Abstract: In the present study, we tried to integrate information from various sources to understand the role of cancer metasignature genes in initiation and progression of neoplasia. We analyzed these cancer metasignature genes for their chromosomes distribution, expression profiles in normal human tissues and the cellular pathways in which they are involved using the relevant data from the biological databases. It is concluded that cancer metasignature genes are needed for proper functioning and maintenance of normal cellular physiology. We report that multiple numbers of these genes were involved in three cellular processes; cell division cycle, antigen processing and presentation and proteasome dependent proteolysis. They are also involved in a myriad of metabolic and genetic processes. We propose that it is possible that almost all cell types of the human body contain a dormant genetic network which enables them to overcome senescence or evade apoptosis. This network when activated due to some effective trigger may lead to cancer generation and progression.
INTRODUCTION
Cancer is a generic term for a group of more than 100 diseases that can affect any part of the body (Stewart and Kleihues, 2003). Basically it is caused due to over expression or mutation of proto-oncogene or deactivation of tumor suppressors or failure of DNA repair. Cancer can result from accumulation of mutations and other heritable changes in susceptible cells. So far, abnormalities in about 350 genes have been implicated in human cancers, but the true number of cancer genes is unknown (Higgins et al., 2007; Forbes et al., 2006). One defining feature of cancer is the rapid creation of abnormal cells which grow beyond their normal tissue boundaries. These cancerous cells can invade adjoining parts of the body and spread to other organs; this process is referred to as metastasis. Metastasis are the major cause of death from cancer.
The battle against cancer has many fronts. But the main focus amongst the researchers has been to understand the origin and progress of this disease at the molecular level. Cancer may arise from mutation in a single cell. The transformation from a normal cell into a tumor cell is a multistage process, typically a progression from a pre-cancerous lesion to malignant tumors. During the progress of the disease, hundreds of different genes are seen activated or deactivated at different times (Bucca et al., 2004; Kaiser, 2005; Greenman et al., 2007). In 1990s, DNA microarray or DNA chip technology was developed, which enabled researchers to measure the expression levels of hundreds of genes simultaneously. Using this technique, a comprehensive understanding of the cell can be achieved (Hanai et al., 2006). Studies have been conducted to identify the gene expression pattern in specific cancers like the lung cancer, colon cancer and leukemia etc. (Bucca et al., 2004; Kim et al., 2005; Halvorsen et al., 2005; Talbot et al., 2005; De Pitta et al., 2005). Meta-analysis of such microarray datasets was done to identify group of genes which were universally activated in all cancers (Rhodes et al., 2004). This means that all cancer types share the common features of unregulated cell proliferation and invasion. In the present study, we analyzed the chromosomal distribution, expression profile in normal body tissues and the cellular pathways of the genes of cancer metasignature. Since the gene sets were derived through statistical procedures, we wanted to investigate whether these signature genes yield biological relevant information regarding fundamental aspects of cancer.
MATERIALS AND METHODS
This study involves investigation of chromosomal location, cellular pathways and expression profile of cancer metasignature genes. Using bioinformatics tools, as described in the methodology below, we have carried out this study in the Bioinformatics Lab of our institute.
The metasignature gene lists were obtained from the Rhodes et al. (2004). There are two datasets of cancer metasiganature genes; the neoplastic and the undifferentiated. The neoplastic cancer metasignature genes dataset contains a list of 69 genes where as the undifferentiated cancer metasignature genes dataset consists of 67 genes. The gene symbols used in the list were from HUGO Gene Nomenclature Committee (HGNC) database, which is the accepted standard for gene names (Bruford et al., 2008). The two metasignature genes datasets were combined to generate a single dataset with no repeating genes. This combined gene list was submitted to web server WEBGESTALT for analyzing their chromosomal distribution in the human genome and their expression in normal body tissues (Zhang et al., 2005). These genes were clustered based on the information from KEGG pathway database.
Web Gestalt (WEB-based Gene Set Analysis Toolkit) is a web-based integrated data mining system to help biologists in exploring large sets of genes. It is composed of four modules: gene set management, information retrieval, organization/visualization and statistics. The management module uploads, saves, retrieves and deletes gene sets, as well as performs boolean operations to generate the unions, intersections or differences between different gene sets. The information retrieval module retrieves information for up to 20 attributes for all genes in a gene set. The organization/visualization module organizes and visualizes gene sets in various biological contexts, including Gene Ontology, tissue expression pattern, chromosome distribution, metabolic and signaling pathways, protein domain information and publications. Web Gestalt can be accessed at http://bioinfo.vanderbilt.edu/webgestalt.
RESULTS AND DISCUSSION
Chromosomal Distribution
The existence of a general cancer metasignature may not be entirely
surprising, because all cancer types share the common features of unregulated
cell proliferation and invasion and it would follow that the genes that
are essential to these processes would be highly expressed in multiple
cancer types. It is interesting that a small number of genes are almost
universally activated, given the vast array of transforming mechanisms
that are known to initiate cancer and the variety of tissue types represented.
Since the neoplastic and the undifferentiated metasignatures contained
an overlap of 16 genes. We combined the metasignatures by removing the
repeating 16 genes and analysed their chromosomal distribution (Fig.
