Purpose: Expression of the type 1 insulin-like growth factor receptor (IGF1R) confers adverse prognosis in clear cell renal cell cancer (CC-RCC). We recently showed that IGF1R expression is inhibited by the von Hippel-Lindau (VHL) tumor suppressor, and the IGF1R is up-regulated in CC-RCC, in which VHL is frequently inactivated. We tested the hypothesis that IGF1R up-regulation mediates resistance to cancer therapeutics, evaluating the effects of IGF1R depletion on sensitivity to cytotoxic drugs, which are ineffective in RCC, and the mammalian target of rapamycin (mTOR) inhibitor rapamycin, analogues of which have clinical activity in this tumor.

Experimental Design: This study used CC-RCC cells harboring mutant VHL, and isogenic cells expressing functional VHL. Cells were transfected with nonsilencing control small interfering RNA (siRNA), or with one of two different IGF1R siRNAs. The more potent siRNA was modified by 2′-O-methyl derivatization for in vivo administration.

Results: CC-RCC cells expressing mutant VHL and higher IGF1R were more chemoresistant than cells expressing functional VHL. IGF1R depletion induced apoptosis, blocked cell survival, and sensitized to 5-fluorouracil and etoposide. These effects were significantly greater in CC-RCC cells expressing mutant VHL, supporting the hypothesis that IGF1R up-regulation makes a major contribution to the chemoresistance associated with VHL loss. IGF1R depletion also enhanced sensitivity to mTOR inhibition, at least in part due to suppression of rapamycin-induced Akt activation. Administration of stabilized IGF1R siRNA was shown to sensitize CC-RCC xenografts to rapamycin in vivo.

Conclusion: These data validate IGF1R as a therapeutic target in CC-RCC, and support the evaluation of IGF1R-inhibitory drugs in patients with renal cancer. [Mol Cancer Ther 2009;8(6):1448–59]

Translational Relevance

The data presented in this article indicate that the type I insulin-like growth factor receptor (IGF1R) is an important mediator of chemoresistance in renal cancer. IGF1R depletion was shown to enhance sensitivity to two cytotoxic drugs and also to rapamycin, analogues of which have clinical activity in patients with this tumor type. These results support the evaluation of drug candidates that target the IGF1R in patients with renal cancer, as a means of enhancing sensitivity to mammalian target of rapamycin inhibition, and potentially also to chemotherapy.

The type 1 insulin-like growth factor receptor (IGF1R) plays a key role in the regulation of transformation, cell survival, and proliferation. These effects are achieved via recruitment to the IGF1R of docking proteins including insulin receptor substrate-1 (IRS-1), and activation of multiple downstream effectors, including the phosphatidylinositol-3-kinase (PI3K) phosphoinositide–dependent kinase-1 AKT and mitogen-activated protein kinase pathways (1). Our recent investigations focused on the regulation of IGF1R expression in clear cell renal cell carcinoma (CC-RCC), a highly chemoresistant tumor characterized by frequent inactivating mutations of the von Hippel-Lindau (VHL) gene (24). Our studies revealed that the VHL protein is capable of suppressing Sp-1–mediated IGF1R promoter activation, and destabilizing IGF1R mRNA via interaction with the RNA-binding protein, HuR (5). This represents a new, non-HIF–dependent role for VHL. Consistent with the frequent loss of functional VHL in CC-RCC, we detected significant overexpression of IGF1R mRNA in CC-RCCs compared with paired samples of benign kidney. Although we could detect IGF1R transcripts in all tested samples of CC-RCC tumors (5), IGF1R protein is reportedly detectable by immunohistochemical staining in only ∼60% of cases (6), indicating further regulation at the posttranscriptional level. IGF1R positivity on immunohistochemistry has been shown to correlate with higher tumor grade, and poor prognosis even in lower stage disease (6, 7). Moreover, IGF signaling is known to regulate survivin expression, identified as a mediator of poor prognosis in CC-RCC (8, 9). These findings suggest that IGF1R expression in CC-RCC is of biological significance.

We hypothesized that the IGF1R makes a significant contribution to the resistance to clinical therapy that characterizes CC-RCC. We tested this hypothesis by evaluating the effects of IGF1R blockade on sensitivity to cytotoxic and biological therapies. Given the known homology between the IGF1R and the insulin receptor (10), and the ability of many IGF1R drug candidates to block insulin signaling (11, 12), we used a sequence-specific approach to target the IGF1R using small interfering RNAs (siRNA) that silence the IGF1R gene by RNA interference (13, 14). We show here that cell survival and chemoresistance of VHL mutant CC-RCC cells is dependent on IGF signaling to a greater extent than in isogenic cells that express functional VHL. In addition, IGF1R depletion was found to enhance sensitivity to rapamycin in vitro and in vivo, via suppression of a negative feedback loop that operates between mammalian target of rapamycin (mTOR) and IRS-1. These findings have a clear relevance for the development of new systemic therapies for patients with CC-RCC.

Cell Culture and siRNA Transfection

Human CC-RCC cell lines, 786-0/EV, 786-0/VHL, RCC4/EV, and RCC4/VHL, were obtained from Cancer Research UK Laboratories, Clare Hall, Hertfordshire, United Kingdom. The cell lines had been stably transfected with HA-tagged wild-type VHL (786-0/VHL, RCC4/VHL) or empty vector (786-0/EV, RCC4/EV; ref. 15). The cells were cultured in DMEM supplemented with 10% FCS and 0.5 mg/mL of G418 (Invitrogen) in a humidified atmosphere of 5% CO2 and 95% air. All cell lines were negative for Mycoplasma infection.

RCC cells were transfected at 30% to 50% confluence with nonsilencing control (NSC) siRNA (sense strand, 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense strand, 5′-ACGUGACACGUUCGGAGAATT-3′) or one of two IGF1R siRNAs targeting different regions of the IGF1R transcript. Duplex R1 was designed by conventional criteria (16) to target nucleotides 168 to 186 of human IGF1R mRNA (10), and has the sequence: sense strand, 5′-CGACUAUCAGCAGCUGAAGTT-3′; antisense strand, 5′-CUUCAGCUGCUGAUAGUCGTT-3′. The duplex designated R4 targeted IGF1R mRNA at nucleotides 639 to 657, identified in an array-based screen as a highly accessible region of the IGF1R transcript (13). This siRNA has the sequence: sense strand, 5′-CAAUGAGUACAACUACCGCTT-3′; antisense strand, 5′-GCGGUAGUUGUACUCAUUGTT-3′. All siRNAs were from Qiagen. Cells were transfected with 100 nmol/L of siRNA using OligofectAMINE (Invitrogen) as described previously (5), and were incubated at 37°C for 24 to 48 h before further analysis.

Immunoblotting

Cells were lysed for 30 min on ice in IGF1R lysis buffer containing 50 mmol/L of HEPES, 100 mmol/L of sodium chloride, 10 mmol/L of EDTA, 1% Triton X-100, 4 mmol/L of sodium pyrophosphate, 2 mmol/L of sodium orthovanadate, 10 mmol/L of sodium fluoride with 1.5 mmol/L of Pefabloc SC Plus (Roche Diagnostics), and EDTA-free protease inhibitor cocktail (Roche). Lysates were centrifuged at 13,000 rpm for 15 min to pellet insoluble material, and were analyzed by SDS-PAGE and immunoblotting. Primary antibodies were to IGF-1Rβ (Cell Signaling Technology), HIF-1α (BD Biosciences), and HIF-2α, (Abcam), HA tag (Covance), phosphorylated (S612) IRS-1 (clone L7B8; Cell Signaling), phosphorylated (S473) and total Akt (Cell Signaling), phosphorylated (T421/S424) p70 S6 kinase (Cell Signaling), phosphorylated (S240/244) and total S6 peptide (Cell Signaling), β-tubulin (clone TUB2.1; Sigma), and actin (Abcam). IGF1R levels were quantified using ImageJ software and were corrected for β-tubulin or actin loading. IGF1R levels in siRNA-transfected cultures were compared by paired t test or ANOVA using GraphPad Prism v.4.0 software (GraphPad), for paired and multiple samples, respectively.

Apoptosis Assay

Forty-eight hours after siRNA transfection, CC-RCC cells were re-seeded at 40,000 cells per well into 96-well plates coated with poly-HEMA as previously described (17) to prevent adherence. Caspase 3/7 activation assays (Apo-1; Promega) were done according to the instructions of the manufacturer, by adding assay substrate, incubating at 37°C for up to 12 h, and analysis on a FluorScan Universal Microplate Spectrophotometer. Apoptosis was expressed as relative fluorescence light units.

