The EL Ca2+ store releases Ca2+ through a variety of Ca2+-permeable channels, including Transient Receptor Potential (TRP) Mucolipin 1-3 (TRPML1-3) and two-pore channels 1-2 (TPC1-2), whereas EL Ca2+ refilling is sustained by the proton gradient across the EL membrane and/or by the endoplasmic reticulum (ER). EL Ca2+ signals may be either spatially restricted to control vesicle trafficking, autophagy and membrane repair or may be amplified into a global Ca2+ signal through the Ca2+-dependent recruitment of ER-embedded channels. Emerging evidence suggested that nicotinic acid adenine dinucleotide phosphate (NAADP)-gated TPCs sustain multiple cancer hallmarks, such as migration, invasiveness and angiogenesis. Herein, we first survey the EL Ca2+ refilling and release mechanisms and then focus on the oncogenic role of EL Ca2+ signaling. While the evidence in favor of TRPML1 involvement in neoplastic transformation is yet to be clearly provided, TPCs are emerging as an alternative target for anticancer therapies.The acidic vesicles of the endolysosomal (EL) system are emerging as an intracellular Ca Typically, extracellular agonists, including growth factors, cytokines, neurotransmitters and hormones, bind to their specific tyrosine kinase receptors (TKRs) or Gq-protein coupled receptors (GPCRs), thereby recruiting, respectively, phospholipase Cγ (PLCγ) and PLCβ, which in turn cleave phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (InsP3) and diacyl-glycerol (DAG) ER) which triggers a signaling cascade leading to the opening of a Ca2+-permeable pathway on the PM, known as store-operated Ca2+ entry (SOCE) ER, which undergoes a dramatic conformational change and sub-cellular repositioning. The depletion of ER Ca2+ induces STIM1 to oligomerize and translocate from its reticular location in the bulk ER to specialized ER-PM junctions (≈20 nm) where it forms STIM1 puncta and interacts with the pore-forming subunit of store-operated channels (SOCs), Orai1 i i ,26, alth see the .2+ exchange with the ER, thereby regulating pleoiotropic functions across the phylogenetic tree i i in a wide number of cells types i ,13. The vacuoles . GPN is ls types ,39,40,41 and LEs ,29. It hs fusion . In addi of TFEB , as desc2+-dependent manner i concentration by further mobilizing lysosomal Ca2+ through TRPML1 i i . Moreoveus [2+]i . Subsequus [2+]i . In thist manner ,102.Arabidopsis thaliana i that drive a variety of cellular processes, ranging from fertilization to muscle contraction i ,14. Acco process ,162,163.al Ca2+) . Ultrastal Ca2+) . As revial Ca2+) ,165. Notal Ca2+) ,166. MCSal Ca2+) ,167 and ent, GPN . Thereforocesses ,51. Thesrocesses , neurotrrocesses , exocytorocesses , neuriterocesses , cardiacrocesses  and vascrocesses  contractrocesses , vasculorocesses , and nitrocesses ,173.2+ release from the EL store can also be effected by other members of the TRP superfamily, such as TRPA1 and TRPM2, ATP-gated ionotropic P2X4 receptors and voltage-gated Ca2+ channels  and hydrogen peroxide (H2O2), and positively modulated by NAADP and cyclic ADP-ribose [2+ release channels in mouse pancreatic β cells, in which it mediates H2O2–induced apoptosis [TRPM2 is a CaP-ribose . Additiopoptosis , and in poptosis .2+-permeable, non-selective cation current that is activated by noxious cold temperatures and mechanical stimuli and by a wealth of irritant compounds in peripheral somatosensory neurons [2+ sparks that stimulate vesicle exocytosis and calcitonin gene-related peptide release. It has, therefore, been proposed that EL TRPA1 further increases the excitability of dorsal root ganglia [TRPA1 belongs to a distinct sub-family of TRP channels and mediates a Ca neurons . Herein, ganglia .2+-permeable, non-selective channels [2- and COOH-terminal tails [2-terminus [P2X receptors are homo- or heterotrimers which serve as ATP-gated ionotropic, Cachannels . Seven sal tails . Earlierterminus . Neverthterminus . A subseterminus . Notablyterminus .2+ channels mediate extracellular Ca2+ entry in response to membrane depolarization to regulate multiple functions, including muscle contraction and gene expression (CaV1), neurotransmitter release (CaV2), and repetitive firing in sino-atrial cells and thalamic neurons (CaV3) [V2.1, which mediates voltage-gated P/Q-type Ca2+ currents at cerebellar terminal synapses and is encoded by the CACNA1A gene [V2.1 prevented autophagy by inhibiting the fusion between lysosomes and endosomes and/or autophagosomes [2+ currents were not measured, this finding raises the possibility that lysosomal Ca2+ release could effectively occur in response to a positive shift in ΔΨ [Voltage-gated Cas (CaV3) . SurprisA1A gene , is alsoA1A gene . Mutatioagosomes . Althougft in ΔΨ .2+ content drive or are tightly involved in the development of neurodegenerative disorders, such as MLIV, Parkinson’s disease, Alzheimer’s disease, and Niemann Pick disease, type C [The lysosomal membrane serves as a signaling platform that regulates multiple functions, including endocytic traffic, autophagy, and lysosomal function, thereby impacting on the metabolism and fate of the entire cell ,185. The, type C ,146,186., type C ,188. Bri, type C . In neop, type C . As a co, type C ,192. Cat, type C . Finally, type C . It shou, type C ,195.2+-permeable channels, i.e., TRPML1, or defects in the acidic Ca2+ store underlie severe neurodegenerative disorders, such as MLIV and Niemann Pick disease, type C, respectively [2+ toolkit supports many cancer hallmarks. For instance, the reduction in ER Ca2+ concentration is responsible for the increased resistance to pro-apoptotic stimuli [2+ machinery was implicated in malignant transformation [2+ channels in malignant transformation [2+-dependence of the subcellular localization of TFEB in cancer cells. TRPML2 is the only member of this sub-family to have been clearly associated to malignant transformation. A recent investigation revealed that TRPML2 transcript and protein were highly expressed in glioma tissues and that their expression increased during tumor progression [2+ signalling in the oncogenic effect of TRPML2 was not explored. Conversely, emerging evidence clearly hints at TPCs as main drivers of multiple cancer hallmarks in a growing number of solid malignancies , which is indispensable for cell migration [The first report about the pro-tumorigenic role of TPCs was, however, provided by Favia and coworkers . This stma cells . Accordima cells . A more 2+ store . NAADP-eigration .2+ response to NAADP stimulated angiogenesis through the paracrine release of growth factors from the cancer cells. In addition, the same group previously reported that NAADP sustains vascular endothelial growth factor (VEGF)-dependent angiogenesis in vitro and neovessel formation in vivo [2+ signals were attenuated by NED-19 and v-ATPase inhibition with bafilomycin A1. Likewise, the pharmacological blockade of TPCs with NED-19 blocked cell proliferation, migration and tube formation by suppressing VEGF-induced phosphorylation of ERK1/2, Akt, JNK and endothelial nitric oxide synthase (eNOS) [−/−TPCN2, but not −/−TPCN1, mice [2+ signals in HUVECs and prevented their assembly into capillary-like bidimensional structures in Matrigel scaffolds [2+ store enriched with TPC1 is also expressed in human endothelial colony forming cell (ECFCs) [2+/CaM-dependent manner [This study further suggested that the Ca in vivo . Accordie (eNOS)   ,171, a s (ECFCs) ,216. Nott manner ,171. Tumt manner ,218,219.2+ response to NAADP was inhibited by antagonists of L-type CaV channels, such as verapamil and diltiazem and dihydropyridines  [V antagonists within their selectivity filter, which is not surprising when considering the strong molecular similarity between TPCs and CaV [V antagonists, such as nitrendipine, nifedipine and verapamil, are routinely employed to treat life-threatening disorders [V antagonists could also be probed in preclinical trials assessing whether they are able to induce cancer shrinkage by blocking TPCs.The studies described strongly suggest that TPC1 and TPC2 represent suitable molecular targets to interfere with tumor growth, vascularization, and metastasis. Pharmacological blockade of TPCs could thus be regarded as a promising, alternative anticancer approach ,21. Thrordipine) ,224. Sub and CaV . As revi and CaV , CaV antisorders , such as2+ release. The most efficient inhibitors were PF-543, a cell permeant blocker of sphingosine kinase 1, SKF96365, a non-specific antagonist of Ca2+-permeable channels that was already known to affect NAADP signalling [2+ signals [2+ release in rat Jurkat T-lymphocytes in vitro and was effective in an EAE model of multiple sclerosis in vivo. Another agent obtained by this screening, termed 3 or BZ194 [2+ response to NAADP in vitro [A larger arsenal of drugs is currently under intense scrutiny to design alternative treatments of diseases associated to mutated or dysfunctional TPCs, including neurodegenerative diseases, or requiring a functional EL trafficking, such as viral infections. A recent screening campaign conducted on sea urchin egg homogenates disclosed a novel pharmacopeia of 18 drugs effectively (>80%) inhibiting NAADP-induced Cagnalling , and racgnalling . The sub signals  and atte signals . Paralle signals . This th signals . Notably signals . Finally signals . One of or BZ194 , blockedin vitro  and cardin vitro .2+-permeable channels, such as TPCs, is now coming of age and is predicted to rapidly enlarge our arsenal of selective TPC inhibitors, which could be also testable for anticancer treatments.Only NED-19, tetrandrin and naringenin were hitherto probed to halt tumor growth and vascularization. Unfortunately, tetrandrine and naringenin may target multiple signalling pathways and are not approved for human use worldwide . However2+ signalling through TRPML1-3 and TPC1-2 finely regulates cellular fate and behavior by driving the EL trafficking machinery and the autophagic flux, thus enabling the cells to adapt to stressful conditions, such as those imposed by lack of nutrients. In addition, local EL Ca2+ signals control trafficking and correct positioning of signaling proteins involved in cell proliferation and migration, such as integrins and EGF receptors, and the endocytosis of bacteria, toxins and viruses, which may cause cell death. Moreover, TPCs may interact with ER-embedded InsP3Rs and RyRs to generate global Ca2+ signals that further expand the repertoire of cellular functions regulated by the EL Ca2+ store. Herein, we discussed the evidences supporting the role of EL Ca2+ signalling in cancer. While the oncogenic function of TRPML1, the main driver of TFEB activation in healthy cells, remains obscure, TPC1 and TPC2 are standing out as crucial drivers of many cancer hallmarks, including excessive proliferation, aberrant migration and sustained vascularization. Many issues remain to be elucidated. For instance, is TPC expression actually altered in cancer cells? Is NAADP the main ligand of tumor TPCs or is there a role also for PIP2? Is the EL Ca2+ store altered by neoplastic transformation, just as it happens for the ER? Which is the main source of Ca2+ responsible for EL Ca2+ refilling in cancer cells: the cytosol, the ER or the extracellular milieu? Does CICR support the Ca2+ response to NAADP also in cancer cells (EL Caer cells ? With al


