Roles of Alix in regulating cell morphology

Mammalian Alix (ALG2-interacting protein X&barbelow;) is a conserved adaptor protein that is involved in endosomal trafficking, apoptosis and growth factor receptor turnover. Accumulating evidence also indicates that Alix plays roles in promoting/maintaining spread and aligned fibroblast morphology in monolayer culture. Since cell morphology is determined by the structure and dynamics of an integrin-mediated transmembrane protein network that links extracellular matrix to intracellular cytoskeleton, we hypothesized that Alix plays direct or indirect roles in regulating certain components or steps in this transmembrane protein network. To test this hypothesis, we first examined the subcellular localization of Alix and discovered that, as a predominantly cytoplasmic protein, Alix is also present on the substratum/cell surface and in the conditioned medium of fibroblast cultures. Further, precoating of culture surfaces with recombinant Alix promotes spreading and fibronectin assembly to NIH/3T3 cells, and siRNA-mediated Alix knockdown in W138 cells has the opposite effects. These findings indicate the extracellular functions of Alix in regulating cell spreading and extracellular matrix assembly. In a separate study, we analyzed Alix immunocomplexes from normal fibroblast W138 cells by mass spectrometry and identified actin as a major partner protein of Alix. Follow-up studies demonstrated that Alix preferentially binds filamentous actin (F-actin) in vitro and is required for maintaining normal F-actin content and proper actin cytoskeleton assembly in W138 cells. These findings establish direct and essential roles of Alix in regulating actin cytoskeleton. Finally, we investigated the effects of Alix knockdown on the activation and subcellular localization of FAK and Pyk2, the focal adhesion kinases required for cell spreading/migration by promoting turnover of integrin-mediated cell adhesions. We discovered that Alix knockdown inhibits FAK and Pyk2 localizations to focal adhesions or plasma membrane, in association with characteristics of reduced turnover of focal adhesions. These findings reveal a positive role of Alix in focal adhesion turnover. Based on these results, we conclude that Alix targets both intracellularly and extracellularly components to regulate extracellular matrix remodeling, actin cytoskeleton assembly and focal adhesion turnover. A combination of these three functions of Alix explains its crucial role in regulating spread and aligned fibroblast morphology. ^

Tumor heterogeneity dictates sensitivity to gefitinib (Iressa(TM)/ZD1839) in solid tumor models

The epidermal growth factor receptor (EGFR) and its ligands are overexpressed in many human tumors, including bladder and pancreas, correlating with a more aggressive tumor phenotype and poor patient prognosis. We initiated the present study to characterize the heterogeneity of gefitinib responsiveness in a panel of human bladder and pancreatic cancer cell lines in order to identify the biological characteristics of EGFR-dependent proliferation that could be used to prospectively identify drug-sensitive tumors. A second objective was to elucidate how to best exploit these results by utilizing gefitinib in combination therapy. To these ends, we examined the effects of the EGFR antagonist gefitinib on proliferation and apoptosis in a panel of 18 human bladder cancer cell lines and 9 human pancreatic cancer cell lines. Our data confirmed the existence of marked heterogeneity in Iressa responsiveness with less than half of the cell lines displaying significant growth inhibition by clinically relevant concentrations of the drug. Gefitinib responsiveness was found to be p27 kip1 dependent as DNA synthesis was restored following exposure to p27siRNA. Unfortunately, Iressa responsiveness was not closely linked to surface EGFR or TGF-α expression in the bladder cancer cells, however, cellular TGF-α expression correlated directly with Iressa sensitivity in the pancreatic cancer cell lines. These findings provide the potential for prospectively identifying patients with drug-sensitive tumors. ^ Further studies aimed at exploiting gefitinib-mediated cell cycle effects led us to investigate if gefitinib-mediated TRAIL sensitization correlated with increased p27kip1 accumulation. We observed that increased TRAIL sensitivity following gefitinib exposure was not dependent on p27 kip1 expression. Additional studies initiated to examine the role(s) of Akt and Erk signaling demonstrated that exposure to PI3K or MEK inhibitors significantly enhanced TRAIL-induced apoptosis at concentrations that block target phosphorylation. Furthermore, combinations of TRAIL and the PI3K or MEK inhibitors increased procaspase-8 processing above levels observed with TRAIL alone, indicating that the effects were exerted at the level of caspase-8 activation, considered the earliest step in the TRAIL pathway. ^

