Triple Negative Breast Tumor Cells Research Articles

The indole alkaloid meleagrin, from the olive tree endophytic fungus Penicillium chrysogenum, as a novel lead for the control of c-Met-dependent breast cancer proliferation, migration and invasion

Fungi of the genus Penicillium produce unique and chemically diverse biologically active secondary metabolites, including indole alkaloids. The role of dysregulated hepatocyte growth factor (HGF) and its receptor, c-Met, in the development and progression of breast carcinoma is documented. The goal of this work is to explore the chemistry and bioactivity of the secondary metabolites of the endophytic Penicillium chrysogenum cultured from the leaf of the olive tree Olea europea, collected in its natural habitat in Egypt. This fungal extract showed good inhibitory activities against the proliferation and migration of several human breast cancer lines. The CH2Cl2 extract of P. chrysogenum mycelia was subjected to bioguided chromatographic separation to afford three known indole alkaloids; meleagrin (1), roquefortine C (2) and DHTD (3). Meleagrin inhibited the growth of the human breast cancer cell lines MDA-MB-231, MDA-468, BT-474, SK BR-3, MCF7 and MCF7-dox, while similar treatment doses were found to have no effect on the growth and viability of the non-tumorigenic human mammary epithelial cells MCF10A. Meleagrin also showed excellent ATP competitive c-Met inhibitory activity in Z-Lyte assay, which was further confirmed via molecular docking studies and Western blot analysis. In addition, meleagrin treatment caused a dose-dependent inhibition of HGF-induced cell migration, and invasion of breast cancer cell lines. Meleagrin treatment potently suppressed the invasive triple negative breast tumor cell growth in an orthotopic athymic nude mice model, promoting this unique natural product from hit to a lead rank. The indole alkaloid meleagrin is a novel lead c-Met inhibitory entity useful for the control of c-Met-dependent metastatic and invasive breast malignancies.

Abstract 5380: Near-infrared-resonant IONP/Au2S core/satellites hybrid nanocomposite for imaging-guided photothermal therapy

Abstract Abstract: Photothermal therapy (PTT) using near-infrared-resonant nanomaterials including multifunctional probes with both therapeutic functions and imaging capabilities has gained great attention in recent years. However, various challenges still exist including the large size of gold nanoshells, the “melting” effect of gold nanorods, lack of imaging capability, and tedious multiple steps for making imaging-capable multifunctional probes. Consequently, it is desired to develop an effective PTT mediator with the following criteria: small size, photo stability, facile synthesis, low toxicity, and imaging capability. We report the design of a core/satellites hybrid nanocomposite with a highly crystallized iron oxide nanoparticle (IONP) core (10-20 nm) and multiple gold sulfide (Au2S) nanoparticle satellites (1-2 nm) attached to the IONP polysiloxane-containing polymer coating for imaging-guided photothermal cancer therapy. In this nanostructure, the multiple satellites (Au2S) enhance PTT, while the IONP core serves as both a photothermal mediator and magnetic resonance imaging (MRI) contrast agent to localize the tumor. To make these core/satellites nanocomposites we first modify the IONP polymer coating with (3-mercaptopropyl) trimethoxysilane followed by mixing with Au2S nanoparticles, which are made by mixing sodium sulfide (Na2S) and chloroauric acid (HAuCl4) at the designed ratio. After purification using a magnet, the TEM image shows that around 8-10 Au2S nanoparticles (1-2 nm average diameter) were attached to each IONP (15 nm in diameter). This core/satellites nanocomposite has obvious absorption in the near infrared (NIR) range and the absorption peak can be adjusted by the molar ratio of Na2S to HAuCl4. The developed nanocomposite exhibits an enhanced photothermal effects without any quenching under NIR laser light. Benefitting from the anti-biofouling polysiloxane-containing polymer coating for in vivo applications, the core/satellites nanocomposites effectively accumulate to xenograft tumor tissue (SUM-159, triple-negative breast tumor cell line) through the enhanced permeability and retention effect after tail vein injection. MRI imaging is used to locate the tumors. NIR triggered PTT, which is enhanced by the core/satellites nanoparticles within the tumor site, destroys the tumor cells, while the tumor tissues in control mice do not show any change under the same laser light irradiation. Our in vitro and in vivo data reveal that NIR-resonant IONP/Au2S core/satellites nanocomposites can be used as effective MRI imaging-guided photothermal cancer therapy. Correspondence should be addressed to: Duxin Sun, PhD, or Hongwei Chen, PhD, Department of Pharmaceutical Sciences, College of Pharmacy, The University of Michigan, 428 Church Street, Ann Arbor, MI 48109, Email: duxins@umich.edu (D. Sun); or hongweic@umich.edu (H. Chen) Citation Format: Hongwei Chen, Joseph Burnett, Xiaoqing Ren, Feng Ni, Yi Wei, Hayley Paholak, Duxin Sun. Near-infrared-resonant IONP/Au2S core/satellites hybrid nanocomposite for imaging-guided photothermal therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5380. doi:10.1158/1538-7445.AM2014-5380

