Oncogenic Gene Research Articles

Construction of a fluorescence sensing platform based on RISC and CRISPR/Cas12a for the assay of Ago2 enzyme activity

PurposeArgonaute 2 (Ago2) is a protein with nucleic acid endonuclease activity and is ubiquitous in eukaryotic cells. Ago2 is a core member of RNA interference and is involved in important gene regulation in the body and the development of multiple tumors by affecting oncogenic or oncogenic gene expression. Ago2 is a potential tumor biomarker. Therefore, the assay of Ago2 activity can help monitor the disease progression and prognosis and contribute to the study of the mechanism of disease development. MethodsAgo2-based nucleic acid endonuclease activity cleaves the target hairpin nucleic acid chain and progressively stimulates the hybridization chain reaction (HCR) in the sensing strategy, causing signal amplification to form specific dsDNA, which then triggers the powerful non-selective single nucleic acid chain cleavage activity of the CRISPR/cas12a system and releases the fluorescence signal in the reporter probe. ResultsThe sensitive assay of Ago2 enzyme activity was achieved using the biofunctional properties of RNA-induced silencing complex (RISC) and CRISPR/Cas12a. Under the optimized conditions, the fluorescence signal intensity of the assay was positively correlated with the activity concentration of Ago2, with an excellent linear response range of 0.02–20 nM and a limit of detection (LOD) of 10 pM. When applied to the assay of Ago2 in cell lysates, the experimental recovery was 93.33%-107.08%, and the sensing platform was able to detect the Ago2 content in the presence of interference specifically. ConclusionTherefore, the fluorescence sensing platform successfully constructed in this paper is suitable for the assay of endogenous Ago2 activity in complex samples, and the assay method has the advantages of high specificity, sensitive detection, easy operation and low instrumentation requirements, which is expected to be applied to the actual sample assay in clinical settings. Meanwhile, it provides an assay direction for the biofunctional analysis of nucleases and a feasible direction for applying RISC and Crispr/Cas12a in sensing analysis.

Overexpressed FAM111B degrades GSDMA to promote esophageal cancer tumorigenesis and cisplatin resistance.

Chemotherapeutic agents such as cisplatin are commonly used in patients with clinically unresectable or recurrent esophageal cancer (ESCA). However, patients often develop resistance to cisplatin, which in turn leads to a poor prognosis. Studies have shown that FAM111B may be involved in the development of tumors as an oncogene or tumor suppressor gene. However, the pathological role and corresponding mechanism of FAM111B in ESCA are still unclear. The GEPIA web tool, ENCORI Pan-Cancer Analysis Platform and UALCAN-TCGA database were used to study the expression of FAM111B in ESCA. CCK-8, angiogenesis, Transwell and xenograft assays were applied to explore the biological function of FAM111B in ESCA. Western blot, RT-qPCR, and RNA-seq analyses were applied to study the FAM111B/GSDMA axis in the progression of ESCA cells. CCK-8 and xenograft assays were used to study the role of the FAM111B/GSDMA axis in determining the sensitivity of ESCA to cisplatin. Our results demonstrated that FAM111B is highly expressed in ESCA tissues compared to normal tissues. We showed that FAM111B promotes the progression of ESCC cells by binding to GSDMA and that the trypsin protease domain is essential for the activity of FAM111B. Furthermore, we showed that the FAM111B/GSDMA axis regulates cisplatin sensitivity in ESCA. Overall, we identified a novel FAM111B/GSDMA axis regulating ESCA tumorigenesis and chemosensitivity, at least in ESCC cells.

RNF149 confers cisplatin resistance in esophageal squamous cell carcinoma via destabilization of PHLPP2 and activating PI3K/AKT signalling.

Chemo-resistance has been identified as a crucial factor contributing to tumor recurrence and a leading cause of worse prognosis in patients with ESCC. Therefore, unravel the critical regulators and effective strategies to overcome drug resistance will have a significant clinical impact on the disease. In our study we found that RNF149 was upregulated in ESCC and high RNF149 expression was associated with poor prognosis with ESCC patients. Functionally, we have demonstrated that overexpression of RNF149 confers CDDP resistance to ESCC; however, inhibition of RNF149 reversed this phenomenon both in vitro and in vivo. Mechanistically, we demonstrated that RNF149 interacts with PH domain and leucine rich repeat protein phosphatase 2 (PHLPP2) and induces E3 ligase-dependent protein degradation of PHLPP2, substantially activating the PI3K/AKT signalling pathway in ESCC. Additionally, we found that inhibition of PI3K/AKT signalling pathway by AKT siRNA or small molecule inhibitor significantly suppressed RNF149-induced CDDP resistance. Importantly, RNF149 locus was also found to be amplified not only in ESCC but also in various human cancer types. Our data suggest that RNF149 might function as an oncogenic gene. Targeting the RNF149/PHLPP2/PI3K/Akt axis may be a promising prognostic factor and valuable therapeutic target for malignant tumours.

