Therapeutic application of oncogene and

tumor suppressor gene

Introduction Proto-oncogenes: A gene which codes for protein that help in the regulation of

normal cell growth. Stimulate cell division Prevent cell differentiation Regulate programmed cell death

Oncogenes: sequence of DNA that have been altered or mutated from its original form, proto-oncogenes.

Unregulated cell division Slower rate of cell differentiation Increased inhibition of cell death

Discovery of Oncogenes Discoverd by Peyton Rous 1909, worked at

the Rockefeller institute for medical research began a series of experiments that started with a chicken that had a lump on its leg, which was a soft-tissue sarcoma.

When Rous ground up the tumour and injected into other chickens, also causes sarcoma in it.

Active agent was identified as virus-retrovirus- was called as Rous sarcoma virus

Analysis of RSV in 1970 showed retrovirus with small single stranded RNA gene, src which is responsible for cancer-inducing properties of RSV and considered as Oncogenes.

In 1975, scientists discovered that healthy cells also carry normal version of src (c-src) genes, which plays an important role in normal cell development.

When it gets mutated will converted into virulant (v-src)

SRC: Proto-oncogene tyrosine-protein kinase Cell morphology, motility Proliferation and survival

Structure: 14-c myristoyl group, SH3 andSH2 domain, protein

tyrosine kinase domain and c-terminal regulatory tail Chief phosphorylation site of src include tyrosine 416

result in activaion from autophosphorylation and tyrosine 527 result in inhibition from phosphorylation by c-terminal src kinase.

Tyrosine kinase signalling and oncogenesis

Normal cells receive and interpret external signals which influence their growth, shape, metabolism and movement. Complex interactions between molecules inside the cell transmit these signals and initiate appropriate cellular responses.

Protein phosphorylation on Tyrosine (Tyr) residues has evolved as an important mechanism to coordinate this intracellular signalling process in multicellular organisms.

Inactivation: Y416 is autophosphorylation site in the activation loop and

negative regulation in tyrosine site by phosphorylation at Y527 in the c-terminal end.

c-src is auto inhibited through intramolecular association between the SH2 and Y527, and SH3 and poly proline motif in the linker.

Activation: Activation is by either dephosphorylation of Y527 to release

the SH2 domain or by binding of peptide ligand to recognise SH2 leading to autophosphorylation of Y416.

When Src goes wrong??? Under normal circumstances, Src is predominantly inactive in cells,

being switched on only at specific times.

fine balance between phosphorylation and dephosphorylation is

disrupted, changes can occur in Src activity with drastic results.

Several cancers, including colon and breast cancer, have been

associated with an increase in Src activity.

v-Src, from the transforming virus, Rous Sarcoma Virus. v-Src was found to lack the region of the cellular protein (c-Src) that contains Tyr527, making it continually active.

In late stage colon cancers, mutations have been reported in the src gene that cause the loss of the region containing Tyr527, leading to Src over-activity.

Proteins that regulate Src have also been found at abnormal levels in cancer cells, including both those that activate and those that inactivate Src.

Proteins such as PTPalpha, SHP-1 and PTP1B that activate Src by dephosphorylating Tyr527 have been detected at elevated levels in various cancer cells, including epidermal and breast carcinoma cells.

Therapeutic applications Control over the metabolic alterations : A decreased oxidative phosphorylation, increased glucose consumubsequent

lactate production are the remarkable features of oncogenic activation .

Over the last decade, evidence has accumulated that the oncogenes myc, AKT, nuclear factor kB (NF-kB) and the tyrosine kinase receptors (insulin-like growth factor 1, epidermal growth factor, EGF; IGF-1; Her-2; etc.), which turn on phosphatidylinositol 3-kinases (PI3Ks), mammalian target of rapamycin (mTOR) pathway and the Ras, RAF–Mitogen-Activated Protein Kinase (MAP kinase) along with hypoxia induced factor (HIF), can activate the transcription of numerous genes that encode the proteins that mediate the various metabolic pathways .

