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Keywords:

  • breast cancer;
  • PTPL1/PTPN13;
  • phosphatase;
  • prognosis

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Although it is well established that some protein tyrosine kinases have a prognostic value in breast cancer, the involvement of protein tyrosine phosphatases (PTPs) is poorly substantiated for breast tumors. Three of these enzymes (PTP-gamma, LAR, and PTPL1) are already known to be regulated by estrogens or their antagonists in human breast cancer cells. We used a real-time reverse transcriptase polymerase chain reaction method to test the expression levels of PTP-gamma, LAR and its neuronal isoform, and PTPL1 in a training set of RNA from 59 breast tumors. We sought correlations between levels of these molecular markers, current tumor markers, and survival. We then quantified the expression level of the selected phosphatase in 232 additional samples, resulting in a testing set of 291 breast tumor RNAs from patients with a median follow-up of 6.4 years. The Spearman nonparametric test revealed correlations between PTPL1 expression and differentiation markers. Cox univariate analysis of the overall survival studies demonstrated that PTPL1 is a prognostic factor [risk ratio (RR) = 0.45], together with the progesterone receptor (PR) (RR = 0.52) and node involvement (RR = 1.58). In multivariate analyses, PTPL1 and PR retained their prognostic value (RRs of 0.48 and 0.55, respectively). This study demonstrates for the first time that PTPL1 expression level is an independent prognostic indicator of favorable outcome for patients with breast cancer. In conjunction with our mechanistic studies, this finding identifies PTPL1 as an important regulatory element of human breast tumor aggressiveness and sensitivity to treatments such as antiestrogens and antiaromatase. © 2008 Wiley-Liss, Inc.

In breast cancer, the clinical and biological variables commonly used to predict the outcome of primary chirurgical treatments include regional lymph node invasion, histological grade, and hormone receptor expression. All of these parameters are well-recognized prognostic and predictive factors. Additionally, the expression of new markers associated with proliferation (Ki-67) and cell cycle (cyclin E, cyclin D1),1 with mitogenic and survival pathways (HER tyrosine kinase receptor family)2 and with invasion processes (urokinase-type plasminogen activator, cathepsin D),3, 4 has also been linked to the survival of breast cancer patients or to their response to hormonal or cytotoxic therapies.

Although it is now well established that some protein tyrosine kinases have a prognostic value in breast cancer, the involvement of protein tyrosine phosphatases (PTPs) is poorly substantiated for breast tumors.5 Initially, we showed that, in a breast cancer cell line model, PTP activity was involved in antiestrogen inhibition of growth factor-stimulated proliferation.6 Furthermore, through mutational analysis of the TP gene superfamily in human cancers, a recent study identified six PTPs that are quite commonly affected.7 Three of these enzymes, consisting of two transmembrane subtypes (PTP gamma and LAR) and one intracellular subtype (PTPL1), are already known to be regulated by estrogens8 or their antagonists9 in human breast cancer cells, and also known to play a role in the growth of these tumors in in vitro models.

PTP gamma, which has been regarded as a potential tumor suppressor gene in kidney and lung adenocarcinoma,10 is more highly expressed in normal breast tissue than in breast tumors or breast cancer cell lines.8, 11 Moreover, Liu et al.12 have recently demonstrated that PTP gamma is able to inhibit anchorage-independent growth of breast cancer cells in soft agar and to reduce the proliferative response of MCF-7 cells to oestradiol, thus suggesting that PTP gamma may be a potential estrogen-regulated tumor suppressor gene in human breast cancer. However, Lamprianou et al. did not describe mammary gland phenotypic effects in PTP gamma knockout mice.13 Hence to verify the tumor suppressor properties of PTP gamma, the susceptibility of these mice to various carcinogens should be tested.

LAR gene deletion in mice suggests an important role for LAR-mediated signaling in mammary gland development and final differentiation.14 Moreover, the inhibitory effect of LAR ectopic expression on the growth of neu-transformed human breast carcinoma cells15 implies a negative role of LAR on the growth or survival of breast cancer cells. On the other hand, Yang et al.16 showed increased expression of a LAR isoform in malignant breast tissues. This LAR isoform, generated by neuronal-type alternative splicing,17 contains an insertion in the extracellular domain and could have potential clinical relevance as a tumor marker.

