Mitogen Activated Protein kinase signal transduction pathways in the prostate

The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy.


Background
Signal transduction via mitogen activated protein (MAP) kinases plays a key role in a variety of cellular responses, including proliferation, differentiation, and cell death. MAP kinases have provided a focal point for remarkably rapid advances in our understanding of the control of cellular events by growth factors and stresses. Since their initial discovery in yeast, over a dozen MAP kinase families have been identified of these highly genetically conserved proteins. MAP kinase signal transduction pathways have not been studied in great detail in the prostate; however over one hundred publications describing the effects of various manipulations, including growth factors, chemical modifiers and androgens on prostatic cells have been described in the literature. Despite these studies, the struc-ture and function of the MAP kinase pathways in prostate are far from clearly understood.
Diseases of the prostate are a tremendous source of morbidity and mortality in aging males. Benign enlargement of the prostate gland is a significant source of discomfort and prostate cancer is the second leading cause of cancer related deaths in males. Most of the prostate cancer deaths result from emergence of an androgen resistant phenotype of prostate cancer. Unfortunately, treatment options for these androgen resistant prostate cancer patients are few and generally ineffective. These facts underline the need to develop new therapies that will improve outlook for hormone-independent prostate cancer. Several lines of evidence suggest a role for MAP kinase signal transduction pathways in prostate cancer. Here we provide a comprehensive review of studies specifically using prostate tissue or cell lines. Admittedly, many more publications may have examined some aspect of MAPKs, but we focused on abstracts including MAPK, ERK, JNK, or p38.
The three major MAP kinase (MAPK) pathways include the extracellular-signal regulated kinase (ERK, also known as p42/44 MAP kinase), c-jun N-terminal kinase (JNK, also known as stress activated protein kinase-1 (SAPK1)) and p38 MAPK (also known as SAPK2/RK). In general, ERK1 and ERK2 are key transducers of proliferation signals and are often activated by mitogens. In contrast, SAPKs/JNKs and p38 are poorly activated by mitogens but strongly activated by cellular stress inducers. After activation, these cytosolic proteins translocate to the nucleus to activate numerous proteins and/or transcription factors.
Each MAPK cascade consists of a core MAPK module, which has no less than three enzymes activated in series: 1) a MAPK, 2) an immediate upstream kinase (Known as Mitogen Activated Protein Kinase Kinase or MAPKK), and 3) an additional kinase upstream of the MAPKK (Known as Mitogen Activated Protein Kinase Kinase Kinase or MAPKKK). These regulatory cascades not only convey information to the target effectors, but also coordinate incoming information from parallel signaling pathways. These mechanisms allow for signal amplification and generate a threshold subject to multiple activation cascades. Then there are elements upstream of the core module. The interactions between MAP kinase and its immediate upstream kinase (MAPKK) are highly specific: for instance, p42/p44 MAP kinases are phosphorylated solely by MAP/ERK kinase (MEK) 1 and 2; p38 MAP kinase is selectively activated by MAP kinase kinases (MKK) 3 and 6, while JNK is activated by MKK7 and MKK4 in most conditions, however MKK4 can sometimes activate p38 MAP kinase when over expressed. The specificity is less clearly defined for elements upstream of the MAPKK modular level. For instance MAPKKK are highly promiscuous and can interact with and activate a number of down stream components. Similarly, signaling cross talk in the transmission levels between the mitogen/stress activator and the core MAPK module understandably adds more complexity to subtle differences in response despite equivalent activation. The specificity upstream of the core module may be regulated by additional components like scaffold proteins that help bring the specific components of the MAPK machinery together or keep various components from interacting with each other. A simplistic view of the MAP kinase signal transduction is presented in Figure 1.

