Serum-starved Cells Research Articles

FC044: Heterozygous Variants in Kinase Domain of NEK8 cause an Autosomal-Dominant Ciliopathy

Abstract BACKGROUND AND AIMS NEK8/NPHP9 encodes a protein that localizes to the primary cilium. Biallelic NEK8 variants are known to cause multiorgan developmental defects, including kidney cystic dysplasia and extensive extra-renal defects, with heterozygous carrier parents being asymptomatic [1]. This autosomal recessive inheritance is the most common inheritance mode for ciliopathies. Complementary to this, we now propose a dominant negative effect for specific heterozygous NEK8 missense variants in the kinase domain resulting in an autosomal-dominant ciliopathy. METHOD We performed genetic testing in patients from several medical centers. To explore the consequences of the identified NEK8 variants, we are performing cilia staining assays in patients' skin fibroblast and kidney cells, as well as in mIMCD3 cells overexpressing the identified variants. Furthermore, we are examining the impact of the NEK8 variants on replication stress response. RESULTS We identified three distinct heterozygous NEK8 variants in 12 families (Table 1), all leading to missense alterations in the kinase domain. Interestingly the p.Arg45Trp variant is a recurrent variant we detected in 10 unrelated families. All patients have a kidney phenotype that varies from mild focal segmental glomerulosclerosis to prenatal presentation with polycystic kidneys. Most patients have kidney failure needing kidney replacement therapy at varying ages of onset. In all patients, we thoroughly checked whether a second variant could be found. Furthermore, the large symptomatic family and de novo occurrences favor a dominant inheritance mode. Our preliminary results from functional studies show abnormal primary cilia formation in serum-starved cells as well as increased replication stress. CONCLUSION We present the first evidence for a pathogenic effect of heterozygous NEK8 variants. Remarkably our patients present with a kidney limited phenotype as compared to the multiorgan defects found in patients with biallelic variants. This reveals a new mode of inheritance for NEK8 variants and expands genotype-phenotype correlations for this gene.

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.

Autolysosomes and caspase-3 control the biogenesis and release of immunogenic apoptotic exosomes

Apoptotic exosome-like vesicles (ApoExos) are a novel type of extracellular vesicle that contribute to the propagation of inflammation at sites of vascular injury when released by dying cells. ApoExos are characterized by the presence of the C-terminal perlecan LG3 fragment and 20S proteasome, and they are produced downstream of caspase-3 activation. In the present study, we assessed the relative roles of autophagy and caspase-3-mediated pathways in controlling the biogenesis and secretion of immunogenic ApoExos. Using electron microscopy and confocal immunofluorescence microscopy in serum-starved endothelial cells, we identified large autolysosomes resulting from the fusion of lysosomes, multivesicular bodies, and autophagosomes as a site of ApoExo biogenesis. Inhibition of autophagy with ATG7 siRNA or biochemical inhibitors (wortmannin and bafilomycin) coupled with proteomics analysis showed that autophagy regulated the processing of perlecan into LG3 and its loading onto ApoExos but was dispensable for ApoExo biogenesis. Caspase-3 activation was identified using caspase-3-deficient endothelial cells or caspase inhibitors as a pivotal regulator of fusion events between autolysosomes and the cell membrane, therefore regulating the release of immunogenic ApoExos. Collectively, these findings identified autolysosomes as a site of ApoExo biogenesis and caspase-3 as a crucial regulator of autolysosome cell membrane interactions involved in the secretion of immunogenic ApoExos.

Assessment of T cell chemotaxis to S1P

Introduction: Migration of leukocytes in response to chemical gradients, chemotaxis, is dependent on many factors, including cell type, surface markers, the chemoattractant, etc. Sphinogsine-1-phosphate (S1P) is a chemoattractant playing a large role in migrating activated T cells out of lymph nodes by binding to their S1P receptor, S1P1. The importance of the egress in T cells from lymph nodes is highlighted by pharmacological disruption of this migration can lead to immune dampening and thus control of multiple sclerosis, an autoimmune disease. In the case of human immunodeficiency virus (HIV), it has been shown that HIV downregulates S1P1 surface expression, effectively inhibiting chemotaxis. Our experiments attempt to study a particular HIV-encoded protein, Nef in S1P-elicited T cell migration, and to optimize the conditions for assessing T cell migration in response to S1P.
 Methods: In our Transwell migration assays, migration of serum-starved SupT1 cells was induced using various concentrations of S1P bound to delipidated bovine serum albumin (BSA). Before migration, cells were labeled using Calcein AM. Cells were allowed to migrate for 2-4 hours at 37°C in serum-free media. After migration, fluorescence intensity was measured using a CLARIOstar microplate reader.
 Results: S1P showed a direct dose-dependent response to SupT1 cell migration from 0 to 100 nM S1P. Optimization of the migration showed that both number of trans-migrated cells and those still present within the transwell filter were significant indicators of SupT1 migration.
 Conclusion: S1P’s chemoattractant ability is prevalent in the migration of SupT1 cells in concentrations lower than 125nM. Because we have inducible systems for HIV-Nef expression established in this cell line, these data are useful for testing the role of Nef in HIV-mediated T cell retention.

