Regulation of the RB1 Gene through DNMT1 by SGI-1027 and its Impact on the Growth and Metastasis of Gastric Cancer Cells.

BACKGROUND: SGI-1027 is a recognized inhibitor of DNA methyltransferase 1 (DNMT1), and earlier investigations have indicated an inverse correlation between dysregulated DNMT1 expression in gastric cancer (GC) and retinoblastoma 1 (RB1) gene expression. Despite this knowledge, the precise mechanisms underlying the action of SGI-1027 in GC cells remain inadequately comprehended. The primary objective of this study is to elucidate the impact of SGI-1027 on the behavior of GC cells, encompassing aspects such as growth and metastatic potential, by intervening in DNMT1, thereby influencing the regulation of RB1 gene expression. METHOD: The acquisition of the normal gastric mucosal cell line GES-1 and the human gastric cancer cell line MKN45 was followed by employing Western blot (WB) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) techniques to evaluate the expression levels of RB1 and DNMT1 in these two cell lines. Subsequently, the MKN45 cell line was cultured in medium containing varying concentrations of SGI-1027, and the impact of SGI-1027 on the regulation of RB1 and DNMT1 in GC cells was reassessed using WB and qRT-PCR techniques. To scrutinize the effect of SGI-1027 on GC cells, we utilized the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay to determine cell proliferation and performed Transwell experiments to assess cell migration and invasion capabilities. Throughout this process, we also employed WB to assess the levels of cell cycle-associated proteins (Cyclin D1, Cyclin E1, and Cyclin B1) and proteins related to apoptosis (BCL-2 associated protein X apoptosis regulator (BAX) and B-cell lymphoma 2 apoptosis regulator (BCL-2)). Furthermore, we injected the MKN45 cell line and MKN45 cell line cultured with the optimal concentration of SGI-1027 for 5 days and 10 days into mice subcutaneously and through the tail vein, dividing them into the Model group, Model+SGI-1027 5d group, and Model+SGI-1027 10d group. We monitored changes in tumor size and volume in mice, and tumor tissues as well as lung tissues were collected for hematoxylin and eosin (HE) staining. Finally, DNMT1 expression levels in GC tissues were detected using both WB and immunohistochemistry (IHC) techniques. Additionally, RB1 expression levels in GC tissues were assessed using WB. RESULT: In contrast to GES-1 cells, MKN45 cells displayed a distinctive profile characterized by increased DNMT1 expression and decreased RB1 expression (p < 0.05). However, upon the introduction of SGI-1027, a notable decrease in DNMT1 levels within GC cells was observed, concomitant with an elevation in RB1 gene expression, with 25 µmol/L SGI-1027 identified as the optimal concentration (p < 0.05). Functional assays demonstrated that SGI-1027-treated GC cells exhibited pronounced features of inhibited proliferation, migration, and invasion when compared to untreated MKN45 cells (p < 0.05). Moreover, in SGI-1027-treated GC cells, the levels of Cyclin D1, Cyclin E1, Cyclin B1, and BCL-2 were significantly reduced, while the expression level of BAX increased (p < 0.05). Notably, the most pronounced impact was observed at 25 µmol/L SGI-1027, further underscoring its regulatory effects on tumor cell behavior (p < 0.05). In animal experiments, the Model group exhibited a substantial increase in tumor volume, with HE staining results indicating extensive necrosis in most gastric tissues and noticeable signs of lung metastasis, accompanied by increased DNMT1 expression and decreased RB1 gene expression. In contrast, the SGI-1027 group displayed a reduction in gastric tumor volume, decreased necrosis, and reduced lung tumor metastasis (p < 0.05). Additionally, the expression of DNMT1 was significantly reduced in SGI-1027-treated GC cells, while RB1 expression increased (p < 0.05), further confirming the inhibitory effects of SGI-1027 on tumor growth and metastasis. CONCLUSIONS: SGI-1027 effectively hinders the proliferation and dissemination of GC cells by downregulating DNMT1 and promoting the expression of RB1.

Molecular monitoring by CDKN2A/p16INK4A and RB1 gene methylation in breast cancer.

