癌症為國人十大死因之首已為期多年，根據國民健康署的統計資料中這些因為癌症去逝的病患又有接近五分之一的人是死於肺癌，因此更加凸顯了對於肺癌的研究重要性。近年來我們實驗室發現泛素特異蛋白脢二十四 (Ubiquitin specific peptidase 24, USP24) 參與了調控腫瘤生成以及癌症的轉移等現象，然而對於其詳細的機制尚未釐清。NF-κB是細胞中很常見的轉錄因子，根據先前的研究顯示它調控發炎、免疫反應、細胞存活與細胞生長等生理現象。此外先前的研究顯示NF-κB訊息傳遞途徑可以調控USP24的轉錄，因此我們想反過來觀察USP24能不能反過來影響NF-κB訊息傳遞途徑本身，所以我們先靜默USP24後發現NF-κB的抑制子IκB的量有明顯的上升而NF-κB本身則沒有明顯的變化，我們更進一步的去確認在靜默USP24後IκB的蛋白質穩定性有所上升而泛素化修飾的程度則有明顯的下降，由於USP24是進行去泛素化與前面看到的結果正好相反，所以我們在靜默USP24後再去觀察IκB的泛素化連接脢β-TrCP是否會因為USP24的靜默有所變化，我們發現β-TrCP蛋白的量在靜默USP24後有明顯的下降而β-TrCP的蛋白質穩定性也有所下降，接著我們利用免疫沈澱的方式發現USP24確實能與β-TrCP有交互作用後再接著觀察到靜默USP24後β-TrCP的泛素化修飾程度有所上升而大量表現USP24時β-TrCP的泛素化修飾則有所下降，接下來為了觀察USP24是否會影響NF-κB的功能，因此我們先利用免疫螢光來觀察在大量表現USP24後NF-κB的入核情形並發現NF-κB的入核有明顯的上升，接著利用報導基因偵測的方式分別觀察大量表現以及靜默USP24後NF-κB的活性差異，結果發現NF-κB的活性會因為大量表現USP24增加而因為靜默USP24下降，綜合這些結果我們可以知道USP24除了先前的報導會受NF-κB調控外，還可以藉由調控β-TrCP反過來影響NF-κB本身的訊息傳遞。接著我們將實驗室先前有無靜默USP24的microarray中表現量有明顯變化的基因與已知受NF-κB調控的452個基因比對，發現靜默USP24後表現量上升的125個基因中有3個受NF-κB調控，而靜默USP24後表現量上升的251個基因中有22個受NF-κB調控，其中在這些因為靜默USP24後表現量有下降的基因裡我們發先有7個已知與癌轉移相關的基因，加上先前實驗室發現USP24會參與調控癌轉移，我們可以合理推斷USP24很可能是透過影響NF-κB訊息傳遞途徑來調控癌轉移的發生。

Ubiquitin specific peptidase 24 (USP24) is one member of the USPs family known with protein ubiquitination-regulating enzyme activity. USP24 is also known for involving in tumorigenesis and metastasis. In this study we found that USP24 can regulate NF-κB signal pathway through regulating β-TrCP, the ubiquitin E3 ligase of IκB. After analyzed shUSP24 cDNA array data, we also found that USP24 may regulate metastasis through NF-κB signal pathway. After knocking down USP24, we found IκB protein level increased and NF-κB level didn’t change. After knocked down USP24 we found the increasing of IκB protein stability and also found the ubiquitination of IκB was decreased. Therefore, we investigated the E3 of IκB, β-TrCP, and found that β-TrCP decreased in both protein level and stability after USP24 knocked down. We used immunoprecipitation and western blotting to clarify the interaction between USP24 and β-TrCP. After confirmed the interaction between USP24 and β-TrCP, we checked the ubiquitination of β-TrCP and found that ubiquitinated β-TrCP signal was decreased when we overexpressed USP24 and ubiquitinated β-TrCP signal was increased when we knocked down USP24. In immunofluorescence assay we found overexpressed USP24 increased the nuclear translocation of NF-κB. Moreover, we used NF-κB response element-containing vector to do reporter assay and found that USP24 overexpression increased NF-κB activity and USP24 knockdown decreased NF-κB activity. These results suggested that USP24 could regulate NF-κB activity through control β-TrCP degradation. Finally, we analyzed shUSP24 cDNA array data and found that USP24 may cause metastasis through regulating NF-κB activity.

