Jorge Martín-Pérez

Cytoplasmic localization of p120ctn and E-cadherin loss characterize lobular breast carcinoma from preinvasive to metastatic lesions

Accumulating evidences indicate that p120 catenin, a member of the E-cadherin (E-CD)/catenin adhesion complex, plays a role in tumor invasion. To establish the expression pattern of p120 in breast cancer, we analysed 326 breast tissue biopsies by tissue microarray. Most of the lobular tumors (88%) showed exclusive cytoplasmic localization, and 6% of them also had p120 nuclear staining. Cytoplasmic p120 strongly associated with complete loss of E-CD and β-catenin not only in lobular carcinoma and its metastases but also in atypical lobular hyperplasias. In the latter, loss of heterozygosity of E-CD gene was also observed. Complete loss of E-CD and cytoplasmic and nuclear p120 staining was also observed in primary lobular cancer cell cultures generated by us. In ductal tumors, by contrast, reduction of p120 and E-CD in membrane was very common (57 and 53%, respectively), whereas cytoplasmic p120 staining was rarely seen. This simultaneous reduction of membranous E-CD and p120 was not associated with increased Src kinase activity. To demonstrate that cytoplasmic p120 localization was a consequence of the absence of E-CD, the endogenous E-CD was re-expressed in MDA-231 cells by 5-Aza-2′-deoxycytidine (5Aza) treatment. After treatment, p120 shifted from the cytoplasm to the membrane, where it colocalized with endogenous E-CD. Additionally, suppressing E-CD expression in Madin–Darby canine kidney cells by stable transfection of the transcriptional repressors Snail, E47 or Slug, provokes p120 cytoplasmic localization and p120 isoform switching. In conclusion, abnormal cytoplasmic and nuclear localization of p120, which are mediated by the absence of E-CD, characteristically occur in the early stages of lobular breast cancer and are maintained during tumor progression to metastasis. Consequently, p120 may be an important mediator of the oncogenic effects derived from E-CD inactivation, including enhanced motility and invasion, in lobular breast cancer.

Protein kinase Akt/PKB phosphorylates heme oxygenase-1 in vitro and in vivo.

Heme oxygenase‐1 (HO‐1) is a stress response protein that protects cells against diverse noxious stimuli. Although regulation of HO‐1 occurs mainly at the transcriptional level, its posttranslational modifications remain unexplored. We have identified a putative consensus sequence for phosphorylation by Akt/PKB of HO‐1 at Ser188. Recombinant human and rat HO‐1, but not mutant HO‐1(S188A), are phosphorylated in vitro by Akt/PKB. Isotopic 32P‐labeling of HEK293T cells confirmed that HO‐1 is a phosphoprotein and that the basal HO‐1 phosphorylation is increased by Akt1 activation. HO‐1(S188D), a single point mutant equivalent to the phosphorylated protein, exhibited over 1.6‐fold higher activity than wild type HO‐1. Fluorescence resonance energy transfer (FRET) studies indicated that HO‐1(S188D) bound to cytochrome P450 reductase (CPR) and biliverdin reductase (BVR) with a slightly lower K d than wild‐type HO‐1. Although the changes in activity are small, this study provides the first evidence for a role of the survival kinase Akt in the regulation of HO‐1.

Role of c-Src in Human MCF7 Breast Cancer Cell Tumorigenesis

To study the role of c-Src in breast cancer tumorigenesis, we generated a cell line derived from MCF7 carrying an inducible dominant negative c-Src (c-SrcDN: K295M/Y527F) under tetracycline control (Tet-On system). c-SrcDN expression caused phenotypic changes, relocation of c-Src, Fak, and paxillin, and loss of correct actin fiber assembly. These alterations were coupled to increased Fak-Tyr397 autophosphorylation and to inhibition of Fak-Tyr925, p130CAS, and paxillin phosphorylation. An increased association of total Src with Fak and a decreased interaction of p130CAS and p85-PI3K with Fak were also observed. SrcDN inhibited cell attachment, spreading, and migration. Serum and EGF-induced stimulation of cell proliferation and Akt phosphorylation were also significantly reduced by SrcDN, whereas p27Kip1 expression was increased. Consistently, silencing c-Src expression by siRNA in MCF7 cells significantly reduced cell migration, attachment, spreading and proliferation. Inoculation of MCF7 cells carrying inducible SrcDN to nude mice generated tumors. However, doxycycline administration to mice significantly reduced tumorigenesis, and when doxycycline treatment was installed after tumor development, a significant tumor regression was observed. In both situations, inhibition of tumorigenesis was associated with decreased Ki67 staining and increased apoptosis in tumors. These data undoubtedly demonstrate the relevance of the Src/Fak complex in breast cancer tumorigenesis.

Inhibitory action of a met-enkephalin on ACTH release in man.

