Serum-starved Conditions Research Articles

Wharton jelly-derived mesenchymal stem cell exosomes induce apoptosis and suppress EMT signaling in cervical cancer cells as an effective drug carrier system of paclitaxel.

Mesenchymal stem cells can be obtained and multiplied from various sources and have a very high capacity to release exosomes. Exosomes are nano-sized extracellular vesicles containing biological signaling molecules. This study aimed to determine the effect of MSC-derived exosomes as a drug delivery system for paclitaxel in cervical cancer cells. In this study, human MSC were isolated from wharton jelly of umbilical cord tissue (WJ-MSC), and cells were characterized by CD44, CD90, CD105, and CD34 staining. Exosomes were released in WJ-MSC cells with serum-starved conditions for 48 hours, and particle sizes and structures were examined with zeta-sizer and TEM. In addition, exosomes CD9, CD63, and CD81 markers were checked by western blot. Paclitaxel was loaded into exosomes (Exo-PAC) by electroporation and then incubated with Hela cervical cancer cells for 24 hours. TGF-β, SMAD, Snail, Slug, β-catenin, Notch, Caspase-3, Caspase-9, Bax, Bcl-2 protein and gene expression levels were analyzed in Hela cells. As a result, low concentration Exo-PAC induced apoptosis, and suppressed epithelial-mesenchymal transition proteins in Hela cells. In this study, it has been demonstrated that WJ-MSCs can be used as drug delivery systems for cervical cancer if exosomes are produced scalably in the future.

Abstract 1119: Non-adherent breast and non-small-cell lung cancer cell cultures as a promising CTCs’ model for evaluation of the anti-tumor effects of artesunate

Abstract Background: Artesunate (AS), besides being widely used as an antimalarial agent, has been also suggested to exert anti-tumor activity in a plethora of cancer types, including breast and lung cancer. Recently, AS has been shown to inhibit the expression of JunB, which is associated with metastatic progression. The aim of this study is to examine the effect of AS on the viability of both adherent and non-adherent (resembling circulating tumor cells; CTCs) breast and lung cancer cell cultures. Methods: Prevention of cell attachment and establishment of the CTCs’ model was achieved via polyHEMA [poly(2-hydroxyethyl methacrylate)] treatment. Viability of adherent and non-adherent MDA-MB-231 (triple negative breast cancer) and H1299 (non-small-cell lung cancer) cells was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT) assay, 48 h following treatment with different concentrations of AS (3 μM to 100 μΜ) in serum starvation conditions. Cell migration was examined using the wound healing assay. Results: Optimization involving the duration (20 min vs. overnight) and concentration (10 mg/ml) of polyHEMA coating, as well as the duration of AS treatment (24 h vs. 48 h) were performed. 5-fluorouracil was used as a positive control. AS decreased viability of both cell lines in a concentration-dependent manner with comparable IC50, whether being adherent (pIC50 = 4.6 ± 0.1 and 5.1 ± 0.1 for MDA-MB-231 and H1299, respectively; n = 8) or non-adherent (pIC50 = 4.5 ± 0.1 and 4.9 ± 0.1 for MDA-MB-231 and H1299, respectively; n =8). AS (10 μΜ, 48 h) inhibited proliferation of both adherent MDA-MB-231 (34% ± 5, n = 8) and H1299 (57% ± 4, n = 8) cells. Interestingly, the effect of AS on MDA-MB-231 and H1299 non-adherent cultures was less prominent (15% ± 6 and 26% ± 6, P<0.001, respectively; n = 8), demonstrating resistance to AS. Furthermore, AS (10 μM, 48 h) decreased cell migration, an effect which was more pronounced in H1299 cells. Conclusions: Evidently, non-adherent, free-floating cells seem to respond differently to AS compared to their attached counterparts. These results suggest that antitumor agents might exert different effects in CTCs compared to adherent tumor cells. Acknowledgements: This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T2ΕΔΚ-01562). Citation Format: Evangelia Pantazaka, Dafni Graikioti, Constantinos M. Athanassopoulos, Galatea Kallergi. Non-adherent breast and non-small-cell lung cancer cell cultures as a promising CTCs’ model for evaluation of the anti-tumor effects of artesunate [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 1119.

