Urothelial carcinomas: a focus on human epidermal receptors signaling

Abstract

Bladder cancer is a common malignancy and a frequent cause of cancer-related death worldwide. The benefit from current chemotherapy has reached a relative plateau, thus identification of molecular targets for better therapy is a high priority. Human epidermal receptors constitute a family of receptor tyrosine kinases, which appear to be implicated in cellular transformation and can be over-expressed in a variety of solid tumors. There is preclinical and clinical data suggesting the role of EGFR and HER2 in urothelial carcinoma, thus prompting clinical investigation of anti-HER targeted therapies attempting to inhibit HER-induced tumor-promoting signaling. There is significant and dynamic cross-talk between HER and other signaling pathways and the identification of the structure and function of such cellular networks in the setting of urothelial cancer is a complex and difficult task. The development of prognostic and predictive biomarkers is needed in order to improve the personalized management of patients with urothelial cancer.

Introduction

Bladder cancer is the fourth most common malignancy, and the eighth leading cause of cancer death in men [1]. The estimated number of new cases in men worldwide is 297,300 with the estimated death rate being 112,300 [2]. In women, the cumulative risk of developing bladder cancer by age 75 is 0.2-0.4% with the cumulative risk of dying from this disease by that age is 0.1% [2]. Based on NCI data, the estimated new cases and deaths from bladder cancer in the United States in 2010 was 70,530 and 14,680, respectively (http://www.cancer.gov/cancertopics/types/bladder). Urothelial carcinoma is the most common histology, accounting for about 90% of cases. At the time of diagnosis, 75-80% of bladder carcinomas are superficial and approximately 20% of those eventually become invasive. A significant number of patients present with or develop ad-vanced/metastatic disease, which can be fatal.

Urothelial carcinoma is considered chemotherapy-sensitive; however, overall survival for patients with advanced disease has not been significantly impacted over the past 3 decades since the introduction of combination meth-otrexate, vinblastine, doxorubicin and cisplatin (MVAC) in the early 1980s. Since then, several regimens have been tested against MVAC including gemcitabine plus cisplatin (GC), which had comparable efficacy with relatively less tox-icity [3-6]. However, irrespective of treatment arm, less than 10% of these patients achieved a long-term disease-free survival [7,8]. The addition of paclitaxel to the GC regimen resulted in higher response rates, but at the expense of increased hematological toxicity, and did not provide significant overall survival benefit [9].

Given the dismal prognosis of advanced urothelial carcinoma, more effective systemic therapy is needed. Bladder cancer has several molecular alterations that regulate cellular processes, such as proliferation, differentiation, angiogenesis, metastasis and apoptosis. Some of these molecular alterations may serve as potential targets for systemic therapy, either as mono-therapy or in combination. Therefore, a thorough understanding of the molecular mechanisms that underlie urothelial cell malignant transformation and progression is critical for the optimization of treatment.

This review will focus on the role that human epidermal receptor (HER) family appears to play in the pathogenesis and prognosis of urothelial carcinomas, and it will summarize preclinical and clinical data regarding HER targeting approaches.

