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
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.
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