Adrenocortical Cancer Cell Research Articles

The Adipose Stem Cell as a Novel Metabolic Actor in Adrenocortical Carcinoma Progression: Evidence from an In Vitro Tumor Microenvironment Crosstalk Model.

Metabolic interplay between the tumor microenvironment and cancer cells is a potential target for novel anti-cancer approaches. Among stromal components, adipocytes and adipose precursors have been shown to actively participate in tumor progression in several solid malignancies. In adrenocortical carcinoma (ACC), a rare endocrine neoplasia with a poor prognosis, cancer cells often infiltrate the fat mass surrounding the adrenal organ, enabling possible crosstalk with the adipose cells. Here, by using an in vitro co-culture system, we show that the interaction between adipose-derived stem cells (ASCs) and the adrenocortical cancer cell line H295R leads to metabolic and functional reprogramming of both cell types: cancer cells limit differentiation and increase proliferation of ASCs, which in turn support tumor growth and invasion. This effect associates with a shift from the paracrine cancer-promoting IGF2 axis towards an ASC-associated leptin axis, along with a shift in the SDF-1 axis towards CXCR7 expression in H295R cells. In conclusion, our findings suggest that adipose precursors, as pivotal components of the ACC microenvironment, promote cancer cell reprogramming and invasion, opening new perspectives for the development of more effective therapeutic approaches.

Activity and safety of temozolomide in advanced adrenocortical carcinoma patients.

Temozolomide has shown a significant anti-proliferative activity on adrenocortical cancer (ACC) cells in vitro. On the basis of these results the drug was prescribed as second/third line in advanced metastatic ACC patients in four referral centers in Italy. We retrospectively collected anagraphic, clinical and pathological data of patients with advanced ACC with disease progression to standard chemotherapy plus mitotane who were treated with temozolomide at the dose of 200 mg/m2/die given for 5 consecutive days every 28 days. The primary endpoint was the disease control rate, defined as objective response or disease stabilization after 3 months. Secondary endpoints were overall survival (OS), progression-free survival (PFS) and drug safety. Twenty-eight patients have been included in the study. Ten patients (35.8%, 95% CI: 17.8-53.8) obtained a disease control from temozolomide treatment. In particular, 1 patient had a complete response, 5 patients a partial response and 4 patients stable disease. Median PFS was 3.5 months and median OS was 7.2 months. Disease response was more frequently observed in patients with methylation of O6-methylguanine-DNA methyltransferase (MGMT) gene. Temozolomide therapy was well tolerated and most toxicities were limited to grade G1-2 according to WHO criteria. Temozolomide was found active in the management of advanced ACC patients. The disease control rate obtained, however, was short-lived and the prognosis of treated patients was poor.

In vitro cytotoxicity of cabazitaxel in adrenocortical carcinoma cell lines and human adrenocortical carcinoma primary cell cultures☆

Adrenocortical cancer (ACC) is a rare and aggressive malignancy with a poor prognosis. The overall 5-year survival rate of patients with ENS@T stage IV ACC is less than 15%. Systemic antineoplastic therapies have a limited efficacy and new drugs are urgently needed.Human ACC primary cultures and cell lines were used to assess the cytotoxic effect of cabazitaxel, and the role of P-glycoprotein in mediating this effect.Cabazitaxel reduced ACC cell viability, both in ACC cell lines and in ACC primary cell cultures. Molecular and pharmacological targeting of ABCB1/P-gp did not modify its cytotoxic effect in NCI–H295R cells, while it increased the paclitaxel-induced toxicity. Cabazitaxel modified the expression of proteins involved in cellular physiology, such as apoptosis and cell cycle regulation. The drug combination cabazitaxel/mitotane exerted an additive/moderate synergism in different ACC cell experimental models. These results provide a rationale for testing cabazitaxel in a clinical study.

