Upregulation Of P21 Research Articles

Benzo[a]pyrene evokes epithelial-mesenchymal transition and pulmonary fibrosis through AhR-mediated Nrf2-p62 signaling

Benzo[a]pyrene (BaP) and its metabolic end product benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide (BPDE), are known toxic environmental pollutants. This study aimed to analyze whether sub-chronic BPDE exposure initiated pulmonary fibrosis and the potential mechanisms. In this work, male C57BL6/J mice were exposed to BPDE by dynamic inhalation exposure for 8 weeks. Our results indicated that sub-chronic BPDE exposure evoked pulmonary fibrosis and epithelial-mesenchymal transition (EMT) in mice. Both in vivo and in vitro, BPDE exposure promoted nuclear translocation of Snail. Further experiments indicated that nuclear factor erythroid 2-related factor 2 (Nrf2) and p62 were upregulated in BPDE-exposed alveolar epithelial cells. Moreover, Nrf2 siRNA transfection evidently attenuated BPDE-induced p62 upregulation. Besides, p62 shRNA inhibited BPDE-incurred Snail nuclear translocation and EMT. Mechanically, BPDE facilitated physical interaction between p62 and Snail in the nucleus, then repressed Snail protein degradation by p62-dependent autophagy-lysosome pathway, and finally upregulated transcriptional activity of Snail. Additionally, aryl hydrocarbon receptor (AhR) was activated in BPDE-treated alveolar epithelial cells. Dual-luciferase assay indicated activating AhR could bind to Nrf2 gene promoter. Moreover, pretreatment with CH223191 or α-naphthoflavone (α-NF), AhR antagonists, inhibited BPDE-activated Nrf2-p62 signaling, and alleviated BPDE-induced EMT and pulmonary fibrosis in mice. Taken together, AhR-mediated Nrf2-p62 signaling contributes to BaP-induced EMT and pulmonary fibrosis.

Synthesized Gold Nanoparticles with Moringa Oleifera leaf Extract Induce Mitotic Arrest (G2/M phase) and Apoptosis in Dalton's Lymphoma Cells.

The therapeutic potential of chemically synthesized AuNPs has been demonstrated in various types of cancer. However, gold nanoparticles (AuNPs) synthesized using typical chemical methods have concerns regarding their environmental safety and adverse impact on human well-being. To overcome this issue, we used an environmentally friendly approach in which gold nanoparticles were synthesized using Moringa oleifera leaf extract (MLE). The present research was mainly focused on the biosynthesis and characterization of gold nanoparticles (AuNPs) using Moringa oleifera leaf extract (MLE-AuNPs) and explore its anticancer potential against Dalton's Lymphoma (DL) cells. Characterization of the MLE-AuNPs was conducted using UV-Vis Spectroscopy to confirm the reduction process, FTIR analysis to ascertain the presence of functional groups, and XRD analysis to confirm the crystallinity. SEM and TEM images were used to examine size and morphology. After characterization, MLE-AuNPs were evaluated for their cytotoxic effects on Dalton's lymphoma cells, and the results showed an IC50 value of 75 ± 2.31 µg/mL; however, there was no discernible cytotoxicity towards normal murine thymocytes. Furthermore, flow cytometric analysis revealed G2/M phase cell cycle arrest mediated by the downregulation of cyclin B1 and Cdc2 and upregulation of p21. Additionally, apoptosis induction was evidenced by Annexin V Staining, accompanied by modulation of apoptosis-related genes including decreased Bcl-2 expression and increased expression of Bax, Cyt-c, and Caspase-3 at both the mRNA and protein levels. Collectively, our findings underscore the promising anti-cancer properties of MLE-AuNPs, advocating their potential as a novel therapeutic avenue for Dalton's lymphoma.

Hernandezine promotes cancer cell apoptosis and disrupts the lysosomal acidic environment and cathepsin D maturation

