Search

Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. Our unbiased proteomics analysis of melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared with those derived from extracranial metastases. We showed that melanoma cells require amyloid beta (Aβ) for growth and survival in the brain parenchyma. Melanoma-secreted Aβ activates surrounding astrocytes to a prometastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacologic inhibition of Aβ decreases brain metastatic burden.Significance:Our results reveal a novel mechanistic connection between brain metastasis and Alzheimer's disease, two previously unrelated pathologies; establish Aβ as a promising therapeutic target for brain metastasis; and demonstrate suppression of neuroinflammation as a critical feature of metastatic adaptation to the brain parenchyma.This article is highlighted in the In This Issue feature, p. 1171

Supplementary Table from Melanoma-Secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

Supplementary Data from Melanoma-Secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

Supplementary Figure from Melanoma-Secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

There is considerable interest in the clinical development of inhibitors of mTOR complexes mTORC1 and 2. Because mTORC1 and its downstream mRNA translation effectors may protect against genotoxic DNA damage, we investigated the inhibition of mTORC1 and mTORC1/2 in the ability to reverse platinum resistance in tissue culture and in animal tumor models of serous ovarian cancer. Cell survival, tumor growth, PI3K–AKT–mTOR pathway signaling, DNA damage and repair response (DDR) gene expression, and translational control were all investigated. We show that platinum-resistant OVCAR-3 ovarian cancer cells are resensitized to low levels of carboplatin in culture by mTOR inhibition, demonstrating reduced survival after treatment with either mTORC1 inhibitor everolimus or mTORC1/2 inhibitor PP242. Platinum resistance is shown to be associated with activating phosphorylation of AKT and CHK1, inactivating phosphorylation of 4E-BP1, the negative regulator of eIF4E, which promotes increased cap-dependent mRNA translation and increased levels of CHK1 and BRCA1 proteins. Animals with platinum-resistant OVCAR-3 tumors treated with carboplatin plus mTORC1/2 inhibition had significantly longer median survival and strikingly reduced metastasis compared with animals treated with carboplatin plus everolimus, which inhibits only mTORC1. Reduced tumor growth, metastasis, and increased survival by mTORC1/2 inhibition with carboplatin treatment was associated with reduced AKT-activating phosphorylation and increased 4E-BP1 hypophosphorylation (activation). We conclude that mTORC1/2 inhibition is superior to mTORC1 inhibition in reversing platinum resistance in tumors and strongly impairs AKT activation, DNA repair responses, and translation, promoting improved survival in the background of platinum resistance. Mol Cancer Ther; 15(7); 1557–67. ©2016 AACR.

Supplementary Figure S1: Growth inhibitory 50% (GI50) determination of single agent everolimus, PP242 and carboplatin using OVCAR-3 cells; Supplementary Figure S2: mTORC1 and mTORC1/2 inhibition sensitizes platinum resistant SKOV3 cells to carboplatin-mediated growth inhibition; Supplementary Figure S3: qRT-PCR analysis of select mRNAs in mTORC1 and mTORC1/2 inhibited cells with and without carboplatin treatment.

Research offers ample insights into how people of different genders could experience transportation systems in equitable ways, but gender equity is still not part of mainstream transportation practice. We propose that Complete Streets could serve as an implementation system to advance gender equity. We provide empirical information, gender concepts, and regional cases from literatures on gender and transportation, multimodal travel, and public space to support this call to action. We find that a gender-aware Complete Streets movement would: 1) implement gender-specific tools and data; 2) address social environments and infrastructure; and 3) establish a gender-inclusive agenda to reform transportation policy.

Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. Our unbiased proteomics analysis of melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared with those derived from extracranial metastases. We showed that melanoma cells require amyloid beta (Aβ) for growth and survival in the brain parenchyma. Melanoma-secreted Aβ activates surrounding astrocytes to a prometastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacologic inhibition of Aβ decreases brain metastatic burden. Significance: Our results reveal a novel mechanistic connection between brain metastasis and Alzheimer's disease, two previously unrelated pathologies; establish Aβ as a promising therapeutic target for brain metastasis; and demonstrate suppression of neuroinflammation as a critical feature of metastatic adaptation to the brain parenchyma. This article is highlighted in the In This Issue feature, p. 1171

