Human SKBR3 Research Articles

USP22 overexpression fails to augment tumor formation in MMTV-ERBB2 mice but loss of function impacts MMTV promoter activity.

The Ubiquitin Specific Peptidase 22 (USP22), a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) histone modifying complex, is overexpressed in multiple human cancers, but how USP22 impacts tumorigenesis is not clear. We reported previously that Usp22 loss in mice impacts execution of several signaling pathways driven by growth factor receptors such as erythroblastic oncogene B b2 (ERBB2). To determine whether changes in USP22 expression affects ERBB2-driven tumorigenesis, we introduced conditional overexpression or deletion alleles of Usp22 into mice bearing the Mouse mammary tumor virus-Neu-Ires-Cre (MMTV-NIC) transgene, which drives both rat ERBB2/NEU expression and Cre recombinase activity from the MMTV promoter resulting in mammary tumor formation. We found that USP22 overexpression in mammary glands did not further enhance primary tumorigenesis in MMTV-NIC female mice, but increased lung metastases were observed. However, deletion of Usp22 significantly decreased tumor burden and increased survival of MMTV-NIC mice. These effects were associated with markedly decreased levels of both Erbb2 mRNA and protein, indicating Usp22 loss impacts MMTV promoter activity. Usp22 loss had no impact on ERBB2 expression in other settings, including MCF10A cells bearing a Cytomegalovirus (CMV)-driven ERBB2 transgene or in human epidermal growth factor receptor 2 (HER2)+ human SKBR3 and HCC1953 cells. Decreased activity of the MMTV promoter in MMTV-NIC mice correlated with decreased expression of known regulatory factors, including the glucocorticoid receptor (GR), the progesterone receptor (PR), and the chromatin remodeling factor Brahma-related gene-1 (BRG1). Together our findings indicate that increased expression of USP22 does not augment the activity of an activated ERBB2/NEU transgene but impacts of Usp22 loss on tumorigenesis cannot be assessed in this model due to unexpected effects on MMTV-driven Erbb2/Neu expression.

Proteolytic Targeting Chimeras with Specificity for Plasma Membrane and Intracellular Estrogen Receptors.

Decisions regarding the assignment of hormonal therapy for breast cancer are based solely upon the presence of nuclear estrogen receptors (ERs) in biopsied tumor tissue. This is despite the fact that the G-protein-coupled estrogen receptor (GPER) is linked to advanced breast cancer and is required for breast cancer stem cell survival, an observation that suggests that effective endocrine therapy should also target this receptor. Here, two ER/GPER-targeting proteolytic chimeras (UI-EP001 and UI-EP002) are described that effectively degrade ERα, ERβ, and GPER. These chimeras form high-affinity interactions with GPER and ER with binding dissociation constants of ∼30 nM and 10-20 nM, respectively. Plasma membrane and intracellular GPER and nuclear ER were degraded by UI-EP001 and UI-EP002, but not by a partial proteolytic targeting chimera (PROTAC) lacking its estrogen-targeting domain. Pretreatment of cells with the proteasomal inhibitor, MG132, blocked UI-EP001 and UI-EP002 proteolysis, while the lysosomotrophic inhibitor, chloroquine, had no effect. The off-target activity was not observed against recombinant β1-adrenergic receptor or CXCR4. Target specificity was further demonstrated in human MCF-7 cells where both drugs effectively degraded ERα, ERβ, and GPER, sparing the progesterone receptor (PR). UI-EP001 and UI-EP002 induced cytotoxicity and G2/M cell cycle arrest in MCF-7 breast cancer and human SKBR3 (ERα-ERβ-GPER+) breast cancer cells but not human MDA-MB-231 breast cancer cells that do not express functional GPER/ER. These results suggest that it is possible to develop a receptor-based strategy of antiestrogen treatment for breast cancer that targets both plasma membrane and intracellular estrogen receptors.

SAT-LB137 Rescue of Misfolded G-Protein Coupled Estrogen Receptor, GPER, from the Endoplasmic Reticulum via Natural and Synthetic Estrogens

