Targeting Acid Ceramidase to Improve the Radiosensitivity of Rectal Cancer

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Article Targeting Acid Ceramidase to Improve the Radiosensitivity of Rectal Cancer

1, , 1, 1 1 1 Rachael E. Cliﬀord * † , Naren Govindarajah †, David Bowden , Paul Sutton , Mark Glenn , Mahnaz Darvish-Damavandi 2,3, Simon Buczacki 2,3, Ultan McDermott 4, Zdzislaw Szulc 5, Besim Ogretmen 5, Jason L. Parsons 1,6 and Dale Vimalachandran 1,7,*

1 Cancer Research Centre, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, 200 London Road, Liverpool L3 9TA, UK; [email protected] (N.G.); [email protected] (D.B.); [email protected] (P.S.); [email protected] (M.G.); [email protected] (J.L.P.) 2 Nuffield Department of Surgical Science, University of Oxford, Oxford OX3 7DQ, UK; [email protected] (M.D.-D.); [email protected] (S.B.) 3 Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0AW, UK 4 Oncology R&D, AstraZeneca, Cambridge CB2 0AW, UK; [email protected] 5 Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA; [email protected] (Z.S.); [email protected] (B.O.) 6 Clatterbridge Cancer Centre NHS Foundation Trust, Clatterbridge Road, Bebington CH63 4JY, UK 7 The Countess of Chester Hospital, Liverpool Road, Chester CH2 1UL, UK * Correspondence: Rachael.cliff[email protected] (R.E.C.); [email protected] (D.V.) Denotes shared first authorship. † Received: 30 November 2020; Accepted: 14 December 2020; Published: 15 December 2020

Abstract: Previous work utilizing proteomic and immunohistochemical analyses has identified that high levels of acid ceramidase (AC) expression confers a poorer response to neoadjuvant treatment in locally advanced rectal cancer. We aimed to assess the radiosensitising effect of biological and pharmacological manipulation of AC and elucidate the underlying mechanism. AC manipulation in three colorectal cancer cell lines (HT29, HCT116 and LIM1215) was achieved using siRNA and plasmid overexpression. Carmofur and a novel small molecular inhibitor (LCL521) were used as pharmacological AC inhibitors. Using clonogenic assays, we demonstrate that an siRNA knockdown of AC enhanced X-ray radiosensitivity across all colorectal cancer cell lines compared to a non-targeting control siRNA, and conversely, AC protein overexpression increased radioresistance. Using CRISPR gene editing, we also generated AC knockout HCT116 cells that were significantly more radiosensitive compared to AC-expressing cells. Similarly, two patient-derived organoid models containing relatively low AC expression were found to be comparatively more radiosensitive than three other models containing higher levels of AC. Additionally, AC inhibition using carmofur and LCL521 in three colorectal cancer cell lines increased cellular radiosensitivity. Decreased AC protein led to significant poly-ADP ribose polymerase-1 (PARP-1) cleavage and apoptosis post-irradiation, which was shown to be executed through a p53-dependent process. Our study demonstrates that expression of AC within colorectal cancer cell lines modulates the cellular response to radiation, and particularly that AC inhibition leads to significantly enhanced radiosensitivity through an elevation in apoptosis. This work further solidifies AC as a target for improving radiotherapy treatment of locally advanced rectal cancer.

Keywords: acid ceramidase; ionizing radiation; LCL521; rectal cancer

Cells 2020, 9, 2693; doi:10.3390/cells9122693 www.mdpi.com/journal/cells Cells 2020, 9, 2693 2 of 14

1. Introduction Neoadjuvant chemoradiotherapy (CRT) remains the mainstay of treatment for locally advanced rectal cancer [1]. However, response to such treatment remains both variable and unpredictable and has led to an ongoing drive to develop novel prognostic and/or therapeutic biomarkers/molecular targets [2]. Successful response to CRT not only improves the likelihood of surgical resection of the tumour with clear margins, but if the pathological response is significant there is an associated improvement in overall long-term survival. Unfortunately, only 12% of patients with locally advanced rectal cancer experience a complete pathological response [3], and a significant proportion develop disease progression locally and/or systemically during CRT. The primary mechanism by which CRT delivers its effect on cancer cells is through the induction of DNA damage, particularly via DNA double strand breaks [4]. However, cancer cells are equipped with cellular DNA damage response mechanisms that typically repair the DNA damage and which can promote radioresistance. Such resistance is also thought to be mediated by cellular sub-populations, particularly cancer stem cells, that possess properties such as efficient DNA damage repair and self-renewal [5]. The combination of radiotherapy with an agent that enhances tumour killing is well-recognised, and typically in rectal cancer a cytotoxic radiation-chemical reaction is employed, most often using a fluorine analogue such as 5-fluorouracil (5-FU) [6]. The exact molecular mechanisms by which 5-FU exerts its radiosensitising effects are not fully understood, but are in part thought to be through its production of fluorodeoxyuridine (FdUrd) and its subsequent effects on both RNA function and DNA repair post irradiation [7]. Despite much effort, there are at present no other novel or more effective radiosensitising agents available for rectal cancer that can improve on currently observed response rates [6]. We have previously performed temporal proteomic analysis of patients undergoing CRT through serial rectal biopsies with associated oncological outcome data [8]. Using this approach, we uniquely identified acid ceramidase (AC) as being over-expressed in patients who responded poorly to CRT. AC is a lysosomal cysteine hydrolase encoded by the asah1 gene, which catalyzes the conversion of ceramide into fatty acid and sphingosine [9]. Ceramide plays a central role in response to cellular stress, and its synthesis and accumulation can mediate cell death through a number of mechanisms, including apoptosis and autophagy. In addition to lowering ceramide levels, this reaction also produces sphingosine-1-phosphate (S1P), which can alternatively lead to an increase in cellular proliferation. This tightly regulated balance between ceramide and S1P is increasingly thought to play a key role in cancer development and progression, and AC has been identified as a potential novel therapeutic target which is overexpressed in a number of human cancers, such as prostate and head and neck cancers [10]. Inhibition of AC has been shown to both inhibit cancer cell formation in melanoma cell lines, but importantly has also been demonstrated to sensitize glioblastoma and prostate cancer cell lines to radiation [11–14]. Historically, pharmacological inhibition of AC has been achieved through the off-target effects of traditional cytotoxic agents [15,16], although the side-effect profile of many of these drugs has limited their use. Specific ceramide analogs, such as B13, will effectively inhibit AC in vivo but have limited in vitro use due to their inability to access the lysosomal compartment where AC resides [17]. Development of structurally altered B13 prodrugs, such as LCL521, are however, able to provide novel, specific subcellular targeted delivery of B13 and thus achieve AC inhibition [18]. In this study, we now further analyse the impact of AC in mediating radioresistance of colorectal cancer cell lines in vitro through both biological (siRNA and CRISPR) manipulation and pharmacological inhibition of AC. To this effect, we demonstrate that targeting AC can enhance the radiosensitivity of colorectal cancer cells through promoting cellular apoptosis. Cells 2020, 9, 2693 3 of 14

