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Stromal control of oncogenic traits expressed in response to the overexpression of GLI2, a pleiotropic oncogene.

Snijders AM, Huey B, Connelly ST, Roy R, Jordan RC, Schmidt BL, Albertson DG - Oncogene (2008)

Bottom Line: We show that GLI2 is a pleiotropic oncogene.The overexpression induces genomic instability and blocks differentiation, likely mediated in part by enhanced expression of the stem cell gene SOX2.GLI2 also induces transforming growth factor (TGF)B1-dependent transdifferentiation of foreskin and tongue, but not gingival fibroblasts into myofibroblasts, creating an environment permissive for invasion by keratinocytes, which are in various stages of differentiation having downregulated GLI2.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Institute, University of California San Francisco, San Francisco, CA 94143-0808, USA.

ABSTRACT
Hedgehog signaling is often activated in tumors, yet it remains unclear how GLI2, a transcription factor activated by this pathway, acts as an oncogene. We show that GLI2 is a pleiotropic oncogene. The overexpression induces genomic instability and blocks differentiation, likely mediated in part by enhanced expression of the stem cell gene SOX2. GLI2 also induces transforming growth factor (TGF)B1-dependent transdifferentiation of foreskin and tongue, but not gingival fibroblasts into myofibroblasts, creating an environment permissive for invasion by keratinocytes, which are in various stages of differentiation having downregulated GLI2. Thus, upregulated GLI2 expression is sufficient to induce a number of the acquired characteristics of tumor cells; however, the stroma, in a tissue-specific manner, determines whether certain GLI2 oncogenic traits are expressed.

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GLI2 induces expression of stem cell genesSections of organotypic cultures of GLI2 expressing HaCaT GLI2 cells stained using HRP and DAB detection and antibodies to SOX2 and TITF1. The locations of keratinocytes in the sections are indicated by staining adjacent sections with antibodies to CAM5.2. Two photomicrographs are shown from each section.
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Figure 5: GLI2 induces expression of stem cell genesSections of organotypic cultures of GLI2 expressing HaCaT GLI2 cells stained using HRP and DAB detection and antibodies to SOX2 and TITF1. The locations of keratinocytes in the sections are indicated by staining adjacent sections with antibodies to CAM5.2. Two photomicrographs are shown from each section.

Mentions: Stem cell gene expression signatures have been reported in poorly differentiated breast and other tumors (Ben-Porath et al., 2008; Chen et al., 2008), as well as in gliomas, which are associated with activated hedgehog signaling and GLI overexpression (Clement et al., 2007), suggesting that GLI2 might block keratinocyte differentiation by up-regulating stem cell genes. To investigate this possibility, expression profiling was carried on RNA extracted from isolated keratinocyte layers from the tissue reconstructs. This analysis revealed elevated expression of SOX2 in the GLI2 expressing keratinocytes (Supplementary Figure 5), which was also confirmed by staining reconstructs with antibodies to SOX2 (Figure 5). These observations suggest that SOX2 is a GLI2 responsive gene. Indeed, infection of primary keratinocytes with a GLI2 retrovirus resulted in induction of SOX2 RNA (Supplementary Figure 6A), and in HaCaT GLI2 cells, addition of doxcycline induced SOX2 expression with the same kinetics as the two known GLI2 downstream targets, GLI1 and PTCH1 (Supplementary Figure 6B). Moreover, a number of potential GLI2 consensus binding sites are present in SOX2 (Supplementary Table 4). In a separate analysis, we found that neither NANOG nor OCT4 (POU5F1) was up-regulated in GLI2 expressing cells (data not shown), whereas elevated RNA and protein expression of TITF1 (Nkx2.1) was observed (Figure 5). TITF1 is reciprocally regulated with SOX2 during early dorsal/ventral foregut patterning (Que et al., 2007). It is frequently amplified in lung cancer (Kendall et al., 2007; Kwei et al., 2008; Weir et al., 2007) and was amplified in one of 89 oral SCC (Snijders et al., 2005). Thus, induction of stem cell genes is likely to underlie the block in differentiation resulting from GLI2 overexpression, and is consistent with disruption of differentiation when SOX2 is expressed at high levels in basal epithelial cells of transgenic mice (Okubo et al., 2006).


