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1.
iScience ; 26(6): 106820, 2023 Jun 16.
Article in English | MEDLINE | ID: mdl-37250781

ABSTRACT

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.

2.
iScience ; 25(5): 104327, 2022 May 20.
Article in English | MEDLINE | ID: mdl-35602933

ABSTRACT

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.

3.
iScience ; 24(1): 102019, 2021 Jan 22.
Article in English | MEDLINE | ID: mdl-33521594

ABSTRACT

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.

4.
Sci Rep ; 9(1): 16588, 2019 11 12.
Article in English | MEDLINE | ID: mdl-31719634

ABSTRACT

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.


Subject(s)
Apoptosis , Gene Expression Regulation, Neoplastic/drug effects , Histones/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Pancreatic Neoplasms/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidine Nucleosides/pharmacology , Survivin/antagonists & inhibitors , Animals , Antibiotics, Antineoplastic/pharmacology , Biomarkers, Tumor , Cell Proliferation , Histones/genetics , Histones/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Phosphorylation , Prognosis , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Survivin/genetics , Survivin/metabolism , Tumor Cells, Cultured , Tumor Microenvironment , Xenograft Model Antitumor Assays
5.
Cancer Res ; 79(7): 1535-1548, 2019 04 01.
Article in English | MEDLINE | ID: mdl-30696657

ABSTRACT

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.


Subject(s)
Immunologic Factors/pharmacology , Macrophages/immunology , Pancreatic Neoplasms/immunology , Precancerous Conditions/immunology , Thalidomide/analogs & derivatives , Animals , Humans , Interferon Regulatory Factors/metabolism , Mice , Pancreatic Neoplasms/metabolism , Precancerous Conditions/metabolism , Thalidomide/pharmacology , Tumor Microenvironment , U937 Cells
6.
Sci Rep ; 7(1): 9524, 2017 08 25.
Article in English | MEDLINE | ID: mdl-28842658

ABSTRACT

Dependent on their cellular localization, Protein Kinase D (PKD) enzymes regulate different processes including Golgi transport, cell signaling and response to oxidative stress. The localization of PKD within cells is mediated by interaction with different lipid or protein binding partners. With the example of PKD2, we here show that phosphorylation events can also contribute to localization of subcellular pools of this kinase. Specifically, in the present study, we show that tyrosine phosphorylation of PKD2 at residue Y87 defines its localization to the focal adhesions and leads to activation. This phosphorylation occurs downstream of RhoA signaling and is mediated via Src. Moreover, mutation of this residue blocks PKD2's interaction with Focal Adhesion Kinase (FAK). The presence and regulation of PKD2 at focal adhesions identifies a novel function for this kinase as a modulator of cell adhesion and migration.


Subject(s)
Cell Adhesion , Focal Adhesions , Protein Kinases/metabolism , src-Family Kinases/metabolism , Cell Movement , Fluorescent Antibody Technique , Immunohistochemistry , Phosphorylation , Protein Kinase D2 , rhoA GTP-Binding Protein/metabolism
7.
Sci Rep ; 6: 35963, 2016 10 24.
Article in English | MEDLINE | ID: mdl-27775029

ABSTRACT

Focal adhesions (FAs) are highly dynamic structures that are assembled and disassembled on a continuous basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion and spreading to directed cell migration. The turnover of FAs is regulated at multiple levels and involves a variety of signaling molecules and adaptor proteins. In the present study, we show that in response to integrin engagement, a subcellular pool of Protein Kinase D1 (PKD1) localizes to the FAs. PKD1 affects FAs by decreasing turnover and promoting maturation, resulting in enhanced cell adhesion. The effects of PKD1 are mediated through direct phosphorylation of FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ) at serine residue 448. This phosphorylation occurs in response to Fibronectin-RhoA signaling and leads to a decrease in PIP5Klγs' lipid kinase activity and binding affinity for Talin. Our data reveal a novel function for PKD1 as a regulator of FA dynamics and by identifying PIP5Klγ as a novel PKD1 substrate provide mechanistic insight into this process.


