Recent research has enhanced our understanding of the diverse biological processes that occur in pediatric gliomas; and molecular genetic analysis has become essential to diagnose and treat these conditions. Because targetable molecular aberrations can be detected in pediatric gliomas, identifying these aberrations is very important. This review provides an overview of pediatric gliomas, and describes recent developments made in strategies for their diagnosis and treatment. Additionally, it presents a current picture of pediatric gliomas in light of advances in molecular genetics, and describes the current scientific progress in gliomas’ treatment using information from recently completed and ongoing clinical trials. The era of incorporating molecular genetic analysis into clinical practice is emerging.Recent research has promoted elucidation of the diverse biological processes that occur in pediatric central nervous system (CNS) tumors. Molecular genetic analysis is essential not only for proper classification, but also for monitoring biological behavior and clinical management of tumors. Ever since the 2016 World Health Organization classification of CNS tumors, molecular profiling has become an indispensable step in the diagnosis, prediction of prognosis, and treatment of pediatric as well as adult CNS tumors. These molecular data are changing diagnosis, leading to new guidelines, and offering novel molecular targeted therapies. The Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT-NOW) makes practical recommendations using recent advances in CNS tumor classification, particularly in molecular discernment of these neoplasms as morphology-based classification of tumors is being replaced by molecular-based classification. In this article, we summarize recent knowledge to provide an overview of pediatric gliomas, which are major pediatric CNS tumors, and describe recent developments in strategies employed for their diagnosis and treatment. Central nervous system (CNS) tumors are the most frequent solid tumors in children, accounting for 21% of pediatric cancers and representing a primary cause of mortality . ImproveRecent research has promoted our understanding of the complex biology of pediatric CNS tumors. Molecular genetic analysis is essential not only for proper classification but also for monitoring biological behavior and clinical management of tumors. Recent genome studies have revealed several unique genomic changes observed in pediatric CNS tumors, which were different from those observed in adults ,5,6. SinIn the past, conventional diagnosis of pediatric CNS tumors involved a pathologist’s histopathologic review, supported by radiological findings, such as magnetic resonance imaging, and standardized immunohistochemical testing for specific biomarkers. However, the importance of molecular genetic analysis has become a focus over the past decade, and the molecular profiling of tumors has revealed a number of important details including prognostic factors and predictive markers of drug sensitivity, resistance, and adverse events. This has allowed personalized therapy to become feasible. Molecular genetic analysis is required in order for pediatric CNS tumors to be incorporated into a histologically based tiered classification system and improve their diagnosis. In the 2016 CNS WHO classification of medulloblastomas, a histologically defined and a genetically defined lists of tumors were combined to produce an integrated list of parameters for improved diagnosis . This prDNA methylation-based classification for CNS tumors is reported to be a reproducible and valuable approach, and is expected to reduce the substantial inter-observer variability that occurs in conventional morphology-based classification . In cIMPIn recent years, advances in next-generation sequencing (NGS) and array-based genomic platforms have transformed analysis of the molecular landscapes of cancers, including pediatric CNS tumors . NGS-basLiquid biopsy is also one of the current topics as a less invasive method using body fluid, such as plasma or cerebrospinal fluid (CSF), for molecular diagnosis. Recent efforts including ours have developed digital PCR-based liquid biopsy targeting cell-free tumor DNA in CSF for detecting glioma-specific diagnostic mutations . NGS-basBecause targetable molecular aberrations can be detected in pediatric CNS tumors, searching for these aberrations is essential for treating children with these tumors. However, most existing cancer multi-gene panel tests using NGS are currently performed only for adult cancer, resulting in undetectability of the rare genetic aberrations specific to pediatric cancer. Although molecular diagnosis is essential in cases of pediatric cancer, current cancer multi-gene panel tests cannot be used for the purpose of diagnosis. To resolve this issue, Kohsaka et al. reported the establishment of a comprehensive assay, the Todai OncoPanel, which consists of DNA and RNA hybridization capture-based NGS panels . In thisCurrently, novel molecular profiling technologies, including whole-proteome, phosphoproteome, metabolome, and single-cell RNA-sequencing are also developing. Many of these technologies are being applied in current research to further characterize tumor biology . In addiNumerous entities comprise pediatric CNS tumors. The Central Brain Tumor Registry of the United States (CBTRUS) Statistical Report showed the frequency of pediatric CNS tumors in a population-based study in the USA . GliomasCompared to adult LGGs, isocitrate dehydrogenase (IDH) mutations are less observed in children, and malignant progression is extremely rare. These gliomas account for approximately 30% of pediatric CNS tumors ,22. The KIAA1549-BRAF fusion by tandem duplication is historically well-known and the most frequently observed (>70%) [In pLGG, the most common entity is pilocytic astrocytoma (PA) comprising >15% of tumors in patients aged 0 to 19 years . PA is cd (>70%) . Howeverd (>70%) . Recent d (>70%) ,31,32,33BRAF V600E mutation: in pLGG, patients with the BRAF V600E mutation demonstrated poor outcomes. Lassaletta et al. reported that the 10-year PFS is 27% and 60.2% for the BRAF V600E mutant and wild-type pLGG, respectively (p < 0.001) [BRAF V600E mutations can be found in CNS tumors in any location, and are often detected in midline tumors, including the optic pathway, brainstem, and spinal cord [BRAF V600E mutation is commonly observed in pleomorphic xanthoastrocytomas (78%), followed by gangliogliomas (49%), and also in diffuse astrocytomas (43%) [BRAF V600E [< 0.001) . BRAF V6nal cord . The BRAas (43%) . AstroblAF V600E . HoweverAF V600E .FGFR1: FGFRs are a family of receptor tyrosine kinases [FGFR1-3 is identified in CNS tumors, including glioma, ependymoma, and medulloblastoma [FGFR1 is well-noted and is expected to be a targetable aberration. FGFR1 aberrations include FGFR1 mutations, FGFR1-TACC1 fusions, and FGFR1-TKD duplications [FGFR1 autophosphorylation, resulting in upregulation of the RAS/MAPK pathway  is a well-known inherited tumor predisposition syndrome caused by a germline mutation in the NF-1 tumor suppressor gene. This gene encodes neurofibromin, a GTPase-activating protein that functions as the negative regulator of RAS [r of RAS ,43. LGGsr of RAS ,45. Mostr of RAS ,46.pLGG, including PA, is considered a single RAS/MAPK pathway disease. However, some aberrations indirectly affect the RAS/MAPK pathway.MYB and MYBL1 alterations: MYB alteration influences on control of proliferation and differentiation of hematopoietic and other progenitor cells, and is associated with proto-oncogenic functions in both human leukemia and solid tumors [MYB alterations, and MYB-QKI fusion was specified as a driver aberration for angiocentric gliomas [MYBL1 is a member of the MYB family. Although the biological function of MYBL1 is less known than that of MYB, MYBL1 is considered to function as a transcriptional regular critical for proliferation and differentiation [MYBL1 alterations are rare and are detected in diffuse astrocytomas [MYB family genes, such as MYB and MYBL1 [MYB and MYBL1 alterations, and Chiang et al. reported that the 10-year OS is 90%, and 10-year PFS is 95% [d tumors ,48. Band gliomas . MYBL1 intiation . MYBL1 aocytomas ,49. pLGGnd MYBL1 ,50. Thesnd MYBL1 . The cliS is 95% . CDKN2A homozygous deletion: CDKN2A is a gene encoding two tumor suppressors, protein p14ARF, and p16INK4A. Homozygous deletion of CDKN2A can contribute to uncontrolled tumor cell proliferation [CDKN2A homozygous deletion is also observed in pediatric gliomas, and is a well-known hallmark lesion of pleomorphic xanthoastrocytoma. CDKN2A homozygous deletion co-occurs with the BRAF V600E mutation, demonstrating poor clinical outcomes [feration , and hasferation ,54,55. Coutcomes ,56.MYB, MYBL1, or FGFR1 alterations or BRAF V600E mutations [BRAF V600E mutation, FGFR alteration, or rearrangement of MYB or MYBL1 were detected in 84% of IDH-wild type/H3-wild type diffuse gliomas in a large pediatric cohort [MYB-altered;Diffuse glioma, MYBL1-altered;Diffuse glioma, FGFR1 TKD-duplicated;Diffuse glioma, FGFR1-mutant;Diffuse glioma, BRAF V600E-mutant;Diffuse glioma, Diffuse glioma, other MAPK pathway alteration.In cIMPACT-NOW update 4, an integrated diagnosis of pediatric diffuse gliomas was reported. In this report, pediatric diffuse gliomas were classified by utations . These ac cohort . The folc cohort :Diffuse BRAF inhibitors for BRAF-mutated gliomas has been recently reported [BRAF-mutated gliomas [BRAF inhibitors occurs in a majority of patients, and one of the most frequent causes of this resistance is reactivation of the MAPK pathway [MEK inhibitor that inhibits the MAPK pathway to BRAF inhibitors, trametinib, overcame BRAF inhibitor resistance, and demonstrated superiority over a BRAF inhibitor alone in phase III clinical trials in patients with BRAF mutant metastatic melanoma [BRAF and MEK inhibitors has also