SUN-216 Investigating the Role of the Liver X Receptor in Potentiating Mitotane Therapy in Adrenocortical Carcinoma

Abstract Introduction: Adrenocortical Carcinoma is a rare aggressive cancer which carries a poor prognosis. Adjuvant mitotane improves survival but is limited by a narrow therapeutic window and severe adverse effects. Liver X receptors (LXRs), part of the nuclear receptor superfamily are highly expressed in adrenal tissue and mediate transcellular and intracellular cholesterol homeostasis. We hypothesise that LXRα inhibition increases toxic lipid accumulation in adrenocortical cancer cells and potentiates the adrenolytic effect of mitotane. Methodology: ATCC-H295R and MUC1 ACC cells and were pre-treated with the LXRα inverse agonist SR9243 5µM and antagonist GSK2033 5µM followed by mitotane treatment (20, 40, 50μΜ) for 6 hours. Cholesterol-methyl-β-cyclodextrin treatment was carried out 1hr prior to mitotane. H295R cells were transfected with a LXR⍺ dominant negative construct using lipofectamine. Cell death was assessed using annexin/PI staining and proliferation using MTT assay. Free cholesterol (FC) levels were assayed using filipin staining and lipid droplets via BODIPY® and analysed on the Amnis ImageStream® imaging cytometer. Downstream targets ABCA1 and ABCG1 were evaluated by qRT-PCR. Lipid droplet associated proteins PLIN 1-4 and hormone sensitive lipase (HSL) expression were evaluated using western blotting. Results: Downstream reduction of ABCA1 and ABCG1 expression confirmed LXRα blockade. Mitotane effectively induced dose-dependent H295R apoptotic cell death which was potentiated pharmacologically and genetically by LXR⍺ inhibition. In line with these findings, cholesterol-methyl-β-cyclodextrin treatment increased cell death in H295R and MUC1 cells. In addition to inducing cell death, LXR⍺ inhibition decreased proliferation of both cell lines. An increase in FC and a decrease in cholesterol esters was observed following mitotane treatment in H295R cells. This was accompanied by decreased lipid droplet numbers confirmed by lower expression of lipid droplet associated proteins, PLIN1-3. These effects were potentiated when mitotane was combined with LXRα inhibition. We demonstrate increased HSL activity, which was associated with higher SOAT-1 expression and increasing toxic FC accumulation. Investigation of lipid droplet content BODIPY® of both cell lines showed H295R cells preferentially store cholesterol esters and MUC1 cells store triacylglycerides.Conclusion: We propose a mechanism for enhancing mitotane’s efficacy as an adrenolytic through increased free cholesterol via LXR⍺inhibition. Targeting the LXRα, its putative ligands, or associated lipid mediators may present a novel therapeutic approach in the setting of primary and metastatic ACC.

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Investigating the Role of Cholesterol and Lipid Trafficking in Mitotane Resistance in Adrenocortical Carcinoma

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.141 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A70-A70

Author(s):

Kate Warde ◽

Yi-Jan Lim ◽

Felix Beuschlein ◽

Constanze Hantel ◽

Michael Conall Dennedy

Keyword(s):

