Oncogene :19!!! 15. iii— 54!
`
`'53 199? Stockton Press All rights reserved 99513-92326? $12.00
`“I
`
`The effect of HER-linen overexpression on chemotherapeutic drug
`sensitivity in human breast and ovarian cancer cells
`
`Mark D Pegram. Richard S Finn. Karo Arzoo. Malgorzata Beryt. Richard J Pictras and
`Dennis J Slamon
`
`Division in" ”alumni-ogr-Onc'oiri‘gi'. Um‘vcrvity of California or Lot Angela's. School of Medicine. Lot: .dnget'cs. ("i'tilform'u magi,
`{3 3.4
`
`Recent studies indicate that oncogenes may he involved
`in determining the sensitivity of human cancers
`to
`chemotherapeutic agents. To define the effect of HER-
`anen oncogene overexprescion on sensitivity to che-
`motherapeutic drugs, a full-length, human HER-Zine»
`cDNA Was introduced into human hrcust and ovarian
`cancer cells.
`In virro dose-response curves
`following
`exposure to 7 different classes of chemotherapeutic
`agents were compared for
`t-tER-Z— and control-trans-
`tected cells. Chemosensitivity was also tested in viva for
`HER-2-
`and control—transfected human breast
`and
`ovarian cancer senogral'ts in athyrnic mice. These studios
`indicate that HER-linen overexpression was not suffi-
`cient
`to induce intrinsiC. plenmorphic drug resistance.
`Furthermore, changes in chemosensitivity profiles result-
`ing from HER-Zines: transfectlon observed in viii-o were
`cell
`line specific.
`in viva. HER-Zineo-overexpressing
`breast and ovarian cancer xenografts were responsive to
`different classes of chemotherapeutic drugs compared to
`control~treated xenografts with no statistically significant
`differences between HER-Zinen-overexpruing and non-
`overespressing scuografts. We found no instance in
`which HER-Zinen-overexpressing xenogrsfts were ren-
`dered more sensitive to chemotherapeutic drugs in vivo.
`ltER-Zinee—overexpressing senogratts consistently ex-
`hibited more rapid regrowth than control xenogrul‘ts
`following initial
`response to chemotherapy suggesting
`that a high rate of tumor cell proliferation rather than
`intrinsic drug resistance may be responsible for
`the
`adverse prognosis associated with HER-Zillion over-
`expression in human cancers.
`
`breast
`Keywords: HER-2mm [c-crbB-Z):
`ovarian cancer; drug resistance; chemotherapy
`
`CEII'ICEI’:
`
`
`
`___,__.___—__.
`
`Introduction
`
`Thc human HER-2mm lc-erbB-E} prom-oncogene
`encodes a
`lliS kD transmemhrnnc receptor
`tyrosine
`kinnsc which is homologous to. but distinct from.
`the
`epidermal growth Factor receptor (EC-FR) as well as
`other members of the type i receptor tyrosine kinasc
`I'tlmily (i.e_ HER-3 and HER—4}. Sequence identity
`betuvccn members of
`this
`receptor
`family in
`their
`extracellular. and intracellular tyrosine kinase domains
`is 40—60% and fifl~80°fm respectively (Rajkurnar and
`Gullick. 1994). Amplification of the HER-linen gene
`
`
`_
`Correspondence: DJ Slamon
`Received l9 August "3'96; tevrscd .’| April 1993'; accepted 31 April
`I99?
`
`occurs in «15-30% of human breast and ovarian
`cancers resulting in overexprcssion of the gene product.
`and this molecular alteration. when present.
`is an
`independent predictor of both relapse-free and overall
`survival in these diseases (Pauletti et at, l996; Slamon
`er at. 1987].
`In breast cancer. overcaprcssion of the
`HE R—Emen gene has been associated with a number 01‘
`other adverse prognostic factors including: advanced
`pathologic stage tSeshadri at at.
`1993}. number of
`axillary lymph node metastasis [Slitmon er at.
`[987),
`absence ol‘ estrogen and progesterone receptors [Queue]
`ct n1. 1995; Qucrzoli or at. 1990; Barbareschi ct m1.
`I992).
`increased S-phasc fraction {Borg ct at. 199k
`Anbnzhagan or at. 1991), DNA ploidy {'Stal er al.,
`I994; Lee et at. 1992). and high nuclear grade (Berger
`et mt. 1983: Pollcr ct oi” 199l}. A role for the. HER-2.I
`non alteration in metastasis has also been suggested
`given the increased occurrence of visceral metastasis
`[Kallionicma cf at. 199“ and higher
`incidence of
`micrometastatic bone marrow disease (Pantcl et at,
`1993} in patients with HER-linen overexprcssion.
