Al-masmoum, Hussain
(2016)
An investigation of chromosome 20q13 amplification in colorectal cancer.
PhD thesis, University of Nottingham.
Abstract
Background: Colorectal cancer (CRC) is the third most prevalent and second most fatal type of cancer in the Western world. 20q amplification has been identified in approximately 50% of CRC cases. 20q amplification and may be associated with liver metastasis. In our lab, a previous array comparative genomic hybridization (aCGH) study of a series of primary CRCs and corresponding liver metastases, found that the 20q13 was frequently amplified in both groups. Several known oncogenes are located at Chromosome 20q (for example SRC, TPX2 and CSE1L) suggesting that 20q amplification may be an important event in the development and metastasis of CRC.
Hypothesis and Aim: Although there are many genes located in 20q identified to have role in developing CRC, other genes with potential role in tumour progression have not been studied in CRC. We hypothesized that 20q harbouring genes have a potential role in CRC progression. Therefore, we aimed to (i) study the frequency of 20q amplification in CRC by validating our aCGH data by alternative method and screen larger sample set for 20q amplification. (ii) Protein expression of candidate genes was investigated. Then, candidate genes were evaluated in several cancer related processes including proliferation and cell motility.
Materials and Methods: The evaluation of 20q amplification was performed in DNA from FFPE of micro-dissected 20 CRCs and their matched liver metastasis, using comparative quantitative PCR (qPCR) based on 6 genes (PTPN1, CSE1L, ADNP, PREX1, ELMO2 and PTGIS), which are located in the same region. It was found that quantification of two genes was sufficient to evaluate amplification and thus separate series of 103 cases were screened for 20q amplification based on qPCR of two genes (ELMO2 and PTPN1). For studying the functional activity, PTPN1, CD40, PREX1, ELMO2 and PTGIS were studied in CRC cell lines. To evaluate gene function, small interference RNAs (siRNAs) were used to knockdown genes of interest in CRC cell lines. Knockdown was validated by western blot and qPCR and the effect of knockdown was evaluated on cellular functions such as proliferation, cell migration, cell invasion and wound healing. Immunohistochemistry was used to investigate protein expression in primary CRCs.
Results: The amplification of 20q in CRC sample was studied by qPCR and aCGH. High concordance was found between the two methods aCGH, and high correlation coefficient between the qPCR results of the genes demonstrated the utility of this method for measuring amplification. In the combined data set, 20q amplification was seen in both primary tumours (51%) and metastases (60%). TP53 mutation was associated with 20q amplification (p =0.010). Then, we investigated the functional activity of 5 genes located on 20q, which might be has a role in CRC progression. (1) PTPN1 has been reported as tumour suppresser and oncogene. Therefore, we studied protein expression and its functional role in CRC cell lines. PTPN1 was overexpressed in 59% of primary tumours. Knockdown of PTPN1 reduced cell motility but did not affect cell proliferation. (2) CD40, previously, has been identified as a tumour suppresser in CRC. However, high CD40 expression was detected 25% of tumours. Functional assays showed CD40 promotes proliferation by altering subG1 phase of the cell cycle. Therefore, CD40 might promote tumour growth by inhibiting apoptosis. (3) PREX1 was expressed in 61% of primary tumours. Functional analysis showed that it promotes both cell proliferation and motility. (4) ELMO2 was expressed in 38% of primary tumours and functional analysis showed it promotes proliferation and cell migrations. (5) Finally, PTGIS is highly expressed in CRC and its expression associated with liver metastasis. However, PTGIS effect on tumour progression has not been studied. PTGIS knockdown had no effect on CRC cell lines growth and motility.
Conclusion: Using alternative methods to evaluate the 20q13 amplification, we have confirmed our previous aCGH and found a frequency similar to that reported in the literature. Amplification of 20q appears frequently in primary tumours but it not positively associated with metastasis. Many genes located in 20q have oncogenic effects which would support the hallmarks of cancer. Understanding their role in CRC development could reveal new avenue in understanding cancer biology and treatment.
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