Glioblastoma is the most aggressive primary brain tumor, characterized by a short survival and by an invariably poor outcome. The clinical management of glioblastoma patients is based on surgery followed by adjuvant radio-chemotherapy. Glioblastoma therapy remained substantially unaltered in the last two decades, due to the lack of significant therapeutic alternatives. Regorafenib, a multikinase inhibitor already used as an anticancer drug for hepatocellular carcinoma, has recently been introduced as a therapy for relapsed glioblastoma, based on the encouraging results of a randomized phase II clinical trial. However, very little is known about the mechanisms governing glioblastoma cells’ response to regorafenib. Here we present an in vitro study, performed on glioblastoma tumor cells and on patient-derived glioma stem cells, aiming at characterizing the cellular response to regorafenib. Overall, the emerging message is that regorafenib limits glioblastoma cell proliferation, but might eventually increase the tumor cells’ migration ability.Glioblastoma (GBM), the most malignant primary brain tumor in adults. Although not frequent, it has a relevant social impact because the peak incidence coincides with the age of professional maturity. A number of novel treatments have been proposed, yet clinical trials have been disappointing. Recently, a phase II clinical trial (REGOMA) demonstrated that the multikinase inhibitor regorafenib significantly increased the median overall survival (OS) of GBM patients when compared to lomustine-treated patients. On this basis, the National Comprehensive Cancer Network (NCCN) 2020 Guidelines included regorafenib as a preferred regimen in relapsed GBM treatment. Despite the use in GBM patients’ therapy, little is known about the molecular mechanisms governing regorafenib effectiveness on the GBM tumor. Here we report an in vitro characterization of GBM tumor cells’ response to regorafenib, performed both on cell lines and on patient-derived glioma stem cells (GSCs). Overall, regorafenib significantly reduced cell growth of 2D tumor cell cultures and of 3D tumor spheroids. Strikingly, this effect was accompanied by transcriptional regulation of epithelial to mesenchymal transition (EMT) genes and by an increased ability of surviving tumor cells to invade the surrounding matrix. Taken together, our data suggest that regorafenib limits cell growth, however, it might induce an invasive phenotype. Glioblastoma (GBM) is the most malignant and, unfortunately, the most frequent primary brain tumor in adults. It is a highly vascularized cancer whereby neo-angiogenesis is one hallmark for histological diagnosis. Standard-of-care treatment consists of surgery followed by chemo-radiation. Nevertheless, median survival is 14.6 months ,3; (iv) Multikinase inhibitors, molecules targeting a panel of different kinases, have been evaluated in several studies for recurrent GBM therapy ,8 with tA very recent phase II study, analyzing patient-reported outcomes of regorafenib compared with lomustine, revealed that regorafenib did not negatively affect the health-related quality of life , furtherDespite the encouraging results from the REGOMA trial, the real benefit of regorafenib use in the clinical management of relapsed GBM remains to be fully addressed. Although it is undoubted that there is a subgroup of patients who respond to regorafenib ,15, the Here we present an in vitro characterization of GBM cells’ response to regorafenib, using both tumor cell lines and GSCs. Our study shows that, in vitro, regorafenib limits tumor cells viability but promotes epithelial to mesenchymal transition (EMT) and tumor cells’ migratory ability.2. All cell lines were regularly checked to exclude mycoplasma contamination by Mycoalert Detection Kit .U87  and A172 were purchased from ATCC and cultured in DMEM 4.5 g/L glucose , supplemented with 10% fetal bovine serum (Thermo Fisher Scientific). GSCs were isolated from patients’ surgical samples at the Institute of Neurosurgery, Catholic University of Rome as previously described  and growRegorafenib (Bay73-4506) was purchased from Selleckem  and resuspended in DMSO.The effect of regorafenib exposure on cell viability was evaluated by the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay  or by CellTiter-Glo™ (Promega), according to the manufacturer’s instructions. Briefly, U87 and A172 were seeded in technical triplicate on a 96-well microplate. Twenty-four hours post-plating, we added regorafenib. MTS was performed 48 and 72 h later. For GSCs, cells were dissociated and seeded in technical triplicate on a 96-well microplate. Twenty-four hours post-plating, we added regorafenib. ATP levels were measured 48 and 72 h later using the CellTiter-Glo™. The mean of the raw luminescence values (LD) from triplicate wells treated with vehicle alone , was used as reference to calculate percent viability from wells treated with drugs (VD), using the following formula: VD = (LD/mLC) × 100, as previously described [Viability assay paragraph was fitted in Prism to determine the IC50, at 48 (U87 and A172) and 72 h (GSCs) of regorafenib exposure, specific for each cell strain.IC50 was calculated for U87, A172, GSC#1, GSC#83, GSC#61, GSC#366, GSC#450 and GSC# 493 by GraphPad Prism . Briefly, the dose-response curve to regorafenib obtained as reported in the Cell-cycle analysis was performed as in . BrieflyU87, A172, GSC#1, and GSC#83 were treated with regorafenib (7.5 µM) for 24 h and 48 h. Apoptosis was analyzed using flow cytometry . Cells were stained with annexin V-FITC (BD Biosciences) and propidium iodide (PI) according to the manufacturer’s instructions.The experiment was performed in triplicate, and the proportions of apoptotic cells were compared between the regorafenib-treated and untreated groups, using FlowJo (ver.10.8) (BD Biosciences).Colony-formation ability of GSCs after regorafenib treatment was evaluated by plating a single cell/well in 96-well plates. The experiments were performed in GSC complete medium in the presence of regorafenib or DMSO as control. He dose of regorafenib was 2.5 µM, and the drug was added again after 1 week. After 2 weeks, each well was examined and the number of spheres/cell aggregates was counted.p < 0.01; ***; p < 0.001 based on Student’s t test.Results are shown as percent colony number values from independent experiments in triplicate, calculated over the correspondent control and are shown as mean ± SD. **, One μg of RNA isolated by TRIzol (Thermo Fisher Scientific) was retrotranscribed with MLV-Reverse Transcriptase (Promega) and amplified by real-time PCR using SYBR Select Master Mix  and gene specific-primers . Real-tiU87, A172, GSC#1, and GSC#83 were treated with 7.5 µM regorafenib for 48 and 72 h. Protein extraction was performed using protein buffer extraction  supplemented with protease and phosphatase inhibitors. Protein concentration was determined using Bradford assay  and 15 µg of protein were resolved by SDS-PAGE, transferred onto nitrocellulose membrane, and then analyzed by immunoblotting using specific antibodies: p-p53 ser15  1:1000; p53  1:500; Actin  1:5000; AKT  1:1000; and p-akt ser473  1:1000; p21  1:1000. Graphs report quantitation of the proteins levels as addressed by Image Lab Software (BioRad).3 cells/spheroid in 100 µL) and plated on ultra-low attachment (ULA) 96-well plates . After 48 h, cellular media and regorafenib or vehicle were added. Media were changed every three days. For collagen-embedded spheroids, 2 × 103 A172 or 103 U87, GSC#1, and GSC#83 were suspended in 20% methylcellulose solution and plated on ULA plates. After 48 h, the spheroids were embedded into collagen matrix as indicated in [Cells were suspended in 20% methylcellulose in complete growth medium , calcein AM , and Hoechst  in culture medium. After incubation, the medium was removed and replaced by PBS solution. Images were acquired by laser confocal microscope .v/v) Lugol solution (Sigma Aldrich) for 72 h at room temperature. Spheroids were individually transferred and analyzed in 0.5 mL microtubes. The acquisition of the 3D datasets was performed using a high-resolution 3D Micro-CT Imaging System , with a camera pixel/size of 4.8 µm, tube voltage peak of 39 kV, exposure time of 170 ms, with no physical filtration. Captured data were reconstructed using built-in NRecon Skyscan Bruker Software (Version:1.6.6.0) and processed with CTvox 3D-Visualization software  to create 3D images; DataViewer v. 1.4.4 (Skyscan Bruker software) was used to generate volume rendering and virtual sectioning views [This technique allows the fine analysis of spheroid volumes, being a bioimaging system that develops a three-dimensional virtual model of the sample in a non-destructive way and with a micrometric spatial resolution. Samples were fixed in 3.7% formaldehyde for 15 min, then treated with potassium iodine contrast agent, 0.1N .Data are expressed as mean ± SD as indicated in figure legends. Mean values and standard deviation were generated from at least three biological replicates. Significance was calculated using a two-tailed t test and As a first step in characterizing GBM cells’ response to regorafenib, we evaluated cell viability by MTS assays on two GBM cell lines, U87 and A172, and on six patient-derived GSCs established in our laboratory  . U87 andTo characterize regorafenib impact on cell-cycle regulation, we analyzed, by FACS, propidium iodide-labeled U87 and A172 cells. We extended the analysis to two GSC lines, GSC#1 and GSC#83. We selected these two GSC lines because they are the better characterized among our collection ,27 and bCells were exposed to 7.5 µM regorafenib for 24 or 48 h before propidium iodide labeling and FACS analysis . OverallFinally, we addressed the impact of regorafenib on the ability of GSCs to form spheres. It has been previously reported that regorafenib impairs this ability , which iWe then wondered if regorafenib negatively affects GBM cells viability through induction of apoptosis. To address this point, we performed fluorescent annexin V/propidium iodide (PI) double-staining assay on U87, A172, GSC#1, and GSC#83 treated with regorafenib for 24  or 48 h To further explore the mechanisms at the basis of regorafenib-induced cell death in our GBM cell models, we examined, by Western blot, the expression of p53 and p21, two proteins fundamental in regulating the cellular apoptotic response. Finally, we looked at AKT phosphorylation as an indirect strategy to confirm regorafenib effectiveness in our cellular models. AKT signaling pathway is indeed common to several tyrosine kinases targeted by regorafenib. Regorafenib targets different pathways, including the pathway of VEGF, which is particularly relevant in GBM growth. Previous attempts to restrain GBM growth targeting VEGF or its receptors, either by bevacizumab , by VEGF-trap (a soluble decoy receptor for VEGF) or by anti-VEGFR antibody, resulted in the improvement of tumor cells’ ability to invade brain parenchyma, a phenotypic change known as infiltrative shift ,36,37. MInterestingly, TEM7, VEGFA, ID1 and SNAI2, were over-expressed in U87 and A172, at both 24 and 48 h of treatment. The regulation of the other genes examined was more variable and cell-line-specific .We then asked whether the involvement of the EMT pathway observed in GBM established cell lines also characterizes GSCs. In comparison with 2D cell-culture models, 3D spheroids are able to accurately mimic some features of solid tumors, such as their spatial architecture, physiological responses, secretion of soluble mediators, gene-expression patterns, and drug resistance mechanism ,41. In oTo further characterize tumor spheroids, we analyzed them by X-ray microtomography (microCT). This technique allows the fine analysis of spheroid volumes, being a bioimaging system which develops a three-dimensional virtual model of the sample in a non-destructive way and with a micrometric spatial resolution. Tumor spheroids were established in 20% methylcellulose or collagen and exposed to 7.5 µM regorafenib for seven days before fixation and microCT analysis see , we obseIn summary, the observations obtained in 3D spheroids further support the idea that regorafenib, besides limiting tumor cells’ growth, might induce an invading phenotype, similarly to that already described in GBM treated with bevacizumab ,38.To further characterize regorafenib-treated tumor spheroids, we stained them with calcein AM, propidium and Hoechst . In moreOverall, data obtained in the 3D GBM tumor and cancer stem cell models confirm the anti-tumor effect of regorafenib addressed in the 2D cell cultures in terms of inhibition of tumor cell growth. Notably, cells migratory ability was enhanced by regorafenib.Regorafenib is a multikinase inhibitor targeting a variety of receptors, such as VEGFR1-3, TIE2, KIT, RET, RAF1, BRAF, PDGFR, and FGFR. It is approved as monotherapy for the treatment of hepatocellular carcinoma, gastrointestinal stromal tumors, and colorectal cancers ,44,45. IThe use of anti-angiogenic drugs, and the following inhibition of angiogenesis, protects the blood–brain barrier (BBB), which is less disrupted, eventually resulting in a decrease of tumor enhancement even in rapidly growing tumors . This meAlthough our data require confirmation by independent studies, they set a word of caution in regorafenib clinical use and strongly support the need for studying the molecular mechanisms involved in regorafenib response of GBM cells.


