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This retrospective case series study, using data obtained through questionnaires and histopathological diagnoses from 656 patients enrolled in the Department of Defense (DoD) Clinical Breast Care Project (CBCP), evaluated associations between hormonal contraceptive use and breast cancer pathology including benign breast pathologies. Three combination hormonal contraceptive agents (COCs) Lo Ovral (LO), Ortho Novum (ON), and Ortho Tri-Cyclen (OTC) were evaluated as they represented the most commonly used hormonal contraceptives in our cohort. The results of this study suggest that the ever use of LO + ON + OTC does not influence the overall incidence of benign breast condition or malignant disease compared to other COCs; however, patients that have used OTC had an association with a diagnosis of benign or luminal A pathologies whereas ON was associated with a diagnosis of benign and DCIS; LO showed no association with any diagnosis-benign or malignant. Patients that have used LO or ON were more likely to be diagnosed with breast cancer at age ≥ 40 years whereas patients that had ever used OTC were likely to be diagnosed before the age of 40. Caucasians were less likely to have used OTC and more likely to have used ON; however, use of either hormonal agent positively correlated with premenopausal status at diagnosis and having a benign condition. Age at diagnosis, ethnicity, BMI, family history, menstruation status, and duration of use were all independent predictors of different histopathological subtypes. We conclude that patient-specific variables should be considered when deciding on which type of hormonal contraceptive to use to minimize the risk of developing breast cancer or a breast-related pathology.

This abstract was not presented at the symposium.

Enhanced invasion and migration into the surrounding tissues are hallmarks of the malignancy of tumor cells. To successfully metastasize, a cancer cell has to detach from the primary tumor, invade into surrounding tissues, and intravasate into blood or lymphatic vessels. These processes are composed of complex mechanisms involving tumor recognition, degradation of extracellular matrix (ECM) proteins and migration into tissue. Triple negative (TN) breast cancers are defined by a lack of expression of estrogen, progesterone, and HER2 receptors. It is widely recognized that TN breast cancers have a poorer prognosis than any other subtype of breast cancer. Given the lack of effective targeted therapies for TN breast cancer patients, understanding of the mechanisms of migration and invasion of these tumors will provide insight into developing novel approaches to lower the mortality from TN breast cancer. Previous studies demonstrated that NEDD9 plays a key role facilitating progression and metastasis of various tumor cells including breast. We previously demonstrated that NEDD9 plays a critical role in promoting migration and growth of MDA-MB-231. In order to further characterize the mechanisms of NEDD9-mediated cancer migration and growth, we established stable cell lines expressing NEDD9 using HCC38 as a parental cell line which expresses low level of endogenous NEDD9. Microarray studies demonstrated that enzymes (CHST11, CHST15, and CSGALNACT1) involved in biosynthesis of chondroitin sulfate (CS) but not heparan sulfate (HS) were markedly upregulated in HCC38(NEDD9) compared to control HCC38(Vector) cells. These results suggest that NEDD9 regulates specific structures of tumor-associated glycans such as chondroitin sulfate. Core proteins of CD44 and Serglycin were markedly upregulated in HCC38(NEDD9) cells compared to HCC38(Vector) cells, while those of Syndecan-1, Syndecan-2, and Versican were downregulated in HCC38(NEDD9). Immunofluorescence studies using specific antibody, GD3G7, confirmed the enhanced expression of CS-E subunit in HCC38(NEDD9). Immunoprecipitation and western blotting analysis demonstrated that CS-E was attached to Serglycin and CD44 core proteins. We demonstrated that removing CS by chondroitinase ABC significantly inhibited anchorage-independent growth of HCC38(NEDD9) in methylcellulose. Importantly, the fact that GD3G7 significantly inhibited colony formation of HCC38(NEDD9) cells suggest that CS-E subunit plays a key role in this process. Furthermore, treatment of HCC38(NEDD9) cells with chondroitinase ABC or GD3G7 significantly inhibited mammosphere formation. Exogenous addition of CS-E enhanced colony formation and mammosphere formation of HCC38 parental and HCC38(Vector) cells. These results suggest that NEDD9 regulates the synthesis and expression of tumor associated glycocalyx structures including CS-E, which plays a key role in promoting and regulating breast cancer progression metastasis and possibly stem cell phenotypes. The opinion and assertions contained herein are the private views of the authors and are not to be construed as official or as representing the views of the Department of the Army or the Department of Defense. Citation Format: Iida J, Dorchak J, Clancy R, Slavik J, Cutler ML, Shriver CD. Tumor-associated glycans as key molecules to promote growth of triple-negative breast cancer cells. [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 P2-05-16.

Molecular heterogeneity in human breast cancer has challenged diagnosis, prognosis, and clinical treatment. It is well known that molecular subtypes of breast tumors are associated with significant differences in prognosis and survival. Assuming that the differences are attributed to subtype-specific pathways, we then suspect that there might be gene regulatory mechanisms that modulate the behavior of the pathways and their interactions. In this study, we proposed an integrated methodology, including machine learning and information theory, to explore the mechanisms. Using existing data from three large cohorts of human breast cancer populations, we have identified an ensemble of 16 master regulator genes (or MR16) that can discriminate breast tumor samples into four major subtypes. Evidence from gene expression across the three cohorts has consistently indicated that the MR16 can be divided into two groups that demonstrate subtype-specific gene expression patterns. For example, group 1 MRs, including ESR1, FOXA1, and GATA3, are overexpressed in luminal A and luminal B subtypes, but lowly expressed in HER2-enriched and basal-like subtypes. In contrast, group 2 MRs, including FOXM1, EZH2, MYBL2, and ZNF695, display an opposite pattern. Furthermore, evidence from mutual information modeling has congruently indicated that the two groups of MRs either up- or down-regulate cancer driver-related genes in opposite directions. Furthermore, integration of somatic mutations with pathway changes leads to identification of canonical genomic alternations in a subtype-specific fashion. Taken together, these studies have implicated a gene regulatory program for breast tumor progression.

Background: Given the lack of effective targeted therapies for triple-negative (TN) breast cancer patients, a better understanding of the mechanisms of growth and invasion of these tumors will provide valuable insight into developing novel approaches to lower the mortality associated with TN breast cancer. There is substantial evidence indicating that the canonical Wnt signaling pathway is activated in TN breast cancer tissues. Previous studies report that FH535, a small molecule inhibitor of the Wnt signaling pathway, decreases growth of cancer cells but not normal fibroblast cells, suggesting this pathway plays a role in tumor progression and metastasis. In this study, we test FH535 as a potential inhibitor of migration, invasion, and growth against malignant phenotypes of breast cancer. Methods and Results: We show that FH535 significantly inhibits growth, migration, and invasion of TN breast cancer cells (MDA-MB-231 and HCC38) using established protocols, implicating the Wnt signaling pathway plays a role in tumor progression and metastasis. FH535 is a potent growth inhibitor for TN breast cancer cells (MDA-MB-231 and HCC38), but not in other breast cancer cells (MCF-7, T-47D, or SK-BR-3) when cultured in three dimensional type I collagen gel. Additionally, western blotting analysis shows that treatment of MDA-MB-231 cells with FH535 markedly inhibits the expression of NEDD9, but not FAK, Src, or p130Cas, suggesting specificity of the Wnt signaling pathway in regulating gene expressions. Co-immunoprecipitation studies suggest that NEDD9 is specifically immunoprecipitated with CSPG4, but not with β1 integrin or CD44, implying CSPG4 and NEDD9 form a molecular complex for facilitating tumor migration, invasion, and growth. Statistical analysis (ANOVA) of The Cancer Genome Atlas (TCGA) project gene expression data (https://tcga-data.nci.nih.gov/) suggests that tumors from patients with basal-type breast cancer had significantly higher CSPG4 expression than tumors from patients with other subtypes, with all the p values Discussion: In this study, we demonstrate the pivotal role of the canonical Wnt signaling pathway in enhancing tumor migration, invasion, and growth of TN breast cancer cells MDA-MB-231 and HCC38. We identified NEDD9 as a potential downstream target of the Wnt signaling pathway and found an association between NEDD9 and CSPG4 in MDA-MB-231 cells. Our results suggest a novel mechanism of migration, invasion, and growth of tumor cells involving a molecular complex between CSPG4 and NEDD9, which could be drug targets in TN and possibly Luminal A breast cancer patients. More comprehensive characterization of canonical Wnt signaling pathway by genomic and biological studies of breast cancer cells are clearly required for generating better treatments. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-09.

