Breast Cancer Expressing the Activated HER2/Neu Is Sensitive to Gefitinib in Vitro and in Vivo and Acquires Resistance Through a Novelpoint Mutation in the HER2/Neu

Research Article

Breast Cancer Expressing the Activated HER2/neu Is Sensitive to Gefitinib In vitro and In vivo and Acquires Resistance through a NovelPoint Mutation in the HER2/neu

Marie P. Piechocki,1 George H. Yoo,1 Susan K. Dibbley,1 and Fulvio Lonardo2

1Department of Otolaryngology-Head and Neck Surgery, Wayne State University and Karmanos Cancer Center and 2Department of Pathology, Wayne State University, Detroit, Michigan

Abstract NOTCH-1, FLT-1, PDGFB, and several other genes that may contribute to the resistant phenotype and sustain signaling The HER2/neu oncogene is an important diagnostic and through MAPK and Akt. This model will be useful in under- prognostic factor and therapeutic target in breast and other standing the differences between intrinsic drug sensitivity cancers. We developed and characterized a breast cancer cell and acquired resistance in the context of therapeutic strate- line (Bam1a) that overexpresses the activated HER2/neu and gies that target oncogene addicted diseases. ErbB-3 and has a gene expression profile consistent with the [Cancer Res 2007; ErbB-2genetic signature. We evaluated the effects of the 67(14):6825–43] epidermal growth factor receptor (EGFR)/HER2inhibitor, gefitinib, on this breast tumor line in vitro and in vivo.We Introduction characterized the effects of gefitinib on EGFR, HER2, and Receptor tyrosine kinases (RTK) play important roles in ErbB-3 phosphorylation by Western blot and determined the regulating normal cell growth by initiating specific intracellular effects on downstream signaling through growth, survival, signaling pathways in response to binding of extracellular growth and stress pathways and the effect on proliferation, cell cycle, factors. A wide variety of cellular functions are modulated by the and apoptosis. Gefitinib treatment diminished phosphoryla- four members of the ErbB [or epidermal growth factor receptor tion of the ErbB-3 > EGFR > HER2/neu and signal transducers (EGFR)] family, including regulation of mitogenesis, cell death, and activators of transcriptions in a dose-dependent fashion. angiogenesis, and cell differentiation (1). The oncogenic potential Downstream mitogenic signaling through mitogen-activated of the ErbB family members has been correlated to overexpression protein (MAP)/extracellular signal regulated kinase kinase, or alterations in a variety of human cancers, including breast, p44/42MAP kinase (MAPK) and stress signaling through ovarian, non–small cell lung, glioblastoma, prostate, pancreas, head c-Jun-NH2-kinase (JNK) 1 and c-Jun was impaired (1 Mmol/L, and neck, and other cancers (2). Thus, targeting the signaling 4–24 h), leading to cytostasis and cell cycle arrest within activity of the receptor has emerged as an attractive approach for 24 h by decreased cyclin D1, cyclin B1, and pSer795Rb and treatment and prevention of RTK-driven malignancies (3) with increased p27. Proliferation and colony formation were inhi- well-appreciated complexity [reviewed by Hynes and Lane (4)]. bited at 0.5 and 1 Mmol/L, respectively, and correlated with Signal transduction from the oncogenic HER2/neu leads to altered gene expression profiles. Diminished survival signal- neoplastic transformation, initiation, cellular immortalization, and ing through Akt, induction of bim, loss of connexin43, and tumor progression. This is in part due to dysregulated signaling decreased production of vascular endothelial growth factor-D through the HER2kinase domain to the growth and survival path- preceded caspase-3 and poly(ADP)ribose polymerase (PARP) ways. In models of spontaneous tumorigenesis (5), overexpressed cleavage and apoptosis (>50% 2 Mmol/L, 48 h). Oral admin- or mutated p185 leads toward the formation of homodimers istration of gefitinib was able to prevent the outgrowth of or heterodimers with other EGFRs. As these dimers transduce Bam1a tumor cells from palpable lesions, shrink established positive growth signals in a ligand-independent way (6), they are tumors, eliminate HER2and HER3 phosphorylation, and involved in the initiation and progression of neoplastic transfor- decrease MAPK and Akt signaling in vivo. A variant of the mation (7). Expression of the activated neu oncogene in transgenic Bam1a cell line, IR-5, with acquired ability to grow in 5 Mmol/L mice has been associated with both the synchronous (single step) gefitinib was developed and characterized. IR-5 bears a novel and the stochastic (ref. 8; multistep) transformation of mammary point mutation in the HER2/neu that corresponds to a L726I epithelium. Sequence analyses revealed that activation of neu in the ATP-binding pocket and correlates with a log decrease occurs through a single amino acid change in the transmembrane in sensitivity to gefitinib, increased heterodimerization with portion of the protein (7). This single point mutation replaces the EGFR and HER3, and impaired down-regulation. Gene expres- valine residue at position 664 in the transmembrane domain of sion profiling of IR-5 showed increased expression of EMP-1, p185 with glutamic acid, favors p185 homodimerization and heterodimerization, and transforms the Her-2/neu proto-oncogene into a dominant transforming oncogene (7). Monoclonal antibodies specific for the EGFR (9) and HER2(10) Note: Supplementary data for this article are available at Cancer Research Online receptors have been developed with successful clinical outcomes (http://cancerres.aacrjournals.org/). Requests for reprints: Marie P. Piechocki, Department of Otolaryngology-Head (10). More recently, several small-molecule inhibitors targeting the and Neck Surgery, Wayne State University, Room 423 Prentis Building of KCI, 110 East tyrosine kinase domains of specific RTKs have been developed as Warren Avenue, Detroit, MI 48201. Phone: 313-833-0715, ext. 2390; Fax: 313-833-7294; therapeutic agents to treat a variety of cancers and include classes E-mail: [email protected]. I2007 American Association for Cancer Research. of quinazolines that act as reversible or irreversible small-molecule doi:10.1158/0008-5472.CAN-07-0765 competitive substrate (ATP) inhibitors (11). www.aacrjournals.org 6825 Cancer Res 2007; 67: (14). July 15, 2007

In the case of the ErbB family, the preclinical efficacy of EGFR- activated rat HER2 oncogene in female BALB-NeuT transgenic selective, small-molecule tyrosine kinase inhibitors [TKI; i.e., mice has been described (21). We characterized the tumorigenic ZD1839 (gefitinib, Iressa; AstraZeneca) and OSI-774] in EGFR- potential of the Bam1a cell line and compared it with the dependent tumor models has been well characterized (12, 13) morphology of the parental. Bam1a cells grown in immunocom- ZD1839 (gefitinib) is an orally available, active, selective EGFR TKI petent BALB/c were evaluated using H&E and immunohistochem- that blocks signal transduction pathways implicated in proliferation istry for expression of the HER2receptor. Histologic features of the and survival of cancer cells and other host-dependent processes mammary tumor that was used to establish the Bam1a cell line are promoting cancer growth (13–15). In tyrosine kinase activity assays, shown in Fig. 1A. EGFR inhibition occurs at IC50 doses of 0.023 to 0.079 Amol/L. When grown from single-cell suspension in the fat pad of female Inhibition of c-erbB-2(IC 50,3Amol/L) and KDR/vascular endothe- BALB/c mice, tumors that formed were morphologically similar to lial growth factor (VEGF) receptor 2(3.7–33 Amol/L) also occurs but the parental and developed well-circumscribed and encapsulated at doses 100-fold higher than EGFR inhibition. In vitro, the effect of nodules, made of smaller acini, separated by fine stroma (Fig. 1A). gefitinib on human breast, ovarian, and colon cell lines expressing Acini have prominent, open lumens so that focally the confluence various amounts of EGFR and/or HER2has been described as of acini creates a cribriform pattern and often are filled with mainly cytostatic with increasing apoptotic activity at the higher eosinophilic secretions. The stroma is, overall, more developed doses (14, 15). Supra-additive antiproliferative effects were observed than in the parental tumor, creating a more pronounced division of with a broad range of cytotoxics (12, 16) and radiation (17) in vitro the acini and prominent comedo-type necrosis is present. and in vivo. The efficacy of gefitinib in HER2-overexpressing human Immunohistochemistry reveals strong and diffuse positivity for breast cancer cell lines has been described and seems to be HER2that is similar to that of the parental with a similar ( f2+) contingent upon the expression levels of both HER2and EGFR as membrane and cytoplasmic intensity. well as the degree to which these receptors are coupled to the Bam1a cells in vitro are uniform and cuboidal in appearance and growth [mitogen-activated protein kinase (MAPK)] and survival grow as monolayers to a high saturation density and are (Akt) pathways (14, 15). morphologically similar to several human breast cancer cell lines In recent studies, a subset of EGFR mutations in lung cancer (22). When evaluated in monolayer cultures in situ, HER2receptors patients was shown to correlate with clinical responsiveness to are diffusely distributed throughout the cytoplasm and as gefitinib therapy (18). Subsequently, several HER2and EGFR aggregates adjacent to the plasma membrane. Established cultures mutations and polymorphisms have been identified in a variety express uniformly high levels of HER2on their cell surface. of human tumors that influence patient prognosis and sensitivity We further characterized the Bam1a cell line using microarray to gefitinib (19) and may alter sensitivity to other RTKIs. analysis. Lobular carcinomas from BALB-NeuT transgenic animals We have already shown the effectiveness of gefitinib on the have been reported to have a gene expression profile that phosphorylation and signaling of the oncogenically activated (rat) resembles the genetic signature of human ErbB-2breast cancers HER2/neu in the context of a salivary gland adenocarcinoma (20). of the basal subtype (23, 24) that correlates with aggressive disease Suppression of HER2signaling by gefitinib induced profound and poor prognosis. The Bam1a cell line has a similar expression cytostasis by silencing growth signaling through MAPK. Neverthe- pattern as determined by whole-genome analysis. Highly expressed less, cells displayed intrinsic resistance to gefitinib-induced genes are reflected in Supplementary Table S1. When compared apoptosis because signaling from the Akt pathway was intact to with Universal mouse RNA on the Agilent whole-mouse genome sustain survival and inhibition of fas was required for apoptosis. chip containing 44,000 genes, 4,823 gene sequences were identified To test the efficacy of gefitinib in breast cancer expressing the as being overexpressed (>2-fold) in Bam1a when compared with mutated HER2/neu oncogene, we isolated and characterized a the universal mouse RNA prep. We defined the Bam1a tran- breast cancer cell line and the mechanisms of gefitinib with respect scriptome of 1,285 (1,045 unique) known genes (Supplementary to HER2signal transduction through the growth and survival Table S1) and compared it with the published profiles of mouse pathways as well as proliferation, anchorage-independent growth, mammary tumor virus (MMTV)-neu mammary tumors from two kinetics of cell cycle progression, cell death through apoptosis, different strains (23, 25) and genes characteristically expressed in and tumor growth and signal transduction in vivo. Further, we human ErbB-2 breast and basal subtype cancers (24, 26). For determined the ability of HER2-overexpressing breast cancer to comparative purposes, Supplementary Table S2lists the genes develop resistance to this agent and characterized mechanisms commonly overexpressed in Bam1a and MMTV-neu tumors and responsible for decreased sensitivity to gefitinib. We determined cell lines as reported by Astolfi (ref. 23; Supplementary Table S2A) that a novel point mutation in the ATP-binding pocket HER2/neu and genes overexpressed in Neu mammary tumors relative to age- receptor was responsible for the resistant phenotype and led to matched glands as reported by Landis (ref. 25; Supplementary additional genetic alterations that are likely to contribute to Table S2B). The table also includes several genes from the Bam1a acquired gefitinib resistance. profile that are present in the ‘‘intrinsic’’ gene list published by Sorlie et al. (24) that was used to define histologic subtypes of Materials and Methods human breast cancers (Supplementary Table S2C) and Bam1a genes associated with the human ErbB-2amplicon as described by Routine methodology was used as we have previously described (20). The Bertucci et al. (ref. 26; Supplementary Table S2D). These data specific experimental details are provided in Supplementary Methods. support the use of the 44K mouse CGH Agilent gene chip and universal mouse RNA as a screen for defining the genetic signature Results and Discussion of HER2/neu–expressing mouse mammary tumor cell lines and Characterization of the Bam1a cell line. The Bam1a cell line shows the similarities among Bam1a, independent Neu-expressing was derived from a mammary gland tumor that developed in a mammary tumors and cell lines, and human ErbB-2breast cancers. BALB-NeuT female mouse. Mammary gland tumorigenesis by the Additional breast cancer and HER2relevant genes that are