TRAIP serves as a potential prognostic biomarker and correlates with immune infiltrates in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is one of the major types of lung cancer with high morbidity and mortality. The TRAF-interacting protein (TRAIP) is a ring-type E3 ubiquitin ligase which has been recently identified to play pivotal roles in various cancers. However, the expression and function of TRAIP in LUAD remain elusive. In this study, we used bioinformatic tools as well as molecular experiments to explore the exact role of TRAIP and the underlying mechanism. Data mining across the UALCAN, GEPIA and GTEx, GEO and HPA databases revealed that TRAIP was significantly overexpressed in LUAD tissues than that in adjacent normal tissues. Kaplan-Meier curve showed that high TRAIP expression was associated with poor overall survival (OS) and relapse-free survival (RFS). Univariate and multivariate cox regression analysis revealed that TRAIP was an independent risk factor in LUAD. And the TRAIP-based nomogram further supported the prognostic role of TRAIP in LUAD. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that TRAIP-associated genes were mainly involved in DNA replication, cell cycle and other processes. The immune infiltration analysis indicated that TRAIP expression was tightly correlated with the infiltration of diverse immune cell types, including B cell, CD8+T cell, neutrophil and dendritic cell. Moreover, TRAIP expression was observed to be significantly associated with tumor infiltrating lymphocytes (TILs) and immune checkpoint molecules. In vitro experiments further confirmed knockdown of TRAIP inhibited cell migration and invasion, as well as decreasing chemokine production and inhibiting M2-like macrophage recruitment. Lastly, CMap analysis identified 10 small molecule compounds that may target TRAIP, providing potential therapies for LUAD. Collectively, our study found that TRAIP is an oncogenic gene in LUAD, which may be a potential prognostic biomarker and promising therapeutic target for LUAD.

Identıfıcatıon of KRAS Mutatıons in Colorectal Carcınoma Patıents at Dr. M. Djamıl Hospıtal, West Sumatra-Indonesıa

Kirsten rat sarcoma viral oncogene (KRAS) gene mutations lead to abnormal activation of the RAS signalling pathway and have been associated with poor prognosis and resistance to some therapeutics. This study aimed to identify mutation characteristics of the KRAS genes codon 12 and 13 in colorectal cancer patients in West Sumatra. KRAS mutations were analyzed in 20 DNA of colorectal cancer patients' tissue samples by using polymerase chain reaction (PCR) with specific primer and direct sequencing analysis. Our findings showed five samples (25%) with mutated KRAS at codons 12 and 13 (including three samples with GGT→GAT, one sample with GGT→GTT in codon 12, and one sample with GGC→GAC in codon 13). In conclusion, we found two variations of amino acid changes at codon 12 (G12D and G12V) and one at codon 13 (G13D). More research with many samples is required to obtain conclusive data on the relationship between these gene mutations and colorectal cancer response to therapy and prognosis.

Circulating microRNAs as Potential Biomarkers in Pancreatic Cancer-Advances and Challenges.

