Adrenocortical Carcinoma Cell Research Articles

Therapeutic potential of targeting the FLNA-regulated Wee1 kinase in adrenocortical carcinomas.

Filamin A (FLNA) is poorly expressed in adrenocortical carcinomas (ACC) compared to adenomas (ACA). Its presence is associated to a less aggressive tumour behaviour, potentially due to its role in negatively regulating IGF1R signalling. Upregulation of G2/M Wee1 kinase was shown in FLNA-deficient mouse neural progenitor cells, and it has been reported in several tumours. This study explored Wee1 expression in ACC and its regulation by FLNA, the effects of Wee1 inhibitor AZD1775, and the impact of FLNA on its efficacy in ACC cell lines and primary cells. Analysis of FLNA and Wee1 proteins revealed elevated Wee1 and reduced FLNA in ACC compared to normal adrenal gland. FLNA knockdown increased Wee1 protein in NCI-H295R, MUC-1, and in primary ACC cells. Higher p-CDK1 and cyclin B1 were shown in FLNA-silenced MUC-1, while decreased Wee1, p-CDK1 and cyclin B1 resulted after FLNA overexpression. Wee1 reduction was reverted by lactacystin treatment and FLNA transfection increased p-Wee1 (Ser123), suggesting FLNA's role in targeting Wee1 for degradation. AZD1775 dose-dependently reduced proliferation and viability in ACC cell lines and primary cultures, and it triggered MUC-1 cell death. Similar effects were induced by Wee1 silencing. FLNA depletion augmented AZD1775's efficacy in reducing proliferation and potentiating apoptosis in MUC-1 and primary cells. In conclusion, we demonstrated that FLNA regulates Wee1 expression by promoting its degradation, suggesting that low FLNA typical of ACC leads to increased Wee1 with consequent cancer cells growth. It proposes Wee1 inhibition as a new potential therapeutic approach for ACC, particularly for those lacking FLNA.

8543 Non-Specific Uptake Of Magnetic Iron Oxide Nanoparticles Into Adrenocortical Carcinoma Cells, Endothelium And Primary Monocytes

Abstract Disclosure: R. Challapalli: None. A. Sorushanova: None. C. Hong: None. N.J. Mullen: None. S. Feely: None. O. Covarrubias-Zambrano: None. J. Covarrubias: None. S. Varghese: None. C. Hantel: None. P. Owens: None. M. O’Halloran: None. P. Prakash: None. S.H. Bossmann: None. M.C. Dennedy: None. Adrenocortical carcinoma (ACC), a rare malignancy with a poor prognosis and a survival rate of up to 24 months, requires varied treatments such as surgery, chemotherapy, or radiation. The high recurrence rate emphasizes the need for innovative therapies. Magnetic iron oxide nanoparticles have emerged as promising candidates for cancer treatment due to their versatility in size and shape and the ability to modify their surface for targeted cellular uptake. In our initial investigation, we assessed the uptake of magnetic iron oxide nanoparticles (IONP) by ACC (H295R, HAC15, and MUC1) cells, HUVECs and primary monocytes, incubated with 0, 5, 10, 20, and 50 µg/ml of IONP for 24 hours. The results demonstrated concentration and time-dependent nanoparticle uptake and its impact on cellular viability. The optimal concentration of IONP was identified as 10 µg/ml, subsequently used to evaluate the rate of uptake and intracellular location. The efficiency of IONP uptake by ACC cells decreased when exposed to primary monocytes and an HUVEC layer, as demonstrated through a Transwell migration system. This study revealed insights into the non-specific uptake of IONP by ACC, primarily attributed to the mechanism of macropinocytosis. These preliminary findings lay the groundwork for exploring potential modifications to enhance specific uptake by ACC cells, particularly for applications like magnet-induced thermal ablation or nanobiocontrast. Presentation: 6/2/2024