Background Previous studies suggest that certain transition metal complexes, such as cisplatin, are efficacious for treating various cancer types, including ovarian, lung, and breast. Methods In order to further evaluate ruthenium (Ru) complexes as potential anti-cancer agents, we synthesized and evaluated Ru-arene complexes. Two complexes with the general formula [Ru (η6-p-cym) (N–N) Cl]+ were tested for their abilities to inhibit cancer cells. Results The complex with o-phenylenediamine as the N–N ligand (o-PDA) significantly inhibited growth of breast (MDA-MB-231, MCF-7, SKBR-3, and SUM149), lymphoma (Raji), melanoma (Bowes), and osteosarcoma (HT1080); however, the complex with o-benzoquinonediimine (o-BQDI) was ineffective except for SUM149. In contrast, o-PDA failed to inhibit growth of human breast epithelial cells, MCF-10A. Treatment of MDA-MBA-231 cells with o-PDA resulted in a significant reduction of productions of PDGF-AA, GM-CSF, and VEGF-A proteins at the transcriptional levels. Finally, we demonstrated that o-PDA synergistically inhibited MDA-MB-231 cell growth with cyclophosphamide but not doxorubicin or paclitaxel. Conclusion These results suggest that Ru-arene complexes are promising anti-cancer drugs that inhibit progression and metastasis by blocking multiple processes for breast and other types of cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0797-9) contains supplementary material, which is available to authorized users.

Triple negative (TN) breast cancers are defined by a lack of expression of estrogen, progesterone, and her-2/neu receptors. It is widely recognized that TN breast cancers have a poorer prognosis than other subtypes of breast caner. Given the lack of effective targeted therapies for TN breast cancer patients, understanding of the mechanisms of growth and invasion of these tumors will provide insight into developing novel approaches to lower the mortality from TN breast cancer. We utilized three TN breast cancer cell lines (HCC38, HCC1806, HCC1937) as model systems to characterize mechanisms of growth, migration, and invasion. FACS analyses demonstrated that these TN cell lines showed a phenotype of CD44+CD24-/lowepithelial specific antigen (ESA)+. An inhibitory antibody against exon v10 of CD44 significantly inhibited three-dimensional (3D) growth of tumor cells. Importantly, this antibody also inhibited alpha2beta1/alpha3beta1 integrins-mediated migration and invasion into matrigel. These results suggest that CD44 harboring exon 10 facilitates progression and metastasis of TN breast cancer cells in vivo by enhancing growth, migration, and invasion. We also demonstrated that this exon interacts with a cell surface tyrosine kinase, EphA2. Thus, we hypothesize that CD44 would form a molecular complex with EphA2 through exon v10 on TN breast cancer cells that plays a key role in facilitating invasion and metastasis. In order to develop novel reagents to inhibit TN breast cancer invasion and metastasis, we have utilized SELEX (systematic evolution of ligands by exponential enrichment) to isolate DNA aptamers that specifically recognize exon v10 of CD44. DNA aptamers are functional molecules with the appropriate sequence and structure to form a complex with a target molecule. Typically, dissociation constants for these aptamer-target complexes are in the high pico-molar to low nano-molar range, which is comparable to antibodies. Given the fact of the ease of chemical synthesis and modifications of oligonucleotides, DNA aptamers that recognize cancer cells will be easier to develop and have a great impact for the next generation of strategies for cancer detection, diagnosis, and therapy. We describe the development of “function-blocking” DNA aptamers specific for exon v10 of CD44 that inhibits TN breast cancer invasion and metastasis. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-08.

Introduction Hyaluronan (HA) and its biosynthetic enzymes hyaluronan synthases (HAS2 and HAS3) mediate Matrigel invasion by SW620 colon carcinoma cells. Because matrix metalloproteinases (MMPs) have been implicated in cancer invasion, we hypothesized that changes in HAS expression would alter MMP expression and activity in these cells. Methods To determine whether an MMP was involved in invasion, Matrigel invasion assays with SW620 cells were performed in the presence or absence of the MMP inhibitors GM6001 or TIMP2. HAS isozymes were inhibited by stably transfecting SW620 cells with vectors that contained antisense HAS2 and/or -3 cDNA; transfection with an empty vector served as a control. MMP-7 transcription was assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR). MMP-7 protein was detected by enzyme-linked immunosorbent assay (ELISA) and enzymatic activity compared using zymography. Results GM6001 and TIMP2 decreased Matrigel invasion, which confirms that an MMP played a key role in this process. MMP-7 expression was then detected in SW620 cells. Finally, MMP-7 expression, protein, and enzymatic activity were significantly lower in antisense HAS tranfectants than in SW620 or vector control cells. Conclusion We have demonstrated previously that inhibition of HAS expression and HA production in SW620 colon carcinoma cells inhibits Matrigel invasion. In the studies presented here, we have demonstrated that SW620 cells express high levels of MMP-7 and that inhibition of HAS isozymes dramatically decreases MMP-7 expression, protein, and enzymatic activity. Taken together, these findings suggest that HAS and HA may mediate cellular invasion via changes in MMP-7 expression.

Tumor cell invasion through basement membranes and into stromal tissue are key steps for promoting growth and metastasis. Tumor cells express various extracellular-matrix-degrading enzymes such as matrix metalloproteinases (MMPs) to degrade extracellular matrix components to facilitate tumor migration and invasion. Histological and clinical studies suggest a role for MMP-1 (collagenase-1) in malignant melanoma invasion. In this study, we evaluated MMP-1 in regulating malignant phenotypes of human melanoma cells by generating human melanoma cells stably transfected with pro-MMP-1 cDNA. The transfectants expressed the active form of MMP-1 associated with cells and showed enhanced invasive and growth abilities in type I collagen gel. Furthermore, MMP-1 expression promoted anchorage-independent growth, which was inhibited in the presence of type II transforming growth factor (TGF)-beta receptor:Fc fusion protein that scavenges TGF-beta receptors. Finally, we demonstrated that MMP-1 directly generated active TGF-beta from its latent form. Thus, these results suggest that MMP-1 produced from melanoma cells would play a role in tumor progression by both degrading matrix proteins and generating active growth factors such as TGF-beta in vivo.

Natural killer (NK) cells play a key role in inflammation and tumor regression through their ability to migrate into tissues. CXCL12 is a chemokine that promotes lymphocyte invasion and migration into tissues; however, the mechanism for this process remains incompletely understood. In this study, we show that CXCL12 significantly enhanced CD16(+)CD56(+) human peripheral NK-cell invasion into type I collagen by the catalytic activity of matrix metalloproteinase-1 (MMP-1). Confocal immunofluorescence and co-immunoprecipitation studies suggest that MMP-1 colocalized with alpha(2)beta(1) integrin on CXCL-12-stimulated NK-cell surface. The binding of pro-MMP-1 with alpha(2)beta(1) integrin required activation of G(i)-coupled pathway. However, the production of MMP-1 from CXCL12-stimulated NK cells was mediated by p38 and mitogen-activated or extracellular signal-regulation protein kinase kinase 1/2 in a manner independent of the G(i)-coupled pathway. These results suggest that CXCL12/CXCR4 interaction transduces the two signaling pathways to promote NK-cell invasion, which stimulates pericellular degradation of extracellular matrix proteins by membrane-associated MMP-1. The mechanisms would thus play a role in facilitating lymphocyte trafficking and accumulation in tissues during physiological and pathological processes.

Background Hyaluronan (HA) is a cell-surface glycosaminoglycan that has been implicated in cancer progression. Cells isolated from metastatic colon carcinoma (SW620) produce greater amounts of pericellular HA than cells isolated from a primary tumor (SW480). Inhibition of hyaluronan synthases (HAS) by transfection with antisense cDNA decreases HA production. Because adhesion to the extracellular matrix (ECM) is required for invasion and metastasis, we hypothesized that pericellular HA mediates adhesion to ECM proteins such as laminin, collagen, and fibronectin and that inhibition of HA production or removal of HA by digestion with hyaluronidase would impair adhesion. Materials and methods SW480, SW620, and antisense transfectants (SW620 cells transfected with vector alone, antisense HAS2, antisense HAS3, and both antisense HAS2 and HAS3) were assessed for adhesion to laminin, Type 1 collagen, or fibronectin-coated plates. To confirm that adhesion was mediated by HA, cells were treated with or without hyaluronidase prior to the assays. Results Metastatic SW620 cells adhered well to laminin; SW480 cells demonstrated 46% less adhesion ( P t test). SW620 cell adhesion to Type 1 collagen and fibronectin was >50% less than adhesion to laminin. Inhibition of HAS2 and/or HAS3 or pretreatment with hyaluronidase significantly decreased adhesion of SW620 cells to laminin ( P Conclusions Metastatic SW620 cells that produce large amounts of pericellular HA adhered well to laminin. Inhibition of HAS2 and/or HAS3 expression, or hyaluronidase digestion of pericellular HA significantly inhibited adhesion. These data suggest that HA promotes adhesion to laminin and may thereby facilitate invasion of the basement membrane and metastasis in colon carcinoma.

Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal–regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with α4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.

Background: Heterogeneity between different breast cancer tumors plays an important role in patients’ survival outcomes, and such tumor heterogeneity unveiled by transcriptome and proteome are often in disagreement. We hypothesize that alternative splicing of mRNA could explain heterogeneity within intrinsic breast cancer subtypes. Methods: Cases used in this study were drawn from the Clinical Breast Care Project where breast cancer patients were consented using an IRB-approved protocol. Fifty breast tumors were selected and processed by laser microdissection. RNA and protein were extracted from tissues using the Illustra triplePrep kit (GE Healthcare). Paired-end mRNA sequencing was performed using the Illumina HiSeq platform. Paired-end reads were preprocessed using PRINSEQ and splice-aligned to the genome using GSNAP software. Gene counts were measured using HTSeq while Exon counts used the DEXSeq. Differential expression was called at 10% false discovery rate. Proteome Discoverer with Byonic node was used for analyzing the quantitative global proteomics dataset, and we were able to quantitate 8600 proteins. All other analyses were performed using Perl and R. Results: The number of sequencing reads for the 50 cases ranged from 60 million to 410 million reads. After preprocessing, an average of 93% of reads was mapped, and 36,700 genes were identified among 50 tumor samples. PAM50 algorithm was used for intrinsic subtype calls, yielding 16 Basal, 8 Her2+, 19 Luminal A (LA) and 7 Luminal B tumors. Unsupervised clustering of the 8 Her2+ cases using the PAM50 genes and highly varying proteins resulted in different clustering patterns, with the latter clustering 6 of the 8 Her2+ cases with two distinct 3-case sub-clusters. Between these two sub-clusters, there were 10 differentially expressed (DEX) genes and 25 DEX proteins, but none of the 25 proteins were mapped to the 10 DEX genes. This motivated us to investigate the DEX exons, and we found 7,076 DEX exons between the Her2+ sub-clusters, and 9 of the 25 DEX proteins were matched to genes bearing DEX exons. For comparison, we performed the same analyses between similarly clustered Basal and LA sub-clusters. Even though the number of DEX genes between Basal (8) and LA (18) sub-clusters were comparable to that between the Her2+ sub-clusters, DEX exons were much lower for both subtypes (616 & 157), and none of the DEX proteins (3 & 7) mapped to DEX exons or genes for either subtype. Conclusions: Our findings imply that there is more proteomic-level heterogeneity in the Her2+ subtype than in Basal and LA subtypes, which could be due to alternative usage of exons (alternative splicing). If such heterogeneity is associated with patients’ survival outcomes then our results will further stress the importance of alternative splicing in breast cancer. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Format: Praveen Kumar Raj Kumar, Tao Liu, Lori A. Sturtz, Albert Kovatich, Marina A. Gritsenko, Vladislav A. Petyuk, Brenda Deyarmin, Viswanadham Sridhara, James Craig, Jason E. McDermott, Anil K. Shukla, Ronald J. Moore, Matthew E. Monroe, Bobbie-Jo M. Webb-Robertson, Jeffrey A. Hooke, Leigh Fantacone-Campbell, Leonid Kvecher, Jianfang Liu, Jennifer Kane, Jennifer Melley, Stella Somiari, Joji Iida, Stephen C. Benz, Justin Golovato, Shahrooz Rabizadeh, Patrick Soon-Shiong, Richard D. Smith, Richard J. Mural, Karin D. Rodland, Craig D. Shriver, Hai Hu. Differential exon usage in the HER2 subtype of breast cancer identified with RNA-seq and proteomic data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3372. doi:10.1158/1538-7445.AM2017-3372

Introduction: Molecular characteristics of breast tumors play an important role in determining patients’ survival outcome. Here, we report preliminary findings of proteogenomic profiling of 50 breast tumors using RNA-Seq and mass-spectrometry (MS) based proteomic technologies. An additional 60 tumors are being analyzed, including WGS for all samples. We are also collecting patient survival outcome data. Methods: Cases used in this study were drawn from the Clinical Breast Care Project, where patients were consented using an IRB-approved protocol. A total of 50 breast tumors were selected and processed by laser capture microdissection (LCM). This cohort includes 36 Caucasian Americans (CA) and 8 African Americans (AA), and the age of the patients is 57 ± 13 years. Protein and RNA were extracted using the Illustra triplePrep kit, which isolates DNA from the same cells as well. Quantitative global proteomics and phosphoproteomics analyses were performed using isobaric TMT 6-plex labeling with the “universal reference” strategy and IMAC enrichment of phosphopeptides. Mass spectrometry data were acquired using a Q-Exactive instrument and analyzed using Proteome Discoverer with Byonic node. Phosphopeptide abundance was normalized to abundance measurements of the parent protein for all of the phosphorylation analyses. Phosphoproteomic data was also searched for the presence of O-GlcNAc modifications. RNA-Seq analyses were done on Illumina HiSeq and the data were analyzed using GSNAP. Results: There were 19 Luminal A, 7 Luminal B, 8 HER2-enriched, and 16 basal-like subtypes based on the PAM50 algorithm. In the global proteomics data, we were able to quantitate >8600 proteins. Unsupervised clustering on the highly varying proteins across the samples resulted in two primary clusters, with one being luminal-enriched. The other cluster contains a basal-like tumor sub-cluster and a sub-cluster of mixed subtypes. Differential protein expression analyses between the two primary clusters confirmed known markers (e.g., overexpression of KRT8/KRT18 in luminal-enriched cluster). The luminal-enriched cluster is primarily CA with post-menopausal status. A similar search of the phosphoproteomic data yielded quantitation of >12500 phosphopeptides. Unsupervised clustering of the phosphoproteins resulted in four primary groups, with one being basal-enriched and another being luminal-enriched. We also observed >50 overexpressed phosphopeptides. While some of these phosphosites have been previously reported (e.g., on RANBP2), other phosphosites appeared to be novel (e.g., on IRF2BP2). Conclusion: Analysis of LCM breast tumors using proteogenomic technologies resulted in basal- and luminal-enriched clusters, thus enabling us to study protein and phosphopeptide markers across multiple platforms. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Format: Viswanadham Sridhara, Tao Liu, Marina A. Gritsenko, Lori A. Sturtz, Albert J. Kovatich, Vladislav A. Petyuk, Brenda Deyarmin, Jason E. McDermott, Anil K. Shukla, Ronald J. Moore, Matthew E. Monroe, Bobbie-Jo M. Webb-Robertson, Jeffrey A. Hooke, Leigh Fantacone-Campbell, Praveen Kumar Raj Kumar, Leonid Kvecher, Jianfang Liu, Jennifer Kane, Jennifer Melley, Stella Somiari, Joji Iida, Stephen C. Benz, Justin Golovato, Shahrooz Rabizadeh, Patrick Soon-Shiong, Richard D. Smith, Richard J. Mural, Craig D. Shriver, Hai Hu, Karin D. Rodland. Integrated proteogenomic analysis of laser capture microdissected breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 213. doi:10.1158/1538-7445.AM2017-213

NEDD9 has been characterized as a metastasis-promoting gene in various cancer cells including breast. We previously reported that NEDD9 promotes malignant phenotypes of breast cancer cells through distinct and non-overlapped domains. For example, the FAT (Focal Adhesion Targeting) domain of NEDD9 promotes cancer cell growth, while the SH-domain facilitates cell migration. These results suggest that NEDD9 promotes tumor metastasis by enhancing dissemination and growth in the tumor-host microenvironments through distinct and non-overlapped domains. Thus, targeting functions of NEDD9 is a promising approach for breast cancer therapies. In order to further characterize NEDD9-mediated breast cancer growth, we performed yeast-two hybrid (Y2H) screening to identify proteins that associate with the FAT domain of NEDD9. Using the FAT domain constructed in pGBKKT7 (Clonetech, CA) as a bait to screen library of human fibroblast (Clonetech, CA), we identified several proteins that associate with the domain. They are small GTPases (i.e. RAB11a and ARF4), cytoskeletal proteins (i.e. Nexilin), and cytosolic proteins (i.e. HAX-1). Among of these potential partner proteins, we focused on the interaction between NEDD9 and HAX-1 in breast cancer cells. Co-immunoprecipitation assays confirm the molecular complex of NEDD9-HAX-1 in both SK-Br3 and SUM149 cells. Importantly, p130cas, which harbors similar domain structures with NEDD9, was not precipitated with NEDD9, suggesting a specific interaction between NEDD9 and HAX-1. Given the fact of NEDD9 as a key metastasis promoting gene, these results suggest that NEDD9-HAX-1 plays a key role breast cancer metastasis by facilitating growth in microenvironments. While the biological function are not clear at present, previous studies demonstrated that HAX-1 localizes in mitochondria in breast cancer cells, Indeed, we demonstrated that NEDD9 was found in both cytosol and mitochondria fractions in malignant breast cancer cell MDA-MB-231, but not non-metastatic HCC38. These results suggest the presence of NEDD9-HAX1 complex in mitochondria and this complex may facilitate breast cancer metastasis. In addition to HAX-1, several mitochondrial proteins such as EFG1, DCTN6, and MMADHC were found in the Y2H screening system as described above. These results suggest that NEDD9 facilitates breast cancer metastasis through regulating multiple pathways including signaling pathways and mitochondrial functions, thus serving as a promising therapeutic target for cancer patients including breast. The view expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S.Government. Citation Format: Iida J, Dorchak J, Slavik J, Clancy R, Cutler ML, Shriver CD. NEDD9 promotes breast cancer metastasis by regulating mitochondrial functions [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-05-02.

