Limits...
Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 dephosphorylation, which is required for transformation and metastasis.

Khapare N, Kundu ST, Sehgal L, Sawant M, Priya R, Gosavi P, Gupta N, Alam H, Karkhanis M, Naik N, Vaidya MM, Dalal SN - PLoS ONE (2012)

Bottom Line: The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8.Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation.These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.

View Article: PubMed Central - PubMed

Affiliation: Tata Memorial Centre, Kharghar Node, Navi Mumbai, Maharashtra, India.

ABSTRACT
The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.

Show MeSH

Related in: MedlinePlus

K8 downregulation leads to an inhibition of transformation in vitro and in vivo.A. The S10 derived K8 (8.21, 8.24 and 8.28) knockdown clones or the vector alone (S10P3) were plated in soft agar and colony formation determined after 2–3 weeks. The number of colonies formed by the clones per 20 low power fields (10X) was counted in triplicate in each experiment and the mean and standard deviation of three independent experiments is plotted as shown. B. 106 cells from the S10 derived K8 (8.21 and 8.28) knockdown clones or the vector alone (S10P3) were injected subcutaneously into 5 different nude mice and tumor size determined every week as described. Tumor volume is plotted on the Y-axis and the time in weeks on the X-axis. C. Protein extracts from primary tumors from mice injected with the S10 derived K8 (8.21 and 8.28) knockdown clones or the vector alone (S10P3) were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K8 and β-actin. The numbers indicate different mice injected with the single or double knockdown clones. All the samples were run on the same gel and the Western blots performed at the same time. D. Haematoxylin and eosin staining of paraffin embedded sections of lung tissue from nude mice injected with 106 cells of the vector alone (S10P3) or the double knockdown clones (8.21 and 8.28). Lung section from mice injected with S10P3 cells show extensive metastasis with thickening of alveolar walls from deposition and aggregation of metastasized tumor cells, whereas lungs from mice injected with the double knockdown cells show normal lungs with thin walled alveoli, with a few metastatic tumor cells. The images in the top row are at magnification x100 and images in the bottom row are at magnification x 400. E. PCR reactions were performed on DNA isolated from paraffin sections for the presence of Alu repeats in genomic DNA. Genomic DNA was purified from normal lung tissue, lung tissue from mice injected with cells with PKP3 knockdown alone (S10P3) and lung tissue from mice injected with the double knockdown clones (8.21 and 8.28). Lung tissues from uninjected mice (N) were used as a negative control for the Alu PCR. A PCR for the mouse patch gene was performed as a loading control. F. Immunohistochemical staining was performed with antibody against K8 on sections of paraffin embedded tissue of tumor and lungs of mice injected with S10P3 vector control cells or the double knockdown clones, 8.21 and 8.28. Images a, b, c, g, h and i are taken at a magnification x 200 and the respective magnified images at magnification x 400, of the indicated areas in the white rectangles, are represented by images d, e, f, j, k and l. Tumor tissue from mice injected with S10P3 (a and d) show K8 staining at levels higher compared to tumors from mice injected with the double knockdown clones, 8.21 and 8.28 (b, c, e and f). In contrast lung tissue, from mice injected with the vector control S10P3 cells (g and j) and the double knockdown clones (8.21 and 8.28) (h, i, k and l) show elevated K8 staining in metastatic areas of the lungs with infiltrated tumor cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368841&req=5

pone-0038561-g004: K8 downregulation leads to an inhibition of transformation in vitro and in vivo.A. The S10 derived K8 (8.21, 8.24 and 8.28) knockdown clones or the vector alone (S10P3) were plated in soft agar and colony formation determined after 2–3 weeks. The number of colonies formed by the clones per 20 low power fields (10X) was counted in triplicate in each experiment and the mean and standard deviation of three independent experiments is plotted as shown. B. 106 cells from the S10 derived K8 (8.21 and 8.28) knockdown clones or the vector alone (S10P3) were injected subcutaneously into 5 different nude mice and tumor size determined every week as described. Tumor volume is plotted on the Y-axis and the time in weeks on the X-axis. C. Protein extracts from primary tumors from mice injected with the S10 derived K8 (8.21 and 8.28) knockdown clones or the vector alone (S10P3) were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K8 and β-actin. The numbers indicate different mice injected with the single or double knockdown clones. All the samples were run on the same gel and the Western blots performed at the same time. D. Haematoxylin and eosin staining of paraffin embedded sections of lung tissue from nude mice injected with 106 cells of the vector alone (S10P3) or the double knockdown clones (8.21 and 8.28). Lung section from mice injected with S10P3 cells show extensive metastasis with thickening of alveolar walls from deposition and aggregation of metastasized tumor cells, whereas lungs from mice injected with the double knockdown cells show normal lungs with thin walled alveoli, with a few metastatic tumor cells. The images in the top row are at magnification x100 and images in the bottom row are at magnification x 400. E. PCR reactions were performed on DNA isolated from paraffin sections for the presence of Alu repeats in genomic DNA. Genomic DNA was purified from normal lung tissue, lung tissue from mice injected with cells with PKP3 knockdown alone (S10P3) and lung tissue from mice injected with the double knockdown clones (8.21 and 8.28). Lung tissues from uninjected mice (N) were used as a negative control for the Alu PCR. A PCR for the mouse patch gene was performed as a loading control. F. Immunohistochemical staining was performed with antibody against K8 on sections of paraffin embedded tissue of tumor and lungs of mice injected with S10P3 vector control cells or the double knockdown clones, 8.21 and 8.28. Images a, b, c, g, h and i are taken at a magnification x 200 and the respective magnified images at magnification x 400, of the indicated areas in the white rectangles, are represented by images d, e, f, j, k and l. Tumor tissue from mice injected with S10P3 (a and d) show K8 staining at levels higher compared to tumors from mice injected with the double knockdown clones, 8.21 and 8.28 (b, c, e and f). In contrast lung tissue, from mice injected with the vector control S10P3 cells (g and j) and the double knockdown clones (8.21 and 8.28) (h, i, k and l) show elevated K8 staining in metastatic areas of the lungs with infiltrated tumor cells.