2. Materials and Methods

2.1. Cell Culture Colorectal cancer cell lines (HCT116, HT29, LIM1215, MDST8, GEO, NCI-H716; Wellcome Sanger Institute, Cambridge, UK) were all short tandem repeat (STR) profiled and tested free of mycoplasma. / p53− − HCT116 cell line was kindly provided by Prof B. Vogelstein. HCT116 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, Gillingham, UK) supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% Penicillin-streptomycin and 1% non-essential amino acids at 37 ◦C in 5% CO2. All other cells were cultured in Roswell Park Memorial Institute (RPMI; Sigma-Aldrich, Gillingham, UK) with the same supplements, but additionally with 10 mM hydroxyethyl piperazineethanesulfonic acid (Hepes). siRNA knockdown of asah1 (using a single siRNA sequence; D-005228-03, Horizon Discovery Ltd., Cambridge, UK) or using a non-targeting control siRNA (AllStars Negative Control siRNA; Qiagen, Manchester, UK) was performed using Lipofectamine RNAiMAX (Life Technologies, Paisley, UK) for 48 h. The mammalian overexpression plasmid for asah1 was kindly provided by Dr D. Krainc, and transfection with this plasmid was performed for 24 h using Lipofectamine 2000 (Life Technologies, Paisley, UK). Carmofur and 5-FU were purchased from Sigma-Aldrich (Gillingham, UK). Organoids were characterised and kindly gifted by The Sanger Institute (Cambridge, UK). They were embedded in 40 µL of Matrigel (Corning, NY, USA) in 24-well plates and grown in Intesticult™ Organoid Media (StemCell, Cambridge, UK) supplemented with 1% Penicillin-streptomycin and 10 µM Y27632 dihydrochloride, Rho kinase (ROCK) inhibitor. For passaging organoids, matrigel domes were submerged in 1 mL TrypLE 1X (Gibco, ThermoFisher Scientific, Waltham, MA, USA) and incubated at 37 ◦C for 10 min. Aspirated cells were suspended in cold PBS and centrifuged at 1200 rpm for 5 min. The pellet was then resuspended in Matrigel and plated onto a pre-warmed 24-well plate.

2.2. Preparation of Whole Cell Extracts and Immunoblotting Whole cell extracts (WCE) were prepared and immunoblotting analysis performed using the Odyssey Image Analysis System (Li-Cor Biosciences, Cambridge, UK), as previously described [19,20]. Primary antibodies raised against ASAH1 (1:500) (BD Biosciences, Wokingham, UK), poly-ADP ribose polymerase-1 (PARP-1) (1:5000) (Santa Cruz Biotechnology, Heidelberg, Germany) or actin/tubulin (1:20,000) (Sigma-Aldrich, Gillingham, UK), along with Alexa Fluor 680 or IR Dye 800 secondary antibodies (1:20,000) (Li-Cor Biosciences, Cambridge, UK) were used for detection.

2.3. asah1 mRNA Analysis Affymetrix Human Genome U219 microarray data were analysed on the Human Genome U219 96-Array Plate using the Gene Titan MC instrument (Affymetrix, ThermoFisher Scientific, Waltham, MA, USA). The robust multi-array analysis (RMA) algorithm was used to establish intensity values for each of 18,562 loci. Raw data were finally deposited in ArrayExpress (accession number: E-MTAB-3610). The RMA processed dataset is available at http://www.cancerrxgene.org/gdsc1000/ [21].

2.4. Detecting AC Using ELISA A sandwich enzyme-linked immunosorbent assay (ELISA) kit (Cloud-Clone Corporation, Houston, TX, USA) was used to establish a dose response relationship for carmofur and LCL521 in inhibiting AC antibody binding in colorectal cancer cell lysates. AC standards and cell lysates were added to a 96-well plate pre-coated with a biotin-conjugated AC speciﬁc antibody and incubated with avidin conjugated to Horseradish Peroxidase. After addition of the tetramethylbenzidine substrate, the colour change was assessed spectrophotometrically at 450 nm and AC detection levels in the cell lysates determined relative to the levels in the standards. Cells 2020, 9, 2693 4 of 14

2.5. Clonogenic Assays Clonogenic assays were performed as previously described [22,23], following irradiation with a CellRad X-ray irradiator (Faxitron Bioptics, Tucson, AZ, USA). For drug treatments, cells were treated as a monolayer for 2 h prior to irradiation and using DMSO as a vehicle only control. Cells were trypsinized, counted and plated into 6-well plates and colonies allowed to grow for 10–14 days. Speciﬁcally for LCL521 treatments, cells were treated with the drug for a further 24 h post-irradiation. Colonies were counted using a GelCount colony counter (Oxford Optronix, Oxford, UK), and relative colony formation (surviving fraction) was expressed as colonies per treatment level versus colonies that appeared in the untreated control. All experiments were performed in triplicate, as independent biological experiments. Plating eﬃciencies for the cells were as followed: - HCT116 (45%), HT29 (40%) and LIM1215 (30%). Statistical analysis was performed using the CFAssay for R package [24]. For the analysis of cell survival curves, the CFAssay uses the linear-quadratic model (LQ model) to compare responses to radiation in the presence of an intervention versus a respective control.

2.6. Necrosis Analysis Cells were stained with 4 µg/mL propidium iodide and 1 µg/mL Hoechst 33,342 in triplicate for 10 min at room temperature before imaging using an EVOS confocal microscope (Life Technologies, Paisley, UK). All experiments were performed in triplicate. Apoptotic cells show an increased uptake of Hoescht 33,342 due to membrane permeability. Propidium iodide (PI) discriminates early apoptotic cells with dye exclusion due to intact cell membrane and necrotic cells which have loss of membrane integrity.

2.7. Flow Cytometry Analysis Cells were trypsinized, pelleted at 100 g for 5 min at 4 C using ice cold PBS along with the × ◦ existing media to ensure no cell loss, and resuspended in 500 µL of Annexin V (1:20,000, diluted with Annexin binding buﬀer) and 0.5 µL of PI (0.5 µg/mL) for 20 min at room temperature whilst being protected from the light. Apoptosis analysis was performed by ﬂow cytometry using the Attune NxT Flow Cytometer (Life Technologies, Paisley, UK).

2.8. CRISPR Gene Editing of asah1

Two guides targeting asah1 (guide 321: 50-GCTCAAGCTCACTCACCGG-30 and guide 421: 50-TAGCAGCCAACGCCACTCCC-30) were designed using CRISPOR (Concordet and Haeussler, 2018) and purchased from Integrated DNA Technologies (IDT, Leuven, Belgium). The individual guides were cloned into pLenti-CRISPRv2 (Feng Zhang, Broad Institute; Addgene plasmid #52961) and the resulting plasmids were transfected into Lenti-X 293T cells together with psPAX2 and pMD2.G using standard PEI transfection techniques. After 72 h, the supernatant was collected and lentivirus particles were concentrated by ultracentrifugation at 100,000 g for 2 h. The purified lentiviral × particles were combined and transduced into HCT116 cells, which were incubated overnight at 37 ◦C at 5% CO2. Following a change of media, cells were then incubated for 72 h and subjected to puromycin selection (1 µg/mL for 72 h) before undergoing the first genotyping screen using asah1 screening primers (50-GCCCAGCACGAGGTGTTCCT-30, 50-TCGGTCCGACTATTGCCCGC-30). This genotyping indicated the presence of deletions corresponding to what would be expected using guides 321 and 421. The cells were subject to limiting dilution, and incubation continued for ~2 weeks. Resulting clones were subject to further genotyping to confirm the presence of the deletion.