Human epidermal receptor (HER) family

The human epidermal receptor (HER) family of receptor tyrosine kinases consists of 4 receptors; HER1 (EGFR, erb-B1), HER2 (neu, erb-B2), HER3 (erb-B3), HER4 (erb-B4), and is implicated in several cellular processes, such as proliferation, growth, and survival. Epidermal Growth Factor Receptor (EGFR) is a 170 Kd membrane-spanning glycoprotein consisting of an extracellular ligand-binding domain, a trans-membrane domain, and an intracellular cytoplasmic domain with tyrosine kinase activity [10]. These receptor proteins belong to subclass I of the super-family of receptor tyrosine kinases (RTKs), classified based on their sequence homology and domain organization. They are expressed in many tissues of epithelial, mesenchymal, and neuronal origin and are critical for cell proliferation and tissue differentiation [11-14]. These receptors are usually monomers, but ligand binding induces the formation of homo- and hetero-dimers, activating the intracellular kinase domain, leading to phosphorylation of tyrosine residues. This can cause stereo-chemical con-formational changes in the receptor structure and increases its affinity with downstream adaptors and transducers, which bind to the receptor and form functional complexes in the cytoplasmic milieu, initiating a “marathon” of signal transduction in the nucleus, thus regulating gene transcription [10]. The target genes and the encoded proteins are essential for cell proliferation, differentiation, apoptosis, invasion, metastasis and angiogenesis, and under certain circumstances can contribute to tumor initiation and progression. The activity of signaling pathways and the genomic effects depend on the relative micro-concentrations of adaptors, transducers and effectors that influence intracellular signal transduction and trafficking, at a certain point in time in the sub-cellular microenvironment. Receptor-ligand complexes can be internalized, resulting in signal termination, while the receptor itself can be either recycled in the membrane or degraded, depending on the biochemical micro-context. Intervention in these processes can influence the receptor expression, modulating its role in cell signaling.

The HER family is stimulated by several growth factors (Table 1), while epigen can act as a ligand that promotes hetero-dimerization [15]. Over-expression of these ligands is considered to promote tumor development via EGFR in vitro [16, 17], and can be produced by a variety of tumors, which also express EGFR, suggesting that autocrine stimulatory mechanisms might participate in EGFR-driven tumor development [17]. It is worth mentioning that HER2 has no known ligand, while HER-3 does not have a functional tyrosine kinase domain, but rather depends upon neuregulin binding and subsequent hetero-dimer formation, particularly with HER2, to be trans-phosphorylated and acquire signaling competence [18]. Human epidermal receptors co-expression can activate cell signaling and tumor cell invasion pathways, not activated by single receptors [19]. Interestingly, human epidermal receptors also appear to interact with cell adhesion proteins. We have previously shown that soluble E-cadherin fragment can form a complex with HER2 and HER3 in breast cancer cells, resulting in stabilization of HER2/ HER3 hetero-dimer, induced receptor activation and signaling via ERK pathway, supporting cell migration and proliferation [20].

Table 1.

Endogenous HER ligands

EGF, TGF-α, Amphiregulin, Heparin – binding EGF, Epiregulin, Betacellulin	EGFR
	HER2
Neuregulins 1 and 2	HER3
Neuregulins 1-4, Amphiregulin, Betacellulin, Epiregulin, Heparin-binding EGF	HER4

HER family over-expression has been reported in a number of solid tumors, such as colorectal, breast, lung, head and neck, pancreatic, urothelial carcinoma, and gliomas [21-23]. Enhanced ligand levels, hetero-dimerization, cross-phosphorylation, and cross-talk with other surface receptors commonly contribute to tumor aggressive behavior. Co-expression of either EGF or TGF-a and EGFR has been associated with poor prognosis in pancreatic cancer [24]. Apart from the known role in breast and gastric cancer, data also suggest that HER2 is an unfavorable prognostic factor in prostate cancer [25,26].

EGFR mutations have been reported in several cancer types [27]. For example, tumor specific EGFRvlll, an EGFR mutant, has a constitutively activated tyrosine kinase domain that can be implicated in cellular transformation. Preclinical studies have implied that EGFR expression is correlated with tumor cell motility, invasiveness, angiogenesis, and metastatic potential [28-31]. The relationship between EGFR expression and survival of patients with several cancer types has been examined in a meta-analysis of data derived from more than 200 studies, involving more than 20,000 patients [32].

EGFR expression and role in bladder cancer

In normal urothelium EGFR is expressed only by the basal cells, and EGF is physiologically excreted in the urine, but a layer of EGFR-negative cells prevents its binding to EGFR. The disruption of this barrier may allow ligand-receptor binding, which may play a role in tumorigenesis. The urinary concentration of EGF in patients with urothelial carcinoma was significantly lower than that of controls, which implies EGF uptake by the tumor receptors [33].

