Personalized Cancer Therapy for Urological Cancers: from Bench to Bedside and Back

Advances in Urology

Personalized Cancer Therapy for Urological Cancers: From Bench to Bedside and Back

Guest Editors: Hirotsugu Uemura, Colleen Nelson, Jack A. Schalken, and Laurence Klotz Personalized Cancer Therapy for Urological Cancers: From Bench to Bedside and Back Advances in Urology

Personalized Cancer Therapy for Urological Cancers: From Bench to Bedside and Back

This is a special issue published in “Advances in Urology.” All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Editorial Board

Sascha A. Ahyai, Germany Christopher M. Gon, USA Sangtae Park, USA M. Hammad Ather, Pakistan Narmada Prasad Gup, India Giorgio Pomara, Italy Darius J. Bagli, Canada Kostis Gyftopoulos, Greece Michael P. Porter, USA Steven B. Brandes, USA Jason M. Hafron, USA Alejandro R. Rodri, USA Robert E. Branniga, USA Daniel W. Lin, USA Jose Rubio Briones, Spain James A. Brown, USA William Lynch, Australia Matthew Rutman, USA Fabio Campodonico, Italy Martin Marszalek, Austria Douglas S. Scherr, USA In Ho Chang, Korea Viraj Master, USA Norm D. Smith, USA Felix Chun, Germany Brian Matlaga, USA A. Stenzl, Germany Peter E. Clark, USA Maxwell V. Meng, USA Nazareno Suardi, Italy Donna Deng, USA Axel S. Merseburge, Germany Flavio Trigo Rocha, Brazil Paddy Dewan, Australia Hiep T. Nguyen, USA Vassilios Tzortzis, Greece Miroslav L. Djordj, Serbia Alan Nieder, USA Willie Underwood, USA Carlos Estrada, USA Matthew Nielsen, USA Ouida L. Westney, USA Walid A. Farhat, Canada William K. Oh, USA Contents

Personalized Cancer Therapy for Urological Cancers: From Bench to Bedside and Back, Hirotsugu Uemura, Colleen Nelson, Jack A. Schalken, and Laurence Klotz Volume 2012, Article ID 298105, 2 pages

The Interactions between Insulin and Androgens in Progression to Castrate-Resistant Prostate Cancer, Jennifer H. Gunter, Amy A. Lubik, Ian McKenzie, Michael Pollak, and Colleen C. Nelson Volume 2012, Article ID 248607, 11 pages

Redeﬁning Hormone Sensitive Disease in Advanced Prostate Cancer,XiaoyuHouandThomasW.Flaig Volume 2012, Article ID 978531, 6 pages

Preclinical Remodeling of Human Prostate Cancer through the PTEN/AKT Pathway, Marco A. De Velasco and Hirotsugu Uemura Volume 2012, Article ID 419348, 12 pages

Progress in Personalizing Chemotherapy for Bladder Cancer, James S. Chang, Primo N. Lara Jr., and Chong-Xian Pan Volume 2012, Article ID 364919, 10 pages

The 5 Alpha-Reductase Isozyme Family: A Review of Basic Biology and Their Role in Human Diseases, Faris Azzouni, Alejandro Godoy, Yun Li, and James Mohler Volume 2012, Article ID 530121, 18 pages

Correlation of Gleason Scores with Diﬀusion-Weighted Imaging Findings of Prostate Cancer, Rajakumar Nagarajan, Daniel Margolis, Steven Raman, Ke Sheng, Christopher King, Robert Reiter, and M. Albert Thomas Volume 2012, Article ID 374805, 5 pages

AReviewofERCC1 Gene in Bladder Cancer: Implications for Carcinogenesis and Resistance to Chemoradiotherapy, Atsunari Kawashima, Hitoshi Takayama, and Akira Tsujimura Volume 2012, Article ID 812398, 6 pages Hindawi Publishing Corporation Advances in Urology Volume 2012, Article ID 298105, 2 pages doi:10.1155/2012/298105

Editorial Personalized Cancer Therapy for Urological Cancers: From Bench to Bedside and Back

Hirotsugu Uemura,1 Colleen Nelson,2 Jack A. Schalken,3 and Laurence Klotz4

1 Department of Urology, Kinki University Faculty of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan 2 Australian Prostate Cancer Research Centre-Queensland, Queensland University of Technology and Princess Alexandra Hospital, Level 1, Building 1, Ipswich Road, Brisbane, QLD 4102, Australia 3 Department of Experimental Urology, The Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands 4 Division of Urology, Sunnybrook Health Sciences Centre, 2075 Bayview Avenue MG 408, Toronto, ON, Canada M4N3M5

Correspondence should be addressed to Hirotsugu Uemura, [email protected]

Received 13 August 2012; Accepted 13 August 2012

Copyright © 2012 Hirotsugu Uemura et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A better understanding of key regulatory pathways perturbed historical account of ADT for metastatic prostate cancer and in cancers has led to the development of a molecularly summarizes current advancements in hormonal therapies targeted therapeutic approach. As a result, new generations using newly developed agents such as MDV3100, TOK-001, of compounds aimed at inhibiting specific pathways that play and TAK-700. prominent roles in urological cancers have been developed The association between insulin and androgens in and continue to evolve. The possibility of being able to prostate cancer progression is detailed by J. H. Gunter et al. perform molecular profiling in tumors would, at least in In this review, the authors describe the inverse relationship theory, provide the clinician with information needed to between insulin and testosterone levels and the metabolic manage a personalized therapeutic regimen according to an crosstalk between the two signaling axes. The authors discuss individual patient’s needs. While this modality is already the effects of ADT-induced hyperinsulinaemia and describe being implemented in other cancer fields, personalized the direct effects of insulin on prostate tumor cells and its medicine for patients suffering from advanced lower urogen- clinical implications. ital tumors is still in the developmental phase. This impetus The 5 α-reductase inhibitors represent an important fam- has prompted a concerted effort from investigators in the ily of enzymes that mediate the conversion of testosterone to fields of basic, translational, and clinical research to develop the more potent dihydrotestosterone (DHT). Consequently, and implement a tailored treatment approach. In this issue, several compounds have been developed to inhibit the we present a collection of seven articles that contribute to our activity of the enzymes to prevent and treat a number of knowledge of personalized medicine for urological cancers. diseases. The paper by F. Azzouni et al. goes into great detail Androgen deprivation therapy (ADT) for advanced to describe the basic biochemical properties and functions of prostate cancer is one of the earliest forms of targeted the 5 α-reductase isozyme family and its clinical significance therapy and has remained the choice of treatment by with regards to prostate cancer prevention and treatment. physicians. Unfortunately, most patients will eventually Gene inactivation of PTEN is a common occurrence in become non-responsive to ADT and succumb to the disease. prostate cancers and is associated to metastatic potential, Since its inception, knowledge for the understanding of androgen independence, and poor prognosis. The review by androgen receptor (AR) signaling and mechanisms driving M. A. De Velasco and H. Uemura chronicles the evolution the resistance to ADT has been significantly improved. As of the PTEN-deficient genetically engineered mouse (GEM) a result, a new generation of therapeutic agents has been and the cooperation between PTEN and other genetic developed. The paper by X. Hou and T. W. Flaig gives a alterations that contribute to tumor progression. The authors 2 Advances in Urology also describe the usefulness of GEM models for biomarker and drug discovery. Also in this issue, two reviews delve into the progress towards personalized medicine for muscle invasive and metastatic bladder cancer. J. S. Chang et al. review potential molecular biomarkers currently under investigation and discuss their future. In the second paper A. Kawashima et al. review the excision repair cross-complementing group 1 (ERCC1) gene and its role in tumor development and therapeutic resistance to cytotoxic DNA-damaging chemotherapy and ionizing radiation. Finally, in the original paper by R. Nagarajan et al. the authors use the apparent diffusion coefficient, derived from diffusion-weighted (DWI) imaging, to identify higher-grade prostate cancer lesions. Although the data is preliminary, the authors’ findings reveal a potential for the use of DWI imaging to differentiate those patients with high-grade lesions.

Acknowledgments We would like to thank all the contributors and hope that this special issue will be informative to our readers. We also wish that the contents included in this issue may in some way encourage others to begin or continue research in the ﬁeld of personalized medicine so that one day the realization of personalized may be fulﬁlled. Hirotsugu Uemura Colleen Nelson Jack A. Schalken Laurence Klotz Hindawi Publishing Corporation Advances in Urology Volume 2012, Article ID 248607, 11 pages doi:10.1155/2012/248607

Review Article The Interactions between Insulin and Androgens in Progression to Castrate-Resistant Prostate Cancer

Jennifer H. Gunter,1 Amy A. Lubik,1 Ian McKenzie,1 Michael Pollak,2 and Colleen C. Nelson1

1 Australian Prostate Cancer Research Centre-Queensland, Queensland University of Technology, Princess Alexandra Hospital, Level 1, Building 1, Ipswich Road, Brisbane, QLD 4102, Australia 2 McGill University, Jewish General Hospital, 3755 Cote-Sainte-Catherineˆ Road, Room E-740, Montreal, QC, Canada H3T 1E2

Correspondence should be addressed to Colleen C. Nelson, [email protected]

Received 27 October 2011; Accepted 6 January 2012

Academic Editor: Hirotsugu Uemura

Copyright © 2012 Jennifer H. Gunter et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

An association between the metabolic syndrome and reduced testosterone levels has been identified, and a specific inverse relationship between insulin and testosterone levels suggests that an important metabolic crosstalk exists between these two hormonal axes; however, the mechanisms by which insulin and androgens may be reciprocally regulated are not well described. Androgen-dependant gene pathways regulate the growth and maintenance of both normal and malignant prostate tissue, and androgen-deprivation therapy (ADT) in patients exploits this dependence when used to treat recurrent and metastatic prostate cancer resulting in tumour regression. A major systemic side effect of ADT includes induction of key features of the metabolic syndrome and the consistent feature of hyperinsulinaemia. Recent studies have specifically identified a correlation between elevated insulin and high-grade PCa and more rapid progression to castrate resistant disease. This paper examines the relationship between insulin and androgens in the context of prostate cancer progression. Prostate cancer patients present a promising cohort for the exploration of insulin stabilising agents as adjunct treatments for hormone deprivation or enhancers of chemosensitivity for treatment of advanced prostate cancer.

1. Prostate Cancer and the Metabolic Syndrome metabolic syndrome favour aggressive behaviour of prostate cancer but also difficulty in detection as the high rate of The metabolic syndrome describes a cluster of comorbidities benign prostatic hyperplasia (BPH) in obese men increases including abdominal obesity, elevated blood glucose, high the likelihood of missing abnormalities with prostate biop- cholesterol, and hypertension, which increase the risk of sies [10] and misdiagnosis following screening using the developing diabetes and cardiovascular disease [1]. Many of prostate-specific antigen (PSA) biomarker; obese men have these factors have also been established as risk factors for been reported to have both lower measured PSA, due to in- prostate cancer, strongly suggesting that there is a metabolic creased blood volume and increased PSA concomitant with component to this malignancy [2–6]. increased prostate volume and BPH [3], making prostate ir- Epidemiological studies have also shown that patients regularities complicated to assess. with existing obesity are more likely to be diagnosed with Dyslipidaemia associated with the metabolic syndrome, higher-grade cancers and higher Gleason scores, have a high- including increased triglycerides and LDL and decreased er rate of positive surgical margins at radical prostatectomy, HDL, is also associated with increased prostate cancer risk and suffer a higher incidence of prostate cancer recurrence [11–13], and cholesterol-lowering medications such as sta- and higher risk of dying of prostate cancer than men with a tins are currently believed to exert most of their positive healthy body mass index (BMI) [7–9]. A number of factors effects via systemic reductions in total cholesterol [14]. Both are thought to contribute to these findings including not only dietary and de novo synthesis of free fatty acids have been abiologiceffect by which the endocrine abnormalities of the shown to promote prostate cancer cell survival [15, 16], 2 Advances in Urology but studies which characterise fatty acid profiles in prostate androgen receptor (AR) give rise to a promiscuous receptor disease remain controversial [17, 18]. which permits activation by other molecules including other The metabolic syndrome is associated with altered hor- steroid hormones and antiandrogens (e.g., flutamide) [38]. monal profiles for testosterone, insulin, IGFs, and oestrogen, Amplification of the AR gene has been reported in 30% of all of which are linked to prostate cancer [19–22]. An asso- tumour samples and is often accompanied by an increase ciation between the metabolic syndrome and reduced testos- in AR stabilization [35]. A third mechanism followed the terone levels exists [9, 23–26] which is not simply related to observation that, despite low circulating androgen levels with age [27, 28]. The specific inverse relationship between insulin ADT, intraprostatic levels of testosterone in CRPC are high and testosterone levels observed across the age range from [39]. We and others subsequently demonstrated that, in the pubertal boys and young men to the elderly (19–90+ years) face of ADT, prostate tumours upregulate expression of the [26, 28, 29] suggests that an important metabolic crosstalk enzymes necessary to synthesise their own androgens de exists between these two hormonal axes. novo [37, 40, 41] resulting in paracrine and autocrine supply of androgens in the prostate tumour microenvironment to sufficient levels to reactivate AR-driven pathways and 2. Androgen-Deprivation Therapy and promote CRPC [40]. Castrate-Resistant Prostate Cancer Current standard cytotoxic chemotherapies have shown limited benefit for the treatment of CRPC, with modest Prostate cancer (PCa) is the most commonly diagnosed le- survival benefits of 2–5 months with docetaxel (Taxotere or thal cancer in men accounting for approximately one-third cabazitaxel) [42]. The discovery of de novo steroidogenesis in of all cancers with a relative lifetime risk of 1 in 7. Its prostate tumours as a mechanism driving CRPC has played a incidence continues to rise with an ageing population, and significant part in rationalising the newly approved steroido- despite improved survival rates, it remains the second leading genic CYP17A1 inhibitor, abiraterone, which is showing cause of cancer deaths in western men [30, 31]. In general, great clinical promise for improved control of CRPC [43, 44]. patients with organ-confined PCa are initially treated with However ∼50% of men treated are or ultimately become radical prostatectomy or radiation-based therapies; however, resistant to abiraterone highlighting the urgent need to un- 25–40% of patients will experience biochemical recurrence derstand the factors driving this resistance to develop alter- defined by a rise in prostate-specific antigen (PSA), an an- native or adjuvant treatment options [45]. drogen-regulated gene which in these patients acts as a bio- What is striking in patients undergoing ADT is the rapid marker of recurrent prostate tumour growth and metastatic onset of several key features of the metabolic syndrome in progression [32]. For decades the most common treatment men with no preexisting metabolic dysfunction. for metastatic PCa has been androgen-deprivation therapy (ADT) which suppresses testicular testosterone production. Androgen supply is controlled centrally via the hypo- 3. Androgen Deprivation Causes thalamic-pituitary-testicular axis. Luteinizing hormone-re- Metabolic Dysfunction leasing hormone (LHRH) is released from the hypothalamus to activate the anterior pituitary to produce Luteinizing hor- While ADT is initially an effective treatment for prostate mone (LH), which stimulates testosterone production from cancer for most patients, the systemic side effects include the Leydig cells of the testes. LHRH production is eventual- key features of the metabolic syndrome. Patients typically ly inhibited by the ligand-mediated activation of the an- experience a loss of muscle mass, increased fat mass, and the drogen receptor [33]. Androgen deprivation is generally development of central adiposity, hyperlipidaemia, increased achieved using the class of LHRH agonists such as goserelin risk of cardiovascular mortality, hyperglycaemia, and the acetate which disrupts pituitary stimulation and causes dra- consistent feature of hyperinsulinaemia [46–48]. These in matic decreases in LH production and subsequent castrate turn are associated with poor outcomes including more rapid testosterone levels. progression to advanced disease and increased cancer mor- Most patients initially respond to ADT; however, after tality [6, 49, 50]. Moreover, recent studies have specifically a median 18–36 months patients recur with rising PSA identified a correlation between elevated insulin/C-peptide levels despite castrate androgen levels in the serum. This is levels (normalized surrogate insulin levels) with high-grade termed castrate-resistant prostate cancer (CRPC) and leads PCa and worse patient prognosis [6, 9, 47, 51–54]. Pre- to significant comorbidities and inevitable mortality [32, 34– diagnostic body mass index (BMI) and C-peptide correlate 36]. The recurrent expression of PSA following the nadir with the risk of prostate cancer-specific mortality, and obese with androgen deprivation therapy implies resumption of men (BMI > 30) were more likely to have extraprostatic or androgen receptor activation during progression to castrate metastatic prostate cancer, or a higher Gleason grade of resistance. Similarly the fact that up to 30% of patients cancer at the time of diagnosis than men with BMI < 30. respond to secondary androgen axis manipulation during Of greater significance, patients with higher C-peptide had castrate resistance implies an ongoing dependence on the an increased prostate cancer-specific mortality compared to androgen receptor pathway in these patients [37]. At least those with low C-peptide levels suggesting that at least part of three mechanisms contribute to the reactivation of the the effect of increased BMI on mortality was related to coin- androgen receptor in prostate cancer progression. Mutations cident hyperinsulinaemia [6]. Similarly, a nested case-control or splicing events to the ligand binding domain of the trial within the Prostate Cancer Prevention Trial found Advances in Urology 3 that while increasing C-peptide level was weakly associated adipose tissue in obesity may increase the rate of conversion with cancer risk, there was a strong association with the of testosterone to oestradiol, a more potent inhibitor of development of high-grade prostate cancer of Gleason grade LHRH secretion from the hypothalamus. Studies investi- 7 or greater. In contrast to the Ma study, this association gating the levels of oestradiol in these patients, however, was found to be independent of BMI [54]. J. Hammarsten show mixed results. Decreased testosterone is coupled with and B.Hogstedt¨ prospectively assessed baseline insulin levels increased oestradiol in some studies [65, 66] while others at time of prostate cancer diagnosis and compared them have shown reduced oestradiol concentrations in hypogo- between men who died from prostate cancer during 5 years nadal men [67, 68], consistent with reduced testosterone of followup and men who survived. Statistically significant substrate. Differences in the sensitivity of detection methods risk factors identified for lethal prostate cancer included both and duration of condition may account for discrepancies in type 2 diabetes and hyperinsulinaemia. But only hyperin- these reports. Hypogonadism as a result of age or in lean men sulinaemia remained significant after adjusting for stage and with type 2 diabetes is likewise associated with reduced levels grade of prostate cancer, factors known to independently of oestradiol suggesting that decreased testosterone levels in affect prognosis, strongly suggesting that hyperinsulinaemia these men is not likely to be due to oestradiol-mediated is the key promoter of prostate cancer progression associated suppression of LHRH secretion [62, 68]. with metabolic dysfunction [53]. Thus, while ADT initially LHRH and LH secretion is suppressed in animal models is effective treatment for prostate cancer, the metabolic by increased circulating cytokines which are elevated in complications of ADT may not only cause multisystem obesity [69, 70]. Related changes to the adipokine secretion morbidity (obesity, increased risk of stroke, and so forth) but profile of men receiving ADT may also contribute to reduc- also lead to an altered hormonal environment that favours tions in insulin sensitivity [47, 71]. Leptin and adiponectin, the development of castrate-resistant behaviour. circulating factors secreted by adipose tissue with known modulatory functions on insulin sensitivity, are both elevated during ADT [71, 72]. Leptin plays an important role in 4. Chicken or the Egg: The Interrelationship appetite and energy balance, immune modulation, and bone between Insulin and Testosterone homeostasis [73] and is secreted from fat tissue in proportion to adiposity. Circulating levels of leptin are increased during While we have just discussed the increased insulin resistance ADT, in line with increased deposition of fat in these men; and hyperinsulinaemia which result from pharmacological however, leptin has been shown to increase even in the inhibition of testicular testosterone production, it is also true absence of discernable weight gain following 28 days of ADT, that pre-existing hyperinsulinaemia, such as that seen in type although relative fat/lean mass was not measured in this II diabetes, is associated with reduced testosterone levels. study [71]. The profile of adiponectin secretion is generally The inverse relationship between testosterone and insulin opposite to leptin with reduced expression and secretion in males without cancer has been well documented; yet the with increasing adiposity [74]. However, adiponectin is nor- mechanisms linking these two hormonal pathways remain mally suppressed by testosterone [75–77], and the resulting poorly understood [24, 55, 56]. increase in adiponectin following ADT is attributed to the Reducedlevelsoffreeandtotaltestosteronehavebeen loss of suppression by androgen. The insulin-sensitising associated with type 2 diabetes (T2DM), central adiposity, effects of adiponectin which may be present following ADT, dyslipidaemia, and hyperinsulinaemia in various studies however, fail to overcome the effects of androgen withdrawal [24–26, 57–61], usually in combination with reduced LHRH on the development of hyperinsulinaemia [71, 72]. and LH levels however, the observation of low testosterone A recently published paper by Rubinow et al. manipu- levels remains relatively rare in men with type 1 diabetes lated testosterone levels in young–middle-aged healthy men, suggesting that hyperglycaemia is not a direct cause. In excluding patients with confounding underlying conditions addition, low androgen levels are observed in lean men with such as prostate cancer, diabetes, and hypogonadism [71]. T2DM suggesting that raised BMI is not necessary for the Groups were randomised to receive ADT alone, ADT persistence of hypogonadism [62]. In population studies, with testosterone replacement, and ADT with testosterone following adjustment for age and adiposity, insulin was replacement and an aromatase inhibitor. Decreased insulin found to be significantly and inversely correlated to free and sensitivity was observed in the men receiving ADT only, in total testosterone levels [24]. Further, a study of middle-aged line with >90% reduction in circulating testosterone levels men followed over 11 years found that low testosterone levels which was observed in the absence of changes to body were predictive for development of the metabolic syndrome weight and fasting glucose concentrations. Insulin sensitivity and heralded as a predictive biomarker of metabolic and was not affected by reduced oestradiol levels in participants diabetic pathogenesis [58]. In contrast, high testosterone receiving the aromatase inhibitor suggesting that testosterone levels are linked to insulin sensitivity, and pharmacological is the major regulator of insulin sensitivity in healthy males. improvements to insulin sensitivity increase testosterone Similar observations have been made of men receiving ADT levels [63, 64]. for prostate cancer. The rapid withdrawal of androgens with Several mechanisms have been proposed which may ADT causes hyperinsulinaemia and loss of insulin sensitivity contribute to reduced testosterone/insulin resistance in these in these patients, reflected by increased homeostatic model cohorts. Testosterone can be converted to oestradiol through assessment (HOMA) score, within 2 weeks [78] suggesting the irreversible action of aromatase in adipose tissue. Excess that this is a direct effect of ADT and not subsequent to 4 Advances in Urology changes in fat mass. On the contrary, studies have shown that insulin resistance, defined by impaired glucose uptake and insulin sensitivity following ADT is independent of fat mass metabolism, as occurs in insulin-sensitive metabolic tissue, and age [48, 79]. hyperinsulinaemia would be expected to increase insulin These observations provide strong evidence for an im- signalling in PCa cells [81] in parallel to the elevated levels portant functional role for hyperinsulinaemia in PCa proof ligand. gression and CRPC following androgen deprivation. Major The molecular bases for the inverse clinical observations findings from recent studies [54, 80]ofmenreceivingADT between levels of insulin and testosterone are less well de- demonstrated a strong trend between an elevated C-peptide scribed. The androgen receptor is a member of the steroid level and more rapid progression to CRPC. Although there hormone receptor family and classically controls transcrip- is mounting epidemiological evidence linking hyperinsuli- tion of androgen-regulated genes in a ligand-dependent naemia and CRPC, the mechanisms of insulin action directly manner; however, androgens can also elicit rapid signalling on PCa cells in the context of ADT has, until recently, not responses independent of the AR [86, 87]. Androgen receptor been the subject of biological scrutiny. signalling plays a role in metabolic function. Direct AR- mediated effects of testosterone on fat metabolism are evidenced by AR knockout mice which have increased 5. Potential Mechanisms of Insulin-Androgen adiposity, accompanied by elevated leptin and adiponectin Crosstalk in the Prostate profiles and altered lipid metabolism [88], and men with genetic androgen resistance due to defective AR expression Traditionally insulin has been primarily considered a hor- have increased central adiposity [89]. AR signalling in muscle mone essential for metabolic regulation; however, insulin can tissue may also affect systemic insulin sensitivity. AR acti- also activate lipogenesis, steroidogenesis, protein synthesis vation favours development of pluripotent stem cells down and antiapoptotic survival pathways in many cell types [81, the myogenic lineage via AR-dependent activity through 82]. Insulin positively and negatively regulates approximately noncanonical Wnt signalling, favouring the formation of 150 genes; however, transcriptional factors act differently in muscle while suppressing the formation of new fat tissue different target tissues; thus, insulin affects transcription by (adipogenesis) [90]. AR activation in muscle tissue also modulation of the level, localization, and activity of tran- increases oxidative metabolism and insulin sensitivity via scription factors differently in specific microenvironments upregulation of the transcription factor PPARγ coactivator [82]. Insulin signals through its cognate receptor of which 1α (PGC1α) which stimulates mitochondrial biogenesis and there are two isoforms, INSR-A and INSR-B [81], that increases the oxidative potential of skeletal muscle; decreased belong to a family of receptor tyrosine kinases that includes testosterone levels are associated with decreased PGC1α lev- the receptor for insulin-like growth factor 1 (IGF-1R). els and increased insulin resistance. The activation of PGC1α Many tumour types have upregulated expression of IGF-1R, presents a potential mechanism for cancer cell survival INSR, and potentially hybrid INSR/IGF-1Rs which facilitate that may confer increased resistance to oxidative stress and increased activation of mitogenic, prosurvival and protein cellular senescence by increasing oxidative phosphorylation synthesis pathways with the increased levels of ligands insu- in the tumour cell [91, 92]. lin, IGF-1, or IGF-2 [83–85]. IC50 values, calculated for each Recent reports have identified previously unappreciated ligand and each receptor, reveal that IGF-1 can bind the crosstalk in prostate cancer cells between the AR pathway INSR-A with ∼2.5% the efficiency of insulin and with even and PI3K signalling pathway, the major signalling pathway weaker affinity to INSR-B. Likewise, insulin, at physiological activated downstream of the INSR [93–96]. The PI3K path- levels, will not activate the IGF-1R [81]. In contrast, insulin way has been implicated in a number of malignancies [97] and IGF-1 can activate signalling with varying potency including prostate cancer. Approximately 40% of primary through the hybrid INSR/IGF-1R. IGF-2 is able to signal and 70% of metastatic prostate cancers have mutations through each of the INSR-A (weakly binds INSR-B) and within the PI3K signalling pathway, mostly associated with IGF-1R as well as hybrid INSR/IGF-1R. Ligand binding to a loss of the negative regulator, PTEN [98–100]. Reciprocal the INSR and IGF-1R activate numerous downstream path- feedback regulation between androgen receptor signalling ways including phosphatidylinositol 3-kinase (PI3K)/Akt and unfettered signalling through the PI3K-AKT-mTOR and Ras/MAPK pathways with many well-characterised pathway in prostate cancer [93, 95] have been shown to downstream effects including increased glucose metabolism, inhibit AR signalling and suggest a possible pathway to inhibition of apoptosis (e.g., via FOXO and BAD-mediated androgen-independent growth of prostate tumours [93], pathways) and stimulation of cell proliferation (e.g., via and conversely, inhibition at each of these signalling nodes mTOR) (Figure 1)[81]. was associated with enhanced AR signalling and increased While the role of IGF-1 in cancer has been recognised for transcription of AR-responsive genes (Figure 1). over 20 years, the presence of the INSR directly on prostate Hyperinsulinaemia and increased insulin signalling in tumour tissue has only recently been reported and show prostate tumour cells as a result of androgen deprivation that increased INSR expression correlates with increasing are likely to activate survival pathways downstream of the Gleason grade and CRPC [51] providing further evidence insulin receptor which have the potential to contribute that insulin and insulin receptor signalling may have a critical to progression to castrate resistance; thus, these candidate role driving progression of advanced prostate cancer. As molecules downstream of insulin receptor signalling may prostate epithelial cells are not subject to development of have therapeutic utility in advanced prostate cancer. Advances in Urology 5

