Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene

Radiotherapy and Oncology xxx (2017) xxx–xxx

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Original article

Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene Vincent Hung-Shu Chang a,1, Yi-Chih Tsai b,1, Ya-Li Tsai b, Shu-Ling Peng c, Su-Liang Chen b, Tsung Ming Chang b, Winston Chun-Yuan Yu a, Hui-Ju Ch’ang a,d,⇑ a The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei; b National Institute of Cancer Research, National Health Research Institutes, Miaoli County ; c Department of Pathology, National Cheng Kung University Hospital, Tainan; and d Department of Radiation Oncology, Taipei Municipal Wanfang Hospital, Taiwan

a r t i c l e

i n f o

Article history: Received 8 August 2016 Received in revised form 29 December 2016 Accepted 1 January 2017 Available online xxxx Keywords: Krüpple-like factor 10 Radio-sensitivity Pancreatic cancer UV radiation resistance-associated gene

a b s t r a c t Background and purpose: Krüpple-like factor 10 (Klf10), an early response gene of TGFb, was reported to be a prognostic biomarker for pancreatic cancer survival. The role of Klf10 in predicting tumor response to cancer treatment is unknown. Materials and methods: Genetically manipulated MiaPaCa and Panc-1 cells were established to evaluate clonogenic survival, autophagy, apoptosis and DNA repair after radiation. The interaction between Klf10 and UV radiation resistance-associated gene (UVRAG) was demonstrated by ChiP-PCR and luciferase reporter assay. Orthotopic murine tumor model and clinical specimens were used to evaluate radiosensitivity of pancreatic cancer. Results: We found Klf10 silencing correlates with enhanced pancreatic cancer clonogenic survival and murine tumor growth after radiation. UVRAG was an essential down-stream mediator transcriptionally suppressed by Klf10. Silencing UVRAG mRNA in Klf10 depleted Panc-1 cells reversed the radioresistant phenotypes including decreased apoptosis and enhanced DNA repair as well as autophagy. Metformin, an anti-diabetic agent, was found to increase Klf10 and suppress UVRAG expression to improve radiation cytotoxicity in pancreatic cancer. The predictive value of Klf10 in radiation response and the inverse correlation with UVRAG were confirmed in cohorts of pancreatic cancer patients. Conclusions: Klf10 is a potential biomarker in predicting and sensitizing radiation effect in pancreatic cancer. Ó 2017 Published by Elsevier Ireland Ltd. Radiotherapy and Oncology xxx (2017) xxx–xxx

Patients with pancreatic cancer are usually diagnosed at late stage with less than 20% being able to receive curative intent surgery. Radiotherapy is considered a staple of therapy for localized pancreatic cancer to either prevent local failure or shrink the tumor to become resectable [1,2]. However randomized clinical trials have shown mixed results of radiotherapy to improve survival or quality of life in pancreatic cancer patients [2,3]. Preclinical studies demonstrated intrinsic radio-resistance of pancreatic cancer cells [4,5]. The molecular mechanisms of pancreatic cancer resistance warrant further exploration. Pancreatic cancer is well-known for its de-regulated TGFb signal pathway due to activated Ras mutation [6]. While the role of TGFb in radiation-induced fibrosis has been extensively investigated, radiation-induced TGFb signals in tumor suppression remains ⇑ Corresponding author at: R1-2034, 35 Keyan road, Zhunan, Miaoli County 35053, Taiwan. E-mail address: [email protected] (H.-J. Ch’ang). 1 Contribute equally to this manuscript.

largely unexplored. The absence or low expression level of TGFbRII or Smad 4 is thought to be the reason for resistance to radiotherapy [7]. Investigators demonstrated that abrogation of endogenous TGFb function causes increased proliferative potential as well as increased resistance to radiation-induced cytotoxicity. Restoration of TGFb signaling pathway in p53 deficient pancreatic cancer cells led to enhanced sensitivity to radiation [8]. Krüppel-like factor 10 (Klf10), originally termed TGFb inducible early gene 1, can be rapidly induced within sixty minutes of TGFb treatment [9]. It contains three zinc finger domains at C-terminal and proline-rich Src homology-3 binding domains at N terminal, which bind SP1 protein for transcriptional regulation [9,10]. Klf10 positively regulates TGFb signaling by promoting Smad 2 phosphorylation and suppressing inhibitory Smad 7 gene transcription. Klf10 has been reported to be a tumor suppressor [11,12] and to be down-regulated in human cancers [13–15]. We have reported the correlation between loss of Klf10 immunolabeling and shortened survival among ninety-five pancreatic cancer patients [16]. In this study, we investigated the role and

http://dx.doi.org/10.1016/j.radonc.2017.01.001 0167-8140/Ó 2017 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Chang V-HS et al. Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene. Radiother Oncol (2017), http://dx.doi.org/10.1016/j.radonc.2017.01.001

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Klf10 regulates radio-sensitivity via UVRAG

mechanisms of Klf10 in predicting treatment response to radiotherapy in pancreatic cancer. Klf10 was found to modulate radiation-induced apoptosis, autophagy and DNA damage repair via regulating UV radiation resistance-associated gene (UVRAG) at the transcriptional level. Metformin, an oral anti-diabetic agent, enhanced Klf10 expression and sensitized pancreatic cancer to radiotherapy by down-regulating UVRAG.

