口腔生物學特論

1. 口腔生物學特論 Oral Cancer: Mechanism & Prevention 口腔癌之機轉與預防 陳玉昆教授: 高雄醫學大學 口腔病理科 07-3121101~2755 yukkwa@kmu.edu.tw

2. 參 考 書 目 • Gibbs WW. Untangling the roots of cancer. Sci Am 2003;289:56-65. • What you need to know about cancer. Sci Am 1996 ;289:28-119. • Hannen EJM, Riediger D. The quantification of angiogenesis in relation to metastasis in oral cancer: a review. Int. J Oral Maxillofac Surg 2004;33:2-7. • Shieh et al. Role of angiogenic and non-angiogenic mechanisms in oral squamous cell carcinoma: correlation with histologic differentiation and tumor progression. J Oral Pathol Med 2004;33:601-6. • Sharma DC. Betel quid and areca nut are carcinogenic without tobacco. Lancet Oncol 2003;4:587. • Sharma DC. Indian betel quid more carcinogenic than anticipated. Lancet Oncol 2001;2:464. • Braakhuis BJM et al. A genetic progression model of oral cancer: current evidence and clinical implications. J Oral Pathol Med 2004;33:317-22. • Braakhuis BJM et al. A Genetic explanation of slaughter’s concept of field cancerization: evidence and clinical implications. Cancer Res 2003;63:1727-30. • Loktionov A. Common gene polymorphisms, cancer progression and prognosis. Cancer Letters 2004;208 :1-33. • Desmaze C et al. Telomere-driven genomic instability in cancer cells. Cancer Letters 2003;194:173-82. • Hiyama E & Hiyama K. Telomerase as tumor marker. Cancer Letters 2003;194:221-33. • Kaohsiung Medical University, Oral Pathology Department • Huang AH et al. Isolation and characterization of normal hamster buccal pouch stem/stromal cells – a potential oral cancer stem/stem-like cell model. Oral Oncol 2009;45: e189-e195. • Umezawa & Gorham. Dueling models in head and neck tumor formation. Lab Investig 2010; 90:1546-8. • Spillane JB, Henderson MA. Cancer stem cells: a review. ANZ J Surg 2007;77:464-8. • Zhou ZT, Jiang WW. Cancer stem cell model in oral squamous cell carcinoma. Curr Stem Cell Res Ther 2008;3:17–20. • Harper LJ et al. Stem cell patterns in cell lines derived from head and neck squamous cell carcinoma. J Oral Pathol Med 2007;36:594-603. • Lim YC et al. Cancer stem cell traits in squamospheres derived from primary head and neck squamous cell carcinomas. Oral Oncol 2011;47:83-91.

3. 學 習 目 標 探索癌症之旅 Field cancerization 癌化的標準教條 5 3 4 癌細胞的六種超能力 四種癌化理論 6 2 Stages of carcinogenesis 癌症的預防 7 始 點 1 終 點 How cancer arise

4. 第一站：How Cancer Arises Stochastic Clonal Evolution Model Interaction between tumor cells and stromal cells Tumor cell In this model, clonal variants, including stromal cells derived from tumor cells, generate a microenvironment (niche) for tumor cells, and support tumor progression after tumor cells undergo clonal evolution. Ref. 14

5. Mutation Only at the Stem Cell Normal Stem Cell Asymmetrical Division Mutation Stem Cell Stem Cell EarlyProgenitor Late Progenitor Tumor Definitive Tissue Line Stem cells create an exact copy of themselves and an EP cell when they divide. The EP cell then progresses to a late progenitor cell and then to the definitive cell line Cancer stem cell replicates forming an exact copy of itself and a continuous supply of heterogeneous tumor cells Ref. 15

6. Traditional Model of Tumor Formation Mutation Only at the Stem Cell Mature Definitive Tissue Cell Stem Cell Mutation Mutation Mutation Mutation Tumor Tumor Tumor Traditional model of tumor formation. A series of mutations affect a mature cell, causing it to become malignant. Any cell has the potential to form a tumor Mutation only at the stem cell or progenitor cell level. The cancer stem cell replicates forming an exact copy of itself and a continuous supply of heterogeneous tumor cells Ref. 15