IGF2 role in adrenocortical carcinoma biology

PurposeClinical outcomes of adrenocortical carcinomas (ACC) could be improved by using novel treatment targets based on the recent advances of tumor biology knowledge. Insulin-like growth factor 2 (IGF2) protein expression is usually 8–80 fold higher in ACC when compared to normal adrenal glands (N-AG) or adrenocortical adenomas (ACA), despite the fact that the biological features of high vs. low IGF2 expressing ACC have not yet been well characterized. Our goal was to understand the IGF2 role in ACC biology by focusing in several cancer hallmarks, including cell proliferation, viability, invasion, and metabolism.MethodsIGF2 immunohistochemistry expression was evaluated in ACC (n = 13), non-functioning adrenocortical adenoma (ACAn) (n = 14), and N-AG (n = 9). The effects of IGF2 (50, 100 ng/mL) in cell proliferation, viability, invasion, and metabolism, as well as in MAPK/ERK and mTOR pathways activation and N-cadherin expression, were evaluated in the ACC human cell line H295R.ResultsIGF2 expression was increased in ACC compared to ACAn and N-AG. Exposure to 100 ng/mL of IGF2 increased H295R cell proliferation and viability. mTOR inhibition reverted IGF2 triggered cell proliferation and viability while MEK/MAPK/ERK inhibition only reverted IGF2 effects on cell proliferation. IGF2 at a 50 ng/mL concentration increased the glycolytic flux and decreased glutamine consumption.ConclusionsIGF2 is an excellent marker to differentiate ACC from ACAn. In addition, IGF2 was demonstrated to influence adrenocortical cancer cell proliferation, metabolism, and viability, but not the cell invasion. These data support that different IGF2 concentrations in ACC can be responsible for different biological behaviors of ACC.

Adrenocortical cancer cell line mutational profile reveals aggressive genetic background.

Adrenocortical carcinomas are rare tumors with poor prognosis and limited treatment options. Although widely used as in vitro models to test novel therapeutic strategies, the adrenocortical carcinoma-derived cell lines NCI-H295R and SW-13 have only partially been described genetically. Our aim was to characterize the mutational landscape of these cells to improve their experimental utility and map them to clinical subtypes of adrenocortical carcinoma. Genomic DNA from NCI-H295R and SW-13 cells was subjected to whole-exome sequencing. Variants were filtered for non-synonymous mutations and curated for validated adrenocortical and pan-cancer driver gene mutations. Genes mutated in the cell lines were mapped using gene ontology and protein pathway tools to determine signaling effects and compared to mutational and clinical characteristics of 92 adrenocortical carcinoma cases from The Cancer Genome Atlas. NCI-H295R and SW-13 cells carried 1325 and 1836 non-synonymous variants, respectively. Of these, 61 and 76 were known cancer driver genes, of which 32 were shared between cell lines. Variant interaction analyses demonstrated dominant TP53 dysregulation in both cell lines complemented by distinct WNT (NCI-H295R) and chromatin remodeling (SW-13) pathway perturbations. Both cell lines genetically resemble more aggressive adrenocortical carcinomas with worse prognosis, for which development of targeted therapies is most critical. Careful incorporation of the genetic landscapes outlined in this study will further the in vitro utility of these cell lines in testing for novel therapeutic approaches for adrenocortical malignancy.

Long non-coding RNA UCA1 promoted the growth of adrenocortical cancer cells via modulating the miR-298-CDK6 axis

Adrenocortical cancer (ACC) is an aggressive malignancy with no available effective treatments; therefore, exploring the molecular mechanisms involved in the initiation and progression of ACC is quite important. Here, we found that the long noncoding RNA urothelial carcinoma-associated 1 (UCA1) was highly expressed in ACC tissues and closely associated with the TNM stage and metastasis of ACC patients. Overexpression of UCA1 significantly promoted the proliferation and suppressed the apoptosis of ACC cells. Mechanism study showed that UCA1 acted as sponge of miR-298 and decreased the expression abundance of miR-298 in ACC cells. Further investigation identified that miR-298 bound the 3’-UTR of the cyclin-dependent kinase 6 (CDK6) and inhibited the expression of CDK6. Consistently, ectopic expressed UCA1 suppressed miR-298 and up-regulated the expression of CDK6, which promoted the cell cycle progression of ACC cells. Taken together, our results identified the potential oncogenic function of UCA1 in ACC by regulating the miR-298-CDK6 axis.