The Spanish translation, cultural adaptation and validation of the Powe Fatalism Inventory

Background/significance. The scarcity of reliable and valid Spanish language instruments for health related research has hindered research with the Hispanic population. Research suggests that fatalistic attitudes are related to poor cancer screening behaviors and may be one reason for low participation of Mexican-Americans in cancer screening. This problem is of major concern because Mexican-Americans constitute the largest Hispanic subgroup in the U.S.^ Purpose. The purposes of this study were: (1) To translate the Powe Fatalism Inventory, (PFI) into Spanish, and culturally adapt the instrument to the Mexican-American culture as found along the U.S.-Mexico border and (2) To test the equivalence between the Spanish translated, culturally adapted version of the PFI and the English version of the PFI to include clarity, content validity, reading level and reliability.^ Design. Descriptive, cross-sectional.^ Methods. The Spanish language translation used a translation model which incorporates a cultural adaptation process. The SPFI was administered to 175 bilingual participants residing in a midsize, U.S-Mexico border city. Data analysis included estimation of Cronbach's alpha, factor analysis, paired samples t-test comparison and multiple regression analysis using SPSS software, as well as measurement of content validity and reading level of the SPFI. ^ Findings. A reliability estimate using Cronbach's alpha coefficient was 0.81 for the SPFI compared to 0.80 for the PFI in this study. Factor Analysis extracted four factors which explained 59% of the variance. Paired t-test comparison revealed no statistically significant differences between the SPFI and PFI total or individual item scores. Content Validity Index was determined to be 1.0. Reading Level was assessed to be less than a 6th grade reading level. The correlation coefficient between the SPFI and PFI was 0.95.^ Conclusions. This study provided strong psychometric evidence that the Spanish translated, culturally adapted SPFI is an equivalent tool to the English version of the PFI in measuring cancer fatalism. This indicates that the two forms of the instrument can be used interchangeably in a single study to accommodate reading and speaking abilities of respondents. ^

Molecular mechanisms of response in gastrointestinal stromal tumors treated with imatinib mesylate

Gastrointestinal stromal tumors (GIST) represent 80% of sarcoma arising from the GI tract. The inciting event in tumor progression is mutation of the kit or, rarely, platelet derived growth factor receptor-α (PDGFR) gene. These mutations encode ligand independent, constitutively active proteins: Kit or PDGFR. ^ These tumors are notoriously chemo and radio resistant. Historically, patients with advanced disease realized a median overall survival of 9 months. However, with modern management of GIST with imatinib mesylate (Novartis), a small molecule inhibitor of the Kit, PDGFR, and Abl tyrosine kinases, patients now realize a median overall survival greater than 30 months. However, almost half of patients present with surgically resectable GIST and the utility of imatinib in this context has not been prospectively studied. Also, therapeutic benefit of imatinib is variable from patient to patient and alternative targeted therapy is emerging as potential alternatives to imatinib. Thus, elucidating prognostic factors for patients with GIST in the imatinib-era is crucial to providing optimal care to each particular patient. Moreover, the exact mechanism of action of imatinib in GIST is not fully understood. Therefore, physicians find difficulty in accurately predicting which patient will benefit from imatinib, how to assess response to therapy, and the time at which to assess response. ^ I have hypothesized that imatinib is tolerable and clinically beneficial in the context of surgery, VEGF expression and kit non-exon 11 genotypes portend poor survival on imatinib therapy, and imatinib's mechanism of action is in part due to anti-vascular effects and inhibition of the Kit/SCF signaling axis of tumor-associated endothelial cells. ^ Results herein demonstrate that imatinib is safe and increases the duration of disease-free survival when combined with surgery. Radiographic and molecular (namely, apoptosis) changes occur within 3 days of imatinib initiation. I illustrate that non-exon 11 mutant genotypes and VEGF are poor prognostic factors for patients treated with imatinib. These findings may allow for patient stratification to emerging therapies rather than imatinib. I show that imatinib has anti-vascular effects via inducing tumor endothelial cell apoptosis perhaps by abrogation of the Kit/SCF signaling axis. ^

