Survivin Research Articles

RIPK2 promotes the progression of colon cancer by regulating BIRC3-mediated ubiquitination of IKBKG

Colon cancer is a cancer with high morbidity and mortality worldwide. Receptor interacting serine/threonine kinase 2 (RIPK2) has been identified as a proto-oncogene, but its role in colon cancer is largely unknown. Herein, we found that RIPK2 interference could inhibit the proliferation and invasion of colon cancer cells, and promote apoptosis. Baculoviral IAP repeat containing 3 (BIRC3) is an E3 ubiquitin ligase, which was found highly expressed in colon cancer cells. Co-immunoprecipitation (Co-IP) experiments showed that RIPK2 could directly bind with BIRC3. Then, we demonstrated that RIPK2 overexpression promoted the expression of BIRC3, BIRC3 interference could eliminate RIPK2-dependent cell proliferation and invasion, and BIRC3 overexpression rescued the suppressive effect of RIPK2 interference on cell proliferation and invasion. We further identified IKBKG, an inhibitor of nuclear factor kappa B, as a ubiquitination substrate targeted by BIRC3. IKBKG interference could eliminate the inhibitory effect of BIRC3 interference on cell invasion. RIPK2 could promote BIRC3-mediated ubiquitination of IKBKG, inhibit the expression of IKBKG protein, and promote the expression of NF-κB subunits p50 and p65 proteins. In addition, DLD-1 cells transfected with sh-RIPK2 or/and sh-BIRC3 were injected into mice to establish a tumor xenograft model, and we found that administration of sh-RIPK2 or sh-BIRC3 impeded the growth of xenograft tumors in vivo, and co-administration displayed a better inhibitory effect. In general, RIPK2 promotes the progression of colon cancer by promoting BIRC3-mediated ubiquitination of IKBKG and activating the NF-κB signaling pathway.

LncRNA CROCCP2/miR-5584-5p/BIRC5 network activates apoptosis to inhibit glioma development

Objective: To detect the role of long non-coding RNA (lncRNA) CROCC Pseudogene 2 (CROCCP2)/miR-5584-5p /Baculoviral IAP Repeat Containing 5 (BIRC5) network in glioma growth. Methods: The Cancer Genome Atlas (TCGA) database was accessed to obtain the gene datasets associated with glioma growth. Bioinformatics techniques was employed to analyze the key network and construct the regulatory network of lncRNA CROCCP2/miR-5584-5p targeting BIRC5. Subsequently, the quantitative Polymerase Chain Reaction (qPCR) experiment was conducted to validate the expression levels of LncRNA CROCCP2, miR-5584-5p, and BIRC5 in both glioma tissues and normal brain tissues. Furthermore, we harnessed RNA interference technology to knock down BIRC5 in U251 cells, and flow cytometry was utilized to assess cell apoptosis. Results: LncRNA CROCCP2 is implicated in the binding of miR-5584-5p, and targeting BIRC5. PCR analysis revealed an elevated expression level of CROCCP2 and BIRC5 in glioma tissues, accompanied by a low expression of miR-5584-5p. Moreover, knockdown of BIRC5 results in an induction of apoptosis. Conclusions: LncRNA CROCCP2 could absorb miR-5584-5p targeting BIRC5 to activate cell apoptosis, so as to inhibit glioma development.

Dysregulation of PLOD2 Promotes Tumor Metastasis and Invasion in Hepatocellular Carcinoma.

Metastasis is a major factor associated with high recurrence and mortality in hepatocellular carcinoma (HCC) patients while the underlying mechanism of metastasis remains elusive. In our study, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) was shown to be involved in the process of metastasis in HCC. The Cancer Genome Atlas (TCGA) database and HCC tissue microarrays were used to evaluate the expression of genes. In vitro migration, invasion, in vivo subcutaneous tumor model and in vivo lung metastasis assays were used to determine the role of PLOD2 in tumor growth and metastasis in HCC. RNA sequencing and gene set enrichment analysis were performed to uncover the downstream factor of PLOD2 in HCC cells. A luciferase reporter assay was performed to evaluate the interaction between PLOD2 and interferon regulatory factor 5 (IRF5). The expression of PLOD2 in HCC tissues was higher than that in adjacent tissues, and increased PLOD2 expression was often found in advanced tumors and was correlated with poor prognosis in HCC patients. In vitro experiments, knockdown of PLOD2 reduced the migration and invasion of human HCC cells. Loss of PLOD2 suppressed human HCC growth and metastasis in a subcutaneous tumor model and a lung metastasis model. Baculoviral IAP repeat containing 3 (BIRC3) was proven to be the downstream factor of PLOD2 in human HCC cells. In addition, PLOD2 was transcriptionally regulated by IRF5 in HCC cells. High expression of PLOD2 was regulated by IRF5, which was correlated with the poor survival of HCC patients. PLOD2 enhanced HCC metastasis via BIRC3, suggesting that PLOD2 might be a valuable prognostic biomarker for HCC treatment.

Tuning Nanobodies' Bioactivity: Coupling to Ultrasmall Gold Nanoparticles Allows the Intracellular Interference with Survivin.

