Annexin A1 in inflammation and breast cancer: a new axis in the tumor microenvironment

ABSTRACT

Targeting inflammation in cancer has shown promise to improve and complement current therapies. The tumor microenvironment plays an important role in cancer growth and metastasis and -tumor associated macrophages possess pro-tumoral and pro-metastatic properties. Annexin A1 (ANXA1) is an immune-modulating protein with diverse functions in the immune system and in cancer. In breast cancer, high ANXA1 expression leads to poor prognosis and increased metastasis. Here, we will review ANXA1 as a modulator of inflammation, and discuss its importance in breast cancer and highlight its new role in alternative macrophage activation in the tumor microenvironment. This review may provide an updated understanding into the various roles of ANXA1 which may enable future therapeutic developments for the treatment of breast cancer.     

Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties

Although there is a growing literature describing the role of macrophages in breast cancer, the role of macrophages in inflammatory breast cancer (IBC) is unclear. The aim of present study was to isolate and characterize tumor associated macrophages of IBC and non-IBC patients and define their role in IBC. Tumor infiltrating monocytes/macrophages (CD14+ and CD68+) were measured by immunohistochemistry using specific monoclonal antibodies. Blood drained from axillary vein tributaries was collected during breast cancer surgery and the percentage of CD14+ in the total isolated leukocytes was assessed by flow cytometric analysis. CD14+ cells were separated from total leukocytes by immuno-magnetic beads technique and were cultured overnight. Media conditioned by CD14+ were collected and subjected to cytokine profiling using cytokine antibody array. Wound healing and invasion assays were used to test whether cytokines highly secreted by tumor drained macrophages induce motility and invasion of breast cancer cells. We found that macrophages highly infiltrate into carcinoma tissues of IBC patients. In addition blood collected from axillary tributaries of IBC patients is highly enriched with CD14+ cells as compared to blood collected from non-IBC patients. Cytokine profiling of CD14+ cells isolated from IBC patients revealed a significant increase in secretion of tumor necrosis factor-α; monocyte chemoattractant protein-1/CC-chemokine ligand 2; interleukin-8 and interleukin-10 as compared to CD14+ cells isolated from non-IBC patients. Tumor necrosis factor-α, interleukin-8 and interleukin-10 significantly increased motility and invasion of IBC cells in vitro. In conclusion, macrophages isolated from the tumor microenvironment of IBC patients secrete chemotactic cytokines that may augment dissemination and metastasis of IBC carcinoma cells.     

Osteoblasts suppress high bone turnover caused by osteolytic breast cancer in-vitro

The skeleton is the most common site of breast cancer metastasis, which can occur in up to 85% of patients during their lifetime. The morbidity associated with bone metastases in patients with breast cancer includes pathological fractures, bone pain, hypercalcaemia, and spinal cord compression. When breast cancer metastasizes to bone, the balance of bone resorption (mediated by osteoclasts) and bone formation (mediated by osteoblasts) favors bone resorption, which leads to net bone destruction (i.e., osteolysis). Anti-resorptive agents such as bisphosphonates are commonly used to treat bone resorption in osteoporosis or osteolytic cancer patients. However, bisphosphonates by themselves are unable to rebuild lost bone tissue, and can cause severe side effects. In this study, we developed a bovine bone explant culture system and have observed that murine osteoblasts can modulate the activity of osteotropic human breast cancer cells on this substrate. Using markers of bone metabolism, we observe diminished bone turnover in organ culture following the addition of exogenous osteoblasts. The data presented in this study supports further investigation into the use of cytotherapies to limit breast cancer mediated osteolysis.     

