Receptor-expressing Breast Cancer Cells Research Articles

Iron oxide nanoparticle encapsulated; folic acid tethered dual metal organic framework-based nanocomposite for MRI and selective targeting of folate receptor expressing breast cancer cells

Metal organic frameworks (MOFs) have been used as an efficient carrier for many therapeutic agents for cancer due to their unique properties. Even though numerous discrete MOFs have been used for biomedical applications, the traditional MOF-based composites are mainly limited to the solitary MOF source and functionalities. Herein, we report a novel and facile strategy for the design and fabrication of iron oxide (IO) nanoparticle encapsulated dual MOF composite, decorated with folic acid (FA) as targeting agent and Quercetin (Q) as drug payload. Due to the presence of superparamagnetic IO nanoparticles, the composites inherently show the potential usage for magnetic resonance imaging (MRI). By integrating dual MOFs (ZIF-8/ZIF-67) with the targeting agents and drug the resultant nanocomposites (IO/Z8-Z67/FA/Q) exhibited effective anticancer activities on FA receptor-positive breast cancer cell culture model (MDA-MB-231). The IO/Z8-Z67/FA/Q nanocomposite enhanced apoptosis and cytotoxicity in the MDA-MB-231 cell line (expressing folate receptors) as compared to the MCF-7 cell line (lacking the folate receptors). The uptake analysis of the nanocomposite was also carried out using flow cytometry to confirm FA targeting. Mechanistically, the folate receptor targeted IO/Z8-Z67/FA/Q nanocomposite delivery to MDA-MB-231 cells caused high ROS generation and nuclear fragmentation, leading to cell death. The results indicate that the IO/Z8-Z67/FA/Q can be a potential theranostics agent for effective cancer therapy and targeting. The proposed IO/Z8-Z67/FA composite was also used to load another anticancer drug like 5-fluorouracil (5FU) and cytotoxicity results show promising outcomes suggesting the current nanocomposite as all-purpose drug delivery carrier.

Tuning the protein corona of PLGA nanoparticles: Characterization of trastuzumab adsorption behavior and its cellular interaction with breast cancer cell lines

The protein corona (PC) that forms around nanoparticles (NPs) upon exposure to biological media can “mask” targeting ligands that have been attached covalently or adsorptively to the NP surface, potentially decreasing targeting efficiency. In this study, NPs composed of the biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) were incubated with human serum albumin (HSA) as a model protein and trastuzumab as a functional antibody and consequently tested for their interaction with HER2/neu receptor-expressing cell lines. Adsorption behavior was investigated in the presence of competing human plasma (HP) and serum (HS) to determine whether trastuzumab remains adsorptively bound in the PC or “masking” and even displacement effects are to be expected. By fluorescence quantification and LC-MS/MS measurements, trastuzumab could be classified as “hard corona” protein, while HSA appeared as a “soft corona” protein. It was shown that while undirected cell uptake occurred upon exposition to HSA-incubated NPs, through the adsorptive decoration with trastuzumab, HER2/neu receptor-expressing breast cancer cell lines can be selectively addressed.

Abstract 1231: Targeting JNK1/2 induces a potent cytostatic response in estrogen receptor expressing breast cancer cells, with induction of autophagy and senescence as measurable outcomes

