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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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Functional Role of SOX1 during invasion. A) The Trans-Lentiviral pTRIPZ system from Open Biosystems was used to introduce shRNA against BMX, SOX1 or a non-silencing control vector in DU145 cells. The cells were selected for 2 weeks in 1 μg/mL of puromycin and single cell clones were generated. To induce expression of the shRNA 1 μg/mL of doxycycline was added. The plasmid is designed to have a TET inducible TurboRFP upstream of the shRNA and they should appear red upon successful infection. B) Lowered expression was confirmed using Western blotting. Follow up experiments were conducted using BMX clone 3 and 5 and SOX1 clone 7 and 8 since they demonstrated the most significant decrease in protein expression. Fold changes represent samples normalized to actin and the control level of expression. C) Proliferation assays were conduced using Cell Titer-Glo kit and assayed on Day 1, 3, 5 and 7. More proliferation is indicated by an increase in relative luciferase units (RLUs). *denotes statistical significant p < 0.05 compared to vector transfected cells. A significant decrease was observed in shSOX1 #7 cells compared to vector transfected cells, and a significant increase was observed in shBMX #5 cell line. D) Matrigel invasion assays were conducted for 24 hours toward SCM. Top cells were removed and bottom cells were stained with the Diff-Quick staining kit from Dade Behring. Cells were counted using 4 independent fields per sample and 2 chambers were used per cell line. *denotes statistical significance p < 0.05 compared to vector transfected cells. Both shSOX #7 and #8 demonstrated significant decreases in invasion toward SCM compared to vector transfected cells.
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Figure 4: Functional Role of SOX1 during invasion. A) The Trans-Lentiviral pTRIPZ system from Open Biosystems was used to introduce shRNA against BMX, SOX1 or a non-silencing control vector in DU145 cells. The cells were selected for 2 weeks in 1 μg/mL of puromycin and single cell clones were generated. To induce expression of the shRNA 1 μg/mL of doxycycline was added. The plasmid is designed to have a TET inducible TurboRFP upstream of the shRNA and they should appear red upon successful infection. B) Lowered expression was confirmed using Western blotting. Follow up experiments were conducted using BMX clone 3 and 5 and SOX1 clone 7 and 8 since they demonstrated the most significant decrease in protein expression. Fold changes represent samples normalized to actin and the control level of expression. C) Proliferation assays were conduced using Cell Titer-Glo kit and assayed on Day 1, 3, 5 and 7. More proliferation is indicated by an increase in relative luciferase units (RLUs). *denotes statistical significant p < 0.05 compared to vector transfected cells. A significant decrease was observed in shSOX1 #7 cells compared to vector transfected cells, and a significant increase was observed in shBMX #5 cell line. D) Matrigel invasion assays were conducted for 24 hours toward SCM. Top cells were removed and bottom cells were stained with the Diff-Quick staining kit from Dade Behring. Cells were counted using 4 independent fields per sample and 2 chambers were used per cell line. *denotes statistical significance p < 0.05 compared to vector transfected cells. Both shSOX #7 and #8 demonstrated significant decreases in invasion toward SCM compared to vector transfected cells.

Mentions: To further determine the role of Bmx and Sox1 during the process of invasion we performed the invasion assay with DU145 cells stably infected with shRNAs directed against Sox1or Bmx (Figure 4). A significant decrease in expression of SOX1 and BMX following induction with 1 μg/mL of doxycycline (Dox) for 24 hours was first verified using western blotting. Upon induction with Dox, the shRNA is turned on and a downstream red fluorescent protein (RFP) demonstrates efficiency of this induction (Figure 4A). Densitometry analysis was performed to compare expression of individual clones with the NS cells, and no significant differences in protein expression were seen using the non-silencing (NS) controls (Figure 4B). In addition, SOX1 shRNA cells demonstrated a significant decrease in proliferation compared to either the parental cell line (total cells) or the NS infected line (Figure 4C), as well as a significant decrease in invasion toward SCM (Figure 4D) (p-value < 0.05). However, there was not a significant difference using the shBMX lines, except for a slight reduction in invasion using clone #3. Interestingly, a small increase in proliferation was seen with the shBMX clones (Figure 4C). Further promoter tiling array analysis using two short term cultures primary prostate tumor cell lines, PCSC1 and PCSC2, determined that Sox1, and not Bmx, was methylated in the invasive population of cells (Additional File 2, Table S2A and B). Overall, we demonstrate that Sox1is differentially methylated within the invasive CSC population and the shRNA studies indicate it could be selectively targeted to block invasion.


