Cell Lines MDA Research Articles

Characterization of the roles of RHOC and RHOA GTPases in invasion, motility, and matrix adhesion in inflammatory and aggressive breast cancers

The 2 closely related small GTPases, RHOC and RHOA, are involved in mammary gland carcinogenesis; however, their specific roles in determining cancer cell adhesion and invasion have not been elucidated. RHOA and RHOC are highly homologous, thereby posing a major challenge to study their individual functions in cancer cells. By selectively knocking down these proteins, we have been able to alternatively inhibit RHOC and RHOA, while preserving expression of the other rho protein. Quantitative analyses of the growth patterns and invasion in the aggressive estrogen receptor negative cell lines MDA-231 and SUM149 were carried out on collagen I and Matrigel substrates. RHOC, and not RHOA, modulates surface expression and colocalization of alpha2 and beta1 integrins in MDA-MB-231 on collagen I. Neither RHOC or RHOA affected integrin expression in the inflammatory breast cancer cell line SUM149, further highlighting the different regulation of adhesion and motility in inflammatory breast cancer. This work shows that RHOC and RHOA play different roles in cell-matrix adhesion, motility, and invasion of MDA-MB-231 and reaffirms the crucial role of RHOC-GTPase in inflammatory breast cancer cell invasion.

Abstract 172: A role for the demethylation of microRNA-496 in MBD2-mediated repression

Abstract BACKGROUND: MicroRNAs are small RNA molecules that have the ability to repress target mRNAs through binding in their 3’ UTR. Prior work has shown that the expression of methyl binding domain protein 2 (MBD2) induces a variety of prometastatic genes through direct demethylation. HYPOTHESIS: MBD2 turns on microRNA expression through promoter demethylation that in turn suppress other genes. RESULTS: Methylated DNA Immunoprecipitation (mDIP) and MBD2 ChIP arrays identified a subset of microRNAs that change with the overexpression and transient knockdown of MBD2 in MCF10A and MCF7 cell lines, respectively. Methylation of these microRNAs with bisulfite sequencing and validation of MBD2 binding with quantitative PCR (qPCR) revealed the possible induction of hsa-mir-496 by MBD2. Expression of mir-496 was shown to be induced in MBD2 transfected MCF10A cells and repressed in MCF7 and MDA-231 cells with qPCR. Luciferase promoter assays revealed hsa-mir-496 promoter to be regulated by methylation and driven forward by co-transfection with MBD2. In silico scans of possible targets of hsa-mir-496 identified CPEB3 as a putative target. Expression of CPEB3 was shown to be inversely proportional to MBD2 in MBD2 transfected MCF10A cell lines and transient knockdowns of MBD2 in MCF7 and MDA-231 cell lines with qPCR. CONCLUSIONS: The discovery of microRNAs has shed a whole new light on transcriptional regulation and with it has raised several questions about specific cell states. Considering that MBD2 causes complete transformation of a cell and plays a large role in tumorigenicity it is essential to characterize the roles of microRNA action with regards to MBD2 and its respective network of action. More importantly to show MBD2 as a key regulator of microRNA expression can further reinforce its role as a DNA demethylase and a key regulator of cancer. FUTURE DIRECTIONS: Future work will involve testing mir-496 action in the cell with its putative target CPEB3 using 2-O-methyl primer assays in conjunction with luciferase reporter assays in order to probe the functionality of the CPEB3 3′UTR. The aforementioned results and directions will give us a refined picture of our model as well as attempt to define a system of MBD2-microRNA action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 172.

Abstract 5426: New suppression strategy for human breast cancer invasion and metastasis

