Non-transformed Cell Line Research Articles

Validation of Babesia proteasome as a drug target

Babesiosis is a tick-transmitted zoonosis caused by apicomplexan parasites of the genus Babesia. Treatment of this emerging malaria-related disease has relied on antimalarial drugs and antibiotics. The proteasome of Plasmodium, the causative agent of malaria, has recently been validated as a target for anti-malarial drug development and therefore, in this study, we investigated the effect of epoxyketone (carfilzomib, ONX-0914 and epoxomicin) and boronic acid (bortezomib and ixazomib) proteasome inhibitors on the growth and survival of Babesia. Testing the compounds against Babesia divergens ex vivo revealed suppressive effects on parasite growth with activity that was higher than the cytotoxic effects on a non-transformed mouse macrophage cell line. Furthermore, we showed that the most-effective compound, carfilzomib, significantly reduces parasite multiplication in a Babesia microti infected mouse model without noticeable adverse effects. In addition, treatment with carfilzomib lead to an ex vivo and in vivo decrease in proteasome activity and accumulation of polyubiquitinated proteins compared to untreated control. Overall, our results demonstrate that the Babesia proteasome is a valid target for drug development and warrants the design of potent and selective B. divergens proteasome inhibitors for the treatment of babesiosis.

Abstract A65: Evaluating an under-studied kinase as a potential druggable target for breast cancer

Abstract Breast cancer is the most frequently diagnosed cancer among women, accounting for 23% of all cancer cases. Triple-negative breast cancer (TNBC) is a very aggressive type of breast cancer that occurs in African-American women at three-fold higher frequency compared to Hispanic and Caucasian women. TNBC has a lower 5-year survival rate compared to other subtypes of breast cancer and no targeted therapeutics. In order to identify novel targets for TNBC, replicate kinome-wide RNAi screens were performed with HCC1806, a basal-like TNBC cell line, using a 4-day cell viability assay. Among the RNAi hits, we identified the COASY gene, encoding Coenzyme A synthase (CoAsy), as an under-studied kinase. Thus, we sought to confirm this RNAi hit and evaluate CoAsy for its potential as a novel druggable target for TNBC. CoAsy is a bifunctional enzyme that carries out the last two steps in the coenzyme A (CoA) biosynthetic pathway, including the phosphorylation of dephospho-CoA to generate CoA. Our hypothesis is that TNBC cells require a higher level of CoA synthase activity for cell proliferation compared to normal cells and thus inhibiting CoA synthase represents a novel therapeutic strategy for TNBC. Using four different individual RNAi targeting COASY, we used transient RNAi transfection of a panel of breast cancer cell lines and a non-transformed breast cell line to determine the effect of CoAsy knock down on cell viability and proliferation. Knockdown of CoAsy protein by RNAi was demonstrated by Western blot. Transient COASY RNAi transfection inhibited the cell viability/proliferation assay for several TNBC cell lines, but not for a normal breast cell line. Thus, these data suggest that knockdown of CoAsy may preferentially inhibit cell proliferation of breast cancer cell lines. In addition, Western blot was performed to determine and compare the expression of CoAsy in a panel of breast cancer cell lines and nontransformed cell lines. Ongoing studies are investigating the dependence of cell line viability and proliferation on the expression level of CoAsy by creating stable inducible shRNA cell lines. Citation Format: Hamzah Kharabsheh, Xiyou Zhou, John Scott. Evaluating an under-studied kinase as a potential druggable target for breast cancer [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr A65.

PO-286 Protein kinase D3: a crucial regulator of breast cancer stem cell survival

