HER2Neu kinase

8/4/2019 HER2Neu kinase

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Introduction to HER 2/Neu

EGFRERBB4 orHER 4

ERBB1

or EGFR

ERBB2 orHER 2 orEGFR 2

ERBB3 orHER 3

Strong kinase activity

No kinase activity

HER 2/Neu proto oncogene located on 17 chromosome. Geneproduct is a transmembrane protein 1255 amino acids. No known

ligands of HER2 .

Cell proliferation

& Differentiation,Motility &

adhesion

Apoptosis resistant& transformation

Enhances tumor cell motility, adhesion andmetastatic potential while normal HER1

signaling disrupts cell cycle control (leadingto proliferation) and apoptosis

lower expression of HER-4 inbreast and prostate tumors

relative to normal tissue.


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Plasma Membrane

Molecular strucutre of Her2

Extracellulardomain

Transmemnbranedomain (22 aa)

Intracellulardomain (580 aa)

cytoplasm

N C

High affinity - low specificityLow affinity - high specificity

Order of dimerization tendencyErbb2 > erbb1 errb4 > erbb 3

(632 aa)

Extracellulardomain

HER 3 HER 2 combination is most potent mitogen and prolonged signal and slow internalization

PPPPP


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Biology of Her2

E A Y V M A S V D N P H V C R L L GEGFR

E A Y V M A G V G S P Y V C R L L GHER2

Oncogenic mutations activate kinases by disrupting the autoinhibitorymechanisms that normally stabilize their inactive forms . They targetstructures around the ATP binding cleft that are involved in phosphorylationevents. These structures include the phosphate binding and activation loops.

In normal cell, HER 2 ligand HER2, few heterodimers, weak responsecontrolled cell growth.

In tumor cell, ligands from stroma or tumor cells recruit HER2. HER2, highchances of heterodimer formation, slow internalization, signal present forlong time enhanced response for growth factor , malignant growth.


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Her2 as target

Trastuzumab (Herceptin), a humanized monoclonal IgG1antibody that binds

the extracellular domain of HER2 , is effective for HER2 overexpressingbreast cancer patients when used with other cytotoxic agents.

In addition to being active as a single agent, trastuzumab potentiates

the anti-tumor activity of paclitaxel and doxorubicin and cisplatin.


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Expression of a HER2 mutant containing a G776YVMA insertion in exon 20was more potent than wild-type HER2 in activating post receptor signaltransducers and in transforming mammary and bronchial epithelial cells.

Aim of this study is to evaluate the potential gain-of-function effects of HER2

mutations.

Hypothesis


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Experimental Procedures

Cell Lines

NCI H1781 cells

BEAS 2B cells

293 cells

Phoenix - Ampho cellsPackaging Cell

Lines

MCF 10 A cells

32D cells

Adenocarcinoma, brochoalveolar

carcinoma. Mutated ERBB2, NM EGFR

Bronchial epithelium, infected with adeno-virus SV-40 12 hybrid , only differentiate

Breast epithelial cells, expresses epithelialsialomucins, cytokeratins and milk fatglobule antigen and express breast specificantigens, XX, genteic abnormalities, p53 &ER -

Epithelial cell derived, embryonic cells,hypotriploid, ch # 64, receptor forvitronectin, contains adenovirus DNA

Mouse - Suspension Lymphoblast

infected with leukemia virus

Retrovirus producer cell lines, based on293 T cell line, high stability and longterm high expression of genetic elements

Loza, 2010 [accessed online] http://www.lonzabio.com/no_cache/extras/cell-transfection-database/cell-details/cell/673/[accessed on 10-9-2011]

ATCC,2011 [accessed online] http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCC

Num=CRL-1573&Template=cellBiology [accessed on 10-9-2011]

