RESUMO
Gene therapy of B16 tumors with a dominant-negative signal transducer and activator of transcription (Stat3) variant, designated Stat3beta, results in inhibition of tumor growth and tumor regression. Although only 10-15% of the tumor cells are transfected in vivo, the Stat3beta-induced antitumor effect is associated with massive apoptosis of B16 tumor cells, indicative of a potent bystander effect. Here, we provide evidence that blocking Stat3 signaling in B16 cells results in release of soluble factors that are capable of inducing apoptosis and cell cycle arrest of nontransfected B16 cells. RNase protection assays using multi-template probes specific for key physiological regulators of apoptosis reveal that overexpression of Stat3beta in B16 tumor cells induces the expression of the apoptotic effector, tumor necrosis factor-related apoptosis-inducing ligand. These in vitro results suggest that the observed in vivo bystander effect leading to tumor cell growth inhibition is mediated, at least in part, by soluble factors produced as a result of overexpression of Stat3beta in tumor cells.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Glicoproteínas de Membrana/biossíntese , Transativadores/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Células 3T3 , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Ciclo Celular/fisiologia , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Glicoproteínas de Membrana/genética , Camundongos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fator de Transcrição STAT3 , Transdução de Sinais/fisiologia , Solubilidade , Ligante Indutor de Apoptose Relacionado a TNF , Transativadores/biossíntese , Transativadores/genética , Transfecção , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/genéticaRESUMO
Multiple myeloma (MM) remains an incurable malignancy due, in part, to the influence of the bone marrow microenvironment on survival and drug response. Identification of microenvironment-specific survival signaling determinants is critical for the rational design of therapy and elimination of MM. Previously, we have shown that collaborative signaling between ß1 integrin-mediated adhesion to fibronectin and interleukin-6 confers a more malignant phenotype via amplification of signal transducer and activator of transcription 3 (STAT3) activation. Further characterization of the events modulated under these conditions with quantitative phosphotyrosine profiling identified 193 differentially phosphorylated peptides. Seventy-seven phosphorylations were upregulated upon adhesion, including PYK2/FAK2, Paxillin, CASL and p130CAS consistent with focal adhesion (FA) formation. We hypothesized that the collaborative signaling between ß1 integrin and gp130 (IL-6 beta receptor, IL-6 signal transducer) was mediated by FA formation and proline-rich tyrosine kinase 2 (PYK2) activity. Both pharmacological and molecular targeting of PYK2 attenuated the amplification of STAT3 phosphorylation under co-stimulatory conditions. Co-culture of MM cells with patient bone marrow stromal cells (BMSC) showed similar ß1 integrin-specific enhancement of PYK2 and STAT3 signaling. Molecular and pharmacological targeting of PYK2 specifically induced cell death and reduced clonogenic growth in BMSC-adherent myeloma cell lines, aldehyde dehydrogenase-positive MM cancer stem cells and patient specimens. Finally, PYK2 inhibition similarly attenuated MM progression in vivo. These data identify a novel PYK2-mediated survival pathway in MM cells and MM cancer stem cells within the context of microenvironmental cues, providing preclinical support for the use of the clinical stage FAK/PYK2 inhibitors for treatment of MM, especially in a minimal residual disease setting.
Assuntos
Quinase 2 de Adesão Focal/metabolismo , Mieloma Múltiplo/patologia , Animais , Morte Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Quinase 2 de Adesão Focal/antagonistas & inibidores , Humanos , Interleucina-6/metabolismo , Janus Quinase 1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Microambiente TumoralRESUMO
CD95 (Fas/APO-1) is a member of the TNFR superfamily that induces apoptosis following cross-linking with its cognate ligand, CD95L (FasL/APO-1L) or agonist antibody. The human myeloma cell line, RPMI 8226, has limited sensitivity to CD95-mediated apoptosis, with a maximum of 65% of the population responding. To determine the source of the limited sensitivity to CD95-mediated apoptosis, we isolated multiple clones from the RPMI-8226 cell line by limiting dilution. Analysis of these clones demonstrated that sensitivity to CD95-mediated cell death directly correlated with CD95 expression. Clones with high levels of CD95 expression had greater than 90% cell death, whereas cells with low levels of expression had less than 10% cell death. In contrast, no correlative differences were identified for other members of the DISC complex, or for members of the anti-apoptotic Bcl-2 family. We further examined the sensitivity of the 8226 clones to various cytotoxic agents. Although modest clonal variability was demonstrated in response to the chemotherapeutic drugs, doxorubicin, etoposide (VP-16), and vincristine, there was no correlation between CD95 function and sensitivity to chemotherapeutic drugs. These results indicate that in this cell line, receptor expression is rate limiting in CD95-mediated apoptosis, whereas CD95 expression was not a determinant in drug-induced programmed cell death.
Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Mieloma Múltiplo/patologia , Transcrição Gênica , Receptor fas/genética , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/genética , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Doxorrubicina/toxicidade , Etoposídeo/toxicidade , Citometria de Fluxo , Humanos , Células Tumorais Cultivadas , Vincristina/toxicidade , Receptor fas/fisiologiaRESUMO
Clinical circumvention of multidrug resistance (MDR) is a Sisyphian task faced in the treatment of many cancers. Identification of several mechanisms of acquired MDR has led to the development of chemosensitizing agents that counter specific mechanisms of MDR. Initial successes in therapy using "chemosensitizers" often culminate in relapse due to the multifactorial nature of acquired MDR. Therefore, it may be important to design therapeutic strategies that focus on mechanisms that allow for cell survival after initial treatments, before the acquisition of MDR. It has been proposed that extracellular effectors such as cytokines, matrix components, and adjacent cells may provide sanctuary to cancer cells by preventing stress-induced cell death. This review focuses on research implicating the cancer cell environment as a particularly important determinant in the emergence of drug resistance. More specifically, we will discuss the role of direct contact between cancer cells and the extracellular matrix or with adjacent cells as extrinsic effectors of de novo MDR. Cell adhesion has been demonstrated to prevent cell death through a number of mechanisms. Identification of cell adhesion-mediated drug resistance as an initial or de novo effector of MDR suggests that therapies targeting interactions between cancer cells and their environment may lead to the sensitization of cancer cells to chemotherapy or radiotherapy before the emergence of acquired mechanisms of MDR.
Assuntos
Adesão Celular/fisiologia , Resistência a Múltiplos Medicamentos/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Animais , Antineoplásicos/farmacologia , Apoptose , Humanos , Neoplasias/tratamento farmacológicoRESUMO
B-cell tumorigenesis results from a host of known and unknown genetic anomalies, including non-random translocations of genes that normally function as determinants of cell proliferation or cell survival to regions juxtaposed to active immunoglobulin heavy chain enhancer elements, chromosomal aneuploidy, somatic mutations that further affect oncogenic signaling and loss of heterozygosity of tumor-suppressor genes. However, it is critical to recognize that even in the setting of a genetic disease, the B-cell/plasma cell tumor microenvironment (TME) contributes significantly to malignant transformation and pathogenesis. Over a decade ago, we proposed the concept of cell adhesion-mediated drug resistance to delineate a form of TME-mediated drug resistance that protects hematopoietic tumor cells from the initial effect of diverse therapies. In the interim, it has been increasingly appreciated that TME also contributes to tumor initiation and progression through sustained growth/proliferation, self-renewal capacity, immune evasion, migration and invasion as well as resistance to cell death in a host of B-cell malignancies, including mantle cell lymphoma, diffuse large B-cell lymphoma, Waldenstroms macroglobulinemia, chronic lymphocytic leukemia and multiple myeloma. Within this review, we propose that TME and the tumor co-evolve as a consequence of bidirectional signaling networks. As such, TME represents an important target and should be considered integral to tumor progression and drug response.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Linfoma Difuso de Grandes Células B/genética , Linfoma de Célula do Manto/genética , Mieloma Múltiplo/genética , Microambiente Tumoral/genética , Linfócitos B/patologia , Carcinogênese/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma Difuso de Grandes Células B/patologia , Linfoma de Célula do Manto/patologia , Mieloma Múltiplo/patologia , Transdução de SinaisRESUMO
