Proc. Nall. Acad. Sci. USA
`Vol. 84, pp. 7159-7163, October 1987
`Cell Biology
`
`Increased expression of the putative growth factor receptor
`p185I
`R2 causes transformation and tumorigenesis of NIH 3T3 cells
`ROBERT M. HUDZIAK*, JOSEPH SCHLESSINGERt, AND AXEL ULLRICH*
`*Department of Developmental Biology, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080; and tBiotechnology Research
`Center, Meloy Laboratories, 4 Research Court, Rockville, MD 20850
`
`Communicated by Hilary Koprowski, July 13, 1987
`
`ABSTRACT
`The HER2 gene encodes a cell-surface glyco-
`protein with extensive homology to the epidermal growth factor
`receptor. Recently it was found to be amplified in about 30%
`of primary human breast malignancies. In experiments de-
`signed to assess the role of the HER2 gene in oncogenesis, we
`found that overexpression of unaltered HER2 coding sequences
`in NIH 3T3 cells resulted in cellular transformation and
`tumorigenesis.
`
`The HER2 gene encodes a transmembrane glycoprotein with
`extensive structural homology to the human epidermal
`growth factor (EGF) receptor and the chicken oncogene
`v-erbB (1-3). Chromosomal mapping and sequence compar-
`ison strongly suggest that the HER2 gene product and the
`ethylnitrosourea-activated, rat neuroblastoma oncogene neu
`represent species variants of the same polypeptide (4). The
`neu oncogene encodes a 185-kDa cell-surface glycoprotein
`that possesses intrinsic tyrosine-specific kinase activity that
`is likely to be activated by an as yet unidentified ligand (5, 6).
`Comparison of the transforming neu oncogene sequence with
`its normal rat protooncogene counterpart suggested that a
`point mutation in the transmembrane domain resulting in
`substitution of a valine residue by glutamate unmasked the
`transforming potential of this putative growth factor receptor
`(7). Analogously, structural alterations have converted nor-
`mal genes coding for the receptors for macrophage colony-
`stimulating factor type 1 and EGF into v-fms (8) and v-erbB
`(9) oncogenes, respectively.
`Southern analysis of primary human tumors and estab-
`lished tumor-derived cell lines revealed amplification and in
`some cases rearrangement of the EGF receptor gene. Am-
`plification was particularly apparent in squamous carcinomas
`(10, 11) and glioblastomas (12). The HER2 gene was also
`found to be amplified in a human salivary gland adenocarci-
`noma (3), a mammary gland carcinoma (2), and a gastric
`cancer cell line (13). Recently, Slamon et al. (14) demon-
`strated that about 30% of primary human breast carcinoma
`tumors contained an amplified HER2 gene. Although a few
`sequence rearrangements were detected, in most tumors
`there were no obvious differences between amplified and
`normal HER2 genes. Furthermore, amplification of the
`HER2 gene correlated significantly with the prognosis of the
`disease and the probability of relapse.
`To investigate the significance of the correlation between
`overexpression and cellular transformation as it has been
`observed for protooncogenes c-mos (15) and c-Ha-rasl (16),
`we employed a HER2 expression vector and a selection
`scheme that permitted sequence amplification after transfec-
`tion of mouse NIH 3T3 cells. We report here that amplifi-
`cation of the unaltered HER2 gene in NIH 3T3 cells leads to
`overexpression ofp185HER2 as well as cellular transformation
`and tumor formation in athymic mice. These findings, in
`
`combination with the results of Slamon et al. (14), suggest
`that mere amplification of the HER2 gene and resulting
`overexpression of its product may play a crucial role in the
`genesis and development of some types of human cancer.
`
`MATERIALS AND METHODS
`Expression Plasmids. The mammalian expression vector
`CVN (17) contained expression units for mouse dihydrofolate
`reductase (DHFR) cDNA (18) and the bacterial neomycin
`phosphotransferase (neo) gene (19), both under simian virus
`40 early promoter control. Transcription of a 4.4-kilobase-
`pair Sal I-Dra I HER2 fragment containing the full-length
`HER2 coding region (1) was driven by the Rous sarcoma
`virus (RSV) long terminal repeat promoter (LTR). The
`poly(A) site was provided by the 3' untranslated sequence of
`the hepatitis B virus surface antigen gene (20). The control
`CVN plasmid was identical but lacked cDNA sequences
`downstream from the RSV LTR.
