頭頸癌是世界上第七大常見癌症，晚期頭頸癌患者即便是在鉑金類化療藥物治療下，仍有百分之十五到四十的患者會產生抗藥性而有合併遠端器官轉移的情況發生。而癌細胞轉移又是一項複雜過程，其中的關鍵步驟便是癌細胞必須在血液循環這種懸浮狀態下，抵抗因失去與細胞外基質黏附而產生的凋亡現象才能生存下來，稱作抗失巢凋亡現象。而這些抗失巢凋亡癌細胞也因較具備癌幹細胞特性，從而具有高度致瘤與轉移能力。然而鉑金類化療藥物對於頭頸癌轉移性復發之分子機制至今仍不清楚。先前實驗室研究發現在順鉑處理後之殘存癌細胞中，PTX3、休眠相關基因及轉移相關基因呈現高度表達，並且在EGF刺激下有更高的表現。而在本篇研究中，將針對探討在鉑金類化療藥物處理前後，PTX3是否參與在調控癌細胞抗失巢凋亡能力，進而促成癌細胞之轉移。在本篇研究中所篩選出具有較高抗失巢凋亡特性癌細胞 (P3)中發現PTX3及MMP-9在P3細胞中表現增加，並且在EGF的刺激下具有更高表現。同時P3細胞也具備較強的球體形成與爬行能力，而PTX3基因致弱則能抑制EGF處理之下誘導的癌細胞爬行能力。此外，動物實驗中也發現P3細胞具備較強的外滲能力，PTX3基因致弱組別中外滲能力有下降趨勢。另一方面，與P3細胞相同，在化療藥物處理過後之殘存癌細胞中也發現具有較高之PTX3及MMPs相關基因表現，而在EGF刺激之下則是呈現更高之基因表現。而休眠相關基因在殘存癌細胞中也呈現高表達的情形。此外，殘存之癌細胞也具備較強球體形成能力，並在EGF處理下有更加乘的效果，PTX3基因致弱則能抑制EGF處理之下誘導的癌細胞球體形成能力。動物實驗結果也證實Oxaliplatin-S細胞具備較強的外滲能力。這些結果顯示，化療後殘存癌細胞可透過PTX3所調控之抗失巢凋亡能力，進而在血液循環中獲得轉移和存活的潛能。
關鍵字：頭頸癌、鉑金類化療藥物、抗失巢凋亡、五聚環蛋白 3、癌轉移

Head and neck squamous cell carcinoma (HNSCC) is the 7th leading cause of cancer-related mortality. Most patients develop chemo drug resistance, which in turn leads to the cancer recurrence and metastasis. During metastasis, tumor cells need to survive in the circulation without attaching with the extracellular matrix, which is called anoikis resistance. Cancer cells with this characteristic may have highly metastatic potential. However, the molecular mechanisms involved in cancer metastatic potential of post-platinum-based drugs surviving HNSCC cells (CDDP-S and Oxaliplatin-S cells) were still not very clear. In this study, we will investigate whether PTX3 is involved in the regulation of anoikis resistance in post-platinum-based drugs surviving HNSCC cells, thereby contributing to the metastasis. Results showed that PTX3 and MMP9 expression were increased in the repeated selected anoikis-resistant cells (P3), and EGF treatment had a synergistic effect on these gene expressions. The sphere formation and migration abilities were all increased in P3 cells. In vivo data showed that P3 cells had stronger extravasation ability than the parental cells. PTX3 knockdown significantly inhibited sphere formation and EGF-induced migration ability and slightly blocked extravasation ability in P3 cells. On the other hand, gene expressions of dormancy-related, PTX3 and MMPs were increased in CDDP-S and Oxaliplatin-S cells, and EGF treatment had a synergistic effect on PTX3 and MMPs gene expressions. Furthermore, we investigated that Oxaliplatin-S cells had acquired sphere formation ability, and PTX3 knockdown significantly suppressed EGF-induced sphere formation ability which was also in consistence with P3 cells. In vivo data revealed that Oxaliplatin-S cells had stronger extravasation ability than the parental cells. These results demonstrated that post-platinum-based drugs surviving HNSCC cells could acquire metastatic and survival potential in the circulation, which may confer EGF-induced metastasis to HNSCC.