There is an urgent unmet need for robust and reliable biomarkers for early diagnosis, prognosis, and prediction of response to specific treatments of many aggressive and deadly cancers, such as pancreatic cancer, and liquid biopsy-based miRNA profiling has the potential for this. MiRNAs are a subset of non-coding RNAs that regulate the expression of a multitude of genes post-transcriptionally and thus are potential diagnostic, prognostic, and predictive biomarkers and have also emerged as potential therapeutics. Because miRNAs are involved in the post-transcriptional regulation of their target mRNAs via repressing gene expression, defects in miRNA biogenesis pathway and miRNA expression perturb the expression of a multitude of oncogenic or tumor-suppressive genes that are involved in the pathogenesis of various cancers. As such, numerous miRNAs have been identified to be downregulated or upregulated in many cancers, functioning as either oncomes or oncosuppressor miRs. Moreover, dysregulation of miRNA biogenesis pathways can also change miRNA expression and function in cancer. Profiling of dysregulated miRNAs in pancreatic cancer has been shown to correlate with disease diagnosis, indicate optimal treatment options and predict response to a specific therapy. Specific miRNA signatures can track the stages of pancreatic cancer and hold potential as diagnostic, prognostic, and predictive markers, as well as therapeutics such as miRNA mimics and miRNA inhibitors (antagomirs). Furthermore, identified specific miRNAs and genes they regulate in pancreatic cancer along with downstream pathways can be used as potential therapeutic targets. However, a limited understanding and validation of the specific roles of miRNAs, lack of tissue specificity, methodological, technical, or analytical reproducibility, harmonization of miRNA isolation and quantification methods, the use of standard operating procedures, and the availability of automated and standardized assays to improve reproducibility between independent studies limit bench-to-bedside translation of the miRNA biomarkers for clinical applications. Here I review recent findings on miRNAs in pancreatic cancer pathogenesis and their potential as diagnostic, prognostic, and predictive markers.

Dissection of tumor-intrinsic and tumor-extrinsic features of MiT/TFE translocation renal cell carcinoma via single-cell RNA sequencing

Abstract Background Translocation renal cell carcinoma (tRCC) is a rare and aggressive subtype of renal cell carcinoma driven by oncogenic gene fusions involving MiT/TFE family transcription factors, most commonly TFE3. Currently, there are no molecularly tailored treatments for tRCC, and standard-of-care therapies utilized for other RCC subtypes are typically less effective in tRCC. Emerging data suggest that tRCC is molecularly distinct from more common RCC subtypes. However, an incomplete understanding of both tumor-intrinsic drivers and the tumor microenvironment (TME) features of tRCC presents barriers to developing effective therapeutics for this cancer. Methods We used single-nucleus RNA sequencing (snRNA) to profile ten samples of tRCC tumors and one adjacent normal tissue. Five of the samples also underwent multiome profiling with snATAC-seq while nine of the tumor samples underwent whole genome sequencing and RNA-sequencing. We preprocessed and obtained the raw fasta files using cellranger and extracted RNA counts and chromatin accessibility peaks using the Seurat/Signac algorithm. Scrublet was used to exclude data from droplets containing more than one cell by performing doublet detection and removal on gene-barcode matrices. Cells with fewer than 200 genes detected or more than 5% of counts attributed to mitochondrially-encoded transcripts were removed before across-sample integration. We also excluded genes detected in fewer than three cells across all samples. The combined cohort was analyzed using the MergeData function in Seurat, and cancer cells were selected based on known tRCC gene markers and inferred transcriptional copy number variations estimated via the inferCNV package and verified from WGS data. Non-tumor cell types were determined through manual annotation via known marker genes. We analyzed patterns of gene expression at the single-cell level using the Seurat V4 package and module score functions. Differential expression analysis comparing cells from different clusters or treatment exposure groups was performed using a two-sided Wilcoxon rank-sum test with Bonferroni FDR correction. Results Following quality control and integration, 71,124 single-cells were used for analysis (67,762 from tumor samples) – representing 43,214 tumor cells, 8,352 monocytes, 4,692 T cells, 4,614 endothelial/stromal cells, and normal kidney cells. The normal-adjacent sample represented multiple cell types from the normal kidney, including cells from distal connecting tubule, connecting duct, proximal tubule, thick ascending limb, podocytes, and endothelial cells. Analysis of chromatin accessibility profiles from snATAC-seq and snRNA-seq data of tumor cells was employed to identify unique cell states and a putative cell of origin for tRCCs. Tumor cell analysis also demonstrated intra- and inter-tumoral transcriptional variability in genetically similar tumor subclusters, each with distinct regulon activity. Immune subpopulation analyses revealed higher proportions of resting T cells in treatment-naive tRCC samples and higher proportions of progenitor-exhausted and terminal-exhausted populations in an immune checkpoint inhibitor (ICI) treated sample, indicating immune reprogramming in response to ICI in this mutationally quiet tumor. To further understand the role of, and factors permissive to, anti-tumor T cell responses in tRCC (which is characterized by a low tumor mutational burden) we explored the hypothesis that MiT/TFE fusions may constitute tumor neoantigens. We computationally identified fusion-associated neoantigens corresponding to these tRCC samples, as well as across a larger aggregate dataset of tRCC samples profiled by bulk RNA-Seq. We predicted binding affinities between fusion-associated neoantigens and HLA types and identified multiple peptide candidates with high HLA-binding affinity, which will be validated for their immunogenicity in vitro. Conclusions Overall, our results highlight tumor-intrinsic and tumor-extrinsic determinants of immunogenicity in tRCC and may guide the development of immunotherapeutic strategies with strong mechanistic rationale in tRCC. CDMRP DOD Funding: yes