Materials and methods

Immuno-fluorescence Assays were described in Supplement Materials and Methods 5. Primary antibodies including Anti-UVRAG (1:50, Santa Cruz sc82115), anti-LC3B (1:1000, Cell Signaling#2775) and anti-cH2AX (1:400, Cell Signaling#9718) were used to detect cancer cells with autophagy activity or DNA double strand breaks.

Annexin V-Propidium Iodide (PI) assay As described in Supplement Materials and Methods 6.

Cell culture and chemicals Human pancreatic cancer cell lines, Panc-1 (60284) and MiaPaCa (60139), were purchased from Bioresource Collection and Research Center, Taiwan, and were authenticated by DNA finger printing. Resistant cell lines were generated as described in Supplement Materials and Methods 1. MiaPaCa and Panc-1 cells labeled with the firefly luciferase plasmid vector (MiaPaCa-Luc, Panc-1-Luc) were kindly provided from Dr. Kelvin K.C. Tsai from NICR, NHRI. Metformin and Compound C were purchased from Sigma–Aldrich.

Subcellular fractionation As described in Supplement Materials and Methods 7.

Acid vesicular organelle (AVO) assay As described in Supplement Materials and Methods 8.

TdT-mediated nick end labeling (TUNEL) assay Animals and orthotopic xenograft Mice were housed at the animal core facility of NHRI, Taiwan. The facility was approved by the National Association for Accreditation of Laboratory Animal Care, Taiwan, and was maintained in accordance with the regulations and standards of NHRI Animal Council’s procedural and ethical guidelines. NOD/SCID mice (NOD.CB17-Prkdcscid/Cr1Nar1) were purchased from National Laboratory Animal Center. Orthotopic tumor studies were described in Supplement Materials and Methods 2.

Radiation and tumor tissue collection Whole abdomen irradiation of 7.5 Gy was delivered using a 160 kV RS 2000 X-ray biological Irradiator (Rad Source Technologies) at a dose rate of 16.55 mGy/s at 25 mA. Mice were put in plexiglass chambers with slight physical restraint to expose abdomen area to the radiation field. The cranium, thorax, pelvis and limbs of mice were protected from lead shield. Animals were observed twice a day, and tissue samples were obtained at specific time points or from animals displaying agonal breathing.

Clonogenic assay and MTT assay As described in Supplement Materials and Methods 3.

Immuno-precipitation and western blotting Assays were described in Supplement Materials and Methods 4. We used anti-LC3B (1:500, Sigma L7543), anti-UVRAG (1:1000, Cell Signaling#5320), ant-Beclin-1 (1:1000, Cell Signaling#3495), antiPARP (1:1000, Cell Signaling#9532), anti-PI3KC3 (1:1000, Invitrogen #PA5-34735), anti-caspase 3 (1:1000, Cell Signaling#9662), anti-cH2AX (1:500, Millipore#05–636), anti-Ku80 (1:1000, Cell Signaling#2180) and anti-DNA-PKcs (phosphoT2609, 1:500, Abcam ab13856) to detect autophagy, apoptosis and DNA damage repair-related markers, respectively. The Klf10 antibody was raised in rabbits using a full-length peptide supplied by LTK BioLaboratories (Taiwan) [17]. A b-actin antibody (MAB1501, C4, Chemicon) at a 1:5,000 dilution was used as control. We used anti-DNP-PKcs (1:100, Santa Cruz sc-101664), anti-beclin (1:100, Cell Signaling #3495) for immune-precipitation.

As described in Supplement Materials and Methods 9.

Plasmid end-joining assay As described in Supplement Materials and Methods 10.

Transfection and transduction As described in Supplement Materials and Methods 11.

Generation of stable doxycycline inducible Klf10 clones The Lenti-XTM Tet-OneTM Inducible Expression Systems (Clontech) was used for generation of stable doxycycline inducible clones of Klf10 insert under the TRE3G promoter in MiaPaCa cells (Supplement Materials and Methods 12). The clone that demonstrated the highest Klf10 protein expression was expanded and labeled as MiaPaCa-Klf10 and used in all of the studies described.

