Cell Transformation Model Research Articles

P53 exerts anticancer effects by regulating enhancer formation and activity.

The abnormality of the p53 tumor suppressor is crucial in lung cancer development, because p53 regulates target gene promoters to combat cancer. Recent studies have shown extensive p53 binding to enhancer elements. However, whether p53 exerts a tumor suppressor role by shaping the enhancer landscape remains poorly understood. In the current study, we employed several functional genomics approaches to assess the enhancer activity at p53 binding sites throughout the genome based on our established TP53 knockout (KO) human bronchial epithelial cells (BEAS-2B). A total of 943 active regular enhancers and 370 super-enhancers (SEs) disappeared upon the deletion of p53, indicating that p53 modulates the activity of hundreds of enhancer elements. We found that one p53-dependent SE, located on chromosome 9 and designated as KLF4-SE, regulated the expression of the Krüppel-like factor 4 ( KLF4) gene. Furthermore, the deletion of p53 significantly decreased the KLF4-SE enhancer activity and the KLF4 expression, but increased colony formation ability in the nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced cell transformation model. Subsequently, in TP53 KO cells, the overexpression of KLF4 partially reversed the increased clonogenic capacity caused by p53 deficiency. Consistently, KLF4 expression also decreased in lung cancer tissues and cell lines. It appeared that overexpression of KLF4 significantly suppressed the proliferation and migration of lung cancer cells. Collectively, our results suggest that the regulation of enhancer formation and activity by p53 is an integral component of the p53 tumor suppressor function. Therefore, our findings offer some novel insights into the regulation mechanism of p53 in lung oncogenesis and introduce a new strategy for screening therapeutic targets.

Receptor for hyaluronan-mediated motility (RHAMM) defines an invasive niche associated with tumor progression and predicts poor outcomes in breast cancer patients.

Breast cancer invasion and metastasis result from a complex interplay between tumor cells and the tumor microenvironment (TME). Key oncogenic changes in the TME include aberrant synthesis, processing, and signaling of hyaluronan (HA). Hyaluronan-mediated motility receptor (RHAMM, CD168; HMMR) is an HA receptor enabling tumor cells to sense and respond to this aberrant TME during breast cancer progression. Previous studies have associated RHAMM expression with breast tumor progression; however, cause and effect mechanisms are incompletely established. Focused gene expression analysis of an internal breast cancer patient cohort confirmed that increased RHAMM expression correlates with aggressive clinicopathological features. To probe mechanisms, we developed a novel 27-gene RHAMM-related signature (RRS) by intersecting differentially expressed genes in lymph node (LN)-positive patient cases with the transcriptome of a RHAMM-dependent model of cell transformation, which we validated in an independent cohort. We demonstrate that the RRS predicts for poor survival and is enriched for cell cycle and TME-interaction pathways. Further analyses using CRISPR/Cas9-generated RHAMM-/- breast cancer cells provided direct evidence that RHAMM promotes invasion in vitro and in vivo. Immunohistochemistry studies highlighted heterogeneous RHAMM protein expression, and spatial transcriptomics associated the RRS with RHAMM-high microanatomic foci. We conclude that RHAMM upregulation leads to the formation of 'invasive niches', which are enriched in RRS-related pathways that drive invasion and could be targeted to limit invasive progression and improve patient outcomes. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

UCP2 Upregulation Increases Fra-1 Expression and S-phase Cell Population without Decreasing Apoptosis during Skin Cell Transformation

Upregulation of uncoupling protein 2 (UCP2) is considered a prosurvival mechanism for cancer cells. This prosurvival function is thought to be mediated by UCP2’s uncoupling activity which reduces the production of superoxide in the mitochondria. However, exactly how highly expressed UCP2 regulates cell proliferation, cell cycle, and cell death during the early stage of tumorigenesis has not been studied thoroughly. For this purpose, we generated UCP2 stably overexpressed JB6 Cl-41 cells (a skin cell transformation model) and performed studies to answer the above questions. Our results demonstrated that UCP2 overexpression enhanced cell proliferation, activation of the oncoprotein Fra-1, anchorage-independent growth, 3D spheroids growth, and glucose uptake during skin cell transformation. Next, our results demonstrated that UCP2 overexpression resulted in marked decreases in the proportion of the cells in the G1 phase and an increase of cells in the S phase of the cell cycle, which was accompanied by increased expression of Cyclin E and Cdk2. Lastly, UCP2 overexpression did not enhance or suppress apoptosis during skin cell transformation, as indicated by Annexin V and active caspase 3/7 staining. Taken together, these data suggest that UCP2 upregulation mainly enhances the Fra-1 oncogenic pathway which drives cell proliferation, without inhibiting apoptosis during skin cell transformation.

