Marta Mendiola

Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation

BackgroundThere is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed.ResultsTo address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) (heat shock protein 90 (HSP90) inhibitor) and CCT130234 (in-house phospholipase C (PLC)γ inhibitor). Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration/invasion compared with proliferation, suggesting their preferential utility in metastatic disease.ConclusionsWe have established and validated a suite of highly reproducible tumor microplate three-dimensional functional assays to enhance the biological relevance of early preclinical cancer studies. We believe these assays will increase the translational predictive value of in vitro drug evaluation studies and reduce the need for in vivo studies by more effective triaging of compounds.

Lysyl Oxidase-Like 2 as a New Poor Prognosis Marker of Squamous Cell Carcinomas

Lysyl oxidase-like 2 (Loxl2) interacts with and stabilizes Snai1 transcription factor, promoting epithelial-mesenchymal transition. Either Loxl2 or Snai1 knock-down blocks tumor growth and induces differentiation, but the specific role of each factor in tumor progression is still unknown. Comparison of the gene expression profiles of the squamous cell carcinoma cell line HaCa4 after knocking-down Loxl2 or Snai1 revealed that a subset of epidermal differentiation genes was specifically up-regulated in Loxl2-silenced cells. In agreement, although both Loxl2- and Snai1-knockdown cells showed reduced in vivo invasion, only Loxl2-silenced cells exhibited a skin-like epidermal differentiation program. In addition, we show that expression of Loxl2 and Snai1 correlates with malignant progression in a two-stage mouse skin carcinogenesis model. Furthermore, we found that increased expression of both LOXL2 and SNAI1 correlates with local recurrence in a cohort of 256 human laryngeal squamous cell carcinomas. We describe for the first time that high levels of LOXL2 are associated with decreased overall and disease-free survival in laryngeal squamous cell carcinomas, lung squamous cell carcinoma, and lymph node-negative (N(0)) breast adenocarcinomas. Altogether, our results show that LOXL2 can be used as a new poor prognosis indicator in human squamous cell carcinomas promoting malignant transformation by both SNAI1-dependent and SNAI1-independent pathways.

Tumour-microenvironmental interactions: paths to progression and targets for treatment.

Primary human tumours can often be eradicated by surgery if detected early; however metastatic disease renders complete cure less likely and the development of resistance to therapy results in tumour escape and increased risk of death. Interactions of tumour cells with each other, surrounding normal cells and extracellular matrix or basement membrane components are crucial to all stages of cancer progression. Changes in both cell-cell and cell-substrate proteins are linked to tumour cell migratory and invasive ability, induction of angiogenesis (on which sustained tumour growth and dissemination depends) and apoptosis resistance in response to drugs or radiotherapy. Hypoxia within solid tumours is a key driver of many aspects of progression, and may also nurture cancer stem-like cells which are increasingly linked to relapse and treatment failure. This review will briefly outline the cellular and molecular mechanisms underlying tumour progression, focussing on the acquisition of metastatic capacity and resistance to therapy.

DNA methylation signatures identify biologically distinct thyroid cancer subtypes.

OBJECTIVE The purpose of this study was to determine the global patterns of aberrant DNA methylation in thyroid cancer. RESEARCH DESIGN AND METHODS We have used DNA methylation arrays to determine, for the first time, the genome-wide promoter methylation status of papillary, follicular, medullary, and anaplastic thyroid tumors. RESULTS We identified 262 and 352 hypermethylated and 13 and 21 hypomethylated genes in differentiated papillary and follicular tumors, respectively. Interestingly, the other tumor types analyzed displayed more hypomethylated genes (280 in anaplastic and 393 in medullary tumors) than aberrantly hypermethylated genes (86 in anaplastic and 131 in medullary tumors). Among the genes indentified, we show that 4 potential tumor suppressor genes (ADAMTS8, HOXB4, ZIC1, and KISS1R) and 4 potential oncogenes (INSL4, DPPA2, TCL1B, and NOTCH4) are frequently regulated by aberrant methylation in primary thyroid tumors. In addition, we show that aberrant promoter hypomethylation-associated overexpression of MAP17 might promote tumor growth in thyroid cancer. CONCLUSIONS Thyroid cancer subtypes present differential promoter methylation signatures, and nondifferentiated subtypes are characterized by aberrant promoter hypomethylation rather than hypermethylation. Additional studies are needed to determine the potential clinical interest of the tumor subtype-specific DNA methylation signatures described herein and the role of aberrant promoter hypomethylation in nondifferentiated thyroid tumors.

Comparison of gene expression profiling by reverse transcription quantitative PCR between fresh frozen and formalin-fixed, paraffin-embedded breast cancer tissues

Recent reports demonstrate the feasibility of quantifying gene expression by using RNA isolated from blocks of formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The development of molecular tests for clinical use based on archival materials would be of great utility in the search for and validation of important genes or gene expression profiles. In this study, we compared the performance of different normalization strategies in the correlation of quantitative data between fresh frozen (FF) and FFPE samples and analyzed the parameters that characterize such correlation for each gene. Total RNA extracted from FFPE samples presented a shift in raw cycle threshold (Cq) values that can be explained by its extensive degradation. Proper normalization can compensate for the effects of RNA degradation in gene expression measurements. We show that correlation between normalized expression values is better for moderately to highly expressed genes whose expression varies significantly between samples. Nevertheless, some genes had no correlation. These genes should not be included in molecular tests for clinical use based on FFPE samples. Our results could serve as a guide when developing clinical diagnostic tests based on RT-qPCR analyses of FFPE tissues in the coming era of treatment decision-making based on gene expression profiling.

