TAS-116, a Novel Hsp90 Inhibitor, Selectively Enhances Radiosensitivity of Human Cancer Cells to X-rays and Carbon Ion Radiation

Heat shock protein 90 (Hsp90) is a chaperone protein that regulates the function of tumor growth–related proteins, and Hsp90 inhibition is considered a useful strategy for treating cancer (1). Importantly, Hsp90 inhibition selectively affects tumor cells (1–3), because Hsp90 proteins in tumor cells are present in complexes with higher ATPase activity than those in normal cells (4, 5). Hsp90 inhibition suppresses levels of key DNA double-strand break (DSB) repair proteins (1–3). Thus, Hsp90 inhibitors are promising agents for cancer therapy, including combined treatment with radiation (1, 6). As adverse side effects have been reported with early Hsp90 inhibitors (1, 7), development of novel Hsp90 inhibitors is urgently needed to overcome these limitations. One of the side effects induced by early-generation Hsp90 inhibitors is visual impairment (8, 9). TAS-116, a novel Hsp90 inhibitor, has shown less ocular toxicity than conventional Hsp90 inhibitors (10), so we explored whether TAS-116 presents a useful alternative with reduced adverse effects in chemoradiotherapy with X-rays or carbon ion radiation. There are only three prior reports of TAS-116 effects, showing antitumor activity against multiple myeloma and lung tumors (10–12). The biological effects of TAS-116 are poorly understood, and the effects of combined TAS-116 and radiotherapy have not been previously investigated.

Radiotherapy using carbon ions has received great attention because it offers improved tumor targeting and increased biological effectiveness (13, 14). Unlike low linear energy transfer (LET) radiation, high LET radiation, such as carbon ions, overcomes radioresistance of S-phase and hypoxic cells (15, 16), and it induces more complex DSBs that are repaired more slowly than X-ray–induced DSBs (14, 17). Because of these and other differences, radiosensitizing agents may differentially influence cellular responses to low and high LET radiation, including DNA repair, DNA damage signaling, cell-cycle arrest, and activation of cell death pathways. These processes are critical determinants of cell and tumor responses to radiotherapy; thus, it is important to study the mechanistic basis of radiosensitization in cells irradiated with low and high LET radiation.

DSB repair is an important determinant of cellular radiosensitivity (18, 19). DSBs are repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ), and cells with defects in HR or NHEJ repair show increased radiosensitivity (20–22). The Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) was found to inhibit HR and enhance radiosensitivity (2, 3). DSB repair pathways and other processes fluctuate during the cell cycle; hence, cell-cycle distribution is another determinant of radiosensitivity (23). Hsp90 inhibitors have been shown to alter cell-cycle distribution by regulating cell-cycle checkpoints. The Hsp90 inhibitors NVP-AUY922 and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) induce G₂–M arrest in A549 cells by downregulating Cdk1 and Cdk4 (24), and geldanamycin (GA) and 17-AAG downregulate the cdc2 and cdc25c cell-cycle regulators in glioblastoma cell lines (25).

Here, we investigate the mechanistic basis for the radiosensitizing effects of TAS-116 to low and high LET irradiation by focusing on DSB repair pathways and cell-cycle distribution. Our results indicate that TAS-116 enhances cell death induced by X-rays or carbon ions in vitro and that it enhances tumor control by carbon ion radiotherapy in a mouse xenograft model. Radiosensitization resulted from TAS-116 inhibition of both HR and NHEJ, and it was associated with marked G₂–M arrest.