MTS and Clonogenic Assays

5-Fluorouracil (5-FU; Sigma) and etoposide (Sigma) were prepared as stock solutions of 42.5 mmol/L, cisplatin (Sigma) was prepared as a 20 mmol/L stock solution, and rapamycin (Sigma), an inhibitor of mTOR (18), was prepared as a 1 mmol/L stock, all in sterile DMSO. These compounds were aliquoted and stored at −20°C, and further diluted in culture medium (DMEM) to the correct final concentration.

Twenty-four hours after siRNA transfection, CC-RCC cells were re-seeded in 96-well plates at 3,000 cells per well, and the following day, were treated with compounds as above or with solvent (DMSO). After 3 days, MTS assays (Promega) were done according to the instructions of the manufacturer, and the absorbance at 490 nm was quantified on a plate reader (Becton Dickinson Labware). The results were expressed as a percentage of OD490 in control cells treated with DMSO without added drug.

Clonogenic assays were done as previously described (14, 17). In brief, 48 h after siRNA transfection, cells were re-seeded into 10 cm dishes at 1,000 to 1,500 cells per dish, depending on the cell line. The remaining cells were pelleted by centrifugation at 13,000 rpm at 4°C, washed with ice-cold PBS, and lysed for immunoblotting to quantify IGF1R gene silencing. Clonogenic assay dishes were incubated at 37°C in 5% CO2 for 10 to 14 days until discreet colonies were visible. For the investigation of drug sensitivity, RCC cells were transfected with siRNAs, re-seeded as above, and the following day, were treated with DMSO or drug in DMEM with 10% FCS. After 24 h, the medium was removed and fresh medium without drug was added. Following incubation for 10 to 14 days, visible colonies were fixed in methanol/acetic acid (3:1), stained with crystal violet (400 μg/mL; Sigma), and counted on an automated colony counter (ColCount, Oxford Optronix). Data from MTS and clonogenic assays were analyzed by GraphPad Prism software, using nonlinear regression to fit the data to a curve, from which IC50 values (dose of drug required to kill 50% of the cells) were calculated. Each experiment used three to four replicate data points and was repeated at least thrice. IC50 values for control-transfected and IGF1R siRNA–transfected cultures were compared by two-tailed paired t test or ANOVA for two or multiple samples, respectively, using Prism v.4.0 software. Changes in drug sensitivity were expressed as fold sensitization, calculated as the ratio of the IC50 value for the control transfectants to the IC50 value for the IGF1R siRNA transfectants.

Serum Stability Assay

To assess siRNA stability, 200 ng of unmodified or 2′-O-methyl (2′-O-Me)–modified siRNAs were added to nine volumes of FCS and incubated at 37°C for 0 to 48 h. Samples were mixed with RNase-free loading buffer and analyzed by electrophoresis on 2% Seakem GTG agarose (NuSieve) gels in parallel with 25 bp DNA size ladder (New England Biolabs). Gels were photographed and digitized using a Flurochem camera and AlphaEaser image analysis software (version 2.0, Alpha Innotech Corporation).

In vivo Assays

In vivo work was carried out at the Cancer Research UK Biological Resources Department, Clare Hall Laboratories, and all procedures were approved by the Cancer Research UK Animal Ethics Committee. RCC xenografts were established by injecting 786-0/EV cells (107 per mouse, mixed with an equal volume of Matrigel; BD Biosciences) into the flanks of 6- to 8-week-old female BALB/c severe combined immunodeficiency mice. When tumors reached 6 to 8 mm in diameter, animals were treated with i.p. injection of PBS or 50 μg of siRNA, complexed with siPORT Amine transfection reagent (Ambion) according to the instructions of the manufacturer. Some animals also received 0.1 mg of rapamycin (Rapamune Oral Solution; Wyeth) by daily gavage. At the end of each experiment, the mice were sacrificed and tumors were snap-frozen in liquid nitrogen. Xenograft tissue was homogenized using Lysing Matrix D beads (Qbiogene) in IGF1R lysis buffer (as above), disruption in a FastPrep FP120 (Bio101, Thermo Electron, Co.) for two periods of 10 s at speed 6. After incubation on ice for 15 min, and centrifugation at 14,000 rpm, 4°C for 5 min to pellet insoluble debris, the lysates were stored at −80°C prior to immunoblotting as above.

IGF1R Depletion Inhibits CC-RCC Cell Survival and Expression of HIF-1α

Assessment of IGF1R levels in each pair of isogenic cell lines confirmed that IGF1R levels were significantly higher in EV-transfected cells that lacked functional VHL (Fig. 1A), as we previously reported (5). In order to investigate the effects of IGF1R depletion, human CC-RCC cells were transfected with IGF1R siRNAs, using two previously validated siRNAs targeting different regions of IGF1R mRNA (13, 14). The siRNA transfection resulted in inhibition of IGF1R expression to 27% and 17% of control levels in cells transfected with the R1 and R4 siRNAs, respectively (P < 0.001 for each comparison with NSC transfectants; Fig. 1A). Within each pair of isogenic cell lines, there was a trend to increased resistance to apoptosis and increased clonogenic survival in empty vector (EV)–transfected cells lacking functional VHL, compared with isogenic cells expressing wild-type VHL, but these differences were not statistically significant (Fig. 1B and C). In all four sublines, IGF1R depletion induced apoptosis upon loss of anchorage, and also inhibited clonogenic survival (Fig. 1B and C). Furthermore, cell survival was inhibited to a greater extent by IGF1R knockdown in the absence of functional VHL: compared with results in NSC transfectants, cell survival was suppressed by the R4 IGF1R siRNA to ∼25% in 786-0/EV cells and ∼50% in isogenic 786-0/VHL cells (P < 0.05). Similarly, IGF1R depletion had a more profound inhibitory effect on survival in RCC4/EV cells than RCC4/VHL (Fig. 1C). These data suggest that the survival of CC-RCC cells expressing mutant VHL was more heavily dependent on the IGF1R compared with the survival of cells expressing functional VHL.

Figure 1.

IGF1R depletion induces apoptosis and blocks cell survival and HIF1-α expression in CC-RCC cells. A, isogenic paired 786-0 and RCC4 CC-RCC cells were transfected with NSC siRNA or two different IGF1R siRNAs, designated R1 and R4. After 48 h, the cells were analyzed by immunoblotting for IGF1Rβ (arrow, IGF1R proreceptor), HA-tagged VHL and β-tubulin. Triplicate immunoblots were corrected for β-tubulin loading. IGF1R levels in NSC-transfected cells expressing functional VHL were significantly lower (18 ± 3% in 786-0; 12 ± 2% in RCC4; P < 0.001) than in EV transfectants. The R1 and R4 IGF1R siRNAs inhibited IGF1R expression to 27 ± 7% and 17 ± 6%, respectively, of levels in NSC-transfected cells (P < 0.001 by ANOVA). B, parallel cultures, transfected as in A, were re-seeded into poly-HEMA coated plates for measurement of apoptosis. Columns, mean from three independent experiments, each in triplicate; bars, SE. Black columns, NSC; hatched columns, R1; open columns, R4. IGF1R siRNA–transfected CC-RCC cells showed significant increase in apoptosis compared with control transfectants (*, P < 0.05; **, P < 0.01; ***, P < 0.001). C, after siRNA transfection as above, cells were re-seeded for clonogenic survival assay. Results are expressed as a percentage of survival in the EV NSC–transfected cells. Columns, mean from three independent experiments, each in triplicate; bars, SE. Black columns, NSC; hatched columns, R1; open columns, R4. Compared with NSC transfectants, both IGF1R siRNAs caused significant suppression of RCC cell survival (*, P < 0.05; **, P < 0.01; and ***, P < 0.001). There was a nonsignificant trend to greater inhibition of survival in cells transfected with R4 siRNA compared with R1. Comparing the effects of IGF1R depletion in EV and VHL-transfected isogenic paired cell lines, cell survival was suppressed in 786-0/EV cells by the R1 siRNA to 38 ± 2% of the value in NSC transfectants and to 63 ± 9% in 786-0/VHL (P < 0.05), and by the R4 siRNA to 26 ± 8% in 786-0/EV and to 51 ± 3% in 786-0/VHL (P < 0.05). Equivalent results in RCC4 cells transfected with R1 and R4 siRNA were 47 ± 6% and 23 ± 3%, respectively, in RCC4/EV, and 76 ± 4% and 50 ± 11%7 in RCC4/VHL (P < 0.05 for comparison of effects of each siRNA in EV and VHL-transfected cells). D, CC-RCC cells transfected as in A were exposed to normoxia (20% O2) or hypoxia (0.1% O2) for 16 h and analyzed by immunoblotting for IGF1R, HA-VHL, HIF1-α, and HIF2-α.

Figure 1.