Nanobodies are highly affine binders, often used to track disease-relevant proteins inside cells. However, they often fail to interfere with pathobiological functions, required for their clinical exploitation. Here, a nanobody targeting the disease-relevant apoptosis inhibitor and mitosis regulator Survivin (SuN) is utilized. Survivin's multifaceted functions are regulated by an interplay of dynamic cellular localization, dimerization, and protein-protein interactions. However, as Survivin harbors no classical "druggable" binding pocket, one must aim at blocking extended protein surface areas. Comprehensive experimental evidence demonstrates that intracellular expression of SuN allows to track Survivin at low nanomolar concentrations but failed to inhibit its biological functions. Small angle X-ray scattering of the Survivin-SuN complex locates the proposed interaction interface between the C-terminus and the globular domain, as such not blocking any pivotal interaction. By clicking multiple SuN to ultrasmall (2nm) gold nanoparticles (SuN-N), not only intracellular uptake is enabled, but additionally, Survivin crosslinking and interference with mitotic progression in living cells are also enabled. In sum, it is demonstrated that coupling of nanobodies to nanosized scaffolds can be universally applicable to improve their function and therapeutic applicability.

Combined expression of WT1/PRAME genes and BIRC5/PRAME genes and their clinical significance in adult patients with acute myeloid leukemia

Abstract Background Several tumor-associated antigens have been recently recognized that qualify to monitor tumor burden of acute myeloid leukemia (AML) or are used as antigen-specific approach for anticancer immunotherapy. Aim The current study was designed to assess the intracellular expression levels of the immunogenic tumor antigens ( WT1 , BIRC5, and PRAME genes) and clarify the prognostic effect of their combined overexpression, that is, WT1 /PRAME and BIRC5 / PRAME , on the clinical course, the outcome, and the response to treatment in patients with AML. Settings and design A total of 61 patients with AML were included in the study. All patients received induction chemotherapy. Detection of intracellular WT1, BIRC5, and PRAME genes in myeloid blast cells was done by real-time PCR on bone marrow samples. Their levels were correlated with the demographic data, the clinical and pathological data, the defined subgroups, the overall survival, and the response to treatment in patients with AML. Results WT1, BIRC5, and PRAME overexpression was detected in 73.8, 28, and 38.6% of patients, respectively. Nearly half of the patients (51.7%) were WT1+/PRAME− and significantly associated with absence of both favorable cytogenetic prognosis and complete remission. In addition, it expression was correlated with female sex, higher count of blasts in peripheral blood, positive CD34 expression, and higher FAB classification (M4) but with no statistically significant difference. Most patients were double negative BIRC5−/PRAME− (45.6%). A significant relation was found between BIRC5+/PRAME− and older age patients (P=0.003) and a lower overall survival (P=0.005) and also associated with higher count of blast cells but not statistically significant. Most BIRC5−/PRAME+ (72.7%) achieved complete remission at 28 days, followed by double negative BIRC5−/PRAME− (56.6%) at 28 days, most of BIRC5+/PRAME− patients died (77.8%). Conclusion In patients with AML, the expression levels of genes WT1, BIRC5, and PRAME were high, and these genes had a substantial correlation with poor prognostic factors, including a poor cytogenetic prognosis, incomplete remission, and a reduced survival rate. Therefore, they could be considered as targets for anticancer immunotherapy as well as possible markers for monitoring AML.

Sex-Mediated Differences in TNF Signaling- and ECM-Related Gene Expression in Aged Rat Kidney.

Aging leads to the functional decline of an organism, which is associated with age and sex. To understand the functional change of kidneys depending on age and sex, we carried out a transcriptome analysis using RNA sequencing (RNA-Seq) data from rat kidneys. Four differentially expressed gene (DEG) sets were generated according to age and sex, and Gene Ontology analysis and overlapping analysis of Kyoto Encyclopedia of Genes and Genomes pathways were performed for the DEG sets. Through the analysis, we revealed that inflammation- and extracellular matrix (ECM)-related genes and pathways were upregulated in both males and females during aging, which was more prominent in old males than in old females. Furthermore, quantitative real-time PCR analysis confirmed that the expression of tumor necrosis factor (TNF) signaling-related genes, Birc3, Socs3, and Tnfrsf1b, and ECM-related genes, Cd44, Col3a1, and Col5a2, which showed that the genes were markedly upregulated in males and not females during aging. Also, hematoxylin-eosin (H&E) staining for histological analysis showed that renal damage was highly shown in old males rather than old females. In conclusion, in the rat kidney, the genes involved in TNF signaling and ECM accumulation are upregulated in males more than in females during aging. These results suggest that the upregulation of the genes may have a higher contribution to age-related kidney inflammation and fibrosis in males than in females.

