Adrenocortical Cancer Cell Research Articles

12407 Assessing Human Monocyte Migration And Co-culture With Adrenocortical Cancer Cells

Abstract Disclosure: C. Hong: None. S. Feely: None. R. Challapalli: None. N. Mullen: None. A. Sorushanova: None. C. Hantel: None. M.D. Griffin: None. M.C. Dennedy: None. Adrenocortical carcinoma (ACC) is a lipid, typically steroidogenic cancer which carries a 5-year prognosis of <10%. Therapeutic options are limited including surgery and mitotane – a poorly tolerated and efficacious insecticide. Recent data have demonstrated that the immune environment of adrenocortical carcinoma is deficient in lymphocytes while demonstrating a relative rich monocyte/macrophage population in the tumour microenvironment. We have investigated the gradient for migration of circulating peripheral monocytes to ACC. ACC cells were seeded in the bottom chamber of the transwell system. Monocytes were isolated from human peripheral blood, by negative selection using magnetic beads with high purity (>95%) and quality. Migration of monocytes at 24/48h was evaluated by identifying those remaining in the top chamber, the floating fraction in the bottom chamber and those adherent to ACC cells in the bottom chamber. Analysis of monocyte/macrophages was undertaken by flow cytometry using the following markers: Sytox Blue, CD45, CD14, CD16 and HLA-DR. At 24h, when compared to control conditions, more monocytes had migrated to the ACC cells seeded lower chamber and attached to the bottom. Similar results were observed in migration at 48h. Predominantly Classical monocytes (CD14++CD16-HLA-DR+) migrated to metastatic cancer cells (MUC1), and Non-Classical monocytes (CD14+CD16+HLA-DR+) migrated to primary cancer cells (H295R/ HAC15) at 48h. We then investigated the polarization cytokine profile of migrated monocytes with or without ACC cells at 72 h by multiplex bead-based assay. Migrated monocytes indicated a significant shift toward the M2 phenotype in the presence of H295R or HAC15. The production of the pro-inflammatory factors of IL-6, TNF-alpha, etc. decreased and immunosuppressive factors of IL-10, TARC, etc. increased. MUC1 didn’t increase M2 or M1 cytokine secretion. Moreover, surface markers of migrated monocytes were evaluated at 72h by flow cytometry. When compared to the control group, all 3 ACC cell lines switched monocytes/ macrophages into M2 phenotype, as evidenced by the decrease in the expression of the M1 marker CD86 and the increase in the expression of the M2 markers CD163 and CD206. We demonstrate monocytes migrate and associate with ACC cells: (i) subpopulations differ in transmigration properties in response to ACC cells (ii) monocytes/macrophages exhibit M2 polarization phenotype. Presentation: 6/2/2024

12407 Assessing Human Monocyte Migration And Co-culture With Adrenocortical Cancer Cells

12407 Assessing Human Monocyte Migration And Co-culture With Adrenocortical Cancer Cells

Evaluating human monocyte migration and co-culture with adrenocortical cancer cells

Evaluating human monocyte migration and co-culture with adrenocortical cancer cells

Enhancing mitotane efficacy in adrenocortical carcinoma by calcineurin inhibition with cyclosporine A

Aims: The aim of this study is to determine the effect of calcineurin (CaN) in adrenocortical cancer (ACC) cells, which is a rare but aggressive type of cancer resistant to mitotane therapy. The intracellular calcium signaling pathway is one of the most important mechanisms for cells. The effect of intracellular calcium concentration [(Ca2+i)] on the function of cancer cells is also known. CaN, activated by the binding of calmodulin and Ca2+, is critical in this pathway. Methods: H295 adrenocortical cancer cells were treated with mitotane, cyclosporine A (CsA), and a combination of both. Cell viability, apoptosis, cell cycle, and gene expression levels of apoptosis-related genes (BCL2, BAX, TP53) were analyzed. Western blotting was used to measure CaN protein levels, and wound healing assays assessed cell migration. Results: CsA significantly suppressed CaN protein levels in a dose-dependent manner, reducing cell viability and increasing apoptosis in H295 cells. Mitotane alone also suppressed CaN protein, but the combination of mitotane and CsA had a synergistic effect, further decreasing cell viability and increasing apoptosis. The combination treatment led to significant suppression of the BCL2 gene and upregulation of TP53. Cell cycle analysis showed increased arrest in the G0/G1 phase with combination treatment. Conclusion: Suppression of CaN by CsA enhances the cytotoxic effects of mitotane on ACC cells, suggesting a potential therapeutic strategy to improve ACC treatment outcomes. This study highlights the importance of targeting intracellular calcium signaling pathways to overcome resistance and enhance the efficacy of existing cancer therapies.

