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The Role of Downstream Signaling Pathways of the Epidermal Growth Factor Receptor for Artesunate’s Activity in Cancer Cells V. Badireenath Konkimalla1, James A. McCubrey2 and Thomas Efferth*,1 1
German Cancer Research Center, Pharmaceutical Biology, Heidelberg, Germany; 2Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, NC, USA Abstract: Epidermal growth factor (EGF) and its receptor (EGFR) as well as the EGFR-coupled Ras>Raf>MEK>ERK pathway are known to affect the survival of cancer cells upon chemotherapeutic treatment. In the present investigation, we analyzed the role of EGFR signaling pathways for the activity of artesunate towards cancer cells. The microarray-based mRNA expression of genes involved in EGFR signaling pathway was correlated with the 50% inhibition concentrations (IC50) of 55 tumor cell lines for artesunate. The log10IC50 values were in a range of -6.609 to -4.0M. Candidate genes identified by this approach were then experimentally validated by transfecting cell lines with corresponding cDNA vectors and treating them with artesunate. Indeed, we observed that the Ras>Raf>MEK>ERK pathway is an important signaling route for the response of tumor cells to artesunate. As exemplarily shown for artesunate, the application of such a combined approach to identify signal transduction pathways involved in the response of tumor cells to cytotoxic compounds might foster the development of novel molecular targeted therapies for cancer treatment.
Keywords: Artesunate, cancer, chemotherapy, EGFR, molecular pharmacology, natural products. INTRODUCTION Cancer is the second leading cause of death in the western world, surpassed only by cardiovascular diseases. Despite considerable advancements in research and medical care during the past decades, there is still a great need for improvement in the treatment options for patients afflicted with cancer. Deciphering of various signaling transduction pathways gave a thorough insight into the processes of how cancer cells escape normal growth control. The concept of molecular targeted chemotherapy has been developed based on the understanding of complex signaling networks. Here, the objective is to specifically alter the function of those target proteins that play a critical role in tumor growth and progression upon binding to antibodies or small molecule inhibitors. Through this approach, we hope that new treatment adjuncts can be developed with higher specificity for tumor cells and less side effects on normal tissues, a problem often encountered with existing conventional chemotherapy. Most cancer-related genetic alterations involve genes encoding signal transducers, particularly protein kinases [1]. Hence, the development of protein kinase inhibitors came into focus as novel cancer therapeutics. During recent years, prominent new treatment options were implemented in clinical setting resulting in a substantial improvement in survival time of cancer patients [2-5]. Recently, ten protein kinase inhibitors have been approved for clinical use, and more than 100 kinase-inhibiting agents are in different phases of clinical trials in the United States [6]. Moreover, deregulated protein kinase activities and imbalanced signaling routes foster the development of resistance towards anti-tumor agents [7]. Growth factors and their receptors, i.e., epidermal growth factor (EGF) and the EGF*Address correspondence to this author at the German Cancer Research Center, Pharmaceutical Biology (C015), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; Tel: 0049-6221- 42 34 26; Fax: 0049-6221- 42 34 33; E-mail:
[email protected] 1568-0096/09 $55.00+.00
receptor (EGFR), can cause multiple drug resistance of anticancer drugs [8]. The classical Ras>Raf>MEK>ERK pathway is involved in driving the proliferation and promoting the survival of cancer cells [9]. Moreover, the role of this pathway to drug resistance has been unambiguously shown during the past years [10]. The PI3K/PTEN/AKT signaling route provides proliferative and anti-apoptotic signals and its deregulation has often been linked with malignant transformation and drug resistance too [11, 12]. The development of drug resistance and severe side effects of currently established anti-cancer drugs urges the need for search of novel drugs with optimized features. It is a well–known fact that many medicinal plants do elicit diverse pharmacological activities and may, thus, serve as a valuable resource for procuring novel lead compounds. In our earlier studies, we have systematically analyzed medicinal plants used in traditional Chinese medicine [13-16]. Here, we found that the active principle of Artemisia annua L., artemisinin, and its semi-synthetic derivative, artesunate, not only exert anti-malarial activity but also profound cytotoxicity against tumor cells. The inhibitory activity of artemisinin and its derivatives towards cancer cells is in the nano- to micromolar range [17, 18]. Candidate genes that may contribute to the sensitivity and resistance of tumor cells to artemisinins were identified by pharmacogenomic and molecular pharmacological approaches [19, 20]. Target validation was performed using cell lines transfected with candidate genes or corresponding knockout cells. These genes are from classes with different biological function; for example, regulation of proliferation (BUB3, cyclins, CDC25A), angiogenesis (vascular endothelial growth factor and its receptor, matrix metalloproteinase-9, angiostatin, thrombospondin-1) or apoptosis (BCL-2, BAX) [15]. Artesunate triggers apoptosis both through p53-dependent and -independent pathways [21]. Anti-oxidant stress genes (thioredoxin, catalase, -glutamylcysteine synthetase, glutathione S-transferases) as well as EGFR confer resistance to artesunate [19, 22]. Cell lines over-expressing genes that confer resistance to established © 2009 Bentham Science Publishers Ltd.
