RESUMO

Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress response programs that counteract the inherent toxicity of such aberrant signaling. Although inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of protein phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor-suppressive resistance.

RESUMO

B cells contribute to the immune system in many ways such as antigen presentation to CD4+ T cells, secretion of cytokines and lymphoid tissue organogenesis. Furthermore, they are the only cell type capable of producing immunoglobulins. B cells also account for critical aspects of the resistance against intracellular pathogens. Trypanosoma cruzi is an intracellular parasite that sabotages humoral response by depletion of immature B cells. Polyclonal activation and secretion of non-specific antibodies are also other mechanisms used by T cruzi to evade and subvert the mammalian host immune system, leading to increased parasitemia and susceptibility to Chagas' disease. It remained unclear whether B cell depletion occurs due to direct contact with T. cruzi or results from a global increase in inflammation. Unlike previous reports, we demonstrated in this study that T. cruzi infects human B cells, resulting in parasite-induced activation of caspase-7 followed by proteolytic cleavage of phospholipase Cγ1 and cell death. These data contribute to explain the mechanisms ruling B-cell depletion and evasion of the immune response by T. cruzi.

Assuntos

RESUMO

B cells contribute to the immune system in many ways such as antigen presentation to CD4+ T cells, secretion of cytokines and lymphoid tissue organogenesis. Furthermore, they are the only cell type capable of producing immunoglobulins. B cells also account for critical aspects of the resistance against intracellular pathogens. Trypanosoma cruzi is an intracellular parasite that sabotages humoral response by depletion of immature B cells. Polyclonal activation and secretion of non-specific antibodies are also other mechanisms used by T cruzi to evade and subvert the mammalian host immune system, leading to increased parasitemia and susceptibility to Chagas’ disease. It remained unclear whether B cell depletion occurs due to direct contact with T. cruzi or results from a global increase in inflammation. Unlike previous reports, we demonstrated in this study that T. cruzi infects human B cells, resulting in parasite-induced activation of caspase-7 followed by proteolytic cleavage of phospholipase Cgama1 and cell death. These data contribute to explain the mechanisms ruling B-cell depletion and evasion of the immune response by T. cruzi.

RESUMO

In malignant transformation, cellular stress-response pathways are dynami-cally mobilized to counterbalance oncogenic activity, keeping cancer cellsviable. Therapeutic disruption of this vulnerable homeostasis might changethe outcome of many human cancers, particularly those for which no effec-tive therapy is available. Here, we report the use of fibroblast growth factor2 (FGF2) to demonstrate that further mitogenic activation disrupts cellularhomeostasis and strongly sensitizes cancer cells to stress-targeted therapeu-tic inhibitors. We show that FGF2 enhanced replication and proteotoxicstresses in a K-Ras-driven murine cancer cell model, and combinations ofFGF2 and proteasome or DNA damage response-checkpoint inhibitorstriggered cell death. CRISPR/Cas9-mediated K-Ras depletion suppressedthe malignant phenotype and prevented these synergic toxicities in thesemurine cells. Moreover, in a panel of human Ewing’s sarcoma family tumorcells, sublethal concentrations of bortezomib (proteasome inhibitor) or VE-821 (ATR inhibitor) induced cell death when combined with FGF2. Sus-tained MAPK-ERK1/2 overactivation induced by FGF2 appears to under-lie these synthetic lethalities, as late pharmacological inhibition of thispathway restored cell homeostasis and prevented these described synergies.Our results highlight how mitotic signaling pathways which are frequentlyoverridden in malignant transformation might be exploited to disrupt therobustness of cancer cells, ultimately sensitizing them to stress-targeted ther-apies. This approach provides a new therapeutic rationale for human can-cers, with important implications for tumors still lacking effectivetreatment, and for those that frequently relapse after treatment with avail-able therapies.

