Home | Volume 3

Transcriptional repression in cancer gene therapy: targeting HER-2/neu overexpression as an example

Dr. , Mien-Chie Hung et. al.

Download as PDF : 10_Hung.pdf : 66539 bytes


Previous Article: Application of recombinant Herpes Simplex Virus-1 (HSV-1) for the treatment of malignancies outside the central nervous system
Next Article: Gene therapy targeting p53

Gene Therapy and Molecular Biology Vol 3, page 91

Gene Ther Mol Biol Vol 3, 91-101. August 1999.

Transcriptional repression in cancer gene therapy:

targeting HER-2/neu overexpression as an example

Review Article

Mien-Chie Hung* and Shao-Chun Wang

University of Texas M. D. Anderson Cancer Center, Department of Cancer Biology, Section of Molecular Cellular
Biology, Box 79, 1515 Holcombe Boulevard, Houston, Texas 77030 .

* To whom correspondence should be addressed. Phone: (713) 792-3668. Fax: (713) 794-4784. E-mail:

mchung@odin.mdacc.tmc.edu

Received: 16 October 1998; accepted: 9 November 1998

Summary

Overexpression of the HER-2/neu oncogene has been well-documented as a frequent event in human cancers. In clinic, overexpression of HER-2/neu indicates a unfavorable prognosis and highly correlated with the low survival rate of patients associated with breast and ovarian cancers. Downregulation of the HER-2/neu gene expression in cancer cells by attenuating the promoter activity of the gene is therefore an attractive strategy to reverse the transformation phenotype induced by HER-2/neu overexpression. We have identified a number of cellular and viral transcriptional regulators, including the ets family member PEA3 , the SV40 large T antigen, and the adenovirus type 5 E1A , which are able to repress the HER-2/neu gene expression. Expression of these transcriptional regulators resulted in downregulation of the HER-2/neu promoter activity and reversed the malignant phenotype of the transformed cells in vitro . These observations were followed by a series of studies to investigate whether these HER-2/neu repressors can act therapeutically as tumor suppressor genes for cancers that overexpress HER-2/neu . The growth of tumors derived from HER-2/neu -overexpressing cancer cells was inhibited by the transcriptional repressors, accompanied by decreased HER-2/neu expression in tumor cells. The results of these preclinical studies clearly indicate that transcriptional repressors which downregulate HER-2/neu can be a promising regimen for cancer treatment in a gene therapy format.

I. Introduction

A. HER2/neu overexpression serves as a critical target for cancer gene therapy

The HER-2/neu (also known as c-erbB2) gene encodes a receptor tyrosine kinase (p185) with significant structural and functional homology to the epidermal growth factor receptor (EGFR) (Bargmann et al, 1986a, Hung et al,
1986, Yamamoto et al, 1986). Each protein member of the erbB receptor family contains an extracellular domain, a transmembrane domain, and an intracellular domain with intrinsic tyrosine kinase activity. Although the ligand for the HER2/neu receptor has not been identified, the HER2/neu receptor is known to mediate lateral signal
transduction through all erbB receptor family members (Craus-Porta et al, 1997, Wallasch et al, 1995, Carraway et al, 1994, Sliwkowski et al, 1994, Plowman et al,
1993), due to the preference for the HER2/neu receptor as a heterodimerization partner for all erbB receptors. After ligand binding, EGFR, HER-3 (also known as erbB3), and HER-4 (also known as erbB4) can heterodimerize with HER2/neu, and can lead to the tyrosine phosphorylation of all of these receptors (Craus-Porta et al, 1997, Wallasch et al, 1995, Sliwkowski et al, 1994).
The oncogenic property of the HER-2/neu proto- oncogene was originally demonstrated in the rat neu oncogene (Hung et al, 1989, Bargmann 1986b, Hung et al,
1986). As a matter of fact, the mutation-activated rat neu
91

Hung and Wang: Targeting HER-2/neu overexpression in cancer gene therapy

oncogene, which contains a point mutation in the transmembrane domain of the protein resulting in a constitutive tyrosine kinase activity, was originally isolated from rat neuroblastoma due to its ability to transform mouse cells (Hung et al, 1989, Bargmann et al,
1986b, Hung et al, 1986). In human, the HER-2/neu proto-oncogene is frequently amplified or overexpressed in many types of cancers including breast (Gusterson et al,
1992, Toikkanen et al, 1992, Slamon et al, 1989, Slamon et al, 1987), ovarian (Slamon et al, 1989, Burchuck et al,
1991, Burchurk et al, 1990), lung (Shi et al, 1992, Weiner et al, 1990, Schneider et al, 1989), stomach (Yokota et al,
1988, Park et al, 1989), and oral (Xia et al, 1997) cancers, suggesting that HER-2/neu overexpression plays a critical role in the development of human cell malignancy. The overall survival rate of cancer patients whose tumors have HER-2/neu overexpression is significantly shorter than those patients whose tumor do not have HER-2/neu overexpression (Slamon et al, 1989, Slamon et al, 1987, Burchurk et al, 1990, Weiner et al, 1990, Xia et al, 1997). Furthermore, increased expression of the HER-2/neu gene has been shown to correlate with the number of lymph node metastases in breast cancer patients (Slamon et al,
1987), an observation consistent with many studies in that the mutation-activated neu gene induced metastatic potential in mouse 3T3 cells and that overexpression of the normal human HER2/neu gene enhanced metastatic potential in human breast, ovarian, and non-small-cell lung carcinoma (NSCLC) cells by promoting multiple steps in the metastatic cascade (Tan et al, 1997, Yu et al,
1994, Benz et al, 1993, Yu et al, 1992a, Chazin et al,
1992, Yu and Hung 1991a, Slamon et al, 1989, Slamon et al, 1987). In addition to metastasis of cancer cells, it is generally believed that HER-2/neu overexpression is correlated to chemoresistance of cancer cells. High level of HER-2/neu expression in human NSCLC appeared to result in enhanced resistance to a panel of chemotherapeutic agents (Tsai et al, 1995. Tsai et al,
1993). Similarly, overexpression of HER-2/neu in breast cancer cells induced chemoresistance to Taxol (Paclitaxel) (Yu et al, 1998, Yu et al, 1996). However, the expression level of HER-2/neu seems to be critical for the development of chemoresistance since in certain cell lines moderate p185 expression level is not accompanied with significant drug resistance (Pegram et al, 1997). It is likely that the HER-2/neu expression has to be higher than a threshold level to induce significant drug resistance. Furthermore, the chemoresistance developed in those HER-

2/neu-overexpressing breast cancer cells is limited to Paclitaxal and Taxotere but not to other drugs (Pegram et al, 1997; Yu et al, 1996; Yu, D. and Hung, M. -C., unpublished results), suggesting a selective mechanism of resistance. it is not yet clear why HER-2/neu overexpression-mediated drug resistance behaves differently between lung and breast cancer cells. However, in the case

of resistance to Paclitaxel by HER-2/neu overexpression in breast cancer cells, a molecular mechanism has recently been suggested (Yu et al, 1998a): upregulation of p21 by HER-2/neu overexpression inhibits cyclin B/cdc2 kinase activity in G2/M phase which is required for Paclitaxel induced apoptosis. This mechanism clearly indicates that HER-2/neu overexpression in breast cancer cells antagonizes Paclitaxel-induced apoptosis.
Since the HER2-neu proto-oncogene overexpression significantly contributes to the malignant development of many types of human cancers in different aspects, molecular strategies which aim to down-regulate the HER-

2/neu gene expression have become highly attractive approaches to fight against human cancer.

