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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 by shRNA induces invasive properties in 3D tissues(A) Western blot analysis of Ras-expressing HaCaT-II-4 cells expressing shRNA against RalA, RalB, or both RalA and RalB. Total Erk is shown as a loading control. (B) Quantification of RalA or RalB knockdown compared to control determined by densitometry in the linear range. (C) Scrambled (a), RalA knockdown (b), RalB knockdown (c), or RalA/RalB double-knockdown (d) HaCaT-II-4 cells were grown on 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 (D). * p < 0.05 for sh-RalA and sh-RalA/B compared to sh-Scram using Mann-Whitney test.
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Figure 3: Suppression of RalA expression by shRNA induces invasive properties in 3D tissues(A) Western blot analysis of Ras-expressing HaCaT-II-4 cells expressing shRNA against RalA, RalB, or both RalA and RalB. Total Erk is shown as a loading control. (B) Quantification of RalA or RalB knockdown compared to control determined by densitometry in the linear range. (C) Scrambled (a), RalA knockdown (b), RalB knockdown (c), or RalA/RalB double-knockdown (d) HaCaT-II-4 cells were grown on 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 (D). * p < 0.05 for sh-RalA and sh-RalA/B compared to sh-Scram using Mann-Whitney test.

Mentions: To test whether direct suppression of RalA expression in Ras-expressing keratinocytes enhances their tumor progression in this bioengineered tissue model of SCC, RalA and RalB expression levels were directly knocked down in II-4 cells. Stable expression of shRNA against each GTPase was generated through lentiviral vector infection, and drug selected pools of II-4 cells were obtained. Knockdown efficiency was ∼60% for either RalA “sh-RalA” or RalB “sh-RalB” II-4 cells (Fig. 3A,B) when compared to control “sh-Scram,” which was comparable to the level of protein reduction found when E-cadherin levels were suppressed (see Fig. 1A). A second sequence for both RalA and RalB was also tested, with similar levels of knockdown (Supplementary Fig. 4A). When these cells were used to populate the epithelia of tissues, sh-Scram and sh-RalB showed typical dysplastic, non-invasive behavior (Fig. 3C panels a,c and Supplementary Fig. 4B panels a,c). Strikingly, both types of II-4 cells expressing either one of sh-RalA sequences (Fig. 3C panel b and Supplementary Fig. 4B panel b) showed clear invasive behavior, as significant numbers of cells (Fig. 3D), usually as clusters, breached the basement membrane to initiate invasion into the dermal layer (Fig. 3C panel b, black arrows) in amounts similar to that seen when E-cadherin function is suppressed (see Fig. 2F). This invasive phenotype was reversed by the expression of RNAi-resistant wild-type RalA but not empty vector (Supplementary Fig. 4C,D). In other systems, knockdown of RalB had the effect of reversing the phenotype of RalA knockdown (Chien & White, 2003; Oxford et al., 2005). This was not the case in the present study, as knockdown of RalB in sh-RalA cells did not abrogate their invasive phenotype (Fig. 3C panel d).


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 by shRNA induces invasive properties in 3D tissues(A) Western blot analysis of Ras-expressing HaCaT-II-4 cells expressing shRNA against RalA, RalB, or both RalA and RalB. Total Erk is shown as a loading control. (B) Quantification of RalA or RalB knockdown compared to control determined by densitometry in the linear range. (C) Scrambled (a), RalA knockdown (b), RalB knockdown (c), or RalA/RalB double-knockdown (d) HaCaT-II-4 cells were grown on 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 (D). * p < 0.05 for sh-RalA and sh-RalA/B compared to sh-Scram using Mann-Whitney test.
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Figure 3: Suppression of RalA expression by shRNA induces invasive properties in 3D tissues(A) Western blot analysis of Ras-expressing HaCaT-II-4 cells expressing shRNA against RalA, RalB, or both RalA and RalB. Total Erk is shown as a loading control. (B) Quantification of RalA or RalB knockdown compared to control determined by densitometry in the linear range. (C) Scrambled (a), RalA knockdown (b), RalB knockdown (c), or RalA/RalB double-knockdown (d) HaCaT-II-4 cells were grown on 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 (D). * p < 0.05 for sh-RalA and sh-RalA/B compared to sh-Scram using Mann-Whitney test.
Mentions: To test whether direct suppression of RalA expression in Ras-expressing keratinocytes enhances their tumor progression in this bioengineered tissue model of SCC, RalA and RalB expression levels were directly knocked down in II-4 cells. Stable expression of shRNA against each GTPase was generated through lentiviral vector infection, and drug selected pools of II-4 cells were obtained. Knockdown efficiency was ∼60% for either RalA “sh-RalA” or RalB “sh-RalB” II-4 cells (Fig. 3A,B) when compared to control “sh-Scram,” which was comparable to the level of protein reduction found when E-cadherin levels were suppressed (see Fig. 1A). A second sequence for both RalA and RalB was also tested, with similar levels of knockdown (Supplementary Fig. 4A). When these cells were used to populate the epithelia of tissues, sh-Scram and sh-RalB showed typical dysplastic, non-invasive behavior (Fig. 3C panels a,c and Supplementary Fig. 4B panels a,c). Strikingly, both types of II-4 cells expressing either one of sh-RalA sequences (Fig. 3C panel b and Supplementary Fig. 4B panel b) showed clear invasive behavior, as significant numbers of cells (Fig. 3D), usually as clusters, breached the basement membrane to initiate invasion into the dermal layer (Fig. 3C panel b, black arrows) in amounts similar to that seen when E-cadherin function is suppressed (see Fig. 2F). This invasive phenotype was reversed by the expression of RNAi-resistant wild-type RalA but not empty vector (Supplementary Fig. 4C,D). In other systems, knockdown of RalB had the effect of reversing the phenotype of RalA knockdown (Chien & White, 2003; Oxford et al., 2005). This was not the case in the present study, as knockdown of RalB in sh-RalA cells did not abrogate their invasive phenotype (Fig. 3C panel d).

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