Significant Reduction In Proliferation Research Articles

DHA-enriched phosphatidylcholine suppressed angiogenesis by activating PPARγ and modulating the VEGFR2/Ras/ERK pathway in human umbilical vein endothelial cells.

Docosahexaenoic acid-enriched phosphatidylcholine (DHA-PC) is a new generation of omega-3 lipids, which contains an ester bond linking DHA at the sn-2 position of phospholipid. DHA-PC has become the interest recently as its better bioavailability and anti-oxidation capacity. In this study, the anti-angiogenic effect of DHA-PC was evaluated. The capacities of proliferation, migration, tube formation of human umbilical vein endothelial cells were significantly declined after DHA-PC treatment. Furthermore, DHA-PC inhibited the neovascularization of the chick chorioallantoic membrane in vivo. Mechanism results indicated that DHA-PC enhances the expression of peroxisome proliferator-activated receptor γ (PPARγ) at transcriptional and translational level, subsequently down-regulates the VEGFR2 expression and VEGFR2-mediated downstream Ras/ERK pathway, resulting in significant reduction in proliferation and differentiation. Additionally, PPARγ-specific antagonist GW9662 partly reversed the inhibition effects of DHA-PC on tube formation and neovascularization, suggesting that DHA-PC exerts anti-angiogenesis effect through activating PPARγ. These findings indicated that DHA-PC has a great prospect of anti-tumor angiogenesis therapy.

Conditions Mimicking the Cancer Microenvironment Modulate the Functional Outcome of Human Chorionic Villus Mesenchymal Stem/Stromal Cells in vitro.

Mesenchymal stem/stromal cells isolated from chorionic villi of human term placentae (CV-MSCs) possess unique biological characters. They exhibit self-renewal, directional migration, differentiation, and immunomodulatory effects on other cell lineages, by virtue of which they can be utilized as therapeutic carriers, for drug targeting, and therapy. Tumors display characteristic features of a damaged tissue microenvironment, which is saturated with conditions such as hypoxia, sustained inflammation, and increased oxidative stress. CV-MSCs function normally in a high oxidative stress environment induced by hydrogen peroxide (H2O2) and glucose and also protect endothelial cells from their damaging effects. For their therapeutic applications in a disease like cancer, it is necessary to ascertain the effects of tumor microenvironment on their functional outcome. In this study, we investigated the functional activities, of CV-MSCs in response to conditioned media (CM) obtained from the culture of breast cancer cell line MDA-231 (CM-MDA231). CV-MSCs were exposed to CM-MDA231 for different spatio-temporal conditions, and their biological functions as well as modulation in gene expression were evaluated. Effect of CM-MDA231 on factors responsible for changes in functional outcome were also investigated at the protein levels. CV-MSCs exhibited significant reduction in proliferation but increased adhesion and migration after CM-MDA231 treatment. Interestingly, there was no change in their invasion potential. CM-MDA231 treatment modulated expression of various genes involved in important cellular events including, integration, survival, message delivery and favorable outcome after transplantation. Analysis of pathways related to cell cycle regulation revealed significant changes in the expression of p53, and increased phosphorylation of Retinoblastoma (Rb) and Checkpoint Kinase 2 in CV-MSCs treated with CM-MDA231. To summarize, these data reveal that CV-MSCs retain the ability to survive, adhere, and migrate after sustained treatment with CM-MDA231, a medium that mimics the cancer microenvironment. These properties of CV-MSCs to withstand the inflammatory tumor like microenvironment prove that they may make useful candidate in a stem cell based therapy against cancer. However, further pre-clinical studies are needed to validate their therapeutic usage.

Effect of Cordyceps polysaccharides on hepatocyte apoptosis induced by tumor necrosis factor-α

Objective To investigate the effect of Cordyceps polysaccharides on hepatocyte apoptosis and its mechanism of action. Methods Normal human L02 hepatocytes without any drug treatment was established as normal group. The L02 hepatocytes were treated with different concentrations of tumor necrosis factor-α (TNF-α) (5, 10, 20, and 40 ng/ml) for 24 hours to screen out the modeling conditions for the model of hepatocyte apoptosis, which were established as model group. According to the experimental design, L02 hepatocytes pretreated with three different concentrations of Cordyceps polysaccharides (50, 100, and 200 μg/ml) for 12 hours, with or without TNF-α treatment for 24 hours at the selected concentration, were established as experimental group. Related samples were collected for analysis. CCK8 assay was used to measure cell proliferation; Annexin V/PI double staining was used to measure the number of apoptotic hepatocytes; RT-PCR was used to measure the mRNA expression of cell apoptosis genes (Bax, caspase-3, caspase-8, caspase-9, and death receptor Fas); Western blotting was used to measure the protein expression of cleaved-caspase-3 and cleaved-caspase-8. A one-way analysis of variance or the Kruskal-Wallis H test was used for comparison of continuous data between multiple groups, and the least significant difference t-test or the Dunnett-t test was used for further comparison between two groups. Results CCK8 assay showed that compared with the normal group, the L02 hepatocytes induced by 40 ng/ml TNF-α for 24 hours had a significant reduction in proliferation (73.54%±14.19% vs 100.00%±23.61%, P Conclusion Cordyceps polysaccharides can effectively inhibit the apoptosis of normal L02 hepatocytes induced by TNF-α.

