Methylation-eQTL analysis in cancer research.

MOTIVATION: DNA methylation is a key epigenetic factor regulating gene expression. While promoter methylation has been well studied, recent publications have revealed that functionally important methylation also occurs in intergenic and distal regions, and varies across genes and tissue types. Given the growing importance of inter-platform integrative genomic analyses, there is an urgent need to develop methods to discover and characterize gene-level relationships between methylation and expression. RESULTS: We introduce a novel sequential penalized regression approach to identify methylation-expression quantitative trait loci (methyl-eQTLs), a term that we have coined to represent, for each gene and tissue type, a sparse set of CpG loci best explaining gene expression and accompanying weights indicating direction and strength of association. Using TCGA and MD Anderson colorectal cohorts to build and validate our models, we demonstrate our strategy better explains expression variability than current commonly used gene-level methylation summaries. The methyl-eQTLs identified by our approach can be used to construct gene-level methylation summaries that are maximally correlated with gene expression for use in integrative models, and produce a tissue-specific summary of which genes appear to be strongly regulated by methylation. Our results introduce an important resource to the biomedical community for integrative genomics analyses involving DNA methylation. AVAILABILITY AND IMPLEMENTATION: We produce an R Shiny app (https://rstudio-prd-c1.pmacs.upenn.edu/methyl-eQTL/) that interactively presents methyl-eQTL results for colorectal, breast and pancreatic cancer. The source R code for this work is provided in the Supplementary Material. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Clinical significance of homologous recombination deficiency score testing in endometrial Cancer.

BACKGROUND: Homologous recombination deficiency (HRD) score is related to chemotherapy response in some cancers, but its role in endometrial cancer in not known. We determined frequency and clinical significance of alterations in the HR pathway in endometrial cancer. METHODS: 253 endometrioid endometrial adenocarcinoma (EEA) samples from two independent cohorts (discovery and replication) were tested for HRD score using the Myriad HRD assay, microsatellite instability (MSI) and tumor mutation burden (TMB) using a next generation sequencing assay. HRD scores were also generated on endometrial cancer cell lines and in vivo response to olaparib was assessed. RESULTS: ROC curves were employed to determine optimal cutoffs of HRD in relation to survival impact in endometrial cancer and a cutoff of HRD ≥ 4 was suggested for DFS using the discovery cohort. Patients from two independent cohorts with HRD score ≥ 4 trended toward worse survival as compared to those with HRD score < 4. Both cohorts were further separated into four groups according to molecular subtypes (TMB positive; MSI positive; HRD positive; all others). When grouped by molecular subtype, there was a significant difference between groups using an HRD ≥4 cutoff in the initial (p = 0.0024) and replication (p = 0.042) cohorts. The Hec1a model (HRD score = 19) was highly sensitive to olaparib in in vitro and in vivo experiments. CONCLUSIONS: High HRD score was associated with worse DFS in our patient cohort. These findings suggest that HRD score may have clinical utility in patients with advanced or recurrent endometrial cancer.

Proteomic Profiling of Signaling Networks Modulated by G-CSF/Plerixafor/Busulfan-Fludarabine Conditioning in Acute Myeloid Leukemia Patients in Remission or with Active Disease prior to Allogeneic Stem Cell Transplantation.

To characterize intracellular signaling in peripheral blood (PB) cells of acute myeloid leukemia (AML) patients undergoing pretransplant conditioning with CXCR4 inhibitor plerixafor, granulocyte colony-stimulating factor (G-CSF), and busulfan plus fludarabine (Bu+Flu) chemotherapy, we profiled 153 proteins in 33 functional groups using reverse phase protein array. CXCR4 inhibition mobilized AML progenitors and clonal AML cells, and this was associated with molecular markers of cell cycle progression. G-CSF/plerixafor and G-CSF/plerixafor/Bu+Flu modulated distinct signaling networks in AML blasts of patients undergoing conditioning with active disease compared to nonleukemic PB cells of patients in remission. We identified AML-specific proteins that remained aberrantly expressed after chemotherapy, representing putative chemoresistance markers in AML.

