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FAM83D promotes cell proliferation and motility by downregulating tumor suppressor gene FBXW7.

Wang Z, Liu Y, Zhang P, Zhang W, Wang W, Curr K, Wei G, Mao JH - Oncotarget (2013)

Bottom Line: The list of candidate oncogenes in 20q has expanded over the past decade.High expression levels of FAM83D are significantly associated with poor clinical outcome and distant metastasis in breast cancer patients.Mechanistic studies reveal that overexpression of FAM83D downregulates FBXW7 expression levels through a physical interaction, which results in elevated protein levels of oncogenic substrates downstream to FBXW7, such as mTOR, whose inhibition by rapamycin can suppress FAM83D-induced cell migration and invasion.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, One Cyclotron Road, Berkeley, CA, USA.

ABSTRACT
Amplification of chromosome 20q is frequently found in various types of human cancers, including breast cancer. The list of candidate oncogenes in 20q has expanded over the past decade. Here, we investigate whether FAM83D (family with sequence similarity 83, member D) on chromosome 20q plays any role in breast cancer development. The expression level of FAM83D is significantly elevated in breast cancer cell lines and primary human breast cancers. High expression levels of FAM83D are significantly associated with poor clinical outcome and distant metastasis in breast cancer patients. We show that ectopic expression of FAM83D in human mammary epithelial cells promotes cell proliferation, migration and invasion along with epithelial-mesenchymal transition (EMT). Ablation of FAM83D in breast cancer cells induces apoptosis and consequently inhibits cell proliferation and colony formation. Mechanistic studies reveal that overexpression of FAM83D downregulates FBXW7 expression levels through a physical interaction, which results in elevated protein levels of oncogenic substrates downstream to FBXW7, such as mTOR, whose inhibition by rapamycin can suppress FAM83D-induced cell migration and invasion. The results demonstrate that FAM83D has prognostic value for breast cancer patients and is a novel oncogene in breast cancer development that at least in part acts through mTOR hyper-activation by inhibiting FBXW7.

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Biological consequences of FAM83D knockdown in BT549 cells(A) FAM83D expression was analyzed by Western blotting using anti-FAM83D antibody in both stable shRNA and transient siRNA knockdown in BT549 cells. Loading was controlled by β-actin levels. (B) BT549 cells with stable FM83D knockdown (shFAM83D) show a significant reduction in proliferation in comparison to control cells (shCtrl). BT549-shFAM83D and control cells were seeded in six-well dish in triplicates and cell numbers were counted by hemocytometer at indicated time points. (C) Knockdown of FAM83D induces cell apoptosis in BT549 cells. BT549 cells were transfected with siFAM83D or control siRNA (siCtrl) in a 96-well plate in triplicates for the indicated times. Apoptosis was determined by a Caspase3/7 assay. (D) Knockdown of FAM83D reduces colony formation in BT549 cells. (E) Knockdown of FAM83D significantly decreases cell invasiveness. Representative photographs from the Matrigel-coated transwell invasion assay of BT549 shCtrl control cells and shFAM83D cells. Data are presented as means ± standard deviation from three independent experiments, each performed in triplicate. * indicates p<0.05 and ** indicates p< 0.01, which were obtained from t-test.
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Figure 5: Biological consequences of FAM83D knockdown in BT549 cells(A) FAM83D expression was analyzed by Western blotting using anti-FAM83D antibody in both stable shRNA and transient siRNA knockdown in BT549 cells. Loading was controlled by β-actin levels. (B) BT549 cells with stable FM83D knockdown (shFAM83D) show a significant reduction in proliferation in comparison to control cells (shCtrl). BT549-shFAM83D and control cells were seeded in six-well dish in triplicates and cell numbers were counted by hemocytometer at indicated time points. (C) Knockdown of FAM83D induces cell apoptosis in BT549 cells. BT549 cells were transfected with siFAM83D or control siRNA (siCtrl) in a 96-well plate in triplicates for the indicated times. Apoptosis was determined by a Caspase3/7 assay. (D) Knockdown of FAM83D reduces colony formation in BT549 cells. (E) Knockdown of FAM83D significantly decreases cell invasiveness. Representative photographs from the Matrigel-coated transwell invasion assay of BT549 shCtrl control cells and shFAM83D cells. Data are presented as means ± standard deviation from three independent experiments, each performed in triplicate. * indicates p<0.05 and ** indicates p< 0.01, which were obtained from t-test.

Mentions: Next we examined whether depletion of the high level of endogenous FAM83D proteins in BT549 cells (Fig. 1B) inhibited the cell proliferation and motility phenotypes. FAM83D was depleted using both stable (shFAM83D) and transient (siFAM83D) knockdown systems, which was verified at the protein level by Western blotting (Fig. 5A). Depletion of FAM83D dramatically inhibited BT549 cell proliferation (Fig. 5B). We observed many floating BT549-shFAM83D cells, implicating a potential apoptotic effect resulting from FAM83D depletion, which was confirmed by the Caspase3/7 apoptosis assay (Fig. 5C). Consistent with this result, we found a dramatic reduction in colony formation of BT549-shFAM83D cells (Fig. 5D). In addition, significantly fewer BT549-shFAM83D cells were able to migrate through Matrigel compared to controls (Fig. 5E), indicating that inhibition of FAM83D dramatically restrained the invasiveness of BT549 cells.


