Date Published: June 21, 2012
Publisher: Hindawi Publishing Corporation
Author(s): Han Han, Erika M. Wolff, Gangning Liang.
Urothelial carcinoma (UC), the most common type of bladder cancer, is one of the most expensive malignancies to treat due to its high rate of recurrence. The characterization of the genetic alterations associated with UC has revealed the presence of two mutually exclusive molecular pathways along which distinct genetic abnormalities contribute to the formation of invasive and noninvasive tumors. Here, we focus on the epigenetic alterations found in UC, including the presence of an epigenetic field defect throughout bladders with tumors. A distinct hypomethylation pattern was found in noninvasive tumors, whereas widespread hypermethylation was found in invasive tumors, indicating the two pathways given rise to two tumor types also differ epigenetically. Since certain epigenetic alterations precede histopathological changes, they can serve as excellent markers for the development of diagnostic, prognostic, and surveillance tools. In addition, their dynamic nature and reversibility with pharmacological interventions open new and exciting avenues for therapies. The epigenetic abnormalities associated with UC would make it an excellent target for epigenetic therapy, which is currently approved for the treatment of a few hematological malignancies. Future research is needed to address efficacy and potential toxicity issues before it can be implemented as a therapeutic strategy for solid tumors.
Bladder cancer is one of the most commonly diagnosed malignancies in the United States, with an estimated number of 73,510 new cases and 14,880 deaths in 2012 . Worldwide, bladder cancer is the seventh most common malignancy . The risk factors associated with development of bladder cancer include cigarette-smoking, exposure to chemicals, such as aromatic amines, chronic bladder inflammation, genetic predisposition, and age [3, 4]. In the United States, more than 90% of bladder tumors are diagnosed as urothelial carcinoma (UC), 5% as squamous-cell carcinoma (SCC), and 2% as adenocarcinomas . In countries, where chronic urinary infection by Schistosoma haematobium is prevalent, most bladder cancers are SCC . Due to the low incidence of SCC in the US as well as the rest of the Western countries, this paper primarily focuses on UC. Of all newly diagnosed UC cases, approximately 80% are noninvasive papillary tumors, which are confined to the urothelium (CIS, Ta) or lamina propria (T1). The remaining 20% of tumors are muscle invasive (T2–T4) and are typically treated by radical cystectomy . Despite the fact that most noninvasive UCs can be successfully treated by transurethral resection of bladder tumor (TURBT), 70% of patients will suffer tumor recurrence after the initial treatment and 10–20% of those recurrent tumors will become invasive. Specific genetic alterations characterize UCs; for instance, noninvasive tumors show frequent mutations in fibroblast growth factor receptor 3 (FGFR3) mutations; whereas invasive tumors often display TP53 mutations. Further progression of noninvasive tumors to invasive tumors requires subsequent mutations in TP53 (Figure 1) [4, 8]. The high rate of recurrence and inability to predict which tumor will progress require frequent and invasive clinical management after the initial treatment.
Many types of invasive carcinomas, including colon cancer , arise from noninvasive carcinomas via the accumulation of mutations over time. However, pioneering work done by our group has demonstrated that such a developmental continuum does not exist in UC. There is substantial evidence for the existence of two mutually exclusive molecular pathways that lead to bladder carcinogenesis in which distinct genetic alterations are responsible for the formation of noninvasive and invasive tumors, resulting in divergent clinical behaviors . Noninvasive tumors usually arise by tissue hyperplasia and show mutations in fibroblast growth factor receptor 3 (FGFR3) [16, 17], which is involved in cell differentiation and angiogenesis . Patients with such tumors usually do not show disease progression, but experience frequent recurrence [8, 19]. Invasive tumors are believed to arise by tissue dysplasia and often harbor mutations in TP53 [15, 20], a critical tumor suppressor gene that initiates cell-cycle arrest upon DNA damage . These tumors are aggressive and associated with high mortality . These two pathways do not occur sequentially and only under rare circumstances, when a subsequent p53 mutation is acquired, noninvasive tumors can progress to invasive tumors . The genetic alterations associated with UC are relatively well defined as compared to its epigenetic alterations. Therefore, this paper mainly focuses on the epigenetic aberrations found in UC.
Epigenetics encompasses the heritable changes in gene expression that are not caused by changes in the underlying DNA sequence . Such epigenetic changes include DNA methylation, histone modifications, and nucleosome positioning [15, 22–24]. Among the three layers of epigenetic regulation, DNA methylation was the first to be identified and is the most extensively studied. It involves the covalent addition of a methyl group to the 5′ position of cytosine residues in the context of CpG dinucleotides. The distribution of CpG sites is asymmetrical and nonrandom throughout the genome, with a high frequency of CpG sites occurring near promoters (CpG islands) and repetitive elements [25, 26]. The majority of promoter-associated CpG islands are usually not methylated under normal conditions, with the exception of imprinted genes [25, 27]. DNA methylation at gene promoters modifies DNA accessibility to transcription factors or helps recruit silencing-associated proteins, resulting in gene silencing [28, 29].
The alarmingly high recurrence rate of bladder cancer is of clinical concern, highlighting the need for physicians and scientists to elucidate its underlying mechanism. The presence of a field defect, an area of tissue that is predisposed to undergo oncogenic transformation, has been postulated to be responsible for such high recurrence rate . This concept was first introduced by Slaughter et al.,who found abnormal tissues composed of epithelial cells of polyclonal origins surrounding oral squamous cell carcinomas . Since then a field defect, as identified by genetic alterations, has been found in tumors arising from various tissues, including upper aerodigestive tract , lung , esophagus , vulva , cervix , colon , skin , and bladder [58, 59].
