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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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PKD1 overexpression decreases tumorigenic phenotypes by inhibiting the nuclear transcriptional activity of β-catenin in SW480 colon cancer cells(A)Cell proliferation. An equal number of SW480-GFP and SW480-PKD1-GFP cells were plated and after 48h the amount of ATP present in the metabolically active cells was measured using CellTiter-Glo Reagent. PKD1 overexpression decreased cell proliferation by over 50%. Mean ± SE; n=3; **p<0.05. (B)Anchorage dependent colony formation. SW480 cells overexpressing either PKD1 or GFP vector (2×103) were plated in 100mm dishes for 12 days and the average number of colonies formed was counted and graphed. PKD1 overexpression inhibited anchorage dependent colony formation in SW480 cells. Mean ± SE; n=3; **p<0.05. (C)Anchorage independent colony formation. SW480-GFP and SW480-PKD1-GFP cells (4×104) were seeded in 0.3% agarose and grown for 14 days. The number of colonies formed was enumerated and plotted. PKD1 overexpression decreased anchorage independent colony formation in SW480 cells. Mean ± SE; n=3; **p<0.05. (D)Effect of PKD1 overexpression on β-catenin transcription activity. Reporter luciferase assay was used to measure β-catenin transcription activity. The SW480-PKD1-GFP and SW480-GFP cells were transiently transfected with either a TCF-promoter-luciferase construct or a mutant TCF-promoter luciferase construct along with an internal control plasmid expressing Renilla luciferase gene. The β-catenin transcription activity was measured, normalized to the control Renilla luciferase activity and expressed as a ratio of TCF-promoter-luciferase activity to mutant TCF-promoter luciferase activity. The β-catenin activity of control SW480-GFP cells was set to 100%. PKD1 overexpression decreased the β-catenin transcription activity by over 70%. The inset depicts representative blots of nuclear lysates isolated from SW480-GFP or SW480-PKD1-GFP cells and probed for β-catenin expression. Histone H1 was used as internal control. PKD1 overexpression decreased nuclear β-catenin expression in SW480 cells. (E)Effect on downstream targets. Total protein isolated from SW480-PKD1-GFP or control SW480-GFP cells was resolved on gel and immunoblotted using specific antibodies. β-actin was used as loading control. PKD1 overexpression decreased Cyclin D1 and TCF-4 levels, both of which are downstream products of β-catenin/TCF transcription activity. (F)Immunoprecipitation (IP). Equal amounts of nuclear extract isolated from the PKD1 overexpressing cells or control cells were subjected to IP using anti-TCF-4 antibody. The immune-complexes were resolved on gel and sequentially probed for β-catenin, TCF-4 and PKD1. PKD1 overexpression decreased the amount of TCF-4/β-catenin complex in the nucleus.
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Figure 2: PKD1 overexpression decreases tumorigenic phenotypes by inhibiting the nuclear transcriptional activity of β-catenin in SW480 colon cancer cells(A)Cell proliferation. An equal number of SW480-GFP and SW480-PKD1-GFP cells were plated and after 48h the amount of ATP present in the metabolically active cells was measured using CellTiter-Glo Reagent. PKD1 overexpression decreased cell proliferation by over 50%. Mean ± SE; n=3; **p<0.05. (B)Anchorage dependent colony formation. SW480 cells overexpressing either PKD1 or GFP vector (2×103) were plated in 100mm dishes for 12 days and the average number of colonies formed was counted and graphed. PKD1 overexpression inhibited anchorage dependent colony formation in SW480 cells. Mean ± SE; n=3; **p<0.05. (C)Anchorage independent colony formation. SW480-GFP and SW480-PKD1-GFP cells (4×104) were seeded in 0.3% agarose and grown for 14 days. The number of colonies formed was enumerated and plotted. PKD1 overexpression decreased anchorage independent colony formation in SW480 cells. Mean ± SE; n=3; **p<0.05. (D)Effect of PKD1 overexpression on β-catenin transcription activity. Reporter luciferase assay was used to measure β-catenin transcription activity. The SW480-PKD1-GFP and SW480-GFP cells were transiently transfected with either a TCF-promoter-luciferase construct or a mutant TCF-promoter luciferase construct along with an internal control plasmid expressing Renilla luciferase gene. The β-catenin transcription activity was measured, normalized to the control Renilla luciferase activity and expressed as a ratio of TCF-promoter-luciferase activity to mutant TCF-promoter luciferase activity. The β-catenin activity of control SW480-GFP cells was set to 100%. PKD1 overexpression decreased the β-catenin transcription activity by over 70%. The inset depicts representative blots of nuclear lysates isolated from SW480-GFP or SW480-PKD1-GFP cells and probed for β-catenin expression. Histone H1 was used as internal control. PKD1 overexpression decreased nuclear β-catenin expression in SW480 cells. (E)Effect on downstream targets. Total protein isolated from SW480-PKD1-GFP or control SW480-GFP cells was resolved on gel and immunoblotted using specific antibodies. β-actin was used as loading control. PKD1 overexpression decreased Cyclin D1 and TCF-4 levels, both of which are downstream products of β-catenin/TCF transcription activity. (F)Immunoprecipitation (IP). Equal amounts of nuclear extract isolated from the PKD1 overexpressing cells or control cells were subjected to IP using anti-TCF-4 antibody. The immune-complexes were resolved on gel and sequentially probed for β-catenin, TCF-4 and PKD1. PKD1 overexpression decreased the amount of TCF-4/β-catenin complex in the nucleus.