Lawrence Berkeley National Laboratory. Accessed online [http://icbp.lbl.gov/ccc/viewline.php?id=37]on [10-9-2011]
http://icbp.lbl.gov/ccc/viewline.php?id=37http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1573&Template=cellBiologyhttp://www.lonzabio.com/no_cache/extras/cell-transfection-database/cell-details/cell/673/http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1573&Template=cellBiologyhttp://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1573&Template=cellBiologyhttp://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1573&Template=cellBiologyhttp://icbp.lbl.gov/ccc/viewline.php?id=37http://icbp.lbl.gov/ccc/viewline.php?id=37http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1573&Template=cellBiologyhttp://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1573&Template=cellBiologyhttp://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1573&Template=cellBiologyhttp://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1573&Template=cellBiologyhttp://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1573&Template=cellBiologyhttp://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1573&Template=cellBiologyhttp://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1573&Template=cellBiologyhttp://www.lonzabio.com/no_cache/extras/cell-transfection-database/cell-details/cell/673/http://www.lonzabio.com/no_cache/extras/cell-transfection-database/cell-details/cell/673/http://www.lonzabio.com/no_cache/extras/cell-transfection-database/cell-details/cell/673/http://www.lonzabio.com/no_cache/extras/cell-transfection-database/cell-details/cell/673/http://www.lonzabio.com/no_cache/extras/cell-transfection-database/cell-details/cell/673/http://www.lonzabio.com/no_cache/extras/cell-transfection-database/cell-details/cell/673/http://www.lonzabio.com/no_cache/extras/cell-transfection-database/cell-details/cell/673/


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Cell Growth assay

Equal numbers ofcells were seededon 12-well plates

allowed to grow infull medium

Maintained at37C at 5% CO2incubator

harvested bytrypsinizationevery 24 hr

Cell number was determinedin a Coulter counter


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Apoptosis assay /Apo BrdU TUNEL assay

labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow or

image cytometry

Nucleaseactivated

BrdUBrdU

BrdU

BrdU

BrdUBrdU

BrdU BrdU

Terminaldeoxynucleotidyl

transferase


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Pull Down assay

A pull-down assay is a small-scale affinity purification technique similar to immunoprecipitation, except that theantibody is replaced by some other affinity system. In this case, the affinity system consists of a glutathione S-transferase (GST)-, polyHis- or streptavidin-tagged protein or binding domain that is captured by glutathione-,metal chelate (cobalt or nickel)- or biotin-coated agarose beads, respectively. The immobilized fusion-taggedprotein acts as the "bait" to capture a putative binding partner (i.e., the "prey"). In a typical pull-down assay, the

immobilized bait protein is incubated with a cell lysate, and after the prescribed washing steps, the compexes areselectively eluted using competitive analytes or low pH or reducing buffers for in-gel or Western blot analysis


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Wound closure assay

cells were

allowed to reachconfluence on a6-well plate

monolayers were

scraped with aplastic pipette tip

replenished with

fresh serum-freemedium.

Phase contrast images were photographedat 0, 10, and 24 hr after wounding.

serum starved

for 24 hr

Events during wound healing or repair

Vacularization by

angiogenic factors

cell proliferation

& extracellularmatrix deposition

infiltration by

inflammatoryimmune cells


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Transwell Migration assay

The transwell migration assay is a commonly used test to study the migratory

response of cells to angiogenic inducers or inhibitors. This assay is also knownas the Boyden or modified Boyden chamber assay

Transwell motility assays were performed utilizing 5 mm pore, 6.5 mm polycarbonate transwell filters . Cells (1.5 X 105 per well)were seeded in serum-free medium onto the upper surface of the filters and allowed to migrate. After 24 hr, the cells on the upper

surface of the filters were wiped off with a cotton swab . Cells that had migrated to the filterunder-side were fixed, stained withDiff-Quik stain set, and counted by bright field microscopy


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Soft agarose colony forming assay

Base layers consisting of growth medium containing 0 .8% low-melting point Agarose and 10 mM HEPES(pH 7.5) were poured onto 6-well plates and allowed to solidify. Cells (3 X 104 per well) were plated in

triplicate in top layers consisting o f growth medium containing 0.4% agarose and 5 mM HEPES. Coloniesmeasuring R 50 mm were photographed after 710 days and counted manually

The Soft Agar Assay for Colony Formation is an anchorage independent growth

assay in soft agar, which is considered the most stringent assay for detectingmalignant transformation of cells.


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Xenograft Studies

Exponentiallygrowing cellswere scraped off& resuspendedin serum-free

medium

2 X 10 6 cells in 0.3

ml were injectedsub-cutaneouslyinto each of 4-week-old female athymicnude mice (HarlanSprague-Dawley)

Tumor formationwas monitoredby palpationtwice a week

Volume of tumors measuring 3 mm in

diameter was calculated by the formula:volume = width2 x length/2.