IGF1R depletion induces apoptosis and blocks cell survival and HIF1-α expression in CC-RCC cells. A, isogenic paired 786-0 and RCC4 CC-RCC cells were transfected with NSC siRNA or two different IGF1R siRNAs, designated R1 and R4. After 48 h, the cells were analyzed by immunoblotting for IGF1Rβ (arrow, IGF1R proreceptor), HA-tagged VHL and β-tubulin. Triplicate immunoblots were corrected for β-tubulin loading. IGF1R levels in NSC-transfected cells expressing functional VHL were significantly lower (18 ± 3% in 786-0; 12 ± 2% in RCC4; P < 0.001) than in EV transfectants. The R1 and R4 IGF1R siRNAs inhibited IGF1R expression to 27 ± 7% and 17 ± 6%, respectively, of levels in NSC-transfected cells (P < 0.001 by ANOVA). B, parallel cultures, transfected as in A, were re-seeded into poly-HEMA coated plates for measurement of apoptosis. Columns, mean from three independent experiments, each in triplicate; bars, SE. Black columns, NSC; hatched columns, R1; open columns, R4. IGF1R siRNA–transfected CC-RCC cells showed significant increase in apoptosis compared with control transfectants (*, P < 0.05; **, P < 0.01; ***, P < 0.001). C, after siRNA transfection as above, cells were re-seeded for clonogenic survival assay. Results are expressed as a percentage of survival in the EV NSC–transfected cells. Columns, mean from three independent experiments, each in triplicate; bars, SE. Black columns, NSC; hatched columns, R1; open columns, R4. Compared with NSC transfectants, both IGF1R siRNAs caused significant suppression of RCC cell survival (*, P < 0.05; **, P < 0.01; and ***, P < 0.001). There was a nonsignificant trend to greater inhibition of survival in cells transfected with R4 siRNA compared with R1. Comparing the effects of IGF1R depletion in EV and VHL-transfected isogenic paired cell lines, cell survival was suppressed in 786-0/EV cells by the R1 siRNA to 38 ± 2% of the value in NSC transfectants and to 63 ± 9% in 786-0/VHL (P < 0.05), and by the R4 siRNA to 26 ± 8% in 786-0/EV and to 51 ± 3% in 786-0/VHL (P < 0.05). Equivalent results in RCC4 cells transfected with R1 and R4 siRNA were 47 ± 6% and 23 ± 3%, respectively, in RCC4/EV, and 76 ± 4% and 50 ± 11%7 in RCC4/VHL (P < 0.05 for comparison of effects of each siRNA in EV and VHL-transfected cells). D, CC-RCC cells transfected as in A were exposed to normoxia (20% O2) or hypoxia (0.1% O2) for 16 h and analyzed by immunoblotting for IGF1R, HA-VHL, HIF1-α, and HIF2-α.

Close modal

RCC4 cells express both HIF-1α and HIF-2α, and it was noted that IGF1R depletion led to HIF1-α down-regulation both basally, in RCC4/EV lacking functional VHL, and in response to hypoxia in RCC/VHL cells in which a functional response to normoxia was restored by re-expression of wild-type VHL. This effect on HIF-1α is consistent with the ability of IGFs to protect against hypoxia and to activate PI3K-AKT and mitogen-activated protein kinase signaling, known to induce HIF-1α translation and stabilization (1921). The 786-0 cell line does not express HIF-1α, but both RCC4 and 786-0 express HIF-2α, the levels of which were not influenced by IGF1R gene silencing (Fig. 1D). This finding is consistent with reports that expression of HIF-2α by breast cancer cells was unaffected by PI3K inhibition (21), although in other cell types, HIF-2α was reported to be IGF-inducible (22, 23).

IGF1R Depletion Enhances the Chemosensitivity of CC-RCC

The ability of IGF1R gene silencing to induce apoptosis and to block CC-RCC cell survival raises the question of whether IGF1R targeting can enhance the sensitivity of CC-RCC cells to cancer therapeutics. In other tumor types, chemosensitivity can be enhanced by blocking IGF signaling (24, 25). Work from our laboratory has shown that IGF1R depletion enhances the chemosensitivity and radiosensitivity of prostate cancer and melanoma (14, 17, 26). However, these tumors are routinely treated with chemotherapy, whereas RCC is intrinsically radioresistant and chemoresistant, and cytotoxic drugs currently play no part in routine treatment (27). The sole chemotherapeutic agent with reported activity in RCC is 5-FU, although recent data suggest that the addition of 5-FU to IFNα and interleukin 2 confers no benefit over IFNα alone (28).

Initial assays investigated the effects of IGF1R depletion on sensitivity to 5-FU in 786-0/EV cells that express higher levels of IGF1R than isogenic 786-0/VHL cells (Fig. 1A). Clonogenic assays showed evidence of dose-dependent killing of 5-FU–treated 786-0/EV cells, with greater killing of IGF1R-siRNA–transfected cells than control transfectants at each drug concentration tested (Fig. 2A). Clonogenic and MTS assays gave similar results, with mean IC50 values in control and IGF1R siRNA transfectants of 26 ± 3 and 13 ± 2 μmol/L by clonogenic assay (2-fold sensitization; P < 0.01 by paired t test), and 23 ± 6 and 9 ± 3 μmol/L by MTS assay (2.5-fold sensitization; P < 0.05). The minor reduction in 5-FU sensitization measured by the clonogenic assay may relate to the fact that clonogenic survival was measured over 10 days, beyond the duration of IGF1R depletion (Fig. 2B), whereas MTS assays were completed within 5 days. Next, we tested the effects of IGF1R depletion on sensitivity to two additional cytotoxic drugs, etoposide and cisplatin, which have no clinical activity in RCC. The 786-0/EV cells were highly resistant to the topoisomerase II inhibitor etoposide, with IC50 values in excess of 50 μmol/L, decreasing to 21 ± 4 μmol/L following transfection with R4 IGF1R siRNA (Fig. 2C). There was little change in sensitivity to cisplatin following IGF1R depletion, with reduction in IC50 values by a factor of 1.2 in clonogenic assay and by a factor of 1.7 in MTS assay, effects that were statistically significant (P < 0.05) but modest. Thus, we found evidence that IGF1R depletion induced significant sensitization to 5-FU and etoposide but only a minor reduction in IC50 for cisplatin. This is a different pattern from that observed previously in prostate cancer, in which we found sensitization to DNA-damaging cytotoxic drugs but not to 5-FU or paclitaxel (14). The reasons for this difference are unclear, but could include differences in the spectrum of intrinsic chemosensitivity in these tumor types, and the observation that the IGF axis can influence the repair of DNA damage in some cells (26, 29).

Figure 2.

RCC chemosensitivity is enhanced by IGF1R depletion. A, following transfection with NSC or IGF1R R4 siRNA, 786-0/EV cells were re-seeded into 10 cm dishes for clonogenic assay (left) or 96-well plates for MTS assay (right) and treated with solvent or 5-FU. MTS assays were done after 3 d, and colonies were fixed, stained, and counted for clonogenic assay after 10 to 14 d. Results were expressed as a percentage of survival (for clonogenic assay) and %OD490 (for MTS assay) in siRNA-transfected cells that had not been treated with 5-FU. The graphs show representative assays: points, mean of triplicate dishes or wells; bars, SE. The data were fitted to curves (unbroken line, NSC; dotted line, R4) from which IC50 values were calculated. Two additional independent assays yielded comparable results, revealing mean IC50 values measured for NSC and R4-transfected cultures of 23 ± 6 and 9 ± 3 μmol/L, respectively, by MTS assay (P < 0.05), and 26 ± 3 and 13 ± 2 μmol/L by clonogenic assay (P < 0.01). B, to assess the duration of IGF1R depletion, 786-0/EV cells were transfected with R4 IGF1R siRNA, parallel cultures were lysed each day, and analyzed by immunoblotting for levels of IGF1R and phosphorylated S473 Akt. IGF1R levels were suppressed for ∼6 d, and phosphorylated Akt for 4 to 5 d, after siRNA transfection. C, 786-0/EV cells were transfected and re-seeded for MTS assay as in A, and were treated with solvent or etoposide. Points, mean of triplicate points in a representative assay; bars, SE. The data were fitted to curves (unbroken line, NSC; dotted line, R4) from which IC50 values were calculated. Triplicate MTS assays revealed IC50 values of >200 μmol/L in control transfectants, decreasing to 21 ± 4 μmol/L following transfection with R4 IGF1R siRNA. D, cells were siRNA-transfected and re-seeded for MTS and clonogenic assays as in A, and were treated with solvent or cisplatin. Points, mean of triplicate data points; bars, SE. The graphs show representative assays (unbroken line, NSC; dotted line, R4). Mean IC50 values in triplicate clonogenic assays were 2.6 ± 0.2 and 2.2 ± 0.1 μmol/L for NSC and R4-transfected cultures, respectively (1.2-fold sensitization; P < 0.05). Equivalent values derived from MTS assays were 4.0 ± 0.5 and 2.3 ± 0.1 μmol/L (1.7-fold sensitization; P < 0.05).

