Clinical Cancer Research Research Funding Stand Up to Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakayama, K.
Right arrow Articles by Takebayashi, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakayama, K.
Right arrow Articles by Takebayashi, Y.
Clinical Cancer Research Vol. 10, 2804-2811, April 15, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Prognostic Value of the Cu-Transporting ATPase in Ovarian Carcinoma Patients Receiving Cisplatin-Based Chemotherapy

Kentaro Nakayama2, Atsuko Kanzaki1, Kunihiko Terada4, Masato Mutoh5, Kenji Ogawa6, Toshihiro Sugiyama4, Seiichi Takenoshita1, Kiyoshi Itoh6, Nobuo Yaegashi6, Kohji Miyazaki2, Nouri Neamati3 and Yuji Takebayashi1

1 Department of Surgery II, Institute of Biomedical Sciences Fukushima Medical, University School of Medicine, Fukushima, Japan; 2 Department of Obstetrics and Gynecology, Shimane Medical University, Izumo, Japan; 3 Department of Pharmaceutical Sciences, University of Southern California, School of Pharmacy, Los Angeles, California; 4 Department of Biochemistry, Akita University School of Medicine, Akita, Japan; 5 Pharmaceutical Research Laboratories, Toray Industries, Kamakura, Kanagawa, Japan; and 6 Department of Obstetrics and Gynecology, Tokohu University, Sendai, Miyagi, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: A major obstacle in the treatment of ovarian carcinoma is the intrinsic/acquired resistance to cisplatin-based chemotherapy. Cu-transporting ATPase (ATP7B) has been reported to be associated with cisplatin resistance in vitro. However, the clinical significance of this transporter has not previously been addressed. Our goal was to investigate ATP7B expression in ovarian carcinoma and whether its expression correlates with prognosis and reduced responsiveness to cisplatin treatment.

Experimental Design: We retrospectively examined the expression of ATP7B and p53 in primary ovarian carcinoma and its association with chemotherapeutic effect. Tissues were surgically removed from 104 ovarian carcinomas patients who received cisplatin-based chemotherapy. We performed immunohistochemical analysis of ATP7B and p53 using a monoclonal antibody against ATP7B and DO7 antibody against p53 protein in 104 ovarian carcinomas and adjacent nonneoplastic tissues. The significance of ATP7B and p53 in the prognosis of patients with ovarian carcinomas was also examined in the survival analysis of mortality follow-up data covering the period between 1988 and 2001. Furthermore, mutation analysis at the six Cu-binding domain and ATP-binding domain, which may be important for cisplatin transport, were performed using single-strand conformational polymorphism after reverse transcriptase-PCR.

Results: A variable degree of cytoplasmic staining of ATP7B in tumor cells was observed in 34.6% (36 of 104 cases) of the analyzed carcinomas. ATP7B expression was not observed in adjacent nonneoplastic tissues. ATP7B positivity in poorly/moderately differentiated carcinoma was significantly higher than that in low malignant potential tumor/well-differentiated carcinoma (P = 0.0276). Patients with ATP7B-positive tumors had a significantly inferior response to chemotherapy compared with the patients with ATP7B-negative tumors (P = 0.025). The multivariate Cox regression analysis revealed that ATP7B expression (hazard ratio, 1.8; 95% confidence interval, 1.0–3.2, P = 0.048), as well as International Federation of Gynecologists and Obstetricians stage (hazard ratio, 2.0; 95% confidence interval, 1.1–3.6, P = 0.018), was prognostic for poor disease outcome after adjustment for p53 expression, grade, and residual tumor. p53 expression was detected in 31.5% (26/104 cases). No mutation was observed on the six Cu-binding domain or ATP-binding domain in human ovarian carcinomas expressing ATP7B gene.

Conclusions: This study demonstrates that overexpression of ATP7B in ovarian carcinoma is correlated with unfavorable clinical outcome in patients treated with cisplatin-based chemotherapy. Therefore, ATP7B expression may be considered as a predictive marker of chemoresistance for cisplatin-based chemotherapy in patients with ovarian carcinoma. We further predict that drugs targeting ATP7B might be useful in combination with cisplatin-based regimen for the improvement of patients with ovarian carcinoma.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian carcinoma is the most lethal gynecological malignancy (1) . The incidence and mortality of ovarian carcinoma has not declined in the past decade. This is due to diagnosis during the later stages of this disease and the lack of effective therapy (2) . The treatment with cisplatin-based chemotherapy after reductive surgery has improved prognosis of patients with ovarian carcinoma; however, the complete pathological response and 5-year survival rates have not improved. One of the most important clinical problems in the treatment of ovarian carcinoma is the intrinsic/acquired resistance to cisplatin-based chemotherapy. Knowledge of the active mechanism of drug resistance may lead to new treatment strategies and allows the selection of those patients for specific treatment modalities.