Abstract 831: Discovery of a novel soluble Neuropilin-2 isoform with antiangiogenic and antitumorigenic activity

Abstract Upregulation of the non-tyrosine kinase receptor neuropilin-2 (NRP2) is described for several tumor entities and correlates with disease progression. Two membrane-anchored isoforms are transcribed from the NRP2 gene that only differ in their terminal exons to encode transmembrane stretches and short cytoplasmic tails, respectively. While NRP2a was shown to support tumor cell proliferation, NRP2b facilitates tumor cell migration, resulting in enhanced metastatic spreading. A third, soluble NRP2 isoform encoding only aminoterminal protein domains exists as dimeric protein and functions in sequestration of the VEGF-C ligand. By using a next-generation sequencing approach to enrich for NRP2 terminal exons, we identified NRP2-Mo83, a novel NRP2 isoform, from a high expressing tumor cell line, and verified secreted NRP2-Mo83 protein by mass spectrometry in cell supernatants. Preliminary mass spectrometric data give an indication of the presence of soluble NRP2-Mo83 protein in human serum samples.NRP2-Mo83 mRNA is expressed at variable quantities in vitro in a panel of bladder carcinoma cell lines, but is downregulated in a small cohort of macrodissected urothelial carcinoma samples. Transcript abundances of a NRP2 transcript classified as non-coding with high sequence similarity to the NRP2-Mo83 3’ terminus confirm the observed downregulation in the TCGA bladder carcinoma cohort.Preliminary data indicate that recombinant NRP2-Mo83 protein exerts a dose-dependent inhibitory effect on tumor cell growth in vitro in all tested bladder carcinoma cell lines in the presence of serum. However, a fraction of cell lines does not respond to NRP2-Mo83 under serum-starved conditions. Proliferation of human endothelial cells (HUVEC) is inhibited in a dose-dependent manner by recombinant NRP2-Mo83 protein in vitro. In vivo, NRP2-Mo83 reveals an anti-angiogenic effect on the chicken chorioallantoic membrane.Recombinant NRP2-Mo83 is secreted by HEK293 cells. Enrichment of endogenous and purification of recombinant NRP2-Mo83 protein is achieved by exploiting its capacity to bind heparin. In contrast to the described s9NRP2 isoform, NRP2-Mo83 is largely monomeric in solution, as verified by size exclusion chromatography. We identified N and O glycosylation as well as the capacity for polysialation as posttranslational modifications of recombinant NRP2-Mo83 protein, similar to membrane anchored NRP2 isoforms. NanoDSF experiments indicate that calcium ions stabilize NRP2-Mo83. We present NRP2-Mo83 as a novel NRP2 splice isoform, validated its expression at the mRNA and protein level. NRP2-Mo83 appears down-regulated during bladder tumorigenesis, congruent with its function in bladder tumor growth inhibition and its anti-angiogenic properties in vitro and in vivo. The identification of NRP2-Mo83 in human serum may suggest a suppressive function even under non-pathological conditions. Citation Format: Thomas Mayr, Paul L. Dix, Sayed-Mohammad Hasheminasab, Chuanpit Hampel, Marc Sylvester, Niels Schneberger, Sarah Foerster, Gregor Hagelueken, Samikshan Dutta, Glen Kristiansen, Regine Schneider-Stock, Kaustubh Datta, Michael H. Muders. Discovery of a novel soluble Neuropilin-2 isoform with antiangiogenic and antitumorigenic activity [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 831.

AGE/RAGE axis regulates reversible transition to quiescent states of ALK-rearranged NSCLC and pancreatic cancer cells in monolayer cultures

Cancer recurrence due to tumor cell quiescence after therapy and long-term remission is associated with cancer-related death. Previous studies have used cell models that are unable to return to a proliferative state; thus, the transition between quiescent and proliferative states is not well understood. Here, we report monolayer cancer cell models wherein the human non-small cell lung carcinoma cell line H2228 and pancreatic cancer cell line AsPC-1 can be reversibly induced to a quiescent state under hypoxic and serum-starved (HSS) conditions. Transcriptome and metabolome dual-omics profiles of these cells were compared with those of the human lung adenocarcinoma cell line A549, which was unable to enter a quiescent state under HSS conditions. The quiescence-inducible cells had substantially lower intracellular pyruvate and ATP levels in the quiescent state than in the proliferative state, and their response to sudden demand for energy was dramatically reduced. Furthermore, in quiescence-inducible cells, the transition between quiescent and proliferative states of these cells was regulated by the balance between the proliferation-promoting Ras and Rap1 signaling and the suppressive AGE/RAGE signaling. These cell models elucidate the transition between quiescent and proliferative states, allowing the development of drug-screening systems for quiescent tumor cells.