HSF-1/miR-145-5p transcriptional axis enhances hyperthermic intraperitoneal chemotherapy efficacy on peritoneal ovarian carcinosis

Hyperthermic intraperitoneal administration of chemotherapy (HIPEC) increases local drug concentrations and reduces systemic side effects associated with prolonged adjuvant intraperitoneal exposure in patients affected by either peritoneal malignancies or metastatic diseases originating from gastric, colon, kidney, and ovarian primary tumors. Mechanistically, the anticancer effects of HIPEC have been poorly explored. Herein we documented that HIPEC treatment promoted miR-145-5p expression paired with a significant downregulation of its oncogenic target genes c-MYC, EGFR, OCT4, and MUC1 in a pilot cohort of patients with ovarian peritoneal metastatic lesions. RNA sequencing analyses of ovarian peritoneal metastatic nodules from HIPEC treated patients unveils HSF-1 as a transcriptional regulator factor of miR-145-5p expression. Notably, either depletion of HSF-1 expression or chemical inhibition of its transcriptional activity impaired miR-145-5p tumor suppressor activity and the response to cisplatin in ovarian cancer cell lines incubated at 42 °C. In aggregate, our findings highlight a novel transcriptional network involving HSF-1, miR145-5p, MYC, EGFR, MUC1, and OCT4 whose proper activity contributes to HIPEC anticancer efficacy in the treatment of ovarian metastatic peritoneal lesions.

AML with complex karyotype: extreme genomic complexity revealed by combined long-read sequencing and Hi-C technology

AbstractAcute myeloid leukemia with complex karyotype (CK-AML) is associated with poor prognosis, which is only in part explained by underlying TP53 mutations. Especially in the presence of complex chromosomal rearrangements, such as chromothripsis, the outcome of CK-AML is dismal. However, this degree of complexity of genomic rearrangements contributes to the leukemogenic phenotype and treatment resistance of CK-AML remains largely unknown. Applying an integrative workflow for the detection of structural variants (SVs) based on Oxford Nanopore (ONT) genomic DNA long-read sequencing (gDNA-LRS) and high-throughput chromosome confirmation capture (Hi-C) in a well-defined cohort of CK-AML identified regions with an extreme density of SVs. These rearrangements consisted to a large degree of focal amplifications enriched in the proximity of mammalian-wide interspersed repeat elements, which often result in oncogenic fusion transcripts, such as USP7::MVD, or the deregulation of oncogenic driver genes as confirmed by RNA-seq and ONT direct complementary DNA sequencing. We termed this novel phenomenon chromocataclysm. Thus, our integrative SV detection workflow combing gDNA-LRS and Hi-C enables to unravel complex genomic rearrangements at a very high resolution in regions hard to analyze by conventional sequencing technology, thereby providing an important tool to identify novel important drivers underlying cancer with complex karyotypic changes.

High G9a Expression in DLBCL and Its Inhibition by Niclosamide to Induce Autophagy as a Therapeutic Approach.

Diffuse large B-cell lymphoma (DLBCL) is a malignant lymphoid tumor disease that is characterized by heterogeneity, but current treatment does not benefit all patients, which highlights the need to identify oncogenic genes and appropriate drugs. G9a is a histone methyltransferase that catalyzes histone H3 lysine 9 (H3K9) methylation to regulate gene function and expression in various cancers. TCGA and GTEx data were analyzed using the GEPIA2 platform. Cell viability under drug treatment was assessed using Alamar Blue reagent; the interaction between G9a and niclosamide was assessed using molecular docking analysis; mRNA and protein expression were quantified in DLBCL cell lines. Finally, G9a expression was quantified in 39 DLBCL patient samples. The TCGA database analysis revealed higher G9a mRNA expression in DLBCL compared to normal tissues. Niclosamide inhibited DLBCL cell line proliferation in a time- and dose-dependent manner, reducing G9a expression and increasing p62, BECN1, and LC3 gene expression by autophagy pathway regulation. There was a correlation between G9a expression in DLBCL samples and clinical data, showing that advanced cancer stages exhibited a higher proportion of G9a-expressing cells. G9a overexpression is associated with tumor progression in DLBCL. Niclosamide effectively inhibits DLBCL growth by reducing G9a expression via the cellular autophagy pathway; therefore, G9a is a potential molecular target for the development of therapeutic strategies for DLBCL.

