Silencing of the IGF1R gene enhances sensitivity to DNA ... - Nature

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Cancer Gene Therapy (2005) 12, 90–100 All rights reserved 0929-1903/05 $30.00

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Silencing of the IGF1R gene enhances sensitivity to DNA-damaging agents in both PTEN wild-type and mutant human prostate cancer Mark A Rochester,1,2 Johann Riedemann,1 Giles O Hellawell,1,2 Simon F Brewster,2 and Valentine M Macaulay1 1

Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK; and 2Department of Urology, Churchill Hospital, Oxford OX3 7LJ, UK. The type 1 insulin-like growth factor receptor (IGF1R) is overexpressed in prostate cancer, and mediates proliferation, motility, and survival. Many prostate cancers harbor inactivating PTEN mutations, enhancing Akt phosphorylation. This activates the principal antiapoptotic pathway downstream of the IGF1R, calling into question the value of IGF1R targeting in this tumor. The aim of the current study was to assess the effect of IGF1R gene silencing in prostate cancer cells that lack functional PTEN protein. In human DU145, LNCaP and PC3 prostate cancer cells, transfection with IGF1R small interfering RNA induced significant enhancement of apoptosis and inhibition of survival, not only in PTEN wild-type DU145 but also in PTEN mutant LNCaP and PC3. This was attributed to attenuation of IGF signaling via Akt, ERKs and p38. In both DU145 and PC3, IGF1R knockdown led to enhancement of sensitivity to mitoxantrone, etoposide, nitrogen mustard and ionizing radiation. There was no sensitization to paclitaxel or 5fluorouracil, which do not damage DNA, suggesting that chemosensitization results from impairment of the DNA damage response, in addition to removal of apoptosis protection. These results support the concept of IGF1R targeting in prostate cancer, and indicate that PTEN loss does not render tumor cells refractory to this strategy. Cancer Gene Therapy (2005) 12, 90–100. doi:10.1038/sj.cgt.7700775 Publihsed online 22 October 2004 Keywords: IGF receptor; prostate cancer; PTEN; RNA interference; siRNA; chemosensitisation

rostate cancer is the most common cancer in men, and the second most common cause of male cancer deaths P in the US and the UK. Metastases are most commonly 1,2

found in bone, and advanced disease is initially responsive to androgen ablation therapy. However the development of androgen independence is inevitable, usually after 12– 18 months of endocrine therapy.3 Prostate cancer is essentially resistant to chemotherapy. Combination treatment with mitoxantrone and prednisolone leads to an improved quality of life, but does not prolong survival.4,5 There is an urgent need for the development of novel treatments for patients with advanced androgen-independent disease. The insulin-like growth factors (IGFs)-I and -II play a critical role in the establishment and maintenance of the transformed phenotype. Elevated levels of plasma IGF-I are associated with an increased risk of prostate cancer.6,7 Overexpression of the type 1 insulin-like growth factor receptor (IGF1R) induces growth, neoplastic transformation, and protection from apoptosis.8,9 IGFs induce Received May 5, 2004.

Address correspondence and reprint requests to: Dr Valentine M Macaulay, Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK. E-mail: [email protected]

tumor cell motility through interaction with integrins, and modulate cell–cell attachment via crosstalk between the IGF axis and cell adhesion molecules such as Ecadherin.10–12 These interactions may influence the propensity for metastasis. We have shown that the IGF1R is overexpressed by prostate cancers compared with benign prostatic epithelium. This finding has recently been confirmed by others.13,14 In addition, we showed that expression of both the IGF1R and its principal docking molecule insulin receptor substrate-1 (IRS-1) commonly persist in metastatic disease.15 Furthermore, the IGF1R is reportedly upregulated during the progression to androgenindependence of prostate cancer in vivo.16 This suggests that the IGF1R may be a potential target for therapy of androgen-independent prostate cancer. Indeed antisense IGF1R expression has been shown to block the growth of rat prostate tumours in vivo.17 The IGF1R influences survival principally via activation of the phosphatidylinositol 3-kinase (PI3 kinase) pathway, leading to phosphorylation of Akt and inhibitory phosphorylation of the proapoptotic molecule Bad.8 The action of PI3 kinase is antagonized by PTEN, a tumour suppressor gene encoding a phosphatase. PTEN function is lost by gene deletion or mutation in over 60% of prostate cancers, notably those with high Gleason score