Role of hypoxia-inducible factor-1α and survivin in breast cancer recurrence and prognosis

ObjectiveTo analyze the expression of hypoxia-inducible factor-1α (HIF-1α) and survivin in breast cancer, and different molecular subtypes of breast cancer and to assess their relationship with recurrence and prognosis. MethodsThe expression levels of HIF-1α and survivin genes in breast cancer were investigated using bioinformatics. Their protein expression levels were then verified through immunohistochemistry (IHC), and their relationship with recurrence and prognosis was assessed. ResultsExpression levels of HIF-1α and survivin genes and proteins were increased in breast cancer tissues compared with normal tissues. Both were associated with clinical features of breast cancer and differentially expressed in different molecular subtypes of breast cancer, and both are related to the signal pathway of breast cancer growth and invasion. HIF-1α and survivin gene and protein expression levels were correlated, and both were associated with breast cancer recurrence (R = 0.380, P < 0.05; R = 0.673, P < 0.05, respectively). According to The Cancer Genome Atlas (TCGA) database, HIF1A and BIRC5 gene were not associated with breast cancer prognosis (P ≥ 0.05); however, HIF-1α and survivin protein were associated with recurrence patient's overall survival (OS) (P < 0.05). ConclusionHIF-1α and survivin are highly expressed in breast cancer and can be used as potential biomarkers to predict recurrence and assess prognosis.

Survivin expression at the mRNA level in tumors and the protein concentration in the serum and peritoneal fluid in patients with serous ovarian tumors.

Ovarian cancer is one of the gynecological cancers that have the worst prognosis. The expression of the proteins from the IAP family (inhibitor of apoptosis protein), including survivin, is observed in many types of cancer. The aim of the study was to evaluate survivin at the mRNA level in tumors and the protein concentration in the serum and peritoneal fluid of patients with serous ovarian cancer in order to assess the relationship between the concentration of survivin and the histological subtypes of cancer. The study group consisted of 55 women, including patients with serous ovarian cancer (n = 30, nine low-grade serous carcinoma LGSC, 21 high-grade serous carcinoma HGSC), serous cysts (n = 10) and the control group (n = 15). The concentration of protein in the peritoneal fluid and serum was assessed using ELISA tests. The expression of survivin gene BIRC5 in the tumors was assessed using the RT-qPCR method. The data that was obtained indicated that the concentration of survivin was higher in the serum of the women with serous ovarian cancer compared those that had benign tumors (p < 0.05) and the control group (p < 0.001). The survivin concentration was also higher in both the serum and peritoneal fluid in the HGSC group compared to the LGSC group (p < 0.001). The mRNA level was highest in the HGSC group, and there was a statistically significant difference compared to those in the benign tumor group and HGSC group ( p < 0.05). The observed changes prove that the expression level increases significantly in HGSC in both the protein and mRNA levels. Based on these findings, it can be assumed that assessing this parameter could be a useful additional indicator of the progression and differentiation of this type of cancer. However, this requires further research in a larger group of patients and possibly in other types of ovarian cancer.

CBX5 loss drives EGFR inhibitor resistance and results in therapeutically actionable vulnerabilities in lung cancer

Although epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFRi) are approved for treating EGFR-mutant lung adenocarcinoma (LUAD), emergence of acquired resistance limits their clinical benefits. Several mechanisms for acquired resistance to EGFRi in LUAD have been identified; however, the molecular basis for this resistance remains unknown in ~30% of LUAD. Chromatin and DNA modifiers and their regulators play important roles in determining response to anticancer therapies. Therefore, to identify nongenetic mechanisms of EGFRi resistance in LUAD, we performed an epigenome-wide shRNA screen targeting 363 human epigenetic regulator genes. This screen identified loss of the transcriptional repressor chromobox homolog 5 (CBX5) as a driver of EGFRi resistance in EGFR-mutant LUAD. Loss of CBX5 confers resistance to multiple EGFRi in both cell culture and mice. We found that CBX5 loss in EGFR-mutant LUAD cells leads to increased expression of the transcription factor E2F1, which in turn stimulates expression of the antiapoptotic gene BIRC5 (survivin). This E2F1-mediated upregulation of BIRC5 in CBX5-knockdown LUAD cells attenuates apoptosis induction following EGFRi treatment. Consistent with these results, knockdown of E2F1 or BIRC5 partly rescues CBX5-knockdown-induced EGFRi resistance in cell culture and mice. EGFRi-resistant LUAD cell lines show reduced CBX5 expression compared to parental lines; however, bromo- and extra-terminal (BET)-domain inhibitors (BETi) restore CBX5 expression in these cells and sensitize them to EGFRi/BETi combination therapy. Similarly, treatment with a BIRC5 inhibitor suppresses growth of EGFRi-resistant LUAD cells. Collectively, these studies identify CBX5 loss as a driver of EGFRi resistance and reveal therapeutic opportunities for treating EGFRi-resistant LUAD.

Looking for Tumor Specific Promoters In Silico

—Previously we demonstrated the tumor-specific activity of several human native and chimeric promoters. Here we have analyzed the DNA sequences of experimentally tested tumor-specific promoters for the presence of recognition matrices of transcription factors and for de novo motif discovery. CiiiDER and MEME Suite software tools were used for this purpose. A number of transcription factor matrices have been identified, which are present more often in tumor-specific promoters than in the promoters of housekeeping genes. New promoter–TF regulatory relationships have been predicted by pathway analysis. A motif of 44 bp characteristic of tumor-specific promoters but not of housekeeping gene promoters has been discovered. The search through 29 598 human promoters from the EPDnew promoter database has revealed a series of promoters with this motif, their genes being associated with unfavorable prognoses in cancer. We suppose that some of these promoters may possess a tumor specific activity. In addition, a close similarity in nucleotide motifs between the promoters of the BIRC5 and MCM2 genes has been shown. The results of the study may contribute to understanding the peculiarities of gene transcription in tumors, as well as to searching for native tumor-specific promoters or creating artificial ones for cancer gene therapy, as well as in the development of anticancer vaccines.