Abstract Identification of agents that are nontoxic but can delay onset and/or progression of breast cancer, which is the main leading cause of cancer-related deaths among women, is highly desirable. Epithelial-mesenchymal transition (EMT) is a key event in the tumor invasion process and poor prognosis of breast cancer is associated with a high potential of cancer cells invasion and metastasis. We have recently uncovered novel evidence that signal transducers and activators of transcription 5a (Stat5a) and its Janus tyrosine kinase, (Jak2), suppress invasion and promote human breast cancer cells differentiation through inhibition of epithelial-to-mesenchymal dedifferentiation. Here, we have further taken advantage of the combination of Jak2/Stat5a overexpression, HDAC inhibitors, Mistletoe (Viscum album) plant extracts, and/or diallyl trisulfide (DATS), a promising cancer chemopreventive constituent of garlic, as an alternative way to inhibit breast cancer invasion and restore differentiation in vitro and in vivo. We have expanded our investigation to a panel of human breast cancer cell lines that display different phenotypic and differentiation patterns ranging from poor to high differentiation, including MDA-231, MDA-468, T-47D, MCF-7, BT-20, SKBr3, and ZR-75-1 cell lines. Cells were pretreated with or without HDAC inhibitors, Mistletoe plant extracts and/or diallyl trisulfide (DATS), and then either mock infected or infected with adenovirus carrying Wild type (Wt-Jak2), Wt-Stat5a, Dominant negative (Dn-Stat5), or a combination of Wt-Jak2 and Wt-Stat5a. Using three-dimensional (3D) Matrigel model system in vitro, the pretreatment with HDAC inhibitors, Mistletoe plant extracts, and /or DATS synergized with co-expression of Wt-Jak2/Stat5a to promote epithelial differentiation by reversing the mesenchymal phenotype morphology, inducing homotypic adhesion, and inhibiting cell motility and invasiveness. We have also established xenograft tumor models of MDA-231 and T-47D cell lines respectively in nude mice for in vivo studies. After 4 week, the growth of MDA-231 and T-47D tumors that co-expressing Wt-Jak2/Stat5a was inhibited by 45.4% and 51.12% compared to the mock group (t=2.410, P<0.05) and by 48.3% and 54.33% respectively compared to the tumors expressing Dn-Stat5 group (t=1.994, P<0.05). Furthermore, the pretreatment with HDAC inhibitors and/or DATS significantly inhibited the growth of MDA-231 and T-47D xenografted tumors expressing Wt-Jak2/Stat5a compared to the tumors expressing Wt- Stat5a or Wt-Jak2 alone. All-in-all, the observations support a novel role of a combination between Jak2/Stat5a overexpression and nontoxic agents that can delay onset and/or progression of breast cancer as a new tool to restore lost differentiation of human breast cancer through EMT inhibition, which could be a new therapeutic strategy to inhibit human breast cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5426.

Abstract 432: Expression of PITX2 is correlated with invasiveness in breast cancer cells and its downregulation reduces invasive potential

Abstract Objective We have previously found that PITX2 expression in the bone marrow (BM) of women newly diagnosed with breast cancer correlates with the development of distant disease. PITX2 is not expressed in the BM of normal volunteers, and thus the source of PITX2 is likely to be disseminated tumor cells which lodge in the bone marrow. PITX2 plays a well-defined role in development. This study was conducted to understand the role of expression of PITX2 in the breast cancer phenotype. Experimental procedures PITX2 has three different transcript variants. The RNA expression level of each transcript variant was determined by qRT-PCR using specific primers designed for each isoforms in a variety of breast cancer cell lines. PITX2 was stably silenced using shRNA lentiviral constructs against all isoforms of PITX2 (Sigma, St. Louis) and selecting with puromycin. Individual clones were selected and tested for PITX2 expression. Those cells with near total knockdown of the gene were used for matrigel invasion assays using manufacturer's protocols. Briefly, about 3 × 104 cells were suspended in growth factor free media and added to each well of invasion chamber and complete growth medium was added to the lower chamber as chemoattractant. The number of cells which invaded was counted at 24h and 48h after seeding. Control cells included empty vector, a non-targeting sequence, and shRNA against the unrelated gene, Beta 2- Microglobulin. The number of invasive cells were counted from five different fields from each well and tallied. Percentage invasion was calculated as the number of cells passed through the Matrigel to the total number of cells before harvest. All experiments were repeated three times. Results PITX2 isoforms A and B were found to be expressed in the invasive cell lines MDA MB 231 and C1A1 while none of the isoforms were expressed in non-invasive cell lines MCF7 and MCF10A. In matrigel invasion assay, the knockdown of PITX2 substantially reduced invasiveness compared to the controls. There was a 63.8 % reduction in invasion in PITX2 deficient cells at 24 hrs and 72.6 % reduction at 48 hrs compared to the parental MDA MB 231 cells. ConclusionPITX2 is expressed in breast cancer cell lines which have the ability invade and is not expressed in cell lines which cannot invade. Silencing of PITX2 in breast cancer cell lines abrogates the ability of breast cancer cells to invade. These findings combined with our clinic data suggest that PITX2 plays an important role in the invasiveness of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 432.

Abstract 711: VEGF121/rGel inhibits prostate cancer-induced osteoblastogenesis by targeting osteoblasts and tumor neovasculature

