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Mol Cancer Ther. 2005;4:814-823
© 2005 American Association for Cancer Research

Transcriptional signature of Ecteinascidin 743 (Yondelis, Trabectedin) in human sarcoma cells explanted from chemo-naïve patients

Nerea Martínez1, Margarita Sánchez-Beato1, Amancio Carnero2, Victoria Moneo2, Juan C. Tercero3, Isabel Fernández1, Mercedes Navarrete1, José Jimeno3 and Miguel A. Piris1

1 Molecular Pathology Programme and 2 Experimental Therapeutics Programme, Centro Nacional de Investigaciones Oncológicas and 3 Pharmamar R&D, Madrid, Spain

Requests for reprints: Miguel Angel Piris, Molecular Pathology Programme, Centro Nacional de Investigaciones Oncológicas, C/ Melchor Fernández Almagro 3, E-28029 Madrid, Spain. Phone: 34-91-224-69-00; Fax: 34-91-224-69-23. E-mail: mapiris{at}cnio.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ecteinascidin 743 (ET-743; Yondelis, Trabectedin) is a marine anticancer agent that induces long-lasting objective remissions and tumor control in a subset of patients with pretreated/resistant soft-tissue sarcoma. Drug-induced tumor control is achievable in 22% of such patients, but there is no clear indication of the molecular features correlated with clinical sensitivity/resistance to ET-743. Nine low-passage, soft-tissue sarcoma cell lines, explanted from chemo-naïve patients with different patterns of sensitivity, have been profiled with a cDNA microarray containing 6,700 cancer-related genes. The molecular signature of these cell lines was analyzed at baseline and at four different times after ET-743 exposure. The association of levels of TP53 mutation and TP73 expression with ET-743 sensitivity and cell cycle kinetics after treatment was also analyzed. Gene expression profile analysis revealed up-regulation of 86 genes and down-regulation of 244 genes in response to ET-743. The ET-743 gene expression signature identified a group of genes related with cell cycle control, stress, and DNA-damage response (JUNB, ATF3, CS-1, SAT, GADD45B, and ID2) that were up-regulated in all the cell lines studied. The transcriptional signature 72 hours after ET-743 administration, associated with ET-743 sensitivity, showed a more efficient induction of genes involved in DNA-damage response and apoptosis, such as RAD17, BRCA1, PAR4, CDKN1A, and P53DINP1, in the sensitive cell line group. The transcriptional signature described here may lead to the identification of ET-743 downstream mediators and transcription regulators and the proposal of strategies by which ET-743–sensitive tumors may be identified.

Key Words: Sarcoma • ET-743 • expression profile • time course


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ecteinascidin 743q (ET-743; Yondelis, Trabectedin) is a tetrahydroisoquinoline alkaloid with antiproliferative effects, isolated from the tunicate Ecteinascidia turbinata (1). This compound binds to the minor groove of DNA and binds the N2 position of guanine (2–4), producing a bend towards the major groove of the DNA, in a way that differs from that of other known alkylating agents. This alteration in the DNA structure could be the cause of the broad inhibition of inducible transcription, previously described at pharmacologically achievable concentrations, probably interfering with transcription factors (5–7). In addition, an efficient DNA repair function is known to be required for ET-743 cytotoxicity. In fact, the mode of action of ET-743 is different from that of all other DNA-interacting antitumorals because nucleotide-excision-repair deficiencies that increase the susceptibility of cell lines to cisplatin, UV, and alkylating reagents are more resistant to ET-743.

At the cellular level, ET-743 produces cell cycle perturbations with a decreased rate of S-phase progression and a G2-M blockade (3, 8–11). Experiments with synchronized cells showed that cells in G1 phase are more sensitive to the cytotoxic effects of ET-743 than are cells in G2 or S phase.

Phase I clinical trials started back in 1996 and phase II and comparative trials are currently in progress (12). The data available thus far confirm a therapeutic potential in patients with advanced breast cancer pretreated with anthracyclines and taxanes and in women with ovarian cancer that is resistant, or has experienced relapse, to platin-taxane–based therapy (13–15). Additionally, a large phase II program, conducted in patients with advanced soft-tissue sarcoma that is resistant, or has undergone relapse, to conventional agents, has shown long-lasting objective responses and tumor control in a clinically significant proportion of cases (16–18); such a rate of ET-743–induced tumor control seems to have an effect on median survival and particularly on the proportion of patients (29%) who were alive after 2 or more years.

This clinical data set suggests major constitutive/molecular differences between a group of patients in whom the compound is able to modulate progression of the disease for very long periods and a cohort that is fully resistant to ET-743.

Given the shown clinical benefit of ET-743 treatment for soft-tissue sarcoma, we decided to investigate the mechanism of action of this drug in a panel of chemo-naïve, low-passage soft-tissue sarcoma cell lines established from biopsies of newly diagnosed patients with different sarcoma histology types. We characterized the expression profiles of those cell lines and estimated their correlation with in vitro sensitivity to ET-743 both in the absence of the drug and in time-course experiments. This should make it possible to develop a predictive ecteinascidin sensitivity model for subsequent validation in tumor samples from patients treated with ET-743.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines
Eleven cell lines were studied, eight of which were directly established from surgical tumor-tissue specimens from sarcoma patients (Table 1). Sensitive and isogenic resistant chondrosarcoma cell lines (CS-1S and CS-R) were kindly provided by Dr. Lawrence Weissbach (Massachusetts General Hospital, Boston, MA). The sarcoma cell line SW872 was obtained from the American Type Culture Collection (Rockville, MD). The in vitro sensitivity of each cell line to ET-743 was previously established.4 Cells were maintained in RPMI medium supplemented with 10% fetal bovine serum, fungizone, and penicillin/streptomycin. CS-1R and CS-1S were grown without antibiotics or fungizone.


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  		Table 1. Details of the cell lines treated with ET-743 and analyzed by cDNA microarrays

 
For time-course analysis, cells were treated with 10 nmol/L ET-743 for 6, 24, 48, and 72 hours.