Insulin Insulin IGF1 IGF1 Testosterone IGF2 IGF2 IGF2 Metformin

INSR IGF-1R Hybrid R Migration Invasion Proliferation Testosterone Altered glucose Ras/MAPK PI3K/Akt metabolism +LKB1 STAT3 DHT AMKK2 +C

+LKB1 AMPK AR Antiapoptosis, survival BAD mTOR resistance to hypoxia Bcl FOXO

4EBP1 +LKB1 eiF4E AR Antiapoptosis SREBP transcription ARE Proliferation mRNA Cell cycle ACC activity progression Protein synthesis control of Nucleus cellular energy Fatty acid synthesis

Figure 1: Insulin receptor (INSR) belongs to a family of receptor tyrosine kinases that includes the receptor for insulin-like growth factor 1 (IGF-1R). Many tumour types have upregulated expression of IGF-1R, INSR, and potentially hybrid INSR/IGF-1Rs which facilitate increased activation of mitogenic, prosurvival and protein synthesis pathways following activation by of ligands insulin, IGF-1 or IGF-2. IGF-1 can bind the INSR with 2.5% the eﬃciency of insulin. Insulin, at physiological levels, will not activate the IGF-1R. Insulin and IGF-1 can activate signalling with varying potency through the hybrid INSR/IGF-1R. IGF2 is able to signal through each of the INSR, IGF-1R, and hybrid receptor (Hybrid R). Ligand binding to the INSR and IGF-1R activate numerous downstream pathways including phosphatidylinositol 3 kinase (PI3K)/Akt and Ras/MAPK pathways with many well-characterised downstream eﬀects including increased glucose metabolism, inhibition of apoptosis (e.g., via FOXO and BAD-mediated pathways), and stimulation of cell proliferation (e.g., via mammalian target of rapamycin; mTOR). Reciprocal feedback regulation occurs in prostate cancer cells between the AR signalling and signalling through the PI3K-AKT-mTOR pathway. Unfettered activity through this pathway, associated with the common PTEN mutation, inhibits AR signalling and suggests a possible pathway to androgen-independent growth of prostate tumours. AMP-activated protein kinase (AMPK) is a potentially bifunctional modulator in prostate cancer cells. Activation of AMPK modulates insulin signalling by negatively regulating mTOR control of protein synthesis and halting cell proliferation. AMPK activation also promotes fatty acid oxidation and downregulates expression of SREBP and activity of acetyl-CoA carboxylase (ACC) resulting in decreased fatty acid synthesis. However, AMPK has complex regulation in prostate cancer cells and may potentiate increased proliferation and migration when activated downstream of the AR under the control of AR responsive regulator calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2). AR directly regulates CAMKK2 and upregulates AMPK which mediates AR-induced migration and invasion in a CAMKK2-dependent manner. In studies where metformin activation of AMPK results in cessation of cancer cell growth, signalling is thought to be through LKB-1 tumour suppressor; therefore, competition for AMPK signalling via LKB1 stimulation versus AR-mediated CAMKK2 activation could result in altered functional outcomes. DHT, dihydrogen testosterone; AR, androgen receptor; ARE, androgen response element; MAPK, mitogen-activated protein kinase.

6. Diabetes, Prostate Cancer, and Metformin primarily via reduced hepatic glucose output, and promising data emerging from clinical studies suggests that metformin Diabetes has been shown to be associated with increased may improve patient outcomes in prostate and other cancers risk of several cancers including colon, pancreatic, and [106–110]. A retrospective cohort study compared diabetic breast cancer [101]. In contrast studies have found that patients on metformin against matched diabetic patients not diabetics have lower PCa risk than nondiabetics [102, 103], receiving metformin and showed a significantly decreased perhaps related to decreased levels of androgens [103– risk of cancer diagnosis of the metformin group with an 105]. Metformin, which works in part by activating AMP- adjusted hazard ratio of 0.63 (95% CI 0.53–0.75) and activated protein kinase (AMPK), is used clinically in obese decreased cancer-related mortality, as well as a prolonged and diabetic patients to normalise circulating insulin levels median time to cancer diagnosis [111]. These results were 6 Advances in Urology supported by an observational study of 11,876 diabetic activation of AMPK was sufficient to increase cell migration patients demonstrating a 33% decreased risk of develop- via potentiation of Rac1 activity, a regulator of cell migration ing cancer with metformin treatment compared to other [122, 125].Alternativecofactorsyettobeidentifiedmay treatments [112]. In contrast, metformin gave no benefit regulate this effect of AMPK, specifically downstream of AR to prostate cancer risk compared to controls in a recent signalling. report; on the contrary, metformin treatment was reported In studies where metformin activation of AMPK results to increase the risk of prostate cancer diagnosis back up in cessation of cancer cell growth, signalling is thought be to normal levels [113] possibly due to the normalisation of through LKB-1 tumour suppressor [126]; therefore, the dif- androgen levels associated with stabilisation of insulin levels. ference in these functional outcomes of AMPK activa- Based on these results, targeting AMP-activated protein tion could be the company it keeps. Competition for kinase (AMPK) has been proposed as a therapeutic strategy AMPK signalling via LKB1 stimulation versus AR-mediated in cancer [114, 115]. Reports indicate that pharmacological CAMKK2 activation could result in decreased mTOR sig- activation of AMPK in cancer cells by either metformin nalling and decreased glucose (fuel) for anabolic pathways. or AICAR results in halting cell proliferation by negatively Taken together, it seems that AMPK at the cellular level is regulating mammalian target of rapamycin (mTOR) control a potentially bi-functional modulator. This latter role for of protein synthesis (Figure 1)[101, 116–118]. In vitro AMPK is in contrast to earlier reports which show that studies of metformin also demonstrate an antitumoral effect metformin reduced cancer cell proliferation [107, 117, 127, in prostate cancer cells by blocking cell cycle progression 128] via inhibition of anabolic pathways such as lipogenesis and decreasing cyclin D1 protein levels resulting in reduced [119, 129] starving the major bioenergetic pathway in LNCaP xenograft tumour growth [107]. AMPK activation prostate cancer cells [130]. Nevertheless, we should interpret also promotes fatty acid oxidation reducing availability of with caution the effect of metformin from men with pre- fatty acids for biosynthetic pathways and downregulating existing obesity or type 2 diabetes where, in contrast to expression of SREBP and phosphorylation/deactivation of other cancers, diabetes appears to protect from cancer, while acetyl-CoA carboxylase (ACC) resulting in decreased fatty metformin may increase the risk of diagnosis or more likely acid synthesis (Figure 1)[119]. mitigate the PCa risk-reducing benefit of diabetes in these Activation of AMPK modulates insulin signalling down- patients. Several clinical trials are currently addressing the stream of the insulin receptor [120], most notably via benefit of metformin treatment in men on ADT without differentially phosphorylating the tuberous sclerosis complex pre-existing diabetes. The outcomes of these trials will be of TSC1-TSC2 to inactivate mTOR [118, 121]. The ability of significant clinical interest. AMPK to potentiate insulin action on cancer cell growth and survival has not been greatly explored in models of prostate cancer. Indeed the effects of metformin have not 7. Targeting the Insulin Axis in Advanced yet been demonstrated to be direct effects on cancer cells Prostate Cancer or an indirect consequence of systemic insulin normalisation [115]. Nevertheless, the intersection between mTOR, insulin Reactivation of the AR following ADT, heralded by rising signaling, and AMPK provides an intriguing, tantalising link serum PSA, is a hallmark of CRPC progression. We and between cellular energy and cancer pathways. others have demonstrated in recent years that intratumoral Recent data suggests that AMPK activation may have androgen synthesis is a major contributor to reactivation more complex regulation in prostate cancer cells and may of AR in CRPC [39, 40, 131, 132]. Moreover, we have potentiate increased prostate cancer cell proliferation and noted that insulin promotes steroidogenesis in other cell migration when activated downstream of the androgen types [133–135]. From these merging hypotheses, we have receptor (AR) [122, 123]. Using elegant integrated bioin- recently reported that insulin, in the absence of androgen, formatic analyses of transcriptional profiling with AR ChIP may drive PCa progression in part through upregulation of analysis, Massie et al. recently identified AR-mediated upreg- SREBP and its downstream enzymes responsible for lipid and ulation of several metabolic pathways including increased steroid synthesis in cell models of prostate cancer, resulting aerobic glycolysis under the control of a master regula- in dramatically increased intratumoral steroid production tor calcium/calmodulin-dependent protein kinase kinase 2 which is sufficient to reactivate the AR to stimulate PSA (CAMKK2) [123]. AR directly regulates CAMKK2 by bind- expression and secretion [136]. Thus, insulin can act directly ing to its promoter and is highly expressed in normal on PCa cells to activate pathways contributing to CRPC prostate with elevated expression in both AR-sensitive and progression. CRPC models of prostate cancer [122, 123]. Intriguingly, In addition to activation of de novo steroid synthesis, AR activation was also shown to upregulate the primary insulin is capable of driving numerous transcriptional pro- CAMKK2 substrate AMPK [122–124] which mediated AR- grams predominantly downstream of PI3K/AKT signalling. induced migration and invasion in a CAMKK2-dependent Pathways activated in response to cell stress (e.g., glucose manner (Figure 1). 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Review Article Redeﬁning Hormone Sensitive Disease in Advanced Prostate Cancer

Xiaoyu Hou and Thomas W. Flaig

Division of Medical Oncology, University of Colorado Denver School of Medicine, 12801 E. 17th Avenue, Room L18-8117, Aurora, CO 80045, USA

Correspondence should be addressed to Thomas W. Flaig, thomas.ﬂ[email protected]

Received 4 August 2011; Accepted 20 December 2011

Academic Editor: Hirotsugu Uemura

Copyright © 2012 X. Hou and T. W. Flaig. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Prostate cancer is the most common cancer among men in the United States. For decades, the cornerstone of medical treatment for advanced prostate cancer has been hormonal therapy, intended to lower testosterone levels, known as Androgen Deprivation Therapy (ADT). The development of hormone-resistant prostate cancer (now termed castration-resistant prostate cancer:CRPC) remains the key roadblock in successful long-term management of prostate cancer. New advancements in medical therapy for prostate cancer have added to the hormonal therapy armamentarium. These new therapeutic agents not only provide a survival beneﬁt but also show potential for reversing hormonal resistance in metastatic CRPC, and thus redeﬁning hormonally sensitive disease.

1. Background (1728–1793) observed the relationship between prostate growth and testicular function for the first time in his Prostate cancer is the 2nd most frequently diagnosed cancer textbook Practical Observations on the Treatment of Diseases and the 6th leading cause of death among males worldwide of the Prostate Gland [4]. Louis Auguste Mercier (1811–1882) [1]. In 2011, it is estimated that there will be 240,890 new of France first performed orchiectomy for the treatment of cases of prostate cancer and an estimated 33,720 deaths due an enlarged prostate in 1857 [4]. In 1941, Huggins first used to prostate cancer in the USA [2]. A variety of primary estrogen to treat metastatic prostate cancer, which led to a treatment modalities exist to treat localized cancer of the Nobel prize in 1966, representing one of the first successful prostate including surgery, external beam radiation therapy, systemic therapies for cancer [5]. brachytherapy, cryosurgery, and High Intensity Focused Similar to breast cancer, prostate cancer is a hormonally Ultrasound (HIFU). However, for metastatic prostate cancer, driven solid malignancy. Androgens are the key driver of the initial treatment has traditionally been hormonal ther- growth for both the normal prostate and prostate cancer ff apy. ADT is e ective for 2-3 years on average, and when cells. Vital in the definition of castrate-resistant disease hormonal therapy fails, chemotherapy provides a survival is the recognition of the potential sources of androgen ff and palliative benefit, at the cost of considerable side e ects. and approaches aimed at the reduction of their levels. ADT has been given standalone and as an adjunct with other While we have historically focused on the testicular pro- treatment modalities such as chemotherapy, radiation, and duction of androgens, other sites of production include the surgery [3]. adrenal glands and the more recently appreciated source of intratumoral androgen production. Normally 90–95% of 2. History of Androgen Deprivation Therapy circulating testosterone is produced by the Leydig cells of the in Prostate Cancer testes. Another 5–10% of systemic testosterone is synthesized by the adrenal glands [6]. While production by the testis Hormonal therapy has long been an integral part of prostate is the main source of androgen in prostate cancer prior to cancer treatment. In 1811, Scottish surgeon John Hunter castration, it has now been appreciated that traditionally 2 Advances in Urology defined CRPC is frequently driven by intratumoral androgen 3. Current Advancement in Hormonal Therapy production and synthesis of testosterone from weak adrenal < androgen precursors [7, 8]. Historically, a serum testosterone level of 50 ng/dL < Given the multiple sources and associated biosynthesis ( 1.74 nmol/L) has been used as the benchmark to assess ffi pathways of androgen productions, hormonal therapy in the e ciency of hormonal therapy, as comparable to the prostate cancer is achieved through multiple mechanisms level of suppression achieved with surgical castration [26]. with several different classes of agents (Table 1). Subsequently, disease progression after achieving castration Though in limited use today, estrogen was initially used levels of serum testosterone has been used as the definition to systematically treat prostate cancer decades ago. Beyond of CRPC. For many years, clinicians lacked rigorously proven an agent simply to induce a reduction in testosterone levels, therapeutic hormonal options for the treatment of prostate Serrate et al. reported in 2009 that diethylstilbestrol (DES) is cancer after the development of castration-resistant disease, with only docetaxel chemotherapy providing a clear survival a reasonable option for salvage therapy for CRPC in patients benefit in this setting [27, 28]. However, the introduction of previously treated with docetaxel chemotherapy, suggesting several new hormonal agents has challenged the traditional a direct anticancer effect and larger “hormone sensitive” definition of CRPC. treatment window [9]. Surgical orchiectomy remains a viable The CYP17 enzyme is a member of the cytochrome option to achieve androgen deprivation to this day [10]. P450 family of enzymes that mediates the biosynthesis of Medical castration can be achieved by using gonadotropin- dehydroepiandrosterone and androstenedione, both precur- releasing hormone (GnRH) agonists such as goserelin, sors of testosterone. Previously, nonselective inhibitors of leuprolide, and histrelin acetate, which induce androgen CYP17suchasketoconazolehavebeenusedinprostate deprivation through persistent overstimulation and subse- cancer treatment [29]. In contrast, abiraterone acetate has quent downregulation at the level of the GnRH receptor been developed to selectively and irreversibly inhibit the [11, 12]. GnRH antagonists such as degarelix can also disrupt CYP17 enzyme (17α-hydroxylase and C17,20-lyase) [30] androgen production and are a more recent addition to and demonstrated significant antitumor activities against our medical treatment options [13]. Antiandrogens such prostate cancer in phase I/II clinical trials [31, 32]. Similar as bicalutamide, nilutamide, and flutamide block the effect to abiraterone, TAK-700 (Orteronel) is an investigational of androgen directly at the androgen receptor, although CYP17 inhibitor, that may offer a more precise targeting of the blockade of the androgen receptor is incomplete and the CYP17 biosynthesis pathway by inhibiting only C17,20- partial agonist properties are observed with these agents [14]. lyase [33]. Clinical trials of TAK-700 are currently ongoing In contrast, MDV3100 is a new generation antiandrogen [34]. TOK-001 (Galeterone) is another promising investi- with great affinity for the androgen receptor and no known gational CYP17 inhibitor that is currently being evaluated agonist effect [15]. Ketoconazole is a nonspecific inhibitor of a phase I/II clinical trial (ARMOR1: Androgen Receptor cytochrome P450 enzyme-mediated androgen biosynthesis Modulation Optimized for Response 1) [35]. Uniquely, α [16]. Glucocorticoids such as prednisone have been used TOK-001 is not only a selective CYP17 (17 -hydroxylase as a palliative agent and likely act by suppressing ACTH and C17,20-lyase) inhibitor analogous to abiraterone acetate secretion and thus reducing adrenal androgen production but also an androgen receptor modulator [36]. These new selective CYP17 enzyme inhibitors represent an important [17](Figure 1). bench-to-bedside advancement, responding to the need Common side effects of hormonal therapy in prostate for more potent and specific inhibitors of extra-gonadal cancer treatment include hot flashes, weight gain, gyneco- androgen production. mastia, and osteoporosis [18]. Estrogen additionally has In a landmark phase III clinical trial from 2011, de cardiovascular side effects including blood clots [19]and Bono et al. reported that abiraterone acetate plus prednisone there is also evidence of increased cardiovascular risk with ff compared to prednisone alone yielded an improved median GnRH agonist in this setting [20]. Side e ects of ketocona- overall survival from 10.4 to 14.8 months in patients zole include elevated liver transaminases and gastrointestinal with metastatic hormonal-resistant prostate cancer after complaints [21]. docetaxel chemotherapy (P<0.0001). Circulating serum Hormonal therapy can produce dramatic clinical re- testosterone levels are reduced to levels of 1-2 ng/dL with sponses when initially used to treat advanced prostate abiraterone acetate—much lower than the previous standard cancer. Unfortunately, prostate cancer patients become re- of 50 ng/dL used to define CRPC. In this trial, abiraterone sistant to androgen deprivation after about 2-3 years on plus prednisone also showed significant improvement in average with progressive disease, despite castrate levels time to PSA progression (10.2 months versus 6.6 months; of testosterone [22, 23]. It is hypothesized that despite P<0.0001), radiographic progression-free survival (PFS) reduced androgen levels, one mechanism of resistance is (5.6 months versus 3.6 months; P<0.0001), and PSA the production of androgen for growth via intratumoral response rate (29.1% versus 5.5%; P<0.0001) when and extragonadal pathways. Other mechanisms of hormonal compared to prednisone alone. The immediate side effects resistance include upregulation of androgen receptors with of abiraterone were manageable and primarily related to increased sensitivity to androgen at the androgen receptors, elevated mineralocorticoid levels, including hypertension, and mutations of androgen receptors itself [24, 25]. fluid retention, and hypokalemia [37]. Advances in Urology 3

Table 1: Hormonal therapy in prostate cancer.

Class Existing drugs Investigational drugs Estrogen Diethylstilbestrol (DES) GnRH agonists Goserelin, leuprolide, triptorelin, histrelin acetate GnRH antagonists Degarelix, ∗abarelix Antiandrogens Bicalutamide, nilutamide, flutamide, ∗∗cyproterone acetate MDV3100, TOK-001 Non-specific,cytochrome P450 enzyme inhibitors Ketoconazole Specific CYP17 inhibitors Abiraterone acetate TAK-700, TOK-001 Glucocorticoids Prednisone, Dexamethasone, others ∗ Abarelix use in the United States was previously limited to a registry program and it is not actively marketed in the United States currently. ∗∗Cyproterone acetate is not currently FDA approved for the treatment of prostate cancer in the United States.

Hypothalamus

Leuprolide GnRH Pituitary Degarelix gland ACTH Prednisone

LH, FSH

Testicle Adrenal gland Orchiectomy Ketoconazole Adrenal Abiraterone, TAK-700, Testosterone androgens Cytoplasm TOK-001

Bicalutamide MDV3100, TOK-001 Androgen receptors Prostate cancer cell DNA

Intratumoral androgen production

Figure 1: Hormonal therapy in prostate cancer. Physiologically relevant androgens for prostate cancer originate from three sources: the testicle, adrenal glands, and via intratumoral production. The sites of activity of clinically relevant hormonal therapies are illustrated here, with new and investigational treatments indicated in red.

While androgen receptor signaling remains an important [39]. In a multicenter, phase I/II study involving 140 patients pathway of growth in CRPC, the currently available antian- from 2010, Scher et al. reported that MDV3100 showed drogens have not been able to completely block androgen antitumor activities in patients with metastatic CRPC, in- receptor signaling [38]. In another area of bench-to-bedside cluding decreases in serum prostate-specific antigen of 50% advancement, a new generation antiandrogen (MDV3100) or more in 78 of 140 (56%) patients, responses in soft tissue is now in clinical development with encouraging results. in 13 of 59 (22%) patients, stabilized bone disease in 61 Unlike currently available antiandrogens, MDV3100 is a pure of 109 (56%) patients, and conversion from unfavorable to androgen receptor antagonist without agonist activity. It is favorable circulating tumor cell counts in 25 of 51 (49%) differentiated from the current antiandrogens by its more patients [40]. Side effects include fatigue, which generally effective blocking of androgen receptor nuclear translocation resolved with dose reduction [40] and seizure at higher doses and coactivator recruitment of the ligand-receptor complex [41]. Phase III studies examining the efficacy of MDV3100 4 Advances in Urology both before and after docetaxel chemotherapy in men with It is exciting to envision a future with additional clinical CRPC are underway [34, 42]. gains from the earlier use of potent CYP17 enzyme inhibitors GnRH plays a key role in the androgen axis. GnRH in the course of disease treatment or with combination agonists have been used to achieve medical castration in therapy of abiraterone acetate and other newer agents such prostate cancer for decades. Unlike the original GnRH recep- as MDV3100. As a general principle, selective pressures from tor agonists, the recently approved degarelix acts as a direct cancer therapy typically yield resistant strains of disease GnRH antagonist, suppressing LH release without the iniital with progressively more aggressive features. Currently, we androgen flare and potential exacerbation of symptoms do not have a good understanding of the phenotypes of noted with GnRH receptor agonists [43]. GnRH antagonists advanced prostate cancer that may emerge after early such as degarelix also have a faster onset of action with less treatment with much more potent hormonal therapies such delay in the suppression of testosterone to castrate levels as MDV3100 and abiraterone acetate. Many unanswered than GnRH agonists [44]. In a phase III clinical trial from questions remain about the prostate cancer phenotype that 2008, Klotz et al. reported that when compared to leuprolide will emerge after early use of CYP17 inhibition in terms of after a 1-year treatment period, degarelix was not inferior to robustness, virulence, speed of disease progression, and the leuprolide at maintaining low testosterone levels. In addition, responsiveness to cytotoxic chemotherapy. degarelix achieved testosterone and PSA suppression more Since John Hunter first described the link between the rapidly than leuprolide with no need for antiandrogen testis and the prostate over 200 years ago, hormonal therapy supplements to prevent the initial clinical flare. Manageable remains one of the mainstays of advanced prostate cancer side effects of degarelix include flushing, injection-site pain, treatment. After initial response, metastatic prostate cancer weight gain, and increased serum transaminase levels [45]. It typically becomes resistant to standard androgen deprivation is important to highlight that when compared to leuprolide, therapy. Improved survival in this setting has been demon- degarelix might offer better suppression of serum alkaline strated in recent clinical trials involving new agents such phosphatase (S-ALP) level and more prolonged control as the CYP17 inhibitor abiraterone acetate and the antian- of skeletal metastases [46]. Furthermore, though degarelix drogen MDV3100, showing that CRPC remains hormonally and leuprolide both act on the GnRH receptor, reports of driven even after treatment with docetaxel chemotherapy, response to degarelix after previous resistance to GnRH challenging our traditional definition of hormone-sensitive agonist therapy with leuprolide have appeared [47], also disease. Even though the survival benefit is incremental in the potentially complicating our traditional definition of CRPC. phase III study of very advanced CRPC patients, a threshold has clearly been crossed in defining and treating advanced prostate cancer. 4. Discussion With so many new agents for advanced prostate cancer, individual patient characteristics may become very The phase III, landmark findings of the abiraterone acetate important in the selection and monitoring of therapy. The study by de Bono et al. have drawn into question our advantages of Degarelix over previous GnRH agonists may traditional definition of CRPC. Previously, phase I/II clinical be incremental for most CRPC patients, but the quicker fall trials of the CYP17 inhibitor abiraterone acetate demon- in testosterone may be very important to those with critical strated clinical activity in CRPC [31, 32]. This phase III metastatic or painful lesions such as spinal metastases or study demonstrates a clear survival benefit from additional near-total urinary obstruction. Likewise, the combination hormonal manipulation in a prostate cancer population of “complete” androgen deprivation with abiraterone along previously described as hormone refractory. Clearly, simply with the required safety medication of low-dose gluco- defining CRPC as progressive disease with a serum testos- corticoid may induce more bone disease via worsening terone level of less than 50 ng/dL is no longer adequate. The osteoporosis. Men with preexisting bone mineral density findings of de Bono et al. are consistent with other inves- loss in this setting may require careful monitoring of the tigations showing ongoing androgen-related activity in the bone mineral density and the use of early bisphosphonates. post-chemotherapy setting. Going forward, the definition of In this same light, the side effect profile of a potent hormone-sensitive prostate cancer will need to incorporate antiandrogen, which will not lower circulating androgen the use of a potent CYP17 enzyme inhibitor such as abi- levels by itself, may become an important consideration if raterone acetate. Accordingly, a much lower level of systemic monotherapy with such agents becomes accepted. Further- testosterone, beyond the traditional 50 ng/dL benchmark more, much like breast cancer is classified and treated based and closer to the 1-2 ng/dL level, must be targeted to on hormonal receptor characterization today, we believe achieve “complete” androgen deprivation. New investiga- that advanced prostate cancer may one day also be treated tional agents such as TAK-700 and TOK-001 represent based on molecular assessment. As more is known about addition agents in this class of CYP17 inhibitors, currently the molecular characterization of advanced prostate cancer, undergoing clinical testing in CRPC [33, 35]. Additionally, it will be imperative to develop more personalized hormonal the use of new generation antiandrogens, such as MDV3100, therapy for individual patients. Taken together, one of the must also be integrated into our definition of hormone- major challenges moving forward will be the personalized sensitive prostate cancer. The available clinical trial data with application of these new agents in those with advanced this drug, although early and limited, suggests substantial CRPC,nowthatwehavemanymorehormonalagentsatour activity in a traditionally defined CRPC population [48]. disposal with different side effects and mechanisms of action. Advances in Urology 5

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Review Article Preclinical Remodeling of Human Prostate Cancer through the PTEN/AKT Pathway

Marco A. De Velasco1, 2 and Hirotsugu Uemura1

1 Department of Urology, Kinki University School of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan 2 Department of Genome Biology, Kinki University School of Medicine, Osaka-Sayama, Osaka 589-8511, Japan

Correspondence should be addressed to Hirotsugu Uemura, [email protected]

Received 1 November 2011; Accepted 4 December 2011

Academic Editor: Jack Schalken

Copyright © 2012 M. A. De Velasco and H. Uemura. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Knowledge gained from the identification of genetic and epigenetic alterations that contribute to the progression of prostate cancer in humans is now being implemented in the development of functionally relevant translational models. GEM (genetically modified mouse) models are being developed to incorporate the same molecular defects associated with human prostate cancer. Haploinsufficiency is common in prostate cancer and homozygous loss of PTEN is strongly correlated with advanced disease. In this paper, we discuss the evolution of the PTEN knockout mouse and the cooperation between PTEN and other genetic alterations in tumor development and progression. Additionally, we will outline key points that make these models key players in the development of personalized medicine, as potential tools for target and biomarker development and validation as well as models for drug discovery.