IntroductionTriple negative breast cancer (TNBC) accounts for 15%–20% of all breast carcinomas in women. TNBC is particularly aggressive, highly metastatic and associated with poor prognosis. We previously identified Protein Kinase D3 (PKD3) to be upregulated in TNBC. PKD3 is a serine-threonine kinase that is best known for its functions in membrane trafficking and actin remodelling. Here we identify a novel role for PKD3 in breast cancer stem cell (CSC) maintenance. CSCs are endowed with tumor-initiating capacity, they possess increased chemoresistance and are known to initiate metastasis, and therefore are responsible for tumour relapse.Material and methodsIn the TNBC cell line MDA-MB-231 with a stable PKD3 knockdown, we performed a flow cytometry based screen of cell surface proteins, revealing the downregulation of several membrane proteins associated with CSCs. The importance of PKD3 for driving a CSC gene expression signature, maintaining CSC numbers and function was tested by qRT-PCR analysis of CSC markers, by measuring aldehyde dehydrogenase (ALDH) activity and performing mammosphere assays, respectively. Paclitaxel sensitivity of TNBC cell lines was assessed in combination with the selective PKD inhibitor CRT0066101. Whether the ectopic expression of PKD3 was sufficient to induce the expansion of the CSC pool was analysed in the non-transformed breast epithelial cell line MCF10A.Results and discussionsIn MDA-MB-231 cells with PKD3 knockdown, the CSC markers (CD184, Notch4, Sox2, Oct3/4) were downregulated. This was associated with reduced CSC numbers based on ALDH positivity and sphere-forming efficiency in mammosphere assays. Pharmacological PKD inhibition in the TNBC cell lines MDA-MB-231, BT549 and MDA-MB-436 verified these results. By contrast, inducible PKD3 expression in MCF10A cells elevated mammosphere formation and stem cell marker expression. Importantly, the combination of paclitaxel with CRT0066101 was superior over the single treatments in suppressing the survival of MDA-MB-231, BT549 and MDA-MB-436 cells in both colony formation and mammosphere assays.ConclusionOur results establish PKD3 as a crucial regulator of CSC maintenance and demonstrate that paclitaxel treatment in combination with PKD inhibition provides a potential novel strategy for future therapeutic applications in TNBC treatment.

Abstract 4092: Glycosyltransferase ST6Gal-I promotes pancreatic ductal adenocarcinoma progression and metastasis

Abstract Sialyltransferase ST6Gal-I adds α2-6 sialic acids to select N-glycosylated cell surface receptors, thereby modulating receptor function and intracellular signaling. ST6Gal-I is upregulated in multiple human cancers, including >70% of pancreatic ductal adenocarcinoma (PDAC) samples. ST6Gal-I confers cancer stem cell (CSC) properties evidenced by tumorspheroid growth, chemoresistance and tumor initiating potential. As an example, we previously reported that ST6Gal-I imparts gemcitabine resistance by dampening DNA damage. One possible mechanism by which ST6Gal-I promotes a CSC phenotype is by increasing the expression of the master stem cell transcription factor, Sox-9. Forced overexpression or knockdown of ST6Gal-I directly regulates Sox9 expression in a wide array of established cancer lines. Sox9 is known to be a potent driver of acinar to ductal metaplasia (ADM), a key PDAC initiating event. To interrogate the role of ST6Gal-I and Sox9 in ADM, ST6Gal-I was overexpressed in the nontransformed acinar cell line, 266-6. ST6Gal-I overexpression was found to induce expression of Sox9 and other ductal markers. Furthermore, ST6Gal-I overexpressing cells spontaneously formed foci in adherent culture, a hallmark of malignant transformation. Contrarily, ST6Gal-I knockdown in 266-6 cells decreased cell viability. Post ADM, PDAC progresses through the formation of PanINs. This process of progressive lesion formation leading to frank PDAC can be replicated in murine models of pancreatic cancer utilizing pancreas specific oncogenic K-Ras (KC). To investigate the role of ST6Gal-I pertaining to PDAC in vivo we generated ST6Gal-I knock-in (KI) mice with oncogenic K-Ras (KC-ST6Gal-I KI). The KC-ST6Gal-I KI mice presented with significantly decreased overall survival (median = 4.3 months) compared to the KC mice (median = 14 months). This was coupled with a marked increase in liver and lung metastasis in KC-ST6Gal-I KI mice compared to KC mice. To better understand the molecular events underlying ST6Gal-I's PDAC-promoting function, we generated organoids from KC, KC-ST6Gal-I KI, ST6Gal-I KI and WT mice. Preliminary data indicate that there is increased Sox-9 expression in ST6Gal-I KI organoids compared to WT controls. As well, the number of organoids formed in culture is increased in ST6Gal-I KI mice compared to the control. Since organoids are derived from the resident stem cells, this data further indicates increased stemness with enhanced ST6Gal-I expression. In ongoing studies we are defining the signaling mechanisms underpinning a ST6Gal-I/Sox9 axis in PDAC initiation events and other aspects of CSC behavior. These collective studies highlight a novel role for tumor glycans in PDAC pathogenesis, and implicate ST6Gal-I as a promising therapeutic target. Citation Format: Asmi Chakraborty, Robert B. Jones, Christopher A. Klug, Lesley E. Smythies, Susan L. Bellis. Glycosyltransferase ST6Gal-I promotes pancreatic ductal adenocarcinoma progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4092.