OBJECTIVE: This prospective study aimed to provide a comprehensive analysis of the methylation status of two pivotal genes, CDKN2A/p16INK4A (cyclin-dependent kinase inhibitor 2A) and RB1 (retinoblastoma transcriptional corepressor 1), in breast cancer patients. METHODS: Samples were obtained from 15 women diagnosed with breast cancer and who underwent a total mastectomy. DNA was extracted from the tumor, non-tumor tissue, and peripheral blood (circulating cell-free DNA). The methylation pattern of cell-free DNA extracted from blood collected on the day of mastectomy was compared with the methylation pattern of cell-free DNA from blood collected 1 year post-surgery. The methylation analysis was carried out by sodium bisulfite conversion and polymerase chain reaction, followed by electrophoresis. RESULTS: Methylation of CDKN2A/p16INK4A was identified in 13 tumor samples and 12 non-tumor tissue samples. Two patients exhibited CDKN2A/p16INK4A methylation in the cell-free DNA of the first blood collection, while another showed methylation only in the cell-free DNA of the subsequent blood collection. Regarding RB1, 11 tumors and 8 non-tumor tissue samples presented methylation of the gene. CONCLUSION: This study presents a novel approach for monitoring breast cancer patients through the analysis of cell-free DNA methylation. This analysis can detect changes in methylation patterns before any visible sign of cancer appears in breast tissue and could help predict the recurrence of malignant breast tumors.

Heterozygous RB1 mutation enhanced ATP production in human iPSC-derived retinal organoids.

BACKGROUND: Recent in vitro studies using RB1+/- fibroblasts and MSCs have shown molecular and functional disruptions without the need for biallelic loss of RB1. However, this was not reflected in the recent in vitro studies employing RB1+/- retinal organoids. To gain further insights into the molecular disruptions in the RB1+/- retinal organoids, we performed a high throughput RNA sequencing analysis. METHODS AND RESULTS: iPSCs were generated from RB1+/+ and RB1+/- OAMSCs derived from retinoblastoma patients. RB1+/+ and RB1+/- iPSCs were subjected to a step-wise retinal differentiation protocol. Retinal differentiation was evaluated by Real-time PCR and flow cytometry analysis of the retinal markers. To gain further insights into the molecular differences in RB1+/- retinal organoids, a high throughput RNA sequencing followed by differential gene expression analysis and gene set enrichment analysis (GSEA) was performed. The analysis revealed a shift from the regular metabolic process of glycolysis to oxidative phosphorylation in the RB1+/- retinal organoids. To investigate further, we performed assays to determine the levels of pyruvate, lactate and ATP in the retinal organoids. The results revealed significant increase in ATP and pyruvate levels in RB1+/- retinal organoids of day 120 compared to that of the RB1+/+. The results thus revealed enhanced ATP production in the RB1+/- retinal organoids. CONCLUSION: The study provides novel insights into the metabolic phenotype of heterozygous RB1 mutant suggesting dysregulation of energy metabolism and glycolytic pathways to be first step even before the changes in cellular proliferation or other phenotypic consequences ensue.

Establishment, characterization, and genetic profiling of patient-derived osteosarcoma cells from a patient with retinoblastoma.

Osteosarcoma is the most common malignant bone cancer in pediatric patients. Patients who respond poorly to chemotherapy experience worse clinical outcomes with a high mortality rate. The major challenge is the lack of effective drugs for these patients. To introduce new drugs for clinical approval, preclinical studies based on in vitro models must demonstrate the potency of the tested drugs, enabling the drugs to enter phase 1 clinical trials. Patient-derived cell culture is a promising testing platform for in vitro studies, as they more accurately recapitulate cancer states and genetic profiles compared to cell lines. In the present study, we established patient-derived osteosarcoma cells (PDC) from a patient who had previously been diagnosed with retinoblastoma. We identified a new variant of a germline mutation in the RB1 gene in the tissue of the patient. The biological effects of this PDC were studied to observe whether the cryopreserved PDC retained a feature of fresh PDC. The cryopreserved PDC preserved the key biological effects, including cell growth, invasive capability, migration, and mineralization, that define the conserved phenotypes compared to fresh PDC. From whole genome sequencing analysis of osteosarcoma tissue and patient-derived cells, we found that cryopreserved PDC was a minor population in the origin tissue and was selectively grown under the culture conditions. The cryopreserved PDC has a high resistance to conventional chemotherapy. This study demonstrated that the established cryopreserved PDC has the aggressive characteristics of osteosarcoma, in particular the chemoresistance phenotype that might be used for further investigation in the chemoresistant mechanism of osteosarcoma. In conclusion, the approach we applied for primary cell culture might be a promising method to generate in vitro models for functional testing of osteosarcoma.