B. Transcriptional repression as an effective means to downregulate HER2/neu expression in cancer cells

HER-2/neu gene amplification can be detected in majority of breast tumor tissues with overexpression of the HER-2/neu-encoded p185 protein (Slamon et al, 1989). In established breast cancer cell lines, both gene amplification and transcriptional upregulation are common scenario accounting for the increased HER-2/neu gene expression in different breast cancer cells (Kraus et al,

1987, Millar et al, 1994, Bosher et al, 1996). Interestingly, it has been shown that in 10-20% of HER2/neu -overexpressing breast tumors and in virtually all HER2/neu-positive lung cancers the HER2/neu mRNA and protein expression can occur in the absence of increased gene copy number (Kameda et al, 1990, Kern et al, 1990, Slamon et al, 1989, King et al, 1989, Tandon et al, 1989, Berger et al, 1988). It is therefore likely that both gene amplification and transcriptional upregulation are involved in HER-2/neu overexpression in cancer cells.
The promoter of the HER-2/neu gene has been well characterized. In the past few years, knowledge about the cis- and trans-acting elements regulating the transcription of the HER-2/neu proto-oncogene have been rapidly accumulated. A number of cis-acting motifs are distributed along the HER-2/neu promoter, including the binding sites of transcription factors Sp1, OTF1, AP2, E4TF1, and PEA3. Another 13-bp sequence in the promoter region has been identified as a positive element for HER-2/neu transactivation (Miller et al, 1994). The corresponding binding transcription factor(s), however, has not yet been identified. AP2 has been shown to be a strong activator of the HER-2/neu gene and is functionally activated in the HER-2/neu-overexpressing breast cancer cell lines such as MDA-MB-361, MDA-MB-175, ZR-75-1, BT-474, and SK-BR-3 (Hollywood et al, 1993). The high activity of AP2 in these cell lines has been correlated with the elevated HER-2/neu gene expression level in these cells.
92

Gene Therapy and Molecular Biology Vol 3, page 93

On the other hand, the HER-2/neu gene is subject to the negative regulation of a number of cellular or viral factors through different mechanisms. For example, PEA3, a member of the ets family (X. Xing, S. -C. Wang, and M.
-C. Hung, unpublished results; Xing et al, 1997), and the retinoblastoma tumor suppressor (RB ) (Yu et al, 1992b) can repress the HER-2/neu gene expression. Interestingly, in addition to the cellular factors, the HER-2/neu gene transcription can also be repressed by a number of viral transcription factors such as the simian virus 40 (SV40) large T antigen and the adenovirus type 5 E1A (Yan et al,
1991a, Yu et al, 1991b). These studies have indicated that repression of transcription is an effective way to reverse the malignant transformation mediated by HER-2/neu overexpression, and have demonstrated the potential application of transcriptional repressors as therapeutic agents targeting HER-2/neu-overexpressing cancer cells.

II. Tumor suppression effects of HER-

2/neu down-regulation mediated by genes

encoding transcriptional regulators

A. Tumor suppression by viral transcriptional regulators

Both E1A and T antigen are viral proteins, and their ability to suppress HER-2/neu-mediated cell transformation is surely a surprising biological phenomenon. The adenovirus genome is about 36 kb in size. Among the proteins encoded by the adenovirus genome, E1A gene products are nuclear-localized phosphoproteins and have special regulatory role in the adenoviral life cycle (Berk 1986). E1A is the first region to be expressed after infection (Tooze 1981). Other late adenoviral genes can then be turned on by E1A proteins through interacting and modifying the host transcriptional apparatus. There are two types of adenovirus E1A . One is the transforming E1A carried by the adenovirus type 12. This type of E1A gene alone can transform normal cell lines (Schrier et al, 1983). The other type of E1A , such as the adenovirus type 2 or type 5 E1A , can not transform cells by itself alone. It is noteworthy that for the purpose of this review E1A refers to the type 5, non-transforming E1A . E1A was classified as an "immortalization oncogene" due to its ability to cooperate with the transforming ras or E1B genes to transform primary embryo cells (Byrd et al, 1988, Montell et al, 1984, Land et al 1983, Ruley 1983). However, expression of the E1A gene itself does not induce transforming phenotypes (Yu et al, 1992a). As a matter of fact, there are a number of studies indicated that E1A is associated with metastasis- or tumor-suppression activities (Pozzatti et al, 1988, Frisch
1991, van Groningen 1996). Recently, E1A has been shown to induce apoptosis under some conditions (Lowe and Ruley 1993, Rao et al, 1992). This property is similar
to the well-known tumor suppressor gene p53 that also has the ability to induce apoptosis (Subramanian et al,
1995, Symonds et al, 1994). All of these observations indicate that tagging E1A as an oncogene is a misconception.
We have first discovered that the adenovirus 5 E1A gene can repress HER-2/neu overexpression through both transient transfection and adenovirus delivery systems (Yu et al, 1991b, Yu et al, 1990). Transfection of the E1A gene into the genomic rat neu oncogene transformed mouse embryo fibroblast cell lines virtually abolishes the tumorigenicity and metastatic potential induced by the HER-2/neu oncogene through repression of HER-2/neu gene expression (Yu et al, 1992a, Yu et al, 1991b). Re- expression of the HER-2/neu-encoded p185 protein in these E1A transfectants by transfection of a HER-2/neu cDNA construct driven by a promoter that cannot be inhibited by E1A recovered virtually all of the transforming phenotypes including tumorigenicity, the ability to grow in soft agar, and higher in vitro growth rate (Yu et al, 1993a, b). Interestingly, the ability to induce experimental metastasis (measured by lung colonization through i. v. injection of the tumor cells) was only partially recovered. The incomplete regeneration of metastatic potential could be accounted for by the fact that E1A inhibits gelatinolytic activity that was critical for invasive activity of metastatic cells. This result indicates that the suppression of metastasis by E1A is through multiple molecular mechanisms in addition to repressing the HER-2/neu gene expression (Yu et al, 1992a). We have also demonstrated that E1A can indeed function as a tumor suppressor in the HER-2/neu-overexpressing human ovarian cancer cell line by down-regulating the expression of the HER-2/neu mRNA and the p185 protein product (Yu et al, 1995, Yu et al, 1993a, b, Yu et al, 1991b, Yu et al, 1990). The E1A-expressing ovarian cancer cell line had reduced malignancy, including a decreased ability to develop tumors in nude mice. Therefore, for the HER-

2/neu-overexpressing transforming cells including fibroblasts and human cancer cells, E1A can function as tumor suppressor. And transcriptional repression of the HER-2/neu oncogene contributes to the tumor suppression function. However, since E1A is not a DNA-binding protein, the transcriptional repression of HER-2/neu by E1A has to be mediated through the targeting of other transcription factors. This is supported by our recent study demonstrating that E1A can abolish HER-2/neu overexpression by targeting the coactivator p300, which is required for efficient expression of HER-2/neu (Chen and Hung 1997).