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3 Dimensional morphogenesis and indirect immunofluorescence

Three-dimensional culture of MCF-10A cells on reconstituted basement membraneGlandular epithelial cells, such as those in the mammary gland, have several

distinguishing histological features including a polarized morphology, specializedcellcell contacts, and attachment to an underlying basement membrane

Epithelial tumors (carcinomas), requires the disruption of this intact, well-orderedarchitecture.

MCF10A cells expressing HER2 WT , HER2 YVMA , or vector were seeded on Growth Factor Reduced Matrigel in 8-wellchamber slides following the protocol described by Debnath et al. (2003) Morphogenesis of acini was photographed every 2days. For cell number counting, cultures growing on Matrigel were trypsinized and cell numbers were measured in a Coultercounter. Invasion assay was performed in BD BioCoat Growth Factor Reduced Matrigel invasion chambers according to themanufacturers protocol. Immunofluorescence staining of 3D acini was performed as described by Debnath et al. (2003) usingantibodies against beta -catenin, cleaved caspase-3, Ki-67, and Myc tag. Confocal analyses were performed with Zeissinverted LSM510 confocal microscopy system. Indirect immunofluorescence assay (IFA) was performed as describedpreviously ( Wang et al., 2005). Fluorescent images were captured using a Princeton Instruments cooled CCD digital camera

from a Zeiss Axiophot upright microscope . Primary antibodies include Ki-67 and cleaved caspase-3. The fluorescentantibodies are Oregon green -a -mouse IgG and Texas red- a -rabbit IgG


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Immunoblotting / Western blotting

Samplepreparation

SDS-PAGE

BlotTransfer

Blotbinding


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Primary Antibodies Used

P-MAPK MAPK P-Akt

5473

Akt P-SrcY416 Myc tag

P-EGFRY1068 EGFR HER3

P-HER2Y1248 P-HER3Y1249 HER 2

Src P-Tyr p85

Shc Actin

HA


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Generation of HER 2YVMA

Wild typeYVMA containing sequence was synthesized

PCR amplified

Inserted into HER 2 cDNA by splicing at NdeI

Amplified HER2 contains YVMA sequence and aMyc tag was added at 3 end

Myc-HER2 YVMA


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Retroviral Mammalian Expression Vector Construct

pBabe Puro5169 bp

XbaI

ORI

KpnI

NotI

FspI

PvuI ScaI PstIXbaI

KpnI

PstI

PstI

Truncated gag

SV 40 IEP

MCS

AmpR

Puro

BamHISnaBIEcoRISalI


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SnaBI SalI

Digestion by REs

Myc HER2 YVMA/ HER2WT

Phoenix Ampho cells transfected with HER2 YVMA/WT containing plasmid

Retrovirus infected cells selected using Puromycin


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Generation of HER 2 chimeras

pMN.HER2

YVMA

.F2.HA

pBabe HER2WT /HER2YVMAwas PCR amplified

pMN.HER2

WT

.F2.HA

MSV 5LTR pMN FKBP FKBP HA

XbaI SpeI Stop

pMN.F2.HA

Inserted into pMN.F2.HA by digestion at Xba I and Spe I

pMN.HER2YVMA.F2.HA or pMN.HER2YVMA.F2.HA


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Immunoprecipitation

Protein Extraction

Cells insuspension

Cell lysiswith NP-40

Washed with PBS

Sonicate &centrifuge

Proteins insupernatant

Proteins estimated by BCAprotein assay reagent

Immunoprecipitation

200 gprotein

Add 50 gProtein A/Gsepharosebeads

Incubate at 4Cfor 2 hours

Boil for 3 minwith 20 l 2Xloading buffer

Perform SDS - PAGE

Incubate at 4C overnight

With Myc Tag Antibody


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Transfection of DNA

DNA Transfection is the process of deliberately introducing deoxyribonucleicacid into eukaryotic cells.

Generalized protocol for DNA transfection in adherent cell lines

Transfection of 32D suspension cells by Electroporation


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Transfection of SiRNA

The RNAi pathway is found in many eukaryotes including animals and isinitiated by the enzyme Dicer, which cleaves long double-stranded

RNA (dsRNA) molecules into short fragments of ~20 nucleotides that arecalled siRNAs.

SiRNA oligos were designed to target the sequence5- AAGCATACGTGATGGCTGTGT -3 for HER2MUT in H1781

or5-AAGCATACGTGATGGCTGGTG for HER2WT. Transfection done by using

cationic liposomes.