Item does not contain fulltext There are lines of evidence demonstrating that NEDD9 (Cas-L, HEF-1) plays a key role in the development, progression, and metastasis of breast cancer cells. We previously reported that NEDD9 plays a critical role for promoting migration and growth of MDA-MB-231. In order to further characterize the mechanisms of NEDD9-mediated cancer migration and growth, stable cells overexpressing NEDD9 were generated using HCC38 as a parental cell line which expresses low level of endogenous NEDD9. Microarray studies demonstrated that core proteins of CD44 and Serglycin were markedly upregulated in HCC38(NEDD9) cells compared to HCC38(Vector) cells, while those of Syndecan-1, Syndecan-2, and Versican were downregulated in HCC38(NEDD9). Importantly, enzymes generating chondroitin sulfate glycosaminoglycans (CS) such as CHST11, CHST15, and CSGALNACT1 were upregulated in HCC38(NEDD9) compared to HCC38(Vector). Immunofluorescence studies using specific antibody, GD3G7, confirmed the enhanced expression of CS-E subunit in HCC38(NEDD9). Immunoprecipitation and western blotting analysis demonstrated that CS-E was attached to CD44 core protein. We demonstrated that removing CS by chondroitinase ABC significantly inhibited anchorage-independent colony formation of HCC38(NEDD9) in methylcellulose. Importantly, the fact that GD3G7 significantly inhibited colony formation of HCC38(NEDD9) cells suggests that CS-E subunit plays a key role in this process. Furthermore, treatment of HCC38(NEDD9) cells with chondroitinase ABC or GD3G7 significantly inhibited mammosphere formation. Exogenous addition of CS-E enhanced colony formation and mammosphere formation of HCC38 parental and HCC38(Vector) cells. These results suggest that NEDD9 regulates the synthesis and expression of tumor associated glycocalyx structures including CS-E, which plays a key role in promoting and regulating breast cancer progression and metastasis and possibly stem cell phenotypes.

Tumor cell adhesion and proteolysis of the extracellular matrix proteins surrounding the cells are tightly linked processes in tumor invasion. In this study, we sought to identify components of the cell surface of a vertical growth phase melanoma cell line, WM1341D, that mediate invasive cellular behavior. We determined by antisense inhibition that melanoma chondroitin sulfate proteoglycan (MCSP) and membrane-type 3 matrix metalloproteinase (MT3-MMP) expressed on WM1341D are required for invasion of type I collagen and degradation of type I gelatin. MT3-MMP co-immunoprecipitated with MCSP in WM1341D melanoma cells cultured on type I collagen or laminin. The association between MT3-MMP and MCSP was largely disrupted by removing chondroitin sulfate glycosaminoglycan (CS) from the cell surface, suggesting CS could mediate the association between the two cell surface core proteins. Recombinant MT3-MMP and MT3-MMP from whole cell lysates of WM1341D cells were specifically eluted from CS- conjugated affinity columns. The results indicate that MT3-MMP possesses the potential to promote melanoma invasion and proteolysis and that the formation of a complex between MT3-MMP and MCSP may be a crucial step in activating these processes.

Melanoma chondroitin sulphate proteoglycan (MCSP) is a cell-surface antigen that has been implicated in the growth and invasion of melanoma tumours. Although this antigen is expressed early in melanoma progression, its biological function is unknown. MCSP can stimulate the integrin-alpha4 beta1-mediated adhesion and spreading of melanoma cells. Here we show that stimulated MCSP recruits tyrosine-phosphorylated p130 cas, an adaptor protein important in tumour cell motility and invasion. MCSP stimulation also results in a pronounced activation and recruitment of the Rho-family GTPase Cdc42. MCSP-induced spreading of melanoma cells is dependent upon active Cdc42, a Cdc42-associated tyrosine kinase (Ack-1) and tyrosine phosphorylation of p130cas. Furthermore, vectors inhibiting Ack-1 or Cdc42 expression and/or function abrogate MCSP-induced tyrosine phosphorylation and recruitment of p130cas. Our findings indicate that MCSP may modify tumour growth or invasion by a unique signal-transduction pathway that links Cdc42 activation to downstream tyrosine phosphorylation and subsequent cytoskeletal reorganization.

CD44 adhesion molecules are expressed in many breast cancer cells and have been demonstrated to play a key role in regulating malignant phenotypes such as growth, migration, and invasion. CD44 is an integral transmembrane protein encoded by a single 20-exon gene. The diversity of the biological functions of CD44 is the result of the various splicing variants of these exons. Previous studies suggest that exon v10 of CD44 plays a key role in promoting cancer invasion and metastasis, however, the molecular mechanisms are not clear. Given the fact that exon v10 is in the ectodomain of CD44, we hypothesized that CD44 forms a molecular complex with other cell surface molecules through exon v10 in order to promote migration of breast cancer cells. In order to test this hypothesis, we selected DNA aptamers that specifically bound to CD44 exon v10 using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). We selected aptamers that inhibited migration of breast cancer cells. Co-immunoprecipitation studies demonstrated that EphA2 was co-precipitated with CD44. Pull-down studies demonstrated that recombinant CD44 exon v10 bound to EphA2 and more importantly aptamers that inhibited migration also prevented the binding of EphA2 to exon v10. These results suggest that CD44 forms a molecular complex with EphA2 on the breast cancer cell surface and this complex plays a key role in enhancing breast cancer migration. These results provide insight not only for characterizing mechanisms of breast cancer migration but also for developing target-specific therapy for breast cancers and possibly other cancer types expressing CD44 exon v10.

Tumor cell invasion and metastasis is highly dependent on dynamic changes in the adhesion and migration of transformed and malignant cells. As with normal cell adhesion, the adhesion of tumor cells influences their cytoskeletal organization, activation of signal transduction pathways within the cell, and nuclear events leading to changes in mRNA transcription and protein synthesis. Furthermore, as tumor cells invade the circulation, they adhere to activated endothelial cells at sites within the vasculature during arrest and extravasation. Studies in the area of tumor cell adhesion and migration have demonstrated that the recognition of extracellular matrix ligands, or adhesion promoting ligands expressed on neighboring cells (i.e. counter-receptors), involves complex molecular recognition mechanisms. The complexity arises, in part, from the multiple recognition sites that are present within adhesion promoting ligands. Some of these structures within ECM components act by binding integrins, whereas others bind additional receptors such as cell surface proteoglycans. In this sense, adhesion promoting ligands may be considered as informational arrays, that function to modulate cell phenotype by engaging specific combinations of adhesion receptors on the cell surface. Understanding the mechanism(s) by which these receptor 'cluster' modify cell adhesion, motility and growth may lead to novel therapeutic strategies to control tumor cell invasion and metastasis formation. This review will highlight the role that cell surface chondroitin sulfate proteoglycans may play in modulating tumor cell adhesion, migration and invasion, with an emphasis on the relationship between cell surface chondroitin sulfate proteoglycans and integrins.

Cell adhesion to extracellular matrices is fundamental for maintaining normal tissue architecture and function. Many diseases are characterized, in part, by molecular changes in cell adhesion. These changes can occur as a result of modifications of the composition or integrity of the extracellular matrix or as a result of disease associated changes in the expression and/or function of adhesion receptors. Such alterations in cell adhesion can have profound effects on the phenotypic traits of cells, and as a result, these changes in cell adhesion can be of primary importance in facilitating disease-associated breakdown of normal tissue function. This is most apparent in a disease such as cancer, where neoplastic transformation can lead to alterations in tumor cell growth, changes in the composition or integrity of tissue proteins, tumor cell migration, invasion, and ultimately metastasis formation. Understanding the molecular basis of cell adhesion could lead to new insights into the molecular basis of many diseases, leading to better therapies. The purpose of this review is to highlight the role that collagens play in mediating cell adhesion, with an emphasis on the structural features of collagen important for cellular recognition and adhesion. Additionally, we briefly review the major receptors and intracellular signals that are important for cellular recognition of, and adhesion to, collagens.