been reported [MEK1/2 inhibitor, was assessed [BRAF aberrations, KIAA1549-BRAF fusion or the BRAF V600E mutation, and NF-1 associated LGG, participated. Selumetinib was found to be active in cases of recurrent or progressive BRAF aberrated PA and NF-1 associated LGG [BRAF V600E inhibitor approved for metastatic melanoma, was determined to be acceptable for recurrent or refractory BRAF V600E mutant glioma [BRAF-mutated tumors.We summarize recently reported clinical trials of targeted therapy for pediatric glioma in reported ,59,60,61 gliomas . Despite pathway . Howevermelanoma ,65. In Creported ,67. To eassessed . In thisated LGG . In Japat glioma ,70. In tFGFR kinase inhibitors are currently in clinical development. AZD4547 is an orally bioavailable FGFR1-3 inhibitor [FGFR1-3 mutations or fusions. In this phase II trial, the CNS tumor was only one case out of a total of 20, and AZD4547 was not found to meet the primary end point. However, this trial showed modest activity of AZD4547 in patients with FGFR mutations and fusions, suggesting the possibility of clinical use in the future [nhibitor . In the e future .K27M histone mutations, including most DIPGs, are classified as WHO grade 4, regardless of histological findings [pHGGs account for 8–12% of pediatric CNS tumors, including anaplastic astrocytoma (WHO grade 3) and GBM (WHO grade 4), both types malignant, diffuse, infiltrating astrocytic tumors ,77,78. Cfindings .G34R/V and K27M mutations are well-known mutations in pHGG. In an integrated molecular meta-analysis of pHGG, the distribution of recurrently mutated genes was 47% TP53, 36% H3.3, 24% ATRX, and 7% BRAF V600E [G34 and K27M mutations, and co-occurring mutations of ATRX with G34R of NF1 [AF V600E . These tR of NF1 ,84.H3K27M mutation: histone H3 (H3F3A and HIST1H3B) pK27M mutations are frequently observed in DIPGs, which arise in the brainstem almost exclusively in children, and in pHGGs in midline structures such as the thalamus and spinal cord [H3K27M-mutant” (WHO grade 4), in the 2016 WHO classification [H3K27M mutation results in a global H3K27me3 reduction by multiple mechanisms, such as aberrant polycomb repressive complex 2 (PRC2) interactions and hampered H3K27me3 spreading. It is also thought to suppress neuroglial differentiation through deregulation of epigenetic modifications [H3K27M mutation is an important key for pHGG treatment.nal cord ,87,88,89ications ,91,92,93H3G34R/V mutation: in cIMPACT-NOW update 6, diffuse glioma, H3.3 G34-mutant was reported as a novel tumor type separated from the established gliomas as well as from diffuse midline glioma, H3K27M-mutant. It was also found to correspond with WHO grade 4 [K27M mutation, how the G34 mutation affects the epigenome remains unclear. G34R/V in H3F3A, which encodes non-canonical histone H3.3, occurs in pHGGs of the cerebral cortex [G34W mutation is prevalent in bone tumors [G34R/V and K27M mutations was 16% and 32%, respectively [H3F3A mutations, of which 4 had G34R mutations and 10 K27M mutations [G34 mutations exerted characteristic methylomic effects, regardless of the tumor tissue of origin, and this mutation could affect chromosome instability [G34R/V mutations are relatively rare in pHGG, this genotype is likely to have specific methylomic signals and show extensive infiltration and various histological phenotypes [ grade 4 . When col cortex ,86,88, we tumors . Neumannectively . Among outations . We recetability . Althougenotypes ,97.H3K27M has been shown to inhibit PRC2, a multiprotein complex responsible for the methylation of H3 at lysin 27, by binding to its catalytic subunit, EZH2 [H3K27M mutated tumors require PRC2 for proliferation, and EZH2 inhibitors cease tumor cell growth [H3K27M mutated tumors, and the pediatric MATCH study included this targeted therapy. In Japan, a phase I clinical trial of a dual EZH1/2 inhibitor for pediatric, adolescent, and young adult patients with malignant solid tumors is in progress.Clinical trials of targeted therapy for pHGG are summarized in it, EZH2 . Mohammal growth . EZH2 isIn the treatment of patients with DIPG, the Individualized Therapy For Relapsed Malignancies in Childhood (INFORM) registry study offered comprehensive molecular profiling of high-risk tumors to identify targetable alterations for precision therapy . In thisVEGFR), platelet-derived growth factor receptor (PDGFR), and stem cell factor receptor (KIT), had no antitumor activity against HGG and ependymoma [AKT inhibitor, and temsirolimus, an mTOR inhibitor, for CNS tumors, including HGG, was well tolerated, sufficient antitumor activity was not demonstrated [PDGFR-A inhibitor, crizotinib, and c-Met inhibitor, for recurrent/progressive HGG or DIPG was poorly tolerated and its antitumor activity was minimal [H3K27M mutation, demonstrated clinical efficacy [H3K27M mutation, dopamine receptor D2 is overexpressed and dopamine receptor D5 is suppressed, resulting in enhancement of sensitivity to a dopamine receptor D2 antagonist [Some recent clinical trials of targeted therapy for pHGG have failed to prove its efficacy and safety. Sunitinib, a multi-targeted tyrosine kinase inhibitor that inhibits vascular endothelial growth factor receptor (endymoma . Althougnstrated . The com minimal . Howeverefficacy . Dopaminefficacy . In gliotagonist .In addition, immune checkpoint inhibitors have been expected to bring a paradigm shift in treatment for gliomas. These have been shown effective in treatments for other malignancies, such as melanoma ,105. HowALK, ROS1, NTRK, and MET fusions, which are enriched for high-grade glioma, and have an intermediate clinical outcome.Group 1, hemispheric receptor tyrosine kinase-driven gliomas, including Group 2, hemispheric RAS/MAPK-driven gliomas, which demonstrate excellent long-term survival with minimal post-surgery clinical intervention.BRAF alternations, and have a poor outcome.Group 3, midline RAS/MAPK-driven gliomas, which are enriched for LGG, such as PA, with Compared to gliomas in children and adults, infantile gliomas have paradoxical clinical behavior. Whereas HGGs have a better clinical course ,110, LGGBecause each subtype indicates the clinical and molecular features, updating the diagnosis and treatment of these gliomas is necessary. Additionally, because these molecular features can be targetable, novel molecular targeted therapies are expected to develop. The involvement of the RAS/MAPK pathway in infantile gliomas has been revealed, and current molecular profiling have identified novel alterations regarding this pathway.NTRK fusions: NTRK1, NTRK2, and NTRK3 are actionable drivers of tumor growth [NTRK fusions affect both the RAS/MAPK and PI3K/AKT/mTOR pathways, and are considered to be associated with tumorigenesis [NTRK1-3 fusion genes [NTRK1-3 fusion genes have not been identified frequently in pLGG and adult GBM [NTRK fusions can be detected in histologically diagnosed glioneuronal tumors [r growth ,116, andr growth . NTRK fuigenesis ,119. Wu on genes . NTRK1-3dult GBM ,40,120. l tumors , and canALK fusions: the ALK gene is thought to be associated with development and function of the nervous system. ALK fusion is reported to cause ectopic expression of the ALK fusion protein [ALK fusion are rare and the published literature is limited to several case studies, CCDC88A-ALK and PPP1CB-ALK were reported as the most frequent alterations [VCL as a novel partner of the ALK fusion gene [ protein ,122, res protein . Althougerations ,124,125.ion gene .ROS1 fusions: ROS1 is an orphan tyrosine receptor, which is considered to be associated with cell proliferation and differentiation. Although ROS1 fusion in glioma is quite rare, several reports have highlighted it as a targetable genetic alteration [GOPC-ROS1 was reported as the most common ROS1 alteration in glioma. In addition, CEP85L-ROS1, ZCCHC8-ROS1, and KLC1-ROS1 have also been identified [ROS1 has shown significant antitumor activity [teration ,128,129.entified ,128,129.activity ,131.Clinical trials of targeted therapy for infantile glioma are summarized in ALK/ROS1/NTRK/MET alterations, and 5-year OS was 53.8, 25.0, and 42.9% for tumors with ALK, ROS1, and NTRK fusions, respectively [NTRK1-3, ROS1, and ALK were found in adult patients with solid tumors, including CNS tumors [NTRK fusion, is planned in the United States. NTRK1-3, ROS1, and ALK fusion genes in infantile glioma are targetable, therefore, nationwide adaptation of NGS to evaluate these fusion genes and more extensive accumulation of clinical data are required.Group 1 tumors: group 1 tumors harbored ectively . EntrectS tumors . ResponsS tumors . A phaseGroup 2 tumors: because group 2 tumors show excellent long-term survival, a safe resection and careful follow-up are recommended .BRAF/MEK inhibitors, should be administered as soon after initial diagnosis as possible [BRAF inhibitor, and selumetinib, an MEK inhibitor, have been reported to be effective and tolerable [Group 3 tumors: Because most group 3 tumors result in poor outcomes after conventional chemotherapy, targeted therapy, such as possible . In the olerable ,133,134.Because the molecular characteristics of pediatric gliomas are different from those of adult gliomas, molecular genetic analysis is essential to diagnose and treat pediatric gliomas. Because targetable molecular aberrations can be detected in pediatric gliomas, searching for these aberrations is very important. Moreover, sufficient evidence of the novel targeted therapies has not been demonstrated in the present clinical trials. However, efficient targeted therapies are expected to be feasible even for rare subtypes in CNS tumors because of multi-gene panel analysis using NGS in the near future. Although the era of incorporating molecular genetic analysis into clinical practice is beginning, the search for specific molecular aberrations in pediatric gliomas is insufficient. How to administer molecular genetic analysis in clinical practice may be a future issue.


