Active episodes of inflammatory bowel disease (IBD), which include ulcerative colitis and Crohn’s disease, coincide with profound shifts in the composition of the microbiota and host metabolic energy demand. Intestinal epithelial cells (IEC) that line the small intestine and colon serve as an initial point for contact for the microbiota and play a central role in innate immunity. In the 1980s, Roediger et al proposed the hypothesis that IBD represented a disease of diminished mucosal nutrition and energy deficiency (“starved gut”) that strongly coincided with the degree of inflammation. These studies informed the scientific community about the important contribution of microbial-derived metabolites, particularly short-chain fatty acids (SCFA) such as butyrate, to overall energy homeostasis. Decades later, it is appreciated that disease-associated shifts in the microbiota, termed dysbiosis, places inordinate demands on energy acquisition within the mucosa, particularly during active inflammation. Here, we review the topic of tissue energetics in mucosal health and disease from the original perspective of that proposed by the starved gut hypothesis. Both Crohn’s disease (CD) and ulcerative colitis (UC) are significantly increasing in severity and prevalence worldwide . IBD is incompletely understood at present, but a number of factors are thought to contribute including changes within the microbiota, environmental exposures, and a complex set of genetic elements . Despite these complex factors, a pattern of abnormal innate immune responses, including within the intestinal epithelium, appear to contribute substantially to disease pathogenesis .Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the intestine affecting more than 3 million individuals in the United States . The gut microbiota consists of the constellation of microorganisms, including bacteria, fungi and viruses, in coexistence with an intact organism. Technological advancements, especially inexpensive DNA sequencing techniques, have made it possible to understand these microbial communities at a detail never previously imagined. Studies in the past 20 years have identified a precise and consistent role for microbial communities as essential for health. Multiple mucosal surfaces, including the gastrointestinal tract, the skin, and the oral cavity provide a close and intimately interaction between the host and the microbiota. While the mammalian microbiota consists of fungi, archaea, protozoa, and bacteria, bacteria vastly outnumber the other microorganisms to the extent that the prokaryotic: eukaryotic ratio is approximately 1:1 . Some believe that our microbial signature is as unique as our fingerprint and only now are we beginning to understand the factors that mold this microbial population. Clearly, differences in lifestyle, diet, environment, and genetics shape the composition and activity of the gut microbiota, and ultimately our health and well-being. In this short review, we will explore how the microbiota contributes to gut energy balance health and how inflammation-associated changes to the microbiota influence disease outcome.Accumulating evidence suggests that microbial dysbiosis and dysfunction of the mucosal innate immune system play central roles in the pathogenesis of IBD –9. In the 1980s and 1990s, Dr. William Roediger published a compelling series of manuscripts that probed the role of short-chain fatty acid (SCFA) transport and utilization as energy sources in human IBD –16. These studies strongly implicated defects in SCFA processing associated with IBD that implicated a loss of energy balance  as a driving force in the pathogenesis of IBD.Ongoing inflammatory responses are associated with profound shifts in tissue metabolism that can fundamentally influence tissue and organ function . It is now understood that the colonic epithelium consistently favors oxidative metabolism of butyrate ,18. In the colon, for example, the insatiable metabolism of butyrate depletes local O2 to the extent that hypoxia-inducible factor (HIF) is stabilized and transcriptional targets of HIF are activated . HIF target genes that regulate metabolism classically include glycolytic genes ,21, major butyrate transporters  ,23 and pyruvate dehydrogenase kinase (PDK) . The induction of PDK within the mucosa inactivates pyruvate dehydrogenase and results in a decreased utilization of glucose as a source of acetyl-CoA within the tricarboxylic acid (TCA) cycle. As a result, the β-oxidation of butyrate becomes the primary energy source of intestinal epithelial metabolism. To compound this metabolic pathway, the functioning of butyrate as an HDAC inhibitor (see later) induces PDK1 expression, thereby further diminishing glucose utilization . Given this unique metabolic adaptation within the mucosa, it is not surprising that more that 70% of oxygen utilized by human colonic epithelial cells occurs through butyrate β-oxidation . Some regional differences were also demonstrated, where the distal colon was found to be more dependent than the proximal colon for butyrate as the major energy source. Second, he suggested that changes in the SCFA reflect the degree of inflammation in UC. This study reported that concentrations of SCFA were increased in severe UC and that butyrate, in particular, was raised in all severities of disease. It was suggested that decreased absorption of SCFA accounted for the observed increase. While this latter observation has been debated (see below), these studies lead to the major hypothesis that IBD is an energy-deficient disease. He reported that colonocytes harvested from UC patients were deficient in butyrate oxidation and postulated that the disease has a substantial “starvation” component . In the backdrop of deficient butyrate oxidation, the harvested colonocytes manifested increased glucose and glutamine oxidation. It is also notable that mitochondria in the epithelium of patients with IBD are reduced in number, altered in subcellular location, and dysmorphic in appearance , thus compounding the multiple defects in overall energy procurement.These studies were systematic. First, using suspensions of isolated human colonocytes, he examined oxygen consumption rates as a primary endpoint with added energy sources, including SCFA, glucose, and glutamine alone or in combination. Through the process of elimination, he concluded that butyrate is the major source of fuel for the colonic mucosa and accounts for more than 70% of the oxygen consumed by the colon +-coupled MCT1 and MCT4 transporters and the Na+-coupled transporters SMCT1 and SMCT2 . MCT1 is expressed on the apical and basolateral IEC membrane, whereas MCT4 exists only basolaterally. SMCT1 and SMCT2 are expressed exclusively on the apical IEC membrane. A number of studies have demonstrated that inflammatory mediators such as TNFα and IFNγ repress MCT1 and SMCT1 expression and function in IEC ,29. A meta-analysis of 12 datasets comparing expression of MCT1, SMCT1 and SMCT2 in healthy and UC colonic tissue revealed a significant (range 17%–95%) decrease in MCT and SMCT expression in 10 of the 12 studies examined . Whether such decreases could account for the deficit in host butyrate is unclear, it is notable that MCT1 itself is upregulated by butyrate  and suggest decreased overall microbiota-derived butyrate likely contributes.Consistent with Roediger’s original studies, transport of butyrate appears to be significantly diminished in UC Figure . Butyrat. Inhibiting the deacetylation of histones allows genomic DNA to remain accessible to transcription machinery. HDAC inhibition impacts expression of ~2% of mammalian genes ,32. Butyrate also stabilizes HIF, a master transcription factor in the mucosa , via direct inhibition of the prolyl hydroxylase (PHD) enzymes that regulatorily degrade HIF  and limitation of oxygen availability necessary for PHD function through increased oxygen utilization through energy-harvesting butyrate metabolism .Beyond energy provision, SCFAs fundamentally regulate gene expression. Butyrate, for example, potently regulates transcription through HDAC inhibition Figure . HDACs r,35. Activation of these receptors on IEC trigger the production and secretion of interleukin-10 (IL-10) and IL-18 to suppress inflammation . Signaling through these GPRs is not limited to IECs. Regulatory T cells (Tregs) express SCFA-responsive G-protein coupled receptors (GPCRs), the ligation of which promotes histone H3 acetylation of the FoxP3 promoter to influence Treg differentiation ,38. Furthermore, butyrate suppresses TH17 cells through GPCR activation to promote Treg and intestinal homeostasis in colitis ,40.There is significant interest in understanding how butyrate and other SCFA influence immune responses. One of the more recent mechanisms of action include SCFA-mediated activation of G-protein–coupled receptor (GPR) signaling. GPR41 or free fatty acid receptor 3 (FFAR3), GPR43 or FFAR2, GPR109A, and GPR164 have been identified as SCFA receptors, with FFAR2 and GPR109A being more specific for butyrate . Based initially on the regulation of barrier function and wound healing, this unbiased approach revealed a prominent induction of gene transcripts important in cell migration, tight junction formation/maintenance and myosin/kinesin-based motor complexes , revealed the novel expression and function of cytoskeletal regulation in the mucosa. Originally described in kidney and post-synaptic densities . Dietary creatine is minimally digested with almost 100% absorbed in passage through the small intestine .Beyond defects in SCFA assimilation and utilization, it is more recently appreciated that storage pools of energy may also be deficient in IBD. These storage pools, or energy reserves, are provided largely by the phosphocreatine (PCr) shuttle, and recent investigations have defined an important role for creatine in intestinal inflammation. Approximately 50% of creatine is obtained through our diet and 50% is synthesized endogenously ,49. Two mitochondrial CK (mtCK) isoforms have also been characterized, the ubiquitous mtCK and the muscle-specific sarcomeric mtCK, both of which localize to the mitochondrial intermembrane space ,49. The CK system is essential for cellular energy homeostasis, especially in tissues with highly dynamic energy demands such as the