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Inhibition of Rho-associated coiled-coil containing protein kinase enhances the activation of epidermal growth factor receptor in pancreatic cancer cells.

Nakashima M, Adachi S, Yasuda I, Yamauchi T, Kawaguchi J, Hanamatsu T, Yoshioka T, Okano Y, Hirose Y, Kozawa O, Moriwaki H - Mol. Cancer (2011)

Bottom Line: As a result, the precise role of ROCK remains controversial.We also obtained similar results using transforming growth factor-α.Taken together, these data indicate that ROCK functions to switch off EGFR signaling by promoting the internalization of the EGFR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.

ABSTRACT

Background: Rho-associated coiled-coil containing protein kinase (Rho-kinase/ROCK) is involved in various cellular functions including cell proliferation, and is generally considered to be oncogenic, while some studies show that ROCK functions as a negative regulator of cancer progression. As a result, the precise role of ROCK remains controversial. We have previously reported that Rho-kinase/ROCK negatively regulates epidermal growth factor (EGF)-induced cell proliferation in SW480 colon cancer cells. In the present study, we investigated the role of ROCK in EGF receptor (EGFR) signaling in the pancreatic cancer cell lines, Panc1, KP3 and AsPc1.

Results: In these cells, Y27632, a specific ROCK inhibitor, enhanced EGF-induced BrdU incorporation. The blockade of EGF stimulation utilizing anti-EGFR-neutralizing antibodies suppressed Panc1 cell proliferation. EGF induced RhoA activity, as well as the phosphorylation of cofilin and myosin light chain (MLC), both targets of ROCK signaling, and Y27632 suppressed both of these processes, indicating that the phosphorylation of cofilin and MLC by EGF occurs through ROCK in Panc1 cells. EGF-induced phosphorylation of EGFR at tyrosine residues was augmented when the cells were pretreated with Y27632 or were subjected to gene silencing using ROCK-siRNA. We also obtained similar results using transforming growth factor-α. In addition, EGF-induced phosphorylation of p44/p42 mitogen-activated protein kinase and Akt were also enhanced by Y27632 or ROCK-siRNA. Moreover, an immunofluorescence microscope study revealed that pretreatment with Y27632 delayed EGF-induced internalization of EGFR. Taken together, these data indicate that ROCK functions to switch off EGFR signaling by promoting the internalization of the EGFR.

Conclusions: While EGF first stimulates the activation of the EGFR and subsequently increases cancer cell proliferation, EGF concurrently induces the activation of ROCK, which then turns off the activated EGFR pathway via a negative feedback system.

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A schematic representation of the involvement of ROCK in the EGFR signaling in pancreatic cancer cells. First, EGF binds to the EGFR on the cell surface, and the EGFR undergoes dimerization and autophosphorylation at tyrosine residues. This triggers EGFR-related downstream signaling, such as activation of the MEK1/2-p44/p42 MAPK or Akt pathways, within 5 min of EGF stimulation (①). Around 5 min after the start of stimulation, EGF starts to induce the activation of RhoA and subsequently ROCK, as demonstrated by upregulation of the phosphorylation of cofilin and MLC (②). Inhibition experiments using Y27632 or siRNA indicated that activated ROCK cancels the persistent activation of the EGFR and the downstream pathways of MEK1/2 and Akt beginning at 5 min after the initiation of stimulation. Thus, ROCK functions to switch off the EGFR signaling that induces pancreatic cancer cell proliferation. EGF: epidermal growth factor, EGFR: EGF receptor, ROCK: Rho-associated coiled-coil containing protein kinase, MAPK: mitogen-activated protein kinase, GSK-3β: glycogen synthase kinase-3β, MLC, myosin light chain, P: phosphorylation, Y: tyrosine.
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Figure 6: A schematic representation of the involvement of ROCK in the EGFR signaling in pancreatic cancer cells. First, EGF binds to the EGFR on the cell surface, and the EGFR undergoes dimerization and autophosphorylation at tyrosine residues. This triggers EGFR-related downstream signaling, such as activation of the MEK1/2-p44/p42 MAPK or Akt pathways, within 5 min of EGF stimulation (①). Around 5 min after the start of stimulation, EGF starts to induce the activation of RhoA and subsequently ROCK, as demonstrated by upregulation of the phosphorylation of cofilin and MLC (②). Inhibition experiments using Y27632 or siRNA indicated that activated ROCK cancels the persistent activation of the EGFR and the downstream pathways of MEK1/2 and Akt beginning at 5 min after the initiation of stimulation. Thus, ROCK functions to switch off the EGFR signaling that induces pancreatic cancer cell proliferation. EGF: epidermal growth factor, EGFR: EGF receptor, ROCK: Rho-associated coiled-coil containing protein kinase, MAPK: mitogen-activated protein kinase, GSK-3β: glycogen synthase kinase-3β, MLC, myosin light chain, P: phosphorylation, Y: tyrosine.

