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Src, PKCalpha, and PKCdelta are required for alphavbeta3 integrin-mediated metastatic melanoma invasion.

Putnam AJ, Schulz VV, Freiter EM, Bill HM, Miranti CK - Cell Commun. Signal (2009)

Bottom Line: Inhibition of Src signaling was sufficient to restore normal actin architecture, and resulted in decreased p190RhoGAP phosphorylation and enhanced RhoA activity.Src had no effect on Rac activity.PKCalpha and Src enhance alphavbeta3-mediated invasion in part by increasing the GTPase activity of Rac relative to RhoA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Integrin Signaling and Tumorigenesis, Van Andel Research Institute, Grand Rapids, Michigan 49503, USA. cindy.miranti@vai.org.

ABSTRACT

Background: Integrins, cell-surface receptors that mediate adhesive interactions between cells and the extracellular matrix (ECM), play an important role in cancer progression. Expression of the vitronectin receptor alphavbeta3 integrin correlates with increased invasive and metastatic capacity of malignant melanomas, yet it remains unclear how expression of this integrin triggers melanoma invasion and metastasis.

Results: Two melanoma cell lines C8161.9 and M14 both express high levels of alphavbeta3 integrin and adhere to vitronectin. However, only the highly metastatic C8161.9 cells are capable of invading vitronectin-enriched Matrigel in an alphavbeta3-depenent manner. Elevated levels of PKCalpha and PKCdelta, and activated Src were detected specifically in the highly metastatic melanoma cells, but not in the low metastatic M14 cells. Inhibition of Src or PKC activity suppressed alphavbeta3-dependent invasion. Furthermore, over expression of Src or PKCalpha and PKCdelta was sufficient to confer alphavbeta3-dependent invasiveness to M14 cells. Stress fiber formation and focal adhesion formation were almost completely absent in C8161.9 cells compared to M14 cells. Inhibition of Src signaling was sufficient to restore normal actin architecture, and resulted in decreased p190RhoGAP phosphorylation and enhanced RhoA activity. Src had no effect on Rac activity. Loss of PKCalpha expression, but not PKCdelta, by siRNA inhibited Rac and PAK activity as well as invasiveness. Loss of PKCalpha restored focal adhesion formation and partially restored stress fiber formation, while loss of PKCdelta primarily restored stress fibers.

Conclusion: The misregulated expression of PKCalpha and PKCdelta and elevated Src activity in metastatic melanoma cells is required for efficient alphavbeta3-mediated invasion. PKCalpha and Src enhance alphavbeta3-mediated invasion in part by increasing the GTPase activity of Rac relative to RhoA. PKCalpha influences focal adhesion formation, while PKCdelta controls stress fibers.

No MeSH data available.


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Src activation prevents RhoA-dependent formation of stress fibers and focal adhesions. A) C8161.9, M14, or normal cultured melanocytes (NHEM) were plated on fibronectin (FN) or vitronectin (VN) for 2 hours and the extent of stress fiber formation was monitored by staining with phalloidin (red). Focal adhesion formation was monitored by immunostaining with paxillin antibodies (green). Cells were counterstained with Hoescht to detect nuclei (blue). B) C8161.9 cells were transiently transfected with GFP expressing plasmid and an active form of RhoA (Q61L). Cells were plated on vitronectin for 2 hours and stained with phalloidin (red) to monitor actin structures in GFP-positive transfected (green) cells. Only the three cells expressing green GFP (i.e. transfected with RhoA (Q61L)) displayed prominent stress fibers. The nuclei of cells not expressing GFP/RhoA Q61L are marked by asterisks. C) C1861.9 cells were left untreated (control), treated with SU6656, or infected with dominant interfering mutant Src (K295M) virus. Cells were plated on vitronectin for 2 hours and stress fiber formation monitored by staining with phalloidin (red) and focal adhesion formation by immunostaining with paxillin antibodies (green).
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Figure 3: Src activation prevents RhoA-dependent formation of stress fibers and focal adhesions. A) C8161.9, M14, or normal cultured melanocytes (NHEM) were plated on fibronectin (FN) or vitronectin (VN) for 2 hours and the extent of stress fiber formation was monitored by staining with phalloidin (red). Focal adhesion formation was monitored by immunostaining with paxillin antibodies (green). Cells were counterstained with Hoescht to detect nuclei (blue). B) C8161.9 cells were transiently transfected with GFP expressing plasmid and an active form of RhoA (Q61L). Cells were plated on vitronectin for 2 hours and stained with phalloidin (red) to monitor actin structures in GFP-positive transfected (green) cells. Only the three cells expressing green GFP (i.e. transfected with RhoA (Q61L)) displayed prominent stress fibers. The nuclei of cells not expressing GFP/RhoA Q61L are marked by asterisks. C) C1861.9 cells were left untreated (control), treated with SU6656, or infected with dominant interfering mutant Src (K295M) virus. Cells were plated on vitronectin for 2 hours and stress fiber formation monitored by staining with phalloidin (red) and focal adhesion formation by immunostaining with paxillin antibodies (green).