`Cell Culture. NIH 3T3 cells were cultured in a 1:1 mixture
`of Dulbecco's modified Eagle's medium and Ham's nutrient
`mixture F-12 supplemented with glutamine (2 mM), penicillin
`(100 units/ml), streptomycin (100 ,g/ml), and 10% HyClone
`(Logan, Utah) calf serum in a humidified incubator under 5%
`CO2 in air atmosphere.
`Transfections and Amplification. Plasmid DNA was intro-
`duced into mammalian cells by the calcium phosphate
`coprecipitation method (21). Half-confluent plates of cells (60
`mm) were exposed to 5 ,ug of plasmid DNA in 1 ml of
`precipitate for 6-8 hr. After a 20% (vol/vol) glycerol shock
`(22), the cells were fed with nonselective medium. Two days
`later, they were passaged into selective medium containing
`Geneticin (G418) at 400 ,ug/ml.
`Clones were picked using glass cloning cylinders with
`petroleum jelly for the bottom seal. Colonies arising from
`transfected cells selected for growth in G418 were picked,
`expanded, and subcultured into medium containing 7%
`dialyzed fetal bovine serum in place of 10% calf serum and the
`appropriate concentration of methotrexate for plasmid am-
`plification (23). The dialysis step removes trace amounts of
`purines and pyrimidines present in serum that decrease the
`efficiency of the methotrexate selection. To apply selective
`pressure, stepwise increasing concentrations of methotrex-
`ate were used with a final concentration of 400 nM. To avoid
`enriching for spontaneously transformed cells, cells were
`kept subconfluent. An additional control was to amplify the
`CVN neo-DHFR vector without the HER2 cDNA insert in
`the NIH 3T3 recipient cell line.
`Immunoprecipitations and Labeling. The G-H2CT17 anti-
`body recognizing the C-terminal 17 amino acids of HER2 was
`prepared in rabbits using a synthetic peptide conjugated with
`soybean trypsin inhibitor.
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`Abbreviations: DHFR, dihydrofolate reductase; R, resistance; EGF,
`epidermal growth factor; RSV, Rous sarcoma virus; LTR, long
`terminal repeat.
`
`7159
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`
`Cells were harvested by trypsinization and counted by
`Coulter Counter, and 1.5 x 106 cells were plated per 60-mm
`culture dish. After 36 hr, the cells were lysed at 4°C with 0.4
`ml of HNEG buffer per plate (50 mM Hepes, pH 7.5/150 mM
`NaCI/1 mM EGTA/10% glycerol) containing 1.0% Triton
`X-100 detergent and 1 mM phenylmethylsulfonyl fluoride.
`After 10 min, 0.8 ml of lysis dilution buffer (HNEG buffer
`with 1% bovine serum albumin and 0.1% Triton X-100) was
`added to each plate and the extracts were pelleted at 12,000
`x g for 5 min.
`HER2 antibody was added to the cell extracts, which were
`then incubated at 4°C for 2 hr; this was followed by incubation
`with protein A-Sepharose beads for 20 min and three washes
`with 1 ml of HNEG buffer with 0.1% Triton X-100.
`Autophosphorylation reactions were carried out at 4°C in 50
`Al of HNEG wash buffer with 5 mM MnCl2 and 3 ,Ci of
`[y-32P]ATP (Amersham, 5000 Ci/mmol; 1 Ci = 37 GBq) for
`20 min. Proteins were separated on 7.5% NaDodSO4/poly-
`acrylamide gels and analyzed by autoradiography.
`Transformation Assays. The efficiency of colony formation
`in soft agar (24) was determined by plating 25,000 cells in 3
`ml of 0.2% agar (Difco, "purified") over 4 ml of 0.4% agar in
`a 60-mm dish. After 2-4 weeks, colonies of about 100 cells or
`more were counted.
`The plating efficiency of cell lines (25) in 1% calf serum was
`determined by plating equal numbers of cells into 100-mm
`plates with either 10% or 1% calf serum. After 2-3 weeks, the
`plates were stained with crystal violet and colonies were
`counted.
`Mouse Tumorigenicity Assays. Athymic (nu/nu) mice were
`obtained from Charles River Breeding Laboratories. Control
`NIH 3T3 and NIH 3T3/CVN cells and experimental HER2-
`3400 cells were harvested by trypsinization and counted with
`a Coulter Counter. They were then collected by low-speed
`centrifugation and resuspended in ice-cold phosphate-buff-
`ered saline to either 2.5 x 106, 5.0 x 106, or 1.0 x 107 cells
`per ml. Animals were injected subcutaneously with 0.1-ml
`volume of the cell suspensions. Tumor occurrence and size
`were monitored twice weekly.
