Embryonic Lethality in Mice Lacking the Nuclear Factor of Activated T Cells 5 Protein Due to Impaired Cardiac Development and Function

Nuclear factor of activated T cells 5 protein (NFAT5) is thought to be important for cellular adaptation to osmotic stress by regulating the transcription of genes responsible for the synthesis or transport of organic osmolytes. It is also thought to play a role in immune function, myogenesis and cancer invasion. To better understand the function of NFAT5, we developed NFAT5 gene knockout mice. Homozygous NFAT5 null (NFAT5−/−) mouse embryos failed to develop normally and died after 14.5 days of embryonic development (E14.5). The embryos showed peripheral edema, and abnormal heart development as indicated by thinner ventricular wall and reduced cell density at the compact and trabecular areas of myocardium. This is associated with reduced level of proliferating cell nuclear antigen and increased caspase-3 in these tissues. Cardiomyocytes from E14.5 NFAT5−/− embryos showed a significant reduction of beating rate and abnormal Ca2+ signaling profile as a consequence of reduced sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and ryanodine receptor (RyR) expressions. Expression of NFAT5 target genes, such as HSP 70 and SMIT were reduced in NFAT5−/− cardiomyocytes. Our findings demonstrated an essential role of NFAT5 in cardiac development and Ca2+ signaling. Cardiac failure is most likely responsible for the peripheral edema and death of NFAT5−/− embryos at E14.5 days

Exclusion of NFAT5 from Mitotic Chromatin Resets Its Nucleo-Cytoplasmic Distribution in Interphase

The transcription factor NFAT5 is a major inducer of osmoprotective genes and is required to maintain the proliferative capacity of cells exposed to hypertonic stress. In response to hypertonicity, NFAT5 translocates to the nucleus, binds to regulatory regions of osmoprotective genes and activates their transcription. Besides stimulus-specific regulatory mechanisms, the activity of transcription factors in cycling cells is also regulated by the passage through mitosis, when most transcriptional processes are downregulated. It was not known whether mitosis could be a point of control for NFAT5.Using confocal microscopy we observed that NFAT5 was excluded from chromatin during mitosis in both isotonic and hypertonic conditions. Analysis of NFAT5 deletions showed that exclusion was mediated by the carboxy-terminal domain (CTD). NFAT5 mutants lacking this domain showed constitutive binding to mitotic chromatin independent of tonicity, which caused them to localize in the nucleus and remain bound to chromatin in the subsequent interphase without hypertonic stimulation. We analyzed the contribution of the CTD, DNA binding, and nuclear import and export signals to the subcellular localization of this factor. Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase. Finally, we identified several regions within the CTD of NFAT5, some of them overlapping with transactivation domains, which were separately capable of causing its exclusion from mitotic chromatin.Our results reveal a multipart mechanism regulating the subcellular localization of NFAT5. The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis. This mechanism, together with export signals acting in interphase, resets the cytoplasmic localization of NFAT5 and prevents its nuclear accumulation and association with DNA in the absence of hypertonic stress

Phenotypic and transcriptomic characterization of canine myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) are key players in immune evasion, tumor progression and metastasis. MDSCs accumulate under various pathological states and fall into two functionally and phenotypically distinct subsets that have been identified in humans and mice: polymorphonuclear (PMN)-MDSCs and monocytic (M)-MDSCs. As dogs are an excellent model for human tumor development and progression, we set out to identify PMN-MDSCs and M-MDSCs in clinical canine oncology patients. Canine hypodense MHC class II-CD5-CD21-CD11b+ cells can be subdivided into polymorphonuclear (CADO48A+CD14-) and monocytic (CADO48A-CD14+) MDSC subsets. The transcriptomic signatures of PMN-MDSCs and M-MDSCs are distinct, and moreover reveal a statistically significant similarity between canine and previously published human PMN-MDSC gene expression patterns. As in humans, peripheral blood frequencies of canine PMN-MDSCs and M-MDSCs are significantly higher in dogs with cancer compared to healthy control dogs (PMN-MDSCs: p < 0.001; M-MDSCs: p < 0.01). By leveraging the power of evolution, we also identified additional conserved genes in PMN-MDSCs of multiple species that may play a role in MDSC function. Our findings therefore validate the dog as a model for studying MDSCs in the context of cancer

