This document summarizes key signaling pathways in muscle-invasive bladder carcinoma. It discusses molecular markers that indicate basal or luminal subtypes of bladder cancer, which differ in response to treatment. Basal cancers often overexpress EGFR and respond to chemotherapy, while luminal cancers involve alterations in genes like FGFR3, ERBB2/3 and are generally less aggressive. The document also reviews markers for cancer stem cells, receptor tyrosine kinase signaling pathways, cytoskeleton proteins, the PI3K-Akt-mTOR pathway, and VEGF/VEGFR pathways that are clinically significant for modeling and optimizing treatment of muscle-invasive bladder cancer.

1. Key signaling pathways in the muscle-invasive bladder
carcinoma: Clinical markers for disease modeling and
optimized 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, Russia
2
Department of Computer Science and Mathematics, Ariel University, Ariel 40700, Israel
3
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 the
transitional 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 of
these cancers ultimately invade the bladder wall and metastize
into neighboring organs or lymph nodes by undergoing radical
molecular and cellular changes. The resulting muscle-invasive
bladder cancer (MIBC) is a heterogeneous group of aggressive
epithelial tumors with a high rate of metastasis and poor 5-year
survival rate of 30–50%. As noticed by numerous clinical
research teams, the number of somatic mutations exhibited by
MIBC is notoriously high.1
This diversity in genetic background
leads 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 to
environmental and biomolecular factors. Several specific exam-
ples include point mutations in genes encoding receptor tyrosine
or cytosolic kinases (e.g., ERBB1-3, FGFR3, MET, PI3KCA) and
alterations in epigenetics machinery including both methylated
genes (e.g., CDH1, FHIT, LAMC2, RASSF1A, TIMP3) and
respective effector enzymes (e.g., DNMT1/3). Considering the
highly aggressive and invasive nature of MIBC, numerous
attempts have been made to both reliably diagnose and to treat
the disease using cystectomy or its combination with adjuvant
chemo- and/or radiotherapy. Numerous authors attempted to
devise a reliable molecular marker panel relevant to clinical and
biochemical manifestations of MIBC2–4
; however, the panel
components change regularly as new clinical and biological evi-
dence becomes available. There is an ample data on both cellular
origins and biological pathways activated in MIBC including
cell–cell/cell–matrix interactions, cytoskeletal dynamics, receptor
tyrosine kinases, cell cycle, and p53 signaling and apoptosis.5
A
fundamental studies of gene expression of high-grade MIBCs
revealed two subsets of cancer exhibiting distinct features of uro-
thelial differentiation and resembling the luminal and basal-like
molecular 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 further
expanded to include p53-like luminal MIBC based on their
respective origin and development route (Fig. 1).
Basal MIBC (ca 25% of invasive BCs) is controlled by the
stem cell transcription factor DNp63a and an activation of the
epidermal 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: alex.kiselyov@geneabiocells.com
MiniReview
Int. J. Cancer: 00, 00–00 (2015) VC 2015 UICC
International Journal of Cancer
IJC

2. 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 to
be 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 the
distinct features of luminal BC. p53-like BC displays distinct
markers of cell cycle progression, proliferation, and stromal
invasion. Basal BC is intrinsically aggressive, but is sensitive to
cisplatin-based combination chemotherapy and anti-EGFR
agents. The luminal subtypes are less aggressive; however, p53-
like tumors are resistant to chemotherapy.7,8
Bladder cancer
stem cells (CSCs) have been introduced as common progenitor
cells resulting in MIBC.9
In the last two decades, great efforts
have been made to introduce specific molecular markers in
MIBC. Considering the complex heterogeneous nature of the
MIBC as well as new molecular and cellular biology evidence,
there is an ongoing need for constructing diverse multimarker
panels that could be used in routine clinical practice to both
diagnose and to suggest optimal therapeutic intervention for the
cancer. 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 this
review, we focus on key signaling pathways contributing to
MIBC pathology (Fig. 2). It is likely that this basal-luminal clas-
sification of MIBC will be instrumental in designing both
umbrella- and basket-type clinical trials based on the presence
of specific molecular markers or matching a specific mutation
and 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 further
suggest a panel of diverse biological markers amenable to
mathematical modeling of MIBC and optimization of the
individual treatment protocol.