In order to study the mechanism of action of a Met-enkephalin (FK 33824) on the pituitary-adrenal axis eight normal male volunteers were subjected to an ACTH stimulation test. Lysine-vasopressing (LVP), 5 IU, was injected intramuscularly after pretreatment with 0.5 mg FK 33824 i.m. or a placebo. In six of the subjects the opiate was again administered preceding a single injection of 0.25 mg ACTH beta 1-24 i.m. Blood was collected at regular intervals and ACTH and cortisol concentrations analyzed in all samples. LVP induced significant plasma ACTH (P < 0.05) and cortisol (P < 0.001) increases. Pretreatment with FK 33824 completely antagonized the effect of LVP. Furthermore, the cortisol elevation after exogenous ACTH was not modified by previous administration of FK 33824. It is concluded that the Met-enkephalin derivative FK 33824 directly suppresses ACTH release from the pituitary without influencing adrenal synthesis of cortisol.

Prolactin induces c-Myc expression and cell survival through activation of Src/Akt pathway in lymphoid cells

Stimulation of resting W53 cells (lymphoid murine cells expressing prolactin (PRL) receptor) by PRL induced expression of growth-related immediate-early genes (IEG), and proliferation through activation of the Src kinases. Since IEG are essential for cell cycle progression, we have studied how PRL controls expression of c-Myc mRNA and c-Fos. Stimulation of W53 cell proliferation by PRL required activation of MAPK, as the Mek1/2 inhibitor PD184352 eliminated Erk1/2 stimulation, cell proliferation, and expression of c-Fos mRNA. In contrast, PD184352 did not alter PRL activation of c-Myc mRNA expression or stimulation of p70S6K, Akt, and the Jak2/Stat5 pathway. Activation of the PI3K by PRL was necessary for the expression of c-MycmRNA and W53 cell proliferation, as the PI3K inhibitor LY294002 abolished them. However, it did not modify PRL stimulation of c-Fos mRNA expression or activation of Erk1/2 and Stat5. Furthermore, rapamycin, an inhibitor of mTOR and consequently of p70S6K, did not alter PRL stimulation of c-Myc and c-Fos mRNA expression and it had a very minor inhibitory effect on PRL stimulation of W53 cell proliferation. In addition, rapamycin did not affect PRL stimulation of Akt or Stat5. However, it reinforced PRL activation of Erk1/2. Overexpression of a constitutively activated Akt (myristoylated Akt) in W53 cells overcame the inhibitory effect of LY294002 on c-Myc expression, as well as cell death upon PRL deprivation. Consistently, inducible expression of Akt-CAAX Box in W53 cells caused inhibition of c-Myc expression. PRL stimulation of W53 cells resulted in Akt translocation to the nucleus, phosphorylation of FKHRL1 transcription factor, and its nuclear exclusion. In contrast, induced expression of Akt-CAAX Box caused inhibition of FKHRL1 phosphorylation. Furthermore, transient expression of nonphosphorylatable FKHRL1-A3 mutant impaired PRL-induced activation of the c-Myc promoter. Akt activation also resulted in phosphorylation and inhibition of glycogen synthetase kinase 3 (GSK3), which in turn promoted c-Myc stability. Consistently, treatment of W53 with selective inhibitors of GSK3 such as SB415286 and lithium salts resulted in increased levels of c-Myc. Also, overexpression of c-Myc in W53 cells overcame the decrease in cell proliferation induced by LY294002. These findings defined a PRL-signalling cascade in W53 cells, involving Src kinases/PI3K/Akt/FKHRL1-GSK3, that mediates stimulation of c-Myc expression.

Phosphorylation of Serine 11 and Serine 92 as New Positive Regulators of Human Snail1 Function: Potential Involvement of Casein Kinase-2 and the cAMP-activated Kinase Protein Kinase A

New phosphorylation sites in Snail1 have been identified with functional implications. Serines 11 and 92 participate in the control of Snail1 stability and positively regulate Snail1 repressive function and its interaction with mSin3A co-repressor. Furthermore, serines 11 and 92 are required for Snail1-mediated EMT and cell viability, respectively.

Protein tyrosine phosphatase 1B modulates GSK3β/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity

Acute hepatic failure secondary to acetaminophen (APAP) poisoning is associated with high mortality. Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of tyrosine kinase growth factor signaling. In the liver, this pathway confers protection against injury. However, the involvement of PTP1B in the intracellular networks activated by APAP is unknown. We have assessed PTP1B expression in APAP-induced liver failure in humans and its role in the molecular mechanisms that regulate the balance between cell death and survival in human and mouse hepatocytes, as well as in a mouse model of APAP-induced hepatotoxicity. PTP1B expression was increased in human liver tissue removed during liver transplant from patients for APAP overdose. PTP1B was upregulated by APAP in primary human and mouse hepatocytes together with the activation of c-jun (NH2) terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), resulting in cell death. Conversely, Akt phosphorylation and the antiapoptotic Bcl2 family members BclxL and Mcl1 were decreased. PTP1B deficiency in mouse protects hepatocytes against APAP-induced cell death, preventing glutathione depletion, reactive oxygen species (ROS) generation and activation of JNK and p38 MAPK. APAP-treated PTP1B−/− hepatocytes showed enhanced antioxidant defense through the glycogen synthase kinase 3 (GSK3)β/Src kinase family (SKF) axis, delaying tyrosine phosphorylation of the transcription factor nuclear factor-erythroid 2-related factor (Nrf2) and its nuclear exclusion, ubiquitination and degradation. Insulin-like growth factor-I receptor-mediated signaling decreased in APAP-treated wild-type hepatocytes, but was maintained in PTP1B−/− cells or in wild-type hepatocytes with reduced PTP1B levels by RNA interference. Likewise, both signaling cascades were modulated in mice, resulting in less severe APAP hepatotoxicity in PTP1B−/− mice. Our results demonstrated that PTP1B is a central player of the mechanisms triggered by APAP in hepatotoxicity, suggesting a novel therapeutic target against APAP-induced liver failure.