The level of EGFR expression has been correlated with higher tumor grade and stage, disease progression, and worse prognosis in bladder carcinomas [33-40]. In two multivariate analyses, EGFR over-expression has shown to be an independent predictor of survival [39, 41]. In a third multivariate analysis, there was a significant correlation between EGFR expression and survival [26]. However, EGFR expression was not predictive of survival independent of stage. EGFR expression has also been associated with disease-specific mortality [40]. The estimated 5-year cancer-specific survival in a group of 121 patients who underwent radical cystectomy with curative intent was 60% in 47 patients with weak or moderate EGFR expression compared to 41% for 45 patients with strong EGFR expression (p=.039) [40].

HER3 and HER4 expression and role in bladder cancer

EGFR and HER2 are often co-expressed and form hetero-dimers with the other two members of HER family, HER3 and HER4. Data from 88 bladder cancer patient biopsies showed that the outcome of patients with EGFR- and HER2- expressing tumors is dependent on the expression of HER3 and HER4 [42]. A recent literature review on prognostic indicators of recurrence, progression, treatment response, and mortality in urothelial carcinoma concluded that EGFR and HER2 expression appears to indicate poor prognosis, while HER4 and Fibroblast Growth Factor Receptor 3 (FGFR3) appear to be favorable prognostic indicators [43]. Another study using urothelial carcinoma tissue arrays, stained for EGFR, HER2, HER3 and HER4, revealed that high EGFR or low HER4 expression was associated with non-papillary, high grade and invasive tumors, as well as with significantly lower recurrence-free and overall survival (p<0.002, p=0.028, p=0.047, respectively). HER2 and HER3 expression was not associated with overall or recurrence-free survival [44].

EGFR targeting in bladder cancer

Preclinical studies

Supportive data for the potential role of EGFR blockade in urothelial cancer comes from preclinical studies. Utilizing bladder cancer cell lines, it was demonstrated that the addition of gefitinib (an EGFR tyrosine kinase inhibitor) to radiation therapy resulted in a significant radio-sensitization effect [45]. Only a modest induction of apoptosis with single agent gefitinib was observed, but there was a marked induction of apoptosis with gefitinib in combination with ionizing radiation. A recent study on bladder cancer cell lines suggested that activation of the EGFR induced a cell-survival function when bladder cancer cells were treated with the DNA-damaging drug etoposide, and that combined treatment with etoposide and the EGFR inhibitor gefitinib might improve the efficacy of treatment [46]. Moreover, dual EGFR and VEGF inhibition with vandetanib was found to sensitize bladder cancer cells to cisplatin in a dose- and sequence-dependent manner [47]. The same dual approach also increased epithelial characteristics and chemotherapy sensitivity in mesenchy-mal bladder cancer cells [48]. In addition, members of the microRNA-200 family were reported to control the epithelial-mesenchymal process and sensitivity to anti-EGFR therapy in bladder cancer cells [49]. The expression of microRNA-200 was found to be sufficient to restore EGFR-dependency at least in a number of mesenchy-mal bladder cancer cells. One of microRNA-200 targets includes ERRFI-1, which appears to be a novel regulator of EGFR-independent growth.

The combination therapy of photodynamic therapy and cetuximab, a monoclonal antibody against the extracellular domain of EGFR, inhibited effectively tumor growth in a bladder tumor xenograft model, and can be considered a promising therapeutic strategy [50]. Additionally, an interesting study attempted to define molecular biomarkers of response to cetuximab in a panel of urothelial carcinoma cell lines [51]. The results suggested that expression of intact HER-4 (p=0.008), E-cadherin (p=0.015), beta-catenin (p=0.015) and loss of expression of platelet-derived growth factor receptor beta (p = 0.167) were associated with response to cetuximab therapy.