Different Ca2+ channels, pumps, or exchangers regulate variations in the duration and levels of intracellular Ca2+, which may be transient or sustained. These changes are then decoded by an elaborate toolkit of Ca2+-sensors, which translate Ca2+ signal to intracellular operational cell machinery, thereby regulating numerous Ca2+-dependent physiological processes. Alterations to Ca2+ homoeostasis and signaling are often deleterious and are associated with certain pathological states, including cancer. Altered Ca2+ transmission has been implicated in a variety of processes fundamental for the uncontrolled proliferation and invasiveness of tumor cells and other processes important for cancer progression, such as the development of resistance to cancer therapies. Here, we review what is known about Ca2+ signaling and how this fundamental second messenger regulates life and death decisions in the context of cancer, with particular attention directed to cell proliferation, apoptosis, and autophagy. We also explore the intersections of Ca2+ and the therapeutic targeting of cancer cells, summarizing the therapeutic opportunities for Ca2+ signal modulators to improve the effectiveness of current anticancer therapies.Calcium (Ca Indeed, there is a 20,000-fold gradient between outside (about 1.2 mM) and inside (approximately 10–100 nM) of cells. Moreover, in the mitochondria and in the nucleus, the concentrations of Ca2+ are similar to those in the cytoplasm. In the endoplasmic reticulum (ER), considered the main intracellular Ca2+ store, the [Ca2+] ranges between 100 and 800 μM [2+ levels show that lysosomes present an internal [Ca2+] of about ≈500 μM  and regulates the propagation of intracellular Ca2+ changes that are fundamental to intracellularly transmitted biological information and important physiologic processes, including metabolism, cell proliferation and death, protein phosphorylation, gene transcription, neurotransmission, contraction, and secretion [2+]i can increase more than twofold at the micromolar level. Different channels situated in the plasma membrane (PM) induce the influx of extracellular Ca2+ into the cells. Among these channels, the most important are transient receptor potential channels (TRPC) i has adverse effects for the cells. Therefore, different channels, pumps, and buffering systems reestablish low [Ca2+]i. The reuptake of Ca2+ into the ER lumen is allowed by the activity of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), which pumps Ca2+ into the ER with a stoichiometry of 2:1 Ca2+/ATP and by the secretory protein calcium ATPase (SPCA), which transports Ca2+into the Golgi apparatus i increases during the G1/S phase transition c levels and the simultaneous activation of pro-survival autophagy in cancer cells. Consistently, addition of intracellular Ca2+ chelators prevented autophagic activation and induced cell death. Different downstream effectors were proposed to regulate Ca2+-dependent autophagy. For example, it was suggested that increases in [Ca2+]c determined activation and phosphorylation of protein kinase Cθ stimulating LC3-II conversion and autophagy [2+]c was also associated to activation of the ERK pathway, which activates mitochondrial depolarization, autophagy, and apoptosis c promotes activation of CAMK2, which, in turn, activates autophagy through the regulation of the AMPK/mTOR pathway [2+]c dynamics are regulated at ER levels by BCL-2 protein, which lower [Ca2+]ER levels, thus reducing the Ca2+ leak from ER [Autophagic dynamics in cancer are modulated by Caic death . ORAI1 isurvival . Additio agents) ,117 and  agents)  provoke utophagy . Rapid epoptosis . However pathway . Importa from ER .2+ mobilization from the ER can also increase autophagic flux. In this scenario, IP3Rs are the primary regulator. Lithium and L-690330 stimulate autophagy by reducing the levels of IP3 and inositol and consequent IP3Rs activities [2+ dynamics and promotes autophagy [2+-dependent mitochondrial activation. Correct IP3R-mediated Ca2+ transfer between ER and mitochondria supports the tricarboxylic acid cycle and consequent ATP production. This signal determines the inhibition of the energy sensor of the ATP/AMP ratio, AMPK, which is also the most investigated positive regulator of autophagic induction mechanism: AMPK-ULK1-mTOR [2+ from the ER to mitochondria with a subsequent decrease in ATP production, which determines the activation of the AMPK pathway, thereby promoting pro-survival autophagy [In contrast, inhibition of Cativities . Consistutophagy . InteresLK1-mTOR . Recentlutophagy . Similarutophagy   for the treatment of various solid tumors [2+, inducing the apoptosis in prostate and bladder cancer cells, respectively [TRPCs are classified into different subfamilies  and are implicated in different diseases, including cancer . Alteratma cells ,129. Thema cells . In addid tumors . This cld tumors , no receectively . Finallyectively . Interesectively .2+-dependent cellular processes, including cell proliferation, survival, and differentiation [2+ channels and are frequently altered in different cancer types. The upregulation of T-type Ca2+ channels has been mainly observed in prostate, breast, and ovarian cancers; however, it has also been found in melanoma, retinoblastoma, glioma, glioblastoma, hepatocellular, colon, and esophageal cancers cells [2+ channels by using the channel blocker mibefradil reduced esophageal and colon cancer cell proliferation by upregulating p53. Moreover, this inhibitor induced the apoptosis in glioblastoma cells and ovarian cancer cells. Furthermore, the administration of mibefradil or NNC-55-096, another T-type Ca2+ channel blocker, decreased tumor growth in xenograft models of glioblastoma and ovarian cancers [VGGCs are classified in high voltage activated (HVA) and low voltage activated (LVA) channels according to their pharmacological and electrophysiological profiles. They regulate various Cantiation . LVA arers cells . The pha cancers .2+ mobilization by the activation of IP3Rs and SOCE channels [Purinergic P2 receptors are classified in two subfamilies named P2X and P2Y. P2X receptors, activated by ATP, are ligand-gated nonselective cation channels formed by homotrimeric or heterotrimeric complexes of seven different subunits (P2X1–7). The P2Y receptor category, comprising eight members , may be activated by ATP, ADP, UTP, UDP, and UDP-glucose. In particular, P2Y2, P2Y4, and P2Y6 receptors are coupled to Gq proteins and their stimulation leads to Cachannels . In cancchannels . Furtherchannels . Among Pchannels . Many anchannels . Consistchannels , meanwhichannels .2+-sensors are molecular SOCE components. Currently, in mammalian cells, three isoforms of ORAI  and two isoforms of STIM (STIM1 and STIM2) have been identified. Remodeling of Ca2+ signals due to SOCE dysregulation may cause various diseases, including cancer. In fact, activating ORAI1 mutations were found in different types of cancer, including colorectal, stomach, and uterine cancers [2+ channels expressed in prostate and colorectal cancers but not in healthy tissue [ORAI channels and STIM Ca cancers . Moreove cancers . Altered cancers . Neverthy tissue ,140.3+ and Gd3+; however, these channel blockers, as well as SKF-96365, are not SOCE-specific inhibitors; therefore, treatment with these compounds may cause side effects. In this regard, DPB-162AE and DPB-163AE, derivatives of 2-APB, have been developed and are potent SOCE inhibitors capable of inhibiting SOC channels without affecting IP3Rs activity. In addition, RO2959, which inhibits the ORAI1-mediated current, may represent an important therapeutic tool since it is able to selectively increase the ORAI1 channel [2+ influx. These mTOR inhibitors are being tested in different clinical trials as anticancer therapy [2+ influx was also found to be crucial for activating the cell death induced by the anti-CD20 monoclonal antibody GA101/obinutuzumab in non-Hodgkin lymphoma and primary B-cell chronic lymphocytic leukemia cells. Moreover, in addition to ORAI1-dependent Ca2+ influx, in this study, it was demonstrated that GA101 determines intracellular Ca2+ elevation by provoking Ca2+ release from lysosomes [The first described inhibitor for ORAI1 channel was SKF-96365, which is able to reduce the growth and migration of breast cancer cells . SOCE ch channel . Another channel . As thes therapy . ORAI1-dysosomes .IP3Rs consist of three isoforms  that are activated by the generation of intracellular IP3. The most isoform