BIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF MURINE-TUMOR SPECIFIC TRANSPLANTATION ANTIGENS (ISOTACHOPHORESIS, IMMUNOGENICITY)

Tumor-specific transplantation antigens (TSTA) are individually distinct neoantigens expressed on the cells of chemically-induced neoplasms. TSTA are operationally defined by immunization of syngeneic mice against challenge with viable tumor cells. Immunization with cell surface or extracted TSTA induces specific resistance to transplanted tumor cells. The biological and biochemical nature of TSTA was investigated in the 3-methylcholanthrene-induced fibrosarcomas of female C3H/HeJ mice, MCA-F and MCA-D. Tumor cell suspensions were extracted by treatment with 3M KCl or 2.5% butanol solutions and the TSTA was partially purified by preparative isoelectric focusing. The isoelectric pH of TSTA purified from 3M KCl extracts was 5.8-6.0, and from butanol extracts was 6.4-6.6. Whereas immunization with 10('5) and 10('6) irradiated tumor cells induces complete rejection of tumor cell challenge over a two-fold-log dose range, immunization with ug quantities within a one-fold-log dose range of extracted TSTA induces only partial resistance to tumor challenge. Reduced immunogenicity of extracted TSTA is hypothesized to result from immunization of mice with insufficiently purified TSTA preparations. The hypothesis predicts that immunization with highly purified TSTA, free from interfering substances, induces complete rejection of tumor challenge over a broad dose range. To test the hypothesis preparative isotachophoresis (pITP) was used to purify TSTA from electrofocused TSTA fractions. Significant purification was achieved, as immunization with 15 pg to 1.5 ug (5 logs) of pITP-purified TSTA extracted from the MCA-F, or with 1 pg to 10 ng (4 logs) of TSTA from the MCA-D tumor induced specific resistance to tumor challenge. Despite 50,000 fold purification of TSTA, immunization induced partial, not complete, rejection of transplanted tumor cells. This suggests a clear dissociation of the immunogenicity and purification of extracted TSTA, indicating that the induction of partial immunity to tumor challenge is an intrinsic property of extracted TSTA.^

Tumor immunization strategies for enhancing the graft versus tumor activity of allogeneic bone marrow transplantation

Allogeneic bone marrow transplantation (BMT) is known to induce a beneficial anti-tumor immune response called graft-versus-tumor (GVT) activity. However, GVT activity is closely associated with graft-versus-host disease (GVHD), a potentially fatal immune response against antigens on normal recipient tissues. The T-cell populations mediating these two processes are often overlapping, but studies have shown that some donor T-cells can be tumor-specific. Therefore, the goal of this study was to develop strategies for preferentially activating donor T-cells capable of mediating GVT activity but not GVHD. The three hypotheses tested were: (1) Pre-transplant immunization of BMT donors with a recipient-derived tumor cell vaccine will induce a relative increase in GVT activity as compared to GVHD. (2) Post-transplant tumor immunization of BMT recipients will enhance GVT activity without exacerbating GVHD. (3) Pre-transplant immunization of BMT donors against a tumor-specific antigen will enhance GVT activity without exacerbating GVHD. ^ To test the first two hypotheses, C3H.SW mice (MHC-matched donors) were immunized with a C57BL/6 (recipient)-derived tumor cell vaccine (leukemia or fibrosarcoma) prior to BMT, or recipients were immunized starting one month after BMT. Both donor and recipient immunization led to a significant increase in GVT activity (enhanced recipient survival and decreased tumor growth). However, donor immunization also increased fatal GVHD, which was at least partially due to activation of alloreactive T-cells recognizing the immunodominant minor histocompatibility antigen B6dom1. GVT immunity following recipient immunization was not associated with an exacerbation of GVHD or a response to B6dom1. ^ To test the third hypothesis, influenza nucleoprotein (NP) was used as a model tumor antigen. C3H.SW donors were immunized against NP prior to BMT, which led to a significant increase in GVT activity. Although recipients were not completely protected against growth of antigen loss variant tumors, there was no increase in GVHD. ^ In conclusion, (1) immunization of allogeneic BMT donors with a recipient-derived tumor cell vaccine substantially increases GVT activity but also exacerbates GVHD, (2) post-transplant tumor immunization of allogeneic BMT recipients significantly increases GVT activity and survival without exacerbating GVHD, and (3) immunization of allogeneic BMT donors against a tumor-specific antigen significantly enhances GVT activity without exacerbating GVHD. ^

