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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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Loss of E-cadherin function induces down-regulation of RalA and RalB in HaCaT keratinocytesWestern blot analysis of HaCaT, Ras-expressing HaCaT-II-4, and HaCaT-II-4 cells expressing the dominant-negative E-cadherin transgene H-2Kd-Ecad (A). Total Erk is shown as a loading control. RalA and RalB protein (B) or mRNA (C) expression were measured in the linear range by densitometry or real-time PCR, respectively. (D) Western blot analysis of HaCaT-II-4 and HaCaT-II-4 cells expressing shRNA against E-cadherin. Total Erk is shown as a loading control. RalA and RalB protein (E) or mRNA (F) expression were measured in the linear range by densitometry or real-time PCR, respectively.
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Figure 1: Loss of E-cadherin function induces down-regulation of RalA and RalB in HaCaT keratinocytesWestern blot analysis of HaCaT, Ras-expressing HaCaT-II-4, and HaCaT-II-4 cells expressing the dominant-negative E-cadherin transgene H-2Kd-Ecad (A). Total Erk is shown as a loading control. RalA and RalB protein (B) or mRNA (C) expression were measured in the linear range by densitometry or real-time PCR, respectively. (D) Western blot analysis of HaCaT-II-4 and HaCaT-II-4 cells expressing shRNA against E-cadherin. Total Erk is shown as a loading control. RalA and RalB protein (E) or mRNA (F) expression were measured in the linear range by densitometry or real-time PCR, respectively.

Mentions: We observed that decreased E-cadherin protein levels in II-4-H-2Kd-Ecad cells was associated with a modest (∼60%) decrease in the expression of both RalA and RalB proteins (Fig. 1A,B). To test whether a similar phenomenon occurs when E-cadherin levels are reduced by down-regulation of E-cadherin mRNA, a more common phenomenon in tumor progression, HaCaT-II-4 cells were stably infected with lentivirus expressing either control shRNA (“sh-Scram”) or shRNA against E-cadherin (“sh-Ecad”) (Fig. 1D). Cells that displayed reductions in E-cadherin levels comparable to those found in II-4-H-2Kd-Ecad cells also displayed comparable down-regulation of Ral proteins: ∼2-fold for RalA and ∼3-fold for RalB (Fig. 1E). Real-time PCR analysis on cDNA derived from H-2Kd-Ecad- and sh-Ecad-expressing II-4 cells showed comparable decreases in mRNA of the RALA and RALB genes, implying that E-cadherin regulates Ral proteins predominantly at the level of transcription (Fig. 1C,F).


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

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

Loss of E-cadherin function induces down-regulation of RalA and RalB in HaCaT keratinocytesWestern blot analysis of HaCaT, Ras-expressing HaCaT-II-4, and HaCaT-II-4 cells expressing the dominant-negative E-cadherin transgene H-2Kd-Ecad (A). Total Erk is shown as a loading control. RalA and RalB protein (B) or mRNA (C) expression were measured in the linear range by densitometry or real-time PCR, respectively. (D) Western blot analysis of HaCaT-II-4 and HaCaT-II-4 cells expressing shRNA against E-cadherin. Total Erk is shown as a loading control. RalA and RalB protein (E) or mRNA (F) expression were measured in the linear range by densitometry or real-time PCR, respectively.
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Figure 1: Loss of E-cadherin function induces down-regulation of RalA and RalB in HaCaT keratinocytesWestern blot analysis of HaCaT, Ras-expressing HaCaT-II-4, and HaCaT-II-4 cells expressing the dominant-negative E-cadherin transgene H-2Kd-Ecad (A). Total Erk is shown as a loading control. RalA and RalB protein (B) or mRNA (C) expression were measured in the linear range by densitometry or real-time PCR, respectively. (D) Western blot analysis of HaCaT-II-4 and HaCaT-II-4 cells expressing shRNA against E-cadherin. Total Erk is shown as a loading control. RalA and RalB protein (E) or mRNA (F) expression were measured in the linear range by densitometry or real-time PCR, respectively.
Mentions: We observed that decreased E-cadherin protein levels in II-4-H-2Kd-Ecad cells was associated with a modest (∼60%) decrease in the expression of both RalA and RalB proteins (Fig. 1A,B). To test whether a similar phenomenon occurs when E-cadherin levels are reduced by down-regulation of E-cadherin mRNA, a more common phenomenon in tumor progression, HaCaT-II-4 cells were stably infected with lentivirus expressing either control shRNA (“sh-Scram”) or shRNA against E-cadherin (“sh-Ecad”) (Fig. 1D). Cells that displayed reductions in E-cadherin levels comparable to those found in II-4-H-2Kd-Ecad cells also displayed comparable down-regulation of Ral proteins: ∼2-fold for RalA and ∼3-fold for RalB (Fig. 1E). Real-time PCR analysis on cDNA derived from H-2Kd-Ecad- and sh-Ecad-expressing II-4 cells showed comparable decreases in mRNA of the RALA and RALB genes, implying that E-cadherin regulates Ral proteins predominantly at the level of transcription (Fig. 1C,F).

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