`
`RESULTS
`For expression ofHER2 sequences in NIH 3T3 cells, a cDNA
`coding for the entire 1255-amino acid polypeptide (1) was
`placed under transcriptional control of the RSV LTR. Tran-
`scriptional termination signals and a poly(A) site were pro-
`vided by 3' sequences of the hepatitis virus surface antigen
`gene (20). In addition, the expression vector contained the
`neo resistance (neoR) gene, which confers cellular resistance
`to the aminoglycoside antibiotic G418 (18) and therefore
`allows selection of primary transfectants, as well as the
`DHFR gene for methotrexate resistance, which was used to
`amplify transfected DNA sequences under selective pres-
`sure. Both drug resistance genes were under simian virus 40
`early promoter transcriptional control. Bacterial plasmid
`sequences, including an origin of replication and the gene for
`ampicillin resistance, allowed replication of the entire expres-
`sion plasmid in Escherichia coli.
`The transforming activity of HER2 sequences was initially
`tested using a conventional NIH 3T3 cell focus-formation
`assay. Under conditions that resulted in about 104 foci per ,g
`of a v-fms viral construct, we were unable to detect any
`HER2-transforming activity. Because of the recently report-
`ed finding that about 30% of mammary carcinomas contain
`amplified HER2 gene sequences without apparent sequence
`rearrangements (14), we investigated whether amplification
`of an unaltered HER2 gene could transform mouse fibro-
`blasts. NIH 3T3 cells were transfected with the pCVN/HER2
`construct. An identical plasmid missing the HER2 expression
`module was used as a control. Four independent primary
`
`Assay for growth in soft agar and 1% calf serum of
`Table 1.
`HER2 primary and amplified cell lines
`
`Plating
`efficiency
`Soft agar
`HER2 gene copies
`in 1% calf
`colonies,
`per haploid
`serum, %
`Cell line
`no.
`genome, no.
`NIH 3T3/CVN
`0.27
`0
`0
`NIH 3T3/CVN400
`0
`0
`0
`3
`1
`3
`HER2-1
`HER2-14m
`38
`424
`60
`1.4
`0
`2
`HER2-3
`HER2-34m
`11.7
`836
`55
`HER2-4
`0.2
`0
`4
`HER2-44
`49
`376
`90
`0.6
`0
`1
`HER2-B3
`HER2-B34w
`50.2
`373
`131
`Two control lines were used. The first one was a NIH 3T3 line
`transfected with a plasmid containing only the neo and DHFR genes.
`The second control line contained the neo-DHFR plasmid and was
`amplified to resistance to 400 mM methotrexate. HER2 gene copy
`numbers were determined using a human DNA standard and
`densitometer scanning of Southern hybridization autoradiograms.
`Equal cell numbers (25,000) were plated in soft agar and colonies
`were counted after 2-4 weeks. The plating efficiency in 1% calf
`serum is relative to the number of colonies arising when an equal
`aliquot was simultaneously plated in medium containing 10% calf
`serum.
`
`G418-resistant clones (HER2-1, HER2-3, HER2-4, HER2-
`B3) were isolated. Cell lines containing amplified HER2
`coding sequences were generated from these parental clones
`by culturing the cells in gradually increasing concentrations
`of methotrexate up to 400 nM (HER2-14m, HER2-3400,
`HER2-4400, HER2-3B4w). Southern hybridization analysis of
`parental and amplified cell lines demonstrated that the HER2
`cDNA copy number increased from 1-4 to 55-131 per haploid
`genome (Table 1).
`To test whether gene amplification resulted in overexpres-
`sion of the HER2 gene product, cell lysates were im-
`munoprecipitated with an antibody against the C-terminal 17
`amino acids of the HER2 sequence. As shown in Fig. 1,
`substantially increased levels of the p185 HER2 gene product
`were found in amplified cell lines relative to their parental
`G418R transfectants. The parental cells had a normal mor-
`phology that was indistinguishable from NIH 3T3 cells.
`However, amplified cells had the typical refractile, spindle-
`1 234 5 6 789
`
`..'
`
`O
`
`200-
`
`116-
`
`Quantitation ofp185HER2 in four primary, unamplified cell
`FIG. 1.
`lines and lines derived from them by amplification to resistance to 400
`nM methotrexate. Lane 1, neo-DHFR control; lanes 2 and 3, HER2-1
`parent and amplified lines; lanes 4 and 5, HER2-3 parent and
`amplified lines; lanes 6 and 7, HER2-4 parent and amplified lines;
`lanes 8 and 9, HER2-B3 parent and amplified lines. Positions of the
`size markers myosin and j8-galactosidase are indicated in kDa.
