Novel cellular systems unveil mucosal melanoma initiating cells and a role for PI3K/Akt/mTOR pathway in mucosal melanoma fitness.

BACKGROUND: Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. METHODS: Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. RESULTS: This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. CONCLUSIONS: Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

Retrospective Analysis of Patient-Reported Adverse Drug Reactions in an Italian Allergy Unit: ALLERG-RAF Study.

INTRODUCTION: The Italian Medicines Agency indicates that about 5% of hospital admissions are due to adverse drug reactions (ADRs). Several factors are recognized to be associated with an increased risk for ADRs, such as the female gender and polytherapy. The aim of this study was to retrospectively analyze the suspected ADRs reported by patients during the anamnestic interview at the Allergy Unit. PATIENTS AND METHODS: ALLERG-RAF study is a retrospective analysis of the medical records of patients evaluated in the Allergy Unit of ASST Spedali Civili and the University of Brescia from 2000 to 2016. The inclusion criteria were age ≥18 years and medical consultation requested for suspected ADRs. Data relating to the patient's intrinsic characteristics, the drug supposed to be the cause, and the prescribed pharmacological therapy were collected. Pseudonymized data from each patient were collected in an informatics database. RESULTS: From 2000 to 2016, 35,817 accesses to the Allergy Unit were made, and 2,171 unique events related to a suspected ADR were collected in 1,840 patients. More than two-thirds of the reports concerned females (70.4%). Antibiotics were involved in the majority of the self-reported suspected ADRs (48.7%), particularly beta-lactams (61.1%). Anti-inflammatory drugs, mainly NSAIDs, were second in incidence and suspected in 25.2% of reports. As a site of ADR manifestation, most of the reported reactions involve the skin. No clinical sequelae were reported. CONCLUSIONS: Our results underline the importance of patient reporting in pharmacovigilance. Furthermore, gender gap data emphasizes the importance of the gender-specific medicine approach.

FSCN1 as a new druggable target in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a high risk of relapse and metastatic spread. The actin-bundling protein fascin (FSCN1) is overexpressed in aggressive ACC and represents a reliable prognostic indicator. FSCN1 has been shown to synergize with VAV2, a guanine nucleotide exchange factor for the Rho/Rac GTPase family, to enhance the invasion properties of ACC cancer cells. Based on those results, we investigated the effects of FSCN1 inactivation by CRISPR/Cas9 or pharmacological blockade on the invasive properties of ACC cells, both in vitro and in an in vivo metastatic ACC zebrafish model. Here, we showed that FSCN1 is a transcriptional target for ß-catenin in H295R ACC cells and that its inactivation resulted in defects in cell attachment and proliferation. FSCN1 knock-out modulated the expression of genes involved in cytoskeleton dynamics and cell adhesion. When Steroidogenic Factor-1 (SF-1) dosage was upregulated in H295R cells, activating their invasive capacities, FSCN1 knock-out reduced the number of filopodia, lamellipodia/ruffles and focal adhesions, while decreasing cell invasion in Matrigel. Similar effects were produced by the FSCN1 inhibitor G2-044, which also diminished the invasion of other ACC cell lines expressing lower levels of FSCN1 than H295R. In the zebrafish model, metastases formation was significantly reduced in FSCN1 knock-out cells and G2-044 significantly reduced the number of metastases formed by ACC cells. Our results indicate that FSCN1 is a new druggable target for ACC and provide the rationale for future clinical trials with FSCN1 inhibitors in patients with ACC.

Preclinical Evidence of Progesterone as a New Pharmacological Strategy in Human Adrenocortical Carcinoma Cell Lines.

BACKGROUND: Adrenocortical cancer (ACC) is a rare malignancy with a dismal prognosis. The treatment includes mitotane and EDP chemotherapy (etoposide, doxorubicin, and cisplatin). However, new therapeutic approaches for advanced ACC are needed, particularly targeting the metastatic process. Here, we deepen the role of progesterone as a new potential drug for ACC, in line with its antitumoral effect in other cancers. METHODS: NCI-H295R, MUC-1, and TVBF-7 cell lines were used and xenografted in zebrafish embryos. Migration and invasion were studied using transwell assays, and MMP2 activity was studied using zymography. Apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Progesterone significantly reduced xenograft tumor area and metastases formation in embryos injected with metastatic lines, MUC-1 and TVBF-7. These results were confirmed in vitro, where the reduction of invasion was mediated, at least in part, by the decrease in MMP2 levels. Progesterone exerted a long-lasting effect in metastatic cells. Progesterone caused apoptosis in NCI-H295R and MUC-1, inducing changes in the cell-cycle distribution, while autophagy was predominantly activated in TVBF-7 cells. CONCLUSION: Our results give support to the role of progesterone in ACC. The involvement of its analog (megestrol acetate) in reducing ACC progression in ACC patients undergoing EDP-M therapy is now under investigation in the PESETA phase II clinical study.

