Attenuation of the DNA Damage Response by Transforming Growth Factor-Beta Inhibitors Enhances Radiation Sensitivity of Non–Small-Cell Lung Cancer Cells In Vitro and In Vivo

International Journal of

Radiation Oncology biology

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Biology Contribution

Attenuation of the DNA Damage Response by Transforming Growth Factor-Beta Inhibitors Enhances Radiation Sensitivity of NoneSmallCell Lung Cancer Cells In Vitro and In Vivo Shisuo Du, MD, PhD,* Sophie Bouquet, PhD,* Chen-Hao Lo, MS,* Ilenia Pellicciotta, PhD,* Shiva Bolourchi, MS,* Renate Parry, PhD,y and Mary Helen Barcellos-Hoff, PhD* *Department of Radiation Oncology, New York University School of Medicine, New York, New York; and yVarian Medical Systems, Palo Alto, California Received Jun 12, 2014, and in revised form Sep 15, 2014. Accepted for publication Sep 18, 2014.

Summary This study shows that TGF-b inhibition synergizes with RT through control of the DNA damage response, which leads to greater tumor cell kill. There are several TGF-b inhibitors in clinical trials. The excellent safety profiles demonstrated in these clinical trials, as well as the possibility of protection from late complications of RT, provide further motivation for assessing TGF-b inhibitors as an adjunct to RT for NSCLC.

Purpose: To determine whether transforming growth factor (TGF)-b inhibition increases the response to radiation therapy in human and mouse nonesmall-cell lung carcinoma (NSCLC) cells in vitro and in vivo. Methods and Materials: TGF-bemediated growth response and pathway activation were examined in human NSCLC NCI-H1299, NCI-H292, and A549 cell lines and murine Lewis lung cancer (LLC) cells. Cells were treated in vitro with LY364947, a small-molecule inhibitor of the TGF-b type 1 receptor kinase, or with the panisoform TGF-b neutralizing monoclonal antibody 1D11 before radiation exposure. The DNA damage response was assessed by ataxia telangiectasia mutated (ATM) or Trp53 protein phosphorylation, gH2AX foci formation, or comet assay in irradiated cells. Radiation sensitivity was determined by clonogenic assay. Mice bearing syngeneic subcutaneous LLC tumors were treated with 5 fractions of 6 Gy and/or neutralizing or control antibody. Results: The NCI-H1299, A549, and LLC NSCLC cell lines pretreated with LY364947 before radiation exposure exhibited compromised DNA damage response, indicated by decreased ATM and p53 phosphorylation, reduced gH2AX foci, and increased radiosensitivity. The NCI-H292 cells were unresponsive. Transforming growth factor-b signaling inhibition in irradiated LLC cells resulted in unresolved DNA damage. Subcutaneous LLC tumors in mice treated with TGF-b neutralizing

Reprint requests to: Mary Helen Barcellos-Hoff, PhD, Department of Radiation Oncology, New York University, School of Medicine, 566 First Ave, New York, NY 10016. Tel: (212) 263-3021; E-mail: [email protected] This research was supported by Varian Medical Systems. Conflict of interest: M.H.B.-H. reports grants from Varian Medical Systems and nonfinancial support from Genzyme during the conduct of the Int J Radiation Oncol Biol Phys, Vol. 91, No. 1, pp. 91e99, 2015 0360-3016/$ - see front matter Ó 2015 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ijrobp.2014.09.026

study; S.B. finished her postdoctoral studies at New York University (and so the experiments in this article) and went to work for Roche, where she is an employee. AcknowledgmentsdThe authors thank Dr Raheleh Hatami for comments on the manuscript.

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antibody exhibited fewer gH2AX foci after irradiation and significantly greater tumor growth delay in combination with fractionated radiation. Conclusions: Inhibition of TGF-b before radiation attenuated DNA damage recognition and increased radiosensitivity in most NSCLC cells in vitro and promoted radiation-induced tumor control in vivo. These data support the rationale for concurrent TGF-b inhibition and RT to provide therapeutic benefit in NSCLC. Ó 2015 Elsevier Inc.