Specific niches within the lymphoma tumor microenvironment (TME) provide sanctuary for subpopulations of tumor cells through stromal cell-tumor cell interactions. These interactions notably dictate growth, response to therapy and resistance of residual malignant B cells to therapeutic agents. This minimal residual disease (MRD) remains a major challenge in the treatment of B-cell malignancies and contributes to subsequent disease relapse. B-cell receptor (BCR) signaling has emerged as essential mediator of B-cell homing, survival and environment-mediated drug resistance (EMDR). Central to EMDR are chemokine- and integrin-mediated interactions between lymphoma and the TME. Further, stromal cell-B cell adhesion confers a sustained BCR signaling leading to chemokine and integrin activation. Recently, the inhibitors of BCR signaling have garnered a substantial clinical interest because of their effectiveness in B-cell disorders. The efficacy of these agents is, at least in part, attributed to attenuation of BCR-dependent lymphoma-TME interactions. In this review, we discuss the pivotal role of BCR signaling in the integration of intrinsic and extrinsic determinants of TME-mediated lymphoma survival and drug resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Linfoma de Células B/fisiopatologia , Linfoma/tratamento farmacológico , Linfoma/mortalidade , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos B/citologia , Adesão Celular , Sobrevivência Celular , Humanos , Neoplasia Residual , Transdução de Sinais , Células Estromais/metabolismoRESUMO
Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative approach in patients with multiple myeloma, but its use for consolidation of first remission has not yet been fully explored. Twenty-two myeloma patients with very good partial response (VGPR) or CR received allogeneic peripheral blood grafts as consolidation from HLA-matched donors between 2007 and 2012. Conditioning regimens were fludarabine (30 mg/m(2) i.v. if with bortezomib and 40 mg/m(2) i.v. when without bortezomib, × 4 days) plus melphalan (70 mg/m(2) intravenously × 2 days) with (n=13) or without (n=9) bortezomib (1.3 mg/m(2)). The cumulative incidence of grades II - IV acute GVHD at day 100 was 45% (95% CI: 24-65%) and moderate-to-severe chronic GVHD at 2 years was 46% (95% CI: 19-69%). With a median follow-up of 18 (range, 2-61) months, the 2-year PFS estimate is 74.8% (95% CI: 45-90%), which compares favorably with the 52% (95% CI: 35-66%) after autologous HCT for similar patients (a median follow-up of 30 (range, 9-55) months). We are conducting a phase 2 study to assess the efficacy of allogeneic HCT as post-remission therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/métodos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/cirurgia , Condicionamento Pré-Transplante/métodos , Adulto , Ácidos Borônicos/administração & dosagem , Bortezomib , Estudos de Coortes , Feminino , Doença Enxerto-Hospedeiro/etiologia , Humanos , Masculino , Melfalan/administração & dosagem , Pessoa de Meia-Idade , Pirazinas/administração & dosagem , Qualidade de Vida , Indução de Remissão , Estudos Retrospectivos , Quimeras de Transplante , Transplante Homólogo , Vidarabina/administração & dosagem , Vidarabina/análogos & derivadosAssuntos
Janus Quinase 2/genética , Melfalan/administração & dosagem , Mieloma Múltiplo/genética , Mieloma Múltiplo/terapia , Trombocitemia Essencial/genética , Trombocitemia Essencial/terapia , Medula Óssea/patologia , Feminino , Fator Estimulador de Colônias de Granulócitos/química , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Humanos , Hidroxiureia/uso terapêutico , Pessoa de Meia-Idade , Mieloma Múltiplo/complicações , Mutação , Agonistas Mieloablativos/administração & dosagem , Contagem de Plaquetas , Trombocitemia Essencial/complicações , Resultado do TratamentoRESUMO
The influence of the microenvironment in the pathogenesis and progression of human cancer has traditionally been considered in the context of solid tumors. More recently, evidence has been accumulating to support the role of the bone marrow microenvironment in hematologic malignancies as well, particularly in multiple myeloma. This review focuses on myeloma as a model to demonstrate that the bone marrow microenvironment provides a sanctuary against programmed cell death and promotes tumor cell survival and progression. Additionally, the protective effects of the bone marrow milieu may confer a protection from cytotoxic drugs, allowing the emergence of drug-resistant tumors. These advances may assist in the design of novel therapeutic approaches to enhance the efficacy of standard chemotherapeutic drugs.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias/metabolismo , Neoplasias/patologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Adesão Celular , Morte Celular , Sobrevivência Celular , Humanos , Integrinas/metabolismo , Modelos Biológicos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Transdução de Sinais , Receptor fas/metabolismoRESUMO