Hernandezine (Her), a bisbenzylisoquinoline alkaloid extracted from Thalictrum flavum, is recognized for its range of biological activities inherent to this herbal medicine. Despite its notable properties, the anti-cancer effects of Her have remained largely unexplored. In this study, we elucidated that Her significantly induced cytotoxicity in cancer cells through the activation of apoptosis and necroptosis mechanisms. Furthermore, Her triggered autophagosome formation by activating the AMPK and ATG5 conjugation systems, leading to LC3 lipidation. Our findings revealed that Her caused damage to the mitochondrial membrane, with the damaged mitochondria undergoing mitophagy, as evidenced by the elevated expression of mitophagy markers. Conversely, Her disrupted autophagic flux, demonstrated by the upregulation of p62 and accumulation of autolysosomes, as observed in the RFP-GFP-LC3 reporter assay. Initially, we determined that Her did not prevent the fusion of autophagosomes and lysosomes. However, it inhibited the maturation of cathepsin D and increased lysosomal pH, indicating an impairment of lysosomal function. The use of the early-stage autophagy inhibitor, 3-methyladenine (3-MA), did not suppress LC3II, suggesting that Her also induces noncanonical autophagy in autophagosome formation. The application of Bafilomycin A1, an inhibitor of noncanonical autophagy, diminished the recruitment of ATG16L1 and the accumulation of LC3II by Her, thereby augmenting Her-induced cell death. These observations imply that while autophagy initially plays a protective role, the disruption of the autophagic process by Her promotes programmed cell death. This study provides the first evidence of Her’s dual role in inducing apoptosis and necroptosis while also initiating and subsequently impairing autophagy to promote apoptotic cell death. These insights contribute to a deeper understanding of the mechanisms underlying programmed cell death, offering potential avenues for enhancing cancer prevention and therapeutic strategies.

Resveratrol mediated the proliferation and apoptosis of gastric cancer cells by modulating the PI3K/Akt/P53 signaling pathway

The aim of this study was to investigate the anti-cancer effects of resveratrol (RES) against gastric cancer (GC) and explore the potential mechanisms. We first measured the anti-cancer effects of RES on GC cell lines (i.e. AGS and HGC-27). Then protein-protein interaction (PPI) network was constructed, followed by GO and KEGG analysis to screen the possible targets. Molecular docking analysis was given to visualize the pharmacological effects of RES on GC cell lines. For the in vivo experiments, xenograft tumor model was established, and Western blot analysis was performed to determine the expression of protein screened by network pharmacology. Our results showed that RES could promote the apoptosis of GC cells. Five hub targets were identified by network pharmacology, including AKT1, TP53, JUN, ESR1 and MAPK14. GO and KEGG analyses revealed the PI3K/Akt/P53 signaling pathway was the most related signaling pathway. Molecular docking analysis indicated that RES could form 3 hydrogen bonds with AKT1 and 3 hydrogen bonds with TP53. The inhibitory effects of RES on the proliferation and promoting effects of RES on the apoptosis of AGS and HGC-27 cells were significantly reversed when blocking the PI3K-Akt signaling pathway using the LY294002. In vivo results showed that RES induced significant decrease of tumor volume and tumor weight without changing the body weight, or inducing significant cytotoxicities. Western blot analysis proved that RES could induce down-regulation of p-Akt and up-regulation of P53 in vivo. In conclusion, RES showed anti-cancer effects in GC by regulating the PI3K/Akt/P53 signaling pathway.

Curcumol reduces lower limb arteriosclerosis in rats by inhibiting human arterial smooth muscle cell activity.

Cardiovascular diseases, particularly those involving arterial stenosis and smooth muscle cell proliferation, pose significant health risks. This study aimed to investigate the therapeutic potential of curcumol in inhibiting platelet-derived growth factor-BB (PDGF-BB)-induced human aortic smooth muscle cell (HASMC) proliferation, migration and autophagy. Using cell viability assays, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays and Western Blot analyses, we observed that curcumol effectively attenuated PDGF-BB-induced HASMC proliferation and migration in a concentration-dependent manner. Furthermore, curcumol mitigated PDGF-BB-induced autophagy, as evidenced by the downregulation of LC3-II/LC3-I ratio and upregulation of P62. In vivo experiments using an arteriosclerosis obliterans model demonstrated that curcumol treatment significantly ameliorated arterial morphology and reduced stenosis. Additionally, curcumol inhibited the activity of the KLF5/COX2 axis, a key pathway in vascular diseases. These findings suggest that curcumol has the potential to serve as a multi-target therapeutic agent for vascular diseases.

Hesperidin alleviates zinc-induced nephrotoxicity via the gut-kidney axis in swine.