In triple negative breast tumor cells, PLC-β2 promotes the conversion of CD133high to CD133low phenotype and reduces the CD133-related invasiveness

BackgroundBeyond its possible correlation with stemness of tumor cells, CD133/prominin1 is considered an important marker in breast cancer, since it correlates with tumor size, metastasis and clinical stage of triple-negative breast cancers (TNBC), to date the highest risk breast neoplasia.MethodsTo study the correlation between the levels of CD133 expression and the biology of breast-derived cells, CD133low and CD133high cell subpopulations isolated from triple negative MDA-MB-231 cells were compared in terms of malignant properties and protein expression.ResultsHigh expression of CD133 characterizes cells with larger adhesion area, lower proliferation rate and reduced migration speed, indicative of a less undifferentiated phenotype. Conversely, when compared with CD133low cells, CD133high cells show higher invasive capability and increased expression of proteins involved in metastasis and drug-resistance of breast tumors. Among the signalling proteins examined, PLC-β2 expression inversely correlates with the levels of CD133 and has a role in inducing the CD133high cells to CD133low cells conversion, suggesting that, in TNBC cells, the de-regulation of this PLC isoform is responsible of the switch from an early to a mature tumoral phenotype also by reducing the expression of CD133.ConclusionsSince CD133 plays a role in determining the invasiveness of CD133high cells, it may constitute an attractive target to reduce the metastatic potential of TNBC. In addition, our data showing that the forced up-regulation of PLC-β2 counteracts the invasiveness of CD133-positive MDA-MB-231 cells might contribute to identify unexplored key steps responsible for the TNBC high malignancy, to be considered for potential therapeutic strategies.

In triple negative breast tumor cells, PLC-β2 promotes the conversion of CD133high to CD133low phenotype and reduces the CD133-related invasiveness

Beyond its possible relationship with stemness of tumor cells, CD133/prominin is a highly glycosylated trans-membrane protein that correlates with tumor size, metastasis and clinical stage of triple negative breast cancers (TNBC), that represent 20% of all breast tumors and have a particularly worse clinical outcome than other tumor subtypes [1]. The correlation between the levels of CD133 expression and the biology of breast tumor cells was studied in CD133low and CD133high cell subpopulations isolated from MDA-MB-231 cells (ER-, PR-, HER2-). High expression of CD133 characterizes a small percentage of cells with larger adhesion area, lower proliferation rate, higher invasion capability and increased expression of proteins involved in metastasis and drug resistance of breast cancers. PLC-b2 expression, that plays a crucial role in malignancy of breast tumor cells [2, 3], inversely correlates with the levels of CD133 and has a role in inducing the CD133high cells to CD133low cells conversion. The forced up-regulation of PLC-b2 counteracts the invasiveness of CD133high MDAMB- 231, suggesting that, in TNBC, the de-regulation of this PLC isozyme is responsible of the switch from an early to a mature tumoral phenotype also by reducing the expression of CD133. These data might contribute to identify unexplored key steps in TNBC malignancy, to be considered for potential therapeutic strategies.

Abstract S3-8: Identification of Novel Synthetic-Lethal Targets for MYC-Driven Triple-Negative Breast Cancer

Abstract Currently, the greatest clinical challenge in treating breast cancer occurs in those patients whose tumors lack expression of the estrogen and progesterone receptors and the HER2 oncoprotein. No targeted agents currently exist against this aggressive type of receptor ‘triple negative’ breast cancer that disproportionately affects young African American women and also a substantial number of white American women. We have discovered that triple-negative tumors frequently express high levels of the MYC proto-oncogene. We sought to identify new “synthetic-lethal” strategies to selectively kill triple-negative breast tumors with MYC overexpression. Synthetic lethality arises when a combination of mutations in two or more genes leads to cell death, whereas a mutation in only one of these genes has little effect. Using this strategy, we can take advantage of the elevated MYC signaling in triple-negative tumors to selectively kill them, while sparing normal tissues in which MYC is expressed at much lower levels. RESULTS: We performed a shRNA synthetic-lethal screen to identify new molecules, such as cell cycle kinases, which when inhibited can preferentially kill triple-negative breast tumor cells. The unbiased shRNA-based screening approach was performed in human mammary epithelial cells (HMECs) that have inducible MYC over-expression. A high-throughput screen of ∼2000 shRNAs, that target the human kinome (∼ 600 kinases) was performed, and identified 13 kinases whose inhibition by >1 shRNAs gave rise to >50% inhibition of cell growth. Two of the kinases identified in our screen, ARK5 and GSK3A, were also recently identified by other studies and shown to have a synthetic-lethal interaction with MYC. These results validated our ability to identify synthetic-lethal targets. We are currently characterizing and validating the 11 novel targets identified in this screen, using human cancer cell lines as well as mouse cancer models to determine the impact of inhibiting these targets on triple-negative breast cancer development and proliferation. Additional synthetic-lethal interactions with MYC over-expression in triple-negative breast cancers will be presented. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S3-8.

