Current and Emerging Strategies in Bladder Cancer

Anti-Cancer Agents in Medicinal Chemistry, 2012, 12, 589-603 589

Current and Emerging Strategies in Bladder Cancer

Simone Carradoria,*, Cristiano Cristinib, Daniela Seccia, Caterina Guliab, Vincenzo Gentileb and Giovanni Battista Di Pierrob,*

aDipartimento di Chimica e Tecnologie del Farmaco and bDipartimento di Scienze Ginecologico-Ostetriche e Scienze Urologiche, “Sapienza” University of Rome, P.le A. Moro 5, 00185 Rome, Italy

Abstract: Urothelial cell carcinoma is one of the most common malignancies of the urinary tract. The standard of care, intravesical chemo- and immunotherapy, while effective, is associated with a considerable side-effect profile and approximately 30% of patients either fail to respond to treatment or suffer recurrent disease within 5 years. In the setting of muscle-invasive urothelial carcinoma, use of neoadjuvant chemotherapy is associated with overall survival benefit. Muscle invasive bladder cancer is life threatening, showing modest chemosensitivity, and usually requires radical cystectomy. Although bladder cancer is fairly well-genetically characterized, clinical trials with molecularly targeted agents have, in comparison to other solid tumors, been few in number and largely unsuccessful. Hence, bladder cancer represents a considerable opportunity and challenge for alternative therapies. In this review, we will focus on promising global or pathway-based approaches (epigenetic modulators, kinase inhibitors, angiogenesis blockage, peroxisome proliferator-activated receptor γ agonists, apoptosis inductors, virus therapy) supported by a deeper understanding of molecular biology of urothelial carcinoma, which have been recently tested in clinical trials.

Keywords: Angiogenesis antagonists, Apoptosis agonists, Bladder cancer, Clinical studies, Epigenetic modulation, Intravesical therapy, Kinase inhibitors, mTOR inhibitors, PPARγ agonists, Target therapy, Virus therapy.

1. INTRODUCTION Bladder carcinoma is the most common malignancy of the urinary tract. An estimated 104,400 incident cases of bladder cancer were diagnosed in Europe in 2006, of which 82,800 were found in men and 21,600 in women. This represents 6.6% of the total cancers in men and 2.1% in women, with an estimated male:female ratio of 3.8:1.0 and resulting in 4.1% of total cancer deaths in men and 1.8% of total cancer deaths in women [1]. Active and passive tobacco smoking is the most well-established risk factor for bladder cancer, causing 50-65% of male cases and 20-30% of female cases. The incidence of bladder cancer is directly related to the duration of smoking and number of cigarettes smoked per day and is also higher in those who start smoking at a young age or who are exposed to environmental tobacco smoke during childhood [2-4]. A recent meta-analysis looked at 216 observational studies on cigarette smoking and cancer from 1961 to 2003 shows an immediate decrease (40%) in the risk of bladder cancer was observed in those who stopped smoking [3]. Occupational exposure is the second most important risk factor for bladder cancer. Work-related cases accounted for 20-25% of all bladder cancer cases. The substances involved in chemical exposure are mainly benzene derivatives and certain arylamines, and it is likely to occur in occupations in which chemicals are used [5]. Increased rates of secondary bladder malignancies have been reported after external beam radiation therapy (EBRT) for gynecological malignancies, with relative risks of 2 to 4 [6]. A recent population cohort study identified 243,082 men treated for prostate cancer between 1988 and 2003 in the Surveillance, Epidemiology and End Results database in the USA. The standardized incidence ratios for bladder cancer developing after radical prostatectomy (RP), EBRT, brachytherapy (BT), and EBRT-BT were 0.99, 1.42, 1.10, and 1.39, respectively, compared with the general US population. The increased risk of bladder cancer in patients undergoing ERBT, BT or ERBT-BT should be

*Address correspondence to these authors at the Dipartimento di Chimica e Tecnologie del Farmaco and Dipartimento di Scienze Ginecologico-Ostetriche e Scienze Urologiche, “Sapienza” University of Rome, P.le A. Moro 5, 00185 Rome, Italy; Tel: +39 06 49913975; Fax: +39 06 49913772; E-mails: [email protected], [email protected]

taken into account during follow-up especially if treated with radiation at a young age [7]. Bladder cancer, particularly invasive squamous cell carcinoma, is directly related to the presence of chronic urinary tract infection. Bladder schistosomiasis (bilharzia) has been considered a definitive cause of urinary bladder cancer with an associated five-fold risk. Schistosomiasis is a very common parasitic infection with about 600 million people exposed to infection [8]. Although there is a well-established relationship between squamous cell carcinoma of the bladder and schistosomiasis, the trends are changing for bladder cancer in endemic zones, such as Egypt [9]. About chemotherapy, the use of cyclophosphamide, an alkylating agent used for treatment of lymphoproliferative diseases and other non-neoplastic diseases, has been correlated with later development of muscle-invasive bladder cancer (MIBC) with a period of latency of 6-13 years. Acrolein, a metabolite of cyclophosphamide, is responsible for the increase in the incidence of bladder cancer. This effect occurs independently of the association of hemorrhagic cystitis with the same treatment [10, 11]. At the initial diagnosis of bladder cancer, 70% of cases are diagnosed as confined to the mucosa (stage Ta, CIS) or submucosa (stage T1) and approximately 30% as muscle-invasive (stage T2-T4) disease (Table 1). Haematuria is the most common finding in bladder tumors, while non-muscle invasive bladder cancer does not usually cause bladder pain and rarely presents with irritation, dysuria or urgency. In patients who do complain of these symptoms CIS (carcinoma in situ) may be suspected. Diagnostic tools include urine cytology, ultrasonography, and cystoscopy with description of the tumor (site, size, number and appearance) and mucosal abnormalities. IVU (intravenous urography) or CT is usually considered in selected cases (tumors located in the trigone). However, a complete and correct TUR (transurethral resection) is essential to make a correct diagnosis and remove all visible lesions [12].