In silico analysis revealed the potential circRNA-miRNA-mRNA regulative network of non-small cell lung cancer (NSCLC)

BackgroundThe primary source of death in the world is non-small cell lung cancer (NSCLC). However, NSCLCs pathophysiology is still not completely understood. The current work sought to study the differential expression of mRNAs involved in NSCLC and their interactions with miRNAs and circRNAs. MethodsWe utilized three microarray datasets (GSE21933, GSE27262, and GSE33532) from the GEO NCBI database to identify the differentially expressed genes (DEGs) in NSCLC. We employed DAVID Functional annotation tool to investigate the underlying GO biological process, molecular functions, and KEGG pathways involved in NSCLC. We performed the Protein-protein interaction (PPI) network, MCODE, and CytoHubba analysis from Cytoscape software to identify the significant DEGs in NSCLC. We utilized miRnet to anticipate and build interaction between miRNAs and mRNAs in NSCLC and ENCORI to predict the miRNA-circRNA relationships and build the ceRNA regulatory network. Finally, we executed the gene expression and Kaplan-Meier survival analysis to validate the significant DEGs in the ceRNA network utilizing TCGA NSCLC and GEPIA data. ResultsWe revealed a total of 156 overlapped DEGs (47 upregulated and 109 downregulated genes) in NSCLC. The PPI network, MCODE, and CytoHubba analysis revealed 12 hub genes (cdkn3, rrm2, ccnb1, aurka, nuf2, tyms, kif11, hmmr, ccnb2, nek2, anln, and birc5) that are associated with NSCLC. We identified that these 12 genes encode 12 mRNAs that are strongly linked with 8 miRNAs, and further, we revealed that 1 circRNA was associated with this 5 miRNA. We constructed the ceRNAs network that contained 1circRNA-5miRNAs-7mRNAs. The expression of these seven significant genes in LUAD & LUSC (NSCLC) was considerably higher in the TCGA database than in normal tissues. Kaplan-Meier survival plot reveals that increased expression of these hub genes was related to a poor survival rate in LUAD. ConclusionOverall, we developed a circRNA-miRNA-mRNA regulation network to study the probable mechanism of NSCLC.

Molecular Landscape of Chronic Lymphocytic Leukemia Using Targeted Gene Panel Sequencing

Introduction Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease with various genetic abnormalities that can affect the treatment and survival of patients. Recently, by the advent of next-generation sequencing (NGS) techniques recurrently mutated genes have been described in CLL. Driver gene mutations such as NOTCH1, SF3B1, TP53, and MYD88 have been considered to have prognostic impact in CLL. The aim of this study was to detect pathogenic variants of commonly mutated genes in CLL patients and to explore their associations with IGHV mutational status and cytogenetic abnormalities. Patients and Methods The samples were collected from bone marrow or peripheral blood of 124 CLL patients. Genomic DNA was isolated using QIAamp Blood Mini kit. NGS analysis was performed using Twist Custom Panel and bidirectional sequencing with a minimum coverage of 1000x on Illumina NextSeq according to the manufacturers’ instructions. Data were analyzed by NextGene software. The following 27 recurrently mutated CLL genes were studied: PIK3CD, PIK3CA, SF3B1, MYD88, FBXW7, BRAF, NOTCH1, PTEN, BIRC3, PTPN6, MAP2K1, TP53, STAT5B, STAT3, CD79B, TCF3, MAPK1, DDX3X,ATM, BCL2, BTK, KRAS, NRAS, CARD11, CXCR4, PLCG2, POT1. Variants below 5% variant allele frequency (VAF) were filtered out from the analysis. Variants were considered subclonal with 5-10% VAF and clonal when VAF was >10%. Classification of variants were performed according to the American College of Medical Genetics and Genomics (ACMG) criteria. Results Our NGS study revealed a total of 96 variants in 124 CLL patients. Overall, 63/124 patients (50.8%) harbored at least one mutation, the majority of them (60/96, 62.5%) was clonal. In 36 patients only one pathogenic variant was detected, while 88 patients carried two or more (2-4) genetic alterations. The most frequently mutated gene was the NOTCH1, followed by TP53, SF3B1, FBXW7, and BRIC3. Subclonal variants were also detected in 36/124 patients (29%), particularly in NOTCH1,FBXW7, BIRC3, and PIK3CA genes. TP53 and NOTCH1 variants occured mostly in unmutated IGHV CLL cases. Patients with mutated IGHV harbored mainly FBXW7 and BIRC3 mutationss. FBXW7 variants were detected in 9/125 (7%) patients, that is a higher frequency compared to other studies. Fluorescence in situ hybridization (FISH) was performed in 113 cases. The majority of these patients (84 cases, 74%) had one or more cytogenetic abnormality. In this cohort, 13q deletion was the most common aberration (65%, 55/84 patients), followed by 11q deletion (26%, 22/84 patients) and 17q deletion (14%, 12/84 patients). Deletion of 17p co-occured with TP53 single nucleotide variants in 10 of 12 patients. SF3B1, NOTCH1, BIRC3 and TP53 variants occured with 11q deletion in 10 cases. Trisomy 12 was detected with NOTCH1, BIRC3 and FBXW7 variants, and showed negative association with unfavorable SF3B1 mutations. Conclusions Our results confirmed the presence of high number of clonal and subclonal variants in the most frequently mutated genes (NOTCH1, TP53, SF3B1, FBXW7, BIRC3). TP53, NOTCH1 and SF3B1 mutations were enriched with unfavorable biologic factors, as they were found to be associated with unmutated IGHV and cytogenetic aberrations, such as del17p, del11q and trisomy 12. Cases with NOTCH1 variants harbored all types of the investigated cytogenetic alterations (del13q, del11q, de17p, trisomy 12) and were associated exclusively with unmutated IGHV. TP53 variants occured mostly in unmutated IGHV CLL cases together with del17p. Patients with SF3B1 variants showed del11q, del13q and mostly unmutated IGHV. FBXW7 variants enriched in both of unmutated and mutated IGHV cases and harbored del13q, de17p and trisomy 12. FBXW7 and NOTCH1 pathogenic variants have the same biological consequences in CLL, therefore presence of FBXW7 mutations may have clinical relevance regarding anti-CD20 therapy. Taken together, these NGS results complemented with the study of IGHV mutational status and cytogenetic data can contribute to better prognostic workup and management of our CLL patients.

