Cathepsin D: newly discovered functions of a long-standing aspartic protease in cancer and apoptosis.

The lysosomal aspartic protease cathepsin D (cath-D) is over-expressed and hyper-secreted by epithelial breast cancer cells. This protease is an independent marker of poor prognosis in breast cancer being correlated with the incidence of clinical metastasis. Cath-D over-expression stimulates tumorigenicity and metastasis. Indeed it plays an essential role in the multiple steps of tumor progression, in stimulating cancer cell proliferation, fibroblast outgrowth and angiogenesis, as well as in inhibiting tumor apoptosis. A mutated cath-D devoid of catalytic activity still proved mitogenic for cancer, endothelial and fibroblastic cells, suggesting an extra-cellular mode of action of cath-D involving a triggering, either directly or indirectly, of an as yet unidentified cell surface receptor. Cath-D is also a key mediator of induced-apoptosis and its proteolytic activity has been involved generally in this event. During apoptosis, mature lysosomal cath-D is translocated to the cytosol. Since cath-D is one of the lysosomal enzymes which requires a more acidic pH to be proteolytically-active relative to the cysteine lysosomal enzymes, such as cath-B and -L, it is open to question whether cytosolic cath-D might be able to cleave substrate(s) implicated in the apoptotic cascade. This review summarises our current knowledge on cath-D action in cancer progression and metastasis, as well as its dual function in apoptosis.

Cdk4 promotes adipogenesis through PPARgamma activation.

Cell cycle regulators such as E2F1 and retinoblastoma (RB) play crucial roles in the control of adipogenesis, mostly by controlling the transition between preadipocyte proliferation and adipocyte differentiation. The serine-threonine kinase cyclin-dependent kinase 4 (cdk4) works in a complex with D-type cyclins to phosphorylate RB, mediating the entry of cells into the cell cycle in response to external stimuli. Because cdk4 is an upstream regulator of the E2F-RB pathway, we tested whether cdk4 was a target for new factors that regulate adipogenesis. Here we find that cdk4 inhibition impairs adipocyte differentiation and function. Disruption of cdk4 or activating mutations in cdk4 in primary mouse embryonic fibroblasts results in reduced and increased adipogenic potential, respectively, of these cells. We show that the effects of cdk4 are not limited to the control of differentiation; cdk4 also participates in adipocyte function through activation of PPARgamma.

The nuclear receptor liver receptor homolog-1 is an estrogen receptor target gene.

Liver receptor homolog-1 (LRH-1) is a nuclear receptor previously known to have distinct functions during mouse development and essential roles in cholesterol homeostasis. Recently, a new role for LRH-1 has been discovered in tumor progression, giving LRH-1 potential transforming functions. In order to identify critical factors stimulating LRH-1 expression leading to deregulated cellular proliferation, we studied its expression and its regulation in several breast cancer cell lines. We observed that LRH-1 expression was increased in estrogen receptor (ER) alpha expressing cell lines, whereas weak-to-no expression was found in nonexpressing ERalpha cell lines. In MCF7, LRH-1 expression was highly induced after treatment with 17beta-estradiol (E2). This transcriptional regulation was the result of a direct binding of the ER to the LRH-1 promoter, as demonstrated by gelshift and chromatin immunoprecipitation assays. Interestingly, siRNA-mediated inactivation of LRH-1 decreased the E2-dependent proliferation of MCF7 cells. Finally, LRH-1 protein expression was detected by immunohistochemistry in tumor cells of human mammary ductal carcinomas. Altogether, these data demonstrate that LRH-1 is transcriptionally regulated by the ER alpha and reinforce the hypothesis that LRH-1 could exert potential oncogenic effects during breast cancer formation.

Involvement of HP1alpha protein in irreversible transcriptional inactivation by antiestrogens in breast cancer cells.

