SEARCH

SEARCH BY CITATION

Keywords:

  • cell cycle regulation;
  • TP53;
  • DNA methylation;
  • chromosomal instability;
  • cancer stem cells

Abstract

  1. Top of page
  2. Abstract
  3. Approaches to the molecular biology of UC
  4. TP53 as a biomarker in UC
  5. Arguments for a “cancer pathway” approach to UC
  6. Cell cycle regulation as a “cancer pathway”
  7. Consequences of cell cycle disturbances in UC
  8. Conclusion and perspective
  9. Acknowledgements
  10. References

Urothelial carcinoma (UC), the common histological subtype of bladder cancer, presents as a papillary tumor or as an invasive, often lethal form. To study UC molecular biology, candidate gene and genome-wide approaches have been followed. Here, it is argued that a ‘cancer pathway’ perspective is useful to integrate findings from both approaches. According to this view, papillary cancers typically exhibit activation of the MAPK pathway, as a consequence of oncogenic mutations in FGFR3 or HRAS, with increased Cyclin D1 expression. In contrast, invasive UC are characterized by severe disturbances in proximate cell cycle regulators, e.g. RB1 and CDKN2A/p16INK4A, which decrease dependency on mitogenic signaling. In addition, these disturbances permit, promote and are in turn exacerbated by chromosomal instability, which is further enhanced by loss of TP53 function. In another vicious cycle, defective cell cycle regulation interacts with DNA methylation alterations. The transition toward invasive UC may require concomitant and interacting defects in cell cycle regulation and the control of genomic stability. Intriguingly, neither canonical WNT/β-Catenin nor hedgehog signaling appear to play major roles in UC. This may reflect its origin from more differentiated urothelial cells possessing a high regenerative potential rather than a stem cell population. © 2006 Wiley-Liss, Inc.

Bladder cancer is the fifth most frequent cancer in industrialized countries, accounting for up to 5% of all cancers. Histologically, urothelial carcinoma (UC), formerly designated ‘transitional cell carcinoma,’ is distinguished from rarer types like squamous cell carcinoma (SCC) and adenocarcinoma. UC are derived from the urothelium, the epithelium lining the urinary tract from the renal pelvis to the urethra. Accordingly, some cases occur outside the bladder in other segments of the urinary tract. Urothelial carcinomas express markers of urothelial differentiation, including CK7 and CK13 cytokeratins, and even uroplakins, proteins of the apical membrane of terminally differentiated umbrella cells in the top urothelial layer. Clinically, the most important distinction is that between papillary and invasive UC. Papillary UC (pTa) are characterized by hyperproliferation leading to lateral and vertical extension of the urothelial layer. As a consequence, the epithelium folds up around connective tissue papillae into a polypous structure protruding into the lumen of the urinary tract. Papillary UC can be treated by local transurethral resection (TUR). Unfortunately, the disease tends to recur in up to 75% of the patients, necessitating long-term monitoring by cystoscopy and repeated treatments. Recurrences can be partly prevented by optimized resection and by adjuvant intravesical instillation therapy with cytotoxic drugs or with BCG, a mycobacterium vaccine preparation. Thus, while papillary UC is seldom lethal, it causes considerable suffering and high expenses. Moreover, a small but significant percentage of papillary UC, usually characterized by poor differentiation, progress to invasive stages. Invasive bladder cancers are mostly derived from another precursor, carcinoma in situ (pTIS), flat highly dysplastic lesions. They are lethal by local progression or metastasis, unless completely removed by cystectomy at early invasive stages. Chemo- and radiotherapy are efficacious, but rarely curative. In the older literature, papillary tumors of various grades, carcinoma in situ, and carcinomas with invasion of the lamina propria only (pT1), were often summarized as ‘superficial.’ This designation is now obsolete, as they are recognized to exhibit considerable clinical and molecular differences.

Molecular biological research on bladder cancer is challenged to yield advances for its prevention, diagnostics and therapy. In fact, several ideas referred in this article have already influenced the latest WHO classification of urothelial cancers and thus contributed to practical improvements.1 In the future, most urgently needed are (1) markers identifying those patients with papillary and early stage invasive tumors at high risk for recurrence and progression, (2) reliable and affordable non-invasive techniques for monitoring, (3) markers distinguishing between metastatic and localized invasive disease and (4) more efficacious therapies for patients with locally progressed and metastatic cancer.

Approaches to the molecular biology of UC

  1. Top of page
  2. Abstract
  3. Approaches to the molecular biology of UC
  4. TP53 as a biomarker in UC
  5. Arguments for a “cancer pathway” approach to UC
  6. Cell cycle regulation as a “cancer pathway”
  7. Consequences of cell cycle disturbances in UC
  8. Conclusion and perspective
  9. Acknowledgements
  10. References

Two major approaches have been followed to analyze UC molecular biology: candidate gene analysis and genome-wide screens. In 1981, the first human oncogene, a mutated HRAS, was isolated from UC cell lines.2 Since then, almost every newly emerging oncogene or tumor suppressor was investigated for a role in UC. Several tumor suppressors were indeed found to be frequently affected, including TP53, RB1 and CDKN2A. Several oncogenes were observed to be activated in a fraction of cases, including HRAS, MYC and CCND1.3, 4 More recently, FGFR35, 6, 7 and E2F38, 9 were identified as oncogenes activated by point mutations and amplification, respectively.

Familial cases of bladder cancer are very rare. In the case of an exceptionally young index patient, an inherited translocation led to the identification of a novel oncogene, CDC91L1 at 20q.10 Its significance in sporadic cases remains to be ascertained.11 In general, identification of genes important in bladder cancer must rely on analyses of somatic alterations in tumor tissues and cell lines.