Abstract The c-Jun N-terminal kinases, JNK1/2 are members of the mitogen activated protein kinase (MAPK) family that can mediate inflammatory responses, cell differentiation, cell proliferation, and/or cell death. In breast cancer, JNK signaling has been associated with increased proliferation, angiogenesis, and tumor metastasis. Also, inhibition of JNK has been shown to promote tumor cytostasis, but this inhibition also attenuates the cytotoxic effects of certain chemotherapeutics, calling into question the therapeutic potential of JNK inhibitors. In this study, we hypothesized that JNK1/2 inhibition induced autophagy, as a mechanism of protection against chemotherapy- and endocrine-induced apoptosis. Autophagy is a normal physiologic process required for the elimination of damaged mitochondria and misfolded proteins that can be induced in cancer cells under certain stresses such as hypoxia and drug insult to provide protection from death. To characterize the role of JNK1/2 in autophagy, we utilized two ERα expressing human breast cancer cell lines, T47-D and MCF-7. Autophagy flux experiments were performed in which the steady state levels of the autophagy protein LC3 II were analyzed in control cells versus cells undergoing JNK1/2 inhibition with the non-reversible JNK-IN8 inhibitor. JNK-IN8 was used as a single agent or in combination with estradiol and/or antiestrogen treatment. These studies reproducibly identified JNK1/2 inhibition as a mechanism of autophagy activation. The induction of autophagy correlated with sustained inhibition of proliferation as determined by cell counts, MTT, and clonogenic assays, and induction of senescence (in T47-D cells) as determined by the expression of β-galactosidase. In contrast, induction of apoptosis was not a major outcome of JNK1/2 inhibition. These pre-clinical studies provide strong evidence that JNK1/2 is an attractive molecular target to improve the treatment of endocrine responsive breast cancer, but they also emphasize that cells are surviving by autophagy and/or senescence. Because cell survival by autophagy and/or senescence could ultimately lead to breast cancer relapse, ongoing studies are addressing approaches to effectively kill these surviving cell populations. Citation Format: Kyler Herrington, Carol Joseph, Samar Abdulmoneim, Allison Lewis, Jingwen Cai, Hannah Youngblood, Yutao Liu, Patricia Schoenlein. Targeting JNK1/2 induces a potent cytostatic response in estrogen receptor expressing breast cancer cells, with induction of autophagy and senescence as measurable outcomes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1231.

Identifying the Responses from the Estrogen Receptor-Expressed MCF7 Cells Treated in Anticancer Drugs of Different Modes of Action Using Live-Cell FTIR Spectroscopy.

Recently, we have shown that changes in Fourier transform infrared (FTIR) spectra of living MDA-MB-231 cells (a triple negative cell line) upon exposure to anticancer drugs reflect the changes in the cellular compositions which are correlated to the modes of action of drugs. In the present study, MCF7 cells (an estrogen receptor expressing breast cancer cell line) were exposed to three anticancer drugs belonging to two well-characterized anticancer classes: selective estrogen receptor modulators (SERMs) and DNA-intercalating agent. First, we evaluated if the changes in the spectrum of cells are according to the modes of action of drugs and the characteristics of the MCF7 cell line in the same way as the MDA-MB-231 cell. Living MCF7 cells were treated in the three drugs at half maximal inhibitory concentration (IC50), and the difference spectra were analyzed using principal component analysis (PCA). The results demonstrated clear separation between tamoxifen/toremifene (SERM)-treated cells from the doxorubicin (DNA-intercalator)-treated and untreated cells (control). Tamoxifen and toremifene induced similar spectral changes in the cellular compositions of MCF7 cells and lead to the clustering of these two drugs in the same quadrant of the principal component 1 (PC1) versus PC2 score plots. The separation is mostly attributed to their similar modes of actions. However, doxorubicin-treated MCF7 cells highlighted spectral changes that mainly occur in bands at 1085 and 1200–1240 cm–1, which could be associated with the DNA-intercalation effects of the drug. Second, the pairwise PCA at various individual time points was employed to investigate whether the spectral changes of MCF7 and MDA-MB-231 cells in response to the IC50 of tamoxifen/toremifene and doxorubicin are dependent on the characteristics of the cell lines. The estrogen-expressing MCF7 cells demonstrated significant differences in response to the SERMs in comparison to the triple negative MDA-MB-231 cells, suggesting that different modes of action have taken place in the two tested cell lines. In contrast, the doxorubicin-treated MDA-MB-231 and MCF7 cells show similar changes in 1150–950 cm–1, which indicates that the DNA intercalation effect of doxorubicin is found in both cell lines. The results have demonstrated that live-cell FTIR analysis is sensitive to the different modes of action from the same drugs on cells with different characteristics.