To further investigate whether the E1A gene can be used as a therapeutic agent for HER-2/neu-overexpressing human breast and ovarian cancers in living host, a tumor- bearing mouse model was established and the E1A gene
93

Hung and Wang: Targeting HER-2/neu overexpression in cancer gene therapy

was delivered by the cationic liposome DC-Chol or a recombinant replication-deficient adenovirus. E1A treatment was able to effectively reduce the mortality of tumor-bearing mice and, in 60-80 % of the treated mice, resulted in tumor-free survival, suggesting that E1A gene therapy is a promising therapeutic regimen for cancers that overexpress HER-2/neu . In addition, the number of mice with distant metastases was significantly reduced even though a local treatment protocol by mammary fat pad injection was used in the orthotopic breast cancer model (Lane and Crawford 1979, Zhang et al, 1995). In addition to the breast and ovarian cancer animal models, we also used a lung cancer animal model to test the therapeutic efficiency of E1A (Chang et al, 1996). In this case, the tumor-bearing mice were established through intratracheal inoculation of lung cancer cells and the E1A gene was delivered by an adenovirus vector through intravenous injection. A significant therapeutic efficacy was observed. Therefore the tumor suppression effect of E1A can be demonstrated through two independent gene delivery systems and three different animal models. Based on these results, a phase I clinical trial, using cationic liposome to deliver the E1A gene was initiated at the M. D. Anderson Cancer Center. Preliminary results suggested a down- regulation of the HER-2/neu p185 oncoprotein concomitant with the detection of the E1A gene expression in treated breast and ovarian cancer patients.
The simian virus 40 (SV40) large T antigen is a multifunctional protein required for the replication of the viral genome and for cell transformation (Lane and Crawford 1979, Linzer and Levine 1979). This viral protein contains transformation domains which can mediate binding to the retinoblastoma protein (pRb) and p53, respectively (Manfredi and Prive 1994). Our previous studies showed that a mutant SV40 large T antigen can repress rat neu transcription in mouse fibroblast NIH 3T3 cells (Matin and Hung 1993). The mutant large T antigen, named K1, contains a single amino acid change within the pRb-binding/transformation domain, which renders the viral protein unable to bind to pRb, and consequently failed to induce cell transformation (Kalderon and Smith
1984, Cherington et al, 1988, DeCaprio et al, 1988). Since the K1 mutant represses HER-2/neu expression as effectively as the wild-type counterpart (Matin and Hung
1993), we further tested whether K1 can function as a tumor suppressor for HER-2/neu-overexpressing ovarian cancer cells. K1 did suppress cancer cell growth, resulting in a significant therapeutic effect on mice with ovarian cancer with about 40% of treated mice were alive after one year (Xing et al, 1996). The autopsies showed that the mice from the control groups had larger volume of ascites and tumors within the peritoneal cavity or diaphragm or metastasis to the lungs. However, the mice that received K1-liposome complex had more locally distributed tumor
nodules in their peritoneal cavities. This difference indicates that K1 suppressed the growth of HER-2/neu- overexpressing tumor cells so that the tumors developed with longer latency. The K1-treated mice survived for one year were sacrificed and examined for residual tumors, but no tumors were observed in the peritoneal cavity.
Our results indicate that both viral transcription factors, E1A and the large T antigen, can suppress tumor cell growth through a HER-2/neu-involved pathway. However, the possibility that E1A can mediate tumor suppression function through a HER-2/neu-independent mechanism should not be excluded.

B. Tumor suppression by cellular DNA- binding transcriptional factor, PEA3

The mouse PEA3 (Polyomavirus Enhancer Activator
3) gene and its human homologue were first cloned from cDNA expression libraries due to the binding ability to the sequence 5’-AGGAAG-3’ (the PEA3 binding motif) within the polyomavirus enhancer promoter element (Xin et al,
1992, Higashino et al, 1993). The PEA3 protein contains a stretch of about 85 amino acids with extensive sequence homology with the ETS domain, a conserved region shared by all ets family members that characteristically bind as monomers to the consensus core sequence GGAA by their ETS DNA-binding domains (Monte et al, 1994, Brown et al, 1992, Xin et al, 1992, Karim et al, 1990), and regulates the expression of target genes including genes involved in cell growth and differentiation (Ma et al, 1998, Taylor et al, 1997). The ets gene family currently contains at least 30 members present in a diverse spectrum of metazoan organisms (Degnan et al, 1993, Laudet et al,
1993) Subfamilies can be identified based on sequence/structure homology and the association with other accessory proteins for DNA binding. The PEA3 subfamily is composed of three members : PEA3 (Xin et al, 1992), ERM (Nakae et al, 1995, Monte et al, 1994), and ER81 (Brown et al, 1992). In addition to the ETS domain, members of this subfamily share significant sequence similarity at an N-terminal acidic transcriptional activation domain (Nakae et al, 1995, Wasylyk et al,
1993, Macleod et al, 1992, Seth et al, 1992, Karim et al,
1990). Expression of the PEA3 gene is ubiquitous in different species and can be identified in mouse, rat, monkey, and human cells. However, PEA3 RNA expression is tissue-specific with highest level detected in brain, and, to a lower level, in pancreas, lung, and mammary gland (Xin et al, 1992). Most members of the ets family express at high levels in hemotopoietic cells. Unlike other Ets proteins, PEA3 is the only member identified to date that is apparently not expressed in cells with hematopoietic origins (Xin et al, 1992). The significance of this tissue specific distribution is not clear.
94