B7-H3 Chimeric Antigen Receptor Redirected T Cells Target Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma.

Simple SummaryAlthough chemotherapy is associated with high relapse rates and numerous side effects, it is still used as the front line treatment of anaplastic large cell lymphoma (ALCL). Therefore, alternative treatment options for ALCL are needed. In this study, we show that B7-H3 is a novel and promising target in ALCLs, and demonstrate that B7-H3 directed chimeric antigen receptor (CAR) T cells have therapeutic potency in controlling ALCL tumor growth.Potent CAR-T therapies that target appropriate antigens can benefit the treatment of anaplastic lymphoma kinase-positive (ALK+) anaplastic large cell lymphoma (ALCL), which is the most common subtype of T cell lymphoma. In this study, we observed overexpression of B7-H3 in ALCL cell lines derived from clinical samples and differential expression of B7-H3 in an ALK-induced T cell transformation model. A B7-H3-redirected CAR based on scFv from mAb 376.96 was developed. B7-H3 CAR-T cells showed strong cytotoxicity and cytokine secretion against target ALCL cells (SUP-M2, SU-DHL-1, and Karpas 299) in vitro. Furthermore, the B7-H3 CAR-T cells exhibited proliferative capacity and a memory phenotype upon repeated antigen stimulation. We demonstrated that B7-H3 CAR-T cells could promptly eradicate ALCL in murine xenografts. Taken together, B7-H3 is a novel and promising target in ALCLs and B7-H3 CAR-T may be a viable treatment option for ALCL.

Abstract PO-116: High resolution single cell microscopy analyses identifies the cellular redox state that supports conversion to a neoplastic stem cell state

Abstract High resolution single cell microscopy analyses identifies the cellular redox state that supports conversion to a neoplastic stem cell state Brian Spurlock and Kasturi Mitra University of Alabama at Birmingham Introduction: Single cell analyses lie at the core of teasing out tumor cell heterogeneity at the level of their self-renewal and differentiation ability, gene mutation and expression, energetics, metabolism, redox status etc. Cellular redox states are modulated by Reactive Oxygen Species (ROS) that are primarily generated by mitochondria. The neoplastic stem cells, which lie at the base of tumor cell hierarchy, are supported by optimally elevated mitochondrial ROS levels. However, induction of higher levels of mitochondrial ROS can eliminate the neoplastic stem cells. Therefore, there appears to be a sweet spot of mitochondrial ROS levels for maintenance of neoplastic stem cells. Given our longstanding interest in mitochondrial biology, we aimed at probing mitochondrial redox modulation at a single cell level towards understanding cellular susceptibility for neoplastic transformation. Experimental procedures: For this study we have used our high resolution confocal microscopy based single cell assay for mitochondrial properties, cell based Seahorse respirometry assay, tumorsphere assay, xenograft assays and shRNA mediated gene knockdown. Summary of data: We have used carcinogen induced cell transformation model to study neoplastic transformation. We found that carcinogen induced enrichment of neoplastic stem cells was associated with a certain level of mitochondrial ROS beyond which the number of neoplastic stem cell frequency decreases dramatically. The increase in the lineage specific stem cell marker, Sox2, happens only in cells with repression of a mitochondrial property called mitochondrial fission. Knockdown of the master regulator of mitochondrial fission, Drp1 (Dynamin Regulated Protein 1), alters mitochondrial ROS levels and causes increase in Sox2 levels and accelerates carcinogen induced cell transformation. Therefore, our data suggested that repression of mitochondrial fission sustains mitochondrial ROS at a certain level to allow enrichment of neoplastic stem cells. Most importantly, our single cell microscopy analyses of mitochondrial ROS and mitochondrial fission in the same cells identifies the intermediate mitochondrial ROS levels that associates with a minimum mito-fission state that supports neoplastic stem cells. Conclusion: We conclude that the cells with a certain quantifiable level of mitochondrial-ROS status are maximally susceptible to (carcinogen induced) conversion to neoplastic stem cells and can be identified by our novel high resolution microscopy based single cell analyses. Currently, we are performing single cell RNAseq experiments with the goal of integrated understanding of gene expression and mitochondrial redox regulation in neoplastic stem cells. Citation Format: Brian Spurlock, Kasturi Mitra. High resolution single cell microscopy analyses identifies the cellular redox state that supports conversion to a neoplastic stem cell state [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-116.

Mechanism of miR-222 and miR-126 regulation and its role in asbestos-induced malignancy.

