Cell Survival Signaling Research Articles

Proteomic alterations associated with residual disease in neoadjuvant chemotherapy treated ovarian cancer tissues

BackgroundOptimal cytoreduction to no residual disease (R0) correlates with improved disease outcome for high-grade serous ovarian cancer (HGSOC) patients. Treatment of HGSOC patients with neoadjuvant chemotherapy, however, may select for tumor cells harboring alterations in hallmark cancer pathways including metastatic potential. This study assessed this hypothesis by performing proteomic analysis of matched, chemotherapy naïve and neoadjuvant chemotherapy (NACT)-treated HGSOC tumors obtained from patients who had suboptimal (R1, n = 6) versus optimal (R0, n = 14) debulking at interval debulking surgery (IDS).MethodsTumor epithelium was harvested by laser microdissection from formalin-fixed, paraffin-embedded tissues from matched, pre- and post-NACT treated tumors for twenty HGSOC patients and analyzed by quantitative mass spectrometry-based proteomics.ResultsDifferential analysis of patient matched pre- and post-NACT treated tumors revealed proteins associated with cell survival and metabolic signaling to be significantly altered in post-NACT treated tumor cells. Comparison of pre-NACT treated tumors from suboptimal (R1) versus optimally (R0) debulked patients identified proteins associated with tumor cell viability and invasion signaling enriched in R1 patients. We identified five proteins altered between R1 and R0 patients in pre- NACT treated tumors that significantly correlated with PFS in an independent cohort of HGSOC patients, including Fermitin family homolog 2 (FERMT2), a protein elevated in R1 that correlated with disease progression in HGSOC patients (multivariate Cox HR = 1.65, Wald p = 0.022) and increased metastatic potential in solid-tumor malignancies.ConclusionsThis study identified distinct proteome profiles in patient matched pre- and post-NACT HGSOC tumors that correlate with NACT resistance and that may predict residual disease status at IDS that collectively warrant further pre-clinical investigation.

Evaluation of the direct protective effects of Canagliflozin on the Isoproterenol-induced cell injury in rat cardiomyocytes

Abstract Background Sodium-glucose cotransporter-2 (SGLT2) inhibitors are agents that act by inhibiting glucose and sodium reabsorption in the proximal renal tubule which promotes urinary glucose excretion. More recently, significant benefit data of SGLT2 inhibitors in patients with heart failure, independent of the presence of type 2 diabetes has been reported. We have previously demonstrated that Canagliflozin (Cana), a SGLT2 inhibitor, reduced the ventricular effective refractory period in isoproterenol (ISP)-induced myocardial injury rat model accompanied with the suppression of reactive oxygen species and the elevation of ketone bodies, suggesting the effect of Cana on electrical cardiac remodeling. The direct effect of Cana to the cardiomyocytes and its underlying molecular mechanism was remained to be clarified. We therefore established an ISP-induced neonatal rat ventricular cardiomyocyte (NRVCM) in vitro model, pretreated with Cana and/or ketone bodies. Methods Primary NRVCM were isolated from Wistar rats, were pretreated by Cana with or without βOHB (the most abundant ketone body in circulation), followed by a stimulation of ISP (10μM). Cells without drug or ketone body pretreatment were used as control. We then analyzed its effect on cell viability, apoptosis, and mitochondrial membrane potential using MTT assay, TUNEL assay, and mitochondrial membrane potential assay, respectively. MTT assay was also performed with or without PI3k inhibitor, LY294002. The end-labeling of DNA fragmentation were labelled with FITC, followed by the nuclei counterstain with DAPI and were observed with confocal microscope. The apoptotic index was defined as the percentage of TUNEL positive cells / total nuclei. Results Cana rescued the reduction of NRVCM cell viability induced by ISP stimulation for 24 hours which was inhibited by LY294002 compared to cells without pretreatment. Interestingly, pretreatment of βOHB with or without Cana improved also the NRCVM cell viability whereas there was no significant difference between these two conditions or with cells treated with Cana only, suggesting the direct protective effect of Cana. In 48 hours of ISP stimulation, the apoptotic index intends to decrease in Cana and/or βOHB compared to cells without pretreatment (Figure 1). Although the mitochondrial function was maintained in Cana-pretreated cells compared to cells without pretreatment, there was no significant difference in βOHB-pretreated cells. Conclusions Cana has a direct protective effect on cardiomyocytes cell viability, apoptosis as well as the mitochondrial function impaired by ISP through the cell survival signaling PI3K/Akt pathway. This brings a new insight to the therapeutic target of cardiovascular disease. Funding Acknowledgement Type of funding sources: None.

