Oncogenes and Cancer Case File

Oncogenes and Cancer Case File

Eugene C.Toy, MD, William E. Seifert, Jr., PHD, Henry W. Strobel, PHD, Konrad P. Harms, MD

❖ CASE 11

A 32-year-old female presents to your clinic with concerns over a recently detected right breast lump. A mammogram performed revealed a right breast mass measuring 3 cm with numerous microcalcifications suggestive of breast cancer. During your discussion with the patient, she revealed that she had a sister who was diagnosed with breast cancer at the age of 39, a mother who passed away with ovarian cancer at age 40 years, and a maternal aunt who had both breast and colon cancer. Patient underwent an examination which revealed a fixed and nontender breast mass on right side measuring 3 cm with mild right axillary lymphadenopathy. No skin involvement is noted. A biopsy was performed and revealed intraductal carcinoma.

◆ What cancer gene might be associated with this clinical scenario?

◆ What is the likely mechanism of the cancer gene in this case?

ANSWERS TO CASE 11: ONCOGENES AND CANCER

Summary: A 32-year-old female with strong family history of breast, colon, and ovarian cancer, who now presents with a fixed breast lesion that is biopsyproven carcinoma.

◆ Most likely cancer gene: Breast cancer (BRCA) gene

◆ Likely mechanism: Inhibition of tumor-suppressor gene

CLINICAL CORRELATION

This young woman has developed breast cancer at age 32 years. Moreover, she has two first-degree relatives with breast and/or ovarian cancer prior to menopause. This makes BRCA gene mutation likely. The BRCA1 gene resides on chromosome 17. This gene encodes a protein which most likely is important in deoxyribonucleic acid (DNA) repair. Thus, a mutation of the BRCA1 gene likely leads to abnormal cells propagating unchecked. A woman with a BRCA1 mutation has a 70 percent lifetime risk of developing breast cancer, and a 30 to 40 percent risk of ovarian cancer. The vast majority of breast cancer is not genetically based, but occurs sporadically. However, familial-based breast cancers are most common because of BRCA1 mutation. BRCA2 is another mutation that is more commonly associated with male breast cancer. Other genetic mechanisms of cancer include oncogenes, which are abnormal genes that cause cancer usually by mutations. Protooncogenes are normal genes that are present in normal cells and involved in normal growth and development, but if mutations occur, they may become oncogenes.

APPROACH TO ONCOGENES

Objectives

1. Know the definitions of oncogenes and protooncogenes.

2. Understand the role of promoter and repressor functions of DNA synthesis.

3. Know the normal DNA replication.

4. Be familiar with DNA mutations (point mutations, insertions, deletions).

5. Know the process of DNA repair.

6. Understand the recombination and transposition of genes.

Definitions

Okazaki fragment: Short segments of DNA (approximately 1000 nucleotides in prokaryotes, 100 to 200 nucleotides in eukaryotes) synthesized on the lagging strand during DNA replication. As the replication fork opens, the ribonucleic acid (RNA) polymerase primase synthesizes a short RNA primer, which is extended by DNA polymerase until it reaches the preceding Okazaki fragment. The RNA primer on the previous Okazaki fragment is removed, the gap filled in with DNA and the strands ligated.

Oncogene: Genes whose products are involved in the transformation of normal cells to tumor cells. Most oncogenes are mutant forms of normal genes (protooncogenes).

Primase: An RNA polymerase that synthesizes a short RNA primer complementary to a DNA template strand that is being replicated. This RNA primer serves as the starting point for addition of nucleotides in the replication of the strand serving as a template.

Recombination: Any process in which DNA strands are cleaved and rejoined resulting in an exchange of material between molecules of DNA.

Tumor suppressor genes: Genes that encode for proteins that normally inhibit the progress of a cell through the cell cycle. If these genes are mutated, a deficiency in the suppressor proteins creates unregulated cell growth, a condition permissive for tumorigenesis.

DISCUSSION

DNA replication normally occurs during the S phase of the cell cycle. DNA replication occurs in a semiconservative fashion and this is because of the intrinsic antiparallel nature of the double helix. All known DNA polymerases synthesize DNA in a 5' to 3' direction; this means that one strand will be continuously synthesized and the other must be made through a discontinuous mechanism and the production of Okazaki fragments. After this process is finished, the cell will divide the newly replicated material in mitosis. In some aberrant cases, the cell will replicate its DNA over and over again without performing an intervening mitosis. This generates cells with abnormally high content of DNA, an abnormal form of DNA replication control.

DNA replication begins by separating the parental DNA strands. This is accomplished by enzymes called helicases. They separate the strands and move along the strands in a fixed direction at the expense of ATP, opening up the strands so that the DNA polymerases can bind to them. To prevent reannealing of the two strands, single-stranded binding proteins bind the two complementary strands. The next step in the replication process is the laying down of an RNA primer, which is catalyzed by the enzyme primase. DNA polymerases extend the chain by adding deoxyribonucleotides to the 5' end of the RNA primer. It does this in a continuous fashion on the leading strand, but for the lagging strand, the one with its 5' end toward the replication fork, a series of short segments termed Okazaki fragments must be synthesized. The RNA primers of the Okazaki fragments are removed as the DNA polymerase reaches the previous Okazaki fragment and the DNA segments are then joined by DNA ligase.