Cell Lines ◽

Adrenocortical Carcinoma ◽

Poor Response ◽

Hormone Sensitive Lipase ◽

Lipid Uptake ◽

Lipid Trafficking ◽

Resistant Disease ◽

Associated Proteins ◽

Cholesterol Supplementation ◽

Cholesterol Storage

Abstract Introduction: Adrenocortical Carcinoma (ACC) is a rare aggressive cancer which carries a poor prognosis. Adjuvant mitotane improves survival but is limited by poor response rates and resistance following tumour recurrence. Mitotane’s efficacy has been attributed to intracellular accumulation of toxic free cholesterol (FC) predominantly through inhibition of cholesterol storage through SOAT1. Yet SOAT1 specific inhibitors demonstrate inferior efficacy to mitotane in inducing ACC cell death. We hypothesize that mitotane’s efficacy to induce toxic FC accumulation in ACC cells is also mediated through enhanced breakdown of stored cholesterol within intracellular lipid droplets (LDs). Methodology: ATCC-H295R (mitotane sensitive) and MUC-1 (mitotane resistant) ACC cells were evaluated for neutral lipid content using BODIPY493/503 under baseline and cholesterol loaded conditions using Amnis ImageStream, additionally cells were treated with mitotane (H295R - 20, 40, 50µM; MUC1 - 50, 100, 200µM) for 6hr. Analysis of LDs using CE-BODIPY and FA-BODIPY identified cholesterol ester (CE) and triacylglycerol (TAG)-containing LDs, respectively. Lipid droplet-associated proteins (LDAPs) Perilipin (PLIN) 1–4 and hormone sensitive lipase (HSL) were evaluated using western blotting and PCR. Lipid uptake receptors; SRB1, LDLR, LRP1 and CD36 were measured by flow cytometry. Results: Mitotane treatment, within therapeutic range, decreased staining for LDs significantly in H295R. This was also reflected by decreased expression of LDAPs, PLIN1 and PLIN3. The decrease in H295R LDs was associated with increased activation of HSL (pHSL and LIPE). However, this effect was only evident in MUC-1 at supratherapeutic mitotane (200µM). H295R and MUC-1 demonstrated similar overall LD numbers at baseline and under cholesterol supplementation. Expression of PLIN3 was high in both cell lines, while PLIN1, PLIN2 and PLIN4 demonstrated distinct LD profiles in each. Investigation of LD content showed that H295R preferentially store CEs while MUC-1 store only TAG, irrespective of cholesterol-loading. Mitotane treatment significantly reduces both CE and TAG LDs in H295R and MUC-1. Expression of lipid uptake receptors also demonstrated significant variability between cell lines including SRB1 and LRP1. Conclusion: We highlight that lipolysis through LD breakdown and activation of HSL represents a putative additional mechanism for mitotane induced FC cytotoxicity in ACC. We also demonstrate significant differences in cholesterol handling and LDAPs between mitotane sensitive and mitotane resistant models, in particular, the absence of CE LDs in MUC-1. We therefore propose a mechanism of resistance to mitotane through absent CE storage. Further understanding of cholesterol and lipid handling in ACC offers novel therapeutic exploitation, especially in the setting of mitotane resistant disease.

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Liver X receptor inhibition potentiates mitotane-induced adrenotoxicity in ACC

Endocrine Related Cancer ◽

10.1530/erc-20-0031 ◽

2020 ◽

Vol 27 (6) ◽

pp. 361-373

Author(s):

Kate M Warde ◽

Erik Schoenmakers ◽

Eduardo Ribes Martinez ◽

Yi Jan Lim ◽

Maeve Leonard ◽

...

Keyword(s):

Cell Death ◽

Cell Lines ◽

Cholesterol Ester ◽

Free Cholesterol ◽

Cholesterol Metabolism ◽

Apoptotic Cell ◽

Treatment Options ◽

Liver X Receptor ◽

Resistant Disease ◽

Cholesterol Storage

Adrenocortical carcinoma (ACC) is a rare aggressive malignancy with a poor outcome largely due to limited treatment options. Here, we propose a novel therapeutic approach through modulating intracellular free cholesterol via the liver X receptor alpha (LXRα) in combination with current first-line pharmacotherapy, mitotane. H295R and MUC-1 ACC cell lines were pretreated with LXRα inhibitors in combination with mitotane. In H295R, mitotane (20, 40 and 50 µM) induced dose-dependent cell death; however, in MUC-1, this only occurred at a supratherapeutic concentration (200 µM). LXRα inhibition potentiated mitotane-induced cytotoxicity in both cell lines. This was confirmed through use of the CompuSyn model which showed moderate pharmacological synergism and was indicative of apoptotic cell death via an increase in annexinV and cleaved-caspase 3 expression. Inhibition of LXRα was confirmed through downregulation of cholesterol efflux pumps ABCA1 and ABCG1; however, combination treatment with mitotane attenuated this effect. Intracellular free-cholesterol levels were associated with increased cytotoxicity in H295R (r2 = 0.5210) and MUC-1 (r2 = 0.9299) cells. While both cell lines exhibited similar levels of free cholesterol at baseline, H295R were cholesterol ester rich, whereas MUC-1 were cholesterol ester poor. We highlight the importance of LXRα mediated cholesterol metabolism in the management of ACC, drawing attention to its role in the therapeutics of mitotane sensitive tumours. We also demonstrate significant differences in cholesterol storage between mitotane sensitive and resistant disease.

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SUN-LB22 PLK1 as a New Treatment Target for Adrenocortical Carcinoma

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.2164 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Gabrielle Smith ◽

