Cellular Proliferative Capacity Research Articles

CDK14 is regulated by IGF2BP2 and involved in osteogenic differentiation via Wnt/β-catenin signaling pathway in vitro

AimsCyclin-dependent kinase (CDK) family proteins involve in various cellular processes via regulating the cell cycle; however, their expression during osteogenic differentiation and postmenopausal osteoporosis remains poorly understood. Main methodsUsing bioinformatics, we screened for CDK14 bound to Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and explored its expression in vitro with time-gradient model and in a mouse model of postmenopausal osteoporosis, building on prior research. Subsequently, we investigated its effect on osteoblast proliferation, cell cycle dynamics, and osteogenic differentiation by administering CDK14 siRNA and the covalent inhibitor FMF-04-159-2. Furthermore, we examined the interaction between IGF2BP2 and CDK14. Finally, we validated the regulatory role of CDK14 on the Wnt/β-catenin pathway. Key findingsOur findings demonstrate a time-dependent CDK14 expression patterns during osteogenic differentiation of MC3T3-E1 cell line, with an initial increase followed by gradual decline over time. Notably, CDK14 expression exhibited significant reduction in bone tissue of postmenopausal osteoporosis mouse model. CDK14 inhibition altered osteoblast cell cycle dynamics, significantly reduced cellular proliferation capacity, and impaired osteogenic differentiation ability. IGF2BP2 interacted with CDK14 mRNA, and stabilizing mRNA's structure and inhibiting its degradation. Additionally, CDK14 facilitated Low-density lipoprotein receptor-related protein 6 (LRP6) and Glycogen synthase kinase 3β (GSK3β) phosphorylation, thus regulating β-catenin levels. SignificanceThese findings provide further insight into the molecular mechanisms governing osteoblast proliferation, differentiation and osteoporosis.

Integrated ATAC-seq and RNA-seq Analysis of In Vitro Cultured Skeletal Muscle Satellite Cells to Understand Changes in Cell Proliferation.

Skeletal muscle satellite cells, the resident stem cells in pig skeletal muscle, undergo proliferation and differentiation to enable muscle tissue repair. The proliferative and differentiative abilities of these cells gradually decrease during in vitro cultivation as the cell passage number increases. Despite extensive research, the precise molecular mechanisms that regulate this process are not fully understood. To bridge this knowledge gap, we conducted transcriptomic analysis of skeletal muscle satellite cells during in vitro cultivation to quantify passage number-dependent changes in the expression of genes associated with proliferation. Additionally, we explored the relationships between gene transcriptional activity and chromatin accessibility using transposase-accessible chromatin sequencing. This revealed the closure of numerous open chromatin regions, which were primarily located in intergenic regions, as the cell passage number increased. Integrated analysis of the transcriptomic and epigenomic data demonstrated a weak correlation between gene transcriptional activity and chromatin openness in expressed genic regions; although some genes (e.g., GNB4 and FGD5) showed consistent relationships between gene expression and chromatin openness, a substantial number of differentially expressed genes had no clear association with chromatin openness in expressed genic regions. The p53-p21-RB signaling pathway may play a critical regulatory role in cell proliferation processes. The combined transcriptomic and epigenomic approach taken here provided key insights into changes in gene expression and chromatin openness during in vitro cultivation of skeletal muscle satellite cells. These findings enhance our understanding of the intricate mechanisms underlying the decline in cellular proliferation capacity in cultured cells.

UBAP2L ensures homeostasis of nuclear pore complexes at the intact nuclear envelope.

Assembly of macromolecular complexes at correct cellular sites is crucial for cell function. Nuclear pore complexes (NPCs) are large cylindrical assemblies with eightfold rotational symmetry, built through hierarchical binding of nucleoporins (Nups) forming distinct subcomplexes. Here, we uncover a role of ubiquitin-associated protein 2-like (UBAP2L) in the assembly and stability of properly organized and functional NPCs at the intact nuclear envelope (NE) in human cells. UBAP2L localizes to the nuclear pores and facilitates the formation of the Y-complex, an essential scaffold component of the NPC, and its localization to the NE. UBAP2L promotes the interaction of the Y-complex with POM121 and Nup153, the critical upstream factors in a well-defined sequential order of Nups assembly onto NE during interphase. Timely localization of the cytoplasmic Nup transport factor fragile X-related protein 1 (FXR1) to the NE and its interaction with the Y-complex are likewise dependent on UBAP2L. Thus, this NPC biogenesis mechanism integrates the cytoplasmic and the nuclear NPC assembly signals and ensures efficient nuclear transport, adaptation to nutrient stress, and cellular proliferative capacity, highlighting the importance of NPC homeostasis at the intact NE.

