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Molecular Oncology Nov 2023The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to...
The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate gene expression in a cell-type- and developmental-stage-specific manner. To date, a combined analysis of essential MYC-bound E-boxes and their downstream target genes important for growth of different types of cancer is missing. In this study, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. In parallel, we used the Brunello library to knock out protein-coding genes. We performed high-throughput screens with these libraries in four MYC-dependent cancer cell lines-K562, ST486, HepG2, and MCF7-which revealed several essential E-boxes and genes. Among them, we pinpointed crucial common and cell-type-specific MYC-regulated genes involved in pathways associated with cancer development. Extensive validation of our approach confirmed that E-box disruption affects MYC binding, target-gene expression, and cell proliferation in vitro as well as tumor growth in vivo. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in cancer cells.
Topics: Humans; CRISPR-Cas Systems; Proto-Oncogene Proteins c-myc; Cell Line; Transcription Factors; Gene Expression Regulation; Neoplasms
PubMed: 37519063
DOI: 10.1002/1878-0261.13493 -
Molecular Oncology Sep 2023ALK, ROS1, and RET fusions and MET∆ex14 variant associate with response to targeted therapies in non-small-cell lung cancer (NSCLC). Technologies for fusion testing in...
ALK, ROS1, and RET fusions and MET∆ex14 variant associate with response to targeted therapies in non-small-cell lung cancer (NSCLC). Technologies for fusion testing in tissue must be adapted to liquid biopsies, which are often the only material available. In this study, circulating-free RNA (cfRNA) and extracellular vesicle RNA (EV-RNA) were purified from liquid biopsies. Fusion and MET∆ex14 transcripts were analyzed by nCounter (Nanostring) and digital PCR (dPCR) using the QuantStudio System (Applied Biosystems). We found that nCounter detected ALK, ROS1, RET, or MET∆ex14 aberrant transcripts in 28/40 cfRNA samples from positive patients and 0/16 of control individuals (70% sensitivity). Regarding dPCR, aberrant transcripts were detected in the cfRNA of 25/40 positive patients. Concordance between the two techniques was 58%. Inferior results were obtained when analyzing EV-RNA, where nCounter often failed due to a low amount of input RNA. Finally, results of dPCR testing in serial liquid biopsies of five patients correlated with response to targeted therapy. We conclude that nCounter can be used for multiplex detection of fusion and MET∆ex14 transcripts in liquid biopsies, showing a performance comparable with next-generation sequencing platforms. dPCR could be employed for disease follow-up in patients with a known alteration. cfRNA should be preferred over EV-RNA for these analyses.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Protein-Tyrosine Kinases; Lung Neoplasms; Anaplastic Lymphoma Kinase; RNA; Proto-Oncogene Proteins c-ret; Proto-Oncogene Proteins; Liquid Biopsy; Cell-Free Nucleic Acids; Oncogene Proteins, Fusion
PubMed: 37243883
DOI: 10.1002/1878-0261.13468 -
Nucleic Acids Research Apr 2024DNA Methylation is a significant epigenetic modification that can modulate chromosome states, but its role in orchestrating chromosome organization has not been well...
DNA Methylation is a significant epigenetic modification that can modulate chromosome states, but its role in orchestrating chromosome organization has not been well elucidated. Here we systematically assessed the effects of DNA Methylation on chromosome organization with a multi-omics strategy to capture DNA Methylation and high-order chromosome interaction simultaneously on mouse embryonic stem cells with DNA methylation dioxygenase Tet triple knock-out (Tet-TKO). Globally, upon Tet-TKO, we observed weakened compartmentalization, corresponding to decreased methylation differences between CpG island (CGI) rich and poor domains. Tet-TKO could also induce hypermethylation for the CTCF binding peaks in TAD boundaries and chromatin loop anchors. Accordingly, CTCF peak generally weakened upon Tet-TKO, which results in weakened TAD structure and depletion of long-range chromatin loops. Genes that lost enhancer-promoter looping upon Tet-TKO showed DNA hypermethylation in their gene bodies, which may compensate for the disruption of gene expression. We also observed distinct effects of Tet1 and Tet2 on chromatin organization and increased DNA methylation correlation on spatially interacted fragments upon Tet inactivation. Our work showed the broad effects of Tet inactivation and DNA methylation dynamics on chromosome organization.