Raimunde Liang ◽

Vasileios Chortis ◽

Sana Khan ◽

Juliane Lippert ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Lines ◽

Adrenocortical Carcinoma ◽

Cell Apoptosis ◽

Cancer Type ◽

Solid Organ ◽

Treatment Target ◽

Significant Difference ◽

H295r Cells ◽

Plk1 Expression

Abstract Background: Adrenocortical carcinoma (ACC) is an aggressive malignancy with limited medical treatment options. We previously identified polo-like kinase 1 (PLK1) as one of most overexpressed genes in ACC; thus PLK1 represents a potential treatment target for this cancer type. Some PLK1 inhibitors are under evaluation in clinical trials for other solid organ malignancies, and seem to be more effective in TP53 mutated tumours. The aim of this study was to evaluate PLK1 protein levels in a large series of ACC and assess the in vitroefficacy of PLK1 inhibitors in two different ACC cell lines. Methods: 104 formalin-fixed paraffin-embedded ACC tissue samples with available genetic data were investigated. Nuclear PLK1 protein expression was evaluated by immunohistochemistry and a semi-quantitative H-score was calculated. PLK1 expression levels were correlated to clinical and histological parameters. Efficacy of PLK1-specific inhibitor Volasertib (0-200 nM) was tested in the standard NCI-H295R ACCcell line, which presents PLK-1 overexpression and a large TP53 deletion, and in the newly established MUC1 cell line, which bears a frameshift mutation in TP53. Cell proliferation was analysed using DNA fluorescence and cell apoptosis by Caspase Glo 3/7 assay. Results: Nuclear PLK1 expression was classified as high in 59% of ACC samples, with a significant difference noted between TP53-mutated (n=24) and wild-type (n=80) cases (87.5 vs 51%, p<0.01). PLK1 levels did not correlate with either progression-free or overall survival. H295R cells showed a significant time- and dose-dependent reduction of cell proliferation compared to vehicle control after 72h of Volasertib treatment (p<0.005 per trend, p=0.01 by 200nM by non-parametric two-way ANOVA). A less pronounced and non-significant trend towards inhibited proliferation was observed in MUC1 cells. Cell apoptosis was significantly higher in the H295R cells treated with 175nM and 200nM Volasertib when compared to control (p<0.05), while there was no significant difference in MUC1 cells. Conclusion:In this pilot study, we propose PLK1 inhibitors as promising candidates for treatment of a subset of ACC patients that may be pre-selected according to the tumour molecular pattern. We plan to extend functional experiments to further PLK1 inhibitors, including additional ACC cell lines with a different molecular profile.

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Antineoplastic Effect of a Combined Mitotane Treatment/Ionizing Radiation in Adrenocortical Carcinoma: A Preclinical Study

Cancers ◽

10.3390/cancers11111768 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1768

Author(s):

Cerquetti ◽

Bucci ◽

Carpinelli ◽

Lardo ◽

Proietti ◽

...

Keyword(s):

Dna Damage ◽

Cell Death ◽

Ionizing Radiation ◽

Tumor Growth ◽

Growth Inhibition ◽

Cell Lines ◽

Adrenocortical Carcinoma ◽

Repair System ◽

Msh2 Gene ◽

Sw13 Cell

Mitotane (MTT) is an adrenolytic drug used in adjuvant and advanced treatments of adrenocortical carcinoma (ACC). Ionizing radiation (IR) is also used in adrenal cancer treatment, even though its biological action remains unknown. To provide a reliable in vivo preclinical model of ACC, we used mouse xenografts bearing human ACC to test the effects of MTT and IR alone and in combination. We evaluated tumor growth inhibition by the RECIST criteria and analyzed the cell cycle by flow cytometry (FCM). In the xenograft ACC model treated with MTT/IR in combination, we observed a marked inhibition of tumor growth, with strong tumor regression (p < 0.0001) compared to MTT and IR given alone (p < 0.05). The MTT results confirm its antisteroidogenic activity (p < 0.05) in the xenograft ACC model, revealing its ability to render cancer cells more prone to radiotherapy treatment. In addition, to explain the biological effect of these treatments on the Mismatch Repair System (MMR), we interfered with the MSH2 gene expression in untreated and MTT/IR-treated H295R and SW13 cell lines. Moreover, we observed that upon treatment with MTT/IR to induce DNA damage, MSH2 gene inhibition in both the H295R and SW13 cell lines did not allow DNA damage repair, thus inducing cell death. In conclusion, MTT seems to have a radiosensitizing property and, when given in combination with IR, is able to promote neoplastic growth inhibition, leading to a significant reduction in tumor size due to cell death.

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Anti-Myeloma Activity of the Small-Molecule Aurora Kinase Inhibitor VE465.

Blood ◽

10.1182/blood.v108.11.3468.3468 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3468-3468 ◽

Cited By ~ 2

Author(s):

Joseph M. Negri ◽

Douglas W. McMillin ◽

Nicholas Mitsiades ◽

Teru Hideshima ◽

Dharminder Chauhan ◽

...

