LIV-1, a zinc transporter, is an effector molecule downstream from soluble growth factors. This protein has been shown to promote epithelial-to-mesenchymal transition (EMT) in human pancreatic, breast, and prostate cancer cells. Despite the implication of LIV-1 in cancer growth and metastasis, there has been no study to determine the role of LIV-1 in prostate cancer progression. Moreover, there is no clear delineation of the molecular mechanism underlying LIV-1 function in cancer cells. In this study, we found increased LIV-1 expression in a progresssive manner in benign, PIN, primary and bone metastatic human prostate cancer. We characterized the mechanism by which LIV-1 drives prostate cancer EMT in an androgen-refractory human prostate cancer cell (ARCaP) bone metastasis model. LIV-1, when overexpressed in ARCaPE cells (derivative cells of ARCaP with epithelial phenotype), promoted EMT irreversibly. LIV-1 overexpressed ARCaPE cells had elevated levels of HB-EGF and matrix metalloproteinase (MMP) 2 and MMP 9 proteolytic enzyme activities, without affecting intracellular zinc concentration. The activation of MMPs resulted in the shedding of heparin binding-epidermal growth factor (HB-EGF) from ARCaPE cells, eliciting constitutive epidermal growth factor receptor (EGFR) phosphorylation and its downstream extracellular signal regulated kinase (ERK) signaling. Further investigation of the HB-EGF promoter revealed that both Stat3 and AP-1 controlled HB-EGF promoter activity. Ectopic LIV-1 overexpression induced AP-1 and Stat3 activation. Blockade of both Stat3 and AP-1 by specific inhibitors or dominant negative expression vectors diminished the HB-EGF promoter activity induced by LIV-1 overexpression. These results suggest that LIV-1 is involved in prostate cancer progression as an intracellular target of growth factor receptor signaling which promotes EMT and cancer metastasis. LIV-1 could be an attractive therapeutic target for the eradication of pre-existing human prostate cancer and bone and soft tissue metastases.

Activated epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations characterize molecular subgroups of non-small cell lung cancer (NSCLC) and have a strong predictive value for response to EGFR inhibitor therapy. Recently, EGFR mutation testing was included in the diagnostic algorithm of NSCLC. However, there is a controversy about the clonal stability of the mutation during the progression of the disease. The aim of this study was to analyze NSCLC tumor tissue for the presence of both EGFR and KRAS mutations in morphologically different parts of the primary tumor. Formaldehyd fixed and paraffin embedded lung cancer specimens from primary resected NSCLC patients were selected; five cases harboring EGFR and five with KRAS mutations. From each tumor, three morphologically different tumor sites were manually micro-dissected and analyzed for the presence of EGFR and KRAS mutations. Additionally, normal lung tissue at a distance from the primary tumor as well as in close vicinity was tested.The EGFR and KRAS status were consistent in the three different areas of the primary tumors of all ten cases. EGFR as well as KRAS mutations were as well detectable in close and in some distant normal lung parenchyma in 7 of 10 analyzed patient samples. In conclusion, we found consistent KRAS and EGFR mutation status in primary NSCLC tumors. This finding is of importance for clinical practice, because it indicates that any part of the tumor, independent of intratumoral histological pattern, is representative for EGFR and KRAS mutation testing.