Low Dose Cyclophosphamide in Combination with Elotuzumab - a Novel Immunotherapeutic Strategy for Multiple Myeloma

IntroductionCyclophosphamide (CTX) is a widely used anti-neoplastic, performing as an alkylating agent at high doses and immunomodulatory agent at low doses 1.. Combining CTX with monoclonal antibody (mAb) therapy has proven beneficial in potentiating relapsed and/or refractory multiple myeloma (RRMM) therapies, with daratumumab-directed MM cell death enhanced in the presence of CTX 2,3.. Elotuzumab (ELO), the second mAb approved for treating RRMM, promotes MM cell clearance by enhancing macrophage-mediated phagocytosis and CD16- and SLAMF7-directed NK cell cytotoxicity. ELO has been approved for use alongside dexamethasone and lenalidomide 4 or pomalidomide (POM) 5.. However, potential therapeutic benefits of ELO in combination with immunomodulatory drugs such as CTX and POM have yet to be examined. Our research investigates, the efficacy of combining low-dose CTX, alone or in combination with POM, and ELO in enhancing macrophage and NK cell infiltration and function in the MM tumour microenvironment.Materials and MethodsMultiple myeloma cells (MM1S and H929) were treated with low-dose CTX and/or POM for 24hrs, washed to remove residual drug and resuspended in fresh media for tumour cell secretome (TCS) generation. Direct effects of CTX and/or POM on surface expression of checkpoint proteins (PD-1 and CD47) on MM cells was assessed by mean fluorescent intensity (MFI) flow cytometry. CD32/CD64 receptor expression on THP-1 macrophages, NKG2D, CD2, DNAM-1, CD96 and KIR2DL1 receptors on KHYG1 and primary NK cells, were measured using flow cytometry as a measure of activation. Migration of serum-starved, CFSE-labelled macrophages and NK cells towards CTX and/or POM TCS was assessed after 4hrs, with total number of migrated cells quantified using the Accuri flow cytometer. Immune cell function following indirect conditioning of macrophages/NK cells with MM cell TCS was measured by quantifying antibody-directed cellular phagocytosis (ADCP) or antibody-directed cellular cytotoxicity (ADCC), respectively. Conditioned immune cells were co-cultured with MM cells in a 2:1 effector to target ratio for 4hrs in the absence/presence of mAbs (ELO, nivolumab and anti-CD47), after which MM cell clearance was quantified by flow cytometry and presented as relative uptake (ADCP) and cytotoxicity (ADCC). One-way ANOVA statistical analysis was performed, followed by Tukey post hoc tests, with significance recognized at p<0.05.ResultsDirect treatment of MM cells with CTX increased surface expression of immune evading checkpoint proteins PD-1 and CD47 (p<0.05,n=3). POM monotherapy did not alter PD-1/CD47 expression, however dual therapy of CTX and POM supported the CTX-driven effect (p<0.001,n=3). Expression of CD32/CD64 macrophage activation markers was significantly increased on THP-1 cells following CTX-TCS conditioning (p<0.001,n=3). POM altered CD32, but not CD64, however dual treatment with CTX and POM significantly increased expression of both CD32 and CD64 (p<0.001, n=3). Migration of macrophages towards CTX-TCS was enhanced in a dose-dependent manner (p<0.01,n=3). CTX and POM dual therapy supported this CTX driven effect (p<0.001,n=3). Migration trends of both primary and KHYG1 NK cells were also increased towards the secretome from CTX treated MM cells. ADCP and ADCC were increased by CTX alone or in combination with POM (p<0.05, n=3). Effects of CTX on ADCP were not significantly enhanced by ELO, however ELO did significantly augment ADCC by CTX-conditioned primary NK cells (p<0.05,n=3). Given the increased expression of PD-1 and CD47, we investigated if the inclusion of nivolumab and anti-CD47 mAbs potentiated ADCC. Although ADCC was increased in all combinations, there was no significant difference between ELO alone versus ELO in combination with either nivolumab or anti-CD47.ConclusionsLow-dose CTX and POM potentiated the immunomodulatory effects of ELO, with NK-directed cytotoxicity of MM cells enhanced in the presence of this mAb. Our data therefore indicates that the inclusion of low-dose CTX and or POM in combination with ELO could be a novel immunotherapeutic strategy for treating RRMM.

Wnt5a Induces ROR1 to Interact Grb2 to Enhance Ras Activation in Chronic Lymphocytic Leukemia

