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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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βIII-tubulin silencing in pancreatic cancer cell linesA) Top panel, Western blot analysis of βIII-tubulin silencing in protein extracts from MiaPaCa-2 cells. Cell lysates were harvested from cells 48h or 72h after transfection with mock, control siRNA (ns-siRNA), or βIII-tubulin siRNA (βIII-Tub siRNA). GAPDH was used as a loading control. Bottom graph, real-time PCR analysis of βIII-tubulin silencing in MiaPaCa-2 cells. RNA was harvested from cells 48h or 72h after transfection with mock, ns-siRNA, or βIII-tub siRNA. βIII-tubulin mRNA levels were normalized to 18S mRNA. B) as per A, except cell extracts were obtained from HPAF-II cells. Asterisks indicate significance (** p≤0.01, ** p≤0.01; n=3). C) Representative Western blots for βI-, βII-, βIII-tubulin and total tubulin in protein extracts from MiaPaCa-2 cells transfected with mock, ns-siRNA, or βIII-Tub siRNA (n=3). GAPDH was used as a loading control.
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Figure 2: βIII-tubulin silencing in pancreatic cancer cell linesA) Top panel, Western blot analysis of βIII-tubulin silencing in protein extracts from MiaPaCa-2 cells. Cell lysates were harvested from cells 48h or 72h after transfection with mock, control siRNA (ns-siRNA), or βIII-tubulin siRNA (βIII-Tub siRNA). GAPDH was used as a loading control. Bottom graph, real-time PCR analysis of βIII-tubulin silencing in MiaPaCa-2 cells. RNA was harvested from cells 48h or 72h after transfection with mock, ns-siRNA, or βIII-tub siRNA. βIII-tubulin mRNA levels were normalized to 18S mRNA. B) as per A, except cell extracts were obtained from HPAF-II cells. Asterisks indicate significance (** p≤0.01, ** p≤0.01; n=3). C) Representative Western blots for βI-, βII-, βIII-tubulin and total tubulin in protein extracts from MiaPaCa-2 cells transfected with mock, ns-siRNA, or βIII-Tub siRNA (n=3). GAPDH was used as a loading control.

Mentions: To examine whether βIII-tubulin could be suppressed in pancreatic cancer cells, we transfected two-independent pancreatic cancer cell lines (MiaPaCa-2 and HPAF-II) with βIII-tubulin siRNA. 48h and 72h post transfection, βIII-tubulin expression was measured. Knockdown of βIII-tubulin was observed at the gene level in both cell lines (MiaPaCa-2, 84.4 ± 2.6% knock-down; HPAF-II, 76.8 ± 1.1% knock-down relative to control-siRNA; 72h post-transfection) (Figure 2A and B). This correlated to knockdown (>90%) of βIII-tubulin at the protein level (Figure 2A and B). Knockdown of βII-tubulin was also observed when pancreatic cancer cells (MiaPaCa-2 and HPAF-II) were treated with βII-tubulin siRNA (Supplementary Figure 2).


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)

βIII-tubulin silencing in pancreatic cancer cell linesA) Top panel, Western blot analysis of βIII-tubulin silencing in protein extracts from MiaPaCa-2 cells. Cell lysates were harvested from cells 48h or 72h after transfection with mock, control siRNA (ns-siRNA), or βIII-tubulin siRNA (βIII-Tub siRNA). GAPDH was used as a loading control. Bottom graph, real-time PCR analysis of βIII-tubulin silencing in MiaPaCa-2 cells. RNA was harvested from cells 48h or 72h after transfection with mock, ns-siRNA, or βIII-tub siRNA. βIII-tubulin mRNA levels were normalized to 18S mRNA. B) as per A, except cell extracts were obtained from HPAF-II cells. Asterisks indicate significance (** p≤0.01, ** p≤0.01; n=3). C) Representative Western blots for βI-, βII-, βIII-tubulin and total tubulin in protein extracts from MiaPaCa-2 cells transfected with mock, ns-siRNA, or βIII-Tub siRNA (n=3). GAPDH was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385848&req=5

Figure 2: βIII-tubulin silencing in pancreatic cancer cell linesA) Top panel, Western blot analysis of βIII-tubulin silencing in protein extracts from MiaPaCa-2 cells. Cell lysates were harvested from cells 48h or 72h after transfection with mock, control siRNA (ns-siRNA), or βIII-tubulin siRNA (βIII-Tub siRNA). GAPDH was used as a loading control. Bottom graph, real-time PCR analysis of βIII-tubulin silencing in MiaPaCa-2 cells. RNA was harvested from cells 48h or 72h after transfection with mock, ns-siRNA, or βIII-tub siRNA. βIII-tubulin mRNA levels were normalized to 18S mRNA. B) as per A, except cell extracts were obtained from HPAF-II cells. Asterisks indicate significance (** p≤0.01, ** p≤0.01; n=3). C) Representative Western blots for βI-, βII-, βIII-tubulin and total tubulin in protein extracts from MiaPaCa-2 cells transfected with mock, ns-siRNA, or βIII-Tub siRNA (n=3). GAPDH was used as a loading control.
Mentions: To examine whether βIII-tubulin could be suppressed in pancreatic cancer cells, we transfected two-independent pancreatic cancer cell lines (MiaPaCa-2 and HPAF-II) with βIII-tubulin siRNA. 48h and 72h post transfection, βIII-tubulin expression was measured. Knockdown of βIII-tubulin was observed at the gene level in both cell lines (MiaPaCa-2, 84.4 ± 2.6% knock-down; HPAF-II, 76.8 ± 1.1% knock-down relative to control-siRNA; 72h post-transfection) (Figure 2A and B). This correlated to knockdown (>90%) of βIII-tubulin at the protein level (Figure 2A and B). Knockdown of βII-tubulin was also observed when pancreatic cancer cells (MiaPaCa-2 and HPAF-II) were treated with βII-tubulin siRNA (Supplementary Figure 2).

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