Figure 2.

RCC chemosensitivity is enhanced by IGF1R depletion. A, following transfection with NSC or IGF1R R4 siRNA, 786-0/EV cells were re-seeded into 10 cm dishes for clonogenic assay (left) or 96-well plates for MTS assay (right) and treated with solvent or 5-FU. MTS assays were done after 3 d, and colonies were fixed, stained, and counted for clonogenic assay after 10 to 14 d. Results were expressed as a percentage of survival (for clonogenic assay) and %OD490 (for MTS assay) in siRNA-transfected cells that had not been treated with 5-FU. The graphs show representative assays: points, mean of triplicate dishes or wells; bars, SE. The data were fitted to curves (unbroken line, NSC; dotted line, R4) from which IC50 values were calculated. Two additional independent assays yielded comparable results, revealing mean IC50 values measured for NSC and R4-transfected cultures of 23 ± 6 and 9 ± 3 μmol/L, respectively, by MTS assay (P < 0.05), and 26 ± 3 and 13 ± 2 μmol/L by clonogenic assay (P < 0.01). B, to assess the duration of IGF1R depletion, 786-0/EV cells were transfected with R4 IGF1R siRNA, parallel cultures were lysed each day, and analyzed by immunoblotting for levels of IGF1R and phosphorylated S473 Akt. IGF1R levels were suppressed for ∼6 d, and phosphorylated Akt for 4 to 5 d, after siRNA transfection. C, 786-0/EV cells were transfected and re-seeded for MTS assay as in A, and were treated with solvent or etoposide. Points, mean of triplicate points in a representative assay; bars, SE. The data were fitted to curves (unbroken line, NSC; dotted line, R4) from which IC50 values were calculated. Triplicate MTS assays revealed IC50 values of >200 μmol/L in control transfectants, decreasing to 21 ± 4 μmol/L following transfection with R4 IGF1R siRNA. D, cells were siRNA-transfected and re-seeded for MTS and clonogenic assays as in A, and were treated with solvent or cisplatin. Points, mean of triplicate data points; bars, SE. The graphs show representative assays (unbroken line, NSC; dotted line, R4). Mean IC50 values in triplicate clonogenic assays were 2.6 ± 0.2 and 2.2 ± 0.1 μmol/L for NSC and R4-transfected cultures, respectively (1.2-fold sensitization; P < 0.05). Equivalent values derived from MTS assays were 4.0 ± 0.5 and 2.3 ± 0.1 μmol/L (1.7-fold sensitization; P < 0.05).

Close modal

Effect of IGF1R Depletion on Chemosensitivity is Greater in CC-RCC Cells Expressing Mutant Inactive VHL than Isogenic Cells Expressing Functional VHL

As a further test of the hypothesis that IGF1R overexpression contributes to CC-RCC chemoresistance, effects of IGF1R depletion were compared in 786-0/EV and 786-0/VHL cells that express relatively high and low IGF1R levels, respectively. These experiments used 5-FU and etoposide because these were the agents to which IGF1R depletion had sensitized 786-0/EV cells (Fig. 2). It was clear that control-transfected 786-0/EV cells were more resistant to 5-FU than 786-0/VHL (Fig. 3A; IC50 34 ± 2 versus 8 ± 1 μmol/L; P < 0.001), consistent with the known effects of VHL loss (4). IGF1R depletion induced 3.4-fold sensitization in 786-0/EV cells (IC50 values of 34 ± 2 in NSC transfectants, 10 ± 2 μmol/L in IGF1R siRNA transfectants; P < 0.001), whereas isogenic 786-0/VHL cells showed no significant change in IC50 upon IGF1R depletion (8 ± 1 versus 5 ± 1 μmol/L, not significant. Next, we evaluated sensitivity to etoposide (Fig. 3B), using higher concentrations of etoposide than had been used previously (Fig. 2C), in an attempt to identify the IC50 value in control transfectants. However, 786-0/EV cells showed striking resistance to this drug, with an IC50 of >200 μmol/L, and as in assays of 5-FU sensitivity (Fig. 3A), the isogenic 786-0/VHL cells were more sensitive (IC50 34 ± 14 μmol/L). Etoposide sensitivity was significantly enhanced following IGF1R depletion, with IC50 values of 31 ± 11 and 19 ± 3 μmol/L in cells transfected with the R1 and R4 siRNAs, respectively, representing at least 6.5-fold and 10.5-fold sensitization. IGF1R depletion also sensitized 786-0/VHL cells to etoposide, with IC50 values in control transfectants of 34 μmol/L, decreasing to 9 μmol/L (P < 0.05) and 5 μmol/L (P < 0.05) using R1 and R4 siRNAs. This represents 3.8-fold and 6.8-fold sensitization, respectively, in 786-0/VHL cells, a significant effect, but of lesser magnitude than that induced in the 786-0/EV cells that lack functional VHL (Fig. 3B). In the clinical setting, maximal plasma concentrations of 20 to 60 μmol/L of etoposide can be attained after short infusions of 50 to 175 mg/m2 etoposide, whereas constant infusional schedules achieve steady state levels of ∼5 μmol/L (30, 31). Thus, following IGF1R depletion, IC50 values for etoposide fell into the range that can be achieved clinically.

Figure 3.

CC-RCC cells expressing mutant VHL show greater IGF1R dependence than isogenic cells expressing functional VHL. A, 786-0/EV (top) and 786-0/VHL (bottom) cells were transfected with NSC (unbroken line) or R4 IGF1R siRNA (dotted line) and treated with 5-FU as described in Fig. 2A. The graphs show the results of individual MTS assays, which were repeated thrice, each with triplicate data points. Mean ± SE IC50 values are shown in the table below the graphs. There was significant reduction in IC50 values upon IGF1R depletion of 786-0/EV cells (3.4-fold sensitization; P < 0.001), but no significant change in 786-0/VHL cells (1.6-fold reduction in IGF1R siRNA transfectants, not significant). B, RCC cells were transfected with NSC, R1, or R4 siRNAs and sensitivity to etoposide was measured by MTS assay. Representative graphs are shown (unbroken line, NSC; dashed line, R1; dotted line, R4) and the table below summarizes mean ± SE IC50 values from three independent assays. In 786-0/EV cells, etoposide sensitivity was enhanced at least 6-fold to 10-fold following IGF1R depletion; this was not amenable to statistical analysis because the IC50 value in control-transfected 786-0/EV cells could not be determined. In 786-0/VHL cells, transfection with R1 and R4 siRNAs resulted in 3.8-fold and 6.8-fold sensitization, respectively (*, P < 0.05 for each comparison with NSC transfectants).

Figure 3.

CC-RCC cells expressing mutant VHL show greater IGF1R dependence than isogenic cells expressing functional VHL. A, 786-0/EV (top) and 786-0/VHL (bottom) cells were transfected with NSC (unbroken line) or R4 IGF1R siRNA (dotted line) and treated with 5-FU as described in Fig. 2A. The graphs show the results of individual MTS assays, which were repeated thrice, each with triplicate data points. Mean ± SE IC50 values are shown in the table below the graphs. There was significant reduction in IC50 values upon IGF1R depletion of 786-0/EV cells (3.4-fold sensitization; P < 0.001), but no significant change in 786-0/VHL cells (1.6-fold reduction in IGF1R siRNA transfectants, not significant). B, RCC cells were transfected with NSC, R1, or R4 siRNAs and sensitivity to etoposide was measured by MTS assay. Representative graphs are shown (unbroken line, NSC; dashed line, R1; dotted line, R4) and the table below summarizes mean ± SE IC50 values from three independent assays. In 786-0/EV cells, etoposide sensitivity was enhanced at least 6-fold to 10-fold following IGF1R depletion; this was not amenable to statistical analysis because the IC50 value in control-transfected 786-0/EV cells could not be determined. In 786-0/VHL cells, transfection with R1 and R4 siRNAs resulted in 3.8-fold and 6.8-fold sensitization, respectively (*, P < 0.05 for each comparison with NSC transfectants).

