Tumor Suppressor Genes

Tumor suppressor genes or anti- oncogenes are the gene that regulates a cell during cell division and replication. These are normal genes that slow down cell division or tell cells to die at the right time (a process known as apoptosis or programmed cell death). When a tumor suppressor gene is mutated, the cell can start to grow out of control, which might lead to cancer.

Identification of Tumor Suppressor Genes:

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. In the most cases, such hybrid cells were not capable of forming tumor in animals. Therefore, it appeared that genes derived from normal parent cell acted to inhibit (suppress) tumor development.

• The first tumor suppressor gene was identified by studies of retinoblastoma (Rb gene), a rare childhood eye tumor. This disease is an inherited condition caused by inactivating mutation in Rb1 gene that cause increased risk of developing retinoblastoma (often in both eyes). Interestingly, about 60% of retinoblastomas occur sporadically (almost in one eye). To explain this dichotomy, Knudson proposed a “two-hit” hypothesis:
• The development of retinoblastoma requires two mutations which are now known to correspond to the loss of both of functional copies of tumor susceptibility gene (the Rb tumor suppressor gene) that would be present on homologous chromosomes of a normal diploid cell.
• In inherited retinoblastoma, one defective copy of Rb is genetically transmitted (‘one-hit’). The loss of this single Rb copy is not by itself sufficient to trigger tumor development, but retinoblastoma almost always develops in these individuals as a result of second mutating (‘second-hit’) leading to the loss of remaining normal Rb allele.
• In sporadic cases of retinoblastoma, both normal Rb alleles must undergo a somatic mutation in the same cell. The Probability of this is low, which explains why retinoblastoma is uncommon in the general population.
• In both the cases, the resulting two hits leads to the development of retinoblastoma.

 Based on these observations, there are three important properties of classic tumor suppressor genes(TSGs)

1.  Classic tumor suppressor genes (TGSs) are recessive at cellular level, with inactivation of both alleles typically found in tumors.
2. Inheritance of a solitary mutant allele increase tumor susceptibility because only a single additional inactivating event is necessary for complete loss of gene function.
3. The same gene is often inactivated in sporadic cancers.

 Types of Tumor Suppressing Genes:

Tumor suppressor genes can be broadly divided into five types functionally:

1. Genes that encode intracellular proteins that are crucial in controlling the progression of cell cycle stages (e.g. pRb and p16).
2. Gene that encode receptors or signal transducers which orchestrate signals that inhibit cell proliferation (e.g. adenomatous polyposis coli (APC) and transforming growth factor (TGF-β))
3. Genes that control the check-point proteins, which are useful in triggering cell cycle arrest in case of DNA damage or chromosomal defects (e.g. p16,p14 and breast cancer type1 susceptibility protein (BRCA1)).
4. Genes that encode proteins useful for the induction of apoptosis(e.g. p53).
5.  Genes that encode proteins involved in the repair of DNA (e.g. DNA mismatch repair protein2 (MSH2) and p53).

Retinoblastoma (Rb):

Rb gene, also called ‘Governor of the cell cycle’, encodes the Rb protein that, when hypophosphorylated, binds and inhibits E2F transcription factors. These transcription factors regulate genes that are essential for cells to pass from G1 to the S phase of the cell cycle. Typical growth factor signaling causes Rb hyperphosphorylation and inactivation, thus causing cell cycle progression. A Variety of mechanisms like loss-of-function mutations affecting Rb, CDK4 and cyclinD gene amplification, cyclin-dependent kinase inhibitors loss (p16/INK4a), and inhibition of Rb by the binding of viral oncoproteins (E7 protein of HPV) can revoke the antiproliferative effect of Rb in cancer.

TP53 Tumor Suppressor Gene:

The TP53 tumor suppressor gene is also known as the ‘guardian of the genome’ as it serves to monitor for cellular stress like anoxia, identify DNA damage or inappropriate signaling by mutated oncoproteins. The TP53 gene encodes for the p53 protein, which controls the expression of proteins and their activity in cell cycle arrest, cellular senescence, DNA repair and apoptosis. Loss of p53 can cause continued cell replication despite DNA damage and failure to activate programmed cell death. The DNA damage is perceived by complexes comprising kinase of the ATM/ATR family. These kinases phosphorylate p53, releasing it from inhibitors such as MDM2. The active p53 unregulated the expressions of important proteins like cyclin-dependent kinase inhibitor p21, which causes G1-S checkpoint arrest of the cell cycle. In instances where the DNA damage is not repairable, p53 induces events like activating the BAX gene, which encodes a pro-apoptotic protein that finally lead to cellular apoptosis or senescence. It also works to inhibit the BCL2 anti-apoptotic gene and stimulates the release of cytochrome c from the mitochondria. Cytochrome c activates Caspases within the cell responsible for its eventual degradation. Similar to Rb,p53 can be inactivated by viral oncoproteins like the E6 protein of HPV, thus revoking the anti-proliferative and other important cellular effects. Most of the cancers demonstrate a biallelic loss of function mutation in TP53. Uncommon patients with Li-Fraumeni syndrome have a very high incidence of a wide variety of cancers like breast cancer, soft-tissue and bone sarcomas, and brain tumors since they inherit one defective copy of TP53.