RESUMO

With recent progress in understanding the pathogenesis of adrenocortical tumors (ACTs), identification of molecular markers to predict their prognosis has become possible. Transcription factor 21 (TCF21)/podocyte-expressed 1 (POD1) is a transcriptional regulatory protein expressed in mesenchymal cells at sites of epithelial-mesenchymal transition during the development of different systems. Adult carcinomas express less TCF21 than adenomas, in addition, the KEGG pathway analysis has shown that BUB1B, among others genes, is negatively correlated with TCF21 expression. The difference between BUB1B and PTEN-induced putative kinase 1 (PINK1) expression has been described previously to be associated with survival in adult but not in pediatric carcinomas. Here, we analyzed the gene expression of TCF21, BUB1B, PINK1, and NR5A1 in adult and pediatric ACTs. We found a negative correlation between the relative expression levels of TCF21 and BUB1B in adult ACTs, but the relative expression levels of TCF21, BUB1B, PINK1, and NR5A1 were similar in childhood ACTs. In addition, we propose using the subtracted expression levels of the TCF21/POD-1 genes as a predictor of overall survival (OS) in adult carcinomas and TCF21-NR5A1 as a predictor of malignancy for pediatric tumors in patients aged <5 years. These results require further validation in different cohorts of both adult and pediatric samples. Finally, we observed that the OS for patients aged <5 years was markedly favorable compared with that for patients >5 years as well as adult patients with carcinoma. In summary, we propose TCF21/POD-1 as a new prognostic marker in adult and pediatric ACTs.

RESUMO

Renal cell carcinoma (RCC) accounts for approximately 2%-3% of human malignancies and is the most aggressive among urologic tumors. Biological heterogeneity, drug resistance, and chemotherapy side effects are the biggest obstacles to the effective treatment of RCC. The NF-κB transcription factor is one of several molecules identified to be responsible for the aggressive phenotype of this tumor. In the past decade, several studies have demonstrated the activation of NF-κB in RCC, and many have implicated NF-κB1 (p50) as an important molecule in tumor progression and metastasis. In the present study, a lentivirus was used to deliver shRNA targeting NF-κB1 into mouse RCC (Renca) cells. It was determined that the knockdown of the NF-κB1 gene led to a reduction in cell proliferation and late apoptosis/necrosis in vitro. Flow cytometry analysis demonstrated G2/M arrest in the cells. In addition, immunoblotting analysis revealed a significant increase in cyclin B1 and Bax. In vivo experiments showed that Renca-shRNA-NF-κB1 cells have significantly diminished tumorigenicity. Moreover, immunohistochemical analysis revealed an increase in necrotic areas of Renca-shRNA-NF-κB1 tumors. Thus, this study indicates that downregulation of NF-κB1 can suppress RCC tumorigenesis by inducing late apoptosis/necrosis. Therefore, NF-κB1 may be a potential therapeutic target for RCC.

Assuntos

RESUMO

Renal cell carcinoma (RCC) accounts for approximately 2%-3% of human malignancies and is the most aggressive among urologic tumors. Biological heterogeneity, drug resistance, and chemotherapy side effects are the biggest obstacles to the effective treatment of RCC. The NF-kappa B transcription factor is one of several molecules identified to be responsible for the aggressive phenotype of this tumor. In the past decade, several studies have demonstrated the activation of NF-kappa B in RCC, and many have implicated NF-kappa B1 (p50) as an important molecule in tumor progression and metastasis. In the present study, a lentivirus was used to deliver shRNA targeting NF-kappa B1 into mouse RCC (Renca) cells. It was determined that the knockdown of the NF-kappa B1 gene led to a reduction in cell proliferation and late apoptosis/necrosis in vitro. Flow cytometry analysis demonstrated G(2)/M arrest in the cells. In addition, immunoblotting analysis revealed a significant increase in cyclin B1 and Bax. In vivo experiments showed that Renca-shRNA-NF-kappa B1 cells have significantly diminished tumori genicity. Moreover, immunohistochemical analysis revealed an increase in necrotic areas of Renca-shRNA-NF-kappa B1 tumors. Thus, this study indicates that downregulation of NF-kappa B1 can suppress RCC tumorigenesis by inducing late apoptosis/necrosis. Therefore, NF-kappa B1 may be a potential therapeutic target for RCC.