Genetic Polymorphisms of CFH and ARMS2 Do Not Predict Response to Antioxidants and Zinc in Patients with Age-Related Macular Degeneration: Independent Statistical Evaluations of Data from the Age-Related Eye Disease Study.

PURPOSE: Considerable controversy has erupted in recent years regarding whether genotyping should be part of standard care for patients with age-related macular degeneration (AMD) who are being considered for treatment with antioxidants and zinc. We aimed to determine whether genotype predicts response to supplements in AMD. DESIGN: Three separate statistical teams reanalyzed data derived from the Age-Related Eye Disease Study (AREDS), receiving data prepared by the AREDS investigators and, separately, data from investigators reporting findings that support the use of genotyping. PARTICIPANTS: The population of interest was AREDS participants with AMD worse than category 1 and genotyping data available. Data from the 2 groups overlap imperfectly with respect to measurements made: the largest common set involved 879 participants for whom the same CFH and ARMS2 single nucleotide polymorphisms were measured by both groups. METHODS: Each team took a separate but complementary approach. One team focused on data concordance between conflicting studies. A second team focused on replicating the key claim of an interaction between genotype and treatment. The third team took a blank slate approach in attempting to find baseline predictors of treatment response. MAIN OUTCOME MEASURES: Progression to advanced AMD. RESULTS: We found errors in the data used to support the initial claim of genotype-treatment interaction. Although we found evidence that high-risk patients had more to gain from treatment, we were unable to replicate any genotype-treatment interactions after adjusting for multiple testing. We tested 1 genotype claim on an independent set of data, with negative results. Even if we assumed that interactions in fact did exist, we did not find evidence to support the claim that supplementation leads to a large increase in the risk of advanced AMD in some genotype subgroups. CONCLUSIONS: Patients who meet criteria for supplements to prevent AMD progression should be offered zinc and antioxidants without consideration of genotype.

High-throughput profiling of signaling networks identifies mechanism-based combination therapy to eliminate microenvironmental resistance in acute myeloid leukemia.

The bone marrow microenvironment is known to provide a survival advantage to residual acute myeloid leukemia cells, possibly contributing to disease recurrence. The mechanisms by which stroma in the microenvironment regulates leukemia survival remain largely unknown. Using reverse-phase protein array technology, we profiled 53 key protein molecules in 11 signaling pathways in 20 primary acute myeloid leukemia samples and two cell lines, aiming to understand stroma-mediated signaling modulation in response to the targeted agents temsirolimus (MTOR), ABT737 (BCL2/BCL-XL), and Nutlin-3a (MDM2), and to identify the effective combination therapy targeting acute myeloid leukemia in the context of the leukemia microenvironment. Stroma reprogrammed signaling networks and modified the sensitivity of acute myeloid leukemia samples to all three targeted inhibitors. Stroma activated AKT at Ser473 in the majority of samples treated with single-agent ABT737 or Nutlin-3a. This survival mechanism was partially abrogated by concomitant treatment with temsirolimus plus ABT737 or Nutlin-3a. Mapping the signaling networks revealed that combinations of two inhibitors increased the number of affected proteins in the targeted pathways and in multiple parallel signaling, translating into facilitated cell death. These results demonstrated that a mechanism-based selection of combined inhibitors can be used to guide clinical drug selection and tailor treatment regimens to eliminate microenvironment-mediated resistance in acute myeloid leukemia.

Quality of life among long-term survivors of advanced stage ovarian cancer: A cross-sectional approach.