Resistance to 4-hydroxy-tamoxifen (OHT), which appears in breast cancer cells after long-term antiestrogen treatment, may involve irreversible changes of gene expression. We previously developed a MCF-7 derived cell line (MVLN), in which OHT rapidly and irreversibly inactivates the expression of an estrogen-regulated luciferase transgene (Vit-tk-luciferase). In chromatin immunoprecipitation experiments, heterochromatin protein 1 (HP1alpha) was found to be associated with the Vit-tk-luciferase transgene, only when it was inactivated by OHT treatment. Chimeras composed of either HP1alpha or the Krupple-associated box (KRAB) module of KOX-1 protein (known to repress gene expression by recruitment of HP1 proteins), fused to the estrogen receptor (ER)-DNA binding domain (DBD) and the androgen receptor (AR)-ligand binding domain (LBD) were generated and appeared as potent transcriptional repressors. In stably transfected MVLN cells, irreversible inactivation of the luciferase transgene expression obtained with HP1alpha-ER(DBD)-AR(LBD) was partial, whereas inactivation obtained with KRAB-ER(DBD)-AR(LBD) was comparable to that obtained with OHT, although with a slower kinetics. Altogether, these data suggest that HP1alpha is involved in the silencing effects associated with long-term OHT treatments.

Differential responses of PPARalpha, PPARdelta, and PPARgamma reporter cell lines to selective PPAR synthetic ligands.

To characterize the specificity of synthetic compounds for peroxisome proliferator-activated receptors (PPARs), three stable cell lines expressing the ligand binding domain (LBD) of human PPARalpha, PPARdelta, or PPARgamma fused to the yeast GAL4 DNA binding domain (DBD) were developed. These reporter cell lines were generated by a two-step transfection procedure. First, a stable cell line, HG5LN, expressing the reporter gene was developed. These cells were then transfected with the different receptor genes. With the help of the three PPAR reporter cell lines, we assessed the selectivity and activity of PPAR agonists GW7647, WY-14-643, L-165041, GW501516, BRL49653, ciglitazone, and pioglitazone. GW7647, L-165041, and BRL49653 were the most potent and selective agonists for hPPARalpha, hPPARdelta, and hPPARgamma, respectively. Two PPAR antagonists, GW9662 and BADGE, were also tested. GW9662 was a selective PPARgamma antagonist, whereas BADGE was a low-affinity PPAR ligand. Furthermore, GW9662 was a full antagonist on PPARgamma and PPARdelta, whereas it showed partial agonism on PPARalpha. We conclude that our stable models allow specific and sensitive measurement of PPAR ligand activities and are a high-throughput, cell-based screening tool for identifying and characterizing PPAR ligands.

SHP represses transcriptional activity via recruitment of histone deacetylases.

The orphan receptor short heterodimer partner (SHP) is a common partner for a great number of nuclear receptors, and it plays an important role in many diverse physiological events. In a previous study, we described SHP as a strong repressor of the androgen receptor (AR). Herein, we addressed the mechanism of action of its negative activity on transcription. We first investigated the intrinsic repressive potential of SHP and mapped two core repressive domains to the amino acids 170-210 and 210-240. From GST pull-down assays, we demonstrated a direct interaction between SHP and diverse histone deacetylases (HDACs) as well as a strong interaction between HDAC1 and SHP inhibitory domains. We further supported the evidence for an interaction between SHP and HDAC1 by showing their co-immunoprecipitation and provided evidence for the existence of a ternary complex comprising AR, SHP, and HDAC1. The use of trichostatin A (TSA), a specific inhibitor of HDAC activity, confirmed that HDACs significantly contribute to the intrinsic transrepressive activity of SHP. Finally, we showed that TSA reversed SHP-induced repression of AR, further emphasizing the relevance of the interaction between SHP and HDACs. This latter action affected in a very similar manner SHP-mediated repression of estrogen receptor alpha (ERalpha) transactivation. Altogether, our results indicate that SHP mediates most of its repressive effect through recruitment of HDACs and suggest that the physiological actions of SHP could be affected by HDAC inhibitors.

In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters.

