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Protein kinase D1 attenuates tumorigenesis in colon cancer by modulating β-catenin/T cell factor activity.

Sundram V, Ganju A, Hughes JE, Khan S, Chauhan SC, Jaggi M - Oncotarget (2014)

Bottom Line: This subcellular modulation of β-catenin results in enhanced membrane localization of β-catenin and thereby increases cell-cell adhesion.Studies in a xenograft mouse model indicate that PKD1 overexpression delayed tumor appearance, enhanced necrosis and lowered tumor hypoxia.Overall, our results demonstrate a putative tumor-suppressor function of PKD1 in colon tumorigenesis via modulation of β-catenin functions in cells.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Research Center, Sanford Research/USD, Sioux Falls, SD, USA. .

ABSTRACT
Over 80% of colon cancer development and progression is a result of the dysregulation of β-catenin signaling pathway. Herein, for the first time, we demonstrate that a serine-threonine kinase, Protein Kinase D1 (PKD1), modulates the functions of β-catenin to suppress colon cancer growth. Analysis of normal and colon cancer tissues reveals downregulation of PKD1 expression in advanced stages of colon cancer and its co-localization with β-catenin in the colon crypts. This PKD1 downregulation corresponds with the aberrant expression and nuclear localization of β-catenin. In-vitro investigation of the PKD1-β-catenin interaction in colon cancer cells reveal that PKD1 overexpression suppresses cell proliferation and clonogenic potential and enhances cell-cell aggregation. We demonstrate that PKD1 directly interacts with β-catenin and attenuates β-catenin transcriptional activity by decreasing nuclear β-catenin levels. Additionally, we show that inhibition of nuclear β-catenin transcriptional activity is predominantly influenced by nucleus targeted PKD1. This subcellular modulation of β-catenin results in enhanced membrane localization of β-catenin and thereby increases cell-cell adhesion. Studies in a xenograft mouse model indicate that PKD1 overexpression delayed tumor appearance, enhanced necrosis and lowered tumor hypoxia. Overall, our results demonstrate a putative tumor-suppressor function of PKD1 in colon tumorigenesis via modulation of β-catenin functions in cells.

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Overexpression of PKD1 inhibits cellular motility(A). Cell migration assay. SW480 cells overexpressing PKD1 or GFP were mixed with agarose and equal volume was placed on fibrinonectin/BSA coated plates. Representative images of an agarose bead edge with motile cells are shown. The inset shows the corresponding whole agarose bead. PKD1 overexpression decreased the motility of SW480 cells. (B)Quantitative analysis of cell migration assay. The number of cells that migrated from the agarose bead was counted and plotted. PKD1 overexpression significantly inhibited migration of SW480 cells. Mean ± SE; n=3; **p<0.05. (C)Scratch assay. Confluent growth of SW480-PKD1-GFP or SW480-GFP cells was ‘wounded’ by a scratch using a micropipette tip. The ‘wound’ was periodically monitored for ‘wound healing’ and photographed. PKD1 overexpression decreased the motility of SW480 cells compared to vector control. (D)Immunoblot analysis. Representative blots of whole cell lysates from SW480-PKD1-GFP and control SW480-GFP cells were probed for proteins regulating cellular motility. PKD1 overexpression enhanced the expression levels of inactive phospho-cofilin and cofilin.
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Figure 5: Overexpression of PKD1 inhibits cellular motility(A). Cell migration assay. SW480 cells overexpressing PKD1 or GFP were mixed with agarose and equal volume was placed on fibrinonectin/BSA coated plates. Representative images of an agarose bead edge with motile cells are shown. The inset shows the corresponding whole agarose bead. PKD1 overexpression decreased the motility of SW480 cells. (B)Quantitative analysis of cell migration assay. The number of cells that migrated from the agarose bead was counted and plotted. PKD1 overexpression significantly inhibited migration of SW480 cells. Mean ± SE; n=3; **p<0.05. (C)Scratch assay. Confluent growth of SW480-PKD1-GFP or SW480-GFP cells was ‘wounded’ by a scratch using a micropipette tip. The ‘wound’ was periodically monitored for ‘wound healing’ and photographed. PKD1 overexpression decreased the motility of SW480 cells compared to vector control. (D)Immunoblot analysis. Representative blots of whole cell lysates from SW480-PKD1-GFP and control SW480-GFP cells were probed for proteins regulating cellular motility. PKD1 overexpression enhanced the expression levels of inactive phospho-cofilin and cofilin.