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Figure 1. A (i–iv), histochemical analysis of lobular carcinoma used to derive the Bam1a cell line (i, iii) and the outgrowth of Bam1a tumor cells in vivo (ii, iv). Histochemical analyses of tumors using H&E (i and ii). Immunohistochemical analyses of the HER2receptor ( iii and iv) were done on serial paraffin sections as described in Supplementary Methods. Histologically, the parental tumor was found to be a large, single proliferation, with vaguely multilobular architecture, each composed of smaller acini, separated by fine stroma, coalescing in varying degrees, and focally forming tightly packed, solid nodules. Acini were mostly devoid of lumens, or have fine ones, and outlines are ill defined, with lobules expanding irregularly adjacent adipose tissue. By immunohistochemistry, tumor cells showed (iii) strong and diffuse expression of HER-2, in a membranous pattern, whereas the other histologic components were negative. Bam1a cell line grown in vivo (ii, iv) in syngenic hosts were morphologically similar to the parental. HER-2staining is strong and diffuse in a membranous pattern. Magnification, Â40. B, effect(s) of gefitinib on ErbB receptor phosphorylation. Bam1a cells were grown to 80% confluence and subsequently treated by changing growth medium with fresh medium containing increasing levels of gefitinib and cultured for 4 or 24 h. Cells were harvested and lysates were extracted and processed as described in Supplementary Methods. Whole-cell lysates (20 Ag/lane) were resolved in 4% to 20% SDS-PAGE. Blots were probed with the indicated phosphospecific antibodies and pan actin was used as a loading control. Doses of gefitinib (Amol/L concentration) are indicated above each lane. C, effect of gefitinib on MAPK signaling. Bam1a cells were grown to 80% confluence and subsequently treated by changing growth medium with fresh medium containing increasing levels of gefitinib and cultured for 4 or24h. Cells were harvested and lysates were extracted and processed as described in Supplementary Methods. Whole-cell lysates (20 Ag/lane) were resolved in 4% to 20% SDS-PAGE. Blots were probed with the indicated phosphospecific antibodies and then reprobed with antibodies against total proteins. These data are representative of at least four independent preparations of whole-cell lysates. The trends are highly reproducible and consistent. Immunofluorescence photomicrographs: In parallel, Bam1a cells were grown on glass coverslips, treated with a medium change containing 0 Amol/L (i–iii)or1Amol/L (iv–vi) gefitinib for 4 h, fixed with methanol, and stained for HER2in green ( i and iii), 4¶,6-diamidino-2-phenylindole in blue (ii and v) or phospho-MAPK in red (iii and vi). Photographs were taken with the Â100 objective under oil immersion. D, effect of gefitinib on SAPK signaling. Bam1a cells were grown to 80% confluence and subsequently treated by changing growth medium with fresh medium containing increasing levels of gefitinib and cultured for 4 or 24 h. Cells were harvested and lysates were extracted and processed as described in Supplementary Methods. Whole-cell lysates (20 Ag/lane) were resolved in 4% to 20% SDS-PAGE. Blots were probed with the indicated phosphospecific antibodies and then reprobed with antibodies against total proteins. Doses of gefitinib (Amol/L concentration) are indicated above each lane. Immunofluorescence photomicrographs: In parallel, Bam1a cells were grown on glass coverslips, treated with a medium change containing 0 Amol/L (i)or1Amol/L (ii) gefitinib for 4 h, fixed with methanol, and stained for phospho-SAPK in red. Photos were taken with the Â100 objective under oil immersion. overexpressed in Bam1a cells and diverse human breast cancer cell known functions, we reduced this list to 504 (416 unique) genes lines (22) include ERBB-3, STARD10, ADAMTS8, FGF1, FGF9, BTC, with gefitinib-induced alterations in expression; 384 with decreased AREG, EPGN, CD44, CYP2J6, ITGB4, LAMA4, and STFA1. expression and 120 with increased expression and is provided as We determined the effect of gefitinib on this basal gene Supplementary Table S3. Table 1 lists unique genes with greatest expression pattern and identified 1,976 genes with a consistent level of modulation by gefitinib. In this gefitinib-sensitive cell line, (P > 0.05) change in expression level that was >2-fold. Based on genes regulating cell cycle and associated processes, HER2tumor www.aacrjournals.org 6827 Cancer Res 2007; 67: (14). July 15, 2007

Table 1. Gefitinib-induced changes in Bam1a gene expression

Agilent Genbank Average fold P (n = 3) Gene description and symbol accession accession difference*

A_51_P123405 NM_009772 0.025215827 0.996 Mus musculus budding uninhibited by benzimidazoles 1 homologue (Saccharomyces cerevisiae; Bub1), mRNA [NM_009772] A_52_P558401 NM_172301 0.029716673 0.999 Mus musculus cyclin B1 (Ccnb1), mRNA [NM_172301] A_52_P25599 NM_009860 0.04001378 1 Mus musculus cell division cycle 25 homologue C (S. cerevisiae; Cdc25c), mRNA [NM_009860] A_52_P415299 NM_011623 0.040513812 0.985 Mus musculus topoisomerase (DNA) II a (Top2a), mRNA [NM_011623] A_51_P415059 NM_011496 0.041450744 0.996 Mus musculus aurora kinase B (Aurkb), mRNA [NM_011496] A_51_P130015 NM_007900 0.042157208 0.999 Mus musculus ect2oncogene ( Ect2), mRNA [NM_007900] A_52_P162099 NM_001004140 0.042787931 0.993 Mus musculus cytoskeleton associated protein 2( Ckap2), mRNA [NM_001004140] A_51_P185688 NM_032006 0.047530595 0.92 Mus musculus matrix metalloproteinase 1a (interstitial collagenase; Mmp1a), mRNA [NM_032006] A_51_P253803 XM_133912 0.051843002 0.999 Mus musculus mRNA for Ki-67 [X82786] A_51_P481398 NM_010615 0.058657388 0.996 Mus musculus kinesin family member 11 (Kif11), mRNA [NM_010615] A_51_P230103 NM_009689 0.06272923 1 Mus musculus baculoviral IAP repeat-containing 5 (Birc5), transcript variant 1, mRNA [NM_009689] A_52_P18267 NM_009764 0.065913226 0.99 Mus musculus breast cancer 1 (Brca1), mRNA [NM_009764] A_51_P279575 NM_009860 0.077246343 1 Mus musculus cell division cycle 25 homologue C (S. cerevisiae; Cdc25c), mRNA [NM_009860] A_51_P326499 NM_007691 0.088213698 0.982 Mus musculus checkpoint kinase 1 homologue (S. pombe; Chek1), mRNA [NM_007691] A_52_P58558 NM_001037134 0.091717737 0.991 Mus musculus cyclin E2, mRNA (cDNA clone MGC:60620 IMAGE:30061057), complete cds. [BC053727] A_52_P243388 NM_008017 0.092834945 0.993 Mus musculus SMC2structural maintenance of chromosomes 2-like 1 (yeast; Smc2l1), mRNA [NM_008017] A_51_P366931 NM_145150 0.107044907 0.998 Mus musculus protein regulator of cytokinesis 1 (Prc1), mRNA [NM_145150] A_51_P441426 NM_019932 0.118613552 0.905 Mus musculus chemokine (C-X-C motif) ligand 4 (Cxcl4), mRNA [NM_019932] A_51_P324287 NM_024245 0.127708811 0.997 Mus musculus kinesin family member 23 (Kif23), mRNA [NM_024245] A_52_P193265 NM_025676 0.132855012 0.966 Mus musculus minichromosome maintenance deficient 8 (S. cerevisiae; Mcm8), mRNA [NM_025676] A_51_P448741 NM_009425 0.135304137 0.932 Mus musculus tumor necrosis factor (ligand) superfamily, member 10 (Tnfsf10), mRNA [NM_009425] A_51_P324934 NM_008563 0.143838287 0.999 Mus musculus minichromosome maintenance deficient 3 (S. cerevisiae; Mcm3), mRNA [NM_008563] A_52_P487686 NM_001001332 0.14482608 0.941 Mus musculus stefin A1 (Stfa1), mRNA [NM_001001332] A_51_P328333 NM_007634 0.14891755 0.999 Mus musculus cyclin F (Ccnf ), mRNA [NM_007634] A_52_P170882 NM_009928 0.152664203 0.989 Mus musculus procollagen, type XV (Col15a1), mRNA [NM_009928] A_52_P350148 NM_017392 0.158192173 0.98 Mus musculus cadherin EGF LAG seven-pass G-type receptor 2 (Celsr2), mRNA [NM_017392] A_52_P219473 NM_011799 0.162001839 0.743 Mus musculus cell division cycle 6 homologue (S. cerevisiae; Cdc6), transcript variant 1, mRNA [NM_011799] A_51_P377094 NM_0077420.171042697 0.921 Mus musculus procollagen, type I, a1(Col1a1), mRNA [NM_007742] A_51_P150912NM_011497 0.17334682 1 Mus musculus aurora kinase A (Aurka), mRNA [NM_011497] A_52_P584302 NM_008696 0.180708637 0.993 Mus musculus mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), mRNA [NM_008696] A_52_P299915 NM_011943 0.180804743 0.978 Mus musculus mitogen-activated protein kinase kinase 6 (Map2k6), mRNA [NM_011943] A_51_P214557 NM_010959 0.2035635 0.881 Mus musculus oncoprotein induced transcript 3 (Oit3), mRNA [NM_010959] A_51_P286665 NM_011249 0.20899749 1 Mus musculus retinoblastoma-like 1 (p107; Rbl1), mRNA [NM_011249]

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Table 1. Gefitinib-induced changes in Bam1a gene expression (Cont’d)

Agilent Genbank Average fold P (n = 3) Gene description and symbol accession accession difference*