Reporter gene technology is widely used to measure activity of hormone analogs, and bioluminescent in vitro assays have allowed rapid screening of numerous chemicals either to identify new agonists or antagonists of hormones or to detect the presence of endocrine disrupters in the environment. Stable bioluminescent cell lines have been established and they provide reproducible dose-response curves and accurate determination of in vitro efficiencies of various chemicals. In vivo, however, these molecules can be metabolized, bound by proteins, or stored in fats and thus could display efficiencies different from those observed in vitro. In vivo assays, such as the uterotrophic bioassay, require numerous sacrificed animals, and responses not only are dependent on an estrogenic action but also imply other factors. For a faster assay and to avoid the use of numerous animals, we developed an in vivo biosensor constituted of stable bioluminescent cells implanted in nude mice. MCF-7 bioluminescent cell lines were chosen since their proliferation is low in the absence of estrogen and the xenograft size can thus be stable for several weeks. Luciferase gene expression was monitored noninvasively with a cooled charge-coupled device camera. Quantitative analysis allowed us to compare in vitro and in vivo actions of different estrogenic compounds (estradiol, estrone) and endocrine disruptors (ethynylestradiol, genistein, octylphenol, and 2,4'-dichlorodiphenyldichloroethylene) in the same cell lines and to follow hormone action on a living animal as a function of time. Different administration protocols have been used and good correlation was observed for most products. However, we found that ethynylestradiol was the most efficient chemical when orally administered.

Binding of estrogenic compounds to recombinant estrogen receptor-alpha: application to environmental analysis.

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-alphaligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-alpha and whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-alpha to separate ligands for ER and AhR that are present in river sediments. Immobilized ER-alpha, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis.

Catalytically inactive human cathepsin D triggers fibroblast invasive growth.

The aspartyl-protease cathepsin D (cath-D) is overexpressed and hypersecreted by epithelial breast cancer cells and stimulates their proliferation. As tumor epithelial-fibroblast cell interactions are important events in cancer progression, we investigated whether cath-D overexpression affects also fibroblast behavior. We demonstrate a requirement of cath-D for fibroblast invasive growth using a three-dimensional (3D) coculture assay with cancer cells secreting or not pro-cath-D. Ectopic expression of cath-D in cath-D-deficient fibroblasts stimulates 3D outgrowth that is associated with a significant increase in fibroblast proliferation, survival, motility, and invasive capacity, accompanied by activation of the ras-MAPK pathway. Interestingly, all these stimulatory effects on fibroblasts are independent of cath-D proteolytic activity. Finally, we show that pro-cath-D secreted by cancer cells is captured by fibroblasts and partially mimics effects of transfected cath-D. We conclude that cath-D is crucial for fibroblast invasive outgrowth and could act as a key paracrine communicator between cancer and stromal cells, independently of its catalytic activity.

Protein tyrosine phosphatases and breast cancer.

Protein tyrosine phosphatases (PTPs) consist of a large family of related enzymes, including the group of classical PTPs with its two main subgroups, the transmembrane receptor-type (RPTPs) and the intracellular or non-transmembrane PTPs. Published data on the expression and function of a panel of these enzymes in normal and cancerous breast tissues are discussed in this review. While most studies, albeit on different enzymes, have tended to agree on the evidence for an increased PTP expression in breast cancer, any connection between PTP expression and the enzymes' role in cancer development and progression remains largely open to interpretation. Concomitant increases of protein tyrosine kinase (PTK) and PTP activities in many cancers further indicate that a complex dysregulation in the balance of tyrosine phosphorylation could be responsible for major alterations in various cellular processes controlling tissue homeostasis. In particular, any relationship between the expression of PTPs and their specific diverse roles in the regulation of cell growth and apoptosis in breast cancer needs to be addressed in major fundamental, preclinical and clinical studies.

Involvement of estrogen receptor beta in ovarian carcinogenesis.

Knockout and expression studies suggest that estrogen receptor beta (ERbeta) plays a prominent role in ovarian function and pathology. Moreover, ovarian cancers are characterized by high morbidity and low responsiveness to anti-estrogens. Here we demonstrate, using quantitative PCR to measure ERalpha and ERbeta levels in 58 ovarian cancer patients, that ERbeta expression decreased in cysts and ovarian carcinomas as compared with normal ovaries and that this decrease is attributable only to a selective loss in ERbeta expression during cancer progression. To address the question of a possible involvement of ERbeta in ovarian cancers, we restored ERalpha and ERbeta expression in two human ovarian cancer cell lines PEO14 (ERalpha-negative) and BG1 (ERalpha-positive) using adenoviral delivery. ERalpha, but not ERbeta, could induce progesterone receptor and fibulin-1C. Moreover, ERalpha and ERbeta had opposite actions on cyclin D1 gene regulation, because ERbeta down-regulated cyclin D1 gene expression, whereas ERalpha increased cyclin D1 levels. Interestingly, ERbeta expression strongly inhibited PEO14 and BG1 cell proliferation and cell motility in a ligand-independent manner, whereas ERalpha had no marked effect. Induction of apoptosis by ERbeta also contributed to the decreased proliferation of ovarian cancer cells, as shown by Annexin V staining. This study shows that ERbeta is an important regulator of proliferation and motility of ovarian cancer and provides the first evidence for a proapoptotic role of ERbeta. The loss of ERbeta expression may thus be an important event leading to the development of ovarian cancer.