Oncogenic activation of c-Abl tyrosine kinase in non-small cell lung cancer: FUS1 tumor suppressor down-regulates c-Abl tyrosine kinase

The FUS1 tumor suppressor gene (TSG) has been found to be deficient in many human non-small cell lung cancer (NSCLC) tissue samples and cell lines (1,2,3). Studies have shown potent anti-tumor activity of FUS1 in animal models where FUS1 was delivered through a liposomal vector (4) and the use of FUS1 as a therapeutic agent is currently being studied in clinical human trials (5). Currently, the mechanisms of FUS1 activity are being investigated and my studies have shown that c-Abl tyrosine kinase is inhibited by the FUS1 TSG.^ Considering that many NSCLC cell lines are FUS1 deficient, my studies further identified that FUS1 deficient NSCLC cells have an activated c-Abl tyrosine kinase. C-Abl is a known proto-oncogene and while c-Abl kinase is tightly regulated in normal cells, constitutively active Abl kinase is known to contribute to the oncogenic phenotype in some types of hematopoietic cancers. My studies show that the active c-Abl kinase contributes to the oncogenicity of NSCLC cells, particularly in tumors that are deficient in FUS1, and that c-Abl may prove to be a viable target in NSCLC therapy.^ Current studies have shown that growth factor receptors play a role in NSCLC. Over-expression of the epidermal growth factor receptor (EGFR) plays a significant role in aggressiveness of NSCLC. Current late stage treatments include EFGR tyrosine kinase inhibitors or EGFR antibodies. Platelet-derived growth factor receptor (PDGFR) also has been shown to play a role in NSCLC. Of note, both growth factor receptors are known upstream activators of c-Abl kinase. My studies indicate that growth factor receptor simulation along deficiency in FUS1 expression contributes to the activation of c-Abl kinase in NSCLC cells. ^

The transcriptional repressor Delta EF1 controls resistance to the EGFR inhibitor erlotinib in human HNSCC lines

Epidermal Growth Factor Receptor (EGFR) overexpression occurs in about 90% of Head and Neck Squamous Cell Carcinoma (HNSCC) cases. Aberrant EGFR signaling has been implicated in the malignant features of HNSCC. Thus, EGFR appears to be a logical therapeutic target with increased tumor specificity for the treatment of HNSCC. Erlotinib, a small molecule tyrosine kinase inhibitor, specifically inhibits aberrant EGFR signaling in HNSCC. Only a minority of HNSCC patients were able to derive a substantial clinical benefit from erlotinib. ^ This dissertation identifies Epithelial to Mesenchymal Transition (EMT) as the biological marker that distinguishes EGFR-dependent (erlotinib-sensitive) tumors from the EGFR-independent (erlotinib-resistant) tumors. This will allow us to prospectively identify the patients who are most likely to benefit from EGFR-directed therapy. More importantly, our data identifies the transcriptional repressor DeltaEF1 as the mesenchymal marker that controls EMT phenotype and resistance to erlotinib in human HNSCC lines. si-RNA mediated knockdown of DeltaEF1 in the erlotinib-resistant lines resulted in reversal of the mesenchymal phenotype to an epithelial phenotype and significant increase in sensitivity to erlotinib. ^ DeltaEF1 represses the expression of the epithelial markers by recruiting HDACs to chromatin. This observation allows us to translate our findings into clinical application. To test whether the transcriptional repression by DeltaEF1 underlines the mechanism responsible for erlotinib resistance, erlotinib-resistant lines were treated with an HDAC inhibitor (SAHA) followed by erlotinib. This resulted in a synergistic effect and substantial increase in sensitivity to erlotinib in the resistant cell lines. Thus, combining an HDAC inhibitor with erlotinib represents a novel promising pharmacologic strategy for reversing resistance to erlotinib in HNSCC patients. ^

Role of activation of the protein tyrosine kinase, Src, in colorectal cancer chemoresistance

