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βIII-tubulin: a novel mediator of chemoresistance and metastases in pancreatic cancer.

McCarroll JA, Sharbeen G, Liu J, Youkhana J, Goldstein D, McCarthy N, Limbri LF, Dischl D, Ceyhan GO, Erkan M, Johns AL, Biankin AV, Kavallaris M, Phillips PA - Oncotarget (2015)

Bottom Line: Evidence suggests that microtubule proteins namely, β-tubulins are dysregulated in tumor cells and are involved in regulating chemosensitivity.Further, we demonstrated that silencing βIII-tubulin expression reduced pancreatic cancer cell growth and tumorigenic potential in the absence and presence of chemotherapeutic drugs.Finally, we demonstrated that suppression of βIII-tubulin reduced tumor growth and metastases in vivo.

View Article: PubMed Central - PubMed

Affiliation: Children's Cancer Institute, Lowy Cancer Research Centre, UNSW Australia, Sydney, Australia.

ABSTRACT
Pancreatic cancer is a leading cause of cancer-related deaths in Western societies. This poor prognosis is due to chemotherapeutic drug resistance and metastatic spread. Evidence suggests that microtubule proteins namely, β-tubulins are dysregulated in tumor cells and are involved in regulating chemosensitivity. However, the role of β-tubulins in pancreatic cancer are unknown. We measured the expression of different β-tubulin isotypes in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Next, we used RNAi to silence βIII-tubulin expression in pancreatic cancer cells, and measured cell growth in the absence and presence of chemotherapeutic drugs. Finally, we assessed the role of βIII-tubulin in regulating tumor growth and metastases using an orthotopic pancreatic cancer mouse model. We found that βIII-tubulin is highly expressed in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Further, we demonstrated that silencing βIII-tubulin expression reduced pancreatic cancer cell growth and tumorigenic potential in the absence and presence of chemotherapeutic drugs. Finally, we demonstrated that suppression of βIII-tubulin reduced tumor growth and metastases in vivo. Our novel data demonstrate that βIII-tubulin is a key player in promoting pancreatic cancer growth and survival, and silencing its expression may be a potential therapeutic strategy to increase the long-term survival of pancreatic cancer patients.

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The effect of βIII-tubulin silencing on orthotopic tumor growth and metastasesA) Representative photomicrographs of primary tumors expressing control (ns-shRNA) or βIII-tubulin (βIII-Tub) shRNA eight weeks post-implantation (n=4 individual mice per group). B) Bars represent tumor volume (mean±s.e.m.) of primary pancreatic tumors eight weeks post-implantation. Asterisks indicate significance (* p≤0.05; n=9 for ns-shRNA and n=10 for βIII-tubulin shRNA). C) Bars represent the number of metastatic sites per mouse (mean±s.e.m.) eight weeks post-implantation. Asterisks indicate significance (* p≤0.05). D) Table showing the total number of metastases detected in each organ for ns-shRNA and βIII-Tub shRNA tumors.
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Figure 8: The effect of βIII-tubulin silencing on orthotopic tumor growth and metastasesA) Representative photomicrographs of primary tumors expressing control (ns-shRNA) or βIII-tubulin (βIII-Tub) shRNA eight weeks post-implantation (n=4 individual mice per group). B) Bars represent tumor volume (mean±s.e.m.) of primary pancreatic tumors eight weeks post-implantation. Asterisks indicate significance (* p≤0.05; n=9 for ns-shRNA and n=10 for βIII-tubulin shRNA). C) Bars represent the number of metastatic sites per mouse (mean±s.e.m.) eight weeks post-implantation. Asterisks indicate significance (* p≤0.05). D) Table showing the total number of metastases detected in each organ for ns-shRNA and βIII-Tub shRNA tumors.

Mentions: To extend our findings in vivo, we generated MiaPaCa-2 cells that stably expressed luciferase and a βIII-tubulin shRNA construct. Cells stably expressing the same construct but with a non-functional shRNA (ns-shRNA) served as controls. These cells possessed potent knockdown of βIII-tubulin compared to control shRNA cells (Supplementary Figure 5A and 5B). Notably, cells with stable suppression of βIII-tubulin had a significant decrease in anchorage-independent growth when compared to controls (ns-shRNA) (Figure 7A). These results concurred with our data using transient knockdown of βIII-tubulin (Figure 6B). Cells were implanted into the pancreas of mice, and allowed to grow for 8 weeks before tumors along with the spleen, liver, kidneys, intestines, and heart/lungs were harvested together with any lymph nodes that exhibited signs of metastases. βIII-tubulin knockdown in the tumors (8 weeks post implantation) containing βIII-tubulin shRNA was confirmed (Figures 7B-7D). Pancreatic tumors expressing βIII-tubulin shRNA had significantly reduced tumor volume relative to control shRNA tumors (Control: 145.0 ± 30.5 mm3, βIII-Tubulin shRNA: 80.6 ± 21.9 mm3, p<0.05; Figure 8A-B). The incidence of metastases was reduced by 30% in mice with tumors expressing βIII-tubulin shRNA (4/10 mice had metastases) versus tumors expressing control shRNA (7/10 mice had metastases). Metastases that were detected by ex vivo imaging of individual organs on a Xenogen IVIS platform (Supplementary Figure 6A) were found to be reduced by 62.5% in the βIII-tubulin shRNA pancreatic tumors, relative to controls (Figure 8C-D). Metastases detected by ex vivo imaging were confirmed by histology (Supplementary Figure 6B) and immunohistochemistry (Supplementary Figure 6C).


