Metabolic enzyme PFKFB3 mediates matrix stiffness‐potentiated tumour growth and radiotherapeutic resistance in HCC

β-TrCP1 facilitates cell cycle checkpoint activation, DNA repair, and cell survival through ablation of β-TrCP2 in response to genotoxic stress

XRCC2 repairs mitochondrial DNA damage and fuels malignant behavior in hepatocellular carcinoma.

The Polycomb Group Protein L3MBTL2 Modulates DNA Double Strand Break Repair and Predicts Response of Triple Negative Breast Cancer Cells to Poly (ADP-ribose) Polymerase Inhibition

BackgroundHepatocellular carcinoma (HCC) is the most common malignant liver tumor with poor clinical outcomes. Increasing amount of long non-coding RNAs (lncRNAs) have been revealed to be implicated in the carcinogenesis and progression of HCC. However, the expressions, clinical significances, and roles of most lncRNAs in HCC are still unknown.MethodsThe expression of lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) in HCC tissues and cell lines was detected by qRT-PCR and fluorescence in situ hybridization. Immunoblotting, CCK-8, EdU, colony formation and flow cytometry were performed to investigate the role of MCM3AP-AS1 in HCC cell proliferation, cell cycle and apoptosis in vitro. A subcutaneous tumor mouse model was constructed to analyze in vivo growth of HCC cells after MCM3AP-AS1 knockdown. The interactions among MCM3AP-AS1, miR-194-5p and FOXA1 were measured by RNA pull-down, RNA immunoprecipitation and luciferase reporter assay.ResultsWe revealed a novel oncogenic lncRNA MCM3AP-AS1, which is overexpressed in HCC and positively correlated with large tumor size, high tumor grade, advanced tumor stage and poor prognosis of HCC patients. MCM3AP-AS1 knockdown suppressed HCC cell proliferation, colony formation and cell cycle progression, and induced apoptosis in vitro, and depletion of MCM3AP-AS1 inhibited tumor growth of HCC in vivo. Mechanistically, MCM3AP-AS1 directly bound to miR-194-5p and acted as competing endogenous RNA (ceRNA), and subsequently facilitated miR-194-5p’s target gene forkhead box A1 (FOXA1) expression in HCC cells. Interestingly, FOXA1 restoration rescued MCM3AP-AS1 knockdown induced proliferation inhibition, G1 arrest and apoptosis of HCC cells.ConclusionsOur results recognized MCM3AP-AS1 as a novel oncogenic lncRNA, which indicated poor clinical outcomes in patients with HCC. MCM3AP-AS1 exerted an oncogenic role in HCC via targeting miR-194-5p and subsequently promoted FOXA1 expression. Our findings suggested that MCM3AP-AS1 could be a potential prognostic biomarker and therapeutic target for HCC.

Myelodysplastic syndrome: An inability to appropriately respond to damaged DNA?

Bcl2 and c-Myc are two major oncogenic proteins that can functionally promote DNA damage, genetic instability, and tumorigenesis. However, the mechanism(s) remains unclear. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen contained in cigarette smoke that induces cellular DNA damage. Here we found that Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions in association with increased c-Myc transcriptional activity. The Bcl2 BH4 domain (amino acids 6-31) was found to bind directly to c-Myc MBII domain (amino acids 106-143), and this interaction is required for Bcl2 to enhance c-Myc transcriptional activity and inhibit DNA repair. In addition to mitochondria, Bcl2 is also expressed in the nucleus, where it co-localizes with c-Myc. Expression of nuclear-targeted Bcl2 enhances c-Myc transcriptional activity with suppression of DNA repair but fails to prolong cell survival. Depletion of c-Myc expression from cells overexpressing Bcl2 significantly accelerates the repair of NNK-induced DNA damage, indicating that c-Myc may be essential for the Bcl2 effect on DNA repair. It is known that apurinic/apyrimidinic endonuclease (APE1) plays a crucial role in the repair of abasic sites of DNA lesions. That overexpression of Bcl2 results in up-regulation of c-Myc and down-regulation of APE1 suggests APE1 may function as the downstream target of Bcl2/c-Myc in the DNA repair machinery. Thus, Bcl2, in addition to its survival function, may also suppress DNA repair in a novel mechanism involving c-Myc and APE1, which may lead to an accumulation of DNA damage in living cells, genetic instability, and tumorigenesis.