MiR-222 and miR-126 are associated with asbestos exposure and the ensuing malignancy, but the mechanism(s) of their regulation remain unclear. We evaluated the mechanism by which asbestos regulates miR-222 and miR-126 expression in the context of cancer etiology. An 'in vitro' model of carcinogen-induced cell transformation was used based on exposing bronchial epithelium BEAS-2B cells to three different carcinogens including asbestos. Involvement of the EGFR pathway and the role of epigenetics have been investigated in carcinogen-transformed cells and in malignant mesothelioma, a neoplastic disease associated with asbestos exposure. Increased expression of miR-222 and miR-126 were found in asbestos-transformed cells, but not in cells exposed to arsenic and chrome. Asbestos-mediated activation of the EGFR pathway and macrophages-induced inflammation resulted in miR-222 upregulation, which was reversed by EGFR inhibition. Conversely, asbestos-induced miR-126 expression was affected neither by EGFR modulation nor inflammation. Rather than methylation of the miR-126 host gene EGFL7, epigenetic mechanism involving DNMT1- and PARP1-mediated chromatin remodeling was found to upregulate of miR-126 in asbestos-exposed cells, while miR-126 was downregulated in malignant cells. Analysis of MM tissue supported the role of PARP1 in miR-126 regulation. Therefore, activation of the EGFR pathway and the PARP1-mediated epigenetic regulation both play a role in asbestos-induced miRNA expression, associated with in asbestos-induced carcinogenesis and tumor progression.

SAMHD1 inhibits epithelial cell transformation in vitro and affects leukemia development in xenograft mice

ABSTRACTSterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a mammalian dNTP hydrolase (dNTPase) and functions as a negative regulator in the efficacy of cytarabine treatment of acute myeloid leukemia (AML). We have reported that SAMHD1 knockout (KO) increased the activity of phosphoinositide 3-kinase (PI3K) in AML-derived THP-1 cells and attenuated their ability to form subcutaneous tumors in xenografted immunodeficient mice. However, the functional significance of SAMHD1 in controlling AML leukemogenesis remains unclear. Previous studies show that in vitro transformation of Madin-Darby canine kidney (MDCK) epithelial cells by the Jaagsiekte sheep retrovirus (JSRV) envelope protein requires activation of the PI3K/Akt oncogenic signaling pathway. Using this cell transformation model, we demonstrated that ectopic expression of wild-type human SAMHD1 or a dNTPase-defective SAMHD1 mutant (HD/AA) significantly inhibited MDCK cell transformation, but did not affect cell proliferation. To visualize and quantify THP-1 cell growth and metastasis in xenografted immunodeficient mice, we generated luciferase-expressing stable SAMHD1 KO THP-1 cells and control THP-1 cells, which were injected intravenously into immunodeficient mice. Bioluminescence imaging and quantification analysis of xenografted mice revealed that SAMHD1 KO cell-derived tumors had similar growth and metastatic potential compared with control cells at 35 days post-injection. However, mice xenografted with SAMHD1 KO cells showed greater survival compared with mice injected with control cells. Our data suggest that exogenous SAMHD1 expression suppresses in vitro cell transformation independently of its dNTPase activity, and that endogenous SAMHD1 affects AML tumorigenicity and disease progression in vivo.

Abstract 3501: Adipocytes sustain pancreatic cancer progression through a non-canonical WNT paracrine network inducing ROR2 nuclear shuttling

Abstract Background: Solid epidemiological evidences connect obesity with incidence, stage, and survival in pancreatic cancer. However, the underlying mechanistic basis linking adipocytes to pancreatic cancer progression remain largely elusive. We hypothesized that factors secreted by adipocytes could be responsible for epithelial-to-mesenchymal transition (EMT) induction and, in turn, a more aggressive phenotype in models of pancreatic preneoplastic lesions. Methods: We studied the role of factors secreted by two adipogenic model systems from primary human Bone Marrow Stromal Cells (hBMSC) in an in vitro experimental cell transformation model system of human pancreatic ductal epithelial (HPDE) cell stably expressing activated KRAS (HPDE/KRAS). Results: We measured a significant induction of EMT and aggressiveness in HPDE and HPDE/KRAS cell lines when cultured with medium conditioned by fully differentiated adipocytes (ADIPOCM) if compared with the same cells cultured with medium conditioned by hBMSC (hBMSCCM) from two different healthy donors. Several genes coding for soluble modulators of the non-canonical WNT signaling pathway, including FRZB, SFRP2, RSPO1, WNT5A and 5B were significantly overexpressed in fully differentiated adipocytes than in their respective in hBMSC. ADIPOCM induced the overexpression and the nuclear translocation of the Frizzled family member Receptor tyrosine kinase-like orphan receptor (Ror) 2 in HPDE and HPDE/KRAS cells. Vantictumab, an anti-Frizzled monoclonal antibody, reduced ROR2 nuclear translocation and in turn the EMT and aggressiveness in HPDE and HPDE/KRAS cells. Conclusions: We demonstrated that adipocytes could induce EMT and aggressiveness in models of pancreatic preneoplastic lesions by orchestrating a complex paracrine signaling of soluble modulators of the non-canonical WNT signaling pathway that determine, in turn, the activation and nuclear translocation of ROR2. This signaling pathway could represent a novel target for pancreatic cancer chemoprevention. Most importantly, these factors could serve as novel biomarkers to select a risk population among obese subjects for screening and, thus, early diagnosis of pancreatic cancer. Citation Format: Carmine Carbone, Geny Piro, Raffaela Santoro, Francesca Simionato, Valeria Merz, Camilla Zecchetto, Aldo Scarpa, Giampaolo Tortora, Davide Melisi. Adipocytes sustain pancreatic cancer progression through a non-canonical WNT paracrine network inducing ROR2 nuclear shuttling [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 3501.