2.9. Organoid Viability Assays in Response to Irradiation Organoids were kindly gifted by the Wellcome Sanger Institute (Cambridge, UK) derived as part of the Cell Model Network UK and Human Cancer Models Initiative; HCM-SANG-0265-C18 (referred to as COLIMV005), HCM-SANG-0266-C18 (referred to as COLO005), HCM-SANG-0280-C18 (referred to as COLO081), COLO109 and COLO131. 10,000 cells from dissociated organoids were embedded Cells 2020, 9, 2693 5 of 14 in 30 µL of matrigel in a 96-well plate and allowed to mature. A single dose of radiation (2 Gy) was delivered via a CellRad X-ray irradiator (Faxitron Bioptics, Tucson, AZ, USA). Organoids were allowed to recover for 5–8 days and cell viability assessed using the CellTiter-Blue (Promega). Data were normalised to the unirradiated control to calculate viability of the irradiated cells [25]. All experiments were performed in triplicate, as independent biological experiments.

3. ResultsCells 2020, 9, x FOR PEER REVIEW 5 of 15 3. Results 3.1. Correlation of Acid Ceramidase Protein Expression and Colorectal Cancer Cell Radiosensitivity Analysis3.1. Correlation of protein of Acid Ceramidase expression Protein of AC Exp inression six colorectal and Colorectal cancer Cancer cell Cell lines Radiosensitivity (NCI-H716, GEO, HT29, HCT116,Analysis MDST8 andof protein LIM1215) expression showed of AC variable in six levelscolorectal (Figure cancer1A). cell Protein lines (NCI-H716, expression GEO, was HT29, generally in keepingHCT116, with MDST8 RNA and expression LIM1215) showed (Figure 1variableB). We levels then examined(Figure 1A). comparative Protein expression radiosensitivity was generally of the cell linesin keeping using with clonogenic RNA expression assays, although (Figure 1B). two We cell th linesen examined (NCI-H716, comparative GEO) grown radiosensitivity in suspension of the were omitted.cell lines MDST8 using cells, clonogenic with the assays, highest although AC protein two cell expression, lines (NCI-H716, displayed GEO) thegrown greatest in suspension cell survival post-irradiation,were omitted. and MDST8 therefore, cells, with radioresistance the highest AC (Figure protein1C,D). expression, Interestingly, displayed LIM1215 the greatest reproducibly cell survival post-irradiation, and therefore, radioresistance (Figure 1C,D). Interestingly, LIM1215 demonstrated reduced survival at 1 Gy, but were generally radioresistant at higher radiation doses reproducibly demonstrated reduced survival at 1 Gy, but were generally radioresistant at higher along with HT29 cells. HCT116 cells were the most radiosensitive. This was conﬁrmed by statistical radiation doses along with HT29 cells. HCT116 cells were the most radiosensitive. This was analysisconfirmed (CFAssay by [24statistical]) that demonstrated analysis (CFAssay signiﬁcantly [24]) increasedthat demonstrated radiosensitivity significantly of HCT116 increased compared to HT29,radiosensitivity LIM1215 and of HCT116 MDST8 compared cell lines to (Table HT29,1 ).LIM1215 and MDST8 cell lines (Table 1).

FigureFigure 1. Protein 1. Protein levels levels of acidof acid ceramidase ceramidase (AC) (AC) in colorectalin colorectal cancer cancer cells cells show show good good correlation correlation with the degreewith the of degree radiosensitivity. of radiosensitivity. (A) AC (A protein) AC protein expression expression in whole in whol celle extractscell extracts from from colorectal colorectal cancer cells wascancer analysed cells was by analysed immunoblotting, by immunoblotting, along with along tubulin with tubulin as a loading as a loading control. control. (B) mRNA(B) mRNA and and protein expressionprotein levels expression of AC levels in colorectal of AC in cancer colorectal cells, cancer which cells, is also which shown is graphically.also shown graphically. (C) Radiosensitivity (C) of colorectalRadiosensitivity cancer cellsof colorectal was analysed cancer cells by clonogenic was analysed assays. by clonogenic (D) Representative assays. (D) imagesRepresentative of colonies in non-irradiatedimages of colonies irradiated in non-irradiated (2 Gy) plates irradiated are (2 shown Gy) plates (the are latter shown were (the seeded latter were with seeded four timeswith the four times the number of cells). number of cells).

TableTable 1. Comparative 1. Comparative survival survival of of colorectalcolorectal cancer cells cells in in response response to X-ray to X-ray radiation. radiation. Cell Line Comparative Cell Line Statistical Analysis Cell Line Comparative Cell Line Statistical Analysis HCT116 HT29 p < 0.0003 HCT116HCT116 LIM1215 HT29 p < 0.000002p < 0.0003 HCT116HCT116 LIM1215MDST8 p < 0.000001p < 0.000002 HCT116 MDST8 p < 0.000001 3.2. Changes in AC Expression Modulate Radiosensitivity of Colorectal Cancer Cells To directly demonstrate the role of AC in modulating colorectal cancer cell radiosensitivity, we firstly depleted the enzyme using siRNA, which was shown to be >70% effective in HCT116, HT29 and LIM1215 cells (Figure 2A). On analysis of cell survival, reduced radiation doses were used for LIM1215 to account for prior variability in colony formation at higher doses. We observed statistically significant increases in radiosensitivity of HT29 (p < 0.00001), LIM1215 (p < 0.002) and HCT116 (p <

Cells 2020, 9, 2693 6 of 14

3.2. Changes in AC Expression Modulate Radiosensitivity of Colorectal Cancer Cells To directly demonstrate the role of AC in modulating colorectal cancer cell radiosensitivity, we firstly depleted the enzyme using siRNA, which was shown to be >70% effective in HCT116, HT29 and LIM1215 cells (Figure2A). On analysis of cell survival, reduced radiation doses were used for LIM1215 to account for prior variability in colony formation at higher doses. We observed statistically significant increases in radiosensitivity of HT29 (p < 0.00001), LIM1215 (p < 0.002) and