BNIP3L-mediated mitophagy is required for mitochondrial remodeling during the differentiation of optic nerve oligodendrocytes

ABSTRACT Retinal ganglion cell axons are heavily myelinated (98%) and myelin damage in the optic nerve (ON) severely affects vision. Understanding the molecular mechanism of oligodendrocyte progenitor cell (OPC) differentiation into mature oligodendrocytes will be essential for developing new therapeutic approaches for ON demyelinating diseases. To this end, we developed a new method for isolation and culture of ON-derived oligodendrocyte lineage cells and used it to study OPC differentiation. A critical aspect of cellular differentiation is macroautophagy/autophagy, a catabolic process that allows for cell remodeling by degradation of excess or damaged cellular molecules and organelles. Knockdown of ATG9A and BECN1 (pro-autophagic proteins involved in the early stages of autophagosome formation) led to a significant reduction in proliferation and survival of OPCs. We also found that autophagy flux (a measure of autophagic degradation activity) is significantly increased during progression of oligodendrocyte differentiation. Additionally, we demonstrate a significant change in mitochondrial dynamics during oligodendrocyte differentiation, which is associated with a significant increase in programmed mitophagy (selective autophagic clearance of mitochondria). This process is mediated by the mitophagy receptor BNIP3L (BCL2/adenovirus E1B interacting protein 3-like). BNIP3L-mediated mitophagy plays a crucial role in the regulation of mitochondrial network formation, mitochondrial function and the viability of newly differentiated oligodendrocytes. Our studies provide novel evidence that proper mitochondrial dynamics is required for establishment of functional mitochondria in mature oligodendrocytes. These findings are significant because targeting BNIP3L-mediated programmed mitophagy may provide a novel therapeutic approach for stimulating myelin repair in ON demyelinating diseases. Abbreviations: A2B5: a surface antigen of oligodendrocytes precursor cells, A2B5 clone 105; ACTB: actin, beta; APC: an antibody to label mature oligodendrocytes, anti-adenomatous polyposis coli clone CC1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG9A: autophagy related 9A; AU: arbitrary units; BafA1: bafilomycin A1; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CNP: 2′,3′-cyclic nucleotide 3′-phosphodiesterase; Ctl: control; COX8: cytochrome c oxidase subunit; CSPG4/NG2: chondroitin sulfate proteoglycan 4; DAPI: 4′6-diamino-2-phenylindole; DNM1L: dynamin 1-like; EGFP: enhanced green fluorescent protein; FACS: fluorescence-activated cell sorting; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; GFP: green fluorescent protein; HsESC: human embryonic stem cell; IEM: immunoelectron microscopy; LAMP1: lysosomal-associated membrane protein 1; LC3B: microtubule-associated protein 1 light chain 3; MBP: myelin basic protein; MFN2: mitofusin 2; Mito-Keima: mitochondria-targeted monomeric keima-red; Mito-GFP: mitochondria-green fluorescent protein; Mito-RFP: mitochondria-red fluorescent protein; MitoSOX: red mitochondrial superoxide probe; MKI67: antigen identified by monoclonal antibody Ki 67; MMP: mitochondrial membrane potential; O4: oligodendrocyte marker O4; OLIG2: oligodendrocyte transcription factor 2; ON: optic nerve; OPA1: OPA1, mitochondrial dynamin like GTPase; OPC: oligodendrocyte progenitor cell; PDL: poly-D-lysine; PINK1: PTEN induced putative kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; RGC: retinal ganglion cell; ROS: reactive oxygen species; RT-PCR: real time polymerase chain reaction; SEM: standard error of the mean; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; TUBB3: tubulin, beta 3 class III.

Retinoic and ascorbic acids induce osteoblast differentiation from human dental pulp mesenchymal stem cells

Previous studies have suggested an important role of retinoic acid (RA) and ascorbic acid (AA) in the stimulation of osteoblastic differentiation; however, the function of RA and AA in the osteogenic differentiation from human dental pulp (hDPSCs) remains unclear. ObjectiveThis in vitro study investigated the effects of RA and AA on the differentiation of osteoblast from hDPSCs. MethodshDPSCs were treated with different doses of RA and AA, separately or in combination (RA ​+ ​AA). Morphology and cell proliferation were assessed. Osteoblast differentiation was evaluated by alizarin red, alkaline phosphatase staining, and RUNX2 gene expression. ResultsA significant reduction was observed in the number of cells treated with RA (26%) and RA ​+ ​AA (30%) after 12 days of treatment. AA treatment alone induced a 12% reduction in the number of cells. Morphologically, the cells treated with RA and RA ​+ ​AA were larger and more elongated than the control cells. A mesh pattern was observed in cells treated with AA. Numerous calcified nodules were present in cells treated with RA, AA, and RA ​+ ​AA. This coincided with increased expression of RUNX2 and high alkaline phosphatase staining levels. ConclusionshDPSCs treated with RA and RA ​+ ​AA showed significant reduction in proliferation, detectable morphological changes, and expression of the key differentiation gene RUNX2, consistent with an osteoblast phenotype. AA induced morphological changes and early formation of calcified nodules. RA had a predominant effect when AA and RA were used together.

