Limits...
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.

Show MeSH

Related in: MedlinePlus

PIK3CA contributed to K-ras initiated transformation in vitro.A and B. Summary of colony numbers of the different combination of oncogenes in soft agar assay.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2629728&req=5

pone-0004295-g008: PIK3CA contributed to K-ras initiated transformation in vitro.A and B. Summary of colony numbers of the different combination of oncogenes in soft agar assay.

Mentions: To further confirm the above in vivo observation, we next tested the role of PIK3CA in OSE transformation in vitro. Primary murine OSE cells (MOSEs) were isolated from ovaries of female mice and cultured short-term (only early passage, <10 passages, cells were used in this study) [40]. Soft agar assay was used to evaluate the potential contribution of PIK3CA in transformation of OSE [41]. Because PI3-kinase/PTEN-ras [8] and PI3-kinase/AKT-myc [5] pathways have been reported involving in OSE transformation, we chose ras and c-myc in our following studies. Myr-PIK3CA (pUSEamp-myr-mPIK3CA, under CMV promoter, G418), K-rasv12 and c-myc (both in pBabe-puro vector, puromycin) expression vectors were transfected to MOSE cells. The pooled stable expression cells were generated by short-term antibiotic selection (10 days for G418 or 3 days for puromycin). The transgene expression was further confirmed by RT-PCR. We found that overexpression of PIK3CA alone did not significantly increase anchorage-independent growth of OSEs (Figure 8A). In contrast, introduce of either mutant K-ras of c-myc resulted in increased colony formation of OSEs (Figure 8A). This result indicates that unlike mutant K-ras or c-myc, PIK3CA is not able to cause complete transformation of OSE in vitro. Next, we tested the combination of PIK3CA with either of the two other oncogenes by co-transfection. The myr-PIK3CA was transfected first. After 10 days G418 selection, K-ras or c-myc was transfected subsequently and selected again by puromycin for 3 days. Interestingly, we found that combining PIK3CA and mutant K-ras significantly increased anchorage-independent growth of cultured OSE cells compared with mutant K-ras alone (Figure 8A). However, PIK3CA did not significantly increase colony numbers of OSE cells transformed by c-myc (Figure 8A). This finding suggests that early activation of PIK3CA might promote transformation of OSE cells in certain cellular and molecular contexts, e.g. in the presence of K-ras mutation. To further confirm that PIK3CA can play a role in transformation induced by mutant K-ras, we blocked endogenous PIK3CA expression in cultured OSE cells transfected with mutant K-ras by RNA interference using small interfering RNAs (siRNA). The efficiency of the siRNA targeting mPIK3CA was confirmed by real-time RT-PCR (mRNA expression of mPIK3CA was knocked down to ∼30% in the siRNA treated cells compared with control cells, data not shown). The specificity of the siRNA was also examined by measuring endogenous mPIK3CB expression (there was no significantly difference of PIK3CB mRNA expression between siRNA treated and control cells, data not shown). We found that in the absence of PIK3CA, the transformed OSEs could not grow in soft agar (Figure 8B), which suggests that the growth of K-ras transfected OSE cells require PIK3CA expression. However, it is still unclear whether PIK3CA contributes to K-ras initiated transformation in vivo. Generation of “bigenic” mouse expressing both activated PIK3CA and mutant Ras specifically in murine OSE using MISIIR promoter is still a technical challenge. Because mutant Ras is able to fully transform epithelium in vivo [42], MISIIR-driven mutant Ras will induce very rapidly reproductive system carcinoma in both female and male animals, and these transgenic mice will lose reproductive ability. Therefore, it will be difficult to generate “bigenic” animals by crossing MISIIR-driven mutant Ras and MISIIR-driven myr-PIK3CA mice. We believe that a novel transgenic strategy based on the Cre-loxp conditional expression and intrabursal administration of adenovirus [6], [8], [9] may allow us to further test PIK3CA and Ras cooperation in vivo in the future.