Myc and HIF-1 transcription factors enhances the rate of transcription of some of the GLUT transporters and hexokinase 2, increasing both glucose uptake and its retention in the cell .

Increases the oxidative phosphorylation and regulate the metabolic pathway.

Tumor suppressor gene: A protective gene that normally limits the growth of tumors. When a tumor

suppressor gene is mutated (altered), it may fail to keep a cancer from growing.

The first insight into the activity of tumor suppressor genes came from somatic cell hybridization experiments, initiated by Henry Harris and his colleagues in 1969.

The fusion of normal cells with tumor cells yielded hybrid cells containing chromosomes from both parents.

The first tumor suppressor gene was identified by studies of retinoblastoma, a rare childhood eye tumor.

Protein that halts cell cycle progression in response to DNA damage may no longer sense damage or trigger a response. Genes that normally block cell cycle progression are known as tumor suppressors.

Tumor suppressors prevent the formation of cancerous tumors when they are working correctly, and tumors may form when they mutate so they no longer work.

One of the most important tumour suppressor gene is p53, which plays a key role in the cellular response to DNA damage.

Mechanism of p53:

Involved in two process

Repairing DNA damage

Apoptosis

DNA repair, which also depends on p53, whose second job is to activate DNA repair enzymes. If the damage is fixed, p53 will release the cell, allowing it to continue through the cell cycle.

If the damage is not fixable, p53 will play its third and final role: triggering apoptosis (programmed cell death) so that damaged DNA is not passed on.

p53 in cell cycle: p53 involved in the checkpoints. Cell cycle check points:

There are a number of checkpoints, but the three most important ones are:

The G1 checkpoint, at the G1/S transition.The G2 checkpoint , at the G2/M transition.The spindle checkpoint, at the transition from

metaphase to anaphase.

Key to the DNA damage response is a protein called p53, a famous tumor suppressor often described as “the guardian of the genome.”

p53 works on multiple levels to ensure that cells do not pass on their damaged DNA through cell division.

it stops the cell cycle at the checkpoint by triggering production of Cdk inhibitor (CKI) proteins. The CKI proteins bind to Cdk-cyclin complexes and block their activity for DNA repair.

cancer cells, p53 is often missing, nonfunctional, or less active than normal. For example, many cancerous tumors have a mutant form of p53 that can no longer bind DNA.

Since p53 acts by binding to target genes and activating their transcription,

the non-binding mutant protein is unable to do its job.

When p53 is defective, a cell with damaged DNA may proceed with cell division. The daughter cells of such a division are likely to inherit mutations due to the unrepaired DNA of the mother cell.

Over generations, cells with faulty p53 tend to accumulate mutations, some of which may turn proto-oncogenes to oncogenes or inactivate other tumor suppressors.

ApoptosisThe cells between your embryonic fingers died in a

process called apoptosis, a common form of

programmed cell death.

In programmed cell death, cells undergo “cellular

suicide” when they receive certain cues. Apoptosis

involves the death of a cell, but it benefits the

organism as a whole.

Cells that undergo apoptosis go through a different and much more orderly process. They shrink and develop bubble-like protrusions (technical name: “blebs”) on their surface.

The DNA in the nucleus gets chopped up into small pieces, and some organelles of the cell, such as the endoplasmic reticulum, break down into fragments.

They release signals that attract debris-eating (phagocytic) immune cells, such as macrophages. Also, the fragments of the dying cell display a lipid molecule called phosphatidylserine on their surface.

Phosphatidyserine is usually hidden on the inside of the membrane, and when it is on the outside, it lets the phagocytes bind and "eat" the cell fragments.

Pathways : Intrinsic and extrinsic pathways

Extrinsic pathway: In this pathway, phosphatidyserine is the death signal, which

activates the pro-caspase 8(inactive) to caspase 8. Activated caspase 8 , which activated the procaspase 3 to

caspase3.