2. NON-MUSCLE INVASIVE (SUPERFICIAL) BLADDER CANCER (NMIBC) The goal of the TUR in TaT1 bladder tumors is to make the correct diagnosis and remove all visible lesions. As Ta and T1 tumors can be removed by transurethral resection, they are grouped

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590 Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 6 Carradori et al.

under the heading of non-muscle invasive (superficial) bladder cancer for therapeutic purposes. Also included under this heading are flat, high-grade tumors confined to the mucosa, classified as carcinoma in situ (CIS). Table 1. 2009 TNM Classification of Urinary Bladder Cancer

T Primary Tumor

TX Primary tumor cannot be assessed

T0 No evidence of primary tumor

Ta Non-invasive papillary carcinoma

Tis Carcinoma in situ: ‘flat tumor’

T1 Tumor invades subepithelial connective tissue

T2 Tumor invades muscle

T2a Tumor invades superficial muscle (inner half)

T2b Tumor invades deep muscle (outer half)

T3 Tumor invades perivesical tissue

T3a Microscopically

T3b Macroscopically (extravesical mass)

T4 Tumor invades any of the following: prostate, uterus, vagina, pelvic wall, abdominal wall

T4a Tumor invades prostate, uterus or vagina

T4b Tumor invades pelvic wall or abdominal wall

N Lymph Nodes

NX Regional lymph nodes cannot be assessed

N0 No regional lymph node metastasis

N1 Metastasis in a single lymph node in the true pelvis (hypogastric, obturator, external iliac or presacral)

N2 Metastasis in multiple lymph nodes in the true pelvis (hypogastric, obturator, external iliac or presacral)

N3 Metastasis in a common iliac lymph node(s)

M Distant Metastasis

M0 No distant metastasis

M1 Distant metastasis

The classic way to categorize patients with TaT1 tumors is to divide them into risk groups (low-risk, intermediate-risk, and high-risk) based on prognostic factors derived from multivariate analyses [13]. When using this technique, however, no difference is usually made between the risk of recurrence and progression. Although prognostic factors may indicate a high risk for recurrence, the risk of progression may still be low and other tumors may have a high risk of both recurrence and progression. In order to separately predict the short-term and long-term risks of both recurrence and progression in individual patients, the European Organization for Research and Treatment of Cancer (EORTC) developed a scoring system and risk tables [14]. The scoring system is based on the five most significant clinical and pathological factors:

• number of tumors; • tumor size and grade; • prior recurrence rate; • T category; • presence of concomitant CIS. Although a state-of-the-art TUR by itself could eradicate a TaT1 tumor completely, these tumors will recur in a high percentage of cases up to 50% and progress to muscle invasive bladder cancer in a limited number of cases up to 10-15%. For these reasons, the result of the first cystoscopy after TUR at 3 months is a very important prognostic factor for recurrence and for progression [14-17]. Moreover, the high variability in the 3-month recurrence rate indicates that TUR is incomplete or provokes recurrences in a considerable percentage of patients [18]. It is therefore necessary to consider adjuvant therapy in almost all patients as intravesical chemotherapy (mitomycin C, epirubicin and doxorubicin) or immunotherapy [bacillus Calmette-Guérin (BCG)] instillations in order to prevent or decrease recurrence and/or progression. Treatment with BCG is considered to have failed in following situations: • whenever muscle invasive tumor is detected during follow-up; • if high-grade, non-muscle invasive tumor is present at both 3

and 6 months [19]. In patients with tumor present at 3 months, an additional BCG course provokes complete response in more than 50% of cases, both in patients with papillary tumors and CIS [19, 20];

• any worsening under BCG treatment, such as a higher number of recurrences, higher T or higher grade, or appearance of CIS, in spite of an initial response (level of evidence: 3).

The subsequent follow up is based on the specific risk class for progression/recurrence: • Patients with low risk of recurrence and progression should

have a cystoscopy at 3 months. If negative, the following cystoscopy is advised at 9 months and consequently yearly for 5 years. (Grade of recommendation: C);

• Patients with high risk of progression should have a cystoscopy and urinary cytology at 3 months. If negative, the following cystoscopies and cytologies should be repeated every 3 months for a period of 2 years, every 4 months in the third year, every 6 months thereafter until 5 years, and yearly thereafter. (Grade of recommendation: C);

• Patients with intermediate-risk of progression (about one-third of all patients) should have an in-between follow-up scheme using cystoscopy and cytology. (Grade of recommendation: C).

3. MUSCLE-INVASIVE AND METASTATIC BLADDER CANCER At the initial diagnosis of bladder cancer, 70% of cases are diagnosed as NMIBC and approximately 30% as muscle-invasive (stage T2-T4) disease. Among patients treated with radical cystectomy because of MIBC, 57% had muscle invasion at presentation, while 43% had been initially diagnosed with NMIBC that progressed despite organ-preserving treatment [21]. Up to one-third of patients diagnosed with MIBC have undetected metastasis at the time of treatment of the primary tumor [22, 23], while 25% of patients subjected to radical cystectomy present with lymph node involvement at the time of surgery. The standard treatment for patients with muscle-invasive (T2-T4a, N0-Nx, M0) bladder cancer is radical cystectomy. However, this ‘gold standard’ only provides 5-year survival in about 50% of patients [24-28]. In order to improve these unsatisfactory results, the use of peri-operative chemotherapy has been introduced. Neoadjuvant cisplatin-containing combination chemotherapy improves overall survival by

Current and Emerging Strategies in Bladder Cancer Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 6 591