The localization and identification of candidate tumor suppressor genes involved in prostate cancer

Frequent loss of heterozygosity (LOH) at specific chromosomal regions are highly associated with the inactivation of tumor suppressor genes (TSGs) (Weinberg, 1991; Bishop, 1989). Chromosome 8p is the most frequently reported site of LOH (∼60%) in prostate cancer (PC), suggesting that there may be inactivated TSG(s) involved in PC on chromosome 8p. (Bergerheim et. al., 1991; Kagan et. al., 1995). In order to identify the smallest common regions of frequent LOH (SCLs) on chromosome 8, we screened 52 PC patient/tumor samples with 39 polymorphic markers in successive screenings. In the course of refining the SCLs, we identified 3 tumors with >6 Mb homozygous deletions (HZDs) at 8p22 and 8p21, suggesting the presence of candidate TSGs at both loci. These HZDs spanned the two SCLs at 8p22 (46%) and 8p21 (45%). The SCLs were narrowed to 3.2 cM at 8p22 and less than 3 cM at 8p21. ^ In order to identify candidate TSGs within the SCLs on 8p, two approaches were used. In the candidate gene approach, thirty genes that mapped to the SCLs were evaluated for expression in normal prostate and in PC cell lines. One of the candidate genes, Clusterin, showed decreased expression in 4/7 (57%) prostate cancer cell lines by Northern blot analysis. Clusterin will be further examined as a candidate TSG. ^ The second approach involved utilizing subtractive hybridization and hybrid affinity capture to generate pools of expressed sequence tags (ESTs) enriched for genes that are downregulated or deleted in PC and that map to specific regions of interest. We took advantage of a prostate cancer cell line (PC3) with a known HZD of a candidate TSG, CTNNA1 on 5q31, to develop and validate a model system. We then developed subtracted libraries enriched for 8p22 and 8p21 ESTs by this method, using two cell lines, MDAPCa-2b and PC3. The ESTs were cloned, and 40 were sequenced and evaluated for expression in normal prostate and PC cell lines. Three ESTs from the subtracted libraries, C2, C17 and F12, showed decreased expression in 29–57% of the prostate tumor cell lines studied, and will be further examined as candidate TSGs. ^