of IP3 receptor isoforms involved in carcinogenesis is IP3R3. The dysfunction of this receptor was found in clear cell renal cell carcinoma cells and in colorectal and ovarian cancer cell lines, where this receptor exerted proliferative and antiapoptotic effects . Further2+-dependent cell proliferation [Altered expression or mutations of SERCA isoforms (SERCA2 and SERCA3) was observed in several cancer types, including colon, gastric, lung, and prostate carcinoma ,146,147.feration . In addiferation ,150. In feration ,152. How2+ levels [2+ levels by inhibiting PMCA in prostate cancer cells. Unfortunately, the function of the latter compounds is exerted by the activation of IP3Rs; therefore, they cannot be considered PMCA-specific inhibitors [In the last years, many compounds able to inhibit SERCA for cancer treatment have been produced. Mipsagargin (G-202), a thapsigargin derivate, has been tested on different solid tumors, including prostate cancers, glioblastoma, kidney, and hepatocellular carcinoma, in phase I and II clinical trials. The results obtained in phase I demonstrate that mipsagargin displays a favorable pharmacokinetic profile and acceptable tolerability. Furthermore, significant disease stabilization was observed, suggesting possible antitumor activity . The res+ levels . Furtherhibitors ,157.Alterations in the MCU complex expression/function were found in different cancer types. High expression levels of MCU were detected in colorectal, ovarian, pancreatic, stomach, and prostate cancers, while genetic mutations were observed mainly in prostate, breast, and uterine cancers. Genetic modifications linked to cancer development were also detected in the other components of the MCU complex . Based o2+-permeable pore located on the outer membrane of mitochondrion, where it regulates the flux of ions and metabolites from cytosol to mitochondria and vice versa. Three isoforms for this channel, VDAC1, VDAC2, and VDAC3, have been identified in mammalian cells [2+ fluxes, it is speculated that VDACs may be involved in the control of cell proliferation and apoptosis; therefore, these channels may affect the fate of cancer cells. VDAC1 is upregulated in a variety of human cancer cell lines, while VDAC2 is overexpressed in melanoma, mesothelioma, and thyroid cancer cells [Another important mitochondrial protein is VDAC; this pore is a nonselective Caan cells . Based oer cells . The tarer cells .2+ signaling is extensively involved in these molecular processes, it is not surprising that an anticancer agent may indirectly modulate Ca2+ dynamics in cancer cells. Various studies demonstrated that chemotherapeutic agents modulate intracellular Ca2+ levels. For example, 5-fluorouracil (5FU) is an approved anticancer treatment for several cancer types. It has been observed that 5FU mediates in hepatocarcinoma cell death by diminishing Ca2+ influx. Indeed, 5FU administration decreased ORAI1 levels and induced autophagic cell death by inhibiting PI3K/AKT/mTOR pathway [2+ amounts to a level necessary to activate calmodulin, which, in turn, phosphorylated p53 to trigger apoptosis [2+-mediated apoptosis [2+ transport. However, in this case, Ca2+ dynamics were associated with chemoresistance. Indeed, both inhibition of TRPCs and Ca2+ chelation increased the sensitivity of human leukemia cells to dexamethasone [2+ signaling. Accordingly, these drugs induced the persistent release of Ca2+ from intracellular stores, provoking mitochondrial Ca2+ accumulation and apoptosis. Interestingly, doxorubicin also promoted the binding of p53 to SERCA in the ER. In this state, p53 increased Ca2+ transmission between the ER and mitochondria to induce apoptosis [2+ oscillations that affect neuronal Ca2+ sensor 1 proteins, leading to Ca2+ release from the ER via an IP3R3-dependent pathway [2+ concentration and activating the apoptotic pathway in a p53-dependent pathway [2+ signaling in tumors, particularly 4-aminoquinoline antimalarial compounds chloroquine (CQ) and hydroxychloroquine (HCQ). Currently, more than 30 clinical studies are evaluating the antitumor efficacy of CQ and HCQ. Publications reporting the clinical trial results are encouraging. Indeed, most of these investigations describe positive and/or partial effects of CQ and HCQ in reducing tumor growth alone or in combination with the conventional therapies used for several cancer types [2+ accumulation by inhibiting the IP3Rs-dependent ER Ca2+ release and the Ca2+ influx mediated by TRPCs, ORAI, and STIM channels [2+ signaling to modulate the immunological response [2+ levels that were necessary to activate p38, NF-kB, and TFEB to reprogram the TAM phenotype [2+ homeostasis also in solid tumors associated with PML absence or downregulation. Loss of PML conferred resistance to chemotherapies due to a reduction in ER-mitochondria Ca2+ transmission that activates autophagy and establishes a metabolic advantage for the cancer cells. As a consequence of blocking autophagy with the specific inhibitors , the apoptotic process was rescued in vitro and in vivo [2+ release, and activation of apoptosis. Interestingly, specific MCU inhibitors or MCU silencing abrogated CQ-dependent effects, thus confirming the importance of mitochondrial Ca2+ overload for cell death induction [2+ mobilization in glioma cells that had previously been rendered sensitive to anticancer therapy [2+ uptake and ATP production and diminishing cancer cell proliferation and migration [2+ signaling, which improves the chemotaxis of T cells by increasing intracellular Ca2+ influx [2+ signaling inhibits PDL1 and PDL2 expression [2+ flux is abolished when T cells express high levels of PD-1 [2+ transmission. However, a greater understanding of the steps involving Ca2+ signaling during cancer immunotherapy is required.Proliferation, invasiveness, cell death, neovascularization, gene transcription, protein production, and phosphorylation/dephosphorylation events are some of the numerous targets of anticancer compounds. Given that Ca pathway . In contpoptosis . Similarpoptosis . Additioethasone ,165. Stupoptosis . Taxane  pathway . Photody pathway . Finallyer types . It has channels . This efresponse . This hyhenotype . CQ exer in vivo . Similarnduction . A reduc therapy ; in addiigration . Anotherigration . PD-1 bi+ influx . Furtherpression  and that of PD-1 . Therefo2+ is a ubiquitous signal that contributes to the control of diverse cellular functions. Uncontrolled remodeling of Ca2+ flux contributes to severe pathophysiology processes and often intersects key aspects of cancer progression, such as tumor proliferation, malignant transformation, escape from cell death, and resistance to anticancer agents. Accumulating preclinical and clinical evidence supports the relationship between Ca2+ and cancer, indicating that Ca2+ signaling is a reliable target for novel anticancer treatments. As summarized in this review, defects in Ca2+ channels/transporters/pumps are typical features of cancerous cells and confer low sensitivity to cell death inducers, thus sustaining the tumor growth and metastasis. Hence, pharmacological modulation of these proteins may be a reliable approach to restore the effectiveness of current cancer treatment regimes (2+-regulators, it is important to consider other critical aspects. Targeting these processes is difficult, and most importantly, the tumor environment presents substantial cellular heterogeneity in which only a subset of cancer cells should be targeted. Recent investigations have made progress in overcoming these problems. For example, by encapsulating pumps and channel agonists in lipid nanocapsules, it is possible to efficiently modulate the activities of these pumps and channels and the related cellular processes [2+ modulators can be solely directed to the cancer cell population [2+ in cancer, this area of study is still in its infancy, with a number of studies starting to explore and highlight the importance of these interconnections. Removing the remaining gap in our knowledge on the intersections between Ca2+ and cancer will help researchers better understand the multiple molecular mechanisms that affect tumor development, maintenance, and metastasis and help clinicians design and develop new-generation drugs with the final aim of breaking all the defense barriers of cancer.Cellular Ca regimes . Howeverrocesses . In addipulation ,180. Fur