Gene Therapy and Molecular Biology Vol 3, page 95

There have been a number of candidate PEA3-regulated genes reported mainly based on the occurrence of putative PEA3 binding motif in their promoter regions. Interestingly, a great portion of these candidates fall in the category of genes encoding matrix metalloproteinases (Higashino et al, 1995), such as collagenase (Gutman et al, 1990), stromelysin (Buttice et al, 1993), and the urokinase-type plasminogen activator (uPA), a serine proteinase (Nerlov et al, 1992). All these enzymes are believed to involve in the regulation of extracelluar proteolysis, both in the normal organisms and in certain pathological conditions including tumor invasion and metastasis (Matrisian 1994). Consistent with this correlation, exogenous expression of PEA3 in the breast cancer cell line MCF-7 resulted in enhanced tumor invasiveness and metastasis (Mitsunori et al, 1996).
Caveats should be taken to interpret these results. It is possible that members of the Ets family can have the same specificity required for DNA binding and share the same binding motif (Xin et al, 1992). As a matter of fact, it has been shown that Ets-2, an Ets protein belonging to the Pointed subfamily (Klambt 1993), is critical for the phobol ester (TPA)-mediated induction of the human stromelysine gene expression through the PEA3 binding motif in the promoter (Buttice et al, 1993). A similar conclusion has been drawn for the promoter of the uPA gene (Pankov et al, 1994). In addition, whether the PEA3 protein directly binds to the putative PEA3 motif in the collagenase promoter is not clear due to the lack of appropriate anti-PEA3 antibodies to confirm the identity of the DNA-binding activity detected on the PEA3 motif (Gutman et al, 1990).
The occurrence of the PEA3 binding motif is not limited to those genes which potentially can enhance invasion and metastasis. Two consensus PEA3 binding motifs, distal and proximal, have been identified in the upstream regulatory region of the tumor suppressor gene maspin (Zhang et al, 1997a, Zhang et al, 1997b). Both motifs are positive regulatory elements for expression of the gene; the proximal site is the major functioning motif of the gene in mammary epithelial cells while both sites are equally critical for maspin expression in prostate cells. Functional studies have demonstrated that maspin functions as a tumor suppressor by inhibiting tumor invasion, metastasis, as well as tumor growth (Sheng et al, 1995, Zou et al, 1994). Even though it is still not clear if the PEA3 protein binds to the PEA3 motif in the maspin promoter, these observations are consistent with the prospect that tumor metastasis may be the result of imbalance between enhancing and suppressing factors (Liotta et al, 1991). This point is especially noteworthy given the large number of the ets family members and the resemblance of their DNA-binding domains and the DNA sequences of their target DNA motifs.
It is interesting to investigate the role of PEA3 in HER-2/neu gene expression and HER-2/neu-mediated transformation since a consensus PEA3- binding motif, 5'- AGGAAG-3', is present 26 nucleotide upstream from the major mRNA start site in the promoter of the human, rat, and mouse HER-2/neu gene (Tal et al, 1987). It has been reported that PEA3 can mediate induction of the HER-

2/neu gene expression through the PEA3 binding motif (Benz et al, 1997). These results, however, were derived from the experiment using the COS monkey cell line. As will be mentioned below, this cell line can be characteristically different from other laboratory human breast and ovarian cancer cell lines, for which the investigation of PEA3's functions would be more biologically relevant. Furthermore, the hypothesis of PEA3-mediated HER-2/neu induction would predict a causal relationship between elevated PEA3 expression and HER-2/neu overexpression in cancer cells. However, analysis of PEA3 gene expression in various breast cancer cells dose not support this hypothesis. In fact, decreased PEA3 RNA expression was detected in breast cancer cell lines with HER-2/neu overexpression (such as BT 474, SK-BR-3, MDA-MB-361), while there was no detectable PEA3 mRNA in other HER-2/neu-overexpressing cell lines (such as MDA-MB-453, ZR-75-1, and MDA-MB-

134-V) (Baert et al, 1997).
Nevertheless, these results suggest a negative role of PEA3 in regulating HER-2/neu expression. This prospect was directly tested in our laboratory and the following results demonstrate that PEA3 is indeed a negative trans- regulator of the proto-oncogene HER-2/neu (Xing et al,
1997).
(1 ) The purified GST-PEA3 fusion protein can specifically recognize and bind to the consensus PEA3 binding motif on the HER-2/neu promoter.
(2 ) Based on the co-transfection experiments performed on HER-2/neu-overexpressing human cancer cell lines, the HER-2/neu promoter activity can be down-regulated by PEA3 in a dose-dependent manner. However, destruction of the PEA3-binding site on the HER-2/neu promoter by site-directed mutagenesis abolished the promoter activity, indicating that PEA3-induced trans-repression of the HER-

2/neu promoter might involve competition between PEA3 and another ets-related transcriptional activator(s), which contributes to the transformed phenotype of HER-2/neu .

(3 ) PEA3 can suppress the focus forming ability of mouse embryonic fibroblast transformed by the genomic mutation-activated genomic rat neu.
(4 ) Expression of PEA3 can suppress the growth of HER-2/neu-overexpressing human cancer cell lines in vitro, but not cell lines with basal level of HER-2/neu expression.
Based on these results, the tumor suppression function
95

Hung and Wang: Targeting HER-2/neu overexpression in cancer gene therapy

of PEA3 is emerging. Trimble et al, have reported that mammary tumors derived from the transgenic mice bearing the rat neu gene under the control of the mouse mammary tumor virus (MMTV) promoter expressed high level of PEA3 mRNA, suggesting that PEA3 may be required for tumorigenesis and metastasis in HER2/neu overexpressing cells (Trimble et al, 1993). However, the data is also consistent with the possibility that there may exist a negative regulatory loop pathway in which the overexpression of HER-2/neu would turn on the expression of PEA3 which then act as a transcriptional repressor of the HER-2/neu gene and resume the homeostatic balance. The rat neu gene in the transgenic mice setting was driven by the heterologous MMTV promoter which is very likely not subject to the negative control by PEA3. Expression levels of both PEA3 and HER-2/neu would be elevated under this situation. In addition to PEA3, other Ets proteins including ERF and Net have been reported to function as transcriptional repressors (Sgouras et al, 1995, Giovan et al, 1994). Other promoters negatively regulated by Ets binding sites have also been reported (Chen and Boxer 1995, Goldberg et al,
1994). Interestingly, the ets family member Ets2 has recently been reported to function as a tumor suppressor by reversing ras-mediated cellular transformation (Foos et al,
1998).
To test whether PEA3 can be used as a therapeutic agent in vivo, tumors were induced in nude mice (nu/nu) with SK-OV-3-ip1, an ovarian cancer cell line derived from SK-OV-3 and has higher HER-2/neu expression. For mice treated with PEA3 -DC-Chol complex, 50% of the mice were alive and healthy without palpable tumors after 12 months. The mice of the control group, however, developed tumors and ascites, and died within 6 months. The tumor suppression activity of PEA3 is correlated with HER-2/neu expression since another cell line 2774 c-10, an ovarian cancer cell line with basal level of HER-2/neu expressed, did not have response to PEA3 treatment and the mice died of tumor with 5 months. Tumor samples were examined for the expression of HER-2/neu with immunoblot analysis. The results confirmed that PEA3 delivered by the cationic liposome downregulated the expression of p185. The correlation between PEA3 expression and HER-2/neu downregulation was further demonstrated by immunohistochemical staining of the tumor samples obtained from the PEA3 -treated, moribund mice. Approximately 30% of the cancer cells in the tumor were positive for PEA3 protein expression, while the p185 staining was negative for about 50% of cells. Similar level of PEA3 expression was observed for PEA3-treated 2774 c-10-derived tumors while no repression of p185 was detected in these tumors. These in vitro and in vivo data clearly demonstrate the tumor suppression activity of PEA3 and indicate the potential clinical application of

PEA3 -cationic liposome targeting the HER-2/neu

overexpressing cancer cells.
Even though PEA3 as well as the viral proteins E1A and SV40 large T can all suppress HER-2/neu transcription, they are very likely functioning through different mechanisms. Both E1A and SV40 large T may suppress HER-2/neu in an indirect manner. Association of E1A with the transcriptional co-activator CBP/p300 inhibits the p300 transactivation activity, which is required for efficient expression of the HER-2/neu gene (Chen and Hung, 1997). On the other hand, PEA3 down-regulates the HER-2/neu gene by directly binding to its cognate binding sequence on the promoter. This feature makes PEA3 a more attractive target for further molecular manipulation to develop therapeutic molecules with higher binding affinity and enhanced specificity.