Cellular differentiation of non-transformed intestinal epithelial cells is regulated by Lactobacillus rhamnosus and L. casei strains.

The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.

Small Molecule Inhibition of IFN‐γ‐Induced Major Histocompatibility Complex Class II Expression by Thyroid Cells

Major histocompatibility complex (MHC) molecules are cell surface receptors involved in host immune response. Several lines of evidence suggest that increased expression of MHC class I (MHC I) and aberrant expression of MHC class II (MHC II) on non‐immune cells contributes to autoimmune disease, including the autoimmune thyroid disease termed Graves' disease. Graves' disease is often treated, especially in Europe, by methimazole (MMI). Previous work has shown that MMI decreases constitutive MHC I expression on the Fisher rat thyroid cell line 5 (FRTL‐5), a non‐transformed cell line often used to gain insights into human thyroid pathology. Further, MMI decreases interferon‐gamma (IFN‐γ) induction of MHC II in FRTL‐5 cells. Our labs, as well as others, have demonstrated that a phenyl‐derivative of MMI, termed C10, is more potent than MMI in a variety of cell models of disease including the FRTL‐5 model described above. Additionally, we have an ongoing molecular screening program that seeks to identify compounds that are more efficacious than MMI and C10. Thus, we initially sought to screen three novel compounds from our library in the FRTL‐5 model. Two of these compounds (COB‐204, COB‐214) were more effective than C10 in other cell models and the third (COB‐187) appears to be a highly specific inhibitor of glycogen synthase kinase ‐3 (GSK‐3). We first investigated the effect of the compounds on basal expression of MHC I. FRTL‐5 cells were treated with each compound for 24 hours and the level of MHC Class I determined via flow cytometric analysis. In each case, no significant effect on MHC Class I was observed at the levels of compound tested (50 μM for COB‐204 and COB‐214 and 20 μM for COB‐187). We next investigated the effect of the compounds on inhibition of IFN‐γ induction of MHC II. In this case, FRTL‐5 cells were treated for 24 hours with 100 U/ml IFN‐γ in the presence of the compounds or carrier control (DMSO) and MHC II expression determined by flow cytometry. 50 μM COB‐214 caused a slight (19%) inhibition, 50 μM COB‐204 caused a marked (54%) inhibition and 20 μM COB‐187 caused a fairly dramatic (80%) inhibition of IFN‐γ induced MHC II expression. Based on these results, we decided to investigate COB‐187 further and also test the effects of two other known GSK‐3 inhibitors, namely Tideglusib and AR‐A014418. All three compounds appeared to inhibit IFN‐γ induction of MHC II albeit with varying potencies. The IC50 for AR‐A014418 was 4.8 μM, for COB‐187 9.6 μM and for Tideglusib > 80 μM. The MHC II inhibition did not appear to be due to non‐specific toxicity since an MTS assay revealed the 24 hour TC50 to be 50 μM for AR‐A014418, and > 80 μM for COB‐187 and Tideglusib. Combined, these results suggest a connection between GSK‐3 and IFN‐γ induced aberrant expression of MHC II on FRTL‐5 cells and provides a possible avenue for the development of molecular therapeutics for Graves' disease.Support or Funding InformationNIH R15GM110602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Benzo[a]pyrene activates an AhR/Src/ERK axis that contributes to CYP1A1 induction and stable DNA adducts formation in lung cells

Benzo[a]pyrene (B[a]P), the most extensively studied carcinogen in cigarette smoke, has been regarded as a critical mediator of lung cancer. It is known that B[a]P-mediated Aryl hydrocarbon Receptor (AhR) activation stimulates the mitogen activated protein kinases (MAPK) signaling cascade in different cell models. MAPK pathway disturbances drive alterations in cellular processes, such as differentiation, proliferation, and apoptosis, and the disturbances may also modify the AhR pathway itself. However, MAPK involvement in B[a]P metabolic activation and toxicity in lung tissues is not well understood. Here, we used a non-transformed human bronchial epithelial lung cell line, BEAS-2B, to study the participation of ERK 1/2 kinases in the metabolic activation of B[a]P and in its related genotoxic effects. Our results indicate that B[a]P is not cytotoxic to BEAS–2B cells at relatively low concentrations, but it enhances CYP1A1 gene transcription and protein induction. Additionally, B[a]P promotes Src and ERK 1/2 phosphorylation. Accordingly, inhibition of both Src and ERK 1/2 phosphorylation decreases CYP1A1 protein induction, AhR nuclear translocation and production of B[a]P adducts. Together, these data suggest a crosstalk between AhR and the members of the MAPK pathway, ERK 1/2 mediated by Src kinase. This interaction is important for the adequate AhR pathway signaling that in turn induces transcription and protein induction of CYP1A1 and B[a]P-induced DNA damage in BEAS–2B cells.

