Jose L. Fernandez-Luna

Constitutive Activation of Stat3 Signaling Confers Resistance to Apoptosis in Human U266 Myeloma Cells

Interleukin 6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the STAT proteins. We report that one STAT family member, Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line U266. Moreover, U266 cells are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-xL. Blocking IL-6 receptor signaling from Janus kinases to the Stat3 protein inhibits Bcl-xL expression and induces apoptosis, demonstrating that Stat3 signaling is essential for the survival of myeloma tumor cells. These findings provide evidence that constitutively activated Stat3 signaling contributes to the pathogenesis of multiple myeloma by preventing apoptosis.

Resistance to chemotherapy via Stat3-dependent overexpression of Bcl-2 in metastatic breast cancer cells

Disruption of apoptosis may allow metastatic cell survival and confer resistance to chemotherapeutic drugs. We have analysed the molecular pathways that activate these survival genes in specific sites of metastasis. Estrogen receptor-negative breast cancer cell line MDA-MB435 and two metastatic sublines derived from lung (435L) and brain (435B) were analysed for the expression of members of the Bcl-2 family of apoptosis regulators. The levels of Bcl-2 were higher in the metastatic sublines than in parental cells, which correlated with the activation of Stat3, but not with the expression and/or activation of known bcl-2 transcription factors (CREB and WT1). In the brain subline, both expression of Bcl-2 and Stat3 activation were induced by epidermal growth factor and abrogated after treatment with kinase inhibitors specific for epidermal growth factor receptor or Jak2. Furthermore, transfection of 435B with a dominant-negative Stat3 markedly reduced the expression of Bcl-2 protein, whereas transient expression of a constitutively active Stat3 increased Bcl-2 in parental 435 cells. In addition, blockade of Stat3 activation by treatment with epidermal growth factor receptor and Jak2 kinase inhibitors or transfection with a dominant negative Stat3, sensitizes 435B cells to chemotherapy-induced apoptosis. Our data suggest that an increased activation of the Stat3–Bcl-2 pathway in estrogen receptor-negative metastatic breast cancer cell lines confer a survival advantage to these cells and contribute to their chemoresistance.

Expression of Bcl-x in Erythroid Precursors from Patients with Polycythemia Vera

BACKGROUND Deregulating the expression of Bcl-xL, an inhibitor of apoptosis, in an erythropoietin-dependent erythroblast cell line averts apoptosis induced by the withdrawal of erythropoietin. Since in polycythemia vera an abnormal clone of erythroid progenitors is independent of erythropoietin, we investigated whether the endogenous expression of Bcl-xL was deregulated in these cells. METHODS Erythroid colonies from patients with polycythemia vera and normal subjects were cultured in the presence and absence of erythropoietin and assessed by immunocytochemical and flow-cytometric analysis with anti-Bcl-x antibodies that recognize the two species of Bcl-x (Bcl-xL and Bcl-xS). Reverse-transcriptase-polymerase-chain-reaction analysis was used to determine which one of the two species was responsible for anti-Bcl-x staining. Bone marrow mononuclear cells from 8 healthy bone marrow donors, 14 patients with polycythemia vera, 19 patients with other myeloproliferative syndromes, and 12 patients with secondary erythrocytosis were analyzed by flow cytometry with antibodies against Bcl-x and glycophorin A, an erythroid marker. RESULTS Erythroid cells from patients with polycythemia vera survived in vitro without erythropoietin, and this finding correlated with the expression of Bcl-x protein (Bcl-xL messenger RNA was the main species of Bcl-x found), even in mature erythroblasts that normally do not express Bcl-x. The mean (+/-SD) percentage of cells positive for both glycophorin A and Bcl-x in the 14 patients with polycythemia vera (21.8+/-3.6 percent) was significantly higher than that in 8 normal donors (6.62+/-1.58 percent), 12 patients with secondary erythrocytosis (6.87+/-1.95 percent), 9 patients with essential thrombocythemia (3.81+/-0.97 percent), and 10 patients with chronic myeloid leukemia (2.7+/-0.41 percent). CONCLUSIONS Deregulated expression of Bcl-x may contribute to the erythropoietin-independent survival of erythroid-lineage cells in polycythemia vera and thereby contribute to the pathogenesis of this disease.