The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) is known to lead to the progressive degeneration of specific neuronal populations, including cerebellar Purkinje cells (PCs), brainstem cranial nerve nuclei and inferior olive nuclei, and spinocerebellar tracts. The disease-causing protein ataxin-1 is fairly ubiquitously expressed throughout the brain and spinal cord, but most studies have primarily focused on the role of ataxin-1 in the cerebellum and brainstem. Therefore, the functions of ataxin-1 and the effects of SCA1 mutations in other brain regions including the cortex are not well-known. Here, we characterized pathology in the motor cortex of a SCA1 mouse model and performed RNA sequencing in this brain region to investigate the impact of mutant ataxin-1 towards transcriptomic alterations. We identified progressive cortical pathology and significant transcriptomic changes in the motor cortex of a SCA1 mouse model. We also identified progressive, region-specific, colocalization of p62 protein with mutant ataxin-1 aggregates in broad brain regions, but not the cerebellum or brainstem. A cross-regional comparison of the SCA1 cortical and cerebellar transcriptomic changes identified both common and unique gene expression changes between the two regions, including shared synaptic dysfunction and region-specific kinase regulation. These findings suggest that the cortex is progressively impacted via both shared and region-specific mechanisms in SCA1. ATAXIN-1 (ATXN1), resulting in a polyglutamine (polyQ) expansion in the ataxin-1 protein  under its endogenous regulators, and replicates many key aspects of SCA1 disease progression and pathology, including progressive behavioral and cognitive impairments, as well as mutant ataxin-1 aggregation, gliosis, and PC pathology  antibody-specific), the ImageJ plugin JaCoP was used [ImageJ  was used for all image processing and quantification. For all images across experiments, the maximum intensity projection was utilized, converted to 8-bit, and thresholded using identical parameters across all images of each experiment. ROI manager was used to measure equal areas across images. To quantify cortical thickness, the length of the cortical layer was measured at three locations in the image approximately 100 μm apart, from layer 1 to layer 6 based on NeuN stain, and averaged per image. To quantify neuron number and size, clumped nuclei were split using the watershed function, and only NeuN-positive nuclei greater than 30 μmwas used . Images t tests was performed. When comparing more than two experimental groups, a one-way analysis of variance (ANOVA) with Tukey’s post hoc analysis was performed. Statistical significance was determined using a significance cutoff of p < 0.05. All statistical analyses were completed using GraphPad Prism software. All data are shown as mean ± SEM. When comparing two experimental groups, a two-tailed unpaired student’s In order to identify region-specific disease signatures between the SCA1 motor cortex and cerebellum, we first aimed to broadly investigate the degree in which the cortex might be affected or altered in mouse models of SCA1. It is known that ataxin-1 is expressed in the cortex , and thaAs cortical thinning has been observed in studies of SCA1 patients ,42, we fNext, since gliosis is reported in the SCA1 KI cerebellum at these time points , we inveATXN1 (Flag-hATXN1) plasmids   D,E. Thisp-value < 0.05 and a log2foldchange (LFC) greater than |0.25| were considered significant and used for further analyses. The top Gene Ontology (GO) terms for the downregulated DEGs were largely related to phosphorylation and kinase regulation, calcium channel activity, synaptic organization and function, and dendrite development, etc.  , Parkinsgregates ,57. Thergregates ,64,65, hgregates ,60,61, Hgregates ,64, but gregates . In addigregates ,55,66,67Our bulk RNA sequencing of the whole SCA1 KI mouse cortex was performed at 12 weeks, as this is considered to be an early to-intermediate stage of disease progression in SCA1 KI mice, with motor and cognitive impairments observed, as well as gliosis and nuclear inclusions, but no major cellular loss in the cerebellum ,27,40,41Lastly, our present study describes pathological and molecular changes in the SCA1 KI mouse cortex and clearly suggests that the cortex is indeed affected in this SCA1 mouse model. Importantly, SCA1 patients also experience a wide range of symptoms related to cortex. These include cognitive impairment and memory decline, specifically executive dysfunction, impaired verbal memory, and decreased visuospatial performance, which tends to correlate with ataxia symptoms ,5,6,7. T


Rod-cone dystrophy (RCD) is the most common inherited retinal disease that is characterised by the progressive degeneration of retinal photoreceptors. RCD genes classification is based exclusively on gene mutations’ prevalence and does not consider the implication of the same gene in different phenotypes. Therefore, we first investigated the mutations occurrence in autosomal recessive RCD (arRCD) and non-arRCD conditions. Then, finally, we identified arRCD enriched mutational patterns in specific genes and coding exons.EYS, IMPG2, RP1L1 and USH2A mutations were enriched in arRCD (p<0.05). The exon-based analysis revealed specific mutation patterns in exons of CRB1, RP1L1 and exons 12, 60 and 62 coding for Laminin EGF and FTIII domains of USH2A.The mutations patterns differed according to arRCD (p=0.001). Specifically, When compared with missense; insertions/deletions , nonsense  and splice-site mutations  increased the OR of arRCD by 20%–60% versus non-arRCD conditions. The gene-based analysis identified that The current analysis showed that many aRCD genes have unique mutational patterns. The current study is the first to investigate all the autosomal recessive rod-cone dystrophy (arRCD) genes to report unique arRCD mutational signatures in exons and genes.Our study has several limitations: (1) Our analysis relied on the number of reported mutations and not the patients carrying them; thus, we could not use the allelic frequencies in all our analysis; (2) No association with specific clinical ocular phenotypes such as the visual field, the electroretinogram and the fundus appearance was performed. Unfortunately, this was not possible in the current study because of the absence of this information; (3) We could not stratify these genotype–phenotype correlations according to the geographic location and (4) For the gene based, 1 test per gene was performed ; thus, a Bonferroni correction might further be used. If applied, USH2A and CRB1 remain highly associated (p<0.001). In the exon-based analysis, one test was performed for all the gene exons, thus abolishing the concern of multiple testing.Rod-cone dystrophy (RCD), also known as retinitis pigmentosa, is an inherited retinal disease (IRD) characterised by the progressive degeneration of the rod and cone photoreceptors.2https://web.sph.uth.edu/RetNet/sum-dis.htm%23A-genes).7The worldwide prevalence of RCD is around 1:4000 individuals.Genotype–phenotype correlations in RCD and other rare diseases have largely been based on cosegregation analysis. Furthermore, RCD genes classification is based exclusively on gene mutations’ prevalence and does not consider the implication of the same gene in different IRDs. Therefore, we first investigated the occurrence of mutations according to arRCD. Then, we searched for specific mutation types highly enriched in arRCD rather than non-arRCD, such as Stargardt disease, Usher syndrome, Leber congenital amaurosis and bestrophinopathies. Finally, we identified unique mutational patterns in specific genes and coding exons.ABCA4, AGBL5, AHR, ARHGEF18, ARL6, ARL2BP, BBS1, BBS2, BEST1, C2orf71, C8orf37, CERKL, CLCC1, CLRN1, CNGA1, CNGB1, CRB1, CYP4V2, DHDDS, DHX38, EMC1, EYS, FAM161A, GPR125, HGSNAT, IDH3B, IFT140, IFT172, IMPG2, KIAA1549, KIZ, LRAT, MAK, MERTK, MVK, NEK2, NEUROD1, NR2E3, NRL, PDE6A, PDE6B, PDE6G, POMGNT1, PRCD, PROM1, RBP3, REEP6, RGR, RHO, RLBP1, RP1, RP1L1, RPE65, SAG, SAMD11, SLC7A14, SPATA7, TRNT1, TTC8, TULP1, USH2A, ZNF408, ZNF513) .The Retinal Information Network (Retnet) is a database that provides tables of genes and loci causing IRDs.Rhodopsin mutations that were reported to have a dominant effect. Furthermore, we have removed the ‘duplicate’ mutations; these are different DNA mutations that lead to the same amino acid (a.a) exchange in a gene. This filtering kept 5868 mutations. For every mutation, we added a type  or splice site) based on the HGMD annotation.The Human Gene Mutation Database (HGMD) is a repository for published gene mutations responsible for human inherited diseases.https://grenada.lumc.nl/LSDB_list/lsdbs/).Similar to HGMD Pro, genetic variations in arRCD genes were also downloaded from the LOVD database .https://www.ncbi.nlm.nih.gov/gene). These databases provided a means to annotate the protein domain and transcript location of each genetic mutation extracted from the HGMD.All a.a domains were retrieved from the universal protein knowledgebase (UniProt)  mutation. In this analysi,s individual mutations but not their frequencies were used to create an integer count or mutation occurrence statistic. As such, if a mutation was associated with disease in more than one person in the database, it was still only counted once. However, if a mutation is genetically heterogeneous—influences more than one trait—then it was counted once for each phenotype studied here. This statistic was defined for three genomic features: (1) ‘global’ or genome wide, (2) ‘genic’ or for each gene of interest and (3) ‘exonic’ or for each exon defined for the longest transcript of each gene of interest. As a sensitivity analysis, the mutations identified in the LOVD databases were also used to derive this mutation occurrence statistic but only in a (1) ‘global’ framework. To test for differences among the arRCD and non-arRCD mutation occurrence statistics, we performed a χ2 and logistic regressions, the null hypothesis of no association was rejected based on p<0.05.The analyses were conducted using SPSS software V.20 (SPSS). All studied variables were expressed as frequencies. The plots were generated using Origin software . χWe first used the RetNet database to identify genes previously known to cause arRCD. Sixty-three genes were found and further investigated, all listed in USH2A were known to cause arRCD. In contrast, >90% of RP1, EYS, CNGB1 and PDE6A mutations were causing arRCD  , p≤0.004ABCA4, BBS1, CRB1, CYP4V2, EYS, IMPG2, RP1L1 and USH2A . In IMPG2, all the InDels and splice sites were enriched in arRCD, whereas two-thirds of the missense mutations were non-arRCD . Noteworthy, these results