Increased peri-ductal collagen micro-organization may contribute to raised mammographic density

BACKGROUND: High mammographic density is a therapeutically modifiable risk factor for breast cancer. Although mammographic density is correlated with the relative abundance of collagen-rich fibroglandular tissue, the causative mechanisms, associated structural remodelling and mechanical consequences remain poorly defined. In this study we have developed a new collaborative bedside-to-bench workflow to determine the relationship between mammographic density, collagen abundance and alignment, tissue stiffness and the expression of extracellular matrix organising proteins. METHODS: Mammographic density was assessed in 22 post-menopausal women (aged 54–66 y). A radiologist and a pathologist identified and excised regions of elevated non-cancerous X-ray density prior to laboratory characterization. Collagen abundance was determined by both Masson’s trichrome and Picrosirius red staining (which enhances collagen birefringence when viewed under polarised light). The structural specificity of these collagen visualisation methods was determined by comparing the relative birefringence and ultrastructure (visualised by atomic force microscopy) of unaligned collagen I fibrils in reconstituted gels with the highly aligned collagen fibrils in rat tail tendon. Localised collagen fibril organisation and stiffness was also evaluated in tissue sections by atomic force microscopy/spectroscopy and the abundance of key extracellular proteins was assessed using mass spectrometry. RESULTS: Mammographic density was positively correlated with the abundance of aligned periductal fibrils rather than with the abundance of amorphous collagen. Compared with matched tissue resected from the breasts of low mammographic density patients, the highly birefringent tissue in mammographically dense breasts was both significantly stiffer and characterised by large (>80 μm long) fibrillar collagen bundles. Subsequent proteomic analyses not only confirmed the absence of collagen fibrosis in high mammographic density tissue, but additionally identified the up-regulation of periostin and collagen XVI (regulators of collagen fibril structure and architecture) as potential mediators of localised mechanical stiffness. CONCLUSIONS: These preliminary data suggest that remodelling, and hence stiffening, of the existing stromal collagen microarchitecture promotes high mammographic density within the breast. In turn, this aberrant mechanical environment may trigger neoplasia-associated mechanotransduction pathways within the epithelial cell population. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13058-015-0664-2) contains supplementary material, which is available to authorized users

Abstract P2-05-07: RAGE-ligand signaling drives breast cancer metastasis through affecting cells of the tumor and microenvironment

