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Expression of activated PIK3CA in ovarian surface epithelium results in hyperplasia but not tumor formation.

Liang S, Yang N, Pan Y, Deng S, Lin X, Yang X, Katsaros D, Roby KF, Hamilton TC, Connolly DC, Coukos G, Zhang L - PLoS ONE (2009)

Bottom Line: The consistent result was also observed in primary cultured OSEs.Although enforced expression of PIK3CA could not induce OSE anchorage-independent growth, it significantly increased anchorage-independent growth of OSE transformed by mutant K-ras.While PIK3CA activation may not be able to initiate OSE transformation, we conclude that activation of PIK3CA may be an important molecular event contributing to the maintenance of OSE transformation initiated by oncogenes such as K-ras.

View Article: PubMed Central - PubMed

Affiliation: Center for Research on the Early Detection and Cure of Ovarian Cancer, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: The Phosphatidylinositol 3'-kinase is a key regulator in various cancer-associated signal transduction pathways. Genetic alterations of its catalytic subunit alpha, PIK3CA, have been identified in ovarian cancer. Our in vivo data suggests that PIK3CA activation is one of the early genetic events in ovarian cancer. However, its role in malignant transformation of ovarian surface epithelium (OSE) is largely unclear.

Methodology/principal findings: Using the Müllerian inhibiting substance type II receptor (MISIIR) promoter, we generated transgenic mice that expressed activated PIK3CA in the Müllerian epithelium. Overexpression of PIK3CA in OSE induced remarkable hyperplasia, but was not able to malignantly transform OSE in vivo. The consistent result was also observed in primary cultured OSEs. Although enforced expression of PIK3CA could not induce OSE anchorage-independent growth, it significantly increased anchorage-independent growth of OSE transformed by mutant K-ras.

Conclusions/significance: While PIK3CA activation may not be able to initiate OSE transformation, we conclude that activation of PIK3CA may be an important molecular event contributing to the maintenance of OSE transformation initiated by oncogenes such as K-ras.

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Related in: MedlinePlus

The illustration of the construct that was used to generate transgenic mouse.The arrows show two sets of the genotyping primers and one set of the RT-PCR primers.
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pone-0004295-g003: The illustration of the construct that was used to generate transgenic mouse.The arrows show two sets of the genotyping primers and one set of the RT-PCR primers.

Mentions: To investigate the role of PIK3CA in malignant transformation of the OSE in vivo, we generated transgenic mice in which activated PIK3CA was specifically overexpressed in the Müllerian epithelium of the female reproductive tract including OSE. In this model, we used the Müllerian epithelium specific promoter, Müllerian inhibiting substance type II receptor promoter (MISIIR) [36], [37], [38], [39], to drive expression of the murine PIK3CA (Figure 3). In the male animal, Müllerian inhibiting substance (MIS) is secreted from Sertoli cells of the developing testes and stimulates the regression of the Mullerian duct. Testosterone is also secreted from the developing testes and induces the differentiation of the Wolffian duct into the secondary structures of the male reproductive tract. In the absence of MIS in the developing female embryo, the Mullerian duct differentiates into the secondary structures of the female reproductive tract [36], [37], [38], [39]. Expression of the MISIIR has been reported to be restricted to mesenchymal cells surrounding the Mullerian duct during embryogenesis, tubular and follicular structures of fetal gonads, Sertoli and Leydig cells of adult testis, and granulosa cells of adult ovary [36], [37], [38], [39]. Above information provides a possible strategy to develop a transgenic model of ovarian carcinoma [39]. Using this promoter, Connolly et al. have successfully developed the very first ovarian cancer transgenic models that develop ovarian carcinomas with metastatic spread to peritoneal organs [39]. In addition, increasing evidence indicates that PIK3CA is activated in a large percentage of human ovarian cancer patients [25], [27], [35]. Therefore, we generated the activating mutation by addition of the avian src myristoylation sequence (MGSSKSKPK) at the N-terminus of the wild type of murine PIK3CA to constitutively activate PI3-kinase pathway in vivo. To demonstrate that our transgenic construct was able to constitutively activate PI3-kinase pathway, we transient transfected myr-PIK3CA, wt-PIK3CA and control vectors to ovarian cancer cell line 2008. 48 hrs of post-transfection, the transfected cells were cultured in low serum overnight. Protein and total RNA were isolated from cells. Real-time RT-PCR demonstrated that cells from wt-PIK3CA and myr-PIK3CA transfections were expressed similar levels of PIK3CA mRNA, which was about 11.5-fold higher comparing to cells from control vector transfection. Total and phosphate AKT, the downstream molecule of PI3-kinase pathway, were examined by western blot. Figure 4 showed that myr-PIK3CA was able to constitutively activate AKT in low serum condition (1%) compared with wt-PIK3CA and control transfection.


