Ceramide nanoliposomes augment the efficacy of venetoclax and cytarabine in models of acute myeloid leukemia.

Despite several new therapeutic options for acute myeloid leukemia (AML), disease relapse remains a significant challenge. We have previously demonstrated that augmenting ceramides can counter various drug-resistance mechanisms, leading to enhanced cell death in cancer cells and extended survival in animal models. Using a nanoscale delivery system for ceramide (ceramide nanoliposomes, CNL), we investigated the effect of CNL within a standard of care venetoclax/cytarabine (Ara-C) regimen. We demonstrate that CNL augmented the efficacy of venetoclax/cytarabine in in vitro, ex vivo, and in vivo models of AML. CNL treatment induced non-apoptotic cytotoxicity, and augmented cell death induced by Ara-C and venetoclax. Mechanistically, CNL reduced both venetoclax (Mcl-1) and cytarabine (Chk1) drug-resistant signaling pathways. Moreover, venetoclax and Ara-C augmented the generation of endogenous pro-death ceramide species, which was intensified with CNL. Taken together, CNL has the potential to be utilized as an adjuvant therapy to improve outcomes, potentially extending survival, in patients with AML.

Harnessing the power of sphingolipids: Prospects for acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive, heterogenous malignancy characterized by clonal expansion of bone marrow-derived myeloid progenitor cells. While our current understanding of the molecular and genomic landscape of AML has evolved dramatically and opened avenues for molecularly targeted therapeutics to improve upon standard intensive induction chemotherapy, curative treatments are elusive, particularly in older patients. Responses to current AML treatments are transient and incomplete, necessitating the development of novel treatment strategies to improve outcomes. To this end, harnessing the power of bioactive sphingolipids to treat cancer shows great promise. Sphingolipids are involved in many hallmarks of cancer of paramount importance in AML. Leukemic blast survival is influenced by cellular levels of ceramide, a bona fide pro-death molecule, and its conversion to signaling molecules such as sphingosine-1-phosphate and glycosphingolipids. Preclinical studies demonstrate the efficacy of therapeutics that target dysregulated sphingolipid metabolism as well as their combinatorial synergy with clinically-relevant therapeutics. Thus, increased understanding of sphingolipid dysregulation may be exploited to improve AML patient care and outcomes. This review summarizes the current knowledge of dysregulated sphingolipid metabolism in AML, evaluates how pro-survival sphingolipids promote AML pathogenesis, and discusses the therapeutic potential of targeting these dysregulated sphingolipid pathways.

Promoter Demethylation Upregulates STEAP1 Gene Expression in Human Prostate Cancer: In Vitro and In Silico Analysis.

The Six Transmembrane Epithelial Antigen of the Prostate (STEAP1) is an oncogene overexpressed in several human tumors, particularly in prostate cancer (PCa). However, the mechanisms involved in its overexpression remain unknown. It is well known that epigenetic modifications may result in abnormal gene expression patterns, contributing to tumor initiation and progression. Therefore, this study aimed to analyze the methylation pattern of the STEAP1 gene in PCa versus non-neoplastic cells. Bisulfite amplicon sequencing of the CpG island at the STEAP1 gene promoter showed a higher methylation level in non-neoplastic PNT1A prostate cells than in human PCa samples. Bioinformatic analysis of the GEO datasets also showed the STEAP1 gene promoter as being demethylated in human PCa, and a negative association with STEAP1 mRNA expression was observed. These results are supported by the treatment of non-neoplastic PNT1A cells with DNMT and HDAC inhibitors, which induced a significant increase in STEAP1 mRNA expression. In addition, the involvement of HDAC in the regulation of STEAP1 mRNA expression was corroborated by a negative association between STEAP1 mRNA expression and HDAC4,5,7 and 9 in human PCa. In conclusion, our work indicates that STEAP1 overexpression in PCa can be driven by the hypomethylation of STEAP1 gene promoter.