muscle, brain, and the intestine –51. Within individual cells, CK facilitates the shuttling of high energy phosphates in the form of PCr between sites of ATP generation in the mitochondria and cytosolic locations of ATP consumption. As such, the diffusion limitations and thermodynamic limitations of using ADP and ATP for the storage of high energy phosphates are overcome by PCr/Cr shuttling –51.The creatine kinase (CK)/PCr shuttle, with Cr as an energy precursor, plays a critical physiological role for cells and tissues with high and fluctuating energy requirements Figure . CK exis. Additionally, a specific creatine transporter (CrT1), belonging to the X-linked gene SLC6A8, as a member of a solute carrier family, is present in the apical cell membrane of IEC . Electrogenic Na+- and Cl−-dependent Cr transport, with high affinity for Cr (Km ~30 µM), allows IEC to import Cr ingested with the diet  as the most significant sources for Cr . As CrT1 function is dependent on Na+, inhibition of the Na+/Ka+-ATPase , also inhibits Cr uptake into cells, as shown recently .Intestinal epithelial cells (IEC) predominantly express the cytosolic brain-type BB-CK and mitochondrial mtCK isoenzymes . Additional analysis revealed that cytosolic BB-CK enzyme localizes to apical intestinal epithelium cell adherence junctions, where it is centrally involved in ATP-dependent tight junction assembly, epithelial integrity, and barrier function. The analysis of transcripts from 30 IBD patient samples (including Crohn’s disease and ulcerative colitis) showed a significant reduction in all isoforms of CK compared to healthy patient controls  and the major CrT SLC6A8 are all coordinately regulated by HIF s Figure . From th. A more recent analysis revealed that serum Cr were significantly lower in UC patients that may be related to amino acid malabsorption . Second, a retrospective study of 131 IBD patients demonstrated significantly higher serum CK (undifferentiated isoform) levels in subsets of patients receiving anti-TNF therapy. High serum CK was greatest in male vs female patients and in the nonsmoker vs smoker cohort. Similar elevations in serum CK levels were noted in patients receiving anti-leukocyte adhesion molecule therapy . Third, a recent clinical trial evaluated the efficacy and safety of an oral selective inhibitor of Janus kinase 1 (JAK1) in UC. This phase 2b trial incorporating 250 patients revealed an increase in circulating CK levels in response to JAK1 inhibition . A 30-month follow-up indicated showed continued elevation of CK with chronic treatment . It remains unclear if CK serves as an adverse event to therapy or as a biomarker of treatment response. Finally, Starr et al recently reported that colon biopsies from pediatric IBD patients with active disease show decreased CKB expression, particularly when biopsies are harvested from areas of active disease vs areas of mucosal healing . It is also notable that Crohn’s patient responders to IBD-targeted therapy also expressed higher levels of CK than drug naive patients. Taken together, these observations provide some evidence that CK could serve as a biomarker of disease and may suggest drug response in some incidences.Studies subsequent to the initial identification of deficiencies in Cr-PCr shuttle among IBD patients have been revealing from the perspective of drug response indicators and biomarker identification. First, an unbiased nuclear magnetic resonance (NMR)-based profiling of urine and serum metabolites from 43 IBD and 17 healthy control subjects revealed a prominent decrease in serum Cr among actively inflamed UC and Crohn’s disease patients . Supporting evidence in cultured IEC and in colonoids from CrT1-deficient revealed that diminished Cr transport results in reduced epithelial barrier function that tracks to defects in metabolism and actin polymerization. Localization of IEC CrT1 revealed that this transporter is expressed specifically in areas of epithelial tight junctions and overexpression or targeted knockdown of CrT1 in an IEC cell line implicated intracellular creatine concentration as a driving force for epithelial barrier formation and ultimately wound healing. In IECs lacking CrT1, metabolism shifted toward a glycolysis-predominant energy metabolism that was reflected by leaky tight junctions, aberrant expression and localization of tight junction proteins and dysregulated actin polymerization similar to findings in CK-deficient IECs. In addition to the above study, a screening of 36,530 germline mutant mice for susceptibility to DSS colitis identified a mutation in glycine amidinotransferase (GATM) enzyme necessary for creatine biosynthesis. GATM-deficient colonocytes similarly showed evidence of metabolic stress and creatine supplementation was protective in GATM-deficient mice . These findings support the concept that energy deficiencies within the mucosa support defects in barrier formation and decreased wound healing responses.Creatine transport occurs primarily in the small intestine via the apical CrT. CrT expression has been examined in IBD patients. In one study of mucosal biopsies from 30 patients with Crohn’s disease and 27 patients with UC both showed lower expression levels of CrT1 gene transcript . In this regard, the unique environment of the mucosa was more clearly delineated with the discovery that the intestinal microbiota is a major source of purines, termed microbiota-sourced purines (MSP) within the salvage pathways  showed a propensity for increased ER stress and a loss of barrier function. Conversely, gain-of-function studies with MSP-sufficient bacterial mutants alleviated ER stress and promoted colonic barrier, thereby establishing MSP and a critical metabolic component for tissue homeostasis.Most recently, it has been shown that the salvage of purine molecules can be a significant source of energy within the mucosa, particularly during stress. In models of inflammation and hypoxia, energy flux results in the pooling and salvage of hypoxanthine, which in turn becomes a major building block for the regeneration of ATP s Figure [70]. The. Patients that did not achieve remission in this trial were enriched in a microbiota that lacked SCFA production and diminished purine production, providing valuable insight into the contribution of tissue energetics to inflammatory resolution. Furthermore, purine metabolism was identified as a novel host-microbial metabolic pathway in patients with irritable bowel syndrome (IBS), a multi-factorial disease of recurrent abdominal pain and discomfort . Stool samples from IBS patients showed significantly lower levels of MSP, with this starvation phenotype shown to be the result of increased degradation by the microbiota and host alike, revealing a potential novel mechanism underlying IBS pathophysiology .Purine salvage have also been shown to be important in human disease. For example, in a double-blind trial of 81 active UC patients with and without FMT, it was shown that the salvage of purine nucleobases  tracked with FMT-associated disease remission n-butyrate is a far more efficient source of energy supply than glycolysis, and the faulty oxidation reflects a state of “energy deficiency” .In his original manuscript, Roediger wrote “To unify the present metabolic findings in colonocytes with clinical features of UC and histological changes in the diseased mucosa, the term “energy-deficiency” is proposed. Low levels of free CoA and diminished oxidation of fatty acids are the basis for using the term “energy deficiency”. Usually, β-oxidation of fatty acids such as . While the cause of IBD is much debated, a common feature is inflammation which follows a clinical course that can be episodic or chronic. Important insight into the role of dysbiosis has been gained through longitudinal studies. For example, IBD-related dysbiosis improves with treatment but nonetheless persists even in patients that have achieved full mucosal healing . Such observations suggest that dysbiosis, as defined by our current understanding of disease-associated microbiota, is a fundamental feature of IBD.In this short review, we summarize changes associated with metabolic deficiencies in active inflammation and IBD. An important question that remains unanswered is whether inflammation-associated dysbiosis is cause or effect in IBD and other related disorders. It is clear that tissue metabolism during inflammation shifts toward a less tolerant environment for strict anaerobes  toward a less diverse microbiota with higher tolerance for the outgrowth of facultative anaerobes  Clostridium difficiile colitis (CdC), where up to 90% of patients are cured with a single treatment . It is also notable that in a small cohort of patients, fecal samples filtered to remove bacteria show nearly equivalent efficacy , implicating a non-bacterial source of disease resolution . Studies assessing FMT for the treatment in UC has been less promising than CdC, but generally suggest that positive FMT outcomes promote energy homeostasis associated with an enrichment of microbiota that promote SCFA production and purine salvage .It is also notable that the dysbiosis associated with active intestinal inflammation has resulted in some clinical progress. Fecal microbiota transplantation (FMT), for example, has shown great success in the treatment of active Taken together, even upon reflection and on the backdrop of more sophisticated technologies and advanced experimental approaches, Roediger’s original “starved gut” hypothesis appears to have survived the test of time. The presumption that energy homeostasis within the mucosa is central to the resolution of active inflammation has proven to provide an essential insight to our understanding the healthy mucosa. A more refined understanding of the composition of the microbiota and the subsequent discovery of other energy metabolites important within this conversation have resulted in previously unimaginable opportunities for the treatment of patients with IBD and likely other mucosal diseases into the future.SPC, RXW, CHTH, GB, and JSL wrote the paper with input from all authors.No conflicts of interest exist for any aspect of the work presented here.This work was supported by the National Institutes of Health , the Crohn’s and Colitis Foundation and by the Veterans Administration (award BX002182).