Circular IRE-type RNAs of the NR5A1 gene are formed in adrenocortical cells

The recently discovered circular RNAs (circRNAs) are mostly formed by back-splicing where the downstream 5′ splice site splices to the upstream 3’ splice site by conventional pre-mRNA splicing. These circRNAs regulate gene expression by acting as sponges for micro-RNAs or RNA-binding proteins. Here we show that the NR5A1 (previously called Ad4BP or SF-1) gene which is exclusively expressed in the adrenal cortex and steroidogenic tissue can form atypical circRNAs by unconventional splicing. Two stem loops with inositol-requiring protein-1α (IRE1α) cleavage sites are connected by an IRE1α cleavage site to form a circRNA (circIRE RNA). From total RNA of normal human adrenal cortex, we detected a circIRE RNA with connected ends by IRE1α cleavage sites in exon 6 and exon 1 (circIRE NR5A1 ex6-1 RNA). circIRE NR5A1 ex6-1 RNA was not detected in the adrenocortical cancer cell line, H295R. When IRE1α was expressed in H295R cells a different circIRE NR5A1 RNA connecting IRE1-cleavage sites in exon 7 and exon 1 was detected (circIRE NR5A1 ex7-1 RNA). The expression of this circIRE RNA was inhibited by the IRE1 inhibitor 1, STF-083010, implicating that it was formed via the ER stress pathway, where IRE1α is a major factor. This is the first report of this type of circular RNA connected by IRE1-cleavage sites found to be expressed in mammalian cells in a tissue-specific manner. To our surprise, the concomitant expression of NR5A1 was increased by IRE1α implicating that NR5A1 was not subjected to IRE1-dependent decay of mRNA (RIDD) but rather activating a transcriptional regulatory network to cope with ER stress in steroidogenic tissue reminiscent to XBP1 in other tissue. We believe this is the first report of such tissue-specific transcriptional cascade responding to ER stress as well as the novel finding of circular RNAs connected by IRE1α cleavage sites expressed in mammalian tissue.

The physiological role of nucleobindin-2/nesfatin-1 and their potential clinical significance

Nucleobindin-2 is a multidomain protein. Nucleobindin-2 can be cleaved into three peptide products: nesfatin-1, nesfatin-2 and nesfatin-3. It has been also shown that both Nucleobindin-2 and nesfatin-1 exhibit anorexigenic effect in rodents. In this review, we focused on a systematic characteristic of Nucleobindin-2, its anorexigenic effect and discussed possible mechanisms of its action. The first one is associated with melanocortin system and is leptin independent. The second – involves neurons that produce orexigenic neuropeptide Y. This has allowed integrating key findings which have important implications for treatment of obesity. We also presented Nucleobindin-2/nesfatin-1 as proteins which might become potentially important for understanding and treatment of such diseases as epilepsy, acute appendicitis or cancer. It has been shown that the Nucleobindin-2 function in cancerogenesis may be dual. The high level of Nucleobindin-2 and nesfatin-1 expression was found in colony, breast and endometrium cancer cells. Additionally, Nucleobindin-2/nesfatin-1 induced the proliferation process of these cells. In the future, Nucleobindin-2/nesfatin-1 may be used as a potential biomarker in diagnosis. However, Nucleobindin-2/nesfatin-1 inhibited ovarian and adrenocortical cancer cells proliferation and stimulated its apoptosis. The action of both proteins involved variety of metabolic pathways. The knowledge about activity control of Nucleobindin- 2/nesfatin-1 may contribute to new breakthrough in medicine.

Fascin-1 Is a Novel Prognostic Biomarker Associated With Tumor Invasiveness in Adrenocortical Carcinoma.

Novel tumor markers are urgently needed to better stratify adrenocortical cancer (ACC) patients and improve therapies for this aggressive neoplasm. To assess the diagnostic and prognostic value of the actin-bundling protein fascin-1 (FSCN1) in adrenocortical tumors. A local series of 37 malignant/37 benign adrenocortical tumors at Careggi University Hospital and two independent validation ACC cohorts (Cochin, TCGA) from the European Network for the Study of Adrenal Tumors were studied. FSCN1 expression was quantified by immunohistochemistry, Western blot and quantitative RT-PCR in ACC specimens; overall and disease-free survival associated with FSCN1 expression were assessed by Kaplan-Meier analysis and compared with that of Ki67 labeling index and tumor stage. Despite the low diagnostic power, in the Florence ACC series, FSCN1 immunohistochemical detection appeared as an independent prognostic factor, also refining results obtained with staging and Ki67 labeling index. The robust prognostic power of FSCN1 levels was further confirmed in two independent ACC cohorts. A positive correlation was found between FSCN1 and steroidogenic factor-1 (SF-1), with a substantially higher expression of both factors in ACCs at advanced stages and with at least one of the three Weiss score parameters associated with invasiveness. Moreover, we demonstrated FSCN1 role in promoting cell invasion in a human ACC cell line only in the case of increased SF-1 dosage. These findings show that FSCN1 is a novel independent prognostic marker in ACC and may serve as a potential therapeutic target to block tumor spread.