Alexandraki, K., Piperi, C., Kalofoutis, C., Singh, J., Alaveras, A., & Kalofoutis, A. (2006). Inflammatory Process in Type 2 Diabetes. Annals of the New York Academy of Sciences, 1084(1), 89-117.
Almeida, B., Fernandes, S., Abreu, I., & Macedo-Ribeiro, S. (2013). Trinucleotide Repeats: A Structural Perspective. Frontiers in Neurology, 4(76).
Amerik, A. Y., & Hochstrasser, M. (2004). Mechanism and function of deubiquitinating enzymes. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1695(1), 189-207.
Apte, R. N., Dotan, S., Elkabets, M., White, M. R., Reich, E., Carmi, Y., . . . Voronov, E. (2006). The involvement of IL-1 in tumorigenesis, tumor invasiveness, metastasis and tumor-host interactions. Cancer and Metastasis Reviews, 25(3), 387-408.
Atkin, G., & Paulson, H. (2014). Ubiquitin pathways in neurodegenerative disease. Frontiers in Molecular Neuroscience, 7, 63.
Ayesha, K., Joy, W., Ilaria, F., Chaofeng, M., Junhua, M., Zhizhou, Y., . . . Haifa, S. (2015). Recent Advances in Discovering the Role of CCL5 in Metastatic Breast Cancer. Mini-Reviews in Medicinal Chemistry, 15(13), 1063-1072.
Bar-Eli, M. (1999). Role of Interleukin-8 in Tumor Growth and Metastasis of Human Melanoma. Pathobiology, 67(1), 12-18.
Bautmans, I., Rose, N., Lambert, M., Demanet, C., & Mets, T. (2005). Circulating Acute Phase Mediators and Skeletal Muscle Performance in Hospitalized Geriatric Patients (Vol. 60).
Bertram, L., Lill, C. M., & Tanzi, R. E. (2010). The Genetics of Alzheimer Disease: Back to the Future. Neuron, 68(2), 270-281.
Chang, L.-C., Fan, C.-W., Tseng, W.-K., Chein, H.-P., Hsieh, T.-Y., Chen, J.-R., . . . Hua, C.-C. (2016). The Ratio of Hmox1/Nrf2 mRNA Level in the Tumor Tissue Is a Predictor of Distant Metastasis in Colorectal Cancer. Disease Markers, 2016, 8143465.
Ding, C., Parameswaran, V., Udayan, R., Burgess, J., & Jones, G. (2008). Circulating Levels of Inflammatory Markers Predict Change in Bone Mineral Density and Resorption in Older Adults: A Longitudinal Study. The Journal of Clinical Endocrinology & Metabolism, 93(5), 1952-1958.
Fuchs, S. Y., Spiegelman, V. S., & Kumar, K. G. (2004). The many faces of beta-TrCP E3 ubiquitin ligases: reflections in the magic mirror of cancer. Oncogene, 23(11), 2028-2036.
Fuchs, S. Y., Spiegelman, V. S., & Suresh Kumar, K. G. (2004). The many faces of β-TrCP E3 ubiquitin ligases: reflections in the magic mirror of cancer. Oncogene, 23, 2028.
Griffin, W. S., Stanley, L. C., Ling, C., White, L., MacLeod, V., Perrot, L. J., . . . Araoz, C. (1989). Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proceedings of the National Academy of Sciences, 86, 7611-7615.
Hammerschmidt, S., & Wirtz, H. (2009). Lung cancer: current diagnosis and treatment. Dtsch Arztebl Int, 106(49), 809-818; quiz 819-820.
Hanpude, P., Bhattacharya, S., Dey Amit, K., & Maiti Tushar, K. (2015). Deubiquitinating enzymes in cellular signaling and disease regulation. IUBMB Life, 67(7), 544-555.