In vitro resistance to cisplatin includes decreased drug accumulation, enhanced detoxification, and increased DNA repair efficiency. Multidrug resistance (MDR) has been noted as an important mechanism of drug resistance. Several genes, including MDR1, MRP1 and LRP, have been identified (3 , 4) . MDR1 and MRP1 function as a drug efflux pump and are classified as ABC transporter gene family (5 , 6) and are expressed in both human solid tumors and hematological malignancies (7 , 8) . The 110-kDa LRP, the major vault protein, is frequently overexpressed in MDR cells and has an important role(s) in transport of drugs from nuclei to cytoplasm and confers to MDR in vitro (4) . Recently, BCRP (MXR/ABCP) gene, another member of the ABC transporter family, has been described in breast, colon, gastric, and fibrosarcoma cell lines (3 , 4 , 9) . However, there is no evidence that these molecules are involved in cisplatin resistance in vitro and in clinic.

Previously, it was shown that ATP7B is associated with cisplatin resistance in vitro (10) . The ATP7B gene was induced by exposure to cisplatin in human prostate cells and the ATP7B-transfected cells showed marked increased in cisplatin efflux (10) . Although an active efflux pump for cisplatin has yet to be identified, it is likely that ATP7B may function to efflux cisplatin from some carcinoma cells.

ATP7B is a member of a class of heavy metal-transporting P-type ATPases that pump copper, cadmium, zinc, silver, or lead (11, 12, 13) . Copper is an essential trace element and is integrated into many enzymatic reactions. Excess level of copper is transported to the extracellular environment by an energy-dependent system (8) , and alteration in copper homeostasis can cause severe problems. For example, Wilson disease, an autosomal recessive disease of copper transport, is characterized by chronic liver and/or neurological disorder, sometimes accompanied by kidney damage (14) . Detailed understanding of ATP7B is therefore crucial in several diseases, including cancer. The fact that such transporters can also transport small molecule drug is intriguing and could potentially have a significant value in the clinic.

The ATP7B gene is highly conserved across species and contains several motifs such as TGEA/S motif (phosphatase domain), DKTGT/S motif (phosphorylation domain), TGDN motif (ATP-binding domain), and the sequence MXGDGXNDXP that connects the ATP binding domain to the transmembrane segment. The six repeated motifs, GMTCXXC located at the NH2 terminus binds six molecules of copper. Human ATP7B protein is able to rescue the yeast strains lacking the CCC2 gene, the yeast homologue of ATP7B. The experiments with the wild-type or mutated ATP7B cDNA demonstrated that the mutant containing only the sixth Cu-binding domain are sufficient to rescue {Delta}ccc2 (15) . This suggests the importance of the sixth Cu-binding for ATP7B function. Therefore, the sixth Cu-binding domain may be important for the transport of cisplatin. However, no mutations at the sixth Cu-binding in human ovarian carcinoma have previously been documented.

Because of the importance of p53 function in apoptosis and cell proliferation (see below) and its role in predicting drug efficacy in various cancers, we extended our studies to include the analysis of p53 gene expression in the same samples.

It is now well established that p53 plays important roles in the regulation of the cell cycle and has been implicated in cell differentiation, apoptosis, DNA synthesis, and repair (16) . Mutations in p53 gene has been observed in {cong}50% of ovarian carcinoma cases (17 , 18) .

Overexpression of p53 corresponds to a cellular accumulation of a biologically inactive protein stabilized either by a decreased rate of degradation of mutated gene product or by complex formation with certain proteins such as viral oncoproteins or heat-shock protein 70 (19 , 20) . Recently, particularly in ovarian carcinomas, special attention was attributed to p53 because tumors bearing p53 aberrations were reported to be less sensitive to cisplatin and doxorubicin exposure (21 , 22) . p53 is one of the most important molecule in the cytotoxicity of DNA-damaging agents (23) . p53 and mismatch repair can cooperate to control sensitivity to the cytotoxic effect of cisplatin and to limit its mutagenic potential in the colon cancer cells (24) . Although some investigators failed to demonstrate a relation between the course of the disease and p53 expression (17 , 25) , the results obtained in a number of studies on the prognostic impact of overexpression of p53 protein in ovarian carcinoma tissue (26, 27, 28) .

