ijc 29918

Key signaling pathways in the muscle-invasive bladdercarcinoma: Clinical markers for disease modeling andoptimized treatment

Alex Kiselyov1, Svetlana Bunimovich-Mendrazitsky2 and Vladimir Startsev3

1 NBIC, Moscow Institute of Physics and Technology (MIPT), 9 Institutsky per, Dolgoprudny, Moscow Region 141700, Russia2 Department of Computer Science and Mathematics, Ariel University, Ariel 40700, Israel3 Department of Oncology, State Pediatric Medical University, St.-Petersburg 194100, Russia

In this review, we evaluate key molecular pathways and markers of muscle-invasive bladder cancer (MIBC). Overexpression

and activation of EGFR, p63, and EMT genes are suggestive of basal MIBC subtype generally responsive to chemotherapy.

Alterations in PPARc, ERBB2/3, and FGFR3 gene products and their signaling along with deregulated p53, cytokeratins KRT5/

6/14 in combination with the cellular proliferation (Ki-67), and cell cycle markers (p16) indicate the need for more radical

treatment protocols. Similarly, the “bell-shape” dynamics of Shh expression levels may suggest aggressive MIBC. A panel of

diverse biological markers may be suitable for simulation studies of MIBC and development of an optimized treatment proto-

col. We conducted a critical evaluation of PubMed/Medline and SciFinder databases related to MIBC covering the period

2009–2015. The free-text search was extended by adding the following keywords and phrases: bladder cancer, metastatic,

muscle-invasive, basal, luminal, epithelial-to-mesenchymal transition, cancer stem cell, mutations, immune response, signal-

ing, biological markers, molecular markers, mathematical models, simulation, epigenetics, transmembrane, transcription factor,

kinase, predictor, prognosis. The resulting selection of ca 500 abstracts was further analyzed in order to select the latest pub-

lications relevant to MIBC molecular markers of immediate clinical significance.

Vast majority (>90%) of bladder carcinomas that arise from thetransitional cells of the bladder mucosal epithelium are noninva-sive papillary tumors that are relatively easy to deal with. How-ever, if not detected and treated properly, at least one-third ofthese cancers ultimately invade the bladder wall and metastizeinto neighboring organs or lymph nodes by undergoing radicalmolecular and cellular changes. The resulting muscle-invasivebladder cancer (MIBC) is a heterogeneous group of aggressiveepithelial tumors with a high rate of metastasis and poor 5-yearsurvival rate of 30–50%. As noticed by numerous clinicalresearch teams, the number of somatic mutations exhibited byMIBC is notoriously high.1 This diversity in genetic backgroundleads to great variability in cancer aggressiveness, progression,and response rates making MIBC particularly difficult to treat.The underlying causes of MIBC have been directly linked toenvironmental and biomolecular factors. Several specific exam-

ples include point mutations in genes encoding receptor tyrosineor cytosolic kinases (e.g., ERBB1-3, FGFR3, MET, PI3KCA) andalterations in epigenetics machinery including both methylatedgenes (e.g., CDH1, FHIT, LAMC2, RASSF1A, TIMP3) andrespective effector enzymes (e.g., DNMT1/3). Considering thehighly aggressive and invasive nature of MIBC, numerousattempts have been made to both reliably diagnose and to treatthe disease using cystectomy or its combination with adjuvantchemo- and/or radiotherapy. Numerous authors attempted todevise a reliable molecular marker panel relevant to clinical andbiochemical manifestations of MIBC2–4; however, the panelcomponents change regularly as new clinical and biological evi-dence becomes available. There is an ample data on both cellularorigins and biological pathways activated in MIBC includingcell–cell/cell–matrix interactions, cytoskeletal dynamics, receptortyrosine kinases, cell cycle, and p53 signaling and apoptosis.5 Afundamental studies of gene expression of high-grade MIBCsrevealed two subsets of cancer exhibiting distinct features of uro-thelial differentiation and resembling the luminal and basal-likemolecular subtypes of breast cancer including a “claudin-low”genotype. As a result, clinically significant panel of 47 genes(BASE47) was introduced as a classifier of high-grade MIBC.6

Subsequently, basal and luminal cancer subsets were furtherexpanded to include p53-like luminal MIBC based on theirrespective origin and development route (Fig. 1).

