RESUMO
The innate immune system has a key role in pancreatic cancer initiation, but the specific contribution of different macrophage populations is still ill-defined. While inflammatory (M1) macrophages have been shown to drive acinar-to-ductal metaplasia (ADM), a cancer initiating event, alternatively activated (M2) macrophages have been attributed to lesion growth and fibrosis. Here, we determined cytokines and chemokines secreted by both macrophage subtypes. Then, we analyzed their role in ADM initiation and lesion growth, finding that while M1 secrete TNF, CCL5, and IL-6 to drive ADM, M2 induce this dedifferentiation process via CCL2, but the effects are not additive. This is because CCL2 induces ADM by generating ROS and upregulating EGFR signaling, thus using the same mechanism as cytokines from inflammatory macrophages. Therefore, while effects on ADM are not additive between macrophage polarization types, both act synergistically on the growth of low-grade lesions by activating different MAPK pathways.
RESUMO
TREM2 is exclusively expressed by microglia in the brain and is strongly linked to the risk for Alzheimer's disease (AD). As microglial responses modulated by TREM2 are central to AD pathogenesis, enhancing TREM2 signaling has been explored as an AD therapeutic strategy. However, the effective therapeutic window targeting TREM2 is unclear. Here, by using microglia-specific inducible mouse models overexpressing human wild-type TREM2 (TREM2-WT) or R47H risk variant (TREM2-R47H), we show that TREM2-WT expression reduces amyloid deposition and neuritic dystrophy only during the early amyloid seeding stage, whereas TREM2-R47H exacerbates amyloid burden during the middle amyloid rapid growth stage. Single-cell RNA sequencing reveals suppressed disease-associated microglia (DAM) signature and reduced DAM population upon TREM2-WT expression in the early stage, whereas upregulated antigen presentation pathway is detected with TREM2-R47H expression in the middle stage. Together, our findings highlight the dynamic effects of TREM2 in modulating AD pathogenesis and emphasize the beneficial effect of enhancing TREM2 function in the early stage of AD development.
Assuntos
Doença de Alzheimer , Amiloidose , Doença de Alzheimer/patologia , Amiloide/metabolismo , Amiloidose/patologia , Animais , Encéfalo/patologia , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Microglia/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismoRESUMO
Desmoplasia around pancreatic lesions is a barrier for immune cells and a hallmark of developing and established pancreatic cancer. However, the contribution of the innate immune system to this process is ill-defined. Using the KC mouse model and primary cells in vitro, we show that alternatively activated macrophages (AAM) crosstalk with pancreatic lesion cells and pancreatic stellate cells (PSCs) to mediate fibrosis and progression of lesions. TGFß1 secreted by AAM not only drives activation of quiescent PSCs but also in activated PSCs upregulates expression of TIMP1, a factor previously shown as crucial in fibrosis. Once activated, PSCs auto-stimulate proliferation via CXCL12. Furthermore, we found that TIMP1/CD63 signaling mediates PanIN lesion growth and TGFß1 contributes to a cadherin switch and drives structural collapse of lesions, indicating a potential progression step. Taken together, our data indicate TGFß1 produced by Ym1+ AAM as a major driver of processes that initiate the development of pancreatic cancer.
RESUMO
The development of pancreatic cancer requires recruitment and activation of different macrophage populations. However, little is known about how macrophages are attracted to the pancreas after injury or an oncogenic event, and how they crosstalk with lesion cells or other cells of the lesion microenvironment. Here, we delineate the importance of CXCL10/CXCR3 signaling during the early phase of murine pancreatic cancer. We show that CXCL10 is produced by pancreatic precancerous lesion cells in response to IFNγ signaling and that inflammatory macrophages are recipients for this chemokine. CXCL10/CXCR3 signaling in macrophages mediates their chemoattraction to the pancreas, enhances their proliferation, and maintains their inflammatory identity. Blocking of CXCL10/CXCR3 signaling in vivo shifts macrophage populations to a tumor-promoting (Ym1+, Fizz+, Arg1+) phenotype, increases fibrosis, and mediates progression of lesions, highlighting the importance of this pathway in PDA development. This is reversed when CXCL10 is overexpressed in PanIN cells.
