The role of platelet-derived growth factor (PDGF) in malignancies involves both autocrine and paracrine stimulation of cells within the tumor. The interstitial fluid pressure (IFP) is one of the forces that govern the transvascular flow of fluids. In both experimental and clinical cancers, the IFP is elevated and is thought to act as a barrier for delivery of drugs. Increasing evidence points to PDGF as a positive regulator of the interstitial fluid pressure in loose connective tissue. In this thesis, the effect of PDGF receptor inhibition on the tumor IFP, transvascular transport and efficacy of anti-cancer drugs is investigated. All studies were performed using tumor models that display extensive tumor stroma and PDGF receptor expression restricted to stroma cells. Blocking of PDGF receptor signaling significantly reduced the tumor IFP in various tumor models. In parallel, pre-treatment with PDGF antagonists increased the tumor content of cytotoxic agents without affecting the uptake in other organs. Moreover, combination treatment with PDGF receptor inhibitors and chemotherapeutic agents dramatically enhanced the anti-tumor effects of the cytotoxic drugs, whereas treatment with only PDGF receptor inhibitors did not affect the growth of the tumors. Beneficial effects on the tumor reponse to radioimmunotherapy were also produced after concomitant administration of PDGF antagonists. Importantly, anti-angiogenic effects, changes in cell composition and increased tumor cell sensitivity to cytotoxic agents were ruled out as the cause for the synergistic effects. Studies with different temporal scheduling of PDGF receptor inhibitors demonstrated a perfect correlation between a reduced IFP, an increased transvascular transport and an enhanced therapeutic effect of cytotoxic drugs, strongly suggesting that the phenomena are causally linked. The studies presented herein illustrate for the first time the potential of cells in the stroma compartment as a target for efforts to treat cancer. In conclusion, a novel, possibly general, strategy to enhance the effects of conventional anti-cancer drugs has been identified.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.

The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

Efficacy of tyrosine kinase inhibitor (TKI) therapy depends on epidermal growth factor receptor (EGFR) mutation status in patients with non-small cell lung cancer (NSCLC). There has been an increasing interest in studying mutation-specific rabbit monoclonal antibodies of delE746-A750 mutation in exon 19 and L858R point mutation in exon 21 for detecting EGFR mutants. These two mutations account for approximately 90% of all EGFR mutations. We evaluated the two mutation-specific monoclonal antibodies for the detection of EGFR mutations by immunohistochemistry (IHC) and the relationship with treatment outcome and survival. Twenty-five patients (58.1%) harbored EGFR mutations. These mutations include delE746-A750 mutation for seven patients, L858R point mutation for in eighteen patients. IHC showed, for the delE746-A750 and L858R mutations, sensitivity (57.1% and 66.7%), specificity (97.3% and 100%), positive predictive value (80.0% and 100%), and negative predictive value (94.7% and 80.6%). Analysis for progression-free survival was not correlated to IHC staining, but the overall survival was correlated to IHC staining. These mutation-specific antibodies for delE746-A750 and L858R mutations have high positive predictive value and specificity for predefined EGFR mutations and may be suitable for screening for these predefined mutations. However, negative IHC results required further mutation analyses before excluding EGFR TKI therapy.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 126-144).

The investigations conducted within this dissertation centers around the use of electric cell-substrate impedance sensing (ECIS). This system is able to characterize in real-time analysis, the adhesion of cells to their substrate and neighboring cells. With this, valuable information can be gathered with in-vitro experiments regarding a tissue culture's response to physiological stimulation. This dissertation has taken advantage of ECIS' ability to analyze toxicology, barrier function, and cancer invasion on a tissue culture. With proper analysis modifications, trans-epethelial resistance (TER) can be used as a cytotoxicity assay with higher sensitivity than previously thought. In vitro assessment of cytotoxicity based on TER needs more quantitative methods to analyze the alteration of cell morphology and motility. Here, we applied ECIS to evaluate dose-dependent responses of human umbilical vein endothelial cells (HUVEC) and mouse embryonic fibroblasts (NIH 3T3) exposed to cytochalasin B and protein kinase inhibitor H7. To detect subtle changes in cell morphology, the frequency-dependent impedance data of the cell monolayer were measured and analyzed with a theoretical cell-electrode model. To detect the alternation of cell micromotion in response to cytochalasin B and H7 challenge, time-series impedance fluctuations of cell-covered electrodes were monitored and the values of power spectrum, variance, and variance of the increment were calculated to verify the difference. While a dose-dependent relationship was generally observed from the overall resistance of the cell monolayer, the analysis of frequency-dependent impedance and impedance fluctuations distinguished cytochalasin B levels as low as 0.1µM and H7 levels as low as 10 µM for HUVEC and 3T3 layers. Even though overall resistance values are relatively small for 3T3 layers, and frequency scan measurements are negligible, impedance fluctuation analysis reveals significant micromotion for cytotoxic detection. Our results show that cytochalasin B and H7 causes a decrease of junctional resistance between cells and an increase of membrane capacitance. Cigarette smoke is cytotoxic and tumorigenic. Initial studies were conducted to evaluate the cytotoxicity of cigarette smoke condensate (CSC) on HUVEC layers. The focus was then turned to investigations involving in vitro cancer invasion assays with CSC on HUVEC layers. ECIS is an excellent investigative device that can be utilized to observe cancer invasion on normal tissue cultures due to the significantly higher impedance signature of cancer cells. The investigation in this dissertation focused on cigarette smoke's influence on cellular mechanics of endothelial cells and the invasive potential of two ovarian cancer cell lines (ALST and OVCA429) against a fully active endothelium. The HUVEC cultures responded to CSC with an increase in junctional binding, where as ALST and OVCA429 relieved adhesion thereby providing an improved motility when evaluated in wound healing assays. Transmigration of the HUVEC layer by ALST cells exhibit a pre-CSC exposure time-dependence affecting the effectiveness of ALST transmigration. The HUVEC layer's decreased tight junction binding that resulted from CSC exposure, allowed for a more aggressive ALST layer formation that occurred during simulated intravasation. Increased HUVEC layer tight junction binding that occurred in the first five hours in response to CSC during extravasation contributes to impeding ALST transmigration at high concentrations of CSC. Overall, CSC has an impeding effect on ALST transmigration during extravasation while causing aggressive transmigration during intravasation.