Anti-Tumor Efficacy of Oleuropein-Loaded ZnO/Au Mesoporous Silica Nanoparticle in 5-FU-Resistant Colorectal Cancer Cells.

5-fluorouracil (5-FU) is a first-line chemotherapeutic agent used to treat colorectal cancer (CRC). However, 5-FU induces drug resistance and activation of cancer stem cells (CSCs). In the present study, we designed a novel biocompatible nanomedicine system with high efficacy and biocompatibility by synthesizing mesoporous silica nanoparticle (MSN)-structured ZnO and gold ions. Oleuropein (OLP) is a phenolic compound derived from olive leaves that exerts anti-cancer effects. Therefore, we synthesized OLP-loaded ZnO/Au MSNs (ZnO/Au/OLP MSNs) and examined their anti-cancer effects on 5-FU-resistant CRC cells. ZnO/Au MSNs were synthesized and functionalized, and their physical and chemical compositions were characterized using UV-visible spectroscopy, dynamic light scattering, and transmission electron microscopy (TEM). Their effects were assessed in terms of cellular proliferation capacity, migration and invasion ability, colony-forming ability, spheroid-forming ability, reactive oxygen species (ROS) production, and mitochondrial membrane depolarization. ZnO/Au MSNs were mostly composed of various ions containing ZnO and gold ions, had a spheroid phenotype, and exhibited no cytotoxicity. ZnO/Au/OLP MSNs reduced cell viability and CSC formation and induced apoptosis of 5-FU-resistant CRC cells via necrosis via ROS accumulation and DNA fragmentation. ZnO/Au/OLP MSNs exhibited anti-cancer activity by upregulating necrosis. These results revealed that ZnO/Au/OLP MSNs are a novel drug delivery system for 5-FU CRC therapy.

Transcription factor E2F7 activates PKMYT1 to partially suppress adriamycin sensitivity in gastric cancer through the MAPK signaling pathway.

Adriamycin resistance remains an obstacle to gastric cancer chemotherapy treatment. Objective: The objective of this study was to study the role and mechanism of transcription factor E2F7 in sensitivity to ADM chemotherapeutic agents in gastric cancer. Cell viability and cell sensitivity were assessed by CCK-8 and IC50 values of ADM were calculated. The impact of ADM on cellular proliferative capacity was assessed through colony formation assay. The binding relationship between E2F7 and PKMYT1 was then verified by dual luciferase assay and chromatin immunoprecipitation assay. ERK1/ERK2 and p-ERK1/p-ERK2 protein expression levels were detected by western blot. In both gastric cancer tissue and ADM-resistant cells, a conspicuous upregulation of E2F7 and PKMYT1 was observed. Upregulated PKMYT1 was notably enriched in the MAPK signaling pathway. Enhanced levels of E2F7 were shown to not only drive gastric cancer cell proliferation but also engender a reduction in the sensitivity of these cells to ADM. Furthermore, PKMYT1 emerged as a downstream target of E2F7. Activation of E2F7 culminated in the transcriptional upregulation of PKMYT1, and silencing E2F7 reversed the inhibitory impact of PKMYT1 overexpression on ADM sensitivity in gastric cancer cells. E2F7/PKMYT1 axis might promote the proliferation and partially inhibit ADM sensitivity of gastric cancer cells by activating the MAPK pathway.

HIV Expression in Infected T Cell Clones.

