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Die Interaktion von ErbB2/Her2 mit Hitzeschockproteinen in Mammakarzinomzellen / The interaction of ErbB2/Her2 with heatshockproteins in breast cancer cellsStreller, Felix 10 February 2015 (has links)
Her2-positiver Brustkrebs, der Subtyp des Mammakarzinoms, bei dem eine Überexpression des epidermalen Wachstumsfaktor-Rezeptors-2 (ErbB2/Her2) vorliegt, hat für die betroffenen Frauen eine besonders schlechte Prognose. Eine positive Korrelation zwischen der ErbB2-Expression in Brusttumoren und der Expression des Makrophagen-Migration-inhibierenden Faktors (MIF), einem inzwischen gut bekannten tumorfördernden Protein, konnte bereits gezeigt werden. Ferner konnte gezeigt werden, dass MIF durch das Hitzeschockprotein 90 (Hsp90) in einem Mausmodell des Her2-positiven Brustkrebses stabilisiert wird. Der zugrundeliegende Mechanismus war bisher unverstanden. In dieser Doktorarbeit konnte erstmalig demonstriert werden, dass Hitzeschockfaktor-1 (HSF-1), der Transkriptionsfaktor der stressinduzierten Hitzeschockproteine (HSP), einschließlich Hsp90, in ErbB2-überexprimierenden SK-BR-3-Brustkrebszellen konstitutiv durch ErbB2 aktiviert wird. Durch eine Behandlung mit dem ErbB2-Inhibitor CP724.714 konnte die aktivierende Serin326-Phosphorylierung von HSF-1 verhindert werden. Als Folge wird, wie durch Western-Blot-Analysen gezeigt, die HSP-Maschinerie inhibiert und tumorfördernde Hsp90-Klienten wie MIF, Akt, mutiertes p53, ErbB2 und HSF-1 destabilisiert. Außerdem konnte die unterbleibende HSF-1-Aktivierung durch quantitative PCR-Analysen und Immunfluoreszenzmikroskopie bestätigt werden. Die mechanistischen Untersuchungen konnten die hier erstmalig beschriebene ErbB2-Akt-HSF-1-Achse aufdecken, über die HSF-1 in SK-BR-3-Zellen reguliert wird. Ferner konnte eine ErbB2-Inhibition sogar die HSF-1-Aktivierung durch einen Hitzeschock unterbinden. Unsere Ergebnisse zeigen zum ersten Mal, dass die ErbB-2-Überexpression in SK-BR-3-Zellen eine konstitutive HSF-1-Aktivierung bewirkt, mit der Folge, dass tumorfördernde Hsp90-Klienten wie MIF, HSF-1 selbst, ErbB2 und mutiertes p53 stabilisiert werden. Die neu entdeckte ErbB2-Akt-HSF-1-Hsp90-Klienten-Achse legt möglicherweise neue Angriffspunkte für zusätzliche Pharmaka bei der Therapie Her2-positiven Brustkrebses offen.
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EGFR and HER2 Targeting for Radionuclide-Based Imaging and Therapy : Preclinical StudiesNordberg, Erika January 2008 (has links)
<p>The optimal way to detect and treat cancer is to target cancer cells exclusively without affecting the surrounding tissue. One promising approach is to use radiolabelled molecules to target receptors that are overexpressed in cancer cells. Since the epidermal growth factor receptor (EGFR) family is overexpressed in many types of cancer, it is an attractive target for both diagnostic and therapeutic applications.</p><p>This thesis can be divided into two parts. In part one (paper I), studies were conducted to modulate radionuclide uptake in tumour cells. The results showed that it was possible to modulate the cellular uptake of <sup>125</sup>I delivered by trastuzumab (targeting HER2) by adding EGF (targeting EGFR).</p><p>In part two (papers II-V) a high affinity EGFR-targeting affibody molecule (Z<sub>EGFR:955</sub>)<sub>2</sub> was selected and analysed both <i>in vitro</i> and <i>in vivo</i>. In papers II, III and V, the results obtained when using (Z<sub>EGFR:955</sub>)<sub>2</sub> were compared with those obtained with the two EGFR-binding molecules, EGF and cetuximab. These studies demonstrated that the affibody molecule bound specifically to EGFR (probably to subdomain III) with high affinity (~50 nM in biosensor analysis and ~1 nM in cellular studies) and produced intracellular signalling changes similar to those with cetuximab. In paper IV, <i>in vivo</i> studies were made, demonstrating that [<sup>111</sup>In](Z<sub>EGFR:955</sub>)<sub>2</sub> gave a tumour-specific <sup>111</sup>In uptake of 3.8±1.4% of injected dose per gram tumour tissue, 4 h post-injection. The tumours could be easily visualized with a gamma camera at this time-point. </p><p>The results of these studies indicated that the affibody molecule (Z<sub>EGFR:955</sub>)<sub>2</sub> is a possible candidate for radionuclide-based imaging of EGFR-expressing tumours. The biological effects of (Z<sub>EGFR:955</sub>)<sub>2</sub> might be of interest for therapy applications.</p>
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Cellular Studies of HER-family Specific Affibody MoleculesGöstring, Lovisa January 2011 (has links)