Sarcomas are rare mesenchymally-derived tumors with limited treatment options. Tyrosine kinases may serve as potential targets for sarcoma therapy because many are mutated or overexpressed in sarcomas and cell lines. One potential molecular target for sarcoma treatment is the Src tyrosine kinase. Three independently synthesized Src kinase inhibitors were evaluated in human sarcoma cell lines. Of the three, dasatinib, provided promising results as a potential sarcoma therapy. Until this study, dasatinib activity had not been characterized in sarcoma cells. Based on our previous findings of Src activation in human sarcomas, we evaluated the effects of dasatinib in twelve sarcoma cell lines. Dasatinib inhibited Src activity and downstream signaling at nanomolar concentrations. Inhibition of Src signaling was accompanied by blockade of cell migration and invasion. Moreover, apoptosis was induced in a subset of bone sarcomas at nanomolar concentrations of dasatinib. Inhibition of Src protein expression by siRNA also induced apoptosis, indicating that these bone sarcoma cell lines are dependent on Src activity for survival. These results demonstrate that dasatinib inhibits migration and invasion of diverse sarcoma cell types, and selectively blocks the survival of bone sarcoma cells. Therefore dasatinib may provide therapeutic benefit by preventing the growth and metastasis of sarcomas. Microarray analysis of the sarcoma cell lines lead to the identification of a molecular signature that successfully predicts response to dasatinib by induction of apoptosis. Components of this molecular signature are expressed in primary human sarcomas. Furthermore, expression of the molecular signature in sarcomas can be utilized to cluster tumors based on theoretical response to dasatinib. While the prediction of response in tumors is theoretical, there is encouraging evidence to support further endeavors into validating the potential of this molecular signature to predict response in patients.Together, these studies reveal that, in cell lines, both constitutive Src activation and the presence of a molecular signature that predicts response to dasatinib are important parameters to consider when selecting dasatinib as a treatment for. Furthermore, novel therapeutic approaches that inhibit Src signaling may selectively induce apoptosis in tumor cells and sensitize to chemotherapy those tumors that contain the relevant molecular signature.

Targeted therapies have begun to be developed and approved in the clinic over the past several decades to treat cancers with specific genetic alterations. In non-small cell lung cancer (NSCLC), patients harboring EGFR activating mutations often respond to the EGFR inhibitors gefitinib/erlotinib, exhibiting down-regulation of central oncogenic pathways and dramatic tumor regressions. Despite initially promising results, the vast majority of patients develop resistance to targeted therapies. Thus far, several mechanisms of resistance including T790M mutation in EGFR, amplification of the MET receptor tyrosine kinase (RTK), activating mutations in downstream signaling molecules, and loss of negative regulators have been identified. As a result, next generation inhibitors and combination therapies continue to be developed and tested in the clinic. There are still many cases in which the cause of resistance to a particular targeted therapy is unknown, or the subset of patients most likely to benefit has not been identified. This thesis describes the ability of the MET ligand, HGF, to activate PI3K signaling and cause gefitinib resistance in EGFR-driven cancers. In addition, detection of a preexisting subpopulation of MET amplified cells (present before treatment with an EGFR inhibitor) is shown to successfully predict the development of MET amplification as a resistance mechanism. These results suggest that it may be possible to prospectively identify patients who will benefit from combined MET/HGF and EGFR inhibitors as initial therapies. Further, this thesis highlights the importance of both PI3K/AKT and MEK/ERK signaling as drivers of cell proliferation and viability, and describes a novel feedback network regulating these pathways. In multiple cancer models, treatment with a single agent MEK inhibitor leads to feedback up-regulation of ERBB3/PI3K/AKT signaling. The mechanism for this feedback involves loss of an inhibitory threonine phosphorylation in the conserved juxtamembrane domains of EGFR and HER2 following MEK inhibition, which leads to increased ERBB receptor activation. These results further elucidate the complex feedback networks that regulate signaling in cancer cells, and suggest possible limitations for the efficacy of single agent RAF/MEK pathway inhibitors. Collectively, this work describes multiple resistance mechanisms to kinase inhibitors, and suggests new biomarkers to define those patients who are likely to benefit from specific targeted therapies.