Karin Leandersson

The presence of tumor associated macrophages in tumor stroma as a prognostic marker for breast cancer patients

BackgroundTumor associated macrophages (TAMs) are alternatively activated macrophages that enhance tumor progression by promoting tumor cell invasion, migration and angiogenesis. TAMs have an anti-inflammatory function resembling M2 macrophages. CD163 is regarded as a highly specific monocyte/macrophage marker for M2 macrophages. In this study we evaluated the specificity of using the M2 macrophage marker CD163 as a TAM marker and compared its prognostic value with the more frequently used pan-macrophage marker CD68. We also analyzed the prognostic value of the localization of CD163+ and CD68+ myeloid cells in human breast cancer.MethodsThe extent of infiltrating CD163+ or CD68+ myeloid cells in tumor nest versus tumor stroma was evaluated by immunohistochemistry in tissue microarrays with tumors from 144 breast cancer cases. Spearman’s Rho and χ2 tests were used to examine the correlations between CD163+ or CD68+ myeloid cells and clinicopathological parameters. Kaplan Meier analysis and Cox proportional hazards modeling were used to assess the impact of CD163+ and CD68+ myeloid cells in tumor stroma and tumor nest, respectively, on recurrence free survival, breast cancer specific and overall survival.ResultsWe found that infiltration of CD163+ and CD68+ macrophages into tumor stroma, but not into tumor nest, were of clinical relevance. CD163+ macrophages in tumor stroma positively correlated with higher grade, larger tumor size, Ki67 positivity, estrogen receptor negativity, progesterone receptor negativity, triple-negative/basal-like breast cancer and inversely correlated with luminal A breast cancer. Some CD163+ areas lacked CD68 expression, suggesting that CD163 could be used as a general anti-inflammatory myeloid marker with prognostic impact. CD68+ macrophages in tumor stroma positively correlated to tumor size and inversely correlated to luminal A breast cancer. More importantly, CD68 in tumor stroma was an independent prognostic factor for reduced breast cancer specific survival.ConclusionThese findings highlight the importance of analyzing the localization rather than merely the presence of TAMs as a prognostic marker for breast cancer patients.

p38-MAPK Signals Survival by Phosphorylation of Caspase-8 and Caspase-3 in Human Neutrophils.

Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38–mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response.

Wnt-5a/Ca2+-induced NFAT activity is counteracted by Wnt-5a/Yes-Cdc42-casein kinase 1alpha signaling in human mammary epithelial cells.

ABSTRACT Wnt-5a has been shown to influence the metastatic behavior of human breast cancer cells, and the loss of Wnt-5a expression is associated with metastatic disease. We show here that NFAT1, a transcription factor connected with breast cancer metastasis, is activated by Wnt-5a through a Ca2+ signaling pathway in human breast epithelial cells. This activation was simultaneously counteracted by a Wnt-5a-induced Yes/Cdc42 signaling pathway. The observation that inhibition of the Wnt-5a/Yes/Cdc42 signal prolonged the duration of ionomycin-induced NFAT1 activation revealed the general importance of this pathway. The Wnt-5a-induced inhibition of NFAT1 did not require glycogen synthase kinase 3β, JNK, or Pak1 activity or modulation of the cytoskeleton. Instead, we observed that Wnt-5a induced a complex formation of NFAT1/casein kinase 1α, even upon treatment with ionomycin, which was blocked upon inhibition of the Wnt-5a/Yes/Cdc42 signaling pathway. Our results explain why Wnt-5a/Ca2+-induced NFAT activity is hard to detect and suggest a novel mechanism by which Wnt-5a can suppress tumor-specific, agonist-induced NFAT activity and thus the metastatic behavior of breast cancer cells.

A formylated hexapeptide ligand mimics the ability of Wnt-5a to impair migration of human breast epithelial cells.

Loss of Wnt-5a protein expression is associated with shorter recurrence-free survival in breast carcinoma patients and increased motility in mammary cell lines. Based on sequence analysis of Wnt-5a, we identified 14 peptide fragments and investigated their ability to mimic the effects of Wnt-5a on mammary cell adhesion and migration. Two of these peptides significantly increased adhesion and impaired migration in the non-tumorigenic HB2 breast epithelial cell line and in the MDA-MB-468 breast cancer cell line, both of which show little endogenous expression of the Wnt-5a protein. We removed two amino acids at a time from the N terminus of the shorter of these two peptides to identify the shortest peptide that still inhibited migration. The influence on tumor cell adhesion was gradually lost and was no longer detectable when only six amino acids remained. However, formylation of the N-terminal methionine of this hexapeptide restored its effect on adhesion and reduced tumor cell motility via a Frizzled-5 receptor-dependent mechanism, even at a low pH such as encountered in breast tumor tissue. This formylated hexapeptide ligand induced a rapid cytosolic calcium signal, whereas it did not affect the cellular levels of unphosphorylated β-catenin or active JNK. The novel formyl-Met-Asp-Gly-Cys-Glu-Leu peptide ligand is not only a valuable experimental tool but has also a potential role in antimetastatic treatment of the 50% of human breast cancer patients that have reduced endogenous Wnt-5a protein expression.