A functional sgRNA-CRISPR screening method for generating murine RET and NTRK1 rearranged oncogenes.

CRISPR/Cas9 gene editing represents a powerful tool for investigating fusion oncogenes in cancer biology. Successful experiments require that sgRNAs correctly associate with their target sequence and initiate double stranded breaks which are subsequently repaired by endogenous DNA repair systems yielding fusion chromosomes. Simple tests to ensure sgRNAs are functional are not generally available and often require single cell cloning to identify successful CRISPR-editing events. Here, we describe a novel method relying on acquisition of IL3-independence in Ba/F3 cells to identify sgRNA pairs that generate oncogenic gene rearrangements of the Ret and Ntrk1 tyrosine kinases. The rearrangements were confirmed with PCR, RT-PCR and sequencing and Ba/F3 cells harboring Ret or Ntrk1 rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNA pairs inducing the Kif5b-Ret and Trim24-Ret rearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) established from a Trim24-Ret positive tumor exhibited high in vitro sensitivity to the RET inhibitors LOXO-292 and BLU-667 and orthotopic TR.1 cell-derived tumors underwent marked shrinkage upon LOXO-292 treatment. Thus, the method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel, syngeneic murine oncogene-driven tumor models.

Long noncoding RNA XLOC_006786 inhibits the proliferation, invasion and metastasis of osteosarcoma cells through NOTCH3 signaling pathway by targeting miR-491-5p.

Recent research has indicated that Long noncoding RNAs (LncRNAs) are crucial in many disorders, especially tumors. However, the exact role of LncRNA XLOC_006786 (LncRNA-SPIDR-2:1) in malignancies, especially in human osteosarcoma, is unclear. The results of RT‒qPCR, western blotting, CCK-8 assays, and Transwell assays showed that LncRNA XLOC_006786 inhibited osteosarcoma cell proliferation, invasion, and migration, indicating that it may be a tumor suppressor gene in osteosarcoma. We found that LncRNA XLOC_006786 negatively regulated NOTCH3, which is an oncogenic gene in osteosarcoma, as we previously reported. Bioinformatics analysis showed that miR-491-5p may be a direct target of LncRNA XLOC_006786, while NOTCH3 is a key target of miR-491-5p. Then, we verified that LncRNA XLOC_006786 could prevent lung metastatic osteosarcoma in vivo. Taken together, our research showed that LncRNA XLOC_006786 suppresses osteosarcoma proliferation, invasion, and metastasis through the NOTCH3 signaling pathway by targeting miR-491-5p.

Identification of cuproptosis and immune-related gene prognostic signature in lung adenocarcinoma.

Cuproptosis is a novel form of programmed cell death that differs from other types such as pyroptosis, ferroptosis, and autophagy. It is a promising new target for cancer therapy. Additionally, immune-related genes play a crucial role in cancer progression and patient prognosis. Therefore, our study aimed to create a survival prediction model for lung adenocarcinoma patients based on cuproptosis and immune-related genes. This model can be utilized to enhance personalized treatment for patients. RNA sequencing (RNA-seq) data of lung adenocarcinoma (LUAD) patients were collected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The levels of immune cell infiltration in the GSE68465 cohort were determined using gene set variation analysis (GSVA), and immune-related genes (IRGs) were identified using weighted gene coexpression network analysis (WGCNA). Additionally, cuproptosis-related genes (CRGs) were identified using unsupervised clustering. Univariate COX regression analysis and least absolute shrinkage selection operator (LASSO) regression analysis were performed to develop a risk prognostic model for cuproptosis and immune-related genes (CIRGs), which was subsequently validated. Various algorithms were utilized to explore the relationship between risk scores and immune infiltration levels, and model genes were analyzed based on single-cell sequencing. Finally, the expression of signature genes was confirmed through quantitative real-time PCR (qRT-PCR), immunohistochemistry (IHC), and Western blotting (WB). We have identified 5 Oncogenic Driver Genes namely CD79B, PEBP1, PTK2B, STXBP1, and ZNF671, and developed proportional hazards regression models. The results of the study indicate significantly reduced survival rates in both the training and validation sets among the high-risk group. Additionally, the high-risk group displayed lower levels of immune cell infiltration and expression of immune checkpoint compared to the low-risk group.

