Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of PKCζ expression significantly reduces orthotopic pancreatic tumor proliferation and increases tumor necrosis.A) Quantitative analysis of tumor proliferation detected by BrdUrd incorporation; *p<0.003. B) Quantitative analysis of tumor apoptosis detected by cleaved caspase-3 staining. C) Representative H&E stained orthotopic Panc-1 NT and PKCζ RNAi pancreatic tumors with areas of necrosis identified (yellow outline) (bar = 1 mm). Green line delineates tumor tissue. D) Quantitative analysis of tumor necrosis plotted as percent of total tumor area; *p<0.0002. E) Quantitative analysis of tumor vascularity, as determined by percent area CD31 staining. A–E) n = 16 NT RNAi tumors and 15 PKCζ RNAi tumors. F) Panc-1 NT and PKCζ RNAi cells (z1 and z2) were assessed for cellular invasion through Matrigel-coated chambers. Bars = average of 3 or more replicates+/−SD and graph is representative of 2 or more independent experiments. *p<0.05 vs NT.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3756013&req=5

pone-0072061-g004: Inhibition of PKCζ expression significantly reduces orthotopic pancreatic tumor proliferation and increases tumor necrosis.A) Quantitative analysis of tumor proliferation detected by BrdUrd incorporation; *p<0.003. B) Quantitative analysis of tumor apoptosis detected by cleaved caspase-3 staining. C) Representative H&E stained orthotopic Panc-1 NT and PKCζ RNAi pancreatic tumors with areas of necrosis identified (yellow outline) (bar = 1 mm). Green line delineates tumor tissue. D) Quantitative analysis of tumor necrosis plotted as percent of total tumor area; *p<0.0002. E) Quantitative analysis of tumor vascularity, as determined by percent area CD31 staining. A–E) n = 16 NT RNAi tumors and 15 PKCζ RNAi tumors. F) Panc-1 NT and PKCζ RNAi cells (z1 and z2) were assessed for cellular invasion through Matrigel-coated chambers. Bars = average of 3 or more replicates+/−SD and graph is representative of 2 or more independent experiments. *p<0.05 vs NT.

Mentions: We next investigated the effect of PKCζ KD on pancreatic tumor formation and growth using a previously described Panc-1 orthotopic tumor model [16]. Panc-1 cells expressing the firefly luciferase gene (pSIN-Fluc) and either NT or PKCζ RNAi were injected into the pancreas of nude mice to form orthotopic tumors. Tumor growth was monitored by bioluminescence detection (Figure 3A), and mice were harvested 5 weeks after inoculation. Tumor formation was observed in all mice injected with Panc-1 cells expressing either RNAi construct; however, final pancreas weight was significantly lower in mice bearing PKCζ RNAi tumors, due to reduced tumor size (Figure 3B and 3C). We hypothesized that, similar to the effect of PKCζ KD in vitro (Figure 2B–D), the reduced tumor size of PKCζ KD Panc-1 cells in vivo was due to reduced tumor cell proliferation and enhanced tumor cell death. The level of BrdUrd incorporation, a measure of tumor proliferation, was evaluated in Panc-1 PKCζ RNAi tumors and compared to the level of BrdUrd incorporation in Panc-1 NT RNAi tumors [16]. As predicted, tumor proliferation was significantly reduced in PKCζ RNAi tumors compared to NT RNAi tumors (Figure 4A). Interestingly, we did not observe a significant effect of PKCζ KD on tumor apoptosis, detected by cleaved caspase-3 (Figure 4B). However, PKCζ RNAi tumors had a significantly higher level of necrosis than NT RNAi tumors (Figure 4C and 4D). Tumor necrosis results from an accumulation of tumor cell death, which can occur when a tumor outgrows its blood supply. Although PKCζ RNAi tumors are drastically smaller than NT RNAi tumors, they do not exhibit a decrease in tumor blood vessel density as quantified by CD31 staining (Figure 4E). These data suggest that the reduced tumor volume of PKCζ RNAi pancreatic tumors is the result of the cumulative effect of decreased cell proliferation and survival over the time course of the in vivo experiment.