In vivo and in vitro investigation of cytotoxic and antitumor activities of polyphenolic leaf extract of Hibiscus sabdariffa against breast cancer cell lines

In folk medicine Hibiscus sabdariffa (HS) L. (Malvaceae) has been used for the treatment of several ailments. HS leaf is highly enriched with polyphenol and many polyphenols is a very promising anticancer bioactive marker as inhibition of cell propagation and induction of differentiation. Therefore the main object of this research is to investigate the cytotoxic and anti-cancer activity of leaf extract of Hibiscus sabdariffa. Cytotoxic effect of polyphenol extract of Hibiscus sabdariffa leaves on breast cancer cell line (MCF-7, T-47-D and MDA-MB-231) were investigated using SRB assay. Total phenolic and flavonoids contents were determined using HPTLC methods. In vitro, antioxidant activity was also determined. Antimetastasis, neovascularization potential of the extract was carried out in vivo study on the zebra fish model compared with standard drug paclitaxel. Extract of Hibiscus sabdariffa showed 50% growth inhibition of MCF-7, MDA-MB-231, and T-47-D cells in dose dependant manner, respectively as compared to standard drug adriamycin. HPTLC analysis revealed the presence of flavonoids and phenolic compounds and Significant (p<0.05) antioxidant activity was observed. Consistent with in vitro data, the polyphenol extract showed significant tumor growth inhibition (45 ± 2.24%) in zebra fish equivalent to paclitaxel. The extract significantly reduced the angiogenesis (15 ± 0.7%) (p<0.05) and inhibit the metastasis compared to the negative control group. The effective dose of 80 μg treatments reduced up to 60% of the tumor cells compared to the model. Similar reductions in neovascularization level and mortality levels were observed. Results reflected cytotoxicity in breast cancer cell lines and inhibits the growth of breast cancer cell lines (MDA-MB-231) and estrogen receptor-expressing breast cancer cell lines (MCF-7 and T-47-D), significant prevention of metastasis, and angiogenesis in zebra fish suggesting Hibiscus sabdariffa has the potential to be a good anti-cancer agent for breast cancer.

Targeting of prostate-specific membrane antigen for radio-ligand therapy of triple-negative breast cancer

BackgroundTriple-negative breast cancer has extremely high risk of relapse due to the lack of targeted therapies, intra- and inter-tumoral heterogeneity, and the inherent and acquired resistance to therapies. In this study, we evaluate the potential of prostate-specific membrane antigen (PSMA) as target for radio-ligand therapy (RLT).MethodsTube formation was investigated after incubation of endothelial HUVEC cells in tumor-conditioned media and monitored after staining using microscopy. A binding study with 68Ga-labeled PSMA-addressing ligand was used to indicate targeting potential of PSMA on tumor-conditioned HUVEC cells. For mimicking of the therapeutic application, tube formation potential and vitality of tumor-conditioned HUVEC cells were assessed following an incubation with radiolabeled PSMA-addressing ligand [177Lu]-PSMA-617. For in vivo experiments, NUDE mice were xenografted with triple-negative breast cancer cells MDA-MB231 or estrogen receptor expressing breast cancer cells MCF-7. Biodistribution and binding behavior of [68Ga]-PSMA-11 was investigated in both tumor models at 30 min post injection using μPET. PSMA- and CD31-specific staining was conducted to visualize PSMA expression and neovascularization in tumor tissue ex vivo.ResultsThe triple-negative breast cancer cells MDA-MB231 showed a high pro-angiogenetic potential on tube formation of endothelial HUVEC cells. The induced endothelial expression of PSMA was efficiently addressed by radiolabeled PSMA-specific ligands. 177Lu-labeled PSMA-617 strongly impaired the vitality and angiogenic potential of HUVEC cells. In vivo, as visualized by μPET, radiolabeled PSMA-ligand accumulated specifically in the triple-negative breast cancer xenograft MDA-MB231 (T/B ratio of 43.3 ± 0.9), while no [68Ga]-PSMA-11 was detected in the estrogen-sensitive MCF-7 xenograft (T/B ratio of 1.1 ± 0.1). An ex vivo immunofluorescence analysis confirmed the localization of PSMA on MDA-MB231 xenograft-associated endothelial cells and also on TNBC cells.ConclusionsHere we demonstrate PSMA as promising target for two-compartment endogenous radio-ligand therapy of triple-negative breast cancer.