Transcription of cytochrome P450 46A1 in NIH3T3 cells is negatively regulated by FBS

Extracellular administration of side-chain oxysterols, such as 24S-hydroxycholesterol (24S-HC), 27-hydroxycholesterol (27-HC) and 25-hydroxycholesterol (25-HC) to cells suppresses HMG-CoA reductase (Hmgcr) and CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) mRNA levels. Oxysterols are enzymatically produced in cells from cholesterol by cytochrome P450 46A1 (Cyp46A1), Cyp27A1, Cyp3A11 and cholesterol 25-hydroxylase (Ch25h). We analyzed which of these oxysterol-producing enzymes are expressed in NIH3T3 cells and found that only Cyp46A1 was expressed. When Cyp46A1 was overexpressed in NIH3T3 cells, intrinsic oxysterols increased in the order 24S-HC > 25-HC > 27-HC. We investigated the mechanism regulating the production of endogenous oxysterols in NIH3T3 cells by Cyp46A1 and found that the mRNA, relative protein levels and enzymatic activity of Cyp46A1, and the amounts of 24S-HC, 25-HC and 27-HC significantly increased under serum-starved conditions, and these increases were suppressed by FBS supplementation. The aqueous phase of FBS obtained by the Bligh & Dyer method significantly suppressed Cyp46A1 mRNA levels. Fractionation of the aqueous phase by HPLC and analysis of the inhibiting fractions by nanoLC and TripleTOF MS/MS identified insulin-like factor-II (IGF-II). Cyp46A1 mRNA levels in serum-starved NIH3T3 cells were significantly suppressed by the addition of IGFs and insulin and endogenous oxysterol levels were decreased. CYP46A1 mRNA levels in the T98G human glioblastoma cell line were also increased by serum starvation but not by FBS supplementation, and the aqueous phase did not inhibit the increase. These results suggest that mRNA levels of Cyp46A1 are regulated by factors in FBS.

A novel isophorone-derived fluorescent probe for detecting sulfite and the application in monitoring the state of hybridoma cells

In this work, a novel isophorone-derived fluorescent probe, ISOP-SFT, was developed for the detection of sulfite, and, for the first time, it was applied into monitoring the state of hybridoma cells. ISOP-SFT could exhibit a red-emission response to sulfite with the excitation at 455 nm. It also indicated practical advantages such as wide pH tolerance within 6.0–9.0 and high storage steadiness within at least one week. The linear range was relatively long (0–20 Eq) and reliable (Pearson's correlation coefficient: 0.9989), while Limit of Detection was determined as 0.0095 Eq (95 nM). For the selectivity, ISOP-SFT could distinguish sulfite from other analytes, and the response of ISOP-SFT towards sulfite was not seriously interfered in co-existence system. With low cytotoxicity, ISOP-SFT achieved monitoring both the exogenous and endogenous sulfite in living MCF-7 cells. As a novel attempt, ISOP-SFT was applied into monitoring the state of hybridoma cells, and associated with the quality control of producing antibodies. The result preliminarily inferred the positive correlation between the production efficiency of antibodies in hybridoma cells and the intracellular sulfite level, including the conditions of serum starvation, oxidative stress, severe hypoxia and endogenous induction. We hope that the information in this work could lead to new applications of fluorescent probes in biological industry in future.

The Influence of Angiotensin Peptides on Survival and Motility of Human High-Grade Serous Ovarian Cancer Cells in Serum Starvation Conditions.