The human epidermal growth-factor like receptor (HER) family of receptor tyrosine kinases are important targets for cancer therapy. The family consists of four members - EGFR, HER2, HER3 and HER4 - that normally transfer stimulatory signals from extracellular growth factors to the intracellular signalling network. Over-activation of these receptors leads to uncontrolled cell proliferation and is seen in several types of tumours. The aim of the studies reported in this thesis was to study the uptake and effects of affibody molecules against EGFR, HER2 and HER3 in cultured cells. Affibody molecules are affinity proteins originally derived from one of the domains of protein A, and their small size and robust structure make them suitable agents for tumour targeting and therapy. Papers I and II of this thesis concern EGFR-specific affibody molecules, which were shown to be more similar to the antibody cetuximab than the natural ligand EGF in terms of cellular uptake, binding site and internalisation rate. In addition, fluorescence-based methods for the quantification of internalisation were evaluated. In the studies reported in papers III and IV, HER2-specific affibody molecules were utilised as carriers of radionuclides. Paper III reports that different cell lines exhibit different radiosensitivities to 211At-labelled affibody molecules; radiosensitivity was found to correlate with cell geometry and the rate of internalisation. Paper IV discusses the use of 17-AAG, an agent that induces HER2 internalisation and degradation, to force the internalisation of 211At- and 111In-labelled affibody molecules. Papers V and VI describe the selection and maturation of HER3-specific affibody molecules, which were found to compete with the receptor’s natural ligand, heregulin, for receptor binding. These affibody molecules were demonstrated to inhibit heregulin-induced HER3 activation and cell proliferation. The studies summarised in this paper will hopefully contribute to a better understanding of these affibody molecules and bring them one step closer to being helpful tools in the diagnosis and treatment of cancer.
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Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions : Resolving How the Epidermal Growth Factor Binds to Its ReceptorBjörkelund, Hanna January 2013 (has links)
Cells are complex biological units with advanced signalling systems, a dynamic capacity to adapt to its environment, and the ability to divide and grow. In fact, they are of such high level of complexity that it has deemed extremely difficult or even impossible to completely understand cells as complete units. The search for comprehending the cell has instead been divided into small, relatively isolated research fields, in which simplified models are used to explain cell biology. The result produced through these reductionistic investigations is integral for our current description of biology. However, there comes a time when it is possible to go beyond such simplifications and investigate cell biology at a higher level of complexity. That time is now. This thesis describes the development of mathematical tools to investigate intricate biological systems, with focus on heterogeneous protein interactions. By the use of simulations, real-time measurements and kinetic fits, standard assays for specificity measurements and receptor quantification were scrutinized in order to find optimal experimental settings and reduce labour time as well as reagent cost. A novel analysis platform, called Interaction Map, was characterized and applied on several types of interactions. Interaction Map decomposes a time-resolved binding curve and presents information on the kinetics and magnitude of each interaction that contributed to the curve. This provides a greater understanding of parallel interactions involved in the same biological system, such as a cell. The heterogeneity of the epidermal growth factor receptor (EGFR) system was investigated with Interaction Map applied on data from the instrument LigandTracer, together with complementing manual assays. By further introducing disturbances to the system, such as tyrosine kinase inhibitors and variation in temperature, information was obtained about dimerization, internalization and degradation rates. In the long term, analysis of binding kinetics and combinations of parallel interactions can improve the understanding of complex biomolecular mechanisms in cells and may explain some of the differences observed between cell lines, medical treatments and groups of patients.
