PPP2CA; transcriptional regulation of PP2A regulators and differential cellular localisation of regulators. The complexity of PP2A regulation warranted the use of biomarkers that reflect PP2A activity or FTY720 sensitivity. Moreover, validation of the 5 candidate PP2A activity markers across in silica data, cellular models and patients, identified AURKA and KIF2C as transcriptional biomarkers of PP2A-dependent growth factor activation (PP2AGFA}. Expression of the PP2AGFA markers is a prerequisite for mitotic entry due to their pivotal role in cell cycle regulation. AURKA and KIF2C are implicated with an aggressive phenotype of malignancies, characterised by increased proliferation and acquired motility. Using AURKA and KIF2C, TCGA and local breast cancer cases were classified into a novel class of tumours (PP2AGFA} that associate with FTY720-sensitive breast cell lines. The PP2AGFA class of tumours were represented by a prevalent overexpression of CIP2A and the TNBC or Basal breast cancer subtypes. The novel class defined by overexpression of AURKA and KIF2C, represents a new therapeutic class of tumours with predicted susceptibility to restoration of PP2A activity.