TP53 gene mutations were analyzed in accordance with described protocols (19).

Real-time Reverse Transcription-PCR
Quantitative reverse transcription-PCR (RT-PCR) was done for ATF3, BRCA1, GADD45B, GAPDH, JUNB, RAD17, SAT, TAp73, and {Delta}Np73 using the Applied Biosystems 7900HT (Applied Biosystems, Foster City, CA). GADPH expression was used as a reference for calculating expression levels of the other genes.

Flow Cytometry Analysis
Cells were cultured for 24 to 72 hours in the absence of the drug. ET-743, 10 nmol/L,was then added to the medium and incubation was continued for 6, 24, 48, and 72 hours. For flow cytometry analysis, cells corresponding to each treatment time were fixed in 70% ethanol, and RNase (0.6 µg/µL final concentration) and propidium iodide (50 µg/mL) were added to the fixed cells. The DNA content was quantified with a flow cytometer (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ).

RNA Extraction and Amplification
Total RNA from cells was extracted in two steps after 6, 24, 48, and 72 hours of treatment using Trizol (Life Technologies, Inc., Grand Island, NY) and then purified with RNeasy (Qiagen, Inc., Valencia, CA). Double-strand cDNA was synthesized from 4 µg of total RNA using the Superscript System for cDNA synthesis (Life Technologies). In vitro transcription was carried out using the T7 Megascript in vitro transcription kit (Ambion, Austin, TX). The quality of the amplified RNA produced was checked by electrophoresis and its concentration was measured.

cDNA Microarray Hybridizations and Analysis
Amplified RNA was hybridized on the CNIO-Oncochip as previously described (20). Briefly, 5 µg of amplified RNA were directly labeled with cyanine 3–conjugated dUTP (Cy3), whereas 5 µg of aRNA from the Universal Human Reference RNA (Stratagene) were labeled with cyanine 5–conjugated dUTP (Cy5) as reference. Hybridizations were done as described (21). Hybridized slides were scanned and analyzed using the Scanarray 5000 XL (GSI Lumonics, Kanata, Ontario, Canada) and GenePix Pro 4.0 software (Axon Instruments, Inc., Union City, CA), respectively.

Data Analysis
Raw microarray data were processed as previously described. Data obtained from each hybridization were stored in a database for analysis. Cy3/Cy5 ratios were normalized with respect to the median ratio value of all of the spots in the array. After normalization, spots with intensities for both channels (sum of medians) less than that of the local background were discarded. The ratios of the remaining spots were log transformed (base 2), and duplicated spots on the OncoChip were averaged to the median (20). After this first processing, results at all treatment times were compared with those in untreated cells, and genes were deemed to be up-regulated or down-regulated if the ratio of the difference with untreated cells was more than twice that at any of the four treatment times studied in at least 72% (8 of 11) of the cell lines analyzed. Biological functions of selected genes were assigned using the GeneCards5 and Gene Ontology6 databases. Sensitivity/resistance signatures were obtained by dividing the median expression level for each gene in all sensitive cell lines by that of all resistant cell lines under basal conditions (i.e., in the absence of the drug) and throughout the time course of treatment with ET-743. Selected genes were defined as those with at least 2-fold up-regulation or down-regulation in the sensitive group of cell lines relative to the resistant group.

Clustering Analysis
To classify the temporal profiles of gene expression, cluster analysis was done using the Self-Organizing Tree Algorithm7 (22), assuming Euclidian distances between genes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gene Signature of ET-743
Among the 11 human sarcoma cell lines studied, the SR2103/1A, SR2103/1B, CA1010, SR2205, SR2410, SR2910, SR0312, and SR0306 lines had been established at the Centro Nacional de Investigaciones Oncológicas from tissue samples of sarcoma patients and are considered low-passage cell lines. Cellular phenotype and ET-743 sensitivity data of all the cell lines are detailed in Table 1. The 11 human sarcoma cell lines were exposed to 10 nmol/L ET-743; this concentration may be achieved and maintained for more than 72 hours in patients plasma below the recommended clinical dose incorporated in the phase II trials in sarcoma and is therefore considered to be therapeutically appropriate for this experimental study (23).

ET-743 in vitro treatment produced deregulation of 330 genes in the majority of these cell lines (8 of 11; 72% of samples). Most of the deregulated genes were down-regulated (244; 74%) and only 86 genes (26%) were up-regulated (Fig. 1; raw data and selected gene data can be found in Table 1 at the website http://redlinfomas.cnio.es/publications/ET743).8 The number of genes of which expression level changed increased with the time elapsed after treatment. Thus, early on (6 hours of treatment), only four genes were noted as being consistently changed in most of the cell lines in response to treatment with ET-743: two genes (SAT and EST) were up-regulated and two genes (BCAR3 and DDX20) were down-regulated. The upward or downward tendency of these genes was constant throughout the treatment.



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  		Figure 1. Expression profile of deregulated genes after treatment with ET-743. Eighty-six genes were up-regulated and 244 genes were down-regulated as a consequence of the treatment.

 
The genes up-regulated after treatment with ET-743 are mainly involved in signal transduction, molecule and ion transport, and energy pathways, whereas the down-regulated genes are predominantly associated with nucleic acid binding, gene transcription, signal transduction, and cell cycle control. Notably, the most significantly overexpressed downstream mediators after treatment with ET-743 were CS-1, ATF3, SAT, JUNB, GADD45, and ID2, all of which are involved in cell cycle arrest and apoptosis (Fig. 2A).



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  		Figure 2. ET-743 downstream genes. A, average of gene expression log-ratios (base 2) of the most significantly up-regulated genes in response to treatment with ET-743 in 11 sarcoma cell lines. The ratio for each time point is calculated by dividing the average expression at the corresponding time by the average of expression before treatment. B, expression pattern of four genes identified as ET-743 downstream genes were validated in two cell lines by RT-PCR.