`
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`Cell Biology: Hudziak et al.
`
`Proc. Natl. Acad. Sci. USA 84 (1987)
`
`7161
`
`FIG. 2.
`Morphology of NIH 3T3 cells transfected with HER2
`expression construct. (1) NIH 3T3 cells transfected with the control
`neo-DHFR plasmid. (2) NIH 3T3 cells with the neo-DHFR plasmid
`amplified to resistance to 400 mM methotrexate. (3) Primary G418R
`cell line HER2-1, an unamplified clone expressing low levels ofp185.
`(4) The same clone amplified to 400 mM methotrexate resistance.
`(x50.)
`
`shaped appearance oftransformed cells and grew in irregular,
`piled-up clumps (Fig. 2).
`NIH 3T3 cell lines with an amplified HER2 gene and high
`levels of HER2 gene expression also displayed other char-
`acteristics associated with a transformed phenotype. As
`shown in Table 1 and Fig. 3, these cells all formed colonies
`in soft agar and were able to grow at low density at low serum
`concentration. In contrast, primary transfectants did not
`grow under these conditions. The primary transfectants did,
`however, grow to a higher saturation density than the
`parental NIH 3T3 cells.
`The correspondence between a transformed phenotype
`and HER2 gene amplification and overexpression was inde-
`pendently confirmed by directly selecting for transformed
`cells and then analyzing the resulting clones. For this purpose
`the parental cell line HER2-3, which contains about two
`copies of the HER2 expression construct, was cultured in
`medium containing a low concentration of fetal calf serum
`(0.5%); control cells containing the expression vector without
`HER2 coding sequences (pCVN) were cultured in parallel.
`After 5 weeks, a few colonies appeared in the control culture
`and roughly 10-fold more colonies appeared in dishes con-
`taining the HER2-3 cell line. These colonies appeared to be
`morphologically transformed and were subsequently ana-
`lyzed for HER2 overexpression. As shown in Fig. 4, three
`individual clones as well as a pool of the remaining colonies
`had elevated levels of p185HER2 compared with the original
`parental G418-resistant HER2-3 cell line. In addition, there
`
`Anchorage-independent growth of HER2-transformed
`FIG. 3.
`cells in soft agar. Cells were plated in 0.2% soft agar over a 0.4% agar
`lower layer. After 3 weeks the plates were photographed at 40x
`magnification using a Nikon microscope with phase-contrast optics.
`(Upper) Control untransformed NIH 3T3 cells. (Lower) Anchorage-
`containing the HER2
`independent growth of the cell line HER2-34
`expression plasmid and amplified to resistance to 400 mM methotrex-
`ate. (X25.)
`
`was a 26-fold increase in the number ofcells plating in 100 nM
`methotrexate in the selected cells compared with the parental
`cells, implying that the unselected but linked DHFR gene had
`also been coamplified.
`The tumorigenicity of cell lines with a high HER2 cDNA
`copy number was tested in nude mice by subcutaneous
`
`1 2 3 4 5 6
`
`wast -185
`
`Quantitation of p185 in the primary line HER2-3 and cell
`FIG. 4.
`lines selected for growth in 0.5% calf serum. HER2-encoded protein
`was immunoprecipitated and labeled. Lanes 1-3, three colonies
`picked and expanded from plates after selection for growth in 0.5%
`serum; lane 4, the starting cell line, HER2-3; lane 5, a pool of 0.5%
`serum-selected colonies of HER2-3; lane 6, a clone derived from a
`G418R control line selected for growth in 0.5% calf serum. The
`position expected for a protein of apparent molecular mass of 185
`kDa is indicated.
`
`PETITIONER'S EXHIBITS
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`
`Cell Biology: Hudziak et al.
`
`Table 2.
`
`Tumorigenicity testing of cell lines
`
`Cell line
`NIH 3T3
`
`NIH 3T3/CVN
`
`HER2-34m
`
`Mice with tumors/
`Cells injected, no.
`mice injected
`0.25 x 106
`0/6
`0/6
`0.5 x 106
`0/6
`1.0 x 106
`0/6
`0.25 x 106
`0.5
`x 106
`0/6
`1.0 x 106
`0/6
`0.25 x 106
`5/5
`0.5 x 106
`6/6
`1.0 X 106
`5/5
`The cell line HER2-3 and two control lines, NIH 3T3 cells and a
`NIH 3T3 line containing the control plasmid, were injected subcu-
`taneously at three different dosages into nude mice. The time after
`injection before tumors became visible was dose related: 22 days
`(average) for 1 x 106 cells, 28 days (average) for 0.5 x 106 cells, and
`34 days (average) for 0.25 X 106 cells.