Subject(s)
Cell Adhesion , Focal Adhesions/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , TRPP Cation Channels/metabolism , Animals , Cell Line , Humans , Integrins/metabolism , Mice , Phosphorylation , Protein Processing, Post-Translational , Signal Transduction
8.
PLoS One ; 9(5): e98090, 2014.
Article in English | MEDLINE | ID: mdl-24840177

ABSTRACT

BACKGROUND: Protein kinase D (PKD) enzymes regulate cofilin-driven actin reorganization and directed cell migration through both p21-activated kinase 4 (PAK4) and the phosphatase slingshot 1L (SSH1L). The relative contributions of different endogenous PKD isoforms to both signaling pathways have not been elucidated, sufficiently. METHODOLOGY/PRINCIPAL FINDINGS: We here analyzed two cell lines (HeLa and MDA-MB-468) that express the subtypes protein kinase D2 (PKD2) and protein kinase D3 (PKD3). We show that under normal growth conditions both isoforms can form a complex, in which PKD3 is basally-active and PKD2 is inactive. Basal activity of PKD3 mediates PAK4 activity and downstream signaling, but does not significantly inhibit SSH1L. This signaling constellation was required for facilitating directed cell migration. Activation of PKD2 and further increase of PKD3 activity leads to additional phosphorylation and inhibition of endogenous SSH1L. Net effect is a dramatic increase in phospho-cofilin and a decrease in cell migration, since now both PAK4 and SSH1L are regulated by the active PKD2/PKD3 complex. CONCLUSIONS/SIGNIFICANCE: Our data suggest that PKD complexes provide an interface for both cofilin regulatory pathways. Dependent on the activity of involved PKD enzymes signaling can be balanced to guarantee a functional cofilin activity cycle and increase cell migration, or imbalanced to decrease cell migration. Our data also provide an explanation of how PKD isoforms mediate different effects on directed cell migration.


Subject(s)
Actin Depolymerizing Factors/metabolism , Cell Movement/physiology , Multiprotein Complexes/metabolism , Protein Isoforms/metabolism , Protein Kinase C/genetics , Signal Transduction/physiology , Cell Movement/genetics , HeLa Cells , Humans , Immunoblotting , Immunoprecipitation , Multiprotein Complexes/genetics , Oligonucleotides/genetics , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Protein Isoforms/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , p21-Activated Kinases/metabolism
9.
J Biol Chem ; 288(34): 24382-93, 2013 Aug 23.
Article in English | MEDLINE | ID: mdl-23846685

ABSTRACT

Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP) protein family members link actin dynamics and cellular signaling pathways. VASP localizes to regions of dynamic actin reorganization such as the focal adhesion contacts, the leading edge or filopodia, where it contributes to F-actin filament elongation. Here we identify VASP as a novel substrate for protein kinase D1 (PKD1). We show that PKD1 directly phosphorylates VASP at two serine residues, Ser-157 and Ser-322. These phosphorylations occur in response to RhoA activation and mediate VASP re-localization from focal contacts to the leading edge region. The net result of this PKD1-mediated phosphorylation switch in VASP is increased filopodia formation and length at the leading edge. However, such signaling when persistent induced membrane ruffling and decreased cell motility.


Subject(s)
Cell Adhesion Molecules/metabolism , Cell Movement/physiology , Microfilament Proteins/metabolism , Phosphoproteins/metabolism , Protein Kinase C/metabolism , Signal Transduction/physiology , Actins/genetics , Actins/metabolism , Cell Adhesion Molecules/genetics , Focal Adhesions/genetics , Focal Adhesions/metabolism , HeLa Cells , Humans , Microfilament Proteins/genetics , Phosphoproteins/genetics , Phosphorylation/physiology , Protein Kinase C/genetics , Protein Transport/physiology , Pseudopodia/genetics , Pseudopodia/metabolism , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism
10.
Biochem J ; 455(2): 251-60, 2013 Oct 15.
Article in English | MEDLINE | ID: mdl-23841590