Abstract 6274: Integrated multi-omics revealed the involvement of MYC and mTOR pathways in radiation resistance of adrenocortical cancer

Abstract The morbidity and mortality of patients with adrenocortical cancer (ACC) are commonly due to locoregional postoperative recurrence and progressive systemic metastasis. Surgical resection remains the only curative treatment option, but even with complete resection, locoregional recurrence rates remain high. Adjuvant radiation therapy (RT) has been shown to reduce recurrence rates and may improve overall survival in selected patients. However, half of the patients receiving postoperative RT still develop locoregional recurrence, clearly demonstrating the heterogeneous nature of ACC. Minimal investigation into the mechanisms underlying radiation response and resistance in ACC has been performed, but elucidating this information is critical to improving outcomes. To address this knowledge gap, NCI-H295R cells were treated with fractionated radiation at 1 Gray (Gy) per day for 5 consecutive days for two consecutive weeks, cumulating in a total radiation dose of 10 Gy. Treated and control ACC cells were harvested on days 7 and 14 for bulk RNA-sequencing (RNA-Seq) and total proteomics by liquid chromatography and tandem mass spectrometry. Significantly differentially expressed genes (DEGs) and proteomes (adj. p. value <0.05) that overlapped between day 7 and day 14 were selected for further analysis and comparison with publicly available gene expression data sets in ACC (The Cancer Genome Atlas (TCGA); Gene Expression Omnibus: GSE12368, GSE33371, GSE75415, GSE90713). There were 3993 DEGs and 423 proteomes identified, respectively. Of these, there were 139 overlapping DEGs with respective proteomes found, 72 of which were differentially expressed in the 4 GEO data sets comparing normal adrenal cortex to ACC. RNA-Seq pathway analysis demonstrated enrichment of E2F targets, G2M checkpoint, mTORC1 signaling, and MYC targets v1, while total proteomics pathway analysis revealed enrichment of cell cycle, transcriptional regulation, cellular stress and injury, alterations of RNA metabolism, and metabolism proteomes. Given that MYC and mTOR are both involved in transcriptional regulation and cell cycle regulation, and mTORC1 in particular is associated with cellular metabolism, the RNA-Seq and proteomics pathway analyses show general consistency with each other. TCGA gene expression analysis showed that overexpression of MYC or mTOR was correlated with shorter overall survival (OS) and disease-free survival (DFS) in the entire ACC cohort (p<0.05). Together, the combination of in vitro pathway analysis and TCGA analysis suggests a potential involvement of the MYC and mTOR pathways in radiation resistance in ACC. Further in vitro and in vivo studies are warranted to identify specific molecular compounds that target MYC and mTOR pathways and improve radiation response in ACC. Citation Format: Bhavishya Ramamoorthy, Prachi Mishra, Myriem Boufraqech, Nicholas Michael, Margaret Cam, Richard Finney, Thomas Meyer, Ronald Holewinski, Thorkell Andresson, Madeline Wong, Steven Shema, Desiree Tillo, Naris Nilubol. Integrated multi-omics revealed the involvement of MYC and mTOR pathways in radiation resistance of adrenocortical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6274.

Low PRKAB2 Expression Is Associated with Poor Outcomes in Pediatric Adrenocortical Tumors, and Treatment with Rottlerin Increases the PRKAB2 Level and Inhibits Tumorigenic Aspects in the NCI-H295R Adrenocortical Cancer Cell Line.