GPER bears structural and functional characteristics shared by members of the G-protein coupled receptor (GPCR) superfamily, the largest class of cell surface receptors, with more than 800 members encoded in the human genome. GPER is localized predominately in intracellular membranes, in many but not all cell types, and its surface expression is modulated by steroid hormones and during tissue homeostasis. An intracellular staining pattern is not unique among GPCRs, which deploy a diverse array of posttranslational regulatory mechanisms to determine cell surface expression, effectively regulating cognate ligand binding and activity.Here, we show nascent GPER undergoes strict quality control via endoplasmic reticulum associated degradation (ERAD) requiring direct poly-ubiquitinylation of GPER and valosin-containing protein VCP/p97-mediated segregation of misfolded proteins from the ER membrane to the cytoplasm for delivery to the 26S proteasome. Specifically, we find that inhibition of p97 using the pharmacological compound, CB-5083, or by doxycycline-inducible p97 shRNA results in the accumulation of immature glycosylated GPER in the ER. Inhibition of proteasome function facilitates anterograde trafficking with the transport of nonfunctional GPER to the plasma membrane as indicated by no increase in specific estrogen binding using 3H-17β-estradiol in a radioreceptor assay. The forward trafficking of misfolded GPER requires transit through the Golgi as treatment with brefeldin A (BFA) prevents GPER plasma membrane expression. Substitution of all three lysines (K333, K342, and K357) encoded in the cytoplasmic tail of GPER with arginines blunts its polyubiquitinylation and allows GPER to evade degradation by quality control but does not result in increased plasma membrane expression suggesting that additional structural motifs encoded within GPER control its anterograde trafficking. In contrast, functional GPER is recovered at the plasma membrane of human SKBR3 breast cancer cells treated with either 17β-estradiol or the GPER selective antagonist, G15, in the presence of cycloheximide resulting in increased surface GPER. Thus, our findings suggest that estrogens, both natural and synthetic, can function as pharmacochaperones capable of promoting the correct folding of GPER and enhanced expression of functional GPER at the plasma membrane.

Promotion on NLRC5 upregulating MHC-I expression by IFN-γ in MHC-I-deficient breast cancer cells.

Breast cancer is the most dominant cancer in women and the second most frequent cancer in the general population worldwide. NLRC5 critically transactivates MHC class I (classically HLA-ABC in human) which is crucial for cancer immunosurveillance. But the expressional and functional impairments of NLRC5 have been found in many cancers as a major mechanism of immune evasion. Promotion of NLRC5 with the enhancement of MHC class I contributes to cancer immunotherapy and counteraction against cancer immune evasion. In many cancers, IFN-γ promotes the expression of MHC class I involving NLRC5; however, it is unclear in breast cancer cells. In this study, qRT-PCR, western blot, and flow cytometry were used to detect the mRNAs and proteins of NLRC5, β2m, and HLA-ABC in MHC class I-deficient human SKBR3 breast cancer cells after IFN-γ treatment. It was shown that the relative levels of NLRC5 mRNA, β2m mRNA, and HLA-ABC α heavy chain mRNA, in concentrations of 50U/ml and 100U/ml IFN-γ groups, were statistically increased (p < 0.05) with dose dependent tendency compared with the control group. The protein levels of NLRC5 and β2m in concentrations of 50U/ml and 100U/ml IFN-γ groups, HLA-ABC (positive rates) in different concentrations of IFN-γ groups, were statistically increased (p < 0.05), with dose dependent tendency for NLRC5 and HLA-ABC, compared with the control group. Promotion of NLRC5 by IFN-γ with upregulation of MHC class I (HLA-ABC) in SKBR3 breast cancer cells, suggesting the contribution to counteracting cancer evasion from immunosurveillance and benefiting cancer immunotherapy.

The synthesis and characterization of a clickable-photoactive NAADP analog active in human cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing second messenger which triggers Ca2+ release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca2+ release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable. This ‘all-in-one-clickable’ NAADP (AIOC-NAADP) elicited Ca2+ release when microinjected into cultured human SKBR3 cells at low concentrations. In contrast, it displayed little activity in sea urchin egg homogenates where very high concentrations were required to elicit Ca2+ release. In mammalian cell homogenates, incubation with low concentrations of [32P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23 kDa protein(s). Competition between [32P]AIOC-NAADP and increasing concentrations of NAADP demonstrated that the labeling was selective. We show that this label recognizes and selectively photodervatizes the 23 kDa NAADP binding protein(s) in cultured human cells identified in previous studies using [32P]5-N3-NAADP.

Muscadine Grape Extract Prevents Proliferation of HER2 Positive Breast Cancer Cells in Association with a Decrease in Phosphorylation of AKT and mTOR

