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Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells.

Mathews LA, Hurt EM, Zhang X, Farrar WL - Mol. Cancer (2010)

Bottom Line: The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion.Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity.Using this method we can begin to understand which genes are epigenetically regulated in the invasive population compared to the bulk tumor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Stem Cell Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA.

ABSTRACT

Background: Recently, much attention has been focused on gaining a better understanding of the different populations of cells within a tumor and their contribution to cancer progression. One of the most commonly used methods to isolate a more aggressive sub-population of cells utilizes cell sorting based on expression of certain cell adhesion molecules. A recently established method we developed is to isolate these more aggressive cells based on their properties of increased invasive ability. These more invasive cells have been previously characterized as tumor initiating cells (TICs) that have a stem-like genomic signature and express a number of stem cell genes including Oct3/4 and Nanog and are more tumorigenic compared to their 'non-invasive' counterpart. They also have a profile reminiscent of cells undergoing a classic pattern of epithelial to mesenchymal transition or EMT. Using this model of invasion, we sought to investigate which genes are under epigenetic control in this rare population of cells. Epigenetic modifications, specifically DNA methylation, are key events regulating the process of normal human development. To determine the specific methylation pattern in these invasive prostate cells, and if any developmental genes were being differentially regulated, we analyzed differences in global CpG promoter methylation.

Results: Differentially methylated genes were determined and select genes were chosen for additional analyses. The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion. Ingenuity pathway analysis revealed the methylated gene list frequently displayed genes from the IL-6/STAT3 pathway. Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity. Finally, using Oncomine, it was determined that more aggressive metastatic prostate cancers in humans also have higher levels of both Stat3 and Sox1.

Conclusions: Using this method we can begin to understand which genes are epigenetically regulated in the invasive population compared to the bulk tumor cells. These aggressive sub-populations of cells may be linked to the cancer stem cell hypothesis, making their patterns of epigenetic regulation very attractive for biomarker analysis.

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Related in: MedlinePlus

Oncomine analysis of overlapping targets methylated in both LNCaP and DU145 cells. Isolated targets from the methylation arrays overlapping in LNCaP and DU145 cells were analyzed in Oncomine 4.2 (Ann Arbor, MI). The heat map represents raw data from the Varambally over-expression in prostate cancer analysis comparing primary tissue and metastatic tissue [17]. Expression is in terms of normalized over-expression units. The P-value represents a student's t-test comparing primary and metastatic expression and the gene ID is provided. Genes of interest included Sox1 (p = 0.024) since it has high homology to the stem cell gene Sox2 and albeit demonstrating significance, Bmx (p = 0.294), since it has previously been implicated in prostate cancer regulation.
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Figure 2: Oncomine analysis of overlapping targets methylated in both LNCaP and DU145 cells. Isolated targets from the methylation arrays overlapping in LNCaP and DU145 cells were analyzed in Oncomine 4.2 (Ann Arbor, MI). The heat map represents raw data from the Varambally over-expression in prostate cancer analysis comparing primary tissue and metastatic tissue [17]. Expression is in terms of normalized over-expression units. The P-value represents a student's t-test comparing primary and metastatic expression and the gene ID is provided. Genes of interest included Sox1 (p = 0.024) since it has high homology to the stem cell gene Sox2 and albeit demonstrating significance, Bmx (p = 0.294), since it has previously been implicated in prostate cancer regulation.

Mentions: Individual promoter tiling arrays were performed to analyze global CpG promoter methylation for both non-invasive and invasive cell isolates from both LNCaP and DU145 (Figure 1). The cells were allowed to invade the Matrigel toward a highly defined media called stem cell media (SCM) [18]. It was then determined which genes were methylated in the non-invasive cells and not in the invasive fraction of cells. This analysis determined that 869 probes were differentially methylated in the non-invasive LNCaP fraction compared with the invasive and 1015 for DU145 (Additional File 1, Tables S1A and S1B). A very small subset of 44 overlapping genes was methylated in the non-invasive cells and not in the invasive population from both of the prostate cancer lines analyzed. These included genes involved in development such as Irx3, Six1 and Sox1, as well as a type-III 5" deiodinase (Dio3), and an embryonic version of myosin (Myh3) (Table 1). Using the Oncomine database we investigated changes in expression patterns for these methylated targets, and we found a significant association between progression of prostate cancer and metastasis with expression of a number of genes including G protein, beta-1 subunit (Gnb1), retinoblastoma binding protein 8 (rbbp8), secretogranin III (Scg3) and Sox1 (Figure 2). Albeit a number of these proteins have been shown to play a role in cancer, we chose to investigate the role of Sox1 in our model since it is very homologous to the induced pluripotent stem cell (iPS) regulator Sox2, and has been shown to play a role in progression of lung and nasopharyngeal cancer [19,20]. We also chose to investigate bone marrow tyrosine kinase gene in chromosome X protein (Bmx) since it has been shown to regulate hematopoiesis [21] and play a role in the regulation of prostate cancer [22]. However, from our Oncomine analysis Bmx was not shown to significantly affect prostate cancer metastasis (Figure 2).


Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells.

