# Patent

## Title
Method for treating muscle-invasive bladder cancer, containing S100A9 and EGFR inhibitors and cisplatin as active ingredients

## Abstract

The present invention relates to a method for predicting the probability of muscle-invasive bladder cancer (MIBC) recurrence or metastasis, a method for providing information on a personalized medicine of MIBC, and a pharmaceutical composition for treating MIBC, containing S100A9 and EGFR inhibitors and cisplatin as active ingredients. According to the present invention, it is possible to accurately predict a prognosis after chemotherapy of an MIBC patient, to provide information on cisplatin sensitivity in the provision of a personalized medicine for chemotherapy of an MIBC patient, and to increase the cisplatin sensitivity of an MIBC patient by concomitantly administering S100A9 and EGFR inhibitors together with a conventional cisplatin.

## Background
TECHNICAL FIELD
The present invention relates to a method for predicting the probability of recurrence or metastasis of muscle invasive bladder cancer (MIBC), a method for providing information about personalized medicine for MIBC, and a pharmaceutical composition for treatment of MIBC comprising inhibitors of S100A9 and EGFR and cisplatin as effective components.
BACKGROUND ART
Bladder cancer is the second most common genitourinary tumor, and more than 90% of these tumors are urothelial carcinomas. Almost 25% of patients with newly diagnosed bladder cancer have MIBC, with the vast majority of these tumors being of high histological grade. Moreover, nearly 50% of patients with MIBC already have occult distant metastases at the time of diagnosis.
Although radical cystectomy is the standard treatment for MIBC, about 50% of these patients develop metastases within 2 years, and the 5 year survival rate after surgery alone is approximately 50%. Systemic cisplatin-based combination chemotherapy is the first-line treatment modality for patients with metastatic bladder cancer. However, despite the initial high response rates of 40 to 70% reported in patients with advanced disease, chemotherapy is usually not curative and the overall 5 year survival is only 5 to 15%. Performance status and the presence of visceral metastases are well-established prognostic markers demonstrated to predict a poor prognosis in patients treated with cisplatin-based chemotherapy. However, while these clinicopathological markers are useful as survival indicators, they are inadequate to predict either the response rate or the survival rate in an individual patient. Accordingly, there is growing interest in the role played by genes in the chemotherapeutic response of patients with MIBC and the predictive power of this relationship in an individual patient.
Thus far, information that would allow the response to chemotherapy to be predicted in an individual patient is lacking in the case of MIBC as well as many other cancers. Consequently, some patients suffer the adverse side effects of these highly toxic drugs without the benefit of their intended action. Perhaps even more important is that, as their physical condition worsens, some of these unnecessarily treated patients may be deprived of additional therapy.
In our previous study, a gene expression profile analysis was carried out with the aim of identifying a genetic signature for progression in MIBC patients. Among the 1,320 genes thus identified by microarray data analysis, four genes (IL1B, S100A8, S100A9, and EGFR) were determined to be important in predicting disease progression. In the present study, we asked whether this four-gene signature could be used to predict disease progression after chemotherapy in patients with locally recurrent or metastatic MIBC.
PRIOR ART DOCUMENT
Patent Document
Patent Document KR 10-2007-0018108
DISCLOSURE
Technical Problem
The present invention is directed to providing a method for predicting the probability of recurrence or metastasis of MIBC.
Also, the present invention is directed to providing a method for providing information about personalized medicine for MIBC.
Further, the present invention is directed to providing a pharmaceutical composition for treatment of MIBC comprising inhibitors of S100A9 and EGFR and cisplatin as effective components.
Technical Solution
One aspect of the present invention provides a method for predicting the probability of recurrence or metastasis of MIBC by measuring expression levels of S100A9 and EGFR after chemotherapy.
Preferably, the expression levels may be mRNA or protein expression levels.
Another aspect of the present invention provides a method for providing information about personalized medicine for chemotherapy by measuring expression levels of S100A9 and EGFR from a patient with MIBC.
Preferably, the expression levels may be mRNA or protein expression levels.
Preferably, the chemotherapy may be cisplatin-based chemotherapy.
Further, still another aspect of the present invention provides a pharmaceutical composition for treatment of MIBC comprising inhibitors of S100A9 and EGFR and cisplatin as effective components.
Preferably, the inhibitors may be siRNAs of S100A9 and EGFR.
Preferably, the inhibitors may be protein inhibitors of S100A9 and EGFR.
Advantageous Effects
According to the present invention, it is possible to accurately predict the prognosis of a patient with MIBC after chemotherapy, and also possible to provide information about the sensitivity to cisplatin when providing personalized medicine for chemotherapy of a patient with MIBC, and still also possible to enhance the sensitivity of a patient with MIBC to cisplatin by a co-administration of inhibitors of S100A9 and EGFR together with an administration of cisplatin.



