Pathway aberrations of murine melanoma cells observed in Paired-End diTag transcriptomes

Kuo Ping Chiu,Han Xu,Adrian Tan,Edison Tak-Bun Liu,Chia-Lin Wei,Pramila Ariyaratne,Patrick Ng,Yijun Ruan,Wing-Kin Ken Sung

doi:10.1186/1471-2407-7-109

Kuo Ping Chiu, Han Xu + Show 7 more

Open Access

https://doi.org/10.1186/1471-2407-7-109

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Abstract

BackgroundMelanoma is the major cause of skin cancer deaths and melanoma incidence doubles every 10 to 20 years. However, little is known about melanoma pathway aberrations. Here we applied the robust Gene Identification Signature Paired End diTag (GIS-PET) approach to investigate the melanoma transcriptome and characterize the global pathway aberrations.MethodsGIS-PET technology directly links 5' mRNA signatures with their corresponding 3' signatures to generate, and then concatenate, PETs for efficient sequencing. We annotated PETs to pathways of KEGG database and compared the murine B16F1 melanoma transcriptome with three non-melanoma murine transcriptomes (Melan-a2 melanocytes, E14 embryonic stem cells, and E17.5 embryo). Gene expression levels as represented by PET counts were compared across melanoma and melanocyte libraries to identify the most significantly altered pathways and investigate the expression levels of crucial cancer genes.ResultsMelanin biosynthesis genes were solely expressed in the cells of melanocytic origin, indicating the feasibility of using the PET approach for transcriptome comparison. The most significantly altered pathways were metabolic pathways, including upregulated pathways: purine metabolism, aminophosphonate metabolism, tyrosine metabolism, selenoamino acid metabolism, galactose utilization, nitrobenzene degradation, and bisphenol A degradation; and downregulated pathways: oxidative phosphorylation, ATPase synthesis, TCA cycle, pyruvate metabolism, and glutathione metabolism. The downregulated pathways concurrently indicated a slowdown of mitochondrial activities. Mitochondrial permeability was also significantly altered, as indicated by transcriptional activation of ATP/ADP, citrate/malate, Mg++, fatty acid and amino acid transporters, and transcriptional repression of zinc and metal ion transporters. Upregulation of cell cycle progression, MAPK, and PI3K/Akt pathways were more limited to certain region(s) of the pathway. Expression levels of c-Myc and Trp53 were also higher in melanoma. Moreover, transcriptional variants resulted from alternative transcription start sites or alternative polyadenylation sites were found in Ras and genes encoding adhesion or cytoskeleton proteins such as integrin, β-catenin, α-catenin, and actin.ConclusionThe highly correlated results unmistakably point to a systematic downregulation of mitochondrial activities, which we hypothesize aims to downgrade the mitochondria-mediated apoptosis and the dependency of cancer cells on angiogenesis. Our results also demonstrate the advantage of using the PET approach in conjunction with KEGG database for systematic pathway analysis.

Highlights

Melanoma is the major cause of skin cancer deaths and melanoma incidence doubles every 10 to 20 years
This study aimed to reveal global pathway aberrations in melanoma cells using the robust Gene Identification Signature Paired End diTag (GIS-PET) technology in conjunction with KEGG pathway database and hypergeometric distribution analysis
The most significantly upregulated melanoma pathways are very diverse as they are involved in a broad spectrum of cellular activities including purine and amino acid biosyntheses, galactose utilization, and detoxification of various harmful compounds

Summary

Introduction

Melanoma is the major cause of skin cancer deaths and melanoma incidence doubles every 10 to 20 years. We applied the robust Gene Identification Signature Paired End diTag (GIS-PET) approach to investigate the melanoma transcriptome and characterize the global pathway aberrations. Cancers are caused by multiple genetic and/or epigenetic alterations [1,2,3,4]. These alterations include activation of oncogenes, inactivation of tumor suppressor genes, mutations that cause chromosome instability [5], and mutations that affect key pathways such as apoptosis, MAPK, cell cycle progression, Wnt/β-catenin, metastasis, and angiogenesis [6,7,8,9]. We applied the robust Gene Identification Signature Paired-End diTag technology (GIS-PET) to reveal the global pathway aberrations in melanoma by using the murine melanoma cell line B16F1 as a model system. Some in vitro and in vivo studies of this cell line, including deletion in Ink4a/ Arf exons and p53 protein expression level, have been well documented and can serve as controls for data validation [12,13]

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