口腔病理 陳玉昆副教授 : 高雄醫學大學 口腔病理科 ~2755 Carcinogenesis 癌 化

References 1.Gibbs WW. Untangling the roots of cancer. Sci Am 2003;289: What you need to know about cancer. Sci Am 1996 ;289: Braakhuis BJM et al. A genetic progression model of oral cancer: current evidence and clinical implications. J Oral Pathol Med 2004;33: Braakhuis BJM et al. A Genetic explanation of slaughter’s concept of field cancerization: evidence and clinical implications. Cancer Res 2003;63: Loktionov A. Common gene polymorphisms, cancer progression and prognosis. Cancer Letters 2004;208 : Kaohsiung Medical University, Oral Pathology Department. 7.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-e Umezawa & Gorham. Dueling models in head and neck tumor formation. Lab Investig 2010; 90: Spillane JB, Henderson MA. Cancer stem cells: a review. ANZ J Surg 2007;77: Zhou ZT, Jiang WW. Cancer stem cell model in oral squamous cell carcinoma. Curr Stem Cell Res Ther 2008;3:17– Harper LJ et al. Stem cell patterns in cell lines derived from head and neck squamous cell carcinoma. J Oral Pathol Med 2007;36: 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.

Carcinogenesis( 癌化 ) How cancer arise - Molecular approach Stages of carcinogenesis 癌化的標準理論 四種癌化理論 Field cancerization 綱 要

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. Stochastic Clonal Evolution Model (1) How Cancer Arises - Molecular Approach Ref. 8 Stochastic clonal expansion Interaction between tumor cells and stromal cells Tumor cell

Definitive Tissue Line Early Progenitor Late Progenitor Stem Cell 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 Normal Stem Cell The cancer stem cell replicates forming an exact copy of itself as well as a continuous supply of heterogeneous tumor cells Tumor Stem Cell Mutation Only at the Stem Cell Mutation Ref. 9 Asymmetrical Division

(a) 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 Traditional Model of Tumor Formation Mature Definitive Tissue Cell Tumor Mutation Ref. 9 (b) Mutation only at the stem cell or progenitor cell level. The cancer stem cell replicates forming an exact copy of itself & a continuous supply of heterogeneous tumor cells Tumor Stem Cell Mutation Mutation Only at the Stem Cell

Cancer Stem Cell Model (1) mutation Self- renewing stem cell Progenitor cell Mature cell Cancer cell Self- renewing cancer stem cell Ref. 9 Mutation only at the stem cell or progenitor cell level

Cancer Stem Cell Model (2) 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 Tumor from an early stem cell Heterogeneous cancer Increased metastatic potential Tumor from a late progenitor cell Homogenous cancer Less metastatic potential Tumor Early Progenitor Late Progenitor Definitive Tissue Line Stem Cell Mutation Tumor Ref. 9

Cancer Stem Cell Model (3) 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 p16 ink4a. 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. (a)In hypoxia (e.g. within niche) (b) In increased O 2 (e.g. outside niche) Stem cell in quiescenceProliferation Stem cell depletion Exhaustion Self-renewing stem cell (normal or cancer) Progenitor or differentiated cell Ref. 9

Comparison of Somatic and Cancer Stem Cells Somatic Stem CellCancer Stem Cell Self renew, highly regulatedSelf-renew, poorly regulated Differentiate, produces mature tissue Differentiate, produces tumor Migrate to distant tissuesMetastasize to distant sites Long lifespan Resistant to apoptosis Ref. 9

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 to accumulate the necessary number of genetic changes for malignancy to develop 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 days, most epithelial cells do not exist long enough to accumulate the genetic changes necessary for the development of an OSCC.

A Schematic Diagram Showing Sites of Origins of Putative CSCs in OSCC Epithelium Connective tissue Ref. 10 CSC might come from: 1. Epithelial SC/progenitor within basal layer with genetic alterations 2. Muscle-derived SCs 3. Fibroblast-derived SCs 4. Vessel wall-derived SCs 5. Blood-derived SCs 6. Adipose derived SCs.

