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microRNA-21 governs TORC1 activation in renal cancer cell proliferation and invasion.

Dey N, Das F, Ghosh-Choudhury N, Mandal CC, Parekh DJ, Block K, Kasinath BS, Abboud HE, Choudhury GG - PLoS ONE (2012)

Bottom Line: Metastatic renal cancer manifests multiple signatures of gene expression.Moreover, we show that miR-21 Sponge inhibited the inactivating phosphorylation of the tumor suppressor protein tuberin and attenuated TORC1 activation.Our results uncover a layer of post-transcriptional regulation of PTEN by transcriptional activation of miR-21 to force the canonical oncogenic Akt/TORC1 signaling conduit to drive renal cancer cell proliferation and invasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

ABSTRACT
Metastatic renal cancer manifests multiple signatures of gene expression. Deviation in expression of mature miRNAs has been linked to human cancers. Importance of miR-21 in renal cell carcinomas is proposed from profiling studies using tumor tissue samples. However, the role of miR-21 function in causing renal cancer cell proliferation and invasion has not yet been shown. Using cultured renal carcinoma cells, we demonstrate enhanced expression of mature miR-21 along with pre-and pri-miR-21 by increased transcription compared to normal proximal tubular epithelial cells. Overexpression of miR-21 Sponge to quench endogenous miR-21 levels inhibited proliferation, migration and invasion of renal cancer cells. In the absence of mutation in the PTEN tumor suppressor gene, PTEN protein levels are frequently downregulated in renal cancer. We show that miR-21 targets PTEN mRNA 3'untranslated region to decrease PTEN protein expression and augments Akt phosphorylation in renal cancer cells. Downregulation of PTEN as well as overexpression of constitutively active Akt kinase prevented miR-21 Sponge-induced inhibition of renal cancer cell proliferation and migration. Moreover, we show that miR-21 Sponge inhibited the inactivating phosphorylation of the tumor suppressor protein tuberin and attenuated TORC1 activation. Finally, we demonstrate that expression of constitutively active TORC1 attenuated miR-21 Sponge-mediated suppression of proliferation and migration of renal cancer cells. Our results uncover a layer of post-transcriptional regulation of PTEN by transcriptional activation of miR-21 to force the canonical oncogenic Akt/TORC1 signaling conduit to drive renal cancer cell proliferation and invasion.

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miR-21 regulates phosphorylation of tuberin and activation of mTOR in renal cancer cells.ACHN cells were transfected with miR-21 Sponge or vector. The cell lysates were immunoblotted with phospho-tuberin (Thr-1462), tuberin (panel A), phospho-S6 kinase (Thr-389), S6 kinase (panel B), phospho-mTOR (Ser-2448) and mTOR (panel C). miR-21 uses Rheb activation for TORC1 activity. ACHN cells were cotransfected with miR-21 Sponge and CA Rheb plasmid. The cell lysates were immunoblotted with phospho-S6 kinase (Thr-389), S6 kinase (panel D), phospho-mTOR (Ser-2448) and mTOR (panel E).
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pone-0037366-g007: miR-21 regulates phosphorylation of tuberin and activation of mTOR in renal cancer cells.ACHN cells were transfected with miR-21 Sponge or vector. The cell lysates were immunoblotted with phospho-tuberin (Thr-1462), tuberin (panel A), phospho-S6 kinase (Thr-389), S6 kinase (panel B), phospho-mTOR (Ser-2448) and mTOR (panel C). miR-21 uses Rheb activation for TORC1 activity. ACHN cells were cotransfected with miR-21 Sponge and CA Rheb plasmid. The cell lysates were immunoblotted with phospho-S6 kinase (Thr-389), S6 kinase (panel D), phospho-mTOR (Ser-2448) and mTOR (panel E).

Mentions: Activated Akt phosphorylates the tumor suppressor protein tuberin at Thr-1462 leading to its inactivation, and Rheb-mediated activation of TORC1 [34], [35], [36]. We have established that downregulation of PTEN due to increased miR-21 expression in renal cancer cells activates Akt (Fig. 3), which contributes to proliferation and migration of these cells (Figs. 5 and 6). We tested the role of miR-21 in tuberin phosphorylation. ACHN cells were transfected with miR-21 Sponge. Due to increased Akt activation, ACHN cells display enhanced phosphorylation of tuberin (Fig. 7A, lane 1). Expression of miR-21 Sponge inhibited the phosphorylation of tuberin (Fig. 7A and Fig. S15A). Similar to increased tuberin phosphorylation, ACHN cells showed augmented phosphorylation of S6 kinase at Thr-389 (Fig. 7B, lane 1), measured as a surrogate for TORC1 activity. miR-21 Sponge blocked S6 kinase phosphorylation (Fig. 7B). This reduction in S6 kinase phosphorylation by miR-21 Sponge was associated with inhibition of mTOR phosphorylation at Ser-2448 (Fig. 7C). These results suggest that miR-21 regulates TORC1 activity in the renal cancer cells. To examine whether miR-21 Sponge-dependent mTOR inhibition is due to inactivation of Rheb, we cotransfected ACHN cells with miR-21 Sponge and a constitutively active Rheb expression plasmid and the results were compared with that in miR-21 Sponge-transfected cells. mTOR activation was examined in the cell lysates. Expression of constitutively active Rheb reversed the inhibitory effect of miR-21 Sponge on S6 kinase phosphorylation (Fig. 7D and Fig. S15B). Similarly, the reduced phosphorylation of mTOR by miR-21 Sponge was also prevented by constitutively active Rheb (Fig. 7E and Fig. S15C). These results conclusively demonstrate that miR-21-mediated phosphorylation/inactivation of tuberin activates Rheb to increase mTOR activity.


microRNA-21 governs TORC1 activation in renal cancer cell proliferation and invasion.