[PTPL1, a proapoptotic protein tyrosine phosphatase in breast cancers]. / PTPL1, une protéine tyrosine phosphatase proapoptotique dans les cancers mammaires.

The protein tyrosine phosphatase L1 (PTPL1), also known as FAP1, has two major types of remarkable structural domains, in addition to its catalytic unit: a FERM domain which is responsible for its localization at the apical pole of the cell plasma membrane and 5 PDZ domains suggestive of numerous possibilities of protein partners and consequently of a role as a cargo protein or an integrator between different signalling pathways. In fact, though it was initially suggested, in 1995, that this enzyme acts as an inhibitor of Fas death receptor several recent studies indicate that PTPL1 plays many other roles. It dephosphorylates Ephrin B (ligand of Eph, a receptor triggering angiogenesis and axonal guidance), it interacts with numerous proteins associated to cytoskeleton plasticity and it is implicated in cytokinesis. We have demonstrated that its expression is regulated by antiestrogens in mammary cancer and shown, with stable antisense transfectants, that PTPL1 plays a key role in the mediation of the inhibitory effects of these antagonists on growth factor signalling by impeding the IRS-I/PI3-K/Akt survival pathway. Altogether PTPL1 has to be regarded as a unique marker of mammary tumor response to antiestrogens and a potential therapeutic target to activate apoptotic stimuli in tumor cells.

Mechanisms underlying differential expression of interleukin-8 in breast cancer cells.

We have recently reported that interleukin-8 (IL-8) expression was inversely correlated to estrogen receptor (ER) status and was overexpressed in invasive breast cancer cells. In the present study, we show that IL-8 overexpression in breast cancer cells involves a higher transcriptional activity of IL-8 gene promoter. Cloning of IL-8 promoter from MDA-MB-231 and MCF-7 cells expressing high and low levels of IL-8, respectively, shows the integrity of the promoter in both cell lines. Deletion and site-directed mutagenesis of the promoter demonstrate that NF-kappaB and AP-1 and to a lesser extent C/EBP binding sites play a crucial role in the control of IL-8 promoter activity in MDA-MB-231 cells. Knockdown of NF-kappaB and AP-1 activities by adenovirus-mediated expression of an NF-kappaB super-repressor and RNA interference, respectively, decreased IL-8 expression in MDA-MB-231 cells. On the contrary, restoration of Fra-1, Fra-2, c-Jun, p50, p65, C/EBPalpha and C/EBPbeta expression levels in MCF-7 cells led to a promoter activity comparable to that observed in MDA-MB-231 cells. Our data constitute the first extensive study of IL-8 gene overexpression in breast cancer cells and suggest that the high expression of IL-8 in invasive cancer cells requires a complex cooperation between NF-kappaB, AP-1 and C/EBP transcription factors.

Multiple domains of the Receptor-Interacting Protein 140 contribute to transcription inhibition.

