Elevated level of PLRG1 is critical for the proliferation and maintenance of genome stability of tumor cells.

Pleiotropic regulator 1 (PLRG1), a highly conserved element in the spliceosome, can form a NineTeen Complex (NTC) with Prp19, SPF27, and CDC5L. This complex plays crucial roles in both pre-mRNA splicing and DNA repair processes. Here, we provide evidence that PLRG1 has a multifaceted impact on cancer cell proliferation. Comparing its expression levels in cancer and normal cells, we observed that PLRG1 was upregulated in various tumor tissues and cell lines. Knockdown of PLRG1 resulted in tumor-specific cell death. Depletion of PLRG1 had notable effects, including mitotic arrest, microtubule instability, endoplasmic reticulum (ER) stress, and accumulation of autophagy, ultimately culminating in apoptosis. Our results also demonstrated that PLRG1 downregulation contributed to DNA damage in cancer cells, which we confirmed through experimental validation as DNA repair impairment. Interestingly, when PLRG1 was decreased in normal cells, it induced G1 arrest as a self-protective mechanism, distinguishing it from effects observed in cancer cells. These results highlight multifaceted impacts of PLRG1 in cancer and underscore its potential as a novel anti-cancer strategy by selectively targeting cancer cells. [BMB Reports 2023; 56(11): 612-617].

Split genome-based retroviral replicating vectors achieve efficient gene delivery and therapeutic effect in a human glioblastoma xenograft model.

The murine leukemia virus-based semi-retroviral replicating vectors (MuLV-based sRRV) had been developed to improve safety and transgene capacity for cancer gene therapy. However, despite the apparent advantages of the sRRV, improvements in the in vivo transduction efficiency are still required to deliver therapeutic genes efficiently for clinical use. In this study, we established a gibbon ape leukemia virus (GaLV) envelopepseudotyped semi-replication-competent retrovirus vector system (spRRV) which is composed of two transcomplementing replication-defective retroviral vectors termed MuLV-Gag-Pol and GaLV-Env. We found that the spRRV shows considerable improvement in efficiencies of gene transfer and spreading in both human glioblastoma cells and pre-established human glioblastoma mouse model compared with an sRRV system. When treated with ganciclovir after intratumoral injection of each vector system into pre-established U-87 MG glioblastomas, the group of mice injected with spRRV expressing the herpes simplex virus type 1-thymidine kinase (HSV1-tk) gene showed a survival rate of 100% for more than 150 days, but all control groups of mice (HSV1-tk/PBS-treated and GFP/GCV-treated groups) died within 45 days after tumor injection. In conclusion, these findings sug-gest that intratumoral delivery of the HSV1-tk gene by the spRRV system is worthy of development in clinical trials for the treatment of malignant solid tumors. [BMB Reports 2022; 55(12): 615-620].

Aß-induced mitochondrial dysfunction in neural progenitors controls KDM5A to influence neuronal differentiation.

Mitochondria in neural progenitors play a crucial role in adult hippocampal neurogenesis by being involved in fate decisions for differentiation. However, the molecular mechanisms by which mitochondria are related to the genetic regulation of neuronal differentiation in neural progenitors are poorly understood. Here, we show that mitochondrial dysfunction induced by amyloid-beta (Aß) in neural progenitors inhibits neuronal differentiation but has no effect on the neural progenitor stage. In line with the phenotypes shown in Alzheimer's disease (AD) model mice, Aß-induced mitochondrial damage in neural progenitors results in deficits in adult hippocampal neurogenesis and cognitive function. Based on hippocampal proteome changes after mitochondrial damage in neural progenitors identified through proteomic analysis, we found that lysine demethylase 5A (KDM5A) in neural progenitors epigenetically suppresses differentiation in response to mitochondrial damage. Mitochondrial damage characteristically causes KDM5A degradation in neural progenitors. Since KDM5A also binds to and activates neuronal genes involved in the early stage of differentiation, functional inhibition of KDM5A consequently inhibits adult hippocampal neurogenesis. We suggest that mitochondria in neural progenitors serve as the checkpoint for neuronal differentiation via KDM5A. Our findings not only reveal a cell-type-specific role of mitochondria but also suggest a new role of KDM5A in neural progenitors as a mediator of retrograde signaling from mitochondria to the nucleus, reflecting the mitochondrial status.

Novel Semi-Replicative Retroviral Vector Mediated Double Suicide Gene Transfer Enhances Antitumor Effects in Patient-Derived Glioblastoma Models.