A Review of Herbal Medicine-Based Phytochemical of Garcinia as Molecular Therapy for Breast Cancer.

Data from globocan statistic in 2020 indicate that breast cancer has become highest incidence rate of cancer. Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) are known immunohistochemistry (IHC) markers that mediate cell growth and survival signaling. Furthermore, regulator proteins, receptors, and their downstream signaling pathways have emerged as critical components in breast cancer formation and proliferation, and have become well-established therapeutic targets and the core focus of breast cancer therapy research. Garcinia is a big genus in the Clusiaceae family that contains a wide spectrum of biologically active metabolites for the chemical composition of their isolated fruits, stem barks, seeds, leaves, and roots, have resulted including polyisoprenylated benzophenones, polyphenols, bioflavonoids, xanthones, lactones, and triterpenes. This review article aimed to analyze the potential of Garcinia phytochemicals as a molecular therapy of breast cancer. The results showed that phytochemicals of Garcinia (i.e., α-mangostin, Cambogin, Gambogic Acid [GA], Garcinol, Griffipavixanthone, Friedolanostane triterpenoid, Hexane, Neobractatin, 7-Epiclusianone, xanthochymol - guttiferone E, and isoxanthochymol - cycloxanthochymol) have anticancer properties, including apoptosis, inhibition of proliferation, and metastasis. This review is important to provide information regarding phytochemicals of Garcinia as an alternative treatment for breast cancer patients. This article selected 28 article researches based on inclusion criteria with the keyword “Garcinia” and “Breast cancer”, in English, and available in full text and abstract searching on PubMed.

Circulation microRNA expression profiles in patients with complete responses to chemoradiotherapy in nasopharyngeal carcinoma.

BackgroundNasopharyngeal carcinoma (NPC) is endemic cancer in Southeast Asia with a relatively poor prognosis. Chemoradiotherapy is a primary treatment that advantages certain patients, particularly in the early stages. New predictive and prognostic biomarkers are required to guide and select the best treatment.AimsTo evaluate the circulation expression profile of microRNAs (miRNAs) associated with responses to chemoradiotherapy in nasopharyngeal carcinoma.MethodsPeripheral blood from 17 patients was collected before and after chemotherapy and radiotherapy. Differential expression circulating miRNAs were analyzed using microRNA Cancer Panels and were compared among patients with complete responses. Differential expression analysis using GenEx 7 Multid, statistic represented by GraphPad Prism 9. Alterations mechanism signaling pathways and biological function using IPA (Ingenuity Pathways Analysis).ResultsUsing microRNAs Cancer Plate consisting of 116 miRNAs, we identified ten circulating miRNAs that were differentially expressed in NPC patients after chemoradiotherapy. Unsupervised clustering and confirmation using qRT-PCR showed that miR-483-5p, miR-584-5p, miR-122-5p, miR-7-5p, miR-150-5p were overexpressed and miRNA are miR-421, miR-133a-3p, miR-18a-5p, miR-106b-3p, miR-339-5p were significantly downregulated after chemoradiotherapy (p < 0.0001). In addition, ROC analysis through AUC (Area Under Curve) with 99% confidence interval (CI) p value < 0.0001. Gene enrichment analysis of microRNAs and the targeted proteins revealed that the main involved pathways for chemoradiotherapy in NPC were cell death and survival signaling pathways.ConclusionqPCR profiling in circulating blood compared before and after chemoradiotherapy in nasopharyngeal carcinoma can identify pathways involved in treatment responses. miR-483-5p, miR-584-5p, miR-122-5p, miR-7-5p, miR-150-5p, miR-421, miR-133a-3p, miR-18a-5p, miR-106b-3p, miR-339-5p are differentially regulated after chemoradiotherapy in NPC.

Neuroprotective Effects of Probiotic Lactobacillus reuteri GMNL-263 in the Hippocampus of Streptozotocin-Induced Diabetic Rats.

