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Protein kinase C zeta regulates human pancreatic cancer cell transformed growth and invasion through a STAT3-dependent mechanism.

Butler AM, Scotti Buzhardt ML, Li S, Smith KE, Fields AP, Murray NR - PLoS ONE (2013)

Bottom Line: Furthermore, PKCζ inhibition reduced orthotopic tumor size in vivo by inhibiting tumor cell proliferation and increasing tumor necrosis.In addition, PKCζ inhibition reduced tumor metastases in vivo, and caused a corresponding reduction in pancreatic cancer cell invasion in vitro.We conclude that PKCζ is required for pancreatic cancer cell transformed growth and invasion in vitro and tumorigenesis in vivo, and that STAT3 is an important downstream mediator of the pro-carcinogenic effects of PKCζ in pancreatic cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, United States of America.

ABSTRACT
Pancreatic cancer is a very aggressive disease with few therapeutic options. In this study, we investigate the role of protein kinase C zeta (PKCζ) in pancreatic cancer cells. PKCζ has been shown to act as either a tumor suppressor or tumor promoter depending upon the cellular context. We find that PKCζ expression is either maintained or elevated in primary human pancreatic tumors, but is never lost, consistent with PKCζ playing a promotive role in the pancreatic cancer phenotype. Genetic inhibition of PKCζ reduced adherent growth, cell survival and anchorage-independent growth of human pancreatic cancer cells in vitro. Furthermore, PKCζ inhibition reduced orthotopic tumor size in vivo by inhibiting tumor cell proliferation and increasing tumor necrosis. In addition, PKCζ inhibition reduced tumor metastases in vivo, and caused a corresponding reduction in pancreatic cancer cell invasion in vitro. Signal transducer and activator of transcription 3 (STAT3) is often constitutively active in pancreatic cancer, and plays an important role in pancreatic cancer cell survival and metastasis. Interestingly, inhibition of PKCζ significantly reduced constitutive STAT3 activation in pancreatic cancer cells in vitro and in vivo. Pharmacologic inhibition of STAT3 mimicked the phenotype of PKCζ inhibition, and expression of a constitutively active STAT3 construct rescued the transformed phenotype in PKCζ-deficient cells. We conclude that PKCζ is required for pancreatic cancer cell transformed growth and invasion in vitro and tumorigenesis in vivo, and that STAT3 is an important downstream mediator of the pro-carcinogenic effects of PKCζ in pancreatic cancer cells.

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Constitutively active STAT3 rescues the transformed phenotype in PKCζ RNAi-expressing cells.Panc-1 cells expressing NT or PKCζ RNAi were infected with adenoviral constructs expressing either  (control), or constitutively active, FLAG-tagged STAT3 (STAT3-C). A) Immunoblot analysis of p-STAT3, STAT3, FLAG, PKCζ and β-actin expression. Cells were assessed for B) anchorage-independent growth in soft agar and C) cellular invasion through Matrigel-coated chambers. For each graph: Bars = average of 3 or more replicates±SD and graph is representative of 2 or more independent experiments. *significantly reduced compared to NT/, p<0.05; **significantly increased compared to -treated, p<0.05.
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pone-0072061-g006: Constitutively active STAT3 rescues the transformed phenotype in PKCζ RNAi-expressing cells.Panc-1 cells expressing NT or PKCζ RNAi were infected with adenoviral constructs expressing either (control), or constitutively active, FLAG-tagged STAT3 (STAT3-C). A) Immunoblot analysis of p-STAT3, STAT3, FLAG, PKCζ and β-actin expression. Cells were assessed for B) anchorage-independent growth in soft agar and C) cellular invasion through Matrigel-coated chambers. For each graph: Bars = average of 3 or more replicates±SD and graph is representative of 2 or more independent experiments. *significantly reduced compared to NT/, p<0.05; **significantly increased compared to -treated, p<0.05.

Mentions: To test the hypothesis that STAT3 is a critical downstream effector of PKCζ in pancreatic cancer cells, we assessed whether expression of a constitutively active STAT3 construct (STAT3-C) could rescue the effects of PKCζ inhibition in Panc-1 cells. Panc-1 NT and PKCζ RNAi cells were infected with adenovirus expressing flag-tagged, STAT3-C or control () adenovirus (Figure 6A). Expression of STAT3-C significantly recovered anchorage-independent growth of Panc-1 PKCζ RNAi cells, without significantly affecting the anchorage-independent growth of NT RNAi cells (Figure 6B). In addition, the reduced cellular invasion phenotype of PKCζ RNAi cells was significantly recovered by expression of STAT3-C (Figure 6C). Taken together, these data demonstrate that increased cellular STAT3 activity can rescue the anti-oncogenic phenotype of PKCζ RNAi cells, and demonstrate that PKCζ mediates pancreatic cancer cell transformation, at least in part, through regulation of STAT3 activity.


