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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Acquired resistance is a major obstacle for conventional cancer chemotherapy, and also for some of the targeted therapies approved to date. Long-term treatment using protein tyrosine kinase inhibitors (TKIs), such as gefitinib and imatinib, gives rise to resistant cancer cells carrying a drug-resistant gatekeeper mutation in the kinase domain of the respective target genes, EGFR and BCRABL. As for the phosphatidylinositol 3-kinase inhibitors (PI3Kis), little is known about their acquired resistance, although some are undergoing clinical trials. To address this issue, we exposed 11 human cancer cell lines to ZSTK474, a PI3Ki we developed previously, for a period of more than 1 year in vitro. Consequently, we established ZSTK474-resistant cells from four of the 11 cancer cell lines tested. The acquired resistance was not only to ZSTK474 but also to other PI3Kis. None of the PI3Ki-resistant cells, however, contained any mutation in the kinase domain of the PIK3CA gene. Instead, we found that insulin-like growth factor 1 receptor (IGF1R) was overexpressed in all four resistant cells. Interestingly, targeted knockdown of IGF1R expression using specific siRNAs or inhibition of IGF1R using IGF1R-TKIs reversed the acquired PI3Ki resistance. These results suggest that long-term treatment with PI3Kis may cause acquired resistance, and targeting IGF1R is a promising strategy to overcome the resistance.

Targeted cancer therapy is thought to be more effective and less harmful than conventional chemotherapy. Accordingly, much effort has been made to develop promising targets for cancer therapy, one of which is phosphatidylinositol 3-kinase (PI3K), a class of lipid kinase that phosphorylates phosphatidylinositol (PI).[1, 2] Among the PI3Ks, PIK3CA encodes p110α, the best-studied PI3K isoform.[3, 4] It mediates growth signal from the upstream receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF1R), to the downstream effectors including Akt and mTOR, which play fundamental roles in tumor proliferation.[5, 6] The PI3K pathway is often activated in cancer and its inhibition is thought to efficiently suppress tumor proliferation.[7] LY294002 and wortmannin are the first generation PI3K inhibitors (PI3Kis), but neither of them went to clinical trials, because of cytotoxicity to liver and skin.[8, 9] We previously reported on a selective PI3K inhibitor ZSTK474, which showed potent in vivo antitumor activity without any severe cytotoxicity in nude mice.[10, 11] Subsequently, numerous PI3Kis have been developed and some of them, including NVP-BEZ235 and ZSTK474, have already been tested in clinical trials.[7]

Acquired resistance is a major obstacle for conventional cancer chemotherapy and also for some of the approved targeted therapies. The most-studied mechanism of drug resistance is the increased drug efflux from cells due to the overexpression of P-glycoprotein.[12] However, there are other known mechanisms for drug resistance. For example, resistance against tyrosine kinase inhibitors (TKIs), including imatinib and gefitinib, commonly occurs as a result of an induced mutation at the “gatekeeper” site of the respective targeted kinases.[13, 14] Long-term treatment with gefitinib also caused drug resistance by a reversible, epigenetic mechanism.[15] Previously, Zunder et al.[16] have shown that a genetically engineered mutation of PIK3CA showed resistance to some PI3Kis. However, it remains unclear whether long-term exposure to PI3Kis would naturally generate drug-resistant cancer cells and whether the resistance was due to a mutation within the kinase domain of the respective PIK3CA genes or by some other mechanism.

To address this issue, we exposed 11 human cancer cell lines to the PI3Ki ZSTK474 in vitro for over 1 year. As a result, we obtained cells showing resistance to PI3Kis by >10-fold from four of the 11 cell lines tested, indicating that long-term use of PI3Kis would indeed lead to acquired resistance. This acquired resistance was not due to a mutation in the kinase domain of the relevant PIK3CA gene. Instead, we found that IGF1R was overexpresssed in these resistant cells. We also showed that overexpression of IGF1R conferred PI3Ki resistance in cancer cells.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Drugs

ZSTK474 was synthesized by the Research Laboratory of Zenyaku Kogyo (Tokyo, Japan). GDC-0941, NVP-BEZ235, OSI-906, NVP-AEW541, and cisplatin were obtained from Symansis (Auckland, New Zealand), Selleck (Houston, TX, USA), ChemieTek (Indianapolis, IN, USA), Cayman (Ann Arbor, MI, USA), and Nippon Kayaku (Tokyo, Japan), respectively. These compounds were dissolved in DMSO.