ROR1 (Receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, oncoembryonic antigen found on chronic lymphocytic leukemia (CLL) cells, but not on healthy postpartum tissues. In prior studies we found that higher-level CLL-cell expression of ROR1 associated with accelerated disease progression and unfavorable clinical outcome (Cui B. et al., Blood, 128:2931, 2016). ROR1 is a receptor for Wnt5a, which is found at high levels in plasma of patients with CLL. We found Wnt5a could enhance ERK1/2 phosphorylation and leukemia-cell proliferation (Hasan MK. et al., Leukemia, 35:1621, 2021). Moreover, Wnt5a-enhanced ERK1/2 phosphorylation and leukemia-cell proliferation could be inhibited by UC-961, a first-in-class humanized mAb specific for a functional epitope of the ROR1 extracellular domain that is undergoing phase II clinical testing (Choi MY. et al., Cell Stem Cell, 22:951, 2018). However, it is not known how Wnt5a/ROR1-signaling induces ERK1/2 activation. We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 from CLL cells and identified Grb2 (Growth Factor Receptor Bound Protein 2), a cytoplasmic protein that may interact with SOS (son of sevenless) to induce activation of Ras, which can activate the mitogen-activated protein kinase (MAPK) pathway, leading to activation of ERK1/2. We found that treatment of serum-starved CLL cells with Wnt5a induced Grb2 to associate with ROR1 and activate Ras and ERK1/2; these effects could be blocked by siRNA silencing of Grb2 or by treatment with UC-961, indicating that Wnt5a induced activation of ERK1/2 is dependent upon expression of ROR1 and Grb2. To examine the structure-function relationship(s) in the interaction(s) between ROR1 and Grb2, we used the CLL-derived leukemia cell line MEC1, which we found expresses high-levels of Wnt5a, but not ROR1. Subclones of MEC1 cells transfected to express ROR1 (MEC1-ROR1) had significantly higher levels of ERK1/2 activation than MEC1 cells; immune-precipitation studies confirmed constitutive association of Grb2 and SOS1 with ROR1. This was not observed in MEC1-ROR1-Wnt5a -/- cells in which we disrupted both alleles of WNT5a via CRISPR-Cas9. Moreover, MEC1-ROR1 cells, or MEC1-ROR1-Wnt5a -/- cells treated with Wnt5a, had significantly higher levels of activated Ras and ERK1/2 than MEC1, MEC1-Wnt5a -/-, or MEC1-ROR1-Wnt5a -/-, demonstrating that activation of Ras, MAPK, and ERK1/2 in MEC1 was dependent upon on expression of ROR1 and Wnt5a. We next examined single amino acid substitutions of proline (P) to alanine (A) in the P-X-X-P- motifs of the proline-rich domain (PRD) of ROR1 at positions 784, 808, 826 or 841. Interestingly we found that P808A of ROR1 (MEC1-ROR1 P808A), a binding site of DOCK2 and critical for activation of ERK1/2, failed to complex with Grb2, SOS1, or enhance activation of Ras compared to MEC1-ROR1 cells. Overall, our results indicate that, upon Wnt5a stimulation, a novel complex of DOCK2/Grb2/SOS1 may form at position 808 of ROR1 that leads to activation of Ras and ERK1/2, which can promote proliferation of CLL cells and potentially contribute to the noted unfavorable prognostic implication of high-level expression of ROR1 in CLL. DisclosuresKipps: VelosBio, Inc.: Research Funding; Celgene: Honoraria, Research Funding; Genentech/Roche: Honoraria; Pharmacyclics/AbbiVie: Honoraria, Research Funding; Oncternal Therapeutics, Inc.: Current equity holder in publicly-traded company, Patents & Royalties: Cirmtuzumab, Research Funding; Janssen, Gilead, Dava Oncology,: Honoraria; Breast Cancer Research Foundation: Honoraria, Research Funding; Md Anderson Cancer Center: Research Funding; Gilead: Honoraria; National Cancer Institute/NIH: Honoraria, Research Funding; OncLive: Honoraria; Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS): Research Funding; California Institute for Regenerative Medicine (CIRM): Research Funding; European Research Initiative on CLL (ERIC): Honoraria; Dava Oncology: Honoraria; Patient Power, LLC: Honoraria; iwNHL: Honoraria; NCCN CLL/SLL Hairy Cell Leukemia Panel Meeting: Honoraria.

Mitogen-induced transcriptional programming in human fibroblasts.

Treatment of serum-starved quiescent human cells with fetal bovine serum (FBS), epidermal growth factor (EGF), or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) activates the RAS-MAPK pathway which initiates a transcriptional program which drives cells toward proliferation. Stimulation of the RAS-MAPK pathway activates mitogen- and stress-activated kinases (MSK) 1 and 2, which phosphorylate histone H3 at S10 (H3S10ph) or S28 (H3S28ph) (nucleosomal response) located at the regulatory regions of immediate-early genes, setting in motion a series of chromatin remodeling events that result in transcription initiation. To investigate immediate-early genes regulated by the MSK, we have completed transcriptome analyses (RNA sequencing) of human normal fibroblast cells (CCD-1070Sk) stimulated with EGF or TPA±H89, a potent MSK/PKA inhibitor. The induction of many immediate-early genes was independent of MSK activity. However, the induction of immediate-early genes attenuated with H89 also had reduced induction with the PKA inhibitor, Rp-cAMPS. Several EGF-induced genes, coding for transcriptional repressors, were further upregulated with H89 but not with Rp-cAMPS, suggesting a role for MSK in modulating the induction level of these genes.

KRAS-dependent cancer cells promote survival by producing exosomes enriched in Survivin.

