Effects of HER2-Binding Affi body Molecules on Intracellular Signaling Pathways
Lina Ekerljung
a Ann-Charlott Steffen
a Jörgen Carlsson
a Johan Lennartsson
b
a Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, and
b Ludwig Institute for Cancer Research, Uppsala University, Uppsala , Sweden
Z HER2: 342 is a promising small agent (7 kDa) that may be used as an alternative, or complement, to trastuzumab. If radiolabelled, it can hopefully also be used for HER2 imaging and radionuclide therapy.
HER2 is a transmembrane tyrosine kinase receptor in the epidermal growth factor receptor (EGFR) family. Members of the EGFR family bind a multitude of li-gands, leading to the formation of both homo- and het-erodimers. As a consequence of dimerization, the intra-cellular part of the receptor becomes tyrosine phosphory-lated. This serves two purposes: fi rst, it will increase the tyrosine kinase activity, and second, phosphorylated ty-rosines function as docking sites for molecules with Src homology 2 or phosphotyrosine-binding domains [1] . Im-portant signal transduction pathways downstream of the EGFR family are the mitogen-activated protein (MAP) kinases Erk1 and Erk2 (Erk1/2), the phosphatidylinositol 3 (PI3) kinase/Akt cascade and PLC � 1 ( fi g. 1 ) [2] . Signal-ing through these molecules is important for prolifera-tion, survival and migration [3–6] . These processes are essential for tumor formation, development and metas-tasis.
Key Words Affi body � Akt � Erk � HER2 � PLC � 1 � Signal transduction
Abstract Background: HER2, which is overexpressed in 25–30% of human breast cancers, is a tyrosine kinase receptor crit-ical for the signal transduction network that regulates proliferation, migration and apoptosis of cells. Method: We report the effects of two novel HER2-binding affi body molecules (Affi body ® ), (Z HER2: 4 ) 2 and Z HER2: 342 , on intra-cellular signal transduction pathways (Erk1/2, Akt and PLC � 1) using quantitative immunoblotting techniques and their biological effects in cell culture. The clinically approved antibody trastuzumab (Herceptin ® ) was used as reference substance. Results: Our data showed that, although all substances target HER2, the effects on the receptor and signaling molecules differed. For example, HER2 phosphorylation was induced by trastuzumab and (Z HER2: 4 ) 2 but inhibited by Z HER2: 342 . The effects these substances had on signal transduction correlated to some degree with changes in growth and migration, e.g. (Z HER2: 4 ) 2 stimulated phosphorylation of Erk1/2 and PLC � 1, as well as growth and migration, while Z HER2: 342 did not. Z HER2: 342 even inhibited phosphorylation of PLC � 1 and migration. Conclusion: Our data suggest that
Received: July 30, 2005 Accepted after revision: October 27, 2005 Published online: April 27, 2006
Johan LennartssonLudwig Institute for Cancer Research, Uppsala UniversityBox 595, Biomedical CenterSE–751 24 Uppsala (Sweden)Tel. +46 18 160406, Fax +46 18 160420, E-Mail [email protected]
HER2 has no natural ligand but is activated through hetero- or homodimerization with members of the EGFR family. HER2 also has an unregulated kinase activity and, when overexpressed, can be considered to be an on-coprotein [7, 8] . HER2 is overexpressed in 25–30% of breast cancers and is associated with resistance to con-ventional cancer therapies, e.g. chemotherapy and radia-tion therapy, and poor patient prognosis [9] . Thus, HER2 is an important target for anti-tumor therapies.
The most extensively studied drug that targets HER2 is trastuzumab (Herceptin ® ). It is a humanized mono-clonal antibody that recognizes an epitope on the extra-cellular domain of HER2 with a K D of 1 n M and blocks downstream signaling [10] . Trastuzumab has been re-ported to downregulate the amount of HER2 at the cell surface and activate the human complement cascade [11, 12] . Today, trastuzumab is used for treatment of HER2-overexpressing breast cancer, but less than 50% of the patients respond to treatment [11, 13] . Thus, there is a need for new HER2-specifi c targeting agents that are alternatives or complements to trastuzumab. In order to predict the therapeutic response of substances that target HER2, it is important to be aware of the effects these substances have on proteins downstream of the receptors. A detailed understanding of which signal transduction pathways are inhibited, activated or not affected by a substance may explain responses to treatments, or some-times the lack of a response, and suggest logical combina-tion therapies to improve effi cacy.
In a previous study from our group, the construction and in vitro testing of (Z HER2: 4 ) 2 for radionuclide-based diagnostics was described [14] . The HER2-binding (Z HER2: 4 ) 2 is a bivalent version of a class of affi nity ligands denoted affi body molecules (Affi body ® ). Affi body mole-cules are based on the Z domain of staphylococcal protein A and their robust structure together with their low mo-lecular weight (7 kDa, in comparison with an antibody of 150 kDa) make them suitable for many different applica-tions, such as receptor-specifi c imaging and targeted ra-diotherapy [14, 15] . In the present study, we have inves-tigated the intracellular effects by two HER2-binding af-fi body molecules, (Z HER2: 4 ) 2 and an affi nity-maturated monovalent version, Z HER2: 342 . Affi nity maturation is a process in which the original affi body is selectively re-randomized at key amino acids in the HER2-binding sur-face and selected based on affi nity for HER2. Z HER2: 342 binds HER2 with three orders of magnitude higher af-fi nity than the original HER2-binding affi body, which binds with a K D of 50 n M [15] . The bivalent affi body (Z HER2: 4 ) 2 has a K D of 3 n M [14] . The high affi nity to-wards HER2, combined with their small size, make the affi body molecules well suited for tumor targeting. Here, we have studied the effects that these two affi body mol-ecules and trastuzumab have on the intracellular signal-ing pathways described above ( fi g. 1 ) and their in vitro effects on growth, migration and apoptosis.