Cancer Stem Cell Markers
Constitutive 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 this
pathway mediated by the sonic hedgehog (Shh) protein, one
of the key regulators of organogenesis and adult stem cells
division.13
A novel insight into the chemical carcinogenesis
model of MIBC suggests the “biphasic” involvement of Shh
protein in aggressive BC. This model is likely to involve a
single 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 to
MIBC 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 associated
with decreased overall survival in MIBC.15
Expression of
E-cadherin and p63 inversely correlate with expression of the
mesenchymal markers Zeb-1, Zeb-2, and vimentin in human
BC lines and primary tumors (N 5 101). 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 to
Erk1/2 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 of
miR-205 contributes to the regulation of EMT in BC cells
identifying miR-205 as a molecular marker of the lethal sub-
set of human BCs.18
There is a significantly higher Notch
ligand Jagged2 expressions in aggressive BC. Jagged2 expres-
sion is positively correlated with histological grade, pT stage,
recurrence, and metastasis (N 5 120).19
The expression levels
of OCT4 responsible for maintaining the pluripotent proper-
ties of embryonic stem are higher for BC with a high grade
and greater aggressiveness (N 5 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.]
MiniReview
2 Key signaling pathways in bladder carcinoma
Int. J. Cancer: 00, 00–00 (2015) VC 2015 UICC

3. aldehyde dehydrogenase 1 A1 (ALDH1A1) are detected in
26% (N 5 56/216) of human BC specimens with advanced
pathological stage, high histological grade, and metastasis.21
Overexpression of Yes-associated protein 1 (YAP 1), the
nuclear effector of the Hippo pathway, correlates with poor
differentiation, higher T/N classifications of BC patients
(N 5 213) and shorter overall survival (OS).22
Cripto-1 is an
embryonic gene involved in self-renewal and maintenance of
pluripotency of stem cells. Its expression is significantly asso-
ciated with tumor size, tumor grade (N 5 130) and poorer
RFS/metastasis-free survival (MFS).23
The AR-mediated splic-
ing of the key urological stem cell protein and hyaluronan
receptor CD44 is proposed to contribute to malignant trans-
formation of bladder cells.24
Receptor Tyrosine Kinase Signaling: ERBB1-3,
FGFR3, and c-Met
MDA-9/synthein expression (N 5 44) has been shown to mod-
ulate EGFR signaling in BC associated with stage, grade, and
invasion. Alterations of b-catenin, E-cadherin, vimentin,
claudin-1, ZO-1, and T-cell factor-4 (TCF4) levels were also
observed.25
Loss of Sh3gl2 (endophilin A1), a regulator of
EGFR endocytosis, is associated with MIBC (N 5 20). Silencing
of Sh3gl2 in RT4 cells by RNAi enhances proliferation and
colony formation in vitro, inhibits EGF-induced EGFR inter-
nalization, and increases EGFR activation.26
Studies on the
ERBB2 overexpression with chemoradiation therapy resistance
in MIBC patients (N 5 119) indicate that the target is an inde-
pendent predictor for shorter cancer-specific survival (CSS).27
FGFR3 protein overexpression is detected in post-RC MIBC
patients (N 5 33/72, 45.8%). In patients treated with adjuvant
chemotherapy, FGFR3 overexpression correlates with shorter
disease-free survival (DFS) and OS. FGFR3 staining is present
in 29% of primary BCs and 49% of metastases and does not
impact OS (N 5 231).28
Analysis of urinary Met levels allows
for differentiation of MIBC from NMIBC patients and control
group. Reduced membranous Met staining is associated with
unfavorable tumor phenotype.29
Cytoskeleton
Keratin 14 (KRT14) marks the most primitive differentiation
state that precedes KRT5 and KRT20 expression. Its expres-
sion has been associated with worse BC prognosis.30
Using
murine 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 controlled
the nuclear translocation of the androgen receptor (AR) in
urothelial cells.31
Loss of expression of ARID1A, a chromatin
remodeling enzyme, increases with higher BC stage/grade. It is
inversely associated with FGFR3 but not p53 overexpression.32
PI3K-Akt-mTOR Pathway
Expression 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 the
tumor stage are independently related to RFS (N 5 49).33
The
activity 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 response
to everolimus.35
A recent study36
identified an exceptional
responder 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.]