CORRIGENDUM FOR "Inhibition of Human Adrenocortical Cancer Cell Growth by Temozolomide in Vitro and the Role of the MGMT Gene".

CORRIGENDUM FOR "Inhibition of Human Adrenocortical Cancer Cell Growth by Temozolomide in Vitro and the Role of the MGMT Gene".

MiR-483-5p and miR-139-5p promote aggressiveness by targeting N-myc downstream-regulated gene family members in adrenocortical cancer.

Adrenocortical carcinoma (ACC) is a tumor with poor prognosis in which overexpression of a panel of microRNAs has been associated with malignancy but a very limited number of investigations on their role in ACC pathogenesis have been conducted. We examined the involvement of miR-483-5p and miR-139-5p in adrenocortical cancer aggressiveness. Using bioinformatics predictions and mRNA/miRNA expression profiles, we performed an integrated analysis to identify inversely correlated miRNA-mRNA pairs in ACC. We identified N-myc downstream-regulated gene family members 2 and 4 (NDRG2 and NDRG4) as targets of miR-483-5p and miR-139-5p, respectively. NDRG2 and NDRG4 expressions were inversely correlated respectively with miR-483-5p and miR-139-5p levels in aggressive ACC samples from two independent cohorts of 20 and 44 ACC. Moreover, upregulation of miR-139-5p and downregulation of NDRG4 demonstrated a striking prognostic value. A direct interaction between miR-483-5p or miR-139-5p and their targets was demonstrated in reporter assays. Downregulation of miR-483-5p or miR-139-5p in the ACC cell lines NCI-H295R and SW13 increased NDRG2 or NDRG4 mRNA and protein expression, compromised adrenocortical cancer cell invasiveness and anchorage-independent growth. MiR-483-5p or miR-139-5p overexpression and NDRG2 or NDRG4 inhibition produce similar changes, which are rescued by NDRG2 or NDRG4 ectopic expression. We established that key factors mediating epithelial-to-mesenchymal transition are downstream effectors of miR-483-5p/NDRG2 and miR-139-5p/NDRG4 pathways. Collectively, our data show for the first time that miR-483-5p/NDRG2 and miR-139-5p/NDRG4 axes promote ACC aggressiveness, with potential implications for prognosis and therapeutic interventions in adrenocortical malignancies.

Development of new preclinical models to advance adrenocortical carcinoma research.

Adrenocortical cancer (ACC) is an orphan malignancy that results in heterogeneous clinical phenotypes and molecular genotypes. There are no curative treatments for this deadly cancer with 35% survival at five years. Our understanding of the underlying pathobiology and our ability to test novel therapeutic targets has been limited due to the lack of preclinical models. Here, we report the establishment of two new ACC cell lines and corresponding patient-derived xenograft (PDX) models. CU-ACC1 cell line and PDX were derived from a perinephric metastasis in a patient whose primary tumor secreted aldosterone. CU-ACC2 cell line and PDX were derived from a liver metastasis in a patient with Lynch syndrome. Short tandem repeat profiling confirmed consistent matches between human samples and models. Both exomic and RNA sequencing profiling were performed on the patient samples and the models, and hormonal secretion was evaluated in the new cell lines. RNA sequencing and immunohistochemistry confirmed the expression of adrenal cortex markers in the PDXs and human tumors. The new cell lines replicate two of the known genetic models of ACC. CU-ACC1 cells had a mutation in CTNNB1 and secreted cortisol but not aldosterone. CU-ACC2 cells had a TP53 mutation and loss of MSH2 consistent with the patient's known germline mutation causing Lynch syndrome. Both cell lines can be transfected and transduced with similar growth rates. These new preclinical models of ACC significantly advance the field by allowing investigation of underlying molecular mechanisms of ACC and the ability to test patient-specific therapeutic targets.