Topics: DNA Methylation; Animals; Mice; DNA-Binding Proteins; Dioxygenases; Proto-Oncogene Proteins; Chromatin; CpG Islands; Mouse Embryonic Stem Cells; CCCTC-Binding Factor; Epigenesis, Genetic; Promoter Regions, Genetic; Chromosomes
PubMed: 38300758
DOI: 10.1093/nar/gkae054 -
Journal of Autoimmunity Jul 2023Hepatocyte nuclear factor 4 α (HNF4α), a transcription factor (TF) essential for embryonic development, has been recently shown to regulate the expression of...
Hepatocyte nuclear factor 4 α (HNF4α), a transcription factor (TF) essential for embryonic development, has been recently shown to regulate the expression of inflammatory genes. To characterize HNF4a function in immunity, we measured the effect of HNF4α antagonists on immune cell responses in vitro and in vivo. HNF4α blockade reduced immune activation in vitro and disease severity in the experimental model of multiple sclerosis (MS). Network biology studies of human immune transcriptomes unraveled HNF4α together with SP1 and c-myc as master TF regulating differential expression at all MS stages. TF expression was boosted by immune cell activation, regulated by environmental MS risk factors and higher in MS immune cells compared to controls. Administration of compounds targeting TF expression or function demonstrated non-synergic, interdependent transcriptional control of CNS autoimmunity in vitro and in vivo. Collectively, we identified a coregulatory transcriptional network sustaining neuroinflammation and representing an attractive therapeutic target for MS and other inflammatory disorders.
Topics: Humans; Autoimmunity; Gene Expression Regulation; Gene Regulatory Networks; Hepatocyte Nuclear Factor 4; Multiple Sclerosis; Transcriptome; Genes, myc
PubMed: 37236124
DOI: 10.1016/j.jaut.2023.103053 -
Journal of Cellular and Molecular... Sep 2023Spi-1 proto-oncogene (SPI1) plays a vital role in carcinogenesis. Our work aimed to investigate the potential regulatory mechanism of SPI1 in melanoma. The mRNA and...
Spi-1 proto-oncogene (SPI1) plays a vital role in carcinogenesis. Our work aimed to investigate the potential regulatory mechanism of SPI1 in melanoma. The mRNA and protein levels were measured via qRT-PCR and Western blotting. Cell viability was assessed by CCK-8 assay. The target relationship between SPI1 and hexokinase 2 (HK2) was determined using dual-luciferase reporter detection. ChIP was conducted to confirm the targeted relationship between SPI1 and the HK2 promoter. Immunohistochemistry analysis was conducted to measure the positive cell number of SPI1 and HK2 in melanoma tissues. The cell migration abilities were determined using a wound healing assay. Glucose consumption, pyruvate dehydrogenase activity, lactate production and ATP levels were measured to assess glycolysis. SPI1 transcription in melanoma cells and tissues was dramatically higher than that in adjacent normal tissues and epidermal melanocyte HEMa-LP, respectively. Knockdown of SPI1 restrained cell viability, metastasis and glycolysis in melanoma cells. SPI1 directly targeted HK2, and knockdown of SPI1 repressed HK2 expression. Overexpression of HK2 weakened the inhibitory effects of SPI1 knockdown on the viability, metastasis and glycolysis of melanoma cells. The serine-threonine kinase 1 (AKT1)/mammalian target of rapamycin (mTOR) axis is involved in melanoma progression. SPI1 knockdown restrained melanoma cell proliferation, metastasis and glycolysis by regulating the AKT1/mTOR pathway.