Chip-PCR and reverse transcription PCR (RT-PCR) analysis The EZ ChIP chromatin immune-precipitation kit (Millipore) was used according to the manufacturer’s protocol (Supplement Materials and Methods 13).

Plasmid construction and promoter luciferase assay As described in Supplement Materials and Methods 14.

In vivo imaging system (IVIS) As described in Supplement Materials and Methods 15.

Tumor regression grade, quantitative histology and immunehistochemistry The tissue slides were examined independently by two observers (S.L.P. and C.C.) who were masked to both the clinical and pathological data. Tumor regression grade, immunehistochemistry and quantification are described in Supplement Materials and Methods 16.

Please cite this article in press as: Chang V-HS et al. Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene. Radiother Oncol (2017), http://dx.doi.org/10.1016/j.radonc.2017.01.001

V.H.-S. Chang et al. / Radiotherapy and Oncology xxx (2017) xxx–xxx

Patient specimens and statistics We retrospectively reviewed twenty pancreatic cancer patients receiving preoperative chemoradiotherapy followed by surgery and another seventy surgically resected pancreatic cancer patients from NCKUH that had been collected during 2003 and 2014. The study was approved by institutional review board of NCKUH (HR-99-033). We used nonparametric tests to compare independent groups of data. Correlations among numerical variables were assessed by Pearson’s correlation analysis. All of the statistical analyses were performed using the SPSS statistical software version 10.0.

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Down-regulated Klf10 correlated with enhanced AVO expression over two-fold (Fig. 3B), decreased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) signals of
10 fold, (Fig. 3C) and better DNA end-joining from 0.8% to 2.3% (Fig. 3D) after radiation. To reverse the radio-resistant phenotype of Panc-1 cells with Klf10 mRNA-silencing, we further down-regulated UVRAG (Suppl. Fig. 3B). The levels of autophagy, apoptosis and DNA repair (Fig. 3B–D) as well as that in orthotopic murine tumor model (Suppl. Fig. 4) were significantly recovered to those of vector control or silencing UVRAG mRNA alone. We concluded that UVRAG might be an essential down-stream mediator of Klf10 in regulating radio-sensitivity of pancreatic cancer.

Results Klf10 transcriptionally suppressed UVRAG promoter activity Klf10 expression correlated with radio-sensitivity of pancreatic cancer Using Panc-1 cells with increasing resistance to radiation (Fig. 1A) or gemcitabine (Suppl. Fig. 1A), we found the protein expression level of Klf10 decreased with increasing resistance. Genetic modulation also showed a two-fold increase in clonogenic cytotoxicity of MiaPaCa cells with doxycycline (Dox)-inducible Klf10 over-expression (Fig. 1B; 45–80% at 2 Gy) and a nearly twofold increase in survival of Panc-1 cells after Klf10 mRNA silencing (Suppl Fig. 1B; 42–78% at 2 Gy). In orthotopically implanted murine model, we demonstrated that over-expressing Klf10 radiosensitized pancreatic tumors of MiaPaCa (Fig. 1C). A tumor growth delay of three weeks was noted after radiation to pancreatic tumors with Klf10 over-expression (Fig. 1D, dotted black line). On the other hand, silencing Klf10 mRNA of Panc-1 cells led to significantly aggressive tumor growth despite irradiation (Fig. 4C, dotted vs. solid black line; Suppl. Fig. 4 second & third panels). Tumor growth was not significantly changed by over-expressing or silencing Klf10 alone.

To demonstrate physical interaction between Klf10 and UVRAG, we designed a ChiP-PCR assay of Panc-1 cells. We found overexpressing Klf10 in Panc-1 cells had enhanced UVRAG promoter binding (Suppl. Fig. 5A, upper panel). RT-PCR also showed that Klf10 negatively regulates mRNA level of UVRAG (Suppl. Fig. 5A, lower panel). In luciferase-labeled UVRAG promoter assay, there was a dose dependent decrease in promoter activity after TGFb or Klf10 treatment in Panc-1 cells (Suppl. Fig. 5B). Two SP/KLF binding sites were predicted in UVRAG promoter region by TFsearch. (http://www.cbrc.jp) These SP/KLF sites were located at 22 to 12 (1BS), and at 48 to 39 (2BS) (Suppl. Fig. 6). The inhibitory effect of Klf10 to UVRAG promoter activity was reversed completely and partially by mutation over 1BSmu and 2BSmu, respectively, on UVRAG (Suppl. Fig. 5C). We also found that SP1 might compete with Klf10 for UVRAG promoter binding and transcription (Suppl. Fig. 5D). From the above observations, we concluded that Klf10 transcriptionally regulated UVRAG to modulate radio-sensitivity of pancreatic cancer cells.