Role of SPTSSB-Regulated de Novo Sphingolipid Synthesis in Prostate Cancer Depends on Androgen Receptor Signaling.

Anti-androgens are a common therapy in prostate cancer (PCa) targeting androgen receptor (AR) signaling. However, these therapies fail due to selection of highly aggressive AR-negative cancer cells that have no therapeutic options available. We demonstrate that elevating endogenous ceramide levels with administration of exogenous ceramide nanoliposomes (CNLs) was efficacious in AR-negative cell lines with limited efficacy in AR-positive cells. This effect is mediated through reduced de novo sphingolipid synthesis in AR-positive cells. We show that anti-androgens elevate de novo generation of sphingolipids via SPTSSB, a rate-limiting mediator of sphingolipid generation. Moreover, pharmacological inhibition of AR increases the efficacy of CNL in AR-positive cells through de novo synthesis, while SPTSSB knockdown limited CNL's efficacy in AR-negative cells. Alluding to clinical relevance, SPTSSB is upregulated in patients with advanced PCa after anti-androgens treatment. These findings emphasize the relevance of AR regulation upon sphingolipid metabolism and the potential of CNL as a PCa therapeutic.

Inhibition of Lysosomal Function Mitigates Protective Mitophagy and Augments Ceramide Nanoliposome-Induced Cell Death in Head and Neck Squamous Cell Carcinoma.

Therapies for head and neck squamous cell carcinoma (HNSCC) are, at best, moderately effective, underscoring the need for new therapeutic strategies. Ceramide treatment leads to cell death as a consequence of mitochondrial damage by generating oxidative stress and causing mitochondrial permeability. However, HNSCC cells are able to resist cell death through mitochondria repair via mitophagy. Through the use of the C6-ceramide nanoliposome (CNL) to deliver therapeutic levels of bioactive ceramide, we demonstrate that the effects of CNL are mitigated in drug-resistant HNSCC via an autophagic/mitophagic response. We also demonstrate that inhibitors of lysosomal function, including chloroquine (CQ), significantly augment CNL-induced death in HNSCC cell lines. Mechanistically, the combination of CQ and CNL results in dysfunctional lysosomal processing of damaged mitochondria. We further demonstrate that exogenous addition of methyl pyruvate rescues cells from CNL + CQ-dependent cell death by restoring mitochondrial functionality via the reduction of CNL- and CQ-induced generation of reactive oxygen species and mitochondria permeability. Taken together, inhibition of late-stage protective autophagy/mitophagy augments the efficacy of CNL through preventing mitochondrial repair. Moreover, the combination of inhibitors of lysosomal function with CNL may provide an efficacious treatment modality for HNSCC.

Glucosylceramide production maintains colon integrity in response to Bacteroides fragilis toxin-induced colon epithelial cell signaling.

Enterotoxigenic Bacteroides fragilis (ETBF) is a commensal bacterium of great importance to human health due to its ability to induce colitis and cause colon tumor formation in mice through the production of B. fragilis toxin (BFT). The formation of tumors is dependent on a pro-inflammatory signaling cascade, which begins with the disruption of epithelial barrier integrity through cleavage of E-cadherin. Here, we show that BFT increases levels of glucosylceramide, a vital intestinal sphingolipid, both in mice and in colon organoids (colonoids) generated from the distal colons of mice. When colonoids are treated with BFT in the presence of an inhibitor of glucosylceramide synthase (GCS), the enzyme responsible for generating glucosylceramide, colonoids become highly permeable, lose structural integrity, and eventually burst, releasing their contents into the extracellular matrix. By increasing glucosylceramide levels in colonoids via an inhibitor of glucocerebrosidase (GBA, the enzyme that degrades glucosylceramide), colonoid permeability was reduced, and bursting was significantly decreased. In the presence of BFT, pharmacological inhibition of GCS caused levels of tight junction protein 1 (TJP1) to decrease. However, when GBA was inhibited, TJP1 levels remained stable, suggesting that BFT-induced production of glucosylceramide helps to stabilize tight junctions. Taken together, our data demonstrate a glucosylceramide-dependent mechanism by which the colon epithelium responds to BFT.

