The injury-induced transcription factor SOX9 alters the expression of LBR, HMGA2, and HIPK3 in the human kidney.

Induction of SRY box transcription factor 9 (SOX9) has been shown to occur in response to kidney injury in rodents, where SOX9-positive cells proliferate and regenerate the proximal tubules of injured kidneys. Additionally, SOX9-positive cells demonstrate a capacity to differentiate toward other nephron segments. Here, we characterized the role of SOX9 in normal and injured human kidneys. SOX9 expression was found to colocalize with a proportion of so-called scattered tubular cells in the uninjured kidney, a cell population previously shown to be involved in kidney injury and regeneration. Following injury and in areas adjacent to inflammatory cell infiltrates, SOX9-positive cells were increased in number. With the use of primary tubular epithelial cells (PTECs) obtained from human kidney tissue, SOX9 expression was spontaneously induced in culture and further increased by transforming growth factor-ß1, whereas it was suppressed by interferon-γ. siRNA-mediated knockdown of SOX9 in PTECs followed by analysis of differential gene expression, immunohistochemical expression, and luciferase promoter assays suggested lamin B receptor (LBR), high mobility group AT-hook 2 (HMGA2), and homeodomain interacting protein kinase 3 (HIPK3) as possible target genes of SOX9. Moreover, a kidney explant model was used to demonstrate that only SOX9-positive cells survive the massive injury associated with kidney ischemia and that the surviving SOX9-positive cells spread and repopulate the tubules. Using a wound healing assay, we also showed that SOX9 positively regulated the migratory capacity of PTECs. These findings shed light on the functional and regulatory aspects of SOX9 activation in the human kidney during injury and regeneration.NEW & NOTEWORTHY Recent studies using murine models have shown that SRY box transcription factor 9 (SOX9) is activated during repair of renal tubular cells. In this study, we showed that SOX9-positive cells represent a proportion of scattered tubular cells found in the uninjured human kidney. Furthermore, we suggest that expression of LBR, HMGA2, and HIPK3 is altered by SOX9 in the kidney tubular epithelium, suggesting the involvement of these gene products in kidney injury and regeneration.

Infiltration of NK and plasma cells is associated with a distinct immune subset in non-small cell lung cancer.

Immune cells of the tumor microenvironment are central but erratic targets for immunotherapy. The aim of this study was to characterize novel patterns of immune cell infiltration in non-small cell lung cancer (NSCLC) in relation to its molecular and clinicopathologic characteristics. Lymphocytes (CD3+, CD4+, CD8+, CD20+, FOXP3+, CD45RO+), macrophages (CD163+), plasma cells (CD138+), NK cells (NKp46+), PD1+, and PD-L1+ were annotated on a tissue microarray including 357 NSCLC cases. Somatic mutations were analyzed by targeted sequencing for 82 genes and a tumor mutational load score was estimated. Transcriptomic immune patterns were established in 197 patients based on RNA sequencing data. The immune cell infiltration was variable and showed only poor association with specific mutations. The previously defined immune phenotypic patterns, desert, inflamed, and immune excluded, comprised 30, 13, and 57% of cases, respectively. Notably, mRNA immune activation and high estimated tumor mutational load were unique only for the inflamed pattern. However, in the unsupervised cluster analysis, including all immune cell markers, these conceptual patterns were only weakly reproduced. Instead, four immune classes were identified: (1) high immune cell infiltration, (2) high immune cell infiltration with abundance of CD20+ B cells, (3) low immune cell infiltration, and (4) a phenotype with an imprint of plasma cells and NK cells. This latter class was linked to better survival despite exhibiting low expression of immune response-related genes (e.g. CXCL9, GZMB, INFG, CTLA4). This compartment-specific immune cell analysis in the context of the molecular and clinical background of NSCLC reveals two previously unrecognized immune classes. A refined immune classification, including traits of the humoral and innate immune response, is important to define the immunogenic potency of NSCLC in the era of immunotherapy. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Deletion of Nemo-like Kinase in T Cells Reduces Single-Positive CD8+ Thymocyte Population.