The purpose of this study was to provide clinical evidence that ATP7B expression is important for cisplatin resistance. This is the first study suggesting that ATP7B expression should be considered as a predictive marker of chemoresistance to cisplatin in patients with ovarian carcinoma. Thus, in conjunction with the previously identified markers for cisplatin resistance, inclusion of ATP7B expression is of beneficial in selecting the right patient for the right treatment.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Samples.
After obtaining informed consent in accordance with each institutional guideline, 104 tumor specimens were collected and embedded in OCT compound (Sakura Fine technical Co., Ltd., Tokyo, Japan). Diagnosis was based on conventional morphological examination of the sections stained with H&E staining, and tumors were classified according to the WHO classification. Staging of tumor was performed according to the International Federation of Gynecology and Obstetrics (FIGO) classification. The clinicopathological characteristics of the patients included in this study are summarized in Table 1Citation . The patients had no other form of malignancies and performance status with grade 0 or 1. All patients were primarily treated with reductive surgery and postoperative chemotherapy, which basically consisted of a cisplatin-based regimen [cisplatin (75 mg/m2), doxorubicin (30 mg/m2), and cyclophosphamide (500 mg/m2)]. All of the cases performed 4–6 course cisplatin regimens. Response to induction chemotherapy was assessed by second-look surgery or clinical and/or radiographic evaluation according to WHO criteria after chemotherapy. Patients with incomplete response to induction chemotherapy and patients with recurrent tumors were treated with a variety of second-line chemotherapy regimens. Follow-up for all of the patients included in the survival analysis was updated in June 1, 2002 (median follow-up was 51 months; range, 26–165 months). At that time, 27 patients had died of ovarian carcinoma and 77 were alive.


View this table:
[in this window]
[in a new window]

  		Table 1 Relationship of ATP7B or p53 expressions and clinicopathological variables in patients with ovarian carcinoma (n = 104)

 
Tissue Staining and Evaluation of Stained Sections.
A 2.5-µm section of each submitted frozen block was first stained with H&E to verify the histopathological diagnosis and the quality of fixation for immunohistochemical analysis. Immunostaining was performed on cryostat sections using the standard immunoperoxidase procedure (Vectastain Elite ABC kit; Vector, Burlingame, CA). After recovering from OCT compound (Sakura Fine Technical Co., Ltd.), the sections were fixed in 10% neutral buffered formalin, incubated in 0.03% H2O2 in absolute methanol for 30 min at room temperature, and blocked in 3% skim milk in PBS for 30 min at room temperature. The sections were then incubated with 100-fold diluted monoclonal antibody against the NH2-terminal region of ATP7B, which included the six Cu-binding domains (amino acid residues 21–623; Ref. 29 ) for 15 h at 4°C. After rinsing with PBS, the sections were incubated in biotinylated horse antimouse IgG at 1:200 in 1.5% normal horse serum for 30 min at room temperature. Sections were then rinsed in PBS and incubated for 30 min at room temperature in the avidin-biotin horseradish peroxidase macromolecular complex. After rinsing with PBS, the sections were incubated for 6 min in 0.05% diaminobenzidine in PBS with 0.03% H2O2. The slides were counterstained with hematoxylin, dehydrated, and mounted. The serial sections were routinely incubated with irrelevant mouse IgG as a negative control.

The slides were examined and scored independently by two observers (K. Nakayama and Y. Takebayashi) without knowledge of clinical information of the patients in the evaluation of ATP7B expression. If >10% of the tumor cells were stained, the samples were considered to be ATP7B-positive carcinomas. The 10% cutoff level was chosen for the following reasons: (a) 10% positive cells were considered the lowest level of expression that could be most consistently detected in cryostat sections; and (b) Chan et al. (30) demonstrated that a small percentage of cells positive for multidrug-resistance-related proteins (i.e., P-glycoprotein) could have clinical significance. The {kappa} test of reliability between the two observers was 0.750, indicating fair to good agreement for the evaluation of ATP7B and p53 expression in ovarian carcinoma (data not shown). When the two observers’ report differed from each other, they would evaluate the images of stained sections on a TV-captured image station. Finally, the evaluation of p53 expression was performed essentially as described previously (31) .