Extracellular
Intracellular
HER
Ras-GTPRaf
Mek Mek
SosGrb2
P
P
Shc
PKC
PI3K
IP3
PIP2 PIP3 PIP3
PIP3
Akt
Akt
PDK
PIP3
Ca2+
DAG
Erk Erk
Ras-GDP
PP
Fig. 1. Schematic drawing of the signaling pathways discussed. Proteins in the path-ways specifi cally analyzed with phosphoan-tibodies are marked with a star symbol (i.e. p-Erk, p-Akt, and p-PLC � 1). Signaling through these pathways is important for proliferation, survival and cell migration. GTP = Guanosine triphosphate; GDP = guanosine diphosphate; DAG = diacylglyc-erol; PIP 2 = phosphatidylinositol 4,5-bisphosphate; PIP 3 = phosphatidylinositol 3,4,5-triphosphate; PKC = protein kinase C; PDK = phosphatidylinositol-dependent protein kinase.
Affi body Molecules and Intracellular Signaling
Tumor Biol 2006;27:201–210 203
Materials and Methods
Antibodies and Reagents Antibodies specifi c for HER2 and phosphorylated Erk1/2 or
Akt were purchased from Cell Signaling Technology (Beverly, Mass., USA). Antibodies recognizing phosphotyrosine and Akt were from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Rabbit antiserum against Erk2 and PLC � 1 were generated by im-munizing rabbits with a peptide corresponding to their carboxyl terminus as previously described [16, 17] .
Cell Culture Human ovarian carcinoma (SKOV-3) cells from American
Type Culture Collection (Manassas, Va., USA) were grown in Mc-Coy’s 5A medium supplemented with 10% fetal bovine serum (FBS), L -glutamine (2 m M ), penicillin (100 IU/ml), and streptomy-cin (100 � g/ml). All reagents were from Biochrom KG (Berlin, Ger-many).
Cell Lysis, Immunoprecipitation and Immunoblotting After starvation in 0.1% FBS overnight, approximately 4 ! 10 6
SKOV-3 cells were treated with trastuzumab (16.6 n M ), (Z HER2: 4 ) 2 (16.6 n M ), Z HER2: 342 (16.4 n M ) or EGF (1.67 n M ) in 6 ml media (0.1% FBS) for different periods of time, ranging from 15 min to 8 h. These concentrations correspond to a ratio of approximately 7.5: 1 between each substance and the receptor. Cells were then washed once with ice-cold PBS and lysed in a lysis buffer (1% Tri-ton X-100, 10% glycerol, 150 m M NaCl, 5 m M EDTA, 20 m M Tris, 1% Trasylol, 1 m M phenylmethylsulfonyl fl uoride, and 1 m M Na 3 VO 4 , pH 7.4). Immunoprecipitations were performed over-night at +4 ° C. Next, protein A-Sepharose beads were added and incubated with end-over-end rotation for an additional 30 min. The beads were washed three times in lysis buffer, boiled with sample buffer containing � -mercaptoethanol, and eluates were separated by SDS-PAGE. For Western blotting, samples were electrotrans-ferred to polyvinylidene difl uoride (Immobilon-P transfer mem-brane, pore size 0.45 � m; Millipore, Billerica, Mass., USA) mem-branes, which were blocked in 5% BSA in Tris-buffered saline solu-tion containing 0.1% Tween-20. Primary antibodies were incubated overnight at +4 ° C or 2 h at room temperature, using concentrations recommended by manufacturers with 1% BSA in Tris-buffered saline solution containing 0.1% Tween-20. Rabbit antiserum was used at a dilution of 1: 300. After washing, the mem-branes were incubated with horseradish peroxidase- conjugated anti-mouse or anti-rabbit IgG antibodies (both from Amersham Biosciences, Sweden), and proteins were visualized using enhanced chemiluminescence Western blotting detection systems from either Roche Applied Science (Indianapolis, Ind., USA) or Santa Cruz Biotechnology on a cooled charge-coupled device camera. Quanti-fi cations were performed using Aida image analyzer software, ver-sion 3.10 (Fuji Film, Minami-Ashigara, Japan).
Proliferation Study A number of 50,000 SKOV-3 cells were seeded in three culture
bottles (25 cm 2 ; Nunc A/S, Denmark) for each condition and treat-ed with (Z HER2: 4 ) 2 (16.6 n M ), Z HER2: 342 (16.4 n M ), trastuzumab (16.6 n M ) or left untreated. The media and substances were re-placed three times a week. Cell counting was performed once a week followed by reseeding of 45,000 cells for continuous culturing. The change in cell number was monitored for 33 days.