EGFR Signaling Pathways and Artesunate
anti-tumor drugs (MDR1, MRP1, BCRP, dihydrofolate reductase, ribonucleotide reductase) were not cross-resistant to artesunate, indicating that artesunate is not involved in multidrug resistance [19, 20]. The anti-cancer activity of artesunate has also been shown in human xenograft tumors in mice [23]. First encouraging observations in the clinical treatment of patients suffering from uveal melanoma [24] suggest further clinical trials with artesunate for cancer treatment in the near future. The observation that EGFR (or Erb-B1) represents a determinant of resistance of tumor cells towards artesunate [21, 25] can be indicative that the EGFR-coupled Ras>Raf> MEK>ERK pathway might also be involved in resistance towards artesunate. The aims of the current investigation are, therefore, to establish the role of protein kinases for artesunate’s activity against cancer cells. Therefore, we correlated the microarray-based mRNA expression of genes involved in EGFR signaling with the 50% inhibition concentration (IC50) for artesunate in 55 cell lines of the National Cancer Institute (NCI), USA. The candidate genes identified by this in silico approach were experimentally validated. Transfected cell lines over-expressing these candidate genes were treated with artesunate to analyze whether these cells reveal resistance towards artesunate. By applying this combined methodology, we observed that the Ras>Raf>MEK>ERK pathway is an important signaling route for the determination of resistance of tumor cells to artesunate.
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ing, Elmira, NY, USA) with initially 104 cells/well that were incubated for one day in the presence of the indicated supplements. During the last 6h of incubation, DNA proliferation was measured by adding [3H]-thymidine (6.7 Ci/mmol; NEN, Boston, MA, USA) as described [27]. Flow Cytometric Analysis The cells were recovered after trypsinization using EDTA-free trypsin (Life Technologies, Inc.). The cells were washed once with EDTA-free PBS and then incubated for 15 min with a mixture containing annexin-V and propidium iodide (Roche Diagnostics, Indianapolis, IN) in binding buffer (19mM HEPES (pH 7.4), 140mM NaCl, and 5mM CaCl2). After the incubation period, the supernatants were removed, and 500l of binding buffer was added to each sample. The fluorescence was measured using a Becton Dickinson FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Compensation on FL1 and FL2 channels was performed as required to account for autofluorescence. Statistical Analysis
MATERIALS AND METHODS
The mRNA expression values of the NCI cell lines of genes of interest were selected from the N.C.I. database (http://dtp.nci.nih.gov). The mRNA expression has been determined by microarray analysis [30, 31]. Fisher’s exact test was used to calculate significance values as a relative measure for the linear dependency of two variables.
