16.5: Tumor Suppressor Genes - Biology

16.5: Tumor Suppressor Genes - Biology

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Tumor suppressor genes normally do what would be expected from their name. Whereas the oncogenes mostly drive the cell cycle forward, the tumor suppressor genes’ primary functions are to temporarily stall the cell cycle so that DNA repair mechanisms can have time to work. However, if repair is unsuccessful after a few attempts, the tumor suppressor gene product may then trigger apoptosis rather than allow a damaged cell to replicate and potentially create another genetically damaged cell. Thus, the presence of an oncogene in a cell will not necessarily lead to development of cancer because a functioning tumor suppressor gene might prevent the cell from replicating. Equally, if a tumor suppressor gene is knocked out but there is no oncogene present, then the cell is unlikely to be immediately cancerous because although a cellular “emergency brake” is nonfunctional, if there is nothing to drive the cell through its cycle any faster or more frequently than usual, then the “brake” is never needed anyway.

Like oncogenes, tumor suppressor genes can work (or not work, as would be the case in cancer) in several ways. Here is an example with the breast cancer-associated genes, BRCA1 and BRCA2. These gene products are involved in DNA repair (chapter 7). When BRCA1 or BRCA2 is knocked out, the cell loses its ability to use that DNA repair pathway. There are other repair pathways, and even if there weren’t there may not be any serious lesions to the DNA, so the cell could behave normally for the time being. What is important from a cancer standpoint, is that each safety/repair mechanism that is lost increases the likelihood that an additional mutation may cause the cell to become cancerous.

It should be clear now how recessive loss-of-function mutations in a tumor suppressor gene can lead to an inherited predisposition to cancer. As diploid organisms, we have two copies of each gene in our cells, so losing one to mutation does not wipe out the protective function. Thus, if nothing happens to the other one, then the cell is fine. It is just a question of probability. Losing the function of one is a very low probability event, but the probability of losing both copies is extremely small. Thus, even though it is “only 1 step” on the way to losing the protection of this particular tumor suppressing function, it is a very large difference in probabilities. Of course, keep in mind that even complete loss of a single tumor suppressor gene is usually not enough to lead immediately to cancer, and still other mutations must occur to take advantage of the weakened cell defenses and push it towards a cancerous state.

The developmental biology of brain tumors

Tumors of the central nervous system (CNS) can be devastating because they often affect children, are difficult to treat, and frequently cause mental impairment or death. New insights into the causes and potential treatment of CNS tumors have come from discovering connections with genes that control cell growth, differentiation, and death during normal development. Links between tumorigenesis and normal development are illustrated by three common CNS tumors: retinoblastoma, glioblastoma, and medulloblastoma. For example, the retinoblastoma (Rb) tumor suppressor protein is crucial for control of normal neuronal differentiation and apoptosis. Excessive activity of the epidermal growth factor receptor and loss of the phosphatase PTEN are associated with glioblastoma, and both genes are required for normal growth and development. The membrane protein Patched1 (Ptc1), which controls cell fate in many tissues, regulates cell growth in the cerebellum, and reduced Ptc1 function contributes to medulloblastoma. Just as elucidating the mechanisms that control normal development can lead to the identification of new cancer-related genes and signaling pathways, studies of tumor biology can increase our understanding of normal development. Learning that Ptc1 is a medulloblastoma tumor suppressor led directly to the identification of the Ptc1 ligand, Sonic hedgehog, as a powerful mitogen for cerebellar granule cell precursors. Much remains to be learned about the genetic events that lead to brain tumors and how each event regulates cell cycle progression, apoptosis, and differentiation. The prospects for beneficial work at the boundary between oncology and developmental biology are great.

Tumour Suppressor Genes | Genetics

In this article we will discuss about the development of tumour suppressor genes.

Inactivation of tumour suppressor genes also contributes to development of tumours. Normally, tumour suppressor genes act to inhibit cell proliferation and tumour development. When these genes are inactivated or lost they lead to abnormal proliferation of tumour cells. The first tumour suppressor gene was identified through studies on retinoblastoma.

Through studies on patients with retinoblastoma that have survived, it was found that some cases of retinoblastoma are inherited, 50% of the children of an affected parent have a chance to develop retinoblastoma. According to Mendelian inheritance, this suggests transmission of retinoblastoma by a single dominant gene.

