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YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1.

Somasekharan SP, El-Naggar A, Leprivier G, Cheng H, Hajee S, Grunewald TG, Zhang F, Ng T, Delattre O, Evdokimova V, Wang Y, Gleave M, Sorensen PH - J. Cell Biol. (2015)

Bottom Line: YB-1 inactivation in human sarcoma cells dramatically reduces G3BP1 and SG formation in vitro.Finally, G3BP1 down-regulation in sarcoma xenografts prevents in vivo SG formation and tumor invasion, and completely blocks lung metastasis in mouse models.Together, these findings demonstrate a critical role for YB-1 in SG formation through translational activation of G3BP1, and highlight novel functions for SGs in tumor progression.

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Affiliation: Department of Pathology and Laboratory Medicine and Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia V5Z 1L3, Canada.

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YB-1 regulates G3BP1 expression in vivo. (A) MNNG shControl or shYB-1 kd cells were implanted under the renal capsules of three independent pairs of NOD/SCID mice, and primary site tumors were collected 4–5 wk after implantation. Viable regions of tumors were cryosectioned and subjected to IF with anti–YB-1 (green) or anti-G3BP1 (red) antibodies, and counterstained with DRAQ5 to detect nuclei. Insets show fivefold higher magnification of indicated areas. 10 high-power fields (HPF) of representative tumor sections from each implantation site tumor (n = 3 per group) were used for quantification of SGs as in Fig. 1 B, as shown below with a bar graph. Mean values ± SD (error bars) are shown for three independent tumors. **, P < 0.01. Bars: (main panels) 10 µm; (enlarged insets) 1 µm. (B) Lysates extracted from tumor tissues from A were subjected to immunoblot analysis using the indicated antibodies.
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fig4: YB-1 regulates G3BP1 expression in vivo. (A) MNNG shControl or shYB-1 kd cells were implanted under the renal capsules of three independent pairs of NOD/SCID mice, and primary site tumors were collected 4–5 wk after implantation. Viable regions of tumors were cryosectioned and subjected to IF with anti–YB-1 (green) or anti-G3BP1 (red) antibodies, and counterstained with DRAQ5 to detect nuclei. Insets show fivefold higher magnification of indicated areas. 10 high-power fields (HPF) of representative tumor sections from each implantation site tumor (n = 3 per group) were used for quantification of SGs as in Fig. 1 B, as shown below with a bar graph. Mean values ± SD (error bars) are shown for three independent tumors. **, P < 0.01. Bars: (main panels) 10 µm; (enlarged insets) 1 µm. (B) Lysates extracted from tumor tissues from A were subjected to immunoblot analysis using the indicated antibodies.

Mentions: SG formation in vivo has previously been documented in pathological tumor and brain tissues (Moeller et al., 2004; Vanderweyde et al., 2012). Given that YB-1 regulates SG formation in cultured sarcoma cells, we next wished to determine the relevance of YB-1 for SG formation in vivo. We therefore used a murine renal subcapsular implantation model to establish human sarcoma xenografts in mice as described previously (Mendoza-Naranjo et al., 2013). MNNG osteosarcoma cell lines transfected with control or YB-1 shRNAs for stable YB-1 kd were implanted under the renal capsules of immunocompromised mice, and implantation site tumors were surgically removed after 4–5 wk. We confirmed that this model is appropriate for analysis of stress responses, as areas of hypoxia (detected by pimonidazole staining; Raleigh et al., 1999), ER stress (detected by increased expression of the ER stress marker, Bip; Boelens et al., 2007), and oxidative stress (detected by protein carbonylation; Dalle-Donne et al., 2006) could be readily detected in tumors but not in normal mouse kidney (Fig. S5, A–C). Viable regions of the excised tumors were examined by immunoblot analysis, in order to assess SG associated proteins, and by cryosectioning to detect SGs (Figs. 4 A and S5 D). This clearly demonstrated cytosolic SGs in control tumor sections, with colocalization of YB-1 and G3BP1, FMRP, and eIF3η. However, SGs were significantly reduced in MNNG tumors with stable YB-1 kd. Reduced expression of YB-1 in tumor lysates was confirmed by immunoblotting, and, importantly, YB-1 kd strongly correlated with reduced G3BP1 expression in the same lysates (Figs. 4 B and S5 E). Second, we assessed whether YB-1 and G3BP1 protein expression are correlated in human sarcoma specimens using tissue microarrays (TMAs) of 153 different human sarcoma cases (Fig. 5, A and B). Serial sections of TMAs were subjected to sequential immunohistochemistry (IHC) using antibodies to YB-1 and G3BP1. YB-1 expression strongly correlated with G3BP1 in human sarcomas (Spearman correlation of 0.4963 and a p-value = 0.0001437), which is consistent with our in vitro and in vivo results and supports a positive role for YB-1 in regulating G3BP1 expression in human sarcomas. Moreover, G3BP1 expression in sarcoma patient samples strongly correlates with survival (Fig. 5, C and D), highlighting G3BP1 as a potential prognostic marker in sarcomas. Together, these results provide compelling evidence that YB-1 is a critical regulator of G3BP1 levels and SG formation in vivo.


YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1.

