Osteosarcoma (OS) is a bone malignant tumor affecting children, adolescents and young adults. Currently, osteosarcoma is treated with chemotherapy regimens established over 40 years ago. The investigation of novel therapeutic strategies for the treatment of osteosarcoma remains an important clinical need. Cyclin-dependent kinases (CDKs) have been considered promising molecular targets in cancer therapy. Among these, CDK12 has been shown to play a crucial role in the pathogenesis of malignancies, but its clinical significance and biological mechanisms in osteosarcoma remain unclear. In the present study, we aim to determine the expression and function of CDK12, and evaluate its prognostic and therapeutic value in metastatic osteosarcoma. We found that overexpression of CDK12 was associated with high tumor grade, tumor progression and reduced patient survival. Underlying mechanism revealed that knockdown of CDK12 expression with siRNA or functional inhibition with the CDK12-targeting agent THZ531 effectively exhibited time- and dose-dependent cytotoxicity. Downregulation of CDK12 paused transcription by reducing RNAP II phosphorylation, interfered with DNA damage repair with increased γH2AX, and decreased cell proliferation through the PI3K-AKT pathway. This was accompanied by the promotion of apoptosis, as evidenced by enhanced Bax expression and reduced Bcl-xL expression. Furthermore, the CDK12 selective inhibitor THZ531 also hindered ex vivo 3D spheroid formation, growth of in vitro 2D cell colony, and prevented cell mobility. Our findings highlight the clinical importance of CDK12 as a potentially valuable prognostic biomarker and therapeutic target in metastatic osteosarcoma.

OBJECTIVE: Radiotherapy has achieved remarkable effects in treating non-small cell lung cancer (NSCLC). However, radioresistance remains the major obstacle to achieving good outcomes. This study aims at identifying potential targets for radiosensitizing NSCLC and elucidating the underlying mechanisms.
METHODS: Lentivirus-based infection and CRISPR/Cas9 technology were used to modulate the expression of microRNA-384 (miR-384). Cell clonogenic formation assays and a xenograft tumor model were used to analyze radiosensitivity in NSCLC cells. Fluorescence-activated cell sorting was used to assess the cell cycle and cell death. Immunofluorescence staining, Comet assays, and homologous recombination or non-homologous end-joining I-SceI/GFP reporter assays were used to study DNA damage and repair. Western blotting and quantitative real-time polymerase chain reaction were used to identify the targets of miR-384. Chromatin immunoprecipitation and polymerase chain reaction were performed to evaluate upstream regulators of miR-384.
RESULTS: MiR-384 was downregulated in NSCLC. Overexpression of miR-384 increased the radiosensitivity of NSCLC cells in vitro and in vivo, whereas knockout of miR-384 led to radioresistance. Upregulation of miR-384 radiosensitized NSCLC cells by decreasing G2/M cell cycle arrest, inhibiting DNA damage repair, and consequently increasing cell death; miR-384 depletion had the opposite effects. Further investigation revealed that ATM, Ku70, and Ku80 were direct targets of miR-384. Moreover, miR-384 was repressed by NF-κB.
CONCLUSIONS: MiR-384 is an ionizing radiation-responsive gene repressed by NF-κB. MiR-384 enhances the radiosensitivity of NSCLC cells via targeting ATM, Ku80, and Ku70, which impairs DNA damage repair. Therefore, miR-384 may serve as a novel radiosensitizer for NSCLC.

Given the widespread applications in industrial and agricultural production, the health effects of rare earth elements (REEs) have garnered public attention, and the genotoxicity of REEs remains unclear. In this study, we evaluated the genetic effects of lanthanum nitrate, a typical representative of REEs, with guideline-compliant in vivo and in vitro methods. Genotoxicity assays, including the Ames test, comet assay, mice bone marrow erythrocyte micronucleus test, spermatogonial chromosomal aberration test, and sperm malformation assay were conducted to assess mutagenicity, chromosomal damage, DNA damage, and sperm malformation. In the Ames test, no statistically significant increase in bacterial reverse mutation frequencies was found as compared with the negative control. Mice exposed to lanthanum nitrate did not exhibit a statistically significant increase in bone marrow erythrocyte micronucleus frequencies, spermatogonial chromosomal aberration frequencies, or sperm malformation frequencies compared to the negative control (P > 0.05). Additionally, after a 24-h treatment with lanthanum nitrate at concentrations of 1.25, 5, and 20 μg/ml, no cytotoxicity was observed in CHL cells. Furthermore, the comet assay results indicate no significant DNA damage was observed even after exposure to high doses of lanthanum nitrate (20 μg/ml). In conclusion, our findings suggest that lanthanum nitrate does not exhibit genotoxicity.

