62
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`KWONG AND HUNG
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`MOLECULAR CARCINOGENESIS 23:62–68 (1998)
`
`A Novel Splice Variant of HER2 With Increased
`Transformation Activity
`
`Ka Yin Kwong and Mien-Chie Hung*
`Department of Tumor Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
`
`The HER2 proto-oncogene (also known as neu or c-erbB-2) belongs to the epidermal growth factor receptor
`family. HER2 is frequently amplified in human carcinomas. Gene amplification or overexpression of HER2 has
`been correlated with poor prognosis in several human cancers. Point mutation in the rat HER2 homolog, neu,
`is involved in the formation of rat neuroblastomas. However, no similar mutation in HER2 has been found in
`human cancers. Here we report the identification of a novel alternative splicing form of HER2 (D HER2) in
`human cell lines. An exon 16 amino acids long in the extracellular domain was deleted in DHER2. Deletion
`mutations in the corresponding region were shown previously to be involved in the formation of mammary
`carcinomas in transgenic mice. In the focus-formation assay, D HER2 showed much stronger transformation
`activity than did wild-type HER2. This result suggests that the deleted 16–amino acid exon may play a regula-
`tory role in HER2 transformation activity. Mol. Carcinog. 23:62–68, 1998.
`© 1998 Wiley-Liss, Inc.
`Key words: HER2; receptor protein-tyrosine kinase; proto-oncogene; alternative splicing; transformation
`
`INTRODUCTION
`The rat neu oncogene was originally identified by
`a chemical mutagenesis experiment in the rat, and
`HER2 is the human counterpart. The HER2/neu gene
`(also known as c-erbB-2) encodes a 185-kDa receptor
`tyrosine kinase that belongs to the epidermal growth
`factor receptor family [1]. A single point mutation
`(valine to glutamic acid) in the transmembrane do-
`main can activate the rat neu proto-oncogene [2]. The
`same amino acid change introduced by in vitro mu-
`tagenesis into human HER2 has also been shown to
`activate the transformation ability [3]. No identical
`mutation in HER2 has been found in human cancers
`[4,5]. However, overexpression and gene amplifica-
`tion of HER2 is frequently observed in human can-
`cers. Poor prognosis was found to correlate with
`overexpression or gene amplification of HER2 in sev-
`eral cancers [6–10]. In vitro experiments demonstrated
`that overexpression of HER2/neu was sufficient to ac-
`tivate the tyrosine kinase activity in the absence of
`mutation or ligand stimulation [11,12]. Transgenic
`mice that overexpressed normal rat neu protein (the
`rat HER2 homolog) in the mammary epithelium de-
`velop mammary carcinomas with high penetrance
`[13]. The formation of tumors in these transgenic
`mice correlates with de novo mutations of the neu
`transgene that activates its tyrosine kinase activity
`in the tumors. A novel class of small deletions in the
`extracellular domain was found in these tumors.
`These deletion mutations were shown to activate the
`in vitro transformation activities of neu by enhanc-
`ing dimerization and tyrosine kinase activities [14].
`Here we report the identification of an alternatively
`spliced mRNA in human cancer cell lines. This al-
`
`© 1998 WILEY-LISS, INC.
`
`ternatively spliced HER2 variant appeared as smaller
`bands in our reverse transcription (RT)–polymerase
`chain reaction (PCR) analysis of HER2 expression
`in human cancer cell lines. Strikingly, the alterna-
`tive splicing form of HER2 (referred to hereafter as
`DHER2) has one exon deleted from the coding se-
`quence, and this deleted exon overlaps with the de-
`letions in the corresponding region of the rat neu
`transgenes. The similarity in the predicted protein
`structure of D HER2 and the deletion mutants in
`transgenic mice suggests that D HER2 may be an ac-
`tivated form of HER2 and therefore may play an
`important role in human cancers.
`
`MATERIALS AND METHODS
`Oligonucleotide Primers Sequences
`The primers used were NP-1 (5¢ -CAT GCC CAT
`CTG GAA GTT TC-3¢ (nt 2004–2023)), NP-2 (5¢ -
`GCT CCA CCA GCT CCG TTT CCT G-3¢ (nt 2269–
`2248)), NP-5 (5¢ -ATG CCA GCC TTG CCC CAT
`CAA CTG C-3¢ (nt 2040–2064)), and NP-6 (5¢ -AGA
`CCA CCC CCA AGA CCA CGA CCA G-3¢ (nt 2185–
`2161)). The nucleotide sequence numbers are
`based on GenBank sequence X03363.