Introduction Improved treatment of nonesmall-cell lung cancer (NSCLC), which is one of the leading causes of deaths from cancer worldwide, is urgently needed (1). Approximately 70% of NSCLC patients receive radiation therapy (RT), either alone or in combination with other treatment modalities, such as surgery or chemotherapy. Biologically augmenting tumor cell radiosensitivity can play a crucial role in determining treatment success (2). Transforming growth factor (TGF)-b ligands are enriched in the tumor microenvironment, where their production by stromal or tumor cells varies according to the tumor phenotype. In NSCLC, increased TGF-b activity correlates with tumor progression and increased tumor angiogenesis (3, 4). Active TGF-b signaling has been identified as a key factor for chemotherapy resistance in NSCLC (4), in addition to its well-recognized impact promoting tumor progression and metastasis. TGF-b activation is efficiently induced by ionizing radiation, in part owing to the presence of a redox sensitive motif in the latency associated peptide (reviewed in ref. [5]). Growing evidence supports a specific role for TGF-b in mediating the rapid execution of the DNA damage response (DDR) (6). Normal Tgfb1 null keratinocyte cell lines irradiated in vitro exhibit reduced autophosphorylation of ataxia telangiectasia mutated (ATM) protein and its kinase activity (7). The latter then results in decreased phosphorylation of substrate proteins upon DNA damage, including DNA damage transducers Chk2, p53, and Rad17, and by decreased gH2AX foci, which is a chromatin modification near DNA double-strand breaks; together these events impair cell survival. Notably, human cells treated with a TGF-b type 1 receptor kinase small-molecule inhibitor phenocopies murine genetic depletion. TGF-b signaling blockade augments response to chemoradiation in glioblastoma (GBM) preclinical models (8, 9) and specifically inhibits GBM cancer stem cell renewal after radiation (10). TGF-b inhibition promotes clonogenic cell death in irradiated mouse and human GBM and breast cancer cell lines in vitro. Systemically neutralizing TGF-b enhances RT actions in GBM and breast tumor preclinical models (8, 10, 11). Little is known about the contribution of TGF-b to NSCLC response to radiation. On the basis of the efficacy in breast and brain tumors, it is plausible that TGF-b inhibition could also improve radiosensitivity of NSCLC.

Currently, TGF-b inhibitors are in clinical trials for several types of cancer, including GBM and breast cancer (reviewed in ref. [12]). Clinically viable inhibitors of TGFb signaling include small-molecule inhibitors of TGF-b type 1 receptor kinase and neutralizing antibodies that have low toxicity. Clinical trials involving TGF-b inhibition in combination with RT in patients with breast cancer and GBM are under way (ClinicalTrials.gov identifiers: NCT01401062, NCT01220271). Here, we determined the effect of TGF-b signaling blockade after irradiation in human NSCLC cell lines and murine Lewis lung carcinoma (LLC) tumors. TGF-b inhibition increased radiosensitivity and compromised DDR in 3 of 4 NSCLC cell lines in vitro, and the combination of TGF-b neutralizing antibody with fractionated RT significantly increased LLC tumor growth control in vivo. These results suggest that concurrent TGF-b inhibition and RT may provide therapeutic benefit in the clinical setting for NSCLC.

Methods and Materials Cell culture Human NCI-H1299, NCI-H292, A549, and murine LLLC cells were purchased from American Type Culture Collection (Manassas, VA). The LLC cells were cultured in Dulbecco’s modified Eagle medium GlutaMAX (Gibco Grand Island, NY) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO). Human NCIH1299, NCI-H292, and A549 cells were cultured in Roswell Park Memorial Institute 1640 medium with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO). Cells were treated in 10% serum replacement medium (Knockout SR; Life Technologies, Carlsbad, CA) containing either 500 pg/mL TGF-b1 (R&D Systems, Minneapolis, MN), 400 nM small-molecule inhibitor of the TGF-b type 1 receptor kinase LY364947 ([3-(Pyridin-2-yl)-4(4-quinonyl)]-1H-pyrazole; Lilly designation HTS466284; catalog no. 616451; Calbiochem, St. Louis, MO), or 10 mg/mL 1D11, a pan-isoform neutralizing TGF-b monoclonal antibody, or 13C4, a murine monoclonal isotype control antibody (kindly provided by Genzyme, Framingham, MA). For growth studies, cells were trypsinized and counted using a Vi-Cell Beckman Coulter counter (Indianapolis, IN) at 24 hours and 48 hours after plating.