As glioblastomas are mostly localized infiltrative lesions, gene therapy based on the retroviral replicating vector (RRV) system is considered an attractive strategy. Combinations of multiple suicide genes can circumvent the limitations associated with each gene, achieving direct and synergistic cytotoxic effects, along with bystander cell killing. In this study, we constructed a semi-and pseudotyped-RRV (sp-RRV) system harboring two suicide genes-herpes simplex virus type 1 thymidine kinase (TK) and yeast cytosine deaminase (CD)-to verify the dissemination and antitumor efficacy of our sp-RRV system (spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD) in seven patient-derived glioblastoma stem-like cells (GSCs). Flow cytometry and high-content analysis revealed a wide range of transduction efficiency and good correlation between the delivery of therapeutic genes and susceptibility to the prodrugs ganciclovir and 5-fluorocytosine in patient-derived GSCs in vitro. Intra-tumoral delivery of spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD, combined with prodrug treatment, synergistically inhibited cell proliferation and angiogenesis while increasing apoptosis and the depletion of tumor-associated macrophages in orthotopic glioblastoma xenografts. Genomic profiling of patient-derived GSCs revealed that the key genes preventing sp-RRV infection and transmission were associated with cell adhesion, migration, development, differentiation, and proliferation. This is the first report demonstrating that a novel sp-RRV-mediated TK/CD double suicide gene transfer system has high oncolytic power against extremely heterogeneous and treatment-refractory glioblastomas.

Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth.

KRAS is the most frequently mutated oncogene in human tumors, and its activating mutations represent important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here, we harnessed CRISPR to specifically target mutant KRAS alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA. The survival, proliferation, and tumorigenicity of cancer cells in vitro and the growth of tumors in vivo were determined after delivery of Cas9 and guide RNA. We identified guide RNAs that efficiently target mutant KRAS without significant alterations of the wild-type allele. Doxycycline-inducible expression of this guide RNA in KRAS-mutant cancer cells transduced with a lentiviral vector encoding Cas9 disrupted the mutant KRAS gene, leading to inhibition of cancer cell proliferation both in vitro and in vivo. Intra-tumoral injection of lentivirus and adeno-associated virus expressing Cas9 and sgRNA suppressed tumor growth in vivo, albeit incompletely, in immunodeficient mice. Expression of Cas9 and the guide RNA in cells containing wild-type KRAS did not alter cell survival or proliferation either in vitro and in vivo. Our study provides a proof-of-concept that CRISPR can be utilized to target driver mutations of cancers in vitro and in vivo.

Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter.

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca(2+) uptake in a stable MCU knockdown HeLa cell line and exerted dominant-negative effects in the wild-type MCU-expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.

EPA attenuates ultraviolet radiation-induced downregulation of aquaporin-3 in human keratinocytes.

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid (ω-3 PUFA) that protects against photodamage and photocarcinogenesis in mammals. Aquaporin-3 (AQP3) is a water/glycerol transport protein that is found in basal layer keratinocytes. In this study, we have investigated the protective effect of EPA against ultraviolet B (UVB)-induced AQP3 downregulation in human keratinocytes. EPA treatment was found to increase AQP3 gene and protein expression in human epidermal keratinocytes (HaCaT). Using a specific inhibitor, we observed that the effect of EPA on AQP3 expression was mediated by extracellular signal-regulated kinase (ERK) activation. UVB radiation induced AQP3 downregulation in HaCaT cells, and it was found that EPA treatment attenuated UVB-induced AQP3 reduction and the associated cell death. UVB-induced downregulation of AQP3 was blocked by EPA and p38 inhibitor SB203580. Collectively, the present results show that EPA increased AQP3 expression and that this led to a reduction UVB-induced photodamage.

Sulfasalazine induces apoptosis of HBx-expressing cells in an NF-kappaB-independent manner.

The Hepatitis B virus (HBV) is a causative agent of acute chronic hepatitis, cirrhosis, and hepatocarcinoma. The Hepatitis B virus X protein (HBx) has pleiotypic functions in the regulation of proliferation and apoptosis. It has been suggested that the anti-inflammatory drug sulfasalazine, which is commonly used to treat rheumatoid arthritis and inflammatory bowel disease, inhibits nuclear factor NF-kappaB and induces cell death in HBx-expressing liver cells. In this study, we demonstrate that sulfasalazine induces cell death via apoptosis in HBx-expressing liver cells, as evidenced by characteristic changes in nuclear morphology, cleavage of poly (ADP-ribose) polymerase (PARP), caspase-3 and caspase-9, and activation of caspase-3. We also demonstrate that inhibition of NF-kappaB by siRNA fails to induce apoptosis of HBx-expressing liver cells, indicating that sulfasalazine modulates apoptosis of HBx-expressing cells in an NF-kappaB-independent manner.