Molecular genotyping of adrenocortical carcinoma: a systematic analysis of published literature 2019-2021.

PURPOSE OF REVIEW: comprehensive molecular characterization of adrenocortical carcinoma (ACC) through next-generation sequencing and bioinformatics analyses is expanding the number of targets with potential prognostic and therapeutic value. We performed a critical review of recent published literature on genotyping of ACC. RECENT FINDINGS: 423 studies were published between 2019 and 2021. After manual curation we summarized selected evidence in two thematic areas: germline deoxyribonucleic acid (DNA) variations, genomic alterations and prognosis. SUMMARY: the evolving genomic landscape of ACC requires target validation in terms of prognostic and predictive value within scientific consortia. Although the existing multiple driver genes are difficult targets in the perspective of precision oncology, alterations in DNA damage repair genes or in promoter hypermethylation could open new venues for repurposing of existing drugs in ACC.

Caffeine Inhibits Direct and Indirect Angiogenesis in Zebrafish Embryos.

In this study, we report the effects of caffeine on angiogenesis in zebrafish embryos both during normal development and after exposure to Fibroblast Growth Factor 2 (FGF2). As markers of angiogenesis, we measured the length and width of intersegmental vessels (ISVs), performed whole-mount in situ hybridization with fli1 and cadh5 vascular markers, and counted the number of interconnecting vessels (ICVs) in sub-intestinal venous plexus (SIVP). In addition, we measured angiogenesis after performing zebrafish yolk membrane (ZFYM) assay with microinjection of fibroblast growth factor 2 (FGF2) and perivitelline tumor xenograft assay with microinjection of tumorigenic FGF2-overexpressing endothelial (FGF2-T-MAE) cells. The results showed that caffeine treatment causes a shortening and thinning of ISVs along with a decreased expression of the vascular marker genes and a decrease in the number of ICVs in the SIVP. Caffeine was also able to block angiogenesis induced by exogenous FGF2 or FGF2-producing cells. Overall, our results are suggestive of the inhibitory effect of caffeine in both direct and indirect angiogenesis.

Localization of TNF alpha in ileocolonic biopsies of patients with inflammatory bowel disease.

BACKGROUND: Although antitumor necrosis factor alfa (TNFα) agents are widely used to treat patients with inflammatory bowel diseases (IBD) - both Crohn's disease (CD) and ulcerative colitis (UC) - there is still some uncertainty in the cell type expressing TNFα in human ileo-colonic segments. AIMS: We investigated the immunohistochemical (IHC) expression of TNFα in the ileo-colonic segments of patients with both active CD and UC, to establish its anatomic and cellular localization in the inflamed sites. Our aim was to identify patients potentially resistant to anti TNFα agents. PATIENTS AND METHODS: Ileo-colonic slides of complete histological mapping of patients with CD and UC before any treatment was started were obtained, and serial sections assessed for TNFα expression, together with IHC markers for lymphocytes, macrophages, and plasma cells. RESULTS: TNFα was expressed in almost all inflamed segments of IBD patients, albeit with different strength, and was present, in addition to lymphocytes and, to a lesser extent, to macrophages, in plasma cells, where it had a strong positivity, as also demonstrated by colocalization of specific IHC staining. The expression of TNFα was mostly focal in CD patients and more diffuse in UC patients, likely due to the different patterns of inflammation (transmural and mucosal) of the two entities. CONCLUSIONS: In IBD, TNFα is strongly expressed also in plasma cells, and it is easily evidenced by conventional IHC techniques. It remains to be established whether this observation might be useful in future to establish in routine biopsy samples whether patients may be responsive to treatments toward this cytokine.

1,25(OH)2 vitamin D(3) contributes to osteoclast-like trans-differentiation of malignant plasma cells.