Identification of tumor suppressors is usually performed through delineation of common regions of deletion followed by mutational and methylation analysis of candidate genes. Genome-wide screens using LOH analyses or cytogenetical techniques revealed several consistently altered chromosomal regions in bladder cancer, but also a high background of chromosomal instability, with essentially each chromosome affected in some cases.12, 13 Some recurrent chromosomal gains or losses reflect alterations in known oncogenes or tumor suppressors. Losses at 9p21, 13q14 and 17p reflect the established inactivation of CDKN2A, RB1 and TP53, respectively. Some targets of less common losses can also be assigned, e.g., 10q losses may usually reflect PTEN inactivation. Gains at 7p12, 8q24 and 11q13 can result in overexpression of EGFR, MYC and Cyclin D1, respectively.3, 4 On a note of caution, additional or alternative genes may be targeted by losses and amplifications in some cases. Recurrent amplifications at 6p22.3 in advanced stage cancers are even more difficult to interpret because they are highly heterogeneous. Their most likely target is E2F3, but several other genes in the region are over-expressed to a comparable or greater extent in individual cases.8, 9, 14, 15 This example illustrates the limitations to identifying targets of chromosomal aberrations in cancers with pronounced genomic instability. Consequently, the significance of many chromosomal gains in UC is uncertain.

This predicament extends to chromosome losses, even highly prevalent ones at 3p and 8p. The most exasperating problem concerns chromosome 9q. Loss of (or LOH at) chromosome 9 is the most frequent chromosomal alteration in bladder cancer throughout all stages and grades, including well-differentiated papillary cancers and occasionally preneoplastic hyperplasia.3, 4, 12, 13 On 9p, loss and recombination are centered around CDKN2A at 9p21. This gene encodes p16INK4A, a CDK inhibitor and thereby activator of RB1, and p14ARF, an indirect activator of TP53, in different reading frames. In UC, this general tumor suppressor is most frequently inactivated by homozygous deletion.16, 17 Point mutations, typically inactivating both reading frames, and hypermethylation of either or both alternative promoters contribute in a smaller fraction of the cases. While often the entire chromosome is affected, allelic losses at 9q occur also independently of changes at 9p. One or several tumor suppressors are therefore assumed to be located on 9q. Candidates comprise PTCH1 and TSC1 already implicated in other cancers, and the novel, cautiously named DBCCR1 (deleted in bladder cancer candidate region 1). Mutations inactivating the second allele, meeting standard expectations for tumor suppressors, have been detected only in TSC1, at a low frequency.18, 19 Inherited TSC1 mutations predispose to several tumor types, but not obviously to bladder cancer. Methylation of DBCCR1 increases in UC, but does not seem consistently associated with gene silencing.20 Nevertheless, DBCCR1 overexpression inhibits cell proliferation.21 The mechanism involved and the effects of physiological expression levels remain to be elucidated.

PTCH1 is a tumor suppressor in the (sonic) hedgehog pathway and is often inactivated in basal cell carcinoma of the skin and in medulloblastoma.22 These cancers are generally characterized by activation of the hedgehog pathway, which can be alternatively brought about by oncogenic mutations in SMO, a membrane signaling protein normally inhibited by PTCH1. Activation of intracellular hedgehog signaling by either alteration results in increased expression of several pathway components including the transcription activators GLI1 and GLI2, and the inhibitory membrane proteins HIP1 and PTCH1, through the increased activity of GLI-dependent promoters. No evidence of such a response was found in UC cell lines.23 In cancer tissues, PTCH1 expression is approximately halved in cases with 9q loss in accord with a pure dosage effect. PTCH1 mutations are extremely rare in UC and none have been reported in other pathway components.24 Taken together, the evidence indicates that PTCH1 hemizygosity is not pathogenetic in UC.

Increasingly, global approaches are being used to investigate molecular changes in UC. These include array CGH and SNP arrays at the DNA level25, 26 microarray expression profiling at the RNA level,27, 28, 29, 30 and proteomics approaches.31 Early studies addressed differences among cell lines, cancer versus normal tissues and papillary versus invasive cancers. More recently, more sophisticated issues have been tackled, such as response to therapy or differential prognosis in cases with similar histopathological parameters, e.g. the clinically problematic pTaG3 tumors. Reassuringly, cluster analyses of expression profiles separate most cases of papillary from muscle-invasive cancers and cancers from normal tissue, with interesting exceptions that deserve further study.

Unfortunately, microarray expression studies have yielded few overlapping results with regard to the expression of individual genes, which makes it difficult to draw any conclusions on molecular mechanisms in UC. Some of the inconsistencies may derive from technical reasons and can presumably be resolved by improved standardization of arrays and of sample preparation, e.g. through microdissection. Furthermore, a significant proportion of the variability may have biological causes. Rampant chromosomal instability in UC ought to lead to variable dose changes and according differences in the expression of many genes, independently of whether they are causally involved in cancer development. For instance, individual bladder cancer cell lines over-express different genes from the 6p22.3 amplification unit up to a 100 fold.15 Moreover, gene expression profiling tends to identify those genes reacting most strongly to primary genetic or epigenetic alterations and not the causative change itself. Obviously, in a heterogeneous cancer like UC, data interpretation needs to be anchored in a conceptual framework to gain the most from genome-wide analyses.

