Amilcar Flores-Morales

SOCS2 negatively regulates growth hormone action in vitro and in vivo

Mice deficient in SOCS2 display an excessive growth phenotype characterized by a 30-50% increase in mature body size. Here we show that the SOCS2-/- phenotype is dependent upon the presence of endogenous growth hormone (GH) and that treatment with exogenous GH induced excessive growth in mice lacking both endogenous GH and SOCS2. This was reflected in terms of overall body weight, body and bone lengths, and the weight of internal organs and tissues. A heightened response to GH was also measured by examining GH-responsive genes expressed in the liver after exogenous GH administration. To further understand the link between SOCS2 and the GH-signaling cascade, we investigated the nature of these interactions using structure/function and biochemical interaction studies. Analysis of the 3 structural motifs of the SOCS2 molecule revealed that each plays a crucial role in SOCS2 function, with the conserved SOCS-box motif being essential for all inhibitory function. SOCS2 was found to bind 2 phosphorylated tyrosines on the GH receptor, and mutational analysis of these amino acids showed that both were essential for SOCS2 function. Together, the data provide clear evidence that SOCS2 is a negative regulator of GH signaling.

Androgen Induction of Prostate Cancer Cell Invasion Is Mediated by Ezrin

Ezrin is a key signaling molecule that regulates cell survival, adhesion migration, and invasion. We have previously shown that ezrin is regulated by androgen in rat prostate and that its expression is increased in prostate cancer and in prostate intraepithelial neoplasia. We have used the androgen-sensitive cell line LNCaP-FGC to investigate the role of ezrin in androgen-induced cell invasion. We found that androgen treatment of LNCaP-FGC cells induces ezrin expression, an effect that is inhibited by the androgen receptor antagonist, bicalutamide. In addition, androgen treatment induces the phosphorylation of ezrin in Thr-567 and Tyr-353 in a sequential manner. This is mediated through protein kinase C α and Src tyrosine kinase, respectively. Androgen treatment induces the translocation of both protein kinase C α and ezrin to the cell membrane and their association. Inhibition of ezrin function using short interference RNA or the overexpression of T567A and Y353F-ezrin mutants significantly reduces androgen-induced Matrigel invasion but does not affect cell proliferation or cell adhesion. Matrigel invasion of the androgen-insensitive prostate cancer cell lines PC-3 and LNCaP-R is also dependent on ezrin. In summary, we have shown that androgens regulate ezrin at transcriptional and posttranscriptional levels. Hormonal regulation of ezrin phosphorylation is required for androgen-induced cell invasion.

REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer

The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds chromatin regions containing well-characterized cis-elements known to mediate REST transcriptional repression, while cell imaging studies confirmed that REST and AR closely co-localize in vivo. Androgen-induced gene repression also involves modulation of REST protein turnover through actions on the ubiquitin ligase β-TRCP. Androgen deprivation or AR blockage with inhibitor MDV3100 (Enzalutamide) leads to neuroendocrine (NE) differentiation, a phenomenon that is mimicked by REST inactivation. Gene expression profiling revealed that REST not only acts to repress neuronal genes but also genes involved in cell cycle progression, including Aurora Kinase A, that has previously been implicated in the growth of NE-like castration-resistant tumors. The analysis of prostate cancer tissue microarrays revealed that tumors with reduced expression of REST have higher probability of early recurrence, independently of their Gleason score. The demonstration that REST modulates AR actions in prostate epithelia and that REST expression is negatively correlated with disease recurrence after prostatectomy, invite a deeper characterization of its role in prostate carcinogenesis.

Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3

Background: Flt3 is an important regulator of hematopoiesis and is often found mutated and constitutively active in patients with acute myeloid leukemia. Results: SOCS6 is up-regulated by Flt3 activation and binds to phosphorylated Flt3. Conclusion: SOCS6 is a negative regulator of Flt3 signaling. Significance: Our results provide a role for SOCS6 in Flt3 signaling. The absence of SOCS6 promotes transformation of cells by Flt3 ITD. The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia. SOCS6 (suppressor of cytokine signaling 6) is a member of the SOCS family of E3 ubiquitin ligases that can regulate receptor tyrosine kinase signal transduction. In this study, we analyzed the role of SOCS6 in Flt3 signal transduction. The results show that ligand stimulation of Flt3 can induce association of SOCS6 and Flt3 and tyrosine phosphorylation of SOCS6. Phosphopeptide fishing indicated that SOCS6 binds directly to phosphotyrosines 591 and 919 of Flt3. By using stably transfected Ba/F3 cells with Flt3 and/or SOCS6, we show that the presence of SOCS6 can enhance ubiquitination of Flt3, as well as internalization and degradation of the receptor. The presence of SOCS6 also induces weaker activation of Erk1/2, but not Akt, in transfected Ba/F3 and UT-7 cells and in OCI-AML-5 cells. The absence of SOCS6 promotes Ba/F3 and UT-7 cell proliferation induced by oncogenic internal tandem duplications of Flt3. Taken together, these results suggest that SOCS6 negatively regulates Flt3 activation, the downstream Erk signaling pathway, and cell proliferation.

Structural Basis for c-KIT Inhibition by the Suppressor of Cytokine Signaling 6 (SOCS6) Ubiquitin Ligase

The c-KIT receptor tyrosine kinase mediates the cellular response to stem cell factor (SCF). Whereas c-KIT activity is important for the proliferation of hematopoietic cells, melanocytes and germ cells, uncontrolled c-KIT activity contributes to the growth of diverse human tumors. Suppressor of cytokine signaling 6 (SOCS6) is a member of the SOCS family of E3 ubiquitin ligases that can interact with c-KIT and suppress c-KIT-dependent pathways. Here, we analyzed the molecular mechanisms that determine SOCS6 substrate recognition. Our results show that the SH2 domain of SOCS6 is essential for its interaction with c-KIT pY568. The 1.45-Å crystal structure of SOCS6 SH2 domain bound to the c-KIT substrate peptide (c-KIT residues 564–574) revealed a highly complementary and specific interface giving rise to a high affinity interaction (Kd = 0.3 μm). Interestingly, the SH2 binding pocket extends to substrate residue position pY+6 and envelopes the c-KIT phosphopeptide with a large BG loop insertion that contributes significantly to substrate interaction. We demonstrate that SOCS6 has ubiquitin ligase activity toward c-KIT and regulates c-KIT protein turnover in cells. Our data support a role of SOCS6 as a feedback inhibitor of SCF-dependent signaling and provides molecular data to account for target specificity within the SOCS family of ubiquitin ligases.

SOCS proteins in regulation of receptor tyrosine kinase signaling.

Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that play critical roles in signal transduction from extracellular stimuli. Many in this family of kinases are overexpressed or mutated in human malignancies and thus became an attractive drug target for cancer treatment. The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar proteins, SOCS1–7, and cytokine-inducible SH2-containing protein (CIS). A key feature of this family of proteins is the presence of an SH2 domain and a SOCS box. Recent studies suggest that SOCS proteins also play a role in RTK signaling. Activation of RTK results in transcriptional activation of SOCS-encoding genes. These proteins associate with RTKs through their SH2 domains and subsequently recruit the E3 ubiquitin machinery through the SOCS box, and thereby limit receptor stability by inducing ubiquitination. In a similar fashion, SOCS proteins negatively regulate mitogenic signaling by RTKs. It is also evident that RTKs can sometimes bypass SOCS regulation and SOCS proteins can even potentiate RTKs-mediated mitogenic signaling. Thus, apart from negative regulation of receptor signaling, SOCS proteins may also influence signaling in other ways.