Cell Lines
RESULTS
The maintenance of the IL-3/GM-CSF-dependent murine myeloid cell line FDC-P1 has been described earlier [26]. Estradiol-dependent FD/v-ErbB:ER, FD/Raf-1:ER, FD/ MEK1:ER, FD/MEK1:ER+Akt1, FD/Bcr-Abl, and FD/ A-Raf:ER, transfectant cell lines were grown in the presence of 1M ß-estradiol (Sigma, St Louis, MO, USA). The transfection of FDC-P1 cells with plasmid cDNAs containing recombinant retroviruses has been described [27]. The panel of 55 human tumor cell lines of the Developmental Therapeutics Program of the NCI consisted of leukemia, melanoma, tumors of the central nervous system, colon, prostate, lung, kidney, ovary, and breast. Their origin and processing have been previously described [28]. Sulforhodamine B Assay The determination of drug sensitivity in the NCI cell lines by the sulforhodamine B assay has been reported [29]. The 50% inhibitory concentration (IC50) values for artesunate have been deposited in the DTP-NCI database (http://dtp.nci.nih.gov). [3H]-Thymidine Incorporation Assay Cells were washed three times with phosphate buffered saline (PBS) before being set up for growth curves or proliferation assays. Growth curves were performed in 5mL of medium with the indicated supplements. Cell proliferation assays were performed in 96-well flat bottom plates (Corn-
We investigated the association of EGFR signalingrelated genes to the response of tumor cells to artesunate. For this reason, genes that belong to ERB-B/EGFR and MAPK pathways were selected, as shown in Fig. (1). We then used the IC50 values of the NCI cell line panel for artesunate [17] and correlated them with the microarray-based mRNA expression values for these EGFR pathway-related genes. The log10IC50 values were in a range of -6.609 to -4.0M. The genome-wide mRNA expression profiling of the NCI cell lines has been reported [30, 31] and deposited at the NCI database (http://dtp.nci.nih.gov). As shown in Table (1), the mRNA expression of genes belonging to the Ras>Raf> MEK>ERK pathway significantly correlated with the IC50 values for artesunate, indicating that this signaling route might play a role for the response of tumor cells to artesunate. Furthermore, the gene expression of the PIK3R2>Akt1>BAD or >RPS6KA5 and CBLC pathways also significantly correlated with response of tumor cell lines to artesunate (Table 1). The mRNA expression of genes belonging to the CRK>ABL1 signaling route did not correlate with artesunate’s activity in cancer cells (see Table (1)). To test this hypothesis experimentally, we investigated whether these signal transducers affect cellular sensitivity to artesunate in cell lines transfected with corresponding cDNA constructs. As shown in Fig. (2), transfected cell lines selectively expressing RAF-1, MEK, or AKT-1 under appropriate conditional stimulation were treated with or without artesu-
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Fig. (1). Putative signaling routes identified by correlation analyses between microarray-based mRNA expression of 55 cell lines of the NCI, USA, and the 50% inhibition concentrations (IC50) of these cell lines for artesunate. Black boxes indicate significant correlations (P -5.336)*
low ( 75.45)
18
10
high (> 75.45)
10
17
low (166.76)
18
9
high (>166.76)
9
18
low (479.04)
17
10
high (>479.04)
10
17
Fisher Exact Test
Alternate Gene Symbols
Description
P=0.039
ERBB. ERBB1.
Epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog. avian)
P=0.014
p85-BETA. P85B
Phosphoinositide-3-kinase. regulatory subunit 2 (p85)
P=0.050
AKT. MGC99656. PKB. PRKBA. RAC. RAC-ALPHA
V-akt murine thymoma viral oncogene homolog 1
Pathway 1: PIK3R2
AKT1
EGFR Signaling Pathways and Artesunate
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(Table 1). Contd.....