DESCRIPTION OF DRAWINGS
FIG. 1 is a graph that compares the progression probability over time in patients with recurrent or metastatic MIBC who received chemotherapy for each signature group.
FIG. 2a and FIG. 2b are graphs that compare the overall survival and cancer-specific survival over time in patients with recurrent or metastatic MIBC who received chemotherapy for each signature group.
FIG. 3a to FIG. 3f are respectively images and graphs showing immunohistochemical staining for S100A9 and EGFR in bladder cancer.
FIG. 4a to FIG. 4d show that overexpressed S100A9 leads to the enhanced migration, proliferation, and resistance to cisplatin-induced cell apoptosis.
FIG. 5a to FIG. 5d show that EGFR expression coincides with apoptosis by cisplatin treatment.
FIG. 6 shows that inhibition of S100A9 and EGFR leads to an increased sensitivity to cisplatin treatment.



MODES OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The present invention provides a method for predicting the probability of recurrence or metastasis of MIBC by measuring expression levels of S100A9 and EGFR after chemotherapy.
The inventors of the present invention confirmed that the probability of local recurrence or metastasis in a patient with MIBC after chemotherapy is correlated to the expression levels of S100A9 and EGFR.
To be more specific, most of the patients showing recurrence or metastasis of MIBC also show an increase in expression levels of S100A9 and EGFR. This means an increase in mRNA or protein expression levels of S100A9 and EGFR.
Further, the present invention provides a method for providing information about personalized medicine for chemotherapy by measuring expression levels of S100A9 and EGFR from a patient with MIBC.
The expression levels are mRNA or protein expression levels of S100A9 and EGFR, and the chemotherapy is cisplatin-based chemotherapy.
According to the above-described method of the present invention, it is possible to provide information about personalized medicine for cisplatin-based chemotherapy of a patient with MIBC.
Furthermore, the present invention provides a pharmaceutical composition for treatment of MIBC comprising inhibitors of S100A9 and EGFR and cisplatin.
Preferably, the inhibitors may be siRNAs or protein inhibitors of S100A9 and EGFR.
The pharmaceutical composition of the present invention remarkably enhances the chemo-sensitivity to cisplatin as compared with chemotherapy such as an administration of cisplatin alone. Thus, an administration of a small dose of medicine can achieve a result equivalent or superior to that of the administration of cisplatin alone.
The term “treatment”, as used herein, unless otherwise indicated, means reversing, palliating, inhibiting the progress of, or preventing the disorder or disease to which such term applies, or one or more symptoms of the disorder or disease. The term “treatment”, as used herein, refers to the act of treating as the term “treating” is defined above. Therefore, the “treatment” or “therapy” of a mammal with MIBC may include at least one of the following:
(1) Inhibiting the growth, i.e. development, of MIBC;
(2) Preventing proliferation, i.e. metastasis of MIBC;
(3) Relieving MIBC;
(4) Preventing recurrence of MIBC; and
(5) Palliating symptoms of MIBC.
Further, the composition for prevention or treatment of MIBC according to the present invention may comprise only the above-described effective components in pharmaceutically effective amounts or may comprise one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutically effective amount means an amount sufficient to prevent, improve, and treat symptoms of the MIBC.
Furthermore, according to the present invention, the pharmaceutically effective amount can be appropriately changed depending on a degree of symptoms of MIBC, an age of a patient, a weight of a patient, a health condition of a patient, a sex of a patient, an administering route, a period of treatment, and the like.
Also, the pharmaceutically acceptable composition means that it is physiologically acceptable and a side effect or the similar effect thereof, such as a gastroenteric trouble, and dizziness, is not caused typically when being administered to humans. Examples of the carrier, excipient, and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and minerals. Further, they may additionally include fillers, anticoagulants, lubricants, wetting agents, flavoring, emulsifying agents, preservatives, and the like.
Further, the composition of the present invention may be formulated by using a method publicly known in the art in order to provide rapid, continuous, or delayed release of an active component after being administered to mammals. A dosage form may be powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, a sterile injection solution, or a sterile powder.
Furthermore, the composition for prevention or treatment of MIBC according to the present invention can be administered in various ways for example, orally, percutaneously, subcutaneously, intravenously or intramuscularly. A dosage of the active component may be selected appropriately depending on various factors, such as an administering route, an age, a sex, and a weight of a patient, severity of a patient, and the like. Further, the composition for prevention or treatment of MIBC according to the present invention may be administered along with a compound publicly known as having an effect of preventing, improving, or treating symptoms of MIBC.