IGF1R gene silencing in prostate cancer MA Rochester et al

and advanced pathological stage.18 In a murine model, prostate-specific PTEN deletion leads to the development of spontaneously metastasizing prostate adenocarcinomas.19 Loss of functional PTEN protein ensures that there is deregulated activation of the Akt pathway for apoptosis protection, even in prostate cancer cells such as LNCaP that lack IRS-1.20,21 Previously, we used antisense strategies to downregulate the IGF1R in androgen-independent DU145 human prostate cancer, showing moderately decreased survival and enhanced sensitivity to cisplatin.22 However, the limited efficacy and nonspecific toxicity associated with phosphorothioate antisense oligonucleotides prevented the demonstration of any effect in other prostate cancer cells. Subsequently, we developed small interfering RNAs (siRNAs) to induce IGF1R gene silencing in human and murine tumor cells.23,24 The greater potency and reduced toxicity of these agents has allowed us to study the effects of IGF1R silencing in the PTEN mutant cell lines PC3 (androgen-resistant) and LNCaP (androgen-sensitive). This has enabled us to investigate whether PTEN loss could negate the effect of IGF1R inhibition; any significant antagonistic effect could represent a significant obstacle to the development of IGF1R targeting in prostate cancer. In fact, we found that IGF1R siRNAs blocked survival of both PTEN wild-type and mutant cells, with evidence of IGF sensitivity in the Akt response of PTEN mutant LNCaP and PC3. Furthermore, IGF1R gene silencing was associated with enhanced sensitivity to DNA-damaging agents, consistent with a role for the IGF1R in the DNA damage response.25,26

Materials and methods

siRNA design This study utilized two IGF1R siRNAs targeting different regions of the IGF1R transcript. The first, R1, was designed using conventional criteria24,27 and was homologous to nucleotides 168–186 of the human IGF1R transcript.28 The sequence of this duplex was: sense strand: 50 -CGACUAUCAGCAGCUGAAGTT-30 , antisense strand 50 -CUUCAGCUGCUGAUAGUCGTT-30 . The sequence of the inverted control duplex (Inv 1) was: sense strand: 50 -GAAGUCGACGACUAUCAGCTT-30 , and antisense strand: 50 -GCUGAUAGUCGUCGACUUCTT-30 . The other duplex, R4, targeted IGF1R mRNA at nucleotides 639–657, identified in our recent array-based screen as a highly accessible region of the transcript.23 This duplex had the sequence: sense strand 50 -CAAUGAGUACAACUACCGCTT-30 , and antisense strand GCGGUAGUUGUACUCAUUGTT-30 . The sequence of the scrambled control duplex (Scr4) was: sense strand: 50 -GUCACACCGAUAAGUCACATT-30 , antisense strand 50 -UGUGACUUAUCGGUGUGACTT-30 . Each strand incorporated 19 bases of RNA with two 30 deoxythymidines. Oligonucletides were synthesized and HPLC purified initially at Transgenomic Laboratories (Glasgow, UK), and latterly at Qiagen (TX, USA). All

sequences were submitted to BLAST search to ensure that only the IGF1R gene was targeted by the IGF1R siRNAs, and that control sequences were not homologous to any known genes. Complementary sense and antisense oligonucleotides were annealed as described.23

Cell culture and transfection This study used human prostate cancer cells DU145 (androgen-independent), PC3 (androgen-independent) and LNCaP (androgen-sensitive, lacking IRS-1;21), obtained from Cancer Research UK Laboratories, Clare Hall, Hertfordshire, UK. DU145 cells carry one wild-type PTEN allelle and a second variant allele, PC3 cells have a homozygous PTEN gene deletion, and LNCaP have a deletion of one PTEN allele and mutation of the other. Neither PC3 nor LNCaP cells express detectable PTEN protein.18,20 All cell lines were negative when tested for Mycoplasma infection. DU145 and LNCaP were cultured in RPMI-1640 and PC3 in Hams F12 all supplemented with 10% fetal calf serum unless otherwise stated. The cells were transfected with 200 nM IGF1R siRNA or inverted or scrambled sequence control duplex using Oligofectamine (Invitrogen) as described.23 Cultures were incubated at 371C for 24–48 hours before further analysis. Some cultures were serum-starved overnight (16 hours), treated with Akt inhibitor SH5 (Alexis Biochemicals UK) for 1 hour and stimulated with 50 nM longR3 IGF-1 (Gropep, Adelaide) or diluent for 10–30 minutes at 371C. Cell lysates were analyzed by immunoblotting as described,22 using antibodies to IGF-1Rb (Santa Cruz), b-tubulin (Sigma), phosphorylated Akt, total Akt, phosphorylated MAPK, total MAPK, phosphorylated p38 and total p38 (Cell Signalling). IGF1R levels were quantified by densitometry after correcting for b-tubulin loading on immunoblots of three sets of independently prepared lysates. Levels in siRNA- and control-transfected cultures were compared by paired t-test (GraphPad Prism).