5-7% at 5 years and should be considered in muscle-invasive bladder cancer, irrespective of definitive treatment. In the most recent meta-analysis, published in 2005 [29], with updated independent patient data of 11 randomized trials (3005 patients), a statistically significant survival benefit in favour of neoadjuvant chemotherapy was also seen. The results of this analysis confirmed the previously published data and showed 5% absolute improvement in survival at 5 years. Of note, only cisplatin combination chemotherapy with at least one additional chemotherapeutic agent resulted in a meaningful benefit [30, 31]; the regimens tested were MVA(E)C (methotrexate, vinblastine, doxoruhicin/epirubicin, cisplatin), CMV (cisplatin, methotrexate, vinblastine), CM (cisplatin, methotrexate), cisplatin/adriamycin, cisplatin/5-fluorouracil (5-FU), and CarboMV (carboplatin, methotrexate, vinblastine). To date, it is unknown if more modern chemotherapy regimens are as effective. Approximately 30% of patients with urothelial cancer present with muscle-invasive disease; about half will relapse after radical cystectomy depending on the pathological stage of the primary tumor and the nodal status. Local recurrence accounts for about 30% of relapses, whereas distant metastases are more common. Varying response rates of single-agent first-line chemotherapy have been reported with only 12% for cisplatin compared to MVAC [32], 12% for carboplatin [33], 42% for paclitaxel [34], 31% for docetaxel [35], 29% for methotrexate, 19% for adriamycin, 15% for epirubicin, 13% for mitomycin C, 35% for 5-FU, 14% for vinblastine, 29% for ifosfamide, and 8% for cyclophosphamide [36, 37]. The most robust single-agent data is a response rate of about 25% for gemcitabine for first- and second-line use in several, larger-sized, phase II trials [38-45]. Responses with single agents are usually short-lived and complete responses are rare. Of note, no long-term disease-free survival has been reported with single-agent chemotherapy (median survival= 6-9 months). Cisplatin-containing combination chemotherapy has been the standard of care since the late 1980s. MVAC has been proven superior to cisplatin monotherapy, CISCA (cisplatin, cyclophosphamide, and adriamycin) [32, 46], and to cisplatin/ docetaxel [47]. MVAC and gemcitabine/cisplatin (GC) have prolonged survival up to 14.8 and 13.8 months, respectively [48-50]. Neither of the two combinations was proven to be superior over the other with response rates of 46% and 49% for MVAC and GC, respectively. The long-term survival results confirmed the anticipated equivalence of the two regimens [46]. The major difference between the above-mentioned combinations was toxicity, with GC being less toxic [50]. High-dose intensity MVAC (HD-MVAC) with GCSF (granulocyte colony-stimulating factor) is less toxic and more efficacious than standard MVAC in terms of dose density, complete response, and 2-year survival rate. However, there is no significant difference in median survival between the two regimens [51, 52]. Carboplatin-containing chemotherapy is not proven to be equivalent to cisplatin combinations. However, it is probably inferior and therefore should not be considered interchangeable or standard. The only randomized phase III study of carboplatin-containing chemotherapy had a disappointing response rate of only 28.2% in the investigational arm (paclitaxel/carboplatin) compared to MVAC and had to be closed down early because of a low accrual rate. There is therefore no evidence that this doublet might have adequate efficacy for first-line use [53]. Various carboplatin versus cisplatin combination chemotherapies in randomised phase II trials have produced lower complete response rates and a shorter overall survival for the carboplatin arms [54-56]. Up to 50% of patients are unfit for cisplatin-containing chemotherapy, either due to a poor PS (performance status) and/or impaired renal function, or due to co-morbidity that forbids high-volume hydration [57, 58]. In such cases, carboplatin combination

or single-agent chemotherapy is reasonable [33, 59]. Non-platinum combinations, such as frontline chemotherapy in patients with two adverse prognostic factors (glomerular filtration rate< 50-60 mL/min and PS≥ 2), should be reserved for investigational use because they have not been tested in purely ‘unfit’ patients and might be too toxic. Trials with clearly defined ‘unfit’ patients or patients with multiple adverse prognostic factors are rare. The first randomized phase II/III trial in this setting was conducted by the EORTC and compared carboplatin/vinblastine/methotrexate and carboplatin/gemcitabine in patients unfit for cisplatin. The phase II analysis of this trial showed that patients with both stratification factors (PS= 2 and impaired renal function) did not benefit from combination chemotherapy [60].

4. EPIGENETICS AND GENE SILENCING IN BLADDER CANCER Histone modifications and DNA methylation are important and reversible epigenetic mechanisms of gene and protein regulation which play essential roles in tumor initiation and progression. Aberrant epigenetic alterations have been also observed in bladder cancer and they could be evaluated as potential tumor biomarkers for diagnosis and risk assessment [61]. In mammals and in lower organisms, these genes are physiologically involved in important cellular pathways (cell-cycle control, apoptosis, cell invasion, putative tumor suppressors, wingless-type antagonists, cell differentiation and adhesion, and DNA damage repair). Gene-expression profiling by microarray analysis revealed that these genes could be upregulated or downregulated in BC compared with normal tissue [62], also if their complete epigenetic modulation and correct molecular mechanisms underlying the development and progression of BC remain to be elucidated. In general, the methylation patterns in BC specimens were found to be altered in 17% of sequences in comparison with normal urothelial epithelia [63]. Another potentially useful application of methylation profiles is in the molecular classification of BC which could be predictive of the cancer behaviour. DNA hypermethylation seems to be a promising biomarker because methylation always occurs in specific regions (CpG islands) and can be detected with high-sensitivity techniques (matching sequence pair and bisulfite genomic sequencing). Among the several cell cycle checkpoints, CDK (cyclin-dependent kinase) inhibitors (CDKIs), such as p14ARF and p16INK4a, are inhibitors of cell-cycle progression (potential tumor suppressor genes) [64]. Then, inactivation of these genes by promoter hypermethylation can contribute to the pathogenesis and progression of different human malignancies. In BC tissues, hypermethylation of p14ARF and p16INK4a is characterized by simultaneous genetic alterations of both of them, including loss of heterozygosity (LOH), homozygous deletion, and point mutation with a significant correlation between their methylation frequencies and the pathological stage or prognosis of patients. Also the cadherin-catenin adhesion is another critical system for the maintenance of normal tissue architecture. In BC, the expression of its gene CDH1 is strongly reduced and has a prognostic significance, because CDH1 promoter hypermethylation is also associated with tumor stage, grade, and prognosis. However, evidence of the methylation frequency of the CDH1 gene promoter has been difficult to interpret because moderate to high levels of methylation have been detected in some studies [65], but very low levels have been registered in others [66]. In addition, age-dependent gene methylation of CDH1 was also physiological in normal bladder epithelium [67]. On the contrary, hypermethylation of genes involved in DNA damage repair, such as gene glutathione S-transferase Pi 1 (GSTP1) and the DNA repairing gene O6-methylguanine DNA methyltransferase (MGMT), has been reported to be involved in BC


[68], albeit the methylation frequency of GSTP1 is low [69]. Tumors with a reduced MGMT expression have a higher incidence of point mutations in genes encoding for example p53 and K-ras, which play a role in cancer progression and sensitivity to chemotherapeutic alkylating agents [70]. Moreover, functional loss of well known tumor suppressor genes, such as the von Hippel-Lindau gene and the mismatch repair gene, mutL homolog, through DNA hypermethylation are not so frequent in BC [71]. However, certain putative tumor suppressor genes have been reported to be silenced by DNA hypermethylation in BC such as ras association domain family 1 gene (RASSF1), deleted in bladder cancer 1, and insulin-like growth factor binding protein 3 [72]. It is unknown, however, whether hypermethylation of these genes is important for bladder carcinogenesis or it can be only considered a predictive biomarker for BC diagnosis. Apoptotic pathways are also targets for epigenetic silencing. Among the apoptosis-related genes in BC, promoter hypermethylation of the DAPK gene (death-associated protein kinase) has been investigated mostly because its hypermethylation has been associated with tumor recurrence in BC. The wingless-type (Wnt) proteins are powerful regulators of cell proliferation and differentiation. Therefore, the inactivation of their related genes by promoter hypermethylation results in the activation of the Wnt/β-catenin signaling pathway, leading to the pathogenesis of various human tumors. CpG hypermethylation of Wnt antagonists (APC, Wif-1, Dkk-3, sFRP-1,-2,-4,-5) is a common event in BC, and methylation-specific polymerase chain reaction for these genes can be a useful diagnostic and staging biomarker. Additionally, aberrant methylation of retinoic acid receptor beta (RARB) is considered to be related to the development of retinoic acid resistance in cancer

