Limits...
Reduced expression of AMPK-β1 during tumor progression enhances the oncogenic capacity of advanced ovarian cancer.

Li C, Liu VW, Chiu PM, Yao KM, Ngan HY, Chan DW - Mol. Cancer (2014)

Bottom Line: AMP-activated protein kinase (AMPK) is a key energy sensor that is involved in regulating cell metabolism.Functionally, enforced expression of AMPK-β1 inhibited ovarian-cancer-cell proliferation, anchorage-independent cell growth, cell migration and invasion.Taken together, these findings suggest that the reduced expression of AMPK-β1 confers lower AMPK activity, which enhances the oncogenic capacity of advanced-stage ovarian cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics & Gynecology, The University of Hong Kong, 6th Floor, Professorial Block, Queen Mary Hospital, Pokfulam, Hong Kong, SAR, People's Republic of China. hysngan@hku.hk.

ABSTRACT
AMP-activated protein kinase (AMPK) is a key energy sensor that is involved in regulating cell metabolism. Our previous study revealed that the subunits of the heterotimeric AMPK enzyme are diversely expressed during ovarian cancer progression. However, the impact of the variable expression of these AMPK subunits in ovarian cancer oncogenesis remains obscure. Here, we provide evidence to show that reduced expression of the AMPK-β1 subunit during tumor progression is associated with the increased oncogenic capacity of advanced ovarian cancer cells. Immunohistochemical analysis revealed that AMPK-β1 levels were reduced in advanced-stage (P = 0.008), high-grade (P = 0.013) and metastatic ovarian cancers (P = 0.008). Intriguingly, down-regulation of AMPK-β1 was progressively reduced from tumor stages 1 to 3 of ovarian cancer. Functionally, enforced expression of AMPK-β1 inhibited ovarian-cancer-cell proliferation, anchorage-independent cell growth, cell migration and invasion. Conversely, depletion of AMPK-β1 by siRNA enhanced the oncogenic capacities of ovarian cancer cells, suggesting that the loss of AMPK-β1 favors the aggressiveness of ovarian cancer. Mechanistically, enforced expression of AMPK-β1 increased AMPK activity, which, in turn, induced cell-cycle arrest via inhibition of AKT/ERK signaling activity as well as impaired cell migration/invasion through the suppression of JNK signaling in ovarian cancer cells. Taken together, these findings suggest that the reduced expression of AMPK-β1 confers lower AMPK activity, which enhances the oncogenic capacity of advanced-stage ovarian cancer.

Show MeSH

Related in: MedlinePlus

Overexpression of AMPK-β1 inhibits cell proliferation and anchorage-independent growth ability. (A) XTT cell proliferation assay showing that enforced expression of AMPK-β1 in A2780cp (C4, C5 and C11) (P < 0.002) and SKOV3 (C1, C2 and C3) (P < 0.001) clones displaying a 45 to 50% decrease in the cell growth rate compared with the empty vector (V1) and the parental (P) cell control. (B) Focus formation assay showing that the size and number of foci was reduced 2.5- to 3-fold in AMPK-β1 stable clones of A2780cp (C4 and C5) (P < 0.001) cells and 3- to 4-fold in AMPK-β1 stable clones of SKOV3 (C1 and C3) (P < 0.001) cells compared with the vector controls. (C) Soft agar assay revealing that the AMPK-β1 stable clones of A2780cp (C4, C5 and C11) (P < 0.04) and SKOV3 (C1, C2 and C3) (P < 0.05) cells had a 2.5- to 3-fold reduction in the size and number of colonies compared with the control. P: parental. V, V1 or V2: empty vector controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4016028&req=5

Figure 2: Overexpression of AMPK-β1 inhibits cell proliferation and anchorage-independent growth ability. (A) XTT cell proliferation assay showing that enforced expression of AMPK-β1 in A2780cp (C4, C5 and C11) (P < 0.002) and SKOV3 (C1, C2 and C3) (P < 0.001) clones displaying a 45 to 50% decrease in the cell growth rate compared with the empty vector (V1) and the parental (P) cell control. (B) Focus formation assay showing that the size and number of foci was reduced 2.5- to 3-fold in AMPK-β1 stable clones of A2780cp (C4 and C5) (P < 0.001) cells and 3- to 4-fold in AMPK-β1 stable clones of SKOV3 (C1 and C3) (P < 0.001) cells compared with the vector controls. (C) Soft agar assay revealing that the AMPK-β1 stable clones of A2780cp (C4, C5 and C11) (P < 0.04) and SKOV3 (C1, C2 and C3) (P < 0.05) cells had a 2.5- to 3-fold reduction in the size and number of colonies compared with the control. P: parental. V, V1 or V2: empty vector controls.