Breast cancer is the most common cancer among American women and the second leading cause of cancer death. Human epidermal growth factor receptor 2 (HER2) positive breast cancer, characterized by overexpression of the HER2 protein, is an aggressive type of breast cancer comprising about 20% of diagnosed cases. Although targeted HER2 drugs are successful in treating HER2 overexpressing breast cancer with up to 70% of patients responding to treatment, less than 10% of the patients achieve total remission and many women become resistant, highlighting the need for additional therapies for this disease. Grape seed and/or skin extracts are popular dietary supplements due to their high polyphenol content and potent anti‐oxidant and anti‐inflammatory properties. Recently, numerous studies showed the cancer chemotherapeutic potential of extracts prepared from or active components isolated from a number of different species of grapes. The muscadine grape (Vitis rotundifolia), found in the warm, humid climate of the southeast United States, has a distinct phytochemical composition as compared to other grape varieties. The muscadine grape contains a high concentration of anthocyanin 3,5‐diglucosides, ellagic acid, ellagic acid precursors, gallic acid, flavan‐3‐ols and flavonols. Extracts from muscadine grapes have not been extensively studied and information regarding their therapeutic effects on cancer is limited. The objective of this study was to determine whether a muscadine grape skin and seed extract (MGE) inhibits the proliferation of HER2 positive breast cancer cells. Treatment with increasing concentrations of MGE (a proprietary MGE from Piedmont Research &amp; Development Corp.; 10 – 40 μg/mL) significantly inhibited proliferation of human SKBR3 HER2 overexpressing breast cancer cells. The reduction in growth was dependent upon the dose of the MGE with an observed maximal effect at 40 μg of phenolics/mL (58.4% inhibition; n=3, p &lt; 0.0001). In addition, incubation of human SKBR3 cells with 30 μg/mL of MGE attenuated cell proliferation in a time‐dependent manner with a maximal 49.2% reduction after 48 h (n=3; p &lt; 0.0001). Protein kinase B (AKT) and its downstream effector, mechanistic target of rapamycin (mTOR), are implicated in the regulation of breast cancer proliferation and cell survival. Treatment of SKBR3 cells with MGE (20 μg of phenolics/mL) for 16 h significantly reduced phospho‐AKT by approximately 70% (n=4, p ≤ 0.01) and phospho‐mTOR by greater than 50% (n = 5, p ≤ 0.05) compared to untreated SKBR3 cells. These results suggest that MGE attenuates proliferation in HER2 positive breast cancer cells by reducing phospho‐AKT which inhibits phosphorylation of mTOR. Thus, MGE may serve as an effective therapeutic either administered singly or in a combination regimen with targeted drugs for the treatment of HER2 positive breast cancer.

Abstract B02: Complex molecular-scale dynamics of HER2 receptor transport in live SKBR3 breast cancer cells revealed by nanoscale quantum dot imaging

Abstract The dynamic behavior of proteins in the spatial and temporal context of a cell is critical for dictating downstream signaling and subsequent cellular effects. Human epidermal growth factor receptor 2 (HER2) is a ligand-less receptor tyrosine kinase whose heterodimerization with other HER receptors (EGFR, HER3, HER4) activates potent downstream signaling to modulate cell proliferation and survival. HER2 represents a major therapeutic target as its overexpression is prevalent in a major subset of invasive breast cancers. Yet while current understanding of HER2 signaling entails a broad sequence of signaling events, the spatiotemporal dynamics of how these signaling mechanisms are executed remains undefined. Here, we describe the molecular-scale motions that underlie HER2 signaling behavior in live cells using a bright and photostable anti-HER2 quantum dot (QD) probe designed and validated to track the receptor for long durations with high spatiotemporal resolution. Methods: HER2 was detected using an anti-HER2 affibody that was fused to a fluorogen activating peptide (FAP) which bound with high affinity to a fluorogen (Malachite Green)-QD complex. HER2-QDs were imaged in live unstimulated and stimulated human SKBR3 breast cancer cells. Cells were serum-starved overnight and: 1) received no treatment during the course of imaging (unstimulated) or 2) were chronically treated with the HER3 and HER4 specific ligand, heregulin-beta1 (HRG) during the course of imaging (stimulated). HRG was administered globally across all cells or locally to a specific cellular region. Trajectory information was analyzed using custom single particle tracking MATLAB software to measure key biophysical parameters. Results: The anti-HER2-QD probe showed robust and increased signal-to-noise compared to conventional organic dyes. Validation was measured by sensitive quantitation of single probes in low and high HER2-expressing cell lines with background non-specific binding of less than five probes per cell. HER2 motions were complex and composed of a heterogeneous composite of diffusive, constrained, and stationary motions. These motions were observed in the trajectories of HER2 receptors traveling along cellular protrusions, cell body, and along protrusions between adjacent cells. HRG stimulation produced morphological changes (e.g. membrane ruffling, protrusion growth) as well as changes in HER2 dynamics. HRG-stimulated cells demonstrated an increase in HER2 mobility (as measured by diffusion constant), increased speed (as measured by instantaneous velocity), and wider exploratory area (as measured by maximum trajectory distance) than in unstimulated cells. Interestingly, increased receptor mobility did not correlate strongly with a wider exploratory area, and more mobile receptors showed more constrained motion. Conclusions: In contrast to the simple diffusive motions previously reported for members of major classes of receptors (e.g. GPCRs, tyrosine kinase receptors), our sensitive measurements of HER2 receptor motion demonstrate complex molecular dynamics in both unstimulated and stimulated cells. Ligand stimulation induces changes in cell morphology such as membrane ruffling and protrusion growth that accompany changes in HER2 mobility and range of motion. The diversity of receptor dynamics suggest a heterogeneous environment in which HER2 engages. Future experiments include understanding the dynamic interplay between HER2 with the cytoskeleton and downstream signaling with HER2-targetting therapies using real-time measurements of HER2 molecular motions. Citation Format: Wai Yan Lam, Yi Wang, Mark J. Olah, Keith A. Lidke, Marcel Bruchez, Joe Gray, Tania Q. Vu. Complex molecular-scale dynamics of HER2 receptor transport in live SKBR3 breast cancer cells revealed by nanoscale quantum dot imaging. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B02.

