Anti-miR-93-5p therapy prolongs sepsis survival by restoring the peripheral immune response.

Sepsis remains a leading cause of death for humans and currently has no pathogenesis-specific therapy. Hampered progress is partly due to a lack of insight into deep mechanistic processes. In the past decade, deciphering the functions of small noncoding miRNAs in sepsis pathogenesis became a dynamic research topic. To screen for new miRNA targets for sepsis therapeutics, we used samples for miRNA array analysis of PBMCs from patients with sepsis and control individuals, blood samples from 2 cohorts of patients with sepsis, and multiple animal models: mouse cecum ligation puncture-induced (CLP-induced) sepsis, mouse viral miRNA challenge, and baboon Gram+ and Gram- sepsis models. miR-93-5p met the criteria for a therapeutic target, as it was overexpressed in baboons that died early after induction of sepsis, was downregulated in patients who survived after sepsis, and correlated with negative clinical prognosticators for sepsis. Therapeutically, inhibition of miR-93-5p prolonged the overall survival of mice with CLP-induced sepsis, with a stronger effect in older mice. Mechanistically, anti-miR-93-5p therapy reduced inflammatory monocytes and increased circulating effector memory T cells, especially the CD4+ subset. AGO2 IP in miR-93-KO T cells identified important regulatory receptors, such as CD28, as direct miR-93-5p target genes. In conclusion, miR-93-5p is a potential therapeutic target in sepsis through the regulation of both innate and adaptive immunity, with possibly a greater benefit for elderly patients than for young patients.

Long noncoding RNA 01534 maintains cancer stemness by downregulating endoplasmic reticulum stress response in colorectal cancer.

Background: Studies have shown that cancer stemness and the endoplasmic reticulum (ER) stress response are inversely regulated in colorectal cancer (CRC), but the mechanism has not been fully clarified. Long noncoding RNAs (lncRNAs) play key roles in cancer progression and metastasis. In this study we investigated lncRNA 01534 (LINC01534) as a possible modulator between cancer stemness and ER stress response. Methods: In vitro experiments using CRC cell lines were performed to explore a possible role of LINC01534. The expression of LINC01534 in clinical CRC samples was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization. Results: Silencing LINC01534 led to suppression of cell proliferation, invasiveness, and cell cycle progression at the G2-M phase, and promoted apoptosis. Moreover, we found that silencing LINC01534 suppressed cancer stemness, while it activated the ER stress response, especially through the PERK/eIF2α signaling pathway. In situ hybridization revealed LINC01534 was expressed in tumor cells and upregulated in CRC tissues compared with normal epithelium. A survival survey indicated that high LINC01534 expression was significantly associated with shorter overall survival in 187 CRC patients. Conclusion: This is the first report on LINC01534 in human cancer. Our findings suggest that LINC01534 may be an important modulator of the maintenance of cancer stemness and suppression of the ER stress response, and that it could be a novel prognostic factor in CRC.

17ß-estradiol promotes extracellular vesicle release and selective miRNA loading in ERα-positive breast cancer.

The causes and consequences of abnormal biogenesis of extracellular vesicles (EVs) are not yet well understood in malignancies, including in breast cancers (BCs). Given the hormonal signaling dependence of estrogen receptor-positive (ER+) BC, we hypothesized that 17ß-estradiol (estrogen) might influence EV production and microRNA (miRNA) loading. We report that physiological doses of 17ß-estradiol promote EV secretion specifically from ER+ BC cells via inhibition of miR-149-5p, hindering its regulatory activity on SP1, a transcription factor that regulates the EV biogenesis factor nSMase2. Additionally, miR-149-5p downregulation promotes hnRNPA1 expression, responsible for the loading of let-7's miRNAs into EVs. In multiple patient cohorts, we observed increased levels of let-7a-5p and let-7d-5p in EVs derived from the blood of premenopausal ER+ BC patients, and elevated EV levels in patients with high BMI, both conditions associated with higher levels of 17ß-estradiol. In brief, we identified a unique estrogen-driven mechanism by which ER+ BC cells eliminate tumor suppressor miRNAs in EVs, with effects on modulating tumor-associated macrophages in the microenvironment.