1. Introduction therapeutic agents that target quintessential factors that are now known to contribute to tumor growth, development A major breakthrough in the developmental strategy for and progression. A large number of novel therapeutics are the treatment of prostate cancer came through in 1941 currently undergoing clinical evaluation for the treatment when Huggins discovered that metastatic prostate cancer of prostate cancer, and small molecule signal transduction responds to androgen ablation thus ushering in a new era inhibitors are a promising class of agents. These inhibitors in the treatment of prostate cancer therapy [1]. Androgen deprivation therapy remains the most powerful treatment have recently become standard therapy and have been FDA for advance prostate cancer and newer generation androgen approved for the treatment of various solid cancers including ff deprivation therapies (ADTs) that more effectively inhibit renal, GIST, breast, pancreas, colon, and NSCLC and o er AR signaling are rapidly being developed and approved significant promise in prostate cancer [8–14]. for patients with metastatic CRPC. However, therapeutic The development of an effective treatment strategy to effects are short lived and eventually all patients succumb treat advanced prostate cancer has been challenging due in to the disease [2]. The prognosis for men with CRPC is fact to the heterogeneity of the disease. Complex genomic bleak as currently available approved treatments only provide aberrations targeting multiple genes through mutation, marginal benefit and systemic treatments for metastatic changes in copy number, and methylation make patient se- CRPC are primarily approved for the management of lection difficult for the use of targeted molecular inhibitors symptoms [3, 4]. Recently, cytotoxic treatments such as the [15]. The first steps have already been taken to identify combinations of prednisone plus docetaxel or cabazitaxel some of the genetic alterations that lead to perturbed cell demonstrated modest improvements in extended survival signaling pathways that contribute to tumor development buthaveyettoproducelong-termbenefits[5–7]. and progression. However, developing effective therapeutic A better understanding of the biology of prostate cancer strategies will require relevant preclinical models of prostate has resulted in the identification of novel therapeutic targets cancer to identify and validate therapeutic targets and bio- and thus encouraged the development of new small molecule markers. 2 Advances in Urology

2. Preclinical Modeling for Prostate Cancer sophisticated and allow for any combination of tissue- specific expression of oncogenes as well as conditional, Traditional medical intervention treatments for prostate tissue-specific deletion of tumor suppressors. The value of cancer are based on data from epidemiological, clinical, or GEM modeling relies on the development of transgenic mice evidence-based medicine. However, this model is optimized that possess most of the clinicopathological and molecular for a large population and not for any one particular individ- characteristics of human prostate cancer. ual. The recent trend in medicine is to employ a personalized treatment approach that is based on molecular profiling 3. Biology of Prostate Cancer and PTEN to determine the best treatment strategy for a particular individual. This approach requires the development of new Prostate cancer progression follows a series of defined states high-throughput technologies to decipher and interrogate characterized by molecular changes associated with disease tumors at various molecular levels and integrates resources progression. The heterogeneity of prostate cancer has pre- from various specialized fields into one system to discover, vented clear identification and correlation of critical genetic coordinate, and extract a strategic approach derived from events contributing to disease progression and treatment multidimensional input data. resistance. However, there are constant genetic alterations Cancer is a complex heterogeneous disease that involves frequently present in prostate cancer such as the loss of genetic events that lead to the disruption of multiple PTEN function. PTEN, located on chromosome 10 (10q23), signaling networks. Consequently, multiple cellular processes is a tumor suppressor gene that is broadly expressed during within the tumor microenvironment within a host system development and adulthood and is essential for embryogen- are involved and may be influenced by any number of envi- esis [24]. PTEN encodes a dual lipid and protein phosphatase ronmental factors over a period of time. Basically, the “one that functions as an inhibitor of PIP3 [25]. Accumulation of gene, one protein, one function” hypothesis is outdated and PIP3 then mimics the effect of phosphatidylinositol 3-kinase not applicable for systems biology. Until recently, preclinical (PI3K) activation resulting in the activation of downstream models for prostate cancer have been limited largely due to effectors including Akt. Activation of Akt phosphorylates the lack of animal models that develop spontaneous prostate various physiological substrates that results in the stim- tumors in a manner similar to humans. Spontaneous animal ulation of cell cycle progression, survival, migration, and models such as dog and rat have been used extensively to metabolism [24–27]. study hormonal carcinogenesis but are impractical and do PTEN function lost through mutations, deletions, or ffi not represent a feasible model for preclinical e cacy evalua- promoter methylation silencing occurs at a high frequency in tion [16, 17]. Mouse xenograft models derived from human many primary and metastatic human cancers in humans and prostate tumor cell have been developed and used extensively is the second most commonly tumor suppressor gene after in academia and pharmaceutical industry. Although the p53 [24, 26–28]. Current estimates suggest that PI3K/Akt/ number of human prostate cancer cell lines is limited, the mTOR signaling is upregulated in 30–50% of prostate convenience and low cost have made the xenograft model cancers, often through loss of PTEN function [29, 30]. popular for gene validation and compound testing [18, 19]. Clinical findings have demonstrated that biallelic deletion During a 10-year period (1991–2000) big pharma com- of PTEN correlates with disease-specific mortality and is panies in the United States and Europe reported that only associated with Akt and AR deregulation [30–32]. Published 5 out of 100 drugs passing preclinical testing achieved reports have shown that heterozygous loss of PTEN occurs drug approval success [20]. The majority of attrition (70%) in as many as 70–80% of primary tumors, and complete occurred in Phase II trials with lack-of-efficacy accounting inactivation occurs in 20% of primary tumors and 63% of for approximately 30% of failures. Lack of treatment efficacy metastasis [29, 33, 34]. A more recent report showed that in Phase II and III trials has been attributed to unpredictable copy-number alterations (CNAs) in prostate cancer were preclinical models [20, 21]. Pharmaceutical companies rou- present in 42% of primary tumors and 100% of metastases tinely use human prostate cancer cell xenografts during [35]. the preclinical testing phase to evaluate new drug efficacy. It has been widely recognized that AR signaling remains However, only three human prostate cancer cells lines (PC3, important even in the presence of reduced androgen levels DU145, and LNCaP) account for vast majority of cells used and thus remains a major target for targeted therapeutic in prostate cancer drug efficacy screens. The use of these interventions [36]. Clinically, the deletion of PTEN and cells to study human prostate cancer in vivo is inadequate as AR expression has been significantly correlated to cancer- they lack many key features found in autochthonous cancers. specific mortality in patients with CRPC [31]. Reports While xenograft models (ectopic or orthotopic) may have suggest that PI3K through AKT may play an important role value in certain situations, they are inappropriate and bear in upregulating AR protein expression in the absence of little relevance to human prostate cancer. PTEN [37, 38]. PTEN can modulate AR activity directly With the development of GEM (genetically engineered or through PI3K/Akt signaling pathways; however, levels mice), preclinical modeling for prostate cancer has evolved of AR are often heterogeneous in late-stage disease [39– significantly in the last 15 years [16–23]. These models 41]. Evidence from published reports has now shown that consist of mice that have been designed to deliberately inhibit alterations in AR develop with sequential hormonal ablation or express a particular gene function through the intro- therapies and tumor progression [42, 43]. Also, reciprocal duction of foreign DNA. Today, GEM have become quite feedbackregulationbetweenARandPTENinprostate Advances in Urology 3 cancer initiates a series of molecular events that contribute to alterations to further characterize the role of PTEN in growth survival and differentiation and may thus participate prostate tumorigenesis. in ADT resistance. It was recently demonstrated that loss Alterations of p53 and retinoblastoma (Rb)oncogenes of AR expression, in the absence of PTEN, can lead to correspond to prostate cancer progression in humans [61, downregulation of Fkbp5 and PHLPP-mediated Akt inhi- 62]. One particular study used the TRAMP mouse model bition resulting in increased cellular proliferation [44, 45]. to investigate the cooperation between PTEN haploinsuf- Therefore, it is essential to develop and test new compounds ficiency and abrogated function of the tumor suppressor that target known compensatory and survival pathways in genes p53 and Rb in prostate cancer development [49]. advanced prostate cancer and identify new targets for po- The TRAMP mouse model is a first generation transgenic ssible interventions. knockout and was one of the first mouse models to effectively induce the development of aggressive prostate tumors 4. Traditional PTEN Knockout Mouse Models of through the expression of large/small SV40 tumor antigens Prostate Cancer (T/tag) under the control of the prostate-specific rat probasin promoter [22]. The transforming activity of T/tag inactivates The strong implication of PTEN in prostate cancer pro- both p53 and Rb tumor suppressor proteins [63]. Prostate /− gression in humans has prompted the development of cancer progression in PTEN+ /TRAMP mutant mice shows genetically GEM models based on PTEN inactivation (see increased rates of tumor development and decreased survival / Table 1). Traditional knockout models of PTEN developed compared to PTEN+ +/TRAMP mice. A different study −/− /− in the late 1990’s were generated by deleting exons 4, or used the Ink4a/Arf PTEN+ model to investigate the 4and5ofthePTEN gene, which codes for the entire cooperation between PTEN haploinsufficiency and RB and α p53 [50]. The Ink4a/Arf gene focus regulates the tumor PTEN-phosphatase domain and part of the two -helix Ink a Ar f motifs flanking the catalytic core [46, 47]. Homozygous suppressor proteins RB and p53 through p16 4 and p19 , −/− /− inactivation of PTEN results in normal appearing, but respectively [64]. Ink4a/Arf PTEN+ mice experienced a nonviable embryos. Heterozygous PTEN knockout mice are much faster rate of PIN development compared to Ink4a/ / /− born viable to develop prostatic intraepithelial neoplasia Arf+ +PTEN+ controls; however, these mice did not develop (PIN) in the prostate as well as a neoplasias in a number adenocarcinomas [50]. of organs including skin, colon, endometrium, liver, thyroid, Deletions of chromosome 12p11-13 (corresponding to and thymus [46, 47]. However, progression to malignant CDKN1B(p27/Kip1I0)) have been identified in advanced adenocarcinoma is not observed in heterozygous mutants human prostate cancer suggesting a tumor suppressor role indicating that inactivation of one allele of PTEN is enough for p27(Kip1) [48]. Loss of p27(Kip1) function has been to initiate tumorigenesis but not progression. It is important implicated with prostate tumor recurrence and poor disease- to note that the viability of the mice is compromised by free survival in humans [65, 66]. p27−/− mice develop lymphoid proliferation and development of tumors arising enlarged hyperplastic prostates and increased fibromuscular in other organs such as intestines, mammary, thyroid, stromal cells closely resembling benign prostatic hyperplasia endometrial, and adrenal glands. (BPH) but fail to develop prostate cancer [67]. However, Increased phosphorylation of Akt occurs as a result of when these mice are bred with heterozygous PTEN mutant −/− /− PTEN inactivation; however, it was uncertain whether hyper- mice, all resulting p27 /Pten+ mutant mice became activation of Akt was enough to drive tumor development susceptible to the development of invasive prostate adeno- in the prostate. To address this question, one group looked carcinomas [68]. These animal models have provided genetic at the effects of Akt overexpression in the mouse prostate evidence to show that collaboration between PTEN haploin- ffi using the MPAKT transgenic mouse [57]. Overexpression of su ciency and inactivation of other tumor suppressor genes Akt1 in MPAKT transgenic mice results in the development by either gain or loss of function promotes prostate cancer of PIN in the ventral prostate but not cancer. Thus activation progression. of Akt signaling alone in the presence of PTEN is insufficient Nkx3.1 is a transcription factor whose expression is to induce prostate cancer although the deletion of Akt1 androgen dependent and limited to the luminal cell com- but not Akt2 (Akt1 is the predominant isoform found in partments in prostate glandular tissue [69]. Although Nkx3.1 mouse prostate) was sufficient to suppress the development mutations are not reported in humans, loss of Nkx3.1 protein of high-grade PIN lesions in PTEN+/− mice [54, 58]. These expression is strongly correlated to CRPC and advanced findings not only cement the role of PTEN in early prostate stage prostate cancer [70, 71]. The cooperative function carcinogenesis but also demonstrate the multifunctional role of PTEN and Nkx3.1 haploinsufficiency was explored in a of PTEN in regulating other biological processes related double knockout transgenic mouse model [51–53]. In this to malignant transformation. Prostate cancer in humans model, double heterozygous mutants demonstrate a propen- displays a range of clinical phenotypes that develops over sity to develop invasive prostate adenocarcinoma after 12 time as a result of gene alterations involving multiple months of age and frequently display iliac lymph node regulatory pathways [59, 60]. In order to achieve clinically metastases. In contrast, Nkx3.1 knockout mice only develop relevant models of human prostate cancer in mice, several PIN lesions [72, 73]. Another interesting observation with investigators have sought to generate bigenic knockout mice Nkx3.1+/−/PTEN+/− mice is the ability for these mice to de- that combine PTEN haploinsufficiency with other genetic velop CRPC after castration. 4 Advances in Urology ] ] ] ] ] ] ] ] ] 53 – 55 49 50 54 48 56 46 47 51 2009 [ 2001 [ 2002 [ 2006 [ 2001 [ 2003 [ 2011 [ 1998 [ 1999 [ − − / / + + Pten Pten ciency leads to ffi progeny were nonviable; progeny were nonviable; − − / / − − invasive adenocarcinoma and reduced cancer latency Increased rate of tumor development and metastases Overexpression of ERG cooperates and Pten haploinsu Early onset of PIN lesionsMultiple organ neoplasia and reduced tumor-free survival Akt deficiency attenuated PIN development Rapid progression of invasive carcinoma and decreased survival Mice developed adenocarcinomas inthedorsolateralprostateat12 months and androgen independent phenotypes following castration Increased incidence of AdCa in the ventral lobe Pten Pten mutants mutants multiple organ neoplasia in multiple organ neoplasia in Comments Year Ref. Yes Not Not Not Not Not Not Not Not reported reported reported reported reported reported reported reported resistance Castration Metastatic neuroendocrine carcinoma Invasive adenocarcinoma Invasive adenocarcinoma Metastatic adenocarcinoma to lymph nodes Invasive adenocarcinoma 1: Traditional Pten knockout mouse models of prostate cancer. Mouse strain Phenotype Table mutation locus Pten level Gene Single Exons 4Single and 5 129SvJy/C57BL/6 Exon PIN 5 129SvJy/C57BL/6 PIN knockout Compound Exon 5Compound C57BL/6 Exon 5Compound Exon FVB/n/C57BL/6Compound 5 Exons 4 PIN and 5 129SvJy/C57BL/6 129SvJy/C57B6Compound PIN Exon 5 FVB/n/C57BL/6 Compound Exon 5 129SvJy/C57BL/6 Compound Exons 4 and 5 129SvJy/C57BL/6 − / + − / − / − + − / − /Pten − / − /TRAMP PB-ERG − − /Cdkn1b /Nkx3.1 /Akt1 / / + + − − − − − − / / / / / / + + + + + + ARR2Pb.Stat3C/ PTEN Description Pten Pten Ink4a/Arf Pten Pten PTEN Pten Pten Advances in Urology 5

The ERG gene is frequently translocated to the TMPRSS2 Inducible promoters used for conditional targeting of the promoter region; the resulting TMPRSS2-ERG fusion pro- mouse prostate include PSACreERT2 and Nkx3.1CreERT2,both tein is positively expressed in half of human prostate inducible with tamoxifen [90, 91]. Floxed PTEN mice have cancer cases [74–76]. Mice expressing the truncated ERG been developed by flanking exons 4, or 4 and 5 with LoxP product from TMPRSS2-ERGa, under the control of the cassettes [84, 92–94]. As in traditional knockouts, these androgen-responsive region (ARR2Pb) probasin promoter sites correspond to the coding regions for the entire PTEN- (functionally analogous to the TMPRSS2-ERGa fusion prod- phosphatase domain and portion of the two α-helix motifs uct), only develop PIN [75]. In the presence of PTEN flanking the catalytic core [46, 47]. haploinsufficiency, overexpression of ARR2Pb-ERG results in We and others have generated prostate-specific condi- the progression of PIN lesions to prostatic adenocarcinoma tional mouse models of prostate cancer to better characterize [55]. This model has confirmed that two common critical full loss of PTEN gene expression and its effect on prostate events, concomitant loss of PTEN and EGR genetic rear- tumor carcinogenesis, summarized in Table 2.Heterozygous rangement, accelerate initiation and progression in human PTENloxp/+ mice develop PIN in a manner similar to tradi- prostate adenocarcinoma. Stat3 has been implicated in tional heterozygous PTEN knockouts [84–86, 89]. However, the promotion and progression of human prostate cancer PTEN inactivation under the control of PSACre or PBCre4 [77]. Transgenic mice designed to constitutively express promoter in PTENloxp/+ mice is largely restricted to the Stat3 under the control of ARR2Pb develop PIN but fail prostate, and trace levels of PTEN deletion are seen in the to progress to malignant adenocarcinoma; however, when seminal vesicles [84–86]. Complete inactivation of PTEN crossed with PTEN+/− mutant mice, the resultant double in traditional knockouts results in embryonic lethality thus knockouts develop invasive adenocarcinomas [56]. Phos- limiting the characterization of total PTEN inactivation. phorylated Stat3 expression was potentiated by the loss of Development of PIN occurs quickly in homozygous PTEN PTEN and subsequent overexpression of Akt. Collectively, knockout mice ranging from 6 to 16 weeks of age, and these studies have shown the crucial relevance of “two latency to the development of prostate adenocarcinoma hits” for the development of prostate adenocarcinoma and varies from 9 to 24 weeks [84–86, 89]. Locally invasive demonstrated how genetic alterations that play subtle roles disease is present in these models and some mice develop in tumor initiation cooperate with PTEN haploinsufficiency metastases to iliac lymph nodes, and occasionally lung [84– to produce malignant phenotypes in mice similar to human 86]. A clinically relevant feature of prostate-specific PTEN prostate adenocarcinoma. conditional knockout mice is the sensitivity to androgen ablation and the ability to develop CRPC [85, 91]. 5. Conditional PTEN Knockout Mouse Models Altogether, these studies have shown that prostate- of Prostate Cancer specific conditional PTEN knockout mice share many features seen in human prostate cancer. Biallelic inactivation Development of conditional gene targeting by the Cre-LoxP of PTEN leads to hyperproliferation that is followed by the system has significantly changed the landscape for transgenic development of PIN which eventually progresses to locally mouse modeling research. In conditional mouse models, invasive adenocarcinoma and eventual metastases. Moreover, the target gene is flanked by LoxP cassettes and remains tumors are initially responsive to androgen ablation and in the germline. Inactivation of this gene is controlled by develop into CRPC. Besides histopathological similarities, Cre recombinase which catalyzes recombination between tumors from these mice also share molecular profiles similar the two LoxP sites [78]. Orientation of the LoxP cassettes to human prostate cancer [96]. Inducible variations of determines type of recombination to produce deletion, the prostate-specific conditional knockout model provide inversions, or chromosomal translocations [79]. Expression spatiotemporal control of induced mutagenesis [90, 91]. The of Cre is dependent on transgene expression of a widespread ability to incorporate bigenic gene alterations to mice with or tissue-specific promoter. A variation of this system uses conditional PTEN haploinsufficiency makes it a relevant an inducible transgene promoter that is inactive until it is preclinical model to study the epigenetic events or LOH that induced by an activating agent [80]. Conditional knockout lead to disease progression. models have the ability to induce the genetic mutation in the target tissue without affecting nontargeted cells. In 6. PTEN Knockout Mice as this manner, both genes can be knocked out in the target Drug Targeting Models cells while the rest of the mouse cells retain normal gene expression and function. GEM models offer several unique advantages over the xeno- Promoter selection is critical for targeting the prostate graft model. The first and probably most important feature gland, and several have been characterized and well described is that through controlled gene disruption, these mice can be in the literature [16, 81–83]. The most common promoters manipulated to develop prostate cancer from phenotypically used in prostate-specific conditional targeting are the prostate normal cells, thus encompassing the whole spectrum of specific antigen-Cre (PSA−Cre), probasin-Cre (PB−Cre), and tumor carcinogenesis. Secondly, tumors develop in situ ARR2PB-Cre (PB−Cre4)promoters[84–88]. The mouse mam- taking into account all the components involved in the mary tumor virus (MMTV−Cre) promoter has also been used carcinogenesis process, including interactions with all tumor to conditional drive mutations in the prostate; however, microenvironment factors that can promote tumor develop- its activity was not specific to the prostate gland [89]. ment. Another key feature of these mice is that they retain 6 Advances in Urology ] ] ] ] ] ] ] ] 87 84 89 88 85 86 90 91 2006 [ 2003 [ 2004 [ 2005 [ 2003 [ 2005 [ 2012 [a] 2008 [ 2009 [ Pten proximal Cre-ERT2 Cre-ERT2 promoter-driven; promoter-driven; promoter-driven; promoter-driven; FGF8b and promoter-driven; PSA promoter promoter-driven; all promoter-driven; 50% Nkx3.1 12 months ARR2PB-Cre activation of heterozygous loss of cooperate in the late-onset induction of metastatic prostate cancer with high incidence ARR2PB-Cre 100% of mice developed invasive carcinoma at 6 months MMTV-Cre focally invasive carcinoma at 10 weeks Mice die from lymphomas at 14 wks ARR2PB-Cre invasive adenocarcinoma at 4–6 months with mean survival ofmonths 5 ARR2PB-Cre Pin lesions develop at 6 weeks and invasive adenocarcimoa by 9 weeks PSA-Cre mice develop adenocarcinoma at 10–14 months with rare metastases PSA-Cre incicence of adenocarcinoma at 10 weeks, lymph node metastasis > Comments Year Ref. promoter Tam-inducible recombinase under the control of the human Tam-inducible recombinase under the control of the Yes Yes Yes resistance Castration Not reported Not reported Not reported Not reported Not reported Homozygous deletion results in invasive adenocarcinoma and metastatic spread to lymph nodes Homozygous deletion results in invasive adenocarcinoma and metastatic spread to lymph nodes Homozygous deletion results in invasive adenocarcinoma Homozygous deletion results in invasive adenocarcinoma and metastatic spread to lymph nodes Homozygous deletion results in invasive adenocarcinoma Homozygous deletion results in invasive adenocarcinoma and metastatic spread to lymph nodes Homozygous deletion results in invasive adenocarcinoma after tamoxifen treatment Homozygous deletion results in invasive adenocarcinoma after tamoxifen treatment FVB/n 129/BALB/c /129/BALB/c Mouse strain Phenotype C57BL/6/DBA2 C57BL/6/DBA2x 129SvJy/C57BL/6/ 2: Conditional Pten knockout mouse models of prostate cancer. Pten locus mutation Table level Gene Single Exons 4 and 5 Single Exon 5 C57BL/6/129/Sv knockout Inducible Inducible conditional conditional Conditional Single Exons 4 and 5 129SvJy/C57BL/6 Conditional SingleConditional Exon 5 Single ExonsConditional 4 and 5 Single C57BL/6 Conditional Exon 5 Single FVB/n/129Ola Exons 4 and Invasive 5Conditional adenocarcinoma Not reported Compound Exons 4 C57BL/6 and 5 129SvJy/C57BL/6 Conditional Compound Exon 5 manipulation PTEN genetic ]. loxp/loxp /FGF8b 95 loxp/loxp / loxP/loxP/ 2 /loxP loxp/loxp loxp/loxp loxp/loxp loxp/loxp + /Pten 2 CreERT /Pten /Pten /Pten /Pten /Pten loxP/loxP 4 4 4 loxp/loxp Cre Cre CreERT Cre Cre Cre De Velasco et al. [ PSA PB PSA PB MMTVCre/PTEN PSA Pb-Cre4/Pten Description PB Trp53 Pten Nkx3.1 a Advances in Urology 7 an intact immune system, thus incorporating all the impor- Although Ras mutations in prostate cancer are infre- tant components of innate and acquired immunity. Lastly, as quent, wild-type Ras is chronically activated in prostate in humans, tumors in these mice show heterogeneity, a key cancer as a result of autocrine and paracrine growth factor feature of cancer. stimulation [66, 106]. Upregulation of MAPK signaling in Despite all of the advantages over xenograft models, prostate cancer is likely due to overexpression of growth concerns exist whether tumors arising from GEM are factor receptors. Several growth factor receptors including homologous to human prostate cancers. Compared to the the epidermal growth factor receptor (EGFR) and insulin- human prostate which is divided into zones, the mouse like growth factor-1 receptor (IGF-1R) have been shown prostate develops as a lobular structure consisting of the to be overexpressed in prostate cancer. EGFR belongs to anterior, dorsal, lateral, and ventral lobes [97]. Some believe the ErbB family of receptor tyrosine kinase proteins and that the dorsolateral lobes of the mouse prostate are the most is highly expressed in primary tumors and metastases. In similar to the human peripheral zone, which is the region prostate cancer, EGFR overexpression is associated with poor where most cancers arise [96–98]. However, the Bar Harbor prognosis and the transition to CRPC status [107, 108]. It has pathology panel for genetically modified mouse models of also been shown that Ras activation can play a causal role in prostate cancer had the consensus opinion that there is no moving PCa cells towards decreased hormone dependence direct relationship between the lobes of mouse prostates and an increased malignant phenotype [109]. The role of and human prostate zones [97]. Nevertheless, GEM offer a MAPK signaling, as a target for prostate cancer therapy, unique tool for biomedical research in the understanding of becomes complicated as others report that MAPK signaling biochemical and disease pathways and the development of may be inhibited in advanced prostate cancer due to the new therapeutic strategies through new target and biomarker deletion of the PTEN [110–112]. Akt activation, through discovery and validation. the deletion of PTEN, can result in the phosphorylation and The evolution of newer generation transgenic mice based inactivation of Raf-1 thus decreasing downstream signaling on the conditional mutation, deletion, or insertion of single of MEK and ERK which then leads to the loss of cellular or multiple targeted genes is becoming an attractive model differentiation [111, 113]. Evidence of crosstalk between for researchers in academia and industry. As a result, mice PI3K/Akt/mTOR and MAPK signaling pathways suggests develop tumors which feature many similarities to human that compensatory survival signaling exists in this network prostate cancer including various pathological and molecular and could therefore be exploited therapeutically [114]. characteristics [84–86, 96]. Since tumors in these mice The transcription factor, signal transducer and activator arise from normal tissues, preclinical trials can be designed of transcription 3 (Stat3), has been implicated in the to target specific points during tumor development that growth and progression of several cancer types including take advantage of the windows of opportunity provided. A prostate [77, 115–118]. Stat3 has been shown to directly developing paradigm for new treatments strategies involves and indirectly regulate the expression of genes required for the use of combination-targeted therapies. Tumor growth proliferation and apoptosis and is also known to negatively is not dependent on one particular signaling pathway, regulate the expression of p53, stimulate tumor angiogenesis, rather, it is an orchestrated event that is driven by complex and suppress antitumor immune responses [77, 119–121]. feedback loops from crosstalk between multiple signaling Stat3 has been shown to induce the metastatic behavior of pathways. PTEN and bigenic knockout mice are excellent prostate cancer cells in vitro and in vivo [77]. Activation models to investigate the preclinical therapeutic effects from of Stat3 occurs by the binding of various cytokines which combinatorial treatment strategies. Treatment strategies can become constitutively activated by their respective ligands be designed as either horizontal or vertical to inhibit targets by an autocrine and paracrine manner [120, 122]. Stat3 involved in altered signaling pathways resulting from PTEN is also activated by growth factors as a downstream tar- inactivation. get of PI3K/Akt/mTOR and MAPK signaling through the PI3K/Akt/mTOR inhibitors are currently being evalu- phosphorylation of Ser-727 [121, 123]. Activated IL-6 has ated in various tumor types. mTORC1 inhibitors such as been shown to be elevated in the sera from patients with rapamycin and rapalogs have demonstrated limited success metastatic prostate cancer. In addition to activating Stat3, IL- as single agent treatments [28, 99–101]. This lack of efficacy 6 can also induce MAPK activation through various distinct is attributed to the inability to maintain reduced levels of mechanisms [124–127]. Combined targeted inhibition of phosphorylated 4E-BPs resulting from upregulation of Akt PI3K/Akt/mTOR, RAS/RAF/MEK, and JAK/STAT signaling through the loss of the S6K to IRS-1 negative feedback may be a promising strategy for the treatment of prostate regulation loop [100, 102, 103]. However, published reports cancer and PTEN knockout mice should play an important also suggest that crosstalk between RAS/RAF/MEK signal- role in the preclinical development and discovery of candi- ing after mTOR inhibition results in resistance to mTOR date agents. inhibitors. Humans with advanced prostate cancer treated with RAD001 show schedule-dependent increases of MAPK 7. PTEN Knockout Mice in signaling activation [104]. Data from two independent stud- Biomarker Discovery ies conducted with PTEN knockout mice demonstrate that dual inhibition of PI3K/Akt/mTOR and MAPK signaling To effectively treat human prostate cancer, one must be able results in synergetic antitumor responses and is at least to identify specific targets that drive molecular and cellular feasible in a preclinical setting [104, 105]. events to tumorigenesis. Cancer-related cellular processes 8 Advances in Urology