Hereinafter, the present invention will be described in more detail with reference to Examples. However, Examples are just preferable examples, but do not limit the scope of the present invention.
EXAMPLE
Patients and Tissue Samples
In the present Example, the population consisted of patients with locally recurrent or metastatic MIBC who received chemotherapy. To minimize bias due to selection or knowledge of previous results, the patient cohort of the present Example was not the same as the one enrolled in the previous study of the inventors of the present invention on MIBC. Patients with locally recurrent MIBC underwent prior radical cystectomy but subsequently developed local or lymph node recurrence. Patients with metastatic MIBC had metastases to visceral organs. Only patients with good performance status (PS) (ECOG 0 or 1) were enrolled, thus removing the confounding effect of this functional parameter. All of the enrolled patients had been treated with at least four cycles of the cisplatin-based chemotherapy. Patients additionally treated with radiation therapy for any reason or patients who had experienced serious complications associated with surgery were excluded. Regardless of radical cystectomy status, patients who had not undergone an imaging work-up, such as CT scan or MRI, at least once per 3 months were also excluded from the study. Primary MIBC samples were obtained at the institute, to which the inventors of the present invention belong, from the 69 patients with histologically verified urothelial carcinoma who were enrolled in the study of the present Example.
All tumors were macro-dissected, typically within 15 minutes of surgical resection. Each bladder cancer specimen was confirmed as representative by analysis of adjacent tissue in fresh frozen sections from cystectomy and transurethral resection specimens. The tumor specimens were then frozen in liquid nitrogen and stored at −80° C. until use. Both the collection and the analysis of all samples were approved by the Institutional Review Board of Chungbuk National University. Informed consent was obtained from each patient enrolled in the study of the present Example (IRB approval number: 2006-01-001).
Tumors were staged according to the 2002 TNM classification and the 1973 WHO grading system. In the present Example, disease progression was defined as newly diagnosed distant metastasis and a 20% increment in the MIBC tumor mass after chemotherapy.
Reagents
TRIzol and First-Strand cDNA synthesis kit were purchased from Invitrogen (Carlsbad, Calif.) and from Amersham Biosciences (Freiburg, Germany). Ventana Ultraview DAB Kit was purchased from Ventana Medical Systems (Tucson, Ariz.). Heat-inactivated fetal bovine serum (FBS) and Lipofectamine 2000 were purchased from Invitrogen. Protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Basel, Switzerland). The Micro BCA protein assay kit was obtained from Pierce (Rockford, Ill.). Premade SDS-PAGE gels, Coomassie Blue R-250 staining solution and destaining solution were purchased from Bio-Rad (Hercules, Calif.). Small interfering RNAs (siRNAs) against EGFR, S100A9 and NON-TARGET controls were purchased from Dharmacon (Chicago, Ill.). Antibodies against EGFR and β-actin were purchased from Cell Signaling Technology (Danvers, Mass.). Antibody against S100A9 was purchased from AbCam (West Grove, Pa.). All other reagents were obtained from Sigma-Aldrich or Promega (Madison, Wis.).
RNA Extraction and cDNA Synthesis
Total RNA isolated from malignant bladder tissue using 1 ml of TRIzol was homogenized in a 5 ml glass tube. The homogenate was transferred to a 1.5 ml tube and then mixed with 200 μl of chloroform. After a 5-min incubation at 4° C., the homogenate was centrifuged for 13 minutes at 13,000×g and 4° C. The upper aqueous phase was transferred to a clean tube to which 500 μl of isopropanol was added. These samples were incubated for 60 minutes at 4° C. and centrifuged for 8 minutes at 13,000×g and 4° C. The upper aqueous phase was discarded and the pellet was resuspended with 500 μl of 75% ethanol, followed by a 5-min centrifugation at 13,000×g and 4° C. The upper aqueous layer was removed and the pellet was dried at room temperature, dissolved in diethylpyrocarbonate (DEPC)-treated water, and then stored at −80° C. The quality and integrity of the RNA were confirmed by agarose gel electrophoresis and ethidium bromide staining followed by visual inspection under ultraviolet light. The cDNA was prepared using 1 μg of random primers and a First-Strand cDNA synthesis kit (Amersham Biosciences Europe GmbH, Freiburg, Germany) according to the manufacturer's protocol.
RT-PCR Analysis
The expression of four genes was quantified by real-time PCR using a Rotor-Gene 6000 system (Corbett Research, Mortlake, Australia). The assays were carried out in micro-reaction tubes (Corbett Research, Mortlake, Australia) using SYBR Premix EX Taq (TAKARA BIO INC., Otsu, Japan). The primers used to amplify the four genes analyzed in the present invention are shown in Table 1.










