[CANCER RESEARCH 53, 5784-5790, December 1, 19931

Reexpression of neu-encoded Oncoprotein Counteracts the Tumor-suppressing but

not the Metastasis-suppressing Function of E 1 A 1

D i h u a Yu, 2 D a r e n Shi , 3 M i c h a e l S c a n l o n , a n d M i e n - C h i e H u n g 4

Department of Tumor Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT

By transfecting the adenovirus 5 E I A gene into neu-transformed

NIH3T3 cells, we previously showed that E I A can dramatically repress neu-encoded p185 expression and, concomitantly, suppress the features of transformation and metastasis of neu+E1A transfectants. From these re- suits we concluded that suppression of transformation and metastasis by E1A in neu-transformed cells may be through repression of neu gene expression. However, EIA has recently been shown to also repress the transformation features of other human cancer cells that do not overex- press neu. This observation raised a possibility that repression of neu gene expression in our neu+E1A cells might not be the only mechanism for transformation and metastasis suppression. To study whether other mo- lecular mechanisms might be involved in suppression of transformation and metastasis by E1A in our neu 4-E1A cells, we reexpressed p185 onco- protein in the neu § cells by transfecting them with a plasmid con- taining activated rat neu complementary DNA and we examined whether E I A can suppress transformation and metastasis when the neu-encoded

p185 protein is reexpressed. All the features of transformed cells including cell morphology, DNA synthesis rate, colony formation in soft agar, and tumorigenicity in nu/nu mice were restored in the cell lines that reex- pressed neu. In addition, the levels of neu reexpression corresponded to the degree of malignant transformation. However, the in vivo metastatic tumor formation by these p185 reexpressing cells was still significantly inhibited by E1A. When metastasis-associated properties were examined in the cell lines that reexpressed p185, we found that cell motility was recovered by reexpression of p185 to the degree corresponding to the p185 reexpression level, but secretion of membrane-degrading gelatinases and invasion through the basement membrane preparation Matrigel by these cells were still inhibited by E1A. The data demonstrated that reexpression of p185 in neu+E1A cells can counteract the tumor-suppressing function of E I A but not completely recover the neu-induced metastatic phenotype. We conclude from these results that (a) repression of neu oncogene expression was indeed the molecular mechanism by which E1A suppressed tumor formation in neu-transformed 3T3 cells,~and (b) suppression of metastasis by E I A in neu-transformed 3T3 cells was via multiple molecular mecha- nisms in addition to repressing neu. Our model system clearly demon- strated that tumorigenicity and metastasis are related but separable phenomena.

INTRODUCTION

The neu oncogene (also known as c-erbB-2, HER-2, and NGL )

encodes a Mr 185,000 transmembrane glycoprotein (p185) that is homologous to the epidermal growth factor receptor (1-5). The rat neu

oncogene activated by a single point mutation in the transmembrane domain of the p185 protein can transform fibroblasts (6--8). Our previous studies demonstrated that amplification of the normal rat neu

Received 6/16/93; accepted 9/21/93. The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

t This research was in part supported by Department of Health and Human Services Cancer Center Core Support Grant CA-16672 from the National Cancer Institute.

2 Supported by Department of Health and Human Services Grant RO3-CA54989 from the National Cancer Institute. To whom requests for reprints should be addressed, at Department of Tumor Biology, Box 79, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.

3 Visiting Scientist from Department of Pathology, Cancer Hospital, Shanghai Medical University, Shanghai, People's Republic of China.