Reverse Transcriptase-PCR.
We examined mutation after analyzed ATP7B mRNA expression level using reverse transcriptase-PCR. Of the total of 104 patients, we could only obtain RNA from 82 patients because of tissue availability.

Total RNA was prepared by Trizol (Life Technologies, Inc., Gaithersburg, MD) from the frozen block after observing whether the tissues contain the carcinoma cells. The tissues containing at least 80% of carcinoma cells were used in this study. cDNA was synthesized with 3 µg of total RNA and random hexadeoxynucleotide primer (Life Technologies, Inc.) in 20 µl of a solution containing reverse transcriptase. After synthesis the cDNA was diluted 1:4 with water and stored at –20°C until use. PCR was performed with cDNA derived from 30 ng of RNA. PCR reactions were carried out in a total volume of 25 µl containing cDNA, dGTP, dATP, dTTP, and dCTP at a concentration of 200 µM, 4 µM of each primer, and 0.25 unit of ExTaq polymerase (Takara Shuzo, Otsu, Shiga, Japan). The PCR cycling parameters were as follows: 10 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C, and a final cycle at 72°C for 10 min. The PCR products were electrophoresed on 1.5%-agarose gel. The PCR primer sequences of ATP7B for ATP-/sixth copper-binding domains were shown in Fig. 4BCitation .



View larger version (36K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. A, sequence of ATP7B gene at ATP-/sixth Cu-binding domain. B, primers to detect PCR products including sixth Cu-binding and ATP-binding domain. C, expression of ATP7B gene determined by reverse transcriptase-PCR, as described in "Materials and Methods." Cu; PCR product including sixth Cu-binding domain. ATP; PCR product including ATP-binding domain. Mutation analysis of ATP7B gene. D, single-strand conformational polymorphism analysis (left panel); sixth Cu-binding domain (right panel); ATP binding domain. E, sequence analysis (left panel); sixth Cu-binding domain, (right panel); ATP binding domain.

 
Single-Strand Conformational Polymorphism and Mutation Analysis of PCR Products.
PCR single-strand conformational polymorphism assays were performed as described. PCR amplification was performed as described above. PCR products were loaded onto a 12% polyacrylamide gel containing 8% glycerol. Electrophoresis was performed at 15°C for 8 h at 100 V. After electrophoresis, gels were stained by silver staining kit (Bio-Rad, Hercules, CA). Sequence analysis of a fraction of each PCR product was performed by 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Statistical Analysis.
Data analysis was performed using Statview version 5 statistical software package. Continuous variables were analyzed using Student t test and {chi}2 test as appropriate. Overall survivals were measured in months from the month of surgery to a reported death. Survival curves were determined using Kaplan-Meier method, and differences in survival between subgroups were compared with log-rank test. Multivariate prognostic analysis was performed using Cox proportional hazards model. Ps < 0.05 were hypothesized to be significant. All reported Ps were two-sided.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of ATP7B Protein in Human Ovarian Carcinoma.
A total of 104 primary ovarian carcinoma tissues was used for the detection of ATP7B protein by immunohistochemistry using an antibody that specifically reacts with ATP7B as evidenced by immunoblotting analysis (32) . A granular staining of ATP7B was observed in the cytoplasm of ovarian carcinoma cells (Fig. 1A)Citation . ATP7B-negative tumor stained with anti-ATP7B monoclonal antibody (Fig. 1B)Citation . Although, a weak to moderate cytoplasmic staining could be observed in some stromal cells (Fig. 1C)Citation , in the normal ovary, the ATP7B expression was not detectable (Fig. 1D)Citation . The immunostaining results are summarized in Fig. 1ECitation . A variable degree of cytoplasmic staining of tumor cells was observed in 35.6% (37 of 104 cases) of the analyzed tumors.



View larger version (125K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Immunohistochemical staining of ovarian carcinoma specimens using antibodies to ATP7B from cryostat sections. A, ATP7B-positive tumor stained with anti-ATP7B monoclonal antibody. Note the distinct cytoplasmic staining in ovarian carcinoma cells (immunoperoxidase stain; original magnification, x200). B, ATP7B-negative tumor stained with anti-ATP7B monoclonal antibody (magnification, x200). C, ATP7B-positive normal ovarian stromal tissue stained with anti-ATP7B monoclonal antibody (immunoperoxidase stain; original magnification, x400). D, normal ovarian tissue stained with anti-ATP7B monoclonal antibody (immunoperoxidase stain; original magnification, x400). E, percentage of cells expressing ATP7B protein in ovarian carcinoma.