SOCS2 deletion protects against hepatic steatosis but worsens insulin resistance in high-fat-diet-fed mice

Hepatic steatosis is a prominent feature in patients with growth hormone (GH) deficiency. The ubiquitin ligase SOCS2 attenuates hepatic GH signaling by inhibiting the Janus kinase 2 (JAK2)‐signal transducer and activator of transcription 5b (STAT5b) axis. Here, we investigated the role of SOCS2 in the development of diet‐induced hepatic steatosis and insulin resistance. SOCS2‐knockout (SOCS2−/−) mice and wild‐type littermates were fed for 4 mo with control or high‐fat diet, followed by assessment of insulin sensitivity, hepatic lipid content, and expression of inflammatory cytokines. SOCS2−/− mice exhibited increased hepatic TG secretion by 77.6% (P< 0.001) as compared with wild‐type control mice and were protected from high‐fat‐diet (HFD)‐induced hepatic steatosis, showing 49.3% (P<0.01) reduction in liver TG levels compared to HFD‐fed wild‐type littermates. In contrast, we found that HFD‐triggered attenuation of systemic insulin sensitivity was more marked in SOCS2−/− mice. Livers from the HFD‐fed SOCS2−/− mice showed increased NF‐κB activity as well as elevated expression of genes for the inflammatory cytokines IFN‐γ and IL‐6. An inhibitory role of SOCS2 on Toll‐like receptor 4 signaling was demonstrated in macrophages obtained from the SOCS2−/− and wild‐type mice. This study identified SOCS2 as an important regulator of hepatic homeostasis under conditions of high‐fat dietary stress.—Zadjali, F., Santana‐Farre, R., Vesterlund, M., Carow, B., Mirecki‐Garrido, M., Hernandez‐Hernandez, I., Flodström‐Tullberg, M., Parini, P., Rottenberg, M., Norstedt, G., Fernandez‐Perez, L., Flores‐Morales, A. SOCS2 deletion protects against hepatic steatosis but worsens insulin resistance in high‐fat‐diet‐fed mice. FASEB J. 26, 3282–3291 (2012). www.fasebj.org

Role of Pituitary Hormones on 17α-Ethinylestradiol-Induced Cholestasis in Rat

Estrogens cause intrahepatic cholestasis in susceptible women during pregnancy, after administration of oral contraceptives, or during postmenopausal hormone replacement therapy. 17α-Ethinylestradiol (EE) is a synthetic estrogen widely used to cause experimental cholestasis in rodents with the aim of examining molecular mechanisms involved in this disease. EE actions on the liver are thought to be mediated by estrogen receptor α (ERα) and pituitary hormones. We tested this hypothesis by analyzing metabolic changes induced by EE in livers from hypophysectomized (HYPOX) and hypothyroid rats. Microarray studies revealed that the number of genes regulated by EE was increased almost 4-fold in HYPOX rat livers compared with intact males. Little overlap was apparent between the effects of EE in intact and HYPOX rats, demonstrating that pituitary hormones play a critical role in the hepatic effects of EE. Consistently, hypophysectomy protects the liver against induction by EE of serum bilirubin and alkaline phosphatase, two markers of cholestasis and hepatotoxicity and modulates the effects of EE on several genes involved in bile acid homeostasis (e.g., FXR, SHP, BSEP, and Cyp8b1). Finally, we demonstrate a novel mechanism of action of EE through binding and negative regulation of glucocorticoid receptor-mediated transcription. In summary, pituitary- and ERα-independent mechanisms contribute to development of EE-induced changes in liver transcriptome. Such mechanisms may be relevant when this model of EE-induced cholestasis is evaluated. The observation that the pharmacological effects of estrogen in liver differ in the absence or presence of the pituitary could be clinically relevant, because different drugs that block actions of pituitary hormones are now available.