Advances in therapy for colorectal cancer have been hampered by development of resistance to chemotherapy. The Src family of protein tyrosine kinases has been associated with colorectal cancer development and progression. Activation of the prototypic member of the family, Src, occurs in advanced colorectal cancer and is associated with a worse outcome. This work tests the hypotheses that Src activation contributes to chemoresistance in some colon tumors and that this resistance can be overcome by use of Src inhibitors. The aims of the proposal were to (1) determine if constitutive Src activation is sufficient to induce oxaliplatin resistance; (2) evaluate the role of reactive oxygen species (ROS) in the activation of Src after oxaliplatin treatment; (3) determine the frequency of Src activation in liver metastases after oxaliplatin treatment; and (4) evaluate the safety, preliminary efficacy, and pharmacodynamics of the combination of dasatinib with oxaliplatin-based therapy in patients with metastatic colorectal cancer. ^ Using a panel of colon cancer cell lines and murine models, I demonstrate that administration of oxaliplatin, a commonly utilized chemotherapy for colorectal cancer, results in an increased activation of Src. The activation occurs acutely in some, but not all, colorectal carcinoma cell lines. Cell lines selected for oxaliplatin resistance are further increased in Src activity. Treatment of cell lines with dasatinib, a non-selective pharmacologic inhibitor of the Src family kinases synergistically killed some, but not all cell lines. Cell lines with the highest acute activation of Src after oxaliplatin administration were the most sensitive to the combination therapy. Previous work demonstrated that siRNA to Src increased sensitivity to oxaliplatin, suggesting that the effects of dasatinib are primarily due to its ability to inhibit Src in these cell lines. ^ To examine the mechanism underlying these results, I examined the effects of reactive oxygen species (ROS), as previous studies have demonstrated that platinum chemotherapeutics result in intracellular oxidative stress. I demonstrated that oxaliplatin-induced reactive oxygen species were higher in the cell lines with Src activation, relative to those in which Src was not activated. This oxaliplatin-induced Src activation was blocked by the administration of anti-oxidants, thereby demonstrating that synergistic killing between dasatinib and oxaliplatin was associated with the ability of the latter to generate ROS. ^ In a murine model of colorectal cancer metastasis to the liver, the combination of dasatinib and oxaliplatin was more effective in reducing tumor volume than either agent alone. However, when oxaliplatin resistant cell lines were treated with a combination of oxaliplatin and AZD0530, an inhibitor in the clinic with increased specificity for Src, no additional benefit was seen, although Src was activated by oxaliplatin and Src substrates were inhibited. The indolent growth of oxaliplatin-resistant cells, unlike the growth of oxaliplatin resistant tumors in patients, precludes definitive interpretation of these results. ^ To further explore Src activation in patients with oxaliplatin exposure and resistance, an immunohistochemistry analysis of tumor tissue from resected liver metastases of colorectal cancer was performed. Utilizing a tissue microarray, staining for phosphorylated Src and FAK demonstrated strong staining of tumor relative to stromal and normal liver. In patients recently exposed to oxaliplatin, there was increased FAK activation, supporting the clinical relevance of the prior preclinical studies. ^ To pursue the potential clinical benefit of the combination of Src inhibition with oxaliplatin, a phase IB clinical trial was completed. Thirty patients with refractory metastatic colorectal cancer were treated with a combination of 5-FU, oxaliplatin, an epidermal-growth factor receptor monoclonal antibody, and dasatinib. The recommended phase II dose of dasatinib was established, and toxicities were quantified. Pharmacodynamic studies demonstrated increased phosphorylation of the Src substrate paxillin after dasatinib therapy. Tumor biopsies were obtained and Src expression levels were quantitated. Clinical benefit was seen with the combination, including a response rate of 20% and disease control rate of 56%, prompting a larger clinical study. ^ In summary, although Src is constitutively activated in metastatic colorectal cancer, administration of oxaliplatin chemotherapy can further increase its activity, through a reactive oxygen species dependent manner. Inhibition of Src in combination with oxaliplatin provides additional benefit in vitro, in preclinical animal models, and in the clinic. Further study of Src inhibition in the clinic and identification of predictive biomarkers of response will be required to further advance this promising therapeutic target. ^