TP53 as a biomarker in UC

  1. Top of page
  2. Abstract
  3. Approaches to the molecular biology of UC
  4. TP53 as a biomarker in UC
  5. Arguments for a “cancer pathway” approach to UC
  6. Cell cycle regulation as a “cancer pathway”
  7. Consequences of cell cycle disturbances in UC
  8. Conclusion and perspective
  9. Acknowledgements
  10. References

An important goal of array investigations of UC is the development of biomarkers for prognostic purposes. This aim has so also been pursued by investigations focused on individual genes, the most prominent one being TP53.32, 33, 34 The TP53 tumor suppressor gene harbors missense mutations in up to 50% of bladder cancers. The frequency of mutations increases with tumor stage and particularly with tumor grade. Specifically, their frequency is enhanced in early stage cancers considered as high-risk from clinical experience, such as carcinoma in situ and poorly differentiated papillary and early stage invasive tumors (pTaG3 and pT1G3). Moreover, LOH at 17p is preferentially found in advanced cases. Evidently, TP53 mutations are associated with an increased risk of progression in bladder cancer.3, 4, 32, 33, 34 The moot point is whether detection of TP53 mutations can predict the natural course of the disease or responses to therapy better than histopathological parameters. This issue, too, is confounded by technical as well as biological factors. Many studies have used techniques incapable of detecting all TP53 mutations, e.g. single-strand conformation polymorphism analysis plus sequencing starting from total tumor tissue. Moreover, accumulation of TP53 protein has often been used as a surrogate parameter for mutation. The correlation between mutations and nuclear accumulation of TP53 is very good in bladder cancer, but not perfect. In addition, TP53 immunohistochemistry poses its own vagaries, although standardized techniques have meanwhile been developed.32, 33, 34

These technical difficulties are exacerbated by the fact that TP53 acts within a molecular network. Loss of TP53 function can therefore not only be caused by mutations and deletions of the gene itself, but also by alterations in the mechanisms acting ‘upstream’ or ‘downstream’ of TP53 in the network. Such alterations may not have as severe effects as loss of TP53 itself, but may suffice to impede crucial functions of the nodal protein in the network. ‘Upstream’ alterations in the network diminish TP53 activation and include loss of p14ARF (encoded by CDKN2A) in a large fraction of cases and amplification of HDM2 in a few.3, 4, 34 Defects in the function of the protein kinases ATM, CHK2 and DNA-PK, which signal DNA damage to TP53, are implicated too.35 Activation of these kinases in pTa and pT1 tumors may prevent or delay their progression, but subsides in high-stage cancers in spite of increased chromosomal instability. ‘Downstream’ alterations diminish the efficacy of TP53 action through cell cycle inhibitors like p21CIP1 (see later) and pro-apoptotic proteins.

Arguments for a “cancer pathway” approach to UC

  1. Top of page
  2. Abstract
  3. Approaches to the molecular biology of UC
  4. TP53 as a biomarker in UC
  5. Arguments for a “cancer pathway” approach to UC
  6. Cell cycle regulation as a “cancer pathway”
  7. Consequences of cell cycle disturbances in UC
  8. Conclusion and perspective
  9. Acknowledgements
  10. References

The example of TP53, like that of PTCH1 and the hedgehog pathway discussed earlier, illustrates how productive it is to consider mutational and epigenetic gene alterations in the context of relevant networks or pathways. This approach may be particularly useful in a cancer with pronounced genomic instability such as UC. Hanahan and Weinberg36 have postulated that the distinctive properties of cancers are brought about by the activation or inactivation of a limited number of regulatory systems termed ‘cancer pathways.’ In addition to the TP53 network and the hedgehog pathway, these include the cell cycle regulatory system and the (canonical) MAPK pathway discussed later, the PI3K, STAT and NFκB pathways and the TGFβ response not discussed here because of space limitations, and the WNT/β-Catenin pathway, which can serve as another example for the value of this approach.

Constitutive activation of this pathway is the ‘gatekeeper’ change in several cancers, notably colorectal carcinoma. Activation is caused by loss of function of the tumor suppressors APC or AXIN1, or by oncogenic mutations in β-Catenin. Either results in the increased activity of promoters regulated by TCF4/β-Catenin.22 The few published studies on this pathway in UC report no or very few genetic alterations in pathway components.37, 38 Indeed, promoter-reporter constructs monitoring TCF/β-Catenin dependent transcription were found to be inactive in all UC lines tested and in normal urothelial cells, indicating lack of pathway activity.39 Furthermore, in most UC lines and in normal cells, transcription could not even be induced by overexpressed oncogenic β-Catenin, suggesting that it is actively repressed. Interestingly, exceptional inducible cell lines lacked expression of E-Cadherin, a modulator of the pathway.39 Thus, some bladder cancers may acquire the ability to respond to strong WNT signaling through loss of E-Cadherin, but constitutive activation is apparently very rare.

Importantly, many cancer pathways interact with each other and the effects of further regulators, e.g. cell adhesion molecules and extracellular proteases, are partly mediated through these pathways. The level of regulatory pathways may thus be optimal to integrate results from single candidate gene analyses and large-scale genomics approaches. Not least, the identification of relevant cancer pathways yields immediate options for pharmacological therapy.