7. Cancer Stem Cell Model (1) Mutation only at the stem cell or progenitor cell level mutation Self- renewing cancer stem cell mutation Cancer cell Self- renewing stem cell Progenitor cell Mature cell Ref. 15

8. Cancer Stem Cell Model (2) Mutation Stem Cell Tumor from an early stem cell Heterogenous cancer Increased metastatic potential Tumor Mutation Early Progenitor Tumor Mutation Late Progenitor Tumor from a late progenitor cell Homogenous cancer Less metastatic potential Tumor Definitive Tissue Line In the stem cell model, only the stem cells or their progenitor cells have the ability to form tumors. Tumor characteristics vary depending on which cell undergoes the malignant transformation Ref. 15

9. Cancer Stem Cell Model (3) In hypoxia (e.g. within niche) In increased O2 (e.g. outside niche) Stem cell in quiescence Progenitor or differentiated cell Ref. 15 Self-renewing stem cell (normal or cancer) Stem cells (normal or cancer) reside in a hypoxic niche where self renewal and differentiation activity is balanced. With an increase in oxygen levels, proliferation becomes a dominant feature mediated by an increase in p38 MAPK and p16ink4a. This transiently leads to the expansion of the progenitors, which results in a long-term decrease in the stem cell pool and its eventual exhaustion. Proliferation Stem cell depletion Exhaustion

10. Comparison of Somatic and Cancer Stem Cells Ref. 15

11. Stem cell - Oral Epithelia • According to the progression model, the development of most of OSCC takes months or years. • As normal human oral epithelia have a rate of renewal estimated to be about 14-24 days, most epithelial cells do not exist long enough to accumulate the genetic changes necessary for the development of an OSCC. • The hierarchical stem cell structure present in human oral epithelia indicates that stem cells are the only long-time residents of oral epithelia and, consequently, the only cells able toaccumulate the necessary number of genetic changes for malignancy to develop

12. A Schematic Diagram Showing Sites of Origins of Putative Cancer Stem Cells (CSCs) in OSCC Epithelium Connective tissue 1, CSC might come from epithelial SC or progenitor within basal layer with genetic alterations; 2, muscle-derived SCs; 3, fibroblast-derived SCs; 4, vessel wall-derived SCs; 5, blood-derived SCs; and 6, adipose derived SCs. Ref. 16

13. Putative Cell Surface Markers of Presumptive CSC Tumor Type Surface Markers SP-C+CCA+ Ref. 16

14. Frequencies of CSCs in Various Human Cancers Human cancer Recipient mice Cancer stem cell frequency (%) Ref. 16

15. A minority population of CD44+ cancer cells (<3%/<10% of the cells in head and neck SCC cell line), but not the CD44- cancer cells, generate new tumors in vivo CD44+CD24- Lineage negative Tumor formed CD44+CD24- CD44+CD24- New tumor formed Ref. 16

16. Potential Mechanisms of CSC Formation A MUTATION Differentiated cells Stem/progenitor cells Progenitors Self renewal Self renewal CSC (A) Mutation. The cancer stem cells might appear after mutations in specific stem cells or early stem cells progenitors. It is also possible that CSC can be derived from differentiated cells. Ref. 16

17. Potential Mechanisms of CSC Formation B MULTIPLE GENETIC HITS Stem/progenitor cells CSC (B) Multiple genetic hits. Progressive genetic alterations drive the transformation of stem/progenitor cells into CSC. Ref. 16

18. C MULTISTEP DEDIFFERENTIATION Cancer cell CSC Cancer cell Potential Mechanisms of CSC Formation (C) Multistep de-differentiation. Multistep dedifferentiation of cancer cells might give rise to CSC. Ref. 16

19. Potential Mechanisms of CSC Formation D FUSION CSC Cancer cell Stem/progenitor cells (D) Cell fusion. Cell fusion between cancer cells and stem/progenitor cells might induce CSC. Ref. 16

20. DMBA-Induced Hamster Buccal Pouch Model • Hamster buccal-pouch mucosa provides one of the most widely-accepted experimental models for oral carcinogenesis. (Gimenez-Conti & Slaga 1993) Carcinogen: DMBA 14-wk Normal DMBA:7,12-dimethylbenz[a]anthracene Ref. 12

