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Molecular and functional characteristics of ovarian surface epithelial cells transformed by KrasG12D and loss of Pten in a mouse model in vivo.

Mullany LK, Fan HY, Liu Z, White LD, Marshall A, Gunaratne P, Anderson ML, Creighton CJ, Xin L, Deavers M, Wong KK, Richards JS - Oncogene (2011)

Bottom Line: Ovarian cancer is a complex and deadly disease that remains difficult to detect at an early curable stage.Furthermore, although some oncogenic (Kras, Pten/PI3K and Trp53) pathways that are frequently mutated, deleted or amplified in ovarian cancer are known, how these pathways initiate and drive specific morphological phenotypes and tumor outcomes remain unclear.We propose that elevated TRP53 and miR-34a-c may exert negatively regulatory effects that reduce the proliferative potential of OSE cells leading to the low-grade serous adenocarcinoma phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
Ovarian cancer is a complex and deadly disease that remains difficult to detect at an early curable stage. Furthermore, although some oncogenic (Kras, Pten/PI3K and Trp53) pathways that are frequently mutated, deleted or amplified in ovarian cancer are known, how these pathways initiate and drive specific morphological phenotypes and tumor outcomes remain unclear. We recently generated Pten(fl/fl); Kras(G12D); Amhr2-Cre mice to disrupt the Pten gene and express a stable mutant form of Kras(G12D) in ovarian surface epithelial (OSE) cells. On the basis of histopathologic criteria, the mutant mice developed low-grade ovarian serous papillary adenocarcinomas at an early age and with 100% penetrance. This highly reproducible phenotype provides the first mouse model in which to study this ovarian cancer subtype. OSE cells isolated from ovaries of mutant mice at 5 and 10 weeks of age exhibit temporal changes in the expression of specific Mullerian epithelial marker genes, grow in soft agar and develop ectopic invasive tumors in recipient mice, indicating that the cells are transformed. Gene profiling identified specific mRNAs and microRNAs differentially expressed in purified OSE cells derived from tumors of the mutant mice compared with wild-type OSE cells. Mapping of transcripts or genes between the mouse OSE mutant data sets, the Kras signature from human cancer cell lines and the human ovarian tumor array data sets, documented significant overlap, indicating that KRAS is a key driver of OSE transformation in this context. Two key hallmarks of the mutant OSE cells in these mice are the elevated expression of the tumor-suppressor Trp53 (p53) and its microRNA target, miR-34a-c. We propose that elevated TRP53 and miR-34a-c may exert negatively regulatory effects that reduce the proliferative potential of OSE cells leading to the low-grade serous adenocarcinoma phenotype.

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The MEK1/ERK1/2 and PI3K/AKT pathways drive gene expression and transformation of the mutant OSE cells. A.) Western blot of lysates prepared from mutant OSE cells cultured in media alone or with either the MEK1 inhibitor UO126 (10 □M) or the PI3K inhibitor LY294002 (10 □M) for 24 hrs. B.) Colony formation of mutant OSE cells grown in soft agar is inhibited by both UO126 and LY294002. C.) Gene expression is altered in cells cultured in media alone or with either UO126 or LY294002 for 24h. D.) TRP53 immunolabeling decreases in mutant cells cultured for 24 hours with or without 10 □M UO126 or LY294002 compared to media alone.
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Figure 5: The MEK1/ERK1/2 and PI3K/AKT pathways drive gene expression and transformation of the mutant OSE cells. A.) Western blot of lysates prepared from mutant OSE cells cultured in media alone or with either the MEK1 inhibitor UO126 (10 □M) or the PI3K inhibitor LY294002 (10 □M) for 24 hrs. B.) Colony formation of mutant OSE cells grown in soft agar is inhibited by both UO126 and LY294002. C.) Gene expression is altered in cells cultured in media alone or with either UO126 or LY294002 for 24h. D.) TRP53 immunolabeling decreases in mutant cells cultured for 24 hours with or without 10 □M UO126 or LY294002 compared to media alone.

Mentions: The disruption of Pten and expression of mutant KRASG12D are expected to increase the activity of the PI3K/AKT and RAS/MEK1/ERK1/2 pathways, respectively. However, there is also important cross-talk between these pathways and increasing evidence indicates that activation of the PI3K pathway is essential to maintain the growth promoting and transformation effects of KRAS (Miller KA, Yeager N, et al., 2009), in part, by blocking negative feedback regulatory loops (Wee S, Jagani Z, et al., 2009). Therefore, we determined if pharmacological disruption of either PI3K or MEK1/ERK1/2 activity would prevent colony formation and/or the expression of selected genes. As shown in Figure 5A, AKT and ERK1/2 are phosphorylated and hence activated in OSE cells isolated from ovaries of tumor-bearing mice at 10 weeks of age and this was blocked by blocked by inhibitors of PI3K (LY294002) and MEK1 (U0126), respectively. PTEN was undetectable and Foxo1 mRNA was markedly reduced in the mutant OSE cell (data not shown). When U0126 and/or LY294002 were added to the soft agar, they inhibited colony formation by approximately 70-90% (Fig. 5B).


