RESUMO
The inducible costimulator (ICOS) plays a key role in the development of Th17 cells, but its role in the development and antitumor activity of IL-17-producing CD8(+) T cells (Tc17) remains unknown. We found that ICOS costimulation was important for the functional maintenance, but not differentiation, of Tc17 cells in vitro. Blocking the ICOS pathway using an antagonist mAb or by using recipient mice genetically deficient in the ICOS ligand reduced the antitumor activity of adoptively transferred Tc17 cells. Conversely, activating Tc17 cells with an ICOS agonist in vitro enhanced their capacity to eradicate melanoma and induce autoimmune vitiligo when infused into mice. However, ICOS stimulation did not augment the antitumor activity of IL-2 expanded T cells. Additional investigation revealed that ICOS stimulation not only increased IL-2Rα, CXCR3, and IL-23R expression on Tc17 cells, but also dampened their expression of suppressive molecule CD39. Although Tc17 cells activated with an ICOS agonist cosecreted heightened IL-17A, IL-9, and IFN-γ, their therapeutic effectiveness was critically dependent on IFN-γ production. Depletion of IL-17A and IL-9 had little impact on antitumor Tc17 cells activated with an ICOS agonist. Collectively, our work reveals that the ICOS pathway potentiates the antitumor activity of adoptively transferred Tc17 cells. This work has major implications for the design of vaccine, Ab and cell-based therapies for autoimmunity, infectious disease, and cancer.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva/métodos , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Melanoma/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Diferenciação Celular/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Interleucina-17/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Metastatic melanoma is an aggressive and deadly disease. The chemokine receptor CXCR4 is active in melanoma metastasis, although the mechanism for the promotion and maintenance of CXCR4 expression in these cells is mostly unknown. Here, we find melanoma cells express two CXCR4 isoforms, the common version and a variant that is normally restricted to cells during development or to mature blood cells. CXCR4 expression is driven through a highly conserved intronic enhancer element by the transcription factors PAX3 and FOXD3. Inhibition of these transcription factors slows melanoma cell growth, migration, and motility, as well as reduces CXCR4 expression. Overexpression of these transcription factors drives the production of increased CXCR4 levels. Loss of PAX3 and FOXD3 transcription factor activity results in a reduction in cell motility, migration, and chemotaxis, all of which are rescued by CXCR4 overexpression. Here, we discover a molecular pathway wherein PAX3 and FOXD3 promote CXCR4 gene expression in melanoma.
Assuntos
Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Box Pareados/genética , Receptores CXCR4/genética , Células 3T3 , Animais , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/genética , Elementos Facilitadores Genéticos/genética , Fatores de Transcrição Forkhead/metabolismo , Células HEK293 , Humanos , Íntrons/genética , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/metabolismo , Ligação Proteica , Receptores CXCR4/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Several key transcription factors regulate cell growth, survival, and differentiation during neural crest and melanoblast development in the embryo, and these same pathways may be reactivated in tumors arising from the progenitors of these cells. The transcription factors PAX3 and FOXD3 have essential roles in melanoblasts and melanoma. In this study, we define a regulatory pathway where FOXD3 promotes the expression of PAX3. Both factors are expressed in melanoma cells and there is a positive correlation between the transcript levels of PAX3 and FOXD3. The PAX3 gene contains two FOX binding motifs within highly conserved enhancer regulatory elements that are essential for neural crest development. FOXD3 binds to both of these motifs in vitro but only one of these sites is preferentially utilized in melanoma cells. Overexpression of FOXD3 upregulates PAX3 levels while inhibition of FOXD3 function does not alter PAX3 protein levels, supporting that FOXD3 is sufficient but not necessary to drive PAX3 expression in melanoma cells. Here, we identify a molecular pathway where FOXD3 upregulates PAX3 expression and therefore contributes to melanoma progression.
Assuntos
Fatores de Transcrição Forkhead/fisiologia , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Box Pareados/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Sequência Conservada , Expressão Gênica , Humanos , Melanoma , Dados de Sequência Molecular , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Regiões Promotoras Genéticas , Ativação TranscricionalRESUMO
BACKGROUND: The development of novel therapeutics for pancreatic cancer has been hindered by a lack of relevant preclinical models. The purpose of this study was to evaluate the clinical relevancy of two pancreatic cancer models using standard-of-care therapeutic agent gemcitabine. MATERIALS AND METHODS: Murine Panc02 cells were injected directly into the spleen or pancreas of C57BL/6 mice to respectively create models of metastatic and locally advanced pancreatic cancer. Beginning 7 d post-Panc02 injection, treated mice received 20 mg/kg gemcitabine i.p. every 3 d. Animals were sacrificed when the untreated mice became moribund and tumor/liver weight used to assess tumor burden. RESULTS: Untreated mice became moribund 22 d after pancreatic Panc02 injection. Gross analysis revealed localized pancreatic tumors weighing 1.063 g. Intrasplenic Panc02 injection produced extensive liver metastasis by d 15 when the untreated mice first became moribund. Liver weights at this time averaged 3.6 g compared with the average non-tumor-bearing weight of 1.23 g. Gemcitabine therapy resulted in a 54% decrease in localized pancreatic tumor weight and 62.5% decrease in metastatic liver weight. Additionally, gemcitabine therapy extended animal survival to 20.5 d compared with 18.0 d average for the untreated mice. CONCLUSIONS: We describe two models depicting both locally advanced and metastatic pancreatic cancer in immunocompetent mice. In efforts to establish baseline therapeutic efficacy, we determined that gemcitabine reduces tumor burden in both models and enhances survival in the metastatic model. These clinically relevant models provide valuable tools to evaluate novel therapeutics in pancreatic cancer.
