One of the most urgent needs in AML is to improve the disease cure rate as relapse still occurs in 60–80% of patients. Recent evidence suggests that dismal clinical outcomes may be improved by a better definition of the tight interaction between the AML cell population and the bone marrow (BM) microenvironment (“the niche”); the latter has been progressively highlighted to have an active role in the disease process. It has now been well established that the leukemic population may misinterpret niche-derived signals and remodel the niche, providing a shelter to AML cells and protecting them from the cytotoxic effects of chemoradiotherapy. Novel imaging technological advances and preclinical disease models have revealed that, due to the finite number of BM niches, leukemic stem cells (LSCs) and normal hematopoietic stem cells (HSCs) compete for the same functional areas. Thus, the removal of LSCs from the BM niche and the promotion of normal HSC engraftment should be the primary goals in antileukemic research. In addition, it is now becoming increasingly clear that AML-niche dynamics are disease stage specific. In AML, the niche has been linked to disease pathogenesis in the preleukemic stage, the niche becomes permissive once leukemic cells are established, and the niche is transformed into a self-reinforcing structure at a later disease stage. These concepts have been fostered by the demonstration that, in unrelated AML types, endosteal vessel loss occurs as a primary AML-induced niche alteration, and additional AML-induced alterations of the niche and normal hematopoiesis evolve focally and in parallel. Obviously, this endosteal vessel loss plays a fundamental role in AML pathogenesis by causing excessive vascular permeability, hypoxia, altered perfusion, and reduced drug delivery. Each of these alterations may be effectively targeted by various therapeutic procedures, but preservation of endosteal vessel integrity might be the best option for any future antileukemic treatment. In AML, there is an urgent need to develop more effective treatments as the current standard of care chemotherapy cures only 40–45% of younger and 10–20% of older patients . An earlα4β1 integrin), CD44, E-selectin, and CD98) together with the exploitation of the aforementioned competition between normal and leukemic stem cells for the same BM niches might be an effective therapeutic strategy in patients with minimal residual disease (MRD) at the time of hematopoietic stem cell transplantation (HSCT). Among the adhesion molecules responsible for normal and leukemic stem cell homing to BM niches, CXCR4 which binds to CXCL12 appears to be the most relevant one as it controls migration, mobilization, homing, and retention of LSCs within the BM and activates apoptosis that is prevented by an as-yet-undefined soluble factor produced by osteoblasts [The demonstration that the number of functional niches within the BM is limited and that niche occupancy is the limiting factor in transplantation of normal HSPCs is the basis for the notion that LSCs and HSCs might share and compete for the same BM niches , 16, 17.eoblasts –24. The eoblasts –27. Thuseoblasts , 29 fromeoblasts , 30. Thieoblasts . Phase Ieoblasts . Importaeoblasts . Thus, fβ1. Recent in vitro studies have revealed that a CD98 antibody significantly reduces adhesion and the colony-forming ability of primary AML cells, and in experimental murine models, it significantly reduces the AML tumor burden [Mobilization of LSCs might also be achieved by targeting the VCAM1/VLA4 axis, which mediates the binding of AML cells to endothelial cells with the development of a quiescent leukemic cell phenotype. These AML cells are physically and functionally integrated within the endothelium but may still cause relapse . Howeverr burden .β2 adrenergic signaling with loss of niche quiescence. In particular, this loss is due to the expansion of mesenchymal stromal progenitor cells committed to osteoblastic differentiation and the reduction of arteriole-associated NG2+ niche cells. Thus, a potential therapeutic approach could be to circumvent this β2 adrenergic receptor damage with receptor agonists. However, to date, these last drugs have provided contradictory results.Sympathetic nervous system (SNS) neuropathy is another mechanism that in the MLL-AF9 AML model may lead to leukemia progression . These lThe BM vascular niche and the major interactions between its cellular components are summarized in α and 2α (Hif-1α and Hif-2α) and the PI3K/Akt/mammalian target of the rapamycin (mTOR) signaling pathway, which provides environmental prosurvival cues to leukemic cells. It was demonstrated that Hif-1α and Hif-2α through their interaction with the “hypoxia responsive elements” (HREs) of various genes  may upregulate the expression of CXCR4 and CXCL12 on AML cells and endothelial cells, thereby promoting LSC maintenance and disease aggressiveness [α, which is stabilized by the AML-associated mutations of the isocitrate dehydrogenase (IDH) 1 and IDH2 genes, is to make leukemic cells completely dependent on mitochondrial oxidative phosphorylation to fulfill their low energy requirements as they are unable to employ glycolysis [in vitro cultures of chemosensitive and chemoresistant AML stem and progenitor cells and inhibited leukemic progression in in vivo murine models [L or MCL-1 and the presence of IDH1/IDH2 mutations as the best predictors of response [β activation and MCL-1 degradation, hence overcoming venetoclax resistance [Evidence coming from rat models has sufficiently demonstrated a close relationship between BM hypoxia and AML cells. In particular, it was demonstrated that AML cells preferentially infiltrate BM hypoxic areas, which expand on leukemia engraftment and disease progression . In addisiveness –48  or microvesicle (larger vesicles produced by the shedding of the plasma membrane) release , 67. miRent mice . Anotherent mice . This trent mice , might bent mice . Reciproent mice . These dent mice . Currentsurvival . Even mosurvival , 76. Thesurvival  are ablesurvival . An LSC survival , 80.versus a significant increase of those associated with arterioles, a scenario that leads to the generation of several hypoperfused BM areas. At an early AML stage, these hypoxic areas, which appeared leakier than areas nonengrafted by AML cells, were localized near AML cells, whereas at a later disease stage, they were scattered throughout the entire BM [α stability and activity, thereby promoting the expansion of endosteal vessels and increasing the number of HSCs within the trabecula-rich metaphysis. However, DFO did not modify the number of BM AML cells and did not improve the survival and disease progression rate unless it was combined with chemotherapy. Interestingly, recent evidence suggests that DFO by modulating reactive oxygen species (ROS) pushes the differentiation of leukemic blasts and normal BM precursors towards monocytes/macrophages. This observation points to a strong role of ROS in endosteal vascular niche remodeling [Various studies have reported that, in murine AML models and AML patients, the number of sinusoidal vessels within the BM central region ) is increased –83. It hntire BM . Anotherntire BM . Moreoventire BM . Thus, intire BM  and to fntire BM . In ordemodeling . In normmodeling  in respoHSCT, which remains the only curative treatment for most AML patients, can be considered an immunological procedure since it causes the potential eradication of recipient LSCs through various immunological mechanisms  cells and production of an inflammatory milieu) that constitute the so-called graft-versus-leukemia (GVL) effect. Peptides presented by self-HLA are recognized as targets by T cells, whereas activation of NK cells depends on the absence or downregulation of self-HLA class I molecules. These two different types of recognition have evolved as complementary strategies to ensure immunological recognition of virus-infected cells in which downregulation of self-HLA class I molecules represents an important mechanism to avoid immunological recognition by virus-specific T cells and promote viral persistence , 90. HowV617F myeloproliferative neoplasms. In these disorders, STAT3 and STAT5 phosphorylation by the oncogene JAK2V617F enhances PD-L1 expression by increasing PD-L1 promoter activity. T cells that interact with these JAK2V617F mutant cells have reduced cell-cycle progression and metabolic activity, explaining why these myeloid cells can escape immune responses [α (SIRPα) expressed on APCs limits their activity and blocks their antigen uptake [+ T-cell and lower Treg counts. Thus, further studies with ipilimumab in AML relapsed after transplant are currently ongoing to establish the effectiveness of this therapeutic strategy in preventing posttransplant immune escape.In addition, LSCs can upregulate the expression of coinhibitory molecules in order to block T-cell activation . Physiolesponses , 96. A sn uptake . In thesn uptake . Howevern uptake . A highen uptake . In patiβ that reduce the expression of MHC class II molecules, an event that renders leukemic cells less immunogenic [γ (IFN-γ), IL-15, IL-1β, and granulocyte colony-stimulating factor (G-CSF), are produced by normal myeloid and lymphoid progenitors and improve leukemic cell recognition and immune cell activation. Therefore, it is reasonable for leukemic cells to block the production of these proinflammatory cytokines, as demonstrated by laboratory and clinical data. In a clinical study performed in 393 patients with B-lineage ALL, lower IFN-γ levels were associated with a younger age at diagnosis and a high-risk profile defined by prednisone response, cytological remission, and minimal residual disease. This finding supports the role of IFN-γ in immunosurveillance and its effect on the early response to steroid therapy [+ T cells and NK cells. More importantly, AML cells carrying the internal tandem duplication of the FLT3 gene (FLT3-ITD) are able to block IL-15 production. In animal studies, FLT3 inhibition together with T-cell transfer promoted AML cell elimination and helped achieve long-term disease control [Another mechanism of LSCs to evade immune control relies on the production of anti-inflammatory cytokines that create an anti-inflammatory microenvironment able to block the immune response by hindering leukemic cell recognition and destruction. Several studies performed in the nontransplant setting have shown that CML cells can produce IL-4 and TGF-unogenic –103. IL-unogenic , 105. Pr therapy . IL-15 i therapy –110. IL- therapy –113. Bas therapy . Apart f control . IL-15 pβ. Further studies are needed to clarify the mechanisms by which these inflammatory cytokines help the immune system recognize and eliminate leukemic cells in order to develop therapeutic strategies. However, it is known that these cells can also evade immune surveillance by producing various immunosuppressive enzymes. One of these, indoleamine 2,3-dioxygenase 1 (IDO1), is responsible for the first step of tryptophan degradation and subsequent kynurenine production. Both the lack of tryptophan and the presence of kynurenine negatively affect T-cell functions and reprogram Treg activity [Moreover, it can be speculated that other mechanisms of immune escape may rely on the block of several proinflammatory cytokines, such as G-CSF (which is involved in APC maturation) and IL-1activity . IDO1 isactivity . Arginasactivity , 117. Ecactivity , 119.AML treatment has remained unchanged over the past twenty-five years and is frequently associated with dismal outcomes. This has not changed with the development of molecular drugs that are mutation specific, as they do not address the genetic heterogeneity of the disease. Thus, there is a need for the development of novel treatments that target the cellular and molecular mechanisms controlling dynamic AML-niche interactions and resolve niche-mediated drug resistance. Current studies have continued to highlight the role of the niche in AML development and progression, and potential targets of niche-directed treatments are now starting to emerge. AML xenograft models have revealed that severe vascular BM damage consisting of increased vascular leakiness occurs as the primary AML-induced niche alteration and leads to mobilization of healthy HSCs to the periphery. This experimental finding may seriously limit the success of allo-HSCT by reducing the outcompetition effect of patient residual and donor-derived HSCs on the AML cell population. Therefore, studies aimed at keeping HSCs in their BM niches are warranted . This go