Stimulation of c-Src by prolactin is independent of Jak2.

Interaction of prolactin (PRL) with its receptor (PRLR) leads to activation of Jak and Src family tyrosine kinases. The PRL/growth hormone/cytokine receptor family conserves a proline-rich sequence in the cytoplasmic juxtamembrane region (Box 1) required for association and subsequent activation of Jaks. In the present work, we studied the mechanisms underlying c-Src kinase activation by PRL and the role that Jak2 plays in this process. PRL addition to chicken embryo fibroblasts (CEF) expressing the rat PRLR long form resulted in activation of c-Src and Jak2 and in tyrosine phosphorylation of the receptor. Receptor phosphorylation was due to associated Jak2, since in cells expressing either a Box 1 mutated PRLR (PRLR(4P-A)), which is unable to interact with Jak2, or a kinase-domain-deleted Jak2 (Jak2Deltak), PRL did not stimulate receptor phosphorylation. Interestingly, addition of PRL to cells expressing PRLR(4P-A) resulted in an activation of c-Src equivalent to that observed with the wild-type receptor. These findings indicate that PRL-mediated stimulation of c-Src was independent of Jak2 activation and of receptor phosphorylation. Our results suggest that PRL-activated Src could send signals to downstream cellular targets independently of Jak2.

MYC antagonizes the differentiation induced by imatinib in chronic myeloid leukemia cells through downregulation of p27 KIP1

Chronic myeloid leukemia (CML) progresses from a chronic to a blastic phase where the leukemic cells are proliferative and undifferentiated. The CML is nowadays successfully treated with BCR-ABL kinase inhibitors as imatinib and dasatinib. In the CML-derived K562 cell line, low concentrations of imatinib induce proliferative arrest and erythroid differentiation. We found that imatinib upregulated the cell cycle inhibitor p27KIP1 (p27) in a time- and -concentration dependent manner, and that the extent of imatinib-mediated differentiation was severely decreased in cells with depleted p27. MYC (c-Myc) is a transcription factor frequently deregulated in human cancer. MYC is overexpressed in untreated CML and is associated to poor response to imatinib. Using K562 sublines with conditional MYC expression (induced by Zn2+ or activated by 4-hydroxy-tamoxifen) we show that MYC prevented the erythroid differentiation induced by imatinib and dasatinib. The differentiation inhibition is not due to increased proliferation of MYC-expressing clones or enhanced apoptosis of differentiated cells. As p27 overexpression is reported to induce erythroid differentiation in K562, we explored the effect of MYC on imatinib-dependent induction of p27. We show that MYC abrogated the imatinib-induced upregulation of p27 concomitantly with the differentiation inhibition, suggesting that MYC inhibits differentiation by antagonizing the imatinib-mediated upregulation of p27. This effect occurs mainly by p27 protein destabilization. This was in part due to MYC-dependent induction of SKP2, a component of the ubiquitin ligase complex that targets p27 for degradation. The results suggest that, although MYC deregulation does not directly confer resistance to imatinib, it might be a factor that contributes to progression of CML through the inhibition of differentiation.

Activation of the unliganded estrogen receptor by prolactin in breast cancer cells

Both prolactin (PRL) and estrogen (E2) are involved in the pathogenesis and progression of mammary neoplasia, but the mechanisms by which these hormones interact to exert their effects in breast cancer cells are not well understood. We show here that PRL is able to activate the unliganded estrogen receptor (ER). In breast cancer cells, PRL activates a reporter plasmid containing estrogen response elements (EREs) and induces the ER target gene pS2. These actions are blocked by the antagonist ICI 182,780, showing that ER is required for the PRL-mediated effect. Moreover, PRL leads to phosphorylation of ERα in serine-118 (P-ERα), a modification related to the potentiation of ligand-independent transcriptional activation. In addition, PRL mimics the effect of E2 on target gene expression by inducing cyclical recruitment of ERα and P-ERα to ERE-containing promoters, resulting in recruitment of co-activators and acetylation of histone H3. Finally, PRL induces expression of c-Myc and Cyclin D1 and leads to increased cell proliferation, which is specifically antagonized by ICI 182,780 or ERα depletion. These results show that ligand-independent ERα activation appears to be an important component of the proliferative and transcriptional actions of PRL in breast cancer cells.