Diabetes-related brain complications have been reported in clinical patients and experimental models. The objective of the present study was to investigate the neuroprotective mechanisms of Lactobacillus reuteri GMNL-263 in streptozotocin (STZ)-induced diabetic rats. In this study, three different groups, namely control group, STZ-induced (55mg/kg streptozotocin intraperitoneally) diabetic rats (DM), and DM rats treated with Lactobacillus reuteri GMNL-263 (1 × 109CFU/rat/day), were utilized to study the protective effect of GMNL-263 in the hippocampus of STZ-induced diabetic rats. The results demonstrated that GMNL-263 attenuated diabetes-induced hippocampal damage by enhancing the cell survival pathways and repressing both inflammatory and apoptotic pathways. Histopathological analysis revealed that GMNL-263 prevented structural changes in the hippocampus in the DM group and decreased the level of inflammation and apoptosis in the hippocampus of DM rats. The IGF1R cell survival signaling pathway also improved after GMNL-263 treatment. These results indicate that probiotic GMNL-263 exerts beneficial effects in the brain of diabetic rats and has potential ability for clinical application.

Palmitoylation of GNAQ/11 is critical for tumor cell proliferation and survival in GNAQ/11-mutant uveal melanoma.

More than 85% of patients with uveal melanoma (UM) carry a GNAQ or GNA11 mutation at a hotspot codon (Q209) that encodes G protein α subunit q/11 polypeptides (Gαq/11). GNAQ/11 relies on palmitoylation for membrane association and signal transduction. Despite the palmitoylation of GNAQ/11 was discovered long before, its implication in UM remains unclear. Here, results of palmitoylation-targeted mutagenesis and chemical interference approaches revealed that the loss of GNAQ/11 palmitoylation substantially affected tumor cell proliferation and survival in UM cells. Palmitoylation inhibition through the mutation of palmitoylation sites suppressed GNAQ/11Q209L-induced malignant transformation in NIH3T3 cells. Importantly, the palmitoylation-deficient oncogenic GNAQ/11 failed to rescue the cell death initiated by the knock down of endogenous GNAQ/11 oncogenes in UM cells, which are much more dependent on Gαq/11 signaling for cell survival and proliferation than other melanoma cells without GNAQ/11 mutations. Furthermore, the palmitoylation inhibitor, 2-bromopalmitate, also specifically disrupted Gαq/11 downstream signaling by interfering with the MAPK pathway and BCL2 survival pathway in GNAQ/11-mutant UM cells and showed a notable synergistic effect when applied in combination with the BCL2 inhibitor, ABT-199, in vitro. The findings validate that GNAQ/11 palmitoylation plays a critical role in UM and may serve as a promising therapeutic target for GNAQ/11-driven UM.

Protosappanin-B suppresses human melanoma cancer cell growth through impeding cell survival, inflammation and proliferative signaling pathways

This study evaluates protosappanin B's (PSB) role in inflammation, proliferation, and subsequent apoptosis of human melanoma A875 cells. The cell lines were treated with PSB (15 and 20 μM) for 24 h of incubation mediated cytotoxicity was assessed by MTT assay; ROS was studied by flow cytometry. PSB treatment mediated nuclear fragmentation and apoptotic morphological features were analyzed by AO, EtBr, PI, and DAPI stainings. The mRNA and protein expression of inflammatory, proliferation, cell survival and apoptosis were studied by RT-PCR and western blotting. PSB (15 and 20 μM) treatment with A875 produces cytotoxicity associated with nuclear fragmentation, and apoptotic changes were observed. PSB inhibits inflammatory proteins (TNF-α, NF-κB, COX-2, IL-6, iNOS, and VEGF) in A875 cells. Furthermore, PSB induces pro-apoptotic protein expressions i.e. Bax, caspase-3; inhibiting anti-apoptotic, cell proliferative proteins (cyclin-D1, Bcl-2, c-Myc, and survivin) in A875 cells. The p-PI3K, p-AKT, and p-GSK-3β contribute to oncogenic progression; hence, inhibition of these signaling events is considered a significant target for the treatment of melanoma. This study observed that PSB treatment magnificently inhibited the p-PI3K, p-AKT, and p-GSK-3β in A875 cells. Thus, PSB inhibits inflammation, proliferation, and survival; promotes apoptosis by suppressing PI3K, AKT, and GSK-3β in human melanoma cancer.

Upregulated PD-1 signaling antagonizes glomerular health in aged kidneys and disease.