The tumor suppressor p53 (TP53) is the most frequently mutated human gene. Mutations in TP53 not only disrupt its tumor suppressor function, but also endow oncogenic gain-of-function (GOF) activities in a manner independent of wild-type TP53 (wtp53). Mutant TP53 (mutp53) GOF is mainly mediated by its binding with other tumor suppressive or oncogenic proteins. Increasing evidence indicates that stabilization of mutp53 is crucial for its GOF activity. However, little is known about factors that alter mutp53 stability and its oncogenic GOF activities. In this review article, we primarily summarize key regulators of mutp53 stability/activities, including genotoxic stress, post-translational modifications, ubiquitin ligases, and molecular chaperones, as well as a single nucleotide polymorphism (SNP) and dimer-forming mutations in mutp53. TP53 has the highest mutation rate, the majority of which are missense mutations with amino acid changes mainly in the DNA binding domain. There are hotspot mutations including R175, G245, R248, R249, R273, and R282, accounting for ~30% of p53 mutations [TP53-null Saos2 cells enhanced soft agar colony formation and tumor development in mice. Since then, considerable evidence has solidified that mutp53 not only loses wild-type TP53 (wtp53) function, but also enhances tumor malignancy in vitro and in vivo independent of wtp53. The primary role of the tumor suppressor p53 (TP53) is to control cell cycle progression, senescence, DNA repair, cell death, and cell metabolism, leading to inhibition of tumorigenesis ,2. Compautations . Such onThe oncogenic function of mutp53 is mainly caused by altered structure and properties of mutp53 to bind with other oncogenic  or tumor suppressive  proteins ,13,14. MGenerally, mutp53 can be roughly divided into two subtypes, DNA contact and conformational (or misfolded/unfolded) mutants. DNA contact mutants have mutations in amino acids where TP53 directly contacts with DNA, leading to impaired TP53’s binding activity while sparing the intact TP53 structure. On the other hand, conformational mutants robustly alter the TP53 structure and disrupt the DNA binding activity ,27. As mLike wtp53, mutp53 stability is regulated by various genotoxic stresses, nutrient depletion, and oncogenic or tumor suppressive proteins ,25, whicMutations in TP53 impair the transcriptional activity (loss of function: LOF). The majority of TP53 mutations are missense mutations. These dysfunctional TP53 mutant proteins are present in the cells and retain or gain abilities to bind with other proteins, resulting in altered function of these binding partners to enhance tumor malignancy, referred to as GOF ,12,13,14−/−TP53 mice [R172HTP53-knockin mice, it remains unclear whether levels of other TP53 mutants also increase by these environmental stresses in mice. Due to mutp53’s inability to transactivate downstream targets including its ubiquitin ligase MDM2, it was considered that mutp53 could be accumulated in both cancer and normal tissues. However, studies using mutp53 knockin mice have demonstrated that mutp53 levels are low in most normal tissues, while mutp53 is accumulated in many cancer tissues ,23,31. T−/− mice ,31. Addi−/− mice ,32,33,34Mutp53 can be degraded by cellular conditions associated with autophagy . SuppresBy mainly focusing on the involvement of PTMs, specific chaperones, and ubiquitin ligases in mutp53 stability/activity, we briefly discuss chemical compounds that can alter mutp53 stability/activity. These include mutp53 accumulators , degraders/depleters , and reactivators  ,41,42. DNA damage agents result in increased levels of mutp53 to alter function of TP53-binding partners, which contributes to chemoresistance ,44,45,46https://clinicaltrials.gov/). Cancer cells are frequently addicted to mutp53, since knockdown of TP53 mutants attenuates malignant properties of cancer cells and tumor development in mice . Tumor shttps://clinicaltrials.gov/). Many TP53 mutants are structurally altered, especially in the DNA binding domain, thereby losing the DNA binding potential . ResearcAccumulated studies demonstrate efficacy of mutp53 degraders/depleters and reactivators to inhibit tumor progression in vitro and in vivo; however, it remains unclear whether these compounds cause PTMs of mutp53 and if the PTMs induced by them play crucial roles in mutp53 degradation or reactivation. Wtp53 is post-translationally modified following a variety of genotoxic and cellular stresses at approximately 15% of amino acid residues, including serine, threonine, and lysine residues, through phosphorylation, acetylation, and ubiquitination ,60,61. MPhosphorylation of proteins is one of the major mechanisms for regulating protein stability or function and transmitting signals in the cell. Following various stresses, wtp53 is phosphorylated at several serine/threonine residues, including serine 15 (S15), threonine 18 (T18), serine 20 (S20), serine 46 (S46), and serine 392 (S392) . IntriguUllrich et al.  report tThe N-terminal serine residues of mutp53 are phosphorylated at multiple sites. Adorno et al.  show thaAnother well-studied phosphorylated serine residue at the N-terminus of mutp53 is S15, a key serine residue to activate wtp53 following genotoxic stress . MelnikoAdditionally, stathmin 1  which plays a role in microtubule destabilization and dynamics contributes to mutp53 stability with phosphorylation at S15 and S37 . KnockdoBesides S15, T18 and S46 are phosphorylated on wtp53, which is enhanced by PML ,113,114.R172HTP53 mutations, concomitant homozygous deletion of Pin1 attenuates tumor progression [S46 of wtp53 is known to be required for TP53-mediated apoptosis ,116,117.gression . Pin1 algression . Thus, iC-terminal serine residues of mutp53 are also phosphorylated. Polo-like kinase 2 (PLK2), which is activated by DNA damage, phosphorylates mutp53  at T377, leading to enhanced binding of mutp53 with p300, increased acetylation of mutp53, and induction of the GOF activity including increase in cell proliferation, NF-Y’s transcriptional activity on cyclin A, cyclin B, cdk1, and cdc25C, and Adriamycin resistance .Additionally, Matsumoto et al.  reveal SOverall, phosphorylation of wtp53 stabilizes and enhances the function of wtp53 as a transcription factor and a tumor suppressor. On the other hand, phosphorylation of mutp53 results in context-dependent changes in the stability and oncogenic GOF. Further detailed studies are needed to clarify the role of each phosphorylation site in mutp53 GOF.CBP/p300, P/CAF (p300/CBP-associated factor), and Tip60 are well-documented acetyltransferases for wtp53 which enhance wtp53 transcriptional activity , while HThirteen lysine (K) residues on wtp53 are reported to be acetylated. Three  are in the DBD, two  are in the domain containing a nuclear localization signal (NLS), and eight  are located in the C-terminal regulatory domain including a tetramerization/oligomerization domain, a nuclear export signal (NES), and a negative regulatory domain  124,125125. NotaMinamoto et al.  report tp21 and PUMA promoters. Additionally, Knowell et al. [Intriguingly, Perez et al.  observe l et al.  find thal et al. .As mentioned in the previous section, glucose restriction triggers mutp53 degradation via autophagic cell death in several mutp53-carrying cancer cell lines, including TOV112D (R175H), MDA-MB-231 (R280K), T47D (L194F), BT-20 (K132Q), and PANC1 (R280T) . IntriguPUMA, NOXA), suggesting reactivation of wtp53 activity through deacetylation. In support of their findings, Zhang et al. [Moreover, Yi et al.  observe g et al.  demonstrThus, some lysine residues contribute to stabilization and activation of GOF mutp53, while other residues can alter the structure of mutp53 to restore wtp53 activity upon acetylation or deacetylation. Detailed analyses for each lysine residue in mutp53 are required to understand the exact role of mutp53 acetylation in its stabilization, GOF activity, and restoration of wtp53 activity.Ubiquitination of mutp53 plays a crucial role in the stability and subcellular localization of mutp53, which impacts its GOF activities. Polyubiquitination of mutp53 leads to its degradation, while its monoubiquitination may alter subcellular localization of mutp53 likewise wtp53 ,131. The−/−TP53−/−MDM2 cells, whereas R273H mutp53 which is not ubiquitinated in the same cells is present in the nucleus [Lukashchuk and Vousden  reveal t nucleus . Similar nucleus  observe Recently, Frum et al.  report tR248W/−TP53 HCT116 cells, they also show that MDM2-B enhances mutp53 GOF including tumor growth and metastasis in vivo. Additionally, MDM2-B is overexpressed in R172H/R172HTP53 mouse lymphomas. Thus, it is postulated that any proteins which inhibit MDM2 activity could stabilize mutp53 to increase the GOF activities.MDM2 has more than 40 splice variants, but it is unclear whether all variants are translated and have functional effects on TP53 . SeveralCHIP is a cofactor that interacts with HSC70 and accelerates ubiquitin-dependent degradation of chaperone substrates through its carboxyl-terminal U-box . Mutp53 Another ubiquitin ligase which can ubiquitinate and degrade wtp53 is Pirh2. Intriguingly, arsenic trioxide (ATO) induces Pirh2-mediated degradation of multiple TP53 mutants in SW480 (R273H), MiaPaCa-2 (R248W), HaCaT (H179Y/R282W) cells, as well as HCT116 cells expressing R175H and R273H .In summary, mutp53 is ubiquitinated by MDM2 and other ubiquitin ligases. Unfolded/misfolded mutp53 may be ubiquitinated by multiple ubiquitin ligases such as MDM2 and CHIP, while ubiquitination of DNA contact mutp53 is mainly executed by MDM2. Additionally, ubiquitinated mutp53 appears be exported to the cytoplasm for degradation likewise wtp53. Thus, ubiquitination and degradation of mutp53 may be dependent on its binding to molecular chaperones and their associated ubiquitin ligases.Deubiquitinases (DUBs) are a group of proteases that cleave ubiquitin from proteins. USP10 is a cytoplasmic ubiquitin-specific protease and is shown to deubiquitinate wtp53 to reverse MDM2-induced wtp53 ubiquitination, nuclear export, and degradation . KnockdoUSP15 is another deubiquitinase that is reported to increase the stability of MDM2 and hence reduce wtp53 activity to inhibit apoptosis in cancer cells . HoweverThus, several ubiquitin ligases and deubiquitinases can regulate ubiquitination and degradation of both wtp53 and mutp53; however, it remains unclear whether a ubiquitin ligase which ubiquitinates both wtp53 and mutp53 modifies the same lysine residues, if different ubiquitin ligases modify the same lysine residues, and which lysine residues are involved in subcellular localization of mutp53. It is also unknown if other ubiquitin-like modifications such as neddylation and SUMOylation occur in mutp53 and whether these modifications alter the stability, GOF activities, and subcellular localization of mutp53.Endogenous and environmental stresses, such as heat shock, oxidative stress, inflammation, infection, chemicals, and irradiation, significantly impact behavior of the cell. Heat shock proteins (HSPs) play central roles in responding to these stresses as molecular chaperones. Molecular chaperones interact with a variety of proteins to promote their proper folding in order to prevent generation of unfolded/misfolded or damaged proteins, causing them to be refolded or degraded .HSP family proteins, including HSP90, HSP70 (DnaK homologue), and HSP40 (DnaJ homologue), play major roles in protein folding, protein maturation, protein localization, proteolysis, and cell signaling . There aR172H/R172HTP53 and R248Q/−TP53 mice with minimal effects on TP53-null mice. Recently, Ingallina et al. [HSP90 plays a crucial role in stabilizing mutp53. Geldanamycin, a HSP90 inhibitor, alters the physical association of mutp53 with the HSP90 complex, leading to reduced protein half-life of several TP53 mutants via ubiquitination and proteasomal degradation, in multiple cancer cell lines, including T47D L194F), MDA-MB-468 (R273H), and SKBR3 (R175H) ,150,151.F, MDA-MBHSP70 is another chaperone which is involved in mutp53 stabilization or degradation. Muller et al.  show thaMortalin is a member of mitochondrial HSP70 and is also known as mtHSP70/Grp75 (mot-2). Mortalin is involved in mitochondrial biogenesis regulating mitochondrial import of nuclear-encoded proteins . Mortalip21, PUMA, and MDM2, as well as mutp53’s binding to the TP53-resposible elements of these genes. Moreover, CTM restores MDM2’s ability to induce mutp53 degradation. Additionally, Tracz-Gaszewska