DNMT3A Mutations Enhance T-ALL Survival, Which Is Reversed By JAK/STAT Inhibition

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm of T-cell progenitors, which accounts for approximately 15% and 25% of pediatric and adult acute lymphoblastic leukemia (ALL) cases respectively. In T-ALL, accumulation of genomic abnormalities often leads to the aberrant expression of select groups of transcription factors that result in increased proliferation, cell survival and impaired differentiation of T-cell progenitors, which give rise to this disease The Notch signaling pathway is essential for T-lymphopoiesis and T-cell differentiation from thymic precursors. Moreover, gain-of-function mutations in this pathway act as the major genetic lesions that drive T-ALL development (over 60% of adult cases). The de novo DNA methyltransferase enzyme (DNMT3A) is one of the three genes in mammals encoding for enzymes with DNA methyltransferase activity and mutations in this gene are frequently associated with NOTCH1 mutations in adult T-ALL. Interestingly, DNMT3A mutations are rare in pediatric and adolescent T-ALL. Prognosis of pediatric and adolescent T-ALL cases has seen major improvements during recent years with the introduction of high-dose, multi-agent chemotherapy regimens which has not carried over to adult T-ALL patients. These factors suggest the involvement of genetic differences between adult and pediatric T-ALL patients for the differential clinical outcomes Previous work in our lab has shown that mouse T-ALL cells genetically deficient for Dnmt3a are less apoptotic and have increased JAK/STAT signaling. Here, we demonstrate that DNMT3A-mutant human T-ALL patient derived xenografts (PDXs) are resistant to the chemotherapeutic drugs dexamethasone (DEX) and doxorubicin compared to T-ALL patient specimens with wild-type DNMT3A. However, when primary DNMT3A-mutant human T-ALL cells were treated with dexamethasone or doxorubicin in combination with the JAK/STAT inhibitor ruxolitinib (RUX), we observed the survival benefit of DNMT3A-mutant T-ALL cells were mitigated, suggesting enhanced JAK/STAT signaling promotes survival in T-ALL patient cells with DNMT3A mutations. To investigate the genes and pathways that endows DNMT3A-mutant T-ALL cells chemoresistance, gene expression profiling was performed by RNA-seq on DNMT3A-mutant, and wild-type human T-ALL cells treated with DEX+RUX. Gene set enrichment analysis (GSEA) showed that MTORC1, IL2-STAT5, E2F and G2M pathways were significantly downregulated in DEX+RUX treated DNMT3A-mutant PDX samples. E2F and G2M pathways are associated with cell cycle progression and further analysis of these two pathways revealed BIRC5 as a common gene that is downregulated in both these pathways following DEX+RUX treatment. Furthermore, our RNA-seq analysis demonstrated that the expression of BIRC5 in DNMT3A-mutant PDXs is downregulated upon DEX+RUX treatment compared to the DNMT3A-wildtype group. Moreover, baseline BIRC5 expression is higher in DNMT3A-mutant T-ALL PDXs compared to the DNMT3A-wildtype group. Following treatment with the BIRC5 inhibitor YM155, the viability of DNMT3A-mutant T-ALL cells decrease significantly compared to DNMT3A-wildtype PDXs, which were relatively unaffected. In addition, we observed a similar reduction in cell viability in DNMT3A-mutant PDXs following BIRC5 siRNA nucleofection compared to the DNMT3A-wildtype group. Cumulatively, these studies reveal that enhanced JAK/STAT signaling promotes survival and chemoresistance in T-ALL patient cells with DNMT3A mutations and identify the JAK/STAT target gene BIRC5 as a specific genetic dependency of DNMT3A-mutant T-ALL cells. These data provide a critical first step towards a novel target for precision medicine approaches for this patient group.