MiniReview
Kiselyov et al. 3
Int. J. Cancer: 00, 00–00 (2015) VC 2015 UICC

4. blocker). Two activating mTOR mutations E2014K and
E2419K within the same MIBC were pinpointed to cause
particular mTOR signaling dependency of MIBC and conse-
quently a specific sensitivity to pathway inhibition with ever-
olimus. This example further validates the “basket” clinical
study design that matches patients with a rare mutation,
regardless of tumor histology, to an agent designed to effect
the mutated pathway. Aberrant nuclear accumulation of
GSK-3b has been detected in 91% (21/23) of MIBC. GSK-3b
nuclear staining is significantly associated with high-grade
tumors, advanced stage of BC, metastasis, and worse CSS.37
VEGF/VEGFR Pathways
Patients with MIBC T2 stage show lower expression levels of
VEGF-C. VEGF-D overexpression correlates with positive
lymph node status (pN1). A higher pT stage, pN1, the pres-
ence of LVI, vascular invasion (VI), and VEGF-D/VEGFR-3
overexpression are significantly associated with reduced
DSS.38
The expression of VEGFR2 is significantly higher in
MIBC (N 5 212). 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 in
MIBC clinical trials. For example, a combination therapy
including 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 antibody
CyramzaVR
(ramucirumab) in combination with docetaxel
yielded a significant median PFS (5.4 vs 2.8 months) and
objective response rate (ORR, 24% vs 12%) compared to
docetaxel alone in patients with advanced MIBC (N 5 140).
Cell Cycle
P16INK4a
(p16) inhibits the activities of cyclin-dependent
kinases (CDKs) and maintains the retinoblastoma protein
(pRb) in its active hypophosphorylated state. Coexpression of
p16/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 of
these markers.41
CDKN2A homozygous deletion is signifi-
cantly more frequent in FGFR3-mutated tumors than in
wild-type FGFR3 tumors. This event is associated with MIBC
within the FGFR3-mutated subgroup but not in wild-type
FGFR3 tumors.42
Recurrent protein-inactivating mutations in
CDKN1A and FAT1 genes along with TP53 mutations or
MDM2 amplification are related to higher tumor stage/grade
and greater clonal diversity.43
Immunological Markers, Interleukins, Chemokines,
and Their Receptors
Multiple 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 respective
receptor 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 was
reported to directly correlate with response to the agent in
pretreated MIBC patients. The highest ORR was achieved in
the IC3 and IC2/3 groups (67% and 50%, respectively). A
significant number of patients (>57%) exhibiting the highest
levels of PD-L1 survived past 1 year, whereas CRs were
reported in 20% of MIBC subjects.45
Analysis of BC tissues
has 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 in
MIBC patients. It is further detected in BC cell lines 5637
and T-24. IL-5 increases migration and MMP-9 expression
via activation of transcription factors NF-jB and AP-1, and
induces activation of ERK1/2 and Jak-Stat signaling and
induction of p21WAF1 in both cell lines.47
High-grade inva-
sive tumors (pT1–pT2) exhibit higher levels of IL-8 and
MMP-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 the
activation 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 Receptors
Human leukocyte antigen (HLA) class I downregulation has
been detected in 22/65 (33.8%) of analyzed MIBC. The RFS
of 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- and
P-cadherins (N-/P-cadherin switching) has been shown to
promote 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 the
involvement 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 involved
in cell migration and adhesion, is higher in noninvasive (Ta,
Tis) vs invasive (T1–T4) tumors. In TUR patients, absent
CD44v6 expression is associated with a 2.3-fold increase in
risk of recurrence.55
A carbohydrate-binding protein,
galectin-3, gene expression levels increase in MIBC. Protein
expression 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 between
NMIBC and MIBC patients (N 5 35).57
Hyaluronic acid
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4 Key signaling pathways in bladder carcinoma
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5. (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 (N 5 72)
reveal HYAL-1 and HAS1 expression to be predictive of BC
metastasis, and HYAL-1 expression of DSS.58
Apoptosis
Positive surviving expression in BC has been associated with
poor RFS and OS. A significant association between expression
of survivin and age as well as stage has been suggested, specifi-
cally expression of survivin indicates poor prognosis in older
patients and MIBC.59
The mean level of serum Smac/DIABLO
in patients with MIBC is lower than that in NMIBC patients.