HCT116Cells (p 2020< 0.03), 9, x FOR cells PEER when REVIEW AC was depleted using siRNA in comparison to non-targeting6 of 15 control siRNA treated cells (Figure2B–D, Figure S1A–C and Table2). siRNA knockdown of AC in MDST8 cells led0.03) to significantcells when AC post-radiation was depleted using cell siRNA death, in even comparison at low to dosing non-targeting (data control not shown). siRNA treated Note that the cells (Figure 2B–D, Figure S1A–C and Table 2). siRNA knockdown of AC in MDST8 cells led to non-targeting (NT) control siRNA also appeared to have an impact on cell survival post-irradiation, significant post-radiation cell death, even at low dosing (data not shown). Note that the non-targeting particularly(NT) control in HCT116 siRNAcells, also appeared as compared to have to an the impact transfection on cell survival reagent post-irradiation, (Lipofectamine) particularly only. in We then increasedHCT116 the proteincells, as compared levels of to AC the through transfection a mammalian reagent (Lipofectamine) expression only. plasmid, We then where increased a 4.4–5.0-fold the proteinprotein increase levels was of observedAC through in a HCT116mammalian and expression HT29 cells, plasmid, respectively where a 4.4–5.0-fold (Figure2E). protein On analysisincrease of cell survivalwas post-irradiation, observed in HCT116 we foundand HT29 that cells, HCT116 respectively cells were (Figure significantly 2E). On analysis more of radioresistant cell survival post- compared to bothirradiation, lipofectamine we found control that HCT116(p < 0.004) cellsand were ASAH1 significantly siRNA more knockdown radioresistant across compared irradiation to both doses lipofectamine control (p < 0.004) and ASAH1 siRNA knockdown across irradiation doses (Figure 2F, (Figure2F, Figure S1A–C and Table2). Similarly, overexpression of AC was found to significantly Figure S1A–C and Table 2). Similarly, overexpression of AC was found to significantly (p < 0.00001) (p < 0.00001)increaseincrease the radioresistance the radioresistance of HT29 and ofLIM1215 HT29 cells and (Figure LIM1215 2G, Figure cells (FigureS1A–C and2G, Table Figure 2). These S1A–C and Table2).data These demonstrate data demonstrate that AC plays that a significant AC plays role a in significant controlling roleradiosensitivity in controlling of colorectal radiosensitivity cancer of colorectalcells. cancer cells.

FigureFigure 2. Modulation 2. Modulation of AC of AC expression expression controls controls the radiosensitivity radiosensitivity of colorectal of colorectal cancer cancer cells. (A cells.) AC (A) AC proteinproteinexpression expression in whole in whole cell cell extracts extracts from from HT29, HT29, HCT116 HCT116 and LI andM1215 LIM1215 cells following cells AC following or non- AC or non-targetingtargeting (NT) (NT) siRNA was was analysed analysed by immunoblotting, by immunoblotting, along with along actin withas a loading actin ascontrol. a loading (B–D) control. HT29, HCT116 or LIM1215 cells were treated with transfection reagent (lipofectamine) only, AC (B–D) HT29, HCT116 or LIM1215 cells were treated with transfection reagent (lipofectamine) only, siRNA or a non-targeting control (NT) siRNA knockdown for 48 h, and radiosensitivity measured AC siRNA or a non-targeting control (NT) siRNA knockdown for 48 h, and radiosensitivity measured using clonogenic assays. (E) AC protein expression in whole cell extracts from HCT116 and HT29 cells using clonogenicfollowing transfection assays. (E reagent) AC protein (Lipofectamine) expression only in, AC whole siRNA cell for extracts 48 h, or a from mammalian HCT116 expression and HT29 cells followingplasmid transfection for AC (AC reagent O/E) for (Lipofectamine) 24 h was analysed only, by immunoblotting, AC siRNA for 48along h, orwith a mammalianactin as a loading expression plasmidcontrol. for AC (F,G (AC) HCT116 O/E) foror HT29 24 h cells was were analysed treated bywith immunoblotting, transfection reagentalong (lipofectamine) with actin only, as AC a loading control.siRNA (F,G for) HCT116 48 h or a ormammalian HT29 cells expression were treatedplasmid withfor AC transfection (AC O/E) for 24 reagent h and radiosensitivity (lipofectamine) only, AC siRNAmeasured for 48 using h or clonogenic a mammalian assays. expression plasmid for AC (AC O/E) for 24 h and radiosensitivity measured using clonogenic assays.

Cells 2020, 9, 2693 7 of 14

Table 2. Comparative survival of colorectal cancer cells with modiﬁed levels of AC in response to X-ray radiation.

Comparative Treatment HCT116 HT29 LIM1215

NTCells siRNA 2020, 9, vs.x FOR AC PEER siRNA REVIEW p < 0.03 p < 0.00001 p < 0.0027 of 15 Lipo vs. AC siRNA p < 0.00001 p < 0.0001 p < 0.0004 LipoTable vs. 2. ACComparative O/E survival of pcolorectal< 0.004 cancer * cells with pmodified< 0.00001 levels *of AC in responsep < 0.00001 to X- * ray radiation. * Denotes increased radioresistance. Comparative Treatment HCT116 HT29 LIM1215 NT siRNA vs. AC siRNA p < 0.03 p < 0.00001 p < 0.002 3.3. CRISPR AC KnockoutLipo vs. AC Confers siRNA Radiosensitivityp < 0.00001 for HCT116p < 0.0001 Colorectal Cancerp < 0.0004 Cell Line Lipo vs. AC O/E p < 0.004 * p < 0.00001 * p < 0.00001 * To further demonstrate the role* Denotes of AC increased in modulating radioresistance.colorectal cancer cell radiosensitivity, we targeted asah1 using CRISPR gene editing within the HCT116 cell line. The genotyping for individual clones3.3. CRISPR was AC analysed Knockout Confers using Radiosensiti PCR tovity assess for HCT116 single Colorectal or double Cancer allele Cell Line deletion. Clones labelled F1, G1, E2, D4, G5,To further D7 and demonstrate F7 yielded the role a of single AC in bandmodulating of approximately colorectal cancer cell 400 radiosensitivity, bp following we genotyping targeted asah1 using CRISPR gene editing within the HCT116 cell line. The genotyping for individual PCR, in keepingclones with was analysed a complete using PCRasah1 to assessdeletion single (Figure or double3 A).allele Clones deletion. C6,Clones A8, labelled D4, A7 F1, andG1, E2, G7 displayed two bands, theD4, secondG5, D7 and at F7 500 yielded bp in a single keeping band with of approximately a wild type 400 (WT)bp followin control,g genotyping and therefore PCR, in indicated a single allele deletionkeeping with only. a complete Clones asah1 C2 deletion and D3 (Figure had only3A). Clones a single C6, A8, band D4, atA7500 and bpG7 displayed indicating twono successful bands, the second at 500 bp in keeping with a wild type (WT) control, and therefore indicated a single deletion (Figureallele3 A).deletion Immunoblotting only. Clones C2 and was D3 performed had only a single on cell band extracts at 500 bp derived indicating from no successful the A7, A8, G1, D7, E2, F1 and F7deletion clones, (Figure which 3A). confirmed Immunoblotting a lack was ofperforme AC proteind on cell expressionextracts derived for from both the the A7, doubleA8, G1, (G1, D7, E2, F1 and F7) andD7, E2, surprisingly F1 and F7 clones, the which single confirmed (A7 and a lack A8) of allele AC protein knockouts expression (Figure for both3 theB). double Focusing (G1, specifically D7, E2, F1 and F7) and surprisingly the single (A7 and A8) allele knockouts (Figure 3B). Focusing on the completespecifically gene on knockouts, the complete and gene on knockouts, analysis and of on cell analysis survival of cell post-irradiation, survival post-irradiation, we found we that asah1 CRISPR cell linesfound werethat asah1 significantly CRISPR cell more lines radiosensitivewere significantlycompared more radiosensitive to asah1-expressing compared to asah1- WT control cells (Figure3C–E)expressing across clones; WT control G1 cells ( p <(Figure0.000001), 3C–E) across F7 (p clones;< 0.000001), G1 (p < 0.000001), F1 (p < F70.000001). (p < 0.000001), These F1 (p < findings were 0.000001). These findings were consistent across each successful CRISPR clone (data not shown). consistent acrossThese eachdata further successful demonstrate CRISPR that AC clone protein (data expression not shown). plays a Thesesignificant data role further in controlling demonstrate that AC protein expressionradiosensitivity plays of colorectal a significant cancer cells. role in controlling radiosensitivity of colorectal cancer cells.