Hansen, M. T., Forst, B., Cremers, N., Quagliata, L., Ambartsumian, N., Grum-Schwensen, B., . . . Grigorian, M. (2014). A link between inflammation and metastasis: serum amyloid A1 and A3 induce metastasis, and are targets of metastasis-inducing S100A4. Oncogene, 34, 424.
Hardy, J. (2003). Impact of genetic analysis on Parkinson's disease research. Movement Disorders, 18(6), 96-98.
Hayashi, F., Smith, K. D., Ozinsky, A., Hawn, T. R., Yi, E. C., Goodlett, D. R., . . . Aderem, A. (2001). The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature, 410, 1099.
Hayden, M. S., & Ghosh, S. (2004). Signaling to NF-κB. Genes & Development, 18(18), 2195-2224.
Hayden, M. S., & Ghosh, S. (2008). Shared Principles in NF-κB Signaling. Cell, 132(3), 344-362.
Hofmann, R. M., & Pickart, C. M. (1999). Noncanonical MMS2-Encoded Ubiquitin-Conjugating Enzyme Functions in Assembly of Novel Polyubiquitin Chains for DNA Repair. Cell, 96(5), 645-653.
Hu, H., & Sun, S.-C. (2016). Ubiquitin signaling in immune responses. Cell Research, 26, 457.
Israel, A. (2010). The IKK complex, a central regulator of NF-kappaB activation. Cold Spring Harb Perspect Biol, 2(3), a000158.
Jin, J., Liu, J., Chen, C., Liu, Z., Jiang, C., Chu, H., . . . Wang, P. (2016). The deubiquitinase USP21 maintains the stemness of mouse embryonic stem cells via stabilization of Nanog. Nature Communications, 7, 13594.
Jung, H.-Y., Fattet, L., & Yang, J. (2015). Molecular Pathways: Linking Tumor Microenvironment to Epithelial–Mesenchymal Transition in Metastasis. Clinical Cancer Research, 21(5), 962.
Kirkby, N. S., Chan, M. V., Zaiss, A. K., Garcia-Vaz, E., Jiao, J., Berglund, L. M., . . . Mitchell, J. A. (2016). Systematic study of constitutive cyclooxygenase-2 expression: Role of NF-kappaB and NFAT transcriptional pathways. Proc Natl Acad Sci U S A, 113(2), 434-439.
Kitagawa, M., Hatakeyama, S., Shirane, M., Matsumoto, M., Ishida, N., Hattori, K., . . . Nakayama, K. (1999). An F‐box protein, FWD1, mediates ubiquitin‐dependent proteolysis of β‐catenin. The EMBO Journal, 18(9), 2401.
Komander, D., & Rape, M. (2012). The ubiquitin code. Annu Rev Biochem, 81, 203-229.
Lambert, A. W., Pattabiraman, D. R., & Weinberg, R. A. (2017). Emerging Biological Principles of Metastasis. Cell, 168(4), 670-691.
Lawrence, T. (2009). The Nuclear Factor NF-κB Pathway in Inflammation. Cold Spring Harbor Perspectives in Biology, 1(6), a001651.
Lindersson, E., Beedholm, R., Højrup, P., Moos, T., Gai, W., Hendil, K. B., & Jensen, P. H. (2004). Proteasomal Inhibition by α-Synuclein Filaments and Oligomers. Journal of Biological Chemistry, 279(13), 12924-12934.
Lücking, C. B., Dürr, A., Bonifati, V., Vaughan, J., De Michele, G., Gasser, T., . . . Brice, A. (2000). Association between Early-Onset Parkinson's Disease and Mutations in the Parkin Gene. New England Journal of Medicine, 342(21), 1560-1567.
Luo, J., Song, J., Feng, P., Wang, Y., Long, W., Liu, M., & Li, L. (2016). Elevated serum apolipoprotein E is associated with metastasis and poor prognosis of non-small cell lung cancer. Tumor Biology, 37(8), 10715-10721.