The ß-catenin/Wnt signaling pathway plays an important role in all stages of T cell development. Nemo-like kinase (NLK) is an evolutionary conserved serine/threonine kinase and a negative regulator of the Wnt signaling pathway. NLK can directly phosphorylate histone deacetylase 1 (HDAC1), as well as T cell factor/lymphoid enhancer-binding factor (TCF/LEF), causing subsequent repression of target gene transcription. By engineering mice lacking NLK in early stages of T cell development, we set out to characterize the role NLK plays in T cell development and found that deletion of NLK does not affect mouse health or lymphoid tissue development. Instead, these mice harbored a reduced number of single-positive (SP) CD8+ thymocytes without any defects in the SP CD4+ thymocyte population. The decrease in SP CD8+ thymocytes was not caused by a block in differentiation from double-positive CD4+CD8+ cells. Neither TCR signaling nor activation was altered in the absence of NLK. Instead, we observed a significant increase in cell death and reduced phosphorylation of LEF1 as well as HDAC1 among NLK-deleted SP CD8+ cells. Thus, NLK seems to play an important role in the survival of CD8+ thymocytes. Our data provide evidence for a new function for NLK with regard to its involvement in T cell development and supporting survival of SP CD8+ thymocytes.

Inflammatory macrophage derived TNFα downregulates estrogen receptor α via FOXO3a inactivation in human breast cancer cells.

Patients with estrogen receptor α positive (ERα+) breast cancer can respond to endocrine therapy, but treatment resistance is common and associated with downregulation of ERα expression in the dormant residual cells. Here we show, using long-term NSG xenograft models of human breast cancer and primary human monocytes, in vitro primary cell cultures and tumors from breast cancer patients, that macrophage derived tumor necrosis factor alpha (TNFα) downregulates ERα in breast cancer cells via inactivation of the transcription factor Forkhead box O transcription factor 3a (FOXO3a). Moreover, presence of tumor associated macrophages in the primary tumor of breast cancer patients, was associated with ERα negativity, and with worse prognosis in patients with ERα+ tumors. We propose that pro-inflammatory macrophages, despite being tumoricidal, may have direct effects on tumor progression and endocrine resistance in breast cancer patients. Our findings suggest that TNFα antagonists should be evaluated for treatment of ERα+ breast cancer.

Quantitative, qualitative and spatial analysis of lymphocyte infiltration in periampullary and pancreatic adenocarcinoma.

Immunotherapeutic modalities are currently revolutionizing cancer treatment. In pancreatic cancer, however, early clinical trials have been disappointing. The optimization of immunotherapeutic strategies requires better understanding of the inflammatory tumor microenvironment. Therefore, the aim of our study was to perform a detailed in situ description of lymphocyte infiltration patterns in resected pancreatic and other periampullary cancers. Multiplexed immunofluorescence imaging was applied to tissue microarrays with tumors from a cohort of 175 patients with resected periampullary adenocarcinoma. A panel of immune cell markers including CD4, CD8α, FoxP3, CD20, CD45RO and pan-cytokeratin was applied to allow for simultaneous spatial analysis of multiple lymphocyte populations. The majority of lymphocyte populations were significantly more abundant in intestinal (I-type) compared to pancreatobiliary (PB-type) tumors. Hierarchical cluster analysis revealed several immune cell signatures of potential clinical relevance. Notably, in the stromal compartment of PB-type tumors, high infiltration of B cells, CD8α+ CD45RO+ and single-positive CD4+ T cells, but low levels of FoxP3+ CD45ROhigh and single-positive CD8α+ T cells were associated with improved overall survival (OS). The study also defined prognostic relevant topographical patterns of lymphocytic infiltration, in particular proximity of CD8α+ cells to cancer cells. Moreover, the presence of lymphocytes with potential T-helper capacities (CD4+ ) in the nearest vicinity to CD8α+ cells was associated with a prolonged OS. Our data demonstrate that the composition and clinical impact of immune infiltrates in periampullary adenocarcinoma differ by morphological type as well as localization. Furthermore, spatial in situ analysis identified potential immunological mechanisms of prognostic significance.

Clinical relevance of systemic monocytic-MDSCs in patients with metastatic breast cancer.