High-grade serous ovarian carcinoma (HGSOC) is the most frequent and malignant form of ovarian cancer. A local renin–angiotensin system (RAS) has been found in the ovary, and changes in selected components of this system were observed in pathological states and also in ovarian cancer. In the present study, we examined the effect of three peptides, Ang-(1-7), Ang-(1-9) and Ang-(3-7), on proliferation and motility of the OVPA8 cell line, a new well-defined and preclinical model of HGSOC. We confirmed the presence of mRNA for all angiotensin receptors in the tested cells. Furthermore, our findings indicate that all tested angiotensin peptides increased the metabolic serum in the medium by activation of cell defense mechanisms such as nuclear factor kappaB signaling pathway andapoptosis. Moreover, tested angiotensin peptides intensified serum starvation-induced cell cycle arrest at the G0/G1 phase. In the case of Ang-(3-7), a significant decrease in the number of Ki67 positive cells (Ki67+) and reduced percentage of activated ERK1/2 levels in ovarian cancer cells were additionally reported. The angiotensin-induced effect of the accumulation of cells in the G0/G1 phase was not observed in OVPA8 cells growing on the medium with 10% FBS. Moreover, in the case of Ang-(3-7), the tendency was quite the opposite. Ang-(1-7) but not Ang-(1-9) or Ang-(3-7) increased the mobility of reluctant-to-migrate OVAP8 cells cultured in the serum-free medium. In any cases, the changes in the expression of VIM and HIF1A gene, associated with epithelial–mesenchymal transition (EMT), were not observed. In conclusion, we speculate that the adaptation to starvation in nutrient-deprived tumors can be modulated by peptides from the renin–angiotensin system. The influence of angiotensin peptides on cancer cells is highly dependent on the availability of growth factors and nutrients.

Interplay between p300 and HDAC1 regulate acetylation and stability of Api5 to regulate cell proliferation

Api5, is a known anti-apoptotic and nuclear protein that is responsible for inhibiting cell death in serum-starved conditions. The only known post-translational modification of Api5 is acetylation at lysine 251 (K251). K251 acetylation of Api5 is responsible for maintaining its stability while the de-acetylated form of Api5 is unstable. This study aimed to find out the enzymes regulating acetylation and deacetylation of Api5 and the effect of acetylation on its function. Our studies suggest that acetylation of Api5 at lysine 251 is mediated by p300 histone acetyltransferase while de-acetylation is carried out by HDAC1. Inhibition of acetylation by p300 leads to a reduction in Api5 levels while inhibition of deacetylation by HDAC1 results in increased levels of Api5. This dynamic switch between acetylation and deacetylation regulates the localisation of Api5 in the cell. This study also demonstrates that the regulation of acetylation and deacetylation of Api5 is an essential factor for the progression of the cell cycle.

Secretory Leukocyte Proteinase Inhibitor Protects Acute Kidney Injury Through Immune and Non-Immune Pathways.

Acute kidney injury (AKI) is characterized by rapid loss of excretory function and is the clinical manifestation of several disorders affecting the kidney. The aim of the present study was to investigate the mechanism of action of Secretory Leukocyte Proteinase Inhibitor (SLPI) that protects the kidneys form AKI. In vivo and in vitro experiments were performed to assess the effect of SLPI on kidney injury. Animal models of kidney injury was generated by 40 min obstruction of kidney artery and vein (ischemia-reperfusion injury model) or daily administration of 60 mg/kg/day of gentamicine for 5 day (gentamicin-associated AKI model). For in vitro assessment, human renal epithelium HK-2 cells were cultured under serum starvation conditions or with tacrolimus. The administration of SLPI (250 μg/kg, i.p.) reduced elevated plasma creatinine and blood urea nitrogen levels, tissue myeloperoxidase content, and acute tubular necrosis induced by kidney damage. Furthermore, SLPI treatment reduced CD86, CD68, CD14, CCL2, TNFα, and IL-10 transcripts in kidney biopsies. To further analyze a direct effect of SLPI on renal epithelial cells, HK-2 cells from human renal epithelium were cultured under serum starvation conditions or with tacrolimus. Both conditions induced apoptosis of HK-2 cells which was reduced when SLPI was present in the culture medium. Furthermore, SLPI favored the proliferation and migration of HK-2 cells. An analysis of the gene profiles of HK-2 cells treated with calcineurin inhibitors affected inflammatory and non-inflammatory pathways that were reversed by SLPI. Among them, SLPI down modulated the expression of CCL2, SLC5A3, and BECN1 but up-regulated the expression of TLR4, ATF4, ATF6, HSP90B, BBC3 SLC2A1, and TNFRSF10B. Overall, these results suggest that SLPI, in addition to its activity on immune cells, may directly target tubular epithelial cells of the kidney to mediate the nephroprotective activity in AKI.

