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Manipulation of prostate cancer metastasis by locus-specific modification of the CRMP4 promoter region using chimeric TALE DNA methyltransferase and demethylase.

Li K, Pang J, Cheng H, Liu WP, Di JM, Xiao HJ, Luo Y, Zhang H, Huang WT, Chen MK, Li LY, Shao CK, Feng YH, Gao X - Oncotarget (2015)

Bottom Line: Transfection of dTALEs of DNA methyltransferase or demethylase induced artificial, yet active locus-specific CpG and subsequent histone modifications.Remarkably, locus-specific CpG demethylation of the CRMP4 promoter in metastatic PC3 cells abolished metastasis, whereas locus-specific CpG methylation of the promoter in non-metastatic 22Rv1 cells induced metastasis.CRMP4-mediated metastasis suppression was found to require activation of Akt/Rac1 signaling and down-regulation of MMP-9 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510630, China.

ABSTRACT
Prostate cancer is the most commonly diagnosed non-cutaneous cancer and one of the leading causes of cancer death for North American men. Whereas localized prostate cancer can be cured, there is currently no cure for metastatic prostate cancer. Here we report a novel approach that utilizes designed chimeric transcription activator-like effectors (dTALEs) to control prostate cancer metastasis. Transfection of dTALEs of DNA methyltransferase or demethylase induced artificial, yet active locus-specific CpG and subsequent histone modifications. These manipulations markedly altered expression of endogenous CRMP4, a metastasis suppressor gene. Remarkably, locus-specific CpG demethylation of the CRMP4 promoter in metastatic PC3 cells abolished metastasis, whereas locus-specific CpG methylation of the promoter in non-metastatic 22Rv1 cells induced metastasis. CRMP4-mediated metastasis suppression was found to require activation of Akt/Rac1 signaling and down-regulation of MMP-9 expression. This proof-of-concept study with dTALEs for locus-specific epigenomic manipulation validates the selected CpG methylation of CRMP4 gene as an independent biomarker for diagnosis and prognosis of prostate cancer metastasis and opens up a novel avenue for mechanistic research on cancer biology.

No MeSH data available.


Related in: MedlinePlus

Akt-Rac1-MMP9 signaling pathway in CRMP4-mediated suppression of metastasis(a) Western blot detection of phosphorylation and expression of Akt and Rac1 in the PC3 cells transfected with CRMP4-TAL-Tet1c or empty vector phCMV1 as control. CRMP4 siRNA was utilized to verify the loss-of-function. (b) Akt and CRMP4 interaction detected in the PC3 cells with differential Co-IP and Western blot. (c) Rac1 and CRMP4 interaction detected in the PC3 cells with differential Co-IP and Western blot. (d) Subcellular co-localization of CRMP4 with Akt and Rac1, respectively, in the CRMP4-TAL-Tet1c-expressing PC3 cells detected using confocal images. (e) Rac1 GTPase activity detected in the CRMP4-TAL-Tet1c-expressing PC3 cells using a Pull-down assay. (f) MMP-9 activity detected in the CRMP4-TAL-Tet1c-expressing PC3 cells using a Gelatin zymography assay. (g) IHC detection of opposite expression of CRMP4 and MMP-9 in the primary tumors from mice injected with PC3 or 22Rv1 cells expressing specified dTALEs. (h) Schematic of CRMP4-mediated signaling pathway involving phosphorylation of Akt and Rac1, activity of Rac1 GTPase and MMP-9, and repressed expression of MMP-9, VEGFB and VEGFC15, collectively leading to suppression of prostate cancer metastasis (Solid-lines: findings of this manuscript. Dashed-lines: previously published data and data not shown). The P values in e and f were determined with the Student's t-test. The error bars in e and f are s.e.m.
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Figure 5: Akt-Rac1-MMP9 signaling pathway in CRMP4-mediated suppression of metastasis(a) Western blot detection of phosphorylation and expression of Akt and Rac1 in the PC3 cells transfected with CRMP4-TAL-Tet1c or empty vector phCMV1 as control. CRMP4 siRNA was utilized to verify the loss-of-function. (b) Akt and CRMP4 interaction detected in the PC3 cells with differential Co-IP and Western blot. (c) Rac1 and CRMP4 interaction detected in the PC3 cells with differential Co-IP and Western blot. (d) Subcellular co-localization of CRMP4 with Akt and Rac1, respectively, in the CRMP4-TAL-Tet1c-expressing PC3 cells detected using confocal images. (e) Rac1 GTPase activity detected in the CRMP4-TAL-Tet1c-expressing PC3 cells using a Pull-down assay. (f) MMP-9 activity detected in the CRMP4-TAL-Tet1c-expressing PC3 cells using a Gelatin zymography assay. (g) IHC detection of opposite expression of CRMP4 and MMP-9 in the primary tumors from mice injected with PC3 or 22Rv1 cells expressing specified dTALEs. (h) Schematic of CRMP4-mediated signaling pathway involving phosphorylation of Akt and Rac1, activity of Rac1 GTPase and MMP-9, and repressed expression of MMP-9, VEGFB and VEGFC15, collectively leading to suppression of prostate cancer metastasis (Solid-lines: findings of this manuscript. Dashed-lines: previously published data and data not shown). The P values in e and f were determined with the Student's t-test. The error bars in e and f are s.e.m.