The NOTCH signaling system regulates a variety of cellular processes during embryonic development and homeostasis maintenance in different tissues and contexts. Hence, dysregulation of NOTCH signaling is associated with a plethora of human cancers, and there have been multiple efforts to target key components of this pathway. In this review, we briefly highlight the latest research advances in understanding HES6, a poorly studied component of the NOTCH pathway. We summarize the role of HES6 in cancers with a focus on uveal melanoma. Finally, we discuss the ongoing efforts to target the NOTCH-HES6 axis in cancers. The NOTCH pathway remains a central focus in both basic and translational research.Its activity is essential for normal embryonic development and tissue homeostasis. Dysregulated NOTCH signaling is associated with a variety of nonmalignant and malignant pathologies. To date, however, relatively little is known about the regulation of the NOTCH signaling pathway and target gene expression in various cellular contexts Drosophila melanogaster, Lag-1 in Caenorhabditis elegans) transcription factor and the transcriptional coactivator Mastermind Like transcriptional coactivator 1 (MAML1) to transactivate target genes  are activated following ligand binding . These ligands contain a DSL (Delta/Serrate/Lag2) domain that is required to interact with NOTCH. NOTCH activation is mediated by successive proteolytic cleavages; the latter involves γ-secretase, which leads to the release of the NOTCH intracellular domain (NICD) -4 are acAll HES proteins have three evolutionarily conserved domains, the basic helix loop helix (bHLH), Orange, and WRPW domains Figure B. In thebHLH factors usually bind to a hexanucleotide consensus sequence called the E box (CANNTG) that is present in the promoter region of their target genes. Although they belong to the bHLH family of transcription factors, some HES proteins have been reported to bind canonical E boxes (CANNTG) but with low affinity only and to bind to a different target sequence, namely, the N box (CACNAG) with high affinity HES factors can exert transcriptional repression through active and passive mechanisms While NOTCH activation can trigger its effects through the canonical pathway that involves the expression of Hes genes, noncanonical NOTCH signaling has also been reported Figure . NoncanoIn this review, we focus on the role, regulation and mechanisms of action of HES6, with a special focus on cancers including uveal melanomas.Hes genes that are associated with the inhibition of neurogenesis Hes6 was isolated by the group of Kageyama Moreover, Hes6 has been reported to be a direct target of the myogenic factors MyoD and Myf5 and to control myoblast fusion, a fundamental step in the differentiation of muscle in most organisms In addition to myogenesis and neurogenesis, HES6 has been implicated in the homeostasis and function of other tissues where its role still remains elusive. For instance, overexpression of Hes6 in the developing retina promotes rod photoreceptor differentiation through Hes1 inhibition, suggesting that they are involved in the activation of canonical NOTCH signaling Despite structural similarity, Hes6 seems to exhibit unique features within the Hes family. These features might rely on a loop region that is shorter than four or five amino acid residues compared to the other Hes factors HES6 showed the highest enrichment in gliomas, suggesting that it might represent a lineage-specific cancer driver The clinical significance and biological role of HES6 in human cancers remain poorly elucidated. Nevertheless, it might play a broad role due to amplification of the genomic region where HES6 is located in several cancers in vitro, and this was confirmed in tumor xenografts in vivoMoreover, functional studies have revealed an important role for HES6 in supporting the growth and motile ability of cancer cells. HES6 increased proliferation in MCF-7 breast cancer cells HES6 has been recently implicated in uveal melanoma progression, which is the main primary intraocular malignancy in adults and the second most common melanoma. Despite successful treatment of primary uveal melanoma by radiotherapy or enucleation, up to 50% of patients will develop metastases, predominantly in the liver BAP1 and recurring chromosomal aberrations, such as chromosome 3 loss or 8q amplification, both associated with a high metastatic risk and a poor prognosis The main known oncogenic drivers in uveal melanomas are mutations in the heterotrimeric G-protein alpha subunit GNAQ or its paralog GNA11 (GNAQ/11). Eighty percent of uveal melanomas harbor a mutation in one of these two genes. Mechanistically, oncogenic GNAQ/11 promotes ARF6 activation, which orchestrates the activation of a broad range of events, including the activation of the PKC/ERK and HIPPO/YAP signaling pathways to control the proliferation of uveal melanoma cells HES6 as a prognostic biomarker has been shown in primary cutaneous melanomas in vitro and in vivoActivation of NOTCH signaling was reported to play a key role downstream of oncogenic GNAQ in uveal melanoma cells. MRK003, a NOTCH signaling inhibitor, reduced the viability and migration induced by mutant GNAQ In summary, much work remains to be done to precisely determine which and how HES/HEY factors are implicated in cancer. HES6 contributes to the metastatic phenotype in tumors, including uveal melanomas, and represents a potentially important avenue of research toward uncovering therapeutic targets.MYC expression or activity HES6 gene promoter in the lymphoblastic leukemia HPB-ALL human cell line Transcriptomic analysis of glioma cells in which the expression of HES6 was reduced or enhanced revealed that the commonly dysregulated genes were associated with biological functions such as migration, invasion, cell-to-cell signaling and cell proliferation MYC is of particular interest in uveal melanomas given its localization on chromosome 8, whose amplification (8q) is one of the most common genetic abnormalities in this disease and is strongly associated with metastatic risk Interestingly, in colorectal cancer, NOTCH activation by DLL4 triggers the induction, through DAB1 and ABL, of the TRIO-RHO module to drive invasion and metastasis The WNT/β-catenin signaling pathway, which can promote cancer metastasis SLUG is a member of the Snail transcription factor family involved in epithelial-mesenchymal transition (EMT), a molecular process by which epithelial cells lose their cell-cell adhesion and gain migratory and invasive capabilities to favor malignant progression. The prototype of the EMT switch is the loss of CDH1, which encodes E-cadherin that mediates cell-cell adhesion. E-cadherin sequesters β-catenin on the cell membrane via its cytoplasmic tail. Consequently, its loss of function results in β-catenin release into the cytoplasm and translocation into the nucleus, where it triggers the expression of EMT-inducing transcription factors that are implicated in cancer and metastasis Several lines of evidence indicate that NOTCH has a role in promoting resistance to both conventional chemotherapy in vitro and in preclinical models. For instance, BMS-906024 enhanced the anticancer efficacy of paclitaxel compared to either drug alone in lung adenocarcinoma Gamma secretase inhibitors (GSIs) have been reported to induce cancer stem-like cell differentiation and apoptosis, to impair EMT and to sensitize cells to traditional chemoradiotherapies To date, NOTCH inhibitors alone or combined with other therapies have failed to translate into beneficial effects in most solid tumors, with the notable exceptions of central nervous system malignancies and desmoid tumors NOTCH also plays an essential role in coordinating vessel development and maintenance Another possibility would be targeting downstream NOTCH effectors. As previously indicated, HES6 expression is increased and associated with a poor prognosis in some cancers. Its detection may help to assess a patient's risk of disease progression, thereby allowing better follow-up and improving clinical decision-making. In the context of uveal melanoma, HES6 detection may also serve patient stratification for administration of adjuvant therapy rendered possible with the recent discovery of tebentafusp HDAC4) and ChaC glutathione-specific γ-glutamylcyclotransferase 1 (CHAC1) expression correlated with poor prognosis (UM-TCGA dataset) . These may represent therapeutic targets in uveal melanomas but also in other tumor types. Among them, histone deacetylase 4  Figure A-B. InhiPatients with uveal melanoma are in therapeutic need. We anticipate that information gained from basic research will lead to a paradigm shift in the management and outcomes of uveal melanomas by identifying new druggable frailties to limit metastatic dissemination and metastatic growth. HES6 appears to be a valuable prognostic biomarker that could enable patient stratification for adjuvant treatment, and it may be a valuable therapeutic target in different cancers.


Prostate cancer (PCa) is one of the most common cancers with increasing rates of incidence. Bone metastasis and drug resistance are the most serious threat faced by patients following a delayed diagnosis of cancer, which might lead to treatment failure and death. The theoretical model of cancer stem cells (CSCs) explains the diverse molecular characteristics of cancer as well as its relapse, metastasis and drug resistance. Prostate cancer involves heterogeneous cells community, including prostate cancer stem cells as an important component. These subtypes of cancer cells are usually monoclonal, expressing specific biomarkers and exhibiting self-renewal and differentiation capacity. Therefore, therapies that target CSCs might be more effective in overcome drug resistance and metastasis. Thus, anti-CSCs therapies differ from the traditional anti-proliferative approach. We focus here on reviewing the effects of prostate CSCs on bone metastasis and resistance to traditional treatment in PCa, and report new clinical strategies that address CSC-based tumorigenesis. Prostate cancer (PCa) is the second most common cancer and the fifth most common cause of cancer-related mortality among men worldwide Early-stage PCa is androgen-dependent and surgical prostate removal is the most common treatment strategy, which can effectively cure localized prostate cancer p21, p18, and p63. When they exit the quiescent state, these cells self-renew and differentiate into other types cells + and CD38-Advances leading to the discovery of hematopoietic stem cells have increased the number of treatment options for cancer The human prostate is composed of stromal cells and three types of epithelial cells: basal cells, luminal secretory cells, and neuroendocrine cells hi, CD44+, and integrin α2β1+ increases when castration resistance occurs, and such cells display potential self-renewal capacity and colony formation, which indicates that castration resistance increase the number of PCSCs +, integrin α2β1+, and CD133+ were isolated from PCa patients, and shown to exhibit self-renewal, suggesting that the three markers can be used to define the CSCs phenotype Prostate cancer is a highly heterogeneous entity, comprised of multiple cancer cell subpopulations (including CSCs) and non-tumoral cells such as epithelial cells, stromal cells, immune cells, vascular endothelial cells, tumor-associated macrophages (TAMs) and cancer associated fibroblasts SPOP gene interferes with this process by degrading the NANOG protein. It was inferred that tumor suppressor genes also play an important role in the evolution of PCSCs. Mutations involving genes such as TP53 and PTEN usually occur in PCSCs BRCA1 and BRCA2 are tumor suppressor genes, while the mutant genotypes are closely related to different cancer types, including breast cancer and PCa It is necessary to understand the molecular basis of CSCs stemness, and the mechanism of reprogramming non-CSCs into CSCs. The important transcription regulators, SOX2, OCT4, and NANOG, contribute to the maintenance of the pluripotent state of CSCs. Some studies indicated that inhibition of androgen receptor (AR) function increases the stemness of PCSCs +/Ly6G+ tumor-associated types to pro-tumor types. As for lymphocytes, TGF-β signaling inhibits the generation of effector T lymphocytes and promote Treg production. It can inhibit B lymphocyte maturation and natural killer cells via mTOR signaling inhibition. Myeloid-derived suppressor cells (MDSCs) can suppress the anti-tumor immunity by inhibiting T cell functions. TGF-β activation can recruit the MDSCs to the prostate cancer microenvironment.Bone is composed of cortical bone and bone marrow in different proportions The phenotypic heterogeneity of metastatic prostate cancer cell is established. CSCs are one important component of bone metastasis, and promote bone metastasis progression via various mechanism. Most cancer-related deaths are attributed to metastasis, and PCa is likely to lead to bone metastasis, with devastating consequences for patients' health Circulating tumor cells (CTCs) have been known for 150 years, since they were first reported by Ashworth in 1869 The CSCs