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Clonogenic assay To assess clonogenic survival of cells irradiated in monolayer culture, human and murine lung cancer cell lines were grown for 48 hours to approximately 70% confluence, at which point media was replaced with serum replacement media. Cells were treated with 400 nmol/L of LY364947 kinase inhibitor or 10 mg/mL pan-specific TGF-beneutralizing antibody 1D11 or control antibody 13C4 for 48 hours before and 3 hours after radiation exposure. Cells were irradiated with graded doses of up to 8 Gy using a Varian Clinac 2300 C/ D linear accelerator with 1-cm bolus below (Varian Medical Systems, Palo Alto, CA). Colony formation of 3 biological replicates was averaged for each treatment and corrected according to plating efficiency of respective controls. Sensitization dose enhancement ratios (DER10) were calculated as the ratio of doses required to achieve 10% surviving fraction for cells without and with TGF-b inhibition.

gH2AX foci Tumor cryosections or cells grown on chamber slides were fixed using 2% paraformaldehyde for 20 minutes at room temperature, followed by permeabilization with 100% methanol for 20 minutes at 20 C. Then specimens were blocked with the supernatant of 0.5% casein/phosphate-buffered saline, stirred for 1 hour, and incubated with a mouse monoclonal antibody against gH2AX (clone JBW301; Upstate Biotechnology, Lake Placid, NY) overnight at 4 C as previously described (11). Specimens were imaged using a 40  objective with 0.95 numerical aperture Zeiss Plan-Apochromat objective on a Zeiss Axiovert (Carl Zeiss, Oberkochen, Germany) equipped with epifluorescence. All images were acquired with a charge-coupled device Hamamatsu Photonics (Hamamatsu City, Japan) monochrome camera at 1392  1040 pixel size, 12 bits per pixel depth, using the Metamorph imaging platform (Molecular Devices, Sunnyvale, CA).

Comet assay The LLC cells were cultured and treated with 400 nmol/L of LY364947 kinase inhibitor as described above and irradiated with 5 Gy. The LLC cells were dissociated 0.5 or 4 hours after irradiation for single-cell gel electrophoresis at 19 V (300 mAM, 40 minutes) and analysis by neutral comet assay (Trevigen, Gaithersburg, MD) according to the manufacturer’s instructions. SYBR green-stained DNA comets were imaged at 400  magnification, and the extent of DNA breaks was quantified as tail moment using CometScore (TriTek Corp, Summerduck, VA) software.

Western analysis Cells were grown in complete media for 48 hours, treated with LY364947, irradiated with 5 Gy, and lysed after 1 hour. Protein estimation was carried out using the BCA protein

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assay kit from Pierce (Rockford, IL). One hundred micrograms of protein was electrophoresed on a 4% to 15% gradient gel from BioRad (Hercules, CA) and transblotted on polyvinylidene difluoride membrane. The immunoblots were incubated with one of the following primary antibodies at 1/500 dilution: Smad2 serine 465/467 phosphorylation (Cell Signaling, Danvers, MA), Smad2/3 (BD, Franklin Lakes, NJ), p53 serine 15 phosphorylation (Cell Signaling), p53 (Neomarkers, Fremont, CA), ATM serine1981 phosporylation (Epitomics, Burlingame, CA), and ATM, clone 2C1 (GeneTex, Irvine, CA) and detected with infrared labeled antibodies using a LiCor Odyssey system (Lincoln, NE).

Tumor studies All animal experiments were carried out in accordance with guidelines specified by the New York University institutional animal care and use committee. Female 6- to 8-weekold C57BL/6 mice obtained from Taconic (Hudson, NY) were housed in a temperature-controlled animal care facility with a 12-hour light/dark cycle and allowed chow and water ad libitum. The LLC cells (105) were injected into the right flank of mice and allowed to grow until the tumors reached an average size of 60-80mm3. Animals were distributed randomly into groups (nZ5-10) to receive 1D11 or 13C4 control antibody (10 mg/kg, intraperitoneal injection) 24 hours before localized irradiation and were injected every other day to termination. Radiation was delivered at 600 cGy/min with 6-MV x-rays with a Varian Clinac 2300 C/D linear accelerator fitted with a 25-mm radiosurgery conical collimator (Varian Medical Systems, Palo Alto, CA). Superflab bolus (1.5-cm tissue equivalent material) was placed over the tumor.