Lead facilitates foci formation in a Balb/c-3T3 two-step cell transformation model: role of Ape1 function

Several possible mechanisms have been examined to gain an understanding on the carcinogenic properties of lead, which include among others, mitogenesis, alteration of gene expression, oxidative damage, and inhibition of DNA repair. The aim of the present study was to explore if low concentrations of lead, relevant for human exposure, interfere with Ape1 function, a base excision repair enzyme, and its role in cell transformation in Balb/c-3T3. Lead acetate 5 and 30μM induced APE1 mRNA and upregulation of protein expression. This increase in mRNA expression is consistent throughout the chronic exposure. Additionally, we also found an impaired function of Ape1 through molecular beacon-based assay. To evaluate the impact of lead on foci formation, a Balb/c-3T3 two-step transformation model was used. Balb/c-3T3 cells were pretreated 1week with low concentrations of lead before induction of transformation with n-methyl-n-nitrosoguanidine (MNNG) (0.5μg/mL) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.1μg/mL) (a classical two-step protocol). Morphological cell transformation increased in response to lead pretreatment that was paralleled with an increase in Ape1 mRNA and protein overexpression and an impairment of Ape1 activity and correlating with foci number. In addition, we found that lead pretreatment and MNNG (transformation initiator) increased DNA damage, determined by comet assay. Our data suggest that low lead concentrations (5, 30μM) could play a facilitating role in cellular transformation, probably through the impaired function of housekeeping genes such as Ape1, leading to DNA damage accumulation and chromosomal instability, one of the most important hallmarks of cancer induced by chronic exposures.

In vitro cell transformation induced by synthetic amorphous silica nanoparticles

Synthetic amorphous silica nanoparticles (SAS) are among the most widely produced and used nanomaterials, but little is known about their carcinogenic potential. This study aims to evaluate the ability of four different SAS, two precipitated, NM-200 and NM-201, and two pyrogenic, NM-202 and NM-203, to induce the transformation process. For this, we used the recently developed in vitro Bhas 42 cell transformation assay (CTA). The genome of the transgenic Bhas 42 cells contains several copies of the v-Ha-ras gene, making them particularly sensitive to tumor-promoter agents. The Bhas 42 CTA, which includes an initiation assay and a promotion assay, was validated in our laboratory using known soluble carcinogenic substances. Its suitability for particle-type substances was verified by using quartz Min-U-Sil 5 (Min-U-Sil) and diatomaceous earth (DE) microparticles. As expected given their known transforming properties, Min-U-Sil responded positively in the Bhas 42 CTA and DE responded negatively. Transformation assays were performed with SAS at concentrations ranging from 2μg/cm2 to 80μg/cm2. Results showed that all SAS have the capacity to induce transformed foci, interestingly only in the promotion assay, suggesting a mode of action similar to tumor-promoter substances. NM-203 exhibited transforming activity at a lower concentration than the other SAS. In conclusion, this study showed for the first time the transforming potential of different SAS, which act as tumor-promoter substances in the Bhas 42 model of cell transformation.

UCP2 upregulation promotes PLCγ-1 signaling during skin cell transformation.