Induced Tamoxifen Resistance is Mediated by Increased Methylation of E-Cadherin in Estrogen Receptor-Expressing Breast Cancer Cells

Estrogen receptor-positive breast cancers are treated with tamoxifen, a drug that competitively inhibits the binding of estrogen to its receptor. Resistance to tamoxifen is a major hurdle in effective management of target breast cancer patient population. A number of dynamic changes within the tumor microenvironment, including the phenomenon of epithelial to mesenchymal transition (EMT), determine the response to endocrine therapy. EMT is marked by silencing or suppression of epithelial marker, E-Cadherin and we found significantly down-regulated E-Cadherin, among other epithelial markers, and a significantly up-regulated mesenchymal marker, Twist, among other mesenchymal markers, in a model system that comprised of tamoxifen sensitive MCF-7 cells and their tamoxifen-resistant counterparts, MCF-7-TAM, developed by chronic and escalating exposure of parental cells to tamoxifen. Further, E-cadherin, but not Twist, was differentially expressed in MCF-7-TAM cells because of differential methylation. Treatment with demethylating agent 5-azacytidine increased the expression of E-cadherin thus verifying a role of methylation in its silencing and, moreover, 5-azacytidine treatment also re-sensitized MCF-7-TAM cells to tamoxifen, as evaluated by assays for viability, apoptosis and migration potential. The 5-azacytidine effects were similar to effects of E-cadherin overexpression in MCF-7-TAM cells. This work describes novel mechanism of E-cadherin downregulation in tamoxifen resistant breast cancer cells. Further studies are needed to exploit this information for betterment of breast cancer therapy.

Highlights of This Issue

Chen and colleagues analyzed next generation sequencing of 216 advanced thyroid carcinomas identifying prevalent targetable MAPK pathway activating mutations. Secondary mutations in PIK3CA which may confer resistant to single agent targeted therapy were 8x more frequent compared to the thyroid TCGA. In this retrospective analysis, when targeted therapy to actionable pathway mutations was used, an improved survival was noted in patients with poorly differentiated thyroid carcinomas compared to similar tumors lacking mutations or showing only tumor suppressor mutations. The combined findings lend support for mutational testing across targetable pathways to ultimately benefit patients with advanced thyroid carcinoma through targeted therapy.Resistance to T-DM1 poses a challenge in therapy for HER2-positive breast cancer. A better understanding of the molecular mechanisms of primary and acquired resistance to T-DM1 is particularly important for the development of new therapeutic strategies. Breed and colleagues established resistant cells and identified different features of T-DM1 resistant cells. Their findings show the complexities of T-DM1 resistance in that the 2 models they studied showed little overlap in identified resistance mechanisms. The data also suggest possible therapeutic strategies using combinations with inhibitors that target signal transduction resistance pathways for overcoming T-DM1 resistance.Brain tumors are the leading cause of cancer-related deaths in children. Hence, there is an unmet need to develop novel therapies to improve outcome. Telomerase-dependent incorporation of 6-thio-dG into telomeres leads to telomere damage and cell death of primary stem-like cells derived from pediatric patients with high-risk brain tumors. The effect of 6-thio-dG is persistent after drug withdrawal. In vivo, 6-thio-dG delays tumor growth, crosses the blood-brain barrier and specifically targets tumor cells in the mouse brain. These findings suggest that 6-thio-dG is a promising novel approach to treat therapy-resistant pediatric brain tumors.5-fluorouracil (5-FU) is often given to breast cancer patients, after curative surgery of primary tumor to prevent tumor recurrence. However, 5-FU accelerated lung metastasis, when it was administered upon the complete resection of the tumors arising from the orthotopic injection of a mouse breast cancer cell line. The acceleration was associated with enhanced intrapulmonary infiltration of neutrophils, which were attracted by tumor cell-derived chemokines and expressed abundantly prokineticin-2 with a capacity to support the proliferation of prokinectin-2 receptor-expressing breast cancer cells. Thus, adjuvant chemotherapy with 5-FU may have adverse effects on some breast cancer patients.