Close modal

These findings indicate that CC-RCC cells harboring mutant VHL and relatively high IGF1R are more chemoresistant than cells expressing wild-type VHL, and can be significantly chemosensitized by IGF1R depletion. This supports the hypothesis that IGF1R up-regulation makes a major contribution to the chemoresistance associated with loss of functional VHL. There are several factors that could contribute to this effect. HIF1-α is known to mediate resistance to cytotoxic drugs, at least in part due to a reduction in drug-induced senescence (32, 33). However, the effects of IGF1R depletion documented here must presumably have been HIF-independent, because 786-0 cells lack HIF1-α, and express only HIF2-α, which is not IGF-regulated in these cells (Fig. 1D). Alternatively, the relatively high IGF1R level in 786-0/EV cells could itself render this cell line more sensitive to IGF1R targeting, given that high IGF1R expression has been linked to sensitivity to IGF1R blockade in other tumor models (34, 35).

Rapamycin-Induced AKT Activation is Abrogated by IGF1R Depletion

We next wished to investigate whether there was any interaction between IGF1R blockade and one of the new generation of kinase inhibitors recently developed for the treatment of RCC (36). Activation of mTOR is a frequent event in CC-RCC (37). Rapamycin analogues have been shown to have objective activity in poor prognosis RCC, inducing significant delay in time to disease progression, although with a low incidence of objective regressions (38, 39). The IGF1R is a potent activator of mTOR via recruitment of IRS-1 and activation of PI3K and phosphoinositide–dependent kinase-1, which phosphorylates Akt on threonine 308 (40). The mTOR kinase exists in two multiprotein complexes, mTOR complexes 1 and 2, in which mTOR is complexed with raptor and rictor, respectively (41). The mTOR complex 2 complex induces S473 phosphorylation and activation of Akt, leading in turn to mTOR complex 1 activation to promote the translation of proteins mediating cellular growth and survival. This is achieved by mTOR complex 1–induced phosphorylation and inactivation of the translational inhibitor 4E-BP1, and by activation of p70 S6 kinase (41). S6 kinase phosphorylates translational targets including the ribosomal S6 peptide (S6), and also serine phosphorylates IRS-1, causing IRS-1 to dissociate from the IGF1R and to undergo proteasomal degradation (42, 43). These molecular events have consequences for the clinical use of mTOR inhibitors, because loss of this negative feedback loop may amplify receptor tyrosine kinase signaling to Akt, potentially mediating resistance to mTOR inhibition.

The effect of rapamycin on mTOR activity was monitored in 786-0/EV cells by measuring phosphorylation of mTOR effectors, revealing the inhibition of S6 kinase and its substrate S6 peptide. This effect was associated with dose-dependent inhibition of IRS-1 serine phosphorylation and increase in S473 Akt phosphorylation (Fig. 4A), suggesting that rapamycin had released mTOR-induced feedback inhibition on IGF1R signaling. Similar enhancement of Akt phosphorylation has been observed following rapamycin treatment in myeloblasts, myeloma, prostate and breast cancer cells in vitro, and in RCC biopsies following clinical mTOR inhibitor treatment (4446), suggesting that this phenomenon is clinically relevant.

Figure 4.

IGF1R depletion suppresses rapamycin-induced Akt activation and sensitizes RCC cells to rapamycin in vitro. A, subconfluent 786-0/EV cultures were treated with rapamycin or solvent for 6 h before lysis and immunoblotting for phosphorylated IRS-1, AKT, p70 S6 kinase, and S6. Similar results were obtained in two sets of independently prepared lysates. B, the 786-0/EV cells were transfected with NSC or IGF1R R4 siRNA. After 48 h, cultures were treated with rapamycin or solvent for 6 h before lysis and immunoblotting for IGF1Rβ and phosphorylated signaling intermediates as in A. Bottom, graph shows the analysis of three sets of independently prepared lysates, quantified by densitometry, in which Akt phosphorylation was corrected for β-actin loading and expressed relative to solvent-treated controls. Points, mean; bars, SE; continuous line, NSC; dotted line, R4 (*, P < 0.05; **, P < 0.01). C, forty-eight hours after transfection with control or IGF1R R4 siRNA, 786-0/EV cells were re-seeded for clonogenic assay and treated with rapamycin. The number of surviving colonies in triplicate dishes were expressed as a percentage of survival in solvent-treated dishes. Points, mean of three replicates from an individual experiment; bars, SE. The data were curve-fitted (continuous line, NSC; dotted line, R4) and IC50 values were calculated. Similar results were obtained in two additional assays, and mean IC50 values in NSC and IGF1R siRNA–transfected cultures were 340 ± 38 and 92 ± 26 nmol/L, respectively (P < 0.05).

Figure 4.

IGF1R depletion suppresses rapamycin-induced Akt activation and sensitizes RCC cells to rapamycin in vitro. A, subconfluent 786-0/EV cultures were treated with rapamycin or solvent for 6 h before lysis and immunoblotting for phosphorylated IRS-1, AKT, p70 S6 kinase, and S6. Similar results were obtained in two sets of independently prepared lysates. B, the 786-0/EV cells were transfected with NSC or IGF1R R4 siRNA. After 48 h, cultures were treated with rapamycin or solvent for 6 h before lysis and immunoblotting for IGF1Rβ and phosphorylated signaling intermediates as in A. Bottom, graph shows the analysis of three sets of independently prepared lysates, quantified by densitometry, in which Akt phosphorylation was corrected for β-actin loading and expressed relative to solvent-treated controls. Points, mean; bars, SE; continuous line, NSC; dotted line, R4 (*, P < 0.05; **, P < 0.01). C, forty-eight hours after transfection with control or IGF1R R4 siRNA, 786-0/EV cells were re-seeded for clonogenic assay and treated with rapamycin. The number of surviving colonies in triplicate dishes were expressed as a percentage of survival in solvent-treated dishes. Points, mean of three replicates from an individual experiment; bars, SE. The data were curve-fitted (continuous line, NSC; dotted line, R4) and IC50 values were calculated. Similar results were obtained in two additional assays, and mean IC50 values in NSC and IGF1R siRNA–transfected cultures were 340 ± 38 and 92 ± 26 nmol/L, respectively (P < 0.05).

Close modal

To assess whether IGF1R knockdown could influence signaling induced by rapamycin, control and IGF1R siRNA–transfected 786-0/EV cells were treated with rapamycin, and analyzed by immunoblotting for Akt phosphorylation. Control transfectants showed an increase in Akt phosphorylation in response to rapamycin, compatible with the effect in untransfected cells (Fig. 4A), and this effect was suppressed by IGF1R gene silencing (Fig. 4B). Clonogenic assays were used to assess the sensitivity of 786-0/EV cells to rapamycin, and it was noted that higher concentrations of this drug were required to inhibit cell survival compared with the low nanomolar levels shown to block signaling. Cell killing by rapamycin was enhanced following IGF1R depletion (Fig. 4C): mean IC50 values for control-transfected and IGF1R siRNA–transfected cells were 340 ± 38 and 92 ± 26 nmol/L, respectively (P < 0.05), representing 3.7-fold sensitization. These findings indicate that IGF1R depletion prevented rapamycin-induced Akt activation, and sensitized CC-RCC cells to mTOR inhibition in vitro.

Administration of Stabilized IGF1R siRNA Sensitizes RCC Xenografts to Rapamycin In vivo

Aiming to assess the effects of IGF1R depletion on the growth of CC-RCC xenografts, we generated modified siRNAs for in vivo delivery. It has been shown that siRNAs can accommodate stabilizing modifications at both base paired and non–base paired positions without significant effects on activity (47, 48). siRNA molecules with one or both strands consisting entirely of 2′-O-Me–modified residues were reported to show enhanced stability but were unable to induce gene silencing, whereas siRNAs incorporating alternate 2′-O-Me modification retained RNA interference activity and stability to nuclease digestion (47). Therefore, we adopted the strategy of replacement of alternate 2′-hydroxy groups of the ribose sugar ring with 2′-O-Me. We synthesized two variants of the R4 IGF1R siRNA, in which the 2′-O-Me groups were placed either in opposing (R4-1) or alternating (R4-2) positions on the sense and antisense strands of the siRNA (Fig. 5A). In order to assess serum stability, siRNAs were incubated in 100% FCS at 37°C, and were analyzed by agarose gel electrophoresis. As shown in Fig. 5B, the half-life of 2′-O-Me–modified IGF1R siRNA was ∼24 hours, compared with ∼4 hours for unmodified siRNA. This indicated that 2′-O-Me modification of IGF1R siRNA had significantly increased stability to serum nucleases, consistent with published data (47). It has been reported that modifications that enhance stability could impair RNA interference activity in vitro (4749). Indeed, there was detectable reduction in efficacy of the modified siRNAs, most notably in the R4-1 duplex (Fig. 5A). The alternating pattern of modification in the R4-2 duplex seemed to be relatively well-tolerated; this sequence retained the ability to silence the IGF1R, and to inhibit clonogenic survival in vitro, comparable with the effect of the unmodified siRNA (Fig. 5C and D). This suggests that 2′-O-Me modification of IGF1R siRNA at specific nucleotides did not compromise the ability of this duplex to induce RNA interference, and therefore, this siRNA was used in subsequent experiments.