Group housing of growing rabbits is currently used in commercial farms to improve animal welfare. However, these systems have shown some weaknesses, mainly associated with the aggressive behavior exhibited among rabbits. Environmental enrichment can offer additional space and a sheltered area in which an animal can protect itself from pen mates, but studies in rabbits under farming conditions are scarce. Thus, this study evaluated the use of two types of enrichment (an elevated plastic-slatted platform and/or a plastic hiding tube) in rabbits housed in collective pens within large groups during the growing period. The use of the platform allowed rabbits to move up/down, to rest in a more comfortable position and to increase explorative behavior, without modifying production performance. However, more injured rabbits were found at the end of the trial in pens with platforms. The presence of the tube impaired growth performance and did not modify behavior. Hence, elevated platforms have been shown to work as a structural enrichment in group-housed rabbits, whereas the usefulness of the tube remains questionable.2) on the performance and welfare of rabbits kept from weaning (at 33 days of age) to slaughter (at 68 or 75 days of age). Growth performance, injuries, and behavior (video recorded for 24 h) of rabbits (n = 504) were recorded. The platform allowed rabbits to adopt the rearing position more frequently (+0.14 events during 2 min every 30 min across 24 h) and to rest with stretched body for longer (+3.8% of observed time) (p ≤ 0.001). Production parameters and reactivity at the open field test were not modified, but the occurrence of injured rabbits at the trial end was higher in pens with platforms . This result was possibly related to the higher group size in pens with platforms (36 rabbits) compared to those without platforms (27 rabbits). The inclusion of the tube decreased growth , whereas it was scarcely used by rabbits and it did not substantially change their behavior or the occurrence of injuries. In conclusion, under the experimental conditions of this study, elevated platforms worked as a useful structural enrichment in view of animal behavior but negatively impacted on the rate of injuries, whereas the usefulness of the tube was not confirmed.This study assessed the effects of an elevated plastic-slatted platform and/or a plastic hiding tube in collective pens with large group sizes (27 or 36 rabbits/pen; 16 rabbits/m To improve animal health and welfare in rabbit farms, the European Parliament has requested a change from conventional cages to collective pens, also called parks ,2. Some Undoubtedly, in comparison with bicellular (2 rabbits) or multi-purpose 4–6 rabbits) cages, collective pens can provide rabbits with more opportunities to satisfy their ethological needs, with special emphasis on free social contacts and increased total available space for movement . In rabb rabbits 2 at 2.5 kg slaughter weight; 40 kg slaughter weight/m2) [2) for rabbit movements in each pen/park. That is why, under commercial conditions, collective systems are moving towards the use of large groups in large pens and research has focused on how to manage aggression in these systems.A group size higher than 7–9 growing rabbits  increases the risk of aggression and wound rates as well as the risk of disease spread , findingight/m2) , the smaTherefore, environmental and structural enrichments of housing systems could provide animals with the opportunity to display different behaviors, reduce abnormal behaviors, limit aggression and minimize animal stress. Among the different enrichments, gnawing materials are most commonly used. In growing rabbits kept in cages and pens with both small  and medium-size  groups, gnawing sticks have often reduced stereotyped behaviors, such as cage bar biting or chewing ,11, aggrThe use of structural enrichments, such as elevated platforms or hiding elements  to offer a shelter and/or more possibilities to escape in case of aggression, has received less attention in rabbits kept in large groups. The presence of elevated platforms can offer additional space for movement and for physical exercise, without impairing productivity and health ,17,18, e2) and slaughtered at 10 or 11 weeks of age.In this context, the present study aimed at evaluating the effect of two structural elements (an elevated plastic-slatted platform and/or a plastic hiding tube) on the growth performance, occurrence of lesions, behavior, and reactivity at the open field test in growing rabbits kept in large collective pens (27–36 rabbits/pen) at the same stocking density  . All animals were handled according to the principles stated in the EC Directive 2010/63/EU regarding the protection of animals used for experimental and other scientific purposes. The trial was performed at the experimental farm of the University of Padova . Temperatures ranged between 13 °C and 24 °C and a natural photoperiod (11–13 h daylight) was used.2) equipped with a plastic-slatted floor  at a stocking density of 16 animals/m2. The sidewalls of the pens (105 cm height) were made of wooden material with the front/back walls of galvanized wire net. Each pen was equipped with four manual feeders and four automatic nipple drinkers (At weaning (33 days of age), a total of 504 Hyplus rabbits  of both sexes were selected from healthy and homogenous litters in a commercial farm and moved to the experimental farm of the University. Rabbits were individually identified by ear mark and housed in 16 open-top pens  and a plastic tube , were used to obtain four types of collective pens (2)  a;2)  b;2)  c;2)  d.Two types of enrichment, a plastic-slatted elevated platform  were slaughtered at 68 days of age and the remainder (2 pens) at 75 days of age.The animals were provided with ad libitum access to fresh water and to a pelleted commercial diet  without coccidiostat. The diet was formulated to meet the nutritional requirements of growing rabbits according to de Blas and Mateos .Individual live weight and pen feed intake were recorded twice per week. Health status was monitored daily to detect any clinical signs of diseases. The day before slaughter, all rabbits were individually examined for the presence of injuries  due to aggression. The behavior of the rabbits was video-recorded for 24 h at 63 days  and at 70 days (8 pens housing rabbits to be slaughtered at 75 days). Two minutes per half-hour were recorded for evaluating the duration of some behaviors and all occurrences of other behaviors as reported in The open-field test was performed to evaluate rabbits reactivity towards a new environment at 65 days of age on 80 rabbits  and at 72 days of age on another 80 rabbits . Within pens, tested animals were homogeneously distributed by sex. The test was conducted in an arena (2 m × 2 m) with 0.8-m-high wooden walls and plastic floor divided into 9 numbered squares. The arena was in a contiguous room in the same stable where the trial was conducted. The total duration of each test was 10 min per animal. Each rabbit was put in a closed wooden box (22 cm length × 30 cm width × 30 cm high) connected to the arena by a sliding door. After one minute, the sliding door was opened and the number of attempts (n) the rabbit made and the time  spent to enter the arena was recorded for another minute. If, after this minute, the rabbit was still in the box, it was gently pushed into the arena, the sliding door was closed and the behavior of the rabbit was video-recorded for 8 min. The behaviors that were evaluated during the open-field test are described in All data were analyzed with SAS 9.4 software . The performance traits  were analyzed separately using the GLM procedure and fitting a generalized linear model with platform (no vs. yes), tube (no vs. yes), slaughter age (68 days vs. 75 days) and their interactions as the main factors, and the pen as the experimental unit. Injury data were coded as a binary variable  for estimating the percentage of animals with injuries using the CATMOD procedure by maximum likelihood analysis with platform, tube, slaughter age and their interactions as main factors.The different behaviors detailed in Data from the open-field test was analyzed using the GLIMMIX procedure with a generalized linear mixed model considering platform, tube, animal age (65 days vs. 72 days), and their interactions as fixed effects, and pen as the random effect. An underlying Poisson distribution was also assumed for these data. The percentage of animals entering the arena spontaneously was coded as a binary variable (Enter = Yes or No) and evaluated by maximum likelihood analysis with the CATMOD procedure which used platform, tube, animal age, and their interactions as the main factors.p-value ≤ 0.05 were accepted as representing statistically significant differences. The complete sets of p-values for the single effects and their interactions are provided in Overall, differences among means with a p ≤ 0.01)  . p ≤ 0.05)  . Even whp ≤ 0.001) and biting or licking the pen elements  (p ≤ 0.001). The differences in resting time with stretched body were higher at 70 d (28.7% vs. 33.5% of observation time in pens without vs. those with a platform) . The time the rabbits were observed drinking , allo-grooming , and moving around the pen , as well as the number of aggressive encounters  were all lower in pens with platforms compared to those without (p ≤ 0.001) (p ≤ 0.001)   and under the platform for an mean of 29.4% of the observed time . In addition, they moved 0.95 times per observation interval to/from the platform  (data not shown in tables). Regarding other behaviors , rabbits were observed for a longer time resting in a stretched position   in pens  without . Time sp≤ 0.001) . Rabbits≤ 0.001) . Rabbits≤ 0.001) . p ≤ 0.05) (Some differences were recorded for the open-field test including the time spent grooming which was higher (+2.18 s) in rabbits from pens with a platform compared to those without ( ≤ 0.05) . The use ≤ 0.05) .p ≤ 0.05)  compared to 0.25% of the time in pens with both a tube and platform  (data not shown in tables). Moreover, those rabbits housed in pens with any form of enrichment  were observed sniffing and moving for a lesser time period compared to those from pens with no enrichment  .p ≤ 0.001), a lower daily weight gain  together with an increased feed intake , which impaired the feed conversion