Abstract 720: Dual MMP-7-proximity-activated and folate-targeted nanoparticles for siRNA delivery

Abstract We have developed a dual, folic acid (FA) and matrix metalloproteinase (MMP), proximity-activated targeting (PAT) smart polymeric nanoparticle (SPN) for tumor-specific siRNA delivery. The nanocarrier was designed to expose concealed FA ligands specifically in regions of elevated MMP activity, resulting in targeted uptake by folate receptor-expressing cells. Site-selective chemistry and reversible addition-fragmentation chain transfer (RAFT) polymerization were used to prepare diblock copolymers {p(DMAEMA)-b-p(DMAEMA-PAA-BMA) (SPN)} terminated with either MMP-7 peptide coupled to a Y-shaped 20 kDa PEG (PAT-SPN) or a 2 kDa linear PEG coupled to FA (FA-SPN). These amphiphilic polymers were co-assembled in aqueous media, forming mixed micelles consisting of 0, 25, 50, 75, 100 mol% FA and presenting zeta potentials of -0.21, -0.20, +1.75, +6.71 and +12.2 mV, respectively. Cleavage by MMP-7 was confirmed by decreased particle diameter (e.g., 25% FA micelle diameter decreased from 54 to 48 nm) and simultaneous increase in zeta potential (-0.20 to +8.11 mV). These data suggest that MMP-7 removal of the PEG corona from the nanocarriers revealed the underlying polycationic charge of the pDMAEMA core. Intracellular uptake of PAT/FA-SPNs was investigated in FA receptor-expressing breast cancer cells by confocal microscopy and flow cytometry. Pre-treatment with exogenous MMP-7 to mimic the proteolytically active microenvironment of human breast cancers, elevated cellular uptake 2.3-fold for single targeting PAT-SPN and 4.6-fold for dual-targeting PAT/FA-SPN compared to intact PAT-SPN. Cell uptake of uncleaved PAT/FA-SPN was statistically equivalent to untreated cells. These data suggest that the 20 kDa Y-shaped PEG effectively shields both the underlying cationic charge and the FA, and also blocks nonspecific cellular interactions of the SPN. 75-100% FA-SPN polymer reduced the dependency of cellular uptake on MMP-7 activity. Excess FA reduced the uptake of FA-SPN-containing micelles, consistent with FA receptor-dependent uptake following MMP activation. Functional protein knockdown delivered by PAT/FA-SPN was validated using luciferase-expressing MDA-MB-231 cells. 50% PAT/FA-SPN, pre-activated with MMP-7and loaded with luciferase siRNA, resulted in significant suppression of luciferase expression relative to controls (no MMP activation or competitive inhibition with free FA or scrambled siRNA), confirming the efficient dual PAT/FA targeting and endosomal escape of this designed nanoparticle. Unlike other similar technologies, this micelle design is optimized for use in vivo. Animal studies confirm an extended blood half-life, appropriate organ distribution, minimal toxicity and significant siRNA activity in mammary tumors. 1. Li, H. et al. Adv. Func. Mater. 2013. 10.1002/adfm.201202215 Support DoD CDMRP (W81XWH-10-1-0445/0446); Vanderbilt Institute of Nanoscale Science &amp; Engineering Citation Format: Todd D. Giorgio, Hongmei Li, Martina Miteva, Kellye C. Kirkbride, Ming Cheng, Chris E. Nelson, Craig L. Duvall. Dual MMP-7-proximity-activated and folate-targeted nanoparticles for siRNA delivery. [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 720. doi:10.1158/1538-7445.AM2014-720