Signal transduction of CEACAM1-L tumor suppressor

CEACAM1-L is an adhesion molecule that suppress the growth of prostate, breast, colon and endometrial tumors. In this study we defined the domain involved in CEACAM1-L tumor suppression activity. DU145 prostate cancer cells were infected with recombinant adenoviruses containing various CEACAM1-L mutant genes, and the effects of the mutant proteins on the growth of DU145 cells were assessed in a nude-mice xenograft model. We found that expression of the CEACAM1-L cytoplasm domain alone led to growth suppression of DU145 cells. These results suggest that the cytoplasmic domain of CEACAM1-L is necessary and sufficient for its growth-suppressive function. ^ The cytoplasmic domain of CEACAM1-L is presumed to be involved in a signaling pathway resulting in the suppression of tumor cell growth. It was not clear whether post-translational modification of CEACAM1-L is required for tumor suppressor function, therefore the importance of phosphorylation in growth-inhibitory signaling pathway was investigated. Full-length CEACAM1-L was found to be phosphorylated in vivo in both tyrosine and serine residues. Mutation of tyrosine 488 to phenylalanine did not abolish the tumor-suppressive activity of CEACAM1-L while mutation of serine 503 to alanine abolished the growth-inhibitory activity. In addition, mutation of serine 503 to aspartic acid produced tumor-suppressive activity similar to that of the wild-type CEACAM1-L. These results suggested that only phosphorylation at serine 503 is essential for CEACAM1-L's growth-inhibitory function in vivo. ^ Phosphorylation of CEACAM1-L may lead to its interaction with molecules in CEACAM1-L's signaling pathway. In the last part of this study we demonstrate that CEACAM1 is able to interact with the adapter protein p66Shc. p66Shc was found to be co-immunoprecipitated with full length CEACAM1-L but not with CEACAM1-L lacking its cytoplasmic tail. Additionally this interaction occurred in the absence of the tyrosine phosphorylation of CEACAM1-L. These results suggest that p66Shc is able to interact with the cytoplasmic domain of CEACAM1-L and this interaction does not require tyrosine phosphorylation. ^ In conclusion, this study suggests that CEACAM1-L signals tumor suppression through its cytoplasmic domain by initially becoming phosphorylated on serine 503. Additionally, the interaction with p66Shc may be involved in CEACAM1-L's signaling pathway. ^

Use of cardiac glycosides for treatment of cancer: Determinants of cancer cell sensitivity

Cardiac glycoside compounds have traditionally been used to treat congestive heart failure. Recently, reports have suggested that cardiac glycosides may also be useful for treatment of malignant disease. Our research with oleandrin, a cardiac glycoside component of Nerium oleander, has shown it to be a potent inducer of human but not murine tumor cell apoptosis. Determinants of tumor sensitivity to cardiac glycosides were therefore studied in order to understand the species selective cytotoxic effects as well as explore differential sensitivity amongst a variety of human tumor cell lines. ^ An initial model system involved a comparison of human (BRO) to murine (B16) melanoma cells. Human BRO cells were found to express both the sensitive α3 as well as the less sensitive α1 isoform subunits of Na+,K +-ATPase while mouse B16 cells expressed only the α1 isoform. Drug uptake and inhibition of Na+,K+-ATPase activity were also different between BRO and B16 cells. Partially purified human Na+,K+-ATPase enzyme was inhibited by cardiac glycosides at a concentration that was 1000-fold less than that required to inhibit mouse B16 enzyme to the same extent. In addition, uptake of oleandrin and ouabain was 3–4 fold greater in human than murine cells. These data indicate that differential expression of Na+,K+-ATPase isoform composition in BRO and B16 cells as well as drug uptake and total enzyme activity may all be important determinants of tumor cell sensitivity to cardiac glycosides. ^ In a second model system, two in vitro cell culture model systems were investigated. The first consisted of HFU251 (low expression of Na+,K+-ATPase) and U251 (high Na+ ,K+-ATPase expression) cell lines. Also investigated were human BRO cells that had undergone stable transfection with the α1 subunit resulting in an increase in total Na+,K+-ATPase expression. Data derived from these model systems have indicated that increased expression of Na+,K+-ATPase is associated with an increased resistance to cardiac glycosides. Over-expression of Na +,K+-ATPase in tumor cells resulted in an increase of total Na+,K+-ATPase activity and, in turn, a decreased inhibition of Na+,K+-ATPase activity by cardiac glycosides. However, of interest was the observation that increased enzyme expression was also associated with an elevated basal level of glutathione (GSH) within cells. Both increased Na+,K+-ATPase activity and elevated GSH content appear to contribute to a delayed as well as diminished release of cytochrome c and caspase activation. In addition, we have noted an increased colony forming ability in cells with a high level of Na+,K+-ATPase expression. This suggests that Na+,K+-ATPase is actively involved in tumor cell growth and survival. ^

Towards the identification of a tumor suppressor gene on chromosome 3p12: Physical mapping and candidate gene screening

Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney. Characterization of RCC tumors indicates that the most frequent genetic event associated with the initiation of tumor formation involves a loss of heterozygosity or cytogenetic aberration on the short arm of human chromosome 3. A tumor suppressor locus Nonpapillary Renal Carcinoma-1 (NRC-1, OMIM ID 604442) has been previously mapped to a 5–7 cM region on chromosome 3p12 and shown to induce rapid tumor cell death in vivo, as demonstrated by functional complementation experiments. ^ To identify the gene that accounts for the tumor suppressor activities of NRC-1, fine-scale physical mapping was conducted with a novel real-time quantitative PCR based method developed in this study. As a result, NRC-1 was mapped within a 4.6-Mb region defined by two unique sequences within UniGene clusters Hs.41407 and Hs.371835 (78,545Kb–83,172Kb in the NCBI build 31 physical map). The involvement of a putative tumor suppressor gene Robo1/Dutt1 was excluded as a candidate for NRC-1. Furthermore, a transcript map containing eleven candidate genes was established for the 4.6-Mb region. Analyses of gene expression patterns with real-time quantitative RT-PCR assays showed that one of the eleven candidate genes in the interval (TSGc28) is down-regulated in 15 out of 20 tumor samples compared with matched normal samples. Three exons of this gene have been identified by RACE experiments, although additional exon(s) seem to exist. Further gene characterization and functional studies are required to confirm the gene as a true tumor suppressor gene. ^ To study the cellular functions of NRC-1, gene expression profiles of three tumor suppressive microcell hybrids, each containing a functional copy of NRC-1, were compared with those of the corresponding parental tumor cell lines using 16K oligonucleotide microarrays. Differentially expressed genes were identified. Analyses based on the Gene Ontology showed that introduction of NRC-1 into tumor cell lines activates genes in multiple cellular pathways, including cell cycle, signal transduction, cytokines and stress response. NRC-1 is likely to induce cell growth arrest indirectly through WEE1. ^

A mechanism of insulin-like growth factor binding protein 2-induced cell mobility in glioma

Insulin-like growth factor binding protein 2 (IGFBP2) is a protein known to be overexpressed in a majority of glioblastoma multiforme (GBM) tumors. While it is known the IGFBP2 is involved in promoting GBM tumor cell invasion, no mechanism exists for how the protein is involved in signal transduction pathways leading to enhanced cell invasion. ^ We follow up on preliminary microarray data on IGFBP2-overexpressing GBM cells and protein sequence analysis of IGFBP2 in generating the hypothesis that IGFBP2 interacts with integnn α5 in regulating cell mobility. Microarray data showing upregulation of integrin α5 by IGFBP2 is validated and evidence of protein-protein interaction between IGFBP2 and integrin α5 is found. The exact binding domain on IGFBP2 responsible for its interaction with integrin α5 is also determined, confirming our initial findings and reaffirming that the IGFBP2/integrin α5 interaction is specific. Disruption of this interaction resulted in attenuation of IGFBP2-enhanced cell mobility. Further, we found that cell mobility is only enhanced when IGFBP2 and integrin α5 are both overexpressed and able to interact with each other. ^ We also determined fibronectin to be a critical player in the activation of the IGFBP2/integrin α5 pathway. The activation of this pathway appears to be progressive and initiates once GBM cells have sufficiently established anchorage. ^

The low molecular weight (LMW) isoforms of cyclin E provide a novel mechanism of cell cycle deregulation