Mentions: Adhesion of normal cultured melanocytes or M14 melanoma cells to VN induced the formation of multiple stress fibers and large focal adhesion complexes from which the stress fibers emanated (Figure 3A). However, adhesion of C8161.9 cells to VN failed to induce stress fiber formation and the number and size of focal adhesions were dramatically reduced. Since RhoA has been shown to regulate the formation of stress fibers and focal adhesions, RhoA signaling may be impaired in C8161.9 cells. To test this C8161.9 cells were transiently cotransfected with a constitutively active form of RhoA and GFP. Only the three cells expressing green GFP (i.e. transfected with and expressing constitutively active RhoA Q61L) displayed prominent stress fibers (Figure 3B). The surrounding cells (asterisks) not expressing GFP and RhoA Q61L do not have prominent stress fibers. Src has been shown to be a negative regulator of RhoA activity [40]. Therefore, it is possible that the high levels of Src activity in C8161.9 cells may be inhibiting RhoA activity. Indeed, inhibition of Src with the specific inhibitor SU6656 or over expression of the K295M Src dominant interfering mutant was sufficient to induce stress fibers and increase focal adhesion formation (Figure 3C). Thus, the aberrant stress fiber and focal adhesion formation in C8161.9 cells plated on VN is likely due to the constitutively high level of active Src, which effectively down regulates RhoA activity.


Src, PKCalpha, and PKCdelta are required for alphavbeta3 integrin-mediated metastatic melanoma invasion.

Putnam AJ, Schulz VV, Freiter EM, Bill HM, Miranti CK - Cell Commun. Signal (2009)

Src activation prevents RhoA-dependent formation of stress fibers and focal adhesions. A) C8161.9, M14, or normal cultured melanocytes (NHEM) were plated on fibronectin (FN) or vitronectin (VN) for 2 hours and the extent of stress fiber formation was monitored by staining with phalloidin (red). Focal adhesion formation was monitored by immunostaining with paxillin antibodies (green). Cells were counterstained with Hoescht to detect nuclei (blue). B) C8161.9 cells were transiently transfected with GFP expressing plasmid and an active form of RhoA (Q61L). Cells were plated on vitronectin for 2 hours and stained with phalloidin (red) to monitor actin structures in GFP-positive transfected (green) cells. Only the three cells expressing green GFP (i.e. transfected with RhoA (Q61L)) displayed prominent stress fibers. The nuclei of cells not expressing GFP/RhoA Q61L are marked by asterisks. C) C1861.9 cells were left untreated (control), treated with SU6656, or infected with dominant interfering mutant Src (K295M) virus. Cells were plated on vitronectin for 2 hours and stress fiber formation monitored by staining with phalloidin (red) and focal adhesion formation by immunostaining with paxillin antibodies (green).
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Figure 3: Src activation prevents RhoA-dependent formation of stress fibers and focal adhesions. A) C8161.9, M14, or normal cultured melanocytes (NHEM) were plated on fibronectin (FN) or vitronectin (VN) for 2 hours and the extent of stress fiber formation was monitored by staining with phalloidin (red). Focal adhesion formation was monitored by immunostaining with paxillin antibodies (green). Cells were counterstained with Hoescht to detect nuclei (blue). B) C8161.9 cells were transiently transfected with GFP expressing plasmid and an active form of RhoA (Q61L). Cells were plated on vitronectin for 2 hours and stained with phalloidin (red) to monitor actin structures in GFP-positive transfected (green) cells. Only the three cells expressing green GFP (i.e. transfected with RhoA (Q61L)) displayed prominent stress fibers. The nuclei of cells not expressing GFP/RhoA Q61L are marked by asterisks. C) C1861.9 cells were left untreated (control), treated with SU6656, or infected with dominant interfering mutant Src (K295M) virus. Cells were plated on vitronectin for 2 hours and stress fiber formation monitored by staining with phalloidin (red) and focal adhesion formation by immunostaining with paxillin antibodies (green).
Mentions: Adhesion of normal cultured melanocytes or M14 melanoma cells to VN induced the formation of multiple stress fibers and large focal adhesion complexes from which the stress fibers emanated (Figure 3A). However, adhesion of C8161.9 cells to VN failed to induce stress fiber formation and the number and size of focal adhesions were dramatically reduced. Since RhoA has been shown to regulate the formation of stress fibers and focal adhesions, RhoA signaling may be impaired in C8161.9 cells. To test this C8161.9 cells were transiently cotransfected with a constitutively active form of RhoA and GFP. Only the three cells expressing green GFP (i.e. transfected with and expressing constitutively active RhoA Q61L) displayed prominent stress fibers (Figure 3B). The surrounding cells (asterisks) not expressing GFP and RhoA Q61L do not have prominent stress fibers. Src has been shown to be a negative regulator of RhoA activity [40]. Therefore, it is possible that the high levels of Src activity in C8161.9 cells may be inhibiting RhoA activity. Indeed, inhibition of Src with the specific inhibitor SU6656 or over expression of the K295M Src dominant interfering mutant was sufficient to induce stress fibers and increase focal adhesion formation (Figure 3C). Thus, the aberrant stress fiber and focal adhesion formation in C8161.9 cells plated on VN is likely due to the constitutively high level of active Src, which effectively down regulates RhoA activity.

