LSM4214: Cancer Pharmacology Lecture 12: …...Tumor suppressor genes are usually recessive (loss of function) • loss of a cellular gene or chromosome region by deletion • loss

Lecture 12:

Cellular Oncogenes/Tumor suppressor

genes

LSM4214: Cancer Pharmacology

LSM4214-Cancer Pharmacology 2016/2017 L12: Oncogenes and Tumor suppressor genes 1

A/P Gautam Sethi

Dept of Pharmacology

Building MD3, #04-01,

16 Medical Drive

E-mail: [email protected]

Learning Objectives on Principles of

Chemotherapy

To be able to grasp the basic concepts on role of

oncogenes in cancer

To understand the different classes of oncogenes and

their mechanism(s) of action

To be able to understand the contribution of gene therapy

in cancer treatment

2 LSM4214-Cancer Pharmacology 2016/2017 L12: Oncogenes and Tumor suppressor genes 2

Normal

colon cell

Increased

cell growth

Adenoma

I

Adenoma

II

Adenoma

III

Carcinoma

Metastasis

Multistep carcinogenesis

Stages in the evolution of colon cancer

Chromosome 5q gene loss or mutation

Ras gene mutation

Chromosome 18 loss or mutation

DCC tumor suppressor gene

Chromosome 17 loss or mutation

p53 tumor suppressor gene

Other chromosome losses

3

DCC-Deleted in colorectal cancer


Protooncogenes and Oncogenes

• Proto-oncogene: A gene that normally functions to control cell division and may become a cancer gene (oncogene) by mutation

• Oncogene: A gene that induces or continues uncontrolled cell proliferation

• act in an autosomal dominant fashion


Oncogenes

1 mutation sufficient for role in cancer development

Protoncogenes

(regulate cell growth)

1st mutation

(Oncogenes= lead to

accelerated cell

division)



Oncogene Discovery

• Rous Sarcoma virus (RSV) was first discovered in 1911 by

Peyton Rous.

• Observed that cancer can be transmitted.

• Induced tumors in health chickens by injecting a preparation of

cell free filtered extract from chicken tumors.

• Later, it was discovered that the causative agent is the rous

sarcoma virus (RSV) - a retrovirus: it reverse transcribes its

RNA genome into cDNA before integration into the host DNA.

• The first confirmed oncogene was discovered in 1970 and was

termed src (pronounced sarc as in sarcoma). Src was in fact

first discovered as an oncogene in a chicken retrovirus.


Tumor Suppressor Genes

• Tumor suppressor gene products inhibit cell

proliferation.

• Mutant versions in cancer cells have lost their

function.

• Both alleles of a tumor suppressor gene must

be inactivated to change the behavior of the

cell.


Normal genes (prevent cancer)

1st mutation (susceptible carrier)

2nd mutation or loss (leads to

cancer)



• Disease examples

–Retinoblastoma, pRB (nuclear

phosphoprotein)

–Wilm’s tumor, WT-1

–Li-Fraumeni syndrome, p53

(transcription factor)

–Colon carcinoma, DCC

–Breast cancer, BRCA1



Function of the Rb protein

E2F Rb E2F

E2F

E2F

p

p

Rb

p

p

Rb

p

p

Rb

p

p

p

p

E1A

Growth suppression Relief of growth suppression

G1 phase phosphorylation

releases E2F

Adenovirus E1A oncoprotein binding

releases E2F

Gene mutation affecting binding pocket

releases E2F

• E2F is a transcription factor

that mediates growth-dependent

activation of genes required to

make the transition into and

through S phase

• Rb binds and inactivates E2F

under conditions of growth

suppression

• There are several ways to

alleviate growth suppression

resulting in controlled or

uncontrolled cell growth


Features of Retinoblastoma

• 1 in 20,000 children

• Most common eye tumor

in children

• Occurs in heritable and

non-heritable forms

• Identifying at-risk infants

substantially reduces

morbidity and mortality

LSM4214-Cancer Pharmacology 2016/2017

L12: Oncogenes and Tumor suppressor genes 12

p53 is the “guardian of the genome”

•p53 is frequently found mutated in human tumors

• the p53 protein functions as a transcription factor that

regulates cell-cycle and DNA repair genes

• UV irradiation causes cell-cycle arrest in G1 that is dependent

on p53; cells that contain a mutated p53 cannot arrest

and go into S phase and replicate damaged DNA

• p53 loss-of-function mutations result in the replication of

cells with damaged DNA and to the further accumulation

of other mutations affecting oncogenes and tumor

suppressor genes, and to an increased

likelihood of cancer


LSM4214-Cancer Pharmacology 2016/2017

L12: Oncogenes and Tumor suppressor genes 14

What are the genes responsible for tumorigenic cell growth?