With an aging population, kidney health becomes an important medical and socioeconomic factor. Kidney aging mechanisms are not well understood. We previously showed that podocytes isolated from aged mice exhibit increased expression of programmed cell death protein 1 (PD-1) surface receptor and its 2 ligands (PD-L1 and PD-L2). PDCD1 transcript increased with age in microdissected human glomeruli, which correlated with lower estimated glomerular filtration rate and higher segmental glomerulosclerosis and vascular arterial intima-to-lumen ratio. In vitro studies in podocytes demonstrated a critical role for PD-1 signaling in cell survival and in the induction of a senescence-associated secretory phenotype. To prove PD-1 signaling was critical to podocyte aging, aged mice were injected with anti–PD-1 antibody. Treatment significantly improved the aging phenotype in both kidney and liver. In the glomerulus, it increased the life span of podocytes, but not that of parietal epithelial, mesangial, or endothelial cells. Transcriptomic and immunohistochemistry studies demonstrated that anti–PD-1 antibody treatment improved the health span of podocytes. Administering the same anti–PD-1 antibody to young mice with experimental focal segmental glomerulosclerosis (FSGS) lowered proteinuria and improved podocyte number. These results suggest a critical contribution of increased PD-1 signaling toward both kidney and liver aging and in FSGS.

Long noncoding RNAs (lncRNAs) in HIV-mediated carcinogenesis: Role in cell homeostasis, cell survival processes and drug resistance.

There is accruing data implicating long non-coding RNAs (lncRNAs) in the development and progression of non-communicable diseases such as cancer. These lncRNAs have been implicated in many diverse HIV-host interactions, some of which are beneficial to HIV propagation. The virus-host interactions induce the expression of HIV-regulated long non-coding RNAs, which are implicated in the carcinogenesis process, therefore, it is critical to understand the molecular mechanisms that underpin these HIV-regulated lncRNAs, especially in cancer formation. Herein, we summarize the role of HIV-regulated lncRNAs targeting cancer development-related processes including apoptosis, cell cycle, cell survival signalling, angiogenesis and drug resistance. It is unclear how lncRNAs regulate cancer development, this review also discuss recent discoveries regarding the functions of lncRNAs in cancer biology. Innovative research in this field will be beneficial for the future development of therapeutic strategies targeting long non-coding RNAs that are regulated by HIV, especially in HIV associated cancers.

Antiproliferative effect of Potentilla fulgens on glioblastoma cancer cells through downregulation of Akt/mTOR signaling pathway.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor that is common among adults. This aggression is due to increased invasion, migration, proliferation, angiogenesis, and decreased apoptosis. Plant-based compounds have a high potential to be used as an anticancer agent due to their various mechanisms and less undesirable side effects. Potentilla fulgens is a medicinal plant, and methanolic root extract of P. fulgens (PRE) has anti-inflammatory and anticancer properties. In this study, we aimed to investigate antiproliferative effect of PRE on U118 and T98G glioblastoma cancer cells and to reveal which molecular signaling pathways regulate this mechanism of action. The effect of PRE on cell viability of GBM cells was investigated by MTT assay. Involvement of PRE with cell growth and survival signaling pathways, phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR and c-Src/signal transducer and activator of transcription 3 (STAT3), was examined using Western Blot. PRE reduced cell viability of GBM and human dermal fibroblast (HDF) cells in a dose-and time-independent manner. PI3K expression/phosphorylation level remained unchanged in both GBM and HDF cells after PRE treatment, but Akt/mTOR signaling pathway was downregulated in PRE-treated cells. PRE treatment did not affect c-Src expression/phosphorylation level in GBM cells; however, expression of c-Src was suppressed in HDF cells. Similar results were observed for STAT3 expression and phosphorylation status. PRE has the ability to suppress cell viability in GBM cells, by targeting the Akt/mTOR signaling pathway.

CD146 increases stemness and aggressiveness in glioblastoma and activates YAP signaling