Increasing interest in studying the role of vitamin D in cancer has been provided by the scientific literature during the last years, although mixed results have been reported. Vitamin D deficiency has been largely associated with various types of solid and non-solid human cancers, and the almost ubiquitous expression of vitamin D receptor (VDR) has always led to suppose a crucial role of vitamin D in cancer. However, the association between vitamin D levels and the risk of solid cancers, such as colorectal, prostate and breast cancer, shows several conflicting results that raise questions about the use of vitamin D supplements in cancer patients. Moreover, studies on vitamin D supplementation do not always show improvements in tumor progression and mortality risk, particularly for prostate and breast cancer. Conversely, several molecular studies are in agreement about the role of vitamin D in inhibiting tumor cell proliferation, growth and invasiveness, cell cycle arrest and inflammatory signaling, through which vitamin D may also regulate cancer microenvironment through the activation of different molecular pathways. More recently, a role in the regulation of cancer stem cells proliferation and short non-coding microRNA (miRNAs) expression has emerged, conferring to vitamin D a more crucial role in cancer development and progression. Interestingly, it has been shown that vitamin D is able not only to potentiate the effects of traditional cancer therapy but can even contribute to overcome the molecular mechanisms of drug resistance—often triggering tumor-spreading. At this regard, vitamin D can act at various levels through the regulation of growth of cancer stem cells and the epithelial–mesenchymal transition (EMT), as well as through the modulation of miRNA gene expression. The current review reconsiders epidemiological and molecular literature concerning the role of vitamin D in cancer risk and tumor development and progression, as well as the action of vitamin D supplementation in potentiating the effects of drug therapy and overcoming the mechanisms of resistance often triggered during cancer therapies, by critically addressing strengths and weaknesses of available data from 2010 to 2020. Vitaminondition . In receondition . Low levondition ,6,7,8, aondition ,12,13,14ondition . This stondition , althougondition . Moreoveondition  and cancondition .More recently, accumulating evidence from epidemiological and experimental studies ,20,21,22The current review critically and extensively discusses the molecular mechanisms underlying the protective effects of vitamin D, the synergistic activity with different cancer drugs and the drug-resistance reversion, evaluated in in vivo and in vitro studies, on different types of solid cancer. Moreover, the review briefly reports the results of the epidemiological evidence linking endogenous levels of vitamin D and supplemented vitamin D doses to cancer risk, cancer development and growth.The relevant literature of the latest 10 years was screened from PubMed (MEDLINE), by using the following search terms: “vitamin D”, “cancer”, “solid cancer”, “colorectal cancer”, “prostate cancer”, “breast cancer”, “drug-resistance”, “reversal effect”.Given the huge amount of epidemiological and in vivo and in vitro experimental data on vitamin D and cancer, this review mainly focuses on the role of vitamin D in a subgroup of solid tumors . Conversely, considering the scarcity of studies investigating the reversal effects of vitamin D in cancer drug resistance, data available in all cancer types are presented and discussed.Circulating vitamin D derives from diet for only 20%, with the remaining 80% being synthesized in the skin where a precursor of cholesterol, 7-dehydrocholesterol, is converted to vitamin D or cholecalciferol by the UVB component of sunlight ,30. IndeVitamin D3, either derived from diet or produced in the skin, represents the inactive form, which requires two hydroxylations to be converted to fully active vitamin D. The first hydroxylation mainly occurs in the liver by the microsomal enzyme with 25-hydroxylase activity, CYP2R1, to obtain 25(OH)D, the major circulating, but less active, form of vitamin D , CYP27A12D, the fully active and most potent metabolite of vitamin D, commonly called calcitriol. The 1α hydroxylase is expressed in the kidney, skin, brain, as well as in placenta, testis, intestine, lung and myeloid cells [A second hydroxylation occurs in the kidney and peripheral tissues by the only enzyme with 1α hydroxylase activity, CYP27B1, which transforms 25(OH)D in 1,25(OH)id cells ,38. In pid cells .2D and 25(OH)D are finely controlled by the balance between production and degradation processes, with the aim to maintain their circulating values into physiological normal levels. Vitamin D physiological status is defined when 25(OH)D circulating levels are between 25 to 70 ng/mL. The enzyme with the 24-hydroxylase activity, CYP24A1, catalyzes the degradation reactions of 25(OH)D and 1,25(OH)2D.Circulating levels of 1,25(OH)2D, and/or 1,25(OH)2D, for which CYP24A1 has higher affinity compared with 25(OH)D, to stop its biologic activity [By multiple hydroxylation reactions on the side chain, CYP24A1 catabolizes 25(OH)D, to prevent its eventual activation in 1,25(OH)activity .2D production [2D downregulates PTH secretion, inhibits renal 1α hydroxylase activity concurrently inducing the 27-hydroxylase. Increased serum calcium and phosphorus levels can antagonize vitamin D action by inducing its degradation via 24-hydroxylase [Calcium requirement is one of the factors involved in the regulation of circulating levels of vitamin D to maintain the correct skeletal integrity. High serum calcium levels suppress renal 1-hydroxylase, while low serum calcium levels stimulate parathyroid hormone (PTH) secretion from parathyroid glands . PTH actroxylase .2D levels and half-life control, it is also considered an oncogene, as demonstrated in several solid cancers, including colorectal [Since the important role of 24-hydroxylase in 1,25(OH)lorectal , prostatlorectal  and brealorectal  cancer.2D is a steroid hormone with a wide range of physiological functions performed by binding specific receptors [2D actions can be divided in classical (genomic actions) and non-classical (non-genomic) actions. Genomic and non-genomic actions of 1,25(OH)2D are mediated by classical nuclear and non-classical membranous VDR, respectively [1,25(OH)eceptors ,47,48. T2D is able to activate non-classical, non-genomic effects, which are not mediated directly by nuclear VDR–ligand and DNA interaction, but dependent on protein–protein interaction mechanisms activating intracellular molecules. Among these molecules, the adenylyl cyclase/cyclicAMP/protein kinase A (AC/cAMP/PKA) system and a phosphoinositide-specific phospholipase C (PLC) trigger the rapid generation of intracellular second messengers [3), promote the activation of protein kinase C (PKC) and rapid release of Ca2+ from the endogenous stores, thus controlling a wide range of intracellular physiological mechanisms [VDR, an intracellular receptor belonging to the nuclear receptor superfamily of ligand-activated transcription factors, is classically associated with calcium and phosphorus homeostasis . Upon bissengers . In factchanisms .As largely demonstrated, VDR is not only present in cells and tissues involved in calcium regulation, but it is also expressed in malignant cells, by suggesting a pivotal role of vitamin D in cancer growth and progression ,55. Sevep = 0.034) [p = 0.041) [Colorectal cancer, particularly in late stages, is associated with vitamin D deficiency ,67,68,69= 0.034)  and West= 0.041) .p = 0.004), therefore inviting to caution in vitamin D supplementation [p = 0.04) [The relationship between vitamin D and prostate cancer risk is still controversial, due to contradictory data reporting an inverse correlation ,76,79,96entation . Discrepentation , vitaminentation ,100 and entation  may be aentation , which m = 0.04) .Similar to prostate model, conflicting data are also reported for breast cancer. As largely described, an inverse correlation between high circulating 25(OH)D levels and breast cancer risk ,84,92,93Besides the importance of vitamin D in cancer risk, vitamin D levels have been further investigated to explore the potential association with cancer prognosis and tumor progression, although present data do not clarify it exactly.p trend = 0.0009 and 0.03), respectively [p = 0.006/HR: 0.91; 95% CI: 0.84 to 0.99, p = 0.027) [p = 0.0021), but not to prognosis [In patients with advanced or metastatic colorectal cancer, higher 