WNT5A induces release of exosomes containing pro-angiogenic and immunosuppressive factors from malignant melanoma cells

BackgroundWnt proteins are important for developmental processes and certain diseases. WNT5A is a non-canonical Wnt protein that previously has been shown to play a role in the progression of malignant melanoma. High expression of WNT5A in melanoma tumors correlates to formation of distant metastasis and poor prognosis. This has partly been described by the findings that WNT5A expression in melanoma cell lines increases migration and invasion.MethodsMalignant melanoma cell lines were treated with rWNT5A or WNT5A siRNA, and mRNA versus protein levels of soluble mediators were measured using RT-PCR, cytokine bead array and ELISA. The induced signaling pathways were analyzed using inhibitors, Rho-GTPase pull down assays and western blot. Ultracentrifugation and electron microscopy was used to analyze microvesicles. Gene expression microarray data obtained from primary malignant melanomas was used to verify our data.ResultsWe show that WNT5A signaling induces a Ca2+-dependent release of exosomes containing the immunomodulatory and pro-angiogenic proteins IL-6, VEGF and MMP2 in melanoma cells. The process was independent of the transcriptional machinery and depletion of WNT5A reduced the levels of the exosome-derived proteins. The WNT5A induced exosomal secretion was neither affected by Tetanus toxin nor Brefeldin A, but was blocked by the calcium chelator Bapta, inhibited by a dominant negative version of the small Rho-GTPase Cdc42 and was accompanied by cytoskeletal reorganization. Co-cultures of melanoma/endothelial cells showed that depletion of WNT5A in melanoma cells decreased endothelial cell branching, while stimulation of endothelial cells with isolated rWNT5A-induced melanoma exosomes increased endothelial cell branching in vitro. Finally, gene expression data analysis of primary malignant melanomas revealed a correlation between WNT5A expression and the angiogenesis marker ESAM.ConclusionsThese data indicate that WNT5A has a broader function on tumor progression and metastatic spread than previously known; by inducing exosome-release of immunomodulatory and pro-angiogenic factors that enhance the immunosuppressive and angiogenic capacity of the tumors thus rendering them more aggressive and more prone to metastasize.

Wnt-5a mRNA translation is suppressed by the Elav-like protein HuR in human breast epithelial cells

Wnt-5a is a non-transforming Wnt protein. Since Wnt-5a mRNA and protein levels differ within and between tumours, the potential of Wnt-5a as a prognostic factor has been debated. We have previously shown that the lack of Wnt-5a protein is a predictor of shorter disease-free survival in human breast cancer. Recently, however, we also showed that the breast tumours lacking Wnt-5a protein had a high or normal level of Wnt-5a mRNA that might explain the discrepancies in previous studies. We here report that Wnt-5a is regulated at the post-transcriptional level. The regulation was mediated by the Embryonic Lethal Abnormal Vision (ELAV)-like protein HuR, which inhibited translation of Wnt-5a when bound to highly conserved AU-rich sequences in the 3′-untranslated region (3′-UTR) of the Wnt-5a mRNA molecule, as shown by both HA-tagged Wnt-5a- and Luciferase-Wnt-5a-3′-UTR reporter assays. The HuR-dependent inhibition of Wnt-5a was supported by the fact that active HuR is located in the cytoplasm in invasive human breast tumours and that hypoxia-induced activation of HuR inhibits translation of both Luciferase-Wnt-5a-3′-UTR and endogenous Wnt-5a protein. We propose that the lack of Wnt-5a protein expression in invasive human breast tumours is caused by a HuR-mediated suppression of Wnt-5a mRNA translation.

Wnt5a Induces a Tolerogenic Phenotype of Macrophages in Sepsis and Breast Cancer Patients

A well-orchestrated inflammatory reaction involves the induction of effector functions and, at a later stage, an active downregulation of this potentially harmful process. In this study we show that under proinflammatory conditions the noncanonical Wnt protein, Wnt5a, induces immunosuppressive macrophages. The suppressive phenotype induced by Wnt5a is associated with induction of IL-10 and inhibition of the classical TLR4-NF-κB signaling. Interestingly, this phenotype closely resembles that observed in reprogrammed monocytes in sepsis patients. The Wnt5a-induced feedback inhibition is active both during in vitro LPS stimulation of macrophages and in patients with sepsis caused by LPS-containing, Gram-negative bacteria. Furthermore, using breast cancer patient tissue microarrays, we find a strong correlation between the expression of Wnt5a in malignant epithelial cells and the frequency of CD163+ anti-inflammatory tumor-associated macrophages. In conclusion, our data point out Wnt5a as a potential target for an efficient therapeutic modality in severe human diseases as diverse as sepsis and malignancy.

