Bladder cancer: the challenge for personalized treatment

Signal transduction
Epithelial growth factor (EGF) receptors
Epidermal growth fibroblast receptor (EGFR) (HER-1) and HER-2/neu are the most studied
growth factor receptors in bladder tumors (TV) [1]. They are part of a family of
transmembrane proteins with tyrosine kinase activity comprising four members:
ErbB1/HER1/EGFR, ErbB2/HER-2/neu, ErbB3/HER-3 and ErbB4/HER-4. Activation of
EGF receptors occurs by homo- or heterodimerization, after ligand binding leading to
autophosphorylation of intracellular tyrosine kinase residues. The cascade of cellular events
that follows involves several molecular pathways including the PI3K/Akt and Ras/Raf/MAPK
pathways. Activation of these signaling pathways leads to activation of cell proliferation and
survival, angiogenesis, tumor progression, development of metastases and resistance of tumor
cells to treatments (Fig. 1). EGFR EGFR overexpression, assessed by immunohistochemistry,
affects 31 to 48% of TVs [2]. All cell layers are affected by this overexpression, which
increases contact between tumor cells and EGF. This overexpression, correlated with tumor
stage and grade, is a powerful prognostic factor for VT with a reduction in recurrence-free
survival in five out of seven studies and a reduction in specific survival in seven out of 11
studies. It was also demonstrated that overexpression of EGFR and its ligands (EGF, TGF
mainly) was a more significant prognostic factor than overexpression of EGFR alone. This
could explain that the high concentration of activated EGF, found in the urine of rats carrying
TV, can explain tumor growth and angiogenesis by increased expression of pro-angiogenic
factors vascular endothelial growth factor (VEGF), interleukin -8 (IL-8) and matrix
metalloproteinases (MMP-2 AND-9) [3]. TV/EGFR+ metastases also overexpress the
receptor, making EGFR a preferred target for metastatic TVs. Inhibition of the EGFR
pathway allows for a downstream cytotoxic effect on the cell cycle in G1/S phase by
inhibiting the cyclinE/cyclin dependent kinase 2 couple, an increase in the expression of the
p27 inhibitor but also an decrease in pro-angiogenic factors VEGF, IL-8 and ßFGF [4]. Two
strategies emerge: monoclonal antibodies which recognize antigenic sites on the extracellular
part of the receptor and compete with specific ligands for binding to the receptor and tyrosine
kinase inhibitors (TKIs) which are small molecules that block the intracellular tyrosine kinase
activity at the ATP binding domain.
Monoclonal antibodies (suffix -ab)
Cetuximab treatment of the infiltrative human TV 253JB-V line, in vivo, at dosages ranging from 200
to 400 mg/m 2 per week, has a dose-dependent cytostatic effect not exceeding 55% inhibition,
suggesting that this Treatment administered alone is partially effective and justifies the joint
administration of a cytotoxic agent. The combination of cetuximab and paclitaxel showed synergistic
action in a mouse model of VT [5]. In addition, an increase in the proapoptotic factor Bax and a
decrease in the anti-apoptotic factor Bcl-2 were found in two colon (DiFi) and breast (ZR75) tumor
lines treated with cetuximab. Finally, cetuximab showed an inhibitory effect on tumor progression and
the appearance of metastases by reducing the expression of certain matrix metalloproteinases. Other
monoclonal antibodies evaluated alone and/or in combination with gemcitabine-cisplatin
chemotherapy are matuzumab (EMD-72000), panitumumab (ABX-EGF, Vectibix ®), pertuzumab
(2C4, Omnitarg ®) and MDX-447 .
Tyrosine kinase inhibitors (TKIs) (suffix -ib)
Gefitinib (ZD1839; Iressa ®), a TKI with reversible action, selective antiEGFR, completely inhibits
the autophosphorylation of treated human tumor cells. In a phase I trial, gefitinib showed low toxicity
(skin rash and type 1 diarrhea) which was reversible upon cessation of treatment [6] . Combined with
cytotoxic chemotherapy, gefitinib significantly reduces tumor proliferation of human TV xenografts
and improves survival, regardless of the level of EGFR expression [7]. Molecularly, gefitinib was
shown to inhibit the activation of the MAPK pathway and reduce tumor cell proliferation, increase the
expression of p27Kip1 (cyclin-dependent kinase inhibitor: cdk2-cyclin E), and stimulate the apoptosis.
It is in lung cancer that this treatment seemed most promising with combinations of gefitinib—
caboplatin—docetaxel or gefitinib—cisplatin—gemcitabine in phase II and III trials but with very
mixed results [8]. However, combinations of gefitinib with platinum salts (cisplatin, carboplatin and
oxaliplatin) or with taxanes (paclitaxel, docetaxel), or even with topoisomerase inhibitors have shown
very encouraging results in human VT models but have not yet been evaluated in clinical trials. The
other TKI under evaluation is erlotinib (OSI-774; Tarceva ®), whose action is reversible and which
has shown efficacy in a phase II trial in non-small cell lung cancer.
Insert diagram here