The principal barrier to an HIV-1 cure is the persistence of infected cells harboring replication-competent proviruses despite antiretroviral therapy (ART). HIV-1 transcriptional suppression, referred to as viral latency, is foremost among persistence determinants, as it allows infected cells to evade the cytopathic effects of virion production and killing by cytotoxic T lymphocytes (CTL) and other immune factors. HIV-1 persistence is also governed by cellular proliferation, an innate and essential capacity of CD4+ T cells that both sustains cell populations over time and enables a robust directed response to immunological threats. However, when HIV-1 infects CD4+ T cells, this capacity for proliferation can enable surreptitious HIV-1 propagation without the deleterious effects of viral gene expression in latently infected cells. Over time on ART, the HIV-1 reservoir is shaped by both persistence determinants, with selective forces most often favoring clonally expanded infected cell populations harboring transcriptionally quiescent proviruses. Moreover, if HIV latency is incomplete or sporadically reversed in clonal infected cell populations that are replenished faster than they are depleted, such populations could both persist indefinitely and contribute to low-level persistent viremia during ART and viremic rebound if treatment is withdrawn. In this review, select genetic, epigenetic, cellular, and immunological determinants of viral transcriptional suppression and clonal expansion of HIV-1 reservoir T cells, interdependencies among these determinants, and implications for HIV-1 persistence will be presented and discussed.

Pten knockout in mouse preosteoblasts leads to changes in bone turnover and strength.

Bone development and remodeling are controlled by the phosphoinositide-3-kinase (Pi3k) signaling pathway. We investigated the effects of downregulation of phosphatase and tensin homolog (Pten), a negative regulator of Pi3k signaling, in a mouse model of Pten deficiency in preosteoblasts. We aimed to identify mechanisms that are involved in the regulation of bone turnover and are linked to bone disorders. Femora, tibiae, and bone marrow stromal cells (BMSCs) isolated from mice with a conditional deletion of Pten (Pten cKO) in Osterix/Sp7-expressing osteoprogenitor cells were compared to Cre-negative controls. Bone phenotyping was performed by μCT measurements, bone histomorphometry, quantification of bone turnover markers CTX and procollagen type 1N propeptide (P1NP), and three-point bending test. Proliferation of BMSCs was measured by counting nuclei and Ki-67-stained cells. In vitro, osteogenic differentiation capacity was determined by ALP staining, as well as by detecting gene expression of osteogenic markers. BMSCs from Pten cKO mice were functionally different from control BMSCs. Osteogenic markers were increased in BMSCs derived from Pten cKO mice, while Pten protein expression was lower and Akt phosphorylation was increased. We detected a higher trabecular bone volume and an altered cortical bone morphology in Pten cKO bones with a progressive decrease in bone and tissue mineral density. Pten cKO bones displayed fewer osteoclasts and more osteoblasts (P = .00095) per trabecular bone surface and a higher trabecular bone formation rate. Biomechanical analysis revealed a significantly higher bone strength (P = .00012 for males) and elasticity of Pten cKO femora. On the cellular level, both proliferation and osteogenic differentiation capacity of Pten cKO BMSCs were significantly increased compared to controls. Our findings suggest that Pten knockout in osteoprogenitor cells increases bone stability and elasticity by increasing trabecular bone mass and leads to increased proliferation and osteogenic differentiation of BMSCs.

Hippo/YAP1 promotes osteoporotic mice bone defect repair via the activating of Wnt signaling pathway

BackgroundThis study is to investigate the role and mechanism of Hippo/YAP1 in the repair of osteoporotic bone defects in aged mice, both in vivo and in vitro. MethodsWe investigated the expression differences of the Hippo signaling in young and aged individuals both in vivo and in vitro. By manipulating the expression of Lats1/2 and Yap1, we investigated the role of Hippo/YAP1 in regulating osteogenic differentiation in aged BMSCs. In vivo, by intervening in the local and systemic expression of Lats1/2 and Yap1 respectively, we sought to demonstrate whether Hippo/YAP1 promotes the repair of bone defects in aged osteoporotic conditions. Finally, we delved into the underlying mechanisms of Hippo/YAP1 in regulating osteogenic differentiation. ResultsWe observed differences in the expression of the Hippo signaling between young and aged individuals. After knocking out Lats1/2 in aged BMSCs, we observed that the upregulation of endogenous YAP1 promotes cellular osteogenic differentiation and proliferation capacity. Through interference with Yap1 expression, we provided strong evidence for the role of Hippo/YAP1 in promoting osteogenic differentiation in aged BMSCs. In vivo, we confirmed that Hippo/YAP1 promotes the repair of bone defects in aging osteoporosis. Moreover, we discovered an interaction relationship among YAP1, β-catenin, and TEAD1. ConclusionThis study elucidates the role of Hippo/YAP1 in promoting the repair of osteoporotic bone defects in aged mice. Mechanistically, YAP1 functions by activating the Wnt/β-catenin pathway, and this process is not independent of TEAD1.