Klf10 regulated radio-sensitivity of pancreatic cancer cells by modulating autophagy, apoptosis and DNA repair

Metformin increased Klf10 expression via AMPK and enhanced radiosensitivity of pancreatic cancer

After radiation, Klf10 expression elevated within half an hour, reached maximum at 4 hours and recovered to baseline by eight hours (Suppl. Fig. 3A, left panel). We choose 4 hours after irradiation in our following experiments if not specified otherwise. In MiaPaCa cells with Klf10 over-expression, we found decreased levels of autophagy-related proteins including beclin and LC3B 2 conversion; increased apoptosis with abundant PARP and caspase 3 cleavages; and elevated DNA damage showing up-regulated c-H2AX and slightly elevated phospho-DNA-PKcs (Fig. 2A, left panel). The findings were further evaluated by fluorescence imaging and flow-cytometry (Suppl. Fig. 2A–C). Down-regulating Klf10 in Panc-1 cells reversed the phenomenon observed (Fig. 2A, right panel). We found Klf10 over-expression interfered with the interaction between beclin and PI3KC3 for autophagosome formation (Fig. 2B). Bax translocation to nuclei was enhanced by up-regulated Klf10 level (Fig. 2C). Binding between DNA-PKcs and Ku70/80 during DNA repair was diminished by overexpressing Klf10 (Fig. 2D). We concluded that Klf10 regulated radio-sensitivity of pancreatic cancer by modulating autophagy, apoptosis and DNA damage repair.

Since Klf 10 knockout mice prone to develop metabolic disease, and 50 adenosine monophosphate activated protein kinase (AMPK) could phosphorylate Klf10 at Thr189, (unpublished data) we tried to up-regulate Klf10 expression via AMPK. Metformin, a first-line oral anti-diabetic agent, an AMPK activator, and also a radiosensitizer, might enhance Klf10 expression in a dose- (Fig. 4A, left panel) and time- (Fig. 4A, right panel) dependent pattern. Using compound C, a phospho-AMPKa inhibitor, we demonstrated that metformin regulated Klf10 expression by modulating AMPK activity (Fig. 4B). Incorporating metformin in radiation to Panc-1 cells, we found elevated Klf10 and suppressed UVRAG expression (Fig. 4D, upper panel). Metformin sensitized shKlf10 Panc-1 tumors, in orthotopic murine tumor model, to radiation (Fig. 4C, black long dash vs black dot line). Tumor tissue lysates collected at six weeks after radiation showed elevated Klf10 and decreased UVRAG expression by metformin (Fig. 4D, lower panel). We demonstrated that pharmacologic manipulation of Klf10 might modulate radio-sensitivity of pancreatic cancer.

Klf10 regulates radio-sensitivity of pancreatic cancer cells via UVRAG Using Chip-Chip assay, we found UVRAG to be one of the candidate genes regulated by Klf10 [17]. UVRAG was induced by radiation in a similar pattern as that of Klf10 to a lesser extent (Suppl. Fig. 3A). However, there was inverse expression of Klf10 in UVRAG in various irradiated cancer cell lines including pancreas, oral (OECM-1) and colon (HCT116) after genetic manipulation (Fig. 3A).

Klf10 immuno-labeling correlated with pancreatic cancer response to radiotherapy To evaluate the predictive value of Klf10 in radiotherapy response in pancreatic cancer, we collected paired surgical specimens from twenty pancreatic cancer patients, receiving chemoradiotherapy followed by curative intent surgery (Suppl. Table 1). Eleven patients had pathological good tumor regression grade (TRG 2/3; Fig. 5A upper panel); while another nine had poor tumor response (TRG 4/5; Fig. 5A lower panel). The patients with better

Please cite this article in press as: Chang V-HS et al. Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene. Radiother Oncol (2017), http://dx.doi.org/10.1016/j.radonc.2017.01.001

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Klf10 regulates radio-sensitivity via UVRAG