Statistics Data are presented as mean  SEM. Statistical comparison between 2 groups was performed using the Student t test. One-way analysis of variance (ANOVA) was used to test for differences among 3 or more groups. Differences were considered statistically significant when the P value was <.05.

Results Response of human and murine NSCLC cell lines to TGF-b Mutational inactivation of the TGF-b signaling pathway in human NSCLC is associated with specific histologic subtypes, more aggressive tumor behavior, and reduced patient survival (2). NCI-H1299 and NCI-H292 are aggressive tumor cell lines established from a lymph node metastasis of a pulmonary carcinoma, the A549 cell line is from a primary human adenocarcinoma, and LLC is a highly metastatic murine lung cancer cell line. Because NSCLC cells can selectively evade TGF-b growth regulation while maintaining signaling, we first

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Fig. 1. TGF-b signaling and growth regulation in nonesmall-cell lung cancer cell lines. (A) Western detection of p-smad2 and total Smad2 in NCI-H1299, NCI-H292, LLC, and A549 cells. TGF-b treatment induced phosphorylation of Smad2, which was blocked by LY364947 pretreatment. Quantitation of the ratios of phosphorylated protein/total protein normalized to TGF-betreated alone is indicated below each lane. All cell lines were competent to signal via TGF-b receptors. (B) Growth of NSCL cancer cell lines 24 and 48 hours after treatment with the small-molecule inhibitor LY364947 (SMI), TGFb, or TGF-b þ SMI. Values represent ratios of viable cells in untreated control to treatment groups (nZ4). Significance was obtained using an unpaired, 2-tailed Student t test; *P<.05, **P<.01. examined whether the TGF-b canonical pathway was intact, as indicated by Smad2 phosphorylation (p-Smad2) upon exposure to TGF-b. Induction of p-Smad2 by a short TGF-b treatment was suppressed by 50% to 75% by the TGF-b smallmolecule inhibitor LY364947 (Fig. 1A). These data indicate that receptor-mediated signaling in response to TGF-b is intact in these 4 NSCLC cell lines. Because cell proliferation status confers an important component of the tumor cell radiosensitivity, we tested NSCLC tumor sensitivity to TGF-bemediated growth inhibition. Only H292 cells were growth-inhibited by TGF-b, whereas NCI-H1299, A549, and LLC cells were refractory to TGF-bemediated growth regulation (Fig. 1B).

Blocking TGF-b signaling attenuates DNA damage response in NSCLC cells in vitro We next assessed components of the DDR pathway previously determined to be affected by TGF-b in nonmalignant human cells and cancer cell lines (7, 10, 11). Phosphorylation of ATM Ser1981 was attenuated after LY364947 small molecule inhibitor (SMI) treatment in 3 of 4 cell lines. No effect of LY364937 was observed in NCI-H292 cells. Although p53 was undetectable in NCI-H1299 cells carrying a Tp53 homozygous deletion, LY364947 significantly decreased phosphorylation of ATM at serine 1981. Both ATM autophosphorylation and p53 phosphorylation at serine 15 were decreased by SMI

treatment of LLC cells before irradiation (Fig. 2A). As found in our prior studies (10, 11), radiation-induced gH2AX foci formation was markedly reduced by pretreatment with LY364947 in NCI-1299, A549, and LLC cells in vitro (Fig. 2B). Quantitation confirmed the absence of response in NCI-H292 cells (Fig. 2C). A compromised DDR could either delay or prevent resolution of DNA double-strand breaks (13). Here we tested the consequences of TGF-b inhibition in LLC cells using neutral comet assays to investigate the effect of TGF-b inhibition on DNA damage induction at 30 minutes and resolution at 4 hours after irradiation with 5 Gy (Fig. 3A). The initial comet tail moment was similar for irradiated cells with or without LY364947, indicating comparable initial DNA damage. Most radiation-induced double-strand breaks are repaired in the first 1 to 6 hours after RT (14). The comet tail moment decreased compared with 30 minutes after 5 Gy. In contrast, the comet tail moment failed to decline in irradiated cells treated with LY364947 (Fig. 3B, C). This persistent DNA damage indicates that treatment with LY364947 impairs repair of DNA damage.

