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RNA-seq profiling of a radiation resistant and radiation sensitive prostate cancer cell line highlights opposing regulation of DNA repair and targets for radiosensitization.

Young A, Berry R, Holloway AF, Blackburn NB, Dickinson JL, Skala M, Phillips JL, Brettingham-Moore KH - BMC Cancer (2014)

Bottom Line: Opposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis.While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1.Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02).

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, University of Tasmania, Private Bag 23, Hobart, TAS 7000, Australia. khmoore@utas.edu.au.

ABSTRACT

Background: Radiotherapy is a chosen treatment option for prostate cancer patients and while some tumours respond well, up to 50% of patients may experience tumour recurrence. Identification of functionally relevant predictive biomarkers for radioresponse in prostate cancer would enable radioresistant patients to be directed to more appropriate treatment options, avoiding the side-effects of radiotherapy.

Methods: Using an in vitro model to screen for novel biomarkers of radioresistance, transcriptome analysis of a radioresistant (PC-3) and radiosensitive (LNCaP) prostate cancer cell line was performed. Following pathway analysis candidate genes were validated using qRT-PCR. The DNA repair pathway in radioresistant PC-3 cells was then targeted for radiation sensitization using the PARP inhibitor, niacinimide.

Results: Opposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis. Candidate genes BRCA1, RAD51, FANCG, MCM7, CDC6 and ORC1 were identified as being significantly differentially regulated post-irradiation. qRT-PCR validation confirmed BRCA1, RAD51 and FANCG as being significantly differentially regulated at 24 hours post radiotherapy (p-value =0.003, 0.045 and 0.003 respectively). While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1. Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02).

Conclusions: These findings suggest that the DNA repair candidates identified via RNA-seq hold potential as both targets for radiation sensitization and predictive biomarkers in prostate cancer.

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Related in: MedlinePlus

Pathway analysis highlights opposing regulation of a DNA repair pathway in radioresistant versus radiosensitive cells. Gene lists determined by RNA-seq for the (A) PC-3 and (B) LNCaP cell lines 24 hours after 2 Gy irradiation were analysed using IPA. DNA repair pathways were identified as being significantly altered in response to RT (q-value 1x10−10). Significantly up-regulated genes are coloured red and down-regulated green, those present within our data set but not significant are shown in grey. Significant genes were defined as reporting a log2 fold change >1 and a q-value <0.05. Arrows indicate gene products which were found to be oppositely regulated.
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Fig2: Pathway analysis highlights opposing regulation of a DNA repair pathway in radioresistant versus radiosensitive cells. Gene lists determined by RNA-seq for the (A) PC-3 and (B) LNCaP cell lines 24 hours after 2 Gy irradiation were analysed using IPA. DNA repair pathways were identified as being significantly altered in response to RT (q-value 1x10−10). Significantly up-regulated genes are coloured red and down-regulated green, those present within our data set but not significant are shown in grey. Significant genes were defined as reporting a log2 fold change >1 and a q-value <0.05. Arrows indicate gene products which were found to be oppositely regulated.

Mentions: The gene lists generated for radiosensitive LNCaP and radioresistant PC-3 cells at 6 and 24 hours following 2 Gy irradiation showed large differences in both the number and type of genes that were transcriptionally activated. Irradiation appeared to impact transcriptional response to a greater extent within the PC-3 cell line with 399 genes significantly differentially regulated by 6 hours (using a 2 fold cut-off). In comparison, at the same time-point only 89 genes were significantly up- or down-regulated for the radiosensitive LNCaP cell line. An unbiased analysis of the gene lists obtained from RNA-seq was then used to uncover pathways involved in radioresponse. Interactions between significantly differentially regulated genes in each cell line were determined using Ingenuity Pathway Analysis (IPA). Two canonical pathways identified were shown to have opposing responses 24 hours after irradiation. The top pathway affected 24 hours post-irradiation for both cell lines was a DNA repair pathway. While key genes within this pathway were significantly up-regulated in the radioresistant PC-3 cell line (Figure 2A) the same subset of genes were oppositely regulated, displaying down-regulation within the radiosensitive LNCaP cell line (Figure 2B). These oppositely regulated genes include BRCA1, RAD51 and FANCG.Figure 2


RNA-seq profiling of a radiation resistant and radiation sensitive prostate cancer cell line highlights opposing regulation of DNA repair and targets for radiosensitization.

