1. The Basics of Cancer Biology
Lucio Miele, M.D., Ph.D.

2. Part II: “Partners in Crime -1”
Tumor Suppressors, Oncogenes, Enablers and Turncoats

3. Cancer is a genetic disease
“Cancer is, in essence, a genetic disease. Although
cancer is complex, and environmental and other
nongenetic factors clearly play a role in many
stages of the neoplastic process, the tremendous
progress made in understanding tumorigenesis in
large part is owing to the discovery of the genes,
that when mutated, lead to cancer.”
Bert Vogelstein (1988) NEJM 1988; 319:

4. Vogelstein B and Kinzler KW
Abstract
Cancer is a distinct type of genetic disease in which not one, but several, mutations are required. Each mutation drives a wave of cellular multiplication associated with gradual increases in tumor size, disorganization and malignancy. Three to six such mutations appear to be required to complete this process.
Vogelstein B and Kinzler KW
Trends Genet. 1993

5. What are Cancer Genes?
From a genetic standpoint: Any gene which, when mutated, increases the risk of cancer is a cancer gene.
From a phenotypical or functional standpoint: Any gene which, when altered, causes the transformation of normal cells into cancerous cells is a cancer gene.
Note: a gene that increases cancer risk when mutated does not NEED to be transforming! Its effect could be indirect

6. Traditional Types of Cancer Genes
Cancer genes include those whose products:
Directly regulate cell proliferation or survival
a. Tumor suppressor genes (cell proliferation inhibitory)
b. Oncogenes (cell proliferation promoting)
Are involved in the repair of damaged DNA
a. DNA repair genes
b. DNA recombination genes (homologous recombination, non-homologous end-joining)
Regulate TSGs or oncogenes
a. Non-coding RNAs (miRNAs, lnRNAs)
b. Epigenetic modifiers

7. Oncogenes and Tumor Suppressors
There are two broad categories of genes to think about when considering cancer-forming mutations:
Oncogene - An oncogene is a gene whose normal activity promotes cellular proliferation or division.
Tumor Suppressor - a gene that inhibits events leading towards cancer. (This type of gene will be discussed in tomorrow’s lecture.)
Oncogene = gas pedal
Tumor Suppressor gene = brakes

8. Sporadic
Small % of cases Relative large % of cases

9. Tumor suppressor genes:
Oncogenes:
Mutation in one copy of the gene - Dominant
promote cell proliferation
Tumor suppressor genes:
Mutations in both copies of the gene - Recessive
DNA repair genes:
Usually recessive, loss-of-function mutations
that increase spontaneously and environmentally
induced mutation rates

11. Cancer Gene Census
Almost 2% of protein coding genes in humans are cancer genes
15% of cancer genes bear germline mutations
90% of them have somatic mutations
10% of them show both germline and somatic mutations
Wellcome Trust Sanger Institute at

12. Tumor Suppressor Genes
Tumor suppressor genes (TSG) are genes that normally slow down cell division, repair DNA damage, or cause cells to die (by apoptosis or programmed cell death, by necrosis, by autophagy or by or by mixed cell death mechanisms).
When tumor suppressor genes are damaged, deleted or epigenetically silenced, cells can grow out of control, which can lead to cancer.
More than 30 tumor suppressor genes have been identified, including p53, BRCA1, BRCA2, APC, and RB1.
An important difference between oncogenes and TSGs is that oncogenes result from the activation (turning on) of proto-oncogenes, but TSGs cause cancer when they are inactivated (turned off).
Another major difference is that while the oncogenes develop from mutations in normal genes (proto-oncogenes) during the life of the individual (acquired mutations), abnormalities of TSGs can be inherited as well as acquired.

13. Tumor suppressor gene
A tumor suppressor gene is like the brake pedal on a car. It normally
keeps the cell from dividing too quickly, just as a brake keeps a car
from going too fast. When something goes wrong with the gene, such
as a mutation, cell division can get out of control.