Pediatric adrenocortical tumors (ACTs) are rare, highly heterogeneous neoplasms with limited therapeutic options, making the investigation of new targets with potential therapeutic or prognostic purposes urgent. The PRKAB2 gene produces one of the subunits of the AMP-activated protein kinase (AMPK) complex and has been associated with cancer. However, little is known about the role AMPK plays in ACTs. We have evaluated how PRKAB2 is associated with clinical and biological characteristics in 63 pediatric patients with ACTs and conducted in vitro studies on the human NCI-H295R ACC cell line. An analysis of our cohort and the public ACC pediatric dataset GSE76019 showed that lower PRKAB2 expression was associated with relapse, death, metastasis, and lower event-free and overall survival rates. Multivariate analysis showed that PRKAB2 expression was an independent prognostic factor when associated with age, tumor weight and volume, and metastasis. In vitro tests on NCI-H295R cells demonstrated that Rottlerin, a drug that can activate AMPK, modulated several pathways in NCI-H295R cells, including AMPK/mTOR, Wnt/β-catenin, SKP2, HH, MAPK, NFKB, and TNF. Treatment with Rottlerin decreased cell proliferation and migration, clonogenic capacity, and steroid production. Together, these results suggest that PRKAB2 is a potential prognostic marker in pediatric ACTs, and that Rottlerin is promising for investigating drugs that can act against ACTs.

Selective non-lipogenic ABCA1 inducer, CL2-57, affects cholesterol efflux pathways and adrenocortical cancer cell migration in 2D and 3D spheroid models

Selective non-lipogenic ABCA1 inducer, CL2-57, affects cholesterol efflux pathways and adrenocortical cancer cell migration in 2D and 3D spheroid models

Biological activities of the aerial and undergound parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) from the Italian Alps

This study investigated the bioactivity of both aerial (GNAR) and underground (GNUG) parts of Gymnadenia nigra Rchb.f. (syn. Nigritella nigra (L.) Rchb. f.) (Orchidaceae). The obtained data proved interesting when the samples were tested in two adrenocortical cancer cell lines (SW13 and H295R). In particular, the GNAR 80% methanol extract distinctly inhibited their viability after 24 h at a concentration of 1 µg/µL by MTT assay and trypan blue dye exclusion method. Cell morphology evaluation by means Wright’s staining also showed significant results, particularly in SW13 cells under the effect of both extracts. GNAR extract was able to scavenge the DPPH radical better than GNUG extract. It also was more active in albumin denaturation (a maximum % denaturation equal to 463.0 ± 8.3 vs 77.3 ± 13.3) and protease inhibition (a maximum % inhibition equal to 138.5 ± 7.0 vs 2.1 ± 2.0) tests. The results highlighted an important antitumor activity of G. nigra in vitro that deserves to be further studied.

Gi/o GPCRs drive the formation of actin-rich tunneling nanotubes in cancer cells via a Gβγ/PKCα/FARP1/Cdc42 axis

The functional association between stimulation of G-protein-coupled receptors (GPCRs) by eicosanoids and actin cytoskeleton reorganization remains largely unexplored. Using a model of human adrenocortical cancer cells, here we established that activation of the GPCR OXER1 by its natural agonist, the eicosanoid 5-oxo-eicosatetraenoic acid, leads to the formation of filopodia-like elongated projections connecting adjacent cells, known as tunneling nanotube (TNT)-like structures. This effect is reduced by pertussis toxin and GUE1654, a biased antagonist for the Gβγ pathway downstream of OXER1 activation. We also observed pertussis toxin-dependent TNT biogenesis in response to lysophosphatidic acid, indicative of a general response driven by Gi/o-coupled GPCRs. TNT generation by either 5-oxo-eicosatetraenoic acid or lysophosphatidic acid is partially dependent on the transactivation of the epidermal growth factor receptor and impaired by phosphoinositide 3-kinase inhibition. Subsequent signaling analysis reveals a strict requirement of phospholipase C β3 and its downstream effector protein kinase Cα. Consistent with the established role of Rho small GTPases in the formation of actin-rich projecting structures, we identified the phosphoinositide 3-kinase-regulated guanine nucleotide exchange factor FARP1 as a GPCR effector essential for TNT formation, acting via Cdc42. Altogether, our study pioneers a link between Gi/o-coupled GPCRs and TNT development and sheds light into the intricate signaling pathways governing the generation of specialized actin-rich elongated structures in response to bioactive signaling lipids.