Supplementary Materials and Methods for additional procedures

Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer that displays profound cancer stem cell (CSC) and mesenchymal features that promote rapid metastasis. Another hallmark of IBC is high infiltration of M2 tumor-associated (immune-suppressing) macrophages. The molecular mechanism that drives these IBC phenotypes is not well understood. Using patient breast tumor specimens, breast cancer cell lines, and a patient-derived xenograft model of IBC, we demonstrate that IBC strongly expresses IL8 and growth-regulated oncogene (GRO) chemokines that activate STAT3, which promotes development of high levels of CSC-like cells and a mesenchymal phenotype. We also show that IBC expresses high levels of many monocyte recruitment and macrophage polarization factors that attract and differentiate monocytes into tumor-promoting, immune-suppressing M2-like macrophages. The M2 macrophages in turn were found to secrete high levels of IL8 and GRO chemokines, thereby creating a feed-forward chemokine loop that further drives an IBC epithelial-to-mesenchymal transition. Our study uncovers an intricate IBC-initiated autocrine–paracrine signaling network between IBC cells and monocytes that facilitates development of this highly aggressive form of breast cancer.Significance:This study uncovers a signaling network in which IBC cells commandeer macrophages to become tumor-promoting, and they in turn drive IBC cells to be more cancer stem-like, mesenchymal, and aggressive.

Figure S5. VEGF-D 5'UTR mRNA exhibits two structural forms that allow the binding of different proteins

Figure S2. Expression of endogenous VEGF-D in mice tissues

Figure S7. Tumor growth after NSAID treatment

The vascular endothelial growth factor VEGF-D promotes metastasis by inducing lymphangiogenesis and dilatation of the lymphatic vasculature, facilitating tumor cell extravasion. Here we report a novel level of control for VEGF-D expression at the level of protein translation. In human tumor cells, VEGF-D colocalized with eIF4GI and 4E-BP1, which can program increased initiation at IRES motifs on mRNA by the translational initiation complex. In murine tumors, the steady-state level of VEGF-D protein was increased despite the overexpression and dephosphorylation of 4E-BP1, which downregulates protein synthesis, suggesting the presence of an internal ribosome entry site (IRES) in the 5′ UTR of VEGF-D mRNA. We found that nucleolin, a nucleolar protein involved in ribosomal maturation, bound directly to the 5′UTR of VEGF-D mRNA, thereby improving its translation following heat shock stress via IRES activation. Nucleolin blockade by RNAi-mediated silencing or pharmacologic inhibition reduced VEGF-D translation along with a subsequent constriction of lymphatic vessels in tumors. Our results identify nucleolin as a key regulator of VEGF-D expression, deepening understanding of lymphangiogenesis control during tumor formation. Cancer Res; 76(15); 4394–405. ©2016 AACR.

Figure S8. Schematic representation of VEGF-D synthesis in tumor cells

Supplementary Data from Identification of Markers of Taxane Sensitivity Using Proteomic and Genomic Analyses of Breast Tumors from Patients Receiving Neoadjuvant Paclitaxel and Radiation

Supplementary figure legend

FS1: Four panels (A-D) demonstrating that SUM190 cells are enriched in cancer stem-like cells, shown by immunoblot analysis, cell flow cytometry and by increased cell invasion. FS2: Three panels (A-C) showing that co-culture of IBC cells with monocytes-macrophages increases IBC cell mesenchymal characteristics, shown by immunoblot analysis and invasion assays. FS3: Three immunoblot panels showing that addition of IL8 and GRO cytokines to the media of IBC cells promotes phosphorylation of STAT3 and cancer stem cell-like and mesenchymal characteristics. FS4: Four panels showing that IBC cells secrete attracting and type II polarization factors for monocytes and macrophages. Shown by function assays, cytokine sandwich blot assays, flow cytometry and immunohistochemistry.

TS1 describes the characteristics of the cell lines used in this study, including hormone receptor status and type of breast cancer. TS2 lists the antibodies used and the method by which immunohistochemistry stained breast cancer specimens were scored. TS3 describes the levels of IL8, Jak2, P-Ja2, STAT3, P-STAT3 and monocyte macrophages (CD68, CD163) in normal breast tissue and IBC specimens. TS4 lists the expression levels of 80 cytokines, chemokines and growth factors in the conditioned media from SUM149 cells. Data were obtained from at least three independent studies. TS5 lists the sequences of siRNAs that were used for gene silencing in this study. TS6 lists the primer sequences and the genes targeted for qPCR studies carried out.