Interleukin 3-dependent activation of DREAM is involved in transcriptional silencing of the apoptotic hrk gene in hematopoietic progenitor cells

The apoptotic protein Hrk is expressed in hematopoietic progenitors after growth factor deprivation. Here we identify a silencer sequence in the 3′ untranslated region of the hrk gene that binds to the transcriptional repressor DREAM in interleukin‐3 (IL‐3)‐dependent hematopoietic progenitor cells, and abrogates the expression of reporter genes when located downstream of the open reading frame. In addition, the binding of DREAM to the hrk gene is reduced or eliminated when cells are cultured in the absence of IL‐3 or treated with a calcium ionophore or a phosphatidylinositol 3‐kinase‐specific inhibitor, suggesting that both calcium mobilization and phosphorylation can regulate the transcriptional activity of DREAM. Furthermore, we have shown that DREAM is phosphorylated by a phosphatidylinositol 3‐kinase‐dependent, but Akt‐independent pathway. In all cases, loss of the DREAM–DNA binding complex was correlated with increased levels of Hrk and apoptosis. These data suggest that IL‐3 may trigger the activation of DREAM through different signaling pathways, which in turn binds to a silencer sequence in the hrk gene and blocks transcription, avoiding inappropriate cell death in hematopoietic progenitors.

Imatinib Inhibits Proliferation of Ewing Tumor Cells Mediated by the Stem Cell Factor/KIT Receptor Pathway, and Sensitizes Cells to Vincristine and Doxorubicin-Induced Apoptosis

Purpose and Experimental Design: The stem cell factor/KIT receptor loop may represent a novel target for molecular-based therapies of Ewing tumor. We analyzed the in vitro impact of KIT blockade by imatinib in Ewing tumor cell lines. Results: KIT expression was detected in 4 of 4 Ewing tumor cell lines and in 49 of 110 patient samples (44.5%) by immunohistochemistry and/or Western blot analysis. KIT expression was stronger in Ewing tumors showing EWS-FLI1 nontype 1 fusions. Despite absence of c-kit mutations, constitutive and ligand-inducible phosphorylation of KIT was found in all tumor cell lines, indicating an active receptor. Treatment with KIT tyrosine kinase inhibitor imatinib (0.5–20 μm) induced down-regulation of KIT phosphorylation and dose response inhibition of cell proliferation (IC50, 12–15 μm). However, imatinib administered alone at doses close to IC50 for growth inhibition (10 μm) did not induce a significant increase in apoptosis. We then analyzed if blockade of KIT loop through imatinib (10 μm) was able to increase the antitumor in vitro effect of doxorubicin (DXR) and vincristine (VCR), drugs usually used in Ewing tumor treatment. Addition of imatinib decreased in 15–20 and 15–36% of the proliferative rate of Ewing tumor cells exposed to DXR and VCR, respectively, and increased in 15 and 30% of the apoptotic rate of Ewing tumor cells exposed to the same drugs. Conclusions: Inhibition of Ewing tumor cell proliferation by imatinib is mediated through blockade of KIT receptor signaling. Inhibition of KIT increases sensitivity of these cells to DXR and VCR. This study supports a potential role for imatinib in the treatment of Ewing tumor.

Antiapoptotic protein Bcl-xL is up-regulated during megakaryocytic differentiation of CD34+ progenitors but is absent from senescent megakaryocytes

OBJECTIVE The expression of Bcl-x(L) has been shown to be regulated during the maturation process of different hematopoietic cell lineages (i.e., erythroid cells, neutrophils, monocytes/macrophages). In the present study, we examined the expression of Bcl-x(L) in megakaryocytes derived from CD34(+) progenitors and in the megakaryoblastic cell line UT7. MATERIALS AND METHODS Expression of Bcl-x(L) was analyzed in CD41(+) cells cultured in the presence of thrombopoietin and in UT7 cells treated with phorbol diester by Western blot, flow cytometry, and immunocytochemistry analysis. Apoptosis was determined at different culture times by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and propidium iodide uptake. RESULTS Bcl-x(L) but not Bcl-2 was up-regulated in the megakaryocytic population (CD41(+)) during the first 15 days of culture, which was consistent with the pattern of Bcl-x(L) expression in UT7 cells differentiated to megakaryocytes by incubation with phorbol diester. However, by day 20 of culture, the levels of Bcl-x(L) in CD41(+) cells were greatly reduced, and this expression pattern was accompanied by an increase in the number of apoptotic cells. At this culture time, we detected the presence of cytoplasmic fragments resembling proplatelets with prominent Bcl-x immunostaining, most likely due to the Bcl-x(L) isoform, in close proximity to Bcl-x(-) senescent megakaryocytes. The presence of Bcl-x(L) but not of Bcl-2 in platelets was confirmed by Western blot analysis. CONCLUSION Although little is known regarding the functional significance of survival proteins within the megakaryocytic compartment, the changes in the Bcl-x(L) expression pattern observed in UT7 and CD41(+) cells may play a role in the survival of developing megakaryocytes and the lifespan of mature platelets.