Mentions: To determine whether K8 loss lead to a decrease in the transformed phenotype of the PKP3 knockdown clones, soft agar assays were performed. As shown in figure 4A, the double knockdown clones formed significantly fewer colonies in soft agar as compared to the PKP3 knockdown clones. In contrast, K18 knockdown did not lead to a significant decrease in colony formation in soft agar (Figure S6A). The ability of the double knockdown clones to form tumors in immunocompromised mice was monitored weekly over a period of four weeks. As shown in figure 4B, the double knockdown clones 8.21 and 8.28 formed smaller tumors as compared to the PKP3 knockdown clone. All five mice injected with the vector control and the 8.28 clone developed tumors while four mice injected with the 8.21 clone developed tumors. The difference in size was statistically significant at all the time points studied for the 8.28 clone and for the first three weeks for the 8.21 clone. These results are similar to those observed in other cell lines [19]. A Western blot analysis for tumors formed in four mice for each cell type demonstrated that tumors derived from the K8 knockdown clones have a lower level of K8 protein than those derived from the vector control (figure 4D). A Western blot for actin was performed as a loading control. A quantitation for these Western blots is shown in Figure S6B.


Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 dephosphorylation, which is required for transformation and metastasis.

Khapare N, Kundu ST, Sehgal L, Sawant M, Priya R, Gosavi P, Gupta N, Alam H, Karkhanis M, Naik N, Vaidya MM, Dalal SN - PLoS ONE (2012)

K8 downregulation leads to an inhibition of transformation in vitro and in vivo.A. The S10 derived K8 (8.21, 8.24 and 8.28) knockdown clones or the vector alone (S10P3) were plated in soft agar and colony formation determined after 2–3 weeks. The number of colonies formed by the clones per 20 low power fields (10X) was counted in triplicate in each experiment and the mean and standard deviation of three independent experiments is plotted as shown. B. 106 cells from the S10 derived K8 (8.21 and 8.28) knockdown clones or the vector alone (S10P3) were injected subcutaneously into 5 different nude mice and tumor size determined every week as described. Tumor volume is plotted on the Y-axis and the time in weeks on the X-axis. C. Protein extracts from primary tumors from mice injected with the S10 derived K8 (8.21 and 8.28) knockdown clones or the vector alone (S10P3) were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K8 and β-actin. The numbers indicate different mice injected with the single or double knockdown clones. All the samples were run on the same gel and the Western blots performed at the same time. D. Haematoxylin and eosin staining of paraffin embedded sections of lung tissue from nude mice injected with 106 cells of the vector alone (S10P3) or the double knockdown clones (8.21 and 8.28). Lung section from mice injected with S10P3 cells show extensive metastasis with thickening of alveolar walls from deposition and aggregation of metastasized tumor cells, whereas lungs from mice injected with the double knockdown cells show normal lungs with thin walled alveoli, with a few metastatic tumor cells. The images in the top row are at magnification x100 and images in the bottom row are at magnification x 400. E. PCR reactions were performed on DNA isolated from paraffin sections for the presence of Alu repeats in genomic DNA. Genomic DNA was purified from normal lung tissue, lung tissue from mice injected with cells with PKP3 knockdown alone (S10P3) and lung tissue from mice injected with the double knockdown clones (8.21 and 8.28). Lung tissues from uninjected mice (N) were used as a negative control for the Alu PCR. A PCR for the mouse patch gene was performed as a loading control. F. Immunohistochemical staining was performed with antibody against K8 on sections of paraffin embedded tissue of tumor and lungs of mice injected with S10P3 vector control cells or the double knockdown clones, 8.21 and 8.28. Images a, b, c, g, h and i are taken at a magnification x 200 and the respective magnified images at magnification x 400, of the indicated areas in the white rectangles, are represented by images d, e, f, j, k and l. Tumor tissue from mice injected with S10P3 (a and d) show K8 staining at levels higher compared to tumors from mice injected with the double knockdown clones, 8.21 and 8.28 (b, c, e and f). In contrast lung tissue, from mice injected with the vector control S10P3 cells (g and j) and the double knockdown clones (8.21 and 8.28) (h, i, k and l) show elevated K8 staining in metastatic areas of the lungs with infiltrated tumor cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368841&req=5