 
Expression of p53 Protein in Human Ovarian Carcinoma.
The central role of p53 in cell growth and differentiation as well as apoptosis and efficacy of drugs in vivo prompted us to investigate its expression in the same ovarian carcinoma tissues as above. As expected, the p53 staining was observed only in the nuclei of carcinoma cells (Fig. 2A)Citation . Of the analyzed tumors, we observed 25.0% (26 of 104) of cases as p53 positive (Fig. 2B)Citation .



View larger version (68K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. A, immunohistochemical staining of ovarian carcinoma specimens using antibodies against p53. (immunoperoxidase stain; original magnification, x100). B, percentage of cells expressing p53 protein in ovarian carcinoma.

 
Relationship between Clinicopathologic Findings, Chemotherapeutic Response, and ATP7B and p53 Expression.
Table 1Citation summarizes the relationship between clinicopathological features and ATP7B and p53 expression in ovarian carcinomas. No significant association was found between ATP7B expression and age, FIGO stage, and histological subtype (Table 1)Citation . ATP7B positivity was correlated with tumor grade. ATP7B positivity in poorly/moderately differentiated carcinoma was significantly higher than that in low malignant potential tumor/well-differentiated carcinoma (Table 1, PCitation = 0.0276). Interestingly, the p53 expression was independent of all of the examined clinicopathological variables (Table 1)Citation .

Patients with ATP7B-positive tumors had a significantly inferior response to chemotherapy (59% complete and partial response and 41% no response) as compared with the patients with ATP7B-negative tumors (80% complete and partial response and 20% no response; Table 2Citation ; P = 0.025). No significant association was found between p53 expression and response to cisplatin-based chemotherapy (Table 2Citation ; P = 0.933).


View this table:
[in this window]
[in a new window]

  		Table 2 The relationship between ATP7B and p53 expression and cisplatin-based chemotherapeutic response

 
Prognostic Relevance of ATP7B Expression.
Kaplan-Meier estimate of overall survival excluding low malignant potential tumor tumors is plotted in Fig. 3Citation . The patients with ATP7B-positive carcinomas had a significantly poorer overall survival than those with ATP7B-negative tumors (P = 0.0161; Fig. 3ACitation ). The median overall survival of ATP7B-positive or negative carcinomas are 33 or 65 months. p53 expression was also prognostic indicator by Kaplan-Meier curve analysis (P = 0.0073; Fig. 3BCitation ). To determine whether ATP7B and/or p53 positivity were a prognostic factor(s) independent of FIGO stage, an established prognostic factor, we conducted an overall survival analysis with the use of the Cox proportional hazards model. Although it was unlikely that the other clinicopathological variables shown in Table 1Citation affected patient survival, we included them in our analysis to confirm the absence of their associations with survival. Univariate analysis revealed that FIGO stage, grade, residual tumor, and ATP7B and p53 positivity significantly affected on the survival of the patients with ovarian carcinomas (Table 3)Citation . Therefore, we performed multivariate analysis with FIGO stage, grade, residual tumor, p53 positivity, and ATP7B positivity, and the results are shown in Table 3Citation . FIGO stage and ATP7B positivity were independent prognostic factors (P = 0.0184 and 0.0477, respectively) in patients with ovarian carcinoma treated with cisplatin-based chemotherapy.



View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Kaplan-Meier overall survival curves of patients with ovarian carcinoma. A, comparison of overall survival curves for patients whose tumors stained positive for ATP7B with those patients whose tumors were classified as negative for ATP7B expression. B, comparison of overall survival curves for patients whose tumors stained positive for p53 with those patients whose tumors were classified as negative for p53 expression. Circles represent the results for all patients. P was conducted by log-rank test.

 

View this table:
[in this window]
[in a new window]

  		Table 3 Univariate and multivariate Cox proportional hazard analysis for overall survival of 98 patients with ovarian carcinoma treated with cisplatin-based chemotherapy