Putative Cell Surface Markers of Presumptive CSC SP-C + CCA + Tumor TypeSurface Markers Ref. 10

Frequencies of CSCs in Various Human Cancers Human cancerRecipient miceCancer stem cell frequency (%) Ref. 10

CD44 + CD24 - Lineage negative CD44 + CD24 - Tumor formed New tumor formed Ref. 10 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

Potential Mechanisms of CSC Formation CSC MUTATION A Progenitors Self renewal Stem/progenitor cells Differentiated cells (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. 10

(B) Multiple genetic hits. Progressive genetic alterations drive the transformation of stem/progenitor cells into CSC. CSC MULTIPLE GENETIC HITS B Stem/progenitor cells Potential Mechanisms of CSC Formation Ref. 10

(C) Multistep de-differentiation. Multistep dedifferentiation of cancer cells might give rise to CSC. Potential Mechanisms of CSC Formation Ref. 10 CSC C Cancer cell MULTISTEP DEDIFFERENTIATION

(D) Cell fusion. Cell fusion between cancer cells and stem/progenitor cells might induce CSC. CSC FUSION D Cancer cell Stem/progenitor cells Potential Mechanisms of CSC Formation Ref. 10

DMBA-Induced Hamster Buccal Pouch Model 14-wk Normal Carcinogen: DMBA Hamster buccal-pouch mucosa provides one of the most widely-accepted experimental models for oral carcinogenesis. (Gimenez-Conti & Slaga 1993) Ref. 6

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) Animal Study Animal Study Human Study Human Study Ref. 6

AB Isolation and Characterization of Stem Cells from Normal Hamster Buccal Pouch (HBPSC) Normal hamster buccal pouch tissues revealed no obvious grossly (A; inset) and histological (B, Hematoxylin & eosin stain,  200) changes. Ref. 7

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

HBPSCs obtained from the normal hamster buccal pouch tissues were spindle-shaped in morphology (  200). Ref. 7

A B HBPSCs obtained from the normal hamster buccal pouch tissues were able to form colonies, stained with crystal violet (A; B,  100). Ref. 7

AB Cytoplasmic keratin (A,  200) and vimentin (B,  200) stainings were noted for the HBPSCs obtained from the normal hamster buccal pouch tissues. Ref. 7

Proliferation rate (# of folds) Pouch 2 Pouch 3 Proliferation rates for the HBPSCs obtained from the three normal hamster buccal pouch tissues (p: passage). Ref. 7

A NM GAPDH PPAR  B bp (A) 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; H 2 O was the negative control (N); M: molecular weight marker. Ref. 7

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). Ref. 7 HBPSCs obtained from the normal hamster buccal pouch tissues were able to differentiate towards osteogenic lineage (×200).

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; H 2 O was the negative control (N); M: molecular weight marker. MN GAPDH Osteonectin Nestin Oct-4 Nanog Rex bp Ref. 7

0.9 CD14 % of Max 100 CD 29 % of Max CD 34 % of Max CD % of Max 100 CD % of Max CD % of Max 100 Ref. 7 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).

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. DMBA-Induced Hamster Buccal Pouch Model

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. Comparison of Morphology Between Our Isolated Cells & Literature Results Our isolated cells from DMBA-induced cancer pouch tissue Refs. 7, 11 squamospheres

Self-renewal, stem cell marker expression, aberrant differentiation, and tumor-initiating potential OSCC-driven squamospheres demonstrated: (1) 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 Hallmarks of CSCs (1) CSCs is known to be significantly resistant to various chemotherapeutic agents (cisplatin, 5-fluorouracil (FU), paclitaxel, and doxetaxel)- side population cells

Hallmarks of CSCs (2) Ref. 12

(1) 小 結 Cancer development: Stochastic clonal evolution model VS Cancer stem cells model 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 3. Accumulated evidences have identified that CSCs in SCCs of head and neck region including oral cavity function in initiation, maintenance, growth, and metastasis of tumors 2. CSCs may originate from normal somatic stem cells, it has been estimated that 3 to 6 genetic events are required to transform a normal human cell into a cancer cell 請注意以下的重點提要