`in
`addition, expression of HER-Zincn has prognostic
`significance in patients with gastric {Yoncrnura or at.
`1991). endometrial {Bcrchuck e: oh, 1991; Hctzel er at.
`1992; Lukes er mt, 1994; Sallhri
`t't mi, 1995). and
`salivary gland cancers (Samba at at. l985; Press at all.
`I994]. The exact role alteration of HER-Zinc” receptor
`expression plays in the pathogenesis of these cancers
`remains unclear.
`Retrospective data from two large clinical trials in
`breast cancer suggests an association between HER—2i
`ncu overexpression and resistance to chemotherapy.
`Results from the lntcrgroup Study Otlll [Allrcd ct at.
`1992) and the International {Ludwig} Breast Cancer
`Study Group {Gustersou or all. 1992} led investigators
`to conclude that node-negative breast cancer patients
`whose tumors contain HER—linen overespression have
`a less favorable prognosis clue to a lack of response to
`adjuvant
`cyclophosphamidc
`(CPA). methotrexate
`(MTX). and S-fluorouraci]
`[S-FUJ-based chemother-
`apy {CM F). in addition, in a study of 68 patients with
`advanced
`breast
`cancer. Wright
`and
`colleagues
`reported a shortened survival for patients with HER-
`Eg'iren orerexpression who were treated with mitoxan-
`tronc despite the fact
`that
`response rates between
`HER-Zinen-overexprcssing
`and
`non-overcxprcssing
`tumors were
`similar.
`50% vs
`58%,
`respectively
`(Wright et oi” N92). A study of HER-linen over-
`cxprccsion in epithelial ovarian cancer demonstrated
`that patients whose tumors had the alteration were
`more
`likely to fail chemotherapy with CPA and
`carboplatin tCBDCAl [Felip ct oi" I995). Conversely.
`in a clinical series reviewed by Klijn er oi. patients with
`Genentech 2139
`Genentech 2139
`Celltrion v. Genentech
`Celltrion v. Genentech
`IPR2017-01122
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`
`

`

`HER-Z/nee and elnmotharaowtic the: sun-Hm
`MD Pegram at at
`538
`
`metastatic breast cancer and amplification of the HER-
`2lnen
`gene
`had
`a
`superior
`response
`to CMF
`chemotherapy 05%) compared to patients without
`HERaneu amplified tumors (45%) and the median
`length of progression-free survival
`from the start of
`chemotherapy was superior in patients whose tumors
`exhibited amplification (Bems et at. I993; Klijn et ch.
`1993). Recently. data from the Cancer and Leukemia
`Group-B demonstrated
`that
`node-positive
`breast
`cancer patients with HER-2mm overexpression de-
`rived
`a
`benefit
`from dosorubicin
`(DOX)-based
`adjuvant
`chemotherapy which
`is
`dose-dependent
`indicating that HER-fluent overexpression may be
`associated with an increased response to DOX {Muss
`et at. 1994). In composite. the clinical data to date are
`somewhat contradictory and do not adequately define
`what role. if any, HER-2mm overexpression plays in
`chemotherapy response. Moreover.
`there
`is
`little
`experimental data to address this potentially impor-
`tant question.
`In one
`study evaluating in
`virro
`chemoseosilivity
`in HER-Elneu-transfected MOP".l
`breast carcinoma cells. no significant difference in
`response to either S-FU or DOX was seen. while
`HER-2 overexpression was associated with a 2 —4-fold
`increase in resistance to cisplatin (CDDP) (Benz at at.
`[992}.
`in another study. HER-2mm transfeetion of
`MDA-MB-435 cells conferred resistance to paclitattel
`(TAX) via an ntdr—l-independent mechanism (Yu er at,
`1996).
`In vitro studies of
`lung cancer
`cell
`lines
`demonstrated
`an
`association
`between HER-linen
`expression levels and intrinsic chemoresistance to six
`different chemotherapeutic drugs (Tsai or at. 1993].
`and transfection of HER-Zlneu CDNA into one lung
`cancer
`cell
`line
`resulted in an increase
`in drug
`resistance {Tsai er all.
`l995l.
`In an attempt to further define the effect of HER-2i
`nee overexpression on sensitivity to chemotherapeutic
`drugs
`in human breast and ovarian cancers. we
`introduced a full-length. human HER-Zl'neu cDNA,
`via a retroviral expression vector,
`into four difl'erent
`breast cancer cell lines: MCF'L MDA-MB—ZJI. MDA-
`M8435 and
`{ST-20.
