Majority of differentially expressed genes are down-regulated during malignant transformation in a four-stage model

Abstract

The transformation of normal cells to malignant, metastatic tumor cells is a multistep process caused by the sequential acquirement of genetic changes. To identify these changes, we compared the transcriptomes and levels and distribution of proteins in a fourstage cell model of isogenically matched normal, immortalized, transformed, and metastatic human cells, using deep transcriptome sequencing and immunofluorescence microscopy. The data show that ∼6% (n = 1,357) of the human protein-coding genes are differentially expressed across the stages in the model. Interestingly, the majority of these genes are down-regulated, linking malignant transformation to dedifferentiation. The up-regulated genes are mainly components that control cellular proliferation, whereas the down-regulated genes consist of proteins exposed on or secreted from the cell surface. As many of the identified gene products control basic cellular functions that are defective in cancers, the data provide candidates for follow-up studies to investigate their functional roles in tumor formation. When we further compared the expression levels of four of the identified proteins in clinical cancer cohorts, similar differences were observed between benign and cancer cells, as in the cell model. This shows that this comprehensive demonstration of the molecular changes underlying malignant transformation is a relevant model to study the process of tumor formation.C ancer development is a multistep process where genetic changes are accumulated, thus progressively transforming cells into a cancerous phenotype (1, 2). Over the last decades, numerous investigators have studied the underlying molecular mechanisms for malignant transformation. This has resulted in many models that explain the development of a malignant phenotype of human cells, for instance the "hallmarks of cancer" by Hanahan and Weinberg (3,4).With new technologies for deep sequencing, novel opportunities to study the underlying molecular events leading to cancers have emerged. A large number of investigations have analyzed mutations occurring in tumors, such as the analysis of mRNA expression, microRNA expression, and DNA copy number in a large number of tumors in the Cancer Genome Atlas (5). Similarly, the Human Protein Atlas project (6) studies human cancers using a proteome-wide collection of antibodies, resulting in publicly available immunohistochemistry images covering 20 different human cancer types.An interesting approach to studying the molecular mechanisms underlying cancer is to use an isogenically matched cell model in which normal cells are progressively transformed into malignant cells. There are several studies where genetic elements such as oncogenes have been introduced to different types of cells in an accumulative order as an attempt to mimic the natural steps of transformation (7,8). One such cell model is the fourstage model based on BJ fibroblasts developed by the Weinberg group, in which primary fibroblast cells were immortalized with telomerase reverse transcriptase (...

The reverse evolution from multicellularity to unicellularity during carcinogenesis

“…However, this should have diluted their overrepresentation in known cancer gene sets. In addition, using an isogenically matched cell model with three oncogenes added sequentially so as to mimic the expression divergences during cancer, a previous study found that B80% of the differentially expressed genes are downregulated 25 . We analysed these genes and found that they are highly enriched in multicellularity-related GO terms ( Supplementary Fig.…”

“…Samples with an RNA integrity number (RIN) 49.5 (Agilent 2100 Bioanalyzer) were used. Poly(A) þ mRNA was isolated with Dynabeads Oligo(dT) 25 (Life Technology 61005). Libraries were constructed following the TruSeq RNA Guide (Illumina) and subject to Illumina GAII or HiSseq sequencing.…”

Vimentin Levels and Serine 71 Phosphorylation in the Control of Cell-Matrix Adhesions, Migration Speed, and Shape of Transformed Human Fibroblasts

“…The cells were cultured as described previously [36]. These cells have been characterized previously with regard to their total gene transcription and protein expression, as well as the cytoskeletal organization and behavior of the cells [21,22,23]. For confocal and single-color STED experiments, the cells were transiently transfected with a plasmid coding for mCherry fused to the protein of interest, 24 h prior to analysis, as described previously [2].…”

“…In contrast, in our study, we have used transformed, highly migratory, metastasizing human fibroblasts, which have a high endogenous level of vimentin. These cells have been well characterized, both due to quantitative analysis of the global transcriptome within these cells, and because the levels and localization of many proteins, as well as the cytoskeletal organization and behavior of the cells, have been described previously [21,22,23]. We also previously showed that these cells have reduced FA size, altered spatial distribution of cell-matrix adhesions, and a more entangled vimentin network compared to normal, primary fibroblast [23].…”

Involvement of Merkel Cell Polyomavirus in the Etiology and Pathogenesis of Merkel Cell Carcinoma: A Systematic Review

“…The transformation process of normal cells into malignant tumor cells is caused by sequence genetic mutations and consists of sequential stages in cascade [1]. The genome sequencing efforts have identified a large number of somatic genomic alterations and numerous germinal mutations associated with cancer predisposition.…”