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

8468 Development And Characterisation Of Three-Dimensional Cell Culture Models Of Adrenocortical Carcinoma

Abstract Disclosure: S. Feely: None. N. Mullen: None. P. Donlon: None. C. Hantel: None. M.C. dennedy: None. Adrenocortical carcinoma (ACC) is a rare malignancy carrying a poor prognosis and limited treatment options. Surgical resection is the only option for cure; but unsuitable for advanced disease. Mitotane is the approved medical therapy, but associated with treatment resistance and adverse effects. Newer therapies, such as immune checkpoint inhibitors, have limited success. Development of improved therapies is limited by current pre-clinical disease models. No available model reflects the tumour micro-environment. Three-dimensional (3D) cell culture models are increasingly used in cancer research (6), with a paucity of developed models in ACC. In the current study, we developed novel 3D models of ACC using a type-1 collagen matrix. We then characterised these as representative ACC disease models . 3D cell culture models of ACC were optimised by embedding ACC cell lines, MUC-1, HAC15 and H295R within a type-1 Collagen matrix at differing cell numbers. Expression of type-1 collagen in ACC primary tumours was determined using immunohistochemistry (n=4). Cell viability for each model was assessed by evaluating sytox blue staining by Flow Cytometry. Metabolic activity of ACC cells within the matrix was determined using AlamarBlue staining. Prolferative activity was assessed using Ki67. Steroidogenesis was evaluated by measuring cortisol, aldosterone and androstenedione using LC-MS/MS.. Expression of CYP11B1, CYP11B2 CYP17, and StAR was measured using RT-qPCR. Type 1 collagen is strongly expressed in ACC tumour microenvironment. MUC-1, H295R and HAC15 cells were successfully cultured in a type 1 collagen matrix. MUC-1 cells cultured in the collagen matrix remain viable throughout the period of experimentation with optimum viability between 14 and 21 days. H295R cells also remain viable with optimum viability at an earlier stage of 7-14 days. HAC15 cells demonstrate high resilience in 3D cell culture with maximum viability throughout the entire period of experimentation up to 21 days. All three models increase their metabolic and proliferative activity over time. All three steroids are expressed by all three models with an increase in aldosterone secretion observed in 3D HAC15 and H295R models compared to monolayer (p>0.001) at days 7 and 14. All models express key steroidogenic enzymes. 3D ACC cell culture models were developed successfully using collagen type 1 abundant in ACC. Typical characteristics of the ACC tumour environment included (i) the presence of live and necrotic cells (ii) high metabolic activity and proliferation, reflective of cell turnover.. Capacity of cells to spread and proliferate within the 3D cell culture models was limited by available collagen substrate. Steroidogenic capacity was demonstrated in 3D cell culture. This model therefore retained key characteristics of ACC and shows promise as an animal-sparing, pre-clinical model. Presentation: 6/1/2024

7888 Mitotane Induces Different Modes Of Cell Death In Treatment Resistant And Sensitive Models Of Adrenocortical Carcinoma