Abstract Prostate cancer metastasis to bone usually produces bone-forming lesions although an osteolytic component is present in most cases. We have previously shown excellent efficacy against prostate cancer growth in bone in an osteolytic model (PC-3) with VEGF121/rGel, a chimeric fusion toxin of VEGF121 and the plant toxin gelonin (rGel) that targets VEGFR-1 and VEGFR-2. In the current study, we demonstrate that VEGF121/rGel systemic administration is also effective in a model of prostate cancer that displays the bone-forming phenotype when growing in the femurs of immunodeficient mice. Both murine osteoblast precursor (MC3T3) cells and primary mouse osteoblasts (PMOs) were shown to express VEGFR-1 but not VEGFR-2. VEGFR-1 mRNA expression was shown to down-regulate during osteoblast (MC3T3) differentiation. In vitro, treatment with VEGF121/rGel showed cytotoxicity against osteoblast precursor cells (IC50 = 15 nM) that was substantially reduced (IC50 > 1000 nM) after MC3T3 had been allowed to undergo differentiation. This suggests that the effect of VEGF121/rGel is specifically mediated by VEGFR-1. Furthermore, immunofluorescence microscopy showed that internalization of VEGF121/rGel into PMOs is VEGF121-receptor driven. In an ex vivo model of osteoblastic disease, 100 nM VEGF121/rGel significantly inhibited prostate cancer-mediated new bone formation in neonatal mouse calvaria. In vivo, systemic (iv, tail vein) administration of VEGF121/rGel significantly inhibited growth of the osteoblastic prostate cancer cell line MDA PCa 118b growing intrafemorally. Only 25% of VEGF121/rGel-treated mice showed the development of osteoblastic lesions compared to 90% of saline-treated mice. Micro-CT (µCT) analysis of isolated femurs demonstrated that intrafemoral growth of MDA PCa118b tumors caused a dramatic increase in bone volume and that treatment with VEGF121/rGel restored the bone volume fraction to normalized levels with no changes to the uninvolved contralateral femurs. H&E results confirmed that the osteoblastic growth of 118b cells was severely impeded. Our results clearly demonstrate that VEGF121/rGel administration may suppress the eventual development of skeletal prostate tumors and may have significant therapeutic effect against prostate cancer-mediated osteoblastic lesions in bone. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 711.

Triple-Negative Breast Cancers Express Receptors for GnRH and Can Be Effectively Targeted by the Orally Active GnRH-Antagonist AEZS 115.

Abstract IntroductionAEZS-115* is an orally active peptidomimetic antagonist of GnRH, which has been shown to block the pituitary GnRH receptor. In various tumors an autocrine growth promoting loop consisting of GnRH secreted by the malignant cells and tumoral GnRH receptors has been described. The current study investigates the expression of GnRH-receptors in tumor samples of triple-negative breast cancers and evaluates the anti-tumor activity of the GnRH antagonist AEZS 115 in in vitro models of triple negative breast cancers.Study design17 human samples of triple negative breast cancers were tested for GnRH-receptor expression by immunohistochemistry. Human triple negative breast cancer cell lines MDA-MB 468, HCC 1806 and HCC 1937 were analyzed for GnRH receptor expression by immunocytochemistry and western blot analysis. These cell lines were incubated with increasing concentrations of AEZS-115, peptidic GnRH-antagonist Cetrorelix and GnRH-agonist Triptorelin (1, 10, 100 µm) for 48 hours and the number of viable cells was determined by crystal violet staining as well as by ATP-dependent luminometric assay.ResultsPositive staining for GnRH-receptors was detected in all tumor samples (2 high, 12 moderate, 3 weak). Cell lines MDA MB 468, HCC 1806 and HCC 1937 expressed GnRH receptors as demonstrated by immunocytochemistry and western blot analysis. GnRH-antagonist AEZS 115 dose dependently inhibited growth of all three cell lines, while GnRH-agonist Triptorelin and peptidic GnRH antagonist Cetrorelix showed marginal growth inhibition. At 10 µm AEZS-115 inhibited cell growth by 40-60%, at 100 µm growth inhibition was 60-80%. These results obtained by crystal violet staining were confirmed by additional luminometric evaluation of the ATP content after treatment with the respective substances.ConclusionsThe orally active, peptdidomimetic GnRH antagonist AEZS-115 showed substantial anti-tumor activity in these cell lines, while GnRH-agonist Triptorelin and peptidic antagonist Cetrorelix showed only minor growth inhibition. Thus, in triple negative breast cancers the peptidomimetic antagonist AEZS-115 was more potent than the decapeptide Cetrorelix. This finding could be explained by atypical GnRH-receptors on tumor tissue to which the different GnRH-antagonists might bind with different affinity. Due to high receptor expression the GnRH receptor is a promising target in triple negative breast cancers. The good antitumor-activity makes AEZS-115 a promising candidate for in vivo studies in this tumor entity.*Aeterna Zentaris GmbH, Frankfurt/M, Germany. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6108.

Cyclin D1 Is a Direct Target of JAG-Mediated Notch Signaling in Breast Cancer.