Cyclin E, in complex with cyclin dependent kinase 2 (CDK2), is a positive regulator of G1 to S phase progression of the cell cycle. Deregulation of G1/S phase transition occurs in the majority of tumors. Cyclin E is overexpressed and post-translationally generates low molecular weight (LMW) isoforms in breast cancer, but not normal cells. Such alteration of cyclin E is linked to poor prognosis. Therefore, we hypothesized that the LMW isoforms of cyclin E provide a novel mechanism of cell cycle de-regulation in cancer cells. Insect cell expression system was used to explore the biochemical properties of the cyclin E isoforms. Non-tumorigenic (76NE6) and tumorigenic (T47D) mammary epithelial cells transfected with the cyclin E isoforms and breast tumor tissue endogenously expressing the LMW isoforms were used to study the biologic consequences of the LMW isoforms of cyclin E. All model systems studied show that the LMW forms (compared to full-length cyclin E) have increased kinase activity when partnered with CDK2. Increases in the percentage of cells in S phase and colony formation were also observed after overexpression of LMW compared to full-length cyclin E. The LMW isoforms of cyclin E utilize several mechanisms to attain their hyper-activity. They bind CDK2 more efficiently, and are resistant to inhibition by cyclin dependent kinase inhibitors (CKIs) as compared to full-length cyclin E. In addition, the LMW isoforms sequester the CKIs from full-length cyclin E abrogating the overall negative regulation of cyclin E. Despite their correlation with adverse biological consequences, the direct role of the LMW isoforms of cyclin E in mediating tumorigenesis remained unanswered. Subsequent to LMW cyclin E expression in 76NE6 cells, they lose their ability to enter quiescence and exhibit genomic instability, both characteristic of a tumor cell phenotype. Furthermore, injection of 76NE6 cells overexpressing each of the cyclin E isoforms into the mammary fat pad of nude mice revealed that the LMW isoforms of cyclin E yield tumors, whereas the full-length cyclin E does not. In conclusion, the LMW isoforms of cyclin E utilize several mechanisms to acquire a hyperactive phenotype that results in deregulation of the cell cycle and initiates the tumorigenic process in otherwise non-transformed mammary epithelial cells. ^

EphA2 expression is regulated by EGFR and KRAS and promotes non-small cell lung cancer progression

Lung cancer is the leading cause of cancer deaths worldwide. The development of improved systemic therapy is needed for the most common form of the disease, non-small cell lung cancer (NSCLC). This will depend on the identification of valid molecular targets. Recent studies point to the receptor tyrosine kinase EphA2 as a novel therapeutic target. Overexpression of EphA2 has been demonstrated in a number of epithelial cancers, and its expression has been associated with more severe disease. Regulation of EphA2 in cancer is poorly understood. Recently, regulation of EphA2 by EGFR and KRAS has been reported in a number of in vitro models, but no examination of this relationship has been undertaken in patient tumors. Because of the established importance of EGFR and KRAS in NSCLC, we have investigated the relationship between these mutations and EphA2 in NSCLC patient tissues and cell lines. The significance of Epha2 expression was further examined by testing for correlation with survival, metastases, histology, and smoking status in patient tissues, and tumor cell proliferation and migration in vitro. EphA2 expression was analyzed in by immunohistochemistry in tissue microarray (TMA) format utilizing surgically resected lung cancer specimens. EGFR and KRAS mutation status was determined for the majority of specimens. EphA2 expression was detected in >90% of NSCLC tumors. High EphA2 expression was associated with decreased time to recurrence and metastases, and predicted poorer progression free and overall survival. Expression of EphA2 was positively correlated with activated EGFR and with KRAS mutation. Expression of EphA2 was also positively correlated with a history of smoking. There was no association between gender or histology and EphA2 expression. In H322 cells, activation of EGFR or KRAS resulted in an increase in EphA2 protein expression. Downregulation of EphA2 resulted in decreased proliferation in a clonal growth assay, and inhibited migration in a wound healing assay, in a panel of cell lines. The decrease in proliferation correlated with a transient decrease in the levels of phospho-ERK, a downstream effector of EGFR and KRAS. Based on these data, the potential of EphA2 as a therapeutic target for NSCLC should be further investigated. ^

