LY2109761

TGF-b/Smads Signaling Affects Radiation Response and Prolongs Survival by Regulating DNA Repair Genes in Malignant Glioma

To understand the molecular mechanism underlying the causal relationship between aberrant upregulation of transforming growth factor beta (TGF-b) and radio-resistance in glioma. The mouse glioma cell GL261 was irradiated, and relative expression of TGF-b/Smad signaling genes was determined by real-time PCR and western blotting. The DNA repair response on exogenous TGF-b or LY2109761 was evaluated by quantification of diverse genes by real-time PCR and western blotting. Xenograft mice were employed for in vivo investigation to assess the response to irradiation and LY2109761 either alone or in combination. The expression of DNA repair genes was further determined in the xenograft tumor. The TGF-b/Smad signaling pathway was activated by radiation in the GL261 cell line. The exogenous complement of TGF-b significantly stimulated DNA repair response. Administration of LY2109761 suppressed DNA repair genes. Simultaneous treatment with LY2109761 abrogated the upregulation of DNA repair genes in GL261. In the xenograft tumor model, LY2109761 syner- gistically improved the therapeutic effect of radiation via improvement of sensitivity. Our data suggested that LY2109761 treatment re-sensitized glioma to radiation via antagonizing TGF-b/Smad-induced DNA repair.

Keywords: LY2109761, TGF-b, glioma, DNA repair, radiation

Introduction

LIOMA Is THE MOsT MALIgNANT and common primary human brain tumor originating from the glial cells in the brain or the spine, which comprise *30% of all brain and central nervous system tumors and 80% of all malignant brain tumors (Weller et al., 2015). The etiology of gliomas is poorly understood currently and deserves to be further pre- cisely defined. Notably, the hereditary genetic disorders such as neurofibromatosis (type 1 and type 2) and tuberous sclerosis complex are well recognized to predispose to incidence of this disease (Reuss and von Deimling, 2009). Epidemiological in- vestigations suggest that dietary N-nitroso compounds might influence the risk of gliomas in both children and adults (Dubrow et al., 2010).

Until recently, the potential causal linkage between elec- tromagnetic radiation from cell phone and brain tumor is in- creasingly suspected, though results from several large-scale studies demonstrated none of conclusive evidences. The cu- mulative evidences indicate that compromised DNA repair capacity is intimately associated with tumorigenesis of gliomas (Adel Fahmideh et al., 2014). Epigenetic repression of DNA repair gene is frequently found in sporadic glioblastoma, which, consequently, relates to the low expression of O 6- methylguanine-DNA methyltransferase (MGMT) due to either promoter methylation or microRNA inhibition in majority of glioblastoma specimens (Skiriute et al., 2012). The epigenetic suppression of ERCC1, a key factor in the nucleotide excision repair pathway, has also been demonstrated in 53% glioma specimens (Chen et al., 2010).

Under the conditions that DNA damage response is com- promised, the spontaneous DNA damages accumulate in cells at a higher level, which, in turn, cause increased mutation burden in glioma(Narayanan et al., 1997). Mutations in glio- mas frequently occur in either isocitrate dehydrogenase (IDH) 1 or 2 genes, with one of the somatic mutations in IDH1 oc- curring in about 80% of low-grade gliomas and secondary high-grade gliomas (Molenaar et al., 2014). Clinical man- agements of glioma predominantly include surgery, radiation therapy, and chemotherapy individually or in combination (Damodaran et al., 2014). However, most glioma is resistant to the conventional treatments and prognosis is generally poor (Osuka and Van Meir, 2017). Therefore, deep insights into the molecular mechanism underlying the resistance occurrence and new strategies to surmount this obstacle are desperately needed for clinical success.

Transforming growth factor beta (TGF-b) is a multifunc- tional cytokine belonging to a superfamily of transforming growth factors that is involved in a variety of biological processes, including cell proliferation, angiogenesis, immune- surveillance, and embryonic stem cell stemness and differ- entiation. Activated TGF-b complexes with other factors to form a serine/threonine kinase complex, which, subsequently, binds to TGF-b receptors, stimulates the downstream phosphorylation signaling cascade, and, eventually, leads to activation of diverse substracts and regulatory proteins (Massague, 2012). The aberrant high level of TGF-b ligands has been characterized in various malignant entities, including gliomas, wherein increased TGF-b is associated with tumor progression and unfavorable outcomes (Kjellman et al., 2000; von Bernstorff et al., 2001; Hawinkels et al., 2009).