Objectives Hypopharyngeal cancer (HPC), originating from the hypopharyngeal mucosa, is associated with a poor prognosis. Extracellular matrix (ECM) stiffness is closely linked to tumor progression and patient outcomes. This study aimed to investigate the effects of matrix stiffness and to identify molecular markers relevant to HPC prognosis, with the goal of improving clinical outcomes.Methods Immunohistochemical analysis and cervical enhanced computed tomography (CT) data were used to evaluate correlations among CT values, matrix stiffness, and prognosis, and to develop a prognostic model for HPC. Cell culture models with varying matrix stiffness were established using polypropylene hydrogel. Western blotting, colony formation, EDU incorporation, and Transwell assays were employed to assess the effects of matrix stiffness on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). High-throughput sequencing identified differentially expressed genes in HPC cells cultured on matrices of differing stiffness. Gene editing, in vivo subcutaneous tumorigenesis studies, and Western blotting were performed to elucidate the molecular mechanisms by which high matrix stiffness influences HPC progression and the role of ephrin A2 (EFNA2) in proliferation, migration, and EMT.Results Arterial phase CT values were positively correlated with matrix stiffness. Increased matrix stiffness was associated with lymph node metastasis, diminished therapeutic response, and poorer prognosis. Furthermore, metastatic lymph nodes in HPC patients exhibited higher CT values than those in patients with nasopharyngeal carcinoma. A high-stiffness ECM promoted proliferation, migration, and EMT in HPC cells. Mechanistically, a stiff ECM enhanced EFNA2 expression, thereby promoting proliferation, migration, EMT, and tumor growth in vivo.Conclusion EFNA2 and elevated matrix stiffness jointly contribute to the malignant phenotype in HPC. EFNA2 may represent a potential therapeutic target for managing HPC progression induced by high matrix stiffness.

DNA damage associated with ultraviolet (UV) radiation exposure causes accelerated skin aging and cancer-initiating mutations. Over-the-counter UVA and UVB filters are required in all sunscreens, but transdermal absorption of these compounds have been reported to impair development and reproduction and metabolize into carcinogenic/mutagenic chemical intermediates. To assess the effects of sunscreen active ingredients on UVR-induced DNA damage and repair, we developed a novel high throughput (HTP) screening platform, UValidate, employing mixed populations of isogenic skin cells, exposed to single or combinations of UV filters in the presence of UVB and UVA. UValidate contains a patented LED UVR DNA damage induction system (LUDIS:V3), facilitating programmable precise induction of DNA damage and repair at multiple time points and replicates, using a 96-well configuration capable of simultaneous delivering UVA and UVB. LUDIS:V3 optimization experiments surprisingly revealed that, unlike keratinocytes, skin fibroblasts only repaired UVA DNA damage in conditioned media. We then tested sunscreen active ingredient combinations for their capacity to alter DNA repair and found that: 1) Some sunscreen ingredients, including dioxybenzone, induce a significant increase in DNA damage above endogenous levels. 2) Fibroblasts and keratinocytes respond differently to the treatment protocols. 3) Some sunscreen active compounds contribute to reduction of UVA-induced DNA damage. 4) No sunscreen completely inhibits all UVA DNA damage. We are currently employing the LUDIS:V3 system with HTP comet assays to compare DNA damage and DNA repair capacities, +/- sunscreen active ingredients, of six isogenic sets of primary epidermal keratinocytes (NHEK), melanocytes (NHEM), and fibroblasts (NHDF)` with different Fitzpatrick phototypes I-VI, representative of the diverse American population. These cells were isolated from donor skin samples, and further purified to establish banks of isogenic NHEK, NHEM, and NHDF cell lines. UVA exposure with the LUDIS:V3 and comet assays revealed higher levels of DNA damage in NHEK and NHDF, compared to NHEM, likely due to the UVR-protective effects of melanin in NHEM. Consistently, NHEK with phototype V (from darkly pigmented skin with higher melanin levels) exhibit significantly less UVA-induced DNA damage compared with phototype II NHEK. To create cellular models of dermal disease for DNA repair assays, we have successfully knocked out XPA in multiple NHDF cell lines with different phototypes using a CRISPR-Cas9 KO strategy, as confirmed by immunoblot and NGS sequence analyses. These and other engineered DNA repair-deficient skin cells will be assayed as single or mixed cell-type 2D cultures, with fluorescent labeling allowing for identification of subpopulations in comet and alkaline diffusion assays. This technology addresses the need for better platforms for rapid genotoxicity testing of safer and more effective chemical UV filters to prevent cutaneous carcinogenesis in diverse populations. Citation Format: Dean S. Rosenthal, Elijah Finn, Devin Teehan, Nusrat Islam, Veerupaxagouda Patil, Bonnie Carney, Scott S. Rosenthal, Lucia Dussan, Cynthia M. Simbulan-Rosenthal, Peter Sykora. Developing the UValidate platform to measure DNA damage and repair capacity in isogenic donor-derived skin keratinocytes, fibroblasts and melanocyte cell-lines with different Fitzpatrick phototypes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B035.