Assays for apoptosis and survival At 24 hours after transfection with 200 nM siRNA or control duplex, prostate cancer cells were treated with 5 mM etoposide or diluent in complete growth medium with serum. On the following day, cells were reseeded into 96-well plates at 20,000 per well in 100 ml medium, and caspase 3/7 activation was determined using Apo-One Caspase 3/7 assay reagent (Promega) according to the manufacturer’s instructions. To measure survival, cells were reseeded 48 hours after transfection at 2000 cells/ 10 cm dish in culture medium supplemented with 10% fetal calf serum. After 10–21 days incubation at 371C, 10% CO2, visible colonies were fixed, stained and counted as described.22 Assays were repeated two to three times, on each occasion with three replicates. Parallel cultures were used for immunoblotting to confirm IGF1R gene silencing. For chemosensitivity assays, transfected cell monolayers were disaggregated and reseeded in complete

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medium with serum at 4000 cells/10 cm dish. On the following day the culture medium was removed and replaced with fresh medium containing etoposide, mitoxantrone, 5-fluorouracil or paclitaxel (Sigma, UK). The drugs were dissolved in solvent (DMSO for etoposide, 5FU and paclitaxel, and ethanol for mitoxantrone) and control cultures were treated with solvent alone. After 24 hours of incubation, monolayers were washed with PBS and fresh culture medium plus serum but without drug was added. The dishes were incubated at 371C, 10% CO2 for 10–21 days, and colonies were stained and counted. Since clonogenic assays could routinely take 20–30 minutes to set up, compared with B5 minutes for MTS assay, and because nitrogen mustard has a half-life of only 20 minutes in aqueous solution,29 we elected to test effects of this drug using MTS assays. Therefore, cultures were reseeded 48 hours after transfection into 24-well

plates at 2000 cells/well. On the following day, nitrogen mustard (diluted in culture medium) was added to the wells, and the growth was measured 5 days later by MTS assay (Promega). To assess the effect of ionizing radiation, cells were irradiated in suspension 48 hours after transfection in a 137Cs source at 3 Gy/min, and were then reseeded at 4000 cells/10 cm dish for clonogenic assay as above. Dose–response graphs plotted using GraphPad Prism software were used to calculate IC50 values, the dose of agent required to kill 50% of the cells. Each experiment was repeated two to three times. The IC50 values for control-transfected and siRNA-transfected cultures were compared by two-tailed paired t-test (GraphPad Prism). The results were expressed as fold sensitization, calculated as the ratio of the IC50 value for the control culture/IC50 value in the siRNA-transfected culture.

Figure 1 IGF1R siRNAs induce profound IGF1R knockdown in human prostate cancer cells. (a) Prostate cancer cells were transfected with 200 nM siRNA R1 homologous to the IGF1R gene or inverted sequence control. Total cell lysates were analyzed by immunoblotting, showing relative IGF1R levels 48 hours after transfection. Similar results were obtained with siRNA R4. (b) IGF1R protein levels in cultures 48 hours after transfection with either siRNA R1 or R4. IGF1R levels were corrected for loading differences, and expressed as percentage of levels in cultures treated with inverted (R1) or scrambled (R4) sequence control duplex. Bars show mean7SEM IGF1R levels from three independent experiments. (c) Time scale of IGF1R downregulation in DU145. Cells were transfected with siRNA R1 and lysed 1–8 days later. IGF1R levels were corrected for loading and plotted as percentage of the level in untransfected cells.