Glioblastoma (GBM), a highly malignant and lethal brain tumor, is characterized by diffuse invasion into the brain and chemo-radiotherapy resistance resulting in poor prognosis. In this study, we examined the involvement of the cell adhesion molecule CD146/MCAM in regulating GBM aggressiveness. Analyses of GBM transcript expression databases revealed correlations of elevated CD146 levels with higher glioma grades, IDH-wildtype and unmethylated MGMT phenotypes, poor response to chemo-radiotherapy and worse overall survival. In a panel of GBM stem cells (GSCs) variable expression levels of CD146 were detected, which strongly increased upon adherent growth. CD146 was linked with mesenchymal transition since expression increased in TGF-ß-treated U-87MG cells. Ectopic overexpression of CD146/GFP in GG16 cells enhanced the mesenchymal phenotype and resulted in increased cell invasion. Conversely, GSC23-CD146 knockouts had decreased mesenchymal marker expression and reduced cell invasion in transwell and GBM-cortical assembloid assays. Moreover, using GSC23 xenografted zebrafish, we found that CD146 depletion resulted in more compact delineated tumor formation and reduced tumor cell dissemination. Stem cell marker expression and neurosphere formation assays showed that CD146 increased the stem cell potential of GSCs. Furthermore, CD146 mediated radioresistance by stimulating cell survival signaling through suppression of p53 expression and activation of NF-κB. Interestingly, CD146 was also identified as an inducer of the oncogenic Yes-associated protein (YAP). In conclusion, CD146 carries out various pro-tumorigenic roles in GBM involving its cell surface receptor function, which include the stimulation of mesenchymal and invasive properties, stemness, and radiotherapy resistance, thus providing an interesting target for therapy.

Context dependent role of p53 during the interaction of hepatocellular carcinoma and endothelial cells.

During the progression of hepatocellular carcinoma (HCC), several angiogenic factors are overexpressed in the hepatic microenvironment, which play a critical role in governing the phenotype of the endothelial cells. Mutation in the p53 gene (TP53) is a common event in HCC that may dysregulate the angiogenic signals. However, their functional messages remain largely unexplored at the onset of metastasis. Role of p53 was studied by siRNA mediated silencing of p53 in HepG2 cells (WTp53), collecting and analyzing their conditioned medium, followed by indirect co-culture with endothelial cells (HUVECs). Gene and protein expression in HCC cells and endothelial cells was studied by RT-qPCR and western blotting respectively. β-catenin protein expression and localization were analyzed by immunocytochemistry. We have studied a cell-to-cell interaction model to investigate the crosstalk of endothelial and hepatoma cells by either knocking down p53 or by using p53 null low metastatic HCC cell line. In the absence of p53, the HCC cells influence the migration and vascular network formation of endothelial cells through paracrine signaling of VEGF. Secretory VEGF activated the VEGF receptor-2 along with the survival signaling in endothelial cells. However, the β-catenin signal is upregulated in endothelial cells only during interaction with metastatic set up irrespective of absence and presence of p53, indicating context-dependent participation of p53 during communication between hepatoma cells and endothelial cells. This study highlights that the role of p53 on cellular responses during interaction of hepatocellular carcinoma and endothelial cells is distinct to cell types and context.

Plumbagin-induced anticancer effects are associated with mitochondrial-encoded respiratory gene downregulation in oral squamous cell carcinoma

Plumbagin (PL) is a known quinoid (5-hydroxyl-2-methyl-1,4-napthoquinone) initially isolated from the roots of Plumbago zeylanica L. It is reported to exert anti-proliferative effects in oral squamous cell carcinoma (OSCC) cells, suggesting that PL would be a promising therapeutic anti-cancer drug for OSCC; however, the molecular basis by which PL suppresses cell survival signaling is poorly understood. In this study, we conducted a comprehensive gene expression analysis to identify the molecular basis by which PL suppresses cell survival signaling. Human OSCC cell lines HSC-3 and SAS were used in this study. Comprehensive gene expression analysis was performed with an Agilent Whole Human Genome DNA microarray 4 × 44 format. Gene Set Enrichment Analysis was conducted to investigate the effect of PL on the oncogenic signaling pathway in the OSCC cells. cDNA microarray and subsequent gene set enrichment analyses showed that PL readily downregulates oxidative phosphorylation, MYC-target genes, and MTORC signaling at 4 h after treatment in HSC-3 and SAS cells. Semi-quantitative PCR analysis showed that gene expression levels of mitochondrial (Mt)-encoded COX-2, COX-3, and ND1 genes are significantly lower in the PL-treated OSCC cells than those in the untreated cells. In addition, an ROS scavenger, NAC, reversed the PL-induced downregulation of expression of Mt-encoded genes. Accordingly, intracellular ATP levels significantly decreased after PL treatment. Collectively, PL downregulates both oxidative phosphorylation and oncogenic signatures and decreases the survival of OSCC cells, thus highlighting its application as a potent treatment for patients with OSCC.