Mutations in KRAS frequently occur in human cancer and are especially prevalent in pancreatic ductal adenocarcinoma (PDAC), where they have been shown to promote aggressive phenotypes. However, targeting this onco-protein has proven to be challenging, highlighting the need to further identify the various mechanisms used by KRAS to drive cancer progression. Here, we considered the role played by exosomes, a specific class of extracellular vesicles (EVs) derived from the endocytic cellular trafficking machinery, in mediating the ability of KRAS to promote cell survival. We found that exosomes isolated from the serum of PDAC patients, as well as from KRAS-transformed fibroblasts and pancreatic cancer cells, were all highly enriched in the cell survival protein Survivin. Exosomes containing Survivin, upon engaging serum-starved cells, strongly enhanced their survival. Moreover, they significantly compromised the effectiveness of the conventional chemotherapy drug paclitaxel, as well as a novel therapy that combines an ERK inhibitor with chloroquine, which is currently in clinical trials for PDAC. The survival benefits provided by oncogenic KRAS-derived exosomes were markedly reduced when depleted of Survivin using siRNA or upon treatment with the Survivin inhibitor YM155. Taken together, these findings demonstrate how KRAS mutations give rise to exosomes that provide a unique form of intercellular communication to promote cancer cell survival and therapy resistance, as well as raise interesting possibilities regarding their potential for serving as therapeutic targets and diagnostic markers for KRAS-dependent cancers.

Effect of serum starvation on rheology of cell monolayers

The rheological properties of cells and tissues are central to embryonic development and homeostasis in adult tissues and organs and are closely related to their physiological activities. This work presents our study of rheological experiments on cell monolayer under serum starvation compared to healthy cell monolayer with full serum. Serum starvation is one of the most widely used procedures in cell biology. However, the effect of deprivation of serum concentration on the material properties of cells is still unknown. Therefore, we performed macro-rheology experiments to investigate the effect of serum starvation on a fully confluent Madin–Darby Canine Kidney cell monolayer. The material properties, such as linear and non-linear viscoelastic moduli, of the monolayer, were measured using oscillatory shear experiments under serum-free [0% fetal bovine serum (FBS)] and full serum (10% FBS) conditions. Our results indicate that a serum-starved cell monolayer shows a different rheological behavior than a healthy cell monolayer. The loss and storage moduli decrease for the step-change in oscillatory strain amplitude experiments for a serum-starved cell monolayer and do not recover fully even after small deformation. In comparison, a healthy cell monolayer under full serum condition remains flexible and can fully recover even from a large deformation at higher strain. The effect of adhesion due to fibronectin was also studied in this work, and we found a significant difference in slip behavior for cell monolayer with and without serum.

Wnt5a Induces Interleukin-6 Via a ROR1-Dependent Pathway in Chronic Lymphocytic Leukemia, Leading to Leukemia-Cell Activation of STAT3

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic cell surface protein, which is expressed on the neoplastic B cells of patients with chronic lymphocytic leukemia (CLL), but not on virtually all normal post-partem tissues. ROR1 functions as a receptor for Wnt5a, which induces activation of Rho-GTPases, AKT, and HS-1 and promotes migration, proliferation, and survival of CLL B cells. We observed that freshly isolated CLL cells also had high levels of pSTAT3 (Y705), which attenuated over time in culture in serum-free media unless the CLL cells were stimulated with exogenous Wnt5a. This effect of Wnt5a could be blocked by cirmtuzumab, a humanized anti-ROR1 mAb that currently is undergoing clinical evaluation in patients with CLL. We examined the capacity of recombinant Wnt5a (rWnt5a) to induce activation of STAT3 in isolated CLL cells by performing a time-course study, evaluating for pSTAT3 in serum-starved CLL cells at time 0, 5', 15', 30' and 1, 2, 3, 4, and 24 hours after stimulation with exogenous Wnt5a. Surprisingly, activation of STAT3 in CLL cells was delayed, appearing first at 3 hours after addition of rWnt5a, and peaking at 24 hours, the last time point examined. In contrast, activation of CLL cells via treatment of CLL cells with interleukin (IL)-6, induced activation of STAT3 within 15' of the addition of this cytokine to the cultures. We hypothesized that the activation of STAT3 in CLL cells by Wnt5a was indirect, being caused by a factor(s) made by isolated CLL cells in response to stimulation with Wnt5a. To test this hypothesis, we examined CLL cells, and harvested supernatants of cultured CLL cells, at various times after stimulation with rWnt5a. RNA isolated from CLL cells was examined via real-time PCR for cytokine transcripts. This revealed a prominent, time-dependent increase in transcripts encoding IL-6 following stimulation of CLL cells with rWnt5a. Consistent with this observation we found Wnt5a induced CLL cells to secrete IL-6, which was detected via ELISA at increasing concentrations over time in culture-supernatants of CLL cells treated with rWnt5a, but not in the culture supernatants of unstimulated CLL cells. Culture supernatants collected from CLL cells stimulated for 24 hours with rWnt5a could induce activation of STAT3 within 15' in serum-starved CLL cells; this effect could be inhibited significantly by the anti-IL-6-receptor mAb, tocilizumab, but not by cirmtuzumab, even when used at concentrations that could block the capacity of concomitantly-added Wnt5a to induce latent STAT3 activation at 24 hours, suggesting that cirmtuzumab could inhibit Wnt5a-induced CLL-cell production of cytokines, such as IL-6. We examined for IL-6 in cultures of CLL cells treated for 24 hours with rWnt5a and cirmtuzumab, or rWnt5a and an antibody of irrelevant specificity. We found the concentration of IL-6 in the supernatants of CLL cells treated with rWnt5a and cirmtuzumab (110.4 ± 18.9 pg/ml) was significantly lower than that found in the supernatants of CLL cells treated with rWnt5a and control antibody (416.2 ± 53.7 pg/ml, p = 0.0007, Students t test). This study reveals that Wnt5a can induce CLL to secrete cytokines, such as IL-6, via a ROR1-dependent pathway, leading to latent, autocrine activation of STAT3. DisclosuresKipps:Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding; Oncternal: Research Funding; Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria.