The overall aim of this prospective study was to delineate the role of monocytic myeloid-derived suppressor cells (Mo-MDSCs) in patients with metastatic breast cancer (MBC). MDSCs are a heterogeneous group of immunosuppressive cells often enriched in different malignancies which hold prognostic and predictive value for clinical outcomes. Here, we assessed the clinical significance of Mo-MDSCs in 54 patients with de novo or distant recurrent MBC. We show that high levels of Mo-MDSCs significantly correlated with de novo MBC (metastatic disease at initial diagnosis), estrogen receptor (ER) negativity, and liver- and bone metastasis. A trend towards an association between high levels of Mo-MDSCs and survival (P = 0.053) was also found in patients with distant recurrent ER-positive MBC. We therefore propose that an increased population of Mo-MDSCs may be related to the metastatic or immunoregulatory switch associated with transition to a more systemic disease. Our data imply that high levels of systemic Mo-MDSCs represent patients with more aggressive disease and worse outcome.

Localization and Regulation of Polymeric Ig Receptor in Healthy and Diseased Human Kidney.

The polymeric Ig receptor (PIgR) constitutes an important part of the immune system by mediating transcytosis of dimeric IgA into mucosal fluids. Although well studied in organs such as the intestine, the regulation and localization of PIgR in human kidney are incompletely characterized. Herein, using immunohistochemistry, we show that in healthy human kidneys, PIgR is expressed by the progenitor-like tubular scattered cells of the proximal tubules and by parietal epithelial cells of glomeruli. We further show that proximal tubular expression of PIgR becomes widespread during kidney disease, correlating to elevated levels of urinary secretory IgA. Urinary secretory IgA levels also correlated to the degree of tubular fibrosis, plasma creatinine, and urea levels. In addition, primary tubular cells were cultured to study the function and regulation of PIgR in vitro. Cellular PIgR expression was induced by conditioned medium from activated human leukocytes, as well as by inflammatory cytokines, whereas transforming growth factor-ß1 caused decreased expression. Furthermore, interferon-γ increased the transcytosis of dimeric IgA in cultured tubular cells. Finally, a correlation study of mRNA data from the Genotype-Tissue Expression portal indicated that PIGR mRNA expression in kidney correlates to the expression of TNFSF13, a cytokine involved in plasma cell class switching to IgA. These results indicate that PIgR induction is an integral part of the injury phenotype of renal tubular cells.

Chemotherapy, host response and molecular dynamics in periampullary cancer: the CHAMP study.

BACKGROUND: Pancreatic cancer is a devastating disease with a dismal prognosis. Despite profound medical advances in systemic therapies for other types of aggressive tumours during recent years, a diagnosis of pancreatic cancer is still often synonymous with a fatal outcome. The term periampullary cancer includes pancreatic cancer and applies to the group of tumours found in proximity to the ampulla of Vater. Molecular events and immune response in the host during chemotherapy remain largely unexplored in this group of tumours. Therefore, the "Chemotherapy, Host Response and Molecular Dynamics in Periampullary Cancer (CHAMP)" study aims to monitor these processes to gain new insight into this perplexing disease. METHODS: The CHAMP study is a prospective, single-arm observational study. All patients diagnosed with pancreatic or other periampullary adenocarcinoma undergoing adjuvant or palliative chemotherapy treatment in the Department of Oncology, Skåne University Hospital, are invited to participate. Clinical and pathological data will be compiled at study entry. A single tissue microarray (TMA) block is constructed for each patient with a resected tumour and blood samples are drawn before, during and after chemotherapy in order to sample peripheral blood mononuclear cells (PBMC), cytokines and circulating tumour DNA (ctDNA). Next generation sequencing will be performed on tumour tissue and ctDNA to detect changes in the clonal landscape over space and time. DISCUSSION: Despite the recent emergence of some promising biomarkers for periampullary cancer, there has been a lack of success in clinical implementation. Cancer cells continuously adapt and become resistant to treatment during chemotherapy. To be able to keep pace with and hopefully overtake this rapid evolution we must, with the help of new diagnostic tools, be ready to adapt and alter treatment accordingly. It seems to us that the only way forward is to gain a better understanding of the dynamics of the disease during treatment. With insights gained from the CHAMP study we hope to find answers to key questions in this largely unexplored territory. TRIAL REGISTRATION: This study has been registered 30th October 2018 at clinicaltrials.gov as NCT03724994.

