Liana Toia

Early G1 cyclin-dependent kinases as prognostic markers and potential therapeutic targets in esophageal adenocarcinoma

Purpose: Chromosomal gain at 7q21 is a frequent event in esophageal adenocarcinoma (EAC). However, this event has not been mapped with fine resolution in a large EAC cohort, and its association with clinical endpoints and functional relevance are unclear. Experimental Design: We used a cohort of 116 patients to fine map the 7q21 amplification using SNP microarrays. Prognostic significance and functional role of 7q21 amplification and its gene expression were explored. Results: Amplification of the 7q21 region was observed in 35% of tumors with a focal, minimal amplicon containing six genes. 7q21 amplification was associated with poor survival and analysis of gene expression identified cyclin-dependent kinase 6 (CDK6) as the only gene in the minimal amplicon whose expression was also associated with poor survival. A low-level amplification (10%) was observed at the 12q13 region containing the CDK6 homologue cyclin-dependent kinase 4 (CDK4). Both amplification and expression of CDK4 correlated with poor survival. A combined model of both CDK6 and CDK4 expressions is a superior predictor of survival than either alone. Specific knockdown of CDK4 and/or CDK6 by siRNAs shows that they are required for proliferation of EAC cells and that their function is additive. PD-0332991 targets the kinase activity of both molecules and suppresses proliferation and anchorage independence of EAC cells through activation of the pRB pathway. Conclusions: We suggest that CDK6 is the driver of 7q21 amplification and that both CDK4 and CDK6 are prognostic markers and bona fide oncogenes in EAC. Targeting these molecules may constitute a viable new therapy for this disease. Clin Cancer Res; 17(13); 4513–22. ©2011 AACR.

CYCLIC STRAIN INDUCES PROLIFERATION OF CULTURED EMBRYONIC HEART CELLS

SummaryEmbryonic heart cells undergo cyclic strain as the developing heart circulates blood to the embryo. Cyclic strain may have an important regulatory role in formation of the adult structure. This study examines the feasibility of a computerized cell-stretching device for applying strain to embryonic cardiocytes to allow measurement of the cellular response. A primary coculture of myocytes and a secondary culture of nonmyocytes from stage-31 (7 d) embryonic chick hearts were grown on collagen-coated membranes that were subsequently strained at 2 Hz to 20% maximal radial strain. After 24 h, total cell number increased by 37±6% in myocyte cocultures and by 26±6% in nonmyocyte cultures over unstrained controls. Lactate dehydrogenase and apoptosis assays showed no significant differences in cell viabilities between strained and unstrained cells. After 2 h strain, bromodeoxyuridine incorporation was 38±1.2% versus 19±0.2% (P<0.01) in strained versus unstrained myocyte cocultures, and 35±2.1% versus 16±0.2% (P=0.01) in nonmyocyte cultures. MF20 antibody labeling and periodic acid-Schiff (PAS) staining estimated the number of myocytes in strained wells as 50–67% larger than in control wells. Tyrosine phosphorylation may play a role in the cellular response to strain, as Western blot analysis showed an increase in tyrosine phosphorylation of two proteins with approximate molecular weights of 63 and 150 kDa within 2 min of strain. The results of this study indicate that embryonic chick cardiocytes can be cultured in an active mechanical environment without significant detachment and damage and that increased proliferation may be a primary response to strain.

Bile exposure inhibits expression of squamous differentiation genes in human esophageal epithelial cells.

Objective:This study aimed to identify pathways and cellular processes that are modulated by exposure of normal esophageal cells to bile and acid. Background:Barretts esophagus most likely develops as a response of esophageal stem cells to the abnormal reflux environment. Although insights into the underlying molecular mechanisms are slowly emerging, much of the metaplastic process remains unknown. Methods:We performed a global analysis of gene expression in normal squamous esophageal cells in response to bile or acid exposure. Differentially expressed genes were classified into major biological functions using pathway analysis and interaction network software. Array data were verified by quantitative PCR and western blot both in vitro and in human esophageal biopsies. Results:Bile modulated expression of 202 genes, and acid modulated expression of 103 genes. Genes involved in squamous differentiation formed the largest functional group (n = 45) all of which were downregulated by bile exposure. This included genes such as involucrin (IVL), keratinocyte differentiation-associated protein (KRTDAP), grainyhead-like 1 (GRHL1), and desmoglein1 (DSG1) the downregulation of which was confirmed by quantitative PCR and western blot. Bile also caused expression changes in genes involved in cell adhesion, DNA repair, oxidative stress, cell cycle, Wnt signaling, and lipid metabolism. Analysis of human esophageal biopsies demonstrated greatly reduced expression of IVL, KRTDAP, DSG1, and GRHL1 in metaplastic compared to squamous epithelia. Conclusions:We report for the first time that bile inhibits the squamous differentiation program of esophageal epithelial cells. This, coordinated with induction of genes driving intestinal differentiation, may be required for the development of Barretts esophagus.