Tumor development 100% PCa > 95% PCa Preclinical drug A 50% PCa efﬁcacy trials 50% PIN 50 20 15 Weeks 10 8 6 Chemoprevention model 4 Intervention model Castration CRPC intervention model Regression model

Drug target and biomarker B discovery

Tumorigenesis C Genomics Proteomics

Compound Environmental mutations dietary Figure 1: Modeling prostate cancer in the PTEN conditional knockout mouse model. (A) PSACre/PTENloxP/loxP can be used to screen for tumor response against targeted therapies in chemoprevention, intervention, or regression models using noncastrated or castrated mice. (B) Comprehensive genomic and proteomic analyses can be performed in PSACre/PTENloxP/+, PSACre/PTENloxP/loxP, or bigenic mutants to identify candidate genes or proteins signatures aberrantly expressed between different pathologic, genomic, or temporal disease conditions. (C) Cooperation between genetic and nongenetic factors can be assessed in tumor development in both homozygous and heterozygous PTEN-conditional mutant mice. are being studied to identify possible targets for new pathobiology of human prostate cancer, differences between drug development and biomarker discovery. However, drug human and mouse kinetics, physiology, and metabolism target and biomarker discovery using human samples is must be considered. Despite these limitations, PTEN knock- difficult and hampered by the amount of genetic variation out mice will continue to be used to further characterize among individuals as well as external influences (lifestyle prostate carcinogenesis. The use of these models in preclin- and environmental factors) that contribute to the patho- ical drug, target, and biomarker discovery and development genesis of the prostate cancer [128–130]. Furthermore, will increase and will most likely become a standard in drug this requires the acquisition of large numbers of samples discovery pipeline. which is time consuming and may be difficult in many instances. Interspecies conservation of genomic aberrations across conserved regions of tumorigenesis provides an References alternative approach to identify genes responsible for tumor developments and progression [15, 131]. Transgenic mice [1] C. Huggins and C. V. Hodges, “Estrogen and of androgen injection on serum phosphatases studies on prostatic cancer. have lower biological variances and can be studied under I. The effect of castration, of in metastatic carcinoma of the controlled situations that better enable the detection of target prostate,” Cancer Research, vol. 1, pp. 293–297, 1941. molecules. Because of this, transgenic mouse models of [2] J. S. De Bono, C. J. Logothetis, A. Molina et al., “Abiraterone prostate cancer, in particular PTEN-mutant mice, provide a and increased survival in metastatic prostate cancer,” The unique opportunity for the discovery of novel targets. New England Journal of Medicine, vol. 364, no. 21, pp. 1995– 2005, 2011. 8. Concluding Remarks [3]I.F.Tannock,D.Osoba,M.R.Stockleretal.,“Chemotherapy with mitoxantrone plus prednisone or prednisone alone for Further advances in the treatment strategies for prostate can- symptomatic hormone-resistant prostate cancer: a Canadian cer are dependent on the development, use, and incorpora- randomized trial with palliative end points,” Journal of tion of clinically relevant faithful animal models of human Clinical Oncology, vol. 14, no. 6, pp. 1756–1764, 1996. prostate cancer (Figure 1). Recent work on PTEN mouse [4]H.Beltran,T.M.Beer,M.A.Carduccietal.,“Newtherapies models has helped characterize human prostate carcinogen- for castration-resistant prostate cancer: efficacy and safety,” esis. Although these models share amazing similarity to the European Urology, vol. 60, no. 2, pp. 279–290, 2011. Advances in Urology 9

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Review Article Progress in Personalizing Chemotherapy for Bladder Cancer

James S. Chang,1 Primo N. Lara Jr.,1 and Chong-Xian Pan1, 2, 3, 4

1 Department of Internal Medicine, Division of Hematology/Oncology, University of California Davis, Sacramento, CA 95817, USA 2 Department of Urology, University of California Davis, Sacramento, CA 95817, USA 3 Department of Medicine, VA Northern California Health Care System, Mather, CA 95655, USA 4 UC Davis Urothelial Carcinoma Initiative, University of California Davis Cancer Center, 4501 X Street, Room 3016, Sacramento, CA 95817, USA

Correspondence should be addressed to Chong-Xian Pan, [email protected]

Received 25 August 2011; Revised 16 November 2011; Accepted 25 November 2011

Academic Editor: Hirotsugu Uemura

Copyright © 2012 James S. Chang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Platinum-based chemotherapy is commonly used for the treatment of locally advanced and metastatic bladder cancer. However, there are currently no methods to predict chemotherapy response in this disease setting. A better understanding of the biology of bladder cancer has led to developments of molecular biomarkers that may help guide clinical decision making. These biomarkers, while promising, have not yet been validated in prospective trials and are not ready for clinical applications. As alkylating agents, platinum drugs kill cancer cells mainly through induction of DNA damage. A microdosing approach is currently being tested to determine if chemoresistance can be identiﬁed by measuring platinum-induced DNA damage using highly sensitive accelerator mass spectrometry technology. The hope is that these emerging strategies will help pave the road towards personalized therapy in advanced bladder cancer.

1. Introduction frequent even after surgery. For example, about 50% of patients with deep, muscle-invasive disease will develop meta- Bladder urothelial cancer is the 4th most common cancer in males and 9th in females and a major cause of morbidity and static disease even after surgery [6]. Thus, systemic platinum- mortality worldwide. In the United States, approximately based chemotherapy, either in a neoadjuvant or adjuvant set- 70,530 individuals were diagnosed with bladder cancer in ting, is considered a component of the standard care for 2010 and 14,680 died from it [1]. Most bladder cancers in the this disease. Metastatic disease is usually treated with chem- developed world are of urothelial origin (transitional cell), otherapy, but the median survival even with the best chemo- arising from the epithelial lining. Bladder cancers are broadly therapy is often only about 14 months [7]. classified as noninvasive or invasive (muscle-invasive and Improving survival outcomes in advanced bladder cancer metastatic) cancers. The noninvasive and invasive subtypes will require moving beyond conventional histopathologic are thought to arise from distinct biological pathways [2]. evaluation such as stage and grade. Molecular biomarkers About 70 to 80% of newly diagnosed bladder cancers are have the potential to more accurately determine prognosis noninvasive. The initial treatment of noninvasive cancer in- and assign patients to appropriate treatments. Such biomark- volves a complete transurethral resection followed by adju- ers are already being used in other solid tumors such as vant intravesical therapy [3]. As many as 70% of noninvasive breast, colon, and lung. For example, high expression of the cancers recur, necessitating life-long surveillance, and up to ERCC1 gene is prognostic of improved survival and predic- 25% will progress to more advanced disease [4, 5]. tive of reduced response to platinum-based therapy in non- For patients with muscle-invasive, nonmetastatic disease, smallcellcancer(NSCLC)[8]. Many promising biomarkers radical cystectomy with bilateral pelvic lymph node dissec- are now being evaluated for bladder cancer, offering the po- tion remains the mainstay of treatment. Recurrence can be tential of improving clinical outcomes. As our understanding 2 Advances in Urology of the molecular pathways in bladder cancer improves, more platinum-based combination chemotherapy compared to personalized therapies can be delivered that are potentially local therapy alone had a 14% reduction in the risk of death more active and hopefully less toxic. In this paper, we will [14]. This was equivalent to a 5% absolute improvement in review the current chemotherapeutic treatments for ad- overall survival from 45 to 50% at 5 years (P = 0.003). vanced disease, highlight the potential role of biomarkers, There was also a significant disease-free survival benefit (P< and remark on the future direction of bladder cancer care. 0.0001), equivalent to a 9% absolute improvement at 5 years. Even though CMV and MVAC were used in the above- mentioned prospective trials, the gemcitabine and cisplatin 2. Treatment of Muscle-Invasive (GC) combination is commonly used in the neoadjuvant set- Bladder Cancer ting. There is no randomized trial supporting the use of the GC regimen in this setting. Clinicians mainly extrapolated The current standard treatment in the United States for the data in the metastatic setting showing similar efficacy but muscle-invasive bladder cancer is radical cystectomy with better tolerability with the GC regimen compared to MVAC. bilateral pelvic lymph node dissection. These patients often Neoadjuvant cisplatin-based chemotherapy is not widely develop metastatic disease despite aggressive surgical inter- used in practice even though there is level I evidence indi- vention. In organ-confined pT2 disease, the 5-year survival cating a significant survival advantage for patients with mus- rate is approximately 68% [9]. Patients with more deeply in- cle-invasive bladder cancer [15]. An analysis of 7,161 patients vasive tumors have lower five-year survival rates of 30 to with stage III bladder cancer from the National Cancer Data- 50% [10]. Relapse is due to the presence of occult micro- base between 1998 to 2003 revealed that perioperative chem- metastases. otherapy was administered to 11.6% of patients with stage III bladder transitional cell carcinoma with 10.4% receiving 2.1. Neoadjuvant Therapy. The rationale for neoadjuvant adjuvant chemotherapy and 1.2% receiving neoadjuvant chemotherapy prior to cystectomy is to treat micrometastatic chemotherapy [16]. A more recent analysis from the same disease that is present at diagnosis. It also helps downstage database between 2003 and 2007 showed a slight increase in the tumor and increases the potential for complete resection the use of neoadjuvant chemotherapy (up to 13% of patients of tumor. Furthermore, neoadjuvant chemotherapy allows in 2007) [17]. One major concern is the potential for disease delivery of systemic therapy through intact blood vessels and progression due to delayed definitive treatment in patients can be better tolerated before the patient is debilitated by who do not respond to neoadjuvant chemotherapy. There is surgery. There is level I evidence with two randomized trials a critical need to identify those who will respond favorably to to support the use of neoadjuvant chemotherapy [11, 12]. neoadjuvant chemotherapy. The largest neoadjuvant chemotherapy trial was conducted by the Medical Research Council/European Organisation for 2.2. Adjuvant Therapy. The postoperative adjuvant approach Research and Treatment of Cancer. In this phase III trial, 976 has several advantages. It allows for selection of patients at patients with high-grade T2-T4a, N0-NX, M0 bladder can- highest risk for surgical failure based on accurate pathologic cer were randomly assigned to three cycles of neoadjuvant staging, avoids delay in potentially curative surgery especially chemotherapy (CMV: cisplatin, methotrexate, and vinblas- in nonresponders to chemotherapy, and prevents overtreat- tine, n = 491) or no chemotherapy (n = 485) then followed ing patients who may have a reasonable outcome from sur- by institution’s choice of therapy with radical cystectomy gery alone. The drawbacks include difficulty in administering and/or radiation therapy [11]. At three years, the pathologic chemotherapy postoperatively as a result of declines in per- complete response (pCR) in the neoadjuvant group was 33%. formance status or development of complications and delays Although there was a 5.5% survival benefit at three years in treating occult metastatic disease. (55.5 for chemotherapy versus 50% for no chemotherapy), it The results from the adjuvant trials have been conflicting did not reach statistical significance. At eight-year followup, and difficult to interpret. Two small trials have shown a sig- results showed a statistically significant 16% reduction in the nificant difference in favor of adjuvant chemotherapy. One risk of death, corresponding to an increase in 10-year sur- trial randomized 91 patients with pT3-T4a or node-positive vival from 30 to 36% after neoadjuvant chemotherapy [13]. bladder cancer to four cycles of adjuvant chemotherapy or to A US Intergroup trial (INT 0080) randomized 307 pa0 observation after radical cystectomy [18]. The chemotherapy tients with stage T2-4, N0, M0 bladder cancer to three cycles regimen employed was cisplatin, cyclophosphamide, and do- of neoadjuvant methotrexate, vinblastine, doxorubicin, and xorubicin. There was a significant improvement in median cisplatin (MVAC) or no chemotherapy followed by cystec- survival in the adjuvant group (4.3 versus 2.4 years, P = tomy [12]. The trial took 13 years to accrue. At a median 0.0062) and percentage free of progression (70 versus 46%, followup of 8.7 years, pCR with MVAC was higher (38 versus P = 0.01) at three years compared to observation. The three- 15%). Patients treated with MVAC showed an improvement year overall survival in patients was not statistically signifi- in median overall survival (77 versus 46 months, P = 0.06) cant though. A German trial randomized 49 similarly high- and five-year overall survival (57 versus 43%, P = 0.06) that risk patients (pT3b, pT4a, and/or positive pelvic lymph were of borderline statistical significance. nodes) to MVAC, MVEC (methotrexate, vinblastine, epiru- The benefit of neoadjuvant chemotherapy was confirmed bicin, and cisplatin), or observation [19]. It was terminated by a meta-analysis of 11 randomized trials with 3005 pa- early when an interim analysis showed significant improve- tients. It was found that those who received neoadjuvant ment in three-year progression-free survival (PFS) (63% Advances in Urology 3 versus 13%, P = 0.002). Ten-year survival data from this trial grade 3 or 4 neutropenia, neutropenic sepsis, and mucositis. still favored adjuvant chemotherapy over surgery alone [20]. An updated analysis showed similar 5-year overall survival Of note, most patients randomly assigned to observation rates of 13.0% for GC and 15.3% for MVAC [7]. Although after cystectomy were not given chemotherapy at the time of the trial was not designed as an equivalence trial, GC has relapse. been adopted by many as the standard first-line treatment Other trials revealed no benefit with adjuvant chem- based on similar efficacy and lesser toxicity. otherapy. A Swiss trial randomized 77 patients with muscle- Another randomized phase III trial assigned 263 patients invasive (pT2), nonmetastatic bladder cancer to observation to high-dose-intensity MVAC (2-week cycles) with granu- or 3 cycles of cisplatin. There was no significant difference in locyte colony-stimulating factor (G-CSF) versus standard theoverallsurvivalat5yearsbetweenthetreatmentandcon- MVAC (4-week cycles) to see if overall survival can be im- trol group (57 versus 54%) [21]. proved [27]. High-dose-intensity MVAC had significantly The positive trials were criticized for having major defi- improved complete response (21 versus 9%), overall res- ciencies including small sample size, early stopping of patient ponse (62 versus 50%), and the median PFS time (9.1 versus entry, inappropriate statistical analyses, and poor reporting 8.2 months), but there was no difference in overall survival. of results [22]. A meta-analysis based on 491 patients from Toxicity was less with high-dose-intensity MVAC which was six trials reported an absolute improvement in survival of 9% attributed to the use of G-CSF. In a subsequent seven-year at 3 years [23]. It did acknowledge that the results were drawn update, there was a significant survival advantage at 5 years from limited data and, therefore, was insufficient to base with high-dose-intensity MVAC (21.8 versus 13.5%) [28]. reliable treatment decisions. Many large cooperative group This regimen is becoming more popular because of improved trials were designed to answer the adjuvant chemotherapy outcomes, shorter duration, and less toxicity compared to question but have been terminated prematurely due to prob- standard MVAC. lems with accrual. A recent large retrospective cohort study consisting of 3,947 patients from 11 centers demonstrated a significant survival benefit with adjuvant chemotherapy in 4. Biomarkers for Personalized Chemotherapy patients at the highest risk of disease progression, such as Currently, chemotherapy for bladder cancer is taking the ap- those with advanced pathologic stage and nodal involvement proach of one formula for all. Most patients presently receive [24]. In the 20% of patients with the highest risk disease, the a platinum-based regimen, usually GC. However, only about median survival was 25 weeks for those receiving adjuvant half of the bladder cancers will respond to chemotherapy. chemotherapy and 19.2 weeks for those who did not. As of Extensive research is ongoing to better understand the bio- now, adjuvant chemotherapy is commonly used in patients logy of the disease process in order to improve clinical out- with pT3-T4 or node-positive disease who have not received comes. Conventional prognostic factors such as the grade neoadjuvant chemotherapy. and stage of the tumor and tools like nomograms are useful in predicting the outcomes associated with surgery and the 3. Treatment of Metastatic Bladder Cancer risk of recurrence but are inadequate in predicting response to chemotherapy [29, 30]. Biomarkers have the potential not The standard treatment for patients with metastatic bladder only to further identify high-risk bladder cancer patients, but cancer is systemic chemotherapy. Although bladder cancer is also to help select therapy for those who will benefit most a chemosensitive tumor, the median survival with chemo- from it. A personalized approach to chemotherapy has the therapy is only around 14 months. The five-year survival rate potential to reduce toxicity and improve clinical outcomes remains poor at about 15% [7]. Cisplatin-based combination (Table 1). therapy is considered first-line based on a prospective randomized trial that compared cisplatin alone to MVAC in 269 patients [25]. Patients treated with MVAC had significant 4.1. Single Gene Markers improvement in response rate (39 versus 12%), PFS (10 versus 4.3 months), and overall survival (12.5 versus 8.2 4.1.1. p53. p53 is the most studied biomarker in bladder months). Toxicity is a major concern with the MVAC regi- cancer and many other cancer types. It plays a critical role in men, particularly leukopenia, febrile neutropenia, mucositis, the regulation of cell cycle and is also involved in DNA da- and nausea/vomiting. Only 24% of the patients received mage and repair, cell cycle arrest, and apoptosis [31]. Altera- full-dose MVAC without dosage modifications. Five patients tion in the p53 leads to a loss of its tumor suppressor function (4%) in the MVAC group plus 2 patients who were switched and is thought to be a key event in carcinogenesis. It has been over to the MVAC regimen died from treatment-related tox- reported that overexpression of p53 in the nucleus, as detect- icity. ed by immunohistochemistry, was associated with increased In the search for a less toxic yet still effective regimen, a risk of recurrence and death in bladder cancer [32, 33]. randomized phase III trial of 405 patients compared GC to However, a meta-analysis of 117 studies comprising of 10,026 MVAC [26]. The overall response rate (49 versus 46%), time patients showed that changes in p53 were only weak-ly to progression (7.4 versus 7.4 months), and median survival predictive of recurrence, progression, and mortality in blad- (13.8 versus 14.8 months) were similar for both regimens. dercancer[34]. p53 overexpression was predictive of recur- Patients treated with GC were more likely to complete treat- rence, progression, and mortality in 27%, 50%, and 29% of ment with fewer dose adjustments. They had experienced less eligible studies, respectively. 4 Advances in Urology

Table 1: Single gene markers for prognosis and prediction of response in bladder cancer.

