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Clinical Cancer Research Vol. 10, 2776-2783, April 15, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Activation of the MDR1 Upstream Promoter in Breast Carcinoma as a Surrogate for Metastatic Invasion

Selina Raguz1, Manuela Tamburo De Bella2, Gopichani Tripuraneni2, Martin J. Slade2, Christopher F. Higgins1, R. Charles Coombes2 and Ernesto Yagüe1

1 Clinical Sciences Centre, Medical Research Council and
2 Department of Cancer Medicine, Imperial College, Hammersmith Hospital, London, United Kingdom


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Activation of the MDR1 upstream promoter (USP) has been described previously in four lymphoblastic leukemia patients, where it is the major MDR1 promoter associated with P-glycoprotein overexpression. We asked whether MDR1 USP-derived transcripts were also present in breast carcinoma and assessed their potential as a biomarker.

Experimental Design: We developed a sensitive method for detecting transcripts derived from the MDR1 USP and used it to identify MDR1 USP-derived transcripts in cell model systems, in 61 breast carcinoma biopsies of the primary tumor, and in isolated malignant epithelial cells both from the primary tumor and from the associated invaded lymph nodes.

Results: The MDR1 USP was not active in several independent leukemic and breast cancer cell lines or nucleated peripheral blood cells (n = 9). However, transcripts derived from the MDR1 USP were detected in some drug-resistant cell lines and a high proportion of primary breast tumors (71.6%; n = 61), whereas they were present at low frequency in normal breast tissue (10%; n = 10). Activation of MDR1 USP was not due to chromosomal amplifications or rearrangements at the MDR1 locus. Transcription from the MDR1 USP correlated with metastatic node invasion [N = 0–3 versus N > 3 (N = number of lymph nodes invaded); Fisher’s exact test, P = 0.011] and was detected in malignant epithelial cells from the primary tumor and those that metastasized to the lymph nodes.

Conclusions: MDR1 USP activation is a surrogate marker for breast carcinoma progression and can be used as a marker to study breast cancer susceptibility.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite advances in understanding the molecular causes of breast cancer, treatment of patients is still based primarily on the evaluation of a series of macroscopic and microscopic prognostic markers such as tumor size, grade of differentiation, histological type, and lymph node invasion (1) . The only molecular marker widely used to predict treatment outcome in the clinic is estrogen receptor (ER) status (2 , 3) . Other markers are currently being evaluated in clinical studies (4, 5, 6, 7) , and progesterone receptor and HER2/neu receptor (8) are already used in some clinics. There is, therefore, a requirement for new molecular markers that could aid in guiding treatments, for example, to predict which tumors will eventually become invasive and metastatic, and which will remain localized and indolent (9, 10, 11) . The use of highly sensitive detection methods, such as PCR-based technologies, may also facilitate the development of detection tools to detect micrometastatic disease in the follow-up of patients after mastectomy (12 , 13) .

The MDR1 gene codes for P-glycoprotein, an ATP-dependent membrane transporter, which pumps many cytotoxic drugs out of the cells and hence, when overexpressed, can confer resistance to chemotherapy (14) . Expression of P-glycoprotein in breast tumors is associated with poor response to chemotherapy (15) . Two MDR1 promoters were originally described in highly drug-resistant derivatives of human cervical carcinoma KB cell lines (16) , the major downstream promoter [DSP (which is used by most cell lines and tissues expressing the MDR1 gene)] and a minor upstream promoter [USP (which is not active in cell lines or healthy human tissues; Refs. 17, 18, 19 )]. However, in acute lymphoblastic leukemic patients who overexpress P-glycoprotein, the MDR1 USP represents the major promoter (and in some patients, the only promoter) used by mononuclear cells (20) .