Basal MIBC (ca 25% of invasive BCs) is controlled by thestem cell transcription factor DNp63a and an activation of theepidermal growth factor receptor (EGFR). They express multiple

Key words: muscle-invasive bladder cancer, biomarker, basal,

luminal, epithelial-to-mesenchymal transition, cancer stem cell

Funding Support: No specific funding was disclosed.

Conflict of Interest Disclosures: The authors made no disclosures.

DOI: 10.1002/ijc.29918

History: Received 28 Aug 2015; Accepted 4 Nov 2015; Online 6

Nov 2015

Correspondence to: Alex Kiselyov, PhD, NBIC, Moscow Institute of

Physics and Technology (MIPT), 9 Institutsky Per., Dolgoprudny,

Moscow Region 141700, Russia, Tel.: 11 858 397 8882,

E-mail: [email protected]

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Int. J. Cancer: 00, 00–00 (2015) VC 2015 UICC

International Journal of Cancer

IJC

molecular markers of epithelial-to-mesenchymal transition(EMT) including Snail family of transcription factors that pro-mote the repression of E-cadherin. Luminal MIBC are likely tobe driven by peroxisome proliferator activator receptor g

(PPARg) and estrogen receptor (ER) activation. ERBB2 amplifi-cations and activating FGFR3/ERBB3 mutations are a few of thedistinct features of luminal BC. p53-like BC displays distinctmarkers of cell cycle progression, proliferation, and stromalinvasion. Basal BC is intrinsically aggressive, but is sensitive tocisplatin-based combination chemotherapy and anti-EGFRagents. The luminal subtypes are less aggressive; however, p53-like tumors are resistant to chemotherapy.7,8 Bladder cancerstem cells (CSCs) have been introduced as common progenitorcells resulting in MIBC.9 In the last two decades, great effortshave been made to introduce specific molecular markers inMIBC. Considering the complex heterogeneous nature of theMIBC as well as new molecular and cellular biology evidence,there is an ongoing need for constructing diverse multimarkerpanels that could be used in routine clinical practice to bothdiagnose and to suggest optimal therapeutic intervention for thecancer. Importantly, MIBC markers should be reflective of clini-cal, cellular, and biomolecular evidence, predictive, reliable, eas-ily accessible, and quantifiable via available techniques.10 In thisreview, we focus on key signaling pathways contributing toMIBC pathology (Fig. 2). It is likely that this basal-luminal clas-sification of MIBC will be instrumental in designing bothumbrella- and basket-type clinical trials based on the presenceof specific molecular markers or matching a specific mutationand respective targeted treatment.11,12

Due to the specific focus of this work, we will not be cov-ering MIBC-specific gene mutations, gene polymorphism/sin-gle nucleotide polymorphism, and RNAi/siRNAs. We furthersuggest a panel of diverse biological markers amenable tomathematical modeling of MIBC and optimization of theindividual treatment protocol.

