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Loss of RASSF2 Enhances Tumorigencity of Lung Cancer Cells and Confers Resistance to Chemotherapy.

Clark J, Freeman J, Donninger H - Mol Biol Int (2012)

Bottom Line: RASSF2 is a novel pro-apoptotic effector of K-Ras that is frequently inactivated in a variety of primary tumors by promoter methylation.In this study, we confirm that RASSF2 and K-Ras form an endogenous complex, validating that RASSF2 is a bona fide K-Ras effector.Loss of RASSF2 expression resulted in a more aggressive phenotype that was characterized by enhanced cell proliferation and invasion, decreased cell adhesion, the ability to grow in an anchorage-independent manner and cell morphological changes.

View Article: PubMed Central - PubMed

Affiliation: Molecular Targets Program, Department of Medicine, James Graham Brown Cancer Center, University of Louisville, 505 S. Hancock Street, Louisville, KY 40202, USA.

ABSTRACT
RASSF2 is a novel pro-apoptotic effector of K-Ras that is frequently inactivated in a variety of primary tumors by promoter methylation. Inactivation of RASSF2 enhances K-Ras-mediated transformation and overexpression of RASSF2 suppresses tumor cell growth. In this study, we confirm that RASSF2 and K-Ras form an endogenous complex, validating that RASSF2 is a bona fide K-Ras effector. We adopted an RNAi approach to determine the effects of inactivation of RASSF2 on the transformed phenotype of lung cancer cells containing an oncogenic K-Ras. Loss of RASSF2 expression resulted in a more aggressive phenotype that was characterized by enhanced cell proliferation and invasion, decreased cell adhesion, the ability to grow in an anchorage-independent manner and cell morphological changes. This enhanced transformed phenotype of the cells correlated with increased levels of activated AKT, indicating that RASSF2 can modulate Ras signaling pathways. Loss of RASSF2 expression also confers resistance to taxol and cisplatin, two frontline therapeutics for the treatment of lung cancer. Thus we have shown that inactivation of RASSF2, a process that occurs frequently in primary tumors, enhances the transforming potential of activated K-Ras and our data suggests that RASSF2 may be a novel candidate for epigenetic-based therapy in lung cancer.

No MeSH data available.


Related in: MedlinePlus

Loss of RASSF2 enhances proliferation and tumorigenicity of lung cancer cells. H441 lung cancer cells were transfected with two independent RASSF2 shRNA constructs and a noneffective shRNA and selected in puromycin for 2 weeks to obtain a population of cells stably expressing the various shRNA constructs. Knockdown of RASSF2 expression was confirmed by Western Blotting (a). Actin was used as a control for protein loading. (b) Growth analysis of the H441 shF2 cells. Cells were harvested and counted at the indicated times to determine cell number. P < 0.05 for both shF2-transfected cells compared to control cells. (c) H441 cells stably expressing the shRNA constructs to RASSF2 or control shRNA were plated in soft agar and colony number determined after 14 days. *Statistically different (P < 0.05) from cells expressing the control shRNA. The panel on the right shows representative images of the colonies.
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fig2: Loss of RASSF2 enhances proliferation and tumorigenicity of lung cancer cells. H441 lung cancer cells were transfected with two independent RASSF2 shRNA constructs and a noneffective shRNA and selected in puromycin for 2 weeks to obtain a population of cells stably expressing the various shRNA constructs. Knockdown of RASSF2 expression was confirmed by Western Blotting (a). Actin was used as a control for protein loading. (b) Growth analysis of the H441 shF2 cells. Cells were harvested and counted at the indicated times to determine cell number. P < 0.05 for both shF2-transfected cells compared to control cells. (c) H441 cells stably expressing the shRNA constructs to RASSF2 or control shRNA were plated in soft agar and colony number determined after 14 days. *Statistically different (P < 0.05) from cells expressing the control shRNA. The panel on the right shows representative images of the colonies.

Mentions: To determine the biological effects of downregulating RASSF2, we used two independent RASSF2 shRNA constructs to generate stable RASSF2 knockdown cell lines in H441 lung cancer cells. An shRNA molecule that did not knockdown RASSF2 was used as a control. Knockdown of RASSF2 expression in the H441 cells was validated by Western Blotting using our RASSF2 antibody (Figure 2(a)). Analysis of cell proliferation confirmed that the RASSF2 knockdown cells exhibited statistically significant (P < 0.05) enhanced proliferation compared to control cells (Figure 2(b)).