Here we show that the presence of transcripts derived from the MDR1 USP in breast cancer tumors correlates with metastatic invasion of lymph nodes. We also detected activation of the MDR1 USP in isolated carcinoma cells, both from the primary tumor and from invaded lymph nodes. Activation of the MDR1 USP may then represent a marker associated with the metastatic phenotype.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
Seventy-one archival breast biopsies were used. Ten were from non-breast carcinoma patients (four normal tissue biopsies obtained after reductive mammoplasty and six from benign breast tumors). Sixty-one were from breast carcinoma patients. The mean age of the breast carcinoma patients was 56 years (range, 29–99 years); 25 of 37 patients had lymph node involvement; 2 patients had grade I disease, 20 patients had grade II disease, and 18 patients had grade III disease; and 23 of 31 patients were ER positive. Only one patient had received neoadjuvant chemotherapy with mitozantrone, methotrexate, and mitomycin C (21) before biopsy. Biopsies were collected at surgery and snap-frozen in liquid nitrogen.

Epithelial cells from five additional invasive ductal carcinoma patients and two reductive mammoplasty patients were purified, and purification was performed immediately after surgery. Three breast carcinoma patients had grade II disease, and two patients had grade III disease; four patients had N > 10 (N = number of lymph nodes invaded) and were ER and progesterone receptor negative, whereas one patient scored N = 3 and was positive for ER and progesterone receptor markers. These patients had not received neoadjuvant therapy.

Research into the MDR1 USP was approved by The Hammersmith Hospital Research Ethics Committee.

Purification of Epithelial Cells from Primary Tumors and Lymph Nodes.
Carcinoma cells were immunoaffinity purified from normal breast tissue, primary tumors, and invaded lymph nodes after collagenase digestion using superparamagnetic polystyrene beads (Dynal, Wirral, United Kingdom) coated with mouse monoclonal antibody Ber-EP4, as described previously (22) . The purity of the preparations was determined by immunohistochemistry for cytokeratins 8 and 18 (22) . More than 98% of the purified cells were of epithelial origin.

Cell Lines.
The vinblastine-selected, multidrug-resistant human cervical carcinoma mutant line KB-V1; leukemic cell line K562; and breast carcinoma cell lines MCF7, MDA-MB-231, MDA-MB-453, BT474, and CAL51 and their multidrug-resistant derivatives MCF7AdrR and KD30 have been described previously (23, 24, 25, 26, 27) . K562 sublines were exposed to doxorubicin in a stepwise fashion (28) to obtain cells resistant to 60, 120, and 225 nM doxorubicin (designated KD60, KD120, and KD225, respectively).

RNA Isolation.
Total RNA was prepared from cells by RNAzol extraction (Biogenesys, Poole, United Kingdom). Frozen breast biopsies were pulverized under liquid nitrogen (Spex CertiPrep Freezer Mill 6750; Glen Creston Ltd., Stanmore, United Kingdom) and resuspended in 3 M LiCl and 6 M urea, and, after precipitation, total RNA was isolated by phenol/chloroform extraction and ethanol precipitation as described previously (29) .