Markers Function Relation to bladder cancer p5334 Tumor suppressor, DNA repair, and apoptosis p53 mutation associated with high recurrence and progression Low expression associated with increased response to platinum-based ERCC141−43 DNA repair chemotherapy High expression with improved survival and possibly resistance to RRM141, 49 Synthesis of deoxyribonucleotides gemcitabine hENT154-55 Nucleoside transporter Sensitivity to gemcitabine BRCA159 DNA repair Low expression related to increased response and prolonged survival MDR142, 60 P-glycoprotein eﬄux pump High expression associated with resistance to chemotherapy Bcl-264 Antiapoptosis protein Associated with more advanced stage and worse prognosis

In addition to having prognostic qualities, studies have P = 0.03). Low ERCC1 level is predictive of the progression- suggested that p53 may be predictive of benefit to chemother- free survival in patients who received adjuvant cisplatin- apy [35]. A retrospective analysis of patients treated with ad- based chemotherapy. At 5 years, only 45% of patients with juvant therapy found that patients with p53 alteration had low ERCC1 level had progressed versus 70% of patients with increased sensitivity to treatment and had more benefit high ERCC1 level (hazard ratio = 0.52, P = 0.03) [42]. There from adjuvant chemotherapy [36]. Since p53 is involved in was also a PFS advantage found in metastatic patients with cell cycle arrest and DNA repair, the lack of a normal p53 low ERCC1 (10.6 versus 8.4 months, P = 0.03) [43]. How- could result in greater cancer cell killing when exposed to ever, prospective randomized controlled clinical trials are DNA-damaging therapy. This hypothesis was put to the test needed to determine the true value of ERCC1 expression in in a phase III trial that focused on patients with pT1 or predicting response to platinum-based chemotherapy. pT2, N0, M0 bladder cancer who had undergone a radical cystectomy and bilateral pelvic lymphadenectomy [37]. This 4.1.3. RRM1. Ribonucleotide reductase subunit M1 (RRM1) group is not usually treated with adjuvant therapy but gene encodes one of two nonidentical subunits of ribonu- has a recurrence rate of about 30%. Those whose tu- cleotide reductase, an essential enzyme involved in the pro- ≥ mors demonstrated 10% nuclear immunoreactivity for duction of deoxyribonucleotides for DNA synthesis and p53 were randomized to three cycles of adjuvant MVAC ver- repair [44]. Another function of RRM1 is suppression of cell sus observation while p53-negative patients were observed. migration and metastasis formation [45]. It is the molecular The trial tried to confirm p53 as a predictive biomarker and target of gemcitabine, an antimetabolite used in several ma- to see whether p53-altered tumors would respond better to lignancies including lung and bladder. Increased expression MVAC. A total of 521 patients were registered, 499 under- of RRM1 is associated with increased survival of patients went p53 assessment, 272 (55%) were positive, and 114 with resected NSCLC [46]. In patients with early stage (42%) were randomly assigned. Unfortunately, due to the NSCLC who had only received surgical treatment, the overall high patient refusal rate, lower than expected event rate, and survival for those with high RRM1 expression was more than failures to receive assigned therapy, accrual was halted and 120 months compared to 60.2 months for those with low questions about p53 as a biomarker remain. RRM1 (P = 0.02) [47]. The survival advantage was limited to patients with tumors that also expressed ERCC1. On the 4.1.2. ERCC1. Nucleotide excision repair pathway plays a other hand, high RRM1 expression appears to be a predictor major role in DNA damage repair, and the excision repair of decreased response to gemcitabine/platinum chemother- cross-complementing group 1 (ERCC1) is key member [38]. apy [48]. The cytotoxic effect of cisplatin is attributed to the formation RRM1 may have utility as a biomarker in bladder cancer of bulky DNA adducts. ERCC1 helps remove these adducts as well. In the Bellmunt study where patients received GC andthusmaycauseresistancetoplatinumagents[39]. In with or without paclitaxel, there was a trend towards longer NSCLC, high ERCC1 is associated with an improved prog- time in progression in tumors with low RRM1 expression nosis and predictive of reduced response to platinum-based [41]. High RRM1 expression was found to be prognostic for < therapy [40]. improved survival in younger patients (aged 70 years) with Studies have also been done to evaluate ERCC1 as a bio- muscle-invasive bladder [49]. The median overall survival marker in patients with advanced bladder cancer treated with was 10.6 years in younger patients high RRM1 expression P = . cisplatin-based chemotherapy. Bellmunt et al. performed versus 1.6 years in older patients ( 0 001), but made no ff gene expression analysis by using real-time quantitative PCR significant di erence for patients with low RRM1 expression in tumors of 57 patients with metastatic or locally advanced, (2.3 versus 1.6 years in younger and older patients, resp.). surgically incurable patients who were treated with either GC or GC plus paclitaxel [41]. At a median followup of 4.1.4. hENT1. Human equilibrative nucleoside transporter 19 months, the median survival was significantly longer in 1 (hENT1) is the major molecule of nucleoside transporter patients with low ERCC1 level (25.4 versus 15.4 months, proteins. Gemcitabine is a pyrimidine nucleoside analogue Advances in Urology 5 that requires plasma membrane nucleoside transporter pro- regulating cellular apoptosis. It was originally identified in teins to enter the cell and exert cytotoxicity. Studies in cul- follicular lymphoma at the site of the t(14; 18) translocation tured cells showed that hENT1 deficiency is associated with [61]. Bc1-2 is an antiapoptotic protein that is localized in in- gemcitabine resistance [50].TheabilityforhENT1topredict tracellular membranes and has been found to have prognos- benefit in patients receiving gemcitabine has been studied in tic value in bladder cancer. Overexpression of Bcl-2 is asso- other cancers such as pancreas and lung [51–53]. ciated with reduced survival in patients with invasive bladder In a small study of 12 patients, hENT1 was detected in 3 cancer and lower response rate to chemotherapy [62]. In pa- patients and two of them presented with a complete response tients with invasive disease treated with radiotherapy only, to gemcitabine [54]. The mean value of hENT1 was signifi- Bcl-2 positivity was found to be related to poor local control cantly higher in the patients who had a pathological complete (36 versus 72%) as well as to shorter disease-specific sur- response. A larger study evaluated 40 patients with metastatic vival (74 versus 94%) at 3 years [63]. In a study of four apop- bladder cancer who received GC-based chemotherapy [55]. tosis markers, including Bcl-2, caspase-3, p53, and survivin, Immunohistochemistry on tumor tissue was performed with in patients treated by radical cystectomy, Bcl-2 was indepen- specific hENT1 antibodies. Eighteen (90%) out of 20 pa- dently associated with higher pathologic stage, probability of tients with high hENT1 expression showed a response to disease recurrence (HR 2.24, P<0.001), and disease-specific chemotherapy whereas only 7 (35%) of 20 patients with low mortality (HR 2.06, P = 0.001) [64]. hENT1 expression responded. A significantly longer median survival was seen in patients with high hENT1 expression 4.1.8. MicroRNA. MicroRNA (miRNA) is small noncoding than those with lower levels (17.3 versus 11.6 months, P = regulatory RNA molecules with the stem-loop secondary 0.003). Therefore, hENT1 might be a relevant biomarker in structure. Its size ranges from 17 to 25 nucleotides. It was metastatic bladder cancer patients receiving GC-based chem- first found in worms but later was found in most eukaryotic otherapy. cells [65, 66]. It works as a posttranscriptional regulator of genes by binding to the complementary 3 untranslated re- 4.1.5. BRCA1. The breast cancer susceptibility gene 1 gion of target mRNA and degrading the target mRNA or sup- (BRCA1) is a tumor suppressor gene that is central in DNA pressing translation. Because one miRNA can bind to and repair pathways. It encodes a nuclear protein that functions regulate the expression of multiple mRNAs, it works as in multiple biological processes, including gene transcrip- a master posttranscriptional regulator of gene expression. tion, DNA damage repair, and apoptosis [56]. Low expres- miRNAs are involved in almost every aspects of oncogenesis. sion of BRCA1 has been found to increase sensitivity to They have been found to be upregulated or downregulated cisplatin-based chemotherapy in ovarian cancer and NSCLC depending on their corresponding functions as a tumor sup- [57, 58]. A similar result was found in bladder cancer where pressororpromoter[67]. Multiple miRNAs have been found a significant pathologic response to neoadjuvant cisplatin- to be involved in bladder cancer [68]. Genome-wide deep se- based chemotherapy was attained in 66% of patients with quencing of clinical specimens revealed that a set of miRNAs low/intermediate BRCA1 levels compared with 22% of was aberrantly expressed in bladder cancer when compared patients with high BRCA1 levels (P = 0.01) [59]. Median to the normal matched control [69]. Many of the miRNA survival was prolonged in patients with low/intermediate dysregulated in bladder cancer were repeatedly identified in compared to high BRCA1 levels (168 versus 34 months, P = other cancer types, but some of them have not been reported ff 0.002). BRCA1 may be a useful tool in the selection of pa- before [70]. Some of these miRNAs a ect several signaling tients for neoadjuvant cisplatin-based chemotherapy. and metabolic pathways that can be potentially targeted for cancer therapy in bladder cancer. Among those miRNAs, MiR-34a is frequently downregulated or deleted in several 4.1.6. MDR1. The multidrug resistance gene 1 (MDR1) en- cancer types [71, 72]. It is a known downstream effector of codes P-glycoprotein (Pgp), a membrane protein that acts as ffl p53 that regulates several components of the p53 pathway an energy-dependent cellular e ux pump. Pgp can reduce such as Cdk6 and E2F3. Researchers at University of Cali- intracellular concentrations of chemotherapy drugs like an- fornia at Davis found that transfection of bladder cancer cell thracyclines and vinca alkaloids which are components of lines with pre-miR-34a followed by cisplatin treatment re- MVAC, resulting in decreased cytotoxicity. Furthermore, it sults in a dramatic reduction in clonogenic potential and in- appears that chemotherapy drugs induce MDR1 and lead to duction of senescence compared to treatment with cisplatin drug resistance [60]. In patients with locally advanced blad- alone. Analysis of 27 preneoadjuvant chemotherapy patient der cancer receiving adjuvant chemotherapy, high MDR1 samples revealed many of the patients who subsequently did expression is associated with inferior survival [42]. After 2 not respond to treatment (based on surgical resection post- years, more than 65% of patients with high MDR1 expression chemotherapy and 5-year survival data) express lower levels had progressed compared to only 25% of patients with low of miR-34a [73]. However, the clinical significance needs to MDR1 expression. After 5 years, only 23% of patients with be defined in prospective trials. high MDR1 expression were still alive versus 62% of patients P = . with low MDR1 expression (HR 0.25, 0 0006). 4.2. Combination of Genetic Markers. Individual gene biomarkers may not adequately capture the complex molecular 4.1.7. Bcl-2. B-cell lymphoma 2 (Bcl-2) is the represent- activities in tumor cells. To accurately predict chemosen- ing member of the large Bcl-2 family that is important in sitivity requires a large body of information. For example, 6 Advances in Urology there are over 150 genes involved in the major DNA repair Platinum chemotherapy pathways that are relevant to platinum-DNA adducts alone. Several studies have been conducted using a combination of Dose, administration genetic markers to predict response to chemotherapy. Takata et al. analyzed the gene expression profile of 27,648 genes Drug metabolism from 27 invasive bladder cancers using a cDNA microarray [74]. Profiles of tumors from patients who responded to Drug delivery, local vasculature MVAC neoadjuvant chemotherapy were compared to nonresponders to develop a scoring system of 14 predictive genes. Cell uptake and efflux This system was able to accurately predict drug response in 8 out of 9 patients. It was applied to 22 additional cases of Intracellular inactivation bladder cancer and correctly predicted clinical response for 19 cases [75]. Another approach uses the coexpression extra- DNA damage polation (COXEN) algorithm derived from expression microarray data of the National Cancer Institute (NCI)-60 cell Cell cycle arrest and DNA repair line panel to predict drug sensitivity of bladder cancer cell lines [76]. The COXEN-based gene expression model was Fast repair/tolerance Slow repair/intolerance able to effectively stratify chemosensitivity and predict the 3- year overall survival in patients treated with MVAC [77]. Survival/resistance Apoptosis/sensitive Figure 1: Pathways leading to chemotherapy-induced cell death 4.3. DNA Damage as a Predictor of Chemoresistance. The ma- and resistance. The major steps are shown in the sequential order. jor limitation of studying individual genes or gene combina- DNA damage is the critical step in this response pathway. Cells with tion in cancer specimens is that in vivo patient pathophys- low chemotherapy-induced DNA damage will survive chemother- iological changes cannot be analyzed. Furthermore, genetic apy and are chemoresistant. We propose that chemoresistance can alterations can be so complicated that they cannot be fully possibly be identified by measuring chemotherapy-induced DNA explored. For example, over 700 genes are involved in cellular damage and that some of the underlying resistance mechanisms can response to platinum chemotherapy [78]. Even though all potentially be elucidated by measuring the other major steps such these genes can be studied with the currently available high as metabolism, cell uptake/efflux, and DNA repair. The underlined throughput analysis, such as microarray and whole genome steps can be determined with AMS. or transcriptome sequencing, the in vivo factors (such as drug metabolism, distribution, local vasculature, and drug delivery) and external factors (such as dose calculation and ad- We have developed a highly sensitive technology to meas- ministration) cannot be analyzed. ure carboplatin or oxaliplatin-induced DNA damage using To overcome these limitations, we have taken a radically accelerator mass spectrometry (AMS). It is an isotope ratio different approach to study chemoresistance to platinum mass spectrometric method that precisely determines the therapy in vivo under the physiological conditions [79, 80]. concentration of very rare (<1:109) isotope in an isolated As alkylating agents, platinum analogs kill cancer cells mainly sample [91, 92]. Compared with the other technologies, the through induction of DNA damage (adducts). We hypothe- advantage of using AMS to detect DNA adducts/damage is its size that cancer cells with low platinum-induced DNA can high sensitivity and precision. It can detect 14C at the zepto- survive chemotherapy and are platinum resistant (Figure 1). mole (zmole, 10−21 mole) level per mg of carbon with the Therefore, measurement of platinum-induced DNA damage precision as low as 0.25% [93]. To measure platinum-in- may allow for identification of chemoresistance. By analyzing duced DNA damage, 14C-labeled platinum analogs are used. some other major steps along the chemotherapy pathway, When DNA damage is induced, platinum analogs together such as drug metabolism and DNA repair, some of the major with 14C are conjugated to genomic DNA. By measuring the underlying chemoresistant mechanisms can be determined amount of 14C on genomic DNA, the amount of DNA da- that help design of personalized chemotherapy to overcome mage can be calculated. Because of the supersensitivity of resistance. For example, if the chemoresistance to platinum AMS, this approach allows the measurement of DNA adducts chemotherapy is secondary to increased DNA repair (as without exposing cancer cells or patients to toxic chemother- measured by fast decrease of DNA adducts over time), plat- apy. To perform this test, cancer cells/patients are first treated inum drugs can be combined with a DNA repair inhibitor, with one nontoxic microdose (1% of therapeutic dose or less) such as a PARP (Poly ADP ribose polymerase) inhibitor, to of 14C-labeled platinum drug before cancer patients undergo overcome resistance. Several studies already showed that low biopsy or surgical resection. Biopsy is required for most can- DNA damage induced by alkylating agents correlated with cer patients before chemotherapy can be administered as the chemoresistance with a few exceptions [81–90]. However, the standard of care. Some of the tumor specimens will be used major limitation for those studies to be in clinical application for the AMS analysis. Only a few milligrams of tumor tissue is that the technologies used in those studies are not sensitive are needed for detection of DNA adducts with AMS. One enough for clinical applications because patients had to major limitation of this approach is that cisplatin cannot receive toxic therapeutic to high-dose chemotherapy before be studied as there is no carbon atom in cisplatin molecule DNA damage could be detected. to allow for 14C labeling. Fortunately, the chemoresistance Advances in Urology 7 spectrum of cisplatin and carboplatin is very similar as these progression,” Urologic Oncology, vol. 28, no. 4, pp. 429–440, two drugs form the same platinum-DNA cross-links [94]. 2010. We have performed extensive preclinical studies to show [3] Z. Kirkali, T. Chan, M. 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Review Article The 5 Alpha-Reductase Isozyme Family: A Review of Basic Biology and Their Role in Human Diseases

Faris Azzouni, Alejandro Godoy, Yun Li, and James Mohler

Department of Urology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buﬀalo, NY 14263, USA

Correspondence should be addressed to Faris Azzouni, [email protected]

Received 15 July 2011; Revised 11 September 2011; Accepted 27 September 2011

Academic Editor: Colleen Nelson

Copyright © 2012 Faris Azzouni et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Despite the discovery of 5 alpha-reduction as an enzymatic step in steroid metabolism in 1951, and the discovery that dihydrotestosterone is more potent than testosterone in 1968, the significance of 5 alpha-reduced steroids in human diseases was not appreciated until the discovery of 5 alpha-reductase type 2 deficiency in 1974. Affected males are born with ambiguous external genitalia, despite normal internal genitalia. The prostate is hypoplastic, nonpalpable on rectal examination and approximately 1/10th the size of age-matched normal glands. Benign prostate hyperplasia or prostate cancer does not develop in these patients. At puberty, the external genitalia virilize partially, however, secondary sexual hair remains sparse and male pattern baldness and acne develop rarely. Several compounds have been developed to inhibit the 5 alpha-reductase isozymes and they play an important role in the prevention and treatment of many common diseases. This review describes the basic biochemical properties, functions, tissue distribution, chromosomal location, and clinical significance of the 5 alpha-reductase isozyme family.

1. Introduction scrotum), and pubertal growth of facial and body hair. DHT plays an important role in several human diseases, Testosterone (T) is the most abundant androgen in serum. which include acne, hirsutism, male pattern baldness, benign Approximately 97% of T is bound to albumen and sex- prostate hyperplasia (BPH), and prostate cancer (CaP) [3]. hormone binding globulin and the remaining 3% is free The role of DHT was discovered after the description of and biologically active. T is synthesized by the Leydig 5α-R2 deficiency in a group of males from the Dominican cells of the testes under the control of the hypothalamus Republic [4]. DHT has 2–5 times higher binding affinity and anterior pituitary gland. In male fetuses, T stimulates the differentiation of the Wolffian duct into male internal for AR than T, and 10-fold higher potency of inducing AR ff genitalia (epididymis, vas deferens, and seminal vesicles) signaling than T [5], which means that their e ects are ff and development of libido, enlargement of the vocal cords, di erent but complementary [6]. skeletal muscles, penis, and scrotum and the initiation Three isozymes of 5α-R are known to exist (5α-R1-3) [7] of spermatogenesis at puberty [1, 2]. T is taken from and two other proteins exhibit 5-alpha reducing capabilities, circulation to cells through processes that remain poorly glycoprotein synaptic 2 (GPSN2), and glycoprotein synaptic understood. Intracellular T is converted to dihydrotestos- 2-like (GPSN2L) proteins. Only one 5 beta-reductase (5β- terone (DHT), the preferred ligand for androgen receptor R) enzyme has been identified. Its products, 5β-isomers, (AR) transactivation, by the enzyme 5 alpha-reductase (5α- are labeled as epi-product, such as 5β-DHT (epi-DHT) R). Upon ligand binding and transactivation, the DHT- [8]. Several compounds have been developed to inhibit the AR complex translocates from cytoplasm to nucleus and 5α-R enzyme system and they play an important role in activates the transcription of certain genes (the androgen the prevention and treatment of many common diseases receptor-regulated genes, ARRG). [9]. This review describes the basic biochemical properties, DHT is important for in utero differentiation and growth functions, tissue distribution, chromosomal location, and of the prostate gland, male external genitalia (penis and clinical significance of this enzyme family. 2 Advances in Urology

2. Background was highest in the primordia of the prostate and external genitalia prior to their virilization, but very low in Wolffian Steroids are a special type of lipid. The backbone of steroids duct structures [27, 28], and from genetic studies on a rare is the compound “gonane”, a 17-carbon molecule composed disorder of male sexual differentiation, originally termed of 4 rings. The three cyclohexane rings are labeled A, B, pseudovaginal perineoscrotal hypospadias and subsequently and C. These 3 rings together are called phenanthrene. Ring referred to as 5α-R deficiency [4]. Analysis of enzyme activity D is a cyclopentane ring. The carbon atoms are numbered in skin samples and of urinary and serum steroids revealed a from 1 to 17. Typically, steroids have a methyl group (– generalized defect in the conversion of T to DHT. CH3) at carbons C-10 and C-13 and an alkyl side chain Studying 5α-R was hampered by the insolubility of the (R) at C-17 (Table 1). Alkanes are saturated hydrocarbons protein, a hurdle which was overcome in 1989. The tech- composed of carbon and hydrogen atoms linked by single nique of expression cloning in Xenopus laevis oocytes bonds. The simplest alkyl group is a methyl group. Steroids was used to isolate a cDNA from rat liver that encoded vary by the configuration of the alkyl side chain, the number 5α-R enzyme, which was used to isolate a human 5α-R of additional methyl groups, and the functional groups by cross-hybridization with a prostate cDNA library. The attached to the steroid nucleus. Carbons number 18 and two expressed proteins had different biochemical properties 19 are attached to carbons number 13 and 10, respectively. and different responses to finasteride. These observations Additional carbon atoms are usually a part of the R side suggested the presence of two 5α-R isozymes that were chain or attached elsewhere to the steroid backbone [22]. confirmed by studies done in patients with 5α-R deficiency. Androgens are derivatives of androstane and contain 19 car- The coding sequence of the gene specifying the rat liver bons and either a keto group (e.g., dehydroepiandrosterone cDNA was isolated and found to be normal in these patients. (DHEA) and androstenedione (ASD)) or a hydroxy group Further genetic studies in these patients identified a different (e.g., T and DHT) at position 17 of the steroid nucleus mutated gene that encoded a 5α-R in normal individuals (Figure 1). with identical biochemical properties to the human prostatic 5α-R. The first cDNA, isolated from rat liver, was named 3. Historical Overview 5α-R1 (SRD5A1) gene, and the second cDNA, which was isolated from human prostate and found defective in 5α-R- Steroid-5-reductases (5α-R and 5β-R) were first discovered, deficient patients, was named 5α-R2 (SRD5A2) gene [29]. purified, and characterized in rat liver homogenates [23]. More recently, with the development of genome-wide These early experiments demonstrated that these enzymes gene expression profile analyses, a third 5α-R (SRD5A3) were capable of irreversibly reducing the delta 4, 5 bond gene was identified. GPSN2 and GPSN2L proteins were (double bond between carbons 4 and 5; Δ4,5)ofC-19and identified using sequence searching and NCBI’s BLAST C-21 steroids to 5α-and5β-stereoisomers. (http://blast.ncbi.nlm.nih.gov/Blast.cgi). All primary species The first androgen isolated was androsterone, a 5α- (from plant, amoeba, yeast, to vertebrate) in Eukaryota reduced androstane, which was isolated by Butenant in 1931 contain all 3 subfamilies [8]. from 25,000 liters of urine from adult men. This steroid was assumed to be the male hormone until 1935 when Ernst 4. Family Members Laquer and his colleagues isolated T from several tons of bull testes. The 5α-R enzyme was characterized initially in the The 5α-R family is composed of 3 subfamilies and 5 members 1950s in rat liver slices based on its ability to convert deoxy- (isozymes) in total. Isozymes are different proteins that per- corticosterone to 5α-reduced metabolites [24]. Tomkins and form the same function: others showed that the enzyme required a reduced pyridine nucleotide cofactor (i.e., NADPH) and could metabolize a (a) 5α-R1 and 5α-R2, variety of steroid substrates [25]. Speculation persisted about (b) 5α-R3, whether a single enzyme or multiple enzymes were involved in 5α-reduction of steroids. The 5α-reduction of steroids (c) GPSN2 and GPSN2L proteins. made them susceptible to further reduction, sulfation, and glucuronidation, modifications that decreased their affinity 5. Functions to bind proteins, made them more hydrophilic and facilitated their excretion. In the 1960s, 5α-reduction was shown to 5.1. 5 Alpha Reduction: (5α-R1-3) [29, 30]. The substrates be an irreversible reaction and DHT was found to be a for 5α-reductases are 3-oxo (3-keto), Δ4,5 C 19/C21 steroids. more potent androgen than T in prostate bioassays [26]. The The group “3-keto” refers to the oxygen-carbon double bond administration of radiolabeled T to rats resulted in a time- at carbon 3. Delta 4, 5 refers to the double bond between dependent accumulation of DHT in the nuclei of ventral carbon atoms 4 and 5. The reaction involves a stereospecific, prostate cells, which subsequently bound to a specific nuclear irreversible breakage of the double bond between carbons (androgen) receptor. These data indicated that 5α-reduction 4 and 5 (delta 4, 5) with the aid of cofactor NADPH and of T is a crucial step in androgen action and focused the insertion of a hydride anion (H−) to the α face at attention on 5α-R. The central role of 5α-R in mammalian carbon C-5 and a proton to the β face at position C-4. male physiology was obtained from developmental studies Examples of substrates are T, progesterone, androstenedione, of mammalian embryos showing that 5α-reduction activity epi-T, cortisol, aldosterone, and deoxycorticosterone. The Advances in Urology 3

NH H R O 12 11 17 H H 13 16 Gonane H Finasteride 1 9 14 2 10 8 15 5 H H 3 7 H H 4 6 O N H H O H

F3C H Dehydroepiandrosterone

CF3 H H O NH HO O H Dutasteride H Dehydroepiandrosterone-sulfate H H O O N H H O S H H HO O Cl OH H

H Testosterone LY 191704

O N H H H O CH3 OH O H N

H Dihydrotestosterone H

Epristeride H H HO H H O H

Figure 1: Structure of various steroids.

physiologic role of 5α-reduction of these steroids (other than of the eye, is synthesized in the lens of the eye, and may T) is unknown but probably related to their degradation play a role in the regulation of aqueous humor formation and excretion or to certain physiologic functions. 5α- [32]. 5α-Dihydroaldosterone is a potent antinatriuretic agent dihydroprogesterone (5α-DHP) is a major hormone in the with somewhat diﬀerent physiologic eﬀects than aldosterone circulation of both normal cycling and pregnant women itself; its formation in the kidney is enhanced by restriction [31]. 5α-dihydrocortisol is present in the aqueous humor of dietary sodium intake, which suggests its importance for 4 Advances in Urology