Zinc (Zn) is an essential trace element in animals, but excessive intake can lead to renal toxicity damage. Thus, the exploration of effective natural antagonists to reduce the toxicity caused by Zn has become a major scientific problem. Here, we found that hesperidin could effectively alleviate the renal toxicity induced by Zn in pigs by using hematoxylin-eosin staining, transmission electron microscope, immunohistochemistry, fluorescence quantitative PCR, and microfloral DNA sequencing. The results showed that hesperidin could effectively attenuate the pathological injury in kidney, and reduce autophagy and apoptosis induced by Zn, which evidenced by the downregulation of LC3, ATG5, Bak1, Bax, Caspase-3 and upregulation of p62 and Bcl2. Additionally, hesperidin could reverse colon injury and the decrease of ZO-1 protein expression. Interestingly, hesperidin restored the intestinal flora structure disturbed by Zn, and significantly reduced the abundance of Tenericutes (phylum level) and Christensenella (genus level). Thus, altered intestinal flora and intestinal barrier function constitute the gut-kidney axis, which is involved in hesperidin alleviating Zn-induced nephrotoxicity. Our study provides theoretical basis and practical significance of hesperidin for the prevention and treatment of Zn-induced nephrotoxicity through gut-kidney axis.

The Role of SARS-CoV-2 Spike Protein in the Growth of Cervical Cancer Cells.

Recently developed vaccines for the SARS-CoV-2 virus utilize endogenous production of the virus' spike protein (SP), allowing the host to develop an immune response. As a result of the novelty of this virus and its vaccines, little is known overall about the potential effects of the SP on the pathogenesis of neoplasia, either from vaccination or from infection. This study was designed to investigate whether SARS-CoV-2 SP has any direct effect on SiHa cervical cancer cells. The effects of SARS-CoV-2 SP on cervical cancer cell proliferation and apoptosis were investigated by using clonogenic cell survival assay, quick cell proliferation assay, and caspase-3 activity kits in a widely-used cervical cancer cell line, SiHa. RT-PCR and immunohistochemistry were also performed to determine the potential molecular mechanisms. The growth and proliferation of SiHa cancer cells were inhibited by SARS-CoV-2 SP. SARS-CoV-2 SP also induced apoptosis in SiHa cancer cells. The anti-proliferative effect of SARS-CoV-2 SP on SiHa cancer cells was associated with the up-regulation of the anti-proliferative molecule p53. The pro-apoptotic effect of SARS-CoV-2 SP on SiHa cells was associated with the up-regulation of the pro-apoptotic molecule TRAIL. SARS-CoV-2 SP inhibits the growth of cervical cancer via up-regulation of p53 and TRAIL. Further studies are needed to elaborate on the potential effects of the SARS-CoV-2 SP on other cancer cell lines and normal physiological cell lines for comparison.

The senescence-associated secretory phenotype and its physiological and pathological implications.

Cellular senescence is a state of terminal growth arrest associated with the upregulation of different cell cycle inhibitors, mainly p16 and p21, structural and metabolic alterations, chronic DNA damage responses, and a hypersecretory state known as the senescence-associated secretory phenotype (SASP). The SASP is the major mediator of the paracrine effects of senescent cells in their tissue microenvironment and of various local and systemic biological functions. In this Review, we discuss the composition, dynamics and heterogeneity of the SASP as well as the mechanisms underlying its induction and regulation. We describe the various biological properties of the SASP, its beneficial and detrimental effects in different physiological and pathological settings, and its impact on overall health span. Finally, we discuss the use of the SASP as a biomarker and of SASP inhibitors as senomorphic interventions to treat cancer and other age-related conditions.

Human Motor Neurons Elicit Pathological Hallmarks of ALS and Reveal Potential Biomarkers of the Disease in Response to Prolonged IFNγ Exposure.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder marked by progressive motor neuron degeneration and muscle denervation. A recent transcriptomic study integrating a wide range of human ALS samples revealed that the upregulation of p53, a downstream target of inflammatory stress, is commonly detected in familial and sporadic ALS cases by a mechanism linked to a transactive response DNA-binding protein 43 (TDP-43) dysfunction. In this study, we show that prolonged interferon-gamma (IFNγ) treatment of human induced pluripotent stem cell-derived spinal motor neurons results in a severe cytoplasmic aggregation of TDP-43. TDP-43 dysfunction resulting from either IFNγ exposure or an ALS-associated TDP-43 mutation was associated with the activation of the p53 pathway. This was accompanied by the hyperactivation of neuronal firing, followed by the complete loss of their electrophysiological function. Through a comparative single-cell transcriptome analysis, we have identified significant alterations in ALS-associated genes in motor neurons exposed to IFNγ, implicating their direct involvement in ALS pathology. Interestingly, IFNγ was found to induce significant levels of programmed death-ligand 1 (PD-L1) expression in motor neurons without affecting the levels of any other immune checkpoint proteins. This finding suggests a potential role of excessive PD-L1 expression in ALS development, given that PD-L1 was recently reported to impair neuronal firing ability in mice. Our findings suggest that exposing motor neurons to IFNγ could directly derive ALS pathogenesis, even without the presence of the inherent genetic mutation or functional glia component. Furthermore, this study provides a comprehensive list of potential candidate genes for future immunotherapeutic targets with which to treat sporadic forms of ALS, which account for 90% of all reported cases.