Determination of mRNA Expression by Reverse Transcription-PCR.
Total RNA (500 ng) was transcribed with avian myeloblastosis virus reverse transcriptase using random hexamers (Roche, Indianapolis, IN) in a 10-µl reaction, and 1 µl was amplified by PCR. Estimation of total MDR1 mRNA (derived from both USPs and DSPs) by amplifying exons 6, 7, and 8 with oligonucleotide RON5 (5'-CACGTGGTTGGAAGCTAACC-3') and RON6 (5'-GAAGGCCAGAGCATAAGATGC-3') was as described previously (30) . Transcripts derived from the MDR1 USP were amplified (94°C for 30 s, 60°C for 1 min, and 72°C for 30 s, 30 cycles) using primers OLCDNA1 (5'-CCTACTCTATTCAGATATTCTCCAGATTCC-3') and OLMDR455 (5'-GCTCCTCCATTGCGGTCCCC-3'). Amplified cDNA was detected by Southern blotting and hybridization using internal oligonucleotides as described previously (31) . The MDR1 USP-derived cDNA was detected with OLEY1 (5'-GGCTTCCGTTGCACCTCTCTTTTATCTGGTTGCTTCC-3') spanning the first intron of the MDR1 gene. Patients were scored as USP positive when a signal was obtained with OLEY1. USP-negative scores were assigned when no hybridization signal was obtained after 4 days of exposition. Total MDR1-derived cDNA was detected with HMDR7Rev (5'-TGCTCCAGCTTTTGCATACG-3'). CD2 transcripts were amplified (94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, 35 cycles) using oligonucleotide OLEY11 (5'-GTCAGCTGTCCAGAGAAAGG-3') and OLAS2 (5'-GAGGGCAGAAATCCACAGT-3') and detected by Southern hybridization with internal oligonucleotide OLEY109 (5'-TCGGAGAAATGATGAGGAGC-3'). GAPDH transcripts were amplified (using the same cycling conditions described for CD2) with primers OLEY8 (5'-ACCACAGTCCATGCCATCAC-3') and OLEY70 (5'-GGTTGAGCACAGGGTACTTT-3') and detected with OLEY9 (5'-TCCACCACCCTGTTGCTGTA-3').

Quantitative real-time reverse transcription-PCR was carried out using an Opticon DNA Engine Continuous Fluorescence Detector (MJ Research) with Quantitect SYBR Green PCR master mix (Qiagen) and specific MDR1 primers as described previously (27) . GAPDH primers were as follows: sense, 5'-GAAGGTGRAGGTCGGAGTC-3'; and antisense, 5'-GAAGATGGTGATGGGATTTC-3'. Relative MDR1 expression levels were calculated by the comparative threshold cycle (CT) method as described previously (27) .

Genomic DNA and Southern Blotting.
Genomic DNA was isolated from cells and some biopsies (both from MDR1 USP-positive and -negative patients) by proteinase K digestion and phenol/chloroform extraction, digested with EcoRI, Southern blotted, and hybridized to nick-translated radioactive probes by standard procedures (31) . MDR1 and ß-globin loci probes have been described previously (27) . MDR1:ß-globin ratios were obtained by quantitation using a PhosphorImager.

Statistical Analysis.
Because the continuous data did not follow normal distributions, a nonparametric approach was used (Mann-Whitney U test). For tabular data, Fisher’s exact test was used. Statistical significance of each test was assumed when P < 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Novel Splice Variants in Transcripts Derived from the MDR1 USP.
By using a reverse transcription-PCR approach, we detected three transcripts derived from the MDR1 USP in KB-V1 cells (Fig. 1A)Citation . Sequence analysis indicated that the longer product (Fig. 1BCitation , Transcript A) corresponded to the transcript originating from the USP, described previously (17) . However, the other two products (Fig. 1BCitation , Transcripts B and C) corresponded to previously unreported splice variants. In transcript B, an intron (Fig. 1BCitation , IVS-1a) is spliced within exon 1, originating exons 1c and 1d, whereas in transcript C, a new intron (IVS-1b, comprising IVS-1a, exon 1d, and IVS-1) is spliced between exon 1c and exon 2.



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  		Fig. 1. MDR1 upstream promoter (UPS) and splice variants. A, reverse transcription-PCR detection of transcripts derived from the MDR1 USP in mutant carcinoma KB-V1 cells. The migration of molecular size markers (in bp) is indicated to the right. B, architecture of the 5'-end of the MDR1 locus. Nomenclature of exons and intervening sequence(s) follows Refs. 17 18 19 . Numbering corresponds to nucleotides from the MDR1 cDNA sequence (GenBank database accession no. M14758). Exons are represented as boxes (black box, coding; white box, noncoding), and intervening sequences are represented as lines. Arrows in exons –1 and 1b represent the USP and downstream promoter (DSP) transcription start points, respectively. Positions of oligonucleotides used for the PCR amplifications from USP-derived transcripts are indicated as arrows at the bottom of the figure. The probe used for Southern hybridization, spanning intervening sequence 1 (IVS-1), is indicated by the hatched lines. Transcript A was originally described by Ueda et al. (17 , 18) ; transcripts B and C are novel splice variants that will appear in the GenBank database with accessions numbers AY425005 and AY425006, respectively.