Although it is a dominant trait, inheritance of the gene is not sufficient to convert a normal retinal cell into a tumour cell. That is because tumour cells have further requirements in addition to inheriting the gene.

In 1971 Knudson found out that development of retinoblastoma requires two mutations that would make both copies of the gene, that is the Rb tumour suppressor gene, to become non-functional. Thus both alleles of Rb on the two homologous chromosomes must be inactivated to induce retinoblastoma. One defective copy of Rb is not sufficient for tumour development.

That Rb gene may be considered as a negative regulator of tumorigenesis was deduced from study of deletions in chromosomes 13 and 14 displaying loss of Rb gene. Gene mapping studies confirmed that the loss of normal alleles of Rb resulted in tumour development, suggesting Rb’s function as a tumour suppressor gene.

Gene transfer experiments made it clear that introduction of a normal Rb gene into retinoblastoma cells reverses their tumorigenecity, thus indicating activity of Rb as a tumor suppressor.

Later studies have shown that Rb is lost or inactivated in many other human cancers, such as bladder, breast and lung carcinomas. Additional tumour suppressor genes that contribute to development of tumours have subsequently been identified.

Studies show that tumour suppressor genes are involved in the development of both inherited and non-inherited cancers of humans. Mutations in the tumour suppressor genes appear to be the most common molecular alterations resulting in human tumour development.

Subsequently,p53 was identified as the second tumour suppressor gene that is inactivated in a wide variety of human cancers, including leukemia’s, lymphomas, brain tumours, sarcomas, and carcinomas of several tissues. Mutations in p53 are said to play a role in about 50% of all cancers, making it the most common target for genetic alterations in human cancers.

Cancer cells that have lost p53 function cannot undergo apoptosis and they become highly resistant to further treatment. This may be the primary reason why tumours that typically lack a functional p53 gene (example melanoma, colon cancer, prostate cancer, pancreatic cancer) respond much more poorly to radiation and chemotherapy than tumours that have a wild-type copy of this gene (example testicular cancer, childhood acute lymphoblastic leukemias).

Like p53, the INK4 and the PTEN tumour suppressor genes are also frequently mutated in several human cancers. Cancer of the colon may have mutation in two other tumour suppressor genes, namely, APC and MADR2. Additional tumour suppressor genes have been indicated in the development of brain tumours, pancreatic cancers and basal cell carcinoma of skin, as well as in several rare inherited cancers.

Survivin Protein and Cell Division:

Survivin is a recently discovered (1997) small-sized protein that is essential for cell divison and also acts as an inhibitor for apoptosis. Because of its involvement in promoting cell proliferation and preventing apoptosis, it is considered to be the protein that interfaces life and death.

Survivin has been found to be abundant in human cancers, where it has the potential as a prognostic marker for cancer, and is also a target for chemotherapy. The survivin gene, about 15 kb long is located on chromosome 17 at position q25.

Survivin is expressed in embryos and juveniles, but has not been detected in quiescent cells and terminally differentiated adult tissue. In actively proliferating cells, survivin expression is regulated through the cell cycle, such that it is absent in G1 and S phases but with a peak level in G2 and in mitosis.

Attempts to localise survivin in proliferating HeLa cells using fluorochromes have indicated presence of survivin during prophase to prometaphase stages of cell division at the centromeres and associated with the microtubules.

Not much is known about its prognostic importance. Importantly however, is the finding that survivin is expressed in many human malignancies, both solid and haematological and seems to be one of the most tumour-specific of all human gene products.

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Functions of Tumor Suppressor Gene Products

In contrast to proto-oncogene and oncogene proteins, the proteins encoded by most tumor suppressor genes inhibit cell proliferation or survival. Inactivation of tumor suppressor genes therefore leads to tumor development by eliminating negative regulatory proteins. In several cases, tumor suppressor proteins inhibit the same cell regulatory pathways that are stimulated by the products of oncogenes.