index   .p ≤ 0.001), at the expenses of resting in the stretched position (−12.8 percentage points) (p ≤ 0.001) and allo-grooming  (p ≤ 0.001), hops , and aggressive encounters  were decreased with age , self-grooming (+5.3 percentage points), and sniffing (+1.81 percentage points) ( ≤ 0.05) . Moreovewith age .p ≤ 0.05), however they crossed the center of the arena less frequently . An interaction between tube and animal age was found for time spent in biting behaviors which increased with age, especially in rabbits housed in pens with a tube compared to those in pens without (p ≤ 0.05)  and running (+2.2 s) (0.01 ≤  ≤ 0.05) .2) on the floor compared to the platform area as well as a higher presence of them on the floor area in front of the platform than under the platform, both at every weekly observation and for the whole period (average of observations between 5 to 11 weeks of age). The elevated platform represents an additional area that animals can use and occupy to a different extent depending on different extrinsic factors  or intrinsic ones . In the present study, rabbits were observed in the floor area in front of the platform  for most of the time (50.0% of observation time on average), followed by the area under the platform  and finally by the area above the platform . Such a distribution of animals is consistent with previous results in collective pens   that rep2 were observed on plastic-mesh platforms than on wire-mesh ones. Similarly, reproducing does and their kits at 28 days of age preferred plastic-mesh platforms over wire-mesh ones [The platform design can also affect rabbit preference and use of the different pen areas. Growing rabbits kept in groups  preferred to stay under the platforms when these had a solid floor or deep-litter floor, rather than a wire-net floor . NeverthIn our trial, the presence of the elevated platform had a positive effect on behavior, in terms of increased observations of comfortable resting (stretched position). The decrease of grooming in favor of biting or licking pen elements may suggest that the platform was also used to re-direct attention. These findings are in line with that of Buijs et al.  who show2), rather than to the presence of the platform itself. In fact, in growing rabbits, aggressive behavior and related injuries have been found to increase with group size. For example, at the same stocking density of the present study, an increased occurrence of agonistic behavior from cages with 2 rabbits to pens with 13 rabbits (average across 46 and 74 days of age) was found [Under the conditions of the present study, the platform did not modify rabbit growth performance, in agreement with previous research in rabbits kept at different groups sizes in collective cages  or collas found . Likewisas found . Althougas found  showed tIn our study, the lack of agreement between aggression observed at 63 and 70 days of age and the rate of injured animals at the end of the trial could be attributed to the fact that behaviors were recorded one week before scoring for injuries. On the other hand, we could also hypothesize that the observation time  during the behavioral analysis may have impeded the detection of events such as aggression.Despite the benefits mentioned above, some authors claim that elevated platforms can make some daily farm management procedures difficult  because animals are less visible and accessible. Moreover, the hygiene in the cage/pen can also be impaired , especiaThe inclusion of a plastic tube as a structural enrichment aimed to provide a place in which rabbits could escape from unwanted social contacts from other pen mates or could recover at the end of a flight response in case of any disturbing event, including aggression. On average, rabbits were observed inside the tube for a relatively short time period , which increased to 4.6% in those pens with only a tube and decreased to 0.25% in pens with both a platform and tube. These results are independent of the area the tube occupied within the pen  and from the group size (26 vs. 37 rabbits/pen) in the different pen types (only tube vs. platform and tube). As indicated rabbits preferred the platform to the tube and only used the tube when no other enrichment item was available. The age at which behavioral recordings were performed (63 days or 70 days) may account for the scarce use of the tube. Young animals soon after weaning are expected to use and share the tube more frequently because they look for close contact , and havLiterature on the use of hiding elements is scarce and mainly limited to laboratory rabbits. Hansen and Berthelsen  found thIn our trial, the tube did not serve as an agent of reducing the rate of injured rabbits or aggressive behavior, consistent with findings in reproducing does housed in part-time collective systems equipped with hiding places (one plastic tube and one platform) . On the Under our conditions, the negative result of the tube is difficult to explain. The hypothesis of greater competition to enter and stay inside the tube at the expense of feeding and drinking, that could be further aggravated as animals grow and body size increases, is not supported by data. The effects of increasing slaughter age on productive performance and final weight observed in the present trial are consistent with a previous study in collective pens and large groups (20–27 rabbits/pen) and confirm previous research with individual or bicellular cages ,39. In tDespite only one week of difference between recordings, rabbit behavioral pattern across 24 h significantly changed towards a more passive attitude  and change in the resting position (more crouched body). These variations can be associated partly with the reduction of the available space per animal with increasing animal age and size. Trocino et al. [On the other hand, during the open-field test, older rabbits increased cautious exploration and running in the new environment, but crossed the center of the arena fewer times, which can be considered to be due to a more fearful attitude. In a previous study , fewer dResults of the present study illustrate that group housing of growing rabbits in pens with an elevated plastic-slatted platform allows rabbits to increase their behavioral repertoire and movement opportunities without negative effects on production traits or animal reactivity at the open field test. Nevertheless, based on the rate of injured rabbits, the higher available surface area offered by the platform and the corresponding higher group size pose great risks for social instability and related aggression among pen mates. On the other hand, the inclusion of a hiding tube impaired growth performance and did not improve rabbit welfare in terms of behavior. The underlying reasons for the negative outcomes of the hiding tube warrants further investigation to evaluate whether the structural characteristics  and/or number of tubes could affect their use.


CEACAM6 gene silencing using an siRNA delivery platform targeting the acidic tumour microenvironment in a lung adenocarcinoma xenograft mouse model. An siRNA delivery vector was constructed by tethering the peptide nucleic acid form of an siRNA targeting CEACAM6 (siCEACAM6) to a peptide with a low pH-induced transmembrane structure (pHLIP) to transport siRNAs across the plasma membrane. Specific binding of the pHLIP-siCEACAM6 conjugate to A549 lung adenocarcinoma cells at low pH was demonstrated by flow cytometry. A549 cells incubated with pHLIP-siCEACAM6 at an acidic pH showed downregulated expression of endogenous CEACAM6 protein and reduced cell viability. The in vivo tumour-suppressing effects of pHLIP-siCEACAM6 in lung adenocarcinoma were assessed in a xenograft model generated by injecting BALB/c nude mice with A549 cells. pHLIP-siCEACAM6 treatment alone resulted in tumour growth inhibition of up to 35.5%. When combined with cisplatin treatment, pHLIP-siCEACAM6 markedly enhanced tumour growth inhibition by up to 47%. In conclusion, the delivery of siCEACAM6 to lung adenocarcinoma using the pHLIP peptide has therapeutic potential as a unique cancer treatment approach.Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) plays an important role in lung cancer progression. Here, we examined the therapeutic efficacy of  Despite improvements in early diagnosis and recent advances in the treatment of lung cancer, the prognosis of patients with NSCLC remains dismal2.Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for 80–85% of lung cancer cases5. CEACAM6 is involved in cell adhesion, proliferation, migration, and invasion, as well as tumour cell metastasis6. It is overexpressed in a wide variety of carcinomas, including NSCLC and pancreatic, colon, breast, gastric, and hepatocellular carcinomas17. In NSCLC, lung adenocarcinomas express higher levels of CEACAM6 protein than other histologic subtypes. Although other CEACAM family proteins, such as CEACAM1 and CEACAM5, are known to be dysregulated in lung adenocarcinoma as well, CEACAM6 is more highly expressed than CEACAM5 and is associated with poor clinical outcomes among lung adenocarcinoma patients19. These data support a critical role of CEACAM6 in lung adenocarcinoma and suggest that it is an attractive therapeutic target. A recent study demonstrated the therapeutic potential of an anti-CEACAM6 monoclonal antibody by increasing the anoikis sensitivity in lung adenocarcinoma20; however, the use of RNA interference (RNAi)-based therapy to inhibit CEACAM6 in lung cancer has not been investigated.Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a single-chain glycosyl phosphoinositol-anchored immunoglobulin-like glycoprotein that, along with other CEACAM family molecules, mediates homotypic and heterotypic cell–cell interactions through integrin receptors21. Since the first report of RNAi in 199822, numerous studies have explored the use of RNAi-based approaches for therapeutic purposes23. siRNAs hold great promise as therapeutic agents in cancer because of their potential for silencing oncogenes24. While liposomes and nanoparticles provide certain advantages over viral-based systems for RNAi delivery, they exhibit low efficiency, as well as toxicity and rapid clearance from circulation25.RNAi is an endogenous cellular mechanism for regulating gene expression in which small-interfering RNAs (siRNAs) bind to the RNA-induced silencing complex to induce target mRNA cleavage and degradation through a catalytic process involving the Argonaute 2 endonuclease27. Because of the acidic pH of the tumour microenvironment28, pHLIP can target a variety of solid tumours and avoid systemic clearance by the liver. Therefore, we decided to use pHLIP as a delivery vector for CEACAM6-targeting siRNA. Furthermore, we utilized a peptide nucleic acid (PNA) as a nucleic acid analogue of siRNA. PNAs are characterized by nucleobases joined by intramolecular amide bonds in which the phosphodiester backbone is replaced by a peptide-like backbone of repeated N-(2-aminoethyl)-glycine units. The lack of anionic phosphodiester groups increases the binding affinity for complementary nucleic acids. In addition, PNAs are resistant to nucleases and proteases and are stable across a wide range of pH values29. Cheng et al. utilized the charge-neutral properties of PNAs to tether PNA antisense oligomers to pHLIP, facilitating the intracellular delivery of the antisense oligomers30. We used the tumour-targeting properties of pHLIP and the potent binding affinity of the PNA to construct a