 
Transcripts derived from the MDR1 USP were not detected in several leukemic or breast cancer-derived cell lines (K562, MCF7, MDA-MB-231, MDA-MB-453, BT474, or CAL51; data not shown), in agreement with previous reports (17) . However, the MDR1 USP was active in MCF7AdrR, a cell line that, like KB-V1, overexpresses P-glycoprotein and is able to grow in the presence of 1 µM doxorubicin (Fig. 2A)Citation . By successive selection with doxorubicin, we obtained K562 sublines able to grow in the presence of toxic concentrations of the drug ranging from 30 to 225 nM (this report and Ref. 27 ). Interestingly, the MDR1 USP was activated only in KD225 (Fig. 2A)Citation . However, real-time reverse transcription-PCR showed up-regulation of total MDR1 mRNA, which detects transcripts derived from both the USPs and DSPs in all drug-resistant lines (Fig. 2B)Citation . Therefore, in these cell lines, as in KB-V1 (18) , activation of MDR1 USP occurs after multiple rounds of selection for higher levels of resistance.



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  		Fig. 2. MDR1 upstream promoter (USP) activation in cell lines. A, transcripts from the MDR1 USP are only detected in highly drug-resistant cell lines. Reverse transcription-PCR detection was performed in MCF7, K562, and their derivatives. Generation of MCF7AdrR, a MCF7 derivative selected for growth on highly toxic concentrations of doxorubicin, has been described previously (23) . K562 cells were treated with increasing doxorubicin concentrations in a stepwise fashion and selected for growth on 30, 60, 120, and 225 nM doxorubicin (KD lines; left panel). Only MCF7AdrR and KD225 showed activation of the MDR1 USP. B, MDR1 mRNA (derived from both USP and downstream promoter) is up-regulated in drug-resistant cell lines. Quantification of MDR1 mRNA (exon 14) was performed by real-time PCR and normalized to endogenous GAPDH mRNA. Amplification does not differentiate between transcripts derived from the USP or downstream promoter, and therefore data are expressed as total MDR1 mRNA. Results are shown as fold difference with respect to the expression in K562 cells. C, activation of the MDR1 USP can occur without gene amplification. Southern blot analysis of the MDR1 and ß-globin loci of K562 cells and drug-resistant derivatives. The KB-V1 cell line was used as a positive control for amplification and rearrangement of the MDR1 locus; arrows show the position of rearranged MDR1 fragments in this cell line. Ratios of MDR1:ß-globin hybridization signals were quantified with a PhosphorImager.

 
Activation of MDR1 USP Is Not Due to Chromosomal Amplification or Rearrangements of the MDR1 Locus.
Because in the MDR1 USP-positive cell line KB-V1 the MDR1 locus is highly amplified (Ref. 32 ; Fig. 2CCitation ), we performed genomic Southern analysis to determine whether amplification or rearrangements of the MDR1 locus had occurred in the other cell lines. MCF7AdrR showed essentially the same pattern of amplification as KB-V1 cells (data not shown), with a MDR1:ß-globin hybridization signal ratio suggesting that the locus was amplified 100–200-fold. A major and several minor chromosomal rearrangements had occurred in these lines such that the MDR1 probe hybridized to several DNA fragments in addition to the bona fide 6-kb genomic EcoRI fragment (Fig. 2B)Citation . In contrast, in the K562 derivative KD225, activation of MDR1 USP occurred without detectable amplification or rearrangement of the locus (Fig. 2C)Citation . Therefore, activation of MDR1 USP can occur without gross genomic abnormalities in the locus, and its transcripts must then be the result of transcriptional activation.