`
`injection ofthree different cell numbers per animal. As shown
`in Table 2, the overexpressing cell line HER2-34m was
`strongly tumorigenic at all dosages tested, whereas the
`control cell lines, NIH 3T3 cells and NIH 3T3 cells
`transfected with CVN vector without the HER2 insert, were
`both negative under the same conditions. Necropsy of three
`mice with well-established tumors failed to identify any
`metastasis. Cell lines reestablished from three excised tu-
`mors still expressed the G418R phenotype, were resistant to
`methotrexate, and expressed high levels of pl85HER2 (not
`shown).
`
`DISCUSSION
`Amplification of the HER2 gene has been reported in a few
`primary human tumors (2, 3) and tumor-derived cell lines (13,
`26). Recently, Slamon et al. (14) found that the HER2 gene
`is amplified in 30% of primary breast tumors, a common
`human malignancy that affects about 7% of all American
`females. Notably, only 3/189 tumors surveyed showed any
`gross rearrangement of the HER2 gene. To assess the role of
`the HER2 gene in the neoplastic process, we characterized
`the transforming potential ofthe HER2 gene in an in vitro cell
`culture system.
`Expression of the full-length cDNA in NIH 3T3 cells did
`not lead to transformation as determined by a standard
`focus-forming transfection assay. However, HER2 overex-
`pression caused by gene amplification transformed these
`cells. Colonies that survived methotrexate selection were
`morphologically transformed and exhibited loss of contact
`inhibition. Such cells also grew in soft agar and would grow
`in 1% calf serum. Furthermore, cells transformed by HER2
`were tumorigenic in athymic mice.
`Selection of cells transfected with the HER2 cDNA for
`growth in low serum provided independent evidence that
`high-level expression of an unaltered HER2 gene product
`caused cellular transformation. Since DNA introduced into
`mammalian cells by transfection is more labile than genomic
`DNA (23, 27), we reasoned that selection for a property
`demonstrated by pharmacologically amplified HER2 lines-
`namely, growth in low serum-might lead to amplification or
`other changes resulting in overexpression of p185HER2.
`Clones derived from an unamplified HER2 line by this selection
`procedure appeared morphologically transformed and exhibit-
`ed elevated levels of p185HER2.
`Taken together, the characteristic morphological changes,
`results of in vitro and in vivo transformation and tumorigenic-
`ity assays, and the elevated levels of p185HER2 in cells
`selected for a transformed phenotype imply that high-level
`expression of HER2 results in transformation of NIH 3T3
`
`Proc. Natl. Acad. Sci. USA 84 (1987)
`
`cells. Another member ofthe tyrosine kinase gene family was
`recently found to be amplified 4- to 8-fold in spontaneously
`arising foci of NIH 3T3 cells (28). Amplification of the met
`gene appears to be a frequent event and, similar to HER2
`amplification in mammary carcinomas, is rarely accompa-
`nied by gross rearrangements. These findings and the dis-
`covery of amplified EGF receptor genes in primary human
`tumors (10-12) suggest that overexpression of other growth
`factor receptor genes will also lead to transformation and
`tumorigenesis. Whether susceptibility to spontaneous ampli-
`fication is caused by characteristics of the genetic loci for
`HER2, met and EGF receptor, or cell-specific selective
`advantages caused by receptor overexpression remains to be
`investigated.
`is not yet clear whether transformation of cells
`It
`overexpressing a growth factor receptor gene is dependent on
`paracrine or autocrine stimulation by the appropriate ligand.
`For HER2, this question cannot yet be addressed since its
`ligand has not been identified. In the case of the EGF
`receptor, however, we were able to demonstrate that primary
`human tumors and tumor-derived cell lines frequently ex-
`press mRNAs for both the receptor and transforming growth
`factor type a ligand (29). The finding that mere overexpres-
`sion of an intact growth factor receptor may subvert normal
`cellular growth control mechanisms and lead to tumorigenic
`growth provides new potential for diagnostic approaches and
`therapeutic strategies for treatment of human malignancies.
`We express appreciation for the superb technical assistance of
`Thomas Dull in constructing the HER2 expression plasmids and Bill
`Lagrimas for help with the mouse tumorigenicity assays. We are
`grateful to Jeanne Arch for her patience and skill in typing this
`manuscript and to Dr. Suzanne Pfeffer for her valuable editorial
`comments.
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`
`PETITIONER'S EXHIBITS
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`Exhibit 1047 Page 5 of 5