4 Supported by Department of Health and Human Services Grant R01-CA58880 from the National Cancer Institute and Smokeless Tobacco Research Council Grant 0287.

protooncogene can facilitate oncogenic activation by the same point mutation (9). Amplification or overexpression of the normal human. neu protooncogene is a frequent event in many types of human can- cers (10-17) and correlates with the number of lymph node metasta- ses in breast cancer patients and with poor prognosis in both breast and ovarian cancer patients (18, 19). In addition, we recently demon- strated that the neu oncogene can induce metastasis in mouse 3T3 cells by promoting multiple steps in the metastatic cascade (20, 21). Therefore, both mutational activation of the rat neu gene and overex- pression of the normal human neu gene are involved in the process of

tumorigenesis and cancer metastasis. The adenovirus E1A proteins are multifunctional transcriptional

regulators that can activate or repress cellular genes as well as adeno- viral genes (22-25). We have shown previously that the adenovirus 5 E I A gene product can repress neu oncogene expression (26, 27) and suppress the malignant transformation and tumor metastasis of mouse NIH3T3 cells transformed with mutation-activated rat neu oncogene (21, 28); therefore, we proposed that E I A is a transformation and metastasis suppressor for neu oncogene-transformed cells. However, it was recently shown that E I A also has an antioncogenic effect in certain human tumor ceils that do not overexpress neu (29). These observations raise an interesting question: is suppression of transfor- mation and metastasis by E I A in neu-transformed NIH3T3 cells through repression of neu gene expression alone or through multiple parameters, including repression of neu and other properties intrinsi- cally associated with E1A? To distinguish these two possibilities, we restored neu gene expression by transfecting into EIA-express ing

neu+E1A cells a plasmid that contains a mutation-activated rat neu

cDNA 5 driven by a heterologous promoter LTR that is not repressed by E I A . Reexpression of neu-encoded oncoprotein in these cells, which still express E I A , restored all the transformation phenotypes tested. However, the metastatic potential of these cells was still sig- nificantly inhibited in vivo and those metastasis-associated properties such as gelatinase IV activity and invasion ability were inhibited in

vitro. Therefore, the mechanism by which E1A functions as a trans- formation suppressor gene in neu-transformed NIH3T3 cells is indeed due to repression of neu gene expression, whereas multiple molecular mechanisms in addition to repressing neu are involved in E I A sup- pression of metastasis in neu-transformed 3T3 cells. Our model sys- tem provides evidence that tumorigenicity and metastasis are related

but separable phenomena.

MATERIALS AND M E T H O D S

Cell Lines and Culture. The B-E1A1 and B-EIA2 cell lines were estab- lished and maintained as described previously (28). B-neo cells are neu- transformed NIH3T3 cells (B104-1-1 cells) transfected with the neomycin resistance marker gene pSV2neo (30). Cells were grown in Dulbecco's modi- fied Eagle's medium:-12 medium (1:1); (GIBCO) supplemented with 10% calf serum and 500/xg/ml G418 in a humidified atmosphere containing 95% air and 5% CO2 at 37~

DNA Transfection. All DNA transfections were carded out using the modified calcium-phosphate precipitation procedure (31). B-E1A1 and B-E1A2 cells (10 6 ceils in 10-cm dishes) were seeded 24 h before transfection.

5 The abbreviations used are: cDNA, complementary DNA; LTR, long terminal repeat; HSE-CM, hepatic sinusoidal endothelial cell-conditioned medium; HPF, high power field.

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T U M O R - A N D M E T A S T A S I S - S U P P R E S S I N G F U N C T I O N O F E I A

The cells were then cotransfected with 10 ~g of pDOL-E plasmid DNA, which contains a mutation-activated rat neu cDNA driven by a heterologous promoter LTR in the pDOL expression vector (32), and 1 /xg of pSV2-hygro plasmid DNA, which carries the hygromycin resistance marker gene (33) (to generate the BE1A.D cell lines) or with 10 /xg of pGEM carrier DNA and 1 /xg of pSV2-hygro DNA (to generate BEIA.Hy cell lines). Approximately 10 h after transfection, the cells were washed and cultured in fresh medium for 24 h and then split 1:10. The cells were then grown in selection medium contain- ing 150 /~g/ml of hygromycin B for 4-6 weeks, after which individual hy- gromycin B-resistant colonies were cloned and expanded to mass culture.