The deleted in oral cancer (DOC1 aka CDK2AP1) tumor suppressor gene is downregulated in oral squamous cell carcinoma by multiple microRNAs.

Cyclin-dependent kinase 2-associated protein 1 (CDK2AP1; also known as deleted in oral cancer or DOC1) is a tumor suppressor gene known to play functional roles in both cell cycle regulation and in the epigenetic control of embryonic stem cell differentiation, the latter as a core subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex. In the vast majority of oral squamous cell carcinomas (OSCC), expression of the CDK2AP1 protein is reduced or lost. Notwithstanding the latter (and the DOC1 acronym), mutations or deletions in its coding sequence are extremely rare. Accordingly, CDK2AP1 protein-deficient oral cancer cell lines express as much CDK2AP1 mRNA as proficient cell lines. Here, by combining in silico and in vitro approaches, and by taking advantage of patient-derived data and tumor material in the analysis of loss of CDK2AP1 expression, we identified a set of microRNAs, namely miR-21-5p, miR-23b-3p, miR-26b-5p, miR-93-5p, and miR-155-5p, which inhibit its translation in both cell lines and patient-derived OSCCs. Of note, no synergistic effects were observed of the different miRs on the CDK2AP1-3-UTR common target. We also developed a novel approach to the combined ISH/IF tissue microarray analysis to study the expression patterns of miRs and their target genes in the context of tumor architecture. Last, we show that CDK2AP1 loss, as the result of miRNA expression, correlates with overall survival, thus highlighting the clinical relevance of these processes for carcinomas of the oral cavity.

Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) pathway is a powerful tool to combat tumor growth and progression; however, drug resistance frequently emerges. We identify CD5L (CD5 antigen-like precursor) as an important gene upregulated in response to antiangiogenic therapy leading to the emergence of adaptive resistance. By using both an RNA-aptamer and a monoclonal antibody targeting CD5L, we are able to abate the pro-angiogenic effects of CD5L overexpression in both in vitro and in vivo settings. In addition, we find that increased expression of vascular CD5L in cancer patients is associated with bevacizumab resistance and worse overall survival. These findings implicate CD5L as an important factor in adaptive resistance to antiangiogenic therapy and suggest that modalities to target CD5L have potentially important clinical utility.

Contributions of Circulating microRNAs for Early Detection of Lung Cancer.

There is unmet need to develop circulating biomarkers that would enable earlier interception of lung cancer when more effective treatment options are available. Here, a set of 30 miRNAs, selected from a review of the published literature were assessed for their predictive performance in identifying lung cancer cases in the pre-diagnostic setting. The 30 miRNAs were assayed using sera collected from 102 individuals diagnosed with lung cancer within one year following blood draw and 212 controls matched for age, sex, and smoking status. The additive performance of top-performing miRNA candidates in combination with a previously validated four-protein marker panel (4MP) consisting of the precursor form of surfactant protein B (Pro-SFTPB), cancer antigen 125 (CA125), carcinoembryonic antigen (CEA) and cytokeratin-19 fragment (CYFRA21-1) was additionally assessed. Of the 30 miRNAs evaluated, five (miR-320a-3p, miR-210-3p, miR-92a-3p, miR-21-5p, and miR-140-3p) were statistically significantly (Wilcoxon rank sum test p < 0.05) elevated in case sera compared to controls, with individual AUCs ranging from 0.57−0.62. Compared to the 4MP alone, the combination of 3-miRNAs + 4MP improved sensitivity at 95% specificity by 19.1% ((95% CI of difference 0.0−28.6); two-sided p: 0.006). Our findings demonstrate utility for miRNAs for early detection of lung cancer in combination with a four-protein marker panel.

SIK2 inhibition enhances PARP inhibitor activity synergistically in ovarian and triple-negative breast cancers.