Fig. 1. Klf10 expression is associated with radiation sensitivity of pancreatic cancer. (A) Upper panel: Representative immunoblots of Klf10 expression levels in parental Panc1 and Panc-1 cells selected by various cumulated dosage of radiation (R10, R22, R32, R55) described in Experimental Procedures. Densitometry results of three experiments were shown as fold change compared with control cells after normalization to b-actin. * denotes p < 0.05. Lower panel: Cumulated data of clonogenic survival of parental Panc-1 (black line) and radiation-resistant Panc-1 cell lines (gray lines). Each point and error bar represents mean value and standard error of at least three experiments. * represents p < 0.05. (B) Clonogenic survival of parental (solid line) and Klf10 over-expressing (dashed line) MiaPaCa cells after various dosage of radiation exposure. Insert immunoblots were the expression level of Klf10 in MiaPaCa cells with inducible Klf10 over-expression. Each point and error bar represents mean value and standard error of at least three experiments. * represents p < 0.05. (C) Representative weekly IVIS of mice orthotopically implanted with luciferase-labeled, inducible Klf10 over-expressing MiaPaCa cells as described in Experimental Procedures. Mice were treated with PBS or doxycycline (Dox) before exposing to mock or 7.5 Gy whole abdominal irradiation. (D) Cumulated data from at least 10 mice of each group from (C). Insert immunoblots were the expression level of Klf10 in inducible system of MiaPaCa cells after treatment with doxycycline. Each point and error bar represents mean value and standard error of at least two experiments. * represents p < 0.05.

tumor regression had higher extent-intensity (EI) score of Klf10 and lower EI score of UVRAG (Fig. 5B, upper panel); while the others with poor tumor regression had lower EI score of Klf10 and higher UVRAG EI score (Fig. 5B, lower panel). The correlation of Klf10 staining with pathologic response was significant (Fig. 5C,

correlation coefficient = 0.69, p = 0.001). Cumulated data of another seventy clinical specimens from pancreatic cancer patients receiving upfront surgery (Suppl. Table 2) showed inverse correlation between UVRAG and Klf10 immuno-staining (Fig. 5D, correlation coefficient = 0.259, p = 0.03). From the above observations,

Please cite this article in press as: Chang V-HS et al. Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene. Radiother Oncol (2017), http://dx.doi.org/10.1016/j.radonc.2017.01.001

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Fig. 2. Klf10 regulates apoptosis, autophagy and DNA damage repair of pancreatic cancer. (A) Left panel: Representative immunoblots of protein expression levels, 4 h after 5 Gy, of MiaPaCa (CTL) and MiaPaCa cells transfected with lipofectamine (Lipo) or Klf10 over-expressing (Klf10) plasmid. Markers denote autophagy (beclin, LC3B1/2), apoptosis (PARP, caspase 3) and DNA repair (cH2AX, phospho-DNA-PKcs) were measured. b-Actin was used as internal control. Right panel: Representative immunoblots of protein expression levels, 4 h after irradiation, of parental Panc-1 (CTL) and Panc-1 cells transfected with vector control (PLKO) or shKlf10 mRNA (shKlf10) using markers described above. The experiments were repeated at least twice. (B) Interaction of beclin and PI3KCIII, 4 h after 5 Gy, in parental MiaPaCa cells with (CTL) or without (N) beclin antibody immunoprecipitation; and MiaPaCa cells transfected with lipofectamine (Lipo) or Klf10 over-expressing (Klf10) plasmids. Densitometry results of three experiments were shown as fold change with standard error compared with control cells after normalization to beclin. * denotes p < 0.05. (C) Cytosol and nuclear distribution of bax protein, 4 h after irradiation, in MiaPaCa (CTL) or MiaPaCa cells transfected with lipofectamine (Lipo) or Klf10 over-expressing plasmids (Klf10). b-Actin and lamin B were used as cytosol and nuclear protein internal control, respectively. Densitometry results of three experiments were shown as fold change with standard error compared with control cells after normalization to lamin B. * denotes p < 0.05. (D) Interaction of DNA-PKcs and Ku80, 4 h after irradiation, in MiaPaCa cells with (CTL) or without (N) DNAPKcs antibody for immune-precipitation; and MiaPaCa cells transfected with lipofectamine (Lipo) or Klf10 over-expressing (Klf10) plasmids. Densitometry results of three experiments were shown as fold change with standard error compared with control cells after normalization to DNA-PKcs. * denotes p < 0.05.

we concluded that loss of Klf10 immuno-staining was associated with up-regulated UVRAG and radio-resistance in pancreatic cancer patients.