Abstract We have previously demonstrated that expression of the Notch ligand, JAG1 is associated with the basal breast cancer phenotype and disease recurrence. Herein we report on a genomics approach to elucidate mechanisms downstream of JAG1 that promote breast cancer growth. In a survey of 46 breast cancer cell lines we found that triple negative (TN; basal and mesenchymal ER-, PR- and Her2-negative) lines express JAG1 at significantly higher levels than do HER2+ or luminal (ER+) Her2- cell lines. The TN breast cancer cell lines HCC1143 and MDA MB231, which express JAG1 and demonstrate growth inhibition with RNA interference-induced down-regulation of JAG1, were selected for further study. We used microarray profiling of tumor cells transfected with JAG1 siRNA to identify JAG1-regulated genes. Differentially expressed genes (p ≤ 0.005; fold change ≥ 1.5) were identified for further study. Among the JAG1-regulated genes identified, cyclin D1 was found to be a direct target of NOTCH1 and NOTCH3. We show that JAG1 down-regulation results in reduced cyclin D1 expression, reduced direct binding of Notch to the cyclin D1 promoter, and inhibition of cell cycle progression through the cyclin D1-dependant G1/S check-point. Furthermore, we show that cyclin D1 and JAG1 expression correlate in basal breast cancer expression data sets. These data suggest a model whereby JAG1 promotes cyclin D1-mediated proliferation of TN breast cancer cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2150.

Synergistic Effect of Lapatinib and the Class 1 HDAC Inhibitor SNDX-275 in Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is a rare but aggressive form of primary breast cancer with high metastasis rates and poor survival outcomes in patients. Currently, no specific targeted therapy is available to improve patient outcomes, although agents (i.e. trastuzumab and lapatinib) targeting the human epidermal growth factor 2 (HER2) have shown promise in clinical trials. Histone deactylases (HDACs) represent another family of proteins for which inhibitors have been clinically validated and shown to inhibit proliferation of breast cancer cells in vitro and in vivo. In these studies we determined the single agent activity of the class 1 selective HDAC inhibitor entinostat (SNDX-275) in IBC cell models and whether SNDX-275 was synergistic with the HER2 targeted agent lapatinib.Methods: SNDX-275 activity was evaluated in SUM190, SUM149 and KPL-4 IBC cell lines using standard proliferation assays and compared to the non-IBC cell lines MDA-MB-231, SKBr3 and MCF-7. Apoptotic activity and cell cycle analysis were analyzed. SNDX-275 combination with lapatinib was initially determined in vivo in a HER2+ breast cancer model and subsequently in the SUM190, SUM149, KPL-4 IBC cells. For xenograft studies, athymic nude mice bearing human breast (BT474) tumor xenografts were treated with SNDX-275 at 15 or 30 mg/kg/day and lapatinib at 30 mg/kg/ 2xday or 75 mg/kg/ 2xday.Results: Significant anti-proliferative activity of SNDX-275 was observed in IBC (IC50, 250–500 nM) when compared with the non-IBC breast cancer cell lines MDA-MB-231, SKBr3, and MCF-7 (IC50 2–5 mM). Cell cycle analysis showed the onset of apoptosis in IBC cell lines (10%-17%); in the non-IBC cell lines, very little apoptosis occurred (0.8%–3.1%), although G1 stage arrest was seen in the non-IBC cell lines MDA-231 and MCF-7. The SNDX-275–induced apoptosis in IBC cell lines was dependent on caspase 9 rather than Caspase 8 cleavage indicating that the intrinsic apoptotic pathway is activated. The experiments with lapatinib demonstrated a significant benefit of the SNDX-275/lapatinib combination in both the BT474 xenograft study as well as the IBC cell lines tested. In the animal group that was treated with 15 mg/kg SNDX-275 plus 75 mg/kg lapatinib, synergistic effects were observed with tumor regression that was continued at least for 4 weeks after treatment was stopped. Similarly, synergistic anti-proliferative activity was found in almost all (4 of the 5) cell lines tested (SUM190, SUM149, KPL-4, and BT474). Investigation into the mechanism of SNDX-275–mediated apoptosis and the combined effects of lapatinib and SNDX-275 in IBC are under way. Our data demonstrate that HDACi as single agents and particularly in combination with HER2 targeted agents represent a promising new approach for clinical development in IBC breast cancer and patients with HER2-overexpressing breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3135.

Modulation of the Membrane Type 1 Matrix Metalloproteinase Cytoplasmic Tail Enhances Tumor Cell Invasion and Proliferation in Three-dimensional Collagen Matrices

Increasing evidence suggests that the cytoplasmic tail of membrane type 1 matrix metalloproteinase (MT1-MMP) is subject to phosphorylation and that this modification may influence its enzymatic activity at the cell surface. In this study, phosphorylated MT1-MMP is detected using a phospho-specific antibody recognizing a protein kinase C consensus sequence (phospho-TXR), and a MT1-MMP tail peptide is phosphorylated by exogenous protein kinase C. To characterize the potential role of cytoplasmic residue Thr(567) in these processes, mutants that mimic a state of either constitutive (T567E) or defective phosphorylation (T567A) were expressed and analyzed for their functional effects on MT1-MMP activity and cellular behavior. Phospho-mimetic mutants of Thr(567) exhibit enhanced matrix invasion as well as more extensive growth within a three-dimensional type I collagen matrix. Together, these findings suggest that MT1-MMP surface action is regulated by phosphorylation at cytoplasmic tail residue Thr(567) and that this modification plays a critical role in processes that are linked to tumor progression.