All cell lines were authenticated by short tandem repeat profiling, and cells were thawed upon arrival, expanded, and stored in a liquid nitrogen tank. Each stocked cell line was thawed, expanded, and used within 3 months after thawing. Human cervical carcinoma cells (HeLa) were obtained from Cell Resource Center for Biomedical Research at Tohoku University (Sendai, Japan) in 2013 and cultured in αMEM (Wako) supplemented with 10% FBS. Human non–small cell lung carcinoma cells (H1299) were purchased from ATCC in 2014 and cultured in RPMI1640 (Wako) supplemented with 10% FBS. HFL1 human fibroblasts were purchased from RIKEN BioResource Center in 2002 and cultured in αMEM supplemented with 15% FBS. The HFL1 cell line is nontransformed and nontumorigenic, and this cell line was used as a normal control, as reported previously (26–28). All cells were grown in a humidified incubator at 37°C and 5% CO₂.

TAS-116 (Supplementary Fig. S1) was purchased from Active Biochem and dissolved into DMSO. Cells were pretreated with TAS-116 or DMSO for 24 hours and irradiated with X-rays or carbon ions. Cells were irradiated with a TITAN-320 X-ray generator (200 kV, 20 mA, Shimadzu) or with 290 MeV/n carbon ions (6 cm spread-out Bragg peak, ∼50 keV/μm) accelerated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Sciences (NIRS; Chiba, Japan). After irradiation, cell culture medium was replaced with fresh medium without drug.

Cell survival was evaluated by colony formation assay. After irradiation, cells were trypsinized and seeded into cell culture dishes at appropriate cell densities. Cells were cultured for 10 to 14 days, and they were fixed with ethanol and stained with 0.2% crystal violet. Colonies with more than 50 cells were counted. Surviving fractions were calculated on the basis of the plating efficiencies of corresponding nonirradiated cells.

Cells were grown on 4-well chamber slides (Nunc). At various time points after irradiation, cells were fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, and blocked in 3% BSA. γ-H2AX and RAD51 foci were detected with primary and secondary antibodies: anti-phospho-histone H2AX (Ser139; Millipore), anti-RAD51 (Santa Cruz Biotechnology), Alexa 488 anti-mouse secondary antibody and Alexa 594 anti-rabbit secondary antibody (Thermo Fisher Scientific). The cells were stained and mounted with ProLong Gold Antifade Agent with DAPI (Life Technologies). γ-H2AX and RAD51 foci were scored in at least 50 cells per condition with an Olympus BX51 fluorescence microscope.

Cell-cycle distributions and the percentage of sub-G₁ cells were determined by fixing cells in 70% cold ethanol 24 or 48 hours after irradiation. Fixed cells were washed in PBS and stained with 50 μg/mL propidium iodide (Sigma-Aldrich) in the presence of RNase (Wako). Cellular DNA content was measured with a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed with CellQuestPro Software (BD Biosciences) and ModFit Software (Verity Software House). At least 10,000 cells were analyzed per sample.

Protein levels were determined in whole-cell lysates prepared by washing cells in cold PBS and lysing with RIPA buffer (Pierce). Protein concentrations of cell lysates were determined by BCA Protein Assay Kit (Pierce). Equal amounts of proteins were loaded onto SDS-PAGE gels, separated by electrophoresis, and transferred onto PVDF membranes. The membranes were blocked with 0.2% I-Block (Tropix) and incubated with following primary and secondary antibodies: RAD51 (Santa Cruz Biotechnology), Ku70 (Thermo Fisher Scientific), DNA-PKcs (Thermo Fisher Scientific), phospho DNA-PKcs (S2056, Abcam), cdc25c (Cell Signaling Technology), cleaved caspase-3 (Cell Signaling Technology), PARP (Cell Signaling Technology), β-actin (Cell Signaling Technology), GAPDH (Cell Signaling Technology), and anti-mouse/rabbit secondary antibodies (Sigma-Aldrich). Protein expression levels were quantified with an ImageQuant LAS-4000 system (FujiFilm).