The STAT3 inhibitor galiellalactone inhibits the generation of MDSC-like monocytes by prostate cancer cells and decreases immunosuppressive and tumorigenic factors.

BACKGROUND: The transcription factor signal transducer and activator of transcription 3 (STAT3) is implicated in cancer drug resistance, metastasis, and immunosuppression and has been identified as a promising therapeutic target for new anticancer drugs. Myeloid-derived suppressor cells (MDSCs) play a major role in the suppression of antitumor immunity and STAT3 is involved in the accumulation, generation, and function of MDSCs. Thus, targeting STAT3 holds the potential of reversing immunosuppression in cancer. This study aims to investigate the effect of the small molecule STAT3 inhibitor galiellalactone on prostate cancer cell- induced generation of MDSCs from monocytes and the effect on immunosuppressive factors and inflammatory cytokines. METHODS: Primary human monocytes were cocultured with prostate cancer cells (DU145, PC3, and LNCaP-IL6) or with conditioned medium (CM) from prostate cancer cells in the presence or absence of the STAT3 inhibitor galiellalactone. Monocytes were analyzed by flow cytometry for an MDSC-like phenotype (CD14+ HLA-DR-/lo ). The secretion and gene expression of immunosuppressive factors and inflammatory cytokines from prostate cancer cells and monocytes were investigated. RESULTS: Galiellalactone blocked the prostate cancer cell-induced generation of MDSC-like monocytes with an immunosuppressive phenotype ex vivo. Monocytes cultured with CM from prostate cancer cells showed increased expression of phosphorylated STAT3. Prostate cancer cells increased the expression of interleukin1ß (IL1ß), IL10, and IL6 in monocytes which was inhibited by galiellalactone. In addition, galiellalactone decreased indoleamine 2,3-dioxygenase gene expression in monocytes. Galiellalactone reduced the levels of IL8 and granulocyte macrophage-colony stimulating factor in prostate cancer cells per se. CONCLUSION: The STAT3 inhibitor galiellalactone may prevent the prostate cancer cell-induced generation of MDSCs and reverse the immunosuppressive mechanisms caused by the interplay between prostate cancer cells and MDSCs. This is a potential new immunotherapeutic approach for the treatment of prostate cancer.

Docetaxel promotes the generation of anti-tumorigenic human macrophages.

The taxanes Docetaxel and Paclitaxel are two of the standard chemotherapies for patients with metastatic breast cancer. The functional effect of Docetaxel and Paclitaxel on human innate immune cells of the myeloid lineage is not well established, nor is the effects these agents have on differentiation of monocytes into macrophages and dendritic cells. Therefore, the aim with this project was to determine the effects of Docetaxel and Paclitaxel on primary human monocyte differentiation, activation and function. For this purpose, primary human monocytes were isolated from healthy donors and cultured with or without Docetaxel and Paclitaxel. We found that Docetaxel promoted the differentiation of primary human monocytes into pro-inflammatory macrophages with an M1 phenotype and an ability to present antigens to T cells. Monocytes treated with Docetaxel also displayed an elevated secretion of IL-8 and IL-1ß, but did not promote generation of monocytic myeloid-derived suppressor cells. In conclusion, Docetaxel appears to have an immune stimulatory effect that would be beneficial for an anti-tumorigenic type of immune response, whereas Paclitaxel seems to have less effect on myeloid cells.

Papillary renal cell carcinoma-derived chemerin, IL-8, and CXCL16 promote monocyte recruitment and differentiation into foam-cell macrophages.