PURPOSE: Long-term survival of women with advanced-stage ovarian cancer is relatively rare. Little is known about quality of life (QOL) and survivorship concerns of these women. Here, we describe QOL of women with advanced-stage ovarian cancer surviving for 8.5 years or longer and compare women with 0-1 recurrence to those with multiple recurrences. METHODS: Participants (n=56) recruited from 5 academic medical centers and the Ovarian Cancer Research Fund Alliance completed surveys regarding QOL (FACT-O), mood (CESD), social support (SPS), physical activity (IPAQ-SF), diet, and clinical characteristics. Median survival was 14.0 years (range 8.8-33.3). RESULTS: QOL and psychological adjustment of long-term survivors was relatively good, with mean FACT-G scores (multiple recurrences: 80.81±13.95; 0-1 recurrence: 89.05 ±10.80) above norms for healthy community samples (80.1±18.1). Survivors with multiple recurrences reported more compromised QOL in domains of physical and emotional well-being (p <.05), and endorsed a variety of physical and emotional concerns compared to survivors with 0-1 recurrence. Difficulties in sexual functioning were common in both groups. Almost half (43%) of the survivors reported low levels of physical activity. CONCLUSIONS: Overall, women with advanced-stage ovarian cancer who have survived at least 8.5 years report good QOL and psychological adjustment. QOL of survivors with multiple recurrences is somewhat impaired compared to those with 0-1 recurrence. Limitations include a possible bias towards participation by healthier survivors, thus under-representing the level of compromise in long-term survivors. Health care practitioners should be alert to psychosocial issues faced by these long-term survivors to provide interventions that enhance QOL.

Effect of Vitamin D and Calcium Supplementation on Cancer Incidence in Older Women: A Randomized Clinical Trial.

Importance: Evidence suggests that low vitamin D status may increase the risk of cancer. Objective: To determine if dietary supplementation with vitamin D3 and calcium reduces the risk of cancer among older women. Design, Setting, and Participants: A 4-year, double-blind, placebo-controlled, population-based randomized clinical trial in 31 rural counties (June 24, 2009, to August 26, 2015-the final date of follow-up). A total of 2303 healthy postmenopausal women 55 years or older were randomized, 1156 to the treatment group and 1147 to the placebo group. Duration of treatment was 4 years. Interventions: The treatment group (vitamin D3 + calcium group) received 2000 IU/d of vitamin D3 and 1500 mg/d of calcium; the placebo group received identical placebos. Main Outcomes and Measures: The primary outcome was the incidence of all-type cancer (excluding nonmelanoma skin cancers), which was evaluated using Kaplan-Meier survival analysis and proportional hazards modeling. Results: Among 2303 randomized women (mean age, 65.2 years [SD, 7.0]; mean baseline serum 25-hydroxyvitamin D level, 32.8 ng/mL [SD, 10.5]), 2064 (90%) completed the study. At year 1, serum 25-hydroxyvitamin D levels were 43.9 ng/mL in the vitamin D3 + calcium group and 31.6 ng/mL in the placebo group. A new diagnosis of cancer was confirmed in 109 participants, 45 (3.89%) in the vitamin D3 + calcium group and 64 (5.58%) in the placebo group (difference, 1.69% [95% CI, -0.06% to 3.46%]; P = .06). Kaplan-Meier incidence over 4 years was 0.042 (95% CI, 0.032 to 0.056) in the vitamin D3 + calcium group and 0.060 (95% CI, 0.048 to 0.076) in the placebo group; P = .06. In unadjusted Cox proportional hazards regression, the hazard ratio was 0.70 (95% CI, 0.47 to 1.02). Adverse events potentially related to the study included renal calculi (16 participants in the vitamin D3 + calcium group and 10 in the placebo group), and elevated serum calcium levels (6 in the vitamin D3 + calcium group and 2 in the placebo group). Conclusions and Relevance: Among healthy postmenopausal older women with a mean baseline serum 25-hydroxyvitamin D level of 32.8 ng/mL, supplementation with vitamin D3 and calcium compared with placebo did not result in a significantly lower risk of all-type cancer at 4 years. Further research is necessary to assess the possible role of vitamin D in cancer prevention. Trial Registration: clinicaltrials.gov Identifier: NCT01052051.