21. DMBA-Induced Hamster Buccal Pouch Model • Despite anatomical and histological differences between (hamster) pouch mucosa and human buccal tissue, experimental carcinogenesis protocols for the former induce premalignant changes and carcinomas that are similar to the development of premalignancy and malignancy in human oral mucosa. (Morris 1961) AnimalStudy HumanStudy Ref. 12

22. Isolation and Characterization of Stem Cells from Normal Hamster Buccal Pouch (HBPSC) A B Representative sample of the normal hamster buccal pouch tissues revealed no obvious grossly (A; inset) and histological (B, Hematoxylin & eosin stain, 200) changes. Ref. 13

23. Minimal Criteria of Stem Cell Capacity • Self-renewal ---Colony forming unit (CFU) ---Proliferation • One or more lineages differentiation ---Adipogenic differentiation ---Osteogenic differentiation ---Chondrogenic differentiation ---Neurogenic differentiation

24. Representative sample of the HBPSCs obtained from the normal hamster buccal pouch tissues were spindle-shaped in morphology (200). Ref. 13

25. Representative sample of the HBPSCs obtained from the normal hamster buccal pouch tissues were able to form colonies, stained with crystal violet (A; B, 100). A B Ref. 13

26. Cytoplasmic keratin (A, 200) and vimentin (B, 200) stainings were noted for the representative sample of the HBPSCs obtained from the normal hamster buccal pouch tissues. A B Ref. 13

27. Proliferation rates for the HBPSCs obtained from the three normal hamster buccal pouch tissues (p: passage). Proliferation rate (# of folds) Pouch 3 Pouch 2 Ref. 13

28. (A) Representative sample of the HBPSCs obtained from the normal hamster buccal pouch tissues were able to differentiate towards adipogenic lineage (×200). (B) Expression of PPARγ mRNA (401-bp) upon RT-PCR also indicates adipogenic lineage of HBPSCs obtained from normal hamster buccal pouch tissues; GAPDH (135-bp) was the positive control; H2O was the negative control (N); M: molecular weight marker. M N GAPDH PPAR bp 400 350 300 250 200 150 100 A 50 B Ref. 13

29. Representative sample of the HBPSCs obtained from the normal hamster buccal pouch tissues were able to differentiate towards chondrogenic lineage (×200); inset: a yellowish chondroid pellet (~3mm in diameter). Representative sample of the HBPSCs obtained from the normal hamster buccal pouch tissues were able to differentiate towards osteogenic lineage (×200). Ref. 13

30. Representative sample of the HBPSCs obtained from the normal hamster buccal pouch tissues expressed the differentiation markers (Osteonectin: 323-bp & Nestin: 416-bp) and stem cell markers (Nanog: 364-bp, Rex-1: 232-bp & Oct-4: 717-bp) upon RT-PCR. GAPDH (135-bp) was the positive control; H2O was the negative control (N); M: molecular weight marker. Osteonectin GAPDH Nanog Rex-1 Nestin Oct-4 M N bp 700 600 500 400 300 200 100 Ref. 13

31. Representative sample of the HBPSCs obtained from the normal hamster buccal pouch tissues showed high expression for surface markers: CD29, CD90, and CD105 but very low expression for CD14, CD34, and CD45 (Black/blue line: isotype control, Red line: marker of interest; Max: maximum). 100 85.8 % of Max 100 % of Max 0.9 CD 90 CD14 100 100 100 % of Max % of Max 51.3 93.6 CD 29 CD 105 100 100 % of Max % of Max 1.5 1.7 CD 45 CD 34 Ref. 13

32. DMBA-Induced Hamster Buccal Pouch Model Isolation of normal HBPSC, we may follow in vitro the sequential changes of the normal HBPSCs during multistep oral carcinogenesis or the alternations of these cells upon irradiation treatment and/or chemotherapy. Hence, the isolated normal HBPSCs, would provide a potential avenue for the future study of CSCs of buccal SCCs.