Interleukin-6 Drives Multiple Myeloma Progression through Upregulating of CD147/Emmprin Expression and Its Sialylation

Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation of plasma cells in the bone marrow (BM). CD147, known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. With regard to MM, it was recently documented that the aberrantly elevated expression of CD147 has been tightly correlated with MM cell colonization and proliferation. During malignant transformation, many glycoproteins undergo a wide range of glycosylation alterations, especially increased sialylation, have been associated with malignant transformation and metastasis. However, it is still unclear the mechanisms of regulating CD147 and its glycosylation in MM. In this study, we found that CD147 and its sialylation can be up-regulated by interleukin-6 (IL-6), which is derived from either autocrine or paracrine sources and plays an essential role in the malignant progression of MM. When serum-starved MM cell lines RPMI8226, MM1.S and NCI-H929 were stimulated with 20ng/mL of IL-6, we found that the expression of CD147 on MM cell membrane was significantly upregulated, as determined by both flow cytometric analysis and Western blotting. Importantly, the results of Western blotting also clearly show that CD147 is a glycoprotein with higher molecular weight bands than core CD147. By using PNGase F and sialidase to digest the protein samples, we further demonstrated that CD147 is typical N-linked glycoprotein with high level of sialylation. Moreover, we found that the expression of α-2,3 and α-2,6 sialic acid on the cell surface were significantly upregulated by IL-6 analyzed by flow cytometry using MAA and SNA lectin binding assay. Next, we investigated the signaling pathway involved in the IL-6 mediated upregulation of CD147 and its sialylation in MM cells. By using Real-Time PCR, we confirmed that CD147 was transcriptionally upregulated by IL-6. Simultaneously, several sialyltransferases like ST3GAL3, ST3GAL6 and ST6GAL1 were also transcriptionally upregulated by IL-6. By using Western blotting, we further confirmed that IL-6 can activate the JAK/STAT3 signaling pathway by inducing the phosphorylation of STAT3 at Tyrosine 705 in MM cells. Further bioinformatics and ChIP analysis demonstrated that the existing of transcription factor STAT3 binding sites in the promoter of CD147 and sialyltransferase (ST3GAL3, ST3GAL6 and ST6GAL1) genes.To further validate the role of JAK/STAT3 in regulating CD147 and its sialylation, we used a specific STAT3 inhibitor Cryptotanshinone to treat the MM cells and found that the expression of CD147 and its sialylation in MM cells was accordingly decreased. Furthermore, we validated the roles of high level of CD147 and its sialylation in MM biology by knocking-down CD147 or inhibiting its sialylation using 3Fax-Peracetyl Neu5Ac, which is a specific sialyltransferase inhibitor. We found that both siRNA mediated knock-down of CD147 and sialylation inhibition did not affect the proliferation in RPMI8226 and MM1.S cells, measured by EDU incorporation assay using flow cytometry. However, knock-down of CD147 and sialylation inhibition were found to significantly reduce the adhesion to BMSCs and HUVECs, and the migration to BMSC-conditioned media in vitro. Finally, to further validate the influence of IL-6 and the related sialyltransferase genes in MM patients, we assessed the effect of expression on survival of patients using GEP data from the CoMMpass trial (n=664), and noted a significantly reduced PFS (progression-free survival) for patients with high levels of expression of IL-6 and ST3GAL6 (P <0.0001). Taken together, our data provide evidence that IL-6 can up-regulate CD147 expression and its sialylation in a STAT3-dependent manner, and offer a compelling rationale for exploring this axis as a therapeutic target for MM. DisclosuresO'Dwyer:GlycoMimetics Inc: Research Funding; Onkimmune Ltd: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Janssen: Consultancy, Honoraria, Research Funding.