Normal

Cancer

Proto-oncogenes

Cell growth and proliferation

Tumor suppressor genes

+

-

Mutated or “activated”

oncogenes Malignant transformation

Loss or mutation of

Tumor suppressor genes

++


Oncogenes are usually dominant (gain of function)

• cellular proto-oncogenes that have been mutated (and “activated”)

• cellular proto-oncogenes that have been captured by retroviruses

and have been mutated in the process (and “activated”)

• virus-specific genes that behave like cellular proto-oncogenes

that have been mutated to oncogenes (i.e., “activated”)

Tumor suppressor genes are usually recessive (loss of function)

• loss of a cellular gene or chromosome region by deletion

• loss of gene function by an inactivating point mutation


Oncogene Activation

• Altered gene function

– Base substitutions

• Amplification

• Altered regulation

• Viral insertion


amino acid position

Ras gene 12 59 61 Tumor

c-ras (H, K, N) Gly Ala Gln normal cells

H-ras Gly Ala Leu lung carcinoma

Val Ala Gln bladder carcinoma

K-ras Cys Ala Gln lung carcinoma

Arg Ala Gln lung carcinoma

Val Ala Gln colon carcinoma

N-ras Gly Ala Lys neuroblastoma

Gly Ala Arg lung carcinoma

Murine sarcoma virus

H-ras Arg Thr Gln Harvey strain

K-ras Ser Thr Gln Kirsten strain

Amino acid substitutions in Ras family proteins


Chromosomal rearrangements or translocations

Neoplasm Translocation Proto-oncogene

Burkitt lymphoma t(8;14) 80% of cases c-myc1

t(8;22) 15% of cases

t(2;8) 5% of cases

Chronic myelogenous t(9;22) 90-95% of cases bcr-abl2

leukemia

1c-myc is translocated to the IgG locus, which results in its activated expression 2bcr-abl fusion protein is produced, which results in a constitutively active abl kinase


Transforming viruses

Viral class Viral genome Oncogenes

adenovirus ds DNA E1A & E1B

papovavirus ds DNA T antigens

SV40 (monkey)

Polyoma (human)

retrovirus ss RNA mutated cellular

proto-oncogenes


Retrovirus oncogenes derived from normal cellular genes

Retrovirus Viral oncogene Cellular proto-oncogene

Rous sarcoma virus v-src c-src (src)

Simian sarcoma v-sis c-sis (sis)

Harvey murine sarcoma v-H-ras c-H-ras (H-ras)

Kirsten murine sarcoma v-K-ras c-K-ras (K-ras)

FBJ murine osteosarcoma v-fos c-fos (fos)

Avian myelocytomatosis v-myc c-myc (myc)

Abelson leukemia virus v-abl c-abl (abl)

Avian erythroblastosis v-erbB c-erbB (erbB)

• viral oncogenes are ~80-99% homologous to cellular proto-oncogenes


Conversion of a regulatory gene into a viral oncogene


Classification of Oncogenes

• Growth factors

– sis

• Growth factor receptors

– erbB, fms,trk

• Intracellular transducers

– src, abl, raf, gsp, ras

• Nuclear transcription factors

– jun, fos, myc, erbA


4. Nuclear

Proteins:

Transcription

Factors

Cell Growth

Genes

3. Cytoplasmic

Signal Transduction

Proteins

1. Secreted Growth Factors

2. Growth Factor Receptors


Oncogene-encoded defective EGF receptor

Breast, stomach, and ovary cancers

The epidermal growth factor

receptor (EGFR; ErbB-1; HER1

in humans) is the cell-surface

receptor for members of the

epidermal growth factor family

(EGF-family) of extracellular

protein ligands.The epidermal

growth factor receptor is a

member of the ErbB family of

receptors, a subfamily of four

closely related receptor tyrosine

kinases: EGFR (ErbB-1),

HER2/c-neu (ErbB-2), Her 3

(ErbB-3) and Her 4 (ErbB-4).