TABLE 1







Gene
Primer









S100A9
S: 5′-CACCCAGACA CCCTGAACCA-3′




AS: 5′-CCTCGAAGCT CAGCTGCTTG-3′



 



S100A8
S; 5′-ATTTCCATGC CGTCTACAGG-3′




AS: 5′-TGCCACGCCC ATCTTTATCA-3′



 



EGFR
S: 5′-TCCAGTGGCG GGACATAGTC-3′




AS: 5′-AGTCACTGGG GGACTTGCCA-3′



 



IL-1B
S: 5′-TGAGCTCGCC AGTGAAATGA-3′




AS: 5′-AAGCCCTTGC TGTAGTGGTG-3′







S: sense, AS: antisense.






The PCR consisted of 5 μl of 2× SYBR Premix EX Taq buffer, 0.5 μl of each of the sense and antisense primers (10 pmol/μl), and 1 μl of the sample cDNA, in a final reaction volume of 10 μl. The amplified products were purified with a QIAquick extraction kit (QIAGEN, Hilden, Germany) and quantified with a spectrophotometer (Perkin Elmer MBA2000, Fremont, Calif.). Fragments were sequenced using an automated laser fluorescence sequencer (ABI PRISM 3100 Genetic Analyzer, Foster City, Wis.). Ten-fold serial dilutions were prepared to obtain known concentrations of product in the range of 100 to 0.1 pg/μl. This dilution series was then used to establish a standard curve for real-time PCR, carried out under the following conditions: (i) denaturation, 1 cycle of 20 seconds at 96° C. followed by 40 cycles of 2 seconds at 96° C.; (ii) annealing, 15 seconds at 60° C.; and (iii) extension, 15 seconds at 72° C. The melting program was performed within a temperature range of 72 to 95° C., with heating at a rate of 1° C. per 45 seconds. Spectral data were captured and analyzed using Rotor-Gene Real-Time Analysis Software 6.0 Build 14 (Corbett Research, Mortlake, Australia). All samples were run in triplicate. Gene expression was normalized to β-globin expression.
Immunohistochemical Staining
Paraffin blocks from 38 bladder cancer cases were used for immunohistochemical analysis. Tissue sections were cut and placed on Superfrost Plus microscope slides (Fisher Scientific). Using the Benchmark XT automated immunohistochemistry stainer (Ventana Medical Systems, Inc., Tucson, Ariz., USA), slides were stained by the following procedure. Detection was done using the Ventana Ultraview DAB Kit (Ventana Medical Systems). Sections were deparaffinized using EZ Prep solution. CC1 standard (pH 8.4 buffer containing Tris/Borate/EDTA) was used for antigen retrieval. DAB inhibitor (3% H2O2, Endogenous peroxidase) was blocked for 4 minutes at 37° C. Sections were incubated with an anti-EGFR (Abcam Inc., San Diego, Calif., dilution 1/100) and anti-S100A9 primary antibody (Abcam Inc., San Diego, Calif., dilution 1/100) for 40 minutes at 37° C., and a secondary antibody of Univeral HRP Multimer for 8 minutes at 37° C. Slides were treated on a DAB+H2O2 substrate for 8 min followed by hematoxylin and bluing reagent counterstain at 37° C. Reaction buffer (pH 7.6 Tris buffer) was used as a washing solution. Staining intensity and proportion of positively-stained cells were evaluated. EGFR and S100A9 localized primarily to the cytoplasm. Staining intensity was classified as follows: none, weak, moderate and strong. Each specimen was examined and scored separately by three investigators, and discrepancies were discussed until agreement was reached.
Cell Culture and Transfection
T24 human bladder cancer cells were purchased from American Type Culture Collection (ATCC, Manassas, Va.) and maintained in RPMI1640 media (Invitrogen, Carlsbad, Calif.) with 10% FBS and 1% Penicillin/Streptomycin at 37° C. under 5% CO2. T24 cells at  ̃80% confluence were transiently transfected with expressing constructs or small interfering RNAs (siRNAs) of EGFR and S100A9 using Lipofactamine 2000. For S100A9 overexpression, pCMV-Sports6-S100A9 and vector constructs were kindly provided by Dr. A. Moon (Duksung University, Korea). As transfection controls, empty (Ctrl) or NON-TARGET control siRNAs (siCtrl) were used.
Western Blot Analysis