`
`*Correspondence to: Department of Tumor Biology, Box 79, The
`University of Texas M. D. Anderson Cancer Center, 1515 Holcombe
`Blvd., Houston, TX 77030.
`Received 11 June 1998; Revised 26 August 1998; Accepted 27
`August 1998
`Abbreviations: RT, reverse transcription; PCR, polymerase chain
`reaction; MAPK, mitogen-activated protein kinase.
`
`IMMUNOGEN 2051, pg. 1
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`ALTERNATIVE SPLICING OF HER2
`
`63
`
`RT-PCR Analysis of Cancer Cell Lines
`RNA was extracted from cancer cell lines with
`TRIZOL (Life Technologies, Gaithersburg, MD). The
`RT reaction was performed with Superscript II reverse
`transcriptase by following the manufacturer’s proto-
`col (Life Technologies). Primers NP-2 and NP-5 were
`used for the RT-PCR analysis (Figure 1B). The ex-
`pected sizes of RT-PCR products were 206 and 158
`bp for wild-type HER2 and DHER2, respectively. The
`RT-PCRs were performed in the presence of 1 m Ci of
`[32P]dCTP in a reaction volume of 50 m L. Ten to
`twenty microliters of the PCR products was separated
`on a 6% polyacrylamide gel. The gel was dried be-
`fore exposure to X-OMAT film (Kodak, Rochester,
`NY) for autoradiography.
`
`Sequencing of RT-PCR Products
`The RT-PCR bands from the dried polyacrylamide
`gel were cut out, and the DNA was eluted by boiling
`in H2O for 2 min. Aliquots of the DNA solutions were
`reamplified with the same primers, and the PCR prod-
`ucts were directly sequenced with a Cyclist sequenc-
`ing kit (Stratagene, La Jolla, CA) by using 32P
`end-labeled primers or an [35S]dATP incorporation
`protocol.
`
`PCR and Sequencing of Genomic DNA
`High-molecular-weight DNA (0.5–1 m g) was am-
`plified with primers NP-5 and NP-6 (Figure 1B) by
`using the Expand Long Template PCR System
`(Boehringer Mannheim, Indianapolis, IN) and fol-
`lowing the manufacturer’s protocol. The PCR prod-
`ucts were separated on both 1% and 3% agarose gels
`to detect large and small PCR products. Only one
`PCR product of 6 kb was observed, and it was ex-
`cised from the 1% agarose gel and purified with Qiaex
`II (Qiagen, Valencia, CA). The purified DNA samples
`were sequenced manually with 32P end-labeled prim-
`ers by using the Cyclist sequencing kit or with an
`automatic sequencer (Applied Biosystems, Inc.,
`Ramsey, NJ) in the core sequencing facility of M. D.
`Anderson Cancer Center.
`
`Plasmid Construction
`The wild-type human HER2 cDNA coding region
`was released from psv2erb2 (kindly provided by Dr.
`T. Yamamoto) by HindIII digestion and subcloned
`into the plasmid Bluescript (Stratagene) for mutagen-
`esis. The RT-PCR fragment of DHER2 from the cell
`line BT-474 was amplified with primers NP-1 and NP-
`2. The 218-bp RT-PCR product was digested with SphI
`and AatII and subcloned into Bluescript/HER2 to
`generate a DHER2 clone. Point-mutated HER2 in
`Bluescript was generated by digesting the vector
`LTR1erb-Glu (kindly provided by Dr. P. P. Di Fiore)
`[3] with SphI and NdeI and was subcloned into the
`same sites in Bluescript/HER2. HindIII fragments
`from these HER2 clones in Bluescript were cut out
`
`and subcloned into pcDNA3 (Invitrogen, Carlsbad,
`CA). The wild-type HER2, DHER2, and point-mutated
`HER2 pcDNA3 clones were named CMV-HER2-WT,
`CMV-HER2-D
`, and CMV-HER2-Glu, respectively. The
`EcoRI fragment from CMV-HER2-D
` was cut out and
`used to replace the corresponding region in LTR1erb-
`Glu to create LTR-HER2-D
`.