Cell lines and cell culture

The following cells from the JFCR39 panel of human cancer cell lines[11, 17-19] were used in this study: glioblastoma SF295, SNB75, and SNB78; ovarian cancer OVCAR3 and OVCAR4; gastric cancer MKN1; breast cancer BSY1, MCF7, and HBC5; lung cancer NCI-H226; and prostate cancer PC3. Cells were grown in RPMI-1640 (Wako Pure Chemical, Osaka, Japan) supplemented with 1 μg/mL kanamycin and 5% (v/v) FBS (Moregate Biotech, Bulimba, Qld, Australia) as described before.[11] ZSTK474-resistant cells were grown in medium containing ZSTK474 at a concentration of 10 μM (SF295-R) or 8 μM (OVCAR3-R, SNB75-R, and SNB78-R).

Establishment of ZSTK474-resistant cancer cell lines

To establish ZSTK474-resistant cell lines from 11 human cancer cell lines, we cultured the cells in medium containing ZSTK474 at a concentration of 1–10 μM for a period of more than 1 year. When passaging the cells, the medium was washed out with PBS and cells were trypsinized using 0.05% trypsin-EDTA (Invitrogen, CA, USA). Cells were then suspended in a ZSTK474-free medium and an aliquot of cell suspension was placed into a new dish and incubated overnight, to allow the cells to attach to the dish. Cells were then exposed to ZSTK474 until the next passage. The resultant cells were a mixture of cells proliferating in the presence of ZSTK474.

Determination of drug efficacy

Drug efficacy was assessed as changes in the total cellular protein after 48 h of drug treatment using a sulforhodamine B (SRB) assay. The drug concentration required for 50% reduction in the net protein increase (GI50) was calculated as described previously.[19, 20]

Nucleotide sequence analysis of PIK3CA cDNA

Total RNA was extracted using the RNeasy RNA purification kit (Qiagen, Valencia, CA, USA). cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The PIK3CA cDNA fragment was amplified by PCR. Sequencing reactions were carried out using the BigDye Terminator version 3.1 (Applied Biosystems). Sequences of PIK3CA cDNA from +1447 to +3364, covering amino acid region 430–1068 of the PI3Kα protein, which includes all helical and kinase domains, were analyzed using a 3130 Genetic Analyzer (Applied Biosystems). Primers used for amplifying the PIK3CA cDNA fragment and for sequencing were described previously.[11]

Assay for RNA interference

The siRNAs against IGF1R were purchased from Invitrogen (siIGF1R-1, IGF1R-HSS105253; siIGF1R-2, NM_000875.3_stealth_1603). Cells were plated on 6-well plates and transfected with 33.3 nmol/L siIGF1R-1, siIGF1R-2 or Stealth RNAi Negative Control Medium GC Duplex using Lipofectamine 2000 (1 μL). Next day, cells were replated on 96-well plates and allowed to attach overnight. Cells were then incubated with or without drugs for an additional 48 h, and cell growth was determined using the SRB assay. For immunoblot analysis, cells were replated on 6-cm dishes after transfection of siRNA and allowed to attach overnight. Cells were then incubated with or without ZSTK474 for 3 h.

Quantitative real-time RT-PCR

To quantify IGF1R mRNA expression, a TaqMan Gene Expression Assay (Applied Biosystems) was carried out using an IGF1R-specific probe (Hs00609566_m1) and an 18S ribosomal RNA specific probe (4319413E) combined with the ABI Prism 7000 system. The relative IGF1R expression for each sample was determined using the formula inline image, which reflected the IGF1R expression normalized to the 18S ribosomal RNA level.

Immunoblot analysis

Immunoblot assays were carried out on cell extracts as described previously[11] using a primary antibody for IGF1R-β, phosphorylated IGF1R-β at Tyr1135, phosphorylated Akt at Thr308 or Ser473, and phosphorylated ERK1/2 at Thr202/Tyr204 (Cell Signaling Technology, Danvers, MA, USA) as the probe. Quantification of the bound antibody was carried out using an anti-rabbit immunoglobulin secondary antibody labeled with Alexa Fluor 680 (Invitrogen) and the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

Isobologram analysis

Concentrations of IGF1R-TKIs and PI3Kis were plotted on the x and y coordinates of the isobologram, respectively. Three isoeffect curves modes (I, IIa, IIb) were created according to the method of Steel and Peckham.[21] The area enclosed by all three lines represents the “envelope of additivity.” Experimental data points falling to the left of the envelope signify synergy, and those falling to the right of the envelope signify a subadditive relationship.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Establishment of PI3Ki-resistant cell lines by long-term exposure to ZSTK474