INTERACTION BETWEEN BRK AND HER2 IN BREAST CANCER

INTERACTION BETWEEN BRK AND HER2 IN BREAST CANCER Midan Ai, Ph.D. Supervisory Professor: Zhen Fan, M.D. Breast tumor kinase (Brk) is a nonreceptor protein-tyrosine kinase that is highly expressed in approximately two thirds of breast cancers but is not detectable or is expressed at very low levels in normal mammary epithelium. Brk plays important roles in promoting proliferation, survival, invasion, and metastasis of breast cancer cells, but the mechanism(s) of which remain largely unknown. Recent studies showed that Brk is frequently co-overexpressed with human epidermal growth factor receptor-2 (HER2) and is physically associated with HER2 in breast cancer. The mechanism needs to be determined. In my studies, I found that high expression of HER2 is correlated with high expression of Brk in breast cancer cell lines. Silencing HER2 expression via RNA interference in HER2 over-expressed breast cancer cells resulted in Brk protein decrease and overexpression of HER2 in HER2 low-expressed breast cancer cells up-regulated Brk expression. The mechanism study indicated that overexpression of HER2 increased Brk protein stability. Brk was degraded through a Ca2+-dependent protease pathway involving calpain and HER2 stimulated Brk expression via inhibiting calpain activity. Calpastatin is a calpain endogenous inhibitor and the calpain-calpastatin system has been implicated in a number of cell physiological functions. HER2 restrained calpain activation via up-regulating calpastatin expression and HER2 downstream signaling, MAPK pathway, was involved in the regulation. Furthermore, silencing of Brk expression by RNA interference in HER2-overexpressing breast cancer cells decreased HER2-mediated cell proliferation, survival, invasion/metastasis potential and increased cell sensitivity to HER2 kinase inhibitor, lapatinib, treatment, indicating that Brk plays important roles in regulating and mediating the oncogenic functions of HER2. The Stat3 pathway played important roles in Brk mediated cell survival and invasion/metastasis in the context of HER2-overexpressing breast cancer cells. However, transgenic mice with inducible expression of constitutively active Brk (CA) in the mammary epithelium failed to develop malignant change in the mammary glands after Brk induction for 15 months which indicated that expression of Brk protein alone was not sufficiently to induce spontaneous breast tumor. Bitransgenic mice with co-expression of HER2/neu and inducible expression of Brk in the mammary epithelium developed multifocal mammary tumors, but there were no significant difference in the tumor occurring time, tumor size, tumor weight and tumor multiplicity between the mouse group with co-expression of Brk and HER2/neu and the mouse group with HER2/neu expression only.

NOVEL THERAPEUTIC TARGETS IDENTIFIED BY HIGH-THROUGHPUT TECHNOLOGIES FOR TRIPLE-NEGATIVE BREAST CANCER

Triple-negative breast cancers (TNBC) are characterized by the lack of or reduced expression of the estrogen and progesterone receptors, and normal expression of the human epidermal growth factor receptor 2. The lack of a well-characterized target for treatment leaves only systemic chemotherapy as the mainstay of treatment. Approximately 60-70% of patients are chemosensitive, while the remaining majority does not respond. Targeted therapies that take advantage of the unique molecular perturbations found in triple-negative breast cancer are needed. The genes that are frequently amplified or overexpressed represent potential therapeutic targets for triple-negative breast cancer. The purpose of this study was to identify and validate novel therapeutic targets for triple-negative breast cancers. 681 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in 2 data sets. For two genes, 3 of the 4 siRNAs showed preferential growth inhibition in TNBC cells. These two genes were the low density lipoprotein receptor-related protein 8 (LRP8) and very low-density lipoprotein receptor (VLDLR). Exposure to their cognate ligands, reelin and apolipoprotein E isoform 4 (ApoE4), stimulated the growth of TNBC cells in vitro. Suppression of the expression of either LRP8 or VLDLR or exposure to RAP (an inhibitor of ligand binding to LRP8 and VLDLR) abolished this ligand-induced proliferation. High-throughput protein and metabolic arrays revealed that ApoE4 stimulation rescued TNBC cells from serum-starvation induced up-regulation of genes involved in lipid biosynthesis, increased protein expression of oncogenes involved in the MAPK/ERK and DNA repair pathways, and reduced the serum-starvation induction of biochemicals involved in oxidative stress response and glycolytic metabolism. shLRP8 MDA-MB-231 xenografts had reduced tumor volume, in comparison to parental and shCON xenografts. These results indicate that LRP8-APOE signaling confers survival advantages to TNBC tumors under reduced nutrient conditions and during cellular environmental stress. We revealed that the LRP8-APOE receptor-ligand system is overexpressed in human TNBC. We also demonstrated that this receptor system mediates a strong growth promoting and survival function in TNBC cells in vitro and helps to sustain the growth of MDA-MD-231 xenografts. We propose that inhibitors of LRP8-APOE signaling may be clinically useful therapeutic agents for triple-negative breast cancer.