Molecular and functional characteristics of ovarian surface epithelial cells transformed by KrasG12D and loss of Pten in a mouse model in vivo.

Mullany LK, Fan HY, Liu Z, White LD, Marshall A, Gunaratne P, Anderson ML, Creighton CJ, Xin L, Deavers M, Wong KK, Richards JS - Oncogene (2011)

The MEK1/ERK1/2 and PI3K/AKT pathways drive gene expression and transformation of the mutant OSE cells. A.) Western blot of lysates prepared from mutant OSE cells cultured in media alone or with either the MEK1 inhibitor UO126 (10 □M) or the PI3K inhibitor LY294002 (10 □M) for 24 hrs. B.) Colony formation of mutant OSE cells grown in soft agar is inhibited by both UO126 and LY294002. C.) Gene expression is altered in cells cultured in media alone or with either UO126 or LY294002 for 24h. D.) TRP53 immunolabeling decreases in mutant cells cultured for 24 hours with or without 10 □M UO126 or LY294002 compared to media alone.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139785&req=5

Figure 5: The MEK1/ERK1/2 and PI3K/AKT pathways drive gene expression and transformation of the mutant OSE cells. A.) Western blot of lysates prepared from mutant OSE cells cultured in media alone or with either the MEK1 inhibitor UO126 (10 □M) or the PI3K inhibitor LY294002 (10 □M) for 24 hrs. B.) Colony formation of mutant OSE cells grown in soft agar is inhibited by both UO126 and LY294002. C.) Gene expression is altered in cells cultured in media alone or with either UO126 or LY294002 for 24h. D.) TRP53 immunolabeling decreases in mutant cells cultured for 24 hours with or without 10 □M UO126 or LY294002 compared to media alone.
Mentions: The disruption of Pten and expression of mutant KRASG12D are expected to increase the activity of the PI3K/AKT and RAS/MEK1/ERK1/2 pathways, respectively. However, there is also important cross-talk between these pathways and increasing evidence indicates that activation of the PI3K pathway is essential to maintain the growth promoting and transformation effects of KRAS (Miller KA, Yeager N, et al., 2009), in part, by blocking negative feedback regulatory loops (Wee S, Jagani Z, et al., 2009). Therefore, we determined if pharmacological disruption of either PI3K or MEK1/ERK1/2 activity would prevent colony formation and/or the expression of selected genes. As shown in Figure 5A, AKT and ERK1/2 are phosphorylated and hence activated in OSE cells isolated from ovaries of tumor-bearing mice at 10 weeks of age and this was blocked by blocked by inhibitors of PI3K (LY294002) and MEK1 (U0126), respectively. PTEN was undetectable and Foxo1 mRNA was markedly reduced in the mutant OSE cell (data not shown). When U0126 and/or LY294002 were added to the soft agar, they inhibited colony formation by approximately 70-90% (Fig. 5B).

Bottom Line: Ovarian cancer is a complex and deadly disease that remains difficult to detect at an early curable stage.Furthermore, although some oncogenic (Kras, Pten/PI3K and Trp53) pathways that are frequently mutated, deleted or amplified in ovarian cancer are known, how these pathways initiate and drive specific morphological phenotypes and tumor outcomes remain unclear.We propose that elevated TRP53 and miR-34a-c may exert negatively regulatory effects that reduce the proliferative potential of OSE cells leading to the low-grade serous adenocarcinoma phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
Ovarian cancer is a complex and deadly disease that remains difficult to detect at an early curable stage. Furthermore, although some oncogenic (Kras, Pten/PI3K and Trp53) pathways that are frequently mutated, deleted or amplified in ovarian cancer are known, how these pathways initiate and drive specific morphological phenotypes and tumor outcomes remain unclear. We recently generated Pten(fl/fl); Kras(G12D); Amhr2-Cre mice to disrupt the Pten gene and express a stable mutant form of Kras(G12D) in ovarian surface epithelial (OSE) cells. On the basis of histopathologic criteria, the mutant mice developed low-grade ovarian serous papillary adenocarcinomas at an early age and with 100% penetrance. This highly reproducible phenotype provides the first mouse model in which to study this ovarian cancer subtype. OSE cells isolated from ovaries of mutant mice at 5 and 10 weeks of age exhibit temporal changes in the expression of specific Mullerian epithelial marker genes, grow in soft agar and develop ectopic invasive tumors in recipient mice, indicating that the cells are transformed. Gene profiling identified specific mRNAs and microRNAs differentially expressed in purified OSE cells derived from tumors of the mutant mice compared with wild-type OSE cells. Mapping of transcripts or genes between the mouse OSE mutant data sets, the Kras signature from human cancer cell lines and the human ovarian tumor array data sets, documented significant overlap, indicating that KRAS is a key driver of OSE transformation in this context. Two key hallmarks of the mutant OSE cells in these mice are the elevated expression of the tumor-suppressor Trp53 (p53) and its microRNA target, miR-34a-c. We propose that elevated TRP53 and miR-34a-c may exert negatively regulatory effects that reduce the proliferative potential of OSE cells leading to the low-grade serous adenocarcinoma phenotype.

Show MeSH
Related in: MedlinePlus