Cancer Stem Cell MarkersConstitutive activation of the Hh pathway leading to tumori-genesis was reported in basal cell carcinomas and medullo-blastoma. Multiple cancers including GI, lung, prostate,brain, and breast cancers display aberrant activation of thispathway mediated by the sonic hedgehog (Shh) protein, oneof the key regulators of organogenesis and adult stem cellsdivision.13 A novel insight into the chemical carcinogenesismodel of MIBC suggests the “biphasic” involvement of Shhprotein in aggressive BC. This model is likely to involve asingle precursor to afford the carcinoma-in-situ (CIS)lesion(s). Basal cells within this region produce Shh and trig-ger tumor development. However, as the tumor progresses toMIBC stage, the Shh levels drop presumably due to the inter-rupted cross-talk with the BMP pathway that controls uro-thelial differentiation.14 High expression levels of PTCH2,miRNA-92A, miRNA-19A, and miRNA-20A are associatedwith decreased overall survival in MIBC.15 Expression ofE-cadherin and p63 inversely correlate with expression of themesenchymal markers Zeb-1, Zeb-2, and vimentin in humanBC lines and primary tumors (N5 101). A subset of MIBC(T2–T4) maintain high levels of E-cadherin and p63 expres-sion.16 In vivo genetic inactivation of Notch signaling leads toErk1/2 phosphorylation and urinary tract tumorigenesis sug-gesting that loss of Notch activity may be a contributing fac-tor in BC development.17 DNp63a-mediated expression ofmiR-205 contributes to the regulation of EMT in BC cellsidentifying miR-205 as a molecular marker of the lethal sub-set of human BCs.18 There is a significantly higher Notchligand Jagged2 expressions in aggressive BC. Jagged2 expres-sion is positively correlated with histological grade, pT stage,recurrence, and metastasis (N5 120).19 The expression levelsof OCT4 responsible for maintaining the pluripotent proper-ties of embryonic stem are higher for BC with a high gradeand greater aggressiveness (N5 90).20 Elevated levels of

Figure 1. (a) Bladder cancer development stages including muscle invasion (T2A-T3 and metastasis into adjacent organs (T4)). (b) Tentative

origins of basal and luminal muscle-invasive bladder carcinomas. [Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]

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2 Key signaling pathways in bladder carcinoma


http://wileyonlinelibrary.com

aldehyde dehydrogenase 1 A1 (ALDH1A1) are detected in26% (N5 56/216) of human BC specimens with advancedpathological stage, high histological grade, and metastasis.21

Overexpression of Yes-associated protein 1 (YAP 1), thenuclear effector of the Hippo pathway, correlates with poordifferentiation, higher T/N classifications of BC patients(N5 213) and shorter overall survival (OS).22 Cripto-1 is anembryonic gene involved in self-renewal and maintenance ofpluripotency of stem cells. Its expression is significantly asso-ciated with tumor size, tumor grade (N5 130) and poorerRFS/metastasis-free survival (MFS).23 The AR-mediated splic-ing of the key urological stem cell protein and hyaluronanreceptor CD44 is proposed to contribute to malignant trans-formation of bladder cells.24

Receptor Tyrosine Kinase Signaling: ERBB1-3,FGFR3, and c-MetMDA-9/synthein expression (N5 44) has been shown to mod-ulate EGFR signaling in BC associated with stage, grade, andinvasion. Alterations of b-catenin, E-cadherin, vimentin,claudin-1, ZO-1, and T-cell factor-4 (TCF4) levels were alsoobserved.25 Loss of Sh3gl2 (endophilin A1), a regulator ofEGFR endocytosis, is associated with MIBC (N5 20). Silencingof Sh3gl2 in RT4 cells by RNAi enhances proliferation andcolony formation in vitro, inhibits EGF-induced EGFR inter-nalization, and increases EGFR activation.26 Studies on theERBB2 overexpression with chemoradiation therapy resistancein MIBC patients (N5 119) indicate that the target is an inde-pendent predictor for shorter cancer-specific survival (CSS).27

FGFR3 protein overexpression is detected in post-RC MIBCpatients (N5 33/72, 45.8%). In patients treated with adjuvantchemotherapy, FGFR3 overexpression correlates with shorter

disease-free survival (DFS) and OS. FGFR3 staining is presentin 29% of primary BCs and 49% of metastases and does notimpact OS (N5 231).28 Analysis of urinary Met levels allowsfor differentiation of MIBC from NMIBC patients and controlgroup. Reduced membranous Met staining is associated withunfavorable tumor phenotype.29