Topics: Humans; MicroRNAs; Hexokinase; TOR Serine-Threonine Kinases; Melanoma; Cell Proliferation; Cell Line, Tumor; Proto-Oncogene Proteins c-akt; Melanoma, Cutaneous Malignant
PubMed: 37539493
DOI: 10.1111/jcmm.17844 -
Drug Resistance Updates : Reviews and... Nov 2023High levels of the estrogen receptor β (ERβ) predict poor prognosis following platinum-containing adjuvant chemotherapies in patients with non-small cell lung cancer...
High levels of the estrogen receptor β (ERβ) predict poor prognosis following platinum-containing adjuvant chemotherapies in patients with non-small cell lung cancer (NSCLC). However, the precise role of ERβ remains elusive. In this study, we demonstrated that targeting ERβ could significantly increase the cytotoxicity of cisplatin both in vitro and in vivo. Mechanically, cisplatin directly binds to ERβ, which facilitates its homodimerization and nuclear translocation. ERβ activation transcriptionally represses the expression of DCAF8, an adaptor of CRL4 E3 ubiquitin ligase, which in turn attenuates the proteasomal degradation of ERβ, leading to ERβ accumulation; this positive feedback loop results in Akt activation and eventually cisplatin resistance in NSCLC through PTEN inhibition. Moreover, low expression of DCAF8 and high expression of ERβ are associated with treatment resistance in patients receiving cisplatin-containing adjuvant chemotherapy. The present results provide insights into the underlying mechanism of ERβ-induced cisplatin resistance and offer an alternative therapeutic strategy to improve the efficacy of platinum-based chemotherapy in patients with NSCLC.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Cisplatin; Proto-Oncogene Proteins c-akt; Estrogen Receptor beta; Lung Neoplasms; Drug Resistance, Neoplasm; Feedback; Cell Line, Tumor; PTEN Phosphohydrolase
PubMed: 37913652
DOI: 10.1016/j.drup.2023.101014 -
Current Oncology (Toronto, Ont.) Apr 2024KRAS is a small GTPase that is among the most commonly mutated oncogenes in cancer. Here, we discuss KRAS biology, therapeutic avenues to target it, and mechanisms of... (Review)
Review
KRAS is a small GTPase that is among the most commonly mutated oncogenes in cancer. Here, we discuss KRAS biology, therapeutic avenues to target it, and mechanisms of resistance that tumors employ in response to KRAS inhibition. Several strategies are under investigation for inhibiting oncogenic KRAS, including small molecule compounds targeting specific KRAS mutations, pan-KRAS inhibitors, PROTACs, siRNAs, PNAs, and mutant KRAS-specific immunostimulatory strategies. A central challenge to therapeutic effectiveness is the frequent development of resistance to these treatments. Direct resistance mechanisms can involve KRAS mutations that reduce drug efficacy or copy number alterations that increase the expression of mutant KRAS. Indirect resistance mechanisms arise from mutations that can rescue mutant KRAS-dependent cells either by reactivating the same signaling or via alternative pathways. Further, non-mutational forms of resistance can take the form of epigenetic marks, transcriptional reprogramming, or alterations within the tumor microenvironment. As the possible strategies to inhibit KRAS expand, understanding the nuances of resistance mechanisms is paramount to the development of both enhanced therapeutics and innovative drug combinations.
