Regulation of TIF1β Phosphorylation in Colorectal Cancer Progression

調控蛋白質轉譯後修飾作用的動態平衡在腫瘤形成與惡化的過程中扮演很重要的角色。近來越來越多文獻報導指出調控磷酸化程度變化的激酶與磷酸酶造成的異常與腫瘤進程有關。蛋白磷酸酶Protein phosphatase 4 (PP4) 是一種絲胺酸/酪胺酸磷酸酶，在乳癌及肺癌末期中有高度表現。然而，高度表現的PP4在腫瘤進程中的功能目前尚不清楚。在DNA修補機制中，PP4會直接去除Transcription Intermediary Factor 1 beta (TIF1β) 在絲胺酸473及絲氨酸824的磷酸化。TIF1β是一個基因表觀遺傳調控者，在許多生理機能上，例如胚胎發育、細胞分化亦或影響腫瘤生成等，都能藉由TIF1β與調控染色質重組的heterochromatin protein 1(HP1)蛋白或其他染色質調控因子的交互作用進一步調控基因的表現。有趣的是，TIF1β的絲胺酸473位點於TIF1β與HP1蛋白結合的序列附近，已知能藉由絲胺酸473的磷酸化與否來調控TIF1β與HP1蛋白的結合能力。在本研究中，我們利用大腸癌為研究模式，探討經由PP4和激酶調控磷酸化TIF1β的動態平衡在大腸癌進程中的分子機制。利用免疫組織染色比較不同病理分期的大腸癌病人組織發現，在高分期大腸癌中PP4的高表現和TIF1β低磷酸化是相符合的，由此可推測在大腸癌進程中PP4會去除TIF1β的磷酸化。磷酸化TIF1β在人類口腔上皮基底層與老鼠腸上皮的crypt region有高度表現，可推測磷酸化TIF1β和上皮細胞的增殖與分化有關。此外，磷酸化TIF1β在老鼠腸腺瘤及致癌HRas誘發的侵入都有高度表現，指出磷酸化TIF1β可能和參與腫瘤進程有關。我們進一步利用探討分化能力不同細胞c-Jun/JunB變異的3D培養模式，發現磷酸化TIF1β只出現在具有分化能力的細胞上。因此我們認為磷酸化TIF1β與具有分化能力的增殖細胞有關進一步影響腫瘤的進程。除此之外，我們證實TIF1β的磷酸化可藉由EGF經RAS-MEK-ERK訊息傳遞路徑刺激，而ERK-1/2可能是TIF1β的激酶之一。根據以上實驗結果發現，PP4和激酶調控TIF1β磷酸化動態平衡可能參與細胞的增殖與分化進而影響大腸癌的進程。Dynamic state of post-translational modifications has been postulated to involve in a wide range of processes in cancer. Notably, dysregulating the balance between kinases and phosphatases leads to change protein phosphorylation status which has amply reported in cancer progression. Protein phosphatase 4 (PP4) is a serine/threonine phosphatase and is highly expressed in terminal stage of breast and lung cancer. However, the function of PP4 in tumor progression remain unclear. PP4 directly acts on serine 473 of Transcription Intermediary Factor 1 beta (TIF1β) phosphorylation during DNA damage response. TIF1β is an intermediary factor of transcription and epigenetic modulator of gene expression in several physiological processes including embryonic development and cell differentiation as well as in establishment and/or progression of some cancers. Interestingly, serine 473 residue of TIF1β affects the binding between TIF1β and heterochromatin protein 1 (HP1) which could regulate chromatin remodeling and epigenetic modulation. In this study, we used human colorectal cancer to investigate the molecular mechanisms regulating phosphorylation/de-phosphorylation of TIF1β through PP4 and its specific kinase in tumor progression. Comparison of immunohistochemistry (IHC) data on different differentiation grades of colon cancer showed low expression of phosphorylated TIF1β which were consistent with high PP4C expression on high grade which provides a link that PP4 may be involved in de-phosphorylation of TIF1β in tumor progression. Our IHC data on basal layer of oral epithelium and crypt region of intestinal epithelium have shown that the phosphorylated TIF1β is an event closely correlated with cellular proliferation and differentiation. In addition, phosphorylated TIF1β was found to be highly expressed in mouse intestine adenoma and oncogenic HRas-induced invasion region indicating that phosphorylated TIF1β is involved in tumor progression. I used 3D culture model with c-Jun/JunB mutants to compare cells in different potentials of differentiation, phosphorylated TIF1β has only been shown in the differentiation proficient cells but not in the differentiation deficient cells. It suggested phosphorylated TIF1β is correlated with cellular differentiation-dependent proliferation to further involve in tumor progression. Furthermore, I confirmed phosphorylated TIF1β could be induced by EGF through RAS-MEK-ERK signaling pathway and ERK-1/2 may be one of the possible kinases. Based on experimental data, I suggest that the phosphorylated TIF1β may be involved in cellular proliferation and differentiation as well as tumor progression through homeostatic balance between PP4 and its kinase