Figure S6. Polysome profiling of 4T1 cells

Figure S4. Lymphangiogenesis and tumor gowth are not affected by 4T1 or 67NR lentiviral transduction

Purpose: To identify molecular markers of pathologic response to neoadjuvant paclitaxel/radiation treatment, protein and gene expression profiling were done on pretreatment biopsies.Experimental Design: Patients with high-risk, operable breast cancer were treated with three cycles of paclitaxel followed by concurrent paclitaxel/radiation. Tumor tissue from pretreatment biopsies was obtained from 19 of the 38 patients enrolled in the study. Protein and gene expression profiling were done on serial sections of the biopsies from patients that achieved a pathologic complete response (pCR) and compared to those with residual disease, non-pCR (NR).Results: Proteomic and validation immunohistochemical analyses revealed that α-defensins (DEFA) were overexpressed in tumors from patients with a pCR. Gene expression analysis revealed that MAP2, a microtubule-associated protein, had significantly higher levels of expression in patients achieving a pCR. Elevation of MAP2 in breast cancer cell lines led to increased paclitaxel sensitivity. Furthermore, expression of genes that are associated with the basal-like, triple-negative phenotype were enriched in tumors from patients with a pCR. Analysis of a larger panel of tumors from patients receiving presurgical taxane-based treatment showed that DEFA and MAP2 expression as well as histologic features of inflammation were all statistically associated with response to therapy at the time of surgery.Conclusion: We show the utility of molecular profiling of pretreatment biopsies to discover markers of response. Our results suggest the potential use of immune signaling molecules such as DEFA as well as MAP2, a microtubule-associated protein, as tumor markers that associate with response to neoadjuvant taxane–based therapy. Clin Cancer Res; 16(2); 681–90

Figure S3. Podoplanin staining of lymphatic vessels

p23 is a heat shock protein 90 (Hsp90) cochaperone located in both the cytoplasm and nucleus that stabilizes unliganded steroid receptors, controls the catalytic activity of certain kinases, regulates protein-DNA dynamics, and is upregulated in several cancers. We had previously shown that p23-overexpressing MCF-7 cells (MCF-7+p23) exhibit increased invasion without affecting the estrogen-dependent proliferative response, which suggests that p23 differentially regulates genes controlling processes linked to breast tumor metastasis. To gain a comprehensive view of the effects of p23 on estrogen receptor (ER)-dependent and -independent gene expression, we profiled mRNA expression from control versus MCF-7+p23 cells in the absence and presence of estrogen. A number of p23-sensitive target genes involved in metastasis and drug resistance were identified. Most striking is that many of these genes are also misregulated in invasive breast cancers, including PMP22, ABCC3, AGR2, Sox3, TM4SF1, and p8 (NUPR1). Upregulation of the ATP-dependent transporter ABCC3 by p23 conferred resistance to the chemotherapeutic agents etoposide and doxorubicin in MCF-7+p23 cells. MCF-7+p23 cells also displayed higher levels of activated Akt and an expanded phosphoproteome relative to control cells, suggesting that elevated p23 also enhances cytoplasmic signaling pathways. For breast cancer patients, tumor stage together with high cytoplasmic p23 expression more accurately predicted disease recurrence and mortality than did stage alone. High nuclear p23 was found to be associated with high cytoplasmic p23, therefore both may promote tumor progression and poor prognosis by increasing metastatic potential and drug resistance in breast cancer patients. Cancer Res; 70(21); 8446–56. ©2010 AACR.

Supplementary Figure 2 - PDF file 19K, S2. Plating efficiency of MB-435 cells under hypoxia on plastic or vitronectin with beta3 silencing

Supplementary Figures 1-8, Tables 1-7 from High Levels of Hsp90 Cochaperone p23 Promote Tumor Progression and Poor Prognosis in Breast Cancer by Increasing Lymph Node Metastases and Drug Resistance

Supplementary Figure Legends - PDF file 59K, Figure legends for Sup. Figures 1 and 2

Supplementary Figure 1 - PDF file 20K, S1. Plating efficiency of MB-435 and MB-231 cells on different ECM matrices