Immunobiotting. Immunoblot analyses were performed according to pub- lished techniques (34) as "previously described (27, 35). The primary mono- clonal antibodies used were c-neu-Ab-3 against the neu-encoded p185 protein (Oncogene Science, Inc., Manhasset, NY) and M73 against E1A proteins (a generous gift from Dr. L-S. Chang, Ohio State University, Columbus, OH). The blots were then incubated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Bio-Rad Laboratories, Richmond, CA) and the proteins were visualized with enhanced chemiluminescent Western blotting detection reagents (Amersham, Arlington Heights, IL).

[3H]Thymidine Incorporation Assay, Colony Formation in Soft Aga- rose, and Tumorigenicity Assay. These assays were performed as described previously (28, 36).

Experimental Metastasis Assay, Chemotaxis Assay, Zymography of Gelatinolytic Activity, in Vitro Chemoinvasion Assay. These assays were performed as described previously (20, 21).

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E,A[ Fig. 1. Immunoblot analysis for the neu-encoded p185 and E1A proteins in the cell

lysates of the indicated cell lines, respectively. A 100-/zg protein from each sample was electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to nitrocellulose. (.4) The filters were incubated with the primary antibody c-neu-Ab-3 against p185. (B) The filters were incubated with the primary antibody M73 which recognizes E1A proteins. Ordinate, positions of the p185 and E1A proteins.

RESULTS

Reexpression of neu-encoded p185 in E I A - e x p r e s s i n g B-E1A Cell Lines. B104-1-1, the mutation-activated rat genomic neu onco- gene-transformed NIH3T3 cell line, contains 10-20 copies of the neu

gene (7), expresses high levels of neu-encoded p185 protein, and is a highly transformed cell line that can induce tumors when injected s.c. into nu/nu mice (9) and induce experimental metastases when injected into the tail veins of nu/nu mice (20). In contrast, the EIA-expres s ing

B-EIA1 and B-EIA2 cell lines, which were established by introduc-

ing E I A gene into B104-1-1 cells, express barely detectable levels of p185 due to transcriptional repression of neu gene expression by E I A

and have dramatically reduced transformation potential and inhibited metastatic potential (21, 28). These observations indicate that E1A

may function as a transformation and metastasis suppressor for neu

oncogene-transformed cells by repressing neu-encoded p185 expres- sion. To examine whether E I A can repress transformation and metas- tasis of neu-transformed cells when neu-encoded p185 is expressed,

we intended to restore neu gene expression in the n e u + E i A (B-E1A) cells and test whether reexpression of p185 could counteract trans- formation and metastasis suppression functions of E I A . To achieve this, B-EIA1 and B-E1A2 cell lines were chosen as recipient cells for transfection with pDOL-E plasmid, which contains an activated rat

neu cDNA driven by a heterologous promoter LTR (32) that is not repressed by E I A (27). We cotransfected into these cells the pSV2- hygro plasmid, which carries the hygromycin resistance marker gene for selection of the stable transfectants (33). Individual hygromycin B-resistant clones were selected and expanded into cell lines that were designated BE1A.D1 and BEIA.D2, etc. pSV2-hygro plasmid was introduced into the B-E1A1 and B-EIA2 cells to generate the BEIA1.Hy and the BE1A2.Hy control cell lines. To identify those BE1A.D transfectants that actually reexpressed high levels of neu-

encoded p185, immunoblot analyses of the BEIA.D transfectants with c-neu-Ab3 anti-p185 antibodies were performed. We identified two cell lines from each of the B-EIA1 and B-EIA2 parental lines that reexpressed p185, i.e., BE1A1.D1, BE1A1.D2, BE1A2.D1, and BEIA2.D2 (Fig. 1A). In comparison with the B.neo cell line, which is a neu-transformed cell line generated by transfecting B104-1-1 cells with the neomycin resistance marker gene pSV2neo (30) and was used as a positive control for p185 expression, the p185 protein levels in

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Fig. 2. (A) Increased [3H]thymidine incorporation of the BE1A.D cell lines compared with BE1A.Hy control cell lines. The cells were incubated for 2 h with addition of 1/xCi [3H]thymidine/well at the indicated time points after plating to label those cells that were synthesizing DNA. The radioactivity in each sample was measured with a scintillation counter. Columns, average cpm calculated from five replicate samples; bars, SD. The experiments were repeated three times. (B) Increased colony formation of BE1A.D cell lines compared with the control BE1A.Hy cells. Soft agar assays were performed as described in "Materials and Methods." Columns, number of soft agar colonies from each cell; bars, SD. The experiments were repeated twice.