Table 1: Different steroid families. Class Example Number of carbon atoms Steroid backbone Gonane 17 Estranes Estradiol 18 Androstanes Testosterone 19 Pregnanes Progesterone 21 Glucocorticoids Cortisol 21 Mineralocorticoids Aldosterone 21 Cholanes Cholic acid 24 Cholestanes Cholesterol 27 the conservation of sodium [33]: compared to AS-BP. 5α-R3 expression was increased in lung, + breast, papillary thyroid, and testicular (seminoma and yolk (Substrate) + (NADPH) + H sac) cancers compared to their benign counterparts. (1) −→ (5α-substrate) + NADP+ 5.4. Erythropoiesis [36]. 5α-C 19 steroids increase the pro- Uemura et al. used small interfering RNA (siRNA) to knock duction of erythropoietin hormone in the kidneys. 5β-C 19 α down the expression of 5 -R3 isozyme in 22RV1 and LNCaP- steroids are important for heme synthesis in the liver. C4-2 CaP cells by transfecting them with several siRNA expression vectors [34]. Subsequently, they studied mRNA α β expression of 5α-R3 (RT-PCR), cell growth and viability, 5.5. Regulation of Bile Synthesis [37]. Both 5 -R and 5 -R are and the ratio of DHT/T using liquid chromatography- involved in bile biosynthesis, where they catalyze the conver- α sion of 7α,12α-dihydroxy-4-cholesten-3-one into 7α,12α- tandem mass spectrometry. Knockdown of 5 -R3 expression α α α β caused decreased cell growth and viability and DHT/T dihydroxy- 5 -cholestan-3-one, and 7 ,12 -dihydroxy-5 - β ratio. Unpublished work from our group has confirmed cholestan-3-one, respectively. Only the 5 -isomer has been the ability of 5α-R3 to 5α-reduce 3-oxo, delta 4,5 C19 shown to be biologically active and is used for bile synthesis. α and C21 (T, androstenedione and progesterone) steroids in The 5 -isomer is inactive and suggested to be an inhibitory lysates of CHO-K1 cells transfected with 5α-R3 cDNA via step in bile biosynthesis regulation in humans. an adenovirus vector, CaP cell lines CWR-22 and CWR- 22R, and clinical human samples of androgen-stimulated 5.6. GPSN2 Family [38]. While the functions of the GPSN2 benign prostate (AS-BP), androgen-stimulated (AS-CaP), subfamily are not understood fully, several reports have and castration-recurrent (CR-CaP) CaP. shown that GPSN2 members are involved in the fourth reaction of fatty acid elongation by reducing a fatty chain 5.2. N-Glycosylation of Proteins: (5α-R3). Congenital defi- double bond in mammals. Although the substrate (fatty ciency of 5α-R3 has been linked to a rare, autosomal recessive acid) of GPSN2 members is structurally different from that disorder in which patients are born with mental retardation, of the other two 5α-R subfamilies, all three subfamilies of 5α- cerebellar, and ophthalmologic defects [35]. The presumed R share a similar biochemical ability of reducing a double defect involves the reduction of the terminal double bond bond of the substrate. of polyprenols to dolichols, an important step in protein N-glycosylation. N-linked protein glycosylation involves the 6. Protein Structure and addition of a 14-sugar glycan to select asparagine residues on Gene Location [8, 29, 39] a nascent protein to facilitate proper folding and trafficking of the protein and occurs in the membranes of endoplasmic 5α-R1 and 2 isozymes are NADPH-dependent, membrane- reticula. This disorder is part of the family of congenital associated (microsomal) enzymes, composed of 259 and disorders of glycosylation and was described for the first time 254 amino acids, and have molecular weights of 29.5 and in a family in the United Arab Emirates by Cantagrel et al. 28.4 kilodaltons, respectively. They contain a high content [35]. of hydrophobic amino acids distributed throughout their sequences, which suggests that they are intrinsic membrane 5.3. Potential Biomarker of Malignancy: (5α-R1-3) [14, 20]. proteins deeply embedded in the lipid bilayer. The bulk of published literature indicates that the expression Even though these two isozymes are intrinsic membrane of 5α-R1 increases and 5α-R2 decreases in CaP compared to proteins and catalyze the same reaction, they only share a benign prostate and BPH. Umera et al. confirmed for the limited degree of homology in protein sequence, are located first time increased expression of 5α-R3 at the mRNA level on different chromosomes, and possess distinctive biochem- in CR-CaP. Godoy et al., confirmed this at the protein level. ical properties. The average sequence identity between these A validated monoclonal antibody showed that expression two isozymes within a given species is approximately 47%, of 5α-R3 was increased similarly in AS-CaP and CR-CaP while the sequence identity between the same isozyme Advances in Urology 5 across species is 60% for 5α-R1 and 77% for 5α-R2. They other steroid receptors. The first inhibitors were steroids that are encoded by the 5α-R1 and 5α-R2 genes. These genes mimicked T and, in many cases, were substrates themselves have similar structures, with five coding exons separated (i.e., not true inhibitors). The inhibitors can be broadly by four introns. The positions of the introns are essentially classified into two categories: steroidal and nonsteroidal. The identical in the two genes. However, SRD5A1 is located steroidal class has more inhibitors thus far. on chromosome 5p15 whereas SRD5A2 is on 2p23. Gene The mechanism of 5α-RI is complex but involves the polymorphisms exist for the two genes and are more binding of NADPH to the enzyme followed by the substrate. common for 5α-R2. More than 850 and more than 550 single The Δ4,5 bond is broken and a hydride anion is transferred nucleotide polymorphisms (SNPs) have been reported for from NADPH directly to the C-5 carbon on the α face 5α-R2 and 5α-R1 genes, respectively [40, 41]. Only a few followed by a proton attacking the C-4 carbon on the β face of these gene polymorphisms affect enzyme activity; some leading to the formation of the product that subsequently decrease (e.g., V89L SRD5A2 variant) and others increase leaves the enzyme-NADP+ complex. NADP+ departs last and (e.g., A49T SRD5A2 variant) enzyme activity [42]. Molecular the enzyme becomes free for further catalysis cycles. Based on epidemiologic studies are inconclusive as to whether altered this, the mechanism of inhibition of 5α-R isozymes is divided 5α-R2 isozyme activity due to 5α-R2 gene polymorphism into three types [30]: affects CaP risk [43]. A variant of 5α-R1 gene was reported to increase risk of polycystic ovary syndrome (PCOS) and (a) competitive with the cofactor (NADPH) and sub- more severe hirsutism in lean women, whereas a variant of strate (bi-substrate inhibitors): the inhibitor binds 5α-R2 gene was associated with decreased risk of PCOS in the the free enzyme, for example, ONO-3805; same cohort [44]. More than 300 SNPs have been reported (b) competitive with the substrate: the inhibitor binds for 5α-R3 gene; however, their clinical significance remains the enzyme-NADPH complex for example, 4-, 6-, uncertain [45]. 5α-R3 is composed of 318 amino acids and and 10-azasteroids; α has only 19% homology with 5 -R1 and 20% homology with + α α (c) uncompetitive with the enzyme-NADP complex: 5 -R2 [39]. 5 -R3 is encoded by SRD5A3, which is located + at 4q12. The genes encoding GPSN2, GPSN2-like, and 5β- the inhibitor binds the enzyme-NADP complex aft- R are located at 19p13.12, 4q13.1, and 7q34, respectively. er the product leaves, for example, epristeride. GPSN2 and GPSN2-like proteins are composed of 308 and 363 amino acids, respectively. The amino acid sequence 8.1. Steroidal 5α-RI (Figure 1). (1) 4-Azasteroids: the 3-oxo, homology for GPSN2 is 15% with 5α-R1, 17% with 5α-R2 5-alpha steroids with a nitrogen atom at position 4 have been and 11% with 5α-R3. GPSN2-like has 6%, 11%, 6%, and the most extensively studied. Examples include finasteride 44% sequence homology with 5α-R1, 5α-R2, 5α-R3, and (MK-906), dutasteride (GG745), 4-MA, turosteride, MK- GPSN2, respectively. 386, MK-434, and MK-963. (a) Finasteride is a synthetic 4-azasteroid and is the first 7. Biochemical Properties [8, 29] 5α-RI approved for treatment of benign prostatic enlargement (BPE) and subsequently male pattern When examined in lysates of transfected cells, 5α-R1 exhibits baldness. Finasteride is a potent (mean inhibitory a broad pH optimum, which ranges between 6.0 and 8.5, concentration [IC50], 69 nM) competitive inhibitor α while 5 -R2 shows a narrow acidic pH optimum (pH 5– of 5α-R2 but inhibits less effectively 5α-R1 (IC50 5.5). However, there is evidence to suggest that inside intact 360 nM) [47]. Finasteride decreases mean serum level human cells, 5α-R2 isozyme functions optimally at a more of DHT by 71% after 24 weeks of use [48]. Seven-day neutral pH range (6.0–7.0). 5α-R1 has a larger turnover treatment with finasteride (1 or 5 mg daily) has been number, as indicated by its Kcat value and a lower substrate reported to suppress intraprostatic DHT in men with affinity for T, Km = 1–5 μM. 5α-R2 has a lower turnover lower urinary tract symptoms (LUTSs) attributed to number (Kcat) and a higher substrate affinity, as indicated BPE by approximately 85% relative to placebo [49], by Km = 0.004–1 μM for T. Under optimal conditions, whereas another study of finasteride 5 mg/d (also in 5α-R2 has a higher 5α-reducing activity than 5α-R1, as men with LUTS attributed to BPE) demonstrated a indicated by its high Vmax/km ratio. Both isozymes contain reductionof68%at6months[49]. Finasteride was an NH2-terminal steroid (ligand) binding domain and a shown in vitro to inhibit 5α-R3 at a similar potency to COOH-terminal NADPH binding domain. The apparent 5α-R2 (IC50 = 17.4 nM, 14.3 nM, resp.) in transfected dissociation constant for NADPH cofactor is similar for HEK-293 cells [21]. both isozymes (3–10 μM). No such comparisons exist for (b) Dutasteride is a synthetic 4-azasteroid with a half-life 5α-R3 except that it appears to be efficient at pH 6.5–6.9 of nearly 5 weeks and is only approved for treatment (unpublished work from our group). of BPH. Dutasteride is a dual 5α-RIsinceitismore effective (more potent) at inhibiting 5α-R1 and 2 α 8. 5α-Reductase Inhibitors [9, 30, 46] than finasteride; IC50 for inhibiting 5 -R1 is 7 nM and 5α-R2 is 6 nM. Dutasteride reduced mean levels Goal of development of 5α-reductase inhibitors (5α-RI) was of serum DHT at 24 weeks better than finasteride to bind to 5α-R with little or no affinity for the androgen or (94.7% versus 70.8% suppression) [50]andcaused 6 Advances in Urology

a 97% reduction in intraprostatic DHT levels in men or specially a Cl) and a methyl group at position with CaP treated with 5 mg/d for 6–10 weeks [51]. 4. LY 191704 is the most potent (IC50 = 8 nM). Another trial of dutasteride 3.5 mg/d for 4 months (b) Piperidones lack B and D rings. prior to RP decreased intraprostatic DHT by 99% (c) Quinolinones lack C ring. [52]. The near-maximal suppression of intraprostatic DHT with dutasteride 3.5–5 mg daily and the report (d) Pyridines lack B and C rings. that dutasteride inhibits 5α-R3 in vitro (IC50 = (e) Benzo[c]quinolinones tricyclic compounds de- 0.33 nM) [21] suggest that the development of a triple rived from 6-azasteroids (no D ring, aromatic 5α-R inhibitor may not be necessary. Table 2 provides ring for the C ring) that have selective but weak α a comparison between finasteride and dutasteride. inhibitory activity against 5 -R1. (f) Benzo[c]quinolizinones are tricyclic compounds (c) 4-MA was a potent dual inhibitor of 5α-R1 (IC = 50 derived from 10-azasteroids (no D ring, aroma- 1.7 nM) and 5α-R2 (IC = 1.9 nM). 4-MA had a 50 tic ring for the C ring) that include some very very low affinity for AR and thus was not expected potent, selective inhibitors of 5α-R1. to produce undesirable antiandrogen effects, such as impotence, impaired muscle growth, or gyneco- (Subgroups (b), (c), and (d)) are very weak 5α-R1I. mastia. However, 4-MA was withdrawn from clinical development after it was shown to be an inhibitor (2) Nonsteroidal aryl acids are tricyclic compounds de- of 3β-hydroxysteroid dehydrogenase and to cause rived from androstanecarboxylic acids that differ hepatotoxicity [9]. from their parent compounds in being selective, noncompetitive 5α-R1I. (d) Turosteride, MK-434, and MK-963 inhibit mainly 5α-R2. MK-386 is a selective 5α-R1 inhibitor [46]. (3) Butanoic acid derivatives contain an aromatic ring (generally benzene or indole) that bears a butanoic (2) 6-Azasteroids (e.g., GIlS7669X) have a heterocyclic B acid chain and aromatic moieties. Examples include ring (nitrogen atom at position 6) and a Δ4,5 bond in the A ONO-3805, demonstrated in vitro to be a selective ring and are potent competitive inhibitors of 5α-R1 and 2 inhibitor of 5α-R1, and FK143, which inhibits 5α-R1 [46]. and 5α-R2 equally and noncompetitively. (3) 10-azasteroids, for example, AS97004, are compet- (4) Polyunsaturated fatty acids, found in vegetable oils, α itive 5 -RI with a similar mechanism of action to 6-azas- have been found to inhibit human and rat microso- teroids [9]. mal 5α-R activity. In this group, y-linolenic acid is the (4) Androstanecarboxylic acids, such as epristeride, are most potent compound tested. Since 5α-R isozymes α noncompetitive, specific inhibitors for 5 -R2 [30]. are intrinsic membrane proteins, their activity may (5) Other steroidal inhibitors include progesterone esters depend on the unique environment of the lipid α such as 4-bromo-17 -(p-fluorobenzoyloxy)-4-pregnene-3, bilayer. Whether and how fatty acids may function as 20-dione [53], 2-azasteroids, 3-azasteroids, 19-nor-10-azas- endogenous regulators of 5α-R remain unknown. teroids, and diazasteroids [9]. (5) Some cations, especially zinc, have been reported to reduce sebum production in vivo and have been used 8.2. Nonsteroidal Inhibitors [9, 30, 46]. Several pharmaceu- to treat acne. In vitro assays have indicated that zinc tical and academic groups have pursued the synthesis of specifically inhibits 5α-R1. This inhibition may be nonsteroidal compounds that inhibit human 5α-reductases ff mediated both by non-competitive inhibition of T due to the undesired hormonal side e ects of steroidal com- binding to 5α-R and by reduced formation of the pounds. Nonsteroidal inhibitors can be classified according NADPH co-factor. to their structure. Most have been derived from azasteroidal inhibitors by removing one or more rings from the aza- (6) Other nonsteroidal inhibitors include epicatechin- steroidal structure. Nonsteroidal inhibitors are thought to 3-gallate and epigallocatechin-3-gallate, which are act as competitive inhibitors with exception of epristeride major constituents of green tea. Also included are analogues, which are noncompetitive inhibitors. The most 7-hydroxycoumarin derivatives, 2,6-disubstituted 4- potent and selective inhibitors of human 5α-R1 are found hydroxy-4-hydroxymethyl biphenyl derivatives, iso- among these classes of compounds and include the follow- flavonoids, and 3,3-diphenylpentane derivatives [9]. ing. 9. Tissue Distribution (1) Benzoquinolines include many subgroups. Numerous reports exist in the literature on the expres- (a) Benzo[f]quinolinones are tricyclic compounds sion pattern of 5α-R1 and 5α-R2inhumantissueat that are derived from 4-azasteroids by removing various stages of development. The results vary due to the D ring and substituting the C ring with an differences in antibody sensitivity and specificity, mRNA aromatic one. These are selective against 5α- analysis (in situ hybridization versus northern blotting versus R1. The potency against 5α-R1 increases by reverse transcriptase-polymerase chain reaction), protein substituting a halogen atom at position 8 (F, Br, analysis (immunohistochemistry versus western blotting), Advances in Urology 7

Table 2: Comparison between ﬁnasteride and dutasteride (referenced in text).

Finasteride Dutasteride Family Steroidal 5α-RI (4-azasteroid) Steroidal 5α-RI (4-azasteroid)

IC50 for 5α-R1, 2 and 3 (nM) 360, 69, 17.4 7, 6, 0.33 Male androgenic alopecia FDA-approved clinical uses Benign prostatic enlargement Benign prostatic enlargement 1 mg daily for male androgenic alopecia Clinical dose 0.5 mg daily 5 mg daily for benign prostatic enlargement Half-life (T 1/2) 6–8 hours 5 weeks ↓Serum DHT by 71% ↓Serum DHT by 95% Suppression of DHT ↓Intraprostatic DHT by 85% ↓Intraprostatic DHT by 97–99%

tissue preparation, nature of tissue, evaluation of results, not detected in the skin and scalp until the onset of puberty. tissue fixation protocols, and control tissue. In addition, At puberty, only 5α-R1 is reexpressed in the skin and scalp normal, benign, and malignant human tissue specimens are and persists thereafter until 81 years. In the prostate gland, heterogeneous with variable expression of proteins among Lunacek et al. reported that both 5α-R1 and 5α-R2 were specimens from different individuals and within the same detectable at the protein level using IHC until approximately specimen, that is, inter- and intraindividual variability. 1 year of age. After that, they were detectable at the mRNA Therefore, a summary of many studies that discussed the level (RT-PCR) until 6 years of age. Thigpen et al. only tissue distribution of 5α-R1-3 in different human tissues was detected 5α-R2 at the protein level using immunoblotting in tabulated to demonstrate differences in results (Table 3). prostatic tissue from a 7-year-old male. Since the methods used by this group did not detect 5α-R1 protein in the newborn, juvenile, or adult prostatic tissues, and since other 9.1. According to Age groups detected 5α-R1 at the protein level in fetal and α α 9.1.1. Fetus. Ellsworth and Harris [54] studied 5α-R activity adult benign prostatic tissue, 5 -R1 and 5 -R2 appear to in fetal scalp, back skin, and prostatic tissues and compared be expressed in the prostate in male fetuses and throughout it to 5α-R activity in adult male scalp and prostatic tissues. postnatal life. They studied the conversion of radio-labeled T into DHT in relation to pH and response to selective 5α-R1 and 5α- 9.1.3. Adulthood-Old Age. 5α-R1-3 is ubiquitously expressed R2 inhibitors and calculated the km of T at pH values of [10, 11, 13, 20, 55]. 5α-R1 and 5α-R2 are expressed 7.0 and 5.5. 5α-R1 is expressed in fetal scalp and nongenital differently in liver, genital and nongenital skin, prostate, (back) skin at levels that are 5–50 times less than adult skin. epididymis, seminal vesicle, testis, ovary, uterus, kidney, 5α-R2 is expressed in the fetal prostate at levels similar to exocrine pancreas, and brain (Table 2, Aumuller et al.). Our adult prostate. Thigpen et al. [13] studied 5α-R expression laboratory described the expression of 5α-R3 using IHC in in fetal liver, adrenal, testis, ovary, brain, scalp, chest, and various benign and malignant tissues. 5α-R3 is overexpressed genital skin, using immunoblotting. They detected 5α-R2 particularly in lung adenocarcinoma, testicular seminoma only in fetal genital skin. Lunacek et al. [55] studied the and yolk sac tumors, papillary thyroid cancer, and androgen- expression of 5α-R 1 and 5α-R2 at the mRNA (RT-PCR) and stimulated and castration-recurrent CaP relative to their protein (immunohistochemistry) levels in fetal and postnatal benign counterparts [20]. When contrasting these data with prostatic tissues until 6 years of age. Both 5α-R1 and 5α-R2 the expressed sequence tag (EST) database from NCBI, both proteins were expressed in prostatic epithelial and stromal sets of data suggest a broad pattern of expression for 5α-R1-3 components, at consistent levels throughout all age groups. in human tissues; ESTs for 5α-R1 (271 sequences) have been 5α-R1 is expressed mainly in the epithelium and 5α-R2 is reported from different human tissues, which include lung, expressed mainly in the stroma of the prostate. At the mRNA brain, intestine, skin, prostate, testis, and stomach [56]. ESTs levels, both are detectable throughout the ages studied and for 5α-R2 (39 sequences) have been reported in prostate, both peak in the second trimester. lung, liver, kidney, brain, testis, and skin [57]. ESTs for 5α- R3 (149 sequences) have been reported in kidney, testis, 9.1.2. Newborn-Onset of Puberty. In newborns, 5α-R1 is intestine, brain, liver, uterus, pancreas, skin, and prostate [58]. Tissue distribution of 5α-R3 protein in several human expressed at the protein level in the liver, skin, scalp [13] α and prostate [55]. 5α-R2 is expressed in prostate, seminal benign tissues was consistent with the tissue origin of the 5 - vesicles, epididymis, liver, and to lesser extent in scalp and R3 EST reported at NCBI [20]. skin [13]. Hepatic expression of 5α-R1 and 2 is present at the protein level (immunoblotting) throughout postnatal life. At 9.2. According to Organs. (See [10–13, 15–20, 54–61] approximately 1.5 years, the expression of both proteins is (Table 3).) 8 Advances in Urology -R2 that α 1-2 is ubiquitous -R1 more α α 5 5 uniformly spread in skin versus 5 is mainly found in inner epith RS )cells ), ZR − − ), PT ), C cells ) − − ) − − ) erences in ff − ) ), CD (+) ) ) − − − − er cells ( ff ) − ) of sweat glands, dermal − ), med ( − ) -R2 Notes α − Cytoplasmic, in epidermis from all sites: stratum spinosum (++), stratum basale (+), absent in stratum granulosum and stratum corneum, inner epithelial RS (++), matrix cells of hair bulb (+), absent in dermal papillae, fibrous and outer epithelial RS, basal (+) and glandular ( Mainly cytoplasmic: Prostate: stroma (+), epithelium (++), specially basal cells Seminal vesicle.: stroma (+), epithelium (++) Epididymis: stroma (+), epithelium (+) Testis: spermatogonia (+), Leydig and Sertoli cell ( Cerebral cortex: pyramidal c (++), glial c ( Adrenal: ZG (+), ZF (+/ Pituitary: prolactin cells (+), others ( Thyroid: thyrocytes ( Ovary: stroma (+), theca and granulosa cells (+/ (++), DT (+/ Kidney: glomerulus ( males and females adipocytes (+) No qualitative di Uterus: endometrium (+), myometrium (+) Liver: hepatocytes (++), bile duct c(+),kup ( Pancreas: exocrine (+), islets of Langerhans ( of sebaceous glands, myoepithelial (+) and secretory cells ( (+/ ) erent authors. ), ff − − > ), bile ), C cells erences in breast − ff − > ), medulla ( ) − er cells (++) − ff axilla ) ) − > − ) − eye lid): ) > -R1 5 α − -reductase 1–3 according to di lip stratum basale (++), stratum spinosum (++), absent in stratum granulosum and stratum corneum, dermal papillae, fibrous and outer epithelial RS (++), inner epithelial RS (+), matrix cells of hair bulb (++), scrotal fibroblast (++), basal and secretory cells of sebaceous glands (++), secretory and myoepithelial cells of sweat glands (++), arrector pili muscles (+), dermal adipocytes (+). No qualitative di males and females. Mainly nuclear: Prostate: stroma (+), epithelium (+) Seminal vesicle: stroma (+), epithelium (+) Epididymis: stroma (+), epithelium (+) Testis: Leydig cells (+), Sertoli cells (+) Ovary: stroma (++), theca and granulosa cells ( Cerebral cortex: pyramidal c (+), glial cells (+/ Nuclear, in epidermis from all sites: (scrotal Thyroid: thyrocytes ( Uterus: endometrium (+), myometrium (+) Liver: hepatocytes (+/ duct c (+), kup Kidney: glomerulus (+), PT ( DT (++), CD (+) Adrenal: cortex ( ( Pituitary: prolactin cells (+), others ( Pancreas: exocrine (+), islets of Langerhans ( α -R1-2 proteins. -R1-2 proteins. α α 3: Tissue distribution of 5 Antibody sensitivity and specificity confirmed by ELISA and WB. Tissues from surgical pts and autopsies, fixed in Bouin’s or formaldehyde Protein expression (IHC) using rabbit polyclonal antibodies against synthetic peptides from C-terminus parts of 5 Antibody sensitivity and specificity confirmed by ELISA and WB on FFPE biopsy or autopsy tissues Protein expression (IHC) using rabbit polyclonal antibodies against synthetic peptides from C-terminus parts of 5 Table Many tissue types, using a semiquantitative visual scale Tissue type Materials and methods 5 Epidermis: genital (scrotum) nongenital (Axilla, breast, lip, eyelid) using a semiquantitative visual scale ] ] 11 10 ¨ uller et al. [ -R1-2 -R1-2 α α 5 Author isozymes studied Eicheler et al. [ Aum 5 Advances in Urology 9 erences are ff -R1 protein was α 5 not detected in any prostate sample All di statistically significant > CR-CaP = HGPIN = -R2 was detected by WB Metastasis α = -R2 protein is expressed in -R2 mRNA was detected in -R2 was detected by WB in -R2 was not detected by WB in -R2 is expressed in -R2 Notes α α α α α α 5 prostate, seminal vesicles, epididymis, and liver. 5 prostate, SV, epididymis. and liver. 5 only in fetal genital skin (not detected in fetal liver, adrenal, testis, ovary, scalp, and brain). 5 newborn prostate, SV, epididymis, liver, skin. and scalp. 5 any sample of balding and nonbalding scalp from one man. It was detected in allprostate, normal BPH, and PC samples Mainly cytoplasmic in all specimens Immunostaining intensity: BPH In balding and nonbalding scalp: 5 infundibula, outer (mainly) and inner epithelial RS of hair follicles. No expression was detected in dermal papillae or in sebaceous glands AS-CaP = -R1 α AS-CaP > CR-CaP > BPH -R1 is expressed only in -R1 was detected by WB > -R1 was not detected by α α α 3: Continued. -R1 was detected by WB in -R1 protein is expressed in -R1 5 α α α mRNA was detected in cerebellum, hypothalamus, pons, medulla oblongata, skin, and liver 5 WB in any fetal tissue. 5 newborn liver, skin, and scalp.5 in all scalp samples from balding and nonbalding men (except one). It was not detected innormal any prostate, BPH, or PC sample 5 liver and chest skin and 5 In balding and non-balding scalp: 5 sebaceous glands No expression was detected in hair follicles or in epidermis Nuclear in BPH, shifts to cytoplasm in HGPIN and CaP Immunostaining intensity: Metastasis HGPIN Table -R2- α H-T 3 -R1- and 5 α -R1-2 and enzyme -R1-2 -R1-2 proteins. ıve, 5 ¨ α α α transfected COS-1 cells and IHC on transfected COS-1 cells-Evaluated by measuring percentage of moderate- and high-intensity immunostaining areas in relation to total epithelial, PIN, or tumor area intissues PE Protein expression (IHC) using validated mouse monoclonal antibodies against peptides from N-terminus parts of 5 activity using Messenger RNA (NB) and protein expression (IHC and WB) using rabbit polyclonal antibodies against peptides from C-terminus parts of 5 Protein expression (IHC) using rabbit polyclonal antibodies against peptides from N-terminus of 5 Antibody specificity confirmed with WB on na Tissue type Materials and methods 5 Scalp biopsies from bald and non-bald men Autopsy and surgical tissue samples Prostate: BPH (TURP), ASCaP (RP), CaP metastasis in androgen deprivation-treated men (autopsy) ] ] ] 13 14 12 -R1-2 -R1-2 -R1-2 α α α 5 5 Author isozymes studied Bayne et al. [ Thigpen et al. [ Thomas et al. [ 5 10 Advances in Urology erent ff Staining in CaP-adjacent benign tissue is not significanty di from low- and high-grade CaP for either isozyme > -R1 α stroma 5 > -R2 α adjacent -R2 mRNA is moderate -R1 mRNA in = α α > 5 > Normal prostate stroma > > -R2 immunostaining α luminal epithelial) and low grade BPH high grade > = = > -actin β -R2 enzyme activity -R2 mRNA -R2 mRNA and enzyme -R1 mRNA expressed in -R2 Notes α α α α α BPH 5 Mainly cytoplasmic (weak), in normal prostate and BPH tissue (basal stroma. In CaP, 5 became nuclear and cytoplasmic and more intense in HGPIN and CaP versus adjacent benign tissue (specially CR-CaP) 5 ISH showed that 5 expressed in epithelium mRNA expression levels by sqRT-PCR: liver Mainly cytoplasmic in all samples (benign and malignant) Immunostaining intensity: BPH grade mRNA as expressed on the basis of 5 activity were higher in BPH than in AS-CaP. Therewasapositivecorrelation between enzyme activity at pH 5.5 and expression of 5 5 epithelium enzyme activity in homogenized BPH tissue benign tissue CaP = -R2 α stroma 5 > > Normal -R1 mRNA is -R1 mRNA α > α moderate grade stroma BPH PC-adjacent benign basal) and in stroma > > > > -R1 immunostaining > -actin α BPH β CaP > > 3: Continued. -R1 immunostaining -R1 mRNA expression is -R1 mRNA expressed in -R1 5 α α α α in BPH (in both epithelium and stroma). In CaP, 5 became nuclear and cytoplasmic and more intense in HGPIN and CaP versus adjacent benign tissue (specially CR-CaP) Mainly nuclear in BPH, nuclear and cytoplasmic in CaP (all grades), and in adjacent benign epithelial tissue Immunostaining intensity: High grade prostate 5 expressed in epithelium mRNA expression levels by sqRT-PCR: liver Mainly nuclear, in normal prostate and BPH tissue (luminal epithelial ISH showed that 5 tissue low grade 5 similar in BPH and AS-CaP. There was no correlation between enzyme activity at pH (5.5 and 7) and 5 expression as expressed on the basis of 5 epithelium Table C ◦ -R α Cin ◦ C-T at 37 14 H-T at 37 3 -R1 and 2, validated by α Protein expression (IHC) using validated rabbit polyclonal antibodies, evaluated by visually estimating percent of total tumor area showing low-, moderate, and high-intensity in relation to Gleason score enzyme activity at pH 5.5 and 7 using Messenger RNA expression (sqRT-PCR) and measurement of 5 Messenger RNA expression (ISH, RT-PCR) and measurement of enzyme activity at pH 5 and 7.5 using Protein expression (IHC) using polyclonal rabbit antibodies against peptides from C-terminus parts of 5 ELISA and WB Messenger RNA expression (ISH, sqRT-PCR) in homogenized frozen pulverized prostate tissue homogenized pulverized frozen prostate tissue 7 > 7, 7, < Tissue type Materials and methods 5 Prostate: AS-CaP (RP) with Gleason score Prostate: BPH (TURP) and AS-CaP via RP or RC BPH tissue (TURP) frozen in liquid nitrogen or in ice-cold RPMI with FBS and archival PE-BPH tissue BPH (TURP), AS-CaP (RP), CR-CaP (channeling TUR) BPH and CaP tissue post prostate biopsy or RP frozen in liquid nitrogen, one human liver sample ] ] 15 ] 17 ] ] 14 16 61 et al. [ ff ¨ om et al. [ -R1-2 -R1-2 -R1-2 -R1-2 -R 1-2 ¨ α α α α α oderst Author isozymes studied Thomas et al. [ 5 S Lehle et al. [ Habib et al. [ Bonkho 5 5 5 5 Advances in Urology 11 -R1 is α in the ↑ in vitro -R2 (in nucleus) -R3 is ubiquitous -RI -R3 protein α α α α In all 3 tissues, expression of 5 consistenty more than 5 but similar in cytoplasm 5 Dutasteride is a triple 5 5 expression cytoplasm of malignant cells versus benign cells in prostate, testis, thyroid, lung and breast CA = 50 -R3 -R1 in ASBP α -R1 mRNA in CR-CaP CR-CaP α α 5 5 > > -R2 staining -R1and2in > < α α -R2 staining intensity: -R2 was undetectable ASBP) α α AS-CaP AS-CaP > = = hybridization; RCC: renal cell carcinoma; HCC: hepatocellular n embedded; RS: root sheath; RP: radical prostatectomy; sqRT- -R2 is mainly cytoplasmic in -R2 protein expression is -R1) -R2 activity -R2 mRNA -R2 Notes ffi α α α α α α in situ 5 all 3 tissues Nuclear 5 ASBP and AS-CaP BPH 5 detected in epithelium and stroma of BPH (less intense than 5 Cytoplasmic 5 Notdetectedinstromainanyof the 3 tissues In WB, 5 intensity: ASBP in CR-CaP 5 5 : CR-CaP = -R3 with similar potency (IC α 0.33 nM) = > -R2 50 α 5 > -R2 and 5 α -R2 in all 3 CR-CaP CR-CaP α = 5 > -R2 protein) -R1 staining -R2 enzyme > α α α -R1 staining intensity: -R1 α α AS-CaP AS-CaP = = 3: Continued. -R1 enzyme activity -R3 was mainly cytoplasmic -R1 is nuclear and cytoplasmic -R3 than finasteride (IC -R3 expression at the mRNA level is higher than 5 -R2 mRNA was the most abundant in BPH and muscle -R1 enzyme activity at pH 7.5 -R1 mRNA copy numbers -R2 mRNA in BPH -R1 protein expression is -R1 5 α α α α α α α α α α α in CR-CaP (3 folds) tissues 5 5 Benign tissue immunostaining: Kidney (PT,DT ++),exocrine liver pancreas (++), (++), skeletal muscle (+),spinosum skin ++), (strata gastric basale epithelial and cells (+),Malignant tissue: myometrium (++) colon adenoCA (++), esophagealRCC adenoCA (++), (++), HCC (++), ovarian mucinousadenoCA CA (++), (++), testis stomach seminoma and YSpapillary tumor CA (++), (++), thyroid endometrioid CAASBP: (++), basal breast epithelial CA cells, (+) HGPIN: benignepithelial basal cells, and AS-CaP neoplastic and CR-CaP: in neoplastic cells (5 immunostaining intensity: AS-CaP Not detected in stroma in anythe of 3 tissues In WB, 5 ASBP 5 in all 3 tissues Nuclear 5 14.3 nM, 17.4 nM, resp.). Dutasteride5 is a more potent inhibitor of 5 frontal cortex, heart, colon, stomach, liver,prostate, pancreas, testis, lung, mammary BPH, gland, brain,epidermis, cervix, total ovary, dermis, skin, small intestine, spleen,5 and kidney Finasteride inhibits 5 5 is similar to 5 activity at pH 5.0 5 5 5 intense in epithelium of BPH (higher than 5 Cytoplasmic 5 intensity: ASBP Table C ◦ n-embedded; RPMI: Roswell Park Memorial Institute; FBS: fetal bovine serum; PT: proximal tubules; DT: distal tubules; CD: -R 1–3 ffi α H-T at 37 C 3 ◦ -R1 and 2, α cystectomy; HGPIN: high-grade prostate intraepithelial neoplasia; ISH -R3 protein and α -R1-3 mRNA expression C-labelled ASD and T in α H-ASD at 37 validated by ELISA and enzyme activities at pH 5 and 7.5 using Messenger RNA (qRT-PCR), protein (IHC) expression using polyclonal rabbit antibodies against peptides from C-terminus parts of 5 quantified by visual scoring and digital image analysis enzyme activity using Protein expression (IHC) using validated monoclonal mouse antibody against peptide from N-terminus of 5 5 (RT-PCR) and measurement of 5 Protein expression (by IHC in TMAs that are quantified by visual scoring and digital image analysis and by WB) and enzyme activity in homogenized pulverized prostate tissue using intact cells in culture 3 14 n-embedded ffi Tissue type Materials and methods 5 BPH (RC) fixed in formaldehyde and para Benign and malignant human tissue TMAs 20 benign human tissues, CaP, and breast cancer cell lines ASBP, AS-CaP and CR-CaP (RP or channeling TURP) tissue that was FFPE or snap frozen in liquid nitrogen ] ] 18 ] 21 ] 20 19 -R3 -R1-2 -R3 -R1-2 α α α α Author isozymes studied Shirakawa et al. [ Titus et al. [ Godoy et al. [ Yamana et5 al. [ 5 5 5 collecting ducts; TURP: transurethral resection of prostate; RC: radical carcinoma; adenoCA: adenocarcinoma; CA: carcinoma; YS: yolk sac; TMA: tissue microarray. PCR: semiquantitative reverse transcriptase-polymerase chain reaction; PE: para ELISA: enzyme-linked immunosorbant assay; IHC: immunohistochemistry; WB: western blot; NB: northern blot; FFPE: formalin fixed para 12 Advances in Urology