Clinical trials

Despite the potential biologic role for EGFR, clinical trials evaluating EGFR targeted therapy have surprisingly been limited. SWOG evaluated the role of gefitinib in 31 patients with metas-tatic transitional cell carcinoma, who had failed one chemotherapeutic regimen [52]. Patients were required to have a pre-treatment biopsy to assess EGFR expression. The median progression-free survival was 2 months; 2 patients survived past 6 months without disease progression. Grade 4 cerebrovascular ischemia and an increase in creatinine level were reported. There was one confirmed partial response in a patient with pulmonary metastases. Recently, a randomized, non-comparative phase II trial evaluated the efficacy of cetuximab combined with paclitaxel in 39 patients with previously treated (with platinum-based chemotherapy) metastatic urothelial carcinoma [53]. Patients were randomized to cetuximab 250mg/m² (after 400 mg/m² loading dose) with or without paclitaxel 80 mg/m² every week. The cetuximab arm closed when 9 of the first 11 patients progressed by 8 weeks. In the combination arm, 35.7% of patients were progression-free for more than 16 weeks. The overall response rate was 28.5%, with 2 complete and 6 partial responses; 4 additional patients had unconfirmed partial responses. The median progression-free survival for the combination arm was 115 days, while the median number of administered cycles was 3. Grade 3 toxicity occurring in more than 2 patients included rash, fatigue, anemia, hypomagnesemia. The authors concluded that this combination merits further evaluation.

Our group is currently conducting a randomized, open-label, phase II clinical trial investigating the potential benefit of adding cetuximab to the standard gemcitabine/cisplatin chemotherapy in patients with locally advanced or metastatic urothelial carcinoma (Clinical Trials.gov identifier: NCT00645593). The trial has completed accrual, and the results are pending.

HER2 expression and role in bladder cancer

HER2 expression in bladder carcinoma is variable between different studies, ranging between 9 and 81% [54-58]. We have previously reported that 28% of primary bladder cancers over-express HER2 by immunohistochemistry (IHC) and that primary tumor over-expression consistently predicts over-expression in a distant or regional metastatic site [59]. However, 45% of HER2-negative primary tumors may show over-expression in their corresponding metastasis. These data suggested that HER2 might play a role in the biological progression of bladder cancer and the development of metastatic disease. The median survival for HER2-positive primary cancers was 33 months compared to 50 months for HER2-negative cancers (p=0.46). HER2 over-expression in the metastatic lymph nodes did not have prognostic value.

In a recent study with 1,005 patients, HER2 protein over-expression was found in 9.2%, while HER2 gene amplification was found in 5.1% of tumor specimens [60]. Variability in IHC assays as well as tumor staining heterogeneity might account for, at least partially, the discordant results among different studies. In a clinical trial, HER2-positive staining was associated with a lower complete response rate after chemotherapy/irradiation (50 vs 81%, p = 0.03), but not with overall survival [61]. This study suggested that HER2 could be more related to the resistance to combined chemo-radiotherapy and thus may be more important in the local control of the disease. Moderate and heavy HER2 expression correlated significantly with aneuploidy, higher grade, and shorter overall survival, in a study with over 14 years of follow-up, while HER2 gene amplification correlated with grade, stage and survival in approximately 25% of patients in a different study [62, 63].

However, in another report, EGFR and HER2 expression was inversely related to tumor invasion in grade III tumors [64]. HER2 status has not consistently correlated with EGFR expression, stage, grade and survival [65-66]. The higher rate of HER2 gene amplification in T1 compared to more invasive tumors that was reported implied a role in the development of early disease, but not in further invasion [67].

