cancers Article A Transgenic Model Reveals the Role of Klotho in Pancreatic Cancer Development and Paves the Way for New Klotho-Based Therapy Tammi Arbel Rubinstein 1,2 , Inbal Reuveni 1,2 , Arkadi Hesin 1,2 , Anat Klein-Goldberg 1 , Hannes Olauson 3 , Tobias E. Larsson 3,4 , Carmela R. Abraham 5,6,7 , Ella Zeldich 5,7,† , Assumpció Bosch 7,8,9,10 , Miguel Chillón 7,8,9,11 , Kenneth Samuel Hollander 1 , Ayelet Shabtay-Orbach 1 , Gilad W. Vainer 12,‡ , Ido Wolf 1,2 and Tami Rubinek 1,2, * 1 Institute of Oncology, Tel Aviv Sourasky Medical Center, Tel Aviv 64239, Israel; rtammi@gmail.com (T.A.R.); inbalreu1@gmail.com (I.R.); arkadih@tlvmc.gov.il (A.H.); anatkgoldberg@gmail.com (A.K.-G.); kennethh@tauex.tau.ac.il (K.S.H.); ayelet_shabtay@yahoo.com (A.S.-O.); idow@tlvmc.gov.il (I.W.) 2 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel 3 Division of Renal Medicine, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, 14186 Stockholm, Sweden; hannes.olauson@gmail.com (H.O.); tobias.larsson@ki.se (T.E.L.) 4 Department of Nephrology, Karolinska University Hospital, 17176 Stockholm, Sweden 5 Boston University School of Medicine, Department of Biochemistry, Boston, MA 02118, USA; cabraham@bu.edu (C.R.A.); ezeldich@bu.edu (E.Z.)  6 Pharmacology & Experimental Therapeutics, Boston, MA 02118, USA  7 Klogenix Therapeutics Inc., Boston, MA 02116, USA; Assumpcio.Bosch@uab.cat (A.B.); Citation: Arbel Rubinstein, T.; Miguel.Chillon@uab.cat (M.C.) 8 Institut de Neurociències, Department of Biochemistry and Molecular Biology, Universitat Autonoma de Reuveni, I.; Hesin, A.; Barcelona, 08035 Barcelona, Spain Klein-Goldberg, A.; Olauson, H.; 9 Unitat Mixta UAB-VHIR, Vall d’Hebron Institut de Recerca (VHIR), 08035 Barcelona, Spain Larsson, T.E.; Abraham, C.R.; Zeldich, 10 CIBERNED, Instituto de Salud Carlos III, 28029 Madrid, Spain E.; Bosch, A.; Chillón, M.; et al. A 11 ICREA, Institut Catalan Recerca Avançada, 08010 Barcelona, Spain Transgenic Model Reveals the Role of 12 Pathology Institute, Tel Aviv Sourasky Medical Center, Tel Aviv 6423906, Israel; giladwv@gmail.com Klotho in Pancreatic Cancer * Correspondence: tamarru@tlvmc.gov.il; Tel.: +972-527466151 Development and Paves the Way for † Ella Zeldich’s present address: Anatomy and Neurobiology, Boston University School of Medicine, New Klotho-Based Therapy. Cancers Boston, MA 02118, USA. 2021, 13, 6297. https://doi.org/ ‡ Gilad W. Vainer’s present address: Department of Pathology, Hadassah Medical Center, Faculty of Medicine, 10.3390/cancers13246297 Hebrew University of Jerusalem, Jerusalem 91120, Israel. Academic Editor: Alexander Arlt Simple Summary: We aimed to study the role of the anti-aging protein klotho and its secreted isoform, sKL, in pancreatic cancer. Three in vivo models, including a novel genetic mouse model and Received: 13 November 2021 bioinformatics analyses, indicated klotho as a tumor suppressor in pancreatic ductal adenocarcinoma, Accepted: 13 December 2021 and unveiled a unique klotho DNA hypermethylation pattern in pancreatic tumors. These results Published: 15 December 2021 possess significant prognostic value, and further suggest that sKL may serve as a therapeutic agent for pancreatic ductal adenocarcinoma. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Abstract: Klotho is an anti-aging transmembrane protein, which can be shed and can function published maps and institutional affil- as a hormone. Accumulating data indicate that klotho is a tumor suppressor in a wide array iations. of malignancies, and designate the subdomain KL1 as the active region of the protein towards this activity. We aimed to study the role of klotho as a tumor suppressor in pancreatic ductal adenocarcinoma (PDAC). Bioinformatics analyses of The Cancer Genome Atlas (TCGA) datasets revealed a correlation between the survival of PDAC patients, levels of klotho expression, and DNA Copyright: © 2021 by the authors. methylation, and demonstrated a unique hypermethylation pattern of klotho in pancreatic tumors. Licensee MDPI, Basel, Switzerland. The in vivo effects of klotho and KL1 were examined using three mouse models. Employing a This article is an open access article novel genetic model, combining pancreatic klotho knockdown with a mutation in Kras, the lack distributed under the terms and conditions of the Creative Commons of klotho contributed to PDAC generation and decreased mousece survival. In a xenograft model, Attribution (CC BY) license (https:// administration of viral particles carrying sKL, a spliced klotho isoform containing the KL1 domain, creativecommons.org/licenses/by/ inhibited pancreatic tumors. Lastly, treatment with soluble sKL prolonged survival of Pdx1-Cre; 4.0/). KrasG12D/+ ;Trp53R172H/+ (KPC) mice, a model known to recapitulate human PDAC. In conclusion, Cancers 2021, 13, 6297. https://doi.org/10.3390/cancers13246297 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 6297 2 of 13 this study provides evidence that klotho is a tumor suppressor in PDAC. Furthermore, these data suggest that the levels of klotho expression and DNA methylation could have prognostic value in PDAC patients, and that administration of exogenous sKL may serve as a novel therapeutic strategy to treat PDAC. Keywords: klotho; KL1; sKL; tumor suppressor; pancreatic cancer; PDAC 1. Introduction Pancreatic cancer is among the most aggressive cancers, with a 5-year survival rate of 9%. Incidence and mortality are rising, with 60,430 new cases and 48,220 deaths expected in 2021 in the US alone [1]. Pancreatic ductal adenocarcinoma (PDAC) represents 85–90% of all malignant pancreatic neoplasms. Current models indicate its development from benign pancreatic intraepithelial neoplasia (PanIN) 1–3 to invasive carcinoma, along with acquisition of genetic mutations. Activation of Kras is considered the first step towards malignancy [2], and in mouse models, leads to the development of PanIN by 9 months [3]. Although overt PDAC in these mice is rare, in the KPC model, combining Kras mutation with loss of the tumor suppressor p53 [4], PDAC usually develops by 3–6 months of age [5]. Klotho (here used as klotho) is a type I transmembrane protein involved in the regula- tion of aging [6]. In mice, klotho deficiency leads to a syndrome resembling accelerated aging, while klotho overexpression extends life span [7,8]. The klotho extracellular region is composed of two homologous domains, KL1 and KL2, which can be cleaved from the membrane and act as circulating hormones [9–11]. A second, differentially spliced klotho isoform is secreted (sKL). The sKL is identical to KL1, plus 15 additional amino acids at the C-terminus. Different activities of klotho have been described, including activation of fibroblast growth factor (FGF) 23 signaling [12,13], regulation of transient receptor potential cation channel subfamily V member (TRPV) 5 calcium channel [14,15], and inhibition of the insulin and insulin-like growth factor (IGF)-1 pathways [8,16,17]. Klotho is expressed predominantly in the kidneys and brain, but also in various tissues, including the exocrine and endocrine pancreas [7,17–20]. Its physiologic activities in the pancreas have yet to be determined; however, there is evidence for its involvement in regulation of glucose homeostasis. Klotho induces insulin production and secretion in vitro and in vivo, attenuates insulin sensitivity in mice, and is depleted in pancreatic islets of type 2 diabetes mellitus (T2DM) patients [8,19–22]. Klotho is a potent tumor suppressor in numerous malignancies, including gastroin- testinal cancers [16,17,23–30]. It is epigenetically silenced in cancer, and both klotho and KL1 reduce growth of cancer cells in vitro and in vivo [16,17,23,25,26,28,31–33]. In pancre- atic cancer, klotho’s downregulation is correlated with patients’ survival. In vitro, klotho reduces growth of pancreatic cancer cells, and inhibits the IGF-I and bFGF pathways [17,34]. In this study, we aimed to decipher the role of klotho as a tumor suppressor in PDAC by utilizing three in vivo models. Employing a novel genetic model, we showed that pancreatic klotho knockdown contributed to PDAC development, and reduced survival of Kras mutant mice. In a second model, we showed that administration of viral particles carrying sKL inhibited pancreatic tumors in a xenograft model. Lastly, treatment with soluble recombinant sKL prolonged survival of KPC mice. These results manifest the role of klotho as an important player in PDAC development, and suggest klotho as a therapeutic strategy to treat PDAC patients. 2. Materials and Methods 2.1. TCGA Analysis Using UCSC Xena Browser Gene expression RNAseq (IlluminaHiSeq) and DNA methylation (Methylation450k) datasets for The Cancer Genome Atlas (TCGA) Pancreatic Cancer (PAAD) cohort were studied using the University of California Santa Cruz (UCSC) Xena Browser (http://xena. Cancers 2021, 13, 6297 3 of 13 ucsc.edu/, accessed on 19 July 2020) [35]. Using the Xena Browser, overall survival and the progression-free interval of pancreatic cancer patients were analyzed according to either klotho expression or KLOTHO DNA methylation, and Kaplan-Meier plots were created. Duplicated individuals’ data were removed by filtering for primary tumors only. Gene expression data, given in log2 (x + 1) units, were divided into two groups according to median; DNA methylation data were divided into two groups according to lower and upper quartiles. For site-specific KLOTHO DNA methylation, samples with low or high levels of klotho expression, as defined as by lower or upper quartiles, respectively, were further analyzed. The β values [36] for each methylation site were calculated, and correlation with klotho expression in the corresponding sample was examined via Pearson’s correlation coefficient (r). 2.2. Chemicals, Antibodies, and Constructs Chemicals used in this study included soluble human splice variant klotho, compris- ing amino acids 34–549 (sKL domain; accession number BAA24941.1), with a 15-amino-acid unique sequence at the C-terminus (100-53; PeproTech Inc, Rocky Hill, NJ, USA); glucose (Floris, Misgav, Israel), human insulin (Actrapid® ; Novo Nordisk A/S, Bagsværd, Den- mark), and luciferin (122799; PerkinElmer, Waltham, MA, USA). Antibodies used in this study included rat IgG2a isotype control (02-9688; Thermo Fisher Scientific, Waltham, MA, USA) and anti-β-actin (A5441; Sigma-Aldrich, St. Louis, MO, USA). Anti-klotho antibodies directed against the KL1 domain (KM2076) were a kind gift from Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan. 2.3. AAV Vector Production and Purification AAV9 vectors, null empty vector, and sKL (containing the secreted klotho splice vari- ant isoform, accession number BAA24941.1) were produced and purified in the biosafety level 2 facilities of the Unitat Mixta UAB-VHIR and the Vector Production Unit (VPU). Briefly, vectors were generated using the triple transfection system in HEK293 cells. Af- ter 48 h, AAV vectors were harvested, treated with benzonase, purified in an iodixanol gradient, and titrated using PicoGreen [37]. Transgene expression was driven by a CMV promoter, as previously described [37]. 