tumour-targeted PNA delivery vector (pHLIP-PNA) by fusing pHLIP to the PNA form of an siRNA targeting CEACAM6 (hereafter siCEACAM6). Here, we report the results of a concept study in a lung adenocarcinoma preclinical model that demonstrated the efficacy of CEACAM6 silencing using pHLIP-mediated PNA siRNA delivery.A peptide with a low pH-induced transmembrane structure (pHLIP) can be inserted into the cell membrane through the formation of an inducible transmembrane α-helix under acidic conditions and can translocate membrane-impermeable molecules into cells via a non-endocytic routehttp://www.cbioportal.org) showed CEACAM6 mRNA overexpression in 50 out of the 515 patients. Kaplan-Meier survival analysis showed that high levels of CEACAM6 mRNA expression were associated with poor overall survival in lung adenocarcinoma patients (P = 0.026)  label, which was attached to the PNA. The generation of pHLIP-PNA was verified by reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry  or pHLIP-scr for 48 h at pH 6.2. Western blotting analyses showed that pHLIP-siCEACAM6 significantly downregulated the expression of endogenous CEACAM6 at low pH in a dose-dependent manner in A549 cells, confirming the intracellular delivery and inhibitory activity of siCEACAM6 Fig. . Flow cyAdditionally, we evaluated the effect of pHLIP-siCEACAM6 in two CEACAM6-epxressing colon cancer cell lines, HT29 and LoVo. As shown in A549 cells, pHLIP-siCEACAM6 downregulated CEACAM6 expression  were injected with different doses (2 mg/kg and 4 mg/kg) twice per week for 3 weeks. Because cisplatin is the backbone of lung cancer treatment, cisplatin-treated mice served as the positive control. Mice received 2 mg/kg cisplatin intraperitoneally two or three times per week. After 3 weeks of treatment, all mice were sacrificed.We first assessed the knockdown efficacy of delivered siCEACAM6 P < 0.001). A significant reduction in tumour growth was also observed in the cisplatin treatment group compared with that in vehicle-treated mice  . Cisplatin-treated tumours also showed a lower proliferation rate than vehicle-treated tumours . These data indicate that in vivo silencing of CEACAM6 in tumours inhibits proliferation. Staining of cleaved caspase 3 (CC3), a marker of apoptosis, was higher in the pHLIP-siCEACAM6 treatment group than in pHLIP-scr tumours (P = 0.025)  Fig. . The abiivo Fig. . Animalsivo Fig. . To inve25) Fig. . These rCEACAM6 targeting. Two weeks after subcutaneous injection of 1.2 × 107 A549 cells, mice were randomly assigned to the following treatment groups (n = 5 mice/group): (i) vehicle, (ii) pHLIP-siCEACAM6, (iii) cisplatin, and (iv) pHLIP-siCEACAM6 + cisplatin. According to the in vivo results, a dose of 2 mg/kg pHLIP-siCEACAM6 was selected for combination treatment. Two weeks after cell line injection, pHLIP-siCEACAM6 was injected into the mice via the tail vein, and cisplatin was intraperitoneally injected twice per week for 3 weeks. Tumour volume was measured every 2–3 days. After 3 weeks of systemic therapy, mice in the pHLIP-siCEACAM6 treatment group exhibited a 21.8% reduction in tumour volume compared with that in vehicle-treated mice (P = 0.004). Cisplatin treatment reduced tumour volume by 29.9% (P < 0.001), whereas pHLIP-siCEACAM6 + cisplatin treatment significantly reduced tumour volume by 47% (P < 0.001)  Fig. . Taken t01) Fig. .Figure 4P = 0.121) or cisplatin alone (P = 0.271) . However, combination-treated tumours showed higher numbers of CC3-positive cells than those treated with siCEACAM6 (P = 0.022) or cisplatin alone (P = 0.016)  Fig. . Taken tIn this study, we demonstrated that the pHLIP-siCEACAM6 conjugate is an effective molecule in treating lung adenocarcinoma that functions by targeting the acidic tumour microenvironment. Moreover, the addition of a chemotherapeutic drug to siCEACAM6 treatment elicited potent therapeutic responses.35. At physiological pH, pHLIP is largely water-soluble, whereas at slightly acidic pH, pHLIP forms an α-helix capable of insertion into a lipid bilayer. Because of this characteristic, pHLIP is an attractive targeting moiety for selectively labelling and tracing acidic tissues in vivo32. As acidosis is a general property of the tumour microenvironment28, the pHLIP peptide localizes to tumours. We generated a pHLIP-siCEACAM6 conjugate by tethering the PNA form of siCEACAM6 to pHLIP, thereby translocating the PNA across the plasma membrane. The PNA and peptide are linked by a disulphide bond that is reduced in the cytosol; therefore, attachment of a PNA to the C-terminus of pHLIP promotes the intracellular delivery of the PNA.A tumour-targeted siRNA delivery vector (pHLIP-siRNA) was constructed on the basis of the ability of pHLIP to transport siRNAs across the plasma membrane in the acidic tumour microenvironment. pHLIP can exist in three states: a water-soluble protein, a protein bound to the membrane surface, and a protein inserted into the lipid membrane as an α-helix7. Because a number of human adenocarcinomas express higher levels of CEACAM6 than other histologic subtypes, these results may have direct translational implications for the treatment of various human adenocarcinomas17. Several studies have explored the therapeutic targeting of CEACAM6 in various cancers using immunotherapeutic agents such as immunotoxin-based therapy36, monoclonal antibodies6, anti-CEACAM6 single chain variable fragment37, and antibody-drug conjugates38. In particular, a phase I/II trial of a protein conjugate named L-DOS47, consisting of a urease conjugated to a monoclonal antibody targeting CEACAM6, in NSCLC was reported. The urease enzyme of L-DOS47 converts urea into cytotoxic ammonia, and the ammonia increases the pH of the tumour microenvironment39.Overexpression of CEACAM6 promotes cancer progression by inducing aberrant cell differentiation, anti-apoptosis, proliferation, and resistance to therapeutic agents40. Wong et al.41 used the L-DOS47 immunoconjugate to augment the extracellular pH and restore the function of a human T lymphoblastoid cell line, promoting cell proliferation, IL-2 production, and cell surface PD-1 expression. The immune evasion property of CEACAM6 supports the use of therapeutic targeting of CEACAM6 as an immune checkpoint therapy.Recently, the role of CEACAM6 in the inhibition of CD8+ T-cell responses in multiple myeloma was identified. Blocking CEACAM6 on the surface of myeloma cells restored T-cell reactivity against malignant plasma cellsCEACAM6 in pancreatic adenocarcinoma was studied by Duxbury et al.42CEACAM6 gene silencing resulted in tumour regression in a pancreatic adenocarcinoma xenograft model, including diminished tumour proliferation and improved survival. The present study is the first to report the therapeutic gene silencing of CEACAM6 in lung adenocarcinoma. Moreover, the pHLIP vector was used to improve tumour-targeting activity and mediate the effective delivery of therapeutic small RNAs.The therapeutic use of siRNA against CEACAM6-targeting siRNA indicate that RNAi may effectively reduce CEACAM6 activity in vitro and induce antitumour effects in A549 human lung xenograft tumours. pHLIP-siCEACAM6 treatment significantly inhibited tumour growth with a superior antitumour response following a combination of RNA therapy and the chemotherapeutic drug cisplatin. The response to chemotherapy can be improved by specifically modulating genes involved in chemoresistance pathways. CEACAM6 overexpression results in chemoresistance through the Src-AKT pathway in various cancers, such as pancreatic adenocarcinoma44, cholangiocarcinoma45, and breast cancer46. Furthermore, CEACAM6 overexpression is associated with paclitaxel resistance in lung adenocarcinoma cells, and anti-CEACAM6 monoclonal antibody treatment enhances chemosensitivity in vitro20. In this study, siCEACAM6 delivery combined with cisplatin-based chemotherapy had an additive effect in suppressing tumour growth. The inhibition of CEACAM6 may regulate chemoresistance-related genes and increase the effect of cisplatin treatment. Therefore, RNA therapy may be combined with chemotherapy to improve the efficacy of existing cancer therapies.The present results using 10; however, because of the tumour-targeting property of pHLIP, siCEACAM6 delivery was limited to tumour tissues. Considering that CEACAM6 is also detected in the sera of cancer patients47, antibody-based therapies against CEACAM6 may be neutralized by the presence of circulating CEACAM6 in the serum before reaching cancer cells and producing effects. However, the pHLIP-siCEACAM6 developed in this study is directly delivered into the cytoplasm of tumour cells, minimizing the loss of therapeutic activity.No mice in our experiments exhibited any overt signs of toxicity. CEACAM6 is expressed on granulocytes and the epithelia of various organs49. Future research will reveal whether treated tumours become resistant to small RNA-based therapies through mechanisms such as reduced nanoparticle uptake, increased nanoparticle exocytosis, and global downregulation of RNAi pathway function.siRNA delivery as a therapeutic approach has many characteristics that make it desirable for further drug development. In first-in-human clinical trials, lipid nanoparticle-formulated siRNA delivery was found to be safe and well tolerated and, in some cases, elicited complete responses in metastatic cancerThe present findings demonstrated that the pHLIP-siCEACAM6 conjugate is an effective antitumour molecule that targets the acidic tumour microenvironment and elicits potent antitumour responses when combined with chemotherapeutic drugs. The effective delivery of small RNAs to lung cancer cells using the pHLIP peptide is a unique approach to the use of small RNA therapies in cancer treatment.PNA oligomers siCEACAM6 (TAMRA–ooo-GATCACAGTCTCTGGAAGT-ooo-Cys) and scrambled siRNA  were purchased from PANAGENE . The PNA oligomer sequences including -ooo-  and cysteine were generated by solid-phase synthesis using an automatic synthesizer with Bts PNA monomers, which are the proprietary PANAGENE building blocks for PNA oligomer synthesis. The oligomers were cleaved from the resin using m-cresol:TFA (1:4) cocktail solution. TAMRA was exclusively conjugated to the amino (N)-terminus of the PNA. The PNA oligomers were purified by RP-HPLC and characterized by matrix-assisted laser desorption/ionization–time of flight. The estimated melting temperatures of the siCEACAM6 and scrambled siRNA PNA oligomers were 79 °C and 75 °C, respectively.