口腔病理
Carcinogenesis
癌 化
陳玉昆副教授: 高雄醫學大學 口腔病理科
07-3121101~2755
yukkwa@kmu.edu.tw
References
1. Gibbs WW. Untangling the roots of cancer. Sci Am 2003;289:56-65.
2. What you need to know about cancer. Sci Am 1996 ;289:28-119.
3. Braakhuis BJM et al. A genetic progression model of oral cancer: current evidence and
clinical implications. J Oral Pathol Med 2004;33:317-22.
4. Braakhuis BJM et al. A Genetic explanation of slaughter’s concept of field
cancerization: evidence and clinical implications. Cancer Res 2003;63:1727-30.
5. Loktionov A. Common gene polymorphisms, cancer progression and prognosis. Cancer
Letters 2004;208 :1-33.
6. 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-e195.
8. Umezawa & Gorham. Dueling models in head and neck tumor formation. Lab Investig
2010; 90:1546-8.
9. Spillane JB, Henderson MA. Cancer stem cells: a review. ANZ J Surg 2007;77:464-8.
10. Zhou ZT, Jiang WW. Cancer stem cell model in oral squamous cell carcinoma. Curr
Stem Cell Res Ther 2008;3:17–20.
11. 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.
12. 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)
綱
癌化的標準理論
3
要
Field cancerization
5
4
四種癌化理論
2
Stages of carcinogenesis
1
How cancer arise - Molecular approach
(1) How Cancer Arises - Molecular Approach
Stochastic Clonal Evolution Model
Stochastic clonal expansion
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. 8
Normal Stem Cell
Asymmetrical Division
Stem Cell
Mutation Only
at the Stem Cell
Mutation
Stem Cell
Early Progenitor
Late Progenitor
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
Tumor
The cancer stem cell replicates forming
an exact copy of itself as well as a
continuous supply of heterogeneous
tumor cells
Ref. 9
Traditional Model of
Tumor Formation
Mature
Definitive
Tissue
Cell
Mutation Only
at the Stem Cell
Mutation
Stem Cell
Mutation
Mutation
Mutation
Tumor
Tumor
(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
Tumor
(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
Ref. 9
Cancer Stem Cell Model (1)
Selfrenewing
mutation cancer
stem cell
Selfrenewing
stem cell
Cancer cell
mutation
Progenitor
cell
Mature cell
Mutation only at the stem cell or progenitor cell level
Ref. 9
Cancer Stem Cell Model (2)
Stem Cell
Mutation
Tumor
Tumor from an early stem cell
Heterogeneous cancer
Increased metastatic potential
Mutation
Early Progenitor
Tumor
Mutation
Late Progenitor
Tumor
Tumor from a late progenitor cell
Homogenous cancer
Less metastatic potential
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. 9
Cancer Stem Cell Model (3)
(a) In hypoxia
(e.g. within niche)
Stem cell in quiescence
(b) In increased O2
(e.g. outside niche)
Proliferation
Self-renewing stem cell
(normal or cancer)
Stem cell depletion
Exhaustion
Progenitor
or differentiated cell
Ref. 9
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.
Comparison of Somatic and Cancer Stem Cells
Somatic Stem Cell
Cancer Stem Cell
Self renew, highly regulated
Self-renew, poorly regulated
Differentiate, produces mature
tissue
Differentiate, produces
tumor
Migrate to distant tissues
Metastasize to distant sites
Long lifespan
Long lifespan
Resistant to apoptosis
Resistant to apoptosis
Ref. 9
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 to accumulate the
necessary number of genetic changes for malignancy to develop
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.
Connective tissue
Epithelium
A Schematic Diagram Showing Sites of Origins of
Putative CSCs in OSCC
Ref. 10
Putative Cell Surface Markers of Presumptive CSC
Tumor Type
Surface Markers
SP-C+CCA+
Ref. 10
Frequencies of CSCs in Various Human Cancers
Human cancer
Recipient mice
Cancer stem cell frequency (%)
Ref. 10
A minority population of CD44+ cancer cells (<3%/<10% of the
cells in head and neck SCC cell line), but not the CD44cancer cells, generate new tumors in vivo
CD44+CD24- Lineage negative
Tumor formed
CD44+CD24-
CD44+CD24-
New tumor formed
Ref. 10
Potential Mechanisms of CSC Formation
MUTATION
A
Stem/progenitor cells
Differentiated 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. 10
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. 10
Potential Mechanisms of CSC Formation
C
MULTISTEP DEDIFFERENTIATION
Cancer cell
CSC
(C) Multistep de-differentiation. Multistep dedifferentiation of cancer cells might give
rise to CSC.