Cymensifin A: a promising pharmaceutical candidate to defeat lung cancer via cellular reactive oxygen species-mediated apoptosis.

Background: The upgrade of natural products for cancer treatment is essential since current anticancer drugs still pose severe side effects. Cymensifin A (Cym A) isolated from an orchid Cymbidium ensifolium has shown its potential to induce the death of several cancer cells; however, its underlying molecular mechanisms are hitherto unknown. Methods: Here, we conducted a set of in vitro preliminary tests to assess the cytotoxic effects of Cym A on non-small-cell lung cancer (NSCLC) cells (A549, H23, H292, and H460). A flow cytometry system and Western blot analyses were employed to unveil molecular mechanisms underlying cancer cell apoptosis caused by Cym A. Results: Cym A at 25-50μM caused the death of all NSCLC cells tested, and its cytotoxicity was comparable to cisplatin, a currently used anticancer drug. The compound induced apoptosis of all NSCLC cells in a dose-dependent manner (5-50μM), proven by flow cytometry, but H460 cells showed more resistance compared to other cells tested. Cym A-treated H460 cells demonstrated increased reactive oxygen species (ROS) and downregulated antioxidants (catalase, superoxide dismutase, and thioredoxin). The compound also upregulated the tumor suppressor P53 and the pro-apoptotic protein BAX but downregulated pro-survival proteins (BCL-2 and MCL-1) and deactivated survival signals (AKT and ERK) in H460 cells. Cym A was proven to trigger cellular ROS formation, but P53 and BAX were 2-fold more activated by Cym A compared to those treated with hydrogen peroxide. Our findings also supported that Cym A exerted its roles in the downregulation of nuclear factor erythroid 2-related factor 2 (a regulator of cellular antioxidant activity) and the increased levels of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase 3/7 during apoptosis. Conclusion: We propose that Cym A induces lung cancer cell death via ROS-mediated apoptosis, while the modulation of cellular ROS/antioxidant activity, the upregulation of P53 and BAX, the downregulation or deactivation of BCL-2, MCL-1, AKT, and ERK, and the increased cleavage of PARP and caspase 3/7, were the elucidated underlying molecular mechanisms of this phytochemical. The compound can be a promising candidate for future anticancer drug development.

Abstract LB048: Unleashing the power of p53 mRNA therapeutics to suppress proliferation of p53 mutant tumors

Abstract The p53 protein, recognized as the guardian of the genome, plays a pivotal role in suppressing tumor formation. The TP53 gene, which encodes the p53 protein, is commonly subject to mutations and deletions in many cancer types resulting in loss of tumor suppressor activity. A significant proportion of patients diagnosed with lung, pancreatic, ovarian, and head and neck cancers exhibit the expression of mutant p53 in their tumors, correlating with unfavorable prognosis and survival outcomes.This study endeavors to assess the efficacy of therapeutic tumor suppressive mRNA which induces expression of wild-type p53 protein across diverse cancer types. Optimization of the 5’ and 3’ untranslated regions (UTRs) and the 5’ cap has been undertaken to enhance the efficient expression of p53 mRNA. The growth inhibitory potential of p53 mRNA therapy has been evaluated across p53 mutant cancer cell lines representing various cancer origins, along with normal or p53 wild-type cancer cell lines. We have substantiated that the p53 mRNA therapeutics can selectively inhibit the proliferation of p53 mutant cancer cells. Furthermore, we have confirmed that p53 mRNA treatment upregulated modulator of apoptosis (PUMA) and B-cell translocation gene 2 (BTG2) and cell cycle arrest via p21 upregulation. In vivo efficacy assessments of the lipid nanoparticle (LNP)-encapsulated p53 mRNA have been conducted in lung, pancreatic, and ovarian cancer xenograft models. Both regional intratumoral (i.t) and systemic intravenous (i.v) administrations of the p53 mRNA therapeutics have shown substantial suppression of tumor growth, leading to prolonged mouse survival. These results underscore the potential of the p53 mRNA therapeutics as a promising avenue for targeted cancer treatment, offering insights into the selective inhibition of p53 mutant cancer cells. Citation Format: Yong Ho Heo, Seung-Hyun Shin, Youngjin Han, Chang Gyu Lim, Innah Kim, Ji Hee Lee, Gunwoo Lee, Seongju Jeong, Yunjae Kim, Jooyun Byun, Daejin Kim, In Young Choi. Unleashing the power of p53 mRNA therapeutics to suppress proliferation of p53 mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB048.

