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RalA suppresses early stages of Ras-induced squamous cell carcinoma progression.

Sowalsky AG, Alt-Holland A, Shamis Y, Garlick JA, Feig LA - Oncogene (2009)

Bottom Line: Conversion of Ras-expressing keratinocytes from a premalignant to malignant state induced by decreasing E-cadherin function was associated with and required an approximately two to threefold decrease in RalA expression.Knockdown of the Ral effector, Exo84, mimicked the effects of decreasing RalA levels in these engineered tissues.These results imply that an important component of the early stages in squamous carcinoma progression may be a modest decrease in RalA gene expression that magnifies the effects of decreased E-cadherin expression by promoting its degradation.

View Article: PubMed Central - PubMed

Affiliation: Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111, USA.

ABSTRACT
Ras proteins activate Raf and PI-3 kinases, as well as exchange factors for RalA and RalB GTPases. Many previous studies have reported that the Ral-signaling cascade contributes positively to Ras-mediated oncogenesis. Here, using a bioengineered tissue model of early steps in Ras-induced human squamous cell carcinoma of the skin, we found the opposite. Conversion of Ras-expressing keratinocytes from a premalignant to malignant state induced by decreasing E-cadherin function was associated with and required an approximately two to threefold decrease in RalA expression. Moreover, direct knockdown of RalA to a similar degree by shRNA expression in these cells reduced E-cadherin levels and also induced progression to a malignant phenotype. Knockdown of the Ral effector, Exo84, mimicked the effects of decreasing RalA levels in these engineered tissues. These phenomena can be explained by our finding that the stability of E-cadherin in Ras-expressing keratinocytes depends upon this RalA signaling cascade. These results imply that an important component of the early stages in squamous carcinoma progression may be a modest decrease in RalA gene expression that magnifies the effects of decreased E-cadherin expression by promoting its degradation.

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Suppression of the Ral effector Exo84 expression by shRNA induces invasive properties of HaCaT II-4 cells in 3D tissues(A) Western blot analysis of Ras-expressing HaCaT-II-4 cells expressing scrambled shRNA or shRNA against Exo84, Sec5, or RalBP-1. Total Erk is shown as a loading control. (B) Scrambled (a), Exo84 knockdown (b), Sec5 knockdown (c), or RalBP-1 knockdown (d) HaCaT-II-4 cells were used to populate tissues as described and stained with H&E. Bar: 100μm. Invading cells in representative images are shown with arrows and quantified from >100 microscope fields in >20 sections from multiple experiments (C) and two different shRNA sequences. * p < 0.01 for sh-Exo84 compared to sh-Scram using Mann-Whitney test.
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Figure 4: Suppression of the Ral effector Exo84 expression by shRNA induces invasive properties of HaCaT II-4 cells in 3D tissues(A) Western blot analysis of Ras-expressing HaCaT-II-4 cells expressing scrambled shRNA or shRNA against Exo84, Sec5, or RalBP-1. Total Erk is shown as a loading control. (B) Scrambled (a), Exo84 knockdown (b), Sec5 knockdown (c), or RalBP-1 knockdown (d) HaCaT-II-4 cells were used to populate tissues as described and stained with H&E. Bar: 100μm. Invading cells in representative images are shown with arrows and quantified from >100 microscope fields in >20 sections from multiple experiments (C) and two different shRNA sequences. * p < 0.01 for sh-Exo84 compared to sh-Scram using Mann-Whitney test.

Mentions: To test which RalA effector is involved, we utilized lentiviral vectors to knock down the well-characterized effectors Exo84, Sec5, and RalBP-1 (Fig. 4A) to similar degrees in Ras-expressing II-4 cells. When these cells were seeded on contracted collagen gels to generate epithelial tissues, only the cells expressing shRNA against Exo84 (“sh-Exo84”) displayed an invasive morphology (Fig. 4B panel b). The number of invasive cells (Fig. 4C) was comparable to when RalA expression was suppressed by shRNA (see Fig. 3D).