Induction of apoptosis by Shikonin through ROS-mediated intrinsic and extrinsic apoptotic pathways in primary effusion lymphoma

Primary effusion lymphoma (PEL) is an incurable non-Hodgkin's lymphoma and novel biology-based treatments are urgently needed in clinical settings. Shikonin (SHK), a napthoquinone derivative, has been used for the treatment of solid tumors. Here, we report that SHK is an effective agent for the treatment of PEL. Treatment with SHK results in significant reduction of proliferation in PEL cells and their rapid apoptosis in vitro. SHK-induced apoptosis of PEL cells is accompanied by the generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential (Δψm), an activation of c-Jun-N-terminal kinase (JNK), p38, as well as caspase-3, -8, and -9. Scavenging of ROS in the presence of N-acetylcysteine (NAC) almost blocks the loss of mitochondrial membrane Δψm, activation of JNK, cleavage of caspase-3, -9, and an induction of apoptosis in SHK treated PEL cells. SP600125, a specific inhibitor of JNK, also rescues a proportion of cells from the apoptotic effect of SHK. In addition, inhibition of caspase activation in the presence of pan-caspase inhibitor, Q-VD-OPh, blocks the SHK-inducing apoptosis, but doesn't completely inhibit SHK-mediated JNK activation. Therefore, ROS is an upstream trigger of SHK-induced caspase dependent apoptosis of PEL cells through disruption of mitochondrial membrane Δψm in an intrinsic pathway and an activation of JNK in an extrinsic pathway. In a PEL xenografted mouse model, SHK treatment suppresses PEL-mediated ascites formation without showing any significant adverse toxicity. These results suggested that SHK could be a potent anti-tumor agent for the treatment of PEL.

Imetelstat Inhibits Telomerase and Prevents Propagation of ADAR1-Activated Myeloproliferative Neoplasm and Leukemia Stem Cells

BACKGROUND Clonal stem cell derived myeloproliferative neoplasms (MPNs) have a propensity to evolve to acute myeloid leukemia (AML). Deregulation of the innate immune deaminase associated with RNA1 (ADAR1) has been linked to malignant progression and therapeutic resistance. Increased expression of the stem cell gene, human telomerase reverse transcriptase (hTERT), has also been linked with malignant transformation. However, the combinatorial role of ADAR1 and hTERT in the evolution of MPN stem cells to therapy resistant acute myeloid leukemia stem cells (LSCs) and the capacity of a telomerase inhibitor, imetelstat, to prevent survival and self-renewal of pre-LSC and LSC had not been established. Recent clinical trials show early signs of efficacy of imetelstat in treatment of myelofibrosis (MF). However, its role in selectively inhibiting pre-LSC transformation to self-renewing LSC has not been elucidated. Here we show that targeting telomerase activity prevents pre-LSC and LSC maintenance both in vitro and in vivo, suggesting telomerase inhibition as an effective strategy for preventing MPN progression. METHODS To quantify hTERT level and ADAR1 activity in the setting of normal HSPC and MPN stem cell evolution, whole genome sequencing (WGS) analysis was performed on 76 normal and MPN blood CD34+ cells and matching saliva samples. Results were compared with RNA-seq of 100 FACS purified young, aged, MPN and AML CD34+CD38- stem cells and CD34+CD38+ progenitor cells. Confocal fluorescence microscopic evaluation of stem cell ADAR1 and hTERT localization, telomere length by Flow-FISH and telomerase activity by TRAP assays, lentiviral ADAR1 overexpression and shRNA knockdown were performed. In vitro stromal co-cultures, and humanized immunocompromised mouse models were established to determine the impact of imetelstat (a oligonucleotide inhibitor of telomerase) on normal, MPN stem cell and LSC maintenance. RESULTS Combined hTERT overexpression, ADAR1 activation and a significant reduction in telomere length correlated with accelerated stem cell aging during MPN progression to AML. Increased ADAR1 mediated adenosine to inosine (A-to-I) transcript editing coincided with accelerated telomere shortening in high risk MPN stem cells. Moreover, lentiviral ADAR1 overexpression enhanced pre-LSC engraftment. Treatment with imetelstat reduced MPN stem cell and LSC propagation in stromal co-cultures as well as in humanized mouse models commensurate with reduced hTERT expression levels and telomerase activity and decreased ADAR1 editing activity. Specifically, stromal co-culture assays revealed that combined treatment with dasatinib at 1 nM, and imetelstat at 1 µM or 5 µM significantly inhibited survival and replating of blast crisis (BC) CML progenitors compared with aged bone marrow progenitors (p < 0.001, ANOVA). As a single agent, imetelstat (5 µM) inhibited survival and replating of pre-LSC derived from myelofibrosis compared with normal bone marrow progenitor samples (p < 0.001, ANOVA). In pre-LSC MPN NSG-SGM mouse models established from 4 different MF samples, a significant reduction in proliferation of human CD45+ cells (p < 0.01, t test) was observed in bone marrow and spleen, when compared with vehicle control. Treatment of humanized LSC mouse models, established with 5 different BC CML, with 30 mg/kg of imetelstat, 3 times a week for 4 weeks resulted in a significant reduction in proliferation of malignant progenitors and human CD45+ cells (p < 0.001, ANOVA). As measured by a Flow-FISH assay, abnormal telomere length was reversed by imetelstat treatment compared with mismatch control (p < 0.05, ANOVA). In addition, FACS analysis revealed a significant reduction in activated beta-catenin expression after imetelstat treatment of LSC engrafted mice compared with vehicle control (p < 0.01, ANOVA). Finally, RNA-seq analysis performed on human CD34+ cells from imetelstat treated LSC mouse models revealed a significant reduction in LSC harboring malignant ADAR1-mediated A-to-I editing at doses that spared normal hematopoietic stem cells. CONCLUSIONS Combined WGS and RNA-Seq analyses, lentiviral ADAR1 overexpression, stromal co-culture assays and humanized pre-LSC and LSC mouse model studies reveal that pre-LSC evolution into LSC coincides with both ADAR1 and hTERT activation, which can be prevented with imetelstat. Disclosures Rizo: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Huang:Geron Corp: Current Employment, Current equity holder in publicly-traded company. Jamieson:Forty Seven Inc: Patents & Royalties; Bristol-Myers Squibb: Other.