III. Conclusions

Overexpression of the proto-oncogene HER-2/neu can lead to cell transformation and tightly correlated with the development of malignant tumor growth in many tissue types. There are molecular approaches to target the promoter of HER-2/neu , which can downregulate the gene expression, reverses the malignant phenotype, and retards tumor growth in animal. The results of our in vivo and in vitro experiments demonstrate using viral or cellular transcriptional repressor genes transferred by safe and efficient molecular vehicles can result in significant therapeutic effects on cancer cells. Since gene overexpression is a common mechanism of cancer as well as other types of diseases such as AIDS, the therapeutic strategy discussed here can have a tremendous potential in clinical application. Finally, the studies of E1A - and PEA3 -mediated HER-2/neu repression have unveiled new areas in cancer biology which is excitingly more complicated than what we used to expect. Studies of these areas would be critical for our understanding of cancer.

Acknowledgment

The authors are supported by NCI RO1 CA 58880 and
CA 77858 (to M.C.H.).

References

Baert, J. L., Monte, D., Musgrove, E. A., Albagli, O., Sutherland, R. L., and Launoit, Y. (1997 ). Expression of the PEA3 group of EST-related transcription factors in human breast cancer cells. Int. J. Cancer 70, 590-597.

Bargmann, C. I. Hung, M. -C., and Weinberg, R. A. (1986a ).

The neu oncogene encodes an epidermal growth factor receptor-related protein. Nature 319, 226-230.

Bargmann, C. I. Hung, M. -C., and Weinberg, R. A. (1986b ).

96

Gene Therapy and Molecular Biology Vol 3, page 97

Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell 45, 649-657.

Benz, C. C., O’Hagan, R. C., Richter, B., et al, (1997 ).

HER2/Neu and the Ets transcription activator PEA3 are coordiately upregulated in human breast cancer. Oncogene 15, 1513-1525.

Benz CC. Scott GK. Sarup JC. Johnson RM. Tripathy D.

Coronado E. Shepard HM. Osborne CK. (1993 ). Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu. Breast Cancer Res. Treat. 24, 85-95.

Berger, M. S., Locher, G. W., Saurer, S., et al, (1988 ).

Correlation of c-erbB-2 gene amplification and protein expression in human breast carcinoma with nodal status and nuclear grading. Cancer Res. 48, 1238-1243.

Berk, A. J. (1986 ). Adenovirus promoters and E1A

transactivation. Ann. Rev. Genet. 20 , 45-79.

Bosher, J. M., Totty, N. F., Hsuan, J. J., Williams, T., and Hurst, H. C. (1996 ). A family of AP-2 proteins regulates c-erbB-2 expression in mammary carcinoma. Oncogene

13, 1701-1707.

Brown, T. A. and McKnight, S. L. (1992 ). Specificities of protein-protein and protein-DNA interaction of GABP alpha and two newly defined ets related proteins. Genes Dev. 6, 2502-2512.

Burchuck, A, Rodriguez, G., Kinney, R., Soper, J., Dodge, R., Clark-Pearson, D., and Bast, R. (1991 ). Overexpression of HER-2/neu in endometrial cancer is associated with advanced stage disease. Am. J. Obstet. Gynecol.

164, 15-21.

Burchuck, A., Kamel, A., Whitaker, R., Kerns, B., Olt, G., Kinney, R., Soper, J., Dodge, R., Clark-Pearson, D., Marks, P., McKenzie, S., Yin, S., and Bast Jr. R. (1990 ). Overexpression of HER-2/neu is associated with poor survival in advanced epithelial ovarian cancer. Cancer Res. 50, 4087-4091.

Buttice, G. and Kurkinen, M. (1993 ). A polyomavirus enhancer A-binding protein-3 site and Ets-2 protein have a major role in the 12-O-tetradecanoylphorbol-13-acetate response of the human stromelysin gene. J. Biol. Chem. 268, 7196-7204.

Byrd, P. J., Grand, R. J. A., and Gallimore, P. H. ( 1988 ).

Differential transformation of primary human embryo retinal cells by adenovirus E1A regions and combination of E1A (+) ras. Oncogene 2, 477-484.

Carraway, K. L., Sliwkowski, M. X., Akita, R. M., et al, (1994 ). The erbB-3 gene product is a receptor for heregulin. J. Biol. Chem. 269, 14303-14306.

Chang JY. Xia W. Shao R. Hung M. -C. (1996 ). Inhibition of intratracheal lung cancer development by systemic delivery of E1A. Oncogene 13, 1405-1412.

Chang, J. Y., Xia, W., Shao, R., Sorgi, F., Hortobagyi, G. N., Huang, L., and Hung, M. -C. (1997 ). The tumor suppression activity of E1A in HER-2/neu-overexpressing

breast cancer. Oncogene 14, 561-568.

Chazin VR. Kaleko M. Miller AD. Slamon DJ. (1992 ).

Transformation mediated by the human HER-2 gene independent of the epidermal growth factor receptor. Oncogene 7, 1859-1866.

Chen, H. and Hung M. -C. (1997 ). Involvement of co- activator p300 in the transcriptional regulation of the HER-2/neu gene. J. Biol. Chem. 272, 6101-6104.

Chen, H. M. and Boxer, L. M. (1995 ). Pi 1 binding sties are negative regulator of bcl-2 expressionin pre-B cells. Mol. Cell Biol. 15, 3840-3847.

Cherington V., Brown, M., Paucha, E., Louis, J., Spiegelman

BM, and Roberts TM. (1988 ). Separation of simian virus

40 large T antigen-transforming and origin-binding functions from the ability to block differentiation. Mol. Cell Biol. 8, 1380-1384.

DeCaprio, J. A., Ludlow, J. W., Figge, J., Shew, J. -Y., Huang, C. -M., Lee, W. -H., Marsilis, E., Paucha, E., and Livingston, D. M. (1988 ). SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell 54, 275-283.

Degnan, B. M., Degnan, S. M., Naganuma, T., and Morese, D.

E. (1993 ). The ets multigene family is conserved throughout the Metazoa. Nucleic Acid Res. 21, 3479-

3484.

Foos, G., Garcia-Ramirez, J. J., Galang, C. K., et al, ( 1998 ).

Elevated expression of Ets2 or distinct portions of Ets2 can reverse Ras-mediated cellular transformation. J. Biol. Chem. 273, 18871-18880.

Frisch, S. M. ( 1991 ). Antioncogenic effect of adenovirus E1A in human tumor cells. Pros. Natl. Acad. Sci. USA 88, 9077-9081.

Giovan, A., Pintzas, A., Maira, S. M., et al, ( 1994 ). Net, a new ets transcription factor that is activated by Ras. Genes Dev. 8, 1502-1513.