Response to Artesunate
Gene Expression
Sensitive
Resistant
( -5.336 )*
(> -5.336)*
low (96.29)
18
9
high (>96.29)
9
18
RPS6KA5 low (121.90)
18
10
high (>121.90)
10
17
low (-0.041)
17
6
high (>-0.041)
11
21
low (5.60)
18
10
high (>5.60)
10
17
low (-15)
14
5
high (>-15)
14
22
low (0.1004)
18
10
high (>0.1004)
10
17
low (-0.017)
20
10
high (>-0.017)
8
17
low (153.90)
10
18
high (>153.90)
18
9
low (72)
17
11
high (>72)
11
16
low (60.06)
17
11
high (>60.06)
10
16
low (253.1)
18
10
high (>253.1)
10
17
BAD
Fisher Exact Test
Alternate Gene Symbols
Description
P=0.014
BBC2. BCL2L8
BCL2-antagonist of cell death
P=0.039
KS6A5. MGC1911. MSK1. MSPK1. RLPK
ribosomal protein S6 kinase. 90kDa. polypeptide 5
P=0.004
FLJ26504. p52SHC. p66. p66SHC. SHC. SHCA
SHC (Src homology 2 domain containing) transforming protein 1
P=0.039
GF1. GGF1. GINGF. HGF
Son of sevenless homolog 1 (Drosophila)
P=0.014
c-Raf. CRAF. Raf-1
V-raf-1 murine leukemia viral oncogene homolog 1
P=0.039
MAPKK1. MEK1. MKK1. MP2K1. PRKMK1
Mitogen-activated protein kinase kinase 1
P=0.010
ERK. ERK2. ERT1. MAPK2. MK01. p38. p40. p41. p41mapk. P42MAPK. PRKM1. PRKM2
Mitogen-activated protein kinase 1
P=0.020
c-Myc
V-myc myelocytomatosis viral oncogene homolog
P=0.11
CRKII. p38
v-crk sarcoma virus CT10 oncogene homolog
P=0.086
ABL. JTK7. c-ABL. p150. v-abl
v-abl Abelson murine leukemia viral oncogene homolog 1
P=0.039
C-CBL. CBL2. RNF55
Cas-Br-M (murine) ecotropic retroviral transforming sequence c
Pathway 2: SHC
SOS1
RAF1
MAP2K1
MAPK1
MYC
Pathway 3: CRK
ABL1
Pathway 4: CBLC
*The median log10IC50 value for artesunate was used as a cut-off to separate tumor cell lines as being “sensitive“ or „resistant“. The normalization has been performed either by pooling equal amounts of mRNA from HL-60, K562, NCI-H226, COLO205, SNB-19, LOX-IMVI, OVCAR-3, OVCAR-4, CAKI-1, PC-3, MCF7 and Hs578T cell lines (Synteni microarrays) and analyzing by ScanAlyze program or using Affymetrix U95Av2 GeneChips. Data were normalized using the Affymetrix Normalization 5.0 software.
nate and analyzed by the annexin-V/propidium iodide apoptosis assay. The RAF-1 gene, which was under the control of the promoter for the estrogen receptor gene was conditionally activated by -estradiol. This resulted in reduced rates of
apoptosis induced by 3 to 300pg/mL artesunate. Similarly, activated MEK or AKT-1 caused reduced rates of apoptosis induced by artesunate (see Fig. (2)).
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Fig. (2). Effects of activated protein kinases on the induction of apoptosis in response to artesunate. Annexin V/propidium iodide apoptosis binding assays of FDC-P1, FD/v-ErbB:ER, FD/Raf-1:ER, FD/MEK1:ER, and FD/MEK1:ER+Akt1 cells measured in the presence of 1M -estradiol treated with or without 3.3 or 33pg/mL artesunate for 24h. The cells were harvested after trypsinization with EDTA-free trypsin and incubated for 20min in a mixture containing annexin V-FITC and propidium iodide. After removing the staining solution, the cells were re-suspended in the binding buffer, and the fluorescence was detected with a Becton Dickinson FACScan flow cytometer. The X axis shows annexin V-FITC fluorescence and the Y axis shows the propidium iodide fluorescence. The percentages of cells in each quadrant are presented. Lower left quadrant, viable cells; lower right quadrant, early apoptotic cells; upper left quadrant, necrotic cells; upper right quadrant, late apoptotic and necrotic cells.