Abstract Disclosure: S. Feely: None. N. Mullen: None. C. Hong: None. C. Hantel: None. W.E. Rainey: None. M.C. Dennedy: None. Adrenocortical carcinoma (ACC) is a rare aggressive cancer with poor survival. Adjuvant mitotane is the only approved drug for treatment of ACC. It improves survival but its use is limited by poor tolerability and drug resistance. For metastatic ACC, combination chemotherapy, using mitotane and etoposide, doxorubicin, and cisplatin improves survival, but efficacy is limited. Better understating the cytotoxic mechanisms of mitotane offers potential to develop improved therapeutic options for ACC. To investigate the underlying mechanisms in vitro, human ACC cells (HAC15 and H295R), in monolayer cell culture were treated with increasing concentrations of mitotane. Cell death was evaluated at 6 and 24 hours using flow cytometry analysis of Annexin V and Sytox Blue. This was complemented by Incucyte ® Live-Cell Analysis System imaging of annexin V and Sytox Blue staining. Expression of the markers of apoptosis, cleaved caspase 3, and necroptosis, phosphorylated MLKL and RIPK1 was measured at 6 and 24 hours. Mitotane significantly reduced the viability of H295R cells at 6 and 24 hours at the therapeutic concentration of 50uM (p>0.0001), thereby demonstrating treatment-sensitivity. In HAC15 cells, a supratherapeutic dose of 200uM was needed to significantly reduce viability (p>0.05) at 24 hours, indicating resistance to mitotane treatment at therapeutic concentrations. There was increasing Annexin V fluorescence with increasing mitotane concentration in H295R and HAC15 cells ar 6 and 24 hours without higher coinciding staining with Sytox Blue. Video analysis demonstrated these findings. Cleaved caspase 3 expression was associated with mitotane-induced cell death in H295R cells at 6 and 24 hours, indicating apoptosis. HAC15 cells demonstrated cleaved caspase 3 expression only at the highest concentration of mitotane exposure (600uM) at 6 hours, without appreciable expression at 24 hours. Phosphorylated-MLKL and RIPK1 expression was seen in controls and all mitotane doses in both cell lines at both timepoints, indicating necroptosis. In this study, H295R cells, representing a treatment-sensitive model of Mitotane undergo both apoptotic and necroptotic cell death at 6 and 24 hours of mitotane exposure. In contrast, HAC15 cells, demonstrated relative treatment resistance to mitotane, and undergo necroptotic cell death only. These results highlight the importance of understanding and targeting non-apoptotic and necroptotic cell death pathways, particularly when evaluating mechanisms of treatment resistance in ACC. Given the difference in the mechanism of cell death in treatment-sensitive and treatment-resistant cells, further exploration of necroptosis is necessary to better understand how ACC cells may manifest treatment resistance. Presentation: 6/1/2024

8123 Identification Of Glutamine Metabolism As A Druggable Therapeutic Vulnerability For Adrenocortical Carcinoma

Abstract Disclosure: V. Chortis: None. C. Yao: None. C. Ribeiro: None. L. Kelley: None. L. Nagano: None. M. Berber: None. S. Raveenthiraraj: None. P. Vendramini: None. K. Kiseljak-Vassiliades: None. D.L. Carlone: None. M.C. Haigis: None. K.S. Borges: None. D.T. Breault: None. Dysregulation of cellular metabolism is a hallmark feature of cancer that can be targeted therapeutically but remains underexplored in adrenocortical carcinoma (ACC), despite the urgent need for improved treatments. We employed a recently developed transgenic mouse model of ACC (BPCre), driven by the two most common mutations in human ACC (β-catenin gain-of-function and p53 loss-of-function), aiming to characterize in vivo ACC metabolism at a tissue level and to identify metabolic vulnerabilities. We employed metabolomics (liquid chromatography-mass spectrometry) and transcriptomics (bulk RNA-seq) to compare tissue metabolism in spontaneous BPCre tumors with control adrenals. Polar metabolites were analyzed using a library that provides a representative cross-section of major carbon and nitrogen-handling pathways. We identified widespread dysregulation of tumor metabolism (64/175 metabolites significantly dysregulated, FDR<0.05), with the most significantly dysregulated pathways including purine and pyrimidine metabolism, proline and glutamate metabolism, glycolysis, and the pentose phosphate pathway (FDR<0.05). Orthogonal analysis by RNA-seq revealed congruent metabolic pathway alterations. Comparison of the most significantly dysregulated pathways between BPCre mouse tumors and human ACC using publicly accessible human transcriptomic datasets (TCGA and GTEx) revealed extensive overlap. Given the prominent dysregulation of several glutamine (Gln)-dependent metabolic pathways in mouse and human ACC, we hypothesized that Gln catabolism is likely to represent a targetable metabolic vulnerability in ACC. Pharmacological targeting of Gln metabolism using the Gln antagonist 6-Diazo-5-Oxo-L-Norleucine (L-DON) induced marked cytotoxicity in two cell lines derived from BPCre tumors and three human ACC cell lines (NCI-H295R, CU-ACC1-2), as assessed by cell viability and clonogenic assays. Remarkably, this effect was rescued only by nucleoside supplementation, suggesting that Gln’s contribution to de novo purine and pyrimidine biosynthesis is the critical metabolic dependency in ACC cells. Treatment with the L-DON pro-drug JHU-083, in vivo, led to marked growth inhibition of subcutaneous mouse ACC tumor implants in both immunocompetent and immunodeficient mice. As expected, tissue metabolomics of JHU-083-treated tumors confirmed extensive depletion of purine metabolites. Tumor immunophenotyping by flow cytometry revealed significantly increased infiltration with Natural Killer cells, likely potentiating the anti-tumor effect of glutamine antagonism in immunocompetent mice. JHU-083 was also effective against human NCI-H295R xenografts, in vivo. This work reveals important new insights into ACC metabolism and provides pre-clinical evidence supporting Gln antagonism as a new metabolic treatment approach for ACC. Presentation: 6/2/2024