Impact of heterogeneous nuclear ribonucleoprotein A/B subtype overexpression on the expression of cancer stem cell markers CD133 and CD44 and cellular proliferation capacity.

The occurrence and progression of intestinal cancer are complex, multifactorial processes in which tumor stem cells are believed to play a crucial role. An in-depth understanding of their molecular mechanisms holds imperative clinical significance for improving intestinal cancer treatment. HT-29 and HCT116intestinal cancer cell lines were utilized as research models. The experimental group (group E) and control group (group C) were established by transfecting the hnRNPA1 subtype and empty vector, respectively. The expression (EP) levels of hnRNPA1 and changes in Wnt/β-catenin signaling-related markers were evaluated using techniques such as RNA extraction, reverse transcription reactions, real-time quantitative PCR (RT qPCR), and protein extraction. The EP of tumor stem cell markers CD133 and CD44 was assessed using immunohistochemistry. Additionally, cell invasion assays, scratch assays, and cell counting kit-8 (CCK-8) proliferation assays were conducted. Furthermore, a mouse tumor model was established to observe the growth of tumors in both groups. overexpression (OP) of hnRNPA1 in group E greatly increased the protein EP levels of β-catenin, Axin2, and Cyclin D1 and exhibited a higher positivity rate for CD133 and CD44. The invasive, migratory, and proliferative abilities of cells in group E were notably enhanced, and tumor growth was observably faster versus group C. OP of hnRNPA1 was closely associated with the activation of Wnt/β-catenin signaling and promoted the proliferation and invasive capacity of tumor cells. hnRNPA1 played an imperative role in intestinal cancer stem cells.

Silver Nanoparticles (AgNPs) as Enhancers of Everolimus and Radiotherapy Sensitivity on Clear Cell Renal Cell Carcinoma.

Nanomedicine's advent has promised to revolutionize different biomedical fields, including oncology. Silver Nanoparticles (AgNPs) showed promising results in different tumor models. Clear cell Renal Cell Carcinoma (ccRCC) is especially challenging due to its late diagnosis, poor prognosis and treatment resistance. Therefore, defining new therapeutic targets and regimens could improve patient management. This study intends to evaluate AgNPs' effect in ccRCC cells and explore their potential combinatory effect with Everolimus and Radiotherapy. AgNPs were synthesized, and their effect was evaluated regarding their entering pathway, cellular proliferation capacity, ROS production, mitochondrial membrane depolarization, cell cycle analysis and apoptosis assessment. AgNPs were combined with Everolimus or used to sensitize cells to radiotherapy. AgNPs are cytotoxic to 786-O cells, a ccRCC cell line, entering through endocytosis, increasing ROS, depolarizing mitochondrial membrane, and blocking the cell cycle, leading to a reduction of proliferation capacity and apoptosis. Combined with Everolimus, AgNPs reduce cell viability and inhibit proliferation capacity. Moreover, 786-O is intrinsically resistant to radiation, but after AgNPs' administration, radiation induces cytotoxicity through mitochondrial membrane depolarization and S phase blockage. These results demonstrate AgNPs' cytotoxic potential against ccRCC and seem promising regarding the combination with Everolimus and sensitization to radiotherapy, which can, in the future, benefit ccRCC patients' management.

MiR-383-5p serves as a tumor suppressor in bladder cancer by suppressing PI3K/AKT signaling pathway.