2.4. Animal Maintenance Mouse maintenance and experiments were carried out in the Sourasky Medical Center (Tel Aviv, Israel), and in accordance with the regulations and standards of the Sourasky Medical Center Animal Care and Use Committee. Mouse strains used for transgenic models were all on a mixed C57BL/6 and JVB/NJ background. Pdx1-Cre, LSL-KrasG12D/+ , and LSL-Trp53R172H/+ mice were kind gifts from Dr. Ziv Gil (Technion-Israel Institute of Technology at Rambam, Haifa, Israel). 2.5. Pancreas-Specific KLOTHO Knockdown Mouse Models In order to target knockdown of KLOTHO to mouse pancreata, mice carrying floxed KLOTHO alleles (KLflox ) were generated, as previously described [38]. KLflox/flox mice were crossbred with Pdx1-Cre mice, expressing Cre recombinase controlled by the promotor of Pdx1, thus obtaining Pdx1-Cre; KL−/− mice. Pancreatic KLOTHO knockdown was confirmed with immunohistochemistry and mRNA levels, as described hereinafter. To generate mice harboring Pdx1 promotor-controlled KLOTHO knockdown and mu- tant Kras expression, LSL-KrasG12D/+ mice [3], carrying a Lox-Stop-Lox sequence followed by a mutant Kras allele, were crossbred with KLflox/flox mice. These mice were further cross- bred with Pdx1-Cre mice, yielding Pdx1-Cre; KL−/− ;KrasG12D/+ mice. LSL-KrasG12D/+ were also crossbred with Pdx1-Cre mice separately, and these Pdx1-Cre; KrasG12D/+ mice were used as the control. Cancers 2021, 13, 6297 4 of 13 Mice were examined daily for signs of suffering, including hard breathing, major weight changes, and large tumors (more than 1 cm), and those that met the criteria set by the ethical committee were sacrificed. Mice were monitored for survival, with death defined either spontaneously or by signs necessitating sacrifice. Due to the rapid digestion of pancreatic tissue by pancreatic fluids, we could only examine pancreata of mice that had been sacrificed. Tissues were harvested and kept in formaldehyde for 24 h, which was then replaced by 70% ethanol. 2.6. Mice Tumor Xenograft Studies Female athymic nude mice (BALB/c background), 4–6 weeks of age, were purchased from Envigo RMS (Jerusalem, Israel). Mice were housed and maintained in laminar flow cabinets under specific pathogen-free conditions. On the first day of the experiment, MIA PaCa-2 cells stably expressing m-Cherry/luciferase were subcutaneously (s.c.) inocu- lated into their flanks (n = 21, 1 × 106 cells in 100 µL of 5% FCS DMEM medium). Ten days following tumor inoculation, a high dose (5 × 1011 GC/mL, n = 7) or a low dose (5 × 1010 GC/mL, n = 6) of human AAV-sKL was injected intramuscularly (i.m.). AAV-null served as the control (5 × 1010 GC/mL, n = 8). Tumors were measured with a digital caliper three times a week, and volume was calculated by the ellipsoid volume calculation formula (0.5 × length × width2 ). On day 20 of the experiment, tumors were evaluated in vivo by monitoring MIA PaCa-2 cells’ luciferase activity by injection of 150 µg/mL luciferin intra-peritoneally, and luciferase intensity was measured by Biospase. On the same day, the experiment was ended and the mice were euthanized. Tumors were removed, weighed, and measured with a digital caliper. Blood was collected prior to euthanization, and levels of human klotho were measured in serum using ELISA (IBL, Minneapolis, MN, USA). Fifty microliters of serum per sample (in duplicates) were used for ELISA, and the assay was conducted according to the manufacturer’s instructions. Correlation between weight of tumors and human klotho blood levels was examined via Pearson’s correlation coefficient. 2.7. KPC Mouse Models Generation of KPC mice was done by crossbreeding LSL-Trp53R172H/+ mice [5] carry- ing a Lox-Stop-Lox sequence, followed by a point-mutant p53 allele that functions as a null mutation, with LSL-KrasG12D/+ mice. These were further crossbred with Pdx1-Cre mice, resulting in Pdx1-Cre; KrasG12D/+ ; Trp53R172H/+ mice. Mice were matched according to sex, age, and weight, and were randomly assigned to receive treatment with intraperitoneal (i.p.) injections of soluble human sKL (15 mg/kg, twice weekly) or a vehicle control (n = 6 for the sKL-treated group; n = 7 for the control group). Treatment continued for up to 30 weeks. Mice were examined daily for signs of suffering, including hard breathing, major weight changes, and large tumors (more than 1 cm), and those that met the criteria set by the ethical committee were sacrificed. Mice were monitored for survival, with death defined either spontaneously or by signs necessitating sacrifice. 2.8. Genotyping Mouse tails were taken at 3 weeks of age for genotyping. DNA was extracted by incubation of the tail in alkaline lysis solution, consisting of 250 µM NaOH and 2 µM disodium EDTA (pH 12.0), for 30–60 min in 95 ◦ C, followed by neutralization with 0.4 mM Tris HCL (pH 5.0). DNA subsequently underwent PCR amplification for the different genotypes, using REDTaq ReadyMix PCR Reaction Mix (Sigma-Aldrich, Rehovot, Israel). Primers were at 1000 nM final concentration. PCR cycles and primers are detailed in Table S1. Products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide. Cancers 2021, 13, 6297 5 of 13 2.9. Immunohistochemistry (IHC) Analysis Tissues were fixed in 4% paraformaldehyde, and embedded tissues were serially sectioned. Sections were either stained with hematoxylin and eosin (H&E) and examined microscopically by an experienced pathologist, or stained by IHC, as described. For klotho IHC, formalin-fixed and paraffin-embedded sections, 4 µm thick, were dewaxed in xylene and rehydrated. Antigen retrieval was performed using a hot bath (95 ◦ C) for 20 min in citrate buffer pH 6.0. After cooling for 30 min, slides were rinsed in TBS-triton (TBS-T) buffer. Subsequently, an endogenous peroxidase block was performed for 10 min in 3% H2 O2 /methanol. After rinsing in TBS-T, sections were blocked for 30 min using 5% BSA in TBS-T, and later were incubated overnight with klotho primary antibody at 4 ◦ C. Detection was performed with ZytoChem Plus (HRP) One-Step Polymer anti- Mouse/Rabbit/Rat (ZUC053; Zytomed Systems, Berlin, Germany). Rat IgG2a served as the isotype control. 