GPER-independent inhibition of adrenocortical cancer growth by G-1 involves ROS/Egr-1/BAX pathway

We previously demonstrated that treatment of the H295R adrenocortical cancer cell line with the non-steroidal, high-affinity GPER (G protein-coupled estrogen receptor 1) agonist G-1 reduced tumor growth in vitro and in vivo through a GPER independent action. Moreover, we observed that G-1 treatment induces cell-cycle arrest and apoptosis following a sustained ERK1/2 activation. However, the precise mechanisms causing these effects were not clarified. Starting from our preliminary published results, we performed a microarray study that clearly evidenced a strong and significative up-regulation of EGR-1 gene in H295R cells treated for 24h with micromolar concentration of G-1. The microarray findings were confirmed by RT-PCR and Western-blot analysis as well as by immunofluorescence that revealed a strong nuclear staining for EGR-1 after G-1 treatment. EGR-1 is a point of convergence of many intracellular signaling cascades that control tumor cell growth and proliferation as well as others that relate to cell death machinery. Here we found that the increased Egr-1 expression was a consequence of G-1-mediated ROS-dependent ERK activation that were promptly reversed by the presence of the antioxidant n-acetyl-cysteine. Finally, we observed that silencing EGR-1 gene expression reversed the main effects induced by G-1 in ACC cells, including upregulation of the negative regulator of cell cycle, p21Waf1/Cip1 and the positive regulator of mitochondrial apoptotic pathway, BAX, as well as the cell growth inhibition. The identified ROS/MAPK/Egr-1/BAX pathway as a potential off-target effect of the G-1 could be useful in implementing the pharmacological approach for ACC therapy.

Role of Scaffold Protein Proline-, Glutamic Acid-, and Leucine-Rich Protein 1 (PELP1) in the Modulation of Adrenocortical Cancer Cell Growth

PELP1 acts as an estrogen receptor (ER) coactivator that exerts an essential role in the ER’s functions. ER coregulators have a critical role in the progression and response to hormonal treatment of estrogen-dependent tumors. We previously demonstrated that, in adrenocortical carcinoma (ACC), ERα is upregulated and that estradiol activates the IGF-II/IGF1R signaling pathways defining the role of this functional cross-talk in H295R ACC cell proliferation. The aim of this study was to determine if PELP1 is expressed in ACC and may play a role in promoting the interaction between ERα and IGF1R allowing the activation of pathways important for ACC cell growth. The expression of PELP1 was detected by Western blot analysis in ACC tissues and in H295R cells. H295R cell proliferation decrease was assessed by A3-(4,5-Dimethylthiaoly)-2,5-diphenyltetrazolium bromide (MTT) assay and [3H] thymidine incorporation. PELP1 is expressed in ACC tissues and in H295R cells. Moreover, treatment of H295R with E2 or IGF-II induced a multiprotein complex formation consisting of PELP1, IGF1R, ERα, and Src that is involved in ERK1/2 rapid activation. PELP1/ER/IGF1R/c-Src complex identification as part of E2- and IGF-II-dependent signaling in ACC suggests PELP1 is a novel and more efficient potential target to reduce ACC growth.

Macrolides selectively inhibit mutant KCNJ5 potassium channels that cause aldosterone-producing adenoma.