Figure 1. Diagram of the transduction pathway involving EGFR and the signaling pathways potentially
involved in tumor progression (Ras/Raf/MAPK, PI-3K/AKT, JAK/STAT and PLC/PKC) as well as the
different targeted therapies available .

The Ras/Raf/MAPK pathway

This important molecular pathway for tumor cell growth is activated in both bladder tumorigenesis
and tumor progression with metastatic development. This pathway can be activated by EGF
receptors. It was in the human bladder tumor line T24 that the first activating mutations of Ras were
discovered [15]. H-Ras is the first oncogene identified in TV with activating mutations identified in 30
to 40% of cases, particularly in superficial tumors, hence its involvement during tumor development.
Ras interacts with the serine/threonine kinase Raf and activates the MAPK pathway in superficial and
infiltrative VTs [16]. The participation of Ras in carcinogenesis depends on the tissue concerned (75—
95% of pancreatic carcinomas and 50% of colon carcinomas). The activation of Ras by the EGF
pathway is done by the Sos-Grb2 proteins which increases the level of the active form of Ras (GTP-
Ras). It is easy to imagine that inhibition of the EGF pathway could partially slow down the Ras
pathway. To block the Ras/Raf/MAPK pathway, farnesylation can be inhibited. Indeed, this step,
essential for the activation of the Ras protein, is the addition of a farnesyl group to the carboxy end
of Ras using the enzyme farnesyltransferase (FT). FT inhibitors act selectively on cancer cells. Among
this therapeutic class, we retain lonafarnib (SCH66336), tipifarnib (R115777), L744832 and BIM-
46228 [17,18]. The effectiveness of lonafarnib and tipifarnib has already been shown in trials
conducted on refractory tumors with an objective response in 10 to 15% of cases. Another way to
neutralize the Ras pathway is to use a monoclonal antibody (scFv fragment) directed against Ras (Ras
Y13-239) which stimulates apoptosis of the targeted cell. No trials have yet been performed against
human bladder tumor cell lines. The use of gene therapy to deliver the dominant negative mutated
H-Ras protein (N116Y: substitution of the tyrosine by an asparginine at 116) into the tumor cell
inhibits the transformation of the inactive RasGDP form into the active Ras-GTP form and has
demonstrated an inhibitory action on the growth of the infiltrating bladder tumor line T24.
Instillation of the adenovirus carrying the mutated protein (AdCMV-N116Y) could be delivered by
endovesical instillation. Finally, MAPK inhibitors (MEK1 and MEK2) can also inhibit this molecular
pathway such as PD32901, ARRY-142886 and CI-1040.
The PI-3K/AKT pathway

Many growth factor receptors such as EGFR, HER-2/neu, or even FGFR3, rather involved in superficial
VT, have the possibility of activating the AKT pathway, which results in stimulating cell mobility,
invasion, inhibit apoptosis and provide resistance to treatments [19]. The tumor suppressor gene
PTEN (or MMAC1/TEP1), located at 10q23, antagonizes this molecular pathway by dephosphorylating
the second messenger (phosphatidylinositol 3,4,5 triphosphate or PIP3). PTEN is mutated or deleted
in 14 to 23% of bladder tumors. Genomic mutations of PTEN cause Cowden disease where patients
present with early bladder tumors. Additionally, PI-3K has been shown to be overexpressed in
bladder tumors conferring a five- to 20-fold increase in activity in tumor cells [20]. These results
suggest that the PI-3K/AKT pathway could be involved in both tumorigenesis and bladder tumor
progression, a hypothesis already verified in prostate, ovarian, pancreatic and breast cancers.
Activation of this signaling pathway has been shown to provide resistance to treatments, particularly
in bladder cancer. Restoring PTEN function becomes a therapeutic alternative to resensitize tumor
cells to cytotoxic chemotherapy. It was shown in a human bladder tumor line (UM-UC-6dox) resistant
to doxorubicin that delivering a wild-type PTEN by gene therapy (Ad-MMAC) inhibited the expression
of the active form of AKT ( phospho-Akt) and again made the tumor cells sensitive to doxorubicin
with a complete disappearance of VT in three out of ten mice for more than 120 days. The mTOR
protein is a downstream target of AKT. Phospho-Akt activates mTOR and allows the synthesis of
ribosomal protein p70s6K, which results in stimulating tumor growth and inhibiting apoptosis via the
EGF pathway. The use of the mTOR inhibitor, rapamicin, has remarkable effectiveness in the
treatment of human cancers in vitro but also on human TV xenografts. Temsirolimus, an analogue of
rapamicin, also has an antitumor effect by inhibiting the activation of p70s6K and also restores
sensitivity to doxorubicin.