Glucosylceramide synthase maintains influenza virus entry and infection.

Influenza virus is an enveloped virus wrapped in a lipid bilayer derived from the host cell plasma membrane. Infection by influenza virus is dependent on these host cell lipids, which include sphingolipids. Here we examined the role of the sphingolipid, glucosylceramide, in influenza virus infection by knocking out the enzyme responsible for its synthesis, glucosylceramide synthase (UGCG). We observed diminished influenza virus infection in HEK 293 and A549 UGCG knockout cells and demonstrated that this is attributed to impaired viral entry. We also observed that entry mediated by the glycoproteins of other enveloped viruses that enter cells by endocytosis is also impaired in UGCG knockout cells, suggesting a broader role for UGCG in viral entry by endocytosis.

Glucosylceramidase Maintains Influenza Virus Infection by Regulating Endocytosis.

Influenza virus is an RNA virus encapsulated in a lipid bilayer derived from the host cell plasma membrane. Previous studies showed that influenza virus infection depends on cellular lipids, including the sphingolipids sphingomyelin and sphingosine. Here we examined the role of a third sphingolipid, glucosylceramide, in influenza virus infection following clustered regularly interspaced short palindromic repeats with Cas9 (CRISPR-Cas9)-mediated knockout (KO) of its metabolizing enzyme glucosylceramidase (GBA). After confirming GBA knockout of HEK 293 and A549 cells by both Western blotting and lipid mass spectrometry, we observed diminished infection in both KO cell lines by a PR8 (H1N1) green fluorescent protein (GFP) reporter virus. We further showed that the reduction in infection correlated with impaired influenza virus trafficking to late endosomes and hence with fusion and entry. To examine whether GBA is required for other enveloped viruses, we compared the results seen with entry mediated by the glycoproteins of Ebola virus, influenza virus, vesicular stomatitis virus (VSV), and measles virus in GBA knockout cells. Entry inhibition was relatively robust for Ebola virus and influenza virus, modest for VSV, and mild for measles virus, suggesting a greater role for viruses that enter cells by fusing with late endosomes. As the virus studies suggested a general role for GBA along the endocytic pathway, we tested that hypothesis and found that trafficking of epidermal growth factor (EGF) to late endosomes and degradation of its receptor were impaired in GBA knockout cells. Collectively, our findings suggest that GBA is critically important for endocytic trafficking of viruses as well as of cellular cargos, including growth factor receptors. Modulation of glucosylceramide levels may therefore represent a novel accompaniment to strategies to antagonize "late-penetrating" viruses, including influenza virus.IMPORTANCE Influenza virus is the pathogen responsible for the second largest pandemic in human history. A better understanding of how influenza virus enters host cells may lead to the development of more-efficacious therapies against emerging strains of the virus. Here we show that the glycosphingolipid metabolizing enzyme glucosylceramidase is required for optimal influenza virus trafficking to late endosomes and for consequent fusion, entry, and infection. We also provide evidence that promotion of influenza virus entry by glucosylceramidase extends to other endosome-entering viruses and is due to a general requirement for this enzyme, and hence for optimal levels of glucosylceramide, for efficient trafficking of endogenous cargos, such as the epidermal growth factor (EGF) receptor, along the endocytic pathway. This work therefore has implications for the basic process of endocytosis as well as for pathogenic processes, including virus entry and Gaucher disease.

Comparing diagnostic and prognostic performance of two-gene promoter methylation panels in tissue biopsies and urines of prostate cancer patients.