cells, leading to cell undifferentiation and proliferation. On the other hand, the degree of methylation of MDR1 (human multidrug resistance gene), which serves as a xenobiotic-efflux pump, has been shown to change during chemotherapy in BC patients, implying a mechanism of drug resistance mediated by gene methylation [73]. A new demethylating agent, zebularine (Fig. (1)), has been tested in BC cells, and it reactivated methylated tumor suppressor genes such as p16INK4a and apoptosis-related genes, such as APAF-1 (apoptotic protease activating factor 1) and DAPK-1. Until we have a full understanding of the pathophysiological pathway of global hypomethylation in BC, therapeutics targeting DNMTs (DNA methyltransferases) in cancer should be used with caution. Ideal treatments should selectively activate a group of methylated genes without inducing undesired demethylation in the rest of the genome [74]. On the contrary, demethylation of normally methylated DNA can alter such a defense mechanism and cause genome alterations, contributing to one of the multiple mechanisms of loss of imprinting. An example of gene regulated by hypomethylation is heparanase (HPSE); this endo-β-D-glucuronidase plays a role in disassembling the extracellular matrix through the cooperation of matrix metalloproteases and urokinase-type plasminogen activator and is often upregulated in malignant tumors. Increased heparanase expression during the pathogenesis of BC is strongly due to promoter hypomethylation which has been reported in BC specimens compared with normal bladder epithelium [75]. Instead, several genes of biological significance to BC are potentially regulated by histone modification. One of these,

Fig. (1). Drugs which target bladder cancer through the modulation of epigenetic factors.

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coxsackie and adenovirus receptor (CAR), is the primary receptor for group C adenoviruses and its decreased expression has been registered in BC and associated with tumor stage and grade. Upregulation of CAR can be seriously enhanced in BC cells through the use of some histone deacetylase inhibitors reported in Fig. (1) such as trichostatin A, sodium phenylbutyrate, and a depsipeptide (FK228). This implies that combining histone deacetylase inhibitor with gene therapy may have a synergistic effect in BC treatment. A novel HDAC inhibitor, belinostat, has been shown to suppress bladder cancer cell growth in vitro and in vivo (mouse). Several phase II studies of HDAC inhibitors in urothelial carcinoma are currently in progress. Cell cycle inhibition and induction of apoptosis have been shown in BC cell line T24 for an experimental deacetylase inhibitor. The clinical effect of this HDAC inhibitor SAHA (suberoylanilide hydroxamic acid, Vorinostat) was tested for patients with solid tumors in a phase I trial and two patients showed a tumor regression [76]. Although, DNA methylation and histone acetylation can separately modulate gene expression, they can also cross-talk to each other in order to produce a transcriptionally inactive chromatin through the binding of methylated DNA binding proteins. This interaction then recruits HDAC activity to methylated promoters, resulting in gene silencing. In addition, DNMTs can directly recruit HDAC activity to silence gene expression [61]. Farnesyl transferase is also responsible for another post-translational modification (farnesylation). Farnesylation is required for the function of a number of proteins involved in signal transduction pathways. Among these proteins, Ras proteins transmit signals from cell surface receptors and mediate a range of cellular effects such as cellular proliferation, survival, and angiogenesis. Farnesyl transferase inhibitors include tipifarnib and lonafarnib. In many preclinical series of urothelial carcinoma, Ras mutations have been found to be prevalent, albeit with different frequencies. However despite their biological importance, in several phase II studies in patients with urothelial carcinoma, farnesyl transferase inhibitors only resulted in very modest responses [77]. The 26S ubiquitin-proteasome, present in both the cytoplasm and the nucleus of all eukaryotic cells, modulates the levels and functions of proteins involved in cell cycle progression, differentiation, apoptosis, and adhesion. Proteasome inhibitors such as bortezomib inhibit proteasomes, but in two phase II studies, no responses were registered when patients with urothelial carcinoma were previously treated with bortezomib [78]. MicroRNAs, small noncoding RNA gene products, are novel epigenetic regulators of gene expression. About 1000 microRNAs have been identified and collected in several databases. They function epigenetically as negative modulators of gene expression by binding to complementary sites in the target mRNA, indicating that microRNAs might also function as tumor suppressor genes or oncogenes. However, little is known about microRNAs in BC. Some authors screened 156 mRNAs and identified 27 microRNAs differentially expressed in BC specimens and in normal bladder epithelia [79].

5. TYROSINE KINASE BLOCKERS Given that protein kinases have emerged as critical key of all aspects of cancer, including proliferation, invasion, angiogenesis, and metastasis, the development of potent and selective inhibitors for targeted cancer treatment must be considered imperative [80]. These compounds can be classified into monoclonal antibodies and small-molecule inhibitors (SMIs). Monoclonal antibodies provided an efficient way to target receptor tyrosine kinases, but their use is limited by their size, heterogeneous antigen expression, and localization of targeted antigens in normal cells, while SMIs offered some advantages such as a known mechanism of action, less adverse effects than common chemotherapeutics, and orally administration.

5.1. PI3K/Akt/mTOR Pathway Inhibitors mTOR is a serine/threonine-specific protein kinase, within the PI3K/Akt/mTOR pathway, involved in the regulation of proliferation, metabolism, and motility through the integration of signals from different sources such as growth factors, nutrients, hormones, and oxygen level. Activation of PI3K in bladder cancer, initiates PIP3 which phosphorylates and activates Akt leading to tumor cell mobility, cell invasion, and chemotherapeutic resistance. Rapamycin or sirolimus, shown in Fig. (2), can selectively inhibit the PI3K/Akt pathway by directly binding to FKBP protein 12, which blocks the mTOR pathway, thus representing one of the possible targets bladder cancer. In fact, mTOR inhibitors have predominantly cytostatic effects, and in clinical trials these agents appear to stabilize disease, although with a limited ability to induce an evident tumor regression. Overall, moderate response rates to treatment with rapamycin analogs (rapalogs) have been observed.