et al. [HSP40 is another chaperone that binds with conformational or misfolded mutp53 (R175H) . Mass spa et al.  observe a et al. . Intrigua et al. .R248W/−TP53 HCT116 cells as well as H1299 and Saos2 cells exogenously expressing R175H, R248W, and R273H mutp53. They observe that BAG2 inhibits MDM2’s activity on mutp53 and accumulate the protein levels of mutp53. Furthermore, BAG2 knockdown enhances sensitivity of mutp53-expressing cells to 5-Fluorouracil, a chemotherapy drug, and reduces migration, tumor growth, and lung colonization of R248W/−TP53 HCT116 cells. Moreover, BAG2 is elevated in a diverse range of human cancers, including colorectal cancers, lung cancers, breast cancers and sarcomas, while high levels of BAG2 are associated with increased mutp53 levels and poor prognosis in these cancer patients [BCL2-associated athanogene (BAG) family proteins are co-chaperones that interact with mutp53. Through mass spectrometry analyses, two BAG family proteins, BAG2 and BAG5, are found to bind with mutp53 (mouse R172H) and promote its GOF activity ,102. Yuepatients . Later, Thus, multiple chaperone and co-chaperone proteins play crucial roles in mutp53 stability and GOF activities as well as restoration of wtp53 structure and activity. Currently, only HSP90 inhibitors are in clinical trials for cancer therapy. Development of inhibitors for other HSPs and chaperones/co-chaperones would greatly help understand mechanisms behind restoration of wtp53 activity and mutp53 stabilization/GOF and may also advance current therapeutic strategies for cancers carrying mutp53.A SNP in wtp53 at codon 72 alters localization and function of wtp53, where arginine 72 (72R)-wtp53 has greater ability to localize to the mitochondria and induce apoptosis more efficiently than proline 72 (72P)-wtp53 . EvidencTP53-null H1299 or PC3 cells, as well as by comparing results between two colon cancer cell lines, HT29 (72P-R273H) and SW620 (72R-R273H). They reveal that 72P-mutp53 more efficiently binds to and inhibits activity of PCG-1α, a master regulator of mitochondrial function, as compared with 72R-mutp53. As a result, cancer cells carrying 72R-mutp53 have increased mitochondrial function, leading to enhanced metastatic capacity. Moreover, 72R-mutp53 is correlated with poorer outcomes in human breast cancer, as compared with 72P-mutp53 [Recently, Basu et al.  reportedP-mutp53 . FurtherP-mutp53 ,164.Oligomerization of TP53 has been implicated in TP53’s DNA binding/transcriptional activity, ubiquitination/degradation, and interactions with TP53 binding partners . RecentlThis article has summarized multiple factors that impact levels, subcellular localization, and GOF activities of mutp53, as well as those inducing reactivation of wtp53 or depletion of mutp53. These include the upstream regulators of mutp53, such as genotoxic stress, PTMs, ubiquitin ligases, and molecular chaperones, as well as different sequences in mutp53 including codon 72 polymorphism and mutations in the oligomerization domain. Although studies examining upstream regulators of mutp53 GOF activities are limited, unlike wtp53, many upstream regulators of wtp53 also appear to impact mutp53 stability and GOF. However, the biological consequences are distinct between wtp53 and mutp53, which can be dependent on types of TP53 mutations, cellular context, and experimental conditions. To clarify these differences, detailed studies using various TP53 mutants in a well-controlled experimental setting should be performed in the future.In conclusion, stability and activity of mutp53 are altered by multiple factors, some of which regulate wtp53 activity. However, the biological consequences are distinct. More studies are required to obtain clear-cut conclusion of the roles of mutp53 PTMs and SNPs in the GOF activity and cancer progression.


Triple negative breast cancer (TNBC), is defined as a type of tumor lacking the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). The ER, PR and HER2 are usually the molecular therapeutic targets for breast cancers, but they are ineffective for TNBC because of their negative expressions, so chemotherapy is currently the main treatment strategy in TNBC. However, drug resistance remains a major impediment to TNBC chemotherapeutic treatment. Recently, the protein phosphatase 2A (PP2A) has been found to regulate the phosphorylation of some substrates involved in the relevant target of TNBC, such as cell cycle control, DNA damage responses, epidermal growth factor receptor, immune modulation and cell death resistance, which may be the effective therapeutic strategies or influence drug sensitivity to TNBCs. Furthermore, PP2A has also been found that could induce ER re-expression in ER-negative breast cancer cells, and which suggests PP2A could promote the sensitivity of tamoxifen to TNBCs as a resistance reversal agent. In this review, we will summarize the potential therapeutic value of PP2A as the main node in developing targeting agents, disrupting resistance or restoring drug sensitivity in TNBC. Breast cancer is the most frequent cancer and the second leading cause of cancer-related mortality among women worldwide ,2. TriplRecently, TNBC molecular subtypes were refined into four (TNBCtype-4) tumor-specific subtypes: basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), and luminal androgen receptor-like (LAR), to demonstrate differences in diagnosis age, grade, local and distant disease progression and histopathology ,8. DespiPhosphoprotein phosphatase 2A (PP2A), a major serine/threonine phosphatase, appears to be critically involved in cellular growth control and potentially in the development of cancer through regulating a broad array of biological processes such as the cell cycle, DNA replication, transcription and translation, signal transduction, cell proliferation, cytoskeleton dynamics and cell mobility and apoptosis ,20,21,22Unlike many kinases , PP2A is a multimer ,30. The The cell cycle covers a broad range of interconnected pathways but is more likely to be deregulated in TNBC. Cyclin E1, an activator of cyclin-dependent kinase 2 (CDK2), is an important regulator of the G1–S-phase transition of the cell division cycle. However, cyclin E1 is aberrantly overexpressed in many human cancers. Approximately 30% of breast cancers overexpress cyclin E1 ,40. CyclIn triple-negative breast cancer, retinoblastoma protein (RB) and its regulatory cyclin-dependent kinases (CDK) have been reported as one of the key regulatory elements in the cell cycle progression. RB is phosphorylated by cyclin E/CDK2 in cooperation with cyclin D/CDK4/CDK6. RB pathway deregulation comprises ~20% of TNBC, and knockdown of retinoblastoma (RB1) in TNBC cell lines showed an increase of sensitivity to gamma-irradiation, doxorubicin and methotrexate treatments . A recenBRCA-1 and BRCA-2 defective cells are markedly sensitive to PARP inhibitor. The incidence of BRCA1 mutation carriers in TNBC patients is very high at about 70% and indicates a particularly poor prognosis in these patients [Poly(ADP-ribose) polymerase (PARP) inhibitors are currently emerging as one of the most promising targeted therapeutics to treat TNBCs. Targeting the DNA repair complex of PARP inhibitor is a novel, biological approach currently being evaluated. PARP-1 is an important enzyme involved in the repair of single-strand DNA breaks as a part of the base excision repair pathway. In BRCA deficient cancer cells, which are deficient in DNA damage-sensing and the homologous recombination (HR) dependent DNA-repair pathway , inhibitpatients ,56. The ® , was approved by the FDA as the first ‘personalized therapy’ for advanced BRCA1/2 mutated ovarian cancer [In 2014, the first drug targeting DNA repair defects, the PARP inhibitor Lynparzan cancer ,58. In Tn cancer . In factn cancer .BRCA1 and RAD51 [The role of PP2A in the tumor cell survival is understudied and some work has been done on PP2A role in DNA damage responses. Reports on the PR72/PR130 subunits show that these PP2A isoforms support pro-survival signaling and metastasis ,61. At lnd RAD51 . Cells wEpidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) that belongs to the ErbB family, and a transmembrane protein comprising an extracellular ligand binding domain, transmembrane domain, and cytoplasmic tyrosine kinase domain ,65,66,67TNBCs have the highest rate of EGFR overexpression with a frequency ranging from 13% to 78% ,74, depeIn fact, suppression of PP2A appeared to be associated with ErbB2-mediated carcinogenesis ,79. WongRapid progression and the development of resistance are the main challenges of the TNBC treatment. Several large retrospective analyses from individual clinical trials have demonstrated that tumor infiltrating lymphocytes (TILs) are prognostic in early-stage TNBC ,82,83,84IL-2 gene transcription by CTLA-4, indicating that PP2A represses CTLA-4 function [Programmed cell death protein 1 (PD-1) is an inhibitory co-receptor expressed on activated and exhausted T cells and B cells, monocytes and natural killer T cells. Further, cytotoxic T-lymphocyte antigen 4 (CTLA-4) and PD-1 are 2 key cell-surface receptors and suppress AKT mediated signaling upon ligation with their associated ligand  . In the function .Additionally, Villagra found that histone deacetylases (HDACs) down-regulated the expression of PD-L1 which activated the inhibitory regulation pathway PD-1 in T-cells . This noThese findings reveal the requirement for proper balance of PP2A activity in immune modulation through multiple regulatory mechanisms, including CTLA-4 and PD-1, and provide a key pre-clinical rationale and justification to further study PP2A regulators as potential immuno-modulatory agents in TNBCs.Inducing apoptosis is the major aim of chemotherapeutic treatments in TNBC, as apoptosis escape is one of the most common causes of therapeutic resistance . Gene muBcl2 is an important protein in regulating apoptosis. Although its role in therapeutic resistance in TNBC was still uncertain, low Bcl2 increased the risk of death and recurrence, and Bcl2 expression seemed to predict the outcome of treatment ,107. It Another approach for triggering apoptosis is targeting TNF-related apoptosis-inducing ligand (TRAIL). Normally, TRAIL binds death receptors 4 and 5 then triggers the death-inducing signaling complex (DISC) which phosphorylates caspase-8 and initiates the extrinsic apoptosis pathway. Many cancers including breast cancer, exhibit disruption of TRAIL-mediated signaling, contributing to chemotherapeutic resistance and tumor metastasis ,114,115.Autophagy, type II programmed cell death, is an evolutionarily conserved, self-degenerative process has been associated with both cell viability and death. It is often expressed at a basal level in cells representing its role in the recycling of proteins and tissue homeostasis . HoweverA recent study reported that B55 alpha was involved in negative regulation of autophagy by serving as a Beclin 1 phosphatase . FujiwarAdditionally, p62/SQSTM1 has an important function in promoting survival signals. In TNBC, p62 was found overexpressed and correlated with advanced stage, higher proportion of lymph nodes and lymphovascular invasion, as well as higher risk of distant metastasis . p62 canPR [TNBCs are characterized by the lack of expression of ER, PR, and HER2. Absence of the estrogen receptor alpha (ERα) in human breast cancer cells is an indicator of poor prognosis, and predictive of lack of response to hormonal therapy. In fact, PP2A has been reported to regulate ER expression through different contributing molecular mechanisms, including not only transcriptional activation at the gene promoter but rather an increased ER mRNA stability and half-life ,127. In PR . In addiPR , and furPR . FTY720 PR ,130. MosPR . TherefoKIAA1524 gene, CIP2A is overexpressed and may be prognostic in lung cancer, breast cancer, and ovarian cancer [c-MYC resulting in c-MYC stabilization and consequential proliferation. Up to now, CIP2A has been targeted using natural compounds, erlotinib derivatives or small molecules, such as celastrol, ethoxysanguinarine, bortezomib and lapatinib [Several previous studies have demonstrated that CIP2A and SET (two main endogenous PP2A inhibitors) play a role in regulating drug sensitivity in TNBCs ,134,135.n cancer ,137,138.apatinib ,140. SETapatinib ,142. Oneapatinib ,143,144.apatinib . Moreoveapatinib . ConsideTNBC is a diverse entity for which additional subclassifications beyond basal and non-basal may be needed. Recently, Lehmann et al. classified TNBC into four molecular subtypes: BL1, BL2, M and LAR based on gene expression profiles ,8. The dEstablishment of an effective therapy for TNBCs due to the restricted therapeutic strategies relative to the lack of expression of ER/PR/HER2 and intrinsic or developed resistance still remains as a great challenge in breast cancer therapy. As mentioned in this review, one of the most important concepts in targeted TNBC chemotherapy is consideration of overlapping molecular tumor-specific alterations induced by cytokines, ROS, and hypoxia. A better understanding of PP2A regulation in cell cycle, DNA damage, EGFR degradation, immune responses, cell death resistance and ER or AR regulation  gives us