Poly(ADP-ribose) polymerase inhibitors (PARP inhibitors) have had an increasing role in the treatment of ovarian and breast cancers. PARP inhibitors are selectively active in cells with homologous recombination DNA repair deficiency caused by mutations in BRCA1/2 and other DNA repair pathway genes. Cancers with homologous recombination DNA repair proficiency respond poorly to PARP inhibitors. Cancers that initially respond to PARP inhibitors eventually develop drug resistance. We have identified salt-inducible kinase 2 (SIK2) inhibitors, ARN3236 and ARN3261, which decreased DNA double-strand break (DSB) repair functions and produced synthetic lethality with multiple PARP inhibitors in both homologous recombination DNA repair deficiency and proficiency cancer cells. SIK2 is required for centrosome splitting and PI3K activation and regulates cancer cell proliferation, metastasis, and sensitivity to chemotherapy. Here, we showed that SIK2 inhibitors sensitized ovarian and triple-negative breast cancer (TNBC) cells and xenografts to PARP inhibitors. SIK2 inhibitors decreased PARP enzyme activity and phosphorylation of class-IIa histone deacetylases (HDAC4/5/7). Furthermore, SIK2 inhibitors abolished class-IIa HDAC4/5/7-associated transcriptional activity of myocyte enhancer factor-2D (MEF2D), decreasing MEF2D binding to regulatory regions with high chromatin accessibility in FANCD2, EXO1, and XRCC4 genes, resulting in repression of their functions in the DNA DSB repair pathway. The combination of PARP inhibitors and SIK2 inhibitors provides a therapeutic strategy to enhance PARP inhibitor sensitivity for ovarian cancer and TNBC.

The Use of Machine Learning Algorithms and the Mass Spectrometry Lipidomic Profile of Serum for the Evaluation of Tacrolimus Exposure and Toxicity in Kidney Transplant Recipients.

Tacrolimus has a narrow therapeutic window; a whole-blood trough target concentration of between 5 and 8 ng/mL is considered a safe level for stable kidney transplant recipients. Tacrolimus serum levels must be closely monitored to obtain a balance between maximizing efficacy and minimizing dose-related toxic effects. Currently, there is no specific tacrolimus toxicity biomarker except a graft biopsy. Our study aimed to identify specific serum metabolites correlated with tacrolinemia levels using serum high-precision liquid chromatography-mass spectrometry and standard laboratory evaluation. Three machine learning algorithms were used (Naïve Bayes, logistic regression, and Random Forest) in 19 patients with high tacrolinemia (8 ng/mL) and 23 patients with low tacrolinemia (5 ng/mL). Using a selected panel of five lipid metabolites (phosphatidylserine, phosphatidylglycerol, phosphatidylethanolamine, arachidyl palmitoleate, and ceramide), Mg2+, and uric acid, all three machine learning algorithms yielded excellent classification accuracies between the two groups. The highest classification accuracy was obtained by Naïve Bayes, with an area under the curve of 0.799 and a classification accuracy of 0.756. Our results show that using our identified five lipid metabolites combined with Mg2+ and uric acid serum levels may provide a novel tool for diagnosing tacrolimus toxicity in kidney transplant recipients. Further validation with targeted MS and biopsy-proven TAC toxicity is needed.

lncRNAs UC.145 and PRKG1-AS1 Determine the Functional Output of DKK1 in Regulating the Wnt Signaling Pathway in Gastric Cancer.

DKK1 inhibits the canonical Wnt signaling pathway that is known to be involved in various cancers. However, whether DKK1 acts as an oncogene or tumor suppressor gene remains controversial. Furthermore, the DKK1-regulating mechanism in gastric cancer has not yet been defined. The aim of this study was to explore whether the ultraconserved region UC.145 regulates epigenetic changes in DKK1 expression in gastric cancer. Microarray analysis revealed that UC.145 exhibited the highest binding affinity to EZH2, a histone methyltransferase. The effects of UC.145 inactivation were assessed in gastric cancer cell lines using siRNA. The results indicated that UC.145 triggers DKK1 methylation via interaction with EZH2 and is involved in the canonical Wnt signaling pathway. Additionally, interaction between UC.145 and another long non-coding RNA adjacent to DKK1, PRKG1-AS1, induced a synergistic effect on Wnt signaling. The regulation of these three genes was closely associated with patient overall survival. Inactivation of UC.145 induced apoptosis and inhibited the growth and migratory, invasive, and colony-forming abilities of gastric cancer cells. The study findings provide insights into Wnt signaling in gastric cancer and support UC.145 as a potential novel predictive biomarker for the disease.