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Metformin Loaded Zein Polymeric Nanoparticles to Augment Antitumor Activity against Ehrlich Carcinoma via Activation of AMPK Pathway: D-Optimal Design Optimization, In Vitro Characterization, and In Vivo Study.

Metformin (MET), an antidiabetic drug, is emerging as a promising anticancer agent. This study was initiated to investigate the antitumor effects and potential molecular targets of MET in mice bearing solid Ehrlich carcinoma (SEC) as a model of breast cancer (BC) and to explore the potential of zein nanoparticles (ZNs) as a carrier for improving the anticancer effect of MET. ZNs were fabricated through ethanol injection followed by probe sonication method. The optimum ZN formulation (ZN8) was spherical and contained 5 mg zein and 30 mg sodium deoxycholate with a small particle size and high entrapment efficiency percentage and zeta potential. A stability study showed that ZN8 was stable for up to three months. In vitro release profiles proved the sustained effect of ZN8 compared to the MET solution. Treatment of SEC-bearing mice with ZN8 produced a more pronounced anticancer effect which was mediated by upregulation of P53 and miRNA-543 as well as downregulation of NF-κB and miRNA-191-5p gene expression. Furthermore, ZN8 produced a marked elevation in pAMPK and caspase-3 levels as well as a significant decrease in cyclin D1, COX-2, and PGE2 levels. The acquired findings verified the potency of MET-loaded ZNs as a treatment approach for BC.

P53 upregulation by USP7-engaging molecular glues

Molecular glues are typically small chemical molecules that act at the interface between a target protein and degradation machinery to trigger ternary complex formation. Identifying molecular glues is challenging. There is a scarcity of target-specific upregulating molecular glues, which are highly anticipated for numerous targets, including P53. P53 is degraded in proteasomes through polyubiquitination by specific E3 ligases, whereas deubiquitinases (DUBs) remove polyubiquitination conjugates to counteract these E3 ligases. Thus, small-molecular glues that enhance P53 anchoring to DUBs may stabilize P53 through deubiquitination. Here, using small-molecule microarray-based technology and unbiased screening, we identified three potential molecular glues that may tether P53 to the DUB, USP7, and elevate the P53 level. Among the molecular glues, bromocriptine (BC) is an FDA-approved drug with the most robust effects. BC was further verified to increase P53 stability via the predicted molecular glue mechanism engaging USP7.Consistent with P53 upregulation in cancer cells, BC was shown to inhibit the proliferation of cancer cells in vitro and suppress tumor growth in a xenograft model. In summary, we established a potential screening platform and identified potential molecular glues upregulating P53. Similar strategies could be applied to the identification of other types of molecular glues that may benefit drug discovery and chemical biology studies.

Discovery of ganoderic acid A (GAA) PROTACs as MDM2 protein degraders for the treatment of breast cancer

Breast cancer is one of the most common female malignant tumors, with triple-negative breast cancer (TNBC) being the most specific, highly invasive, metastatic and associated with a poor prognosis. Our previous study showed that the natural product ganoderic acid A (GAA) has a certain affinity for MDM2. In this study, two series of novel GAA PROTACs C1–C10 and V1–V10 were designed and synthesized for the treatment of breast cancer. The antitumor activity of these compounds was evaluated against four human tumor cell lines (MCF-7, MDA-MB-231, SJSA-1, and HepG2). Among them, V9 and V10 showed stronger anti-proliferative effects against breast cancer cells, and V10 showed the best selectivity in MDA-MB-231 cells (TNBC), which was 5-fold higher than that of the lead compound GAA. Preliminary structure-activity analysis revealed that V-series GAA PROTACs had better effects than C-series, and the introduction of 2O–4O PEG linkers could significantly improve the antitumor activity. Molecular docking, surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), and Western blot researches showed that both V9 and V10 could bind with MDM2, and degrade the protein through the ubiquitin-proteasome system. Molecular dynamics simulation (MD) revealed that V10 is a bifunctional molecule that can bind to von Hippel-Lindau (VHL) at one end and target MDM2 at the other. In addition, V10 promoted the upregulation of p21 in p53-mutant MDA-MB-231 cells, and induced apoptosis via down-regulation of the bcl-2/bax ratio and the expression of cyclin B1. Finally, in vivo experiments showed that, V10 also exhibited good tumor inhibitory activity in xenografted TNBC zebrafish models, with an inhibition rate of 27.2% at 50 μg/mL. In conclusion, our results suggested that V10 has anti-tumor effects on p53-mutant breast cancer in vitro and in vivo, and may be used as a novel lead compound for the future development of TNBC.