Cell-specific expression of fibronectin and EIIIA and EIIIB splice variants after oxygen injury

Cellular fibronectin (cFN) expression is characteristic of injured tissues. Unlike plasma FN, cFN mRNA often contains the EIIIA or EIIIB domains. We examined the lung cell-specific expression of total cFN mRNA and the EIIIA and EIIIB splice variants in rabbits after acute oxygen injury. By in situ hybridization, control lung had low cFN mRNA. After exposure to > 95% oxygen, mRNAs for total cFN and EIIIA were noted primarily in alveolar macrophages and large-vessel endothelial cells. By 3-5 days recovery, cFN and EIIIA mRNA abundance was increased in alveolar septal cells (i.e., alveolar epithelial, interstitial, or endothelial cells) and in some large-vessel endothelial cells but was low in bronchial epithelial cells. During recovery, EIIIB mRNA was low in alveolar septal cells but was noted mainly in chondrocytes. Immunostaining for EIIIA increased during recovery, paralleling the in situ hybridizations. Because FN may modulate alveolar type II cell phenotype, we investigated type II cell cFN mRNA expression in vivo. During recovery, neither isolated type II cells nor cells with surfactant protein C mRNA in vivo contained FN mRNA. In summary, these data suggest that cFN with the EIIIA domain has a role in alveolar cell recovery from oxygen injury and that type II cells do not express cFN during recovery.

Epidermal Growth Factor and/or Growth Hormone Induce Differential, Side-Specific Signal Transduction Protein Phosphorylation in Enterocytes

BACKGROUND Epidermal growth factor (EGF) plus growth hormone (GH) enhances luminal glutamine transport into rabbit and human intestinal cells. Our objective was to screen for activation status of signal proteins in C2(BBe)1 cells (enterocyte-like cell line) in response to side-specific EGF or GH treatment and to investigate the dependence of EGF receptor (EGFR) phosphorylation status on its tyrosine kinase. METHODS C2(BBe)1 cells on Transwells were treated for 15 minutes on either the basolateral or apical-side with EGF or GH. Lysates underwent Kinetworks phospho site-screen-2.1 analysis (duplicate experiments). In addition, lysates from cells treated as above with or without tyrphostin AG1478 (a specific EGFR tyrosine kinase inhibitor) underwent Western blot analysis for total EGFR and EGFR phosphorylated on tyrosine 1173, 1086 or 1068 (4-7 experiments). RESULTS Kinetworks phospho-screening demonstrated a broad range of interactions dependent on both side of exposure and protein studied. From this screen, it appears that ErbB2, Met, and insulin receptor (R)/insulin-like growth factor 1 R are not involved in the growth factors signals. For EGFR phosphorylation, basolateral, but not apical, EGF was a strong activator. Synergism was seen, but only with apical EGF plus basolateral GH. All EGFR phosphorylations were EGFR tyrosine kinase dependent. In contradistinction, apical EGF phosphorylated FAK and MAPKs. CONCLUSIONS Kinetworks phosphoprotein screens can suggest pathways involved in side-specific and synergistic interaction between EGF and GH. For EGFR, synergism by EGF + GH was noticed only with Ap EGF plus Bl GH and was EGFR tyrosine kinase dependent. Adaptive intestinal responses due to enterally administrated EGF might be accelerated by the availability of parenteral GH.

Macroarray analysis reveals a strain-induced oxidant response in pulmonary epithelial cells

Mechanical strain initiates a variety of responses in pulmonary epithelial cells. The signaling pathways and molecular alterations leading to these responses remain unclear. To identify novel signal transduction pathways activated by strain, macroarray analysis was performed on strained pulmonary epithelial cells. Glutathione S-transferase (GST) pi, GST mu, and heat shock protein (HSP)-27 were increased by strain. Western blotting verified that increases in cDNA of these redox-related molecules resulted in an increase in protein. Phosphorylation of HSP-27 was increased after strain, further supporting the role of HSP-27 in strain-induced signal transduction. Strain-induced oxidative stress was verified with the oxidant-sensitive dye dichlorodihydrofluorescein diacetate.