Discussion Pancreatic cancer was shown to have poor response to radiotherapy due to its intrinsic resistance and desmoplastic stroma

[18]. Clinically, the role of radiotherapy is still undefined in pancreatic cancer [2,3]. However, around thirty percent of pancreatic cancer patients died from local destructive disease despite adequate multi-modality therapy [19]. It is urgent to find novel biomarkers to predict treatment response to help clinicians establish personalized treatment; or to develop therapeutic targets to sensitize pancreatic cancer to radiotherapy. Pancreatic cancer cells with defective components of TGFb signaling pathway are radio-resistant. Restoration of TGFb signaling

Please cite this article in press as: Chang V-HS et al. Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene. Radiother Oncol (2017), http://dx.doi.org/10.1016/j.radonc.2017.01.001

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Fig. 3. Klf10 regulates apoptosis, autophagy and DNA damage repair of pancreatic cancer via UVRAG. (A) Representative immunoblots of Klf10 and UVRAG at 4 h after 5 Gy irradiation in various cancer cells including pancreatic (MiaPaCa, upper left panel; Panc-1, upper right panel), oral (OECM-1, lower left panel), and colon (HCT116, lower right panel) cancer cells. Control (CTL) cancer cells transfected with lipofectamine (Lipo), Klf10 over-expressing plasmid (Klf10) and Klf10 shRNA (shKlf10) were evaluated. Densitometry results of three experiments were shown as fold change with standard error compared with control cells after normalization to b-actin. * denotes p < 0.05. (A) AVO assay. Representative histograms (upper panel) and cumulative results (lower panel) of positive acridine orange staining, 4 h after 5 Gy irradiation, by flow cytometry in parental Panc-1 cells (Panc-1) or Panc-1 cells with stable transfection of control vector (PLKO), shRNA of Klf10 alone (shKlf10); shKlf10 Panc-1 cells with lipofectamine (shKlf10(Lipo)), or with shRNA of UVRAG plasmid (shKlf10 + shUVRAG); and Panc-1 cells transfected with UVRAG shRNA alone (shUVRAG). * denotes p < 0.05. Numeric data on right upper quadrant of histograms are percentages of AVO cells co-expressing green and red signals. (C) TUNEL assay. Representative histograms (upper panel; red line: experiment; black line: control) and cumulative results (lower panel) of DNA DSB, 4hr after irradiation, in Panc-1 cells described as in (B). Numeric data in histograms are percentages of TUNEL positive cells. (D) Plasmid end-joining assay. Panc-1 cells were transfected with HindIII-or Narl-digested DNA and assayed for luciferase activity. Repair efficiency was calculated from the luciferase activities of linearized reporter constructs compared with that of the uncut circular plasmid at 4 h after irradiation. * denotes p < 0.05. The experiments were repeated independently at least twice and presented as mean fold change and standard error.

might sensitize pancreatic cancer cells to radiation [8]. However, there is a non-canonical signal of TGFb involving inhibitory Smad 7 which stabilizes b-catenin and enhances tumor proliferation [20]. Pancreatic cancer cells with intact TGFb pathway tend to have reduced TGFb response to radiation due to induction of inhibitory Smad 7, leading to negative feedback repression of this signaling [21]. Klf10, transcriptionally activated early after TGFb signaling, provides positive feedback to the signaling not only by activating

phospho-Smad 2 but also by repressing Smad 7 expression [7]. To overcome the impediment in radiation-induced TGFb signaling, we evaluated Klf10 as a molecular target in predicting radiation response and modulating radio-sensitivity. From Chip-Chip assay [17], we found UVRAG to be one of the candidate genes transcriptionally controlled by Klf10. UVRAG was identified through its ability to complement UV sensitivity in xeroderma pigmentosum cells [22]. It activates

Please cite this article in press as: Chang V-HS et al. Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene. Radiother Oncol (2017), http://dx.doi.org/10.1016/j.radonc.2017.01.001

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Fig. 4. Metformin stabilized Klf10 and enhanced radio-sensitivity of pancreatic tumor. (A) Representative immunoblots of Klf10 expression level in Panc-1 cells after treatment with increasing dosage of metformin for 24 h (left panel); and 50 lM metformin for increasing time duration (right panel). (B) Representative immunoblots of Klf10 and phospho-AMPKa expression of Panc-1 cells after 50 lM metformin with concomitant Compound C at increasing dosages for 24 h (left panel); and 5 lM compound C for increasing time duration (right panel). b-Actin was used as internal control. The experiments were repeated independently at least twice. (C) Cumulated data of weekly IVIS signals of mice orthotopically implanted with luciferase-labeled Panc-1 cells or Panc-1 cells transfected with vector control (PLKO), Klf10 shRNA plasmid (Klf10shRNA). One week later, mice were exposed to mock (gray lines) or whole abdominal irradiation (black lines) and daily PBS or metformin (
1.25 mg/50 ll ip) as described in Experimental Procedures. There were at least six mice in each group from two independent experiments. Each point represents mean ± SE. * represents p < 0.05. (D) Upper panel: Representative immunoblots of Klf10 and UVRAG expression in Panc-1 cells treated with irradiation alone (IR), 50 lM metformin without (Met) or with 5 Gy radiation (Met + IR). Densitometry results of three experiments were shown as fold change and standard error compared with control cells after normalization to b-actin. * denotes p < 0.05. Lower panel: Representative immunoblots of tumor lysates from mice six weeks after exposing to whole abdominal irradiation in (C). The experiments were repeated independently at least twice.