 
Mutation Analysis of ATP7B Gene at ATP-/Sixth Cu-Binding Domain in Ovarian Carcinomas.
Mutation analysis of ATP7B gene at ATP-binding motif and the sixth Cu-binding domain were performed and shown in Fig. 4, A and BCitation , respectively. The PCR products from the ATP-binding motif and the sixth Cu-binding domain (Fig. 4C)Citation were subjected to single-strand conformational polymorphism analysis. PCR products from cDNA of normal placenta were used as control. No case with mutation of ATP7B gene at ATP-/sixth Cu-binding domain was observed by single-strand conformational polymorphism analysis (Fig. 4D)Citation . To confirm these results, direct sequence analysis of these PCR products from 10 ovarian carcinomas, which were selected randomly, was performed. Again we did not find any cases with mutation by sequence analysis (Fig. 4E)Citation .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The observation that ATP7B is associated with cisplatin resistance in vitro as reported by Komatsu et al. (10) has now been confirmed in tissues from ovarian carcinoma patients treated with cisplatin-based chemotherapy. Herein, we demonstrated that ATP7B protein is expressed in human ovarian carcinoma as assessed by immunohistochemistry (Fig. 1)Citation . A significant ATP7B protein expression 35.6% (37 of 104 cases) was observed in ovarian carcinoma (Fig. 1E)Citation . ATP7B immunoreactivity was detected as characteristic granular cytoplasmic staining. In agreement with this observation, ATP7B has been reported to be abundant in the Golgi apparatus (9) . These findings are the first evidence that ATP7B, which is involved in Wilson disease, is expressed in human ovarian carcinoma.

The present study demonstrates that the ATP7B expression in poorly differentiated ovarian carcinoma is more frequent than well to moderately differentiated carcinoma (Table 1)Citation . Very low expression of ATP7B protein could be detected in adjacent nonneoplastic tissues. The fact that the expression level of ATP7B is very low in normal ovarian tissue raises the possibility that ATP7B might be involved in transformation of a normal cell to a malignant tumor cell and/or differentiation of carcinoma cell.

Patients with ATP7B-positive tumors had a significantly inferior response to chemotherapy as compared with patients with ATP7B-negative tumors (Table 2)Citation . The fact that ATP7B expression assessed by immunohistochemistry was an independent prognostic factor in ovarian carcinoma attests to the diagnostic value as a marker for prediction of response to cisplatin-based chemotherapy. However, this study had one limitation that the patients were treated with combination regimen, including cisplatin, doxorubicin, and cyclophosphamide. Therefore, it is not entirely sure that the effect of ATP7B on cisplatin plays the primary role in this study. To explore this limitation, additional studies with analyzing ATP7B function for other anticancer agents such as doxorubicin and cyclophosphamide should be required.

Because of its central role in a variety of cellular mechanisms, analysis of p53 status in our sample was of paramount importance. In fact, it was shown previously that p53 is a very important molecule in the activity of DNA-damaging agents, including cisplatin (23) . However, the results obtained in a number of studies on the prognostic impact of overexpression of p53 protein in ovarian carcinoma tissue were controversial. Whereas some investigators reported impaired clinical outcome in patients with p53 overexpressing tumors (22 , 27 , 28) , others failed to demonstrate a relation between the course of the disease and p53 expression (17) . Actually, p53 expression was a prognostic indicator in univariate analysis but was not in multivariate analysis. This suggests that the detection of p53 by immunohistochemistry may be difficult in clinical samples. Because unlike p53 missense mutations, which result in a stable mutant protein detected by immunohistochemistry, other mutations such as deletions, additions, or splice site mutations do not result in a stable protein (33 , 34) .

The sixth Cu-binding domain is very important for ATP7B function (15) , and by extension, it might also be important for cisplatin transport. We were interested to see whether there are any significant polymorphisms among patients in the regions highly critical for ATP7B function and possibly cisplatin efflux. When we looked for mutations in either ATP-binding motif or the sixth copper-binding domain, we did not observe any (Fig. 4, D and E)Citation .

In conclusion, we demonstrated that ATP7B, a transporter associated with chemoresistance, is expressed in a subset of ovarian carcinomas. Of especial interest is the finding that the expression is more frequent in undifferentiated carcinomas that are usually more refractory to therapy. Furthermore, ATP7B expression is an independent prognostic factor in human ovarian carcinoma treated with cisplatin-based chemotherapy. Therefore, ATP7B expression may be considered as a predictive marker of chemoresistance for cisplatin-based regimen in patients with ovarian carcinoma. We further predict that drugs targeting ATP7B might be useful in combination with cisplatin-based chemotherapy for the improvement of patients with ovarian carcinoma.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Yuji Takebayahshi, Department of Surgery II, Institute of Biomedical Sciences Fukushima Medical, University School of Medicine, Fukushima 960-3249, Japan. Phone: 81-24-547-1259; Fax: 81-24-548-3249; E-mail: tyuji{at}fmu.ac.jp