The protein encoded by the PTEN tumor suppressor gene is an interesting example of antagonism between oncogene and tumor suppressor gene products (Figure 15.37). The PTEN protein is a lipid phosphatase that dephosphorylates the 3 position of phosphatidylinositides, such as phosphatidylinositol 3,4,5-bisphosphate (PIP3). By dephosphorylating PIP3, PTEN antagonizes the activities of PI 3-kinase and Akt, which can act as oncogenes by promoting cell survival. Conversely, inactivation or loss of the PTEN tumor suppressor protein can contribute to tumor development as a result of increased levels of PIP3, activation of Akt, and inhibition of programmed cell death.

Figure 15.37

Suppression of cell survival by PTEN. The tumor suppressor protein PTEN is a lipid phosphatase that dephosphorylates PIP3 at the 3 position of inositol, yielding PIP2. PTEN thus counters the action of the oncogenes PI 3-kinase and Akt, which promote cell (more. )

Proteins encoded by both oncogenes and tumor suppressor genes also function in the Hedgehog signaling pathway (see Figure 13.44). The receptor Smoothened is an oncogene in basal cell carcinomas, whereas Patched (the negative regulator of Smoothened) is a tumor suppressor gene. In addition, the Gli proteins (the mammalian homologs of the Drosophila Ci transcription factor activated by Smoothened) were first identified as the products of an amplified oncogene.

Several tumor suppressor genes encode transcriptional regulatory proteins. A good example is provided by the product of WT1, which is frequently inactivated in Wilms' tumors (a childhood kidney tumor). The WT1 protein is a repressor that appears to suppress transcription of a number of growth factor-inducible genes. One of the targets of WT1 is thought to be the gene that encodes insulin-like growth factor II, which is overexpressed in Wilms' 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. Two other tumor suppressor genes, DPC4 and MADR2, encode SMAD family transcription factors that are activated by TGF-β signaling and lead to inhibition of 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 (Figure 15.38). 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 (see Figure 14.20). 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. As discussed in Chapter 14, p16 inhibits Cdk4/cyclin D activity. Inactivation of INK4 therefore leads to elevated activity of Cdk4/cyclin D complexes, resulting in uncontrolled phosphorylation of Rb.

Figure 15.38

Inhibition of cell cycle progression by Rb and p16. Rb inhibits progression past the restriction point in G1. Cdk4/cyclin D complexes promote passage through the restriction point by phosphorylating and inactivating Rb. The activity of Cdk4/cyclin D is (more. )

The p53 gene product regulates both cell cycle progression and apoptosis (Figure 15.39). DNA damage leads to rapid induction of p53, which activates transcription of the Cdk inhibitor p21 (see Figure 14.21). The inhibitor p21 blocks cell cycle progression, both by acting as a general inhibitor of Cdk/cyclin 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 damage-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.

Figure 15.39

Action of p53. Wild-type p53 is required for both cell cycle arrest and apoptosis induced by DNA damage.

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 cells carrying potentially deleterious mutations (e.g., cells that might develop into cancer cells). Cells lacking p53 fail to undergo apoptosis in response to agents that damage DNA, including radiation and many of the drugs used in cancer chemotherapy. This failure to undergo apoptosis in response to DNA damage 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.

Slow-Acting Carcinogenic Retroviruses Can Activate Cellular Proto-Oncogenes

Because its genome carries the v-src oncogene, Rous sarcoma virus induces tumors within days. Most oncogenic retroviruses, however, induce cancer only after a period of months or years. The genomes of the slow-acting retroviruses differ from those of transducing viruses such as RSV in one crucial respect: they lack an oncogene. Thus, slow-acting, or “long latency,” retroviruses have no direct affect on growth of cells in culture.

The mechanism by which avian leukosis viruses cause cancer appears to operate in all slow-acting retroviruses. Like other retroviruses, avian leukosis virus DNA generally integrates into cellular chromosomes more or less at random. However, the finding that the site of integration in the cells from tumors caused by these viruses is near the c-myc gene suggested that these slow-acting viruses cause disease by activating expression of c-Myc. As noted earlier, c-Myc is required for transcription of many genes that encode cellcycle proteins. These viruses act slowly both because integration near c-myc is a random, rare event and because additional mutations have to occur before a full-fledged tumor becomes evident.