RalA suppresses early stages of Ras-induced squamous cell carcinoma progression.

Sowalsky AG, Alt-Holland A, Shamis Y, Garlick JA, Feig LA - Oncogene (2009)

Suppression of the Ral effector Exo84 expression by shRNA induces invasive properties of HaCaT II-4 cells in 3D tissues(A) Western blot analysis of Ras-expressing HaCaT-II-4 cells expressing scrambled shRNA or shRNA against Exo84, Sec5, or RalBP-1. Total Erk is shown as a loading control. (B) Scrambled (a), Exo84 knockdown (b), Sec5 knockdown (c), or RalBP-1 knockdown (d) HaCaT-II-4 cells were used to populate tissues as described and stained with H&E. Bar: 100μm. Invading cells in representative images are shown with arrows and quantified from >100 microscope fields in >20 sections from multiple experiments (C) and two different shRNA sequences. * p < 0.01 for sh-Exo84 compared to sh-Scram using Mann-Whitney test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803332&req=5

Figure 4: Suppression of the Ral effector Exo84 expression by shRNA induces invasive properties of HaCaT II-4 cells in 3D tissues(A) Western blot analysis of Ras-expressing HaCaT-II-4 cells expressing scrambled shRNA or shRNA against Exo84, Sec5, or RalBP-1. Total Erk is shown as a loading control. (B) Scrambled (a), Exo84 knockdown (b), Sec5 knockdown (c), or RalBP-1 knockdown (d) HaCaT-II-4 cells were used to populate tissues as described and stained with H&E. Bar: 100μm. Invading cells in representative images are shown with arrows and quantified from >100 microscope fields in >20 sections from multiple experiments (C) and two different shRNA sequences. * p < 0.01 for sh-Exo84 compared to sh-Scram using Mann-Whitney test.
Mentions: To test which RalA effector is involved, we utilized lentiviral vectors to knock down the well-characterized effectors Exo84, Sec5, and RalBP-1 (Fig. 4A) to similar degrees in Ras-expressing II-4 cells. When these cells were seeded on contracted collagen gels to generate epithelial tissues, only the cells expressing shRNA against Exo84 (“sh-Exo84”) displayed an invasive morphology (Fig. 4B panel b). The number of invasive cells (Fig. 4C) was comparable to when RalA expression was suppressed by shRNA (see Fig. 3D).

Bottom Line: Conversion of Ras-expressing keratinocytes from a premalignant to malignant state induced by decreasing E-cadherin function was associated with and required an approximately two to threefold decrease in RalA expression.Knockdown of the Ral effector, Exo84, mimicked the effects of decreasing RalA levels in these engineered tissues.These results imply that an important component of the early stages in squamous carcinoma progression may be a modest decrease in RalA gene expression that magnifies the effects of decreased E-cadherin expression by promoting its degradation.

View Article: PubMed Central - PubMed

Affiliation: Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111, USA.

ABSTRACT
Ras proteins activate Raf and PI-3 kinases, as well as exchange factors for RalA and RalB GTPases. Many previous studies have reported that the Ral-signaling cascade contributes positively to Ras-mediated oncogenesis. Here, using a bioengineered tissue model of early steps in Ras-induced human squamous cell carcinoma of the skin, we found the opposite. Conversion of Ras-expressing keratinocytes from a premalignant to malignant state induced by decreasing E-cadherin function was associated with and required an approximately two to threefold decrease in RalA expression. Moreover, direct knockdown of RalA to a similar degree by shRNA expression in these cells reduced E-cadherin levels and also induced progression to a malignant phenotype. Knockdown of the Ral effector, Exo84, mimicked the effects of decreasing RalA levels in these engineered tissues. These phenomena can be explained by our finding that the stability of E-cadherin in Ras-expressing keratinocytes depends upon this RalA signaling cascade. These results imply that an important component of the early stages in squamous carcinoma progression may be a modest decrease in RalA gene expression that magnifies the effects of decreased E-cadherin expression by promoting its degradation.

Show MeSH
Related in: MedlinePlus