To test whether long-term exposure to PI3Kis causes drug resistance, human cancer cell lines were exposed in vitro to ZSTK474 for a period of more than 1 year and their sensitivities to ZSTK474 were examined. Of 11 cell lines tested, four showed resistance to ZSTK474 by >10-fold (Fig. 1), and the resistance became stronger in a time-dependent manner. For example, SF295 cells became resistant to ZSTK474 by 3-fold in 5 months, by ninefold in 12 months, and by >30-fold in 26 months (Fig. 2A). Similarly, SNB75, SNB78, and OVCAR3 cells acquired resistance to ZSTK474 by 10–100-fold compared to their respective untreated control cells (Figs 1,S1). The resultant cells showed cross-resistance to PI3Kis NVP-BEZ235 and GDC-0941, but not to cisplatin (Fig. S2). The results indicate that long-term treatment with PI3Kis may cause therapeutic inefficacy.

image

Figure 1. Sensitivities to ZSTK474 in ZSTK474-resistant cell lines and their parental cell lines. Acquisition of resistance following long-term exposure to ZSTK474. SF295 (26 months), OVCAR3, SNB75, and SNB78 (12 months) were exposed to ZSTK474 for the period indicated in parentheses. Sensitivities to ZSTK474 in drug-treated cells and their parental cells were then examined. Assays were carried out in triplicate and the data are the average of three independent experiments. Error bars = SD.

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image

Figure 2. Changes in sensitivity to ZSTK474 after exposure to or removal of ZSTK474. (A) SF295 cells were treated with ZSTK474 (10 μM) for the indicated period. The GI50 values of drug-treated and untreated control cells were calculated by using dose response curves of ZSTK474 as described in 'Materials and Methods'. Relative ZSTK474 resistance was then calculated as GI50 sample/GI50 control. Drug-treated cells were a mixture of cells proliferating in the presence of ZSTK474. Assays were carried out in quadruplicate. (B) Loss of ZSTK474 resistance after drug withdrawal. SF295-R cells were cultured in the absence of ZSTK474 for the indicated period. Drug resistance was gradually weakened and finally cells restored sensitivity to ZSTK474 to a level comparable to that of the parental SF295 cells. The resistant cells after drug withdrawal for each period were a mixture of cells grown in the absence of ZSTK474. Sample designations SF295-R/df3, SF295-R/df14, and SF295-R/df100 represent SF295-R cells incubated in the absence of ZSTK474 for 3, 14, and >100 days, respectively. Assays were carried out in triplicate and the present data are representative of two independent experiments.

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We next examined whether the acquired resistance to PI3Kis was reversible. As shown, the acquired resistance gradually weakened by culturing the SF295-R cells in the absence of ZSTK474, and finally, after a 3-month culture in drug-free medium, sensitivity to ZSTK474 was restored to a level that was comparable to the parental SF295 cells (SF295-R/df100 in Figs 2B,S1). Similar results were also obtained with the other three cell lines (data not shown). From these results, we concluded that the acquired resistance observed here is a reversible event.

Acquired PI3Ki resistance is not due to a PIK3CA mutation

We next examined whether the acquired resistance was due to a mutation within the kinase domain of the PIK3CA gene. Sequence analysis of the full-length kinase domain of PIK3CA cDNA derived from the resistant cells (SF295-R, SNB75-R, SNB78-R, and OVCAR3-R) did not show any sequence changes compared to the respective control cells (data not shown). These results indicate that the observed drug resistance is not due to an acquired mutation in the PIK3CA gene.

Overexpression of IGF1R mRNA and protein in PI3Ki-resistant cell lines

To identify genes involved in acquired PI3Ki resistance, we analyzed the expression of upregulated genes in SF295-R cells using the Affymetrix (Santa Clara, CA, USA) GeneChip (Human Genome U133 Plus 2.0 Array; data not shown). Among these upregulated genes, we focused our attention on the IGF1R gene, whose expression would most likely be associated with the PI3Ki resistance.