14. Non-inherited cancers
Tumor suppressors are mutated in both inherited and non-inherited cancers
Inherited cancer
(Germline mutation)
Abnormal gene
Non-inherited cancers
(Somatic mutations)
Retinoblastoma
RB1
Retinoblastoma, osteosarcoma, lung cancer
Li-Fraumeni Syndrome (sarcomas, brain tumors, leukemias)
p53
Brain tumors, skin cancers, lung cancer, head and neck cancers, others
Melanoma
INK4a
Many different cancers
Colorectal cancer (due to familial polyposis)
APC
Many GI cancers
Colorectal cancer (without polyposis), Lynch Syndrome
MLH1, MSH2, or MSH6
Colorectal, gastric, endometrial cancers
Breast and/or ovarian
BRCA1, BRCA2, PALB2, CHEK2
Breast cancer, ovarian cancers
Wilms Tumor
WT1
Wilms tumors
Nerve tumors, including CNS
NF1, NF2
Small numbers of colon cancers, melanomas, neuroblastoma
Von Hippel-Lindau Syndrome
VHL
Certain types of kidney cancers

15. Examples of Tumor Suppressor Genes
BRCA 1, BRCA 2:
Both proteins involved in DNA repair pathway (usually double strand break repair).
Loss of these proteins can increase the acquisition of subsequent mutations and chromosomal instability.
Mutations greatly increase the susceptibility to develop breast or ovarian cancer in women.
Wilms’ Tumor Suppressor (WT1):
This gene encodes a transcription factor with multiple functions
Mutation is associated with Wilms’ tumor (a renal cancer called nephroblastoma).
Von Hippel‐Lindau (VHL):
VHL protein degrades protein important for promoting angiogenesis, thereby inhibiting the ability to form new blood vessels.
Mutation is associated with renal cell carcinoma and other types of cancers.

16. Functions of Tumor Suppressor genes
1. Regulator of cell cycle:
Serve as genes that insure proper progression through the cell cycle to mediate proper cell division.
Rb gene, INK-4 gene, P53
2. Inducer of apoptosis:
Proteins that promote apoptosis in response to improper signals, presence of DNA damage, etc.
Bim, a pro-apoptotic member of the Bcl-2 family of genes
3. Transcription factors:
Repressor transcription factors: WT1 is a repressor that appears to suppress a factor (Insulin like growth factor) that can contribute to the development of a tumor.
Activator transcription factors: Members of the SMAD family are activated by TGF-β, leading to inhibition of cell proliferation
4. Regulator of angiogenesis response (VHL)

17. Hereditary cancer susceptibility syndromes
About 10% of the most common cancers are due to a hereditary predisposition
Examples:
Breast/ovarian cancer
Colorectal
Retinoblastoma

18. RB - the first tumor suppressor gene cloned (1986)
Loss of both alleles of RB leads to the development of Retinoblastoma in humans
RB is seldom mutated in sporadic human cancers
Inhibits proliferation, promotes differentiation but also inhibits apoptosis

19. Children with RB1 mutations will develop retinoblastoma very early
in life because retinoblastoma originates from neuroblasts during retinal development. Therefore, retinoblastoma is not observed in adults

22. Burkhart D et al., Nat Rev Can 2008

24. Current model of Rb in G1 cell cycle regulation
Narasimha AM et al., eLife, 2014

25. Rb functions as a protein adaptor with at least 4 mechanisms of action: multitasking proteins
Burkhart D et al., Nat Rev Can 2008

26. Burkhart D et al., Nat Rev Can 2008

27. Burkhart D et al., Nat Rev Can 2008

28. Burkhart D et al., Nat Rev Can 2008

30. Two-Hit Hypothesis
The relatively high frequency of the second hit explains the early age of onset of tumors and that they are often bilateral.
In sporadic cancers two independent rare events within the target tissue must occur to form a tumor; hence, the later age of onset.
Loss of Heterozygosity (LOH) analysis can help identify tumor suppressor genes.

31. 1st hit 2nd hit 1st hit LOH T/T T/C C/C D10S107 D17S855
Knudson two-hit model
1st hit nd hit
1st hit
D10S D17S855
LOH
T/T T/C C/C

32. Loss of Heterozygosity (LOH)Mm mm Mm Mm mm mm Mm

33. Mechanisms leading to LOH

34. p53: the most commonly mutated TSG in human cancers
DNA damage
Oncogenic signals
GADD45
DNA repair
p53R2
p53
Active P
p21
Cell cycle arrest
p53
post-translational
modifications
Inactive
Apoptosis
Bax
PUMA
Noxa
Pigs 1

35. p53 - a Classic Tumor Suppressor
Point mutations account for nearly 75% of all of the mutated forms of p53 found in tumors. (Compare to the other commonly mutated genes found in cancer.)
These mutations almost always lead to amino acid substitutions.
A majority of the identified point mutations (from over 15,000 mutated p53 genes sequenced in cancers) are present in the DNA-binding domain of the gene.
Therefore, these mutations inhibit the ability of p53 to bind to DNA and therefore inhibit its ability to activate (or repress) the genes listed in the previous table.