and bone marrow microenvironment are closely linked, and signals from bone marrow are very important for the function of CSCs. We will briefly elaborate the most crucial signaling pathway, including those mediated by Notch, Wnt, Hedgehog, FGF, and TGF Notch is a transmembrane receptor expressed in CSCs that plays an important role in stem cell self-renewal and differentiation, and inhibition of this signaling pathway appears to be a therapeutic strategy for CSCs elimination ATOH1, CCND1, CD44, LGR5 and SnailDAB2IP was identified as a new tumor suppressor gene in PCa. It regulates the stemness of PCSCs through modulation of CD117 transcription, in which the Wnt/β-catenin signaling pathway plays an important role DAB2IP also inhibits the EMT and PCa bone metastasis OCT4, SOX2, NANOG, CD44, KLF4, c-MYC, and MMP2, are upregulated by miR-1301-3p, while GSK3β and SFRP1 genes are downregulated Wnt signaling pathway is very important for embryonic development and adult tissue homeostasis. The canonical Wnt signaling pathway also plays an important role in stem cell self-renewal, maintenance, and differentiation, partly via enhancement of human telomerase reverse transcriptase (hTERT) activity Hedgehog (Hh) signaling pathway is highly conserved. It plays an important role in embryonic development, stem cells maintenance, and tissue regeneration Hh signaling pathway is critically important for CSC functions, and therefore strategies to inhibit the pathway using pharmacological or genetic methods were studied. Cyclopamine, vismodegib, saridegib, and sonidegib are potent and specific SMO inhibitors, with clinical trials indicating therapeutic benefit for patients diagnosed with cancer In fact, the interaction between PCSCs and bone microenvironment is a dynamic process that drives CSC evolution. Rapid division of PCa cells leads to a hypoxic environment, which affects the PCSCs function by enhance their stemness and self-renewal Disseminated PCa cells can be converted to CSCs in the bone environment, depending on the supply of cytokines and growth factors in the stem cell niches. The CSC phenotype in the bone metastasis is usually defined by the dual expression of CD133 and CD44 PTEN-gene knockout PCa mouse model. They also activate Wnt and Notch signaling pathways in CSCs to maintain their stemness Stemness of CSCs is not stable and changes dynamically. It is a transient state that is influenced by genetic, epigenetic, and bone microenvironment. The characteristics of CSCs are affected by their interactions with the bone microenvironment, while factors released by them affect stromal cells in the microenvironment, hijacking them to promote tumor growth. Stromal cells include fibroblast, endothelial, and immune cells, as well as other cell types. The extracellular matrix produced by the stromal cells can also influence CSC function Recent studies reported that the hypoxic cancer environment plays an important role in CSC evolution. Hypoxia was found to be conducive to establish an acidic tumor microenvironment and activate several proteases facilitating prostate cancer metastasis. The hypoxic environment can induce cancer cells, including CSCs, to express HIF, which enables cellular adaptation to hypoxia 0 phase of cell cycle), and are highly conserved. In this state, they can bypass traditional therapies. Actually, cell quiescence is closely related to cell dormancy, and CSCs usually utilize these characteristics to evade harsh environment stimuli The dormant state of PCSCs can be induced under a foreign environment of the bone marrow. The dormant state can be maintained for prolonged durations, and might be related to their anti-apoptotic and DNA repair capability The HSC niche plays an important role in inducing HSCs dormancy. When the PCSCs arrive to the bone marrow, they reside in the HSCs niche and become dormant 1 phase of the cell cycle might be an effective approach for future investigation.Dormant PCSCs are also an important factor contributing to treatment failure. During therapeutic intervention, the PCSCs often become dormant to evade death induced by anti-proliferating agents, such as docetaxel. Therefore, new strategies should be developed to target the dormant PCSCs, which is a challenge. Strategies that redirect the dormant PCSCs to enter the GBcl-2, Bcl-xl, and survivin overexpressed in some CSCs. These cells have active DNA damage detection and repair systems. Further, PCSCs were shown to be resistant to radiation therapy, which might be related to activation of Chk1 and Chk2 Traditional treatment can effectively destroy the non-stem cells cancer cells but not the dormant CSCs - and therefore do not respond well to androgen deprivation therapy (ADT). Isolated CD44+/ integrin α2β1+/CD133+ CSCs from several human PCa samples expressed ABCG2, but AR expression was almost undetectable + and AR- clones exist in CRPCs. Most PCSCs do not express AR, while some do, and both clones exhibit distinct biological features Androgen receptor (AR) is very important for the survival of normal prostate cells and prostate cancer cells. In primary and untreated PCa, the PCSCs are ARIt is evident that cancer cells are driven and maintained by a set of stem cells. Treatment strategies should be directed to CSCs, which are proposed to be the root cause of cancer. A study of PCSCs showed that 5-lipoxygenase inhibition induced apoptosis of PCSCs by activating c-jun N-terminal kinase and downregulating their stemness Novel treatment strategies and assay systems should be adapted to effectively identify the CSCs, such as those expressing high levels of CD44. When miR-34a was used to inhibit the expression of CD44 in PCSCs, the progression and metastasis of the PCa were remarkably reduced.PCSCs have gained increasing attention in recent years and are supposed to play an important role in prostate cancer bone metastasis and drug resistance. PCSCs are believed to the precursor of bone metastasis. It is proposed that PCSCs occupy the HSCs niche and remain dormant for extended duration, followed by activation by some signals resulting in bone metastasis. The origin of PCSCs needs to be further studied. How to identify the PCSCs in the samples of bone prostate cancer bone metastasis, and how to accurately discriminate the dormant PCSCs using the proper molecular biology techniques are two important challenges that we are faced. Additionally, the gene instability of PCSCs is closely related to drug resistance. Therefore, the in-depth research for the mechanism of PCSCs gene instability will shed some light on prostate cancer bone metastasis.The importance of CSCs in cancer initiation, progression, recurrence, and metastasis is gaining increasing attention. We have discussed here a number of important aspects of PCa: 1) the role of CSCs in PCa bone metastasis and their drug resistance; 2) the complex interactions between PCSCs and the bone microenvironment following bone metastasis of PCa; 3) relevant signaling pathways related to this interaction; 4) the dynamic evolution of PCSCs in the bone microenvironment, as this is a dynamic rather than static process; 5) the dormant state of PCSCs that is vital for the successful formation of bone metastasis; 6) the role of PCSCs in drug resistance, including castration resistance. We hope that this review will advance our understanding of bone metastasis and drug resistance of PCa.


DNA methylation is an important epigenetic mechanism that regulates gene expression. To date, most DNA methylation studies have focussed on CpG islands in the gene promoter region, and the mechanism of methylation and the regulation of gene expression after methylation have been clearly elucidated. However, genome-wide methylation studies have shown that DNA methylation is widespread not only in promoters but also in gene bodies. Gene body methylation is widely involved in the expression regulation of many genes and is closely related to the occurrence and progression of malignant tumours. This review focusses on the formation of gene body methylation patterns, its regulation of transcription, and its relationship with tumours, providing clues to explore the mechanism of gene body methylation in regulating gene transcription and its significance and application in the field of oncology. In the 1970s, Riggs AD and Holliday R proposed that methylation of the 5′-terminal cytosine (5′-C) of CpG dinucleotides is an important epigenetic marker of gene silencing that can cause changes in chromatin structure and DNA stability without altering the DNA sequence, thereby regulating gene expression . By the With the rise of technologies such as genomic bisulfite sequencing, genome-wide DNA methylation mapping has been possible, and more extensive DNA methylation in the gene body region has attracted attention. Gene body methylation, which refers to the methylation of CpG sites in the transcribed regions of genes, including the methylation of scattered CpG sites and CGIs , was firAlthough DNA methylation is commonly thought to lead to gene silencing, in the case of genomic methylation, it appears to be regulated by active transcription while affecting gene transcription. Following genetic and acquired genomic rearrangements, aberrant methylation of CGIs occurs when an unmethylated CGI is aberrantly recombined into another transcriptionally active gene in vivo , 20. ThuHowever, in ES cells, the CGIs through which transcription extends are not methylated. This is because, in ES cells, CGIs are usually bound by CpG binding factors such as CFP1, a component of the Set1 complex that mediates trimethylation of H3K4, a marker known to block the recruitment of DNMT 3A/3B/3L –25  and alternative promoter CGIs (~ 85%) are unmethylated (≤ 45% methylation). Their hypomethylated state is associated with H3K4me3 enrichment and lack of H3K36me3. In contrast, the majority (~ 65%) of gene body CGIs are DNA-methylated (≥ 55% methylation). The hypermethylated states of these CGIs are associated with traversing transcription elongation, H3K36me3 enrichment, and lack of H3K4me3 and H3K27me3 . This phH3K4 is normally distributed on promoter CGIs and is regulated by the competitive binding of the CXXC domain of H3K4 methyltransferase and the ADD domain of DNMTs , 28. ThuH3K9me3 binds primarily to heterochromatin and mediates DNA methylation of heterochromatin regions by the methyltransferase DNMT3A/B: 1. DNMT3A/B is recruited to heterochromatin by interacting with heterochromatin protein 1 (HP1) bound to H3K9me3 ; 2. DNMTH4K20me3 is a repressive mark found in constitutive heterochromatin and at imprinted genes where it is selectively enriched on the DNA-methylated allele –38. In pH3K36me3 is mainly marked on the gene body and is formed by the histone methylation enzyme SETD2 and in conjunction with RNA Pol II catalyses the methylation of H3K36me0 , which iH3K27me3 is an epigenetic marker that inhibits the transcriptional elongation of RNA Pol II and promotes chromatin densification. H3K27me3 whole gene mapping shows that H3K27me3 is widely enriched in the gene body . RemovalIn conclusion, there is an inextricable relationship between histone modifications and DNA methylation, with histone modifications regulating DNA methylation and DNA methylation influencing histone modifications and acting further through them.DNA methylation near the transcription start site usually has a repressive effect on gene expression, but methylation of the gene body has the opposite effect –52. ThisSpurious transcription is the process by which RNA polymerase binds to an ectopic promoter rather than a normal promoter to produce an abnormal transcript . Some ofAcetylation of the N-terminal lysine of histones is closely associated with transcriptional activation, and this modification neutralizes the positive charge of the lysine, thereby inducing a change in nucleosome conformation and making it easier for nucleosomes to participate in transcription , 61. GenIn eukaryotes, pre-messenger RNAs (pre-mRNAs) undergo splicing, a process of maturation by which large intervening sequences (introns) are removed, leaving a mature transcript composed only of exons spliced together . This prTwo mechanisms that are not mutually exclusive have been proposed to explain how epigenetic modifications work: the kinetic model and the recruitment model. In the kinetic model, epigenetic modifications influence the kinetics of transcriptional elongation, which in turn affects splicing. This includes affecting the pace at which splice sites and regulatory sequences emerge in nascent precursor mRNAs during transcription, the rate of movement of RNA Pol II, and the open state of chromatin . During It has been found that demethylation of the genome results in the appearance of many new active enhancers, evenly distributed across gene bodies, and mediates the upregulation of gene expression, suggesting that gene body methylation silences enhancer function . This isRecent studies have revealed that many genes specifically expressed in tissues and involved in encoding key regulators of morphogenesis and organ development contain genomic CGIs, which may function as cis-acting elements controlling tissue-specific transcription . By compGene