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Targeted Molecular MR Imaging of HER2 and EGFR Using De Novo Designed Protein Contrast AgentsQiao, Jingjuan 08 December 2011 (has links)
The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We developed a novel MRI probe which targets HER2, a biomarker for various cancers and a target for anti-cancer therapies. This multimodal HER2-targeted MRI probe integrates a rationally designed protein contrast agent with a high affinity HER2 affibody and near IR dye. Our probe can differentially monitor tumors with different HER2 levels in both cells and xenograft mice. In addition to its 10-fold higher dose efficiency compared to clinically-approved agent DTPA, our developed agent also exhibits advantages in crossing the endothelial boundary, tissue distribution, and tumor tissue retention as demonstrated by even distribution of the imaging probe across the entire tumor mass. Additionally, a second series of protein contrast agents that included affibody against EFGR developed with the capability to specifically target EGFR. These contrast agents have been utilized to monitor drug treatments and quantitatively analyze biomarker expression level. Furthermore, we anticipate these agents will provide powerful tools for quantitative assessment of molecular markers, and improved resolution for diagnosis, prognosis and drug discovery.
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The Concordance between Immunohistochemical Staining and Silver In Situ Hybridization for HER2 Status in Breast Cancer Tissue SamplesKardeby, Caroline January 2011 (has links)
The human epidermal growth factor receptor-2 (HER2) protein has been associated with breast cancer progression and the HER2 status can be used to determine the type of treatment for each breast cancer patient. The purpose of this study was to examine the HER2 protein and gene statuses in breast cancer tissue samples using two methods and analyze the concordance between them. Ten paraffin-embedded, formaldehyde-fixed breast cancer tissue samples from the Biobank at the Department of Pathology and Cytology at Sundsvall Hospital were analyzed in this study. All samples were from women born between 1931 and 1976. The methods used were immunohistochemistry (IHC) to visualise the HER2 protein and silver in situ hybridization (SISH) to detect gene amplification. The IHC staining method is an indirect detection of the HER2 protein using antibodies. The SISH method used in this study is a Dual ISH which detects both the HER2 gene and the centromere region of Chromosome 17 on the same tissue slide. A HER2 gene/Chromosome 17 ratio was calculated according to the manufacturer’s instructions. This ratio was used to determine HER2 gene status. Out of ten samples, seven were positive with IHC and three were negative. The results from the SISH staining exposed a gene amplification in three of the IHC positive samples, while seven samples did not contain any amplified HER2 genes. The conclusion was that the concordance between IHC and SISH for HER2 was 60 percent.
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Affibody Molecules for PET ImagingStrand, Joanna January 2015 (has links)
Optimization of Affibody molecules would allow for high contrast imaging of cancer associated surface receptors using molecular imaging. The primary aim of the thesis was to develop Affibody-based PET imaging agents to provide the highest possible sensitivity of RTK detection in vivo. The thesis evaluates the effect of radiolabelling chemistry on biodistribution and targeting properties of Affibody molecules directed against HER2 and PDGFRβ. The thesis is based on five published papers (I-V). Paper I. The targeting properties of maleimido derivatives of DOTA and NODAGA for site-specific labelling of a recombinant HER2-binding Affibody molecule radiolabelled with 68Ga were compared in vivo. Favourable in vivo properties were seen for the Affibody molecule with the combination of 68Ga with NODAGA. Paper II. The aim was to compare the biodistribution of 68Ga- and 111In-labelled HER2-targeting Affibody molecules containing DOTA, NOTA and NODAGA at the N-terminus. This paper also demonstrated favourable in vivo properties for Affibody molecules in combination with 68Ga and NODAGA placed on the N-terminus. Paper III. The influence of chelator positioning on the synthetic anti-HER2 affibody molecule labelled with 68Ga was investigated. The chelator DOTA was conjugated either at the N-terminus, the middle of helix-3 or at the C-terminus of the Affibody molecules. The N-terminus placement provided the highest tumour uptake and tumour-to-organ ratios. Paper IV. The aim of this study was to evaluate if the 68Ga labelled PDGFRβ-targeting Affibody would provide an imaging agent suitable for PDGFRβ visualization using PET. The 68Ga labelled conjugate provided high-contrast imaging of PDGFRβ-expressing tumours in vivo using microPET as early as 2h after injection. Paper V. This paper investigated if the replacement of IHPEM with IPEM as a linker molecule for radioiodination of Affibody molecules would reduce renal retention of radioactivity. Results showed that the use of the more lipophilic linker IPEM reduced the renal radioactivity retention for radioiodinated Affibody molecules. In conclusion, this thesis clearly demonstrates that the labelling strategy is of great importance with a substantial influence on the targeting properties of Affibody molecules and should be taken under serious considerations when developing new imaging agents.