Cell cycle and gene therapy

Molecular cell cycle abnormalities are the most frequent and most studied aberrations in
bladder tumor pathology. The p53 tumor suppressor gene encodes a protein that regulates the
course of the cell cycle by controlling the G1/S point and activating the expression of
p21WAF1/CIP1. The loss of function of p53 in TV occurs in two stages with the loss of one
of the two alleles at 17p then the mutation of the remaining allele. This loss of heterozygosity
of chromosome 17 is a late event occurring in the advanced stages. The mutation of the p53
gene has a primordial role in the acquisition of an aggressive phenotype since it is considered
a major cause of tumor progression with invasion of the lamina propria and the bladder
muscle and considerably worsens the prognosis [22,23] ( Fig. 2). Restoring the function of the
tumor suppressor gene constitutes an interesting therapeutic alternative. This function can best
be delivered using a viral vector which delivers the wild-type p53 gene into p53-deficient
tumor cells. Adenovirus was used for this purpose (AdCMV-TP53) and showed effectiveness
in inhibiting tumor proliferation and significantly reducing tumor volume in human TV
xenograft models. It was in 2002 that the transfer of AdCMV-TP53 by endovesical route in
patients with bladder tumors was successfully carried out [24]. Phase I trials have shown a
high degree of tolerance for this type of treatment [25]. A synergy of action was even
demonstrated when AdCMV-TP53 was associated with cisplatin with an accentuation of
apoptosis of tumor cells, requiring a study on a larger cohort in order to validate this
therapeutic strategy. The other virus evaluated to restore p53 function is Vaccinia virus (rVV-
TK-53) which showed promising results in a phase I trial [26]. The use of a low molecular
weight molecule like p53 reactivation and induction of massive apoptosis (PRIMA-1) has also
been successfully evaluated in VT with inhibition of tumor proliferation and synergistic
action with cisplatin to induce the apoptosis of tumor cells. The restoration of Rb gene
function follows the same logic as p53. The truncated Rb94 protein (112 amino acids fewer at
the N-terminal) is a more potent tumor growth inhibitor than the normal protein. Using a viral
vector to deliver it showed enhanced apoptosis in both Rb-positive and Rb-negative tumor
cells. This treatment has a high degree of selectivity because normal urothelial cells are not
affected by this agent. One of the mechanisms of Rb inactivation in tumor cells is
hyperphosphorylation. Thus, the use of inhibitors of this phosphorylation (cyclin-dependent
kinase inhibitor:CDKI) such as staurosporine, flavopiridol (L86-8275) or even UCN01 (7-
hydroxystaurosporine) in clinical trials in human tumors seems legal.
Apoptosis: anti-Bcl2 treatment It has been shown that the anti-apoptotic protein Bcl-2 is
directly involved in bladder tumorigenesis. Its overexpression in tumor cells has already been
linked to resistance to radio- and/or chemotherapy in gastric and lung cancers [27].
Transfection of Bcl-2 into human TV cell lines renders them resistant to cisplatin. At present,
the anti-apoptotic biochemical mechanism of Bcl-2 remains unknown but the treatment of TV
cell lines with a Bcl-2 antisense oligonucleotide (oblimersen: Genasene®) strongly reduces
the expression of Bcl-2 and restores chemosensitivity.
Angiogenesis

The place of physiological and tumoral neo-angiogenesis is based on the work of J. Folkman. In the
tumor process, there is a disruption in the balance between pro- and anti-angiogenic factors,
allowing the tumor to acquire its own vascular network to grow without restriction and have direct
access to the blood circulation. The acquisition of the angiogenic phenotype is a key step in tumor
progression and metastatic development and is measured by the density of tumor microvessels
(microvascular density: MVD). It is acquired either by increased expression of pro-VEGF factors, -
bFGF, -thymidine phosphorylase..., or by reduction of anti-thrombospondin-1 factors, -angiostatin, -
endostatin..., or both combined [28]. Pro-factors are synthesized by tumor cells but also by stromal
cells and those of the immune system. Angiogenesis constitutes a target of choice in the case of
advanced and/or metastatic VT (Fig. 3). MVD has shown its prognostic value in VT. Tumors at high
risk of progression T1G3, with high MVD, recur and progress more frequently than T1G3 with low
MVD. Activation of the EGFR signaling pathway causes an increase in the expression of pro-
angiogenic factors VEGF, interleukin-8 (IL-8) and certain matrix metalloproteinases (MMPs) involved