Mathews LA, Hurt EM, Zhang X, Farrar WL - Mol. Cancer (2010)

Oncomine analysis of overlapping targets methylated in both LNCaP and DU145 cells. Isolated targets from the methylation arrays overlapping in LNCaP and DU145 cells were analyzed in Oncomine 4.2 (Ann Arbor, MI). The heat map represents raw data from the Varambally over-expression in prostate cancer analysis comparing primary tissue and metastatic tissue [17]. Expression is in terms of normalized over-expression units. The P-value represents a student's t-test comparing primary and metastatic expression and the gene ID is provided. Genes of interest included Sox1 (p = 0.024) since it has high homology to the stem cell gene Sox2 and albeit demonstrating significance, Bmx (p = 0.294), since it has previously been implicated in prostate cancer regulation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2958982&req=5

Figure 2: Oncomine analysis of overlapping targets methylated in both LNCaP and DU145 cells. Isolated targets from the methylation arrays overlapping in LNCaP and DU145 cells were analyzed in Oncomine 4.2 (Ann Arbor, MI). The heat map represents raw data from the Varambally over-expression in prostate cancer analysis comparing primary tissue and metastatic tissue [17]. Expression is in terms of normalized over-expression units. The P-value represents a student's t-test comparing primary and metastatic expression and the gene ID is provided. Genes of interest included Sox1 (p = 0.024) since it has high homology to the stem cell gene Sox2 and albeit demonstrating significance, Bmx (p = 0.294), since it has previously been implicated in prostate cancer regulation.
Mentions: Individual promoter tiling arrays were performed to analyze global CpG promoter methylation for both non-invasive and invasive cell isolates from both LNCaP and DU145 (Figure 1). The cells were allowed to invade the Matrigel toward a highly defined media called stem cell media (SCM) [18]. It was then determined which genes were methylated in the non-invasive cells and not in the invasive fraction of cells. This analysis determined that 869 probes were differentially methylated in the non-invasive LNCaP fraction compared with the invasive and 1015 for DU145 (Additional File 1, Tables S1A and S1B). A very small subset of 44 overlapping genes was methylated in the non-invasive cells and not in the invasive population from both of the prostate cancer lines analyzed. These included genes involved in development such as Irx3, Six1 and Sox1, as well as a type-III 5" deiodinase (Dio3), and an embryonic version of myosin (Myh3) (Table 1). Using the Oncomine database we investigated changes in expression patterns for these methylated targets, and we found a significant association between progression of prostate cancer and metastasis with expression of a number of genes including G protein, beta-1 subunit (Gnb1), retinoblastoma binding protein 8 (rbbp8), secretogranin III (Scg3) and Sox1 (Figure 2). Albeit a number of these proteins have been shown to play a role in cancer, we chose to investigate the role of Sox1 in our model since it is very homologous to the induced pluripotent stem cell (iPS) regulator Sox2, and has been shown to play a role in progression of lung and nasopharyngeal cancer [19,20]. We also chose to investigate bone marrow tyrosine kinase gene in chromosome X protein (Bmx) since it has been shown to regulate hematopoiesis [21] and play a role in the regulation of prostate cancer [22]. However, from our Oncomine analysis Bmx was not shown to significantly affect prostate cancer metastasis (Figure 2).

Bottom Line: The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion.Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity.Using this method we can begin to understand which genes are epigenetically regulated in the invasive population compared to the bulk tumor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Stem Cell Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA.

ABSTRACT

Background: Recently, much attention has been focused on gaining a better understanding of the different populations of cells within a tumor and their contribution to cancer progression. One of the most commonly used methods to isolate a more aggressive sub-population of cells utilizes cell sorting based on expression of certain cell adhesion molecules. A recently established method we developed is to isolate these more aggressive cells based on their properties of increased invasive ability. These more invasive cells have been previously characterized as tumor initiating cells (TICs) that have a stem-like genomic signature and express a number of stem cell genes including Oct3/4 and Nanog and are more tumorigenic compared to their 'non-invasive' counterpart. They also have a profile reminiscent of cells undergoing a classic pattern of epithelial to mesenchymal transition or EMT. Using this model of invasion, we sought to investigate which genes are under epigenetic control in this rare population of cells. Epigenetic modifications, specifically DNA methylation, are key events regulating the process of normal human development. To determine the specific methylation pattern in these invasive prostate cells, and if any developmental genes were being differentially regulated, we analyzed differences in global CpG promoter methylation.

Results: Differentially methylated genes were determined and select genes were chosen for additional analyses. The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion. Ingenuity pathway analysis revealed the methylated gene list frequently displayed genes from the IL-6/STAT3 pathway. Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity. Finally, using Oncomine, it was determined that more aggressive metastatic prostate cancers in humans also have higher levels of both Stat3 and Sox1.

Conclusions: Using this method we can begin to understand which genes are epigenetically regulated in the invasive population compared to the bulk tumor cells. These aggressive sub-populations of cells may be linked to the cancer stem cell hypothesis, making their patterns of epigenetic regulation very attractive for biomarker analysis.

Show MeSH
Related in: MedlinePlus