Fig. (2). PI3K/Akt/mTOR pathway inhibitors tested for the treatment of bladder cancer. Mansure et al. showed the effects of everolimus on bladder cancer in vitro, on a variety of cell lines, including two metastatic ones, and observed cytostatic effects of this drug in vivo models; cell cycle analysis and TUNEL assays revealed no increase of the apoptotic SubG

1 fraction, but they registered significantly reduced

VEGF (vascular endothelial growth factor) levels after treatment with everolimus, suggesting that the biological effect might be due to inhibition of tumor angiogenesis [81]. Chen et al. performed one of the first studies concerning the role of germ cells genetic variations in the PI3K pathway in bladder cancer [82]. They evaluated a large number of single-nucleotide polymorphisms (SNPs) in 19 genes in PI3K/Akt/mTOR-signaling searching for cancer risk predictors. They found four SNPs that were significantly associated with developing bladder cancer and hypothesized that heavy smoking was a strong environmental risk factor. More in detail, smokers had high nicotine concentration in their blood and urine, which stimulates the nicotinic acetylcholine receptors (nAChRs) in the brain and promotes cancer cell growth by activating EGFR (epidermal growth factor receptor) and β-adenoreceptor and involving ERK1/2 (extracellular signal-regulated kinases) and STAT 3 (signal transducer and activator of transcription 3) which disrupt the cell cycle progression. PTEN (phosphatase and tensin homolog) is a potent endogenous inhibitor of the PI3K/Akt/mTOR pathway and its alterations have been also described in bladder cancer [83]. For instance, in invasive UCC, PTEN was mutated or showed loss of heterozygosity in up to 30% of cases, whereas this percentage is

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reduced in superficial tumors (pTa/pT1) [84]. In addition, PTEN status might be important in influencing response to treatment, because it appears to mediate resistance to PI3K pathway-directed therapy [85]. In addition to its direct effects on cancer cell processes, PTEN may also have downstream effects via interaction with the p53 pathway, which is commonly mutated in bladder cancer as stated above. More in detail, both PTEN and p53 inactivation (but not alone) may promote bladder tumorigenesis and poor patient outcome. Although the PI3K/Akt pathway can be altered by loss of PTEN, mutations in PI3K itself may result in its constitutive activity. One of the most common mutations specific to PI3K is in PIK3CA, which encodes the catalytic p110a subunit of PI3K. Mutations are almost exclusively gain-of-function and have been identified in up to 27% of UCCs. The mutations are strongly associated with FGFR3 mutations found prevalent in superficial bladder tumors and are restricted to certain ‘hot spots’ in the gene, specifically in the helical (E542K and E545K) and in the kinase (H1047R) domains [86]. Akt activity appears significantly increased in UCC, although Akt and P-Akt levels are not significantly associated with tumor grade or stage but they affect mTOR activation. Elevated levels of active Akt have been proposed to mediate resistance to the pro-apoptotic cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Under phisiological conditions, TRAIL activity induces caspase activation, mitochondrial depolarization, and apoptosis [87]. Some studies confirmed this theory because they found that enhanced levels of Akt protected bladder cancer cell lines from TRAIL-induced apoptosis, limited mitochondrial cytochrome-c release and caspase-mediated apoptosis. Finally, increased activity of Akt may also be due to single point mutations in bladder cancer, but, given the rarity of these mutations, they are unlikely to contribute to the development of UCC. Conversely, Akt inhibition sensitizes these cells to chemotherapy treatment [88], because Akt may also play a role in DNA methylation, which is catalyzed by DNA methyltransferase 1, thus affecting gene expression and chromatin stability. Lastly, the TSC complex (TSC1/TSC2) has effects directly downstream of Akt and is important in mTOR inhibition. Not surprisingly, loss of TSC1 function occurs in 14.5% of UCCs, resulting in increased mTOR signaling and resistance to apoptosis. The TSC2 portion of this inhibitory complex has a critical role in mTOR inhibition but, so far, mutations in TSC2 have not been characterized in detail in UCC [89].

5.2. EGFR Inhibitors In recent years, several agents targeting the EGFR signaling pathway have been developed because EGFR is over-expressed up to 48% in bladder cancer [90]. These drugs include small molecule tyrosine kinase inhibitors (gefitinib, lapatinib, and sorafenib) shown in Fig. (3) as well as monoclonal antibodies (cetuximab and trastuzumab). In a phase II study, Philips et al. treated 27 patients with advanced urothelial carcinoma with GC plus gefitinib, an oral EGFR tyrosine kinase inhibitor, but this regimen was associated with excessive toxicity [91]. Sorafenib, which mainly targets VEGF and PDGF (platelet-derived growth factor) receptors, is currently under phase II investigation for advanced and metastatic bladder cancer [92]. Cetuximab is a recombinant, human/murine chimeric monoclonal antibody that binds specifically to the extracellular domain of the human EGFR/HER1. Treatment with cetuximab results in EGFR inhibition and downregulation. From the analysis of the preclinical and clinical data, it is possible to demonstrate the importance of the epidermal growth factor pathway in urothelial carcinoma, and the enhanced clinical efficacy of associating cetuximab with standard chemotherapy (paclitaxel) for its inhibitory effects in metastatic bladder cancer [93]. Human epidermal growth factors are involved in oncogenesis through its effects on several pathways leading to proliferation, angiogenesis, cell survival, and metastatic potential. Her-2 (human epidermal growth factor receptor 2) expression in bladder carcinoma is variable, ranging from 9 to 81%. The real incidence of Her-2 amplification or overexpression is not well known, ranging from 23 to 80% for overexpression and from 0 to 32% for amplification. These variations can be explained, at least in part, by variability in immunochemistry (IHC) assays, regarding cut-off values, antibodies, kits, and protocols. This is why FISH (fluorescent in situ hybridization) seems to be a better approach, as it provides more objective results. Therefore, standardized methods are needed to avoid this heterogeneity in Her-2 testing in bladder cancer. Although there are some interesting preclinical data, there are no phase III trials in bladder cancer exploring the use of anti-Her-2 therapies, and the clinical experience with these agents is still very limited. The best benefit from an anti-Her-2 therapy is restricted to those cases with a recognized Her-2 amplification [94]. Activation of the EGFR pathway has been correlated with clinical outcome in bladder cancer, but its influence in prognosis

Fig. (3). Small molecules as tyrosine kinase blockers.