body methylation also plays an important role in the aberrant differentiation of tumour cells. In prostate cancer cells, DNMT1 is reduced to promote epithelial–mesenchymal transition (EMT) and conversion to tumour stem cells by promoting PKCα expression, which in turn promotes prostate cancer growth and metastasis . In tripThe majority of CpG sites are found in repeat elements (REs) that occupy two-thirds of the genome, including interspersed repeats and tandem repeats , 83. RepHuang et al. found that intestinal epithelial saprophytic cells exhibit aberrant hypermethylation in the early stages, but global gene body hypomethylation occurs in the transition from intestinal epithelial saprophytic cells to cancer cells . This suIn summary, gene body methylation, similar to promoter DNA methylation, has been shown to play an important role in the occurrence and development of tumours. However, unlike promoter DNA methylation, which mediates tumour development by suppressing oncogene expression, gene body methylation plays a complex role, and the relevant research results are relatively one-sided. As a result, the role of gene body methylation in different tumours still needs to be analysed in specific genes. It is expected that with the gradual exploration of gene body methylation, there will be a complete and accurate theory to explain its abnormal distribution and mechanism of action in tumours.There are two main types of DNMT inhibitors. One is a nucleotide analogue that inhibits its activity by replacing cytosine during DNA replication and forming a covalent bond with DNMT. Currently, nucleotide analogues of DNMT inhibitors are approved for use by the U.S. Food and Drug Administration (FDA) and show great clinical value in haematologic and solid tumours , 116. PrAnother DNMT inhibitor, which is still in the research stage, is a non-nucleotide analogue that binds directly to the region on DNMT where its methylation acts to inhibit its function . Recent DNMT inhibitors, as widely used antitumor drugs at present, have not received much attention for their demethylation effects on gene body regions. The above studies have provided clues to the role of DNMT inhibitors in gene body methylation, which may inhibit abnormally overexpressed genes in tumours and have a certain therapeutic effect on tumours. Therefore, future studies can investigate gene body methylation levels as a new therapeutic target, focussing on the role and effects of DNMT inhibitors on gene body methylation and identifying combination drugs or antitumor drugs that specifically target gene body methylation.GenE body methylation abnormalities, as a cause of tumorigenesis, are of great significance as prognostic biomarkers in tumours. There is currently widespread interest in comparing the impact of methylation differences on survival through the analysis of biological information and finding corresponding biomarkers.In head and neck squamous cell carcinoma, lincRNA C5orf66-AS1 methylation levels are significantly negatively correlated with overall survival, which is a promising prognostic biomarker in head and neck squamous cell carcinoma and can be used to identify HPV-negative patients at risk of recurrence and metastasis . In glioIn gastrointestinal tumours, Liu et al. identified methylation changes in the GFRA1, SRF and ZNF382 genes as potential synergistic biomarkers for predicting gastric cancer metastasis . DebernaThe above studies show that gene body methylation provides a clear indication of prognosis for a variety of tumours and can be an important supplement for tumour prognosis judgement. Unfortunately, the relationship between gene body methylation and tumour prognosis is still at the level of bioinformatics analysis, and its specific mechanism has not been fully explained, which makes gene body methylation not convincing enough as a biomarker and cannot be widely used in different tumours. Future research needs to focus more on exploring the mechanism of gene body methylation and tumour invasion and development, seeking clearer and more definite pathophysiological mechanisms, and translating them into clinical applications to provide a more accurate judgement for tumour treatment and prognosis.The study of genome-wide methylation has led to the initial elucidation of the mechanism of gene body methylation occurrence and the regulation of gene expression. However, the specific role of gene body methylation in gene transcription is still widely debated. Gene body methylation suppresses gene expression by promoting chromatin densification  and inteIn the field of oncology, current studies have shown that gene body methylation is essential for maintaining the stability of gene transcription and genomes. Tumour genomic hypomethylation can reduce chromosomal stability, disrupt genomic imprinting, and lead to abnormal tumour metabolism and differentiation, which are closely related to tumour occurrence, treatment, and prognosis. However, the mechanism of gene body hypomethylation in tumours is still unclear, and there are mainly two conjectures: (1) one conjecture suggests that tumour gene body hypomethylation is an active demethylation process, and TET family proteins, AICDA, and ELP3 are associated with the demethylation of methylated cytosines –128. HowCurrent research on promoter DNA methylation is still just the tip of the DNA methylation iceberg. With the iterative update of DNA methylation sequencing technology and the decreasing cost of sequencing, research on DNA methylation will boom. The discovery of oxidative bisulfite sequencing (oxBS-Seq) in recent years has solved the previous problem of being unable to distinguish DNA methylation from hydroxymethylation, making the study of DNA methylation more accurate. With the availability of large amounts of sequencing data, the challenge ahead is to ensure the depth of sequence reads and the optimization of computational methods. By solving the above challenges, the mechanism and function of DNA methylation can be systematically elaborated and refined, and DNA methylation will represent a new generation of epigenetic biomarkers with unprecedented clinical value.


Phonotrauma is considered the main cause of vocal fold polyps (VFP). However, the authors believe that an underlying anatomical deviation could render the vocal folds more susceptible to such trauma.To prove this hypothesis a retrospective chart review was carried out to correlate the surgical findings of patients with VFP.The charts of thirty-three patients who underwent surgery for excision of VFP were reviewed: 21 had right VFP, 10 had left VFP and 2 had bilateral lesions.Associated lesions were reported in 27 patients : 10 opposite nodules, 12 sulcus vocalis, 3 cysts, and 2 capillary engorgement.The high incidence of associated anatomical lesions to the VF (63%) suggests that patients with these minor underlying anatomical deviations are more vulnerable to vocal abuse, probably because they present abnormal glottic closure and an irregular vibratory margin. Current literature states that the main etiology of vocal fold polyps is sudden vocal trauma, which causes hematoma formation in Reinke's space that can initiate an inflammatory process, which would be associated with polyp formationVocal fold polyps are usually located in the anterior two-thirds of the vocal foldSpeech trauma is very common in our country, however individuals respond differently to such trauma. There are those who develop polyps, others develop nodules and others do not develop injuries on the vocal folds after a speech trauma. Things like glottic configuration and exposure to chemical products and allergens have been used to explain the different types of speech-trauma lesionsIn order to contribute to the pathophysiology of vocal fold polyps, the present investigation tries to find a correlation between vocal fold polyps and the structural alterations that happen to the contralateral vocal fold or to the ipsilateral one.We carried out a retrospective study, based on the charts of 33 consecutive patients submitted to laryngeal micro-surgery to remove a vocal fold polyp between July of 1997 and August of 2003, in a tertiary University Hospital, after being approved by the Research Ethics Committee of the institution (CEP 39/03). Of all the patients, 18 were men (55%) and 15 (45%) were women, with mean age of 42 years.The polyp was clinically diagnosed through video-laryngoscopy and confirmed during surgery. The patients who had infiltrative processes or that of deposit diseases were taken off the study.During surgery in our institution, we always look for minimum structural alterations on the vocal folds, by manipulating them with a spatula or another long blunt instrument.By analyzing the charts, the lesions were grouped in the following fashion: the associated lesions were described as reactional lesions on the contralateral fold, epidermoid cyst, mucous cyst, minor stria sulcus, major stria sulcus, pocket-type sulcus, capillary ectasia and anterior commissure microdiaphragm, and the lesion side was described as ipsi or contralateral to the polyp.Of all our patients, 21 (63.6%) had polyps on their right vocal fold (RVF), 10 (30.3%) had polyps on their left vocal fold, and 2 (6.1%) had polyps on both vocal folds .Figure 1We studied 27 associated lesions on vocal folds during the surgery: 10 (37%) of them were of the nodular thickening reaction-type lesions, 6 (22.2%) were stria major sulcus, 3 (11.1%) were epidermoid-type cyst, minor stria sulcus and pocket-type sulcus, 2 (7.4%) capillary ectasia. We did not find mucous-type cysts, or microdiaphragm on the anterior commissure . Except Of the patients with description of stria major-type sulcus, half had polyps on their vocal folds and the other half had it on their contralateral fold.The epidermoid-type cyst was found in one case, on the contralateral vocal fold, and in two cases on the ipsilateral vocal fold. The stria minor sulcus and the pocket-type sulcus surprisingly had the same statistics and incidence.Capillary ectasia were found in the same vocal fold where the polyps were found.None of the lesions were found bilaterally.Of all the structural lesions found (17), 6 were contralateral and 11 were ipsilateral .Figure 3Our initial hypothesis that a pre-existing lesion, because it alters vocal fold vibration dynamics, would render the patient more vulnerable to vocal trauma proved to be very much justifiable, because of the 27 lesions found, 17 were considered pre-existing lesions . Therefore, 51% of the patients with polyps already have some pre-existing structural alteration.This preliminary study is part of a larger one, which is still ongoing, aiming at providing a greater understanding of the factors associated with polyps on the vocal folds. We noticed structural lesions in 63% of those patients with polyps who were operated. We believe these pre-existing lesions participate in an aerodynamic change in vocal fold vibration patterns and, coupled to vocal abuse, lead to hematoma in Reinke's area, thus creating vocal fold polyps. These structural lesions change vocal fold layers that already have its own anatomical structure adapted to the patient's vibratory pattern9-10. These lesions could interfere in vocal fold coaptation, creating an irregular mucosal wave during speech production. An irregular mucosal wave could expose Reinke's area to a structural attackThe predominance of male patients in our study matches the pattern found in the literature; however, there was no overwhelming predominance of males, maybe because women go to doctors more often than men. Moreover, females, because they have more mass in their vocal folds, have a lower pitch, which impacts further their social lives, more than it would impact their male counterparts.Surprisingly, the predominance of polyps found on the right vocal fold is twice that on the left vocal fold. This data could be associated with a cerebral dominance; however that was not the scope of this study.The contralateral contact lesions found in 37% of the patients confirm the suspicion that the polyp's impact on the contralateral otherwise healthy vocal fold, on the long run, may change the fold's epithelial layerAmong minimum structural lesions, sulcus were the more commonly found lesions (70%), being found 4 times more often than cysts (17%), and 6 times more than capillary ectasia (12%). Capillary ectasia that could predispose the patient to a higher risk of hematomasThe present investigation attempts to show the importance of evaluating the polyp-related lesions during surgery, thus detecting a patient's vulnerability as to disease recurrenceAlthough this study is still preliminary, it suggests a close relation between vocal fold polyps and the minimum structural lesions, thus better explaining the higher susceptibility of some individuals towards developing vocal fold polyps after vocal trauma.The rate of structural lesions associated with vocal fold polyps suggests that anatomical changes in the vocal fold can be associated with the pathophysiology of vocal fold polyps. These associated structural lesions must be always investigated in order to achieve a better treatment planning.