CytoskeletonKeratin 14 (KRT14) marks the most primitive differentiationstate that precedes KRT5 and KRT20 expression. Its expres-sion has been associated with worse BC prognosis.30 Usingmurine model, the authors identify KRT5-positive/KRT7-nega-tive basal cells as the putative cells-of-origin for b-catenin-induced luminal tumor predominant in males and controlledthe nuclear translocation of the androgen receptor (AR) inurothelial cells.31 Loss of expression of ARID1A, a chromatinremodeling enzyme, increases with higher BC stage/grade. It isinversely associated with FGFR3 but not p53 overexpression.32

PI3K-Akt-mTOR PathwayExpression levels of PTEN, phosphorylated p-Akt, p-mTOR,p-p70 ribosomal S6 kinase, and p-4E-binding protein 1 (4E-BP1) have been assessed to reveal that p-4E-BP1 and thetumor stage are independently related to RFS (N5 49).33 Theactivity of mammalian target of rapamycin complex 2(mTORC2) is significantly elevated in specimens of MIBC.34

Whole-genome targeted sequencing identified a loss of func-tion tuberous sclerosis complex 1 (TSC1) activating muta-tions (8% of 109 BC cases) to correlate with MIBC responseto everolimus.35 A recent study36 identified an exceptionalresponder to a combination of everolimus (PTEN-AKT-mTOR pathway inhibitor) and pazopanib (angiogenesis

Figure 2. Key signaling pathways in the muscle-invasive bladder cancer. [Color figure can be viewed in the online issue, which is available

at wileyonlinelibrary.com.]

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Kiselyov et al. 3



blocker). Two activating mTOR mutations E2014K andE2419K within the same MIBC were pinpointed to causeparticular mTOR signaling dependency of MIBC and conse-quently a specific sensitivity to pathway inhibition with ever-olimus. This example further validates the “basket” clinicalstudy design that matches patients with a rare mutation,regardless of tumor histology, to an agent designed to effectthe mutated pathway. Aberrant nuclear accumulation ofGSK-3b has been detected in 91% (21/23) of MIBC. GSK-3b

nuclear staining is significantly associated with high-gradetumors, advanced stage of BC, metastasis, and worse CSS.37

VEGF/VEGFR PathwaysPatients with MIBC T2 stage show lower expression levels ofVEGF-C. VEGF-D overexpression correlates with positivelymph node status (pN1). A higher pT stage, pN1, the pres-ence of LVI, vascular invasion (VI), and VEGF-D/VEGFR-3overexpression are significantly associated with reducedDSS.38 The expression of VEGFR2 is significantly higher inMIBC (N5 212). Patients with higher levels of VEGF,VEGFR1, and VEGFR2 tend to have poorer RFS.39 Accord-ingly, agents that block the molecular machinery of VEGF–VEGFR signaling cascade show considerable promise inMIBC clinical trials. For example, a combination therapyincluding cisplatin, gemcitabine, and a recombinant monoclo-nal antibody bevacizumab targeting circulating VEGF-A(CGB) resulted in CR and PR in 8 and 23 patients, respec-tively40. A Phase II study of VEGFR2 antiangiogenic antibodyCyramzaVR (ramucirumab) in combination with docetaxelyielded a significant median PFS (5.4 vs 2.8 months) andobjective response rate (ORR, 24% vs 12%) compared todocetaxel alone in patients with advanced MIBC (N5 140).