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TUMOR- AND METASTASIS-SUPPRESSING FUNCTION OF E1A

BE1A.D transfectants were as follows: BE1A1.D2 > B.neo > BE1A1.D1 = BEIA2.D1 > BEtAI.D2 > BEIAHy. To confirm that the restored p185 expression was not due to loss ofEIA gene expres- sion, immunoblot analyses with M73 anti-E1A antibodies were per- formed (Fig. 1B). All four of the BEIA.D transfectants expressed levels of E1A proteins similar to those of the BE1A1.Hy and BE1A2.Hy transfectants and the parental B-E1A1 and B-E1A2 cell lines, whereas the B.neo cell line, as expected, did not express E1A protein (Fig. 1B). Thus we have established stable cell lines that reexpress neu-encoded p185 in the presence of EIA expression.

Reexpression of p185 in neu +E1A Cells Can Counteract the Tumor-suppressing Function of E1A. To determine whether re- stored expression of p185 in BEIA.D cells could antagonize the transformation suppression function of EIA and lead to the recurrence of malignant transformation, we examined transformation phenotypes of B.neo, BE1A.Hy, and BE1A.D ceils both in vitro and in vivo, including DNA synthesis, anchorage-independent growth, and the ability to induce tumors in nu/nu mice.

We first noted the morphological difference between BEIA.Hy and BE1A.D cells in culture. BEIA.D cells had a highly transformed morphology as did B.neo cells, whereas BE1A.Hy cells remained flat like the parental B-EIA cells (data not shown). This suggested that the fiat morphology of B-E1A cells can be reversed by reexpression of neu-encoded p185. We next determined the DNA synthesis levels by measuring [3H]thymidine incorporation into DNA in these cells. As expected, the neu-transformed B.neo cells had a high [3H]thymidine incorporation level similar to that of B104-1-1 cells (data not shown), and the control BE1A.Hy cells had very low levels of [3H]thymidine incorporation like the B-E1A parental cells (Fig. 2A). Although the [3H]thymidine incorporation levels of the four BE1A.D cell lines differed among themselves, the DNA synthesis rates were generally

Table 1 Experimental metastasis in neu cDNA transfectants that reexpressed high levels of p185

Cell line

Experimental metastasis

Frequency No. of lung nodules p185 Mice with metastases neu /no. of mice given injections Range Av.

B. neo + + + a 6/6 6--28 21 BE1A1.Hy --- 0/6 0 0 BE1A1.DI + + + 1/6 0--1 0 BE1A1.D2 + 1/6 0-1 0 BE1A2.Hy ___ 0/5 0 0 BE1A2.DI + + + 3/6 1-5 2 BE1A2.D2 + + + + 4/7 3--i7 6

,+++, p185 level in B. neo control lines; +, one-third of the p185 level in B. neo; _% below one-sixth of the p185 level in B. neo cells; +++% p185 level is higher than that in the B. neo cells.

much higher than those of the control BEIA.Hy cell lines, which correlated with their restored p185 level. These data suggest that reduced DNA synthesis in B-E1A cells can be restored by reexpres- sion of neu-encoded p185.

We then examined the transformation feature of anchorage-inde- pendent growth in soft agar, which is an indicator of the transforming potential of transformed cells in vitro. As shown in Fig. 2B, BE1A.D ceils had a high efficiency of colony formation in 0.35% soft agar, as did B.neo-transformed cells, significantly higher than that of the BE1A.Hy control cells. Therefore, restoring p185 expression in BE1A.D cell lines also restored the colony-forming efficiency that was originally reduced in B-E1A cell lines by E1A expression.