Assuntos
Quimiocina CXCL10/imunologia , Quimiocina CXCL10/metabolismo , Inflamação/etiologia , Neoplasias Pancreáticas/fisiopatologia , Receptores CXCR3/imunologia , Receptores CXCR3/metabolismo , Microambiente Tumoral/imunologia , Animais , Células Cultivadas , Quimiocina CXCL10/antagonistas & inibidores , Quimiocina CXCL10/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Inflamação/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Pâncreas/citologia , Pâncreas/imunologia , Pâncreas/patologia , Neoplasias Pancreáticas/imunologia , Receptores CXCR3/antagonistas & inibidores , Receptores CXCR3/genética , Transdução de SinaisRESUMO
Doublecortin-like kinase 1 (DCLK1)-positive pancreatic cancer stem cells develop at a precancerous stage and may contribute to the lack of efficacy of pancreatic cancer therapy. Although PanIN cells express oncogenic KRas and have an increased activity of epidermal growth factor receptor (EGFR), we demonstrate that, in DCLK1+ PanIN cells, EGFR signaling is not propagated to the nucleus. Mimicking blockage of EGFR with erlotinib in PanIN organoid culture or in p48cre;KrasG12D mice led to a significant increase in DCLK1+ PanIN cells. As a mechanism of how EGFR inhibition leads to formation of DCLK1+ cells, we identify an increase in hydrogen peroxide contributing to activation of Protein Kinase D1 (PKD1). Active PKD1 then drives stemness and abundance of DCLK1+ cells in lesions. Our data suggest a signaling mechanism that leads to the development of DCLK1+ pancreatic cancer stem cells, which can be exploited to target this population in potential therapeutic approaches.
RESUMO
Current treatment options for patients with pancreatic cancer are suboptimal, resulting in a five year survival rate of about 9%. Difficulties with treatment are due to an immunosuppressive, fibrotic tumor microenvironment that prevents drugs from reaching tumor cells, but also to the limited efficacy of existing FDA-approved chemotherapeutic compounds. We here show that the nucleoside analog Sangivamycin and its closely-related compound Toyocamycin target PDA cell lines, and are significantly more efficient than Gemcitabine. Using KINOMEscan screening, we identified the kinase Haspin, which is overexpressed in PDA cell lines and human PDA samples, as a main target for both compounds. Inhibition of Haspin leads to a decrease in Histone H3 phosphorylation and prevents Histone H3 binding to survivin, thus providing mechanistic insight of how Sangivamycin targets cell proliferation, mitosis and induces apoptotic cell death. In orthotopically implanted tumors in mice, Sangivamycin was efficient in decreasing the growth of established tumors. In summary, we show that Sangivamycin and derivatives can be an efficient new option for treatment of PDA.
Assuntos
Apoptose , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pancreáticas/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Nucleosídeos de Pirimidina/farmacologia , Survivina/antagonistas & inibidores , Animais , Antibióticos Antineoplásicos/farmacologia , Biomarcadores Tumorais , Proliferação de Células , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Fosforilação , Prognóstico , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Survivina/genética , Survivina/metabolismo , Células Tumorais Cultivadas , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
During development of pancreatic cancer, alternatively activated macrophages contribute to fibrogenesis, pancreatic intraepithelial neoplasia (PanIN) lesion growth, and generation of an immunosuppressive environment. Here, we show that the immunomodulatory agent pomalidomide depletes pancreatic lesion areas of alternatively activated macrophage populations. Pomalidomide treatment resulted in downregulation of interferon regulatory factor 4, a transcription factor for M2 macrophage polarization. Pomalidomide-induced absence of alternatively activated macrophages led to a decrease in fibrosis at PanIN lesions and in syngeneic tumors; this was due to generation of an inflammatory, immune-responsive environment with increased expression of IL1α and presence of activated (IFNγ-positive) CD4+ and CD8+ T-cell populations. Our results indicate that pomalidomide could be used to decrease fibrogenesis in pancreatic cancer and may be ideal as a combination treatment with chemotherapeutic drugs or other immunotherapies. SIGNIFICANCE: These findings reveal new insights into how macrophage populations within the pancreatic cancer microenvironment can be modulated, providing the means to turn the microenvironment from immunosuppressive to immune-responsive.