Cell CycleP16INK4a (p16) inhibits the activities of cyclin-dependentkinases (CDKs) and maintains the retinoblastoma protein(pRb) in its active hypophosphorylated state. Coexpression ofp16/Ki-67 in the same cells is observed in high-grade tumors(80/101, 79.2%). High-grade intraurothelial lesions (13/14,92.8%) are dual labeled showing congruent association ofthese markers.41 CDKN2A homozygous deletion is signifi-cantly more frequent in FGFR3-mutated tumors than inwild-type FGFR3 tumors. This event is associated with MIBCwithin the FGFR3-mutated subgroup but not in wild-typeFGFR3 tumors.42 Recurrent protein-inactivating mutations inCDKN1A and FAT1 genes along with TP53 mutations orMDM2 amplification are related to higher tumor stage/gradeand greater clonal diversity.43

Immunological Markers, Interleukins, Chemokines,and Their ReceptorsMultiple immunomodulating molecules play role in recogniz-ing somatic mutations observed in MIBC. Of these, the pro-grammed death-ligand 1 (PD-L1 or B7-H1) and its respectivereceptor PD-1 are of particular clinical significance. The anti-

PDL1 antibody atezolizumab (MPDL3280A) that blocks PD-L1/PD-1 interaction was successfully introduced for the treat-ment of metastatic MIBC.44 PD-L1 expression level wasreported to directly correlate with response to the agent inpretreated MIBC patients. The highest ORR was achieved inthe IC3 and IC2/3 groups (67% and 50%, respectively). Asignificant number of patients (>57%) exhibiting the highestlevels of PD-L1 survived past 1 year, whereas CRs werereported in 20% of MIBC subjects.45 Analysis of BC tissueshas revealed high IL-4Ra immunostaining (�21) in Grade 2(85%) and Grade 3 (97%) compared to Grade 1 tumors(0%). Similarly, only 9% stage I tumors are positive for IL-4Ra (�21) compared to 84% stage II and 100% stages III–IV tumors.46 The expression of IL-5/IL-5Ra is elevated inMIBC patients. It is further detected in BC cell lines 5637and T-24. IL-5 increases migration and MMP-9 expressionvia activation of transcription factors NF-jB and AP-1, andinduces activation of ERK1/2 and Jak-Stat signaling andinduction of p21WAF1 in both cell lines.47 High-grade inva-sive tumors (pT1–pT2) exhibit higher levels of IL-8 andMMP-9 than pTa tumors.48 The expressions of IL-20 and IL-20R1 are assessed in BC 5637 and T-24 cells. IL-20 signifi-cantly increases the expression of MMP-9 and stimulates theactivation of ERK1/2, JNK, p38 MAPK, and JAK-STAT sig-naling.49 TRAIL/osteoprotegerin (OPG) combination is coex-pressed in 96.6% of BC cases and positively interrelated.TRAIL/OPG both display an inverse relationship with histo-logical grade, T-category and LVI.50

Other Cellular ReceptorsHuman leukocyte antigen (HLA) class I downregulation hasbeen detected in 22/65 (33.8%) of analyzed MIBC. The RFSof post-RC patients with HLA class I-positive tumors is sig-nificantly better.51 Cadherins are mediators of cell–cell adhe-sion in epithelial tissues. The abnormal expression of N- andP-cadherins (N-/P-cadherin switching) has been shown topromote more invasive and malignant BC phenotypes.52 Lev-els of urinary epithelial cell adhesion molecule (EpCAM)have ben found to increase with stage and grade of BC.53

Loss of the single transmembrane protein syndecan-1 (SDC1)in tumor cells and the parallel increase of serum SDC1 ecto-domain in high-stage, high-grade BC is associated with theinvolvement of SDC1 shedding in BC progression. High pre-operative SDC1 serum levels are associated with LN metasta-ses.54 Expression of CD44v6, a cell surface protein involvedin cell migration and adhesion, is higher in noninvasive (Ta,Tis) vs invasive (T1–T4) tumors. In TUR patients, absentCD44v6 expression is associated with a 2.3-fold increase inrisk of recurrence.55 A carbohydrate-binding protein,galectin-3, gene expression levels increase in MIBC. Proteinexpression patterns correlate galectin-3 with tumor stage,grade, Ki67, and OS in T1G3 patients.56 Significant differen-ces in the levels of B1 domain-containing tenascin-C (Tn-C),a glycoprotein expressed in the ECM, are detected betweenNMIBC and MIBC patients (N5 35).57 Hyaluronic acid