−1cm−1 (A), 6,600 M−1cm−1 (C), 11,700 M−1cm−1 (G), 8,800 M−1cm−1 (T), 200 M−1cm−1 , and 32,300 M−1cm−1 (TAMRA).To generate pHLIP-PNA constructs, the following pHLIP sequence was synthesized: AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG. The C-terminus of each PNA oligomer was conjugated to pHLIP through a disulphide bond. To synthesize pHLIP-PNA constructs, pHLIP and PNA  were reacted overnight in the dark in a mixture of DMF/Tris (pH 7.5). After conjugation, pHLIP-PNA was purified by RP-HPLC and characterized by matrix-assisted laser desorption/ionization–time of flight. The pHLIP-PNAs were freeze-dried after quantification by UV/VIS spectrophotometry at 260 nm using the following extinction coefficients: 13,700 M2 atmosphere. LoVo cells were maintained in F-12K containing penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% FBS at 37 °C in a 5% CO2 atmosphere. For all pH-controlled cell culture experiments, cells were incubated with 10% FBS in the culture media buffered at pH 7.4 with HEPES or pH 6.2 with MES.A549 human lung adenocarcinoma cell lines and HT-29 and LoVo human colon adenocarcinoma cell lines were purchased from the American Type Culture Collection. A549 and HT-29 were maintained in RPMI containing l-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% FBS at 37 °C in a 5% COsiRNA duplexes were purchased from Dharmacon Research, Inc. . To confirm the specificity of the inhibition, the control, non-target siRNA was used as the negative control. The siRNA sequences used for CEACAM6 siRNA experiments are as follows:Negative control: UGGUUUACAUGUCGACUAASeq #1: GAUCACAGUCUCUGGAAGU,Seq #2: GAACAUGGCUAAAUACAAU,Seq #3: GAGGGUAACUUAACAGAGU,Seq #4: CUACAUACUCCAACUGAAA.A549 cells were transfected with 10 nM of siRNA duplexes using Lipofectamine RNAiMax  according to the manufacturer’s instructions. After 48 h, knockdown of the CEACAM6 gene was quantified by real-time RT-PCR.Total RNA was isolated using the TRIzol reagent , and cDNA was synthesized using the iScript™ cDNA synthesis kit  according to the manufacturer’s protocols. Quantitative real-time RT-PCR (qRT-PCR) was performed using iQ SYBR Green supermix (Bio-Rad). The primer sequences were as follows: GAPDH ; CEACAM6 . CEACAM1 . CEACAM5 . Thermal cycling and fluorescence detection were performed using the S1000 thermal cycler real-time PCR system (Bio-Rad). Reactions were run in a CFX96 Real-Time PCR System (Bio-Rad) using the following thermal conditions: an initial denaturation step at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 55 °C for 30 s. Gene expression levels were evaluated relative to GAPDH expression.A549, HT-29, and LoVo cells were incubated with 10% FBS in in the culture media buffered at pH 7.4 with HEPES or pH 6.2 with MES and were treated with pHLIP-PNA suspended in reaction buffer for 48 h. Proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Thermo Fisher Scientific). The membranes were blocked with 5% nonfat milk and then incubated with a mouse anti-CEACAM6 monoclonal antibody  or a mouse anti-GAPDH monoclonal antibody  as a loading control. The protein complexes were detected with enhanced chemiluminescence reagents (Thermo Fisher Scientific).A549 cells were incubated with 10% FBS in RPMI buffered at pH 7.4 with HEPES or pH 6.2 with MES and were treated with pHLIP-PNA suspended in reaction buffer for 48 h. After washing the cells to remove free pHLIP-PNA, they were resuspended in 500 μl PBS (pH 7.4) and analysed by flow cytometry .4 cells/ml) were treated with pHLIP-siCEACAM6 at doses of 0, 100, 250, or 500 nM in 96-well plates for 48 h; pHLIP-scr served as the control. All treatments were performed at the indicated pH. After 48 h, the extent of cell growth was assessed using CCK-8 assay (Dojindo). CCK-8 solution (10 μl) was added to each well, followed by incubation for 2 h at 37 °C. The absorbance at 450 nm was determined by a multiplate reader . Cell viability was expressed as a percentage of that in control cells.A Cell Counting Kit-8 (CCK-8) assay  was used to measure the effects of CEACAM6 inhibition on A549, HT-29, and LoVo cells. Cells (5 × 107 cells/mouse). Tumour growth was determined by measuring the length (L), width (W), and height (H) with callipers and using the formula V = (L × W × H) × 0.5. Upon tumour formation (approximately 44.8 mm3), mice were injected with pHLIP-PNA constructs intravenously and with cisplatin intraperitoneally as indicated.All animal work was performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology. A549 cells were subcutaneously injected into the right flanks of 5-week-old BALB/c athymic nude mice  and processed using Zen software .Mice were killed by carbon dioxide asphyxiation. Staining was performed in formalin-fixed, paraffin-embedded tumour sections (4-μm thickness). Fully automated immunostaining was achieved using a BenchMark XT autostainer . The following antibodies were used: monoclonal mouse anti-human CEACAM6 , anti-CC3 , and anti-Ki-67 . The number of positive cells per tumour area was quantified.http://tcga-data.nci.nih.gov/) containing multidimensional genomic data combined with clinicopathological reports from 515 patients with lung adenocarcinoma was analysed using cBioPortal tools 31. RNA sequence data from lung adenocarcinoma patients from the TCGA were used for the analysis of CEACAM6 mRNA expression. For univariate survival analysis, Kaplan-Meier plots were created with the log-rank test using the overall survival data of lung adenocarcinoma patients from the TCGA.A TCGA dataset .Two-way analysis of variance was performed for comparisons of cell viability data from three groups. For animal experiments, mouse and tumour weights for each group were compared using the Student’s Supplementary information


SOX family genes have been proved to be involved in multiple essential processes during carcinogenesis and act as potential biomarkers for cancer diagnosis, staging, prediction of prognosis, and monitoring of response to therapy. Herein, we revealed SOX30 methylation and its clinical implication in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS).Methylation-associated SOX30 methylation, a frequent event in AML, was negatively associated with SOX30 expression and correlated with overall survival (OS) and leukemia-free survival (LFS) in cytogenetically normal AML among SOX family members from The Cancer Genome Atlas (TCGA) datasets. In the validation stage, we verified that SOX30 methylation level was significantly higher in AML even in MDS-derived AML compared to controls, whereas SOX30 hypermethylation was not a frequent event in MDS. SOX30 methylation was inversely correlated with SOX30 expression in AML patients. Survival analysis showed that SOX30 hypermethylation was negatively associated with complete remission (CR), OS, and LFS in AML, where it only affected LFS in MDS. Notably, among MDS/AML paired patients, SOX30 methylation level was significantly increased in AML stage than in MDS stage. In addition, SOX30 methylation was found to be significantly decreased in AML achieved CR when compared to diagnosis time and markedly increased in relapsed AML when compared to the CR population.In the discovery stage, we identified that SOX30 methylation was associated with disease progression in MDS and acted as an independent prognostic and predictive biomarker in AML.Our findings revealed that The online version of this article (10.1186/s13148-018-0523-y) contains supplementary material, which is available to authorized users. Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are common clonal disorders in myeloid malignancies. AML is etiologically, biologically, and clinically heterogeneous disease characterized by the accumulation of excessive blasts , whereasID4, which exhibited anti-proliferation and pro-apoptosis effects in leukemia cells, and predicted disease progression and treatment outcome in myeloid malignancies  genes encode transcription factors belonging to the HMG (High Mobility Group) superfamily . There an humans . The SOXn humans . Althoug cancers . For inst of AML , 14. Man pathway . Our preSOX30 in AML and MDS. SOX30 methylation was a novel biomarker associated with prognosis and disease recurrence in AML and correlated with disease evolution in MDS. The results might provide us with novel insights into the mechanisms of MDS/AML leukemogenesis.In this study, we examined the methylation pattern and clinical significance of A total of 196 AML patients (184 de novo AML and 12 MDS-derived AML), 104 MDS patients, and 28 healthy donors were enrolled in the present study approved by the Institutional Ethics Committee of the Affiliated People’s Hospital of Jiangsu University. The diagnosis and classification of MDS and AML patients were based on the 2016 World Health Organization (WHO) criteria . Secondly, by Kaplan-Meier analysis, SOX30 hypermethylated patients had significantly shorter OS and LFS than SOX30 non-hypermethylated patients , and also higher than in MDS patients with low/Int IPSS risks (P = 0.068)  and WHO classifications (MDS-EB2). In addition, among gene mutations, we observed the association of SOX30 hypermethylation with U2AF1 mutation.We further investigated ols Fig. . Neverth68) Fig. . We alsoSOX30 methylation in MDS patients was performed in 96 patients with available follow-up data . Kaplan-Meier analysis showed that SOX30 hypermethylated patients had a tendency of shorter OS . Significantly, SOX30 methylation level in CR stage was significantly decreased than in diagnosis time and was markedly increased in relapsed population when compared to CR stage , which may be caused by disease entity (less blasts in BM). Notably, SOX30 methylation seemed to be associated with IDH1/2 mutations despite that the P did not attach statistical significance. As is well known, cancer-associated IDH mutations are characterized by neomorphic enzyme activity and resultant 2-hydroxyglutarate production [IDH1/2-mutant AMLs displayed global DNA hypermethylation and a specific hypermethylation signature [SOX30 methylation during leukemogenesis may be caused by IDH1/2 mutations. For MDS, we did not observe the significant association of SOX30 methylation with IDH1/2 mutations and older age, which may be caused by limited cases in MDS. However, we found the association of SOX30 methylation with U2AF1 mutations. U2AF1 mutations altered sequence specificity of pre-mRNA binding and splicing and was an important feature of the pathogenesis of MDS and related myeloid neoplasms [U2AF1 mutations caused differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B) [U2AF1 mutation and SOX30 methylation needs further studies. Importantly, SOX30 methylation was associated with higher blasts, high-risk MDS, and shorter LFS time. Moreover, SOX30 methylation showed a higher methylation level in MDS-derived AML compared to de novo AML, and the detection of SOX30 methylation in 12 paired MDS/sAML patients showed that SOX30 methylation level was significantly increased in AML stage than in MDS stage. These results suggested SOX30 methylation might play a crucial role in MDS progression. By gene array technologies, Jiang et al. demonstrated that aberrant DNA methylation, more frequently than chromosome aberrations, was the dominant mechanism for tumor suppressor gene silencing and clonal variation in MDS evolution to AML [SOX30 methylation in myeloid malignancies, whether SOX30 functioned as a progression-related driver in MDS needed further studies.In this study, we first identified oduction  and alsooduction , 40. Mutignature . These ieoplasms . Studies(DNMT3B) . Howevern to AML . HoweverH19 overexpression promoted leukemogenesis and predicted unfavorable prognosis in AML [CEBPA caused by its methylation was also regarded as a prognostic biomarker to guide treatment plan for AML patients [SOX30 as a prognostic biomarker has been reported in lung cancer [SOX30 methylation was not an independent indicator in MDS, we revealed that SOX30 methylation could act as a promising biomarker in AML. Firstly, SOX30 hypermethylation was associated with lower CR rate, which indicated SOX30 methylation was associated with chemotherapy response in AML. Secondly, SOX30 methylation was associated with shorter LFS/OS and acted as independent prognostic factor in AML. Lastly, the dynamic changes of SOX30 methylation in the surveillance of AML showed it could act as a predictor in monitoring disease recurrence. All these results suggested that determination of the SOX30 methylation may be useful to predict long-term survival and to guide post-remission therapy in MDS and AML. Interestingly, Božić et al. also used DNA methylation profiles of AML patients from TCGA and identified a CpG site in complement component 1 subcomponent R (C1R) as best suited biomarker to further complement risk assessment in AML [Epigenetic modifications not only played crucial roles in cancer biology but also acted as biomarkers for cancer diagnosis and prognosis especially in blood cancer. Our previous study showed that the long non-coding RNA s in AML . Moreovepatients . SOX30 ag cancer . From out in AML . ObviousSOX30 methylation was associated with disease progression in MDS and acted as an independent prognostic and predictive biomarker in AML.Taken together, Additional file 1:Figure S1. The prognostic value of SOX genes methylation for OS and LFS among whole-cohort AML patients from TCGA databases. SOX genes methylation (HM450) data was downloaded via cBioPortal . AML patients were divided into two groups by the median methylation level of each gene respectively. Figure S2. ROC curve analysis of SOX30 methylation for discriminating AML patients form controls. Table S1. Univariate and multivariate analyses of prognostic factors for overall survival in AML patients. Table S2. Univariate and multivariate analyses of prognostic factors for overall survival in non-M3 AML patients. Table S3. Univariate and multivariate analyses of prognostic factors for overall survival in CN-AML patients. Table S4. Univariate and multivariate analyses of prognostic factors for overall survival and leukemia free survival in MDS patients. (DOCX 660 kb)


