Eftihia Cocolakis

Activin/TGF-β induce apoptosis through Smad-dependent expression of the lipid phosphatase SHIP

Members of the transforming growth factor β (TGF-β) family regulate fundamental physiological processes, such as cell growth, differentiation and apoptosis, in almost all cell types. As a result, defects in TGF-β signalling pathways have been linked to uncontrolled cellular proliferation and carcinogenesis. Here, we explored the signal transduction mechanisms downstream of the activin/TGF-β receptors that result in cell growth arrest and apoptosis. We show that in haematopoietic cells, TGF-β family members regulate apoptosis through expression of the inositol phosphatase SHIP (Src homology 2 (SH2) domain-containing 5′ inositol phosphatase), a central regulator of phospholipid metabolism. We also demonstrated that the Smad pathway is required in the transcriptional regulation of the SHIP gene. Activin/TGF-β-induced expression of SHIP results in intracellular changes in the pool of phospholipids, as well as in inhibition of both Akt/PKB (protein kinase B) phosphorylation and cell survival. Our results link phospholipid metabolism to activin/TGF-β-mediated apoptosis and define TGF-β family members as potent inducers of SHIP expression.

The p38 MAPK Pathway Is Required for Cell Growth Inhibition of Human Breast Cancer Cells in Response to Activin

Activin, a member of the TGFβ family inhibits cell growth in various target tissues. Activin interacts with a complex of two receptors that upon activation phosphorylate specific intracellular mediators, the Smad proteins. The activated Smads interact with diverse DNA binding proteins and co-activators of transcription in a cell-specific manner, thus leading to various activin biological effects. In this study, we investigated the role and mechanism of action of activin in the human breast cancer T47D cells. We found that activin treatment of T47D cells leads to a dramatic decrease in cell growth. Thus activin appears as a potent cell growth inhibitor of these breast cancer cells. We show that activin induces the Smad pathway in these cells but also activates the p38-mitogen-activated protein kinase pathway, further leading to phosphorylation of the transcription factor ATF2. Finally, specific inhibitors of the p38 kinase (SB202190, SB203580, and PD169316) but not an inactive analogue (SB202474) or the MEK-1 inhibitor PD98059 completely abolish the activin-mediated cell growth inhibition of T47D cells. Together, these results define a new role for activin in human breast cancer T47D cells and highlight a new pathway utilized by this growth factor in the mediation of its biological effects in cell growth arrest.

Defining the role of prolactin as an invasion suppressor hormone in breast cancer cells

Prolactin hormone (PRL) is well characterized as a terminal differentiation factor for mammary epithelial cells and as an autocrine growth/survival factor in breast cancer cells. However, this function of PRL may not fully signify its role in breast tumorigenesis. Cancer is a complex multistep progressive disease resulting not only from defects in cell growth but also in cell differentiation. Indeed, dedifferentiation of tumor cells is now recognized as a crucial event in invasion and metastasis. PRL plays a critical role in inducing/maintaining differentiation of mammary epithelial cells, suggesting that PRL signaling could serve to inhibit tumor progression. We show here that in breast cancer cells, PRL and Janus-activated kinase 2, a major kinase involved in PRL signaling, play a critical role in regulating epithelial-mesenchymal transformation (EMT), an essential process associated with tumor metastasis. Activation of the PRL receptor (PRLR), achieved by restoring PRL/JAK2 signaling in mesenchymal-like breast cancer cells, MDA-MB-231, suppressed their mesenchymal properties and reduced their invasive behavior. While blocking PRL autocrine function in epithelial-like breast cancer cells, T47D, using pharmacologic and genetic approaches induced mesenchymal-like phenotypic changes and enhanced their invasive propensity. Moreover, our results indicate that blocking PRL signaling led to activation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) and transforming growth factor-beta/Smad signaling pathways, two major prometastatic pathways. Furthermore, our results indicate that following PRL/JAK2 inhibition, ERK1/2 activation precedes and is required for Smad2 activation and EMT induction in breast cancer cells. Together, these results highlight PRL as a critical regulator of epithelial plasticity and implicate PRL as an invasion suppressor hormone in breast cancer.

The G protein‐coupled receptor kinase‐2 is a TGFβ‐inducible antagonist of TGFβ signal transduction

Signaling from the activin/transforming growth factor β (TGFβ) family of cytokines is a tightly regulated process. Disregulation of TGFβ signaling is often the underlying basis for various cancers, tumor metastasis, inflammatory and autoimmune diseases. In this study, we identify the protein G‐coupled receptor kinase 2 (GRK2), a kinase involved in the desensitization of G protein‐coupled receptors (GPCR), as a downstream target and regulator of the TGFβ‐signaling cascade. TGFβ‐induced expression of GRK2 acts in a negative feedback loop to control TGFβ biological responses. Upon TGFβ stimulation, GRK2 associates with the receptor‐regulated Smads (R‐Smads) through their MH1 and MH2 domains and phosphorylates their linker region. GRK2 phosphorylation of the R‐Smads inhibits their carboxyl‐terminal, activating phosphorylation by the type I receptor kinase, thus preventing nuclear translocation of the Smad complex, leading to the inhibition of TGFβ‐mediated target gene expression, cell growth inhibition and apoptosis. Furthermore, we demonstrate that GRK2 antagonizes TGFβ‐induced target gene expression and apoptosis ex vivo in primary hepatocytes, establishing a new role for GRK2 in modulating single‐transmembrane serine/threonine kinase receptor‐mediated signal transduction.