Expression of activated PIK3CA in ovarian surface epithelium results in hyperplasia but not tumor formation.

Liang S, Yang N, Pan Y, Deng S, Lin X, Yang X, Katsaros D, Roby KF, Hamilton TC, Connolly DC, Coukos G, Zhang L - PLoS ONE (2009)

The illustration of the construct that was used to generate transgenic mouse.The arrows show two sets of the genotyping primers and one set of the RT-PCR primers.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2629728&req=5

pone-0004295-g003: The illustration of the construct that was used to generate transgenic mouse.The arrows show two sets of the genotyping primers and one set of the RT-PCR primers.
Mentions: To investigate the role of PIK3CA in malignant transformation of the OSE in vivo, we generated transgenic mice in which activated PIK3CA was specifically overexpressed in the Müllerian epithelium of the female reproductive tract including OSE. In this model, we used the Müllerian epithelium specific promoter, Müllerian inhibiting substance type II receptor promoter (MISIIR) [36], [37], [38], [39], to drive expression of the murine PIK3CA (Figure 3). In the male animal, Müllerian inhibiting substance (MIS) is secreted from Sertoli cells of the developing testes and stimulates the regression of the Mullerian duct. Testosterone is also secreted from the developing testes and induces the differentiation of the Wolffian duct into the secondary structures of the male reproductive tract. In the absence of MIS in the developing female embryo, the Mullerian duct differentiates into the secondary structures of the female reproductive tract [36], [37], [38], [39]. Expression of the MISIIR has been reported to be restricted to mesenchymal cells surrounding the Mullerian duct during embryogenesis, tubular and follicular structures of fetal gonads, Sertoli and Leydig cells of adult testis, and granulosa cells of adult ovary [36], [37], [38], [39]. Above information provides a possible strategy to develop a transgenic model of ovarian carcinoma [39]. Using this promoter, Connolly et al. have successfully developed the very first ovarian cancer transgenic models that develop ovarian carcinomas with metastatic spread to peritoneal organs [39]. In addition, increasing evidence indicates that PIK3CA is activated in a large percentage of human ovarian cancer patients [25], [27], [35]. Therefore, we generated the activating mutation by addition of the avian src myristoylation sequence (MGSSKSKPK) at the N-terminus of the wild type of murine PIK3CA to constitutively activate PI3-kinase pathway in vivo. To demonstrate that our transgenic construct was able to constitutively activate PI3-kinase pathway, we transient transfected myr-PIK3CA, wt-PIK3CA and control vectors to ovarian cancer cell line 2008. 48 hrs of post-transfection, the transfected cells were cultured in low serum overnight. Protein and total RNA were isolated from cells. Real-time RT-PCR demonstrated that cells from wt-PIK3CA and myr-PIK3CA transfections were expressed similar levels of PIK3CA mRNA, which was about 11.5-fold higher comparing to cells from control vector transfection. Total and phosphate AKT, the downstream molecule of PI3-kinase pathway, were examined by western blot. Figure 4 showed that myr-PIK3CA was able to constitutively activate AKT in low serum condition (1%) compared with wt-PIK3CA and control transfection.

Bottom Line: The consistent result was also observed in primary cultured OSEs.Although enforced expression of PIK3CA could not induce OSE anchorage-independent growth, it significantly increased anchorage-independent growth of OSE transformed by mutant K-ras.While PIK3CA activation may not be able to initiate OSE transformation, we conclude that activation of PIK3CA may be an important molecular event contributing to the maintenance of OSE transformation initiated by oncogenes such as K-ras.

View Article: PubMed Central - PubMed

Affiliation: Center for Research on the Early Detection and Cure of Ovarian Cancer, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: The Phosphatidylinositol 3'-kinase is a key regulator in various cancer-associated signal transduction pathways. Genetic alterations of its catalytic subunit alpha, PIK3CA, have been identified in ovarian cancer. Our in vivo data suggests that PIK3CA activation is one of the early genetic events in ovarian cancer. However, its role in malignant transformation of ovarian surface epithelium (OSE) is largely unclear.

Methodology/principal findings: Using the Müllerian inhibiting substance type II receptor (MISIIR) promoter, we generated transgenic mice that expressed activated PIK3CA in the Müllerian epithelium. Overexpression of PIK3CA in OSE induced remarkable hyperplasia, but was not able to malignantly transform OSE in vivo. The consistent result was also observed in primary cultured OSEs. Although enforced expression of PIK3CA could not induce OSE anchorage-independent growth, it significantly increased anchorage-independent growth of OSE transformed by mutant K-ras.

Conclusions/significance: While PIK3CA activation may not be able to initiate OSE transformation, we conclude that activation of PIK3CA may be an important molecular event contributing to the maintenance of OSE transformation initiated by oncogenes such as K-ras.

Show MeSH
Related in: MedlinePlus