Abstract 1126: Concurrent degradation of BTK and IMiD neosubstrates by NX-2127 enhances multiple mechanisms of tumor killing

Abstract B-cell receptor (BCR) signaling is integral for the development, adhesion, growth, and survival of human B cells. Chronic activation of BTK-mediated BCR signaling is a hallmark of many B cell lymphoid malignancies, making BTK an attractive therapeutic target. While covalent inhibitors of BTK have proven effective, they have been associated with the occurrence of acquired resistance mutations. IMiDs (immunomodulatory imide drugs) exert direct anti-cancer effects in B cell malignancies as well as enhance the activity of T cells. Based on clinical data of BTK covalent inhibitors and IMiDs in B-cell malignancies, a strategy combining BTK and IMiD targeting in a single oral, small molecule may improve anti-tumor activity. Here we describe NX-2127, a bifunctional molecule that catalyzes the degradation of BTK and IMiD neosubstrates Aiolos and Ikaros. In primary human T cells, NX-2127 degrades Aiolos (DC50=25 nM) and Ikaros (DC50=54 nM) and enhances IL-2 secretion. NX-2127 promotes superior in vitro killing of a DLBCL cell line, TMD8, and a MCL cell line, REC-1, as compared to BTK inhibitors ibrutinib, acalabrutinib, and pirtobrutinib, or IMiDs pomalidomide and lenalidomide. BTK inhibitors promote partial death of TMD8 cells (Emax = 17-33% viable) and REC-1 cells (Emax = 46-52% viable). By contrast, the dual BTK and IMiD activity of NX-2127 promotes complete killing of TMD8 and REC-1 cells and does so more potently than IMiDs alone. In REC-1 cells, suppression of Ikaros protein levels by IMiDs, BTK inhibitors, and NX-2127 correlates closely with downstream viability reduction, independent of drug mechanism. In vivo, oral administration of NX-2127 demonstrates dose proportional degradation of BTK and Aiolos protein in a TMD8 xenograft model, closely correlating with the degree of tumor control. RNA-seq analysis performed on REC-1 cells demonstrates that NX-2127 exhibits distinct regulation of gene expression when compared to ibrutinib or pomalidomide. Gene set enrichment analysis (GSEA) of these uniquely modulated genes reveals that NX-2127 downregulates gene sets involved in cell cycle, DNA replication, DNA repair, and survival signaling pathways. Furthermore, a key mediator of T cell recognition is more strongly upregulated by NX-2127 than pomalidomide and ibrutinib at both the transcript and cell surface expression levels in REC-1 cells, implying a potential immune-mediated anti-neoplastic mechanism. The gene expression signature of NX-2127 exposure supports the hypothesis that its anti-tumor effects include both tumor cell-intrinsic and tumor cell-extrinsic mechanisms and suggests that the combination of BTK degradation and IMiD activity may be more efficacious in treating certain B-cell malignancies than either activity alone. A phase 1a/b trial of NX-2127 for patients with relapsed or refractory B-cell malignancies is ongoing. Citation Format: Mark A. Noviski, Jun Ma, Ernestine Lee, Nivetha Brathaban, May Tan, Luz Perez, Daniel W. Robbins, Austin Tenn-McClellan, Janine Powers, Gwenn Hansen, Cristiana Guiducci, Ryan Rountree. Concurrent degradation of BTK and IMiD neosubstrates by NX-2127 enhances multiple mechanisms of tumor killing [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 1126.

Abstract 1008: Cell density-related variability in chemotherapeutic resistance patterns in human cancer cells