The G protein-coupled estrogen receptor-1, GPER-1, promotes fibrillogenesis via a Shc-dependent pathway resulting in anchorage-independent growth.

The G protein-coupled estrogen receptor-1, GPER-1, coordinates fibronectin (FN) matrix assembly and release of heparan-bound epidermal growth factor (HB-EGF). This mechanism of action results in the recruitment of FN-engaged integrin α5β1 to fibrillar adhesions and the formation of integrin α5β1-Shc adaptor protein complexes. Here, we show that GPER-1 stimulation of murine 4T1 or human SKBR3 breast cancer cells with 17β-estradiol (E2β) promotes the formation of focal adhesions and actin stress fibers and results in increased cellular adhesion and haptotaxis on FN, but not collagen. These actions are also induced by the xenoestrogen, bisphenol A, and the estrogen receptor (ER) antagonist, ICI 182, 780, but not the inactive stereoisomer, 17α-estradiol (E2α). In addition, we show that GPER-1 stimulation of breast cancer cells allows for FN-dependent, anchorage-independent growth and FN fibril formation in "hanging drop" assays, indicating that these GPER-1-mediated actions occur independently of adhesion to solid substrata. Stable expression of Shc mutant Y317F lacking its primary tyrosyl phosphorylation site disrupts E2β-induced focal adhesion and actin stress fiber formation and abolishes E2β-enhanced haptotaxis on FN and anchorage-dependent growth. Collectively, these data demonstrate that E2β action via GPER-1 enhances cellular adhesivity and FN matrix assembly and allows for anchorage-independent growth, cellular events that may allow for cellular survival, and tumor progression.

A Novel Mechanism by Which Tissue Transglutaminase Activates Signaling Events That Promote Cell Survival

Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein cross-links. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvation-induced apoptosis and triggered the activation of the PI3-kinase/mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase (LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer.

Rapid optical imaging of human breast tumour xenografts using anti-HER2 VHHs site-directly conjugated to IRDye 800CW for image-guided surgery

Molecular optical imaging using monoclonal antibodies is slow with low tumour to background ratio. We used anti-HER2 VHHs conjugated to IRDye 800CW to investigate their potential as probes for rapid optical molecular imaging of HER2-positive tumours by the determination of tumour accumulation and tumour to background levels. Three anti-HER2 VHHs (11A4, 18C3, 22G12) were selected with phage display and produced in Escherichia coli. Binding affinities of these probes to SKBR3 cells were determined before and after site-specific conjugation to IRDye 800CW. To determine the potential of VHH-IR as imaging probes, serial optical imaging studies were carried out using human SKBR3 and human MDA-MB-231 xenograft breast cancer models. Performance of the anti-HER2 VHH-IR was compared to that of trastuzumab-IR and a non-HER2-specific VHH-IR. Image-guided surgery was performed during which SKBR3 tumour was removed under the guidance of the VHH-IR signal. Site-specific conjugation of IRDye 800CW to three anti-HER2 VHHs preserved high affinity binding with the following dissociation constants (KD): 11A4 1.9 ± 0.03, 18C3 14.3 ± 1.8 and 22G12 3.2 ± 0.5 nM. Based upon different criteria such as binding, production yield and tumour accumulation, 11A4 was selected for further studies. Comparison of 11A4-IR with trastuzumab-IR showed ∼20 times faster tumour accumulation of the anti-HER2 VHH, with a much higher contrast between tumour and background tissue (11A4-IR 2.5 ± 0.3, trastuzumab-IR 1.4 ± 0.4, 4 h post-injection). 11A4-IR was demonstrated to be a useful tool in image-guided surgery. VHH-IR led to a much faster tumour accumulation with high tumour to background ratios as compared to trastuzumab-IR allowing same-day imaging for clinical investigation as well as image-guided surgery.