Phosphatase andTension Homolog (PTEN) Gene:

PTEN Gene encodes a lipid phosphatase that negatively regulates the phosphoinositide-3-kinase (PI3K)-AKT and that target of mTOR signaling pathways. These pathways are vital for cell proliferation, cell cycle progression and apoptosis. The PTEN protein also functions to keep migration, adhesion and angiogenesis in check. It also plays a role in the overall stabilization of the genome. A biallelic loss of function is common in diverse cancers. Cowden syndrome is an autosomal dominant disorder resulting from germline loss-of -function mutations of this gene and correlates with a higher risk of breast and endometrial cancer.

CDH1 (E-cadherin):

Normal calls stop proliferating once they come into contact with neighbouring cells, which helps to maintain structure and architecture of the tissue, referred to as contact inhibition.Mediation of cell-to-cell contact in many tissues is the function of a group of proteins called cadherins. E-Cadherin (epithelial cadherin) regulates contact inhibition by binding to a key componenet of the WNT signaling pathway,β-catenin. This binding prevents E-cadherin from translocating to the nucleus of the cell, stopping it from activating transcription of pro-growth target genes.Overall, this interaction regulates the morphology and organization of epithelial cell linings. Autosomal dominant familial carcinoma is associated with a germline loss-of-function in this gene.

NF1 & NF2:

NF1 gene encodes for neurofibromin1, which is a GTPase that function as a negative regulator of RAS. A germline loss-of-function mutation of this gene cause Neurofibromatosis type1, an autosomal dominant disorder associated with the formation of neurofibromas, brain tumors like optic gliomas and malignant never sheath tumors peripherally.

 NF2 encode neurofibromin2 (also known as merlin) which is a cytoskeletal protein associated with contact inhibition.Loss of function mutation of this gene leads to neurofibromarosis type2, which is also an autosomal dominant disorder associated with an increased risk of bilateral schwannomas among other tumors.

BRCA1,BRCA2,PARP-1:

BRCA1&BRCA2 are tumor suppressor genes that encode proteins involved in the repair of DNA double-strand breaks through the homologous recombination repair pathway.PARP-1 encodes a protein that assists with the repair of single-stranded breaks in the DNA. Without functional proteins that repair DNA, the cell cycle continues to pass along defective and mutated genetic material that leads to aberrant daughter cells.

APC:

The APC gene encodes a tumor suppressor gene that negatively regulates the WNT(wingless and Int-1) signaling pathway.This regulation leads to enhancement of the formation of a complex that degrades β-catenin that is involved in the regulation and co-ordination of cell adhesion and gene transcription. The APC mutation is  presentr in familial adenomatous polyposis,  an autosomal dominat disorder where thousands of colonic polyps develop with early onset of colon carcinoma.The tumor development is associated with a loss of a single normal APC allele.

CDKN2A:

This Complex encodes two tumor suppressor proteins,p16/Ink4a and ARF, which augments Rb function and stabilizes p53, respectively.Loss of function germline mutations inn this gene occur in autosomal dominant familial melanoma. A biallelic loss-of -function presents in multiple cancers including,melanomas,leukemias and carcinomas.

WT1:

WT1 gene encodes for transcription factors required for normal genitourinary tissue development. Wilms tumor, Pediatric kidney cancer, is associated with a germline loss-of-function mutation in this gene. Sporadic Wilm’s tumor also correlates with similar WT1 mutations.

 PTCH1:

PTCH1 tumor suppressor gene encodes protein patched homolog1 that negatively regulated the hedgehog signaling pathway. Gorlin syndrome is an autosomal domination disorder that correlates with a germline loss-of function mutation in this gene and a high risk of developing basal cell carcinom and medulloblastoma.