Alteration in Lactate Production and Decrease in MTT Dye Reduction in Serum-starved Human Peripheral Blood Mononuclear Cells (PBMCs)

Background: Immunometabolism targeting therapy of auto-inflammatory diseases is an emerging strategy compared to immune system global suppression. However, our knowledge in this field needs promotion. Objectives: We examined the effects of serum starvation stress on metabolic activity in human peripheral blood mononuclear cells (PBMCs). Methods: Fresh immune cells were isolated from four healthy adult volunteers and cultivated with or without fetal bovine serum (FBS) at various time points under standard conditions. Glucose and intra- and extracellular lactate levels were assessed using routine techniques, and 3-(4, 5 -dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) reduction assay was used to determine mitochondrial function. Results: Spindle shape macrophage-like cells, which appeared early, were replaced at 96 h by large round monocytes/macrophage-like cells, with more frequency in the non-starved group. Interestingly, serum starvation dictated a status, especially in monocyte/macrophage-like cells, that led to prolong decrement in mitochondrial dehydrogenase-mediated reduction of MTT. This difference was confirmed with the MTT assay quantitatively (P &lt; 0.05). Moreover, the intra- and extracellular lactate concentrations were lower in starved cells than in non-starved controls (P &lt; 0.05), and glucose levels were higher in 72 h starved cell culture supernatants than in non-starved control cells (P &lt; 0.05). Conclusions: This study showed that under serum starvation-induced metabolic stress, lactate production is altered in immune cells, and total oxidative mitochondrial activity is reduced in macrophage-like cells. These findings open a new window to target immune cell metabolism for the treatment of autoinflammatory and autoimmune diseases.

Effects of 1800 MHz radiofrequency fields on signal transduction and antioxidant proteins in human A172 glioblastoma cells

Purpose To assess the effects of 1800 MHz radiofrequency electromagnetic field (RF-EMF) exposure on the expression of signal transduction and antioxidant proteins in a human-derived A172 glioblastoma cell line. Materials and methods Adherent human-derived A172 glioblastoma cells (1.0 × 105 cells per 35 mm culture dish, containing 2 mL DMEM media) were exposed to 1800 MHz continuous-wave (CW) or GSM-modulated RF fields, in the presence or absence of serum for 5, 30 or 240 min at a specific absorption rate (SAR) of 0 (sham) or 2.0 W/kg. Concurrent negative (vehicle) and positive controls (1 µg/mL anisomycin) were included in each experiment. Cell lysates were collected immediately after exposure, stabilized by protease and phosphatase inhibitors in lysis buffer, then frozen and maintained at −80 °C until analysis. The relative expression levels of phosphorylated- and total-signal transduction proteins (CREB, JNK, NF-κB, ERK1/2, Akt, p70S6K, STAT3 and STAT5) and antioxidant proteins (SOD1, SOD2, CAT, TRX1, PRX2) were assessed using Milliplex magnetic bead array panels and a MagPix Multiplex imaging system. Results In cells exposed to 1800 MHz continuous-wave RF-EMF with the presence of serum in the culture medium, CAT expression was statistically significantly decreased after a 30 min exposure, total JNK was decreased at both 30 and 240 min of exposure, STAT3 was decreased after 240 min of exposure and phosphorylated-CREB expression was decreased after 30 min of exposure. In cells exposed to 1800 MHz GSM-modulated RF-EMF in serum-free cultures, the expression level of total STAT5 was decreased after 30 and 240 min of exposure. These observed changes were detected sporadically across time-points, culture conditions and RF-EMF exposure conditions indicating the likelihood of false positive events. When cells were treated with anisomycin for 15 min as a positive control, dramatic increases in the expression of phosphorylated signaling proteins were observed in both serum-starved and serum-fed A172 cells, with larger fold change increases in the serum-free cultures. No statistically significant differences in the expression levels of SOD1, SOD2 or TRX1 were observed under any tested conditions after exposure to RF-EMF. Conclusions The current study found no consistent evidence of changes in the expression of antioxidant proteins (SOD1, SOD2, CAT or TRX2) or a variety of signal transductions proteins (CREB, JNK, NF-κB, ERK1/2, Akt, p70S6K, STAT3, STAT5) in a human-derived glioblastoma A172 cell line in response to exposure to 1800 MHz continuous-wave or GSM-modulated RF-EMF for 5, 30 or 240 min in either serum-free or serum-containing cultures.

Effects of Lysophosphatidic Acid Receptor and Free Fatty Acid Receptor Agonists on CCN1/Cyr61 Expression in Prostate Cancer Cells

Expression of CCN1/Cyr61, a member of the CCN family of matricellular proteins, can be induced by growth factors in cancer cells. CCN1 participates in cell migration and other regulatory processes in various cancers. However, the role of CCN1 in prostate cancer remains poorly understood. In previous work we observed that lysophosphatidic acid (LPA), a bioactive lipid, stimulates proliferation and migration in prostate cancer cells via the G protein-coupled receptor (GPCR) LPAR1, and also induces CCN1 expression. We also showed that activation of free fatty acid receptor 4 (FFAR4), another GPCR, inhibits LPA-induced proliferation and migration. For the current study, we hypothesized that CCN1 contributes to adhesion and migration of prostate cancer cells. We first confirmed that CCN1 protein levels were increased by LPA after 2-5 hours in PC-3 human prostate cancer cells. This response was inhibited by TUG-891, an FFA4 agonist. When serum-starved PC-3 cells were treated with LPA in an extended time course from 2 minutes to 24 hours, Erk, Akt, and FAK were initially activated within 30 minutes of LPA addition, as detected by immunoblotting for the phosphorylated kinases. However, all three kinases exhibited a later phase of activation after ~6 hours. Thus, CCN1 induction is downstream of early signals initiated by LPAR1, but potentially upstream of later events. For adhesion assays, serum-starved PC-3 cells were incubated with and without 10 μM LPA ± 1 μM TUG-891. Enhanced adhesion was prominent 1 hour after LPA addition, and was sustained for at least 8 hours. For migration assays, serum-starved cells were incubated with the same agents in modified Boyden chambers. Enhanced cell migration was observed by 2 hours after LPA addition, and continued for at least 24 hours. LPA-induced adhesion and migration were blocked by TUG-891. In other experiments, extracellular matrix was subjected to immunoblotting and to immunofluorescence microscopy for CCN1. In the immunoblotting experiments, CCN1 protein was increased in the extracted extracellular matrix 2-5 hours after LPA addition. In the immunohistochemistry experiments, CCN1 was detected in the extracellular space of PC-3 cells treated with LPA for 0-5 hours. In conclusion, the results suggest a role for LPA-induced CCN1 in adhesion and migration in prostate cancer cells, making CCN1 a potential therapeutic target. In addition, the results further indicate that FFAR activation is a strategy to suppress both short- and long-term LPA responses.