The Nucleolar Protein Nucleophosmin Is Physiologically Secreted by Endothelial Cells in Response to Stress Exerting Proangiogenic Activity Both In Vitro and In Vivo.

Nucleophosmin (NPM), a nucleolar multifunctional phosphoprotein, acts as a stress sensor in different cell types. NPM can be actively secreted by inflammatory cells, however its biology on endothelium remains unexplored. In this study, we show for the first time that NPM is secreted by human vein endothelial cells (HUVEC) in the early response to serum deprivation and that NPM acts as a pro-inflammatory and angiogenic molecule both in vitro and in vivo. Accordingly, 24 h of serum starvation condition induced NPM relocalization from the nucleus to cytoplasm. Interestingly, NPM was increasingly excreted in HUVEC-derived conditioned media in a time dependent fashion upon stress conditions up to 24 h. The secretion of NPM was unrelated to cell necrosis within 24 h. The treatment with exogenous and recombinant NPM (rNPM) enhanced migration as well as the Intercellular Adhesion Molecule 1 (ICAM-1) but not Vascular cell adhesion protein 1 (VCAM-1) expression and it did not affect cell proliferation. Notably, in vitro tube formation by Matrigel assay was significantly increased in HUVEC treated with rNPM compared to controls. This result was confirmed by the in vivo injection of Matrigel plug assay upon stimulation with rNPM, displaying significant enhanced number of functional capillaries in the plugs. The stimulation with rNPM in HUVEC was also associated to the increased expression of master genes regulating angiogenesis and migration, including Vascular Endothelial Growth Factor-A (VEGF-A), Hepatocyte Growth Factor (HGF), Stromal derived factor-1 (SDF-1), Fibroblast growth factor-2 (FGF-2), Platelet Derived Growth Factor-B (PDGF-B), and Matrix metallopeptidase 9 (MMP9). Our study demonstrates for the first time that NPM is physiologically secreted by somatic cells under stress condition and in the absence of cell necrosis. The analysis of the biological effects induced by NPM mainly related to a pro-angiogenic and inflammatory activity might suggest an important autocrine/paracrine role for NPM in the regulation of both phenomena.

SOX2 enhances cell survival and induces resistance to apoptosis under serum starvation conditions through the AKT/GSK-3β signaling pathway in esophageal squamous cell carcinoma.

The human SOX2 gene was recently identified as a novel major oncogene, recurrently amplified and overexpressed in esophageal squamous cell carcinoma (ESCC). However, the role and molecular mechanism of SOX2 in the carcinogenesis of ESCC remain to be elucidated. The present study investigated the effect of SOX2 on ESCC cell survival and resistance to apoptosis under serum starvation conditions. An adenoviral vector-mediated expression system and RNA interference were used to study the effect of SOX2. The present results revealed that SOX2 promoted ESCC cell survival and enhanced resistance to apoptosis under serum starvation conditions, but not in culture conditions with serum. Mechanistically, SOX2 increased the expression levels of phosphorylated AKT and glycogen synthase kinase-3β (GSK-3β), a downstream factor of AKT, under serum starvation conditions, leading to the promotion of ESCC cell survival. Additionally, SOX2 activated AKT through the PTEN/PI3K/phosphoinositide-dependent protein kinase 1 and mammalian target of rapamycin complex 2 signaling pathways. Therefore, SOX2 may facilitate the survival of ESCC cells under poor nutrient conditions by activating the AKT/GSK-3β signaling pathway.

