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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 RalA expression is necessary for the migratory and invasive properties associated with the loss of E-cadherin function(A) Western blot analysis of Ras-expressing HaCaT-II-4 cells expressing empty vector or H-2Kd-Ecad transgene infected with retroviruses encoding wild-type RalA or RalB. Total Erk is shown as a loading control. (B) Phase-contrast and immunofluorescent micrographs showing colony morphology and E-cadherin membrane localization in HaCaT-II-4 (a, e), HaCaT-II-4-H-2Kd-Ecad (b, f), HaCaT-II-4-H-2Kd-Ecad-RalAwt (c, g), and HaCaT-II-4-H-2Kd-Ecad-RalBwt (d, h) cells. Bar: 100μm. (C) The indicated cell lines were incubated with 10μM cycloheximide for the indicated amount of time (n≥3). Endogenous E-cadherin protein expression was analyzed by Western blot and quantified by densitometry. (D) E-cadherin mRNA expression in the indicated cell lines was analyzed by real-time PCR. (E) Cells from the indicated lines were grown on fibroblast-populated organotypic collagen gels and tissues were 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 (F). * p < 0.05 for H-2Kd-Ecad/RalAwt compared to H-2Kd-Ecad/Bleo using Mann-Whitney test.
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Figure 2: Suppression of RalA expression is necessary for the migratory and invasive properties associated with the loss of E-cadherin function(A) Western blot analysis of Ras-expressing HaCaT-II-4 cells expressing empty vector or H-2Kd-Ecad transgene infected with retroviruses encoding wild-type RalA or RalB. Total Erk is shown as a loading control. (B) Phase-contrast and immunofluorescent micrographs showing colony morphology and E-cadherin membrane localization in HaCaT-II-4 (a, e), HaCaT-II-4-H-2Kd-Ecad (b, f), HaCaT-II-4-H-2Kd-Ecad-RalAwt (c, g), and HaCaT-II-4-H-2Kd-Ecad-RalBwt (d, h) cells. Bar: 100μm. (C) The indicated cell lines were incubated with 10μM cycloheximide for the indicated amount of time (n≥3). Endogenous E-cadherin protein expression was analyzed by Western blot and quantified by densitometry. (D) E-cadherin mRNA expression in the indicated cell lines was analyzed by real-time PCR. (E) Cells from the indicated lines were grown on fibroblast-populated organotypic collagen gels and tissues were 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 (F). * p < 0.05 for H-2Kd-Ecad/RalAwt compared to H-2Kd-Ecad/Bleo using Mann-Whitney test.

Mentions: The finding that E-cadherin loss leads to the down-regulation of RalA and RalB raised the possibility that these GTPases normally suppress, rather than promote, the transition of Ras-expressing, non-invasive keratinocytes to a malignant state. It also suggested that removal of the tumor-suppressive effect of Ral proteins by a decrease in their expression by ∼50% is required for tumor progression to occur in this model system. To test these hypotheses, RalA or RalB levels in invasive II-4-H-2Kd-Ecad cells were restored back to those found in parental non-invasive II-4 cells by infection with a wild-type RalA or RalB-expressing retrovirus (Fig. 2A). While II-4 cells formed tightly packed colonies where E-cadherin was localized to cell-cell borders in 2-D cultures (Fig. 2B panels a,e), II-4 cells expressing dominant negative E-cadherin did not, and instead displayed a scattered phenotype consistent with their low E-cadherin levels (Fig. 2B panels b,f). Strikingly, II-4-H-2Kd cells in which RalA expression was restored to levels found in parental II-4 cells did form tightly packed colonies, and displayed E-cadherin at cell-cell borders (Fig. 2B panels c,g). Immunoblotting lysates from these cells showed that endogenous levels of E-cadherin are similar to those found in parental II-4 cells despite the continued expression of the dominant-negative transgene (Fig. 2A). In contrast, re-expressing RalB at levels similar to those found in control II-4 cells had no effect on cell scattering, the level of endogenous E-cadherin expression, or its subcellular localization (Fig. 2B panels d,h). We repeated these experiments in cells where E-cadherin levels were reduced (∼5-fold) by shRNA expression (Supplementary Fig. 2A) and found the same results. (Supplementary Fig. 2A and B).