36. p53 - a Classic Tumor Suppressor
In addition to loss of or mutation to p53, p53 activity can be altered through deletion or mutations to other genes.
MDM2 binds to p53, inhibits the ability of p53 to act as a transcription factor, and targets it for ubiquitylation and subsequent degradation.
The action of MDM2 insures the 20 minutes half life of p53 in normal cells.
The amplification of the MDM2 gene in cancer would therefore interfere with the normal ability of p53 to respond to extracellular stimuli and prevent the required cell cycle arrest and promotion of apoptosis.

37. The effects of p53 are DOSE-DEPENDENT
When activated at low levels, p53 binds differentially to promoters that encode genes that suppress cell cycle progression and allow DNA repair (e.g., p21)
When DNA damage is not promptly repaired and active p53 accumulates, it binds lower affinity chromatin sites that contain the promoters of genes that cause apoptosis (e.g., NOXA, DAXX)
This mechanism permits DNA repair and survival if the damage is not irreparable, and causes the cell to commit suicide if the damage is irreparable
Hence, p53 has been called “the guardian of the genome”

38. DNA
Damage
E2F
(1) (2)
1) Wt-p53 binds to p21 promoter; 2) Elevated p21 inhibits CDK2/4 kinases, reducing Rb1 phosphorylation; 3) unphosphorylated Rb1 forms complex with E2F; 4) E2F cannot activate genes important for G1/S transition - no tumor.
2) Mut-p53 is unable to bind to p21 promoter, causing reduced p21 level; 2) CDK2/4 kinases induce Rb1 phosphorylation; 3) more E2F free from Rb1 complex; 4) more E2F will promote cell cycle progression - Tumor formation.

39. Dominant-negative mechanism
WTp53
WTp53
Transcriptional activation
WTp53
WTp53
Tetramer formation
WTp53
Mutant
p53
WTp53
Mutant
p53
WTp53

40. p53 mutations More than 50% of human tumors contain p53 mutations.
80% are missense mutations in the DNA binding domain.
“Hot spot” mutations at pp175, 248, 249, 273, and 281
Mutations of p53 are more frequent events and show worse prognosis compared to deletion of p53 gene.
248
273
175
281
249 N I II
III IV V C
Activation domain
DNA binding domain
Tetramerization domain

41. Some mutations turn p53 from a TSG into an oncogene: a Turncoat!
Over 50% of human cancers either have lost p53 protein or have mutations in the gene.
About 70% of Li-Fraumeni syndrome patients carry p53 alterations (heterozygous).
Loss of p53 in mice causes tumors dependent on gene dosage.
Mutant p53 accumulates as a nuclear phosphoroprotein.
The mutant p53 functions as dominant-negative that inactivates wild-type p53 function through a tetramer formation.
However, some forms of mutant p53 show unexpected oncogenic functions which can not be explained simply by loss of p53. These mutants activate transcription at some chromatin sites
Therefore, p53 (mutant p53) was originally believed to be an oncogene.

42. Identification of TSG Before 2007
- Linkage analysis of familial cancer families and positional cloning of TSG
- Loss of heterozygosity analysis to clone TSG
After 2007
- Exome sequencing of familial cancer family members and bioinformatics analysis
- Mutation co-segragates with phenotype in family

43. Cancer gene (TSG) discovery
by Exome sequencing
Exclude
variants
in control

44. Exome capture

45. Exome sequencing

46. Some tumor suppressors don’t encode proteins

47. miRNAs and lncRNAs can be TSGs
By downregulating the expression of oncogenes
mRNA stability
mRNA translation arrest
By regulating the expression of cancer risk genes that are not themselves oncogenes
E.g., genes in the immune system that control cancer immune surveillance
By regulating the splicing of transcripts or the activity of regulatory elements in DNA