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GRIM-19 inhibits v-Src-induced cell motility by interfering with cytoskeletal restructuring.

Sun P, Nallar SC, Kalakonda S, Lindner DJ, Martin SS, Kalvakolanu DV - Oncogene (2009)

Bottom Line: We also show that the N terminus of GRIM-19 played a major role in this process and identified critical residues in this region.More importantly, we show that tumor-associated GRIM-19 mutations disrupted its ability to inhibit v-Src-induced cell motility.These actions appear to occur independently of STAT3, a known target of GRIM-19-mediated inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

ABSTRACT
GRIM-19 (Gene associated with Retinoid-Interferon-induced Mortality 19) is a novel tumor suppressor regulated by interferon/retinoid combination. We have recently shown that GRIM-19 inhibits v-Src-induced oncogenic transformation and metastatic behavior of cells. Oncogenic v-Src induces cell motility by cytoskeletal remodeling, especially the formation of podosomes and. Here, we show that GRIM-19 inhibited the v-Src-induced cell motility by inhibiting cytoskeletal remodeling, that is, podosome formation. We also show that the N terminus of GRIM-19 played a major role in this process and identified critical residues in this region. More importantly, we show that tumor-associated GRIM-19 mutations disrupted its ability to inhibit v-Src-induced cell motility. These actions appear to occur independently of STAT3, a known target of GRIM-19-mediated inhibition. Lastly, tumor-associated GRIM-19 mutants significantly lost their ability to control v-Src-induced metastases in vivo, indicating the biological and pathological significance of these observations.

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GRIM-19 suppressed v-Src-induced morphologic changes and cytoskeleton remodeling(A) Phase contrast photomicrographs of 3Y1 cells expressing GRIM-19, v-Src, v-Src/GRIM-19 and control vector. The graph below show the percentage of cells with rounded morphology as quantified from various fields. Magnification: 10×. Mean percentages and SE were plotted. (B) Immunofluorescent images of cells expressing, control vector GRIM-19, v-Src, and v-Src/GRIM-19 showing actin network (Red), exogenous GRIM-19 (green) and nucleus (Blue). Magnification: 100×. Scale bar = 50µm (C) Immunofluorescent images of cells expressing control vector, GRIM-19, v-Src, and v-Src/GRIM-19 showing dynamic microtubules (green), stable microtubules (red) and nucleus (blue). Magnification: 100×. Scale bar = 50µm.
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Figure 1: GRIM-19 suppressed v-Src-induced morphologic changes and cytoskeleton remodeling(A) Phase contrast photomicrographs of 3Y1 cells expressing GRIM-19, v-Src, v-Src/GRIM-19 and control vector. The graph below show the percentage of cells with rounded morphology as quantified from various fields. Magnification: 10×. Mean percentages and SE were plotted. (B) Immunofluorescent images of cells expressing, control vector GRIM-19, v-Src, and v-Src/GRIM-19 showing actin network (Red), exogenous GRIM-19 (green) and nucleus (Blue). Magnification: 100×. Scale bar = 50µm (C) Immunofluorescent images of cells expressing control vector, GRIM-19, v-Src, and v-Src/GRIM-19 showing dynamic microtubules (green), stable microtubules (red) and nucleus (blue). Magnification: 100×. Scale bar = 50µm.

Mentions: Our previous study demonstrated that GRIM-19 inhibits v-Src-induced transformation at multiple levels, including cell motility. Cells transformed by v-Src go through a series of morphologic and cytoskeletal changes, which permits loss of cell adhesion and a potential for motility. Therefore, we first studied whether GRIM-19 affected v-Src-induced morphologic change and cytoskeletal restructuring. Since most cancer cells possess multiple oncogenic changes, we used a non-oncogenic rat fibroblast line 3Y1, where introduction of a single oncogene like v-Src is sufficient to cause cellular transformation. Cells transfected with expression vector coding v-Src and control vector were used for this study. After infecting these cells with lentivirus coding for GRIM-19, cells were selected for 5 days with puromycin to eliminate uninfected cells (usually less than 5% under these conditions). In presence of v-Src around 70% of cells appeared rounded and ready to detach from the substratum compared to the controls that were well-spread in shape indicative of strong adherence (Fig. 1A). This rounded appearance of v-Src-transformed cells returned to the morphology of naïve 3Y1 cells upon expression of GRIM-19. However, expression of GRIM-19 alone did not cause any morphologic change (Fig. 1A). The expression of exogenous v-Src and GRIM-19 were confirmed by Western blot analysis (Fig. 2).