Identifying the personalized driver gene sets maximally contributing to abnormality of transcriptome phenotype in glioblastoma multiforme individuals.

High heterogeneity in genome and phenotype of cancer populations made it difficult to apply population-based common driver genes to the diagnosis and treatment of cancer individuals. Characterizing and identifying the personalized driver mechanism for glioblastoma multiforme (GBM) individuals were pivotal for the realization of precision medicine. We proposed an integrative method to identify the personalized driver gene sets by integrating the profiles of gene expression and genetic alterations in cancer individuals. This method coupled genetic algorithm and random walk to identify the optimal gene sets that could explain abnormality of transcriptome phenotype to the maximum extent. The personalized driver gene sets were identified for 99 GBM individuals using our method. We found that genomic alterations in between one and seven driver genes could maximally and cumulatively explain the dysfunction of cancer hallmarks across GBM individuals. The driver gene sets were distinct even in GBM individuals with significantly similar transcriptomic phenotypes. Our method identified MCM4 with rare genetic alterations as previously unknown oncogenic genes, the high expression of which were significantly associated with poor GBM prognosis. The functional experiments confirmed that knockdown of could significantly inhibit proliferation, invasion, migration and clone formation of the GBM cell lines U251 and U118MG, and overexpression of MCM4 significantly promoted the proliferation, invasion, migration and clone formation of the GBM cell line U87MG. Our method could dissect the personalized driver genetic alteration sets that are pivotal for developing targeted therapy strategies and precision medicine. Our method could be extended to identify key drivers from other levels, and could be applied to more cancer types.

Nuclear circRNA: Impairing genome stability via circR loops

AbstractConn et al. have recently reported circRNA: DNA hybrids (circR loops) to promote oncogenic gene translocations and impair genome stability which drive leukemia. Herein, we will summarize potential molecular mechanisms and discuss clinical significance of uncovering circR loops as endogenous RNA carcinogens.

Prevalence and Landscape of Pathogenic or Likely Pathogenic Germline Variants and Their Association With Somatic Phenotype in Unselected Chinese Patients With Gynecologic Cancers

Understanding germline and somatic status in patients with gynecologic cancers could improve risk assessment and guide therapeutic decision-making. To evaluate the prevalence and landscape of germline pathogenic or likely pathogenic (P/LP) variants and explore whether these variants are associated with somatic phenotypes and cancer risk in unselected patients with gynecologic cancers. This cross-sectional study retrospectively enrolled unselected patients in China with a gynecologic cancer, including ovarian, cervical, and endometrial, who underwent tumor-normal sequencing using a 520-gene panel from October 1, 2017, through May 31, 2021. Germline variants in gynecologic cancers. The P/LP germline variant rates in 62 cancer predisposition genes were assessed using descriptive statistics. The associations of P/LP variant status with age, somatic profiles, and cancer risk were also investigated using the Fisher exact test or Student t test. A total of 1610 women (median [IQR] age, 54 [47-62] years; 1201 [74.6%] with stage III-IV disease) were included (945 with ovarian cancer, 307 with endometrial cancer, and 358 with cervical cancer). The prevalence of patients with P/LP variants was 20.5% (194 of 945) for ovarian cancer, 13.4% (41 of 307) for endometrial cancer, and 6.4% (23 of 358) for cervical cancer; 95.1% of the germline findings (n = 252) were potentially actionable, mainly in homologous recombination repair (HRR) and mismatch repair genes. Chinese patients with endometrial cancer had a higher rate of P/LP variants than a White population from The Cancer Genome Atlas (42 of 307 [13.7%] vs 24 of 367 [6.5%]; P = .003). In endometrial and cervical cancers, the prevalence of P/LP variants was 12.7% (30 of 237) and 4.8% (13 of 270), respectively, in patients diagnosed at age 45 years or older and increased to 25.0% (9 of 36; P = .09) and 12.0% (10 of 83; P = .04), respectively, for those with an onset age of less than 45 years. Mismatch repair P/LP variants were associated with a younger age at onset for ovarian cancer (46 vs 54 years; P = .02) and endometrial cancer (48 vs 57 years; P < .001), while HRR P/LP variants were associated with a younger age at onset for cervical cancer (46 vs 52 years; P = .04). Carriers of HRR P/LP variants had more prevalent somatic TP53 variants and less common somatic variants in oncogenic driver genes vs noncarriers. BRCA1/2 P/LP variants were also associated with moderate risks for endometrial and cervical cancer. This study delineates the landscape of germline P/LP variants in Chinese women with gynecologic cancers. The findings highlight the hereditary factor in cervical cancer that has long been neglected and suggest the importance of next-generation sequencing-based genetic testing with a large gene panel for gynecologic cancers.