Migration Assay SKOV-3 cells were cultivated to a confl uent layer in culture
bottles (25 cm 2 ; Nunc A/S) after which they were starved in serum-free medium for 40 h. Next, wounds (three per group) were intro-duced in the confl uent cell layers by scraping a 10- � l pipette tip against the bottom of the bottles. The cells were then rinsed to re-move cell debris and new serum-free medium was added. The cells were treated with (Z HER2: 4 ) 2 (16.6 n M ), Z HER2: 342 (16.4 n M ), trastu-zumab (16.6 n M ) or left untreated. Phase-contrast microscopic pic-tures were taken at 0, 24, 32, 48, 55 and 72 h after introduction of the wounds. After 72 h, the cells were fi xated and colored with he-matoxylin to detect mitosis. This was done to estimate the level of proliferation in each group.
Apoptosis Assay SKOV-3 cells were treated with (Z HER2: 4 ) 2 (16.6 n M ), Z HER2: 342
(16.4 n M ), trastuzumab (16.6 n M ) or left untreated for 4 h. Then, camptothecin (30 � M ) from Sigma-Aldrich (Munich, Germany) was added to one bottle from each group. After 24 h, the cells, at-tached or in the medium, were harvested. The cells were centri-fuged at 165 g for 5 min and the pellets were resuspended in an-nexin-binding buffer (10 m M HEPES, 140 m M NaCl, 2.5 m M CaCl 2 , pH 7.4) to a concentration of approximately 2 ! 10 6 cells/ml. An amount of 100 � l was taken from each sample and mixed with 5 � l annexin V (Alexa Flour 488 conjugate; Molecular Probes, Eugene, Oreg., USA). Propidium iodide was added to a fi nal con-centration of 1.3 � g/ml. The samples were incubated at room tem-perature for 15 min. Then, 400 � l annexin-binding buffer was add-ed and the samples were kept on ice before fl ow cytometric analysis. The samples were analyzed on a FACSort instrument. Data on 10,000 events were acquired and processed using the Cell Quest™ Software.
Results
Effects on HER2 Phosphorylation by (Z HER2: 4 ) 2 , Z HER2: 342 and Trastuzumab SKOV-3 cells, expressing high levels of HER2 (ap-
proximately 2 ! 10 6 HER2 per cell), were treated with trastuzumab, (Z HER2: 4 ) 2 or Z HER2: 342 for different periods of time, as indicated in fi gure 2 . Results from three inde-pendent experiments were analyzed by densitometry and the mean ratio between phosphorylation and total protein content was plotted ( fi g. 2 ). Trastuzumab induced a rapid, 15-fold increase in HER2 phosphorylation ( fi g. 2 a), while treatment with (Z HER2: 4 ) 2 led to a gradual increase in phosphorylation over time (9-fold) ( fi g. 2 b). In contrast, Z HER2: 342 reduced HER2 phosphorylation by 30–40% compared with untreated cells ( fi g. 2 c). These results agree with earlier observations that trastuzumab induc-es phosphorylation of HER2 [12] . Interestingly, the two affi body reagents affected HER2 phosphorylation dif-ferently: (Z HER2: 4 ) 2 led to a progressive increase in phos-phorylation, whereas Z HER2: 342 reduced phosphoryla-
Ekerljung /Steffen /Carlsson /Lennartsson
Tumor Biol 2006;27:201–210 204
tion of HER2. Thus, although all substances target HER2, they affect receptor phosphorylation in different ways.
Baseline Activity of Signal Transduction Proteins The above data demonstrated that trastuzumab,
(Z HER2: 4 ) 2 and Z HER2: 342 affected HER2 phosphorylation differently. Therefore, we analyzed these fi ndings on signal transduction. Initially, we analyzed the baseline activity of downstream signal transduction, i.e. the background phos-phorylation levels of the MAP kinases Erk1/2, the serine/threonine kinase Akt and the phospholipase PLC � 1 in SKOV-3 cells. This was done by stimulating serum-starved cells with EGF for 1 h, using non-treated cells as controls. SKOV-3 cells also express EGFR (approximately 2 ! 10 5 EGFR per cell) in addition to HER2. We know from pre-vious experiments that EGF treatment leads to a robust activation of the aforementioned proteins in glioma U343 cells [18] . Typical results are shown in fi gure 3 . Erk1/2 could not be further activated by EGF ( fi g. 3 a), whereas Akt ( fi g. 3 b) and PLC � 1 ( fi g. 3 c) could be somewhat more activated by treatment with EGF. Incubation for 1 h with EGF reduced the level of HER2 phosphorylation below that detected in untreated cells ( fi g. 3 d). The latter is prob-ably due to EGF-dependent downregulation of EGFR-HER2 heterodimers. Note that there was a signifi cant phosphorylation of Erk1/2, HER2 and Akt in the absence of treatment, consistent with the fact that SKOV-3 is a cancer cell line expressing HER2 at oncogenic levels.