1,25-dihydroxyvitamin D (1,25(OH)2D) exerts pleiotropic effects including bone turnover and immune system regulation. It inhibits both T and B cell proliferation while decreasing the secretion of inflammatory cytokines and immunoglobulins. 1,25(OH)2D also modulates monocyte-macrophage and osteoclast (OC) maturation. Since we have previously described that malignant plasma cells may trans-differentiate towards the myeloid lineage participating to skeletal devastation in multiple myeloma (MM), we here evaluated in vitro the role of 1,25(OH)2D in this lineage switch. We investigated the gene and protein expression of vitamin D receptor (VDR) in MM cell lines. Thus, after cell treatment with 1,25(OH)2D, we analyzed their morphology and the expression of myeloid and OC markers. Finally, we assessed their bone resorption property on calcium phosphate slices. All MM cells expressed VDR in nuclear and perinuclear sites. Treatment with 1,25(OH)2D altered their morphology from round to fusiform, while inducing paxillin focalization. 1,25(OH)2D administration also up-regulated myeloid and OC genes, including C/EBPα, RANK, M-CSFR and V-ATPase, whose promoters contain potential 1,25(OH)2D responsive elements. Finally, 1,25(OH)2D increased MM cell capability to generate pits of erosion on calcium phosphate discs. This data suggest that myeloma cells may undergo a functional trans-differentiation into OCs and, under appropriate experimental conditions, 1,25(OH)2D triggers this lineage switch.

Amiloride Is Effective in the Management of Abiraterone-Induced Mineralocorticoid Excess Syndrome without Interfering with Its Antineoplastic Activity.

BACKGROUND: The administration of abiraterone acetate (abiraterone) leads to an adrenocorticotropic hormone (ACTH)-driven increase in mineralocorticoid hormones, requiring glucocorticoid supplementation that may stimulate the growth of prostate cancer (PCa). Amiloride is a drug that selectively reduces the aldosterone-sensitive Na+/K+ exchange and could be effective in the management of mineralocorticoid excess syndrome (MCES). METHODS: The efficacy of amiloride + hydrochlorothiazide (HCT) in the clinical management of abiraterone-induced MCES was assessed in 5 consecutive patients with castration-resistant PCa (CRPC). Then, using the in vitro experimental model of PCa cell lines, the possible effects of drugs usually used in the clinical management of CRPC patients on PCa cell viability were investigated. RESULTS: Amiloride/HCT led to a complete disappearance of all clinical and biochemical signs of abiraterone-induced MCES in the 5 treated patients. The in vitro study showed that abiraterone treatment significantly decreased cell viability of both androgen receptor (AR)-expressing VCaP (vertebral-cancer of the prostate) and LNCaP (lymph node carcinoma of the prostate) cells, with no effect on AR-negative PC-3 cells. Prednisolone, spironolactone, and eplerenone increased LNCaP cell viability, while amiloride reduced it. The non-steroid aldosterone antagonist PF-03882845 did not modify PCa cell viability. CONCLUSIONS: The combination of amiloride/HCT was effective in the management of abiraterone-induced MCES. Amiloride did not negatively interfere with the abiraterone inhibition of PCa cell viability in vitro.

Computational and functional analysis of biopharmaceutical drugs in zebrafish: Erythropoietin as a test model.

The zebrafish (Danio rerio) is a very popular vertebrate model system, especially embryos represent a valuable tool for in vivo pharmacological assays. This is mainly due to the zebrafish advantages when compared to other animal models. Erythropoietin is a glycoprotein hormone that acts principally on erythroid progenitors, stimulating their survival, proliferation and differentiation. Recombinant human erythropoietin (rhEPO) has been widely used in medicine to treat anemia and it is one of the best-selling biotherapeutics worldwide. The recombinant molecule, industrially produced in CHO cells, has the same amino acid sequence of endogenous human erythropoietin, but differs in the glycosylation pattern. This may influence efficacy and safety, particularly immunogenicity, of the final product. We employed the zebrafish embryo as a vertebrate animal model to perform in vivo pharmacological assays. We conducted a functional analysis of rhEPO alpha Eprex(®) and two biosimilars, the erythropoietin alpha Binocrit(®) and zeta Retacrit(®). By in silico analysis and 3D modeling we proved the interaction between recombinant human erythropoietin and zebrafish endogenous erythropoietin receptor. Then we treated zebrafish embryos with the 3 rhEPOs and we investigated their effect on erythrocytes production with different assays. By real time-PCR we observed the relative upregulation of gata1 (2.4 ± 0.3 fold), embryonic α-Hb (1.9 ± 0.2 fold) and ß-Hb (1.6 ± 0.1 fold) transcripts. A significant increase in Stat5 phosphorylation was also assessed in embryos treated with rhEPOs when compared with the negative controls. Live imaging in tg (kdrl:EGFP; gata1:ds-red) embryos, o-dianisidine positive area quantification and cyanomethemoglobin content quantification revealed a 1.8 ± 0.3 fold increase of erythrocytes amount in embryos treated with rhEPOs when compared with the negative controls. Finally, we verified that recombinant human erythropoietins did not cause any inflammatory response in the treated embryos. Our data showed that zebrafish embryo can be a valuable tool to study in vivo effects of complex pharmacological compounds, such as recombinant human glycoproteins, allowing to perform fast and reproducible pharmacological assays with excellent results.