Mammalian Cells Undergo Endoreduplication in Response to Lactic Acidosis

Polyploidization, a common event during the evolution of different tumours, has been proposed to confer selective advantages to tumour cells by increasing the occurrence of mutations promoting cancer progression and by conferring chemotherapy resistance. While conditions leading to polyploidy in cancer cells have been described, a general mechanism explaining the incidence of this karyotypic change in tumours is still missing. In this study, we tested whether a widespread tumour microenvironmental condition, low pH, could induce polyploidization in mammalian cells. We found that an acidic microenvironment, in the range of what is commonly observed in tumours, together with the addition of lactic acid, induced polyploidization in transformed and non-transformed human cell lines in vitro. In addition, we provide evidence that polyploidization was mainly driven through the process of endoreduplication, i.e. the complete skipping of mitosis in-between two S-phases. These findings suggest that acidic environments, which characterize solid tumours, are a plausible path leading to polyploidization of cancer cells.

Fibroblast growth factor receptor signaling plays a key role in transformation induced by the TMPRSS2/ERG fusion gene and decreased PTEN.

Prostate cancer is the most common visceral malignancy and the second leading cause of cancer deaths in US men. Correlative studies in human prostate cancers reveal a frequent association of the TMPRSS2/ERG (TE) fusion gene with loss of PTEN and studies in mouse models reveal that ERG expression and PTEN loss synergistically promote prostate cancer progression. To determine the mechanism by which ERG overexpression and PTEN loss leads to transformation, we overexpressed the TE fusion gene and knocked down PTEN in an immortalized but non-transformed prostate epithelial cell line. We show that ERG overexpression in combination with PTEN loss can transform these immortalized but non-tumorigenic cells, while either alteration alone was not sufficient to fully transform these cells. Expression microarray analysis revealed extensive changes in gene expression in cells expressing the TE fusion with loss of PTEN. Among these gene expression changes was increased expression of multiple FGF ligands and receptors. We show that activation of fibroblast growth factor receptor signaling plays a key role in transformation induced by TE fusion gene expression in association with PTEN loss. In addition, in vitro and in silico analysis reveals PTEN loss is associated with widespread increases in FGF ligands and receptors in prostate cancer. Inhibitors of FGF receptor signaling are currently entering the clinic and our results suggests that FGF receptor signaling is a therapeutic target in cancers with TE fusion gene expression and PTEN loss.

N4 -aryl substituted thiosemicarbazones derived from 1-indanones as potential anti-tumor agents for breast cancer treatment.

Breast cancer is the first cause of cancer death in women. Many patients are resistant to current therapies, and even those were sensitive at first may eventually become resistant later. Thiosemicarbazones (TSCs) are synthetic compounds that exhibit several pharmacological activities. In this study, we investigated the potential anti-tumor activity of a set of N4 -arylsubstituted TSCs (N4 -TSCs) on human breast cancer cell lines. Studies on the effect of N4 -TSCs (T1, T2, and T3) were carried on MCF-7, MDA-MB 231, and BT 474 cell lines which differ in their expression of ER, PR, and Her2/neu. Non-transformed MCF-10A breast cell line were used as normal cells. Action of N4 -TSCs were evaluated by proliferation assay, quantification of apoptosis and cell cycle analysis. Modulation of clonogenic efficiency and migratory capacity by N4 -TSCs were also evaluated. We further investigated the effects of N4 -TSCs on ROS level and Ribonucleotide Reductase (RR) activity. We analyzed the action of these compounds on cellular mammosphere-forming capacity. We found that T1 and T2 had specific anti-tumor effect on all breast cancer cell lines based on their pro-apoptotic action and inhibitory effect on clonogenic efficiency and cell migration capacity. We also showed that both compounds increased ROS level and inhibited RR activity. Finally, we found that all N4 -TSCs diminished mammospehere-forming capacity of MCF-7 and BT 474 cells. N4 -TSCs showed specific anti-tumor action on human breast cancer cells independently their biomarkers expression pattern. Our results place these compounds as promising novel anti-tumor drugs with potential therapeutic application against different types of breast cancer.