Persistent epigenetic signals propel a senescence-associated secretory phenotype and trained innate immunity in CD34+ hematopoietic stem cells from diabetic patients

BackgroundDiabetes-induced trained immunity contributes to the development of atherosclerosis and its complications. This study aimed to investigate in humans whether epigenetic signals involved in immune cell activation and inflammation are initiated in hematopoietic stem/progenitor cells (HSPCs) and transferred to differentiated progeny.Methods and resultsHigh glucose (HG)-exposure of cord blood (CB)-derived HSPCs induced a senescent-associated secretory phenotype (SASP) characterized by cell proliferation lowering, ROS production, telomere shortening, up-regulation of p21 and p27genes, upregulation of NFkB-p65 transcription factor and increased secretion of the inflammatory cytokines TNFα and IL6. Chromatin immunoprecipitation assay (ChIP) of p65 promoter revealed that H3K4me1 histone mark accumulation and methyltransferase SetD7 recruitment, along with the reduction of repressive H3K9me3 histone modification, were involved in NFkB-p65 upregulation of HG-HSPCs, as confirmed by increased RNA polymerase II engagement at gene level. The differentiation of HG-HSPCs into myeloid cells generated highly responsive monocytes, mainly composed of intermediate subsets (CD14hiCD16+), that like the cells from which they derive, were characterized by SASP features and similar epigenetic patterns at the p65 promoter. The clinical relevance of our findings was confirmed in sternal BM-derived HSPCs of T2DM patients. In line with our in vitro model, T2DM HSPCs were characterized by SASP profile and SETD7 upregulation. Additionally, they generated, after myeloid differentiation, senescent monocytes mainly composed of proinflammatory intermediates (CD14hiCD16+) characterized by H3K4me1 accumulation at NFkB-p65 promoter.ConclusionsHyperglycemia induces marked chromatin modifications in HSPCs, which, once transmitted to the cell progeny, contributes to persistent and pathogenic changes in immune cell function and composition.

Abstract 2960: Deciphering the role of miRNA146-a and its crosstalk with cellular redox state in senescence escape in breast cancer cells

Abstract Senescence serves as a double-edged sword in cancer as studies revealed that senescence exhibits both tumor-suppressing and tumor-promoting effects. A subpopulation of cancer cells that enters senescence can evade senescence to proliferate, thereby resulting in tumor regrowth, which has been associated with chemoresistance and tumor aggressiveness. Unlike senescent cells, the senescence-escaped cells harbor low levels of reactive oxygen species (ROS), however, the mechanism(s) underlying this redox dichotomy in senescence and senescence evasion remains elusive. In this regard, recent reports have highlighted the pivotal role of miRNAs in regulating cellular senescence. To that end, miR-146a has been reported as a biomarker of senescence probably via modulating pro-inflammatory and antioxidant pathways. We set up a model of doxorubicin-induced senescence in MDA-MB-231 breast carcinoma cells to study the role and involvement of miRNA146a in senescence induction and its regulation. Senescence induction was confirmed by upregulation of p21, increase in senescence-associated beta galactosidase and C12FDG staining, as well as propidium iodide positivity. We show that, following the initial phase of apoptotic cell death, as evidenced by increased yH2AX and cleaved caspase 3, a population of senescent cells escaped growth arrest to re-enter cell cycle and proliferate. Senescence-escaped cells harbored lower ROS levels compared to senescent cells, which correlated with upregulation of antioxidant enzymes such as MnSOD and CuZnSOD as well as increased transcription of NRF2 and NQO1. Our results also provide evidence for a novel senescence associated redox state, which implicates intracellular peroxynitrite as a driver of cellular senescence. Interestingly, in our model system, p21 upregulation was not associated with intracellular ROS accumulation, which was reported in earlier findings. Interestingly, these changes coincided with a significant increase in miR-146 expression (during senescence), which was previously shown to downregulate ROS in other model systems. While modulating miR-146a did not seem to change ROS levels upon doxorubicin-induced senescence in MDA-MB231 cells, the precise crosstalk between cellular ROS, miR146-a and senescence evasion is currently being pursued. Citation Format: Shazib Pervaiz, Angeline Q. Liew, Marie-Veronique Clement. Deciphering the role of miRNA146-a and its crosstalk with cellular redox state in senescence escape in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2960.

