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Myc-dependent purine biosynthesis affects nucleolar stress and therapy response in prostate cancer.

Barfeld SJ, Fazli L, Persson M, Marjavaara L, Urbanucci A, Kaukoniemi KM, Rennie PS, Ceder Y, Chabes A, Visakorpi T, Mills IG - Oncotarget (2015)

Bottom Line: In this study, we report that in PCa cells de novo purine biosynthesis and the subsequent conversion to XMP is tightly regulated by MYC and independent of AR activity.We characterized two enzymes, PAICS and IMPDH2, within the pathway as PCa biomarkers in tissue samples and report increased efficacy of established anti-androgens in combination with a clinically approved IMPDH inhibitor, mycophenolic acid (MPA).In conclusion, targeting purine biosynthesis provides an opportunity to perturb PCa metabolism and enhance tumour suppressive stress responses.

View Article: PubMed Central - PubMed

Affiliation: Prostate Research Group, Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo, Oslo, Norway.

ABSTRACT
The androgen receptor is a key transcription factor contributing to the development of all stages of prostate cancer (PCa). In addition, other transcription factors have been associated with poor prognosis in PCa, amongst which c-Myc (MYC) is a well-established oncogene in many other cancers. We have previously reported that the AR promotes glycolysis and anabolic metabolism; many of these metabolic pathways are also MYC-regulated in other cancers. In this study, we report that in PCa cells de novo purine biosynthesis and the subsequent conversion to XMP is tightly regulated by MYC and independent of AR activity. We characterized two enzymes, PAICS and IMPDH2, within the pathway as PCa biomarkers in tissue samples and report increased efficacy of established anti-androgens in combination with a clinically approved IMPDH inhibitor, mycophenolic acid (MPA). Treatment with MPA led to a significant reduction in cellular guanosine triphosphate (GTP) levels accompanied by nucleolar stress and p53 stabilization. In conclusion, targeting purine biosynthesis provides an opportunity to perturb PCa metabolism and enhance tumour suppressive stress responses.

No MeSH data available.


Related in: MedlinePlus

The expression of purine biosynthesis enzymes in prostate cancer cells is directly regulated by MYCA. Illumina beadarray results. LNCaP MYC cells were hormone-deprived for 72 h prior to stimulation with 2 μg/ml Doxycycline or vehicle for the indicated time points to induce MYC-overexpression. Total RNA of three independent experiments was isolated and subjected to expression array analysis. Genes with significantly altered expression (q < 0.05) compared to vehicle treated cells are displayed using unsupervised hierarchical clustering (left). Functional annotation using KEGG pathway analysis of 266 genes defined as ‘upregulated by MYC-overexpression at both time points’ is shown (right). B. Expression data from a previously published study (Taylor et al.) was used to define candidate MYC-regulated genes. Only patients marked as ‘biochemical recurrence (BCR)’ or ‘metastases (METS)’ were included in the analysis. Similarity search using Pearson correlation (1% tolerance) was performed on MYC expression and displayed using unsupervised hierarchical clustering with genes on the Y-axis and patients on the X-axis (left). Functional annotation of the 264 MYC-correlated genes using KEGG pathway analysis at 1% tolerance is shown (right) C. Real-time PCR results of MYC-overexpressing cells. LNCaP MYC cells were hormone-deprived for 72 h prior to stimulation with 2 μg/ml Doxycycline to induce MYC-overexpression. Total RNA was isolated, reverse transcribed and used for qRT-PCR. n = 3 (D-E) Cells were transfected with 50 nM of control or MYC siRNA for 72 h. D. Real-time PCR results of MYC-depleted cells. Total RNA was isolated, reverse transcribed and used for qRT-PCR. n = 3–4 E. Western Blot results of MYC-depleted cells. Protein lysates were harvested, separated by SDS-PAGE and blotted for the indicated proteins. Protein levels were normalized to siCTRL and β-actin levels. Densitometry analysis of three biological replicates with SEM can be found in Figure S1H. F. ChIP-qPCR detection of promoter regions of purine biosynthesis genes in MYC ChIP samples. Cells were hormone-deprived for 72 h prior to stimulation with 1 nM R1881 or vehicle for 4 h. Subsequently, chromatin was crosslinked, sonicated and subjected to ChIP using an antibody against MYC or an unspecific IgG control. n = 3.
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Figure 1: The expression of purine biosynthesis enzymes in prostate cancer cells is directly regulated by MYCA. Illumina beadarray results. LNCaP MYC cells were hormone-deprived for 72 h prior to stimulation with 2 μg/ml Doxycycline or vehicle for the indicated time points to induce MYC-overexpression. Total RNA of three independent experiments was isolated and subjected to expression array analysis. Genes with significantly altered expression (q < 0.05) compared to vehicle treated cells are displayed using unsupervised hierarchical clustering (left). Functional annotation using KEGG pathway analysis of 266 genes defined as ‘upregulated by MYC-overexpression at both time points’ is shown (right). B. Expression data from a previously published study (Taylor et al.) was used to define candidate MYC-regulated genes. Only patients marked as ‘biochemical recurrence (BCR)’ or ‘metastases (METS)’ were included in the analysis. Similarity search using Pearson correlation (1% tolerance) was performed on MYC expression and displayed using unsupervised hierarchical clustering with genes on the Y-axis and patients on the X-axis (left). Functional annotation of the 264 MYC-correlated genes using KEGG pathway analysis at 1% tolerance is shown (right) C. Real-time PCR results of MYC-overexpressing cells. LNCaP MYC cells were hormone-deprived for 72 h prior to stimulation with 2 μg/ml Doxycycline to induce MYC-overexpression. Total RNA was isolated, reverse transcribed and used for qRT-PCR. n = 3 (D-E) Cells were transfected with 50 nM of control or MYC siRNA for 72 h. D. Real-time PCR results of MYC-depleted cells. Total RNA was isolated, reverse transcribed and used for qRT-PCR. n = 3–4 E. Western Blot results of MYC-depleted cells. Protein lysates were harvested, separated by SDS-PAGE and blotted for the indicated proteins. Protein levels were normalized to siCTRL and β-actin levels. Densitometry analysis of three biological replicates with SEM can be found in Figure S1H. F. ChIP-qPCR detection of promoter regions of purine biosynthesis genes in MYC ChIP samples. Cells were hormone-deprived for 72 h prior to stimulation with 1 nM R1881 or vehicle for 4 h. Subsequently, chromatin was crosslinked, sonicated and subjected to ChIP using an antibody against MYC or an unspecific IgG control. n = 3.