Whole cell lysates were extracted with NP40-containing lysis buffer (1% Nonidet P-40, 50 mM Tris pH 7.4, 10 mM NaCl, 1 mM NaF, 5 mM MgCl2, 0.1 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail) on ice. After vigorous vortexing, lysates were centrifuged at 12,000×g for 15 minutes to remove debris. Protein concentration was determined by microBCA (Pierce/Thermo Scientific) and 25 μg of proteins were subjected to SDS-PAGE separation and following western blot analysis. Secondary antibodies and the Micro BCA protein assay kit were obtained from Pierce (Rockford, Ill., USA). The ECLTM detection system was used for blotting signal detection, which was followed by densitometry (Amersham Biosciences, Little Chalfont, UK) to measure band intensities.
Wound-Healing Assay
Wound healing assays were performed in 6-well plates after cell density reached to about 90%. Gentle scratch on T24 cells was done with a sharp tip, and the plates were incubated for additional 8 hours before observation under microscope.
Cell Proliferation Assay
T24 bladder cancer cells were plated onto 6-well culture plates at a density of 5×103 cells per well in 10% FBS-containing growth medium. To overexpress S100A9, transient transfection was performed using Lipofectamine 2000. 24 hours after transfection, cells were serum-starved for 16 hours. Medium was replaced with normal growth medium for proliferation assay. A cell proliferation rate was determined by crystal violet staining at 0, 1 and 2 days. Briefly, cells were stained with a crystal violet solution and quantified by dissolving the stained cells in a 10% acetic acid solution. Colorimetric measurement was done by reading absorbances at 570 nm.
Cell Viability Assay
T24 cells were transfected with various constructs or siRNAs, and the cells were incubated with drugs (e.g. cisplatin or Iressa) containing serum free medium for the indicated time. Cell viability was determined using MTS reagents as instructed by the manufacturer's protocol (Promega Corporation, Madison, Wis.).
Data and Statistical Analysis
Due to the highly skewed distribution of the mRNA expression levels of each gene, the data were natural-log-transformed and then back-transformed for the interpretation of the results. The association between disease progression after chemotherapy and the gene signatures was evaluated using univariate Cox regression analysis. Time to progression was calculated according to the Kaplan-Meier method, with differences between the times assessed using log-rank statistics.
After univariate Cox analysis of the four progression-related genes, two were used to calculate a risk score of disease progression for each patient, defined as the sum of the levels of expression of each gene multiplied by the corresponding regression coefficient. ROC curves identified the optimal cutoff point of each risk score that yielded the highest combined sensitivity and specificity for disease progression. Based on these values, the patients were classified into either the good- or the poor-prognostic gene signature group. The prognostic value of the gene expression signature was determined by multivariate Cox proportional hazard regression models. The association between disease progression after chemotherapy and results of the immunohistochemical staining was evaluated using Fisher's exact test, and the correlation between the mRNA expression level and the immunohistochemical staining intensity was evaluated by Spearman rank correlation. In in vitro experiments, p-values were calculated using unpaired Student's t-test. Statistical analysis was performed using SPSS 19.0 software (SPSS Inc., Chicago, Ill.), with p<0.05 considered statistically significant.
Results
Baseline Characteristics
The mean age of the 69 patients who received chemotherapy was 64.74±8.97 years, and the mean follow-up period was 46.24±56.24 months. 54 (78.3%) patients had local recurrence without visceral metastasis and 15 (21.7%) patients had metastases to other organs. 40 (58.0%) patients underwent prior radical cystectomy. Other baseline characteristics of the patients are presented in Table 2.