Since these data point to a role of signal transducers connected to the ERB-B/EGFR signaling network, we directly tested the effect of artesunate on v-ERB-b:ER constructs. 3 Using a [ H]-thymidine incorporation assay to measure proliferative activity of cells, we found that the activation of vERB-B expression in FD cells by -estradiol-induced stimu3 lation led to a reduced inhibition of [ H]-thymidine incorporation by artesunate compared to cells without conditionally activated v-ERB-B (see Fig. (3A)). Furthermore, conditional expression of RAF-1 also induced resistance to artesunate (see Fig. (3B)), an observation that confirms the results obtained with the annexin-V/propidium iodide apoptosis assay. DISCUSSION Since protein kinases play a crucial role for many fundamental cellular processes such as proliferation, apoptosis, differentiation etc. [32, 33], it is noteworthy to speculate that they are also involved in drug resistance. Indeed, the association of several kinases involved in signaling downstream of EGFR with resistance towards established anti-cancer drugs is well documented [7, 34]. In the present investigation, we applied a combination of a biostatistical approach followed by experimental validation to study the role of EGFR downstream pathways for the re-
sponse of tumor cells to artesunate. Initially developed as anti-malarial drug, artesunate turned out to reveal profound inhibitory activity against cancer cells [17, 18]. We focused on four major pathways, which are linked to EGFR signalling. The key molecules of pathway 1, AKT1 is well known from the literature to be involved in resistance to standard chemotherapy [35]. Its role in artesunate resistance is new. BAD belongs to the Bcl-2 family of apoptosis regulators, whose function for sensitivity and resistance to standard chemotherapy and also to artesunate is well known [14, 19, 36]. The PIK3R2 and RPS6KA5 gene have not been assigned to drug resistance yet. It is, however, plausible that these genes also contribute to resistance to artesunate as parts of the entire signaling cascade. The second pathway included SHC, RAF1, MAP2K1, and MAPK1, all of which have been linked to therapy unresponsiveness [37-40]. The SOS1 gene found in the present investigation have not been described in the context of chemotherapy resistance yet. MYC, an important transcription factor and oncogene, which regulates the cell cycle machinery also affect endocrine and cytotoxic cancer therapy [41, 42]. Our results enlarge MYC’s role for therapy response to artesunate. EGFR-related pathway 3 contained ABL1 and CRK genes, both of which were not related to artesunate resistance
EGFR Signaling Pathways and Artesunate
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Fig. (3). Effects of artesunate on DNA synthesis in oncogene-transformed and -estradiol-activated FD cells. [3H]-Thymidine incorporation in cells over-expressing (A) v-ERB-B or (B) RAF-1. The cells were deprived of -estradiol for 24h. Three 100L aliquots per data point were cultured at the indicated conditions and incubated in three wells of 96 well plates. Cells were incubated in medium supplemented with (squares) or without 1M -estradiol (triangles) and artesunate at a concentration range of 0.1 to 100pg/mL.
in our investigation. ABL1 represents the non-activated wild-type form og the gene. While the bcr-abl fusion gene is constitutively activated and mediates drug resistance by the Raf/MEK/ERK pathway signalling [7], the wild-type ABL1 gene does not. We also did not find a correlation between CRK and artesunate resistance. The v-CRK gene has been shown to protect against apoptosis [43]. Therefore, it could be speculated that it should also play a role for drug resistance. The lack of resistance induction in our study may indicate that the influence of CRK in the entire regulatory signalling network leading to cell death or survival after drug exposure might not be as strong as other genes involved in our analysis. CBLC belongs to another EGFR-connected pathway, which was significantly correlated to artesunate responsiveness. There are no reports in the literature that CBLC plays a role for chemosensitivity or resistance, indicating that this gene might be of minor importance. From the data presented in the present investigation and the reports in the literature we conclude that the AKT1- and MAPK-pathways (pathways 1 and 2) were the most relevant ones associated with resistance of cancer cells to artesunate. The fact that we identified mainly genes belonging to the MAPK pathway speaks for some specificity of artesunate’s mode of action towards tumor cells. The correlation of microarray-based mRNA expression profiles with the IC50 values of cancer cell lines for artesunate represents a hunting strategy for the identification of candidate genes. It does, however, not provide evidence for a causative link. For this reason, we treated cell lines transfected with the corresponding candidate genes with artesunate. Indeed, we could verify that the genes identified by our correlation analysis mediated reduced rates of apoptosis by artesunate. We and other authors have shown that artesunate-type drugs kill cancer cells by the induction of apoptosis [14, 16, 21, 44, 45]. Re-