Characterization of Jak, STAT, and Src interactions in Head and Neck Squamous Cell Carcinoma

Recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is common; thus, it is essential to improve the effectiveness and reduce toxicity of current treatments. Proteins in the Src/Jak/STAT pathway represent potential therapeutic targets, as this pathway is hyperactive in HNSCC and it has roles in cell migration, metastasis, proliferation, survival, and angiogenesis. During short-term Src inhibition, Janus kinase (Jak) 2, and signal transducer and activator of transcription (STAT) 3 and STAT5 are dephosphorylated and inactivated. Following sustained Src inhibition, STAT5 remains inactive, but Jak2 and STAT3 are reactivated following their early inhibition. To further characterize the mechanism of this novel feedback pathway we performed several experiments to look at the interactions between Src, Jak2, STAT5 and STAT3. We attempted to develop a non-radioactive kinase assay using purified recombinant Jak2 and Src proteins, but found that phospho-tyrosine antibodies were non-specifically binding to purified recombinant proteins. We then performed in vitro kinase assays (IVKAs) using purified recombinant Jak2, Src, STAT3, and STAT5 proteins with and without Src and Jak2 pharmacologic inhibitors. We also examined the interactions of these proteins in intact HNSCC cells. We found that recombinant Jak2, STAT3, and STAT5 are direct substrates of Src and that recombinant Src, STAT3, and STAT5 are direct substrates of Jak2 in the IVKA. To our knowledge, the finding that Src is a Jak substrate is novel and has not been shown before. In intact HNSCC cells we find that STAT3 can be reactivated despite continuous Src inhibition and that STAT5 continues to be inhibited despite Jak2 reactivation. Also, Jak2 inhibition did not affect Src or STAT5 activity but it did cause STAT3 inhibition. We hypothesized that the differences between the intact cells and the IVKA assays were due to a potential need for binding partners in intact HNSCC cells. One potential binding partner that we examined is the epidermal growth factor receptor (EGFR). We found that EGFR activation caused increased activation of Src and STAT5 but not Jak2. Our results demonstrate that although STAT3 and STAT5 are capable of being Src and Jak2 substrates, in intact HNSCC cells Src predominantly regulates STAT5 and Jak2 regulates STAT3. Regulation of STAT5 by Src may involve interactions between Src and EGFR. This knowledge along with future studies will better define the mechanisms of STAT regulation in HNSCC cells and ultimately result in an ideal combination of therapeutic agents for HNSCC.

Transcriptional regulation of the {\it neu\/} oncogene and its negative regulation by the c-{\it myc\/} oncogene

The first part of my research involved the characterization of the neu gene promoter. I subcloned a 2.2-kb sequence located upstream to the extreme 5$\sp\prime$ end of the neu gene, in front of the bacterial reporter gene, chloramphenicol acetyltransferase (CAT). Transfection of this construct into different cell lines and subsequent CAT assays demonstrated that this 2.2-kb fragment was functional as a promoter. A series of deletion constructs was engineered to study the contribution of different fragments to transcription. Subcloning of individual fragments was followed by a cotransfection competition experiment, which demonstrated the involvement of protein factors interacting with the promoter. A gel retardation assay was also performed to show the physical binding of protein factors to the promoter. The combined results suggested that both positively and negatively acting protein factors are involved in interacting with different regions of the promoter, contributing to the overall transcription activity. My findings provide an insight into the regulation of neu gene expression, which in turn provides the tools to understand the molecular mechanisms of overexpression of the neu gene in some breast cancer and ovarian cancer cell lines.^ In the second part of my research, I discovered that another oncogene, c-myc, was able to reverse the transformed morphology that was induced by the neu oncogene. Utilizing the promoter constructs that I made, I was able to show that the c-myc oncogene has a negative regulatory effect on the expression of the neu oncogene. Further studies suggested that c-myc is able to lower the effective concentration of a positive factor(s) that interact with a 139-bp fragment of the neu gene promoter. These findings may provide a direct evidence of the long suspected role of the c-myc gene in transcriptional regulation. The neu gene may very well be the first identified mammalian target gene that is regulated by the c-myc oncogene. Since c-myc is known to be stimulated by various mitogenic signals and the neu gene is likely to be a growth factor receptor, it is possible that c-myc, when stimulated by the signal transduction pathway of the neu gene, would function as a negative feedback regulator on the neu gene receptor. (Abstract shortened with permission of author.) ^

