Tumor Initiating Cells, Cell Cycle Control and Cancer Stem Cells

Tumor Initiating Cells, Cell Cycle Control and Cancer Stem 

Cells 

Eldad Zacksenaus 

MGY-425 

MSB 3171 

March 5, 2012

Cancer is a highly heterogeneous disease 

1. Distinct oncogenic networks 

2. Distinct tumor subtypes due to different cell of origin 

(and transforming oncogenic networks) 

3. Hierarchical organization with Cancer Stem cells (CSC)/ 

Tumor initiating cells (TICs) capable of self-renewal and 

tumorigenicity at its apex, and non-TICs which were derived from 

TICs but have lost their tumorigenic potential forming tumor bulk 

4. Clonal evolution.

Hallmarks of Cancer: The Next Generation 

Douglas Hanahan and Robert A. Weinberg 

Cell 

Volume 144, Issue 5, Pages 646-674 (March 2011)

Stochastic versus hierarchical models of cancer 

Primary tumors 

Transplantation 

Secondary tumors 

Stochastic: all cells 

have the capacity 

to induce tumors but 

the process is not efficient 

(e.g. most cell die after 

transplantation) 

Hierarchical: only a small fraction 

of tumor cells – cancer stem cells/ 

tumor initiating cells – is capable 

of inducing tumors.

Isolation of tumorigenic cells. 

Al-Hajj M et al. PNAS 2003;100:3983-3988 

©2003 by National Academy of Sciences

Histology from the CD24+ injection site (a; ×20 objective magnification) revealed only normal 

mouse tissue, whereas the CD24−/low injection site (b; ×40 objective magnification) 

contained malignant cells. 



Phenotypic diversity in tumors arising from CD44+CD24−/lowLineage− cells. 



Breast cancer stem cells revealed 

John E. Dick 

PNAS April 1, 2003 vol. 100 no. 7 3547–3549

A model depicting the hierarchical organization of the breast cancer tumor. A rare and primitive 

breast cancer-initiating cell (BrCa-IC) maintains the breast cancer tumor. BrCa-IC possess high self-renewal 

capacity; however, they can also mature or differentiate into breast cancer cells that have lost the ability 

to sustain the tumor. Some of these cells may still possess extensive proliferative capacity indicative of a 

progenitor cell, but have lost the ability to sustain the tumor after transplantation. The breast cancer cells 

that comprise the bulk of the tumor tissue retain remnants of normal differentiation genetic programs.

Can non-TICs spontaneously reverse into TICs 

Primary tumors 

Transplantation 

Secondary tumors 

Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state. 

Chaffer CL, Brueckmann I, Scheel C, Kaestli AJ, Wiggins PA, Rodrigues LO, Brooks M, 

Reinhardt F, Su Y, Polyak K, Arendt LM, Kuperwasser C, Bierie B, Weinberg RA. 

Proc Natl Acad Sci U S A. 2011 May 10;108(19):7950-5.

Implications of CSC model to chemotherapy 

 

Reya T et al . Nature 

2001:414

Model of the human mammary epithelial hierarchy linked to cancer subtype 

Prat & Perou, Nature Medicine 2009

Proto-oncogenes promote hallmarks of cancer 

and are activated in human cancer 

Tumor suppressors (TS) inhibit hallmarks of cancer 

and are lost in human cancer

For example, evasion of apoptosis: 

• Bcl-2 is a survival factor (It inhibits mitochondrial outer 

membrane permeabilization (MOMP) and the release of 

cytochrome c, which is required for caspase activation and cell death). 

- Bcl-2 is amplified in cancer - oncogene 

• p53 is an anti-survival factor (among other functions, it transcriptionally 

activates Bax which induces MOMP and cell death) 

-p53 is lost/mutated in cancer hence a TS (however most mutations in p53 

are not null but dominant negative that also inhibit p63 - hence oncogenic) 

• The TS RB inhibits cell proliferation - but also apoptosis. Its loss leads 

to ectopic proliferation (oncogenic) but also so apoptosis (tumor 

suppression).