Integrated MicroRNA-mRNA Profiling Identifies Oncostatin M as a Marker of Mesenchymal-Like ER-Negative/HER2-Negative Breast Cancer.

MicroRNAs (miRNAs) simultaneously modulate different oncogenic networks, establishing a dynamic system of gene expression and pathway regulation. In this study, we analyzed global miRNA and messenger RNA (mRNA) expression profiles of 17 cell lines representing different molecular breast cancer subtypes. Spearman's rank correlation test was used to evaluate the correlation between miRNA and mRNA expression. Hierarchical clustering and pathway analysis were also performed. Publicly available gene expression profiles (n = 699) and tumor tissues (n = 80) were analyzed to assess the relevance of key miRNA-regulated pathways in human breast cancer. We identified 39 significantly deregulated miRNAs, and the integration between miRNA and mRNA data revealed the importance of immune-related pathways, particularly the Oncostatin M (OSM) signaling, associated with mesenchymal-like breast cancer cells. OSM levels correlated with genes involved in the inflammatory response, epithelial-to-mesenchymal transition (EMT), and epidermal growth factor (EGF) signaling in human estrogen receptor (ER)-negative/human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Our results suggest that the deregulation of specific miRNAs may cooperatively impair immune and EMT pathways. The identification of the OSM inflammatory pathway as an important mediator of EMT in triple-negative breast cancer (TNBC) may provide a novel potential opportunity to improve therapeutic strategies.

A genome-scale screen reveals context-dependent ovarian cancer sensitivity to miRNA overexpression.

Large-scale molecular annotation of epithelial ovarian cancer (EOC) indicates remarkable heterogeneity in the etiology of that disease. This diversity presents a significant obstacle against intervention target discovery. However, inactivation of miRNA biogenesis is commonly associated with advanced disease. Thus, restoration of miRNA activity may represent a common vulnerability among diverse EOC oncogenotypes. To test this, we employed genome-scale, gain-of-function, miRNA mimic toxicity screens in a large, diverse spectrum of EOC cell lines. We found that all cell lines responded to at least some miRNA mimics, but that the nature of the miRNA mimics provoking a response was highly selective within the panel. These selective toxicity profiles were leveraged to define modes of action and molecular response indicators for miRNA mimics with tumor-suppressive characteristics in vivo. A mechanistic principle emerging from this analysis was sensitivity of EOC to miRNA-mediated release of cell fate specification programs, loss of which may be a prerequisite for development of this disease.

Tackling the widespread and critical impact of batch effects in high-throughput data.

High-throughput technologies are widely used, for example to assay genetic variants, gene and protein expression, and epigenetic modifications. One often overlooked complication with such studies is batch effects, which occur because measurements are affected by laboratory conditions, reagent lots and personnel differences. This becomes a major problem when batch effects are correlated with an outcome of interest and lead to incorrect conclusions. Using both published studies and our own analyses, we argue that batch effects (as well as other technical and biological artefacts) are widespread and critical to address. We review experimental and computational approaches for doing so.

Intratumoral morphologic and molecular heterogeneity of rhabdoid renal cell carcinoma: challenges for personalized therapy.