Cell cycle regulation as a “cancer pathway”

  1. Top of page
  2. Abstract
  3. Approaches to the molecular biology of UC
  4. TP53 as a biomarker in UC
  5. Arguments for a “cancer pathway” approach to UC
  6. Cell cycle regulation as a “cancer pathway”
  7. Consequences of cell cycle disturbances in UC
  8. Conclusion and perspective
  9. Acknowledgements
  10. References

Many cancer pathways converge towards cell cycle regulation, a regulatory network in itself (Fig. 1). Since enhanced cell proliferation is a property of human cancers by definition, altered cell cycle regulation seems a prerequisite for cancer development. However, cancer types differ in the mechanisms that lead to altered cell cycle regulation and it its degree. In bladder cancer, genetic and epigenetic alterations of proximate regulators of the cell cycle abound.3, 4 Deletion and mutational inactivation of RB1 is found in a significant fraction of invasive cancers.33, 40 A large fraction of cancers of all stages harbor defects in CDKN2A, with loss of the CDK4 inhibitor p16INK4A.16, 17 In exceptional cases CDK4 is overexpressed due to amplification of the gene at 12q1341 or mutations interfere with p16INK4A binding. In contrast, overexpression of Cyclin D1 is widely found. Overexpression of MYC could also be counted as a cell-cycle related alteration. Like overexpression of Cyclin D1, it is caused by chromosomal gains or gene amplification only in a small number of tumors, but is more regularly a consequence of deregulation.37, 42 The fact that overexpression of MYC is caused by amplification in some cases and by deregulation in others could explain why overexpression is not entirely consistently associated with tumor stage and grade. These alterations are compounded by additional changes in the CIP/KIP family of CDK inhibitors. The p21CIP1 and p27KIP1 inhibitory proteins are down-regulated in the majority of invasive cancers18, 33, 43, 44 and expression of p57KIP2 is diminished or ablated by a combination of genetic and epigenetic mechanisms.45

thumbnail image

Figure 1. Signaling pathways converging on cell cycle regulation in normal urothelial cells (a), typical papillary superficial UC (b) and typical invasive UC (c). Inactive pathways: white, properly regulated active pathways: plain grey; deregulated pathways or proteins: patchy grey; aberrantly inactivated pathways or proteins: hatched.

Download figure to PowerPoint

While disturbances of cell cycle regulation are doubtless crucial in all UC, closer analyses reveal interesting variations. Alterations of RB1 are almost exclusively observed in muscle-invasive cancers and are associated with a significantly worse prognosis.33, 39 In contrast, Cyclin D1 overexpression is characteristic of early stage and papillary cancers and rather associated with less malignant behavior.3, 4CDKN2A alterations are overall independent of stage, grade and clinical course.16, 17 Obviously, the degree of disturbance of cell cycle regulation parallels the biological and clinical aggressiveness of bladder cancers. In line with this supposition, losses of p21CIP1, p27KIP1, and p57KIP2 are more frequent in muscle-invasive tumors,18, 33, 43, 44, 45 although their independent prognostic value is debated. In contrast, many papillary tumors over-express p21CIP1.

There is thus convincing evidence that ‘primary’ alterations in cell cycle regulators such as inactivation of RB1 and p16INK4A, usually by genetic mechanisms, together with down-regulation of CIP/KIP CDK inhibitors, predominantly by epigenetic mechanisms, are crucial changes in invasive UC. It is not quite as clear whether this conclusion extends to papillary cancers. A large proportion of well-differentiated papillary cancers harbor oncogenic mutations in FGFR3 which encodes a receptor for fibroblast growth factors like FGF1 and FGF8. As a rule, tumors exhibiting this change show fewer recurrences and their proportion decreases strongly at higher stages.5, 6, 7, 46FGFR3 mutations are inversely associated with TP53 mutations, but positively with Cyclin D1 overexpression.46 Overactivity of FGFR3 would be expected to lead to activation of the MAPK pathway through RAS and RAF and hence to induction of Cyclin D1 and cell cycle activation. Indeed, HRAS mutations were reported to occur in a complementary pattern to those in FGFR3 suggesting they belong to the same pathway.47 Thus, the growth of typical papillary cancers could be driven mainly by activation of the MAPK pathway, either through FGFR3 mutations, HRAS mutations, or through activation of other tyrosine kinase receptors, e.g. EGFR (Fig. 1).

The proliferation of normal urothelium, e.g. in response to tissue damage, is likewise thought to be stimulated by growth factors acting on tyrosine kinase receptors, in particular by EGF family members acting in an autocrine or paracrine fashion.48 These mechanisms can be studied in primary cultures of urothelial cells which proliferate spontaneously when placed in appropriate serum-free culture media. Their proliferation is elicited by autocrine growth factors such as HB-EGF and further enhanced by addition of exogeneous EGF. Inhibition of MAPK signaling decreases proliferation of normal urothelial cells, but cell lines from invasive UC are much less sensitive to MAP kinase inhibitors.49 Accordingly, while phosphorylated ERK and MEK can be detected in some UC lines, their transcriptional response to MAPK activation is muted.49, 50 These findings suggest several interesting conclusions. Firstly, the more massive alterations in cell cycle regulators found in invasive bladder cancers apparently diminish the dependence on exogenous growth factors and MAPK signaling, at least with regard to cell proliferation. Secondly, MAPK signaling is known to not only stimulate cell proliferation, but also cell differentiation and to induce inhibitors such as p21CIP1 which can induce cell cycle arrest and senescence. Therefore, activation of the pathway in papillary cancers could lead to a self-limiting tumor growth. This would explain the generally lower malignancy of papillary cancers with FGFR3 mutations. Continuous tumor growth would require additional alterations that prevent terminal differentiation and replicative senescence induced eventually by activation of G1 checkpoints through TP53 and RB1. Alterations compromising these networks in UC may therefore also prevent replicative senescence. Specifically, complete or even partial inactivation of CDKN2A also common in pTa cases17 may complement activation of the MAPK pathway. Interestingly, mutant RAS proteins may more efficiently induce premature senescence through a different pathway dependent on p38MAPK.51 Tumors with mutant HRAS could therefore behave differently from those with FGFR mutations, in spite of similar effects on canonical MAPK pathway activity. Thirdly and importantly, papillary and invasive UC may respond quite differently to therapeutic MAPK pathway inhibition, with additional differences within each group.