In some tumors, the avian leukosis proviral DNA is found at the 5′ end of the myc gene in the same transcriptional orientation. In such cases, the right-hand LTR of the integrated retrovirus — which usually serves as a terminator — is believed to act as a promoter, initiating synthesis of RNA transcripts from the c-myc gene (Figure 24-10a). In other tumors, the proviral DNA is found in the opposite transcriptional orientation in this case, it is thought to exert an indirect enhancer activity (Figure 24-10b). Whether the inserted proviral DNA acts as a promoter or enhancer of c-myc transcription, the expressed c-Myc protein apparently is perfectly normal. The enhanced level of c-Myc resulting from the strong promoting or enhancing activity of the retroviral LTR partly explains the oncogenic effect of avian leukosis viruses. A second aspect is that c-myc expression is usually down-regulated when cells are induced to differentiate, but the LTR-driven expression of c-myc does not respond to such signals, and thus cells that normally would differentiate instead undergo DNA replication and cell division. These mechanisms of oncogene activation —�lled promoter insertion and enhancer insertion — operate in a variety of oncogenes and have been implicated in many animal tumors induced by slow-acting retroviruses.

Figure 24-10

Activation of the c-myc proto-oncogene by retroviral promoter and enhancer insertions. (a) The promoter can be activated when the retrovirus inserts upstream (5′) of the c-myc exons. The right-hand LTR may then act as a promoter if the provirus (more. )

In natural bird and mouse populations, slow-acting retroviruses are much more common than oncogenecontaining retroviruses such as Rous sarcoma virus. Thus, insertional oncogene activation is probably the major mechanism whereby retroviruses cause cancer.

Tumor Suppressor genes

Tumor-suppressor genes generally encode proteins that in one way or another inhibit cell proliferation.Five broad classes of proteins are generally recognized as being encoded by tumor-suppressor genes:

  1. Intracellular proteins that regulate or inhibit progression through a specific stage of the cell cycle (e.g., p16 and Rb, retinoblastoma).
  2. Receptors or signal transducers for secreted hormones or developmental signals that inhibit cell proliferation (e.g., TGF-β, the hedgehog receptor patched).
  3. Checkpoint-control proteins that arrest the cell cycle if DNA is damaged or chromosomes are abnormal (e.g., p53).
  4. Proteins that promote apoptosis.
  5. Enzymes that participate in DNA repair. Although DNA-repair enzymes do not directly inhibit cell proliferation, cells that have lost the ability to repair errors, gaps, or broken ends in DNA accumulate mutations in many genes, including those that are critical in controlling cell growth and proliferation. Thus loss-of-function mutations in the genes encoding DNA-repair enzymes prevent cells from correcting mutations that inactivate tumor suppressor genes or activate oncogenes. Generally one copy of a tumor-suppressor gene suffices to control cell proliferation, therefore, both alleles of a tumor suppressor gene must be lost or inactivated in order to promote tumor development. Thus, oncogenic loss-of function mutations in tumor-suppressor genes are genetically recessive. In many cancers, tumor-suppressor genes have deletions or point mutations that prevent production of any protein orlead to production of a non functional protein Examples of tumor suppressor genes

i) Rb (Retinoblastoma)

Rb is a tumor suppressor gene. The Rb protein controls cell cycle moving past the G1 checkpoint. Rb protein binds to the regulatory transcription factor E2F. The factor E2F is required for synthesis of replication enzymes. Upon binding of Rb to E2F,no transcription/ replication can take place. Rb restricts the cell’s ability to replicate DNA by preventing its progression from the G1 (first gap phase) to S (synthesis phase) phase of the cell division cycle.Rb is phosphorylated to pRb by certain Cyclin Dependent Kinases (CDKs). The phosphorylated/ mutated form of Rb (pRb) is unable to complex E2F and therefore, unable to restrict progression from the G1 phase to the S phase of the cell cycle. When E2F is free it activates factors like cyclins (e.g. Cyclin E and A), which push the cell through the cell cycle by activating cyclin-dependent kinases, this lead to cell division and progression of cancer.