A_51_P497332 NM_008709 0.209301423 0.967 Mus musculus neuroblastoma myc-related oncogene 1 (Nmyc1), mRNA [NM_008709] A_51_P255699 NM_010809 0.211435537 0.986 Mus musculus matrix metalloproteinase 3 (Mmp3), mRNA [NM_010809] A_52_P662752 NM_020570 0.211797223 0.938 Mus musculus X-ray repair complementing defective repair in Chinese hamster cells 2(Xrcc2 ), mRNA [NM_020570] A_52_P482897 NM_009704 0.21556259 0.986 Mus musculus amphiregulin (Areg), mRNA [NM_009704] A_52_P582374 NM_178825 0.217340253 0.887 Mus musculus epithelial stromal interaction 1 (breast; Epsti1), transcript variant b, mRNA [NM_178825] A_52_P329197 NM_010049 0.218103373 0.996 Mus musculus dihydrofolate reductase (Dhfr), mRNA [NM_010049] A_52_P54218 NM_008371 0.24977048 0.979 Mus musculus interleukin 7 (Il7), mRNA [NM_008371] A_51_P249957 NM_008005 0.256306007 0.93 Mus musculus fibroblast growth factor 18 (Fgf18), mRNA [NM_008005] A_52_P796682 NM_007633 0.266689223 0.931 Mus musculus cyclin E1 (Ccne1), mRNA [NM_007633] A_52_P117090 NM_016681 0.269876487 0.982 Mus musculus CHK2checkpoint homologue (S. pombe ; Chek2), mRNA [NM_016681] A_51_P122356 NM_009878 0.281024733 0.996 Mus musculus cyclin-dependent kinase inhibitor 2D (p19, inhibits CDK4; Cdkn2d), mRNA [NM_009878] A_51_P139978 NM_020612 0.284976833 0.949 Mus musculus cell matrix adhesion regulator (Cmar), mRNA [NM_020612] A_51_P204454 NM_013787 0.288494197 0.991 Mus musculus SCF complex protein Skp2mRNA, complete cds. [AF083215] A_52_P463340 NM_178596 0.289607373 0.987 Mus musculus gap junction membrane channel protein m1(Gjc1), mRNA [NM_178596] A_51_P120275 NM_017378 0.300594087 0.983 Mus musculus protocadherin 12( Pcdh12), mRNA [NM_017378] A_52_P498608 NM_016710 0.302066697 0.995 Mus musculus nucleosome-binding protein 1 (Nsbp1), mRNA [NM_016710] A_51_P202714 NM_018761 0.30690475 0.999 Mus musculus catenin (cadherin-associated protein), a-like 1 (Ctnnal1), mRNA [NM_018761] A_51_P480855 NM_021385 0.3082394 0.992 Mus musculus RAD18 homologue (S. cerevisiae; Rad18), mRNA [NM_021385] A_51_P290290 NM_011102 0.314272293 0.976 Mus musculus protein kinase C, g (Prkcc), mRNA [NM_011102] A_52_P377750 NM_011053 0.320462427 1 Mus musculus programmed cell death protein 11 (Pdcd11), mRNA [NM_011053] A_51_P298790 NM_010896 0.320510883 0.997 Mus musculus neurogenin 1 (Neurog1), mRNA [NM_010896] A_52_P73307 NM_134092 0.323017367 1 Mus musculus Mdm2, transformed 3T3 cell double minute p53 binding protein (Mtbp), mRNA [NM_134092] A_51_P113182NM_013584 0.327713863 0.974 Mus musculus leukemia inhibitory factor receptor (Lifr), mRNA [NM_013584] A_52_P590546 NM_011631 0.334020433 0.998 Mus musculus tumor rejection antigen gp96 (Tra1), mRNA [NM_011631] A_52_P282058 NM_007739 0.336306323 0.972 Mus musculus procollagen, type VIII, a 1(Col8a1), mRNA [NM_007739] A_51_P112405 NM_011113 0.34067499 0.993 Mus musculus urokinase plasminogen activator receptor (Plaur), mRNA [NM_011113] A_52_P327236 NM_148922 0.353719653 0.996 Mus musculus transformed mouse 3T3 cell double minute 1 (Mdm1), transcript variant 2, mRNA [NM_148922] A_52_P646684 NM_178381 0.362261213 0.961 Mus musculus Trp53 inducible protein 5 (Trp53i5), mRNA [NM_178381] A_52_P70255 NM_009283 0.37297335 0.979 Mus musculus signal transducer and activator of transcription 1 (Stat1), mRNA [NM_009283] A_51_P144303 NM_013641 0.375085977 0.998 Mus musculus prostaglandin E receptor 1 (subtype EP1; Ptger1), mRNA [NM_013641] A_51_P230382 NM_008815 0.375166887 0.984 Mus musculus ets variant gene 4 (E1A enhancer binding protein, E1AF; Etv4), mRNA [NM_008815] A_51_P419226 NM_025393 0.38857333 0.98 Mus musculus S100 calcium binding protein A14 (S100a14), mRNA [NM_025393] A_52_P213932 NM_009621 0.39318495 0.982 Mus musculus a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1 (Adamts1), mRNA [NM_009621] A_52_P76988 NM_030220 0.399363717 0.973 Mus musculus Sp2transcription factor (Sp2 ), mRNA [NM_030220] A_51_P493649 NM_023135 0.399434357 0.994 Mus musculus sulfotransferase family 1E, member 1 (Sult1e1), mRNA [NM_023135] A_51_P187491 NM_053087 0.402711757 0.968 Mus musculus epithelial mitogen (Epgn), mRNA [NM_053087] A_51_P500813 NM_010708 0.415638603 0.997 Mus musculus lectin, galactose binding, soluble 9 (Lgals9), mRNA [NM_010708] A_52_P651298 NM_183417 0.42817818 0.99 Mus musculus cyclin-dependent kinase 2( Cdk2), transcript variant 1, mRNA [NM_183417]

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Table 1. Gefitinib-induced changes in Bam1a gene expression (Cont’d)

Agilent Genbank Average fold P (n = 3) Gene description and symbol accession accession difference*

A_51_P311038 NM_001024139 2.017729033 0.999 Mus musculus a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 15 (Adamts15), mRNA [NM_001024139] A_52_P680941 NM_010284 2.125057033 0.933 Mus musculus growth hormone receptor (Ghr), mRNA [NM_010284] A_52_P534870 NM_009741 2.1289126 0.567 Mus musculus B-cell leukemia/lymphoma 2(Bcl2 ), transcript variant 1, mRNA [NM_009741] A_52_P672803 NM_008906 2.129077267 0.88 Mus musculus protective protein for h-galactosidase (Ppgb), mRNA [NM_008906] A_52_P433889 NM_007798 2.130360467 0.951 Mus musculus cathepsin B (Ctsb), mRNA [NM_007798] A_51_P417077 NM_011700 2.135687533 1 Mus musculus villin-like (Vill), mRNA [NM_011700] A_52_P404533 NM_013560 2.158545233 0.995 Mus musculus heat shock protein 1 (Hspb1), mRNA [NM_013560] A_51_P390480 NM_178020 2.162897367 0.821 Mus musculus hyaluronidase 3 (Hyal3), mRNA [NM_178020] A_51_P319460 NM_011019 2.174328267 0.984 Mus musculus oncostatin M receptor (Osmr), mRNA [NM_011019] A_51_P196605 NM_026880 2.177814133 0.995 Mus musculus PTEN induced putative kinase 1 (Pink1), mRNA [NM_026880] A_52_P93910 NM_010939 2.1833415 0.951 Mus musculus neuropilin 2, mRNA (cDNA clone IMAGE:6827742) [BC057028] A_52_P533809 NM_010495 2.189329533 0.938 Mus musculus inhibitor of DNA binding 1 (Id1), mRNA [NM_010495] A_51_P291361 NM_001013365 2.193070567 0.991 Mus musculus oncostatin M (Osm), mRNA [NM_001013365] A_51_P220062 NM_008609 2.213941267 0.876 Mus musculus matrix metalloproteinase 15 (Mmp15), mRNA [NM_008609] A_51_P263965 NM_010442 2.263528067 0.996 Mus musculus heme oxygenase (decycling) 1 (Hmox1), mRNA [NM_010442] A_52_P183088 NM_008714 2.342757133 0.835 Mus musculus Notch gene homologue 1 (Drosophila; Notch1), mRNA [NM_008714] A_52_P144173 NM_008317 2.352243767 0.898 Mus musculus hyaluronidase 1 (Hyal1), mRNA [NM_008317] A_52_P925197 NM_007570 2.3584059 0.826 Mus musculus B-cell translocation gene 2, antiproliferative (Btg2), mRNA [NM_007570] A_51_P479865 2.463538333 0.849 Mus musculus galactosidase, h 1-like, mRNA (cDNA clone MGC:28635 IMAGE:4222994), complete cds. [BC021773] A_52_P162957 NM_177603 2.464996233 0.973 Mus musculus frequently rearranged in advanced T-cell lymphomas 2( Frat2), mRNA [NM_177603] A_52_P329207 NM_007969 2.482193867 0.863 Mus musculus extracellular proteinase inhibitor (Expi), mRNA [NM_007969] A_51_P500984 NM_008655 2.5388139 0.643 Mus musculus growth arrest and DNA-damage-inducible 45h (Gadd45b), mRNA [NM_008655] A_51_P345649 NM_011058 2.5982837 0.28 Mus musculus platelet-derived growth factor receptor, a polypeptide (Pdgfra), mRNA [NM_011058] A_51_P494430 NM_031166 2.665149833 0.996 Mus musculus inhibitor of DNA binding 4 (Id4), mRNA [NM_031166] A_52_P58208 NM_013605 2.704789033 0.243 Mus musculus mucin 1, transmembrane (Muc1), mRNA [NM_013605] A_51_P398723 NM_010228 2.726919833 0.976 Mus musculus FMS-like tyrosine kinase 1 (Flt1), mRNA [NM_010228] A_51_P175580 NM_021897 2.754532467 0.852 Mus musculus transformation related protein 53 inducible nuclear protein 1 (Trp53inp1), mRNA [NM_021897] A_51_P369311 NM_015760 2.764042933 0.912 Mus musculus NADPH oxidase 4 (Nox4), mRNA [NM_015760] A_52_P273891 NM_018881 2.8159229 0.592 Mus musculus flavin containing monooxygenase 2( Fmo2), mRNA [NM_018881] A_51_P266683 NM_010107 2.866408533 0.89 Mus musculus ephrin A1 (Efna1), mRNA [NM_010107] A_51_P293087 NM_008606 2.9066167 0.808 Mus musculus matrix metalloproteinase 11 (Mmp11), mRNA [NM_008606] A_52_P465012 NM_028392 3.0081877 0.901 Mus musculus protein phosphatase 2(formerly 2A),regulatory subunit B (PR 52), h isoform (Ppp2r2b), transcript variant 2, mRNA [NM_028392] A_51_P343833 NM_009421 3.230854067 0.484 Mus musculus Tnf receptor-associated factor 1 (Traf1), mRNA [NM_009421] A_51_P355906 NM_021099 3.2519368 0.323 Mus musculus kit oncogene (Kit), mRNA [NM_021099] A_51_P463765 NM_011595 3.285614467 0.571 Mus musculus tissue inhibitor of metalloproteinase 3 (Timp3), mRNA [NM_011595] A_52_P15388 NM_008522 3.340970833 0.865 Mus musculus lactotransferrin (Ltf ), mRNA [NM_008522] A_51_P137991 NM_009525 3.3973666 0.885 Mus musculus wingless-related MMTV integration site 5B (Wnt5b), mRNA [NM_009525] A_52_P363110 NM_010207 3.4384949 0.822 Mus musculus fibroblast growth factor receptor 2, mRNA (cDNA clone MGC:102519 IMAGE:5349249), complete cds [BC091652] A_51_P444447 NM_007679 3.797682933 0.968 Mus musculus CCAAT/enhancer binding protein (C/EBP), y (Cebpd), mRNA [NM_007679]

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Table 1. Gefitinib-induced changes in Bam1a gene expression (Cont’d)

Agilent Genbank Average fold P (n = 3) Gene description and symbol accession accession difference*

A_51_P204153 NM_010518 3.8213841 0.399 Mus musculus insulin-like growth factor binding protein 5 (Igfbp5), mRNA [NM_010518] A_52_P384394 NM_138313 3.8343365 0.822 Mus musculus Bcl2modifying factor, mRNA (cDNA clone MGC:90616 IMAGE:6847314), complete cds. [BC079650] A_52_P584188 NM_008043 4.0804498 0.669 Mus musculus frequently rearranged in advanced T-cell lymphomas (Frat1), mRNA [NM_008043] A_51_P452629 NM_011905 4.686056867 0.814 Mus musculus toll-like receptor 2(Tlr2 ), mRNA [NM_011905] A_52_P472324 NM_011414 4.766305433 0.407 Mus musculus secretory leukocyte protease inhibitor (Slpi), mRNA [NM_011414] A_52_P540219 NM_011594 4.911111233 0.324 Mus musculus tissue inhibitor of metalloproteinase 2( Timp2), mRNA [NM_011594] A_51_P321341 NM_133670 5.3052078 0.765 Mus musculus sulfotransferase family 1A, phenol-preferring, member 1 (Sult1a1), mRNA [NM_133670] A_51_P470935 NM_027208 7.642287667 0.294 Mus musculus dehydrogenase/reductase (SDR family) member 6 (Dhrs6), mRNA [NM_027208] A_51_P453909 NM_007817 11.02520367 0.265 Mus musculus cytochrome P450, family 2, subfamily of, polypeptide 2 (Cyp2f2), mRNA [NM_007817] A_51_P110301 NM_009778 12.12553833 0.729 Mus musculus complement component 3 (C3), mRNA [NM_009778]

*Average fold difference is the ratio (n =3,P value cutoff >0.05) of Bam1a RNA from gefitinib-treated cells (1 Amol/L, 24 h) to Bam1a RNA from sham-treated cells. RNA from gefitinib-treated cells was labeled in red (cy5); RNA from sham-treated cells was labeled in green (cy3). Three independent RNA preparations per condition were analyzed on three separate chips and averaged; a confidence cutoff of >0.95 was used for the raw values of red/green before normalization. The fold change (**treated/**control) = normalized ratio value(cy5/cy3), fold change cutoff >2fold (up-regul ate), or <1/2(down-regulate). The P value represents the consistency between the three replicate samples.

