Limits...
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.


Related in: MedlinePlus

Both PKCα and PKCδ are required for αvβ3-mediated invasion. A) C8161.9 cells were transfected with scrambled (Scram), PKCδ siRNA (si1δ), or two PKCα siRNAs (si2α, si3α) and siRNA-transfected cells were plated on vitronectin for 2 hours and the extent of stress fiber and focal adhesion formation was monitored by staining with phalloidin (red) and immunostaining with anti-paxillin (green) respectively. Nuclei were counterstained with Hoescht (blue). White arrows indicate actin stress fibers around the perinuclear area in PKCα siRNA-treated cells. B) C8161.9 cells were transfected with scrambled (Sc), PKCδ (i1δ), si2α PKCα (i2α), or si3α PKCα (i3α) siRNAs. The levels of PKCα, PKCδ, and PKCε in siRNA transfected cells were measured by immunoblotting with isoform-specific PKC antibodies. Total levels of tubulin were also monitored (tubulin). C) C8161.9 cells were transfected with scrambled (Sc), PKCα siRNAs si3α (si3a), si2α (si2a), or PKCδ siRNA (si1d) and tested for their ability to invade VN-enriched Matrigel. Data represents at least 4 independent experiments. D) M14 cells were infected with viruses expressing nothing (V), PKCα (α), PKCδ (δ), Src, or PKCα and PKCδ (α + δ). Total levels of each protein were monitored by immunoblotting. E) Ability of the over expressing M14 cells to invade VN-enriched Matrigel was monitored. Data represents 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2683837&req=5

Figure 6: Both PKCα and PKCδ are required for αvβ3-mediated invasion. A) C8161.9 cells were transfected with scrambled (Scram), PKCδ siRNA (si1δ), or two PKCα siRNAs (si2α, si3α) and siRNA-transfected cells were plated on vitronectin for 2 hours and the extent of stress fiber and focal adhesion formation was monitored by staining with phalloidin (red) and immunostaining with anti-paxillin (green) respectively. Nuclei were counterstained with Hoescht (blue). White arrows indicate actin stress fibers around the perinuclear area in PKCα siRNA-treated cells. B) C8161.9 cells were transfected with scrambled (Sc), PKCδ (i1δ), si2α PKCα (i2α), or si3α PKCα (i3α) siRNAs. The levels of PKCα, PKCδ, and PKCε in siRNA transfected cells were measured by immunoblotting with isoform-specific PKC antibodies. Total levels of tubulin were also monitored (tubulin). C) C8161.9 cells were transfected with scrambled (Sc), PKCα siRNAs si3α (si3a), si2α (si2a), or PKCδ siRNA (si1d) and tested for their ability to invade VN-enriched Matrigel. Data represents at least 4 independent experiments. D) M14 cells were infected with viruses expressing nothing (V), PKCα (α), PKCδ (δ), Src, or PKCα and PKCδ (α + δ). Total levels of each protein were monitored by immunoblotting. E) Ability of the over expressing M14 cells to invade VN-enriched Matrigel was monitored. Data represents 3 independent experiments.

Mentions: Inhibition of Src was sufficient to induce stress fibers and increase focal adhesion formation in C8161.9 cells adherent to VN (see Figure 3C). Inhibition of PKCα expression by siRNA increased focal adhesion formation, but only partially restored stress fibers – mostly around the nucleus (Figure 6A). Inhibition of PKCδ primarily increased stress fibers, with some increase in the size, but not number of focal adhesions. However, neither response was as strong as seen with inhibition of Src (see Figure 3C). Thus PKCα appears to more strongly influence focal adhesions, while PKCδ primarily targets stress fibers.


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)

Both PKCα and PKCδ are required for αvβ3-mediated invasion. A) C8161.9 cells were transfected with scrambled (Scram), PKCδ siRNA (si1δ), or two PKCα siRNAs (si2α, si3α) and siRNA-transfected cells were plated on vitronectin for 2 hours and the extent of stress fiber and focal adhesion formation was monitored by staining with phalloidin (red) and immunostaining with anti-paxillin (green) respectively. Nuclei were counterstained with Hoescht (blue). White arrows indicate actin stress fibers around the perinuclear area in PKCα siRNA-treated cells. B) C8161.9 cells were transfected with scrambled (Sc), PKCδ (i1δ), si2α PKCα (i2α), or si3α PKCα (i3α) siRNAs. The levels of PKCα, PKCδ, and PKCε in siRNA transfected cells were measured by immunoblotting with isoform-specific PKC antibodies. Total levels of tubulin were also monitored (tubulin). C) C8161.9 cells were transfected with scrambled (Sc), PKCα siRNAs si3α (si3a), si2α (si2a), or PKCδ siRNA (si1d) and tested for their ability to invade VN-enriched Matrigel. Data represents at least 4 independent experiments. D) M14 cells were infected with viruses expressing nothing (V), PKCα (α), PKCδ (δ), Src, or PKCα and PKCδ (α + δ). Total levels of each protein were monitored by immunoblotting. E) Ability of the over expressing M14 cells to invade VN-enriched Matrigel was monitored. Data represents 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683837&req=5

Figure 6: Both PKCα and PKCδ are required for αvβ3-mediated invasion. A) C8161.9 cells were transfected with scrambled (Scram), PKCδ siRNA (si1δ), or two PKCα siRNAs (si2α, si3α) and siRNA-transfected cells were plated on vitronectin for 2 hours and the extent of stress fiber and focal adhesion formation was monitored by staining with phalloidin (red) and immunostaining with anti-paxillin (green) respectively. Nuclei were counterstained with Hoescht (blue). White arrows indicate actin stress fibers around the perinuclear area in PKCα siRNA-treated cells. B) C8161.9 cells were transfected with scrambled (Sc), PKCδ (i1δ), si2α PKCα (i2α), or si3α PKCα (i3α) siRNAs. The levels of PKCα, PKCδ, and PKCε in siRNA transfected cells were measured by immunoblotting with isoform-specific PKC antibodies. Total levels of tubulin were also monitored (tubulin). C) C8161.9 cells were transfected with scrambled (Sc), PKCα siRNAs si3α (si3a), si2α (si2a), or PKCδ siRNA (si1d) and tested for their ability to invade VN-enriched Matrigel. Data represents at least 4 independent experiments. D) M14 cells were infected with viruses expressing nothing (V), PKCα (α), PKCδ (δ), Src, or PKCα and PKCδ (α + δ). Total levels of each protein were monitored by immunoblotting. E) Ability of the over expressing M14 cells to invade VN-enriched Matrigel was monitored. Data represents 3 independent experiments.
Mentions: Inhibition of Src was sufficient to induce stress fibers and increase focal adhesion formation in C8161.9 cells adherent to VN (see Figure 3C). Inhibition of PKCα expression by siRNA increased focal adhesion formation, but only partially restored stress fibers – mostly around the nucleus (Figure 6A). Inhibition of PKCδ primarily increased stress fibers, with some increase in the size, but not number of focal adhesions. However, neither response was as strong as seen with inhibition of Src (see Figure 3C). Thus PKCα appears to more strongly influence focal adhesions, while PKCδ primarily targets stress fibers.

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