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 RalA expression is necessary for the migratory and invasive properties associated with the loss of E-cadherin function(A) Western blot analysis of Ras-expressing HaCaT-II-4 cells expressing empty vector or H-2Kd-Ecad transgene infected with retroviruses encoding wild-type RalA or RalB. Total Erk is shown as a loading control. (B) Phase-contrast and immunofluorescent micrographs showing colony morphology and E-cadherin membrane localization in HaCaT-II-4 (a, e), HaCaT-II-4-H-2Kd-Ecad (b, f), HaCaT-II-4-H-2Kd-Ecad-RalAwt (c, g), and HaCaT-II-4-H-2Kd-Ecad-RalBwt (d, h) cells. Bar: 100μm. (C) The indicated cell lines were incubated with 10μM cycloheximide for the indicated amount of time (n≥3). Endogenous E-cadherin protein expression was analyzed by Western blot and quantified by densitometry. (D) E-cadherin mRNA expression in the indicated cell lines was analyzed by real-time PCR. (E) Cells from the indicated lines were grown on fibroblast-populated organotypic collagen gels and tissues were 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 (F). * p < 0.05 for H-2Kd-Ecad/RalAwt compared to H-2Kd-Ecad/Bleo using Mann-Whitney test.
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Related In: Results  -  Collection

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Figure 2: Suppression of RalA expression is necessary for the migratory and invasive properties associated with the loss of E-cadherin function(A) Western blot analysis of Ras-expressing HaCaT-II-4 cells expressing empty vector or H-2Kd-Ecad transgene infected with retroviruses encoding wild-type RalA or RalB. Total Erk is shown as a loading control. (B) Phase-contrast and immunofluorescent micrographs showing colony morphology and E-cadherin membrane localization in HaCaT-II-4 (a, e), HaCaT-II-4-H-2Kd-Ecad (b, f), HaCaT-II-4-H-2Kd-Ecad-RalAwt (c, g), and HaCaT-II-4-H-2Kd-Ecad-RalBwt (d, h) cells. Bar: 100μm. (C) The indicated cell lines were incubated with 10μM cycloheximide for the indicated amount of time (n≥3). Endogenous E-cadherin protein expression was analyzed by Western blot and quantified by densitometry. (D) E-cadherin mRNA expression in the indicated cell lines was analyzed by real-time PCR. (E) Cells from the indicated lines were grown on fibroblast-populated organotypic collagen gels and tissues were 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 (F). * p < 0.05 for H-2Kd-Ecad/RalAwt compared to H-2Kd-Ecad/Bleo using Mann-Whitney test.
Mentions: The finding that E-cadherin loss leads to the down-regulation of RalA and RalB raised the possibility that these GTPases normally suppress, rather than promote, the transition of Ras-expressing, non-invasive keratinocytes to a malignant state. It also suggested that removal of the tumor-suppressive effect of Ral proteins by a decrease in their expression by ∼50% is required for tumor progression to occur in this model system. To test these hypotheses, RalA or RalB levels in invasive II-4-H-2Kd-Ecad cells were restored back to those found in parental non-invasive II-4 cells by infection with a wild-type RalA or RalB-expressing retrovirus (Fig. 2A). While II-4 cells formed tightly packed colonies where E-cadherin was localized to cell-cell borders in 2-D cultures (Fig. 2B panels a,e), II-4 cells expressing dominant negative E-cadherin did not, and instead displayed a scattered phenotype consistent with their low E-cadherin levels (Fig. 2B panels b,f). Strikingly, II-4-H-2Kd cells in which RalA expression was restored to levels found in parental II-4 cells did form tightly packed colonies, and displayed E-cadherin at cell-cell borders (Fig. 2B panels c,g). Immunoblotting lysates from these cells showed that endogenous levels of E-cadherin are similar to those found in parental II-4 cells despite the continued expression of the dominant-negative transgene (Fig. 2A). In contrast, re-expressing RalB at levels similar to those found in control II-4 cells had no effect on cell scattering, the level of endogenous E-cadherin expression, or its subcellular localization (Fig. 2B panels d,h). We repeated these experiments in cells where E-cadherin levels were reduced (∼5-fold) by shRNA expression (Supplementary Fig. 2A) and found the same results. (Supplementary Fig. 2A and B).

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