Genomic profiling of pleomorphic rhabdomyosarcoma reveals a genomic signature distinct from that of embryonal rhabdomyosarcoma.

Pleomorphic rhabdomyosarcoma (PRMS) is a rare and highly aggressive sarcoma, occurring mostly in the deep soft tissues of middle-aged adults and showing a variable degree of skeletal muscle differentiation. The diagnosis is challenging as pathologic features overlap with embryonal rhabdomyosarcoma (ERMS), malignant Triton tumor, and other pleomorphic sarcomas. As recurrent genetic alterations underlying PRMS have not been described to date, ancillary molecular diagnostic testing is not useful in subclassification. Herein, we perform genomic profiling of a well-characterized cohort of 14 PRMS, compared to a control group of 23 ERMS and other pleomorphic sarcomas (undifferentiated pleomorphic sarcoma and pleomorphic liposarcoma) using clinically validated DNA-targeted Next generation sequencing (NGS) panels (MSK-IMPACT). The PRMS cohort included eight males and six females, with a median age of 53 years (range 31-76 years). Despite similar tumor mutation burdens, the genomic landscape of PRMS, with a high frequency of TP53 (79%) and RB1 (43%) alterations, stood in stark contrast to ERMS, with 4% and 0%, respectively. CDKN2A deletions were more common in PRMS (43%), compared to ERMS (13%). In contrast, ERMS harbored somatic driver mutations in the RAS pathway and loss of function mutations in BCOR, which were absent in PRMS. Copy number variations in PRMS showed multiple chromosomal arm-level changes, most commonly gains of chr17p and chr22q and loss of chr6q. Notably, gain of chr8, commonly seen in ERMS (61%) was conspicuously absent in PRMS. The genomic profiles of other pleomorphic sarcomas were overall analogous to PRMS, showing shared alterations in TP53, RB1, and CDKN2A. Overall survival and progression-free survival of PRMS were significantly worse (p < 0.0005) than that of ERMS. Our findings revealed that the molecular landscape of PRMS aligns with other adult pleomorphic sarcomas and is distinct from that of ERMS. Thus, NGS assays may be applied in select challenging cases toward a refined classification. Finally, our data corroborate the inclusion of PRMS in the therapeutic bracket of pleomorphic sarcomas, given that their clinical outcomes are comparable.

A synthetic lethal dependency on casein kinase 2 in response to replication-perturbing therapeutics in RB1-deficient cancer cells.

Resistance to therapy commonly develops in patients with high-grade serous ovarian carcinoma (HGSC) and triple-negative breast cancer (TNBC), urging the search for improved therapeutic combinations and their predictive biomarkers. Starting from a CRISPR knockout screen, we identified that loss of RB1 in TNBC or HGSC cells generates a synthetic lethal dependency on casein kinase 2 (CK2) for surviving the treatment with replication-perturbing therapeutics such as carboplatin, gemcitabine, or PARP inhibitors. CK2 inhibition in RB1-deficient cells resulted in the degradation of another RB family cell cycle regulator, p130, which led to S phase accumulation, micronuclei formation, and accelerated PARP inhibition-induced aneuploidy and mitotic cell death. CK2 inhibition was also effective in primary patient-derived cells. It selectively prevented the regrowth of RB1-deficient patient HGSC organoids after treatment with carboplatin or niraparib. As about 25% of HGSCs and 40% of TNBCs have lost RB1 expression, CK2 inhibition is a promising approach to overcome resistance to standard therapeutics in large strata of patients.