Topics: Humans; Proto-Oncogene Proteins p21(ras); Drug Resistance, Neoplasm; Neoplasms; Antineoplastic Agents; Mutation
PubMed: 38668053
DOI: 10.3390/curroncol31040150 -
Aging and Disease Apr 2024Various diseases, including cancers, age-associated disorders, and acute liver failure, have been linked to the oncogene, MYC. Animal testing and clinical trials have... (Review)
Review
Various diseases, including cancers, age-associated disorders, and acute liver failure, have been linked to the oncogene, MYC. Animal testing and clinical trials have shown that sustained tumor volume reduction can be achieved when MYC is inactivated, and different combinations of therapeutic agents including MYC inhibitors are currently being developed. In this review, we first provide a summary of the multiple biological functions of the MYC oncoprotein in cancer treatment, highlighting that the equilibrium points of the MYC/MAX, MIZ1/MYC/MAX, and MAD (MNT)/MAX complexes have further potential in cancer treatment that could be used to restrain MYC oncogene expression and its functions in tumorigenesis. We also discuss the multifunctional capacity of MYC in various cellular cancer processes, including its influences on immune response, metabolism, cell cycle, apoptosis, autophagy, pyroptosis, metastasis, angiogenesis, multidrug resistance, and intestinal flora. Moreover, we summarize the MYC therapy patent landscape and emphasize the potential of MYC as a druggable target, using herbal medicine modulators. Finally, we describe pending challenges and future perspectives in biomedical research, involving the development of therapeutic approaches to modulate MYC or its targeted genes. Patients with cancers driven by MYC signaling may benefit from therapies targeting these pathways, which could delay cancerous growth and recover antitumor immune responses.
Topics: Animals; Humans; Genes, myc; Proto-Oncogene Proteins c-myc; Repressor Proteins; Biological Products; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Signal Transduction; Neoplasms
PubMed: 37450923
DOI: 10.14336/AD.2023.0520 -
Journal of Cellular and Molecular... Sep 2023Hairy cell leukaemia (HCL) diagnosis is based on the morphologic detection of circulating abnormal hairy cells in the peripheral blood and/or bone marrow, an HCL...
Hairy cell leukaemia (HCL) diagnosis is based on the morphologic detection of circulating abnormal hairy cells in the peripheral blood and/or bone marrow, an HCL immunological score of 3 or 4 based on the expression of the CD11c, CD25, CD103 and CD123 and also the presence of a BRAF V600E activating mutation in the B-raf proto-oncogene (BRAF gene) (7q34). When using new generation sequencing of 21 targeted genes in 124 HCL patients, we identified a cohort of 6/124 (2%) patients with unusual BRAF mutations: two patients presented non-V600 mutations (BRAF F595L, BRAF W604L respectively) and four other patients silent BRAF mutations. When using droplet digital PCR (ddPCR) three of the four patients with concomitant BRAF V600E and silent mutation were negative. The respective role of these mutations in the occurrence of HCL or its progression remains to be clarified, but BRAF sequencing is necessary in case of negative BRAF V600E by ddPCR.
Topics: Humans; Leukemia, Hairy Cell; Proto-Oncogene Proteins B-raf; Mutation; Bone Marrow; Exons
PubMed: 37530550
DOI: 10.1111/jcmm.17890 -
International Journal of Biological... 2024Kirsten rat sarcoma viral oncogene homolog (KRAS) is an oncogene implicated in the pathophysiology of many cancers. Increasing evidence shows that KRAS mutation is... (Review)
Review
Kirsten rat sarcoma viral oncogene homolog (KRAS) is an oncogene implicated in the pathophysiology of many cancers. Increasing evidence shows that KRAS mutation is correlated with poor prognosis in numerous cancers, including colorectal cancer (CRC), breast cancer, and melanoma. KRAS also participates in regulating the CRC microenvironment. However, the direct and indirect therapeutic targets of KRAS in CRC have not been identified; thus, elucidating the mechanisms and interactions between KRAS and the tumor microenvironment (TME) in-depth is paramount. Herein, we present some of the major roles KRAS plays in shaping the heterogeneity of the TME and propose a potential strategy for targeting the downstream components of the KRAS signaling pathway and the TME in CRC.
Topics: Humans; Proto-Oncogene Proteins p21(ras); Tumor Microenvironment; Mutation; Signal Transduction; Colorectal Neoplasms
PubMed: 38481800
DOI: 10.7150/ijbs.88779