Tumor protein p53, also known as p53, cellular tumor antigen p53, phosphoprotein p53 is a tumor suppressor gene. The name p53 is in reference to its apparent molecular mass: SDS-PAGE analysis indicates that it is a 53- kilodalton (kDa) protein. p53 has many mechanisms of anticancer function, and also plays a role in apoptosis, genomic stability, and inhibition of angiogenesis.p53 acts as a transcription factor for gene p21. It activates p21 which in turn bindsto CDK I (cyclin dependent kinase 1). When p21is complexed with CDK1 the cell cannot continue to the next stage of cell division. A mutant p53 can not bind DNA in an effective way, and, as a consequence, the p21 protein will not be available to act as the “stop signal” for cell division. This leads to uncontrolled cell proliferation and cancer. More than 50% cases of cancer involve mutation of p53 gene.

Tumor supressor genes

A Tumor Suppressor Enhancer of PTEN in T-cell development and leukemia

Long-range oncogenic enhancers play an important role in cancer. Yet, whether similar regulation of tumor suppressor genes is relevant remains unclear. Loss of expression of PTEN is associated with the pathogenesis of various cancers, including T-cell leukemia (T-ALL). Here, we identify a highly conserved distal enhancer (PE) that interacts with the PTEN promoter in multiple hematopoietic populations, including T-cells, and acts as a hub of relevant transcription factors in T-ALL. Consistently, loss of PE leads to reduced PTEN levels in T-ALL cells. Moreover, PE-null mice show reduced Pten levels in thymocytes and accelerated development of NOTCH1-induced T-ALL. Furthermore, secondary loss of PE in established leukemias leads to accelerated progression and a gene expression signature driven by Pten loss. Finally, we uncovered recurrent deletions encompassing PE in T-ALL, which are associated with decreased PTEN levels. Altogether, our results identify PE as the first long-range tumor suppressor enhancer directly implicated in cancer.

Keywords: NOTCH1 PTEN T-ALL T-cell acute lymphoblastic leukemia enhancer.

Conflict of interest statement

Conflict of interest disclosure: The authors declare no competing financial interests.


Identification of PE, a PTEN…

Identification of PE, a PTEN enhancer in T-ALL. A, H3K27ac Hi-ChIP, 4C-seq, ChIP-seq,…

Functional characterization of the PE…

Functional characterization of the PE enhancer. A, Luciferase reporter activity in JURKAT T-ALL…

PE-deficient mice show reduced Pten…

PE-deficient mice show reduced Pten levels in the thymus. A-C, Pten and/or Rnls…

Effects of PE loss in thymic T-cell development. A, Thymus weight in 6-week-old…

PE loss leads to accelerated…

PE loss leads to accelerated NOTCH1-induced T-ALL development. A, Schematic of retroviral-transduction protocol…

Secondary loss of PE leads…

Secondary loss of PE leads to accelerated NOTCH1-induced T-ALL progression and reduced levels…

The molecular biology of cervical cancer

Cervical cancer remains a major worldwide health problem, especially in developing countries. Over the last few decades many advancements have been made in determining the molecular genetics of the development of cancer. This paper attempts to summarize the major disturbances in cellular function known to date to play a role in the development of cervical cancer. The role of human papillomavirus (HPV) infection in the development of cervical cancer is a major player in the genetic abnormalities described thus far. The effects of HPV E6 and E7 on important cell cycle genes are discussed. As oncogenes and tumor suppressor genes have been described in the different types of cancer, their possible role in cervical cancer has been investigated. The possible role of angiogenesis and angiogenic factors is described. Because of the importance of HPV infection in the development of cervical cancer, the role of the body's immune function in this cancer is also under study, and the results of these findings are summarized. Although a complete paradigm of the development of cervical cancer from normal cervical epithelium is not yet known, continued study in this area will hopefully lead to a defined progression of molecular and immunologic abnormalities that cause the disease. The goal would be to use this information to help prevent and/or treat cervical cancer in the future.


The discovery of oncogenes and their ability to deregulate cellular processes related to cell proliferation and development appeared first in the literature as opposed to the idea of tumor suppressor genes. [5] However, the idea of genetic mutation leading to increased tumor growth gave way to another possible genetic idea of genes playing a role in decreasing cellular growth and development of cells. This idea was not solidified until experiments by Henry Harris were conducted with somatic cell hybridization in 1969. [6]

