Rafal Zielinski

The crosstalk between EGF, IGF, and Insulin cell signaling pathways - computational and experimental analysis

BackgroundCellular response to external stimuli requires propagation of corresponding signals through molecular signaling pathways. However, signaling pathways are not isolated information highways, but rather interact in a number of ways forming sophisticated signaling networks. Since defects in signaling pathways are associated with many serious diseases, understanding of the crosstalk between them is fundamental for designing molecularly targeted therapy. Unfortunately, we still lack technology that would allow high throughput detailed measurement of activity of individual signaling molecules and their interactions. This necessitates developing methods to prioritize selection of the molecules such that measuring their activity would be most informative for understanding the crosstalk. Furthermore, absence of the reaction coefficients necessary for detailed modeling of signal propagation raises the question whether simple parameter-free models could provide useful information about such pathways.ResultsWe study the combined signaling network of three major pro-survival signaling pathways: E pidermal G rowth F actor R eceptor (EGFR), I nsulin-like G rowth F actor-1 R eceptor (IGF-1R), and I nsulin R eceptor (IR). Our study involves static analysis and dynamic modeling of this network, as well as an experimental verification of the model by measuring the response of selected signaling molecules to differential stimulation of EGF, IGF and insulin receptors. We introduced two novel measures of the importance of a node in the context of such crosstalk. Based on these measures several molecules, namely Erk1/2, Akt1, Jnk, p70S6K, were selected for monitoring in the network simulation and for experimental studies. Our simulation method relies on the Boolean network model combined with stochastic propagation of the signal. Most (although not all) trends suggested by the simulations have been confirmed by experiments.ConclusionThe simple model implemented in this paper provides a valuable first step in modeling signaling networks. However, to obtain a fully predictive model, a more detailed knowledge regarding parameters of individual interactions might be necessary.

Affitoxin—A Novel Recombinant, HER2-specific, Anticancer Agent for Targeted Therapy of HER2-positive Tumors

Expression of the human epidermal growth factor receptor 2 (HER2) is amplified in 25% to 30% of breast cancers and has been associated with an unfavorable prognosis. Here we report the construction, purification, and characterization of Affitoxin—a novel class of HER2-specific cytotoxic molecules combining HER2-specific Affibody molecule as a targeting moiety and PE38KDEL, which is a truncated version of Pseudomonas exotoxin A, as a cell killing agent. It is highly soluble and does not require additional refolding, oxidation, or reduction steps during its purification. Using surface plasmon resonance technology and competitive binding assays, we have shown that Affitoxin binds specifically to HER2 with nanomolar affinity. We have also observed a high correlation between HER2 expression and retention of Affitoxin bound to the cell surface. Affitoxin binding and internalization is followed by Pseudomonas exotoxin A activity domain-mediated ADP-ribosylation of translation elongation factor 2 and, consequently, inhibition of protein synthesis as shown by protein expression analysis of HER2-positive cells treated with Affitoxin. Measured IC50 value for HER2-negative cells MDA-MB468 (65±2.63 pM) was more than 20 times higher than the value for low HER2 level-expressing MCF7 cells (2.56±0.1 pM), and almost 3 orders of magnitude higher for its HER2-overexpressing derivative MCF7/HER2 (62.7±5.9 fM). These studies suggest that Affitoxin is an attractive PE38-based candidate for treatment of HER2-positive tumors.

Active Targeting Using HER‐2‐Affibody‐Conjugated Nanoparticles Enabled Sensitive and Specific Imaging of Orthotopic HER‐2 Positive Ovarian Tumors

Despite advances in cancer diagnosis and treatment, ovarian cancer remains one of the most fatal cancer types. The development of targeted nanoparticle imaging probes and therapeutics offers promising approaches for early detection and effective treatment of ovarian cancer. In this study, HER-2 targeted magnetic iron oxide nanoparticles (IONPs) are developed by conjugating a high affinity and small size HER-2 affibody that is labeled with a unique near infrared dye (NIR-830) to the nanoparticles. Using a clinically relevant orthotopic human ovarian tumor xenograft model, it is shown that HER-2 targeted IONPs are selectively delivered into both primary and disseminated ovarian tumors, enabling non-invasive optical and MR imaging of the tumors as small as 1 mm in the peritoneal cavity. It is determined that HER-2 targeted delivery of the IONPs is essential for specific and sensitive imaging of the HER-2 positive tumor since we are unable to detect the imaging signal in the tumors following systemic delivery of non-targeted IONPs into the mice bearing HER-2 positive SKOV3 tumors. Furthermore, imaging signals and the IONPs are not detected in HER-2 low expressing OVCAR3 tumors after systemic delivery of HER-2 targeted-IONPs. Since HER-2 is expressed in a high percentage of ovarian cancers, the HER-2 targeted dual imaging modality IONPs have potential for the development of novel targeted imaging and therapeutic nanoparticles for ovarian cancer detection, targeted drug delivery, and image-guided therapy and surgery.