were replicated in the LOVD database since the latter showed the same trend observed in the HGMD database.USH2A were detected in all syndromic patients with more severe phenotypes than non-syndromic ones;USH2A, Zhu et al reported that individuals with truncating mutations experienced an earlier decline in visual function.Our findings point out that the InDels, nonsense and splice-site mutations increased the OR of USHER syndrome at least twice compared with missense . These fEYS, CNGB1 and PDE6A whose functions appear to be uniquely tied to arRCD, genes such as ABCA4 and USH2A seem to play a broader physiological role because mutations in it are commonly associated with conditions other than just arRCD. A possible explanation for being arRCD ‘specific’ is their expression site, as they are predominantly expressed in the eye and specifically involved in the biology of rods and cones. RP1 is a microtubule-associated protein crucial for photoreceptors' function, It encodes a photoreceptor-specific protein expressed in rods and cones.EYS is the largest gene expressed in the human eye. It is expressed in the human retina with minor expression in other tissues.CNGB1 encodes an ion channel needed for phototransduction by regulating the ion flow into the rod photoreceptor in response to light-induced alterations.PDE6A lead to excessive accumulation of cGMP and subsequent rod, followed by cone photoreceptors death.17The differences in the mutation types between arRCD and non-arRCD phenotypes, directed us to a gene-based analysis to identify the genes responsible for these differences. Of note, this analysis revealed that unlike USH2A has been shown to harbour mutations causing Usher syndrome type II and non-syndromic arRCD. Mutations in exons 12, 60 and 62 coding for Laminin EGF and FTIII domains in USH2A showed an interesting pattern of implication in arRCD: missense mutations were mainly arRCD specific, whereas InDels, nonsense and splice site mutations were abundant in non-arRCD. The mechanisms involved in this phenotypic variation are usually presented as assumptions and only exceptionally rely on proven data.USH2A mutations underlines the complex relationship between the disease-causing mutations and the retinal phenotype, rendering the Mendelian concept of monogenic diseases not applicable for a growing number of diseases.For decades, the genotype–phenotype correlations were based on cosegregation analysis inside small pedigrees. Our study is the first to use statistical tests to investigate the mutations patterns globally, per gene and per exons according to arRCD. On the other hand, this study has several limitations: (1) Our analysis relied on the number of reported mutations and not the patients carrying them; thus, we could not use the allelic frequencies in all our analysis; (2) No association with specific clinical ocular phenotypes such as the visual field, the electroretinogram and the fundus appearance was performed; (3) We could not stratify these genotype–phenotype correlations according to the geographical location and (4) For the gene based, one test per gene was performed ; thus, a Bonferroni correction might further be used. If applied, USH2A and CRB1 remain highly associated (p<0.001). In the exon-based analysis, one test was performed for all the gene exons, thus abolishing the concern of multiple testing.In conclusion, the current approach showed specific mutational patterns specifically enriched in arRCD.


The transient melastatin receptor potential (TRPM) ion channel subfamily functions as cellular sensors and transducers of critical biological signal pathways by regulating ion homeostasis. Some members of TRPM have been cloned from cancerous tissues, and their abnormal expressions in various solid malignancies have been correlated with cancer cell growth, survival, or death. Recent evidence also highlights the mechanisms underlying the role of TRPMs in tumor epithelial-mesenchymal transition (EMT), autophagy, and cancer metabolic reprogramming. These implications support TRPM channels as potential molecular targets and their modulation as an innovative therapeutic approach against cancer. Here, we discuss the general characteristics of the different TRPMs, focusing on current knowledge about the connection between TRPM channels and critical features of cancer. We also cover TRPM modulators used as pharmaceutical tools in biological trials and an indication of the only clinical trial with a TRPM modulator about cancer. To conclude, the authors describe the prospects for TRPM channels in oncology. TRPMs are very different in terms of selectivity, activation, and physiological functions. The members of this family share some common characteristics: 1) they are non-selective calcium-permeable cation channels, and almost all family-members conduct Ca-8, repre2+ in three of these channels, TRPM2, TRPM4, and TRPM8, as well as the PIP2 and other pharmacological compounds in TRPM8 , which contributes to the functional tetrameric assembly , 20. In After the CC sequence, TRPM2 includes an additional motif, NUDT9-H, which has high homology (50% sequence identity) to the mitochondrial NUDT9 ADPR pyrophosphatase. This region, indispensable for channel function, does not show enzymatic activity functioning as an intracellular ligand-binding site for nucleotides , 23. TRP2+ signaling, ion homeostasis, interactomes, and/or kinase activity. Many of the TRPMs are involved in cell/organ sensory transduction processes, including the perception of temperature  and pain  (2+ uptake and reabsorption (TRPM6 and TRPM7)  –32, visid TRPM8) , taste (d TRPM8) , 34, andd TRPM8) . Some ofd TRPM7) , and besd TRPM7) , 37–40. d TRPM7) –47. In t2+, Mn2+, and Mg2+. TRPM3 shows tissue-specific permeability to mono- and divalent cations related to alternative splicing  and encodes five protein isoforms, containing between 1516 and 1643 amino acids, and an intronic microRNA (miR-211) co-expressed with TRPM1 proteins , encodes many protein isoforms, most of which have unknown functionality yet, and, analogously to TRPM1, also encodes a microRNA (miR-204) –57. TRPM2+, and an alternative permeation pathway that mediated monovalent cation current and that involves the voltage-sensing domain of TRPM3 , nifedipine, the synthetic ligand CIM0216, as well as by noxious heat , 31, 40.of TRPM3 , 65. 17βnd Egr-1 –69. Intractivity , 70. Anaberation , 71  and encodes a 1503 amino acid protein in humans  species activate TRPM2 (2+ influx after activation by ROS and responds to RNS releasing adenosine diphosphate ribose (ADPR) from mitochondria and overproducing TNF-α  identified as the physiological sensor of environmental cold  cold temperatures , 98, 99,2+ , encodes an 1165 amino acids protein . TRPM5 irization , 136. Thrization , 138. TRrization , 140.TRPM5 is physiologically activated by intracellular calcium and heat , 141–1432+ and Mg2+ and regulated by intracellular levels of Mg2+ , phospholipase C gamma 2 (PLCγ2), and stromal interaction molecule 2 (STIM2)  –182. TRP (STIM2) , 184. Pr (STIM2) . In addient mode , 186.2+, conductance, and pore structure. In these channels, the different sensitivity to low pH depends on the difference between the number of negatively charged residues in the pore domain, 7 in TRPM7 and 8 in TRPM6 , promoting AKT activation after CaMKIIδ/AKT interaction, cell mobility, colony formation, and tumor growth in an in vivo (xenograft) melanoma cell model  anthranilic acid , TRPM2 showed a mechanism of action similar to that observed in neuroblastoma. TRPM2 deletion in cells provokes a decrease in mitochondrial potential and calcium uptake, with a reduced antioxidant response , autophagy inhibition , and bioenergetic modifications (reduced ATF4 and CREB levels). These effects inhibited leukemia proliferation and increased sensitivity to doxorubicin, an effect that was reversed after reconstitution with TRPM2 (In th TRPM2 .In a Trpm2(-/-) mouse model of AML driven by MLL-AF9, the loss of TRPM2 had neither a substantial effect on the progression of leukemic disease nor a synergistic effect with the cytotoxic therapy . These dN-(p-amylcinnamoyl) anthranilic acid (ACA), a TRPM2 inhibitor, followed by irradiation (IR), reduced the CAMKII phosphorylation and hampered the radiation-assisted inactivation of cdc2, which is dependent on the indicated phosphorylation , compared to the standard control group. Upregulation of TRPM2 channels could stimulate pancreatic cancer processes , independently of the tumor cell type , and of the tumor-bearing mice model. TRPM2 level is significantly increased in PA tissues and negatively correlated to overall survival , 235. Th2O2 addition triggers intracellular Ca2+ increase, decreased autophagy marker LC3-II, and induced apoptotic cell death  compared to the normal prostate epithelium, and its expression fuels in parallel with increasing clinical tumor grade , 236. Inll death . The TRPsurvival . Silencisurvival . Also, tsurvival Table 1.2O2 is Ca2+- and TRPM2-dependent in several tumor cell lines , and inhibition with 2-APB, or silencing through RNAi, reduced tumor cell multiplication Table 1.ll lines . Silenci2+-CAMK2 cascade by oxidative stress resulted in phosphorylation of the BECN1 protein, which ultimately inhibited autophagy in liver cells and promotes cell death , also contributes to GC cell invasion and metastasis  patients has negatively been correlated to the TRPM2 expression . In MKN-ll death . TRPM2, tastasis . TRPM2 ctastasis .2O2 for 24 hours induced an increased number of apoptotic cells, while TRPM2 knockdown inhibited SCC9 survival and migration, independently of the apoptotic p53-p21 pathway (In the tongue carcinoma (SCC) cell line SCC9, it was observed an enhanced expression of TRPM2 located at the nucleus of cancer cells, in contrast with non-malignant human tongue samples. Treatment of SCC9 with H pathway .2+ ion homeostasis  . Low TRP2+ entry (SOCE). In PC3 and DUA145 androgen-insensitive PCa cell lines, silencing the TRPM4 ion channel decreased cell migration, but not proliferation. Stable CRISPR/Cas9-mediated TRPM4 knockdown DU145 cells showed a rounder shape, lower proliferation, migration, and viability, as well as reduced cell adhesion. However, current TRPM4 inhibitors  did not elicit specific TRPM4 effects in DU145 cells, questioning the function of the ion conductivity of TRPM4 in PCa. In line with the HeLa study  . IncreasLa study , decreasLa study . TRPM4 ootential .in vitro, while in vivo restrains tumor growth and metastasis  necessary for contractility and migration . Blanco + channel tetramerization domain 5 (KCTD5) protein expressions are increased in different breast cancer samples. KCTD5 positively regulates TRPM4 activity by enhancing its