Mentions: PKD1 plays a significant role in regulating cellular motility [18, 27, 28]. PKD1 has been shown to inhibit cellular motility by interacting with proteins at the leading edge of the motile cells. It negatively regulates cellular motility in part by indirectly maintaining the depolymerizing factor cofilin in its inactive phosphorylated form. PKD1 achieves this by enhancing cofilin phosphorylation (through PAK4-LIMK pathway) or by inhibiting its de-phosphorylation (through the direct phosphorylation and inhibition of SS1L phosphatase function) to shift the equilibrium towards maintaining the phosphorylated and inactive form of cofilin [14, 18]. Therefore, we evaluated the effect of PKD1 overexpression on motility of SW480 colon cancer cells using the agarose bead assay. In this test, the SW480-PKD1-GFP or SW480-GFP cells were embedded within agarose beads and spotted on cell culture plates pre-coated with fibrinonectin to examine the ability of cells to escape the beads and migrate on the surface of the plate (Figure 5A). PKD1 overexpression inhibited the motility of colon cancer cells compared to control SW480-GFP cells (Figure 5B). The ability of PKD1 to inhibit motility of SW480 colon cancer cells was also confirmed using a wound-healing assay (Figure 5C). A wound (or scratch) was created using the pointed edge of a sterile pipette tip on the surface of confluent plates of SW480-PKD1-GFP or SW480-GFP cells and the plates were examined at regular intervals to document and evaluate wound healing (or gap closure) by motile cells. SW480-GFP cells more effectively closed the gap/wound compared to PKD1 overexpressing cells (SW480-PKD1-GFP). In order to examine the molecular mechanisms regulating the cellular motility of the PKD1 overexpressing colon cancer cells, total protein lysates from SW480-PKD1-GFP or SW480-GFP cells were immunoblotted and examined for the expression of various motility related proteins (Figure 5D). We observed enhanced expression and phosphorylation of cofilin in PKD1 overexpressing cells compared to control cells. Little to no change was observed either in the expression or phosphorylation of other proteins involved in actin remodeling, including LIMK, Arp2 and Arp3. Thus PKD1 overexpression appeared to inhibit cellular motility partly by inhibiting the activity of cofilin, a protein critical for depolymerization of filamentous actin filaments to generate new monomeric actin for formation/extension of actin fibers at the leading edge.


Protein kinase D1 attenuates tumorigenesis in colon cancer by modulating β-catenin/T cell factor activity.

Sundram V, Ganju A, Hughes JE, Khan S, Chauhan SC, Jaggi M - Oncotarget (2014)