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has not been clearly stated yet. One study, published in 2005, proved the activity of trastuzumab in six patients with metastatic urothelial carcinoma. These patients had a positive IHC for Her-2 and achieved a partial response, ranging from 30 to 80% reduction in the size of the metastatic lesions [95]. In 2007, a phase II trial conducted by Hussain et al. tested, on 44 patients with Her-2-positive tumors, the combination of carboplatin, paclitaxel, gemcitabine, and trastuzumab in advanced urothelial carcinoma. Five of these patients achieved a complete response, 26 had a partial response, 5 had stable disease, and 5 had no response assessment (overall response rate of 70%) [96]. Finally, the clinical development of anti-Her-2 therapies, mainly trastuzumab, has shown us mostly the same problems. Although some studies with chemotherapy combined with trastuzumab achieved high response rates in advanced disease, no current phase III trial with this schedule is ongoing. Perhaps this might be due to inappropriate patient selection or lack of standardized Her-2 assessment by FISH technique. Lastly lapatinib, a tyrosine kinase inhibitor of EGFR and Her-2, has also been tested in BC, but with disappointing results. Some preclinical data with this drug showed promising activity in transitional carcinoma cell lines, enhancing the activity of concomitant chemotherapy in a dose-dependent way [97].

6. VIRUS THERAPY Superficial bladder cancer is a very attractive target for virus-based gene and local immunotherapy due to the confined bladder area, direct delivery, evaluation via transurethral approach, and immunosensitivity of BC as established by BCG therapy. Transgene/GM-CSF (granulocyte-colony stimulating factor) production, possibly virus replication, and anti-tumor activity have been reported in early trials. Innovative studies with dendritic cell-based therapy have been approved by FDA (Food and Drug Administration) and focused on establishing activity versus a standard control group (chemotherapy or BCG). Despite these advantages, virus-based antineoplastics have only been explored in a few studies so far [98]. The first trial of virus-based therapy for bladder cancer started ten years ago with a vaccine strain of replication-competent vaccinia virus. The study examined the treatment of four patients with advanced bladder cancer (invading muscle or tissues surrounding the bladder) with three instillations of vaccinia virus at maximum doses of 108 PFU (plaque forming unit) prior to cystectomy and serious side effects were not reported after this therapy. Post-resection evaluation showed inflammatory infiltration including dendritic cell migration to the site of virus infection, as well as evidence of virus infection in both normal and tumor cells [99]. Pagliaro et al. investigated the use of a replication-defective serotype 5 adenovirus for bladder cancer. In this investigation, 13 patients with locally advanced bladder cancer were treated with a replication-defective CMV-driven P53 adenoviral construct, INGN 201. Multiple doses of 1010-1012 virus particles were administered and no dose limiting toxicity was encountered. In terms of vector delivery and gene expression, 2 of 7 patients showed evidence of transgene expression via RT PCR (reverse transcriptase-polymerase chain reaction); however, immunohistochemistry did not reveal changes in p53 apoptotic pathway regulation. The authors reported a single patient showing possible evidence of response [100]. SCH 58500, another replication-defective adenovirus serotype 5 expressing P53, was infused, before cystectomy, to patients with advanced bladder cancer at single doses up to 7.5 x 1013 virus particles in conjunction with the transduction-enhancing agent Big CHAP. The improved infection observed with BIG CHAP was attributed to the modification or removal of the glycosaminoglycan protective layer that covers the internal surface of the bladder. Also

with this approach, no clinically significant toxicities were reported [101]. Mckiernan et al. reported preliminary results of a trial utilizing a replication-competent, E2F promoter-controlled, GM-CSF armed serotype 5 adenovirus, CG0070, for superficial BC after at least one prior BCG treatment course. Use of the E2F promoter to control E1a gene expression was designed to limit virus replication and GM-CSF production to RB gene and pathway-defective bladder tumors. The phase I-II trial focused on superficial bladder cancer, CIS, Ta, and T1 subgroups. Also in this evaluation to improve virus access to the urothelium, a glycosaminoglycan layer solubilizing agent, dodecylmaltoside, was used. Studies revealed high concentrations of GM-CSF and viral genomes in urine samples following treatment in all patients. Conversely, very low transient virus and GM-CSF levels were detected in the blood. Local toxicities (dysuria, bladder pain, and frequency) and flu-like symptoms were the most common adverse events observed [102].

7. APOPTOSIS AGONISTS One of the prerequisite for the correct use of compounds which target specific dysregulations in apoptotic pathways of BC cells is the altered expression of the apoptotic pathways regulators in many if not most cells of a particular clone constituting BC disease. The administration of pro-apoptotic drugs offers potential options for the improvement of BC treatment that works in vivo and in animal models. Unfortunately, none of these principles has so far successfully been translated into clinical trials for BC [103]. Because mutations and altered gene expression of p53 are very common in BC, the stimulation of the extrinsic pathway in order to reactivate the apoptotic capability of BC cells seems to be reasonable. This suggestion is supported by the finding that there is an upregulation of TRAIL in patients with intravesical BCG treatment for BC. Because high TRAIL levels in urine were associated with a good response to this local treatment, BCG therapy might be regarded as the first used pro-apoptotic treatment in BC therapy [104]. The death receptors (DR) have been one of the main targets of pro-apoptotic research in BC. Expression of DR4 and DR5 is an essential point for the sensitivity to treatments and polymorphisms, especially in DR4, might be useful markers. There are only few preclinical studies about the possible effect of mAb (monoclonal antibody) against death receptors in BC. For example, the anti-DR5 mAb lexatumumab induced apoptotic cell death in J82 and T24 human BC cells in vitro. This effect was enhanced by the combination of lexatumumab with cisplatin [105]. Administration of the death receptor-ligand FasL induced in vitro apoptosis in BC cell lines. The resistance to FasL induced apoptosis was increased by the in vitro induced overexpression of c-Flip (inhibitor of death receptor signaling) in BC cell lines, thus confirming that another possible therapeutic option is the inhibition of c-Flip. The combination of PI3K pathway inhibitors, gefitinib and wortmannin is mentioned in Figs. (3 and 4), respectively, with TRAIL showed synergistic effects in downregulation of active Akt, XIAP (X-linked inhibitor of apoptosis protein), and c-Flip and enhanced the TRAIL-induced apoptosis in EGFR-dependent human BC cells in vitro [106]. The dramatic prognostic role of Bcl-2 (B-cell lymphoma 2) overexpression in BC supported its role as a possible target for pro-apoptotic drugs. The treatment with antisense oligonucleotides against Bcl-2 or with BH3 (Bcl-2 homology domain 3) mimetics seems to be promising especially in those cases with TRAIL resistance. The BH3-mimetic (-)-gossypol induced apoptosis in chemosensitive and chemoresistant BC cell lines. In addition, a combined treatment with gossypol and gemcitabine/carboplatin induced apoptosis also in primarily chemoresistant BC cell lines. Downregulation of anti-apoptotic Bcl-2 and Bcl-XL