Ciliary Assembly/Disassembly Assay in Non-transformed Cell Lines.

The primary cilium is a non-motile sensory organelle whose assembly and disassembly are closely associated with cell cycle progression. The primary cilium is elongated from the basal body in quiescent cells and is resorbed as the cells re-enter the cell cycle. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human diseases. The in vitro serum-stimulated ciliary assembly/disassembly assay has gained popularity in addressing the functions of the protein-of-interest in ciliary dynamics. Here, we describe a well-tested protocol for transfecting human retinal pigment epithelial cells (RPE-1) and performing ciliary assembly/disassembly assays on the transfected cells.

Epithelial Smad4 Deletion Up-Regulates Inflammation and Promotes Inflammation-Associated Cancer.

Background & AimsChronic inflammation is a predisposing condition for colorectal cancer. Many studies to date have focused on proinflammatory signaling pathways in the colon. Understanding the mechanisms that suppress inflammation, particularly in epithelial cells, is critical for developing therapeutic interventions. Here, we explored the roles of transforming growth factor β (TGFβ) family signaling through SMAD4 in colonic epithelial cells.MethodsThe Smad4 gene was deleted specifically in adult murine intestinal epithelium. Colitis was induced by 3 rounds of dextran sodium sulfate in drinking water, after which mice were observed for up to 3 months. Nontransformed mouse colonocyte cell lines and colonoid cultures and human colorectal cancer cell lines were analyzed for responses to TGFβ1 and bone morphogenetic protein 2.ResultsDextran sodium sulfate treatment was sufficient to drive carcinogenesis in mice lacking colonic Smad4 expression, with resulting tumors bearing striking resemblance to human colitis–associated carcinoma. Loss of SMAD4 protein was observed in 48% of human colitis–associated carcinoma samples as compared with 19% of sporadic colorectal carcinomas. Loss of Smad4 increased the expression of inflammatory mediators within nontransformed mouse colon epithelial cells in vivo. In vitro analysis of mouse and human colonic epithelial cell lines and organoids indicated that much of this regulation was cell autonomous. Furthermore, TGFβ signaling inhibited the epithelial inflammatory response to proinflammatory cytokines.ConclusionsTGFβ suppresses the expression of proinflammatory genes in the colon epithelium, and loss of its downstream mediator, SMAD4, is sufficient to initiate inflammation-driven colon cancer. Transcript profiling: GSE100082.

Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers.

Histone deacetylase inhibitors (HDACIs) are known to alter gene expression by both up- and down-regulation of protein-coding genes in normal and cancer cells. However, the exact regulatory mechanisms of action remain uncharacterized. Here we investigated genome wide dose-dependent epigenetic and transcriptome changes in response to HDACI largazole in a transformed and a non-transformed cell line. Exposure to low nanomolar largazole concentrations (<GI50) predominantly resulted in upregulation of gene transcripts whereas higher largazole doses (≥GI50) triggered a general decrease in mRNA accumulation. Largazole induces elevation of histone H3 acetylation at Lys-9 and Lys-27 along many gene bodies but does not correlate with up- or down-regulation of the associated transcripts. A higher dose of largazole results in more RNA polymerase II pausing at the promoters of actively transcribed genes and cell death. The most prevalent changes associated with transcriptional regulation occur at distal enhancer elements. Largazole promotes H3K27 acetylation at a subset of poised enhancers and unexpectedly, we also found active enhancers that become decommissioned in a dose and cell type-dependent manner. In particular, largazole decreases RNA polymerase II accumulation at super-enhancers (SEs) and preferentially suppresses SE-driven transcripts that are associated with oncogenic activities in transformed cells.

Hypoxia modulates the stem cell population and induces EMT in the MCF-10A breast epithelial cell line.