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(HA), the respective HA synthases (HA1, HA2, and HA3),HYAL-1 hyaluronidase and HA receptors (CD44s, CD44v,and RHAMM) play key role in tumor growth and progres-sion. IHC and qPCR analyses of the BC tissues (N5 72)reveal HYAL-1 and HAS1 expression to be predictive of BCmetastasis, and HYAL-1 expression of DSS.58

ApoptosisPositive surviving expression in BC has been associated withpoor RFS and OS. A significant association between expressionof survivin and age as well as stage has been suggested, specifi-cally expression of survivin indicates poor prognosis in olderpatients and MIBC.59 The mean level of serum Smac/DIABLOin patients with MIBC is lower than that in NMIBC patients.Patients with T2–T4 MIBC with high-serum Smac/DIABLOlevel have a higher DFS.60 Both the mean OS and mean RFSare significantly decreased in the high cIAP1-N group. cIAP1-N expression correlates strongly with Ki-67 expression.61

Transcription FactorsSnail family of zinc finger transcription factors plays a keyrole in EMT. Upregulation of Snail2 (Slug) has been signifi-cantly correlated with a higher tumor stage and the E- to N-cadherin switch in BC cells and tissues. Ectopic expression ofSlug in BC 5637 and RT-4 cell lines promote EMT, increasedcell invasiveness, and chemoresistance.62 The protein Twist isa transcriptional repressor of E-cadherin, tumor progression,and metastasis. Twist1 and Y-box-binding protein-1 (YB-1)are positively correlated with invasiveness of BC (N5 75).Patients with high Twist1 and YB-1 expression levels exhibitlower OS.63 ZEB1 and ZEB2 (SIP1) inhibit transcription ofthe E-cadherin gene and induce EMT in vitro. SIP1 has beendescribed as an independent factor of poor prognosis in BCspecimens obtained from MIBC patients treated with radio-therapy.64 MIBC tissues are characterized by elevated nuclearexpression of phosphorylated STAT1, 3, and 5. Knockdownof STAT3 induces G0/G1 arrest and inhibited adhesion in J82cells. Knockdown of STAT1 inhibits migration in J82 andRT112 lines.65 Loss of FOXA1 expression is associated withaggressive BC, as well as increased tumor proliferation andinvasion. The female FOXA1 ko mouse model reveals a sig-nificant increase in KT14 expression in the urothelium. Ele-vated genes associated with keratinocyte differentiation andenrichment of KRT14-positive basal cells have been detectedin the hyperplastic urothelial mucosa in male ko mice.66

GATA-binding protein 3 (GATA3) is a zinc finger transcrip-tion factor and an ER-regulated gene. Its high expression hasbeen found to be a strong prognosticator for progression andCSS of MIBC (N5 65). Lower expression of GATA3 hasbeen found in pN0 tumors (32/47) than in node-positivetumors (20/21). There are significant correlations betweenGATA3 vs AR, ERa or ERb expression levels.67 Overexpres-sion of the eukaryotic translation initiation factor 5A2(EIF5A2) is an independent predictor for poor MFS of local-ized invasive BC patients treated with RC. Knockdown of

EIF5A2 inhibits BC cell migratory and invasive capacities invitro and metastatic potential in vivo presumably via block-ade of TGF-b1 expression.68 A key role for CD24 (heat stableantigen, HSA) in BC and metastasis in vivo has been con-firmed and found to be androgen regulated.69