TABLE 2





Variables
Incidence or mean value (%)







Age (years)
64.74 ± 8.97 


Median follow-up (months)
46.24 ± 56.24


Gender


Male
57 (82.6)


Female
12 (17.4)


Grade


2
25 (36.2)


3
44 (63.8)


Stage


Advanced (any T, N1 or N2, and M0)
54 (78.3)


Metastasis (any T, any N, and M1)
15 (21.7)


Prior radical cystectomy


No
29 (42.0)


Yes
40 (58.0)









Identification of Genes Associated with Disease Progression in Locally Recurrent or Metastatic MIBC
Univariate Cox regression analysis of four genes (IL1B, S100A8, S100A9, and EGFR), which were previously determined to play an important role in MIBC progression, was performed. Two of them, S100A9 and EGFR, significantly correlated to disease progression (p=0.023, p=0.045, respectively). These two genes were then used to calculate a risk score of disease progression after chemotherapy in MIBC patients. The risk score identified two groups of patients. A good-prognostic signature group represented relatively low expression levels of the two genes, while a poor-prognostic signature group had significantly higher expression. A cutoff value (36.1683) was determined for disease progression with the highest combined sensitivity (91.9%) and specificity (56.3%) based on the ROC curve.
Prognostic Value of the Two-Gene Signature for Disease Progression in Patients with Locally Recurrent or Metastatic MIBC
A comparison of the two groups showed that the time to progression was significantly longer in the good-prognostic signature group (p<0.001) (FIG. 1). Univariate Cox regression analysis showed that metastasis, prior cystectomy, and combined gene signature were significant influential factors for disease progression after chemotherapy (Table 3). In multivariate Cox regression analysis, only combined gene signature was a significant influential factor for disease progression in patients with locally recurrent or metastatic MIBC after chemotherapy (Table 3).