MicroRNAs have been proven to be key molecules in human malignancy. However, to our knowledge, there is no study reporting miR-383-5p expression level and the role it plays in bladder cancer (BC). We identified miR-383-5p to be one of the tumor-suppressing genes through using data from The Cancer Genome Atlas (TCGA) and GEO database. We evaluate the expression and activity of miR-383-5p in both BC tissue and cell lines. The impacts of miR-383-5p on proliferative, migratory ability and apoptotic rate in BC cell were evaluated by utilizing CCK-8 kits, flow cytometry, and Transwell assays. qRT-PCR, western blot, and luciferase reporter assays have been adopted to investigate the underlying mechanisms. In vivo tumorigenicity testing was conducted to determine the impact of miR-383-5p on BC cellular proliferative capacity. Reduced miR-383-5p expression has been determined in BC tissue than in normal bladder tissue. Furthermore, BC cell proliferative, migratory ability was inhibited while apoptosis enhanced in vitro and in vivo by miR-383-5p up-regulation. In vitro and in vivo, silencing miR-383-5p considerably improved the growth and invasive capacity of cell, while decreased the apoptotic rates of BC cells. miR-383-5p plays its role as a tumor-suppressing gene by suppressing the PI3K/AKT signaling, hence preventing the development of BC.

Shelter in place: Live CLL cells inside the bone marrow fibroblasts and its implication in drug resistance

Introduction: Cell-in-cell (CIC) describes a century old observation where an intact cell can be seen inside another whole cell. It has been associated with human cancers, especially hematopoietic malignancies. However, most observations have been published as case reports. In the absence of systematic studies of case cohorts, understanding of the pathological significance of live CIC remains challenging. Methods: In this investigation, we studied primary CLL samples using an ex vivo co-culture model system which mimics the CLL tumor microenvironment (TME). In these TME models, CLL cells were cocultured with bone marrow fibroblasts (BMF) in the presence of B-cell growth factors. The tumor cells survive for weeks, become larger, downregulate surface CXCR4 and upregulate Ki67 expression. Phenotypically, tumor cells proliferate and divide into daughter cells. Collectively, these features recapitulate the behavior of lymph node-resident CLL cells. In this study, cells were observed and analyzed with confocal microscopy and 3D reconstruction. Results: We observed that CLL cells were actively internalized by BMF in 41 CLL cases studied. The internalized CLL cells were alive and mobile within the BMF’s cytoplasm. Moreover, the tumor cells were seen leaving their BMF host and re-entering into other BMFs. The number of internalized CLL cells were quantified and correlated with patients’ clinical and pathological parameters. No correlations were found between cell internalization and widely used CLL prognostic indicators, including cytogenetic features and IGHV mutational status. However, we have found that treated patients had higher level of CIC than those not treated. We hypothesized that live CIC may contribute to drug resistance and internalization may be an important mechanism for tumor cells to evade harmful drug insult and survive as residual disease. To test the hypothesis, we exposed CLL cells in the TME models to BTK inhibitors including ibrutinib and pirtobrutinib. Our results showed that the drug exposure drove CLL cells into BMF. Additionally, we observed that increased CIC was associated with higher cellular proliferation capacity. Importantly, chemokine CXCL12-CXCR4 axis played a critical role in this process. Specifically, CXCL12 ligand promoted cell internalization, while a CXCR4 antagonist partially prevented CIC from occurring. The research was funded by: NCI Keywords: Chronic Lymphocytic Leukemia (CLL), Microenvironment, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.

Impairments in SHMT2 expression or cellular folate availability reduce oxidative phosphorylation and pyruvate kinase activity