Novel repurposing of sulfasalazine for the treatment of adrenocortical carcinomas, probably through the SLC7A11/xCT-hsa-miR-92a-3p-OIP5-AS1 network pathway

BackgroundRecent multigenomic analysis of adrenocortical carcinomas (ACCs) identified SLC7A11/xCT as a novel biomarker. The Food and Drug Administration–approved anti-inflammatory drug, sulfasalazine (SAS), induces ferroptosis by blocking SLC7A11 expression. We hypothesize that SAS could be repurposed to target ACC cells. MethodsExpression of SLC7A11 and its association with ACC survival was analyzed using Gene Expression Profiling Interactive Analysis (GEPIA). The validated ACC cell lines NCI-H295R, ACC1, and ACC2 were grown in 2D culture. In vitro studies included the CellTiter-Glo assay to calculate viability, Western blot (WB) analysis for apoptosis and other target protein changes, reverse transcriptase polymerase chain reaction for steroidogenic enzyme changes, C11BODIPY for lipid peroxidation, and mass spectrometry for changes in lipids. ResultsThe Cancer Genome Atlas Program database analysis in GEPIA showed that SLC7A11 and linked long noncoding RNA OAP5-AS1 are highly expressed in ACC tumors versus normal adrenals (n = 77 vs 128; P < .05). This was associated with poor overall and disease-free survival with hazard ratios of 4.3 and 5.2 for SLC7A11 and 4.8 and 2.7 for OAP5-AS1, respectively. ACC cell line half-inhibitory maximum concentration values after 72-hour SAS treatment ranged from 412 nM (ACC1) to 799 nM (ACC2), and all showed cleavage of poly (ADP-ribose) polymerase, upregulation of p-Akt and p-ERK, and downregulation of GPX4 and SLC7A11 (P < .05) by WB analysis. Sphere formation, migration, and invasion assay showed inhibition, and lipid peroxidation using C11BODIPY, increase in intracellular iron, induction of oxidative stress, and significant upregulation of oxidized polyunsaturated fatty acid phospholipids (P < .05 each) by mass spectrometry suggests induction of ferroptosis. ConclusionSAS downregulates tSLC7A11 in ACCs, targets the Akt/ERK pathway and lipid metabolism, and induces cell death in vitro, warranting additional translational studies to define its therapeutic potential in ACC.

Uptake of K20-coated iron oxide nanoparticles by adrenocortical carcinoma cells

Uptake of K20-coated iron oxide nanoparticles by adrenocortical carcinoma cells

A Database Tool Integrating Genomic and Pharmacologic Data from Adrenocortical Carcinoma Cell Lines, PDX, and Patient Samples.

ACC_CellMinerCDB, a comprehensive database of cell lines, patient-derived xenografts, surgical samples, and drug responses, reveals shared genomic pathways and treatment-relevant markers in ACC. This resource offers insights into potential therapeutic targets and the opportunity to repurpose existing drugs for ACC therapy.