A common feature among pre-malignant lesions is the induction of hypoxia through increased cell propagation and reduced access to blood flow. Hypoxia in breast cancer has been associated with poor patient prognosis, resistance to chemotherapy and increased metastasis. Although hypoxia has been correlated with factors associated with the latter stages of cancer progression, it is not well documented how hypoxia influences cells in the earliest stages of transformation. Using the immortalized MCF-10A breast epithelial cell line, we used hypoxic culture conditions to mimic reduced O2 levels found within early pre-malignant lesions and assessed various cellular parameters. In this non-transformed mammary cell line, O2 deprivation led to some changes not immediately associated with cancer progression, such as decreased proliferation, cell cycle arrest and increased apoptosis. In contrast, hypoxia did induce other changes more consistent with an increased metastatic potential. A rise in the CD44+CD24−/low-labeled cell sub-population along with increased colony forming capability indicated an expanded stem cell population. Hypoxia also induced cellular and molecular changes consistent with an epithelial-to-mesenchymal transition (EMT). Furthermore, these cells now exhibited increased migratory and invasive abilities. These results underscore the contribution of the hypoxic tumour microenvironment in cancer progression and dissemination.

BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination

Mutations in the tumor suppressor BRCA2 predominantly predispose to breast cancer. Paradoxically, while loss of BRCA2 promotes tumor formation, it also causes cell lethality, although how lethality is triggered is unclear. Here, we generate BRCA2 conditional non-transformed human mammary epithelial cell lines using CRISPR-Cas9. Cells are inviable upon BRCA2 loss, which leads to replication stress associated with under replication, causing mitotic abnormalities, 53BP1 nuclear body formation in the ensuing G1 phase, and G1 arrest. Unexpected from other systems, the role of BRCA2 in homologous recombination, but not in stalled replication fork protection, is primarily associated with supporting human mammary epithelial cell viability, and, moreover, preventing replication stress, a hallmark of pre-cancerous lesions. Thus, we uncover a DNA under replication-53BP1 nuclear body formation-G1 arrest axis as an unanticipated outcome of homologous recombination deficiency, which triggers cell lethality and, we propose, serves as a barrier that must be overcome for tumor formation.

Genotoxic effects in transformed and non-transformed human breast cell lines after exposure to silver nanoparticles in combination with aluminium chloride, butylparaben or di-n-butylphthalate

In the present study genotoxic effects after combined exposure of human breast cell lines (MCF-10A, MCF-7 and MDB-MB-231) to silver nanoparticles (AgNP, citrate stabilized, 15 and 45nm by STEM, Ag15 and Ag45, respectively) with aluminium chloride, butylparaben, or di-n-butylphthalate were studied. In MCF-10A cells exposed for 24h to Ag15 at the concentration of 23.5μg/mL a statistically significant increase in DNA damage in comet assay (SSB) was observed. In the presence of the test chemicals the genotoxic effect was decreased to a level comparable to control values. In MCF-7 cells a significant increase in SSB level was observed after exposure to Ag15 at 16.3μg/mL. The effect was also diminished in the presence of the three test chemicals. In MDA-MB-231 cells no significant increase in SSB was observed, however increased level of oxidative DNA damage (incubation with Fpg enzyme) was observed after exposure to combinations of both AgNP with aluminium chloride. No increase in micronuclei formation was observed in neither cell line after the single nor combined treatments. Our results point to a low risk of increased genotoxic effects of AgNP when used in combination with aluminium salts, butylparaben or di-n-butylphthalate in consumer products.