Ca2+ sensitivity and promoting cell migration and contractility , the overexpression of TRPM4 has been related to characteristic adverse tumor patterns, such as epithelial-mesenchymal transition and hence infiltrative growth . In HCT1henotype . Silencipression , 263. Fupression , 263. TRproteins –266. Genproteins . Functioproteins .Transcriptome analysis on the impact of azacitidine treatment on four acute myeloid leukemia cell lines identified five up-regulated coding genes, among which TRPM4 is the only surface protein up-regulated . In MLL-In patients with CD5+ subtypes of diffuse large B-cell lymphoma (DLBCL), the upregulation of TRPM4 mRNA was related to a poorer prognosis compared to CD5− patients . The TRPSingle nucleotide polymorphisms (SNP) present in TRPM5 (CG or GG genotype) have been associated with a reduced risk of suffering childhood leukemia compared to the CC genotype . In cont2+ homeostasis and its kinase functions , the expression of Mg studies , 275. TR studies , 278. Th studies . Always  studies . A studying path .2+/Mg2+ intake diet (One of the first studies about the implication of TRPM7 in cancer progression informs on the association of an SNP TRPM7 polymorphism (Thr1482Ile) and an increased risk of developing hyperplastic polyps or colorectal adenoma, particularly in patients with a high Caake diet . Recent ake diet , 283.2+/Mg2+ ratio in serum, promoting cell proliferation in PCa  cells and tissues and this upregulation has been correlated with poor survival of patients . The actn in PCa . Knockdon in PCa . TRPM7 wn in PCa . Under hn in PCa .2+ entry, which has no effects on cell viability but was a requisite for cell invasion (2+ entry prompted heat‐shock protein 90α (Hsp90α) secretion, with the subsequent stabilization of both urokinase plasminogen and pro‐MMP2 activator pathways, stimulating the extracellular matrix degeneration and the PDAC cell invasiveness. In consequence, silencing TRPM7 in PDAC cell lines reduced cancer cell invasion  progression and invasion. The over-expression of these channels has been correlated with the increase in tumor size and the advancement of tumor stages, and hence, inversely connected to patient survival , 289. Ininvasion . This Mginvasion . Analogoinvasion . TRPM7 cinvasion , 292. Th2+ entry and involves the channel α-kinase activity, which is required for the phosphorylation of myosin-IIA heavy-chain , the high levels of TRPM7 expression have been interconnected with the Ki67 proliferation index, Scarff-Bloom-Richardson (SBR) cancer grade, and tumor size . High TRvy-chain , 297. TRvy-chain . In thisvy-chain . These rvy-chain , 298.2+]i. TRPM7 silencing also attenuated calcium-related PI3K/AKT activation, which was enhanced by treatment with LY2904002, a PI3K inhibitor  tissues, especially in invasive tissues, and overexpressed in hypopharynx squamous carcinoma (FaDu), tongue squamous carcinoma (SAS), and buccal carcinoma (TW2.6) cell lines, which was associated with poor overall survival rates. The knockdown of TRPM7 negatively regulates the expression of genes and proteins correlated to the calcineurin/NFAT pathway  and reduced the percentage of migrating and invasive SAS cells . Silenciin vitro, and delayed tumor growth in vivo (Different works related TRPM7 upregulation to bladder cancer (Bca) cells’ proliferation, motility, and apoptosis , 306. TR in vivo Table 1.In vitro and in experimental lung metastasis in vivo, when TRPM7 inhibition repressed O-GlcNAcylation, c-Myc and Cav-1, ubiquitination and then their proteasomal degradation increased, therefore inhibiting cell migration and invasion  cell line A549, the TRPM7 channel is over-expressed after stimulation of epidermal growth factor (EGF), and an increased cell migration is observed. TRPM7-knockout attenuates the effects of EGF stimulation . In TRPMinvasion .2+influx/O-GlcNAcylation axis, which directly targets ITGA4 and ITGB7 human integrins, as a potential target against the motility and dissemination of myeloma (MM) cells  cells . Silencitreat MM .2+ concentration, required for optimal kinase activity, which is essential for both productive TRPM7-RhoA interaction and MRTF transcriptional activity. Treatment of HuH7 and HuH6 cells with NS8593, a TRPM7 blocker, strongly reduced proliferation by cell cycle arrest in G1 phase. This fact, together with the increase in senescence-associated ß-galactosidase activity, ERK phosphorylation, GTP loading of Ras, and TNFSF10 and p16INK4a expression, indicated that TRPM7 lack of function contributes to oncogene-induced senescence of hepatocarcinoma (HCC) cells. The same results were obtained in vivo in the corresponding mice model of HCC xenograft, derived from HuH7 cells (Inhibition of the TRPM7/myocardin-related transcription factors A and B (MRTFs) axis is another promising strategy to curb hepatocellular carcinoma (HCC) growth . The expH7 cells .2+, and weak sensitivity to 2-APB inhibition , two soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNARE) located, respectively, in the lysosome and in the autophagosome, and therefore arresting autophagy flux  was also described . In PC3 FGC cells overexpressing the channel, treatment with WS-12 (48 h) in combination with either enzalutamide or docetaxel increases cell death percentage, from about 20% after single treatments to nearly 60%  reached a clinical trial (phase I) for the management of advanced solid tumors  expression. These findings indicate that the TRPM8 channel is essential for sustaining uncontrolled cancer cell growth, by regulating cell cycle phases and replicative senescence  cell lines overexpressed TRPM8 channels, and this fact has been correlated to advanced TNM, vast tumor size, and distant metastasis. Patients having high TRPM8 expression show worse DFS and OS than patients with low TRPM8 levels , 338. Innescence . TRPM8 rnescence . The pho8 tumors .via AKT/GSK-3β pathway activation , could be a possible mechanism behind the cell migration in glioma, induced by TRPM8-mediated Ca2+ influx  isoforms, and phosphatase CDC25C (TRPM8 affects glioblastoma (GBM) cell migration rate by stimulation with specific agonists, such as menthol and icilin, which mediated a substantial increase in [Ca2+]i , 346, wh2+]i . In T98G2+]i Table 1.+ influx . TRPM8 a+ influx , 346, wh2+]i, and decreased cyclinD1, Cdk4 expression, therefore blocking cell cycle in G0/G1, and also p-GSK-3β and p-Akt expression, inhibiting metastasis. In silencing TRPM8 cells treated with epirubicin, the inhibition of Akt-GSK-3β pathway with suppression of FAK and p44/p42 phosphorylation, amplified epirubicin-induced cell apoptosis (The proliferative role of TRPM8 in osteosarcoma (OSS) is demonstrated in osteosarcoma cancer cells lines, MG-63 and U2OS, where TRPM8 is aberrantly over-expressed . TRPM8 kpoptosis . Overexppoptosis .Treatment of several osteosarcoma cell lines , with AMTB, a TRPM8 antagonist, results in suppressed proliferation and metastasis, and induces cellular apoptosis . In 143BTRPM8 is overexpressed at the plasma membrane and in the intracellular region of oral squamous cell carcinoma (SCC), promoting the proliferation of these cancer cells. RQ-00203078, a potent and selective antagonist of TRPM8, strongly inhibits migration and invasion capability of HSC3 and HSC4 SCC lines, markedly reducing clonogenic potential and calcium entry Table 1.Recently, Huang et al. observed that the overexpression of TRPM8 channels increased basal autophagy levels, while TRPM8 knockdown reduced it, in different types of mammalian cancer cells, including cervical cancer (HeLa), breast cancer (MDA-MB-231 and MCF7), embryonic kidney HEK293) and colorectal carcinoma (HCT116) 3 and col.During the last decade, accumulative research indicates the close relationship between the cancer hallmarks and the deregulation of one or more ion channels. TRPM proteins are part of that family that plays a crucial role in the mechanisms of cancer cell proliferation, invasion, and survival. However, forthcoming treatments with TRPM modulators still need more comprehensive and coordinated basic and applied investigations.We think that the recently disclosed structural data, and the new structures that undoubtedly will come soon, will aid in a more rational design of modulators. As proof, we refer to the channels with more information on structural data, including right now an open channel state , and a mTRPM channels may represent key support for the success of more conventional anticancer strategies such as chemotherapy and radiotherapy. By damaging target cells, these therapies trigger many stress responses, such as ion channel activation. Stimulation of sensitizing channels (TRPM2 or TRPM8) can intensify insults provoked by these therapies, whereas modulation of other channels can circumvent intrinsic chemo(radio)resistance.https://atikapharma.com/es/products/alodia-manos). The preparation of TRPM-based biosensors for cancer diagnosis and the development of tumor-targeted nanomedicines containing TRPM modulators, alone or in combination with known chemotherapeutic agents, could also be contemplated soon in this field.Since the TRPM8 receptor is closely related to the control of core body temperature, the possibility of oral or systemic treatments based on TRPM8 antagonists is minimal, as it has been shown with compounds that have reached, but not passed, early clinical trials . HoweverAs described in this review, several miRNA/TRP channel pairs seem to play a key role in tumor biology such as TRPM1, TRPM3, and TRPM4. New therapies based on siRNA and antisense oligonucleotides should be considered in the search for specific anti-tumor targets and modulators centered on TRPM channels .Improved knowledge of the TRPM/receptors interactome as well as on the functional association between different ion channels, is another fundamental aspect to discover cellular pathways operating in TRPM-associated cancers. The search for synergy between channels/receptors/proteins or between different channels involved in the regulation of the same or complementary cancer signaling pathways can be the basis for the design of more effective TRPM modulators.Another issue is finding the most suitable animal model to validate TRPMs as potential targets and translate efficacy and safety discoveries to human studies. It is commonly accepted that integrating relevant data from humans at the early stages of the drug discovery process strengthens the probability of success , 356. InIn summary, the functional modulation of TRPM channels will provide promising opportunities for new antitumor agents’ development, and behind cancer, for peripheral neuropathies and other disabling diseases.All authors have read and agreed to the published version of the manuscript.