autophagy-related Beclin-PI(3)KC3 complex [23], and regulates not only autophagosome formation but also maturation [24]. Later, UVRAG was demonstrated to regulate BAX-mediated mitochondrial apoptosis, and led to resistance to anticancer therapy [25]. Recent investigation also revealed that UVRAG maintains chromosome stability by activating DNA-PK in nonhomologous end joining to promote DNA damage repair; as well as physically binding with CEP63 to protect centrosome integrity [26]. Cancer-related UVRAG frameshift was found to render cells more sensitive to standard chemotherapy due to a DNA repair defect [27]. UVRAG coordinates activities of apoptosis, autophagy and chromosome stability to maintain cell survival. In this study, we demonstrated that Klf10 transcriptionally suppressed UVRAG in pancreatic cancer cells to enhance radiation induced cytotoxicity. The inverse correlation of Klf10 and UVRAG was also demonstrated in pancreatic cancer patients.

TGFb signaling was demonstrated to interact with ATM and contributes to irradiation-induced DSB [28]. After radiation, the activated TGFb signaling cooperates with p53 which leads to cell cycle arrest or apoptosis [7]. Severe DNA damage can induce both the extrinsic and intrinsic apoptosis pathway which stabilized p53 to trigger apoptosis. Several reports indicated that autophagy pathway contributes to the growth-inhibitory effect of TGFb [29,30]. It involves mammalian target of rapamycin complex 1 (mTORC1) which represses autophagy via phosphorylation of the ULK1/2Atg13-FIP200 complex thus preventing maturation of preautophagosomal structures [31]. When DNA damage occurs, it is recognized by some proteins or their complexes, such as PARP-1, Mre11-Rad50-Nbs1 complex or FOXO3, which activate repressors of mTORC1. However, our study showed that although silencing Klf10, as well as TGFb signal, suppressed apoptosis and DNA damage response, the level of autophagy protein expression was

Please cite this article in press as: Chang V-HS et al. Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene. Radiother Oncol (2017), http://dx.doi.org/10.1016/j.radonc.2017.01.001

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Klf10 regulates radio-sensitivity via UVRAG

Fig. 5. Klf10 expression inversely correlated with UVRAG and predicted pancreatic cancer response to radiotherapy. (A) Representative photos of H&E stain of pancreatic cancer of various tumor regression grade (TRG) after chemo-radiotherapy. TRG2: few cancer cells; TRG3: tumor cells < stroma cells; TRG4: tumor cells > stroma cells; TRG5: no response. Original magnification was 400. (B) Representative photos of Klf10 and UVRAG immune-staining of two patients. The patient (upper panel) with high Klf10 extent intensity score (EI) had low EI score of UVRAG. Another patient (lower panel) with low Klf10 EI score had high EI score of UVRAG. Original magnification was 400. (C) EI scores of Klf10 in patient groups of TRG1/2/3 and TRG4/5, respectively. Correlation of Klf10 EI score with TRG was 0.69 (p = 0.001) (D) Correlation of Klf10 and UVRAG in 70 pancreatic cancer patients after surgery. The correlation coefficient is 0.259. (p = 0.03) The size of symbol represents the number of patient specimens evaluated.

elevated after radiation in pancreatic cancer. The discordance between autophagy and DNA damage in pancreatic cancer compared to those of other cancers we observed (HCT116 and OECM1, unpublished) or previously reported was unclear. It might be explained by the fact that the autophagic response of cancer cells to radiotherapy may be cell and tissue specific [32]. Our results were in line with the observations that loss of Klf10 immunolabeling [16] and high autophagic transcription factors expression were noted in advanced pancreatic cancer compared with those in other solid tumors [33]. Furthermore, the relationship between autophagy and apoptosis is, in general, mutual inhibition [34]. Our finding argued that loss of Klf10 leads to radio-resistance of pancreatic cancer cells by suppressing apoptosis, enhancing autophagy as well as DNA damage repair. TGF b related response in apoptosis, autophagy and DNA repair after radiation was coordinated by Klf10 via transcriptional control of UVRAG. Metformin, a widely used drug for type 2 diabetes mellitus, was found to radio-sensitize cancer cells and killed radio-resistant