In vitro vascularized tumor platform for modeling tumor-vasculature interactions of inflammatory breast cancer

Inflammatory breast cancer (IBC), a rare form of breast cancer associated with increased angiogenesis and metastasis, is largely driven by tumor-stromal interactions with the vasculature and the extracellular matrix (ECM). However, there is currently a lack of understanding of the role these interactions play in initiation and progression of the disease. In this study, we developed the first three-dimensional, in vitro, vascularized, microfluidic IBC platform to quantify the spatial and temporal dynamics of tumor-vasculature and tumor-ECM interactions specific to IBC. Platforms consisting of collagen type 1 ECM with an endothelialized blood vessel were cultured with IBC cells, MDA-IBC3 (HER2+) or SUM149 (triple negative), and for comparison to non-IBC cells, MDA-MB-231 (triple negative). Acellular collagen platforms with endothelialized blood vessels served as controls. SUM149 and MDA-MB-231 platforms exhibited a significantly (p < .05) higher vessel permeability and decreased endothelial coverage of the vessel lumen compared to the control. Both IBC platforms, MDA-IBC3 and SUM149, expressed higher levels of vascular endothelial growth factor (p < .05) and increased collagen ECM porosity compared to non-IBCMDA-MB-231 (p < .05) and control (p < .01) platforms. Additionally, unique to the MDA-IBC3 platform, we observed progressive sprouting of the endothelium over time resulting in viable vessels with lumen. The newly sprouted vessels encircled clusters of MDA-IBC3 cells replicating a key feature of in vivo IBC. The IBC in vitro vascularized platforms introduced in this study model well-described in vivo and clinical IBC phenotypes and provide an adaptable, high throughput tool for systematically and quantitatively investigating tumor-stromal mechanisms and dynamics of tumor progression.     

Stem cell transplantation for metastatic breast cancer: analysis of tumor contamination

The purpose of this study was to determine the efficacy, engraftment kinetics, effect of bone marrow tumor contamination, and safety of high-dose therapy and granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood progenitor cell (PBPC) support for patients with responding metastatic breast cancer. Forty two patients underwent G-CSF (10 μg/kg) stimulated PBPC harvest. PBPC and bone marrow aspirates were analyzed by histologic and immunocytochemical methods for tumor contamination. Thirty-seven patients received high-dose therapy consisting of cyclophosphamide 6 g/m², thiotepa 500 mg/m², and carboplatin 800 mg/m² (CTCb) given as an infusion over 4 d followed by PBPC reinfusion and G-CSF (5 μg/kg) support. No transplant related deaths or grade 4 toxicity was recorded. CD34+ cells/kg infused was predictive of neutrophil and platelet recovery. With a median follow-up of 38 months, three year survival was 44% with relapse-free survival of 19%. Histological bone marrow involvement, found in 10 patients, was a negative prognostic factor and was associated with a median relapse-free survival of 3.5 months. Tumor contamination of PBPC by immunohistochemical staining was present in 22.5% of patients and found not to be correlated with decreased survival. G-CSF stimulated PBPC collection followed by a single course of high dose chemotherapy and stem cell infusion with G-CSF stimulated marrow recovery leads to rapid, reliable engraftment with low toxicity and promising outcome in women with responding metastatic breast cancer.     

Interleukin (IL)-17 enhances tumor necrosis factor-alpha-stimulated IL-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

Inflammatory cytokines are well known to play crucial roles in the pathogenesis of rheumatoid arthritis. Among them, interleukin (IL)-17 is a cytokine that is mainly synthesized by activated T cells and its receptors are present in osteoblasts. The synthesis of IL-6, known to stimulate osteoclastic bone resorption, is reportedly responded to bone resorptive agents such as tumor necrosis factor-alpha (TNF-alpha) in osteoblasts. It has been reported that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We previously showed that sphingosine 1-phosphate (S1-P) mediates TNF-alpha-stimulated IL-6 synthesis in these cells. In the present study, we investigated the mechanism of IL-17 underlying enhancement of IL-6 synthesis in MC3T3-E1 cells. IL-17 induced phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, significantly reduced the enhancement by IL-17 of TNF-alpha-stimulated IL-6 synthesis. IL-17 also amplified S1-P-stimulated IL-6 synthesis, and the amplification by IL-17 was suppressed by SB203580. Anisomycin, an activator of p38 MAP kinase, which alone had no effect on IL-6 level, enhanced the IL-6 synthesis stimulated by TNF-alpha. SB203580 and PD169316 inhibited the amplification by anisomycin of the TNF-alpha-induced IL-6 synthesis. Taken together, our results strongly suggest that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.     