In view of the elementary and multifaceted roles of TGF- b in gliomas, the target therapeutics has been extensively exploited for clinical applications (Uhl et al., 2004; Ikush- ima et al., 2009). A novel TGF-bR-I kinase inhibitor, LY2109761, features SMAD2-selective inhibitory profile and possesses wide-spectrum anti-tumor properties in vari- ous tumor models (Fransvea et al., 2008; Melisi et al., 2008; Zhang et al., 2009). However, the potential association be- tween high TGF-b signaling and radio-resistance of glioma is still elusive so far, which prompts us to clarify this pos- sibility and elucidate the underlying molecular mechanism.

Methods

Cell culture

The mouse glioma cell line GL261 was purchased from and authenticated by the American Type Culture Collection (ATCC, VA). The cells were maintained in RPMI-1640 medium (Hyclone, MO) containing 10% fetal bovine serum (Gibco, CA) and 1% penicillin/streptomycin. Cells were cultured in a humidified CO2 (5%) incubator at 37°C. The exponentially growing cells were harvested by trypsin di- gestion for the following analysis.

Irradiation

For cell irradiation assay, totally 500 indicated cells were seeded in a 60-mm petri dish and followed by 4 h of attachment. The irradiation was administrated at 8 Gy for 30 min with a 200 kV X-ray source (RS-2100 Biological irradiator; Rad Source Technologies, MO). The irradiated cells were har- vested, and protein and total RNA was extracted respectively. For xenograft tumor irradiation, the recipient mice received 5 · 2 Gy of irradiation treatments from day 0 for 5 consecutive days postinoculation. The xenograft tumor growth in irradi- ated animals was monitored daily and measured with digital calipers. The tumor volume was calculated by the formula: volume (V) = length · width2/2.

Real-time PCR

The total RNA from indicated cells was isolated with Trizol reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s instructions. The concentration was measured by Nanodrop 2000 (ThermoFisher, MA), and the integrity was determined by agarose gel. One microgram RNA was then reversely transcribed into cDNA by using a commercial High-Capacity cDNA Reverse Transcription Kit (ThermoFisher) according to the manufacturer’s in- structions. The real-time PCR was performed with PowerUp SYBR Green Master Mix (ThermoFisher) on ABI PRISM 7900HT. Relative expression was calculated by the 2-DDCt method and normalized to GAPDH. The primer sequences used for real-time PCR were available on request.

Western blotting

The protein lysates were prepared in RIPA lysis buffer on ice for 30 min, and cell debris were completely discarded via centrifugation at 4°C. The protein concentration was deter- mined by BCA Protein Assay Kit (ThermoFisher). An equal amount of protein was resolved by SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membrane on ice. The PVDF membrane was briefly blocked with skim milk (5% in TBST buffer with 0.05% Tween-20) and hy- bridized with primary antibodies at 4°C overnight. After rigorous washing with TBST for 30 min and incubation with secondary antibodies, the protein was visualized by using the enhanced chemiluminescence (ECL; Millipore, CA) method.

Xenograft mouse model

The immunodeficient NPG mice (4–6 weeks ole, 20 per group) were purchased from Vitalstar (China) and accli- mated for 1 week in a pathogen-free environment. All the animal-related experiments were performed in strict accor- dance with the protocol preapproved by the Animal Care and Use Committee of Yidu Central Hospital of Weifang (20160809). Briefly, the single-cell suspension (107 cells/mL) was prepared in HEPES buffer via trypsin digestion and mixed with an equal volume of Basement Membrane Ex- tracts (R&D System, MN) on ice. The mixture was subcu- taneously inoculated into bilateral flanks immediately. The tumor growth was monitored regularly.