520 Unconjugated but Not Conjugated Bile Acid Reduces Squamous Differentiation in Esophageal Cells Possibly Through Intracellular Mechanisms

Background: Barretts Esophagus (BE) is a precancerous lesion in which the normal squamous epithelium of the esophagus undergoes columnar metaplasia as a result of protracted duodeno-esophageal reflux. Bile acid (BA) both conjugated and unconjugated at an acidic pH, forms the chief component of reflux and has the ability to affect cellular processes by virtue of its anionic detergent action or acting as a signaling molecule. Previously, our group had shown that the esophageal squamous epithelium loses the expression of genes involved in squamous differentiation following exposure to a cocktail containing conjugated and unconjugated BA at pH5. In this study, we aimed determining which BA component, whether conjugated or unconjugated, predominantly contributed to the down-regulation of squamous differentiation. Methods: Human telomerase-immortalized primary esophageal squamous cells (EPC1) were grown on polyester transwells and treated separately with conjugated or unconjugated BA at both pH7.4 and pH5, in 15 min pulses three times a day for six days. Trans-epithelial Electrical Resistance (TEER) was measured daily, and at the end of the treatment cells were harvested for mRNA and protein. Results: Unconjugated BA at pH5 reduced the expression of genes involved in squamous differentiation as well as the TEER in the EPC1 in transwells culture, whereas conjugated BA did not have any significant effect. Moreover, unconjugated BA at pH5 induced the expression of FGF-19, a transcription target of BA nuclear receptor FXR. Conclusions: Unconjugated BA at pH5 is unionized and can penetrate through cell membrane to enter EPC1 cells. On the other hand, EPC1 cell membrane is impermeable to conjugated BA due to the absence of apical BA transporters. Since the anionic detergent properties of BA increases with conjugation and ionization, these results implicate a possible intracellular signaling mechanism of action of BA to reduce the squamous differentiation in primary esophageal cells, as opposed to detergent action. Long term gastric acid suppression therapy might be putting patients at risk to BA induced altered cellular differentiation, as the therapy is known to increase bacterial deconjugation of BA.

Abstract 3051: CDC28 protein kinase subunit 1B (CKS1B) is a potential therapeutic target in esophageal adenocarcinomas overexpressing the CDK inhibitor p21Cip1/Waf1

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: The incidence of adenocarcinoma of the esophagus and gastroesophageal junction (EAC) has grown faster than any tumor type in the United States and westernized countries, outpacing the next closest cancer by almost 3 times. This rising incidence of EAC is associated with the increasing prevalence of obesity and gastroesophageal reflux disease (GERD). Due to the limited effect of current chemotherapy for this cancer, the search for new therapeutic targets is disparately needed. Chromosomal amplification is one mechanism by which cancer cells acquire and maintain neoplastic phenotype and genes that are amplified and overexpressed may represent potential therapeutic targets. Methods: DNA and RNA from a cohort of 116 patients were analyzed using SNP and mRNA expression microarrays respectively. Gene amplification was explored for correlation with expression and patient survival. Results: 25% of tumors harbor amplification at chromosome 1q which is significantly associated with poor survival. Although the amplicon is non-focal spanning almost the entire arm of chromosome 1q, only a subset of amplified genes (including The CDC28 protein kinase subunit 1B, CKS1B) are significantly overexpressed. CKS1B is an auxiliary protein required for ubiquitination and cytosomal degradation of the CDK inhibitors p27Kip1 and p21Cip1/Waf1 by the SCFSKP2 E3 ubiquitin ligase complex. Both p27Kip1 and p21Cip1/Waf1 inhibits CDK2/Cyclin E complex during the G1/S cell cycle transition. The endogenous protein expression of p27Kip1 is low among EAC cell lines. However the expression of p21Cip1/Waf1 is differential being very high in a subset of cells and low or undetectable in others. Knocking down CKS1B by siRNA in six EAC cell lines results in accumulation of p27 Kip1 with little or no effect on p21Cip1/Waf1 expression. CKS1B knockdown inhibits monolayer cell proliferation only in cells with high endogenous expression of p21Cip1/Waf1. Conclusion: We suggest that the combined action of accumulation of p27 Kip1 and the endogenously highly expressed p21Cip1/Waf1 triggers the response to CKS1B knockdown in EAC cell lines. We thus propose the potential use of CKS1B as a novel therapeutic target in esophageal adenocarcinomas with high p21Cip1/Waf1 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3051. doi:1538-7445.AM2012-3051