LncRNA ZFHX4-AS1 as a novel biomarker in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare and highly aggressive malignant tumor. Currently, there is a lack of reliable prognostic markers in clinical practice. Extensive research has shown that long non-coding RNA (lncRNA) are critical factors in the initiation and progression of cancer, closely associated with early diagnosis and prognosis. Previous studies have identified that ZFHX4 antisense RNA 1 (ZFHX4-AS1) is aberrantly expressed in various cancers and is associated with poor outcomes. This study investigates whether ZFHX4-AS1 affects the prognosis of ACC patients and, if so, the potential mechanisms involved. In this study, utilizing four multi-center cohorts from The Cancer Genome Atlas (TCGA) program and Gene Expression Omnibus (GEO), we validated the prognostic capability of ZFHX4-AS1 in ACC patients through Kaplan-Meier survival analysis, cox regression models, and nomograms. Then, we explored the biological functions of ZFHX4-AS1 using gene set enrichment analysis (GSEA), competing endogenous RNA (ceRNA) networks, and analyses of somatic mutations and copy number variation (CNV). Finally, in vitro experiments were conducted to further validate the impact of ZFHX4-AS1 on proliferation and migration capabilities of ACC cell lines. Survival analysis indicated that patients in the high ZFHX4-AS1 expression group of ACC had worse prognosis. Cox regression analyses suggested that ZFHX4-AS1 levels were independent risk factors for prognosis. Subsequently, we constructed nomograms based on clinical features and ZFHX4-AS1 levels, demonstrating good predictive performance under the time-dependent receiver operating characteristic (ROC) curve. Analysis based on somatic mutations and CNV revealed that CTNNB1 and 9p21.3-Del drove the expression of ZFHX4-AS1. Cell Counting Kit-8 (CCK-8), colony formation, and Transwell assays confirmed that knockdown of ZFHX4-AS1 inhibited proliferation and migration of ACC cells. This study demonstrates that ZFHX4-AS1 has a reliable predictive value for the prognosis of ACC patients and is a promising biomarker.

Pharmacogenomic analysis in adrenocortical carcinoma reveals genetic features associated with mitotane sensitivity and potential therapeutics.

Adrenocortical carcinoma (ACC) is an aggressive endocrine malignancy with limited therapeutic options. Treating advanced ACC with mitotane, the cornerstone therapy, remains challenging, thus underscoring the significance to predict mitotane response prior to treatment and seek other effective therapeutic strategies. We aimed to determine the efficacy of mitotane via an in vitro assay using patient-derived ACC cells (PDCs), identify molecular biomarkers associated with mitotane response and preliminarily explore potential agents for ACC. In vitro mitotane sensitivity testing was performed in 17 PDCs and high-throughput screening against 40 compounds was conducted in 8 PDCs. Genetic features were evaluated in 9 samples using exomic and transcriptomic sequencing. PDCs exhibited variable sensitivity to mitotane treatment. The median cell viability inhibition rate was 48.4% (IQR: 39.3-59.3%) and -1.2% (IQR: -26.4-22.1%) in responders (n=8) and non-responders (n=9), respectively. Median IC50 and AUC were remarkably lower in responders (IC50: 53.4 µM vs 74.7 µM, P<0.0001; AUC: 158.0 vs 213.5, P<0.0001). Genomic analysis revealed CTNNB1 somatic alterations were only found in responders (3/5) while ZNRF3 alterations only in non-responders (3/4). Transcriptomic profiling found pathways associated with lipid metabolism were upregulated in responder tumors whilst CYP27A1 and ABCA1 expression were positively correlated to in vitro mitotane sensitivity. Furthermore, pharmacologic analysis identified that compounds including disulfiram, niclosamide and bortezomib exhibited efficacy against PDCs. ACC PDCs could be useful for testing drug response, drug repurposing and guiding personalized therapies. Our results suggested response to mitotane might be associated with the dependency on lipid metabolism. CYP27A1 and ABCA1 expression could be predictive markers for mitotane response, and disulfiram, niclosamide and bortezomib could be potential therapeutics, both warranting further investigation.