Insert diagram here

Figure 2.Molecular events in angiogenesis. Activation of the EGFR transduction pathway
stimulates the expression of VEGF, metalloproteinases (MMPs) and interleukin 8 (IL-8).
VEGF activates angiogenesis and MMPs and IL-8 stimulate endothelial cell migration.
Activation of VEGFR stimulates the activation of angiogenesis. p53 inhibits angiogenesis by
increasing the expression of the anti-angiogenic factor TSP-1 (thrombospondin-1).
Potential role of viruses in bladder

carcinogenesis While viral infections are involved in 16% of cancers (more particularly HPV and
cervical cancer, EBV and nasopharyngeal cancer, HHV8 and Kaposi's sarcoma, HTLV-1 and T-cell
lymphoma, hepatitis B and C viruses and hepatocarcinoma)[34], no formal data existed in bladder
tumors until recent data from the Cancer Genome Atlas. It seems that viral expression is found in
7.3% of cases, involving certain HPV viruses (HPV16, HPV45, HPV56, HPV6b) or CMV/HHV5, with, in
the majority of cases, an integration mechanism. viral genetic material within the genome[35].
However, it is difficult to precisely assess the role of these oncogenic viruses and to establish the link
between viral infection and the appearance of cancer due to the importance of potential cofactors.

Epigenetic regulatory mechanisms

Numerous articles, published over the last 10 years, suggest the major importance of epigenetic
alterations (DNA methylation, chromatin remodeling by biochemical modification of histones,
regulation by non-coding RNAs) which can modify the expression of coding genes. proteins in human
tumors, and particularly in urothelial carcinomas [36.37]. The word “epigenetics” refers to
phenomena that modulate the activity of the genome without changing its sequence. These
mechanisms are dynamic and reversible and respond to events, such as embryogenesis or
environmental factors. These phenomena have been demonstrated for several cancer suppressor
genes, which in certain tumors showed a total absence of transcriptional or protein expression while
being free from any alteration at the DNA level.

Histone modification

The NH2-terminal region of histones is accessible outside the nucleosome. Histone modifications in
mammals take place in this region. These modifications (acetylation, but also ubiquitination,
phosphorylation, etc.) determine the structure of chromatin and therefore the expression or
repression of genes. Some of these alterations could be associated with the aggressiveness and
prognosis of bladder tumors. Recently, genetic aberrations involving chromatin remodeling genes
(KDM6A, CREBBP, EP300, ARID1A) were found with a frequency of more than 10%, suggesting a
major role for chromatin regulation in bladder urothelial carcinogenesis.[8.9]. Some of them appear
to have prognostic value; this is the case ofARID1Aloss of expression of which is associated with more
aggressive disease and increased risk of recurrence[39]. Finally, current data from whole genome
analysis as part of the Cancer Genome Atlas have made it possible to confirm the major importance
of mutations concerning chromatin regulation genes in bladder urothelial carcinogenesis, mutations
which seem to be more frequent in the 131 bladder tumors analyzed than in the other types of
cancers studied. Indeed, 76% of bladder tumors presented at least one mutation and 41% two
mutations among chromatin remodeling genes.[9]. Similarly, the SWI/SNF chromatin remodeling
complex, which usually participates in the regulation of variant exon inclusion by reducing the rate of
RNA polymerase II elongation, also appears altered in 64% of cases. This could indicate the possibility
of developing new therapies targeting epigenetic regulatory pathways development of bladder
cancer[43]. Likewise, the Cancer Genome Atlas made it possible to associate the expression of CIMP
(CpG island methylator phenotype)with smoking status [9]. It should be noted that in the short term,
the analysis of "methylomes" will be carried out simultaneously with the analysis of genomes using
"third generation" sequencing methods allowing both the uninterrupted reading of thousands of
nucleotide bases and detection of the methylation state of cytosines.
miRNAs