FAM83D promotes cell proliferation and motility by downregulating tumor suppressor gene FBXW7.

Wang Z, Liu Y, Zhang P, Zhang W, Wang W, Curr K, Wei G, Mao JH - Oncotarget (2013)

Biological consequences of FAM83D knockdown in BT549 cells(A) FAM83D expression was analyzed by Western blotting using anti-FAM83D antibody in both stable shRNA and transient siRNA knockdown in BT549 cells. Loading was controlled by β-actin levels. (B) BT549 cells with stable FM83D knockdown (shFAM83D) show a significant reduction in proliferation in comparison to control cells (shCtrl). BT549-shFAM83D and control cells were seeded in six-well dish in triplicates and cell numbers were counted by hemocytometer at indicated time points. (C) Knockdown of FAM83D induces cell apoptosis in BT549 cells. BT549 cells were transfected with siFAM83D or control siRNA (siCtrl) in a 96-well plate in triplicates for the indicated times. Apoptosis was determined by a Caspase3/7 assay. (D) Knockdown of FAM83D reduces colony formation in BT549 cells. (E) Knockdown of FAM83D significantly decreases cell invasiveness. Representative photographs from the Matrigel-coated transwell invasion assay of BT549 shCtrl control cells and shFAM83D cells. Data are presented as means ± standard deviation from three independent experiments, each performed in triplicate. * indicates p<0.05 and ** indicates p< 0.01, which were obtained from t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Biological consequences of FAM83D knockdown in BT549 cells(A) FAM83D expression was analyzed by Western blotting using anti-FAM83D antibody in both stable shRNA and transient siRNA knockdown in BT549 cells. Loading was controlled by β-actin levels. (B) BT549 cells with stable FM83D knockdown (shFAM83D) show a significant reduction in proliferation in comparison to control cells (shCtrl). BT549-shFAM83D and control cells were seeded in six-well dish in triplicates and cell numbers were counted by hemocytometer at indicated time points. (C) Knockdown of FAM83D induces cell apoptosis in BT549 cells. BT549 cells were transfected with siFAM83D or control siRNA (siCtrl) in a 96-well plate in triplicates for the indicated times. Apoptosis was determined by a Caspase3/7 assay. (D) Knockdown of FAM83D reduces colony formation in BT549 cells. (E) Knockdown of FAM83D significantly decreases cell invasiveness. Representative photographs from the Matrigel-coated transwell invasion assay of BT549 shCtrl control cells and shFAM83D cells. Data are presented as means ± standard deviation from three independent experiments, each performed in triplicate. * indicates p<0.05 and ** indicates p< 0.01, which were obtained from t-test.
Mentions: Next we examined whether depletion of the high level of endogenous FAM83D proteins in BT549 cells (Fig. 1B) inhibited the cell proliferation and motility phenotypes. FAM83D was depleted using both stable (shFAM83D) and transient (siFAM83D) knockdown systems, which was verified at the protein level by Western blotting (Fig. 5A). Depletion of FAM83D dramatically inhibited BT549 cell proliferation (Fig. 5B). We observed many floating BT549-shFAM83D cells, implicating a potential apoptotic effect resulting from FAM83D depletion, which was confirmed by the Caspase3/7 apoptosis assay (Fig. 5C). Consistent with this result, we found a dramatic reduction in colony formation of BT549-shFAM83D cells (Fig. 5D). In addition, significantly fewer BT549-shFAM83D cells were able to migrate through Matrigel compared to controls (Fig. 5E), indicating that inhibition of FAM83D dramatically restrained the invasiveness of BT549 cells.

Bottom Line: The list of candidate oncogenes in 20q has expanded over the past decade.High expression levels of FAM83D are significantly associated with poor clinical outcome and distant metastasis in breast cancer patients.Mechanistic studies reveal that overexpression of FAM83D downregulates FBXW7 expression levels through a physical interaction, which results in elevated protein levels of oncogenic substrates downstream to FBXW7, such as mTOR, whose inhibition by rapamycin can suppress FAM83D-induced cell migration and invasion.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, One Cyclotron Road, Berkeley, CA, USA.

ABSTRACT
Amplification of chromosome 20q is frequently found in various types of human cancers, including breast cancer. The list of candidate oncogenes in 20q has expanded over the past decade. Here, we investigate whether FAM83D (family with sequence similarity 83, member D) on chromosome 20q plays any role in breast cancer development. The expression level of FAM83D is significantly elevated in breast cancer cell lines and primary human breast cancers. High expression levels of FAM83D are significantly associated with poor clinical outcome and distant metastasis in breast cancer patients. We show that ectopic expression of FAM83D in human mammary epithelial cells promotes cell proliferation, migration and invasion along with epithelial-mesenchymal transition (EMT). Ablation of FAM83D in breast cancer cells induces apoptosis and consequently inhibits cell proliferation and colony formation. Mechanistic studies reveal that overexpression of FAM83D downregulates FBXW7 expression levels through a physical interaction, which results in elevated protein levels of oncogenic substrates downstream to FBXW7, such as mTOR, whose inhibition by rapamycin can suppress FAM83D-induced cell migration and invasion. The results demonstrate that FAM83D has prognostic value for breast cancer patients and is a novel oncogene in breast cancer development that at least in part acts through mTOR hyper-activation by inhibiting FBXW7.

Show MeSH
Related in: MedlinePlus