biology and progression are the primary targets for down- autophosphorylation of the activated HER2signaling domain regulation. In particular, KI67, CYCLINB1, CHEK1, MMP1A, KIF11, decreased in a dose-dependent fashion. Phosphorylation of Tyr- CKAP2, AREG, SKP2, COL1A1, FGF18, MAP2K6, STFA1, ETV4, 1248 and Tyr-1221 of the HER2 was reduced by f50% between the S100A14, LGALS9, BIRC5, and CELSR2. Genes that were up- doses of 1 to 2 Amol/L, as one would expect based on the IC50 regulated by gefitinib included oncogenes, and genes involved in (Fig. 1B). EGFR is expressed at a lower level than the HER2 matrix remodeling, drug metabolism, antiproliferative genes, heat oncogene in these cells and is, as expected, more sensitive to shock, and DNA damage. These included ADAMTS15, CTSB, HYAL3, gefitinib (Fig. 1B). Phosphorylation on EGFR Tyr-1173 and Tyr-992 HSPB1, OSMR, OSM, NOTCH1, BGAL1L, BTG2, BMF, DHRS6, was diminished by f50% by 1 Amol/L but was not completely GADD45B, PDGFRA, TRP53INP1, FLT1, NOX4, FMO2, TIMP3, eliminated. WNT5B, KIT, LTF, FGFR2, CEBPD, CYP2F2, C3, SULT1A1, and SLPI. It has been well documented that in tissues where ErbB2is Several have been confirmed by independent microarray Western mutated or overexpressed, it serves as the dominant signaling blot. Gene signatures are becoming increasingly important in receptor due to its promiscuous heterodimerization and impaired designing treatment strategies that use agents that target specific endocytosis (32). The residual phosphorylation that is detected on signal transduction pathways (27). In the case of gefitinib, several the EGFR may represent phosphorylation that persists in HER2/ studies have identified sensitive and resistant profiles that correlate EGFR heterodimers. Phosphorylation on Tyr-1289 of HER3, the with responsiveness (28, 29). These profiles will be discussed when kinase inactive family member was most sensitive probably due to we compare Bam1a with its resistant variant. its dependence on heterodimerization and transphosphorylation. Effect of gefitinib on HER2, ErbB-3, and EGFR receptor At higher doses of gefitinib, total ErbB-3 receptor levels were clearly phosphorylation and downstream signaling. Although EGFR is up-regulated, suggesting enhanced receptor stabilization in the the preferred target for gefitinib, several groups have shown the presence of gefitinib. This was even more pronounced after 24 h. effectiveness of this agent against a variety of HER2-overexpressing Tyrosine phosphorylation on HER2Tyr-877, the Src phosphoryla- human cell lines, including lung (30) and breast (31). We first tion site was least sensitive to gefitinib-induced suppression, determined the concentration of gefitinib necessary to eliminate consistent with retention of Tyr-416 phosphorylation on Src phosphorylation of the HER2/neu, EGFR, and ErbB-3 in Bam1a (the active state; Fig. 3A) at the same concentrations of gefitinib. cells. Cross-talk from another signaling pathway that is insensitive to As shown in Fig. 1B, treatment with gefitinib for 4 or 24 h had a gefitinib suppression may modulate these interactions. Although dramatic effect on phosphorylation of all ErbB family members. significant decrease in phosphorylation was evident with 1 Amol/L Distinct down-regulation of receptor autophosphorylation sites IR on various tyrosine residues in all three receptors, 6 Amol/L IR is evident with 1 Amol/L IR in whole-cell lysates at 4 h of treat- was required for near-complete elimination of phosphotyrosines. ment. When treated for 24 h with increasing doses of gefitinib, After 24 h, phosphorylation recovered slightly at the lowest doses www.aacrjournals.org 6831 Cancer Res 2007; 67: (14). July 15, 2007

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Cancer Research of gefitinib (1–2 Amol/L) but dropped sharply as total HER2and is downstream of both MAPK and JNK, was greatly impaired. EGFR levels both decreased coincident with their ubiquitination Failure to activate c-Jun would eliminate transcription of genes and proteasomal degradation (not shown). modulated by serum and growth factor signaling. Downstream of the ErbBs, mitogenic signaling through MAP/ Gefitinib induces cytostasis through multiple mechanisms. extracellular signal regulated kinase (ERK) kinase 1/2(MEK1/2) Consistent with the observed suppression of mitogenic signaling, and p44/42MAPK was greatly diminished. Phosphorylation of gefitinib effectively inhibited proliferation of Bam1a cells in a MEK1/2and MAPK was completely eliminated between 1 and time- and dose-dependent manner. Within 48 h, a 50% reduction 2 Amol/L within 4 h in whole-cell lysates and cytoplasm and in the rate of proliferation was observed in the presence of 250 to remained absent over 24 h (Fig. 1C) despite the retention of 500 nmol/L gefitinib (Fig. 2A). Inhibition of anchorage-independent phosphotyrosines in the autophosphorylation sites of the ErbBs growth required 1 Amol/L gefitinib (Fig. 2B). Cell cycle analysis (Fig. 1B). We also detected a 2-fold increase in cell surface showed the onset of cytostasis within 24 h of treatment with HER2/neu expression in gefitinib-treated cells by flow cytometry gefitinib (Fig. 2C). Most studies have characterized the effects (not shown) and membrane accentuation by immunofluorescence of gefitinib on cell cycle as G0 arrest (14). Similar to our observa- (Fig. 1C). This is similar to our finding in salivary gland tions in salivary gland carcinoma overexpressing HER2/neu (20), carcinoma (20). the effect of gefitinib on Bam1a cell cycle kinetics is primarily We next evaluated the effect of gefitinib on stress signaling due to inhibition of S-phase entry and execution. At 1 Amol/L, through the stress-activated protein kinase (SAPK) pathway and when maximum inhibition is achieved within 24 h, there is observed a rapid activation of SAPK and JNK in the presence of retention of cells in G0 (49–71%), a sharp decrease in the S-phase gefitinib within 4 h. Using 1 Amol/L gefitinib for 4 h, we observed population (23–2%) but no change in the G2M fraction (28–28%). that active JNK and c-Jun (not shown) were eliminated from the This cell cycle profile is consistent with the effects of gefitinib cytoplasm and translocated to the nucleus (Fig. 1D). After 24 h, on cell cycle regulators controlling the various phases of the active JNK (p46) was no longer observed but activation of SAPK cell cycle as shown in Fig. 2D. We have obtained qualitatively (p54) persisted. Signaling to the serum response factor c-Jun, which and mechanistically similar results using the irreversible inhibitor

Figure 2. Effect of gefitinib on proliferation (A) and anchorage-independent growth (B) in Bam1a cells. Bam1a cells were monodispersed and seeded in the presence of increasing concentrations of gefitinib or diluent (DMSO). Metabolic activity of quadruplicate wells was evaluated at 24 h intervals by the addition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Points, mean for each respective concentration of gefitinib at each time point; bars, SE. Cytostasis occurs between 250 and 500 nmol/L of gefitinib. Similarly, Bam1a were monodispersed and suspended in soft agar containing varying amounts of gefitinib and subcultured for 14 d. Box, mean for the total number of colonies per well; bars, SE; solid symbols, individual data (n = 3). C and D, cell cycle distribution of Bam1a treated for 24 h with gefitinib. Percentage of cells in G0-G1 (shaded columns); S phase (solid columns); and G2-M (striped columns). There is a sharp decline in S-phase activity between the doses of 0.25, 0.5, and 1.0 Amol/L gefitinib (solid columns; 22%, 9%, and 1.4%, respectively). Cell cycle histograms depict cell cycle distribution after 24 h in the absence (left) or presence (right)of1Amol/L gefitinib. The increase in G0-G1 from 49% to 71% coincides with a proportional decrease in S phase from 23% to 1.4%, whereas G2-M activity is unaffected (i.e., 28–28%). D, Western blot analysis of whole-cell lysates for cell cycle control proteins in Bam1a cells after 4 or 24 h in the presence of increasing concentrations (Amol/L) of gefitinib as indicated above each lane.

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Figure 3. A and B, effect of gefitinib on survival signaling and apoptosis. Bam1a cells were grown to 80% confluence and subsequently treated by changing growth medium with fresh medium containing increasing levels of gefitinib and cultured for 4 or 24 h. Cells were harvested and lysates extracted and processed as described in Supplementary Methods. Whole-cell lysates (20 Ag/lane) were resolved in 4% to 20% SDS-PAGE. Blots were probed with the indicated phosphospecific or cleavage-specific antibodies and then reprobed with antibodies against total proteins. Doses of gefitinib (Amol/L concentration) are indicated above each lane. B, gefitinib suppresses cx43 and VEGF-D, alters bcl-2family members, and induces proteolytic cleavage leading to apoptosis in Bam1a cells. Cell treat ment, lysate preparation, and Western blot were as described (above). For apoptosis assays, cells were treated for 24 to 48 h with various doses of gefitinib and evaluated by Annexin staining. Columns, percentage Annexin V–positive cells detected by flow cytometry after 24 h (open columns)or48h(solid columns). C and D, the effect of gefitinib on Bam1a tumor growth and signal transduction in vivo. Tumor-bearing mice were treated with 100 mg/kg gefitinib by oral gavage for 5 consecutive days per week for 4 wks beginning on day 25 after tumor cell injection (50,000 cells) when palpable tumor lesions reached 5 mm. Tumor growth was monitored weekly and measured with calipers in two perpendicular diameters. Boxes, mean size range of five animals per group; solid symbols, individual tumor area measurements; whiskers, SE. D, Western blot analysis of total tumor lysates. Female Balb-NeuT transgenic mice bearing multiple macroscopic lobular carcinomas were treated for 5 consecutive days with 100 mg/kg gefitinib (IR) or diluent (0) by oral gavage and sacrificed 2h after the last dose. Tumor lysates (50 Ag) were resolved in SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed for the indicated phosphospecific antigens.

PD168393 (11) with respect to HER2/neu phosphorylation, signal within 4 h of treatment. By 24 h, complete inhibition of these transduction, proliferation, and cell cycle (data not shown). We regulators and cyclin B1 is achieved with 1 Amol/L gefitinib, the suspect that similar small-molecule inhibitors with specificities for dose and time at which MAPK and Akt signaling is silenced the ATP-binding pocket of the EGFR and/or ErbB-2(i.e., erlotinib, (Figs. 1C and 3A) and cytostasis is achieved (Fig. 2A and C). lapatinib) will have efficacy against this intrinsically sensitive breast Changes in these cell cycle regulators explain the distribution of cancer cell line. cells in the different phases of the cell cycle. Cyclin D1 levels drop Cell cycle arrest was multifactorial and somewhat complex. To significantly due to ubiquitin-mediated degradation (data not determine mechanisms involved in cytostasis, we evaluated the key shown) and increased levels p27 reinforce inhibition of cyclin D1 cell cycle regulators controlling each phase and checkpoint of the and progression of the G0-G1 phase. Furthermore, loss of cyclin cell cycle (Fig. 2D). We observed a significant decrease in the levels D1–dependent activities (by cdk4/6) and suppression cdk2by of cell cycle regulators cyclin D1, phosphoSer795Rb, phosphoY15cdc2 increased levels of p27 would result in hypophosphorylation of Rb www.aacrjournals.org 6833 Cancer Res 2007; 67: (14). July 15, 2007