`
`Elk-GAL4 Luciferase Assay
`CHO cells were transfected with 0.5 m g of Elk-GAL4,
`6 m g of GAL4-LUC (kindly provided by Dr. C. J. Der)
`[15], 1.5 m g of RSVb -gal, and 2 m g of cytomegalovirus
`promoter–driven HER2 expression vectors by the cal-
`cium phosphate method. Luciferase activities were
`normalized to b -galactosidase activities. The same
`experiment was repeated with CMV b -gal as a nor-
`malization control. The experiment was also repeated
`in NIH/3T3 cells by following the same procedures.
`The cells were harvested 36–48 h after transfection
`and assayed for luciferase activity by using the Lu-
`ciferase Assay System (Promega Corp., Madison, WI).
`
`Western Blotting
`COS-7 cells were resuspended at 107 cells/mL of
`Dulbecco’s modified Eagle’s medium/F-12 medium
`with 10% fetal bovine serum. Four hundred microli-
`ters of cell suspension was mixed with DNA at room
`temperature and pulsed at 260 V and 960 m F in a
`0.4-cm cuvette by using Gene Pulser equipment (Bio-
`Rad Labs., Hercules, CA). After transfection, the COS-
`7 cells were allowed to grow for 24–36 h before being
`harvested. Fifty micrograms of lysate was separated
`on a 6% sodium dodecyl sulfate–polyacrylamide gel,
`transferred to a nitrocellulose membrane by using
`the Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad
`Labs.), and blotted with PY20 anti-phosphotyrosine
`antibody (Santa Cruz Biotechnology, Santa Cruz,
`CA). The same membrane was stripped by following
`the procedures recommended in the ECL kit
`(Amersham Corp.) and reprobed with c-neu antibody
`Ab3 (Oncogene Science, Cambridge, MA). Signals
`were detected by the enhanced chemiluminescence
`method (Amersham Corp.).
`
`Focus Formation Assay
`NIH/3T3 cells (2-3x105) were plated in 6-cm tis-
`sue-culture dishes and grown overnight. Ten micro-
`grams of test plasmids (or 2 m g of test plasmids plus
`8 m g of Bluescript plasmid in a lower-concentration
`experiment) was transfected by the calcium phos-
`phate protocol. Two or three days after transfection,
`the cells were split 1:25 into 10-cm tissue-culture
`dishes and were grown in Dulbecco’s modified Eagle’s
`medium/F-12 medium supplemented with 10% fe-
`tal bovine serum. After 14–21 d, the cells were stained
`and fixed for focus counting. The experiment was
`repeated three times. Two independent preparations
`of plasmids were used to avoid bias due to variation
`in the quality of different DNA preparations.
`
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`64
`
`KWONG AND HUNG
`
`RESULTS
`While studying the expression of HER2 in human
`cancer cell lines by RT-PCR analysis, we unexpect-
`edly, obtained additional smaller bands (Figure 1A).
`The same results were also observed with other hu-
`man cancer cell lines, including SK-OV3, BT-474, and
`SK-BR3 (data not shown). The region amplified by
`this set of primers contains the transmembrane and
`juxtamembrane regions of HER2. To determine
`whether these RT-PCR products came from nonspe-
`cific amplification, the bands were excised from the
`gel, reamplified, and then sequenced directly. The
`sequences of the small products matched the pre-
`dicted sequence of HER2 except for a deletion of 48
`bp in the extracellular domain (Figure 1B). The 48-
`bp deletion created an in-frame deletion of 16 amino
`acids. To determine whether the small products re-
`sulted from mutation in the genomic DNA in these
`cancer cell lines, primers NP-5 and NP-6 (Figure 1B)
`were used to amplify genomic DNA extracted from
`the cell lines A431, MDA-MB-361, and HBL-100. The
`PCR products from the three cell lines were directly
`sequenced. By comparing the DNA sequences, it be-
`came clear that the deleted 48 bp was one exon (Fig-
`ure 1C). The mRNA variant with the 48-bp deletion
`will be referred to hereafter as DHER2. We propose
`that DHER2 came from alternative mRNA splicing
`because the sequences of the RT-PCR products of
`DHER2 were consistent with the predicted alterna-
`tive splicing products. Furthermore, no point muta-
`tion in the splice site junctions was observed (data
`not shown). This type of point mutation has been
`shown to account for most mRNA mis-splicing or
`exon skipping in human diseases [16]. The primers
`NP-5 and NP-6 amplified PCR products of the same
`size from the genomic DNA of all three cell lines.