Figure 2 Enhancement of apoptosis following IGF1R siRNA transfection. DU145 (a) and PC3 cells (b) were transfected with 200 nM siRNA R4 (open bars) or scrambled control (black bars). After 24 hours, the cells were treated with 5 mM etoposide or diluent. Apoptosis was detected by caspase 3/7 activation assay. RFLU: relative fluorescent light units. For both cell lines, levels of basal and etoposide-induced apoptosis were significantly greater in cells transfected with IGF1R siRNA (***Po.001 by one-way analysis of variance and Bonferonni’s test). Similar results were obtained in duplicate experiments for each cell line, and also when repeated with 200 nM siRNA R1.

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Results and discussion

IGF1R gene silencing enhances apoptosis susceptibility and blocks survival of both PTEN wild-type and mutant prostate cancer cells IGF1R levels were higher in DU145 cells than in PC3 or LNCaP cells, as previously reported.30 In DU145, transfection with IGF1R siRNA led to significant IGF1R knockdown in DU145 (Fig 1a, b), a more profound effect than we had previously achieved using antisense oligonucleotides.22 Furthermore, we were able to show significant IGF1R downregulation in both PC3 and LNCaP (Fig 1a, b), which had not been possible using antisense oligonucleotides (G Hellawell, unpublished data). A time course of receptor downregulation in DU145 indicated that levels of IGF1R protein were lowest 48–72 h after transfection (approximately 6% of levels in cultures transfected with inverted sequence control), and remained below 60% of control levels for 6 days (Fig 1c), compared with 3 days for antisense oligonucleotides.22 There was no evidence of rebound IGF1R overexpression at later timepoints (10–12 days, not shown). We wished to compare the effects of IGF1R gene silencing on apoptosis susceptibility and survival in PTEN wild-type and mutant cells. Transfection with either R1 or R4 IGF1R siRNA resulted in enhancement of basal apoptosis, compared with results in control-transfected cells (Fig 2). Results were similar in DU145 and PC3, although compared with PC3, the siRNA-transfected DU145 cells showed a proportionately greater increase in apoptosis after etoposide. We note that similar enhancement of apoptosis susceptibility was induced in PC3 cells by forced expression of PTEN, a manoeuvre that also led

to both reduction in cell surface IGF1R expression, and ability of IGF-1 to rescue cells from induced apoptosis. Indeed that study indicated that in PC3 cells, IGF signalling provides a more pronounced survival effect in the absence of PTEN.31 Suppression of PTEN expression in PC12 cells with PTEN antisense has also been shown to enhance the protective effect of IGF-1 against etoposideinduced apoptosis.32 As discussed previously22 and indicated in Figure 1c, profound IGF1R downregulation does not persist for the duration of the clonogenic survival assay, but levels at the time of cell seeding are likely to be important in determining survival. IGF1R gene silencing reduced survival of DU145 cells to 30% of levels in controltransfected cultures (Fig 3). This was a greater effect than we had previously seen using antisense,22 paralleling the more profound IGF1R downregulation induced by siRNA. We compared this effect with results in PTEN mutant PC3 and LNCaP cells, anticipating that deregulated Akt activation might render these cells refractory to the effects of IGF1R gene silencing. In fact, survival inhibition was at least as profound in LNCaP and PC3, compared with results in DU145 (Fig 3). Similar effects on survival were induced by the two siRNAs that targeted different regions of the IGF1R transcript, suggesting that effects on survival were indeed due to IGF1R gene silencing rather than to ‘off-target’ effects on the expression of other partially homologous genes.33 Our previous work in DU145 cells transfected with IGF1R antisense oligonucleotides had shown that a proportion of the survival inhibition was clearly attributable to the sequence-unrelated toxicity of phosphorothioate oligonucleotides.22 In contrast, there was no

Figure 3 IGF1R gene silencing inhibits survival of PTEN wild-type and mutant prostate cancer cells. Prostate cancer cells were transfected with IGF1R siRNAs R1 or R4, or inverted or scrambled sequence controls. Nil, untransfected PC3 cells. Bars show mean7SEM colony numbers from three experiments, each in triplicate (nine data points). Compared with control-transfected cells, the survival of siRNA-transfected cells was significantly reduced, in PTEN wild-type DU145 and also in PTEN mutant PC3 and LNCaP (***Po.001).

