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Loss of Scribble Promotes Snail Translation through Translocation of HuR and Enhances Cancer Drug Resistance.

Zhou Y, Chang R, Ji W, Wang N, Qi M, Xu Y, Guo J, Zhan L - J. Biol. Chem. (2015)

Bottom Line: Furthermore, we demonstrate HuR can recognize AU-rich elements of the Snail-encoding mRNA, thereby regulating Snail translation.Moreover, loss of Scribble-induced HuR translocation mediates the accumulation of Snail via activation of the p38 MAPK pathway.Thus, this work clarifies the role of polarity protein Scribble, which is directly implicated in the regulation of developmental transcription factor Snail, and suggesting a mechanism for Scribble mediating cancer drug resistance.

View Article: PubMed Central - PubMed

Affiliation: From the Key Laboratory of Nutrition and Metabolism, Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.

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Scribble regulation of drug resistance is mediated by Snail.A, phase images of day 12 control and Scribble KD cells in the CRL-1848 and HeLa cell lines in three-dimensional cell culture (×200 magnified). B, EMT markers were examined in Scribble KD cells or control cells in the CRL-1848 cell line by Western blot. C, EMT transcription factors Snail, Slug, and Twist mRNA levels were examined in CRL-1848 Scribble KD cells and control cells by RT-PCR. D, two different siRNAs were used to inhibit Scribble expression in the CRL-1848 cell line. The accumulation of EMT transcription factors Snail, Slug, and Twist were examined by Western blotting. β-Actin was used as a loading control. E, the protein levels of Scribble, Snail, and Hsp90 (loading control) were examined by Western blotting in three different Scribble KD cancer cell lines (A549, CRL-5803, and HeLa). F, apoptosis was measured in cisplatin-treated (15 μg/ml, 24 h) cells using the annexin V apoptosis assay. Cells were transfected with constructs for control; Scribble KD; control + siSnail; or Scribble KD + siSnail.
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Figure 2: Scribble regulation of drug resistance is mediated by Snail.A, phase images of day 12 control and Scribble KD cells in the CRL-1848 and HeLa cell lines in three-dimensional cell culture (×200 magnified). B, EMT markers were examined in Scribble KD cells or control cells in the CRL-1848 cell line by Western blot. C, EMT transcription factors Snail, Slug, and Twist mRNA levels were examined in CRL-1848 Scribble KD cells and control cells by RT-PCR. D, two different siRNAs were used to inhibit Scribble expression in the CRL-1848 cell line. The accumulation of EMT transcription factors Snail, Slug, and Twist were examined by Western blotting. β-Actin was used as a loading control. E, the protein levels of Scribble, Snail, and Hsp90 (loading control) were examined by Western blotting in three different Scribble KD cancer cell lines (A549, CRL-5803, and HeLa). F, apoptosis was measured in cisplatin-treated (15 μg/ml, 24 h) cells using the annexin V apoptosis assay. Cells were transfected with constructs for control; Scribble KD; control + siSnail; or Scribble KD + siSnail.

Mentions: To investigate the cellular effects of Scribble in epithelial cells, we established cell lines stably expressing Scribble shRNA and developed a three-dimensional culture model (22). Surprisingly, when propagated in this three-dimensional culture system, Scribble KD cells exhibited an enhanced ability to invade through Matrigel-impregnated membranes, and disrupted three-dimensional acinar structures generated in a non-small lung cancer cell (NSLCC) line (CRL-1848) and HeLa cells (Fig. 2A). However, Scribble KD lines did not exhibit apparent morphological differences compared with control cells when grown in two-dimensional cultures (data not shown). Despite an apparent lack of morphological effect, Scribble KD lines showed changes in E-cadherin and N-cadherin protein levels in a two-dimensional culture (Fig. 2B), with Scribble KD lines exhibiting a mesenchymal phenotype for production of these two cadherins production, a signature that serves as a marker of EMT fate.


