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miR-335 Targets SIAH2 and Confers Sensitivity to Anti-Cancer Drugs by Increasing the Expression of HDAC3.

Kim Y, Kim H, Park D, Jeoung D - Mol. Cells (2015)

Bottom Line: In this study, we investigated molecular mechanisms associated with regulation of HDAC3 expression.The down-regulation of SIAH2 conferred sensitivity to anti-cancer drugs.The results of the study indicated that the miR-335/SIAH2/HDAC3 axis regulates the response to anti-cancer drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chunchon 200-701, Korea.

ABSTRACT
We previously reported the role of histone deacetylase 3 (HDAC3) in response to anti-cancer drugs. The decreased expression of HDAC3 in anti-cancer drug-resistant cancer cell line is responsible for the resistance to anti-cancer drugs. In this study, we investigated molecular mechanisms associated with regulation of HDAC3 expression. MG132, an inhibitor of proteasomal degradation, induced the expression of HDAC3 in various anti-cancer drug-resistant cancer cell lines. Ubiquitination of HDAC3 was observed in various anti-cancer drug-resistant cancer cell lines. HDAC3 showed an interaction with SIAH2, an ubiquitin E3 ligase, that has increased expression in various anti-cancer drug-resistant cancer cell lines. miRNA array analysis showed the decreased expression of miR-335 in these cells. Targetscan analysis predicted the binding of miR-335 to the 3'-UTR of SIAH2. miR-335-mediated increased sensitivity to anti-cancer drugs was associated with its effect on HDAC3 and SIAH2 expression. miR-335 exerted apoptotic effects and inhibited ubiquitination of HDAC3 in anti-cancer drug-resistant cancer cell lines. miR-335 negatively regulated the invasion, migration, and growth rate of cancer cells. The mouse xenograft model showed that miR-335 negatively regulated the tumorigenic potential of cancer cells. The down-regulation of SIAH2 conferred sensitivity to anti-cancer drugs. The results of the study indicated that the miR-335/SIAH2/HDAC3 axis regulates the response to anti-cancer drugs.

No MeSH data available.


Related in: MedlinePlus

HDAC3 is subjected to ubiquitin-dependent proteasomal degradation. (A) SNU387R or Malme3MR cells were treated with various concentrations of MG132 for 6 h or 1 μM MG132 for various time intervals. SNU387R-Taxol, SNU387R-Vinblastine, Malme3MR-Taxol or WM266-4 cells were also treated with various concentration of MG132 for 6 h. (B) Malme3M cells treated with celastrol or taxol as indicated were subjected to Western blot analysis. (C) Cell lysates isolated from the indicated cancer cells were subjected to Western blot analysis. (D) The indicated cancer cells were transiently transfected with 10 nM of the indicated siRNA. At 48 h after transfection, cell lysates were isolated and subjected to Western blot analysis. Scr. denotes scrambled siRNA.
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f1-molce-38-6-562: HDAC3 is subjected to ubiquitin-dependent proteasomal degradation. (A) SNU387R or Malme3MR cells were treated with various concentrations of MG132 for 6 h or 1 μM MG132 for various time intervals. SNU387R-Taxol, SNU387R-Vinblastine, Malme3MR-Taxol or WM266-4 cells were also treated with various concentration of MG132 for 6 h. (B) Malme3M cells treated with celastrol or taxol as indicated were subjected to Western blot analysis. (C) Cell lysates isolated from the indicated cancer cells were subjected to Western blot analysis. (D) The indicated cancer cells were transiently transfected with 10 nM of the indicated siRNA. At 48 h after transfection, cell lysates were isolated and subjected to Western blot analysis. Scr. denotes scrambled siRNA.