Extracellular vesicle-mediated transport: Reprogramming a tumor microenvironment conducive with breast cancer progression and metastasis

Breast cancer metastatic progression to critical secondary sites is the second leading cause of cancer-related mortality in women. While existing therapies are highly effective in combating primary tumors, metastatic disease is generally deemed incurable with a median survival of only 2, 3 years. Extensive efforts have focused on identifying metastatic contributory targets for therapeutic antagonism and prevention to improve patient survivability. Excessive breast cancer release of extracellular vesicles (EVs), whose contents stimulate a metastatic phenotype, represents a promising target. Complex breast cancer intercellular communication networks are based on EV transport and transference of molecular information is in bulk resulting in complete reprogramming events within recipient cells. Other breast cancer cells can acquire aggressive phenotypes, endothelial cells can be induced to undergo tubule formation, and immune cells can be neutralized. Recent advancements continue to implicate the critical role EVs play in cultivating a tumor microenvironment tailored to cancer proliferation, metastasis, immune evasion, and conference of drug resistance. This literature review serves to frame the role of EV transport in breast cancer progression and metastasis. The following five sections will be addressed: (1) Intercellular communication in developing a tumor microenvironment & pre-metastatic niche. (2) Induction of the epithelial-to-mesenchymal transition (EMT). (3). Immune suppression & evasion. (4) Transmission of drug resistance mechanisms. (5) Precision medicine: clinical applications of EVs.     

Protein kinase CK2 modulates IL-6 expression in inflammatory breast cancer

Inflammatory breast cancer is driven by pro-angiogenic and pro-inflammatory cytokines. One of them Interleukin-6 (IL-6) is implicated in cancer cell proliferation and survival, and promotes angiogenesis, inflammation and metastasis. While IL-6 has been shown to be upregulated by several oncogenes, the mechanism behind this phenomenon is not well characterized. Here we demonstrate that the pleotropic Serine/Threonine kinase CK2 is implicated in the regulation of IL-6 expression in a model of inflammatory breast cancer. We used siRNAs targeted toward CK2 and a selective small molecule inhibitor of CK2, CX-4945, to inhibit the expression and thus suppress the secretion of IL-6 in in vitro as well as in vivo models. Moreover, we report that in a clinical trial, CX-4945 was able to dramatically reduce IL-6 levels in plasma of an inflammatory breast cancer patient. Our data shed a new light on the regulation of IL-6 expression and position CX-4945 and potentially other inhibitors of CK2, for the treatment of IL-6-driven cancers and possibly other diseases where IL-6 is instrumental, including rheumatoid arthritis.     

DLC1在乳腺癌中的肿瘤抑制作用机理

DLC1是1998年在对原发性肝癌进行研究时首次分离和报道的,它不仅在肝癌中表达缺失,而且在人类多种恶性肿瘤中也表达低下或缺失,是近年来研究较热门的肿瘤抑制基因。乳腺癌是女性常见的恶性肿瘤,极大影响女性身心健康,在乳腺癌等恶性肿瘤中,DLC1具有抑制癌细胞增殖、迁移并诱导凋亡的作用。     

Characterization of the relationship between FLI1 and immune infiltrate level in tumour immune microenvironment for breast cancer