Knock out of ELAVL1 affects steroid synthesis in adrenocortical carcinoma cell line NCI-H295R and leads to a less malignant phenotype

Introduction: Adrenocortical carcinoma (ACC) is a rare malignancy of the adrenal gland. With a 5-year-survival rate below 50% and no curative treatment options in advanced stages new therapeutic options in ACC are needed. Analyzing the ACC cohort of the cancer genome atlas we found a significant negative correlation of the expression level of a gene called Embryonic Lethal Abnormal Vision Like 1 (ELAVL1) and ACC patient survival. ELAVL1 has been described to be unfavorable in a lot of different tumor entities, but its function in ACC progression is still unknown. Methods: To evaluate the function of ELAVL1 in ACC progression we performed a CRISPR/Cas9 mediated knock out (KO) of the ELAVL1 gene in the ACC cell line NCI-H295R. The KO was confirmed on DNA level via Sanger sequencing and on protein level via immunofluorescence and western blot. Transcriptome changes were analyzed by next generation RNA sequencing (NGS). To characterize changes in cell function we performed soft agar colony forming assays and evaluated changes in steroid production via mass spectrometry. Results: Via CRISPR/Cas9 we knocked out ELAVL1 in NCI-H295R cells. NGS data showed 6926 significantly deregulated genes in ELAVL1-KO cells compared to control cells. There was an upregulation of 3468 genes, while 3458 genes showed a lower expression in ELAVL1 KO cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed a significant upregulation of 29 genes associated with aldosterone synthesis and a significant downregulation of 87 genes associated with pathways in cancer. Analyzing cell function via soft agar colony forming assays an impaired colony forming ability in ELAVL1 KO cells was seen. Furthermore, we could prove significant changes in steroid production. ELAVL1 KO led to an inhibition of androgen and glucocorticoid synthesis and an enhancement in mineralocorticoid synthesis. These changes could not only be proven by measuring steroid concentrations in cell culture supernatants, but also by NGS showing a downregulation of the genes CYP17A1 and CYP11B1 and an upregulation of CYP21A2 and CYP11B2. Conclusion: Our study showed that ELAVL1 affects the regulation of around 7000 genes in the ACC cell line NCI-H295R. Especially the inhibition of glucocorticoid synthesis shows that ELAVL1 may be a new target for therapeutic approaches in ACC treatment since hypercortisolism is one of the main symptoms in ACC and a negative prognostic factor for patient survival. The authors have nothing to disclose. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Distinct roles of CD244 expression in cancer diagnosis and prognosis: A pan-cancer analysis

The abnormal expression of tumor associated genes in pan-cancer is closely related to the clinicopathological features of distinct cancer types. Thus, identifying the role of specific genes in pan-cancer is needed for developing effective anti-cancer strategies. However, the function of CD244 in pan-cancer has not been fully understood. In this study, we explored the CD244 expression profile across 33 tumor types based on The Cancer Genome Atlas project, the Gene Expression Omnibus database, and other bioinformatics tools. We found down-regulated expression levels in seven tumor types and up-regulated expression levels in two tumor types. We subsequently explored the relationship between survival rate and CD244 expression, and found the positive relationship in patients with adrenocortical carcinoma (ACC), head and neck squamous cell carcinoma (HNSC), skin cutaneous melanoma (SKCM), and uterine corpus endometrial carcinoma (UCEC). We further investigated the association between CD244 expression and tumor-infiltrating immune cells, and discovered their positive correlation in different tumors. We found that CD244 expression level was higher in normal samples than in UCEC samples, and was positively associated with CD8+ T cells infiltrating. The mutation status, promoter methylation, CD244-related molecules and signaling pathways were also employed to study the potential function of CD244 in tumor initiation and progression. Our study offers a comprehensive overview of CD244 in human tumors, revealing CD244 as a potential prognostic biomarker and immunotherapeutic target in cancers.

Activity of the Ubiquitin-activating Enzyme Inhibitor TAK-243 in Adrenocortical Carcinoma Cell Lines, Patient-derived Organoids, and Murine Xenografts.

ACC is a rare endocrine cancer with poor prognosis and limited therapeutic options. We report that TAK-243 is active alone and in combination with currently used therapies and with BCL2 and mTOR inhibitors in ACC preclinical models. Our results suggest implementation of TAK-243 in clinical trials for patients with advanced and metastatic ACC.

