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Genetic instability in urinary bladder cancer: An evolving hallmark N Wadhwa1, BB Mathew2, SK Jatawa1, A Tiwari11 School of Biotechnology, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, Madhya Pradesh, India 2 Department of Biotechnology, Sapthagiri College of Engineering, Bangalore, Karnataka, India
Correspondence Address: Source of Support: Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal,, Conflict of Interest: None DOI: 10.4103/0022-3859.123156
Bladder cancer is a major health-care concern. A successful treatment of bladder cancer depends on its early diagnosis at the initial stage. Genetic instability is an essential early step toward the development of bladder cancer. This instability is found more often at the chromosomal level than at the nucleotide level. Microsatellite and chromosomal instability markers can be used as a prognostic marker for screening bladder cancer. Bladder cancer can be distinguished in two different categories according to genetic instability: Cancers with chromosomal level instability and cancers with nucleotide level instability. Deoxyribonucleic acid (DNA) mismatch repair (MMR) system and its correlation with other biologic pathway, both are essential to understand the basic mechanisms of cancer development. Microsatellite instability occurs due to defects in DNA MMR genes, including human mutL homolog 1 and human mutL homolog 2. Chromosomal alterations including deletions on chromosome 3, 8, 9, 11, 13, 17 have been detected in bladder cancer. In the current review, the most recent literature of genetic instability in urinary bladder cancer has been summarized. Keywords: Chromosomal instability bladder cancer, genomic instability, microsatellite instability
Bladder cancer is one of the most common causes of death in India. [1] It is the fourth most common cancer in men and the eighth most common cancer in women. [2] It is seen largely in the middle and older age group. [3] It is characterized by recurrent hematuria, dysuria and increased frequency. [2] Associated risk factors include tobacco abuse, smoking, industrial carcinogens, occupational exposure and certain other non-occupational risk factors. [3],[4] Low-grade tumors (70-80%) are usually non-invasive and don't extend into the epithelial basement membrane and muscles while high grade tumors obtrude into the muscle resulting in mortality. [3] Clinical studies of transitional cell carcinoma (TCC) identify two tumor types: Superficial tumors (those that do not invade the lamina propria and are moderately differentiated) and invasive tumors (invading into and beyond the lamina propria). [5] Superficial bladder cancer is an early stage TCC, which can progress to invasive bladder cancer due to an increase in genetic instability. [1] Microsatellite instability (MSI) has been found in many cancers including bladder cancer, gall bladder cancer, ovarian cancer, colorectal cancer and has been used to detect tumor cells in bodily fluids. [6],[7],[8],[9] Continuous mutations activate oncogenes and disorganize tumor suppressor genes, which facilitates the process of oncogenesis. [10] Superficial bladder cancer at different stages and grades show distinct behavior due to genetic alteration. [1] Genomic instability is a characteristic feature of almost all major human tumors. Numerous genetic and molecular alterations occur in TCC of the bladder. [5] Genetic instability (spontaneous and mutagen - induced) can itself lead to the development of bladder cancer. [11] It causes unscheduled mutations in deoxyribonucleic acid (DNA) repair genes and occurs at two distinct levels - chromosomal level and nucleotide level [Figure 1]. [12] Instability at the nucleotide level occurs due to faulty DNA repair systems such as base excision repair and nucleotide excision repair and results in base substitutions, deletions or insertions of a few nucleotides. [13] Microsatellites are regions of repetitive DNA sequences found ubiquitously throughout the genome. [14] MSI is caused due to defects in mismatch repair (MMR) pathway. [13] MSI can also be used as a prognostic marker for the detection of many human cancers. [15]
Chromosomal instability (CIN) defines the existence of an accelerated rate of chromosomal alterations. Mutation in CIN genes results in gains or losses of whole chromosomes during cell division as well as inversions, deletions, duplications and translocations of large chromosomal segments. The effect of CIN results in aneuploidy and an increase in the rate of loss of heterozygosity (LOH). [16] LOH is the loss of a maternal or paternal allele in a tumor and are often accompanied by a gain of the opposite allele. For example - a tumor could lose the maternal chromosome 8 while duplicating the paternal chromosome 8, resulting in a normal chromosome 8 karyotype, but an abnormal chromosome 8 "allelotype" [Figure 2]. [17] CIN - related molecular markers can be used to predict progression of cancer. [15]