Abstract 5982: Cannabinoid derived product is a potential novel therapeutic for papillary thyroid carcinoma

Abstract Papillary thyroid carcinoma (PTC) is the most common thyroid cancer that usually affects women ages 20-50, presenting as an asymptomatic neck mass. Total or partial thyroidectomy shows an excellent prognosis; however, investigation into non-invasive therapeutics with minimal adverse effects is ongoing. In addition to driver mutations such as BRAF, RAS, and RET-PTC, p53 mutations have also been identified. The loss of function of p53 is linked to cancer progression from PTC to poorly differentiated thyroid cancer and possibly anaplastic thyroid cancer. The disruption of the Wnt cell pathway is documented to be related to malignant cell transformation in thyroid cancer, leading to remarkable upregulation of c-myc, resulting in enhancement of tumor growth and drug resistance. Previous studies revealed that upregulation of BCL-2 occurs at later stages of tumorigenesis and is involved in chemotherapy resistance in some forms of cancer. In PTC, the downregulation of BCL-2 is linked to its favorable prognosis. Cannabinoid Derived Products (CDPs) have shown effectiveness in inhibiting different types of cancer, with research dating back to the 1970s. Ongoing in-vitro and in-vivo studies show that CDPs can effectively modulate tumor growth. However, the apoptotic properties of CDPs are largely dependent on the cancer type and drug dose/concentration. Our investigation focuses on CDPs and its potential anti-cancer effects by modulation of gene expression. We investigated its effects on gene expression of p53, c-Myc, and BCL-2 in K1 cell line which consists of PTC cells obtained from metastasis of well-differentiated PTC. K1 PTC cells (1 x106 cells/mL) were cultured with CDPs and incubated at 37°C under 5% CO2 for 24 and 48 hours. Cells were harvested, and cell viability was determined via trypan blue exclusion assay. Using qRT-PCR, we determined the effects of CDPs on the expression of TP53, c-Myc, and BCL-2. Results show that the CDPs decreased the viability (p< 0.001) of K1 PTC cells in a dose and time-dependent manner. Within 24 hours, the cannabinoid-containing product increased the gene expression of TP53 (p < 0.01) and decreased the gene expression of BCL-2 (p< 0.01) and c-Myc (p< 0.05) in K1 PTC cells. The results suggest that CDPs interact as a potential regulator in cancer with the upregulation of p53 and downregulation of BCL-2 and c-Myc. Further in-vitro and in-vivo studies are needed to understand the mechanism and therapeutic potential of cannabinoid-containing products in papillary thyroid cancer. Citation Format: Carolina Taico, Ibrahim Musa, Fariba Ardalani, Anish Maskey, Nan Yang, Joseph Breslin, Raj Tiwari, Jan Geliebter, Xiu Min Li. Cannabinoid derived product is a potential novel therapeutic for papillary thyroid carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5982.

Abstract 4593: A synergy between MDM2 and AURKB pathways in driving cell cycle and cell survival in lung cancer cells