We have demonstrated increased PTPL1 mRNA levels after antiestrogen treatment9 and have demonstrated by using an antisense strategy that PTPL1 expression and resulting regulation are crucial for mediation of 4-hydroxytamoxifen inhibitory effects on growth factor activity.18 In addition, we have shown that PTPL1 induces apoptosis by inhibiting the PI3-kinase/Akt survival pathway through IRS-1 dephosphorylation.19 It is interesting to note that the PTPL1/PTPN13 gene presents the characteristics of a tumor suppressor gene.20, 21 It is located on chromosome 4q21, a region frequently deleted in ovarian and liver cancers,22 and its expression is frequently down-regulated or silenced through promoter hypermethylation in several tumor types.23, 24

In this study, we compared the expression level and prognostic value of three PTPs (PTP gamma, PTPL1, and the two LAR splicing variants) in a training set of 59 breast tumors. We confirmed the expression of the LAR neuronal variant in 58 of 59 tumors, and we demonstrated that the level of PTPL1 expression is a prognostic indicator of favorable outcome for breast cancer patients. In the testing set of 291 patients that included 232 complementary tumors and had a median follow-up of 6.4 years, we confirmed that the level of PTPL1 expression is an independent prognostic marker of increased overall survival (OS) for breast cancer patients.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Patients

The pilot study involved 59 breast tumor samples; the mean age of the patients was 59.7 years (range 35–81). The median follow-up duration in living patients was 7.5 years. The number of deaths was 21, and the number of relapses was 27.

To confirm the results in a larger sample set, 232 complementary breast tumor samples were added, resulting in a testing set of 291 patients (Table I). All these patients underwent surgery for locoregional disease in the Centre Oscar Lambret, the Anticancer Centre of the North of France, between May 1989 and December 1991. The mean age of the patients was 57.9 years (range 26–90).

Table I. Patients and Tumor Characteristics (n = 291)
CharacteristicsNo. of cases
Age (years) 
 <5069
 ≥50222
Node involvement 
 Negative134
 Positive155
 Unknown2
HPG 
 I42
 II111
 III109
 Unknown29
Tumor type 
 Ductular212
 Lobular22
 Others57
Tumor diameter (cm) 
 <221
 2–5188
 >568
 Unknown14
ER (fmol/mg protein) 
 <1088
 ≥10201
 Unknown2
PgR (fmol/mg protein) 
 <1088
 ≥10200
 Unknown3

Patients were treated by segmentectomy when tumors were smaller than 3 cm in width and by total mastectomy if tumors were larger or centrally located. Surgery was followed by radiation therapy. Node-positive premenopausal patients, estrogen receptor (ER)-negative and progesterone receptor (PR)-negative postmenopausal patients received adjuvant treatment consisting of six cycles of chemotherapy. The node-positive, ER-positive, and PR-positive postmenopausal patients received tamoxifen for 2 years. Node-negative patients received no adjuvant treatment. The median follow-up duration in living patients was 6.4 years. The number of deaths was 83, and the number of relapses was 108. The biopsies were obtained with the agreement of the Institutional Review Board.

Tumor samples

All tumors were adenocarcinomas. At the time of collection, fat and necrotic tissues were discarded, and two adjacent tumor pieces were removed. The first piece was submitted to histological studies. The second was immediately frozen in liquid nitrogen for hormone receptor (ER, PR) and RT-PCR assays. Both ER and PR levels were determined by the dextran-coated charcoal method as described previously.25 The Laboratoire d'Oncologie Moléculaire Humaine is affiliated with the European Organization for Research and Treatment of Cancer Receptor Study Group, which undertook quality control of the assays.26

PTP mRNA expression

Total RNA was isolated (RNeasy Mini Kit, Qiagen, France) from 40 mg of each tumor sample. Disruption and homogenization of the tumor samples were performed using a Rotor-Stator Homogenizer (Ribolyzer, Hybaid). The amount of extracted RNA was quantified by measuring the absorbance at 260 nm. The quality of the RNA was checked by assessing the ratio between the absorbance values at 260 nm and 280 nm, and it was then confirmed by electrophoresis of the RNA on a 1.5% agarose gel containing ethidium bromide. Total RNA (2 μg) was reverse transcribed into cDNA using 5 μM of random hexamers (Roche) and Superscript II RNAse H-reverse transcriptase according to the manufacturer's instructions (Invitrogen).