The transcriptional regulation of the {\it neu\/} gene: Examples of activation, repression, and cell-specific expression

The neu oncogene encodes a growth factor receptor-like protein, p185, with an intrinsic tyrosine kinase activity. A single point mutation, an A to T transversion resulting in an amino acid substitution from valine to glutamic acid, in the transmembrane domain of the rat neu gene was found to be responsible for the transforming and tumorigenic phenotype of the cells that carry it. In contrast, the human proto-neu oncogene is frequently amplified in tumors and cell lines derived from tumors and the human neu gene overexpression/amplification in breast and ovarian cancers is known to correlate with poor patient prognosis. Examples of the human neu gene overexpression in the absence of gene amplification have been observed, which may suggest the significant role of the transcriptional and/or post-transcriptional control of the neu gene in the oncogenic process. However, little is known about the transcriptional mechanisms which regulate the neu gene expression. In this study, three examples are presented to demonstrate the positive and negative control of the neu gene expression.^ First, by using band shift assays and methylation interference analyses, I have identified a specific protein-binding sequence, AAGATAAAACC ($-$466 to $-$456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.^ Second, it has been reported that the E3 region of adenovirus 5 induces down-regulation of epidermal growth factor (EGF) receptor through endocytosis. I found that the human neu gene product, p185, (an EGF receptor-related protein) is also down-regulated by adenovirus 5, but via a different mechanism. I demonstrate that the adenovirus E1a gene is responsible for the repression of the human neu gene at the transcriptional level.^ Third, a differential expression of the neu gene has been found in two cell model systems: between the mouse fibroblast Swiss-Webster 3T3 (SW3T3) and its variant NR-6 cells; and between the mouse liver tumor cell line, Hep1-a, and the mouse pancreas tumor cell line, 266-6. Both NR-6 and 266-6 cell lines are not able to express the neu gene product, p185. I demonstrate that, in both cases, the transcriptional repression of the neu gene may account for the lack of the p185 expression in these two cell lines. ^

Autoregulation of the {\it neu\/} oncogene

The neu gene encodes a 185,000-Da membrane glycoprotein that is highly homologous to epidermal growth factor receptor. It is frequently overexpressed or amplified in human breast carcinomas and ovarian cancers, which correlates with a poor prognosis for patients. The importance of neu gene regulation is noted by the fact that many breast cancer cells overexpress the neu gene without proportional gene amplification. The mechanism for that is unclear. My initial finding of neu autoregulation led to a realization that defects in neu autoregulation pathway may contribute to neu overexpression in tumor cells. I have found in the nontransformed NIH 3T3 model system that (i) the neu gene product autorepresses its own promoter activity, (ii) the neu gene promoter contains a novel enhancer, (iii) neu autorepression is mediated through this enhancer by inhibition of the enhancer activity, and (iv) c-myc expression serves as an intermediate step downstream from the membrane bound neu-encoded receptor in this complicated feedback inhibition pathway.^ In addition, a part of my research is studying the neu-encoded receptor molecule. I have generated a construct coding the neu ligand-binding domain and demonstrated that (i) the neu ligand-binding domain is a secretory peptide, (ii) it inhibits the normal neu-associated tyrosine kinase but not activated neu-associated tyrosine kinase. My study provided experimental evidence for the mechanisms of neu gene activation. ^