The role if insulin-like growth factor binding protein-2 (IGFBP-2) in mediating chemoresistance in breast cancer cells has been demonstrated, but the mechanism of action is unclear. This study aimed to further investigate the role of IGFBP-2 in the DNA damage response induced by etoposide in MCF-7, T47D (ER+ve), and MDA-MB-231 (ER-ve) breast cancer cell lines. In the presence or absence of etoposide, IGFBP-2 was silenced using siRNA in the ER-positive cell lines, or exogenous IGFBP-2 was added to the ER-negative MDA-MB-231 cells. Cell number and death were assessed using trypan blue dye exclusion assay, changes in abundance of proteins were monitored using Western blotting of whole cell lysates, and localization and abundance were determined using immunofluorescence and cell fractionation. Results from ER-positive cell lines demonstrated that upon exposure to etoposide, loss of IGFBP-2 enhanced cell death, and this was associated with a reduction in P-DNA-PKcs and an increase in γH2AX. Conversely, with ER-negative cells, the addition of IGFBP-2 in the presence of etoposide resulted in cell survival, an increase in P-DNA-PKcs, and a reduction in γH2AX. In summary, IGFBP-2 is a survival factor for breast cancer cells that is associated with enhancement of the DNA repair mechanism.

Malignant rhabdoid tumors (MRTs) are among the most aggressive and treatment-resistant malignancies affecting infants, originating in the kidney, brain, liver, and soft tissues. The 5-year event-free survival rate for these cancers is a mere 20%. In nearly all cases of MRT, the SMARCB1 gene (occasionally SMARCA4)-a pivotal component of the SWI/SNF chromatin remodeling complex-is homozygously deleted, although the precise etiology of these tumors remains unknown. While young patients with localized MRT generally show improved outcomes, especially those who are older and have early-stage disease, the overall prognosis remains poor despite optimal standard treatments. This highlights the urgent need for more effective treatment strategies. We investigated the antitumor activity of a PARP1 inhibitor (talazoparib, TLZ) combined with a DNA alkylating agent (temozolomide, TMZ) in MRT xenograft models. PARP1 is a widely targeted molecule in cancer treatment and, beyond its role in DNA repair, it participates in transcriptional regulation by recruiting chromatin remodeling complexes to modulate DNA accessibility for RNA polymerases. To widen the therapeutic window of the drug combination, we employed PEGylated TLZ (PEG~TLZ), which has been reported to reduce systemic toxicity through slow drug release. Remarkably, our findings indicate that five out of six MRT xenografts exhibited an objective response to PEG~TLZ+TMZ therapy. Significantly, the loss of SMARCB1 was found to confer a protective effect, correlating with higher expression levels of DNA damage and repair proteins in SMARCB1-deficient MRT cells. Additionally, we identified MGMT as a potential biomarker indicative of in vivo MRT response to PEG~TLZ+TMZ therapy. Moreover, our analysis revealed alterations in signaling pathways associated with the observed antitumor efficacy. This study presents a novel and efficacious therapeutic approach for MRT, along with a promising candidate biomarker for predicting tumor response.

Radiation therapy, a standard treatment option for many cancer patients, induces DNA double-strand breaks (DSBs), leading to cell death. Ataxia telangiectasia mutated (ATM) kinase is a key regulator of DSB repair, and ATM inhibitors are being explored as radiosensitizers for various tumors, including primary and metastatic brain tumors. Efficacy of radiosensitizers for brain tumors may be influenced by a lack of effective drug delivery across the blood-brain barrier. The objective of this study was to evaluate the systemic pharmacokinetics and mechanisms that influence the central nervous system (CNS) distribution of WSD0628, a novel and potent ATM inhibitor, in the mouse. Further, we have used these observations to form the basis of predicting effective exposures for clinical application. We observed a greater than dose proportional increase in exposure, likely due to saturation of clearance processes. Our results show that WSD0628 is orally bioavailable and CNS penetrant, with unbound partitioning in CNS (i.e., unbound tissue partition coefficient) between 0.15 and 0.3. CNS distribution is not limited by the efflux transporters P-glycoprotein and breast cancer resistant protein. WSD0628 is distributed uniformly among different brain regions. Thus, WSD0628 has favorable pharmacokinetic properties and potential for further exploration to determine the pharmacodynamics-pharmacokinetics efficacy relationship in CNS tumors. This approach will provide critical insights for the clinical translation of WSD0628 for the treatment of primary and secondary brain tumors. SIGNIFICANCE STATEMENT: This study evaluates the preclinical systemic pharmacokinetics, dose proportionality, and mechanisms influencing CNS distribution of WSD0628, a novel ATM inhibitor for the treatment of brain tumors. Results indicate that WSD0628 is orally bioavailable and CNS penetrant without efflux transporter liability. We also observed a greater than dose proportional increase in exposure in both the plasma and brain. These favorable pharmacokinetic properties indicate WSD0628 has potential for further exploration for use as a radiosensitizer in the treatment of brain tumors.