Breast cancer is the most common cancer and the leading cause of cancer death among women in the world. Tumour-infiltrating lymphocytes were defined as the white blood cells left in the vasculature and localized in tumours. Recently, tumour-infiltrating lymphocytes were found to be associated with good prognosis and response to immunotherapy in tumours. In this study, to examine the influence of FLI1 in immune system in breast cancer, we interrogated the relationship between the FLI1 expression levels with infiltration levels of 28 immune cell types. By splitting the breast cancer samples into high and low expression FLI1 subtypes, we found that the high expression FLI1 subtype was enriched in many immune cell types, and the up-regulated differentially expressed genes between them were enriched in immune system processes, immune-related KEGG pathways and biological processes. In addition, many important immune-related features were found to be positively correlated with the FLI1 expression level. Furthermore, we found that the FLI1 was correlated with the immune-related genes. Our findings may provide useful help for recognizing the relationship between tumour immune microenvironment and FLI1, and may unravel clinical outcomes and immunotherapy utility for FLI1 in breast cancer.     

Mesenchymal stem cell-derived interleukin-6 and vascular endothelial growth factor promote breast cancer cell migration

Several different cytokines and growth factors secreted by mesenchymal stem cells (MSCs) have been hypothesized to play a role in breast cancer progression. By using a small panel of breast cancer cell lines (MCF‐7, T47D, and SK‐Br‐3 cells), we analyzed the role of interleukin‐6 (IL‐6) and vascular endothelial growth factor A (VEGF) in the cross‐talk between MSCs and breast cancer cells. We performed migration assays in which breast cancer cells were allowed to migrate in response to conditioned medium from MSCs (MSCs‐CM), in absence or in presence of the anti‐VEGF antibody bevacizumab or an anti‐IL‐6 antibody, alone or in combination. We found that anti‐VEGF and anti‐IL‐6 antibodies inhibited the migration of breast cancer cells and that the combination had an higher inhibitory effect. We next evaluated the effects of recombinant VEGF and IL‐6 proteins on breast cancer cell growth and migration. IL‐6 and VEGF had not significant effects on the proliferation of breast carcinoma cells. In contrast, both VEGF and IL‐6 significantly increased the ability to migrate of MCF‐7, T47D and SK‐Br‐3 cells, with the combination showing a greater effect as compared with treatment with a single protein. The combination of VEGF and IL‐6 produced in breast cancer cells a more significant and more persistent activation of MAPK, AKT, and p38MAPK intracellular signaling pathways. These results suggest that MSC‐secreted IL‐6 and VEGF may act as paracrine factors to sustain breast cancer cell migration. J. Cell. Biochem. 113: 3363–3370, 2012. © 2012 Wiley Periodicals, Inc.     

miR-139-5p is a regulator of metastatic pathways in breast cancer

Metastasis is a complex, multistep process involved in the progression of cancer from a localized primary tissue to distant sites, often characteristic of the more aggressive forms of this disease. Despite being studied in great detail in recent years, the mechanisms that govern this process remain poorly understood. In this study, we identify a novel role for miR-139-5p in the inhibition of breast cancer progression. We highlight its clinical relevance by reviewing miR-139-5p expression across a wide variety of breast cancer subtypes using in-house generated and online data sets to show that it is most frequently lost in invasive tumors. A biotin pull-down approach was then used to identify the mRNA targets of miR-139-5p in the breast cancer cell line MCF7. Functional enrichment analysis of the pulled-down targets showed significant enrichment of genes in pathways previously implicated in breast cancer metastasis (P < 0.05). Further bioinformatic analysis revealed a predicted disruption to the TGFβ, Wnt, Rho, and MAPK/PI3K signaling cascades, implying a potential role for miR-139-5p in regulating the ability of cells to invade and migrate. To corroborate this finding, using the MDA-MB-231 breast cancer cell line, we show that overexpression of miR-139-5p results in suppression of these cellular phenotypes. Furthermore, we validate the interaction between miR-139-5p and predicted targets involved in these pathways. Collectively, these results suggest a significant functional role for miR-139-5p in breast cancer cell motility and invasion and its potential to be used as a prognostic marker for the aggressive forms of breast cancer.     