Mentions: The stable SW480-PKD1-GFP and control SW480-GFP cells were examined for the effect of PKD1 overexpression on tumorigenic characteristics like cell proliferation and colony formation. PKD1 overexpression significantly (p<0.05) decreased cell proliferation compared to control SW480-GFP cells (Figure 2A). We next examined the clonogenic potential of these cells, an important parameter that reflects the ability of single cancer cells to survive, grow and colonize. PKD1 overexpression (SW480-PKD1-GFP) significantly reduced the ability of colon cancer cells to form anchorage dependent colonies, compared to control cells (Figure 2B). The anchorage independent clonogenic assay attempts to mimic the in-vivo situation and evaluates the ability of cells to form independent colonies when suspended in a gel or viscous medium in the absence of any anchor. Similar to results observed in anchorage dependent assay, SW480-PKD1-GFP cells formed fewer number of colonies compared to control SW480-GFP cells in anchorage independent assay. These results indicate a tumor suppressor function for PKD1 in colon cancer.


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)

PKD1 overexpression decreases tumorigenic phenotypes by inhibiting the nuclear transcriptional activity of β-catenin in SW480 colon cancer cells(A)Cell proliferation. An equal number of SW480-GFP and SW480-PKD1-GFP cells were plated and after 48h the amount of ATP present in the metabolically active cells was measured using CellTiter-Glo Reagent. PKD1 overexpression decreased cell proliferation by over 50%. Mean ± SE; n=3; **p<0.05. (B)Anchorage dependent colony formation. SW480 cells overexpressing either PKD1 or GFP vector (2×103) were plated in 100mm dishes for 12 days and the average number of colonies formed was counted and graphed. PKD1 overexpression inhibited anchorage dependent colony formation in SW480 cells. Mean ± SE; n=3; **p<0.05. (C)Anchorage independent colony formation. SW480-GFP and SW480-PKD1-GFP cells (4×104) were seeded in 0.3% agarose and grown for 14 days. The number of colonies formed was enumerated and plotted. PKD1 overexpression decreased anchorage independent colony formation in SW480 cells. Mean ± SE; n=3; **p<0.05. (D)Effect of PKD1 overexpression on β-catenin transcription activity. Reporter luciferase assay was used to measure β-catenin transcription activity. The SW480-PKD1-GFP and SW480-GFP cells were transiently transfected with either a TCF-promoter-luciferase construct or a mutant TCF-promoter luciferase construct along with an internal control plasmid expressing Renilla luciferase gene. The β-catenin transcription activity was measured, normalized to the control Renilla luciferase activity and expressed as a ratio of TCF-promoter-luciferase activity to mutant TCF-promoter luciferase activity. The β-catenin activity of control SW480-GFP cells was set to 100%. PKD1 overexpression decreased the β-catenin transcription activity by over 70%. The inset depicts representative blots of nuclear lysates isolated from SW480-GFP or SW480-PKD1-GFP cells and probed for β-catenin expression. Histone H1 was used as internal control. PKD1 overexpression decreased nuclear β-catenin expression in SW480 cells. (E)Effect on downstream targets. Total protein isolated from SW480-PKD1-GFP or control SW480-GFP cells was resolved on gel and immunoblotted using specific antibodies. β-actin was used as loading control. PKD1 overexpression decreased Cyclin D1 and TCF-4 levels, both of which are downstream products of β-catenin/TCF transcription activity. (F)Immunoprecipitation (IP). Equal amounts of nuclear extract isolated from the PKD1 overexpressing cells or control cells were subjected to IP using anti-TCF-4 antibody. The immune-complexes were resolved on gel and sequentially probed for β-catenin, TCF-4 and PKD1. PKD1 overexpression decreased the amount of TCF-4/β-catenin complex in the nucleus.