Effects of (Z HER2: 4 ) 2 , Z HER2: 342 and Trastuzumab on the Signal Transduction Proteins, Erk1/2, Akt and PLC � 1 To assess the effect of (Z HER2: 4 ) 2 , Z HER2: 342 and trastu-
zumab on the signal transduction proteins, serum-starved cells were treated with these substances for different pe-riods of time, as indicated in fi gures 4–6 . None of the substances had a dramatic effect on Erk1/2 phosphoryla-tion. Trastuzumab lowered Erk1/2 phosphorylation after 4–8 h incubation by 25–35% ( fi g. 4 a), while (Z HER2: 4 ) 2 treatment led to about 1.5-fold increase in Erk1/2 phos-phorylation ( fi g. 4 b). Z HER2: 342 reduced phosphorylation of Erk1/2 by 15–20% ( fi g. 4 c). In summary, the effects of these substances on Erk1/2 are modest and not correlated with their ability to induce HER2 phosphorylation, indi-cating a complex regulation of Erk1/2 activity. It is pos-sible that also other genetic changes in SKOV-3 cells re-sult in Erk1/2 activation independently of HER2, thus explaining the relative insensitivity to the HER2-target-ing substances.
Phosphorylation of Akt was reduced by 30–40% after treatment with trastuzumab ( fi g. 5 a), whereas treatment with (Z HER2: 4 ) 2 and Z HER2: 342 led to about 2- and 1.5-fold increases, respectively ( fi g. 5 b, c). Increased phosphoryla-tion of Akt is associated with protection from apoptosis,
Trastuzumab2018161412108
p-H
ER
2/H
ER
2 (A
U)
6420
0 0.25 2
Time (h)
4 8
ZHER2:3422018161412108
p-H
ER
2/H
ER
2 (A
U)
6420
0 0.25 2
Time (h)
4 8
(ZHER2:4)220
18
16
14
12
10
8
p-H
ER
2/H
ER
2 (A
U)
6
4
2
00 0.25 2
Time (h)
4 8
a
b
c
Fig. 2. Phosphorylation of HER2. SKOV-3 cells were left untreat-ed or incubated with trastuzumab ( a ), (Z HER2: 4 ) 2 ( b ) or Z HER2: 342 ( c ) for the indicated periods of time. Immunoprecipitation with an anti-HER2 antibody was followed by immunoblotting with anti-bodies specifi c for phosphotyrosine or total HER2. Results from three independent experiments were analyzed by densitometry and the average ratio plotted. Untreated cells were used as reference.
Affi body Molecules and Intracellular Signaling
Tumor Biol 2006;27:201–210 205
and activation of Akt has been suggested to contribute to trastuzumab resistance [19] .
Treatment with trastuzumab strongly ( 1 20-fold) in-duced phosphorylation of PLC � 1 ( fi g. 6 a) and so did (Z HER2: 4 ) 2 , but to a lower extent (2-fold increase) ( fi g. 6 b). In contrast, treatment with Z HER2: 342 gradually reduced the level of PLC � 1 phosphorylation to about half of the level in untreated cells ( fi g. 6 c). Note that in fi gure 6 a, the 15-min treatment with trastuzumab is used for normal-ization. This is due to the fact that in one experiment, the untreated value for PLC � 1 phosphorylation was zero, thus making it impossible to normalize to this value. The phosphorylation levels of PLC � 1 for the untreated sam-ples were generally very low, as shown in fi gure 3 c.
Proliferation Study To evaluate the effects on proliferation after treatment
with trastuzumab, (Z HER2: 4 ) 2 and Z HER2: 342 in cell culture, we performed a cell growth study. Cells were seeded into parallel cultures and incubated with (Z HER2: 4 ) 2 , Z HER2: 342 or trastuzumab throughout the experiment. Untreated cells were used as a control. Cells were counted once a week and a fraction of the cells were then reseeded. As can be seen in table 1 , treatment with (Z HER2: 4 ) 2 had a signifi cant (p ! 0.01) growth-promoting activity com-pared with untreated cells. In contrast, treatment with Z HER2: 342 or trastuzumab showed a tendency for modest growth inhibitory effects. However, the reductions in cell number for these groups were not signifi cant.
Migration Assay In order to study the effects of the three substances on
migration, we performed an in vitro ‘wound-healing’ as-say. In this experiment, a ‘wound’ was introduced into a confl uent layer of SKOV-3 cells, which were treated with (Z HER2: 4 ) 2 , Z HER2: 342 , trastuzumab or left untreated. The healing of the wounds was then followed for 72 h to mea-sure migration. Phase-contrast microscopic pictures were taken immediately after the wounds were introduced, and then after 24, 32, 48, 55 and 72 h. The experiment was performed in serum-free medium to minimize the infl uence of proliferation. After 72 h, the cells were fi x-ated and colored with hematoxylin to detect mitoses, to estimate the extent of proliferation in each group. The amount of mitoses in each group was low compared with control cultures (cells grown in 10% FCS), indicating that proliferation was inhibited (data not shown). Representa-tive pictures of each group are shown ( fi g. 7 ). The cells
– +
EGF
TCLIb: p-Erk1/2
TCLIb: Erk2
TCLIb: p-Akt
TCLIb: Akt
Ip: HER2Ib: pTyr
Ip: HER2Ib: HER2
– + EGF – +
EGF – +EGF
a b
c d
Fig. 3. Background levels of protein phos-phorylation. SKOV-3 cells were left un-treated or incubated with EGF for 1 h. To-tal cell lysate (TCL) was prepared for the analysis of Erk1/2 ( a ) and Akt ( b ). For PLC � 1 ( c ) and HER2 ( d ), the proteins were fi rst immunoprecipitated (Ip) with specifi c antibodies. Immunoblotting (Ib) was done with antibodies specifi c for phosphorylated or total Akt, Erk1/2, PLC � 1 and HER2 as indicated. The result from one representa-tive experiment is shown. pTyr = Phospho-tyrosine.
treated with (Z HER2: 4 ) 2 closed the wound faster than the untreated cells. Both treatment with trastuzumab and Z HER2: 342 decreased the rate of healing, trastuzumab more than Z HER2: 342 .