Intraductal carcinoma of the prostate (IDC-P) is an aggressive subtype of prostate cancer characterized by the growth of tumor cells within the prostate ducts. It is often found alongside invasive carcinoma and is associated with poor prognosis. Understanding the molecular mechanisms driving IDC-P is crucial for improved diagnosis, prognosis, and treatment strategies. This review summarizes the molecular characteristics of IDC-P and their prognostic indications, comparing them to conventional prostate acinar adenocarcinoma, to gain insights into its unique behavior and identify potential therapeutic targets.

Gastric cancer (GACA) is a complex and multifaceted disease influenced by a variety of environmental and genetic factors. Somatic mutations play a major role in its development, and their characteristics, including the asymmetry between two DNA strands, are of great interest and appear as a signal of information and guidance, revealing mechanisms of DNA damage and repair. Here, we analyzed the impact of High-frequency mutated genes on patient prognosis and found that the effect of expression levels of tumor protein p53 (TP53) and lysine methyltransferase 2C (KMT2C) genes remained high throughout the development of GACA, with similar expression patterns. After investigating mutation asymmetry across mutagenic processes, we found that transcriptional asymmetry was dominated by T > G mutations under the influence of transcription couples repair and damage. The apolipoprotein B mRNA editing enzyme catalytic polypeptide like (APOBEC) enzyme that induces mutations during DNA replication has been identified here and we identified a replicative asymmetry, which was dominated by C > A mutations in left-replicating. Strand bias in different mutation classes at transcription factor binding sites and enhancer regions were also confirmed, which implies the important role of non-coding regulatory elements in the occurrence of mutations. This work systematically describes mutational strand asymmetries in specific genomic regions, shedding light on the DNA damage and repair mechanisms underlying somatic mutations in cohorts of GACA patients with gastric cancer.

Although widely known as a tumor suppressor, the breast cancer 1 susceptibility protein (BRCA1) is also important in development, where it regulates fetal DNA repair pathways that protect against DNA damage caused by physiological and drug-enhanced levels of reactive oxygen species (ROS). We previously showed that conditional heterozygous (+/-) knockout (cKO) mouse embryos with a minor 28% BRCA1 deficiency developed normally in culture, but when exposed to the ROS-initiating drug, alcohol (ethanol, EtOH), exhibited embryopathies not evident in wild-type (+/+) littermates. Herein, we characterized a directBrca1 +/- knockout (KO) model with a 2-fold greater (58%) reduction in BRCA1 protein vs. the cKO model. We also characterized and compared learning & memory deficits in both the cKO and KO models. Even saline-exposed Brca1 +/- vs. +/+ KO progeny exhibited enhanced oxidative DNA damage and embryopathies in embryo culture and learning & memory deficits in females in vivo, which were not observed in the cKO model, revealing the potential pathogenicity of physiological ROS levels. The embryopathic EtOH concentration for cultured direct KO embryos was half that for cKO embryos, and EtOH affected Brca1 +/+ embryos only in the direct KO model. The spectrum and severity of EtOH embryopathies in culture were greater in both Brca1 +/- vs. +/+ embryos, and direct KO vs. cKO +/- embryos. Motor coordination deficits were evident in both male and female Brca1 +/- KO progeny exposed in utero to EtOH. The results in our direct KO model with a greater BRCA1 deficiency vs. cKO mice provide the first evidence for BRCA1 protein dose-dependent susceptibility to developmental disorders caused by physiological and drug-enhanced oxidative stress.

Long-term spaceflights affect the structural changes in brain, alter motor or cognitive function and associated development of neuro-optic syndrome in astronauts. Studies addressing the impact of microgravity on brain cells are very limited. Herein, we employed microglial (CHME3) and glioblastoma (U87MG and A172) cells to study their molecular and functional adaptations under simulated microgravity (SMG) exposure. A reduction in cell viability and proliferation with decreased levels of PCNA were observed in these cells. SMG caused extensive DNA damage with an increase in γH2A.X (ser139) phosphorylation and differential activation/expression of DNA damage response (DDR) proteins including ATM, ATR, Chk1, Chk2 and p53 in all the three cell lines. Unlike CHME3, the ATM/Chk2-dependent DDR pathway was activated in glioblastoma cells suggesting a marked difference in the adaptation between normal and cancer cells to SMG. Five different classes of DNA repair pathways including BER, NER, MMR, NHEJ and HR were suppressed in both cell lines with the notable exception of NHEJ (Ku70/80 and DNA-PK) activation in U87MG cells. SMG induced mitochondrial apoptosis with increased expression of Bax, cleaved caspase-3 and cleaved poly-(ADP-ribose) polymerase, and reduced Bcl-2 level. SMG triggered apoptosis simultaneously via ERK1/2 and AKT activation, and inhibition of GSK3β activity which was reversed by MEK1 and PI3K inhibitors. Taken together, our study revealed that microgravity is a strong stressor to trigger DNA damage and apoptosis through activation of ERK1/2 and AKT, and impairment of DNA repair capacity, albeit with a cell-type difference in DDR and NHEJ regulation, in microglial and glioblastoma cells.