Aldosterone-producing adenomas (APAs) are benign tumors of the adrenal gland that constitutively produce the salt-retaining steroid hormone aldosterone and cause millions of cases of severe hypertension worldwide. Either of 2 somatic mutations in the potassium channel KCNJ5 (G151R and L168R, hereafter referred to as KCNJ5MUT) in adrenocortical cells account for half of APAs worldwide. These mutations alter channel selectivity to allow abnormal Na+ conductance, resulting in membrane depolarization, calcium influx, aldosterone production, and cell proliferation. Because APA diagnosis requires a difficult invasive procedure, patients often remain undiagnosed and inadequately treated. Inhibitors of KCNJ5MUT could allow noninvasive diagnosis and therapy of APAs carrying KCNJ5 mutations. Here, we developed a high-throughput screen for rescue of KCNJ5MUT-induced lethality and identified a series of macrolide antibiotics, including roxithromycin, that potently inhibit KCNJ5MUT, but not KCNJ5WT. Electrophysiology demonstrated direct KCNJ5MUT inhibition. In human aldosterone-producing adrenocortical cancer cell lines, roxithromycin inhibited KCNJ5MUT-induced induction of CYP11B2 (encoding aldosterone synthase) expression and aldosterone production. Further exploration of macrolides showed that KCNJ5MUT was similarly selectively inhibited by idremcinal, a macrolide motilin receptor agonist, and by synthesized macrolide derivatives lacking antibiotic or motilide activity. Macrolide-derived selective KCNJ5MUT inhibitors thus have the potential to advance the diagnosis and treatment of APAs harboring KCNJ5MUT.

Jun Dimerization Protein 2 Activates Mc2r Transcriptional Activity: Role of Phosphorylation and SUMOylation.

Jun dimerization protein 2 (JDP2), a basic leucine zipper transcription factor, is involved in numerous biological and cellular processes such as cancer development and regulation, cell-cycle regulation, skeletal muscle and osteoclast differentiation, progesterone receptor signaling, and antibacterial immunity. Though JDP2 is widely expressed in mammalian tissues, its function in gonads and adrenals (such as regulation of steroidogenesis and adrenal development) is largely unknown. Herein, we find that JDP2 mRNA and proteins are expressed in mouse adrenal gland tissues. Moreover, overexpression of JDP2 in Y1 mouse adrenocortical cancer cells increases the level of melanocortin 2 receptor (MC2R) protein. Notably, Mc2r promoter activity is activated by JDP2 in a dose-dependent manner. Next, by mapping the Mc2r promoter, we show that cAMP response elements (between −1320 and −720-bp) are mainly required for Mc2r activation by JDP2 and demonstrate that −830-bp is the major JDP2 binding site by real-time chromatin immunoprecipitation (ChIP) analysis. Mutations of cAMP response elements on Mc2r promoter disrupts JDP2 effect. Furthermore, we demonstrate that removal of phosphorylation of JDP2 results in attenuated transcriptional activity of Mc2r. Finally, we show that JDP2 is a candidate for SUMOylation and SUMOylation affects JDP2-mediated Mc2r transcriptional activity. Taken together, JDP2 acts as a novel transcriptional activator of the mouse Mc2r gene, suggesting that JDP2 may have physiological functions as a novel player in MC2R-mediated steroidogenesis as well as cell signaling in adrenal glands.

Metformin alters an anti-proliferative effect of Mitotane in a human adrenocortical cancer (H295R) cell line: preliminary results

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Inhibition of Human Adrenocortical Cancer Cell Growth by Temozolomide in Vitro and the Role of the MGMT Gene.

Treatment of patients with adrenocortical carcinomas (ACC) with mitotane and/or chemotherapy is often associated with toxicity and poor tumor response. New therapeutic options are urgently needed. The objectives of the study were to evaluate the therapeutic possibilities of temozolomide (TMZ) in ACC cells and to assess the potential predictive role of the DNA repair gene O6-Methylguanine-DNA methyltransferase (MGMT) in adrenocortical tumors. Three human ACC cell lines and eight primary ACC cultures were used to assess effects of TMZ in vitro. In the cell lines, 11 normal adrenals, 16 adrenocortical adenomas, and 29 ACC, MGMT promoter methylation and expression were determined. IC50 values of TMZ on cell growth were 39 μM, 38 μM, and 44 μM for H295R, HAC15, and SW13, respectively. TMZ induced apoptosis and provoked cytotoxic and cytostatic effects by reducing the surviving fraction of ACC colonies and the colony size. TMZ thereby induced cell cycle arrests in ACC cell lines. TMZ and mitotane both inhibited growth of ACC cells cultured as three-dimensional spheroids. TMZ inhibited cell amount in five of eight primary ACC cultures and induced apoptosis in seven of eight primary ACC cultures. In ACC cell lines and adrenal tissues, MGMT promoter methylation was low. In ACCs, methylation was inversely correlated with MGMT mRNA expression. MGMT protein expression was not correlated with MGMT methylation. For the first time, we show the therapeutic potential of temozolomide for ACC, offering an urgently needed potential alternative for patients not responding to mitotane alone or with etoposide, doxorubicin, and cisplatin. (Pre-)clinical studies are warranted to assess efficacy in vivo.