American Ginseng Inhibits Induced COX-2 and NFKB Activation in Breast Cancer Cells

Epidemiologic evidence suggests reduced breast cancer mortality in users of American Ginseng (AG) (Panax quinquefolium). We hypothesized that AG extract decreases proliferation of human breast cancer cells via an anti-inflammatory effect applicable to the prevention of breast and other cancers. A defined lyophilized aqueous extract of AG (LEAG) was dissolved in DMSO 1mg/mL, and serially diluted in saline. The cell lines MDA MB 231 and MCF7 were stimulated with the phorbol ester PDBu and treated with 100-500 mcg/mL LEAG. Proliferation was measured by MDA assay. Induced COX-2 expression was assayed by ELISA. Activation of NFkappaB by phosphorylation of the p65 subunit was quantified by CASE (cellular activation of signaling ELISA). Both cell lines had reduced proliferation when treated with LEAG. PDBu stimulation of MDA MB 231 increased expression of the COX-2 protein 20-fold at 48 hours (P<0.005). COX-2 protein expression remained at baseline concentrations in PDBu- treated MDA MB 231 cells exposed to 100 mcg/mL LEAG. The CASE assay showed a 4-fold increase in p65 activation 24 hours after PDBu treatment in normal medium, while phosphorylated p65 dropped below baseline in the cells treated with PDBu plus LEAG. In MDA MB 231, COX-2 was inducible with PDBu. This induced COX-2 expression was blocked by 100 microgram/mL LEAG in a time course consistent with the decline in the activated p65 subunit of NFkappaB. These results provide an anti-inflammatory mechanism for a possible anti-cancer effect of American Ginseng.

Effects of altered zinc availability on proliferation and oxidative stress in cultured cells

Previous studies from our laboratory have demonstrated that primary and transformed cells differ in their homeostatic response to zinc deprivation. Altered zinc availability might also influence cellular proliferation and antioxidant defenses. In the present study, we examined the effects of zinc supplementation (15‐100 µM) and zinc deficiency induced by diethylenetriaminepentaacetic acid (DTPA, 25‐200 µM) on cell proliferation and reactive oxygen species (ROS). DTPA (50 µM) reduced cell proliferation in both MCF‐7 and MDA‐MB‐231 human breast cancer cells, but effects were greater and seen more quickly in MCF7 cells. 50 µM DTPA also reduced proliferation of H4IIE rat hepatoma cells, but did not affect cell number in primary hepatocytes. Zinc treatment (up to 50 µM) enhanced cell proliferation in MCF‐7 but not in MDA‐MB‐231 cells. It also increased proliferation of H4IIE cells, whereas a decline in cell number was found with primary hepatocytes. Cells were also observed for the generation of ROS. 48 hr incubation with DTPA significantly increased ROS in primary hepatocytes and MCF‐7 cells but not in H4IIE or MDA‐MB‐231 cells. Zinc induced ROS in both MCF‐7 and MDA cell lines but not in primary hepatocytes. Overall, while cellular responses differed, reduction of available zinc limited proliferation and addition stimulated growth. In some cells ROS were enhanced under both circumstances.

Activity and mechanism of action of histone deacetylase inhibitors in trastuzumab resistant breast cancer.

Abstract Abstract #3060 Introduction: The Her2 antibody trastuzumab (T) is the first biological therapy that has proven the value of Her2 blockade in breast cancer. However, not all patients respond to trastuzumab and relapses occur after this treatment. Several studies suggest that histone deacetylase inhibitors (HDACis) are active in Her2-amplified breast cancer but none of them have addressed the role of HDACis in trastuzumab-resistant breast cancer. HDACis act by inhibiting the deacetylation of histones and other non-histone proteins such as Hsp90. Our goal is to study the activity, optimal sequencing and mechanism of action of HDACis in Her2-amplified cell lines, which demonstrate either de novo or acquired resistance to trastuzumab.&amp;#x2028; Methods: T-sensitive and Her2-overexpressing BT474 &amp; SkBr3, T-resistant BT474 and de novo T-resistant cell lines MDA-MB361, MDA-MB453, UACC812 and UACC893 were used for all experiments. The HDACi TSA (0-1uM) and trastuzumab at 100ug/mL were used for proliferation assays applying WST1 reagent (Roche). Western-immunoblotting was performed using cell lysates from untreated and treated cells. Proteolytic cleavage of caspase-3 and PARP was analyzed as indicator for apoptosis, Akt expression and phosphorylation for survival, Her2 and Erk1/2 expression and phosphorylation for proliferation and Hsp90 and Hsp70 were analyzed as key enzymes for chaperone activity.&amp;#x2028; Results: TSA inhibited proliferation in all cell lines [IC50 100-300 nM]. The addition of trastuzumab was antagonistic in de novo T-resistant cell lines MDA MB 453, UACC 812 and 893 but did not change the effect induced by TSA in acquired T-resistant cells (BT474_H100). Her2 expression decreased in TSA and TSA+T treated cells but not in cells treated with trastuzumab alone. Erk1/2 phosphorylation decreased in TSA and TSA+T treated cells but was not affected by trastuzumab alone. Similar phosphorylation patterns were observed regarding Akt. Of note, Akt, Erk1/2, Hsp90 and Hsp70 levels did not change. Finally, TSA induced caspase-3 and PARP cleavage when given as single agent or in combination with trastuzumab.&amp;#x2028; Discussion: This study suggests that HDACis are active in both trastuzumab-sensitive and de novo or acquired trastuzumab-resistant cell lines. However, use of trastuzumab together with TSA may be antagonistic in tumors that are resistant against trastuzumab. Further studies are required in these cell lines in order to elucidate the mechanism of resistance against trastuzumab and the mechanism of the here observed antagonistic effect between trastuzumab and TSA. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3060.