Figure 3. CRISPRFigure 3. gene CRISPR editing gene editing of asah1 of asah1leads leads to to enhanced radiosensitivi radiosensitivityty of HCT116 of colorectal HCT116 cancer colorectal cancer cells. (A) Asah1cells.PCR (A) Asah1 genotyping PCR genotyping of CRISPR of CRISPR clones clones confirms conﬁrms complete complete gene deletions genedeletions in clones with in aclones with a single DNA band at 400 bp (F1, G1, E2, D4, G5, D7, F7), single allele deletions for clones displaying single DNADNA band bands at 400 at 400 bp and (F1, 500 G1, bp (C6, E2, A8, D4, D4, G5, A7 and D7, G7) F7), and single unsuccessful allele deletion deletions for clones for cloneswith a displaying DNA bands at 400 and 500 bp (C6, A8, D4, A7 and G7) and unsuccessful deletion for clones with a DNA band at 500 bp only (C2, D3), in keeping with a WT-expressing asah1 clone. (B) AC protein expression in whole cell extracts from CRISPR clones compared to a WT control was analysed by immunoblotting using AC antibodies, along with actin as a loading control. (C–E) Asah1 CRISPR knockout clones (G1, F7 and F1) were treated with escalating doses of radiation (1–4 Gy) and cellular radiosensitivity compared to the WT asah1-expressing control, assessed using clonogenic assays. Cells 2020, 9, x FOR PEER REVIEW 8 of 15

DNA band at 500 bp only (C2, D3), in keeping with a WT-expressing asah1 clone. (B) AC protein Cells 2020, 9, 2693 8 of 14 expression in whole cell extracts from CRISPR clones compared to a WT control was analysed by immunoblotting using AC antibodies, along with actin as a loading control. (C–E) Asah1 CRISPR knockout clones (G1, F7 and F1) were treated with escalating doses of radiation (1–4 Gy) and cellular 3.4. Correlation of AC Protein Expression and Rectal Cancer Organoid Radiosensitivity radiosensitivity compared to the WT asah1-expressing control, assessed using clonogenic assays. Colorectal cancer primary tissue organoids were sourced (Dr. Hayley Francies, Wellcome Sanger 3.4. Correlation of AC Protein Expression and Rectal Cancer Organoid Radiosensitivity Institute, Cambridge, UK) containing either low or high asah1 mRNA expression and which was conﬁrmed byColorectal immunoblotting, cancer primary as shown tissue organoids by a 1.36–4.45 were sourced AC/actin (Dr. ratioHayley in Francies, high expressors Wellcome versusSanger those Institute, Cambridge, UK) containing either low or high asah1 mRNA expression and which was containing low AC (0.94–1.00 AC/actin ratio) (Figure4A). Organoids were embedded in matrigel and confirmed by immunoblotting, as shown by a 1.36–4.45 AC/actin ratio in high expressors versus those allowed tocontaining grow for low 7–11 AC days (0.94–1.00 between AC/act passagesin ratio) (Figure to develop 4A). Organoids their characteristic were embedded appearances in matrigel (Figure and 4B). Organoidsallowed were to treated grow for with 7–11a days single between radiation passages dose to develop (2 Gy) their in a 96-wellcharacteristic plate appearances and cellviability (Figure was analysed4B). after Organoids 5–8 days were compared treated with to unirradiated a single radiatio controln dose organoids. (2 Gy) in a We96-well observed plate and that cell organoids viability with a relativelywas high analysed baseline after AC 5–8 expression days compared (COLO005, to unirradiated COLO109, control COLO131) organoids. had We signiﬁcantly observed that increased viability,organoids and therefore with a wererelatively more high radioresistant, baseline AC expression compared (COLO005, to those COLO109, with a lower COLO131) AC expression had significantly increased viability, and therefore were more radioresistant, compared to those with a (COLIVM005, COLO081) (Figure4C and Table3). lower AC expression (COLIVM005, COLO081) (Figure 4C and Table 3).

Figure 4.FigureIncreased 4. Increased expression expression of AC of AC expression expression in in colorectal colorectal organoids organoids is isassociated associated with withincreased increased resistanceresistance to radiation. to radiation. (A) Western (A) Western blotting blotting demonstrating demonstrating varyin varyingg levels levels of baseline of baseline AC expression AC expression (B) Representative(B) Representative images images of primary of primary organoids organoids at at 10× 10 andand 20× 20magnificationmagniﬁcation during growth during (C growth) Organoids were treated with a single dose of radiation (2 ×Gy) and viability× assessed after 5–8 days. (C) Organoids were treated with a single dose of radiation (2 Gy) and viability assessed after 5–8 days.

Table 3. Comparative cell viability of colorectal cancer organoids in response to 2Gy X-ray radiation.

Organoid Line Comparative Organoid Line Statistical Analysis COLIVM005 COLO005 p < 0.03 COLIVM005 COLO109 p < 0.05 COLIVM005 COLO131 p < 0.001 COLO081 COLO005 p < 0.001 COLO081 COLO109 p < 0.02 COLO081 COLO131 p < 0.002

3.5. Pharmacological Inhibition of AC Improves Radiosensitivity of Colorectal Cancer Cells We compared the radiosensitivity of colorectal cancer cells in combination with 5-FU, a non-speciﬁc pharmacological inhibitor of AC (carmofur), in addition to a speciﬁc AC inhibitor (LCL521). Using an Cells 2020, 9, x FOR PEER REVIEW 9 of 15

Table 3. Comparative cell viability of colorectal cancer organoids in response to 2Gy X-ray radiation.