Mentions: Androgen treatment of LNCaP reduced MYC expression levels as previously reported (Figure S1F and S1G) [20]. Consequently, to determine the MYC-regulated transcriptome, we cultured LNCaP MYC cells in the absence of androgens prior to stimulation with Doxycycline for 5 h and 12 h. These conditions were chosen to capture primarily direct MYC-mediated effects on transcription whilst limiting AR activation. Using an unbiased Gene Set Enrichment Analysis (GSEA) approach, we found SCHUHMACHER_MYC_TARGETS_UP to be the top upregulated gene set at 12 h amongst the entire c2: curated gene sets (Figure S1C and Table S3 for Top 10 up- and downregulated gene sets). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis using a web-based tool [21] (http://genecodis.cnb.csic.es/) on the 266 genes significantly upregulated by MYC overexpression at both time points (Table S1) revealed pathway enrichment for ribosome biogenesis, amino acid metabolism and nucleotide metabolism (Figure 1A).


Myc-dependent purine biosynthesis affects nucleolar stress and therapy response in prostate cancer.

Barfeld SJ, Fazli L, Persson M, Marjavaara L, Urbanucci A, Kaukoniemi KM, Rennie PS, Ceder Y, Chabes A, Visakorpi T, Mills IG - Oncotarget (2015)

The expression of purine biosynthesis enzymes in prostate cancer cells is directly regulated by MYCA. Illumina beadarray results. LNCaP MYC cells were hormone-deprived for 72 h prior to stimulation with 2 μg/ml Doxycycline or vehicle for the indicated time points to induce MYC-overexpression. Total RNA of three independent experiments was isolated and subjected to expression array analysis. Genes with significantly altered expression (q < 0.05) compared to vehicle treated cells are displayed using unsupervised hierarchical clustering (left). Functional annotation using KEGG pathway analysis of 266 genes defined as ‘upregulated by MYC-overexpression at both time points’ is shown (right). B. Expression data from a previously published study (Taylor et al.) was used to define candidate MYC-regulated genes. Only patients marked as ‘biochemical recurrence (BCR)’ or ‘metastases (METS)’ were included in the analysis. Similarity search using Pearson correlation (1% tolerance) was performed on MYC expression and displayed using unsupervised hierarchical clustering with genes on the Y-axis and patients on the X-axis (left). Functional annotation of the 264 MYC-correlated genes using KEGG pathway analysis at 1% tolerance is shown (right) C. Real-time PCR results of MYC-overexpressing cells. LNCaP MYC cells were hormone-deprived for 72 h prior to stimulation with 2 μg/ml Doxycycline to induce MYC-overexpression. Total RNA was isolated, reverse transcribed and used for qRT-PCR. n = 3 (D-E) Cells were transfected with 50 nM of control or MYC siRNA for 72 h. D. Real-time PCR results of MYC-depleted cells. Total RNA was isolated, reverse transcribed and used for qRT-PCR. n = 3–4 E. Western Blot results of MYC-depleted cells. Protein lysates were harvested, separated by SDS-PAGE and blotted for the indicated proteins. Protein levels were normalized to siCTRL and β-actin levels. Densitometry analysis of three biological replicates with SEM can be found in Figure S1H. F. ChIP-qPCR detection of promoter regions of purine biosynthesis genes in MYC ChIP samples. Cells were hormone-deprived for 72 h prior to stimulation with 1 nM R1881 or vehicle for 4 h. Subsequently, chromatin was crosslinked, sonicated and subjected to ChIP using an antibody against MYC or an unspecific IgG control. n = 3.
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Related In: Results  -  Collection