The long noncoding RNA ANRIL has been found to be abnormally expressed and play important roles in different cancers. However, the expression and function of ANRIL in acute myeloid leukemia (AML) remain to be declared. In this study, we found that ANRIL is up-regulated in AML patients at diagnosis and down-regulated in patients after complete remission (CR). Functional studies showed that knockdown of ANRIL expression resulted in a decline in glucose uptake and inhibition of AML cell maintenance in vitro and in vivo. Mechanically, ANRIL was found to repress the expression of Adiponectin receptor (AdipoR1), a key regulator of glucose metabolism. Both ANRIL and AdipoR1 knockdown reduced the expression levels of phosphorylation of AMPK and SIRT1, implying a previously unappreciated ANRIL-AdipoR1-AMPK/SIRT1 signaling pathway in regulating cell glucose metabolism and survival in AML. The study is the first to demonstrate that ANRIL promotes malignant cell survival and cell glucose metabolism to accelerate AML progression and is a potential prognostic marker and therapeutic target in AML treatment.The online version of this article (10.1186/s12943-018-0879-9) contains supplementary material, which is available to authorized users. AML is a heterogeneous malignancy characterized by uncontrollable proliferation of leukemia cells in bone marrow, and accounts for 30% of leukemia-related pediatric deaths , 2. DespINK4 locus), was highly expressed in acute leukemia patients compared to that in normal controls [INK4B and p16INK4A in multiple solid tumors [In recent years, long non-coding RNAs (lncRNAs) have been found to be dysregulated in cancer –5. Howevcontrols . ANRIL wd tumors , 7. Mored tumors . TherefoTo validate the expression pattern of ANRIL in AML, we recruited 109 with newly diagnosed AML and 14 controls to evaluate the clinical relevance of ANRIL. The detailed clinical parameters are presented in Additional file To demonstrate the oncogenic role of ANRIL, we applied RNA interference (RNAi) to knock down the expression of ANRIL in AML cell lines HL-60 and MOLM-13  and negative control short hairpin RNA (named as sh-NC) were intravenously (tail vein) implanted into NOD/SCID mice. The knockdown efficiency was shown in Additional file Next, a mouse model was used to further validate the function of ANRIL in vivop value, the “Metabolic process” (Marked in red) and “cell death regulation” (Marked in blue) were significantly enriched


We observed that inhibition of UPRmt aggravated cytotoxic effects of Aβ25–35 in SHSY5Y cells. Our research suggests that the UPRmt activation and two pathways necessary for this response, and further provides evidence for the cytoprotective effect of UPRmt during the AD process.Alzheimer disease (AD) is the most common form of dementia. Amyloid β-peptide (Aβ) deposition is a major neuropathologic feature of AD. When unfolded or misfolded proteins accumulate in mitochondria, the unfolded protein responses (UPRmt) is initiated. Numerous lines of evidence show that AD pathogenesis involves mitochondrial dysfunction. However little is known about whether the UPRmt is engaged in the process of AD development. In this study, we investigated the UPRmt in mouse and cell models of AD. We found that UPRmt was activated in the brain of 3 and 9 months old APP/PS1 mice, and in the SHSY5Y cells after exposure to Aβ Upon treatment with statins, C. elegans fail to sense mitochondrial damage and to activate the UPRmt  was purchased from Sigma-Aldrich. The Aβ25–35 was first dissolved in tri-distilled water to 1 mM and then incubated at 37°C for 7 days. The solution was aliquoted and stored at −20°C, until use. Simvastatin was purchased from MedChem Express .Amyloid β protein fragment 25–35 (AβAntibodies specific for APP (126732), HtrA2/Omi (75982), β-Actin (11132), CLPP (124822), GAPDH (9485), respectively, were purchased from Abcam. Antibodies for LONP1 (15440), Hsp60 (15282), HMGCS-1 (17643), SPTLC-1 (15376) were obtained from Proteintech.n = 10 per group) and purchased from Beijing HFK Bioscience Co., Limited . When reaching the age of 3 and 9 months, mice were anesthetized by 10% of chloral hydrate and then sacrificed by cervical dislocation and decapitation. Fresh hippocampal tissues were harvested and stored at −80°C until use for studies. All applicable Shandong University and the ethical committee of the Second Hospital of Shandong University guidelines for the care and use of animals were followed.All procedures regarding the use of animals were conducted according to the guidelines and approved by the Ethical Committee for Animal Experiments of Shandong University. We used the APPsw/PS1dE9 double transgenic mice at the age of 3 and 9 months, age-matched C57BL/6 mice as a control. All mice were male  supplemented with 10% fetal bovine serum (Gibco) at 37°C and 5% C025–35 for another 4 h. After treatments, cells in each well were incubated with Cell Counting Kit 8 (CCK8) solution (MedCjemExpress) at 37°C for 3 h and then used for measuring the absorbance at 450 nm with a microplate reader . Experiments were repeated for at least three times.Cells were seeded into 96-well plates. After culturing for 24 h, cells were treated with simvastatin or SPTLC-1 siRNA for 48 h and then treated with Aβg for 10 min. The supernatants were collected and the protein concentration was measured with the bicinchoninic acid (BCA) method. After SDS-PAGE, proteins were blotted onto polyvinylidene difluoride (PVDF) membranes. Blots were blocked in 5% skimmed milk powder in TBST and incubated with corresponding primary antibodies at 4°C for 12–16 h. After extensive washes in TBST, blots were incubated with HRP conjugated secondary antibodies for 1–2 h at room temperature. Proteins were detected with ECL (enhanced chemiluminescence) regents.The Hippocampal tissues and cells after treatments were homogenized in RIPA lysis buffer (Beyotime Biotechnology). Cell debris and nuclei were discarded after a centrifugation at 4°C 12,000 Cells were collected and fixed in pre-cooled glutaraldehyde for at least 2 h. After postfixation in osmic acid solutions and sequential dehydration, cells were embedded in EPON812 resins. Ultrathin sections were cut and collected onto grids, stained with uranium and lead citrate, and observed under a JEM-1200EX electron microscope .Intracellular reactive oxygen species (ROS) levels were detected using DCFH-DA , which is non-fluorescent and generates fluorescent signals after being oxidized into DCF in the presence of intracellular ROS. The day before experiments, cells were planted into 12-well plates and cultured for 24 h. After brief washes in PBS, cells were incubated with DCFH-DA (10 μmol/L) at 37°C for 20 min, then washed three times with RPMI-1640 medium. Fluorescence images were captured using a fluorescence microscope (OLYMPUS BX43) at excitation and emission wavelengths of 488 and 525 nm. The amount of ROS was quantified with the use of a fluorescence microplate reader. All experiments were repeated for at least three times.SHSY5Y cells were transfected with target siRNAs using INTERFRERin . Corresponding scrambled siRNAs were used as controls. After transfection with siRNA for 48 h, cells were treated with or without Aβ as indicated and then collected cells for examinations.t-test was used for determining statistical significance. Data were analyzed using SPSS 20.0 . A p-value of less than 0.05 was considered to be statistically significant.All data were expressed as mean ± SEM. One-way analysis of variance (ANOVA) or Two-tailed student’s 25–35 treatment can cause the above UPRmt reactions, we treated SHSY5Y cells with different concentrations of Aβ25–35 for 24 h and then examined expression levels of UPRmt related proteins. We found that expression levels of mitochondrial matrix chaperone Hsp60, AAA proteases CLPP and the IMS-localized quality control protease HtrA2/Omi were increased after exposure to 2.5, 10 and 20 μM Aβ25–35 for 24 h. Levels of Hsp60 and CLPP, but not HtrA2/Omi, were also significantly increased in cells treated with 5 μM Aβ25–35 for 24 h. Under the same conditions, the upregulation of the LONP1 protein was detected only in cells exposed to 10 and 20 μM Aβ25–35  and APPsw/PS1dE9 transgenic mice, levels of APP in hippocampal lysates of both 3 and 9 months old transgenic mice was significantly increased compared with those in hippocampal lysates of age-matched WT mice . Western25–35 for 4 h (25–35 on the activation of the UPRmt reaction. We first treated cells with or without 1 μM or 10 μM of simvastatin for 2 h, 6 h, and 24 h, and then added 20 μM Aβ25–35 to cell culture media to treat cells for 4 h. The data in 25–35.There was upregulated expression of HMGCS-1, an enzyme that mediates the first committed step of the mevalonate pathway, in cells treated with 20 μM Aβ for 4 h . This ob for 4 h . We then25–35 (25–35 (25–35.During experiments, we observed a phenomenon with a microscope, that 24 h treatment of 10 μM simvastatin changed the cell’s morphology and promoted the apoptosis of cells treated with Aβ25–35 . The ele25–35 . We furt5 (25–35 . The celC. elegans. To detect whether it happens in SHSY5Y cells treated with Aβ25–35, we used the SPTLC-1 siRNAs to hinder sphingolipid biosynthesis. As shown in 25–35 for 4 h led to the upregulation of SPTLC-1. The expression level of SPTLC-1 was decreased in cells transfected with SPTLC-1 siRNAs for 48 h, compared with the SPTLC-1 level in cells transfected with scrambled siRNA. As shown in 25–35.In a previous study, RNAi mediated inactivation of the sptl-1 gene, which encodes SPT, disrupted response to mitochondrial dysfunction in 25–35 increased the intracellular ROS level and SPTLC-1 siRNAs transfection aggravated the ROS accumulation (25–35 (To determine whether inhibition of the sphingolipid biosynthesis pathway can cause a similar effect like