Mentions: The mechanisms underlying CRMP4-mediated metastasis suppression remain largely unknown. Samples prepared from PC3 cells expressing CRMP4-TAL-Tet1c were arrayed on 1318 tumor-associated protein targets with their site-specific phospho-antibodies. The antibody microarray revealed that CRMP4-TAL-Tet1c significantly activated the phosphorylation status for a variety of targeted signaling proteins (Supplementary S7a and Supplementary Table S1), in particular Akt and Rac1. Interestingly, Akt and Rac1 expressions were not altered as shown in qRT-PCR (Supplementary Figure S7b) and Western blot assays (Figure 5a). Consistently, Figure 5a shows that CRMP4-TAL-Tet1c did significantly elevate the phosphorylation of Akt Ser473 and Rac1 Ser71. Such elevation was subsequently blocked by siRNA that repressed the CRMP4 expression (Figure 5a).


Manipulation of prostate cancer metastasis by locus-specific modification of the CRMP4 promoter region using chimeric TALE DNA methyltransferase and demethylase.

Li K, Pang J, Cheng H, Liu WP, Di JM, Xiao HJ, Luo Y, Zhang H, Huang WT, Chen MK, Li LY, Shao CK, Feng YH, Gao X - Oncotarget (2015)

Akt-Rac1-MMP9 signaling pathway in CRMP4-mediated suppression of metastasis(a) Western blot detection of phosphorylation and expression of Akt and Rac1 in the PC3 cells transfected with CRMP4-TAL-Tet1c or empty vector phCMV1 as control. CRMP4 siRNA was utilized to verify the loss-of-function. (b) Akt and CRMP4 interaction detected in the PC3 cells with differential Co-IP and Western blot. (c) Rac1 and CRMP4 interaction detected in the PC3 cells with differential Co-IP and Western blot. (d) Subcellular co-localization of CRMP4 with Akt and Rac1, respectively, in the CRMP4-TAL-Tet1c-expressing PC3 cells detected using confocal images. (e) Rac1 GTPase activity detected in the CRMP4-TAL-Tet1c-expressing PC3 cells using a Pull-down assay. (f) MMP-9 activity detected in the CRMP4-TAL-Tet1c-expressing PC3 cells using a Gelatin zymography assay. (g) IHC detection of opposite expression of CRMP4 and MMP-9 in the primary tumors from mice injected with PC3 or 22Rv1 cells expressing specified dTALEs. (h) Schematic of CRMP4-mediated signaling pathway involving phosphorylation of Akt and Rac1, activity of Rac1 GTPase and MMP-9, and repressed expression of MMP-9, VEGFB and VEGFC15, collectively leading to suppression of prostate cancer metastasis (Solid-lines: findings of this manuscript. Dashed-lines: previously published data and data not shown). The P values in e and f were determined with the Student's t-test. The error bars in e and f are s.e.m.
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Figure 5: Akt-Rac1-MMP9 signaling pathway in CRMP4-mediated suppression of metastasis(a) Western blot detection of phosphorylation and expression of Akt and Rac1 in the PC3 cells transfected with CRMP4-TAL-Tet1c or empty vector phCMV1 as control. CRMP4 siRNA was utilized to verify the loss-of-function. (b) Akt and CRMP4 interaction detected in the PC3 cells with differential Co-IP and Western blot. (c) Rac1 and CRMP4 interaction detected in the PC3 cells with differential Co-IP and Western blot. (d) Subcellular co-localization of CRMP4 with Akt and Rac1, respectively, in the CRMP4-TAL-Tet1c-expressing PC3 cells detected using confocal images. (e) Rac1 GTPase activity detected in the CRMP4-TAL-Tet1c-expressing PC3 cells using a Pull-down assay. (f) MMP-9 activity detected in the