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OBJECTIVES/SPECIFIC AIMS: Intestinal stem cells (ISC) primarily act in the repair of ulcerated epithelium, and their proliferative capacity relies on Wnt/β-catenin signaling. However, the role of GCs on basal epithelial cell signaling has not been fully characterized. The objective of this study was to interrogate a mechanism by which steroids may limit ISC activation. GCs inhibit NFκB signaling, which has been shown to play a role in nuclear β-catenin activation in epithelial cells. We hypothesized that GCs limit Wnt/β-catenin signaling required for ISC activation and epithelial restitution by inhibiting NFκB activation in epithelial cells. METHODS/STUDY POPULATION: To examine the effects of GCs on intestinal epithelial cells, we treated a nontransformed human colonic epithelial cell line (NCM460) with dexamethasone and observed the effects on NFκB and Wnt/β-catenin signaling events. We isolated mouse epithelial cells from the distal colon for stem cell culture as 3D “organoids.” We obtained pure epithelial cell preparations from mucosal biopsies isolated from patients treated at GI clinics at the University of Kentucky Chandler Hospital and VA Medical Center, Lexington. Steroid treated patients with equivalent levels of inflammation, but no mucosal ulceration were used as controls. RESULTS/ANTICIPATED RESULTS: In steroid-treated NCM460 cells, we saw an increase in steroid-responsive genes GILZ and SGK1. We saw a significant decrease in transcripts for Wnt target genes, including Axin2 and cmyc; NFκB target genes, including IFNG and IL6; and the shared NFκB and Wnt pathway co-activator CREBBP, despite unchanged transcript levels for β-catenin (CTNNB1). This data was corroborated in 3D stem cell cultures from cells isolated from mouse colon tissue, which had significant decreases in transcripts for stem cell markers Lgr5 and Ascl2, proliferative markers KI67 and PCNA, and Wnt target Axin2. NCM460s transfected with a lentivirus carrying a TCF/LEF luciferase construct showed a 2.5-fold decrease in TNF-stimulated luciferase activity with dexamethasone treatment. Interestingly, this effect can be rescued by glucocorticoid receptor (GR) blockade with RU-486. Intestinal epithelial cells from patient biopsies showed significant decreases in colitis-induced Axin2, p-LRP6 (a positive marker of Wnt Signaling) and nuclear β-catenin, which correlated with decreased p-p65 protein levels. DISCUSSION/SIGNIFICANCE OF IMPACT: Together, these data suggest that steroid therapy inhibits Wnt/β-catenin signaling at multiple levels, and effects stem cell proliferation in pure stem cell cultures. Decreases in TCF/LEF transcriptional activation (nuclear β-catenin’s DNA binding target) can be reversed with steroid receptor blockade with RU-486, suggesting that a receptor level interaction may be occurring. Interestingly, the required co-activator CBP, shared between NFκB and Wnt pathways, has decreased transcription following steroid treatment, which may provide a mechanism for limited Wnt activation following steroid therapy. Although steroids play a significant role in regulating the amount of inflammatory damage that occurs during IBD treatment, our data suggest that they may be limiting pathways required for effective healing as well.

Potential mechanisms of calcium dependent regulation of the mammalian cell cycle revealed by comprehensive unbiased label-free nLC-MS/MS quantitative proteomics

Calcium (Ca2+) controls progression through the mammalian cell cycle by engaging a diverse range of molecular pathways. While the essential role of spatio-temporal Ca2+ signalling in the cell cycle is well established, the precise mechanisms by which it regulates cell cycle entry and progression through G1 are not particularly well understood.Here, high-resolution label-free semi-quantitative nLC-MS/MS analysis has been used to support a highly reproducible unbiased analysis of Ca2+ influx dependent growth factor induced protein expression early in G1 in human fibroblasts. Using this strategy a panel of 182 proteins whose expression was Ca2+ dependent were identified. Pathway analysis has indicated that Ca2+ likely regulates cell proliferation via PI3K/AKT pathway and its downstream target mTOR. In addition to cell proliferation found proteins were involved in the regulation of cell morphology and cellular assembly and organization, the environmental clues, which are known to regulate G1 progression.Reported here data represents one of the most comprehensive proteomic datasets of growth factor and Ca2+ dependent protein expression in the mammalian cell cycle and provides a rich source of publically available data to support continued investigation of the role of Ca2+ in G1 progression at both the molecular and systems level. Biological significanceThe results of this study provide new insight into Ca2+ dependent regulation of cell cycle. This manuscript reports first to date global analysis of Ca2+ regulated protein expression changes early in G1 in non-transformed human fibroblast cell line. It also highlights canonical signalling pathways and biological processes that are regulated by the inhibition of Ca2+ influx. Importantly, it appears that Ca2+ may be the factor that links cell division with environmental cues, cell morphology and cellular assembly and organization, on which cell proliferation depends. Hence, the findings presented here provide numerous opportunities for more detailed investigations of the mechanism of Ca2+ dependent regulation of cell cycle at the molecular and systemic level.