 
SAT. SAT [spermidine/spermine N1-acetyltransferase (SSAT)] was up-regulated shortly after the start of treatment (6 hours) and the amount of expression grew over time (from 1.17 at 6 hours to 2.63 at 48 and 72 hours, log scale). This gene codes for a protein involved in the regulation of the intracellular concentration of polyamines (24), a key factor required for tumor cell growth, regulated by a complex system that involves homeostatic balancing of biosynthesis catabolism, uptake, and export of polyamines. Experiments producing selective down-regulation of SSAT by small interfering RNA revealed the requirement for SSAT for apoptosis induction after treatment with polyamines analogues (25). The induction of SSAT after treatment with polyamine analogues has been proposed as a prognostic indicator of drug response (26) because it is known to be induced by 5-fluorouracil, TDX, cisplatin, and oxaliplatin (27, 28). It has also been shown that overexpression of SSAT, which is dependent on p53 status (27), produces cell cycle arrest in G2-M (29).

ATF3. ATF3 was up-regulated after 24 hours of treatment with ET-743 in 8 of 11 cell lines. ATF3 is a stress-inducible transcriptional repressor of which levels are dramatically induced in response to a variety of stress conditions in many different tissues, such as toxic chemicals, anticancer drugs, proteasome inhibitors, and genotoxic agents. This p53 target is involved in drug-induced apoptosis, specifically after treatment with DNA topoisomerase inhibitors, such as camptothecin or etoposide, inducing activation of caspase proteases (30, 31).

ID2. ID2 is a member of a family of helix-loop-helix transcription factors that regulate cell differentiation and proliferation (32–34). Id2 binds to Rb family members, abolishing their growth-suppressing function. It has been shown that trichostatin A, a histone deacetylase inhibitor, induces the expression of ID2 (35–37). Overexpression of ID2 augments apoptosis in myeloid and osteogenic sarcoma-derived cell lines (38).

CS-1. CS-1 is a calcineurin-binding protein. Calcineurin is a calcium- and calmodulin-dependent serine/threonine phosphatase that plays an important role in transducing calcium-dependent signals in a variety of cell types. This enzyme is required for G1 progression and it was recently identified as an important link between Ca2+ signaling and NF-{kappa}B activation in response to mitochondrial stress (39). Overexpression of Cs-1 inhibits calcineurin activity, producing cell cycle arrest (40).

JUNB. Activator protein 1 is composed of homodimers or heterodimers formed by related Jun (c-Jun, JunB, and JunD), Fos (c-Fos, FosB, Fra-1, and Fra-2), and ATF family proteins (41). Jun is known to be up-regulated in response to the exposure to genotoxic stress, such as alkylating agents or short-wavelength UV radiation (42, 43).

GADD45B. Gadd45 proteins cooperate in the activation of S and G2-M checkpoints following exposure of cells to UV irradiation and other genotoxic stresses, producing growth arrest and apoptosis (8, 44).

The expression changes for four selected genes were verified by real-time RT-PCR (Fig. 2B) in two cell lines. The pattern of gene expression modulation was comparable for real-time RT-PCR and microarray results, although the former technique was more sensitive in detecting gene expression changes (Fig. 2B).

Differences between ET-743–Sensitive and ET-743–Resistant Sarcoma Primary Cell Lines
As shown in Table 1, six cell lines were considered as ET-743 sensitive (those with LC50 < 1 nmol/L), and three were resistant to ET-743.

Effect of ET-743 on the Cell Cycle of Resistant and Sensitive Cell Lines
All cell lines were analyzed by flow cytometry at different treatment times with ET-743. The cell cycle of resistant cell lines was undisturbed throughout treatment (data not shown). A cytotoxic effect of ET-743 was revealed in sensitive cell lines as a G2-M blockade, or apoptosis. These effects varied among the cell lines studied, thereby confirming previous observations (8).

Transcriptional Signature Associated with ET-743 sensitivity
The gene expression profiles of the sensitive and resistant cell lines were obtained under basal conditions (i.e., in the absence of the drug). The median expression in sensitive cell lines was compared with the corresponding median expression of the resistant cell lines by way of the S/R expression ratio. Differences between resistant and sensitive cell lines were also reflected in changes of the expression level of several genes (Table 2). The results at each time for each cell line and the median ratios and SDs are presented in Table 3 at the website (http://redlinfomas.cnio.es/publications/ET743). These data represent a mixture of signals concerning cell differentiation and molecular alterations that lead to ecteinascidin sensitivity model resistance. Thus, ET-743–sensitive sarcoma cells constitutively express the following markers: IL-13, MMP3, SAMSN1, EDG1, IGFBP3, and PRKAG2, whereas ecteinascidin-resistant cells show increased expression of PTPRM, PCDH7, NID2, CDH6, CCND1, NBL1, AHR, TIMP3, and PDGFRA (Table 2).


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  		Table 2. Genes constitutively overexpressed or down-regulated in sensitive compared with resistant cell lines

 

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  		Table 3. Genes with a higher level of expression in sensitive than resistant cell lines after 72 hours of treatment

 
Interestingly, matrix metalloproteinase 3 and its inhibitor, tissue inhibitor of metalloproteinase 3, are differentially expressed in the sensitive and resistant cell lines, respectively. Metalloproteinase activity influences cellular sensitivity to extrinsic death in different cell types through proteolytic shedding of cell-surface signaling molecules. Metalloproteinase defective expression has already been described in the resistant chondrosarcoma cell line although its role in ET-743 resistance has not been elucidated (45). Matrix metalloproteinase 3, a regulator of matrix remodeling, plays a role in tumor metastasis through the degradation of fibronectin, laminin, gelatins, collagens, and cartilage proteoglycans. Matrix metalloproteinase 3 has been shown to selectively modulate Fas-mediated neuronal apoptosis induced by doxorubicin (46) and induces apoptosis when overexpressed in mammary epithelial cells (47). On the other hand, tissue inhibitor of metalloproteinase 3, which acts as an inhibitor of matrix metalloproteinase 3, was expressed at higher levels in the resistant cell lines. This inhibitor of metalloproteinase can either enhance or suppress apoptosis, depending on the cell type or context analyzed (48–50).