and impaired cell cycle progression through G0. Downstream of induction of bim, a rheostat for sensing growth factor deprivation. these events, hypophosphorylated Rb binds to E2F and impairs DNA We previously reported the induction of bim by gefitinib in salivary synthesis and S-phase progression. As a result, the S-G2 transition gland carcinoma (20). The early induction of bim by gefitinib in does not occur. The hypophosphorylation of cdc2leads to cyclin B1 Bam1a cells at 4 h (Fig. 3B) could be mediated through inhibition ubiquitination and degradation (Fig. 2D) to impair the G2-M of MAPK as is seen with MEK inhibitors. Bim has also been transition. Cell cycle histograms depict cell cycle distribution after implicated in anoikis and was recently shown to be sensitive to 24 h in the absence (Fig. 2D, left) or presence (Fig. 2D, right)of modulation by ERK and MEK inhibitors (35). Transcript levels for 1 Amol/L gefitinib. Cytostasis is characterized by (a) a decrease in BTG2 and BMF were also increased by gefitinib (Table 1) whereas the percentage of cells in S phase (23–1.4%), (b) accumulation of survivin transcript levels were decreased. Diminished phosphory- cells in G0 (49–71%), and (c) retention of cells G2-M (i.e., 28–28%). lation of bad on serine residue 112, which is under MAPK control, Therefore, G0 arrest and impaired G-S phase transition was due to provides additional support for a dominant role of the mitogenic decreased levels of cyclin D1 and increased levels of p27 and pathway in regulation of the bcl-2family. As a result of the hypophosphorylation of Rb causing a retention of cells in G0. increased mitochondrial stress generated by gefitinib exposure, the However, accumulation of additional cells in G0 (true G0 arrest) did onset of apoptosis through the caspase cleavage pathway is not occur due to impaired transitioning through the G2-M detectable within 24 h over a range of doses (Fig. 3B). Within 24 h checkpoint, due to decreased cyclin B1 levels. of treatment (1 Amol/L gefitinib), 33% of cells undergo apoptosis, We also have overwhelming support from our microarray data as determined by Annexin binding. Apoptosis increased in a time- that cell cycle regulators are significantly modulated at the and dose-dependent manner coincident with caspase-3 and PARP transcriptional level. CYCLIN B1 is a primary target of down- cleavage observed within 24 h at 2 Amol/L gefitinib in whole-cell regulation and is decreased >30-fold by a 24 h treatment with lysates (Fig. 3B). 1 Amol/L gefitinib relative to cells receiving control medium The ability of gefitinib to efficiently induce apoptosis in HER2/ (Table 1; Supplementary Table S3). Other cyclins, including A2, B2, neu–overexpressing breast cancer is in contrast to our observations E1, and E2, and the gene encoding KI67 were also transcriptionally in salivary gland carcinoma where induction of bim and suppressed. Because this treatment is effective in reducing hypophosphorylation of bad by gefitinib did not result in caspase phosphorylation of HER2/neu, MAPK signaling, proliferation, and cleavage and apoptosis. Salivary gland carcinoma exhibited an cell cycle, one would expect alterations in gene expression levels to intrinsic resistance to gefitinib. The primary difference between have a major contribution to the growth and cell cycle suppression these two activated HER2/neu–overexpressing models is in survival induced by gefitinib and vice versa. signaling through the Akt pathway. Our data indicate that in Effects of gefitinib on survival signaling and apoptosis. Bam1a breast cancer cells, which express high levels of HER3, Concentrations of gefitinib >1 Amol/L failed to generate additional unlike salivary gland carcinoma, HER2signaling is tightly coupled therapeutic benefit with respect to proliferation and cell cycle to the Akt survival pathway. Several studies have shown that arrest but higher concentrations were needed to effectively impair coexpression of HER2and HER3 improves tumor cell sensitivity to anchorage-independent growth and survival. Inactivation of gefitinib and other RTKIs by coupling it to the phosphatidylinositol survival signaling by gefitinib is usually associated with inhibition 3-kinase pathway to facilitate down regulation of the survival of HER2/EGFR and HER2/HER3 activity (30, 31) and phosphatidy- signaling pathway (30). In cell lines where gefitinib impedes the linositol 3-kinase leading to apoptosis through the intrinsic Akt/survival signaling pathway (33) apoptosis occurs through the (mitochondrial) pathway (33). intrinsic pathway. The activity of the serine/threonine protein kinase Akt/PKB is Novel targets of gefitinib in breast cancer. We also observed modulated by various growth and survival factors. Akt promotes that Bam1a cells express hyperphosphorylated form of the gap cell survival through two distinct pathways: inhibition of junction protein connexin43 (cx43) in culture. Hyperphosphory- proapoptotic (death) signals and activation of IKK-a signaling lated cx43 impairs GJIC and cell-cell coupling and this form of the to p65 nuclear factor-nh (NF-nh). In Fig. 3A, within 4 h of protein has been shown to be up-regulated in breast hyperplasias treatment with gefitinib, we observed reduction of Akt phosphor- and carcinomas and neoformed capillaries (36). Upon treatment ylation. At this early time point, the doses (4–6 Amol/L) required with gefitinib, there is a marked reduction in the phosphorylated to diminish Akt phosphorylation were higher than those needed form of cx43 within 4 h (Fig. 3B). By 24 h, cx43 protein is no longer to inhibit HER2/neu (1–2 Amol/L) or MAPK (1 Amol/L). However, detected. Regulation of cx43 by gefitinib probably represents within 24 h, complete inhibition of Akt phosphorylation was several distinct mechanisms. Direct mechanisms of gefitinib that achieved with 1 Amol/L. Inhibition of phosphorylation of signal are mediated through tyrosine kinase and MAPK activities can transducers and activators of transcription 1 (STAT1) and STAT3 alter cx43 phosphorylation, transcription, and degradation. Indirect was similar to that of the EGFR and probably represents cross- mechanisms that can modulate cx43 may occur through the effects talk between these receptors through the MAPK pathway. In of gefitinib on the dynamics of cell cycle, cell growth, adhesion, and some instances, STATs can serve an autocrine function to induce cytoskeletal remodeling. Our microarray analyses did not reveal a cytokine expression and rescue cells undergoing growth factor change in cx43 expression but did indicate that two other gap deprivation or EGFR inhibition (34). Phosphorylation of NF-nh junction genes (GJB4 and GJC1) were sensitive to modulation at p65 was not adversely affected by gefitinib and perhaps slightly the transcriptional level along with multiple adhesion molecules enhanced. and cytoskeletal kinases that affect connexin trafficking, stability, Impaired growth and survival signaling was tightly coupled to and assembly. alterations in proapoptotic BH3-only mitochondrial proteins bim The c-fos–inducible lymphangiogenic factor, VEGF-D (37), is also and bad. In the context of HER2overexpression, bim is dramatically reduced at 1 Amol/L gefitinib within 24 h. Loss of constitutively suppressed through a MAPK-dependent mechanism activated c-Jun in the cytoplasm and nucleus of Bam1a treated with (35). Suppression of EGFR/HER2signaling by gefitinib leads to gefitinib may contribute to decreased expression of VEGF-D. Many

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Mechanisms of Gefitinib in Mammary Gland Cancer groups have reported the antiangiogenic effects of gefitinib as activation of factors that enhance the transcriptional activity of the decreases in VEGF (13, 38) and/or interleukin-8 (39), but there have estrogen receptor (46). been no reports of gefitinib effects on expression of VEGF-D. In the case of gefitinib resistance, EGFR receptor mutations that These two novel targets of gefitinib, cx43 and VEGF-D, may have alter the ATP-binding cleft differentially stabilize or destabilize important implications in the clinical management of breast cancer. gefitinib binding and competition for ATP, leading to altered Disruption of cx43 inhibits breast cancer diapedesis (40) and hydrolysis and catalytic rate of the kinase (47). The L858A mutation disruption of VEGF-D impairs lymphangiogenesis, providing a novel in the activation loop increases kinase activity and sensitivity to mechanism for abolishing tumor cell extravasation and metastasis. gefitinib (41). In subpopulations, patients with the L858A mutation Effectiveness of gefitinib in vivo. To determine our ability to that originally respond to gefitinib, outgrowth of resistant tumors effectively target HER2growth and survival signaling in vivo,we harbor a second mutation at codon 790. This T790M mutation in challenged mice with Bam1a tumor cells and tested the ability of the hinge region decreases sensitivity to gefitinib f100-fold (48). gefitinib to suppress tumor outgrowth and shrink established Several somatic exon 20 mutations in HER2 have also been tumors. We originally observed that 50 mg/kg gefitinib was able to identified in lung adenocarcinomas. Insertion of YVMA at codon suppress the outgrowth of small palpable Bam1a nodules when 776 in the HER2kinase domain confers resistance to gefitinib and a administered daily for 5 days a week. This dose was effective for gain of function (49). 3 weeks, at which time nodules began to increase in size in the Acquired resistance to gefitinib in Bam1a cells. We developed 50 mg/kg treatment group, subsequent treatment of these animals a variant of the Bam1a cell line from a soft agar colony that grew in with 100 mg/kg was able to impede tumor growth (not shown). the presence of gefitinib. Cells that were recovered from this colony Animals bearing established Bam1a tumors were effectively treated were continuously exposed to medium supplemented with with 100 mg/kg gefitinib as this dose administered 5 days a gefitinib. Concentrations of gefitinib were gradually increased over week for 4 weeks eliminated tumor burden (Fig. 3C). Finally, we time. The clone that we developed, designated IR-5, grows in established that we could effectively target HER2in naturally 5 Amol/L gefitinib. Compared with the parental cell line, IR-5 cells occurring mammary LCIS tumor-bearing female transgenic mice display a disorganized growth pattern in culture and a lack of and detected reduced levels of phosphorylated HER2, HER3, MAPK, contact inhibition. Cells express lower levels of HER2/neu on their and Akt phosphorylation in tumor biopsies taken from animals cell surface compared with the parental cell line (Fig. 4A). To treated for 5 consecutive days with 100 mg/kg gefitinib (Fig. 3D). characterize the mechanisms associated with the acquired These findings establish that we can indeed target HER2/neu signal resistance to gefitinib, we first compared the effect of gefitinib on transduction pathways in naturally occurring tumors in vivo. HER2receptor phosphorylation and heterodimerization in the These preliminary findings using the Bam1a cell line show the parental Bam1a cell line and the resistant IR-5 cell line. As shown effectiveness of gefitinib in targeting the signaling pathways in Fig. 4B, HER2phosphorylation is eliminated in Bam1a cells downstream of the HER2/neu oncogene and mechanisms of treated with 5 Amol/L gefitinib and preserved in IR-5 cells up to action. The acquisition of resistance in this intrinsically sensitive 15 Amol/L gefitinib. Decreased phosphorylation of HER2and EGFR cell line could represent the development of mutations in the correlated with an increase receptor migration in SDS-PAGE in target receptor that reduce drug binding, biochemical uncoupling whole-cell lysates and HER2immunoprecipitates. Phosphorylation of receptor kinase activity from specific growth and survival of ErbB-3 is not detected in Bam1a treated with gefitinib. In control signaling pathways, and/or alterations in gene expression that Bam1a cells, ErbB-3 that is present in HER2/ErbB-3 heterodimers is minimize the role of RTKs in tumor cell survival. The issues of weakly phosphorylated and gefitinib completely eliminates this intrinsic and acquired resistance or hypersensitivity to RTK phosphorylation. In IR-5, phosphorylation of ErbB-3 is detected at inhibitors have been addressed by several investigations. Recently, all concentrations of gefitinib. HER2/ErbB-3 heterodimers are more several somatic and acquired mutations in the EGFR (41, 42) and abundant in IR-5 cells compared with Bam1a cells. Phosphoryla- HER2(43) have been identified, which alter responsiveness to tion of ErbB-3 in HER2/neu heterodimers is slightly reduced in IR-5 gefitinib and other RTKIs (44, 45). These mutations dictate levels of treated with 15 Amol/L gefitinib and correlates with the reduction intrinsic sensitivity or resistance at the level of the target receptor. in HER2/neu phosphorylation observed at this dose. Similarly, we In some instances, the outgrowth of resistant subpopulations in observe increased migration of EGFR in HER2heterodimers in IR-5 previously sensitive tumors has shown the presence of additional treated with 15 Amol/L gefitinib, suggesting decreased EGFR receptor mutations that dramatically reduce the IC50 (44). The phosphorylation. In Bam1a cells, EGFR and HER2/EGFR hetero- intrinsic resistance in tumors lacking receptor mutations occurs dimers show increased migration upon treatment with gefitinib. through other biochemical mechanisms that uncouple receptor Others have shown that HER2kinase domain mutations show phosphorylation and catalytic activity from downstream signaling increased kinase activity over the wild-type receptor and increased to growth and survival targets and usually relate to constitutively association with and phosphorylation of EGFR and ErbB-3 (49). active survival signaling intermediates or loss of negative These mutant HER2also form gefitinib-resistant ErbB-3 and EGFR regulatory factors. heterodimers and confer a malignant phenotype. Similar to these Characterizing the mechanism(s) responsible for acquired studies, we found that EGFR homodimers in IR-5 cells were also resistance also generates a valuable tool for dissecting and less sensitive to inhibition by gefitinib than in Bam1a cells (Fig. 4C). mapping interactions and the potential for the development of Phosphotyrosine is not detected in EGFR homodimers from Bam1a autocrine or paracrine pathways that emerge as a consequence of treated with gefitinib and total EGFR is dramatically reduced and chronic pathway suppression or attenuation. This is supported by coimmunoprecipitates with hsp60, indicative of receptor down- the observation that the mechanisms responsible for the acquired regulation. The HER2that coimmunoprecipitates with EGFR in resistance to the ErbB2TKI, lapatinib, in breast cancer cells, these cells is also down-regulated. In IR-5 cells, EGFR homodimers reflects a shift in the sole dependence of cell survival on the ErbB-2 are less sensitive to gefitinib-induced down-regulation than the to a codependence on the ErbB-2and estrogen receptor via parental cell line. We observe a slight decrease in EGFR www.aacrjournals.org 6835 Cancer Res 2007; 67: (14). July 15, 2007