To confirm overexpression of the IGF1R gene in the resistant cells, we next carried out real-time RT-PCR analysis. As expected, IGF1R mRNA was upregulated in SF295-R (4-fold), SF295-R/df3 (2.5-fold) and SF295-R/df14 (2.5-fold) cells, but not in SF295-R/df100 cells (Fig. 3A). Similar increases in IGF1R expression in SF295-R cells were also observed at the protein level and the expression finally went back to the basal level in SF295-R/df100 cells (Fig. 3B). IGF1R protein was also upregulated in other ZSTK474-resistant cells derived from SNB75, SNB78, and OVCAR3 cells (Fig. 3C). These results suggested that overexpression of IGF1R is commonly associated with the ZSTK474-resistant phenotype.

image

Figure 3. Overexpression of insulin-like growth factor 1 receptor (IGF1R) in ZSTK474-resistant cells. (A,B) Expression levels of IGF1R in SF295, SF295-R, SF295-R/df3, SF295-R/df14, and SF295-R/df100 cells. IGF1R mRNA levels were quantified by real-time quantitative PCR (A) and IGF1R protein levels were analyzed by immunoblot assay (B). Expression levels of IGF1R correlated well with resistance levels to ZSTK474. The present data are representative of two independent experiments. (C) Immunoblot analysis of IGF1R expression in three parental and their respective ZSTK474-resistant cells. Expression of β-actin was also determined to ensure equal loading of protein in each lane (control). The data are representative of two independent experiments.

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Insulin-like growth factor 1 receptor as a therapeutic target for acquired PI3Ki resistance

We next examined the functional involvement of IGF1R in PI3Ki-resistant phenotype. Treatment of SF295-R cells with IGF1R siRNA dramatically reduced IGF1R protein expression, and concomitantly restored sensitivity to ZSTK474 comparable to that of the parental SF295 cells (Fig. 4). In contrast, sensitivity of SF295 cells to ZSTK474 remained unchanged after treatment with IGF1R siRNA (Fig. 4B).

image

Figure 4. Effect of insulin-like growth factor 1 receptor (IGF1R) siRNAs on protein expression levels of IGF1R and sensitivity to ZSTK474 in SF295 and SF295-R cells. (A) Expression levels of IGF1R protein were analyzed by immunoblot assay in SF295-R and parental SF295 cells transfected with control siRNA (si-cont) or IGF1R-siRNA (siIGF1R-1, 5′-UUAAUGAGCAAAUUGCCCUUGAAGA-3′; siIGF1R-2, 5′-UAAACGGUGAAGCUGAUGAGAUCCC-3′) and incubated for 48 h. Protein levels were dramatically reduced in SF295-R cells after 48 h incubation following transfection with IGF1R-siRNA. (B) Effects of IGF1R siRNAs on ZSTK474 sensitivity of SF295-R and parental SF295 cells. After transfection with siRNA, cells were incubated overnight, replated on 96-well plates, allowed to attach overnight, then treated with the indicated concentration of ZSTK474 for 48 h. Cell growth was assessed by sulforhodamine B assay and the relative growth of the ZSTK474 treated cells compared to that of the untreated cells was calculated. SF295-R cells transfected with IGF1R-siRNA showed restored sensitivity to ZSTK474 comparable to that of the parental SF295 cells. Assays were carried out in duplicate and the data are representative of two independent experiments.

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We next examined the effect of IGF1R-TKIs on the PI3Ki-resistant phenotype. Sensitivities to IGF1R-TKIs alone in SF295-R cells were comparable to their parental SF295 cells (Fig. S2, Table S1). However, after treatment with IGF1R-TKIs, SF295-R cells, but not the parental SF295 cells, became more sensitive to ZSTK474 in a dose-dependent manner (Figs 5A,S3A). Isobologram analysis of the data revealed that the antitumor effect of ZSTK474 in combination with IGF1R-TKI was much greater than their calculated additive effect, thus suggesting that this drug combination has a synergistic effect (Figs 5B,S3B). A similar effect was observed for another PI3Ki, GDC-0941 (Fig. S4). From these results, we concluded that the tyrosine kinase activity of overexpressed IGF1R plays a key role in acquiring resistance to PI3Kis and, therefore, it is a promising therapeutic target for overcoming the resistance.

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Figure 5. Effects of OSI-906 and ZSTK474 in combination on the growth of SF295-R and parental SF295 cells. (A) Dose–response curves of ZSTK474 in the presence or absence of OSI-906. SF295-R and parental SF295 cells were cotreated with the indicated doses of ZSTK474 and OSI-906 for 48 h. Relative growth at each dose of OSI-906 in the absence of ZSTK474 was normalized as 100%. (B) Evaluation of the effects of ZSTK474 and OSI-906 combination by isobologram analysis of the same data as in (A). This drug combination showed a superadditive (synergistic) effect on SF295-R cells but only an additive effect on parental SF295 cells. Assays were carried out in duplicate.

