Bone Cancer Associated Genes: An Analysis of Transcriptome and Methylation level

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Genomic Study of Bone Cancer Associated Genes: An Analysis of Transcriptome and Methylation level



Genomics (Genetics, Epigenetics, Proteomics)


One of the most serious problems of human health is cancer which is responsible for huge number of deaths each year (approx. 1 million each year). There are many kinds of cancer like lung, liver, brain, bone, breast, prostate, pancreatic etc. Some of them are well studied at genomic level while others are not; one of them is bone cancer. There are two main types of cancer i.e. primary bone cancer and secondary bone cancer. Primary bone cancer arises due to uncontrolled growth of own cells of bone while secondary bone cancer arises due to metastases of other cancers to bone like lung, prostate and breast etc. Both of these cancers are developed due to many reasons and two of them are genetic and epigenetic mutations. Genetic mutation arises due to change in DNA sequence which may result in producing no protein or changed protein. While epigenetic mutation arises due to change in modification of same DNA sequence which may result in producing no protein or altered level of protein. Both of these mutations are not studied yet in bone cancer patients and this study will help to do so by different genomics’ approaches which will finally open new horizons to cure bone cancer patients by suppressing the cause as well as outcome of these mutations. Mostly there is change at epigenetic level in both of these bone cancer types.

Project Details


This project will be dealing with the mechanism of bone cancer development. Bone cancer cells will be thoroughly analyzed at different onset of disease occurrence to get genetic and transcriptomic data which will be compared with that of healthy individual’s bone cell. Genetic mutations will be checked out to grow bone cancer cells in cell culture for 10-20 generations while epigenetic mutations will be checked out by analyzing methylation level with the help of bisulfite sequencing approach. On top of that, transcriptomic analysis of mRNA will further help us to validate our data in terms of expression level of genes.



In both kinds of bone cancer, primary as well as secondary, genetic and epigenetic mutations arise resulting in no expression of genes, altered expression level of genes, or different protein formation.


1.To study methylation pattern of primary as well as secondary bone cancer cells

2.To study transcripts or mRNA level of primary as well as secondary bone cancer cells

3.To study genetic mutations in primary and secondary bone cancer cells

4. Data comparison of primary bone cancer with that of secondary bone cancer

5. Data comparison of primary as well as secondary bone cancer with that of normal cells

6. Evaluation of hot markers of bone cancers


In Europe, predicted number associated with cancer deaths was 1, 314, 296 (576, 489 women and 737, 747 men), in 2013 (Malvezzi, Bertuccio, Levi, La Vecchia, & Negri, 2013). In 2014, a total of 585, 720 cancer deaths and 1, 665, 540 new cancer cases are projected to arise in United States (Siegel, Ma, Zou, & Jemal, 2014). Cancer may be caused by chemicals (Sasco, Secretan, & Straif, 2004), diet (Anand et al., 2008), infection (Samaras, Rafailidis, Mourtzoukou, Peppas, & Falagas, 2010), radiation (Brenner & Hall, 2007), heredity (Roukos, 2009), physical agents (Ungefroren, Sebens, Seidl, Lehnert, & Hass, 2011) or harmones (Rowlands et al., 2009) etc. All of these responsible factors are associated directly or indirectly with genetic as well as epigenetic mutations which in turn result in cancer.

This project involves the discipline of “genomics” in which transcriptomic, genetic and epigenetic study will be covered. At genetic level, there may be change in DNA sequence. While at epigenetic level, there may be up-regulation of oncogenes and down-regulation of tumor suppressor genes. This alteration in expression is mainly associated with epimutations by change in methylation level. Methylation binding proteins remain associated with methylated sequence and do no permit RNA polymerase along with transcription machinery to form normal level of transcripts or mRNA. So, higher the methylation at promoter region more will be down-regulation of gene expression and vice versa.

As it is clear from above two paragraphs that there is a strong correlation between epimutations and cancer. While epimutations arise due to difference in methylation level of particular genes involved in cancer development. This project will be covering the methylation level, genetic mutation level and transcript level of bone cancer cells’ genes in comparison with that of normal bone cells. Two main techniques, bisulfite sequencing and illumine sequencing, will allow us to get an idea about it. Once, we have complete information at genomics level then such alterations in expression level can be prevented by designing certain drugs.


Uncontrolled division of cells, due to some biotic or abiotic factors, accounts for cancer which may be either benign (localized) or malignant (non-localized). Out of all advanced cancer carrying patients, one third may develop related skeletal metastasis at any stage of their disease (Thürlimann & de Stoutz, 1996). It is recognized that, after lung and liver, third most common site of metastasis development is bone (Rubens, 1998). Patients carrying lung, prostate and breast are more prone to bone metastases, leading to secondary bone metastases (accounting for 80%) (Coleman & Rubens, 1987). It is also reported that 90% deaths of breast tumor carrying patients are due to bone metastases (Mundy & Yoneda, 1995).