cancer stem cells via AMPK/mTOR signaling [35,36]. Although recent prospective clinical trials showed no survival benefit of concomitant metformin with chemotherapy in advanced pancreatic cancer [37,38], the role of metformin to enhance radiation response is still unknown. Existing evidence supports that the radio-sensitizing effect of metformin is multifaceted and depends on certain genetic backgrounds, such as in cells with loss of p53 and LKB1 [39]. We found metformin upregulated Klf10 expression in a time- and dose-dependent manner by activating AMPK. Concomitant decrease in UVRAG expression was noted in orthotopic tumor tissue when Klf10 expression was enhanced by metformin. Our results demonstrated that the TGFb/Klf10/UVRAG signal pathway contributes to the radio-sensitizing effect of metformin to pancreatic cancer cells. In conclusion, our study showed that Klf10 radio-sensitized pancreatic cancer not only by positive feedback to the cytotoxic effect of TGFb, but also by transcriptional suppression of UVRAG which led to increased apoptosis, DNA damage, and suppressed

Please cite this article in press as: Chang V-HS et al. Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene. Radiother Oncol (2017), http://dx.doi.org/10.1016/j.radonc.2017.01.001

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autophagy. Clinical specimens of pancreatic cancer recapitulated the inverse relationship of Klf10 and UVRAG as well as the predictive value of Klf10 in radiation response. Metformin activated AMPK which upregulated Klf10 expression and contributed to enhanced radiation cytotoxicity in pancreatic cancer. Klf10 is a potential target in predicting response and improving sensitivity of pancreatic cancer to radiation. Conflict of Interest statement Vincent Hung-Shu Chang: nothing to declare. Yi-Chih Tsai, Ya-Li Tsai: nothing to declare. Shu-Ling Peng: nothing to declare. Su-Liang Chen: nothing to declare. Tsung Ming Chang: nothing to declare. Winston Chun-Yuan Yu: nothing to declare. Hui-Ju Ch’ang: nothing to declare. Acknowledgement We thank Dr. Yan-Shen Shan from Department of Surgery, NCKUH, for providing clinical specimens of pancreatic cancer for immune-histochemical studies. We thank Dr. Chen Chang from Department of Pathology, NCKUH for interpreting immunehistologic score in pancreatic clinical specimens. We thank Dr. Jieh-Yuan Liu and Kelvin K.C. Tsai from NHRI for providing gemcitabine resistant and luciferase-labeled pancreatic cancer cells. We are grateful to Dr. Jeffry SM Chang from NHRI for English editing of this manuscript. The study is supported by grants from National Health Research Institutes (NHRI, CA-104-PP-11), Taiwan and Ministry of Science and Technology (MOST 104-2314-B-400-017-MY3), Taiwan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.radonc.2017.01. 001. References [1] Callery MP, Chang KJ, Fishman EK, et al. Pretreatment assessment of resectable and borderline resectable pancreatic cancer: expert consensus statement. Ann Surg Oncol 2009;16:1727–33. [2] Hazard L. The role of radiation therapy in pancreas cancer. Gastrointest Cancer Res 2009;3:20–8. [3] Goodman KA, Hajj C. Role of radiation therapy in the management of pancreatic cancer. J Surg Oncol 2013;107:86–96. [4] Yang Y, Liu H, Li Z, et al. Role of fatty acid synthase in gemcitabine and radiation resistance of pancreatic cancers. Int J Biochem Mol Biol 2011;2:89–98. [5] Long J, Zhang Y, Yu X, et al. Overcoming drug resistance in pancreatic cancer. Expert Opin Ther Targets 2011;15:817–28. [6] Calonge MJ, Massague J. Smad4/DPC4 silencing and hyperactive Ras jointly disrupt transforming growth factor-beta antiproliferative responses in colon cancer cells. J Biol Chem 1999;274:33637–43. [7] Dancea HC, Shareef MM, Ahmed MM. Role of radiation-induced TGF-beta signaling in cancer therapy. Mol Cell Pharmacol 2009;1:44–56. [8] Ahmed MM, Alcock RA, Chendil D, et al. Restoration of transforming growth factor-beta signaling enhances radiosensitivity by altering the Bcl-2/Bax ratio in the p53 mutant pancreatic cancer cell line MIA PaCa-2. J Biol Chem 2002;277:2234–46. [9] Subramaniam M, Harris SA, Oursler MJ, et al. Identification of a novel TGFbeta-regulated gene encoding a putative zinc finger protein in human osteoblasts. Nucleic Acids Res 1995;23:4907–12. [10] Ellenrieder V, Fernandez Zapico ME, Urrutia R. TGFbeta-mediated signaling and transcriptional regulation in pancreatic development and cancer. Curr Opin Gastroenterol 2001;17:434–40.

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Please cite this article in press as: Chang V-HS et al. Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene. Radiother Oncol (2017), http://dx.doi.org/10.1016/j.radonc.2017.01.001