DNA mutations arise from a variety of intrinsic and extrinsic factors. Our genome is constantly under the assault of various genotoxic agents such as ionizing radiation, oxygen free radicals, and UV light. These agents serve to introduce DNA double-strand breaks (DSBs) as well as thymine–

thymine dimers into the DNA. DSBs may generate deletion or insertion mutations and could alter the reading frame of the genetic code, an event that can easily lead to the malfunction of a protein. UV-induced dimers may generate point mutations that also alter the reading frame.

To respond to the various forms of DNA damage, cells have evolved a host of DNA repair mechanisms that serve to restore the genetic material. Depending on the nature of the DNA damage (DSB versus ultraviolet [UV]-induced thymine dimers, etc.), the cell will invoke a different mechanism of repair. In addition, the stage of the cell cycle at which the lesion is detected and processed can activate independent DNA repair pathways. For example, if a daughter chromatid template is present in S or G2 phase of the cell cycle, the cell will use this unperturbed partner molecule to fix a DSB. This process is referred to as homologous recombination (HR) and represents a major branch of the DNA repair process. However, if the damage occurs during the G1 phase of the cell cycle, a period devoid of an existing chromatid template that can be used for repair, a general end joining process will be used. This process, referred to as nonhomologous end joining (NHEJ), ligates the broken ends together with little to no regard for the loss of intervening sequences. Therefore, NHEJ is considered an error prone process, but given the large size of the genome and the presence of many forms of “junk DNA,” the NHEJ process may not necessarily disrupt DNA sequences that encode proteins. In contrast to NHEJ, HR is a process that is error free by virtue of the fact that the daughter chromatid is used as a template for repair.

Recombination and transposition of genes are two processes that mutate the genetic material. As discussed above, recombination is integral to the process of DNA repair. When DNA recombination is impaired through mutation of specific genes (like BRCA1 or Rad51), aberrant recombination takes

place, generating abnormal chromosomes that possess translocations from two or more chromosomes. Translocations transpose genes from one chromosomal environment to another and often this leads to a disruption in gene expression. Such events are known to cause gene amplification, a phenomenon often associated with cancer. They may also generate chromosomes that possess two centromeres (dicentrics), leading to a variety of cellular defects in mitosis.

DNA replication is tightly controlled by a variety of proteins that act to promote the process (i.e., DNA polymerases and cis-acting elements that bind to DNA and recruit factors involved in the process) as well as ones that inhibit the synthesis of DNA, either directly or indirectly. One factor that indirectly inhibits DNA synthesis is the p53 tumor suppressor. Tumor suppressors refer to a general class of proteins that function to slow and alter cell growth and development through a variety of mechanisms. In the absence of these factors, cells will have a reduced capacity to perform a variety of functions essential to maintain genomic stability. The p53 functions to control the G1 S boundary of the cell cycle, and if the cell is not prepared to enter S phase, then DNA synthesis will be negatively regulated.

A major cause of familial breast cancer results from mutation in the breast cancer susceptibility gene, BRCA1. Originally identified in 1994, it was not until 1997 when David Livingston and colleagues demonstrated that BRCA1 is a nuclear protein that its function began to be understood. They found that after treatment of cells with agents known to generate DNA damage (i.e., hydroxyurea, UV light, ionizing radiation), BRCA1 was found to localize to discrete nuclear structures typically called foci. Such foci are known to correlate with sites of DNA damage or the sites of stalled replication forks in S phase of the cell cycle. In addition to the striking nuclear localization of BRCA1 in response to DNA damaging agents, it was also observed that the BRCA1 protein was phosphorylated in response to these agents. Given this and the wealth of data obtained from studying the cell cycle in yeast model systems, it was concluded that BRCA1 functioned as a cell cycle regulator in response to DNA damage.

Further studies have implicated BRCA1 in the cellular response to DNA double-stranded breaks (DSBs), a potentially lethal form of DNA damage. Cells defective in BRCA1 possess numerous cytological and biological features that have been known for years to be correlated with perturbation in the maintenance of chromosome stability. This includes aneuploidy, centrosome amplification, spontaneous chromosome breakage, aberrant recombination events, sensitivity to ionizing radiation, and impaired cell cycle checkpoints. In addition, a variety of experiments have demonstrated roles for BRCA1 in enforcing the G2/M cell cycle transition, homologous recombination between sister chromatids, as well as the restart of stalled replication in S phase.

