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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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Acquisition of invasive properties of II-4 cells upon RalA or Exo84 knockdown is associated with a decrease in E-cadherin protein expression(A) Scrambled (a), RalA knockdown (b), RalB knockdown (c), RalA/RalB double-knockdown (d), Exo84 knockdown (e), Sec5 knockdown (f), or RalBP-1 knockdown (g) HaCaT-II-4 cells were used to populate tissues and frozen sections were subjected to immunofluorescent analysis with anti-E-cadherin antibody (red). DAPI was used to counterstain the nuclei. Invading cells in representative images are shown with arrows. (B) Western blot analysis of homogenized epithelium from tissues shown in (A). Total Erk is shown as a loading control. E-cadherin expression protein (C) and mRNA (D) were measured in the linear range by densitometry or real-time PCR, respectively. * p < 0.05 for sh-RalA, shRalA/B, and sh-Exo84 compared to sh-Scram using Mann-Whitney test.
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Figure 5: Acquisition of invasive properties of II-4 cells upon RalA or Exo84 knockdown is associated with a decrease in E-cadherin protein expression(A) Scrambled (a), RalA knockdown (b), RalB knockdown (c), RalA/RalB double-knockdown (d), Exo84 knockdown (e), Sec5 knockdown (f), or RalBP-1 knockdown (g) HaCaT-II-4 cells were used to populate tissues and frozen sections were subjected to immunofluorescent analysis with anti-E-cadherin antibody (red). DAPI was used to counterstain the nuclei. Invading cells in representative images are shown with arrows. (B) Western blot analysis of homogenized epithelium from tissues shown in (A). Total Erk is shown as a loading control. E-cadherin expression protein (C) and mRNA (D) were measured in the linear range by densitometry or real-time PCR, respectively. * p < 0.05 for sh-RalA, shRalA/B, and sh-Exo84 compared to sh-Scram using Mann-Whitney test.

Mentions: The results from Figure 2 implied that decreased RalA levels reduce E-cadherin levels. To further test this model and the notion that RalA functions through Exo84, immunofluorescent analysis of E-cadherin protein was performed on frozen sections from these engineered tissues. We observed that in tissues containing sh-RalA or sh-Exo84 expressing II-4 cells, E-cadherin was weakly expressed at cell-cell borders and was largely absent in invading cells (Fig. 5A, white arrows). By peeling the stratified epithelium away from the dermal compartment, we were able to directly assay protein and RNA expression in the epidermis. E-cadherin protein expression was reduced by ∼50% in both cases (Fig. 5B,C). Real-time PCR revealed that E-cadherin mRNA expression was not reduced, consistent with the model described in Fig. 3 showing that RalA (and Exo84) regulate E-cadherin by promoting its recycling back to the plasma membrane and thereby targeting it away from the degradation pathway.


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

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

Acquisition of invasive properties of II-4 cells upon RalA or Exo84 knockdown is associated with a decrease in E-cadherin protein expression(A) Scrambled (a), RalA knockdown (b), RalB knockdown (c), RalA/RalB double-knockdown (d), Exo84 knockdown (e), Sec5 knockdown (f), or RalBP-1 knockdown (g) HaCaT-II-4 cells were used to populate tissues and frozen sections were subjected to immunofluorescent analysis with anti-E-cadherin antibody (red). DAPI was used to counterstain the nuclei. Invading cells in representative images are shown with arrows. (B) Western blot analysis of homogenized epithelium from tissues shown in (A). Total Erk is shown as a loading control. E-cadherin expression protein (C) and mRNA (D) were measured in the linear range by densitometry or real-time PCR, respectively. * p < 0.05 for sh-RalA, shRalA/B, and sh-Exo84 compared to sh-Scram using Mann-Whitney test.
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Related In: Results  -  Collection

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Figure 5: Acquisition of invasive properties of II-4 cells upon RalA or Exo84 knockdown is associated with a decrease in E-cadherin protein expression(A) Scrambled (a), RalA knockdown (b), RalB knockdown (c), RalA/RalB double-knockdown (d), Exo84 knockdown (e), Sec5 knockdown (f), or RalBP-1 knockdown (g) HaCaT-II-4 cells were used to populate tissues and frozen sections were subjected to immunofluorescent analysis with anti-E-cadherin antibody (red). DAPI was used to counterstain the nuclei. Invading cells in representative images are shown with arrows. (B) Western blot analysis of homogenized epithelium from tissues shown in (A). Total Erk is shown as a loading control. E-cadherin expression protein (C) and mRNA (D) were measured in the linear range by densitometry or real-time PCR, respectively. * p < 0.05 for sh-RalA, shRalA/B, and sh-Exo84 compared to sh-Scram using Mann-Whitney test.
Mentions: The results from Figure 2 implied that decreased RalA levels reduce E-cadherin levels. To further test this model and the notion that RalA functions through Exo84, immunofluorescent analysis of E-cadherin protein was performed on frozen sections from these engineered tissues. We observed that in tissues containing sh-RalA or sh-Exo84 expressing II-4 cells, E-cadherin was weakly expressed at cell-cell borders and was largely absent in invading cells (Fig. 5A, white arrows). By peeling the stratified epithelium away from the dermal compartment, we were able to directly assay protein and RNA expression in the epidermis. E-cadherin protein expression was reduced by ∼50% in both cases (Fig. 5B,C). Real-time PCR revealed that E-cadherin mRNA expression was not reduced, consistent with the model described in Fig. 3 showing that RalA (and Exo84) regulate E-cadherin by promoting its recycling back to the plasma membrane and thereby targeting it away from the degradation pathway.

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