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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 or Exo84 complements oncogenic Ras for the conversion to high-grade malignant carcinomaScrambled (a, e, i, m), RalA knockdown (b, f, j, n), RalB knockdown (c, g, k, o) or Exo84 knockdown (d, h, l, p) HaCaT-II-4 cells were used to generate bioengineered tissues that were transplanted to the dorsa of nude mice. n=5 for each cohort. (a-d) Clinical appearance of surface tumor after 8 weeks. (e-l) Representative image of H&E staining from sections at 4× and 20× magnifications. (m-p) Ki-67 immunohistochemistry on serial sections of tissue. Bar: 100μm.
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Figure 6: Suppression of RalA or Exo84 complements oncogenic Ras for the conversion to high-grade malignant carcinomaScrambled (a, e, i, m), RalA knockdown (b, f, j, n), RalB knockdown (c, g, k, o) or Exo84 knockdown (d, h, l, p) HaCaT-II-4 cells were used to generate bioengineered tissues that were transplanted to the dorsa of nude mice. n=5 for each cohort. (a-d) Clinical appearance of surface tumor after 8 weeks. (e-l) Representative image of H&E staining from sections at 4× and 20× magnifications. (m-p) Ki-67 immunohistochemistry on serial sections of tissue. Bar: 100μm.

Mentions: We have previously shown that when Ras-expressing II-4 cells in fabricated 3D tissues are transplanted onto the dorsal fascia of nude mice, the transplants form hyperkeratotic surface lesions with low-grade, well-differentiated invasive keratin “pearls,” while cells with reduced E-cadherin function form large nodular and erythematous tumors with aggressively-invasive, high-grade, malignant carcinoma (Margulis et al., 2005a). When tissues comprised of RalA or Exo84 knockdown II-4 cells were transplanted to an in vivo microenvironment, they showed properties similar to transplanted tissues populated with E-cadherin-suppressed II-4 cells (Fig. 6, panels b,d), forming large nodular tumors. H&E analysis of tumor sections revealed tumor cells that had infiltrated the stroma with little evidence of morphologic differentiation (Fig. 6 panels f,h,j,l). This loss of differentiation was associated with tumor cell proliferation throughout the tumor mass as well as at the leading edge of the invasive front (Fig. 6 panels n,p). Thus, knockdown of RalA or Exo84 not only leads to suppressed levels of E-cadherin and enhanced invasive properties in these cells, but it also permits Ras-transformed cells to form aggressive carcinomas after invasion into the dermis. In contrast, transplanted tissues comprised of sh-Scram or sh-RalB cells formed small surface lesions (Fig. 6 panels a,c) harboring well-differentiated tumor islands (Fig. 6 panels e,g,i,k). Proliferating cells, identified by Ki-67 immunohistochemistry, were restricted to the basal cells at the edges of the tumor islands (Fig. 6 panels m,o). Overall, these findings imply that RalA functions through Exo84 to suppress not only Ras-induced cell invasion into the dermal compartment of engineered skin but also progression to aggressive cancer in vivo.


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 or Exo84 complements oncogenic Ras for the conversion to high-grade malignant carcinomaScrambled (a, e, i, m), RalA knockdown (b, f, j, n), RalB knockdown (c, g, k, o) or Exo84 knockdown (d, h, l, p) HaCaT-II-4 cells were used to generate bioengineered tissues that were transplanted to the dorsa of nude mice. n=5 for each cohort. (a-d) Clinical appearance of surface tumor after 8 weeks. (e-l) Representative image of H&E staining from sections at 4× and 20× magnifications. (m-p) Ki-67 immunohistochemistry on serial sections of tissue. Bar: 100μm.
© Copyright Policy
Related In: Results  -  Collection

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Figure 6: Suppression of RalA or Exo84 complements oncogenic Ras for the conversion to high-grade malignant carcinomaScrambled (a, e, i, m), RalA knockdown (b, f, j, n), RalB knockdown (c, g, k, o) or Exo84 knockdown (d, h, l, p) HaCaT-II-4 cells were used to generate bioengineered tissues that were transplanted to the dorsa of nude mice. n=5 for each cohort. (a-d) Clinical appearance of surface tumor after 8 weeks. (e-l) Representative image of H&E staining from sections at 4× and 20× magnifications. (m-p) Ki-67 immunohistochemistry on serial sections of tissue. Bar: 100μm.
Mentions: We have previously shown that when Ras-expressing II-4 cells in fabricated 3D tissues are transplanted onto the dorsal fascia of nude mice, the transplants form hyperkeratotic surface lesions with low-grade, well-differentiated invasive keratin “pearls,” while cells with reduced E-cadherin function form large nodular and erythematous tumors with aggressively-invasive, high-grade, malignant carcinoma (Margulis et al., 2005a). When tissues comprised of RalA or Exo84 knockdown II-4 cells were transplanted to an in vivo microenvironment, they showed properties similar to transplanted tissues populated with E-cadherin-suppressed II-4 cells (Fig. 6, panels b,d), forming large nodular tumors. H&E analysis of tumor sections revealed tumor cells that had infiltrated the stroma with little evidence of morphologic differentiation (Fig. 6 panels f,h,j,l). This loss of differentiation was associated with tumor cell proliferation throughout the tumor mass as well as at the leading edge of the invasive front (Fig. 6 panels n,p). Thus, knockdown of RalA or Exo84 not only leads to suppressed levels of E-cadherin and enhanced invasive properties in these cells, but it also permits Ras-transformed cells to form aggressive carcinomas after invasion into the dermis. In contrast, transplanted tissues comprised of sh-Scram or sh-RalB cells formed small surface lesions (Fig. 6 panels a,c) harboring well-differentiated tumor islands (Fig. 6 panels e,g,i,k). Proliferating cells, identified by Ki-67 immunohistochemistry, were restricted to the basal cells at the edges of the tumor islands (Fig. 6 panels m,o). Overall, these findings imply that RalA functions through Exo84 to suppress not only Ras-induced cell invasion into the dermal compartment of engineered skin but also progression to aggressive cancer in vivo.

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