Abstract Assessment of chemosensitivity and resistance varies with cancer cell of origin, drug and culture conditions. However, it is also not uncommon to find multiple different IC50 values for the same chemotherapeutic treatments acting on the same tumor cell lines. The aim of this research was to investigate this discrepancy in phenotypes and elucidate relevant variables that influence them. We assessed chemosensivity as a function of tumor cell density by plating TOV-21G ovarian, HT29 colorectal, and U2OS osteosarcoma cell lines in 96-well plates at various seeding densities per well and treated them with various concentrations of cisplatin, 5-fluorouracil (5-FU), and etoposide, respectively. CellTiter Glo assays were performed at 72 hours of treatment to determine IC50. Cisplatin IC50 for TOV-21G cells plated at 50,000, 20,000, 10,000, 5,000, 2,000, 1,000, and 500 cells/well were 12.00 μM, 5.47 μM, 3.16 μM, 2.45 μM, 2.54 μM, 2.83 μM, and 2.66 μM, respectively. Etoposide IC50 for U2OS cells plated at the same seeding densities were 6.50 μM, 5.19 μM, 3.23 μM, 2.98 μM, 2.49 μM, 2.13 μM, and 1.88 μM, respectively. IC50 values for HT29 cells treated with 5-FU at 2,000, 1,000, and 500 cells/well were 35.89 μM, 19.74 μM, and 13.42 μM, respectively. TOV-21G, U2OS, and HT29 cells all showed differences in IC50 values depending on cell plating density. TOV-21G and U2OS cells both showed a gradual decrease and plateau in IC50 as the cancer cell density decreased from 50,000 cells/well to 500 cells/well. HT29 cells displayed a sharper IC50 decrease as the cell density decreased from 2,000 cells/well to 500 cells/well. Our experiments demonstrate that IC50 values are not static at 72 hours and that tumor cell density is an important variable in influencing observed tumor resistance to traditionally effective drugs. As the density of cancer cells increases, the cells develop more resistance to the associated chemotherapy and require higher quantities of drug to achieve the same level of growth inhibition. We are currently exploring underlying molecular mechanisms that may influence these changes in IC50 including cell survival signaling proteins, cell-cell contact or growth factors that may influence cell density-related chemoresistance. Citation Format: Thomas Brown, Ujwal Punyamurtula, Jill Strandberg, Wafik S. El-Deiry. Cell density-related variability in chemotherapeutic resistance patterns in human 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 1008.

Interplay between symmetric arginine dimethylation and ubiquitylation regulates TDP1 proteostasis for the repair of topoisomerase I-DNA adducts.

Tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety and is implicated in the repair of trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation of TDP1 at the residues R361 and R586. Here, we establish mechanistic crosstalk between TDP1 arginine methylation and ubiquitylation, which is critical for TDP1 homeostasis and cellular responses to Top1 poisons. We show that R586 methylation promotes TDP1 ubiquitylation, which facilitates ubiquitin/proteasome-dependent TDP1 turnover by impeding the binding of UCHL3 (deubiquitylase enzyme) with TDP1. TDP1-R586 also promotes TDP1-XRCC1 binding and XRCC1 foci formation at Top1cc-damage sites. Intriguingly, R361 methylation enhances the 3'-phosphodiesterase activity of TDP1 in real-time fluorescence-based cleavage assays, and this was rationalized using structural modeling. Together, our findings establish arginine methylation as a co-regulator of TDP1 proteostasis and activity, which modulates the repair of trapped Top1cc.

Data-Driven Mathematical Model of Apoptosis Regulation in Memory Plasma Cells.

Memory plasma cells constitutively produce copious amounts of antibodies, imposing a critical risk factor for autoimmune disease. We previously found that plasma cell survival requires secreted factors such as APRIL and direct contact to stromal cells, which act in concert to activate NF-κB- and PI3K-dependent signaling pathways to prevent cell death. However, the regulatory properties of the underlying biochemical network are confounded by the complexity of potential interaction and cross-regulation pathways. Here, based on flow-cytometric quantification of key signaling proteins in the presence or absence of the survival signals APRIL and contact to the stromal cell line ST2, we generated a quantitative model of plasma cell survival. Our model emphasizes the non-redundant nature of the two plasma cell survival signals APRIL and stromal cell contact, and highlights a requirement for differential regulation of individual caspases. The modeling approach allowed us to unify distinct data sets and derive a consistent picture of the intertwined signaling and apoptosis pathways regulating plasma cell survival.