Protein kinase C zeta regulates human pancreatic cancer cell transformed growth and invasion through a STAT3-dependent mechanism.

Butler AM, Scotti Buzhardt ML, Li S, Smith KE, Fields AP, Murray NR - PLoS ONE (2013)

Constitutively active STAT3 rescues the transformed phenotype in PKCζ RNAi-expressing cells.Panc-1 cells expressing NT or PKCζ RNAi were infected with adenoviral constructs expressing either  (control), or constitutively active, FLAG-tagged STAT3 (STAT3-C). A) Immunoblot analysis of p-STAT3, STAT3, FLAG, PKCζ and β-actin expression. Cells were assessed for B) anchorage-independent growth in soft agar and C) cellular invasion through Matrigel-coated chambers. For each graph: Bars = average of 3 or more replicates±SD and graph is representative of 2 or more independent experiments. *significantly reduced compared to NT/, p<0.05; **significantly increased compared to -treated, p<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756013&req=5

pone-0072061-g006: Constitutively active STAT3 rescues the transformed phenotype in PKCζ RNAi-expressing cells.Panc-1 cells expressing NT or PKCζ RNAi were infected with adenoviral constructs expressing either (control), or constitutively active, FLAG-tagged STAT3 (STAT3-C). A) Immunoblot analysis of p-STAT3, STAT3, FLAG, PKCζ and β-actin expression. Cells were assessed for B) anchorage-independent growth in soft agar and C) cellular invasion through Matrigel-coated chambers. For each graph: Bars = average of 3 or more replicates±SD and graph is representative of 2 or more independent experiments. *significantly reduced compared to NT/, p<0.05; **significantly increased compared to -treated, p<0.05.
Mentions: To test the hypothesis that STAT3 is a critical downstream effector of PKCζ in pancreatic cancer cells, we assessed whether expression of a constitutively active STAT3 construct (STAT3-C) could rescue the effects of PKCζ inhibition in Panc-1 cells. Panc-1 NT and PKCζ RNAi cells were infected with adenovirus expressing flag-tagged, STAT3-C or control () adenovirus (Figure 6A). Expression of STAT3-C significantly recovered anchorage-independent growth of Panc-1 PKCζ RNAi cells, without significantly affecting the anchorage-independent growth of NT RNAi cells (Figure 6B). In addition, the reduced cellular invasion phenotype of PKCζ RNAi cells was significantly recovered by expression of STAT3-C (Figure 6C). Taken together, these data demonstrate that increased cellular STAT3 activity can rescue the anti-oncogenic phenotype of PKCζ RNAi cells, and demonstrate that PKCζ mediates pancreatic cancer cell transformation, at least in part, through regulation of STAT3 activity.

Bottom Line: Furthermore, PKCζ inhibition reduced orthotopic tumor size in vivo by inhibiting tumor cell proliferation and increasing tumor necrosis.In addition, PKCζ inhibition reduced tumor metastases in vivo, and caused a corresponding reduction in pancreatic cancer cell invasion in vitro.We conclude that PKCζ is required for pancreatic cancer cell transformed growth and invasion in vitro and tumorigenesis in vivo, and that STAT3 is an important downstream mediator of the pro-carcinogenic effects of PKCζ in pancreatic cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, United States of America.

ABSTRACT
Pancreatic cancer is a very aggressive disease with few therapeutic options. In this study, we investigate the role of protein kinase C zeta (PKCζ) in pancreatic cancer cells. PKCζ has been shown to act as either a tumor suppressor or tumor promoter depending upon the cellular context. We find that PKCζ expression is either maintained or elevated in primary human pancreatic tumors, but is never lost, consistent with PKCζ playing a promotive role in the pancreatic cancer phenotype. Genetic inhibition of PKCζ reduced adherent growth, cell survival and anchorage-independent growth of human pancreatic cancer cells in vitro. Furthermore, PKCζ inhibition reduced orthotopic tumor size in vivo by inhibiting tumor cell proliferation and increasing tumor necrosis. In addition, PKCζ inhibition reduced tumor metastases in vivo, and caused a corresponding reduction in pancreatic cancer cell invasion in vitro. Signal transducer and activator of transcription 3 (STAT3) is often constitutively active in pancreatic cancer, and plays an important role in pancreatic cancer cell survival and metastasis. Interestingly, inhibition of PKCζ significantly reduced constitutive STAT3 activation in pancreatic cancer cells in vitro and in vivo. Pharmacologic inhibition of STAT3 mimicked the phenotype of PKCζ inhibition, and expression of a constitutively active STAT3 construct rescued the transformed phenotype in PKCζ-deficient cells. We conclude that PKCζ is required for pancreatic cancer cell transformed growth and invasion in vitro and tumorigenesis in vivo, and that STAT3 is an important downstream mediator of the pro-carcinogenic effects of PKCζ in pancreatic cancer cells.

Show MeSH
Related in: MedlinePlus