Mutations affecting EGFR

expression or activity could

result in cancer


Ras family proteins

• the c-ras family contains three genes: H-ras, K-ras, and N-ras

• the Ras proteins encoded by these genes are small G-proteins

• the proteins transmit growth signals from cell surface receptors

• the Ras proteins are activated by binding GTP

• the proteins are inactivated by GTP to GDP hydrolysis

• mutations in the c-ras genes inactivate the Ras GTPase

• mutated Ras proteins are constitutively active

• constitutively active Ras proteins result in uncontrolled cell growth



Oncogene Addiction

The continued activity of the specific over

expressed oncogene is necessary for the

maintenance of malignant phenotype.


Experimental evidence

1. Transgenic mouse is over expressed with myc

oncogene, which induces the formation of malignant

osteogenic sarcoma.

2. Loss of over expression leads to differentiation and the

formation of normal osteocytes.

3. Similarly conditional activation of Bcr-Abl gene in

transgenic mouse resulted in development of leukemia

and subsequent deactivation leads to apoptosis of

cancer cells.


4. On the contrary it is not always necessary that

continued over expression is required for maintenance

of malignant phenotype .

5. Over expressed oncogenes shows their effects by

causing genomic instability.

6. A subset of a c-Myc dependent tumor cells escape Myc

dependence by activating endogenous ras oncogenes.


Molecular alterations in chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML). Deletion or

downregulation of microRNA (miR)-15a/miR-16-1 cluster, located at chromosome 13q14.3 and directly involved in the

regulation of BCL2 and MCL1 expression, represent an early event in the pathogenesis of CLL. During the evolution of

malignant clones, other microRNAs (miRs) can be deleted (such as miR-29) or overexpressed (such as miR-155),

contributing to the aggressiveness of B-cell CLL. Such abnormalities can influence the expression of other protein-

coding genes (PCGs), as TCL1 oncogene, directly regulated by miR-29 and miR-181, or affect other noncoding RNAs

(ncRNAs). The consequences of this steady accumulation of abnormalities are represented by the reduction of

apoptosis and the induction of survival and proliferation of malignant B cells, leading to the evolution of more

aggressive clones. Members of the miR-29 family, lost in AML and in other tumor types as lung cancer, have also been

shown to directly target MCL1 and DNMT3A and B.

MicroRNA Genes (encode for single strand RNA of about 21-23 nucleotides)


Gene Therapy


What is Gene Therapy

Gene therapy is the insertion of genes into an

individual's cells and tissues to treat a disease,

such as a hereditary disease in which a

deleterious mutant allele is replaced with a

functional one.

Basic process for gene therapy: 1. A “corrected’’ gene is inserted into the genome to

replace an “abnormal” disease-causing gene.

2. A carrier called a vector must be used to deliver the

therapeutic gene to the patient’s target cells.


1. A vector delivers the therapeutic gene into a

patient’s target cell.

2. The target cells become infected with the viral

vector.

3. The vector’s genetic material is inserted into the

target cell.

4. Functional proteins are created from the

therapeutic gene causing the cell to return to a

normal state.

How it works?


Uses of gene therapy

1.Replace missing or defective genes;

2.Deliver genes that speed the destruction of cancer

cells;

3.Supply genes that cause cancer cells to revert back

to normal cells;

4.Deliver bacterial or viral genes as a form of

vaccination;

5.Provide genes that promote or impede the growth

of new tissue; and;

6. Deliver genes that stimulate the healing of

damaged tissue.


Gene therapy can target somatic (body cells) or

germline (sperm cells, ova, stem cell precursors of

sperm and ova) cells. In somatic gene therapy the

recipient's genome is changed, but the change is

not passed on to the next generation; whereas with

germ line gene therapy the newly introduced gene

is passed on to the offspring.