Overexpression of PKD1 inhibits cellular motility(A). Cell migration assay. SW480 cells overexpressing PKD1 or GFP were mixed with agarose and equal volume was placed on fibrinonectin/BSA coated plates. Representative images of an agarose bead edge with motile cells are shown. The inset shows the corresponding whole agarose bead. PKD1 overexpression decreased the motility of SW480 cells. (B)Quantitative analysis of cell migration assay. The number of cells that migrated from the agarose bead was counted and plotted. PKD1 overexpression significantly inhibited migration of SW480 cells. Mean ± SE; n=3; **p<0.05. (C)Scratch assay. Confluent growth of SW480-PKD1-GFP or SW480-GFP cells was ‘wounded’ by a scratch using a micropipette tip. The ‘wound’ was periodically monitored for ‘wound healing’ and photographed. PKD1 overexpression decreased the motility of SW480 cells compared to vector control. (D)Immunoblot analysis. Representative blots of whole cell lysates from SW480-PKD1-GFP and control SW480-GFP cells were probed for proteins regulating cellular motility. PKD1 overexpression enhanced the expression levels of inactive phospho-cofilin and cofilin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Overexpression of PKD1 inhibits cellular motility(A). Cell migration assay. SW480 cells overexpressing PKD1 or GFP were mixed with agarose and equal volume was placed on fibrinonectin/BSA coated plates. Representative images of an agarose bead edge with motile cells are shown. The inset shows the corresponding whole agarose bead. PKD1 overexpression decreased the motility of SW480 cells. (B)Quantitative analysis of cell migration assay. The number of cells that migrated from the agarose bead was counted and plotted. PKD1 overexpression significantly inhibited migration of SW480 cells. Mean ± SE; n=3; **p<0.05. (C)Scratch assay. Confluent growth of SW480-PKD1-GFP or SW480-GFP cells was ‘wounded’ by a scratch using a micropipette tip. The ‘wound’ was periodically monitored for ‘wound healing’ and photographed. PKD1 overexpression decreased the motility of SW480 cells compared to vector control. (D)Immunoblot analysis. Representative blots of whole cell lysates from SW480-PKD1-GFP and control SW480-GFP cells were probed for proteins regulating cellular motility. PKD1 overexpression enhanced the expression levels of inactive phospho-cofilin and cofilin.
Mentions: PKD1 plays a significant role in regulating cellular motility [18, 27, 28]. PKD1 has been shown to inhibit cellular motility by interacting with proteins at the leading edge of the motile cells. It negatively regulates cellular motility in part by indirectly maintaining the depolymerizing factor cofilin in its inactive phosphorylated form. PKD1 achieves this by enhancing cofilin phosphorylation (through PAK4-LIMK pathway) or by inhibiting its de-phosphorylation (through the direct phosphorylation and inhibition of SS1L phosphatase function) to shift the equilibrium towards maintaining the phosphorylated and inactive form of cofilin [14, 18]. Therefore, we evaluated the effect of PKD1 overexpression on motility of SW480 colon cancer cells using the agarose bead assay. In this test, the SW480-PKD1-GFP or SW480-GFP cells were embedded within agarose beads and spotted on cell culture plates pre-coated with fibrinonectin to examine the ability of cells to escape the beads and migrate on the surface of the plate (Figure 5A). PKD1 overexpression inhibited the motility of colon cancer cells compared to control SW480-GFP cells (Figure 5B). The ability of PKD1 to inhibit motility of SW480 colon cancer cells was also confirmed using a wound-healing assay (Figure 5C). A wound (or scratch) was created using the pointed edge of a sterile pipette tip on the surface of confluent plates of SW480-PKD1-GFP or SW480-GFP cells and the plates were examined at regular intervals to document and evaluate wound healing (or gap closure) by motile cells. SW480-GFP cells more effectively closed the gap/wound compared to PKD1 overexpressing cells (SW480-PKD1-GFP). In order to examine the molecular mechanisms regulating the cellular motility of the PKD1 overexpressing colon cancer cells, total protein lysates from SW480-PKD1-GFP or SW480-GFP cells were immunoblotted and examined for the expression of various motility related proteins (Figure 5D). We observed enhanced expression and phosphorylation of cofilin in PKD1 overexpressing cells compared to control cells. Little to no change was observed either in the expression or phosphorylation of other proteins involved in actin remodeling, including LIMK, Arp2 and Arp3. Thus PKD1 overexpression appeared to inhibit cellular motility partly by inhibiting the activity of cofilin, a protein critical for depolymerization of filamentous actin filaments to generate new monomeric actin for formation/extension of actin fibers at the leading edge.

Bottom Line: This subcellular modulation of β-catenin results in enhanced membrane localization of β-catenin and thereby increases cell-cell adhesion.Studies in a xenograft mouse model indicate that PKD1 overexpression delayed tumor appearance, enhanced necrosis and lowered tumor hypoxia.Overall, our results demonstrate a putative tumor-suppressor function of PKD1 in colon tumorigenesis via modulation of β-catenin functions in cells.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Research Center, Sanford Research/USD, Sioux Falls, SD, USA. .

ABSTRACT
Over 80% of colon cancer development and progression is a result of the dysregulation of β-catenin signaling pathway. Herein, for the first time, we demonstrate that a serine-threonine kinase, Protein Kinase D1 (PKD1), modulates the functions of β-catenin to suppress colon cancer growth. Analysis of normal and colon cancer tissues reveals downregulation of PKD1 expression in advanced stages of colon cancer and its co-localization with β-catenin in the colon crypts. This PKD1 downregulation corresponds with the aberrant expression and nuclear localization of β-catenin. In-vitro investigation of the PKD1-β-catenin interaction in colon cancer cells reveal that PKD1 overexpression suppresses cell proliferation and clonogenic potential and enhances cell-cell aggregation. We demonstrate that PKD1 directly interacts with β-catenin and attenuates β-catenin transcriptional activity by decreasing nuclear β-catenin levels. Additionally, we show that inhibition of nuclear β-catenin transcriptional activity is predominantly influenced by nucleus targeted PKD1. This subcellular modulation of β-catenin results in enhanced membrane localization of β-catenin and thereby increases cell-cell adhesion. Studies in a xenograft mouse model indicate that PKD1 overexpression delayed tumor appearance, enhanced necrosis and lowered tumor hypoxia. Overall, our results demonstrate a putative tumor-suppressor function of PKD1 in colon tumorigenesis via modulation of β-catenin functions in cells.

Show MeSH
Related in: MedlinePlus