Expression of the putative tumour suppressor gene PTPN13/PTPL1 is an independent prognostic marker for overall survival in breast cancer

Françoise Révillion¹, Carole Puech², Fanja Rabenoelina², Dany Chalbos², Jean-Philippe Peyrat¹, and Gilles Freiss²

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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.

Though 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 tumours (5). Initially, we showed that, in a breast cancer cell line model, PTP activity was involved in anti-estrogen inhibition of growth factor-stimulated proliferation (6). Furthermore, through mutational analysis of the tyrosine phosphatase 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 estrogens (8) or their antagonists (9) in human breast cancer cells, and they are known to play a role in the growth of these tumours in in vitro models.

PTP gamma, which has been regarded as a potential tumour suppressor gene in kidney and lung adenocarcinoma (10), is more highly expressed in normal breast tissue than in breast tumours 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 tumour suppressor gene in human breast cancer. However, Lamprianou et al. did not describe mammary gland phenotypic effects in PTP gamma knockout mice (13). Thus, in order to verify the tumour 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 signalling 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 cells (15) 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 tumour marker.

We have demonstrated increased PTPL1 mRNA levels after anti-estrogen treatment (9) 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 tumour 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 tumour types (23;24).

 In the present 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 tumours. We confirmed the expression of the LAR neuronal variant in 58 of 59 tumours, and we demonstrated that the level of PTPL1 expression is a prognostic indicator of favourable outcome for breast cancer patients. In the testing set of 291 patients that included 232 complementary tumours 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.

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 tumour 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 clinico-pathological features or survival did not support an effect of these PTPs in tumour growth or invasiveness; rather, it suggests the possible importance of these enzymes in the early steps of tumour development. However, translational and post-translational 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 anti-growth factor activity of the anti-estrogens, we found that PTPL1 mRNA levels were increased by non-steroidal partial antagonist (4-hydroxytamoxifen) or steroidal pure antagonist (ICI 182, 780) (9), as well as by benzothiophenes (29) 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 signalling 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 tumour susceptibility. Indeed none phenotypic consequences have been reported for PTPN13 KO mice (35) and studies of mice that lack PTPN13 phosphatase activity have focused on haematopoietic 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 tumour model mice could be used to evaluate the role of PTPL1 in tumour progression and susceptibility.

Considering its links with classical clinico-pathological 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 post-transcriptionnal downregulation by microRNAs (39). Mammary epithelial tumour cells are the major tissue component in primary breast cancer, which also contains stromal cells 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 PTPN13 showed specific staining of tumour 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 tumour cells.

Univariate and multivariate Cox analyses of our results revealed that PTPL1 mRNA expression is a favourable prognostic indicator of OS with a median duration of follow-up of 6.4 years. It is not unexpected that tumours containing high levels of PTPL1, which induces apoptosis of breast cancer cells, have a better prognosis than tumours 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 tumours in spite 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 favourable 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 tumour aggressiveness and sensitivity to treatments such as anti-estrogens and anti-aromatase.