A series of studies have reported a negative prognostic value of HER2 expression in urothelial carcinoma (Table 2). Three retrospective analyses of patients with urothelial carcinoma revealed an association between HER2 expression and poor outcome [68-70]. A study of patients with locally advanced urothelial carcinoma, receiving surgery alone or with adjuvant MVEC chemotherapy reported that bladder carcinoma had significantly higher HER2 staining compared to the upper urinary tract disease [71]. In the adjuvant MVEC arm only, HER2 im-munoreactivity correlated with shorter progression-free and disease-specific overall survival in the univariate analysis. In another study, HER2 protein was over-expressed in 41 out of 80 tumors, corresponding to advanced stage and grade, shorter specific and overall survival, but not to disease recurrence [72]. A cohort of 198 patients undergoing radical cystectomy with lymphadenectomy reported HER2 staining in a 27.8% of primary tumors compared to 44.2% of metastatic lymph nodes; HER2 expression correlated with lymphovascular invasion and higher risk for recurrence and cancer-specific mortality [73].

Table 2.

Studies reporting negative prognostic value of HER2 protein expression or gene amplification in urothelial carcinoma

Author/reference	Correlative outcome/HER2 marker	Patients/Cases
Chakravarti et al [61]	lower complete response rate to chemoradiation / protein expression	73
Lönn et al [63]	aneuploidy, higher grade, shorter overall survival / protein expression	91
Lipponen et al [62]	higher stage and grade, shorter overall survival / gene amplification	178
Masliukova et al [68]	shorter relapse-free survival/ protein expression / protein expression	63
Kolla et al [69]	higher stage and grade, positive lymph node status, shorter disease-free and disease-related survival / protein expression	90
Krüger et al [70]	higher grade, shorter disease-free and disease-related survival / protein expression	138
Tsai et al [71]	site (bladder vs upper urinary tract), shorter progression-free and disease-related survival / protein expression	114
Skagias et al [72]	higher stage, grade, shorter disease-specific and overall survival / protein expression	80
Bolenz et al [73]	lymph nodes (vs primary site), lymphovascular invasion, higher recurrence risk, shorter disease-specific survival / protein expression	198
Alexa et al [74]	higher grade/ protein expression	59

In another study with 59 cases, normal urothe-lium and the neighboring renal parenchyma were HER2-negative [74]. HER2-negative tumors were for the most part well-differentiated, low grade, papillary, or rarely infiltrative. HER2 over-expression did not correlate with stage or lymph node status; however, high staining intensity was associated with high grade. Another study did not identify a strong association between HER2 protein over-expression and gene amplification in high grade invasive urothelial carcinomas; polysomy of chromosome 17 was reported in 9 out of 27 tumors [75]. A similar study suggested that although HER2 gene amplification was detected in high grade and invasive tumors, it was a rare event, and that polysomy of chromosome 17 was associated with tumor stage and grade and thus could be considered a biomarker of tumor progression [76]. Co-amplification of HER2 and MYC was reported in a subset of patients with metastatic urothelial cancer, while in a different study, invasive mi-cropapillary carcinoma showed higher immuno-reactivity for MUC1, CA125, and HER2 compared to invasive urothelial carcinoma with retraction artifact [77, 78]. A tissue microarray study with 100 upper urinary tract urothelial carcinomas revealed 10 cases with HER2 expression, 84 cases with cytoplasmic phospho-AKT and 6 cases with nuclear phospho-AKT; the latter was found to be an independent prognostic factor [79].

HER2 targeting in bladder cancer

A study evaluated the efficacy of trastuzumab in 6 patients with HER2 positive metastatic urothelial carcinoma; all patients achieved a partial response, with 30-80% reduction in the size of the metastatic lesions [80].

We prospectively evaluated the rate of HER2 expression and feasibility of anti-HER2 targeting in a multicenter phase II trial in patients with urothelial cancer [81]. In this study, 109 patients were evaluated for HER2 status by immu-nohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH); 57 were considered HER2 positive at least by one method (either IHC or FISH), and 44/57 received combination of car-boplatin, paclitaxel, gemcitabine, and trastuzumab. Five patients (11%) achieved a complete response, 26 (59%) a partial response, 5 (11%) had stable disease, and 5 (11%) had no response assessment, with an overall response rate of 70%. Median time to progression was 9.3 months, and median survival 14.1 months. Patients with HER2 positive tumors had a higher rate of liver/bone metastases, higher median number of metastatic sites and a higher incidence of two or more metastatic sites, implying a negative prognostic role of the receptor. Additionally, there are 3 recruiting and 4 completed clinical trials investigating an anti-HER2 strategy in various settings and stages in patients with bladder cancer, as reported in the U.S. NIH Clinical Trial Registry (http://clinicaltrials.gov).