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

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

Functional Role of SOX1 during invasion. A) The Trans-Lentiviral pTRIPZ system from Open Biosystems was used to introduce shRNA against BMX, SOX1 or a non-silencing control vector in DU145 cells. The cells were selected for 2 weeks in 1 μg/mL of puromycin and single cell clones were generated. To induce expression of the shRNA 1 μg/mL of doxycycline was added. The plasmid is designed to have a TET inducible TurboRFP upstream of the shRNA and they should appear red upon successful infection. B) Lowered expression was confirmed using Western blotting. Follow up experiments were conducted using BMX clone 3 and 5 and SOX1 clone 7 and 8 since they demonstrated the most significant decrease in protein expression. Fold changes represent samples normalized to actin and the control level of expression. C) Proliferation assays were conduced using Cell Titer-Glo kit and assayed on Day 1, 3, 5 and 7. More proliferation is indicated by an increase in relative luciferase units (RLUs). *denotes statistical significant p < 0.05 compared to vector transfected cells. A significant decrease was observed in shSOX1 #7 cells compared to vector transfected cells, and a significant increase was observed in shBMX #5 cell line. D) Matrigel invasion assays were conducted for 24 hours toward SCM. Top cells were removed and bottom cells were stained with the Diff-Quick staining kit from Dade Behring. Cells were counted using 4 independent fields per sample and 2 chambers were used per cell line. *denotes statistical significance p < 0.05 compared to vector transfected cells. Both shSOX #7 and #8 demonstrated significant decreases in invasion toward SCM compared to vector transfected cells.
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Figure 4: Functional Role of SOX1 during invasion. A) The Trans-Lentiviral pTRIPZ system from Open Biosystems was used to introduce shRNA against BMX, SOX1 or a non-silencing control vector in DU145 cells. The cells were selected for 2 weeks in 1 μg/mL of puromycin and single cell clones were generated. To induce expression of the shRNA 1 μg/mL of doxycycline was added. The plasmid is designed to have a TET inducible TurboRFP upstream of the shRNA and they should appear red upon successful infection. B) Lowered expression was confirmed using Western blotting. Follow up experiments were conducted using BMX clone 3 and 5 and SOX1 clone 7 and 8 since they demonstrated the most significant decrease in protein expression. Fold changes represent samples normalized to actin and the control level of expression. C) Proliferation assays were conduced using Cell Titer-Glo kit and assayed on Day 1, 3, 5 and 7. More proliferation is indicated by an increase in relative luciferase units (RLUs). *denotes statistical significant p < 0.05 compared to vector transfected cells. A significant decrease was observed in shSOX1 #7 cells compared to vector transfected cells, and a significant increase was observed in shBMX #5 cell line. D) Matrigel invasion assays were conducted for 24 hours toward SCM. Top cells were removed and bottom cells were stained with the Diff-Quick staining kit from Dade Behring. Cells were counted using 4 independent fields per sample and 2 chambers were used per cell line. *denotes statistical significance p < 0.05 compared to vector transfected cells. Both shSOX #7 and #8 demonstrated significant decreases in invasion toward SCM compared to vector transfected cells.
Mentions: To further determine the role of Bmx and Sox1 during the process of invasion we performed the invasion assay with DU145 cells stably infected with shRNAs directed against Sox1or Bmx (Figure 4). A significant decrease in expression of SOX1 and BMX following induction with 1 μg/mL of doxycycline (Dox) for 24 hours was first verified using western blotting. Upon induction with Dox, the shRNA is turned on and a downstream red fluorescent protein (RFP) demonstrates efficiency of this induction (Figure 4A). Densitometry analysis was performed to compare expression of individual clones with the NS cells, and no significant differences in protein expression were seen using the non-silencing (NS) controls (Figure 4B). In addition, SOX1 shRNA cells demonstrated a significant decrease in proliferation compared to either the parental cell line (total cells) or the NS infected line (Figure 4C), as well as a significant decrease in invasion toward SCM (Figure 4D) (p-value < 0.05). However, there was not a significant difference using the shBMX lines, except for a slight reduction in invasion using clone #3. Interestingly, a small increase in proliferation was seen with the shBMX clones (Figure 4C). Further promoter tiling array analysis using two short term cultures primary prostate tumor cell lines, PCSC1 and PCSC2, determined that Sox1, and not Bmx, was methylated in the invasive population of cells (Additional File 2, Table S2A and B). Overall, we demonstrate that Sox1is differentially methylated within the invasive CSC population and the shRNA studies indicate it could be selectively targeted to block invasion.

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