There is substantial evidence indicating that the WNT signaling pathway is activated in various cancer cell types including breast cancer. Previous studies reported that FH535, a small molecule inhibitor of the WNT signaling pathway, decreased growth of cancer cells but not normal fibroblasts, suggesting this pathway plays a role in tumor progression and metastasis. In this study, we tested FH535 as a potential inhibitor for malignant phenotypes of breast cancer cells including migration, invasion, and growth. FH535 significantly inhibited growth, migration, and invasion of triple negative (TN) breast cancer cell lines (MDA-MB231 and HCC38) in vitro. We demonstrate that FH535 was a potent growth inhibitor for TN breast cancer cell lines (HCC38 and MDA-MB-231) but not for other, non-TN breast cancer cell lines (MCF-7, T47D or SK-Br3) when cultured in three dimensional (3D) type I collagen gels. Western blotting analyses suggest that treatment of MDA-MB-231 cells with FH535 markedly inhibited the expression of NEDD9 but not activations of FAK, Src, or downstream targets such as p38 and Erk1/2. We demonstrated that NEDD9 was specifically associated with CSPG4 but not with β1 integrin or CD44 in MDA-MB-231 cells. Analyses of gene expression profiles in breast cancer tissues suggest that CSPG4 expression is higher in Basal-type breast cancers, many of which are TN, than any other subtypes. These results suggest not only a mechanism for migration and invasion involving the canonical WNT-signaling pathways but also novel strategies for treating patients who develop TN breast cancer.

細胞膜プロテオグリカン(PG)は、増殖あるいは分化因子に対する細胞の反応更に細胞のECM上における接着移動において重要な働きをしている。細胞膜PGはECM成分として存在するPGと同様にコアタンパク質とそれに結合した一本或いは数本のグリコサミノグリカン鎖からなる。細胞膜PGの最も大きな特徴はPGとして細胞膜上に発現されたグリコサミノグリカン鎖の糖鎖構造と種々のコアタンパク質に基づくその構造の多様性である。本総説ではPGのECM成分への細胞接着における重要性をまとめ、かつ今まで明らかにされてきた細胞膜PGの構造と機能を述べ、更にECM成分であるファイブロネクチン (FN) への細胞接着におけるインテグリンの機能に言及する。現在、細胞膜PGがインテグリンの様な他の細胞接着レセプターと共同しECMへの細胞の接着を調節していることを示唆する多くの報告がある。FNは細胞膜PGとインテグリンに認識される多様の細胞認識部位がお互いに近傍に存在しているため、FNへの細胞接着においては、これらの細胞認識部位が共同して細胞膜上に細胞膜PGとインテグリンからなるレセプター群 (クラスター) を形成させると仮定される。このようなレセプター群は更に細胞の接着に必要とされるシグナルを形成する。この作業仮説はFN、或いは他のECM成分が細胞に与える情報を部分的に説明するための概念的基礎となり, 更に腫瘍細胞の侵潤と転移を初めとした多くの病態に関与する細胞型特異性を理解するために役立つと思われる。

Cellular recognition and adhesion to the extracellular matrix (ECM) has a complex molecular basis, involving both integrins and cell surface proteoglycans (PG). The current studies have used specific inhibitors of chondroitin sulfate proteoglycan (CSPG) synthesis along with anti-alpha 4 integrin subunit monoclonal antibodies to demonstrate that human melanoma cell adhesion to an A-chain derived, 33-kD carboxyl-terminal heparin binding fragment of human plasma fibronectin (FN) involves both cell surface CSPG and alpha 4 beta 1 integrin. A direct role for cell surface CSPG in mediating melanoma cell adhesion to this FN fragment was demonstrated by the identification of a cationic synthetic peptide, termed FN-C/H-III, within the fragment. FN-C/H-III is located close to the amino terminal end of the fragment, representing residues #1721-1736 of intact FN. FN-C/H-III binds CSPG directly, can inhibit CSPG binding to the fragment, and promotes melanoma cell adhesion by a CSPG-dependent, alpha 4 beta 1 integrin-independent mechanism. A scrambled version of FN-C/H-III does not inhibit CSPG binding or cell adhesion to the fragment or to FN-C/H-III, indicating that the primary sequence of FN-C/H-III is important for its biological properties. Previous studies have identified three other synthetic peptides from within this 33-kD FN fragment that promote cell adhesion by an arginyl-glycyl-aspartic acid (RGD) independent mechanism. Two of these synthetic peptides (FN-C/H-I and FN-C/H-II) bind heparin and promote cell adhesion, implicating cell surface PG in mediating cellular recognition of these two peptides. Additionally, a third synthetic peptide, CS1, is located in close proximity to FN-C/H-I and FN-C/H-II and it promotes cell adhesion by an alpha 4 beta 1 integrin-dependent mechanism. In contrast to FN-C/H-III, cellular recognition of these three peptides involved contributions from both CSPG and alpha 4 integrin subunits. Of particular importance are observations demonstrating that CS1-mediated melanoma cell adhesion could be inhibited by interfering with CSPG synthesis or expression. Since CS1 does not bind CSPG, the results suggest that CSPG may modify the function and/or activity of alpha 4 beta 1 integrin on the surface of human melanoma cells. Together, these results support a model in which the PG and integrin binding sites within the 33-kD fragment may act in concert to focus these two cell adhesion receptors into close proximity on the cell surface, thereby influencing initial cellular recognition events that contribute to melanoma cell adhesion on this fragment.

Enamel matrix derivative (EMD) protein has been clinically used for periodontal regeneration, but the molecular mechanisms are not clear. Previous studies suggested that the activation of phosphoinositide 3-kinase (PI 3-kinase) plays a key role in facilitating cell migration. Given that the migration of osteoblasts is one of the key steps in the wound-healing processes, we hypothesized that EMD protein would stimulate osteoblast migration by activating PI 3-kinase. In this study, we tested this hypothesis using MG-63 cells as model systems to evaluate mechanisms of migration by stimulation with EMD protein.Confluent MG-63 cells were mechanically scratched using a sterilized 1-mm pipette tip that removed the cells within a circular area. The wells were incubated for 24 hours in various stimulation conditions (25, 50, or 100 microg/ml EMD protein) with or without the PI 3-kinase inhibitor wortmannin (1, 10, and 100 nM) or LY294002 (1, 10, and 100 microM). Migrated cells in the wound section were counted by randomly selecting three areas from one well. The activation of PI 3-kinase by EMD protein was evaluated by the phosphorylation of Akt using Western blot analysis.Although EMD protein did not affect proliferation, it enhanced migration into wounds on MG-63 cells. We showed that EMD protein enhanced the phosphorylation of Akt in a dose-dependent manner. We demonstrated that the PI 3-kinase inhibitors wortmannin and LY294002 blocked migration into wounds and the phosphorylation of Akt enhanced by EMD protein in MG-63 cells.These results demonstrated that the activation of PI 3-kinase plays a key role in the EMD protein-stimulated migration of osteoblasts.

We previously reported that CS (chondroitin sulfate) GAG (glycosaminoglycan), expressed on MCSP (melanoma-specific CS proteoglycan), is important for regulating MT3-MMP [membrane-type 3 MMP (matrix metalloproteinase)]-mediated human melanoma invasion and gelatinolytic activity in vitro. In the present study, we sought to determine if CS can directly enhance MT3-MMP-mediated activation of pro-MMP-2. Co-immunoprecipitation studies suggest that MCSP forms a complex with MT3-MMP and MMP-2 on melanoma cell surface. When melanoma cells were treated with βDX (p-nitro-β-D-xylopyranoside) to inhibit coupling of CS on the core protein, both active form and proform of MMP-2 were no longer co-immunoprecipitated with either MCSP or MT3-MMP, suggesting a model in which CS directly binds to MMP-2 and presents the gelatinase to MT3-MMP to be activated. By using recombinant proteins, we determined that MT3-MMP directly activates pro-MMP-2 and that this activation requires the interaction of the C-terminal domain of pro-MMP-2 with MT3-MMP. Activation of pro-MMP-2 by suboptimal concentrations of MT3-MMP is also significantly enhanced in the presence of excess C4S (chondroitin 4-sulfate), whereas C6S (chondroitin 6-sulfate) or low-molecular-mass hyaluronan was ineffective. Affinity chromatography studies using CS isolated from aggrecan indicate that the catalytic domain of MT3-MMP and the C-terminal domain of MMP-2 directly bind to the GAG. Thus the direct binding of pro-MMP-2 with CS through the C-domain would present the catalytic domain of pro-MMP-2 to MT3-MMP, which facilitates the generation of the active form of MMP-2. These results suggest that C4S, which is expressed on tumour cell surface, can function to bind to pro-MMP-2 and facilitate its activation by MT3-MMP-expressing tumour cells to enhance invasion and metastasis.