Expression of MDA7/IL-24 human breast cancer and the molecular impact.

Abstract Abstract #2060 INTRODUCTION. MDA-7 (melanoma differentiation associated gene-7), also known as IL-24, was initially identified from cancer cells and was found to be up-regulated in melanoma cells. Forced expression of MDA7 in cancer cells was found to be growth inhibitory. MDA7/IL-24 operates in cells via its receptor, MDA7R/IL-24R, which includes at least the IL-20Rα and IL-20Rβ complex and the IL-22R and IL20Rβ complex. The present study investigated the impact of MDA7 on the growth and motility of breast cancer cells and the clinical relevance of MDA7 expression in mammary tumour tissues.&amp;#x2028; MATERIALS AND METHODS. Human breast cancer cell lines MDA MB-231 and MCF-7 were used for in vitro testing. The response of cancer cells to rhMDA7 in growth and cellular motility was determined using growth assay and ECIS (electric cell impedance sensing, and confirmed by wounding assay). Localisation of MDA7 in mammary issues was assessed using conventional immunohistochemical method. Levels of MDA7 transcript in fresh frozen breast tissues were determined using quantitative PCR analysis and analysed against clinical and pathological information.&amp;#x2028; RESULTS. Recombinant human MDA-7 only had a marginal effect on the in vitro growth of the breast cancer cell lines used. However, rhMDA had a significant effect on the migration of breast cancer cells. Cells treated with rhMDA-7 showed a slower rate of migration (electrical resistance 80.1±24.3Ω), when compared with control cells (130±24.3Ω) (p=0.024). Normal mammary epithelial cells showed a good degree of staining of MDA-7, a staining greatly reduced in cancer cells. Using the Nottingham Prognostic Index as a predictor of prognosis, tumours from patients with poor prognosis showed a significantly lower level of MDA-7 transcript compared with tumours from those with good prognosis (p=0.049). A significant difference was seen between tumours from patients who died of breast cancer and tumours from patients who remained disease free (p=0.035), the later showing a higher levels of MDA7 transcript. Using the Kaplan-Meier survival analysis, it was revealed that low levels of MDA-7 were significantly correlated with a shorter disease free survival (mean survival for low level group 121.7 (108.5-134.9) months compared with high levels 140.4 (133.7 – 147.1, 95% CI) months, p=0.0287. ER positive tumours showed a lower but levels of MDA-7 expression compared with ER-negative tumours, although this was not statistically significant (p=0.094).&amp;#x2028; CONCLUSIONS. MDA-7 is a potential regulator of cellular motility of breast cancer cells. The aberrant expression of the molecule is linked to the prognosis and long term survival of patients, indicating the potential clinical implication of MDA-7 in cancer therapies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2060.

Focal Adhesion Kinase-Related Proline-Rich Tyrosine Kinase 2 and Focal Adhesion Kinase Are Co-Overexpressed in Early-Stage and Invasive ErbB-2-Positive Breast Cancer and Cooperate for Breast Cancer Cell Tumorigenesis and Invasiveness