MOVAS Cells: A Versatile Cell Line for Studying Vascular Smooth Muscle Cell Cholesterol Metabolism.

Cholesterol metabolism is paramount to cells. Aberrations to cholesterol metabolism affects cholesterol homeostasis, which may impact the risk of several diseases. Recent evidence has suggested that vascular smooth muscle cell (VSMC) cholesterol metabolism may play a role in atherosclerosis. However, there is scant in vitro mechanistic data involving primary VSMC that directly tests how VSMC cholesterol metabolism may impact atherosclerosis. One reason for this lack of data is due to the impracticality of gene manipulation studies in primary VSMC, as cultured primary VSMC become senescent and lose their morphology rapidly. However, there are no immortalized VSMC lines known to be suitable for studying VSMC cholesterol metabolism. The purpose of this study was to determine whether MOVAS cells, a commercially available VSMC line, are suitable to use for studying VSMC cholesterol metabolism. Using immunoblotting and immunofluorescence, we showed that MOVAS cells express ABCA1, ABCG1, and SREBP-2. We also determined that MOVAS cells efflux cholesterol to apoAI and HDL, which indicates functionality of ABCA1/ABCG1. In serum-starved MOVAS cells, SREBP-2 target gene expression was increased, confirming SREBP-2 functionality. We detected miR-33a expression in MOVAS cells and determined this microRNA can silence ABCA1 and ABCG1 via identifying conserved miR-33a binding sites within ABCA1/ABCG1 3'UTR in MOVAS cells. We showed that cholesterol-loading MOVAS cells results in this cell line to transdifferentiate into a macrophage-like cell, which also occurs when VSMC accumulate cholesterol. Our characterization of MOVAS cells sufficiently demonstrates that they are suitable to use for studying VSMC cholesterol metabolism in the context of atherosclerosis.

DNA repair and cholesterol-mediated drug efflux induce dose-dependent chemoresistance in nutrient-deprived neuroblastoma cells

SummaryNeuroblastoma is a solid, heterogeneous pediatric tumor. Chemotherapy is widely used to treat neuroblastoma. However, dose-dependent responses and chemoresistance mechanisms of neuroblastoma cells to anticancer drugs remain challenging. Here, we investigated the dose-dependent effects of topotecan on human neuroblastoma cells (SK-N-SH, SH-SY5Y, and SK-N-BE) under various nutrient supply conditions. Serum-starved human neuroblastoma cells showed reduced toxicity. Their survival rate increased upon treatment with a high concentration (1 μM) of topotecan. Quantitative profiling of global and phosphoproteome identified 12,959 proteins and 48,812 phosphosites, respectively, from SK-N-SH cells. Network analysis revealed that topotecan upregulated DNA repair and cholesterol-mediated topotecan efflux, resulting in topotecan resistance. Results of DNA damage assay, cell cycle, and quantitative analyses of membrane cholesterol supported the validity of these resistance factors and their applicability to all neuroblastoma cells. Our results provide a model for high dose-dependent chemoresistance in neuroblastoma cells that could enable a patient-dependent chemotherapy screening strategy.

CD43 (sialophorin) is involved in the induction of extracellular matrix remodeling and angiogenesis by lung cancer cells.

Aberrant expression of CD43 in malignant tumors of nonhematopoietic origin such as those from lung, cervix, colon, and breast has been shown to correlate with poor prognosis, providing tumor cells with enhanced motility, anchorage-independent growth, and in vivo tumor size, while protecting the cells of NK lysis and apoptosis. To further characterize the role of CD43 in cell transformation, we tested whether interfering its expression modified the capacity of the A549 non-small cell lung cancer cells to secrete molecules contributing to malignancy. The proteomic analysis of the secretome of serum-starved A549 cells revealed that cells expressing normal levels of CD43 released significantly high levels of molecules involved in extracellular matrix organization, angiogenesis, platelet degranulation, collagen degradation, and inflammation, as compared to CD43 RNAi cells. This data reveals a novel and unexpected role for CD43 in lung cancer development, mainly in remodeling the tumor microenvironment.

