Elsevier

Experimental Cell Research

Volume 319, Issue 12, 15 July 2013, Pages 1732-1743
Experimental Cell Research

Research Article
AKT upregulates B-Raf Ser445 phosphorylation and ERK1/2 activation in prostate cancer cells in response to androgen depletion

https://doi.org/10.1016/j.yexcr.2013.05.008Get rights and content

Highlights

  • Androgen depletion activates a novel AKT-ERK crosstalk via B-Raf in prostate cancer.

  • AKT activity is sufficient to upregulate B-Raf and its Ser445 phosphorylation.

  • Subsequent MEK/ERK activation requires additional signals independent of AKT.

  • AKT downregulates AR levels partly via B-Raf/MEK/ERK.

Abstract

Upregulated ERK1/2 activity is often correlated with AKT activation during prostate cancer (PCa) progression, yet their functional relation needs elucidation. Using androgen-deprived LNCaP cells, in which ERK1/2 activation occurs in strong correlation with AKT activation, we found that AKT-mediated B-Raf regulation is necessary for ERK1/2 activation. Specifically, in response to androgen deprivation, AKT upregulated B-Raf phosphorylation at Ser445 without affecting A-Raf or C-Raf-1. This effect of AKT was abolished by Arg25 to Ala mutation or truncating (∆4-129) the pleckstrin homology domain of AKT, indicating that the canonical AKT regulation is important for this signaling. Intriguingly, although a constitutively active AKT containing N-terminal myristoylation signal could sufficiently upregulate B-Raf phosphorylation at Ser445 in LNCaP cells, subsequent MEK/ERK activation still required hormone deprivation. In contrast, AKT activity was sufficient to induce not only B-Raf phosphorylation but also MEK/ERK activation in the hormone refractory LNCaP variant, C4-2. These data indicate that androgen depletion may induce MEK/ERK activation through a synergy between AKT-dependent and -independent mechanisms and that the latter may become deregulated in association with castration resistance. In support, consistent AKT-mediated B-Raf regulation was also detected in a panel of PCa lines derived from the cPten−/− L mice before and after castration. Our results also demonstrate that AKT regulates androgen receptor levels partly via the Raf/MEK/ERK pathway. This study reveals a novel crosstalk between ERK1/2 and AKT in PCa cells.

Introduction

Prostate cancer (PCa) is the most commonly diagnosed cancer among men and the second leading cause of male cancer death. PCa often reprogram their signal transduction pathways to develop higher malignancy and therapy resistance [1], [2], [3], [4], [5]. For example, increased activity of extracellular signal-regulated kinase (ERK) 1 and its homolog, ERK2, (collectively referred to as ERK1/2) is implicated in the progression and poor prognosis of PCa [6], [7], [8], [9], [10], [11]. Because activation mutations of Ras or Raf are relatively rare in human PCa [12], [13], [14], [15], [16], [17], [18], ERK1/2 activation should also be attributed to other mechanisms. Notably, ERK1/2 activity is upregulated in strong correlation with AKT (also known as protein kinase B) activation in PCa that arises in prostate-specific phosphatase and tensin homolog (Pten) knockout mice, especially during PCa conversion into a hormone-refractory state [19]. Moreover, in a large cohort study (n=535), ERK1/2 activation was correlated with deregulated activation of all components of the AKT pathway in 21% of human PCa cases and with deregulation of at least one component of the pathway in 42% of the cases, respectively [11]. These studies strongly implicate AKT in ERK1/2 activation in PCa, although the underlying mechanism is yet unclear.

The Raf/MEK/ERK and PTEN/phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathways have pivotal roles in cell survival, cell cycle progression, metabolism, and differentiation, and their deregulation is a central signature of many epithelial cancers [20], [21]. In response to Ras signals, the Ser/Thr kinase Raf (A-Raf, B-Raf or C-Raf-1) activates the dual-specificity kinases MEK1 and MEK2 (collectively referred to as MEK1/2) which, in turn, sequentially phosphorylate Tyr and Thr in the activation loop of the ubiquitously expressed Ser/Thr kinase, ERK1/2. ERK1/2, currently the only known substrate of MEK1/2, serves as the focal point of Raf/MEK/ERK signaling by regulating a wide variety of proteins [22]. The PI3K/AKT pathway can also mediate Ras signaling [23]. Upon activation, PI3K produces phosphatidylinositol-3,4,5-trisphosphate, which is then dephosphorylated by PTEN. Phosphatidylinositol-3,4,5-trisphosphate interacts with the N-terminal pleckstrin homology (PH) domain of the Ser/Thr kinase AKT to facilitate AKT recruitment to the plasma membrane, where AKT is activated through Thr308 and Ser473 phosphorylation in its activation segment [21].