Bone tumor is categorized into two types depending upon its origin i.e. primary bone tumor (due to uncontrolled growth of bone cells) (Keedy, 2012) and secondary bone tumor (due to metastases of cancerous cells associated with other cancers like breast, lung, prostate etc.) (Buijs & van der Pluijm, 2009; G. A. Clines & Guise, 2008). Primary bone cancer exists in three most common types (Osteosarcoma, Ewing’s sarcoma, and Chndrosarcoma) and two less common types (Spindle cell sarcoma and Chordoma). Spindle cell sarcoma is further sub-divided into four subtypes: Undifferentiated bone sarcoma, Fibrosarcoma, Malignant fibrous histiocytoma, and Leiomyosarcoma.

Potentially curable disease is converted into unlikely curable state of malignancy by the escaping of tumor cells from localized area followed by invasion to distinct tissue. The metastasis to skeleton by cancer cells is a most severe case of malignancy which leads to disruption of normal bone remodeling and homeostasis, bone weakening and pathological complications and fractures (G. Clines & Guise, 2005). This metastasis is composed of three main mechanisms: 1) Escape of cancer cell from primary tumour with the help of some substances like matrix metalloproteinsases (MMPs) (Bachmeier, Nerlich, Lichtinghagen, & Sommerhoff, 2000; Egeblad & Werb, 2002; Lynch et al., 2005; Nakopoulou et al., 2003; Ranuncolo, Armanasco, Cresta, Bal de Kier Joffe, & Puricelli, 2003; Upadhyay et al., 1999) or platelets (Boucharaba et al., 2004; Palumbo et al., 2005); 2) Adhesion and invasion of distant organ due to the presence of certain receptors on the surface of targeted bone cells like stromal-cell derived factor 1a (SDF1a or CXCL12) (Kang et al., 2003; Müller et al., 2001; Sun et al., 2005; Taichman et al., 2002; Wang, Loberg, & Taichman, 2006) or αvβ3 integrin (Felding-Habermann et al., 2001; Mishra, Shiozawa, Pienta, & Taichman, 2011; Sung, Stubbs, Fisher, Aaron, & Thompson, 1998); 3) Propagation in invaded environment to form either osteolytic or osteoblastic bone disease. Osteolytic bone disease involves parathyroid harmone related protein (PTHrP), transforming growth factor β (TGFβ) (Guise et al., 1996; Thomas et al., 1999), IL-6, IL-8, IL-11, vascular endothelial growth factor (VEGF) (Bendre et al., 2002; De La Mata et al., 1995; Kakonen et al., 2002) and insulin like growth factors (IGFs) (Sachdev & Yee, 2001; Yoneda, Williams, Hiraga, Niewolna, & Nishimura, 2001). Osteoblastic bone disease involves endothelin-1 (ET-1), DKK-1 etc. (G. A. Clines et al., 2007; Yin et al., 2003).

Cure from bone cancer is a major problem throughout the world. Many attempts have been done to be safe from bone metastases by: 1) inhibiting interaction between cancer cells and platelets (Boucharaba et al., 2004; Palumbo et al., 2005) 2) blocking of SDF1a-CXCR4 interaction (Sun et al., 2005; Taichman et al., 2002) 3) using avβ3 antagonists (Sloan et al., 2006) 4) using TGFβ inhibitor SD-208 (Mohammad et al., 2011) 4) SMAD-7 overexpression (Javelaud et al., 2007) 5) blocking RUNX-2 activity (Javed et al., 2005) 6) reducing GLI-2 expression(Sterling et al., 2006).

Cancer usually arises due to genetic or epigenetic mutations which are associated with activation and silencing of certain genes (Dobrovic & Kristensen, 2009). YET there is no complete genomic data of bone cancer cells by which we can get an idea that which particular genes are activated or silenced in this type of cancer. Once, we have comparison of genomic data between cancerous and normal cells then it will help us to find new prospects to cure bone cancer progression.


  1. Getting consent of patients and individuals for sampling according to proper laws
  2. Isolation of tumor section from affected patients at different times of onset by either needle biopsy or surgical bone biopsy (Saifuddin & Clarke, 2014)
  3. Confirmation of isolated bone cancer section by certain markers e.g. Bone Specific Alkaline Phosphatase (BSAP)
  4. Isolation of single cell from isolated tumor section by micromanipulation, FACS, serial dilution, LCM or microfluidic devices (Adams & Strasser, 2008)
  5. Isolation and amplification of RNA by using the Illumina Totalprep RNA amplification kit from primary and secondary bone cancer cells as well as normal bone cells (Ambion UK)
  6. RNA-seq transcriptomic analysis by hybridizing labelled cRNA to Illumina Sentrix-human 6 version 2 expression bead-chips
  7. Raw values of gene expression were normalized with use of Bioconductor lumi package ( packages/2.3/bioc/html/lumi.html) in R
  8. Estimating methylation level by bisulfite sequencing to check epigenetic mutations
  9. Growing isolated cancer cells in tissue culture lab for 10-20 generations under examination
  10. Isolating genomic DNA of replicated cells
  11. Estimating genetic mutations occurred on replication by sequencing isolated DNA and comparing with control.