Figure 4. A, comparison of parental (Bam1a, right) and gefitinib resistant (IR-5, left) cells by photomicroscopy of cell growth and morphology in vitro (top) and by flow cytometric analysis of cell surface HER2/neu expression (bottom). Phase-contrast photomicrographs (Â20 objective) were taken of confluent cultures of Bam1a (right) and IR-5 (left) cells. Histograms of Bam1a (right) or IR-5 (left) stained for an epitope in the extracellular domain of the rat HER2/neu. Filled histograms, specific antibody-stained cells detected with phycoerythrin-labeled secondary; clear overlay, cells stained with nonimmune IgG and phycoerythrin-conjugated secondary. The mean channel fluorescent values for Bam1a and IR-5 were 1,185 and 432, respectively. B and C, effect of gefitinib on ErbB receptor phosphorylation and heterodimerization in Bam1a and IR-5 cells. Bam1a and IR-5 cells were treated for 4 h by replacing medium with the indicated concentrations of gefitinib. Whole-cell lysates (25 Ag, right) or ErbB-2immunoprecipitates ( IP; left) were resolved in 4% to 12% SDS-PAGE. Western blots were probed for the indicated antigens. C, Western blot analysis of EGFR immunoprecipitated lysates from Bam1a (right) or IR-5 (left) treated as described above and probed for the indicated antigens. D, identification of a HER2/neu point mutation in IR-5 cells. Genomic DNA from Bam1a (top) or IR-5 (bottom) was PCR amplified and sequenced using ABI PRISM 3100. Electropherograms compare nucleotide sequences of the antisense strand of the rat HER2/neu kinase domain from the parental Bam1a cell line (top) and the gefitinib-resistant IR-5 variant (bottom). A single point mutation is detected (C/G to A/T) encoding a leucine to isoleucine mutation at the conserved codon 726 of the human ErbB-2orthologue. Homologous sequences from the human EGFR and human ErbB-2(identical in the rat HER2/neuorthologue) are aligned. Dashes, mismatches; *, point mutations at specific codons that have been reported to correlate with responsiveness to gefitinib (50, 51). R, resistance; S, sensitizing. phosphorylation with 5 Amol/L gefitinib but this does not result in in Fig. 4D) that changes the conserved leucine at codon 726 to an increased EGFR migration or association with hsp60. Treatment isoleucine relative to the human erbB-2(Fig. 4 D). This sequence is with 15 Amol/L was effective in decreasing EGFR phosphorylation conserved between human and rat and aligns with a homologous and increasing receptor migration and coupling to hsp60. ErbB-2 region in exon 18 of the human EGFR. These regions encode a that coimmunoprecipitates with EGFR are also down-regulated. portion of the ATP-binding pocket that is highly conserved among These data show a log-shift in the HER2/neu sensitivity to gefitinib the human EGFR and erbB-2and the orthologous rat HER2/neu in IR-5 cells and resembles resistance that occurs through receptor transgene (Fig. 4D). No additional mutations in or around the mutations. Acquired resistance to imatinib is associated with T334I transmembrane region or the kinase domain were detected. When mutation of c-Abl. Acquired resistance to gefitinib is associated aligned with the EGFR, this residue is within hydrophobic region II with T790M mutation of the EGFR (48). Pharmacophore modeling that represents the hydrophobic slot of the ATP-binding pocket. It has shown that the sites of these mutations align. It is proposed is possible that this mutation is sufficient to alter the competition that T790M in the hinge region of the ATP-binding pocket causes between ATP and gefitinib in favor of ATP, leading to hydrolysis, ligand to escape (47). receptor autophosphorylation, and kinase activity. Gefitinib-induced mutation of the HER2/neu. We sequenced Several mutations have been detected in this region of the EGFR the HER2/neu from the parental and IR-5 cell lines to determine (50). We denote two in particular that correspond to enhanced whether similar mutations had emerged that could explain the sensitivity (i.e., G719S) and resistance (i.e., E709G) to gefitinib in the observed resistance. We detected a single nucleotide point clinic and in vitro (50, 51). Thus, our novel mutation in the HER2/ mutation of C to A (Note: G to T on the antisense strand shown neu corresponds to a site in the EGFR that is frequently mutated in

Cancer Res 2007; 67: (14). July 15, 2007 6836 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Mechanisms of Gefitinib in Mammary Gland Cancer human cancers with differential sensitivity and resistance to of these specific markers, as they predict drug sensitivity in diverse gefitinib. We are not aware of any reports of a similar mutation in tumor targets (lung and breast) of differing etiologies and primary this region of ErbB-2that correlate with intrinsic or acquired oncogene dependence (EGFR and HER2). The three genes that sensitivity or resistance to specific inhibitors. We suspect that were commonly up-regulated in IR-5 and gefitinib resistant lung decreased sensitivity of the EGFR and ErbB-3 to gefitinib (in IR-5 cancers included MAP1B, CDH2, and GAS5, and two genes that cells Fig. 4B and C) is mediated through the mutated HER2/neu were preferentially expressed in Bam1a and gefitinib-sensitive lung and not the result of mutations in either of these receptors. This is cancers included TMEM30B and ALDH1A3. These similarities supported by the fact that mutant HER2with resistance to EGFR suggest that resistance developed by exposure to gefitinib amplifies RTKIs harbor a gain of function that endows them with the genes involved in intrinsic resistance to gefitinib. capacity to heterodimerize and transphosphorylate kinase-dead In the case of acquired resistance, one biomarker, EMP-1, has EGFR and ErbB-3 (49). been identified in vivo and validated as a clinical marker of de novo Altered signaling activity of the mutated HER2/neu and/or and acquired resistance to gefitinib (29). Serial passage and selection in the presence of gefitinib may have lead to additional continued exposure of a HER2-overexpressing adenocarcinoma to genetic changes that are commonly seen in HER2-expressing cell gefitinib in vivo resulted in the emergence of a gefitinib-resistant lines with intrinsic resistance to gefitinib. To characterize the variant with a >10-fold increase in expression of EMP-1. Clinical mutated receptor and resistant phenotype, we compared the gene samples from gefitinib-treated patients correlated with this expression profiles of the parental Bam1a cell line and IR-5 under observation. We found that relative to the parental Bam1a cell various treatment conditions and the effects of gefitinib on signal line, IR-5 cells with acquired in vitro resistance to gefitinib have a transduction pathways, cell cycle, and apoptosis in IR-5 cells. 6.6-fold increase EMP-1 and a 2.1-fold increase in EMP-2. Our data We used the 44K mouse CGH array to generate an expression support a role for EMP-1 expression in the acquisition of resistance profile of the IR-5 cell line and compared it with the gene to gefitinib in a HER-2–overexpressing adenocarcinoma. expression profile of the sensitive, parental cell line, Bam1a. Using a Reviewing trends in gene expression data, we observed that 2-fold cutoff limit and P > 0.05, we identified 1,642divergent genes several genes that were differentially expressed between Bam1a and between the two cell lines. From this, we generated a list of 475 IR-5 were also targets that were sensitive to modulation by gefitinib (430 unique) known genes that were differentially expressed by in Bam1a cells. We found that 57 genes (11.2%) that were 2-fold or greater. This comprehensive gene list is provided as modulated by gefitinib in Bam1a cells were also differentially Supplementary Table S4 and a selection of these genes is given in expressed between Bam1a and IR-5. In this comparison (Supple- Table 2. Several striking differences in the gene expression profiles mentary Table S5), 33 of the gefitinib-induced changes in Bam1a of these two cell lines include the overrepresentation of Notch corresponded to the difference between Bam1a and IR-5, whereas signaling–related genes in IR-5 cells as well as the presence of 24 were inconsistent. It is interesting to conceive that this subset several genes that have recently been linked to clinical resistance to of gefitinib-responsive genes may have an active role in driving and gefitinib. Notch pathway genes that are increased in IR-5 include maintaining resistance to gefitinib. NOTCH-1 (6-fold), NOTCH-3 (2.3-fold), JAG-1 (3.5-fold), JAG-2 (5.6- Because resistance to gefitinib in the IR-5 cell line is not complete fold), MTAP1B (3.8-fold), MMP7 (7.7-fold), CBL (2.9-fold), NFjb1 with respect to the phosphorylation of the HER2/neu, we used high (2.1-fold), CD44 (2.1-fold), and b-CATENIN (2.8-fold). Overexpres- doses of gefitinib to characterize mechanism(s) of responsiveness at sion of activated Notch-1 and Notch-3 has been shown to induce the transcriptional and signal transduction levels to determine the mammary tumor formation in mice (52). Additional up-regulated similarities and differences between Bam1a and IR-5. Treatment of genes that may contribute to the resistant phenotype include FLT-1, IR-5 cells with 10 Amol/L gefitinib induced change in expression PDGFB, MAF, CAV1, and EFNA1. In breast cancer models with levels of 338 genes, 200 known genes, and 190 unique when acquired resistance to the ErbB-2–specific kinase inhibitor, compared with basal gene expression of cells cultured in 5 Amol/L lapatinib, resistant tumor cells amplify signaling through the gefitinib. This complete list is provided as Supplementary Table S6 estrogen receptor pathway, leading to the up-regulation of Foxo3a and includes 14 up-regulated genes and 184 down-regulated genes. and caveolin (46). Although we also detect a 3.5-fold increase in We found that several genes that were sensitive to high-dose caveolin in gefitinib-resistant IR-5 cells, a functional role for this gefitinib in IR-5 cells were also sensitive when an irreversible EGFR protein in our estrogen receptor–negative cell line is not predicted. inhibitor was tested against gefitinib-resistant, EGFR mutant cell Thus, in the context of HER-2–overexpressing estrogen receptor– lines (53). These genes included AREG, CENPA, CCNB1, DEPDC1A, negative breast cancer, acquired resistance to gefitinib amplifies FOSL1, HMMR, KNTC2, NEK2, NUSAP1, and SHCBP1 and are genes involved in the Notch signaling pathway. deemed to be critical in antiproliferative and antisurvival response By examining the genes that are down-regulated in IR-5 and in gefitinib-resistant cells bearing mutant HER family members. abundantly expressed in the parental cell line, it is possible to Although gefitinib-resistant cells can still respond to EGFR/HER-2– detect a trend in the genes that correlate with intrinsic sensitivity targeted therapies, the extent of the response is blunted due to to gefitinib. For instance, elevated expression of the EGFR, compromised signaling activities of the mutant receptors per se and phosphatases (DUSP18, PPP2R2B); drug-metabolizing enzymes genetic changes that provide cells with compensatory mechanisms (ABCG2, EPHX2, CYP2J6, and NOX4) and apoptosis and cell cycle and survival pathways similar to those associated with intrinsic regulators (TRAF1, STK17B, P15, and GSPT2) in Bam1a cells may drug resistance. The extent to which these cell lines still depend on facilitate the antiproliferative and proapototic activity of gefitinib. the HER2/EGFR for growth and survival will determine the utility of Indeed, gene profiling of NSCLC cell lines with mutant and wild- targeting the HER2axis to control these diseases and understanding type EGFR was used to identify genes that could globally predict the mechanism(s) of resistance will indicate treatment modalities gefitinib sensitivity or resistance (28). We compared our list of that could be successful. differentially expressed genes to this data set and found Relative to the parental Bam1a cell line, the number of gefitinib- concordance between several genes, attesting to the robustness responsive genes in IR-5 cells and the level of modulation is www.aacrjournals.org 6837 Cancer Res 2007; 67: (14). July 15, 2007

Table 2. Differential gene expression between gefitinib-sensitive (Bam1a) and gefitinib-resistant (IR-5) breast cancer cells

Agilent Genbank Average fold P (n = 3) Gene description and symbol accession accession difference*