Lymphomagenic Roles of MYC, E2F and Mir-150-5p in Plasmablastic Lymphoma: Therapeutic and Prognostic Implications

Introduction: Plasmablastic lymphoma (PBL) is an uncommon non-Hodgkin lymphoma which is characterized by an aggressive clinical behavior, very poor prognosis and which mostly affects immunosuppressed individuals. Although different studies have described genetic alterations in PBL, its transcriptome and epigenome have been scarcely studied. Our goal was to describe the gene and miRNA expression profiles and to identify new therapeutic targets and new prognostic factors in PBL. Methods: Formalin fixed paraffin embedded (FFPE) samples from 66 patients with PBL and samples from 14 reactive adenopathies, as a control group, were included for miRNA and gene expression studies (microarrays and RNA-seq, respectively). Enrichment analyses of miRNAs and genes differentially expressed, using hallmark data set, were carried out. The role of miR-150-5p in PBL lymphomagenesis was studied in vitro upon introduction of specific miRNA mimic in the PBL-1 cell line (Mine S, Sci Rep. 2017). Expression of the miRNA and its target genes was analyzed by RT-qPCR and Western Blot. Its role in cell proliferation, cell cycle progression and apoptosis induction, was assessed respectively by MTT assay and flow cytometry. Results: Patients with PBL showed 36 differentially expressed miRNAs compared to controls. Two expression profiles of these miRNAs were identified in PBL, related with the presence of MYC-translocation. The functional enrichment analysis of genes targeted by deregulated miRNAs revealed an enrichment of MYC and E2F targets, genes involved in G2M checkpoint and mTORC1 signaling pathway. This latter observation was confirmed by gene set enrichment analysis (GSEA) of genes differentially expressed in PBL (Fig.1). Different genes are shared among the top 3 enriched hallmark gene sets (Fig.2), this data suggests that both MYC and E2F are involved in the deregulation of cell cycle in PBL. Additionally, the upregulation of E2F1, E2F3 and E2F8 correlates significantly with an increased expression of MKI67 (Ki67), indicating that E2F exerts a crucial role in the abnormal proliferation in PBL. To identify deregulated miRNA-target interactions related with the top 3 enriched hallmark gene sets, we studied the correlation between the most differentially expressed miRNAs (miR-148a-3p, -4417, -150-5p, -342-5p, -342-3p, -3609) and their differentially expressed target genes. Only miR-150-5p showed a significant negative correlation with the anti-apoptotic gene BIRC5, coding for survivin, in PBL patients (p. 0.0063) (Fig.2). Of interest, high expression of BIRC5 and low expression of miR-150-5p correlated with high expression of MKI67 (p. 0.0006 and p. 0.04, respectively) indicating that the deregulation of miR-150-5p and BIRC5 may favor the proliferation of PBL cells. Low expression of miR-150-5p (lower than median= 6.56) was associated with shorter overall survival (p <0.0001), suggesting that this miRNA may represent a potential prognostic factor in PBL. We further studied the role of miR-150-5p in PBL lymphomagenesis by mimicking this miRNA in PBL-1 cell line. Upregulation of miR-150-5p decreased the expression of survivin at both mRNA and protein level, promoted apoptosis and reduced viability and proliferation, pointing out a pro-apoptotic role of miR-150-5p via survivin inhibition. Consistent with the known role of survivin in the control of mitosis, the upregulation of miR-150-5p and the consequent reduction in survivin levels produced a slight arrest of cells in G1 cell cycle phase and lower numbers of cells in G2-M phase. Finally, a positive tendency between BIRC5 and E2F3 expression was observed (p. 0.06), being this latter gene negatively correlated with miR-150-5p (p. 0.017). Indeed, a greater expression of E2F3 was encountered in those patients with low expression of miR-150-5p. Thus, these results suggest that E2F3 could repress miR-150-5p and mediate apoptosis via BIRC5 induction. Conclusions: We identified miR-150-5p as a new promising prognostic factor, and MYC and E2F as important regulators of global gene expression in PBL. Among these genes, E2F3 could represent a lymphomagenic-driver in PBL by repressing miR-150-5p and inducing BIRC5 expression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Custom gene expression panel for evaluation of potential molecular markers in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. It is a highly heterogeneous disease with poor prognosis and limited treatment options, which highlights the need for reliable biomarkers. This study aims to explore molecular markers that allow stratification of HCC and may lead to better prognosis and treatment prediction.Materials and methodsWe studied 20 candidate genes (HCC hub genes, potential drug target genes, predominant somatic mutant genes) retrieved from literature and public databases with potential to be used as the molecular markers. We analysed expression of the genes by RT-qPCR in 30 HCC tumour and adjacent non-tumour paired samples from Vietnamese patients. Fold changes in expression were then determined using the 2−∆∆CT method, and unsupervised hierarchical clustering was generated using Cluster v3.0 software.ResultsClustering of expression data revealed two subtypes of tumours (proliferative and normal-like) and four clusters for genes. The expression profiles of the genes TOP2A, CDK1, BIRC5, GPC3, IGF2, and AFP were strongly correlated. Proliferative tumours were characterized by high expression of the c-MET, ARID1A, CTNNB1, RAF1, LGR5, and GLUL1 genes. TOP2A, CDK1, and BIRC5 HCC hub genes were highly expressed (> twofold) in 90% (27/30), 83% (25/30), and 83% (24/30) in the tissue samples, respectively. Among the drug target genes, high expression was observed in the GPC3, IGF2 and c-MET genes in 77% (23/30), 63% (19/30), and 37% (11/30), respectively. The somatic mutant Wnt/ß-catenin genes (CTNNB1, GLUL and LGR5) and TERT were highly expressed in 40% and 33% of HCCs, respectively. Among the HCC marker genes, a higher percentage of tumours showed GPC3 expression compared to AFP expression [73% (23/30) vs. 43% (13/30)].ConclusionThe custom panel and molecular markers from this study may be useful for diagnosis, prognosis, biomarker-guided clinical trial design, and prediction of treatment outcomes.