Gentically altered cell Hyperlasia Dysplasia Tumor development occurs in stages Genetically altered cell (CSC): initiated cell ( 起始細胞 ) Hyperplasia Dysplasia Oral potentially malignant disorders (OPMD) Leukoplakia, Erythroplakia, Oral submucous fibrosis, Verrucous hyperplasia, Erosive lichen planus 基底層完整 (2) Stages of Carcinogenesis Ref. 1

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

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

Endothelial/lymphatic lining Basement membrane Metastatic cell in circulation Secondary tumor site Tumor cell adhering to capillary Ref. 1 How Cancer Spreads

Initiation Phase (Early) (2) Further look on stages of carcinogenesis 去毒 Ref. 5

Initiation Phase (Late) Ref. 5

Promotion Phase (Early) Mutant clone establishment & appearance of phenotypically transformed cells Ref. 5

Promotion Phase (Late) Establishment of phenotypically transformed cell population (dysplasia) Ref. 5

Progression Phase (Early) Malignisation Ref. 5

Progression Phase (Middle) Microinvasion Ref. 5

Progression Phase (Late) Advanced invasion and metastasis Chemotherapy Ref. 5

(2) 小 結 Tumor development occurs in stages Normal cell has self-defense 癌症形成是階段性的 vs 正常細胞有自衛能力 Initiation (early, late) Genetically altered cell (CSC) Promotion (early) Hyperplasia Promotion (late) Dysplaisa Progression (early) In situ cancer Progression (middle) Microinvasion Progression (late) Invasive cancer Progression (late) Metastasis 請注意以下的重點提要

Normal Cell Cycle Cell enlarges and makes new proteins Beginning of cycle Cell divides (mitosis) Cell prepares to divide Cell replicates as DNA Cell rests Restriction point: cell decides whether to commit itself to the complete cycle 崗 哨崗 哨 (3) 癌化的標準理論 G1 arrest Ref. 2

Inhibitory pathways Normal Cell Inhibitory abnormality Stimulatory abnormality Stimulatory pathways 標準理論 致癌基因 Oncogene 抑癌基因 Tumor suppressor gene Ref. 2

Activation of oncogene Inactivation of tumor suppressor gene Cell Cycle 失 控失 控 失 控失 控 下 坡 剎車失靈 油門全開 Aberrant cell cycle —Accelerated car downslope without brake Ref. 2

Oncogene (1) Genes for growth factors or their receptors PDGFCodes for platelet-derived growth factor Involved in glioma (a brain cancer) erb-BCodes for the receptor for epidermal growth factor Involved in glioblastoma (a brain cancer) and breast cancer erb-B2Also called HER-2 or neu. Codes for a growth factor receptor involved in breast, salivary gland and ovarian cancers RETCodes for a growth factor receptor Involved in thyroid cancer Genes for growth factors or their receptors Ki-rasInvolved in lung, ovarian, colon and pancreatic cancers N-rasInvolved in leukemia Ref. 2

Oncogene (2) Genes for growth factors or their receptors c-mycInvolved in leukemia and breast, stomach and lung cancers N-mycInvolved in neuroblastoma (a nerve cell cancer) and glioblastoma L-mycInvolved in lung cancer Genes for growth factors or their receptors Bcl-2Codes for a protein that normally blocks cell suicide. Involved in follicular B cell lymphoma Bcl-1Also called PRAD1. Codes for cyclin D1, a stimulatory component of the cell cycle clock Involved in breast, head and neck cancers MDM2Codes for an antagonist of the p53 tumor suppressor protein. Involved in sarcomas and other cancers Ref. 2