Inhibition of TGF-b signaling sensitizes NSCLC cell lines to irradiation The persistence of DNA damage should compromise cell survival. The clonogenic assay is a gold standard for

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Fig. 2. TGF-b inhibition impedes radiation-induced DNA damage response. (A) Protein phosphorylation of ATM at serine 1981 and Trp53 at serine 15 was assessed by Western blot for human NCI-H1299, NCI-H292, and A549 cells and murine LLC cells. Cells were treated with the TGF-b inhibitor LY364947 for 24 hours, irradiated with 2 Gy, and lysed 30 minutes later. Phosphorylation was reduced by at least 25% in 3 of 4 cell lines. NCI-H292 did not show a reduction of phosphorylation. (B) TGF-b inhibition with LY364947 significantly decreased gH2AX foci (green) in human NCI-H1299, A549, and NCI-H292 and murine LLC. Nuclei are counterstained with 40 ,6_Diamidino_2_Phenylindole (blue). (C) Quantitation of gH2AX foci demonstrates a significant reduction in the number of radiation-induced gH2AX foci with LY354947 in 3 of 4 irradiated NCSLC cell lines (P<.0001; analysis of variance). NCI-H292 did not show a reduction of foci. Magnification: 40  . estimating radiosensitivity. Human cell lines NCI-H1299 and A549 were radiosensitized by pretreatment with LY364947, consistent with the DDR-related proteins analysis. Consistent with the lack of SMI effect on DDR, radiosensitization was not evidenced in NCI-H292 cells (Fig. 4A-C). Radiosensitization was evident in a large-cell

lung cancer carcinoma cell line, NCI-H460, when TGF-b was inhibited (data not shown). We next compared the SMI with a pan-isoform TGF-b neutralizing antibody, 1D11, using the LLC cell line. Murine LLC cells were comparably radiosensitized by pretreatment with either SMI (Fig. 4D) or 1D11 (Fig. 4E).

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Fig. 3. Unrepaired DNA damage assessed by neutral comet assay. (A) A representative image is shown for each treatment from the neutral comet assay in LLC cells 4 hours after 5 Gy and/or TGF-b inhibitor LY364947 (magnification, 40  ). (B, C) The comet tail moment was measured in LLC cells 30 minutes (B) and 4 hours (C) after 5 Gy. Significance was determined using an unpaired 2-tailed Student t test; *P<.05, **P<.01. NS Z nonsignificant. Cells were exposed to 5 Gy ionizing radiation (IR).

Combination of TGF-b inhibitor with fractionated RT increases LLC tumor growth delay The TGF-b neutralizing antibodies currently in clinical development have demonstrated safety and efficacy in several studies (15, 16). We first determined whether the antibody achieved a biologically effective distribution to affect DDR in tumors by assessing gH2AX foci staining in tumors harvested 1 hour after irradiation with 2 Gy. Similar to the in vitro data from LLC cells, induction of gH2AX foci by radiation was reduced in irradiated tumors excised from mice treated with 1D11 compared with control antibody (Fig. 5A, B). To test the efficacy in combination with RT, we next examined mice bearing LLC subcutaneous tumors treated with TGF-b neutralizing antibodies or in combination with fractionated RT. Subcutaneous LLC tumors were treated with 5 daily fractions of 6 Gy and/or 1D11 or 13C4 (10 mg/kg, intraperitoneal). Mice treated with 1D11 alone exhibited no significant change in tumor growth compared with control 13C4 antibodyetreated mice. However, tumor growth delay was significantly enhanced by 1D11 when combined with RT compared with mice receiving RT and 13C4 antibody (Fig. 5C). Note that tumor regrowth initiated approximately 5 days after RT was considerably reduced in 1D11-treated irradiated tumors (Fig. 5D). Tumor volume at the experiment termination

(day 27) was significantly smaller for mice treated with 1D11 and RT when compared with RT and 13C4 antibody (Fig. 5E). Interestingly, more than half the mice (6 of 11) showed tumor growth rate decrease after the first 2 fractions of 6 Gy when treated with 1D11, in which the mean volume increment was 7.9  6.9 mm3 (tumor weight 0.74  0.10 g). However, tumor growth in 13C4 antibodyetreated mice had the same growth rate as the nonirradiated tumors, with a mean volume increase of 34.8  9.7 mm3 (tumor weight 0.34  0.04 g) (Fig. 5F). These data support the potential benefit of TGF-b inhibition in the context of RT, which attenuates the DDR, radiosensitizes tumor cells, and promotes tumor control in vivo.