HER2-Affitoxin: A Potent Therapeutic Agent for the Treatment of HER2-Overexpressing Tumors

Purpose: Cancers overexpressing the HER2/neu gene are usually more aggressive and are associated with poor prognosis. Although trastuzumab has significantly improved the outcome, many tumors do not respond or acquire resistance to current therapies. To provide an alternative HER2-targeted therapy, we have developed and characterized a novel recombinant protein combining an HER2-specific Affibody and modified Pseudomonas aeruginosa exotoxin A (PE 38), which, after binding to HER2, is internalized and delivered to the cytosol of the tumor cell, where it blocks protein synthesis by ADP ribosylation of eEF-2. Experimental Design: The effect of the Affitoxin on cell viability was assessed using CellTiter-Glo (Promega). To assess HER2-specific efficacy, athymic nude mice bearing BT-474 breast cancer, SK-OV-3 ovarian cancer, and NCI-N87 gastric carcinoma xenografts were treated with the Affitoxin (HER2- or Tag-specific), which was injected every third day. Affitoxin immunogenicity in female BALB/c mice was investigated using standard antibody production and splenocyte proliferation assays. Results:In vitro experiments proved that HER2-Affitoxin is a potent agent that eliminates HER2-overexpressing cells at low picomolar concentrations. Therapeutic efficacy studies showed complete eradication of relatively large BT-474 tumors and significant effects on SK-OV-3 and NCI-N87 tumors. HER2-Affitoxin cleared quickly from circulation (T1/2 < 10 minutes) and was well tolerated by mice at doses of 0.5 mg/kg and below. Immunogenicity studies indicated that HER2-Affitoxin induced antibody development after the third injected dose. Conclusions: Our findings showed that HER2-Affitoxin is an effective anticancer agent and a potential candidate for clinical studies. Clin Cancer Res; 17(15); 5071–81. ©2011 AACR.

Quantitative Analysis of HER2 Receptor Expression In Vivo by Near-Infrared Optical Imaging

Human epidermal growth factor receptor 2 (HER2) overexpression in breast cancers is associated with poor prognosis and resistance to therapy. Current techniques for estimating this important characteristic use ex vivo assays that require tissue biopsies. We suggest a novel noninvasive method to characterize HER2 expression in vivo, using optical imaging, based on HER2-specific probes (albumin-binding domain–fused-(ZHER2:342)2-Cys Affibody molecules [Affibody AB, Solna, Sweden], labeled with Alexa Fluor 750 [Molecular Probes, Invitrogen, Carlsbad, CA]) that could be used concomitantly with HER2-targeted therapy. Subcutaneous tumor xenografts, expressing different levels of HER2, were imaged with a near-infrared fluorescence small-animal imaging system at several times postinjection of the probe. The compartmental ligand-receptor model was used to calculate HER2 expression from imaging data. Correlation between HER2 amplification/overexpression in tumor cells and parameters, directly estimated from the sequence of optical images, was observed (eg, experimental data for BT474 xenografts indicate that initial slope, characterizing the temporal dependence of the fluorescence intensity detected in the tumor, linearly depends on the HER2 expression, as measured ex vivo by an enzyme-linked immunosorbent assay for the same tumor). The results obtained from tumors expressing different levels of HER2 substantiate a similar relationship between the initial slope and HER2 amplification/overexpression. This work shows that optical imaging, combined with mathematical modeling, allows noninvasive monitoring of HER2 expression in vivo.

HER2-and EGFR-specific Affiprobes—Novel Recombinant Optical Probes for Cell Imaging

The human epidermal growth factor receptors, EGFR and HER2, are members of the EGFR family of cell‐surface receptors/tyrosine kinases. EGFR‐ and HER2‐positive cancers represent a more aggressive disease with greater likelihood of recurrence, poorer prognosis, and decreased survival rate, compared to EGFR‐ or HER2‐negative cancers. The details of HER2 proto‐oncogenic functions are not deeply understood, partially because of a restricted availability of tools for EGFR and HER2 detection (A. Sorkin and L. K. Goh, Exp. Cell Res. 2009, 315, 683–696). We have created photostable and relatively simple‐to‐produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called affiprobes, consist of a targeting moiety, a HER2‐ or EGFR‐specific Affibody® molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of affiprobes. Affiprobes are able to stain both live cells and frozen tumor xenograph sections. This type of optical probe can easily be extended for targeting other cell‐surface antigens/ receptors.

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy.