BACKGROUND: Prostate cancer (PCa) is one of the most common cancers among men worldwide. Current screening methods for PCa display limited sensitivity and specificity, not stratifying for disease aggressiveness. Hence, development and validation of new molecular markers is needed. Aberrant gene promoter methylation is common in PCa and has shown promise as clinical biomarker. Herein, we assessed and compared the diagnostic and prognostic performance of two-gene panel promoter methylation in the same sample sets. METHODS: Promoter methylation of panel #1 (singleplex-miR-34b/c and miR-193b) and panel #2 (multiplex-APC, GSTP1, and RARß2) was evaluated using MethyLight methodology in two different cohorts [prostate biopsy (#1) and urine sediment (#2)]. Biomarkers' diagnostic (validity estimates) and prognostic (disease-specific survival, disease-free survival, and progression-free survival) performance was assessed. RESULTS: Promoter methylation levels of both panels showed the highest levels in PCa samples in both cohorts. In tissue samples, methylation panel #1 and panel #2 detected PCa with AUC of 0.9775 and 1.0, respectively, whereas in urine samples, panel #2 demonstrated superior performance although a combination of miR-34b/c, miR-193b, APC, and RARß2 disclosed the best results (AUC = 0.9817). Furthermore, higher mir-34b/c and panel #2 methylation independently predicted for shorter DSS. Furthermore, time-dependent ROC curves showed that both miR-34b/c and GSTP1 methylation levels identify with impressive performance patients that relapse up to 15 years after diagnosis (AUC = 0.751 and AUC = 0.765, respectively). CONCLUSIONS: We concluded that quantitative gene panel promoter methylation might be a clinically useful tool for PCa non-invasive detection and risk stratification for disease aggressiveness in both tissue biopsies and urines.

Novel Sphingolipid-Based Cancer Therapeutics in the Personalized Medicine Era.

Sphingolipids are bioactive lipids that participate in a wide variety of biological mechanisms, including cell death and proliferation. The myriad of pro-death and pro-survival cellular pathways involving sphingolipids provide a plethora of opportunities for dysregulation in cancers. In recent years, modulation of these sphingolipid metabolic pathways has been in the forefront of drug discovery for cancer therapeutics. About two decades ago, researchers first showed that standard of care treatments, e.g., chemotherapeutics and radiation, modulate sphingolipid metabolism to increase endogenous ceramides, which kill cancer cells. Strikingly, resistance to these treatments has also been linked to altered sphingolipid metabolism, favoring lipid species that ultimately lead to cell survival. To this end, many inhibitors of sphingolipid metabolism have been developed to further define not only our understanding of these pathways but also to potentially serve as therapeutic interventions. Therefore, understanding how to better use these new drugs that target sphingolipid metabolism, either alone or in combination with current cancer treatments, holds great potential for cancer control. While sphingolipids in cancer have been reviewed previously (Hannun & Obeid, 2018; Lee & Kolesnick, 2017; Morad & Cabot, 2013; Newton, Lima, Maceyka, & Spiegel, 2015; Ogretmen, 2018; Ryland, Fox, Liu, Loughran, & Kester, 2011) in this chapter, we present a comprehensive review on how standard of care therapeutics affects sphingolipid metabolism, the current landscape of sphingolipid inhibitors, and the clinical utility of sphingolipid-based cancer therapeutics.

MicroRNA-27a-5p regulation by promoter methylation and MYC signaling in prostate carcinogenesis.

