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Abstract Background Protein phosphatase 2A (PP2A) is one of the major protein phosphatases in eukaryotic cells and is essential for cellular homeostasis. PP2A is a heterotrimer comprising the dimeric AC core enzyme and a highly variable regulatory B subunit. Distinct B subunits help the core enzyme gain full activity toward specific substrates and contribute to diverse cellular roles of PP2A. PP2A has been thought to play a tumor suppressor and the B56γ3 regulatory subunit was shown to play a key tumor suppressor regulatory subunit of PP2A. Nevertheless, we uncovered a molecular mechanism of how B56γ3 may act as an oncogene in colorectal cancer (CRC). Methods Polyclonal pools of CRC cells with stable B56γ3 overexpression or knockdown were generated by retroviral or lentiviral infection and subsequent drug selection. Co-immunoprecipitation(co-IP) and in vitro pull-down analysis were applied to analyze the protein–protein interaction. Transwell migration and invasion assays were applied to investigate the role of B56γ3 in affecting motility and invasive capability of CRC cells. The sensitivity of CRC cells to 5-fluorouracil (5-FU) was analyzed using the PrestoBlue reagent assay for cell viability. Immunohistochemistry (IHC) was applied to investigate the expression levels of phospho-AKT and B56γ3 in paired tumor and normal tissue specimens of CRC. DataSets of TCGA and GEO were analyzed to investigate the correlation of B56γ3 expression with overall survival rates of CRC patients. Results We showed that B56γ3 promoted epithelial-mesenchymal transition (EMT) and reduced the sensitivity of CRC cells to 5-FU through upregulating AKT activity. Mechanistically, B56γ3 upregulates AKT activity by targeting PP2A to attenuate the p70S6K-mediated negative feedback loop regulation on PI3K/AKT activation. B56γ3 was highly expressed and positively correlated with the level of phospho-AKT in tumor tissues of CRC. Moreover, high B56γ3 expression is associated with poor prognosis of a subset of patients with CRC. Conclusions Our finding reveals that the B56γ3 regulatory subunit-containing PP2A plays an oncogenic role in CRC cells by sustaining AKT activation through suppressing p70S6K activity and suggests that the interaction between B56γ3 and p70S6K may serve as a therapeutic target for CRC. Video Abstract

Background: Protein phosphatase 2A (PP2A) is one of the major protein phosphatases in eukaryotic cells and is essential for cellular homeostasis. PP2A is a heterotrimer comprising the dimeric AC core enzyme and a highly variable regulatory B subunit. Distinct B subunits help the core enzyme gain full activity toward specific substrates and contribute to diverse cellular roles of PP2A. PP2A has been thought to play a tumor suppressor and the B56γ3 regulatory subunit was shown to play a key tumor suppressor regulatory subunit of PP2A. Nevertheless, we uncovered a molecular mechanism of how B56γ3 may act as an oncogene in colorectal cancer (CRC). Methods: Polyclonal pools of CRC cells with stable B56γ3 overexpression or knockdown were generated by retroviral or lentiviral infection and subsequent drug selection. Co-immunoprecipitation(co-IP) and in vitro pull-down analysis were applied to analyze the protein–protein interaction. Transwell migration and invasion assays were applied to investigate the role of B56γ3 in affecting motility and invasive capability of CRC cells. The sensitivity of CRC cells to 5-fluorouracil (5-FU) was analyzed using the PrestoBlue reagent assay for cell viability. Immunohistochemistry (IHC) was applied to investigate the expression levels of phospho-AKT and B56γ3 in paired tumor and normal tissue specimens of CRC. DataSets of TCGA and GEO were analyzed to investigate the correlation of B56γ3 expression with overall survival rates of CRC patients. Results: We showed that B56γ3 promoted epithelial-mesenchymal transition (EMT) and reduced the sensitivity of CRC cells to 5-FU through upregulating AKT activity. Mechanistically, B56γ3 upregulates AKT activity by targeting PP2A to attenuate the p70S6K-mediated negative feedback loop regulation on PI3K/AKT activation. B56γ3 was highly expressed and positively correlated with the level of phospho-AKT in tumor tissues of CRC. Moreover, high B56γ3 expression is associated with poor prognosis of a subset of patients with CRC. Conclusions: Our finding reveals that the B56γ3 regulatory subunit-containing PP2A plays an oncogenic role in CRC cells by sustaining AKT activation through suppressing p70S6K activity and suggests that the interaction between B56γ3 and p70S6K may serve as a therapeutic target for CRC. CUkGCcvqWD-y5KjYaB3kRR Video Abstract [ABSTRACT FROM AUTHOR]

The B56γ-containing protein phosphatase 2A (PP2A-B56γ) has been postulated to have tumor suppressive functions. Here, we report regulation of p27KIP1 subcellular localization by PP2A-B56γ3. B56γ3 overexpression enhanced nuclear localization of p27KIP1, whereas knockdown of B56γ3 decreased p27KIP1 nuclear localization. B56γ3 overexpression decreased phosphorylation at Thr157 (phospho-Thr157), whose phosphorylation promotes cytoplasmic localization of p27KIP1, whereas B56γ3 knockdown significantly increased the level of phospho-Thr157. In vitro, PP2A-B56γ3 catalyzed dephosphorylation of phospho-Thr157 in a dose-dependent and okadaic acid-sensitive manner. B56γ3 did not increase p27KIP1 nuclear localization by down-regulating the upstream kinase Akt activity and outcompeted a myristoylated constitutively active Akt (Aktca) in regulating Thr157 phosphorylation and subcellular localization of p27KIP1. In addition, results of interaction domain mapping revealed that both the N-terminal and C-terminal domains of p27 and a domain at the C-terminus of B56γ3 are required for interaction between p27 and B56γ3. Furthermore, we demonstrated that p27KIP1 levels are positively correlated with B56γ levels in both non-tumor and tumor parts of a set of human colon tissue specimens. However, positive correlation between nuclear p27KIP1 levels and B56γ levels was found only in the non-tumor parts, but not in tumor parts of these tissues, implicating a dysregulation in PP2A-B56γ3-regulated p27KIP1 nuclear localization in these tumor tissues. Altogether, this study provides a new mechanism by which the PP2A-B56γ3 holoenzyme plays its tumor suppressor role.