Serum Starvation Sensitizes Anticancer Effect of Anemarrhena asphodeloides via p38/JNK-Induced Cell Cycle Arrest and Apoptosis in Colorectal Cancer Cells.

The molecular mechanism underlying the anticancer effects of Anemarrhena asphodeloides (A. asphodeloides) on colon cancer is unknown. This is the first study evaluating the anticancer effect of A. asphodeloides extract (AA-Ex) in serum-starved colorectal cancer cells. Changes in cell proliferation and morphology in serum-starved MC38 and HCT116 colorectal cancer cells were investigated using MTS assay. Cell cycle and apoptosis were investigated using flow cytometry, and cell cycle regulator expression was determined using qRT-PCR. Apoptosis regulator protein levels and mitogen-activated protein kinase (MAPK) phosphorylation were assessed using western blotting. AA-Ex sensitively suppressed proliferation of serum-starved colorectal cancer cells, with MC38 and HCT116 cells showing greater changes in proliferation after treatment with AA-Ex under serum starvation than HaCaT and RAW 264.7 cells. AA-Ex inhibited cell cycle progression in serum-starved MC38 and HCT116 cells and increased the expression of cell cycle inhibitors (p53, p21, and p27). Furthermore, AA-Ex induced apoptosis in serum-starved MC38 and HCT116 cells. Consistently, AA-Ex suppressed the expression of the anti-apoptotic molecule Bcl-2 and upregulated pro-apoptotic molecules (cytochrome c, cleaved caspase-9, cleaved caspase-3, and cleaved-PARP) in serum-starved cells. AA-Ex treatment under serum starvation decreased AKT and ERK1/2 phosphorylation in the cell survival signaling pathway but increased p38 and JNK phosphorylation. Furthermore, AA-Ex treatment with serum starvation increased the levels of the transcription factors of the p38 and JNK pathway. Serum starvation sensitizes colorectal cancer cells to the anticancer effect of A. asphodeloidesvia p38/JNK-induced cell cycle arrest and apoptosis. Hence, AA-Ex possesses therapeutic potential for colon cancer treatment.

Regulation of human β-galactoside α2,6-sialyltransferase (hST6Gal I) gene expression during differentiation of human osteoblastic MG-63 cells.

In this study, we found that gene expression of the human β-galactoside α2,6-sialyltransferase (hST6Gal I) was specifically increased during differentiation of human MG-63 osteoblastic cells by serum starvation (SS). In parallel, a distinct increase in binding to SNA, the α2,6-sialyl-specific lectin, was observed in serum-starved cells, as demonstrated by FACS analysis. 5'-Rapid amplification of cDNA ends analysis demonstrated that the increase of hST6Gal I transcript by SS is mediated by P1 promoter. To elucidate transcriptional regulation of hST6Gal I in SS-induced MG-63 cells, we functionally characterized the P1 promoter region of the hST6Gal I gene. The 5'-deletion analysis of P1 promoter region revealed that the 189bp upstream region of transcription start site is critical for transcriptional activity of hST6Gal I gene in SS-induced MG-63 cells. This region contains the predicted binding sites for several transcription factors, including AREB6, FOXP1, SIX3, HNF1, YY2, and MOK2. The mutagenesis analysis for these sites and chromatin immunoprecipitation assay demonstrated that the YY2 binding site at -98 to -77 was essential for the SS-induced hST6Gal I gene expression during differentiation of MG-63 cells.

TNF-α Induces URG-4/URGCP Gene Expression inHepatoma Cells through Starvation Dependent Manner.

URG-4/URGCP is a gene that may be associated with the onset of tumorigenesis and cell cycle regulation. In the literature, there is no study about inflammatory cytokine-mediated URG-4/URGCP regulation. In this study, the effect of TNF-α cytokine was investigated on URG-4/URGCP expression in serum-starved and serum-cultured hepatoma cells. The effect of TNF-α on hepatoma cells was shown using MTT and Annexin-V/PI staining with flow cytometer analyses. As a result, TNF-α leads to the cytotoxicity of hepatoma cells in serum-starved condition whereas no decrease was detected from serum-cultured condition. TNF-α-mediated URG-4/URGCP expression was determined at mRNA andprotein level with qRT-PCR analyses and Western blotting method. URG-4URGCP mRNA expression was upregulated in both serum-starved and serum-cultured hepatoma cells. The transfection studies were carried out with URG-4/URGCP promoter constructs for determining the transcriptional activity. TNF-α caused to the upregulation of the activities of URG/URGCP promoter constructs. The basal activities of the URG-4/URGCP promoter conditions are differential according to serum conditions. In addition, some pathway inhibitors were added into hepatoma cells for blocking specific pathways to find out TNF-α-mediated URG-4/URGCP upregulation at mRNA and protein level. TNF-α used JNK and PI3K pathways for regulating URG-4/URGCP gene at serum-starved Hep3B cells. In serum-cultured condition, wortmannin (PI3K inhibitor), MEK-1 (MAPK inhibitor), and SP600125 (JNK inhibitor) did not inhibit the activation response of TNF-α on URGCP.