Over the last decade, molecular markers have become an integral part in the management of Central Nervous System (CNS) tumors. Somatic mutations that identify and prognosticate tumors are also detected in the bio-fluids especially the serum and CSF; the sampling of which is known as liquid biopsy (LB). These tumor-derived biomarkers include plasma circulating tumor cells (CTCs), cell-free DNA (cf/ctDNAs), circulating cell-free microRNAs (cfmiRNAs), circulating extracellular vesicles, or exosomes (EVs), proteins, and tumor educated platelets. Established in the management of other malignancies, liquid biopsy is becoming an important tool in the management of CNS tumors as well. This review presents a snapshot of the current state of LB research its potential and the possible pitfalls. However, several tests like the cobas EGFR Mutation Test 2 (Roche) that detect EGFR mutations in Non-Small Cell Lung Cancer (NSCLC), the Epi pro Colon (Epigenomics AG) for colorectal cancer patients,4 the Guardant 360 CDx comprehensive genomic profiling (CGP) for the simultaneous assessment of single nucleotide variations (SNVs) or insertion and deletions in 55 tumor-associated genes5 and the 324 gene F1 Liquid CDx for use in (NSCLC) or prostate cancer meet all three criteria.6The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) initiative had recommended analytical validity, clinical validity, and clinical utility essential in adoption of liquid biopsies in clinical care.This review presents a summary of the analytes  in liquid biopsy as they apply to brain tumors, enumerates the challenges of adoption in clinical practice.7 CSF CTCs have been implicated in the prognostication of medulloblastomas for many years,8 but the exact role of CSF CTCs in the management of other primary and secondary malignant tumors remain much less clear. Though the specificity and sensitivity of CSF CTCs are much greater than that in the serum,10 the absolute contraindication in performing CSF draining procedures in patients with cranial tumors has severely limited its use. CTCs found in the serum are also known to form clusters with fibroblasts, parent tumor cells, endothelial cells, and platelets to protect against oxidative and immune stress that confer a survival advantage when compared to solitary cells.11 To increase the specificity and sensitivity of detection, these cells are captured using antibody‐based isolation techniques that have targeted specific cell surface proteins (EpCAM). However, this approach fails to detect the heterogeneity of surface proteins that is present on the CTCs12 or detect CTCs where EpCAMS are either absent or downregulated as in glial tumors.13 The frequency of release of CTCs into the bloodstream has not yet been established and it may not be ubiquitous throughout therapy.12 Negative selection such as erythrocyte lysis and density gradient separation, novel integrated approaches using subtraction enrichment and immunostaining along with fluorescent in situ hybridization and microfluidic systems using the dielectric properties of these malignant cells are now being used for CTC isolation.15 Though the role of CTCs in brain tumors is still being defined, CTCs have been extensively studied in breast cancer‐ its role in prognosis, and functions of clonal subtypes including roles of EpCAM positive and negative cells have been very well validated.16 Muller, et al. identified CTCs in the blood of glioma patients using Glial Fibrillary Acidic Protein (GFAP) as a marker in 29 out of 141 patients. CTCs detected had shared mutations with the tumor, confirming their tumor origins.17 Sullivan et al. used a micro‐fluidic system to deplete the circulating hemopoietic cells with antibodies captured CTCs in 13 out of 33 patients- CTCs exhibiting more mesenchymal than neural differentiation, suggesting a more aggressive origin.18 Gao et al. detected circulating tumor cells in 77% of glioma patients using selective enrichment and Fluorescent In Situ Hybridization (FISH),19 while Macarthur described the isolation of CTCs based on increased telomerase using an adenoviral detection system.20In a more recent article, Bang Christiansen et al. used a recombinant malarial protein rVAR2, to detect glioma cell lines by binding to cancer-specific oncofetal chondroitin sulfate (ofCS).21 In spite of this promise, the fact that CTCs are rare in brain tumors, have no specific cell surface marker, are difficult to harness, and require almost immediate processing do present barriers in its adoption. However, with the advent of single cell transcriptomics, live CTCs may provide vital additional information that aids the management of brain tumors in the future (6Ashworth discovered the presence of tumor cells in the circulation of a cancer patient- that now referred to as Circulating Tumor Cells (CTCs). These cells had been initially assessed as nonleucocytic and implicated in the development of distant metastases.e future .624 cfDNAs are not exclusively present in patients with tumors, but there seem to be an increase in the amount of ctDNA with an increase in the tumor size,25 the increase being possibly caused by an exhaustion of macrophage phagocytosis.26 In spite of low sensitivities27 caused by variable release and a relatively short half-life, the promise of sequencing the entire tumor genome piece meal, attracts interest.cfDNA are cell-derived fragmented DNA usually 170–340 base pairs in length that are found in serum, CSF, and other body fluids, probably from dead and dying cells. ctDNA refers to those released exclusively from dead and dying tumor cells, but detection of ctDNA of active clones suggest active release by viable tumor cells as well.KRAS mutations were demonstrated in a patient with pancreatic cancer.28 The presence of ctDNA in gliomas was first demonstrated by Lavon and then by Marchrzak-Celinska. In the study of 70 tumors, Lavon et al. were able to demonstrate loss of heterozygosity of 1p19q or methylation of MGMT or PTEN in 62 out of 70 samples with a sensitivity of 55% for methylation and 51% for LOH and a specificity of 100%.29 Majchrzak-Celinska et al. also successfully detected methylation of MGMT, RSSFIA, P14ARF, and P15INK4B in the serum with similar specificity and sensitivity.30 IDH mutations were detected with 100% specificity and variable sensitivity in 80 glioma patients using COld PCR.31 Using the Gardent360 cell-free DNA detection assay, Piccioni et al. showed more than one somatic mutation in more than 50% of glioblastoma (GBM) patients.32 In a recently published study, Nassiri et al., using cell‐free methylated DNA immunoprecipitation and high‐throughput sequencing (cfMeDIP‐seq), demonstrated that methylation profiling of ctDNA could detect and discriminate different intracranial tumors, with similar cells of origin.33Studying the methylation of Alu elements in ctDNA of glioma patients, Chen et al. showed significant hypomethylation in the higher grade gliomas when compared to healthy controls, but no significant difference between the tumor grades or types of tumors.34Using targeted sequencing on a 50 gene chip in 27 high-grade gliomas Ahmed et al. found at least one mutation in EGFR, VHL, KIT, PTEN, TP53 and PIK3CA genes, a possible low-cost solution for the developing world.35The presence of ctDNA in the serum was first reported by Mandell and Mathias in 1948, but it was only in the 1970s that EGFR, PTEN, ESR1, IDH1, ERBB2 and FGRR2 in CSF of 12 glioma patients.36Huang et al. isolated ctDNA from patients with Diffuse Intrinsic Pontine Glioma (DIPG) and was able to detect H3K27M mutations in 4 of 5 patients.37 Izqueierdo et al. using digital droplet PCR detected H3F3A_K27M and BRAF_V600E mutations in DIPG and hemispheric pediatric gliomas.38 Pan et al. showed that mutations detected in ctDNA not were not in the tumor biopsy- but neither the ctDNA nor the stereotactic biopsy possessed absolute specificity and sensitivity.39The specificity and sensitivity of ctDNA in CSF has been shown to be greater than that of serum in multiple studies. De‐Mattos Arruda also demonstrated the presence of mutations of et al.40 in a series of 110 liquid biopsies in 48 patients of pediatric DIPG, later corroborated by a similar study of 42 patients of adult GBMs by Bagley et al.41 MRI imaging correlated well with the ctDNA levels but CSF ctDNA was detected in 2 patients who previously did not have ctDNA in CSF prior to radiation- attributed to an increase in ctDNA release following cell damage. Increase in the CSF ctDNA with disease progression, reaching the maximum levels near-death was also demonstrated.40 In a similar study Fontanilles et al. showed that there was significant increase in the amount of cfDNA in the serum in patients with progressive disease, though the two targeted TERT promoter mutations were not reliably detected.42 However, using a novel digital droplet PCR (ddPCR) Muralidharan et al. were able to detect TERT mutations with a sensitivity and specificity of 69% and 100% respectively.43 In a study of 130 terminal patients of GBM and metastatic brain tumors being treated with intra nasal perillyl acohol based therapy, Faria et al. showed that the amount of cfDNA was significantly higher in these patients when compared to the healthy controls and increased levels of cfDNA following cessation of therapy corroborated MRI findings  have also been widely investigated in the exosomal cargo- both in the serum and the CSF. Manterola 22 In addition, exosomes help regulate the adaptation of the tumor cells to the induced hypoxic environment by upregulating transcription of small nucleolar RNA, C/D box 116-21, and downregulating potassium voltage-gated channels.72 What has been of increasing therapeutic interest is the ability of exosomes to home-in on particular cells and modify their behavior by off-loading their nucleic acid cargo-which may be modified to carry drugs or vaccines. Exosomes derived from Natural Killer Cells have already been shown to exhibit anti-tumor properties, though the exact mechanism of such actions is unclear.73 When combined with modalities like Focused Ultrasound Therapy that transiently open the blood–brain barrier, exosomes may have a therapeutic role in brain tumors.74Recently, exosomes have also been shown to actively induce the loss of tumor suppressors like pTEN in metastatic tumors.et al. were able to differentiate patients with brain cancer form the control population with a specificity and sensitivity exceeding 90%.75 Platelets also have been recently in the spotlight for their ability to transfer tumor-associated molecules using their RNA processing machinery. Analysis of such tumor “educated” platelets(TEPs) from 228 samples including GBMs differentiated cancer patients from healthy controls with an accuracy of 84-96%.76 Several plasma protein levels like interleukin 2 (IL-2) and its receptor, tumor necrosis factor alpha (TNFa), transforming growth factor beta (TGFb), chitinase-3-like protein 1 , neural cell adhesion molecule (NCAM), and neuropeptide Y (NPY) have been investigated as potential biomarkers in brain tumors.77Using total reflection(ATR)- Fourier transformed Infrared Spectroscopy (FITR) alongside machine learning algorithms, Butler 78 Clinical trials need to be designed where liquid biopsy is used to fill a specific gap in the care pathway, complementary to radiological or other established investigations. While multi-analyte testing will improve specificity and sensitivity,79 the additional data does increase the complexity of interpretation, increasing cost and sometimes confounding results. For health services around the world grappling with the pandemic and its aftermath, even a small increase in cost may be more than a significant hurdle in its adoption.The widespread adoption of liquid biopsy in clinical practice will require more than just clinical and analytical validation but proven clinical benefit. Though some of the problems with clonal proliferation have largely been answered, questions about the exact function of exosomes and the origin of cfDNA remain. The variability of outcomes will have to be reconciled by guidelines defining preanalytical conditions including blood collection, plasma preparation, and analyte extraction.Despite the present-day challenges, the potential benefits of a tool that provides real-time assessment of patients cutting through the maze of heterogeneity is there for all to see. As global collaboration increases, it is only a matter of time before this tool plays a significant role in our fight against cancer.