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide and non-small cell lung cancer (NSCLC) is the most prevalent type among lung cancers, contributing to the high mortality seen with this deadly disease. Despite aggressive treatments, the 5-year survival rate for lung cancer is only 23.7% in the United States. MDM2 and AURKB are two cancer-driving genes that are highly expressed in a broad range of cancer types including lung cancers. Furthermore, MDM2 overexpression can positively impact AURKB expression and contribute to oncogenesis and cancer progression. Therefore, blocking MDM2 with specific inhibitors has become a promising strategy for the restoration of p53 function that could be used for the treatment of different types of cancers including lung cancers. In this regard, both MDM2 and AURKB inhibitors have shown promising antitumor activity against NSCLC. Therefore, the main objective of this study was to understand the molecular mechanisms linking MDM2 and ARKB using RG7388, CM272, BMS-582949 (p38 inhibitor), and Ralimetinib (p38 inhibitor) in A549 and H460 lung cancer cells. We analyzed the individual or combination treatment effects of these drugs on epigenetic modifications, cell cycle arrest, and cell death mechanisms, using the in vitro experimental model. When RG7388 was used individually, or in combination with BMS-582949 or Ralimetinib, a significant increase in the cytotoxic effect was observed in A549 and H460 cells. Induction of cell cycle arrest by RG7388 in cancer cells was evidenced by elevated p21 expression levels. In addition, RG7388 and combination treatments in NSCLC cells showed significant upregulation of p53, p27, Bax, cleaved PARP, phospho-FOXO3a and down-regulation in DNMT3a, DNMT1, FOXM1, AURKB, CDC25, and CDK4 levels. It appears that during treatments with a combination of MDM2 with DNMT or p38 inhibitor, two pathways might be blocked simultaneously, which could lead to increased cancer cell death. RG7388 appears to induce cell death in a p21-dependent manner and CM272, BMS-582949, and Ralimetinib seem to induce cell death via a p21-independent manner by inhibiting a downstream pathway. However, further studies are needed to fully understand the intracellular link between MDM2 and AURKB that is disrupted by the anti-cancer agents used in our study. (This project was supported by the PFRDG Grant of Nova Southeastern University and by the Generous support from the Royal Dames of Cancer Research Ft. Lauderdale, FL) Citation Format: Umamaheswari Natarajan, Appu Rathinavelu. A synergy between MDM2 and AURKB pathways in driving cell cycle and cell survival in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4593.

Abstract 1272: Spatial profiling of human colorectal cancer brain metastasis identifies chromosomal instability with adaptive niche cellular reorganization and reprograming

Abstract Brain metastasis in colorectal cancer (mCRC) poses a significant clinical challenge with poor outcomes. Our study, the largest spatial analysis reported to date, aimed to uncover unique cellular and genomic features facilitating metastatic lesion development. Employing spatial transcriptomics on 60 patients' surgical resections, we validated our findings with single-cell RNA sequencing (scRNA-seq), whole-genome sequencing (WGS), and ex vivo functional studies in tumor slice cultures. We obtained spatially distinct gene expression profiles of metastatic tumor cells, tumor microenvironment (TME) and brain parenchymal lineages. Our analysis identified spatially heterogenous upregulation of PI3K, MAPK, EGFR, p53, and TGFβ signaling in metastatic tumor cells. Tumor cells had high levels of chromosomal instability (CIN) marked by recurrent amplifications in chromosomes 6, 7, 17, and 20. WGS from three independent cohorts revealed significantly higher CIN in mCRC brain compared to primary CRC or mCRC from other organs. Spatial neighborhood analysis identified an immune desert phenotype with lack of proximity between lymphocytes and metastatic tumor cells. However, mCRC brain contained abundant plasma cells identifying a unique TME niche feature. Significant proximity between tumor cells and endothelial cells underscored their potential role in tumor seeding and growth. Spatially aware differential expression analysis demonstrated adaptive responses in the brain parenchyma. This included increased Golgi-ER processing and Rho GTPase signaling in neurons and astrocytes adjacent to metastatic tumor cells, respectively. Macrophages were characterized by high expression of pro-fibrogenic genes. Increased SPP1 ligand expression from these macrophages interacted with corresponding receptors on spatially proximal fibroblasts, identifying a key niche reorganization feature that can promote metastatic lesion growth. To evaluate the translational potential of our findings, we examined the effects of perturbating the metastatic niche using scRNA-seq. We established a tumor slice culture from a mCRC brain surgical resection, which maintained all original cell states. Despite elevated signaling pathway activity, metastatic tumor cells were resistant to regorafenib, a multi-receptor tyrosine kinase inhibitor. In contrast, pirfenidone, an anti-fibrotic and anti-inflammatory small molecule, successfully reduced extracellular matrix-associated gene expression programs in fibroblasts in the TME. Our analysis identified distinct properties of mCRC brain that can serve as therapeutic targets, including high levels of chromosomal instability, remodeling of brain parenchyma, lymphocyte evasion, plasma cell abundance and macrophage-fibroblast interactions. Citation Format: Anuja Sathe, Aparajita Khan, Ji In Kang, Rithika Meka, Susan M. Grimes, Andrew S. Luksik, Michael Lim, Claudia Petrisch, Christopher M. Jackson, Hannes Vogel, Melanie Gephart, Summer Han, Hanlee P. Ji. Spatial profiling of human colorectal cancer brain metastasis identifies chromosomal instability with adaptive niche cellular reorganization and reprograming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1272.