Abstract 3151: Active immunization with PD1-derived mimotope-Combination immunotherapy against Her-2/neu-expressing tumors

Abstract Application of monoclonal antibodies (mAbs) as immune checkpoint inhibitors (ICIs) has demonstrated a tremendous effect in cancer immunotherapy. However, high costs for and frequent applications of mono- or combination-therapies or possible development of resistance justify modifications of this approach. In this line, active immunization with mimotopes (B cell peptides) of ICIs, activating the patients' own immune system, rather than application of the corresponding therapeutic mAbs, may provide advantages over the costly treatment of the mAbs. Overlapping peptides spanning the extracellular domains of human PD1 (hPD1) were used to identify hPD1-derived mimotope, using the therapeutic mAb Nivolumab as a proof of concept. Additionally, for in vivo evaluation in a tumor mouse model, a mouse PD1 (mPD1)-derived mimotope was identified using an anti-mPD1 mAb with mPD1/mPDL-1 blocking capacity. The identified mimotopes were characterized by in vitro assays, including a reporter cell-based assay, and their anti-tumor effect was evaluated in a syngeneic mouse model involving grafting with human Her-2/neu-expressing tumors. The PD1-mimotopes were shown to specifically inhibit the binding of the corresponding mAbs, and also the mAbs capacity in blocking the respective PD1-PDL1 interactions. Applying the syngeneic tumor mouse model, a significant tumor growth reduction following active immunization with the mPD1-mimotope was shown. Importantly, combined vaccination with the mimotope and a multiple B-cell epitope Her-2/neu vaccine potentiated the anti-tumor effect in the syngeneic mice. Tumor growth reduction in the mice immunized with the mimotope was associated with a significant increase of the apoptotic (CC3) and significant reduction of proliferation (Ki67) markers, as evaluated by immunohistochemistry (IHC) staining. However, the tumor growth reduction following active immunization with the cancer vaccine (alone or in combination with the checkpoint mimotope) showed a different IHC picture, namely highly increased levels of CC3 without reduction of proliferation (Ki67 levels) in the tumor cells. In depth studies on the underlying mechanisms of tumor growth reduction are currently ongoing. Our results suggest active immunization with B cell mimotopes of ICIs as either monovalent vaccine or a combination therapy with tumor specific vaccines to enhance the anti-tumor efficacy. Such strategies may potentially lead to multipurpose treatment regimens adapted to the type, stage and progression phase of the various tumors. Citation Format: Joshua Tobias, Claire Battin, Annika De Sousa Linhares, Michael Lebens, Karin Baier, Katharina Ambroz, Mirjana Drinić, Sandra Högler, Aleksandra Inic-Kanada, Erika Garner-Spitzer, Matthias Preusser, Lukas Kenner, Michael Kundi, Christoph C. Zielinski, Peter Steinberger, Ursula Wiedermann. Active immunization with PD1-derived mimotope-Combination immunotherapy against Her-2/neu-expressing tumors [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 3151.

NHD2-15, a novel antagonist of Growth Factor Receptor-Bound Protein-2 (GRB2), inhibits leukemic proliferation.