Upregulation of MYC and miRNAs deregulation are common in prostate cancer (PCa). Overactive MYC may cause miRNAs' expression deregulation through transcriptional and post-transcriptional mechanisms and epigenetic alterations are also involved in miRNAs dysregulation. Herein, we aimed to elucidate the role of regulatory network between MYC and miRNAs in prostate carcinogenesis. MYC expression was found upregulated in PCa cases and matched precursor lesions. MicroRNA's microarray analysis of PCa samples with opposed MYC levels identified miRNAs significantly overexpressed in high-MYC PCa. However, validation of miR-27a-5p in primary prostate tissues disclosed downregulation in PCa, instead, correlating with aberrant promoter methylation. In a series of castration-resistant PCa (CRPC) cases, miR-27a-5p was upregulated, along with promoter hypomethylation. MYC and miR-27a-5p expression levels in LNCaP and PC3 cells mirrored those observed in hormone-naíve PCa and CRPC, respectively. ChIP analysis showed that miR-27a-5p expression is only regulated by c-Myc in the absence of aberrant promoter methylation. MiR-27a-5p knockdown in PC3 cells promoted cell growth, whereas miRNA forced expression in LNCaP and stable MYC-knockdown PC3 cells attenuated the malignant phenotype, suggesting a tumor suppressive role for miR-27a-5p. Furthermore, miR-27a-5p upregulation decreased EGFR/Akt1/mTOR signaling. We concluded that miR-27a-5p is positively regulated by MYC, and its silencing due to aberrant promoter methylation occurs early in prostate carcinogenesis, concomitantly with loss of MYC regulatory activity. Our results further suggest that along PCa progression, miR-27a-5p promoter becomes hypomethylated, allowing for MYC to resume its regulatory activity. However, the altered cellular context averts miR-27a-5p from successfully accomplishing its tumor suppressive function at this stage of disease.

SETDB2 and RIOX2 are differentially expressed among renal cell tumor subtypes, associating with prognosis and metastization.

Increasing detection of small renal masses by imaging techniques entails the need for accurate discrimination between benign and malignant renal cell tumors (RCTs) as well as among malignant RCTs, owing to differential risk of progression through metastization. Although histone methylation has been implicated in renal tumorigenesis, its potential as biomarker for renal cell carcinoma (RCC) progression remains largely unexplored. Thus, we aimed to characterize the differential expression of histone methyltransferases (HMTs) and histone demethylases (HDMs) in RCTs to assess their potential as metastasis biomarkers. We found that SETDB2 and RIOX2 (encoding for an HMT and an HDM, respectively) expression levels was significantly altered in RCTs; these genes were further selected for validation by quantitative RT-PCR in 160 RCTs. Moreover, SETDB2, RIOX2, and three genes encoding for enzymes involved in histone methylation (NO66, SETD3, and SMYD2), previously reported by our group, were quantified (RT-PCR) in an independent series of 62 clear cell renal cell carcinoma (ccRCC) to assess its potential role in ccRCC metastasis development. Additional validation was performed using TCGA dataset. SETDB2 and RIOX2 transcripts were overexpressed in RCTs compared to renal normal tissues (RNTs) and in oncocytomas vs. RCCs, with ccRCC and papillary renal cell carcinoma (pRCC) displaying the lowest levels. Low SETDB2 expression levels and higher stage independently predicted shorter disease-free survival. In our 62 ccRCC cohort, significantly higher RIOX2, but not SETDB2, expression levels were depicted in cases that developed metastasis during follow-up. These findings were not apparent in TCGA dataset. We concluded that SETDB2 and RIOX2 might be involved in renal tumorigenesis and RCC progression, especially in metastatic spread. Moreover, SETDB2 expression levels might independently discriminate among RCC subgroups with distinct outcome, whereas higher RIOX2 transcript levels might identify ccRCC cases with more propensity to endure metastatic dissemination.

Identification of clear cell renal cell carcinoma and oncocytoma using a three-gene promoter methylation panel.