Hepatocellular carcinoma occurs frequently in prosimians, but the cause of these liver cancers in this group is unknown. Characterizing the genetic changes associated with hepatocellular carcinoma in prosimians may point to possible causes, treatments and methods of prevention, aiding conservation efforts that are particularly crucial to the survival of endangered lemurs. Although genomic studies of cancer in non-human primates have been hampered by a lack of tools, recent studies have demonstrated the efficacy of using human exome capture reagents across primates.In this proof-of-principle study, we applied human exome capture reagents to tumor–normal pairs from five lemurs with hepatocellular carcinoma to characterize the mutational landscape of this disease in lemurs.ARID1A, TP53 and CTNNB1, were mutated in multiple lemurs, and analysis of cancer driver genes mutated in these samples identified enrichment of genes involved with TP53 degradation and regulation. In addition to these similarities with human hepatocellular carcinoma, we also noted unique features, including six genes that contain mutations in all five lemurs. Interestingly, these genes are infrequently mutated in human hepatocellular carcinoma, suggesting potential differences in the etiology and/or progression of this cancer in lemurs and humans.Several genes implicated in human hepatocellular carcinoma, including Collectively, this pilot study suggests that human exome capture reagents are a promising tool for genomic studies of cancer in lemurs and other non-human primates.Hepatocellular carcinoma occurs frequently in prosimians, but the cause of these liver cancers is unknown. In this proof-of-principle study, we applied human DNA sequencing tools to tumor–normal pairs from five lemurs with hepatocellular carcinoma and compared the lemur mutation profiles to those of human hepatocellular carcinomas. Endemic to Madagascar, lemurs are a diverse group of primates comprised of five families and dozens of species . AccordiTP53 gene has been pinpointed as a mutational hot spot in hepatocarcinogenesis frequently associated with AFB1 exposure . SB. SB19]. Given the limited sample size available for this study, we cannot draw broad conclusions about the mutational signatures of hepatocellular carcinoma in lemurs. However, plotting the mutational profiles of the individual lemurs revealed similarities across the lemurs, particularly in the relative absence of T>A mutations . HannibaWe next applied a modified version of the sysSVM2 algorithm [22] for sample-specific identification of cancer driver genes. Molecular properties of mutated genes in lemurs were obtained from their variant annotation, while systems-level properties were retrieved from their corresponding human orthologs. This enabled us to apply a support vector machine that was trained on the properties of known cancer genes of human hepatocellular carcinoma to rank mutated genes with the most similar properties in each lemur .MLH1 and MSH6 genes, and/or clock-like mutational processes  human exome capture reagents provide a useful tool set to evaluate the genomic landscape of lemur hepatocellular carcinoma at low cost without the need to develop custom libraries and (ii) the mutational landscape of hepatocellular carcinoma in lemurs shares key similarities and differences with human hepatocellular carcinoma.The mutational spectrum in human germlines is characterized by a high frequency of C>T and T>C transitions, particularly at CpG sites . A similARID1A and TP53 and the oncogene CTNNB1 [TP53 regulation and degradation. Studies of human hepatocellular carcinoma have identified etiologies with potential links to mutations in these genes: ARID1A mutations are common in hepatocellular carcinoma related to alcohol intake, G>T mutations at codon 249 of TP53 are common in regions with dietary AFB1 exposure and HBV infection, and CTNNB1 mutations are common in HCV-related hepatocellular carcinomas [MLH1 and MSH6 contained missense mutations in Hannibal, who had more than 20 times more mutations than the other lemurs. Besides Bastet, who had a mutation in an MLH1 intron, none of the other lemurs had mutations in these mismatch repair genes. Further studies of cancer genetics in lemurs may uncover a prognostic or predictive role of mutated mismatch repair pathways in non-human primate cancers and reveal similarities and differences in the role of repair mechanisms across primate cancers. Additionally, three lemurs had an inframe deletion at the same chromosome position (11:96169749–96169752) in the gene HECW1, and two lemurs had a single high impact nonsense mutation at the position 19:68353624–68353624. In the same way that the third position of codon 249 of the TP53 gene is a mutational hot spot in human hepatocarcinogenesis, further sequencing studies may uncover mutational hot spots in lemur hepatocellular carcinoma.Among the identified mutations in lemur hepatocellular carcinoma with similarity to human hepatocellular carcinoma, we observed mutations in the tumor suppressors e CTNNB1 ; cancer rcinomas . Althougras gene in lemurs with hepatocellular carcinoma [TPM3, which is frequently overexpressed in human hepatocellular carcinoma, and the histone demethylase KDM6B, both of which may be involved in the epithelial–mesenchymal transition in hepatocarcinogenesis [While another study found a relatively high incidence of mutations in the H-arcinoma , this geogenesis , 47. Theogenesis , 49. TheWhile there remains much to be learned about the molecular pathogenesis of hepatocellular carcinoma in lemurs, this study suggests that human exome sequencing platforms may provide a low-cost alternative to evaluate the mutational landscape of lemur cancers and uncover mutational signatures in this species. Human liver cancer is characterized by distinct mutational signatures, with five COSMIC signatures  accounting for 97% of mutations in human hepatocellular carcinoma . COSMIC EMPH online.eoac016_Supplementary_DataClick here for additional data file.


Picornaviridae family and the genus Enterovirus, which also includes poliovirus (PV), the best known of the enteroviruses (EV). They are mainly transmitted by the fecal–oral route and are a major cause of viral infection each year worldwide, especially in children. They were first isolated in the late 1940s from the feces of paralyzed children living in Coxsackie  and were later classified into CV groups A (CVA) and B (CVB) based on the nature of their pathogenicity in mice. CV infections are considered to be asymptomatic or to induce subclinical or mild symptoms; nevertheless, they are also associated with various acute and chronic pathologies. For example, outbreaks of hand, foot, and mouth disease are mainly caused by enterovirus A71 and CVA, whereas CVB are often associated with the pathogenesis of chronic myocarditis, dilated cardiomyopathy, and autoimmune diseases such as type 1 diabetes (T1D). The knowledge of the cellular and molecular mechanisms of CV pathogenesis in human disease has progressed considerably in recent years, although questions still remain. This Special Issue presents the latest findings on CV virulence, mechanisms involved in the pathogenesis of CV-associated diseases, and therapeutic strategies to combat CV infections.Coxsackieviruses (CV) are ubiquitous and widespread single-stranded RNA viruses belonging to the EV, including CV, initially replicate in the gastrointestinal tract or in the upper respiratory tract before spreading to various target organs  through the lymphatic and blood systems. It has long been thought that the primary replication of all EV takes place in Peyer’s patches (PPs)  according to the results of the PV studies on chimpanzees , but it Igf2 (Insulin-like growth factor 2) expression (a major self-peptide of the insulin family found in the thymus) and disruption of negative selection of autoreactive T cells [Aire (autoimmune regulator) and Fezf2 (forebrain expressed zinc finger 2) in enriched mouse TECs [It has been suggested that the onset of islet autoimmunity and T1D results from impaired central tolerance to β-cell antigens following CVB infection of the thymus, particularly during fetal and neonatal life. Indeed, CVB4 can infect and persist in the thymus of humans and mice, especially in thymic epithelial cells (TECs), resulting in the alteration of  T cells . Furtheruse TECs . It is wuse TECs .Various preventive and therapeutic strategies to combat EV infection, especially CVB, have been reported. Antiviral drugs against CVB targeting viral or host proteins and formalin-inactivated or virus-like particles vaccines have been developed . InterfeIn summary, the complex virus–host interactions have been addressed in original articles and reviews published in the Special Issue “Coxsackievirus Infection and Associated Diseases”, and it provides readers with a better understanding of some aspects of this topic. Further investigation could benefit the research of innovative therapeutic strategies to combat CV infections and associated diseases.


