Spatio-temporäre Analyse des epidermalen Wachstumsfaktor-Rezeptors : Modulation der Signaltransduktion durch das humane Papillomavirus Typ 16 E5-Protein

Ursächlich für die Entstehung eines Zervixkarzinoms ist eine persistierende Infektion mit humanpathogenen Papillomaviren (HPV) des sogenannten Hochrisiko-Typs, deren DNA-Sequenzen in mehr als 65% aller Zervixkarzinome nachgewiesen werden konnten. Die HPV-Familie besteht aus mehr als 100 Typen, von denen vorwiegend die Typen 16 und 18 mit der tumoralen Entwicklung assoziiert sind. Verantwortlich für die Malignisierung des Epithels sind drei Onkogene des HPV-Typs 16, dazu zählt das kleine, hydrophobe Protein E5, welches hauptsächlich in der Membran von Golgi-Apparat und endosomaler Kompartimente lokalisiert ist. Das Protein besitzt selbst nur schwache transformierende Eigenschaften, ist aber in der Lage die Onkogenizität der beiden anderen Onkogene E6 und E7 zu potenzieren. Der Haupteffekt des E5-Onkogens ist eine Überaktivierung des EGF-Rezeptors (EGFR), deren Folge eine gesteigerte Transkription mit anschließender Mitose ist. Ziel dieser Arbeit war die Identifizierung der zellbiologischen Mechanismen, die durch E5 moduliert werden und eine verstärkte Aktivierung der EGF-Rezeptoren bewirken. Die quantitative Analyse mittels Immunblot und On-Cell Western Blot zeigte in Anwesenheit von HPV16 E5 eine Zunahme an Oberflächen-assoziierten EGF-Rezeptoren. Folglich wurde im Vergleich zu den Kontroll-Zellen eine Verstärkung der Liganden-vermittelten EGFR-Aktivierung hervorgerufen, die über einen langen Zeitraum nach Zugabe von EGF anhielt und unabhängig von der Internalisierungsrate war. Zusätzlich zu den klassischen molekular- und zellbiologischen Methoden wurde eine neue Technologie eingesetzt, die eine spatio-temporäre Analyse der E5-bedingten Modulation der EGFR-Signaltransduktion ermöglichte. Mit Hilfe der automatisierten, quantitativen Analyse von drei-dimensionalen Multikanalbildern, die am konfokalen Mikroskop generiert wurden, konnte eine genaue Charakterisierung des endozytotischen Transports von aktivierten und Gesamt-EGFR in Leervektor- und E5-transduzierten Zellen erfolgen. Der Einsatz von Vesikel-spezifischen Markern ermöglichte zudem die genaue Lokalisation der Rezeptoren innerhalb eines spezifischen zellulären Kompartiments. In Anwesenheit von E5 konnte eine stärkere und schnellere Fusion von EGFR-positiven Vesikeln mit dem frühen Endosomen nachgewiesen werden. Durch das zügige Fortschreiten des endozytotischen Transports erreichte eine deutliche höhere Anzahl von phospho-EGFR-positiven Vesikeln frühzeitig die perinuklear lokalisierten multivesikulären Körperchen (MVB), ohne zuvor die zytoplasmatischen MVBs zu passieren. Zu den späten Zeitpunkten der EGF-Stimulation konnte eine Akkumulation von aktivierten Rezeptoren in perinuklearen Endosomen-ähnlichen Kompartimenten heterogener Struktur dokumentiert werden, die mit einer verminderten Dephosphorylierung von aktivierten Rezeptoren verbunden war und schließlich zu einer langanhaltenden Überaktivierung von EGF-Rezeptoren führte. Durch die Behandlung mit dem Recycling-Inhibitor Monensin konnte im Vergleich zu den Kontrollen keine Beeinträchtigung der Phosphorylierung von EGFR in E5-exprimierenden Zellen dokumentiert werden. Damit kann eine potentielle Zunahme des Recyclings von EGF-Rezeptoren als Ursache der E5-vermittelten Überaktivierung ausgeschlossen werden. Die hier dargestellten Ergebnisse beweisen, dass die verstärkte und langanhaltende Aktivierung von EGFR durch eine modifizierte Endozytose und Dephosphorylierung während des Transports der Rezeptoren von frühen Endosomen zu den multivesikulären Körperchen verursacht wird. Dadurch umgehen die Rezeptoren den Abbau in den Lysosomen und bewirken eine gesteigerte Transkription früher Gene im Nukleus, die in einer EGFR-vermittelten Deregulation der Zellproliferation, -differenzierung und Apoptose resultiert