`and
`two
`different
`ovarian
`carcinoma cell
`lines: 2008 and Caov-3. All of the
`parental cell lines used for this study contain a single
`copy of the HER-2mm gene and express basal levels of
`the gene product while
`the matched HER-Zines:
`retroviral
`transfeetants overexpress
`the gene. Dose-
`respcnse
`curves using seven different
`classes of
`chemotherapeutic agents were constructed for
`the
`HER-Zlneu-overexpressing cell
`lines as well as their
`mock-transfectcd parental controls. The rationale for
`this
`ettperitnetttal
`approach was
`to allow direct
`comparison of genetically identical parentldaughter
`cells which differ only in that one member of the pair
`overenpresses
`the human HER—2,.“nett
`gene. This
`approach was taken to circumvent
`the difficulty of
`comparing cell
`lines derived from separate sources
`which may inherently differ
`in. characterislics other
`than HER-2mm overexpression which could impact on
`drug sensitivity. The rationale for evaluating more than
`one cell
`line representing each of these two human
`malignancies is to avoid the possibility that any given
`observation could be unique to an individual cell
`line
`rather than being representative of a more generic
`biologic effect associated with HER-2mm: overexpres-
`sion. Finally. to avoid the possibility that any observed
`
`effects might be restricted to an in vitro setting and
`because monolayer cell culture assays may not detect
`important multicellular mechanisms of drug resistance
`(Kerbel er of" 1994; Kerbel. 1995). chemosensitivity
`was tested in vivo for breast and ovarian cancer parent!
`daughter xenogrsfts in an athymic mouse model.
`
`Results
`
`Characterization of human breast and ovarian cancer
`cells engineered to over-express the HER-2mm] gene
`
`A Full-length HER-flow cDNA was introduced via
`retrovira] vector into a panel of human breast and
`ovarian carcinoma cells which are known to have a
`single copy of the HER—Zines: gene and to express
`‘nomtal' levels of the gene product. Breast cell lines RT-
`20 and MDA-MB-435 were established from previously
`untreated patients making them less
`likely to have
`treatment-induced chemotherapeutic drug resistance
`while the MCF‘? cell
`line was established from a
`patient with prior radiation and hormonal therapy and
`the MDA-MB-ZSI cell line was derived from a patient
`previously treated with tnultidrug chemotherapy (S-FU.
`CPA. DOX. MTX. and prednisone). The ovarian
`carcinoma cell line 2003 was established from a patient
`who had not had prior chemotherapy, whereas the
`Cami-3 cell
`line was derived from a patient whose
`tumor had been exposed to prior S-FU. DOX. and
`CPA in viva. This spectrum of cell
`lines allows for
`response data representative of a diverse group of
`human
`breast
`and
`ovarian
`cancers. HER-25mm-
`euglnecred and control cells were identically infected
`using a neomycin phosphotransl‘erase-based vector
`which either contained. or did not contain. a Full—
`length HER-Zinen cDNA. Retroviral
`infectants were
`selected for neomycin resistance and subjected to
`fluorescence activated cell sorting (FACS) analysis for
`detection of the plSS"E"" protein. Western blot analysis
`confirmed a marked increase in plli5“““'2 espression in
`cells engineered to overexpress the gene relative to
`mock (NEW-infected controls {Figure la and b). SK~
`Bil-3 human breast carcinoma cells and SK-OV-It
`human ovarian carcinoma cells naturally overexpress
`the HER-2 receptor and were included in these studies
`for comparison of nou-manipulated overexpressing
`
`0
`
`U'
`
`MDA«MB-231KNEG
`
`BT-ZDIHEFt—Z
`
`MCFTl'HEFt-2
`
`SKHFt‘J
`
`E. 8iii3 i
`
`«l
`
`MDA—ME-ZEWHEH-EMBA-M343“E0MDA-MB—HSJ'HER-Z
`'I ItSK-OV-3
`
`i
`
`Caov~3iHEFlA2
`
`Caov-3HNEO
`
`
`
`ZOOWHER—Z
`
`ZDOWEO
`
`MCF‘HNEO
`BT—ZWNEO
`U C O
`Figure I Western blot analysis of HER-Zlnt-u- and mock {NEG}-
`vuctor
`infected breast int and ovarian [bi carcinoma cell
`line-é
`demonstrating high-level expression of leS'u'R': in ttunsiected
`cell
`lines. SK-BR-J breast cells and SK-OV-J ovarian cells have
`native amplificationfovempression of the HER-1 one; gene and
`are shown as positive cottlrols
`
`

`

`‘A
`HER-z/nlu and chemotherapeutic drug sensitivity
`('3'
`I'll) Pegrarn et at
`——-——-——.—.—_—____
`539
`
`reproducibility. all sets of in vitro assays were repeated
`at
`least
`two times. This assay yielded 4~paranieten
`sigmoidal curve tits With correlation coefficients ranging
`from 0.938 41.999. Differences between dose-response
`curves were assessed using 2-factor analysis of variance
`{ANOVA} of data points which fell between the Kim
`and ICE“. Representative data from these experiments
`
`:IMDA-MBv231iNEO ]MDA—MB—231IHEFtv2 ]MDA-Ms.tssmeo
`
`-+-
`
`+
`
`-+—
`
`+
`
`HRG-B‘I
`
`+—1-—
`
`4
`
`,.