Abstract 3825: Prognosis of esophageal adenocarcinoma is dependent on early G1 cyclin-dependent kinase activity

Background: Chromosomal gain at 7q21 is a frequent event in esophageal adenocarcinoma (EAC). However, this event has not been mapped with fine resolution in a large EAC cohort and its associations with clinical endpoints and functional relevance are unclear. Methods: We used a cohort of 116 patients to fine map the 7q21 amplification using SNP microarrays. 7q21 amplification and its gene expression were explored for associations with patient survival. Effects of siRNA-mediated knockdown of CDK6, its homolog CDK4 and the CDK4/6 inhibitor PD-0332991 were studied in vitro. Results: 34.5% of tumors harbor 7q21 amplification which spans 17 genes with a focal amplicon of 6 genes. The amplification is associated with patient overall survival. Correlation of gene expression and survival identified CDK6 as the only gene in the core amplicon whose expression strongly associates with poor survival. In addition, the expression of CDK4, which is an unamplified homolog of CDK6, correlates with poor survival. The combined expression of both genes is a superior predictor of survival than either alone. siRNA-mediated knockdown of CDK4 and/or CDK6 suggests that they are required for proliferation of EAC cells and that their function is additive. PD-0332991 targets the kinase activity of both molecules and suppresses proliferation and anchorage-independence of EAC cells through activation of the pRB tumor suppressor pathway. Conclusion: We refined 7q21 amplification in EAC and demonstrate its association with poor survival. We suggest that CDK6 is the driver of the amplicon and that prognosis of EAC is dependent on its expression along with its homolog CDK4. Targeting these molecules may thus constitute a viable new therapy for EAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3825. doi:10.1158/1538-7445.AM2011-3825

Abstract 2157: Is CDK6 the target of amplification at 7q21 in esophageal adenocarcinoma

Introduction: The cyclin-dependent kinase 6 (CDK6) is a positive regulator of the cell cycle. It binds to D-type cyclins and phosphorylates the pRb tumor suppressor proteins allowing progression through the G1-phase of the cell cycle. Amplification of the 7q21 chromosomal region has been reported in esophageal adenocarcinoma (EAC) and it has been proposed that CDK6 is the key gene targeted by this event. However, overexpression of CDK6 in vitro suppresses proliferation of breast cancer cells and causes nuclear accumulation of pRb2 tumor suppressor protein. Thus, it remains unclear whether amplification and overexpression of CDK6 is truly driving the 7q21 amplicon in EAC. Methods: For DNA copy number analysis we analyzed genomic DNA from 83 EAC tumors using Affymetrix 6.0 SNP arrays. Protein expression was analyzed in tissue microarrays comprised of 116 independent EAC samples and pre-EAC lesions (15 high grade dysplasia (HGD), 18 low grade dysplasia (LGD), and 283 cases of columnar cell change (CCM)) using a monoclonal antibody against CDK6. CDK6 was overexpressed by stable transfection of OE19, OE33 and Flo1A esophageal cancer cells using CDK6 cDNA under the control of the pCMV promoter. Results: The microarray DNA copy number analysis showed amplification of the 7q21 region in at least 25% of cases. The minimal amplicon contained CDK6 along with 4 other genes. Immunohistochemistry showed both cytoplasmic and nuclear CDK6 staining in EAC (36, 31.3%), HGD (6, 40.0%), LGD (5, 27.7%), and the CCM (29, 10.2%). Overexpression of CDK6 in EAC cell lines caused either suppression of proliferation and anchorage independence (OE19 cells) or had no effect on proliferation (OE33 and Flo1A cells). The suppression of proliferation in OE19/CDK6 cells is dependent on the kinase activity of CDK6 as OE19 cells overexpressing a kinase-deficient form of CDK6 proliferate normally. As previously reported in breast cancer cells, pRb2 (p130) tumor suppressor protein is upregulated in OE19/CDK6 cells. The upregulation of pRb2, however, does not seem to account for the hypo-proliferative effect of OE19/CDK6 cells as it is also upregulated in normally proliferative cells that overexpress the kinase-negative form of CDK6. Conclusion: Although CDK6 is amplified and overexpressed in EAC and its preneoplastic lesions, it does not seem to account for the tumorigenic importance of the 7q21 amplicon. CDK6 overexpression in vitro does not show any mitogenic phenotype in EAC tumor cells. The presence of other genes within the minimal amplicon raises the possibility that one or more of them might drive such a potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2157.