PeMKK2a gene was 1571 bp, including a 1068 bp open reading frame (ORF) encoding 355 amino acids, and the putative PeMKK2a protein belongs to the PKc_like (protein kinase domain) family (70–336 amino acids) in the PKc_MAPKK_plant subfamily and contains 62 sites of possible phosphorylation and two conserved domains, DLK and S/T-xxxxx-S/T. Detailed information about its gene structure, sequence similarities, subcellular localization, and transcript profiles under salt-stress conditions was revealed. Transgenic poplar lines overexpressing PeMKK2a exhibited higher activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) than non-transgenic poplar under salt stress conditions. These results will provide insight into the roles of MAPK signaling cascades in poplar response to salt stress.Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction modules, which transmit environmental signals in plant cells through stepwise phosphorylation and play indispensable roles in a wide range of physiological and biochemical processes. Here, we isolated and characterized a gene encoding MKK2 protein from poplar through the rapid amplification of cDNA ends (RACE). The full-length  Soil salinization is also the main obstacle that affects the survival rate of poplar afforestation and the high yield of plantation. A better understanding of poplar salt-stress response mechanisms is crucial for breeding salt-tolerant varieties. Transient activation of the mitogen-activated protein kinase (MAPK) signaling cascades, which plays a necessary role in regulating the active adaptation of organisms to the environment, is one of the early triggered conservative defense responses [As one of the major abiotic stresses, soil salinization is an increasingly serious issue that hampers plant growth and development mainly due to osmotic stress and ion toxicity. Under salt stress, excessive sodium , mitogen-activated protein kinase kinase (MAPKK/MAP2K/MKK), and mitogen-activated protein kinase (MAPK/MPK) . Their aArabidopsis thaliana [Populus trichocarpa contains 21 MAPKs and 11 MAPKKs [Zea mays has 19 MAPKs and 6 MAPKK members [Oryza sativa [A. thaliana can be divided into four groups, and each protein performs different functions in different signal transduction pathways [O. sativa, in A. thaliana, the MEKK1-MKK2-MPK4/6 cascades pathway is activated in response to salt to improve salt-stress resistance [Lycopersicon esculentum [A. thaliana, the stress resistance of mek1 and mek2 single mutants and the activity of MPK4 were not difference compared with those of the wild-type plants. However, in mek1/mek2 double mutants, MPK4-dependent disease-resistance signal transduction pathway was significantly affected [A. thaliana, and MPK11 is also involved in cytokinesis [O. sativa, involvement of the OsMKK3-OsMPK7-OsWRKY30 signaling pathway in response to biotic stresses [OsMFT [There are 20 MAPKs, 10 MAPKKs, and 60 MAPKKKs in thaliana . Populus1 MAPKKs . Zea may members , whereasa sativa . Much kna sativa ,29. The pathways . Among tpathways . The OsMpathways . Unlike sistance . The LeMculentum . In A. taffected . These raffected ,36. AtMEaffected . HINKEL okinesis ,38. In gokinesis ,40. In Ostresses  and the s [OsMFT . Group Cs [OsMFT ,19. MKK7s [OsMFT . At press [OsMFT ,44. The s [OsMFT . ZmMKK10s [OsMFT . Using as [OsMFT .PeMKK2a were generated using an Agrobacterium-mediated leaf disk transformation, and their phenotypic characteristics under salt stress were assessed. These results will provide insight into the roles of MAPK signaling cascades in poplar response to salt stress.Although there have been more studies on MAPK signaling in different herbaceous organisms in recent years, little is known on the involvement of MAPK signaling cascades in poplar response to abiotic stress ,49,50. IPeMKK2a was successfully isolated and identified by the rapid-amplification of cDNA ends (RACE) method, yielding an open reading frame (ORF) of 1068 bp and flanked by 80 bp of 5′-untranslated region (UTR) and 423 bp of 3′-UTR. The exon-intron structure of the PeMKK2a gene was obtained by comparing cDNA and genomic sequences, which has eight exons with lengths of 83 bp, 85 bp, 135 bp, 225 bp, 183 bp, 224 bp, 46 bp, and 84 bp  family (70–336 amino acids) in the PKc_MAPKK_plant subfamily. NCBI BLASTp search for sequences with high homology to PeMKK2a protein in different species. In all the homologous sequences, the similarity in sequence identity with sequences in woody plant species such as xxxx-S/T b, unlikebgroup D a. PeMKK2PtMKK genes in different tissues: one-week-old roots, two-week-old roots, root tips, and stem tips. The homologous genes MKK2-1 and MKK2-2 were highly expressed in all tissues and the most active in the one-week-old roots. Similarly, the expression of MKK10, MKK11-1, and MKK11-2 in the different tissues was very low or even nonexistent. In addition, some genes, such as MKK4, MKK6, and MKK7, were more highly expressed in the stem tips. MKK9 was relatively active in the two-week-old roots  technology. In the roots, stems, and leaves under the salt stress of 100 mM, the expression of PeMKK2a showed an upwards trend over time   a. Howeverol (CK) c, and throl (CK) , which wfor 72 h . It is hCaMV promoter was constructed and transformed into poplar protoplasts. Through microscopy observations, the green fluorescence expressed by the 35S::GFP control vector was distributed throughout nearly the whole cell, and the green fluorescence signal could be observed in the nucleus, cytoplasm, and cell membrane  under the control of the 35S membrane . On the  tabacum . Thus, tPeMKK2a gene in transgenic lines were all higher than those in the non-transgenic lines; the expression level in T4 was the highest, followed by that in T37, and the level in T52 was the lowest among these transgenic lines  fluctuated to a certain extent in the media without NaCl , catalase (CAT), and peroxidase (POD) . Here, wout NaCl . It is sP. trichocarpa were identified according to sequences of 10 MKK A. thaliana. Homologous sequences of AtMKK8 are not present in poplar, which may be a precursor of the AtMKK7/8/9 and PtMKK7/9/11 evolutionary branches caused by ancestral gene duplication events after monocotyledonous and dicotyledonous plant differentiation. Thereafter, PtMKK11-1, PtMKK11-2, and AtMKK8 may drift to a nonfunctional state [MKK8 gene in A. thaliana and P. trichocarpa. Initially, in the study of the 10 MKK proteins sequences in A. thaliana, it was considered that AtMKK10 lacks a correct target for constructing the activation loop and has no actual function. In 2018, it was proven that MKK10 in A. thaliana is a kinase that controls leaf opening by regulating downstream MPK6 [A. thaliana members have functions. The transcriptomic data showed that MKK10, MKK11-1, and MKK11-2 were expressed at low or no levels in various tissues, which may be because only 8 of the 11 MKK members of poplar may have functions.MAPK signaling cascades are indispensable pathways in plant growth and development and in response to stress by gradually phosphorylating intracellular environmental signals and mediating the expression and regulation of functional genes. Here, 11 MKK of al state . To dateeam MPK6 . It is pPeMKK2a gene has an effect on plant growth under salt stress.Under higher concentrations of salt-stress treatments, the CK line samples were inhibited in growth, wilted, or even died, while transgenic lines suffered less damage c. Under A. thaliana protoplasts, and the yeast two-hybrid technique and in vitro protein kinase assays revealed that MKK2 targets MPK4 and MPK6, respectively. AtMPK4 and AtMPK6 are constitutively activated in AtMKK2-overexpressing transgenic plants with enhanced salt tolerance in AtMKK2-deficient mutants, and the activation of AtMPK4 and AtMPK6 was blocked and exhibited hyper-salinity sensitivity [MPK4 and MPK6. The results showed that the expression levels of the MPK4 and MPK6 genes were higher than those of the CK line  in this study were cultivated on Murashige and Skoog (MS) medium (pH 5.8) containing 3% (w/v) sucrose and 0.2% (w/v) Gelrite in a growth chamber  at 25/18 °C (day/night), daily photoperiod of 16/8 h , and relative humidity of 60–80%. Transformation acceptor plantlets (P. davidiana × P. bolleanan) were maintained, transformed, and regenerated as previously described.The sampled plantlets of the elite clone  from 40-day-old plants were harvested and stored at −80 °C until RNA extraction. For the salt-stress treatment, 40-day-old plants were transferred from initial solid MS medium (without NaCl) to liquid MS media containing either 100 mM and 300 mM NaCl for different times: 0, 2, 6, 12, 24, and 72 h. Two treatments of plants were used for growing, as previously described. After treatment, various tissues were harvested from three clonal plants of each treatment at each time point, frozen immediately in liquid nitrogen, and then stored at –80 °C for RNA isolation.TM RT Master Mix Kit .Genomic DNA was extracted from “Nanlin895” poplar using the Plant Genomic DNA Kit  following the manufacturer’s instructions. Total RNA was isolated using RNAprep Pure Plant Plus kit , and the RNA was purified by RNase-Free DNase I . To ensure the quality of the extracted total RNA, the NanoDrop 2000 c ultraviolet–visible spectrophotometer  was used to measure the concentration and purity. Analysis of results that the concentration of the sample should not be less than 500 ng/μL and the OD260/280 value around 2.0. Additionally, the integrity was determined by 1% agarose gel electrophoresis and guaranteed clear bands on agarose gels at 18 S and 28 S, with 28 S approximately twice as bright as 18 S. Qualified RNA samples was reverse transcribed into cDNA using TaKaRa PrimeScriptA. thaliana were used as query sequences for BLAST homology comparisons with P. trichocarpa protein sequences within a database  and the 5′-Full RACE Kit with TAP  were used to amplify the full-length PeMKK2a. The isolated fragment was ligated to a pMD-19T vector and transformed into TOP10. Positive detection and sequencing were performed to obtain the nucleotide sequence of the target fragment. The 3′ RACE and 5′ RACE sequences were compared and spliced, and the NCBI ORFfinder  was used to predict open reading frame (ORF). Afterward, we designed specific primers targeting both ends of the potential ORF , SWISS-MODEL software , SOPMA software , and NetPhos 3.1 software  were used to obtain physicochemical properties of the PeMKK2a protein. The conserved domain of PeMKK2a was determined according to information within the NCBI Conserved Domain Database (CDD). ClustalX v2.1.1 software  was performed multiple protein sequences comparison of MKK2 protein from various plant species, and we manually adjusted these acids sequence by GeneDoc 2.7 software . Figtree