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Figure 2: PKD1 overexpression decreases tumorigenic phenotypes by inhibiting the nuclear transcriptional activity of β-catenin in SW480 colon cancer cells(A)Cell proliferation. An equal number of SW480-GFP and SW480-PKD1-GFP cells were plated and after 48h the amount of ATP present in the metabolically active cells was measured using CellTiter-Glo Reagent. PKD1 overexpression decreased cell proliferation by over 50%. Mean ± SE; n=3; **p<0.05. (B)Anchorage dependent colony formation. SW480 cells overexpressing either PKD1 or GFP vector (2×103) were plated in 100mm dishes for 12 days and the average number of colonies formed was counted and graphed. PKD1 overexpression inhibited anchorage dependent colony formation in SW480 cells. Mean ± SE; n=3; **p<0.05. (C)Anchorage independent colony formation. SW480-GFP and SW480-PKD1-GFP cells (4×104) were seeded in 0.3% agarose and grown for 14 days. The number of colonies formed was enumerated and plotted. PKD1 overexpression decreased anchorage independent colony formation in SW480 cells. Mean ± SE; n=3; **p<0.05. (D)Effect of PKD1 overexpression on β-catenin transcription activity. Reporter luciferase assay was used to measure β-catenin transcription activity. The SW480-PKD1-GFP and SW480-GFP cells were transiently transfected with either a TCF-promoter-luciferase construct or a mutant TCF-promoter luciferase construct along with an internal control plasmid expressing Renilla luciferase gene. The β-catenin transcription activity was measured, normalized to the control Renilla luciferase activity and expressed as a ratio of TCF-promoter-luciferase activity to mutant TCF-promoter luciferase activity. The β-catenin activity of control SW480-GFP cells was set to 100%. PKD1 overexpression decreased the β-catenin transcription activity by over 70%. The inset depicts representative blots of nuclear lysates isolated from SW480-GFP or SW480-PKD1-GFP cells and probed for β-catenin expression. Histone H1 was used as internal control. PKD1 overexpression decreased nuclear β-catenin expression in SW480 cells. (E)Effect on downstream targets. Total protein isolated from SW480-PKD1-GFP or control SW480-GFP cells was resolved on gel and immunoblotted using specific antibodies. β-actin was used as loading control. PKD1 overexpression decreased Cyclin D1 and TCF-4 levels, both of which are downstream products of β-catenin/TCF transcription activity. (F)Immunoprecipitation (IP). Equal amounts of nuclear extract isolated from the PKD1 overexpressing cells or control cells were subjected to IP using anti-TCF-4 antibody. The immune-complexes were resolved on gel and sequentially probed for β-catenin, TCF-4 and PKD1. PKD1 overexpression decreased the amount of TCF-4/β-catenin complex in the nucleus.
Mentions: The stable SW480-PKD1-GFP and control SW480-GFP cells were examined for the effect of PKD1 overexpression on tumorigenic characteristics like cell proliferation and colony formation. PKD1 overexpression significantly (p<0.05) decreased cell proliferation compared to control SW480-GFP cells (Figure 2A). We next examined the clonogenic potential of these cells, an important parameter that reflects the ability of single cancer cells to survive, grow and colonize. PKD1 overexpression (SW480-PKD1-GFP) significantly reduced the ability of colon cancer cells to form anchorage dependent colonies, compared to control cells (Figure 2B). The anchorage independent clonogenic assay attempts to mimic the in-vivo situation and evaluates the ability of cells to form independent colonies when suspended in a gel or viscous medium in the absence of any anchor. Similar to results observed in anchorage dependent assay, SW480-PKD1-GFP cells formed fewer number of colonies compared to control SW480-GFP cells in anchorage independent assay. These results indicate a tumor suppressor function for PKD1 in colon cancer.

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