A more stringent parameter of transformation potential is in vivo tumorigenicity after s.c. injection of cells into nu/nu mice. Therefore we also compared the tumorigenicity of these cell lines by testing the ability of s.c. injected B.neo, BE1A.Hy, and BE1A.D cells to form tumors in nude mice (Fig. 3). The two BE1A.Hy cell lines did not

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Days Fig. 3. Tumor formation by B.neo, BE1AI.Hy, BEIA2.Hy, BEIA1.DI, BEIA1.D2, BEIA2.D1, and BEIA.2.D2 cells. Viable cells (105) cells were injected s.c. into right and left

flanks of female homozygous nude mice. Six injections were used for each cell line. Tumor formation was scored on the indicated days and tumor volumes were estimated to be the product of three caliper measurements.

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Fig. 4. Motility of B.neo, BE1A1.Hy, BEIA2.Hy, BEIA1.D1, BE1A1.D2, BE1A2.D1, and BE1A2.D2 cell lines. Migrated cells were counted in at least four fields. X200 HP. The number of cells that migrated in response to Dulbecco's modified Eagle's medi- um:F-12 medium alone was subtracted from each sample; the assays [• (bars)] were done in triplicate.

induce tumors until 56 days after injection. On the other hand, small solid tumors had developed after 12 days in mice given injections of B.neo-transformed cells and BE1A1.D1, BE1A2.D1, and BE1A2.D2 cells. BE1AI.D2 cells induced tumors in nude mice 21 days after injection, probably because of the lower level of p185 in this cell line, and the tumor volume at 56 days was 2360 __. 820 (SD) mm 3. Notably, at every time point the average tumor volumes of mice given injec- tions of BE1A2.D2 cells were larger than those of mice given injec- tions of B.neo-transformed cells (Fig. 3), which correlates with the higher p185 expression level in this cell line. Taken together, these data indicate that the reexpression of neu-encoded p185 can retrans- form B-E1A cells and restore their tumorigenic potential despite the expression of E1A proteins in these cells. In addition, the level of neu expression reflected the degree of malignancy in the BE1A.D cell lines.

To examine whether the tumors induced by injection of BE1A.D cells were biochemically similar to that induced by injection of non- E1A-expressing B.neo cells, we excised the tumors from mice given injections of B.neo and BE1A.D cells and examined them for the neu-encoded p185 expression and E1A expression by immunoblot analysis. The p185 protein was readily detectable in all tumor samples, and the p185 levels in tumors were higher than those of B.neo and BE1A.D cells originally injected (data not shown). All tumors induced by injection of BE1A.D cell lines produced readily detectable levels of E1A protein comparable to those of the control BE1A.Hy cell lines (data not shown). Therefore, the increased tu- morigenic potential of BE1A.D cells was indeed due to the restored expression of neu-encoded p185 by transfection of neu cDNA but not to loss of E1A expression.

EIA Can Still Function as a Metastasis Suppressor in neu +E1A cells when neu-encoded p185 Is Reexpressed. The formation of experimental metastatic tumor nodules requires most of the charac- teristics essential for metastasis from a primary tumor site. To inves- tigate whether E1A can inhibit the metastatic potential of BE1A.D cell lines, we compared the ability of B.neo, BE1A.Hy, and BEIA.D cells to induce experimental metastases (37, 38). Five to 7 mice/ experimental group were inoculated with 2 x 105 B.neo, BE1A.Hy, or BE1A.D cells in 0.1 ml phosphate-buffered saline via the lateral tail veins of nude mice at day 0. The mice were killed 3 weeks after injection, and the numbers of pulmonary and extrapulmonary metas-

tases were counted (Table 1). As expected, all of the mice given injections of neu-transformed B.neo cells developed metastatic lung nodules, and the two BE1A.Hy transfectants were unable to produce experimental metastatic lung nodules. Interestingly, the mice given injections of the BE1A1.D1 and BE1A1.D2 lines had very low inci- dences of lung colonization and the mice given injections of the BE1A2.D1 and BE1A2.D2 lines, which express lower levels of E1A protein than BE1A1.D1 and BE1A1.D2 lines, had approximately 50% incidences of lung colonization; and the number of lung nodules in those mice that had metastatic tumors was significantly lower than mice given injections of B.neo cells. The differences between B.neo cells and BEIA.D cells in inducing experimental metastasis indicate that E1A can still significantly inhibit the metastatic potential of BEIA.D cells even though the neu-encoded p185 is reexpressed.