BACKGROUND: Promoter methylation has emerged as a promising class of epigenetic biomarkers for diagnosis and prognosis of renal cell tumors (RCTs). Although differential gene promoter methylation patterns have been reported for the major subtypes (clear cell, papillary and chromophobe renal cell carcinoma, and oncocytoma), validation of diagnostic performance in independent series have been seldom performed. Herein, we aimed at assessing the diagnostic performance of genes previously shown to be hypermethylated in RCTs in different clinical settings. METHODS: Promoter methylation levels of HOXA9 and OXR1 were assessed by quantitative methylation specific PCR. ROC curves were generated for OXR1, OXR1 combined with MST1R and HOXA9. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy were computed, maximizing specificity. Methylation levels were also correlated with clinical and pathological relevant parameters. RESULTS: HOXA9 and OXR1 promoter methylation was disclosed in 73 and 87% of RCTs, respectively. A two-gene methylation panel comprising OXR1 and MST1R identified malignancy with 98% sensitivity and 100% specificity, and clear cell renal cell carcinoma with 90% sensitivity and 98% specificity. HOXA9 promoter methylation allowed for discrimination between oncocytoma and both papillary and chromophobe renal cell carcinoma but only with 77% sensitivity and 73% specificity. Significantly higher OXR1 promoter methylation levels (p = 0.005) were associated with high nuclear grade in ccRCC. CONCLUSIONS: A panel including OXR1 and MST1R promoter methylation allows specific and sensitive identification of renal cell tumors, and, especially, of clear cell renal cell carcinoma. Moreover, higher OXR1 promoter methylation levels associate with clear cell renal cell carcinoma nuclear grade, a surrogate for tumor aggressiveness. Thus, gene promoter methylation analysis might a useful ancillary tool in diagnostic management of renal masses.

Epigenetic disruption of miR-130a promotes prostate cancer by targeting SEC23B and DEPDC1.

MicroRNAs (miRNAs) are small, non-coding RNAs that mediate post-transcriptional gene silencing, fine tuning gene expression. In an initial screen, miRNAs were found to be globally down-regulated in prostate cancer (PCa) cell lines and primary tumours. Exposure of PCa cell lines to a demethylating agent, 5-Aza-CdR resulted in an increase in the expression levels of miRNAs in general. Using stringent filtering criteria miR-130a was identified as the most promising candidate and selected for validation analyses in our patient series. Down-regulation of miR-130a was associated with promoter hypermethylation. MiR-130a methylation levels discriminated PCa from non-malignant tissues (AUC = 0.956), and urine samples revealed high specificity for non-invasive detection of patients with PCa (AUC = 0.89). Additionally, repressive histone marks were also found in the promoter of miR-130a. Over-expression of miR-130a in PCa cells reduced cell viability and invasion capability, and increased apoptosis. Putative targets of miR-130a were assessed by microarray expression profiling and DEPD1C and SEC23B were selected for validation. Silencing of both genes resembled the effect of over-expressing miR-130a in PCa cells. Our data indicate that miR-130a is an epigenetically regulated miRNA involved in regulation of key molecular and phenotypic features of prostate carcinogenesis, acting as a tumour suppressor miRNA.

Prognostic significance of MST1R dysregulation in renal cell tumors.

Macrophage stimulating 1 receptor (MST1R) is a C-MET proto-oncogene family receptor tyrosine kinase. Promoter methylation patterns determine transcription of MST1R variants as hypermethylation of a region upstream of transcription start site (TSS) is associated with lack of MST1R long transcript (MST1R long) and expression of a short transcript with oncogenic potential. Thus, we aimed to investigate MST1R variant transcript regulation in renal cell tumors (RCT) and assess their prognostic potential. We found, in a series of 120 RCT comprising the four main subtypes (clear cell, papillary and chromophobe renal cell carcinoma, and oncocytoma), that higher methylation levels close to TSS were associated with total MST1R expression levels (MST1R total) in primary tumors (p=0.049) and renal cancer cell lines. After demethylating treatment, MST1R long/MST1R total ratio increased, as expected, in two renal cell carcinoma cell lines tested. However, in primary tumors with hypermethylation upstream of TSS, a decrease in MST1R long/MST1R total ratio was not detected, although higher expression ratio of nuclear factor-κB was apparent. Furthermore, survival analysis demonstrated that MST1R long/MST1R total ratio was independently associated with shorter disease-specific and disease-free survival, whereas MST1R total expression associated with shorter disease-specific survival. In conclusion, although promoter methylation patterns seem to determine MST1R global transcription regulation in renal cell carcinoma, other mechanisms might contribute to deregulate MST1R variant expression in RCT. Nevertheless, MST1R total expression and MST1R long/MST1R total ratio modulate the biological and clinical aggressiveness of renal cell carcinoma, as depicted by its prognostic significance, a finding that requires validation in a larger independent series.