The Clinicopathological Significance of the Cyclin D1/E1-Cyclin-Dependent Kinase (CDK2/4/6)-Retinoblastoma (RB1/pRB1) Pathway in Epithelial Ovarian Cancers.

Cyclin-dependent kinases (CDK2, CDK4, CDK6), cyclin D1, cyclin E1 and phosphorylated retinoblastoma (pRB1) are key regulators of the G1/S cell cycle checkpoint and may influence platinum response in ovarian cancers. CDK2/4/6 inhibitors are emerging targets in ovarian cancer therapeutics. In the current study, we evaluated the prognostic and predictive significance of the CDK2/4/6-cyclin D1/E1-pRB1 axis in clinical ovarian cancers (OC). The CDK2/4/6, cyclin D1/E1 and RB1/pRB1 protein expression were investigated in 300 ovarian cancers and correlated with clinicopathological parameters and patient outcomes. CDK2/4/6, cyclin D1/E1 and RB1 mRNA expression were evaluated in the publicly available ovarian TCGA dataset. We observed nuclear and cytoplasmic staining for CDK2/4/6, cyclins D1/E1 and RB1/pRB1 in OCs with varying percentages. Increased nuclear CDK2 and nuclear cyclin E1 expression was linked with poor progression-free survival (PFS) and a shorter overall survival (OS). Nuclear CDK6 was associated with poor OS. The cytoplasmic expression of CDK4, cyclin D1 and cyclin E1 also has predictive and/or prognostic significance in OCs. In the multivariate analysis, nuclear cyclin E1 was an independent predictor of poor PFS. Tumours with high nuclear cyclin E1/high nuclear CDK2 have a worse PFS and OS. Detailed bioinformatics in the TCGA cohort showed a positive correlation between cyclin E1 and CDK2. We also showed that cyclin-E1-overexpressing tumours are enriched for genes involved in insulin signalling and release. Our data not only identified the prognostic/predictive significance of these key cell cycle regulators but also demonstrate the importance of sub-cellular localisation. CDK2 targeting in cyclin-E1-amplified OCs could be a rational approach.

The clinical value of optical genome mapping in the rapid characterization of RB1 duplication and 15q23q24.2 triplication, for more appropriate prenatal genetic counselling.

BACKGROUND: Despite recent advances in prenatal genetic diagnosis, medical geneticists still face considerable difficulty in interpreting the clinical outcome of copy-number-variant duplications and defining the mechanisms underlying the formation of certain chromosomal rearrangements. Optical genome mapping (OGM) is an emerging cytogenomic tool with proved ability to identify the full spectrum of cytogenetic aberrations. METHODS: Here, we report on the use of OGM in a prenatal diagnosis setting. Detailed breakpoint mapping was used to determine the relative orientations of triplicated and duplicated segments in two unrelated foetuses harbouring chromosomal aberrations: a de novo 15q23q24.2 triplication and a paternally inherited 13q14.2 duplication that overlapped partially with the RB1 gene. RESULTS: OGM enabled us to suggest a plausible mechanism for the triplication and confirmed that the RB1 duplication was direct oriented and in tandem. This enabled us to predict the pathogenic consequences, refine the prognosis and adapt the follow-up and familial screening appropriately. CONCLUSION: Along with an increase in diagnostic rates, OGM can rapidly highlight genotype-phenotype correlations, improve genetic counselling and significantly influence prenatal management.

Generation and characterization of three CRISPR/Cas9 edited RB1 null hiPSC lines for retinoblastoma disease modelling.

Complete loss of RB1 causes retinoblastoma. Here, we report the generation of three RB1-/- iPSC lines using CRISPR/Cas9 based editing at exon 18 of RB1 in a healthy control hiPSC line. The edited cells were clonally expanded, genotyped and characterized to establish the mutant lines. Two of the mutant lines are compound heterozygous, with different in-del mutations in each of their alleles, while the third mutant line is homozygous, with identical edits in both alleles. All lines maintained their stemness, pluripotency, formed embryoid bodies with cell types of all three lineages, displayed a normal karyotype and lost RB1 expression.