Focal delivery of AAV2/1-transgenes into the rat brain by localized ultrasound-induced BBB opening

Delivery of drugs and macromolecules to the central nervous system (CNS) is hindered by the blood-brain barrier (BBB). Several approaches have been used to overcome this hindrance to facilitate the treatment of various CNS diseases. We now present results showing that chimeric adeno-associated virus 2/1 (AAV2/1) particles containing the coding region for the LacZ gene are efficiently delivered into the rat brain upon intravenous (IV) administration after BBB opening by focused ultrasound in the presence of vascular acoustic resonators. We show that the transgene is correctly and efficiently expressed in cells located in the neighborhood of the insonated focus, especially in the vicinity of small vessels and capillaries. Histochemical LacZ staining allows the identification of large amounts of cells expressing the enzymatically active protein. Using double immunofluorescence (IF) with antibodies against tubulinIII and bacterial LacZ, we identified these cells to be mostly neurons. A small proportion of the transduced cells was recognized as glial cells, reacting positive in the IF with antibodies against astrocytic markers. These results demonstrate that our approach allows a very specific, localized, and efficient expression of intravenously administered transgenes in the brain of rats upon ultrasound-induced BBB opening

Multiparametric image analysis reveals role of Caveolin1 in endosomal progression rather than internalization of EGFR

AbstractEndosomes constitute a central layer in the regulation of growth factor signaling. We applied flow cytometry, confocal microscopy and automated image quantification to define the role of Caveolin1 (Cav1) in epidermal growth factor (EGF) receptor (i) internalization and (ii) endosomal trafficking. Antisense-downregulation of Cav1 did not affect internalization of EGF:EGFR-complexes from the plasma membrane. Instead, Cav1-knockdown had a profound effect on endosomal trafficking and caused a shift in EGF vesicle distribution towards Rab7-negative compartments at late timepoints. Moreover, image quantification with single-endosome resolution revealed that EGF:Cav1-complexes undergo a maturation pattern reminiscent of late endosomes. Our data suggest a model in which Caveolin1 acts upon EGF endosomes internalized via the Clathrin-pathway and functions at the transition from early to late endosomes

Abstract 3329: Combinatorial blockade of ERBB receptors in HER2 low breast cancer

Abstract Large number of breast cancer patients clinically classified as HER2 negative, show low/moderate levels of HER2 along with other ERBB receptors. Concomitant blockade of EGFR, ERBB2 and ERBB3 with specific therapeutic antibodies (cetuximab, trastuzumab/pertuzumab and lumretuzumab, respectively) appears as a beneficial approach to improve survival of patients who have failed to previous treatment strategies. Additionally, ERBB3 expression has been reported as a trastuzumab resistance mechanism in HER2 positive subtypes.We have confirmed a specific pattern of ERBB receptors expression in different breast cancer subtypes. Individual and combined blockade of the receptors with therapeutic antibodies has been tested in vitro using a metabolic viability assay (Cell Titer Glo) and their effects in cell cycle status measured by BrdU staining. All four therapeutic antibodies bind to the respective extracellular domains inhibiting downstream signaling pathways. Response to individual treatment is cell dependent and correlates with EGFR expression in the triple negative MDA-MB-468 but not in luminal T47D cancer cell line. Response of T47D cells to lumretuzumab is higher when combined with pertuzumab or trastuzumab, suggesting ERBB2/ERBB3 dimer as the most relevant one, an effect more evident upon ectopic addition of NRG1 (only ligand for ERBB3). Long exposure of MDA-MB468 cells to cetuximab induces ERBB3 expression and increases its sensitivity to cetuximab when combined. As well as, in the HER2+ BT474 cancer cell line resistant to trastuzumab, higher levels of ERBB3 also make cells more sensitive to alternative anti-ERBB therapies. To establish the role of ERBB4 (no specific therapeutic antibody) in the whole ERBB network, stable knock-down regulation (shRNA) has been performed in T47D cells. Although treatment of HER2 overexpressing breast tumors has been successful, targeting other ERBB members in a HER2 moderate/low scenario seems to be a promising approach in combinatorial therapies; even in resistant cell lines. Thus, better characterization of ERBB network should help to pave the way for a personalized treatment of HER2 low breast cancer. [U.K.: Deceased March, 2016] Citation Format: Mireia Berdiel-Acer, Eileen Reinz, Khalid Abnaof, Sara Burmester, Rainer Will, Ulrike Korf, Stefan Wiemann. Combinatorial blockade of ERBB receptors in HER2 low breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3329. doi:10.1158/1538-7445.AM2017-3329</jats:p

Reconstruction of Different Modes of WNT Dependent Protein Networks from Time Series Protein Quantification