Figure 5.

IGF1R gene silencing and inhibition of clonogenic survival by 2′-O-Me–modified IGF1R siRNA. A, the sequences of NSC, R4-1, and R4-2 siRNAs, with 2′-O-Me–modified bases underlined. B, to measure serum stability, siRNAs were incubated with 100% FCS at 37°C for the indicated times, and analyzed by agarose gel electrophoresis. Images from three independently prepared sets of samples were quantified, and the results expressed as siRNA remaining as a percentage of the 0 h time point. Points, mean; bars, SE; continuous line, unmodified siRNA; dotted line, 2′-O-Me–modified siRNA. C, the 786-0 cells were transfected with 2′-O-Me–modified NSC, unmodified R4 IGF1R siRNA, or 2′-O-Me–modified R4-1 or R4-2 IGF1R siRNAs. Cells were lysed 48 h later to assess IGF1R levels by immunoblotting. M, mock-transfected; UT, untransfected. D, forty-eight hours after transfection with 100 nmol/L of unmodified or 2′-O-Me–modified control (C) or R4 or R4-2 IGF1R siRNA, cells were re-seeded for clonogenic assay, and remaining cells were lysed for immunoblotting. Left, IGF1R quantification from three sets of independent lysates (individual immunoblot, inset), corrected for loading and expressed as a percentage of IGF1R (columns, mean; bars, SE) in NSC siRNA transfectants. Black column, NSC siRNA; open column, IGF1R siRNA R4 or R4-2. For both unmodified and modified siRNAs, mean IGF1R levels were lower in cells treated with IGF1R siRNA (*, P < 0.001). The effect was slightly greater in cells transfected with unmodified R4 siRNA, compared with 2′-O-Me–modified R4-2, but this difference was not significant. Right, results of clonogenic assay. Columns, mean colony counts from two independent experiments, each in triplicate (six data points); bars, SE. Both modified and unmodified IGF1R siRNAs inhibited clonogenic survival compared with NSC siRNA (*, P < 0.001), with no significant difference between results for unmodified (R4) and 2′-O-Me–modified (R4-2) IGF1R siRNAs by ANOVA.

Figure 5.

IGF1R gene silencing and inhibition of clonogenic survival by 2′-O-Me–modified IGF1R siRNA. A, the sequences of NSC, R4-1, and R4-2 siRNAs, with 2′-O-Me–modified bases underlined. B, to measure serum stability, siRNAs were incubated with 100% FCS at 37°C for the indicated times, and analyzed by agarose gel electrophoresis. Images from three independently prepared sets of samples were quantified, and the results expressed as siRNA remaining as a percentage of the 0 h time point. Points, mean; bars, SE; continuous line, unmodified siRNA; dotted line, 2′-O-Me–modified siRNA. C, the 786-0 cells were transfected with 2′-O-Me–modified NSC, unmodified R4 IGF1R siRNA, or 2′-O-Me–modified R4-1 or R4-2 IGF1R siRNAs. Cells were lysed 48 h later to assess IGF1R levels by immunoblotting. M, mock-transfected; UT, untransfected. D, forty-eight hours after transfection with 100 nmol/L of unmodified or 2′-O-Me–modified control (C) or R4 or R4-2 IGF1R siRNA, cells were re-seeded for clonogenic assay, and remaining cells were lysed for immunoblotting. Left, IGF1R quantification from three sets of independent lysates (individual immunoblot, inset), corrected for loading and expressed as a percentage of IGF1R (columns, mean; bars, SE) in NSC siRNA transfectants. Black column, NSC siRNA; open column, IGF1R siRNA R4 or R4-2. For both unmodified and modified siRNAs, mean IGF1R levels were lower in cells treated with IGF1R siRNA (*, P < 0.001). The effect was slightly greater in cells transfected with unmodified R4 siRNA, compared with 2′-O-Me–modified R4-2, but this difference was not significant. Right, results of clonogenic assay. Columns, mean colony counts from two independent experiments, each in triplicate (six data points); bars, SE. Both modified and unmodified IGF1R siRNAs inhibited clonogenic survival compared with NSC siRNA (*, P < 0.001), with no significant difference between results for unmodified (R4) and 2′-O-Me–modified (R4-2) IGF1R siRNAs by ANOVA.

Close modal

To assess the ability of 2′-O-Me–modified and unmodified IGF1R siRNAs to effect in vivo IGF1R gene silencing, 786-0/EV cells were grown as xenografts in immunodeficient mice. When tumors were 6 to 8 mm in diameter, animals were treated with a single i.p. injection of 50 μg unmodified or 2′-O-Me–stabilized control or IGF1R siRNAs, in complex with siPORT Amine delivery agent. After 48 hours, the mice were sacrificed and tumor IGF1R was analyzed by immunoblotting. There was no reduction in IGF1R levels in xenografts from animals treated with unmodified IGF1R siRNA (Fig. 6A). In these small groups of animals, there was some variation in IGF1R levels in xenografts of mice treated with 2′-O-Me–stabilized siRNAs; the mean IGF1R level in animals treated with 2′-O-Me–stabilized R4-2 IGF1R siRNA was ∼55% of levels in control-treated xenografts (Fig. 6A). Therefore, we proceeded to evaluate the effects of repeated administration of siRNA, alone and in combination with mTOR inhibition.

Figure 6.

Administration of stabilized IGF1R siRNA sensitizes RCC tumors to rapamycin. A, mice bearing 786-0/EV xenografts were treated with a single i.p. injection of 50 μg unmodified or 2′-O-Me–modified siRNA. After 48 h, the mice were sacrificed, and tumors were snap-frozen, lysed, and IGF1R levels were analyzed by immunoblotting. IGF1R levels in tumor lysates were corrected for loading and expressed as a percentage of IGF1R in control siRNA–treated tumors. Columns, mean IGF1R values; bars, SE; black columns, NSC; white columns, IGF1R siRNA (unmodified R4 or 2′-O-Me–modified R4-2). There was no difference between IGF1R levels in animals treated with unmodified NSC or IGF1R siRNAs. There was evidence of a reduction in IGF1R levels following the injection of 2′-O-Me–modified IGF1R siRNA (*, P = 0.059). B, groups of five tumor-bearing mice were treated with PBS (continuous line) or with 50 μg 2′-O-Me NSC (dashed line) or IGF1R siRNA R4-2 (dotted line) by i.p. injection for 5 days a week over 2 weeks. In the second week, all siRNA-treated animals also received daily rapamycin. There was evidence of modest tumor growth delay following administration of IGF1R siRNA, and a more significant effect in combination with rapamycin (*, P < 0.05; **, P < 0.01). C, one day after completion of treatment in B, animals were sacrificed and tumors were lysed for immunoblotting. D, Bar chart shows tumor volumes at end of treatment, and quantification of immunoblots for IGF1R, phospho-Akt and phospho-S6, corrected for levels of β-tubulin, total Akt and total S6 respectively. Columns, mean values; bars, SE, expressed as % of PBS-treated controls. Tumor volumes and IGF1R levels were similar in NSC siRNA-treated and PBS-treated animals, but were significantly lower in xenografts from animals treated with IGF1R siRNA. Compared with PBS-treated controls, tumors from animals treated with rapamycin and non-silencing siRNA had higher levels of phospho-Akt (not significant, probably due to small sample size and variation between tumors). Levels of phospho-Akt were significantly lower in tumors treated with rapamycin and IGF1R siRNA, compare with those that received rapamycin and control siRNA. S6 phosphorylation was significantly suppressed in all rapamycin-treated animals. * P < 0.05); ** P < 0.01; *** P < 0.001.

Figure 6.

