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Targeting Acid Ceramidase to Improve the Radiosensitivity of Rectal Cancer

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Targeting Acid Ceramidase to Improve the Radiosensitivity of Rectal Cancer cells Article Targeting Acid Ceramidase to Improve the Radiosensitivity of Rectal Cancer 1, , 1, 1 1 1 Rachael E. Clifford * y , Naren Govindarajah y, 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. y 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
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