Activated receptor tyrosine kinase (RTK), such as the epidermal growth factor (EGF) receptor (EGFR) and the platelet-derived growth factor (PDGF) receptor (PDGFR), recruit downstream signalling proteins, including phosphatidylinositol 3-kinase (PI3K). PI3K, composed of a regulatory p85 subunit and a catalytic p110 subunit, phosphorylates phosphatidylinositol 4,5-bisphosphate at the 3 position to generate phosphatidylinositol 3,4,5-trisphosphate. This lipid second messenger activates Akt, which promotes cell growth, cell cycle entry and progression, as well as cell survival and cellular migration. PTEN, a tumor suppressor protein, dephosphorylates phosphatidylinositol 3,4,5-trisphosphate at the 3 position, turning off Akt signalling. PTEN contains a C-terminal PDZ binding motif that binds to the PDZ2 domain of MAGI-2, a scaffolding protein that localizes signalling molecules to the plasma membrane. MAGI-2 has ten domains that potentially mediate multiple protein-protein interactions simultaneously. A PTEN associated-complex (PAC) has been described and may contain MAGI-2, PTEN and p85. The PAC is hypothesized to form at the plasma membrane at appropriate sites for PTEN to gain access to its lipid substrates, since the binding of PTEN to MAGI-2 has been shown to enhance the suppression of PI3K-mediated Akt signalling. In order to better understand the role of the PAC in attenuation of the Akt signalling pathway, regions of the MAGI-2 scaffolding protein were mapped to identify the interactions taking place in the PAC. MAGI-2, and its individual domains, were expressed as GST fusion proteins. These were immobilized onto beads and allowed to bind to cellular proteins including PTEN, p85, PDGFR and EGFR using a GST pull-down experiment. The proteins bound to GST-MAGI-2 were identified using an immunoblot analysis. In vitro pull-down experiments revealed that MAGI-2 PDZ2 and PDZ5 domains bind to PTEN, and both MAGI-2 WW domains were shown to bind to p85. EGFR and PDGFR did not bind to the PDZ domains of MAGI-2 under the conditions studied. In order to study protein-protein interactions in cells, immunoprecipitation assays were also performed. Full length MAGI-2 was expressed tagged to a Myc epitope. This was used in immunoprecipitation assays to determine if MAGI-2 could co-immunoprecipitate with proteins involved in the Akt signalling pathway, such as PTEN, p85, PDGFR and EGFR. MAGI-2 can co-immunoprecipitate with PTEN upon 5 min EGF stimulation however, this result was inconclusive because replicate experiments did not verify this initial observation. MAGI-2 does not co-immunoprecipitate with the EGFR nor p85, under the conditions tested. We examined for these interactions after 5 min of growth factor stimulation and more experiments that test different time points after growth factor stimulation would reveal if these interactions are present at shorter time points. MAGI-2 has been shown to bind to PTEN and p85 in vitro and therefore has the potential to regulate the attenuation of the PI3K/Akt signalling pathway in response to activated EGFR and/or PDGFR.

<p>Noble metal nanoparticles exhibit unique optical properties that are beneficial to a variety of applications, including molecular imaging. The large scattering cross sections of nanoparticles provide high contrast necessary for biomarkers. Unlike alternative contrast agents, nanoparticles provide refractive index sensitivity revealing information regarding the local cellular environment. Altering the shape and composition of the nanoparticle shifts the peak resonant wavelength of scattered light, allowing for implementation of multiple spectrally distinct tags. In this project, nanoparticles that scatter in different spectral windows are functionalized with various antibodies recognizing extra-cellular receptors integral to cancer progression. A hyperspectral imaging system is developed, allowing for visualization and spectral characterization of cells labeled with these conjugates. Various molecular imaging and microspectroscopy applications of plasmonic nanoparticles are then investigated. First, anti-EGFR gold nanospheres are shown to quantitatively measure receptor expression with similar performance to fluorescence assays. Second, anti-EGFR gold nanorods and novel anti-IGF-1R silver nanospheres are implemented to indicate local cellular refractive indices. Third, because biosensing capabilities of nanoparticle tags may be limited by plasmonic coupling, polarization mapping is investigated as a method to discern these effects. Fourth, plasmonic coupling is tested to monitor HER-2 dimerization. Experiments reveal the interparticle conformation of proximal HER-2 bound labels, required for plasmonic coupling-enhanced dielectric sensing. Fifth, all three functionalized plasmonic tags are implemented simultaneously to indicate clinically relevant cell immunophenotype information and changes in the cellular dielectric environment. Finally, flow cytometry experiments are conducted utilizing the anti-EGFR nanorod tag to demonstrate profiling of receptor expression distribution and potential increased multiplexing capability.</p> / Dissertation

LRBA (LPS Responsive Beige-like Protein Kinase A anchor) gene expression is induced by the mitogen LPS and is a member of the WBW gene family member which is comprised of genes that are involved in cellular proliferation and differentiation. This work provides evidence for the over-expression of LRBA in certain cancers, and that LRBA promoter activity and endogenous LRBA mRNA levels are negatively regulated by the tumor suppressor p53 and positively regulated by E2F transactivators. Furthermore, we demonstrate that inhibition of LRBA expression or function leads to decreased proliferation of cancer cells and that LRBA plays a role in the EGFR signal transduction pathway. In addition to the findings of LRBA's role in carcinogenesis, this work also shows evidence of the knockdown of the SH2-containing Inositol 5' Phosphatase (SHIP) in both mouse and human cells. Furthermore, we provide evidence that SHIP-1 is involved in the AKT signal transduction pathway in human Natural Killer cells.