McCrory, D. C., Lewis, S. Z., Heitzer, J., Colice, G., & Alberts, W. M. (2007). Methodology for Lung Cancer Evidence Review and Guideline Development: ACCP Evidence-Based Clinical Practice Guidelines (2nd Edition). Chest, 132(3, Supplement), 23S-28S.
Mogi, M., Harada, M., Riederer, P., Narabayashi, H., Fujita, K., & Nagatsu, T. (1994). Tumor necrosis factor-α (TNF-α) increases both in the brain and in the cerebrospinal fluid from parkinsonian patients. Neuroscience Letters, 165(1), 208-210.
Molina, J. R., Yang, P., Cassivi, S. D., Schild, S. E., & Adjei, A. A. (2008). Non–Small Cell Lung Cancer: Epidemiology, Risk Factors, Treatment, and Survivorship. Mayo Clinic proceedings. Mayo Clinic, 83(5), 584-594.
Monaco, C., Andreakos, E., Kiriakidis, S., Mauri, C., Bicknell, C., Foxwell, B., . . . Feldmann, M. (2004). Canonical pathway of nuclear factor κB activation selectively regulates proinflammatory and prothrombotic responses in human atherosclerosis. Proceedings of the National Academy of Sciences of the United States of America, 101(15), 5634.
Moretti, J., Chastagner, P., Gastaldello, S., Heuss, S. F., Dirac, A. M., Bernards, R., . . . Brou, C. (2010). The Translation Initiation Factor 3f (eIF3f) Exhibits a Deubiquitinase Activity Regulating Notch Activation. PLOS Biology, 8(11), e1000545.
Morreale, F. E., & Walden, H. (2016). Types of Ubiquitin Ligases. Cell, 165(1), 248-248.e241.
Mustafa, M., Azizi, J., Iiizam, E. L., Azizan, N., Sharifa, S., & Abbas, S. A. (2016). Lung Cancer: Risk Factors, Management, And Prognosis (Vol. 15).
Napetschnig, J., & Wu, H. (2013). Molecular basis of NF-kappaB signaling. Annu Rev Biophys, 42, 443-468.
Pan, H., Jia, R., Zhang, L., Xu, S., Wu, Q., Song, X., . . . Fan, X. (2015). BAP1 regulates cell cycle progression through E2F1 target genes and mediates transcriptional silencing via H2A monoubiquitination in uveal melanoma cells. The International Journal of Biochemistry & Cell Biology, 60, 176-184.
Peters, J.-M. (2003). Emi1 Proteolysis: How SCFβ-Trcp1 Helps to Activate the Anaphase-Promoting Complex. Molecular Cell, 11(6), 1420-1421.
Petrucelli, L., Dickson, D., Kehoe, K., Taylor, J., Snyder, H., Grover, A., . . . Hutton, M. (2004). CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation. Human Molecular Genetics, 13(7), 703-714.
Pickart, C. M., & Eddins, M. J. (2004). Ubiquitin: structures, functions, mechanisms. Biochim Biophys Acta, 1695(1-3), 55-72.
Piper, R. C., Dikic, I., & Lukacs, G. L. (2014). Ubiquitin-Dependent Sorting in Endocytosis. Cold Spring Harbor Perspectives in Biology, 6(1), a016808.
Premarathne, S., Murtaza, M., Matigian, N., Jolly, L. A., & Wood, S. A. (2017). Loss of Usp9x disrupts cell adhesion, and components of the Wnt and Notch signaling pathways in neural progenitors. Scientific Reports, 7(1), 8109.
Shembade, N., & Harhaj, E. W. (2012). Regulation of NF-κB signaling by the A20 deubiquitinase. Cellular And Molecular Immunology, 9, 123.
Steeg, P. S. (2016). Targeting metastasis. Nature Reviews Cancer, 16, 201.
Tilstra, J. S., Clauson, C. L., Niedernhofer, L. J., & Robbins, P. D. (2011). NF-κB in Aging and Disease. Aging and Disease, 2(6), 449-465.