IGFBP-3, which is also overexpressed in sensitive cell lines, enhances the p53-dependent apoptotic response of colorectal cells to DNA damage (51).

The aryl hydrocarbon receptor is a ubiquitously expressed transcriptional regulatory protein, which induces the transcription of CYP1A1, CYP1A2, and CYP1B1, which are genes that encode different enzyme isoforms of cytochrome P-450. These enzymes catalyze the oxidation of certain classes of xenobiotics, resulting in the generation of highly reactive electrophilic metabolites that bind specific residues of DNA, causing DNA-adduct formation that may lead to mutation and subsequent cellular transformation (52). The findings described here are consistent with those previously published showing ET-743 to be metabolized by P450, most predominantly by the cytochrome P-450 3A subfamily (53). Additionally, the higher AHR expression values observed here in the ET-743–resistant cell lines are consistent with the previous observation of an increased aryl hydrocarbon receptor expression associated with an adverse outcome in soft-tissue sarcoma patients (16).

Platelet-derived growth factor receptor A, which is more strongly expressed in the resistant cell lines, is a receptor tyrosine kinase overexpressed in a subset of solid tumors, and is therefore the target of drugs inhibiting this function, such as imatinib mesylate (Gleevec). Platelet-derived growth factor A selectively binds to platelet-derived growth factor receptor A, regulating cell proliferation, survival, and chemotaxis (54–56).

Ecteinascidin-Inducible Genes in the Sensitive Cell Lines
In addition to the constitutive differences that exist between ET-743–sensitive and ET-743–resistant cells, the soft-tissue sarcoma cell lines studied showed variable expression of a series of genes in response to treatment with ET-743. Thus, Table 3 shows a group of genes of which expression level increased in the sensitive cell lines after 72 hours of treatment with ET-743. By contrast, Table 4 illustrates a group of genes of which expression increased in the resistant cell lines after 72 hours of treatment with ET-743, and decreased in the sensitive cell lines.


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  		Table 4. Genes with a higher level of expression in resistant than in sensitive cell lines after 72 hours of treatment

 
RAD17. RAD17 is a component of the DNA damage G2-M checkpoint, interacting with RAD1 (protein-protein interaction in the cell cycle checkpoint machinery), and is required for S-phase and G2-M arrest in response to DNA damage or incomplete DNA replication (57, 58).

BRCA1. BRCA1 has been purified as part of a large protein complex known as BRCA1-associated genome surveillance complex, which contains a wide range of DNA repair and replication proteins (44, 59). An interaction with p53-BRCA1 (60) has been noted in the repair after DNA damage.

PAR4. PAR4 (PAWR) is a cell death modulator that inhibits the atypical protein kinase C isoforms, leading to reduced cellular NF-{kappa}B activation and increased apoptosis (61).

CASP8AP2. CASP8AP2 is involved in Fas-mediated activation of caspase 8 during apoptosis.

The transcription of TP53DINP1 is increased during cellular stress by p53-mediated activation of transcription. TP53DINP1, in association with homeodomain-interacting protein kinase 2, regulates p53 transcriptional activity on P21, MDM2, PIG3, and BAX promoters, inducing G1 arrest and increasing p53-mediated apoptosis (62). Homeodomain-interacting protein kinase has been shown to play a critical role in triggering p53-dependent apoptosis in response to the antineoplastic drug cisplatin (63).

CDKN1A. CDKN1A (p21) wild-type p53-activated fragment 1, p21, regulator of cell cycle, cyclin-dependent kinases 2 and 4 (CDK2/CDK4) inhibitor protein, inhibiting cell cycle progression by interacting with G1 cyclin/CDK complexes and proliferating cell nuclear antigen.

The expression levels of Rad17 and BRCA1 were verified by real-time RT-PCR (Fig. 3) in one sensitive and one resistant cell line, confirming the results obtained by cDNA microarray analysis.



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  		Figure 3. Quantitative RT-PCR of RAD17 and BRCA1 genes in one resistant cell line (SR0306) and one sensitive cell line (SR2103).

 
Ecteinascidin-Inducible Genes in the Resistant Cell Lines
MAL and CAV1 are components of membrane rafts. These rafts are membrane microdomains that play a central role in signal transduction by acting as a scaffold in which molecules involved in signal transduction pathways can interact.

Analysis of TP53 Status
TP53 is a critical cellular gatekeeper that mediates DNA damage–induced cell cycle arrest and apoptosis. As time-course experiments reveal the expression in ET-743–sensitive cell lines of p53 targets, we tested TP53 status in this panel of cell lines by sequencing exons 5 to 8. As shown in Table 5, only three cell lines (SR2205, SR2910, and SR2410) had wild-type TP53, whereas all other cell lines featured mutated TP53. In this panel of cell lines, TP53 status did not correlate with ET-743 sensitivity, thereby confirming previous observations (64).


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  		Table 5. TP53 status in the cell lines studied

 
TP73 Expression
To determine whether TP73 expression was induced in response to treatment with ET-743, and to study a possible relation of TP73 expression with sensitivity to ET-743, quantitative PCR was done at all the time points studied (0, 6, 24, 48, and 72 hours).

Basal levels of TAp73 and {Delta}Np73 varied among cell lines, and did not correlate with sensitivity or resistance to ET-743 (see Fig. 4). ET-743 seems to induce a decrease in the level of expression of full-length TAp73, unlike other chemotherapeutic drugs inducing DNA damage (65). By contrast, three of the studied cell lines showed a striking increase in the expression of {Delta}Np73, which is derived from an alternative promoter in intron 3 and lacks the transactivation domain of full-length TAp73. {Delta}Np73 is known to counteract apoptosis and growth suppression mediated by wild-type p53 and TAp73, inducing drug resistance to wild-type p53-harboring tumor cells (66). No relation was observed between the induction of {Delta}Np73 and ET-743 sensitivity.