HUMAN ENDOGENOUS RETROVIRUS K AS A NOVEL TUMOR-ASSOCIATED ANTIGEN FOR DEVELOPMENT OF AN OVARIAN CANCER VACCINE

HUMAN ENDOGENOUS RETROVIRUS K AS A NOVEL TUMOR-ASSOCIATED ANTIGEN FOR DEVELOPMENT OF AN OVARIAN CANCER VACCINE Publication No.________Kiera Rycaj, B.S.Supervisory Professor: Feng Wang-Johanning, Ph.D., M.D. Ovarian cancer (OC) is the fourth most common cancer in women, and the most lethal gynecologic malignancy in the United States. Adequate screening methodologies are currently lacking and most women first present with either stage III or IV disease. To date, there has been no substantial decrease in death rates and the majorities of patients relapse and die from their disease despite response to first-line therapy. Several proteins, such as CA-125, are elevated in OC, but none has proven specific and sensitive enough to serve as a screening tool or for tumor cell recognition and lysis. It has been proposed that human endogenous retrovirus sequences (HERVs) may play a role in the etiology of certain cancers. In a previous study, we showed that HERV-K envelope (env) proteins are widely expressed in human invasive breast cancer (BC) and ductal carcinoma in situ (DCIS), and elicit both serologic and cell-mediated immune responses in BC patients. We also reported the expression of multiple HERV genes and proteins in OC cell lines and tissues. In this study, we strengthened our previous data by determining that HERV-K env mRNAs are expressed in 69% of primary OC tissues (n=29), but in only 24% of benign tissues (N=17). Immmunohistochemistry (IHC) staining revealed HERV-Kpositivecancer cells detected in endometrioid adenocarcinoma and serous adenocarcinoma but not in benign cyst or normal epithelium biopsies. Immunofluorescence staining (IFS) showed greater cell surface expression of HERV-K in OC samples compared to adjacent uninvolved samples. Enzyme-linked immunosorbent assay (ELISA) data confirmed that a humoral immune response is elicited against HERV-K in OC patients. T-cell responses against HERV-K in lymphocytes from OC patients stimulated with autologous HERV-K pulsed dendritic cells included induction of T-cell proliferation and IFN-γ production. HERV-K–specific cytolytic T cells induced greater specific lysis of OC target cells compared to benign and adjacent uninvolved target cells. Finally, upon T regulatory cell (T-reg) depletion, 64% of OC patients displayed an increase in the specific lysis of target cells expressing HERV-K env protein. These findings suggest that HERV-K env protein is a tumor-associated antigen capable of activating both T-cell and B-cell responses in OC patients, and has great potential in the development of immunotherapy regimens against OC.

Chronic stress promotes tumor growth through increased BDNF production and neo-innervation

Background: Activation of the sympathetic nervous system (SNS) in response to chronic biobehavioral stress results in high levels of catecholamines and persistent activation of adrenergic signaling, which promotes tumor growth and progression. However it is unknown how catecholamine levels within the tumor exceed systemic levels in circulation. I hypothesized that neo-innervation of tumors is required for stress-mediated effects on tumor growth. Results: First, I examined whether sympathetic nerves are present in human ovarian cancer samples as well as orthotopic ovarian cancer models. Immunohistochemical (IHC) staining for neurofilament revealed that catecholaminergic neurons are present within tumor tissue. In order to determine whether chronic stress affects the density of nerves in the tumor, I utilized an orthotopic mouse model of ovarian cancer that was exposed to daily restraint stress. IHC analysis revealed that nerve density in tumors increased by more than three-fold in stressed animals versus non-stressed controls. IHC analysis suggested that this results from both recruitment of existing neurons (axonogenesis) as well as new neuron formation (neurogenesis) within the tumor. To determine how tumors are recruiting nerve growth, I utilized a PCR array analysis of 84 nerve growth related genes and their receptors, which showed that stimulation of the SKOV3 ovarian cancer cell line with norepinephrine (NE) leads to increased expression of several neurotrophins, including brain-derived neurotrophic factor (BDNF). Neurite extension assays showed that media conditioned by ovarian cancer cell lines is capable of inducing neurite outgrowth in differentiated neuron-like PC12 cells, and NE treatment of cancer cells potentiates this effect. Norepinephrine-induced neurite extension was abolished after BDNF silencing by siRNA, suggesting that BDNF is critical to tumor cell-induced nerve growth. in vivo BDNF inhibition resulted in complete abrogation of stress-induced increases in tumor weight and nerve density, as well as downstream markers of stress. Discussion: These studies indicate that adrenergic signalling induced by chronic stress promotes neo-innervation in the tumor microenvironment. This results in a mutually beneficial relationship between the tumor cells and neurons. This work is crucial for providing a link between chronic stress and its effects on the tumor and its microenvironment. The data shown here aims to open new venues that can be used in development of therapies designed to block the stress effects on tumor growth.