Previous studies demonstrated that transition metal complexes inhibit cancer cell growth. We have examined a series of ruthenium (Ru (II)) complexes for their ability to inhibit growth of breast cancer cells. Among three complexes with the general formula [Ru(η6-p-cym)(N-N)Cl]+ tested for their abilities to inhibit MDA-MB-231 cell growth, the complex with o-phenalyenediamine (o-pda) as the bidentate nitrogen donor (N-N) ligand, [Ru(η6-p-cym)(o-pda)Cl]+, showed significant inhibitory activity in both a concentration- and a time-dependent manners. Although [Ru(η6-p-cym)(o-pda)Cl]+ inhibited growth of various human cancer types such as MCF-7 (breast), SK-Br3 (breast), B-cell lymphoma (Raji), osteosarcoma (HT1080), and melanoma (Bowes) cells, two TN breast cancer cells, HCC1806 and HCC38, were resistant to this treatment, suggesting cell-type specific functions of [Ru(η6-p-cym)(o-pda)Cl]+ for inhibiting cell growth. When MDA-MB-231 cells were treated with [Ru(η6-p-cym)(o-pda)Cl]+, both cleavage of caspase-3 and release of HMGB-1 into conditioned medium increased in a concentration dependent manner, suggesting [Ru(η6-p-cym)(o-pda)Cl]+ induced both apoptosis and necrosis processes. Importantly, [Ru(η6-p-cym)(o-pda)Cl]+ synergistically inhibited MDA-MB-231 cell growth with cyclophosphamide but not doxorubicin and paclitaxel. These results suggest that [Ru(η6-p-cym)(o-pda)Cl]+ is a potent tumor growth inhibitor per se and enhances tumoricidal activity of chemotherapeutic agents such as cyclophosphamide. Thus, Ru (II) complexes are promising anti-cancer drugs which could be used alone and/or in combination with chemotherapeutic agents for breast cancer patients. The opinion and assertions contained herein are the private views of the authors and are not to be construed as official or as representing the views of the Department of the Army or the Department of Defense. Citation Format: Joji Iida, Marc L Prazo, Yifeng Lu, Elisabeth T Bell-Loncella, Craig D Shriver. Structure-activity relationship of ruthenium (Ru) complexes to inhibit breast cancer growth and metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-02-09.

BackgroundThe tetraspan protein CD63, originally described as a stage-specific melanoma antigen but also present in a number of normal cells, regulates melanoma cell growth in nude mice, motility in serum containing media, and adhesion to several extracellular matrix proteins. CD63 has been reported to associate with β1 and β2 integrins, but the mechanism of signal transduction by CD63 is not clear. This study examined whether CD63 is associated with protein kinase and can transmit signals in melanoma cells.MethodsImmunoprecipitation and radiolabeling were used to test for association of protein kinase activity with CD63. Adhesion of cells to monoclonal antibodies immobilized to microtiter plates was used to examine the ability of CD63 to transmit signals.ResultsCD63 was capable of transmitting a signal in melanoma cells that required extracellular calcium. In the absence of extracellular calcium at the time of binding to the CD63 mAb, the cell was no longer responsive to stimulation by CD63. Immunoprecipitation studies demonstrated protein kinase activity associated with CD63, and phosphoamino acid analysis revealed that most of this protein kinase activity was due to serine kinase activity.ConclusionThe current study suggests that serine protein kinase activity associated with CD63 may play a role in signaling by CD63 in melanoma cells.

Hyaluronan (HA) and its biosynthetic enzymes, HA synthases (HAS1, 2, and 3) are thought to participate in cancer progression. We have shown previously that HA production and HAS3 expression are increased in metastatic colon carcinoma cells (SW620) when compared with cells isolated from a primary tumor (SW480). Because invasion of the extracellular matrix is a fundamental event in tumor growth and metastasis, we hypothesized that SW620 cells would show greater invasive capability than SW480 cells, that invasion is HA dependent, and that HA mediates invasion via interaction with a cell-surface receptor. Invasion into artificial basement membrane (Matrigel) was assessed in vitro. To assess HA functionality, HAS expression was inhibited in SW620 cells by transfection with antisense HAS constructs. Decreased HA secretion and retention in the transfectants were confirmed using competitive binding and particle exclusion assays. SW620 cells demonstrated greater invasion through Matrigel than did SW480 cells. Antisense transfection decreased Matrigel invasion by SW620 cells by >60%; addition of exogenous HA restored invasion. Because the cell-surface HA receptor CD44 has been implicated in cancer progression, HA-CD44 interaction was then inhibited by incubation with an anti-CD44 antibody. Anti-CD44 antibody impaired invasion into Matrigel by 95%. Taken together, these data suggest that pericellular HA is critical for colon carcinoma cell invasion and that this invasive capability is dependent on interaction with CD44.

Hyaluronan (HA) is a cell-surface glycosaminoglycan that has been implicated in cancer progression. Cells isolated from metastatic colon carcinoma (SW620) produce greater amounts of pericellular HA than cells isolated from a primary tumor (SW480). Inhibition of hyaluronan synthases (HAS) by transfection with antisense cDNA decreases HA production. Because adhesion to the extracellular matrix (ECM) is required for invasion and metastasis, we hypothesized that pericellular HA mediates adhesion to ECM proteins such as laminin, collagen, and fibronectin and that inhibition of HA production or removal of HA by digestion with hyaluronidase would impair adhesion.SW480, SW620, and antisense transfectants (SW620 cells transfected with vector alone, antisense HAS2, antisense HAS3, and both antisense HAS2 and HAS3) were assessed for adhesion to laminin, Type 1 collagen, or fibronectin-coated plates. To confirm that adhesion was mediated by HA, cells were treated with or without hyaluronidase prior to the assays.Metastatic SW620 cells adhered well to laminin; SW480 cells demonstrated 46% less adhesion (P0.05; Student's t test). SW620 cell adhesion to Type 1 collagen and fibronectin was50% less than adhesion to laminin. Inhibition of HAS2 and/or HAS3 or pretreatment with hyaluronidase significantly decreased adhesion of SW620 cells to laminin (P0.05), suggesting that adhesion was dependent upon pericellular HA.Metastatic SW620 cells that produce large amounts of pericellular HA adhered well to laminin. Inhibition of HAS2 and/or HAS3 expression, or hyaluronidase digestion of pericellular HA significantly inhibited adhesion. These data suggest that HA promotes adhesion to laminin and may thereby facilitate invasion of the basement membrane and metastasis in colon carcinoma.

Membrane type-I metalloproteinase (MT1-MMP) is a transmembrane metalloproteinase that is critical for tumor cell invasion. MT1-MMP can degrade extracellular matrix (ECM) proteins directly and/or indirectly by activating soluble MMPs such as pro-MMP-2. Although MT1-MMP is upregulated in malignant melanoma, the biological consequences of elevated MT1-MMP expression for tumor progression are not entirely understood. In the current study, we have utilized the Bowes melanoma line for evaluating MT1-MMP in invasion and growth. Our studies extend the earlier observations to demonstrate that MT1-MMP expression in Bowes melanoma cells promotes selective invasion into matrigel but not matrices consisting of type-I collagen. Furthermore, MT1-MMP expressing melanoma cells exhibit increased migration in response to laminin 1 but not to type-I or type-IV collagen. MT1-MMP expression results in enhanced 3 dimensional growth in agarose gels and in long-term cultures within matrigel. The hydroxymate inhibitor BB94 inhibits MT1-MMP enhanced invasion and growth in 3 dimensional culture systems, but had no effect on increased motility. We demonstrated that MT1-MMP expression significantly facilitated tumorigenicity and growth by intradermal injection. The results suggest a more general role for elevated MT1-MMP in promoting both the selective invasion and increased growth of malignant melanoma in vivo.