Multi-level functional genomics reveals molecular and cellular oncogenicity of patient-based 3′ untranslated region mutations

3' untranslated region (3' UTR) somatic mutations represent a largely unexplored avenue of alternative oncogenic gene dysregulation. To determine the significance of 3' UTR mutations in disease, we identify 3' UTR somatic variants across 185 advanced prostate tumors, discovering 14,497 single-nucleotide mutations enriched in oncogenic pathways and 3' UTR regulatory elements. By developing two complementary massively parallel reporter assays, we measure how thousands of patient-based mutations affect mRNA translation and stability and identify hundreds of functional variants that allow us to define determinants of mutation significance. We demonstrate the clinical relevance of these mutations, observing that CRISPR-Cas9 endogenous editing of distinct variants increases cellular stress resistance and that patients harboring oncogenic 3' UTR mutations have a particularly poor prognosis. This work represents an expansive view of the extent to which disease-relevant 3' UTR mutations affect mRNA stability, translation, and cancer progression, uncovering principles of regulatory functionality and potential therapeutic targets in previously unexplored regulatory regions.

Prokaryotic expression, polyclonal antibody preparation, spatio-temporal expression profile and functional analysis of c-Myc of Helicoverpa armigera (Lepidoptera: Noctuidae)

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni2+-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.

Approach to Functions of BHLHE41/DEC2 in Non-Small Lung Cancer Development.

The circadian rhythm-related genes BHLHE40/DEC1 and BHLHE41/DEC2 have various functions under different cell and tissue conditions. BHLHE41/DEC2 has been reported to be both a cancer-suppressive and an oncogenic gene during cancer development. The effects of BHLHE41/DEC2 on differentiation have been examined using Bhlhe41/Dec2 knockout mice and/or in vitro differentiation models, and research has been conducted using genetic analysis of tumor cells, in vitro analysis of cancer cell lines, and immunohistochemical studies of the clinical samples. We summarize some of these studies, detail several problems, and consider possible reasons for contradictory results and the needs for further research.

Oxygen-independent stabilization of HIF-2α in breast cancer through direct interaction with peptidyl-prolyl cis-trans isomerase NIMA-interacting 1

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) isomerizes the nearby proline (Pro) residue when it detects phosphorylated serine (Ser) or threonine (Thr) of target proteins, altering their structure, stability, function, and interaction with other proteins. Hypoxia-inducible factor 2α (HIF-2α), a transcription factor that transactivates many oncogenic genes under hypoxic conditions, harbours the pSer/Thr-Pro motif. We found for the first time that Pin1 binds to HIF-2α physically in normoxic as well as hypoxic conditions in human breast cancer cells. The level of ubiquitinated HIF-2α was significantly raised by Pin1 knockdown, while expression of its mRNA transcript was unaffected. In agreement with this observation, the cycloheximide chase assay demonstrated that Pin1 prolonged the stability of HIF-2α. Serine 672, 696, and 790 of HIF-2α were found to undergo phosphorylation. Of these, the main amino acid involved in the Pin1 binding and HIF-2α stabilization was identified as serine 790, located in the nuclear export signal region of HIF-2α. The tissue array with human breast cancer specimens showed elevated expression of HIF-2α as well as Pin1 compared to adjacent normal tissues. Knockdown of Pin1 or HIF-2α diminished breast cancer cell migration and colony formation. In conclusion, Pin1 stabilizes HIF-2α through direct interaction, which contributes to the growth of breast cancer.