Mentions: The expression level of HDAC3 is lower in anti-cancer drug-resistant cancer cell lines than in anti-cancer drug-sensitive cancer cell lines (Kim et al., 2014). We therefore investigated the mechanism of expression regulation of HDAC3. In most of the anti-cancer drug-resistant cancer cell lines, the expression level of HDAC3 did not show difference from anti-cancer drug-sensitive cancer cell lines (data not shown). We therefore examined the possibility of proteasomal degradation of HDAC3 in anti-cancer drug-resistant cancer cell lines. MG132, an inhibitor of proteasomal degradation, restored the expression of HDAC3 in anti-cancer drug-resistant cancer cell lines such as SNU387R, Malme3MR, SN387R-taxol, SNU387R-Vinblastine, Malme3MR-Taxol, and WM266-4 cells (Fig. 1A). This suggests that the expression level of HDAC3 may be under proteasomal regulation. Celastrol and taxol decreased the expression of HDAC3 while increasing the expression of SIAH2 in Malme3M cells (Fig. 1B). SIAH2, E3 ubiquitin ligase, induces proteasomal degradation of target proteins (Christian et al., 2011) suggestive of its regulatory role in the response to anti-cancer drugs. Because HDAC3 expression level is regulated by proteasome-dependent ubiquitination, we hypothesized that the expression of E3 ubiquitin ligases, including SIAHs are increased in cancer cell lines resistant to anti-cancer drugs. The E2 ubiquitin conjugase UBCH8 (ubiquitin conjugating enzyme [human] 8) cooperates with the E3 ubiquitin ligases SIAH1 and SIAH2 (seven in absentia homolog 1/2) to mediate the proteasomal degradation of oncoproteins (Pietschmann et al., 2012). SIAH2 mediates HDAC3 degradation, and Ski protein exerts a negative effect on SIAH2-mediated HDAC3 degradation by interaction with SIAH2 (Zhao et al., 2010). Ski is necessary for proper chromosome segregation and interacts with aurora kinase A at the centrosome (Mosquera et al., 2011). HDAC3 serves as a substrate of Src (Longworth and Laimins, 2006) and Src activates SIAH2 E3 ubiquitin ligase activity (Sarkar et al., 2012). These reports imply a role for SIAH2 in the regulation of HDAC3 expression. SIAH1 and SIAH2 showed relatively higher expression in anti-cancer drug-resistant cancer cell lines vs. anti-cancer drug-sensitive cancer cell lines (Fig. 1C). The down-regulation of SIAH2 increased the expression of HDAC3 (Fig. 1D), suggesting that SIAH2 may act as a negative regulator of HDAC3. Taken together, these results suggested that proteasomal degradation is responsible for the decreased expression of HDAC3 in anti-cancer drug-resistant cancer cells.


miR-335 Targets SIAH2 and Confers Sensitivity to Anti-Cancer Drugs by Increasing the Expression of HDAC3.

Kim Y, Kim H, Park D, Jeoung D - Mol. Cells (2015)

HDAC3 is subjected to ubiquitin-dependent proteasomal degradation. (A) SNU387R or Malme3MR cells were treated with various concentrations of MG132 for 6 h or 1 μM MG132 for various time intervals. SNU387R-Taxol, SNU387R-Vinblastine, Malme3MR-Taxol or WM266-4 cells were also treated with various concentration of MG132 for 6 h. (B) Malme3M cells treated with celastrol or taxol as indicated were subjected to Western blot analysis. (C) Cell lysates isolated from the indicated cancer cells were subjected to Western blot analysis. (D) The indicated cancer cells were transiently transfected with 10 nM of the indicated siRNA. At 48 h after transfection, cell lysates were isolated and subjected to Western blot analysis. Scr. denotes scrambled siRNA.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4469914&req=5