All experimental procedures were approved by the Institutional Animal Care and Use Committees at NIRS, conforming to the NIH (Bethesda, MD) Guide for the Care and Use of Laboratory Animals (29). HeLa cells (10⁶ cells) were subcutaneously injected into right hind legs of BALB/c-nu/nu mice (male, 7 weeks old). One week after cell inoculation, TAS-116 was administered via intraperitoneal injection at a dose of 20 mg/kg three times daily before irradiation. The tumors were irradiated locally with carbon ions, and tumor size and mice body weight were measured twice a week. Tumor volume (mm³) was calculated (by the formula: length × width × height × π/6], and tumor volume and body weight following treatments were determined relative to measurements performed before drug injection.

Statistical analysis was performed by Student t tests to evaluate significant differences between TAS-116–treated and mock-treated groups using SAS 9.3 (SAS Institute Inc.).

We performed colony formation assays to evaluate the radiosensitizing effects of TAS-116. As shown in Fig. 1A, pretreatment of human cancer cells with 1 μmol/L TAS-116 significantly enhanced sensitivity to X-rays. The sensitizing enhancement ratio (SER) of TAS-116 (1μmol/L) calculated at the D₁₀ value (dose required to reduce surviving fraction to 10%) was 1.87 and 1.34 for HeLa and H1299 cells, respectively. Importantly, TAS-116 did not radiosensitize noncancerous human fibroblast HFL1 cells. The relative biological effectiveness of carbon ions compared with X-rays calculated on the basis of the D₁₀ value was 1.67 and 1.77 for mock-treated HeLa and H1299 cells, and 1.36 and 1.46 for TAS-116–treated HeLa and H1299 cells. Similar to X-irradiated cells, TAS-116 also enhanced cell death in carbon ion–irradiated cancer cells (Fig. 1B); in these cases, SER values calculated from D₁₀ values were 1.53 and 1.11 for HeLa and H1299 cells, respectively. As with X-rays, TAS-116 did not sensitize noncancerous HFL1 cells to carbon ions. The cancer-selective effects of TAS-116 are consistent with prior studies of other Hsp90 inhibitors, which are known to have lower toxicity in normal cells than cancer cells, and do not affect radiosensitivity of normal cells (3, 30). Because TAS-116 sensitized cancer cells to both X-rays and carbon ions, this agent is a promising radiosensitizer to augment cancer therapy with low or high LET radiation, and its minimal toxicity to normal cells will serve to increase the therapeutic index.

To determine the mechanisms by which TAS-116 achieves its effects, we first assessed the ability of this drug to inhibit DSB repair. The number of γ-H2AX foci per nucleus, a marker of DSBs, was determined in HeLa and H1299 cells irradiated with X-rays or carbon ion with or without pretreatment with TAS-116. As shown in Fig. 2 and Supplementary Fig. S2, TAS-116 significantly increased the number of γ-H2AX foci per nucleus compared with mock (DMSO)-treated cells. In mock-treated cells, γ-H2AX foci were at maximum levels 1 hour after irradiation, returning to background levels of nonirradiated control cells within 24 hours. In contrast, γ-H2AX foci persisted for longer periods in TAS-116–treated cells. Although TAS-116 treatment alone slightly increased γ-H2AX foci, TAS-116 clearly increased radiation-induced γ-H2AX foci whether measured as absolute values (Fig. 2; Supplementary Fig. S2) or when background levels were subtracted from each measurement (data not shown). Inhibition of DSB repair is a well-known cause of radiosensitization (19), and these results suggest that TAS-116 inhibition of DSB repair is one mechanism by which it sensitizes cancer cells to X-rays and carbon ion radiation.

To further investigate how TAS-116 inhibits DSB repair, we tested whether it affects RAD51, a key protein involved in DSB repair by HR. We pretreated HeLa cells with TAS-116 prior to irradiation, then replaced growth medium with drug-free medium to mimic the conditions used in colony-forming assays. Treatment of HeLa cells with TAS-116 alone suppressed RAD51 levels, and it also suppressed RAD51 induction in response to radiation at various time points after treatment with X-rays or carbon ions (Fig. 3A). We scored the number of RAD51 foci per nucleus after irradiation in HeLa cells with or without pretreatment with TAS-116. The number of RAD51 foci increased dramatically within 1 hour of X-ray or carbon ion irradiation in mock-treated cells and then decreased to near background levels after 24 hours. Consistent with TAS-116 suppression of RAD51, radiation-induced RAD51 foci were sharply reduced by TAS-116 treatment, and RAD51 recruitment to damage was delayed (Fig. 3B). These results indicate that TAS-116 represses RAD51 expression and recruitment to DNA damage, suggesting that TAS-116 inhibits RAD51-mediated DSB repair by the HR pathway.