Gene expression is also regulated through epigenetic silencing mechanisms involving small non-
coding RNAs of 21–24 nucleotides (miRNAs), leading mainly to repression of translation. There are
more than 1000 genes encoding microRNAs in the human genome, and each microRNA can regulate
the expression of a large number of target genes. (around 200). MicroRNAs are involved in many
physiological processes such as proliferation, apoptosis, differentiation, development and cellular
metabolism. MicroRNAs have also been implicated in cancers. Certain genes, coding for these
microRNAs, are located in regions showing chromosomal translocations and/or deletions in tumors.
MicroRNAs can function as oncogenes or tumor suppressor genes. Several large-scale studies have
made it possible to identify expression profiles associated with bladder tumors, and varying
depending on the degree of infiltration and/or tumor grade.[44]. THETable IIIindicates the microRNAs
most frequently reported as altered in bladder cancer, their main target genes, and their
functions[44–57]. The deregulations described in bladder tumors are essentially under-expressions,
reflecting the probable tumor suppressor effect of these miRNAs, and concern notably miR-145,
involved in apoptosis, the miR-200 family, involved in the epithelial-mesenchymal transition of
highgrade tumors, and miR-99a/100[45]. The latter is known to target FGFR3; thus, the
underexpression of miR-99a/100 would be responsible for an overexpression of FGFR3 in low-grade
tumors[46]. Some microRNA overexpressions are also described but are more frequently observed in
infiltrating tumors. This is the case for miR-21, overexpression of which would be associated with
inhibition of the p53 and PTEN pathway.[46,47]. Few studies have so far reported a correlation
between miRNA expression and the prognosis of bladder tumors.[47]. The molecular signature
associating miRNAsmiR-9, miR-182 AndmiR-200bwas recently identified as associated with the
aggressiveness of bladder tumors[4

List of miRNAs described in the literature as altered in bladder cancer, with their target messenger
RNA and their function

miRNA Deregulation target mRNA Function References

miR-1 Down LASP1, TAGLN2 Cytoskeleton,
differentiation
miR-21 Up TP53, PTEN, Bcl-2, Cell cycle control,
MSH2, E2F3 apoptosis
miR-24 Down FOXM1 Proliferation
miR-31 Down FGFR3 Proliferation
miR-101 Down EZH2 Gene expression
miR-125b Down E2F3 Apoptosis and
proliferation
miR-129 Up SOX4, GALNT1 Signal
transduction,
expression protein
miR-133a Down KRT7, TAGLN2, Differentiation,
EGFR proliferation
miR-143 Down HK2, MMP13, Proliferation and
PRKCE, ERK5, migration
MAPK7, AKT
miR-145 Down CBFB, PPP3CA, Signal
CLINT1 transduction,
 apoptosis
Family miR-183 : Up miR-96, 182 FOXO1, FOXO3 and Proliferation and
miR-96, miR-182 Down miR-183 EZR migration
and miR-183
miR-195 Down CDK-4, GLUT3 Cell cycle control,
proliferation
miR-200 family : Down ERRFl-1 ZEB1, ZEB2
miR-200a, miR-
200b, miR-200c,
miR-141 and miR-
429)
miR-205 Down ACSL4, PTEN Cell cycle control,
apoptosis
miR-221 Up TRAIL Apoptosis

Alterations in gene expression

If the nature of the gene alterations is variable (gene amplifications, mutations, loss of alleles,
hypermethylations, etc.), these alterations mainly result in a quantitative modification of the
expression of the genes in question, which can be measured both at the level of the transcript than
of the protein.

Alterations in gene expression at the messenger RNA level: the transcriptome

At the messenger RNA level, changes in gene expression at the individual level were initially
highlighted by Northern Blot. This method, cumbersome and requiring large quantities of biological
material, was quickly supplanted by real-time quantitative RT-PCR. The introduction of technology
cDNA microarrays then made it possible to analyze gene expression and/or the expression of
microRNAs, no longer at the individual level, but at the genome level (transcriptome) in tumor
samples and to highlight new clinical phenotypes urothelial tumors. The ideal would be to be able to
identify tumors with different evolutionary potential in order to be able to adapt the therapeutic
strategy in a personalized manner. For NIMVT, several studies have sought to identify gene
expression signatures to predict the risk of recurrence or progression to an infiltrating tumor.
However, there is currently no reliable prognostic marker for NIMVT. For IMVT, on the other hand,
certain genes and molecular signatures seem to be associated with tumor aggressiveness, risk of
progression and survival. This is the case, for example, of molecules involved in the angiogenesis
pathway, whose role during urothelialbladder carcinogenesis has been particularly studied in recent
years, like most tumor pathologies. Among the angiogenic factors, vascular endothelial growth factor
(VEGF) is a particularly interesting molecule.

