子宮頸神經內分泌腫瘤是子宮頸癌中相當罕見的一種。和過去常見的子宮頸癌類型，如鱗狀上皮癌及腺癌相比，子宮頸神經內分泌腫瘤大多較具侵犯性，疾病的進展相當快速且預後不佳。我們研究團隊與新竹馬偕醫院婦癌科合作，於先前計畫中得知了一位75歲的婦人疑似患有這種罕見的癌症，也在病人的同意下取得了這個子宮頸腫瘤組織活體樣本。我們透過細胞分離選殖方式，成功的將此子宮頸腫瘤組織分離成原代培養細胞，並且命名此細胞株為HM-1，而STR鑑定的結果顯示，HM-1確實為一個全新的子宮頸腫瘤細胞株。HM-1的細胞型態不同於過去常見的子宮頸癌細胞，較近似於纖維母細胞，呈現較細長的紡錘狀。其細胞倍增時間約為34小時，相較於過去常見的子宮頸癌HeLa細胞株的19小時高出許多。此外我們發現HM-1細胞表現神經內分泌細胞的生物標記SYP，說明了HM-1可能是由神經內分泌細胞演化而來。我們也發現HM-1表現HPV16，這也代表了HM-1這個子宮頸神經內分泌腫瘤的發生可能和HPV感染具有關聯性。
為了觀察HM-1形成腫瘤的能力，我們將HM-1細胞以皮下注射的方式打入小鼠體內。結果顯示HM-1確實可以在小鼠體內形成腫瘤，其腫瘤倍增時間約為13天，較過去文獻指出HeLa細胞株的腫瘤倍增時間5天緩慢許多。而在抗癌藥物反應方面，測試結果顯示Docetaxel對於抑制HM-1細胞存活率具有最好的效果，依次為Cisplatin和Etoposide，而Gemcitabine則幾乎沒有效果。此外我們也驗證Cisplatin和Etoposide兩種藥物的結合能夠有效的抑制HM-1細胞的存活率，與前人研究相符，顯示了多種藥物結合在臨床上對於治療子宮頸神經內分泌腫瘤的可能性。
進一步探討多重藥物療法的增益方案，我們發現以nutlin-3去抑制MDM2，會使子宮頸癌細胞Caski、HeLa以及HM-1細胞中FoxM1的表現量被抑制。且發現Caski和HeLa細胞的移動能力也會受到抑制。因此我們認為MDM2和FoxM1的抑制劑在未來治療子宮頸癌甚至是子宮頸神經內分泌腫瘤或許會是個相當重要的候選藥物。相信我們所建立的子宮頸神經內分泌腫瘤細胞株HM-1於未來可以提供我們進行更多子宮頸神經內分泌腫瘤的相關研究，也期望在未來能夠找到對這種罕見疾病最有效的治療方法。

Neuroendocrine tumor of uterine cervix (NTUC) is a relative rare subtype of cervical cancer. In comparison to other subtypes of gynecologic cancers, such as squamous cell carcinoma and adenocarcinoma, the NTUC is much more aggressive. Consequently, patients of NTUC have poor prognosis.
In collaboration with gynecologic oncologists at Hsinchu branch of MacKay memorial hospital, we obtained a tissue sample of suspected NTUC from a 75-year-old patient. Using enzymatic and mechanical dissociation methods, we established the primary culture (HM-1) from this specimen, and the STR analysis result showed that HM-1 is a novel cancer cell line. HM-1 cells are bipolar or multipolar and showed fibroblast-like, elongated and spindle shape, which is different to common types of cervical cancer. In addition, we found HM-1 has a cell doubling time of 34 hours, which is significantly longer than the 19 hours cell doubling time of the common cervical cancer HeLa cells. Immunocytochemical staining validated that high expression level of the neuroendocrine marker, neuroendocrine synaptophysin (SYP), was present in the HM-1 cells, which suggested that HM-1 cells were originated from neuroendocrine cells. In addition, the presence of human papillomavirus 16 (HPV16) was detected in HM-1 cells, suggesting its potential association with HPV infection. To test its tumorigenesis capacity, the HM-1 cells were subcutaneously inoculated into BALB/c female nude mice. The result showed the HM-1 cells could proliferate in vivo, with a tumor volume doubling time of 13 days, which was higher than that HeLa.
In term of anti-cancer drug sensitivity, Docetaxel has the highest inhibitory effect on HM-1 cell viability, followed by Cisplatin and Etoposide respectively, while the cells was shown to be least sensitive to Gemcitabine. On the other hand, HM-1 cells were highly sensitive to the combination treatment of etoposide and cisplatin.
In order to develop a more effective treatment, we found that inhibition of MDM2 by nutlin-3 would decreased FoxM1 expression in cervical cancer cell lines CasKi, HeLa and NTUC. Inhibition of MDM2 can also decrease the migration ability of cervical cancer cells. Our findings suggested that the inhibition of MDM2 pathways and FoxM1-dependent pathway may be novel therapeutic targets for cervical cancers, including NTUC. We believe this new neuroendocrine cancer cell line HM-1 will be useful as a research model of NTUC. HM-1-based assay may help in developing effective diagnosis test or therapy for this rare subtype of gynecological malignancy.