Quantitative Analysis of the Anti-Proliferative Activity of Combinations of Selected Iron-Chelating Agents and Clinically Used Anti-Neoplastic Drugs

Recent studies have demonstrated that several chelators possess marked potential as potent anti-neoplastic drugs and as agents that can ameliorate some of the adverse effects associated with standard chemotherapy. Anti-cancer treatment employs combinations of several drugs that have different mechanisms of action. However, data regarding the potential interactions between iron chelators and established chemotherapeutics are lacking. Using estrogen receptor-positive MCF-7 breast cancer cells, we explored the combined anti-proliferative potential of four iron chelators, namely: desferrioxamine (DFO), salicylaldehyde isonicotinoyl hydrazone (SIH), (E)-N′-[1-(2-hydroxy-5-nitrophenyl)ethyliden] isonicotinoyl hydrazone (NHAPI), and di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), plus six selected anti-neoplastic drugs. These six agents are used for breast cancer treatment and include: paclitaxel, 5-fluorouracil, doxorubicin, methotrexate, tamoxifen and 4-hydroperoxycyclophosphamide (an active metabolite of cyclophosphamide). Our quantitative chelator-drug analyses were designed according to the Chou-Talalay method for drug combination assessment. All combinations of these agents yielded concentration-dependent, anti-proliferative effects. The hydrophilic siderophore, DFO, imposed antagonism when used in combination with all six anti-tumor agents and this antagonistic effect increased with increasing dose. Conversely, synergistic interactions were observed with combinations of the lipophilic chelators, NHAPI or Dp44mT, with doxorubicin and also the combinations of SIH, NHAPI or Dp44mT with tamoxifen. The combination of Dp44mT with anti-neoplastic agents was further enhanced following formation of its redox-active iron and especially copper complexes. The most potent combinations of Dp44mT and NHAPI with tamoxifen were confirmed as synergistic using another estrogen receptor-expressing breast cancer cell line, T47D, but not estrogen receptor-negative MDA-MB-231 cells. Furthermore, the synergy of NHAPI and tamoxifen was confirmed using MCF-7 cells by electrical impedance data, a mitochondrial inner membrane potential assay and cell cycle analyses. This is the first systematic investigation to quantitatively assess interactions between Fe chelators and standard chemotherapies using breast cancer cells. These studies are vital for their future clinical development.

Long chain lipid based tamoxifen NLC. Part II: Pharmacokinetic, biodistribution and in vitro anticancer efficacy studies

Long chain lipid (LCL) based tamoxifen loaded nanostructured lipid carriers (Tmx-NLCs) meant to target intestinal lymphatic systems (ILSs) was developed and characterized previously. The aim of the present work was to evaluate in vitro efficacy of developed Tmx-NLC against breast cancer cell lines and to confirm the hypothesis of targeting ILS after single dose oral administration. In vitro anticancer activity of Tmx-NLC was assessed in human estrogen receptor expressing breast cancer cell lines viz. MCF-7 and ZR-75-1. The study revealed relatively improved activity for Tmx-NLC compared to free Tmx against MCF-7 cells. However, the activity was compromised against ZR-75-1 cells which could be attributed to its up regulation of MUC1 gene. Confocal and flow cytometric analysis revealed remarkable intracellular uptake of Tmx-NLC and its localization in nuclear and perinuclear region of cells. Tmx-NLC exhibited distinctly different pharmacokinetic profile compared to Tamoxifen suspension (Tmx-susp) and exhibited an increment in the bioavailability by 2.71-fold and prolonged the T1/2 by 7.10-fold. Moreover, detectable drug concentration in mesenteric lymph nodes justifies our hypothesis of targeting ILS and explains the major uptake of Tmx to occur via lymphatic system.