Neuroendocrine tumors (NETs) are heterogeneous, widely distributed tumors arising from neuroendocrine cells. Gastrointestinal (GI)-NETs are the most common and NETs of the rectum represent 15, 2% of gastrointestinal malignancies. Poorly differentiated neuroendocrine carcinomas of the GI tract are uncommon. We report a rare case of poorly differentiated locally advanced rectal neuroendocrine carcinoma with nodal and a subcutaneous metastasis, with a cytoplasmic staining positive for Synaptophysin and Thyroid Transcription Factor-1.A 72-year-old male presented to hospital, due to lumbar, abdominal, perineal pain, and severe constipation. A whole-body computed tomography scan showed a mass of the right lateral wall of the rectum, determining significant reduction of lumen caliber. It also showed a subcutaneous metastasis of the posterior abdominal wall. Patient underwent a multidisciplinary evaluation, diagnostic and therapeutic plan was shared and defined. The pathological examination of rectal biopsy and subcutaneous nodule revealed features consistent with small-cell poorly differentiated neuroendocrine carcinoma. First line medical treatment with triplet chemotherapy and bevacizumab, according to FIr-B/FOx intensive regimen, administered for the first time in this young elderly patient affected by metastatic rectal NEC was highly active and tolerable, as previously reported in metastatic colo-rectal carcinoma (MCRC). A consistent rapid improvement in clinical conditions were observed during treatment. After 6 cycles of treatment, CT scan and endoscopic evaluation showed clinical complete response of rectal mass and lymph nodes; patient underwent curative surgery confirming the pathologic complete response at PFS 9 months.This case report of a locally advanced rectal NEC with an unusual subcutaneous metastasis deserves further investigation of triplet chemotherapy-based intensive regimens in metastatic GEP NEC. Neuroendocrine tumors (NETs) are heterogeneous, widely distributed tumors arising from neuroendocrine cells –4. GastrNETs show a metastatic spread in 30% of cases, more commonly liver, while cutaneous metastases are considered rare –16. In cEven if patients with metastatic high-grade neuroendocrine carcinomas (HGNEC) were prevalently treated with platinum-based chemotherapy, combining cisplatin or carboplatin with etoposide or irinotecan, no standard treatments and clinical management are recommended to date, nor clinical implications according to the primary site of origin, suggesting platinum-based chemotherapy as the treatment of choice .Here we describe a case report of a poorly differentiated NEC originating from rectum, with uncommon immunohistochemical features, and clinical presentation with a subcutaneous metastasis, treated with first line intensive triplet chemotherapy  plus bevacizumab according to FIr-B/FOx schedule, previously developed by our group, highly active in metastatic colorectal cancer (MCRC) patients, as other reported intensive schedules, such as FOLFOXIRI/bevacizumab –20.A 72-year-old male presented to the Emergency Room of our hospital, due to persistent lumbar, abdominal, perineal pain, and severe constipation. Due to the evidence of increased levels of pancreatic and hepatic enzymes, patient was admitted to a medical ward, and the diagnostic pathway was planned. A whole-body computed tomography (CT) scan was performed Fig. a, b and KRAS, NRAS, and BRAF genes were analysed and no mutations were detected.Patient underwent a multidisciplinary evaluation involving medical oncologist, abdominal surgeon, radiotherapist, and diagnostic and therapeutic plan was shared and defined. Endoscopic evaluation of rectum confirmed an ulcerated proliferation, located 7 cm from the anal verge, involving the bowel semi-circumference, and extending for 5 cm in the cranial direction. The pathological examination of the bioptic specimens revealed features consistent with poorly differentiated NEC, with cytoplasmic staining negative for Chromogranin A and positive for CK AE1/AE3, CD56, Synaptophysin and Thyroid Transcription Factor-1 (TTF-1). Staining for Ki-67 revealed high expression of this proliferation marker in cell nuclei, consistent with a high proliferation rate in tumor cells  day 1,15 - irinotecan (160 mg/m2) day 1,15 – oxaliplatin (80 mg/m2) day 8,22 – 5fluorouracil (900 mg/mq/day) day 1–2, 8–9, 15–16, 22–23, cycles repeated every 28 days [Due to metastatic disease, involving a rare site such as subcutaneous tissue and suspected bone metastasis, and locally infiltrating mesorectal, muscular, pre-sacral tissues, prostate gland, with lymph nodes involvement, multidisciplinary team shared the indication to first line medical treatment. Patient was young-elderly (72 years old), with intermediate Cumulative Illness Rating Scale (CIRS) score , due to  28 days . A centrA consistent rapid improvement in clinical conditions, particularly constipation and pain, were observed during treatment, and patient discontinued symptomatic administered therapies. Treatment was well tolerated, with maximum toxicities represented by G2 diarrhea, G2 asthenia, G2 nausea. Re-evaluation of disease was performed, as planned, after the first three cycles of treatment. CT scan showed a marked reduction of the rectal mass of about 70–80%, with reduction also of lymph nodes and the prostatic involvement Fig. c, d. No Due to good safety profile of administered treatment, the marked and rapid improvement of symptoms related to primary tumor, and the metastatic extension of disease, multidisciplinary team shared the indication to continue the same medical treatment for other three cycles. Median received dose-intensity was 100% for all associated drugs. Maximum toxicities were represented by G1 nausea, G1 rhinitis, G1 epistaxis, G1 neurotoxicity, G1 asthenia, G3 alopecia, and G3 neutropenia. CT scan showed further improvement of the marked reduction of the rectal mass, and lymph nodes and prostatic involvement disappeared  , distingIncidence of NECs is 1000 cases annually; 11% within the GI tract , with a Pathological diagnosis, including immunohistochemical and molecular markers, is of key relevance: currently, CgA and Synaptophysin are the most specific immunohistochemical markers for NETs. CgA may have limited sensitivity with some of them, such as tumors of the transverse and distal colon, rectum and anus, that have been found to stain in only 20–50% of cases –43. In tIn addition to degree of differentiation and proliferation of the tumor, also the presence of metastases or lymph node involvement are reliable markers to predict tumor growth and survival. Approximately 50% of the patients shows synchronous metastatic disease , 52, 53.Here, we reported a case of a small-cell rectal NEC with a very uncommon, subcutaneous tissue metastatic site. Cutaneous metastases are more commonly present in breast, lung, colon, stomach, uterus and kidney neoplasia , while sAmong gastroenteropancreatic (GEP) NEC, more studies suggested a worse prognosis of colorectal compared to pancreatic NEC –64, evenp < 0,001) [Ki-67 seemed to play a role to define the proper treatment strategy of GEP and pancreatic NEC. A multicenter, retrospective study of the NORDIC group identified among a population of 305 advanced GEP-NEC (71 pancreatic NEC) treated with first line platinum-based chemotherapy, two subgroups with different prognosis: the former with Ki-67 ≤ 55%, reported objective response rate (ORR) 15%, median overall survival (OS) 14 months; the latter with Ki-67 > 55%, higher ORR 42%, and significantly worse OS 10 months  .A retrospective, multicenter, analysis among 136 patients affected by NEC of different origins, defined three subgroups with different prognosis: well differentiated and Ki-67 20–55%, poorly differentiated and Ki-67% 20–55%, poorly differentiated and Ki-67 > 55%, reporting median OS 43.6, 24.5, and 5.3 months, respectively [To date, no standard treatments and clinical management are recommended in high-grade neuroendocrine carcinomas (HGNEC), nor clinical implications according to the primary site of origin, suggesting platinum-based chemotherapy as the treatment of choice . In a reKRAS/NRAS/BRAF genotype [To date, clinical management of metastatic colorectal cancer (MCRC) patients faces with different options and lines of treatment , 71, accgenotype , 77. Eldgenotype .KRAS exon 2 wild-type and mutant, 38 and 21 months, respectively [RAS mutant, 13.4 months in BRAF mutant [KRAS2–4/NRAS2–4/BRAF15 wild-type and mutant patients treated with FIr-B/FOx, median PFS was 18 and 12 months, median OS 28 and 22 months, respectively, not significantly different [We previously developed FIr-B/FOx schedule adding bevacizumab (BEV) to triplet chemotherapy, reaching objective response rate (ORR) 82%, progression-free survival (PFS) 12 months, overall survival (OS) 28 months, even effective in young elderly patients , 79, 80,ectively , 76, 77,ectively : median F mutant . Among KFIr-B/FOx schedule may achieve preferable toxicity profile, particularly in terms of limiting neutropenia, compared to FOLFOXIRI/BEV , 78, 79.KRAS/NRAS/BRAF wild-type, treated with intensive first line FIr-B/FOx regimen. Patient was symptomatic for the presence of primary locally advanced rectal carcinoma, and rapidly experienced a consistent clinical benefit. Received DI was 100% of planned DI, safety profile was acceptable, with no reported LTS. Complete response was reported after 6 cycles of treatment: CT scan showed > 85% reduction of the rectal mass, disappearance of lymph nodes and prostatic involvement; no evidence of the rectal mass at the endoscopic evaluation. Patient underwent curative surgery that confirmed a pathologic complete response without residual neoplastic cells at the level of distal rectum and 32 resected locoregional lymph nodes, at the actual PFS 9 months.Here, we reported clinical management of a young-elderly patient, with intermediate CIRS stage, PS1, subcutaneous metastasis with lymph nodes and prostatic gland involvement, affected by an undifferentiated rectal carcinoma with neuroendocrine phenotype, Thus, our present case reported an unusual locally advanced rectal NEC with nodal and a subcutaneous metastasis; FIr-B/FOx intensive regimen administered for the first time in this young- elderly patient affected by metastatic rectal NEC was highly active and tolerable as previously reported in MCRC. This case report confirmed the need for further investigation of triplet chemotherapy-based intensive regimens in metastatic GEP-NEC, reporting high activity and increased clinical outcome in metastatic GI cancers , 83, 84.