2.10. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Pancreata from sacrificed Pdx1-Cre; KL−/− and KLflox/flox control mice were excised, frozen in liquid nitrogen, and stored at −80 ◦ C. Following homogenization of the pancreata, total RNA was prepared using the RNA isolation kit (Sigma), and reverse transcribed using qScript cDNA SuperMix (Quanta BioSciences, Gaithersburg, MD, USA). The cDNA was amplified for klotho and β-actin (as the loading control) using REDTaq ReadyMix PCR Reaction Mix (Sigma). The PCR was optimized at 94 ◦ C for 5 min, followed by cycles of 30 s at 94 ◦ C, 90 s at 52.5 ◦ C, and 20 s at 72 ◦ C (45 cycles for klotho, 25 cycles for β-actin), and 10 min at 72 ◦ C for extension, using 12.5 pmol klotho primers (F, 50 - ACGTTCAAGTGGACACTACTCT-30 and R, 50 - TTCTTGGCTACAACCCCGTC-30 ) and 5 pmol β-actin primers (F, 50 -TGTTACCAACTGGGACGACA-30 and R, 50 - GGGGTGTTGA AGGTCTCAAA-30 ). Products were electrophoresed on 1.5% agarose gels stained with ethidium bromide. Quantification was done using ImageJ software, National Institutes of Health, Bethesda, MD, USA. 2.11. Statistical Analysis Results are presented as the mean ± SD or SEM, as mentioned. Continuous variables were compared using the t test, unless otherwise mentioned. All significance tests were two-tailed, and a p value of ≤0.05 was considered as statistically significant. Correlation was assessed using Pearson’s correlation coefficient (r). Survival analysis was done using the log-rank test. 3. Results 3.1. Levels of Klotho Expression and DNA Methylation in Pancreatic Tumors Correlate with Survival of Cancer Patients The Cancer Genome Browser (TCGA) Pancreatic Cancer (PAAD) cohort was examined using the University of California Santa Cruz (UCSC) Xena Browser [35]. Gene expression analyses revealed reduced overall survival (OS) and a progression-free interval (PFI) for pancreatic cancer patients with low klotho expressing tumors, compared to high (n = 89 for each group; Figure 1A,B). Examination of methylation data showed a compatible trend of hampered OS and PFI in patients who had tumors with high KLOTHO DNA methylation levels, compared to low (n = 45 and n = 46, respectively; Figure 2A,B). Further analyses of gene expression and methylation data (Table S2) revealed three specific sites, evaluated by probes cg23282559, cg02441765, and cg25650964, which were hy- permethylated (|Delta β| > 0.15) and negatively correlated to klotho expression (|r| > 0.3) in human pancreatic cancer (Figure 2C–E). Cancers 2021,13, Cancers2021, 13,6297 x 66 of of13 14 Cancers 2021, 13, x 6 of 14 Figure1.1.Low Figure Lowklotho klothoexpression expressionreduces reducessurvival survivalofofpancreatic pancreaticcancer cancerpatients. patients.(A,B) (A,B)The TheUCSC UCSC Figure 1. Low klotho expression reduces survival of pancreatic cancer patients. (A,B) The UCSC Xena Xena Browser was used to examine The Cancer Genome Atlas (TCGA) Pancreatic Cancer (PAAD) XenaBrowser Browserwas wasused usedtotoexamine examineTheTheCancer CancerGenome GenomeAtlasAtlas(TCGA) (TCGA)Pancreatic PancreaticCancer Cancer(PAAD) (PAAD) cohort. Gene cohort. Gene expression expression RNAseq RNAseq(IlluminaHiSeq) (IlluminaHiSeq) dataset dataset was wasanalyzed analyzedfor foroverall overallsurvival, survival, and cohort. Gene expression RNAseq (IlluminaHiSeq) dataset was analyzed for overall survival,and and progression-free interval progression-free interval of of patients patientswith withlow low(<5.675, n =n89) (<5.675, vs. high = 89) (≥5.675, vs. high n = 89)nklotho (≥5.675, = 89) express- klotho progression-free interval of patients with low (<5.675, n = 89) vs. high (≥5.675, n = 89) klotho express- ing tumors. expressing ing tumors.tumors. Figure2.2.KLOTHO Figure KLOTHOisishypermethylated hypermethylatedin inpancreatic pancreaticcancer. cancer.(A–E) (A–E)The TheUCSC UCSCXenaXenaBrowser Browserwaswas Figure 2. KLOTHO is hypermethylated in pancreatic cancer. (A–E) The UCSC Xena Browser was used to examine TCGA PAAD cohort. (A,B) RNA Methylation450k dataset was analyzed used to examine TCGA PAAD cohort. (A,B) RNA Methylation450k dataset was analyzed for overall for overall used to examine TCGA PAAD cohort. (A,B) RNA Methylation450k dataset was analyzed for overall survivaland and progression-freeinterval interval of patientswithwith tumorsshowing showing low(<0.5152, (<0.5152, n = 46)vs. vs. high survival survival andprogression-free progression-free intervalofofpatients patients withtumors tumors showinglow low (<0.5152,n n==46) 46) vs.high high (>0.5830, n = 45) KLOTHO methylation. (C–E) Gene expression RNAseq (IlluminaHiSeq) and RNA (>0.5830,nn== 45) (>0.5830, 45) KLOTHO KLOTHOmethylation. methylation. (C–E) (C–E)Gene Geneexpression expressionRNAseq RNAseq(IlluminaHiSeq) (IlluminaHiSeq)and andRNA RNA Methylation450k datasets were analyzed for correlation between klotho expression and methylation