Ginsenoside Rh2 represses autophagy to promote cervical cancer cell apoptosis during starvation

BackgroundCancer cells through autophagy-mediated recycling to meet the metabolic demands of growth and proliferation. The steroidal saponin 20(S)-ginsenoside Rh2 effectively inhibits the growth and survival of a variety of tumor cell lines and animal models, but the effects of Rh2 on autophagy remain elusive.MethodsCell viability was measured by CCK-8 (cell counting kit-8) assays. Apoptosis, ROS generation and mitochondrial membrane potential were analyzed by flow cytometry. Western blot analyses were used to determine changes in protein levels. Morphology of apoptotic cells and autophagosome accumulation were analyzed by DAPI staining and transmission electron microscopy. Autophagy induction was monitored by acidic vesicular organelle staining, EGFP-LC3 and mRFP-GFP-LC3 transfection. Atg7 siRNA and autophagy regulator was used to assess the effect of autophagy on apoptosis induced by G-Rh2.ResultsIn this study, we found that low concentration G-Rh2 attenuated cancer cell growth and induced apoptosis upon serum-free starvation. Caspase 3 inhibitors failed to block apoptosis in G-Rh2-treated cells, indicating a caspase-independent mechanism. G-Rh2-treated cells in serum-deprived conditions showed impaired mitochondrial function, increased release and nuclear translocation of apoptosis-inducing factor, but little changes in the mitochondrial and cytoplasmic distributions of cytochrome C. Annexin A2 overexpression in 293T cells inhibited G-Rh2-induced apoptosis under serum-starved conditions. Meanwhile, G-Rh2 reduced lysosomal activity and inhibited the fusion of autophagosome and lysosome, leading to a block of autophagic flux. Knockdown Atg7 significantly inhibited autophagy and triggered AIF-induced apoptosis in serm free condition. The autophagy inducer significantly decreased the apoptosis levels of G-Rh2-treated cells in serum-free conditions.ConclusionsUnder nutrient deficient conditions, G-Rh2 represses autophagy in cervical cancer cells and enhanced apoptosis through an apoptosis-inducing factor mediated pathway.

TMOD-33. THE ROLE OF BAFF-R SIGNALING IN THE GROWTH OF PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA

Abstract Primary CNS lymphoma (PCNSL) is an aggressive brain tumor. Despite improvements in therapeutic algorithms, long-term survival remains rare, illustrating an urgent need for novel therapeutic targets. BAFF-R is a pro-survival receptor expressed on most malignant B cells, including PCNSL. To date, its role in PCNSL growth remains elusive. Here, we created a BAFF-R knockout lymphoma cell line (BAFF-R-KO) using CRISPR-Cas9. In serum-starved conditions, BAFF-R-KO cells exhibit decreased viability in vitro compared to BAFF-R+ cells. Combining an orthotopic mouse model of PCNSL with chronic cranial windows and intravital microscopy, we demonstrate a significant delay in tumor growth in mice inoculated with BAFF-R-KO cells compared to BAFF-R+ PCNSL. Additionally, median survival of BAFF-R-KO mice was significantly prolonged. Altogether, our results indicate a potential of BAFF-R as a novel treatment target for PCNSL.

CSIG-12. HDACs-Sp1-BMI1 PATHWAY IS INVOLVED IN THE ATTENUATION OF STRESS- AND THERAPY-INDUCED DEATH IN GLIOMA

Abstract BMI-1 is a transcriptional repressor and overexpresses in various human cancer tissues, which is also known to involve in the maintenance of “stem cell” features in a cell sub-population within the tumor. Here, we investigate the roles of BMI1 oncogene in brain tumors, notably involving drug-resistant metabolism. Interestingly, microarray datasets in OMCOMINE indicated that BMI1 is not commonly increased in clinical brain tumors, and survival plots in PROGgeneV2 also showed that BMI1 expression is not statistically associated with glioma patients. However, stressful serum starvation and anchorage independence growth conditions enabled to induce upregulation of BMI1. Further investigation, we identified HDAC-Sp1 is a stress-responsive pathway, critically promoting BMI1 expression. HDAC inhibition via vorinostat treatment prevented the binding of Sp1 to BMI1 promoter, leading to decreased BMI1 expression and attenuated tumor growth of TMZ-resistant glioma xenografts. Importantly, increased expression of the HDAC-Sp1-BMI1 axis but not BMI1 alone showed an increased risk of death in both high- and low-grade glioma. Thus, the HDAC-Sp1-BMI1 axis is critical to attenuate stress- and therapy-induced death in glioma cells and the axis inhibition may prevent the recurrence of malignant cells persisting after primary therapy.