HAND2-AS1, XIST, RP1-97J1.2, AC010084.1, TTTY15) were identified by a sex-bias comparison. Splicing events were detected within 37 genes and 4 lncRNA loci. Functional analysis of cis-related potential targets for lncRNAs identified 2021 enriched genes. It is presumed that the obtained data will expand the current knowledge of lncRNAs in placenta and human non-coding catalogs, making them more contemporary and specific.Development of particular structures and proper functioning of the placenta are under the influence of sophisticated pathways, controlled by the expression of substantial genes that are additionally regulated by long non-coding RNAs (lncRNAs). To date, the expression profile of lncRNA in human term placenta has not been fully established. This study was conducted to characterize the lncRNA expression profile in human term placenta and to verify whether there are differences in the transcriptomic profile between the sex of the fetus and pregnancy multiplicity. RNA-Seq data were used to profile, quantify, and classify lncRNAs in human term placenta. The applied methodology enabled detection of the expression of 4463 isoforms from 2899 annotated lncRNA loci, plus 990 putative lncRNA transcripts from 607 intergenic regions. Those placentally expressed lncRNAs displayed features such as shorter transcript length, longer exon length, fewer exons, and lower expression levels compared to messenger RNAs (mRNAs). Among all placental transcripts, 175,268 were classified as mRNAs and 15,819 as lncRNAs, and 56,727 variants were discovered within unannotated regions. Five differentially expressed lncRNAs ( The placenta serves as a metabolic, respiratory, excretory, and endocrine organ, whose proper functioning is required for adequate embryonic development during pregnancy . Fetal gSpatiotemporal expression is a huge impediment in any transcriptome analysis, especially in the placenta, an organ that constantly adapts to feto-maternal environmental alterations. Comprehensive analysis of messenger RNA (mRNA) expression in first- and second-trimester placentas compared to term placentas by microarray assay revealed more genes with increasing than decreasing expression . FurtherPrevious studies implied that risk factor profiles for various pathologies are different between singleton and twin births . FurtherThe great progress of RNA sequencing (RNA-Seq) and the capabilities of numerous bioinformatic approaches make it a powerful technology for thorough transcriptome analysis, which enables characterization of gene expression, alternative splicing events, large-scale discovery of novel transcripts, Single Nucleotide Variant (SNV) prediction, and functional annotation ,3,11,12.Disruptions to adaptive changes in the placental transcriptome as a response to altering the feto-maternal environment may be associated with pregnancy complications and compromised fetal outcomes. In this context, defining differences in placenta-specific gene expression regarding the sex of the fetus and the multiplicity of gestation could contribute to the understanding of placental development and function. Since revealing factors that influence the placental expression profile is necessary, this study was conducted to examine whether there are differences in the transcriptomic profile of the human placenta compared for sex of the fetus and number of fetuses. A stringent pathway was applied to identify, analyze, and compare placental transcriptome from male and female fetuses during single and twin pregnancies. This study focused on the lncRNA profile to investigate possible mechanisms regulating the expression profile of the human placenta.In total, 2 × 119,560,140 raw paired-end reads were generated, and subsequently 2 × 109,363,183 reads were acquired after trimming. The 218,726,366 clean reads were mapped to a reference human genome, and an average of 86.14% reads were mapped uniquely. Among all mapped transcripts 258,353; , 67.84% Moreover, 1.48% of the expressed transcripts were derived from other RNAs  and 21.96% originated from unannotated regions, which included potentially new lncRNA transcripts a. After The dynamic range of the expression values was calculated and is presented as a box plot of logarithmic transformed FPKM values for each sample separately a, and thAn lncRNA profile of human term placenta was identified and characterized by applying a stringent pathway . GENCODEExpression levels of antisense, lincRNA biotype classes, and newly discovered lncRNAs were comparable .In the current study, 5453 lncRNA and 65,024 mRNA transcripts with FPKM were identified >1. The lncRNA and mRNA transcripts were compared for their total length, exon length, exon number, and expression level . The avep-adjusted < 0.05) and 21 protein-coding genes (The expression level (FPKM) of long non-coding transcripts was estimated for both sex and multiplicity biases. A sex-bias comparison revealed five differentially expressed lncRNAs (ng genes . Among tng genes . The mulPSG4). Three transcripts with multiple distinct exonic regions, Rho GTPase activating protein 45 (ARHGAP45); GATA binding protein 2 (GATA2), and long non-coding RNA (RP11-440I14.3), were also indicated. Four genes, peptidylprolyl isomerase G (PPIG), HLA class II histocompatibility antigen DRB5 beta chain (HLA-DRB5), torsin 1A interacting protein 1 (TOR1AIP1), and cysteine and serine rich nuclear protein 1 (CSRNP1), displayed simultaneous differential exon and splice-junction usage. Among all AIR/DTU events, four significant differential usages of exons were localized within lncRNA loci: H19, AC132217.4, RP11-440I14.3, and AC005154.6  genes, also known as differential transcript usage (DTU). As a result, differentially expressed exons and altered spliced patterns of placental transcripts were detected . Comparing the placental transcriptome from male and female samples revealed 37 AIR/DTU genes displaying 38 and 8 statistically significant differential exon and splice-junction usages, respectively. The use of the JunctionSeq analysis tool led to the detection of new splice junctions in the gene encoding pregnancy-specific β-1-glycoprotein 4  from the identified lncRNAs were considered as target genes, and the approach produced 2021 genes. Those genes closely related to lncRNAs were analyzed for Gene Ontology (GO) enrichment, as shown in cis-target genes were enriched (p < 0.05) to biological process (148 terms), cellular component (56 terms), and molecular function (20 terms) according to GO classification. GO annotation showed that 61 and 107 protein-coding cis-target genes were enriched in in utero embryonic development and vasculature development, respectively. Within the cellular component GO category, 1772 and 1684 cis-targets were assigned according to cell and intracellular compartments  and known (2899) lncRNA loci predicted in this study . For extPlacenta fine-tunes the expression of various genes involved in major molecular mechanisms essential in pregnancy maintenance and fetal development ,3,28. FoAmong the placental transcripts obtained in our study, 67.84% 175,268) were classified as mRNA, 6.12%  as lncRNA, and 2.60% (6726) as pseudogenes a. In all5,268 werHAND2-AS1 and X chromosome inactive–specific transcript (XIST), displayed higher expression levels in female libraries and three others, RP1-97J1.2, AC010084.1, and TTTY15, were expressed solely in male libraries. XIST as X chromosome–specific was highly enriched in the female libraries. XIST is a kind of functional lncRNA uniquely involved in the formation of repressive chromatin and regulation of the X chromosome inactivation process by cis action [XIST’s expression occurs in a spatiotemporal manner, regulating and influencing female development [HAND2-AS, as antisense to HAND2, may regulate its expression. HAND2 is a kind of transcription factor that plays a key role, e.g., in vascularization, development, and differentiation of sympathetic neurons [HAND2 fosters a level of fibulin-1, which contributes to progesterone action during implantation [HAND2 and XIST exhibit more variants: 11 and 9, respectively. Therefore, fetal sex-specific expression of the aforementioned lncRNAs and their variants in the placenta might impact proper placental development and function. That is why further molecular insights into their function must be gained to fully discover their implication in pregnancy outcome. There were 21 protein-coding genes differentially expressed in female and male term placentas. Among them, microsomal glutathione transferase 1 (MGST1) was identified to have a confirmed role in oxidative stress protection [RXFP1) a receptor for relaxin, a key hormone in mammalian pregnancy [SEMA3A) play essential roles in preventing nerve fiber growth in the placenta to protect the fetus from external stress [The expression of mammalian lncRNAs is strictly associated with their regulatory role in a tissue-specific manner. Among various tissues, the testis and ovary were indicated as the most enriched in lncRNAs , suggests action ,42,43,44elopment . HAND2-A neurons ,47. Moreantation ,49,50. Uantation , but HANotection ; relaxinregnancy , and seml stress .Previous transcriptomic studies performed on beaver discoid placenta revealed that there are differences in gene expression between twin and triple pregnancies and that the number of fetuses may affect pregnancy outcome . It was PSG4). Pregnancy-specific glycoproteins (PSGs) are a specific group of highly expressed trophoblast genes crucial for placentation, acting as regulators of trophoblast cell migration, cytokine secretion, and the establishment of uteroplacental circulation [PSG loci (PSG1–PSG11) are enriched with various types of copy number variations, which may be linked with impaired fertility and pregnancy complications such as preeclampsia [Alternative isoform regulation (AIR) can enhance transcriptome diversity and gain another biological function of a single gene by events such as alternative splice sites, alternative transcription start sites, methylation, nucleosome occupancy, internal promoters, nonsense-mediated decay, and/or transcript switching . Alternaculation . PSGs arculation . A decreculation ,59,60. Hclampsia .ArhGAP45 , which functions as a Rho GTPase [ArhGAP45 mRNA expression is elevated in preeclamptic placentas and is under the control of oxygen accessibility [GATA2) regulates stage-specific trophoblastic gene expression of the preimplantation human embryo [Multiple distinct exonic regions were detected in o GTPase ,63. Rho o GTPase , embryogo GTPase , neural o GTPase , cytokino GTPase . ArhGAP4sibility . GATA bin embryo ,70,71.H19, a placenta-specific lncRNA highly expressed during mammalian embryonic development [H19 is implicated in the regulation of human placenta trophoblast proliferation, placental development [H19 expression may support normal pregnancy, while its impaired regulation might promote preeclampsia, early-onset preeclampsia (EOPE), and IUGR [AC132217.4 lncRNA, because it affects 3′UTR and enhances expression level, fosters mRNA stability and upregulates expression of IGF2 circulating growth factor, which acts during pregnancy to promote both fetal and placental growth [A substantial contribution of lncRNAs in placental formation and function is well known; an evident example is elopment ,73,74. Helopment ,76, and elopment ,78. Moreand IUGR ,79,80. Al growth .RP11-440I14.3, localized in cis position to hydroxyprostaglandin dehydrogenase (HPGD). Hydroxyprostaglandin dehydrogenase inactivates prostaglandins E2 (PGE2) and D2 (PGD2), which affect several biological processes, such as reproduction, differentiation, and inflammation [Differential usage of exons was also detected in lincRNA ammation . In the cis-target genes. A variety of subclasses of ncRNAs, like piRNA, miRNA, siRNA, and lncRNA, have regulatory roles in gene expression [cis-related potential targets for lncRNAs identified 2021 genes. The 61 protein-coding genes were found to be regulated by lncRNA transcripts, and GO enrichment showed that they were enriched in in utero embryonic development (GO:0001701), suggesting that predicted lncRNA functions during pregnancy are linked with developmental, growth, and regulation related processes. Generally, annotation with GO terms displays many of the placentally