Loss of RASSF2 Enhances Tumorigencity of Lung Cancer Cells and Confers Resistance to Chemotherapy.

Clark J, Freeman J, Donninger H - Mol Biol Int (2012)

Loss of RASSF2 enhances proliferation and tumorigenicity of lung cancer cells. H441 lung cancer cells were transfected with two independent RASSF2 shRNA constructs and a noneffective shRNA and selected in puromycin for 2 weeks to obtain a population of cells stably expressing the various shRNA constructs. Knockdown of RASSF2 expression was confirmed by Western Blotting (a). Actin was used as a control for protein loading. (b) Growth analysis of the H441 shF2 cells. Cells were harvested and counted at the indicated times to determine cell number. P < 0.05 for both shF2-transfected cells compared to control cells. (c) H441 cells stably expressing the shRNA constructs to RASSF2 or control shRNA were plated in soft agar and colony number determined after 14 days. *Statistically different (P < 0.05) from cells expressing the control shRNA. The panel on the right shows representative images of the colonies.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368207&req=5

fig2: Loss of RASSF2 enhances proliferation and tumorigenicity of lung cancer cells. H441 lung cancer cells were transfected with two independent RASSF2 shRNA constructs and a noneffective shRNA and selected in puromycin for 2 weeks to obtain a population of cells stably expressing the various shRNA constructs. Knockdown of RASSF2 expression was confirmed by Western Blotting (a). Actin was used as a control for protein loading. (b) Growth analysis of the H441 shF2 cells. Cells were harvested and counted at the indicated times to determine cell number. P < 0.05 for both shF2-transfected cells compared to control cells. (c) H441 cells stably expressing the shRNA constructs to RASSF2 or control shRNA were plated in soft agar and colony number determined after 14 days. *Statistically different (P < 0.05) from cells expressing the control shRNA. The panel on the right shows representative images of the colonies.
Mentions: To determine the biological effects of downregulating RASSF2, we used two independent RASSF2 shRNA constructs to generate stable RASSF2 knockdown cell lines in H441 lung cancer cells. An shRNA molecule that did not knockdown RASSF2 was used as a control. Knockdown of RASSF2 expression in the H441 cells was validated by Western Blotting using our RASSF2 antibody (Figure 2(a)). Analysis of cell proliferation confirmed that the RASSF2 knockdown cells exhibited statistically significant (P < 0.05) enhanced proliferation compared to control cells (Figure 2(b)).

Bottom Line: RASSF2 is a novel pro-apoptotic effector of K-Ras that is frequently inactivated in a variety of primary tumors by promoter methylation.In this study, we confirm that RASSF2 and K-Ras form an endogenous complex, validating that RASSF2 is a bona fide K-Ras effector.Loss of RASSF2 expression resulted in a more aggressive phenotype that was characterized by enhanced cell proliferation and invasion, decreased cell adhesion, the ability to grow in an anchorage-independent manner and cell morphological changes.

View Article: PubMed Central - PubMed

Affiliation: Molecular Targets Program, Department of Medicine, James Graham Brown Cancer Center, University of Louisville, 505 S. Hancock Street, Louisville, KY 40202, USA.

ABSTRACT
RASSF2 is a novel pro-apoptotic effector of K-Ras that is frequently inactivated in a variety of primary tumors by promoter methylation. Inactivation of RASSF2 enhances K-Ras-mediated transformation and overexpression of RASSF2 suppresses tumor cell growth. In this study, we confirm that RASSF2 and K-Ras form an endogenous complex, validating that RASSF2 is a bona fide K-Ras effector. We adopted an RNAi approach to determine the effects of inactivation of RASSF2 on the transformed phenotype of lung cancer cells containing an oncogenic K-Ras. Loss of RASSF2 expression resulted in a more aggressive phenotype that was characterized by enhanced cell proliferation and invasion, decreased cell adhesion, the ability to grow in an anchorage-independent manner and cell morphological changes. This enhanced transformed phenotype of the cells correlated with increased levels of activated AKT, indicating that RASSF2 can modulate Ras signaling pathways. Loss of RASSF2 expression also confers resistance to taxol and cisplatin, two frontline therapeutics for the treatment of lung cancer. Thus we have shown that inactivation of RASSF2, a process that occurs frequently in primary tumors, enhances the transforming potential of activated K-Ras and our data suggests that RASSF2 may be a novel candidate for epigenetic-based therapy in lung cancer.

No MeSH data available.


Related in: MedlinePlus