Since bladder cancer may remain asymptomatic until a relatively late stage, ideal clinical management would be comprised of early detection, accurate prediction of disease progression, and frequent monitoring. However, unlike many other types of cancers, there is no standard and effective noninvasive strategy for early detection . Currently, conventional histopathological evaluations that are used for the categorization of tumor grade and stage are also used to predict the potential behavior of tumors. Such histopathological evaluations are not accurate in predicting the behaviors of heterogeneous tumors, resulting in significant differences in clinical outcomes for patients with tumors of similar stages . Therefore, patients undergo frequent and long-term surveillance after the initial treatment. There is a strong need to develop economically viable, noninvasive methods with high sensitivity and specificity for diagnosis, prognosis, and monitoring of UC. A better understanding from both a genetic and an epigenetic perspective of how UC arises and progresses has greatly contributed to the ongoing efforts to create these new assays.
Although epigenetic modifications are heritable, their dynamic nature and reversibility through pharmacological interventions make them excellent targets for anticancer therapies. Over the past few decades, various drugs aimed at targeting different types of epigenetic alterations observed in cancer, including DNA methylation and histone modifications, have been developed, with the goal of reactivating aberrantly silenced genes. In addition to having genetic abnormalities, UC is also driven by progressive alterations in the epigenome, resulting in changes in chromatin packaging and aberrant gene expression . Epigenetic changes in UC have been well elucidated and their significance has been demonstrated, making UC a suitable candidate for epigenetic therapy. Due to the presence of an epigenetic field defect in UC, epigenetic therapies may also prevent recurrence by reversing the epigenetic aberrations occurring in histological normal tissues that remain after TURBT.
The widespread hypermethylation at promoters in UC, particularly in invasive tumors [40, 41, 46] suggests that restoration of a normal epigenome through the use of DNA hypomethylating agents would be clinically beneficial. Many of these agents are nucleoside analogues, which get incorporated into DNA and sequester DNA methyltransferases (DNMTs), resulting in depletion of DNMTs and global hypomethylation upon subsequent cell divisions .
Another layer of epigenetic regulation includes posttranslational modifications of histones, which play an important role in gene expression by altering chromatin structure . The type and location of histone modifications determine the conformation of chromatin. Certain modifications, such as H3K4me3 and H3K9 acetylation, are associated with euchromatin and make the DNA more accessible to the transcriptional machinery. Other modifications, such as H3K9me3 and H3K27me3, are associated with heterochromatin and make the DNA more condensed and less accessible to the transcriptional machinery [93, 94]. Cytosine methylation is associated with increased H3K9me3 and decreased H3 acetylation and H3K4me3 at gene promoters, leading to chromatin condensation and subsequent transcriptional silencing [95, 96]. The level of histone modifications is orchestrated by histone modifying enzymes, which add or remove specific histone marks to promote or hinder gene expression. A balance between these enzymes is necessary to maintain normal physiological conditions. Cancer cells lack this balance, as they typically overexpress histone deacetylases (HDAC), which results in a global reduction in histone acetylation .
The epigenome of UC is highly disrupted, featuring aberrant gene silencing either through the acquisition of DNA methylation or the repressive histone mark H3K27 trimethylation (Figure 2). The existence of these mechanisms suggests that the combination of DNMTi and HDACi may result in higher therapeutic efficacy. Both additive and synergistic effects have been reported with the combination of these two classes of epigenetic agents in patients with advanced hematological malignancies and solid tumors [32, 102]. However, the clinical utilization of combined epigenetic therapies is still in its early stages and more work is needed to elucidate the mechanism behind the increased clinical efficacy of sequential administration of DNMTi and HDACi in order to achieve an even greater synergistic effect.
UC is as much a disease of disrupted epigenome as it is a disease of genetic mutations. Here, we have summarized the epigenetic abnormalities associated with UC, with an emphasis on DNA methylation. The presence of an epigenetic field defect, where DNA methylation of a significant number of genes is altered not only in primary tumors but also in the surrounding normal-appearing tissues, provides a plausible explanation for the high rate of UC recurrence. Since certain epigenetic alterations precede disease pathology, they have the potential to serve as excellent biomarkers for diagnosis, prognosis, and monitoring. Although a large number of highly specific markers, both genetic and epigenetic, have already been identified, they suffer from low sensitivity. The ability to detect methylation changes in readily obtainable urine samples opens the door for the development of sensitive and specific noninvasive methods for early detection and monitoring of UC. In addition to serving as biomarkers, epigenetic alterations are also excellent therapeutic targets. Epigenetic therapies, such as DNMTi and HDACi, aim at restoring the diseased epigenome to its normal state by reactivating aberrantly silenced genes. While they have shown promising results in both preclinical and clinical settings, their efficacy is still limited to a few hematological malignancies. Epigenetic therapies also reactivate cancer germline antigens, which can be recognized by the immune system, and, therefore, they could potentially enhance the therapeutic value of cancer germline antigen vaccines. Future work, including obtaining a greater understanding of the mechanisms of DNMTi and HDACi, is necessary to determine the extent of their utility in treating solid tumors. With the aid of readily available genome-wide DNA methylation and expression analyses and our rapidly accumulating knowledge regarding epigenetic regulation, the translation of these findings from the bench to the bedside in the near future is an obtainable goal.