Received 10/23/03; revised 12/24/03; accepted 1/ 7/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wingo PA, Tong T, Bolden S. Cancer statistics, 1995. CA Cancer J Clin, 45: 8-30, 1995.[Abstract/Free Full Text]
  2. Boente MP, Schilder R, Ozols RF. Gynecologic cancers. Cancer Chemother Biol Response Modif, 17: 536-61, 1997.[Medline]
  3. Miyake K, Mickley L, Litman T, et al Molecular cloning of cDNAs which are highly overexpressed in mitoxantrone-resistant cells: demonstration of homology to ABC transport genes. Cancer Res, 59: 8-13, 1999.[Abstract/Free Full Text]
  4. Doyle LA, Yang W, Abruzzo LV, et al A multidrug resistance transporter from human MCF-7 breast cancer cells[published erratum appears in Proc Natl Acad Sci USA 1999;96(5):2569]. Proc Natl Acad Sci USA, 95: 15665-70, 1998.[Abstract/Free Full Text]
  5. Allen JD, Brinkhuis RF, Wijnholds J, et al The mouse Bcrp1/Mxr/Abcp gene: amplification and overexpression in cell lines selected for resistance to topotecan, mitoxantrone, or doxorubicin. Cancer Res, 59: 4237-41, 1999.[Abstract/Free Full Text]
  6. Volk EL, Rohde K, Rhee M, et al Methotrexate cross-resistance in a mitoxantrone-selected multidrug-resistant MCF7 breast cancer cell line is attributable to enhanced energy-dependent drug efflux[in process citation]. Cancer Res, 60: 3514-21, 2000.[Abstract/Free Full Text]
  7. Brangi M, Litman T, Ciotti M, et al Camptothecin resistance: role of the ATP-binding cassette (ABC), mitoxantrone-resistance half-transporter (MXR), and potential for glucuronidation in MXR-expressing cells. Cancer Res, 59: 5938-46, 1999.[Abstract/Free Full Text]
  8. Harris ED, Quian Y, Tiffany-Castiglioni E, Lacy AR, Reddy MC. Functional analysis of copper homeostasis in cell culture models: a new perspective on internal copper transport. Am J Clin Nutr, 67 (Suppl): 988-95S, 1998.
  9. Petrukhin K, Lutsenko S, Chernov I, et al Characterization of the Wilson disease gene encoding a P-type copper transporting ATPase: genomic organization, alternative splicing, and structure/function predictions. Hum Mol Genet, 3: 1647-56, 1994.[Abstract/Free Full Text]
  10. Komatsu M, Sumizawa T, Mutoh M, et al Copper-transporting P-type adenosine triphosphatase (ATP7B) is associated with cisplatin resistance. Cancer Res, 60: 1312-6, 2000.[Abstract/Free Full Text]
  11. Terada K, Schilsky ML, Miura N, et al ATP7B (WND) protein. Int J Biochem Cell Biol, 30: 1063-7, 1998.[CrossRef][Medline]
  12. Solioz M, Vulpe C. CPx-type ATPases: a class of P-type ATPases that pump heavy metals. Trends Biochem Sci, 21: 237-41, 1996.[CrossRef][Medline]
  13. Gupta A, Matsui K, Lo JF, et al Molecular basis for resistance to silver cations in Salmonella. Nat Med, 5: 183-8, 1999.[CrossRef][Medline]
  14. Roberts EA, Cox DW Wilson disease. Baillieres Clin Gastroenterol, 12: 237-56, 1998.[CrossRef]
  15. Iida M, Terada K, Sambongi Y, et al Analysis of functional domains of Wilson disease protein (ATP7B) in Saccharomyces cerevisiae. FEBS Lett, 428: 281-5, 1998.[CrossRef][Medline]
  16. Linzer DI, Levine AJ. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell, 17: 43-52, 1979.[CrossRef][Medline]
  17. Marks JR, Davidoff AM, Kerns BJ, et al Overexpression and mutation of p53 in epithelial ovarian cancer. Cancer Res, 51: 2979-84, 1991.[Abstract/Free Full Text]
  18. Goff BA, Shy K, Greer BE, et al Overexpression and relationships of HER-2/neu, epidermal growth factor receptor, p53, Ki-67, and tumor necrosis factor {alpha} in epithelial ovarian cancer. Eur J Gynaecol Oncol, 17: 487-92, 1996.[Medline]
  19. Kupryjanczyk J, Thor AD, Beauchamp R, et al p53 gene mutations and protein accumulation in human ovarian cancer. Proc Natl Acad Sci USA, 90: 4961-5, 1993.[Abstract/Free Full Text]