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difference in survival between PC3 cells treated with inverted sequence control duplex and untreated cultures (Fig 3), suggesting that siRNA transfection was not associated with significant nonspecific toxicity, at least in these cells.

Effects of IGF1R knockdown on phosphorylation of signalling intermediates We have shown that IGF1R targeting was capable of inducing significant impairment of survival even in cells with a PTEN mutation. This suggested either that the PI3 kinase pathway retained sensitivity to IGFs, and/or that IGF1R gene silencing was blocking signalling via other pathways such as MAPK that also influence survival.34 Therefore, we investigated the effect of IGF1R siRNA transfection on the phosphorylation of downstream signalling intermediates in DU145 cells, which express wild-type PTEN protein, and in PC3 and LNCaP cells which lack PTEN protein.18,20 Following an overnight serum starve, we observed phosphorylation of Akt in unstimulated PC3 and LNCaP cells, of ERKs in DU145, and of p38 in all three cell lines (Fig 4a). The detectable basal level of phospho-Akt in PC3 and LNCaP cells has been previously reported31 and is a predictable consequence of inactivating PTEN mutation. Detection of other phosphorylated intermediates suggested persisting IGF1R activation in serum-starved cells, perhaps due to autocrine IGF-II production.35 However, immunoprecipitation/immunoblot analysis of the IGF1R showed detectable phosphorylated receptor only in IGF-stimulated cells (data not shown). Phosphorylation of MAPK isoforms in serum-starved cells could result from activation of other receptor tyrosine kinases such as EGF family ligands and receptors, which are known to be expressed by these prostate cancer cells.36 Alternatively, growth factor receptor-independent activation of signalling intermediates may result from interactions between integrins and extracellular matrix components.37,38 In control-transfected DU145 cells, IGF stimulation induced phosphorylation of Akt, ERKs, and p38 (Fig 4a). At 48 hours after IGF1R siRNA transfection, DU145 cells showed significant attenuation of IGF-induced Akt and ERK phosphorylation, comparable to results we previously reported in breast cancer cells,23 although there was no suppression of the p38 response to IGF-I. While phosphorylated Akt was clearly detectable in serumstarved PC3 and LNCaP, there was an unequivocal Akt response to IGF-I in both cell types. The upstream mediator(s) of this effect in LNCaP are unclear given the lack of IRS-1, but could include another IRS isoform. We were unable to detect significant changes in phospho-JNK levels after IGF stimulation or following IGF1R Figure 4 Effects of IGF1R siRNA transfection on IGF signalling. (a) Prostate cancer cells were transfected with 200 nM siRNA R1 or inverted sequence control. After 24 hours, the cultures were serumstarved overnight prior to a 30 minutes IGF-1 stimulation. Levels of signalling intermediates were assessed by immunoblotting for total and phosphorylated Akt, p38, and MAPK. Similar results were obtained in two further sets of independently prepared lystates. (b) Serum-starved PC3 cells were treated with the Akt inhibitor SH5 for 1 hour, stimulated with IGF-I for 30 minutes, and cell lysates were analyzed by immunoblotting for total and phosphorylated Akt and MAPK. (c) At intervals after transfection with 200 nM siRNA R1, PC3 cells were serum-starved overnight and stimulated with 50 nM IGF-I for 30 minutes prior to analysis as in (a).

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knockdown in any cell line (data not shown). However, IGF1R gene silencing completely or partially blocked the Akt response to IGF-I, and also inhibited phosphorylation of ERKs and p38 in LNCaP. In contrast, siRNAtransfected PC3 cells showed an increase in basal levels of ERK phosphorylation with a further increase after IGF stimulation (Fig 4a). We investigated whether this effect could be a consequence of inhibition of IGF-induced Akt activation in siRNA transfectants. Treatment of PC3 cells with a specific Akt inhibitor caused a marked dosedependent increase in ERK phosphorylation (Fig 4b), compatible with an element of crosstalk between the ERK and PI3kinase pathways.39,40 In the context of the

previous results (Fig 2) showing reduced survival of IGF1R siRNA-transfected PC3 cells, the enhancement of ERK phosphorylation may be consistent with a role for MAPK in apoptosis induction in these cells.41 Serial monitoring of the IGF response indicated that this was a transient effect, apparent 48 hours after transfection, and followed by profound inhibition of IGF-induced activation of ERKs, p38, and Akt (Fig 4c). Clearly, there were some differences in IGF responses between these cell lines, likely to be due to factors other than PTEN status. Further functional studies will be required to clarify the molecular basis for these differences. However, it is also clear that IGF1R gene silencing was capable of influencing