Key word：Head and neck cancer, platinum-based drugs, anoikis resistance, PTX3, metastasis

1 Dhull, A. K., Atri, R., Dhankhar, R., Chauhan, A. K. & Kaushal, V. Major risk factors in head and neck Cancer: a retrospective analysis of 12-year experiences. World journal of oncology 9, 80 (2018).
2 Nigro, C. L., Denaro, N., Merlotti, A. & Merlano, M. Head and neck cancer: improving outcomes with a multidisciplinary approach. Cancer management and research 9, 363 (2017).
3 Huang, S. H. & O’Sullivan, B. Overview of the 8th edition TNM classification for head and neck cancer. Current treatment options in oncology 18, 1-13 (2017).
4 Johnson, D. E. et al. Head and neck squamous cell carcinoma. Nature Reviews Disease Primers 6, 92 (2020).
5 Network, C. G. A. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature 517, 576 (2015).
6 Kalyankrishna, S. & Grandis, J. R. Epidermal growth factor receptor biology in head and neck cancer. Journal of clinical oncology 24, 2666-2672 (2006).
7 Bossi, P. et al. Prognostic and predictive value of EGFR in head and neck squamous cell carcinoma. Oncotarget 7, 74362 (2016).
8 Yokota, J. Tumor progression and metastasis. Carcinogenesis 21, 497-503 (2000).
9 Fares, J., Fares, M. Y., Khachfe, H. H., Salhab, H. A. & Fares, Y. Molecular principles of metastasis: a hallmark of cancer revisited. Signal transduction and targeted therapy 5, 1-17 (2020).
10 Loh, C.-Y. et al. The E-cadherin and N-cadherin switch in epithelial-to-mesenchymal transition: signaling, therapeutic implications, and challenges. Cells 8, 1118 (2019).
11 Peinado, H., Ballestar, E., Esteller, M. & Cano, A. Snail mediates E-cadherin repression by the recruitment of the Sin3A/histone deacetylase 1 (HDAC1)/HDAC2 complex. Molecular and cellular biology 24, 306-319 (2004).
12 Engbring, J. A. & Kleinman, H. K. The basement membrane matrix in malignancy. The Journal of Pathology: A Journal of the Pathological Society of Great Britain and Ireland 200, 465-470 (2003).
13 Gonzalez-Avila, G. et al. Matrix metalloproteinases participation in the metastatic process and their diagnostic and therapeutic applications in cancer. Critical reviews in oncology/hematology 137, 57-83 (2019).
14 Itoh, T. et al. Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer research 58, 1048-1051 (1998).
15 Wilson, C. L., Heppner, K. J., Labosky, P. A., Hogan, B. L. & Matrisian, L. M. Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin. Proceedings of the National Academy of Sciences 94, 1402-1407 (1997).
16 Liu, Z. et al. Gelatinase B–deficient mice are resistant to experimental bullous pemphigoid. The Journal of experimental medicine 188, 475-482 (1998).
17 Katayama, A. et al. Expressions of matrix metalloproteinases in early-stage oral squamous cell carcinoma as predictive indicators for tumor metastases and prognosis. Clinical Cancer Research 10, 634-640 (2004).
18 Zavyalova, M. et al. Intravasation as a key step in cancer metastasis. Biochemistry (Moscow) 84, 762-772 (2019).
19 Paoli, P., Giannoni, E. & Chiarugi, P. Anoikis molecular pathways and its role in cancer progression. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1833, 3481-3498 (2013).
20 Sun, T. et al. Anoikis resistant mediated by FASN promoted growth and metastasis of osteosarcoma. Cell death & disease 10, 1-13 (2019).
21 Fofaria, N. M. & Srivastava, S. K. STAT3 induces anoikis resistance, promotes cell invasion and metastatic potential in pancreatic cancer cells. Carcinogenesis 36, 142-150 (2015).
22 Weiswald, L.-B., Bellet, D. & Dangles-Marie, V. Spherical cancer models in tumor biology. Neoplasia 17, 1-15 (2015).
23 Schumacher, D., Strilic, B., Sivaraj, K. K., Wettschureck, N. & Offermanns, S. Platelet-derived nucleotides promote tumor-cell transendothelial migration and metastasis via P2Y2 receptor. Cancer cell 24, 130-137 (2013).
24 Spano, J. et al. Epidermal growth factor receptor signaling in colorectal cancer: preclinical data and therapeutic perspectives. Annals of oncology 16, 189-194 (2005).
25 Maiti, G. P. et al. Overexpression of EGFR in head and neck squamous cell carcinoma is associated with inactivation of SH3GL2 and CDC25A genes. PloS one 8, e63440 (2013).