Abstract 1568: Predicting stochastic proliferation and death in response to drugs with mechanistic models tailored to genomic, transcriptomic, and proteomic data

Abstract Over the past decade we have seen a shift in cancer therapy from broadly cytotoxic drugs to molecular therapies targeting “driver” mutations. Although targeted therapy has seen great success for some cancers (e.g. imatinib for leukemia), it has struggled with poor efficacy in treating other cancers that can sometimes possess multiple “driver” mutations. This highlights the complex, and at times non-intuitive, interplay between multiple players in a signaling cascade, which can be highly dependent on the biological context – that is, gene expression levels and mutational architecture – of a tumor or cell line. A quantitative, mechanistic, biologically-tailored understanding of how these signaling dynamics drive proliferation and death could improve precision pharmacology approaches to treat cancer. Here, we constructed the first highly detailed, large-scale ordinary differential equation (ODE) mechanistic mathematical model depicting the most commonly mutated cancer signaling pathways across human cancers, as indicated by a pan-cancer analysis by The Cancer Genome Atlas (TCGA). The model includes the RTK/Ras/MAPK, PI3K/AKT/mTOR, CDK/RB cell cycle, p53/MDM2 DNA damage response, and BCL/Caspases apoptosis pathways. The adjustable parameters of the model can be informed by measurements from patients or cell lines, including copy number alterations, mutations, and gene expression levels. This single-cell model links stochastic gene expression processes to quantitative signaling dynamics, and once tailored to a biological context can be used to simulate the effect of various anti-cancer therapies on cell fate behavior such as proliferation and death for a population of cells. The first instance of the model integrated genomic, transcriptomic, and proteomic data from the MCF10A cell line, a non-transformed cell line with predictable phenotypic behaviors. We trained the model using western blot and flow cytometry experiments to refine various biochemical parameters and phenotypic outcomes. Many fundamental questions in signal transduction arose during this process, such as how EGF and insulin synergize to drive S-phase entry or how a specific biological context confers sensitivity or resistance to inhibitors of the ERK and AKT pathways. Simultaneously, we are tailoring the model to patient-derived genetic information from primary glioblastoma tumors and screening brain-penetrable compounds in a patient-specific manner. In conclusion, a quantitative, mechanistic, biologically-tailored mathematical model depicting the major cancer pathways allows us to probe the mechanisms that underlie how signaling dynamics drive proliferation and death in response to various perturbations, and gain insight into their dependence on the biological context of cell lines and patient tumors. Note: This abstract was not presented at the meeting. Citation Format: Mehdi Bouhaddou, Anne Marie Barrette, Rick J. Koch, Marc R. Birtwistle. Predicting stochastic proliferation and death in response to drugs with mechanistic models tailored to genomic, transcriptomic, and proteomic data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1568. doi:10.1158/1538-7445.AM2017-1568

Abstract 2326: A high-throughput screen identified cancer selective small molecules that kill independent of mitochondrial apoptosis

Abstract Triple negative breast cancer (TNBC) lacks expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2). Importantly, there have been fewer advances in the treatment of TNBC with the mainstay of treatment being cytotoxic chemotherapy. All too often though tumor responds poorly to chemotherapy, or relapse and resistance follow an initial response. We previously found in a range of hematological and solid tumors that chemoresistant cancers are far from the mitochondrial apoptotic threshold. To identify novel therapeutics for the treatment of chemoresistant breast cancer, we performed a high-throughput screen to identify small molecules that are cancer selective but can kill independent of the mitochondrial pathway of apoptosis. To perform this screen we used TNBC line MDA-MB-231 in which the pro-death proteins BAX/BAK were knocked down. BAX/BAK are essential for mitochondrial apoptosis. To enrich for compounds that were not non-specifically toxic, we simultaneously counter-screened against the non-transformed breast epithelial cell line MCF10a. We screened a total of 30,000 compounds in duplicate across the two cell lines. There was a hit rate of 0.3% in the screen and 85 compounds were retested in the validation cherry pick screen. From the cherry pick screen 18 compounds were further validated with low-throughput assessment for mitochondrial independent killing and selectivity for cancer cells. To identify the mechanism of action of the lead compound we used a genetic approach to generate an RNAi signature for the compound. This signature enables classification of the compound into known or novel class of compounds. The lead compound signature was most like that of histone deacetylase inhibitors (HDAC). Using an in vitro HDAC inhibitor screen, we identified that the compound selectively inhibited HDAC6. Importantly, we validated that the lead compound caused an increase in acetylated tubulin in cells, a target of HDAC6. In conclusion, we have identified a novel HDAC6 specific inhibitor that selectively kills cancer cells independent of mitochondrial apoptosis. Citation Format: Catriona M. Dowling, Micheal Hemann, James Bradner, Anthony Letai, Triona Ni Chonghaile. A high-throughput screen identified cancer selective small molecules that kill independent of mitochondrial apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2326. doi:10.1158/1538-7445.AM2017-2326