f1-molce-38-6-562: HDAC3 is subjected to ubiquitin-dependent proteasomal degradation. (A) SNU387R or Malme3MR cells were treated with various concentrations of MG132 for 6 h or 1 μM MG132 for various time intervals. SNU387R-Taxol, SNU387R-Vinblastine, Malme3MR-Taxol or WM266-4 cells were also treated with various concentration of MG132 for 6 h. (B) Malme3M cells treated with celastrol or taxol as indicated were subjected to Western blot analysis. (C) Cell lysates isolated from the indicated cancer cells were subjected to Western blot analysis. (D) The indicated cancer cells were transiently transfected with 10 nM of the indicated siRNA. At 48 h after transfection, cell lysates were isolated and subjected to Western blot analysis. Scr. denotes scrambled siRNA.
Mentions: The expression level of HDAC3 is lower in anti-cancer drug-resistant cancer cell lines than in anti-cancer drug-sensitive cancer cell lines (Kim et al., 2014). We therefore investigated the mechanism of expression regulation of HDAC3. In most of the anti-cancer drug-resistant cancer cell lines, the expression level of HDAC3 did not show difference from anti-cancer drug-sensitive cancer cell lines (data not shown). We therefore examined the possibility of proteasomal degradation of HDAC3 in anti-cancer drug-resistant cancer cell lines. MG132, an inhibitor of proteasomal degradation, restored the expression of HDAC3 in anti-cancer drug-resistant cancer cell lines such as SNU387R, Malme3MR, SN387R-taxol, SNU387R-Vinblastine, Malme3MR-Taxol, and WM266-4 cells (Fig. 1A). This suggests that the expression level of HDAC3 may be under proteasomal regulation. Celastrol and taxol decreased the expression of HDAC3 while increasing the expression of SIAH2 in Malme3M cells (Fig. 1B). SIAH2, E3 ubiquitin ligase, induces proteasomal degradation of target proteins (Christian et al., 2011) suggestive of its regulatory role in the response to anti-cancer drugs. Because HDAC3 expression level is regulated by proteasome-dependent ubiquitination, we hypothesized that the expression of E3 ubiquitin ligases, including SIAHs are increased in cancer cell lines resistant to anti-cancer drugs. The E2 ubiquitin conjugase UBCH8 (ubiquitin conjugating enzyme [human] 8) cooperates with the E3 ubiquitin ligases SIAH1 and SIAH2 (seven in absentia homolog 1/2) to mediate the proteasomal degradation of oncoproteins (Pietschmann et al., 2012). SIAH2 mediates HDAC3 degradation, and Ski protein exerts a negative effect on SIAH2-mediated HDAC3 degradation by interaction with SIAH2 (Zhao et al., 2010). Ski is necessary for proper chromosome segregation and interacts with aurora kinase A at the centrosome (Mosquera et al., 2011). HDAC3 serves as a substrate of Src (Longworth and Laimins, 2006) and Src activates SIAH2 E3 ubiquitin ligase activity (Sarkar et al., 2012). These reports imply a role for SIAH2 in the regulation of HDAC3 expression. SIAH1 and SIAH2 showed relatively higher expression in anti-cancer drug-resistant cancer cell lines vs. anti-cancer drug-sensitive cancer cell lines (Fig. 1C). The down-regulation of SIAH2 increased the expression of HDAC3 (Fig. 1D), suggesting that SIAH2 may act as a negative regulator of HDAC3. Taken together, these results suggested that proteasomal degradation is responsible for the decreased expression of HDAC3 in anti-cancer drug-resistant cancer cells.

Bottom Line: In this study, we investigated molecular mechanisms associated with regulation of HDAC3 expression.The down-regulation of SIAH2 conferred sensitivity to anti-cancer drugs.The results of the study indicated that the miR-335/SIAH2/HDAC3 axis regulates the response to anti-cancer drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chunchon 200-701, Korea.

ABSTRACT
We previously reported the role of histone deacetylase 3 (HDAC3) in response to anti-cancer drugs. The decreased expression of HDAC3 in anti-cancer drug-resistant cancer cell line is responsible for the resistance to anti-cancer drugs. In this study, we investigated molecular mechanisms associated with regulation of HDAC3 expression. MG132, an inhibitor of proteasomal degradation, induced the expression of HDAC3 in various anti-cancer drug-resistant cancer cell lines. Ubiquitination of HDAC3 was observed in various anti-cancer drug-resistant cancer cell lines. HDAC3 showed an interaction with SIAH2, an ubiquitin E3 ligase, that has increased expression in various anti-cancer drug-resistant cancer cell lines. miRNA array analysis showed the decreased expression of miR-335 in these cells. Targetscan analysis predicted the binding of miR-335 to the 3'-UTR of SIAH2. miR-335-mediated increased sensitivity to anti-cancer drugs was associated with its effect on HDAC3 and SIAH2 expression. miR-335 exerted apoptotic effects and inhibited ubiquitination of HDAC3 in anti-cancer drug-resistant cancer cell lines. miR-335 negatively regulated the invasion, migration, and growth rate of cancer cells. The mouse xenograft model showed that miR-335 negatively regulated the tumorigenic potential of cancer cells. The down-regulation of SIAH2 conferred sensitivity to anti-cancer drugs. The results of the study indicated that the miR-335/SIAH2/HDAC3 axis regulates the response to anti-cancer drugs.

No MeSH data available.


Related in: MedlinePlus