RB1 loss induces quiescent state through downregulation of RAS signaling in mammary epithelial cells.

While loss of function (LOF) of retinoblastoma 1 (RB1) tumor suppressor is known to drive initiation of small-cell lung cancer and retinoblastoma, RB1 mutation is rarely observed in breast cancers at their initiation. In this study, we investigated the impact on untransformed mammary epithelial cells given by RB1 LOF. Depletion of RB1 in anon-tumorigenic MCF10A cells induced reversible growth arrest (quiescence) featured by downregulation of multiple cyclins and MYC, upregulation of p27KIP1, and lack of expression of markers which indicate cellular senescence or epithelial-mesenchymal transition (EMT). We observed a similar phenomenon in human mammary epithelial cells (HMEC) as well. Additionally, we found that RB1 depletion attenuated the activity of RAS and the downstream MAPK pathway in an RBL2/p130-dependent manner. The expression of farnesyltransferase ß, which is essential for RAS maturation, was found to be downregulated following RB1 depletion also in an RBL2/p130-dependent manner. These findings unveiled an unexpected mechanism whereby normal mammary epithelial cells resist to tumor initiation upon RB1 LOF.

Impact of RB1 gene screening from blood collected on a single day from 411 family members of 113 Retinoblastoma survivors in India.

OBJECTIVES: To analyse the profile and implication of genetic testing in a cohort of retinoblastoma (RB) patients and their families conducted on a single day during World Retinoblastoma Awareness Week 2017. METHODS: Retrospective analysis of blood samples were collected from 411 subjects, including 113 probands at a camp organised for RB awareness and were analysed for RB1 mutations by Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). If germline mutations were detected, the parents and siblings of the proband were tested for the same mutation. RESULTS: Germline RB1 mutations were identified in 61/113(54%) probands with a mutation detection rate of 96% (47/49) and 22% (14/64) for bilateral and unilateral RB, respectively. Ten novel pathogenic mutations were identified. Splice mutation was most common (31%) followed by nonsense mutation (26%). The mean age at RB diagnosis was significantly lower in patients having germline RB1 mutation (mean 10.7 months ±2.5) compared to those without (mean 27.2 months ±6.5) (p = <0.0001). Parental transmission of the mutant allele was detected in 15/61(25%) cases of which 11(18%) parents were unaffected indicating incomplete penetrance. The origin of the variant allele was both paternal (n = 7) and maternal (n = 4) wherein 5 were bilateral and 6 unilateral. CONCLUSIONS: The detection of a germline mutation impacts the proband and family members due to its implications on change in prognosis, frequency of subsequent evaluations, screening for ocular and non-ocular cancers, and surveillance of family and future progeny.

Imaging predictors of 4q12 amplified and RB1 mutated glioblastoma IDH-wildtype.

INTRODUCTION: Recent studies have identified that glioblastoma IDH-wildtype consists of different molecular subgroups with distinct prognoses. In order to accurately describe and classify gliomas, the Visually AcceSAble Rembrandt Images (VASARI) system was developed. The goal of this study was to evaluate the VASARI characteristics in molecular subgroups of IDH-wildtype glioblastoma. METHODS: A retrospective analysis of glioblastoma IDH- wildtype with comprehensive next-generation sequencing and pre-operative and post-operative MRI was performed. VASARI characteristics and 205 genes were evaluated. Multiple comparison adjustment by the Bejamin-Hochberg false discovery rate (BH-FDR) was performed. A 1:3 propensity score match (PSM) with a Caliper of 0.2 was done. RESULTS: 178 patients with GBM IDH-WT met the inclusion criteria. 4q12 amplified patients (n = 20) were associated with cyst presence (30% vs. 12%, p = 0.042), decreased hemorrhage (35% vs. 62%, p = 0.028), and non-restricting/mixed (35%/60%) rather than restricting diffusion pattern (5%), meanwhile, 4q12 non-amplified patients had mostly restricting (47.4%) rather than a non-restricting/mixed diffusion pattern (28.4%/23.4%). This remained statistically significant after BH-FDR adjustment (p = 0.002). PSM by 4q12 amplification showed that diffusion characteristics continued to be significantly different. Among RB1-mutant patients, 96% had well-defined enhancing margins vs. 70.6% of RB1-WT (p = 0.018), however, this was not significant after BH-FDR or PSM. CONCLUSIONS: Patients with glioblastoma IDH-wildtype harboring 4q12 amplification rarely have restricting DWI patterns compared to their wildtype counterparts, in which this DWI pattern is present in ~ 50% of patients. This suggests that some phenotypic imaging characteristics can be identified among molecular subtypes of IDH-wildtype glioblastoma.