Ref. 10
Potential Mechanisms of CSC Formation
D
FUSION
Cancer cell
CSC
Stem/progenitor cells
(D) Cell fusion. Cell fusion between cancer cells and stem/progenitor cells might induce CSC.
Ref. 10
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
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
Human Study
Ref. 6
Isolation and Characterization of Stem Cells
from Normal Hamster Buccal Pouch (HBPSC)
A
B
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
HBPSCs obtained from the normal hamster buccal pouch tissues were
able to form colonies, stained with crystal violet (A; B, 100).
A
B
Ref. 7
Cytoplasmic keratin (A, 200) and vimentin (B, 200) stainings were
noted for the HBPSCs obtained from the normal hamster buccal pouch
tissues.
A
B
Ref. 7
Proliferation rate (# of folds)
Proliferation rates for the HBPSCs obtained from the three normal hamster
buccal pouch tissues (p: passage).
Pouch 2
Pouch 3
Ref. 7
(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; 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. 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).
HBPSCs obtained from the normal hamster buccal pouch
tissues were able to differentiate towards osteogenic lineage
(×200).
Ref. 7
Rex-1
Nanog
Oct-4
Nestin
N
Osteonectin
M
GAPDH
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.
bp
700
600
500
400
300
200
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).
85.8
% of Max
93.6
CD 29
CD 90
% of Max
% of Max
CD 105
100
100
1.5
51.3
1.7
CD 34
% of Max
100
CD 45
% of Max
100
100
100
% of Max
100
0.9
CD14
Ref. 7
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.
Comparison of Morphology Between Our Isolated
Cells & Literature Results
Our isolated cells from DMBA-induced cancer pouch tissue
squamospheres
squamospheres
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. 7, 11
Hallmarks of CSCs (1)
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
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
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 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
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
Cancer development:
Stochastic clonal evolution model
VS Cancer stem cells model
(2) Stages of Carcinogenesis
Tumor development
occurs in stages
Oral potentially malignant disorders (OPMD)
Leukoplakia, Erythroplakia, Oral submucous fibrosis, Verrucous
hyperplasia, Erosive lichen planus
Genetically altered cell (CSC):
initiated cell (起始細胞)
Gentically
altered cell
Hyperplasia
Hyperlasia
Dysplasia
Dysplasia
基底層完整
Ref. 1
Invasive cancer
How Cancer Spreads
In situ cancer
Blood vessel/
lymphatic vessel
Ref. 1
Primary
tumor
How Cancer Spreads
Normal
epithelial cell
Basement membrane
Invasive tumor cell
Blood vessel/
lymphatic channel
Ref. 1
How Cancer Spreads
Secondary tumor site
Endothelial/lymphatic
lining
Basement membrane
Metastatic cell
in circulation
Tumor cell
adhering
to capillary
Ref. 1
(2) Further look on stages of carcinogenesis
Initiation Phase (Early)
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
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)
請注意以下的重點提要
Progression (middle) Progression (late)
Promotion (early)
Hyperplasia
Microinvasion
Invasive cancer
Promotion (late)
Dysplaisa
Progression (early)
In situ cancer
Progression (late)
Metastasis
Tumor development occurs in stages
Normal cell has self-defense
(3) 癌化的標準理論
Cell
divides
(mitosis)
Cell prepares
to divide
Beginning
of cycle
Normal
Cell Cycle
Cell enlarges
and makes
new proteins
Cell rests
G1 arrest
崗 哨
Cell
replicates
as DNA
Restriction point: cell
decides whether
to commit itself to
the complete cycle
Ref. 2
Stimulatory
pathways
Stimulatory
abnormality
致癌基因
Oncogene
標準理論
Normal Cell
Inhibitory
pathways
Inhibitory
抑癌基因
Tumor suppressor abnormality
gene
Ref. 2
Aberrant cell cycle —Accelerated car downslope
without brake
Cell Cycle
油門全開
Activation of
oncogene
剎車失靈
Inactivation of
tumor suppressor gene
Ref. 2
Oncogene (1)
Genes for growth factors or their receptors
PDGF
Codes for platelet-derived growth factor
Involved in glioma (a brain cancer)
erb-B
Codes for the receptor for epidermal growth factor
Involved in glioblastoma (a brain cancer) and breast cancer
erb-B2
Also called HER-2 or neu. Codes for a growth factor receptor involved in
breast, salivary gland and ovarian cancers
RET
Codes for a growth factor receptor
Involved in thyroid cancer
Genes for growth factors or their receptors
Ki-ras
Involved in lung, ovarian, colon and pancreatic cancers
N-ras
Involved in leukemia
Ref. 2
Oncogene (2)
Genes for growth factors or their receptors
c-myc
Involved in leukemia and breast, stomach and lung cancers
N-myc
Involved in neuroblastoma (a nerve cell cancer) and glioblastoma
L-myc
Involved in lung cancer
Genes for growth factors or their receptors
Bcl-2
Codes for a protein that normally blocks cell suicide.