We next examined the effects of TAS-116 on the expression of NHEJ repair proteins. In the NHEJ repair pathway, DNA-PKcs is recruited to DSBs by the Ku70/Ku80 heterodimer and activated to facilitate repair DSBs. As above, growth medium was replaced with drug-free medium following irradiation. We found that TAS-116 treatment alone decreased Ku70 and DNA-PKcs expression, and it also suppressed DNA-PKcs levels after irradiation (Fig. 4A and B). In addition, TAS-116 inhibited activation of DNA-PKcs induced by X-rays or carbon ions, as revealed with phospho-specific antibodies (Fig. 4C). A more substantial reduction in phosphorylated DNA-PKcs was observed with X-rays than carbon ions (Fig. 4C). Together, these results indicate that TAS-116 suppresses two key NHEJ proteins, consistent with the significant inhibition of DSB repair in irradiated tumor cells.

DNA repair efficiency varies during the cell cycle; thus, we investigated whether TAS-116 altered cell-cycle distributions of HeLa and H1299 cells to further investigate potential mechanisms of TAS-116–induced radiosensitization. We found that TAS-116 treatment alone increased the percentage of cells in the G₂–M phase and that combined treatment with TAS-116 and irradiation sharply increased G₂–M arrest compared with either treatment alone (Fig. 5A and B; Supplementary Figs. S3 and S4). G₂–M arrest was particularly prominent 24 hours after cells were transferred to drug-free medium, and it was still apparent even 48 hours after drug removal. Cdc25c plays an important role in G₂–M progression, so we examined cdc25c protein expression levels in response to TAS-116. As with the DSB repair proteins above, TAS-116 suppressed cdc25c, and this effect was apparent in both nonirradiated cells and in cells irradiated with X-rays or carbon ions (Fig. 5C). It appears that cdc25c expression recovered as a function of time after cells were transferred to drug-free medium, consistent with the reductions in the percentage of G₂–M cells at later time points. These results indicate that TAS-116 suppresses cdc25c expression, causing G₂–M arrest, and this is the additional mechanism by which TAS-116 might confer radiosensitivity to cancer cells.

To investigate the mode of cell death by TAS-116 treatment, we evaluated the percentage of sub-G₁ cells and expression of apoptosis-related proteins. TAS-116 increased the percentage of sub-G₁ cells and increased expression of cleaved PARP and cleaved caspase-3 (Supplementary Figs. S5 and S6), indicating that TAS-116 induces apoptotic cell death. However, there appeared to be no synergistic increase in these apoptosis markers after combined treatment with TAS-116 and irradiation (Supplementary Figs. S5 and S6). Thus, apoptosis may not be the principal mode of cell death in combined treated cells, and other modes, such as mitotic catastrophe, might account for the radiosensitization by TAS-116.

