SOCE and cancer: Recent progress and new perspectives

Ca²⁺ acts as a universal and versatile second messenger in the regulation of a myriad of biological processes, including cell proliferation, differentiation, migration and apoptosis. Store‐operated Ca²⁺ entry (SOCE) mediated by ORAI and the stromal interaction molecule (STIM) constitutes one of the major routes of calcium entry in nonexcitable cells, in which the depletion of intracellular Ca²⁺ stores triggers activation of the endoplasmic reticulum (ER)‐resident Ca²⁺ sensor protein STIM to gate and open the ORAI Ca²⁺ channels in the plasma membrane (PM). Accumulating evidence indicates that SOCE plays critical roles in cancer cell proliferation, metastasis and tumor neovascularization, as well as in antitumor immunity. We summarize herein the recent advances in our understanding of the function of SOCE in various types of tumor cells, vascular endothelial cells and cells of the immune system. Finally, the therapeutic potential of SOCE inhibitors in the treatment of cancer is also discussed.     

Blockade of store-operated Ca entry inhibits hepatocarcinoma cell migration and invasion by regulating focal adhesion turnover

Store-operated Ca²⁺ entry (SOCE) is a main Ca²⁺ influx pathway controlling the intracellular Ca²⁺ concentration in normal hepatocytes and hepatocellular carcinoma (HCC) cells. Ca²⁺ influx has been demonstrated to be involved in liver oncogenesis. Stromal interacting molecule (STIM) 1 acts as a sensor for the level of Ca²⁺ stored in the endoplasmic reticulum, and Orai1 protein constitutes the pore-forming subunit of the store-operated channels. Recently, STIM1 and Orai1 were found critical for breast tumor cell migration and metastasis. However, the effects of Ca²⁺ influx pathway on migration and metastasis have not been studied in hepatocellular carcinoma. Here, we found that STIM1 had a higher expression in hepatoma tissues than in precancerous tissues of the same patients. In general, STIM expression is elevated in HCC cell lines compared to a normal hepatocyte cell line. HCC-LM3 cell, which has a higher migration ability, expresses five times higher level of STIM than other HCC cell lines. STIM1 could then be explored as a prognostic marker to screen liver cancer patients with high metastatic potential. Inhibition of SOCE and STIM1 enhance focal adhesions and decrease the focal adhesion turnover, suggesting the therapeutic potential of SOCE and STIM1 as new molecular targets for metastatic HCC.     

New findings of kinase switching in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis

Despite the revolutionary effects of imatinib on advanced gastrointestinal stromal tumors (GISTs), most patients eventually develop disease progression following primary resistance or acquired resistance driven by secondary‐resistant mutations. Even in radiographically vanishing lesions, pathology has revealed persistent viable cells during imatinib therapy, which could lead to the emergence of drug‐resistant clones. To uncover the mechanisms underlying these clinical issues, here we examined imatinib‐induced phosphoproteomic alterations in GIST‐T1 cells, using our quantitative tyrosine phosphoproteomic analysis method, which combined immunoaffinity enrichment of phosphotyrosine‐containing peptides with isobaric tags for relative and absolute quantitation (iTRAQ) technology. Using this approach, we identified 171 tyrosine phosphorylation sites spanning 134 proteins, with 11 proteins exhibiting greater than 1.5‐fold increases in tyrosine phosphorylation. Among them, we evaluated FYN and focal adhesion kinase (FAK), both of which are reportedly involved in proliferation and malignant alteration of tumors. We confirmed increased tyrosine phosphorylation of both kinases by western blotting. Inhibition of FYN and FAK phosphorylation each increased tumor cell sensitivity to imatinib. Furthermore, a FAK‐selective inhibitor (TAG372) induced apoptosis of imatinib‐resistant GIST‐T1 cells and decreased the imatinib IC₅₀. These results indicate that FYN or FAK might be potential therapeutic targets to overcome resistance to imatinib in GISTs. Additionally, we showed that the iTRAQ‐based quantitative phosphotyrosine‐focused phosphoproteomic approach is a powerful method for screening phosphoproteins associated with drug resistance.     