Since its crucial angiogenic activity, in-vitro and in-vivo, makes it the preferred target of current
therapies. There is extensive literature on the role of the VEGF pathway in different cancers, with
more recent interest in urothelial tumors. The different series seem to indicate that VEGF is mainly
involved in the early stages of carcinogenesis and that its expression decreases with the progression
of the disease. [58.59]. However, its prognostic value, particularly concerning the risk of recurrence
or progression, remains very controversial. [59.60]. The next decade will be even more prolific in
terms of discoveries with the advent of high-throughput sequencing (NGS) techniques. next-
generation sequencing)making it possible to obtain, quickly and at low cost, all of the RNA sequences
of a tumor (RNAseq)at the quantitative (transcriptome) and qualitative (mutations, fusion
transcripts) level.
This is particularly the case of recent data obtained as part of the Cancer Genome Atlas (TCGA) which
made it possible to highlight different molecular expression profiles defining new tumor phenotypes
within urothelial carcinomas with potential prognostic and therapeutic implications.[9]. These tumor
profiles are also observed at the microRNA sequencing level and at the protein expression level.
Among infiltrative tumors, we can thus distinguish the “papillary-like” or “luminal” subtype, which
corresponds to tumors of papillary architecture, with strong expression of FGFR3, associated with
mutations or amplifications of the gene.FGFR3.We also note a decrease in the expression of
microRNAs regulating FGFR3, and in particular miR-99a and miR-100, as well as miR-145 and miR-
125b. Conversely, the “basal/squamous-like” subtype is characterized by the expression of epithelial
or stem cell differentiation genes, such as cytokeratins (KRT14, KRT5, KRT6A),as well as an activation
ofp63and theEGFR.We find the same “basal-like” expression profile for certain breast cancers or for
squamous tumors of the lung or head and neck. These tumors would be associated with a more
aggressive urothelial disease at diagnosis and a poor prognosis. Finally, “p53-like” tumors would be
characterized by resistance to conventional chemotherapies, but a potential response to
immunotherapies.

Alterations in gene expression at the protein level: proteome

To meaningfully understand the somatic alterations of a tumor cell, it is now essential to look at the
analysis of the proteins it contains. Indeed, proteins are the main molecules involved in the
functioning of the cellular machinery, while genes are only information carriers. The level of
expression of messenger RNA does not necessarily reflect the abundance, nor the nature, of the
proteins which will be found in the tumor cell. Indeed, the translation of messenger RNAs into
proteins is subject to a large number of regulations, in particular to post-transcriptional (alternative
splicing, microRNAs, etc.) and post-translational (glycosylation, phosphorylation, acetylation,
degradation, etc.) modifications. .) whose role is determining for their functionality (or their
oncogenic power). These posttranslational modifications are not detected by analysis of DNA or
messenger RNA. Proteomics (study of the proteome) owes its current growth in oncology to the
emergence of highperformance analytical technologies making it possible to analyze thousands of
proteins simultaneously: twodimensional electrophoresis and especially Maldi-Tof or SeldiTof type
mass spectrometry. Finally, more recent techniques for analyzing several hundred candidate proteins
from a small quantity of biological material by RPPA (Reverse Phase Protein Array) seem promising.
As part of the Cancer Genome Atlas, these RPPA techniques made it possible to analyze 130 proteins,
phosphorylated or not, and thus to identify protein expression profiles associated with certain tumor
subtypes, alongside RNA expression profiles. previously described [9]. Thus, “papillary-like” tumors
seem associated with high protein expression of HER2, but also of ESR2 (estrogen receptor
beta),suggesting new therapeutic possibilities such as hormone therapies (tamoxifen, raloxifene).