The notion that papillary UC are typically characterized by MAPK activation, and invasive UC by defects in the TP53 and RB1 networks is corroborated by animal models. Transgenic mice that overexpress a mutated Ha-Ras in urothelial cells develop urothelial hyperplasia and papillary UC,52 whereas animals expressing the SV40 large-T antigen, which inactivates Rb1 and Tp53, present with carcinoma in situ or invasive cancers.53

Consequences of cell cycle disturbances in UC

  1. Top of page
  2. Abstract
  3. Approaches to the molecular biology of UC
  4. TP53 as a biomarker in UC
  5. Arguments for a “cancer pathway” approach to UC
  6. Cell cycle regulation as a “cancer pathway”
  7. Consequences of cell cycle disturbances in UC
  8. Conclusion and perspective
  9. Acknowledgements
  10. References

The almost universal disturbance of the cell cycle regulatory network in bladder cancers has consequences beyond increased cell proliferation (Fig. 2). Hyperproliferative signals elicited by activated oncogenes induce cellular senescence through CDKN2A. Inactivation of this locus and down-regulation of other CDK inhibitors that can mediate cellular senescence, p21CIP1 and p57KIP2, would thus allow bladder cancers to tolerate persistently high proliferation signals, emerging from mutated FGFR3, mutated RAS, over-expressed EGFR, or amplified MYC.

thumbnail image

Figure 2. Mutual interaction of defective cell cycle regulation with mitogenic signaling, chromosomal stability and maintenance of DNA methylation patterns in UC.

Download figure to PowerPoint

Proper cell cycle control is also required for the function of cellular checkpoints responding to DNA damage and mitotic disturbances. Chromosomal instability in invasive bladder cancers indicates a dysfunction of these checkpoints. The prevalent loss of TP53 function in these carcinomas certainly contributes to this dysfunction. In cancers retaining intact TP53, down-regulation of p21CIP1 or loss of RB1 should compromise its ability to arrest the cell cycle upon check point activation. In fact, wild-type TP53 transfected into UC lines elicits apoptosis rather than cell cycle arrest.54 Accordingly, recent evidence suggests that disturbances of checkpoint regulation are not due to TP53 loss only. The checkpoint kinases ATM, CHK2 and CHK1 were observed in activated states in papillary UC, but more weakly active in invasive cancers.35 Apparently, dysregulation of DNA replication caused by oncogenic FGFR3, RAS or MYC or by defects in the RB1 cell cycle regulatory network elicit activation of S-phase and G2/M checkpoints. Overcoming these checkpoints through severe defects in cell cycle regulation or inactivation of checkpoint effectors is therefore a prerequisite for continued tumor growth and progression. Indeed, checkpoint responses become increasingly compromised as bladder cancers progress. While the underlying mechanisms are incompletely understood, loss of RB1 in particular has been linked to chromosomal instability.40 This relationship may have several reasons. Firstly, checkpoint signaling may not elicit G1 arrest in cells lacking functional RB1. Secondly, defects in replication and mitosis may occur more frequently during shorter and less coordinated cell cycles. Thirdly, RB1 is implied directly in the control of mitosis.40 In summary, therefore, the chromosomal instability characteristic of invasive bladder cancers may partly represent a consequence of severe disturbances of cell cycle regulation. Conversely, chromosomal instability causes genetic alterations that exacerbate cell cycle deregulation (Fig. 2).

Another vicious cycle in UC links cell cycle deregulation to alterations of DNA methylation, which promote tumor growth and genomic instability (Fig. 2). As in other cancers, 2 kinds of methylation alterations are observed, hypermethylation at CpG-rich promoter regions of selected genes and a genome-wide decrease in overall methylation, which is most evident at retrotransposons and satellites sequences densely methylated in normal cells. Hypermethylation events are more frequent in invasive than in papillary UC,55 while hypomethylation is essentially universal in invasive cancers.56 The mechanisms leading to these changes are complex and far from understood, but clearly proper coordination between the activity of DNA methyltransferases and DNA replication is crucial for the maintenance of methylation patterns in normal cells. Specifically, the major enzyme in somatic cells, DNMT1, is coordinated with the cell cycle by RB1 via E2F transcription factors. Loss of proper RB1 function would be expected to disturb this coordination. Indeed, DNMT1 expression was found to become uncoupled from increased cell proliferation in UC.57 Thus, while DNMT1 expression even increases in some UC, the enzyme cannot maintain normal methylation patterns. In turn, methylation alterations affect cell cycle regulators. The CDK inhibitor p57KIP2, e.g., is down-regulated during UC progression by promoter hypermethylation as well as hypomethylation of a more distant region controlling its imprinting.45

Conclusion and perspective

  1. Top of page
  2. Abstract
  3. Approaches to the molecular biology of UC
  4. TP53 as a biomarker in UC
  5. Arguments for a “cancer pathway” approach to UC
  6. Cell cycle regulation as a “cancer pathway”
  7. Consequences of cell cycle disturbances in UC
  8. Conclusion and perspective
  9. Acknowledgements
  10. References