Generation of an induced pluripotent stem cell line (LVPEIi002-A) with heterozygous RB1 mutation using peri-orbital fat derived mesenchymal cells of a patient with inherited retinoblastoma.

Retinoblastoma is a pediatric intraocular cancer caused by biallelic inactivation of RB1 gene in retinal progenitor cells. Here, we report the generation of a patient-specific induced pluripotent stem cell (iPSC) line (LVPEIi002-A) from a patient diagnosed with retinoblastoma and showing familial inheritance of a nonsense mutation (c.1735C > T) within exon 18 of one of the two alleles. This RB1+/- iPSC line, LVPEIi002-A was generated by reprogramming the peri-orbital fat tissue derived mesenchymal cells and was stably expanded and characterized. It maintains the stemness, pluripotency, normal karyotype, and forms embryoid bodies comprising of all three lineage committed progenitor cells.

Impact of anthracycline-based chemotherapy on RB1 gene methylation in peripheral blood leukocytes and biomarkers of oxidative stress and inflammation in sarcoma patients.

PURPOSE: We investigated the impact of anthracycline-based chemotherapy on methylation status of RB1 gene in peripheral blood leukocytes together with parameters of oxidative stress and inflammation in sarcoma patients. PATIENTS/METHODS: Blood samples were collected from 51 consecutive newly diagnosed sarcoma patients admitted to University Hospital Center Zagreb (Zagreb, Croatia) for first-line chemotherapy before the first cycle and post-chemotherapy. Methylation and copy number variation (CNV) of leukocyte RB1 gene were assessed using MS-MLPA probes. In addition, in blood samples, parameters of oxidative stress (ROS, MDA, SOD, and GSH) and inflammation (CRP, WBC, and NBC) were followed. RESULTS: In pre-chemotherapy samples, no CNVs and aberrant methylation of CpG106 promoter region of RB1 gene were detected; however, one patient had hypermethylation (by approximately 10%) of imprinted locus CpG85 in intron 2 of RB1 gene. In addition, a very good correlation of the tumor burden and CRP and tumor burden and GSH was found. The anthracycline-based chemotherapy reverts methylation of RB1 gene-imprinted locus CpG85 to normal level. Moreover, inflammation and oxidative stress parameters such as CRP, WBC, ROS, and MDA were significantly decreased in post-chemotherapy samples. CONCLUSION: This single-centered study on a cohort of consecutive sarcoma patients indicates that sarcoma patients can have aberrant germline DNA methylation and confirms the relationship of tumor burden with inflammation and oxidative stress. The applied chemotherapy protocols reverted RB1 gene methylation to normal level and decreased the level of inflammation and oxidative damage, thus indicating chemotherapy benefit to the patient's health status.

Dual Inhibition of CDK4/6 and XPO1 Induces Senescence With Acquired Vulnerability to CRBN-Based PROTAC Drugs.