DNA MMR system plays a major role in upkeep of genomic stability. MMR removes mispaired nucleotides and insertion-deletion loops that may form during DNA replication. Insertion-deletion loops affect microsatellite sequences and involve gains or losses of short repeat units. In humans, at least six different MMR proteins are required for mismatch recognition. MutS alpha (MSH2-MSH6 heterodimer) recognizes and binds single base mispairs and insertion-deletion loops for their correction. MutS beta (MSH2-MSH3 heterodimer) also binds to insertion-deletion loops for their correction [Figure 3]. [18]
Similarly, MutL alpha (MLH1-PMS2 heterodimer) coordinates the interface between the mismatch recognition complex and other MMR proteins. It also recognizes and correct insertion deletion loops. MLH1 protein may also dimerise with PMS1 (its role is still unknown) and MLH3 protein (for the correction of insertion deletion loops). MSH4 and MSH5 are additional human MMR proteins required for meiotic recombination, but have no role in MMR system. [18],[19] Loss of MMR genes lead to genetic instability in the form of chromosomal or MSI. [20],[21] Mutation of MMR genes result in the insertion or deletion of microsatellite sequences. DNA hypermethylation inactivates these genes leading to MSI. MSI is also a marker for MMR deficiency. [22]
Microsatellites, also known as short tandem repeats are repeating sequences of 2-6 base pairs of DNA. Microsatellite analysis is a promising marker for the detection and prognosis of bladder cancer. [2] Mutations within microsatellite sequence can be used to identify the clonal evolution of tumor cells. [6] Abnormalities in microsatellites such as MSI and LOH are commonly found in tumor cells and can be detected by polymerase chain reaction (PCR). [2] MSI occurs due to defects in MMR genes, including human mutL homolog 1 and human mutL homolog 2. Reduced expression of MMR protein is linked to MSI in bladder cancer and can be used as a new biomarker for the detection of bladder cancer. [15],[23] Allelic loss of chromosome 9 is the most frequent event in 50-60% of bladder tumors. [14] LOH is frequently found on the chromosomal arms 4p, 8p, 9p, 11p and 17p and plays an important role in the development of bladder cancer. The reported sensitivity and specificity of this test is 85% and 90%, respectively. [24] In 80% of the cases, microsatellite analysis can indicate frequency of low-grade tumors. [2] MSI plays a significant role in evolution, initiation and progression in bladder tumors. [1] Deformity in MMR genes results in incorrect MMR causing replication errors and genetic imbalance. [1],[25] [Table 1] lists some microsatellite markers used for the detection of MSI in bladder cancer. [14],[26],[27]
There is a need to standardize MSI for clinical purpose as well as, new reliable methods must be developed for MSI testing, which are useful for determining MSI and its biological relationship to the disease. [28] MSI testing is an important marker for the detection of bladder cancer due to its increased sensitivity and methodological simplicity. [1] MSI can be used to examine different samples exposed to toxic compounds and risk factors. [29] Microsatellite markers can be used to figure reoccurrence of bladder tumor in a high risk population. After validating the results at a larger scale, particular set of microsatellite markers can be used to target high risk population. [30]
Oxidative damage to DNA causes chromosomal aberration leading to genetic modification in cells and tissues. Bladder cancer develops due to two different genetic pathways-one forming noninvasive, papillary tumors that reoccur frequently and the other resulting in the development of carcinoma in situ, invasive high-grade cancer. [3] Such genetic instability can be assessed by using karyotyping, microsatellite analysis for PCR based techniques, comparative genomic hybridization, fluorescence in situ hybridization (FISH) or single-nucleotide polymorphism arrays. [31] Preliminary alterations in chromosome are associated with tumor ontogeny while secondary chromosomal alterations leads to its growth to a more advanced stage. [1],[25] Chromosome 9 alterations are found in early stages of the urinary bladder cancer as LOH found in both the chromosomal arms does not depend on its stage and grade. Invasive bladder cancer is found to be associated with LOH at 11p, 3p, 13q, and 17p. [6] Genetic modification in these tumors involve deletion of 8p, 9p, 11p, 11q and Y chromosome arms and gain of 1q, 8q, 17q, and 20q chromosome arms [Table 2]. [30],[32],[33],[34],[35],[36] Mutations in p53 gene are observed in 50% of bladder cancer. The recurrence of genetic instability in invasive bladder cancer enhances the detection of urinary bladder urothelial cells in urine using FISH. [33]