TABLE 3









Disease progression after chemotherapy










Univariate Cox regression
Multivariate Cox regression











Variables
HR (95% CI)
p-values
HR (95% CI)
p-values





Age (years)
1.006 (0.969-1.045)
0.743
0.998 (0.962-1.036)
0.922


Gender (male vs female)
0.597 (0.272-1.313)
0.200
0.780 (0.342-1.776)
0.554


Grade (grade2 vs grade3)
0.955 (0.484-1.884)
0.894
1.462 (0.649-3.293)
0.360


Stage (advanced vs metastatic)
2.762 (1.332-5.771)
0.007
2.157 (0.948-4.906)
0.067


Prior radical cystectomy (no vs yes)
0.378 (0.190-0.751)
0.005
0.533 (0.233-1.217)
0.135


combined gene signature (good vs poor)
 6.862 (2.088-22.553)
0.002
 5.380 (1.570-18.436)
0.007









The good-prognostic signature group had a significantly longer cancer-specific survival time than the poor-prognostic signature group (p=0.010) (FIG. 2b). However, the overall survival time was only marginally enhanced in the good-prognostic signature group (p=0.072) (FIG. 2a).
Immunohistochemical Staining of S100A9 and EGFR in Bladder Cancer
To expand the present inventors' findings suggesting that S100A9/EGFR may be a novel diagnostic marker for MIBC with local recurrence or metastasis after chemotherapy, the inventors of the present invention assessed the value of S100A9/EGFR protein as a prediction marker. The protein expression levels of the S100A9 and EGFR protein were assessed in 38 bladder cancer samples by immunohistochemical analysis. Various intensities (from negative to strong) of S100A9 and EGFR were observed in cancer tissues (FIG. 3a to FIG. 3f). S100A9 were detected in cytoplasm as well as nucleus (FIG. 3a), while EGFR was detected in cytoplasm, nucleus and plasma membrane (FIG. 3c). EGFR positivity was marginally associated with disease progression after chemotherapy in our MIBC patients (p=0.106). However, S100A9 positivity was strongly correlated to disease progression (p=0.047). 65% (15 among 23) of bladder cancer patients with progression after chemotherapy exhibited high S100A9 levels, while only 26% (4 among 15) of patients without progression showed high intensities (Table 4). Furthermore, the combined approach of S100A9 and EGFR were much significantly associated with progression (p=0.018). 73.9% (17 among 23) patients with progression showed high S100A9/EGFR staining, and no patient has negative staining of S100A9/EGFR (Table 4). Notably, we found that protein expression intensities of S100A9 and EGFR showed good correlation with mRNA levels of S100A9 and EGFR (r=0.395, p=0.014 and r=0.453, p=0.004).











TABLE 4







Disease
Disease




progression (+)
progression (−)



after chemotherapy
after chemotherapy



N = 23 (%)
N = 15 (%)
p-value



















EGFR staining


0.106


High
16 (76.2) 
5 (23.8)


Low
4 (33.3)
6 (66.7)


Negative
3 (42.9)
4 (57.1)


S100A9 staining


0.047


High
15 (78.9) 
4 (21.1)


Low
6 (50.0)
6 (50.0)


Negative
2 (28.6)
5 (71.4)


Combined


0.018


(EGFR + S100A9)


High†
17 (77.3) 
5 (22.7)


Intermediate‡
6 (42.9)
8 (57.1)


Negative
0 (0)  
2 (100) 