Tokunaga, F., Sakata, S.-i., Saeki, Y., Satomi, Y., Kirisako, T., Kamei, K., . . . Iwai, K. (2009). Involvement of linear polyubiquitylation of NEMO in NF-κB activation. Nature Cell Biology, 11, 123.
van Zijl, F., Krupitza, G., & Mikulits, W. (2011). Initial steps of metastasis: Cell invasion and endothelial transmigration. Mutation Research, 728(1-2), 23-34.
Wan, J. Y., Edwards, K. L., Hutter, C. M., Mata, I. F., Samii, A., Roberts, J. W., . . . Zabetian, C. P. (2014). Association mapping of the PARK10 region for Parkinson's disease susceptibility genes. Parkinsonism and Related Disorders, 20(1), 93-98.
Wang, K., Liu, S., Wang, J., Wu, Y., Cai, F., & Song, W. (2014). Transcriptional regulation of human USP24 gene expression by NF-kappa B. J Neurochem, 128(6), 818-828.
Wang, S. A., Wang, Y. C., Chuang, Y. P., Huang, Y. H., Su, W. C., Chang, W. C., & Hung, J. J. (2017). EGF-mediated inhibition of ubiquitin-specific peptidase 24 expression has a crucial role in tumorigenesis. Oncogene, 36(21), 2930-2945.
Wang, Y. C., Wang, S. A., Chen, P. H., Hsu, T. I., Yang, W. B., Chuang, Y. P., . . . Hung, J. J. (2016). Variants of ubiquitin-specific peptidase 24 play a crucial role in lung cancer malignancy. Oncogene, 35(28), 3669-3680.
Wertz, I. E., & Dixit, V. M. (2010). Signaling to NF-kappaB: regulation by ubiquitination. Cold Spring Harb Perspect Biol, 2(3), a003350.
Whiteside, S. T., & Israël, A. (1997). IκB proteins: structure, function and regulation. Seminars in Cancer Biology, 8(2), 75-82.
Wu, Q. W. (2016). Serpine2, a potential novel target for combating melanoma metastasis. American Journal of Translational Research, 8(5), 1985-1997.
Wu, Z.-H., Shi, Y., Tibbetts, R. S., & Miyamoto, S. (2006). Molecular Linkage Between the Kinase ATM and NF-κB Signaling in Response to Genotoxic Stimuli. Science, 311(5764), 1141.
Xia, Y., Padre, R. C., De Mendoza, T. H., Bottero, V., Tergaonkar, V. B., & Verma, I. M. (2009). Phosphorylation of p53 by IκB kinase 2 promotes its degradation by β-TrCP. Proceedings of the National Academy of Sciences of the United States of America, 106(8), 2629-2634.
Yang, B., Zhang, S., Wang, Z., Yang, C., Ouyang, W., Zhou, F., . . . Xie, C. (2016). Deubiquitinase USP9X deubiquitinates β-catenin and promotes high grade glioma cell growth. Oncotarget, 7(48), 79515-79525.
Yi, Y.-J., Manandhar, G., Sutovsky, M., Li, R., Jonáková, V., Oko, R., . . . Sutovsky, P. (2007). Ubiquitin C-Terminal Hydrolase-Activity Is Involved in Sperm Acrosomal Function and Anti-Polyspermy Defense During Porcine Fertilization1. Biology of Reproduction, 77(5), 780-793.
Zhang, L., Lubin, A., Chen, H., Sun, Z., & Gong, F. (2012). The deubiquitinating protein USP24 interacts with DDB2 and regulates DDB2 stability. Cell Cycle, 11(23), 4378-4384.
Zhang, L., Nemzow, L., Chen, H., Lubin, A., Rong, X., Sun, Z., . . . Gong, F. (2015). The deubiquitinating enzyme USP24 is a regulator of the UV damage response. Cell Reports, 10(2), 140-147.
Ziaee, S., Chu, G. C.-Y., Huang, J.-M., Sieh, S., & Chung, L. W. K. (2015). Prostate cancer metastasis: roles of recruitment and reprogramming, cell signal network and three-dimensional growth characteristics. Translational Andrology and Urology, 4(4), 438-454.