WNT-5a-CKIalpha signaling promotes beta -catenin/E-cadherin complex formation and intercellular adhesion in human breast epithelial cells.

Wnt-5a is a non-transforming Wnt protein that is implicated in cell polarity, adhesion, and motility. We have previously shown that low expression of Wnt-5a is a predictor of shorter disease-free survival in human breast cancer. Here, we investigated whether β-catenin/E-cadherin-mediated cell-cell adhesion was affected by loss of Wnt-5a in breast carcinomas, thereby promoting a metastatic behavior of the tumor. We show that Wnt-5a stimulation of human breast epithelial cells leads to an increased Ca2+-dependent cell-cell adhesion. Furthermore, Wnt-5a/casein kinase Iα (CKIα)-specific Ser-45 phosphorylation of β-catenin is associated with an increased complex formation of β-catenin/E-cadherin. Mutation of Ser-45 decreases the β-catenin/E-cadherin association. Also, the inhibitory effect of Wnt-5a on breast epithelial cell invasion is reduced upon mutation of β-catenin-Ser-45. The Wnt-5a-CKIα-induced Ser-45 phosphorylation does not lead to degradation of β-catenin. Finally we show that human breast cancers lacking Wnt-5a protein have a significantly lower level of membrane-associated β-catenin. Down-regulation of Wnt-5a expression and subsequent reduction of membrane-associated β-catenin in invasive breast cancer, can therefore contribute to a decreased cell-cell adhesion and increased motility resulting in a higher probability for metastatic disease.

A high frequency of MDSCs in sepsis patients, with the granulocytic subtype dominating in gram-positive cases.

The causative microorganisms dictate the type of MDSC generated in sepsis patients, and a large proportion of PMN‐MDSCs in gram‐positive sepsis includes immunosuppressive myeloid blasts. MDSCs constitute a heterogeneous population of immature myeloid cells that potently suppress immune responses. They were identified originally in cancer patients and have since been reported to occur also in chronic inflammation, autoimmunity, and even bacterial infections. Human MDSCs are commonly divided into Mo‐MDSCs and granulocytic (PMN‐MDSCs) subtypes. To what extent the bona fide cancer MDSCs are representative of the proposed MDSCs found in other diseases is not well known. PMN‐MDSCs have been found previously to be enriched among LDGs in density gradient‐centrifuged blood. In this study, we analyzed potential MDSCs in sepsis patients with different causative microorganisms, using total peripheral blood compared with density gradient‐centrifuged blood. We found a high frequency of typical CD14+HLA‐DRlow Mo‐MDSCs in all sepsis patients, whereas the typical PMN‐MDSCs, as well as a prominent CD14low PMN‐MDSC‐like population, appeared preferentially in gram‐positive cases. The CD14low PMN‐MDSC variant was demonstrated to suppress T cell proliferation in vitro via a ROS‐dependent mechanism, to display an increased IL‐10:TNF‐α ratio, and to present with signs of immaturity: blast morphology and low cytokine levels. We conclude that a spectrum of cells with MDSC features is enriched in sepsis and that the microbial origin of sepsis contributes to the substantial interindividual patient variation in the MDSC pattern.

Systemic Monocytic-MDSCs Are Generated from Monocytes and Correlate with Disease Progression in Breast Cancer Patients.

Myeloid-derived suppressor cells (MDSCs) are highly immunosuppressive myeloid cells, which increase in cancer patients. The molecular mechanism behind their generation and function is unclear. Whereas granulocytic-MDSCs correlate with poor overall survival in breast cancer, the presence and relevance of monocytic-MDSCs (Mo-MDSCs) is unknown. Here we report for the first time an enrichment of functional blood Mo-MDSCs in breast cancer patients before they acquire a typical Mo-MDSC surface phenotype. A clear population of Mo-MDSCs with the typical cell surface phenotype (CD14+HLA-DRlow/-CD86low/-CD80low/-CD163low/-) increased significantly first during disease progression and correlated to metastasis to lymph nodes and visceral organs. Furthermore, monocytes, comprising the Mo-MDSC population, from patients with metastatic breast cancer resemble the reprogrammed immunosuppressive monocytes in patients with severe infections, both by their surface and functional phenotype but also at their molecular gene expression profile. Our data suggest that monitoring the Mo-MDSC levels in breast cancer patients may represent a novel and simple biomarker for assessing disease progression.