BackgroundSerine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine-producing THF-conjugated one-carbon units and glycine in the mitochondria. Biallelic SHMT2 variants were identified in humans and suggested to alter the protein’s active site, potentially disrupting enzymatic function. SHMT2 expression has also been shown to decrease with aging in human fibroblasts. Immortalized cell models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels, suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. This study examined the role of SHMT2 and folate availability in regulating mitochondrial function, energy metabolism, and cellular proliferative capacity in both heterozygous and homozygous cell models of reduced SHMT2 expression. In this study, primary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6J dam crossed with a heterozygous Shmt2+/− male to generate Shmt2+/+ (wild-type) or Shmt2+/− (HET) MEF cells. In addition, haploid chronic myeloid leukemia cells (HAP1, wild-type) or HAP1 cells lacking SHMT2 expression (ΔSHMT2) were cultured for 4 doublings in either low-folate or folate-sufficient culture media. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, mitochondrial membrane potential, and mitochondrial function.ResultsHomozygous loss of SHMT2 in HAP1 cells impaired cellular folate accumulation and altered mitochondrial DNA content, formate production, membrane potential, and basal respiration. Formate rescued proliferation in HAP1, but not ΔSHMT2, cells cultured in low-folate medium. Pyruvate kinase activity and protein levels were impaired in ΔSHMT2 cells and in MEF cells exposed to low-folate medium. Mitochondrial biogenesis protein levels were elevated in Shmt2+/− MEF cells, while mitochondrial mass was increased in both homozygous and heterozygous models of SHMT2 loss.ConclusionsThe results from this study indicate disrupted mitochondrial FOCM impairs mitochondrial folate accumulation and respiration, mitochondrial formate production, glycolytic activity, and cellular proliferation. These changes persist even after a potentially compensatory increase in mitochondrial biogenesis as a result of decreased SHMT2 levels.

IRAK2 Downregulation in Triple-Negative Breast Cancer Cells Decreases Cellular Growth In Vitro and Delays Tumour Progression in Murine Models

Breast cancer stem cells (BCSCs) are responsible for tumour recurrence and therapy resistance. We have established primary BCSC cultures from human tumours of triple-negative breast cancer (TNBC), a subgroup of breast cancer likely driven by BCSCs. Primary BCSCs produce xenografts that phenocopy the tumours of origin, making them an ideal model for studying breast cancer treatment options. In the TNBC cell line MDA-MB-468, we previously screened kinases whose depletion elicited a differentiation response, among which IRAK2 was identified. Because primary BCSCs are enriched in IRAK2, we wondered whether IRAK2 downregulation might affect cellular growth. IRAK2 was downregulated in primary BCSCs and MDA-MB-468 by lentiviral delivery of shRNA, causing a decrease in cellular proliferation and sphere-forming capacity. When orthotopically transplanted into immunocompromised mice, IRAK2 knockdown cells produced smaller xenografts than control cells. At the molecular level, IRAK2 downregulation reduced NF-κB and ERK phosphorylation, IL-6 and cyclin D1 expression, ERN1 signalling and autophagy in a cell line-dependent way. Overall, IRAK2 downregulation decreased cellular aggressive growth and pathways often exploited by cancer cells to endure stress; therefore, IRAK2 may be considered an interesting target to compromise TNBC progression.

Generation of Functional Immortalized Human Corneal Stromal Stem Cells.

In addition to their therapeutic potential in regenerative medicine, human corneal stromal stem cells (CSSCs) could serve as a powerful tool for drug discovery and development. Variations from different donors, their isolation method, and their limited life span in culture hinder the utility of primary human CSSCs. To address these limitations, this study aims to establish and characterize immortalized CSSC lines (imCSSC) generated from primary human CSSCs. Primary CSSCs (pCSSC), isolated from human adult corneoscleral tissue, were transduced with ectopic expression of hTERT, c-MYC, or the large T antigen of the Simian virus 40 (SV40T) to generate imCSSC. Cellular morphology, proliferation capacity, and expression of CSSCs specific surface markers were investigated in all cell lines, including TNFAIP6 gene expression levels in vitro, a known biomarker of in vivo anti-inflammatory efficacy. SV40T-overexpressing imCSSC successfully extended the lifespan of pCSSC while retaining a similar morphology, proliferative capacity, multilineage differentiation potential, and anti-inflammatory properties. The current study serves as a proof-of-concept that immortalization of CSSCs could enable a large-scale source of CSSC for use in regenerative medicine.

Nuclear stabilization of p53 requires a functional nucleolar surveillance pathway.