Lamin B1: a novel biomarker in adult and pediatric adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a malignancy with a poor prognosis and high mortality rate. A high tumor mutational burden (TMB) has been found to be associated with poor prognosis in ACC. Thus, exploring ACC biomarkers based on TMB holds significant importance for patient risk stratification. In our research, we utilized weighted gene coexpression network analysis and an assay for transposase-accessible chromatin with high-throughput sequencing to identify genes associated with TMB. Through the comprehensive analysis of various public datasets, Lamin B1 (LMNB1) was identified as a biomarker associated with a high TMB and low chromatin accessibility. Immunohistochemical staining demonstrated high expression of LMNB1 in ACC compared to noncancerous tissues. Functional enrichment analyses revealed that the function of LMNB1 is associated with cell proliferation and division. Furthermore, cell assays suggested that LMNB1 promotes tumor proliferation and invasion. In addition, mutation analysis suggested that the high expression of LMNB1 is associated with TP53 mutations. Additionally, LMNB1 was highly expressed in the vast majority of solid tumors across cancers. In our immune analysis, we discovered that the high expression of LMNB1 might suppress the infiltration of CD8+ T cells in the ACC microenvironment. In summary, LMNB1 is a predictive factor for the poor prognosis of adult and pediatric ACC. Its high expression in ACC is positively associated with high TMB and lower chromatin accessibility, and it promotes ACC cell proliferation and invasion. Therefore, LMNB1 holds promise as a novel biomarker and potential therapeutic target for ACC.

Mitotane-Driven Apoptosis in Adrenocortical Carcinoma: A Molecular Insight

Objective: This study aimed to investigate the responses of antiapoptotic BCL-2 and proapoptotic BID genes to Mitotane and other chemotherapy drugs in Adrenocortical Carcinoma (ACC) cells. The objective was to understand the impact of these drugs on apoptosis-related genes in ACC, shedding light on potential treatment pathways. Materials and Methods: The study involved the use of ACC cells to assess gene expressions in response to Mitotane, Etoposide, and Cisplatin treatment. Gene expression levels of BCL-2 and BID were measured after drug exposure, providing insights into the modulation of apoptosis pathways by Rt-qPCR. Results: The results demonstrated that Mitotane notably affected the expression of the proapoptotic BID gene in ACC cells, promoting apoptosis. In contrast, Etoposide and Cisplatin exhibited less consistent effects, with Cisplatin increasing the antiapoptotic BCL-2 gene and decreasing the proapoptotic BID gene compared to Mitotane. Conclusion: This study revealed that Mitotane, as well as other chemotherapy drugs, influences the expression of key apoptosis-related genes in ACC cells. Mitotane showed a significant effect on the proapoptotic BID gene, indicating its potential as a treatment option for ACC. The findings suggest that Mitotane impacts ACC cells through multiple apoptosis pathways, highlighting its significance in ACC therapy.

Comparison of the Effect of BPA and Related Bisphenols on Membrane Integrity, Mitochondrial Activity, and Steroidogenesis of H295R Cells In Vitro.

Bisphenol A (BPA) is an endocrine-disruptive chemical that is widely utilized in the production of polycarbonate plastic and epoxy resin, which are used to make a wide range of consumer products, food and drink containers, and medical equipment. When the potential risk of BPA emerged, it was substituted by allegedly less harmful substitutes such as bisphenols S, F, B, and AF. However, evidence suggests that all bisphenols can have endocrine-disruptive effects, while the extent of these effects is unknown. This study aimed to determine effect of BPA, BPAF, BPB, BPF, and BPS on viability and steroidogenesis in human adrenocortical carcinoma cell line in vitro. The cytotoxicity of bisphenols was shown to be considerable at higher doses. However, at low concentrations, it improved viability as well as steroid hormone secretion, indicating that bisphenols have a biphasic, hormetic effect in biological systems. The results are consistent with the hypothesis that bisphenols selectively inhibit some steroidogenic enzymes. These findings suggest that bisphenols have the potential to disrupt cellular steroidogenesis in humans, but substantially more detailed and systematic research is needed to gain a better understanding of the risks associated with bisphenols and their endocrine-disrupting effect on humans and wildlife.

A reappraisal of transcriptional regulation by NR5A1 and beta-catenin in adrenocortical carcinoma.