Administration of stabilized IGF1R siRNA sensitizes RCC tumors to rapamycin. A, mice bearing 786-0/EV xenografts were treated with a single i.p. injection of 50 μg unmodified or 2′-O-Me–modified siRNA. After 48 h, the mice were sacrificed, and tumors were snap-frozen, lysed, and IGF1R levels were analyzed by immunoblotting. IGF1R levels in tumor lysates were corrected for loading and expressed as a percentage of IGF1R in control siRNA–treated tumors. Columns, mean IGF1R values; bars, SE; black columns, NSC; white columns, IGF1R siRNA (unmodified R4 or 2′-O-Me–modified R4-2). There was no difference between IGF1R levels in animals treated with unmodified NSC or IGF1R siRNAs. There was evidence of a reduction in IGF1R levels following the injection of 2′-O-Me–modified IGF1R siRNA (*, P = 0.059). B, groups of five tumor-bearing mice were treated with PBS (continuous line) or with 50 μg 2′-O-Me NSC (dashed line) or IGF1R siRNA R4-2 (dotted line) by i.p. injection for 5 days a week over 2 weeks. In the second week, all siRNA-treated animals also received daily rapamycin. There was evidence of modest tumor growth delay following administration of IGF1R siRNA, and a more significant effect in combination with rapamycin (*, P < 0.05; **, P < 0.01). C, one day after completion of treatment in B, animals were sacrificed and tumors were lysed for immunoblotting. D, Bar chart shows tumor volumes at end of treatment, and quantification of immunoblots for IGF1R, phospho-Akt and phospho-S6, corrected for levels of β-tubulin, total Akt and total S6 respectively. Columns, mean values; bars, SE, expressed as % of PBS-treated controls. Tumor volumes and IGF1R levels were similar in NSC siRNA-treated and PBS-treated animals, but were significantly lower in xenografts from animals treated with IGF1R siRNA. Compared with PBS-treated controls, tumors from animals treated with rapamycin and non-silencing siRNA had higher levels of phospho-Akt (not significant, probably due to small sample size and variation between tumors). Levels of phospho-Akt were significantly lower in tumors treated with rapamycin and IGF1R siRNA, compare with those that received rapamycin and control siRNA. S6 phosphorylation was significantly suppressed in all rapamycin-treated animals. * P < 0.05); ** P < 0.01; *** P < 0.001.

Close modal

Tumor-bearing mice were treated with intraperitoneal 2′-O-Me–modified control or R4-2 IGF1R siRNAs, or with PBS. The administration of siRNA with delivery agent had a minor (nonsignificant) inhibitory effect on tumor growth compared with PBS-treated controls (Fig. 6B). After 1 week, there was evidence of modest tumor growth delay in the group that received 2′-O-Me–stabilized IGF1R siRNA compared with the control siRNA–treated group (P < 0.05). In the second week, all siRNA-treated animals also received daily rapamycin. This intervention had no detectable effect on the growth of control siRNA–treated tumors, but induced the cessation of growth in the group treated with 2′-O-Me–modified IGF1R siRNA (P < 0.01; Fig. 6B). On the day following the final treatment, all animals were sacrificed, and tumors were analyzed for IGF1R levels and Akt-mTOR pathway activation. IGF1R levels in tumors from control siRNA–treated animals were not significantly different from those in PBS-treated mice. As after a single siRNA administration (Fig. 6A), there was considerable variation in IGF1R levels in tumor tissue from animals treated with 2′-O-Me–IGF1R siRNA, and mean IGF1R levels were 52 ± 10% of levels in control siRNA–treated animals (P < 0.05; Fig. 6C and D). This provides some evidence of in vivo gene silencing, but effects were variable and probably limited by delivery, which is a major issue for the in vivo use of siRNA-based therapeutics (50). There was evidence that rapamycin had blocked mTOR activity, with reduction in phosphorylation of S6 in the tumor tissue of all rapamycin-treated animals (Fig. 6C and D). Rapamycin induced higher levels of Akt phosphorylation in the NSC siRNA–treated animals than were detected in PBS-treated controls, consistent with rapamycin-induced loss of the negative feedback that limits PI3K-Akt activation. However, levels of phosphorylated Akt were significantly lower in rapamycin-treated animals that received IGF1R-siRNA compared with NSC-treated animals (P < 0.05). This suggests that IGF1R depletion sensitized to rapamycin in vivo, and suppressed rapamycin-induced Akt activation, consistent with the in vitro data (Fig. 4). These findings support the evaluation of novel IGF1R-inhibitory drugs in combination with rapamycin analogues in patients with CC-RCC.

In summary, human CC-RCC cells that lack functional VHL showed IGF1R up-regulation, and the survival of these cells seemed more heavily dependent on the IGF1R than were isogenic cells expressing functional VHL. IGF1R depletion enhanced the sensitivity of CC-RCC cells to 5-FU and etoposide, with significantly greater sensitization in cells expressing mutant inactive VHL. Finally, IGF1R gene silencing sensitized RCC cells to rapamycin in vitro. The relatively modest degree of IGF1R depletion achieved in vivo had only a minor effect on tumor growth. However, the combination of IGF1R depletion with rapamycin was capable of arresting the growth of CC-RCC xenografts, at least in part by suppressing the rapamycin-induced Akt activation that followed loss of the negative feedback loop operating via IRS-1. The results of this study validate IGF1R as a therapeutic target in CC-RCC, and highlight the importance of selecting treatment combinations with reference to the biological properties of the target.

V. Macaulay: Pfizer and Roche expert panel; trials collaboration, Sanofi-Aventis, OSI-Pharmaceuticals. No other potential conflicts of interest were disclosed.

The authors are grateful to Sarah Scott, Sarah Groom, and Samantha Johnson for technical assistance; and to Ian Hickson, Bass Hassan, and Denis Talbot for comments on the manuscript.

