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miR-93-directed downregulation of DAB2 defines a novel oncogenic pathway in lung cancer.

Du L, Zhao Z, Ma X, Hsiao TH, Chen Y, Young E, Suraokar M, Wistuba I, Minna JD, Pertsemlidis A - Oncogene (2013)

Bottom Line: The disabled homolog 2 (DAB2) gene was recently identified as a tumor suppressor gene with its expression downregulated in multiple cancer types.Here we show that low DAB2 levels in lung tumor specimens are significantly correlated with poor patient survival, and that DAB2 overexpression significantly inhibits cell growth in cultured lung cancer cells, indicating its potent tumor suppressor function.We next identify that microRNA miR-93 functions as a potent repressor of DAB2 expression by directly targeting the 3'UTR of the DAB2 mRNA.

View Article: PubMed Central - PubMed

Affiliation: 1] Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio, San Antonio, TX, USA [2] Department of Cellular and Structural Biology, UT Health Science Center at San Antonio, San Antonio, TX, USA.

ABSTRACT
The disabled homolog 2 (DAB2) gene was recently identified as a tumor suppressor gene with its expression downregulated in multiple cancer types. The role of DAB2 in lung tumorigenesis, however, is not fully characterized, and the mechanisms of DAB2 dysregulation in lung cancer are not defined. Here we show that low DAB2 levels in lung tumor specimens are significantly correlated with poor patient survival, and that DAB2 overexpression significantly inhibits cell growth in cultured lung cancer cells, indicating its potent tumor suppressor function. We next identify that microRNA miR-93 functions as a potent repressor of DAB2 expression by directly targeting the 3'UTR of the DAB2 mRNA. Using in vitro and in vivo approaches, we demonstrate that miR-93 overexpression has an important role in promoting lung cancer cell growth, and that its oncogenic function is primarily mediated by downregulating DAB2 expression. Our clinical investigations further indicate that high tumor levels of miR-93 are correlated with poor survival of lung cancer patients. The correlations of both low DAB2 and high miR-93 expression levels with poor patient survival strongly support the critical role of the miR-93/DAB2 pathway in determining lung cancer progression.

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miR-93 over-expression abrogates the inhibitory effect of DAB2-3′UTR on AKT phosphorylation and on cell proliferationA, H1993 cells were transiently transfected with equal amounts of either control pcDNA3.1 vectors, pcDNA3.1-DAB2-3′UTR, pCMV6-miR-93, pcDNA3.1-DAB2-3′UTR/pCMV6-miR-93, or pcDNA3.1-DAB2/pCMV6-miR-93 as indicated. Cell lysates were harvested after 48 hrs. Protein expression levels of Dab2, total Akt, and pAkt were measured by western blot, with β-actin as a loading control. B, H1993 cells were transfected as above. After 48 hrs, cell proliferation was measured by BrdU incorporation assay. C–D, Effect of miR-93 and DAB2 over-expression on Dab2 and Akt protein expression (C) and on cell proliferation (D) in SCLC cell line H524, as above. E, Effect of Akt kinase inhibitor and DAB2 over-expression on Dab2 and Akt protein expression. F, Effect of Akt kinase inhibitor and DAB2 over-expression on cell proliferation in H1993 cells. G–H, Effect of DAB2 over-expression (F) or knockdown (G) on cell viability in a panel of lung cancer cell lines. I–J, Effect of miR-93 over-expression (I) or knockdown (J) on cell viability in a panel lung cancer cell lines. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
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Figure 6: miR-93 over-expression abrogates the inhibitory effect of DAB2-3′UTR on AKT phosphorylation and on cell proliferationA, H1993 cells were transiently transfected with equal amounts of either control pcDNA3.1 vectors, pcDNA3.1-DAB2-3′UTR, pCMV6-miR-93, pcDNA3.1-DAB2-3′UTR/pCMV6-miR-93, or pcDNA3.1-DAB2/pCMV6-miR-93 as indicated. Cell lysates were harvested after 48 hrs. Protein expression levels of Dab2, total Akt, and pAkt were measured by western blot, with β-actin as a loading control. B, H1993 cells were transfected as above. After 48 hrs, cell proliferation was measured by BrdU incorporation assay. C–D, Effect of miR-93 and DAB2 over-expression on Dab2 and Akt protein expression (C) and on cell proliferation (D) in SCLC cell line H524, as above. E, Effect of Akt kinase inhibitor and DAB2 over-expression on Dab2 and Akt protein expression. F, Effect of Akt kinase inhibitor and DAB2 over-expression on cell proliferation in H1993 cells. G–H, Effect of DAB2 over-expression (F) or knockdown (G) on cell viability in a panel of lung cancer cell lines. I–J, Effect of miR-93 over-expression (I) or knockdown (J) on cell viability in a panel lung cancer cell lines. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Mentions: We next investigated whether miR-93 directly regulates DAB2 expression. As shown in Figure 5A, miR-93 is predicted to interact with a specific sequence in the 3′UTR of DAB2. We confirmed this interaction by luciferase reporter assay (Figure 5B). We further examined whether miR-93 down-regulates Dab2 protein expression and inhibits its function. Zhoul, et al. showed that Dab2 directly binds and inhibits c-Src phosphorylation in prostate cancer, which subsequently leads to inhibition of Akt phosphorylation and cell proliferation (27), We therefore examined the biological function of the miR-93/DAB2 interaction by assessing the effect of miR-93 over-expression on Dab2-mediated inhibition of Akt phosphorylation and cell proliferation. As shown in Figure 6A–B, DAB2 over-expression by means of a DAB2-3′UTR expression construct dramatically decreases the levels of phosphorylated Akt (pAkt) and significantly inhibits cell proliferation. miR-93 over-expression increases pAkt levels, and importantly, rescues the inhibitory effect of DAB2-3′UTR on Akt phosphorylation and on cell proliferation, but does not dramatically rescue the inhibitory effects of the DAB2 construct with no 3′UTR. These results indicate that miR-93 functions as a repressor of DAB2 expression by directly targeting the 3′UTR of DAB2, and suggest that DAB2 is the major target of miR-93 that mediates its growth-promoting function. Similar results were obtained with the SCLC cell line H524 (Figure 6C–D). Figure 6E–F shows that inhibiting Akt phosphorylation with an Akt1/2 kinase inhibitor (Sigma) significantly decreases cell viability in control H1993 cells, and that combination of DAB2 over-expression and Akt inhibitor show an additive effect on inhibiting Akt phosphorylation and cell growth. These results further support an important role for Akt phosphorylation in promoting H1993 proliferation and demonstrate that the growth-inhibiting function of Dab2 is mediated by its inhibition of Akt phosphorylation.