P3-18-02: A Combination of Pathway-Targeted Inhibitor with DNA-Repair Inhibitor: Preclinical Efficacy of Zactima and Olaparib in Triple Negative Subset of Breast Cancer.

Abstract Introduction: Pathway-targeted therapy has not been established for the treatment of Triple Negative (TN) subset of breast cancer (BC). Despite emerging data supporting the use of polyADP-ribose polymerase (PARP) inhibitors, complete and durable responses are rare in TNBC and exploration of additional pathway-targeted therapies is needed. The high frequency (72-75%) of EGFR expression in TNBCs suggests that loss of BRCA1 may be coupled, either directly or indirectly, with EGFR overexpression in breast cancer (Van der Groep et al., 2006), and a recent report shows that even a partial loss of BRCA1 leads to an overall increase in EGFR expression in mammary epithelial cells (Burga et al., 2011). We hypothesize that the combined inhibition of growth factor driven EGFR pathway (by Zactima, a small molecule kinase inhibitor of EGFR and VEGFR-2) and DNA-repair pathway (by Olaparib, PARP inhibitor) in the presence of DNA-damaging agent (carboplatin) will have anti-proliferative and anti-migratory effects on triple negative breast tumor (TNBT) cells. Methods: The effects of Zactima, Olaparib (single or in combination) and carboplatin were studied on: (a) the cell survival/proliferation (MTT, SRB, & cell titer-GLO assay), (b) EGF-induced upregulation of downstream proliferation markers, (c) the cellular signals for apoptosis, (d) fibronectin-directed migration (scratch-assay), (e) the vascular mimicry, and (f) the clonogenic survival (3D-ON-TOP assay) in different TNBT cell lines. Results: Our in vitro data show that, (1) Zactima (10 uM) blocked EGF-induced phosphorylation of AKT (S473 & T308), S6RP, 4EBP1 and ERK as early as 1 hr (after the treatment), (2) the range of EC50s for Zactima (96 hrs) varied from 5 uM −15 uM (HCC70, HCC1937, MDA-MB231, MDA-MB468, BT20), (3) EGFR over-expressing, PTEN-null, and ATM kinase mutated MDA-MB468 cell line exhibited lowest EC50 for Zactima as compared to other TNBT cell lines and was found to be the most sensitive cell line to Zactima (∼ 0.05 uM) when combined with 10 uM fixed dose of Olaparib, (4) a combination of Zactima with Olaparib plus carboplatin altered an adhesion-dependent cell-survival, increased caspase3 activity (expression of cleaved caspase3 and cleaved PARP) time dependently, inhibited vascular mimicry (at 6 and 24 hrs), blocked fibronectin-directed migration (scratch assay, 24 hrs), changed the organization of actin-cytoskeleton, and suppressed clonogenic growth in different TNBT cells lines. Conclusion: Zactima alone showed anti-proliferative/pro-apoptotic, and anti-migratory effects in TNBT cells. The combination of Zactima with Olaparib plus carboplatin was most effective in blocking the clonogenic growth of TNBT cells. We are currently pursuing studies to, (1) delineate the relationship between the anti-proliferative effects (clonogenic assay) of Zactima (alone or in combination) and the status of the EGFR/PI3K pathway using PIK3CA-mutated, PTEN-null and EGFR overexpressed TNBT cell lines, and (2) determine the effect of Zactima on angiogenesis using HUVEC cells, the results of which will be presented in the meeting. This work was generously supported by AVON foundation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-18-02.