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Effect of IGF1R overexpression on activation status of PI3K-Akt pathway in SF295-R cells

As IGF/IGF1R signaling is known to activate the PI3K/Akt and MEK/ERK pathways,[22] we carried out immunoblot analysis to examine whether Akt and ERK were activated in SF295-R cells. As a result, the basal phosphorylation level of Akt at Thr308 was upregulated in SF295-R cells, but ERK was not (Fig. 6). We then examined the effect of ZSTK474 on the phosphorylation status of Akt in SF295-R cells. Interestingly, a higher concentration of ZSTK474 (10 μM) was needed to dephosphorylate Akt in SF295-R cells compared to their parental SF295 cells and SF295-R/df100 cells (0.4 μM; Fig. 6). Furthermore, specific knockdown of IGF1R made it easier to dephosphorylate Akt after ZSTK474 treatment (Fig. 7). From these results, we concluded that overexpression of IGF1R made the PI3K-Akt pathway resistant to inactivation by PI3Kis in SF295-R cells.

image

Figure 6. Activation status of the phosphatidylinositol 3-kinase–Akt pathway in SF295, SF295-R, and SF295-R/df100 cells after exposure to ZSTK474. The cells were treated with the indicated concentrations of ZSTK474 for 3 h. Cells were harvested and immunoblot analysis of insulin-like growth factor 1 receptor (IGF1R-β), phosphorylated IGF1R (p-IGF1R-β), phosphorylated Akt (p-Akt) at Thr308 and Ser473, and phosphorylated ERK1/2 (p-ERK1/2) were carried out.

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image

Figure 7. Effect of insulin-like growth factor 1 receptor (IGF1R) siRNA on expression levels of phosho-Akt in SF295-R cells after exposure to ZSTK474. SF295 and SF295-R cells were transfected with control siRNA (sicont) or IGF1R siRNA (siIGF1R-1). Cells were then exposed to ZSTK474 at the indicated concentrations for 3 h. Cells were harvested and immunoblot analysis of IGF1R and phosphorylated Akt at Thr308 and Ser473 were carried out.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

In this study, we showed for the first time that long-term exposure of cancer cells to a PI3K inhibitor ZSTK474 in vitro led to acquired resistance to PI3Kis. The present result strongly suggested that long-term treatment of cancer patients with PI3Kis might cause therapeutic inefficacy in future clinical applications. Therefore, it is very important to examine acquired resistance of PI3Kis and to develop therapeutic strategies for overcoming the resistance. Acquired resistance is a major obstacle for conventional cancer chemotherapy. This is also true for some of the targeted drugs, especially for the TKIs, where the acquired resistance is due to a mutation at the “gatekeeper” site in the kinase domain of the respective kinase gene.[23, 24] Although PI3K is a lipid kinase, a genetically engineered point mutation at I800 residue in the kinase domain of PIK3CA was shown to be resistant to PI3Kis.[16] It is, however, unclear whether such a drug-resistant mutation naturally occurred in cancer cells following prolonged exposure to the drug. Our results suggest that the acquired resistance observed here is not due to any mutational change in the PIK3CA gene. Our findings that the acquired resistance was a reversible event is also consistent with this observation (Fig. 2B), as genetic alterations including mutational changes are irreversible events.

In this study, we focused on how cancer cells acquire resistance to PI3Kis after long-term exposure and how to overcome the acquired resistance. We clearly showed that overexpression of IGF1R is a common event in all of the four cancer cell lines that acquired PI3Ki resistance after long-term exposure to ZSTK474 (Fig. 3). We also showed that the acquired resistance could be overcome by inhibiting IGF1R through knockdown by siRNA (Fig. 4). We further examined the effect of IGF1R-TKIs and a similar result was obtained (Figs 5,S3,S4). From these results we concluded that overexpression of IGF1R is most likely to be one of the common mechanisms by which cancer cells acquire resistance to PI3Kis, and a promising target for overcoming the resistance.