Early cancer cell migration and invasion of neighboring tissues are mediated by multiple events, including activation of focal adhesion signaling. Key regulators include the focal adhesion kinase (FAK) and FAK-related proline-rich tyrosine kinase 2 (Pyk2), whose distinct functions in cancer progression remain unclear. Here, we compared Pyk2 and FAK expression in breast cancer and their effects on ErbB-2-induced tumorigenesis and the potential therapeutic utility of targeting Pyk2 compared with FAK in preclinical models of breast cancer. Pyk2 is overexpressed in tissues from early and advanced breast cancers and overexpressed with both FAK and epidermal growth factor receptor-2 (ErbB-2) in a subset of breast cancer cases. Down-regulation of Pyk2 in ErbB-2-positive, FAK-proficient, and FAK-deficient cells reduced cell proliferation, which correlated with reduced mitogen-activated protein kinase (MAPK) activity. In contrast, Pyk2 silencing had little impact on cell migration and invasion. In vivo, Pyk2 down-regulation reduced primary tumor growth induced by a metastatic variant of ErbB-2-positive MDA 231 breast cancer cells but had little effect on lung metastases in contrast to FAK down-regulation. Dual reduction of Pyk2 and FAK expression resulted in strong inhibition of both primary tumor growth and lung metastases. Together, these data support the cooperative function of Pyk2 and FAK in breast cancer progression and suggest that dual inhibition of FAK and Pyk2 is an efficient therapeutic approach for targeting invasive breast cancer.

Combination of imatinib and vinorelbine enhances cell growth inhibition in breast cancer cells via PDGFR β signalling

Introduction Imatinib mesylate is a tyrosine kinase receptor inhibitor targeted against PDGFR α and β, c-kit and bcr-abl. These receptors regulate cellular processes such as proliferation, differentiation, and survival. This study was performed to evaluate the effects of imatinib on breast cancer cell lines with respect to the activity of PDGFR β and Akt: a downstream modulator of cell growth and survival. Methods Expression of imatinib targets was analyzed with reverse transciptase PCR and immunoblotting assays in the breast cell lines MDA MB 231, MCF 7, ZR 75-1, and T 47-D. Changes on receptor expression and phosphorylation status under imatinib were evaluated using drug concentrations of 2 to 10 μM. The anti-proliferative and pro-apoptotic effects of imatinib alone and in combination with vinorelbine were investigated with an MTT and TUNEL assay. Results Imatinib inhibited growth and induced apoptosis of all cell lines examined. This effect was increased when combined with vinorelbine. A dose-dependent inhibitory effect on the phosphorylation of PDGFR β and Akt was detected. Conclusions The growth inhibitory effect of imatinib on breast cell lines may be caused by inhibiting the activity of the tyrosine kinases PDGFR β and Akt. Imatinib is a promising novel drug for targeted therapy of breast cancer patients.

TVP1022 and Propargylamine Protect Neonatal Rat Ventricular Myocytes Against Doxorubicin-Induced and Serum Starvation-Induced Cardiotoxicity

We recently reported that propargylamine derivatives such as rasagiline (Azilect) and its S-isomer TVP1022 are neuroprotective. The aim of this study was to test the hypothesis that the neuroprotective agents TVP1022 and propargylamine (the active moiety of propargylamine derivatives) are also cardioprotective. We specifically investigated the protective efficacy of TVP1022 and propargylamine in neonatal rat ventricular myocytes (NRVM) against apoptosis induced by the anthracycline chemotherapeutic agent doxorubicin and by serum starvation. We demonstrated that pretreatment of NRVM cultures with TVP1022 or propargylamine attenuated doxorubicin-induced and serum starvation-induced apoptosis, inhibited the increase in cleaved caspase 3 levels, and reversed the decline in Bcl-2/Bax ratio. These cytoprotective effects were shown to reside in the propargylamine moiety. Finally, we showed that TVP1022 neither caused proliferation of the human cancer cell lines HeLa and MDA-231 nor interfered with the anti-cancer efficacy of doxorubicin. These results suggest that TVP1022 should be considered as a novel cardioprotective agent against ischemic insults and against anthracycline cardiotoxicity and can be coadministered with doxorubicin in the treatment of human malignancies.

In vitro testing of biological action of Origanum species from Greek flora in various cancer cell lines

The Origanum species (Lamiaceae family), which are commonly used herbs, contain phenolic compounds; however, their estrogenic effects remain unknown. In this study, we investigated the possible estrogenic/antiestrogenic effects of extracts (dichloromethanic, methanolic, aqueous) derived from three Origanum species of Greek origin, (O. dictamnus, O. scabrum, O. microphyllum) by using the appropriate biological markers in various estrogen-dependent cellular systems. We evaluated the potential of (i) dichloromethanic, methanolic and aqueous extracts (at a concentration range of 0.2–125µg/ml), to modulate cell viability of MCF-7 and MDA breast cancer cell lines, Ishikawa endometrial and PC-3 prostate cancer cell lines, by use of the MTT assay and (ii) of methanolic extracts (at a concentration range of 0.1–100µg/ml) to influence the activity of estrogen receptor (ER) in MCF-7 cells transfected with an estrogen response element (ERE)-driven luciferase (Luc) reporter gene. Ishikawa, MDA and PC-3 cell lines showed no response to all Origanum extracts tested. The viability of MCF-7 breast cancer cells showed a small but significant increase when exposed to methanolic extracts. O. dictamnus and O. scabrum extracts (at a concentration range of 2–100µg/ml), in the absence and in the presence of 17β-estradiol (E2), reduced the luciferase activity significantly. O. microphyllum methanolic extract, when alone, increased the basal luciferase activity, whereas its co-incubation with estradiol inhibited significantly the E2-stimulated gene induction. Concluding, the methanolic extracts of the three Origanum species modulate the cellular growth and estrogenic/antiestrogenic potency in MCF-7 cells indicating an impact of this dietary substance on human health.