Rhabdoid histology in clear-cell renal cell carcinoma is associated with a poor prognosis. The prognosis of patients with clear-cell renal cell carcinoma may also be influenced by molecular alterations. The aim of this study was to evaluate the association between histologic features and salient molecular changes in rhabdoid clear-cell renal cell carcinoma. We macrodissected the rhabdoid and clear-cell epithelioid components from 12 cases of rhabdoid clear-cell renal cell carcinoma. We assessed cancer-related mutations from eight cases using a clinical next-generation exome-sequencing platform. The transcriptome of rhabdoid clear-cell renal cell carcinoma (n=8) and non-rhabdoid clear-cell renal cell carcinoma (n=37) was assessed by RNA-seq and gene expression microarray. VHL (63%) showed identical mutations in all regions from the same tumor. BAP1 (38%) and PBRM1 (13%) mutations were identified in the rhabdoid but not in the epithelioid component and were mutually exclusive in 3/3 cases and 1 case, respectively. SETD2 (63%) mutations were discordant between different histologic regions in 2/5 cases, with mutations called only in the epithelioid and rhabdoid components, respectively. The transcriptome of rhabdoid clear-cell renal cell carcinoma was distinct from advanced-stage and high-grade clear-cell renal cell carcinoma. The diverse histologic components of rhabdoid clear-cell renal cell carcinoma, however, showed a similar transcriptomic program, including a similar prognostic gene expression signature. Rhabdoid clear-cell renal cell carcinoma is transcriptomically distinct and shows a high rate of SETD2 and BAP1 mutations and a low rate of PBRM1 mutations. Driver mutations in clear-cell renal cell carcinoma are often discordant across different morphologic regions, whereas the gene expression program is relatively stable. Molecular profiling of clear-cell renal cell carcinoma may improve by assessing for gene expression and sampling tumor foci from different histologic regions.

Reverse-phase protein array for prediction of patients at low risk of developing bone metastasis from breast cancer.

BACKGROUND: A biomarker that predicts bone metastasis based on a protein laboratory assay has not been demonstrated. Reverse-phase protein array (RPPA) enables quantification of total and phosphorylated proteins, providing information about their functional status. The aim of this study was to identify bone-metastasis-related markers in patients with primary breast cancer using RPPA analysis. PATIENTS AND METHODS: Tumor samples were obtained from 169 patients with primary invasive breast carcinoma who underwent surgery. The patients were categorized by whether they developed breast cancer bone metastasis (BCBM) during follow-up. Clinical characteristics and protein expression by RPPA were compared and verified by leave-one-out cross-validation. RESULTS: Lymph node status (p = .023) and expression level of 22 proteins by RPPA were significantly correlated with BCBM in logistic regression analysis. These variables were used to build a logistic regression model. After filtering the variables through a stepwise algorithm, the final model, consisting of 8 proteins and lymph node status, had sensitivity of 30.0%, specificity of 90.5%, positive predictive value of 30.0%, and negative predictive value of 90.5% in the cross-validation. Most of the identified proteins were associated with cell cycle or signal transduction (CDK2, CDKN1A, Rb1, Src, phosphorylated-ribosomal S6 kinase, HER2, BCL11A, and MYH11). CONCLUSION: Our validated model, in which the primary tumor is tested with RPPA, can predict patients who are at low risk of developing BCBM and thus who likely would not benefit from receiving a bisphosphonate in the adjuvant setting. Clinical trials excluding these patients have the potential to clarify the benefit of bisphosphonates in the adjuvant setting.

Targeting of mTORC1/2 by the mTOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment.

The interactions between the bone marrow (BM) microenvironment and acute myeloid leukemia (AML) is known to promote survival of AML cells. In this study, we used reverse phase-protein array (RPPA) technology to measure changes in multiple proteins induced by stroma in leukemic cells. We then investigated the potential of an mTOR kinase inhibitor, PP242, to disrupt leukemia/stroma interactions, and examined the effects of PP242 in vivo using a mouse model. Using RPPA, we confirmed that multiple survival signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), were up-regulated in primary AML cells cocultured with stroma. PP242 effectively induced apoptosis in primary samples cultured with or without stroma. Mechanistically, PP242 attenuated the activities of mTORC1 and mTORC2, sequentially inhibited phosphorylated AKT, S6K, and 4EBP1, and concurrently suppressed chemokine receptor CXCR4 expression in primary leukemic cells and in stromal cells cultured alone or cocultured with leukemic cells. In the in vivo leukemia mouse model, PP242 inhibited mTOR signaling in leukemic cells and demonstrated a greater antileukemia effect than rapamycin. Our findings indicate that disrupting mTOR/AKT signaling with a selective mTOR kinase inhibitor can effectively target leukemic cells within the BM microenvironment.