SOCS2 mediates the cross talk between androgen and growth hormone signaling in prostate cancer

UNLABELLEDnAnabolic signals such as androgens and the growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis play an essential role in the normal development of the prostate but also in its malignant transformation. In this study, we investigated the role of suppressor of cytokine signaling 2 (SOCS2) as mediator of the cross talk between androgens and GH signals in the prostate and its potential role as tumor suppressor in prostate cancer (PCa). We observed that SOCS2 protein levels assayed by immunohistochemistry are elevated in hormone therapy-naive localized prostatic adenocarcinoma in comparison with benign tissue. In contrast, however, castration-resistant bone metastases exhibit reduced levels of SOCS2 in comparison with localized or hormone naive, untreated metastatic tumors. In PCa cells, SOCS2 expression is induced by androgens through a mechanism that requires signal transducer and activator of transcription 5 protein (STAT5) and androgen receptor-dependent transcription. Consequentially, SOCS2 inhibits GH activation of Janus kinase 2, Src and STAT5 as well as both cell invasion and cell proliferation in vitro. In vivo, SOCS2 limits proliferation and production of IGF-1 in the prostate in response to GH. Our results suggest that the use of GH-signaling inhibitors could be of value as a complementary treatment for castration-resistant PCa.nnnSUMMARYnAndrogen induced SOCS2 ubiquitin ligase expression and inhibited GH signaling as well as cell proliferation and invasion in PCa, whereas reduced SOCS2 was present in castration-resistant cases. GH-signaling inhibitors might be a complementary therapeutic option for advanced PCa.

The Proteome of Primary Prostate Cancer.

BACKGROUNDnClinical management of the prostate needs improved prognostic tests and treatment strategies. Because proteins are the ultimate effectors of most cellular reactions, are targets for drug actions and constitute potential biomarkers; a quantitative systemic overview of the proteome changes occurring during prostate cancer (PCa) initiation and progression can result in clinically relevant discoveries.nnnOBJECTIVESnTo study cellular processes altered in PCa using system-wide quantitative analysis of changes in protein expression in clinical samples and to identify prognostic biomarkers for disease aggressiveness.nnnDESIGN, SETTING, AND PARTICIPANTSnMass spectrometry was used for genome-scale quantitative proteomic profiling of 28 prostate tumors (Gleason score 6-9) and neighboring nonmalignant tissue in eight cases, obtained from formalin-fixed paraffin-embedded prostatectomy samples. Two independent cohorts of PCa patients (summing 752 cases) managed by expectancy were used for immunohistochemical evaluation of proneuropeptide-Y (pro-NPY) as a prognostic biomarker.nnnRESULTS AND LIMITATIONSnOver 9000 proteins were identified as expressed in the human prostate. Tumor tissue exhibited elevated expression of proteins involved in multiple anabolic processes including fatty acid and protein synthesis, ribosomal biogenesis and protein secretion but no overt evidence of increased proliferation was observed. Tumors also showed increased levels of mitochondrial proteins, which was associated with elevated oxidative phosphorylation capacity measured in situ. Molecular analysis indicated that some of the proteins overexpressed in tumors, such as carnitine palmitoyltransferase 2 (CPT2, fatty acid transporter), coatomer protein complex, subunit alpha (COPA, vesicle secretion), and mitogen- and stress-activated protein kinase 1 and 2 (MSK1/2, protein kinase) regulate the proliferation of PCa cells. Additionally, pro-NPY was found overexpressed in PCa (5-fold, p<0.05), but largely absent in other solid tumor types. Pro-NPY expression, alone or in combination with the ERG status of the tumor, was associated with an increased risk of PCa specific mortality, especially in patients with Gleason score ≤ 7 tumors.nnnCONCLUSIONSnThis study represents the first system-wide quantitative analysis of proteome changes associated to localized prostate cancer and as such constitutes a valuable resource for understanding the complex metabolic changes occurring in this disease. We also demonstrated that pro-NPY, a protein that showed differential expression between high and low risk tumors in our proteomic analysis, is also a PCa specific prognostic biomarker associated with increased risk for disease specific death in patients carrying low risk tumors.nnnPATIENT SUMMARYnThe identification of proteins whose expression change in prostate cancer provides novel mechanistic information related to the disease etiology. We hope that future studies will prove the value of this proteome dataset for development of novel therapies and biomarkers.