RESUMO

With recent progress in understanding the pathogenesis of adrenocortical tumors (ACTs), identification of molecular markers to predict their prognosis has become possible. Transcription factor 21 (TCF21)/podocyte-expressed 1 (POD1) is a transcriptional regulatory protein expressed in mesenchymal cells at sites of epithelial-mesenchymal transition during the development of different systems. Adult carcinomas express less TCF21 than adenomas, in addition, the KEGG pathway analysis has shown that BUB1B, among others genes, is negatively correlated with TCF21 expression. The difference between BUB1B and PTEN-induced putative kinase 1 (PINK1) expression has been described previously to be associated with survival in adult but not in pediatric carcinomas. Here, we analyzed the gene expression of TCF21, BUB1B, PINK1, and NR5A1 in adult and pediatric ACTs. We found a negative correlation between the relative expression levels of TCF21 and BUB1B in adult ACTs, but the relative expression levels of TCF21, BUB1B, PINK1, and NR5A1 were similar in childhood ACTs. In addition, we propose using the subtracted expression levels of the TCF21/POD-1 genes as a predictor of overall survival (OS) in adult carcinomas and TCF21-NR5A1 as a predictor of malignancy for pediatric tumors in patients aged <5 years. These results require further validation in different cohorts of both adult and pediatric samples. Finally, we observed that the OS for patients aged <5 years was markedly favorable compared with that for patients >5 years as well as adult patients with carcinoma. In summary, we propose TCF21/POD-1 as a new prognostic marker in adult and pediatric ACTs.

RESUMO

FGF2 (Fibroblast Growth Factor 2) é o membro fundador de uma grande família de fatores de crescimento protéicos. Sua atividade se dá através da ligação e ativação de receptores específicos de membrana (FGFRs) com atividade de tirosina quinase. No organismo adulto, a sinalização de FGF2 está envolvida na indução de processos de sobrevivência, proliferação e diferenciação celular; além de cicatrização e angiogênese. Por atuar como um clássico fator de crescimento, a atividade de FGF2 está freqüentemente implicada em mecanismos pró-tumorais. Entretanto, alguns grupos, incluindo o nosso, têm reportado que FGF2 também pode apresentar efeitos antiproliferativos a até citotóxicos seletivamente em células malignas. Em 2008, publicamos um compreensivo relato mostrando que FGF2 bloqueia irreversivelmente a proliferação de linhagens murinas malignas dependentes de Ras. Alterações que levem a atividade aumentada de proteínas Ras estão presentes em diversos cânceres humanos e, freqüentemente, resultando em problemas no tratamento e prognóstico ruim. No presente trabalho, utilizamos principalmente a linhagem murina maligna dependente de Ras Y1 D1G, que apresenta um controle estrito de quiescência/proliferação em função da presença de soro; e é por isso mesmo um bom modelo para a análise dos efeitos de FGF2 sobre o ciclo celular. Análises por citometria de fluxo mostraram que, nessas células, apesar de disparar a transição G0→G1→S, FGF2 provoca um atraso na fase S seguido de um bloqueio do ciclo em G2. Embora bloqueie a progressão no ciclo (proliferação), FGF2 induz em Y1 D1G o crescimento celular em termos de massa e volume. Assim, nessas células FGF2 "desconecta" crescimento celular de proliferação. Esse desarranjo do ciclo celular provocado por FGF2 nas células Y1 D1G tem como resultado a instabilidade genotípica e morte celular; evidenciada pela perda da integridade de membrana plasmática e altas taxas de fragmentação de DNA observadas após o estímulo por esse fator. Esse efeito tóxico de FGF2 depende da atividade da proteína Src; porque a inibição química dessa proteína apresentou proteção total frente aos efeitos tóxicos de FGF2. Análises por espectrometria de massas mostraram que FGF2 induz aumento dos níveis de proteínas relacionadas à síntese protéica, e também de proteínas relacionadas ao estresse proteotóxico. Sabe-se que células malignas lidam com níveis basais altos de diferentes tipos de estresse; incluindo o estresse proteotóxico. Esse quadro mostra que o efeito tóxico disparado por FGF2 em Y1 D1G está relacionado a um acumulo de proteínas/célula, perda da homeostase de proteínas e estresse proteotóxico. Corrobora essas proposições o fato de que a inibição química de Src, que protege totalmente as células do efeito tóxico de FGF2, impede completamente o acúmulo de proteínas/célula. Além disso, em células Y1 D1G resistentes ao efeito tóxico de FGF2, e que inclusive dependem deste para proliferar em cultura, a atividade de FGF2 tem efeito oposto; ou seja, provoca diminuição dos níveis estacionários de proteínas/célula. Juntos, esses resultados demonstram que FGF2 é capaz de atacar uma vulnerabilidade de células malignas dependentes de Ras; e no caso estudado, essa vulnerabilidade decorre do desequilíbrio na homeostase de proteínas