10. Clinical Role of 5α-R by the administration of DHT, but is reversed by 3α-androstanediol treatment. The biochemical features of this syn- Alterations in the conversion of T into DHT by the enzyme drome include the following: 5α-R are associated with a number of human disorders: (a) high normal to elevated levels of plasma T, 10.1. 5α-R2 Deficiency (Pseudovaginal Perineoscrotal Hypo- (b) low normal to decreased levels of plasma DHT, spadias) [3, 62]. Studies in rabbit, rat, and human fetuses α (c) increased T to DHT ratio at baseline and following have shown that 5 -R activity is present in the urogenital hCG stimulation, sinus and external genital anlage prior to prostate and external genital differentiation. However, 5α-R activity is not (d) normal metabolic clearance of T and DHT, present in the Wolffian duct, at the time of epididymal, (e) decreased levels of urinary 5α-reduced metabolites of vas deferens, and seminal vesicle differentiation. Thus, T C19 and C21 steroids, with increased 5β/5α urinary and DHT have selective roles in male sexual differentiation metabolite ratios, during embryogenesis. T mediates Wolffian ductal differenti- (f) decreased plasma and urinary 3α-androstanediol ation, while DHT mediates male external genital and prostate glucuronide, a major metabolite of DHT, differentiation. 5α-R2 deficiency is caused by decreased synthesis of (g) increased plasma levels of LH and/or FSH. DHT due to mutations in the 5α-reductase 2 gene. At Phenotype, development and reproductive function in least 50 mutations have been reported and is autosomal- human females with 5α-R2 deficiency are unaffected. recessive in the majority of patients. 5α-R2 deficiency results in a 46,XY disorder of sexual development (formerly male pseudohermaphroditism). Affected males are born with 10.2. LUTS Attributed to BPE. BPE results in a significant normal male internal reproductive structures (epididymis, morbidity due to urethral obstruction and secondary detru- seminal vesicles, and vasa deferentia); however, their external sor dysfunction. Histological evidence of BPH is found in genitalia resemble those of females, that is, ambiguous 50% of males by the age of 50 and 90% of males by genitalia. These individuals have a small penis that resembles the age of 80 [63]. The development of BPH depends an enlarged clitoris, labioscrotal fusion, and a urogenital on androgens, and BPH does not occur in men castrated α sinus in which there are two separate urethral and vaginal prior to puberty [64]. 5 -R isozymes play significant roles openings. The vagina is short and blind ending. The testes in BPH development since DHT is the major androgen are either in the labia, or inguinal canals or intra-abdominal. in the prostate. Patients with decreased DHT production α The vasa terminate at the blind-ending vaginal pouch. The due to 5 -R2 deficiency have a small prostate and BPH prostate is hypoplastic, nonpalpable on rectal examination has not been reported [4]. In castrated dogs, treatment and is found to be rudimentary on transrectal ultrasound with either DHT or T results increased intraprostatic DHT and MRI. Prostatic volumes are approximately (1/10)th and BPH [65]. However, coadministration of T with a α of age-matched normal controls. Prostate biopsy reveals 5 -R inhibitor decreased DHT formation and prevented fibrous connective tissue, smooth muscle, and no identifiable BPH [66]. Finasteride and dutasteride have been shown epithelial tissue, which suggests atrophic epithelium or lack to decrease circulating and intraprostatic DHT by 60– of epithelial differentiation. Plasma PSA is low or unde- 90%, and 90–98%, respectively [48–52]. Finasteride and tectable in these patients. Administration of DHT results dutasteride result in a decrease in prostate size by 20–25% in enlargement of the prostate. Neither BPH nor CaP has through prostatic epithelial cell apoptosis and a significant been reported in these patients. At puberty, these individuals improvement in LUTS. Furthermore, they change the natural undergo partial virilization of the external genitalia, although history of the disease by decreasing the risk of acute urinary their secondary sexual hair remains sparse and they develop retention by 57–79% and decreasing BPH-related surgery by less male pattern baldness and acne despite normal sebum 48–69% [67, 68]. Although both isozymes are overexpressed production. They undergo an increase in muscle mass, in prostate tissues from patients with BPH [15], inhibition α phallic growth, development of male body habitus, and of 5 -R2 activity is the major contributor in the treatment α deepening of the voice. Their libido is normal and they of BPH as the additional inhibition of 5 -R1 activity by are capable of erections. Sperm production and fertility dutasteride does not appear to be of any further benefit in have been reported but depend on testicular location. The BPH treatment [69]. mechanism of partial virilization at puberty is through either the androgen receptor binding very high levels of serum T, 10.3. Primary Prevention of CaP [67, 68]. Both finasteride albeit at lower affinity, or the increased expression of skin and dutasteride have been tested in large, prospective, 5α-R1 at puberty, which results in peripheral synthesis of randomized, placebo-controlled, double-blind studies as DHT from T or via the action of 5α-R3 through unknown primary preventive therapies for CaP. The Prostate Cancer mechanisms. 5α-R1 gene is normal in these subjects. No Prevention Trial (PCPT) randomized nearly 19,000 men at genetic deficiencies of the type 1 enzyme have yet been low risk for CaP into a treatment group, given finasteride reported. Inactivation of the 5α-R1geneinmiceadversely at 5 mg/d and a control group, given placebo, who were affected reproduction in females. In addition, mice deficient followed up for 7 years. At the end of the study, participants in 5α-R1 manifest a parturition defect that is not reversed were offered a prostate biopsy. For-cause biopsies were done Advances in Urology 13

Table 4: Comparison between PCPT and REDUCE.

PCPT REDUCE Sponsor South West Oncology Group GalxoSmithKline Duration 7 years 4 years Risk of CaP in participants lower higher 18,882 randomized No. of participants 8122 randomized 9060 included in final analysis Age ≥55 years 50–75 years Entry serum PSA ≤3.0 ng/mL 2.5–10 ng/mL Yes (6–12 cores) within 6 months prior to Baseline biopsies No enrollment Study-mandated biopsies Year 7 Years 2 and 4 Study-mandated biopsy cores ≥6 (6 cores in nearly 80%) 10 (83% had at least 1 biopsy) Overall relative risk reduction in CaP 25% 23% versus placebo ↑26% (6.4% in finasteride versus 5.1% in Incidence of Gleason sum ≥7CaP Same (6.7% in dutasteride versus 6.8%: in placebo) placebo), P<0.05 Same over 4 years (0.9% in dutasteride versus 0.6% ↑91% (2.1% in finasteride versus 1.1% in in placebo); however, in years 3-4, there were 12 GS Incidence of Gleason sum ≥8CaP placebo), P<0.05 ≥8 CaP in dutasteride group (0.5%) versus 1 in placebo group (<0.1%), P<0.05 for abnormal DRE and/or PSA > 4.0 ng/mL. PCPT showed American Urological Association and the American Cancer that finasteride was effective at reducing the overall risk of Society issued a “cautious” joint statement that accepted biopsy detectable CaP by nearly 25% and this was due mainly these drugs as an option to prevent CaP provided that to reduction in the risk of low-grade disease (Gleason sum they are used mainly after a thorough discussion of risks <7). The reduction in the risk of CaP was seen across all and benefits. In January 2011, the FDA’s Oncologic Drug subgroups, such as age, race, family history, and baseline Advisory Committee voted against recommending either PSA. Finasteride users also had improved BPH outcomes. dutasteride or finasteride for the specific indication of CaP The benefits of finasteride treatment occurred at the expense risk reduction because of the potential increased risk of high- of a higher rate of diagnosis of moderate- and high-grade grade disease. Table 4 provides a comparison between PCPT CaP (Gleason score ≥7) and more sexual adverse effects. and REDUCE. The Reduction by Dutasteride of Prostate Cancer Events trial (REDUCE) studied the effect of dutasteride versus placebo in a large group of men at higher risk of CaP than in 10.4. Treatment of CaP PCPT who had at least one negative prostate biopsy at (a) Biochemical Failure after Local Therapy with Curative baseline. The study lasted 4 years and participants received Intent [70–73]. Finasteride and dutasteride have been tried, mandatory prostate biopsy at 2 and 4 years. Dutasteride singly and in combination, in patients with biochemical fail- decreased the risk of biopsy detectable CaP by nearly 24% ure after radical prostatectomy or radiotherapy. The most and this reduction in risk was evident across all subgroups common combination was a 5α-RI and a nonsteroidal an- tested. The frequency of diagnosis of moderate- and high- tiandrogen. Finasteride and dutasteride monotherapy de- grade CaP was unchanged over the entire length of the creased serum PSA to variable extent. PSA decrease was more study and beneficial effects were observed on BPH outcomes. frequent and of greater magnitude in patients treated with However, 12 Gleason score 8–10 cancers were detected in an antiandrogen and 5α-RIversus5α-RI alone. However, the dutasteride group at years 3-4 versus only one in the none of these trials studied the impact on disease-specific placebo group and dutasteride treatment was associated with or overall survival and none compared 5α-RI mono- or more sexual adverse effects. The benefit of finasteride or combination therapy against 1st line androgen deprivation dutasteride in reducing the risk of low-grade CaP is clear. treatment in a randomized fashion. Low-grade CaP is unlikely to be lethal and patients may reduce their risk of overtreatment. However, these drugs may induce high-grade CaP, a concern that has prevented (b) CR-CaP. CR-CaP was thought for many years to be FDA approval of finasteride and dutasteride use for CaP androgen-independent or hormone-refractory but CR-CaP prevention. Several secondary analyses of these two trials remains AR-dependent and probably AR-ligand dependent have concluded that these drugs actually reduce the risk in almost all cases [74]. Despite castrate serum levels of of moderate- and high-grade disease but these analyses are T(<50 ng/dL), CR-CaP tissue levels of T and DHT were hypothesis-generating and not definitive. In March 2009, the similar and 80–90% lower compared to their levels in benign 14 Advances in Urology prostatic tissue, respectively [75]. CR-CaP tissue synthesizes and adrenal tumors, congenital adrenal hyperplasia, and testicular androgens (T and DHT) in an intracrine fashion exogenous androgenic hormone administration. Regardless from several substrates such as cholesterol, progesterone, of the cause, overproduction of either T or T precursors adrenal androgens, and androstandione [76–79]. Other phe- leads to exaggerated T action in target tissues such as skin. In nomena intrinsically acquired by CR-CaP tissue in response skin, T is converted to DHT by the enzyme 5α-R, which acts to castration include the continuous expression of AR directly on hair follicles and the sebaceous glands. The most [80], upregulation of the synthesis of enzymes necessary frequent dermatologic manifestations of androgen excess are for steroidogenesis [76], AR hypersensitivity (up to 10,000 hirsutism, acne, and androgenic alopecia [91]. times) to low levels of ligands by alteration of its co-activator Androgen surge can miniaturize hair follicles resulting profile from SRC1 to TIF2 and through its phosphorylation in male androgenic alopecia at puberty and in the scalp by SRC and Ack1 tyrosine kinases [81–83] and AR functional of genetically predisposed individuals. In women, androgen mutations, which broaden ligand specificity in 5–30% of excess plays a role in scalp hair loss; however, this process cases [84]. is different than in men and is referred to as female pattern New second-line hormonal therapeutic agents that have hair loss. Hirsutism is a disorder of excessive growth of shown better performance in CR-CaP compared to the terminal hair in women, in a male-like distribution, which old generation of second-line hormonal therapies are abi- is stimulated by androgen excess [92]. In acne, the pubertal raterone acetate and MDV3100, among others [77, 85]. androgen surge increases the stimulation of sebaceous glands Abiraterone acetate is a potent, selective, and irreversible resulting in increased sebum production and acne formation inhibitor of CYP17A1 enzyme, which is an important in susceptible individuals [93]. enzyme in the intracrine synthesis of testicular androgens. In all androgen-stimulated skin disorders, the activity MDV3100 inhibits ligand binding to the AR and nuclear of 5α-R enzyme system is increased such as in the hair translocation of AR-ligand complex. The clinical response follicles of hirsute women [93], in balding scalps [94], and in to these new drugs is indirect proof that CR-CaP remains acne-prone skin [95]. Inhibition of the 5α-R enzyme system androgen stimulated. 5α-R isozymes are important in the appears to be a target for treatment of androgen-stimulated growth of CR-CaP tissue since they are upregulated in CR- skin disorders, since 5α-R inhibitors may result in fewer side- CaP and may contribute to intracrine synthesis of testicular effects by not blocking the action of T, unlike classical antiandrogens. These enzymes convert progesterone, ASD, and T androgens such as cyproterone acetate or spironolactone. into pregnanldione, androstandione, and DHT, respectively Finasteride and dutasteride reduce scalp DHT levels by [74, 79]. Pregnanldione is further converted via several steps 64% and 51%, respectively [96, 97]. In men with androgenic into androstandiol which is oxidized by 17β-hydroxysteroid alopecia, finasteride and dutasteride significantly increased dehydrogenase 2 and 10 (17β-HSD2 and 10) to DHT (the hair count after a minimum of 6-month treatment [98, 99]. backdoor pathway to DHT synthesis). Androstandione is While finasteride 1 mg daily was superior to 5% topical converted by 17βHSD3 into DHT. minoxidil in inducing hair growth [100], finasteride 5 mg Clinical trials of finasteride [70] and dutasteride [71]as daily was inferior to dutasteride 2.5 mg daily in a phase monotherapy in patients with advanced CaP showed no II study as treatment for male androgenic alopecia [97]. improvement of clinical end points. The presence of 5α-R3 Finasteride and topical minoxidil (but not dutasteride) are in CR-CaP is a potential explanation, until such time that an FDA-approved for male androgenic alopecia. In women inhibitor has been proven effective clinically. Combination with androgenic alopecia, neither finasteride nor dutasteride therapy of 5α-RI with antiandrogen or ketoconazole and is FDA-approved treatment options due to teratogenicity. hydrocortisone was tried in CR-CaP as second or third line Finasteride has been tested in postmenopausal women at the hormonal therapies [86–89]. PSA decreases of variable mag- 1 mg dose without success. However, finasteride was shown nitudes and durations were achieved in more than half to be effective in 4 women with elevated serum T levels [101]. the patients. However, none of the combination trials were Whether finasteride is only effective in women with hair loss designed to test the effect on disease-specific and overall sur- and hyperandrogenism or that higher doses are needed in vival. In a phase II, single arm study of 57 patients with CR- women remains to be tested. A single case report showed CaP, dutasteride added to ketoconazole, and hydrocortisone improvement with dutasteride in a woman who had failed reduced PSA ≥ 50% of baseline value in 56% of patients, to respond to finasteride [102]. responses that lasted for a median of 20 months. Median time 5α-RI also is acceptable therapy for hirsutism. Finasteride to disease progression was 14.5 months that was better than 5 mg/day was superior to placebo and similar to spirono- all prior studies of ketoconazole and hydrocortisone in CR- lactone and flutamide in reducing the severity of hirsutism CaP [89]. [103]; however, in a different study, finasteride was inferior to flutamide as a treatment for hirsutism [104]. Dutasteride 10.5. Androgen-Stimulated Skin Disorders (Acne, Androgenic has not been tested as a treatment for hirsutism. Alopecia and Hirsutism). Hyperandrogenism, or excessive Theroleof5α-RI is unclear for treatment of acne. MK- androgen production, is primarily a disorder of females. 386, a selective 5α-R1 inhibitor, decreased sebum DHT levels Polycystic ovary syndrome (PCOS) is the most common in a dose-dependent fashion [105]. MK-386 was examined cause of female hyperandrogenism with a prevalence of 6% as a treatment for acne in a placebo-controlled trial and to 10% in women of childbearing age [90]. Other causes wasfoundtobesimilartoplaceboandinferiortosys- of hyperandrogenism include androgen-secreting ovarian temic minocycline therapy. Furthermore, MK-386 did not Advances in Urology 15 enhance the therapeutic benefit of minocycline when used in [10] W. Eicheler, M. Dreher, R. Hoffmann, R. Happle, and G. combination [106]. In another study, finasteride decreased Aumuller, “Immunohistochemical evidence for differential the severity of acne but was inferior to flutamide and distribution of 5α-reductase isoenzymes in human skin,” cyproterone acetate with ethinyl estradiol [107]. 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Research Article Correlation of Gleason Scores with Diffusion-Weighted Imaging Findings of Prostate Cancer

Rajakumar Nagarajan,1 Daniel Margolis,1 Steven Raman,1 Ke Sheng,2 Christopher King,2 Robert Reiter,3 and M. Albert Thomas1

1 Department of Radiological Sciences, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1721, USA 2 Department of Radiation Oncology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1721, USA 3 Department of Urology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1721, USA

Correspondence should be addressed to M. Albert Thomas, [email protected]

Received 15 August 2011; Revised 5 October 2011; Accepted 5 October 2011

Academic Editor: Jack Schalken

Copyright © 2012 Rajakumar Nagarajan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The purpose of our study was to compare the apparent diffusion coefficient (ADC) derived from diffusion-weighted imaging (DWI) of prostate cancer (PCa) patients with three classes of pathological Gleason scores (GS). Patients whose GS met these criteria (GS 3 + 3, GS 3 + 4, and GS 4 + 3) were included in this study. The DWI was performed using b values of 0, 50, and 400 s/mm2 in 44 patients using an endorectal coil on a 1.5T MRI scanner. The apparent diffusion coefficient (ADC) values were calculated from the DWI data of patients with three different Gleason scores. In patients with a high-grade Gleason score (4 + 3), the ADC values were lower in the peripheral gland tissue, pathologically determined as tumor compared to low grade (3 + 3 and 3 + 4). The mean and standard deviation of the ADC values for patients with GS 3 + 3, GS 3 + 4, and GS 4 + 3 were 1.135 ± 0.119, 0.976 ± 0.103 and 0.831 ± 0.087 mm2/sec. The ADC values were statistically significant (P<0.05) between the three different scores with a trend of decreasing ADC values with increasing Gleason scores by one-way ANOVA method. This study shows that the DWI-derived ADC values may help differentiate aggressive from low-grade PCa.