 VHL:

VHL gene encodes a component of a ubiquitin ligase, which is involved in the degradation of hypoxia-induced factors (HIF8). These are transcription factors that alter the expression of genes is response to hypoxia. Von Hippel-Lindau syndrome, an autosomal dominant disorder is associated with the loss-of-functiongermline mutations of this gene and poses a high risk of developing renal cell carcinoma and pheochromocytoma.

Representative Tumor Suppressor Genes:

S.No.	Gene	Function	Familial cancer association	Other major tumor
1.	P53	Transcription factor	Li-Fraumeni Syndrome	>50% of cancer
2.	Rb	Transcriptional co repression	Retinoblasto–ma	Many
3.	INK4a(p16)	Cdk inhibitor (Rb activation)	Melanoma	Many
4.	ARF	Mdm2 antagonist(p53activat-ion)	Melanoma	Many
5.	APC	Wnt/Wingless signaling	Familial adenomatous polyposis	Colorectal cancer
6.	PTEN	Lipid phosphatase (phosphinositide metabolism)	Cowden syndrome	Glioblastoma, endomaterial thyroid and Prostate Cancer
7.	PTCH	Hedgehog signaling(receptor-r)	Basal cell nerve(Gorlin) syndrome	Medulloblastom-a, basal cell carcinoma
8.	NF1	GTPase activating protein for RAS	Neurofibromatosis	Sarcoma, glioma-s
9.	WT1	Transcription factor	Wilm’s tumor

Functions of Tumor Suppressor Gene Products:

1. The protein encoded by the PTEN tumor suppressor genes is an interesting example of antagonism between oncogene and tumor suppressor gene products. The PTEN protein is a lipid phosphate that dephosphorylates the 3 position of phosphatidylinositides , such as phosphatidylinositol 3,4,5- bisphosphate(PIP3). By dephosphorylating PIP3 , PTEN antagonize the activities of PIP3- kinase and Akt , which can act as oncogenes by promoting cell survival.

• Several tumor suppressor genes encode transcriptional regulatory proteins e.g. product of WT1, which is a repressor that appears to suppress transcription of a number of growth factor – inducible genes. One of the target of WT1 is thought to be the gene that encodes insulin-like growth factor II, which is over expressed in Wilm’s tumors and may contribute to tumor development by acting as an autocrine growth factor. Inactivation of WT1 may thus lead to abnormal growth factor expression, which in turn drives tumor cell proliferation.
• The products of the Rb and INK4 tumor suppressor genes regulate cell cycle progression at the same point as that affected by Cyclin D1 .Rb inhibits passage through the restriction point in G1 by repressing transcription of a number of genes involved in cell cycle progression and DNA synthesis.  In normal cells, passage through the restriction point is regulated by Cdk4/Cyclin D complexes, which phosphorylate and inactivate Rb. Mutational inactivation of Rb in tumors thus removes a key negative regulator of cell cycle progression. The INK4 tumor suppressor gene, which encodes the Cdk inhibitor p16, also regulates passage through the restriction point. Inactivation of INK4 therefore leads to elevated activity of Cdk/cyclinD complexes, resulting in uncontrolled phosphorylation of Rb.

• The p53 gene product regulates both cell cycle progression and apoptosis. DNA damage leads to rapid induction of p53, which activates transcription of the Cdk inhibitor p21. The inhibitor p21 blocks cell cycle progression, both by acting as a general inhibitor of Cdk/CyclinD complexes and by inhibiting DNA replication by binding to PCNA (Proliferating Cell Nuclear Antigen). The resulting cell cycle arrest presumably allows time for damaged DNA to be repaired before it is replicated. Loss of p53 prevents this damaged-induced cell cycle arrest, leading to increased mutation frequencies and a general instability of the cell genome. Such genetic instability is a common property of cancer cells, and it may contribute to further alterations in oncogenes and tumor suppressor genes during tumor progression.

•  In addition to mediating cell cycle arrest, p53 is required for apoptosis induced by DNA damage. Unrepaired DNA damage normally induces apoptosis of mammalian cells, a response that is presumably advantageous to the organism because it eliminates cell carrying potentially deleterious mutations. Cell lacking p53 fails to undergo apoptosis in response to agents that damage DNA, including radiations. This failure to undergo apoptosis in response to damage DNA contributes to the resistance of many tumors to chemotherapy.

In addition, loss of p53 appears to interfere with apoptosis induced by other stimuli, such as growth factor deprivation and oxygen deprivation. These effects of p53 inactivation on cell survival are thought to account for the high frequency of p53 mutations in human tumors.