Mentions: Because AMPK-β1 was obviously reduced in advanced-stage ovarian cancer, we investigated the effect of AMPK-β1 on ovarian cancer cell growth and anchorage-independent growth. Stable clones overexpressing AMPK-β1 in two ovarian cancer cell lines (A2780cp and SKOV3) with relatively lower AMPK-β1 level or depleted of AMPK-β1 by shRNAi-mediated gene silencing in another two ovarian cancer cell lines (OVCA433 and OV2008) with relatively higher AMPK-β1 expression were generated (Additional file 2: Figure S2). The XTT cell proliferation assay demonstrated that enhanced expression of AMPK-β1 significantly inhibited ovarian cancer cell growth by 45 to 50% in A2780cp (C4, C5 and C11) (P < 0.002) and SKOV3 stable clones (C1, C2 and C3) (P < 0.001) compared with the parental lines and vector controls (Figure 2A). Furthermore, transient upregulation of AMPK-β1 elevated pAMPKα and mitigated cell proliferation in ovarian cancer cells in a dose-dependent manner (Additional file 3: Figure S3). Additionally, we demonstrated that enforced expression of AMPK-β1 exhibited 60 to 70% less foci in A2780cp and SKOV3 stable clones (P < 0.001) by the focus formation assay (Figure 2B), and we demonstrated that the AMPK-β1 overexpressed clones of A2780cp (C4, C5 and C11) (P < 0.04) and SKOV3 (C1, C2 and C3) (P < 0.05) cells showed a ~70% to 75% reduction in the number and size of colonies compared with the vector controls by the focus formation assay (Figure 2C). Conversely, by depleting endogenous AMPK-β1 in OV2008 (P < 0.002) and OVCA433 (P < 0.001) cells, which highly express AMPK-β1, using the sh-β1 shRNA, we demonstrated that cell proliferation increased 20-25% in all stable clones that overexpressed the sh-β1 shRNA (Figure 3A). Similarly, the stable AMPK-β1 knockdown clones exhibited a 2-3-fold increase in cell growth based on the focus formation assay (P < 0.001) (Figure 3B) and a 4-5-fold increase in colony formation using the anchorage-independent growth ability assay (P < 0.03) (Figure 3C).


Reduced expression of AMPK-β1 during tumor progression enhances the oncogenic capacity of advanced ovarian cancer.

Li C, Liu VW, Chiu PM, Yao KM, Ngan HY, Chan DW - Mol. Cancer (2014)

Overexpression of AMPK-β1 inhibits cell proliferation and anchorage-independent growth ability. (A) XTT cell proliferation assay showing that enforced expression of AMPK-β1 in A2780cp (C4, C5 and C11) (P < 0.002) and SKOV3 (C1, C2 and C3) (P < 0.001) clones displaying a 45 to 50% decrease in the cell growth rate compared with the empty vector (V1) and the parental (P) cell control. (B) Focus formation assay showing that the size and number of foci was reduced 2.5- to 3-fold in AMPK-β1 stable clones of A2780cp (C4 and C5) (P < 0.001) cells and 3- to 4-fold in AMPK-β1 stable clones of SKOV3 (C1 and C3) (P < 0.001) cells compared with the vector controls. (C) Soft agar assay revealing that the AMPK-β1 stable clones of A2780cp (C4, C5 and C11) (P < 0.04) and SKOV3 (C1, C2 and C3) (P < 0.05) cells had a 2.5- to 3-fold reduction in the size and number of colonies compared with the control. P: parental. V, V1 or V2: empty vector controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4016028&req=5