The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood. Using genome-wide loss-of-function screens, we demonstrate the ribosome biogenesis axis as the most potent class of genes whose disruption stabilizes p53. Mechanistically, we identify genes critical for regulation of this pathway, including HEATR3. By selectively disabling the nucleolar surveillance pathway, we demonstrate that it is essential for the ability of all nuclear-acting stresses, including DNA damage, to induce p53 accumulation. Our data support a paradigm whereby the nucleolar surveillance pathway is the central integrator of stresses that regulate nuclear p53 abundance, ensuring that ribosome biogenesis is hardwired to cellular proliferative capacity.

Glucose-Functionalized Silver Nanoparticles as a Potential New Therapy Agent Targeting Hormone-Resistant Prostate Cancer cells.

PurposeSilver nanoparticles (AgNPs) have shown great potential as anticancer agents, namely in therapies’ resistant forms of cancer. The progression of prostate cancer (PCa) to resistant forms of the disease (castration-resistant PCa, CRPC) is associated with poor prognosis and life quality, with current limited therapeutic options. CRPC is characterized by a high glucose consumption, which poses as an opportunity to direct AgNPs to these cancer cells. Thus, this study explores the effect of glucose functionalization of AgNPs in PCa and CRPC cell lines (LNCaP, Du-145 and PC-3).MethodsAgNPs were synthesized, further functionalized, and their physical and chemical composition was characterized both in water and in culture medium, through UV-visible spectrum, dynamic light scattering (DLS), transmission electron microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR). Their effect was assessed in the cell lines regarding AgNPs’ entering pathway, cellular proliferation capacity, ROS production, mitochondrial membrane depolarization, cell cycle analysis and apoptosis evaluation.ResultsAgNPs displayed an average size of 61nm and moderate monodispersity with a slight increase after functionalization, and a round shape. These characteristics remained stable when redispersed in culture medium. Both AgNPs and G-AgNPs were cytotoxic only to CRPC cells and not to hormone-sensitive ones and their effect was higher after functionalization showing the potential of glucose to favor AgNPs’ uptake by cancer cells. Entering through endocytosis and being encapsulated in lysosomes, the NPs increased the ROS, inducing mitochondrial damage, and arresting cell cycle in S Phase, therefore blocking proliferation, and inducing apoptosis.ConclusionThe nanoparticles synthesized in the present study revealed good characteristics and stability for administration to cancer cells. Their uptake through endocytosis leads to promising cytotoxic effects towards CRPC cells, revealing the potential of G-AgNPs as a future therapeutic approach to improve the management of patients with PCa resistant to hormone therapy or metastatic disease.

Meis1 plays roles in cortical development through regulation of cellular proliferative capacity in the embryonic cerebrum.

Meis1 (myeloid ecotropic insertion site 1) is known to be related to embryonic development and cancer. In this study, to analyze the function of Meis1 in neural stem cells, we crossed Meis1fl/fl (Meis1 floxed) mice with Nestin-Cre mice. The results showed that Meis1-conditional knockout mice showed cerebral cortex malformation. The mice had a significantly thinner cortex than wildtype mice. At E14.5, BrdU incorporation and Pax6-positive radial glial cells were significantly decreased in the cerebral cortex of Meis1 knockout embryos as compared with wild-type embryos, whereas Tbr2-positive intermediate progenitors and NeuN-positive differentiated neurons were not. Cell death detected by immunostaining with cleaved caspase3 antibody showed no difference in the cortex between knockout and wild-type embryos. Furthermore, knockout of Meis1 in embryo by in utero electroporation showed that cellular migration was disturbed during cortical development. Therefore, Meis1 could play important roles during cortical development through the regulation of cell proliferation and migration in the embryonic cerebral cortex.