High†: high + high and high + low


Intermediate‡: high + negative, low + low and low + negative






Hyper-Proliferation Caused by S100A9 Overexpression and Cisplatin-Induced Apoptosis
Two independent expression analyses (based on qRT-PCR and IHC) performed by the inventors of the present invention suggest S100A9/EGFR as a novel prognostic marker in bladder cancer for disease progression after cisplatin-based chemotherapy. To assess the hypothesis that S100A9 and EGFR have important function in chemo-resistance, we have attempted in vitro functional analysis to test whether the altered gene expression of S100A9 or EGFR regulates chemo-sensitivity to cisplatin treatment. Data from previous papers from the inventors of the present invention and other laboratories suggested that S100A9 may play an essential role during bladder cancer progression. Recent proteomics analysis revealed that a protein level of S100A9 is correlated to bladder tumor grade (p<0.05). S100A9 was also reported by the inventors of the present invention as one of four-gene signature of diagnostic markers in MIBC. To uncover whether S100A9 has a functional link to chemo-sensitivity of bladder cancer and to test this hypothesis in vitro, T24 cells were transfected with S100A9 overexpressing construct or control, and it was found that S100A9 involves in migration and proliferation of bladder cancer cells (B and C of FIG. 4). As shown in B of FIG. 4, the wound-healing assay revealed that transfected bladder tumor cells with S100A9 construct moved faster and filled the path earlier than control cells. Enforced S100A9 significantly enhanced proliferation rate of T24 cells in growth medium, compared to control cells (Ctrl, transfected T24 cells with a vector only). The increased S100A9 level allowed T24 cells more viable in the presence of cisplatin. Cell viability of control cells was reduced to about 20% after 2 days treatment with 10 μM cisplatin. In contrast, S100A9 expressing cells showed about 60% of viable cells by same treatment (D of FIG. 4). Increased expression level of S100A9 was confirmed by western blot analysis (A of FIG. 4).
Change in Response to Cisplatin-Induced Apoptosis Depending on EGFR Expression Level
The inventors of the present invention performed gain- and loss-of functional studies in T24 bladder cancer cells to assess the biological role of EGFR. Overexpression of EGFR enhanced cell viability in the presence of 10 μM cisplatin in serum free medium. Control T24 cells transfected with empty vector showed 50% viability after 18 h-cisplatin treatment, while EGFR overexpressing cells did not show significant apoptosis until 24 h-treatment (B of FIG. 5). Knockdown of EGFR using RNAi sensitizes T24 cells to cisplatin-induced apoptosis (D of FIG. 5). Control RNAi (siCtrl) showed about 80% of cell viability 6 hours after cisplatin treatment, while two sets of EGFR knockdown (siEGFR-1 and siEGFR-2) both displayed only 30 to 40% of viability of controls (D of FIG. 5). Protein levels after overexpression or knockdown of EGFR were confirmed by western blot analysis (A and C of FIG. 5).
Synergistic Effect on Sensitization of T24 Bladder Cancer Cells to Cisplatin-Induced Apoptosis Caused by Inhibition of Both S100A9 and EGFR
Experimental data in FIG. 6 suggest that S100A9 and EGFR play roles in response to a chemotherapeutic reagent, cisplatin, in bladder cancer cells. The inventors of the present invention tested whether downregulation of EGFR and S100A9 alters levels of cisplatin-induced apoptosis. EGFR kinase activity was downregulated by Iressa (ZD1839, gefitinib), an EGFR kinase inhibitor. S100A9 was silenced using RNAi approach. Control siRNA was used for a control for non-target effect by siRNA. EGFR inhibition (with Iressa) significantly reduced viability, and enhanced chemo-sensitivity to cisplatin (FIG. 6, line 2). Moreover, combined inhibition of S100A9 and EGFR synergistically enhanced chemo-sensitivity of T24 bladder cancer cells (FIG. 6, line 4), suggesting the potential therapeutic strategy overcoming the chemo-resistance, which is often observed during cisplatin-based chemotherapy on bladder cancer patients.
While the present invention has been shown and described with reference to preferable Examples thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims. Therefore, the disclosed Examples should not be considered in view of explanation, but no limitation. The technical scope of the present invention is taught in the claims, but not the detailed description, and all the differences in the equivalent scope thereof should be construed as falling within the present invention.

## Claims

1. A method for treating muscle invasive bladder cancer (MIBC) comprising administering to a subject in need thereof a therapeutically effective amount of antibodies against S100A9 and EGFR, and cisplatin.