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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

RASSF2 and K-Ras form an endogenous complex. Lysates from H441 and H1299 lung cancer cells were immunoprecipitated with a pan Ras antibody, fractionated on SDS gels, and immunoblotted with an anti-RASSF2 antibody. The endogenous interaction between Ras and RASSF2 was confirmed by the presence of RASSF2 in the proteins precipitated from the H441 cells but not the RASSF2-negative H1299 cells.
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fig1: RASSF2 and K-Ras form an endogenous complex. Lysates from H441 and H1299 lung cancer cells were immunoprecipitated with a pan Ras antibody, fractionated on SDS gels, and immunoblotted with an anti-RASSF2 antibody. The endogenous interaction between Ras and RASSF2 was confirmed by the presence of RASSF2 in the proteins precipitated from the H441 cells but not the RASSF2-negative H1299 cells.

Mentions: RASSF2 has previously been shown to directly bind to K-Ras in vitro in a GTP-dependent manner [11]. To confirm that RASSF2 and K-Ras can form an endogenous complex, we serum-starved then briefly serum-stimulated H441 lung cancer cells that express mutant K-Ras and retain RASSF2 expression [11]. The cells were then lysed and immunoprecipitated with a pan-Ras antibody conjugated to sepharose beads and the immunoprecipitate subjected to Western Blotting with a RASSF2 antibody [11] (Figure 1). The presence of RASSF2 in the immunoprecipitate confirmed that the interaction between RASSF2 and K-Ras is physiologically relevant and RASSF2 is a bone fide Ras effector.


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

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

RASSF2 and K-Ras form an endogenous complex. Lysates from H441 and H1299 lung cancer cells were immunoprecipitated with a pan Ras antibody, fractionated on SDS gels, and immunoblotted with an anti-RASSF2 antibody. The endogenous interaction between Ras and RASSF2 was confirmed by the presence of RASSF2 in the proteins precipitated from the H441 cells but not the RASSF2-negative H1299 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: RASSF2 and K-Ras form an endogenous complex. Lysates from H441 and H1299 lung cancer cells were immunoprecipitated with a pan Ras antibody, fractionated on SDS gels, and immunoblotted with an anti-RASSF2 antibody. The endogenous interaction between Ras and RASSF2 was confirmed by the presence of RASSF2 in the proteins precipitated from the H441 cells but not the RASSF2-negative H1299 cells.
Mentions: RASSF2 has previously been shown to directly bind to K-Ras in vitro in a GTP-dependent manner [11]. To confirm that RASSF2 and K-Ras can form an endogenous complex, we serum-starved then briefly serum-stimulated H441 lung cancer cells that express mutant K-Ras and retain RASSF2 expression [11]. The cells were then lysed and immunoprecipitated with a pan-Ras antibody conjugated to sepharose beads and the immunoprecipitate subjected to Western Blotting with a RASSF2 antibody [11] (Figure 1). The presence of RASSF2 in the immunoprecipitate confirmed that the interaction between RASSF2 and K-Ras is physiologically relevant and RASSF2 is a bone fide Ras effector.

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