Apoptosis Assay To determine the effects on apoptosis after treatment
with (Z HER2: 4 ) 2 , Z HER2: 342 or trastuzumab, the apoptosis levels were quantifi ed by fl ow cytometry using annexin V/propidium iodide. Binding of annexin V to phospha-tidylserine residues in the cell membrane is an early
Trastuzumab
2
1
p-E
rk/E
rk2
(AU
)
00 0.25 2
Time (h)
4 8
a
(ZHER2:4)2
2
1
p-E
rk/E
rk2
(AU
)
00 0.25 2
Time (h)
4 8
b
ZHER2:342
2
1
p-E
rk/E
rk2
(AU
)
00 0.25 2
Time (h)
4 8
c
Trastuzumab
2
3
1
p-A
kt/A
kt (
AU
)
00 0.25 2
Time (h)
4 8
a
(ZHER2:4)2
2
3
1
p-A
kt/A
kt (
AU
)
00 0.25 2
Time (h)
4 8
b
ZHER2:342
2
3
1
p-A
kt/A
kt (
AU
)
00 0.25 2
Time (h)
4 8
c
Fig. 4. Phosphorylation of Erk1/2. SKOV-3 cells were left untreat-ed or incubated with trastuzumab ( a ), (Z HER2: 4 ) 2 ( b ) or Z HER2: 342 ( c ) for the indicated periods of time. Immunoblotting was done with antibodies specifi c for phospho-Erk1/2 (pErk) or total Erk2. Results from three independent experiments were analyzed by den-sitometry and the average ratio plotted. Untreated cells were used as reference.
Fig. 5. Phosphorylation of Akt. SKOV-3 cells were left untreated or incubated with trastuzumab ( a ), (Z HER2: 4 ) 2 ( b ) or Z HER2: 342 ( c ) for the indicated periods of time. Immunoblotting was done with antibodies specifi c for phospho-Akt (pAkt) or total Akt. Results from three independent experiments were analyzed by densitom-etry and the average ratio plotted. Untreated cells were used as reference.
Affi body Molecules and Intracellular Signaling
Tumor Biol 2006;27:201–210 207
marker of apoptosis. Apoptosis was induced by camp-tothecin, which works as a type I DNA topoisomerase inhibitor and is known to induce apoptosis [20] . The apoptosis levels were measured both after treatment with only (Z HER2: 4 ) 2 , Z HER2: 342 or trastuzumab and after treatment with these substances together with camptoth-
ecin. The samples treated with only (Z HER2: 4 ) 2 , Z HER2: 342 or trastuzumab showed no deviation from the baseline level of apoptosis of the untreated sample, which was about 0.3%. Treatment with camptothecin induced a no-ticeable percentage of apoptosis, about 5.7%. Neither (Z HER2: 4 ) 2 , Z HER2: 342 nor trastuzumab did signifi cantly
Trastuzumab
1
2
00 0.25 2
Time (h)
4 8
a
(ZHER2:4)2
2
3
1
00 0.25 2
Time (h)
4 8
b
ZHER2:342
2
3
1
00 0.25 2
Time (h)
4 8
c
p-P
LCγ1
/PLC
γ1 (
AU
)p
-PLC
γ1/P
LCγ1
(A
U)
p-P
LCγ1
/PLC
γ1 (
AU
)
Fig. 6. Phosphorylation of PLC � 1. SKOV-3 cells were left untreat-ed or incubated with trastuzumab ( a ), (Z HER2: 4 ) 2 ( b ) or Z HER2: 342 ( c ) for the indicated periods of time. Immunoprecipitation was made with PLC � 1-specifi c antibodies prior to immunoblotting, which was done with antibodies specifi c for phosphotyrosine or total PLC � 1. Results from three independent experiments were analyzed by densitometry and the average ratio plotted. a Note that the 15-min treatment value is used as reference.
Untreated 0 h
Trastuzumab 0 h
(ZHER2:4)2 0 h
ZHER2:342 0 h ZHER2:342 48 h
(ZHER2:4)2 48 h
Trastuzumab 48 h
Untreated 48 h
Fig. 7. Migration assay. SKOV-3 cells were cultivated in serum-free media after which wounds were introduced to the confl uent layers of cells. The cells were left untreated ( a , b ) or incubated with trastu-zumab ( c , d ), (Z HER2: 4 ) 2 ( e , f ) or Z HER2: 342 ( g , h ). Phase-contrast microscopic pictures were taken at the starting point and after 24, 33, 48, 55 and 72 h. The 0-hour ( a , c , e , g ) and 48-hour ( b , d , f , h ) pictures were selected as representatives to show the overall trends in the four groups. Scale bars: 100 � m.