Figure 2: Overexpression of AMPK-β1 inhibits cell proliferation and anchorage-independent growth ability. (A) XTT cell proliferation assay showing that enforced expression of AMPK-β1 in A2780cp (C4, C5 and C11) (P < 0.002) and SKOV3 (C1, C2 and C3) (P < 0.001) clones displaying a 45 to 50% decrease in the cell growth rate compared with the empty vector (V1) and the parental (P) cell control. (B) Focus formation assay showing that the size and number of foci was reduced 2.5- to 3-fold in AMPK-β1 stable clones of A2780cp (C4 and C5) (P < 0.001) cells and 3- to 4-fold in AMPK-β1 stable clones of SKOV3 (C1 and C3) (P < 0.001) cells compared with the vector controls. (C) Soft agar assay revealing that the AMPK-β1 stable clones of A2780cp (C4, C5 and C11) (P < 0.04) and SKOV3 (C1, C2 and C3) (P < 0.05) cells had a 2.5- to 3-fold reduction in the size and number of colonies compared with the control. P: parental. V, V1 or V2: empty vector controls.
Mentions: Because AMPK-β1 was obviously reduced in advanced-stage ovarian cancer, we investigated the effect of AMPK-β1 on ovarian cancer cell growth and anchorage-independent growth. Stable clones overexpressing AMPK-β1 in two ovarian cancer cell lines (A2780cp and SKOV3) with relatively lower AMPK-β1 level or depleted of AMPK-β1 by shRNAi-mediated gene silencing in another two ovarian cancer cell lines (OVCA433 and OV2008) with relatively higher AMPK-β1 expression were generated (Additional file 2: Figure S2). The XTT cell proliferation assay demonstrated that enhanced expression of AMPK-β1 significantly inhibited ovarian cancer cell growth by 45 to 50% in A2780cp (C4, C5 and C11) (P < 0.002) and SKOV3 stable clones (C1, C2 and C3) (P < 0.001) compared with the parental lines and vector controls (Figure 2A). Furthermore, transient upregulation of AMPK-β1 elevated pAMPKα and mitigated cell proliferation in ovarian cancer cells in a dose-dependent manner (Additional file 3: Figure S3). Additionally, we demonstrated that enforced expression of AMPK-β1 exhibited 60 to 70% less foci in A2780cp and SKOV3 stable clones (P < 0.001) by the focus formation assay (Figure 2B), and we demonstrated that the AMPK-β1 overexpressed clones of A2780cp (C4, C5 and C11) (P < 0.04) and SKOV3 (C1, C2 and C3) (P < 0.05) cells showed a ~70% to 75% reduction in the number and size of colonies compared with the vector controls by the focus formation assay (Figure 2C). Conversely, by depleting endogenous AMPK-β1 in OV2008 (P < 0.002) and OVCA433 (P < 0.001) cells, which highly express AMPK-β1, using the sh-β1 shRNA, we demonstrated that cell proliferation increased 20-25% in all stable clones that overexpressed the sh-β1 shRNA (Figure 3A). Similarly, the stable AMPK-β1 knockdown clones exhibited a 2-3-fold increase in cell growth based on the focus formation assay (P < 0.001) (Figure 3B) and a 4-5-fold increase in colony formation using the anchorage-independent growth ability assay (P < 0.03) (Figure 3C).

Bottom Line: AMP-activated protein kinase (AMPK) is a key energy sensor that is involved in regulating cell metabolism.Functionally, enforced expression of AMPK-β1 inhibited ovarian-cancer-cell proliferation, anchorage-independent cell growth, cell migration and invasion.Taken together, these findings suggest that the reduced expression of AMPK-β1 confers lower AMPK activity, which enhances the oncogenic capacity of advanced-stage ovarian cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics & Gynecology, The University of Hong Kong, 6th Floor, Professorial Block, Queen Mary Hospital, Pokfulam, Hong Kong, SAR, People's Republic of China. hysngan@hku.hk.

ABSTRACT
AMP-activated protein kinase (AMPK) is a key energy sensor that is involved in regulating cell metabolism. Our previous study revealed that the subunits of the heterotimeric AMPK enzyme are diversely expressed during ovarian cancer progression. However, the impact of the variable expression of these AMPK subunits in ovarian cancer oncogenesis remains obscure. Here, we provide evidence to show that reduced expression of the AMPK-β1 subunit during tumor progression is associated with the increased oncogenic capacity of advanced ovarian cancer cells. Immunohistochemical analysis revealed that AMPK-β1 levels were reduced in advanced-stage (P = 0.008), high-grade (P = 0.013) and metastatic ovarian cancers (P = 0.008). Intriguingly, down-regulation of AMPK-β1 was progressively reduced from tumor stages 1 to 3 of ovarian cancer. Functionally, enforced expression of AMPK-β1 inhibited ovarian-cancer-cell proliferation, anchorage-independent cell growth, cell migration and invasion. Conversely, depletion of AMPK-β1 by siRNA enhanced the oncogenic capacities of ovarian cancer cells, suggesting that the loss of AMPK-β1 favors the aggressiveness of ovarian cancer. Mechanistically, enforced expression of AMPK-β1 increased AMPK activity, which, in turn, induced cell-cycle arrest via inhibition of AKT/ERK signaling activity as well as impaired cell migration/invasion through the suppression of JNK signaling in ovarian cancer cells. Taken together, these findings suggest that the reduced expression of AMPK-β1 confers lower AMPK activity, which enhances the oncogenic capacity of advanced-stage ovarian cancer.

Show MeSH
Related in: MedlinePlus