Real-time polymerase chain reaction (PCR) quantification of the expression of each gene was carried out on cDNA corresponding to 12.5 ng of total RNA using a LightCycler 3 device (Roche) with LightCycler FastStart DNA MasterPLUS SYBR GreenI Kit (Roche) according to the manufacturer's instructions. Primer sequences, amplification position, product length, time and temperature used for each gene are indicated in Table II.

Table II. PCR Conditions and Primers
GenePrimer sequenceExonProduct size (bp)Annealing temperature (°C)
PTP gammaUpper 5′ GCA ACT CGA TGG CTT CGA CAA 3′38461
Lower 5′ TCT TTC AGA AGG ATG GCG ACT GTT 3′3–4  
LAR-NeuUpper 5′ TGG ACT CCC CAT CAT CCA AGA 3′138566
Lower 5′ CCG CTG ATA GTG GTT TCA TAG TCC T 3′14–15  
LARUpper 5′ TAG CCG AGG CCC AGG AAA C 3′13–158761
Lower 5′ CCC TTG GTG GTA TAG GCA GCA 3′15  
PTPL1Upper 5′ CAA AGG TGA TCG CGT CCT A 3′26–2714861
Lower 5′ CGG GAC ATG TTC TTT AGA TGT T 3′28  
HPRTUpper 5′ CTG ACC TGC TGG ATT ACA 3′325655
Lower 5′ GCG ACC TTG ACC ATC TTT 3′5  

To confirm the specificity of the sequences chosen for the primers, we performed nucleotide-nucleotide BLASTn against a database of expressed sequence tags and nr (the nonredundant set of the GenBank, EMBL and DDBJ database sequences). To avoid amplification of contaminating genomic DNA, one of the two primers consisted of sequences derived from two adjacent exons.

The relative quantification of PTP gene expression was performed using the comparative cycle threshold (CT) method where the CT parameter is defined as the cycle number at which the fluorescent signal is first detectable. This method is based on the use of a calibrator sample and an endogenous RNA control, which permits the quantification of unknown samples. The human breast cancer cell line MCF7, which is known to express the three PTPs, was chosen as the calibrator sample (PTP expression = 1), and hypoxanthine phosphoribosyltransferase 1 (HPRT) mRNA was used as the endogenous RNA internal control. Relative PTP expression was given by the 2−ΔΔC, where ΔΔCT = ΔCT patient sample − ΔCT calibrator sample; with ΔCT = CT PTPCT HPRT.

Statistical analyses

All statistical analyses were performed using SPSS software (version 11.0). Correlations between parameters were assessed using the Spearman nonparametric test. Overall survival and relapse-free survival (RFS) were analyzed using the Kaplan Meier method. Comparison between curves was carried out using the logrank test. The proportional hazard regression method of Cox27 was used to assess the prognostic significance of parameters taken in association.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Training set

The distribution of the expression levels of the three PTPs in breast cancer samples was not Gaussian (Fig. 1). PTPL1, PTP gamma and LAR were expressed in all tumors, with median values that were 2.46 (ranging from 0.31 to 19.56)-fold, 5.16 (ranging from 0.21 to 34.06)-fold and 4.01 (ranging from 0.99 to 22.32)-fold greater than the expression levels in MCF7 cells, respectively (Fig. 1). The LAR neuronal isoform, which comprised of 3 to 4% of the total LAR mRNA in MCF7 cells, was detected in 58 of 59 tumors (median 0.40, ranging from 0.01 to 17.91) (Fig. 1). Its expression in 13 tumors was higher than in MCF7 cells but very low (at least 10-fold lower than in MCF7 cells) in 17 tumors.

thumbnail image

Figure 1. Distribution of breast cancer samples (n = 59) as a function of their PTP transcript levels.

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Classical correlations observed in breast cancer were found between ER and PR (p < 0.001, r = 0.761), the histoprognostic grade and ER (p = 0.05, r = −0.256) or PR (p = 0.045, r = −0.274), and tumor size and ER (p = 0.05, r = −0.271).

Among the PTPs, we observed several positive correlations. The expression of PTP gamma was correlated to that of the three other PTPs (p < 0.001, r = 0.705 for LAR; p < 0.001, r = 0.644 for PTPL1; and p = 0.04, r = 0.266 for LAR neuronal isoform), and LAR expression was correlated with those of both its neuronal isoform and PTPL1 (p = 0.029, r = 0.284 and p < 0.001, r = 0.418, respectively). No correlation was observed between PTP expression and bio-clinical parameters except that of PTPL1, which was positively correlated with hormonal receptor status (p = 0.02, r = 0.307 between PTPL1 and PR).

