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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

Inhibition of IMPDH2 impairs the proliferation of prostate cancer cells and shows additive effects with established anti-androgensA. Cell viability results of siRNA treated cells. Cells were transfected with 25 nM (control 25 nM, IMPDH1 and IMPDH2 #1) or 50 nM (control 50 nM, PAICS, IMPDH2 #2 and MYC) for 72 h and cell viability relative to siCTRL 50 nM was determined using a MTS-based assay. n = 2–4 B. Cell viability results of MPA treated cells. Cells were allowed to attach for 48 h prior to treatment with indicated doses of MPA for 72 h. Cell viability relative to vehicle control was determined using a MTS-based assay. n = 2–4 C. Cell viability results of siRNA and Abiraterone/MDV3100 treated cells. Cells were transfected with 25 nM IMPDH2 #1, 50 nM IMPDH2 #2 siRNA or equal amounts of siCTRL for 48 h. Following treatment with the indicated drugs for another 72 h, viability relative to DMSO and siCTRL was assessed using a MTS-based assay. Doses for LNCaP were 1 μM Abiraterone and 1 μM MDV3100, and for VCaP 1 μM Abiraterone and 100 nM MDV3100 n = 1–4 D. Cell viability results of MPA and Abiraterone/MDV3100 treated cells. Cells were allowed to attach for 48 h prior to treatment with indicated drug combinations for 72 h. Cell viability relative to vehicle control was determined using a MTS-based assay. Doses for LNCaP were 10 μM MPA, 1 μM Abiraterone and 1 μM MDV3100, and for VCaP 5 μM MPA, 1 μM Abiraterone and 100 nM MDV3100 n = 3–4 E. Cell viability results of MPA treated parental and MDV3100-resistant LNCaP. Cells were allowed to attach for 48 h prior to treatment with the indicated doses of MPA for 72 h. Cell viability relative to vehicle control was determined using a MTS-based assay. n = 4 F. Cell viability results of siRNA and MPA treated cells. Cells were transfected with 50 nM MYC or control siRNA for 48 h. Following treatment with the indicated doses of MPA (LNCaP 5 and 10 μM, VCaP 5 and 10 μM and LNCaP abl 1 and 10 μM) for another 72 h, viability was assessed using a MTS-based assay and normalized to the respective DMSO control. n = 3.
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Figure 3: Inhibition of IMPDH2 impairs the proliferation of prostate cancer cells and shows additive effects with established anti-androgensA. Cell viability results of siRNA treated cells. Cells were transfected with 25 nM (control 25 nM, IMPDH1 and IMPDH2 #1) or 50 nM (control 50 nM, PAICS, IMPDH2 #2 and MYC) for 72 h and cell viability relative to siCTRL 50 nM was determined using a MTS-based assay. n = 2–4 B. Cell viability results of MPA treated cells. Cells were allowed to attach for 48 h prior to treatment with indicated doses of MPA for 72 h. Cell viability relative to vehicle control was determined using a MTS-based assay. n = 2–4 C. Cell viability results of siRNA and Abiraterone/MDV3100 treated cells. Cells were transfected with 25 nM IMPDH2 #1, 50 nM IMPDH2 #2 siRNA or equal amounts of siCTRL for 48 h. Following treatment with the indicated drugs for another 72 h, viability relative to DMSO and siCTRL was assessed using a MTS-based assay. Doses for LNCaP were 1 μM Abiraterone and 1 μM MDV3100, and for VCaP 1 μM Abiraterone and 100 nM MDV3100 n = 1–4 D. Cell viability results of MPA and Abiraterone/MDV3100 treated cells. Cells were allowed to attach for 48 h prior to treatment with indicated drug combinations for 72 h. Cell viability relative to vehicle control was determined using a MTS-based assay. Doses for LNCaP were 10 μM MPA, 1 μM Abiraterone and 1 μM MDV3100, and for VCaP 5 μM MPA, 1 μM Abiraterone and 100 nM MDV3100 n = 3–4 E. Cell viability results of MPA treated parental and MDV3100-resistant LNCaP. Cells were allowed to attach for 48 h prior to treatment with the indicated doses of MPA for 72 h. Cell viability relative to vehicle control was determined using a MTS-based assay. n = 4 F. Cell viability results of siRNA and MPA treated cells. Cells were transfected with 50 nM MYC or control siRNA for 48 h. Following treatment with the indicated doses of MPA (LNCaP 5 and 10 μM, VCaP 5 and 10 μM and LNCaP abl 1 and 10 μM) for another 72 h, viability was assessed using a MTS-based assay and normalized to the respective DMSO control. n = 3.