Goldberg, Y., Treier, M., Ghysdael, J., et al, (1994 ).

Repression of AP-1-stimulated transcription by c-Ets-1.

J. Biol. Chem. 269, 16566-16573.

Graus-Porta, D., Beerli, R. R., Daly, J. M., et al, ( 1997 ).

ErbB-2, the preferred heterodimerazation partner of all ErbB receptors, is a mediator of lateral signalling. EMBO J. 16, 1647-1655.

Gusterson, B., Gelber, R., Goldhirsch, A., Price, K., Save- Soderborgh, J., Anbazhagan, R., Styles, J., Rudenstam, C. -M., R., Reed, R., Martinez-Tello, F., Tiltman, A., Torhorst, J., Grigolato, P., Bettelheim, R., Neville, A., Burki, K., Castiglione, M., Collins, J., Lindtner, J., and Senn, H. -J. (1992 ). Prognostic importance of c-erbB-2 expression in breast cancer. J. Clin. Onc. 10, 1049-

1056.

Gutman, A. and Wasylyk, B. (1990 ). The collagenase gene promoter contains a TPA and oncogene-responsive unit emcomapassing the PEA3 and AP-1 binding sites. EMBO J. 9, 2241-2246.

Higashino, F., Yoshida, K., Noumi, T., Seiki, M., and

97

Hung and Wang: Targeting HER-2/neu overexpression in cancer gene therapy

Fujinaga K. (1995 ). Ets-related protein E1A-F can activate three different matrix metallo-proteinase gene promoters. Oncogene 10 , 1461-1463.

Higashino, F., Yoshida, K., Fujinaga, Y, et al, (1993 ).

Isolation of a cDNA encoding the adenovirus E1A enhancer binding protein: a new human member of the ets oncongene family. Nucleic Acid Res. 21, 547-553.

Hollywood, D. P., and Hurst, H. C. (1993 ). A novel transcription factor, OB2-1, is required for overexpression of the proto-oncogene c-erbB-2 in mammary tumor cell lines. EMBO J. 12, 2369-2375.

Hung, M. -C., Yan, D., and Xhao, X. (1989 ). Amplification of the proto-neu gene facilitates oncogenic activation by a single point mutation. Proc. Natl. Acad. Sci.USA

86, 2545-2548.

Hung, M. -C., Schechter, A. L., Chevray, P. L., Stern, D. F., and Weinberg, R. A. (1986 ).Molecular cloning of the neu gene: absence of gross structural alteration in oncogenic alleles. Proc. Natl. Acad. Sci. USA 83, 261-264.

Kalderon, D., and Smith, A. E. (1984 ). In vitro mutagenesis of a putative DNA binding domain of SV40 large T. Virology 39, 109-137.

Kameda, T., Yasui, W., Yoshida, K., et al, (1990 ).

Expression of ERBB2 in human gastric carcinomas: relationship between p185ERBB2 expression and the gene amplification. Cacner Res. 50, 8002-8009.

Karim, F. D., Urness, L. D., Thummel, C. S., et al, ( 1990 ).

The ETS-domain: a new DNA-binding motif that recognizes a purine-rich core DNA sequence. Genes Dev. 4, 1451-1453.

Kern, J. A., Schwartz, D., Nordberg, J. E., et al, (1990 ). p185neu expression in human lung adenocarcinomas predicts shortened survival. Cancer Res. 50, 5184-

5191.

King, C. R., Swain, S. M., Porter, L., et al, (1989 ).

Heterogeneous expression of erbB-2 messenger RNA in human breast cancer. Cancer Res. 49, 4185-4191.

Klambt, C. (1993 ). The Drosophila gene pointed encodes two ETS-likeproteins which are involved in the development of the midline glial cells. Development

117, 163-176.

Kraus, M. H., Popescu, N. C., Ambaugh, S. C., King, C. R. (1987 ). Overexpression of the EGF receptor-related proto-oncogene erbB-2 in human mammary tumor cell lines by different molecular mechanisms. EMBO J. 6 ,

605-610.

Land, H., Parada, L. F., and Weinberg, R. A. (1983 ).

Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature 304,

596-602.

Lane, D. P., and Crawford, L. V. (1979 ). T antigen is bound to a host protein in SV40-transformed cells. Nature 278,

261-262.

Laudet, V., Niel, C., Deuterque-Coquillard, M., Le Prince, D.,

and Stehelin, D. (1993 ). Evolution of the ets gene family. Biochem. Biophys. Res. Comm. 190, 8-

14.

Linzer, D. I. H., and Levine, A. J. (1979 ). Characterization of a 54K Dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell 17, 43-52.

Liotta, L. A., Steeg, P. S., and Stetler-Stevenson, W. G. (1991 ). Cancer metastasis and angiogenesis: imbalance of positive and negative regulation. Cell 64, 327-336.

Lowe, S. W. and Ruley, H. E. (1993 ). Stabilization of the p53 tumor suppressor is induced by adenovirus 5 E1A and accompanies apoptosis. Genes Dev. 7, 535-545.

Ma, Y., Su, Q., and Tempst, P. (1998 ). Differentiation- stimulated activity binds an ETS-like, essential regulatory element in the human promyelocytic defensin-1 promoter. J. Biol. Chem. 273, 8727-8740.

Macleod, K., Leprince, D., and Stehelin, D. (1992 ). The ets

gene family. Trends Biochem. Sci. 17, 251-256.

Manfredi, J. J., and Prive, C. ( 1994 ). The transformation activity of simian virus 40 large tumor antigen. Biochim. Biophys. Acta. 1198, 65-83.

Matin A., and Hung M. -C. (1993 ). Negative regulation of the neu promoter by the SV40 large T antigen. Cell Growth Differ. 4, 1051-1056.

Matrisian, L. M. (1994 ). Matrix metalloproteinase gene expression. Ann. N. Y. Acad. Sci. USA 732, 42-50.

Miller, S. J., Suen, T. C., Sexton, T. B., Hung, M. -C. (1994 ). Mechanisms of deregulated HER2/nue expression in breast cancer cell lines. Int. J. Oncol. 4, 599-608.

Mitsunori, K., Yoshida, K., Higashino, F., Mitaka, T., Ishii, S., and Fujinaga, K. (1996 ). A single ets-related transcription factor, E1AF, confers invasive phenotype on human cancer cells. Oncogene 12, 221-227.

Monte, D., Baert, J. L., Defossez, P. A., et al, , (1994 ).

Molecular cloning and characterization of human ERM, a new member of the Ets family closely related to mouse PEA3 and ER81 transcription factors. Oncogene 9,

1397-1406.

Montell, C., Courtois, G., Eng, C. et al, ( 1984 ). Complete transformation by adenovirus 2 requires both E1A proteins. Cell 36, 951-961.

Nakae, K., Nakajima, K., Inazawa, J., Kitaoka, T., and Hirano, T. (1995 ). ERM, a PEA3 subfamily of Ets transcription factors, can cooperate with c-Jun. J. Biol. Chem. 270,

23795-23800.

