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Targeting a ribonucleoprotein complex containing the caprin-1 protein and the c-Myc mRNA suppresses tumor growth in mice: an identification of a novel oncotarget.

Qiu YQ, Yang CW, Lee YZ, Yang RB, Lee CH, Hsu HY, Chang CC, Lee SJ - Oncotarget (2015)

Bottom Line: Gene expression profiling and gain-of-c-Myc-function experiments also revealed that the downregulated c-Myc contributes to the anti-oncogenic effects of tylophorine compounds.Furthermore, the potent tylophorine derivative dibenzoquinoline-33b elicited a similar effect, as c-Myc protein levels were also decreased in xenograft tumors treated with dibenzoquinoline-33b.Thus, tylophorine compounds exert anti-cancer activity predominantly by targeting and sequestering the caprin-1 protein and c-Myc mRNA associated ribonucleoprotein complex.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan.

ABSTRACT
Tylophorine compounds have been the focus of drug development for decades. Tylophorine derivatives exhibit anti-cancer activities but their cellular targets remain unknown. We used a biotinylated tylophorine derivative to probe for the interacting cellular target(s) of tylophorine. Tylophorine directly binds to caprin-1 and consequently enhances the recruitment of G3BP1, c-Myc mRNA, and cyclin D2 mRNA to form a ribonucleoprotein complex. Subsequently, this tylophorine targeted ribonucleoprotein complex is sequestered to the polysomal fractions and the protein expressions of the associated mRNA-transcripts are repressed. Caprin-1 depleted carcinoma cells become more resistant to tylophorine, associated with decreased formation of the ribonucleoprotein complex targeted by tylophorine. Consequently, tylophorine downregulates c-Myc and cyclins D1/D2, causing hypophosphorylation of Rb and suppression of both processing-body formation and the Warburg effect. Gene expression profiling and gain-of-c-Myc-function experiments also revealed that the downregulated c-Myc contributes to the anti-oncogenic effects of tylophorine compounds. Furthermore, the potent tylophorine derivative dibenzoquinoline-33b elicited a similar effect, as c-Myc protein levels were also decreased in xenograft tumors treated with dibenzoquinoline-33b. Thus, tylophorine compounds exert anti-cancer activity predominantly by targeting and sequestering the caprin-1 protein and c-Myc mRNA associated ribonucleoprotein complex.

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The effect of tylophorine through caprin-1 on c-Myc, cyclin D1, and cyclin D2 expression in carcinoma cellsA. Semi-quantitative RT-PCR analyses of the effect of tylophorine on the mRNA levels of c-Myc, cyclin D1, and cyclin D2. The relative expression levels of each mRNA were normalized with their respective internal loading control GAPDH. B. Immunoblot analyses of the effects of tylophorine on protein expressions related to the caprin-1-associated RNP complex and their common downstream target pRb. The carcinoma cells were treated with tylophorine (2 μM) for 24 h prior to semi-quantitative RT-PCR or western blotting analyses with the indicated gene primer pairs or antibodies. C. Tylophorine repressed the de novo protein syntheses of c-Myc and cyclins D1/D2. TAMRA-labeled newly synthesized proteins from tylophorine treated HONE-1 cells were immunoprecipitated with specific antibody as indicated and then detected by western blotting with indicated antibody. D. Depletion of caprin-1 increased carcinoma cells' resistance to tylophorine or DBQ 33b treatments. HONE-1 cells were transfected with control shRNA or CAPRIN1 shRNA plasmids respectively, and then selected with puromycin. The resultant cells were analyzed by western blotting for validation of caprin-1 depletion before subjected to measurement of cell growth IC50 values by tylophorine or DBQ 33b. A 2-tailed unpaired Student's t test was used to evaluate the p-value between two groups. E & F. The effect of caprin-1 depletion on the association of tylophorine targeted RNP complex. Caprin-1 depleted HONE-1 lysates described in D were subjected to pull-down assays with biotinylated tylophorine or biotin-X-SSE. Immunoblot (E) and Semi-RT-qPCR analyses (F) were used to detect the protein and mRNA components in the tylophorine-associated RNP complex. The sequences of the gene primer pairs used are listed in Supplemental Table 3. BT, biotinylated tylophorine. The results shown are representative of 3 independent experiments. *, p<0.05.
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Figure 3: The effect of tylophorine through caprin-1 on c-Myc, cyclin D1, and cyclin D2 expression in carcinoma cellsA. Semi-quantitative RT-PCR analyses of the effect of tylophorine on the mRNA levels of c-Myc, cyclin D1, and cyclin D2. The relative expression levels of each mRNA were normalized with their respective internal loading control GAPDH. B. Immunoblot analyses of the effects of tylophorine on protein expressions related to the caprin-1-associated RNP complex and their common downstream target pRb. The carcinoma cells were treated with tylophorine (2 μM) for 24 h prior to semi-quantitative RT-PCR or western blotting analyses with the indicated gene primer pairs or antibodies. C. Tylophorine repressed the de novo protein syntheses of c-Myc and cyclins D1/D2. TAMRA-labeled newly synthesized proteins from tylophorine treated HONE-1 cells were immunoprecipitated with specific antibody as indicated and then detected by western blotting with indicated antibody. D. Depletion of caprin-1 increased carcinoma cells' resistance to tylophorine or DBQ 33b treatments. HONE-1 cells were transfected with control shRNA or CAPRIN1 shRNA plasmids respectively, and then selected with puromycin. The resultant cells were analyzed by western blotting for validation of caprin-1 depletion before subjected to measurement of cell growth IC50 values by tylophorine or DBQ 33b. A 2-tailed unpaired Student's t test was used to evaluate the p-value between two groups. E & F. The effect of caprin-1 depletion on the association of tylophorine targeted RNP complex. Caprin-1 depleted HONE-1 lysates described in D were subjected to pull-down assays with biotinylated tylophorine or biotin-X-SSE. Immunoblot (E) and Semi-RT-qPCR analyses (F) were used to detect the protein and mRNA components in the tylophorine-associated RNP complex. The sequences of the gene primer pairs used are listed in Supplemental Table 3. BT, biotinylated tylophorine. The results shown are representative of 3 independent experiments. *, p<0.05.