Discussion Our study demonstrates that TGF-b inhibition by either pharmaceutical or biological means compromises the DNA damage recognition, repair, and ultimately tumor cell survival in the majority of NSCLC preclinical cell lines. The murine tumor cell line and 2 of 3 human NCSCL cell lines were radiosensitized independent of sensitivity to TGF-bemediated growth inhibition. NCI-H1299 cells, which carry a homozygous deletion of the p53 protein, were still sensitized by TGF-b inhibition, whereas TGF-b

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Fig. 4. TGF-b inhibition increases radiosensitivity in vitro. Radiation survival curves are shown for untreated cells (black squares line) and cells pretreated with a TGF-b inhibitor (gray circles) for 48 hours before exposure to graded doses of radiation. Cells were plated 3 hours after irradiation for clonogenic survival analysis. (A) NCI-H1299, (B) A549, (C) NCI-H292, and (D) LLC cells treated with LY364947. (E) LLC cells treated with 1D11. The dose enhancement ratio at 10% survival (DER10) is indicated on the graphs. Mean  SEM values of triplicate determinations are shown. NCIH292 was not radiosensitized. Significance was determined using analysis of variance with Tukey posttest; *P<.05. NS Z nonsignificant. inhibition did not alter DDR or radiosensitivity of NCIH292, despite the apparent activity of TGF-b signaling. In vivo efficacy was demonstrated using LLC syngeneic subcutaneous tumors, in which tumor growth control was significantly improved by use of neutralizing antibodies concurrent with fractionated RT. These data suggest that this strategy might be effective in many lung cancer patients. Deoxyribonucleic acid is the main target for radiationinduced cell killing. Because there is considerable redundancy in the cell’s ability to repair DNA damage, modulating the response to ionizing radiation through the inhibition of DNA repair has been a longstanding aim in translational RT research (17). Previously, we demonstrated that TGF-b is required for efficient ATM activation in epithelial cells (7), that either chemical or genetic inhibition of TGF-b signaling in cells led to reduced ATM activation, and that TGF-b inhibition increased tumor cell radiosensitivity in breast and brain tumor cell lines (10, 11). Consistent with our earlier studies, here we show that TGF-b inhibition before irradiation resulted in reduced phosphorylation of ATM, H2AX, and p53 in most cultured NSCLC cells. Moreover, LLC tumors in mice treated with

TGF-b inhibitors before radiation exposure in vivo exhibited less gH2AX foci formation, a nuclear marker of the rapid molecular radiation response and significantly greater growth control after fractionated RT. Interestingly, 1D11 neutralizing antibodies showed immediate tumor growth inhibition after 2 fractions. At this time point after IR, tumor cell death results mainly from aberrant repair of radiation-induced DNA damage. This is consistent with a direct effect on radiosensitvity due to compromising the DNA damage recognition. Nevertheless, the exact mechanism or pathways involving this TGF-b inhibition that attenuates IR-induced DDR has not been determined but seems to be p53 independent (11). Nonesmall-cell lung cancer consists of heterogeneous histologies, with the most common types being adenocarcinoma, large-cell carcinoma, and squamous cell carcinoma. The differential response to antitumor treatments among these different histologies is a general problem in treating NSCLC (18). Because most NSCLC harbor mutations with different clinical characteristics, it is not surprising that until now only one drug, crizotinib, used in EML4-ALKepositive patients, has proven to be particularly effective for this specific subpopulation. This