Figure 5 IGF1R knockdown enhances sensitivity to DNA-damaging agents. DU145 (a, c, e) and PC3 cells (b, d, f) were treated with mitoxantrone (a, b), etoposide (c, d), or ionizing radiation (e, f). Clonogenic survival was plotted as percentage of result in control dishes treated with diluent alone (a–d), or mock-irradiated cultures (e, f). Continuous line, inverted control; dotted line, IGF1R siRNA R1. Points represent the means7SEM of three replicates from individual experiments. Similar results were obtained in two further assays for each agent. Mean IC50 values are given in Table 1.

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signalling via multiple pathways that influence survival.34,42,43 even in cells lacking functional PTEN protein where Akt regulation is compromised.

IGF1R gene silencing enhances sensitivity to DNA-damaging agents The IGF axis has a well-recognized role in protection from apoptosis induced by cytotoxic drugs.44–47 Given the intrinsic chemoresistance of prostate cancer, we wanted to assess the effect of siRNA-induced IGF1R gene silencing on sensitivity to conventional anticancer treatments. Representative survival curves are shown in Figures 5–7, and the sensitivity data are summarized in Table 1. Mitoxantrone is a topoisomerase II inhibitor which causes double-strand DNA (dsDNA) breaks.48 In combination with prednisolone it has been shown to enhance quality of life in prostate cancer.4,5 Mitoxantrone caused dose-dependent killing of both DU145 and PC3 cells, and at all drug concentrations the siRNA transfectants showed reduced survival compared with controls (Fig 5a and Table 1). In DU145, the mean IC50 values for control-transfected and siRNA-transfected cells were 3.6 and 2.1 nM, respectively (Po.001), representing 1.7-fold sensitization to mitoxantrone. Equivalent values in PC3 were 3.3 and 1.8 nM (Po.01), representing 1.9-fold sensitization (Fig 5b, Table 1). Similar results were

obtained in cultures treated with etoposide, another topoisomerase II inhibitor (Fig 5c, d), where IGF1R knockdown was associated with significant reduction in IC50 values in both DU145 (1.9-fold sensitization, Po.01) and PC3 cells (1.7-fold sensitization Po.05). In general, the PTEN wild-type DU145 and PTEN-null PC3 cells showed comparable chemosensitivity, with similar IC50s upon treatment with mitoxantrone and etoposide, and similar changes in sensitivity following IGF1R knockdown. Previously we observed moderate antisense-IGF1R induced sensitization to cisplatin (1.7-fold), mitoxantrone (2-fold) and paclitaxel (1.5-fold) in DU145 cells transfected with IGF1R antisense RNA.22 Using IGF1R siRNA we were able to achieve more profound inhibition of both IGF1R expression and survival. Despite this the degree of chemosensitization was no greater. This may reflect a threshold effect of residual IGF1R expression, or the fact that stable expression of antisense RNA induced durable IGF1R downregulation compared with the effect of transient siRNA transfection. It is plausible, however, that a two-fold increase in sensitivity may be clinically useful, especially if it applied to all drugs in a combination. Following induction of IGF1R gene silencing, all three cell lines showed approximately two-fold sensitization to ionizing radiation (Fig 5e, f, Figure 7a, Table 1). This

Figure 6 Effects of IGF1R siRNA transfection on sensitivity to nitrogen mustard, 5FU, and paclitaxel. Graphs show cell survival following treatment with nitrogen mustard (a, b), 5FU (c) or paclitaxel (d) in DU145 (a, c) and PC3 cells (b, d). Nitrogen mustard was evaluated in MTS assay, and the other agents in clonogenic assay. The results are plotted as % OD490 or % survival of cells treated with diluent alone. Continuous line, inverted control; dotted line, IGF1R siRNA R1. Points represent the means7SEM of three replicates from single experiments. Similar results were obtained in two further assays for nitrogen mustard, and one each for 5FU and paclitaxel.