Selectivity of osilodrostat as an inhibitor of human steroidogenic cytochromes P450

Osilodrostat (LCI699) is a potent inhibitor of the human steroidogenic cytochromes P450 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). LCI699 is FDA-approved for the treatment of Cushing disease, which is characterized by chronic overproduction of cortisol. While phase II and III clinical studies have proven the clinical efficacy and tolerability of LCI699 for treating Cushing disease, few studies have attempted to fully assess the effects of LCI699 on adrenal steroidogenesis. To this end, we first comprehensively analyzed LCI699-mediated inhibition of steroid synthesis in the NCI-H295R human adrenocortical cancer cell line. We then studied LCI699 inhibition using HEK-293 or V79 cells stably expressing individual human steroidogenic P450 enzymes. Our studies using intact cells confirm the potent inhibition of CYP11B1 and CYP11B2 with negligible inhibition of 17-hydroxylase/17,20-lyase (CYP17A1) and 21-hydroxylase (CYP21A2). Furthermore, partial inhibition of the cholesterol side-chain cleavage enzyme (CYP11A1) was observed. To calculate the dissociation constant (Kd) of LCI699 with the adrenal mitochondrial P450 enzymes, we successfully incorporated P450s into lipid nanodiscs and carried out spectrophotometric equilibrium and competition binding assays. Our binding experiments confirm the high affinity of LCI699 to CYP11B1 and CYP11B2 (Kd ≈ 1 nM or less) and much weaker binding for CYP11A1 (Kd = 18.8 μM). Our results confirm the selectivity of LCI699 for CYP11B1 and CYP11B2 and demonstrate partial inhibition of CYP11A1 but not CYP17A1 and CYP21A2.

PS-B04-9: ELUCIDATING EFFECT OF INHIBITING ALDOSTERONE SYNTHESIS ON HUMAN ADRENOCORTICAL CELLS

Introduction: Modern human diet is high in salt. High salt stimulates decrease of aldosterone by down-regulation of aldosterone synthase (encoded by the gene CYP11B2) in the zona glomerulosa (ZG). Aldosterone regulates blood pressure and volume by regulating salt secretion. Interestingly, adult human adrenal ZG is sparse and confined to discrete islets of cells unlike rodent adrenal where the zone encompasses the whole periphery of the adrenal. We therefore hypothesize that ZG cell function change from synthesis to apoptosis/migration mode when aldosterone is not required due to the high salt diet. Objective: Herein, we intend to characterise human adrenocortical cells silenced for CYP11B2. Methods: We transfected HAC15, a subclone of the H295R human adrenocortical cancer cell line with scrambled siRNA (siScrambled) or siRNA targeting CYP11B2 (siCYP11B2) using the Neon Transfection System. 48-h post-transfection, cell apoptosis rate was assessed using the Pacific Blue Annexin V/SYTOX AADvanced apoptosis kit. The supernatants and cells were harvested for aldosterone and cortisol measurements, and RNA isolation. A representative sample of siCYP11B2 and siScrambled from each independent experiment was selected for RNA-Sequencing (RNAseq). The EZMTT Proliferation Assay was performed on equally seeded HAC15 cells, 72-h post-transfection with either siScrambled or siCYP11B2 using the Lipofectamin 3000 Transfection reagent. Experiments were repeated 3 times independently in triplicates. Results: siCYP11B2 transfection limited the production of aldosterone by 69.8% (p = 0.001) and down-regulated the expression of CYP11B2 by 83.0% (p = 0.001). We confirmed that the silencing was specific to CYP11B2 despite the gene being highly homologous to CYP11B1 as there were no significant change on cortisol production and CYP11B1 mRNA expression. RNAseq analysis found 3 genes, CYP11B2, HNRNPA1, and UHMK1, consistently down-regulated in siCYP11B2 by 32%, 65% and 67% respectively. The most enriched pathways among the differentially expressed genes (DEG) were mitophagy and autophagy. No difference in cell apoptosis was seen in siCYP11B2 cells. Instead, an increase in cell proliferation was detected with the EZMTT assay. Conclusion: Our findings suggest that silencing of CYP11B2 does not affect apoptosis but does affect cell proliferation. Interestingly, the mitophagy and autophagy pathways were enriched in the DEGs of siCYP11B2 cells.

