Joji Iida

Role for chondroitin sulfate glycosaminoglycan in NEDD9-mediated breast cancer cell growth

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.

FH535 Inhibited Migration and Growth of Breast Cancer Cells

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.

DNA Aptamers against Exon v10 of CD44 Inhibit Breast Cancer Cell Migration

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.

Inhibition of cancer cell growth by ruthenium complexes

BackgroundPrevious studies suggest that certain transition metal complexes, such as cisplatin, are efficacious for treating various cancer types, including ovarian, lung, and breast.MethodsIn 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.ResultsThe 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.ConclusionThese 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.

A Gene Regulatory Program in Human Breast Cancer

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.

Abstract P1-05-09: FH535 inhibited migration and growth of breast cancer cells

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.

Abstract P2-05-16: Tumor-associated glycans as key molecules to promote growth of triple-negative breast cancer cells

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.

Abstract P6-02-09: Structure-activity relationship of ruthenium (Ru) complexes to inhibit breast cancer growth 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.

Abstract P3-04-06: Higher gene expression of CSPG4 in the basal-like subtype of invasive breast cancer and its negative association with lymph node metastasis

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 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 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.

Role for CD44 in Enhancing Invasion, Migration, and Growth of Triple Negative (TN) Breast Cancer Cells.

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.