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represents a similar enhancement of radiosensitivity to the effect seen previously in murine melanoma cells following antisense-mediated IGF1R downregulation.25 In those cells, we had also observed a defect in the activation of Atm,25 a multifunctional serine–threonine kinase that

Figure 7 IGF1R knockdown sensitizes LNCaP cells to ionizing radiation but not to cytotoxic drugs. LNCaP cells were irradiated in suspension (a) or treated with etoposide (b), 48 hours after transfection with inverted control duplex (continuous line) or IGF1R siRNA R1 (dotted line), and were then reseeded for clonogenic assay. Graphs show survival as a percentage of mock-irradiated or untreated control cultures. The points represent the means7SEM of three replicates from individual experiments. Similar results were obtained in two assays for each agent.

plays a critical role in the initiation of cell cycle checkpoints and DNA repair pathways following induction of dsDNA breaks.49 Mitoxantrone, etoposide, and ionizing radiation share the capacity to induce dsDNA breaks, and induce cell death by apoptosis.47,50,51 The ability of IGF1R gene silencing to enhance tumor cell sensitivity to cytotoxic drugs and irradiation could therefore be due to removal of apoptosis protection, and/or withdrawal of elements of the DNA damage response. To clarify this issue, we investigated the extent to which IGF1R siRNA transfection could influence prostate cancer cell sensitivity to drugs that cause alternative DNA lesions, or that kill cells without damaging DNA. Nitrogen mustard is an interstrand crosslinking (ICL) agent that does not directly cause strand breaks. However, the structural distortion imposed by ICLs can cause collapse of replication forks, resulting in formation of dsDNA breaks.52 Nitrogen mustard is not used in the clinical management of prostate cancer but none-the-less caused dose-dependent inhibition of survival in both DU145 (Fig 6a) and PC3 cells (Fig 5b). Furthermore, we observed an increase in sensitivity to this agent in both DU145 (Po.001) and PC3 cells (Po.001) after transfection with IGF1R siRNA (Fig 6, Table 1). We selected two drugs that induce apoptosis of tumor cells by mechanisms other than direct DNA damage. Taxanes block cells in mitosis by inhibiting microtubular disassembly. They have in vitro activity against prostate cancer4 and have recently been reported to have greater clinical activity than mitoxantrone in Phase III trials of patients with hormone-refractory prostate cancer.53 The antimetabolite 5-fluorouracil (5FU) exerts its cytotoxicity through conversion by a two-step route to 5-fluorouridine monophosphate (5-FUMP). 5-FUMP is further transformed to 5-FdUMP, an irreversible inhibitor of thymidylate synthase, resulting in dTTP starvation and subsequent apoptosis.48 Prolonged exposure to 5FU, or its use in combination, has been reported to induce DNA strand breaks, although DNA damage has not been detected after 24 hours exposure to 5FU alone.54 We were unable to detect any significant enhancement of sensitivity

Table 1 Sensitivity to cytotoxic drugs and irradiation in human prostate cancer cells transfected with control duplex or IGF1R siRNA DU145

Treatment

Control IC50

PC3

Fold siRNA IC50 sensitization

Mitoxantrone (nM) 3.670.1 2.170.1*** Etoposide (mM) 3.970.8 1.9770.3** Irradiation (Gy) 3.970.1 2.070.1*** Nitrogen mustard (mM) 2.170.1 1.570.1*** Paclitaxel (nM) 2.270.1 2.570.2 5FU (mM) 10.970.1 9.670.2

1.770.1 1.970.1 1.970.1 1.570.1 0.970.1 1.170.1

Control IC50

LNCaP

Fold siRNA IC50 sensitization

3.370.4 1.870.2** 7.571.1 4.771.5* 2.770.1 1.470.1*** 1.470.2 0.7570.2* 2.970.1 2.770.1 10.070.5 9.570.6

1.970.2 1.770.1 1.970.1 1.970.1 1.170.1 1.170.1

Control IC50

siRNA IC50

Fold sensitization

4.3570.2 1.771.1 2.370.1 1.970.1 2.370.2 10.370.8

4.070.1 1.971.2 1.270.1*** 1.970.1 3.070.1 9.970.1

1.170.1 0.870.1 1.970.1 0.970.1 0.870.1 1.070.1

The IC50 values were calculated from three independent assays, each of which included triplicate data points. *Po.05, **Po.01, ***Po.001 for comparison of IC50 values in siRNA transfected versus control-transfected cultures. For each assay, fold sensitization was calculated as a ratio of IC50 in cultures transfected with control duplex to IC50 in cultures transfected with IGF1R siRNA R1. The value shown is the mean7SEM of three IC50 ratios.