Abstract Breast cancer is most common malignant state in women, with 20% of these patients developing metastasis during the course of their disease. Further understanding is needed of the process and mechanisms of metastasis. Our lab and others have been shown that Receptor of Advanced-Glycation End-products (RAGE) plays a role in tumorigenesis and metastasis. RAGE is highly expressed in various cancers including breast cancer and its protein levels correlate with poor patient outcome in breast cancer and other cancers. Activation of RAGE results in increased proliferation, migration and invasion of cancer cells. Further studies in mice have shown it may be a therapeutic target to reduce tumor growth and the resulting metastasis. Further understanding is needed of the role of RAGE in driving metastasis through affecting cells of both the tumor and tumor stroma to design novel therapeutics. Using the breast cancer cell model (MDA-MB-231) and its organotropic sister cells lines selected in vivo for increased metastasis to lung (4175) and bone (1833), we tested the role of RAGE in driving tumor metastasis in vitro and in vivo with xenograft mouse models. To test the role of RAGE in the tumor microenvironment we used the AT-3 syngeneic breast cancer cell model in C57BL6 wild-type and RAGE knockout mice. We demonstrated that the highly metastatic variant of 231 cells (4175 and 1833) have increased expression level of RAGE compared to MDA-MB-231 parental cells. Ectopic over-expression of RAGE in parental 231 cells led to increased migratory and invasive properties compared to vector control cells, without affecting cell proliferation or viability. RAGE knockdown by shRNA in 4175 and 231 parental cells showed decreased cell invasion in transwell assays compared to control scramble shRNA. To validate our data in vivo, we performed mammary fat pad injection of 4175 cells (RAGE and scr shRNA) in NOD SCID gamma mice. Tumor growth and weight was impaired in RAGE gene knockdown 4175 cells compared to scramble (scr) controls. Analysis of lung and liver tissue retrieved from mice revealed RAGE knockdown in 4175 cells prevented metastasis compared to 4175 scr control cells. To test the role of RAGE on non-tumor cells of the breast stroma we next performed syngeneic studies with AT-3 cells (MMTV-PyMT spontaneous BC cell model), by injection into the mammary fat pad of wild-type and RAGE knockout C57BL6 immunocompetent mice. RAGE knockout mice (RAGE -/-) displayed striking impairment of tumor cell growth compared to wild-type (RAGE +/+) mice. We are currently testing whether novel RAGE inhibitors impact breast cancer progression and metastasis. These data highlight RAGE drives breast cancer progression and metastasis through affecting both tumor cell intrinsic and non-tumor cell microenvironment effects. Future studies will demonstrate the potential of RAGE inhibition as a novel therapeutic approach for preventing and treating metastatic disease in breast and other cancers. Citation Format: Kwak T, Drews-Elger K, Ergonul A, Zhao D, Besser A, Slingerland JM, Lippman ME, Hudson BI. RAGE-ligand signaling drives breast cancer metastasis through affecting cells of the tumor and microenvironment. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-07.</jats:p

VEGF drives cancer-initiating stem cells through VEGFR-2/Stat3 signaling to upregulate Myc and Sox2

Vascular endothelial growth factor-A (VEGF), a potent angiogenic factor, is also implicated in self-renewal in several normal tissue types. VEGF has been shown to drive malignant stem cells but mechanisms thereof and tumor types affected are not fully characterized. Here, we show VEGF promotes breast and lung cancer stem cell (CSC) self-renewal via VEGF receptor-2 (VEGFR-2)/STAT3-mediated upregulation of Myc and Sox2. VEGF increased tumor spheres and aldehyde dehydrogenase activity, both proxies for stem cell function in vitro, in triple-negative breast cancer (TNBC) lines and dissociated primary cancers, and in lung cancer lines. VEGF exposure before injection increased breast cancer-initiating cell abundance in vivo yielding increased orthotopic tumors, and increased metastasis from orthotopic primaries and following tail vein injection without further VEGF treatment. VEGF rapidly stimulated VEGFR-2/JAK2/STAT3 binding and activated STAT3 to bind MYC and SOX2 promoters and induce their expression. VEGFR-2 knockdown or inhibition abrogated VEGF-mediated STAT3 activation, MYC and SOX2 induction and sphere formation. Notably, knockdown of either STAT3, MYC or SOX2 impaired VEGF-upregulation of pSTAT3, MYC and SOX2 expression and sphere formation. Each transcription factor, once upregulated, appears to promote sustained activation of the others, creating a feed-forward loop to drive self-renewal. Thus, in addition to angiogenic effects, VEGF promotes tumor-initiating cell self-renewal through VEGFR-2/STAT3 signaling. Analysis of primary breast and lung cancers (>1300 each) showed high VEGF expression, was prognostic of poor outcome and strongly associated with STAT3 and MYC expression, supporting the link between VEGF and CSC self-renewal. High-VEGF tumors may be most likely to escape anti-angiogenics by upregulating VEGF, driving CSC self-renewal to re-populate post-treatment. Our work highlights the need to better define VEGF-driven cancer subsets and supports further investigation of combined therapeutic blockade of VEGF or VEGFR-2 and JAK2/STAT3

Targeting of RAGE-ligand signaling impairs breast cancer cell invasion and metastasis