Mentions: Furthermore, sedimentation fractionation revealed that caprin-1, G3BP1, p-S6, c-Myc mRNA, and cyclin D2 mRNA co-localized within polysomal fractions upon tylophorine treatment (Fig. 2D-a); this colocalization did not occur in vehicle-treated HONE-1 cells (Fig. 2D-b). Moreover, a low abundance of cyclin D2 has been observed in certain carcinoma cell lines [30-32], such as MCF7 (Fig. 3A); under certain conditions, the loss of a specific cyclin can be compensated by the presence of others [33, 34]. Further investigation revealed that cyclin D1 mRNA co-localizes with the caprin-1, G3BP1, and c-Myc mRNA-associated RNP complex (Fig. 2B), as well as within polysomal fractions (Fig. 2D). These results indicate that either cyclin D1 or D2 mRNAs might be sequestered by biotinylated tylophorine (Fig. 2B & 2D) or associated with a tylophorine-targeted RNP complex in tylophorine-treated HONE-1 cells (Fig. 2D).


Targeting a ribonucleoprotein complex containing the caprin-1 protein and the c-Myc mRNA suppresses tumor growth in mice: an identification of a novel oncotarget.

Qiu YQ, Yang CW, Lee YZ, Yang RB, Lee CH, Hsu HY, Chang CC, Lee SJ - Oncotarget (2015)