Papillary renal cell carcinoma (pRCC) is the second most common type of renal cell carcinoma. The only curative treatment available for pRCC is radical surgery. If the disease becomes widespread, neither chemo- nor radiotherapy will have therapeutic effect, hence further research on pRCC is of utmost importance. Histologically, pRCC is characterized by a papillary growth pattern with focal aggregation of macrophages of the foam cell phenotype. In other forms of cancer, a clear role for tumor-associated macrophages during cancer growth and progression has been shown. Although the presence of foamy macrophages is a histological hallmark of pRCC tumors, little is known regarding their role in pRCC biology. In order to study the interaction between pRCC tumor and myeloid cells, we established primary cultures from pRCC tissue. We show that human pRCC cells secrete the chemokines IL-8, CXCL16, and chemerin, and that these factors attract primary human monocytes in vitro. RNAseq data from The Cancer Genome Atlas confirmed a high expression of these factors in pRCC tissue. Conditioned medium from pRCC cultures induced a shift in human monocytes toward the M2 macrophage phenotype. In extended cultures, these macrophages became enlarged and loaded with lipids, adopting the foam cell morphology found in pRCC tissue. These results show for the first time that pRCC primary tumor cells secrete factors that attract and differentiate monocytes into anti-inflammatory tumor-associated macrophages with foam cell histology.

The clinical impact of tumour-infiltrating lymphocytes in colorectal cancer differs by anatomical subsite: A cohort study.

Accumulating evidence demonstrates an association between dense infiltration of lymphocytes and prognosis in colorectal cancer (CRC), but whether this prognostic impact differs by tumour location remains unknown. This study investigated the prognostic impact of cytotoxic and regulatory T cells in CRC, with particular reference to the anatomical subsite of the primary tumour. The density of CD3+ , CD8+ and FoxP3+ tumour-infiltrating T cells was calculated in tissue microarrays with tumours from 557 incident CRC cases from a prospective population-based cohort. Kaplan-Meier and Cox regression analyses were applied to determine the impact of high and low lymphocyte density on 5-year overall survival, in subgroup analysis of right colon, left colon and rectum. High CD8+ cell density was a favourable prognostic factor for patients with right-sided colon tumours (hazard ratio [HR]=0.53, 95% confidence interval [CI] 0.29-0.95), independent of age, sex, TNM stage, differentiation grade and vascular invasion, with a significant prognostic interaction between CD8+ cells and right-sidedness (p = 0.031). High FoxP3+ cell density was an independent favourable prognostic factor only in patients with rectal tumours (HR = 0.54, 95% CI 0.30-0.99), and CD3+ cell density was an independent favourable prognostic factor for tumours in the right colon and rectum, but there was no significant prognostic interaction between CD3+ or FoxP3+ cells and sidedness. These results demonstrate that the prognostic impact of tumour-infiltrating lymphocytes in CRC differs by primary tumour site, further indicating that tumour location may be an important factor to take into consideration in therapeutic decisions, including eligibility for immunotherapy.

The clinical importance of tumour-infiltrating macrophages and dendritic cells in periampullary adenocarcinoma differs by morphological subtype.

BACKGROUND: Dendritic cells (DC) and tumour-associated macrophages (TAM) are essential in linking the innate and adaptive immune response against tumour cells and tumour progression. These cells are also potential target for immunotherapy as well as providing a handle to investigate immune status in the tumour microenvironment. The aim of the present study was to examine their impact on prognosis and chemotherapy response in periampullary adenocarcinoma, including pancreatic cancer, with particular reference to morphological subtype. METHODS: The density of tolerogenic immature CD1a+ dendritic cells (DC), and MARCO+, CD68+ and CD163+ tissue-associated macrophages (TAM) was analysed by immunohistochemistry in tissue micro arrays with tumours from 175 consecutive cases of periampullary adenocarcinoma who had undergone pancreaticoduodenectomy, 110 with pancreatobiliary type (PB-type) and 65 with intestinal type (I-type) morphology. Kaplan-Meier and Cox regression analyses were applied to determine the impact of immune cell infiltration on 5-year overall survival (OS). RESULTS: High density of CD1a+ DCs was an independent prognostic factor for a reduced OS in PB-type but not in I-type tumours (adjusted HR = 2.35; 95% CI 1.13-4.87). High density of CD68+ and CD163+ TAM was significantly associated with poor OS in the whole cohort, however only in unadjusted analysis (HR = 1.67; 95% CI 1.06-2.63, and HR = 1.84; 95% CI 1.09-3.09, respectively) and not in strata according to morphological subtype. High density of MARCO+ macrophages was significantly associated with poor prognosis in I-type but not in PB-type tumours (HR = 2.14 95% CI 1.03-4.44), and this association was only evident in patients treated with adjuvant chemotherapy. The prognostic value of the other investigated immune cells did not differ significantly in strata according to adjuvant chemotherapy. CONCLUSIONS: The results from this study demonstrate that high infiltration of tolerogenic immature DCs independently predicts a shorter survival in patients with PB-type periampullary adenocarcinoma, and that high density of the MARCO+ subtype of TAMs predicts a shorter survival in patients with I-type tumours. These results emphasise the importance of taking morphological subtype into account in biomarker studies related to periampullary cancer, and indicate that therapies targeting dendritic cells may be of value in the treatment of PB-type tumours, which are associated with the worst prognosis.