Methylation450k Methylation450kdatasets datasetswere wereanalyzed analyzedfor forcorrelation correlationbetween betweenklotho klothoexpression expressionand andmethylation methylation at the indicated methylation sites (n = 178). ** p < 0.005. r, Pearson’s correlation coefficient. at the indicated methylation sites (n = 178). ** p < 0.005. r, Pearson’s correlation coefficient. at the indicated methylation sites (n = 178). ** p < 0.005. r, Pearson’s correlation coefficient. Cancers 2021, 13, x 7 of 14 Further analyses of gene expression and methylation data (Table S2) revealed three Cancers 2021, 13, 6297 specific sites, evaluated by probes cg23282559, cg02441765, and cg25650964, which were 7 of 13 hypermethylated (|Delta β| > 0.15) and negatively correlated to klotho expression (|r| > 0.3) in human pancreatic cancer (Figure 2C–E). 3.2. Generation 3.2. Generation of Pancreatic KLOTHO KLOTHO Knockdown Knockdown Mice Mice Non-conditional KLOTHO Non-conditional KLOTHO knockout knockout mice micedie dieat ataround around8–9 8–9weeks weeksofofage age[7], [7],hence, hence, they cannot they cannot serve serve asas models models for for cancer cancer development. development. Therefore, Therefore,we weaimed aimedtototarget target KLOTHO knockdown KLOTHO knockdowntotomice micepancreata. pancreata. ForFor thisthis aim,aim, Pdx1-Cre mice mice Pdx1-Cre were crossbred with were crossbred mice mice with carrying floxedfloxed carrying KLOTHO alleles, alleles, KLOTHO KLflox/floxKL flox/flox [38], thus obtaining [38], thusPdx1-Cre; obtainingKL −/− mice Pdx1-Cre; KL −/− mice (Figure 3A,B).(Figure RNA and protein 3A,B). RNAlevels validated and protein pancreatic levels validated klotho knockdown pancreatic klotho(Figure knock- 3C–E).(Figure down Pdx1-Cre; KL−/− 3C–E). Pdx1-Cre; KL−/− mice showed a slight increasea in mice showed weight slight over in increase time, compared weight to over time, the control (Figure 3F), but no differences in serum glucose or insulin and compared to the control (Figure 3F), but no differences in serum glucose or insulin and glucose toler- ance (Figure glucose S1). (Figure S1). tolerance Figure3.3.Generation Figure Generation of of pancreatic pancreaticKLOTHO KLOTHOknockdownknockdown mice. mice.(A)(A) Schematic diagram Schematic diagram of KL flox allele: of KL LoxP sites flox allele: LoxPflank sitesexon flank 2 of 2KLOTHO, exon of KLOTHO, leading to itstotargeted leading knockdown its targeted knockdown in pancreata of Pdx1-Cre; in pancreata KL KL of Pdx1-Cre; −/− mice. −/−Top mice. panel Topshows panel floxed shows allele, floxed bottom panel shows expected results of Cre recombination. (B) Representative gel showing PCR products of mouse allele, bottom panel shows +/+ expected results of Cre recombination. (B) Representative gel showing PCR products of mouse KLOTHO genotyping: KL (WT; 370 bp), KLflox/+ (470 and 370 bp), and KLflox/flox (470 bp). (C,D) Pancreatic RNA was ex- KLOTHO genotyping: KL+/+ (WT; 370 bp), KLflox/+ (470 and 370 bp), and KLflox/flox (470 bp). (C,D) Pancreatic RNA was tracted from Pdx1-Cre; KL (n = 7) and control mice (n = 4). Klotho mRNA levels were determined by semi-quantitative −/− extracted −/− (n = 7) and control mice (n = 4). Klotho mRNA levels were determined by semi-quantitative RT-PCR from Pdx1-Cre; KL and quantified. Representative gel shown (original uncropped blots are available as Figure S2). (E) Representative RT-PCR and quantified. klotho immunohistochemical Representative gelpancreata staining of shown (original excised uncropped blots from Pdx1-Cre; KLare available −/− and asmice. control Figure S2). (E) Representative Magnification: X20. W/O, without. (F) Weight of mice immunohistochemical in staining klotho both groups (n = 5 perexcised of pancreata group). from Analyzed − / − using repeated-measures Pdx1-Cre; KL and controlANOVA. p = 0.01. Data are mice. Magnification: X20. presented W/O, as the(F) without. mean ± SEM. Weight Control, of mice KLflox/flox in both groups mice.(n = 5 per group). Analyzed using repeated-measures ANOVA. p = 0.01. Data are presented as the mean ± SEM. Control, KLflox/flox mice. Mice harboring pancreatic KLOTHO knockdown and mutant Kras expression were generated by crossbreeding Mice harboring Pdx1-Cre pancreatic KLOTHOwith LSL-KrasG12D/+ knockdown and[3] mice Kras mutant (Pdx1-Cre; KrasG12D/+ expression were mice), andbyfurther generated crossbreeding crossbreeding them Pdx1-Cre withwith KL LSL-Kras G12D/+ flox/flox mice [3](Pdx1-Cre; KrasG12D/+ KL ; Kras mice (Pdx1-Cre; −/− G12D/+ mice). and mice), Analyses furtherwere conductedthem crossbreeding usingwith maleKL flox/flox mice. Female mice mice KL−/− ; Kras were excluded, (Pdx1-Cre; dueG12D/+ to a high rate mice). of anal were Analyses lesions in both Pdx1-Cre; conducted Kras using male mice. Female G12D/+ and Pdx1-Cre; KL excluded, mice were −/− ; Kras due G12D/+ mice, to a which high is consistent rate with in of anal lesions previous reports ofKras both Pdx1-Cre; G12D/+ and Pdx1-Cre; mucocutaneous papillomasKL in−such /− ; Kras mice G12D/+ [3]. mice, which is consistent with previous reports of mucocutaneous papillomas in such mice [3]. 3.3. Loss of Pancreatic Klotho Contributes to Reduced Survival in Mice There was no difference in survival of Pdx1-Cre; KL−/− mice, compared to the control). We proceeded to study the effect of combined pancreatic KLOTHO knockdown and Kras mutation on survival by monitoring the mice for 65 weeks. Death occurred either spontaneously or by euthanasia when signs of severe suffering, or tumor burden that necessitated sacrifice, started. Survival of Pdx1-Cre; KL−/− ; KrasG12D/+ mice (n = 21) was decreased, compared to the control Pdx1-Cre; KrasG12D/+ mice (n = 18; p = 0.02; Figure 4A). 