BackgroundHepatocellular carcinoma (HCC) is the most common type of liver cancer with increasing incidence and poor prognosis. Ubiquitination regulators are reported to play crucial roles in HCC carcinogenesis. UBE2D1, one of family member of E2 ubiquitin conjugating enzyme, mediates the ubiquitination and degradation of tumor suppressor protein p53. However, the expression and functional roles of UBE2D1 in HCC was unknown.MethodsImmunohistochemistry (IHC), western blotting, and real-time PCR were used to detect the protein, transcription and genomic levels of UBE2D1 in HCC tissues with paired nontumor tissues, precancerous lesions and hepatitis liver tissues. Four HCC cell lines and two immortalized hepatic cell lines were used to evaluate the functional roles and underlying mechanisms of UBE2D1 in HCC initiation and progression in vitro and in vivo. The contributors to UBE2D1 genomic amplification were first evaluated by performing a correlation analysis between UBE2D1 genomic levels with clinical data of HCC patients, and then evaluated in HCC and hepatic cell lines.ResultsExpression of UBE2D1 was significantly increased in HCC tissues and precancerous lesions and was associated with reduced survival of HCC patients. Upregulation of UBE2D1 promoted HCC growth in vitro and in vivo by decreasing the p53 in ubiquitination-dependent pathway. High expression of UBE2D1 was attributed to the recurrent genomic copy number gain, which was associated with high serum IL-6 level of HCC patients. Further experiments showed that continuous IL-6 activated the DNA damage response and genomic instability by repressing DNA damage checkpoint protein RAD51B. Moreover, continuous IL-6 could significantly facilitate the HCC growth especially with the genomic gain of UBE2D1.ConclusionsOur findings showed that UBE2D1 played a crucial role in HCC progression, and suggested a novel pattern of continuous IL-6 to promote cancers by inducing the genomic alterations of specific oncogenes.

SIRT1 Regulates UV-Induced DNA Repair through Deacetylating XPA

Circ_0000854 regulates the progression of hepatocellular carcinoma through miR-1294 /IRGQ axis

Persistent DNA Damage and Senescence in the Placenta Impacts Developmental Outcomes of Embryos.

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types' responses to matrix stiffness have been characterized, how adherent cells' susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types' responses to matrix stiffness have been characterized, how adherent cells' susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

Terminal differentiation induction as DNA damage response in hematopoietic stem cells by GADD45A.