Mentions: It is well known that EGF induces the internalization of the EGFR, and this is associated with subsequent ubiquitin-mediated degradation of the EGFR [35]. We showed in Figures 3 and 4 that Y27632 remarkably prolonged the EGF-induced activation of EGFR and subsequent signaling via MEK1/2 and Akt. Therefore, we next examined whether Y27632 affects the EGFR internalization by performing an immunofluorescence microscope study. In this assay, the cells were not permeabilized using Triton X-100 in order to observe the remaining EGFR on the cell surface. In unstimulated Panc1 cells, antibody-tagged EGFR was observed on the cell membrane (Figure 5A, panel 1), and the cell surface EGFR was significantly decreased when the cells were treated with EGF (Figure 5A, panel 2), which is consistent with our previous study [36]. Interestingly, when the cells were pretreated with increasing doses of Y27632, antibody-tagged EGFR still remained on the cells surface even after EGF stimulation for 10 min (Figure 5A, panels 4 and 6), while Y27632 alone had no effect on the localization of the EGFR (Figure 6A, panels 3 and 5). Quantification of the green fluorescence intensities of cell surface EGFR labeled with Alexa 488 revealed that the EGF-induced decrease in cell surface EGFR was restored by pretreatment with Y27632 in a dose-dependent manner (Figure 5B). These results strongly suggest that the inhibition of ROCK delayed the internalization of the EGFR induced by EGF in Panc1 cells.


Inhibition of Rho-associated coiled-coil containing protein kinase enhances the activation of epidermal growth factor receptor in pancreatic cancer cells.

Nakashima M, Adachi S, Yasuda I, Yamauchi T, Kawaguchi J, Hanamatsu T, Yoshioka T, Okano Y, Hirose Y, Kozawa O, Moriwaki H - Mol. Cancer (2011)