A_51_P408346 NM_008329 0.07 7.37EÀ06 Mus musculus IFN activated gene 204 (Ifi204), mRNA [NM_008329] A_51_P408595 NM_016960 0.088 0.01 Mus musculus chemokine (C-C motif) ligand 20 (Ccl20), mRNA [NM_016960] A_51_P377526 NM_009293 0.119 1.59EÀ05 Mus musculus steroid sulfatase (Sts), mRNA [NM_009293] A_51_P398723 NM_010228 0.128 0.000491 Mus musculus FMS-like tyrosine kinase 1 (Flt1), mRNA [NM_010228] A_51_P426096 NM_010810 0.13 0.00523 Mus musculus matrix metalloproteinase 7 (Mmp7), mRNA [NM_010810] A_51_P293401 AK089315 0.142 0.0077 Mus musculus clone:F730004F21 product: thrombospondin 1, full insert sequence. [AK089315] A_51_P508510 Z11886 0.15 9.41EÀ05 Mus musculus notch-1 mRNA. [Z11886] A_51_P368012NM_010783 0.15 0.028 Mus musculus MyoD family inhibitor (Mdfi), mRNA [NM_010783] A_52_P120037 NM_010128 0.152 6.00EÀ05 Mus musculus epithelial membrane protein 1 (Emp1), mRNA [NM_010128] A_51_P253279 NM_017383 0.164 0.00354 Mus musculus contactin 6 (Cntn6), mRNA [NM_017383] A_52_P183088 NM_008714 0.172 0.00152 Mus musculus Notch gene homologue 1 (Drosophila; Notch1), mRNA [NM_008714] A_52_P618427 NM_011057 0.173 0.0314 Mus musculus platelet derived growth factor, B polypeptide (Pdgfb), mRNA [NM_011057] A_51_P451338 NM_010588 0.177 0.00135 Mus musculus jagged 2( Jag2), mRNA [NM_010588] A_51_P253074 NM_027979 0.193 0.00388 Mus musculus chitinase 1 (chitotriosidase; Chit1), mRNA [NM_027979] A_51_P463765 NM_011595 0.201 4.71EÀ05 Mus musculus tissue inhibitor of metalloproteinase 3 (Timp3), mRNA [NM_011595] A_51_P322265 NM_027817 0.219 0.00268 Mus musculus GRB2-related adaptor protein (Grap), mRNA [NM_027817] A_51_P302273 NM_007887 0.226 0.000982 Mus musculus deubiquitinating enzyme 1 (Dub1), mRNA [NM_007887] A_52_P423810 BC027262 0.235 0.000967 Mus musculus metallothionein 1, mRNA (cDNA clone MGC:27821 IMAGE:3483861), complete cds. [BC027262] A_51_P405167 NM_001025577 0.243 0.00098 Mus musculus avian musculoaponeurotic fibrosarcoma (v-maf) AS42oncogene homologue (Maf ), mRNA [NM_001025577] A_52_P540219 NM_011594 0.247 8.33EÀ05 Mus musculus tissue inhibitor of metalloproteinase 2( Timp2), mRNA [NM_011594] A_52_P371237 NM_008737 0.263 0.000175 Mus musculus neuropilin 1 (Nrp1), mRNA [NM_008737] A_51_P510782 NM_130859 0.267 1.27EÀ07 Mus musculus caspase recruitment domain family, member 10 (Card10), mRNA [NM_130859] A_52_P332081 NM_177274 0.268 0.0202 Mus musculus neuronal growth regulator 1 (Negr1), mRNA [NM_177274] A_52_P282058 NM_007739 0.276 0.00077 Mus musculus procollagen, type VIII, a 1(Col8a1), mRNA [NM_007739] A_52_P306845 NM_007616 0.282 0.001 Mus musculus caveolin, caveolae protein 1 (Cav1), mRNA [NM_007616] A_51_P322138 NM_024441 0.282 0.0312 Mus musculus heat shock protein 2( Hspb2), mRNA [NM_024441] A_51_P280906 NM_013822 0.289 0.00616 Mus musculus jagged 1 (Jag1), mRNA [NM_013822] A_51_P266683 NM_010107 0.295 3.27EÀ05 Mus musculus ephrin A1 (Efna1), mRNA [NM_010107] A_52_P180741 NM_053142 0.315 2.12EÀ05 Mus musculus protocadherin h 17 (Pcdhb17), mRNA [NM_053142] A_51_P469433 NM_011659 0.320.000148 Mus musculus tumor necrosis factor receptor superfamily, member 4 (Tnfrsf4), mRNA [NM_011659] A_51_P187121 NM_008127 0.32 0.00033 Mus musculus gap junction membrane channel protein h 4(Gjb4), mRNA [NM_008127] A_51_P421418 NM_008293 0.32 0.0491 Mus musculus hydroxysteroid dehydrogenase-1, y<5>-3-h (Hsd3b1), mRNA [NM_008293] A_51_P466229 NM_026840 0.329 1.60EÀ05 Mus musculus platelet-derived growth factor receptor-like (Pdgfrl), mRNA [NM_026840] A_51_P323011 AK045005 0.339 0.00394 Mus musculus clone:B130018P07 product: hypothetical CBL

proto-oncogene NH2-terminal domain containing protein, full insert sequence. [AK045005] A_52_P644465 AK050511 0.347 0.00597 Mus musculus clone:C820005J08 product:14-3-3 protein ~/y (protein kinase C inhibitor protein-1; kcip-1; mitochondrial import stimulation factor S1 subunit), full insert... A_51_P444447 NM_007679 0.358 2.26EÀ07 Mus musculus CCAAT/enhancer binding protein (C/EBP), y (Cebpd), mRNA [NM_007679]

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Table 2. Differential gene expression between gefitinib-sensitive (Bam1a) and gefitinib-resistant (IR-5) breast cancer cells (Cont’d)

Agilent Genbank Average fold P (n = 3) Gene description and symbol accession accession difference*

A_52_P430886 NM_010662 0.379 0.0317 Mus musculus keratin complex 1, acidic, gene 13 (Krt1-13), mRNA [NM_010662] A_51_P449878 AK006128 0.386 0.000404 Mus musculus clone:1700019L09 product: ATP-binding cassette, subfamily C (CFTR/MRP), member 3, full insert sequence. [AK006128] A_51_P419226 NM_025393 0.391 0.00738 Mus musculus S100 calcium binding protein A14 (S100a14), mRNA [NM_025393] A_52_P70949 NM_183322 0.401 0.0387 Mus musculus Nur77 downstream gene 1 (Ndg1), mRNA [NM_183322] A_52_P70381 AK028772 0.408 0.00647 Mus musculus clone:4732455E07 product: mitogen-activated protein kinase kinase (MKK7) mRNA, full insert sequence. [AK028772] A_52_P174915 NM_010288 0.409 3.64EÀ05 Mus musculus gap junction membrane channel protein a1(Gja1), mRNA [NM_010288] A_51_P220162 NM_008716 0.44 0.00112 Mus musculus Notch gene homologue 3 (Drosophila; Notch3), mRNA [NM_008716] A_51_P170807 NM_016693 0.441 0.00144 Mus musculus mitogen-activated protein kinase kinase kinase 6 (Map3k6), mRNA [NM_016693] A_52_P301591 XM_194344 0.451 0.00333 Predicted: mitogen-activated protein kinase kinase kinase 10 [Mus musculus], mRNA sequence [XM_194344] A_51_P285027 NM_017392 0.46 0.0374 Mus musculus cadherin EGF LAG seven-pass G-type receptor 2( Celsr2), mRNA [NM_017392] A_51_P159194 NM_008416 0.4620.00037 Mus musculus Jun-B oncogene (Junb), mRNA [NM_008416] A_51_P110301 NM_009778 0.476 1.09EÀ05 Mus musculus complement component 3 (C3), mRNA [NM_009778] A_51_P437240 NM_007929 0.479 0.000102 Mus musculus epithelial membrane protein 2(Emp2 ), mRNA [NM_007929] A_52_P229536 AK045226 0.482 9.14EÀ06 Mus musculus clone:B130049I05 product:CD44 antigen, full insert sequence. [AK045226] A_51_P312327 NM_022415 0.491 0.00405 Mus musculus prostaglandin E synthase (Ptges), mRNA [NM_022415] A_51_P184886 NM_011920 2.302 2.92EÀ06 Mus musculus ATP-binding cassette, subfamily G (white), member 2( Abcg2), mRNA [NM_011920] A_52_P106259 NM_207655 2.603 0.000609 Mus musculus epidermal growth factor receptor (Egfr), transcript variant 1, mRNA [NM_207655] A_51_P214197 NM_133810 2.802 0.000115 Mus musculus serine/threonine kinase 17b (apoptosis-inducing; Stk17b), mRNA [NM_133810] A_51_P196113 NM_019945 3.79 0.0437 Mus musculus microtubule associated serine/threonine kinase 1 (Mast1), mRNA [NM_019945] A_52_P220573 NM_009250 3.848 0.0266 Mus musculus serine (or cysteine) proteinase inhibitor, clade I, member 1 (Serpini1), mRNA [NM_009250] A_51_P497332NM_008709 5.120.0303 Mus musculus neuroblastoma myc-related oncogene 1 (Nmyc1), mRNA [NM_008709] A_51_P162272 NM_009524 5.247 0.0359 Mus musculus wingless-related MMTV integration site 5A (Wnt5a), mRNA [NM_009524] A_51_P164530 NM_010210 5.314 0.0151 Mus musculus fragile histidine triad gene (Fhit), mRNA [NM_010210] A_51_P245895 NM_172632 5.326 0.0251 Mus musculus mitogen-activated protein kinase 4 (Mapk4), mRNA [NM_172632] A_51_P418375 NM_023844 5.438 0.00283 Mus musculus junction adhesion molecule 2( Jam2), mRNA [NM_023844] A_51_P172054 NM_019521 5.502 1.58EÀ05 Mus musculus growth arrest specific 6 (Gas6), mRNA [NM_019521] A_51_P343833 NM_009421 5.837 0.0005 Mus musculus Tnf receptor-associated factor 1 (Traf1), mRNA [NM_009421] A_51_P404193 NM_022435 6.112 0.0148 Mus musculus trans-acting transcription factor 5 (Sp5), mRNA [NM_022435]

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Table 2. Differential gene expression between gefitinib-sensitive (Bam1a) and gefitinib-resistant (IR-5) breast cancer cells (Cont’d)

Agilent Genbank Average fold P (n = 3) Gene description and symbol accession accession difference*

A_51_P307840 NM_009978 6.261 0.00198 Mus musculus cystatin 8 (cystatin-related epididymal spermatogenic; Cst8), mRNA [NM_009978] A_51_P500135 NM_1456026.444 6.30EÀ05 Mus musculus N-myc downstream regulated gene 4 (Ndrg4), mRNA [NM_145602] A_51_P449171 NM_007464 6.4620.00744 Mus musculus baculoviral IAP repeat-containing 3 (Birc3), mRNA [NM_007464] A_51_P125355 NM_173740 6.945 4.16EÀ05 Mus musculus monoamine oxidase A (Maoa), mRNA [NM_173740] A_51_P497152NM_013518 7.71 0.0362 Mus musculus fibroblast growth factor 9 (Fgf9), mRNA [NM_013518] A_52_P132165 NM_053262 8.275 0.00186 Mus musculus dehydrogenase/reductase (SDR family) member 8 (Dhrs8), mRNA [NM_053262] A_51_P287418 NM_008548 8.991 5.59EÀ06 Mus musculus mannosidase 1, a (Man1a), mRNA [NM_008548] A_52_P64356 NM_010097 9.349 0.00261 Mus musculus SPARC-like 1 (mast9, hevin; Sparcl1), mRNA [NM_010097] A_51_P355906 NM_021099 11.97 0.000278 Mus musculus kit oncogene (Kit), mRNA [NM_021099] A_51_P215849 NM_019508 12.57 0.0048 Mus musculus interleukin 17B (Il17b), mRNA [NM_019508] A_52_P498241 NM_007670 13.74 0.000219 Mus musculus cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4; Cdkn2b), mRNA [NM_007670] A_51_P116940 NM_007940 13.74 0.000261 Mus musculus epoxide hydrolase 2, cytoplasmic (Ephx2), mRNA [NM_007940] A_51_P220278 NM_028392 16.28 0.00976 Mus musculus protein phosphatase 2(formerly 2A), regulatory subunit B (PR 52), h isoform (Ppp2r2b), transcript variant 2, mRNA [NM_028392] A_51_P445562NM_145142 16.53 0.00506 Mus musculus carbohydrate sulfotransferase 10 (Chst10), mRNA [NM_145142] A_52_P89064 NM_173745 21.72 0.00855 Mus musculus dual specificity phosphatase 18 (Dusp18), mRNA [NM_173745] A_51_P506328 NM_010008 25.17 0.013 Mus musculus cytochrome P450, family 2, subfamily j, polypeptide 6 (Cyp2j6), mRNA [NM_010008] A_51_P376050 NM_029495 40.98 0.000461 Mus musculus epithelial stromal interaction 1 (breast; Epsti1), transcript variant a, mRNA [NM_029495]