expressed lncRNA transcripts involved in the regulation of various biological processes also implicated in the gestation course. GO analysis was applied to explore the function of the pression ,85,86,87Taken together, since the functions of the majority of lncRNAs have yet to be uncovered, tremendous effort should be made to decipher their implication in the course of gestation, placental development, and reproductive disorders. The present research may be used as a resource for functional studies, which is a huge challenge in determining the influence of lncRNAs on reproductive processes. The authors’ previous study  establisn = 2) and pregnancy multiplicity (n = 2). All procedures regarding tissue collection, the characteristics of placental samples (n = 4), RNA extraction, and RNA-Seq were described previously [The lncRNA expression profile of human term placenta was compared between the sex of the fetus  mapper. As a result, a BAM file alignment of the trimmed reads to the reference genome was obtained for each sample. StringTie v. 1.0.4 (https://ccb.jhu.edu/software/stringtie) [http://cole-trapnell-lab.github.io/cufflinks) [The quality of reads was checked using the FastQC tool. Preprocessing using a Trimmomatic tool v. 0.32  includedringtie)  and Cufffflinks) , were aphttps://www.gencodegenes.org/) “lincRNA”, “antisense”, “sense_intronic”, “sense_overlapping”, “bidirectional_promoter_lncRNA”, “non_coding”, “macro_lncRNA”, “TEC” , and “3prime_overlapping_ncRNA”); and (2) potentially non-coding RNA (unannotated transcripts and other than lncRNAs). The second dataset, including unknown non-coding sequences, was reduced by removing a transcript assigned to the “protein_coding” Ensembl class code. Next, these transcripts were subjected to multi-exon filtering. Transcript coding potential was assessed by several tools: The Coding Potential Calculator (CPC) (http://cpc.cbi.pku.edu.cn) [https://pfam.xfam.org) [http://rna-cpat.sourceforge.net) [https://github.com/www-bioinfo-org/CNCI) [https://sourceforge.net/projects/plek/files) [e-value 10−5; release 27), CPAT (cutoff < 0.43), CNCI (cutoff < 0), and PLEK (cutoff < 0). Further, surviving transcripts were searched in Rfam using Blast2GO software (https://www.blast2go.com) [Low-expressed transcripts with FPKM values ≤ 1 (expression sum of 4 libraries) were excluded from the set of merged transcripts. Next, all transcripts longer than 200 nt were passed for further analysis . Selecte.edu.cn) , Pfam (hfam.org) , CPAT (hrge.net) , Coding-rg/CNCI) , and PLEk/files) . CPC (sc2go.com) , to exclp-values were determined for 2 comparisons: sex and multiplicity bias in placental tissue. Thresholds for significantly different expression were set as follows: p-adjusted < 0.05 and log2 fold change (log2FC) ≥ 1.0. A structural comparison between lncRNA and mRNA transcripts was performed by custom R bioconductor scripts. The QoRTs/JunctionSeq pipeline [p-adjusted < 0.05).The reads assembled to mRNA and lncRNA sequences were normalized to FPKM values using Cuffnorm. Applying Cuffdiff, the corresponding pipeline  was adopcis interactions were predicted, since the cis role refers to the influence of lncRNAs on vicinity target genes localized within 2000 nt upstream or downstream of each protein coding gene on the same chromosome. Functional enrichment analysis (p-adjusted < 0.05) of the potential cis target genes was performed by Kobas 3.0 software (http://www.kobas.cbi.pku.edu.cn) [http://www.geneontology.org).Based on the localization of lncRNA in relation to mRNA, .edu.cn)  includin


Chronic pancreatitis presents a high risk of inflammation-related progression to pancreatic cancer. Pancreatic cancer is the fourth leading cause of cancer-related death worldwide. The high mortality rate is directly related to the difficulty in promptly diagnosing the disease, which often presents as overt and advanced. Hence, early diagnosis for pancreatic cancer becomes crucial, propelling research into the molecular and epigenetic landscape of the disease.Recent studies have shown that cell-free DNA methylation profiles from inflammatory diseases or cancer can vary, thus opening a new venue for the development of biomarkers for early diagnosis. In particular, cell-free DNA methylation could be employed in the identification of pre-neoplastic signatures in individuals with suspected pancreatic conditions, representing a specific and non-invasive method of early diagnosis of pancreatic cancer. In this review, we describe the molecular determinants of pancreatic cancer and how these are related to chronic pancreatitis. We will then present an overview of differential methylated genes in the two conditions, highlighting their diagnostic or prognostic potential.Exploiting the relation between abnormally methylated cell-free DNA and pre-neoplastic lesions or chronic pancreatitis may become a game-changing approach for the development of tools for the early diagnosis of pancreatic cancer.The online version of this article (10.1186/s13148-019-0728-8) contains supplementary material, which is available to authorized users. Chronic pancreatitis (CP) represents a spectrum of persisting fibro-inflammatory disorders of the exocrine pancreas that alter the organ’s typical structure and functions, and significantly reduce patients’ quality of life . GeneticThe persistent inflammatory state in CP promotes accelerated tissue repair, which may result in neoplastic (trans)formation. For this reason, CP represents one of the highest risk factors for the development of pancreatic tumors (PTs) , 7. PT iIn the absence of an underlying PT, the lag period between CP diagnosis and tumor is usually one or two decades , 12. ThiKRAS already in the early 90s [The best-known biomarker for PT is the serum protein-carbohydrate antigen 19-9  . When rearly 90s , and imparly 90s , 17. A marly 90s –20. Its arly 90s  and are arly 90s –24. FurtIn this review, we provide an overview of the molecular determinants of PT and the genes showing differential DNA methylation in CP, pre-neoplastic lesions of the pancreas, and PT. In particular, we will compare studies conducted either on primary tissue biopsies or from biological fluids. We aim to decipher disease-specific methylation patterns in pancreatic diseases to serve as a novel diagnostic or prognostic tool for PT. The identification of a “pancreatic disease signature” to distinguish between inflammation and cancer could hopefully enhance non-invasive tools for the early diagnosis of PTs.The pancreatic adenocarcinoma develops almost exclusively from the exocrine pancreatic ductal epithelium cells accounting for 85% of all PTs. Other PTs include the acinar cell carcinoma originating from the exocrine acini of the pancreas, the pancreatic neuroendocrine tumors (PNETs) arising from neuroendocrine cells, and other minorities. For its abundance, we will hereafter use the term PT to refer to pancreatic ductal adenocarcinoma, unless otherwise specified.Molecularly, a variety of genetic and epigenetic events underlie the development of PT Fig. .Fig. 1GKRAS in about 90% of the cases [CDKN2A and TP53 are found [KRAS, they often present mutations in genes involved in the Wnt signalling pathway  [Pre-neoplastic non-invasive lesions are thought to be the first stage of PT etiopathogenesis. Low-grade pancreatic intraepithelial neoplasia (PIN) is the most frequent microscopic lesion of the pancreas, harboring mutations in the he cases , 32. As re found . Intradud RNF43) . Other td RNF43) .KRAS mutation is absent in most PNETs, while frequent mutations in these tumors occur in DAXX , MTOR , and MEN1 genes [Genes mutated in PNETs are generally different from those found in exocrine PTs . For exaN1 genes .SMAD4 (about 55% of the cases) and genes of the SWI/SNF chromatin remodeling family (∼ 15% of the cases) [At the invasive stage, PTs present mutations in e cases) , 8. A mee cases) , and thee cases) , 37. Thee cases) –41.Inflammatory stimuli may lead to aberrant DNA methylation homeostasis and, coherently, to gene expression changes , 42–44. KRAS mutation at known tumor-promoting sites has so far been observed in AIP [STK11, a known risk factor for PTs, has recently been described in CP [Conversely, no d in AIP . Finallyed in CP . In the ed in CP . For theCACNA1G, CDH1, CDKN2A, DAPK1, MGMT, MINT1-2-31-32, MLH1, RARB, THBS1, and TIMP3) was analyzed in PT xenografts [MGMT, which was non-methylated in either neoplastic or normal samples. Simultaneous methylation of at least four loci was a feature of ∼ 14% of PT xenografts. Overall, the most frequently methylated loci were MINT32, MINT1, and MINT28. Moreover, seven CpG islands (CGIs), of which three associated to the known CCNG1, PENK, ZBP genes, were found differentially methylated in pancreatic-derived cell lines compared with the healthy pancreas and validated in PTs [PENK, CGI was found methylated in 91% of cases. Of note, five specimens of CP were also analyzed, showing PENK CGI methylation in two cases [The first studies comparing cancerous and healthy pancreatic tissues analyzed the methylated status of known tumor-suppressors and cancer-associated genes. Also, specific CpG islands spread within the genome were found preferentially methylated in tumors (MINT loci). In particular, the DNA methylation status of CpG islands embedded in several gene promoters , and CP specimens [PENK methylation was present in 93% of invasive PTs, of which 27% presented CDKN2A methylated. In contrast, non-neoplastic specimens resected from matched healthy tissues did not harbor methylation of either gene. Noteworthy, the prevalence of PENK methylation increased significantly with increasing lesion grade . Only one of the extra-ductal PTs presented CDKN2A methylation, while CP specimens had neither gene methylated. Interestingly, as regarding CDKN2A, available data suggest that hypermethylation associated with the loss of CDKN2A expression might occur only in CP in which low-grade PINs were observed [CDKN2A expression was found in this study independently of methylation status.pecimens , 54. PENobserved . In lineThese data support the disease stage-specific DNA methylation model and qualify these genes as potential biomarkers of early pancreatic carcinogenesis.SOCS1 methylation. In contrast, none of the pancreatic normal ductal epithelia and the PINs that were analyzed presented SOCS1 methylation [CDKN2A (p16INK4a specific region) and TP73 methylation, independently of their grade of invasiveness. Conversely, CDKN2B, ESR1, and TIMP3 were methylated in none of the examined specimens. More frequent methylation of APC (50% vs. 10%), CDH1 (38% vs. 10%), MGMT (45% vs. 20%), and MLH1 (38% vs. 10%) was observed in invasive IPMNs, compared to non-invasive samples. Unfortunately, the weakness of these findings results from the limiting size of the non-invasive IPMN subpopulations [Comparing invasive PTs to IPMNs, 6% of IPMNs and 22% of invasive PTs presented hylation . In a prhylation , providi%), MGMT % vs. 20%A progressive increase of the DNA methylation of at least one gene and the average number of methylated genes was observed from PIN to PT in 58 patients who had undergone resection surgery for invasive PTs.BRCA1, APC, CDKN2A, and TIMP3 were methylated in 60%, 59%, 39%, and 31% of the PT cases, respectively [CCND2, CDKN2A, FOXE1, NPTX2, PENK, TFPI2) were analyzed in AIP specimens [The impact of the inflammatory environment on DNA methylation was assessed. The data showed that DNA methylation also increases with inflammation of pancreatic tissue. ectively . Finallypecimens , showingCLDN4, LCN2, MSLN, PSCA, S100A4, TFF2, and YWHAS) were frequently hypomethylated in PT xenografts. In particular, five or six genes showed simultaneous DNA hypomethylation in 92% or 61% of the cases, respectively. Moreover, from 379 identified loci hypomethylated in PT with respect to healthy pancreas [FOS, JUNB, and MYB; the genes NDN and SMARCA1 [CTR9, EP400, HIRIP3, KDM6A, KMT5A, and PRMT1 were identified. These genes presented increased expression in PT specimens playing a role in core signalling pathways of PT [Evidence of DNA hypomethylation events in PTs is also reported –60. A pa SMARCA1 ; and theIn Additional file CDH3, CLDN5, FOXE1, LHX1, NPTX2, RPRM, SFRP1, ST14, TJP2, UCHL1, and WNT7A) markedly induced after treatment