Hallmark 

Tumor 

suppressor 

Function 

Evading apoptosis p53 Induces apoptotic genes (Bax) 

Self-sufficiency in Pten Dephosphorylates PIP3, counteracting PI3K 

growth signal pRb Inhibits E2F - cell proliferation 

Insensitivity to anti- SMAD4 Induces CDK4/6 inhibitor p16 ink4a in response to TGF 

growth signals p16 ink4a Inhibits cyclins 

DNA damage stress ATM Induces p53 and DNA repair machinery 

Mitotic stress LATS2 Inhibits MDM2 (activates p53) 

Normoxia VHL E3 ligase of HIF-1 (induces angiogenesis)

signal 

+ 

Signaling, oncogenes, tumor-suppressors 

and non-oncogene addiction 

Oncogene 

- 

Tumor suppressor 

- 

Negative regulator that is not mutated/lost in cancer 

+ 

Positive regulator that is not activated in cancer 

but its inactivation blocks signaling - non-oncogene 

addiction

For example: The phosphatidylinositol 3-kinase (PI3K) signaling cascade 

Receptor tyrosine 

kinase 

G protein 

couple receptor 

RTK, +, onc 

Ras, +, onc 

PI3K, +, onc 

Pten, - , TS 

Akt, +, onc 

MDM2, +, onc 

P53, - , TS/onc 

GSK3, - 

B-cat, +, onc 

Myc, +, onc 

CyclinD1, +, onc 

TSC, -, TS 

+ 

-catenin 

myc 

cyclin D 

Courtney, K. D. et al. J Clin Oncol; 28:1075-1083 2010 

Copyright © American Society of Clinical Oncology

The landscape of somatic copy-number alteration across human cancers 

High-resolution Affimetric analysis (250K array containing probes for 238,270 single nucleotode polymorphisms 

(SNPs) for somatic copy-number alterations (SCNAs) from 3,131 cancer specimens 

Ink4-Arf 

Beroukhim R., et al Nature 463, 899-905 (18 February 2010)

Notes: 

• On average: 24 gains and 18 losses 

• Most frequent - Myc amplification and Cdkn2A/B (p16ink4a and Arf) 

deletion. 

• At least 10 known tumor suppressors not identified by this analysis - 

Brca2, Fbxw7, Nf2, Ptch1, Smarcb1, Stk11, Sufu, Vhl, Wt1, and Wtx 

- Some of these are specific to cancer types (e.g. Nf2, Wt1) 

- Other primarily suffer arm-level deletions (e.g. Brca2) 

- Some TS genes undergo point mutations not deletions

Tumor Initiating Cells, Cell Cycle Control and Cancer Stem 

Cells 

Eldad Zacksenaus 

MGY-425 

MSB 3171 

March 7, 2012

Regulation of cell cycle progression: To cycle or not to cycle 

R.A. Weinberg Biology of Cancer 2006. Figure 8.1 The Biology of Cancer (© Garland Science 2007)

Figure 8.6 The Biology of Cancer (© Garland Science 2007) 

The restriction point

Phosphorylation of the retinoblastoma gene product is modulated during 

the cell cycle and cellular differentiation 

Stages during the cell 

cycle when pRb in 

under-phosphorylated 

Chen PL, Scully P, Shew JY, Wang JY, Lee WH. Cell. 1989 Sep 22;58(6):1193-8. 

Karen Buchkovich, Linda A. Duffy and Ed Harlow Cell. 1989 Sep 22;58(6):1097-105. 

James A. DeCaprio, John W. Ludlow, Dennis Lynch, Yusuke Furukawa, James Griffin, Helen Piwnica-Worms, Chun-Ming Huang 

and David M. Livingston 1989 Cell ;58(6):1085-95.

The retinoblastoma gene product regulates progression through the 

G1 phase of the cell cycle. 

Micro-injection of Rb 

protein during this 

period blocks entry 

into S phase 

Goodrich DW, Wang NP, Qian YW, Lee EY, Lee WH.Cell. 1991 18;67(2):293-302. 

Goodrich DW, Wang NP, Qian YW, Lee EY, Lee WH.Cell. 1991 67(2):293-302.

Fluctuation of cyclin levels during the cell cycle 

Figure 8.10 The Biology of Cancer (© Garland Science 2007)

D and E type cyclins, which are active in early and late G1, are thought to 

sequentially phosphorylate pRb 

(Cyclins A/B maintain pRb phosphorylation in S/G2) 


Cell cycle-dependent phosphorylation of pRb – new model

Control of cyclin levels during the cell cycle 

CDC2 = Cdk1 is sufficient to drive the mammalian cell cycle - CDK4/6/3/2 are not essential 

for cell proliferation and early embryogenesis - till midgestation) 

Santamar D, Barrie C, Cerqueira A, Hunt S, Tardy C, Newton K, Ceres JF, Dubus P, 

Malumbres M, Barbacid M. 

Nature. 2007 Aug 16;448(7155):811-5. 