simvastatin, we tested the cells’ mitochondrial structure, intracellular ROS level, and cell viability after transfected with SPTLC-1 siRNAs. As shown in mulation . The celn (25–35 .25–35 treatment and APPsw/PS1dE9 transgenic mice, and inhibition of the mevalonate and sphingolipid biosynthesis pathway disrupted the activation of UPRmt in SHSY5Y cells induced by Aβ25–35. We further found that inhibition of these two pathways through simvastatin and SPTLC-1 siRNAs aggravated the mitochondrial injury, ROS accumulation and the decrease of cell viability in SHSY5Y cells induced by Aβ25–35.This study showed that the UPRmt was activated in SHSY5Y cells with Aβ25–35 for 24 h. As shown in results, the UPRmt related proteins Hsp60, HtrA2/Omi and CLPP were upregulated in APPsw/PS1dE9 transgenic mice at 3 months, while to 9 months, Hsp60 and LONP1 proteins were increased compared with age-matched WT mice. Thus, we conclude that the UPRmt response was activated during the AD process in vitro and in vivo.Numerous diseases are associated with UPRmt, including spastic paraplegia, Parkinson’s disease, Friedreich’s ataxia and cancer (Haynes and Ron, 25–35 for 24 h, but increased after 10 or 20 μM Aβ25–35 treatment. Combined with the results in the mouse model, the LONP1 protein level was unchanged in APPsw/PS1dE9 transgenic mice at 3 months but increased at 9 months compared with age-matched WT mice. Hence we speculate that the activation of LONP1 protein may need a prolonged or a large dose of Aβ stimulation, but further research is needed to confirm this. From the result in Sorrentino’s research that several UPRmt genes were down-regulated in the cortex samples of 9 months old AD mice compared with those of 6 months old AD mice (Sorrentino et al., Among the UPRmt related proteins, LONP1 as a mitochondrial matrix protease, maintain the mitochondrial homeostasis by degrading misfolded or oxidized polypeptides. As shown in 25–35 for 4 h, and after inhibiting the mevalonate pathway by HMGCS-1 siRNA or simvastatin in SHSY5Y cells treated with Aβ25–35, the expression of UPRmt related proteins were decreased. This is consistent with the researches in C. elegans that the UPRmt upregulates the expression of HMGS-1 (Oks et al., hmgr-1 deletion mutant, hmgs-1 gene inactivation or statins prevents the activation of UPRmt (Liu et al., The mevalonate pathway is a key metabolic cascade that catalyzes the synthesis of sterol and nonsterol isoprenoids, which plays important effects on many cellular processes and physiological states (Goldstein and Brown, 25–35 for 4 h. Inhibition of the ceramide synthesis by SPTLC-1 siRNA decreased the UPRmt related proteins levels in SHSY5Y cells treated with Aβ25–35 transiently, which provides evidence for an involvement of ceramide synthase SPTLC-1 in the activation of UPRmt response in AD cell models. The result is consistent with the researches in C. elegans that the UPRmt upregulates the transcript level of sptl-1, and inhibition of the sphingolipid biosynthesis pathway through the sptl-1 gene inactivation prevents the activation of UPRmt (Liu et al., in vitro and in vivo (Hojjati et al., 25–35 treatment increases the protein levels of SPTLC-1, and the sphingolipid biosynthesis pathway is necessary to the activation of UPRmt induced by Aβ and this effect of the mevalonate pathway was Aβ-dependent.Several reports have revealed a novel connection between the sphingolipid biosynthesis and the endoplasmic reticulum UPR activation (Spassieva et al., 25–35 in SHSY5Y cells, which suggest that the UPRmt can protect SHSY5Y cells from the damage of Aβ25–35. The UPRmt has been proved cytoprotective effects in several reports (Baker et al., C. elegans (Bennett et al., As shown in our results, inhibition of the mevalonate pathway or the sphingolipid biosynthesis pathway can prevent the activation of UPRmt, change the mitochondrial structure, increase the intracellular ROS level and aggravate the cytotoxic effect of Aβ25–35 in SHSY5Y cells were aggravated. Hence our research helps us understanding the role of UPRmt in the pathogenesis of AD, which may provide new sights for the treatment of AD.Together, these observations imply that the UPRmt activated in AD cell and mice models, but the levels vary according to the ages of mice. We further observed that the mevalonate and sphingolipid biosynthesis pathways were necessary for the activation of UPRmt in AD. When inhibiting the two pathways, the cytotoxic effects of AβAll datasets generated for this study are included in the article.The animal study was reviewed and approved by the Ethical Committee for Animal Experiments of the Second Hospital of Shandong University.JB, PW, and YS designed the research. YS performed the experiments. MD, ZX, and XL provided technical support and helped with mouse dissections. HY, SJ, SX, ZZ, YW, DW, LX, and XZ analyzed the data. JB, PW, and YS wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Finally, some knowledge gaps in the field that may need to be further considered are also discussed.Calcium signaling, in addition to its numerous physiological roles, is also implicated in several pathological conditions including cancer. An increasing body of evidence suggest critical roles of calcium signaling in the promotion of different aspects of cancer, including cell proliferation, therapy resistance and metastatic-related processes. In many cases, this is associated with altered expression and/or activity of some calcium channels and pumps. Brain cancers have also been the subject of many of these studies. In addition to diverse roles of calcium signals in normal brain function, a number of proteins involved in calcium transport are implicated to have specific roles in some brain cancers including gliomas, medulloblastoma, neuroblastoma and meningioma. This review discusses research that has been conducted so far to understand diverse roles of Ca Diverse cellular processes critical for cancer progression are Ca2+ signal-dependent, including proliferation, angiogenesis, invasion and metastasis [2+ signaling has also been shown to promote growth and tumorigenesis of brain cancers. Although, most of these studies so far have been in glioblastomas, an increasing body of evidence suggest crucial role of Ca2+ signaling in other types of brain cancers too. While the calcium signal is ubiquitous, the proteins that control Ca2+ transport are not ubiquitously expressed. Indeed, altered expression and/or activity of specific Ca2+ channels and pumps have been shown in cancers. These proteins regulate highly specialized and selective processes which contribute to cancer initiation or progression [2+ transporting proteins in brain cancers. Calcium , plasma membrane Ca2+-ATPase (PMCA) and Golgi network secretory pathway Ca2+/Mn2+-ATPase (SPCA) are the main pumps found in mammals. An upward or downregulated expression of Ca2+ pumps is observed in various cancers and is believed to promote differentiation and apoptosis resistance in tumor cells [2+ signals are mediated by inositol 1,4,5-trisphosphate (IP3) receptor and ryanodine receptors (RyRs) in sarco and endoplasmic reticulum. Voltage-gated Ca2+ channels (VGCC), transient receptor potential (TRP) ion channels, store-operated Ca2+ entry (SOCE) channels, and ligand-gated Ca2+ channels are involved in movement of Ca2+ ion across plasma membrane. For instance, TRP channels, also known as cellular sensors [2+ into the cell in response to extracellular ATP [2+ signaling has been reviewed extensively elsewhere [2+ can be derived from both internal (such as endoplasmic or sarcoplasmic reticulum) and external sources (such as via plasma membrane ion channels). Ca2+ produces signals in the form of waves and spikes. Spikes activate localized cellular processes, while Ca2+ waves occur when individual channels communicate with each other to produce global signals leading to activation of both intercellular  and intracellular (such as gene transcription and cell proliferation) functions [2+ signals for longer periods are relayed via repetitive Ca2+ pulses known as oscillations. Both Ca2+ spikes and waves can produce oscillating signals triggering different processes depending upon their signal duration [Normal cell functioning requires maintenance of varied distribution of Catrations . Sarcoplor cells . Ca2+ si sensors , consist sensors . Ligand-ular ATP . Piezo clsewhere ,11. Intrunctions . Ca2+ siduration .2+ channels in regulation of epithelial to mesenchymal transition (EMT) [In recent years, studies elucidating the molecular mechanism involved in cancer have led to an enhanced understanding of various factors that play a role in disease progression and complexity . In an aon (EMT) , a proceon (EMT) ,19 and aon (EMT) . 2+ movement often involves aberrant expressions, cellular localization and activity of Ca2+-transporting proteins contributing towards tumor specific characteristics such as unlimited growth, metastasis and resistance to apoptosis [2+ signals are vital for normal cell function, alteration in these signals may lead to modified calcium codes that can trigger tumorigenic behavior. Ca2+ pulses serve as “calcium codes” differing in magnitude, time and frequency. Prolonged Ca2+ pulses are involved in cell division process, angiogenesis, differentiation and genomic instability in cancer cells [In tumor cells, altered Capoptosis ,4. Sinceer cells . Brain cancers are neoplasms that originate within the brain and spinal cord mainly from neurons or glial cells , and occasionally from pituitary gland, meninges, lymphatic tissue and cranial nerves . These t2 showing metastasis, whereas low risk patients have residual tumor with the size of < 1.5 cm2 without metastasis. Current therapies for medulloblastomas include surgery, radiotherapy and chemotherapy. The use of chemotherapeutic agents as an adjuvant has led to improved results and radiation dose reduction [Medulloblastomas (MBs), ependymomas and high-grade gliomas are the most prevalent tumors affecting children and adults . Medulloeduction .Ependymomas, accounting for approximately 10% of pediatric brain tumors, originate from ependymal cells that form the lining of the spinal cord and ventricles of the brain . Recent In adults, the most common type of brain tumor is high-grade gliomas. It includes glioblastoma and anaplastic astrocytoma . CurrentDespite improving the survival rate of patients, current therapies pose serious complications that impact quality of life in patients. Traumatic injuries such as damage to blood vessels and ischemia are common problems associated with surgical resectioning, while radiation and chemotherapy cause neurocognitive deficits, cerebral necrosis, peripheral neuropathy, myelopathy and secondary neoplasms . Current2+ channels play crucial role in nervous system development. Short-term rise in Ca2+ levels, known as calcium transients, are believed to play an important role in mediating various neuronal development stages such as growth, migration, differentiation, survival and formation of neuronal network by maintaining different influx patterns [Capatterns . 2+ between the intracellular compartments and across plasma membrane in brain. The movement of Ca2+ is triggered in response to stimulus such as membrane depolarization, intracellular Ca2+ store depletion, mechanical stretch, extracellular agonists and intracellular messengers [In brain, key functions of neurons are regulated by flow of Cassengers . 