Tumor Suppressor Gene (1) Genes for proteins in the cytoplasm APCInvolved in colon and stomach cancers DPC4Codes for a relay molecule in a signaling pathway that inhibits cell division Involved in pancreatic cancer NF-1Codes for a protein that inhibits a stimulatory (Ras) protein Involved in neurofibroma and pheochromocytoma (cancers of the peripheral nervous system) and myeloid leukemia NF-2Involved in meningioma and ependymoma (brain cancers) and schwannoma (affecting the wrapping around peripheral nerves) Ref. 2

Tumor Suppressor Gene (2) Genes for proteins whose cellular locations is not yet clear BRCA1Involved in breast and ovarian cancers BRCA2Involved in breast cancer VHLInvolved in renal cell cancer Genes for proteins in the nucleus MTS1Codes for the p16 protein, a braking component of the cell cycle clock Involved in a wide range of cancers RBCodes for the pRB protein, a master brake of the cell cycle. Involved in retinoblastoma and bone, bladder, small cell lung and breast cancer p53Codes for p53 protein, which can halt cell division and induce abnormal cells to kill themselves. Involved in a wide range of cancers WT1Involved in Wilms’ tumor of the kidney Ref. 2

基因突變地圖 在各種癌症中發現超過百種以上的突變基因 癌化的標準理論： Cell cycle 中，正常促進細胞形成基因 o 過度 活化 ，變成致癌基因；而抑制細胞形成基 因 o 發生突變，失去功能 X ，成為抑癌基因 A Subway Map for Cancer Pathways Ref. 2

(3) 小 結 Tumor development occurs due to formations of oncogene & tumor suppressor gene 癌化理論 → 標準教條： 細胞循環中，原來正常的腫瘤致癌 基因與抑癌基因發生突變而失控； 造成致癌基因過度活化及抑癌基因 失去功能 請注意以下的重點提要

(4) 癌化的四個理論 Ref. 2 標準理論：癌症相關基 因被致癌物影響而發生 突變，無法製造腫瘤抑 制蛋白，並活化致癌蛋 白，導致產生癌症

修正理論：在癌化前期的細胞基因 組當中，累積的隨機突變有顯著的 增加，終於影響到癌症相關基因 Ref. 2

早期不穩定理論：認為細胞分裂的 主控基因受致癌物質影響而關閉， 造成子代細胞染色體數目異常 早期不穩定理論 其餘兩個理論專注 在非整倍體所扮演的 角色，也就是染色體 上大規模的變異 Ref. 2

全盤非整倍體理論：非整倍體細胞的基因組非常 不穩定，使得癌症基因極易發生突變而形成腫瘤 Ref. 2

癌症是一種基因的疾病 然而癌症的複雜情況， 卻不能用簡單的「基因 突變」來描述。 最近理論認為，染色體 的異常可能才是細胞邁 向癌症之路的第一步。 隨染色體起舞 Ref. 2

正常癌 症癌 症 Ref. 2 Normal & Cancer Chromosomes

(4) 小 結 癌化的四個理論： (1) 致癌基因、抑癌基因； (2) 修 正 教 條； (3) 早期不穩定理論； (4) 全盤非整倍體理論 請注意以下的重點提要

(5) Field Cancerization (1) Patch phase Precursor lesions develop within field Carcinoma excised, field and precursor lesion remains Expanding field phase Field Second field tumor develops from precursor lesion Precursor lesions becomes carcinoma and new precursor becomes develop Epithelium Connective tissue Basal layer with stem cells Genetic altered Ref. 3

Field Cancerization (2) Carcinoma 11q Field Patch Histological Proof Chromosomal Proof p arm q arm centromere Normal 17p 3p, 9p, 8p, 18q Ref. 4

(5) 小 結 Formation of field cancerization Importance of field cancerization 瞭解 Field cancerization 的形成： Normal→Patch→Field→Cancer 瞭解 Field cancerization 的重要： 腫瘤切除要有足夠的 safe margin 請注意以下的重點提要

Carcinogenesis( 癌化 ) How cancer arise - Molecular approach Stages of carcinogenesis 癌化的標準理論 四種癌化理論 Field cancerization Summary( 總結 )