Potential of PET to Predict the Response to Trastuzumab Treatment in an ErbB2-Positive Human Xenograft Tumor Model

Currently, an alteration in the gross volume of a tumor is used to assess its response to trastuzumab; however, this approach provides only a late indication of response. Tissue-sample ex vivo assays are potentially valuable, but their procurement through biopsies is invasive and might be biased by tumor heterogeneity. We studied the feasibility of using PET to quantify changes in ErbB2 (HER2/neu) expression and to predict the response to trastuzumab in BT474 breast cancer xenografts with N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM)-HER2:342 Affibody. Methods: Mice bearing BT474 tumors were given trastuzumab (50 mg/kg loading dose, 25 mg/kg maintenance dose, administered intraperitoneally twice a week) or saline (control) for a total of 5 doses. Tumor size was monitored twice a week. Animals were scanned before the treatment, at 48 h, and 2 wk after the beginning of therapy. After the final scan, PET results were correlated with tumor response and immunohistochemical assessment of ErbB2 level, as well as with vasculature in the treated tumors. Results: Analysis of PET images indicated that tracer uptake was significantly reduced after 1 dose of trastuzumab, compared with baseline, suggesting applicability as an early indicator of changes in ErbB2 expression. After 5 doses of trastuzumab, the overall decrease in 18F-FBEM-HER2:342 Affibody uptake also correlated with tumor response and downregulation of ErbB2 expression by immunohistochemical assessment. However, individual animals had different responses. There was a correlation between bigger PET changes and a higher vessel count in the tumors, suggesting that an increased number of vessels could lead to better trastuzumab delivery. We confirmed that the difference in average vessel count in the tumors was not related to the size of the tumors and therefore was not due to the selection of more vascular tumors. This finding is consistent with previous findings demonstrating that the number of vessels in a tumor could be a useful prognostic marker for treatment response. Conclusion: Our data suggest that Affibody-based PET can noninvasively provide specific information on changes in receptor expression and could be a valuable strategy for predicting tumor response to trastuzumab.

PET of HER2-Positive Pulmonary Metastases with 18F-ZHER2:342 Affibody in a Murine Model of Breast Cancer: Comparison with 18F-FDG

Targeted therapies often depend on the expression of the target present in the tumor. This expression can be difficult to ascertain in widespread metastases. 18F-FDG PET/CT, although sensitive, is nonspecific for particular tumor markers. Here, we compare the use of a human epidermal growth factor receptor 2 (HER2)–specific 18F-ZHER2:342-Affibody and 18F-FDG in HER2-expressing pulmonary metastases in a murine model of breast cancer. Methods: The lung metastasis model was established by intravenous injection of MDA-MB-231HER2-Luc human breast cancer cells into the tail vein. Bioluminescence imaging was used to evaluate metastasis progression. Uptake of 18F-ZHER2:342-Affibody and 18F-FDG was confirmed by coregistration of the PET images with MR and CT images. At the end of the study, the presence of neoplastic cells and HER2 expression in lung tissues, and distribution of the tracer, were assessed ex vivo by immunohistochemistry and autoradiography. Results: 18F-ZHER2:342-Affibody successfully targeted HER2-positive lesions in the lung and allowed detection of metastases as early as 9 wk after injection of cells. In contrast, 18F-FDG uptake was often masked by surrounding inflammatory changes and was nonspecific for HER2 expression. HER2 expression at a cellular level correlated well with tracer uptake on autoradiography. Conclusion: 18F-ZHER2:342-Affibody is a promising tracer for evaluation of HER2 status of breast cancer metastases and is more specific for detecting HER2-positive lesions than 18F-FDG.

Affibody-DyLight Conjugates for In Vivo Assessment of HER2 Expression by Near-Infrared Optical Imaging

Purpose Amplification of the HER2/neu gene and/or overexpression of the corresponding protein have been identified in approximately 20% of invasive breast carcinomas. Assessment of HER2 expression in vivo would advance development of new HER2-targeted therapeutic agents and, potentially, facilitate choice of the proper treatment strategy offered to the individual patient. We present novel HER2-specific probes for in vivo evaluation of the receptor status by near-infrared (NIR) optical imaging. Experimental Design Affibody molecules were expressed, purified, and labeled with NIR-fluorescent dyes. The binding affinity and specificity of the obtained probe were tested in vitro. For in vivo validation, the relationship of the measured NIR signal and HER2 expression was characterized in four breast cancer xenograft models, expressing different levels of HER2. Accumulation of Affibody molecules in tumor tissue was further confirmed by ex vivo analysis. Results Affibody-DyLight conjugates showed high affinity to HER2 (KD = 3.66±0.26). No acute toxicity resulted from injection of the probes (up to 0.5 mg/kg) into mice. Pharmacokinetic studies revealed a relatively short (37.53±2.8 min) half-life of the tracer in blood. Fluorescence accumulation in HER2-positive BT-474 xenografts was evident as soon as a few minutes post injection and reached its maximum at 90 minutes. On the other hand, no signal retention was observed in HER2-negative MDA-MB-468 xenografts. Immunostaining of extracted tumor tissue confirmed penetration of the tracer into tumor tissue. Conclusions The results of our studies suggest that Affibody-DyLight-750 conjugate is a powerful tool to monitor HER2 status in a preclinical setting. Following clinical validation, it might provide complementary means for assessment of HER2 expression in breast cancer patients (assuming availability of proper NIR scanners) and/or be used to facilitate detection of HER2-positive metastatic lesions during NIR-assisted surgery.