GRIM-19 inhibits v-Src-induced cell motility by interfering with cytoskeletal restructuring.

Sun P, Nallar SC, Kalakonda S, Lindner DJ, Martin SS, Kalvakolanu DV - Oncogene (2009)

GRIM-19 suppressed v-Src-induced morphologic changes and cytoskeleton remodeling(A) Phase contrast photomicrographs of 3Y1 cells expressing GRIM-19, v-Src, v-Src/GRIM-19 and control vector. The graph below show the percentage of cells with rounded morphology as quantified from various fields. Magnification: 10×. Mean percentages and SE were plotted. (B) Immunofluorescent images of cells expressing, control vector GRIM-19, v-Src, and v-Src/GRIM-19 showing actin network (Red), exogenous GRIM-19 (green) and nucleus (Blue). Magnification: 100×. Scale bar = 50µm (C) Immunofluorescent images of cells expressing control vector, GRIM-19, v-Src, and v-Src/GRIM-19 showing dynamic microtubules (green), stable microtubules (red) and nucleus (blue). Magnification: 100×. Scale bar = 50µm.
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Related In: Results  -  Collection

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Figure 1: GRIM-19 suppressed v-Src-induced morphologic changes and cytoskeleton remodeling(A) Phase contrast photomicrographs of 3Y1 cells expressing GRIM-19, v-Src, v-Src/GRIM-19 and control vector. The graph below show the percentage of cells with rounded morphology as quantified from various fields. Magnification: 10×. Mean percentages and SE were plotted. (B) Immunofluorescent images of cells expressing, control vector GRIM-19, v-Src, and v-Src/GRIM-19 showing actin network (Red), exogenous GRIM-19 (green) and nucleus (Blue). Magnification: 100×. Scale bar = 50µm (C) Immunofluorescent images of cells expressing control vector, GRIM-19, v-Src, and v-Src/GRIM-19 showing dynamic microtubules (green), stable microtubules (red) and nucleus (blue). Magnification: 100×. Scale bar = 50µm.
Mentions: Our previous study demonstrated that GRIM-19 inhibits v-Src-induced transformation at multiple levels, including cell motility. Cells transformed by v-Src go through a series of morphologic and cytoskeletal changes, which permits loss of cell adhesion and a potential for motility. Therefore, we first studied whether GRIM-19 affected v-Src-induced morphologic change and cytoskeletal restructuring. Since most cancer cells possess multiple oncogenic changes, we used a non-oncogenic rat fibroblast line 3Y1, where introduction of a single oncogene like v-Src is sufficient to cause cellular transformation. Cells transfected with expression vector coding v-Src and control vector were used for this study. After infecting these cells with lentivirus coding for GRIM-19, cells were selected for 5 days with puromycin to eliminate uninfected cells (usually less than 5% under these conditions). In presence of v-Src around 70% of cells appeared rounded and ready to detach from the substratum compared to the controls that were well-spread in shape indicative of strong adherence (Fig. 1A). This rounded appearance of v-Src-transformed cells returned to the morphology of naïve 3Y1 cells upon expression of GRIM-19. However, expression of GRIM-19 alone did not cause any morphologic change (Fig. 1A). The expression of exogenous v-Src and GRIM-19 were confirmed by Western blot analysis (Fig. 2).

Bottom Line: We also show that the N terminus of GRIM-19 played a major role in this process and identified critical residues in this region.More importantly, we show that tumor-associated GRIM-19 mutations disrupted its ability to inhibit v-Src-induced cell motility.These actions appear to occur independently of STAT3, a known target of GRIM-19-mediated inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

ABSTRACT
GRIM-19 (Gene associated with Retinoid-Interferon-induced Mortality 19) is a novel tumor suppressor regulated by interferon/retinoid combination. We have recently shown that GRIM-19 inhibits v-Src-induced oncogenic transformation and metastatic behavior of cells. Oncogenic v-Src induces cell motility by cytoskeletal remodeling, especially the formation of podosomes and. Here, we show that GRIM-19 inhibited the v-Src-induced cell motility by inhibiting cytoskeletal remodeling, that is, podosome formation. We also show that the N terminus of GRIM-19 played a major role in this process and identified critical residues in this region. More importantly, we show that tumor-associated GRIM-19 mutations disrupted its ability to inhibit v-Src-induced cell motility. These actions appear to occur independently of STAT3, a known target of GRIM-19-mediated inhibition. Lastly, tumor-associated GRIM-19 mutants significantly lost their ability to control v-Src-induced metastases in vivo, indicating the biological and pathological significance of these observations.

Show MeSH
Related in: MedlinePlus