Deletions on chromosome 3 Deletions on 3p chromosome arm have been examined in 25% cases of bladder. Deletion on this chromosome is most frequently observed at 3p12-14 in bladder cancer. At 3p14.2 region, alteration can take place during DNA replication stress, which leads to gaps or chromosome break at this site. [31] Reduced fragile histidine triad (FHIT) expression is associated with promoter hypermethylation leading to bladder cancer. [37] FHIT is a haploinsufficient tumor suppressor gene, but its role in the tumor formation is still not known. [34] Deletions on chromosome 8 Deletion on chromosome 8 in bladder carcinoma is mostly found in its short arm (8p) which recur at the rate of 25-50% in urinary bladder cancer. [31] Deletion mapping using PCR based techniques was used to determine deletions at 8p21-22 loci. [38] Four tumor suppressor genes are reported in this region. Fasciculation and elongation protein zeta 1/leucine zipper putative tumor suppressor 1 gene located on loci 8p22 gets inactivated by transcriptional silencing in bladder cancer. The methylation status of the leucine zipper putative tumor suppressor 1 (LZTS1) promoter has not been analyzed yet. Deleted in breast cancer 2 has low mutation rate as compared to LZTS1.[31] Deletions on chromosome 9 Chromosome 9 deletions have been found in 50% cases of bladder cancer. [6] Deactivation of tumor suppressor gene by deletion of chromosome 9 is a primary event in bladder carcinogenesis. Losses of chromosome arms are associated with progression of bladder cancer. Deletion on chromosome 9 is found at the early stages (T0 and T1) of bladder cancer while at subsequent stages, deletions on other chromosomes are also found. [31] High rate of genetic modification is found in all stages of bladder cancer. Chromosomes 9 losses like deletion of the P arm, deletion of the q arm, isochromosome 9q, monosomy of chromosome 9 are explained through molecular approaches in bladder cancer. [30] Deletion of chromosome 9 is frequently found in stage Ta grade I bladder tumors. [39] Inactivation of a reported tumor suppressor gene site on chromosome 9 is involved in the initiation of bladder cancer. [30] Five tumor suppressor genes loci have been reported on chromosome 9 which includes 9p21, 9q22.3, 9q31, 9q33, and 9q34 [Figure 4]. [31]
Deletion on 9p chromosome arm affects the cyclin-dependent kinase inhibitor 2A locus, resulting in deletion or promoter hypermethylation. The detection of tumor suppressor genes on 9q chromosome arm is possible due to the fact that in many cases, LOH are found on the entire chromosome arm. [35] Deletion in 9q22.3 region results in mutation of protein patched homolog gene in bladder cancer. At 9q31 region, no tumor suppressor gene is reported till date. The tumor suppressor gene in 9q33 region is deleted in bladder cancer chromosome region candidate 1. Deletions in 9q34 region results in the mutation of the prominent tumor suppressor tuberous sclerosis complex 1 gene leading to the formation of tuberous sclerosis complex. [31] Deletions on chromosome 11 Chromosome 11 deletions have been found in 71.43% cases of bladder cancer. Numerical changes of chromosome 11 including monosomy are commonly seen in bladder cancer. Loss of 11p chromosome arm results in the mutation of the H-RAs gene in bladder cancer. [40] Numerical aberrations on chromosome 11 results in the increase of cyclin-D copy number and its protein expression. [41] Chromosome 11 anomaly might reveal other genomic aberrations involved in the initiation and development of bladder cancer. [40] Deletions on chromosome 13 Chromosome 13 possesses the tumor suppressor gene retinoblastoma (Rb) at 13q14 locus. LOH is reported in 30% of all the cases and its association with the advancement of tumor stages as well as its recurrence has been described. [31] p53 and Rb mutations are seen in the same neoplasm and collectively increase the frequency of tumor occurrence and shortens the survival time. [36] Rb inactivation is caused due to high and low level of Rb expression. Increase expression of Rb results in its hyperphosphorylation due to which Rb is maintained in the cytoplasm. This blocks the inhibition of the E2F transcription factors by Rb in the nucleus, thereby terminating the cell cycle. [31] Deletions on chromosome 17 Loss of chromosome 17 has been found in 60% cases of bladder cancer. Numerical aberration on 17p chromosome arm is associated with the advancement of bladder cancer. The most important tumor suppressor gene on this chromosome is p53. [31] Mutations in p53 gene are associated with LOH at 17p13.1. Mutations in p53 gene are more commonly seen in high grade tumors than in low grade tumors. [42] Clinical trials need to be done to evaluate the effect of p53 mutation on results of therapy. Many other tumor suppressor genes are also found on chromosome 17p, but their role in bladder cancer remains unknown. [31]
Successful treatment of bladder cancer depends on its early detection. MSI is becoming an important and useful marker for risk assessment and prognosis. In order to use a particular set of microsatellite markers in screening high risk populations, there is a need to validate results seen on a much larger scale. Different combinations of chromosomal lesions and expression tumor markers can be used to classify different grades and stages of bladder cancer. Interpreting the effect of CIN will affirm the basic mechanism of development and progression of bladder cancer, eventually providing hope for those afflicted. Further large scale studies on this biomarker are required to make it clinically meaningful and evidence based.
The author would like to thank Dr. Swati Jain (Post-Doctoral Fellow, Anschutz Medical Campus Research Center, University of Colorado, Denver) for her constant encouragement throughout the study.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]
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