The majority of chronic myeloid leukemia (CML) cases are caused by a chromosomal translocation linking the breakpoint cluster region (BCR) gene to the Abelson murine leukemia viral oncogene-1 (ABL1), creating the mutant fusion protein BCR-ABL1. Downstream of BCR-ABL1 is growth factor receptor-bound protein-2 (GRB2), an intracellular adapter protein that binds to BCR-ABL1 via its src-homology-2 (SH2) domain. This binding constitutively activates growth pathways, downregulates apoptosis, and leads to an over proliferation of immature and dysfunctional myeloid cells. Utilizing novel synthetic methods, we developed four furo-quinoxaline compounds as GRB2 SH2 domain antagonists with the goal of disrupting this leukemogenic signaling. One of the four antagonists, NHD2-15, showed a significant reduction in proliferation of K562 cells, a human BCR-ABL1+ leukemic cell line. To elucidate the mode of action of these compounds, various biophysical, in vitro, and in vivo assays were performed. Surface plasmon resonance (SPR) assays indicated that NHD2-15 antagonized GRB2, binding with a KD value of 119 ± 2 μM. Cellulose nitrate (CN) assays indicated that the compound selectively bound the SH2 domain of GRB2. Western blot assays suggested the antagonist downregulated proteins involved in leukemic transformation. Finally, NHD2-15 was nontoxic to primary cells and adult zebrafish, indicating that it may be an effective clinical treatment for CML.

MiR-489-3p inhibits proliferation and migration of bladder cancer cells through downregulation of histone deacetylase 2.

Since human bladder cancer (BC) is a common malignancy of the urinary system with poor prognosis, it is crucial to clarify the molecular mechanisms of BC development and progression. To the best of our knowledge, the current study demonstrated for the first time that miR-489-3p suppressed BC cell-derived tumor growth in vivo via the downregulation of histone deacetylase 2 (HDAC2). According to the results, expression levels of miR-489-3p were lower in BC tissues compared with corresponding normal tissues. Expression of miR-489-3p mimics in BC-derived T24 and 5637 cells resulted in a significant reduction in proliferation and migration rates. Furthermore, bioinformatics analyses indicated that HDAC2 may be a potential downstream target of miR-489-3p. In contrast to miR-489-3p, HDAC2 was expressed at higher levels in BC tissues compared with corresponding normal tissues. Additionally, small interfering RNA-mediated knockdown of HDAC2 caused a marked decrease in the proliferation and migration rates of T24 and 5637 cells. Consistent with these observations, expression of miR-489-3p mimics attenuated the growth of xenograft tumors arising from T24 cells and resulted in HDAC2 downregulation. In conclusion, the results of the current study indicated that the miR-489-3p/HDAC2 axis serves a role in the development and/or the progression of BC and may be a potential molecular target for the development of a novel strategy to treat patients with BC.

TAF15 contributes to the radiation-inducible stress response in cancer.

Resistance to radiation therapy is a significant problem in the treatment of non-small cell lung cancer (NSCLC). There is an unmet need to discover new molecular targets for drug development in combination with standard of care cancer therapy. We found that TAF15 was radiation-inducible using phage-displayed peptide libraries. In this study, we report that overexpression of TAF15 is correlated with worsened survival in NSCLC patients. Radiation treatment led to surface induction of TAF15 in vitro and in vivo. We genetically silenced TAF15 which led to a significant reduction in proliferation of NSCLC cells. Cells depleted of TAF15 exhibited cell cycle arrest and enhanced apoptosis through activation and accumulation of p53. In combination with radiation, TAF15 knockdown led to a significant reduction in the surviving fraction of NSCLC cell lines. To determine the importance of TAF15 surface expression, we targeted TAF15 with an antibody. In combination with radiation, the anti-TAF15 antibody led to a reduction in the surviving fraction of cancer cells. These studies show that TAF15 is a radiation-inducible molecular target that is accessible to anti-cancer antibodies and enhances cell viability in response to radiation.

A New Strategy Toward B Cell-Based Cancer Vaccines by Active Immunization With Mimotopes of Immune Checkpoint Inhibitors.

Therapeutic monoclonal antibodies (mAbs), targeting tumor antigens, or immune checkpoints, have demonstrated a remarkable anti-tumor effect against various malignancies. However, high costs for mono- or combination therapies, associated with adverse effects or possible development of resistance in some patients, warrant further development and modification to gain more flexibility for this immunotherapy approach. An attractive alternative to passive immunization with therapeutic antibodies might be active immunization with mimotopes (B-cell peptides) representing the mAbs' binding epitopes, to activate the patient's own anti-tumor immune response following immunization. Here, we identified and examined the feasibility of inducing anti-tumor effects in vivo following active immunization with a mimotope of the immune checkpoint programmed cell death 1 (PD1), alone or in combination with a Her-2/neu B-cell peptide vaccine. Overlapping peptides spanning the extracellular domains of human PD1 (hPD1) were used to identify hPD1-derived mimotopes, using the therapeutic mAb Nivolumab as a proof of concept. Additionally, for in vivo evaluation in a tumor mouse model, a mouse PD1 (mPD1)-derived mimotope was identified using an anti-mPD1 mAb with mPD1/mPDL-1 blocking capacity. The identified mimotopes were characterized by in vitro assays, including a reporter cell-based assay, and their anti-tumor effects were evaluated in a syngeneic tumor mouse model stably expressing human Her-2/neu. The identified PD1-derived mimotopes were shown to significantly block the mAbs' capacity in inhibiting the respective PD1/PD-L1 interactions. A significant reduction in tumor growth in vivo was observed following active immunization with the mPD1-derived mimotope, associated with a significant reduction in proliferation and increased apoptotic rates in the tumors. Particularly, combined vaccination with the mPD1-derived mimotope and a multiple B-cell epitope Her-2/neu vaccine potentiated the vaccine's anti-tumor effect. Our results suggest active immunization with mimotopes of immune checkpoint inhibitors either as monotherapy or as combination therapy with tumor-specific vaccines, as a new strategy for cancer treatment.