miR-93-directed downregulation of DAB2 defines a novel oncogenic pathway in lung cancer.

Du L, Zhao Z, Ma X, Hsiao TH, Chen Y, Young E, Suraokar M, Wistuba I, Minna JD, Pertsemlidis A - Oncogene (2013)

miR-93 over-expression abrogates the inhibitory effect of DAB2-3′UTR on AKT phosphorylation and on cell proliferationA, H1993 cells were transiently transfected with equal amounts of either control pcDNA3.1 vectors, pcDNA3.1-DAB2-3′UTR, pCMV6-miR-93, pcDNA3.1-DAB2-3′UTR/pCMV6-miR-93, or pcDNA3.1-DAB2/pCMV6-miR-93 as indicated. Cell lysates were harvested after 48 hrs. Protein expression levels of Dab2, total Akt, and pAkt were measured by western blot, with β-actin as a loading control. B, H1993 cells were transfected as above. After 48 hrs, cell proliferation was measured by BrdU incorporation assay. C–D, Effect of miR-93 and DAB2 over-expression on Dab2 and Akt protein expression (C) and on cell proliferation (D) in SCLC cell line H524, as above. E, Effect of Akt kinase inhibitor and DAB2 over-expression on Dab2 and Akt protein expression. F, Effect of Akt kinase inhibitor and DAB2 over-expression on cell proliferation in H1993 cells. G–H, Effect of DAB2 over-expression (F) or knockdown (G) on cell viability in a panel of lung cancer cell lines. I–J, Effect of miR-93 over-expression (I) or knockdown (J) on cell viability in a panel lung cancer cell lines. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
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Figure 6: miR-93 over-expression abrogates the inhibitory effect of DAB2-3′UTR on AKT phosphorylation and on cell proliferationA, H1993 cells were transiently transfected with equal amounts of either control pcDNA3.1 vectors, pcDNA3.1-DAB2-3′UTR, pCMV6-miR-93, pcDNA3.1-DAB2-3′UTR/pCMV6-miR-93, or pcDNA3.1-DAB2/pCMV6-miR-93 as indicated. Cell lysates were harvested after 48 hrs. Protein expression levels of Dab2, total Akt, and pAkt were measured by western blot, with β-actin as a loading control. B, H1993 cells were transfected as above. After 48 hrs, cell proliferation was measured by BrdU incorporation assay. C–D, Effect of miR-93 and DAB2 over-expression on Dab2 and Akt protein expression (C) and on cell proliferation (D) in SCLC cell line H524, as above. E, Effect of Akt kinase inhibitor and DAB2 over-expression on Dab2 and Akt protein expression. F, Effect of Akt kinase inhibitor and DAB2 over-expression on cell proliferation in H1993 cells. G–H, Effect of DAB2 over-expression (F) or knockdown (G) on cell viability in a panel of lung cancer cell lines. I–J, Effect of miR-93 over-expression (I) or knockdown (J) on cell viability in a panel lung cancer cell lines. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
Mentions: We next investigated whether miR-93 directly regulates DAB2 expression. As shown in Figure 5A, miR-93 is predicted to interact with a specific sequence in the 3′UTR of DAB2. We confirmed this interaction by luciferase reporter assay (Figure 5B). We further examined whether miR-93 down-regulates Dab2 protein expression and inhibits its function. Zhoul, et al. showed that Dab2 directly binds and inhibits c-Src phosphorylation in prostate cancer, which subsequently leads to inhibition of Akt phosphorylation and cell proliferation (27), We therefore examined the biological function of the miR-93/DAB2 interaction by assessing the effect of miR-93 over-expression on Dab2-mediated inhibition of Akt