`The sequences of the PCR products corresponding
`to the 48-bp exon and the exon-intron junctions were
`identical. Running different percentage agarose gels
`revealed no additional PCR products indicating de-
`letion in genomic DNA. This evidence suggests that
`DHER2 is an alternative splicing variant rather than
`a product of mutation in the genomic DNA.
`It is known that tyrosine phosphorylation of the
`HER2-encoded receptor and its tyrosine kinase ac-
`tivity are important indicators of its activity. To de-
`termine the tyrosine phosphorylation activity of
`different forms of HER2, HER2 cDNA expression vec-
`tors were transfected into COS-7 cells, and western
`blotting was performed with an antibody against
`phosphorylated tyrosine residues (PY20) 24 h after
`transfection (Figure 2). DHER2 and the point-mutated
`HER2 produced a much stronger tyrosine phospho-
`rylation signal in the predicted position of the HER2
`protein than did the wild-type HER2. The same ni-
`trocellulose membrane was stripped and reprobed
`with anti-HER2–specific antibody c-neu Ab-3, and
`the expression levels of HER2 in the COS-7 cells were
`
`comparable. It is clear that the transfection of HER2
`caused tyrosine phosphorylation of a 185-kDa pro-
`tein and that D HER2 had much stronger activity than
`did wild-type HER2 in this assay.
`The ras-dependent pathway is one of the down-
`stream signaling pathways of HER2/neu. To examine
`whether D HER2 was able to activate its downstream
`signals, activation of the ras pathway was tested by
`transient transfection of CHO cells and NIH/3T3 cells
`with an Elk-GAL4/GAL4-luc reporter system [15]. The
`expression of luciferase in this system depends on
`the phosphorylation of the Elk-GAL4 fusion protein
`by the mitogen-activated protein kinases (MAPKs).
`The ras pathways activate the MAPKs, which in turn
`activate luciferase activity in this system. The results
`from this assay indicated that D HER2 was indeed
`more active in activating MAPKs than was wild-type
`HER2. D HER2 activated MAPKs sixfold more strongly
`than did wild-type HER2 in CHO cells (Figure 3). The
`same experiments were repeated with NIH/3T3 cells,
`and the results were essentially the same (data not
`shown). Interestingly, the activity of DHER2 in this
`assay was comparable to that of the point-mutated
`HER2, which is known to be a potent transforming
`oncogene.
`One of the most important properties of activated
`HER2/neu is its strong transformation ability. To test
`the transforming ability of D HER2, focus formation
`of NIH/3T3 cells was assayed. DHER2 was subcloned
`into an expression vector driven by the murine leu-
`kemia virus long terminal repeat promoter. The trans-
`formation ability of this construct was compared to
`that of a wild-type HER2 construct and a point-mu-
`tated HER2 construct in the same expression vector.
`Wild-type HER2 produced no significant increase in
`the number of foci above the levels of the negative
`control (Bluescript plasmid) in multiple experiments,
`whereas DHER2 and point-mutated HER2 induced
`foci efficiently under these conditions (Table 1).
`D HER2 formed as many foci as point-mutated HER2
`did, but the foci formation was slower and the foci
`were generally slightly smaller (Figure 4). The experi-
`ments were repeated three times, and two indepen-
`dent preparations of plasmids were used. There was
`variation in the number of foci formed between dif-
`ferent sets of experiments, but the difference between
`wild-type HER2 and D HER2 was so dramatic that we
`conclude that the deletion in the extracellular do-
`main in D HER2 strongly enhanced the transforma-
`tion ability of HER2.
`
`DISCUSSION
`Here we reported the identification of a deletion
`form of the human HER2 mRNA in cancer cell lines
`by RT-PCR analysis. The human HER2 genomic se-
`quence flanking the deleted region was partially de-
`termined by direct sequencing of PCR products. Our
`results suggested that D HER2 was generated by alter-
`native splicing of mRNA rather than mutation. Re-
`
`IMMUNOGEN 2051, pg. 3
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`ALTERNATIVE SPLICING OF HER2
`
`65
`
`Figure 1. Detection of a novel HER2 mRNA (DHER2) in hu-
`man cancer cell lines. (A) RT-PCR was performed with primers
`NP-2 and NP-5 in the presence of [32P]dCTP. The upper bands
`are the wild-type HER2 RT-PCR products, and the lower bands
`are the alternative splicing products. The reactions were per-
`
`formed in the presence (RT+) or absence (RT–) of reverse tran-
`scriptase. The leftmost two lanes contain cloned cDNAs that
`served as positive and size controls for the RT-PCR reactions.