Uncoupling protein 2 (UCP2), whose physiological role is to decrease mitochondrial membrane potential and reactive oxygen species (ROS) production, is often overexpressed in human cancers. UCP2 upregulation has recently been proposed as a novel survival mechanism for cancer cells. However, until now, how exactly UCP2 promotes tumorigenesis remains inconclusive. Based on a widely used skin cell transformation model, our data demonstrated that UCP2 differentially regulated ROS. UCP2 upregulation decreased superoxide whereas it increased hydrogen peroxide production with concomitant increase in the expression and activity of manganese superoxide dismutase (MnSOD), the primary mitochondrial antioxidant enzyme. Furthermore, hydrogen peroxide was responsible for induction of lipid peroxidation, and PLCγ-1 activation in UCP2 overexpressed cells. Additionally, PLCγ-1 activation enhanced skin cell transformation, and pharmacological, and siRNA mediated inhibition of PLCγ-1, markedly reduced colony formation, and 3D cell growth. Moreover, hydrogen peroxide scavenger, catalase, suppressed lipid peroxidation, and dampened PLCγ-1 activity. Taken together, our data suggest that (i) UCP2 is an important regulator of mitochondrial redox status and lipid signaling; (ii) hydrogen peroxide might mediate UCP2's tumor promoting activity; and (iii) pharmacological disruption of PLCγ-1 and/or hydrogen peroxide may have clinical utility for UCP2 overexpressed cancers.

A Tox21 Approach to Altered Epigenetic Landscapes: Assessing Epigenetic Toxicity Pathways Leading to Altered Gene Expression and Oncogenic Transformation In Vitro.

An emerging vision for toxicity testing in the 21st century foresees in vitro assays assuming the leading role in testing for chemical hazards, including testing for carcinogenicity. Toxicity will be determined by monitoring key steps in functionally validated molecular pathways, using tests designed to reveal chemically-induced perturbations that lead to adverse phenotypic endpoints in cultured human cells. Risk assessments would subsequently be derived from the causal in vitro endpoints and concentration vs. effect data extrapolated to human in vivo concentrations. Much direct experimental evidence now shows that disruption of epigenetic processes by chemicals is a carcinogenic mode of action that leads to altered gene functions playing causal roles in cancer initiation and progression. In assessing chemical safety, it would therefore be advantageous to consider an emerging class of carcinogens, the epigenotoxicants, with the ability to change chromatin and/or DNA marks by direct or indirect effects on the activities of enzymes (writers, erasers/editors, remodelers and readers) that convey the epigenetic information. Evidence is reviewed supporting a strategy for in vitro hazard identification of carcinogens that induce toxicity through disturbance of functional epigenetic pathways in human somatic cells, leading to inactivated tumour suppressor genes and carcinogenesis. In the context of human cell transformation models, these in vitro pathway measurements ensure high biological relevance to the apical endpoint of cancer. Four causal mechanisms participating in pathways to persistent epigenetic gene silencing were considered: covalent histone modification, nucleosome remodeling, non-coding RNA interaction and DNA methylation. Within these four interacting mechanisms, 25 epigenetic toxicity pathway components (SET1, MLL1, KDM5, G9A, SUV39H1, SETDB1, EZH2, JMJD3, CBX7, CBX8, BMI, SUZ12, HP1, MPP8, DNMT1, DNMT3A, DNMT3B, TET1, MeCP2, SETDB2, BAZ2A, UHRF1, CTCF, HOTAIR and ANRIL) were found to have experimental evidence showing that functional perturbations played “driver” roles in human cellular transformation. Measurement of epigenotoxicants presents challenges for short-term carcinogenicity testing, especially in the high-throughput modes emphasized in the Tox21 chemicals testing approach. There is need to develop and validate in vitro tests to detect both, locus-specific, and genome-wide, epigenetic alterations with causal links to oncogenic cellular phenotypes. Some recent examples of cell-based high throughput chemical screening assays are presented that have been applied or have shown potential for application to epigenetic endpoints.

Differential KrasV12 protein levels control a switch regulating lung cancer cell morphology and motility.

Oncogenic Kras mutations are important drivers of lung cancer development and metastasis. They are known to activate numerous cellular signaling pathways implicated in enhanced proliferation, survival, tumorigenicity and motility during malignant progression. Most previous studies of Kras in cancer have focused on the comparison of cell states in the absence or presence of oncogenic Kras mutations. Here we show that differential expression of the constitutively active mutation KrasV12 has profound effects on cell morphology and motility that drive metastatic processes. The study relies on lung cancer cell transformation models, patient-derived lung cancer cell lines, and human lung tumor sections combined with molecular biology techniques, live-cell imaging and staining methods. Our analysis shows two cell functional states driven by KrasV12 protein levels: a non-motile state associated with high KrasV12 levels and tumorigenicity, and a motile state associated with low KrasV12 levels and cell dissemination. Conversion between the states is conferred by differential activation of a mechano-sensitive double-negative feedback between KrasV12/ERK/Myosin II and matrix-adhesion signaling. KrasV12 expression levels change upon cues such as hypoxia and integrin-mediated cell-matrix adhesion, rendering KrasV12 levels an integrator of micro-environmental signals that translate into cellular function. By live cell imaging of tumor models we observe shedding of mixed high and low KrasV12 expressers forming multi-functional collectives with potentially optimal metastatic properties composed of a highly mobile and a highly tumorigenic unit. Together these data highlight previously unappreciated roles for the quantitative effects of expression level variation of oncogenic signaling molecules in conferring fundamental alterations in cell function regulation required for cancer progression.