Types of Gene Therapy


Types of Gene Therapy (continued)

• Most gene therapy treatments in humans has been directed at somatic cells.

• Germline gene therapy remains controversial.

• For germline gene therapy, the introduced gene must be incorporated into the

chromosomes by genetic recombination.

Somatic gene therapy:

1. ex vivo: cells are modified outside the body and then transplanted back again.

2. in vivo: genes are changed in cells still in the body.

Recombination based approaches in vivo are uncommon as most DNA constructs

have a very low probability.


Types


Example of ex vivo GT

• Usually done with blood cells because

they are easiest to remove and return.

• Sickle cell anemia


Example of in situ somatic cell

gene therapy

• Infusion of adenoviral vectors into the

trachea and bronchi of cystic fibrosis

patients.

• Injection of a tumor mass with a vector

carrying the gene for a cytokine or toxin.

• Injection of a dystrophin gene directly into

the muscle of muscular dystrophy patients.


Example of in-vivo somatic cell

gene therapy

• No clinical examples.

• In vivo injectable vectors must be

developed. Liposomes can be used.


In vivo techniques usually

utilize viral vectors

– Virus = carrier of desired gene (vector).

– Virus genome is manipulated to remove disease-causing genes and introduce therapeutic genes.

– Viral methods have proved to be the most efficient to date.

– Many viral vectors can stable integrate the desired gene into the target cell’s genome.


Ideal vector characteristics:

1. Insert size: one or more genes.

2. Targeted: limited to a cell type.

3. No immune response.

4. Stable: not mutated.

5. Production: easy to produce high concentrations

6. Can be Regulated: produce enough protein to cause an

effect.


Types of vectors

• RNA viruses (Retroviruses)

1. Murine leukemia virus (MuLV)

2. Lentivirus e.g. Human immunodeficiency viruses (HIV)

3. Human T-cell lymphotropic viruses (HTLV)

• DNA viruses

1. Adenoviruses

2. Adeno-associated viruses (AAV)

3. Herpes simplex virus (HSV)

4. Pox viruses

Non-viral vectors

1. Liposomes

2. Naked DNA

3. Liposome-polycation complexes

4. Peptide delivery systems




Retroviruses can infect only dividing cells. The viral genome in the form of RNA is reverse-transcribed when the

virus enters the cell to produce DNA, which is then inserted into the genome at a random position by the viral

integrase enzyme.


Adenoviral DNA dose not integrate into the genome and is not replicated during cell division.

The simplest method is the direct introduction of the therapeutic DNA into target cells. This approach is limited in its

application because it can be used with only certain tissues and requires large amounts of DNA.

Non-viral approaches

Nonviral approach

involves the creation of

an artificial lipid sphere

with an aqueous core.

This liposome, which

carries the therapeutic

DNA, is capable of

passing the DNA through

the target cell's

membrane


Nonviral substances

such as Ormosil have

been used as DNA

vectors and can

deliver DNA loads to

specifically targeted

cells in living animals.

(Ormosil stands for

organically modified

silica or silicate)

Nano-engineered substances


1. Artificial killing of cancer cells

- Insert a gene encoding a toxin (e.g. diphtheria A chain) or a gene conferring sensitivity to a

drug (e.g. herpes simplex thymidine kinase) into tumor cells. Virus-originated HSV-TK is

different from that of mammals, its product thymidine kinase can metabolize the non-toxic

prodrug ganciclovir (GCV) into a monophosphate derivative, then phosphorylate it further into

GVV triphosphate. This metabolite is incorporated into replicating DNA and acts as a DNA

synthesis inhibitor.

Cancer Gene Therapy

2. Stimulate natural killing of cancer cells

- Enhance the immunogenicity of the tumor by (for e.g. inserting genes encoding foreign antigens).

- Increase anti-tumor activity of immune system cells by (for e.g. inserting genes that encode

cytokines).

- Induce normal tissues to produce anti-tumor substances (e.g. interleukins, interferon).

- Protect surrounding normal tissue from side effects of chemotherapy. One example is the

multidrug-resistant protein-1, which is encoded by the human ABCBI gene named as MDR1 gene.