3.3. Loss of Pancreatic Klotho Contributes to Reduced Survival in Mice There was no difference in survival of Pdx1-Cre; KL−/− mice, compared to the control). We proceeded to study the effect of combined pancreatic KLOTHO knockdown and Kras Cancers 2021, 13, 6297 mutation on survival by monitoring the mice for 65 weeks. Death occurred either sponta- 8 of 13 neously or by euthanasia when signs of severe suffering, or tumor burden that necessi- tated sacrifice, started. Survival of Pdx1-Cre; KL−/−; KrasG12D/+ mice (n = 21) was decreased, compared to the control Pdx1-Cre; KrasG12D/+ mice (n = 18; p = 0.02; Figure 4A). Thus, by the age by Thus, of 22 theweeks, age of100% (18/18) of 22 weeks, Pdx1-Cre; 100% (18/18) Kras of G12D/+ mice were Pdx1-Cre; G12D/+ Krasalive, compared to alive, mice were 71% (15/21) of Pdx1-Cre; KL − / −/−; KrasG12D/+ mice. compared to 71% (15/21) of Pdx1-Cre; KL −No G12D/+ significant ; Kras changes in weight were mice. No significant changes in noted weightbetween the groups were noted (Figure between the4B). groups (Figure 4B). Pancreatic Figure4.4.Pancreatic Figure KLOTHO KLOTHO knockdown knockdown contributes contributes to reduced to reduced survivalsurvival and inducesandPDACinduces in PDAC vivo. (A) Kaplan-Meier curves of Pdx1-Cre; in vivo. (A) Kaplan-Meier curves of Pdx1-Cre; KL KL − −/−; KrasG12D/+ / −(n = 21) ; Kras G12D/+ compared to control Pdx1-Cre; (n = 21) compared to control Kras G12D/+ (n = 18) mice. p = 0.02. (B) Weight of mice in both groups (n = 19 per group). (C–E) Pancreata Pdx1-Cre; KrasG12D/+ (n = 18)−/−mice. pG12D/+= 0.02. (B) Weight of mice in both groups (n = 19 per group). were excised from Pdx1-Cre; KL ; Kras (n = 7, average age 36 weeks) and control Pdx1-Cre; (C–E) Pancreata were excised from Pdx1-Cre; KL−/− ; KrasG12D/+ (n = 7, average age 36 weeks) and KrasG12D/+ (n = 8, average age 49 weeks) mice upon sacrifice. (C) Comparison between pathological control Pdx1-Cre; G12D/+ Kras pancreata (n =of8,both average age 49Representative weeks) mice H&E uponstaining sacrifice. (C) Comparison assessment of H&E stained groups. (D) of pancreata between pathological assessment of H&E harvested from 24-week-old mice. Pdx1-Cre; KL ; Kras stained −/− pancreata G12D/+ of both groups. (D) (right): complete loss of cellular Representative polar- ity, significant nuclear atypia, and budding of cell clusters into H&E staining of pancreata harvested from 24-week-old mice. Pdx1-Cre; KL the ductal lumen, − / along − G12D/+ (right): ; Krasprofuse with fibrosis completeandlosskeratin. Controlpolarity, of cellular Pdx1-Cre; KrasG12D/+ nuclear significant (left): noatypia, lesions.andMagnification: budding of×10. cell(E) Repre-into the clusters sentative PanIN lesions of both groups. Magnification: ×10. G12D/+ ductal lumen, along with profuse fibrosis and keratin. Control Pdx1-Cre; Kras (left): no lesions. Magnification: ×10. (E) Representative PanIN lesions of both groups. Magnification: ×10. 3.4. Klotho Cooperates in the Development of PDAC In Vivo Based on previous studies showing PDAC development in mice harboring both Kras mutation and loss of tumor suppressor activity [4], we examined Pdx1-Cre; KL−/− ; KrasG12D/+ mouse pancreata for lesions. Due to the rapid digestion of pancreatic tissue by pancreatic fluids, we could only use pancreata of mice that had been sacrificed. Samples of Pdx1-Cre; KL−/− ; KrasG12D/+ (n = 7) and control Pdx1-Cre; KrasG12D/+ (n = 8) mice underwent pathological evaluation (average age of death: 36 and 49 weeks, respectively). PDAC was identified in two Pdx1-Cre; KL−/− ; KrasG12D/+ mice, but in none of the Pdx1- Cre; KrasG12D/+ mice, while PanIN 2 was noted in two of the Pdx1-Cre; KL−/− ;KrasG12D/+ mice and in four of the Pdx1-Cre;KrasG12D/+ mice (Figure 4C). Representative images are shown (Figure 4D–E). 3.4. Klotho Cooperates in the Development of PDAC In Vivo Based on previous studies showing PDAC development in mice harboring both Kras mutation and loss of tumor suppressor activity [4], we examined Pdx1-Cre; KL−/−; KrasG12D/+ mouse pancreata for lesions. Due to the rapid digestion of pancreatic tissue by Cancers 2021, 13, 6297 9 of 13 pancreatic fluids, we could only use pancreata of mice that had been sacrificed. Samples of Pdx1-Cre; KL−/−; KrasG12D/+ (n = 7) and control Pdx1-Cre; KrasG12D/+ (n = 8) mice underwent pathological evaluation (average age of death: 36 and 49 weeks, respectively). PDAC was identified 3.5. in two Treatment withPdx1-Cre; KLPancreatic sKL Inhibits −/−; KrasG12D/+ mice, but in none of the Pdx1-Cre; KrasG12D/+ Tumors and Prolongs Survival In Vivo mice,The while potential of sKL treatment as the PanIN 2 was noted in two of Pdx1-Cre; KL a therapeutic −/−;KrasG12D/+ mice and in four of strategy for PDAC was studied the Pdx1-Cre;Kras using G12D/+ two mouse models, micea (Figure 4C).model xenograft Representative images treated with are sKL a viral shown (Figure vector, and4D–the E). transgenic KPC model treated with soluble, recombinant sKL. For the xenograft model, m- Cherry/luciferase-labeled MIA PaCa-2 cells were s.c. inoculated into nude mice. Ten days 3.5. Treatment later, mice were with sKL Inhibits injected Pancreatic Tumorsviruses with adeno-associated and Prolongs (AAV) Survival encoding InsKL Vivo(AAV-sKL) at The potential two doses. of sKL Tumor load treatment in mice as a therapeutic inoculated