DNA transfection

siRNA transfections were performed by using Lipo- fectAMINE 2000 (Invitrogen) according to the manufac- turer’s instructions. Analyses of vehicle groups were performed by using empty LipofectAMINE 2000. The cells were transfected with control siRNA, MGMT siRNA (Santa Cruz Biotechnology) at a final concentration of 10 nM. Overexpression of MGMT in GL261 was transfected by adenovirus vector. To produce adenovirus, the linearized construct DNA was transfected into 293 cells by using Lipo- fectamine 2000™ following the manufacturer’s instruc- tions. After 24 h, the cells were fed fresh medium, and incubation was continued for an additional 5 days.

Virus was released from the cells by freezing and thawing for three consecutive cycles. After the third freeze–thaw cycle, the cells were briefly centrifuged to pellet the debris and the lysate was kept in clean and sterile centrifuge tubes and stored at -20°C for future use. For adenovirus trans- duction, GL261 were plated in six-well plates, and the next day the adenovirus was added with an MOI of 10 in 2 mL of medium. The plate was centrifuged at 220 g for 90 min at 37°C. Then, the cells were incubated in a 5% CO2 incubator for an additional 4 h. Next, the medium was removed and fresh complete growth medium was added. The cells were incubated for another 24 h and were then ready for analysis.

Clonogenic assay

Cells were plated in 25 cm2 flasks, irradiated with 6 MV X-rays (Mevatron; Siemens) for a single dose of 2–8 Gy at a dose rate of 2.5 Gy/min, and returned to the incubator for 10 to 14 days. Colonies formed were stained with crystal violet (Sigma, MO) and those with at least 50 cells were counted by microscopic inspection. The linear quadratic equation was fitted to data sets to generate survival curves, and dose enhancement factor for drugs was calculated at 10% sur- viving fraction (DEF0.1).