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  		Figure 4. Quantitative RT-PCR showing the expression levels of Tap73 and {Delta}Np73 in different sensitive and resistant cell lines during ET-743 treatment. The level of expression is heterogeneous but there is a general inhibition of full-length p73, whereas {Delta}Np73 seems to be induced after treatment with ET-743.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ET-743 is a potent cytotoxic agent that acts as a transcription-interfering agent (5). Different clinical and experimental studies have begun to provide an explanation of the mechanism underlying the cytotoxic effect of this drug, although further effort is still necessary to fully understand its mechanism of action.

One of the most striking findings here is the massive down-regulation of gene transcription, confirming previous studies done with a smaller selection of genes (5). We have used a cDNA Oncochip, enriched in cancer-relevant genes, containing a total of 6,800 genes. Analysis of the changes induced by ET-743 revealed a common signature of 244 down-regulated and 86 up-regulated genes in the majority (8 of 11) of these cell lines.

High-throughput screening has allowed the identification of genes that are induced after treatment with ET-743. These genes (CS-1, ATF3, SAT, JUNB, GADD45, and ID2) have a common role as transcription regulators, leading to cell cycle arrest in G2-M and apoptosis (25, 29–31, 38, 40, 42–44), which can be correlated with the cell cycle arrest and apoptosis induced after treatment of different cell types with ET-743 in our experiments and those of others (3, 8–11).

The approach taken here, using low-passage sarcoma cell lines, makes this study of even greater interest because such cell lines have characteristics that are closest to those of tumors.

This study has also attempted to identify specific drug-induced genes of which expression is associated with ET-743 sensitivity. To this end, tumor cells were treated with ET-743, and basal and ET-743–induced genes were compared in sensitive and resistant cells. Although the cell lines display a constellation of ET-743–induced changes at several time intervals, at 72 hours, all the cells lines showed a relatively uniform signature induced by the drug, which also revealed a set of genes that were differentially expressed in sensitive and resistant cells. Thus, after 72 hours of treatment, ET-743–sensitive soft-tissue sarcoma cells expressed a set of DNA damage sensors, including RAD17 and BRCA1, which underlies the unique mechanism of ET-743 action. ET-743 is known to subvert normal nucleotide excision repair by generating lethal DNA breaks during transcription-coupled nucleotide excision repair (67). ET-743–induced adducts are recognized by the nucleotide excision repair system (67), which leads to apoptosis of the tumor cells, instead of repairing the damage caused by the adducts. The increased expression of both RAD17 and BRCA1 indicates the activation of a sensor for DNA damage including both proteins. Our results are consistent with an explanation whereby BRCA1 functions as a molecular determinant of response to a range of different chemotherapeutic agents (68), now including ET-743.

Genes found to be overexpressed in sensitive soft-tissue sarcoma cells after ET-743 treatment include apoptosis inducers, such as PAR4 and CASP8AP2, and some known p53 targets, such as TP53DINP1 and CDKN1A. This prompted us to investigate further the potential role of p53 and p73 in regulating apoptosis in response to ET-743 treatment. Cell lines harboring TP53 mutations showed no detectable changes in ET-743 sensitivity, confirming previous observations (3, 9, 11), although p21 induction was greater in the TP53 wild-type cell lines.

The confirmation in this series that ET-743 is not associated with TP53 status led us to investigate whether the p73 pathway is involved in the response to ET-743. We hypothesized that p73 might substitute the apoptosis-inducer role of p53, as has been shown in cells lacking functional p53 (65). Thus, we measured the expression levels of p73 throughout the treatment with ET-743 in sensitive and resistant cell lines. Treatment of the panel of human soft-tissue sarcoma cell lines with ET-743 resulted in the inhibition of TAp73 and the induction of the oncogenic form {Delta}Np73, unlike what has been shown for cisplatinum and other DNA-damaging drugs. The expression levels of TAp73 and {Delta}Np73 genes were not related to TP53 status or sensitivity to ET-743, which differs from the observations for other DNA-damaging drugs (65, 69–71). These results strongly suggest that the response of the sarcoma cells to ET-743 does not occur through p53/p73 pathways. The significance of p73 down-regulation after treatment with ET-743 is not yet clear, and functional experiments with p73 are needed to clarify the effect of different p73 transcripts on ET-743 response.

The comparison of sensitive and resistant cells has also revealed a set of genes, including MMP3, AHR, and TIMP3, which are differentially expressed in the absence of the drug. Results obtained here for this panel of low-passage soft-tissue sarcoma cell lines need to be corroborated in tumoral specimens before they are translated to clinical trials. This method may help to identify the subset of soft-tissue sarcoma tumors that potentially show an increased sensitivity to ET-743 (17).


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 V. Moneo and A. Carnero et al., unpublished data Back