CC-486 Mechanism Imparted By Extended Exposure of Azacitidine Upregulates Myeloid Differentiation Markers and Induces Cell Death in AML Cells

CC-486 Mechanism Imparted By Extended Exposure of Azacitidine Upregulates Myeloid Differentiation Markers and Induces Cell Death in AML Cells

Secretory clusterin promotes oral cancer cell survival via inhibiting apoptosis by activation of autophagy in AMPK/mTOR/ULK1 dependent pathway

AimsSecretory clusterin (sCLU) plays an important role in tumor development and cancer progression. However, the molecular mechanisms and physiological functions of sCLU in oral cancer is unclear. We examined the impact of sCLU-mediated autophagy in cell survival and apoptosis inhibition in oral cancer. Main methodsImmunohistochemical analysis was performed to analyze protein expression in patient samples. Autophagy and mitophagy was studied by immunofluorescence microscopy and Western blot. The gain and loss of function was studied by overexpression of plasmid and siRNA approaches respectively. Cellular protection against nutrient starvation and therapeutic stress by sCLU was studied by cell viability, caspase assay and meta-analysis. Key findingsThe data from oral cancer patients showed that the expression levels of sCLU, ATG14, ULK1, and PARKIN increased in grade-wise manners. Interestingly, sCLU overexpression promoted autophagy through AMPK/Akt/mTOR signaling pathway leading to cell survival and protection from long exposure serum starvation induced-apoptosis. Additionally, sCLU was demonstrated to interact with ULK1 and inhibition of ULK1 activity by SBI206965 was found to abolish sCLU-induced autophagy indicating critical role of ULK1 in induction of autophagy. Furthermore, sCLU was observed to promote expression of mitophagy-associated proteins in serum starvation conditions to protect cells from nutrient deprivation. The meta-analysis elucidated that high CLU expression is associated with therapy resistance in cancer and we demonstrated that sCLU-mediated mitophagy was revealed to inhibit cell death by cisplatin. SignificanceThe present investigation has highlighted the probable implications of the clusterin-induced autophagy in cell survival and inhibition of apoptosis in oral cancer.

Abstract 2951: Knockdown of SCAMP3 suppresses the proliferation and emboli formation capacity of inflammatory breast cancer cells through EGFR signaling

Abstract Inflammatory Breast Cancer (IBC) is one of the most lethal forms of breast cancer (BC), where patients have a 43% increased risk of death compared to women with non-IBC advanced BC. Approximately 30% of triple-negative (TN) IBC cases overexpress the Epidermal Growth Factor Receptor (EGFR). EGFR amplification is associated with a poor overall survival rate compared with EGFR-negative IBC cases. We previously identified increased expression of the secretory carrier membrane protein 3 (SCAMP3) in EGFR overexpressing TN SUM149 IBC cells, tissues and in the IBC metastasis promoting structure, the tumor emboli. SCAMP3 is an endosome-associated protein known to regulate the degradation of EGFR. Thus, we aim to elucidate the role of SCAMP3 in the regulation of EGFR signaling and IBC progression. To investigate how SCAMP3 contributes to the IBC cellular response, we knocked out (KO) the expression of SCAMP3 in IBC SUM149 cells using the CRISPR/Cas9 technique. Wild type (WT) and KO IBC cells treated with EGF for 30 min in serum-starved conditions were used to assess cell viability after 24, 48, 72, and 96h, colony, and emboli formation capacity. Our results demonstrate a reduction in the proliferation, colony, and emboli formation capacity of SCAMP3 KO cells in comparison with the WT. Taken together our results show that SCAMP3 plays a promoting role in the progression of IBC through EGFR signaling modulation. Citation Format: Ivette J. Suarez-Arroyo, Jael Reyes-Chea, Michelle M. Martinez-Montemayor. Knockdown of SCAMP3 suppresses the proliferation and emboli formation capacity of inflammatory breast cancer cells through EGFR signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2951.

LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling.

Controlled cell growth and proliferation are essential for tissue homeostasis and development. Wnt and Hippo signaling are well known as positive and negative regulators of cell proliferation, respectively. The regulation of Hippo signaling by the Wnt pathway has been shown, but how and which components of Wnt signaling are involved in the activation of Hippo signaling during nutrient starvation are unknown. Here, we report that a reduction in the level of low-density lipoprotein receptor-related protein 6 (LRP6) during nutrient starvation induces phosphorylation and cytoplasmic localization of YAP, inhibiting YAP-dependent transcription. Phosphorylation of YAP via loss of LRP6 is mediated by large tumor suppressor kinases 1/2 (LATS1/2) and Merlin. We found that O-GlcNAcylation of LRP6 was reduced, and the overall amount of LRP6 was decreased via endocytosis-mediated lysosomal degradation during nutrient starvation. Merlin binds to LRP6; when LRP6 is less O-GlcNAcylated, Merlin dissociates from it and becomes capable of interacting with LATS1 to induce phosphorylation of YAP. Our data suggest that LRP6 has unexpected roles as a nutrient sensor and Hippo signaling regulator.

The Role of BAFF-R Signaling in the Growth of Primary Central Nervous System Lymphoma.

Primary CNS lymphoma (PCNSL) is an aggressive brain tumor. Despite improvements in therapeutic algorithms, long-term survival remains rare, illustrating an urgent need for novel therapeutic targets. BAFF-R is a pro-survival receptor expressed on most malignant B cells, including PCNSL. To date, its role in PCNSL growth remains elusive. Here, we have created a BAFF-R knockout lymphoma cell line (BAFF-R-KO) using CRISPR-Cas9. In serum-starved conditions, BAFF-R-KO cells exhibit decreased viability in vitro compared to BAFF-R+ cells. Combining an orthotopic mouse model of PCNSL with chronic cranial windows and intravital microscopy, we have demonstrated a significant delay in tumor growth in mice inoculated with BAFF-R-KO cells compared to BAFF-R+ PCNSL. Additionally, median survival of BAFF-R-KO mice was significantly prolonged. Altogether, our results indicate the high potential of BAFF-R as a novel treatment target for PCNSL.

Palmitic acid- and/or palmitoleic acid-containing phosphatidic acids are generated by diacylglycerol kinase α in starved Jurkat T cells

Diacylglycerol kinase (DGK) α enhances the proliferation of melanoma and hepatocellular carcinoma cells whereas, in contrast, DGKα induces a nonproliferative state in T cells. We previously found that DGKα produces palmitic acid (16:0)-containing PA species, such as 16:0/16:0- and 16:0/18:0-PA, in melanoma cells under serum-starved (nonproliferative) conditions. In the present study, we identified the PA species generated by DGKα in T cells under serum-starved (nonproliferative) conditions. We found that serum starvation markedly increased the levels of many PA species, such as 14:1/16:1-, 14:0/16:1-, 14:0/16:0-, 16:1/16:2-, 16:1/16:1-, 16:0/16:1-, 16:0/16:0-, 16:1/18:2-, 16:1/18:1-, 16:0/18:1-, 16:0/18:0-, 18:1/18:2-, 18:1/18:1- and 18:0/18:1-PA, in Jurkat T cells. In lysates from serum-starved Jurkat T cells, DGKα activity, which was Ca2+-dependent and sensitive to a DGKα-specific inhibitor (CU-3), was substantially increased, indicating its activation. Moreover, CU-3 (1–10 μM) significantly reduced the amounts of palmitic acid- and/or palmitoleic acid (16:1)-containing PA species, such as 14:1/16:1-, 14:0/16:1-, 14:0/16:0-, 16:1/16:2-, 16:1/16:1-, 16:0/16:1-, 16:0/16:0-, 16:0/18:1- and 16:0/18:0-PA, which were increased by serum starvation. These results indicate that DGKα generates different PA species in starved melanoma cells (palmitic acid-containing PA species) and T cells (palmitic acid- and/or palmitoleic acid (16:1)-containing PA species). Therefore, the differences in the PA molecular species may account for the opposing functions of DGKα in melanoma and T cells.