Loss of Scribble Promotes Snail Translation through Translocation of HuR and Enhances Cancer Drug Resistance.

Zhou Y, Chang R, Ji W, Wang N, Qi M, Xu Y, Guo J, Zhan L - J. Biol. Chem. (2015)

Scribble regulation of drug resistance is mediated by Snail.A, phase images of day 12 control and Scribble KD cells in the CRL-1848 and HeLa cell lines in three-dimensional cell culture (×200 magnified). B, EMT markers were examined in Scribble KD cells or control cells in the CRL-1848 cell line by Western blot. C, EMT transcription factors Snail, Slug, and Twist mRNA levels were examined in CRL-1848 Scribble KD cells and control cells by RT-PCR. D, two different siRNAs were used to inhibit Scribble expression in the CRL-1848 cell line. The accumulation of EMT transcription factors Snail, Slug, and Twist were examined by Western blotting. β-Actin was used as a loading control. E, the protein levels of Scribble, Snail, and Hsp90 (loading control) were examined by Western blotting in three different Scribble KD cancer cell lines (A549, CRL-5803, and HeLa). F, apoptosis was measured in cisplatin-treated (15 μg/ml, 24 h) cells using the annexin V apoptosis assay. Cells were transfected with constructs for control; Scribble KD; control + siSnail; or Scribble KD + siSnail.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697165&req=5

Figure 2: Scribble regulation of drug resistance is mediated by Snail.A, phase images of day 12 control and Scribble KD cells in the CRL-1848 and HeLa cell lines in three-dimensional cell culture (×200 magnified). B, EMT markers were examined in Scribble KD cells or control cells in the CRL-1848 cell line by Western blot. C, EMT transcription factors Snail, Slug, and Twist mRNA levels were examined in CRL-1848 Scribble KD cells and control cells by RT-PCR. D, two different siRNAs were used to inhibit Scribble expression in the CRL-1848 cell line. The accumulation of EMT transcription factors Snail, Slug, and Twist were examined by Western blotting. β-Actin was used as a loading control. E, the protein levels of Scribble, Snail, and Hsp90 (loading control) were examined by Western blotting in three different Scribble KD cancer cell lines (A549, CRL-5803, and HeLa). F, apoptosis was measured in cisplatin-treated (15 μg/ml, 24 h) cells using the annexin V apoptosis assay. Cells were transfected with constructs for control; Scribble KD; control + siSnail; or Scribble KD + siSnail.
Mentions: To investigate the cellular effects of Scribble in epithelial cells, we established cell lines stably expressing Scribble shRNA and developed a three-dimensional culture model (22). Surprisingly, when propagated in this three-dimensional culture system, Scribble KD cells exhibited an enhanced ability to invade through Matrigel-impregnated membranes, and disrupted three-dimensional acinar structures generated in a non-small lung cancer cell (NSLCC) line (CRL-1848) and HeLa cells (Fig. 2A). However, Scribble KD lines did not exhibit apparent morphological differences compared with control cells when grown in two-dimensional cultures (data not shown). Despite an apparent lack of morphological effect, Scribble KD lines showed changes in E-cadherin and N-cadherin protein levels in a two-dimensional culture (Fig. 2B), with Scribble KD lines exhibiting a mesenchymal phenotype for production of these two cadherins production, a signature that serves as a marker of EMT fate.

Bottom Line: Furthermore, we demonstrate HuR can recognize AU-rich elements of the Snail-encoding mRNA, thereby regulating Snail translation.Moreover, loss of Scribble-induced HuR translocation mediates the accumulation of Snail via activation of the p38 MAPK pathway.Thus, this work clarifies the role of polarity protein Scribble, which is directly implicated in the regulation of developmental transcription factor Snail, and suggesting a mechanism for Scribble mediating cancer drug resistance.

View Article: PubMed Central - PubMed

Affiliation: From the Key Laboratory of Nutrition and Metabolism, Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.

Show MeSH
Related in: MedlinePlus