Within Dr. Harris’s experiments, tumor cells were fused with normal somatic cells to make hybrid cells. Each cell had chromosomes from both parents and upon growth, a majority of these hybrid cells did not have the capability of developing tumors within animals. [6] The suppression of tumorigenicity in these hybrid cells prompted researchers to hypothesize that genes within the normal somatic cell had inhibitory actions to stop tumor growth. [6] This initial hypothesis eventually lead to the discovery of the first classic tumor suppressor gene by Alfred Knudson, known as the Rb gene, which codes for the retinoblastoma tumor suppressor protein. [5]

Alfred Knudson, a pediatrician and cancer geneticist, proposed that in order to develop retinoblastoma, two allelic mutations are required to lose functional copies of both the Rb genes to lead to tumorigenicity. [6] Knudson observed that retinoblastoma often developed early in life for younger patients in both eyes, while in some rarer cases retinoblastoma would develop later in life and only be unilateral. [5] This unique development pattern allowed Knudson and several other scientific groups in 1971 to correctly hypothesize that the early development of retinoblastoma was caused by inheritance of one loss of function mutation to an RB germ-line gene followed by a later de novo mutation on its functional Rb gene allele. The more sporadic occurrence of unilateral development of retinoblastoma was hypothesized to develop much later in life due to two de novo mutations that were needed to fully lose tumor suppressor properties. [5] This finding formed the basis of the two-hit hypothesis. In order to verify that the loss of function of tumor suppressor genes causes increased tumorigenicity, interstitial deletion experiments on chromosome 13q14 were conducted to observe the effect of deleting the loci for the Rb gene. This deletion caused increased tumor growth in retinoblastoma, suggesting that loss or inactivation of a tumor suppressor gene can increase tumorigenicity. [6]

Unlike oncogenes, tumor suppressor genes generally follow the two-hit hypothesis, which states both alleles that code for a particular protein must be affected before an effect is manifested. [7] If only one allele for the gene is damaged, the other can still produce enough of the correct protein to retain the appropriate function. In other words, mutant tumor suppressor alleles are usually recessive, whereas mutant oncogene alleles are typically dominant.

Proposed by A.G. Knudson for cases of retinoblastoma. [7] He observed that 40% of U.S cases were caused by a mutation in the germ-line. However, affected parents could have children without the disease, but the unaffected children became parents of children with retinoblastoma. [8] This indicates that one could inherit a mutated germ-line but not display the disease. Knudson observed that the age of onset of retinoblastoma followed 2nd order kinetics, implying that two independent genetic events were necessary. He recognized that this was consistent with a recessive mutation involving a single gene, but requiring bi-allelic mutation. Hereditary cases involve an inherited mutation and a single mutation in the normal allele. [8] Non-hereditary retinoblastoma involves two mutations, one on each allele. [8] Knudson also noted that hereditary cases often developed bilateral tumors and would develop them earlier in life, compared to non-hereditary cases where individuals were only affected by a single tumor. [8]

There are exceptions to the two-hit rule for tumor suppressors, such as certain mutations in the p53 gene product. p53 mutations can function as a dominant negative, meaning that a mutated p53 protein can prevent the function of the natural protein produced from the non-mutated allele. [9] Other tumor-suppressor genes that do not follow the two-hit rule are those that exhibit haploinsufficiency, including PTCH in medulloblastoma and NF1 in neurofibroma. Another example is p27, a cell-cycle inhibitor, that when one allele is mutated causes increased carcinogen susceptibility. [10]

The proteins encoded by most tumor suppressor genes inhibit cell proliferation or survival. Inactivation of tumor suppressor genes therefore leads to tumor development by eliminating negative regulatory proteins. In most cases, tumor suppressor proteins inhibit the same cell regulatory pathways that are stimulated by the products of oncogenes. [11] While tumor suppressor genes have the same main function, they have various mechanisms of action, that their transcribed products perform, which include the following: [12]