Stromal control of oncogenic traits expressed in response to the overexpression of GLI2, a pleiotropic oncogene.

Snijders AM, Huey B, Connelly ST, Roy R, Jordan RC, Schmidt BL, Albertson DG - Oncogene (2008)

GLI2 induces expression of stem cell genesSections of organotypic cultures of GLI2 expressing HaCaT GLI2 cells stained using HRP and DAB detection and antibodies to SOX2 and TITF1. The locations of keratinocytes in the sections are indicated by staining adjacent sections with antibodies to CAM5.2. Two photomicrographs are shown from each section.
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Related In: Results  -  Collection

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Figure 5: GLI2 induces expression of stem cell genesSections of organotypic cultures of GLI2 expressing HaCaT GLI2 cells stained using HRP and DAB detection and antibodies to SOX2 and TITF1. The locations of keratinocytes in the sections are indicated by staining adjacent sections with antibodies to CAM5.2. Two photomicrographs are shown from each section.
Mentions: Stem cell gene expression signatures have been reported in poorly differentiated breast and other tumors (Ben-Porath et al., 2008; Chen et al., 2008), as well as in gliomas, which are associated with activated hedgehog signaling and GLI overexpression (Clement et al., 2007), suggesting that GLI2 might block keratinocyte differentiation by up-regulating stem cell genes. To investigate this possibility, expression profiling was carried on RNA extracted from isolated keratinocyte layers from the tissue reconstructs. This analysis revealed elevated expression of SOX2 in the GLI2 expressing keratinocytes (Supplementary Figure 5), which was also confirmed by staining reconstructs with antibodies to SOX2 (Figure 5). These observations suggest that SOX2 is a GLI2 responsive gene. Indeed, infection of primary keratinocytes with a GLI2 retrovirus resulted in induction of SOX2 RNA (Supplementary Figure 6A), and in HaCaT GLI2 cells, addition of doxcycline induced SOX2 expression with the same kinetics as the two known GLI2 downstream targets, GLI1 and PTCH1 (Supplementary Figure 6B). Moreover, a number of potential GLI2 consensus binding sites are present in SOX2 (Supplementary Table 4). In a separate analysis, we found that neither NANOG nor OCT4 (POU5F1) was up-regulated in GLI2 expressing cells (data not shown), whereas elevated RNA and protein expression of TITF1 (Nkx2.1) was observed (Figure 5). TITF1 is reciprocally regulated with SOX2 during early dorsal/ventral foregut patterning (Que et al., 2007). It is frequently amplified in lung cancer (Kendall et al., 2007; Kwei et al., 2008; Weir et al., 2007) and was amplified in one of 89 oral SCC (Snijders et al., 2005). Thus, induction of stem cell genes is likely to underlie the block in differentiation resulting from GLI2 overexpression, and is consistent with disruption of differentiation when SOX2 is expressed at high levels in basal epithelial cells of transgenic mice (Okubo et al., 2006).

Bottom Line: We show that GLI2 is a pleiotropic oncogene.The overexpression induces genomic instability and blocks differentiation, likely mediated in part by enhanced expression of the stem cell gene SOX2.GLI2 also induces transforming growth factor (TGF)B1-dependent transdifferentiation of foreskin and tongue, but not gingival fibroblasts into myofibroblasts, creating an environment permissive for invasion by keratinocytes, which are in various stages of differentiation having downregulated GLI2.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Institute, University of California San Francisco, San Francisco, CA 94143-0808, USA.

ABSTRACT
Hedgehog signaling is often activated in tumors, yet it remains unclear how GLI2, a transcription factor activated by this pathway, acts as an oncogene. We show that GLI2 is a pleiotropic oncogene. The overexpression induces genomic instability and blocks differentiation, likely mediated in part by enhanced expression of the stem cell gene SOX2. GLI2 also induces transforming growth factor (TGF)B1-dependent transdifferentiation of foreskin and tongue, but not gingival fibroblasts into myofibroblasts, creating an environment permissive for invasion by keratinocytes, which are in various stages of differentiation having downregulated GLI2. Thus, upregulated GLI2 expression is sufficient to induce a number of the acquired characteristics of tumor cells; however, the stroma, in a tissue-specific manner, determines whether certain GLI2 oncogenic traits are expressed.

Show MeSH
Related in: MedlinePlus