Withaferin A suppresses the up-regulation of acetyl-coA carboxylase 1 and skin tumor formation in a skin carcinogenesis mouse model.

Withaferin A (WA), a natural product derived from Withania somnifera, has been used in traditional oriental medicines to treat neurological disorders. Recent studies have demonstrated that this compound may have a potential for cancer treatment and a clinical trial has been launched to test WA in treating melanoma. Herein, WA's chemopreventive potential was tested in a chemically-induced skin carcinogenesis mouse model. Pathological examinations revealed that WA significantly suppressed skin tumor formation. Morphological observations of the skin tissues suggest that WA suppressed cell proliferation rather than inducing apoptosis during skin carcinogenesis. Antibody Micro array analysis demonstrated that WA blocked carcinogen-induced up-regulation of acetyl-CoA carboxylase 1 (ACC1), which was further confirmed in a skin cell transformation model. Overexpression of ACC1 promoted whereas knockdown of ACC1 suppressed anchorage-independent growth and oncogene activation of transformable skin cells. Further studies demonstrated that WA inhibited tumor promotor-induced ACC1 gene transcription by suppressing the activation of activator protein 1. In melanoma cells, WA was also able to suppress the expression levels of ACC1. Finally, results using human skin cancer tissues confirmed the up-regulation of ACC1 in tumors than adjacent normal tissues. In summary, our results suggest that withaferin A may have a potential in chemoprevention and ACC1 may serve as a critical target of WA. © 2015 Wiley Periodicals, Inc.

Pre-stimulation of CD81 expression by resting B cells increases proliferation following EBV infection, but the overexpression of CD81 induces the apoptosis of EBV-transformed B cells

Hepatitis C virus (HCV) E2 protein binds to CD81, which is a component of the B cell co-stimulatory complex. The E2-CD81 interaction leads to B cell proliferation, protein tyrosine phosphorylation and to the hypermutation of immunoglobulin genes. Epidemiological studies have reported a high prevalence of B cell non-Hodgkin lymphoma (NHL) in HCV-positive patients, suggesting a potential association between HCV and Epstein-Barr virus (EBV) in the genesis of B lymphocyte proliferative disorders. In the present study, in order to investigate the association between EBV and HCV in B cells, we created an in vitro EBV-induced B cell transformation model. CD81 was gradually overexpressed during transformation by EBV. B cells isolated from HCV-positive patients grew more rapidly and clumped together earlier than B cells isolated from healthy donors following EBV infection. Pre-stimulation of CD81 expressed by resting B cells with anti-CD81 monoclonal antibody (mAb) or HCV E2 accelerated the generation of lymphoblastoid cell lines (LCLs) by EBV infection. These cells proliferated prominently through the early expression of interleukin-10 and intracellular latent membrane protein (LMP)-l. By contrast, the overexpression of CD81 on EBV-transformed B cells by anti-CD81 mAb or HCV E2 protein induced apoptosis through reactive oxygen species (ROS)-mediated mitochondrial dysfunction. These results suggest that the engagement of CD81 expressed by B cells has differential effects on B cell fate (proliferation or apoptosis) according to EBV infection and the expression level of CD81.

PHLPP2 Downregulation Contributes to Lung Carcinogenesis Following B[a]P/B[a]PDE Exposure.