Bottom Line: Inhibition of Src signaling was sufficient to restore normal actin architecture, and resulted in decreased p190RhoGAP phosphorylation and enhanced RhoA activity.Src had no effect on Rac activity.PKCalpha and Src enhance alphavbeta3-mediated invasion in part by increasing the GTPase activity of Rac relative to RhoA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Integrin Signaling and Tumorigenesis, Van Andel Research Institute, Grand Rapids, Michigan 49503, USA. cindy.miranti@vai.org.

ABSTRACT

Background: Integrins, cell-surface receptors that mediate adhesive interactions between cells and the extracellular matrix (ECM), play an important role in cancer progression. Expression of the vitronectin receptor alphavbeta3 integrin correlates with increased invasive and metastatic capacity of malignant melanomas, yet it remains unclear how expression of this integrin triggers melanoma invasion and metastasis.

Results: Two melanoma cell lines C8161.9 and M14 both express high levels of alphavbeta3 integrin and adhere to vitronectin. However, only the highly metastatic C8161.9 cells are capable of invading vitronectin-enriched Matrigel in an alphavbeta3-depenent manner. Elevated levels of PKCalpha and PKCdelta, and activated Src were detected specifically in the highly metastatic melanoma cells, but not in the low metastatic M14 cells. Inhibition of Src or PKC activity suppressed alphavbeta3-dependent invasion. Furthermore, over expression of Src or PKCalpha and PKCdelta was sufficient to confer alphavbeta3-dependent invasiveness to M14 cells. Stress fiber formation and focal adhesion formation were almost completely absent in C8161.9 cells compared to M14 cells. Inhibition of Src signaling was sufficient to restore normal actin architecture, and resulted in decreased p190RhoGAP phosphorylation and enhanced RhoA activity. Src had no effect on Rac activity. Loss of PKCalpha expression, but not PKCdelta, by siRNA inhibited Rac and PAK activity as well as invasiveness. Loss of PKCalpha restored focal adhesion formation and partially restored stress fiber formation, while loss of PKCdelta primarily restored stress fibers.

Conclusion: The misregulated expression of PKCalpha and PKCdelta and elevated Src activity in metastatic melanoma cells is required for efficient alphavbeta3-mediated invasion. PKCalpha and Src enhance alphavbeta3-mediated invasion in part by increasing the GTPase activity of Rac relative to RhoA. PKCalpha influences focal adhesion formation, while PKCdelta controls stress fibers.

No MeSH data available.


Related in: MedlinePlus