BACKGROUND & AIMS: Despite the increasing number of treatment options available for liver cancer, only a small proportion of patients achieve long-term clinical benefits. Here, we aim to develop new therapeutic approaches for liver cancer. METHODS: A compound screen was conducted to identify inhibitors that could synergistically induce senescence when combined with cyclin-dependent kinase (CDK) 4/6 inhibitor. The combination effects of CDK4/6 inhibitor and exportin 1 (XPO1) inhibitor on cellular senescence were investigated in a panel of human liver cancer cell lines and multiple liver cancer models. A senolytic drug screen was performed to identify drugs that selectively killed senescent liver cancer cells. RESULTS: The combination of CDK4/6 inhibitor and XPO1 inhibitor synergistically induces senescence of liver cancer cells in vitro and in vivo. The XPO1 inhibitor acts by causing accumulation of RB1 in the nucleus, leading to decreased E2F signaling and promoting senescence induction by the CDK4/6 inhibitor. Through a senolytic drug screen, cereblon (CRBN)-based proteolysis targeting chimera (PROTAC) ARV-825 was identified as an agent that can selectively kill senescent liver cancer cells. Up-regulation of CRBN was a vulnerability of senescent liver cancer cells, making them sensitive to CRBN-based PROTAC drugs. Mechanistically, we find that ubiquitin specific peptidase 2 (USP2) directly interacts with CRBN, leading to the deubiquitination and stabilization of CRBN in senescent liver cancer cells. CONCLUSIONS: Our study demonstrates a striking synergy in senescence induction of liver cancer cells through the combination of CDK4/6 inhibitor and XPO1 inhibitor. These findings also shed light on the molecular processes underlying the vulnerability of senescent liver cancer cells to CRBN-based PROTAC therapy.

Transformation to small cell lung cancer is irrespective of EGFR and accelerated by SMAD4-mediated ASCL1 transcription independently of RB1 in non-small cell lung cancer.

OBJECTIVES: Histological transformation to small cell lung cancer (SCLC) has been identified as a mechanism of TKIs resistance in EGFR-mutant non-small cell lung cancer (NSCLC). We aim to explore the prevalence of transformation in EGFR-wildtype NSCLC and the mechanism of SCLC transformation, which are rarely understood. METHODS: We reviewed 1474 NSCLC patients to investigate the NSCLC-to-SCLC transformed cases and the basic clinical characteristics, driver gene status and disease course of them. To explore the potential functional genes in SCLC transformation, we obtained pre- and post-transformation specimens and subjected them to a multigene NGS panel involving 416 cancer-related genes. To validate the putative gene function, we established knocked-out models by CRISPR-Cas 9 in HCC827 and A549-TP53-/- cells and investigated the effects on tumor growth, drug sensitivity and neuroendocrine phenotype in vitro and in vivo. We also detected the expression level of protein and mRNA to explore the molecular mechanism involved. RESULTS: We firstly reported an incidence rate of 9.73% (11/113) of SCLC transformation in EGFR-wildtype NSCLC and demonstrated that SCLC transformation is irrespective of EGFR mutation status (P = 0.16). We sequenced 8 paired tumors and identified a series of mutant genes specially in transformed SCLC such as SMAD4, RICTOR and RET. We firstly demonstrated that SMAD4 deficiency can accelerate SCLC transition by inducing neuroendocrine phenotype regardless of RB1 status in TP53-deficient NSCLC cells. Further mechanical experiments identified the SMAD4 can regulate ASCL1 transcription competitively with Myc in NSCLC cells and Myc inhibitor acts as a potential subsequent treatment agent. CONCLUSIONS: Transformation to SCLC is irrespective of EFGR status and can be accelerated by SMAD4 in non-small cell lung cancer. Myc inhibitor acts as a potential therapeutic drug for SMAD4-mediated resistant lung cancer. Video Abstract.

miR-4529-3p Promotes the Progression of Retinoblastoma by Inhibiting RB1 Expression and Activating the ERK Signaling Pathway.