duced rates of apoptosis in artesunate-treated transfected cell lines, therefore, indicate that genes of the MAPK pathway confer resistance to artesunate. The MAPK pathway represents an important signaling route for receptors of the ERB family [8]. To prove whether there is a direct connection between ERB genes and the MAPK pathway, we analyzed cell constructs carrying cDNAs for v-ERB or RAF-1 for their responsiveness to artesunate. For this analysis, we used an [3H]-thymidine incorporation assay as an independent method in addition to the annexin-V/propidium iodide apoptosis assay. [3H]-thymidine incorporation is a measure for cell proliferation of tumor cells, and tumor growth is the net result of cell proliferation and apoptosis. Therefore, this assay is a supplement to further validate the results obtained by the in silico correlation analyses and annexin-V/propidium iodide apoptosis assays. Addressing the question, whether expression of these enzymes leads to the activation of the enzymes of the signal pathway, we determined the protein levels of the key signaling proteins and found that they did not increase upon activation of the v-Erb-B:ER, Raf-1:ER, MEK1:ER and Akt:ER constructs in the cells. However, importantly their activity did increase upon addition of the appropriate ligand (estrogen or tamoxifen) [46-75], which was linked to a proliferative response. This has been determined by western blot analysis which examined the phosphorylation states and total protein levels of downstream substrates such as MEK1 and ERK1,2 [46-48] and correlated with increase in proliferation (cell division and DNA synthesis). Furthermore, we have established the effects of cytokines such as IL-3 and the conditional v-Erb-B:ER [46-48], Raf1ER (4-24), Akt:ER [54, 56, 58, 60], and MEK1:ER [69-75] constructs on the induction of downstream signaling cascades in these cells (FDC-P1) [46-48, 50-53, 55, 56, 59-63, 70, 71, 73-75] as well as other hematopoietic (FL5.12, TF-1)
78 Current Cancer Drug Targets, 2009, Vol. 9, No. 1
[46, 49, 51, 53, 54, 57, 58, 71, 72], fibroblast (NIH-3T3) [46, 64-66], prostate (DU145) [67] and breast (MCF-7) [68, 69] cells. In all cases, introduction of the conditional oncogene resulted in activation of the downstream signaling cascade. Thus, the conditional oncogene system is an effective tool of monitor the effects of a drug on the growth in response to activation by a particular oncogene, frequently mutated in human cancer. In our system, we have selected for cells which are able to efficiently recognize the different viral promoter systems (retroviral LTRs, SV40 and CMV encoded promoters/enhancers). We have determined in the v-Erb-B:ER, Raf1:ER, MEK1:ER and Akt:ER transfected cells, the chimeric kinases are essentially inactive in the absence of ligand (e.g. estrogen, tamoxifen). Upon addition of estrogen or tamoxifen, they become active. Therefore in our system we are not examining the effects of different levels of mRNA expression, however, we are examining the effects of activation of the chimeric proteins upon either addition or removal of estrogen or tamoxifen. This represents a valid model to examine the effects of drugs such as artesunate. We observed that both v-ERB- or RAF-1-transfected cells were more resistant to artesunate than control cells. This indicates that v-Erb may confer artesunate resistance via the MAPK pathway. The relevance of the ERB family for responsiveness towards artesunate is further substantiated by previous results demonstrating that transfected U87.MGEGFR glioblastoma cells expressing a constitutively activated deletion mutant human Erb-B1 construct is also more resistant to artesunate than mock vector transfected U87.MG control cells [25]. The identification of members of the EGFR family and associated signaling pathways as determinants of artesunate resistance poses the question as to whether silencing of ERBrelated signaling routes renders tumor cells sensitive to artesunate. We have previously shown by isobologram analyses that treatment of U-87. MGEGFR cells treated with the EGFR tyrosine kinase inhibitor erlotinib (OSI-774) sensitizes tumor cells to artesunate in a synergistic manner [25].
Konkimalla et al. [5]
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ACKNOWLEDGEMENT This work was supported by a grant of the Dietmar Hopp-Stiftung to V.B.K.
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Revised: November 03, 2008
Accepted: November 25, 2008