Smad Signaling Antagonizes STAT5-mediated Gene Transcription and Mammary Epithelial Cell Differentiation

Both the transforming growth factor-β (TGFβ)/Smad and the prolactin/JAK/STAT pathway are critical to the proper development, maintenance, and function of the mammary epithelial tissue. Interestingly, opposing physiological effects between these two signaling pathways are prominent in the regulation of mammary gland development. However, the exact nature of the biological network existing between the Smad and STAT signal transduction pathways has remained elusive. We identified a novel regulatory cross-talk mechanism by which TGFβ-induced Smad signaling acts to antagonize prolactin-mediated JAK/STAT signaling and expression of target genes. Furthermore, we found activin, another member of the TGFβ family, to also efficiently block STAT5 signaling and β-casein expression in mammary epithelial cells. Our results indicate that ligand-induced activation of Smad2, -3, and -4 by activin and TGFβ leads to a direct inhibition of STAT5 transactivation and STAT5-mediated transcription of the downstream target genes, β-casein and cyclin D1, thereby blocking vital processes for mammary gland growth and differentiation. Finally, we unveiled the mechanism by which these two signaling cascades antagonize their effects, and we found that activated Smads inhibit STAT5 association with its co-activator CREB-binding protein, thus blocking STAT5 transactivation of its target genes and leading to inhibition of mammary gland differentiation and lactation.

Tyrosine Phosphorylation of Grb2: Role in Prolactin/Epidermal Growth Factor Cross Talk in Mammary Epithelial Cell Growth and Differentiation∇

ABSTRACT Characterizing mechanisms regulating mammary cell growth and differentiation is vital, as they may contribute to breast carcinogenesis. Here, we examine a cross talk mechanism(s) downstream of prolactin (PRL), a primary differentiation hormone, and epidermal growth factor (EGF), an important proliferative factor, in mammary epithelial cell growth and differentiation. Our data indicate that EGF exerts inhibitory effects on PRL-induced cellular differentiation by interfering with Stat5a-mediated gene expression independent of the PRL-proximal signaling cascade. Additionally, our data show that PRL is a potent inhibitor of EGF-induced cell proliferation. We identify tyrosine phosphorylation of the growth factor receptor-bound protein 2 (Grb2) as a critical mechanism by which PRL antagonizes EGF-induced cell proliferation by attenuating the activation of the Ras/mitogen-activated protein kinase (MAPK) pathway. Together, our results define a novel negative cross-regulation between PRL and EGF involving the Jak2/Stat5a and Ras/MAPK pathways through tyrosine phosphorylation of Grb2.

Peripheral and local predictive immune signatures identified in a phase II trial of ipilimumab with carboplatin/paclitaxel in unresectable stage III or stage IV melanoma

BackgroundCheckpoint blockade with ipilimumab provides long-term survival to a significant proportion of patients with metastatic melanoma. New approaches to increase survival and to predict which patients will benefit from treatment are needed. This phase II trial combined ipilimumab with carboplatin/paclitaxel (CP) to assess its safety, efficacy, and to search for peripheral and tumor-based predictive biomarkers.MethodsThirty patients with untreated unresectable/metastatic melanoma were treated with ipilimumab and CP. Adverse events (AEs) were monitored and response to treatment was evaluated. Tumor tissue and peripheral blood were collected at specified time points to characterize tumor immune markers by immunohistochemistry and systemic immune activity by multiplex assays and flow cytometry.ResultsEighty three percent of patients received all 5xa0cycles of CP and 93% completed ipilimumab induction. Serious AEs occurred in 13% of patients, and no treatment-related deaths were observed. Best Overall Response Rate (BORR) and Disease Control Rate (DCR) were 27 and 57%, respectively. Median overall survival was 16.2xa0months. Response to treatment was positively correlated with a higher tumor CD3+ infiltrate (immune score) at baseline. NRAS and BRAF mutations were less frequent in patients who experienced clinical benefit. Assessment of peripheral blood revealed that non-responders had elevated baseline levels of CXCL8 and CCL4, and a higher proportion of circulating late differentiated B cells. Pre-existing high levels of chemokines (CCL3, CCL4 and CXCL8) and advanced B cell differentiation were strongly associated with worse patient overall survival. Elevated proportions of circulating CD8+/PD-1+ T cells during treatment were associated with worse survival.ConclusionsThe combination of ipilimumab and CP was well tolerated and revealed novel characteristics associated with patients likely to benefit from treatment. A pre-existing systemic inflammatory state characterized by elevation of selected chemokines and advanced B cell differentiation, was strongly associated with poor patient outcomes, revealing potential predictive circulating biomarkers.Trial registrationClinicaltrials.gov, NCT01676649, registered on August 29, 2012.

The Development of Novel Therapies for the Treatment of Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is nearly always a fatal malignancy. For the past 40 years, the standard of care remains a combination of cytarabine and an anthracycline known as 7 + 3. This treatment regimen is troubled by both low survival rates (10% at 5 years) and deaths due to toxicity. Substantial new laboratory findings over the past decade have identified many cellular pathways that contribute to leukemogenesis. These studies have led to the development of novel agents designed to target these pathways. Here we discuss the molecular underpinnings and clinical benefits of these novel treatment strategies. Most importantly these studies demonstrate that clinical response is best achieved by stratifying each patient based on a detailed understanding of their molecular abnormalities.

Immune biomarker correlates from a Phase II study of ipilimumab (IPI) with carboplatin and paclitaxel (CP) in patients with unresectable stage III or IV metastatic melanoma (MM)

Meeting abstractsnnPivotal studies with anti-CTLA-4 (Ipilimumab or IPI) demonstrated increased overall survival (OS) both as single agent (10.1 months) and with DTIC (11.2 months). Weve previously reported an encouraging safety profile with IPI plus CP and early efficacy results. We now report