Tumor metastasis is a complex process involving a sequential series of critical steps that include cellular-adhesive interactions, chemotac- tic responses, solubilization of specific basement membrane compo- nents, and invasion of basement membrane (39-41). To investigate which of these steps in the metastatic cascade can be inhibited by E1A in the p185 reexpressing BE1A.D cells that may account for the inhibited metastatic potential in the above in vivo experimental me- tastasis assays, we compared all these metastasis-associated properties among the B.neo, BE1A.Hy, and BE1A.D cells.

Directed cell movement (chemotaxis) mediated by tissue-specific chemotactic factors is important in malignant cell invasion and me- tastasis. Previously, we showed that neu-transformed B104-1-1 cells have higher chemotactic activities than parental NIH3T3 cells and expression of E1A in B104-1-1 cells can inhibit their chemotactic activity (20, 21). To examine whether E1A can still inhibit chemotaxis of BE1A.D cells that reexpressed p185, we measured the migration of the B.neo, BE1A.Hy, and BE1A.D cells to the chemoattractant HSE-CM (42, 43). Approximately 40 B.neo cells/HPF migrated through the filter during the 6-h assay using HSE-CM as chemoat- tractant, whereas only - 10 BE1A.Hy cells/I-IPF were observed on the underside of the filter under the same assay conditions (Fig. 4). However, all the BE1A.D transfectants showed increased chemotactic activity to HSE-CM compared to the BE1A.Hy cell line (range, 15-45 cells/HPF). Interestingly, the chemotactic activity or cell motility of the p185 reexpressing BE1A.D cells correlates very well with their reexpressed p185 level, in that the BE1A1.D2 cells that expressed lowest p185 among the 4 BE1A.D cell lines showed the lowest cell motility; while the BE1A2D.2 cells that contained highest p185 level among the 4 BE1A.D cell lines had the highest cell motility. These results demonstrate that reexpression of p185 in the BEIA.D cells can restore the chemotactic activity of these cells; i.e., E1A did not inhibit in vivo experimental metastasis by inhibiting chemotactic activity of these cells.

It has been shown that gelatinases (type IV collagenases) are the important participants in the solubilization of basement membrane collagen during tumor cell invasion and metastasis (39, 44, 45). We have previously demonstrated that neu-transformed B104-1-1 cells have increased gelatinase activities compared with parental NIH3T3 cells and expression of EIA in B104-1-1 cells can inhibit their gelati- nase enzyme activities (20, 21). To determine the activities of gelati- nases in the p185-reexpressing BE1A.D cells, we performed zymo- graphic analysis on the conditioned media from the BE1A.D cell lines and compared their gelatinase activities with that of B.neo and BE1A.Hy cell lines (Fig. 5). High amounts of Mr 68,000 and Mr 64,000 gelatinase were detected in conditioned medium from the neu-transformed B.neo cells, whereas dramatically inhibited Mr 64,000 and nondetectable amounts of Mr 68,000 gelatinase activities were observed in conditioned medium from the two EIA-expressing BE1A.Hy cell lines, which is consistent with our previous observation

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that expression of E1A in the B-E1A cell lines can lead to a 50-70% reduction in gelatinolytic activity of the Mr 68,000 and M~ 64,000 gelatinases in neu-transformed cells (20, 21). Interestingly, all four of the p 185-reexpressing BE1A.D cell lines contained similar low levels of Mr 64,000 and nondetectable amounts of Mr 68,000 gelatinases like that of the BEIA.Hy cell lines. The data indicate that E1A can inhibit the gelatinase activity in the p185-reexpressing BE1A.D cells which may account for their inhibited metastatic potential (cf. Table 1 and Fig. 5).

