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Interfering with TGFbeta-induced Smad3 nuclear accumulation differentially affects TGFbeta-dependent gene expression.

Lindemann RK, Nordheim A, Dittmer J - Mol. Cancer (2003)

Bottom Line: Our results show that these inhibitors delay the onset of TGFbeta-induced nuclear accumulation of Smad3 and reduces its amplitude.This effect was accompanied by a strong reduction in TGFbeta-responsivess of the slow-responder genes pthrp, pai-1 and upa, while the reactivity of the fast-responder gene smad7 to TGFbeta remained almost unchanged.Neither was the TGFbeta response of the fast-responder ese-1/esx gene, whose expression we found to be strongly downregulated by TGFbeta, affected by the inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Zellbiologie, Abteilung Molekularbiologie, Universität Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany. ralph.lindemann@uni-tuebingen.de

ABSTRACT

Background: Transforming growth factor-beta (TGFbeta) plays an important role in late-stage carcinogenesis by stimulating invasive behavior of cancer cells, promoting neo-angiogenesis and by helping cancer cells to escape surveillance by the immune system. It also supports colonization of the bone by metastatic breast cancer cells by increasing expression of osteolytic parathyroid hormone-related protein (PTHrP). Interfering with TGFbeta signalling may thus weaken the malignant properties of cancer cells. We investigated to what extent two inhibitors, SB-202190 and SB-203580, interfere with TGFbeta-signalling in invasive MDA-MB-231 breast cancer cells. These compounds, formerly used as p38-MAPK-specific inhibitors, were recently also demonstrated to inhibit TGFbeta type I receptor kinase.

Results: Our results show that these inhibitors delay the onset of TGFbeta-induced nuclear accumulation of Smad3 and reduces its amplitude. This effect was accompanied by a strong reduction in TGFbeta-responsivess of the slow-responder genes pthrp, pai-1 and upa, while the reactivity of the fast-responder gene smad7 to TGFbeta remained almost unchanged. Neither was the TGFbeta response of the fast-responder ese-1/esx gene, whose expression we found to be strongly downregulated by TGFbeta, affected by the inhibitors.

Conclusion: The data show that SB-202190 and SB-203580 suppress TGFbeta-dependent activation of genes that are important for the acquisition of invasive behavior, while having no effect on the expression of the natural TGFbeta inhibitor Smad7. This suggests that these compounds are potent inhibitors of malignant behavior of cancer cells.

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TGFβ target genes respond differentially to SB-203580. Real-time RT-PCR analyses of mRNA derived from MDA-MB-231 cells treated with TGFβ for 15, 30, 60, 120, 180 and 240 min. Filled line indicates TGFβ-induced activation in the absence of SB-203580, dotted line represents activation by TGFβ in the presence of this inhibitor. Relative transcript level was calculated by normalizing transcript levels in induced relative to uninduced samples. A-E, known TGFβ target genes. F-H, ets family members.
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Figure 3: TGFβ target genes respond differentially to SB-203580. Real-time RT-PCR analyses of mRNA derived from MDA-MB-231 cells treated with TGFβ for 15, 30, 60, 120, 180 and 240 min. Filled line indicates TGFβ-induced activation in the absence of SB-203580, dotted line represents activation by TGFβ in the presence of this inhibitor. Relative transcript level was calculated by normalizing transcript levels in induced relative to uninduced samples. A-E, known TGFβ target genes. F-H, ets family members.

Mentions: The delayed TGFβ-mediated entry of Smad3 into the nucleus in the presence of SB-203580 prompted us to examine the effect of SB-203580 on TGFβ-induced gene expression in a time-course experiment. We isolated total RNA from the same cell suspension that was used to prepare nuclear extracts for time-course analysis in Fig. 2B. cDNA was synthesized and subjected to quantitative RT-PCR in order to measure the transcript levels of several TGFβ target genes. The genes being examined were divided into two groups: I, known TGFβ target genes: pai-1, smad7, c-myc, upa and pthrp (Fig. 3A,3B,3C,3D,3E) and II, ets family genes: ets-1, ets-2 and ese-1/esx. Smad7 was of particular interest as it belongs to the inhibitory Smads, which can counteract TGFβ-induced gene activation [23]. The response of the smad7 gene to TGFβ is unusually rapid [24] which is believed to stem from the peculiar nature of the Smad7 promoter that contains a "perfect" Smad binding site [25]. Another inhibitory Smad protein is Smad6. We have not analyzed the expression of this protein, since it preferentially inhibit bone morphogenetic protein (BMP)-induced expression [26]. In addition, Smad6 shows varying effects on TGFβ signalling [27]. Nor were TGFβ-responsive genes, such as p21, studied that are involved in cell cycle regulation. Such genes are important targets at early-stage carcinogenesis, where TGFβ has a tumor-suppressive rather than a tumor-promoting function [28]. In addition, e.g. p21 protein is not dectable in MDA-MB-231 cells [29].