A mechanism of regulation for the ubiquitin‐sensing kinase TNK1

TNK1 is a poorly understood non‐receptor tyrosine kinase that by mutation can be converted into an oncogenic driver. While little is known about the normal function of TNK1, it possesses a C‐terminal ubiquitin binding domain (UBA) that interacts with high affinity to multiple poly‐ubiquitin linkages. To our knowledge, this ability to directly interact with poly‐ubiquitin makes TNK1 unique across the human kinome. Our recent data suggest that the UBA domain of TNK1 homes the kinase to phase‐separated condensates of poly‐ubiquitinated proteins where TNK1 becomes fully active. We hypothesize that the interaction between the TNK1 UBA and poly‐ubiquitin at these ubiquitin‐rich condensates tethers the kinase to its substrates, which are involved in condensate biology and cell survival signaling. These findings led us to question how the interaction between TNK1 and ubiquitin is regulated. We have focused on a phosphorylation at Y661 within the TNK1 UBA domain, which has been identified in high throughput PTM mass spectrometry (phosphosite.org) but is of unknown function. Here we show that intrinsic TNK1 kinase activity is required for phosphorylation of Y661, suggesting that it may be an autophosphorylation. Furthermore, our preliminary data suggest that a phosphomimic mutation at Y661 leads to an increase in ubiquitin binding, suggesting that this phosphorylation may enhance the affinity of the TNK1 UBA for ubiquitin. Together, our data suggest a feed‐forward mechanism of TNK1 regulation in which activation of TNK1 kinase activity primes the UBA for binding ubiquitin via phosphorylation at Y661. In turn, the UBA domain helps cluster active TNK1 at ubiquitin‐rich condensates where TNK1 is brought into proximity to its substrates.

Soluble Isoform of ST2 is a Cardiac Inducer of Collagen Deposition in an Ex Vivo Model of Obesity

IntroductionOne of the main cardiac mechanosensor is the IL‐33/ST2L complex composed by the allarmin IL‐33 released into extracellular space by damaged cells during cardiac overload and its receptor, ST2L, which their binding promotes cell survival and anti‐fibrotic signaling. After damage, to stop IL‐33/ST2L signal, resident cardiac cells release sST2, the soluble isoform of transmembrane receptor of ST2, which sequesters IL‐33, functioning as a decoy receptor and blocking its signal. In obesity, the sST2 become over express especially by hypertrophic fat cells. The aim of our study is to evaluate in an ex vivo model of obesity the role of sST2 in the promotion of collagen production by cardiac cells.MethodsTen obese nondiabetic male Zucker rats (OB) (fa/fa‐, 10 weeks of age) and 10 lean littermates (L) (Fa/+) at the age of 25 weeks were weighted and scarified according to Italian Ministry of Health Authorization (N°325/2015PR of 2015/04/05) and their heart and visceral adipose tissue (VAT) were collected for the analysis. VAT biopsies were cut in 30mg pieces and added into a co‐culture system with H9C2 for 6‐24 and 48 hours. At each time points, cells and culture medium were collected. In all samples were evaluated ST2 by ELISA and collagen were quantified by quantitative Sircol assay for measurement of both acid‐soluble and pepsin‐soluble collagens.ResultsOB cardiac biopsies showed higher protein level of both ST2 and collagen than L rats (p&lt;0.02 and p&lt;0.002 respectively) and the rat weight positively correlates with ST2 cardiac protein level (p&lt;0.05). Co‐culture data demonstrated that OB VAT promotes ST2 releases in both cardiac and fat cells, resulting an incremental increase of sST2 release in cell culture medium than L VAT in time. sST2 quantification in correspondent H9C2 co‐cultured lysate is more incremented in OB than L H9C2 after 48h.total collagen quantification in co‐culture medium resulted higher in OB than L (p&lt;0.05).ConclusionOur results demonstrated in a co‐culture system that obesity promotes sST2 increase in the heart and this promotes collagen releases by cardiac cells, suggesting sST2 role in activation of pro‐fibrotic signaling and detrimental heart remodeling.

RSK as a Therapeutic Target for ER+ Breast Cancer

We focus on identification of kinase signaling pathways, inhibition of which could be used in combination with endocrine therapy for treatment of Estrogen Receptor (ER)‐positive breast cancer. The p90 Ribosomal S6 Kinase (RSK), an effector of the MAPK signaling pathway, plays a key role in cell growth and survival, through the regulation of many downstream targets, and may serve a role in promoting response to endocrine therapy. To further elucidate the role of RSK in crosstalk with estrogen signaling pathways, we first examined the expression of RSK in ER‐positive breast cancer subtype and correlation with phosphopeptide abundance using phosphoproteomics analysis. Our results suggest that RSK is highly expressed in ER‐positive breast cancer and also promotes the phosphorylation of many targets involved in cell growth and survival signaling pathways. Our ongoing investigation characterizes the role of new targets of RSK in ER‐positive breast cancer, with the long‐term goal to develop further targeted therapies that can increase sensitivity to endocrine therapy and prevent the development of therapeutic resistance.