Patients with T2–T4 MIBC with high-serum Smac/DIABLO
level have a higher DFS.60
Both the mean OS and mean RFS
are significantly decreased in the high cIAP1-N group. cIAP1-
N expression correlates strongly with Ki-67 expression.61
Transcription Factors
Snail family of zinc finger transcription factors plays a key
role 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 of
Slug in BC 5637 and RT-4 cell lines promote EMT, increased
cell invasiveness, and chemoresistance.62
The protein Twist is
a 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 (N 5 75).
Patients with high Twist1 and YB-1 expression levels exhibit
lower OS.63
ZEB1 and ZEB2 (SIP1) inhibit transcription of
the E-cadherin gene and induce EMT in vitro. SIP1 has been
described as an independent factor of poor prognosis in BC
specimens obtained from MIBC patients treated with radio-
therapy.64
MIBC tissues are characterized by elevated nuclear
expression of phosphorylated STAT1, 3, and 5. Knockdown
of STAT3 induces G0/G1 arrest and inhibited adhesion in J82
cells. Knockdown of STAT1 inhibits migration in J82 and
RT112 lines.65
Loss of FOXA1 expression is associated with
aggressive BC, as well as increased tumor proliferation and
invasion. The female FOXA1 ko mouse model reveals a sig-
nificant increase in KT14 expression in the urothelium. Ele-
vated genes associated with keratinocyte differentiation and
enrichment of KRT14-positive basal cells have been detected
in 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 has
been found to be a strong prognosticator for progression and
CSS of MIBC (N 5 65). Lower expression of GATA3 has
been found in pN0 tumors (32/47) than in node-positive
tumors (20/21). There are significant correlations between
GATA3 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 in
vitro and metastatic potential in vivo presumably via block-
ade of TGF-b1 expression.68
A key role for CD24 (heat stable
antigen, HSA) in BC and metastasis in vivo has been con-
firmed and found to be androgen regulated.69
Epigenetics
Epigenetics alterations including DNA methylation and post-
translational protein modifications are known to modulate key
biological 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 is
frequently found in pT2–4 tumors (53.6% (22/41)), whereas
normal urothelium remains unmethylated.71
Silencing of Disks
large homolog 5 (Dlg5), a guanylate kinase adaptor family of
scaffolding proteins via methylation increases BC cell invasion
in vitro and promotes metastasis in vivo. Downregulation of
Dlg5 is significantly associated with reduced overall survival in
BC patients.72
Methylation of TBX2, TBX3, and ZIC4 are
independent predictors of progression. The combination of
TBX2 and TBX3 methylation is a reliable marker for predict-
ing progression to MIBC in patients with primary pTaG1/2
BC.73
Promoter hypermethylation has been detected in
RASSF1A, APC, and MGMT gene promoters. The methylation
is 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 endogenous
expression of B-cell translocation gene 2 (BTG2) has been
detected in MIBC compared to matched normal tissues and
NMIBC. BTG2 expression is inversely correlated with
increased expression of DNA methyltransferases DNMT1 and
DNMT3a. 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
Nuclear
immunoreactivity of DNA methyltransferase 1 (DNMT1) in
BC 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 is
significantly 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 LSD1
have been examined to reveal that all proteins are significantly
lowered 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 worse
OS. Notably, inhibition of LSD1 suppresses BC cell prolifera-
tion and androgen-induced transcription.76
SIRT6 has been
shown to inhibit glycolysis and promote DNA double-strand
break repairs. Lower expression levels of SIRT6 have been
detected in MIBC upon tumor progression from T2 to T4.77
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6. Mathematical Models of Cancer Therapy
Considering the complexity of cancer, several authors have
attempted to model its dynamics and treatment regiments
using computational models. A simulation of glioma develop-
ment has been introduced to predict the time to relapse using
radiation and radiation–chemotherapy combination.78
Breast
cancer modeling has been used to select high-risk population,
cancer screening strategies, estimate tumor growth, and opti-
mized cancer treatment.79
Simulation studies of NIMBC
treatment with Bacillus Calmette Guerin (BCG) or
BCG 1 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 that
a mathematical model could be of immediate clinical use to
(i) select a treatment protocol including both reduced BCG
dosing and maintenance scheduling to minimize side effect(s)
of vaccination; (ii) predict the outcome; and (iii) assess the
need for synergistic agents on an individual basis. A compu-
tational model describing the initiation and progression of
MIBC has been reported81
; however, it does not take into
consideration tumor heterogeneity and may need further
refinement.