5 http://bioinfo.cnio.es/genecards/index.html Back

6 http://fatigo.bioinfo.cnio.es/ Back

7 http://gepas.bioinfo.cnio.es/cgi-bin/sotarray Back

8 http://redlinfomas.cnio.es/publications/ET743 Back

Received 11/29/04; revised 2/22/05; accepted 3/ 9/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Guan Y, Sakai R, Rinehart KL, Wang AH. Molecular and crystal structures of ecteinascidins: potent antitumor compounds from the Caribbean tunicate Ecteinascidia turbinata. J Biomol Struct Dyn 1993;10:793–818.[Medline]
  2. Pommier Y, Kohlhagen G, Bailly C, Waring M, Mazumder A, Kohn KW. DNA sequence- and structure-selective alkylation of guanine N2 in the DNA minor groove by ecteinascidin 743, a potent antitumor compound from the Caribbean tunicate Ecteinascidia turbinata. Biochemistry 1996;35:13303–9.[CrossRef][Medline]
  3. Takebayashi Y, Goldwasser F, Urasaki Y, Kohlhagen G, Pommier Y. Ecteinascidin 743 induces protein-linked DNA breaks in human colon carcinoma HCT116 cells and is cytotoxic independently of topoisomerase I expression. Clin Cancer Res 2001;7:185–91.[Abstract/Free Full Text]
  4. Aune GJ, Furuta T, Pommier Y. Ecteinascidin 743: a novel anticancer drug with a unique mechanism of action. Anticancer Drugs 2002;13:545–55.[CrossRef][Medline]
  5. Minuzzo M, Marchini S, Broggini M, Faircloth G, D'Incalci M, Mantovani R. Interference of transcriptional activation by the antineoplastic drug ecteinascidin-743. Proc Natl Acad Sci U S A 2000;97:6780–4.[Abstract/Free Full Text]
  6. Jin S, Gorfajn B, Faircloth G, Scotto KW. Ecteinascidin 743, a transcription-targeted chemotherapeutic that inhibits MDR1 activation. Proc Natl Acad Sci U S A 2000;97:6775–9.[Abstract/Free Full Text]
  7. Friedman D, Hu Z, Kolb EA, Gorfajn B, Scotto KW. Ecteinascidin-743 inhibits activated but not constitutive transcription. Cancer Res 2002;62:3377–81.[Abstract/Free Full Text]
  8. Gajate C, An F, Mollinedo F. Differential cytostatic and apoptotic effects of ecteinascidin-743 in cancer cells. Transcription-dependent cell cycle arrest and transcription-independent JNK and mitochondrial mediated apoptosis. J Biol Chem 2002;277:41580–9.[Abstract/Free Full Text]
  9. Erba E, Bergamaschi D, Bassano L, et al. Ecteinascidin-743 (ET-743), a natural marine compound, with a unique mechanism of action. Eur J Cancer 2001;37:97–105.
  10. Li WW, Takahashi N, Jhanwar S, et al. Sensitivity of soft tissue sarcoma cell lines to chemotherapeutic agents: identification of ecteinascidin-743 as a potent cytotoxic agent. Clin Cancer Res 2001;7:2908–11.[Abstract/Free Full Text]
  11. Martinez EJ, Corey EJ, Owa T. Antitumor activity- and gene expression-based profiling of ecteinascidin Et 743 and phthalascidin Pt 650. Chem Biol 2001;8:1151–60.[CrossRef][Medline]
  12. Jimeno J, Lopez-Martin JA, Ruiz-Casado A, Izquierdo MA, Scheuer PJ, Rinehart K. Progress in the clinical development of new marine-derived anticancer compounds. Anticancer Drugs 2004;15:321–9.[CrossRef][Medline]
  13. Hendriks HR, Fiebig HH, Giavazzi R, Langdon SP, Jimeno JM, Faircloth GT. High antitumour activity of ET743 against human tumour xenografts from melanoma, non-small-cell lung and ovarian cancer. Ann Oncol 1999;10:1233–40.[Abstract/Free Full Text]
  14. Izbicka E, Lawrence R, Raymond E, et al. In vitro antitumor activity of the novel marine agent, ecteinascidin-743 (ET-743, NSC-648766) against human tumors explanted from patients. Ann Oncol 1998;9:981–7.[Abstract/Free Full Text]
  15. Valoti G, Nicoletti MI, Pellegrino A, et al. Ecteinascidin-743, a new marine natural product with potent antitumor activity on human ovarian carcinoma xenografts. Clin Cancer Res 1998;4:1977–83.[Abstract]
  16. Delaloge S, Yovine A, Taamma A, et al. Ecteinascidin-743: a marine-derived compound in advanced, pretreated sarcoma patients—preliminary evidence of activity. J Clin Oncol 2001;19:1248–55.[Abstract/Free Full Text]
  17. Garcia-Carbonero R, Supko JG, Manola J, et al. Phase II and pharmacokinetic study of ecteinascidin 743 in patients with progressive sarcomas of soft tissues refractory to chemotherapy. J Clin Oncol 2004;22:1480–90.[Abstract/Free Full Text]
  18. Yovine A, Riofrio M, Blay JY, et al. Phase II study of ecteinascidin-743 in advanced pretreated soft tissue sarcoma patients. J Clin Oncol 2004;22:890–9.[Abstract/Free Full Text]
  19. Saez AI, Artiga MJ, Romero C, et al. Development of a real-time reverse transcription polymerase chain reaction assay for c-myc expression that allows the identification of a subset of c-myc+ diffuse large B-cell lymphoma. Lab Invest 2003;83:143–52.[Medline]
  20. Martinez N, Camacho FI, Algara P, et al. The molecular signature of mantle cell lymphoma reveals multiple signals favoring cell survival. Cancer Res 2003;63:8226–32.[Abstract/Free Full Text]
  21. Tracey L, Villuendas R, Ortiz P, et al. Identification of genes involved in resistance to interferon-{alpha} in cutaneous T-cell lymphoma. Am J Pathol 2002;161:1825–37.[Abstract/Free Full Text]
  22. Herrero J, Valencia A, Dopazo J. A hierarchical unsupervised growing neural network for clustering gene expression patterns. Bioinformatics 2001;17:126–36.[Abstract/Free Full Text]