v1.4.4 software , was used to construct a systematic evolutionary tree, and transcriptome data (RNA-seq) were used to analyze the expression of the PtMKK gene family. The online software ExPASy ProPARma . The qRT-PCR-specific primers were designed by Beacon Designer 8 software .The transient expression vector was constructed by Gateway technology. The BP reaction was used to ligate the coding region of the Using a well-established poplar leaf pulp protoplast transformation system , we tranPeMKK2a was used to design specific primers  plants. The specific amplification primers used are shown in P. davidiana × P. bolleana and CK plants were placed in semisolid rooting screening media supplemented with 0 mM, 100 mM, 200 mM, and 300 mM NaCl for 2 h, 6 h, 12 h, 24 h, and 72 h, respectively. Three samples were taken as biological duplicates and stored at −80 °C for subsequent physiological index testing after being flash frozen in liquid nitrogen. After the treated transgenic plants were removed from the media, the intact plants were scanned to compare and analyze their phenotypes.After screening for hygromycin B (Hyg) and temetine (Tim), positive identification of transformed We precisely weighed 0.05 g of the above transgenic plant samples and CK samples to prepare tissue homogenate and used a total protein quantitative test kit (BCA method), SOD test kit, CAT test kit, and POD test kit to detect the total protein content, SOD activity, CAT activity, and POD activity, respectively, in transgenic plant samples and the CK samples. We referred to the operating instructions of the kits for all our experimental methods.PeMKK2a gene is a positive regulator of salt tolerance in poplar, consistent with the function of the MKK2 gene in A. thaliana. This result enriches our understanding of the salt-stress signaling pathway in poplar and provides a reference for the study of salt-resistance mechanisms in other woody plants. Meanwhile, the downstream genes involved in the salt-stress pathway were not identified, which provides a guide for our future studies.In our study, we demonstrated that the


The polycomb group (PcG) proteins are a subset of transcription regulators highly conserved throughout evolution. Their principal role is to epigenetically modify chromatin landscapes and control the expression of master transcriptional programs to determine cellular identity. The two mayor PcG protein complexes that have been identified in mammals to date are Polycomb Repressive Complex 1 (PRC1) and 2 (PRC2). These protein complexes selectively repress gene expression via the induction of covalent post-translational histone modifications, promoting chromatin structure stabilization. PRC2 catalyzes the histone H3 methylation at lysine 27 (H3K27me1/2/3), inducing heterochromatin structures. This activity is controlled by the formation of a multi-subunit complex, which includes enhancer of zeste (EZH2), embryonic ectoderm development protein (EED), and suppressor of zeste 12 (SUZ12). This review will summarize the latest insights into how PRC2 in mammalian cells regulates transcription to orchestrate the temporal and tissue-specific expression of genes to determine cell identity and cell-fate decisions. We will specifically describe how PRC2 dysregulation in different cell types can promote phenotypic plasticity and/or non-mutational epigenetic reprogramming, inducing the development of highly aggressive epithelial neuroendocrine carcinomas, including prostate, small cell lung, and Merkel cell cancer. With this, EZH2 has emerged as an important actionable therapeutic target in such cancers. The studied . The sec studied . A third studied ,9. ncRNA studied ,11,12 by studied , and cur studied , are und studied ,16,17,18Otx2 , are repressed during pluripotency by polycomb repressor complex 2 (PRC2), suggesting that epigenetic marks have fundamental roles in developmental decisions [The polycomb group (PcG) and trithorax group (trxG) are large protein multimers that are highly conserved throughout evolution, initially discovered in Drosophila melanogaster as transcriptional modulators of the Homeobox (Hox) ,20,21. Tecisions . More reecisions . In addiecisions . For exaecisions  and in aecisions  affects In this review, we first describe the molecular mechanisms underlying the function and recruitment of PcG proteins in gene regulation during normal development, with a special focus on PRC2. We then summarize the involvement of the PRC2 complex in cell differentiation during development and cancer, with a special focus on the description of the latest discovered mechanisms linking polycomb to cell fate determination, metabolism, and immune response in prostate cancer (PCa), small cell lung cancer (SCLC) and Merkel cell carcinoma (MCC). Moreover, due to the low efficiency of epigenetic modulators as monotherapy, we summarize the results of clinical trials using epigenetic treatments combined with other therapeutical options in cancer. Finally, we provide a deeper insight into the clinical potential of the subsequent inhibition of epigenetic mechanisms in combination with checkpoint inhibitors (CPI) to increase tumor immunity and the success of therapies against tumors.The two main PcG complexes identified in mammals are polycomb repressive complex 1 (PRC1), principally composed of RING1A/B and B lymphoma Mo-MLV insertion region 1 (BMI1), and 2 (PRC2) ,32. ThesThe core PRC2 complex, which is conserved from flies to mammals and plants, is composed by the catalytic subunit enhancer zeste homolog 1/2 (EZH1/EZH2), the scaffolding subunits embryonic ectoderm development (EED) (which contains a WD40 repeat domain that recognizes trimethylated peptides), the suppressor of zeste 12 (SUZ12) (which is a Zinc finger-containing protein), and the retinoblastoma-associated protein (RBBP) 4/7  . InH3K27me2 ,121,122.and E2F2 .Rb1 or Trp53) accompanied by significant chromatin remodeling [Lkb1, Apc and PRC2-related H3K27me3 repressive chromatin marks [Ngfr, Sox2, ΔNp63, and Krt5/6 [As described in the latest Hallmarks of Cancer review, phenotypic plasticity is a critical component of cancer pathogenesis, and the loss of cellular differentiation leads to the acquisition of a set of functional capabilities and a stem cell-like phenotype characterized by uncontrolled growth, intratumoral heterogeneity and metastasis . Therefomodeling ,125,126.modeling ,129,130.modeling ,131,132.modeling ,134 , the gene responsible for the expression of INK4 and ARF proteins, which restrict cellular proliferation [BMI1, which is implicated in the transformation of normal human myoblasts into alveolar rhabdomyosarcoma (ARMS), delays tumor growth via activation of the Hippo pathway by LATS1/2 phosphorylation, leading to reduction in YAP levels and YAP/TAZ target genes [BIM expression [ZEB1 (zinc finger E box-binding homeobox 1) promoter site [p16Ink4a and controls the proliferative potential of pancreas/duodenum homeobox protein 1 (PDX1), positive progenitor cells that accumulate transiently in metaplastic lesions [let-7b expression [CD44 and NOTCH, is crucial to maintaining cancer stem cell phenotypes [AR promoter [In B and T-cell lymphomas, it has been shown that BMI1  acts asferation . Therefoferation ,152. Moret genes ,154. Morpression . In additer site . In panc lesions . In PCa pression ,158. Theenotypes . Initialpromoter ,160. Thepromoter ,162. Propromoter . In summSUZ12 and EED hematopoiesis-specific KO models induce exhaustion in HSC at the fetal or adult stage, rather than hyperproliferation, suggesting that the effect of PRC2 on HSC activity is dosage-dependent [Gata2, Fli1, Myb, and Stat5b [Filaggrin. Moreover, it has been shown that EZH2 expression is downregulated in aged keratinocytes, accompanied by the upregulation of senescence-associated genes, suggesting EZH2 involvement in epidermal aging [PRC has been demonstrated to be critical for the repression of developmental genes to prevent cell differentiation. In fact, it has been reported that the loss of PRC2 in ESCs is enough to induce spontaneous differentiation through the meso-endoderm germ layers without neural ectoderm . Genome-ependent ,169,170.ependent ,172. Morependent ,174,175.d Stat5b . Thus, tal aging . Howeveral aging .MYCN  and AURKA (Aurora-A) [PTEN, TP53, and RB1 [RB1 and TP53 in treatment resistance. The gene expression analysis of triple KO mouse tumors and human PCa patient samples with these mutations revealed altered expressions of E2F target genes and neuroendocrine lineage genes, together with increased expressions of stemness and epigenetic reprogramming-related genes, such as SOX2 and EZH2 [FOXA1 binding site, related to the promotion of neuroendocrine lineage-defining genes [ASCL1, SOX2 and NEUROD1 expression are shared master regulators between neuroendocrine cancers. ASCL1 and NEUROD1 expressions are mutually exclusive, and are drivers of intra-tumoral heterogeneity, a program that occurs due to distinct H3K27ac genome-wide de novo deposition in SCLC and PCa [In PCa, CRPC tumors can continue the transition to more aggressive disease states via lineage plasticity, and adopt a phenotype no longer reliant on AR expression and signaling. It has been reported that the EZH2-T350 phosphorylation and consequent AR interaction plays an important role in the transition and differentiation from CRPC to more aggressive PCa phenotypes . Therefourora-A) ,184,185.urora-A) ,186,187, and RB1 , suggestand EZH2 ,190. In ng genes . Recentlng genes . In addi and PCa ,193. TheEZH2-conditional KO mice [E-Cadherin expression as well as the tumor suppressor p16INK4A [E-cadherin promoter and deacetylates histone H3, which leads to reduced RNA polymerase II binding, leading to E-cadherin transcriptional loss [PcG proteins suppress alternate cell lineages when cells commit to differentiation by activating specific gene loci, and stem cells take on a new fate . In this KO mice . EMT is  KO mice , and occ KO mice  and is a KO mice ,199,200. KO mice , while Bp16INK4A . Moreovenal loss . In geneIn addition to the direct impact of epigenetic changes on tumor cell growth and survival, it has been shown that epigenetic changes in tumoral cells mediate the immune responses of cells presented in the TME against the tumor. These mechanisms promote an immunosuppressive network, including the loss of expression of tumor-associated antigens or neoantigens, the impairment of cell surface antigen presentation, changes in the expression of immunosuppressive molecules and proinflammatory cytokines, and the aberrant expression of proteins associated with checkpoint pathways such as programmed cell death protein 1 (PD-L1) ,204,205.MHC-I genes are silenced by the recruitment of EZH2 by lincRNA EPIC1, inducing PD-1 treatment resistance [EZH2 and PD-L1 was reported, which may result from the