1. Introduction prostate. A small number with high-grade tumors will progress to develop local, extracapsular tumor extension and Prostate cancer (PCa) is the most common malignancy distant metastases. among men in the USA, with an estimated 217,730 new cases Prostate tumors are graded according to their patholog- and 32,050 PCa-related deaths in 2010 [1]. The incidence ical appearance with a Gleason score (GS), which represents of PCa increases with age, and it is very uncommon in the sum of the dominant and subdominant histological men younger than 50 years old. With greater longevity and patterns (grades). High GSs indicate aggressive tumors with increased awareness of the disease leading to more men increased potential for local and distant spread; Gleason requesting screening, it is to be expected that there will be an grading has been shown to provide a spectrum of risk for increase in the number of patients diagnosed with PCa in the all patients [3]. Magnetic resonance imaging (MRI) provides future. Most men diagnosed with PCa ultimately survive the incremental value to clinical findings in staging patients disease and die of other causes. The overall 5-year survival of intermediate risk. For example, organ-confined disease rate is 99% for all stages, but only 34% when there are implies that the patient may benefit from local therapy such distant metastases [2]. The aim of PCa management is to as surgery [4]. MRI is more accurate than either digital identify, treat, and cure patients with aggressive disease that rectal examination (DRE) or transrectal ultrasound (TRUS) may prove fatal but to avoid overtreating those in whom biopsy in preoperative anatomical localization of PCa [5]. the disease is unlikely to be life threatening. Most patients The sensitivity and specificity of T2-weighted imaging for diagnosed with PCa have localized disease confined to the PCa vary widely due to differences in imaging techniques, 2 Advances in Urology reference standards, criteria for defining disease involvement MR Images were initially reviewed without clinical infor- on MRI, and interobserver variability [6]. In a meta-analysis mation, but the final report was generated after the clinical by Sonnad et al. T2-weighted imaging showed a maximum information was reviewed. The histology was reviewed by joint sensitivity and specificity rate of 74% for the staging an experienced pathologist. The edge and the contour char- of PCa [7]. In T2-weighted imaging, regions of PCa show acteristics of the lesions were defined using the same slices decreased signal intensity relative to normal peripheral (PZ) on which regions-of-interest (ROI) analyses were performed. tissue because of increased cell density and a loss of prostatic ROIs were drawn independently on the ADC maps, and ducts [8]. This finding is nonspecific, however, because other differences in measurement were resolved by consensus. diseases such as prostatitis or hyperplasia can also cause low ROIs were drawn in the tumor PZ of all the 44 PCa patients. signal intensity in T2-weighted imaging [9–12]. ADCs were calculated for all slices by Diffusion-weighted imaging (DWI) is another MR-based S − S technique that probes functional characteristics of tissues. =−ln( 1 0) 2/ ADC b − b mm s, (1) The clinical success of DWI has led to a broadening ap- ( 1 0) plication in the prostate gland. Rapid changes in diffusion where S is the signal intensity of a voxel after application of properties can be identified by calculating the apparent 1 adiffusion gradient and S is the echo magnitude without diffusion coefficient (ADC). Dickinson et al. [13] showed the 0 diffusion gradients applied (b = 0s/mm2). Diffusion standardizing multiparametric magnetic resonance imaging sensitivity is determined by the difference between b and b . (mpMRI) for PCa detection, localization, and characteri- 1 0 If multiple tumors were present in the peripheral zone, the zation. The use of DWI as a tool for the evaluation and average ADC value was recorded for each lesion. The MRI management of prostatic cancer has grown steadily in the sections and histological slices were matched on the basis of past decades [14–16]. The purpose of the study was to record the sextant level, anterior/posterior, and peripheral/central DWIandtocompareADCvaluesderivedfromDWIinPCa (transitional). patients with three different Gleason scores (3 + 3, 3 + 4, and At the time of these examinations, other sequences 4+3). performed as part of the routine prostate MRI protocol at our institution but not assessed in this study included sagittal 2. Materials and Methods and coronal TSE T2-weighted imaging sequences through the prostate and seminal vesicles. A total of 44 clinically localized PCa patients who underwent radical retropubic prostatectomy between January, 2007 and 3. Statistical Analysis May, 2008 were selected for this study. The entire protocol was approved by the institutional review board (IRB), and Statistical analyses were performed to assess the statistical an informed consent was obtained from each human subject. differences between ADC values for the three different The ages of the patients ranged from 47 to 75 years, and the Gleason scores (GS 3 + 3, GS 3 + 4, and GS 4 + 3) using patients fell into three differentgroupsbysurgeryGS:3+3 one-way analysis of variance (ANOVA) with SPSS software (mean ± SD, 60.1 ± 6.7years),3+4(mean± SD, 58.1 ± 4.2 package assuming parameters were normally distributed. A years),and4+3(mean± SD, 60.3 ± 3.9 years). The mean P value of less than 0.05 was considered to indicate a statis- prostate-specific antigen (PSA) value for the patients with GS tically significant difference. To explore for any relationship 3 + 3, GS 3 + 4, and GS 4 + 3, respectively, were 5.0 ng/mL, between the ADC value, tumor volumes, and the Gleason 6.8 ng/mL, and 7.4 ng/mL. score, Pearson correlation was performed on the data. All patients underwent prostate imaging with an en- Also, analysis of covariance (ANCOVA) was done on ADC dorectal coil on a 1.5 Tesla Avanto-Tim MRI scanner with values of different Gleason scores with tumor volume as a high-performance gradients (Siemens Medical Solutions, covariate to see its effect. Erlangen, Germany). Sequences included axial turbo spin- echo (TSE) T2-weighted imaging through the prostate and 4. Results seminal vesicles (TR/TE, 3800/101 ms; slice thickness, 3 mm; no interslice gap; field of view (FOV), 140 mm, matrix 205 × The patients mean and standard deviation (SD) of age and 256, slice thickness 3 mm, interslice gap 0 mm, echo-train PSA and ADC values for tumor PZ regions of three Gleason number 32, turbo factor 13). In addition, echo-planar scores are shown in Table 1. Figure 1(a) shows the T2- diffusion-weighted sequences sensitized in three orthogonal weighted MRI of a 68-year-old PCa patient with GS 3 + 4 planes (TR/TE 2000/83 ms, bandwidth 1396 Hz in the EPI and Figure 1(b), corresponding ADC map with low signal on frequency direction) with b values of 0, 50, 400 s/mm2 were the left base PZ. Figure 2 illustrates a box plot of ADC values obtained at the same slice positions as the axial T2-weighted for PCa in the peripheral zone tissue categorized by the three images. Twelve 4-mm-thick slices with no interslice gap Gleason scores. In 13 patients with GS 3 + 3, the (mean ± (27 cm FOV) with three averages provided coverage of the SD) ADC value was 1.135 ± 0.119 mm2/sec using 32 ROIs. In prostate with an image acquisition time of less than a minute. 22 patients with GS 3 + 4, the (mean ± SD) ADC value was Isotropic ADC maps were generated with the system software 0.976 ± 0.103 mm2/sec using 52 ROIs. In 9 patients with GS using all b values and taking an average value for the two 4 + 3, the (mean ± SD) ADC value was 0.831±0.087 mm2/sec directions of diffusion sensitization. using 24 ROIs. Although a statistically significant difference Advances in Urology 3

(a) (b)

Figure 1: (a) T2-weighted MRI of 68 yo prostate cancer patient with GS 3 + 4 and (b) corresponding ADC map with low signal on the left base PZ.

1.6 We did not have the biopsy report for 14 patients out of 44 patients. Out of which 15 patients whose biopsy 1.4 and prostatectomy Gleason scores were the same. For the remaining 15 patients, We had four patients with GS 3 + 1.2 3, eight patients with GS 3 + 4, and 3 patients with GS 4 + ﬀ

/sec) 3 whose biopsy Gleason scores were di erent from prosta- 2 1 tectomy. Hence, we have not done the correlation between mm 3 biopsy and prostatectomy Gleason scores. Out of 44 patients, − 0.8 in 35 patients (GS 3 + 3 (n = 13), GS 3 + 4 (n = 13), and GS 4 + 3 (n = 9)) tumors were detected by the DWI 0.6 technique. Nine patients (GS 3 + 4 (n = 9)) were missed by the MRI technique. To evaluate the association between ADC values (10 0.4 ADC value, tumor volumes, and the Gleason grade, all the ADC values and tumor volumes were first summarized at 0.2 the individual patient level, followed by applying Pearson’s correlation coefficients. We observed negative correlation 0 between Gleason score and ADC values and positive cor- GS 3 + 3 GS 3 + 4 GS 4 + 3 relation between Gleason score and tumor volume. In the Gleason scores ANCOVA analysis, the results were statistically significant P = . Figure 2: Box-Whisker plots of ADC values of Gleason score (GS) ( 0 0001) between the Gleason score and ADC values. 3 + 3, (GS) 3 + 4 and (GS) 4 + 3 patients. The center horizontal line indicates the median. 5. Discussion To increase the accuracy of MRI, a number of authors have Table 1: Patient clinical information and ADC values. used special techniques to study a particular characteristic of ADC values the prostate tumor and surrounding tissues such as dynamic PSA mean ± Gleason scores Age mean ± SD mean ± SD contrast-enhanced (DCE) MRI [17–19] and MR spec- SD (ng/mL) (mm2/sec) troscopy (MRS) [20–24]. MR spectroscopy requires a sub- 3+3(n = 13) 60.1 ± 6.75.0 ± 2.81.135 ± 0.119 stantially longer examination time than DWI, and, addition- 3+4(n = 22) 58.1 ± 4.26.8 ± 1.70.976 ± 0.103 ally, shimming process and placement of saturation bands 4+3(n = 9) 60.3 ± 3.97.4 ± 2.80.831 ± 0.087 during the examination are time consuming. For evaluation of MRS, baseline correction and phase correction have to P value NS NS <0.05∗∗∗ be performed in some cases. NS—Nonsignificant DWI is the only functional imaging technique that is ∗∗∗—Significant. able to assess molecular diffusion in vivo and provides information about biophysical properties of tissues such as cell organization, density, and microstructure [25]. DWI may be existed between the three groups (P<0.05), a certain degree helpful in differentiating high-risk patients from those at low of overlap between tissue types was evident. There was no and intermediate risks, since there is a significant correlation statistical significance between the PSA and patients ages between the ADC values from patients with three different with three different Gleason scores. Gleason scores. 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Review Article AReviewofERCC1 Gene in Bladder Cancer: Implications for Carcinogenesis and Resistance to Chemoradiotherapy

Atsunari Kawashima, Hitoshi Takayama, and Akira Tsujimura

The Department of Urology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita City, Osaka 565-0871, Japan

Correspondence should be addressed to Atsunari Kawashima, [email protected]

Received 17 July 2011; Accepted 30 August 2011

Academic Editor: Jack Schalken

Copyright © 2012 Atsunari Kawashima et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The excision repair cross-complementing group 1 (ERCC1) gene performs a critical incision step in DNA repair and is reported to be correlated with carcinogenesis and resistance to drug or ionizing radiation therapy. We reviewed the correlation between ERCC1 and bladder cancer. In carcinogenesis, several reports discussed the relation between ERCC1 single nucleotide polymorphisms and carcinogenesis in bladder cancer only in case-control studies. Regarding the relation between ERCC1 and resistance to chemoradiotherapy, in vitro and clinical studies indicate that ERCC1 might be related to resistance to radiation therapy rather than cisplatin therapy. It is controversial whether ERCC1 predicts prognosis of bladder cancer treated with cisplatin-based chemotherapy. Tyrosine kinase receptors or endothelial-mesenchymal transition are reported to regulate the expression of ERCC1, and further study is needed to clarify the mechanism of ERCC1 expression and resistance to chemoradiotherapy in vitro and to discover novel therapies for advanced and metastatic bladder cancer.

1. Introduction excision repair (NER) pathway. An overall association was reported between genetic variation in the NER pathway and Bladder cancer is the fourth most common cancer in men bladder cancer risk, suggesting the presence of gene-gene and in the United States [1, 2].Bladdercancerismoreprevalent gene-smoking interactions [6]. in men than women, with men accounting for around 80% On the other hand, systemic radiotherapy and chemo- of cases. Up to one-half of bladder cancer cases in men and therapy including cisplatin are used for locally advanced one-third in women are caused by cigarette smoking [3, 4], or metastatic bladder cancer, but their response rates are and another important risk factor is occupational exposure approximately 50%–60% [7, 8]. DNA-damaging chemother- to various chemical carcinogens [5]. A common property apeutic drugs and ionizing radiation (IR) induce a variety of of these exposures is the presence of carcinogens that can DNA lesions in cancer cells as well as in normal cells. The induce DNA damage in the bladder epithelium. Genotoxic mechanisms of cisplatin and radiation resistance have been compounds derived from the metabolism of chemical car- studied in various bladder cancer cell lines, and it has been cinogens can contribute to the accumulation of several forms shown that various DNA repair genes play important roles of DNA damage, such as bulky adducts, single-strand breaks in resistance to various therapies [9, 10]. (SSBs) and double-strand breaks (DSBs), abasic sites, and The excision repair cross-complementing group 1 modified bases. DNA repair mechanisms exist to prevent (ERCC1) gene is located on chromosome 19q13.2-q13.3 detrimental consequences of these types of DNA damage. [11]. ERCC1 performs a critical incision step in NER Specifically, base damage, abasic sites, and SSBs are repaired and is also involved in the repair of DNA interstrand through the base excision repair (BER) pathway, whereas crosslinks and some DSBs [12–14]. In clinical studies, the DSBs are repaired by either nonhomologous end joining expression of ERCC1 influenced the prognosis of the patients or the homologous recombination repair (HRR) pathways. treated with cisplatin-based chemotherapy and chemora- Bulky adducts are generally repaired by the nucleotide diotherapy (CRT) in various cancers such as lung cancer 2 Advances in Urology

[15, 16]. Recently, several reports addressed the relation association of different cancer cell lines with resistance to between ERCC1 expression and prognosis for cisplatin- platinum compounds has been suggested [28, 29]. Welsh based chemotherapy for bladder cancer [17–19], and we et al. reported that ERCC1 expression in bladder cancer also reported that ERCC1 might become a good factor for cell lines was higher than that in testis tumor cell lines, predicting the efficacy of CRT for muscle-invasive bladder and it led to less sensitivity to cisplatin-based chemotherapy cancer (MIBC) [20]. In accordance with these previous in bladder cancer than that in testicular cancer [30]. We reports, we review the role of ERCC1 in bladder cancer from examined ERCC1 expression in four bladder cancer cell lines carcinogenesis to therapeutic resistance. including two cisplatin-resistant cell lines. The cells most resistant to cisplatin had the highest ERCC1 expression, but sensitivity to cisplatin was not significantly recovered 2. ERCC1 and Carcinogenesis of Bladder Cancer by ERCC1 knocked down by siRNA [20]. Usanova et al. Chemical carcinogens, such as tobacco smoke and aromatic reported that downregulation of ERCC1-XPF slightly but amines, can undergo metabolic activation and detoxification significantly increased the sensitivity to cisplatin in one in the liver, and polymorphisms in the relevant genes bladder cancer cell line [10]. These reports indicate that have been shown to be associated with bladder cancer risk ERCC1 might play a slight role in the resistance to cisplatin [21–23]. Additionally, DNA repair enzymes are required therapy in bladder cancer. to repair the DNA damage associated with exposure to In regard to the association between ERCC1 and sensitiv- carcinogens. This suggests that common genetic variation ity to IR, Ahmad et al. reported that ERCC1-XPF is required in DNA repair genes might influence the risk of bladder for DNA DSB repair, and ERCC1-deficient cells are sensitive cancer, and several reports examined the relation between to IR exposure [13]. Liu et al. reported that methylation single nucleotide polymorphisms (SNPs) in DNA repair of the ERCC1 promoter correlates with radiosensitivity in genes and carcinogenesis. In case-control studies, several glioma cell lines [31]. We reported that of four bladder cancer reports discussed the relation between ERCC1 SNPs and cell lines, the cell line with the highest ERCC1 expression carcinogenesis in bladder cancer [6, 24–26]. Matullo et al. was also the most resistant to IR exposure. Moreover, the reported that the ERCC1-19007C variant allele (CC+CT sensitivity to IR exposure recovered significantly in the two versus TT: odds ratio (OR), 0.62; 95% confidence interval cells lines in which ERCC1 was knocked down [20]. To our (CI), 0.41–0.95) decreased the risk of bladder cancer, which knowledge, there are no other reports addressing the relation was consistent across smoking groups, although its SNP between ERCC1 expression and IR resistance in bladder did not affect carcinogenesis in nonsmoking groups. They cancer. DSBs are the most lethal form of IR-induced DNA mentioned that the ERCC1-19007 C>T polymorphism leads damage, and recent studies have observed a close correlation to a silent Asn118Asn change, but other polymorphisms between the number of phospho-H2A.X foci and the number and a combination of SNPs in the ERCC1 gene could be of expected DSBs after irradiation. In our study, two cell lines more important than single SNPs [24]. Garc´ıa-Closas et al. in which ERCC1 was knocked down recovered more slowly examined the SNPs in the NER pathway and bladder cancer in terms of the number of phospho-H2A.X foci than did the risk. Compared with homozygous wild-type individuals, control, suggesting continued accumulation or persistence of those carrying genotypes with variant alleles for ERCC1 DSBs and an increase the sensitivity to IR exposure. Based IVS5+33A>C had a significant increase in risk (OR, 1.2; 95% on our in vitro data, ERCC1 might play greater roles in CI, 1.0–1.5; P(trend) = 0.04), and the association of ERCC1 IR resistance in some bladder cancers; however, we did not with bladder cancer risk seemed to be stronger for cigarette show a direct correlation between ERCC1 and IR resistance smokers than for never-smokers [6]. Recently, Ricceri et al. in bladder cancer cells. Yacoub et al. reported that EGFR- reported that a single SNP analysis showed a protective effect ERK-signaling-induced IR-regulated DNA repair proteins of the rare alleles of 3 ERCC1 SNPs: rs967591 (OR, 0.66; CI XRCC1 and ERCC1 in prostate carcinoma cells [32]. Ko et 95%, 0.46–0.95), rs735482 (OR, 0.62; CI 95%, 0.42–0.90), al. reported that the level of ERK1/2 correlated with DNA and rs2336219 (OR, 0.63; CI 95%, 0.43–0.93). Moreover, repair genes, such as ERCC1 and Rad51 [33]. In clinical haplotype analysis revealed that cases of bladder cancer had studies, EGFR played the key role in drug and radiation a statistically significant excess of ERCC1-GAT haplotypes resistance [34, 35], and further study is needed to examine [26]. These reports were only case-control studies, and the difference between cisplatin and IR resistance associated ERCC1 was not included in other meta-analyses [27]. From with ERCC1, as well as other molecular pathways regulating now forward, a detailed characterization of ERCC1 variation ERCC1. is warranted, and it is necessary to pool comparable data and identify multiple susceptibility variants that could jointly affect risk. 4. ERCC1 and the Efficacy of CRT for MIBC The gold standard treatment for MIBC is radical cystectomy 3. The Role of ERCC1 in Cisplatin and and urinary diversion. Concurrent CRT using cisplatin or IR Resistance in Bladder Cancer In Vitro other agents is an alternative therapy that preserves bladder function [7, 36–40]. Because the complete response (CR) ERCC1 is a crucial gene in the NER pathway. Cisplatin- rate of CRT for MIBC is 60–70%, a simple procedure is DNA adducts are removed via the NER pathway, and an needed to select patients with MIBC who can be expected Advances in Urology 3 to have a good response to CRT so that they will not miss the in primary bladder tumors [45]. It will be important to chance to be cured by immediate cystectomy. assess novel CRTs targeting the Her-2/DNA repair pathway In terms of a predictor of the clinical response of CRT in advanced bladder cancer with metastasis. for MIBC by immunohistochemical study, Chakravarti et al. reported that in a multivariate analysis, only Her-2 expression was significantly associated with a reduced rate of 5. ERCC1 and the Efficacy of CR after CRT [34]. Rodel¨ et al. reported that the apoptotic Cisplatin-Based Chemotherapy index and Ki-67 expression, but not p53 or bcl-2 expression, were significantly related to an initial CR after CRT [41]. In a clinical study using immunohistochemistry, Olaussen Matsumoto et al. suggested that only the Bax/Bcl-2 ratio et al. reported that patients with ERCC1-negative nonsmall was associated with the CR rate, although Bax and Bcl-2 cell lung cancer appeared to benefit from adjuvant cisplatin- individually were not significantly associated with the CR based chemotherapy, whereas patients with ERCC1-positive rate [42]. In terms of DNA repair genes, Sakano et al. studied tumors did not [15]. In advanced bladder cancer, Bell- NER, BER, and HRR SNPs in 78 patients, looking at the munt et al. were the first to report that patients with effect on response and prognosis following platinum-based a high mRNA level of ERCC1 had poorer prognosis for CRT. They found the recurrence rate to be significantly lower cisplatin-based chemotherapy than did patients with a low in patients with a greater number of total variant alleles mRNA level of ERCC1 [17]. However, the authors did in all DNA repair genes and in NER genes, not including not make a general statement concerning the influence ERCC1 (P = 0.03), and the total number of variant alleles of ERCC1 expression on the outcome of other treatment were significantly associated with improved cancer-specific regimens including radiotherapy. In adjuvant cisplatin- survival in a univariate analysis. These findings suggested based chemotherapy for MIBC, Hoffmann et al. reported that NER genes might play an important role in the outcome that high mRNA levels of ERCC1 and MDR1 predicted of CRT for MIBC [43]. inferior progression-free survival [18].Variousregimensof In terms of our previously mentioned in vitro data, we cisplatin-based chemotherapy were used in these previous examined ERCC1 expression level by immunohistochem- reports. ERCC1 expression was not different between the two istry in all 22 patients who underwent CRT for MIBC. All platinum-based treatment arms (CMV versus M-VAC), and patients were treated with aggressive transurethral resection there was no significant relation between ERCC1 expression of bladder tumor before CRT in Osaka University Graduate and progression-free survival in either therapeutic regimen school of Medicine and its affiliated hospital. With standard (P = 0.21, P = 0.07) [18]. Matsumura et al. reported that the fractionation (2 Gy/fraction), the median total radiation expression of not ERCC1 but human equilibrative nucleoside dose is 50 Gy (40–66). Moreover, concurrent platinum transporter 1 (hENT1) could predict prognosis in patients chemotherapy with cisplatin (n = 13) and nedaplatin (n = treated with gemcitabine and cisplatin (GC) therapy in an 9) was intravenously administered within the first and fourth immunohistochemical study [46]. As the major molecular week and administered doses were ranged from 100 mg to component of nucleoside transporter proteins, hENT1 has 280 mg. Six of the 8 ERCC1-positive patients showed a non- been shown to predict benefit in patients undergoing gem- CR after CRT, whereas 12 of the 14 ERCC1-negative patients citabine chemotherapy for several solid malignancies [47, showed a CR. The efficacy of CRT as determined by ERCC1 48]. Matsumura et al. reported that only hENT1 expression expression level had a sensitivity of 75% and specificity of (P = 0.004) and not ERCC1 expression (P = 0.182) was 85.7% (P = 0.008). Finally, we examined the correlation associated with the prognosis of overall survival by multivari- between ERCC1 immunoreactivity and 5-year survival in ate analysis. Kim et al. reported that immunohistochemical the MIBC patients undergoing CRT. Overall 5-year survival expression of ERCC1 could predict only progression-free was 31.2% in the ERRC1-positive and 69.2% in the ERRC1- survival and not overall survival for advanced bladder cancer negative patients (P = 0.088). Although the sample size of treated with GC and M-VAC therapies. They indicated that our study was small, our in vitro data showed that lack of ERCC1 negativity was a statistically significant independent ERCC1 expression may predict the efficacy of CRT for MIBC prognostic marker for progression-free survival (OR, 1.62; [20]. 95% CI, 1.03–2.54; P = 0.003) [19]. The role of ERCC1 In breast cancer, the nuclear expression of Her-2 modu- as it relates to resistance to cisplatin was controversial in lated the interstrand crosslink repair of specific DNA lesions clinical studies of bladder cancer. Recently, we examined produced by chemotherapy [44]. Although we did not exam- the prognosis of cisplatin-based neoadjuvant chemotherapy ine Her-2 expression in our previous study, further study is in 58 bladder cancer patients treated with M-VAC and GC needed to examine the relation between Her-2 expression therapy using the results of an immunohistochemical study and DNA repair genes, such as ERCC1, in bladder cancer. of ERCC1. The expression of ERCC1 could not predict CR One personalized randomized phase I/II study (RTOG- or the prognosis for either disease-free or overall survival 0524) was conducted using Her-2 immunohistochemistry (data not shown). Hsu et al. reported that Snail, one of in bladder cancer treated with radiotherapy combined with the endothelial-mesenchymal transition (EMT) markers, chemotherapy. The results of this clinical study warrant regulated the expression of ERCC1, and coexpression of further investigation about the relation between them. Snail and ERCC1 predicted the poor prognosis of cisplatin- Moreover, Fleischmann et al. recently reported that Her-2 based chemotherapy for head and neck cancer [49]. EMT expression in lymph node metastasis was higher than that is reported to play an important role in progression and 4 Advances in Urology resistance to radiotherapy and chemotherapy in bladder tract cancer risk: a meta-analysis of epidemiologic studies,” cancer and other malignancies [35, 50–53]. We previously Cancer, vol. 89, no. 3, pp. 630–639, 2000. examined Snail expression and found that it correlated with [5] M. Kogevinas, A. t Mannetje, S. Cordier et al., “Occupation ERCC1 expression (P = 0.001). Moreover, coexpression of and bladder cancer among men in Western Europe,” Cancer ERCC1 and Snail were also found to be the prognostic factors Causes and Control, vol. 14, no. 10, pp. 907–914, 2003. to predict poorer disease-free and overall survival by both [6] M. Garc´ıa-Closas, N. Malats, F. X. 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