Retinoblastoma (RB) is a malignant ocular cancer that affects children. Several microRNAs (miRNAs) have been implicated in RB regulation. The present study aimed to investigate the role of miR-4529-3p in RB pathogenesis. Scratch, Transwell, and Cell Counting Kit (CCK)-8 assays were conducted to assess the migratory, invasive, and proliferative abilities of RB cells. The expression levels of miR-4529-3p, RB1, and ERK pathway-related proteins were analyzed using western blotting and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Target relationships were verified using dual-luciferase reporter experiments. A murine RB model was developed to analyze the effects of miR-4529-3p on RB tumor growth in vivo. Our experiments revealed high levels of miR-4529-3p and low levels of RB1 in RB tissues. Functional analyses revealed that the migratory, invasive, and proliferative abilities of RB cells were repressed by miR-4529-3p inhibition. Similarly, p-ERK 1/2 protein levels were suppressed by miR-4529-3p inhibition. Furthermore, downregulation of miR-4529-3p limited tumor growth in vivo. Mechanistically, miR-4259-3p targets RB1. Interestingly, RB1 silencing abrogated the alleviative effects of miR-4529-3p downregulation in RB cells. MiR-4529-3p promotes RB progression by inhibiting RB1 and activating the ERK pathway. This evidence suggests that the miR-4529-3p/RB1 regulatory axis may be a prospective target for RB treatment in clinical settings.

Inhibition of high level E2F in a RB1 proficient MYCN overexpressing chicken retinoblastoma model normalizes neoplastic behaviour.

PURPOSE: Retinoblastoma, a childhood cancer, is most frequently caused by bi-allelic inactivation of RB1 gene. However, other oncogenic mutations such as MYCN amplification can induce retinoblastoma with proficient RB1. Previously, we established RB1-proficient MYCN-overexpressing retinoblastoma models both in human organoids and chicken. Here, we investigate the regulatory events in MYCN-induced retinoblastoma carcinogenesis based on the model in chicken. METHODS: MYCN transformed retinal cells in culture were obtained from in vivo MYCN electroporated chicken embryo retina. The expression profiles were analysed by RNA sequencing. Chemical treatments, qRT-PCR, flow cytometry, immunohisto- and immunocytochemistry and western blot were applied to study the properties and function of these cells. RESULTS: The expression profile of MYCN-transformed retinal cells in culture showed cone photoreceptor progenitor signature and robustly increased levels of E2Fs. This expression profile was consistently observed in long-term culture. Chemical treatments confirmed RB1 proficiency in these cells. The cells were insensitive to p53 activation but inhibition of E2f efficiently induced cell cycle arrest followed by apoptosis. CONCLUSION: In conclusion, with proficient RB1, MYCN-induced high level of E2F expression dysregulates the cell cycle and contributes to retinoblastoma carcinogenesis. The increased level of E2f renders the cells to adopt a similar mechanistic phenotype to a RB1-deficient tumour.

Investigation of the methylation changes in the promoter region of RB1 gene in retinoblastoma: Unraveling the epigenetic puzzle in retinoblastoma.

Retinoblastoma is an infrequent neoplasm that arises during childhood from retinal nerve cells and is attributed to the biallelic inactivation of the RB1 gene. In conjunction with anatomical anomalies, it is widely acknowledged that epigenetic modifications play a significant role in the pathogenesis of cancer. The association between methylation of the RB1 gene promoter and tumor formation has been established; however, there is currently no scholarly evidence to substantiate the claim that it is responsible for the inheritance of retinoblastoma. The initial hypothesis posited for this work was that familial retinoblastoma disease would be similarly observed in cases with RB1 promotor gene methylation, akin to RB1 mutations. The RB1 gene promoter region was subjected to methylation screening using real-time PCR in individuals diagnosed with familial retinoblastoma but lacking RB1 mutations. The study involved a comparison of the germline methylation status of the RB1 gene in the peripheral blood samples of 50 retinoblastoma patients and 52 healthy individuals. The healthy individuals were carefully selected to match the retinoblastoma patients in terms of age, sex, and ethnicity. The data obtained from both groups were subjected to statistical analysis. The study revealed that the methylation level in a cohort of 50 individuals diagnosed with retinoblastoma and 52 healthy control participants was determined to be 36.1% and 33.9%, respectively. As a result, there was no statistically significant disparity observed in RB1 promoter methylation between the patient and control groups (p = 0.126). The methylation of the promoter region of the RB1 gene in familial retinoblastoma does not exert any influence on the hereditary transmission of the disease.