`
`jmoA-Mo-tastHEH-z
`]mcnmso ]MCFrtHEa-z Jtit—20mm
`:]eT-zotHEn-z
`jsxeas
`ten-um and
`tB:HEH-2-+ “- ”Olfipu‘u! E.“
`
`‘1-
`
`Figure 1 To demonstrate the Phosphorylation state of plss'm‘"
`lti HER-Zirten—transfected breast carcinoma cell
`lines.
`anti-
`rlhosphotyrctsinc immunobtcts were performed following immu-
`noprm‘pitaticn with u monoclonal anti-plasmz'?“1 antibody both
`“1 the presence [— l or absence iv] of recombinant heregutin 8-1
`tall. The same hlnt is reprobed with anti-pisst'E“‘= (In. These data
`demonstrate constitutive tyrosine phosphorylation of plitSHF"H
`in SKBRS, MCWIHER-l and B‘f—EWHER-I even in the absence
`of heregulin EH. In mock [NEOl-transteeted MCF? and BT—ltl
`cells. hcrcgulin an induced both an Increase in piss“-J
`tyrosine phosphorylalion (a) and downrcgulation ol' pitting”
`expression lb). MDA-MB-l'il cells exhibited neither basal nor
`heregulin-inducod tyrosine phosphorylation of pltti’m‘” despite
`high expression levels of the protein
`
`l
`
`O
`u.t
`
`‘2‘
`o:
`in
`
`N
`.
`
`3
`:2
`to
`t—t
`—
`+
`
`s
`to
`U
`l—‘I
`-
`+
`
`s
`on
`U
`1—1
`—
`+
`
`s
`8
`N
`t—I
`—
`+
`
`s
`a
`N
`i—‘l
`-
`t-
`
`HRG-Bl
`
`lP:HEfi-2
`IB:PY
`
`h i
`
`s: HER-2 ---—b
`
`Figure 3 Anti-phosphotyrosine immuuo'olot ot‘ HER-lineu- or
`mock
`iNEO)-tmnsfeeled ovarian carcinoma
`cells
`following
`immunoprocipllutlon with an anti-plfifi'm” specific monoclonal
`antibody either in the presence ( + p or absence I — t of exogenous
`recombinant heregulin B—l
`tilt. The same hiot
`is repruhed with
`anti-EllisHEM Hit. The
`data demonstrate an
`increase
`in
`piss 5’” tyrosine phosphorytntton and downregulation of
`plESI'F‘R': expression on exposure to Itcregulin B-I
`in Cami-3t
`NEO cells. Cativ-JiHER—l all!- demonstrate hoth b‘asal and
`lteregulin-induced tyrosine phosphorylarion of leSHF‘R" whereas
`ElmalHER-Z have neither
`increased basal or tieregulin-induced
`pllifil’m“ phosphorylation despite over-expression of the protein
`
`cells to the engineered cells. The levels of HER-Einstt
`overexpression in the engineered cells are comparable
`to. but do not exceed. the levels found in actual human
`tumors circumventing the possibility that any observed
`biologic changes are artifacts of levels of overexpression
`which do not occur
`in nature. As
`a measure of
`functional activity of pl85“"".
`the phosphorylation
`state of plSS’m“ was assessed using immunoblotting
`techniques. Protein lysatcs From each of the transfected
`cell lines were subjected to immunoprecipitation with a
`pldfil'e'“ specific monoclonal antibody. These experi-
`ments were performed on cell
`lines both with and
`without prior exposure to heregulin 3-]. a growth
`factor ligand cloned on the basis of its ability to induce
`tyrosine phosphtérylalion oi" pl85"‘“ through the
`formation of
`' R-EIHER-J andior HER-2iHER-4
`heterodimeric
`omplexcs
`(Sliwkowslti
`at
`all.