Mentions: Next, to determine whether PAICS or IMPDH2 are essential for PCa cell proliferation, we applied siRNA-mediated knockdown and assessed the proliferation of LNCaP, VCaP and an derivative of LNCaP that has acquired resistance to androgen deprivation, LNCaP-abl [29] using a colourimetric-based assay (MTS). Whilst depleting these cells of PAICS had only a modest effect on proliferation, knockdown of IMPDH2 using two different siRNAs significantly (p < 0.05) reduced the number of viable cells in all three lines by about 15–20% (Figure 3A, knockdown efficacy was assessed in Figure S5A). We also included siRNA against IMPDH1, the other IMPDH isoform, and observed cell-line specific differences; LNCaP and LNCaP-abl were unaffected by IMPDH1 knockdown whilst VCaP exhibited effects comparable to IMPDH2 knockdown (Figure 3A). Intriguingly, a selective and uncompetitive inhibitor for IMPDH1 and 2, Mycophenolic acid (MPA) is a clinically approved immunosuppressant, commonly used to prevent organ transplant rejection [30]. MPA induced a dose-dependent inhibition of proliferation in all three lines (Figure 3B) and caused predominantly cytostatic effects as determined using a fluorescence-based assay to measure caspase cleavage in cultured cells over time (Figure S5C). For further experiments, we chose doses that reduced cell proliferation by about 20–30% (LNCaP 10 μM and VCaP 5 μM).


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)

Inhibition of IMPDH2 impairs the proliferation of prostate cancer cells and shows additive effects with established anti-androgensA. Cell viability results of siRNA treated cells. Cells were transfected with 25 nM (control 25 nM, IMPDH1 and IMPDH2 #1) or 50 nM (control 50 nM, PAICS, IMPDH2 #2 and MYC) for 72 h and cell viability relative to siCTRL 50 nM was determined using a MTS-based assay. n = 2–4 B. Cell viability results of MPA treated cells. Cells were allowed to attach for 48 h prior to treatment with indicated doses of MPA for 72 h. Cell viability relative to vehicle control was determined using a MTS-based assay. n = 2–4 C. Cell viability results of siRNA and Abiraterone/MDV3100 treated cells. Cells were transfected with 25 nM IMPDH2 #1, 50 nM IMPDH2 #2 siRNA or equal amounts of siCTRL for 48 h. Following treatment with the indicated drugs for another 72 h, viability relative to DMSO and siCTRL was assessed using a MTS-based assay. Doses for LNCaP were 1 μM Abiraterone and 1 μM MDV3100, and for VCaP 1 μM Abiraterone and 100 nM MDV3100 n = 1–4 D. Cell viability results of MPA and Abiraterone/MDV3100 treated cells. Cells were allowed to attach for 48 h prior to treatment with indicated drug combinations for 72 h. Cell viability relative to vehicle control was determined using a MTS-based assay. Doses for LNCaP were 10 μM MPA, 1 μM Abiraterone and 1 μM MDV3100, and for VCaP 5 μM MPA, 1 μM Abiraterone and 100 nM MDV3100 n = 3–4 E. Cell viability results of MPA treated parental and MDV3100-resistant LNCaP. Cells were allowed to attach for 48 h prior to treatment with the indicated doses of MPA for 72 h. Cell viability relative to vehicle control was determined using a MTS-based assay. n = 4 F. Cell viability results of siRNA and MPA treated cells. Cells were transfected with 50 nM MYC or control siRNA for 48 h. Following treatment with the indicated doses of MPA (LNCaP 5 and 10 μM, VCaP 5 and 10 μM and LNCaP abl 1 and 10 μM) for another 72 h, viability was assessed using a MTS-based assay and normalized to the respective DMSO control. n = 3.
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Related In: Results  -  Collection