Molecular characterization of ovarian cancer by gene-expression profiling.

Ovarian cancer is the second most common gynecologic malignancy, and represents the fifth most common cause of cancer death in women in the United States. The age at diagnosis, extent of disease, success of primary surgery, and the histopathological features of the tumor are important prognostic markers. Epithelial ovarian carcinomas are classified into four major categories: serous, mucinous, endometrioid, and clear cell. Each subtypes of ovarian carcinoma are known to have different clinical characteristics and biological behaviour and response to chemotherapy. Molecular studies have supported for the notion that the different histological types of ovarian cancer likely represent histopathologically, genetically, and biologically distinct diseases. Microarray-based profiling technologies have provided an opportunity to simultaneously examine the relationship between thousands of genes and clinical phenotypes. In this review, we will summarise the current gene-expression profiles that address the classification of ovarian cancer into molecular subtypes with different outcomes.

The PTEN/NRF2 axis promotes human carcinogenesis.

AIMS A recent study conducted in mice reported that liver-specific knockout of tumor suppressor Pten augments nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcriptional activity. Here, we further investigated how phosphatase and tensin homolog deleted on chromosome 10 (PTEN) controls NRF2 and the relevance of this pathway in human carcin ogenesis. RESULTS Drug and genetic targeting to PTEN and phosphoproteomics approaches indicated that PTEN leads to glycogen synthase kinase-3 (GSK-3)-mediated phosphorylation of NRF2 at residues Ser(335) and Ser(338) and subsequent beta-transducin repeat containing protein (β-TrCP)-dependent but Kelch-like ECH-associated protein 1 (KEAP1)-independent degradation. Rescue experiments in PTEN-deficient cells and xerographs in athymic mice indicated that loss of PTEN leads to increased NRF2 signature which provides a proliferating and tumorigenic advantage. Tissue microarrays from endometrioid carcinomas showed that 80% of PTEN-negative tumors expressed high levels of NRF2 or its target heme oxygenase-1 (HO-1). INNOVATION These results uncover a new mechanism of oncogenic activation of NRF2 by loss of its negative regulation by PTEN/GSK-3/β-TrCP that may be relevant to a large number of tumors, including endometrioid carcinomas. CONCLUSION Increased activity of NRF2 due to loss of PTEN is instrumental in human carcinogenesis and represents a novel therapeutic target.

Developmental and tumoral vascularization is regulated by G protein–coupled receptor kinase 2

Tumor vessel dysfunction is a pivotal event in cancer progression. Using an in vivo neovascularization model, we identified G protein-coupled receptor kinase 2 (GRK2) as a key angiogenesis regulator. An impaired angiogenic response involving immature vessels was observed in mice hemizygous for Grk2 or in animals with endothelium-specific Grk2 silencing. ECs isolated from these animals displayed intrinsic alterations in migration, TGF-β signaling, and formation of tubular networks. Remarkably, an altered pattern of vessel growth and maturation was detected in postnatal retinas from endothelium-specific Grk2 knockout animals. Mouse embryos with systemic or endothelium-selective Grk2 ablation had marked vascular malformations involving impaired recruitment of mural cells. Moreover, decreased endothelial Grk2 dosage accelerated tumor growth in mice, along with reduced pericyte vessel coverage and enhanced macrophage infiltration, and this transformed environment promoted decreased GRK2 in ECs and human breast cancer vessels. Our study suggests that GRK2 downregulation is a relevant event in the tumoral angiogenic switch.

Aurora kinases as prognostic biomarkers in ovarian carcinoma.

We investigated the expression of Aurora kinases A and B by immunohistochemistry in 68 ovarian carcinomas to analyze their prognostic value. The amplification of AURKA gene by fluorescence in situ hybridization was also assessed. Overall, 58.8% and 85.3% of ovarian carcinomas showed expression of Aurora A and B, respectively. Amplification of AURKA was found in 27.6% of cases examined. Tumors with Aurora A expression showed a lower rate of recurrence than those tumors without Aurora A expression (65% versus 91.7%, P = .019). In the univariate analysis, patients with Aurora A and B expression showed an increased progression-free survival (P = .023 and .06, respectively, log-rank test) and overall survival (P = .03 and .02, respectively, log-rank test). The multivariate analysis adjusted to optimal surgery by Cox proportional hazards regression showed Aurora A expression as an independent prognostic factor for progression-free survival (P = .03) and overall survival (P = .02). In conclusion, Aurora A expression seems to have a prognostic value in ovarian carcinoma.

Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles

Extensive cross-linking introduced during routine tissue fixation of clinical pathology specimens severely hampers chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis from archived tissue samples. This limits the ability to study the epigenomes of valuable, clinically annotated tissue resources. Here we describe fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq), a method that enables reliable extraction of soluble chromatin from formalin-fixed paraffin-embedded (FFPE) tissue samples for accurate detection of histone marks. We demonstrate that FiT-seq data from FFPE specimens are concordant with ChIP-seq data from fresh-frozen samples of the same tumors. By using multiple histone marks, we generate chromatin-state maps and identify cis-regulatory elements in clinical samples from various tumor types that can readily allow us to distinguish between cancers by the tissue of origin. Tumor-specific enhancers and superenhancers that are elucidated by FiT-seq analysis correlate with known oncogenic drivers in different tissues and can assist in the understanding of how chromatin states affect gene regulation.