Prostate cancer ETS rearrangements switch a cell migration gene expression program from RAS/ERK to PI3K/AKT regulation

Abstract

Background: The RAS/ERK and PI3K/AKT pathways induce oncogenic gene expression programs and are commonly activated together in cancer cells. Often, RAS/ERK signaling is activated by mutation of the RAS or RAF oncogenes, and PI3K/AKT is activated by loss of the tumor suppressor PTEN. In prostate cancer, PTEN deletions are common, but, unlike other carcinomas, RAS and RAF mutations are rare. We have previously shown that over-expression of "oncogenic" ETS transcription factors, which occurs in about one-half of prostate tumors due to chromosome rearrangement, can bypass the need for RAS/ERK signaling in the activation of a cell migration gene expression program. In this study we test the role of RAS/ERK and PI3K/AKT signaling in the function of oncogenic ETS proteins.

Results: We find that oncogenic ETS expression negatively correlates with RAS and RAF mutations in prostate tumors. Furthermore, the oncogenic ETS transcription factors only increased cell migration in the absence of RAS/ERK activation. In contrast to RAS/ERK, it has been reported that oncogenic ETS expression positively correlates with PI3K/AKT activation. We identified a mechanistic explanation for this finding by showing that oncogenic ETS proteins required AKT signaling to activate a cell migration gene expression program through ETS/AP-1 binding sequences. Levels of pAKT correlated with the ability of oncogenic ETS proteins to increase cell migration, but this process did not require mTORC1.

Conclusions: Our findings indicate that oncogenic ETS rearrangements cause a cell migration gene expression program to switch from RAS/ERK control to PI3K/AKT control and provide a possible explanation for the high frequency of PTEN, but not RAS/RAF mutations in prostate cancer.

Figure 1

Prostate cell lines vary in oncogenic ETS expression and RAS/ERK pathway activation. (A) The sensitivity and specificity of antibodies detecting oncogenic ETS proteins were tested by immunoblot of the indicated amount of purified full-length proteins. (B) Immunoblots show levels of four oncogenic ETS proteins, pAKT (PI3K/AKT activation), pERK (RAS/ERK activation), total ERK, and tubulin control in six prostate cancer cell lines (left) and three cell lines derived from normal prostate (right). (C) Immunoblots show levels of ETV4 and pERK in the indicated cell lines with or without U0126 (10 μM, 10 hr). The same cell extracts are loaded on one gel above the dashed line, and a second gel below. ETV4 is only visible in DU145 cells after a very long exposure, hence it is not observed in (A).

Figure 2

ETS expression and RAS activation induce migration of prostate cells via the same pathway. (A) A transwell assay measured relative number of migrating RWPE cells expressing ERG or activated KRAS relative to normal RWPE cells (first lane). (B, C) Transwell assays measured migration of (B) RWPE cells, or (C) RWPE-KRAS cells expressing oncogenic (Black bars) or non-oncogenic (Grey bars) ETS proteins. Number of cells migrated is reported relative to the same cell line transduced with an empty vector (white bar). Mean and SEM of three biological replicates (each mean of two technical replicates) are shown for (A) and five biological replicates for (B) and (C). P-values compare indicated value to the hypothetical mean (1) and are calculated by t test: * < 0.05, ** < 0.005, unmarked > 0.05.

Figure 3

An active PI3K/AKT pathway is required for oncogenic ETS, but not KRAS, to induce prostate cell migration. (A) An immunoblot shows the levels of pAKT, pMEK (activator of ERK), or tubulin (control) after LY294002 (20 μM; 24 h) or ZSTK474 (2 μM; 24 h) treatment in RWPE-ERG or RWPE-KRAS cells. (B) A transwell assay measured cell migration of RWPE prostate cells with or without ERG and KRAS overexpression and in the presence or absence of the PI3K inhibitors LY294002 (20 μM) or ZSTK474 (2 μM). The number of migrated cells is shown as the mean and SEM of six biological replicates (except for ZSTK474 treated cells which have three replicates) relative to RWPE-empty vector. (C) A transwell assay, as in (A), tested the role of PI3K inhibition on ETV1 and ETV5 expressing RWPE cells and shows the mean and SEM of three biological replicates. (D) Results of the scratch assay performed in the presence or absence of LY294002 (20 μM) and AKT inhibitor VIII (10 μM) in RWPE-ERG (Grey bar) and RWPE-KRAS (white bar) cells. The percentage of scratch filled is shown as the mean and SEM of three biological replicates (each mean of three technical replicates) relative to no treatment. P-values are calculated by t test: * < 0.05, ** < 0.005, *** < 0.0005, unmarked > 0.05.

Figure 4

The PI3K pathway can alter the expression of cell migration genes via ETS/AP-1 sites in oncogenic ETS overexpressing cells. mRNA expression of (A)ARHGAP29 or (B)SMAD3, in the presence and absence of PI3K inhibitor (LY294002, 20 μM), in RWPE-ERG and RWPE-KRAS cells was measured by qRT-PCR and compared to RWPE cells. Mean and SEM of seven biological replicates are shown. (C) Firefly luciferase activity from a vector with the indicated sequences (3 copies of neighboring ETS and AP-1 binding sequences or versions of the same with point mutations) is shown relative to Renilla luciferase from a control vector transfected in RWPE cells. The ERK pathway is inhibited by UO126 where indicated. Mean and SEM of six biological replicates (each mean of two technical replicates) are shown. Luciferase reporter activity measured as in (C) is shown in (D) RWPE-ERG, or (E) RWPE-KRAS cells as activity in LY294002 treated cells (20 μM) relative to untreated. P-values are calculated by t test: n.s > 0.10, * < 0.05.

Figure 5

PI3K/AKT signaling in oncogenic ETS function is not through mTORC1. (A) shRNA knock down of mTOR, Raptor (mTORC1 complex) and Rictor (mTORC2 complex) in RWPE-ERG cells was confirmed by qRT-PCR analysis. Mean and SEM of two biological replicates (each mean of two technical replicates) are shown. (B) A transwell assay measured cell migration of RWPE-ERG cells stably expressing the indicated shRNA relative to a negative control (shRNA targeting luciferase, which is not expressed in this cell line). Results are the mean and SEM of four independent experiments, each the mean of two technical replicates. (C) Immunoblot showing the expression level of pAKT and tubulin in RWPE-ERG cells expressing the indicated shRNA. P-values are calculated by t test: *** < 0.0005.