  23. van Kesteren C, Mathjt RA, Lopez-Lazaro L, et al. A comparison of limited sampling strategies for prediction of ecteinascidin 743 clearance when administered as a 24-h infusion. Cancer Chemother Pharmacol 2001;48:459–66.[CrossRef][Medline]
  24. Vujcic S, Halmekyto M, Diegelman P, et al. Effects of conditional overexpression of spermidine/spermine N1-acetyltransferase on polyamine pool dynamics, cell growth, and sensitivity to polyamine analogs. J Biol Chem 2000;275:38319–28.[Abstract/Free Full Text]
  25. Chen Y, Kramer DL, Jell J, Vujcic S, Porter CW. Small interfering RNA suppression of polyamine analog-induced spermidine/spermine N1-acetyltransferase. Mol Pharmacol 2003;64:1153–9.[Abstract/Free Full Text]
  26. Gabrielson EW, Pegg AE, Casero RA Jr. The induction of spermidine/spermine N1-acetyltransferase (SSAT) is a common event in the response of human primary non-small cell lung carcinomas to exposure to the new antitumor polyamine analogue N1,N11-bis(ethyl)norspermine. Clin Cancer Res 1999;5:1638–41.[Abstract/Free Full Text]
  27. Maxwell PJ, Longley DB, Latif T, et al. Identification of 5-fluorouracil-inducible target genes using cDNA microarray profiling. Cancer Res 2003;63:4602–6.[Abstract/Free Full Text]
  28. Marverti G, Bettuzzi S, Astancolle S, Pinna C, Monti MG, Moruzzi MS. Differential induction of spermidine/spermine N1-acetyltransferase activity in cisplatin-sensitive and -resistant ovarian cancer cells in response to N1,N12-bis(ethyl)spermine involves transcriptional and post-transcriptional regulation. Eur J Cancer 2001;37:281–9.
  29. Scorcioni F, Corti A, Davalli P, Astancolle S, Bettuzzi S. Manipulation of the expression of regulatory genes of polyamine metabolism results in specific alterations of the cell-cycle progression. Biochem J 2001;354:217–23.[CrossRef][Medline]
  30. Amundson SA, Bittner M, Chen Y, Trent J, Meltzer P, Fornace AJ Jr. Fluorescent cDNA microarray hybridization reveals complexity and heterogeneity of cellular genotoxic stress responses. Oncogene 1999;18:3666–72.[CrossRef][Medline]
  31. Mashima T, Udagawa S, Tsuruo T. Involvement of transcriptional repressor ATF3 in acceleration of caspase protease activation during DNA damaging agent-induced apoptosis. J Cell Physiol 2001;188:352–8.[CrossRef][Medline]
  32. Sun XH. Constitutive expression of the Id1 gene impairs mouse B cell development. Cell 1994;79:893–900.[CrossRef][Medline]
  33. Benezra R, Davis RL, Lassar A, et al. Id: a negative regulator of helix-loop-helix DNA binding proteins. Control of terminal myogenic differentiation. Ann N Y Acad Sci 1990;599:1–11.
  34. Christy BA, Sanders LK, Lau LF, Copeland NG, Jenkins NA, Nathans D. An Id-related helix-loop-helix protein encoded by a growth factor-inducible gene. Proc Natl Acad Sci U S A 1991;88:1815–9.[Abstract/Free Full Text]
  35. Strait KA, Dabbas B, Hammond EH, Warnick CT, Iistrup SJ, Ford CD. Cell cycle blockade and differentiation of ovarian cancer cells by the histone deacetylase inhibitor trichostatin A are associated with changes in p21, Rb, and Id proteins. Mol Cancer Ther 2002;1:1181–90.[Abstract/Free Full Text]
  36. Eickhoff B, Germeroth L, Stahl C, et al. Trichostatin A-mediated regulation of gene expression and protein kinase activities: reprogramming tumor cells for ribotoxic stress-induced apoptosis. Biol Chem 2000;381:1127–32.[CrossRef][Medline]
  37. Eickhoff B, Ruller S, Laue T, et al. Trichostatin A modulates expression of p21waf1/cip1, Bcl-xL, ID1, ID2, ID3, CRAB2, GATA-2, hsp86 and TFIID/TAFII31 mRNA in human lung adenocarcinoma cells. Biol Chem 2000;381:107–12.[CrossRef][Medline]
  38. Florio M, Hernandez MC, Yang H, Shu HK, Cleveland JL, Israel MA. Id2 promotes apoptosis by a novel mechanism independent of dimerization to basic helix-loop-helix factors. Mol Cell Biol 1998;18:5435–44.[Abstract/Free Full Text]
  39. Biswas G, Anandatheerthavarada HK, Zaidi M, Avadhani NG. Mitochondria to nucleus stress signaling: a distinctive mechanism of NF{kappa}B/Rel activation through calcineurin-mediated inactivation of I{kappa}Bß. J Cell Biol 2003;161:507–19.[Abstract/Free Full Text]
  40. Frey N, Richardson JA, Olson EN. Calsarcins, a novel family of sarcomeric calcineurin-binding proteins. Proc Natl Acad Sci U S A 2000;97:14632–7.[Abstract/Free Full Text]
  41. Shaulian E, Karin M. AP-1 as a regulator of cell life and death. Nat Cell Biol 2002;4:E131–6.[CrossRef][Medline]
  42. Shaulian E, Schreiber M, Piu F, Beeche M, Wagner EF, Karin M. The mammalian UV response: c-Jun induction is required for exit from p53-imposed growth arrest. Cell 2000;103:897–907.[CrossRef][Medline]
  43. Karin M. Mitogen-activated protein kinase cascades as regulators of stress responses. Ann N Y Acad Sci 1998;851:139–46.[Free Full Text]
  44. Vairapandi M, Balliet AG, Hoffman B, Liebermann DA. GADD45b and GADD45g are cdc2/cyclinB1 kinase inhibitors with a role in S and G2/M cell cycle checkpoints induced by genotoxic stress. J Cell Physiol 2002;192:327–38.[CrossRef][Medline]
  45. Shao L, Kasanov J, Hornicek FJ, Morii T, Fondren G, Weissbach L. Ecteinascidin-743 drug resistance in sarcoma cells: transcriptional and cellular alterations. Biochem Pharmacol 2003;66:2381–95.[CrossRef][Medline]