CRMP4-TAL-Tet1c-expressing PC3 cells using a Gelatin zymography assay. (g) IHC detection of opposite expression of CRMP4 and MMP-9 in the primary tumors from mice injected with PC3 or 22Rv1 cells expressing specified dTALEs. (h) Schematic of CRMP4-mediated signaling pathway involving phosphorylation of Akt and Rac1, activity of Rac1 GTPase and MMP-9, and repressed expression of MMP-9, VEGFB and VEGFC15, collectively leading to suppression of prostate cancer metastasis (Solid-lines: findings of this manuscript. Dashed-lines: previously published data and data not shown). The P values in e and f were determined with the Student's t-test. The error bars in e and f are s.e.m.
Mentions: The mechanisms underlying CRMP4-mediated metastasis suppression remain largely unknown. Samples prepared from PC3 cells expressing CRMP4-TAL-Tet1c were arrayed on 1318 tumor-associated protein targets with their site-specific phospho-antibodies. The antibody microarray revealed that CRMP4-TAL-Tet1c significantly activated the phosphorylation status for a variety of targeted signaling proteins (Supplementary S7a and Supplementary Table S1), in particular Akt and Rac1. Interestingly, Akt and Rac1 expressions were not altered as shown in qRT-PCR (Supplementary Figure S7b) and Western blot assays (Figure 5a). Consistently, Figure 5a shows that CRMP4-TAL-Tet1c did significantly elevate the phosphorylation of Akt Ser473 and Rac1 Ser71. Such elevation was subsequently blocked by siRNA that repressed the CRMP4 expression (Figure 5a).

Bottom Line: Transfection of dTALEs of DNA methyltransferase or demethylase induced artificial, yet active locus-specific CpG and subsequent histone modifications.Remarkably, locus-specific CpG demethylation of the CRMP4 promoter in metastatic PC3 cells abolished metastasis, whereas locus-specific CpG methylation of the promoter in non-metastatic 22Rv1 cells induced metastasis.CRMP4-mediated metastasis suppression was found to require activation of Akt/Rac1 signaling and down-regulation of MMP-9 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510630, China.

ABSTRACT
Prostate cancer is the most commonly diagnosed non-cutaneous cancer and one of the leading causes of cancer death for North American men. Whereas localized prostate cancer can be cured, there is currently no cure for metastatic prostate cancer. Here we report a novel approach that utilizes designed chimeric transcription activator-like effectors (dTALEs) to control prostate cancer metastasis. Transfection of dTALEs of DNA methyltransferase or demethylase induced artificial, yet active locus-specific CpG and subsequent histone modifications. These manipulations markedly altered expression of endogenous CRMP4, a metastasis suppressor gene. Remarkably, locus-specific CpG demethylation of the CRMP4 promoter in metastatic PC3 cells abolished metastasis, whereas locus-specific CpG methylation of the promoter in non-metastatic 22Rv1 cells induced metastasis. CRMP4-mediated metastasis suppression was found to require activation of Akt/Rac1 signaling and down-regulation of MMP-9 expression. This proof-of-concept study with dTALEs for locus-specific epigenomic manipulation validates the selected CpG methylation of CRMP4 gene as an independent biomarker for diagnosis and prognosis of prostate cancer metastasis and opens up a novel avenue for mechanistic research on cancer biology.

No MeSH data available.


Related in: MedlinePlus