PSUN14 Elucidating Effect of Modulating Aldosterone Synthesis on Cell Fate in Human Adrenal Cells

Abstract Introduction Aldosterone regulates sodium homeostasis thereby affecting blood volume and blood pressure. Physiologically, aldosterone is synthesized in the zona glomerulosa (ZG) by aldosterone synthase (CYP11B2) when there is a lack of salt. However, in the adult human adrenal ZG is sparse and confined to discrete islets (1). We hypothesize that the high salt in modern diets change ZG cell function from synthesis to apoptosis/migration mode when aldosterone is not required. This hypothesis is based on: [i] CYP11B2−/− mice, where ZG cells migrate and apoptose (2); [ii] the disappearance from most of human ZG of CYP11B2, which became apparent with selective antisera (3); [iii] apoptosis in monkeys following aldosterone synthase inhibition (4). Herein we aim to test our hypothesis by silencing CYP11B2 in human adrenal cells. Methods The HAC15, a subclone of the H295R human adrenocortical cancer cell line, was transfected with scrambled siRNA (siScrambled) or siRNA targeting CYP11B2 (siCYP11B2) using the Neon™ Transfection System (Thermofisher Scientific, USA). An apoptosis assay 48-h post-transfection was performed using the Pacific Blue™ Annexin V/SYTOX™ AADvanced™ apoptosis kit on the BD FACSVerseTM system (BD Biosciences, USA). The supernatants and cells were harvested for aldosterone and cortisol, and RNA isolation. Experiments were repeated 3 times independently in triplicates. RNA-Sequencing (RNAseq) of siCYP11B2 and siScrambled was performed on a representative sample from each independent experiment (BGI Genomics, China). Results Transfected HAC15 cells with siCYP11B2 showed reduction of aldosterone production by 69.8% (p=0.001) and suppression of CYP11B2 mRNA by 8 folds (p=0.001) compared to control, with no significant change on cortisol production and CYP11B1 mRNA expression. This confirmed that the silencing was specific to CYP11B2 despite the gene being highly homologous to CYP11B1. In all 3 independent experiments, 3 genes were significantly downregulated; CYP11B2, HNRNPA1, and UHMK1 expression in siCYP11B2 cells were 32%, 65% and 67% of matched control siScramble cells. Result of KEGG pathway from each independent experiment showed the most enriched pathways among the differentially expressed genes (DEG) were mitophagy and autophagy. However, analysis of flow cytometric apoptosis assay showed silencing of CYP11B2 did not induce cell apoptosis. Conclusion Our finding showed that silencing of CYP11B2 can affect mitophagy and autophagy but does not affect cell apoptosis in the HAC15 human adrenocortical cancer cell line. Further investigation on cell proliferation and in non-cancerous adrenal cells may be needed to identify the effect of modulating aldosterone synthesis on ZG cell fate. Reference (1) Vinson G., Front Neurosci 2016; 10: 238. (2) Lee G, et al., Endocrinology 2005; 146: 2650–2656. (3) Gomez-Sanchez CE, et al., Mol Cell Endocrinol 2014; 383: 111–117. (4) Bogman K, et al., Hypertension. 2017; 69(1): 189–196. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

MicroRNA-149-3p expression correlates with outcomes of adrenocortical tumor patients and affects proliferation and cell cycle progression of H295A adrenocortical cancer cell line.