Secondary bile acids have long been postulated to be tumor promoters in the colon but their mechanism of action are yet to be delineated. Though most bile acids are chemically similar, they have been found to exert contrasting signaling effects in the colonic epithelium. Particularly, hydrophobic bile acids such as deoxycholic acid (DCA) are found to be tumor promoters while their hydrophilic counterparts such as ursodeoxycholic acid (UDCA) are chemopreventive. Given the fact that colon cells do not possess bile acid transporters, the question that arises is how do bile acids activate intracellular signaling? In our studies, we examined the actions of bile acids at the cell membrane and found that hydrophobic bile acids can perturb membrane structure. This membrane perturbation was found to be characterized by a change in membrane fluidity and by cholesterol aggregation. Additionally, several membrane associated proteins were found to be deregulated in response to DCA further supporting the above conclusion regarding membrane perturbation. Moreover, caveolin, a negative regulator of membrane microdomains was seen to be dephosphorylated and disassociated from the membrane microdomains, implicating membrane microdomains as a possible target of the effects of DCA on the membrane. Consistent with this, we found that DCA was able to cause rapid and sustained activation of the receptor tyrosine kinase, EGFR and that this activation was ligand-independent. Using fluorescent-tagged bile acids we showed increased aggregation and clustering in the membranes treated with FITC-DCA in a manner that was reminiscent of receptor activation in immune cells. Collectively, these data suggest that bile-acid induced signaling is likely to be initiated through alterations of the plasma membrane structure in colon cancer cells.

The epidermal growth factor family of receptors is important in the development and progression of many types of cancers including, breast, lung, and glioblastoma. The family consists of 4 members (EGFR/erbB1, Her2/erbB2, erbB3, and erbB4). In all breast cancer cases, EGFR expression is deregulated 20 to 30% of the time; however in the most aggressive form of breast cancer (basal-like) EGFR expression is upregulated in 60% of cases. EGFR's expression and activity can be altered in transformed cells through a variety of mechanisms, such as novel protein-protein interactions, gene amplification, mutations, and loss of regulatory proteins. In this work we have examined the role of cancer specific protein interactions of EGFR with MUC1 and beta-catenin in the progression of breast cancer.Herein I report that the interaction of MUC1 and EGFR in breast cancer cells alters EGFR localization by promoting EGFR nuclear translocation. Importantly, I discovered that the presence of MUC1 mediates EGFR's interaction with chromatin. More specifically, I found that EGFR interacts with the cyclin D1 promoter region in a MUC1-dependent fashion which resulted in a significant increase in cyclin D1 protein expression. Nuclear EGFR localization has been shown to correlate with resistance to anti-EGFR therapies, which indicates that MUC1's interaction with EGFR could be a mechanism of resistance.MUC1's interaction with both EGFR and beta-catenin can promote transformation therefore a peptide therapy was developed, PMIP, which mimics the hypothesized interaction domains of MUC1's cytoplasmic tail. PMIP was designed to inhibit the interaction of MUC1/EGFR and MUC1/beta-catenin thereby regulating EGFR expression and promoting beta-catenin localization to adherens junctions. PMIP effectively enters the cytosol of cells and inhibits the target interactions. Importantly, PMIP inhibited invasion and proliferation of breast cancer cells and in mice significantly reduced the growth rate of breast cancer xenograft and genetically-driven tumors. This study demonstrated that the use of peptides to inhibit intracellular protein interactions is a viable option that would have limited toxic side-effects. Overall, this work reveals a new regulatory role of EGFR localization and activity by MUC1 and that this mechanism is viable therapeutic breast cancer target.Lastly, in a mouse model of breast cancer I examined the role of EGFR tyrosine kinase activity in beta-catenin dependent tumorigenesis. A transgenic mouse model of breast cancer, MMTV-Wnt-1, was bred onto an EGFR kinase deficient background. I discovered that the loss of EGFR kinase activity in this model resulted in a significant delay in tumor onset and inhibited tumor growth. These findings indicate a cooperation of EGFR and beta-catenin dependent signaling pathways, which promote transformation of glandular epithelial cells.

No description available.