Ekerljung /Steffen /Carlsson /Lennartsson
Tumor Biol 2006;27:201–210 208
change this amount of camptothecin-induced apoptosis and all results with these substances were within 5.4–5.9% of apoptosis.
Discussion
In the present study, we investigated the effects of the novel HER2-targeting agents (Z HER2: 4 ) 2 and Z HER2: 342 at a molecular level. The clinically approved monoclonal antibody trastuzumab (Herceptin) was used as reference substance. The results showed that although these sub-stances all target HER2 in the same cellular context, they had dramatically different effects on intracellular signal-ing, which to some degree also translated to cell prolif-eration and migration but not to apoptosis.
The pathways investigated, besides HER2 itself, were: the MAP kinases Erk 1/2, Akt, which is a serine/threonine kinase activated in a PI3-kinase-dependent manner, and the phospholipase PLC � 1 ( fi g. 1 ). Since phosphorylation of Akt, Erk1/2, PLC � 1 and HER2 correlates well with their enzymatic activity, we evaluated their phosphoryla-tion as a measure of activity. Notably, HER2, Erk1/2 and Akt were also reproducibly phosphorylated in the absence of stimulation, which is in concurrence with the fact that SKOV-3 is a transformed cell line.
Our results suggest that phosphorylation of HER2 and PLC � 1 followed the same overall trends, i.e. induced by trastuzumab and (Z HER2: 4 ) 2 , whereas treatment with Z HER2: 342 led to a reduction in phosphorylation. One can speculate that the two bivalent molecules, trastuzumab and (Z HER2: 4 ) 2 , might induce formation of receptor di-mers and thus increase phosphorylation of HER2, lead-ing to more effi cient activation of signal transduction compared with the monovalent Z HER2: 342 . However, we have no data showing whether the bivalent substances bind simultaneously to two HER2 molecules. The impor-tance of bivalent binding capacity is not well known, and further studies on this subject are planned. To the knowl-edge of the authors, there is only one report on this topic [21] . That study was focusing on two anti-HER2 antibod-ies using mono- and bivalent trastuzumab as control. The authors were not able to show any correlation between epitope affi nity or valency on HER2 internalization or cell growth. However, Erk phosphorylation was, after 8 h of exposure, increased by treatment with the bivalent an-tibody as compared with treatment with the monovalent form. In addition, we have recently shown that a bivalent affi body is internalized more effi ciently than the corre-sponding monovalent form [14] .
The phosphorylation of Akt and Erk1/2 did not cor-relate with that of HER2. Trastuzumab inhibited both Akt and Erk1/2 while (Z HER2: 4 ) 2 increased their phos-phorylation. On the other hand, Z HER2: 342 treatment led to an increase in Akt phosphorylation but a minor reduc-tion in phosphorylation of Erk1/2. In JIMT-1 cells, it has been suggested that activation of Akt is a possible mech-anism for trastuzumab resistance [19] . Moreover, inhibi-tion of Akt is an important way for trastuzumab to induce cell cycle arrest [22] . Table 2 summarizes the results from our analyses of the signal transduction proteins HER2, PLC � 1, Akt and Erk1/2.
Previous investigations of the molecular mechanism behind the anti-tumor activity of trastuzumab have main-ly demonstrated a critical role of Akt, and to some extent Erk, inhibition in accordance with our results [11, 22, 23] . Regarding the involvement of PLC � 1 there is no avail-able information. Thus, the molecular events by which trastuzumab acts to inhibit tumor growth is still not com-pletely understood. Further studies on cell lines express-ing different levels of HER2 are planned to determine their responses to affi body molecules (as well as trastu-zumab).
For a cell to respond to treatment from the outside it must integrate the inputs from all activated signal trans-duction pathways. The data from the proliferation study showed that (Z HER2: 4 ) 2 had a robust growth-promoting activity compared with untreated cells, while both trastu-zumab and Z HER2: 342 showed a weak (but not signifi cant) tendency to growth inhibition. These results are in agree-ment with the data from the signaling pathway assays, showing that (Z HER2: 4 ) 2 had a stimulatory effect on Erk1/2, as well as the other two signaling molecules. In contrast, Z HER2: 342 resulted in inhibition of all pathways except Akt. It has been demonstrated that interfering with the Erk1/2 cascade inhibits growth of SKOV-3 xenografts in
+ + + = More than 10-fold increase; + + = 3- to 10-fold increase; + = 1.5- to 3-fold increase; – – – = more than 45% reduction; – – = 25–45% reduction; – = 15–24% reduction.
Affi body Molecules and Intracellular Signaling
Tumor Biol 2006;27:201–210 209
mice [24] . Thus, the minor effects of Z HER2: 342 and trastu-zumab on proliferation may be explained by the insensi-tivity of Erk1/2 phosphorylation to these substances.