The malignant cells must extravasate from the circulation, invade the basement membrane, and colonize distant sites to be metastatic. Since tumor cell invasion is important in these events, we next com- pared the invasiveness of B.neo, BE1A.Hy, and BE1A.D cells to determine whether reexpression of p185 in the BE1A.D cells can lead to an increase of cell invasion or if E1A can still inhibit the invasive properties in BE1A.D cells. In vitro invasion assays can be used to quantitate the invasive potential of B.neo, BE1A.Hy, and BE1A.D cells, and we used the assay of Albini et al. (46) which uses a layer of reconstituted basement membrane (Matrigel) on the surface of porous polycarbonate filters present in the transwell unit as the invasion substrate (42, 46, 47). HSE-CM was placed in the lower chamber beneath the filter as a chemoattractant to stimulate penetration of the cells. The neu-transformed B.neo cells showed very high rates of invasion similar to that of B104-1-1 cells (20), whereas the two BE1A.Hy transfectants showed significantly reduced abilities to in- vade the Matrigel layer (Fig. 6). Notably, we found that the invasive- hess of the p185-reexpressing BE1A.D cells, although slightly in- creased compared with the invasion found with BE1A.Hy cells, was significantly inhibited compared with that of non-E1A-expressing neu-transformed B.neo cells (Fig. 6). These results demonstrate that E1A can still inhibit the invasiveness of p185 reexpressing BE1A.D cells in vitro which correlates with the lower in vivo metastatic potential of BEtA.D cells.

DISCUSSION

Although we have shown previously that E I A products can repress neu gene expression and act as transformation and metastasis sup- pressors in the NIH3T3 cells transformed with mutation-activated rat neu (21, 27, 28), the precise molecular mechanism by which this occurs is complicated by the fact that E1A proteins are well-known multifunctional transcriptional regulators that induce many changes in cellular gene expression (48). EIA has been reported to convert cer- tain types of human cancer cells that do not overexpress neu to an

untransformed state (29), and E1A can also suppress transformation and metastasis of r a s + E 1 A primary rat cells (49, 50). By introducing neu cDNAs that cannot be repressed by E1A into E1A-expressing cells and examining their transformation potential, we have now clearly demonstrated that reexpression of p185 can still transform NIH3T3 even in the presence of E1A proteins. Therefore, transformation sup- pression of neu-transformed NIH3T3 cells by E IA is indeed due to repression of neu oncogene expression, but not to irreversible effects on downstream signals in the neu-induced transformation pathway that may be shared by other transformation (e.g., ras) pathways. The molecular mechanisms for the antioncogenic effect of E IA on human tumor cells that do not overexpress neu have not been deduced (29). It is interesting to note that one of the human cancer cell lines, HT1080, was shown to secret the ligand for neu-encoded p185 (51) and to express a low level of p185. If an autocrine pathway for neu-encoded p185 and its ligand is involved in the transformation of HT1080 cells, it is conceivable that E I A may repress p185 expression and block this pathway. Thus, the transformation-suppression activity of E1A in HT1080 might still be due to repression of neu. It is, of course, possible that E IA may also function as a transformation- suppressor gene independent from repressing neu expression. For example, E IA has been shown to induce apoptosis that may lead to reduced transformation (52) and functional interaction of E I A with the cyclic AMP-responsive transcription factor and other cellular proteins has been proposed to play a role in ElA-mediated transformation suppression in human cancer cells (29, 53, 54). Further studies are certainly required to determine other possible molecular mechanisms of E1A-mediated antioncogenic effects in those human tumor cells that do not overexpress neu. Nevertheless, our current studies and previous reports (20, 21, 27, 28) have provided solid experimental evidence for one mechanism of EIA-mediated antioncogenic effects, repression of neu oncogene expression. The biological significance of the antioncogenic effect of E IA via repression of neu expression has been demonstrated in our recent studies on p185-overexpressing hu- man ovarian cancer cells (36). In this study, we have shown that enhanced p185 expression in human ovarian cancer cells correlates with more severe malignancy. However, when we introduced E I A in to the highly malignant p185-overexpressing human ovarian cancer cells, we found that the EIA-expressing ovarian cancer cell lines had dramatically decreased p185 expression and significantly reduced ma- lignancy, including decreased tumor formation and tumor dissemina- tion after injection into nude mice. These data, in addition to the