Pediatric adrenocortical tumor (ACT) is a rare and aggressive neoplasm, with incidence in southern and southeastern Brazil 10-15 times higher than worldwide. Although microRNAs (miRNAs) have been reported to act as tumor suppressors or oncogenes in several cancers, the role of miR-149-3p in ACT remains unknown. In this study, we evaluated the expression of miR-149-3p in 67 pediatric ACT samples and 19 non-neoplastic adrenal tissues. The overexpression of miR-149-3p was induced in H295A cell line, and cell viability, proliferation, colony formation, and cell cycle were assessed by in miR-149-3p mimic or mimic control. In silico analysis were used to predict miR-149-3p putative target genes. CDKN1A expression at the mRNA and protein levels was evaluated by qRT-PCR and western blot, respectively. Higher miR-149-3p expression was associated with unfavorable ACT outcomes. Compared to the mimic control, miR-149-3p overexpression increased cell viability and colony formation, and affected cell cycle progression. Also, we identified CDKN1A as a potential miR-149-3p target gene, with decreased expression at both the gene and protein levels in miR-149-3p mimic cells. Collectively, these findings suggest that miR-149-3p promotes H295A cell viability by downregulating CDKN1A and provide evidence that miR-149-3p may be useful as a novel therapeutic target for pediatric ACT.

Estrogen Related Receptor Alpha (ERRα) a Bridge between Metabolism and Adrenocortical Cancer Progression.

Simple SummaryAdrenocortical carcinoma (ACC) is a rare and highly aggressive tumor associated with a very poor prognosis, mostly due to a high risk of recurrence and limited therapeutic options. The identification of “master regulators” of the metabolic changes occurring in cancer cells could offer new targets for innovative therapies. Such a strategy has never been used against ACC progression. In this study, we identify ERRα as key player in ACC metabolism and its targeting can prevent progression to a more aggressive phenotype. The development of new therapeutic strategies to selectively target ERRα in the adrenal with a selective antagonist would hinder ACC progression, avoiding off-target effects.The aim of this study was to investigate the metabolic changes that occur in adrenocortical cancer (ACC) cells in response to the modulation of Estrogen Related Receptor (ERR)α expression and the impact on ACC progression. Proteomics analysis and metabolic profiling highlighted an important role for ERRα in the regulation of ACC metabolism. Stable ERRα overexpression in H295R cells promoted a better mitochondrial fitness and prompted toward a more aggressive phenotype characterized by higher Vimentin expression, enhanced cell migration and spheroids formation. By contrast, a decrease in ERRα protein levels, by molecular (short hairpin RNA) and pharmacological (inverse agonist XCT790) approaches modified the energetic status toward a low energy profile and reduced Vimentin expression and ability to form spheroids. XCT790 produced similar effects on two additional ACC cell lines, SW13 and mitotane-resistant MUC-1 cells. Our findings show that ERRα is able to modulate the metabolic profile of ACC cells, and its inhibition can strongly prevent the growth of mitotane-resistant ACC cells and the progression of ACC cell models to a highly migratory phenotype. Consequently, ERRα can be considered an important target for the design of new therapeutic strategies to fight ACC progression.

Bioinformatic analyses of hydroxylated polybrominated diphenyl ethers toxicities on impairment of adrenocortical secretory function.