`CMV-HER2-WT is a wild-type HER2 cDNA expression vector, and
`CMV-HER2-D
` was engineered by replacing the wild-type HER2
`
`IMMUNOGEN 2051, pg. 4
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`66
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`KWONG AND HUNG
`
`Figure 2. Expression of D HER2 was associated with strong
`tyrosine phosphorylation. HER2 cDNAs were subcloned into
`pcDNA3 and transfected into COS-7 cell by electroporation. The
`transfected cells were harvested 24 h later and analyzed by
`western blotting with anti-phosphotyrosine antibody PY-20. PY-
`20 was then stripped, and the same membrane was reprobed
`with anti-HER2 antibody c-neu Ab-3. Note that the D HER2 pro-
`tein ran slightly faster than the wild-type HER2 protein. CMV-
`HER2-Glu is the point-mutated human HER2 cDNA, which is a
`well-characterized oncogenic form of HER2.
`
`moval of the same 48 bp deleted in D HER2 from the
`wild-type HER2 cDNA activated its transformation
`ability in NIH/3T3 cells.
`Mutations in rat neu gene have been shown to be
`responsible for the formation of tumors in animal
`experiments, and clinical studies have clearly demon-
`strated the importance of HER2 in human cancers.
`HER2 overexpression or gene amplification is observed
`in 20–30% of human breast and ovarian cancers and
`is associated with poor prognosis in these patients [6,7].
`The rat neu proto-oncogene can be activated by a single
`point mutation in the transmembrane domain or by
`deletion of the entire extracellular ligand binding do-
`main [11,17]. However, no equivalent mutations have
`been found in human cancers. The absence of point
`mutations in HER2 in human cancers could be ex-
`plained by the fact that the equivalent amino acid
`change from valine to glutamic acid require a 2-bp
`change in human HER2 (GTTfi GAG) and therefore
`requires two point mutations. On the other hand, in
`the rat neu gene, a 1-bp change is sufficient for the
`same amino acid change (GTGfi GAG). It has been
`suggested that the probability of having both point
`mutations required for the valine to glutamic acid
`change in the human HER2 gene is too low to be ob-
`served in human cancers.
`Transgenic mouse models have been established to
`
`sequence with the RT-PCR product from DHER2 mRNA. (B) RT-
`PCR products from BT-474 and MDA-MB-361 were directly se-
`quenced, and the difference between wild-type HER2 and DHER2
`was determined to be the absence of one 48-bp exon (nt 2073–
`2120) in DHER2. The solid bars below the HER2 sequence indi-
`cated the published deletion regions in the tumors of neu
`transgenic mice [14]. (C) The exon and intron junctions in hu-
`man HER2 in the cell line HBL-100. Capital letters indicates exon
`sequences, and lowercase letters indicate intron sequences.
`
`DHER2 was a potent MAPK activator. CHO cells
`Figure 3.
`were transfected with 0.5 m g of Elk-GAL4, 1.5 m g of RSVb -gal, 6
`m g of GAL4-LUC, and 2 m g of HER2 cDNAs in pcDNA3. The lu-
`ciferase activity reflects the phosphorylation of the Elk-GAL4
`fusion protein by the MAPKs. The result showed here is from
`one experiment. Transfections of each HER2 vector was tripli-
`cated. Luciferase activities are normalized to b -galactosidase
`activities. The activity of the empty vector was set to one. The
`results indicated that D HER2 activated MAPK activity as strongly
`as did point-mutated HER2 and about sixfold more strongly
`than did wild-type HER2.
`
`study the role of neu in mammary carcinogenesis.
`Expression of the wild-type rat neu cDNA transgene
`induces mammary carcinomas in the animals. Small
`de novo deletions in the neu extracellular domain
`were observed very frequently in these tumors [14].