26 Huang, P. et al. The role of EGF‐EGFR signalling pathway in hepatocellular carcinoma inflammatory microenvironment. Journal of cellular and molecular medicine 18, 218-230 (2014).
27 Zhang, Z., Dong, Z., Lauxen, I. S., Sant'Ana Filho, M. & Nör, J. E. Endothelial cell-secreted EGF induces epithelial to mesenchymal transition and endows head and neck cancer cells with stem-like phenotype. Cancer research 74, 2869-2881 (2014).
28 Chang, W.-C. et al. PTX3 gene activation in EGF-induced head and neck cancer cell metastasis. Oncotarget 6, 7741 (2015).
29 Liao, Y. et al. Epidermal growth factor-induced ANGPTL4 enhances anoikis resistance and tumour metastasis in head and neck squamous cell carcinoma. Oncogene 36, 2228-2242 (2017).
30 Baba, Y., Fujii, M., Tokumaru, Y. & Kato, Y. Present and future of EGFR inhibitors for head and neck squamous cell cancer. Journal of Oncology 2012 (2012).
31 Sindhu, S. K. & Bauman, J. E. Current concepts in chemotherapy for head and neck cancer. Oral and maxillofacial surgery clinics of North America 31, 145 (2019).
32 Huang, J. et al. Comparative effectiveness and safety between oxaliplatin-based and cisplatin-based therapy in advanced gastric cancer: A meta-analysis of randomized controlled trials. Oncotarget 7, 34824 (2016).
33 Espinosa, M. et al. Oxaliplatin activity in head and neck cancer cell lines. Cancer chemotherapy and pharmacology 55, 301-305 (2005).
34 Gilbert, J. et al. Phase 2 trial of oxaliplatin and pemetrexed as an induction regimen in locally advanced head and neck cancer. Cancer 118, 1007-1013 (2012).
35 Marur, S. & Forastiere, A. A. Head and neck squamous cell carcinoma: update on epidemiology, diagnosis, and treatment. Mayo Clinic Proceedings 91, 386-396 (2016).
36 Specenier, P. & Vermorken, J. B. Cetuximab: its unique place in head and neck cancer treatment. Biologics: targets & therapy 7, 77 (2013).
37 Vermorken, J. B. et al. Platinum-based chemotherapy plus cetuximab in head and neck cancer. New England Journal of Medicine 359, 1116-1127 (2008).
38 Bonner, J. A. et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. New England Journal of Medicine 354, 567-578 (2006).
39 Chang, J.-H., Wu, C.-C., Yuan, K. S.-P., Wu, A. T. & Wu, S.-Y. Locoregionally recurrent head and neck squamous cell carcinoma: incidence, survival, prognostic factors, and treatment outcomes. Oncotarget 8, 55600 (2017).
40 Yeh, T.-J. et al. The Overall Efficacy and Outcomes of Metronomic Tegafur-Uracil Chemotherapy on Locally Advanced Head and Neck Squamous Cell Carcinoma: A Real-World Cohort Experience. Biology 10, 168 (2021).
41 Burtness, B., Goldwasser, M. A., Flood, W., Mattar, B. & Forastiere, A. A. Phase III randomized trial of cisplatin plus placebo compared with cisplatin plus cetuximab in metastatic/recurrent head and neck cancer: an Eastern Cooperative Oncology Group study. Journal of Clinical Oncology 23, 8646-8654 (2005).
42 Kurppa, K. J. et al. Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway. Cancer cell 37, 104-122. e112 (2020).
43 Tetsu, O., Hangauer, M. J., Phuchareon, J., Eisele, D. W. & McCormick, F. Drug resistance to EGFR inhibitors in lung cancer. Chemotherapy 61, 223-235 (2016).
44 Gao, X.-l., Zhang, M., Tang, Y.-l. & Liang, X.-h. Cancer cell dormancy: mechanisms and implications of cancer recurrence and metastasis. OncoTargets and therapy 10, 5219 (2017).
45 Willis, R. A. The spread of tumors in the human body. (1952).
46 Hadfield, G. The dormant cancer cell. British medical journal 2, 607 (1954).
47 Aguirre-Ghiso, J. A. Models, mechanisms and clinical evidence for cancer dormancy. Nature Reviews Cancer 7, 834-846 (2007).