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)

PIK3CA contributed to K-ras initiated transformation in vitro.A and B. Summary of colony numbers of the different combination of oncogenes in soft agar assay.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004295-g008: PIK3CA contributed to K-ras initiated transformation in vitro.A and B. Summary of colony numbers of the different combination of oncogenes in soft agar assay.
Mentions: To further confirm the above in vivo observation, we next tested the role of PIK3CA in OSE transformation in vitro. Primary murine OSE cells (MOSEs) were isolated from ovaries of female mice and cultured short-term (only early passage, <10 passages, cells were used in this study) [40]. Soft agar assay was used to evaluate the potential contribution of PIK3CA in transformation of OSE [41]. Because PI3-kinase/PTEN-ras [8] and PI3-kinase/AKT-myc [5] pathways have been reported involving in OSE transformation, we chose ras and c-myc in our following studies. Myr-PIK3CA (pUSEamp-myr-mPIK3CA, under CMV promoter, G418), K-rasv12 and c-myc (both in pBabe-puro vector, puromycin) expression vectors were transfected to MOSE cells. The pooled stable expression cells were generated by short-term antibiotic selection (10 days for G418 or 3 days for puromycin). The transgene expression was further confirmed by RT-PCR. We found that overexpression of PIK3CA alone did not significantly increase anchorage-independent growth of OSEs (Figure 8A). In contrast, introduce of either mutant K-ras of c-myc resulted in increased colony formation of OSEs (Figure 8A). This result indicates that unlike mutant K-ras or c-myc, PIK3CA is not able to cause complete transformation of OSE in vitro. Next, we tested the combination of PIK3CA with either of the two other oncogenes by co-transfection. The myr-PIK3CA was transfected first. After 10 days G418 selection, K-ras or c-myc was transfected subsequently and selected again by puromycin for 3 days. Interestingly, we found that combining PIK3CA and mutant K-ras significantly increased anchorage-independent growth of cultured OSE cells compared with mutant K-ras alone (Figure 8A). However, PIK3CA did not significantly increase colony numbers of OSE cells transformed by c-myc (Figure 8A). This finding suggests that early activation of PIK3CA might promote transformation of OSE cells in certain cellular and molecular contexts, e.g. in the presence of K-ras mutation. To further confirm that PIK3CA can play a role in transformation induced by mutant K-ras, we blocked endogenous PIK3CA expression in cultured OSE cells transfected with mutant K-ras by RNA interference using small interfering RNAs (siRNA). The efficiency of the siRNA targeting mPIK3CA was confirmed by real-time RT-PCR (mRNA expression of mPIK3CA was knocked down to ∼30% in the siRNA treated cells compared with control cells, data not shown). The specificity of the siRNA was also examined by measuring endogenous mPIK3CB expression (there was no significantly difference of PIK3CB mRNA expression between siRNA treated and control cells, data not shown). We found that in the absence of PIK3CA, the transformed OSEs could not grow in soft agar (Figure 8B), which suggests that the growth of K-ras transfected OSE cells require PIK3CA expression. However, it is still unclear whether PIK3CA contributes to K-ras initiated transformation in vivo. Generation of “bigenic” mouse expressing both activated PIK3CA and mutant Ras specifically in murine OSE using MISIIR promoter is still a technical challenge. Because mutant Ras is able to fully transform epithelium in vivo [42], MISIIR-driven mutant Ras will induce very rapidly reproductive system carcinoma in both female and male animals, and these transgenic mice will lose reproductive ability. Therefore, it will be difficult to generate “bigenic” animals by crossing MISIIR-driven mutant Ras and MISIIR-driven myr-PIK3CA mice. We believe that a novel transgenic strategy based on the Cre-loxp conditional expression and intrabursal administration of adenovirus [6], [8], [9] may allow us to further test PIK3CA and Ras cooperation in vivo in the future.

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