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RT - PCR


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in vitroKinase assay

500 g protein

(MCF 10AHER2WT/YVMA)

Wash withlysis buffer& Kinasebuffer

Aliquoted into two

equal portions

ATP wasadded to onlyone aliquot

Kinase reaction was done at

30C for 5 min and thenterminated by adding 5xloading buffer and boiling for3 minutes

Immunoprecipitation

with a Myc tagantibody

Separation bySDS-PAGE


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Selection of Stably transduced BEAS 2B and MCF 10A

A: 293 cells were transduced with retroviral vectors encoding HER2WT , HER2YVMA or empty vector. At 24 h aftertransfection, cells were lysed and subjected to SDS-PAGE (left) or immunoprecipitation using a Myc tag antibody, followed bythe indicated immunoblotsB: BEAS2B cells were infected with retroviruses encoding HER2WT , HER2YVMA or empty vector. After puromycinselection, single colonies from each group were separated and analyzed for HER2 expression by immunoblot. Clonesselected for subsequent experiments are indicated by circles.

C: Immunoblot analysis of single colonies from retrovirus-infected MCF10A cells. Clones selected for subsequentexperiments are indicated by circles.

Results


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Stable and Transient expression of HER2 YVMA Mutant in BEAS 2B

A) BEAS2B cells stably expressing HER2WT ,HER2YVMA , or vector were seeded on 12-wellplates and allowed to grow in full growth medium orserum-free medium. Cells were trypsinized andcounted every 24 hr. Each data point representsthe mean 6 SD of four wells.

B) Stably transduced BEAS2B cells were maintained in growthmedium or serum starved for 3 days before being subjected toApo-BrdU assay. The percentage of FITC+ (apoptotic) cells isindicated. Quantitative data represent the mean 6 SD of threeexperiments.


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C) Stably transduced BEAS2B cells were seeded incolony-forming assay. Colonies were photographed atday 10.

D) The indicated BEAS2B cells (2 X 106 ) wereinjected s.c. in athymic nude mice. Detectable tumorsof 3 mm in minimal diameter are shown (n = 6 per

group).

E) close-to-confluent monolayers of BEAS2B cells in serum-freemedium were wounded with a pipette tip; wound closure wasmonitored at the indicated times.Right panel: transwell assays. Cells that had migrated to theunderside of transwell filters are shown after fixation.

Quantitative data are depicted below, with each bar representingthe mean 6 SD of six fields from three independent experiments.


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Stable and Transient expression of HER2 YVMA Mutant in MCF 10A

A) MCF 10A cells stably expressing HER2WT ,HER2YVMA , or vector were seeded on 12-wellplates and allowed to grow in full growth medium orserum-free medium. Cells were trypsinized and

counted every 24 hr. Each data point representsthe mean 6 SD of four wells.

B: Phase contrast images of HER2WT -orHER2YVMAexpressing MCF10A acini cultured on basement membrane in 8-well chambers and followed every 2 days. Scale bars, 50 mm.

C: MCF10A acini (from B ) were trypsinized and counted every

48 hr. Each data point represents the mean 6 SD of four wells.


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D: Stably transduced MCF10A cells were seeded at 2.5 3 104cells/well on Matrigel-coated transwells and allowed to invadetoward growth medium. At 24 hr invading cells were counted;each bar represents the mean 6 SD of three wells.

E: MCF10A cells were maintained in growth medium orserum starved for 3 days before being subjected to Apo-BrdU assay as described in Experimental Procedures.The mean percentage 6 SD of FITC+ apoptotic cells fromthree wells is indicated.


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3 Dimensional morphogenesis and indirect immunofluorescence

F: MCF10A cells expressing HER2WT or HER2YVMA were cultured on basement membrane for 8 days and stained with antibodiesa ainst b -catenin reen and cleaved cas ase-3 red .


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G: Transduced MCF10A cells were cultured on basement membrane for 14 days and stained with Ki-67 (green) and Myc tag (red) antibodies.Scale bars, 50 mm. In both panels, serial confocal sections of the stained acini were photographed at 2.96 m m intervals.


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A: MCF10A cells stably expressing HER2WT or HER2YVMA chimeras or empty vector were serum starved for 24 hr beforetreatment with 1 mM AP1510 for 15 min. Cell lysates were precipitated with an HA antibody; the HA pull-downs weresubjected to P-Tyr and HA immunoblot analysis as indicated in Experimental Procedures.