Background: Chondroitin sulfate proteoglycan 4 (CSPG4) was first identified as a melanoma specific antigen and demonstrated to stabilize cell-substratum interactions during early events of tumor cell spreading on endothelial cells and matrix proteins. In invasive breast cancers (IBCs), CSPG4 has been reported to be highly expressed in the triple-negative (TN) subtype. Western blotting analyses suggest that CSPG4 is expressed on TN IBC lines. Here we further explored the specific gene expression (GE) profiles associated with CSPG4 using the available IBC cases from The Cancer Genome Atlas (TCGA) project towards identification of potential drug targets for patients with TN IBC. Methods: The GE data (Agilent, log2 transformed) of 459 IBC tumors were downloaded from the TCGA data portal. PAM50 classification results of all samples were obtained from the TCGA breast cancer AWG group, including 203 Luminal A (LA), 113 Luminal B (LB), 51 HER2+, 84 basal-like (Basal), and 8 Normal-like (not used). Clinicopathologic data (age, race, T-, N-, and M-stages) were also obtained from the AWG group. Non-parametric Kruskal-Wallis test was used for cross-subtype analysis of CSPG4 expression, followed by Wilcoxon-Mann-Whitney test for pairwise comparison with Bonferroni adjustment. Chi-square tests were performed for categorical variation analyses. Differentially Expressed Gene (DEG) analysis was performed using the Bioconductor “samr” package for two class unpaired comparison, with p < 0.05 and FDR < 0.10 as the cut-off for DEGs. All other statistical analyses were performed using SAS. Results: GE level of CSPG4 was highest in the Basal subtypes (mean±SD, −0.92±1.37) compared to all other subtypes of IBCs, including LA (−1.50±0.66, p = 0.0013), LB (−1.86±0.68, p < 0.0001), and HER2 (−1.69±0.60, p = 0.0014). GE in LA was also significantly higher compared to LB (p < 0.0001). The Basal subtype exhibited a much wider variation of the expression level compared to other subtypes. To better understand this variation, we analyzed the age, T, N, and M across the 4 quartiles of the CSPG4 GE levels in the Basal subtype. Only N was significantly associated with the quartiles (p = 0.04), with N-positive rates of 45.8%, 54.2%, 32.0%, and 16.7% from the lowest to the highest quartiles respectively. Further DEG analysis between the top and bottom quartiles of the samples identified 46 genes, including MIA, SOX10, MSMB etc. Many of these genes showed a strikingly similar expression distribution pattern to CSPG4. Discussion: Our study confirmed the higher GE of CSPG4 in Basal subtype. In this subtype, patients expressing high CSPG4 in the tumor show a lower rate of local lymph node metastasis, implying that these tumors may not metastasize via the lymphatic vascular system. A number of DEGs were identified that are positively associated with CSPG4 expression. Although the roles these molecules play in promoting IBC progression needs to be further evaluated, our study may lead to the identification of a drug target for the Basal subtype of IBC patients. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-06.

We have previously reported that alpha4beta1 (but not alpha5beta1) integrin-mediated melanoma cell adhesion is inhibited by removal of cell surface chondroitin sulfate glycosaminoglycan (CSGAG), suggesting that melanoma chondroitin sulfate proteoglycan plays a role in modulating the adhesive function of alpha4beta1 integrin. In the current study, we demonstrated that alpha4beta1 integrin binds to CSGAG. We have identified a peptide from within alpha4 integrin termed SG1 (KKEKDIMKKTI) that binds to cell surface melanoma chondroitin sulfate proteoglycan, indicating that SG1 represents a CSGAG binding site within the alpha4 integrin subunit. Soluble SG1 inhibits alpha4beta1 integrin-mediated human melanoma cell adhesion to CS1. Polyclonal antibody generated against the peptide inhibits melanoma cell adhesion to CS1, and the inhibition is reversed by Mn2+ and an activating monoclonal antibody anti-beta1 (8A2). Additionally, pretreatment of cells with anti-SG1 IgG inhibits the expression of the monoclonal antibody 15/7 epitope in the presence of soluble CS1 peptide, suggesting that anti-SG1 IgG prevents ligand binding by alpha4beta1 integrin. These results demonstrate that alpha4beta1 integrin interacts directly with CSGAG through SG1 site, and that this site can affect the ligand binding properties of the integrin.

Tumor cell invasion of basement membranes (BM) represents one of the critical steps in the metastatic process. Tumor cell recognition of individual BM matrix components may involve individual cell adhesion receptors, such as integrins or cell surface proteoglycans, or may involve a coordinate action of both types of receptors. In this study, we have focused on the identification of a cell surface CD44/chondroitin sulfate proteoglycan (CSPG) and alpha 2 beta 1 integrin on human melanoma cells that are both directly involved in the in vitro invasion of reconstituted BM via a type IV collagen-dependent mechanism. Interfering with cell surface expression of human melanoma CSPG with either p-nitro-phenyl-beta-D-xylopyranoside treatment or anti-CD44 monoclonal antibody (mAb) preincubation (mAb) preincubation inhibits melanoma cell invasion through reconstituted BM. These treatments also strongly inhibit melanoma cell migration on type IV collagen, however, they are ineffective at inhibiting cell adhesion to type IV collagen. Purified melanoma cell surface CD44/CSPG, or purified chondroitin sulfate, bind to type IV collagen affinity columns, consistent with a role for CD44/CSPG-type IV collagen interactions in mediating tumor cell invasion. In contrast, melanoma cell migration on laminin (LM) does not involve CD44/CSPG, nor does CD44/CSPG bind to LM, suggesting that CD44/CSPG-type IV collagen interactions are specific in nature. Additionally, anti-alpha 2 and anti-beta 1 integrin mAbs are capable of blocking melanoma cell invasion of reconstituted BM. Both of these anti-integrin mAbs inhibit melanoma cell adhesion and migration on type IV collagen, whereas only anti-beta 1 mAb inhibits cell adhesion to LM. Collectively, these results indicate that melanoma cell adhesion to type IV collagen is an important consideration in invasion of reconstituted BM in vitro, and suggest that CD44/CSPG and alpha 2 beta 1 integrin may collaborate to promote human melanoma cell adhesion, migration, and invasion in vivo.

Cellular recognition of the extracellular matrix (ECM) is fundamentally important for development, the maintenance of normal adult tissue architecture and function, and for the pathogensis of diesease. The ECM exerts its effects in large part, by serving as an exoskeleton for cells within tissues, thereby providing structural, and other, information to cells that serve to regulate normal cell fuction. Understanding subtle changes in the cellular recognition of the ECM that occur as a result of malignant cell transformation can provide important information regarding the altered biology of tumor celss and could lead to novel therapeutic and/or diagnostic strategies for the altered biology of tumor invasion and metastasis, two features of many malignancies that contribute to the mortality of cancer patients.

The efficacy of synthetic immunoadjuvants and recombinant cytokines for the potentiation of host-resistance against virus infection was investigated using mouse models infected with Sendai virus and herpes simplex type 1 virus (HSV). The synthetic MDP derivative, MDP-Lys(L18), and recombinant cytokines, IL-1 beta, IFN-gamma, G-CSF and GM-CSF were shown to be effective for the stimulation of nonspecific protection against Sendai virus infection in mice. Both MDP-Lys(L18) and GM-CSF were effective for the protection against HSV infection in cyclophosphamide (CY)-treated mice. B30-MDP was suggested to be useful as an immunoadjuvant for the potentiation of antigenicity of recombinant or component vaccines.

Triple negative (TN) breast cancers are defined by a lack of expression of estrogen, progesterone, and her-2/neu receptors. It is widely recognized that TN breast cancers have a poorer prognosis than any other subtypes of breast caner. Given the lack of effective targeted therapies for TN breast cancer patients, understanding of the mechanisms of growth and invasion in the tissues provides insight into developing novel approaches to lower the mortality from TN breast cancer.Neoplastic epithelial cells in breast carcinomas interact with various components in the tissue microenvironment including extracellular matrix (ECM) and mesenchymal cells. Recent studies identified CD44 as a metastasis-related molecule with multiple functions by promoting cell-cell and cell-ECM interactions. CD44 is an integral transmembrane protein encoded by a single 20-exon gene. In the standard form (CD44s), 10 of the 20 exons are translated. Multiple variant isoforms exist (CD44v1-10) which arises from alternate mRNA splicing of the remaining 10 exons. In contrast to the ubiquitous expression of the standard form of CD44, splice variants are highly restricted in their expression in normal or malignant tissues. Indeed, CD44 variants containing v3, v5, v6, v7-8, v10 exons are expressed in malignant breast cancer tissues. However, there is limited information regarding the biological functions of these exons to promote tumor invasion and metastasis. The goal of this study is to evaluate specific exon(s) of CD44 expressed on TN breast cancer cells for promoting tumor progression and metastasis.In order to approach this goal, we utilized three TN cell lines (HCC38, HCC1937, and HCC1806) as model systems to evaluate CD44 in regulating invasion, migration, and growth in extracellular matrix (ECM) environments. Reverse transcriptase (RT)-PCR analysis using exon specific primers indicate that these cells expressed CD44v8-v10 and CD44s. We demonstrated that an inhibitory antibody against exon v10 of CD44 significantly inhibited b1 integrin-mediated migration and invasion into Matrigel and type I collagen gel. Importantly, this antibody also inhibited three dimensional (3D) growth which is a b1 integrin-independent process. The significant inhibition of these processes was also achieved when a FLAG-fusion exon v10 peptide (FLAG-v10, in which FLAG is tagged at the N-terminal of the peptide) was used as an inhibitor, implying that this exon would function to assemble molecular complexes on TN breast cancer cells that facilitate invasion, migration, and growth. Thus, these results suggest that generation of small synthetic molecules that block the functions of exon v10 of CD44 is promising approaches to inhibit invasion and metastasis of TN breast cancer cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6161.