EpigeneticsEpigenetics alterations including DNA methylation and post-translational protein modifications are known to modulate keybiological processes like proliferation and apoptosis. Methyl-ated genes including CDH1, FHIT, LAMC2, RASSF1A,TIMP3, SFRP1, SOX9, PMF1, and RUNX3 have been associ-ated with poor survival in patients with MIBC.70 Tumor-specific DNA methylation of the ST6GAL1 promoter region isfrequently found in pT2–4 tumors (53.6% (22/41)), whereasnormal urothelium remains unmethylated.71 Silencing of Diskslarge homolog 5 (Dlg5), a guanylate kinase adaptor family ofscaffolding proteins via methylation increases BC cell invasionin vitro and promotes metastasis in vivo. Downregulation ofDlg5 is significantly associated with reduced overall survival inBC patients.72 Methylation of TBX2, TBX3, and ZIC4 areindependent predictors of progression. The combination ofTBX2 and TBX3 methylation is a reliable marker for predict-ing progression to MIBC in patients with primary pTaG1/2BC.73 Promoter hypermethylation has been detected inRASSF1A, APC, and MGMT gene promoters. The methylationis more prominent in MIBC. RNA expression of RASSF1A,APC, and MGMT is also found to be decreased in MIBC, sug-gestive of epigenetic silencing. Significantly lower endogenousexpression of B-cell translocation gene 2 (BTG2) has beendetected in MIBC compared to matched normal tissues andNMIBC. BTG2 expression is inversely correlated withincreased expression of DNA methyltransferases DNMT1 andDNMT3a. Over 90% of tumor tissues reveal strong methyla-tion at CpG islands of the BTG2 gene implying epigenetic reg-ulation of BTG2 expression in BC.74 Nuclearimmunoreactivity of DNA methyltransferase 1 (DNMT1) inBC is significantly higher than in nonmalignant urothelium.The incidence of cancer is positively linked to clinical stage(24% in�T1 vs 55% in T2–T4). The staining of DNMT1 issignificantly linked to lower CR rates and reduced OS rates.The authors suggest an EFGR–PI3K–Akt pathway as the acti-vator of DNMT1 in BC.75 The expression levels of the andro-gen receptor (AR) and AR coregulators JMJD2A and LSD1have been examined to reveal that all proteins are significantlylowered in BC. This reduction correlates with stage progres-sion, including MIBC (JMJD2A/LSD1/AR), extravesical exten-sion (JMJD2A/LSD1), and LN metastasis (JMJD2A/AR).Lower JMJD2A intensity correlates with LVI, CIS, and worseOS. Notably, inhibition of LSD1 suppresses BC cell prolifera-tion and androgen-induced transcription.76 SIRT6 has beenshown to inhibit glycolysis and promote DNA double-strandbreak repairs. Lower expression levels of SIRT6 have beendetected in MIBC upon tumor progression from T2 to T4.

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Mathematical Models of Cancer TherapyConsidering the complexity of cancer, several authors haveattempted to model its dynamics and treatment regimentsusing computational models. A simulation of glioma develop-ment has been introduced to predict the time to relapse usingradiation and radiation–chemotherapy combination.78 Breastcancer modeling has been used to select high-risk population,cancer screening strategies, estimate tumor growth, and opti-mized cancer treatment.79 Simulation studies of NIMBCtreatment with Bacillus Calmette Guerin (BCG) orBCG1 IL-2 combo have used in situ data on tumor size,growth rate and immune response assessment from the clini-cal set of estimated parameters.80 The authors conclude thata mathematical model could be of immediate clinical use to(i) select a treatment protocol including both reduced BCGdosing and maintenance scheduling to minimize side effect(s)of vaccination; (ii) predict the outcome; and (iii) assess theneed for synergistic agents on an individual basis. A compu-tational model describing the initiation and progression ofMIBC has been reported81; however, it does not take intoconsideration tumor heterogeneity and may need furtherrefinement.