RelB, an NF-kappaB/Rel-related transacting factor, was initially identified as an immediate-early gene product in fibroblasts and subsequently shown to exhibit constitutive DNA binding activity in lymphoid cells. The data presented in this report show that RelB is also constitutively active, as monitored by electrophoretic mobility shift assay, in the v-Src-transformed fibroblast cell line, SR1. By contrast, nontransformed parental (3Y1) cells displayed inducible NF-kappaB activity; RelB activity was also observed, although to a lesser extent, in two additional v-Src-transformed fibroblast lines. RelB activation in SR1 cells did not require an increase in RelB expression or result from a decrease in the levels of IkappaB alpha or p105, proteins previously shown to bind to and inhibit the activity of the Rel proteins. Numerous studies have shown that stimulus-dependent Rel activation requires degradation of IkappaB alpha, p105 or other member of the IkappaB family, and that this process is precluded by agents that inhibit proteasome activity. We show that treatment of SR1 cells with proteasome inhibitors abolishes RelB activity and thus suggest that RelB in these cells is associated with IkappaB and that v-Src transformation activates RelB by accelerating IkappaB proteolysis. Additional data show that serum and tumor necrosis factor-alpha (TNF-alpha) increase RelB protein levels in 3Y1 cells and that this process is blocked by proteasome inhibitors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteína Oncogênica pp60(v-src)/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Linhagem Celular Transformada , Hidrólise , Proteínas I-kappa B , Camundongos , Camundongos Endogâmicos BALB C , Fator de Transcrição RelBRESUMO
We describe here the conditional expression of the hepatitis B virus X protein using the inducible system controlled by a tet-responsive promoter. Induction of the X protein in Rat-2 fibroblasts activated transcription from a heterologous gene promoter and stimulated the DNA-binding activity of NFkB. The ability to produce this biologically active X protein in a stable cell line will accelerate the elucidation of the function and mechanisms of X and eventually help us understand the role of X in natural viral infection and carcinogenesis.
Assuntos
Transativadores/genética , Ativação Transcricional , Animais , Linhagem Celular , Cloranfenicol O-Acetiltransferase/metabolismo , Fibroblastos , Vírus da Hepatite B , Proteínas Nucleares/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Tetraciclina/farmacologia , Transativadores/biossíntese , Transcrição Gênica , Proteínas Virais Reguladoras e AcessóriasRESUMO
We have previously shown that selection for resistance to the anthracenes, doxorubicin or mitoxantrone, results in coselection for resistance to CD95-mediated apoptosis (Landowski et al: Blood 89:1854, 1997). In the present study, we were interested in determining if the converse is also true; that is, does selection for CD95 resistance coselect for resistance to chemotherapeutic drugs. To address this question, we used two isogenic models of CD95-resistant versus CD95-sensitive cell lines: 8226/S myeloma cells selected for resistance to CD95-mediated apoptosis; and K562 cells expressing ectopic CD95. Repeated exposure of the CD95-sensitive human myeloma cell line, 8226/S, to agonistic anti-CD95 antibody resulted in a cell line devoid of CD95 receptor surface expression and completely resistant to CD95-mediated apoptosis. Multiple clonal populations derived from the CD95-resistant cell line showed no difference in sensitivity to doxorubicin, mitoxantrone, Ara-C, or etoposide, demonstrating that cross-resistance between Fas-mediated apoptosis and drug-induced apoptosis occurs only when cytotoxic drugs are used as the selecting agent. Using the inverse approach, we transfected the CD95-negative cell line, K562, with a CD95 expression vector. Clones expressing variable levels of cell-surface CD95 were isolated by limiting dilution, and analyzed for sensitivity to CD95-mediated apoptosis and response to chemotherapeutic drugs. We show that CD95 surface expression confers sensitivity to CD95-mediated apoptosis; however, it does not alter response to chemotherapeutic drugs. Similarly, doxorubicin-induced activation of caspases 3 and 8 was identical in the CD95-sensitive and CD95-resistant cell lines in both isogenic cell systems. In addition, prior treatment with the CD95 receptor-blocking antibody, ZB4, inhibited CD95-activated apoptosis in 8226/S cells, but had no effect on doxorubicin cytotoxicity. These results show that CD95 and chemotherapeutic drugs use common apoptotic effectors, but the point of convergence in these two pathways is downstream of CD95 receptor/ligand interaction.