Abstract 1196: FZD7 plays a critical role in triple negative breast cancer proliferation

Abstract Breast cancer is genetically and clinically heterogeneous. Triple negative cancer is a subtype of breast cancer, most of which is associated with poor outcome and the lack of benefit from target therapy. A pathway analysis in a microarray study was performed using triple negative breast tumor cells compared with non-triple negative breast tumor cells. Overexpression among several Wnt pathway genes, such as FZD7, LRP6 and TCF7 has been observed in triple negative breast tumors. To validate their function, inhibition of FZD7 using FZD7-shRNA was carried out. Noticeably decreased cell proliferation, suppressed invasiveness and colony formation in triple negative MDA-MB-231 and BT-20 cells were observed. Mechanism study indicated that these effects occurred through silencing the canonical Wnt signaling pathway, as evidenced by loss of nuclear accumulation of β-catenin and decreased transcriptional activity of TCF7. Our finding suggests that FZD7 involved canonical Wnt signaling pathway is essential for tumorigenesis of triple negative breast tumor. Thus, FZD7 may be a biomarker and a potential therapeutic target for triple negative breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1196. doi:10.1158/1538-7445.AM2011-1196

Enhanced Anticancer Effect of the Combination of Cisplatin and TRAIL in Triple-Negative Breast Tumor Cells

Women with triple-negative breast cancer (TNBC) have a worse prognosis compared with other breast cancer subtypes. Hormonal or Herceptin-based therapies were found to be ineffective because of the loss of target receptors, such as ER, PR, and HER-2 amplification. Conventional chemo- and/ or radiation therapy also seems to have limited efficacy in TNBC patients. We studied the effects of cisplatin plus TRAIL on 1 normal and 2 TNBC cells in vitro. The in vitro studies indicate that cisplatin plus TRAIL significantly enhanced cell death in TNBC cell lines CRL2335 and MDA-MB-468 by approximately 60%-70% compared with approximately 10%-15% in CRL8799 normal breast cell line. Treatment with cisplatin/TRAIL also inhibited the expression of EGFR, p63, survivin, Bcl-2, and Bcl-xL in TNBC cells. Specific inhibition of EGFR and/or p63 protein in TNBC cells by small interfering RNA (siRNA) does not increase TRAIL-induced apoptosis. However, inhibition of survivin by siRNA enhances TRAIL-induced apoptosis. These observations suggested the possibility that survivin played an important role in cisplatin plus TRAIL-induced apoptosis in TNBC cells. In vivo experiments, treatment of mice with cisplatin plus TRAIL resulted in a significant inhibition of CRL2335 xenograft tumors compared with untreated control tumors. Taken together the data suggest that cisplatin plus TRAIL treatment have the potential of providing a new strategy for improving the therapeutic outcome in TNBC patients.

Abstract S5-4: Synthetic-Lethality of Triple-Negative Breast Cancers Via the MYC Oncogene Pathway

Abstract Background: Breast cancer lacking the estrogen, progesterone and ERBB2 receptors, (“triple-negative”) encompasses an aggressive sub-type that is associated with a higher incidence of early tumor recurrence and poor patient outcome, particularly after distant relapse. The lack of validated targets means that only conventional cytotoxic chemotherapy offers significant benefits, albeit with associated toxicities and lack of specificity. Among many potential therapeutic targets in triple-negative breast cancer, MYC may be important as its function is elevated in many breast cancers with aggressive phenotypes. Methods: In this study we investigate the association between MYC abundance in primary breast tumors using two large clinical cohorts and examined MYC mRNA and protein abundance as well as the expression of a MYC gene signature. We next examined the association between the MYC gene signature and patient response to neoadjuvant chemotherapy as well as disease-free survival in the ISPY TRIAL. Furthermore, we examined whether high MYC expression in triple-negative tumors might provide a new therapeutic approach in this tumor sub-type. We tested a synthetic-lethal strategy to kill triple-negative breast cells with abundant MYC expression by inhibiting cyclin-dependent kinases (CDKs). Results: We find that human triple-negative breast cell lines have significantly elevated MYC mRNA and protein expression. These results were validated using primary patient tumor samples from the neoadjuvant ISPY TRIAL (N= 138 patients) as well as a separate cohort of primary tumor samples for which quantitative protein expression was determined (N= 208 patients). In the ISPY TRIAL an increased expression of the MYC gene signature was associated with worse outcome in patients with substantial residual tumor burden after neoadjuvant chemotherapy (RCB Class II and III). We sought to exploit this increased MYC activity in triple-negative tumors using a synthetic-lethal approach dependent on cyclin-dependent kinase (CDK) inhibition. We found that triple-negative breast tumor cells undergo cell death in a MYC-dependent manner following treatment with several CDK inhibitors, including SCH72796 that is in Phase II clinical trials. We find that CDK inhibition effectively induces tumor regression in human tumor xenografts. CDK inhibition in triple-negative breast cells up-regulates the pro-apoptotic BCL2 family member BIM. Our findings support the exploration of CDK inhibitors as treatment for triple-negative breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S5-4.