2+ leads to induction of a number of Ca2+ dependent processes such as neurotransmitter release, neuronal excitability, synaptic plasticity, memory storage and gene expression [2+ signal is a result of distinctive signals that differ in their scale, temporal and spatial properties [2+ concentration results in distinct function modification in the same neuron types depending upon the short, medium and long duration and distance travelled by the signal [The influx of Capression ,36,37. Toperties . Specifie signal . 2+ regulated functions involve a set of ion channels, exchangers and pumps present in plasma membrane, mitochondria, endoplasmic reticulum, golgi apparatus and nucleus, collectively known as Ca2+ toolkit. These Ca2+-transporting proteins together with G protein-coupled receptors, Ca2+ binding proteins, and related transcriptional networks mediate functions important for brain physiology [Caysiology .2+-transporting proteins in brain cancers. Most of these studies are conducted in glioma cells, with some research focusing on medulloblastoma, neuroblastoma and meningioma cells. 2+-transporting proteins in the progression of brain cancers. It should be noted that in this review, we only discuss studies that show direct association of Ca2+ transporters and not the ones based on other ion transporters that are Ca2+ dependent. This is, exemplified, by the role of calcium-activated potassium channel KCa3.1 in glioma cell migration, invasion and temozolomide resistance [The following section summarizes the specific roles of Casistance . 2+ influx to refill the endoplasmic reticulum’s (ER) Ca2+ after depletion. This process is initiated when the reduced levels of ER Ca2+ are sensed by STIM1 leading to its oligomerization and interaction with ORAI1 Ca2+ channel on the plasma membrane consequently resulting in the opening of the channel and influx of Ca2+ [SOCE is a process of Ca of Ca2+ . In prim of Ca2+ . While s of Ca2+ . In anot of Ca2+ .2+ influx is regulated by VGCCs. Opening of these channels results in high intracellular Ca2+ levels that activate processes such as neurotransmitter release, neuron growth and gene expression [V3 channels) [2+ channels control processes such as cell proliferation and differentiation by regulating Ca2+ levels at low voltage through creation of resting inward calcium current. Aberrant expression of T-type Ca2+ channels is observed in diverse tumor types [2+ channel blocker with a weak L-type channel inhibiting activity [V.3.1) and CACNA1H (CaV3.2) reduces cell proliferation, induces apoptotic cell death, and sensitizes cells to ionizing radiation via AKT/mTORC2 axis [V3.2 or mibefradil treatment was shown to inhibit the proliferation, survival and stemness features of glioblastoma stem-like cells (GSC) as well as sensitize them to temozolomide chemotherapy [In neurons, Capression . VGCCs ahannels) . T-type or types . In humaactivity , and siRRC2 axis . In anotRC2 axis . More reotherapy . This reotherapy . Mibefraotherapy . TRP channels are expressed in a number of tissues where they control diverse cellular functions such as cell growth, proliferation, differentiation, migration and apoptosis . TRP cha2+-transporting proteins are shown to interplay with hypoxia-inducible factor-1 (HIF-1), which is master regulator of hypoxia transcriptional responses [2+. Inhibition of TRPC6, by shRNA or expression of its dominant-negative mutant (DNC6), abolished hypoxia-induced HIF-1α protein accumulation in U251 human glioblastoma cells via promotion of HIF-1α hydroxylation [Calcium signaling regulates hypoxia-associated pathways, and several Caesponses ,56. In gxylation . Hypoxicxylation . In anotxylation .2+-activated K+ membrane ion channels (BK channels), regulates migration of DBTRG glioblastoma cells [A number of TRP-melastatin (TRPM) channels have been implicated in glioblastoma progression: reactive oxygen species (ROS)-activated TRPM2 channel induces cell death in A172 human glioblastoma cells ; TRPM7 cma cells ,62.2+ influx and induction of apoptosis in U373 human glioblastoma astrocytoma cells via p38 mitogen-activated protein kinases (MAPK) activation [TRP-vanilloid (TRPV) channels also have important functions in gliomas. Activation of TRPV1 by capsaicin, leads to Cativation . Interestivation . Cannabitivation . TRPV2 wtivation . Anothertivation .2+ signals of diverse spatial and temporal patterns such as waves and oscillations [IP3 receptors (IP3R) convert external stimulus to intracellular Callations . In gliollations .Members of the P2X receptors have been shown to play roles in different cancer types . In glioIn medulloblastoma cells, proton-sensing ovarian cancer G protein-coupled receptor 1 (OGR1) promotes the expression of TRPC4 channels in transformed granule cells (DAOY cells). TRPC4 channels in these cells , enhances cell motility in wound healing and transwell migration assays .V3.1 and CaV3.2 promote cell proliferation [V3.2 also stimulates differentiation of NG108-15 cells via an autocrine mechanism of facilitating extracellular secretion of differentiation-promoting factors [In mouse N1E-115 neuroblastoma cells, using plasmid overexpression, siRNA-mediated silencing or mibefradil treatment, it was shown that Caferation . CaV3.2  factors .Full-length and short isoforms of the TRPM2 channels (TRPM2-L and TRPM2-S respectively) are shown to be upregulated in neuroblastoma tissues compared to adrenal glands . SH-SY5YP2X7 receptors, in addition to glioma cells that was discussed above, plays role in neuroblastoma cells too. In neuroblastoma cells lacking trophic support (serum-deprived), P2X7 receptor expression is enhanced via EGFR and PI3K/AKT pathway, where it facilitates cell proliferation .2+ efflux and expression of PMCA2, PMCA3 and PMCA4 was observed in differentiated IMR-32 neuroblastoma cells compared to undifferentiated cells [Retinoic acid-mediated differentiation of neuroblastic (N-type) SH-SY5Y cells was shown to be associated with downregulation of SOCE and expression of STIM1 and ORAI1 proteins . Similared cells . In both2+ levels over time. Furthermore, expression of S100A6, IP3R1, IP3R3, RYR1, RYR3 were altered upon treatment with cisplatin and topotecan, and pharmacological modulators of Ca2+-transporting proteins in combination with these two agents enhanced cytotoxicity [2+-transporting proteins in neuroblastoma progression. Treatment of neuroblastoma cells with chemotherapeutic agents, cisplatin and topotecan, increased intracellular Catoxicity . FurtherVGCCs are shown to promote meningioma cells. The addition of diltiazem and verapamil  to meningioma chemotherapy drug hydroxyurea (HU), and the antiprogesterone, mifepristone RU486), enhanced cell growth inhibition through induction of apoptosis and G1 cell-cycle arrest in vitro. This approach also decreased tumor size in meningioma subcutaneous mouse flank tumors through suppression of proliferation and microvascular density ,86. , enhance2+ channel/regulator into its relevant process where it has shown involvement. These processes include proliferation, migration, invasion, therapy resistance/therapy sensitivity, differentiation, angiogenesis, inflammation and cell death. Many of these processes are involved in cancer hallmarks.2+-transporting proteins in the promotion of different aspects of brain cancer progression. In this section, we discuss studies that are more translational and are conducted in in vivo preclinical models or in clinical trials. In the previous section, we discussed studies that have shown important roles of Ca2+-transporting proteins and outlines the outcomes of these studies. Perhaps one of the most important studies, is recent completion of phase II clinical trial of Mipsagargin  for recurrent or progressive glioblastoma (NCT02067156), as well as for prostate cancer [2+ influx and release from intracellular stores), for recurrent and newly diagnosed glioblastoma and other anaplastic gliomas  or hypoe cancer ) for rece cancer ,89. A ph1107522, ,91). Thi1107522, ,91. A Ph1107522, . 2+ channels with niguldipine and mibefradil, induced selective cell death of glioma-initiating cells and enhanced host survival in an orthotopic mouse model of human glioma [2+-binding protein, calmodulin subtype 2 (CaM2), leading to dissociation of CaM2 from IP3R and subsequent extensive and irreversible Ca2+ release from endoplasmic reticulum by IP3R1 and IP3R2. Interestingly, this study also showed that TFP has less toxic effects on neural stem cells compared to glioblastoma cells [2+ release from internal stores, which ultimately led to increase of glioma invasion. This was shown to be mediated by inducing amoeboid migration via contraction of the cytoskeleton and activation of Ca2+-dependent K+ and Cl− channels [V3.2 in glioblastoma stem-like cells and further supported the use of mibefradil in combination with temozolomide for glioblastoma therapy [Apart from these clinical studies, there have also been some exciting pre-clinical studies in in vivo models with translational potential. In a recent study, pharmacological targeting of T-type Can glioma . In anotma cells . Bradykichannels . As also therapy . Further therapy , while a therapy . P2X7 re therapy  and in v therapy . 2+ signaling pathways for the control of brain cancers. It is interesting to note that the therapeutic targeting of plasma membrane Ca2+ channels in brain cancers has been focused on the use of blockers of these channels and no study has used channel activators to induce cytosolic Ca2+ overload and subsequent cell death, as was recently shown in breast cancer cells through activation of TRPV4 channel [Altogether, these studies showcase promising approaches of therapeutic targeting Ca channel .2+-transporting proteins in different aspects of brain cancer progression. Interestingly, all the proteins that were identified in these studies were Ca2+ channels of different classes (except from STIM1 that is an ER Ca2+ sensor). These Ca2+ channels included T-type voltage-gated channels, number of TRP channels, ORAI1, IP3R3 and P2X7R. As shown in 2+ signaling and specific transporting proteins in the progression of cancer, however it would be interesting and critical to also assess if calcium signaling and its regulating proteins are involved in the initiation of brain cancers. Furthermore, many of these studies are conducted in in vitro models of cancer cells (in most cases continuous cell lines). While these in vitro continuous cell line models provide very important information, these studies need to be also conducted in primary brain cancer cells and in vivo models to have a better understanding of potential translational impact. It is also imperative that in addition to cancer cells, the effect of targeting identified proteins be assessed in normal brain cells, highly sensitive cells that when damaged can result in debilitating permanent side-effects. As discussed throughout the paper, a number of studies suggest involvement of Ca2+-transporting proteins in transmitting the signals from the brain tumor microenvironment to cancer cells, is an area of research that requires further investigation. As was shown, majority of the research conducted so far in the field of calcium signaling in brain cancer has been undertaken in glioma cells with most of it in the past few years, underlining that there remain many unanswered research questions in this emerging field. In particular, role of Ca2+-regulating proteins in coming years.Ion channels are reported to represent 18% of all FDA-approved small-molecule drug targets . Given t