Dual EGFR and HER2 targeting in bladder cancer

The expression of EGFR and HER2 in malignant urothelial cells is biologically attractive for combined targeting. This can be achieved by either the combined use of agents such as the antibodies cetuximab and trastuzumab that target each receptor separately, or an agent like lapatinib, which is an oral reversible non-covalent dual inhibitor of the EGFR and HER2 tyrosine kinases. The latter approach has the advantage of a single oral drug that could, at least in theory, be easier and less toxic.

Preclinical data with lapatinib showed promising activity in transitional carcinoma cell lines, enhancing the activity of concomitant chemotherapy in a dose-dependent fashion [82, 83]. Specifically, synergistic effects have been demonstrated in bladder cancer cell lines treated with lapatinib in combination with gemcitabine and cisplatin. Lapatinib was also shown to reverse multidrug resistance in cancer cell lines by inhibiting the activity of ATP-binding cassette proteins, suggesting that it may reverse chemo-resistance in the clinical setting [84].

Lapatinib has been evaluated in the clinical setting (Table 3). A recent phase II trial tested lapatinib monotherapy at the dose of 1,250 mg daily in 59 patients with urothelial carcinoma who have progressed on a prior platinum-containing chemotherapy schedule [85]. Of the 34 patients evaluable for response, only 1 patient achieved an objective response, and 18 had stable disease, with a median time to progression of 8.6 weeks and a median overall survival of 17.9 weeks. Lapatinib was well tolerated. Although this study was initially considered negative, further analysis showed that clinical benefit (response and stable disease rate) was found to correlate with EGFR over-expression (p=.029), and, to some extent, HER2 over-expression. EGFR and HER2 were evaluated by immunostaining and positivity was defined as any membranous staining above the background level of the cell in >10% of tumor cells; intensity staining of 2+ or 3+ was characterized as over-expression. Of all samples, 52% over-expressed EGFR and 44% HER2. Of the 19 patients with clinical benefit, 17 patients had EGFR and/or HER-2 over-expressing tumors. Patients with EGFR and/or HER-2 over-expressing tumors had a median survival of 30.3 weeks compared to 10.6 weeks in patients with tumors with negative/low expression (p=.0001), implying that this subset of patients may benefit from the inhibitor.

Table 3.

Clinical trials with lapatinib in bladder cancer

Wülfing et al [85]	Phase II, single arm, multicenter, open-label, lapatinib monotherapy in urothelial carcinoma progressed on platinum-containing chemotherapy (results reported)
NCT00447226	Phase II, placebo controlled, double-blind, randomized, discontinuation study in HER2 positive solid tumors (terminated)
NCT00623064	Phase I, cisplatin, gemcitabine and lapatinib as first line therapy in advanced/metastatic urothelial cancer (unknown recruitment status)
NCT00949455	Phase II/III, randomized comparison of maintenance lapatinib vs placebo after first line chemotherapy in EGFR- and/or HER2- over-expressing locally advanced or metastatic blad-der cancer (recruiting)
NCT00313599	Phase I dose-escalation of a 2-day lapatinib chemo-sensitization pulse prior to weekly IV Abraxane in advanced solid tumors (ongoing, not recruiting)
NCT01245660	Pilot (phase 0), lapatinib as neo-adjuvant treatment in local bladder cancer (open, not recruiting yet)