Resveratrol activates autophagic cell death in prostate cancer cells via downregulation of STIM1 and the mTOR pathway

Resveratrol (RSV), a natural polyphenol, has been suggested to induce cell cycle arrest and activate apoptosis‐mediated cell death in several cancer cells, including prostate cancer. However, several molecular mechanisms have been proposed on its chemopreventive action, the precise mechanisms by which RSV exerts its anti‐proliferative effect in androgen‐independent prostate cancer cells remain questionable. In the present study, we show that RSV activates autophagic cell death in PC3 and DU145 cells, which was dependent on stromal interaction molecule 1 (STIM1) expression. RSV treatment decreases STIM1 expression in a time‐dependent manner and attenuates STIM1 association with TRPC1 and Orai1. Furthermore, RSV treatment also decreases ER calcium storage and store operated calcium entry (SOCE), which induces endoplasmic reticulum (ER) stress, thereby, activating AMPK and inhibiting the AKT/mTOR pathway. Similarly, inhibition of SOCE by SKF‐96365 decreases the survival and proliferation of PC3 and DU145 cells and inhibits AKT/mTOR pathway and induces autophagic cell death. Importantly, SOCE inhibition and subsequent autophagic cell death caused by RSV was reversed by STIM1 overexpression. STIM1 overexpression restored SOCE, prevents the loss of mTOR phosphorylation and decreased the expression of CHOP and LC3A in PC3 cells. Taken together, for the first time, our results revealed that RSV induces autophagy‐mediated cell death in PC3 and DU145 cells through regulation of SOCE mechanisms, including downregulating STIM1 expression and trigger ER stress by depleting ER calcium pool. © 2015 The Authors. Molecular Carcinogenesis, published by Wiley Periodicals, Inc.     

Anti‐inflammatory M2 type macrophages characterize metastasized and tyrosine kinase inhibitor‐treated gastrointestinal stromal tumors

We have made a detailed inventory of the immune infiltrate of gastrointestinal stromal tumors (GISTs), which originate from mesenchymal cells in the intestinal tract. These sarcomas are heavily infiltrated with macrophages and T cells, while immune cells of other lineages were much less abundant. Dissecting the functional subtypes of T cells with multicolor fluorescent microscopy revealed substantial populations of cytotoxic T cells, helper T cells and FoxP3⁺ regulatory T cells. The balance of cytotoxic T cells and FoxP3⁺ T cells was toward immune suppression. Analysis of the macrophage population also showed a dominance of anti‐inflammatory cells, as the M2 type scavenger receptor CD163 was abundantly present. Other subsets of macrophages (CD14⁺CD163⁻) were occasionally detected. M2 type CD163⁺ macrophages were associated with the number of infiltrating FoxP3⁺ regulatory T cells and twice as many macrophages were found in metastatic GIST compared to primary lesions. Most metastatic GISTs had been treated with the tyrosine kinase inhibitors imatinib and sunitinib, but the high macrophage infiltrate was not related to this treatment. However, imatinib and sunitinib did induce secretion of anti‐inflammatory IL‐10 in macrophage cultures, indicating that treatment with these inhibitors might contribute to an immune suppressive microenvironment in GIST. Overall, our data reveal a picture of GIST as an active site of tumor‐immune interaction in which suppressive mechanisms overrule potential antitumor responses. Tyrosine kinase inhibitors might promote this negative balance.     

Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours

Drug resistance remains a major challenge in the clinical treatment of gastrointestinal stromal tumours (GISTs). While acquired on‐target mutations of mast/stem cell growth factor receptor (KIT) kinase is the major resistance mechanism, activation of alternative signalling pathways may also play a role. Although several second‐ and third‐generation KIT kinase inhibitors have been developed that could overcome some of the KIT mutations conferring resistance, the low clinical responses and narrow safety window have limited their broad application. The present study revealed that nintedanib not only overcame resistance induced by a panel of KIT primary and secondary mutations, but also overcame ERK‐reactivation‐mediated resistance caused by the upregulation of fibroblast growth factor (FGF) activity. In preclinical models of GISTs, nintedanib significantly inhibited the proliferation of imatinib‐resistant cells, including GIST‐5R, GIST‐T1/T670I and GIST patient‐derived primary cells. In addition, it also exhibited dose‐dependent inhibition of ERK phosphorylation upon FGF ligand stimulation. In vivo antitumour activity was also observed in several xenograft GIST models. Considering the well‐documented safety and pharmacokinetic profiles of nintedanib, this finding provides evidence for the repurposing of nintedanib as a new therapy for the treatment of GIST patients with de novo or acquired resistance to imatinib.     

Suppression of STIM1 inhibits human glioblastoma cell proliferation and induces G0/G1 phase arrest

Background

Depletion of calcium (Ca²⁺) from the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca²⁺ entry (SOCE) pathway which sustains long-term Ca²⁺ signals and is critical for cellular functions. Stromal interacting molecule 1 (STIM1) serves a dual role as an ER Ca²⁺ sensor and activator of SOCE. Aberrant expression of STIM1 could be observed in several human cancer cells. However, the role of STIM1 in regulating tumorigenesis of human glioblastoma still remains unclear.

Methods

Expression of STIM1 protein in a panel of human glioblastoma cell lines (U251, U87 and U373) in different transformation level were evaluated by Western blot method. STIM1 loss of function was performed on U251 cells, derived from grade IV astrocytomas-glioblastoma multiforme with a lentvirus-mediated short harpin RNA (shRNA) method. The biological impacts after knock down of STIM1 on glioblastoma cells were investigated in vitro and in vivo.

Results

We discovered that STIM1 protein was expressed in U251, U87 and U373 cells, and especially higher in U251 cells. RNA interference efficiently downregulated the expression of STIM1 in U251 cells at both mRNA and protein levels. Specific downregulation of STIM1 inhibited U251 cell proliferation by inducing cell cycle arrest in G0/G1 phase through regulation of cell cycle-related genes, such as p21^Waf1/Cip1_, cyclin D1 and cyclin-dependent kinase 4 (CDK4), and the antiproliferative effect of STIM1 silencing was also observed in U251 glioma xenograft tumor model.

Conclusion

Our findings confirm STIM1 as a rational therapeutic target in human glioblastoma, and also indicate that lentivirus-mediated STIM1 silencing is a promising therapeutic strategy for human glioblastoma.     

Secondary mutations in the kinase domain of the KIT gene are predominant in imatinib-resistant gastrointestinal stromal tumor

Although imatinib showed high activity for advanced gastrointestinal stromal tumor (GIST) and improved the prognosis of GIST patients, resistance to the drug appears with prolonged use. Mechanisms of acquired resistance are still under investigation. In the present study, we carried out histologic and genetic analysis of 45 secondary resistant lesions obtained from 25 Japanese GIST patients treated with imatinib. All resistant lesions showed viable tumor cells expressing KIT protein, whereas imatinib-sensitive lesions did not. All pre-imatinib samples have KIT mutations either in exon 9 ( n  = 3) or exon 11 ( n  = 22), identified in the KIT gene of corresponding resistant tumors. In addition to primary mutations, 33 out of 45 tumors (73%) showed secondary KIT mutations in the kinase domain of the KIT gene. Secondary mutations are missense mutations and are mostly located in the kinase domains of the same allele to the primary mutations ( cis -position). Resistant lesions showed monoclonal development of tumor cells. Taken together, additional cis -positioned mutations in the kinase domains are a major cause of secondary resistance to imatinib in Japanese GIST patients. ( Cancer Sci 2008; 99: 799–804)     

Flumatinib,a selective inhibitor of BCR‐ABL/PDGFR/KIT,effectively overcomes drug resistance of certain KIT mutants