BackgroundPolybrominated diphenyl ethers (PBDEs) and their metabolites have severe impact on human health, but few studies focus on their nephrotoxicity. This study was conceived to explore hub genes that may be involved in two hydroxylated polybrominated diphenyl ethers toxicities on impairment of adrenocortical secretory function.MethodsGene dataset was obtained from Gene Expression Omnibus (GEO). Principal component analysis and correlation analysis were used to classify the samples. Differentially expressed genes (DEGs) were screened using the limma package in RStudio (version 4.1.0). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome enrichment analyses of DEGs were conducted. Protein-protein interaction (PPI) network was established using STRING network, and genes were filtered by Cytoscape (version 3.8.2). Finally, the hub genes were integrated by plug-in CytoHubba and RobustRankAggreg, and were preliminarily verified by the Comparative Toxicogenomics Database (CTD).ResultsGSE8588 dataset was selected in this study. About 190 upregulated and 224 downregulated DEGs in 2-OH-BDE47 group, and 244 upregulated and 276 downregulated DEGs in 2-OH-BDE85 group. Functional enrichment analyses in the GO, KEGG and Reactome indicated the potential involvement of DEGs in endocrine metabolism, oxidative stress mechanisms, regulation of abnormal cell proliferation, apoptosis, DNA damage and repair. 2-OH-BDE85 is more cytotoxic in a dose-dependent manner than 2-OH-BDE47. A total of 98 hub genes were filtered, and 91 nodes and 359 edges composed the PPI network. Besides, 9 direct-acting genes were filtered for the intersection of hub genes by CTD.ConclusionsOH-PBDEs may induce H295R adrenocortical cancer cells in the disorder of endocrine metabolism, regulation of abnormal cell proliferation, DNA damage and repair. The screened hub genes may play an important role in this dysfunction.Supplementary informationThe online version contains supplementary material available at https://doi.org/10.1265/ehpm.22-00023.

Abstract 412: Blockade of beta-catenin pathway combined with Aurora kinase activity inhibition enhances antitumor effects in adrenocortical cancer cells

Abstract Adrenocortical cancer (ACC) is a rare and aggressive type of endocrine tumor with high risk of recurrence and metastasis. The overall survival of patients diagnosed with ACC is very low and current treatment for metastatic stages remain limited to mitotane, which has low efficiency in advanced stages of the disease and is associated with high toxicity and side effects. Therefore, identification of new biological targets to improve ACC treatment is crucial. Beta-catenin alterations have been found in approximately 30% of ACCs and blockade of Wnt-beta-catenin showed to decrease adrenal steroidogenesis and increase apoptosis of ACC cells, NCI-H295, in vitro and in a mouse model. Aurora kinases are known to play an important role in cell division during G1-M phase and their aberrant expression is correlated with poor prognosis in different types of tumors. Hence, we hypothesized that inhibition of aurora kinase activity combined with beta-catenin pathway blockade could promote better antitumor effects in NCI-H295 cells. We studied the in vitro effects of AMG 900, an aurora kinase activity inhibitor and PNU-74654, a beta-catenin pathway blocker, in proliferation, migration, colony establishment and invasion of NCI-H295 and SW-13 ACC cell lines. Our results show that the combination of AMG 900 with PNU-74654, decreases cell proliferation and viability, compared to either one of the single treatments. Regarding cell invasion and clonogenesis inhibition, AMG 900 was more efficient than PNU-74654, and its combination with the latter did not improve these effects. On the other hand, PNU-74654 was more effective in decreasing cortisol secretion. Taken together, our data suggest that inhibition of aurora kinases activity combined with blockade of the beta-catenin pathway could bring new approaches for designing new drugs to treat certain ACCs. Citation Format: Andrea G. Maria, Kleiton S. Borges, Regia C. Lira, Carolina H. Thome, Annabel Berthon, Ludivine Drougat, Fabio R. Faucz, Vitor M. Faca, Luiz G. Tone, Constantine A. Stratakis. Blockade of beta-catenin pathway combined with Aurora kinase activity inhibition enhances antitumor effects in adrenocortical cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 412.

Inhibition of Aurora kinase A activity enhances the antitumor response of beta-catenin blockade in human adrenocortical cancer cells