with 5-aza-2-deoxycytidine (5Aza-dC), a DNA demethylation agent, were specifically identified in pancreatic tumor cell lines [CLDN5, NPTX2, SFRP1, UCHL1) were methylated in at least 93% of the screened specimens, while only two (CDH3 and ST14) presented methylation in less than 20% of samples.The advent of genomic technologies improved the screening capability by several orders of magnitude, providing promising results. Using a high-throughput microarray approach, 11 genes  was validated in PT showing that for 20 genes DNA methylation was, on average, 50% more frequent in PTs. Only for EFNA4, BTBD6, and FZD2-7 DNA methylation levels were comparable between the two conditions [BNC1 and ADAMTS1. Quantitative MSP (qMSP) validation confirmed the results. Interestingly, BNC1 methylation was detected in PINs, while ADAMTS1 methylation was exclusively found in invasive PTs. Importantly, these two genes showed increased DNA methylation in pre-neoplastic PINs, with little to no DNA methylation in CP or healthy pancreas [CD1D, CLEC11A, IKZF1, KCNK12, NDRG4, PRKCB) were further selected and tested as diagnostic biomarkers in cfDNA specimens (as described in next section) [Further application of this strategy resulted in the identification of > 1200 known loci that presenting specific methylation in PT. Many of these loci belonged to Wnt signalling pathway or the homeobox and the cadherin superfamilies . Analysinditions . A transnditions . This gepancreas , thus unpancreas . Analysisection) .C5orf38, DLX4, ELAVL2, EMX1, IRX1, NPR3, PITX2, SIM2, TBX5, TFAP2C, and VSTM2B) were further validated by target-specific methods  [NGS was also utilized to identify differentially DMRs in healthy and neoplastic samples. Aberrantly methylated CGIs were more frequent in PT, compared to the healthy pancreas, and DNA hypermethylation events in PTs typically occurred in the vicinity of the transcription start site (TSS). Several individual DMRs (including ase PCR) . It is wMIR124 genes and MIR10B showed a higher mean CpG methylation fraction in PTs compared with matched non-cancerous tissues. Hypermethylation induced the silencing of MIR124, which was associated with poor prognosis. The functional implications of such findings derive from MIR124 role in inhibiting cell proliferation, invasion, and metastasis. Among MIR124 direct targets, stem Rac1, a putative PT-promoting factor that activates JNK-dependent cell motility [Three motility –70.MIR210 and MIR130B displayed PT-specific DNA-hypomethylation. Supporting evidence relates to high levels of MIR210 to PT progression, epithelial-to-mesenchymal transition, and adverse prognosis in cancer patients [MIR130B, its DNA methylation levels [MIR130B is reported to inhibit cell proliferation and invasion in PTs by direct targeting of STAT3 [Also, patients , 72. Forn levels  and the of STAT3 , increasof STAT3 .To find a diagnostic or prognostic tool relevant for PT, several studies, rather than analyzing only one gene, combined many candidate genes into aggregate biomarkers. The methylation levels of the different genes in the aggregate were adopted to differentially diagnose pre-neoplastic conditions or PT from different biological fluids, such as pancreatic juice and blood, as indicated in Table CLDN5, NPTX2, and SFRP1) showed the potential to be employed as aggregate biomarkers [While in PT solid biopsies, six methylated genes were identified to be predictive of advanced disease; in pancreatic juice, only three of them , compared to the normal pancreas (n = 11). Further, the number of methylated genes in the high-risk PT group (n = 44) was more abundant than in healthy controls but similar to patients with chronic pancreatitis (n = 11). Combination of the five most discriminating assays  scoring DNA methylation in more than one gene was highly predictive of PT: nine of 11 patients with PT but none of the 64 individuals without detectable pancreatic neoplasia presented DNA methylation signal (82% sensitivity and 100% specificity for PT). Interestingly, these genes were previously identified in several studies conducted on pancreatic biopsies. Although none of the target genes was useful to distinguish CP from healthy pancreases, this aggregate biomarker successfully discriminated PTs from other pancreatic lesions.For example, Matsubayashi et al. investigated pancreatic juice endoscopically or surgically collected from individuals with suspected pancreatic diseases, including CP and benign pancreatic lesions . QuantitKRAS status, on secretin-stimulated pancreatic juice samples from 61 PT patients, 22 CP patients, and 19 with healthy pancreas [KRAS had a sensitivity of 56% and 39% (at 90% specificity) in discriminating PTs or CP from healthy pancreases, respectively. Overall, the other biomarkers performed better: the sensitivity (at 90% specificity) in discriminating PT from healthy pancreas was 79% for CD1D, 67% for CLEC11A, 62% for IKZF1, 79% for KCNK12, 72% for NDRG4, and 67% for PKRCB. The sensitivity (at 90% specificity) in discriminating PT from CP was generally lower: 53% for CLEC11A, 54% for IKZF1, 46% for KCNK12, and 67% for NDRG4. Of note, CD1D displayed a better discriminating potential between PT and CP (84%), while PKRCB methylation was worse (38%) than mutant KRAS at distinguishing PTs from CPs.In their clinical pilot testing, Kisiel et al. assessed the DNA methylation levels of six previously identified candidate biomarkers, together with mutant The collection of pancreatic juice is a relatively complex procedure, and it might not be appropriate as a standard approach for early detection in asymptomatic individuals. For this, the analysis of cfDNA in plasma specimens received increasing inputs.CCND2, PLAU, SOCS1, THBS1, and VHL) and combined in an aggregate biomarker. This test assesses DNA hypomethylation events, which makes comparison with techniques that detect cancer-related hypermethylation difficult. The assay distinguished healthy pancreas from PTs with 76% sensitivity and 59% specificity. Previous findings had reported CCND2, SOCS1, and THBS1 hypermethylation as informative for cancer detection in pancreatic juice or tissues [Melnikov et al. examined plasma specimens of PT patients with a panel of 56 frequently methylated genes . By mean tissues , 55, 79.BRCA1, CCND2, CDKN1C, MLH1, proximal and distal PGR promoter regions, SYK, and VHL) were useful to distinguish healthy pancreas from CP, with a sensitivity and a specificity of 82% and 78%, respectively. For all genes in this panel, promoter DNA methylation was more frequent in individuals with CP. Fourteen genes  were able to distinguish CP from PTs with a sensitivity and a specificity of 91%. It is worth noting that all genes that were hypermethylated in CP displayed hypomethylation in PT. As CP often precedes PT, dynamic DNA methylation patterns for a given set of genes might underlie the progression of the disease. Validation in larger independent cohorts would be critical to confirm these findings.In a follow-up study by Liggett et al., 17 gene promoters were examined to identify differential cfDNA methylation in PT and CP patients . Eight gCDKN2A, NPTX2, PENK, SFRP1, RASSF1A, and UCHL1) in the plasma of individuals with PT and CP [CDKN2A, specifically methylated in PT but not in CP.Park et al. investigated the DNA methylation levels of six genes (T and CP . The invn = 42) would harbor BNC1 and ADAMTS1 gene methylation while healthy subjects (n = 26) would not [BNC1 methylation, while 20 of 42 showed ADAMTS1 methylation, with a sensitivity of 79% and 48% for BNC1 and ADAMTS1, respectively. Specificity was 89% for BNC1 and 92% for ADAMTS1. Combining both genes slightly improved the overall sensitivity (81%) but worsened the overall specificity (85%).Scarcely abundant cfDNA is often a limiting factor for many studies. To overcome such issue and improve the sensitivity of DNA methylation detection, Yi et al. employed a single-tube high-yield collection method, termed methylation on beads (MOB) . Based oould not . Furthern = 95), CP (n = 124), and acute pancreatitis (n = 59) from plasma specimens [APC, BMP3, BNC1, MESTv2, RASSF1A, TFPI2, and SFRP1-2) and patient’s age (> 65). Although not quantitative, this model successfully distinguished malign from benign conditions with a sensitivity of 76% and a specificity of 83%, independently of the tumor stage.A recent prospective study from Henriksen et al. employed a panel of 28 genes to assess the DNA methylation state in patients with PT  [CCND2) [APC, BRCA1, CLDN5, LHX1, RPRM, SFRP1, and TJP2, that progress to a state of increased methylation in invasive cancer, can be considered biomarkers of an aggressive stage of the disease [For some genes, DNA methylation may indeed mirror different stages of the disease progression  , 56, 65.ctively) , yet the [CCND2) . On the  disease , 63.This review aimed to focus on the present advances in liquid biopsy for PT to establish predictive aggregate biomarkers that provide benefits to patients. Many candidate genes have been discussed in this review, the majority of which are hypermethylated as the pancreatic disease progresses. Interestingly, the use of new technologies and array-based screening employed to analyze DNA methylation status indicated that hypomethylation events, rather than hypermethylation, could be more informative because their occurrence appears to be independent of the inner tumor heterogeneity . BesidesAdditional file 1:Table S1. List of genes presenting differential DNA methylation between healthy pancreas, chronic pancreatitis or pancreatic cancer. CP: chronic pancreatitis; HiR: high PT-risk; IPMN: papillary mucinous neoplasms; PIN: pancreatic intraepithelial neoplasias; PT: pancreatic adenocarcinoma; PTX: pancreatic adenocarcinoma xenografts. Hyper:DNA hypermethylation; hypo: DNA hypomethylation; –: no change; inv.: invasive; *: non-significant; #: cohort size <10; §: p16INK4a specific region; $: p14ARF specific region; £: associated with longer survival. Grey background: genes analysed in both primary and liquid biopsies (XLS 57 kb)


Mangifera indica L. traditionally used in Cuba for its analgesic, anti-inflammatory, antioxidant and immunomodulatory properties. Several formulations are available, and also for mangiferin, its major component. Preclinical studies demonstrated that these products prevented tumor necrosis factor α -induced IκB degradation and the binding of nuclear factor κB to DNA, which induces the transcription of genes implicated in the expression of some mediators and enzymes involved in inflammation, pain, oxidative stress and synaptic plasticity. In this paper we propose its potential utility in the neuropathic pain treatment. This hypothesis is supported in the cumulus of preclinical and clinical evidence around the extract and mangiferin, its major component, and speculates about the possible mechanism of action according to recent advances in the physiopathology of neuropathic pain.It has been accepted that neuroinflammation, oxidative stress and glial activation are involved in the central sensitization underlying neuropathic pain. Vimang is an aqueous extract of  In linethan 50% . On the than 50% . Tumor n as well ,11. Thishibitors ,13. Moreic cleft ,14. SubsMangifera indica L.; MSBE), which contains a definite mixture of components including poly-phenols, triterpenes, phytosterols, fatty acids and microelements [2 (PGE2) production, free radicals formation and reduction of cyclooxygenase-2 (COX-2) synthesis induced by lipopolysaccharide [Vimang is the brand name of a standard aqueous stem bark extract of selected varieties of mango  of nociception in the 1% formalin model was 8.4 mg/kg b.w., but the early phase (0-5 min) was not affected. In acetic acid-induced writhing in mice, a somato-visceral pain model, the ED50 was 54.5 mg/kg b.w. [The ED/kg b.w. . Both li/kg b.w. . 