Tumorerkrankungen stellen in der westlichen Welt eines der wichtigsten Gesundheitsprobleme dar. Das kolorektale Karzinom ist dabei die dritthäufigste Tumorneuerkrankung. Bei fortgeschrittenem Krankheitsverlauf wird zumeist eine kombinierte Radiochemotherapie durchgeführt, bei der zusätzlich zur Bestrahlung Zytostatika wie 5-Fluoruracil oder Oxaliplatin verabreicht werden. Da die eingesetzten Zytostatika nicht ausschließlich gegen Tumorzellen wirken, führt der Einsatz dieser oft zu massiven Nebenwirkungen wie Magen- und Darmproblemen, Myelosuppression und Haarausfall. Neue Therapieansätze versuchen daher Ziele in die Behandlung mit aufzunehmen die stärker karzinomspezifisch sind, wie z.B. verschiedenen Rezeptortyrosinkinasen. Viele Rezeptortyrosinkinasen und deren Liganden liegen im Tumor und umliegenden Gewebe oft dereguliert vor und spielen eine wichtige Rolle bei der Regulierung des Tumorwachstums, der Tumorangiogenese und der Metastasenbildung. In dieser Arbeit konnte für die drei kolorektalen Karzinomzelllinien DLD-1, SW837 und Caco 2 gezeigt werden, dass die gleichzeitige Inhibition des Insulin-like Growth Factor-I Receptor (IGF-IR) und des Epidermal Growth Factor Receptor (EGFR) mit den Tyrosinkinaseinhibitoren AEW-541 (IGF-IR-Inhibitor) und Erlotinib (EGFR-Inhibitor) in vitro zu einem deutlich verstärkten Therapieeffekt der 5-Fluoruracil-basierten Radiochemotherapie führt. Für Xenografttumore der Zelllinie SW837 konnte dieser Effekt auch in vivo bestätigt werden. Mit Hilfe der Co Immunpräzipitation und eines Proximity Ligation Assays konnten in den Kolonkarzinomzelllinien SW480 und DLD-1 Hybridrezeptoren zwischen dem EGFR und dem IGF-IR nachgewiesen werden. Zusätzlich konnte gezeigt werden, dass eine Ligandenstimulation der Rezeptoren zu einer vermehrten EGFR/IGF-IR-Hybridrezeptorbildung führt. Weitere Analysen zeigten, dass für die induzierte Heterodimerisierung beide Liganden notwendig sind und beide Rezeptoren funktionsfähig sein müssen. Mit Hilfe des Proximity Ligation Assays konnten IGF-IR/EGFR-Hybridrezeptoren auch in humanen Rektumtumoren nachgewiesen werden. Im letzten Teil der Arbeit wurde die Bedeutung des Platelet-derived Growth Factor Receptor β (PDGFR-β) in kolorektalen Karzinomzellen untersucht. In SW480- und DLD-1-Zellen führte die Inhibition des PDGFR-β mit Hilfe von spezifischer siRNA zu einer, über den PI3K-Signalweg vermittelten, moderat verminderten Proliferationsrate. Die Verwendung des PDGFR-β-Inhibitors Ki11502 führte in den Zelllinien zu einem starken Rückgang in der Proliferationsrate und zu Veränderungen im Zellzyklus der Zellen. Diese wurden durch eine verminderte Cyclin-B1-Expression hervorgerufen. Weitere Analysen zeigten, dass der Inhibitor Ki11502 neben dem PDGFR-β auch den Rezeptor cKIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) und die Zellmembran-assoziierte zytoplasmatische Tyrosinkinase SRC (v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog) inhibiert.

The primary treatment for colorectal cancer is surgery. Radiotherapy and chemotherapy, sometimes combined, are also frequently used to diminish recurrence risk. In response to radiation exposure, several cellular signaling cascades are activated to repair DNA breaks, prevent apoptosis and to keep the cells proliferating. Several proteins in the radiation response and cell survival pathways are potential targets to enhance the effects of radiation. The epidermal growth factor receptor (EGFR), which is frequently upregulated in colorectal cancer and exhibits a radiation protective function, is an attractive target for treatment. EGFR is activated by radiation which in turn activates numerous signaling pathways such as the PI3 kinase/AKT cascade, the RAS/RAF/ERK pathway and STAT leading to tumor cell proliferation. EGFR is also believed to interact with proteins in the DNA repair process, such as DNA-PKcs and MRE11. The cytotoxic effect of an affibody molecule (ZEGFR:1907)2, with high affinity to EGFR,  in combination with radiation produced a small, but significant, reduction in survival in a KRAS mutated cell line. However, not in the BRAF mutated cell line. The next step was therefore to target proteins downstream of EGFR such as AKT. There was an interaction between AKT and the DNA repair proteins DNA-PKcs and MRE11 and both AKT1 and AKT2 were involved in the radiation response. The knockout of both AKT isoforms impaired the DNA double strand break rejoining after radiation and suppression of DNA-PKcs increased the radiations sensitivity and decreased the DNA repair further. The AKT isoforms also affected the expression of cancer stem cell markers CD133 and CD44 which are associated with the formation of metastasis as well as radiation and drug resistance. The CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. AKT was also involved in cell migration, cell-adhesion and metabolism. Overall, these results illustrate the complexity in response to radiation and drugs in cells with different mutations and the need for combining inhibitors against several targets such as EGFR, AKT, DNA-PKcs, CD133 or CD44.