Nerlov, C., De Cesare, D., Pergola, F., et al, , (1992 ). A regulatory element that mediates co-operation between a PEA3-PA-1 element and an AP-1 stie is required for phorbol ester induction of urokinase enhancer activity in HepG2 hepatoma cells. EMBO J. 11, 4573-4582.

Park, J. B., Rhim, J. S., Park, S. C., Kimm, S. W., and Kraus, M. H. (1989 ). Amplification, overexpression, and rearrangement of the c-erbB-2 proto-oncogene in primary

98

Gene Therapy and Molecular Biology Vol 3, page 99

human stomach carcinomas. Cancer Res. 49, 6005-

6009.

Pankov, R., Umezawa, A., Maki, R., et al, ( 1994 ). Keratin

18 activation by Ha-ras is mediated through Ets and Jun binding sites. Pros. Natl. Acad. Sci. USA 91, 873-

877.

Pegram, M. D., Finn, R. S., Arzoo, K., Beryt, M., Pietras, R.

J., and Slamon, D. J. ( 1997 ). The effect of HER-2/neu overexpression on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells. Oncogene 15,

537-547.

Plowman, G. D., Grenn, J. M., Culouscou, J. M., et al, (1993 ). Heregulin induces tyrosine phosphorylation of HER4/p180 erbB-4. Nature 366, 473-475.

Pozzatti, R., McCormick, M., Thompson, M. A., and Khoury, G. (1988 ). The E1A gene of adenovirus type 2 reduces the metastatic potential of ras-transformed rat embryo cells. Mol. Cell Biol. 8, 2984-2988.

Rao, L., Debbas, M., Sabbatini, P., Hockenbery, D., Korsmeyer, S., and White, E. (1992 ). The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins. Pros. Natl. Acad. Sci. USA 89, 7742-7746.

Ruley, H. E. (1983 ). Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature 304, 602-606.

Schneider P. M., Hung, M. -C., Chiocca, S. M., Manning, J., Zhao, X. Y., Fang, K., and Roth, J. A. (1989 ). Differential expression of the c-erbB-2 gene in human small cell and non-small cell lung cancer. Cancer Res.

49, 4968-4971.

Seth, A., Ascione, R., Fisher, R. J., et al, ( 1992 ). The ets

gene family. Cell Growth Diff. 3 , 327-334.

Sgouras, D. N., Athanasiou, M. A., Beal, G. J., Jr., et al, (1995 ). ERF: an ETS domain protein with strong transcriptional repressor activity, can suppress ets- associated tumorigenesis and is regulated by phosphorylation during cell cycle and mitogenic stimulation. EMBO J. 14, 4781-4793.

Sheng, S., Pemberton, P. A., and Sager, R. (1995 ).

Production, purification, and characterization of recombinant maspin proteins. J. Biol. Chem. 269,

30988-30993.

Shi, D., He, G., Cao, S., Pan, W., Zhang, H. Z., Yu., D., and

Hung, M. -C. (1992 ). Overexpression of the c-erbB-

2/neu-encoded p185 protein in primary lung cancer. Mol. Carcinog. 5, 213-218.

Schrier, P. I., Bernards, R., Vaessen, R. T. M. J et al, ( 1983 ).

Expression of class I major histocompatibility antigens switched off by highly oncogenic adenovirus 12 in transformed rat cells. Nature 305, 771-775.

Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J., Ullrich, A, and McGuire, W. L. (1989 ). Studies of the HER-2/neu proto-oncogene in human breast and

ovarian cancer. Science 244, 707-712.

Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich A., and McGuire, W. L. (1987 ). Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235,

177-182.

Sliwkowski, M. X, Schaefer, G., Akita, R. W., et al, ( 1994 ).

Coexpression of erbB2 and erbB3 proteins reconstitutes a high affinity receptor for heregulin. J. Biol. Chem.

269, 14661-14665.

Subramanian, T., Tarodi, B., and Chinnadurai, G. (1995 ). p53-independent apoptotic and necrotic cell deaths induced by adenovirus infection: suppression by E1B 19K and Bcl-2 proteins. Cell Growth Diff. 6, 131-137.

Symonds, H., Krall, L., Remington, L., Saenz-Robles, M., Lowe, S., Jacks, T., and Van Dyke, T. (1994 ). p53- dependent apoptosis suppresses tumor growth and progression in vivo . Cell 78, 703-711.

Tal, M., King, C. R., Kraus, M. H., Ullrich, A., Schlessinger, J,. Givol, D. (1987 ). Human HER2 (neu) promoter: evidence for multiple mechanisms for transcriptional initiation. Mol. Cell. Biol. 7, 2597-601.

Tan, M., Yao, J., and Yu, D. (1997 ). Overexpression of the c- erbB-2 gene enhanced intrinsic metastasis potential in human breast cancer cells without increasing their transformation abilities. Cancer Res. 57, 1199-1205.

Tandon, A. K., Clark, G. M., Chamness, G. C., et al, ( 1989 ).

HER-2/neu oncogene protein and prognosis in breast cancer. J. Clin. Oncol. 7, 1120-1128.

Taylor, J. M., Dupont-Versteegden, E. E., Davies, J. D., Hassell, J. A., Houle, J. D., Gurley, C. M., and Peterson, C. A. (1997 ). A role for the ETS domain transcription factor PEA3 in myogenic differentiation. Mol. Cell. Biol. 17, 5550-5558.

Toikkanen, S., Helin, H, Isola, J., and Joensuu, H. ( 1992 ) Prognostic significance of HER-2 oncoprotein expression in breast cancer: a 30 year follow-up. J. Clin. Onc. 10,

1044-1048.

Tooze, J. (1981 ). DNA tumor viruses. in Molecular biology of tumor viruses, 2nd Edition, ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Trimble, M. S., Xin, J. -H., Guy, C. T. et al, ( 1993 ). PEA3 is overexpressed in mouse metastatic mammary adenocarcinomas. Oncogene 8, 3037-3042.

Tsai, C. M., Yu, D., Chang, K. T., Wu, L. H., Perng, R. P., Ibrahim, N. K., and Hung, M. -C. (1995 ). Enhanced chemoresistance by elevation of the level of p185 neu in HER-2/neu transfected human lung cancer cells. J. Natl. Cancer Inst. 87, 682-684.

Tsai, C. M., Chang, K. -T., Perng, R. -P., Mitsudomi, T., Chen, M. -H., and Gazdar, A. F. (1993 ). Correlation of intrinsic chemoresistance of non-small-cell lung cancer cell lines with HER-2/neu gene expression but not with ras gene mutations. J. Natl. Cancer Inst. 85, 897-

901.

99

Hung and Wang: Targeting HER-2/neu overexpression in cancer gene therapy

van Groningen, J. J., Cornelissen, I. M., van Muijen, G. N., et al, ( 1996 ). Simultaneous suppression of progression marker genes in the highly malignant human melanoma cell line BLM after transfection with the adenovirus-5 E1A gene. Biochem. Biophys. Res. Commun. 225,

808-816.