Somasekharan SP, El-Naggar A, Leprivier G, Cheng H, Hajee S, Grunewald TG, Zhang F, Ng T, Delattre O, Evdokimova V, Wang Y, Gleave M, Sorensen PH - J. Cell Biol. (2015)

YB-1 regulates G3BP1 expression in vivo. (A) MNNG shControl or shYB-1 kd cells were implanted under the renal capsules of three independent pairs of NOD/SCID mice, and primary site tumors were collected 4–5 wk after implantation. Viable regions of tumors were cryosectioned and subjected to IF with anti–YB-1 (green) or anti-G3BP1 (red) antibodies, and counterstained with DRAQ5 to detect nuclei. Insets show fivefold higher magnification of indicated areas. 10 high-power fields (HPF) of representative tumor sections from each implantation site tumor (n = 3 per group) were used for quantification of SGs as in Fig. 1 B, as shown below with a bar graph. Mean values ± SD (error bars) are shown for three independent tumors. **, P < 0.01. Bars: (main panels) 10 µm; (enlarged insets) 1 µm. (B) Lysates extracted from tumor tissues from A were subjected to immunoblot analysis using the indicated antibodies.
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Related In: Results  -  Collection

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fig4: YB-1 regulates G3BP1 expression in vivo. (A) MNNG shControl or shYB-1 kd cells were implanted under the renal capsules of three independent pairs of NOD/SCID mice, and primary site tumors were collected 4–5 wk after implantation. Viable regions of tumors were cryosectioned and subjected to IF with anti–YB-1 (green) or anti-G3BP1 (red) antibodies, and counterstained with DRAQ5 to detect nuclei. Insets show fivefold higher magnification of indicated areas. 10 high-power fields (HPF) of representative tumor sections from each implantation site tumor (n = 3 per group) were used for quantification of SGs as in Fig. 1 B, as shown below with a bar graph. Mean values ± SD (error bars) are shown for three independent tumors. **, P < 0.01. Bars: (main panels) 10 µm; (enlarged insets) 1 µm. (B) Lysates extracted from tumor tissues from A were subjected to immunoblot analysis using the indicated antibodies.
Mentions: SG formation in vivo has previously been documented in pathological tumor and brain tissues (Moeller et al., 2004; Vanderweyde et al., 2012). Given that YB-1 regulates SG formation in cultured sarcoma cells, we next wished to determine the relevance of YB-1 for SG formation in vivo. We therefore used a murine renal subcapsular implantation model to establish human sarcoma xenografts in mice as described previously (Mendoza-Naranjo et al., 2013). MNNG osteosarcoma cell lines transfected with control or YB-1 shRNAs for stable YB-1 kd were implanted under the renal capsules of immunocompromised mice, and implantation site tumors were surgically removed after 4–5 wk. We confirmed that this model is appropriate for analysis of stress responses, as areas of hypoxia (detected by pimonidazole staining; Raleigh et al., 1999), ER stress (detected by increased expression of the ER stress marker, Bip; Boelens et al., 2007), and oxidative stress (detected by protein carbonylation; Dalle-Donne et al., 2006) could be readily detected in tumors but not in normal mouse kidney (Fig. S5, A–C). Viable regions of the excised tumors were examined by immunoblot analysis, in order to assess SG associated proteins, and by cryosectioning to detect SGs (Figs. 4 A and S5 D). This clearly demonstrated cytosolic SGs in control tumor sections, with colocalization of YB-1 and G3BP1, FMRP, and eIF3η. However, SGs were significantly reduced in MNNG tumors with stable YB-1 kd. Reduced expression of YB-1 in tumor lysates was confirmed by immunoblotting, and, importantly, YB-1 kd strongly correlated with reduced G3BP1 expression in the same lysates (Figs. 4 B and S5 E). Second, we assessed whether YB-1 and G3BP1 protein expression are correlated in human sarcoma specimens using tissue microarrays (TMAs) of 153 different human sarcoma cases (Fig. 5, A and B). Serial sections of TMAs were subjected to sequential immunohistochemistry (IHC) using antibodies to YB-1 and G3BP1. YB-1 expression strongly correlated with G3BP1 in human sarcomas (Spearman correlation of 0.4963 and a p-value = 0.0001437), which is consistent with our in vitro and in vivo results and supports a positive role for YB-1 in regulating G3BP1 expression in human sarcomas. Moreover, G3BP1 expression in sarcoma patient samples strongly correlates with survival (Fig. 5, C and D), highlighting G3BP1 as a potential prognostic marker in sarcomas. Together, these results provide compelling evidence that YB-1 is a critical regulator of G3BP1 levels and SG formation in vivo.

Bottom Line: YB-1 inactivation in human sarcoma cells dramatically reduces G3BP1 and SG formation in vitro.Finally, G3BP1 down-regulation in sarcoma xenografts prevents in vivo SG formation and tumor invasion, and completely blocks lung metastasis in mouse models.Together, these findings demonstrate a critical role for YB-1 in SG formation through translational activation of G3BP1, and highlight novel functions for SGs in tumor progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Laboratory Medicine and Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia V5Z 1L3, Canada.

Show MeSH
Related in: MedlinePlus