The receptor for advanced glycation end products (RAGE) is highly expressed in various cancers and is correlated with poorer outcome in breast and other cancers. Here we tested the role of targeting RAGE by multiple approaches in the tumor and tumor microenvironment, to inhibit the metastatic process. We first tested how RAGE impacts tumor cell-intrinsic mechanisms using either RAGE overexpression or knockdown with short hairpin RNAs (shRNAs). RAGE ectopic overexpression in breast cancer cells increased MEK-EMT (MEK-epithelial-to-mesenchymal transition) signaling, transwell invasion and soft agar colony formation, and in vivo promoted lung metastasis independent of tumor growth. RAGE knockdown with multiple independent shRNAs in breast cancer cells led to decreased transwell invasion and soft agar colony formation, without affecting proliferation. In vivo, targeting RAGE shRNA knockdown in human and mouse breast cancer cells, decreased orthotopic tumor growth, reduced tumor angiogenesis and recruitment of inflammatory cells, and markedly decreased metastasis to the lung and liver in multiple xenograft and syngeneic mouse models. To test the non-tumor cell microenvironment role of RAGE, we performed syngeneic studies with orthotopically injected breast cancer cells in wild-type and RAGE-knockout C57BL6 mice. RAGE-knockout mice displayed striking impairment of tumor cell growth compared with wild-type mice, along with decreased mitogen-activated protein kinase signaling, tumor angiogenesis and inflammatory cell recruitment. To test the combined inhibition of RAGE in both tumor cell-intrinsic and non-tumor cells of the microenvironment, we performed in vivo treatment of xenografted tumors with FPS-ZM1 (1 mg/kg, two times per week). Compared with vehicle, FPS-ZM1 inhibited primary tumor growth, inhibited tumor angiogenesis and inflammatory cell recruitment and, most importantly, prevented metastasis to the lung and liver. These data demonstrate that RAGE drives tumor progression and metastasis through distinct tumor cell-intrinsic and -extrinsic mechanisms, and may represent a novel and therapeutically viable approach for treating metastatic cancers

Infiltrating S100A8+myeloid cells promote metastatic spread of human breast cancer and predict poor clinical outcome

The mechanisms by which breast cancer (BrC) can successfully metastasize are complex and not yet fully understood. Our goal was to identify tumor-induced stromal changes that influence metastatic cell behavior, and may serve as better targets for therapy. To identify stromal changes in cancer-bearing tissue, dual-species gene expression analysis was performed for three different metastatic BrC xenograft models. Results were confirmed by immunohistochemistry, flow cytometry, and protein knockdown. These results were validated in human clinical samples at the mRNA and protein level by retrospective analysis of cohorts of human BrC specimens. In pre-clinical models of BrC, systemic recruitment of S100A8+ myeloid cells-including myeloid-derived suppressor cells (MDSCs)-was promoted by tumor-derived factors. Recruitment of S100A8+ myeloid cells was diminished by inhibition of tumor-derived factors or depletion of MDSCs, resulting in fewer metastases and smaller primary tumors. Importantly, these MDSCs retain their ability to suppress T cell proliferation upon co-culture. Secretion of macrophage inhibitory factor (MIF) activated the recruitment of S100A8+ myeloid cells systemically. Inhibition of MIF, or depletion of MDSCs resulted in delayed tumor growth and lower metastatic burden. In human BrC specimens, increased mRNA and protein levels of S100A8+ infiltrating cells are highly associated with poor overall survival and shorter metastasis free survival of BrC patients, respectively. Furthermore, analysis of nine different human gene expression datasets confirms the association of increased levels of S100A8 transcripts with an increased risk of death. Recruitment of S100A8+ myeloid cells to primary tumors and secondary sites in xenograft models of BrC enhances cancer progression independent of their suppressive activity on T cells. In clinical samples, infiltrating S100A8+ cells are associated with poor overall survival. Targeting these molecules or associated pathways in cells of the tumor microenvironment may translate into novel therapeutic interventions and benefit patient outcome