Assuntos
Fatores Imunológicos/farmacologia , Macrófagos/imunologia , Neoplasias Pancreáticas/imunologia , Lesões Pré-Cancerosas/imunologia , Talidomida/análogos & derivados , Animais , Humanos , Fatores Reguladores de Interferon/metabolismo , Camundongos , Neoplasias Pancreáticas/metabolismo , Lesões Pré-Cancerosas/metabolismo , Talidomida/farmacologia , Microambiente Tumoral , Células U937RESUMO
Phosphatidylinositol-4-phosphate 5-kinase type-1C (PIP5K1C) is a lipid kinase that regulates focal adhesion dynamics and cell attachment through site-specific formation of phosphatidylinositol-4,5-bisphosphate (PI4,5P2). By comparing normal breast tissue to carcinoma in situ and invasive ductal carcinoma subtypes, we here show that the phosphorylation status of PIP5K1C at serine residue 448 (S448) can be predictive for breast cancer progression to an aggressive phenotype, while PIP5K1C expression levels are not indicative for this event. PIP5K1C phosphorylation at S448 is downregulated in invasive ductal carcinoma, and similarly, the expression levels of PKD1, the kinase that phosphorylates PIP5K1C at this site, are decreased. Overall, since PKD1 is a negative regulator of cell migration and invasion in breast cancer, the phosphorylation status of this residue may serve as an indicator of aggressiveness of breast tumors.
RESUMO
The contributions of the innate immune system to the development of pancreatic cancer are still ill defined. Inflammatory macrophages can initiate metaplasia of pancreatic acinar cells to a duct-like phenotype (acinar-to-ductal metaplasia [ADM]), which then gives rise to pancreatic intraepithelial neoplasia (PanIN) when oncogenic KRas is present. However, it remains unclear when and how this inflammatory macrophage population is replaced by tumor-promoting macrophages. Here, we demonstrate the presence of interleukin-13 (IL-13), which can convert inflammatory into Ym1+ alternatively activated macrophages, at ADM/PanIN lesions. We further show that Ym1+ macrophages release factors, such as IL-1ra and CCL2, to drive pancreatic fibrogenesis and tumorigenesis. Treatment of mice expressing oncogenic KRas under an acinar cell-specific promoter with a neutralizing antibody for IL-13 significantly decreased the accumulation of alternatively activated macrophages at these lesions, resulting in decreased fibrosis and lesion growth.
Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Interleucina-13/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Linhagem Celular Tumoral , Polaridade Celular , Proliferação de Células , Fibrose , Inflamação/patologia , Macrófagos/patologia , Metaplasia , Camundongos , Testes de Neutralização , Ductos Pancreáticos/metabolismoRESUMO
Increased expression of PRKD1 and its gene product protein kinase D1 (PKD1) are linked to oncogenic signaling in pancreatic ductal adenocarcinoma, but a direct functional relationship to oncogenic KRas has not been established so far. We here describe the PRKD1 gene promoter as a target for oncogenic KRas signaling. We demonstrate that KRas-induced activation of the canonical NF-κB pathway is one mechanism of how PRKD1 expression is increased and identify the binding sites for NF-κB in the PRKD1 promoter. Altogether, these results describe a novel mechanism governing PRKD1 gene expression in PDA and provide a functional link between oncogenic KRas, NF-κB and expression of PRKD1.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , NF-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Regiões Promotoras Genéticas , Proteína Quinase C/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Humanos , NF-kappa B/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteína Quinase C/genética , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Vasodilator-stimulated phosphoprotein (VASP) signaling is critical for dynamic actin reorganization processes that define the motile phenotype of cells. Here we show that VASP is generally highly expressed in normal breast tissue and breast cancer. We also show that the phosphorylation status of VASP at S322 can be predictive for breast cancer progression to an aggressive phenotype. Our data indicate that phosphorylation at S322 is gradually decreased from normal breast to DCIS, luminal/ER+, HER2+ and basal-like/TN phenotypes. Similarly, the expression levels of PKD2, the kinase that phosphorylates VASP at this site, are decreased in invasive ductal carcinoma samples of all three groups. Overall, the phosphorylation status of this residue may serve as an indicator of aggressiveness of breast tumors.