We observed that expression of the two LAR isoforms and PTP gamma did not have any prognostic value. On the other hand, the overall survival (OS) was significantly longer among patients with a high level of PTPL1 expression when compared with patients whose tumors had low levels of this protein (p = 0.04 by the logrank test).

Testing set

Based on the results of the training set, the PTPL1 expression level was next determined in a complementary cohort of 232 breast cancer patients. In the total cohort (291 patients), classical correlations observed in breast cancer were found between ER and PR (p < 0.001, r = 0.626), ER and age (p < 0.001, r = 0.268) and between PR and age (p = 0.01, r = 0.141). Expression of hormone receptors was negatively correlated with histoprognostic grade (p < 0.001, r = −0.366 and r = −0.342 for ER and PR respectively) and tumor size (p < 0.001, r = −0.229 and p = 0.037, r = −0.126 for ER and PR, respectively). Node involvement was positively correlated with tumor size (p = 0.0015, r = 0.19). PTPL1 expression was positively correlated with hormonal receptor status and negatively correlated with the histoprognostic grade and node invasion (Table III).

Table III. Correlation Between PTPL1 and Clinical, Histological or Biological Parameters
 pr
AgeNS 
Histoprognostic grade<0.001−0.333
Node involvement0.016−0.142
Tumor sizeNS 
ER<0.0010.246
PR<0.0010.262

OS was found to be significantly longer among patients with a high level of PTPL1 expression (p = 0.01 by the logrank test) (Fig. 2).

thumbnail image

Figure 2. Overall survival curve based on relative PTPL1 expression levels (clinical cut-off, upper quartile) in the whole population of patients. Numbers in parentheses indicate failures/total number of patients in each group.

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Cox univariate analyses revealed that, similarly to PTPL1 expression, ER and PR expression, tumor size and node invasion were prognostic factors for global survival (Table IV). Multivariate analyses identified PTPL1 as an independent prognostic factor for OS; PR and tumor size, but not node invasion, also preserved their prognostic value. However, in univariate and multivariate analyses, only node involvement and tumor size were prognostic factors for RFS (Table IV).

Table IV. Prognostic Factors in Cox Univariate and Multivariate Analyses
 Overall survivalRelapse-free survival
UnivariateMultivariateUnivariateMultivariate
pRRpRRpRRpRR
Histoprognostic grade (I, II, III)0.23   0.24   
Node involvement (0; >0)0.0431.58NS 0.0041.780.0201.62
Tumor size (≤2; 2–5; >5 cm)0.0031.850.0241.620.0051.670.0121.59
ER (<10; ≥10 fmol/mg protein)0.0470.63NS 0.13   
PR (<10; ≥10 fmol/mg protein)0.0040.520.0130.550.086   
PTPL1 (<5.7; ≥5.7)0.0120.450.0350.480.075   

Interestingly, for ER+ (n = 201) but not ER- patients, OS was significantly longer among patients with high levels of PTPL1 expression (p = 0.034 by the logrank test for ER+, p = 0.3 for ER-) (Fig. 3).

thumbnail image

Figure 3. Overall survival curve based on relative PTPL1 expression levels (clinical cut-off, upper quartile) in the ER+ subgroup of patients. Numbers in parentheses indicate failures/total number of patients in each group.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this study, we demonstrated that transcripts of PTP gamma, LAR and its neuronal isoform, and PTPL1 are expressed in almost all human breast cancers. These results confirm previous studies demonstrating the expression of phosphatases in breast cancer.8, 16 Using the Spearman test, we showed that PTPL1 expression was positively correlated with that of ER and PR. These results are in agreement with our previous observations.28

PTP gamma and LAR are differently expressed in breast tumor and normal tissue,8, 16 and they have been shown to influence the growth of breast cancer cell lines after ectopic surexpression.12, 15 The absence of correlations between their expression and classical clinicopathological features or survival did not support an effect of these PTPs in tumor growth or invasiveness; rather, it suggests the possible importance of these enzymes in the early steps of tumor development. However, translational and posttranslational modifications in addition to mutations and differential splicing can also regulate the expression and activity of these two PTPs and may have caused the divergence between the findings of the previous in vitro study and our present results.