As trastuzumab increased the phosphorylation of PLC � 1, one could expect cells treated with trastuzumab to have increased motility, as overactivity of PLC � 1 in other model systems has been shown to increase cell motility [6, 17, 25] . However, the migration study did not indicate this. On the contrary, treatment with trastu-zumab reduced the tendency of the cells to migrate. Also Z HER2: 342 resulted in a decrease in migration while (Z HER2: 4 ) 2 increased migration compared with untreated cells. For the two affi body molecules, this result correlates with the phosphorylation of PLC � 1, while for trastuzum-ab, the migration assay and the phosphorylation scheme of PLC � 1 contradict each other. However, PI3 kinase, which lies upstream of Akt, also has an established role in chemotaxis [26] . Thus, it is possible that the reduction in PI3 kinase activity (indirectly measured as Akt phos-phorylation) by trastuzumab inhibits cell migration even in the presence of robust PLC � 1 phosphorylation. Fur-ther work is needed to clarify these effects and the result-ing in vivo impact.
The apoptosis assay did not show any differences in apoptosis levels after treatment with camptothecin alone or together with (Z HER2: 4 ) 2 , Z HER2: 342 or trastuzumab. This indicates that there is no relation between the level of phosphorylated Akt and the extent to which campto-thecin induces apoptosis in SKOV-3 cells. One can there-fore speculate that camptothecin-induced apoptosis in SKOV-3 cells does not involve the PI3 kinase/Akt path-way. However, studies on other cell lines have shown that resistance to camptothecin, or camptothecin derivates, at least partly, occurs through the PI3 kinase/Akt pathway [27, 28] .
General conclusions which can be drawn from this work are: (1) the affi body molecules (Z HER2: 4 ) 2 and Z HER2: 342 (as well as trastuzumab) are biologically active molecules that affect intracellular signaling pathways, cell proliferation and migration, (2) targeting HER2 with dif-ferent substances affects the cell differently, and (3) the effect which a substance has on HER2 itself does not nec-essarily refl ect the effects on downstream signaling, e.g. trastuzumab activates HER2 but inhibits Akt. Possible explanations for these differences could be that each sub-stance, after internalization, results in distinctive subcel-lular localization of HER2, which may affect coupling to various signal transduction pathways. Another possibil-ity is that the different substances result in HER2 com-plexes with differential use of autophosphorylation sites
and consequently variations in interactions with down-stream effector molecules. The latter possibility has been demonstrated for EGFR stimulated with EGF or a dex-tran conjugate of EGF [18] . In addition, it is possible that other oncoproteins are also involved in regulating these signaling pathways independently of HER2.
In order to design effective treatment regimens, it is vital to have a detailed understanding of the effects a sub-stance has on the molecular level in a cell. This informa-tion can be used to rationally combine different therapeu-tic agents. The present study shows that Z HER2: 342 in many ways behaves similar to trastuzumab on cultured SKOV-3 cells. Because of its relatively low molecular weight, it is expected that Z HER2: 342 will have a better tu-mor penetrance than trastuzumab. We propose that Z HER2: 342 is a promising new HER2-targeting molecule to be used for tumor imaging and/or therapy.
Acknowledgements
The authors thank Affi body AB for the generous gift of (Z HER2: 4 ) 2 and Z HER2: 342 . This study was fi nancially supported by the Swedish Cancer Society, grant No. 0980-B04-17XCC (040171), and the Swedish Agency for Innovation Systems (VINNOVA), grant No. 2004-02159, P25882-1.
Ekerljung /Steffen /Carlsson /Lennartsson
Tumor Biol 2006;27:201–210 210
References
1 Schlessinger J, Lemmon MA: SH2 and PTB domains in tyrosine kinase signaling. Sci STKE 2003; 2003:RE12.
2 Marmor MD, Skaria KB, Yarden Y: Signal transduction and oncogenesis by ErbB/HER receptors. Int J Radiat Oncol Biol Phys 2004;
58: 903–913. 3 Datta SR, Brunet A, Greenberg ME: Cellular
survival: a play in three Akts. Genes Dev 1999;
13: 2905–2927. 4 Zhang W, Liu HT: MAPK signal pathways in
the regulation of cell proliferation in mamma-lian cells. Cell Res 2002; 12: 9–18.
5 Wells A, Grandis JR: Phospholipase C-gam-ma1 in tumor progression. Clin Exp Metastasis 2003; 20: 285–290.
6 Thomas SM, Coppelli FM, Wells A, Gooding WE, Song J, Kassis J, Drenning SD, Grandis JR: Epidermal growth factor receptor-stimu-lated activation of phospholipase Cgamma-1 promotes invasion of head and neck squamous cell carcinoma. Cancer Res 2003; 63: 5629–5635.
7 Di Fiore PP, Pierce JH, Kraus MH, Segatto O, King CR, Aaronson SA: erbB-2 is a potent on-cogene when overexpressed in NIH/3T3 cells. Science 1987; 237: 178–182.
8 Lonardo F, Di Marco E, King CR, Pierce JH, Segatto O, Aaronson SA, Di Fiore PP: The nor-mal erbB-2 product is an atypical receptor-like tyrosine kinase with constitutive activity in the absence of ligand. New Biol 1990; 2: 992–1003.
9 Chen JS, Lan K, Hung MC: Strategies to target HER2/neu overexpression for cancer therapy. Drug Resist Updat 2003; 6: 129–136.