Activating mutations in KIT have been associated with gastrointestinal stromal tumors (GISTs). The tyrosine kinase inhibitor imatinib mesylate has revolutionized the treatment of GISTs. Unfortunately, primary or acquired resistance to imatinib does occur in GISTs and forms a major problem. Although sunitinib malate, a multi‐kinase inhibitor, has shown effectiveness against imatinib‐resistant GISTs, recent studies have indicated that some imatinib‐resistant GISTs harboring secondary mutations in the KIT activation loop were also resistant to sunitinib. Therefore, new drugs capable of overcoming the dual drug resistance of GISTs probably have potential clinical utility. In this study, we investigated the efficacy of flumatinib, an inhibitor of BCR‐ABL/PDGFR/KIT, against 32D cells transformed by various KIT mutants and evaluated its potency to overcome the drug resistance of certain mutants. Interestingly, our in vitro study revealed that flumatinib effectively overcame the drug resistance of certain KIT mutants with activation loop mutations (i.e., D820G, N822K, Y823D, and A829P). Our in vivo study consistently suggested that flumatinib had superior efficacy compared with imatinib or sunitinib against 32D cells with the secondary mutation Y823D. Molecular modeling of flumatinib docked to the KIT kinase domain suggested a special mechanism underlying the capability of flumatinib to overcome the drug‐resistance conferred by activation loop mutations. These findings suggest that flumatinib could be a promising therapeutic agent against GISTs resistant to both imatinib and sunitinib because of secondary mutations in the activation loop.     

Acquired resistance to imatinib in gastrointestinal stromal tumor occurs through secondary gene mutation.

Most gastrointestinal stromal tumors (GIST) have an activating mutation in either KIT or PDGFRA. Imatinib is a selective tyrosine kinase inhibitor and achieves a partial response or stable disease in about 80% of patients with metastatic GIST. It is now clear that some patients with GIST develop resistance to imatinib during chronic therapy. To identify the mechanism of resistance, we studied 31 patients with GIST who were treated with imatinib and then underwent surgical resection. There were 13 patients who were nonresistant to imatinib, 3 with primary resistance, and 15 with acquired resistance after initial benefit from the drug. There were no secondary mutations in KIT or PDGFRA in the nonresistant or primary resistance groups. In contrast, secondary mutations were found in 7 of 15 (46%) patients with acquired resistance, each of whom had a primary mutation in KIT exon 11. Most secondary mutations were located in KIT exon 17. KIT phosphorylation was heterogeneous and did not correlate with clinical response to imatinib or mutation status. That acquired resistance to imatinib in GIST commonly occurs via secondary gene mutation in the KIT kinase domain has implications for strategies to delay or prevent imatinib resistance and to employ newer targeted therapies.     

Establishment of phosphatidylinositol 3‐kinase inhibitor‐resistant cancer cell lines and therapeutic strategies for overcoming the resistance

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 BCR–ABL. 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.     

Synergistic induction of apoptosis by the Bcl-2 inhibitor ABT-737 and imatinib mesylate in gastrointestinal stromal tumor cells

Background: Although imatinib mesylate has revolutionized the management of patients with gastrointestinal stromal tumor (GIST), resistance and progression almost inevitably develop with long‐term monotherapy. To enhance imatinib‐induced cytotoxicity and overcome imatinib‐resistance in GIST cells, we examined the antitumor effects of the pro‐apoptotic Bcl‐2/Bcl‐xL inhibitor ABT‐737, alone and in combination with imatinib.Methods: We treated imatinib‐sensitive, GIST‐T1 and GIST882, and imatinib‐resistant cells with ABT‐737 alone and with imatinib. We determined the anti‐proliferative and apoptotic effects by cell viability assay, flow cytometric apoptosis and cell cycle analysis, immunoblotting, and nuclear morphology. Synergism was determined by isobologram analysis.Results: The IC50 of single‐agent ABT‐737 at 72 h was 10 μM in imatinib‐sensitive GIST‐T1 and GIST882 cells, and 1 μM in imatinib‐resistant GIST48IM cells. ABT‐737 and imatinib combined synergistically in a time‐ and dose‐dependent manner to inhibit the proliferation and induce apoptosis of all GIST cells, as evidenced by cell viability and apoptosis assays, caspase activation, PARP cleavage, and morphologic changes. Isobologram analyses revealed strongly synergistic drug interactions, with combination indices <0.5 for most ABT‐737/imatinib combinations. Thus, clinically relevant in vitro concentrations of ABT‐737 have single‐agent antitumor activity and are synergistic in combination with imatinib.Conclusion: We provide the first preclinical evidence that Bcl‐2/Bcl‐xL inhibition with ABT‐737 synergistically enhances imatinib‐induced cytotoxicity via apoptosis, and that direct engagement of apoptotic cell death may be an effective approach to circumvent imatinib‐resistance in GIST.     

