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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.


Related in: MedlinePlus

αvβ3 integrin-dependent invasion of C8161.9 cells. A) Cell surface expression levels of αvβ3 (clear bar) and αvβ5 (black bar) integrins in two metastatic melanoma cell lines, C8161.9 and M14-mel, normal cultured melanocytes (NHEM), and the endothelial cell line HUVEC, were measured using FACS after surface labeling with fluorescently-labeled anti-integrin antibodies. B) Vitronectin-induced Matrigel invasion of C8161.9, M14, or normal cultured melanocytes (NHEM) in Boyden chambers in the absence of serum or growth factors was assessed in the absence (IgG) or presence of anti-integrin blocking antibodies (anti-αvβ3 or anti-αvβ5). In the absence of vitronectin (no VN), cells were unable to invade the Matrigel.
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Figure 1: αvβ3 integrin-dependent invasion of C8161.9 cells. A) Cell surface expression levels of αvβ3 (clear bar) and αvβ5 (black bar) integrins in two metastatic melanoma cell lines, C8161.9 and M14-mel, normal cultured melanocytes (NHEM), and the endothelial cell line HUVEC, were measured using FACS after surface labeling with fluorescently-labeled anti-integrin antibodies. B) Vitronectin-induced Matrigel invasion of C8161.9, M14, or normal cultured melanocytes (NHEM) in Boyden chambers in the absence of serum or growth factors was assessed in the absence (IgG) or presence of anti-integrin blocking antibodies (anti-αvβ3 or anti-αvβ5). In the absence of vitronectin (no VN), cells were unable to invade the Matrigel.

Mentions: The primary ligand for αvβ3 is vitronectin (VN); however, another integrin, αvβ5, also binds VN. To determine which integrin is responsible for the biological effects mediated by adhesion to VN, αvβ3 and αvβ5 integrin expression was measured in the melanoma cells. The cell surface levels of αvβ3 and αvβ5 in cultured melanocytes and in a non-invasive cell line, M14 [38], and a highly metastatic melanoma cell line, C8161.9 [29], were quantified by FACS. Irrespective of their metastatic potential, both melanoma cell lines expressed similar levels of αvβ3, while normal cultured melanocytes and HUVECs (positive control) expressed over 2 times more αvβ3. On the other hand, the more highly metastatic melanoma cell line, C8161.9, expressed over 2× higher levels of αvβ5 than M14 (Figure 1A). To determine which integrin is required for the migratory and invasive properties of the melanoma cell lines, cells were treated with or without VN integrin-blocking antibodies [26,27] or IgG and their ability to invade and migrate through vitronectin-enriched Matrigel to the lower chamber of a Boyden chamber in the absence of serum or growth factors was evaluated. M14 and normal melanocytes both failed to invade through VN/Matrigel, while invasion of the highly metastatic C8161.9 cells was dramatically diminished in the presence of αvβ3, but not αvβ5 blocking antibodies (Figure 1B). If VN is omitted C8161.9 cells are unable to invade. Thus, expression of αvβ3 is required for invasion of highly metastatic melanoma cells. However, αvβ3 expression alone is not sufficient, since αvβ3 is expressed on both M14 and normal cultured melanocytes and these cells were unable to invade. Therefore, additional signaling events must also be required. These signaling events must be intrinsic to the cells, since no exogenous growth factors, serum, or additional external stimuli other than extracellular matrix (ECM) proteins, were added to the invasion assays.


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)

αvβ3 integrin-dependent invasion of C8161.9 cells. A) Cell surface expression levels of αvβ3 (clear bar) and αvβ5 (black bar) integrins in two metastatic melanoma cell lines, C8161.9 and M14-mel, normal cultured melanocytes (NHEM), and the endothelial cell line HUVEC, were measured using FACS after surface labeling with fluorescently-labeled anti-integrin antibodies. B) Vitronectin-induced Matrigel invasion of C8161.9, M14, or normal cultured melanocytes (NHEM) in Boyden chambers in the absence of serum or growth factors was assessed in the absence (IgG) or presence of anti-integrin blocking antibodies (anti-αvβ3 or anti-αvβ5). In the absence of vitronectin (no VN), cells were unable to invade the Matrigel.
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Related In: Results  -  Collection

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Figure 1: αvβ3 integrin-dependent invasion of C8161.9 cells. A) Cell surface expression levels of αvβ3 (clear bar) and αvβ5 (black bar) integrins in two metastatic melanoma cell lines, C8161.9 and M14-mel, normal cultured melanocytes (NHEM), and the endothelial cell line HUVEC, were measured using FACS after surface labeling with fluorescently-labeled anti-integrin antibodies. B) Vitronectin-induced Matrigel invasion of C8161.9, M14, or normal cultured melanocytes (NHEM) in Boyden chambers in the absence of serum or growth factors was assessed in the absence (IgG) or presence of anti-integrin blocking antibodies (anti-αvβ3 or anti-αvβ5). In the absence of vitronectin (no VN), cells were unable to invade the Matrigel.
Mentions: The primary ligand for αvβ3 is vitronectin (VN); however, another integrin, αvβ5, also binds VN. To determine which integrin is responsible for the biological effects mediated by adhesion to VN, αvβ3 and αvβ5 integrin expression was measured in the melanoma cells. The cell surface levels of αvβ3 and αvβ5 in cultured melanocytes and in a non-invasive cell line, M14 [38], and a highly metastatic melanoma cell line, C8161.9 [29], were quantified by FACS. Irrespective of their metastatic potential, both melanoma cell lines expressed similar levels of αvβ3, while normal cultured melanocytes and HUVECs (positive control) expressed over 2 times more αvβ3. On the other hand, the more highly metastatic melanoma cell line, C8161.9, expressed over 2× higher levels of αvβ5 than M14 (Figure 1A). To determine which integrin is required for the migratory and invasive properties of the melanoma cell lines, cells were treated with or without VN integrin-blocking antibodies [26,27] or IgG and their ability to invade and migrate through vitronectin-enriched Matrigel to the lower chamber of a Boyden chamber in the absence of serum or growth factors was evaluated. M14 and normal melanocytes both failed to invade through VN/Matrigel, while invasion of the highly metastatic C8161.9 cells was dramatically diminished in the presence of αvβ3, but not αvβ5 blocking antibodies (Figure 1B). If VN is omitted C8161.9 cells are unable to invade. Thus, expression of αvβ3 is required for invasion of highly metastatic melanoma cells. However, αvβ3 expression alone is not sufficient, since αvβ3 is expressed on both M14 and normal cultured melanocytes and these cells were unable to invade. Therefore, additional signaling events must also be required. These signaling events must be intrinsic to the cells, since no exogenous growth factors, serum, or additional external stimuli other than extracellular matrix (ECM) proteins, were added to the invasion assays.

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