The BRCA1 tumor suppressor interacts with numerous cellular proteins in large complexes. This includes a variety of proteins implicated in various DNA repair and cell cycle processes. In fact, BRCA1 has been reported to interact with as many as 50 proteins! Moreover, BRCA1 has been shown to function in various transcriptional mechanisms, suggesting that the function of this important protein may go well beyond its well-documented role in DSB repair. How the inactivation of BRCA1, a gene that appears to operate in DNA repair pathways that appear generic to various cell types and predisposes women to inherited forms of breast cancer, remains a mystery and the subject of much debate. Given that BRCA1 appears to have key roles in transcriptional regulation, it has been suggested that BRCA1 could influence mammary tissue through pathways that impinge on the biology of estrogen and estrogen-related metabolites. In addition, it has been suggested that breast tissue differs from

other tissues in the types of DNA repair processes that are used. Perhaps there are redundant DNA repair pathways in non-mammary tissue. In any event, the complex behavior of BRCA1 promises to challenge future researchers as they search for the underlying mechanisms that initiate familial breast cancer.

COMPREHENSION QUESTIONS

[11.1] Hereditary retinoblastoma is a genetic disease that is inherited as an autosomal dominant trait. Patients with hereditary retinoblastoma develop tumors of the retina early in life, usually in both eyes. The affected gene (RB1) was the first tumor suppressor gene to be identified. Which of the following best describes the function of the protein encoded by the RB1 gene?

A. It binds transcription factors required for expression of DNA replication enzymes.

B. It allosterically inhibits DNA polymerase.

C. It binds to the promoter region of DNA and prevents transcription.

D. It phosphorylates signal-transduction proteins.

[11.2] Mutations in the tumor suppressor gene BRCA1 are transmitted in an autosomal dominant fashion. When a cell is transformed to a tumor cell in individuals who have inherited one mutant allele of this tumor suppressor gene, which of the following most likely occurs?

A. A transcription factor is over expressed.

B. Deletion or mutation of the normal gene on the other chromosome.

C. Chromosomal translocation.

D. Gene duplication of the mutant gene.

[11.3] Women who inherit one mutant BRCA1 gene have a 60 percent chance of developing breast cancer by the age of 50. The protein produced by the BRCA1 gene has been found to be involved in the repair of DNA double-strand breaks. Which of the following processes is most likely to be adversely affected by a deficiency in the BRCA1 protein?

A. Removal of thymine dimers

B. Removal of RNA primers

C. Removal of carcinogen adducts

D. Homologous recombination

E. Correction of mismatch errors

Answers

[11.1] A. The RB1 protein binds to E2F transcription factors, preventing them from activating transcription of the genes encoding enzymes required for DNA replication, such as DNA polymerase. The RB1–E2F complex acts as a transcription repressor. Midway through G1 phase, RB1 is phosphorylated by cyclin-dependent kinases, releasing E2F to activate transcription. A deficiency of RB1 leads to unregulated transcription of DNA replicatory enzymes and DNA synthesis.

[11.2] B. Since BRCA1 is inherited in an autosomal dominant fashion, then one allele is sufficient to produce the BRCA1 protein. However, if the normal allele is somatically mutated or deleted, then the affected cell cannot produce the BRCA1 protein and can be transformed into a tumor cell.

[11.3] D. BRCA1 plays a significant role in the repair of double-strand breaks, therefore, since homologous recombination requires a cleavage of both strands of a DNA molecule, this event is most likely to be affected by a deficiency in this protein. Thymine dimers, mismatches, and adducts of DNA with carcinogens are effectively removed by a process of excision repair, in which a section of one strand of DNA is removed.

BIOCHEMISTRY PEARLS

❖ DNA replication normally occurs during the S phase of the cell cycle.

❖ DNA replication occurs in a semiconservative fashion and this is because of the intrinsic antiparallel nature of the double helix.

❖ All known DNA polymerases synthesize DNA in a 5′ to 3′ direction.

❖ To respond to the various forms of DNA damage, cells have evolved a host of DNA repair mechanisms that serve to restore the genetic code.

❖ Tumor suppressors refer to a general class of proteins that function to slow and alter cell growth and development through a variety of mechanisms. BRCA tumor suppressor gene when mutated, increases the risk of breast cancer.

❖ Inactivation of BRCA1, a gene that appears to operate in DNA repair pathways that appear generic to various cell types, predisposes women to inherited forms of breast cancer, but the exact mechanism has been elusive.

References

Couch FJ, Weber BL. Breast cancer. In: Scriver CR, Beaudet AL, Sly WS, et al., eds. The Metabolic and Molecular Basis of Inherited Disease, 8th ed. New York: McGraw-Hill, 2001:999–1031. 

Edenberg HJ. DNA replication, recombination, and repair. In: Devlin TM, ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley- Liss, 2002. 

Scully R Chen J, Ochs RL. Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damage. Cell 1997;90:425–35. 

Scully R, Livingston DM. In search of the tumor-suppressor functions of BRCA1 and BRCA2. Nature 2000;408:429–32.

HomeCase FilesOncogenes and Cancer Case File