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Figure 1: The expression of purine biosynthesis enzymes in prostate cancer cells is directly regulated by MYCA. Illumina beadarray results. LNCaP MYC cells were hormone-deprived for 72 h prior to stimulation with 2 μg/ml Doxycycline or vehicle for the indicated time points to induce MYC-overexpression. Total RNA of three independent experiments was isolated and subjected to expression array analysis. Genes with significantly altered expression (q < 0.05) compared to vehicle treated cells are displayed using unsupervised hierarchical clustering (left). Functional annotation using KEGG pathway analysis of 266 genes defined as ‘upregulated by MYC-overexpression at both time points’ is shown (right). B. Expression data from a previously published study (Taylor et al.) was used to define candidate MYC-regulated genes. Only patients marked as ‘biochemical recurrence (BCR)’ or ‘metastases (METS)’ were included in the analysis. Similarity search using Pearson correlation (1% tolerance) was performed on MYC expression and displayed using unsupervised hierarchical clustering with genes on the Y-axis and patients on the X-axis (left). Functional annotation of the 264 MYC-correlated genes using KEGG pathway analysis at 1% tolerance is shown (right) C. Real-time PCR results of MYC-overexpressing cells. LNCaP MYC cells were hormone-deprived for 72 h prior to stimulation with 2 μg/ml Doxycycline to induce MYC-overexpression. Total RNA was isolated, reverse transcribed and used for qRT-PCR. n = 3 (D-E) Cells were transfected with 50 nM of control or MYC siRNA for 72 h. D. Real-time PCR results of MYC-depleted cells. Total RNA was isolated, reverse transcribed and used for qRT-PCR. n = 3–4 E. Western Blot results of MYC-depleted cells. Protein lysates were harvested, separated by SDS-PAGE and blotted for the indicated proteins. Protein levels were normalized to siCTRL and β-actin levels. Densitometry analysis of three biological replicates with SEM can be found in Figure S1H. F. ChIP-qPCR detection of promoter regions of purine biosynthesis genes in MYC ChIP samples. Cells were hormone-deprived for 72 h prior to stimulation with 1 nM R1881 or vehicle for 4 h. Subsequently, chromatin was crosslinked, sonicated and subjected to ChIP using an antibody against MYC or an unspecific IgG control. n = 3.
Mentions: Androgen treatment of LNCaP reduced MYC expression levels as previously reported (Figure S1F and S1G) [20]. Consequently, to determine the MYC-regulated transcriptome, we cultured LNCaP MYC cells in the absence of androgens prior to stimulation with Doxycycline for 5 h and 12 h. These conditions were chosen to capture primarily direct MYC-mediated effects on transcription whilst limiting AR activation. Using an unbiased Gene Set Enrichment Analysis (GSEA) approach, we found SCHUHMACHER_MYC_TARGETS_UP to be the top upregulated gene set at 12 h amongst the entire c2: curated gene sets (Figure S1C and Table S3 for Top 10 up- and downregulated gene sets). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis using a web-based tool [21] (http://genecodis.cnb.csic.es/) on the 266 genes significantly upregulated by MYC overexpression at both time points (Table S1) revealed pathway enrichment for ribosome biogenesis, amino acid metabolism and nucleotide metabolism (Figure 1A).

Bottom Line: In this study, we report that in PCa cells de novo purine biosynthesis and the subsequent conversion to XMP is tightly regulated by MYC and independent of AR activity.We characterized two enzymes, PAICS and IMPDH2, within the pathway as PCa biomarkers in tissue samples and report increased efficacy of established anti-androgens in combination with a clinically approved IMPDH inhibitor, mycophenolic acid (MPA).In conclusion, targeting purine biosynthesis provides an opportunity to perturb PCa metabolism and enhance tumour suppressive stress responses.

View Article: PubMed Central - PubMed

Affiliation: Prostate Research Group, Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo, Oslo, Norway.

ABSTRACT
The androgen receptor is a key transcription factor contributing to the development of all stages of prostate cancer (PCa). In addition, other transcription factors have been associated with poor prognosis in PCa, amongst which c-Myc (MYC) is a well-established oncogene in many other cancers. We have previously reported that the AR promotes glycolysis and anabolic metabolism; many of these metabolic pathways are also MYC-regulated in other cancers. In this study, we report that in PCa cells de novo purine biosynthesis and the subsequent conversion to XMP is tightly regulated by MYC and independent of AR activity. We characterized two enzymes, PAICS and IMPDH2, within the pathway as PCa biomarkers in tissue samples and report increased efficacy of established anti-androgens in combination with a clinically approved IMPDH inhibitor, mycophenolic acid (MPA). Treatment with MPA led to a significant reduction in cellular guanosine triphosphate (GTP) levels accompanied by nucleolar stress and p53 stabilization. In conclusion, targeting purine biosynthesis provides an opportunity to perturb PCa metabolism and enhance tumour suppressive stress responses.

No MeSH data available.


Related in: MedlinePlus