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to either of these agents in PC3, DU145, or LNCaP cells following transfection with IGF1R siRNA (Fig 6 and Table 1). The cultures used in these experiments were taken from the same pool of transfected cells used to analyze mitoxantrone and etoposide sensitivity, and showed identical suppression of IGF1R expression (not shown). Therefore, the inability to sensitize to these agents could not be attributed to any alteration in cellular characteristics or degree of IGF1R gene silencing. IGF1R targeting has been linked with enhancement of chemosensitivity in several tumour cell types. In bladder cancer, IGF1R antisense oligonucletide transfection was shown to enhance sensitivity to mitomycin-C.55 Scotlandi et al56,57 demonstrated dramatic (410-fold) sensitization to doxorubicin in Ewing’s sarcoma cells using IGF1R antisense, dominant negative receptor or monoclonal antibody.58 It is possible that the greater magnitude of chemosensitization in this tumor may relate to the fact that, unlike prostate cancer, Ewing’s sarcoma is inherently chemosensitive.59 Both mitomycin-C and doxorubicin induce dsDNA breaks, the former via interstrand crosslinking of DNA, and the latter via inhibition of topoisomerase II.48 Recently, the growth of pancreatic xenografts was shown to be significantly inhibited by adenoviral expression of dominant negative IGF1R in combination with 5FU, although it was not clear whether this represented enhancement of chemosensitivity per se.60 In androgen-responsive, PTEN mutant LNCaP cells, we were able to show effective IGF1R gene silencing (Fig 1) and profound inhibition of clonogenic survival (Fig 2), comparable to that observed in DU145 and PC3. While there was a consistent increase in sensitivity to ionizing radiation in IGF1R siRNA-transfected LNCaP cells (Fig 7a), we could not detect enhancement of sensitivity to any cytotoxic drug (Fig 7b and Table 1). Unlike DU145 and PC3, LNCaP cells express the androgen receptor but have lost expression of IRS-1, the principal IGF1R-docking molecule.20 These differences may impose variation in signalling via pathways dependent on these molecules. Indeed, a recent report has identified IRS-1 as a mediator of IGF effects on DNA repair, by interacting with Rad51 to influence the rate of homologous recombination (HR26). It is plausible that the loss of IRS-1 could remove a key link between the IGF1R and the HR machinery, rendering LNCaP cells relatively insensitive to changes in IGF1R expression or function. However, as we previously reported, the absence of IRS-1 is unusual in the setting of both primary and metastatic prostate cancer.15 Given that IGFR signalling provides protection from apoptosis8,9 and that conventional anticancer treatments kill by apoptosis,47,50 we had anticipated that IGF1R gene silencing would enhance sensitivity to a wide range of chemotherapeutic agents. Our results indicate that IGF1R knockdown was capable of sensitizing both PTEN wild-type and PTEN-null prostate cancer cells to agents which induce dsDNA breaks. However, there was no evidence of sensitization to agents that kill tumor cells without causing DNA damage. It is possible that differences in response to individual drugs could be attributed to different kinetics of drug action relative to

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IGF1R knockdown; this could also explain, at least in part, why we previously observed modest sensitization to paclitaxel using stable antisense-RNA expression22 but no sensitization following transient siRNA transfection in the current study. However, these results are also compatible with a key role for the IGF1R in the cellular response to DNA damage, as we and others have reported.25,26 This could indicate that the ability to chemosensitize by IGF1R gene silencing was related to attenuation of the DNA damage response, in addition to withdrawal of apoptosis protection. The ability to sensitize a chemoresistant tumor type offers hope for the future development of IGF1R targeting as treatment for patients with hormone-escaped prostate cancer. Acknowledgments

This work was supported by Cancer Research UK and by a Health Foundation Fellowship to MR. We are grateful to Ian Hickson, Peter McHugh, and Andrew Protheroe for comments on the manuscript.

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