Androgen Receptor and Invasion in Prostate Cancer

Activation of androgen receptor (AR) stimulates the growth of not only androgen-dependent but also of androgen-refractory prostate cancer. However, neither the role of AR in invasion/metastasis nor the relationship between invasiveness and androgen-refractory status has been established. In this study, we used the androgen-dependent prostate cancer cell line MDA PCa 2b, derived from a human bone metastasis, to generate an invasive subline (MDA-I) using a Matrigel chamber. MDA-I cells expressed higher levels of AR and prostate-specific antigen than their less invasive parental cells. Blocking AR function or removal of androgen suppressed the invasion of MDA-I cells, whereas stimulating AR increased invasion. In addition, forced AR overexpression increased the invasiveness of MDA PCa 2b cells. Next, we showed that an androgen-refractory subline (MDA-hr) of MDA PCa 2b cells also expressed higher levels of AR and were more invasive than their parental androgen-dependent cells. Blocking AR function suppressed the invasiveness of MDA-hr cells. Gelatin zymography indicated that matrix metalloproteinase 2 (MMP-2) and MMP-9 activities were regulated by AR signaling and closely correlated with the invasiveness of the androgen-dependent and androgen-refractory prostate cancer cells. These data suggest that AR promotes the invasiveness of both androgen-dependent and androgen-refractory prostate cancer and that a more invasive phenotype might develop through AR activation during cancer progression. These findings potentially support the use of adjuvant hormonal therapy and the future development of more potent androgen blockade therapy.

Mithramycin Analogues Generated by Combinatorial Biosynthesis Show Improved Bioactivity

Plasmid pLNBIV was used to overexpress the biosynthetic pathway of nucleoside-diphosphate (NDP)-activated l-digitoxose in the mithramycin producer Streptomyces argillaceus. This led to a "flooding" of the biosynthetic pathway of the antitumor drug mithramycin (MTM) with NDP-activated deoxysugars, which do not normally occur in the pathway, and consequently to the production of the four new mithramycin derivatives 1- 4 with altered saccharide patterns. Their structures reflect that NDP sugars produced by pLNBIV, namely, l-digitoxose and its biosynthetic intermediates, influenced the glycosyl transfer to positions B, D, and E, while positions A and C remained unaffected. All four new structures have unique, previously not found sugar decoration patterns, which arise from either overcoming the substrate specificity or inhibition of certain glycosyltransferases (GTs) of the MTM pathway with the foreign NDP sugars expressed by pLNBIV. An apoptosis TUNEL (=terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay revealed that compounds 1 (demycarosyl-3D-beta- d-digitoxosyl-MTM) and 3 (deoliosyl-3C-beta- d-mycarosyl-MTM) show improved activity (64.8 +/- 2% and 50.3 +/- 2.5% induction of apoptosis, respectively) against the estrogen receptor (ER)-positive human breast cancer cell line MCF-7 compared with the parent drug MTM (37.8 +/- 2.5% induction of apoptosis). In addition, compounds 1 and 4 (3A-deolivosyl-MTM) show significant effects on the ER-negative human breast cancer cell line MDA-231 (63.6 +/- 2% and 12.6 +/- 2.5% induction of apoptosis, respectively), which is not inhibited by the parent drug MTM itself (2.6 +/- 1.5% induction of apoptosis), but for which chemotherapeutic agents are urgently needed.

Synthesis and antitumour evaluation of novel 2-phenylbenzimidazoles

A new series of fluorinated and non-fluorinated 2-phenylbenzimidazoles bearing oxygenated substituents on the phenyl ring has been synthesized. Synthesis of the new series was based on our previous discovery of 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX 610) as a potent and selective antitumour agent in vitro (sub-nanomolar GI50 in sensitive human cancer cell lines), but with poor aqueous solubility and lack of a definitive cellular target limiting further development. In this study we test the hypothesis that 2-phenylbenzimidazoles with similar substitution patterns to PMX 610 would retain potent antitumour activity but with potentially superior pharmaceutical properties. In general the new compounds were less active than the former benzothiazole series in vitro when tested against the breast cancer cell lines MCF-7 and MDA 468; however the two most active compounds in the present series (3j and 3k) exhibit low micromolar GI50 values in both cell lines and provide the opportunity for further chemical derivatization with a view to target identification.