Inflammatory and alternatively activated macrophages independently induce metaplasia but cooperatively drive pancreatic precancerous lesion growth.

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.

Elevating microglia TREM2 reduces amyloid seeding and suppresses disease-associated microglia.

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.

Peripheral apoE4 enhances Alzheimer's pathology and impairs cognition by compromising cerebrovascular function.

The ε4 allele of the apolipoprotein E (APOE) gene, a genetic risk factor for Alzheimer's disease, is abundantly expressed in both the brain and periphery. Here, we present evidence that peripheral apoE isoforms, separated from those in the brain by the blood-brain barrier, differentially impact Alzheimer's disease pathogenesis and cognition. To evaluate the function of peripheral apoE, we developed conditional mouse models expressing human APOE3 or APOE4 in the liver with no detectable apoE in the brain. Liver-expressed apoE4 compromised synaptic plasticity and cognition by impairing cerebrovascular functions. Plasma proteome profiling revealed apoE isoform-dependent functional pathways highlighting cell adhesion, lipoprotein metabolism and complement activation. ApoE3 plasma from young mice improved cognition and reduced vessel-associated gliosis when transfused into aged mice, whereas apoE4 compromised the beneficial effects of young plasma. A human induced pluripotent stem cell-derived endothelial cell model recapitulated the plasma apoE isoform-specific effect on endothelial integrity, further supporting a vascular-related mechanism. Upon breeding with amyloid model mice, liver-expressed apoE4 exacerbated brain amyloid pathology, whereas apoE3 reduced it. Our findings demonstrate pathogenic effects of peripheral apoE4, providing a strong rationale for targeting peripheral apoE to treat Alzheimer's disease.

Ym1+ macrophages orchestrate fibrosis, lesion growth, and progression during development of murine pancreatic cancer.

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.

CXCL10/CXCR3 signaling contributes to an inflammatory microenvironment and its blockade enhances progression of murine pancreatic precancerous lesions.

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.

Dysfunctional EGFR and oxidative stress-induced PKD1 signaling drive formation of DCLK1+ pancreatic stem cells.

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.

Alzheimer's Risk Factors Age, APOE Genotype, and Sex Drive Distinct Molecular Pathways.

Evidence suggests interplay among the three major risk factors for Alzheimer's disease (AD): age, APOE genotype, and sex. Here, we present comprehensive datasets and analyses of brain transcriptomes and blood metabolomes from human apoE2-, apoE3-, and apoE4-targeted replacement mice across young, middle, and old ages with both sexes. We found that age had the greatest impact on brain transcriptomes highlighted by an immune module led by Trem2 and Tyrobp, whereas APOE4 was associated with upregulation of multiple Serpina3 genes. Importantly, these networks and gene expression changes were mostly conserved in human brains. Finally, we observed a significant interaction between age, APOE genotype, and sex on unfolded protein response pathway. In the periphery, APOE2 drove distinct blood metabolome profile highlighted by the upregulation of lipid metabolites. Our work identifies unique and interactive molecular pathways underlying AD risk factors providing valuable resources for discovery and validation research in model systems and humans.

Sangivamycin and its derivatives inhibit Haspin-Histone H3-survivin signaling and induce pancreatic cancer cell death.

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.

Pomalidomide Alters Pancreatic Macrophage Populations to Generate an Immune-Responsive Environment at Precancerous and Cancerous Lesions.

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.

The phosphorylation status of PIP5K1C at serine 448 can be predictive for invasive ductal carcinoma of the breast.

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.

Src-mediated tyrosine phosphorylation of Protein Kinase D2 at focal adhesions regulates cell adhesion.

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.

The Presence of Interleukin-13 at Pancreatic ADM/PanIN Lesions Alters Macrophage Populations and Mediates Pancreatic Tumorigenesis.

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.

Protein Kinase D1 regulates focal adhesion dynamics and cell adhesion through Phosphatidylinositol-4-phosphate 5-kinase type-l γ.

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.

The PRKD1 promoter is a target of the KRas-NF-κB pathway in pancreatic cancer.

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.

The phosphorylation status of VASP at serine 322 can be predictive for aggressiveness of invasive ductal carcinoma.

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.

Protein kinase d isoforms differentially modulate cofilin-driven directed cell migration.

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.

Protein kinase D1-mediated phosphorylations regulate vasodilator-stimulated phosphoprotein (VASP) localization and cell migration.

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.

Protein kinase D-mediated phosphorylation at Ser99 regulates localization of p21-activated kinase 4.

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.

Neuregulin mediates F-actin-driven cell migration through inhibition of protein kinase D1 via Rac1 protein.

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.

Protein kinase D1 maintains the epithelial phenotype by inducing a DNA-bound, inactive SNAI1 transcriptional repressor complex.

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.