48 Yeh, A. C. & Ramaswamy, S. Mechanisms of cancer cell dormancy—another hallmark of cancer? Cancer research 75, 5014-5022 (2015).
49 Endo, H., Okuyama, H., Ohue, M. & Inoue, M. Dormancy of cancer cells with suppression of AKT activity contributes to survival in chronic hypoxia. PloS one 9, e98858 (2014).
50 Ferrer, A. et al. Hypoxia-mediated changes in bone marrow microenvironment in breast cancer dormancy. Cancer Letters 488, 9-17 (2020).
51 Sosa, M. S. et al. NR2F1 controls tumour cell dormancy via SOX9-and RARβ-driven quiescence programmes. Nature communications 6, 1-14 (2015).
52 Fluegen, G. et al. Phenotypic heterogeneity of disseminated tumour cells is preset by primary tumour hypoxic microenvironments. Nature cell biology 19, 120-132 (2017).
53 Sosnoski, D. M., Norgard, R. J., Grove, C. D., Foster, S. J. & Mastro, A. M. Dormancy and growth of metastatic breast cancer cells in a bone-like microenvironment. Clinical & experimental metastasis 32, 335-344 (2015).
54 Li, S. et al. Model of tumor dormancy/recurrence after short-term chemotherapy. PloS one 9, e98021 (2014).
55 Osisami, M. & Keller, E. T. Mechanisms of metastatic tumor dormancy. Journal of clinical medicine 2, 136-150 (2013).
56 Breviario, F. et al. Interleukin-1-inducible genes in endothelial cells. Cloning of a new gene related to C-reactive protein and serum amyloid P component. Journal of Biological Chemistry 267, 22190-22197 (1992).
57 Han, B. et al. TNFα-induced long pentraxin PTX3 expression in human lung epithelial cells via JNK. The Journal of Immunology 175, 8303-8311 (2005).
58 Bottazzi, B. et al. Multimer formation and ligand recognition by the long pentraxin PTX3: similarities and differences with the short pentraxins C-reactive protein and serum amyloid P component. Journal of Biological Chemistry 272, 32817-32823 (1997).
59 Kunes, P., Holubcova, Z., Kolackova, M. & Krejsek, J. Pentraxin 3 (PTX 3): an endogenous modulator of the inflammatory response. Mediators of inflammation 2012 (2012).
60 Doni, A. et al. Production of the soluble pattern recognition receptor PTX3 by myeloid, but not plasmacytoid, dendritic cells. European journal of immunology 33, 2886-2893 (2003).
61 Klouche, M. et al. Modified atherogenic lipoproteins induce expression of pentraxin-3 by human vascular smooth muscle cells. Atherosclerosis 175, 221-228 (2004).
62 Abderrahim-Ferkoune, A. et al. Characterization of the long pentraxin PTX3 as a TNFα-induced secreted protein of adipose cells. Journal of lipid research 44, 994-1000 (2003).
63 Qi, S., Zhao, F., Li, Z., Liang, F. & Yu, S. Silencing of PTX3 alleviates LPS-induced inflammatory pain by regulating TLR4/NF-κB signaling pathway in mice. Bioscience reports 40, BSR20194208 (2020).
64 Norata, G. D. et al. Long pentraxin 3, a key component of innate immunity, is modulated by high-density lipoproteins in endothelial cells. Arteriosclerosis, thrombosis, and vascular biology 28, 925-931 (2008).
65 Bottazzi, B., Doni, A., Garlanda, C. & Mantovani, A. An integrated view of humoral innate immunity: pentraxins as a paradigm. Annual review of immunology 28, 157-183 (2009).
66 Inforzato, A. et al. Structure and function of the long pentraxin PTX3 glycosidic moiety: fine-tuning of the interaction with C1q and complement activation. Biochemistry 45, 11540-11551 (2006).
67 Deban, L. et al. Regulation of leukocyte recruitment by the long pentraxin PTX3. Nature immunology 11, 328-334 (2010).
68 Cappuzzello, C. et al. Mesenchymal stromal cell-derived PTX3 promotes wound healing via fibrin remodeling. Journal of Investigative Dermatology 136, 293-300 (2016).
69 Russell, D. L. & Salustri, A. Extracellular matrix of the cumulus-oocyte complex. Seminars in reproductive medicine 24, 217-227 (2006).
70 Presta, M., Camozzi, M., Salvatori, G. & Rusnati, M. Role of the soluble pattern recognition receptor PTX3 in vascular biology. Journal of cellular and molecular medicine 11, 723-738 (2007).