We have demonstrated that PTPL1 has a pro-apoptotic role in breast cancer cell lines. Indeed, studying antigrowth factor activity of the antiestrogens, we found that PTPL1 mRNA levels were increased by nonsteroidal partial antagonist (4-hydroxytamoxifen) or steroidal pure antagonist (ICI 182, 780),9 as well as by benzothiophenes29 without regulation by estrogens.9 PTPL1 suppression using an antisense strategy completely abrogated the antagonistic effect of 4-hydroxytamoxifen on growth factor activity, thus demonstrating that PTPL1 and its resulting regulation are crucial for the mediation of this inhibitory effect.9 In addition, PTPL1 affected apoptosis by inhibition of the IRS-1/PI3K survival pathway;18 this inhibition was sufficient to induce apoptosis and necessary for UV-induced cell death in MCF7, HEK 293 and HeLa cells.19

PTPL1 has also been implicated in the regulation of biological phenomena associated with the cytoskeleton such as cell motility and cellular adhesion;30, 31 these processes play a fundamental role in invasion and metastasis. Furthermore, PTPL1 has been implicated in the regulation of cytokinesis in HeLa cells,32 and in the control of the meiotic cell cycle,33 clearly supporting its importance in cell growth regulation. More recently, Zhu et al.34 demonstrated that PTPL1 can inhibit HER2/Neu, a signaling pathway that is frequently deregulated in breast cancer. Published studies using mutant mice that lack PTPN13 protein product or phosphatase activity did not report any effect on tumor susceptibility. Indeed none phenotypic consequences have been reported for PTPN13 KO mice35 and studies of mice that lack PTPN13 phosphatase activity have focused on hematopoietic cell lineages and the peripheral nervous system,36 which were previously shown to express this phosphatase.37, 38 Thus, crossbreeding of these mice with mammary tumor model mice could be used to evaluate the role of PTPL1 in tumor progression and susceptibility.

Considering its links with classical clinicopathological features, we observed that PTPL1 expression was negatively correlated with node involvement and histoprognostic grade. This indicates that elevated expression of PTPL1 may be a molecular marker of a more differentiated phenotype.

It is well established that mRNA expression does not necessarily reflect protein expression. Indeed, gene expression is regulated at many levels, including posttranscriptionnal downregulation by microRNAs.39 Mammary epithelial tumor cells are the major tissue component in primary breast cancer, which also contains stromal and endothelial cells. It should be noted that PTP-BL, the PTPL1 mouse orthologue, is predominantly expressed in epithelial and neuronal cells.40 In addition, our in vitro studies have demonstrated that human breast cancer cell lines express and produce PTPL1.18 Furthermore, in the Human Protein Atlas program, immunochemical studies of breast cancer using a specific antibody against PTPL1 showed specific staining of tumor cells with little or no signal in the stromal cells (www.proteinatlas.org). Taken together, these observations support the hypothesis that the PTPL1 transcripts that we quantified by real-time RT PCR were produced by the tumor cells.

Univariate and multivariate Cox analyses of our results revealed that PTPL1 mRNA expression is a favorable prognostic indicator of OS with a median duration of follow-up of 6.4 years. It is not unexpected that tumors containing high levels of PTPL1, which induces apoptosis of breast cancer cells, have a better prognosis than tumors without this phosphatase. This observation is in line with the positive links observed between PTPL1 and steroid hormone receptors or low histoprognostic grading, which are parameters associated with a better prognosis.

It is interesting that PTPL1 retains its prognostic value for OS in patients with ER-positive tumors in spite of the strong correlation of its expression with that of ER. The presence of ER/PR is typically used as a rational basis for hormonal treatment. Our results suggest that PTPL1 could provide an additional criterion for implementation of such therapies.

In conclusion, this study demonstrates for the first time that PTPL1 expression is an independent prognostic factor of favorable outcome for patients with breast cancer. In conjunction with our mechanistic studies, this finding suggests that PTPL1 is an important regulatory element of human breast tumor aggressiveness and sensitivity to treatments such as antiestrogens and antiaromatase.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors are grateful to Françoise Vignon who played an essential role in the initiation of this work. A patent application concerning the work presented in this manuscript was deposited by “INSERM Transfert”.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References