The endoplasmic reticulum (ER) has evolved specific mechanisms to ensure protein folding as well as the maintenance of its own homeostasis. When these functions are not achieved, specific ER stress ...signals are triggered to activate either adaptive or apoptotic responses. Here, we demonstrate that MCF-7 cells are resistant to tunicamycin-induced apoptosis. We show that the expression level of the ER chaperone calnexin can directly influence tunicamycin sensitivity in this cell line. Interestingly, the expression of a calnexin lacking the chaperone domain (DeltaE) partially restores their sensitivity to tunicamycin-induced apoptosis. Indeed, we show that DeltaE acts as a scaffold molecule to allow the cleavage of Bap31 and thus generate the proapoptotic p20 fragment. Utilizing the ability of MCF-7 cells to resist tunicamycin-induced apoptosis, we have characterized a molecular mechanism by which calnexin regulates ER-stress-mediated apoptosis in a manner independent of its chaperone functions but dependent of its binding to Bap31.
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.
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.
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.
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.
We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential biomarker and therapeutic target in breast cancer. eIF4E facilitates nuclear export and translation of ...specific, growth-stimulatory mRNAs and is frequently overexpressed in cancer.
Breast cancer cells were treated with ribavirin, an inhibitor of eIF4E, and effects on cell proliferation and on known mRNA targets of eIF4E were determined. eIF4E expression was assessed, at the mRNA and protein level, in breast cancer cell lines and in skin biopsies from patients with metastatic disease. Additionally, pooled microarray data from 621 adjuvant untreated, node-negative breast cancers were analyzed for eIF4E expression levels and correlation with distant metastasis-free survival (DMFS), overall and within each intrinsic breast cancer subtype.
At clinically relevant concentrations, ribavirin reduced cell proliferation and suppressed clonogenic potential, correlating with reduced mRNA export and protein expression of important eIF4E targets. This effect was suppressed by knockdown of eIF4E. Although eIF4E expression is elevated in all breast cancer cell lines, variability in ribavirin responsiveness was observed, indicating that other factors contribute to an eIF4E-dependent phenotype. Assessment of the prognostic value of high eIF4E mRNA in patient tumors found that significant discrimination between good and poor outcome groups was observed only in luminal B cases, suggesting that a specific molecular profile may predict response to eIF4E-targeted therapy.
Inhibition of eIF4E is a potential breast cancer therapeutic strategy that may be especially promising against specific molecular subtypes and in metastatic as well as primary tumors.
Checkpoint 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.
Thirty 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.
Eighty three percent of patients received all 5 cycles 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 16.2 months. 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.
The 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.
Clinicaltrials.gov , NCT01676649 , registered on August 29, 2012.
Acute myeloid leukemia (AML) with inversion of chromosome 3 is characterized by overexpression of EVI1 and carries a dismal prognosis. Arsenic-containing compounds have been described to be ...efficacious in malignancies overexpressing EVI1. Here, we describe a case of AML with inv(3)(q21q26.2) treated with the organic arsenical darinaparsin. Using a "personalized medicine approach," two different arsenicals were screened for anti-leukemic effect against the patient's cells ex vivo. The most promising compound, darinaparsin, was selected for in vivo treatment. Clinical effect was almost immediate, with a normalization of temperature, a stabilization of white blood cell (WBC) counts and an increased quality of life. Longitudinal monitoring of patient response and resistance incorporating significant correlative studies on patient-derived blood samples over the two cycles of darinaparsin given to this patient allowed us to evaluate potential mechanisms of response and resistance. The anti-leukemic effects of darinaparsin correlated with inhibition of the alternative NF-κB pathway and production of the inflammatory cytokine IL-8. Emergence of resistance was suspected during treatment cycle 2 and supported by xenograft studies in nude mice. Darinaparsin resistance correlated with an attenuation of the effect of treatment on the alternative NF-κB pathway. The results from this patient indicate that darinaparsin may be a good treatment option for inv(3) AML and that inhibition of the alternative NF-κB pathway may be predictive of response. Longitudinal monitoring of disease response as well as several correlative parameters allowed for the generation of novel correlations and predictors of response to experimental therapy in a heavily pretreated patient.
Abstract 2085
Poster Board II-62
Over the past 10 years, the incidence of acute myeloid leukemia (AML) has increased significantly with approximately 15 000 new cases annually. Standard induction ...chemotherapy consisting of cytarabine (Ara-C) and an anthracycline induces remission rates between 50% and 85%. Unfortunately, the majority of patients who achieve remission will relapse and die from their disease within 2 years, highlighting the need for novel therapeutic targets. The eukaryotic translation factor (eIF4E) is overexpressed in many human malignancies, including AML, and is associated with poor prognosis as well as clinical progression. Ribavirin, an anti-viral molecule, is classically used in the treatment of hepatitis C (with interferon), SARS, RSV, Lassa fever and influenza. Its structure physically mimics the m(7)G cap of mRNA, thus inhibiting eIF4E-induced export and translation of sensitive transcripts. We are carrying out the first clinical trial targeting eIF4E with ribavirin in AML patients. Clinical and molecular efficacy has been evaluated in 13 patients to date. The treatment was well tolerated by all patients with no marked toxicity observed. Importantly, no patients developed hemolytic anemia. We demonstrated that ribavirin effectively induces the relocalization of nuclear eIF4E to the cytoplasm and the reduction of eIF4E as well as its target proteins, including suppression of Akt activation. This led to dramatic clinical improvement, including one complete remission, two partial remissions, two blast responses and four patients with stable disease. Final response data will be presented along with translational correlates. Notably, lack of response or relapse after remission was associated with lack of molecular response in leukemic blasts. Despite the encouraging responses of patient on ribavirin, all patients acquired resistance to therapy and eventually relapsed. Hence, we sought novel therapies to combine with ribavirin in order to overcome resistance and maintain remissions. Using a cell line that overexpresses eIF4E, we screened a library of 5000 known drugs and searched for compounds that synergize with ribavirin to suppress tumor growth. We identified nearly 50 lead compounds, many of which are structurally related, with similar biological activity, and are currently used medically for indications other than cancer. Early clinical observations suggest that combinations of cytotoxic agents lead to substantially better clinical outcomes relative to monotherapies. Furthermore, various drugs that suppress the PI3/Akt pathway were found to sensitize leukemia cells to Ara-C. Thus, we combined Ara-C with ribavirin in vitro, and observed an improved reduction in colony growth of AML specimens. Combination therapy with ribavirin and Ara-C in patients with acute myelocytic leukemia is currently ongoing. Preliminary results will be presented.
Borden:Translational Therapeutics: Equity Ownership.
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