A transformation in the mechanism by which the urokinase receptor signals provides a selection advantage for estrogen receptor-expressing breast cancer cells in the absence of estrogen

Binding of urokinase-type plasminogen activator (uPA) to its receptor, uPAR, in estrogen receptor-α (ERα) expressing breast cancer cells, transiently activates ERK downstream of FAK, Src family kinases, and H-Ras. Herein, we show that when uPAR is over-expressed, in two separate ERα-positive breast cancer cell lines, ERK activation occurs autonomously of uPA and is sustained. Autonomous ERK activation by uPAR requires H-Ras and Rac1. A mutated form of uPAR, which does not bind vitronectin (uPAR-W32A), failed to induce autonomous ERK activation. Expression of human uPAR or mouse uPAR but not uPAR-W32A in MCF-7 cells provided a selection advantage when these cells were deprived of estrogen in cell culture for two weeks. Similarly, MCF-7 cells that express mouse uPAR formed xenografts in SCID mice that survived and increased in volume in the absence of estrogen supplementation, probably reflecting the pro-survival activity of phospho-ERK. Autonomous uPAR signaling to ERK was sensitive to the EGFR tyrosine kinase inhibitors, Erlotinib and Gefitinib. The transition in uPAR signaling from uPA-dependent and transient to autonomous and sustained is reminiscent of the transformation in ErbB2/HER2 signaling observed when this gene is amplified in breast cancer. uPAR over-expression may provide a pathway for escape of breast cancer cells from ERα-targeting therapeutics.

Triple negative breast cancer cell lines: One tool in the search for better treatment of triple negative breast cancer

Breast cancer is the most frequently diagnosed cancer in women and one of the leading causes of cancer death for women. Worldwide, over 1.3 million cases of invasive breast cancer are diagnosed, and more than 450,000 women die from breast cancer annually [1]. In the US, approximately 200,000 cases of invasive breast cancer and 50,000 cases of in situ breast cancer will be diagnosed annually, and more than 40,000 women die from breast cancer each year – second only to lung cancer [2]. The mortality due to breast cancer has been declining in the US since 1990 [2]. The continuing decrease in mortality from breast cancer has been attributed to early detection due to screening, improved adjuvant therapy, and more recently to decreases in the incidence due to lowered rates of usage of hormone replacement therapy [3, 4]. Despite the decreased incidence and mortality, breast cancer remains a major cause of cancer mortality for women and accounts for 15% of all cancer deaths in women in the US [2]. Clinically, breast cancer can be divided into distinct subtypes that have prognostic and therapeutic implications. Breast cancer patients routinely have the expression of estrogen receptor (ER), progesterone receptor (PR), and amplification of HER-2/Neu evaluated [5]. These markers allow classification of breast cancer tumors as hormone receptor positive tumors, HER-2/Neu amplified tumors, and those tumors which do not express ER, PR, and do not have HER-2/Neu amplification. The latter group is referred to as triple-negative breast cancer (TNBC) based on the lack of these three molecular markers. Generally, hormone receptor expressing breast cancers have a more favorable prognosis than either those with HER-2/Neu amplification or those that are triple-negative [5]. While all breast tumor types may be treated with chemotherapy, therapeutic options in both early and late stage breast cancer are affected significantly by the expression of these three markers. Tumors that express ER and PR are treated with agents that interfere with hormone production or action [5]. Tumors that have amplified HER-2/Neu are treated with agents that inhibit HER-2/Neu [5]. These targeted therapies are the mainstay of the successful outcomes seen in hormone receptor positive and HER-2/Neu amplified tumors. Both early stage and advanced TNBC tumors are treated with predominantly chemotherapy [5]. TNBC represents approximately 10–15% of all breast cancers and patients with TNBC have a poor outcome compared to the other subtypes of breast cancer [6]. Interestingly, the incidence of TNBC in African American women is two to three times higher than other ethnic groups, although the reason for this has not been elucidated [6, 7]. Given the lack of validated molecular targets and the poor outcome in patients with TNBC, there is a clear need for a greater understanding of TNBC at all levels and for the development of better therapies. Cancer cell lines have proved useful in laboratory and preclinical investigations since the first cell line was established more than 50 years ago [8]. For example, the anti-HER-2/Neu 4D5 mouse monoclonal antibody (which was humanized to create trastuzumab) had anti-tumor effects as a single agent and acted synergistically with chemotherapeutic agents in breast cancer cell lines that had amplified HER-2/Neu but showed no effect in cell lines lacking amplified HER-2/Neu [9–12]. These results formed the basis for clinical trials of trastuzmab and predicted the outcomes of these clinical trials. Similarly, preclinical studies using xenografts of a hormone receptor expressing breast cancer cell line have accurately predicted the outcomes of clinical trials comparing aromatase inhibitors to tamoxifen and to the combination of tamoxifen and aromatase inhibitors [13]. In this review, we will describe triple negative breast cancer cell lines and discuss their utility and the limitations of these cell lines in the investigation of TNBC (mindful of Harry Callahan’s admonition cited above).