`[994;
`Plowman or rat. 1993). The resulting immunoprecipi—
`tales were
`then resolved
`by polyacrylumide
`gel
`electrophoresis (SDS—PAGE] and probed with an
`anti-phosphotyrosinc antibody [Figures 2a and 311].
`These results indicate that HER-2mm cDNA transfec-
`tion results in expression ofa pl85"“‘= protein which is
`either constitutively tyrosine phosphorylated or can be
`phosphorylated on exposure to heregulin 8—1 in each of
`the breast cell lines with the exception of MBA-MB-
`231 {Figure 2a}. Similarly. ovarian C'aov-BH-IER-I! cells
`exhibited heregulin-induced tyrosine phosphorylation
`of p135"UH while 2008iHER-2 cells did not (Figure
`3a}. In Figures 2b and 3b the same blots from Figures
`3a and 33 have been probed with the same anti-
`plfifil'l‘“ antibody used for the immunoprecipitations.
`These
`results confirm overcxpression of p185""”
`protein in the HER-Ei'iteu-transfected cell lines. and in
`addition. demonstrate that exposure of the mock-vector
`(NED) transfected cell lines to hercgulin 8-1 in most
`cases resulted in tyrosine phosphorylation as well as
`down-regulation of plEtSWE'” expression (Figures 2b
`and 3b}. The relative degree of heregulin induced
`tyrosine phosphorylation of pl85"“"" correlated with
`the expression level of HER-3 in these ceils. For
`example. MCF7 cells have 2.5 x In" HER-3 molecules
`per cell whereas MDA-MB-ZM and 2003 cells have
`only 1.4 it l0-‘. and l.l}x IU' HER-3 molecules per cell.
`respectively by quantitative ELISA (Aguilar et ul.
`manuscript
`in preparation}. HER-4 expression levels
`are very low.<10‘ tnoleculesicell, relative to HER-2 or
`HER-3 in this panel of cell lines. therefore heregulin-
`induced HER-2
`phosphorylation
`appears
`to
`be
`predominantly influenced by the abundance of HER-
`ZJHERJ heterodimers in these cells. Having success—
`fully engineered the breast
`and ovarian cells
`to
`overexpress pISS'W“. we next evaluated the effects of
`overexpression on their sensitivity to chemotherapeutic
`drugs in vim: and in viva.
`
`officer of HER~2incu ovei'expresst‘on on sensitivity of
`human breast and ovarian veils to t‘hemothertrpeuttc
`agents in vitro
`
`The efl‘ects ot' HER-Zines everexpression in human
`breast and ovarian carcinoma cell lines on sensitivity to
`a variety of chemotherapeutic agents was determined in
`vitm. The effective dose range for each drug (1C 19—
`Icon) was identified using a range of ten different doses.
`each tested in quintuplicatc. To assure accuracy and
`
`
`
`__._._,—.__._".__.___.__
`
`
`

`

`Han-2mm Ind eMmtttuflpouttc drug sensitivity
`MD Pegram at at
`I!
`
`
`
`—t.—.—.__—'_—.——.-———_-'—-——
`
`are shown in Tables 1 and 2. These data include the
`ICmione standard deviation and the significance icvcl
`for differences between control (NEOl and HER-2-
`engineered
`cell
`lines.
`Introduction of neomycin
`phosphotransferase gene via the NEO control vector
`and selection in neomyc'tn resulted in no change in
`chemosensitivity in MCF7 cells {data not
`shown)
`indicating that neomycin resistance does not confer
`cross-resistanCe lo chemotherapeutic agents in t'tt't'd'.
`Clinically achievable peak plasma levels of chemother-
`apeutic drugs from standard dosing schedules used in
`humans are shown for reference in Table l.
`HER-linen overexpression in MCI“:I breast carcino-
`ma cells resulted in a 2.5-foid decrease in sensitivity to
`the platinum analog CBDCA, as well as a twofold
`decrease in S-F U sensitivity. Conversely. a twofold
`increase in sensitivity to TAX was noted while no
`change in sensitivity to the other four drugs tested was
`found (Table 1). These results are similar
`to those
`reported by Benz ct at. who noted a 2—3-fold decrease in
`sensitivity to CDDP but no change in sensitivity to DOX
`or S-FU in MCF7 cells which overexpress HER-linen
`{Benz er cl'., 1992).
`in contrast. MDA-MB-ZBlfHER-Z
`cells were rendered more sensitive to four of the seven
`drugs tested (Table I}. This increase in sensitivity ranged
`from Lit-fold for thiotepa {TSPM to > IOU-fold for
`TAX. The BT-ZOJHER-Z cells were also 2-4-t'old more
`sensitive to TSPA and S-FU. but
`like MCFWHER-E
`cells. they were more resistant to platinum compounds.