ABSTRACT

PAKs (p21-activated kinases) are effectors of RhoGTPases. PAK4 contributes to regulation of cofilin at the leading edge of migrating cells through activation of LIMK (Lin-11/Isl-1/Mec-3 kinase). PAK4 activity is regulated by an autoinhibitory domain that is released upon RhoGTPase binding as well as phosphorylation at Ser474 in the activation loop of the kinase domain. In the present study, we add another level of complexity to PAK4 regulation by showing that phosphorylation at Ser99 is required for its targeting to the leading edge. This phosphorylation is mediated by PKD1 (protein kinase D1). Phosphorylation of PAK4 at Ser99 also mediates binding to 14-3-3 protein, and is required for the formation of a PAK4-LIMK-PKD1 complex that regulates cofilin activity and directed cell migration.


Subject(s)
Protein Kinase C/metabolism , Serine/genetics , p21-Activated Kinases/analysis , p21-Activated Kinases/metabolism , 14-3-3 Proteins/metabolism , Cell Movement , HEK293 Cells , HeLa Cells , Humans , Phosphorylation , Serine/metabolism , Signal Transduction , Transfection
11.
J Biol Chem ; 288(1): 455-65, 2013 Jan 04.
Article in English | MEDLINE | ID: mdl-23148218

ABSTRACT

Neuregulin (NRG; heregulin) is overexpressed in ∼30% 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.


Subject(s)
Actins/metabolism , Neuregulin-1/metabolism , Protein Kinase C/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Breast Neoplasms/metabolism , Cell Movement , Chemotaxis , Disease Progression , Female , Humans , Mice , Microscopy, Fluorescence/methods , Neoplasm Invasiveness , Neoplasm Metastasis , RNA, Small Interfering/metabolism , Signal Transduction , Wound Healing
12.
PLoS One ; 7(1): e30459, 2012.
Article in English | MEDLINE | ID: mdl-22276203

ABSTRACT

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.


Subject(s)
DNA/metabolism , Epithelial Cells/metabolism , Protein Kinase C/metabolism , Transcription Factors/metabolism , Animals , Cadherins/metabolism , Cell Line , Cell Line, Tumor , Chromatin Immunoprecipitation , Dogs , Female , Humans , Immunoblotting , Immunoprecipitation , In Vitro Techniques , LIM Domain Proteins/metabolism , Mammary Glands, Animal/cytology , Mice , Microscopy, Fluorescence , Phosphorylation , Snail Family Transcription Factors , Tissue Array Analysis
13.
J Biol Chem ; 286(39): 34254-61, 2011 Sep 30.
Article in English | MEDLINE | ID: mdl-21832093

ABSTRACT

Dynamic reorganization of the actin cytoskeleton at the leading edge is required for directed cell migration. Cofilin, a small actin-binding protein with F-actin severing activities, is a key enzyme initiating such actin remodeling processes. Cofilin activity is tightly regulated by phosphorylation and dephosphorylation events that are mediated by LIM kinase (LIMK) and the phosphatase slingshot (SSH), respectively. Protein kinase D (PKD) is a serine/threonine kinase that inhibits actin-driven directed cell migration by phosphorylation and inactivation of SSH. Here, we show that PKD can also regulate LIMK through direct phosphorylation and activation of its upstream kinase p21-activated kinase 4 (PAK4). Therefore, active PKD increases the net amount of phosphorylated inactive cofilin in cells through both pathways. The regulation of cofilin activity at multiple levels may explain the inhibitory effects of PKD on barbed end formation as well as on directed cell migration.


Subject(s)
Actin Depolymerizing Factors/metabolism , Actins/metabolism , Cell Movement/physiology , Protein Kinase C/metabolism , p21-Activated Kinases/metabolism , Actin Depolymerizing Factors/genetics , Actins/genetics , HeLa Cells , Humans , Lim Kinases/genetics , Lim Kinases/metabolism , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/metabolism , Protein Kinase C/genetics , p21-Activated Kinases/genetics
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