Mitochondrial‐dependent anticancer activity of δ‐tocotrienol and its synthetic derivatives in HER‐2/neu overexpressing breast adenocarcinoma cells

Anticancer activity and mitochondrial mechanism of the vitamin E form δ-tocotrienol (δ-T3) was investigated in HER-2/neu-overexpressing human SKBR3 and murine TUBO breast cancer cells. δ-T3 was confirmed to possess high cytotoxic and apoptotic activity in SKBR3 cells as compared with all natural forms of vitamin E and several synthetic forms that included novel derivatives with the same backbone of δ-T3 such as δ-tocotrienyl-succinyl amide (δ-T3AS) and the redox-active analogue δ-tocotrienyl amine (δ-T3NH2). As observed in the case of alpha-TOS, a prototypical anticancer drug derived from α-tocopherol, succinylation of δ-T3 enhanced citotoxicity and apoptotic activity of the vitamer. δ-T3 induced apoptosis of SKBR3 cells was associated with mitochondrial destabilization, energy failure, and unbalanced activity of stress/survival MAPKs, namely p38 and ERK1/2 pathways. An increased generation of ROS followed to such a series of early events. Enhanced activity of δ-T3 in this human carcinoma cell line was characterized by the sustained uptake and oxidative transformation to the quinone derivative δ-T3Q, thereby suggesting redox effects in SKBR3 mitochondria by this vitamer. Viability and uptake data show a different pattern of responses in TUBO cells with higher response to synthetic derivatives of δ-T3 than in SKBR3 cells. In conclusion, synthetic derivatives of δ-T3 with enhanced apoptotic activity in breast carcinoma cells are investigated for the first time in this study also describing mechanistic aspects of mitochondrial effects of δ-T3. Further investigation in preclinical models of HER2/neu-high breast adenocarcinoma is underway to identify other and more effective forms of VE in this type of cancer.

Lapatinib and 17AAG Reduce 89Zr-Trastuzumab-F(ab′)2 Uptake in SKBR3 Tumor Xenografts

Human epidermal growth factor receptor-2 (HER2) directed therapy potentially can be improved by insight in drug effects on HER2 expression. This study evaluates the effects of the EGFR/HER2 tyrosine kinase inhibitor lapatinib, the heat shock protein-90 inhibitor 17AAG, and their combination, on HER2 expression with in vivo HER2-PET imaging. Lapatinib and 17AAG effects on EGFR and HER2 membrane expression were determined in vitro using flow cytometry of human SKBR3 tumor cells. Effect of lapatinib on HER2 internalization was studied in vitro by (89)Zr-trastuzumab-F(ab')(2) internalization. For in vivo evaluation, (89)Zr-trastuzumab-F(ab')(2) μPET imaging was performed two times with a 7 day interval. Lapatinib was administered for 6 days, starting 1 day after the baseline scan. 17AAG was given 1 day before the second (89)Zr-trastuzumab-F(ab')(2) injection. Imaging data were compared with ex vivo biodistribution analysis and HER2 immunohistochemical staining. 17AAG treatment lowered EGFR expression by 41% (P = 0.016) and HER2 by 76% (P = 0.022). EGFR/HER2 downregulation by 17AAG was inhibited by lapatinib pretreatment. Lapatinib reduced internalization of (89)Zr-trastuzumab-F(ab')(2) with 25% (P = 0.0022). (89)Zr-trastuzumab-F(ab')(2) tumor to blood ratio was lowered 32% by lapatinib (P = 0.00004), 34% by 17AAG (P = 0.0022) and even 53% by the combination (P = 0.011). Lapatinib inhibits HER2 internalization and 17AAG lowers HER2 membrane expression. Both drugs reduce (89)Zr-trastuzumab-F(ab')(2) tumor uptake. Based on our findings, supported by previous preclinical data indicating the antitumor potency of lapatinib in combination with HSP90 inhibition, combination of these drugs deserves further investigation.

Short‐ and long‐term toxicities of multi‐walled carbon nanotubes in vivo and in vitro

As nanomaterials are developed and applied, their potential for health hazards needs to be determined. In the present study, we used commercial nude multi-walled carbon nanotubes (MWCNTs) trimmed to a short length (50-200 nm; s-MWCNTs) and synthesized functionalized MWCNTs with polyethylene glycol (PEG) (s-MWCNTs-PEG). We then studied the toxic effects of s-MWCNTs and s-MWCNTs-PEG on cultured cells and in a mouse model. Peripheral haemograms and various biochemical markers of the heart, liver and kidney were measured. We found no toxicity of either type of nanotube on the viability of human SKBR-3 breast carcinoma cells or control cells. There were no differences in vivo on inflammatory responses, the coagulation system, haemograms or vital organ functions between the test and control groups. Additionally, we found no toxicity of these nanotubes on male mouse sperm production or mutagenesis in the long term. In conclusion, both s-MWCNTs and s-MWCNTs-PEG displayed good in vitro and in vivo biocompatibility, making future applications in biology and clinical therapy as a carrier for drug delivery feasible.

Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting

To facilitate comparative oncology trials we compared the biological and molecular homologies of canine (dog; Canis lupus familiaris) and human tumor-associated antigens ErbB-1 and -2. Further, we investigated whether they could serve as targets for anti-ErbB-1 (cetuximab) and anti-ErbB-2 antibodies (trastuzumab), which are highly relevant in human clinical oncology.Immunohistochemistry of canine mammary cancer showed ErbB-1 overexpression in 3/10 patients and ErbB-2 in 4/10. We report 91% amino acid homology for ErbB-1 and 92% for ErbB-2 between canine and human molecules. Modeling of canine on human ErbB-1 revealed that the cetuximab epitope only differs by 4 amino acids: Lys443 is replaced by Arg, Ser468 by Asn, Gly471 by Asp, and Asn473 by Lys in canines. The trastuzumab binding site is identical in human and canine ErbB-2 apart from a single amino acid change (Pro557 to Ser). Binding of cetuximab and trastuzumab to canine mammary carcinoma cells CF33, CF41, Sh1b and P114 was confirmed by flow cytometry. Both antibodies significantly inhibited canine tumor cell proliferation partly due to growth arrest in G0/G1 phase. We explain the lower efficiency on the tested canine than on human SKBR3 and A431 cells, by a 2-log lower expression level of the canine ErbB-1 and -2 molecules.Our results indicate significant homology of human and canine Erb-1 and -2 tumor associated antigens. The fact that the canine homologues express the cetuximab and trastuzumab epitopes may facilitate antibody-based immunotherapy in dogs. Importantly, the striking similarities of ErbB-1 and -2 molecules open up avenues towards comparative strategies for targeted drug development.

The effect of siRNA-Egr-1 and camptothecin on growth and chemosensitivity of breast cancer cell lines

We have examined the effects of a siRNA targeting the Egr-1, alone or in combination with the breast cancer therapeutic camptothecin (Cpt), in suppressing breast cancer cell survival and anchorage-independent growth in the breast cancer cell lines SK-BR3 and MCF-7. In mammary and lung tumors, as well as most normal tissues, Egr-1 expression is low, suggesting a possible relation between the low levels of Egr-1 and the development of mammary neoplasias. However, analyses of the expression of Egr-1 in breast carcinoma cells, SK-BR3 and MCF-7 demonstrated a relatively high expression of the endogenous Egr-1 in these cells. To investigate the effect of the blocking of the endogenous Egr-1 in breast cancer cells, we used small interfering RNA (siRNA) against Egr-1 alone or in combination with Cpt, and expected that the cell sensitivity to chemotherapeutic drug would increase, when blocked with the Egr-1 gene and treated with Cpt. Thus, we performed in vitro experiment to clarify the effect of Egr-1 on tumor cell lines growth. We made control and siRNA-Egr-1 using vector plasmids and then transfected SK-BR3 and MCF-7 cells. After treating the cells with siRNA-Egr-1, the cell lines were assayed with Cpt to confirm the effect of Egr-1 siRNA using the cell expression of mRNA and protein, proliferation assay and anchorage activity with soft agar. Human SK-BR3 and MCF-7 breast carcinoma cell growth and capacity of anchorage transfected with siRNA-Egr-1 or treated with cpt was slower than that of the control group. This effect was increased when the cells were given simultaneously siRNA and camptothecin. The results strongly suggest that siRNA-Egr-1 alone or in combination with camptothecin could be a potent antineoplastic agent in suppressing the growth of breast tumor despite the known role of Egr-1 as a tumor-suppressor in several other types of human cancers.

The membrane estrogen receptor GPR30 mediates cadmium-induced proliferation of breast cancer cells

Cadmium (Cd) is a nonessential metal that is dispersed throughout the environment. It is an endocrine-disrupting element which mimics estrogen, binds to estrogen receptor alpha (ERα), and promotes cell proliferation in breast cancer cells. We have previously published that Cd promotes activation of the extracellular regulated kinases, erk-1 and -2 in both ER-positive and ER-negative human breast cancer cells, suggesting that this estrogen-like effect of Cd is not associated with the ER. Here, we have investigated whether the newly appreciated transmembrane estrogen receptor, G-protein coupled receptor 30 (GPR30), may be involved in Cd-induced cell proliferation. Towards this end, we compared the effects of Cd in ER-negative human SKBR3 breast cancer cells in which endogenous GPR30 signaling was selectively inhibited using a GPR30 interfering mutant. We found that Cd concentrations from 50 to 500 nM induced a proliferative response in control vector-transfected SKBR3 cells but not in SKBR3 cells stably expressing interfering mutant. Similarly, intracellular cAMP levels increased about 2.4-fold in the vector transfectants but not in cells in which GPR30 was inactivated within 2.5 min after treatment with 500 nM Cd. Furthermore, Cd treatment rapidly activated (within 2.5 min) raf-1, mitogen-activated protein kinase kinase, mek-1, extracellular signal regulated kinases, erk-1/2, ribosomal S6 kinase, rsk, and E-26 like protein kinase, elk, about 4-fold in vector transfectants. In contrast, the activation of these signaling molecules in SKBR3 cells expressing the GPR30 mutant was only about 1.4-fold. These results demonstrate that Cd-induced breast cancer cell proliferation occurs through GPR30-mediated activation in a manner that is similar to that achieved by estrogen in these cells.