Intraoperative Right Colic Graft Ischemia, Followed by Delayed Oesophagoplasty Including an Ileo-Transverse Anastomosis and the Cervical Revascularisation of the Ileum: Case Report.

The ischemic complications during the isolation of the substituting oesophageal graft placement and after its placement may lead to graft necrosis and to the need to find a different reconstructive procedure. The most frequent reports of graft necroses occur in the days following the reconstruction. We are presenting the case of a 27-y.o. with full dysphagia as a result of caustic stenosis, in whose case the oesophageal reconstruction was abandoned following the irreversible ischemia of the right colic graft during the vascular isolation, followed by right-side hemicolectomy and ileo-transverse anastomosis. 4 years post the ingestion of a caustic substance and 2 years post the right colic graft ischemic necrosis, we performed an oesophageal reconstruction using a pediculated, cervically revascularized, ileo-colic graft on the left colic vessels. The graft's particularity is that is formed from left and transverse colon and ileum portions, including the ileo-transverse anastomosis performed 2 years prior to the oesophageal reconstruction.

Molecular Correlates of Venous Thromboembolism (VTE) in Ovarian Cancer.

BACKGROUND: The incidence of venous thromboembolism (VTE) in patients with ovarian cancer is higher than most solid tumors, ranging between 10-30%, and a diagnosis of VTE in this patient population is associated with worse oncologic outcomes. The tumor-specific molecular factors that may lead to the development of VTE are not well understood. OBJECTIVES: The aim of this study was to identify molecular features present in ovarian tumors of patients with VTE compared to those without. METHODS: We performed a multiplatform omics analysis incorporating RNA and DNA sequencing, quantitative proteomics, as well as immune cell profiling of high-grade serous ovarian carcinoma (HGSC) samples from a cohort of 32 patients with or without VTE. RESULTS: Pathway analyses revealed upregulation of both inflammatory and coagulation pathways in the VTE group. While DNA whole-exome sequencing failed to identify significant coding alterations between the groups, the results of an integrated proteomic and RNA sequencing analysis indicated that there is a relationship between VTE and the expression of platelet-derived growth factor subunit B (PDGFB) and extracellular proteins in tumor cells, namely collagens, that are correlated with the formation of thrombosis. CONCLUSIONS: In this comprehensive analysis of HGSC tumor tissues from patients with and without VTE, we identified markers unique to the VTE group that could contribute to development of thrombosis. Our findings provide additional insights into the molecular alterations underlying the development of VTE in ovarian cancer patients and invite further investigation into potential predictive biomarkers of VTE in ovarian cancer.

Lenalidomide enhances CD23.CAR T cell therapy in chronic lymphocytic leukemia.

Chimeric antigen receptors (CAR)-modified T cells are an emerging therapeutic tool for chronic lymphocytic leukemia (CLL). However, in patients with CLL, well-known T-cell defects and the inhibitory properties of the tumor microenvironment (TME) hinder the efficacy of CAR T cells. We explored a novel approach combining CARs with lenalidomide, an immunomodulatory drug that tempers the immunosuppressive activity of the CLL TME. T cells from patients with CLL were engineered to express a CAR specific for CD23, a promising target antigen. Lenalidomide maintained the in vitro effector functions of CD23.CAR+ T cells effector functions in terms of antigen-specific cytotoxicity, cytokine release and proliferation. Overall, lenalidomide preserved functional CAR T-CLL cell immune synapses. In a Rag2-/-γc-/--based xenograft model of CLL, we demonstrated that, when combined with low-dose lenalidomide, CD23.CAR+ T cells efficiently migrated to leukemic sites and delayed disease progression when compared to CD23.CAR+ T cells given with rhIL-2. These observations underline the therapeutic potential of this novel CAR-based combination strategy in CLL.

Transcribed Ultraconserved Regions Are Associated with Clinicopathological Features in Breast Cancer.