Isolation and characterization of revertant cell lines expressing oncogenic rat {\it neu\/}

The neu gene encodes the transmembrane tyrosine kinase growth factor receptor, p185. To study neu induced cellular transformation, we developed revertant cells from the neu transformed NIH 3T3 cell line, B104-1-1, by treating the cells with the chemical mutagen ethylmethane sulfonate. The morphologically normal revertant cells were first selected by their ability to either attach to culture plates or survive in the presence of the cytotoxic reagents colchicine or 5-fluoro-2deoxyuridine. Two of the 21 candidate revertant cell lines isolated were further characterized and were found to lose their anchorage independence and ability to grow in 1% calf serum, indicating that they were nontransformed even though they still expressed p185 oncoprotein. The tyrosine residues of p185 in these two revertants were underphosphorylated, which may have contributed to their nontransformed status. Also, the p185 oncoprotein lacked significant tyrosine kinase activity. In addition, these revertants also resisted transformation by neu and several additional oncogenes (H-ras, N-ras, v-mos, v-abl, and v-fos) as determined by focus forming assays. These results indicated that we had successfully developed, from neu transformed cells, revertants which exhibited defective tyrosine phosphorylation and kinase activity of the neu oncoprotein. The results also suggested that neu and several other oncogenes may share common elements in their pathways for the induction of cellular transformation. ^

Biological effects of PEA3 expression in {\it HER-2\/}/{\it neu\/}-overexpressing human cancer cells and the molecular mechanisms of PEA3-mediated transcriptional repression on {\it HER-2\/}/{\it neu\/}

HER-2/neu is a receptor tyrosine kinase highly homologous with epidermal growth factor receptor. Overexpression and/or amplification of HER-2/neu has been implicated in the genesis of a number of human cancers, especially breast and ovarian cancers. Transcriptional upregulation has been shown to contribute significantly to the overexpression of this gene. Studies on the transcriptional regulation of HER-2/neu gene are important for understanding the mechanism of cell transformation and developing the therapeutic strategies to block HER-2/neu-mediated cancers. PEA3 is a DNA binding transcriptional factor and its consensus sequence exists on the HER-2/neu promoter. To examine the role of PEA3 in HER-2/neu expression and cell transformation, we transfected PEA3 into the human breast and ovarian cancer cells that overexpress HER-2/neu and showed that PEA3 dramatically represses HER-2/neu transcription. PEA3 suppresses the oncogenic neu-mediated transformation in mouse fibroblast NIH 3T3 cells. Expression of PEA3 selectively blocks the growth of human cancer cells that overexpress HER-2/neu and inhibits their colony formation. It does not occur in the cancer cells expressing basal level of HER-2/neu. Further studies in the orthotopic ovarian cancer model demonstrated that expression of PEA3 preferentially inhibits growth and tumor development of human cancer cells that overexpress HER-2/neu, the tumor-bearing mice survived significantly longer if treated by injection of the PEA3-liposome complex intraperitoneally. Immunoblotting and immunohistochemical analysis of the tumor tissues indicated that PEA3 mediates the tumor suppression activity through targeting HER-2/neu-p185. Thus, PEA3 is a negative regulator of HER-2/neu gene expression and functions as a tumor suppressor gene in the HER-2/neu-overexpressing human cancer cells.^ The molecular mechanisms of PEA3 mediated transcriptional repression were investigated. PEA3 binds specifically at the PEA3 site on HER-2/neu promoter and this promoter-binding is required for the PEA3 mediated transcriptional repression. Mutation of the PEA3 binding site on HER-2/neu promoter causes decreased transcriptional activity, indicating that the PEA3 binding site is an enhancer-like element in the HER-2/neu-overexpressing cells. We therefore hypothesized that in the HER-2/neu-overexpressing cells, PEA3 competes with a transactivator for binding to the PEA3 site, preventing the putative factor from activating the transcription of HER-2/neu. This hypothesis was supported by the data which demonstrate that PEA3 competes with another nuclear protein for binding to the HER-2/neu promoter in vitro, and expression of a truncated protein which encodes the DNA binding domain of PEA3 is sufficient to repress HER-2/neu transcription in the HER-2/neu-overexpressing human cancer cells. ^