αvβ8 Integrin Interacts with RhoGDI1 to Regulate Rac1 and Cdc42 Activation and Drive Glioblastoma Cell Invasion

As an interface between the circulatory and central nervous systems, the neurovascular unit is vital to the development and survival of tumors. The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are major impediments to surgical resection and targeted therapies. Adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we have utilized human GBM cell lines, primary patient samples, and pre-clinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin associates with Rho GDP Dissociation Inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin-RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, suppresses activation of Rho proteins to promote GBM cell invasiveness. Hence, targeting the αvβ8 integrin-RhoGDI1 signaling axis may be an effective strategy for blocking GBM cell invasion.

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. ^

Aberrant regulation of the Src -FAK signaling pathway in the human colon adenocarcinoma cell line HT-29

c-Src, a protein tyrosine kinase (PTK) the specific activity of which is increased $>$20-fold in $\sim$80% of colon tumors and colon tumor cell lines, plays a role in both growth regulation and tumorigenicity of colon tumor cells. To examine the effect of increased c-Src specific activity on colon tumor cells, coumarin-derived tyrosine analog PTK inhibitors were assessed in a standard colon tumor cell line, HT-29. Of the nine compounds tested for inhibiting c-Src activity in a standard immune complex kinase assay from c-Src precipitated from HT-29 cells, the 7,8-dihydroxy-containing compounds daphnetin and fraxetin were most effective, with IC$\sb{50}$s of 0.6 $\pm$ 0.2 mM and 0.6 $\pm$ 0.3 mM, respectively. Treatment of HT-29 cells with daphnetin resulted in inhibition of cell growth in a dose-dependent manner. In contrast, scopoletin, a relatively poor Src inhibitor in vitro, did not inhibit HT-29 cell growth in the concentration range tested. In daphnetin treated cells, a dose-dependent decrease of c-Src activity paralleling cell growth inhibition was also observed; the IC$\sb{50}$ was 0.3 $\pm$ 0.1 mM for c-Src autophosphorylation. In contrast, the IC$\sb{50}$ for c-Src protein level was $>$ 0.6 mM, indicating that the effects of daphnetin were primarily an enzymatic activity of c-Src, rather than protein level in HT-29 cells. These results are the first to demonstrate that c-Src specific activity regulates colon tumor cell growth.^ To elucidate the signaling pathways activated by c-Src in colon tumor cells, the Src family substrate FAK, which has been shown to play a role in both extracellular matrix-dependent cell growth and survival, was examined. Coprecipitation assays showed Src-FAK association in detergent insoluble fractions of both attached and detached HT-29 cells, indicating that Src-FAK association in HT-29 cells is stable and, unlike untransformed cells, not dependent on cell-substratum contact. FAK also coprecipitated with Grb2, an adaptor protein also playing a role in cell proliferation and survival, in both attached and detached HT-29 cells, suggesting that a Src-FAK-Grb2-mediated signaling pathway(s) in HT-29 cells is/are constitutively activated.^ FAK was also analyzed in c-src antisense HT-29 clones AS15 and AS33 in which c-Src is specifically reduced by transfection of an antisense expression vector. FAK protein level is unexpectedly decreased in both AS15 and AS33 cells by 5-fold and 1.5-fold compared to HT-29, respectively, corresponding with the decreased expression of c-Src observed in these cells. FAK protein level was not decreased compared to parental in the c-src "sense" clone S8. Northern blot analyses showed decreased FAK mRNA levels compared to parental in AS15 and AS33, correlating with decreased FAK protein level, indicating that FAK activity in the antisense cells is regulated, at least in part, by altering FAK expression, and that this regulation is Src dependent. Because FAK has been implicated in anoikis, the ability of c-src antisense cells to survive in the absence of cell-substratum contact was examined. Decreased cell survival is seen in both AS15 and AS33, correlating with the decreases in c-Src and FAK levels and tumorigenicity in these cells. These results suggest that at least one mechanism by which activation of c-Src contributes to tumorigenic phenotype of colon tumor cells is by aberrantly promoting a survival signal through unregulated Src-FAK-Grb2 complexes. (Abstract shortened by UMI.) ^