New therapeutic strategies

Advances in the knowledge of the molecular mechanisms of urothelial cancers are currently leading
to new therapeutic strategies. This is particularly important for bladder cancer since conventional
chemotherapies (based on platinum salts) have only a modest effect on overall survival, with median
survival not exceeding 15 months in the metastatic stage. Targeted therapies aim to specifically
target certain genes involved in carcinogenesis (or their products) with the aim of “re-educating”
these tumor cells or selectively eliminating them. The notion of therapeutic targets in oncology has
always existed. The presence of the tumor thus constituted the first target, for which the associated
therapeutic procedure was primarily surgical excision. Then, the identification of the uncontrolled
proliferation capacities of cancer cells led to the development of treatments targeting this property,
so-called conventional cytotoxic chemotherapies. The notions of target and targeted therapy have
naturally evolved due to a better understanding of the molecular mechanisms of carcinogenesis, in
particular, linked to major technological advances. The identification of key alterations inherent to
the tumor phenotype served as support for the research and selection of relevant targets in
oncology, at the origin of the development of new targeted molecular therapies which have truly
revolutionized the therapeutic management of cancer patients. Among the cellular targets, the most
used currently are membrane receptors (mainly with tyrosine kinase activity). More rarely, these will
be cytoplasmic targets (signaling pathway proteins) or nuclear targets (transcription factors). The
targets of the tumor environment mainly revolve around the phenomenon of angiogenesis, a major
element for the survival of the tumor, its development and its dissemination, including in urothelial
tumors. More recently, the concept of immunotherapy has broadened the spectrum of new targeted
therapies. The identification of a molecular target involved in the tumor process is necessary as a
starting point for targeted therapy but is not sufficient. Indeed, this target must be “druggable”,that
is to say that the chemical properties of the target biomolecule must in particular allow binding of
the candidate drug with sufficient affinity. Potential therapeutic targets are therefore at the
intersection between the genome “druggable »and key genes in carcinogenesis. Recent genome-
wide data from the Cancer Genome Atlas showed that 69% of bladder tumors harbor potential
therapeutic targets, and most of them are “druggable”,either with already existing treatments or
with molecules currently under development[9]. For example, potential targets were identified in
42% of cases within the PI3K/AKT/mTOR pathway and in 45% of cases within the MAP kinase
pathway.

Among the potential molecular targets that may have therapeutic applications in bladder cancer,
surface receptors with tyrosine kinase activity have been particularly studied: EGFR and ErbB2,
VEGFR, FGFR3. . . Another avenue of research is to target proteins controlling the cell cycle, either by
modulating the activity of cyclins and cyclin-dependent kinases, or by restoring p53 and/or RB
functions to stimulate apoptosis. Finally, it is also possible to target chromatin modifications thanks
to the recent development of agents that bind to acetyl-lysine binding motifs (bromodomains). All
these innovative drugs, having an original mechanism of action and rapid release to the market,
however, have the major disadvantage of being extremely expensive, out of all proportion to the
standard treatments known to date. Each drug newly released to the market is likely to exponentially
increase the spending curve in healthcare establishments. It will therefore be essential to evaluate
the optimal conditions of use and the expected benefits for each of these drugs. Thus, it is necessary
to standardize molecular diagnostic methods and to develop “companion” tests allowing, from the
analysis of theranostic biomarkers, to target patients likely to benefit the most from different
treatments.

Anti-angiogenic therapies

Tumor neo-angiogenesis is currently at the cœheart of the development of new targeted therapies in
oncology. The idea is to be able, by inhibiting tumor neovascularization, to prevent the supply of
oxygen and nutrients within the tumor and thus induce hypoxia and ischemic necrosis of cancer cells.
However, it seems unlikely that an antiangiogenic agent alone would be sufficient for disease control.
Use in combination with standard chemotherapies could be more relevant, in the absence of toxicity.

These molecules have been evaluated in phase II and III clinical trials with good results and now have
a validated indication as first or second line treatment of several cancers. In bladder cancer, several
clinical trials have been conducted with controversial results. The first phase II results concerning an
anti-VEGF, bevacizumab (in combination with gemcitabine–cisplatin chemotherapy in first line
treatment of metastatic bladder cancer) were encouraging with 58% of patients in partial or
complete response, but at a price significant toxicity, particularly in terms of thromboembolic (21%)
and hemorrhagic (7%) complications[62]. Trials conducted with tyrosine kinase inhibitors,
sunitinib[63], sorafenib[64]and pazopanib[65], in the first or second line, gave disappointing results.
Other trials are underway with vandetanib, which acts on both the VEGF and EGFR pathway. The
effectiveness of anti-angiogenic therapies may be limited by the fact that spatio-temporal differences
in tumor neovascularization are likely to exist, in relation to the extreme heterogeneity of bladder
tumors. This heterogeneity can be observed within the bladder tumor itself, but

also between the primary tumor and the metastatic locations. More recently, the concept of
angiogenic regulation has been discussed. Rather than blocking the mechanisms of tumor
neoangiogenesis, the idea would be to reorganize and use these vascular networks to improve the
bioavailability of administered cytotoxic treatments, through modulation of vascularization at the
tumor level. Anti-angiogenic treatments have the effect of normalizing tumor vascularization, at least
at the start of their administration, creating a therapeutic window during which tumor diffusion of
cytotoxic drugs is improved. Tumor neovascularization ensures heterogeneous tumor blood flow,
leaving regions that are probably hypoxic, poorly accessible to cytotoxic agents, and radioresistant.
Antiangiogenic treatment makes it possible to homogenize the tumor vascularization through a
vascular remodeling effect, in particular by blocking the VEGF pathway on which tumor vessels are
dependent, and improving the oxygenation of tumor cells; this action can lead to a potentiation of
the effect of cytotoxic chemotherapy during the first weeks of treatment, as has been shown in
bronchial cancers. The combination of the two therapeutic strategies has only given disappointing
results and exposes us to a sometimes significant risk of cumulative toxicity, particularly in fragile
patients representing a significant proportion of patients with bladder cancer.