Protein signaling networks are crucial cornerstones in cellular responses. Deregulation causes various diseases, including cancer. One pathway that is frequently deregulated in cancer is the WNT signaling pathway. It has been shown that WNT signaling is highly context-dependent and the availability of receptors and ligands determines downstream signaling. In order to reveal which signaling pathways are activated by a specific receptor-ligand combination, we overexpressed the non-canonical WNT receptor ROR2 in the human breast cancer cell line MCF-7 and stimulated it with its putative ligand WNT11. Based on characterization of the cells by Reverse Phase Protein Array (RPPA), we integrated the proteomic data by network reconstruction analysis with prior knowledge from a pathway database. Using this approach, we were able to identify novel edges that differed upon ROR2 overexpression and WNT11 stimulation

Focal delivery of AAV2/1-transgenes into the rat brain by localized ultrasound-induced BBB Opening

Delivery of drugs and macromolecules to the central nervous system (CNS) is hindered by the blood–brain barrier (BBB). Several approaches have been used to overcome this hindrance to facilitate the treatment of various CNS diseases. We now present results showing that chimeric adeno-associated virus 2/1 (AAV2/1) particles containing the coding region for the LacZ gene are efficiently delivered into the rat brain upon intravenous (IV) administration after BBB opening by focused ultrasound in the presence of vascular acoustic resonators. We show that the transgene is correctly and efficiently expressed in cells located in the neighborhood of the insonated focus, especially in the vicinity of small vessels and capillaries. Histochemical LacZ staining allows the identification of large amounts of cells expressing the enzymatically active protein. Using double immunofluorescence (IF) with antibodies against tubulinIII and bacterial LacZ, we identified these cells to be mostly neurons. A small proportion of the transduced cells was recognized as glial cells, reacting positive in the IF with antibodies against astrocytic markers. These results demonstrate that our approach allows a very specific, localized, and efficient expression of intravenously administered transgenes in the brain of rats upon ultrasound-induced BBB opening

Disentangling ERBB Signaling in Breast Cancer Subtypes—A Model-Based Analysis

Targeted therapies have shown striking success in the treatment of cancer over the last years. However, their specific effects on an individual tumor appear to be varying and difficult to predict. Using an integrative modeling approach that combines mechanistic and regression modeling, we gained insights into the response mechanisms of breast cancer cells due to different ligand–drug combinations. The multi-pathway model, capturing ERBB receptor signaling as well as downstream MAPK and PI3K pathways was calibrated on time-resolved data of the luminal breast cancer cell lines MCF7 and T47D across an array of four ligands and five drugs. The same model was then successfully applied to triple negative and HER2-positive breast cancer cell lines, requiring adjustments mostly for the respective receptor compositions within these cell lines. The additional relevance of cell-line-specific mutations in the MAPK and PI3K pathway components was identified via L1 regularization, where the impact of these mutations on pathway activation was uncovered. Finally, we predicted and experimentally validated the proliferation response of cells to drug co-treatments. We developed a unified mathematical model that can describe the ERBB receptor and downstream signaling in response to therapeutic drugs targeting this clinically relevant signaling network in cell line that represent three major subtypes of breast cancer. Our data and model suggest that alterations in this network could render anti-HER therapies relevant beyond the HER2-positive subtype.</jats:p

Disentangling ERBB Signaling in Breast Cancer Subtypes&mdash;A Model-Based Analysis

Targeted therapies have shown striking success in the treatment of cancer over the last years. However, their specific effects on an individual tumor appear to be varying and difficult to predict. Using an integrative modeling approach that combines mechanistic and regression modeling, we gained insights into the response mechanisms of breast cancer cells due to different ligand&ndash;drug combinations. The multi-pathway model, capturing ERBB receptor signaling as well as downstream MAPK and PI3K pathways was calibrated on time-resolved data of the luminal breast cancer cell lines MCF7 and T47D across an array of four ligands and five drugs. The same model was then successfully applied to triple negative and HER2-positive breast cancer cell lines, requiring adjustments mostly for the respective receptor compositions within these cell lines. The additional relevance of cell-line-specific mutations in the MAPK and PI3K pathway components was identified via L1 regularization, where the impact of these mutations on pathway activation was uncovered. Finally, we predicted and experimentally validated the proliferation response of cells to drug co-treatments. We developed a unified mathematical model that can describe the ERBB receptor and downstream signaling in response to therapeutic drugs targeting this clinically relevant signaling network in cell line that represent three major subtypes of breast cancer. Our data and model suggest that alterations in this network could render anti-HER therapies relevant beyond the HER2-positive subtype