Delphinidin inhibits cell proliferation and invasion via modulation of Met receptor phosphorylation

The HGF/Met signaling pathway is deregulated in majority of cancers and is associated with poor prognosis in breast cancer. Delphinidin, present in pigmented fruits and vegetables possesses potent anti-oxidant, anti-inflammatory and anti-angiogenic properties. Here, we assessed the anti-proliferative and anti-invasive effects of delphinidin on HGF-mediated responses in the immortalized MCF-10A breast cell line. Treatment of cells with delphinidin prior to exposure to exogenous HGF resulted in the inhibition of HGF-mediated (i) tyrosyl-phosphorylation and increased expression of Met receptor, (ii) phosphorylation of downstream regulators such as FAK and Src and (iii) induction of adaptor proteins including paxillin, Gab-1 and GRB-2. In addition, delphinidin treatment resulted in significant inhibition of HGF-activated (i) Ras-ERK MAPKs and (ii) PI3K/AKT/mTOR/p70S6K pathways. Delphinidin was found to repress HGF-activated NFκB transcription with a decrease in (i) phosphorylation of IKKα/β and IκBα, and (ii) activation and nuclear translocation of NFκB/p65. Inhibition of HGF-mediated membrane translocation of PKCα as well as decreased phosphorylation of STAT3 was further observed in delphinidin treated cells. Finally, decreased cell viability of Met receptor expressing breast cancer cells treated with delphinidin argues for a potential role of the agent in the prevention of HGF-mediated activation of various signaling pathways implicated in breast cancer.

Restoration of Transforming Growth Factor-β Signaling through Receptor RI Induction by Histone Deacetylase Activity Inhibition in Breast Cancer Cells

The loss of transforming growth factor-beta (TGF-beta) response due to the dysregulation of TGF-beta receptors type I (RI) and type II (RII) is well known for its contribution to oncogenesis. Estrogen receptor-expressing breast cancer cells are refractory to TGF-beta-mediated growth control because of the reduced expression of TGF-beta receptors. Although RII is required for the binding of TGF-beta to RI, RI is responsible for directly transducing TGF-beta signals through the Smad protein family. Treatment of estrogen receptor-expressing MCF-7L and ZR75 breast cancer cells with the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) led to a dramatic induction of RI. Accumulation of acetylated histones H3 and H4 was observed in the SAHA-treated cells. Chromatin immunoprecipitation analysis followed by PCR with RI promoter-specific primers indicated an accumulation of acetylated histones in chromatin associated with the RI gene, suggesting that histone deacetylation was involved in the transcriptional inactivation of RI. SAHA treatment stimulated RI promoter activity through the inhibition of Sp1/Sp3-associated HDAC activity. Histone acetyltransferase p300 stimulated RI promoter activity, thus further confirming the involvement of HDAC activity in the transcriptional repression of RI. Significantly, SAHA-mediated RI regeneration restored the TGF-beta response in breast cancer cells.