βIII-tubulin: a novel mediator of chemoresistance and metastases in pancreatic cancer.

McCarroll JA, Sharbeen G, Liu J, Youkhana J, Goldstein D, McCarthy N, Limbri LF, Dischl D, Ceyhan GO, Erkan M, Johns AL, Biankin AV, Kavallaris M, Phillips PA - Oncotarget (2015)

The effect of βIII-tubulin silencing on orthotopic tumor growth and metastasesA) Representative photomicrographs of primary tumors expressing control (ns-shRNA) or βIII-tubulin (βIII-Tub) shRNA eight weeks post-implantation (n=4 individual mice per group). B) Bars represent tumor volume (mean±s.e.m.) of primary pancreatic tumors eight weeks post-implantation. Asterisks indicate significance (* p≤0.05; n=9 for ns-shRNA and n=10 for βIII-tubulin shRNA). C) Bars represent the number of metastatic sites per mouse (mean±s.e.m.) eight weeks post-implantation. Asterisks indicate significance (* p≤0.05). D) Table showing the total number of metastases detected in each organ for ns-shRNA and βIII-Tub shRNA tumors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4385848&req=5

Figure 8: The effect of βIII-tubulin silencing on orthotopic tumor growth and metastasesA) Representative photomicrographs of primary tumors expressing control (ns-shRNA) or βIII-tubulin (βIII-Tub) shRNA eight weeks post-implantation (n=4 individual mice per group). B) Bars represent tumor volume (mean±s.e.m.) of primary pancreatic tumors eight weeks post-implantation. Asterisks indicate significance (* p≤0.05; n=9 for ns-shRNA and n=10 for βIII-tubulin shRNA). C) Bars represent the number of metastatic sites per mouse (mean±s.e.m.) eight weeks post-implantation. Asterisks indicate significance (* p≤0.05). D) Table showing the total number of metastases detected in each organ for ns-shRNA and βIII-Tub shRNA tumors.
Mentions: To extend our findings in vivo, we generated MiaPaCa-2 cells that stably expressed luciferase and a βIII-tubulin shRNA construct. Cells stably expressing the same construct but with a non-functional shRNA (ns-shRNA) served as controls. These cells possessed potent knockdown of βIII-tubulin compared to control shRNA cells (Supplementary Figure 5A and 5B). Notably, cells with stable suppression of βIII-tubulin had a significant decrease in anchorage-independent growth when compared to controls (ns-shRNA) (Figure 7A). These results concurred with our data using transient knockdown of βIII-tubulin (Figure 6B). Cells were implanted into the pancreas of mice, and allowed to grow for 8 weeks before tumors along with the spleen, liver, kidneys, intestines, and heart/lungs were harvested together with any lymph nodes that exhibited signs of metastases. βIII-tubulin knockdown in the tumors (8 weeks post implantation) containing βIII-tubulin shRNA was confirmed (Figures 7B-7D). Pancreatic tumors expressing βIII-tubulin shRNA had significantly reduced tumor volume relative to control shRNA tumors (Control: 145.0 ± 30.5 mm3, βIII-Tubulin shRNA: 80.6 ± 21.9 mm3, p<0.05; Figure 8A-B). The incidence of metastases was reduced by 30% in mice with tumors expressing βIII-tubulin shRNA (4/10 mice had metastases) versus tumors expressing control shRNA (7/10 mice had metastases). Metastases that were detected by ex vivo imaging of individual organs on a Xenogen IVIS platform (Supplementary Figure 6A) were found to be reduced by 62.5% in the βIII-tubulin shRNA pancreatic tumors, relative to controls (Figure 8C-D). Metastases detected by ex vivo imaging were confirmed by histology (Supplementary Figure 6B) and immunohistochemistry (Supplementary Figure 6C).

Bottom Line: Evidence suggests that microtubule proteins namely, β-tubulins are dysregulated in tumor cells and are involved in regulating chemosensitivity.Further, we demonstrated that silencing βIII-tubulin expression reduced pancreatic cancer cell growth and tumorigenic potential in the absence and presence of chemotherapeutic drugs.Finally, we demonstrated that suppression of βIII-tubulin reduced tumor growth and metastases in vivo.

View Article: PubMed Central - PubMed

Affiliation: Children's Cancer Institute, Lowy Cancer Research Centre, UNSW Australia, Sydney, Australia.

ABSTRACT
Pancreatic cancer is a leading cause of cancer-related deaths in Western societies. This poor prognosis is due to chemotherapeutic drug resistance and metastatic spread. Evidence suggests that microtubule proteins namely, β-tubulins are dysregulated in tumor cells and are involved in regulating chemosensitivity. However, the role of β-tubulins in pancreatic cancer are unknown. We measured the expression of different β-tubulin isotypes in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Next, we used RNAi to silence βIII-tubulin expression in pancreatic cancer cells, and measured cell growth in the absence and presence of chemotherapeutic drugs. Finally, we assessed the role of βIII-tubulin in regulating tumor growth and metastases using an orthotopic pancreatic cancer mouse model. We found that βIII-tubulin is highly expressed in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Further, we demonstrated that silencing βIII-tubulin expression reduced pancreatic cancer cell growth and tumorigenic potential in the absence and presence of chemotherapeutic drugs. Finally, we demonstrated that suppression of βIII-tubulin reduced tumor growth and metastases in vivo. Our novel data demonstrate that βIII-tubulin is a key player in promoting pancreatic cancer growth and survival, and silencing its expression may be a potential therapeutic strategy to increase the long-term survival of pancreatic cancer patients.

Show MeSH
Related in: MedlinePlus