Loss of LPAR6 and CAB39L dysregulates the basal-to-luminal urothelial differentiation program, contributing to bladder carcinogenesis.

We describe a strategy that combines histologic and molecular mapping that permits interrogation of the chronology of changes associated with cancer development on a whole-organ scale. Using this approach, we present the sequence of alterations around RB1 in the development of bladder cancer. We show that RB1 is not involved in initial expansion of the preneoplastic clone. Instead, we found a set of contiguous genes that we term "forerunner" genes whose silencing is associated with the development of plaque-like field effects initiating carcinogenesis. Specifically, we identified five candidate forerunner genes (ITM2B, LPAR6, MLNR, CAB39L, and ARL11) mapping near RB1. Two of these genes, LPAR6 and CAB39L, are preferentially downregulated in the luminal and basal subtypes of bladder cancer, respectively. Their loss of function dysregulates urothelial differentiation, sensitizing the urothelium to N-butyl-N-(4-hydroxybutyl)nitrosamine-induced cancers, which recapitulate the luminal and basal subtypes of human bladder cancer.

Comparison of molecular subtype distribution in triple-negative inflammatory and non-inflammatory breast cancers.

INTRODUCTION: Because of its high rate of metastasis, inflammatory breast cancer (IBC) has a poor prognosis compared with non-inflammatory types of breast cancer (non-IBC). In a recent study, Lehmann and colleagues identified seven subtypes of triple-negative breast cancer (TNBC). We hypothesized that the distribution of TNBC subtypes differs between TN-IBC and TN-non-IBC. We determined the subtypes and compared clinical outcomes by subtype in TN-IBC and TN-non-IBC patients. METHODS: We determined TNBC subtypes in a TNBC cohort from the World IBC Consortium for which IBC status was known (39 cases of TN-IBC; 49 cases of TN-non-IBC). We then determined the associations between TNBC subtypes and IBC status and compared clinical outcomes between TNBC subtypes. RESULTS: We found the seven subtypes exist in both TN-IBC and TN-non-IBC. We found no association between TNBC subtype and IBC status (P = 0.47). TNBC subtype did not predict recurrence-free survival. IBC status was not a significant predictor of recurrence-free or overall survival in the TNBC cohort. CONCLUSIONS: Our data show that, like TN-non-IBC, TN-IBC is a heterogeneous disease. Although clinical characteristics differ significantly between IBC and non-IBC, no unique IBC-specific TNBC subtypes were identified by mRNA gene-expression profiles of the tumor. Studies are needed to identify the subtle molecular or microenvironmental differences that contribute to the differing clinical behaviors between TN-IBC and TN-non-IBC.

Epigenetic analysis of the Notch superfamily in high-grade serous ovarian cancer.

OBJECTIVES: Gene methylation and other epigenetic modifications of gene regulation have been implicated in the growth of ovarian cancer, but the clinical significance of such modifications in the Notch pathway in high-grade serous ovarian cancer (HGS-OvCa) is not well understood. We used The Cancer Genome Atlas (TCGA) data to study the clinical relevance of epigenetic modifications of Notch superfamily genes. METHODS: We analyzed the interaction of DNA methylation and miRNAs with gene expression data for Notch superfamily members with the Spearman rank correlation test and explored potential relationships with overall survival (OS) with the log-rank test. We downloaded clinical data, level 3 gene expression data, and level 3 DNA methylation data for 480 patients with stage II-IV HGS-OvCa from the TCGA data portal. Patients were randomly divided into training and validation cohorts for survival analyses. In each set, patients were grouped into percentiles according to methylation and microRNA (miRNA) or messenger RNA (mRNA) levels. We used several algorithms to predict miRNA-mRNA interaction. RESULTS: There were significant inverse relationships between methylation status and mRNA expression for PPARG, CCND1, and RUNX1. For each of these genes, patients with a lower methylation level and higher expression level had significantly poorer OS than did patients with a higher methylation level and lower expression level. We also found a significant inverse relationship between miRNAs and mRNA expression for CCND1, PPARG, and RUNX1. By further analyzing the effect of miRNAs on gene expression and OS, we found that patients with higher levels of CCND1, PPARG, and RUNX1 expression and lower expression levels of their respective miRNAs (502-5p, 128, and 215/625) had significantly poorer OS. CONCLUSIONS: Epigenetic alterations of multiple Notch target genes and pathway interacting genes (PPARG, CCND1, and RUNX1) may relate to activation of this pathway and poor survival of patients with HGS-OvCa.