The carcinogenic capacity of B[a]P/B[a]PDE is supported by epidemiologic studies. However, the molecular mechanisms responsible for B[a]P/B[a]PDE-caused lung cancer have not been well investigated. We evaluated here the role of novel target PHLPP2 in lung inflammation and carcinogenesis upon B[a]P/B[a]PDE exposure. We used the Western blotting, RT-PCR, [(35)S]methionine pulse and immunohistochemistry staining to determine PHLPP2 downregulation following B[a]P/B[a]PDE exposure. Both B[a]PDE-induced Beas-2B cell transformation model and B[a]P-caused mouse lung cancer model were used to elucidate the mechanisms leading to PHLPP2 downregulation and lung carcinogenesis. The important findings were also extended to in vivo human studies. We found that B[a]P/B[a]PDE exposure downregulated PHLPP2 expression in human lung epithelial cells in vitro and in mouse lung tissues in vivo. The ectopic expression of PHLPP2 dramatically inhibited cell transformation upon B[a]PDE exposure. Mechanistic studies showed that miR-205 induction was crucial for inhibition of PHLPP2 protein translation by targeting PHLPP2-3'-UTR. Interestingly, PHLPP2 expression was inversely associated with tumor necrosis factor alpha (TNFα) expression, with low PHLPP2 and high TNFα expression in lung cancer tissues compared with the paired adjacent normal lung tissues. Additional studies revealed that PHLPP2 exhibited its antitumorigenic effect of B[a]P/B[a]PDE through the repression of inflammatory TNFα transcription. Our studies not only first time identify PHLPP2 downregulation by lung carcinogen B[a]P/B[a]PDE, but also elucidate a novel molecular mechanisms underlying lung inflammation and carcinogenesis upon B[a]P/B[a]PDE exposure.

Metabolomic changes during cellular transformation monitored by metabolite-metabolite correlation analysis and correlated with gene expression.

To investigate metabolic changes during cellular transformation, we used a 1H NMR based metabolite–metabolite correlation analysis (MMCA) method, which permits analysis of homeostatic mechanisms in cells at the steady state, in an inducible cell transformation model. Transcriptomic data were used to further explain the results. Transformed cells showed many more metabolite–metabolite correlations than control cells. Some had intuitively plausible explanations: a shift from glycolysis to amino acid oxidation after transformation was accompanied by a strongly positive correlation between glucose and glutamine and a strongly negative one between lactate and glutamate; there were also many correlations between the branched chain amino acids and the aromatic amino acids. Others remain puzzling: after transformation strong positive correlations developed between choline and a group of five amino acids, whereas the same amino acids showed negative correlations with phosphocholine, a membrane phospholipid precursor. MMCA in conjunction with transcriptome analysis has opened a new window into the metabolome.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-015-0838-z) contains supplementary material, which is available to authorized users.

Abstract 451: A novel algorithm for prioritizing candidate genes driving malignant transformation of MCF10F cells and basal-like breast cancer

Abstract We have developed a model of malignant transformation of MCF10F cells to identify the temporal acquisition of changes in genome structure and gene expression that correspond to the progressive transformed phenotype culminating in tumorigenesis. Genomic DNA and total RNA were isolated from MCF10F, trMCF (MCF10F transformed by 70 nM 17-beta estradiol), bsMCF (trMCF selected by Boyden chamber, tumorigenic), bcMCF (clones of bsMCF), and caMCF (cells from tumors of bsMCF grown in SCID mice). The molecular changes accompanying this progression were interrogated using high resolution methods of analysis for loss of heretozygosity (LOH), chromosome copy number (CCN) and gene expression profiles. The observed genomic changes, along with the tumor phenotype of a poorly differentiated adenocarcinoma, indicate an underlying mechanism common to basal-like breast cancer development in women. As examples, similarities were found at chromosomes 4 and 5 between this ER negative cell model and the basal-like tumors, and progression was accompanied by changes in gene expression consistent with epithelial mesenchymal transition. Here we describe an algorithm for prioritizing candidate oncogenes and tumor suppressor genes. Venn analysis of CCN and gene expression (Venn_input, > 1100 genes) was input into GeneIndexer, a text mining tool, and queried by key words or genes that were selected for relevance to breast cancer and then neutralized for gene-by-word correlation bias. The sum of the output cosine values for the Venn_input list was then used to prioritize candidate genes of interest in relation to breast cancer (Venn_output), and further, in relation to their first appearance during the progression of cell transformation. To determine the relevance of these genes to clinical breast cancer, the Venn_output list was used to perform hierarchical cluster analysis of several clinical studies of breast cancer. Of interest, the prioritized 113 genes identified only the basal-like subtype in four separate clinical sample sets and one set of 51 breast cancer cell lines. Further analysis using a support vector machine technique to train and query these same data predicted the tumor class (basal/non basal) with a sensitivity range of 71-87 percent, and a specificity range of 77-100 percent. These results support the use of this approach for combining complex data for the prioritization of candidate genes, and indicate that this model of malignant cell transformation recapitulates many key aspects of basal breast cancer. Citation Format: Sangjun Lee, Behrouz Madahian, Carrie Sutter, Charles Dickens, Irma Russo, Jose Russo, Ramin Homayouni, Thomas Sutter. A novel algorithm for prioritizing candidate genes driving malignant transformation of MCF10F cells and basal-like breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 451. doi:10.1158/1538-7445.AM2014-451

Abstract 3147: Low oxygen-driven expression of KrasV12 controls a switch between lung tumor growth and migration