Experimental Markers for the Mathematical Model
of MIBC
In view of significant advances in the identification of origins
and biological markers of MIBC, several of them hold promise
as clinically relevant and accurate predictors of progression, sur-
vival, and treatment protocol(s). However, several additional
hurdles need to be addressed, namely (i) relatively limited access
to 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 and
markers panel, number of treatments, adjuvant therapy; and (vi)
patient-specific “fingerprint” of the disease.
As summarized above, signaling pathways induced by
MIBC involve multiple cell types and molecules. In addition,
time-resolved changes of these entities pre-/post-treatment
need to be considered. We have selected several markers that
could be used as standalone parameters in the clinic and/or
in the mathematical models to determine an optimized treat-
ment regimen (Fig. 3, bolded) using individual data from
MIBC patients.
Since the aforementioned cellular and molecular markers
are likely to be deregulated regardless of their pre-/post-treat-
ment collection point, we recommend to analyze these as a
panel throughout a patient’s individual history. Overexpres-
sion and activation of EGFR, p63, and EMT genes including
Snail, Slug, Twist, ZEB1/2, and vimentin are suggestive of
basal MIBC subtype generally responsive to chemotherapeutic
intervention. Alterations in PPARg, ERBB2/3, FGFR3 gene
products, and their signaling along with deregulated p53,
cytokeratins KRT5/6/14, especially in combination with the
cellular proliferation markers Ki-67 and cell cycle markers
(e.g., p16) may indicate the need for more radical treatment
protocols. Similarly, the “bell-shape” dynamics of Shh expres-
sion levels, one of the key cancer stem cell markers, may
indicate 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 markers
become more standardized and mainstream in the clinic,
they will be included in designing individual regiments for
the 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.]
MiniReview
6 Key signaling pathways in bladder carcinoma
Int. J. Cancer: 00, 00–00 (2015) VC 2015 UICC

7. of clinical parameters (“entry markers” and “treatment
markers 1”) will allow for the proper calibration of the math-
ematical model and customization of the treatment protocol.
A subsequent comparison of extrapolated and experimental
outcome will enable further refinements in the simulation
process and, more importantly, allow for optimization of a
patient-specific therapeutic approach (“post-treatment
markers 1 and 2” and “treatment markers 2”).
Conclusions
In 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, protein
synthesis and degradation, and epigenetics need further insight
despite the recent significant progress in this area and may
yield new data on MIBC-related markers of the disease. The
suggested 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 insight
into (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, there
is a real need for the rational selection of (i) dose, (ii) fre-
quency of therapeutic intervention, (iii) synergistic adjuvant
therapy, and (iv) a reliable set of biochemical markers related
to tumor response. Addressing these challenges via a multidis-
ciplinary approach involving simulation, molecular biology,
and clinical science may yield a real opportunity to increase
disease-free and overall survival of patients. The resulting
markers may empower both umbrella- and basket-type clinical
trials. In umbrella trials, a combination of a flexible biomarker
panel and carefully selected targeted treatments could provide
a sound alternative to the current randomized clinical trial
design. Basket-type clinical trials that merge both traditional
design and genomic data could provide new insight into the
specific molecular profile of MIBC.
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