GPNMB/OA protein increases the invasiveness of human metastatic prostate cancer cell lines DU145 and PC3 through MMP-2 and MMP-9 activity.

Non-metastatic glycoprotein melanoma protein B (GPNMB), also known as osteoactivin (OA) is expressed in a wide array of tumors and represents an emerging target for drug development. In this study, we investigated the role of GPNMB/OA in the progression of human metastatic DU145 and PC3 prostate cancer cells. GPNMB/OA contribution in PCa malignant phenotype has been analyzed by small interfering RNA-induced GPNMB/OA silencing. We found that following GPNMB/OA silencing the migration capability of both DU145 and PC3 cells, evaluated by using in vitro invasivity assay, as well as the metalloproteinases MMP-2 and MMP-9 activity were equally strongly inhibited. By contrast knocking down GPNMB/OA weakly attenuated cell proliferation rate of DU145, an effect that paralleled with an increase number of apoptotic cells. However, PC3 cell growth seems to be not affected by GPNMB/OA. Together, these data reveal that GPNMB/OA acts as a critical molecular mediator promoting the acquisition of the more aggressive, pro-metastatic phenotype distinctive of human DU145 and PC3 cell lines.

The CDK Inhibitor Dinaciclib Improves Cisplatin Response in Nonseminomatous Testicular Cancer: A Preclinical Study.

BACKGROUND: Most patients with testicular germ cell tumors (GCTs) are treated with cisplatin (CP)-based chemotherapy. However, some of them may develop CP resistance and therefore represent a clinical challenge. Cyclin-dependent kinase 5 (CDK5) is involved in chemotherapy resistance in different types of cancer. Here, we investigated the possible role of CDK5 and other CDKs targeted by dinaciclib in nonseminoma cell models (both CP-sensitive and CP-resistant), evaluating the potential of the CDK inhibitor dinaciclib as a single/combined agent for the treatment of advanced/metastatic testicular cancer (TC). METHODS: The effects of dinaciclib and CP on sensitive and resistant NT2/D1 and NCCIT cell viability and proliferation were evaluated using MTT assays and direct count methods. Flow cytometry cell-cycle analysis was performed. The protein expression was assessed via Western blotting. The in vivo experiments were conducted in zebrafish embryos xenografted with TC cells. RESULTS: Among all the CDKs analyzed, CDK5 protein expression was significantly higher in CP-resistant models. Dinaciclib reduced the cell viability and proliferation in each cell model, inducing changes in cell-cycle distribution. In drug combination experiments, dinaciclib enhances the CP effect both in vitro and in the zebrafish model. CONCLUSIONS: Dinaciclib, when combined with CP, could be useful for improving nonseminoma TC response to CP.