Comparative Cells 2020, 9, 2693 Organoid Line Statistical Analysis 9 of 14 Organoid Line COLIVM005 COLO005 p < 0.03 COLIVM005 COLO109 p < 0.05 µ ELISA assay, we found thatCOLIVM005 AC detection was reducedCOLO131 in HT29 cellsp by< 0.001>80% with 4 M carmofur, whilst 8 µM carmofur was requiredCOLO081 for the same levelCOLO005 in HCT116 cells (Figurep < 0.001 S2A,B). To limit off-target effects, particularly in HCT116COLO081 cells, the dose wasCOLO109 reduced to 4 µMp carmofur< 0.02 (~55% reduction in AC detection). HCT116 cellsCOLO081 showed increasedCOLO131 radiosensitivity inp the< 0.002 presence of carmofur and 5-FU (Table4 and Figure S5A), which were less e ffective in comparison to siRNA knockdown of AC 3.5. Pharmacological Inhibition of AC Improves Radiosensitivity of Colorectal Cancer Cells (Figure5A). Similarly, HT29 cells were significantly radiosensitised in the presence of carmofur and 5-FU (TableWe4 and compared Figure the S5A), radiosensitivity whereas the impactof colorectal of AC cancer siRNA cells appeared in combination to be relatively with 5-FU, more a non- e ffective in reducingspecific cell pharmacological survival (Figure inhibitor5B). On of AC utilizing (carmofur), the specific in addition inhibitor to a specific for AC, AC LCL521, inhibitor an (LCL521). ELISA assay Using an ELISA assay, we found that AC detection was reduced in HT29 cells by >80% with 4 μM demonstrated a >80% reduction in AC detection in both HCT116 and HT29 cells using 10 µM LCL521 carmofur, whilst 8 μM carmofur was required for the same level in HCT116 cells (Figure S2A,B). To (Figure S5A,B). We demonstrate that LCL521 significantly increased the radiosensitivity of HT29 cells limit off-target effects, particularly in HCT116 cells, the dose was reduced to 4 μM carmofur (~55% (p < 0.03),reduction and alsoin AC enhanced detection). the HCT116 radiosensitivity cells showed of HCT116 increased cells radiosensitivity although this in wasthe presence not statistically of significantcarmofur compared and 5-FU to (Table a vehicle 4 and only Figure (DMSO) S5A), control which (Figurewere less5C,D, effective Figure in S3A,Bcomparison and Tableto siRNA4). knockdown of AC (Figure 5A). Similarly, HT29 cells were significantly radiosensitised in the Tablepresence 4. Comparative of carmofur and survival 5-FU (Table of colorectal 4 and Figure cancer S5A), cells whereas following the drug impact treatments of AC siRNA in response appeared to X-rayto be relatively radiation. more effective in reducing cell survival (Figure 5B). On utilizing the specific inhibitor for AC, LCL521, an ELISA assay demonstrated a >80% reduction in AC detection in both HCT116 Comparative Treatment HCT116 HT29 and HT29 cells using 10 μM LCL521 (Figure S5A,B). We demonstrate that LCL521 significantly increased the radiosensitivityDMSO vs. of carmofur HT29 cells (p

FigureFigure 5. Pharmacological 5. Pharmacological inhibition inhibition of AC of expressionAC expression increases increases the the radiosensitivity radiosensitivity of colorectalof colorectal cancer cells.cancer (A) HCT116 cells. (A or) HCT116 (B) HT29 or cells (B) HT29 were cells pretreated were pretreated with a vehicle with a control vehicle (DMSO),control (DMSO), 5 µM 5-ﬂuorouracil 5 μM 5- (5-FU),fluorouracil or 4 µM carmofur (5-FU), or for 4 μ 2M h, carmofur or with ACfor 2 siRNA h, or with for 48AC h, si andRNA radiosensitivity for 48 h, and radiosensitivity measured using

clonogenic assays. (C) HCT116 or (D) HT29 cells were pretreated with a vehicle control (DMSO), or 10 µM LCL521 for 2 h, and also for a further 24 h post-plating, and radiosensitivity was measured using clonogenic assays. 3.6. Inhibition of AC Increases Apoptosis Post-Irradiation in a p53-Dependent Manner To examine the mechanism of increased radiosensitivity of colorectal cancer cells in the absence of AC, we analysed the levels of apoptotic and necrotic cells. Using a 4–12 Gy dose of radiation to stimulate cell death, we demonstrate that there are no significant increases in the levels of necrotic HT29 Cells 2020, 9, 2693 10 of 14 cells at 24–72 h post-irradiation following AC siRNA knockdown in comparison to a non-targeting control siRNA (Figure6A,B). On immunoblotting analysis of PARP-1 cleavage, as a marker of apoptosis, siRNA knockdown independent of AC in HT29 cells appeared to cause a minimal increase in PARP-1 cleavage, compared to a non-targeting control siRNA (Figure4C; compare lanes one and two). However, in response to radiation, there was dramatic PARP-1 cleavage (and associated PARP-1 poly(ADP-ribosylation)) in AC siRNA-treated cells, which was not observed in non-targeting control siRNA-treated cells (Figure6C; compare lanes three and four). This demonstrates that the mechanism of cell death in these cells is through apoptosis. This is supported by flow cytometry analysis which confirmed a significantly higher level of apoptosis in AC siRNA-treated cells compared with a non-targeting control siRNA at both 8 (p < 0.01) and 24 h (p < 0.02) post-irradiation (Figure6D). It is well established that the tumour suppressor protein p53 is essential for cells to undergo apoptosis. / We therefore analysed the impact of AC expression on the radiosensitivity of a p53− − HCT116 cell line. We observed that an siRNA knockdown of AC, or overexpression of AC, had no impact on the radiosensitivity of these p53-deficient cells using clonogenic assays (Figure6D,F and Figure S5). We also found no statistically significant difference in the radiosensitivity of these cells to 5-FU, carmofur or LCL521 (Figure6E,G and Figure S5). This demonstrates that the decrease in cell survival post-irradiation following inhibition of AC is achieved via apoptosis in a p53-dependent manner. Cells 2020, 9, x FOR PEER REVIEW 11 of 15

