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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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Expression of PKD1 is downregulated in colon cancer(A)Immunohistochemical analysis of PKD1 and β-catenin in colon tissues: Normal colon tissues were immunostained for PKD1 (red) and β-catenin (green). PKD1 expression was detected in the cytoplasm and membrane of colon cells, while β-catenin was primarily localized to the membrane. Co-localization of PKD1 with β-catenin was also detected (yellow). A magnified image of a single colon gland is shown to demonstrate co-localization of PKD1 and β-catenin (white arrows). Original magnification 200X. (B)Tissue microarray (TMA): Colon cancer TMA slides were stained for β-catenin (brown) and PKD1 (red). β-catenin staining revealed distinct change in subcellular localization in colon cancer. It was primarily localized on the membrane of non-neoplastic samples (1a), while distinct cytoplasmic and prominent nuclear staining was detected in Duke's stage B (2a) and Duke's stage C colon cancer, respectively (3a). PKD1 expression was strongly detected in non-neoplastic samples (1b, red). However, PKD1 was progressively downregulated in Duke's stage B (2b) and Duke's stage C (3b) colon cancer. Original magnification 400X.
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Figure 1: Expression of PKD1 is downregulated in colon cancer(A)Immunohistochemical analysis of PKD1 and β-catenin in colon tissues: Normal colon tissues were immunostained for PKD1 (red) and β-catenin (green). PKD1 expression was detected in the cytoplasm and membrane of colon cells, while β-catenin was primarily localized to the membrane. Co-localization of PKD1 with β-catenin was also detected (yellow). A magnified image of a single colon gland is shown to demonstrate co-localization of PKD1 and β-catenin (white arrows). Original magnification 200X. (B)Tissue microarray (TMA): Colon cancer TMA slides were stained for β-catenin (brown) and PKD1 (red). β-catenin staining revealed distinct change in subcellular localization in colon cancer. It was primarily localized on the membrane of non-neoplastic samples (1a), while distinct cytoplasmic and prominent nuclear staining was detected in Duke's stage B (2a) and Duke's stage C colon cancer, respectively (3a). PKD1 expression was strongly detected in non-neoplastic samples (1b, red). However, PKD1 was progressively downregulated in Duke's stage B (2b) and Duke's stage C (3b) colon cancer. Original magnification 400X.

Mentions: The deregulation of PKD1 expression is associated with various cancers including prostate and breast cancer [10, 13, 19, 20]. However, the expression profile of PKD1 in colon cancer is not known. Therefore, we investigated the expression pattern of PKD1 by immunofluorescence staining of colon tissues using anti-PKD1 antibody and fluorescently labeled secondary antibodies (red) (Figure 1A). Additionally, tissues were also simultaneously co-stained for β-catenin expression using anti-β-catenin antibody. Representative images from normal colon tissue stained for PKD1 and β-catenin are shown in Figure 1A. PKD1 expression (red staining) was predominantly detected in the cytoplasm with some expression on the membrane and in the nucleus of the cells, while β-catenin expression (green staining) was primarily localized to the membrane of the cells. The immunohistochemical (IHC) staining also revealed co-localization of PKD1 and β-catenin in colon tissues (Figure 1A, lower panel). This suggests a role for PKD1-β-catenin interaction in colon tissues. In order to investigate the expression profile of PKD1 in colon cancer tissues and quantitatively analyze changes in PKD1 or β-catenin expression, IHC analysis was performed on tissue microarray (TMA) slides containing normal (n=8) and colon cancer tissues (n=45) using chromogenic dyes (Figure 1B). The tissue samples were grouped based on the Dukes' staging of colon cancer into non-neoplastic, Duke's stage B (wherein the cancer has invaded the bowel walls, but has not spread to the lymph nodes) and Duke's stage C colon cancer (wherein the cancer has spread to the nearby lymph node) and analyzed for the levels of expression and the localization pattern of the proteins. PKD1 expression was significantly (p<0.05) downregulated in the cancerous tissues compared to normal tissues (Figure S1A). We also detected a trend in the progressive downregulation of PKD1 expression from non-neoplastic stage to Duke's stage B and Duke's stage C colon cancer (Figure 1B). The suppression of PKD1 expression coincided with the distinct change in the β-catenin localization in cancer tissues. While β-catenin was primarily localized on the membrane of normal colon glandular cells, a higher β-catenin staining was detected in the cytoplasm and the nucleus as the cancer progressed from Duke's stage B to Duke's stage C colon cancer, when the cancer had spread to the nearby lymph node. The association between downregulation of PKD1 expression with the change in β-catenin localization in colon cancer seems to suggest a role for PKD1 in regulating β-catenin functions in colon cancer. Based on these results, we proposed that PKD1 functions as a tumor suppressor via modulating β-catenin signaling pathway and inhibiting nuclear β-catenin function to suppress colon cancer growth. Thus an increase in PKD1 levels in colon cancer cells can inhibit the progression of 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)