Folate-bearing doxorubicin-loaded magnetic poly(N-isopropylacrylamide) microspheres as a new strategy for cancer therapy

Doxorubicin is a classic anticancer agent. Recently, numerous strategies have been used to enhance efficacy of drug delivery for cancer treatment. For example, by modifying poly(N-isopropylacrylamide) microspheres, a nanocarrier, makes it more effective. Conjugation with folic acid increases specific targeted drug delivery towards folate receptor-bearing cancer cells to improve anticancer effectiveness by increasing the tissue's local concentration of drugs. In the current studies, we synthesized folate-bearing, doxorubicin-loaded, magnetic, poly(N-isopropylacrylamide) microspheres (FDMPM) to treat breast cancer cells (human SKBR-3). We found efficiency of drug encapsulation very high (95%) at pH above 7.4. Reverse transcription-PCR showed that cancer cells highly expressed folate receptors. Confocal laser scanning microscopy and flow cytometry revealed internalization of the carrier by SKBR-3 in treatments with FDMPM, which was not the case with any other combination for drug delivery (MPM, FMPM, and DMPM). Similarly, SKBR-3 cell growth was inhibited more (assessed by methylthiazolyldiphenyl-tetrazolium bromide and trypan blue exclusion assays) when treated with FDMPM than with any other combinations. Current results confirm our predication and demonstrate that FDMPM has potential as a new targeting strategy in cancer therapy.

567 POSTER GSK1120212 is a novel Mek inhibitor demonstrating sustained inhibition of ERK phosphorylation and selective inhibition of B-Raf and RAS mutant cells in preclinical models

To facilitate comparative oncology trials we compared the biological and molecular homologies of canine (dog; Canis lupus familiaris) and human tumor-associated antigens ErbB-1 and -2. Further, we investigated whether they could serve as targets for anti-ErbB-1 (cetuximab) and anti-ErbB-2 antibodies (trastuzumab), which are highly relevant in human clinical oncology.Immunohistochemistry of canine mammary cancer showed ErbB-1 overexpression in 3/10 patients and ErbB-2 in 4/10. We report 91% amino acid homology for ErbB-1 and 92% for ErbB-2 between canine and human molecules. Modeling of canine on human ErbB-1 revealed that the cetuximab epitope only differs by 4 amino acids: Lys443 is replaced by Arg, Ser468 by Asn, Gly471 by Asp, and Asn473 by Lys in canines. The trastuzumab binding site is identical in human and canine ErbB-2 apart from a single amino acid change (Pro557 to Ser). Binding of cetuximab and trastuzumab to canine mammary carcinoma cells CF33, CF41, Sh1b and P114 was confirmed by flow cytometry. Both antibodies significantly inhibited canine tumor cell proliferation partly due to growth arrest in G0/G1 phase. We explain the lower efficiency on the tested canine than on human SKBR3 and A431 cells, by a 2-log lower expression level of the canine ErbB-1 and -2 molecules.Our results indicate significant homology of human and canine Erb-1 and -2 tumor associated antigens. The fact that the canine homologues express the cetuximab and trastuzumab epitopes may facilitate antibody-based immunotherapy in dogs. Importantly, the striking similarities of ErbB-1 and -2 molecules open up avenues towards comparative strategies for targeted drug development.

Does HER‐2/neu antigen loss in metastatic breast tumors occur under immune pressure?