In this study, we have investigated the role of C-terminal binding proteins (CtBPs) and histone deacetylases (HDACs) in the repressive activity of the nuclear receptor cofactor Receptor-Interacting Protein 140 (RIP140). We have defined the interaction of both CtBP1 and CtBP2 with RIP140 and delineated two motifs (PIDLS and PINLS) differentially required for in vitro interaction. Using different approaches (titration of endogenous CtBPs, mutagenesis and transfection in CtBP knock-out cells), we find that recruitment of CtBPs only partially explains the negative regulation exerted by RIP140. We then demonstrate that RIP140 associates in vitro not only with class I HDACs but also with class II enzymes such as HDAC5. This interaction mainly involves the N-terminus of RIP140 (residues 27-199) and two domains of HDAC5. Moreover, the two proteins functionally interfere in transfection experiments, and confocal microscopy indicates that they co-localize in the nucleus. Interestingly, using the specific HDAC inhibitor trichostatin A, we show that HDAC activity is dispensable for active transrepression by RIP140. Finally, we demonstrate that the C-terminal region of RIP140 contains two additional silencing domains and confers strong active transrepression independently of HDAC activity and CtBPs. Altogether, these data indicate that transcriptional inhibition by the cofactor RIP140 involves complex mechanisms relying on multiple domains and partners.

Identification of genes involved in growth inhibition of breast cancer cells transduced with estrogen receptor.

Estrogen receptor alpha (ERalpha)-negative breast cancer cells display an aggressive phenotype. We previously showed that adenoviral expression of ERalpha in ER-negative breast cancer cells leads to an estrogen-dependent down-regulation of the proliferation, which could be of interest to control the growth of such cells. In this study, we observed an increase in protein levels of p21 and p27 cyclin-dependent kinase inhibitors, whereas pRb phosphorylation is strongly decreased. Flow cytometry experiments showed a slower transit of cells in G1 (hormone-independent), a hormone-induced accelerated transit through S phase and a possible arrest in G2/M phase. In addition, ERalpha-expressing cells were undergoing apoptosis. By using cDNA macroarrays, we identified a novel collection of genes regulated by liganded ERalpha potentially regulating cell cycle, apoptosis, cell signalling, stress response and DNA repair.

Comparative transductions of breast cancer cells by three DNA viruses.

Defining the ideal vectors to transduce breast cancer using viruses is currently under intense pre-clinical evaluation. Our study constitutes the first direct comparison of the infection efficiencies of a human serotype 5 (Ad5), a canine serotype 2 (CAV-2) adenovirus, and a human serotype 2 adeno-associated virus (AAV-2) in breast cancer cells. We observed an excellent infection efficiency for Ad5 vector, whereas both CAV-2 and AAV-2 vectors lead to low infection of these cells. Real-time PCR, flow cytometry, and antibody blocking studies suggest that Ad5 and CAV-2 infection ability is not strictly dependent on coxsackie adenovirus receptor (CAR) or alpha(v) integrin levels. In conclusion, our data suggest that human adenoviruses are excellent transducers of breast cancer cells, though it may be difficult to predict the extent of infection solely on CAR or alpha(v) integrin levels.

Localization of BRCA1 protein in human breast cancer cells.

There is still an ongoing debate concerning the cellular localization of BRCA1 protein in breast cancer. To address this question, we compared the localization of BRCA1 protein using several monoclonal (Ab-1) or polyclonal (C20, D20, I20) antibodies under different technical conditions on human breast cancer cell lines. We worked on the fixation and permeabilization conditions in order to preserve the morphological structures of the cells, as confirmed by transmission electron microscopy studies. As expected from the gene sequence analysis and the biochemical features, both nucleus and cytoplasmic BRCA1 protein staining were detected in cells fixed for 60 min in 4% paraformaldehyde and permeabilized with either 0.3% saponin or 0.02% Triton. In these conditions, the same results were obtained: (i) with the four antibodies tested, (ii) with several dilutions (up to tenfold) of the monoclonal antibody, and (iii) in all the tested breast cancer cell lines. In addition, we validated the functionality of these conditions by quantifying the effects of estrogens and their antagonists on the regulation of BRCA1 protein expression in the MCF7 cell line.

Membrane targeting of protein tyrosine phosphatase PTPL1 through its FERM domain via binding to phosphatidylinositol 4,5-biphosphate.