Table of contents
中文摘要 I
Abstract III
致謝 V
List of Abbreviations XI
1. Introduction 1
1.1 Cervical cancer 1
1.2 Neuroendocrine tumor of uterine cervix and its treatment strategies 2
1.3 Forkhead box M1 (FoxM1) is a tumor progressive and metastatic factor associated with chemotherapy resistance 3
1.4 MDM2/p53/FoxO3 pathway are potential therapeutic targets for various types of cancer 4
1.5 Objectives and significant finding of this study 5
2. Materials and methods 7
2.1 Cancer cell isolation 7
2.2 Cell and culture 8
2.3 STR (Short tandem repeat) analysis 8
2.4 Doubling time assay 8
2.5 Reverse transcription-PCR (RT-PCR for HPV detection) 9
2.6 Western blot 9
2.7 Immunocytochemistry (ICC for HM-1) 9
2.8 Xenotransplantation 10
2.9 Histological analysis 11
2.10 Drug treatment 11
2.11 MTT assay 11
2.12 Transwell migration assay 12
2.13 Wound healing assay 12
3. Results 14
3.1 Establishment of in vitro HM-1 cells culture 14
3.2 HM-1 is a novel cervical cancer cell line 14
3.3 Morphologic and growth characteristics of HM-1 cells 16
3.4 HM-1 cells show expression of HPV-16 and neuroendocrine markers 16
3.5 Xenotransplantation of HM-1 17
3.6 Combination of etoposide and cisplatin may be potential NETCx treatment 18
3.6.1 Drug sensitivity of HM-1 to anti-cancer drugs 18
3.6.2 Drug sensitivity of HM-1 to combination treatment of etoposide and cisplatin 20
3.7 MDM2 regulate FoxM1 expression to affect mobility of cervical cancer cells 20
4. Discussion 23
4.1 Neuroendocrine tumor are rare neoplasm arising from cells of neuroendocrine system 24
4.2 Specific biochemical markers for neuroendocrine tumor 25
4.3 HPV is the most important risk factor of cervical cancer 26
4.4 Potential chemotherapeutic drugs to NTUC 27
4.5 MDM2/FoxO3/FoxM1 pathway may be a novel treatment strategy for neuroendocrine tumor of uterine cervix. 28
4.6 Perspective of this study 29
Reference : 30
 
List of figures
Figure 1. : Morphological presentation of HM-1 and other common cervical cancer cells. 33
Figure 2. : A cell growth curve analysis of HM-1. 34
Figure 3. : HPV detection. 35
Figure 4. Western blot and immuno-cytochemical staining showed that HM-1 cells express the neuroendocrine marker, neuroendocrine synaptophysin (SYP). 36
Figure 5. Xeno-transplantation and in vivo tumor growth curve of HM-1 cells. 37
Figure 6. HM-1 xenograft showed histological morphology similar to that of squamous cell carcinoma, Caski. 38
Figure 7. Dose response curves of HM-1 cells exposed to anti-cancer drug. 39
Figure 8. Drug response assay of HM-1 cells exposed to combination treatment of anti-cancer drugs. 40
Figure 9. Hypothesis for the relationship between MDM2-p53 pathway and FoxM1 axis in cancer progression 41
Figure 10. Inhibition of p53-Mdm2 interaction would decrease 2D cell mobility in cervical cancer. 43
Figure 11. Inhibition of MDM2-p53 and MDM2-FoxO3 interaction would decrease 3D cell mobility in cervical cancer. 45
Figure 12. Inhibition of MDM2-p53 and MDM2-FoxO3 interaction would decrease FoxM1 expression in cervical cancer and HM-1 cells. 47

 
List of Tables
Table 1. RT-PCR primers list 48
Table 2. Antibodies list 49
Table 3. Allele table for the HM-1 cells 50
Table 4. Comparison the STR profile between HM-1 cells and other NET cell lines 51
Table 5. Comparison the STR profile between HM-1 cells and ATCC database 52