Comprehensive Analysis of Secreted Protein, Acidic and Rich in Cysteine in Prostate Carcinogenesis: Development of a 3D Nanostructured Bone-Like Model.

Most aggressive prostate cancer (PCa) types tend to metastasize frequently to bone and SPARC, a matricellular protein, might participate in such biological processes. The objective of this study was to evaluate the effect of SPARC in prostate carcinogenesis and bone metastization. This was explored assessing the morphology, metabolic activity and SPARC expression of different PCa cell lines resembling different stages of carcinogenesis, using a 3D bone-biomimetic model (collagen nanofibers/nanohydroxyapatite) grafted with SPARC. Our findings highlight distinct cellular behavior depending on cell type and presence of exogenous SPARC. In fact, SPARC addition contributed to the survival and significant growth of a non-bone metastatic PCa cell line (LNCaP) on bone-like biomaterial. Moreover, SPARC expression levels were evaluated in a series of prostatic tissues, comparing normal prostate, pre-neoplastic prostate intraepithelial neoplasias and overtly malignant tumors, and also metastasis to its correspondent primary prostate tumors, ascertaining potential association between SPARC and clinicopathological data. Remarkably, SPARC was overexpressed in patients with higher Gleason Score, indicating tumors with poor prognosis, as well as in metastasis, particularly from bone sites, compared with their respective primary tumors. The results suggest a potential role of SPARC as a clinical target on PCa, due to its association with bone metastization.

Diagnostic and prognostic epigenetic biomarkers in cancer.

Growing cancer incidence and mortality worldwide demands development of accurate biomarkers to perfect detection, diagnosis, prognostication and monitoring. Urologic (prostate, bladder, kidney), lung, breast and colorectal cancers are the most common and despite major advances in their characterization, this has seldom translated into biomarkers amenable for clinical practice. Epigenetic alterations are innovative cancer biomarkers owing to stability, frequency, reversibility and accessibility in body fluids, entailing great potential of assay development to assist in patient management. Several studies identified putative epigenetic cancer biomarkers, some of which have been commercialized. However, large multicenter validation studies are required to foster translation to the clinics. Herein we review the most promising epigenetic detection, diagnostic, prognostic and predictive biomarkers for the most common cancers.

Expression of histone methyltransferases as novel biomarkers for renal cell tumor diagnosis and prognostication.

Renal cell tumors (RCTs) are the most lethal of the common urological cancers. The widespread use of imaging entailed an increased detection of small renal masses, emphasizing the need for accurate distinction between benign and malignant RCTs, which is critical for adequate therapeutic management. Histone methylation has been implicated in renal tumorigenesis, but its potential clinical value as RCT biomarker remains mostly unexplored. Hence, the main goal of this study was to identify differentially expressed histone methyltransferases (HMTs) and histone demethylases (HDMs) that might prove useful for RCT diagnosis and prognostication, emphasizing the discrimination between oncocytoma (a benign tumor) and renal cell carcinoma (RCC), especially the chromophobe subtype (chRCC). We found that the expression levels of 3 genes--SMYD2, SETD3, and NO66--was significantly altered in a set of RCTs, which was further validated in a large independent cohort. Higher expression levels were found in RCTs compared to normal renal tissues (RNTs) and in chRCCs comparatively to oncocytomas. SMYD2 and SETD3 mRNA levels correlated with protein expression assessed by immunohistochemistry. SMYD2 transcript levels discriminated RCTs from RNT, with 82.1% sensitivity and 100% specificity [area under curve (AUC) = 0.959], and distinguished chRCCs from oncocytomas, with 71.0% sensitivity and 73.3% specificity (AUC = 0.784). Low expression levels of SMYD2, SETD3, and NO66 were significantly associated with shorter disease-specific and disease-free survival, especially in patients with non-organ confined tumors. We conclude that expression of selected HMTs and HDMs might constitute novel biomarkers to assist in RCT diagnosis and assessment of tumor aggressiveness.