Developing and Validating an Autophagy Gene-Set-Based Prognostic Signature in Hepatocellular Carcinoma Patients.

Hepatocellular carcinoma (HCC), a major type of cancer affecting the lives of people across the world, is one of many diseases whose advancement is thought to be influenced by autophagy dysfunction. Here, the prognostic significance of autophagy in HCC will be investigated. The Cancer Genome Atlas (TCGA) database was employed in this work to identify 62 differentially-expressed Autophagy-Related Genes (ARGs) in HCC patients. Functional enrichment studies revealed that autophagy played a tumor-promoting role in the advancement of HCC. Based on RNA sequencing of 116 tumor samples and 114 paracancerous tissue samples acquired from HCC patients at the Guangxi Medical University Cancer Hospital, 866 differentially expressed prognosis-related genes were identified. Using lasso regression analysis, two ARGs (BIRC5 and BAK1) linked to prognosis were discovered after intersecting the differential genes derived from the prognosis-related groups. A risk score based on ARGs was developed using a Cox proportional hazards regression model. RNA sequencing data were used to construct this model. Finally, the TCGA and The Human Protein Atlas Databases (THPA) were used to validate the clinical data of 116 HCC patients. Elevated expression level of the BAK1 and BIRC5 genes is linked to poor prognosis. The two ARGs were used to calculate the risk score as follows: (0.0253*BAK1) + (0.0051*BIRC5). Risk score served as the independent prognostic factor as per the multivariate analysis. TCGA, THPA, and data from the Guangxi Medical University Cancer Hospital were used to confirm the predictive validity of risk scores for the prognosis of HCC patients. This study offers molecular insights regarding the involvement of autophagy in HCC patients, along with a probable prognostic signature for determining the outcome (prognosis) of HCC patients.

The Prognostic and Therapeutic Implications of the Chemoresistance Gene BIRC5 in Triple-Negative Breast Cancer.

Chemoresistance affects TNBC patient treatment responses. Therefore, identifying the chemoresistant gene provides a new approach to understanding chemoresistance in TNBC. BIRC5 was examined in the current study as a tool for predicting the prognosis of TNBC patients and assisting in developing alternative therapies using online database tools. According to the examined studies, BIRC5 was highly expressed in 45 to 90% of TNBC patients. BIRC5 is not only abundantly expressed but also contributes to resistance to chemotherapy, anti-HER2 therapy, and radiotherapy. Patients with increased expression of BIRC5 had a median survival of 31.2 months compared to 85.8 months in low-expression counterparts (HR, 1.73; CI, 1.4-2.13; p = 2.5 × 10-7). The overall survival, disease-free survival, relapse-free survival, distant metastasis-free survival, and the complete pathological response of TNBC patients with high expression of BIRC5 who received any chemotherapy (Taxane, Ixabepilone, FAC, CMF, FEC, Anthracycline) and anti-HER2 therapy (Trastuzumab, Lapatinib) did not differ significantly from those patients receiving any other treatment. Data obtained indicate that the BIRC5 promoter region was substantially methylated, and hypermethylation was associated with higher BIRC5 mRNA expression (p &lt; 0.05). The findings of this study outline the role of BIRC5 in chemotherapy-induced resistance of TNBC, further indicating that BIRC5 may serve as a promising prognostic biomarker that contributes to chemoresistance and could be a possible therapeutic target. Meanwhile, several in vitro studies show that flavonoids were highly effective in inhibiting BIRC5 in genetically diverse TNBC cells. Therefore, flavonoids would be a promising strategy for preventing and treating TNBC patients with the BIRC5 molecule.