The effect of tylophorine through caprin-1 on c-Myc, cyclin D1, and cyclin D2 expression in carcinoma cellsA. Semi-quantitative RT-PCR analyses of the effect of tylophorine on the mRNA levels of c-Myc, cyclin D1, and cyclin D2. The relative expression levels of each mRNA were normalized with their respective internal loading control GAPDH. B. Immunoblot analyses of the effects of tylophorine on protein expressions related to the caprin-1-associated RNP complex and their common downstream target pRb. The carcinoma cells were treated with tylophorine (2 μM) for 24 h prior to semi-quantitative RT-PCR or western blotting analyses with the indicated gene primer pairs or antibodies. C. Tylophorine repressed the de novo protein syntheses of c-Myc and cyclins D1/D2. TAMRA-labeled newly synthesized proteins from tylophorine treated HONE-1 cells were immunoprecipitated with specific antibody as indicated and then detected by western blotting with indicated antibody. D. Depletion of caprin-1 increased carcinoma cells' resistance to tylophorine or DBQ 33b treatments. HONE-1 cells were transfected with control shRNA or CAPRIN1 shRNA plasmids respectively, and then selected with puromycin. The resultant cells were analyzed by western blotting for validation of caprin-1 depletion before subjected to measurement of cell growth IC50 values by tylophorine or DBQ 33b. A 2-tailed unpaired Student's t test was used to evaluate the p-value between two groups. E & F. The effect of caprin-1 depletion on the association of tylophorine targeted RNP complex. Caprin-1 depleted HONE-1 lysates described in D were subjected to pull-down assays with biotinylated tylophorine or biotin-X-SSE. Immunoblot (E) and Semi-RT-qPCR analyses (F) were used to detect the protein and mRNA components in the tylophorine-associated RNP complex. The sequences of the gene primer pairs used are listed in Supplemental Table 3. BT, biotinylated tylophorine. The results shown are representative of 3 independent experiments. *, p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: The effect of tylophorine through caprin-1 on c-Myc, cyclin D1, and cyclin D2 expression in carcinoma cellsA. Semi-quantitative RT-PCR analyses of the effect of tylophorine on the mRNA levels of c-Myc, cyclin D1, and cyclin D2. The relative expression levels of each mRNA were normalized with their respective internal loading control GAPDH. B. Immunoblot analyses of the effects of tylophorine on protein expressions related to the caprin-1-associated RNP complex and their common downstream target pRb. The carcinoma cells were treated with tylophorine (2 μM) for 24 h prior to semi-quantitative RT-PCR or western blotting analyses with the indicated gene primer pairs or antibodies. C. Tylophorine repressed the de novo protein syntheses of c-Myc and cyclins D1/D2. TAMRA-labeled newly synthesized proteins from tylophorine treated HONE-1 cells were immunoprecipitated with specific antibody as indicated and then detected by western blotting with indicated antibody. D. Depletion of caprin-1 increased carcinoma cells' resistance to tylophorine or DBQ 33b treatments. HONE-1 cells were transfected with control shRNA or CAPRIN1 shRNA plasmids respectively, and then selected with puromycin. The resultant cells were analyzed by western blotting for validation of caprin-1 depletion before subjected to measurement of cell growth IC50 values by tylophorine or DBQ 33b. A 2-tailed unpaired Student's t test was used to evaluate the p-value between two groups. E & F. The effect of caprin-1 depletion on the association of tylophorine targeted RNP complex. Caprin-1 depleted HONE-1 lysates described in D were subjected to pull-down assays with biotinylated tylophorine or biotin-X-SSE. Immunoblot (E) and Semi-RT-qPCR analyses (F) were used to detect the protein and mRNA components in the tylophorine-associated RNP complex. The sequences of the gene primer pairs used are listed in Supplemental Table 3. BT, biotinylated tylophorine. The results shown are representative of 3 independent experiments. *, p<0.05.
Mentions: Furthermore, sedimentation fractionation revealed that caprin-1, G3BP1, p-S6, c-Myc mRNA, and cyclin D2 mRNA co-localized within polysomal fractions upon tylophorine treatment (Fig. 2D-a); this colocalization did not occur in vehicle-treated HONE-1 cells (Fig. 2D-b). Moreover, a low abundance of cyclin D2 has been observed in certain carcinoma cell lines [30-32], such as MCF7 (Fig. 3A); under certain conditions, the loss of a specific cyclin can be compensated by the presence of others [33, 34]. Further investigation revealed that cyclin D1 mRNA co-localizes with the caprin-1, G3BP1, and c-Myc mRNA-associated RNP complex (Fig. 2B), as well as within polysomal fractions (Fig. 2D). These results indicate that either cyclin D1 or D2 mRNAs might be sequestered by biotinylated tylophorine (Fig. 2B & 2D) or associated with a tylophorine-targeted RNP complex in tylophorine-treated HONE-1 cells (Fig. 2D).

Bottom Line: Gene expression profiling and gain-of-c-Myc-function experiments also revealed that the downregulated c-Myc contributes to the anti-oncogenic effects of tylophorine compounds.Furthermore, the potent tylophorine derivative dibenzoquinoline-33b elicited a similar effect, as c-Myc protein levels were also decreased in xenograft tumors treated with dibenzoquinoline-33b.Thus, tylophorine compounds exert anti-cancer activity predominantly by targeting and sequestering the caprin-1 protein and c-Myc mRNA associated ribonucleoprotein complex.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan.

ABSTRACT
Tylophorine compounds have been the focus of drug development for decades. Tylophorine derivatives exhibit anti-cancer activities but their cellular targets remain unknown. We used a biotinylated tylophorine derivative to probe for the interacting cellular target(s) of tylophorine. Tylophorine directly binds to caprin-1 and consequently enhances the recruitment of G3BP1, c-Myc mRNA, and cyclin D2 mRNA to form a ribonucleoprotein complex. Subsequently, this tylophorine targeted ribonucleoprotein complex is sequestered to the polysomal fractions and the protein expressions of the associated mRNA-transcripts are repressed. Caprin-1 depleted carcinoma cells become more resistant to tylophorine, associated with decreased formation of the ribonucleoprotein complex targeted by tylophorine. Consequently, tylophorine downregulates c-Myc and cyclins D1/D2, causing hypophosphorylation of Rb and suppression of both processing-body formation and the Warburg effect. Gene expression profiling and gain-of-c-Myc-function experiments also revealed that the downregulated c-Myc contributes to the anti-oncogenic effects of tylophorine compounds. Furthermore, the potent tylophorine derivative dibenzoquinoline-33b elicited a similar effect, as c-Myc protein levels were also decreased in xenograft tumors treated with dibenzoquinoline-33b. Thus, tylophorine compounds exert anti-cancer activity predominantly by targeting and sequestering the caprin-1 protein and c-Myc mRNA associated ribonucleoprotein complex.

Show MeSH
Related in: MedlinePlus