B: 32D cells stably expressing full-length HER2WT , HER2YVMA , or vector control were serum starved for 4 hr, lysed, andsubjected to the indicated immunoblot analyses.


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C: HER2WT or HER2YVMA receptors expressed in MCF10A cells wereprecipitated with a Myc antibody. An in vitro kinase assay was performed asdescribed in Experimental Procedures, and the products were analyzed by 7.5%

SDS-PAGE followed by P-Tyr and Myc immunoblots.

HER2YVMA i d li d i d d EGFR h h l i


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HER2YVMA induces ligand-independent EGFR phosphorylation

A: BEAS2B and MCF10A cells expressing HER2WT , HER2YVMA , and vector were serum starved for 24 hr, lysed, and then

subjected to SDS-PAGE or precipitation with a Myc tag antibody. Total cell lysates (left) and Myc pull-downs were tested byimmunoblots with the indicated antibodies


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B: MCF10A cells were serum starved for 24 hr and treated with 10 ng/ml EGF. At 030 min, the monolayers were lysed andthe cell lysates subjected to the indicated immunoblot analyses. For P-HER2, lysates were immunoprecipitated with HER2antibody followed by immunoblot with P-Tyr monoclonal antibody. Where indicated, 1 mM gefitinib was added 4 hr prior toEGF.C: HER2WT or HER2YVMA receptors expressed in MCF10A cells were precipitated with a Myc antibody. EGFRK721R(EGFRKD )-GFP expressed in 32D cells was pulled down with a GFP antibody. An in vitro kinase assay was performed asdescribed in Experimental Procedures, and the products were analyzed by 7.5% SDS-PAGE followed by P-Tyr, Myc, and

EGFR immunoblot analyses. The molecular weights (in kDa) are shown at the left of the gel. Positions of EGFRKD-GFP and HER2 receptors are indicated by arrows.


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A & B) MCF10A cells expressing HER2WT or HER2YVMA were grown on 12-well plates in completemedium with or without erlotinib or gefitinib and counted every day for 4 daysC) Cell lysates were prepared, separated by SDS-PAGE and subjected to immunoblot analysis using the

indicated antibodies.


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A: MCF10A cells expressing HER2WT or HER2YVMA weretreated for 24 hr with erlotinib, gefitinib, or lapatinib at theindicated concentrations. Cell lysates were prepared, separated

by SDS-PAGE, and subjected to immunoblots using theindicated antibodies.B: Cells were treated with trastuzumab (10 m g/ml) followed byacid wash to remove bound antibody. Cell surface proteins werethen biotinylated at 4C as indicated in Experimental Procedures.Cell lysates were precipitated with Streptavidin MagneticSpheres followed by HER2 immunoblot. To control for gelloading, whole-cell lysates were subjected to HER2 and actinimmunoblots (bottom panels).


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D: At day 8 and day 14, HER2YVMA acini treated with the combination of lapatinib and trastuzumab were stained for b -catenin (green) and cleaved caspase-3 (red) (day 8), or Ki-67 (green) and Myc tag (red) (day 14). DAPI (blue), nuclear

staining. Scale bars, 50 mm.

RNA interference of mutant HER2 inhibits survival of H1781 lung cancer cells


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RNA interference of mutant HER2 inhibits survival of H1781 lung cancer cells

A: H1781 cells grown on 6-well plates were transfected by siRNA oligonucleotides targeting HER2WT , HER2MUT , or acontrol sequence. At day 3 after transfection, total RNA was extracted. Two hundred nanograms of RNA were used in the RT-PCR reaction as described in Experimental Procedures. Gene-specific primer pairs were used to amplify HER2MUT ,

HER2WT , and b -actin cDNA (control). / lane indicates mock-transfected cells.B: H1781 cells were lysed 3 days after transfection and analyzed by immunoblot using the indicated antibodies.


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C: H1781 cells growing on glass coverslips were transfected by specific siRNA. At day 4 after transfection, cells were stainedusing antibodies against cleaved caspase-3 (red) and Ki-67 (green). DAPI (blue), nuclear staining. Scale bar, 50 mm.D: H1781 cells transfected with the indicated siRNA oligonucleotides were harvested by trypsinization 1 day posttransfectionand seeded in soft agarose con-taining the indicated inhibitors alone or in combination at the same concentrations used inFigure 5C. Colonies measuring R50 mm were counted at day 7.