Activation of the MDR1 USP in Primary Breast Tumors Correlates with Metastatic Spread.
MDR1 USP usage was found in 44 of 61 breast carcinoma biopsies (Fig. 3A)Citation . Only one breast tissue sample originated from a patient who had received neoadjuvant chemotherapy (which scored USP positive), and, therefore, the percentage of MDR1 USP usage at the time of surgery was high (71.6%). In contrast, only 1 of the 10 nonmalignant breast tissue samples showed MDR1 USP activation (Fig. 3A)Citation . This sample was from a reductive mammoplasty and did not show any histological abnormalities. Transcript A was the most abundant in all positive samples, whereas transcript C was absent in some patients (Fig. 3B)Citation . Total MDR1 mRNA, detected by amplification of exons 6–8, was present in most samples and was probably due to lymphocyte infiltration (because lymphocyte marker CD2 was also expressed in most samples) or to small amounts of blood in the sample (data not shown). It is well established that several subpopulations of lymphocytes express MDR1 mRNA to different extents (33) . We have not detected activation of the MDR1 USP in lymphocytes from healthy volunteers (n = 9), thus all of the MDR1 transcripts in these cells originate from the DSP. The fact that lymphocytes are present in most tissue samples obtained from patients highlights the technical problems in determining the MDR1 expression status of tissues by a highly sensitive technique such as PCR, which does not distinguish between tissue-derived and lymphocyte-derived transcripts.



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  		Fig. 3. Activation of the MDR1 upstream promoter (USP) in breast carcinoma patients. A, histogram showing the MDR1 USP score of the 61 biopsies from breast carcinoma patients analyzed. All but one (which had received neoadjuvant chemotherapy and scored USP positive, black bars) were from patients at time of surgery. Gray bars show MDR1 USP-negative scores. Fisher’s exact test indicated that the activation of the MDR1 USP in breast carcinoma was statistically significant (P = 0.0006) when compared with normal tissue. B, reverse transcription-PCR detection of transcripts derived from the MDR1 USP. Sixty-one patient biopsies were analyzed; only 10 are shown for clarity. All 10 normal breast tissue samples analyzed are shown. A–C correspond to the different splice variants described in Fig. 1Citation . Transcript A was the most abundant, and not all USP-positive samples showed the least abundant transcript, transcript C. C, activation of MDR1 USP in breast carcinoma patients is not a consequence of gene amplification. Southern blot analysis (as in Fig. 2BCitation ) of eight biopsies from breast carcinoma patients (four were positive for MDR1 USP activation, and four were negative for MDR1 USP activation) is shown. MDR1:ß-globin loci ratios did not change significantly between MDR1 USP-positive and -negative patients.

 
We performed genomic Southern analysis to ascertain whether amplification of the MDR1 locus had occurred in the MDR1 USP-positive tumors, as observed for highly drug-resistant cell lines. Four USP-positive and four USP-negative samples were analyzed, and the MDR1:ß-globin hybridization signal ratio was the same in the two groups (Fig. 3C)Citation , showing that, in breast carcinoma patients, activation of the MDR1 USP is not a consequence of DNA amplification.

There was no significant correlation between MDR1 USP activation and patient age (P = 0.58), tumor size (P = 0.94), grade of differentiation (P = 0.66), and ER status (P = 0.19). However, MDR1 USP usage in the primary tumor did correlate with the number of lymph nodes with metastatic cells. All (n = 10) patients with four or more lymph nodes with metastasis showed activation of the MDR1 USP (Fig. 4A)Citation . Fisher’s exact test indicated a difference (P = 0.011) between this group of patients and those who had none or one to three nodes invaded. Interestingly, patients with less than four nodes invaded showed activation of MDR1 USP in approximately 50% of cases.