(B-cell lymphoma-extra large) has been the major subject of investigation. The inhibition of Bcl-XL by AS-ODNs (antisense-oligodeoxynucleotides) led to a sensitization to different chemotherapeutics in vitro. The same effect was described for Bcl-2 silencing in BC [107]. The overexpression of IAPs (inhibitor of apoptosis proteins, e.g., survivin) and downregulation of caspases in BC also make IAPs another interesting target for pro-apoptotic treatment. The importance of downregulation of IAPs in BC treatment in vitro was shown by AS-ODNs and, better, by siRNAs. Similarly, XIAP (X-linked inhibitor of apoptosis protein) was also downregulated by AS-ODNs followed by an increased sensitivity of BC cell lines to adriamycin. The synthetic SmacN7 penetratin peptide enhanced chemosensitivity for mitomycin C in BC cells in vitro by downregulation of XIAP and activation of caspase 3, while flavoprirdol induced apoptosis in T24 human BC cells and tumor reduction in a rat BC model. Another target for pro-apoptotic treatment in BC is clusterin. This chaperone protein plays an important role in apoptosis inhibition and chemoresistance in BC. Silencing of clusterin by AS-ODNs led to significant enhancement of cisplatin and gemcitabine effects in vitro. The combination of clusterin AS-ODN, cisplatin, and p53 gene transfer showed significant therapeutic effects of primary tumors and metastases in human BC model. A chemosensitization was achieved in gemcitabine-resistant BC cells by the administration of clusterin AS-ODN in vitro and in vivo [108]. NF-κB pathway inhibitors were also tested in human BC cell lines in vitro. The combination of bortezomib is shown in Fig. (1) with IFN-α induced apoptosis in human UM-UC-5 cells, while the compound curcumin is shown in Fig. (4), which increases expression of DR4, enhanced the apoptotic effects of paclitaxel, gemcitabine, TRAIL, and TNF [109].

8. ANGIOGENESIS INHIBITORS In tumoral tissues, neovascularization is required for tumors to grow and to metastasize to new sites. Bladder tumors produce high levels of angiogenic stimulatory factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and interleukin-8, and it is possible to correlate their levels with tumor stage and outcome. Microvessel density and levels of VEGF and bFGF are inversely associated with prognosis in TCC. Based on these findings, it has been hypothesized that targeting

angiogenesis pathways alone or in combination with standard chemotherapeutic regimens in TCC of the bladder would lead to an improvement in patient outcomes [110]. VEGF is the primary proangiogenic mediator of this process because its mRNA and protein are overexpressed in advanced BC compared with normal bladder epithelium. Recent in vitro experiments suggest a role for VEGF signaling as autocrine and paracrine growth factor which directly promotes bladder cancer growth, abnormal microvasculature, and interstitial fluid pressure. By reducing VEGF levels, not only the aberrant blood vessels are eliminated, but the microvasculature also appears to be remodeled [111]. Preclinical studies have shown that anti-VEGF strategies improved TCC chemotherapy in several human cancers in nude mice. For example, bevacizumab has been tested in combination with gemcitabine and carboplatin in patients not eligible for cisplatin chemotherapy providing important information on the role of bevacizumab and angiogenesis inhibition in advanced TCC. HOG (Hoosier Oncology Group) has completed in 2007 a phase II study using bevacizumab in combination with GC as first-line therapy in metastatic urothelial carcinoma, while CALGB (Cancer and Leukemia Group B) has planned a phase III randomized study of GC with and without bevacizumab as first-line therapy [112]. Within the same pathway, VEGF acts on two principle tyrosine kinase receptors, VEGFR1 and VEGFR2, both of which are overexpressed in most tumor vasculature and therefore represent attractive therapeutic targets. A more recent evidence showed that VEGFR2 is expressed in urothelial carcinoma cell lines and bladder tumors and that its expression level correlates to pathological stage. Targeting VEGFR2, therefore, has the potential to enhance apoptosis both in the tumor cells and the blood vessels. Several polymorphisms have been identified in the VEGF gene promoter associated with altered VEGF levels. Because bevacizumab directly inhibits VEGF, it appears that polymorphisms associated with higher VEGF levels influence response to antiangiogenic therapy [113]. Agents targeting the VEGF signaling pathway available for clinic use include bevacizumab, sunitinib in Fig. (5), and sorafenib as depicted in Fig. (3). Sorafenib is a Raf kinase inhibitor that also inhibits VEGFR2, VEGFR3, and platelet-derived growth factor receptor (PDGFR-β). A phase II multicenter trial with sorafenib in advanced or metastatic TCC demonstrated that, although the drug

Fig. (4). Compounds involved in modulation of apoptotic pathways.

O

O

OO

H3COO

O

O

H

Wortmannin

OHO

HO

HOOH

O

OH

OH

Gossipol

O

OOH

HO

Cl

N

HOH

Flavoprirdol

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H3CO

OH

OCH3

O OH

Curcumin


was well tolerated, it failed to show significant responses in the first-line setting. Another phase II trial in patients with advanced urothelial cancer, whose disease had progressed after treatment with platinum-based chemotherapy, also demonstrated that sorafenib as a single agent had minimal activity in the second-line setting in patients with advanced urothelial cancer. On the contrary, sunitinib only targets VEGFR2 and PDGFR-β and preliminary data from a phase II trial using sunitinib as second-line therapy for patients with progressive metastatic urothelial carcinoma showed clinical activity with this agent, as measured by partial responses in a minority of patients [114]. In a phase II study, Gallagher et al. administered sunitinib to 45 patients, with previously treated advanced urothelial carcinoma. Three of them had a partial response. A phase II trial is currently ongoing and is investigating the role of sunitinib as maintenance therapy in patients with advanced urothelial cancer [115].

Fig. (5). Agents targeting vascular endothelial growth factor (VEGF).

9. ACTIVATION OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ (PPARγ) PPARγ is highly expressed in a large number of solid malignancies, including bladder cancer. It is known that its activation inhibits cancer cell proliferation and angiogenesis, thus inducing apoptosis and playing an important role in carcinogenesis. PPARγ agonists are clinically and usually used as anti-diabetic drugs endowed with limited toxicity (cardiac failure, potential carcinogenicity in rodents). The effects of PPARγ agonists are