Managing progressive disease in patients with GIST: factors to consider besides acquired secondary tyrosine kinase inhibitor resistance

The use of tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of patients with unresectable and/or metastatic gastrointestinal stromal tumors (GIST). Currently, imatinib mesylate is the standard first-line treatment for unresectable and/or metastatic GIST, extending recurrence-free and overall survival for many patients. Nonetheless, eventual progression during imatinib therapy is prevalent, and the development of treatment paradigms for managing GIST progression is of importance. Sunitinib malate has been approved as a second-line treatment for unresectable and/or metastatic GIST and is an option for patients who are intolerant to imatinib or experience disease progression due to acquired resistance, otherwise referred to as secondary resistance. In many cases, however, there may be other causes for GIST progression besides secondary resistance, and consideration of these factors is necessary before switching to second-line treatment. This review presents a treatment strategy for GIST patients who have progressed after initial imatinib responsiveness and addresses necessary considerations that include instances of false progression, insufficient TKI plasma levels, and patient non-adherence. In situations where true progression has occurred, patients may benefit from imatinib dose escalation. Surgery also provides a viable option for patients with stable disease or limited progression, and may prevent and/or delay the development of resistant clones by reducing tumor burden. Switching to second-line therapy with sunitinib should be considered for imatinib-intolerant or -resistant GIST patients.     

Dose optimization of tyrosine kinase inhibitors to improve outcomes in GIST

Tyrosine kinase inhibitors such as imatinib and sunitinib have greatly improved clinical outcomes for patients with gastrointestinal stromal tumors (GIST). Dose optimization of these agents is critical and involves multiple considerations, including ensuring a durable response, monitoring drug blood levels to confirm adequate dosing, deciding whether to use high‐dose imatinib or switch to second‐line sunitinib in the event of disease progression and appropriately managing treatment‐associated side effects. Imatinib is the standard first‐line therapy for unresectable or metastatic GIST and is also an option for the adjuvant treatment of resected disease. Despite the efficacy and safety of imatinib in patients with advanced GIST, some individuals develop primary or secondary resistance or intolerance to the drug. For patients with advanced disease, imatinib dose escalation to 800 mg/day is warranted in cases of disease progression on imatinib 400 mg/day. In addition, patients with documented KIT exon 9 mutations are likely to derive benefit from initial treatment with high‐dose imatinib to improve clinical outcomes. For patients who fail imatinib, sunitinib is an effective treatment option. However, the decision to use either high‐dose imatinib or sunitinib should be based on the underlying cause of failure on imatinib, KIT mutational status and on whether the patient is intolerant of or has developed a resistance to imatinib. In this article we review the existing literature supporting the use of imatinib and sunitinib in GIST to provide a current clinical perspective on how best to use these agents in the management of GIST to optimize patient outcomes.     

Response to Nilotinib as a First-Line Treatment for Metastatic Gastrointestinal Stromal Tumors

Purpose  

Effective and safe treatment options are needed for patients with advanced gastrointestinal stromal tumors (GIST) who are initially unresponsive to the tyrosine kinase inhibitor (TKI) imatinib, or develop acquired secondary imatinib resistance.     