`The sequences deleted in these tumors all overlap with
`the deleted exon in human DHER2 reported here. Be-
`cause the rat neu cDNA construct used in the
`transgenic mouse experiment did not contain introns,
`the deletions in the mice tumors represent mutations
`instead of alternative splicing. The investigators who
`performed these experiments also studied the activa-
`tion mechanism of the mutant neu transgenes and
`demonstrated that the deleted region contains criti-
`cal cysteine residues. Removal of these cysteine resi-
`dues in the rat neu cDNA by deletions strongly
`enhances the receptor dimerization, and, as a result,
`the tyrosine kinase activity of neu is increased. In vitro
`mutagenesis by the same group showed that disrupt-
`ing these cysteine residues in the wild-type rat neu
`gene by point mutations is sufficient to activate the
`transformation ability of neu [18]. In the human
`DHER2 gene reported here, the deleted exon contains
`two of these conserved cysteines residues. It is pos-
`sible that the strong transformation activity of D HER2
`we observed was also due to enhanced receptor dimer-
`ization and increased tyrosine kinase activity.
`There is a previous report of another alternative
`splicing form of human HER2. This alternative splic-
`
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`ALTERNATIVE SPLICING OF HER2
`
`Table 1. Transformation of NIH/3T3 Cells*
`
`67
`
`Expression
`plasmids
`
`Bluescript (control)
`LTR-HER2-WT
`LTR-HER2-D
`LTR-HER2-Glu
`
`Focus assay 1
`10 m g of plasmid
`Set 1
`
`0
`0
`68
`Not
`available
`
`Set 2
`
`0
`0
`89
`71
`
`Focus assay 2
`
`10 m g of plasmid
`Set 1
`Set 2
`
`2 m g of plasmid†
`Set 1
`Set 2
`
`2
`0
`75
`62
`
`2
`1
`63
`73
`
`2
`0
`25
`21
`
`2
`3
`14
`15
`
`*Results from two independent focus formation assays. The expression vectors in the focus formation assay were driven by the murine
`leukemia virus long terminal repeat promoter. LTR-HER-WT, LTR-HER2-D, and LTR-HER-Glu are wild-type HER2, D HER2, and point-
`mutated HER2, respectively.
`†Two micrograms of test plasmids plus 8 m g of Bluescript vector were used in this set of experiments to ensure that the experiment was
`performed within the linear range.
`
`ing of HER2 resulted in the inclusion of an intron
`sequence that contains a stop codon. The premature
`termination of translation produces a truncated HER2
`protein lacking both the entire cytoplasmic domain
`and the transmembrane domain [19]. The functional
`significance of this truncated human HER2 is still
`unknown. It is very unlikely that this C-terminal–
`truncated form of HER2 has transformation ability
`because removal of the cytoplasmic domain abol-
`ishes the tyrosine kinase activity of HER2.
`
`Figure 4. DHER2 was an activated transforming oncogene.
`Ten micrograms of HER2 expression vectors driven by the mu-
`rine leukemia virus long terminal repeat promoter was trans-
`fected into NIH/3T3 cells by the calcium phosphate transfection
`method. The cells were split 1:25 after 2 d and grown for 14–21
`d in Dulbecco’s modified Eagle’s medium/F-12 medium with
`10% fetal bovine serum to test foci formation. One set of typi-
`cal results is shown here. Transfection with either Bluescript or
`wild-type HER2 (LTR-HER2-WT) produced numbers of foci close
`to the background level, whereas transfection with DHER2 (LTR-
`HER2-D ) generated as many foci as did transfection with the
`point-mutated HER2 (LTR-HER2-Glu).
`
`neu expression is required for the normal develop-
`ment of the heart and nervous system in mice [20]
`and is regulated spatially and temporally during de-
`velopment of the embryo [21]. It is not yet clear
`whether D HER2 expression is regulated to control
`these developmental processes. Further investigation
`is required to determine the role of D HER2 in hu-
`man cancer cells and development, and the results
`from these studies may lead to a better understand-
`ing of the pathological and physiological functions
`of the HER2/neu oncogene.
`ACKNOWLEDGMENTS
`We thank Dr. Agnes Pui-Yee Chan and Dr. Ralph
`Zinner for critically reading the manuscript. This
`work was supported by National Institutes of Health
`grants CA 58880 and CA 60856, grants DAMD17-
`94-J-4315 and DAMD17-96-I-6253 from the Depart-
`ment of Defense, the Nellie Connally Breast Cancer
`Research Fund, and the Faculty Achievement Award
`from M. D. Anderson Cancer Center (to M-CH) and
`by Cancer Center Core Grant CA 16672 (to M. D.
`Anderson Cancer Center).
`
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`IMMUNOGEN 2051, pg. 7
`Phigenix v. Immunogen
`IPR2014-00676
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