phosphorylation and cell proliferation. As shown in Figure 6A–B, DAB2 over-expression by means of a DAB2-3′UTR expression construct dramatically decreases the levels of phosphorylated Akt (pAkt) and significantly inhibits cell proliferation. miR-93 over-expression increases pAkt levels, and importantly, rescues the inhibitory effect of DAB2-3′UTR on Akt phosphorylation and on cell proliferation, but does not dramatically rescue the inhibitory effects of the DAB2 construct with no 3′UTR. These results indicate that miR-93 functions as a repressor of DAB2 expression by directly targeting the 3′UTR of DAB2, and suggest that DAB2 is the major target of miR-93 that mediates its growth-promoting function. Similar results were obtained with the SCLC cell line H524 (Figure 6C–D). Figure 6E–F shows that inhibiting Akt phosphorylation with an Akt1/2 kinase inhibitor (Sigma) significantly decreases cell viability in control H1993 cells, and that combination of DAB2 over-expression and Akt inhibitor show an additive effect on inhibiting Akt phosphorylation and cell growth. These results further support an important role for Akt phosphorylation in promoting H1993 proliferation and demonstrate that the growth-inhibiting function of Dab2 is mediated by its inhibition of Akt phosphorylation.

Bottom Line: The disabled homolog 2 (DAB2) gene was recently identified as a tumor suppressor gene with its expression downregulated in multiple cancer types.Here we show that low DAB2 levels in lung tumor specimens are significantly correlated with poor patient survival, and that DAB2 overexpression significantly inhibits cell growth in cultured lung cancer cells, indicating its potent tumor suppressor function.We next identify that microRNA miR-93 functions as a potent repressor of DAB2 expression by directly targeting the 3'UTR of the DAB2 mRNA.

View Article: PubMed Central - PubMed

Affiliation: 1] Greehey Children's Cancer Research Institute, UT Health Science Center at San Antonio, San Antonio, TX, USA [2] Department of Cellular and Structural Biology, UT Health Science Center at San Antonio, San Antonio, TX, USA.

ABSTRACT
The disabled homolog 2 (DAB2) gene was recently identified as a tumor suppressor gene with its expression downregulated in multiple cancer types. The role of DAB2 in lung tumorigenesis, however, is not fully characterized, and the mechanisms of DAB2 dysregulation in lung cancer are not defined. Here we show that low DAB2 levels in lung tumor specimens are significantly correlated with poor patient survival, and that DAB2 overexpression significantly inhibits cell growth in cultured lung cancer cells, indicating its potent tumor suppressor function. We next identify that microRNA miR-93 functions as a potent repressor of DAB2 expression by directly targeting the 3'UTR of the DAB2 mRNA. Using in vitro and in vivo approaches, we demonstrate that miR-93 overexpression has an important role in promoting lung cancer cell growth, and that its oncogenic function is primarily mediated by downregulating DAB2 expression. Our clinical investigations further indicate that high tumor levels of miR-93 are correlated with poor survival of lung cancer patients. The correlations of both low DAB2 and high miR-93 expression levels with poor patient survival strongly support the critical role of the miR-93/DAB2 pathway in determining lung cancer progression.

Show MeSH
Related in: MedlinePlus