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Alcohol promotes mammary tumor development through regulation of estrogen signalingWong, Amy W. 08 July 2013 (has links)
Breast cancer is the most common malignancy affecting women and the second leading cause of death among women in the United States. Alcohol consumption is one of the few modifiable risk factors for breast cancer development but the mechanism by which it contributes to mammary cancer development and progression remains unclear, although it has been suggested that estrogen is critical for this process. To determine if alcohol promotes mammary tumor development via the estrogen pathway, estrogen receptor alpha-negative (ER[alpha]-negative) MMTV-neu mice were treated with various doses of ethanol and activation of estrogen signaling was measured. Our results showed that alcohol consumption increased estrogen signaling activation, serum estrogen levels and, most interestingly, expression of ER[alpha] in tumor tissue in the ER[alpha]-negative mice. Several lines of evidence in literature suggest that ER[alpha] expression in ER[alpha]-negative cancer cells is inhibited through epigenetic regulation. Epigenetics is the study of heritable changes in gene expression caused by mechanisms other than DNA sequence changes. Thus, to determine whether alcohol may regulate ER[alpha] re-expression in ER[alpha]-negative breast cancer cells through epigenetic mechanisms, we examined the effects of ethanol on CpG methylation and histone modifications (acetylation and methylation) of two ER[alpha]-negative breast cancer cell lines, MDA-MB-231 (human) and MMTV-neu (mouse). We also examined whether the epigenetic modifications subsequently affect the recruitment of transcriptional regulation complexes to the ER[alpha] promoter to regulate ER[alpha] transcription. Results showed that alcohol promotes ER[alpha] re-expression in these ER[alpha]-negative cell lines and that this effect was associated with decreased CpG methylation, an overall increase of histone acetylation and decrease of histone methylation, and an alteration in the enrichment of the ER[alpha] transcriptional regulation complexes (pRb2/p130-E2F4/5-HDAC1-SUV39H1-p300 and pRb2/p130-E2F4/5-HDAC1-SUV39H1-DNMT1) at the ER[alpha] promoter, which may contribute to cancer cell progression. In addition, we found that the inhibition of ER[alpha] by tamoxifen specifically blocks the effects of alcohol on ER[alpha] reactivation. To determine how alcohol promotes cell invasive ability, a critical process for cancer progression, we examined the role of two genes, metastasis suppressor Nm23 and integrin alpha-5 ITGA5, which we identified to be important for alcohol-induced breast cancer cell invasion. It has previously been shown that estrogen may regulate Nm23 expression and that estrogen regulation may be important for ITGA5-mediated cancer progression. Our results showed that alcohol promotes cancer cell invasion through the down-regulation of Nm23, which led to the subsequent increase of ITGA5 and increase of cell invasion. Collectively, data from my research strongly supports and provides evidence that alcohol promotes breast cancer development and progression through the regulation of estrogen signaling. / text
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Targeted Auger Electron Radiotherapy of HER2-amplified Breast CancerCostantini, Danny 23 September 2009 (has links)
Monoclonal antibodies (mAbs) conjugated to nuclear localization sequences (NLS) and labeled with Auger electron-emitters have great potential for targeted radiotherapy of cancer. This approach may be especially appropriate for the 25-30% of patients with breast cancer whose tumors display overexpression of HER2. Trastuzumab (Herceptin) is a humanized anti-HER2 mAb approved for immunotherapy of HER2-amplified breast cancer. The goal of this research was to radiolabel trastuzumab with [111]In, and to modify it with peptides harboring the NLS (CGYGPKKKRKVGG) of the simian virus 40 large-T antigen for targeted radiotherapy of breast cancer. It was hypothesized that the NLS-peptides would mediate the translocation of covalently linked [111]In-trastuzumab molecules into the nuclei of HER2-overexpressing breast cancer cells where subcellular-range Auger electrons are most damaging to DNA and lethal to cells.
Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate for reaction with NLS-peptides and labeled with [111]In using diethylenetriaminepentaacetic acid. The dissociation constant for binding of [111]In-NLS-trastuzumab to HER2-overexpressing SK-BR-3 human breast cancer cells was reduced < 3-fold compared to [111]In-trastuzumab, demonstrating relatively preserved receptor-binding affinity. The NLS-peptides did not affect the biodistribution of [111]In-trastuzumab, but promoted its nuclear uptake in HER2-overexpressing MDA-MB-361 xenografts. The cytotoxicity of [111]In-NLS-trastuzumab on breast cancer cells correlated with their HER2 expression. Moreover, [111]In-NLS-trastuzumab was 2-fold and 5-fold more potent at killing MDA-MB-361 and SK-BR-3 cells compared to [111]In-trastuzumab, and nearly 3-fold and 6-fold more effective than unlabeled trastuzumab, respectively. Methotrexate is a known radiosensitizer that can amplify the lethal effects of ionizing radiation on tumor cells. Non-cytotoxic, but radiosensitizing doses of methotrexate were therefore combined with [111]In-NLS-trastuzumab; this enhanced the sensitivity of HER2-overexpressing breast cancer cells to [111]In-NLS-trastuzumab. The blood t1/2 of [111]In-NLS-trastuzumab in non-tumor bearing BALB/c mice was 23-34 h when administered intravenously or intraperitoneally. The maximum tolerated dose was 9.2-18.5 MBq; doses >18.5 MBq caused decreased leukocyte and platelet counts. [111]In-NLS-trastuzumab exhibited strong anti-tumor effects against HER2-overexpressing MDA-MB-361 xenografts, reducing their growth rate 2-fold and 3-fold compared to mice administered [111]In-trastuzumab or unlabeled trastuzumab, respectively.
These promising results suggest that [111]In-NLS-trastuzumab may be a useful Auger electron radioimmunotherapeutic agent for HER2-positive breast cancer in humans.
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Molecular Imaging as a Tool for Predicting and Monitoring Response of Breast Cancer to Trastuzumab (Herceptin(R))McLarty, Kristin 08 March 2011 (has links)
The human epidermal growth factor receptor 2 (HER2) is overexpressed in 20% of breast cancers (BCs) and confers an aggressive tumour phenotype with a poor prognosis. Trastuzumab (Herceptin®) is a humanized IgG1 monoclonal antibody (mAb) approved for treatment of HER2-positive breast cancer (BC), however many eligible patients do not respond. The hypothesis was that molecular imaging strategies that probe: i) the expression of HER2; ii) one of the mechanisms of action of trastuzumab or iii) evaluate the viability of tumour cells by their glucose utilization would be useful in predicting and monitoring the response of BC to treatment with trastuzumab. The relationship between tumour HER2 density, uptake of 111In-DTPA-trastuzumab and response to trastuzumab was evaluated by gamma camera imaging, biodistribution studies and monitoring tumour growth in mice implanted with BC xenografts. There was a non-linear relationship between HER2 expression and uptake of this radiopharmaceutical when tumour uptake was corrected for non-specific IgG accumulation and/or circulating blood radioactivity (r2=0.87-0.99). Tumour response corresponded better with the uncorrected tumour uptake of 111In-DTPA-trastuzumab. HER2 downregulation, a putative mechanism of action of trastuzumab, was noted as decreased tumour uptake on microSPECT/CT of mice bearing MDA-MB-361 xenografts administered 111In-DTPA-pertuzumab. Tumour uptake of 111In-DTPA-pertuzumab was reduced by 53% in mice treated for 3 days with trastuzumab (P<0.05) associated with an early molecular response to the drug. Furthermore, tumour uptake of 111In-DTPA-pertuzumab was reduced by 78% (P<0.001) in mice treated for 3 weeks, which corresponded with a reduction in HER2-positive tumour cells, indicating a therapeutic response. The relationship between changes in tumour uptake of 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) and response to trastuzumab was examined in mice bearing MDA-MB-361 and MDA-MB-231 BC xenografts, with high or very low HER2 expression, treated with trastuzumab. MicroPET imaging and biodistribution studies detected a 43-60% (P<0.03) reduction in tumour uptake of 18F-FDG in mice with MDA-MB-361 xenografts, treated with trastuzumab compared to PBS-treated controls. In contrast, there was no change in 18F-FDG uptake in MDA-MB-231 xenografts, that did not respond to trastuzumab. I conclude that molecular imaging is a promising tool for monitoring response of BC to treatment with trastuzumab.
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