MicroRNA-375 plays a dual role in prostate carcinogenesis.

BACKGROUND: Prostate cancer (PCa), a highly incident and heterogeneous malignancy, mostly affects men from developed countries. Increased knowledge of the biological mechanisms underlying PCa onset and progression are critical for improved clinical management. MicroRNAs (miRNAs) deregulation is common in human cancers, and understanding how it impacts in PCa is of major importance. MiRNAs are mostly downregulated in cancer, although some are overexpressed, playing a critical role in tumor initiation and progression. We aimed to identify miRNAs overexpressed in PCa and subsequently determine its impact in tumorigenesis. RESULTS: MicroRNA expression profiling in primary PCa and morphological normal prostate (MNPT) tissues identified 17 miRNAs significantly overexpressed in PCa. Expression of three miRNAs, not previously associated with PCa, was subsequently assessed in large independent sets of primary tumors, in which miR-182 and miR-375 were validated, but not miR-32. Significantly higher expression levels of miR-375 were depicted in patients with higher Gleason score and more advanced pathological stage, as well as with regional lymph nodes metastases. Forced expression of miR-375 in PC-3 cells, which display the lowest miR-375 levels among PCa cell lines, increased apoptosis and reduced invasion ability and cell viability. Intriguingly, in 22Rv1 cells, which displayed the highest miR-375 expression, knockdown experiments also attenuated the malignant phenotype. Gene ontology analysis implicated miR-375 in several key pathways deregulated in PCa, including cell cycle and cell differentiation. Moreover, CCND2 was identified as putative miR-375 target in PCa, confirmed by luciferase assay. CONCLUSIONS: A dual role for miR-375 in prostate cancer progression is suggested, highlighting the importance of cellular context on microRNA targeting.

SMYD3 contributes to a more aggressive phenotype of prostate cancer and targets Cyclin D2 through H4K20me3.

Prostate cancer (PCa) is one of the most incident cancers worldwide but clinical and pathological parameters have limited ability to discriminate between clinically significant and indolent PCa. Altered expression of histone methyltransferases and histone methylation patterns are involved in prostate carcinogenesis. SMYD3 transcript levels have prognostic value and discriminate among PCa with different clinical aggressiveness, so we decided to investigate its putative oncogenic role on PCa.We silenced SMYD3 and assess its impact through in vitro (cell viability, cell cycle, apoptosis, migration, invasion assays) and in vivo (tumor formation, angiogenesis). We evaluated SET domain's impact in PCa cells' phenotype. Histone marks deposition on SMYD3 putative target genes was assessed by ChIP analysis.Knockdown of SMYD3 attenuated malignant phenotype of LNCaP and PC3 cell lines. Deletions affecting the SET domain showed phenotypic impact similar to SMYD3 silencing, suggesting that tumorigenic effect is mediated through its histone methyltransferase activity. Moreover, CCND2 was identified as a putative target gene for SMYD3 transcriptional regulation, through trimethylation of H4K20.Our results support a proto-oncogenic role for SMYD3 in prostate carcinogenesis, mainly due to its methyltransferase enzymatic activity. Thus, SMYD3 overexpression is a potential biomarker for clinically aggressive disease and an attractive therapeutic target in PCa.