Prognostic stromal gene signatures in breast cancer.

INTRODUCTION: Global gene expression analysis of tumor samples has been a valuable tool to subgroup tumors and has the potential to be of prognostic and predictive value. However, tumors are heterogeneous, and homogenates will consist of several different cell types. This study was designed to obtain more refined expression data representing different compartments of the tumor. METHODS: Formalin-fixed paraffin-embedded stroma-rich triple-negative breast cancer tumors were laser-microdissected, and RNA was extracted and processed to enable microarray hybridization. Genes enriched in stroma were identified and used to generate signatures by identifying correlating genes in publicly available data sets. The prognostic implications of the signature were analyzed. RESULTS: Comparison of the expression pattern from stromal and cancer cell compartments from three tumors revealed a number of genes that were essentially specifically expressed in the respective compartments. The stroma-specific genes indicated contribution from fibroblasts, endothelial cells, and immune/inflammatory cells. The gene set was expanded by identifying correlating mRNAs using breast cancer mRNA expression data from The Cancer Genome Atlas. By iterative analyses, 16 gene signatures of highly correlating genes were characterized. Based on the gene composition, they seem to represent different cell types. In multivariate Cox proportional hazard models, two immune/inflammatory signatures had opposing hazard ratios for breast cancer recurrence also after adjusting for clinicopathological variables and molecular subgroup. The signature associated with poor prognosis consisted mainly of C1Q genes and the one associated with good prognosis contained HLA genes. This association with prognosis was seen for other cancers as well as in other breast cancer data sets. CONCLUSIONS: Our data indicate that the molecular composition of the immune response in a tumor may be a powerful predictor of cancer prognosis.

Expression of functional toll like receptor 4 in estrogen receptor/progesterone receptor-negative breast cancer.

INTRODUCTION: Toll-like receptors (TLRs) are a family of pattern recognition receptors that are expressed on cells of the innate immune system. The ligands can be pathogen derived (pathogen associated molecular patterns; PAMPs) or endogenous (damage associated molecular patterns; DAMPs) that when bound induces activation of nuclear factor kappa B (NF-κB) and transcription of pro-inflammatory genes. TLRs have also been discovered in various malignant cell types, but with unknown function. METHODS: In this study we performed a detailed analysis of TLR and co-receptor expression pattern and function in breast cancer. Expression patterns were examined using real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) on three estrogen receptor-positive (ER(+)) and four estrogen receptor/progesterone receptor-negative (ER(-)/PR(-); ER/PR-negative) breast cancer cell lines, and a breast cancer cohort consisting of 144 primary breast cancer samples. The function was investigated using in vitro assays comprising PAMP/DAMP-stimulation, downstream signaling and TLR-silencing experiments. RESULTS: We found that TLR4 was expressed in a biologically active form and responded to both PAMPs and DAMPs primarily in ER/PR-negative breast cancers. Stimulation of TLR2/4 in vitro induced expression of pro-inflammatory genes and a gene expression analysis of primary breast cancers showed a strong correlation between TLR4 expression and expression of pro-inflammatory mediators. In line with this, TLR4 protein expression correlated with a decreased survival. CONCLUSIONS: These findings suggest that TLR4 is expressed in a functional form in ER/PR-negative breast cancers. Studies regarding TLR4-antagonist therapies should be focusing on ER/PR-negative breast cancer particularly.