`Lastly, MDA~MB~435IHERQ cells exhibited no change
`in chemoscnsitivity to any of the seven classes of
`chemotherapeutic agents tested. Among the ovarian
`carcinoma cell
`lines. Caov-StHER-Z cells were slightly
`more sensitive to [30X and vinblastine tVBL) compared
`to Caov-JIN E0; however, HER-Zines: overexpression in
`2008 cells resulted in a threefold and 7.5-l'old increase in
`resistance to CBDCA and TSPA. respectively (Table 2}.
`
`These results indicate that HER-Etireu overcxpression
`does not produce any consistent or predictable change in
`drug sensitivity profiles in vim: across the various cell
`lines tested and underscore the necessity of evaluating
`more than one ccll
`iine prior
`to drawing general
`conclusions on the
`effect of
`this alteration on
`chemotherapeutic response
`in human cancer
`cells.
`Moreover.
`the difierences in chemosensitivity patterns
`among
`the HER-ZIhwu-Lransfectcd
`cell
`lines
`did
`not appear to correlate with basal or heregulin 3-]-
`induced tyrosine phosphorylation oi“ pl85"""3. Despite
`the fact that chemosensilivity in HER-2incn-overexpres~
`sing cells was cell line specific. some trends did emerge
`from the data. HER-Zi’ncn-overexpression had no major
`effect on sensitivity to 00X in any of the six cell lines
`tested with the exception of Caov-JlHER-E cells where
`it was associated with a
`small
`(0.5mm to 0.] ,uMl
`but
`statistically significant
`increase
`in
`sensitivity.
`Similarly, HER-Zines overexpression had minimal
`efiects on response to etoposidc (VP-Ital with only
`one cell line. MDA—MB-Bl. exhibiting a slight increase
`in
`sensitivity
`after
`transfection with HER-2pm“.
`increased resistance to platinum analogs was observed
`in three of the six cell
`lines with HER-2mm over-
`exprcssion compared to their controls. Finally. when
`agents which interfere with microtuhule formation (VBL
`and TAX) were studied.
`three of six HER~2funu-
`overexpressing cell
`lines demonstrated an increase in
`sensitivity.
`
`expression on t-hemosensitivitj' of
`Effect ofHER-Ett
`breast and ovarian .venogrttfis in vivo
`
`To further evaluate and expand drug sensitivity
`studies associated with HER-linen overexpression.
`we developed
`an
`in
`viva chemotherapeutic drug
`sensitivity assay which utilized serial measurements
`
`Table 1 Effect oi HER-2mm ov‘crttttpression on sensitivity of human breast cells to chemotherapeutic agents in i-itro“
`coo? turn)" BOX (tint)
`5-th {itMl
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`TSPA (31M)
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`plasma levels of each drug achievable in humans with standard dosing schedules are shown for reference
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`

`

`HER-van: Ind chemotherapeutic drug sensitivity
`ND Pegram et al
`541
`
`and MCFTJHER-l Was not statistically significant
`(P-=0.12}. Treatment with TAX. also resulted in
`significant
`responses
`for MCFTIHEO and MCF't'iI
`HER-2 tumors compared to vehicle-treated controls.
`Mean TAX-treated WC ratios at maximum response
`were 0.1910119 and D.3ll:t-_0.l8 for MCP‘TlNEO and
`MCFWHERJ tumors.
`respectively (Figure 5d). and
`this difference was marginally significant
`tP=ll.ll'9‘i.
`Finally.
`response
`to
`treatment with TSPA was
`significant for both MCFTXNEO and MCF‘NHERJ
`tumors computed to control (Figure 5e}. but there was
`no significant difi'erence between response of MCI-"ll
`NEO senogral‘ts compared to MCFTlHER-E xeno-
`gral‘ts
`in
`response to TSPA lP=ll.l7'l. Additional
`analysis
`iii
`a E-I'actor ANOVA model
`failed to
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`llER-2,'nclr. or control {NED}
`Figure ll Tumorigenictty of
`rc-ctorint‘eetcd human breast (MCI-‘1] s_c. xenografts in female
`athymic mice (tr-=l3-14.tgroupl. Error bars indicate standard
`error. MCFTEHERQ sonogrut'ts l‘] have a significant growth
`advantage ovar MCW;NEO[U_I[P=IJ.DODI1m rim. Mic: in this
`experiment were treated with a vehicle control solution i.p.