Although a phase II, placebo-controlled, double-blind, randomized, discontinuation study of lapatinib in patients with HER2 positive solid tumors, including bladder, failed to meet the primary objective of tumor response rate at 12 weeks from first dose and was closed (Clinical Trials.gov identifier: NCT00447226), several trials are evaluating the efficacy of lapatinib in different settings. A phase I trial is evaluating lapatinib with cisplatin and gemcitabine as first-line therapy in locally advanced or metastatic urothelial cancer patients in Europe (Clinical Trials.gov identifier: NCT00623064); a randomized, multicenter, phase ll/lll clinical trial is comparing maintenance lapatinib monotherapy to placebo, after first-line chemotherapy that resulted in no disease progression, in patients with EGFR and/or HER2 over-expressing locally advanced or metastatic bladder cancer (Clinical Trials.gov identifier: NCT00949455). A phase I dose-escalation study of a 2-day oral lapatinib chemosensitization pulse, given prior to weekly abraxane chemotherapy in patients with advanced solid tumors, including bladder, completed accrual (Clinical Trials.gov identifier: NCT00313599). Moreover, a pilot study of lapatinib as neoadjuvant treatment in patients with local bladder carcinoma before cystectomy was just launched (Clinical Trials.gov identifier: NCT01245660).

A recent study compared global genome-wide microarray to EGFR-pathway microarray profiling to identify predictive models of lapatinib sensitivity in bladder cancer [86]. The top-performing combination model included the phosphorylated EGFR (pTyr-1173), with a mean predictive accuracy for response to lapatinib of 98.3%. Two of the three phosphoproteomic models in this study included phosphorylated HER2 (pY1248), in a site associated with activation, poor prognosis, and therapeutic response to trastuzumab in breast cancer patients [87-89]. This phosphory-lation site was also found to be regulated in vitro and in vivo by lapatinib [90, 91].

HER signaling cross-talk with other pathways

As discussed above based on available data, HER family appears to play a role in the patho-genesis and prognosis of urothelial carcinoma. However, there is significant multiplicity, complexity and dynamic cross-talk in the biochemical pathways involved in the initiation and progression of cancer in general. For example, there is evidence of significant cross-talk between the hepatocyte growth factor (HGF)/ mesenchymal-epithelial transition factor (MET) pathway and HER signaling proteins. HGF/MET axis can potentially substitute HER components activity, thus conferring resistance to EGFR-targeting drugs [92]. Insulin growth factor-1 (IGF1) pathway also appears to interact with HER signaling activity. Specifically, IGF1-mediated “trans-activation” of EGFR mediates the IGF1-stimulated phosphorylation of Src homology-2 domain (Sh2) and thus the subsequent activation of the extracellular signal-regulated kinase (Erk) cascade [93]. Additionally, EGFR interaction with adhesion molecules, such as integrins, can influence the activity of transcription factors. EGFR/beta1-integrin complex interaction can drive integrin-dependent PI3K/Akt activation, Akt translocation into the nucleus and phosphorylation of FoxO1, which is a Forkhead transcription factor; FoxO1 inactivation results in increased levels of the transcription factor Egr-1, thus influencing gene transcription [94]. Overall, there appears to be a “chaotic model’ of molecular interactions that is difficult to be precisely determined and predicted with conventional informatics approaches.

Conclusion

HER family appears to be biologically implicated in urothelial carcinoma. The expression pattern of these receptors in a specific tumor as well their dynamic interactions with multiple signaling pathways might dictate cancer-related events. However, limited data is available on the potential efficacy of this approach hence therapeutic targeting of EGFR and HER2 has not been incorporated into the standard oncologist's armamentarium in this disease. Critical to such approach is tumor profiling for better patient selection. Recently, the International Consensus Panel on Bladder Tumor Markers concluded that, based on the current evidence, none of the current prognostic molecular markers, including microsatellite-associated markers, oncogenes, tumor suppressor genes, cell-cycle regulators and extracellular matrix-adhesion molecules, are sufficiently validated to be implemented in the management of patients with urothelial carcinoma [95]. Therefore, there is an unmet need for robust prospective validation of candidate biomarkers and investigation of their prognostic value and predictive role in response to molecular targeted therapies.