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TUMOR- AND METASTASIS-SUPPRESSING FUNCTION OF E1A

results presented in this paper, strongly support the notion that E1A can function as a tumor suppressor gene for p185-overexpressing cancer cells via repression of neu expression.

It has been shown that expression of the second exon of the EIA gene can suppress transformation in the ras+ E IA primary baby rat kidney cells (50). We are currently localizing the regions required for the E1A tumor-suppressing function and it will provide us additional information on whether there are common tumor-suppressing domains for neu- and ras-induced tumorigenicity or EIA suppressed neu- induced tumorigenicity and ras-induced tumorigenicity via different molecular mechanisms that target different pathways.

In contrast to the effect on transformation, reexpression of p185 in n e u + E 1 A cells did not lead to a dramatic increase of experimental metastatic tumor formation; i.e., EIA can still inhibit the metastatic potential of BE1A.D cells even though the neu-encoded p185 is reexpressed. The results indicate that EIA suppressed metastasis via multiple molecular mechanisms in addition to repressing-p185 ex- pression. Our zymography results showed that gelatinase activities of the p185-reexpressing BE1A.D cells are still inhibited by EIA in the B.E1A.D cells and inhibition of gelatinase gene expression in human tumor cell and rat embryo fibroblast was shown to accompany reduc- tion in metastatic properties (55, 56). Thus, inhibition of gelatinase activities in our BE1A.D cells may be one of the important mecha- nisms by which EIA suppressed invasion and metastasis by BE1A.D cells. Previously, EIA was shown to inhibit gene expression of gelati- nase at the transcriptional level in human colon cancer cells (55). It is conceivable that E1A may also suppress this protease expression at the transcriptional level in BE1A.D cells.

It has been proposed that invasion and metastasis require activation of a set of effector genes above those required for tumorigenesis and some of the metastasis effector genes can be regulated independently from those that confer tumorigenicity (57). For example, certain types of cells are capable of being transformed by ras, but do not metasta- size, presumably due to a deficiency in some of the metastasis effector proteins (58). The differential effect of reexpression of p185 on trans- formation and metastasis in the n e u + E 1 A 3T3 cells, i.e., these cells can be transformed by reexpression of p185 but their metastatic po- tential are still inhibited by EIA, clearly demonstrates that tumorige- nicity and metastasis are interrelated but separable phenotypes and metastasis indeed requires activation of signals additional to those needed for tumorigenicity.

Several proteinases including gelatinases (56, 59, 60), cathepsin L (61), and motility-associated cytokines (62) have been indicated as candidate effector proteins associated with metastasis in ras transfec- tion models. Here we demonstrate that in the p185 reexpressing BE1A.D cells, gelatinase activities were suppressed by EIA which correlates with inhibited metastatic potential of these cells. These results together with previous observations indicate that gelatinase is one of the metastasis effector proteins that can be regulated by EIA in both neu-induced and ras-induced metastasis but that has no effect on tumorigenicity. In addition to suppressing the metastatic potential of neu-transformed 3T3 ceils and ras-transformed rat embryo fibroblast cells, E1A has been shown to inhibit certain properties related to the metastatic phenotype of human colon cancer cells (55). These obser- vations suggest that E1A may be a metastasis suppressor gene for diverse types of tumor cells.

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1993;53:5784-5790. Cancer Res   Dihua Yu, Daren Shi, Michael Scanlon, et al.  

E1AFunction of Tumor-suppressing but not the Metastasis-suppressing

-encoded Oncoprotein Counteracts theneuReexpression of

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