In conclusion, 2 major “cancer pathways,” cell cycle regulation and the TP53 network, appear to be affected in UC and the extent of their respective deregulation in each tumor significantly determines its biological and clinical characteristics (Fig. 1). Activation of the canonical MAPK pathway may characterize an important subclass of papillary tumors.1, 4, 58 If so, this subclass may be more responsive to appropriate drug treatment than UC in general. Intriguingly, neither hedgehog nor WNT/β-Catenin signaling appear to play major parts in UC, in spite of their fundamental importance in other carcinomas. Apparently, stimulation of cell proliferation in UC is accomplished by other alterations, notably severe defects in proximate cell cycle regulation. In addition, the lack of importance of these pathways might relate to the urothelial origin of UC. Urothelium is a quiescent tissue, but capable of rapid proliferation in response to injury. The proliferating cells come from the basal and intermediate layers of the tissue. During acute injury, stem cell recruitment does not seem to be required. Thus, the functions of WNT and hedgehog signaling in maintaining stem cell compartments may not be as important in normal urothelial tissue as in continuously proliferating tissues like the skin or the gut epithelium. Consequentially, UC may arise from more differentiated cells, in which these pathways are suppressed. Indeed, UC usually express proteins characteristic of differentiated urothelial cells. According to this view, the property of unlimited growth, which cancer cells share with stem cells, would be acquired secondarily in UC, most likely by inactivation of TP53 and RB1 function. Obviously, to even better understand bladder cancer, the cancer pathway perspective sketched here should be widened to encompass the context of urothelial tissue organization.