S100A9 expressed in ER(-)PgR(-) breast cancers induces inflammatory cytokines and is associated with an impaired overall survival.

BACKGROUND: Breast cancer is the most common cancer form among women today. Depending on hormone receptor status, breast cancers are divided into different subtypes with vastly varying prognosis. S100A9 is a calcium-binding protein that is associated with inflammation and expressed not only in myeloid cells but also in some tumours. The role for S100A9 in the malignant cells is not well characterised; however, previous studies have shown that the protein could have important immune-modulating properties. METHODS: Using a human breast cancer cohort consisting of 144 tumour samples and in vitro analysis of human breast cancer cell lines, we investigated the expression and function of S100A9 in human breast cancer. RESULTS: We show that S100A9 expression in breast cancer correlated with the ER(-)PgR(-) breast tumour subtype (P<0.001) and with Ki67 (P=0.024) and was expressed both in the malignant cells and in the tumour-infiltrating anti-inflammatory CD163(+) myeloid cells (P<0.001). Stromal expression of S100A9 also correlated to nodal stage, tumour size and Her2 positivity. Within the ER(-)PgR(-) subgroup, all Her2(+) and EGFR(+) tumours expressed S100A9 in the cytoplasm. Both cytoplasmic staining in the malignant cells as well as stromal S100A9 expression in myeloid cells correlated with a decreased overall survival in breast cancer patients. Furthermore, rS100A9 homodimers induced expression of pro-inflammatory cytokines (IL-6, IL-8 and IL-1ß) in a TLR4- and EGFR-dependent manner in human breast cancer cells in vitro. CONCLUSION: We suggest that S100A9 could be viewed as a novel therapeutic target for patients with ER(-)PgR(-) breast cancers.

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

BACKGROUND: Wnt 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. METHODS: Malignant 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. RESULTS: We 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. CONCLUSIONS: These 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.

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.

AIRE is expressed in breast cancer TANs and TAMs to regulate the extrinsic apoptotic pathway and inflammation.

The autoimmune regulator (AIRE) is a transcriptional regulator expressed in the thymus and is necessary for maintaining immunological self-tolerance. Extrathymic AIRE expression is rare, and a role for AIRE in tumor-associated innate immune cells has not yet been established. In this study, we show that AIRE is expressed in human pro-tumor neutrophils. In breast cancer, AIRE was primarily located to tumor-associated neutrophils (TANs), and to a lesser extent to tumor-associated macrophages (TAMs) and tumor cells. Expression of AIRE in TAN/TAMs, but not in cancer cells, was associated with an adverse prognosis. We show that the functional role for AIRE in neutrophils and macrophages is to regulate expression of immune mediators and the extrinsic apoptotic pathway involving the Fas/TNFR death receptors and cathepsin G. Here, we propose that the role for AIRE in TAN/TAMs in breast tumors is to regulate cell death and inflammation, thus promoting tumor progression.

Regulation of PMP22 mRNA by G3BP1 affects cell proliferation in breast cancer cells.

BACKGROUND: Regulation of mRNAs is one way to control protein levels and thereby important cellular processes such as growth, invasion and apoptosis. G3BPs constitute a family of mRNA-binding proteins, shown to be overexpressed in several cancer types, including breast, colon and pancreas cancer. G3BP has been reported to both stabilize and induce degradation of specific mRNAs. RESULTS: Here, we show that G3BP1, but not G3BP2, supports proliferation of several breast cancer cell lines. Global gene expression analyses of G3BP1- and G3BP2-depleted cells indicate that primarily G3BP1, and much less G3BP2, influences mRNA expression levels. Peripheral myelin protein 22 (PMP22) was one gene that was significantly influenced by G3BP1 depletion which led to a 2-3 fold increased expression. Depletion of PMP22 resulted in increased proliferation and the G3BP1-mediated effect on proliferation was not seen upon PMP22-depletion. CONCLUSIONS: This indicates a novel role for G3BP1 in the regulation of cell proliferation in breast cancer cells, perhaps via a regulatory effect on PMP22 expression.