Interfering with TGFbeta-induced Smad3 nuclear accumulation differentially affects TGFbeta-dependent gene expression.

Lindemann RK, Nordheim A, Dittmer J - Mol. Cancer (2003)

TGFβ target genes respond differentially to SB-203580. Real-time RT-PCR analyses of mRNA derived from MDA-MB-231 cells treated with TGFβ for 15, 30, 60, 120, 180 and 240 min. Filled line indicates TGFβ-induced activation in the absence of SB-203580, dotted line represents activation by TGFβ in the presence of this inhibitor. Relative transcript level was calculated by normalizing transcript levels in induced relative to uninduced samples. A-E, known TGFβ target genes. F-H, ets family members.
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: TGFβ target genes respond differentially to SB-203580. Real-time RT-PCR analyses of mRNA derived from MDA-MB-231 cells treated with TGFβ for 15, 30, 60, 120, 180 and 240 min. Filled line indicates TGFβ-induced activation in the absence of SB-203580, dotted line represents activation by TGFβ in the presence of this inhibitor. Relative transcript level was calculated by normalizing transcript levels in induced relative to uninduced samples. A-E, known TGFβ target genes. F-H, ets family members.
Mentions: The delayed TGFβ-mediated entry of Smad3 into the nucleus in the presence of SB-203580 prompted us to examine the effect of SB-203580 on TGFβ-induced gene expression in a time-course experiment. We isolated total RNA from the same cell suspension that was used to prepare nuclear extracts for time-course analysis in Fig. 2B. cDNA was synthesized and subjected to quantitative RT-PCR in order to measure the transcript levels of several TGFβ target genes. The genes being examined were divided into two groups: I, known TGFβ target genes: pai-1, smad7, c-myc, upa and pthrp (Fig. 3A,3B,3C,3D,3E) and II, ets family genes: ets-1, ets-2 and ese-1/esx. Smad7 was of particular interest as it belongs to the inhibitory Smads, which can counteract TGFβ-induced gene activation [23]. The response of the smad7 gene to TGFβ is unusually rapid [24] which is believed to stem from the peculiar nature of the Smad7 promoter that contains a "perfect" Smad binding site [25]. Another inhibitory Smad protein is Smad6. We have not analyzed the expression of this protein, since it preferentially inhibit bone morphogenetic protein (BMP)-induced expression [26]. In addition, Smad6 shows varying effects on TGFβ signalling [27]. Nor were TGFβ-responsive genes, such as p21, studied that are involved in cell cycle regulation. Such genes are important targets at early-stage carcinogenesis, where TGFβ has a tumor-suppressive rather than a tumor-promoting function [28]. In addition, e.g. p21 protein is not dectable in MDA-MB-231 cells [29].

Bottom Line: Our results show that these inhibitors delay the onset of TGFbeta-induced nuclear accumulation of Smad3 and reduces its amplitude.This effect was accompanied by a strong reduction in TGFbeta-responsivess of the slow-responder genes pthrp, pai-1 and upa, while the reactivity of the fast-responder gene smad7 to TGFbeta remained almost unchanged.Neither was the TGFbeta response of the fast-responder ese-1/esx gene, whose expression we found to be strongly downregulated by TGFbeta, affected by the inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Zellbiologie, Abteilung Molekularbiologie, Universität Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany. ralph.lindemann@uni-tuebingen.de

ABSTRACT

Background: Transforming growth factor-beta (TGFbeta) plays an important role in late-stage carcinogenesis by stimulating invasive behavior of cancer cells, promoting neo-angiogenesis and by helping cancer cells to escape surveillance by the immune system. It also supports colonization of the bone by metastatic breast cancer cells by increasing expression of osteolytic parathyroid hormone-related protein (PTHrP). Interfering with TGFbeta signalling may thus weaken the malignant properties of cancer cells. We investigated to what extent two inhibitors, SB-202190 and SB-203580, interfere with TGFbeta-signalling in invasive MDA-MB-231 breast cancer cells. These compounds, formerly used as p38-MAPK-specific inhibitors, were recently also demonstrated to inhibit TGFbeta type I receptor kinase.

Results: Our results show that these inhibitors delay the onset of TGFbeta-induced nuclear accumulation of Smad3 and reduces its amplitude. This effect was accompanied by a strong reduction in TGFbeta-responsivess of the slow-responder genes pthrp, pai-1 and upa, while the reactivity of the fast-responder gene smad7 to TGFbeta remained almost unchanged. Neither was the TGFbeta response of the fast-responder ese-1/esx gene, whose expression we found to be strongly downregulated by TGFbeta, affected by the inhibitors.

Conclusion: The data show that SB-202190 and SB-203580 suppress TGFbeta-dependent activation of genes that are important for the acquisition of invasive behavior, while having no effect on the expression of the natural TGFbeta inhibitor Smad7. This suggests that these compounds are potent inhibitors of malignant behavior of cancer cells.

Show MeSH
Related in: MedlinePlus