  46. Wetzel M, Rosenberg GA, Cunningham LA. Tissue inhibitor of metalloproteinases-3 and matrix metalloproteinase-3 regulate neuronal sensitivity to doxorubicin-induced apoptosis. Eur J Neurosci 2003;18:1050–60.[CrossRef][Medline]
  47. Alexander CM, Howard EW, Bissell MJ, Werb Z. Rescue of mammary epithelial cell apoptosis and entactin degradation by a tissue inhibitor of metalloproteinases-1 transgene. J Cell Biol 1996;135:1669–77.[Abstract/Free Full Text]
  48. Fata JE, Leco KJ, Voura EB, et al. Accelerated apoptosis in the Timp-3-deficient mammary gland. J Clin Invest 2001;108:831–41.[CrossRef][Medline]
  49. Smith MR, Kung H, Durum SK, Colburn NH, Sun Y. TIMP-3 induces cell death by stabilizing TNF-{alpha} receptors on the surface of human colon carcinoma cells. Cytokine 1997;9:770–80.[CrossRef][Medline]
  50. Bond M, Murphy G, Bennett MR, Newby AC, Baker AH. Tissue inhibitor of metalloproteinase-3 induces a Fas-associated death domain-dependent type II apoptotic pathway. J Biol Chem 2002;277:13787–95.[Abstract/Free Full Text]
  51. Williams AC, Collard TJ, Perks CM, et al. Increased p53-dependent apoptosis by the insulin-like growth factor binding protein IGFBP-3 in human colonic adenoma-derived cells. Cancer Res 2000;60:22–7.[Abstract/Free Full Text]
  52. MacDonald CJ, Ciolino HP, Yeh GC. The drug salicylamide is an antagonist of the aryl hydrocarbon receptor that inhibits signal transduction induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cancer Res 2004;64:429–34.[Abstract/Free Full Text]
  53. Reid JM, Kuffel MJ, Ruben SL, et al. Rat and human liver cytochrome P-450 isoform metabolism of ecteinascidin 743 does not predict gender-dependent toxicity in humans. Clin Cancer Res 2002;8:2952–62.[Abstract/Free Full Text]
  54. Heldin CH, Ostman A, Ronnstrand L. Signal transduction via platelet-derived growth factor receptors. Biochim Biophys Acta 1998;1378:F79–113.[Medline]
  55. Lindahl P, Bostrom H, Karlsson L, Hellstrom M, Kalen M, Betsholtz C. Role of platelet-derived growth factors in angiogenesis and alveogenesis. Curr Top Pathol 1999;93:27–33.[Medline]
  56. Lindahl P, Betsholtz C. Not all myofibroblasts are alike: revisiting the role of PDGF-A and PDGF-B using PDGF-targeted mice. Curr Opin Nephrol Hypertens 1998;7:21–6.[Medline]
  57. Post SM, Tomkinson AE, Lee EY. The human checkpoint Rad protein Rad17 is chromatin-associated throughout the cell cycle, localizes to DNA replication sites, and interacts with DNA polymerase epsilon. Nucleic Acids Res 2003;31:5568–75.[Abstract/Free Full Text]
  58. Post S, Weng YC, Cimprich K, Chen LB, Xu Y, Lee EY. Phosphorylation of serines 635 and 645 of human Rad17 is cell cycle regulated and is required for G(1)/S checkpoint activation in response to DNA damage. Proc Natl Acad Sci U S A 2001;98:13102–7.[Abstract/Free Full Text]
  59. Wang Y, Cortez D, Yazdi P, Neff N, Elledge SJ, Qin J. BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev 2000;14:927–39.[Abstract/Free Full Text]
  60. MacLachlan TK, Takimoto R, El-Deiry WS. BRCA1 directs a selective p53-dependent transcriptional response towards growth arrest and DNA repair targets. Mol Cell Biol 2002;22:4280–92.[Abstract/Free Full Text]
  61. Garcia-Cao I, Lafuente MJ, Criado LM, Diaz-Meco MT, Serrano M, Moscat J. Genetic inactivation of Par4 results in hyperactivation of NF-{kappa}B and impairment of JNK and p38. EMBO Rep 2003;4:307–12.[CrossRef][Medline]
  62. Tomasini R, Samir AA, Carrier A, et al. TP53INP1s and homeodomain-interacting protein kinase-2 (HIPK2) are partners in regulating p53 activity. J Biol Chem 2003;278:37722–9.[Abstract/Free Full Text]
  63. Di Stefano V, Rinaldo C, Sacchi A, Soddu S, D'Orazi G. Homeodomain-interacting protein kinase-2 activity and p53 phosphorylation are critical events for cisplatin-mediated apoptosis. Exp Cell Res 2004;293:311–20.[CrossRef][Medline]
  64. Poindessous V, Koeppel F, Raymond E, Comisso M, Waters SJ, Larsen AK. Marked activity of irofulven toward human carcinoma cells: comparison with cisplatin and ecteinascidin. Clin Cancer Res 2003;9:2817–25.[Abstract/Free Full Text]
  65. Irwin MS, Kondo K, Marin MC, Cheng LS, Hahn WC, Kaelin WG Jr. Chemosensitivity linked to p73 function. Cancer Cell 2003;3:403–10.[CrossRef][Medline]
  66. Zaika AI, Slade N, Erster SH, et al. {Delta}Np73, a dominant-negative inhibitor of wild-type p53 and TAp73, is up-regulated in human tumors. J Exp Med 2002;196:765–80.[Abstract/Free Full Text]
  67. Takebayashi Y, Pourquier P, Zimonjic DB, et al. Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair. Nat Med 2001;7:961–6.[CrossRef][Medline]
  68. Quinn JE, Kennedy RD, Mullan PB, et al. BRCA1 functions as a differential modulator of chemotherapy-induced apoptosis. Cancer Res 2003;63:6221–8.[Abstract/Free Full Text]
  69. Costanzo A, Merlo P, Pediconi N, et al. DNA damage-dependent acetylation of p73 dictates the selective activation of apoptotic target genes. Mol Cell 2002;9:175–86.[CrossRef][Medline]
  70. Gong JG, Costanzo A, Yang HQ, et al. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature 1999;399:806–9.[CrossRef][Medline]
  71. Yuan ZM, Shioya H, Ishiko T, et al. p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage. Nature 1999;399:814–7.[CrossRef][Medline]



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