  1. Intracellular proteins, that control gene expression of a specific stage of the cell cycle. If these genes are not expressed, the cell cycle does not continue, effectively inhibiting cell division. (e.g., pRB and p16) [13]
  2. Receptors or signal transducers for secreted hormones or developmental signals that inhibit cell proliferation (e.g., transforming growth factor (TGF)-β and adenomatous polyposis coli (APC)). [14]
  3. Checkpoint-control proteins that trigger cell cycle arrest in response to DNA damage or chromosomal defects (e.g., breast cancer type 1 susceptibility protein (BRCA1), p16, and p14). [15]
  4. Proteins that induce apoptosis. If damage cannot be repaired, the cell initiates programmed cell death to remove the threat it poses to the organism as a whole. (e.g., p53). [16] . Some proteins involved in cell adhesion prevent tumor cells from dispersing, block loss of contact inhibition, and inhibit metastasis. These proteins are known as metastasis suppressors. (e.g., CADM1) [17][18]
  5. Proteins involved in repairing mistakes in DNA. Caretaker genes encode proteins that function in repairing mutations in the genome, preventing cells from replicating with mutations. Furthermore, increased mutation rate from decreased DNA repair leads to increased inactivation of other tumor suppressors and activation of oncogenes. [19] (e.g., p53 and DNA mismatch repair protein 2 (MSH2)). [20]
  6. Certain genes can also act as tumor suppressors and oncogenes. Dubbed Proto-oncogenes with Tumor suppressor function, these genes act as “double agents” that both positively and negatively regulate transcription. (e.g., NOTCH receptors, TP53 and FAS). [21]

Scientists Shahjehan A. Wajed et al. state the expression of genes, including tumor suppressors, can be altered through biochemical alterations known as DNA methylation. [22] Methylation is an example of epigenetic modifications, which commonly regulate expression in mammalian genes. The addition of a methyl group to either histone tails or directly on DNA causes the nucleosome to pack tightly together restricting the transcription of any genes in this region. This process not only has the capabilities to inhibit gene expression, it can also increase the chance of mutations. Stephen Baylin observed that if promoter regions experience a phenomenon known as hypermethylation, it could result in later transcriptional errors, tumor suppressor gene silencing, protein misfolding, and eventually cancer growth. Baylin et al. found methylation inhibitors known as azacitidine and decitabine. These compounds can actually help prevent cancer growth by inducing re-expression of previously silenced genes, arresting the cell cycle of the tumor cell and forcing it into apoptosis. [23]

There are further clinical trials under current investigation regarding treatments for hypermethylation as well as alternate tumor suppression therapies that include prevention of tissue hyperplasia, tumor development, or metastatic spread of tumors. [24] The team working with Wajed have investigated neoplastic tissue methylation in order to one day identify early treatment options for gene modification that can silence the tumor suppressor gene. [25] In addition to DNA methylation, other epigenetic modifications like histone deacetylation or chromatin-binding proteins can prevent DNA polymerase from effectively transcribing desired sequences, such as ones containing tumor suppressor genes.

Gene therapy is used to reinstate the function of a mutated or deleted gene type. When tumor suppressor genes are altered in a way that results in less or no expression, several severe problems can arise for the host. This is why tumor suppressor genes have commonly been studied and used for gene therapy. The two main approaches used currently to introduce genetic material into cells are viral and non-viral delivery methods. [25]

Viral methods Edit

The viral method of transferring genetic material harnesses the power of viruses. [25] By using viruses that are durable to genetic material alterations, viral methods of gene therapy for tumor suppressor genes have shown to be successful. [26] In this method, vectors from viruses are used. The two most commonly used vectors are adenoviral vectors and adeno-associated vectors. In vitro genetic manipulation of these types of vectors is easy and in vivo application is relatively safe compared to other vectors. [25] [27] Before the vectors are inserted into the tumors of the host, they are prepared by having the parts of their genome that control replication either mutated or deleted. This makes them safer for insertion. Then, the desired genetic material is inserted and ligated to the vector. [26] In the case with tumor suppressor genes, genetic material which encodes p53 has been used successfully, which after application, has shown reduction in tumor growth or proliferation. [27] [28]