25(OH)D circulating levels were significantly associated with higher overall survival and progression-free survival (ectively  and high= 0.027) ,61. Convrognosis . A differognosis , whereasrognosis . Conversrognosis . Moreoverognosis ,110.A recent study highlighted the correlation between normal 25(OH)D levels and breast cancer patient’s survival. Patients were divided into two groups on the basis of circulating levels of vitamin D (inadequate or deficiency group vs. insufficient or sufficient group) and their survival calculated of about 60.65 months in inadequate or deficiency group compared to 76.24 months of insufficient or sufficient group . MoreoveCurrent evidence about 25(OH)D levels and cancer considers not only the endogenous levels of vitamin D, but also the detectable circulating levels following adequate supplementation.Due to its stability and its capability to produce higher serum 25(OH)D levels than ergocalciferol, cholecalciferol is generally used to supplement vitamin D deficiency .It has emerged that cholecalciferol supplementation increased progression free survival and relapse-free survival in patients with colorectal cancer in chemotherapy regimen, even in advanced or metastatic condition, compared to patients undergoing chemotherapy or surgery alone, demonstrating that vitamin D has a crucial role in prognosis improvement ,13,112. The supplementation of 2000 IU/d cholecalciferol in patients with digestive tract cancer, including colorectal cancer, did not significantly increase the 5-year relapse-free survival compared to placebo group . No signNevertheless, the only open randomized prospective study in patients with metastatic colorectal cancer showed that all enrolled patients had insufficient 25(OH)D levels. The daily 2000 IU/d cholecalciferol supplementation to chemotherapy for two years was not able to normalize circulating levels of 25(OH)D, to modify overall survival and progression free survival in colorectal patients in chemotherapy regimen .2D significantly increased in supplemented patients from baseline as well as circulating and prostate tissue levels of 25(OH)D were significantly higher in 40,000 IU/die treated group [As recently emerged, contradictory data are reported about the effects of vitamin D supplementation in prostate cancer progression . Althouged group . Conversed group ,117. Eveed group . Slight ed group ,117. The2D stimulate 25(OH)D or 1,25(OH)2D degradation and, consequently, reduce rather than stimulate the beneficial effects of vitamin D [Surprisingly, in a retrospective study evaluating human epidermal growth factor receptor 2 positive (HER2+) non-metastatic breast cancer patients treated with trastuzumab from 2006 to 2012 and supplemented with low  and high  doses of cholecalciferol, a significant improvement of disease-free survival has been shown, compared to the group treated with trastuzumab alone [itamin D .Taken together, the results of epidemiological studies suggest that circulating 25(OH)D levels may be inversely associated with colorectal, prostate and breast cancer risk and prognosis, although some controversial data have been reported. Moreover, whether vitamin D supplementation may exert a beneficial role in colorectal, prostate and breast cancer progression has yet to be completely clarified.Several molecular mechanisms are involved in vitamin D anticancer effects as evaluated in animal as well as in cultured cell models.Preclinical studies in animal models have demonstrated that vitamin D treatment may inhibit colorectal ,123,124,In a mouse model of colorectal cancer, chemically induced by azoxymethane (AOM) exposure, dietary intake of high doses of vitamin D  reduced colorectal tumor incidence, number of premalignant lesions and number of visible cancerous lesions ; these eInterestingly, in a colorectal cancer xenograft mouse model, dietary intake of high doses of cholecalciferol increased renal expression of the vitamin D catabolizing enzyme 24-hydroxylase, by inducing significantly larger, heavier and capable to penetrate the skin tumor lesions. The addition of soy containing genistein, a natural 24-hydroxylase inhibitor, to cholecalciferol dietary scheme restores sensitivity to the antitumor effects of vitamin D .Vitamin D may also act in combination with different therapies, by inducing an antiproliferative additive effect. Indeed, in both mouse and rat chemically induced colorectal cancer models -induced and DMH-dextran sodium sulfate (DSS)-induced) and xenografts models, vitamin D analogs, such as paricalcitol and PRI-2191, in combination with 5-fluorouracile (5-FU) or metformin, diminished the risk to develop early colon neoplasia by controlling the early stage of carcinogenesis and reducing or delaying cancer growth through a specific cell cycle arrest and apoptosis induction, when compared to the 5-FU and metformin treatment alone ,123,124.2D, and consequently its anticancer effect, although the risk of hypercalcemic adverse effects is relevant for prostate cancer [2D treatment was able to achieve a further reduction of tumor growth, associated with upregulated messenger expression levels of antiproliferative  and pro-apoptotic  genes and downregulation of antiapoptotic  and cell cycle promoting gene cyclin D1. These results confirmed that the increase of circulating vitamin D levels may control prostate cancer growth by inhibiting cell division and by inducing apoptosis [2D, acts as coactivator and corepressor of VDR activity thus modulating 1,25(OH)2D effects induced by target genes transcription [2D, intraperitoneally injected 18 h before cryotherapy, caused a synergistic antiproliferative effect [In prostate cancer xenograft mice receiving cholecalciferol supplemented diet, the reduction in tumor volume started to be evident after one-week treatment and became significant after three-weeks treatment, with the antiinflammatory and antiproliferative effects of vitamin D controlled by a tight gene modulation of anti-inflammatory and antiproliferative related genes, therefore upregulating COX-2, p21 and insulin like growth factor binding protein-3 (IGFBP-3) . As obsee cancer . The respoptosis . In addipoptosis . Using apoptosis . Interespoptosis . As showcription . Moreovee effect ,129. Thee effect ,129.2D supplementation have been explored in breast cancer xenograft models [2D (12 pmol/24) inhibited tumor growth and lung metastasis development by downregulating cell cycle progression markers expression, such as cyclin D1, without increased blood calcemia [2D [2D seems to accelerate photodynamic therapy in a breast cancer xenograft model. Indeed, photodynamic therapy resulted more efficient in vitamin D pretreated tumors, than vehicle-pretreated tumors, therefore representing a pivotal photosensitizer for breast cancer cells [The beneficial effects of dietary cholecalciferol and injected 1,25(OH)t models ,132. Diet models , althougt models . In partcalcemia . Moreoveemia [2D . Also, 1er cells .Considering these in vivo studies, both dietary and injected vitamin D supplementation may reduce colorectal, prostate and breast cancer growth through several antiproliferative and antiinflammatory molecular processes, mainly involving NF-kB and COX-2 inhibition, Wnt/β catenin pathway deregulation, apoptosis induction and cell cycle arrest. Moreover, vitamin D antiproliferative effects can be potentiated by chemotherapeutic drugs and by target therapy molecules.Anticancer activity of vitamin D has been largely studied in different types of tumors, elucidating its pivotal role in cancer progression and invasion . It has 2D, therefore blocking the cell cycle progression in S phase in prostate [2D exposure increased p21 messenger and protein levels [2D may regulate p21 and p27 transcription is not well clarified, although as observed for p27 transcription, the role of VDR is fundamental [2D is able to induce apoptosis by stimulating pro-apoptotic factor Bax and Bad and by inhibiting antiapoptotic factors such as Bcl-2 [2D inhibition of TACE , a metalloproteinase overexpressed in several cancer types [2D and metformin showed cell growth arrest, by demonstrating the synergistic effect of the treatment and the main effect on autophagy process induction, due to an increase in microtubule-associated protein 1A/1B-light chain 3 II:I (LC3II/LC3I) ratio, the most widely used autophagosome marker, reflecting the number of autophagosomes and autophagy-related structures, in cells treated with a combination therapy compared to mono-treatments [Cytofluorimetric and protein analysis revealed that vitamin D-nanoemulsion causes a cell cycle arrest in G0/G1 phase in colorectal , breast prostate  and breaprostate ,151 celln levels ,158. Thin levels . The exadamental . In prosdamental . The inhdamental ,153 and damental ,155. In as Bcl-2 ,160, andas Bcl-2 . Wnt/β-cas Bcl-2 . Moreoveas Bcl-2 , since cas Bcl-2 ,163. Theer types , may furer types . ADAM17,er types , enhanceer types . It has er types . Intereser types . The proer types . Vitaminer types  or gefiner types , omega-3er types  and metfeatments . MoreoveConsidering the fundamental role of inflammation in oncogenesis , and tha2D displayed lower expression levels of interleukin-6 (IL-6), accompanied to p38 MAPK inactivation [2D and stimulated with lipopolysaccharide, NF-kB activity was markedly inhibited by a significant increase of messenger and protein levels of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkBα), the inhibitory protein of NF-kB [2D on IkBα phosphorylation led to IkBα half-life prolongation, which in turn can influence NF-kB nuclear translocation and, consequently, activity on stimulation of cytokines transcription such as tumor necrosis factor-α (TNF-α) secretion [Primary prostatic cultures of normal and adenocarcinoma cells treated with 1,25(OH)tivation . Moreoveof NF-kB . The inhecretion .2D showed antiproliferative and antimigratory effects by inhibition of TNF-α production [2D influenced both IkBα expression and phosphorylation via phosphoinositide 3-kinases/protein kinase B (PI3K/Akt) pathway [2D and TNF-α inhibited metalloprotease-9 (MMP-9) and monocyte chemoattractant protein-1 (MCP-1) protein expression, otherwise hyperactivated in presence of TNF-α alone, by demonstrating a further role of vitamin D in the regulation of infiltration, migration and cancer invasiveness [2D showed decreased proliferation, in part induced by the reduced TNF-α and IL-1β synthesis, by demonstrating that even drastic deregulation of immune modulators may contribute to cancer progression [Similarly, human colorectal cancer cells exposed to 1,25(OH)oduction , and, in pathway . Moreovesiveness . Conversgression .More recently, the important role of vitamin D in cancer onset and progression has been explored by studying its association with cancer stem cells ,175,176 2D treatment [2D [Cancer stem cells are a subset of cells with the characteristics of self-renewal and differentiation into multiple cell types that confer them the possibility to actively participate in tumor progression . Recentlreatment ; similarment [2D . Last, iment [2D . Moreovement [2D .In conclusion, considering the examined in vitro studies on colorectal, prostate and breast cancer cells, vitamin D acts as anticancer agent by controlling several processes involved in cancer growth and progression. The inhibition of cancer cells proliferation, the arrest of cell cycle and the induction of cell death, such as apoptosis and autophagy, are the main molecular mechanisms by which vitamin D exerts its anticancer action. Moreover, vitamin D anti-inflammatory properties may regulate tumor microenvironment by suppressing inflammatory mediators implied in cancer invasion.More recently, the capability of vitamin D in modulating cancer stem cells proliferation and miRNAs expression has shed new light on the vitamin D role in cancer, by underpinning regulatory properties in the stem and epigenetic fields.Different cell molecular mechanisms are involved in acquired and intrinsic resistance of cancer cells to cancer therapy. Unlike the intrinsic resistance, which appears upon the first exposure to cancer therapy, the acquired resistance occurs during the chemotherapeutic treatments or during a second attempt of treatment, after cancer recurrence .In the last years, scientific evidence reported a large number of anticancer drugs capable to enhance chemotherapy or target therapy efficacy or even to overcome multidrug cancer resistance including that to xenobiotics and nanoparticles ,189. RecControl of cell death, abnormal functionality of cell cycle check-points and epithelial–mesenchymal transition are only a part of a wide range of proliferation and differentiation molecular events in cancer, which are implied in acquired multitarget cancer cell resistance . In the 2D, also downregulated VDR expression may contribute to vitamin D resistance [In addition to all these mechanisms of drug resistance in cancer, in which vitamin D interferes at various levels, there are mechanisms of resistance to the vitamin D itself, which have also been observed in colorectal, prostate and breast cancer. In addition to the previously mentioned oncogenic activity of 24-hydroxylase, capable to control half-life of 1,25(OH)sistance , as wellsistance . HoweverIn the following paragraph, due to the scant available data, a systematic review of the current literature about the role of vitamin D to reverse drug resistance mechanisms in cancer are detailed and represented in Cancer pharmacological resistance represents the major cause of tumor progression therefore, overcoming drug resistance is now the greatest challenge for scientists. Vitamin D has been reported to reverse resistance mechanisms to chemotherapy ,24, TKI 2D, similarly to different cancer models, showed an increased percentage of apoptotic cells with a cell cycle arrest in G0/G1 phase associated with an upregulation of VDR protein expression [2D induced a VDR overexpression and inhibited MCF-7 cancer stem cells proliferation by Wnt/β-catenin proteins downregulation, demonstrating that vitamin D reverts tamoxifen resistance by inhibiting Wnt/β-catenin pathway in breast cancer cells [Human breast cancer cell line MCF-7 treated with 1,25(OH)pression . Surprispression . Last, wer cells . Similarer cells . Chronicer cells . Surpriser cells .R1 Cells) with epidermal growth factor receptor (EGFR) mutation that made these cells erlotinib-resistant. Indeed, HCC827R1 cells developed epithelial–mesenchymal transition (EMT) following prolonged treatment with erlotinib, an EGFR inhibitor. The switch to a more aggressive phenotype of HCC827R1 cells, associated with erlotinib resistance, was also characterized by the overexpression of VDR [2D reverted EMT and restored erlotinib sensitivity, probably by counteracting tumor growth factor β (TGF-β), which is typically induced during EMT in HCC827R1 cells. In this study, authors treated erlotinib-resistant HCC827R1 cells with liposomal nanoparticles (LP) nanocarriers systems which co-deliver vitamin D, and a 24-hydroxylase inhibitor, CTA091. This strategy, eventually translated in vivo, could avoid systemic and amplify local effects [Moreover, reversal effect of vitamin D has been also observed in a non-small cell lung cancer cell line  cell lines resistant to everolimus, an mTOR inhibitor. The authors showed that the treatment with vitamin D restored sensitivity to everolimus by regulation of EMT process and miR-375/oncogenes circuit . Specififunction . Similaractivity .Taken into consideration the few available molecular data, it can be concluded that vitamin D may act on multidrug cancer resistance at different levels. The studied molecular mechanisms involve EMT transition and cancer stem cells inhibition. Moreover, the role of vitamin D on modulation of miRNA expression and, subsequently, on oncogenes regulation, could be a promising field for future investigation.In conclusion, the review of scientific literature concerning the role of vitamin D in the risk of developing colorectal, prostate and breast cancer from 2010 to 2020 has revealed that although there are still conflicting data in this field, a pivotal role of this hormone in cancer progression and mortality exists. Molecular scenario induced by vitamin D in cancer context is traditionally linked to its antiproliferative and apoptotic effects, exerted also in combination with different drugs in order to empower medical treatment. Notable evidence reported by studies on animal and cell culture models also highlighted the importance of its antiinflammatory role in cancer.Last, despite the few works present in the literature, new compelling evidence about the role of vitamin D in the reversion of multidrug resistance is emerging, showing that through miRNA and EMT regulation vitamin D may overcome the increasingly widespread mechanisms of drug resistance in the oncological field, and suggesting its use in the therapeutic approaches for targeting tumors with the multi drug resistance phenotype.