Figure 6. ReductionFigure 6. inReduction survival in ofsurvival colorectal of colorectal cancer cancer cells cells through through inhibition inhibition of AC is is caused caused by by apoptosis. apoptosis. (A,B) HT29 cells were treated with AC siRNA or a non-targeted control (NT) for 48 h prior (A,B) HT29 cells were treated with AC siRNA or a non-targeted control (NT) for 48 h prior to irradiation to irradiation and stained at 24 h intervals with Hoescht 33,342 and propidium iodide stain. (A) and stainedRepresentative at 24 h intervals images withof cells Hoescht irradiated with 33,342 12 Gy and and propidium(B) percentage iodideof necrotic stain. cells at ( A24–72) Representative h images of cellspost-irradiation irradiated with with 4–12 12 Gy. Gy (C and) PARP-1 (B) protein percentage expression, of necroticand PARP-1 cells cleavage, at 24–72 in whole h post-irradiation cell with 4–12 Gy.extracts (C) PARP-1 from HT29 protein cells in the expression, absence and and presence PARP-1 of 4 Gy cleavage, radiation after in whole 24 h was cell analysed extracts by from HT29 immunoblotting, along with actin as a loading control. (D) HCT116 cells treated with AC siRNA and cells in the absencea non-targeted and presencecontrol siRNA of after 4 Gy 8 or radiation 24 h post irradiation after 24 (4 h Gy) was were analysed analysed by flow immunoblotting, cytometry along with actin asto a determine loading levels control. of cellular (D) apoptosis HCT116 (E) cellsp53−/− HCT116 treated cells with were AC treated siRNA with transfection and a non-targeted reagent control siRNA after(lipofectamine) 8 or 24 h post only, irradiation AC siRNA or (4 a non-targetin Gy) wereg analysedcontrol (NT) by siRNA ﬂow knockdown cytometry for 48 to h, determine or a levels mammalian expression plasmid for AC (AC O/E) for 24 h and radiosensitivity measured using / of cellular apoptosisclonogenic assays. (E) p53 (F)− p53− HCT116−/− HCT116 cells cells were were treated treated with a vehicle with transfectioncontrol (DMSO), reagent5 μM 5-FU, (lipofectamine) or only, AC siRNA4 μM or carmofur a non-targeting for 2 h, or withcontrol LCL521 (NT) for 24 siRNA h, and knockdown radiosensitivity for measured 48 h, or using a mammalian clonogenic expression assays. (G,H) Representative images of colonies in non-irradiated irradiated (2 Gy) plates are shown / plasmid for AC (AC O/E) for 24 h and radiosensitivity measured using clonogenic assays. (F) p53− − (the latter were seeded with 2 times the number of cells). HCT116 cells were treated with a vehicle control (DMSO), 5 µM 5-FU, or 4 µM carmofur for 2 h, or with LCL521 for4. Discussion 24 h, and radiosensitivity measured using clonogenic assays. (G,H) Representative images of colonies inColorectal non-irradiated cancer is irradiated the second (2commonest Gy) plates cause are of shown cancer (therelated latter death were in the seeded UK, and with rectal 2 times the number ofcancer cells). accounts for approximately 25% of these cases. Surgical resection of advanced rectal cancer represents a particular surgical challenge due to the anatomical confines of the bony pelvis and adjacent neurovascular structures. Neoadjuvant CRT is employed widely in order to reduce the tumour bulk and improve the chances of obtaining a clear surgical resection margin, however, the effects are still very variable and unpredictable. Those patients who do experience a complete clinical response have a more favorable long-term prognosis, and may also be candidates for organ preserving treatment, although the correlation between complete clinical (radiological) and pathological (post resection) response is unclear. CRT is not without complications, both surgically and also in terms of long-term anorectal function in those patients undergoing resection. Furthermore, the delay to systemic treatment introduced with CRT means that that those patients

Cells 2020, 9, 2693 11 of 14

4. Discussion Colorectal cancer is the second commonest cause of cancer related death in the UK, and rectal cancer accounts for approximately 25% of these cases. Surgical resection of advanced rectal cancer represents a particular surgical challenge due to the anatomical confines of the bony pelvis and adjacent neurovascular structures. Neoadjuvant CRT is employed widely in order to reduce the tumour bulk and improve the chances of obtaining a clear surgical resection margin, however, the effects are still very variable and unpredictable. Those patients who do experience a complete clinical response have a more favorable long-term prognosis, and may also be candidates for organ preserving treatment, although the correlation between complete clinical (radiological) and pathological (post resection) response is unclear. CRT is not without complications, both surgically and also in terms of long-term anorectal function in those patients undergoing resection. Furthermore, the delay to systemic treatment introduced with CRT means that that those patients who respond poorly are at increased risk of long-term treatment failure. Biomarker studies in rectal cancer are thus desperately needed both to identify those patients that will undergo good responses and those that will not, so that unnecessary delays to systemic treatment can be avoided. Given the favorable prognosis associated with complete pathological response (~12%), therapeutic biomarker studies that might increase this rate are also highlyCells 2020 desirable., 9, x FOR PEER REVIEW 13 of 15 We have recently identified a novel finding that AC is associated with a poor response to CRT in patientsLCL521 withhas not advanced yet been rectal evaluated cancer. in Whilstthe clinical the initial setting, exploratory there is an work increasing analysed interest the pre-treatmentin developing biopsiesdrugs that of patients, act on the the validationsphingolipid work pathway. analysed The only biological surgical resectiondata demonstra specimenste andthe impact thus its utilityof AC asmanipulation a predictive on biomarker radiosensitivity is unknown. as a proof Overexpression of principle, of and AC therefore, in human its cancers potential is well as a describedclinical target. in a varietyHowever, of cancers, further ofresearch particular into relevance the identification to this study of more are prostate,potent and CNS effect andive head drug(s) and neckor an cancers, siRNA wheretherapy manipulation would be ofrequired AC has beento address shown similarlyinter-tumour to influence variability chemo in and response radiosensitivity to radiation, through in pro-apoptoticcomparison to pathways that demonstrated [10–14] (Figure with7). However, our pharmacological there has been data little workusing exploring LCL521. theProposed role of ACmechanisms in rectal cancer. through In thiswhich work, this wewill have be achieved now explored include whether nanotechnology acid AC may drug represent delivery, a therapeutic along with biomarkermore traditional to increase targeted the radiosensitivityantibodies [32,33]. of rectalThese cancer. mechanisms We have are employed similar to standard the ones colorectal we have cancerdemonstrated cell lines with which pharmacological are routinely usedinhibition, for investigation which ultimately of rectal will cancer be more response transferrable to radiotherapy, to clinical howeverpractice. we advanced our work by also utilising patient-derived rectal cancer 3D organoid models.

Figure 7. A schematic diagram summarizing the ceramide metabolism pathway and eﬀects of acid

ceramidase (ASAH1) manipulation. Figure 7. A schematic diagram summarizing the ceramide metabolism pathway and effects of acid Weceramidase have confirmed (ASAH1) manipulation. that there is differential expression of AC across our 2D colorectal cancer cell lines (HCT116, HT29 and LIM1215) and 3D organoid models, but that higher expression largely correlates5. Conclusions with increased radioresistance in these ex vivo models. We have shown that biological (bothIn siRNA conclusion, and CRISPR) this work and continues pharmacological to highlight (carmofur the importance and LCL521) of the inhibition sphingolipid of AC pathway in 2D cell in linescancer, increases in particular, radiosensitivity, the role whichthat AC appears may toplay be consistentin mediating across the a numberresponse of of di ffrectalerent colorectalcancer to cancerradiotherapy. cell lines. Further Conversely, work with overexpression more advanced of ACpre-clinical enhanced models cellular (patient-derived radioresistance, organoids, confirming but more so mouse models) are required to confirm our in vitro findings, along with novel drug development and/or delivery that will allow the accurate compartmental delivery of agents, such as LCL521.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Biological manipulation of AC effects colony formation efficiency post-irradiation; Figure S2: ELISA assay demonstrating effect of Carmofur dosing on AC detection; Figure S3: Pharmacological targeting of AC effects colony formation efficiency post-irradiation; Figure S4: ELISA assay demonstrating effect of LCL521 dosing on AC detection; Figure S5: Biological and pharmacological targeting of AC in HCT116 p53−/− cells does not affect colony formation efficiency post-irradiation.