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  		Fig. 4. MDR1 upstream promoter (USP) activation and breast cancer metastasis. A, correlation with lymph node invasion. Breast carcinoma patients were distributed into three groups according to the degree to lymph node metastatic invasion and the score for the MDR1 USP status plotted (negative, ; positive, {blacksquare}). All patients (n = 10) with a high number of lymph nodes invaded (poor prognostic indicator) showed activation of the MDR1 USP. Fisher’s exact test indicated a statistical difference between the N > 3 and N = 0–3 groups (N = number of lymph nodes invaded; P = 0.011). B, activation of the MDR1 USP in isolated epithelial cells. Cells were isolated by immunoaffinity to the epithelial marker Ber-EP4 from primary tumors from five patients (P1–P5) showing lymph node invasion, from the lymph nodes of two patients (P4 and P5), and from two normal breast tissue biopsies from reductive mammoplasty. Detection of MDR1 USP-derived transcripts A–C was performed as described in Fig. 1ACitation .

 
Malignant Metastatic Breast Epithelial Cells Have an Activated MDR1 USP.
To ascertain whether carcinoma cells showed activation of the MDR1 USP, we purified them from fresh tumor biopsies by binding to a monoclonal antibody against the Ber-EP4 epithelial marker (34) . Five patients with 3–23 invaded lymph nodes were selected, and all scored positive for MDR1 USP activation (Fig. 4B)Citation . Because of the strong correlation between MDR1 USP activation and lymph node invasion, we asked whether the metastatic cells present in the lymph nodes from two of the above patients had an activated MDR1 USP. Both were positive (Fig. 4B)Citation . Interestingly, we detected activation of the MDR1 USP in one of two control samples derived from reductive mammoplasty.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Activation of the MDR1 USP, first described in a highly drug-resistant cell line (17 , 18) , has been detected previously in 4 of 28 patients with acute lymphoblastic leukemia (20) , where it becomes the major and sometimes sole active MDR1 promoter. In this study we demonstrate the presence of transcripts, some of which are novel splice variants, derived from the MDR1 USP in a high proportion (71.6%) of primary tumors from breast carcinoma patients. Changes in global gene expression in breast cancer have been reported using microarray technology (10 , 11 , 35) , and overexpression of MDR1 has been associated with the ER-negative/HER2-neu-positive subgroup (36) . However, the probes used to detect MDR1 mRNA in these array experiments do not differentiate between the transcripts derived from either the USP or the DSP, making it difficult to compare our data.

Although MDR1 locus amplification is a common event in cell lines (23 , 37) , it has never been specifically described in human neoplasms (38 , 39) . However, amplification of 7q21, the chromosomal region where the MDR1 locus lies, has been described in one head and neck squamous cell carcinoma (40) and four malignant glioma (41) cases. In addition, random chromosomal rearrangements between MDR1 and other sequences have been described in two patients with drug-refractory acute lymphoblastic leukemia (42) . In this study, we have eliminated MDR1 USP activation as a consequence of gene amplification. Southern analysis of both cell lines and patient biopsies indicated that MDR1 USP-derived transcripts were present when no amplification of the MDR1 locus had taken place (Figs. 2BCitation and 3C)Citation . However, we cannot rule out the possibility that breast cancer patients with activation of the MDR1 USP have microdeletions or that the percentage of tumor cells containing amplifications was so low as to be beyond the detection limits of the techniques used.