Abstract Cancer cell migration in vivo occurs at the single cell level or as a chain of motile cells leaving the primary tumor site. Mutation of Kras to a constituitively active form of Kras, is a common alteration in lung cancer. Kras controls many cellular processes such as cell migration and proliferation. Being constantly active results in a hyper activation of downstream signaling cascades, which lead to enhanced tumorigenicity and high metastatic potential. How constitutive Kras activation translates into these cell functions, however, has remained unclear. Using a lung cancer cell transformation model, which expresses oncogenic KrasV12, we discovered that Kras expression got substantially upregulated under long-term culture in 2% O2. This was accompanied by a significant increase in growth of tumor-like aggregates compared to the same cells cultured under atmospheric 21% O2 conditions. Up-regulation of KrasV12 also correlated with a morphological change from adherent single-cells into sphere-like aggregates. This morphology was reversible upon knock-down of Kras. Separation of single clones confirmed the relation between adherent and sphere-like morphologies and the level of KrasV12 expression. The expression of Kras in these two morphological different types of clones correlated with migration behavior. Low Kras expressers were mobile whereas high Kras expressers remained stationary in aggregates. In agreement, protein analysis of these cells forced to form spheres by culture in low adhesion dishes dramatically increased their Kras content. Most interestingly, this increase was reversible by stimulation of adhesion and migration on collagen coated substrates. Under these conditions, cells at the periphery of the aggregate migrate onto the surrounding extracellular matrix (ECM). Curiously, they often stayed attached to a second cell that did not actively migrate. Using immunofluorescence imaging we could identify that in such pairs the actively migrating cell had a low expression of Kras whereas the passive ‘cargo cell’ retained high Kras levels. As both cells originated from the same aggregate of high Kras expressers we conclude that interaction of these lung cancer cells with the ECM negatively regulates Kras expression. This new form of combined migration was also observed in a 3D collagen matrix. By co-culturing high- and low-Kras clones we showed that low Kras expressers actively haul non-motile, high Kras expressers through a collagen scaffold. Based on these findings we propose a novel mechanism for controlling cancer cell migration by a low O2-driven, intrinsic Kras switch. Cells expressing high levels of Kras show a more sphere-like, non-motile phenotype and can give rise to motile cells upon down-regulation of Kras in contact with ECM. These cells carry high Kras expressers with high tumorigenic potential as cargo generating a dangerous team able to migrate out, spread, and regrow elsewhere. Citation Format: Anette C. Schafer, Jerry W. Shay, Gaudenz Danuser. Low oxygen-driven expression of KrasV12 controls a switch between lung tumor growth and migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2014-3147

Oncogenic transformation of mesenchymal stem cells decreases Nrf2 expression favoring in vivo tumor growth and poorer survival

BackgroundThe transcription factor Nrf2 is a key regulator of the cellular antioxidant response, and its activation by chemoprotective agents has been proposed as a potential strategy to prevent cancer. However, activating mutations in the Nrf2 pathway have been found to promote tumorigenesis in certain models. Therefore, the role of Nrf2 in cancer remains contentious.MethodsWe employed a well-characterized model of stepwise human mesenchymal stem cell (MSC) transformation and breast cancer cell lines to investigate oxidative stress and the role of Nrf2 during tumorigenesis. The Nrf2 pathway was studied by microarray analyses, qRT-PCR, and western-blotting. To assess the contribution of Nrf2 to transformation, we established tumor xenografts with transformed MSC expressing Nrf2 (n = 6 mice per group). Expression and survival data for Nrf2 in different cancers were obtained from GEO and TCGA databases. All statistical tests were two-sided.ResultsWe found an accumulation of reactive oxygen species during MSC transformation that correlated with the transcriptional down-regulation of antioxidants and Nrf2-downstream genes. Nrf2 was repressed in transformed MSC and in breast cancer cells via oncogene-induced activation of the RAS/RAF/ERK pathway. Furthermore, restoration of Nrf2 function in transformed cells decreased reactive oxygen species and impaired in vivo tumor growth (P = 0.001) by mechanisms that included sensitization to apoptosis, and a decreased hypoxic/angiogenic response through HIF-1α destabilization and VEGFA repression. Microarray analyses showed down-regulation of Nrf2 in a panel of human tumors and, strikingly, low Nrf2 expression correlated with poorer survival in patients with melanoma (P = 0.0341), kidney (P = 0.0203) and prostate (P = 0.00279) cancers.ConclusionsOur data indicate that oncogene-induced Nrf2 repression is an adaptive response for certain cancers to acquire a pro-oxidant state that favors cell survival and in vivo tumor growth.