Abstract 933: Protein kinase a regulation of malignant brain tumor migration

Abstract Glioblastoma multiforme (GBM) is the most common and deadly type of primary brain tumor, accounting for over 13,000 new cases in the U.S. annually and a two-year survival rate of < 5%. GBMs are highly invasive and classified by the World Health Organization as a grade IV astrocytoma. Recent studies have shown that protein kinase A (PKA), a key regulator of signal transduction and various biological functions, is essential for maintaining polarity during cell migration and that localized activation of PKA at the leading edge is an early event in directional cell migration of other cell types. Though PKA has previously been shown to promote proliferation and migration in lung, colorectal, adrenocortical, and prostate cancer cell lines, the molecular mechanisms that link PKA activity with GBM physiology remain unclear. Wound healing assays in human GBM cell lines U-251 MG, U-87 MG and A172, demonstrated that GBM cell migration increased following the addition of 20 μM epidermal growth factor (EGF). Similar results were found with 5 μM treatment of the adenylate cyclase activator, forskolin, and inhibited by pre-treatment with 10 μM of the PKA inhibitor, H89. In order to assay the amount of PKA activity, we measured the phosphorylation of cAMP response element binding protein (CREB) in these cell lines. Western blot studies revealed an increase in CREB phosphorylation in cells treated with EGF and forskolin. Phosphorylation of CREB was decreased with the addition of H89 treatment. Our western blot analysis supports our wound healing data demonstrating a correlation between increased PKA-mediated CREB phosphorylation and migration in the absence of serum. Taken together, these data suggest that PKA activation by EGF is involved in GBM cell migration. Moreover, our findings describe a mechanism by which PKA regulates the migration of human brain tumors. Citation Format: Jennifer N. Ausland, Robert H. Newman, Patrick M. Martin. Protein kinase a regulation of malignant brain tumor migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 933.

CACNA1H(M1549V) Mutant Calcium Channel Causes Autonomous Aldosterone Production in HAC15 Cells and Is Inhibited by Mibefradil.

We recently demonstrated that a recurrent gain-of-function mutation in a T-type calcium channel, CACNA1H(M1549V), causes a novel Mendelian disorder featuring early-onset primary aldosteronism and hypertension. This variant was found independently in five families. CACNA1H(M1549V) leads to impaired channel inactivation and activation at more hyperpolarized potentials, inferred to cause increased calcium entry. We here aimed to study the effect of this variant on aldosterone production. We heterologously expressed empty vector, CACNA1H(WT) and CACNA1H(M1549V) in the aldosterone-producing adrenocortical cancer cell line H295R and its subclone HAC15. Transfection rates, expression levels, and subcellular distribution of the channel were similar between CACNA1H(WT) and CACNA1H(M1549V). We measured aldosterone production by an ELISA and CYP11B2 (aldosterone synthase) expression by real-time PCR. In unstimulated cells, transfection of CACNA1H(WT) led to a 2-fold increase in aldosterone levels compared with vector-transfected cells. Expression of CACNA1H(M1549V) caused a 7-fold increase in aldosterone levels. Treatment with angiotensin II or increased extracellular potassium levels further stimulated aldosterone production in both CACNA1H(WT)- and CACNA1H(M1549V)-transfected cells. Similar results were obtained for CYP11B2 expression. Inhibition of CACNA1H channels with the T-type calcium channel blocker Mibefradil completely abrogated the effects of CACNA1H(WT) and CACNA1H(M1549V) on CYP11B2 expression. These results directly link CACNA1H(M1549V) to increased aldosterone production. They suggest that calcium channel blockers may be beneficial in the treatment of a subset of patients with primary aldosteronism. Such blockers could target CACNA1H or both CACNA1H and the L-type calcium channel CACNA1D that is also expressed in the adrenal gland and mutated in patients with primary aldosteronism.