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)

Inhibition of PKCζ expression significantly reduces orthotopic pancreatic tumor proliferation and increases tumor necrosis.A) Quantitative analysis of tumor proliferation detected by BrdUrd incorporation; *p<0.003. B) Quantitative analysis of tumor apoptosis detected by cleaved caspase-3 staining. C) Representative H&E stained orthotopic Panc-1 NT and PKCζ RNAi pancreatic tumors with areas of necrosis identified (yellow outline) (bar = 1 mm). Green line delineates tumor tissue. D) Quantitative analysis of tumor necrosis plotted as percent of total tumor area; *p<0.0002. E) Quantitative analysis of tumor vascularity, as determined by percent area CD31 staining. A–E) n = 16 NT RNAi tumors and 15 PKCζ RNAi tumors. F) Panc-1 NT and PKCζ RNAi cells (z1 and z2) were assessed for cellular invasion through Matrigel-coated chambers. Bars = average of 3 or more replicates+/−SD and graph is representative of 2 or more independent experiments. *p<0.05 vs NT.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756013&req=5

pone-0072061-g004: Inhibition of PKCζ expression significantly reduces orthotopic pancreatic tumor proliferation and increases tumor necrosis.A) Quantitative analysis of tumor proliferation detected by BrdUrd incorporation; *p<0.003. B) Quantitative analysis of tumor apoptosis detected by cleaved caspase-3 staining. C) Representative H&E stained orthotopic Panc-1 NT and PKCζ RNAi pancreatic tumors with areas of necrosis identified (yellow outline) (bar = 1 mm). Green line delineates tumor tissue. D) Quantitative analysis of tumor necrosis plotted as percent of total tumor area; *p<0.0002. E) Quantitative analysis of tumor vascularity, as determined by percent area CD31 staining. A–E) n = 16 NT RNAi tumors and 15 PKCζ RNAi tumors. F) Panc-1 NT and PKCζ RNAi cells (z1 and z2) were assessed for cellular invasion through Matrigel-coated chambers. Bars = average of 3 or more replicates+/−SD and graph is representative of 2 or more independent experiments. *p<0.05 vs NT.
Mentions: We next investigated the effect of PKCζ KD on pancreatic tumor formation and growth using a previously described Panc-1 orthotopic tumor model [16]. Panc-1 cells expressing the firefly luciferase gene (pSIN-Fluc) and either NT or PKCζ RNAi were injected into the pancreas of nude mice to form orthotopic tumors. Tumor growth was monitored by bioluminescence detection (Figure 3A), and mice were harvested 5 weeks after inoculation. Tumor formation was observed in all mice injected with Panc-1 cells expressing either RNAi construct; however, final pancreas weight was significantly lower in mice bearing PKCζ RNAi tumors, due to reduced tumor size (Figure 3B and 3C). We hypothesized that, similar to the effect of PKCζ KD in vitro (Figure 2B–D), the reduced tumor size of PKCζ KD Panc-1 cells in vivo was due to reduced tumor cell proliferation and enhanced tumor cell death. The level of BrdUrd incorporation, a measure of tumor proliferation, was evaluated in Panc-1 PKCζ RNAi tumors and compared to the level of BrdUrd incorporation in Panc-1 NT RNAi tumors [16]. As predicted, tumor proliferation was significantly reduced in PKCζ RNAi tumors compared to NT RNAi tumors (Figure 4A). Interestingly, we did not observe a significant effect of PKCζ KD on tumor apoptosis, detected by cleaved caspase-3 (Figure 4B). However, PKCζ RNAi tumors had a significantly higher level of necrosis than NT RNAi tumors (Figure 4C and 4D). Tumor necrosis results from an accumulation of tumor cell death, which can occur when a tumor outgrows its blood supply. Although PKCζ RNAi tumors are drastically smaller than NT RNAi tumors, they do not exhibit a decrease in tumor blood vessel density as quantified by CD31 staining (Figure 4E). These data suggest that the reduced tumor volume of PKCζ RNAi pancreatic tumors is the result of the cumulative effect of decreased cell proliferation and survival over the time course of the in vivo experiment.

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