The emerging role of HE4 in the evaluation of epithelial ovarian and endometrial carcinomas.

HE4 (human epididymis protein 4) is overexpressed in both ovarian and endometrial cancers. Levels of the shed HE4 protein are elevated in sera from ovarian and endometrial cancer patients. HE4 is less frequently elevated than cancer antigen 125 (CA 125) in benign gynecologic conditions and is found in a fraction of endometrial and ovarian cancers that lack CA 125 expression. Consequently, HE4 has emerged as an important biomarker that complements CA 125 in discriminating between benign and malignant pelvic masses, monitoring response to treatment, and detecting recurrences of both ovarian and endometrial cancer. The "risk of ovarian malignancy algorithm" (ROMA) incorporates CA 125, HE4, and menopausal status to distinguish benign from malignant adnexal masses, and has been approved by the US Food and Drug Administration to aid in referring patients who are likely to have ovarian cancer to specially trained gynecologic oncologists for surgery. HE4 also promises to augment the sensitivity of CA 125 for detecting early-stage ovarian cancer. In this review, we discuss the discovery and biologic significance of HE4 and evaluate available evidence regarding the utility of HE4 as a biomarker for ovarian and endometrial cancer.

Protein and phosphoprotein levels in glioma and adenocarcinoma cell lines grown in normoxia and hypoxia in monolayer and three-dimensional cultures.

BACKGROUND: Three dimensional (3D) growths of cancer cells in vitro are more reflective of in situ cancer cell growth than growth in monolayer (2D). The present study is designed to determine changes in protein and phosphoprotein that reflect adaptation of tumor cells to 3D as compared to 2D. Since relative hypoxia is a common feature of most solid tumors, the present study also aims to look at the impact of transition from normoxia to hypoxia in these two growth conditions. RESULTS: Using reverse-phase protein arrays, we compared levels of 121 different phosphorylated and non-phosphorylated proteins in 5 glioma and 6 adenocarcinoma lines under conditions of 3D and monolayer culture in normoxia and hypoxia. A three-way analysis of variance showed levels of 82 antibodies differed between media (2D vs. 3D) and 49 differed between treatments (hypoxia vs. normoxia). Comparing 2D to 3D growth, 7 proteins were commonly (i.e., > 50% of tumors) elevated in 3D: FAK, AKT, Src, GSK3αß, TSC2, p38, and NFκßp65. Conversely, 7 other proteins are commonly decreased: ATRIP, ATR, ß-catenin, BCL-X, cyclin B1, Egr-1, and HIF-1α. Comparing normoxia to hypoxia, only NCKIPSD was commonly elevated in hypoxia; 6 proteins were decreased: cyclin B1, 4EBP1(Ser65), c-Myc, SMAD3(Ser423), S6(Ser235), and S6(Ser240). Hypoxia affected glioma cell lines differently from adenocarcinoma cell lines: 8 proteins were increased in gliomas (BAX, caspase 7, HIF-1α, c-JUN, MEK1, PARP 1 cleaved, Src, and VEGFR2) and none in adenocarcinomas. CONCLUSIONS: We identified subsets of proteins with clearly concordant/discordant behavior between gliomas and adenocarcinomas. In general, monolayer to 3D culture differences are clearer than normoxia to hypoxia differences, with anti-apoptotic, cytoskeletal rearrangement and cell survival pathways emphasized in the former and mTOR pathway, transcription, cell-cycle arrest modulation, and increased cell motility in the latter.