1). About 35% of metasignature genes were located on chromosomes 1,
7 and 2, whereas chromosomes 22 and Y did not contain any genes. It is
to be noted that about 9 chromosomes (x, 15, 16, 10, 13, 18, 21, 22, Y)
contained only 5% of the metasignature genes. According to Fig.
1, the chromosomes 1, 7 and 2 contain high number of genes. But any
conclusions drawn from this could be erroneous since the chromosomes vary
widely amongst themselves in content and size. Hence we took into consideration
the chromosome length, number of genes it contained and the number of
metasignature genes that were located on it for a given chromosome. These
numbers were converted into percentages from the total length of human
genome i.e., number of base pairs according to the human genome database
at NCBI (Wheeler et al., 2008) and overall number of genes located
on that chromosome from the Vega human genome browser (Wilming et al.,
2008). These percentages were plotted as graph (Fig. 2).
| Fig. 1: | Chromosomal distribution of the cancer metasignature genes in the human genome |
| Fig. 2: | Percentage of cancer metasignature genes per chromosome compared with the percentages of chromosome length and its gene density. The blue bars represent the percentage of genomic length of the chromosome, the red bars represent the percentage of total number of genes present in the given chromosome and the green bars represent the percentage of cancer metasignature genes in the given chromosomes. All the numbers on the top of the bars are given in percentages |
It is interesting to note that about 30% of the metasignature genes are located on chromosomes 7, 17, 19 and 20 which contribute only 12.5% to the total genomic length and contain about 22% of the total genes. The chromosomes 2, 4, 5 and 11 contain less number of total genes when compared to their genomic lengths. These chromosomes contain about 22% of the metasignature genes. Overall eight chromosomes, irrespective of their genomic length and gene density, contain more than 50% of the metasignature genes.
Many genes exist as gene families which are defined as groups of genes with sequence homology and related overlapping functions. Such genes known to exhibit pattern in their location on the chromosomes like the HOX genes, human α-globin gene cluster, multiple members of the MDR-ADH (MDR: medium-chain dehydrogenases/reductases; ADH: alcohol dehydrogenase) family, flavin-containing monooxygenase genes (Abbasi and Grzeschik, 2007; Hernandez et al., 2004; Lai et al., 2005: Lim and Bowles, 2004; Tang et al., 2006; Gonzalez-Duarte and Albalat, 2005). In the case of the cancer metasignature genes, their chromosomal distribution is biased but it is evident that these genes do not exhibit any related pattern in their chromosomal location. We assume that in cancer progression genes from varied regions of the human genome are expressed and the genomic loci of these genes are certainly not related to the genomic length or the gene density of the chromosomes.
| Fig. 3: | Expression of the cancer metasignature genes in the normal human body tissues. The tissues were broadly classified based on their embryonic germ layer. The ectoderm derived tissues are colored blue, mesoderm derived tissues are colored orange and the endoderm derived tissues are colored green. The embryonic tissue is colored red. The numbers at the end of the bars represent the number of metasignature genes |
Expression in Normal Human Tissues
We analysed the expression of the metasignature genes in the normal
tissues. These tissues were classified based on the embryonic germ layers
that were derived from; ectoderm, mesoderm and endoderm. We carried out
this exercise in order to check any bias in the expression of the metasignature
genes in any germ layer derived tissues. There are many different types
of cancer. All cancers, however, fall into one of four broad categories
based on the tissue of origin. Carcinomas are tumors that arise in the
tissues that line the body`s organs. These tissues are derived from the
ectoderm and the endoderm. About 80% of all cancer cases are carcinomas.
Hence we expected biased over expression of the metasignature genes in
these tissues. The sarcomas, leukemia`s and lymphomas are cancers of the
tissues derived from the mesoderm. Sarcomas are tumors that originate
in bone, muscle, cartilage, fibrous tissue or fat. Leukemias are cancers
of the blood or blood-forming organs and the lymphomas affect the lymphatic
system.
We found that the metasignature genes which depict the common transcriptional profile of cancer are expressed in most of the normal tissues (Fig. 3). About 80% of the metasignature genes are expressed in 30 out of 47 tissues. Only 4 tissues showed 50% or less of metasignature genes. Basing on the germ layers it was seen that the ectoderm, mesoderm and endoderm contained similar expression profile of these genes. Only the ectoderm derived tissue showed a drop in the expression when compared to the other two. Even with this reduced expression levels the most of the tissues showed more than 50% of genes expressed. By this we infer that the cancer metasignature are universal in most of the tissues of the normal human body. It is plausible that almost every cell in the human body has a dormant genetic network which enables it to overcome senescence or evade apoptosis. This may lead to cancer generation and progression through uncontrolled cell division.
KEGG Pathways Based Clustering
KEGG is a database of biological systems, consisting of genetic building
blocks of genes and proteins (KEGG GENES), chemical building blocks of
both endogenous and exogenous substances (KEGG LIGAND) and molecular wiring
diagrams of interaction and reaction networks (KEGG PATHWAY) (Kanehisa
et al., 2008). KEGG PATHWAY is a collection of manually drawn pathway
maps representing the knowledge on the molecular interaction and reaction
networks for Metabolism, Genetic Information Processing, Environmental
Information Processing, Cellular Processes, Human Diseases and the structure
relationships in Drug Development. We used the information of cellular
pathways from this database to cluster the metasignature genes.