Tumor immune editing has been demonstrated in the preclinical studies that underscore counter-regulatory function of antitumor immune responses in both tumor rejection and tumor escape or recurrence. We were greatly interested in the article by Santinelli et al.1 in this journal on discrepancy between primary breast tumors and their metastatic lesions on the status of HER-2/neu expression. Their work clearly demonstrates that a fraction of patients with HER-2/neu positive primary tumors appeared to lose HER-2/neu expression in their distant metastatic lesions. However, there was a concordance in HER-2/neu expression between primary tumors and metachronous lymph node metastasis. Similar observations were made by Lower et al.,2 where 382 patients with metastatic breast cancer were studied. A fraction of patients with HER-2/neu positive primary lesions also had HER-2/neu negative metastatic lesions. These observations suggest that breast tumors are not stable in the expression of HER-2/neu in vivo. Therefore, it is important to understand the mechanisms by which tumors lose or gain HER-2/neu expression in vivo. Our preclinical results complement their findings by demonstrating that HER-2/neu antigen loss occurred under the immune pressure in vivo and in vitro.3 Our studies underscored the role of HER-2/neu-specific IFN-γ-secreting T cells in the induction of HER-2/neu antigen loss or downregulation, which facilitated emergence of an invasive HER-2/neu negative tumor variant. Interestingly, microarray analysis showed that expression of IFN-γ Rα in the relapsed tumor lesion, which had lost HER-2/neu expression under immune pressure, was higher than that in the regressing tumor lesion.4 We also showed that presence of the inflammatory cytokine, IL-6, would upregulate expression of IFN-γ Rα in human breast tumor line, SKBR3, rendering it more susceptible to the IFN-γ-induced downregulation of HER-2/neu expression (Fig. 1). Based on these preclinical studies and other reports emphasizing on counter-regulatory role of IFN-γ in tumor rejection and antigen loss,5-7 and the fact that breast tumors are heterogeneous in the expression of receptors, we suggest that IFN-γ Rα positive tumor clones had lost HER-2/neu expression in the presence of HER-2/neu-specific immune responses, IFN-γ-producing T cells in particular, and became metastatic. On the other hand, IFN-γ Rα negative tumor clones continued to express HER-2/neu antigen in primary tumor lesions. Availability of tumor samples from the patients studied by Santinelli et al.1 would allow them to determine whether such discordance between primary and metastatic breast tumors in the expression of HER-2/neu may be correlated with the status of IFN-γ Rα expression in the tumors. Further studies are required to determine whether expression of HER-2/neu in primary tumors and loss of this antigen in metastatic lesions may also be associated with the preexisting HER-2/neu-specific T-cell responses in these patients. IL-6 renders SKBR3 tumors sensitive to the IFN-γ-induced downregulation of HER-2/neu. The HER-2/neu positive human SKBR3 cells were cultured in the presence or absence of IFN-γ (50 ng/ml) for 5 days (a), or cultured with IL-6 for 16 hr and then cultured in the presence or absence of IFN-γ for 5 days (b). Expression of HER-2/neu was evaluated using flow cytometry analysis on the indicated cells (a, b) and RT-PCR analysis of mRNA extracted from the IL-6-pretreated SKBR3 cells that were cultured in the presence or absence of IFN-γfor 5 days (c). The authors gratefully acknowledge the support of VCU Massey Cancer Centre and the Commonwealth Foundation for Cancer Research. Yours sincerely, Masoud H. Manjili*, Maciej Kmieciak*, * Department of Microbiology and Immunology, VCU School of Medicine, Massey Cancer Center, Richmond, VA.

Role of peroxidases, thiols and Bak/Bax in tumor cell susceptibility to Cu[DEDTC] 2

Copper and two molecules of diethyl dithiocarbamate [DEDTC] form the Cu[DEDTC] 2 complex, which shows cytotoxicity against melanoma and carcinoma cells, making it a potentially useful anti-cancer agent. The differential response to Cu[DEDTC] 2 in susceptible human SKBR3 carcinoma and C8161 melanoma cell variants of moderate and high resistance to this organometallic complex was evaluated in this study. Both cell lines underwent apoptosis-associated PARP cleavage, changes in expression of nuclear NFkB p65, p21WAF1 and cyclin A, with loss of clonogenicity in response to this agent. However, a threefold greater concentration [IC 50 0.6 μM DEDTC: 0.3 μM Cu] was required to kill moderately resistant C8161 melanoma compared to highly susceptible SKBR3 cells. Decreased susceptibility to Cu[DEDTC] 2 in C8161 melanoma correlated with greater levels of glutathione peroxidase and catalase, and a fourfold lower requirement for N-acetyl cysteine (1 mM) to overcome toxicity. Whereas melanoma cells selected for resistance to [0.8 μM DEDTC: 0.4 μM Cu] showed persistent catalase and GPx activity, melanoma cells with moderate susceptibility showed decreased catalase and Gpx when responding to treatment. Cytotoxic response in moderately susceptible C8161 melanoma cells involved an early accumulation of pro-apoptotic Bax in the G2 cell cycle phase, followed by an increased ratio of pro-apoptotic Bak to anti-apoptotic Mcl-1 in mitochondria. Our data suggests that Cu[DEDTC] 2 toxicity is mediated through an increase in pro-apoptotic Bak/Bax via disruption of the peroxide and thiol metabolism.