Assuntos
Modelos Animais de Doenças , Imunocompetência/imunologia , Camundongos , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/secundário , Animais , Antimetabólitos Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Camundongos Endogâmicos C57BL , Transplante de Neoplasias/métodos , Pâncreas/imunologia , Neoplasias Pancreáticas/tratamento farmacológico , Índice de Gravidade de Doença , Baço/imunologia , Taxa de Sobrevida , GencitabinaRESUMO
The failure of once promising target-specific therapeutic strategies often arises from redundancies in gene expression pathways. Even with new melanoma treatments, many patients are not responsive or develop resistance, leading to disease progression in terms of growth and metastasis. We previously discovered that the transcription factors ETS1 and PAX3 drive melanoma growth and metastasis by promoting the expression of the MET receptor. Here, we find that there are multiple ETS family members expressed in melanoma and that these factors have redundant functions. The small molecule YK-4-279, initially developed to target the ETS gene-containing translocation product EWS-FLI1, significantly inhibited cellular growth, invasion, and ETS factor function in melanoma cell lines and a clinically relevant transgenic mouse model, BrafCA;Tyr-CreERT2;Ptenf/f. One of the antitumor effects of YK-4-279 in melanoma is achieved via interference of multiple ETS family members with PAX3 and the expression of the PAX3-ETS downstream gene MET. Expression of exogenous MET provided partial rescue of the effects of YK-4-279, further supporting that MET loss is a significant contributor to the antitumor effects of the drug. This is the first study identifying multiple overlapping functions of the ETS family promoting melanoma. In addition, targeting all factors, rather than individual members, demonstrated impactful deleterious consequences in melanoma progression. Given that multiple ETS factors are known to have oncogenic functions in other malignancies, these findings have a high therapeutic impact. SIGNIFICANCE: These findings identify YK-4-279 as a promising therapeutic agent against melanoma by targeting multiple ETS family members and blocking their ability to act as transcription factors.
Assuntos
Indóis/farmacologia , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-ets/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Humanos , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Fator de Transcrição PAX3/antagonistas & inibidores , Fator de Transcrição PAX3/metabolismo , Proteína Proto-Oncogênica c-ets-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteína EWS de Ligação a RNA/antagonistas & inibidores , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
Yes Associated Protein 1 (YAP) and Transcriptional coactivator with PDZ-Binding Motif (TAZ) have gained notoriety for their ability to drive tumor initiation and progression in a wide variety of cancers, including melanoma. YAP and TAZ act as drivers of melanoma through its interaction with the TEAD family of transcription factors. Verteporfin is a benzoporphyrin derivative that is used clinically for photodynamic treatment of macular degeneration. Recently it has emerged as a potential inhibitor of YAP/TAZ-TEAD interaction independent of light activation. In this study we determine if verteporfin has clinical potential by testing this compound on human melanoma cell cultures and in a clinically significant mouse model, BrafCA; Tyr-CreERT2; Ptenf/f, which parallels human melanoma in terms of disease progression, genetics, and histopathology. In culture, Verteporfin treatment induces a rapid drop in YAP and TAZ protein levels and cell numbers. In the transgenic model, utilizing drug levels that correspond to previously determined safe doses in human patients and with a dosing regimen calculated in this study, Verteporfin did not inhibit melanoma initiation or progression in comparison to mock treated controls. Taken together, our study suggests that although Verteporfin induces YAP/TAZ degradation in melanoma cell lines, Verteporfin was not effective as a YAP/TAZ-TEAD specific inhibitor of melanoma in our studies that aimed to mimic conditions found in clinic in terms of treatment regimen and disease model.
RESUMO
Pancreatic cancer is a lethal disease with a propensity for invading and metastasizing into the surrounding tissues, including the liver and intestines. A number of factors are aberrantly overexpressed in this tumor type and actively promote cancer progression and metastasis. The present study demonstrates that paired box transcription factor 6 (PAX6) and C-X-C chemokine receptor 4 (CXCR4) are frequently co-expressed in primary pancreatic adenocarcinoma tumors and established cell lines. Expression analysis methods used in the present study included evaluation of protein expression by western blot analysis and immunofluorescence, transcript expression levels by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and luciferase assays utilizing regulatory elements from the CXCR4 gene locus. Canonical PAX6 and alternative splice variant PAX6(5a) proteins are expressed in pancreatic cancer and can drive gene expression through a conserved enhancer element within the first intron of the CXCR4 gene. As demonstrated by the introduction of an exogenous reporter construct with or without the intronic enhancer, loss of this element inhibited gene expression within numerous pancreatic cancer cell lines including Panc1, MIA-PaCa2 and BxPC3. All of the pancreatic cancer cell lines expressed the canonical CXCR4B transcript in addition to the alternatively spliced variant CXCR4A as determined by RT-qPCR experiments. The discovery of variant transcripts in pancreatic cancer cells may provide new candidates for future targeted therapies.