Overexpression of the transcription factor NR5A1 and constitutive activation of canonical Wnt signalling leading to nuclear translocation of beta-catenin are hallmarks of malignancy in adrenocortical carcinoma (ACC). Based on the analysis of genomic profiles in H295R ACC cells, Mohan etal. (Cancer Res. 2023; 83: 2123-2141) recently suggested that a major determinant driving proliferation and differentiation in malignant ACC is the interaction of NR5A1 and beta-catenin on chromatin to regulate gene expression. I reanalyzed the same set of data generated by Mohan etal. and other published data of knockdown-validated NR5A1 and beta-catenin target genes. Beta-catenin is mainly found in association to canonical T cell factor/lymphoid enhancer factor (TCF/LEF) motifs in genomic DNA. NR5A1 and beta-catenin regulate distinct target gene sets in ACC cells. Overall, my analysis suggests a model where NR5A1 overexpression and beta-catenin activation principally act independently, rather than functionally interacting, to drive ACC malignancy.

Albumin/Mitotane Interaction Affects Drug Activity in Adrenocortical Carcinoma Cells: Smoke and Mirrors on Mitotane Effect with Possible Implications for Patients' Management.

Mitotane is the only drug approved for the treatment of adrenocortical carcinoma (ACC). Although it has been used for many years, its mechanism of action remains elusive. H295R cells are, in ACC, an essential tool to evaluate drug mechanisms, although they often lead to conflicting results. Using different in vitro biomolecular technologies and biochemical/biophysical experiments, we evaluated how the presence of "confounding factors" in culture media and patient sera could reduce the pharmacological effect of mitotane and its metabolites. We discovered that albumin, the most abundant protein in the blood, was able to bind mitotane. This interaction altered the effect of the drug by blocking its biological activity. This blocking effect was independent of the albumin source or methodology used and altered the assessment of drug sensitivity of the cell lines. In conclusion, we have for the first time demonstrated that albumin does not only act as an inert drug carrier when mitotane or its metabolites are present. Indeed, our experiments clearly indicated that both albumin and human serum were able to suppress the pharmacological effect of mitotane in vitro. These experiments could represent a first step towards the individualization of mitotane treatment in this rare tumor.

PLK1 inhibitors as a new targeted treatment for adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is an aggressive malignancy with limited treatment options. Polo-like kinase 1 (PLK1) is a promising drug target; PLK1 inhibitors (PLK1i) have been investigated in solid cancers and are more effective in TP53-mutated cases. We evaluated PLK1 expression in ACC samples and the efficacy of two PLK1i in ACC cell lines with different genetic backgrounds. PLK1 protein expression was investigated by immunohistochemistry in tissue samples and correlated with clinical data. The efficacy of rigosertib (RGS), targeting RAS/PI3K, CDKs and PLKs, and poloxin (Pol), specifically targeting the PLK1 polo-box domain, was tested in TP53-mutated NCI-H295R, MUC-1, and CU-ACC2 cells and in TP53 wild-type CU-ACC1. Effects on proliferation, apoptosis, and viability were determined. PLK1 immunostaining was stronger in TP53-mutated ACC samples vs wild-type (P = 0.0017). High PLK1 expression together with TP53 mutations correlated with shorter progression-free survival (P= 0.041). NCI-H295R showed a time- and dose-dependent reduction in proliferation with both PLK1i (P< 0.05at 100 nM RGS and 30 µM Pol). In MUC-1, a less pronounced decrease was observed (P< 0.05at 1000 nM RGS and 100 µM Pol). 100 nM RGS increased apoptosis in NCI-H295R (P< 0.001), with no effect on MUC-1. CU-ACC2 apoptosis was induced only at high concentrations (P < 0.05 at 3000 nM RGS and 100 µM Pol), while proliferation decreased at 1000 nM RGS and 30 µM Pol. CU-ACC1 proliferation reduced, and apoptosis increased, only at 100 µM Pol. TP53-mutated ACC cell lines demonstrated better response to PLK1i than wild-type CU-ACC1. These data suggest PLK1i may be a promising targeted treatment of a subset of ACC patients, pre-selected according to tumour genetic signature.