`beginning on day it til days status post xenogral‘t inoc elation}. at
`which time objectively measurable senograt‘ts had tot-tried
`
`
`
`response (relative to
`illustrating tumor
`Figure 5 Bot: Plots
`control] For MCFTINEO tunshadcd hoses] and MCFT.'HER-l
`Ishadcd l‘lfl‘lfl} “some; (a = ll- 14 per group] in response to
`treatment with: m 130th mil-tel. tln C'DDP t5 melkel. let 5-
`FL'
`llflt‘l mgikgt. Id] TAX tlS mgi'lrg a 3:. and let TSPA t5 mg:
`in; at 3}. Error bars indicate "1'” to 90'" percentiles. hora indicate
`15‘“ to 15‘“ percentiles. and notches indicate 95% confidence
`Intervals. Group mean TIC ratios and significance levels [Mann-
`Whitncy U test) for dittcrcnccs between MCF‘HNEO and MCF'li
`HER-2 are as follows:
`
`Grater-law MCP‘hNEO htCF7.-'HER-.'t
`3
`0.43
`0.62
`h
`0.30
`0.44
`e
`0.31
`0.41
`d
`0.19
`0.10
`e
`0.27
`033
`
`Significance Level
`9-0.1:
`9-0.1:
`etc-.1:
`ream
`than
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`
`of subcutaneous human tumor xenogral'ts growing in
`athyrnic mice. For the in i'fl'fl studies, human breast
`(MCF?) and ovarian (2008) carcinoma cells were
`selected for testing because of their predictable tumor
`formation in
`athymic mice.
`Immunohistochemical
`analysis of sections from these tumors and Western
`blot analysis
`from cell
`lines derived
`From these
`sonografts confirmed that the relative expression level
`or HER-Zine“ was maintained during the course of the
`study tdata not shown). Ovcrexpression of HER-2mm
`in MCF?
`breast
`carcinoma
`cells
`resulted
`in
`a
`significant change in their
`in l'fl't) growth character-
`istics {Figure 3}. By day 50. MCFilHER-Q tumors
`were
`17-fold
`larger
`than MCFWNEO tumors
`{P=0.0l}[il). At
`the onset of chemotherapy adminis-
`tration. animals were assigned to treatment groups
`such that initial tumor volumes were the same in each
`group (5514 mm‘]. Because the MCFWNEO xeno~
`grafts have a significant difference in inherent growth
`rate compared to MCFYiHER-Z xenOgral'ts. the ratio
`of chemotherapy-treated to untreated control
`tumor
`volume [TIC ratio) was calculated for each tumor. The
`maximum response to chemotherapy. defined as the
`point at which the TC ratio was at a minimum. was
`determined for each individual tumor. The maximum
`drug responses For
`the MC F‘HNEO sonografts were
`then directly compared to responses found in the
`MCFYiHER-l scnografts.
`in the human breast cancer scnogral't model. all
`five drugs tested resulted in significant responses for
`both MCFWNEO and MCFTIHER-Z tumors com-
`pared to their
`respective untreated control
`tumors
`[P-clLOS'}
`indicating
`that HER-Zi'aru-transl'ectcd
`MCF?
`senografts maintain
`sensitivity
`to
`these
`chemotherapeutic drugs in viva [Figure 5]. The mean
`time to point of maximum response was [its days
`and was independent of the drug tested or tumor type
`(Le. NEO tar HER-l). Tumor regrowth following day
`2] uniformly occurred indicating a lack of prolonged
`response to the initial treatment. 01' note was the fact
`that
`there was a significant difi'erence in regrowth
`rates
`following responses
`to chemotherapy when
`comparing MCF'HNEO to MCFllHER-B tumors.
`The mean tumor doubling time Following chemother-
`apy was
`14.6
`days
`for MCFTIHER-l
`tumors
`compared to 23.8 days
`For MCF'UNEO tumors
`tPiDDODIJ. This demonstrates that HER-llitcu-over—
`expressing tumors maintain their prolitierative advan-
`tage Following exposure to chemotherapy in viva. The
`TIC ratios at
`the point of maximum response are
`represented graphically by hos plots
`[Figure
`5).
`Treatment with DOX resulted in significant responses
`for both MCFWNEO and MCF‘lgHER-E tumors
`compared to their respective untreated control groups
`(Figure
`5a]. The difference
`in
`the magnitude of
`maximum response to DOX between MCF'HNEO
`and MCF'llHER-E tumors was
`not
`statistically
`significant
`(P=0.IJJ. Treatment with CDDP also
`resulted in significant
`responses
`for both l'l/tCt-‘T’tl
`NEO and MCF'UHER-Z tumors and again mean
`CDDP-treated TIC ratios were
`not
`significantly
`diiierent at
`the point of maximum response (Figure
`5h. P=U.12t. Similarly. treatment with S-FU resulted
`in significant responses compared to controls For both
`MCFTINEO and MCFIFHER-E tumors {Figure Sc);
`but
`the difference in response between MCF7.“NEO
`
`

`

`\ N
`HER-Z/mm Inc dismount-penile drug sensitivity
`M0 Pegram el al'
`542
`
`demonstrate significant dificrences in the magnitude of
`response between MCF'NNEO and MCFifl-lER-Z
`xenogrnfls to any chemotherapeutic agent tested over
`the time period during which responses were seen.