Intrinsic pathway: p53, which activates the bax, and bax activates cyt C, binds to

apaf1 to form apoptosome. Activation of apoptosome activates procaspase 9 to active form

of caspase 9

Applications of p53-Based Cancer Therapy

Reactivating Mutant p53: PhiKan083 is a carbazole derivative found from in silico screening of

the crystal structure of p53. By binding mutated p53, PhiKan083 raises the melting temperature

of mutated p53, which results in the reactivation of its function. PRIMA-1 -Restored sequence-specific DNA binding and active the

conformation. CP-31398- Restore the protein folding of mutated p53 to a more

natural conformation that permits a wild-type function

p53 Stabilization: MDM2 is an E3 ubiquitin ligase which controls p53

degradation. Many tumors overexpress MDM2, even tumors without p53 mutations.

Nutlins are cis-imidazoline compounds that act as antagonists of the MDM2-p53 interaction.

MI-219 is another small molecule that inhibits the MDM2-p53 interaction. MI-219 also activates the p53 pathway in cells with wild-type p53. Apoptosis and cell cycle arrest were observed in xenograft tumors which resulted in tumor regression .

Classes of Drugs for p53 Stabilization: Tenovin was found by a cell-based drug screen

to activate p53. Tenovin acts as an inhibitor of the protein-deacetylating activities of SirT1 and SirT2.

The intraperitoneal administration of tenovin-6 has been demonstrated to induce a regression of xenograft tumors in a mouse mode

p53 Gene Therapy: p53-based gene therapy was reported in 1996. A

retroviral vector containing the wild-type p53 gene under the control of an actin promoter was injected directly into tumors of nonsmall cell lung cancer patients.

After development of a replication-defective recombinant p53 virus (Ad5CMV-p53) , many clinical trials have been performed, including one in esophageal cancer patients .

Conclusion Cytoplasmic p53 can activate a transcription-

independent apoptotic program. The next generation of p53-based cancer therapeutic approaches should therefore be developed to take advantage of this cytosolic function.

This may be safer than regulating the transcription modulation of wild-type p53, which can induce both prosurvival and proapoptotic effects in tumor cells.

Recent reports have showed that p53 regulates the process of

self-renewal of neural stem cells and hematopoietic stem cells.

The cancer stem model insists that tumors are maintained by a small population of cancer stem cells that can divide both symmetrically and asymmetrically.

Mammary stem cells with the targeted mutation of p53 have been reported to show the same properties as cancer stem cells. The reactivation of p53 restored the asymmetric cell division of cancer stem cells and induced tumor growth inhibition .

References T. Soussi and C. Béroud, “Assessing TP53 status in human

tumours to evaluate clinical outcome,” Nature Reviews Cancer, vol. 1, no. 3, pp. 233–240, 2001. View at Google Scholar · View at Scopus

K. H. Vousden and C. Prives, “Blinded by the light: the growing complexity of p53,” Cell, vol. 137, no. 3, pp. 413–431, 2009. View at Publisher · View at Google Scholar · View at Scopus

T. Riley, E. Sontag, P. Chen, and A. Levine, “Transcriptional control of human p53-regulated genes,” Nature Reviews Molecular Cell Biology, vol. 9, no. 5, pp. 402–412, 2008. View at Publisher · View at Google Scholar · View at Scopus

Bjorn MJ, Groetsema G, Scalapino L. Antibody-Pseudomonas exotoxin A conjugates cytotoxic to human breast cancer cells in vitro. Cancer Res 46:3262, 1986.PubMed

Cline M, Slamon DJ, Lipsick JS. Oncogenes: implications for the diagnosis and treatment of cancer. Ann Intern Med 101:223, 1984.PubMed

Cote RJ, Houghton AN. The generation of human monoclonal antibodies and their use in the analysis of the humoral immune response to cancer. In: Engleman EG, Foung SKH, Larrick JW, Raubitschek A (eds), Human Hybridomas and Monoclonal Antibodies. New York: Plenum Press, 1985, p. 189.

Douillard JY, Lehur PA, Aillet G, et al. Immunohistochemical antigenic expression and in vivo tumor uptake of monoclonal antibodies with specificity for tumors of the gastrointestinal tract. Cancer Res 46:4221, 1986.