complex and do not always correlate with PPARγ activation, and for this reason, several authors have been searching for ligand-dependent and ligand-independent pathways responsible for the anti-cancer activities of these compounds in BC [116]. Peroxisome proliferator-activated receptors are ligand-activated intracellular transcription factors, which consist of three subtypes denoted PPARα, PPARβ/δ, and PPARγ. They exhibit distinct tissue distribution and inhibitor specificity reflecting their multiple biological functions. These nuclear receptors are phosphoproteins and their transcriptional activity is affected by cross-talk with phosphatases and kinases. In particular, PPARγ is highly expressed in many physiological tissues but also in tumors. More in detail, PPARγ is commonly expressed in bladder cancer and its level of expression is correlated with tumor grade and stage, suggesting that PPARγ agonists may mediate more potent anti-tumor effects in the more aggressive types of bladder cancer [117]. The prostaglandin J2 derivative, 15d-PGJ2 shown in Fig. (6), is the most potent endogenous ligand for the PPARγ receptor and is the most commonly used PPARγ-agonist. Other synthetic compounds, that can act as ligands, include the anti-diabetic thiazolinidinedione (TZD) such as troglitazone (TGZ), rosiglitazone, and some non steroidal anti-inflammatory drugs. In conclusion, the wide expression of PPARγ in many tumors and the ability of PPARγ ligands to inhibit cellular proliferation and angiogenesis and to promote differentiation and apoptosis, compelled researchers to postulate that PPARγ may play an important role in carcinogenesis [118]. The first studies reported that PPARγ agonists stimulated TCC differentiation and survival by increasing expression of adipocyte-type fatty acid binding proteins and loss of PPARγ has been associated with progression of bladder cancer. In addition, in bladder cancer, gene polymorphism concerning inflammatory factors such as IL-6 and PPARγ is associated with recurrence risk, progression, and survival. Patients with specific PPARγ variant alleles (SNP-Pro12Ala) show reduced PPARγ receptor activity and may provide favorable conditions for tumor growth. Recent studies have also demonstrated that in bladder tumor cells, which had weak or absent PPARγ expression, standard therapy with BCG improved expression of PPARγ. Additionally, it is noteworthy that the inhibition of cell viability by 15d-PGJ2 was only detected in the presence of BCG, and the effect of 15d-PGJ2

Fig. (6). Agonists of the peroxisome proliferator-activated receptor γ.

NH

O

NH

NH

O N

F

Sunitinib

O

COOH

15d-PGJ2

O

HO

ONH

SO

O

Troglitazone

ONH

SO

O

N

N

Rosiglitazone

O OO O

BADGE (bisphenol-A-diglycidyl-ether)

NH

HN

R

1,1-bis(3'-indolyl)-1-(substituted-phenyl)methanes

O

O OOCH3

COOH

Naveglitazar


was PPARγ-dependent, since BADGE (bisphenol-A-diglycidyl-ether), a specific PPARγ antagonist, reversed the BCG-mediated cell cytotoxicity [119]. Lastly, PPARγ may also target vascular neogenesis in BC as demonstrated by Possati et al. in a large number of human bladder tumor specimens [120]. The level of platelet-derived endothelial cell growth factor (PDECGF) is significantly correlated with tumor recurrence and poor prognosis. On the opposite, the concomitant expression of PPARγ was associated with significantly low incidence of tumor recurrence or progression suggesting a protective effect of PPARγ against PDECGF. Chaffer et al. investigated the effects of endogenous and synthetic PPARγ ligands on proliferation, growth arrest, and apoptosis in a series of transitional cell carcinoma of the bladder with increasing metastatic potential. They found that TGZ and 15d-PGJ2 induced growth inhibition in all bladder carcinoma cell lines although via PPARγ-independent mechanism. The former induced G0/G1 growth arrest, while the latter induced cell apoptosis [121]. Recently, Safe et al. has synthesized a new class of PPARγ agonists, 1,1-bis(3’-indolyl)-1-(substituted-phenyl)methanes, which activate PPARγ in different cancer cell lines (colon, pancreas, prostate, bladder, breast, endometrium, and kidney). Specifically, in bladder cancer cells in vitro and in vivo, these PPARγ agonists showed significant anti-tumorigenic activity and were more potent inhibitors of bladder cancer growth when compared with rosiglitazone. They decreased cell survival in bladder cancer cells and inhibited tumor growth in animal models through the induction of caveolin-1 and p21 expression, also if not all their effects are promoted by PPARγ activation but also by a cross-talk between PPARγ and EGFR signaling pathways through the modulation of glycogen synthase kinase-3 beta and cyclin D1. Taken together, combined targeting of EGFR and PPARγ can offer promising molecules to target bladder cancer [122]. On the opposite, there are studies which demonstrated the potential carcinogenic effects of some PPARγ agonists in rodents. For example, Long et al. investigated in rats, the effect of naveglitazar, a peroxisome proliferator-activated receptor (PPAR)α/γ dual agonist, in carcinogenicity. A significant increase in bladder cancer was observed only in the high-dose group of females after two years treatment. In another study the combination of rosiglitazone and hydroxybutyl(butyl)nitrosamine (OH-BBN), a urinary bladder specific carcinogen, developed more cancers as compared with rats treated with OH-BBN alone. Lastly, it is noteworthy to state that many of the carcinogenic effects of the PPARγ receptor agonists are highly species-specific, as observed in rodents but not in humans or primates [123]. Despite the therapeutic importance, the side effects associated with PPARγ agonist must be tested in large clinical trials. Some preclinical studies have suggested that ligand activation of PPARγ can promote carcinogenesis. However, these effects are controversial because the data depend on different parameters such as PPARγ subtype, animal model (rodent, non-rodent, non-human primate) and cancer type.

CONCLUSIONS AND FUTURE DEVELOPMENTS Bladder carcinoma is a disease with very limited therapeutic options in the advanced setting; moreover, adjuvant therapies in localized high-risk patients have not clearly demonstrated their efficacy, and neoadjuvant chemotherapy has not been largely implemented. All the treatment options discussed above have been given with intent to achieve a degree of disease control and aid symptom management. In addition, palliative cares are an integral part of the management of patients with urological cancers and should provide symptom control and psychological support [124]. The standard of care for first-line therapy of advanced urothelial carcinoma patients who are candidates for cisplatin

therapy is GC or M-VAC. Both of these therapies are not curative and better treatments are strongly needed. A large number of patients with metastatic urothelial carcinoma, following treatment with first-line therapy, eventually develop recurrences and have a poor long-term survival. Further emphasis on drug development research is needed for the discovery of more effective systemic therapies. Several novel drugs seem particularly promising including inhibitors of the EGFR pathway, such as cetuximab, and inhibitors of tumor angiogenesis, such as bevacizumab and sunitinib. These are currently being tested in clinical trials. Development of molecular predictive markers will also help to personalize therapy of urothelial cancer and is expected to improve responses to chemotherapeutic and biological agents.

DECLARATION OF INTEREST The authors state no conflict of interest and have received no payment in preparation of this manuscript.

ACKNOWLEDGEMENT Declared none.

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Received: July 22, 2011 Revised: October 06, 2011 Accepted: October 09, 2011

Current and Emerging Strategies in Bladder Cancer

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