The role of tumor microenvironment in prostate cancer bone metastasis

Prostate cancer (PCa) epithelial cells require a number of factors to facilitate their establishment and growth at a distant site of metastasis. Their ability to adapt to their microenvironment, proliferate and recruit an underlying stroma is integral to the survival and growth of the metastasis. PCa predominantly metastasizes to the bone, and bone metastases are the main cause of morbidity. The bone marrow provides a permissive environment for the formation of a metastasis. In some cases, the cells may remain dormant for some time, eventually proliferating in response to an unknown "trigger." The marrow is rich in progenitor cells that differentiate into numerous cell types, producing new blood vessels, supporting fibroblasts, and an underlying extracellular matrix (ECM) that form the reactive stroma. By secreting a number of cytokines, growth factors and proteases they recruit auxiliary cells required to produce a functional stroma. These components are involved in a reciprocal interaction between the stroma and the PCa cells, allowing for the growth and survival of the tumor. Left unchecked, once a PCa tumor has established itself in the bone marrow it will eventually replace the marrow, interrupting bone homeostasis and typically promoting an osteoblastic response in the bone including osteoclastic events. The abundant deposition of new woven bone results in nerve compression, bone pain and an increase in fractures in patients with PCa bone metastases. This review will examine the tumor microenvironment, its role in facilitating tumor dissemination, growth and the resultant pathologies associated with PCa bone metastasis.     

BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment

Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary breast cancer. Similarly, downregulation of BRCA1 protein expression is observed in the majority of basal-like breast cancers. Here, we set out to study the effects of BRCA1 mutations on oxidative stress in the tumor microenvironment. To mimic the breast tumor microenvironment, we utilized an in vitro co-culture model of human BRCA1-mutated HCC1937 breast cancer cells and hTERT-immortalized human fibroblasts. Notably, HCC1937 cells induce the generation of hydrogen peroxide in the fibroblast compartment during co-culture, which can be inhibited by genetic complementation with the wild-type BRCA1 gene. Importantly, treatment with powerful antioxidants, such as NAC and Tempol, induces apoptosis in HCC1937 cells, suggesting that microenvironmental oxidative stress supports cancer cell survival. In addition, Tempol treatment increases the apoptotic rates of MDA-MB-231 cells, which have wild-type BRCA1, but share a basal-like breast cancer phenotype with HCC1937 cells. MCT4 is the main exporter of L-lactate out of cells and is a marker for oxidative stress and glycolytic metabolism. Co-culture with HCC1937 cells dramatically induces MCT4 protein expression in fibroblasts, and this can be prevented by either BRCA1 overexpression or by pharmacological treatment with NAC. We next evaluated caveolin-1 (Cav-1) expression in stromal fibroblasts. Loss of Cav-1 is a marker of the cancer-associated fibroblast (CAF) phenotype, which is linked to high stromal glycolysis, and is associated with a poor prognosis in numerous types of human cancers, including breast cancers. Remarkably, HCC1937 cells induce a loss of Cav-1 in adjacent stromal cells during co-culture. Conversely, Cav-1 expression in fibroblasts can be rescued by administration of NAC or by overexpression of BRCA1 in HCC1937 cells. Notably, BRCA1-deficient human breast cancer samples (9 out of 10) also showed a glycolytic stromal phenotype, with intense mitochondrial staining specifically in BRCA1-deficient breast cancer cells. In summary, loss of BRCA1 function leads to hydrogen peroxide generation in both epithelial breast cancer cells and neighboring stromal fibroblasts, and promotes the onset of a reactive glycolytic stroma, with increased MCT4 and decreased Cav-1 expression. Importantly, these metabolic changes can be reversed by antioxidants, which potently induce cancer cell death. Thus, antioxidant therapy appears to be synthetically lethal with a BRCA1-deficiency in breast cancer cells and should be considered for future cancer prevention trials. In this regard, immunostaining with Cav-1 and MCT4 could be used as cost-effective biomarkers to monitor the response to antioxidant therapy.