Statistics

Data presented in this study were acquired from at least three independent experiments unless indicated. Data were processed and analyzed with PRISM 7.0, and they were expressed as mean – standard error of mean (SEM). Stu- dent’s t-test and one-way analysis of variance (ANOVA) followed by multiple t-test were employed for statistical comparison. The statistical significances were calculated as p-values, and p < 0.05 was considered statistically different. Results The genes of TGF-b/Smads signaling pathway were upregulated by radiation in GL261 cell line We first evaluated the alteration of TGF-b/Smads sig- naling in response to irradiation in vitro. The mouse glioma cell GL261 was employed for this purpose, and irradiation was administrated at 30 Gy for 30 min. The key genes along this pathway including TGF-b1, TGF-b2, TGF-b3, TGF-bR I, TGF-bR II, TGF-bR III, Smad3, Smad4, and Smad7 were determined by real-time PCR. Although no notable changes in TGF-b1 (Fig. 1A) and TGF-bR III (Fig. 1F) were de- tected, a significant induction of TGF-b2 (Fig. 1B), TGF-b3 (Fig. 1C), TGF-bR I (Fig. 1D), TGF-bR II (Fig. 1E), Smad3 (Fig. 1G), and Smad4 (Fig. 1H) was observed in GL261 cells postirradiation. In contrast, Smad7, the inhibitory factor of TGF-b/Smad signaling, was greatly suppressed (Fig. 1I), which consistently indicated the activation of the TGF-b/Smad pathway on irradiation in GL261 cells. The genes of DNA repair were upregulated by additional TGF-b2 in GL261 cell line The DNA repair capacity was increasingly recognized to relate with radiotherapy resistance in a variety of human ma- lignancies (Frosina, 2009). Next, we challenged GL261 cell with TGF-b2 to mimic the activated TGF-b/Smad, and the potential influence on DNA repair was interrogated by mea- surement of specific genes involved in DNA repair. Arrays of DNA repair genes, including MGMT, OGG1, MLH1, RAD50, and MSH2, were quantified by real-time PCR and western blotting. As shown in Figure 2A–E, all the earlier mentioned genes were significantly upregulated at transcriptional level on TGF-b2 treatment. The increased functional proteins were further consolidated by western blotting, as shown in Figure 2F and Supplementary Fig. S1 (Supplementary Data are available online at www.liebertpub.com/dna). Our results suggested a significantly provoked DNA repair response in the presence of TGF-b2, which might eventually contribute to occurrence of radio-resistance. The genes of DNA repair were downregulated by LY2109761 in GL261 cell line Our previous results demonstrated that an exogenous complement of TGF-b2 stimulated DNA damage response in GL261 cells. Next, we sought to further consolidate this phe- nomenon via specific inhibition of TGF-b/Smad signaling. To this purpose, here we employed LY2109761, the dual inhibitor of both TGF-bR I and TGF-bR II. In contrast to exogenous administration of TGF-b2, treatment with LY2109761 re- markably suppressed DNA repair gene expression in both transcripts and proteins (Fig. 3A–F, Supplementary Fig. S2). Our data confirmed the positive correlation between DNA repair and TGF-b/Smad signaling in glioma cells. FIG. 1. The genes of the TGF-b/Smads signaling pathway were upregulated by radi- ation in the GL261 cell line. The cells were irradiated with 8 Gy for 30 min. Control group (Ctrl) was without irradiation. (A–I) qPCR was used to test the mRNA level of the genes. Data are reported as means – SEM values and representative of at least three independent experiments. *p < 0.05;**p < 0.01 versus the control group (Stu- dent’s t-test). SEM, standard error of mean; TGF-b, transforming growth factor beta. FIG. 2. The genes of DNA repair were upregulated by additional TGF-b2 in the GL261 cell line. The cells were treated with or without (Veh) TGF-b2 (0.5 ng/mL) for 24 h. (A–E) qPCR was used to test the mRNA level of the genes. (F) Western blotting was used to test the protein level of the genes. The bar graph showed the intensities of gene expression compared with GAPDH. Data are reported as means – SEM values and represen- tative of at least three independent experiments. *p < 0.05; **p < 0.01 versus the vehicle group. The effects of LY2109761 with radiation on DNA repair genes in GL261 cell line Next, we applied LY2109761 on the irradiated GL261 cells and characterized the relative changes of the DNA repair gene. Consistent with previous observations, radiation elicited a significant increase of all examined DNA repair genes, which clearly indicated the stimulated DNA repair capacity. Likewise, treatment with LY2019761 alone greatly sup- pressed endogenous TGF-b/Smad signaling and, in turn, DNA repair response. Simultaneous administration of LY2109761 and irradiation significantly compromised the activation of DNA repair genes in comparison with irradiation alone (Fig. 4A–E). The relative changes of the earlier mentioned genes were further verified at protein level (Fig. 4F and Sup- plementary Fig. S3). Our results highlighted the potential of LY2109761 to antagonize the activation of DNA damage response elicited by irradiation