Ultraconserved regions (UCRs) are 481 genome segments, with length longer than 200 bp, that are 100% conserved among humans, mice, and rats. The majority of UCRs are transcriptionally active (T-UCRs) as many of them produce non-coding RNAs. In a previous study, we evaluated the expression level of T-UCRs in breast cancer (BC) patients and found that 63% of transcripts correlated with some clinical and/or molecular parameter of BC. In this study, we delved into the expression levels of 12 T-UCRs and correlated them with clinicopathological parameters, immunohistochemical markers, and overall survival in two breast cancer cohorts: TCGA and Brazilian patients. We found that uc.268 is more expressed in TCGA patients under 40 years of age, associated with progesterone receptor (PR) and estrogen receptor (ER), and its high expression is found in luminal A. Lower uc.84 and uc.376 were respectively observed in metastatic and stage IV tumors associated with good prognostic in luminal B. Moreover, uc.84 was only related to the HER2+, while uc.376 was related to ER+ and PR+, and HER2+. A panel composed of uc.147, uc.271, and uc.427 distinguished luminal A from triple negative patients with an AUC of 0.9531 (sensitivity 92.19% and specificity 86.76%). These results highlight the potential role of T-UCRs in BC and provide insights into the potential application of T-UCRs as biomarkers.

Targeting CDK7 reverses tamoxifen resistance through regulating stemness in ER+ breast cancer.

BACKGROUND: Although tamoxifen is the mainstay endocrine therapy for estrogen receptor-positive (ER+) breast cancer patients, the emergence of tamoxifen resistance is still the major challenge that results in treatment failure. Tamoxifen is very effective in halting breast cancer cell proliferation; nonetheless, the ability of tamoxifen to target cancer stem and progenitor cell populations (CSCs), a major key player for the emergence of tamoxifen resistance, has not been adequately investigated yet. Thus, we explored whether targeting CDK7 modulates CSCs subpopulation and tamoxifen resistance in ER+ breast cancer cells. METHODS: Mammosphere-formation assay, stem cell biomarkers and tamoxifen sensitivity were analyzed in MCF7 tamoxifen-sensitive cell line and its resistant counterpart, LCC2, following CDK7 targeting by THZ1 or siRNA. RESULTS: Analysis of clinically relevant data indicated that expression of stemness factor, SOX2, was positively correlated with CDK7 expression in tamoxifen-treated patients. Moreover, overexpression of the stemness gene, SOX2, was associated with shorter overall survival in those patients. Importantly, the number of CSC populations and the expression of CDK7, P-Ser118-ER-α and c-MYC were significantly higher in LCC2 cells compared with parental MCF-7 cells. Moreover, targeting CDK7 inhibited mammosphere formation, CSC-regulating genes, and CSC biomarkers expression in MCF-7 and LCC2 cells. CONCLUSION: Our data indicate, for the first time, that CDK7-targeted therapy in ER+ breast cancer ameliorates tamoxifen resistance, at least in part, by inhibiting cancer stemness. Thus, targeting CDK7 might represent a potential approach for relieving tamoxifen resistance in ER+ breast cancer.

Analysis of the circRNA and T-UCR populations identifies convergent pathways in mouse and human models of Rett syndrome.

Noncoding RNAs play regulatory roles in physiopathology, but their involvement in neurodevelopmental diseases is poorly understood. Rett syndrome is a severe, progressive neurodevelopmental disorder linked to loss-of-function mutations of the MeCP2 gene for which no cure is yet available. Analysis of the noncoding RNA profile corresponding to the brain-abundant circular RNA (circRNA) and transcribed-ultraconserved region (T-UCR) populations in a mouse model of the disease reveals widespread dysregulation and enrichment in glutamatergic excitatory signaling and microtubule cytoskeleton pathways of the corresponding host genes. Proteomic analysis of hippocampal samples from affected individuals confirms abnormal levels of several cytoskeleton-related proteins together with key alterations in neurotransmission. Importantly, the glutamate receptor GRIA3 gene displays altered biogenesis in affected individuals and in vitro human cells and is influenced by expression of two ultraconserved RNAs. We also describe post-transcriptional regulation of SIRT2 by circRNAs, which modulates acetylation and total protein levels of GluR-1. As a consequence, both regulatory mechanisms converge on the biogenesis of AMPA receptors, with an effect on neuronal differentiation. In both cases, the noncoding RNAs antagonize MeCP2-directed regulation. Our findings indicate that noncoding transcripts may contribute to key alterations in Rett syndrome and are not only useful tools for revealing dysregulated processes but also molecules of biomarker value.