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Figure 3: Inhibition of IMPDH2 impairs the proliferation of prostate cancer cells and shows additive effects with established anti-androgensA. Cell viability results of siRNA treated cells. Cells were transfected with 25 nM (control 25 nM, IMPDH1 and IMPDH2 #1) or 50 nM (control 50 nM, PAICS, IMPDH2 #2 and MYC) for 72 h and cell viability relative to siCTRL 50 nM was determined using a MTS-based assay. n = 2–4 B. Cell viability results of MPA treated cells. Cells were allowed to attach for 48 h prior to treatment with indicated doses of MPA for 72 h. Cell viability relative to vehicle control was determined using a MTS-based assay. n = 2–4 C. Cell viability results of siRNA and Abiraterone/MDV3100 treated cells. Cells were transfected with 25 nM IMPDH2 #1, 50 nM IMPDH2 #2 siRNA or equal amounts of siCTRL for 48 h. Following treatment with the indicated drugs for another 72 h, viability relative to DMSO and siCTRL was assessed using a MTS-based assay. Doses for LNCaP were 1 μM Abiraterone and 1 μM MDV3100, and for VCaP 1 μM Abiraterone and 100 nM MDV3100 n = 1–4 D. Cell viability results of MPA and Abiraterone/MDV3100 treated cells. Cells were allowed to attach for 48 h prior to treatment with indicated drug combinations for 72 h. Cell viability relative to vehicle control was determined using a MTS-based assay. Doses for LNCaP were 10 μM MPA, 1 μM Abiraterone and 1 μM MDV3100, and for VCaP 5 μM MPA, 1 μM Abiraterone and 100 nM MDV3100 n = 3–4 E. Cell viability results of MPA treated parental and MDV3100-resistant LNCaP. Cells were allowed to attach for 48 h prior to treatment with the indicated doses of MPA for 72 h. Cell viability relative to vehicle control was determined using a MTS-based assay. n = 4 F. Cell viability results of siRNA and MPA treated cells. Cells were transfected with 50 nM MYC or control siRNA for 48 h. Following treatment with the indicated doses of MPA (LNCaP 5 and 10 μM, VCaP 5 and 10 μM and LNCaP abl 1 and 10 μM) for another 72 h, viability was assessed using a MTS-based assay and normalized to the respective DMSO control. n = 3.
Mentions: Next, to determine whether PAICS or IMPDH2 are essential for PCa cell proliferation, we applied siRNA-mediated knockdown and assessed the proliferation of LNCaP, VCaP and an derivative of LNCaP that has acquired resistance to androgen deprivation, LNCaP-abl [29] using a colourimetric-based assay (MTS). Whilst depleting these cells of PAICS had only a modest effect on proliferation, knockdown of IMPDH2 using two different siRNAs significantly (p < 0.05) reduced the number of viable cells in all three lines by about 15–20% (Figure 3A, knockdown efficacy was assessed in Figure S5A). We also included siRNA against IMPDH1, the other IMPDH isoform, and observed cell-line specific differences; LNCaP and LNCaP-abl were unaffected by IMPDH1 knockdown whilst VCaP exhibited effects comparable to IMPDH2 knockdown (Figure 3A). Intriguingly, a selective and uncompetitive inhibitor for IMPDH1 and 2, Mycophenolic acid (MPA) is a clinically approved immunosuppressant, commonly used to prevent organ transplant rejection [30]. MPA induced a dose-dependent inhibition of proliferation in all three lines (Figure 3B) and caused predominantly cytostatic effects as determined using a fluorescence-based assay to measure caspase cleavage in cultured cells over time (Figure S5C). For further experiments, we chose doses that reduced cell proliferation by about 20–30% (LNCaP 10 μM and VCaP 5 μM).

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