Germline NGS targeted analysis in adult patients with sporadic adrenocortical carcinoma.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare cancer that arises sporadically or due to hereditary syndromes. Data on germline variants (GVs) in sporadic ACC are limited. Our aim was to characterize GVs of genes potentially related to adrenal diseases in 150 adult patients with sporadic ACC. METHODS: This was a retrospective analysis of stage I-IV ACC patients with sporadic ACC from two reference centers for ACC in Italy. Patients were included in the analysis if they had confirmed diagnosis of ACC, a frozen peripheral blood sample and complete clinical and follow-up data. Next generation sequencing technology was used to analyze the prevalence of GVs in a custom panel of 17 genes belonging to either cancer-predisposition genes or adrenocortical-differentiation genes categories. RESULTS: We identified 18 GVs based on their frequency, enrichment and predicted functional characteristics. We found six pathogenic (P) or likely pathogenic (LP) variants in ARMC5, CTNNB1, MSH2, PDE11A and TP53 genes; and twelve variants lacking evidence of pathogenicity. New unique P/LP variants were identified in TP53 (p.G105D) and, for the first time, in ARMC5 (p.P731R). The presence of P/LP GVs was associated with reduced survival outcomes and had a significant and independent impact on both progression-free survival and overall survival. CONCLUSIONS: GVs were present in 6.7 % of patients with sporadic ACC, and we identified novel variants of ARMC5 and TP53. These findings may improve understanding of ACC pathogenesis and enable genetic counseling of patients and their families.

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

Adrenocortical carcinoma (ACC) is a rare and highly heterogeneous disease with a notably poor prognosis due to significant challenges in diagnosis and treatment. Emphasizing on the importance of precision medicine, there is an increasing need for comprehensive genomic resources alongside well-developed experimental models to devise personalized therapeutic strategies. We present ACC_CellMinerCDB, a substantive genomic and drug sensitivity database (available at https://discover.nci.nih.gov/acc_cellminercdb) comprising ACC cell lines, patient-derived xenografts, surgical samples, and responses to more than 2,400 drugs examined by the NCI and National Center for Advancing Translational Sciences. This database exposes shared genomic pathways among ACC cell lines and surgical samples, thus authenticating the cell lines as research models. It also allows exploration of pertinent treatment markers such as MDR-1, SOAT1, MGMT, MMR, and SLFN11 and introduces the potential to repurpose agents like temozolomide for ACC therapy. ACC_CellMinerCDB provides the foundation for exploring larger preclinical ACC models. SIGNIFICANCE: 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.

Dlk1 is a novel adrenocortical stem/progenitor cell marker that predicts malignancy in adrenocortical carcinoma.

Disruption of processes involved in tissue development and homeostatic self-renewal is increasingly implicated in cancer initiation, progression, and recurrence. The adrenal cortex is a dynamic tissue that undergoes life-long turnover. Here, using genetic fate mapping and murine adrenocortical carcinoma (ACC) models, we have identified a population of adrenocortical stem cells that express delta-like non-canonical Notch ligand 1 (DLK1). These cells are active during development, near dormant postnatally but are re-expressed in ACC. In a study of over 200 human ACC samples, we have shown DLK1 expression is ubiquitous and is an independent prognostic marker of recurrence-free survival. Paradoxically, despite its progenitor role, spatial transcriptomic analysis has identified DLK1 expressing cell populations to have increased steroidogenic potential in human ACC, a finding also observed in four human and one murine ACC cell lines. Finally, the cleavable DLK1 ectodomain is measurable in patients' serum and can discriminate between ACC and other adrenal pathologies with high sensitivity and specificity to aid in diagnosis and follow-up of ACC patients. These data demonstrate a prognostic role for DLK1 in ACC, detail its hierarchical expression in homeostasis and oncogenic transformation and propose a role for its use as a biomarker in this malignancy.

Different muscarinic receptor subtypes modulate proliferation of primary human detrusor smooth muscle cells via Akt/PI3K and map kinases.

While acetylcholine (ACh) and muscarinic receptors in the bladder are mainly known for their role in the regulation of smooth muscle contractility, in other tissues they are involved in tissue remodelling and promote cell growth and proliferation. In the present study we have used primary cultures of human detrusor smooth muscle cells (HDSMCs), in order to investigate the role of muscarinic receptors in HDSMC proliferation. Samples were obtained as discarded tissue from men >65 years undergoing radical cystectomy for bladder cancer and cut in pieces that were either immediately frozen or placed in culture medium for the cell culture establishment. HDSMCs were isolated from samples, propagated and maintained in culture. [(3)H]-QNB radioligand binding on biopsies revealed the presence of muscarinic receptors, with a Kd of 0.10±0.02nM and a Bmax of 72.8±0.1fmol/mg protein. The relative expression of muscarinic receptor subtypes, based on Q-RT-PCR, was similar in biopsies and HDSMC with a rank order of M2≥M3>M1>M4>M5. The cholinergic agonist carbachol (CCh, 1-100µM) concentration-dependently increased [(3)H]-thymidine incorporation (up to 46±4%). This was concentration-dependently inhibited by the general muscarinic receptor antagonist atropine and by subtype-preferring antagonists with an order of potency of darifenacin >4-DAMP>AF-DX 116. The CCh-induced cell proliferation was blocked by selective PI-3 kinase and ERK activation inhibitors, strongly suggesting that these intracellular pathways mediate, at least in part, the muscarinic receptor-mediated cell proliferation. This work shows that M2 and M3 receptors can mediate not only HDSM contraction but also proliferation; they may also contribute bladder remodelling including detrusor hypertrophy.