upregulation of H3K27me3 levels in the promoters of CD274 and IRF1 [PD-L1 expression, indicating that EZH2 is involved in T-cell exhaustion mechanisms [+ T-cells, and this recruitment is mediated by CXCL10-CXC chemokine receptor 3 (CXCR3) [EZH2 is negatively associated with major histocompatibility complex (MHC) class I expression in different types of cancer . In addisistance . These rand IRF1 ,213. Conchanisms   ,219,220.CCL2, correlating with low macrophage infiltration [TNF-α, Nos2, and IL-6) with the concurrent loss of M2 TAMs (characterized by the expression of Arg1 and CD206) [Further, the polarization of tumor-associated macrophages (TAMs), which play an important role in tumoral development, is influenced by cytokines and chemokines derived from tumoral cells A. Ongoinltration , and proltration . Moreoved CD206) ,226  and T-cells, macrophages, myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs) and natural killer (NK) cells. Such chromatin remodeling within immune cells is demonstrated to promote the gradual loss of mature antigen-presenting functions and cytotoxic activity ,228. In + T-cells, the expression of transcriptional factors T-bet and GATA3, and Th1 and Th2 cytokines such as IFN-γ, IL-4 and IL-5, is repressed by EZH2, accompanied by the consequent inhibition of Th1 and Th2 polarization , we have seen loss-of function mutations and deletions affecting EZH2 and SUZ12, leading to the hypomethylation of H3K27 target genes, including Notch, thereby contributing to oncogenesis. In addition, 25% of the T-ALL present global epigenetic remodeling towards H3K27ac, which activates mutations of the JAK/STAT signaling pathway [Accumulating experimental evidence has shown that EZH2 mediates transcriptional regulation in T-cells, contributing to their development, divergent cell functions and proliferation, and may act as an immunomodulatory factor ,144,230.eview in ) , ENO2 and PCK2, are among the PRC-active target genes [EZH2 depletion in PCa inhibited aerobic glycolysis, which was accompanied by the upregulation of miR-181b and lipoprotein-dependent lipid accumulation via the induction of ApoE expression in adipocytes [PGC-1α via the interaction between EZH2 and the mutant telomerase reverse transcriptase (TERT) [Tumoral cells have a high level of metabolic requirements, different from those of normal cells, and upon tumoral growth the normal vasculature is not able to supply sufficient nutrients and oxygen. In the last decade, increasing evidence has suggested that epigenetic and metabolic alterations in cancer cells are interconnected and directly impact tumoral development and TME . Under aet genes . Supportet genes . In addiet genes ,251, andet genes ). For exipocytes . In glioe (TERT) . Therefoe (TERT) ,253.+ ratio, and promotes NAD(H)-sensitive transcriptional binding with the corepressor CtBP to p300, which blocks the binding of p300 and NF-κB with the proinflammatory gene promoters through the regulation of p65/RelA acetylation [+T-cells under tumoral conditions present disrupted methionine metabolisms and high expression levels of the transporter SLC43A2. Moreover, low intracellular levels of methionine and the methyl donor SAM in CD8+ T-cells were observed, and resulted in the loss of H3K79me2, which decreased STAT5 expression levels and impaired T-cell immunity [Like other cells, in immune cells, changes in the metabolism can lead to alterations in their functions and characteristics . In the tylation . In gliotylation , and Yantylation . In additylation . Recentlimmunity . In addiimmunity . In concDue to the key roles played by PRC2 in the pathophysiology of cancer development, controlling cellular plasticity in both tumoral and immune cells, several small molecules that modulate methyltransferase activity have been developed. The first pharmacological agents released directly targeted EZH2 activity. Since 2012, several potent and highly selective S-adenosyl-methionine-competitive inhibitors of EZH2 methyltransferase activity have been developed, and over 35 clinical trials have been initiated in different types of cancer (reviewed in ). HoweveEZH2 gene expression, and PRC2 target genes are broadly H3K27-trimethylated [the FDA in January 2020 for adults and pediatric patients with epithelioid sarcoma not eligible for complete resection, and patients with relapsed or refractory follicular lymphoma [MHC-I, despite TNBC cells being only mildly sensitive to PRC2 inhibition. EZH2 and EED inhibition restored MHC-I expression and enhanced the efficacy of chemotherapy in mouse and in vitro models [+ T-cells, and increased M1 tumor-associated macrophages [Different clinical trials have been undertaken to evaluate the efficacy of EZH2 inhibitors, dual agents, EED inhibitors and PRC2 degraders (thylated . The firlymphoma ,269,270.lymphoma . In addilymphoma ,273,274,lymphoma . Moreovelymphoma . These slymphoma . Thus, so models . In addio models . Theraperophages . These srophages . This strophages , providirophages ,277,278 rophages .In this review, we have summarized emerging evidence showing that EZH2 is expressed in cancer, and most of the immune cells forming the TME. These discoveries suggest that immunotherapy resistance could be associated with the coordinated silencing of genes involved in antigen processing, macrophage polarization, T-cell reprograming and cytokine secretion, which can be reversed by EZH2 inhibitors. Therefore, the combination of drugs targeting the pro-oncogenic role of EZH2 in tumoral cells and its role promoting an immunosuppressive TME, with immunotherapy, could have a synergistic effect, improving the therapeutic responses of some types of cancer. Despite preclinical studies in hepatocellular carcinoma , PCa 20, head an


There are diverse reports of the dual role of sirtuin genes and proteins in breast and prostate cancers. This review discusses the current information on the tumor promotion or suppression roles of SIRT1–7 in breast and prostate cancers. Precisely, we highlight that sirtuins regulate various proteins implicated in proliferation, apoptosis, autophagy, chemoresistance, invasion, migration, and metastasis of both breast and prostate cancer. We also provide evidence of the direct regulation of sirtuins by miRNAs, highlighting the consequences of this regulation in breast and prostate cancer. Overall, this review reveals the potential value of sirtuins as biomarkers and/or targets for improved treatment of breast and prostate cancers.Mammalian sirtuins (SIRT1–7) are involved in a myriad of cellular processes, including apoptosis, proliferation, differentiation, epithelial-mesenchymal transition, aging, DNA repair, senescence, viability, survival, and stress response. In this review, we discuss the current information on the mechanistic roles of SIRT1–7 and their downstream effects (tumor promotion or suppression) in cancers of the breast and prostate. Specifically, we highlight the involvement of sirtuins in the regulation of various proteins implicated in proliferation, apoptosis, autophagy, chemoresistance, invasion, migration, and metastasis of breast and prostate cancer. Additionally, we highlight the available information regarding SIRT1–7 regulation by miRNAs, laying much emphasis on the consequences in the progression of breast and prostate cancer. Caenorhabditis elegans [Drosophila melanogaster [The silent information regulator 2 (sir2) family of proteins, simply known as sirtuins (sir-two-ins), was first discovered in yeast, where its upregulation increased lifespan . Consistnogaster . As membnogaster ,5. By trnogaster ,7  have been identified, which primarily function as NAD-dependent deacetylases (SIRT1–3 and SIRT5–7)  and ADP-By virtue of the above enzymatic functions, mammalian sirtuins are involved in a myriad of biological processes, including apoptosis, proliferation, differentiation, epithelial-mesenchymal transition (EMT), aging, DNA repair, senescence, viability, survival, and stress response, among other biological functions ,31,32,33Given that cancer is an increasing cause of death worldwide, numerous research efforts have been channeled toward understanding the molecular basis of the disease to develop effective treatments. In this regard, several studies have explored the role of sirtuins in the progression, migration, and metastasis of various types of cancer ,36,37,38Sirtuins are reported to play a dual role in the pathogenesis of breast and prostate cancer via the targeting of different proteins, including transcription factors, signaling molecules, and catalytic proteins. Consequently, sirtuins have exhibited both tumor-suppressing and tumor-promoting effects in breast and prostate cancer cells . In thisSIRT1 gene silencing in BT474 breast cancer cells, suggesting that SIRT1 deacetylates and inhibits FOXO3 activity to promote breast cancer [SIRT1 gene silencing increases BT474 cells’ sensitivity to lapatinib treatment, further suggesting that SIRT1 promotes BT474 cells’ resistance to lapatinib treatment. The protein expression of PRRX1, a transcription factor that induces epithelial-to-mesenchymal transition (EMT) and inhibits cancer stemness, was shown to be downregulated by SIRT1 gene silencing in mouse 4T1 breast cancer cells [SIRT1 gene silencing upregulated the expression of the EMT-related protein E-cadherin and cancer stemness markers kruppel-like factor 4 (KLF4) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) [cyclin G2 and p53, in estrogen-dependent breast cancer cells [Recent studies have revealed that SIRT1 is constitutively upregulated in breast cancer cells ,50,58. At cancer . They aler cells . InteresALDH1A1) . Based oALDH1A1) . Moreoveer cells .SIRT1 has been demonstrated to modulate several intracellular signaling protein molecules, including protein kinase B (PKB), B-cell lymphoma 2 (Bcl-2), metadherin (MTDH), Frizzled 7, EMT-related proteins, and the cluster of differentiation 36 (CD36), in breast cancer cells. In a study by Jin et al., SIRT1 was shown to directly interact with and phosphorylate PKB to promote subsequent downstream proliferative effects in MDA-MB-231 and BT-549 breast cancer cells . To subsInhibition of SIRT1 with either EX-527 or SIRT1 siRNA resulted in the upregulation of c-Myc (a pro-oncogenic protein) and MTDH (an oncogenic protein) in MDA-MB-231 and BT-549 breast cancer cells. Further, in the presence of c-Myc siRNA, EX-527 could not upregulate MTDH in breast cancer cells . As a meCYP19A1 (a gene encoding the aromatase enzyme) was downregulated in MDA-MB-231 breast cancer cells expressing aromatase following the inhibition of SIRT1 by treatment with both cambinol and inhibitor VII [CYP19A1 expression. Additionally, another study demonstrated that the knock-out of SIRT1 by shRNA reduced the proliferation, migration, and invasion of MCF-7 breast