ILK supports RhoA/ROCK-mediated contractility of human intestinal epithelial crypt cells by inducing the fibrillogenesis of endogenous soluble fibronectin during the spreading process

BackgroundFibronectin (FN) assembly into an insoluble fibrillar matrix is a crucial step in many cell responses to extracellular matrix (ECM) properties, especially with regards to the integrin-related mechanosensitive signaling pathway. We have previously reported that the silencing of expression of integrin-linked kinase (ILK) in human intestinal epithelial crypt (HIEC) cells causes significant reductions in proliferation and spreading through concomitantly acquired impairment of soluble FN deposition. These defects in ILK-depleted cells are rescued by growth on exogenous FN. In the present study we investigated the contribution of ILK in the fibrillogenesis of FN and its relation to integrin-actin axis signaling and organization.ResultsWe show that de novo fibrillogenesis of endogenous soluble FN is ILK-dependent. This function seemingly induces the assembly of an ECM that supports increased cytoskeletal tension and the development of a fully spread contractile cell phenotype. We observed that HIEC cell adhesion to exogenous FN or collagen-I (Col-I) is sufficient to restore fibrillogenesis of endogenous FN in ILK-depleted cells. We also found that optimal engagement of the Ras homolog gene family member A (RhoA) GTPase/Rho-associated kinase (ROCK-1, ROCK-2)/myosin light chain (MLC) pathway, actin ventral stress fiber formation, and integrin adhesion complex (IAC) maturation rely primarily upon the cell’s capacity to execute FN fibrillogenesis, independent of any significant ILK input. Lastly, we confirm the integrin α5β1 as the main integrin responsible for FN assembly, although in ILK-depleted cells αV-class integrins expression is needed to allow the rescue of FN fibrillogenesis on exogenous substrate.ConclusionOur study demonstrates that ILK specifically induces the initiation of FN fibrillogenesis during cell spreading, which promotes RhoA/ROCK-dependent cell contractility and maturation of the integrin-actin axis structures. However, the fibrillogenesis process and its downstream effect on RhoA signaling, cell contractility and spreading are ILK-independent in human intestinal epithelial crypt cells.

Evaluation of Gene Expression Data From Cybrids and Tumours Highlights Elevated NDRG1-Driven Proliferation in Triple-Negative Breast Cancer.

Background:Triple-negative breast cancer is an aggressive type of breast cancer with high risk of recurrence. It is still poorly understood and lacks any targeted therapy, which makes it difficult to treat. Thus, it is important to understand the underlying mechanisms and pathways that are dysregulated in triple-negative breast cancer.Methods:To investigate the role of mitochondria in triple-negative breast cancer progression, we analysed previously reported gene expression data from triple-negative breast cancer cybrids with SUM-159 as the nuclear donor cell and SUM-159 or A1N4 (c-SUM-159, c-A1N4) as the mitochondrial donor cells and with 143B as the nuclear donor cell and MCF-10A or MDA-MB-231 (c-MCF-10A, c-MDA-MB-231) as the mitochondrial donor cells. The role of potential biomarkers in cell proliferation and migration was examined in SUM-159 and MDA-MB-231 cells using sulforhodamine B and wound healing assays.Results:Rank product analysis of cybrid gene expression data identified 149 genes which were significantly up-regulated in the cybrids with mitochondria from the cancer cell line. Analysis of previously reported breast tumour gene expression datasets confirmed 9 of the 149 genes were amplified, up-regulated, or down-regulated in more than 10% of the patients. The genes included NDRG1, PVT1, and EXT1, which are co-located in cytoband 8q24, which is frequently amplified in breast cancer. NDRG1 showed the largest down-regulation in the cybrids with benign mitochondria and was associated with poor prognosis in a breast cancer clinical dataset. Knockdown of NDRG1 expression significantly decreased proliferation of SUM-159 triple-negative breast cancer cells.Conclusions:These results indicate that mitochondria-regulated nuclear gene expression helps breast cancer cells survive and proliferate, consistent with previous work focusing on an Src gene signature which is mitochondria regulated and drives malignancy in breast cancer cybrids. This is the first study to show that mitochondria in triple-negative breast cancer mediate significant up-regulation of a number of genes, and silencing of NDRG1 leads to significant reduction in proliferation.

Integrin αvβ6-specific therapy for pancreatic cancer developed from foot-and-mouth-disease virus.

