Phosphatase and tensin homolog (PTEN) pseudogene expression in endometrial cancer: a conserved regulatory mechanism important in tumorigenesis?

Abstract

Objectives: The PTEN pseudogene, PTENP1, was recently shown to play a role in cell proliferation in a prostate cancer model. In the present study, we sought to determine whether PTENP1 is expressed in endometrial cancer (EMCA) cell lines and primary tumors along with the microRNAs (miRNAs) that are predicted to regulate PTEN and PTENP1 transcript levels.

Methods: RNA was prepared from six EMCA cell lines, three normal endometrial samples, and 61 primary tumors. TaqMan® RT-PCR was used to quantitate PTEN expression in all specimens and PTENP1 expression in cell lines, and normal endometrial (NE) samples. PTENP1 expression was evaluated using conventional RT-PCR in primary tumors. MicroRNA profiling was undertaken using NanoString(TM) technology in AN3CA and KLE cell lines. The relationship between PTEN transcript levels, PTENP1 expression, and PTEN mutation status was investigated.

Results: All NE samples, cell lines, and primary tumors expressed PTEN. PTENP1 transcript was expressed in NE, cell lines, and 34/61 (56%) primary tumors. The median relative PTEN level was 2.9 arbitrary expression units in PTENP1-positive tumors and 2.3 in PTENP1-negative tumors (p=0.09). PTEN levels in wild-type and haploinsufficient tumors were variable compared to PTEN-null tumors (p=0.015). Four microRNAs predicted to bind PTEN/PTENP1 ranked in the top 20 most abundant microRNA subtypes in the AN3CA and KLE cell lines.

Conclusions: PTENP1 is expressed in NE and EMCA cell lines, as are PTEN/PTENP1 targeting inhibitory miRNAs (cell lines). Further studies are needed to evaluate the impact of PTEN/PTENP1/miRNA interactions on tumorigenesis regulation in EMCA.

Fig. 1

PTEN and PTENP1 expression in endometrial carcinoma cell lines and normal endometrium. A. Quantitative expression of PTEN and PTENP1. Log₁₀-transformed averages of replicate experiments and standard errors of the mean are presented. Relative expression values are normalized to DU145 (prostate cancer cell line). *: PTEN or PTENP1 not expressed (exponential amplification cut off C_t ≥ 35). B. Representative RT-PCR analysis. PCR products (conventional RT-PCR 35 cycles) resolved on 10% polyacrylamide gels. The 246 bp PTENP1 PCR product is present in positive controls (DU145 and PC3 cell lines) and the Ishikawa, KLE, and MFE296 cell lines. Minus RT controls confirm that the PCR product is derived from the PTENP1 transcript. NE1 and NE3 samples are also positive for the 246 bp amplimer. Φx: Φx174 RF DNA/Hae III molecular size marker.

Fig. 2

PTEN and PTENP1 expression in primary endometrial adenocarcinomas. A. Frequency distribution of average PTEN levels (expressed in arbitrary expression units) in primary tumors, normal endometria, and endometrial cancer cell lines. Relative expression normalized to DU145. Primary carcinomas show ∼ 135-fold range of expression. B. Representative RT-PCR PTENP1 expression analysis. The 246 bp PTENP1 PCR product is present in the positive control cell lines DU145, PC3 and Ishikawa (lanes 2–4) and in six primary tumors (lanes 6–11). The PTENP1 246 bp RT-PCR product is absent in two tumors that do not express PTENP1 (lanes 5 and 12). No template control is shown in lane 1. PCR products resolved on 10% polyacrylamide gels with Φx174 RF DNA/Hae III size standards. Variable intensity of the PCR product in positive controls and tumors may reflect differences in transcript levels. C. Relative PTEN expression in PTENP1- postivie and PTENP1-negative primary tumors. Graph of log₁₀ transformed mean relative PTEN expression values of PTENP1-positive and PTENP1-negative tumors as assessed by real time PCR normalized to the DU145 cell line. Data points represent log₁₀ transformed averages of three replicate experiments. Median PTEN levels for each group are indicated with horizontal bars. Dashed line: DU145 reference. *: Two tailed Mann–Whitney test.

Fig. 3

PTENP1 expression and PTEN transcript levels in primary tumors stratified by PTEN mutation status. A. Relative PTEN expression in PTEN wild type (+/+), haploinsufficient (+/−), and PTEN null (−/−) tumors. PTEN levels were more variable in PTEN wild-type (+/+) and haploinsufficient (+/−) tumors than in tumors with two mutations (−/−). *: Test for equality of variances. B. Log₁₀-transformed averages of PTEN expression in wild-type (+/+) and haploinsufficient (+/−) PTENP1-positive and PTENP1-negative tumors are presented. Bars represent mean PTEN transcript values. Error bars represent standard errors of the mean. Dashed line: DU145 reference. †: One-tailed t-test.