A_51_P301394 NM_008179 47.49 0.0023 Mus musculus G1-S phase transition 2( Gspt2), mRNA [NM_008179] A_52_P82741 NM_010479 57.03 0.000194 Mus musculus heat shock protein 1A (Hspa1a), mRNA [NM_010479] A_52_P636948 BC030896 61.84 0.00398 Mus musculus platelet-derived growth factor, D polypeptide, mRNA (cDNA clone MGC:31518 IMAGE:4489485), complete cds [BC030896] A_51_P369311 NM_015760 63.1 1.13EÀ06 Mus musculus NADPH oxidase 4 (Nox4), mRNA [NM_015760] A_51_P353221 NM_011582 64.58 8.35EÀ06 Mus musculus thrombospondin 4 (Thbs4), mRNA [NM_011582] A_52_P232637 NM_007857 92.05 0.00968 Mus musculus desert hedgehog (Dhh), mRNA [NM_007857]

*Average fold difference compares the ratios (n =3,P value cutoff > 0.05) of genes expressed by Bam1a versus IR-5 cells using the data obtained from Bam1a RNA normalized to Universal Mouse RNA versus IR-5 RNA normalized to Universal Mouse RNA. RNA from the cell lines was labeled in red (cy5), Universal RNA is labeled in green (cy3). Three independent RNA preparations per condition were analyzed on three separate chips and averaged; a confidence cutoff of >0.95 was used for the raw values of red/green before normalization. The fold change (**Bam1a/**Universal Mouse) = normalized ratio value(cy5/cy3).

dramatically reduced. Nevertheless, we were able to determine that mechanism(s) of responsiveness to gefitinib despite the other several genes remained responsive to gefitinib in IR-5 cells, genetic changes that have evolved during the acquisition of indicating the preservation of specific signaling pathways involved resistance. in transcriptional responses. Supplementary Table S7 lists the 26 To evaluate the effect of gefitinib in the culture medium of IR-5 genes that remain responsive to gefitinib in IR-5 cells. Only one cells, we determined the gene expression pattern of IR-5 cells after gene, V-MAF, is consistently up-regulated. Twenty-five genes, gefitinib washout. We observed 382genetic changes consisting of including, AREG, BCHE, CXCL4, CYCLINB1, SHCBP1, MMP1A, 176 known and 168 unique genes. To our surprise, the majority, MSH5, and PI16, are consistently down-regulated by gefitinib. This 154 genes, were down-regulated and only 22 genes were found to set of genes probably plays a functional role in preserving the be increased. These data suggest that the presence of gefitinib is

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Mechanisms of Gefitinib in Mammary Gland Cancer required for maintaining the expression of several genes. If the with the increased (2.9-fold) gene expression levels in IR-5 cells presence of gefitinib in the culture medium was responsible for the (Table 2). However, phosphorylation of cbl was dramatically suppression of specific genes, we would expect that elimination of reduced in IR-5, even in the absence of gefitinib. Dephosphorylated gefitinib would restore gene expression. This was the case for cbl abrogates CIN85 binding and receptor internalization, leading TOPIIA (3.25), LPD (2.31), MMP10 (3.65), MMP1A (2.32), SERPINA11 to enhanced receptor stabilization. These data may indicate altered (4.14), and USP26 (2.89). Two of these genes, MMP1A and LPD, are trafficking of the mutated HER2in IR-5 cells. Mutant EGFR has also sensitive to suppression by high-dose gefitinib. The phenotypic been shown to preferentially associate with underphosphorylated consequences of these genetic changes was further evaluated by cbl to impede internalization (54). Although we are able to achieve characterizing the effects of high-dose gefitinib treatment on IR-5 considerable down-regulation of receptor phosphorylation with compared with the response pattern profile of the parental cell line. 15 Amol/L of gefitinib in IR-5 cells, MAPK and STAT3 phosphor- Effects of gefitinib on IR-5 cells. In IR-5 cells, treatment with ylation persist (Fig. 5B). Cross-talk between pathways or establish- 12 Amol/L gefitinib for 24 h was required to diminish phosphor- ment of an autocrine loop (34) capable of signaling through these ylation of HER2(Fig. 5 A). At this dose, ErbB-3 phosphorylation and coupled effectors are likely to be responsible for this observation. EGFR levels were also reduced. Interestingly, c-Cbl levels were This in contrast to the profile generated by Bam1a at 2 Amol/L increased in IR-5 cell lysates compared with Bam1a. This correlates where residual phosphorylation on HER2is still detected and the

Figure 5. A, sensitivity of ErbB receptor phosphorylation to increasing doses of gefitinib in IR-5. Western blot analysis of whole-cell lysates from IR-5 or Bam1a cells treated for 24 h with the indicated concentrations of gefitinib after medium change. Lysates (25 Ag/well) were resolved in 4% to 20% SDS-PAGE and membranes were probed for the indicated antigens. B, effect of gefitinib on mitogenic and survival signaling in IR-5 cells. Western blot analysis of whole-cell lysates from IR-5 or Bam1a cells treated for 24 h with the indicated concentrations of gefitinib after medium change. Lysates (25 Ag/well) were resolved in 4% to 20% SDS-PAGE and membranes were probed for the indicated antigens. C, effect of gefitinib on cell cycle and apoptosis proteins in IR-5 cells. Western blot analysis (top) of whole-cell lysates from IR-5 or Bam1a cells treated for 24 h with the indicated concentrations of gefitinib after medium change. Lysates (25 Ag/well) were resolved in 4% to 20% SDS-PAGE and membranes were probed for the indicated antigens. D, cell cycle distribution of IR-5 treated for 24 h with gefitinib (top). Percentage of cells in G0-G1 (shaded columns), S phase (solid columns), and G2-M (striped columns). Induction of apoptosis in IR-5 cells treated for 48 and 72h with gefitinib ( bottom). Cells were treated for 48 to 72h with various doses of gefitinib and evaluated for apoptosis by Annexin staining. Percentage Annexin V–positive cells detec ted by flow cytometry after 48 h (open columns)or72h(solid columns). www.aacrjournals.org 6841 Cancer Res 2007; 67: (14). July 15, 2007

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Cancer Research phosphorylation of MAPK is eliminated. On the other hand, in IR-5 were not sensitive to changes in gefitinib concentration 0 or cells, decreased phosphorylation of c-Jun and S6 still correspond to 10 Amol/L gefitinib compared with 5 Amol/L gefitinib. Finally, the dose-response for HER2phosphorylation, suggesting that these induction of apoptosis in IR-5 cells required treatment with signaling pathways remain tightly coupled to HER2activity. >15 Amol/L gefitinib for at least 48 h compared (Fig. 5D) with Preserved signaling through the MAPK pathways suggests de- 24-h incubation of Bam1a cells with 1 Amol/L gefitinib (Fig. 3B). creased dependence on the HER2for growth signals and the The equivalent level of apoptosis that is achieved with 1 to 2 Amol/L amplification/development of another source coupled to the gefitinib between 24 and 48 h in Bam1a cells requires 15 Amol/L MAPK pathway. gefitinib for 48 to 72h in IR-5 cells. We observe differential sensitivity of several downstream targets These data show that mammary tumor cells overexpressing the of HER2in response to the gefitinib-induced decrease in HER2 activated rat HER2-neu have a high level of intrinsic sensitivity to phosphorylation. For example, VEGF-D modulation by gefitinib is gefitinib via inhibition of receptor signaling through the MAPK and no longer observed; this may be secondary to retention of MAPK Akt pathways in vitro and in vivo. Continuous exposure of these signaling (Fig. 5B). Similarly, changes in protein levels of the cell tumor cells results in the acquisition of gefitinib and the ability to cycle regulators pSer795Rb, cyclin D1, cyclin B1, and p27 were less grow in the presence of 5 Amol/L gefitinib. Acquired resistance to dramatic relative to the parental cell line (Fig. 5C). CYCLIN B1 gefitinib in Bam1a cells is associated with a novel mutation in the transcript levels were reduced 2.2-fold in IR-5 treated for 24 h with ATP-binding pocket of the HER2that alters its sensitivity to 10 Amol/L gefitinib. This reduction in cyclin B1 is 11-fold lower gefitinib. Resistance is characterized by a decreased fidelity in the than that achieved by 1 Amol/L gefitinib in Bam1a cells. Cell cycle signaling pathways from HER2to MAPK and Akt and is analysis shows that 9 Amol/L IR is required to impair cell cycle phenotypically similar to aberrant signal transduction that has kinetics of IR-5 cells (Fig. 5D) when compared with the cytostasis been observed in cells with intrinsic resistance to gefitinib. The up- achieved with 1 Amol/L IR in Bam1a (Fig. 2C). Consistent with regulation of constitutively active survival factors also contribute to retention of MAPK signaling and cell cycle regulators, a complete the resistant phenotype observed in IR-5 cells. Genes that are elimination of the S-phase population and G2 block was not differentially expressed and regulated by gefitinib in Bam1a and observed in IR-5 cells. This cytostatic response in IR-5 cells was IR-5 cells have been associated with intrinsic and acquired incomplete and transient, as an increase in the S-phase population resistance to gefitinib and the amplification of genes involved in was observed at all doses of gefitinib within 48 h (not shown). We Notch signaling. These data suggest that acquired resistance to also evaluated the integrity/fidelity of signaling to the survival gefitinib can be treated by strategies that target genes/pathways pathway through Akt and apoptosis. Akt phosphorylation was used to achieve HER2independence. modestly reduced at 15 Amol/L gefitinib at 24 h in IR-5 cells The relationship between the receptor mutation and resistant compared with complete elimination in Bam1a with 2 Amol/L phenotype will be tested by cloning and transfection of the wild- (Fig. 5B). The observed induction of bim expression at this dose type and resistant receptors in a genetic background that has not (15 Amol/L gefitinib; Fig. 5C) in IR-5 cells suggests a connection been selected for resistance. Aspects of acquired and intrinsic between bim expression, HER2/neu phosphorylation, and the Akt resistance are being investigated by using specific chemical pathway. inhibitors and small interfering RNA that target NOTCH-1, PDGFB, Acquired resistance to gefitinib in HER2-overexpressing breast, FLT-1, and several other genes to validate their functional role(s) in prostate, and gastric cancers has been attributed to the up- mediating resistance to gefitinib. regulation of growth factor receptors [i.e., insulin-like growth factor-IR (55) and EGFR (56)] to create compensatory signaling Acknowledgments pathways that couple to MAPK and drive cell growth. Under this paradigm, it is reasonable to suggest that the coupling of Notch Received 2/23/2007; revised 4/4/2007; accepted 5/10/2007. Grant support: American Cancer Society grant RSG-03-086-01TBE (M.P. signaling pathways to MAPK and Akt (57) is a potential mechanism Piechocki). M.P. Piechocki is a Research Scholar of the American Cancer Society. contributing to the resistance observed in IR-5 cells. 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 Elimination of gefitinib from the culture medium also restored with 18 U.S.C. Section 1734 solely to indicate this fact. cx43 expression in whole-cell lysates extracted from IR-5 cells, but We thank Dr. Guido Forni (Department of Clinical and Biological Sciences, this expression level is substantially lower than that observed in the University of Turin, Orbassano, Italy and Center for Experimental Research and Medical Studies, Ospedale San Giovanni Battista, Turin, Italy) for generously providing parental Bam1a cells, suggesting altered regulation of cx43 in IR-5 the Balb-NeuT transgenic mice and the Scientists at SuperArray for superb analysis cells. Microarray analysis suggested that transcript levels of GJA-1 and discussion.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Breast Cancer Expressing the Activated HER2/neu Is Sensitive to Gefitinib In vitro and In vivo and Acquires Resistance through a Novel Point Mutation in the HER2/neu

Marie P. Piechocki, George H. Yoo, Susan K. Dibbley, et al.

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