in glioma cells. FIG. 3. The genes of DNA repair were downregulated by LY2109761 in the GL261 cell line. The cells were treated with or without (Veh) LY2109761(LY, 10 M) for 1 h. (A–E) qPCR was used to test the mRNA level of the genes. (F) Western blotting was used to test the protein level of the genes. The bar graph showed the intensities of gene expression compared with GAPDH. Data are reported as means – SEM values and representative of at least three independent experiments. *p < 0.05; **p < 0.01 versus the vehicle group. FIG. 4. The effects of LY2109761 with radiation on DNA repair genes in the GL261 cell line. The cells were treated with or without LY2109761 (LY, 10 M) for 1 h, and they were irradiated with 8 Gy for 30 min. Veh: neither LY2109761 nor radiation; Veh+RT: without LY2109761, but irradiated with 8 Gy for 30 min; LY: with LY2109761, but without radiation; LY+RT: with LY2109761 and irradiated with 8 Gy and lysed 30 min later. (A–E) qPCR was used to test the mRNA level of the genes, related to the vehicle group. Data are reported as means – SEM values and representative of at least three independent experiments. *p < 0.05; **p < 0.01 versus the without RT groups. (F) Western blotting was used to test the protein level of the genes. FIG. 5. The effects of LY2109761 with radiation in vivo. (A) The tumor size with the growth of GL261 cells treated with or without LY2109761 (LY, 50 mg/kg/day twice daily), and irradiated with a fractionated schedule (5 · 2 Gy) starting on day 0 for 5 consecutive days. (B) Survival data in the different groups. Veh: neither LY2109761 nor radiation; Veh+RT: without LY2109761, but with radiation; LY: with LY2109761, but without radiation; LY+RT: with LY2109761 and radiation. *p < 0.05; **p < 0.01 versus the Veh groups. (C) qPCR was used to test the mRNA level for the DNA repair genes, related to the vehicle group. Data are reported as means – SEM values and representative of at least three independent experiments, n = 5. *p < 0.05; **p < 0.01 versus the without RT groups. The effects of LY2109761 with radiation in vivo Our previous results indicated the potential action of LY2109761 to re-sensitize glioma cells to irradiation in vitro. Next, we sought to consolidate this effect in vivo via intro- duction of a xenograft tumor model. The tumor-bearing mice were irradiated at 2 Gy for 5 consecutive days, and they were administrated with either vehicle or LY2109761 (50 mg/kg/ day, twice daily). As shown in Figure 5A, the xenograft tumor growth was significantly suppressed by either radiation or LY2109761 treatment individually. Notably, the synergistic effect was observed while mice were challenged with irradiation and LY2109761 simultaneously. The overall survival was de- termined in response to the indicated treatment as well. As shown in Figure 5B, both irradiation and LY2109761 signifi- cantly prolonged the overall survival of glioma-bearing mice with median survival time increased from 19 to 29 days and 27 days, respectively. Simultaneous administration of irradia- tion and LY2109761 remarkably improved survival extension. We further confirmed the involvement of DNA repair genes in response to either irradiation or LY2109761 alone or in combination. Consistent with previous in vitro observa- tions, DNA damage response was significantly stimulated in irradiated xenograft mice, which was subsequently suppressed by cotreatment with LY2109761 (Fig. 5C). Our data suggested that LY2109761 contributed to the re-sensitization of glioma cells to irradiation via suppression of DNA repair gene expression. Meanwhile, LY210976-enhanced radio-sensitivity in GL261 cells was completely blocked by simultaneous overexpression of MGMT (Fig. 6A). Likewise, the compromised radio-sensitivity conferred by TGF-b2 supplementation was restored by MGMT-knockdown (Fig. 6B). Our data further highlighted the importance of DNA repair capacity underlying radio-resistance in glioma. FIG. 6. The relationship between the TGF-b pathway and MGMT with radiation in GL261 cells. (A) The effects of LY2109761 in enhancing radio-sensitivity were blocked by overexpression of MGMT in GL261 cells. (B) The effects of TGF-b2 in reducing radio-sensitivity were blocked by siMGMT in GL261 cells. Clonogenic survival analysis was conducted. Data are reported as means – SEM values and rep- resentative of at least three independent experiments. *p < 0.05;**p < 0.01 versus the vehicle or siRNA group. MGMT, O 6- methylguanine-DNA methyltransferase.. Discussion The TGF-b/Smad signaling pathway physiologically plays critical roles in inflammatory reaction, damage repair, and embryonic development, which fundamentally regulates cell proliferation, differentiation, and immune functioning as well ( Massague, 2012). The emerging evidences dem- onstrated the significant elevation of TGF-b contents in serum and tissues from cancer patients, which positively correlates with tumor progression and indicates its intimate linkage with tumorigenesis in a variety of human malig- nancies (von Bernstorff et al., 2001). In this study, we cautiously