Goals of investigation: The 5-year survival rate for pancreatic ductal adenocarcinoma (PDAC) has remained at <5% for decades because no effective therapies have been identified. Integrin αvβ6 is overexpressed in most PDAC and represents a promising therapeutic target. Thus, we attempted to develop an αvβ6-specific peptide-drug conjugate (PDC) for therapy of PDAC.Methodology: We conjugated the DNA-binding pyrrolobenzodiazepine (PBD)-based payload SG3249 (tesirine) to an αvβ6-specific 20mer peptide from the VP1 coat protein of foot-and-mouth-disease virus (FMDV) (forming conjugate SG3299) or to a non-targeting peptide (forming conjugate SG3511). PDCs were tested for specificity and toxicity on αvβ6-negative versus-positive PDAC cells, patient-derived cell lines from tumor xenografts, and on two different in vivo models of PDAC. Immunohistochemical analyses were performed to establish therapeutic mechanism.Results: The αvβ6-targeted PDC SG3299 was significantly more toxic (up to 78-fold) for αvβ6-expressing versus αvβ6-negative PDAC cell lines in vitro, and achieved significantly higher toxicity at equal dose than the non-targeted PDC SG3511 (up to 15-fold better). Moreover, SG3299 eliminated established (100mm3) Capan-1 PDAC human xenografts, extending the lifespan of mice significantly (P=0.005). Immunohistochemistry revealed SG3299 induced DNA damage and apoptosis (increased γH2AX and cleaved caspase 3, respectively) associated with significant reductions in proliferation (Ki67), β6 expression and PDAC tumour growth.Conclusions: The FMDV-peptide drug conjugate SG3299 showed αvβ6-selectivity in vitro and in vivo and can specifically eliminate αvβ6-positive cancers, providing a promising new molecular- specific therapy for pancreatic cancer.

CBMT-21. TERT PROMOTER-MUTANT GLIOBLASTOMAS EXHIBIT DEPENDENCY ON TELOMERASE

Abstract TERT promoter mutations are among the most common somatic alterations in cancer and they occur in about 80% of IDH-wildtype glioblastomas. TERT promoter mutations were found to reactivate telomerase by providing a novel binding site for the GABP transcription factor. While the effects of telomerase ablation are well understood in mice and somatic human cells, these effects in cancer are yet to be fully elucidated. In this study, we used a genetic approach with CRISPR-interference to knock down telomerase in TERT promoter-mutant glioblastoma cell lines. We show that this leads to a gradual and significant reduction in proliferation. This phenotype ultimately culminates in telomere crisis, with telomere shortening, activation of the DNA damage response pathway and formation of chromatin bridges. These data suggest that anti-telomerase therapy is a potential effective approach for glioblastoma tumors.

The Klf6-related super enhancer regulates Klf6-SV2 expression mediated proliferation in human hepatoma (HepG2) cells

BackgroundThe Klf6 gene, which belongs to Krüppel-like family of C2H2 zinc finger transcription factors, is greatly related to tumorigenesis via a high rate of somatic mutation in the carcinomas of prostate, liver, colon, stomach, lung, neck, pituitary, and nervous system: Furthermore, the pathways regulating the expressions of Klf6 splice variants termed Klf6-SV1, -SV2, and -SV3 remain obscure although their functional outcomes have been clear. In this study, the functional roles of Klf6 variants in the inhibition of cell proliferation induced by the disruption of Klf6-related super enhancer in human hepatoma (HepG2) cells were evaluated.ResultsAs a result, the disruption of Klf6-related super enhancer not only induced the upregulation of Klf6-SV2 but also led to a significant reduction of proliferation in HepG2 cells. In addition, the disruption of Klf6-related super enhancer led to the induction of p21 and Bax genes mediated by the upregulation of Klf6-SV2.ConclusionIn conclusion, it was demonstrated that Klf6-related super enhancer modulates cell proliferation via the regulation of Klf6-SV2 expression in human hepatoma (HepG2) cells. The results provide the functional significance of Klf6-related super enhancer in understanding the transcriptional regulation mechanism of Klf6.

Clinical parameters affecting multipotent adult progenitor cells in vitro

BackgroundHuman multipotent adult progenitor cells (MAPC®) are an emerging therapy for traumatic brain injury (TBI); however, clinically translating a therapy involves overcoming many factors in vivo which are not present in pre-clinical testing. In this study we examined clinical parameters in vitro that may impact cell therapy efficacy. MethodsMAPC were infused through varying gauged needles and catheters with and without chlorhexidine, and their viability tested with trypan blue exclusion. MAPC were co-cultured with phenytoin and celecoxib at relevant clinical concentrations for 1 h and 24 h. Anti-inflammatory potency was tested using a stimulated rat splenocyte co-culture and ELISA for TNF-α production. MAPC were cultured under different osmolar concentrations and stained with propidium iodide for viability. Anti-inflammatory potency was tested by co-culture of MAPC with naïve lymphocytes activated by CD3/CD28 beads, and Click-iT® Plus EdU was used to quantify proliferation by flow cytometry. ResultsThe mean viability of the MAPC infused via needles was 95 ± 1%; no difference was seen with varying flow rate, but viability was notably reduced by chlorhexidine. MAPC function was not impaired by co-culture with phenytoin, celecoxib, or combination with both. Co-culture with phenytoin showed a decrease in TNF-α production as compared to the MAPC control. MAPC cultured at varying osmolar concentrations all had viabilities greater than 90% with no statistical difference between them. Co-culture of MAPC with CD3/CD28 activated PBMCs showed a significant reduction in proliferation as measured by EdU uptake. DiscussionNeedle diameter, phenytoin, celecoxib, and a relevant range of osmolarities do not impair MAPC viability or anti-inflammatory potency in vitro.