Abstract 2177: Myeloid-derived arginase depletes microenvironmental arginine in PDAC tumors and leads to activation of arginine de novo biosynthesis in cancer cells

Abstract Arginine is an amino acid critical for various cellular processes, not only protein synthesis but also metabolism of other essential metabolites, like polyamines, as well as a signaling factor for pathways such as the growth regulator mTOR. Previously, our group measured arginine levels in the interstitial fluid of tumors (TIF) of pancreatic ductal adenocarcinoma (PDAC) murine models and found extremely low arginine levels (2-5 uM) in the tumor microenvironment (TME). Despite near complete absence of this critical nutrient in the TME, pancreatic tumors exhibit aggressive growth. We have sought to understand both how the PDAC TME becomes arginine limited and how PDAC cells adapt to proliferate in the absence of arginine. Using genetically engineered mice, we find that arginase activity in the myeloid compartment of PDAC tumors is responsible for arginine depletion in the TME. Staining of Arg1+ myeloid populations in human PDAC samples suggest a similar mechanism reduces arginine availability in human PDAC tumors as well. We then leveraged our newfound knowledge of PDAC TIF composition to develop a novel ex vivo cell culture media formulation with physiologically relevant nutrient levels and monitored arginine acquisition pathways using isotope tracing and metabolomics assays to determine how PDAC cells cope with arginine deprivation. Under TME nutrient conditions, PDAC cells consume available citrulline and use it to produce arginine by de novo synthesis. Starving cells of citrulline or genetically perturbing arginosuccinate synthase (ASS1), key enzyme in arginine biosynthesis, significantly reduces PDAC cellular arginine and proliferative capacity. Immunohistochemical analysis of both human and mouse PDAC tumors indicates that the de novo arginine synthesis pathway is highly expressed in PDAC but not in untransformed pancreas, suggesting a key role for this pathway in PDAC progression. Altogether, we find that myeloid-derived arginase challenges PDAC cells by limiting arginine availability and suggest that de novo arginine synthesis may be a critical metabolic pathway that enables PDAC tumors to cope with this metabolic challenge. Citation Format: Juan Apiz-Saab, Alex Muir. Myeloid-derived arginase depletes microenvironmental arginine in PDAC tumors and leads to activation of arginine de novo biosynthesis in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2177.

Abstract 2959: Investigating the oncogenic effects and mechanisms of the SMYD3 methyltransferase in HPV-negative head and neck squamous cell carcinoma

Abstract Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world and is associated with an overall poor prognosis. The protein methyltransferase SET and MYND domain-containing 3 (SMYD3), which trimethylates H3K4 and H4K20, can stimulate gene transcription and activate several oncogenic pathways, including epithelial-mesenchymal transition, and cell cycle related pathways. Additionally, its expression is associated with a poor prognosis in various cancer types, such as colon and ovarian cancer. This study aims to decipher whether SMYD3 has direct oncogenic effects and relevant mechanisms in HPV-negative HNSCC. Our preliminary results have demonstrated that SMYD3 is significantly overexpressed in HPV-negative HNSCC compared to normal and dysplastic epithelium. SMYD3 knockdown decreased cellular proliferation and clonal capacity in CFAs after siRNA-mediated SMYD3 knockdown in several HPV-negative HNSCC cell lines or CRISPR-mediated SMYD3 knockout. Cell cycle analysis of CRISPR SMYD3 KO cell lines showed a significant decrease in the S phase, supporting that cell cycle arrest may be a mechanism through which this decreased cellular proliferation occurs. Nuclear/cytoplasmic dissociation and western blotting of 6 HPV-negative HNSCC cell lines, and immunohistochemistry for SMYD3 in primary and recurrent/metastatic HPV-negative HNSCC tumor tissues have identified the expression of SMYD3 in both the cytoplasm and nucleus, with greater expression in the cytoplasm than the nucleus. Ongoing experiments aim to investigate the effects of SMYD3 knockdown on the global proteome, as well as its effects on histone post-translational modifications in HPV-negative HNSCC cell lines. Genome-wide mapping for SMYD3, H3K4me3 and H4K20me3 in HPV- negative HNSCC cells using CUT&RUN assays are ongoing with the goal to identify direct downstream gene targets regulated by human SMYD3. Finally, we plan to investigate if SMYD3 knockout decreases tumor growth in NSG xenografts. This study may provide the biological rational to target SMYD3 as a novel therapeutic avenue in HPV-negative HNSCC. Citation Format: Madhavi Murali, Daniel Tsai, Nupur Nigam, Kyunghee Burkitt, Sohyoung Kim, Hui Cheng, Vassiliki Saloura. Investigating the oncogenic effects and mechanisms of the SMYD3 methyltransferase in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2959.