Experimental Markers for the Mathematical Modelof MIBCIn view of significant advances in the identification of originsand biological markers of MIBC, several of them hold promiseas clinically relevant and accurate predictors of progression, sur-vival, and treatment protocol(s). However, several additionalhurdles need to be addressed, namely (i) relatively limited accessto clinical data and patients; (ii) lack of standardized bioanalyti-cal procedures to evaluate biomarker levels; (iii) ethnic, epige-netic, treatment backgrounds affecting gene polymorphism and

epigenetic markers; (iv) opportunistic urogenital conditions; (v)longitudinal relationship between disease progression andmarkers panel, number of treatments, adjuvant therapy; and (vi)patient-specific “fingerprint” of the disease.

As summarized above, signaling pathways induced byMIBC involve multiple cell types and molecules. In addition,time-resolved changes of these entities pre-/post-treatmentneed to be considered. We have selected several markers thatcould be used as standalone parameters in the clinic and/orin the mathematical models to determine an optimized treat-ment regimen (Fig. 3, bolded) using individual data fromMIBC patients.

Since the aforementioned cellular and molecular markersare likely to be deregulated regardless of their pre-/post-treat-ment collection point, we recommend to analyze these as apanel throughout a patient’s individual history. Overexpres-sion and activation of EGFR, p63, and EMT genes includingSnail, Slug, Twist, ZEB1/2, and vimentin are suggestive ofbasal MIBC subtype generally responsive to chemotherapeuticintervention. Alterations in PPARg, ERBB2/3, FGFR3 geneproducts, and their signaling along with deregulated p53,cytokeratins KRT5/6/14, especially in combination with thecellular proliferation markers Ki-67 and cell cycle markers(e.g., p16) may indicate the need for more radical treatmentprotocols. Similarly, the “bell-shape” dynamics of Shh expres-sion levels, one of the key cancer stem cell markers, mayindicate aggressive MIBC. A longitudinal assessment of sev-eral relevant membrane receptors including claudins, SDC1,and hyaluronic acid signaling mediators is also warranted.Once gene polymorphism, miRNAs, and epigenetics markersbecome more standardized and mainstream in the clinic,they will be included in designing individual regiments forthe treatment of MIBC. It is anticipated that the initial panel

Figure 3. A summary of biological markers based on key signaling pathways in MIBC described in the text. The most promising predictive

markers and/or their combination are in blue and bolded. [Color figure can be viewed in the online issue, which is available at wileyonline

library.com.]

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of clinical parameters (“entry markers” and “treatmentmarkers 1”) will allow for the proper calibration of the math-ematical model and customization of the treatment protocol.A subsequent comparison of extrapolated and experimentaloutcome will enable further refinements in the simulationprocess and, more importantly, allow for optimization of apatient-specific therapeutic approach (“post-treatmentmarkers 1 and 2” and “treatment markers 2”).

ConclusionsIn the past decade, we have witnessed a growing understand-ing of both cellular and molecular origins of the MIBC. How-ever, areas of MIBC including cancer metabolism, proteinsynthesis and degradation, and epigenetics need further insightdespite the recent significant progress in this area and mayyield new data on MIBC-related markers of the disease. Thesuggested basal/luminal origins of the cancer may prompt bet-ter selection of therapeutic intervention protocols for individ-

ual patients. Moreover, we are likely to witness better insightinto (i) key molecular targets suitable for intervention, (ii)design and optimization of treatment protocols, and (iii)reduced regimen-related toxicities. In the area of MIBC, thereis a real need for the rational selection of (i) dose, (ii) fre-quency of therapeutic intervention, (iii) synergistic adjuvanttherapy, and (iv) a reliable set of biochemical markers relatedto tumor response. Addressing these challenges via a multidis-ciplinary approach involving simulation, molecular biology,and clinical science may yield a real opportunity to increasedisease-free and overall survival of patients. The resultingmarkers may empower both umbrella- and basket-type clinicaltrials. In umbrella trials, a combination of a flexible biomarkerpanel and carefully selected targeted treatments could providea sound alternative to the current randomized clinical trialdesign. Basket-type clinical trials that merge both traditionaldesign and genomic data could provide new insight into thespecific molecular profile of MIBC.

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