Insulin-like growth factor 1 receptor is one of the RTKs, which has been implicated in several types of cancers, including breast, prostate, and lung cancers.[25-27] On the binding of IGF1, IGF1R is activated and phosphorylates itself and insulin receptor substrate 1 and 2 (IRS1/2), and subsequently the phosphorylated IRS1/2 activates PI3K.[22, 28] Activation of RTKs is reported to be involved in resistance to targeted therapy; for example, activation of the HGF/c-MET pathway contributes to resistance against gefitinib in lung cancer cells harboring EGFR-activating mutations.[29-31] Activation of IGF1R also causes acquired resistance to gefitinib. [15, 32] In these cases, activation of the HGF/c-MET or IGF1/IGF1R pathways would likely play as alternatives to the EGF/EGFR pathway to circumvent the antitumor effect of gefitinib. However, as PI3K is a downstream effector of RTKs, they would not work as alternatives to PI3K. In the present study, our results suggested that overexpression of IGF1R made its downstream PI3K-Akt pathway resistant to inactivation by PI3Kis in SF295-R cells, and that this might be the mechanism by which SF295-R cells acquired resistance to PI3Kis.

It is reported that IGF1R/IRS works as a target of negative feedback loop of PI3K/mTOR pathway.[33] Activation of the PI3K/mTOR pathway causes inhibition of IGF1R signal through phosphorylation of IRS1/2 by S6K1 at Ser307 residue, and inhibition of the PI3K/mTOR pathway would activate IGF1R signal through inhibition of negative feedback.[34-36] The involvement of IGF1R and the effect of IGF1R-TKI on this feedback loop and on the efficacy of PI3Kis are currently under investigation.

In this study, we showed a synergistic effect of the IGF1R-TKIs and PI3Kis combination in SF295-R cells overexpressing IGF1R (Figs 5,S3,S4). However, sensitivities to IGF1R-TKIs alone in SF295-R cells were comparable to their parental SF295 cells (Fig. S2). Similar results were obtained in three other resistant cells (Table S1). Consistent with this observation, expression of phospho-Akt remained unchanged after knockdown of IGF1R by siRNA (Fig. 7). These results suggested that the growth of the resistant cells is not addicted to IGF1R but is dependent on other molecules upstream of the PI3K pathway in addition to IGF1R. This might be the reason why IGF1R-TKIs exert a strong antitumor effect on the resistant cells only when they were combined with PI3Kis. However, we found that IGF1R was upregulated in all four resistant cell lines, whereas other RTKs including EGFR, fibroblast growth factor receptor, platelet-derived growth factor receptor, and c-MET were not upregulated by GeneChip analysis (data not shown). Further studies are needed to clarify the upstream molecules on which the growth of the resistant cells depends.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) has been shown to negatively regulate the PI3K pathway. Loss of PTEN triggers sequential phosphorylation of the PI3K downstream signal cascade including Akt and mTOR.[6] Of 11 cell lines used in this study, four cell lines do not express PTEN protein by deletion or mutation of the PTEN gene.[11] Of four PTEN-negative cell lines, SF295 and SNB78 acquired resistance, but PC-3 and BSY-1 did not. Therefore, PTEN status was not likely to be involved in resistance to PI3Kis.

In summary, our in vitro results suggested that long-term exposure to PI3Kis would induce therapeutic inefficacy, and the combination of IGF1R-TKI with PI3Ki could overcome the acquired resistance. We hope that our findings contribute to the improvement of the therapeutic efficacy of PI3Ki in future clinical applications.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

We thank Zenyaku Kogyo for providing us with ZSTK474. This work was supported in part by Grants-in-Aid for Scientific Research (A) from the Japan Society for the Promotion of Science to T. Yamori (22240092), Grants-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science to S. Dan (22700929), and a Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from The Ministry of Education, Culture, Sports, Science and Technology of Japan to T. Yamori (221S0001).

Disclosure Statement

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Takao Yamori (2010 and 2011) and Shingo Dan (2012) have a research fund from Zenyaku Kogyo, which is the proprietary company of ZSTK474; S. Isoyama is an employee of Zenyaku Kogyo.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
cas12004-sup-0001-FigS1.tifimage/TIF126KFig. S1. Growth response curves of ZSTK474-resistant and parental cells.
cas12004-sup-0002-FigS2.tifimage/TIF125KFig. S2. Growth response curves of SF295-R and parental SF295 cells against phosphatidylinositol 3-kinase inhibitors, insulin-like growth factor 1 receptor–tyrosine kinase inhibitor, and cisplatin.
cas12004-sup-0003-FigS3.tifimage/TIF146KFig. S3. Effects of ZSTK474 and AEW541 in combination on SF295-R and SF295 cells.
cas12004-sup-0004-FigS4.tifimage/TIF142KFig. S4. Effects of GDC-0941 and OSI-906 in combination on SF295-R and SF295 cells.
cas12004-sup-0005-TableS1.docxWord document16KTable S1. Sensitivities to NVP-AEW541 in ZSTK474-resistant cell lines and their parental cell lines.

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