Piwi-interacting RNAs (piRNAs) are a class of short chain noncoding RNAs that are constituted by 26-30 nucleotides (nt) and can couple with PIWI protein family. piRNAs were initially described in germline cells and are believed to be critical regulators of the maintenance of reproductive line. Increasing evidence has extended our perspectives on the biological significance of piRNAs and indicated that they could still affect somatic gene expression through DNA methylation, chromatin modification and transposon silencing, etc. Many studies have revealed that the dysregulation of piRNAs might contribute to diverse diseases through epigenetic changes represented by DNA methylation and chromatin modification. In this review, we summarized piRNA/PIWI protein-mediated DNA methylation regulation mechanisms and methylation changes caused by piRNA/PIWI proteins in different diseases, especially cancers. Since DNA methylation and inhibitory chromatin marks represented by histone H3 lysine 9 (H3K9) methylation frequently cooperate to silence genomic regions, we also included methylation in chromatin modification within this discussion. Furthermore, we discussed the potential clinical applications of piRNAs as a new type promising biomarkers for cancer diagnosis, as well as the significance of piRNA/PIWI protein-associated methylation changes in treatment, providing disparate insights into the potential applications of them. Piwi-interacting RNAs (piRNAs) are a class of short chain noncoding RNAs, that are constituted by 26-30 nucleotides (nt) and have a 5'-terminal uridine or tenth position adenosine bias, lacking explicit secondary structure motifs Mature piRNAs are 26-30 nt in size with a 2'-O-methylation at the 3' end, which are distinct and unique features of all piRNAs piRNAs are generated from piRNA clusters, a relatively few and characteristic genomic regions In the cytoplasm, after the generation of primary piRNAs, piRNAs are amplified with the ping-pong mechanism Across different species, the PIWI protein family is highly conservative According to previous studies, piRNAs function at the transcriptional, posttranscriptional and posttranslational modifications levels (nontransposon loci) by recruiting DNA methyltransferase (DNMT) and alters transcriptional activity By binding to the coding regions of gene promoters, piRNAs are involved in the gene regulation process After discovering TGS, researchers found that piRNAs could suppress the function of target genes by regulating posttranscriptional networks, which is similar to what miRNAs form, namely, piRNA-RNA interactions. lncRNAs The piRNA/PIWI complex can directly interact with certain transcriptional factors (TFs) and regulates their posttranslational modifications such as phosphorylation Fig. d. For exBased on the existing research evidence, at the transcriptional level, piRNAs undergo epigenetic changes mainly via sequence-specific aberrant DNA methylation and histone modification in somatic cells Ago family proteins assemble with small RNAs into RISC, such as miRNAs, siRNAs or piRNAs, mediating sequence-specific target gene silencing TEs are thought to be kept silent by DNA methylation and packaging into heterochromatin Moreover, piRNAs can induce and affect DNA methylation not only at transposons, but also at nontransposon loci Recently, second-generation sequencing technology has provided an extremely effective way to research disease-related genes and noncoding RNAs including piRNAs In women, breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer-related death First, it was revealed that the changes in DNA methylation caused by piRNAs in BC could promote tumor development. For example, piR-823 was reported to increase the expression of DNMTs including DNMT1, DNMT3A, and DNMT3B, and promote DNA methylation level of the adenomatous polyposis coli (APC) gene, a tumor suppressor, thereby motivating WNT signaling pathway and inducing the formation of cancer cell stemness (CSCs) in the luminal subtype of BC cells, ultimately contributing to BC tumorigenesis In addition to piRNAs, PIWI was also significantly associated with DNA methylation in BC Lung cancer (LC) has the highest cancer-related mortality among men and the second-highest cancer-related mortality (after breast cancer) among women worldwide Firstly, studies have shown that the DLK1-DIO3 locus is methylated aberrantly in current and former smoker patients with NSCLC and aberrant expression of DLK1-DIO3 might contribute to tumorigenesis in LC Hematological malignancies include leukemia, multiple myeloma (MM) and lymphoma In MM, CSCs and myeloid-derived suppressor cells (MDSCs) are two important cellular components in the tumor microenvironment, that may change the cancer phenotype and affect patient survival Digestive malignancies are a group of cancers that occur in the gastrointestinal tract and its related organs. Cancers of the colon and rectum, stomach, liver, esophagus, and pancreas are five of the ten most commonly diagnosed cancers and causes of cancer-related death in vitro and in vivoRecently, a study revealed that piR-823 induced aberrant DNA methylation through the epigenetic pathway DNMT3B and exerted an oncogenic role in esophageal squamous cell carcinoma (ESCC). Additionally, the expression of piR-823 has high specificity in ESCC, and a higher level of piR-823 signified a higher risk of lymph node metastasis, suggesting its potential as a biomarker for diagnosis and prognosis Genitourinary cancers typically refer to neoplasms of the kidneys, bladder, prostate, testicular, uterus, ovary and so on. Prostate cancer is the most common tumor in men worldwide It was demonstrated that piRNAs were associated with prostate cancer via specific hypermethylation, which led to their transcriptional inactivation in prostate cancer. First, piR-31470 could form a piR-31470/PIWIL4 complex by binding to PIWIL4. Afterward, this complex coupled with the glutathione S-transferase pi 1 (GSTP1) RNA transcripts and recruited DNMT 1 and DNMT 3A and methylated the CpG island of GSTP1 to activate and sustain the hypermethylation and inactivation of GSTP1. In the process of prostate carcinogenesis, silence of GSTP1 via hypermethylation is an early and common event and functional inactivation of GSTP1 enhanced the susceptibility to oxidative stress and the risk of prostatic cancer progression Regarding to the function of PIWI protein and DNA methylation in genitourinary neoplasms, there are two main types of tumors that are common in women: cervical cancer and endometrial cancer. First, HPV oncoproteins E6 and E7 have been reported to reactivate PIWIL2 during cervical cancer tumorigenesis. Then PIWIL2 induced H3K9 acetylation but reduced H3K9 trimethylation, resulting in epigenetic reprogramming and the maintenance of embryonic stem cells (ESCs) by affecting the expression of genes in c-Myc, Nanog, Oct4, Sox2 and Klf4 Melanoma is still a potentially lethal skin malignancy and the occurrence of malignant melanoma has continued to increase for many years MILI, also named PIWIL2 in humans, inhibited melanoma cell metastasis, probably providing a novel perspective on melanoma treatment. Specifically, MILI could methylate the LINE-1 repetitive sequence to regulate MAGEA expression and restrain melanoma cell migration Infertility remains a global problem what is estimated to affect almost 186 million people worldwide, and human infertility contributes to more than 50% of all cases of global childlessness Transposons pose a serious challenge to the germline, and mechanisms that inhibit their activity are crucial for transgenerational genomic integrity; thereinto LINE-1 is the most important retrotransposon and is epigenetically repressed by CpG island DNA methylation Many publications have revealed that piRNAs exist extensively in the central and peripheral nervous systems and participate in regulating the physiological and pathological processes of various neurological diseases including anxiety disorders and Parkinson's disease Researchers demonstrated for the first time that Aplysia piRNA/PIWI complex facilitated serotonin-dependent methylation at the promoter CpG island of CREB2, a major inhibitory limitation of memory in Aplysia, enhancing long-term synaptic facilitation Cardiovascular diseases (CVDs) are the main causes of human mortality worldwide Recent research has indicated that the piRNA/PIWI complex might be a new class of molecules that are potentially useful as biomarkers for the classification of pulmonary hypertension as well as therapeutic targets. The expression of piR-63076 was upregulated in pulmonary vessels under hypoxia and partly attributed to DNA methylation of the acyl-CoA dehydrogenase (Acadm) promoter region. Acadm deficiency ultimately increased hypoxia-induced pulmonary-artery smooth muscle cells (PASMCs) proliferation Although little is known about how piRNAs function in the human immune system, studies have suggested that piRNAs could act as signal transduction mediators in well differentiated immune cells First, a previous study showed that piR-30840 interacted with PIWIL4 and Ago4 that targeted the intron of IL-4, which inversely caused the degradation of IL-4 pre-mRNA In terms of mortality, globally, cancer is the second cause leading to death (8.97 million deaths) after ischemic heart disease but is expected to become the most common cause of death in 2060. Several cancers, such as esophageal, liver, and pancreatic cancers, have the worst prognosis, usually less than 20% in 5 years More and more evidence point to the fact that piRNAs could represent attractive diagnostic and prognostic biomarker candidates, as some piRNAs have been involved in the development of many diseases Beyond the role of piRNAs and piRNA-associated methylation as biomarkers, several studies have put forward their potential role as therapeutic tools Due to the advent of next-generation sequencing technologies, the expression of piRNAs can be easily observed. In fact, the study of piRNAs has deepened our understanding of the complex regulatory network of non-coding RNAs. Numerous studies have reported dysregulated expression of piRNAs in different diseases samples and proposed the underlying mechanisms. In this review, we summarized that piRNAs could guide PIWI proteins to regulate gene expression in a transcriptional way including DNA methylation and histone methylation Fig. a, b and/However, the potential applications of piRNAs for clinical medicine still face plenty of challenges. First of all, it remains unclear that whether the abnormal expression of piRNAs really participate in the development of these diseases or just are a byproduct of other molecular activities. Secondly, to a large extent, the molecular mechanisms of physiological and pathological action of piRNAs are largely unknown, while some studies have proposed potential molecular mechanisms. Thirdly, in clinical practice, the potential application of piRNAs as a diagnostic and therapeutic target is currently at a speculative and theoretical stage, such as how to distinguish between healthy individuals and patients and how to resolve potential side effects, drug resistance, therapeutic effects, and administration mode. Accordingly, in the future, further studies and multi-center clinical trials should be conducted to fully aware of the regulatory mechanism and clinical application of piRNAs.