Suppression of GNAI2 message in ovarian cancer

Abstract

Background: Understanding the integration of hormone signaling and how it impacts oncogenesis is critical for improved cancer treatments. Here we elucidate GNAI2 message alterations in ovarian cancer (OvCa). GNAI2 is a heterotrimeric G protein which couples cell surface hormone receptors to intracellular enzymes, and is best characterized for its direct role in regulating cAMP response element-binding protein (CREB) function by decreasing intracellular cAMP through inhibiting adenylyl cyclase.

Methods: We probed the Origene human OvCa array for the presence of polymorphisms and gene expression alterations of GNAI2 using directing sequencing and qPCR. These data were supported by database mining of the [NCBI NIH GSE:6008, GSE:14764, GSE:29450, GDS:4066, GDS:3297, GSE:32474, and GSE:2003] datasets.

Results: No significant polymorphisms were found, including an absence of the gip2 oncogene. However, 85.9% of (506 of 589) OvCa patients had decreased GNAI2 message. Further characterization demonstrated that the GNAI2 message was on average decreased 54% and maximally decreased by 2.8 fold in clear cell carcinoma. GNAI2 message decreased in early stage cancer while message was increased compared to normal in advanced cancers. The changes in GNAI2 also correlated to deregulation of CREB, Fos, Myc, cyclins, Arf, the transition from estrogen dependence to independence, and metastatic potential.

Conclusion: These data strongly implicate GNAI2 as a critical regulator of oncogenesis and an upstream driver of cancer progression in OvCa.

Figure 1

GNAI2 message is suppressed in ovarian cancer. A. Rank order of GNAI2 expression of Origene OCA. Normal n = 27, Cancer n = 165. B. GNAI2 expression from NIH NCBI TCGA, Nature 2011. Normal n = 8, Cancer n = 315. C. Scatter plot of A. * = cancer specific underexpression in 58% of patients. p < 0.004. D. Scatter plot of B. * = Nonparametric p < 0.003 Normal n = 8, Cancer n = 315. E. and F. GNAI2 expression of NCI60 samples. E. Data derived from GSE32374. F. Data derived from GSE2003. Expression presented as normalized gene centric values derived from ∆∆CT with variance presented as 95% CI.

Figure 2

GNAI2 expression correlates with ovarian cancer tissue type. A. Data derived from GSE6008 normal n = 4, mucinous n = 13, clear cell n = 8, endometrioid n = 37, serous n = 41. Nonparametric normal and clear p < 0.005, normal and mucinous p < 0.03, normal and endometrioid p < 0.02, normal and serous p < 0.05. Expression is presented as HPRT1 normalized ∆∆CT. B. Data derived from GSE14764. Clear cell n = 2, endometrial ovarian n = 7, serous n = 68. Kruskal-Wallis ANOVA p < 0.005. Expression is presented as Affymetrix MAS 5.0 signal intensity C. Data derived from GSE29450. Normal n = 10. Clear Cell n = 10. Nonparametric p < 0.0001. Values reported as RMA signal intensity. D. GNAI2 expression correlates with estrogen sensitivity. Data derived from GDS4066, ER + n = 7, ER- n = 8. Nonparametric p < 0.006. Values reported as RMA signal intensity.

Figure 3

GNAI2 expression correlates with tumor stage. A. Data derived from GDS3297. IIIB n = 9, IIIC n = 23. Nonparametric p < 0.04 B. Data derived from TCGA, Nature 2011. Normal n = 8, IIA n = 2, IIB n = 4, IIC n = 8, IIIA n = 4, IIIB n = 14, IIIC n = 230, IV n = 53. Kruskal-Wallis p < 0.02. Normal versus IIIA nonparametric p < 0.009, IIIB Nonparametric p < 0.006, IIIC Nonparametric p < 0.004, IV Nonparametric p < 0.007. C. Data derived from GSE6008: stage III n = 2, IIIC n = 25, IV n = 5. Kruskal-Wallis ANOVA p < 0.03, Gaussian ANOVA p < 0.05, Gaussian III vs. IV p < 0.04, Gaussian IIIC vs. IV p < 0.05. Anderson-Darling Normality for IIIC p > 0.6 and for IV p > 0.9. Stage IIIC and stage IV were also statistically different using a parametric T-test. Expression is presented as HPRT1 normalized ∆∆CT. D. Methylation of GNAI2 gene correlates inversely with GNAI2 message expression. Methylation values reported as beta values.

Figure 4

CRE effects in ovarian cancer. Data derived from GSE6008. Normal n = 4, mucinous n = 13, clear cell n = 8, endometrioid n = 37, serous n = 41. A. Nonparametric normal and clear p < 0.005, normal and mucinous p < 0.004, normal and endometrioid p < 0.003, normal and serous p < 0.002. B. Nonparametric normal versus endometrioid p < 0.03, normal and serous p < 0.004 C. Nonparametric normal and clear p < 0.005, normal and mucinous p < 0.004, normal and endometrioid p < 0.002, normal and serous p < 0.003. D. Nonparametric normal and endometroioid p < 0.027, normal and serous p < 0.004 E. Nonparametric normal and clear p < 0.005, normal and mucinous p < 0.004, normal and endometrioid p < 0.002, normal and serous p < 0.003. F. Nonparametric normal and clear p = 0.004. G. Nonparametric normal and clear p < 0.05, normal and mucinous p < 0.02, normal and endometrioid p < 0.008, normal and serous p < 0.002. H. Data derived from from NIH NCBI TCGA, Nature 2011. Normal n = 8, stage IIA n = 3, IIB n = 4, IIC n = 16, stage IIIA n = 7, stage IIIB n = 21, stage IIIC n = 353, stage IV n = 79. Nonparametric normal and stage IIA p < 0.01, normal and stage IIB p < 0.005, normal and stage IIC p < 0.001, normal and stage IIIA was p < 0.03, normal and stage IIIB p < 0.002, normal and stage IIIC p < 0.0001, normal and stage IV p < 0.0001. I. Data derived from NIH NCBI TCGA, Nature 2011. Stage IIB n = 4, IIC n = 8, stage IIIA n = 4, stage IIIB n = 14, stage IIIC n = 230, stage IV n = 53.