cancer cells [Interestingly, SIRT1 has also been demonstrated to regulate the expression of certain catalytic proteins in breast cancer. For instance, the transcription of itor VII . This fier cells . The shRer cells . The autAlthough previous studies have demonstrated that SIRT6 expression is downregulated in several human cancers , includiThe mRNA expression level of SIRT7 was found to be higher in patients with metastatic breast cancer . More reSeveral studies have shown that SIRT1 is highly expressed in prostate cancer ,118,119,E-cadherin gene silencing and EMT of tumor cells.The tumor-promoting effects of SIRT1 in prostate cancer have been demonstrated through the regulation of EMT-related protein expression. A 2012 study revealed that SIRT1 promotes the migration and metastasis of DU145 and PC3 prostate cancer cells by downregulating the expression of the epithelial marker E-cadherin and upregulating the expression of mesenchymal markers (N-cadherin and fibronectin) and the EMT-inducing transcription factor ZEB1 . SimilarIt is well-established that androgen receptors (AR) play a pivotal role in prostate carcinogenesis . In thisSIRT1 has been shown to modulate catalytic protein activity as a part of its mechanisms of action in prostate cancer. Notably, it has been revealed that SIRT1 deacetylates and upregulates the expression of matrix metalloproteinase-2 (MMP2) , a zinc-Unlike in other types of cancer, including breast cancer, SIRT6 is overexpressed in prostate tumor tissues and cells , with high SIRT6 expression correlating with poor overall survival of prostate cancer patients ,103,104.SIRT7 is overexpressed in various human cancers , includiMore recently, Ding et al. demonstrated that SIRT7 depletion in LNCaP and 22RV1 prostate cancer cells significantly reduces the conversion of LC3B-I to LC3B-II, a vital process that results in autophagy, and that SIRT7 knockdown downregulates vimentin, slug, MMP2, and MMP9 in 22Rv1 prostate cancer cells . They fuStudies have shown that SIRT2 expression is downregulated in breast cancer tissues compared to normal breast tissues ,132,133.Conversely, Park et al. demonstrated that SIRT2 deacetylates the lysine 305 (K305) residue of the pyruvate kinase M2 (PKM2) isoform to direct glycolysis and promote tumor growth . To corrUnlike in breast cancer, SIRT2 was previously shown to be highly expressed in prostate cancer cells compared with normal prostate cells . HoweverThe primary mitochondrial-localized deacetylase SIRT3 is known to play a vital role in mitochondrial metabolism . Loss ofSIRT3 gene is deleted in approximately 20 percent of human cancers and 40 percent of breast cancers [SIRT3 gene is deleted in breast cancer more than in other cancers [The expression of SIRT3 is decreased in human breast cancers ,149. To  cancers . Armed wde novo overexpression of SIRT3 downregulates the expression of vegfr1 (a proangiogenic protein involved in cell proliferation), EMT markers (vimentin and slug), lactate dehydrogenase A , peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), SIRT1, superoxide dismutase 2 (SOD2), and catalase (CAT) in MCF-7 breast cancer cells, thus indicating a tumor suppressor effect of SIRT3 in breast cancer [Additionally, Zou et al. demonstrated that the loss of SIRT3 (following treatment with SIRT3 shRNA) in MCF-7 breast cancer cells resulted in increased acetylation at lysine 413 of isocitrate dehydrogenase 2 (IDH2), a key enzyme in the Krebs cycle that oxidizes and decarboxylates isocitrate into α-ketoglutarate, and that SIRT3 loss decreases the level of IDH2 dimerization . The autt cancer . Supportt cancer .Intriguingly, another recent study showed that while SIRT3 expression improved mitochondrial mass and potential metabolism (Warburg effect) and increased SOD2 and CAT expression, it also increased mitochondrial ROS, DNA damage, and expression of tissue inhibitor of metalloproteinase 1 (a major inhibitor of MMP9), which have been shown to promote malignant progression and metastasis . FurtherFurther supporting the tumor suppressor role of SIRT3 in breast cancer, SIRT3 suppresses estrogen-induced cytosolic and mitochondrial ROS production, diminishes estrogen-induced DNA synthesis, and upregulates p53 expression in MCF-7 breast cancer cells , despiteTo date, only one study has shown a possible tumor-promoting effect of SIRT3 in breast cancer. SIRT3 knockdown in MCF-7 cells by siRNA increased ROS production (196%), formation of autophagic bodies (47%), and apoptosis (23%); it also decreased MnSOD (20%), IDH2 (50%), PGC1α (12%), and mitochondrial transcription factor A (27%) levels and reduced cell viability (20%) . These fLike other mitochondrial sirtuins, SIRT4 is a mitochondrial deacetylase involved in energy metabolism . HoweverIn breast cancer, the expression pattern of SIRT4 is controversial. While some studies report that SIRT4 expression is upregulated in breast cancer ,158, othMYC and CNDD1), to enhance the sensitivity of MCF7 and T47D breast cancer cells to tamoxifen, thus potentiating the effect of this chemotherapy against breast cancer cells [BRCA1 expression at both mRNA and protein levels [On the contrary, another study demonstrated that SIRT4 overexpression downregulates IL-6 expression and STAT3 Y705 phosphorylation, as well as the transcription and translation of STAT3 target genes  . The autImportantly, an in vivo study of SIRT5 knockout MMTV-PyMT mice supported the tumor-promoting effect of SIRT5 in breast cancer. SIRT5 knockout MMTV-PyMT mice had higher survival and reduced tumor size and lung metastases compared to the SIRT5 wild-type MMTV-PyMT mice . The autThe expression of SIRT3 in prostate cancer remains controversial. SIRT3 expression has been reported to be decreased in prostate cancer by two studies: Quan et al.  found thFOXO3A target genes [Supporting a tumor suppressive function, SIRT3 expression in C42B and PC3 prostate cancer cells downregulated the expression of c-Myc , a well-et genes . Put toget genes .In contrast with the above-mentioned studies indicating the tumor suppressor function of SIRT3 in prostate cancer, Singh et al. demonstrated that SIRT3 expression inhibits the cleavage of poly (ADP-ribose) polymerase , a knownUnlike in other human cancers, including breast cancer, the expression pattern of SIRT4 and its mechanistic roles in prostate cancer have been sparsely investigated. Nevertheless, a study found that SIRT4 suppresses the proliferation of DU145 prostate cancer cells by inhibiting the uptake of glutamine , a metabAs in breast cancer, SIRT5 was found to be highly expressed in prostate tumor tissues compared to normal prostate tissues . HoweverConversely, a more recent study demonstrated that SIRT5 expression reduces the migration and invasion of PC3 prostate cancer cells . This walin-4 was reported to exist in Caenorhabditis elegans [lin-4 was not an RNA-encoded protein but a non-coding RNA [lin-4 post-transcriptionally downregulates lin-14 (a gene found in Caenorhabditis elegans) through its 3′ untranslated region (UTR) and that the 3′UTR of lin-14 was complementary to that of lin-4 [lin-4 post-transcriptionally regulates lin-14 [In 1993, the first miRNA  elegans ,175. Aftding RNA ,177. Furs lin-14 . Followis lin-14 .miRNAs are small non-coding RNAs composed of an average of 22 nucleotides that suppress gene expression by interacting with the 3′ UTR of target mRNAs. Moreover, several studies have demonstrated that miRNAs repress mRNA translation and induce mRNA deadenylation and decapping by binding to the 3′ UTR of their target mRNAs ,180. In miRNAs have been implicated in normal biological processes ,185,186,Reportedly, approximately 30% of genes are regulated by miRNAs. Consistent with this report, the regulatory functions of miRNAs in the progression, metastasis, and drug-resistance of breast tumors and other cancers have been documented ,205. AltSeveral miRNAs have been found to regulate SIRT1 expression in breast cancer, serving as either tumor suppressors or tumor promoters through an indirect mechanism, depending on their effect on SIRT1 expression. For instance, Eades et al. demonstrated that miR-200a epigenetic silencing contributes, at least in part, to the overexpression of SIRT1, and the SIRT1 transcript is subject to regulation by miR-200a via SIRT1 3′-UTR . They fuFurthermore, a study demonstrated that miR-22 is downregulated in MCF-7 and MDA-MB-231 breast cancer cells, and the overexpression of miR-22 or SIRT1 knockdown induces apoptosis and decreases the survival of breast cancer cells . The autBased on our literature search, there is limited information on the regulation of SIRT7 by miRNAs in breast cancer. Being the only available data at the time of our literature search, Li and Li  demonstrAlthough several studies have investigated the role of miRNAs, including miR-1, -21, -106b, -141, -145, -182, -187, -205, -221, -375, -490-3p, and -Let-7c in the prognosis, diagnosis, and progression of prostate cancer ,228,229,miR-212 was found to be downregulated in prostate cancer tissues compared with normal prostate tissues . Its expSo far, most of the above-mentioned studies suggest that miRNAs directly interact with SIRT1 to potentiate their tumor-suppressing effect in prostate cancer. On the contrary, Yang et al. demonstrated that miR-221 and miR-222 are highly expressed in PC3 compared to LNCaP prostate cancer cells and do not target or directly interact with SIRT1 mRNA via its 3′UTR . InteresThis extensive review of the current knowledge on the mechanistic roles of nuclear sirtuins , cytoplasmic sirtuins (SIRT2), and mitochondrial sirtuins  has shown that breast and prostate cancer cells or tissues portray different expression patterns of the various types of sirtuins to aid their tumorigenic phenotypes and potentiate their progression through various carcinogenic stages. Furthermore, we have also shown that sirtuins regulate various proteins to promote or suppress breast and prostate cancer. Specifically, we highlighted that sirtuins regulate various proteins implicated in proliferation, apoptosis, autophagy, chemoresistance, invasion, migration, and metastasis of both breast and prostate cancer. To further illustrate the relevance of sirtuins expression in breast and prostate cancer, we have provided available evidence of the direct regulation of sirtuins by miRNAs (via direct interaction with sirtuins mRNAs), highlighting the consequences of this regulation in breast and prostate cancer. While many studies have begun to investigate the specific mechanisms by which sirtuins influence either breast or prostate cancer, no study has investigated both in the same study. As a result, the specific investigation of either of these cancers has focused on different key genes and/or proteins involved in various molecular pathways. Comparing