Young A, Berry R, Holloway AF, Blackburn NB, Dickinson JL, Skala M, Phillips JL, Brettingham-Moore KH - BMC Cancer (2014)

Pathway analysis highlights opposing regulation of a DNA repair pathway in radioresistant versus radiosensitive cells. Gene lists determined by RNA-seq for the (A) PC-3 and (B) LNCaP cell lines 24 hours after 2 Gy irradiation were analysed using IPA. DNA repair pathways were identified as being significantly altered in response to RT (q-value 1x10−10). Significantly up-regulated genes are coloured red and down-regulated green, those present within our data set but not significant are shown in grey. Significant genes were defined as reporting a log2 fold change >1 and a q-value <0.05. Arrows indicate gene products which were found to be oppositely regulated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4233036&req=5

Fig2: Pathway analysis highlights opposing regulation of a DNA repair pathway in radioresistant versus radiosensitive cells. Gene lists determined by RNA-seq for the (A) PC-3 and (B) LNCaP cell lines 24 hours after 2 Gy irradiation were analysed using IPA. DNA repair pathways were identified as being significantly altered in response to RT (q-value 1x10−10). Significantly up-regulated genes are coloured red and down-regulated green, those present within our data set but not significant are shown in grey. Significant genes were defined as reporting a log2 fold change >1 and a q-value <0.05. Arrows indicate gene products which were found to be oppositely regulated.
Mentions: The gene lists generated for radiosensitive LNCaP and radioresistant PC-3 cells at 6 and 24 hours following 2 Gy irradiation showed large differences in both the number and type of genes that were transcriptionally activated. Irradiation appeared to impact transcriptional response to a greater extent within the PC-3 cell line with 399 genes significantly differentially regulated by 6 hours (using a 2 fold cut-off). In comparison, at the same time-point only 89 genes were significantly up- or down-regulated for the radiosensitive LNCaP cell line. An unbiased analysis of the gene lists obtained from RNA-seq was then used to uncover pathways involved in radioresponse. Interactions between significantly differentially regulated genes in each cell line were determined using Ingenuity Pathway Analysis (IPA). Two canonical pathways identified were shown to have opposing responses 24 hours after irradiation. The top pathway affected 24 hours post-irradiation for both cell lines was a DNA repair pathway. While key genes within this pathway were significantly up-regulated in the radioresistant PC-3 cell line (Figure 2A) the same subset of genes were oppositely regulated, displaying down-regulation within the radiosensitive LNCaP cell line (Figure 2B). These oppositely regulated genes include BRCA1, RAD51 and FANCG.Figure 2

Bottom Line: Opposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis.While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1.Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02).

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, University of Tasmania, Private Bag 23, Hobart, TAS 7000, Australia. khmoore@utas.edu.au.

ABSTRACT

Background: Radiotherapy is a chosen treatment option for prostate cancer patients and while some tumours respond well, up to 50% of patients may experience tumour recurrence. Identification of functionally relevant predictive biomarkers for radioresponse in prostate cancer would enable radioresistant patients to be directed to more appropriate treatment options, avoiding the side-effects of radiotherapy.

Methods: Using an in vitro model to screen for novel biomarkers of radioresistance, transcriptome analysis of a radioresistant (PC-3) and radiosensitive (LNCaP) prostate cancer cell line was performed. Following pathway analysis candidate genes were validated using qRT-PCR. The DNA repair pathway in radioresistant PC-3 cells was then targeted for radiation sensitization using the PARP inhibitor, niacinimide.

Results: Opposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis. Candidate genes BRCA1, RAD51, FANCG, MCM7, CDC6 and ORC1 were identified as being significantly differentially regulated post-irradiation. qRT-PCR validation confirmed BRCA1, RAD51 and FANCG as being significantly differentially regulated at 24 hours post radiotherapy (p-value =0.003, 0.045 and 0.003 respectively). While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1. Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02).

Conclusions: These findings suggest that the DNA repair candidates identified via RNA-seq hold potential as both targets for radiation sensitization and predictive biomarkers in prostate cancer.

Show MeSH
Related in: MedlinePlus