Lines of evidence have shown the embryogenic and transgenerational impact of bisphenol A (BPA), an endocrine-disrupting chemical, on immune-metabolic alterations, inflammation, and oxidative stress, while BPA toxic effects in adult obese mice are still overlooked. Here, we evaluate BPA’s worsening effect on several hepatic maladaptive processes associated to high-fat diet (HFD)-induced obesity in mice. After 12 weeks HFD feeding, C57Bl/6J male mice were exposed daily to BPA (50 μg/kg per os) along with HFD for 3 weeks. Glucose tolerance and lipid metabolism were examined in serum and/or liver. Hepatic oxidative damage , and mitochondrial respiratory capacity were evaluated. Moreover, liver damage progression and inflammatory/immune response were determined by histological and molecular analysis. BPA amplified HFD-induced alteration of key factors involved in glucose and lipid metabolism, liver triglycerides accumulation, and worsened mitochondrial dysfunction by increasing oxidative stress and reducing antioxidant defense. The exacerbation by BPA of hepatic immune-metabolic dysfunction induced by HFD was shown by increased toll-like receptor-4 and its downstream pathways  amplifying inflammatory cytokine transcription and promoting fibrosis progression. This study evidences that BPA exposure represents an additional risk factor for the progression of fatty liver diseases strictly related to the cross-talk between oxidative stress and immune-metabolic impairment due to obesity. Bisphenol A (BPA) is considered one of the most widespread endocrine-disrupting chemicals (EDCs), substances affecting human health and impacting not only the endocrine system but also immune and metabolic functions ,2. Many An increase in oxidative stress-associated inflammation has been hypothesized to be one of the major mechanisms in the pathogenesis of obesity-related diseases . As knowBPA, like several EDCs, can affect mitochondrial function, targets hepatic mitochondria , and preIncreasing data have reported the adverse effects of BPA exposure, particularly during gestation and development ,14. BPA Perinatal exposure to BPA worsens hepatic alterations caused by a high-fat diet (HFD) in rat offspring . MoreoveSimilar evidence has emerged in humans, where higher urinary concentrations of BPA were associated with morbidity in an adult population characterized by IR or central obesity . In an eThis study was focused on the evaluation of BPA’s exacerbating effect on liver immune-metabolic and mitochondrial dysfunction in adult HFD-fed obese mice, identifying the critical correlation and cross-talk between the oxidative stress and inflammasome pathway activation. We analyzed the impairment of glucose and lipid metabolism, the mitochondrial oxidative stress, the worsening of hepatic inflammatory damage in fibrosis, and the link between BPA and liver immunotoxicity related to obesity.https://www.arriveguidelines.org/resources, the Basel Declaration, and the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) concept, and were approved by the Institutional Committee on the Ethics of Animal Experiments (CSV) of the University of Naples Federico II and by the Italian Ministry of Health under protocol No. 371/2017-PR.Male C57Bl/6J mice (20 ± 0.3 g/bw)  at 6 weeks of age, were housed in stainless steel cages in a room kept at 22 ± 1 °C with a 12:12 h lights-dark cycle. All procedures involving the animals were carried out in conformity with international and national law and policies, including European Union (EU) Directive 2010/63/EU for animal experiments, Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines 2.0, The standard chow diet had 17% fat without sucrose, while the HFD  had 45% of energy derived from fat, 7% of sucrose. Standard chow diet (STD) and HFD contained 3.3 and 5.24 kcal/g, respectively. Six-week-old mice were randomly divided into three groups ensuring no differences in body weight mean , as follows: (1) a control group receiving chow diet and vehicle per os (STD); (2) HFD group receiving vehicle; and (3) HFD group exposed daily to BPA . Until 2015, the daily dose of BPA used in our experimental conditions was considered by the scientific community as the TDI for humans. The treatment started after 12 weeks of feeding with HFD, the time by which obesity was full-blown, and continued for 3 weeks. During the treatment, body weight and food intake were monitored weekly. Before sacrifice, bioelectrical impedance analysis (BIA) was performed to measure fat body composition using BIA 101 analyzer, modified for the mouse . Fat-free mass was calculated, and then fat mass content was determined as the difference between body weight and fat-free mass.Blood obtained from STD, HFD, and HFD + BPA mice was collected by cardiac puncture and centrifuged at 2500 rpm at 4 °C for 12 min, and serum samples were stored at −80 °C for following biochemical and hormonal determinations. Alanine aminotransferase (ALT), alkaline phosphatase (ALP), triglycerides (TGL), and monocyte chemoattractant protein 1 (MCP1) were quantified by colorimetric enzymatic method using commercial kits . Serum leptin, adiponectin, lipopolysaccharide (LPS), and IL-10, , and fasting insulin  concentrations were measured using commercially available ELISA kits. As an index of insulin resistance (IR), HOMA-IR (homeostasis model assessment) was calculated, using the formula (HOMA = fasting glucose (mmol/L) × fasting insulin (μU/mL)/22.5). Liver tissues were homogenized in saline solution and then centrifuged at 5000 rpm for 5 min. Supernatants were collected and centrifugated at 14,000 rpm at 4 °C for 15 min and triglycerides quantified .At the end of the experimental period, oral glucose tolerance test (OGTT) was performed as previously described  on 16 h Mitochondrial isolation and oxygen consumption were performed by high-resolution respirometry Hansatech oxygraph , as previously reported . Oxygen g per 10 min at 4 °C. The pellet was mixed at ice-cold temperatures in 5 mL of 100 mM potassium phosphate buffer (pH 7.4) and then incubated for 60 min at 37 °C. The fluorescence was measured by the HTS-7000 Plus-plate-reader spectrofluorometer  at 488 nm for excitation and 525 nm for emission wavelengths. ROS were quantified from the dichloro-fluorescein standard curve in dimethyl sulfoxide (0–1 mM). For MDA assay, tissues were homogenized in 1.15% KCl solution. A small amount of the homogenate (200 μL) was added to a reaction mixture containing 200 μL of 8.1% SDS, 1.5 mL of 20% acetic acid (pH 3.5), 1.5 mL of 0.8% thiobarbituric acid, and 600 μL of distilled water. The supernatant absorbance was spectrophotometrically measured at 550 nm and the concentration of MDA was expressed as micromoles of MDA normalized on mg of protein of tissue homogenate (µM/mg protein). A standard curve was prepared using MDA bis  as the source of MDA.ROS and malondialdehyde (MDA) assays were performed as previously reported . In ROS The liver was dissected and further fixed by immersion in 4% paraformaldehyde in PB overnight at 4 °C. Thus, the tissues were dehydrated in ethanol, cleared in xylene, and embedded in paraffin. Hematoxylin & eosin (H&E) staining was performed on paraffin-embedded sections (4 μm thick), as previously described  to assesLivers were homogenized and total protein lysates were subjected to SDS-PAGE as previously described . The blo®, MACHEREY-NAGEL GmbH & Co, Düren, Germany), according to the manufacturer’s instructions. cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit  as previously described [G6pc, Pck1, Fasn, Srebf1, Pparg, Tnfa, Ifng, Il6, Tgfb, Col1a1, Col3a1, Myd88, NACHT, LRR and PYD domain-containing protein (Nlrp3), Pycard, Casp1, Il1b, Nfkb1, Ccl2, and Itgax . The relative amount of each studied mRNA was normalized to Actb as a housekeeping gene, and the data were analyzed according to the 2−ΔΔCt method.Total RNA, isolated from the liver , was obtained by the extraction using TRIzol Reagent (Bio-Rad Laboratories) and following a specific RNA extraction kit  analysis of variance (ANOVA) for multiple comparisons followed by Bonferroni’s post hoc test, using GraphPad Prism 8 . Statistical significance was set at p < 0.0001 vs. STD; HFD + BPA = 13.91 ± 0.72 g, ##p < 0.01 vs. HFD). Consistently, the adipokine (leptin and adiponectin) profile was altered by the HFD and further modified by BPA  was performed at the end of the experimental time. As expected, HFD increased glycemia during OGTT compared to control; this alteration was amplified by BPA A. Consishe liver E,F.Fasn, Srebf1, Pparg  A. BPA ex1, Pparg E, alreadIn hepatic mitochondria, the evaluation of oxygen consumption using succinate as substrate, was performed A. State Myd88 , and caspase 1 (Casp1) mRNAs, and the strictly related IL-1β (Il1b) transcription (Ccl2) and macrophage recruitment factor Cd11c (Itgax) mRNAs  mRNAs G.Col1A1, Col3a1, and Tgfb, slightly altered by HFD  activation . Here, BTo date, no evidence regarding BPA toxicity on hepatic mitochondria in adult obese animals is available. Our previous studies showed that HFD causes the alteration of liver mitochondrial function and dynamics in mice, also reducing the activity of antioxidant scavengers ,38. HereOur data agree with previous studies reporting that BPA interferes with mitochondrial functions in the liver and other tissues ,39 comprIn our experimental condition, BPA increases systemic inflammation induced by fat overnutrition. Indeed, higher levels of LPS and MCP1, and the reduction of anti-inflammatory IL-10 were observed in serum. LPS is one of the most crucial factors contributing to low-grade inflammation, also called “metainflammation”, associated with HFD feeding, responsible for the induction of inflammatory cytokines by immune cells and adipocytes .Several lines of evidence have previously demonstrated BPA’s capability of inducing the dysregulation of cytokines, hepatocyte apoptosis, and oxidative stress in the liver ,42. MoreDuring lipid overnutrition, circulating free fatty acids, whose levels are commonly increased in obesity, accumulate in the liver as fat storage, and at the same time, in concert with LPS, trigger TLR4/NF-κB pathway . TherefoTherefore, our data demonstrate that BPA exposure aggravates liver damage induced by HFD, not only causing azonal steatosis but also increasing steatohepatitis, accelerating the fibrotic process proven by marked collagen deposition and pro-fibrotic factors expression. Indeed, steatosis progression to steatohepatitis and fibrosis originates from the uncontrolled increase of oxidative damage and inflammatory cascade  and thenHowever, we cannot exclude the involvement of other mechanisms underlying BPA liver toxicity. Among these, oxidative stress caused by BPA exposure can induce epigenetic changes , includiIn summary, BPA aggravates liver immune-metabolic and mitochondrial dysfunction in adult obese mice magnifying the cross-talk among hepatic oxidative stress, cytokine network, and fibrosis progression, and highlighting the critical role of inflammasome activation. Therefore, our data point out that BPA exposure represents an additional risk factor for the progression of fatty liver diseases and the other pathological features strictly related to obesity in adulthood.