Ginsenoside Rg3, a promising agent for NSCLC patients in the pandemic: a large-scale data mining and systemic biological analysis

IntroductionNon-small cell lung cancer (NSCLC) patients are particularly vulnerable to the Coronavirus Disease-2019 (COVID-19). Currently, no anti-NSCLC/COVID-19 treatment options are available. As ginsenoside Rg3 is beneficial to NSCLC patients and has been identified as an entry inhibitor of the virus, this study aims to explore underlying pharmacological mechanisms of ginsenoside Rg3 for the treatment of NSCLC patients with COVID-19. MethodsBased on a large-scale data mining and systemic biological analysis, this study investigated target genes, biological processes, pharmacological mechanisms, and underlying immune implications of ginsenoside Rg3 for NSCLC patients with COVID-19. ResultsAn important gene set containing 26 target genes was built. Target genes with significant prognostic value were identified, including baculoviral IAP repeat containing 5 (BIRC5), carbonic anhydrase 9 (CA9), endothelin receptor type B (EDNRB), glucagon receptor (GCGR), interleukin 2 (IL2), peptidyl arginine deiminase 4 (PADI4), and solute carrier organic anion transporter family member 1B1 (SLCO1B1). The expression of target genes was significantly correlated with the infiltration level of macrophages, eosinophils, natural killer cells, and T lymphocytes. Ginsenoside Rg3 may benefit NSCLC patients with COVID-19 by regulating signaling pathways primarily involved in anti-inflammation, immunomodulation, cell cycle, cell fate, carcinogenesis, and hemodynamics. ConclusionsThis study provided a comprehensive strategy for drug discovery in NSCLC and COVID-19 based on systemic biology approaches. Ginsenoside Rg3 may be a prospective drug for NSCLC patients with COVID-19. Future studies are needed to determine the value of ginsenoside Rg3 for NSCLC patients with COVID-19.

A Metabolism-Related Gene Prognostic Index for Prediction of Response to Immunotherapy in Lung Adenocarcinoma.

Immunotherapy, such as immune checkpoint inhibitors (ICIs), is a validated strategy for treating lung adenocarcinoma (LUAD) patients. One of the main challenges in ICIs treatment is the lack of efficient biomarkers for predicting response or resistance. Metabolic reprogramming has been proven to remodel the tumor microenvironment, altering the response to ICIs. We constructed a prognostic model as metabolism-related gene (MRG) of four genes by using weighted gene co-expression network analysis (WGCNA), the nonnegative matrix factorization (NMF), and Cox regression analysis of a LUAD dataset (n = 500) from The Cancer Genome Atlas (TCGA), which was validated with three Gene Expression Omnibus (GEO) datasets (n = 442, n = 226 and n = 127). The MRG was constructed based on BIRC5, PLK1, CDKN3, and CYP4B1 genes. MRG-high patients had a worse survival probability than MRG-low patients. Furthermore, the MRG-high subgroup was more associated with cell cycle-related pathways; high infiltration of activated memory CD4+T cells, M0 macrophages, and neutrophils; and showed better response to ICIs. Contrarily, the MRG-low subgroup was associated with fatty acid metabolism, high infiltration of dendric cells, and resting mast cells, and showed poor response to ICIs. MRG is a promising prognostic index for predicting survival and response to ICIs and other therapeutic agents in LUAD, which might provide insights on strategies with ICIs alone or combined with other agents.

Identification and Analysis of Immune-Related Gene Signature in Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) originates from the hepatocytes and accounts for 90% of liver cancer. The study intends to identify novel prognostic biomarkers for predicting the prognosis of HCC patients based on TCGA and GSE14520 cohorts. Methods: Differential analysis was employed to obtain the DEGs (Differentially Expressed Genes) of the TCGA-LIHC-TPM cohort. The lasso regression analysis was applied to build the prognosis model through using the TCGA cohort as the training group and the GSE14520 cohort as the testing group. Next, based on the prognosis model, we performed the following analyses: the survival analysis, the independent prognosis analysis, the clinical feature analysis, the mutation analysis, the immune cell infiltration analysis, the tumor microenvironment analysis, and the drug sensitivity analysis. Finally, the survival time of HCC patients was predicted by constructing nomograms. Results: Through the lasso regression analysis, we obtained a prognosis model of ten genes including BIRC5 (baculoviral IAP repeat containing 5), CDK4 (cyclin-dependent kinase 4), DCK (deoxycytidine kinase), HSPA4 (heat shock protein family A member 4), HSP90AA1 (heat shock protein 90 α family class A member 1), PSMD2 (Proteasome 26S Subunit Ubiquitin Receptor, Non-ATPase 2), IL1RN (interleukin 1 receptor antagonist), PGF (placental growth factor), SPP1 (secreted phosphoprotein 1), and STC2 (stanniocalcin 2). First, we found that the risk score is an independent prognosis factor and is related to the clinical features of HCC patients, covering AFP (α-fetoprotein) and stage. Second, we observed that the p53 mutation was the most obvious mutation between the high-risk and low-risk groups. Third, we also discovered that the risk score is related to some immune cells, covering B cells, T cells, dendritic, macrophages, neutrophils, etc. Fourth, the high-risk group possesses a lower TIDE score, a higher expression of immune checkpoints, and higher ESTIMATE score. Finally, nomograms include the clinical features and risk signatures, displaying the clinical utility of the signature in the survival prediction of HCC patients. Conclusions: Through the comprehensive analysis, we constructed an immune-related prognosis model to predict the survival of HCC patients. In addition to predicting the survival time of HCC patients, this model significantly correlates with the tumor microenvironment. Furthermore, we concluded that these ten immune-related genes (BIRC5, CDK4, DCK, HSPA4, HSP90AA1, PSMD2, IL1RN, PGF, SPP1, and STC2) serve as novel targets for antitumor immunity. Therefore, this study plays a significant role in exploring the clinical application of immune-related genes.