1
Chitnis
MM
,
Yuen
JS
,
Protheroe
AS
,
Pollak
M
,
Macaulay
VM
. 
The type 1 insulin-like growth factor receptor pathway
.
Clin Cancer Res
2008
;
14
:
6364
70
.
2
Gnarra
JR
,
Tory
K
,
Weng
Y
, et al
. 
Mutations of the VHL tumour suppressor gene in renal carcinoma
.
Nat Genet
1994
;
7
:
85
90
.
3
Banks
RE
,
Tirukonda
P
,
Taylor
C
, et al
. 
Genetic and epigenetic analysis of von Hippel-Lindau (VHL) gene alterations and relationship with clinical variables in sporadic renal cancer
.
Cancer Res
2006
;
66
:
2000
11
.
4
Kaelin
WG
 Jr
. 
The von Hippel-Lindau tumor suppressor protein and clear cell renal carcinoma
.
Clin Cancer Res
2007
;
13
:
680
4s
.
5
Yuen
JS
,
Cockman
ME
,
Sullivan
M
, et al
. 
The VHL tumor suppressor inhibits expression of the IGF1R and its loss induces IGF1R upregulation in human clear cell renal carcinoma
.
Oncogene
2007
;
26
:
6499
508
.
6
Parker
A
,
Cheville
JC
,
Lohse
C
,
Cerhan
JR
,
Blute
ML
. 
Expression of insulin-like growth factor I receptor and survival in patients with clear cell renal cell carcinoma
.
J Urol
2003
;
170
:
420
4
.
7
Ahmad
N
,
Keehn
CA
,
Coppola
D
. 
The expression of insulin-like growth factor-I receptor correlates with Fuhrman grading of renal cell carcinomas
.
Hum Pathol
2004
;
35
:
1132
6
.
8
Vaira
V
,
Lee
CW
,
Goel
HL
,
Bosari
S
,
Languino
LR
,
Altieri
DC
. 
Regulation of survivin expression by IGF-1/mTOR signaling
.
Oncogene
2007
;
26
:
2678
84
.
9
Parker
AS
,
Kosari
F
,
Lohse
CM
, et al
. 
High expression levels of survivin protein independently predict a poor outcome for patients who undergo surgery for clear cell renal cell carcinoma
.
Cancer
2006
;
107
:
37
45
.
10
Ullrich
A
,
Gray
A
,
Tam
AW
, et al
. 
Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity
.
EMBO J
1986
;
5
:
2503
12
.
11
Sachdev
D
,
Singh
R
,
Fujita-Yamaguchi
Y
,
Yee
D
. 
Down-regulation of insulin receptor by antibodies against the type I insulin-like growth factor receptor: implications for anti-insulin-like growth factor therapy in breast cancer
.
Cancer Res
2006
;
66
:
2391
402
.
12
Haluska
P
,
Carboni
JM
,
Loegering
DA
, et al
. 
In vitro and in vivo antitumor effects of the dual insulin-like growth factor-I/insulin receptor inhibitor, BMS-554417
.
Cancer Res
2006
;
66
:
362
71
.
13
Bohula
EA
,
Salisbury
AJ
,
Sohail
M
, et al
. 
The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript
.
J Biol Chem
2003
;
278
:
15991
7
.
14
Rochester
MA
,
Riedemann
J
,
Hellawell
GO
,
Brewster
SF
,
Macaulay
VM
. 
Silencing of the IGF1R gene enhances sensitivity to DNA-damaging agents in both PTEN wild-type and mutant human prostate cancer
.
Cancer Gene Ther
2005
;
12
:
90
100
.
15
Maxwell
PH
,
Wiesener
MS
,
Chang
GW
, et al
. 
The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis
.
Nature
1999
;
399
:
271
5
.
16
Elbashir
SM
,
Harborth
J
,
Lendeckel
W
,
Yalcin
A
,
Weber
K
,
Tuschl
T
. 
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells
.
Nature
2001
;
411
:
494
8
.
17
Yeh
AH
,
Bohula
EA
,
Macaulay
VM
. 
Human melanoma cells expressing V600E B-RAF are susceptible to IGF1R targeting by small interfering RNAs
.
Oncogene
2006
;
25
:
6574
81
.
18
Huang
S
,
Bjornsti
MA
,
Houghton
PJ
. 
Rapamycins: mechanism of action and cellular resistance
.
Cancer Biol Ther
2003
;
2
:
222
32
.
19
Peretz
S
,
Kim
C
,
Rockwell
S
,
Baserga
R
,
Glazer
PM
. 
IGF1 receptor expression protects against microenvironmental stress found in the solid tumor
.
Radiat Res
2002
;
158
:
174
80
.
20
Fukuda
R
,
Hirota
K
,
Fan
F
,
Jung
YD
,
Ellis
LM
,
Semenza
GL
. 
Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells
.
J Biol Chem
2002
;
277
:
38205
11
.
21
Blancher
C
,
Moore
JW
,
Robertson
N
,
Harris
AL
. 
Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1α, HIF-2α, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3′-kinase/Akt signaling pathway
.
Cancer Res
2001
;
61
:
7349
55
.
22
Akeno
N
,
Robins
J
,
Zhang
M
,
Czyzyk-Krzeska
MF
,
Clemens
TL
. 
Induction of vascular endothelial growth factor by IGF-I in osteoblast-like cells is mediated by the PI3K signaling pathway through the hypoxia-inducible factor-2α
.
Endocrinology
2002
;
143
:
420
5
.
23
Catrina
SB
,
Botusan
IR
,
Rantanen
A
, et al
. 
Hypoxia-inducible factor-1α and hypoxia-inducible factor-2α are expressed in Kaposi sarcoma and modulated by insulin-like growth factor-I
.
Clin Cancer Res
2006
;
12
:
4506
14
.
24
Benini
S
,
Manara
MC
,
Baldini
N
, et al
. 
Inhibition of insulin-like growth factor I receptor increases the antitumor activity of doxorubicin and vincristine against Ewing's sarcoma cells
.
Clin Cancer Res
2001
;
7
:
1790
7
.
25
Warshamana-Greene
GS
,
Litz
J
,
Buchdunger
E
,
Garcia-Echeverria
C
,
Hofmann
F
,
Krystal
GW
. 
The insulin-like growth factor-I receptor kinase inhibitor, NVP-ADW742, sensitizes small cell lung cancer cell lines to the effects of chemotherapy
.
Clin Cancer Res
2005
;
11
:
1563
71
.
26
Macaulay
VM
,
Salisbury
AJ
,
Bohula
EA
,
Playford
MP
,
Smorodinsky
NI
,
Shiloh
Y
. 
Downregulation of the type 1 insulin-like growth factor receptor in mouse melanoma cells is associated with enhanced radiosensitivity and impaired activation of Atm kinase
.
Oncogene
2001
;
20
:
4029
40
.
27
Motzer
RJ
. 
Prognostic factors and clinical trials of new agents in patients with metastatic renal cell carcinoma
.
Crit Rev Oncol Hematol
2003
;
46 Suppl
:
S33
9
.
28
Gore
ME
. 
Interferon-α (IFN), interleukin-2 (IL2) and 5-fluorouracil (5FU) vs IFN alone in patients with metastatic renal cell carcinoma (mRCC): results of the randomised MRC/EORTC RE04 trial
.
J Clin Oncol
2008
;
26
:
abstr 5039
.
29
Trojanek
J
,
Ho
T
,
Del Valle
L
, et al
. 
Role of the insulin-like growth factor I/insulin receptor substrate 1 axis in Rad51 trafficking and DNA repair by homologous recombination
.
Mol Cell Biol
2003
;
23
:
7510
24
.
30
Minami
H
,
Shimokata
K
,
Saka
H
, et al
. 
Phase I clinical and pharmacokinetic study of a 14-day infusion of etoposide in patients with lung cancer
.
J Clin Oncol
1993
;
11
:
1602
8
.
31
Joel
S
. 
The clinical pharmacology of etoposide: an update
.
Cancer Treat Rev
1996
;
22
:
179
221
.
32
Brown
LM
,
Cowen
RL
,
Debray
C
, et al
. 
Reversing hypoxic cell chemoresistance in vitro using genetic and small molecule approaches targeting hypoxia inducible factor-1
.
Mol Pharmacol
2006
;
69
:
411
8
.
33
Sullivan
R
,
Pare
GC
,
Frederiksen
LJ
,
Semenza
GL
,
Graham
CH
. 
Hypoxia-induced resistance to anticancer drugs is associated with decreased senescence and requires hypoxia-inducible factor-1 activity
.
Mol Cancer Ther
2008
;
7
:
1961
73
.
34
Cao
L
,
Yu
Y
,
Darko
I
, et al
. 
Addiction to elevated insulin-like growth factor I receptor and initial modulation of the AKT pathway define the responsiveness of rhabdomyosarcoma to the targeting antibody
.
Cancer Res
2008
;
68
:
8039
48
.
35
Huang
F
,
Greer
A
,
Hurlburt
W
, et al
. 
The mechanisms of differential sensitivity to an insulin-like growth factor-1 receptor inhibitor (BMS-536924) and rationale for combining with EGFR/HER2 inhibitors
.
Cancer Res
2009
;
69
:
161
70
.
36
Heng
DY
,
Bukowski
RM
. 
Anti-angiogenic targets in the treatment of advanced renal cell carcinoma
.
Curr Cancer Drug Targets
2008
;
8
:
676
82
.
37
Robb
VA
,
Karbowniczek
M
,
Klein-Szanto
AJ
,
Henske
EP
. 
Activation of the mTOR signaling pathway in renal clear cell carcinoma
.
J Urol
2007
;
177
:
346
52
.
38
Hudes
G
,
Carducci
M
,
Tomczak
P
, et al
. 
Temsirolimus, interferon α, or both for advanced renal-cell carcinoma
.
N Engl J Med
2007
;
356
:
2271
81
.
39
Figlin
RA
. 
Mechanisms of Disease: survival benefit of temsirolimus validates a role for mTOR in the management of advanced RCC
.
Nat Clin Pract
2008
;
5
:
601
9
.
40
Manning
BD
,
Cantley
LC
. 
AKT/PKB signaling: navigating downstream
.
Cell
2007
;
129
:
1261
74
.
41
Abraham
RT
. 
Identification of TOR signaling complexes: more TORC for the cell growth engine
.
Cell
2002
;
111
:
9
12
.
42
Haruta
T
,
Uno
T
,
Kawahara
J
, et al
. 
A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1
.
Mol Endocrinol
2000
;
14
:
783
94
.
43
Takano
A
,
Usui
I
,
Haruta
T
, et al
. 
Mammalian target of rapamycin pathway regulates insulin signaling via subcellular redistribution of insulin receptor substrate 1 and integrates nutritional signals and metabolic signals of insulin
.
Mol Cell Biol
2001
;
21
:
5050
62
.
44
O'Reilly
KE
,
Rojo
F
,
She
QB
, et al
. 
mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt
.
Cancer Res
2006
;
66
:
1500
8
.
45
Shi
Y
,
Yan
H
,
Frost
P
,
Gera
J
,
Lichtenstein
A
. 
Mammalian target of rapamycin inhibitors activate the AKT kinase in multiple myeloma cells by up-regulating the insulin-like growth factor receptor/insulin receptor substrate-1/phosphatidylinositol 3-kinase cascade
.
Mol Cancer Ther
2005
;
4
:
1533
40
.
46
Wan
X
,
Harkavy
B
,
Shen
N
,
Grohar
P
,
Helman
LJ
. 
Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism
.
Oncogene
2007
;
26
:
1932
40
.
47
Czauderna
F
,
Fechtner
M
,
Dames
S
, et al
. 
Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells
.
Nucleic Acids Res
2003
;
31
:
2705
16
.
48
Amarzguioui
M
,
Holen
T
,
Babaie
E
,
Prydz
H
. 
Tolerance for mutations and chemical modifications in a siRNA
.
Nucleic Acids Res
2003
;
31
:
589
95
.
49
Soutschek
J
,
Akinc
A
,
Bramlage
B
, et al
. 
Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs
.
Nature
2004
;
432
:
173
8
.
50
Aigner
A
. 
Cellular delivery in vivo of siRNA-based therapeutics
.
Curr Pharm Des
2008
;
14
:
3603
19
.

Competing Interests

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.