33. Comparison of Morphology Between Our Isolated Cells & Literature Results Our isolated cells from DMBA-induced cancer pouch tissue A colony with holoclone characteristics of circular outline and tightly packed cobblestone’ cells (h) is surrounded by cells with a spaced and fusiform paraclone morphology (p). A small colony (m) perhaps corresponds to a meroclone. Refs. 13, 17

34. Hallmarks of CSCs (1) • Self-renewal, stem cell marker expression, aberrant differentiation, and tumor-initiating potential • OSCC-driven squamospheres demonstrated: • A number of stem cell markers, such as CK5, OCT4, • SOX2, nestin, and CD44, Bmi-1, CD133, ALDH1 • (2) Single-dissociated squamosphere cells were able to form • new squamospheres within 1 week of reseeding • (3) Serum treatment led HNSCC-driven squamospheres to • be non-tumorigenic differentiated cancer cells • (4) Injection of as few as 100 undifferentiated squamosphere • cells in nude mice gave rise to tumor formation CSCs is known to be significantly resistant to various chemotherapeutic agents (cisplatin, 5-fluorouracil (FU), paclitaxel, and doxetaxel)

35. Hallmarks of CSCs (2) Ref. 18

36. 第一站總結 1. In stochastic model, clonal variants, including stromal cells derived from tumor cells, generate a microenvironment for tumor cells, and support tumor progression after tumor cells undergo clonal evolution 請注意以下的重點提要 2. CSCs may originate from normalsomatic stem cells, it has been estimated that 3 to 6 genetic events are required totransform a normal human cell into a cancercell 3. Accumulated evidences have identified that CSCs in SCCs of head and neck region including oral cavity function in initiation,maintenance,growth, and metastasisof tumors Cancer development: Stochastic clonal evolution model VS cancer stem cells

37. Gentically altered cell Hyperlasia Dysplasia 第二站: Stages of Carcinogenesis Oral premalignant lesions Leukoplakia, Erythroplakia, Oral submucous fibrosis, Verrucous hyperplasia, Erosive lichen planus Tumor development occurs in stages Ref. 1 Genetically altered cell (CSC) Hyperplasia Initiated cell 起始細胞 Dysplasia 基底層完整 基底層完整

38. Invasive cancer In situ cancer Blood vessel/ lymphatic vessel Ref. 1 How Cancer Spreads

39. Primary tumor How Cancer Spreads Normal epithelial cell Basement membrane Invasive tumor cell Blood vessel/ lymphatic channel Ref. 1

40. Secondary tumor site Endothelial/lymphatic lining Basement membrane Tumor cell adhering to capillary Metastatic cell in circulation How Cancer Spreads Ref. 1 (Breach: 突破口)

41. 第二站: Further look on stages of carcinogenesis Initiation Phase (Early) 去毒 Ref. 9

42. Initiation Phase (Late) Ref. 9

43. Promotion Phase (Early) Mutant clone establishment & appearance of phenotypically transformed cells Ref. 9

44. Promotion Phase (Late) Establishment of phenotypically transformed cell population (dysplasia) Ref. 9

45. Progression Phase (Early) Malignisation Ref. 9

46. Progression Phase (Middle) Microinvasion Ref. 9

47. Progression Phase (Late) Advanced invasion and metastasis Chemotherapy Ref. 9

48. Initiation (early, late) Genetically altered cell (CSC) Progression (late) Invasive cancer Progression (middle) Microinvasion Promotion (early) Hyperplasia Promotion (late) Dysplaisa Progression (early) In situ cancer Progression (late) Metastasis 第二站總結 癌症形成是階段性的vs正常細胞有自衛能力 請注意以下的重點提要 Tumor developmentoccurs instages Normal cellhas self-defense

49. 第三站: 癌化理論的標準教條 Ref. 2 Normal Cell Cycle Beginning of cycle Cell divides (mitosis) Cell enlarges and makes new proteins Cell prepares to divide Cell rests G1 arrest 崗 哨 Restriction point:cell decides whether to commit itself to the complete cycle Cell replicates as DNA

50. Inhibitory pathways Stimulatory pathways Normal Cell Inhibitory abnormality Stimulatory abnormality 標準教條 Ref. 2 致癌基因 抑癌基因