  20. Momand J, Zambetti GP, Olson DC, et al The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell, 69: 1237-45, 1992.[CrossRef][Medline]
  21. Vasey PA, Jones NA, Jenkins S, et al Cisplatin, camptothecin, and Taxol sensitivities of cells with p53-associated multidrug resistance. Mol Pharmacol, 50: 1536-40, 1996.[Abstract]
  22. Buttitta F, Marchetti A, Gadducci A, et al p53 alterations are predictive of chemoresistance and aggressiveness in ovarian carcinomas: a molecular and immunohistochemical study. Br J Cancer, 75: 230-5, 1997.[Medline]
  23. O’Connor PM, Jackman J, Bae I, et al Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth inhibitory potency of 123 anticancer agents. Cancer Res, 57: 4285-300, 1997.[Abstract/Free Full Text]
  24. Lin X, Ramamurthi K, Mishima M, et al P53 modulates the effect of loss of DNA mismatch repair on the sensitivity of human colon cancer cells to the cytotoxic and mutagenic effects of cisplatin. Cancer Res, 61: 1508-16, 2001.[Abstract/Free Full Text]
  25. Green JA, Robertson LJ, Campbell IR, et al Expression of the p53 gene and presence of serum autoantibodies in ovarian cancer: correlation with differentiation. Cancer Detect Prev, 19: 151-5, 1995.[Medline]
  26. Diebold J, Baretton G, Felchner M, et al Bcl-2 expression, p53 accumulation, and apoptosis in ovarian carcinomas. Am J Clin Pathol, 105: 341-9, 1996.[Medline]
  27. Klemi PJ, Pylkkanen L, Kiilholma P, et al p53 protein detected by immunohistochemistry as a prognostic factor in patients with epithelial ovarian carcinoma. Cancer, 76: 1201-8, 1995.[CrossRef][Medline]
  28. Henriksen R, Strang P, Wilander E, et al p53 expression in epithelial ovarian neoplasms: relationship to clinical and pathological parameters, Ki-67 expression and flow cytometry. Gynecol Oncol, 53: 301-6, 1994.[CrossRef][Medline]
  29. Yang XL, Miura N, Kawarada Y, et al Two forms of Wilson disease protein produced by alternative splicing are localized in distinct cellular compartments. Biochem J, 326: 897-902, 1997.
  30. Chan HS, Haddad G, Thorner PS, et al P-glycoprotein expression as a predictor of the outcome of therapy for neuroblastoma. N Engl J Med, 325: 1608-14, 1991.[Abstract]
  31. Nakayama K, Konno M, Kanzaki A, et al Allelotype analysis of gallbladder carcinoma associated with anomalous junction of pancreaticobiliary duct. Cancer Lett, 166: 135-41, 2001.[CrossRef][Medline]
  32. Weber-Hall S, Anderson J, McManus A, et al Gains, losses, and amplification of genomic material in rhabdomyosarcoma analyzed by comparative genomic hybridization. Cancer Res, 56: 3220-4, 1996.[Abstract/Free Full Text]
  33. Casey G, Lopez ME, Ramos JC, et al DNA sequence analysis of exons 2 through 11 and immunohistochemical staining are required to detect all known p53 alterations in human malignancies. Oncogene, 13: 1971-81, 1996.[Medline]
  34. Sjogren S, Inganas M, Norberg T, et al The p53 gene in breast cancer: prognostic value of complementary DNA sequencing versus immunohistochemistry. J Natl Cancer Inst (Bethesda), 88: 173-82, 1996.



This article has been cited by other articles:


Home page
 Ann. Surg. Oncol.Home page
S. Motoyama, T. Sugiyama, Y. Ueno, H. Okamoto, S. Takasawa, H. Nanjo, H. Watanabe, K. Maruyama, M. Okuyama, and J.-i. Ogawa
REG I Expression Predicts Long-term Survival among Locally Advanced Thoracic Squamous Cell Esophageal Cancer Patients Treated with Neoadjuvant Chemoradiotherapy Followed by Esophagectomy
Ann. Surg. Oncol., December 1, 2006; 13(12): 1724 - 1731.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakayama, K.
Right arrow Articles by Takebayashi, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakayama, K.
Right arrow Articles by Takebayashi, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online