Dey N, Das F, Ghosh-Choudhury N, Mandal CC, Parekh DJ, Block K, Kasinath BS, Abboud HE, Choudhury GG - PLoS ONE (2012)

miR-21 regulates phosphorylation of tuberin and activation of mTOR in renal cancer cells.ACHN cells were transfected with miR-21 Sponge or vector. The cell lysates were immunoblotted with phospho-tuberin (Thr-1462), tuberin (panel A), phospho-S6 kinase (Thr-389), S6 kinase (panel B), phospho-mTOR (Ser-2448) and mTOR (panel C). miR-21 uses Rheb activation for TORC1 activity. ACHN cells were cotransfected with miR-21 Sponge and CA Rheb plasmid. The cell lysates were immunoblotted with phospho-S6 kinase (Thr-389), S6 kinase (panel D), phospho-mTOR (Ser-2448) and mTOR (panel E).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368259&req=5

pone-0037366-g007: miR-21 regulates phosphorylation of tuberin and activation of mTOR in renal cancer cells.ACHN cells were transfected with miR-21 Sponge or vector. The cell lysates were immunoblotted with phospho-tuberin (Thr-1462), tuberin (panel A), phospho-S6 kinase (Thr-389), S6 kinase (panel B), phospho-mTOR (Ser-2448) and mTOR (panel C). miR-21 uses Rheb activation for TORC1 activity. ACHN cells were cotransfected with miR-21 Sponge and CA Rheb plasmid. The cell lysates were immunoblotted with phospho-S6 kinase (Thr-389), S6 kinase (panel D), phospho-mTOR (Ser-2448) and mTOR (panel E).
Mentions: Activated Akt phosphorylates the tumor suppressor protein tuberin at Thr-1462 leading to its inactivation, and Rheb-mediated activation of TORC1 [34], [35], [36]. We have established that downregulation of PTEN due to increased miR-21 expression in renal cancer cells activates Akt (Fig. 3), which contributes to proliferation and migration of these cells (Figs. 5 and 6). We tested the role of miR-21 in tuberin phosphorylation. ACHN cells were transfected with miR-21 Sponge. Due to increased Akt activation, ACHN cells display enhanced phosphorylation of tuberin (Fig. 7A, lane 1). Expression of miR-21 Sponge inhibited the phosphorylation of tuberin (Fig. 7A and Fig. S15A). Similar to increased tuberin phosphorylation, ACHN cells showed augmented phosphorylation of S6 kinase at Thr-389 (Fig. 7B, lane 1), measured as a surrogate for TORC1 activity. miR-21 Sponge blocked S6 kinase phosphorylation (Fig. 7B). This reduction in S6 kinase phosphorylation by miR-21 Sponge was associated with inhibition of mTOR phosphorylation at Ser-2448 (Fig. 7C). These results suggest that miR-21 regulates TORC1 activity in the renal cancer cells. To examine whether miR-21 Sponge-dependent mTOR inhibition is due to inactivation of Rheb, we cotransfected ACHN cells with miR-21 Sponge and a constitutively active Rheb expression plasmid and the results were compared with that in miR-21 Sponge-transfected cells. mTOR activation was examined in the cell lysates. Expression of constitutively active Rheb reversed the inhibitory effect of miR-21 Sponge on S6 kinase phosphorylation (Fig. 7D and Fig. S15B). Similarly, the reduced phosphorylation of mTOR by miR-21 Sponge was also prevented by constitutively active Rheb (Fig. 7E and Fig. S15C). These results conclusively demonstrate that miR-21-mediated phosphorylation/inactivation of tuberin activates Rheb to increase mTOR activity.

Bottom Line: Metastatic renal cancer manifests multiple signatures of gene expression.Moreover, we show that miR-21 Sponge inhibited the inactivating phosphorylation of the tumor suppressor protein tuberin and attenuated TORC1 activation.Our results uncover a layer of post-transcriptional regulation of PTEN by transcriptional activation of miR-21 to force the canonical oncogenic Akt/TORC1 signaling conduit to drive renal cancer cell proliferation and invasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

ABSTRACT
Metastatic renal cancer manifests multiple signatures of gene expression. Deviation in expression of mature miRNAs has been linked to human cancers. Importance of miR-21 in renal cell carcinomas is proposed from profiling studies using tumor tissue samples. However, the role of miR-21 function in causing renal cancer cell proliferation and invasion has not yet been shown. Using cultured renal carcinoma cells, we demonstrate enhanced expression of mature miR-21 along with pre-and pri-miR-21 by increased transcription compared to normal proximal tubular epithelial cells. Overexpression of miR-21 Sponge to quench endogenous miR-21 levels inhibited proliferation, migration and invasion of renal cancer cells. In the absence of mutation in the PTEN tumor suppressor gene, PTEN protein levels are frequently downregulated in renal cancer. We show that miR-21 targets PTEN mRNA 3'untranslated region to decrease PTEN protein expression and augments Akt phosphorylation in renal cancer cells. Downregulation of PTEN as well as overexpression of constitutively active Akt kinase prevented miR-21 Sponge-induced inhibition of renal cancer cell proliferation and migration. Moreover, we show that miR-21 Sponge inhibited the inactivating phosphorylation of the tumor suppressor protein tuberin and attenuated TORC1 activation. Finally, we demonstrate that expression of constitutively active TORC1 attenuated miR-21 Sponge-mediated suppression of proliferation and migration of renal cancer cells. Our results uncover a layer of post-transcriptional regulation of PTEN by transcriptional activation of miR-21 to force the canonical oncogenic Akt/TORC1 signaling conduit to drive renal cancer cell proliferation and invasion.

Show MeSH
Related in: MedlinePlus