71 Mantovani, A., Allavena, P., Sica, A. & Balkwill, F. Cancer-related inflammation. nature 454, 436-444 (2008).
72 Kondo, S. et al. Clinical impact of pentraxin family expression on prognosis of pancreatic carcinoma. British journal of cancer 109, 739-746 (2013).
73 Chan, S.-H., Tsai, J.-P., Shen, C.-J., Liao, Y.-H. & Chen, B.-K. Oleate-induced PTX3 promotes head and neck squamous cell carcinoma metastasis through the up-regulation of vimentin. Oncotarget 8, 41364 (2017).
74 Choi, B. et al. Pentraxin-3 silencing suppresses gastric cancer-related inflammation by inhibiting chemotactic migration of macrophages. Anticancer research 35, 2663-2668 (2015).
75 Choi, B. et al. Elevated Pentraxin 3 in bone metastatic breast cancer is correlated with osteolytic function. Oncotarget 5, 481 (2014).
76 Ying, T.-H. et al. Knockdown of Pentraxin 3 suppresses tumorigenicity and metastasis of human cervical cancer cells. Scientific reports 6, 1-12 (2016).
77 Tung, J.-N. et al. Inhibition of pentraxin 3 in glioma cells impairs proliferation and invasion in vitro and in vivo. Journal of neuro-oncology 129, 201-209 (2016).
78 Ronca, R. et al. Long pentraxin-3 inhibits epithelial–mesenchymal transition in melanoma cells. Molecular cancer therapeutics 12, 2760-2771 (2013).
79 Ronca, R. et al. Long pentraxin‐3 as an epithelial–stromal fibroblast growth factor‐targeting inhibitor in prostate cancer. The Journal of pathology 230, 228-238 (2013).
80 Inforzato, A. et al. The “sweet” side of a long pentraxin: how glycosylation affects PTX3 functions in innate immunity and inflammation. Frontiers in immunology 3, 407 (2013).
81 Kim, Y.-N., Koo, K. H., Sung, J. Y., Yun, U.-J. & Kim, H. Anoikis resistance: an essential prerequisite for tumor metastasis. International journal of cell biology 2012 (2012).
82 Kim, R. S. et al. Dormancy Signatures and Metastasis in Estrogen Receptor Positive and Negative Breast Cancer. PLOS ONE 7, e35569 (2012).
83 Fang, W. T. et al. Downregulation of a putative tumor suppressor BMP4 by SOX2 promotes growth of lung squamous cell carcinoma. International journal of cancer 135, 809-819 (2014).
84 Bendas, G. & Borsig, L. Cancer cell adhesion and metastasis: selectins, integrins, and the inhibitory potential of heparins. International journal of cell biology 2012 (2012).
85 Sökeland, G. & Schumacher, U. The functional role of integrins during intra-and extravasation within the metastatic cascade. Molecular cancer 18, 1-19 (2019).
86 Park, S.-Y. & Nam, J.-S. The force awakens: metastatic dormant cancer cells. Experimental & Molecular Medicine 52, 569-581 (2020).
87 Brognard, J., Clark, A. S., Ni, Y. & Dennis, P. A. Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer research 61, 3986-3997 (2001).
88 Taddei, M. L., Giannoni, E., Fiaschi, T. & Chiarugi, P. Anoikis: an emerging hallmark in health and diseases. J Pathol 226, 380-393 (2012).
89 Terada, L. S. & Nwariaku, F. E. Escaping Anoikis through ROS: ANGPTL4 controls integrin signaling through Nox1. Cancer cell 19, 297-299 (2011).
90 Oliveira-Ferrer, L., Legler, K. & Milde-Langosch, K. Role of protein glycosylation in cancer metastasis. Semin Cancer Biol 44, 141-152 (2017).
91 Hu, P. et al. E-cadherin core fucosylation regulates nuclear beta-catenin accumulation in lung cancer cells. Glycoconj J 25, 843-850 (2008).
92 Teng, M. W., Swann, J. B., Koebel, C. M., Schreiber, R. D. & Smyth, M. J. Immune-mediated dormancy: an equilibrium with cancer. J Leukoc Biol 84, 988-993 (2008).
93 Weaver, V. M. et al. Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J Cell Biol 137, 231-245 (1997).
94 Albrengues, J. et al. Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice. Science 361 (2018).