Retinoid -induced regression of human head and neck squamous cell carcinomas is associated with the induction of a pro -inflammatory response

Retinoids are Vitamin A derivatives that are effective chemopreventative and chemotherapeutic agents for head and neck squamous cell carcinomas (HNSCC). Despite the wide application of retinoids in cancer treatment, the mechanism by which retinoids inhibit head and neck squamous cell carcinomas is not completely understood. While in vitro models show that drugs affect cell proliferation and differentiation, in vivo models, such as tumor xenografts in nude mice drugs affect more complex parameters such as extracellular matrix formation, angiogenesis and inflammation. Therefore, we studied the effects of retinoids on the growth of the 22B HNSCC tumors using a xenograft model. In this system, retinoids had no effect on tumor cell differentiation but caused invasion of the tumor by inflammatory cells. Retinoid induced inflammation lead to tumor cell death and tumor regression. Therefore, we hypothesized that retinoids stimulated the 22B HNSCC xenografts to produce a pro-inflammatory signal such as chemokines that in turn activated host inflammatory responses. ^ We used real time quantitative RT-PCR to measure cytokine and chemokine expression in retinoid treated tumors. Treatment of tumors with an RAR-specific retinoid, LGD1550, had no effect on the expression of TNFα, IL-1α, GROα, IP-10, Rantes, MCP-1 and MIP-1α but induced IL-8 mRNA 5-fold. We further characterized the retinoid effect on IL-8 expression on the 22B HNSCC and 1483 HNSCC cells in vitro. Retinoids increased IL-8 expression and enhanced TNFα-dependent IL-8 induction. In addition, retinoids increased the basal and TNFα-dependent expression of MCP-1 but decreased the basal and TNFα dependent expression of IP-10. The effect of retinoids on IL-8 and MCP-1 expression was very rapid with increased levels of mRNA detected within 1–2 hours. This effect did not require new protein synthesis and did not result from mRNA stabilization. Both RAR and RXR ligands increased IL-8 expression whereas only RAR ligands activated MCP-1 expression. ^ We identified a functional retinoid response element in the IL-8 promoter that was located adjacent to the C/EBP-NFkB response element. TNFα treatment of the 22B cells caused rapid, transient and selective acetylation of regions of the IL-8 promoter associated with the NFkB response element. Co-treatment of the cells with retinoids plus TNF increased the acetylation of chromatin in this region without altering the kinetics of acetylation. These results demonstrate that ligand activated retinoid receptors can cooperate with NFkB in histone acetylation and chromatin remodeling. We believe that in certain HNSCC tumors this cooperation and the resulting enhancement of IL-8 expression can induce an inflammatory response that leads to tumor regression. We believe that the induction of inflammation in susceptible tumors, possibly coupled with cytotoxic interventions may be an important component in the use of retinoids to treat human squamous cancers. ^