The analysis showed that multiple numbers of genes were involved in three cellular processes; cell division cycle, antigen processing and presentation and proteosome dependent proteolysis. There were about 13 genes relating to cell cycle (Table 1). It is known that the cancer cells undergo continuous and uncontrolled cell division. This analysis further confirms this fact since the metasignatures contain prominent genes which are essential for proper functioning of the cell cycle like the Cyclins, Cyclin dependent Kinases, Histone deacetylases, Mitotic checkpoint protein coding gene, Mini-chromosome maintenance protein coding genes and Polo-like Kinases.
The cyclins and cyclin dependent kinases are required for progression through the various stages of the mitotic cell cycle (Malumbres, 2007). Histone deacetylase deacetylates the histones which is important process in chromosomal remodeling (Kim et al., 2006). It also interacts with retinoblastoma tumor-suppressor protein and this complex is a key element in the control of cell proliferation and differentiation. Together with metastasis-associated protein-2, it deacetylates p53 and modulates its effect on cell growth and apoptosis (Wolffe, 1996). The mitotic checkpoint protein coding gene (MAD2L1) is essential for preventing the onset of anaphase until all chromosomes are properly aligned at the metaphase plate (Taylor et al., 2004). The Mini-Chromosome Maintenance (MCM) proteins are required for establishing pre-replication complexes which trigger the activation of cyclin-dependent kinases for progression of the cell cycle (Forsburg, 2008). Polo-Like Kinases (PLK1) is an important regulator of several events during mitosis. Recent reports show that PLK1 is involved in both G2 and mitotic DNA damage checkpoints (Ferrari, 2006). It can be seen from the above brief description of the genes that the metasignature of cancer contains an essential set of cell cycle related genes.
| Table 1: | Cellular processes in which the metasignature genes are involved with the No. of genes and their entrez gene id |
There are about five genes in the metasignature whose protein products are involved in major Histocompatibility Component (MHC) pathway one and two; where the antigens are presented to the CD8 and CD4 T-cells and the natural killer cell (Goodsell, 2005). It is becoming increasingly clear that MHC may also play a role in the natural control of cancer cells. Cancer cells contain many mutated proteins that may be displayed by MHC to alert the immune system. Tumor cells may also express normal proteins but in unusual places or in abnormal amounts, providing a potential signal to mobilize an immune response. The possibility of enhancing this response with vaccines is an exciting goal of current research (Goodsell, 2005).
The ubiquitin mediated protein modification or degradation through proteasome is a versatile system employed by cell for regulation of various processes like transcription, histone modification and degradation of unwanted or misfolded proteins (Hershko, 2005). There are about seven genes in the metasignature which code for proteins required for this system. This pathway comes under the genetic information processing KEGG pathways (Table 2). The metasignature genes were involved in a numerous metabolic pathways ranging from the energy generation, nucleotide metabolism and biosynthesis of amino acids, glycans and folate biosynthesis (Table 3). Eight metasignature genes were involved in the environmental information processing like the Notch, Jak stat, WNT and TGF Beta signalling pathways and about 4 genes were involved in pathways related to other diseases like the prion disease, type 1 diabetes mellitus and the E. coli infection (Table 4, 5).
| Table 2: | Genetic Information processing pathways in which the metasignature genes are involved with the No. of genes and their entrez gene id |
| Table 3: | Metabolic pathways in which the metasignature genes are involved with the No. of genes and their entrez gene id |
| Table 4: | Environmental information processing pathways in which the metasignature genes are involved with the No. of genes and their entrez gene id |
| Table 5: | Pathways relating to human diseases in which the metasignature genes are involved with the No. of genes and their entrez gene id |
From this exercise, it is evident that the metasignature genes play key roles in a multitude of cellular pathways. In cancer, the whole cellular machinery is thrown out of control into chaos leading to uncontrolled growth and division of the cell. We believe, from the KEGG pathway based analysis, that the sustained proliferation of the cancer cell is only possible due to involvement of various genes which are important in many fundamental cellular pathways needed for the cell to survive.
CONCLUSION
We found that cancer metasignature genes are scattered throughout the human genome and their chromosomal distribution does not show any relation to the genomic length and the gene density of the chromosomes. Most of the genes are generally expressed in almost all normal body tissues derived from the three embryonic germ layers. The KEGG pathway based analysis showed that these genes are involved in a myriad of metabolic and genetic processes in the cell. From the present investigation, we conclude the cancer metasignature genes are vital for proper functioning and maintenance of the normal cellular physiology. It is possible that almost all cell types of the human body contain a dormant genetic network which enables them to overcome senescence or evade apoptosis. The sustained proliferation of the cancer cell is only possible due to involvement of various genes which are important in many fundamental cellular pathways needed for the cell to survive. This may lead to cancer generation and progression through uncontrolled cell division.