Therapies targeting the PI3K/AKT/mTOR pathway

The PI3K/AKT/mTOR pathway is an essential signaling pathway in signal transduction within the
cancer cell. It is notably involved in the regulation of cell proliferation, apoptosis and angiogenesis. It
is frequently dysregulated in many cancers. In bladder cancer, recent TCGA data suggest a more
important role than initially assumed for this signaling pathway in urothelial carcinogenesis. [9]. It
could therefore be an interesting indication for inhibitors of the PI3K/AKT/mTOR pathway, more
particularly in the event of activation of certain components of the pathway. Everolimus, for
example, has shown effectiveness in patients with metastatic urothelial carcinomas and presenting a
TSC1 mutation (found in 8% of cases)[68]. Conversely, the existence of an inactivating mutation or a
deletion of PTENcould be associated with resistance to treatment[69]. However, the results of the
first phase II trials with everolimus are disappointing and other inhibitors of the PI3K/AKT/mTOR
pathway are still being evaluated.[70,71]. The use of “companion” tests to guide indications based on
the analysis of theranostic biomarkers should also allow for more rational use by restricting the use
of these treatments to the subgroup of patients with activation of the pathway ( up to 20% for
PI3KCA mutations in Cancer Genome Atlas analysis.

Therapies targeting FGFR3

There are currently no clinical trial data available for antiFGFR3 targeted therapies in urothelial
carcinomas. Since mutations are mainly observed in non-infiltrative and low-grade tumors (with
more than 45% of mutations described), it is difficult to envisage launching a trial in this subgroup
with a good prognosis or risk. progression towards infiltration is low. However, it appears that a
subpopulation of infiltrating tumors presents mutations in FGFR3 (12% according to the Cancer
Genome Atlas analysis) or translocationsFGFR3/ TACC3 (5%), suggesting a potential benefit of
targeted anti-FGFR3 therapy in certain well-selected patients[78]. Antagonistic antibodies specific for
FGFR3 generally act by interrupting dimerization, and therefore activation of the receptor for R248C
and S249C mutants. Preclinical studies seem to suggest the effectiveness of anti-FGFR3 in terms of
reducing cell proliferation and tumor growth.[79]. These data deserve to be confirmed in prospective
clinical trials in well-selected patients, that is to say, those carrying activating alterations of FGFR3.

Immunotherapy

Beyond the genetic and epigenetic regulatory systems, there is an interaction between the immune
system and tumor development, according to the concept of immunosurveillance. Bladder cancer
remains one of the rare examples of the effectiveness of immunotherapy, even if the exact
mechanism of action of the response to BCG therapy in NIMVT has not yet been established.
Dysfunctions of the immune response have been specifically highlighted in bladder cancer[80]. This
immunosuppression could be explained by the synthesis of molecules inhibiting the activity of T
lymphocytes by the tumor cells themselves. These co-regulatory molecules generate a reduction in
the immune response through anergy or apoptosis of T lymphocytes. This is the case in particular of
PD-L1, a surface glycoprotein that acts as a co-regulatory ligand by binding to the transmembrane
receptor PD-1. Its expression appears aberrant in many cancers, suggesting the benefit of blocking
this pathway with monoclonal antibodies.[81,82]. Furthermore, the overexpression of other
inhibitory costimulatory molecules, such as CTLA-4, has been reported in other cancers, again with a
potential therapeutic implication.

Immunotherapy should soon find a new place in the therapeutic management of bladder cancers,
since there appears to be protein overexpression of PD-L1 correlated with tumor stage and grade.
[84]with a controversial prognostic role particularly in IMVT[85,86]. Finally, it would appear that
tumors with microsatellite instability (MSI) present a particular immune microenvironment with
overexpression of PD-1, PD-L1, CTLA-4 and LAG-3 molecules which may contribute to their cellular
development by escaping control. immune [87]. Initially highlighted in colorectal cancers, this
information could prove useful to optimize the management of MSI+ urothelial carcinomas,
particularly in the upper excretory tract.