Adrenocortical cancer (ACC) is a rare and aggressive type of endocrine tumor with high risk of recurrence and metastasis. The overall survival of patients diagnosed with ACC is low and treatment for metastatic stages remain limited to mitotane, which has low efficiency in advanced stages of the disease and is associated with high toxicity. Therefore, identification of new biological targets to improve ACC treatment is crucial. Blockade of the Wnt/beta-catenin pathway decreased adrenal steroidogenesis and increased apoptosis of NCI–H295 human ACC cells, in vitro and in a xenograft mouse model. Aurora kinases play important roles in cell division during the G1-M phase and their aberrant expression is correlated with a poor prognosis in different types of tumors. Hence, we hypothesized that inhibition of aurora kinases activity combined with the beta-catenin pathway blockade would improve the impairment of ACC cell growth in vitro. We studied the combinatorial effects of AMG 900, an aurora kinase inhibitor and PNU-74654, a beta-catenin pathway blocker, on proliferation, survival and tumor progression in multiple ACC cell lines: NCI–H295, CU-ACC1 and CU-ACC2. Exposure of ACC cells to the combination of AMG 900 with PNU-74654 decreased cell proliferation and viability compared to either treatment alone. In addition, AMG 900 inhibited cell invasion and clonogenesis compared to PNU-74654, and the combination showed no greater effects. In contrast, PNU-74654 was more effective in decreasing cortisol secretion. These data suggest that inhibition of aurora kinases activity combined with blockade of the beta-catenin pathway may provide a combinatorial approach for targeting ACC tumors.

SIRT1 is involved in adrenocortical cancer growth and motility.

Adrenocortical cancer (ACC) is a rare tumour with unfavourable prognosis, lacking an effective treatment. This tumour is characterized by IGF‐II (insulin‐like growth factor II) overproduction, aromatase and ERα (oestrogen receptor alpha) up‐regulation. Previous reports suggest that ERα expression can be regulated by sirt1 (sirtuin 1), a nicotinamide adenine dinucleotide (NAD+)‐dependent class III histone deacetylases that modulates activity of several substrates involved in cellular stress, metabolism, proliferation, senescence, protein degradation and apoptosis. Nevertheless, sirt1 can act as a tumour suppressor or oncogenic protein. In this study, we found that in H295R and SW13 cell lines, sirt1 expression is inhibited by sirtinol, a potent inhibitor of sirt1 activity. In addition, sirtinol is able to decrease ACC cell proliferation, colony and spheroids formation and to activate the intrinsic apoptotic mechanism. Particularly, we observed that sirtinol interferes with E2/ERα and IGF1R (insulin growth factor 1 receptor) pathways by decreasing receptors expression. Sirt1 involvement was confirmed by using a specific sirt1 siRNA. More importantly, we observed that sirtinol can synergize with mitotane, a selective adrenolitic drug, in inhibiting adrenocortical cancer cell growth. Collectively, our data reveal an oncogenic role for sirt1 in ACC and its targeting could implement treatment options for this type of cancer.

The cytoskeleton actin binding protein filamin A impairs both IGF2 mitogenic effects and the efficacy of IGF1R inhibitors in adrenocortical cancer cells.

Adrenocortical carcinomas (ACCs) overexpress insulin-like growth factor 2 (IGF2), that drives a proliferative autocrine loop by binding to IGF1R and IR, but IGF1R/IR-targeted therapies failed in ACC patients. The cytoskeleton actin-binding protein filamin A (FLNA) impairs IR signalling in melanoma cells. Aims of this study were to test FLNA involvement in regulating IGF1R and IR responsiveness to both IGF2 and inhibitors in ACC. In ACC cells H295R and SW13 and primary cultures (1ACC, 4 adenomas) we found that IGF1R and IR interacted with FLNA, and FLNA silencing increased IGF1R and reduced IR expression, with a downstream effect of increased cell proliferation and ERK phosphorylation. In addition, FLNA knockdown potentiated antiproliferative effects of IGF1R/IR inhibitor Linsitinib and IGF1R inhibitor NVP-ADW742 in H295R. Finally, Western blot showed lower FLNA expression in ACCs (n=10) than in ACAs (n=10) and an inverse correlation of FLNA/IGF1R ratio with ERK phosphorylation in ACCs only. In conclusion, we demonstrated that low FLNA levels enhance both IGF2 proliferative effects and IGF1R/IR inhibitors efficacy in ACC cells, suggesting FLNA as a new factor influencing tumor clinical behavior and the response to the therapy with IGF1R/IR-targeted drugs.

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

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.