PTPL1 is the largest known cytoplasmic protein tyrosine phosphatase (PTP) containing a FERM (four point-1, ezrin, radixin and moesin) domain. Enzyme localization and PTP-substrate specificity are thought to play crucial roles in the regulation of PTP activity, which determines their functions. Here we report that PTPL1 is predominantly localized at the apical face of plasma membrane enriched in dorsal microvilli when expressed in HeLa cells. By comparing localization of the full-length enzyme with its FERM domain or FERM-deleted PTPL1 construct, we first concluded that PTPL1-FERM domain is necessary and sufficient to address the wild-type enzyme at the membrane. Two potential phosphatidylinositol 4,5-biphosphate [PtdIns(4,5)P2]-binding motifs were identified within the PTPL1-FERM sequence. We further showed that mutation of both sites altered PTPL1 localization similarly to FERM domain deletion, and impaired its subcellular distribution as confirmed biochemically by cell-fractionation experiments. Using protein-lipid overlays, we demonstrated an interaction of the FERM domain of PTPL1 with PtdIns(4,5)P2, which was lost after mutation of potential PtdIns(4,5)P2-binding motifs. Moreover, neomycin, which masks PtdIns(4,5)P2 polar heads, was shown to decrease by 50% the association of PTPL1 with the cytoskeletal fraction. These results identify the crucial role of the FERM domain in PTPL1 intracellular targeting and demonstrate that localization of PTPL1 is regulated by phosphoinositide metabolism.

IL-8 expression and its possible relationship with estrogen-receptor-negative status of breast cancer cells.

Estrogen-receptor (ER) status is an important parameter in breast cancer management as ER-positive breast cancers have a better prognosis than ER-negative tumors. This difference comes essentially from the lower aggressiveness and invasiveness of ER-positive tumors. Here, we demonstrate, that interleukin-8 (IL-8) was clearly overexpressed in most ER-negative breast, ovary cell lines and breast tumor samples tested, whereas no significant IL-8 level could be detected in ER-positive breast or ovarian cell lines. We have also cloned human IL-8 from ER-negative MDA-MB-231 cells, and we show that IL-8 produced by breast cancer cells is identical to monocyte-derived IL-8. Interestingly, the invasion potential of ER-negative breast cancer cells is associated at least in part with expression of IL-8, but not with IL-8 receptor levels. Moreover, IL-8 increases the invasiveness of ER-positive breast cancer cells by two fold, thus confirming the invasion-promoting role of IL-8. On the other hand, exogenous expression of estrogen receptors in ER-negative cells led to a decrease of IL-8 levels. In summary, our data show that IL-8 expression is negatively linked to ER status of breast and ovarian cancer cells. We also support the idea that IL-8 expression is associated with a higher invasiveness potential of cancer cells in vitro, which suggests that IL-8 could be a novel marker of tumor aggressiveness.

Protein-tyrosine phosphatase PTPL1/FAP-1 triggers apoptosis in human breast cancer cells.

Studies in Jurkat leukemia cells have suggested that protein-tyrosine phosphatase PTPL1/FAP-1 rescues Fas-induced cell death. However, we have previously shown that this enzyme triggers 4-hydroxytamoxifen-induced growth inhibition in human breast cancer cells. The present study addresses the role of PTPL1/FAP-1 in antiestrogen-regulated apoptotic effect and insulin-like growth factor-I survival action in MCF7 cells and further identifies the impacted signaling pathway. By terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and cytoplasmic nucleosome enzyme-linked immunosorbent assay, we demonstrated that 4-hydroxytamoxifen-induced apoptosis was totally lost in PTPL1/FAP-1 antisense transfectants in which enzyme expression was abrogated, revealing the crucial role of this phosphatase in the apoptotic process in human breast cancer cells. Time-dependent expression of PTPL1/FAP-1 in MCF7 cells completely abolished the survival action of insulin-like growth factor-I. This effect occurred through a highly significant reduction in phosphatidylinositol 3-kinase/Akt pathway activation (80% reduction in phosphatidylinositol 3-kinase activity, 55% inhibition of Akt activation) accompanied by a 65% decrease in insulin receptor substrate-1 growth factor-induced tyrosine phosphorylation. These results provide the first evidence that PTPL1/FAP-1 has a key role in the apoptotic process in human breast cancer cells independent of Fas but associated with an early inhibition of the insulin receptor substrate-1/phosphatidylinositol 3-kinase pathway. Our data therefore suggest new therapeutic routes and strengthen the importance of identifying endogenous regulators and substrates of this phosphatase in breast tumors.