profiled the TGF-b/Smad signaling pathway in response to irradiation in mouse gli- oma cell line GL261. Consistent with previous reports, we observed a significant increase of TGF-b2, TGF-b3, TGF- bR I, TGF-bR II, Smad3, and Smad4 alone with suppression of Smad7 postirradiation, which clearly demonstrated the activation of the TGF-b/Smad pathway in the radiotherapy recipient cells. The elegant studies performed by Zhang et al. (2011b) provided the proof of concept that TGF-bR I/ II inhibitor LY2109761 significantly improved the radio- resistance in human malignant glioma, which indicated the potential contribution of TGF-b/Smad to the irradiation sensitivity. To experimentally verify this notion and eluci- date its underlying molecular mechanism, here we further mimicked the activated TGF-b/Smad signaling in vitro via an exogenous complement with TGF-b2 in GL261 cells and observed remarkable upregulation of an array of DNA repair genes, including MGMT, OGG1, MLH1, RAD50, and MSH2. Since these genes are distributed in several canonical DNA repair pathways including direct repair, base excision re- pair, and non-homologous end joining repair, we, therefore, speculate multiple repair pathways, rather than a single one, convergently contributed to radio-resistance of glioma cells. The stimulated DNA damage response in response to TGF-b/ Smad mimicking eventually antagonized irradiation-imposed cell apoptotic effect and contributed to the radio-resistance. In another way, we employed the TGF-b/Smad-specific inhibi- tor, LY2109761, to inhibit endogenous TGF-b/Smad activity, wherein the physiological DNA repair capacity was greatly compromised. Our data unambiguously disclosed the directly regulatory effect of TGF-b/Smad on DNA repair and impli- cated the TGF-b/Smad pathway as the potential target for re-sensitization of glioma to radiotherapy. To better recapitulate the in vivo conditions, we further employed xenograft tumor mice to examine the anti-tumor effect of LY2109761 in combination with radiation. Significant delayed tumor progression and prolonged overall survival was observed on administration with LY2109761 in comparison with irradiation alone. We also consolidated the regulatory effect of LY2109761 on DNA repair genes in vivo. Therefore, our study highlighted the synergistic effect of LY2109761 with radiotherapy in resistant glioma, which might hold great clin- ical promise for further investigations. Notably, our data highlighted the fundamentally regulatory effect of TGF-b/Smad signaling on DNA repair response on irradiation. This action has been previously observed in several investigations. For instance, Kanamoto et al. (2016) performed a functional proteomics analysis in TGF-b1-stimulated MvlLu epithelial cells and identified Rad51 as a target of TGF-b1- dependent regulation of DNA repair, wherein decreased expression of Rad51 correlated with a decrease in DNA repair efficiency. Liu et al. (2014) proposed that TGF-b induced ‘‘BRCAness’’ and sensitivity to PARP inhibition in breast cancer by regulating DNA-repair genes, especially identified breast cancer 1, early onset (BRCA1) as a target down- regulated by TGF-b through the miR-181 family. In contrast, Lu et al. (2015) reported that TGF-b1 transiently triggered upregulation of mitochondrial MUTYH and induced expression of nuclear isoforms, which positively correlated with the severity of kidney fibrosis and responses to oxidative DNA repair. Mitra et al. (2013) demonstrated that Smad4 loss in mouse keratinocytes led to increased susceptibility to UV carcinogenesis with reduced ERCC1-mediated DNA repair. Consistent with the latter, here we consolidated the stimu- latory actions of TGF-b/Smad on DNA repair capacity via a modulation array of key genes underlying this cellu- lar process. Noteworthily, although we solidified the phe- nomenon that TGF-b/Smad stimulated DNA repair genes via both exogenous complement and specific inhibition, the fundamental molecular mechanism is still to be elucidated in future investigations. Conclusion The therapeutic values of LY2109761 were intensely exploited in a variety of human malignancies, and several studies suggested its modifying effect on radiation response. For example, Zhang et al. (2011a) demonstrated that addi- tion of LY2109761 in combination of radiation plus TMZ significantly improved clinical outcome in human glioblas- toma, especially in patients with unmethylated MGMT promoter status. Bouquet et al. (2011) reported that TGF-b inhibition increased the radio-sensitivity of breast cancer cells in vitro and promoted tumor control by radiation in vivo. Zhang et al. (2011b) found that blockade of TGF-b signaling by the TGF-bR I kinase inhibitor LY2109761 enhanced radiation response and prolonged survival in glioblastoma. Both our in vitro and in vivo studies consol- idated the beneficial effect of LY2109761 on radiation in treatment of human cancer. Further, we elucidated the un- derlying mechanism that LY2109761 improved the radio- sensitivity via antagonizing TGF-b/Smad-stimulated acti- vation of DNA repair response.