Involved in follicular B cell lymphoma
Bcl-1
Also called PRAD1. Codes for cyclin D1, a stimulatory component of the
cell cycle clock
Involved in breast, head and neck cancers
MDM2
Codes 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
APC
Involved in colon and stomach cancers
DPC4
Codes for a relay molecule in a signaling pathway that inhibits cell division
Involved in pancreatic cancer
NF-1
Codes for a protein that inhibits a stimulatory (Ras) protein
Involved in neurofibroma and pheochromocytoma (cancers of the peripheral
nervous system) and myeloid leukemia
NF-2
Involved in meningioma and ependymoma (brain cancers) and schwannoma
(affecting the wrapping around peripheral nerves)
Ref. 2
Tumor Suppressor Gene (2)
Genes for proteins in the nucleus
MTS1
Codes for the p16 protein, a braking component of the cell cycle clock Involved
in a wide range of cancers
RB
Codes for the pRB protein, a master brake of the cell cycle. Involved in
retinoblastoma and bone, bladder, small cell lung and breast cancer
p53
Codes for p53 protein, which can halt cell division and induce abnormal cells
to kill themselves. Involved in a wide range of cancers
WT1
Involved in Wilms’ tumor of the kidney
Genes for proteins whose cellular locations is not yet clear
BRCA1
Involved in breast and ovarian cancers
BRCA2
Involved in breast cancer
VHL
Involved in renal cell cancer
Ref. 2
基因突變地圖
Ref. 2
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
(3) 小 結
癌化理論 → 標準教條：
細胞循環中，原來正常的腫瘤致癌
請注意以下的重點提要
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
Tumor development occurs due to
formations of oncogene & tumor
suppressor gene
(4) 癌化的四個理論
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
Ref. 2
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
隨染色體起舞
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。
Ref. 2
Normal & Cancer Chromosomes
正常
癌症
Ref. 2
(4) 小 結
請注意以下的重點提要
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
(5) Field Cancerization (1)
Connective tissue
Ref. 3
Epithelium
Basal layer with
stem cells
Genetic altered
Patch phase
Field
Expanding
field phase
Precursor lesions
develop within field
Precursor lesions
becomes carcinoma
and new precursor
becomes develop
Carcinoma excised,
field and precursor
lesion remains
Second field tumor
develops from
precursor lesion
Field Cancerization (2)
Histological Proof
Normal
Field
Patch
Carcinoma
Chromosomal
Proof
17p
3p, 9p, 8p, 18q
11q
centromere
q arm
p arm
Ref. 4
(5) 小 結
瞭解Field cancerization的形成：
Normal→Patch→Field→Cancer
請注意以下的重點提要
瞭解Field cancerization的重要：
腫瘤切除要有足夠的safe margin
Formation of field cancerization
Importance of field cancerization
Carcinogenesis(癌化)
Summary(總結)
癌化的標準理論
3
Field cancerization
5
4
四種癌化理論
2
Stages of carcinogenesis
1
How cancer arise - Molecular approach