Abstract 5264: Metabolic reprogramming with β-alanine to overcome chemotherapy resistance in pancreatic cancer

Abstract Background: Increased aerobic glycolysis (Warburg effect) leading to acidic microenvironment confers chemoresistance to weakly basic Gemcitabine, the standard treatment in Pancreatic ductal adenocarcinoma (PDAC). Metabolomic perturbations in two PDAC cell lines having differential sensitivity to Gemcitabine was investigated by profiling over 40 metabolites using 1H-NMR spectroscopy. Notably, a significant decrease in the concentration of β-alanine (βA) was observed in Gem resistant compared to Gem sensitive cell line, suggesting βA may have a role in chemotherapy response. We aimed to evaluate the role of βA supplementation in improving Gem efficacy in Gem resistant cell lines Methods: PDAC Panc-1 cell line extracts treated with Gem with and without βA supplementation were analysed via 1H-NMR spectroscopy. Aerobic glycolysis was quantified by measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using the Seahorse XF analyser platform. Global mRNA profiling was done by Next Generation Sequencing. Proliferation, migration and cell cycle analysis was measured by Xcelligence real time cell analysis system and flow cytometry, respectively. PANC-1 and BxPC-3 tumor xenografts treated with Gemcitabine with and without βA for 4 weeks and the tumor volumes and weights were measured. Results: βA supplementation in Gem resistant cells results in: (1) decreased basal glycolytic rate, proton leak and ATP production; (2) increased pyruvate, decreased lactic acid and NAD+ concentrations with decreased LDH-A transcription; also increased carnosine levels with a more basic pH in the conditioned media; (3) enhanced sensitivity to Gem treatment, with a significant reduction in proliferation, cell migration, and increased apoptosis and relative hENt-1 mRNA levels and (5) xenograft tumor volumes and weights were significantly reduced upon βA supplementation. Conclusion: βA supplementation reprograms cell metabolism by normalizing energy production and reducing microenvironment acidification, thus enhancing chemo-sensitivity in Gem resistant PDAC cells and xenografts. βA can be potentially used as a co-therapeutic in PDAC. Note: This abstract was not presented at the meeting. Citation Format: Danny Yakoub, Smitha T. Totiger, Alexandra Moran, Sujit Suwal, Julio Pimentel, Lauren O'Donell, Jamie Walls, Nagaraj Nagathihalli, Nipun Merchant. Metabolic reprogramming with β-alanine to overcome chemotherapy resistance in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5264.

Sulforaphane Impacts Invasion Capacity and Proliferation of Triple Negative Breast Cancer Cells Grown in the Presence of Tumor-associated Macrophages (P05-003-19)

ObjectivesTriple negative breast cancer (TNBC) accounts for 12% to 24% of all breast cancer cases and is characterized by higher proliferation rates and an increased likelihood of tissue invasion. Tumor cells and cells in the tumor microenvironment (TME), specifically tumor associated macrophages (TAMs), interact through signaling (e.g., cytokines) to promote cancer progression by increasing proliferation and invasion capacity. Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables (i.e., broccoli) that has shown promising results in hindering TNBC progression. SFN reduced proliferation in breast cancer cells and altered cytokine signaling between breast cancer cells and cells in the TME (adipocytes). SFN also decreased invasion markers in TNBC cells grown in isolation. However, TAMs promote tumor cell aggression, and SFN’s effect in multicellular environments is unclear. To determine SFN’s potential in cancer treatment, it is critical to investigate SFN’s effect on proliferation and invasion capacity of TNBC cells grown under TAMs influence, not just grown in isolation. The objective of this study was to determine if SFN can reduce proliferation and invasion capacity of TNBC cells grown in TAM secretions. MethodsFor cell proliferation, TNBC cells (MDA-MB-231) were exposed to TAM secretions using conditioned media and were then treated with SFN (10 μM) or DMSO vehicle control for 24 and 48 hours. Proliferation was measured using a MTT-based assay and cell counts. For invasion, TAMS and TNBC cells were co-cultured for 48 hours in transwell plates. Prior to co-culture period, cells were treated with SFN (15 μM) or vehicle control. Invasion capacity was measured through a transwell invasion assay with collagen representing the tumor basement membrane. ANOVA and t-tests were used to determine statistical differences with significance at P = 0.05. ResultsPreliminary analysis revealed significant reductions in proliferation for TAM exposed TNBC cells after 24 hours (P = 0. 0132) and 48 hours (P = 0. 0190) of SFN treatment. ConclusionsSFN reduced proliferation and can potentially reduce invasion capacity of TNBC cells influenced by TAM secretions thus enhancing future utilization of SFN in TNBC treatment regimens. Funding SourcesCalifornia State University, Chico.