pone-0038561-g004: K8 downregulation leads to an inhibition of transformation in vitro and in vivo.A. The S10 derived K8 (8.21, 8.24 and 8.28) knockdown clones or the vector alone (S10P3) were plated in soft agar and colony formation determined after 2–3 weeks. The number of colonies formed by the clones per 20 low power fields (10X) was counted in triplicate in each experiment and the mean and standard deviation of three independent experiments is plotted as shown. B. 106 cells from the S10 derived K8 (8.21 and 8.28) knockdown clones or the vector alone (S10P3) were injected subcutaneously into 5 different nude mice and tumor size determined every week as described. Tumor volume is plotted on the Y-axis and the time in weeks on the X-axis. C. Protein extracts from primary tumors from mice injected with the S10 derived K8 (8.21 and 8.28) knockdown clones or the vector alone (S10P3) were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K8 and β-actin. The numbers indicate different mice injected with the single or double knockdown clones. All the samples were run on the same gel and the Western blots performed at the same time. D. Haematoxylin and eosin staining of paraffin embedded sections of lung tissue from nude mice injected with 106 cells of the vector alone (S10P3) or the double knockdown clones (8.21 and 8.28). Lung section from mice injected with S10P3 cells show extensive metastasis with thickening of alveolar walls from deposition and aggregation of metastasized tumor cells, whereas lungs from mice injected with the double knockdown cells show normal lungs with thin walled alveoli, with a few metastatic tumor cells. The images in the top row are at magnification x100 and images in the bottom row are at magnification x 400. E. PCR reactions were performed on DNA isolated from paraffin sections for the presence of Alu repeats in genomic DNA. Genomic DNA was purified from normal lung tissue, lung tissue from mice injected with cells with PKP3 knockdown alone (S10P3) and lung tissue from mice injected with the double knockdown clones (8.21 and 8.28). Lung tissues from uninjected mice (N) were used as a negative control for the Alu PCR. A PCR for the mouse patch gene was performed as a loading control. F. Immunohistochemical staining was performed with antibody against K8 on sections of paraffin embedded tissue of tumor and lungs of mice injected with S10P3 vector control cells or the double knockdown clones, 8.21 and 8.28. Images a, b, c, g, h and i are taken at a magnification x 200 and the respective magnified images at magnification x 400, of the indicated areas in the white rectangles, are represented by images d, e, f, j, k and l. Tumor tissue from mice injected with S10P3 (a and d) show K8 staining at levels higher compared to tumors from mice injected with the double knockdown clones, 8.21 and 8.28 (b, c, e and f). In contrast lung tissue, from mice injected with the vector control S10P3 cells (g and j) and the double knockdown clones (8.21 and 8.28) (h, i, k and l) show elevated K8 staining in metastatic areas of the lungs with infiltrated tumor cells.
Mentions: To determine whether K8 loss lead to a decrease in the transformed phenotype of the PKP3 knockdown clones, soft agar assays were performed. As shown in figure 4A, the double knockdown clones formed significantly fewer colonies in soft agar as compared to the PKP3 knockdown clones. In contrast, K18 knockdown did not lead to a significant decrease in colony formation in soft agar (Figure S6A). The ability of the double knockdown clones to form tumors in immunocompromised mice was monitored weekly over a period of four weeks. As shown in figure 4B, the double knockdown clones 8.21 and 8.28 formed smaller tumors as compared to the PKP3 knockdown clone. All five mice injected with the vector control and the 8.28 clone developed tumors while four mice injected with the 8.21 clone developed tumors. The difference in size was statistically significant at all the time points studied for the 8.28 clone and for the first three weeks for the 8.21 clone. These results are similar to those observed in other cell lines [19]. A Western blot analysis for tumors formed in four mice for each cell type demonstrated that tumors derived from the K8 knockdown clones have a lower level of K8 protein than those derived from the vector control (figure 4D). A Western blot for actin was performed as a loading control. A quantitation for these Western blots is shown in Figure S6B.

Bottom Line: The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8.Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation.These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.

View Article: PubMed Central - PubMed

Affiliation: Tata Memorial Centre, Kharghar Node, Navi Mumbai, Maharashtra, India.

ABSTRACT
The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.

Show MeSH
Related in: MedlinePlus