A schematic representation of the involvement of ROCK in the EGFR signaling in pancreatic cancer cells. First, EGF binds to the EGFR on the cell surface, and the EGFR undergoes dimerization and autophosphorylation at tyrosine residues. This triggers EGFR-related downstream signaling, such as activation of the MEK1/2-p44/p42 MAPK or Akt pathways, within 5 min of EGF stimulation (①). Around 5 min after the start of stimulation, EGF starts to induce the activation of RhoA and subsequently ROCK, as demonstrated by upregulation of the phosphorylation of cofilin and MLC (②). Inhibition experiments using Y27632 or siRNA indicated that activated ROCK cancels the persistent activation of the EGFR and the downstream pathways of MEK1/2 and Akt beginning at 5 min after the initiation of stimulation. Thus, ROCK functions to switch off the EGFR signaling that induces pancreatic cancer cell proliferation. EGF: epidermal growth factor, EGFR: EGF receptor, ROCK: Rho-associated coiled-coil containing protein kinase, MAPK: mitogen-activated protein kinase, GSK-3β: glycogen synthase kinase-3β, MLC, myosin light chain, P: phosphorylation, Y: tyrosine.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: A schematic representation of the involvement of ROCK in the EGFR signaling in pancreatic cancer cells. First, EGF binds to the EGFR on the cell surface, and the EGFR undergoes dimerization and autophosphorylation at tyrosine residues. This triggers EGFR-related downstream signaling, such as activation of the MEK1/2-p44/p42 MAPK or Akt pathways, within 5 min of EGF stimulation (①). Around 5 min after the start of stimulation, EGF starts to induce the activation of RhoA and subsequently ROCK, as demonstrated by upregulation of the phosphorylation of cofilin and MLC (②). Inhibition experiments using Y27632 or siRNA indicated that activated ROCK cancels the persistent activation of the EGFR and the downstream pathways of MEK1/2 and Akt beginning at 5 min after the initiation of stimulation. Thus, ROCK functions to switch off the EGFR signaling that induces pancreatic cancer cell proliferation. EGF: epidermal growth factor, EGFR: EGF receptor, ROCK: Rho-associated coiled-coil containing protein kinase, MAPK: mitogen-activated protein kinase, GSK-3β: glycogen synthase kinase-3β, MLC, myosin light chain, P: phosphorylation, Y: tyrosine.
Mentions: It is well known that EGF induces the internalization of the EGFR, and this is associated with subsequent ubiquitin-mediated degradation of the EGFR [35]. We showed in Figures 3 and 4 that Y27632 remarkably prolonged the EGF-induced activation of EGFR and subsequent signaling via MEK1/2 and Akt. Therefore, we next examined whether Y27632 affects the EGFR internalization by performing an immunofluorescence microscope study. In this assay, the cells were not permeabilized using Triton X-100 in order to observe the remaining EGFR on the cell surface. In unstimulated Panc1 cells, antibody-tagged EGFR was observed on the cell membrane (Figure 5A, panel 1), and the cell surface EGFR was significantly decreased when the cells were treated with EGF (Figure 5A, panel 2), which is consistent with our previous study [36]. Interestingly, when the cells were pretreated with increasing doses of Y27632, antibody-tagged EGFR still remained on the cells surface even after EGF stimulation for 10 min (Figure 5A, panels 4 and 6), while Y27632 alone had no effect on the localization of the EGFR (Figure 6A, panels 3 and 5). Quantification of the green fluorescence intensities of cell surface EGFR labeled with Alexa 488 revealed that the EGF-induced decrease in cell surface EGFR was restored by pretreatment with Y27632 in a dose-dependent manner (Figure 5B). These results strongly suggest that the inhibition of ROCK delayed the internalization of the EGFR induced by EGF in Panc1 cells.

Bottom Line: As a result, the precise role of ROCK remains controversial.We also obtained similar results using transforming growth factor-α.Taken together, these data indicate that ROCK functions to switch off EGFR signaling by promoting the internalization of the EGFR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.

ABSTRACT

Background: Rho-associated coiled-coil containing protein kinase (Rho-kinase/ROCK) is involved in various cellular functions including cell proliferation, and is generally considered to be oncogenic, while some studies show that ROCK functions as a negative regulator of cancer progression. As a result, the precise role of ROCK remains controversial. We have previously reported that Rho-kinase/ROCK negatively regulates epidermal growth factor (EGF)-induced cell proliferation in SW480 colon cancer cells. In the present study, we investigated the role of ROCK in EGF receptor (EGFR) signaling in the pancreatic cancer cell lines, Panc1, KP3 and AsPc1.

Results: In these cells, Y27632, a specific ROCK inhibitor, enhanced EGF-induced BrdU incorporation. The blockade of EGF stimulation utilizing anti-EGFR-neutralizing antibodies suppressed Panc1 cell proliferation. EGF induced RhoA activity, as well as the phosphorylation of cofilin and myosin light chain (MLC), both targets of ROCK signaling, and Y27632 suppressed both of these processes, indicating that the phosphorylation of cofilin and MLC by EGF occurs through ROCK in Panc1 cells. EGF-induced phosphorylation of EGFR at tyrosine residues was augmented when the cells were pretreated with Y27632 or were subjected to gene silencing using ROCK-siRNA. We also obtained similar results using transforming growth factor-α. In addition, EGF-induced phosphorylation of p44/p42 mitogen-activated protein kinase and Akt were also enhanced by Y27632 or ROCK-siRNA. Moreover, an immunofluorescence microscope study revealed that pretreatment with Y27632 delayed EGF-induced internalization of EGFR. Taken together, these data indicate that ROCK functions to switch off EGFR signaling by promoting the internalization of the EGFR.

Conclusions: While EGF first stimulates the activation of the EGFR and subsequently increases cancer cell proliferation, EGF concurrently induces the activation of ROCK, which then turns off the activated EGFR pathway via a negative feedback system.

Show MeSH
Related in: MedlinePlus