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Fig. 5. TGF-b inhibition combined with fractionated radiation therapy increases LLC tumor growth delay. (A) Immunostaining of gH2AX (green; blue, 40 ,6_Diamidino_2_Phenylindole) foci 1 hour after 2 Gy in tumors of mice receiving 1D11 TGF-b neutralizing antibody or control antibody 13C4 administered 24 hours before tumor irradiation. Fewer foci are evident after RT and 1D11 compared with RT and 13C4. (B) Quantification of mean intensity of gH2AX inside mask defined by nuclear staining. (C) Averaged tumor growth curves (nZ7-8) for mice after 5  6 Gy daily fractions and/or treated with 1D11 or 13C4 (mean  SEM). (D) Individual tumor growth curves for each treatment. Note delayed regrowth of tumors treated with RT and 1D11 compared with RT alone. (E) Individual tumor weights at termination. (F) Tumor volume change after the first 2 fractions of 6 Gy RT was evaluated using an unpaired 2-tailed Student t test. Asterisks denote significant differences (*P<.05, **P<.01). IR Z ionizing radiation. “tailored treatment” has resulted in an unprecedented survival benefit (19, 20). Here, we found that TGF-b inhibition had little effect on IR-induced DDR-related proteins in NCI-H292, which were not radiosensitized. This suggests that that benefit will be restricted to certain tumors; however, further studies are critical to identify the molecular biomarkers that will indicate the potential benefits from inhibiting TGF-b in RT. Therapeutic resistance and normal lung damage are major challenges for effective RT in NSCLC patients. Hypofractionated stereotactic body RT allows for escalation of the fractional dose. This is important for improving not only the local control rate of tumors but also the overall survival for medically inoperable patients with early-stage NSCLC (21). Radiation-induced normal tissue damage is still the biggest obstacle, which limits the dose escalation of stereotactic body RT in NSCLC (22-24). An approach with enormous appeal involves the development of nontoxic, yet effective, molecularly targeted radiosensitizers that could reduce normal tissue toxicity without affecting control. It is expected that chemotherapymediated inhibition of DNA repair mechanisms would synergize the TGF-b inhibition, as shown in combination with temozolomide and IR in GBM preclinical models (8).

If a drug selectively sensitizes the tumor response to radiation, one could use selective escalation of the effective biological dose to the tumor, thereby improving local tumor control without increasing morbidity. Drugs such as gemcitabine, cisplatin, and docetaxel have been demonstrated to radiosensitize tumors in NSCLC (25). Although these sensitizers increase the rates of local tumor control and in some cases overall survival (26), they are not specific for tumor cells and can also increase radiation toxicity to normal tissues. This nonspecific mechanism of action is one of the major limiting factors of many radiation modulators. Because the lung represents the most RT-sensitive tissue, this results in fatal complications. Among the many cytokines involved in this process, TGF-b is thought to play a pivotal role (27, 28). TGF-b is overexpressed at sites of injury, which contributes to delayed tissue fibrosis after radiation and chemotherapy. Various interventional therapies that aim to block TGF-b signaling or decrease tissue levels of TGF-b have proven to be effective in reducing the development of fibrosis in the lung, liver, and intestines (27-29). Reduced normal tissue injury and increased tumor control probability by TGF-b inhibition has significant appeal in RT dose escalation for lung cancer.

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Conclusions Achieving effective targeting the TGF-b pathway is a promising novel treatment option for NSCLC patients, because it selectively increases tumor cell kill and local tumor control while potentially limiting late effects in the surrounding normal tissue.

References 1. Kruse JJ, Floot BG, te Poele JA, et al. Radiation-induced activation of TGF-beta signaling pathways in relation to vascular damage in mouse kidneys. Radiat Res 2009;171:188-197. 2. Begg AC, Stewart FA, Vens C. Strategies to improve radiotherapy with targeted drugs. Nat Cancer Rev 2011;11:239-253. 3. Hasegawa Y, Takanashi S, Kanehira Y, et al. Transforming growth factor-beta1 level correlates with angiogenesis, tumor progression, and prognosis in patients with nonsmall cell lung carcinoma. Cancer 2001; 91:964-971. 4. Huang S, Ho¨lzel M, Knijnenburg T, et al. Med12 controls the response to multiple cancer drugs through regulation of TGF-b receptor signaling. Cell 2012;151:937-950. 5. Du S, Barcellos-Hoff MH. Biologically augmenting radiation therapy by inhibiting TGF-b actions in carcinomas. Sem Radiat Oncol 2013; 23:242-251. 6. Barcellos-Hoff MH, Cucinotta FA. New tricks for an old fox: Impact of TGFbeta on the DNA damage response and genomic stability. Sci Signal 2014;7:re5. 7. Kirshner J, Jobling MF, Pajares MJ, et al. Inhibition of TGFb1 signaling attenuates ATM activity in response to genotoxic stress. Cancer Res 2006;66:10861-10868. 8. Zhang M, Herion TW, Timke C, et al. Trimodal glioblastoma treatment consisting of concurrent radiotherapy, temozolomide, and the novel TGF-b receptor I kinase inhibitor LY2109761. Neoplasia 2011; 13:537-549. 9. Mengxian Z, Kleber S, Ro¨hrich M, et al. Blockade of TGF-b signaling by the TGFbR-I kinase inhibitor LY2109761 enhances radiation response and prolongs survival in glioblastoma. Cancer Res 2011;71: 7155-7167. 10. Hardee ME, Marciscano AE, MedinaRamirez CM, et al. Resistance of glioblastoma-initiating cells to radiation mediated by the tumor microenvironment can be abolished by inhibiting transforming growth factor-b. Cancer Res 2012;72. Epub 2012 Jun 2012. 11. Bouquet SF, Pal A, Pilones KA, Demaria S, Hann B, Akhurst RJ, Babb JS, Lonning SM, DeWyngaert JK, Formenti S, BarcellosHoff MH. Transforming growth factor b1 inhibition increases the radiosensitivity of breast cancer cells in vitro and promotes tumor control by radiation in vivo. Clin Cancer Res 2011;17:6754-6765. 12. Akhurst RJ, Hata A. Targeting the TGFb signalling pathway in disease. Nat Rev Drug Discov 2012;11:790-811.