Each bar represents the mean colony number 6 SD of 3 wells. p values were calculated using Students t test.

CI 1033 inhibits mutant HER2 driven growth and signaling


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CI-1033 inhibits mutant HER2-driven growth and signaling

A: MCF10A cells expressing HER2WT or HER2YVMA and H1781 cells were treated for 16 hr with CI-1033 at the indicated concentrations. Cell lysateswere pre-pared, separated by SDS-PAGE, and subjected to immunoblots using the indicated antibodies. B: Top: HER2WT - or HER2YVMA -expressing MCF10A cells were plated on basement membrane in 8-well chambers with or without CI-1033. Inhibitors were replen-ished every 2 days.Phase contrast image shown was recorded 12 days after the initial seeding of cells. Scale bars, 50 mm. Bottom: 12-day acini were trypsi-nized, and

total cell number was determined in a Coulter counter. Each bar graph represents the mean 6 SD of four wells. C: H1781 cells were seeded in softagarose containing the indicated concentration of CI-1033. Colonies measuring R50 mm were counted at day 7. Each bar represents the mean colonynumber 6 SD of three wells.

Conclusion


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Conclusion

Expression of in-frame YVMA insertion at residue 776 in nontumorigenic BEAS2B andMCF10A epithelial cells was markedly more transforming than wild-type HER2. HER2YVMA

was more potent in inducing serum-free cell proliferation, motility, and tumorigenicity in vivoas well as preventing apoptosis compared to HER2 WT

.

HER2YVMA induced transphosphorylation of kinase-dead EGFR and exhibited higherligand-independent tyrosine phosphorylation and stronger association with

signal transducers that mediate proliferative and prosurvival responses than HER2 WT

Another possible mechanism for activation of the HER2 kinase is transactivation by ligandbound EGFR or HER3/4. I ndeed, coexpression of the EGFR l ig and T GFa with Neu in themammary gland of transgenic mice markedly accelerates tumor onset and progression

compared to mice expressing the Neu or TGF alpha transgenes alone.

HER2YVMA induced transphosphorylation of kinase-dead EGFR and exhibited higherligand-independent tyrosine phosphorylation and stronger association withsignal transducers that mediate proliferative and prosurvival responses than HER2 WT


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Inhibition of mutant HER2 with RNA interference in H1781 lung cancer cells, which contain aVC insertion at G776 in exon 20 of the HER2 gene, inhibited P-MAPK and P-Akt, re-duced

proliferation, and induced cell death. These cells have highly phosphorylated wild-typeEGFR and are insensitive to gefitinib and erlotinib.

EGFR inhibitors would be effective against tumors driven by heterodimers of EGFR andwild-type HER2. In these cancers, HER2 amplifies signals by EGFR/HER2 heterodimers,which remain dependent on the EGFR kinase

cancers expressing mutation/insertions in exon 20 of HER2 would be insensitive to smallmolecule inhibitors of the EGFR tyrosine kinase. a combination of trastuzumab and lapatinibor the irreversible HER2 inhibitor CI-1033 may have clinical activity and is worthy ofprospective investigation.

I

In BEAS2B and MCF10A cells, HER2 YVMA potently induced EGFR phosphorylation. Thistransactivation did not require the EGFR tyrosine kinase in that it was also observed with

catalyti-cally dead K721R EGFR and was not blocked by the EGFR TKIs erlotinib andgefitinib.

References


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References

Debnath, J., Muthuswamy, S.K. and Brugge, J. S. (2003). Morphogenesis and

oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensionalbasement membrane cultures. Methods. 30. 256268

Barbieri, M. A., Roberts, R.A., Gumusboga, A., Highfield, H., Dominguez, C.A.,Wells,A., and Stahl, P.D. (2000). Epidermal Growth Factor and Membrane Trafficking:

EGF Receptor Activation of Endocytosis Requires Rab5a. J. Cell Bio. 30.

Hisayuki Shigematsu, Takao Takahashi, Masaharu Nomura, et al.(2005). SomaticMutations of the HER2 Kinase domain in the lung adenocarcinoma. Americanassociation for Cancer research

I. Rubin & Y. Yarden (2005). The basic biology of HER2. Annals of Oncology 12(Suppl. I): S3-S8, 2001.


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HER2Neu kinase

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