References

  1. Top of page
  2. Abstract
  3. Approaches to the molecular biology of UC
  4. TP53 as a biomarker in UC
  5. Arguments for a “cancer pathway” approach to UC
  6. Cell cycle regulation as a “cancer pathway”
  7. Consequences of cell cycle disturbances in UC
  8. Conclusion and perspective
  9. Acknowledgements
  10. References
  • 1
    EpleJN, SauterG, EpsteinJI, SesterhennIA, eds. Pathology and genetics: tumours of the urinary system and male genital organs. Lyon: IARC Press, 2004. 359p.
  • 2
    Parada LF, Tabin CJ, Shih C, Weinberg RA. Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virus ras gene. Nature 1982; 297: 4748.
  • 3
    Knowles MA. What we could do now: molecular pathology of bladder cancer. Mol Pathol 2001; 54: 21521.
  • 4
    Dinney CP, McConkey DJ, Millikan RE, Wu X, Bar-Eli M, Adam L, Kamat AM, Siefker-Radtke AO, Tuziak T, Sabichi AL, Grossman HB, Benedict WF. Focus on bladder cancer. Cancer Cell 2004; 6: 1116.
  • 5
    Cappellen D, De Oliveira C, Ricol D, de Medina S, Bourdin J, Sastre-Garau X, Chopin D, Thiery JP, Radvanyi F. Frequent activating mutations of FGFR3 in human bladder and cervix carcinomas. Nat Genet 1999; 23: 1820.
  • 6
    Sibley K, Cuthbert-Heavens D, Knowles MA. Loss of heterozygosity at 4p16.3 and mutation of FGFR3 in transitional cell carcinoma. Oncogene 2001; 20: 68691.
  • 7
    van Rhijn BW, Lurkin I, Radvanyi F, Kirkels WJ, van der Kwast TH, Zwarthoff EC. The fibroblast growth factor receptor 3 (FGFR3) mutation is a strong indicator of superficial bladder cancer with low recurrence rate. Cancer Res 2001; 61: 12658.
  • 8
    Feber A, Clark J, Goodwin G, Dodson AR, Smith PH, Fletcher A, Edwards S, Flohr P, Falconer A, Roe T, Kovacs G, Dennis N. Amplification and overexpression of E2F3 in human bladder cancer. Oncogene 2004; 23: 162730.
  • 9
    Oeggerli M, Tomovska S, Schraml P, Calvano-Forte D, Schafroth S, Simon R, Gasser T, Mihatsch MJ, Sauter G. E2F3 amplification and overexpression is associated with invasive tumor growth and rapid tumor cell proliferation in urinary bladder cancer. Oncogene 2004; 23: 561623.
  • 10
    Guo Z, Linn JF, Wu G, Anzick SL, Eisenberger CF, Halachmi S, Cohen Y, Fomenkov A, Hoque MO, Okami K, Steiner G, Engles JM, et al. CDC91L1 is a newly discovered oncogene in human bladder cancer. Nat Med 2004; 10: 37481.
  • 11
    Schultz IJ, Kiemeney LA, Witjes JA, Schalken JA, Willems JL, Swinkels DW, de Kok JB. CDC91L1 (PIG-U) mRNA expression in urothelial cell carcinomas. Int J Cancer 2005; 116: 2824.
  • 12
    Knowles MA, Elder PA, Williamson M, Cairns JP, Shaw ME, Law MG. Allelotype of human bladder cancer. Cancer Res 1994; 54: 531538.
  • 13
    Hovey RM, Chu L, Balazs M, DeVries S, Moore D, Sauter G, Carroll PR, Waldman FM. Genetic alterations in primary bladder cancers and their metastases. Cancer Res 1998; 58: 355560.
  • 14
    Bruch J, Schulz WA, Melzner I, Kemmerling R, Brüderlein S, Möller P, Vogel W, Hameister H. Concurrent gain of chromosomes 5p, 6p, and 20q in bladder carcinoma cell lines: delineation of the 6p22 amplification unit. Cancer Res 2000; 60: 452630.
  • 15
    Wu Q, Hoffmann MJ, Hartmann FH, Schulz WA. Amplification and overexpression of the ID4 gene at 6p22.3 in bladder cancer. BMC Mol Cancer 2005; 4: 16.
  • 16
    Florl AR, Franke KH, Niederacher D, Gerharz CD, Seifert HH, Schulz WA. DNA methylation and the mechanisms of CDKN2A inactivation in transitional cell carcinoma of the urinary bladder. Lab Invest 2000; 80: 151322.
  • 17
    Chapman EJ, Harnden P, Chambers P, Johnston C, Knowles MA. Comprehensive analysis of CDKN2A status in microdissected urothelial cell carcinoma reveals potential haploinsufficiency, a high frequency of homozygous co-deletion and associations with clinical phenotype. Clin Cancer Res 2005; 11: 57407.
  • 18
    Adachi H, Igawa M, Shiina H, Urakami S, Shigeno K, Hino O. Human bladder tumors with 2-hit mutations of tumor suppressor gene TSC1 and decreased expression of p27. J Urol 2003; 170: 6014.
  • 19
    Knowles MA, Habuchi T, Kennedy W, Cuthbert-Heavens D. Mutation spectrum of the 9q34 tuberous sclerosis gene TSC1 in transitional cell carcinoma of the bladder. Cancer Res 2003; 63: 76526.
  • 20
    Habuchi T, Takahashi T, Kakinuma H, Wang L, Tsuchiya N, Satoh S, Akao T, Sato K, Ogawa O, Knowles MA, Kato T. Hypermethylation at 9q32-33 tumour suppressor region is age-related in normal urothelium and an early and frequent alteration in bladder cancer. Oncogene 2001; 20: 5317.
  • 21
    Wright KO, Messing EM, Reeder JE. DBCCR1 mediates death in cultured bladder tumor cells. Oncogene 2004; 23: 8290.
  • 22
    Beachy PA, Karhadkar SS, Berman DM. Tissue repair and stem cell renewal in carcinogenesis. Nature 2004; 432: 32431.
  • 23
    Thievessen I, Wolter M, Prior A, Seifert HH, Schulz WA. Hedgehog signaling in normal urothelial cells and in urothelial carcinoma cell lines. J Cell Physiol 2005; 203: 3727.
  • 24
    Aboulkassim TO, LaRue H, Lemieux P, Rousseau F, Fradet Y. Alteration of the PATCHED locus in superficial bladder cancer. Oncogene 2003; 22: 296771.
  • 25
    Veltman JA, Fridlyand J, Pejavar S, Olshen AB, Korkola JE, DeVries S, Carroll P, Kuo WL, Pinkel D, Albertson D, Cordon-Cardo C, Jian AN. Array-based comparative genomic hybridization for genome-wide screening of DNA copy number in bladder tumors. Cancer Res 2003; 63: 287280.
  • 26
    Primdahl H, Wikman FP, von der Maase H, Zhou XG, Wolf H, Orntoft TF. Allelic imbalances in human bladder cancer: genome-wide detection with high-density single-nucleotide polymorphism arrays. J Natl Cancer Inst 2002; 94: 21623.
  • 27
    Dyrskjot L, Thykjaer T, Kruhoffer M, Jensen JL, Marcussen N, Hamilton-Dutoit S, Wolf H, Orntoft TF. Identifying distinct classes of bladder carcinoma using microarrays. Nat Genet 2003; 33: 906.
  • 28
    Modlich O, Prisack HB, Pitschke G, Ramp U, Ackermann R, Bojar H, Vögeli TA, Grimm MO. Identifying superficial, muscle-invasive, and metastasizing transitional cell carcinoma of the bladder: use of cDNA array analysis of gene expression profiles. Clin Cancer Res 2004; 10: 341021.
  • 29
    Blaveri E, Simko J, Korkola JE, Brewer JL, Baehner F, Mehta K, deVries S, Koppie T, Pejavar S, Carroll P, Waldman FM. Bladder cancer outcome and subtype classification by gene expression. Clin Cancer Res 2005; 11: 404455.