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13. Thompson LH. Recognition, signaling, and repair of DNA doublestrand breaks produced by ionizing radiation in mammalian cells: The molecular choreography. Mutat Res 2012;751:158-246. 14. Bristow RG, Hill RP. Hypoxia and metabolism: Hypoxia, DNA repair and genetic instability. Nat Rev Cancer 2008;8:180-192. 15. Yingling JM, Blanchard KL, Sawyer JS. Development of TGF-beta signalling inhibitors for cancer therapy. Nat Rev Drug Discov 2004; 3:1011-1022. 16. Akhurst RJ. Large- and small-molecule inhibitors of transforming growth factor-beta signaling. Curr Opin Investig Drugs 2006;7: 513-521. 17. Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature 2009;461:1071-1078. 18. Scagliotti GV, Parikh P, von Pawel J, et al. Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer. J Clin Oncol 2008;26:3543-3551. 19. Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol 2009;27:4247-4253. 20. Villaruz LC, Socinski MA. Personalized therapy for non-small cell lung cancer: Which drug for which patient? Semin Thorac Cardiovasc Surg 2011;23:281-290. 21. Lo SS, Fakiris AJ, Chang EL, et al. Stereotactic body radiation therapy: A novel treatment modality. Nat Rev Clin Oncol 2010;7:44-54. 22. Asai K, Shioyama Y, Nakamura K, et al. Radiation-induced rib fractures after hypofractionated stereotactic body radiation therapy: Risk factors and doseevolume relationship. Int J Radiat Oncol Biol Phys 2012;84:768-773. 23. Welcsh P, Lee M, Gonzalez-Hernandez R, et al. BRCA1 transcriptionally regulates genes involved in breast tumorigenesis. Proc Natl Acad Sci U S A 2002;99:7560-7565. 24. Marquis S, Rajan J, Wynshaw-Boris A, et al. The developmental pattern of brca1 expression implies a role in differentiation of the breast and other tissues. Nat Genet 1995;11:17-26. 25. Segawa Y, Kiura K, Takigawa N, et al. Phase III trial comparing docetaxel and cisplatin combination chemotherapy with mitomycin, vindesine, and cisplatin combination chemotherapy with concurrent thoracic radiotherapy in locally advanced non-small-cell lung cancer: OLCSG 0007. J Clin Oncol 2010;28:3299-3306. 26. Topkan E, Parlak C, Topuk S, et al. Outcomes of aggressive concurrent radiochemotherapy in highly selected septuagenarians with stage IIIb non-small cell lung carcinoma: Retrospective analysis of 89 patients. Lung Cancer 2013;81:226-230. 27. Anscher MS, Kong F, Murase T, et al. Normal tissue injury after cancer therapy is a local response exacerbated by an endocrine effect of TGF beta. Br J Radiat 1995;68:331-333. 28. Anscher MS, Thrasher B, Zgonjanin L, et al. Small molecular inhibitor of transforming growth factor-beta protects against development of radiation-induced lung injury. Int J Radiat Oncol Biol Phys 2008; 71:829-837. 29. Du S-S, Qiang M, Zeng Z-C, et al. Radiation-induced liver fibrosis is mitigated by gene therapy inhibiting transforming growth factor b signaling in the rat. Int J Radiat Oncol Biol Phys 2010;78:1513-1523.