`Unlike MCF? cells.
`the ovarian carcinoma cells
`2008/HER-2 had only a small growth advantage over
`ZOOBINEO cells (Figures case. vehicle controls}.
`In
`this model. both the 2008MB.) and EDEISIHER-Z
`xenograt'ts were refractory to treatment with DOX
`using two different
`treatment schedules (5 mgfltg on
`day l or 3 mgfltg on days I and 14. data not shown}.
`Higher doses of DOX resulted in substantial toxicity.
`Similarly. VP-16 at a dost: of 25 mgfltg on days 0, 3.
`and 7 had no effect on ZOOBINEO or ZGlIBfl-IER-E
`tumor growth during the 21 day observation period.
`A dose of 50 I't‘tgy'kg on day 0 and day 3 did result in
`a significant response compared to untreated control
`tumors by day ti (data not shown), however there was
`no response difference between EUUSfNEO and 2003i
`HER—2 tumors. and the higher dose of VP”) resulted
`in substantial mortality beyond day 5. Treatment of
`ovarian
`2008
`tumors with
`(DB?
`resulted
`in
`significant responses by day 6 at which time tumor
`volumes of CDDP-treated tumors were 37% of
`controls and significant dilterences were maintained
`during a 2] day observation period (Figure be}. There
`was no difference. however.
`in the degree of response
`between 20080150 and ZDUBJHER-Z ovarian xeno-
`grafts.
`thus
`the
`threefold shift
`in 1C“.
`suggesting
`platinum resistance in the HER-Electi-overexprcssing
`cells in vitro was not observed in viva. Treatment of
`ovarian 2008 NED and HER-2 tumors with TAX
`resulted
`in
`a 53% reduction in
`tumor volume
`compared to control which was apparent at day 6.
`However.
`there was no difference in response when
`comparing ZOOBINEO and 2008fHER-2 tumors in—
`dicating that HER-2mm overexpression in these cells
`had no impact on sensitivity to TAX in viva [Figure
`6b}. Treatment of ovarian 3003 xenograt'ts with TSPA
`also resulted in a significant
`response compared to
`untreated control tumors. For this drug. a significant
`difference between TSPfiHreated 20083’NEO and 2008}
`HER-2 tumors did emerge by day 2] with TSPA—
`treated 2008lHER-2 tumors measuring 100% larger
`than TSPA-treated
`2008,0150 tumors
`tP=fl.flt]2)
`(Figure 6e}. Moreover.
`this result paralleled the in
`vitro results where a 15-fold increase in resistance to
`TSPA was noted in 2008 cells overexpressing HER-2i
`urn. This dilierencc. however. appeared to be due to
`more rapid tumor regrowth for ZUDSIHERQ xeno—
`grafts following response to chemotherapy rather than
`intrinsic resistance to TSPA.
`in fact. at
`the time of
`
`there was no
`response to TSPA (day 10}.
`maximal
`significant difi‘ercnce between 2008fNEO and 2003i
`HER-2 xenografts [Pr-0.170. These data paralleled the
`results seen with MCFHHERZ xenograi’ts where rapid
`tumor
`regrowth
`occurred
`following
`response
`to
`chemotherapy in vii-o.
`
`Discussion
`
`The involvement ol‘ some oncogenes in the develop-
`ment of chemotherapeutic drug resistance is suggested
`by experimental data demonstrating. increased expres‘
`sion of c-finr. e-nn-e. and c-H-ms gene transcripts in
`
`
`Tmmnn:
`mutt!- a";
`
`WER-fi’l’il's
`m‘fi’d
`m-m null-I
`
`m»
`
`Figure ti Response of human ovarian xenograils littlttiNECI and
`EUUNHER-J to treatment wttlt {It} CDDP i5 mgi'kg}. {hi TAX
`[15 mgjkg stilt. and {e} TSPA (5 nigtltgxai
`in tit-male uihymie
`mice. Injection of a single cycle of these Ihrec drugs resulted in
`significant responses compared in a vehicle control for both zlttttti
`NED and zotati'HER-E renegratts: however.
`the magnitude of
`res