Author Contributions: conceptualization, J.L.P., and D.V.; methodology, R.E.C., N.G., D.B., P.S., M.G., M.D.-D., S.B., U.M., and J.L.P.; reagents, B.O., and Z.S.; validation, R.E.C., and N.G.; formal analysis, R.E.C., and N.G.; writing—original draft preparation, R.E.C., and N.G.; writing—review and editing, R.E.C., N.G., M.D.-D., S.B., U.M., B.O., J.L.P., and D.V.; Supervision, J.L.P., and D.V.; project administration, J.L.P., and D.V.; funding acquisition, J.L.P., and D.V. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by The Royal College of Surgeons (RCS) and Bowel Cancer UK (BCUK).

Acknowledgments: The authors thank D. Krainc for kindly providing the asah1 mammalian expression plasmid. We thank Hayley Francies and Mathew Garnett from the Wellcome Sanger Institute for kind provision of the organoid models generated as part of the Cell Model Network UK and the Human Cancer Models Initiative. A particular thank you to Andrew Beggs (University of Birmingham, Birmingham, UK), The

Cells 2020, 9, 2693 12 of 14 the direct role for AC in modulating radiosensitivity. We discovered that enhanced radiosensitivity of AC knockdown cells was caused by increases in apoptosis, as demonstrated by PARP-1 cleavage and flow cytometry analysis, and that this mediated in a p53-dependent manner due to the lack of change in radiosensitivity of p53-deficient HCT116 cells through AC modulation. Noteworthy, is that HCT116 and LIM1215 cells both harbor wild type p53, whereas HT29 cells contain mutant p53 but which is still radiosensitised by AC knockdown/inhibition. Nevertheless, this correlates with recent work demonstrating that ceramide, the cellular target for degradation by AC, binds to p53 leading to its cellular accumulation and stress response activation through disruption of interaction with the E3 ubiquitin ligase MDM2 that targets p53 for degradation [25]. Ceramides have long been known to be mediators of apoptosis in response to stimuli, including tumour necrosis factor α and Fas ligand, and which is triggered through effector protein kinases including JNK and the pro-apoptotic Bcl-2 family members Bid and Bad [26,27]. Therefore, our work strengthens the importance of AC and the ceramide pathway in controlling cell survival in response to exogenously-induced stress, but more importantly that AC is a cellular target for radiosensitisation in colorectal cancer cells. In this study we employed two pharmacological AC inhibitors. Carmofur (1-hexylcarbamoyl- 5-fluoruracil) has been in clinical use in the adjuvant setting since 1981, where it appears to augment the disease free and overall survival in breast, gastric, bladder and colon cancers [28]. Carmofur is a 5-FU oral prodrug, however until recently, the mechanism of its independent 5-FU anti-tumour activity were unknown. Recent work has, however, confirmed that Carmofur does directly bind to AC [29], and this may explain it synergistic effects with chemotherapy agents, such as 5-FU and oxaliplatin [30]. Unfortunately, Carmofur may be limited use in a wider clinical context due to its potential CNS side effects of leukoencephalopathy, which may explain its restricted use [16]. In view of this, we chose to explore the effects of a specific small molecular inhibitor of AC, LCL521. LCL521 is a prodrug of the AC inhibitor B13, which has been designed to provide efficient lysosomal delivery of the drug, which shows more effectiveness in inhibiting AC compared to non-compartmentalized drugs [18]. Our results suggested that LCL521 was associated with an increase in radiosensitivity in the HT29 and HCT116 cell lines. However, this effect was not statistically significant in HCT116 cells, which may be due to the fact that we were only able to achieve an ~80% reduction in AC detection at a 10 µM dose of LCL521, in line with other studies [31]. Although LCL521 has not yet been evaluated in the clinical setting, there is an increasing interest in developing drugs that act on the sphingolipid pathway. The biological data demonstrate the impact of AC manipulation on radiosensitivity as a proof of principle, and therefore, its potential as a clinical target. However, further research into the identification of more potent and effective drug(s) or an siRNA therapy would be required to address inter-tumour variability in response to radiation, in comparison to that demonstrated with our pharmacological data using LCL521. Proposed mechanisms through which this will be achieved include nanotechnology drug delivery, along with more traditional targeted antibodies [32,33]. These mechanisms are similar to the ones we have demonstrated with pharmacological inhibition, which ultimately will be more transferrable to clinical practice.

5. Conclusions In conclusion, this work continues to highlight the importance of the sphingolipid pathway in cancer, in particular, the role that AC may play in mediating the response of rectal cancer to radiotherapy. Further work with more advanced pre-clinical models (patient-derived organoids, but more so mouse models) are required to conﬁrm our in vitro ﬁndings, along with novel drug development and/or delivery that will allow the accurate compartmental delivery of agents, such as LCL521.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/9/12/2693/s1, Figure S1: Biological manipulation of AC effects colony formation efficiency post-irradiation; Figure S2: ELISA assay demonstrating effect of Carmofur dosing on AC detection; Figure S3: Pharmacological targeting of AC effects colony formation efficiency post-irradiation; Figure S4: ELISA assay demonstrating effect of LCL521 dosing / on AC detection; Figure S5: Biological and pharmacological targeting of AC in HCT116 p53− − cells does not affect colony formation efficiency post-irradiation. Cells 2020, 9, 2693 13 of 14

Author Contributions: Conceptualization, J.L.P., and D.V.; methodology, R.E.C., N.G., D.B., P.S., M.G., M.D.-D., S.B., U.M., and J.L.P.; reagents, B.O., and Z.S.; validation, R.E.C., and N.G.; formal analysis, R.E.C., and N.G.; writing—original draft preparation, R.E.C., and N.G.; writing—review and editing, R.E.C., N.G., M.D.-D., S.B., U.M., B.O., J.L.P., and D.V.; Supervision, J.L.P., and D.V.; project administration, J.L.P., and D.V.; funding acquisition, J.L.P., and D.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by The Royal College of Surgeons (RCS) and Bowel Cancer UK (BCUK). Acknowledgments: The authors thank D. Krainc for kindly providing the asah1 mammalian expression plasmid. We thank Hayley Francies and Mathew Garnett from the Wellcome Sanger Institute for kind provision of the organoid models generated as part of the Cell Model Network UK and the Human Cancer Models Initiative. A particular thank you to Andrew Beggs (University of Birmingham, Birmingham, UK), The University Hospital Birmingham, Owen Sansom (The Beatson Institute, Glasgow, UK), The Queen Elizabeth Hospital Glasgow and the respective biomaterial centres at each of these hospitals for providing patient tissue for organoid derivation. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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