GGGGCC (G4C2) repeat expansion in the first intron of C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-containing RNA is translated into dipeptide repeat (DPR) proteins, some of which are neurotoxic. Using dynamic ribosome profiling, we identified three translation initiation sites in the intron upstream of (G4C2) repeats; these sites are detected irrespective of the presence or absence of the repeats. During translocation, ribosomes appear to be stalled on the repeats. An AUG in the preceding C9ORF72 exon initiates a uORF that inhibits downstream translation. Polysome isolation indicates that unspliced (G4C2) repeat-containing RNA is a substrate for DPR protein synthesis. (G4C2) repeat-containing RNA translation is 5′ cap-independent but inhibited by the initiation factor DAP5, suggesting an interplay with uORF function. These results define novel translational mechanisms of expanded (G4C2) repeat-containing RNA in disease.

Background: Cross-talk between sterol metabolism and inflammatory pathways has been demonstrated to significantly affect the development of atherosclerosis. Cholesterol biosynthetic intermediates and derivatives are increasingly recognized as key immune regulators of macrophages in response to innate immune activation and lipid overloading. 25-Hydroxycholesterol (25-HC) is produced as an oxidation product of cholesterol by the enzyme cholesterol 25-hydroxylase (CH25H) and belongs to a family of bioactive cholesterol derivatives produced by cells in response to fluctuating cholesterol levels and immune activation. Despite the major role of 25-HC as a mediator of innate and adaptive immune responses, its contribution during the progression of atherosclerosis remains unclear. Methods: The levels of 25-HC were analyzed by liquid chromatography-mass spectrometry, and the expression of CH25H in different macrophage populations of human or mouse atherosclerotic plaques, respectively. The effect of CH25H on atherosclerosis progression was analyzed by bone marrow adoptive transfer of cells from wild-type or Ch25h –/– mice to lethally irradiated Ldlr –/– mice, followed by a Western diet feeding for 12 weeks. Lipidomic, transcriptomic analysis and effects on macrophage function and signaling were analyzed in vitro from lipid-loaded macrophage isolated from Ldlr –/– or Ch25h–/–;Ldlr–/– mice . The contribution of secreted 25-HC to fibrous cap formation was analyzed using a smooth muscle cell lineage–tracing mouse model, Myh11 ERT2CRE mT/mG;Ldlr –/– , adoptively transferred with wild-type or Ch25h –/– mice bone marrow followed by 12 weeks of Western diet feeding. Results: We found that 25-HC accumulated in human coronary atherosclerotic lesions and that macrophage-derived 25-HC accelerated atherosclerosis progression, promoting plaque instability through autocrine and paracrine actions. 25-HC amplified the inflammatory response of lipid-loaded macrophages and inhibited the migration of smooth muscle cells within the plaque. 25-HC intensified inflammatory responses of lipid-laden macrophages by modifying the pool of accessible cholesterol in the plasma membrane, which altered Toll-like receptor 4 signaling, promoted nuclear factor-κB–mediated proinflammatory gene expression, and increased apoptosis susceptibility. These effects were independent of 25-HC–mediated modulation of liver X receptor or SREBP (sterol regulatory element–binding protein) transcriptional activity. Conclusions: Production of 25-HC by activated macrophages amplifies their inflammatory phenotype, thus promoting atherogenesis.

Co-opting host cell protein synthesis is a hallmark of many virus infections. In response, certain host defense proteins limit mRNA translation globally, albeit at the cost of the host cell's own protein synthesis. Here, we describe an interferon-stimulated helicase, DDX60, that decreases translation from viral internal ribosome entry sites (IRESs). DDX60 acts selectively on type II IRESs of encephalomyocarditis virus (EMCV) and foot and mouth disease virus (FMDV), but not by other IRES types or by 5' cap. Correspondingly, DDX60 reduces EMCV and FMDV (type II IRES) replication, but not that of poliovirus or bovine enterovirus 1 (BEV-1; type I IRES). Furthermore, replacing the IRES of poliovirus with a type II IRES is sufficient for DDX60 to inhibit viral replication. Finally, DDX60 selectively modulates the amount of translating ribosomes on viral and in vitro transcribed type II IRES mRNAs, but not 5' capped mRNA. Our study identifies a novel facet in the repertoire of interferon-stimulated effector genes, the selective downregulation of translation from viral type II IRES elements.