It stimulates the cellular pump to remove cytotoxic drugs from normal cell cytoplasm to the outside,

thus protecting normal cells from chemotherapy’s side effects. The MDR1 gene is minimally

expressed in malignant cells; thus, chemotherapeutic medications entering the cytoplasm will

remain at a higher concentration, leading to apoptosis. LSM4214-Cancer Pharmacology 2016/2017 L12: Oncogenes and Tumor suppressor genes 50

3. Tumors resulting from oncogene activation

- Selectively inhibit the expression of the oncogene.

- Deliver gene specific antisense oligonucleotide or ribozyme to inactivate/cleave

oncogene mRNA.

4. Tumors arising from inactivation of tumor suppressor genes

- Insert wild type tumor suppressor gene.


Problems with Gene Therapy

1. 1. Short Lived 2. Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing

nature of cells prevent gene therapy from long time.

Would have to have multiple rounds of therapy.

2. Immune Response new things introduced leads to immune response.

increased response when a repeat offender enters.

3. Viral Vectors patient could have toxic, immune, inflammatory response.

also may cause disease once inside.

4. Multigene Disorders Cancer, Heart disease, and diabetes are difficult to treat because you

need to introduce more than one gene.

May induce a tumor if integrated in a tumor suppressor gene

because insertional mutagenesis.


Technical Difficulties in Gene

Therapy

Gene delivery: Successful gene delivery is not easy or predictable, even in single-gene disorders. For example, although the genetic basis of cystic fibrosis is well known, the presence of mucus in the lungs makes it physically difficult to deliver genes to the target lung cells. Delivery of genes for cancer therapy may also be complicated by the disease being present at several sites. Gene-therapy trials for X-linked severe combined immunodeficiency (X-SCID), however, have been more successful


First Approved Gene Therapy

On September 14, 1990 at the U.S. National Institutes

of Health, W. French Anderson M.D. and his

colleagues R. Michael Blaese, M.D., C. Bouzaid, M.D.,

and Kenneth Culver, M.D., performed the first

approved gene therapy procedure on four-year old

Ashanthi DeSilva. Born with a rare genetic disease

called severe combined immunodeficiency (SCID).


What did they do In Ashanthi's gene

therapy procedure,

doctors removed white

blood cells from the

child's body, let the cells

grow in the laboratory,

inserted the missing

gene into the cells, and

then infused the

genetically modified

blood cells back into the

patient's bloodstream.


A success story

As of early 2007, she was still in good

health, and she was attending college.

Some would state that the study is of great

importance despite its indefinite results, if

only because it demonstrated that gene

therapy could be practically attempted

without adverse consequences.


What Gene therapy can Achieve

1. Replacing a mutated

gene that causes

disease with a healthy

copy of the gene.

2.Inactivating, or

“knocking out,” a

mutated gene that is

functioning improperly.

3. Introducing a new

gene into the body to

help fight a disease.


The Future of Gene Therapy

Current uses of gene therapy focus on treating or

curing existing conditions. In the future, the focus

could shift to prevention. As more of the human

genome is understood, medicine will know more

about which genes contribute to or cause disease.

With that knowledge in hand, gene therapy could be

used to head off problems before they occur.


Last two decades made rapid

progress

Over the last 20 years,

the initial thoughts of

gene therapy have been

transformed into reality

with more than 175

clinical trials and 2,000

patients already treated .

Yet with all the trials,

there is still no

conclusive evidence for

efficacy.


1. How can “good” and “bad” uses of gene therapy be distinguished?

2. Who decides which traits are normal and which constitute a disability or disorder?

3. Will the high costs of gene therapy make it available only to the wealthy?

4. Could the widespread use of gene therapy make society less accepting of people who are different?

5. Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability?

What are the ethical issues surrounding

gene therapy?


Understanding Genome and Human Genome

Project is a boost to Gene Therapy


Do not forget Genes can be

Unpredictable ?


Model questions for Final examination

1. Discuss advantages and disadvantages of various types of vectors used for gene therapy?

2. Describe various classes of oncogenes giving relevant examples of each class?

Long essay questions

MCQs


LSM4214: Cancer Pharmacology Lecture 12: …...Tumor suppressor genes are usually recessive (loss of function) • loss of a cellular gene or chromosome region by deletion • loss

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