Molecular basis for primary and secondary tyrosine kinase inhibitor resistance in gastrointestinal stromal tumor

Small molecule kinase inhibitors have irrevocably altered cancer treatment. March 2010 marks the 10th anniversary of using imatinib in gastrointestinal stromal tumors (GIST), a cardinal example of the utility of such targeted therapy in a solid tumor. Before imatinib, metastatic GIST was frustrating to treat due to its resistance to standard cytotoxic chemotherapy. Median survival for patients with metastatic GIST improved from 19 to 60?months with imatinib. In treating patients with GIST, two patterns of tyrosine kinase inhibitor resistance have been observed. In the first, ~9-14% of patients have progression within 3?months of starting imatinib. These patients are classified as having primary or early resistance. Median progression-free survival (PFS) on imatinib is approximately 24?months; patients with later progression are classified as having secondary or acquired resistance. Primary studies and a meta-analysis of studies of imatinib in GIST patients have identified prognostic features that contribute to treatment failure. One of the strongest predictors for success of therapy is KIT or PDGFRA mutational status. Patients with KIT exon 11 mutant GIST have better response rates, PFS, and overall survival compared to other mutations. A great deal has been learned in the last decade about sensitivity and resistance of GIST to imatinib; however, many unanswered questions remain about secondary resistance mechanisms and clinical management in the third- and fourth-line setting. This review will discuss the role of dose effects, and early and late resistance to imatinib and their clinical implications. Patients intolerant to imatinib (5%) and those who progress on imatinib are treated with sunitinib. The mechanism of resistance to sunitinib is unknown at this time but is also appears related to growth of clones with secondary mutations in KIT. Third- and fourth-line treatments of GIST and with future treatment strategies are also discussed.     

GIST中imatinib耐药机制研究进展

在功能获得性突变所致的KIT或PDGFRA受体酪氨酸激酶异常活化与胃肠间质瘤（GIST）的发病关系阐明后,酪氨酸激酶抑制剂甲璜酸伊马替尼（imatinib）在进展期GIST中显现出很高的有效性,使GIST的治疗发生了革命性的改变。然而,GIST中对imatinib的早期或晚期耐药已经成为日益严重的临床问题。因此,对耐药的分子机制的进一步了解就成为当前研究的焦点,本文综述了目前对imatinib耐药的分子机制的认识,这些认识可能引导新的治疗策略的产生。     

Pharmacological interaction with sunitinib is abolished by a germ‐line mutation (1291T>C) of BCRP/ABCG2 gene

Sunitinib malate (Sutent, SU11248) is a small‐molecule multitargeted tyrosine kinase inhibitor (TKI) used for the treatment of renal cell carcinoma and imatinib‐resistant gastrointestinal stromal tumors. Some TKIs can overcome multidrug resistance conferred by ATP‐binding cassette transporter, P‐glycoprotein (P‐gp)/ABCB1, multidrug resistance‐associated protein 1 (MRP1)/ABCC1, and breast cancer resistance protein (BCRP)/ABCG2. Here, we analyzed the effects of sunitinib on P‐gp and on wild‐type and germ‐line mutant BCRPs. Sunitinib remarkably reversed BCRP‐mediated and partially reversed P‐gp‐mediated drug resistance in the respective transfectants. The in vitro vesicle transport assay indicated that sunitinib competitively inhibited BCRP‐mediated estrone 3‐sulfate transport and P‐gp‐mediated vincristine transport. These inhibitory effects of sunitinib were further analyzed in Q141K‐, R482G‐, R482S‐, and F431L‐variant BCRPs. Intriguingly, the F431L‐variant BCRP, which is expressed by a germ‐line mutant allele 1291T>C, was almost insensitive to both sunitinib‐ and fumitremorgin C (FTC)‐mediated inhibition in a cell proliferation assay. Sunitinib and FTC did not inhibit ¹²⁵I‐iodoarylazidoprazosin‐binding to F431L‐BCRP. Thus, residue Phe‐431 of BCRP is important for the pharmacological interaction with sunitinib and FTC. Collectively, this is the first report showing a differential effect of a germ‐line variation of the BCRP/ABCG2 gene on the pharmacological interaction between small‐molecule TKIs and BCRP. These findings would be useful for improving our understanding of the pharmaceutical effects of sunitinib in personalized chemotherapy. (Cancer Sci 2010; 00: 000–000)