Non-viral methods Edit

The non-viral method of transferring genetic material is used less often than the viral method. [25] [27] However, the non-viral method is a more cost-effective, safer, available method of gene delivery not to mention that non-viral methods have shown to induce fewer host immune responses and possess no restrictions on size or length of the transferable genetic material. [25] Non-viral gene therapy uses either chemical or physical methods to introduce genetic material to the desired cells. [25] [27] The chemical methods are used primarily for tumor suppressor gene introduction and are divided into two categories which are naked plasmid or liposome-coated plasmids. [27] The naked plasmid strategy has garnered interest because of its easy to use methods. [25] Direct injection into the muscles allows for the plasmid to be taken up into the cell of possible tumors where the genetic material of the plasmid can be incorporated into the genetic material of the tumor cells and revert any previous damage done to tumor suppressor genes. [25] [27] The liposome-coated plasmid method has recently also been of interest since they produce relatively low host immune response and are efficient with cellular targeting. [27] The positively charged capsule in which the genetic material is packaged helps with electrostatic attraction to the negatively charged membranes of the cells as well as the negatively charged DNA of the tumor cells. [25] [27] In this way, non-viral methods of gene therapy are highly effective in restoring tumor suppressor gene function to tumor cells that have either partially or entirely lost this function.

Limitations Edit

The viral and non-viral gene therapies mentioned above are commonly used but each has some limitations which must be considered. The most important limitation these methods have is the efficacy at which the adenoviral and adeno-associated vectors, naked plasmids, or liposome-coated plasmids are taken in by the host’s tumor cells. If proper uptake by the host’s tumor cells is not achieved, re-insertion introduces problems such as the host’s immune system recognizing these vectors or plasmids and destroying them which impairs the overall effectiveness of the gene therapy treatment further. [28]

Gene Original Function Two-Hit? Associated Carcinomas
Rb DNA Replication, cell division and death Yes Retinoblastoma [5]
p53 Apoptosis No [ citation needed ] Half of all known malignancies [5]
VHL Cell division, death, and differentiation Yes Kidney Cancer [25]
APC DNA damage, cell division, migration, adhesion, death Yes Colorectal Cancer [25]
BRCA2 Cell division and death, and repair of double-stranded DNA breaks Yes Breast/Ovarian Cancer [5]
NF1 Cell differentiation, division, development, RAS signal transduction No Nerve tumors, Neuroblastoma [25]
PTCH Hedgehog signaling No Medulloblastoma, Basal Cell Carcinoma [5]
  • Retinoblastoma protein (pRb). pRb was the first tumor-suppressor protein discovered in human retinoblastoma however, recent evidence has also implicated pRb as a tumor-survival factor. RB1 gene is a gatekeeper gene that blocks cell proliferation, regulates cell division and cell death. [8] Specifically pRb prevents the cell cycle progression from G1 phase into the S phase by binding to E2F and repressing the necessary gene transcription. [29] This prevents the cell from replicating its DNA if there is damage.
  • p53.TP53, a caretaker gene, encodes the protein p53, which is nicknamed "the guardian of the genome". p53 has many different functions in the cell including DNA repair, inducing apoptosis, transcription, and regulating the cell cycle. [30] Mutated p53 is involved in many human cancers, of the 6.5 million cancer diagnoses each year about 37% are connected to p53 mutations. [30] This makes it a popular target for new cancer therapies. Homozygous loss of p53 is found in 65% of colon cancers, 30–50% of breast cancers, and 50% of lung cancers. Mutated p53 is also involved in the pathophysiology of leukemias, lymphomas, sarcomas, and neurogenic tumors. Abnormalities of the p53 gene can be inherited in Li-Fraumeni syndrome (LFS), which increases the risk of developing various types of cancers.
  • BCL2.BCL2 is a family of proteins that are involved in either inducing or inhibiting apoptosis. [31] The main function is involved in maintaining the composition of the mitochondria membrane, and preventing cytochrome c release into the cytosol. [31] When cytochrome c is released from the mitochondria it starts a signaling cascade to begin apoptosis. [32]
  • SWI/SNF. SWI/SNF is a chromatin remodeling complex, which is lost in about 20% of tumors. [33] The complex consists of 10-15 subunits encoded by 20 different genes. [33] Mutations in the individual complexes can lead to misfolding, which compromises the ability of the complex to work together as a whole. SWI/SNF has the ability move nucleosomes, which condenses DNA, allowing for transcription or block transcription from occurring for certain genes. [33] Mutating this ability could cause genes to be turned on or off at the wrong times.

As the cost of DNA sequencing continues to diminish, more cancers can be sequenced. This allows for the discovery of novel tumor suppressors and can give insight on how to treat and cure different cancers in the future. Other examples of tumor suppressors include pVHL, APC, CD95, ST5, YPEL3, ST7, and ST14, p16, BRCA2. [34]