It has been known that AKT can negatively regulate MEK/ERK signaling by directly inhibiting C-Raf via Ser259 phosphorylation, which promotes Raf interaction with the inhibitory scaffold 14-3-3ζ [24], [25]. Similarly, AKT can also negatively regulate B-Raf [26]. In contrast, recent studies have demonstrated that AKT can positively regulate MEK/ERK signaling by promoting Raf activation through the Rac/p21-activated kinase (PAK) pathway, wherein c-Raf Ser338 phosphorylation is a key regulatory mechanism [27], [28]. Therefore, AKT can differentially regulate Raf/MEK/ERK signaling depending upon biological contexts. Because Ser338 of c-Raf is conserved in B-Raf and A-Raf, these Raf proteins may also be subject to AKT-mediated positive regulation. Further, it has not been tested whether positive regulation of Raf by AKT is involved in the upregulation of ERK1/2 activity in PCa.

Previously, we and others reported that hormone depletion upregulates AKT and ERK1/2 activity in the androgen-dependent human PCa line, LNCaP, to mediate androgen receptor (AR) downregulation and subsequent neuroendocrine differentiation [29], [30], [31]. AR is a nuclear transcription factor pivotal to prostate carcinogenesis [4], [5], and its downregulation is implicated in neuroendocrine differentiation of PCa, a process associated with the development of castration resistance [2], [32]. Using this model, this study investigates a crosstalk between AKT and ERK1/2 pathways. Our results suggest that MEK/ERK activation is a downstream event of AKT activation, which is mediated by B-Raf and is partly required for AKT-mediated AR downregulation. Specifically, AKT could sufficiently upregulate Ser445 phosphorylation of B-Raf, but subsequent MEK/ERK activation required additional signals from androgen depletion. These findings were also validated using an androgen refractory variant of LNCaP, C4-2, and the PCa cell lines derived from the prostate specific cPten−/− L mice.

Section snippets

Cell culture and reagents

LNCaP (ATCC) and CWR22Rv1 (ATCC) were maintained in phenol red-deficient RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 100 U of penicillin and 100 μg of streptomycin per ml. LAPC4 (ATCC) was grown in Iscove's medium with 10% FBS. LNCaP C4-2 cells were maintained in phenol red-deficient RPMI 1640 supplemented with 10% charcoal/dextran-stripped FBS (c.s.FBS). The mouse PCa lines, E8, E2, E4, CE1 and CE2, derived from the cPten−/− L mouse [33], were previously

Correlative activation of AKT and ERK1/2 in LNCaP cells deprived of androgen

To determine whether AKT activation occurs in correlation with ERK1/2 activation in LNCaP cells deprived of androgen, we monitored these kinases over 12 days in LNCaP cells maintained in the RPMI medium containing c.s.FBS (c.s.FBS culture). During this period, AKT phosphorylation at Ser473, an indication of AKT activation [21], gradually increased and was accompanied by increasing ERK1/2 phosphorylation in its activation loop (Thr202/Tyr204 of ERK1; Thr183/Tyr185 of ERK2), indicating a strong

Discussion

Based upon the known correlation between AKT and ERK activation during PCa conversion into hormone-refractory state [11], [19] and recently reported AKT-mediated positive regulation of Raf [27], [28], we postulated a role for AKT in Raf/MEK/ERK regulation in PCa cells. Our results demonstrate that AKT can positively regulate the Raf/MEK/ERK pathway at the level of B-Raf in a subset of PCa cells, particularly in response to androgen depletion. Specifically, AKT catalytic activity was sufficient

Conflict of interest

The authors declare no conflict of interest for this article.

Acknowledgments

We thank Pradip Roy-Burman for cPten−/− L mouse PCa cell lines; Michael Robinson for AKT cDNA; Amy Hudson, Stephen Duncan, and Richard Mulligan for lentiviral vectors; Karen Knudson for C4-2. This work was supported by the National Cancer Institute (1R01CA138441), American Cancer Society (RSGM-10–189-01-TBE), FAMRI Young Investigator Award (062438) to J.P.

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