The radiosensitizing effects of TAS-116 on cancer cells prompted us to investigate whether this drug could be useful as an adjunct to radiotherapy. We established HeLa tumor xenografts in mice to study TAS-116 effects in an in vivo tumor model. Mice were assigned to four groups: DMSO treated and nonirradiated (n = 6); DMSO treated and carbon irradiated (n = 6); TAS-116 treated and nonirradiated (n = 6); and TAS-116 treated and carbon irradiated (n = 7). The mice were treated with TAS-116 and/or irradiated with carbon ions by using the protocol diagrammed in Fig. 6A, and tumor volume and body weight were measured twice a week for approximately 6 weeks. Treatment with TAS-116 alone did not affect tumor growth, but combined treatment with TAS-116 and carbon ions inhibited tumor growth to a significantly greater extent than carbon ions alone within 26 days after tumor injection (Fig. 6B, P < 0.05). Beyond 29 days after tumor injection, this trend continued but was not statistically significant with these sample sizes. Representative tumors at 26 and 45 days after tumor injection with mono- and combination therapy are shown in Supplementary Fig. S7. These promising preliminary radiotherapy results justify further studies aimed at enhancing therapeutic efficacy by optimizing drug and radiation doses. It is reasonable to expect that a study with larger sample sizes, higher TAS-116 concentrations, and/or a more frequent dosing schedule will confirm that TAS-116 radiosensitization is a useful strategy to enhance the efficacy of radiotherapy.

We also measured mouse body weight to determine whether TAS-116 induced severe side effects. No significant changes were observed in body weights of TAS-116–treated groups (Fig. 6C); thus, it is likely that TAS-116 has minimal effect on normal tissue, consistent with our finding that TAS-116 did not radiosensitize nontransformed HFL1 cells. The ability of TAS-116 to induce radiosensitivity to tumors without causing noticeable damage to host mice supports the use of this drug as a safe and effective adjunct to radiotherapy.

Hsp90 inhibitors have shown promise as monotherapy antitumor agents (31, 32). Hsp90 inhibitors modulate oncogenic/growth-regulatory proteins (32) and DNA damage response proteins, which enhances radiosensitivity and suggests their use as adjuncts to radiotherapy (6, 19, 33–35). It has been proposed that by targeting multiple cancer relevant pathways, Hsp90 inhibitors might be effective against multiple cancer types and highly heterogeneous cancers (19, 32). However, use of early Hsp90 inhibitors has been limited because of serious side effects, including ocular toxicity, spurring the development of new Hsp90 inhibitors with improved bioavailability, greater specificity to Hsp90 in cancer cells, and reduced side effects. TAS-116 is a recently developed Hsp90 inhibitor with lower ocular toxicity than other Hsp90 inhibitors (10). This study is the first to investigate the radiosensitizing effects of TAS-116, and we demonstrate that TAS-116 sensitizes cancer cells to both low and high LET radiation.

Moreover, TAS-116 has little or no effect on radiosensitivity of noncancerous cells. Although we used nontransformed HFL1 cells as a normal cell control, rather than primary human cells, the selective effects of TAS-116 we observed are consistent with previously reported cancer-specific effects of Hsp90 inhibitors (3, 30). This selectivity of Hsp90 inhibitors has been traced to their high binding affinity to the altered conformation of Hsp90 expressed in cancer cells (4, 5, 31). Thus, TAS-116 follows this pattern as it did not affect the radiosensitivity of noncancerous cells, suggesting TAS-116 as a promising radiosensitizer with selective effects toward cancer cells. DSB repair inhibition is an important mechanism for radiosensitization, and prior studies with other Hsp90 inhibitors have demonstrated their effects on DSB repair pathways. For example, 17-AAG decreased RAD51 expression following irradiation of cancer cells with X-rays or carbon ions (2, 3), and 17DMAG attenuated X-ray–induced DNA-PKcs activation (34). It is well established that impairment of these proteins significantly inhibits DSB repair and enhances ionizing radiation–induced cell death (18, 19). Most research on DSB repair inhibition by Hsp90 inhibitors has focused on either HR or NHEJ, not both pathways. Takahashi and colleagues (22), however, suggested that the inhibition of both HR and NHEJ repair pathways will cause greater radiosensitization than the impairment of either repair pathway alone (22). A unique feature of the current study is the demonstration that TAS-116 selectively radiosensitizes cancer cells by downregulating expression of RAD51, Ku70, and DNA-PKcs, three proteins with critical roles in DSB repair by HR and NHEJ repair pathways. Thus, TAS-116 is a very promising candidate radiosensitizer that is likely to be more effective than agents that affect only one DSB repair pathway.