Late-onset Parkinsonism in NFκB/c-Rel-deficient mice.

Activation of the nuclear factor κB/c-Rel can increase neuronal resilience to pathological noxae by regulating the expression of pro-survival manganese superoxide dismutase (MnSOD, now known as SOD2) and Bcl-xL genes. We show here that c-Rel-deficient (c-rel(-/-)) mice developed a Parkinson's disease-like neuropathology with ageing. At 18 months of age, c-rel(-/-) mice exhibited a significant loss of dopaminergic neurons in the substantia nigra pars compacta, as assessed by tyrosine hydroxylase-immunoreactivity and Nissl staining. Nigral degeneration was accompanied by a significant loss of dopaminergic terminals and a significant reduction of dopamine and homovanillic acid levels in the striatum. Mice deficient of the c-Rel factor exhibited a marked immunoreactivity for fibrillary α-synuclein in the substantia nigra pars compacta as well as increased expression of divalent metal transporter 1 (DMT1) and iron staining in both the substantia nigra pars compacta and striatum. Aged c-rel(-/-) mouse brain were characterized by increased microglial reactivity in the basal ganglia, but no astrocytic reaction. In addition, c-rel(-/-) mice showed age-dependent deficits in locomotor and total activity and various gait-related deficits during a catwalk analysis that were reminiscent of bradykinesia and muscle rigidity. Both locomotor and gait-related deficits recovered in c-rel(-/-) mice treated with l-3,4-dihydroxyphenylalanine. These data suggest that c-Rel may act as a regulator of the substantia nigra pars compacta resilience to ageing and that aged c-rel(-/-) mice may be a suitable model of Parkinson's disease.

Correction: Sigala et al. A Comprehensive Investigation of Steroidogenic Signaling in Classical and New Experimental Cell Models of Adrenocortical Carcinoma. Cells 2022, 11, 1439.

The authors made the following changes to their paper [...].

Trabectedin impairs invasiveness and metastasis in adrenocortical carcinoma preclinical models.

The pharmacological approach to adrenocortical carcinoma (ACC) is based on mitotane with/without etoposide, doxorubicin, and cisplatin, according to the disease stage. Considering the limited efficacy and toxicity of this treatment, new strategies are required. Trabectedin is a marine-derivated antitumoral agent that inhibits oncogenic transcription. We have already demonstrated trabectedin cytotoxic activity at sub-nanomolar concentrations in ACC cells. Here, we expanded the investigation of trabectedin effect on ACC preclinical models, evaluating whether trabectedin could affect ACC cells' invasiveness and metastasis formation. NCI-H295R, MUC-1, and TVBF-7 cell lines were used. Cell tumor xenografts in Danio rerio embryos were performed. The tumor mass areas and the number of embryos with metastasis were evaluated. The in vitro invasiveness of cells was evaluated. Effects of trabectedin of MMP2, TIMP1, and TIMP2 were evaluated at gene level qRT-PCR. MMP2 secreted in the cell medium was evaluated by Western blot and by zymography. Xenograft experiments demonstrated that trabectedin significantly reduced the tumor area in each ACC cell model and metastasis formation in embryos injected with metastasis-derived cell lines. Trabectedin treatment reduced the invasiveness of ACC cells across the matrix, which was greater at baseline for the metastatic models. In metastatic cell models, protein analysis demonstrated a reduction of MMP2 secretion and activity in the culture medium after treatment. Our results indicate that trabectedin interferes with invasiveness and metastasis processes, both dramatic features of ACC. Furthermore, these results support those previously published in providing the rationale for a clinical evaluation of the efficacy of trabectedin in ACC patients.