10 Park BW, Zhang HT, Wu C, Berezov A, Zhang X, Dua R, Wang Q, Kao G, O’Rourke DM, Greene MI, Murali R: Rationally designed anti-HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases in vitro and in vivo. Nat Biotechnol 2000; 18: 194–198.
11 Albanell J, Codony J, Rovira A, Mellado B, Gascon P: Mechanism of action of anti-HER2 monoclonal antibodies: scientifi c update on trastuzumab and 2C4. Adv Exp Med Biol 2003; 532: 253–268.
12 Sliwkowski MX, Lofgren JA, Lewis GD, Ho-taling TE, Fendly BM, Fox JA: Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin). Semin Oncol 1999;
Villman K, Bengtsson NO, Ostenstad B, Lund-qvist H, Blomqvist C: HER2 expression in breast cancer primary tumours and corre-sponding metastases. Original data and litera-ture review. Br J Cancer 2004; 90: 2344–2348.
14 Steffen AC, Wikman M, Tolmachev V, Adams G, Ståhl S, Carlsson J: In vitro characterization of a bivalent anti-HER-2 affi body with poten-tial for radionuclide based diagnostics. Cancer Biother Radiopharm 2005; 20: 233–242.
15 Wikman M, Steffen AC, Gunneriusson E, Tol-machev V, Adams GP, Carlsson J, Stahl S: Se-lection and characterization of HER2/neu-binding affi body ligands. Protein Eng Des Sel 2004; 17: 455–462.
16 Lennartsson J, Blume-Jensen P, Hermanson M, Ponten E, Carlberg M, Ronnstrand L: Phos-phorylation of Shc by Src family kinases is nec-essary for stem cell factor receptor/c-kit medi-ated activation of the Ras/MAP kinase pathway and c-fos induction. Oncogene 1999; 18: 5546–5553.
17 Ronnstrand L, Siegbahn A, Rorsman C, Joh-nell M, Hansen K, Heldin CH: Overactivation of phospholipase C-gamma1 renders platelet-derived growth factor beta-receptor-expressing cells independent of the phosphatidylinositol 3-kinase pathway for chemotaxis. J Biol Chem 1999; 274: 22089–22094.
18 Hagg M, Liljegren A, Carlsson J, Ronnstrand L, Lennartsson J: EGF and dextran-conjugated EGF induces differential phosphorylation of the EGF receptor. Int J Mol Med 2002; 10: 655–659.
19 Tanner M, Kapanen AI, Junttila T, Raheem O, Grenman S, Elo J, Elenius K, Isola J: Charac-terization of a novel cell line established from a patient with Herceptin-resistant breast can-cer. Mol Cancer Ther 2004; 3: 1585–1592.
20 Kaufmann SH, Earnshaw WC: Induction of apoptosis by cancer chemotherapy. Exp Cell Res 2000; 256: 42–49.
21 Neve RM, Nielsen UB, Kirpotin DB, Poul MA, Marks JD, Benz CC: Biological effects of anti-ErbB2 single chain antibodies selected for internalizing function. Biochem Biophys Res Commun 2001; 280: 274–279.
22 Le XF, Lammayot A, Gold D, Lu Y, Mao W, Chang T, Patel A, Mills GB, Bast RC Jr: Genes affecting the cell cycle, growth, maintenance, and drug sensitivity are preferentially regulat-ed by anti-HER2 antibody through phosphati-dylinositol 3-kinase-AKT signaling. J Biol Chem 2005; 280: 2092–2104.
23 Liang K, Lu Y, Jin W, Ang KK, Milas L, Fan Z: Sensitization of breast cancer cells to radia-tion by trastuzumab. Mol Cancer Ther 2003; 2:
1113–1120. 24 McPhillips F, Mullen P, Monia BP, Ritchie
AA, Dorr FA, Smyth JF, Langdon SP: Asso-ciation of c-Raf expression with survival and its targeting with antisense oligonucleotides in ovarian cancer. Br J Cancer 2001; 85: 1753–1758.
25 Hansen K, Johnell M, Siegbahn A, Rorsman C, Engstrom U, Wernstedt C, Heldin CH, Ronnstrand L: Mutation of a Src phosphoryla-tion site in the PDGF beta-receptor leads to increased PDGF-stimulated chemotaxis but decreased mitogenesis. EMBO J 1996; 15:
5299–5313. 26 Ward SG: Do phosphoinositide 3-kinases di-
rect lymphocyte navigation? Trends Immunol 2004; 25: 67–74.
27 Koizumi N, Hatano E, Nitta T, Tada M, Ha-rada N, Taura K, Ikai I, Shimahara Y: Blocking of PI3K/Akt pathway enhances apoptosis in-duced by SN-38, an active form of CPT-11, in human hepatoma cells. Int J Oncol 2005; 26:
1301–1306. 28 Reinhold WC, Kouros-Mehr H, Kohn KW,
Maunakea AK, Lababidi S, Roschke A, Stover K, Alexander J, Pantazis P, Miller L, Liu E, Kirsch IR, Urasaki Y, Pommier Y, Weinstein JN: Apoptotic susceptibility of cancer cells se-lected for camptothecin resistance: gene ex-pression profi ling, functional analysis, and mo-lecular interaction mapping. Cancer Res 2003;