2+ influx induced for NMDA activity triggers enzymatic cascades, which finally also evoke the release of prostaglandins [4 (LTB4) and PGE2 synthesis in vitro and showed an inhibitory effect of topical arachidonic acid- or PMA-induced edemas, in in vivo models sensitive to test inhibitors of lipoxygenase and cyclooxygenase, respectively [The exclusive inhibition of phase II of formalin 1% test for the MSBE is an element that suggests its capacity to modulate a pathological and persistent painful status more than acute nociceptive pain. The formalin test is widely used as a model of acute inflammatory pain, as formalin activates peripheral sensory nerves and produces pain behaviors that involve ongoing peripheral activity (phase I) and peripheral and central sensitization (phase II) underlying in hyperalgesia . It has glandins ,39. Subsglandins ,41. On tglandins ,43. In aglandins . Consequglandins ,46, whicglandins . The forglandins . MSBE shglandins  and Vimaglandins ,34. Thisglandins . This exectively ,49,50. Tectively . Moreoveectively . Severalectively . There cin vivo and in vitro models in addition to its antioxidant effects [2 (PLA2) activity and downstream, the activation of arachidonic acid derived-species biosynthesis, PGE2 and LTB4 [MSBE shows anti-inflammatory activity in different inflammatory  effects ,22,49,50 effects  and adhe effects , chemotaand LTB4 ,49,50. Tand LTB4 . But, thand LTB4 ,24.in vitro models of neuronal and glial glutamate-induced injury [2 production in activated rat microglial cells [It is possible to speculate that MSBE could modulate some molecular targets implicated in central sensitization. Perhaps, its ability to inhibit TNFα synthesis, make this molecule attractive for this hypothesis. There is some evidence that involves this pro-inflammatory cytokine in this process. TNFα level increases dramatically after nerve injury and would thus cross the axonal membrane. The resulting cation channel would allow sodium to leak into the fibre and this would probably create sufficient depolarization to trigger an impulse . Correspd injury ,54. Howed injury ,28. Thisal cells . AccordiIn addition, TNFα induces LTP of C fibres in SDH in rats with nerve injury and NF-κB inhibitors blocked this effect . NF-κB iIn addition, ROS are also involved in central sensitization, and it has been assumed that elevated levels of ROS play an important role in the phosphorylation of NMDA receptor subunit 1 (pNR1) through to PKA and PKC activation in the spinal cord ,8. MSBE Mangifera indica L extract (like Vimang formulations) and its major component mangiferin, we hypothesize that these formulations could be useful in treating and preventing neuropathic pain. The possible mechanism of action may be modulating several molecular targets implicated in central sensitization  and prostaglandin E2 (PGE2). NO produced by neuronal NOS (nNOS) and inducible nitric oxide synthase (iNOS) penetrates membranes and may travel to neighbouring neurons, glial cells and pre-synaptic nerve terminals where it may activate second messenger systems. This results in increased level of cGMP with the subsequent activation of cGMP-dependent kinases, which in turn may lead to further glutamate release. PGE2 may activate adenylyl cyclase (AC) via binding to EP2 receptors resulting in the formation of cAMP, that it may activate cAMP-dependent PKA, which may induce further glutamate release [alpha (TNFα) is inhibited by MSBE and MG. TNFα stimulates the degradation of NF-κB cytoplasmatic inhibitor (IκB). After this, NF-κB is translocated to the cell nucleus to induce the mRNA synthesis of some pro-inflammatory mediators such as TNFα, interleukin 1 beta (IL-1β), granulocyte/macrophage colony stimulating factor (GM-CSF), iNOS, inducible isoform of cyclooxygenase (COX-2) and the adhesion molecule ICAM-1 that are inhibited by MSBE and MG. The extract and its xanthone also inhibit PLA2 activity, PGE2 and leukotriene B4 production. MSBE and MG may inhibit glutamate release in pre-synaptic neuron and regulate microglia activity by means of NF-κB signalling pathway inhibition. Pre-synaptic neuron stimulation leads to an enhanced release of excitatory neurotransmitters such as glutamate (Glu). Glutamate binding to post-synaptic NMDA receptors induces Ca release . Activat


SLC26A4 gene (chr7q22. 3) and characterized by sensorineural hearing loss and variable thyroid phenotype. Silver-Russell syndrome (SRS) is a heterogeneous imprinting disorder including severe intrauterine and postnatal growth retardation, and dysmorphic features. Maternal uniparental disomy of either the whole chromosome 7 (upd(7)mat) or 7q (upd(7q)mat) is one of the multiple mechanisms impacting the expression of imprinted genes in SRS, and is associated with milder clinical features. Here, we report genetic and clinical characterization of a female child with PS, postnatal growth retardation, and minor dysmorphic features. A gross homozygous deletion of SLC26A4 exons 17-20 was suspected by Sanger sequencing and then confirmed by array-CGH. Moreover, an insertion of about 1 kb of the CCDC126 gene (7p15.3), which does not appear to be clinically relevant, was detected. The possible occurrence of a balanced rearrangement between 7p and 7q was excluded. The absence of the deletion in the father led to the investigation of upd, and microsatellite segregation analysis revealed a segmental 7q (upd(7q)mat), leading to SLC26A4 homozygosity and responsible for both PS and SRS-like traits. The proband matched 3 out of 6 major SRS criteria. In conclusion, this is the first report of uniparental isodisomy encompassing almost the whole long arm of chromosome 7 resulting in PS and SRS-like features. Whereas, the inner ear phenotype of PS is typical, the clinical features suggestive of SRS might have been overlooked.Pendred syndrome (PS) is an autosomal recessive disorder due to mutations in the  SLC26A4 gene  . The vast majority of them involves a single nucleotide, while only 5 genomic gross deletions are reported in the human gene mutation database (http://www.hgmd.org/).Pendred syndrome (PS [MIM: 274600]) is an autosomal recessive disorder, characterized by the association of sensorineural hearing loss (SNHL), inner ear malformations, and a partial iodide organification defect leading to an extremely variable thyroid phenotype  of a region including a heterozygous pathogenic variant. Chromosome 7 upd has been reported in patients with cystic fibrosis, primary ciliary dyskinesia, and osteogenesis imperfecta type III . Feeding difficulties and delayed growth were recorded during the perinatal period and first months of life. At 8 months , she was diagnosed with bilateral SNHL, and mutations in both GJB2 and GJB4 genes were ruled out. Magnetic resonance revealed a bilateral dilatation of both the vestibular aqueduct and the membranous labyrinth. Upon PS suspicion, appropriate genetic analysis was requested. At 26 months, weight was 9.2 kg (−2.09 SDS), height 79.5 cm (−2.51 SDS), and OFC 46.5 cm (−0.64 SDS), while at the last visit (34 months) weight 10.5 Kg (−2.09 SDS), height 86.5 cm (−2.20 SDS), and OFC was 47 cm (−0.98 SDS). Post-natal SDS were calculated according to the WHO Child Growth Standard (http://www.who.int/childgrowth/en/). Cranio-facial dysmorphic features included high forehead, mild frontal bossing, low-set posteriorly rotated ears, and thin lips. The patient also displayed brachydactyly of both hands and feet and clinodactyly of the V finger , length 49 cm (−0.1 SDS), and occipitofrontal circumference (OFC) 32.5 cm (−0.94 SDS). Neonatal SDSs were calculated according to the Italian Neonatal Study (INeS) charts , according to cycle conditions suggested by the manufacturer. The following intronic primers were used: F 5′-TCTTTTTTGGCAGGATAGC-3′ and R 5′-TCGTCTGAATAATTCTAGCC-3′. The resulting LR-PCR products were sequenced and the sequences were aligned to the human reference genome sequence (human genome assembly GRCh37/hg19). cDNAs were obtained using the High-Capacity cDNA Reverse Transcription Kit  and RT-PCR was performed using the following primers: F 5′-GAGTTCAGTTTCCTTCTTGGA-3′ and R 5′-TCCCTTGCTCATAGAGACCTC-3′. The fragments obtained were purified and sequenced.Genomic DNA (gDNA) and total RNA were extracted from whole blood samples of the patient and her parents with the Wizard Genomic DNA purification kit  and the Tempus kit , respectively, according to the manufacturer's instructions. The entire coding sequence and intron-exon junctions of the http://projects.tcag.ca/variation/) release: March 2016]. For the CNV not maternal in origin, inheritance was identified by performing quantitative PCR analysis on gDNA of both the proband and her parents using SYBR Green methodology. Two amplicons were chosen within non-repeated DNA segments using Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/):dupX_1F 5′-GAAGCCGTAGCAAGGAATGT-3′ and dupX_1R 5′-ATGGGAAAGCGACACAAATC-3′; dupX_2F 5′-GGAGGTGTTTCCTGGTGTGT-3′ and dupX_2R 5′-ACCGCCCTCAATCTCCAC-3′. A control amplicon was selected with the same parameters in the PCNT gene at 11q14.1 . Amplification and detection were performed on ABI PRISM 7900HT Sequence Detection System . Each experiment was performed in triplicate on patient, parents and three controls known to not carry CNVs affecting the investigated locus. Relative quantification of the amount of DNA was obtained using the 2Ct−ΔΔ method.High-resolution chromosomal Q-banding was performed on cultured peripheral blood lymphocytes of both the patient and her mother, according to standard cytogenetic procedures. For each sample at least 16 metaphases were analyzed. High-resolution array Comparative Genomic Hybridization (CGH) analysis was performed on genomic blood DNA of the patient and her mother, using the SurePrint G3 Human CGH Microarray 2 × 400K Kit  following the manufacturer's protocol. Data were extracted and analyzed for copy number changes using Agilent CytoGenomics software v.3.0.6.6 (Agilent Technologies). Coordinates of Copy Number Variants (CNVs) referred to the Human Genome assembly GRCh37/hg19. CNVs classification was performed according to the Database of Genomic Variants (DGV) .Starting from the patient's gDNA, we successfully amplified and found wild-type all r Figure . We concn Figure , resulti) Figure . AccordiCCDC126 IVS3 insertion. The detected short transcript corresponds to a truncated protein of 608 amino acids. The mother had a short and a long transcript, while the father had only the long transcript corresponding to the wt pendrin  spanning from 7q11.23 to qter, named arr[GRCh37] 7q11.23q36.3(75620641_159119486)x2 hmz mat according to the International System for Human Cytogenomic Nomenclature (ISCN 2016). The region includes the maternal mutated allele of n Figure .SLC26A4 allele of maternal origin. Sequencing of the aligned junction fragments revealed the insertion of about 1 kb of the IVS3 of the CCDC126 gene, mapping to 7p15.3, which does not appear to be clinically relevant. The segmental isodisomy is probably due to a postzygotic mitotic recombination, occurring during the first zygotic division and followed by the loss of one daughter cell  harboring a maternal segmental isodisomy of chromosome 7 (upd(7)mat). Upd may be causative of autosomal recessive diseases due to the loss of heterozygosity of recessive pathogenic variants, as reported for patients harboring a upd(7)mat and affected with cystic fibrosis  and neurosensorial hearing loss, associated to inner ear malformations was previously reported, though no In conclusion, this is the first report of uniparental isodisomy encompassing almost the whole long arm of chromosome 7 leading to PS and SRS-like features. While the inner ear phenotype of PS is always typical and the diagnosis easily achieved, the clinical features of the associated SRS are subtle and might have been undiagnosed in our case. The finding of a patient presenting without a relative macrocephaly at birth and without SGA, but with protruding forehead, postnatal growth failure, and feeding difficulties, usually accounted for by upd(7q)mat, recommends chromosome 7 investigation. In case of homozygous mutations inherited from one single carrier parent, such as in the case here presented, screening for upd is advisable.PF, SR, and LF conception and design. SM and MP provision of study materials or patients. VC, VG, CC, EM, AS, and MR collection and assembly of data. VC, VG, CC, EM, AS, MR, PF, SR, and LF data analysis and interpretation. VC, VG, CC, SM, EM, AS, MR, MP, DG, LL, LP, PF, SR, and LF manuscript writing. LL and LP final approval of manuscript. VC, VG, CC, SM, EM, AS, MR, MP, DG, LL, LP, PF, SR, and LF accountable for all aspects of the work.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.