Blockade of epidermal growth factor receptors chemosensitizes breast cancer cells through up-regulation of Bnip3L

Epidermal growth factor receptor-1 (EGFR) and EGFR-2 (HER2) have become major targets for cancer treatment. Blocking antibodies and small-molecule inhibitors are being used to silence the activity of these receptors in different tumors with varying efficacy. Thus, a better knowledge on the signaling pathways activated by EGFR and HER2 may help unravel novel therapeutic targets and molecular markers of response. Here, we show that treatment of breast cancer cell lines with blocking antibodies against EGFR (cetuximab) or HER2 (trastuzumab) promotes the specific induction of proapoptotic Bnip3L and chemosensitization. Moreover, we found that the Bnip3L gene is transcriptionally activated by FoxO3a. Trastuzumab-mediated induction of Bnip3L and nuclear translocation of FoxO3a was also shown in pleural effusion cells from a breast cancer patient. Transfection of breast cancer cells with constitutively active FoxO3a or with Bnip3L promotes sensitization to chemotherapy-induced apoptosis. On the contrary, blockade of Bnip3L expression by a small interfering RNA strategy significantly diminished the chemosensitizing effect of cetuximab. We found also an inverse correlation between EGFR and Bnip3L expression in surgical specimens from patients with breast cancer. Therefore, blockading EGFR or HER2 specifically up-regulates Bnip3L, which is required for chemosensitization of breast cancer cells. This novel pathway provides also the rationale for therapeutic strategies aimed to induce the expression of Bnip3L.

Deficiency of the NF-κB Inhibitor Caspase Activating and Recruitment Domain 8 in Patients with Rheumatoid Arthritis Is Associated with Disease Severity

Caspase activating and recruitment domain 8 (CARD8) potently inhibits NF-κB signaling, which plays a key role in inflammation, and may contribute to avoid a pathologic activation of NF-κB; however, the transcriptional mechanisms regulating CARD8 expression and the relevance of this protein in inflammatory diseases are poorly understood. We found a NF-κB-binding element within the human CARD8 promoter that was required for transcriptional activity in response to TNF-α and the p65 subunit of NF-κB. Moreover, TNF-α and overexpression of p65 induced the formation of NF-κB-CARD8 promoter complexes. Thus, CARD8 may control NF-κB activation through a regulatory loop. To study the relevance of CARD8 in chronic inflammatory disorders, we functionally characterized a deleterious polymorphism (p.C10X) and studied its association with rheumatoid arthritis (RA). Transfection of cell lines with the allelic variants of CARD8 revealed that full-length (CARD8-L) but not truncated (CARD8-S) protein inhibits NF-κB transcriptional activity, and abrogates the binding of NF-κB to its consensus site. Furthermore, in contrast to the full-length protein, CARD8-S did not modify the expression of NF-κB target genes (cIAP, A1), in response to TNF-α. We analyzed the p.C10X polymorphism in 200 patients with RA, and found that homozygous carriers of the CARD8-S allele have higher disease activity score (p = 0.014), more extra-articular manifestations (p = 0.03), and a lower probability of clinical remission (p = 0.03) than the CARD8-L allele carriers. Overall, our findings provide molecular insight into the expression of CARD8 by NF-κB, and suggest that a deleterious polymorphism of CARD8 may help predict the severity of RA.

Case-control study and meta-analysis of low density lipoprotein receptor-related protein gene exon 3 polymorphism in Alzheimer's disease

The low density lipoprotein receptor-related protein (LRP) may influence both the clearance and the production of beta-amyloid peptide and thus plays a role in Alzheimers disease (AD) pathogenesis. Previous studies, although inconsistent, have suggested that the LRP exon 3 CC genotype contributes to the risk of AD. A case-control study utilizing a clinically well-defined group of 305 sporadic AD patients and 304 control subjects was performed to test this association in an ethnically homogeneous population from Spain. In the current study, the LRP CC genotype was not over-represented in AD patients compared to non-demented controls. A meta-analysis of previous studies revealed a weak correlation of LRP CC genotype with AD (odds ratio of 1.35, P=0.01).

Involvement of miRNAs in the Differentiation of Human Glioblastoma Multiforme Stem-Like Cells

Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process.