Keyword(s):

Cell Death ◽

Cell Lines ◽

Kinase Activity ◽

Kinase Inhibitor ◽

Aurora Kinase ◽

Therapeutic Window ◽

Significant Shift ◽

Aurora Kinase Inhibitor ◽

Ic50 Values

Abstract Multiple Myeloma (MM) remains an incurable plasma cell neoplasia, despite recent additions in the therapeutic arsenal for its management. Aurora kinases play integral roles in the orchestration of chromosomes and cytoskeletal mobility during the process of cell division. Aurora kinase activity has been implicated in several tumor types, including ovarian, colon, and prostate cancers. To determine whether inhibition of Aurora kinase activity could attenuate myeloma cell survival, we performed studies of the Aurora kinase inhibitor VE465 (Vertex Pharmaceuticals / Merck & Co., Inc.). VE465 inhibits all 3 Aurora isoforms (Aur A, B and C) with approximate Ki values of 1, 26, and 8.7 nM respectively. MTT colormetric survival assays (72–96hrs exposure) showed that VE465 is active against a wide panel of human MM cell lines: 26 of 38 MM cell lines had IC50 values at or < 100 nM, which are significantly lower than IC50 values for normal hematopoietic cells, e.g. unstimulated or PHA-stimatuled PBMCs. Importantly, VE465 was active in vitro against MM cell lines and/or primary MM tumor cells resistant to various anti-MM therapeutics, including dexamethasone, alkylating agents, anthracyclines, the proteasome inhibitor bortezomib, and/or immunomodulatory thalidomide derivatives (IMiDs). Moreover, VE465 maintained its activity despite the presence of protective bone marrow-derived cytokines (e.g. IL-6). PI cell cycle analyses showed that VE465 causes (even within 8 hrs of treatment) caused pronounced G2 arrest, followed by significant shift of MM cells to sub-G1 gate, consistent with cell death. Immunoblotting analyses confirmed that VE465 treatment induces cleavage of PARP, as well as cleavage of caspases-8 and -9, without significant changes in the expression levels of several key molecular effectors (e.g. Mcl-1, Bax, p53, hsp70, hsp90, hs27) which have been previously implicated in the mechanism of anti-MM activity of diverse other therapeutics. Screening of VE465-based combination regimens with other anti-MM agents showed additive effects of VE465 with the histone deacetylase inhibitor Vorinostat (SAHA) (Merck & Co., Inc). Ongoing studies in our Center are addressing the identification of specific molecular markers correlating with the degree of sensitivity of MM cells to VE465. Our in vitro evidence for induction of MM cell death and therapeutic window for the anti-MM effect of VE465, its ability to overcome protective effect of BM-derived cytokines, and the clearly distinct pattern of molecular sequelae of VE465 compared to several other agents in our current anti-MM therapeutic armamentarium, all suggest that Aurora kinase inhibition represents an intriguing novel targeted treatment strategy in MM. Importantly, these studies, particularly the identification of a sizeable subset of MM cell lines with higher sensitivity to VE465 than normal cells, provide the framework for in vivo VE465 studies in progress, alone and in combination with other anti-MM agents, to inform the design of potential clinical trials of this class of agents for MM.

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3,6’-dithiopomalidomide reduces neural loss, inflammation, behavioral deficits in brain injury and microglial activation

eLife ◽

10.7554/elife.54726 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Chih-Tung Lin ◽

Daniela Lecca ◽

Ling-Yu Yang ◽

Weiming Luo ◽

Michael T Scerba ◽

...

Keyword(s):

Cell Death ◽

Brain Injury ◽

Microglial Activation ◽

Therapeutic Window ◽

Post Injury ◽

Behavioral Deficits ◽

Immunomodulatory Agents ◽

Associated Proteins ◽

Behavioral Impairments ◽

Pro Inflammatory Cytokines

Traumatic brain injury (TBI) causes mortality and disability worldwide. It can initiate acute cell death followed by secondary injury induced by microglial activation, oxidative stress, inflammation and autophagy in brain tissue, resulting in cognitive and behavioral deficits. We evaluated a new pomalidomide (Pom) analog, 3,6’-dithioPom (DP), and Pom as immunomodulatory agents to mitigate TBI-induced cell death, neuroinflammation, astrogliosis and behavioral impairments in rats challenged with controlled cortical impact TBI. Both agents significantly reduced the injury contusion volume and degenerating neuron number evaluated histochemically and by MRI at 24 hr and 7 days, with a therapeutic window of 5 hr post-injury. TBI-induced upregulated markers of microglial activation, astrogliosis and the expression of pro-inflammatory cytokines, iNOS, COX-2, and autophagy-associated proteins were suppressed, leading to an amelioration of behavioral deficits with DP providing greater efficacy. Complementary animal and cellular studies demonstrated DP and Pom mediated reductions in markers of neuroinflammation and α-synuclein-induced toxicity.

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