Wallasch, C., Weiss, F. U., Niederfellner, G., et al, ( 1995 ).

Heregulin-dependent regulation of HER2/neu oncogenic signaling by heterodimerization with HER3. EMBO J.

14, 4267-4275.

Wasylyk, B., Hahn, S. L., and Giovane, A. (1993 ). The Ets family of transcription factors. Eur. J. Biochem. 211,

7-18.

Weiner, D. B., Nordberg, J., Robinson, R., Nowell, P. C., Gazdar, A, Green, M. I., Williams, W. V., Cohen, J. A., and Kern, J. A. (1990 ). Expression of the neu gene- encoded protein (p185neu) in human non-small cell carcinomas of the lung. Cancer Res. 50, 421-425.

Xia, W., Lau, Y. -K.,, Zhang, H. -Z., Liu, A. -R., Kiyokawa, N., Clayman, G. L., Katz, R., L., and Hung, M. -C. (1997 ). Strong correlation between c-erbB-2 overexpression and overall survival of patients with oral squamous cell carcinoma. Clin. Cancer Res. 3, 3-9.

Xin, J. H., Cowie, A., Lachance, P., and Hassell, J. A. (1992 ). Molecular cloning and characterization of PEA3, a new member of the Ets oncogene family that is differentially expressed in mouse embryonic cells. Genes Dev. 6, 481-496.

Xing, X., Miller, S. J., Xia, W., and Hung, M. -C. ( 1997 ) PEA3 as a therapeutic agent for HER-2/neu- overexpressing human cancers. Abstract of the Department of Defense Breast Cancar Research Program Meeting, Washington, DC, 1997. Volume II, 549-550.

Xing, X., Matin, A., Yu, D., Xia, W., Sorgi, F., Huang, L., and Hung, M. -C. (1996 ). Mutant SV40 large T antigen as a therapeutic agent for HER-2/neu-overexpressing ovarian cancer. Cancer Gene Therapy 3, 168-174.

Yamamoto, T. M., Ikawa, S., Akjiyana, T., Semba, K., Normura, N., Miyajima, N., Saito, T., and Toyoshiman, K. (1986 ). Similarity of protein encoded by the human c- erbB-2 gene to the epidermal growth factor receptor. Nature 319, 230-234.

Yan, D. H., Chang, L. S., Hung, M. -C. (1991). Repressed expression of the HER-2/neu/c-erbB-2 proto-oncogene by the adenovirus E1A gene products. Oncogene 6, 343-

345.

Yokota, J., Yamamoto, T., Miyajima, N., Toyoshima, K., Nomura, N., Sakamoto, H., Yoshida, T., Terada, M., and Sugimura, T. (1988 ). Genetic alterations of the c-erbB-2 oncogene occur frequently in tubular adenocarcinoma of the stomach and are often accompanied by amplification of the v-erbA homologue. Oncogene 2, 283-287.

Yu, D., Liu, B., Jing, T., McDommell, T. J., Sun, D., El- Deiry, W. S., and Hung, M. -C. 1998a ). Overexpression of c-erbB2 blocks Taxol-induced apoptosis by

upregulation of p21cip1 which inhibits p34 cdc2 kinase.

Molecular Cell , in press.

Yu, D., Liu, B., Jing, T., Sun, D., Price, J. E., Singletary, S.

E., Ibrahim, N., Hortobagyi, G. N., and Hung, M. -C. (1998b ). Overexpression of both p185c-erbB-2 and p170mdr-1 renders breast cancer cells highly resistant to taxol. Oncogene 16, 2087-2094.

Yu, D., Liu, B., Tan, M., Li, J., Wang, S. -S., and Hung, M. - C. (1996 ). Overexpression of c-erbB-2/neu in breast cancer cells confers increased resistance to Taxol via mdr-

1-independent mechanisms. Oncogene 13, 1359-1365.

Yu, D., Matin, A., Xia, W., Sorgi, F., Huang, L., and Hung, M. -C. (1995 ). Liposome-mediated E1A gene transfer as therapy for ovarian cancers that overexpress HER-2/neu. Oncogene 11, 1383-1388.

Yu. D, Wang, S. S., Dulski, K. M., Tsai, C. -M., Nicolson, G.

L., and Hung, M. -C. (1994 ). c-erbB2/neu overexpression enhances metastatic potential of human lung cancer cells by induction of metastasis-associated properties. Cancer Res. 54, 3260-3266.

Yu, D., Shi, D., Scanlon, M., and Hung, M. -C. (1993a ). Re- expression of neu-encoded oncoprotein counteracts the tumor-suppressing activity of E1A. Cancer Res. 53,

5784-5790.

Yu, D., Wolf, J. K., Scanlon, M., Price, J. E., and Hung, M. - C. (1993b ). Enhanced c-erbB-2/neu expression in human ovarian cancer cells correlates with more severe malignancy that can be suppressed by E1A. Cancer Res. 53, 891-898.

Yu, D., Hamada, J., Zhang, H., Nicolson, G. L., and Hung, M.

-C. (1992a ). Mechanisms of c-erbB2/neu oncogene- induced metastasis and repression of metastatic properties by adenovirus 5 E1A gene products. Oncogene 6, 2263-

2270.

Yu, D., Matin, A., and Hung, M. -C. (1992b ). The retinoblastoma gene product suppresses neu oncogene- induced transformation via transcriptional repression of neu*. J. Biol. Chem. 267, 10203-10206.

Yu, D., and Hung, M. -C. (1991a ). Expression of activated rat neu oncogene is sufficient to induce experimental metastasis in NIH3T3 cells. Oncogene 6, 1991-1996.

Yu, D., Scorsone, K., and Hung, M. -C. (1991b ). Adenovirus Type 5 E1A products acts as transformation suppressors of the neu oncogene. Mol. Cell Biol. 11, 1745-1750.

Yu, D., Suen T. C., Yan, D. H., Chang, L. S., and Hung, M. -C. (1990 ). Transcriptional repression of the neu proto- oncogene by the Adenovirus 5 E1A gene products. Proc. Natl. Acad. Sci. USA 87, 4499-4503.

Zhang, M., Maass, N., Magit, D., and Sager, R. ( 1997a ).

Transactivation through Ets and Ap1 transcription sites determines the expression of the tumor-suppressing gene maspin. Cell Growth Differentiation 8, 179-86.

Zhang, M., Magit, D., and Sager, R. (1997b ). Expression of maspin in prostate cells isregulated by a positive Ets

100

Gene Therapy and Molecular Biology Vol 3, page 101

element and a negative hormonal reponsive element site recognized by androgen receptor. Pros. Natl. Acad. Sci. USA 94, 5673-5678.

Zhang, Y. J., Yu, D. H., Xia, W. Y., and Hung, M. C. (1995 ).

HER-2/neu-targeting cancer therapy via adenovirus- mediated E1A delivery in an animal model. Oncogene

10, 1947-1954.

Zou, Z., Anisowicz,A., Hendrix, M. J., et al, (1994 ).

Identification of a novel serpin with tumor suppressing activity in human mammary epithelial cells. Science

263, 526-529.

101