磷酸水解酶2A型（Protein phosphatase 2A簡稱PP2A）是主要的絲氨酸/蘇氨酸磷酸酶之一，含有B56γ3調節次單元的PP2A全酶（以下稱為PP2A-B56γ3）在腫瘤抑制中扮演著重要的角色。p70S6K是PI3K/AKT/mTOR訊息傳導路徑下游的重要訊息傳導分子，受到營養物質和生長因子信號所調控，並且在多種類型的癌症中均發現具有致癌性。然而，PP2A-B56γ3則對p70S6K的Thr389位點的去磷酸反應有關。在本篇研究中發現在幾種癌細胞中，過度表現B56γ3會提高AKT磷酸化程度，B56γ3表現量降低則AKT磷酸化也降低的情況。我們研究了腫瘤細胞的磷酸化AKT、磷酸化p70S6K、IRS-1以及B56γ3表現量之間的相關性。在這些不同的結直腸癌細胞株當中，LS147T的B56γ3表達量明顯高於其他細胞株。B56γ3的表現量與Ser473位點的磷酸化相較於Thr308位點的磷酸化較有正相關性。此外，我們還進行免疫共沉澱（Co-IP）以證實在HeLa、HCT116和SW480等細胞中p70S6K與B56γ3之間的相互作用。為了研究B56γ3 與p70S6K間互相作用的調控，我們利用接種不同密度的細胞以模擬營養物質及生長因子之可用性，我們發現當以較高密度條件培養細胞時，B56γ3穩定降低表現對p70S6K的磷酸化有明顯上調，而細胞以低密度培養時，B56γ3過度表達會明顯降低p70S6K的磷酸化。在Co-IP 實驗中顯示出一致的結果，當細胞在高密度的培養條件下B56γ3與p70S6K間的互動有減少的情形。共軛焦免疫螢光顯微鏡分析中發現，當細胞於高密度培養時，p70S6K與B56γ3的共定位（colocalization）減少。在細胞實驗當中我們發現B56γ3正向調控p70S6K蛋白表現量，與資料探勘（data mining）的結果一致，兩分子在各類癌症組織檢體的mRNA表現呈現正相關。與我們在大腸直腸癌細胞中的發現相同，免疫組織染色（immunohistochemistry）分析發現在臨床大腸直腸癌症病人檢體中的B56γ3與磷酸化AKT也有正向關聯性。再者，B56γ3的表現量與大腸直腸癌以及其他癌症類型中較差的預後有關。綜合上述，與已知扮演腫瘤抑制的角色相反，PP2A的B56γ3調節次單元參與了正向調控AKT活性以及p70S6K表現，或許在癌症發展的過程中扮演致癌的角色。

The PP2A holoenzyme containing the B56γ3 regulatory subunit (hereinafter referred to as PP2A-B56γ3) has been reported to play an important role in tumor suppression. p70S6K is a crucial signaling molecule downstream of the PI3K/AKT/mTOR signaling pathway in response to nutrient and growth factor signals and is found to be oncogenic in several types of cancer. Here, we found that B56γ3 overexpression enhances AKT phosphorylation, whereas knockdown of B56γ3 expression decreases AKT phosphorylation to various extents in several cancer cell lines, including HeLa, HCT116, SW480, and Hep3B cells. We investigated the correlation of endogenous levels of phospho-AKT, phospho-p70S6K, IRS-1 and B56γ3 in several colon cancer cell lines. The level of phospho-AKT at Ser473 is more positively correlated with the level of B56γ3 among most of the cell lines compared to phospho-AKT at Thr308. Furthermore, we confirmed p70S6K physical interaction with B56γ3 in HeLa, HCT116 and SW480 cells by performing co-immunoprecipitation (Co-IP) analysis. To investigate the regulation of interaction between B56γ3 and p70S6K, we cultured cells at different cell densities and found that upregulation of phospho-p70S6K by B56γ3 knockdown was more prominent when cells were seeded at high density, whereas more significant downregulation of phospho-p70S6K by B56γ3 overexpression was found when cells were seeded at low density. Consistently, co-IP analysis showed that interaction between p70S6K and B56γ3 was reduced when cells were grown in high density. Confocal immunofluorescence microscopy analysis showed that colocalization of p70S6K and B56γ3 was reduced in cells grown in high density. In cells, we found that B56γ3 positively regulated the endogenous p70S6K protein level, which was consistent with the result of our data mining showing a positive association of mRNA levels of these two molecules in tissue specimens of various types of cancer. In addition, immunohistochemistry (IHC) analysis indicated that expression of B56γ3 was positively correlated with expression of phospho-AKT in human colorectal cancer specimens. Importantly, high expression level of B56γ3 was associated with poor prognosis of CRC and other cancer types. Taken together, in contrast to its well-known role as a tumor suppressor, the B56γ3 regulatory subunit of PP2A up-regulates both AKT activation and p70S6K expression and may play an oncogenic role in tumor progression.

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