The effect of a chemically synthesized polyprenol derivative, dihydroheptaprenol (DHP), on the non-specific resistance of mice to Sendai virus infection was investigated. The mice that received 200 micrograms of DHP intranasally twice, at 3 days and 1 day before the infection, showed a significant protection against Sendai virus infection. Treatment of mice twice even with as much as 2000 micrograms of DHP through the subcutaneous route, however, had no protective effect against infection. Excess interferon and tumour necrosis factor production in intranasally DHP-treated mice was seen 1 day after the infection when compared with Sendai virus alone controls or with DHP alone controls. Variance analysis of these findings indicates a prophylactic activity of DHP in pulmonary viral infections.

Previously, we have established the experimental model using Sendai virus in mice for the screening of immunostimulants which were effective for the enhancement of nonspecific host resistance against viral infection (Ishihara 1985). By using this experimental model, we have reported that synthetic derivatives of N-acetylmuramyl-L-alanylD-isoglutamine (MDP), especially Nα-acetylmuramyl-L-alanyl-D-isoglutaminyl-Ne-stearoyl-L-lysine [MDP-Lys(L18)], chitin derivatives, polypeptides and dihydroheptaprenyl were potent immunostimulants for the potentiation of host resistance against bacterial and viral infections in experimental models (Iida 1987, 1989a, 1990; Ishihara 1985, 1987, 1989). More recently the efficacy of MDP-Lys(L18) was proved by clinical trials for the increase of the number of leukocytes and platelets of cancer patients who received chemotherapy and other conventional therapy (Tsubura 1988).

The immunological properties of six kinds of multiprenylacetic acids and six kinds of multiprenylacetyl muramyldipeptide (MDP) derivatives were examined by using experimental models in mice and guinea-pigs. All the multiprenylacetyl MDP derivatives, particularly TMD-232, showed potent adjuvant activity on the circulating antibody formation against bacterial alpha-amylase in mice, and induction of delayed-type hypersensitivity to monoazobenzenearsonate N-acetyl-L-tyrosine in guinea-pigs. All multiprenylacetic acid preparations tested in this study, however, showed no adjuvant activity in these immune systems. Both TMD-17 and TMD-232 entrapped into multilamellar vesicles showed potent host stimulation activity against Sendai virus infection in mice.

The biological activities of synthetic glycolipids, which were chemically synthesized and based on the structure of lipid A of the lipopolysaccharide (LPS) from Escherichia coli, were examined with special reference to their adjuvant activity on the induction of delayed-type hypersensitivity, activity on the induction of tumour necrotic factor (TNF) and tumour regressive activity on line 10 hepatoma in strain 2 guinea pigs. Among them, a compound structurally corresponding to free E. coli lipid A (compound 506) as well as LPS exhibited potent adjuvant activity in the induction of delayed-type hypersensitivity in guinea pigs and TNF inducing activity in the sera of mice which were presensitized with Propionibacterium acnes. Compound 506 showed potent lethal toxicity in the intravenous administration of BALB/c mice presensitized with P. acnes. The regressive activity on line 10 hepatoma was observed by the multiple intralesional injection of squalane-treated compounds 504 and 505 in strain 2 guinea pigs.

The inhibition of murine lung metastasis by synthetic polypeptides [poly(arg-gly-asp) and poly(tyr-ile-gly-ser-arg)] with a core sequence of cell adhesion molecules

The efficacy of N-acetylglucosaminyl-beta (1----4)-N-acetylmuramyl tri- or tetrapeptides (GM) and the lipophilic derivatives for host augmentation against Sendai virus infection and for macrophage activation in vitro was examined. The anti-infectious activities of GM derivatives were shown to increase with the chain length of the fatty acid combined with the diaminopimelyl group. When the macrophages were activated with 1 U ml-1 murine interferon gamma (IFN-gamma) and 0.001 microgram ml-1 GM derivatives, the cytocidal ability of macrophages depended on the length of the side chain, and exhibited a positive relationship with the anti-infectious activity of GM derivatives against Sendai virus infection. These results indicated that the increment of lipophilicity of GM derivatives would play an important role in the anti-infectious activity and macrophage activation in vitro.

We have investigated the inhibitory effect on experimental or spontaneous lung metastases of polypeptides which contain repetitive structures of the Arg-Gly-Asp (RGD) or Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence derived from adhesion molecules, and studied their biological characterisation after administration. In the spontaneous metastasis model, multiple intravenous (i.v.) administrations of poly (RGD) and poly (YIGSR) resulted in a reduction of lung tumour colonies, although the monomer peptides, RGD or YIGSR, had no effect under these conditions. The treatment with poly(RGD) substantially prolonged the survival time for mice injected i.v. with B16-BL6 cells as compared to the treatment with RGD and random poly(R, G, D). Tumour cell adhesion to the fibronectin-substrates was remarkably inhibited by adding poly(RGD) freely in solution. Poly(RGD) was found to inhibit completely the ability of platelets to enhance tumour cell adhesion to fibronectin-substrate and tumour cell-elicited platelet aggregation in vitro, but poly(R, G, D) had no such effect. We also found that poly(RGD) led to a decrease in the arrest and retention of tumour cells after its co-injection with radiolabelled tumour cells and that the radiolabelled polypeptide can be at least decomposed into small fragments during circulation. Poly(RGD) was found to be still active in inhibiting experimental lung metastasis even when the contributions of NK cells or macrophages were removed from this system after pretreatment with anti-asialo GM1 serum, 2-chloroadenosine or carrageenan. The results indicate that the poly(RGD)-mediated inhibition of tumour metastasis may be due to the interference of the adhesive interaction of tumour cells with a specific site in the target organs. Derivatives of polypeptides which contain RGD and/or YIGSR sequences derived from cell adhesion proteins may thus provide a promising approach for the control and prevention of cancer metastasis.

Poly-L-Lys (mean mol. wt; 12,000), poly-L-Arg (5000) and poly-L-Orn were found to activate peritoneal macrophages effectively in vivo in 14 synthetic homo polypeptides. The ability of sequential poly(L-Arg-L-X) (5000) to activate macrophages was less than that of poly-L-Arg. Neither (L-Arg)12 nor (L-Arg)6 by themselves activated macrophages, but poly-D-Arg (5000) did, as also did poly-L-Arg; this suggests that the polycationic character of poly-L-Arg plays a role in the activation of macrophages. The intranasal administration of poly-L-Lys, -L-Arg, -L-Orn, -D-Arg, all of which activated macrophages, augmented host resistance against Sendai virus infection in mice. The protection afforded by poly-L-Arg seemed to depend on its mol. wt: the order of protection was poly-L-Arg greater than (L-Arg)12 greater than (L-Arg)6. The intranasal administration of poly-L-Arg 3 days before the infection was effective, while that 1 day before infection was not. There was no difference between the groups in the titer of interferon produced by the infection of Sendai virus given with poly-L-Arg either 3 days before or 1 day before the infection. The administration of poly-L-Arg 3 days before the infection caused a significant decrease of the virus titer in the lung 6 days after the infection when compared with the control or the mice given 1 day before. The intravenous administration of 2-chloroadenosine (2-Cl-Ade), which is a selective inhibitor of macrophages, into the mice which had received poly-L-Arg intranasally 3 days before the infection caused a significant decrease in the survival rate of the mice, indicating that the macrophages activated with poly-L-Arg are likely to be an important element in affording the protection. Subcutaneous administration of poly-L-Arg revealed protective activity against systematic infection with herpes virus-type 1.

Mice that received N alpha-acetylmuramyl-L-alanyl-D-isoglutaminyl-N epsilon-stearoyl-L-lysine [MDP-Lys (L18)] were resistant to Sendai virus infection. In these protected mice, a significant growth inhibition of the virus was confirmed repeatedly at 10(0.2) to 10(0.4) of haemadsorbing units at an early non-specific phase but not at a late virus-eliminating phase of the infection. Virus growth was enhanced by treatment with silica but not by treatment with anti-asialo GM1 serum in MDP-Lys (L18)-treated mice. Peritoneal adherent cells activated by MDP-Lys(L18) showed an enhanced uptake and ability to inactivate Sendai virus in vitro. Excess interferon production in MDP-Lys (L18)-treated mice was seen on day 1 but not on days 2 to 7 of the infection. The possible role of macrophages and interferon in providing non-specific protection against Sendai virus in the MDP-Lys (L18)-treated mice is discussed.