FGF2 is the first member of a large family of peptide growth factors. It binds and activates specific membrane receptors (FGFRs) belonging to a family of tyrosine kinase receptors (RTK). In adult organisms, FGF2 signaling is involved in the induction of cell surveillance, proliferation and differentiation; and also wound healing and angiogenesis. FGF2 is a bona fide growth factor and, as such, it is often implicated in pro-tumor mechanisms. However, several groups, including ours, have reported that FGF2 can also display antiproliferative and even cytotoxic effects selectively in malignant cells. In 2008, we fully reported that FGF2 irreversibly blocks the proliferation of Ras-driven mouse malignant lineages. Alterations leading to Ras proteins overactivity are present in many human cancers frequently with bad prognosis. In the present work, we used mainly the Ras-driven mouse malignant lineage Y1 D1G that shows a strict control of quiescence/proliferation by serum factors, making it a great model to analyze the FGF2 effects upon cell cycle control. Flow cytometry analyses showed that in these cells, in spite of triggering G0→G1→S transition, FGF2 causes a delay on S phase followed by cell cycle arrest in G2. Despite blocking cell division, FGF2 induces cell growth in terms of mass and volume. Therefore, in these cells FGF2 "disconnects" cell growth from proliferation. This malfunction of cell cycle control caused by FGF2 on Y1 D1G cells leads to genotypic instability and cell death, highlighted by loss of plasma membrane integrity and high rates of DNA fragmentation. This FGF2 toxic effect depends on the activity of Src protein, because Src chemical inhibition completely protects cells from the FGF2 toxic effects. Mass spec analyses showed that FGF2 increases the levels of proteins involved in the protein synthesis machinery, and also of proteins active in proteostasis, indicating proteotoxic stress. It is known that malignant cells deal with high basal levels of different stresses, including the proteotoxic stress. This picture shows that the toxic effects triggered by FGF2 in Y1 D1G involve accumulation of proteins/cell, loss of protein homeostasis and proteotoxic stress. Corroborating these propositions, chemical inhibition of Src, which completely protects the cells from FGF2 toxic effects, totally abrogates the accumulation of proteins/cell. Moreover, in FGF2-resistant Y1 D1G cells, which depend on this factor for proliferation, FGF2 shows the opposite effect, causing decrease in steady state levels of protein/cell. Altogether, these results show that FGF2 causes a severe proteostasis imbalance in these Ras-driven mouse malignant cells

Assuntos

RESUMO

FGF2 ( Fibroblast Growth Factor 2) é o membro fundador de uma grande família de fatores de crescimento proteícos. Sua atividade se dá através da ligação e ativação de receptores especificos da membrana (FGFRs) com atividade de tirosina quimase. No organismo adulto, a simalização de FGF2 está envolvida na indução de processos de sobrevivência, proliferação e diferenciação celular; além de cicatrização e angiogênese. Por atuar como um clássico fator de crescimento, a atividade de FGF2 está frequentemente implicada em mecanismos pró-tumorais....

FGF2 is the first member of a large family of peptide growth factors. It binds and activates specific membrane receptors ( FGFRs) belonging to a family of tyrosine kinase receptors (RTK). in adult organisms, FGF2 signaling is involved in the induction of cell surveillance, proliferation and differentation; and also wound healing and angiogenesis. FGF2 is a bona fide growth factor and, as such, it is often implicated in pro-tumor mechanisms...

Assuntos