Because no transcripts derived from the MDR1 USP are found in cell lines and most nonmalignant breast tissue samples, we can conclude that this promoter is located in a normally silent region of the genome. Yet in cells exposed to high doxorubicin concentrations, like KD225 and MCF7AdrR, the normally silent MDR1 USP is activated (Fig. 2A)Citation . Although many drugs produce DNA hypermethylation, at the concentrations used here doxorubicin induces DNA hypomethylation (43 , 44) , a phenomenon associated with gene activation (45 , 46) . Our data indicate that the MDR1 USP region becomes transcriptionally competent in a high proportion of breast cancer patients. All patients examined with a high number of invaded nodes (N > 3; n = 10) scored positive for the MDR1 USP in the primary tumor, whereas in patients with N < 3, the marker was present in 52% of cases (14 of 27 cases); these facts indicate that activation of MDR1 USP is associated with tumor progression and metastatic spread. One of the characteristics of breast cancer development is a progressive global DNA hypomethylation, and the DNA methylation status of a tumor has been suggested as a biological marker with prognostic significance (47 , 48) . Transcriptional activation of MDR1 by hypomethylation of the DSP has been demonstrated in acute myeloid leukemia (49) . However, the MDR1 DSP is located in a CpG island, a region of the genome rich in the dinucleotide CpG and associated with many gene promoters (45 , 46) , whereas the MDR1 USP is not. Therefore, although the progressive global demethylation occurring in breast cancer might have a role in activating the MDR1 USP, we cannot rule out the possibility of activation of transcription factors specific to the metastatic phenotype (50) .

Although we do not know the proportion of MDR1 transcripts originating from the DSP and USP in breast cancer patients before chemotherapy, in acute lymphoblastic leukemic patients, who overexpress P-glycoprotein after chemotherapy, the MDR1 USP represents the major promoter used by mononuclear cells (20) . In a cell expressing MDR1 mRNA from the DSP, the presence or absence of transcripts derived from the USP would not notably change its P-glycoprotein status because the start codon present in exon 2 is not affected in either of the splice variants or the transcript originating from the DSP (Fig. 1B)Citation . Although the role of P-glycoprotein in response to chemotherapy in breast cancer is still debated (51) , many studies indicate that patients expressing P-glycoprotein are more likely to fail to respond to chemotherapy than patients whose tumors are P-glycoprotein negative (15 , 52) . Moreover, in MCF7AdrR cells, drugs transported by P-glycoprotein induce membrane ruffling, which is an early indicator of cellular motility and metastatic potential (53) . Therefore, the possibility that some chemotherapeutic agents may promote metastasis cannot be ruled out. However, no correlation between P-glycoprotein expression and lymph node metastasis before chemotherapy has been found previously (15) . We favor the interpretation that the association between MDR1 USP activation and metastatic lymph node invasion reported here represents a surrogate to mark other biological processes such as global demethylation or activation of transcription factors associated with the metastatic phenotype and acting on the MDR1 USP. Expression of both P-glycoprotein and mutant p53 has been reported to be associated with poor prognosis of breast cancer (54) , which could also suggest that the MDR1 USP activation is linked to the expression of a gene or genes involved in tumor progression and metastasis. Which of these mechanisms is correct will require additional studies on the MDR1 USP.

Although we have analyzed a relatively small sample of normal breast tissues for population studies, the results shown here would suggest a relatively low frequency for MDR1 USP activation. In the group of breast carcinoma patients analyzed, frequency significantly increases to 71.6% and becomes 100% in those with a high number of metastasized lymph nodes. Therefore, we can conclude that activation of the MDR1 USP can be used as a surrogate marker for breast cancer progression and metastatic spread, opening the possibility of using it to study breast cancer susceptibility.


   ACKNOWLEDGMENTS
 
We thank Ana Arance for helpful discussions, Robert Tolhust and Alan McCay for obtaining some archival samples, and Carlo Palmieri for obtaining some of the patients’ clinical data.


   FOOTNOTES
 
Grant support: United Kingdom Medical Research Council and Cancer Research UK.

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: Ernesto Yagüe, Medical Research Council Clinical Sciences Centre, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, United Kingdom. Phone: 44-20-8383-8270; Fax: 44-20-8383-8337; E-mail: ernesto.yague{at}csc.mrc.ac.uk

Received 11/ 3/03; revised 1/ 2/04; accepted 1/13/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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