Expression of PKD1 is downregulated in colon cancer(A)Immunohistochemical analysis of PKD1 and β-catenin in colon tissues: Normal colon tissues were immunostained for PKD1 (red) and β-catenin (green). PKD1 expression was detected in the cytoplasm and membrane of colon cells, while β-catenin was primarily localized to the membrane. Co-localization of PKD1 with β-catenin was also detected (yellow). A magnified image of a single colon gland is shown to demonstrate co-localization of PKD1 and β-catenin (white arrows). Original magnification 200X. (B)Tissue microarray (TMA): Colon cancer TMA slides were stained for β-catenin (brown) and PKD1 (red). β-catenin staining revealed distinct change in subcellular localization in colon cancer. It was primarily localized on the membrane of non-neoplastic samples (1a), while distinct cytoplasmic and prominent nuclear staining was detected in Duke's stage B (2a) and Duke's stage C colon cancer, respectively (3a). PKD1 expression was strongly detected in non-neoplastic samples (1b, red). However, PKD1 was progressively downregulated in Duke's stage B (2b) and Duke's stage C (3b) colon cancer. Original magnification 400X.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Expression of PKD1 is downregulated in colon cancer(A)Immunohistochemical analysis of PKD1 and β-catenin in colon tissues: Normal colon tissues were immunostained for PKD1 (red) and β-catenin (green). PKD1 expression was detected in the cytoplasm and membrane of colon cells, while β-catenin was primarily localized to the membrane. Co-localization of PKD1 with β-catenin was also detected (yellow). A magnified image of a single colon gland is shown to demonstrate co-localization of PKD1 and β-catenin (white arrows). Original magnification 200X. (B)Tissue microarray (TMA): Colon cancer TMA slides were stained for β-catenin (brown) and PKD1 (red). β-catenin staining revealed distinct change in subcellular localization in colon cancer. It was primarily localized on the membrane of non-neoplastic samples (1a), while distinct cytoplasmic and prominent nuclear staining was detected in Duke's stage B (2a) and Duke's stage C colon cancer, respectively (3a). PKD1 expression was strongly detected in non-neoplastic samples (1b, red). However, PKD1 was progressively downregulated in Duke's stage B (2b) and Duke's stage C (3b) colon cancer. Original magnification 400X.
Mentions: The deregulation of PKD1 expression is associated with various cancers including prostate and breast cancer [10, 13, 19, 20]. However, the expression profile of PKD1 in colon cancer is not known. Therefore, we investigated the expression pattern of PKD1 by immunofluorescence staining of colon tissues using anti-PKD1 antibody and fluorescently labeled secondary antibodies (red) (Figure 1A). Additionally, tissues were also simultaneously co-stained for β-catenin expression using anti-β-catenin antibody. Representative images from normal colon tissue stained for PKD1 and β-catenin are shown in Figure 1A. PKD1 expression (red staining) was predominantly detected in the cytoplasm with some expression on the membrane and in the nucleus of the cells, while β-catenin expression (green staining) was primarily localized to the membrane of the cells. The immunohistochemical (IHC) staining also revealed co-localization of PKD1 and β-catenin in colon tissues (Figure 1A, lower panel). This suggests a role for PKD1-β-catenin interaction in colon tissues. In order to investigate the expression profile of PKD1 in colon cancer tissues and quantitatively analyze changes in PKD1 or β-catenin expression, IHC analysis was performed on tissue microarray (TMA) slides containing normal (n=8) and colon cancer tissues (n=45) using chromogenic dyes (Figure 1B). The tissue samples were grouped based on the Dukes' staging of colon cancer into non-neoplastic, Duke's stage B (wherein the cancer has invaded the bowel walls, but has not spread to the lymph nodes) and Duke's stage C colon cancer (wherein the cancer has spread to the nearby lymph node) and analyzed for the levels of expression and the localization pattern of the proteins. PKD1 expression was significantly (p<0.05) downregulated in the cancerous tissues compared to normal tissues (Figure S1A). We also detected a trend in the progressive downregulation of PKD1 expression from non-neoplastic stage to Duke's stage B and Duke's stage C colon cancer (Figure 1B). The suppression of PKD1 expression coincided with the distinct change in the β-catenin localization in cancer tissues. While β-catenin was primarily localized on the membrane of normal colon glandular cells, a higher β-catenin staining was detected in the cytoplasm and the nucleus as the cancer progressed from Duke's stage B to Duke's stage C colon cancer, when the cancer had spread to the nearby lymph node. The association between downregulation of PKD1 expression with the change in β-catenin localization in colon cancer seems to suggest a role for PKD1 in regulating β-catenin functions in colon cancer. Based on these results, we proposed that PKD1 functions as a tumor suppressor via modulating β-catenin signaling pathway and inhibiting nuclear β-catenin function to suppress colon cancer growth. Thus an increase in PKD1 levels in colon cancer cells can inhibit the progression of 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