  • 30
    Wild PJ, Herr A, Wissmann C, Stoehr R, Rosenthal A, Zaak D, Simon R, Knuechel R, Pilarsky C, Hartmann A. Gene expression profiling of progressive papillary noninvasive carcinomas of the urinary bladder. Clin Cancer Res 2005; 11: 441529.
  • 31
    Celis JE, Gromova I, Moreira JM, Cabezon T, Gromov P. Impact of proteomics on bladder cancer research. Pharmacogenomics 2004; 5: 38194.
  • 32
    Helpap B, Schmitz-Drager BJ, Hamilton PW, Muzzonigro G, Galosi AB, Kurth KH, Lubaroff D, Waters DJ, Droller MJ. Molecular pathology of non-invasive urothelial carcinomas. Virchows Arch 2003; 442: 30916.
  • 33
    Chatterjee SJ, Datar R, Youssefzadeh D, George B, Goebell PJ, Stein JP, Young L, Shi SR, Gee C, Groshen S, Skinner DG, Cote RJ. Combined effects of p53, p21, and pRb expression in the progression of bladder transitional cell carcinoma. J Clin Oncol 2004; 22: 100713.
  • 34
    Malats N, Bustos A, Nascimento CM, Fernandez F, Rivas M, Puente D, Kogevinas M, Real FX. P53 as a prognostic marker for bladder cancer: a metaanalysis and review. Lancet Oncol 2005; 6: 678686.
  • 35
    Bartkova J, Horejsi Z, Koed K, Krämer A, Tort F, Zieger K, Guldberg P, Sehested M, Nesland JM, Lukas C, Orntoft T, Lukas J. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 2005; 434: 86470.
  • 36
    Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100: 5770.
  • 37
    Shiina H, Igawa M, Shigeno K, Terashima M, Deguchi M, Yamanaka M, Ribeiro-Filho L, Kane CJ, Dahiya R. Beta-catenin mutations correlate with over expression of C-myc and cyclin D1 genes in bladder cancer. J Urol 2002; 168: 22206.
  • 38
    Stoehr R, Krieg RC, Knuechel R, Hofstaedter R, Pilarsky C, Zaak D, Schmitt R, Hartmann A. No evidence for involvement of beta-catenin and APC in urothelial carcinomas. Int J Oncol 2002; 20: 90511.
  • 39
    Thievessen I, Seifert HH, Swiatkowski S, Florl AR, Schulz WA. E-cadherin involved in inactivation of WNT/β-catenin signalling in urothelial carcinoma and normal urothelial cells. Br J Cancer 2003; 88: 19328.
  • 40
    Hernando E, Nahle Z, Juan G, Diaz-Rodriguez E, Alaminos M, Hemann M, Michel L, Mittal V, Gerald W, Benezra R, Lowe SW, Cordon-Cardo C. Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control. Nature 2004; 430: 797802.
  • 41
    Simon R, Struckmann K, Schraml P, Wagner U, Forster T, Moch H, Fijan A, Bruderer J, Wilber K, Mihatsch MJ, Gasser T, Sauter G. Amplification pattern of 12q13-q15 genes (MDM2, CDK4, GLI) in urinary bladder cancer. Oncogene 2002; 21: 247683.
  • 42
    Christoph F, Schmidt B, Schmitz-Dräger BJ, Schulz WA. Overexpression and amplification of the c-myc gene in human urothelial carcinoma. Int J Cancer 1999; 84: 16973.
  • 43
    Clasen S, Schulz WA, Gerharz CD, Grimm MO, Christoph F, Schmitz-Dräger BJ. Frequent and heterogenous expression of cyclin-dependent kinase inhibitor WAF1/p21 protein and mRNA in urothelial carcinoma. Br J Cancer 1998; 77: 51521.
  • 44
    Franke KH, Miklosi M, Goebell P, Clasen S, Steinhoff C, Anastasiadis AG, Gerharz CD, Schulz WA. Cyclin-dependent kinase inhibitor p27KIP1 is expressed preferentially in early stages of urothelial carcinoma. Urology 2000; 56: 68995.
  • 45
    Hoffmann MJ, Florl AR, Seifert HH, Schulz WA. Multiple mechanisms inactivating CDKN1C in bladder cancer. Int J Cancer 2005; 114: 40613.
  • 46
    Bakkar AA, Wallerand H, Radvanyi F, Lahaye JB, Pissard S, Lecerf L, Kouyoumdjian JC, Abbou CC, Pairon JC, Jaurand MC, Thiery JP, Chopin OK. FGFR3 and TP53 gene mutations define two distinct pathways in urothelial cell carcinoma of the bladder. Cancer Res 2003; 63: 810812.
  • 47
    Jebar AH, Hurst CD, Tomlinson DC, Johnston C, Taylor CF, Knowles MA. FGFR3 and Ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma. Oncogene 2005; 24: 521825.
  • 48
    Varley C, Hill G, Pellegrin S, Shaw NJ, Selby PJ, Trejdosiewicz LK, Southgate J. Autocrine regulation of human urothelial proliferation and migration during regenerative responses in vitro. Exp Cell Res 2005; 306: 21629.
  • 49
    Swiatkowski S, Seifert HH, Steinhoff C, Prior A, Thievessen I, Schliess F, Schulz WA. Activities of MAP-kinase pathways in normal uroepithelial cells and urothelial carcinoma cell lines. Exp Cell Res 2003; 282: 4857.
  • 50
    Hoshino R, Chatani Y, Yamori T, Tsuruo T, Oka H, Yoshida O, Shimada Y, Ari-i S, Wada H, Fujimoto J, Kohno M. Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors. Oncogene 1999; 18: 81322.
  • 51
    Deng Q, Liao R, Wu BL, Sun P. High intensity RAS signaling induces premature senescence by activating p38 pathway in primary human fibroblasts. J Biol Chem 2004; 279: 10509.
  • 52
    Zhang ZT, Pak J, Huang HY, Shapiro E, Sun TT, Pellicer A, Wu XR. Role of Ha-ras activation in superficial papillary pathway of urothelial tumor formation. Oncogene 2001; 20: 197380.
  • 53
    Zhang ZT, Pak J, Shapiro E, Sun TT, Wu XR. Urothelium-specific expression of an oncogene in transgenic mice induced the formation of carcinoma in situ and invasive transitional cell carcinoma. Cancer Res 1999; 59: 35127.
  • 54
    Makri D, Schulz WA, Grimm MO, Clasen S, Bojar H, Schmitz-Dräger BJ. WAF1/p21 regulates proliferation but does not mediate p53-dependent apoptosis in urothelial carcinoma cell lines. Int J Oncol 1998; 12: 6218.
  • 55
    Catto JW, Azzouzi AR, Rehman I, Feeley KM, Cross SS, Amira N, Fromont G, Sibony M, Cussenot O, Meuth M, Hamdy FC. Promoter hypermethylation is associated with tumor location, stage, and subsequent progression in transitional cell carcinoma. J Clin Oncol 2005; 23: 290310.
  • 56
    Florl AR, Loewer R, Schmitz-Dräger BJ, Schulz WA. DNA methylation and expression of L1 LINE and HERV-K provirus sequences in urothelial and renal cell carcinoma. Br J Cancer 1999; 80: 131221.
  • 57
    Kimura F, Seifert HH, Florl AR, Santourlidis S, Steinhoff C, Swiatkowski S, Mahotka C, Gerharz CD, Schulz WA. Decreased DNA methyltransferase 1 expression relative to cell proliferation in transitional cell carcinoma. Int J Cancer 2003; 104: 56878.
  • 58
    Wu XR. Urothelial tumorigenesis: a tale of divergent pathways. Nat Rev Cancer 2005; 5: 71325.