Another mechanism by which Hsp90 inhibitors may radiosensitize tumor cells is by altering cell-cycle distributions. Hsp90 inhibitors are known to affect cell-cycle distributions by impairing cell-cycle checkpoint proteins (24, 36). The Hsp90 inhibitors GA and 17-AAG were shown to downregulate cdc2 and cdc25c, induce G₂–M arrest, and inhibit cell growth of glioblastoma and lung cancer cell lines (25, 37). These studies demonstrated that cdc25c and cdc2 are client proteins of Hsp90 and that GA, and its derivative 17-AAG, alters chaperone complexes, leading to degradation of these key cell-cycle proteins. Consistent with these reports, we found that TAS-116 decreases cdc25c expression in HeLa cells, leading to G₂–M arrest and inhibition of cell growth. Treatment with X-rays or carbon ions also induced G₂–M arrest, and combined treatment with TAS-116 and radiation caused more pronounced G₂–M arrest than either individual treatment. These results suggest another potential mechanism by which TAS-116 radiosensitizes tumor cells by altering cell-cycle distribution and enhancing radiation-induced growth arrest.

Because low and high LET radiation induce different types of DNA damage that appear to differentially engage DSB repair pathways (17, 38, 39), it is not surprising that radiosensitizing agents sometimes show differential effects with low versus high LET radiation. For example, gemcitabine sensitizes human myeloma cell lines to low LET (⁶⁰Co) γ rays, but not high-LET α particles (40). This result is consistent with the finding that gemcitabine only affects HR, not NHEJ repair of DSBs. High LET radiation induces more complex DSB damage than low LET radiation, and repair of complex DSB damage appears to require the interplay of both HR and NHEJ repair pathways (41). Importantly, we found that TAS-116 sensitizes cancer cells to the cytotoxic effects of both low LET X-rays and high LET carbon ions, consistent with its suppression of key HR and NHEJ repair proteins. These results indicate that TAS-116 is an excellent candidate for tumor radiosensitization as an adjunct to low and high LET radiotherapy.

In summary, we demonstrated that the novel Hsp90 inhibitor TAS-116 sensitizes cancer cells to carbon ions as well as X-rays and that it has no radiosensitizing effect on noncancerous cells. TAS-116 suppressed the two major DSB repair pathways that are central to tumor radioresistance, by decreasing expression of the key HR and NHEJ proteins RAD51, Ku70, and DNA-PKcs, by suppressing phosphorylation/activation of DNA-PKcs, and by stimulating G₂–M arrest via suppression of cdc25c. TAS-116 gave favorable results in a preliminary in vivo study of tumor radiotherapy response, justifying further optimization of this promising radiosensitizer to augment radiotherapy with low or high LET radiation.

No potential conflicts of interest were disclosed.

Conception and design: Y. Lee, S. Sunada, R. Okayasu

Development of methodology: S. Sunada

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Lee, H. Hirakawa, A. Fujimori, R. Okayasu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Lee, S. Sunada, H. Hirakawa, J.A. Nickoloff, R. Okayasu

Writing, review, and/or revision of the manuscript: Y. Lee, S. Sunada, J.A. Nickoloff, R. Okayasu

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Sunada, H. Hirakawa, J.A. Nickoloff, R. Okayasu

Study supervision: R. Okayasu

We thank Drs. Shuichi Ohkubo and Hiromi Muraoka for valuable discussions during the preparation of this manuscript. We are thankful to the HIMAC operators for their support during carbon ion irradiation.

This work was supported by JSPS KAKENHI grants (nos. 24249067 and 23390301; to R. Okayasu), NIHR01 GM084020 (to J.A.Nickoloff), and was also a part of Research Project with Heavy Ions at NIRS-HIMAC.

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.