Figure 8.23d The Biology of Cancer (© Garland Science 2007) 

E2F1, 2 and 3a are major targets of pRb

Figure 8.23a The Biology of Cancer (© Garland Science 2007)

LxCxE binding 

LxCxE motif 


Figure 8.24b The Biology of Cancer (© Garland Science 2007)

Actions of CDK inhibitors 


Control of cell cycle progression by 

TGF- 



Regulation of p21 localization 


Regulation of p27 localization 

AKT phosphorylates p27 on T157, causing it to localize to the cytoplasm 

Nat Med 2002;8:1145–52; Nat Med 2002;8:1136–44 

Figure 8.15c The Biology of Cancer (© Garland Science 2007)

Nuclear p27 accumulation in tumors inversely correlates with pAKT 


How do p21 and p27 regulate G1 cyclins 

As opposed to their inhibitory effects on E-CDK2, 

P21 and p27 are required for D-CDK4 complex formation & activity 


Figure 8.17b The Biology of Cancer (© Garland Science 2007) 

In addition, Cyclin E-CDK2 facilitates its own 

activation by phosphorylating p27 on Thr-187, 

inducing its degradation

As D type cyclins accumulate during G1 they serve two functions: 

1. Catalytic: Cyclin D-dependent kinases phosphorylate Rb, contributing to its 

inactivation. 

2. Non-catalytic: 

- CyclinD-CDK complexes bind and sequester p21/p27 proteins, 

leading to free E-CDK2 activity, which further inactivates pRb.

Would cells lacking all D cyclins undergo cell cycle arrest or 

phosphorylate pRb through other means and progress through 

the cell cycle

Mouse development and cell proliferation in the absence of D-cyclins. 

Near normal BrdU incorporation 

Heart defect in TKO 

Mouse development and cell proliferation in the absence of D-cyclins. 

Kozar K, Ciemerych MA, Rebel VI, Shigematsu H, Zagozdzon A, Sicinska E, Geng Y, Yu Q, Bhattacharya S, Bronson RT, Akashi K, 

Sicinski P. 

Cell. 2004 Aug 20;118(4):477-91.

Proliferation of Cyclin D1 −/− D2 −/− D3 −/− Cells Is Resistant to p16 INK4a but not p27 

and Sensitive to CDK2 siRNA 

(over-expression of p16 

- no effect) 

>>> In the absence of D cyclins 

pRb is phosphorylated by 

Cyclin-E-CDK2

Molecular Analyses of G1 Phase Progression in Cyclin D1 −/− D2 −/− D3 −/− Cells 

pRb and p107 are phosphorylated with a delayed kinetics 

and not to maximal level 

P27 is degraded faster in TKO, 

Perhaps it is phosphorylated more efficiently on Thr-187 

by Cyclin E-CDK2 - but this is not shown. 

Cyclin E associated kinase activity increases in TKO 

E2F regulated genes are induced but not to maximal level

Positive-feedback loops and the irreversibility of cell cycle advance; 

E2F - cyclin E - Rb loop

cyclin E phosphorylates p27 on Thr-187 leading to its degradation 


The Rb - E2F - Skp2 - p27 autoinduction loop 

The Skp2 autoinduction loop. The model shows the core components of the Skp2 autoinduction loop. 

Arrowheads in (A) indicate the direction of flow through the loop that results in Skp2 autoinduction and promotes 

progression through the restriction point. Sequential repressive effects are indicated in (B). (B) also shows the two 

bidirectional events within the loop. Cyclin E-cdk2 catalyzes inactivating phosphorylations of Rb while Rb 

Sequestration of E2F represses transcription of cyclin E. Similarly, p27 inhibits cyclin E-cdk2 activity while 

cyclin E-cdk2 downregulates p27 levels by catalyzing p27 phosphorylation on T187A.

Positive and negative regulation of the Skp2 autoinduction loop.

Summary 

1. D type cyclins respond to extra cellular signals to induce Rb phosphorylation 

and transition through the R point. 

2. Other cyclins (E, A, B) act autonomously to drive S phase and mitosis. 

3. CDK inhibitors p21 and p27 are required to assemble D-CDK4/6 and inhibit cyclins E, A 

and B-CDK complexes. 

4. As D cyclins become elevated they phosphorylate pRb (catalytic) and also sequester p21/p27 

(non-catalytic), thereby allowing E-CDK2 to further phosphorylate and inactivate pRb. 

5. Cyclin D1 also act as a transcriptional co-factor to induce or suppress transcription of various 

genes such as Notch1 during retinal development (non-Catalytic), 

6. Cyclin A and B maintain hyper-phosphorylated ppRb through S and G2/M. At mid-mitosis 

pRb is dephosphorylated by type 1 phosphatase. 

7. The pathways that regulate CDKI, the cyclins and associated kinases are often 

deregulated in human cancer. 

8. Rb-E2F suppress both cell cycle genes (cyclin E, Thymidylate synthase, Cdk1) as well 

as pro-apoptotic genes such as caspases, Puma, and Noxa. The effect of pRb-E2F 

deregulation (cell proliferation vs apoptosis) is context dependent.

Tumor Initiating Cells, Cell Cycle Control and Cancer Stem Cells

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