strategy with AAV-sKL was for PDAC was significantly studied lower using compared two mouse models, a xenograft model treated with a viral sKL vector, and to the control, reflected by smaller size, weight, and luciferase signals (Figure 5A–E). the transgenic KPC model treated Importantly, tumor with soluble, weights recombinant negatively sKL.with correlated For the xenograft human klothomodel, blood m-Cherry/lu- levels in mice ciferase-labeled (|r| MIA5F–G). > 0.729; Figure PaCa-2 cells were s.c. inoculated into nude mice. Ten days later, mice wereTheinjected second with adeno-associated model viruses utilized for this (AAV) aim was the encoding KPC model. sKL Mice (AAV-sKL) at two were matched doses. Tumor according load to sex, in and age, miceweight, inoculated with AAV-sKL and treated with i.p. was significantly injections lower of soluble humancompared sKL or atovehicle the control, reflected control for up toby30smaller weeks.size, weight, Death andeither occurred luciferase signals (Figure spontaneously or by5A–E). Im- euthanasia portantly, when signstumor weights of severe negatively suffering, correlated or tumor with burden that human klotho necessitated blood sacrifice, levels Survival started. in mice (|r| of > 0.729; mice Figure treated with5F–G). sKL was increased, compared to the control (p = 0.005; Figure 5H). Figure 5. sKL treatment decreases local tumor growth and prolongs survival in vivo. (A–G) Athymic nude BALB/c mice were s.c. inoculated with MIA PaCa-2 cells stably expressing m-Cherry/luciferase (1 × 106 cells per mouse). Ten days later, mice were injected i.m. with high dose AAV-sKL (5×1011 GC/mL, n = 7), low dose AAV-sKL (5 × 1010 GC/mL, n = 6), or control AAV-null (5 × 1010 GC/mL, Cancers 2021, 13, 6297 10 of 13 n = 8). (A) Tumor volume was measured in vivo with a digital caliper. (B) Representative pictures of luciferase activity bioimaging of the local tumors. (C) Tumors’ luciferase activity was measured by counts per minute. (D) Representative images of tumors harvested on day of sacrifice. (E) Weight of harvested tumors. (F) Human klotho blood levels. * p < 0.05, was calculated using Kruskal-Wallis test. (G) Correlation between tumor weights and human klotho blood levels. * p < 0.05. r, Pearson’s correlation coefficient. (H) KPC mice were matched according to sex, age, and weight, and randomly assigned to receive treatment with i.p. injections of soluble human sKL (15 mg/kg, twice weekly) or a vehicle control (n = 6 for the sKL-treated group; n = 7 for the control group) for up to 30 weeks. Kaplan-Meier curves of sKL-treated and control mice are presented. p = 0.005. * p < 0.05; ** p < 0.005; *** p < 0.0005 compared to control. Error bars, mean ± SEM. 4. Discussion The present study establishes the role of klotho as a tumor suppressor in PDAC. Bioin- formatics analyses revealed a correlation between the survival of PDAC patients, and levels of klotho expression and DNA methylation, and demonstrated a unique hypermethyla- tion pattern of KLOTHO in pancreatic tumors. Using a novel mouse model, we showed that pancreatic klotho knockdown cooperates with Kras mutation to decrease survival and generate PDAC in vivo. Moreover, sKL inhibited growth of tumors originating from pancreatic cells in a xenograft model, and prolonged survival of KPC mice. Klotho is epigenetically silenced through promoter hypermethylation in a wide array of malignancies [16,23,25,26,28–31,33,39,40]. This has also been reported in small cohorts in PDAC, as well as in a correlation between survival of PDAC patients and levels of klotho expression and DNA methylation [17,34]. In accordance, our evaluation of OS, as well as PFI using data comprising 178 PDAC samples, showed a positive association with klotho expression and a corresponding negative association with KLOTHO DNA methylation. These data further indicate that klotho expression may be regulated by DNA methylation, and reinforce the notion that these parameters could serve to assess the prognosis of PDAC patients. Previous studies of KLOTHO DNA methylation in PDAC [17,34] were based on a limited subset of patients and in vitro experiments, and did not analyze the methylation pattern of specific sites within the entire KLOTHO gene. The current analysis, based on the aforementioned extensive dataset, revealed that three specific sites, two of which are located within a CpG island in the 1st exon of KLOTHO, act as negative regulators of klotho expression by hypermethylation. We previously found that one of these sites, cg23282559, is also hypermethylated in colorectal cancer [25]. It is yet to be determined whether these sites play a role in klotho regulation in other malignancies as well. Klotho is known to be expressed in the pancreas; however, its role in normal pancreatic development and function is still unknown. While klotho-deficient mice show decreased insulin production, along with a dramatic increase in insulin sensitivity [22], overexpres- sion of klotho in mice results in increased fasting blood insulin, accompanied by insulin resistance [8]. Surprisingly, the mouse model presented in this study, harboring pancreatic klotho knockdown, did not show an overt phenotype of diabetes or altered sensitivity to insulin. Furthermore, there was no effect on pancreatic morphology, tumor formation, nor survival. These results suggest that loss of pancreatic klotho may be compensated by peripheral effects of systemic klotho or by other mechanisms. Moreover, diabetes develop- ment is a complex process, which depends not only