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Serina/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal/genética , Carcinoma Ductal/patologia , Moléculas de Adesão Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Progressão da Doença , Células HeLa , Humanos , Immunoblotting , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Proteínas dos Microfilamentos/genética , Microscopia Confocal , Mutação , Invasividade Neoplásica , Fosfoproteínas/genética , Fosforilação , Prognóstico , Proteína Quinase D2 , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Serina/genética , Análise Serial de TecidosRESUMO
Neuregulin (NRG; heregulin) is overexpressed in â¼30% of breast cancers and mediates various processes involved in tumor progression, including tumor cell migration and invasion. Here, we show that NRG mediates its effects on tumor cell migration via PKD1. Downstream of RhoA, PKD1 can prevent directed cell migration through phosphorylation of its substrate SSH1L. NRG exerts its inhibitory effects on PKD1 through Rac1/NADPH oxidase, leading to decreased PKD1 activation loop phosphorylation and decreased activity toward SSH1L. The consequence of PKD1 inhibition by NRG is decreased binding of 14-3-3 to SSH1L, localization of SSH1L to F-actin at the leading edge, and increased cofilin activity, resulting in increased reorganization of the actin cytoskeleton and cell motility. Our data provide a mechanism through which the Rho GTPase Rac1 cross-talks with PKD1 signaling pathways to facilitate directed cell migration.
Assuntos
Actinas/metabolismo , Neuregulina-1/metabolismo , Proteína Quinase C/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Neoplasias da Mama/metabolismo , Movimento Celular , Quimiotaxia , Progressão da Doença , Feminino , Humanos , Camundongos , Microscopia de Fluorescência/métodos , Invasividade Neoplásica , Metástase Neoplásica , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , CicatrizaçãoRESUMO
BACKGROUND: Protein kinase D1 is downregulated in its expression in invasive ductal carcinoma of the breast and in invasive breast cancer cells, but its functions in normal breast epithelial cells is largely unknown. The epithelial phenotype is maintained by cell-cell junctions formed by E-cadherin. In cancer cells loss of E-cadherin expression contributes to an invasive phenotype. This can be mediated by SNAI1, a transcriptional repressor for E-cadherin that contributes to epithelial-to-mesenchymal transition (EMT). METHODOLOGY/PRINCIPAL FINDINGS: Here we show that PKD1 in normal murine mammary gland (NMuMG) epithelial cells is constitutively-active in its basal state and prevents a transition to a mesenchymal phenotype. Investigation of the involved mechanism suggested that PKD1 regulates the expression of E-cadherin at the promoter level through direct phosphorylation of the transcriptional repressor SNAI1. PKD1-mediated phosphorylation of SNAI1 occurs in the nucleus and generates a nuclear, inactive DNA/SNAI1 complex that shows decreased interaction with its co-repressor Ajuba. Analysis of human tissue samples with a newly-generated phosphospecific antibody for PKD1-phosphorylated SNAI1 showed that regulation of SNAI1 through PKD1 occurs in vivo in normal breast ductal tissue and is decreased or lost in invasive ductal carcinoma. CONCLUSIONS/SIGNIFICANCE: Our data describe a mechanism of how PKD1 maintains the breast epithelial phenotype. Moreover, they suggest, that the analysis of breast tissue for PKD-mediated phosphorylation of SNAI1 using our novel phosphoS11-SNAI1-specific antibody may allow predicting the invasive potential of breast cancer cells.