Reference :
1. Jemal, A., et al., Global cancer statistics. CA Cancer J Clin, 2011. 61(2): p. 69-90.
2. Cervical cancer screening. 2014; Available from: http://www.cancerresearchuk.org/cancer-help/type/cervical-cancer/about/cervical-cancer-screening.
3. Burd, E.M., Human papillomavirus and cervical cancer. Clin Microbiol Rev, 2003. 16(1): p. 1-17.
4. Christopher Dolinsky, M.a.C.H.-K., MD. Cervical cancerThe Basics. 2013 December 18; Available from: http://www.oncolink.org/types/article.cfm?c=179&id=8226.
5. Manchana, T., et al., Targeted therapies for rare gynaecological cancers. Lancet Oncol, 2010. 11(7): p. 685-93.
6. Gardner, G.J., D. Reidy-Lagunes, and P.A. Gehrig, Neuroendocrine tumors of the gynecologic tract: A Society of Gynecologic Oncology (SGO) clinical document. Gynecol Oncol, 2011. 122(1): p. 190-8.
7. Peng, P., et al., Neuroendocrine tumor of the uterine cervix: a clinicopathologic study of 14 cases. Arch Gynecol Obstet, 2012. 286(5): p. 1247-53.
8. Chen, J., O.K. Macdonald, and D.K. Gaffney, Incidence, mortality, and prognostic factors of small cell carcinoma of the cervix. Obstet Gynecol, 2008. 111(6): p. 1394-402.
9. Wang, K.L., et al., Primary treatment and prognostic factors of small cell neuroendocrine carcinoma of the uterine cervix: a Taiwanese Gynecologic Oncology Group study. Eur J Cancer, 2012. 48(10): p. 1484-94.
10. Wang, I.C., et al., Forkhead box M1 regulates the transcriptional network of genes essential for mitotic progression and genes encoding the SCF (Skp2-Cks1) ubiquitin ligase. Mol Cell Biol, 2005. 25(24): p. 10875-94.
11. Raychaudhuri, P. and H.J. Park, FoxM1: a master regulator of tumor metastasis. Cancer Res, 2011. 71(13): p. 4329-33.
12. Viswanathan, A.N., et al., Small cell neuroendocrine carcinoma of the cervix: outcome and patterns of recurrence. Gynecol Oncol, 2004. 93(1): p. 27-33.
13. Chan, D.W., et al., Over-expression of FOXM1 transcription factor is associated with cervical cancer progression and pathogenesis. J Pathol, 2008. 215(3): p. 245-52.
14. Gomes, A.R., F. Zhao, and E.W. Lam, Role and regulation of the forkhead transcription factors FOXO3a and FOXM1 in carcinogenesis and drug resistance. Chin J Cancer, 2013. 32(7): p. 365-70.
15. Pandit, B., M. Halasi, and A.L. Gartel, p53 negatively regulates expression of FoxM1. Cell Cycle, 2009. 8(20): p. 3425-7.
16. Haupt, Y., et al., Mdm2 promotes the rapid degradation of p53. Nature, 1997. 387(6630): p. 296-9.
17. Fu, W., et al., MDM2 acts downstream of p53 as an E3 ligase to promote FOXO ubiquitination and degradation. J Biol Chem, 2009. 284(21): p. 13987-4000.
18. Vassilev, L.T., et al., In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science, 2004. 303(5659): p. 844-8.
19. Tovar, C., et al., Small-molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: implications for therapy. Proc Natl Acad Sci U S A, 2006. 103(6): p. 1888-93.
20. Zhang, W., Blockade of MEK and MDM2 synergistically induces apoptosis in acute myeloid leukemia via BH3-only proteins Puma and Bim. Cancer Res, 2010.
21. Strober, W., Trypan blue exclusion test of cell viability. Curr Protoc Immunol, 2001. Appendix 3: p. Appendix 3B.
22. Growth of HeLa Cells. Available from: http://www.percell.se/116.pdf.
23. V, P., et al., Small cell neuroendocrine carcinoma of the cervix: a rare entity. J Clin Diagn Res, 2014. 8(2): p. 147-8.
24. Yoshida, Y., et al., Atypical metastatic carcinoid of the uterine cervix and review of the literature. J Obstet Gynaecol Res, 2011. 37(6): p. 636-40.
25. Pommier, Y., et al., DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem Biol, 2010. 17(5): p. 421-33.
26. Hande, K.R., Etoposide: four decades of development of a topoisomerase II inhibitor. Eur J Cancer, 1998. 34(10): p. 1514-21.
27. Gordaliza, M., et al., Podophyllotoxin: distribution, sources, applications and new cytotoxic derivatives. Toxicon, 2004. 44(4): p. 441-59.
28. Siddik, Z.H., Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene, 2003. 22(47): p. 7265-79.
29. Lyseng-Williamson, K.A. and C. Fenton, Docetaxel: a review of its use in metastatic breast cancer. Drugs, 2005. 65(17): p. 2513-31.
30. Eisenhauer, E.A. and J.B. Vermorken, The taxoids. Comparative clinical pharmacology and therapeutic potential. Drugs, 1998. 55(1): p. 5-30.
31. Mini, E., et al., Cellular pharmacology of gemcitabine. Ann Oncol, 2006. 17 Suppl 5: p. v7-12.
32. What are neuroendocrine tumors (NETs)? ; Available from: http://www.thenetalliance.com/About-NET.jsp?id=0.
33. Where do neuroendocrine tumors (NETs) occur?
34. Klimstra, D.S., et al., The pathologic classification of neuroendocrine tumors: a review of nomenclature, grading, and staging systems. Pancreas, 2010. 39(6): p. 707-12.
35. Vinik, A.I., et al., Biochemical testing for neuroendocrine tumors. Pancreas, 2009. 38(8): p. 876-89.
36. Koenraad Van Doorslaera, R.D.B., Association between hTERT activation by HPV E6 proteins and oncogenic risk. Virology, 2012. 433(1): p. 216–219.
37. Hengstermann, A., et al., Complete switch from Mdm2 to human papillomavirus E6-mediated degradation of p53 in cervical cancer cells. Proc Natl Acad Sci U S A, 2001. 98(3): p. 1218-23.