on the pancreas, but also on the liver, muscles, and fat tissues. As in our model, klotho was knockdown mainly in the pancreas, it may explain the lack of diabetes in this mouse model. Consistent with the clinical results presented, Pdx1-Cre; KL−/− ; KrasG12D/+ mice had shortened life spans compared to the control Pdx1-Cre; KrasG12D/+ mice (48 vs. 60 weeks, respectively). Although we were unable to pathologically examine all the mouse pancreata, a higher rate of overt PDAC was seen in Pdx1-Cre; KL−/− ; KrasG12D/+ mice, compared to the control Pdx1-Cre; KrasG12D/+ mice. These results demonstrate the significance of klotho in the development of pancreatic malignancies and survival in vivo. The phenotype Cancers 2021, 13, 6297 11 of 13 of Pdx1-Cre; KL−/− ;KrasG12D/+ mice is less pronounced, compared to models combining loss of other tumor suppressors with Kras mutations. As an example, KPC mice have a median survival of only 5 months [5]. It is possible that this is due to partial compensation of loss of local klotho by circulating klotho. Alternatively, residual expression of klotho in the pancreas may have affected the phenotype. Achieving complete targeted silencing of klotho in future studies would enable a better understating of its role in different tissues, as well as the counterbalance between local and systemic effects of klotho. We presented two models demonstrating the therapeutic potential of klotho. In a xenograft mouse model, pancreatic cancer injections of a viral sKL vector were highly effective, not only in inhibiting tumor growth, but also in reducing the size of tumors. Treatment began when tumors were already visible and established, resembling the time of treatment initiation in most patients. The results suggest that sKL may be utilized for gene therapy, thus bypassing major obstacles of stability and efficacious delivery of protein and peptide-based drugs. Moreover, a negative correlation between tumor weight and human klotho blood levels was noted. Further research of klotho blood levels in respect to its effect may assist in predicting who would benefit from treatment with klotho or sKL, as well as in keeping levels within the therapeutic window. The development of klotho-based therapies is a goal sought by many research groups, as well as pharmaceutical companies. In regard to cancer treatment, there are several avenues that are explored. One can increase the endogenous levels of klotho by taking advantage of epigenetic mechanisms shown to affect klotho expression. It has been reported that the klotho promoter is heavily methylated in many cancers, and is also a function of age. Thus, compounds that inhibit DNA methyl transferases could become a therapeutic target to increase klotho expression. On the other hand, one can increase the levels by adding klotho exogenously, for example, by employing gene therapy or delivery of a recombinant klotho protein. Here, we reported the introduction of the active region of klotho (sKL) using the AAV expression system, which resulted in a therapeutic effect in a model of pancreatic cancer. Next, we showed that treatment with soluble sKL prolonged survival of KPC mice, a model known to recapitulate human PDAC. This demonstrated the effect of sKL in an immune-competent host and native tumor environment, further supporting the possible use of sKL in clinical settings. 5. Conclusions In conclusion, this study identified klotho as a potent tumor suppressor in PDAC. Current treatment regimens for PDAC are not sufficient, and administration of exogenous sKL may serve as a novel strategy for the treatment of PDAC. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cancers13246297/s1, Figure S1: Pancreatic klotho knockdown does not affect insulin sen- sitivity in mice, Figure S2: Original uncropped blots of klotho (A) and β-actin (B) from Figure 3C, Table S1: PCR cycles and primers’ sequences used for genotyping, Table S2: Differential methylation, correlation with klotho expression, and characteristics of KLOTHO DNA methylation sites. Author Contributions: Conceptualization, I.W., T.R., and T.A.R.; methodology, I.W., T.R., T.A.R., H.O., C.R.A., T.E.L., A.B., and M.C.; formal analysis, I.W., T.R., T.A.R., A.K.-G., H.O., C.R.A., E.Z., A.B., M.C., and G.W.V.; investigation, T.A.R., I.R., A.H., A.K.-G., E.Z., K.S.H., and A.S.-O.; resources, T.E.L., H.O., A.B., M.C., I.W., and T.R.; data curation, T.A.R., I.R., A.H., A.K.-G., E.Z., K.S.H., and A.S.-O.; writing—original draft preparation, T.A.R.; writing—review and editing, I.W., T.R., C.R.A., H.O., A.B., and M.C.; supervision, I.W. and T.R.; funding acquisition, I.W. All authors have read and agreed to the published version of the manuscript. Funding: This project was funded by the The Sami and Tova Sagol Foundation for the Study of Aging, the Margaret Stultz foundation for Pancreatic Cancer Research, the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Ministerio de Ciencia e Innovación ‘Proyectos I+D+I 2019, to M.C., (grant number PID2019-104034RB-I00) and by the TASMC excellence fund. to I.W. Cancers 2021, 13, 6297 12 of 13 Institutional Review Board Statement: Mouse maintenance and experiments were carried out in accordance with the regulations and standards of the Sourasky Medical Center Animal Care and Use Committee. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest. References 1. Siegel, R.L.; Miller, K.D.; Fuchs, H.E.; Jemal, A. Cancer Statistics, 2021. CA Cancer J. Clin. 2021, 71, 7–33. [CrossRef] 2. 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