p42/p44 MAP kinase and the prostate Expression and activation of p42/p44 MAP kinase in tissue
In normal noncancerous tissue from radical prostatectomy specimens, immunohistochemistry localizes ERK to the cytoplasm of most cells of the prostate including the epithelial, basal, and stromal cells [1,2]. Despite the abundance of ERK it does not appear to be active in the epithelial layer of normal prostatic tissue, but up to 80% of cells in the stroma and basal layers will stain positively for phosphorylated ERK (p-ERK) within the nucleus [3]. Gioeli et al also described p-ERK staining in normal prostate tissue adjacent to areas of prostate cancer and found that ERK activation was directly related to poor histologic/ prognostic features [4].
Nearly all studies involving this pathway have been examined using prostate cancer cell lines. There are 40 prostate cell lines available in the ATCC catalog of both normal and cancerous tissue. The most commonly used cell lines are the androgen sensitive LNCaP cells, isolated from a cancerous supraclavicular lymph node, and the androgen insensitive cell lines DU145 and PC3, derived from brain and bone metastasis respectively. Of note, DU145 cell lines have basal ERK activation from paracrine/autocrine factors that is not demonstrated in other cell lines. Karyotypes have been described for these lines and more recently for a variety of the other cells lines available [5]. Studies of kinase activation in normal prostate tissue cell lines are remarkably absent from the literature.
The most well studied ligands/mitogens in prostatic cells are epidermal-derived growth factor (EGF), transforming growth factor (TGF)-α, and insulin-like growth factor (IGF). The mechanism of action of these proteins is well described in many reviews [6][7][8]. Generally, the ligands interact with a membrane receptor and transmit a signal to a cytosolic tyrosine kinase. EGF and TGF-α share about 35% sequence homology and bind to the same receptor, the epidermal growth factor receptor (EGFR). The ERK module appears to be a primary signal relay as inhibition of this activation prevents cellular proliferation induced by EGF as well as numerous other mitogens, which operate by transactivating the EGF receptor [9]. There are numerous effectors of the ERK pathway in prostate cancer cells and these are described in Table 1 and 2. These stresses and agents are of considerable interest in that they are potential manipulators of this cascade.
Androgenic manipulation has been a mainstay of prostate cancer treatment for over 60 years, but the clinically challenging cancers will grow aggressively in the absence of androgens. Of considerable interest in prostate physiology is the relationship between androgens, the androgen receptor, and the MAP kinase cascade. The effect of the potent androgen dihydrotestosterone (DHT) is still unclear as it activated ERK in one study but not in numerous others with LNCaP cells [9][10][11]. Regardless of the effect on ERK, the contribution of MAPK to cellular proliferation due to DHT appears to be small relative to other pathways. With the apparent poor evidence suggesting direct androgen stimulation of ERK, the focus has been redirected on Interleukin (IL) -6 and the communication links between this important cytokine and the androgen receptor.
The interaction of IL-6 with the MAPK pathways is of particular interest for this is suspected to be a major autocrine factor in the progression of hormone refractory prostate cancer. There is an excellent review of the intracellular activities initiated by IL-6 [12]. IL-6 appears to be able to transactivate the androgen receptor in the absence of androgens at the N-terminal domain as well as increase the mRNA for the androgen receptor. Activation of androgen receptor by IL-6 involves the ERK pathway among others [13,14]. ERK also appears to be involved in the phosphorylation of steroid receptor co-activator (SRC) -1, which binds to the androgen receptor [15]. LNCaP cells are also sensitive to IL-6 as an interesting study demonstrated that IL-6 exposed tumors injected into nude mice demonstrated an abrogation of proteins involved in cell cycle control. Inhibition with PD98059 was able to retard the cancer growth of these IL-6 exposed cells [16]. Our preliminary studies demonstrate that IL-6 expression by PC3, a line of hormone refractory prostate cancer cells is in part regulated by ERK signal transduction pathway aforementioned means can affect prostate cancer cell growth and invasion in PC3 and DU145 cells [17]. Tyrphostin AG825 and ZM252868 are two promising molecules that act in this fashion [18,19]. G protein coupled receptors appeared to be very important in communicating with the EGF receptor in prostate physiology [20]. G proteins can transactivate the EGFR through a variety of means, which include cytosolic protein activation and metallomatrix protein pro-ligand cleavage. Numerous physiologic molecules normally activate this pathway including lysophosphatidic acid, bombesin, adenosine triphosphate (ATP), and 5-HETE [21-26]. A number of other metabolites appear to operate specifically via the metallomatrix protein pathway and these pathways can be inhibited with methyl selenium molecules [27].

Protein signaling
Numerous cytosolic proteins are involved in the intracellular events leading to ERK activation. Among these important molecules are ras, PTEN, ID-1, DOC-2/DAB2, Protein kinase C (PKC) epsilon and some of the integrin subtypes [28-32]. One of the intracellular proteins that transmit signals from the EGF receptor to the modular MAPK pathway is ras. Isoprenylation allows ras to approach the membrane, a requirement for interaction with the EGFR. This chemical reaction is facilitated by HMG-COA reductase and the statin-family of drugs and phenyl acetate can inhibit this process [33,34].
The first part of the modular MAPK cascade resulting in ERK activation is the Raf molecule, which includes three isoforms, Raf-1, RafA, and RafB. These molecules have not been extensively studied with regards to prostate cancer. One interesting study used the LNCaP cell line transfected with a constitutively active form of Raf-1, which increased ERK activity and decreased plating and cloning efficiency [35]. This observation is contrary to other studies by suggesting that ERK activation may have tumor suppressor effects or perhaps chronic high level activation may have different responses. Raf kinase inhibitor protein (RKIP) is a protein that inhibits activation of MEK/ERK and appears to be a metastasis suppressor gene. Fu et al investigated clinical samples of local and metastatic prostate tissue and demonstrated immunohistochemical presence of RKIP was inversely related to histological grade. Additionally, no RKIP antigen was found in metastatic deposits. These investigators also demonstrated no effect of this protein on tumor growth, but in vitro and in vivo evidence of decreased metastasis [36]. These results support results from our lab that show inhibition of p42/p44 MAP kinase affects in vitro clonogenic potential in PC3 cells. Taken together these results suggest that inhibition of this pathway may be effective in preventing metastatic deposits, but not gross tumor growth. There is a growing body of evidence that identify ERK as an enzyme responsible for increased invasive and metastatic potential in prostate cancer. Our studies in PC3 cells demonstrate that ERK signal transduction pathway plays only a minor role in growth and proliferation of these cells, but is essential for clonogenic activity, cell migration and invasion [Koul S et al in preparation]. Our studies demonstrate that ERK signal transduction does not appear to play a role in cell growth, but is essential for new colony formation. This raises interesting aspects in modulation for oncologic control. Clearly, invasion and metastasis are the elements of tumors that make them malignant and the cause of patient suffering. However, ERK inhibition may not affect already developed metastatic sites. This might lend to early use of inhibitors or modulators of this pathway while disease burden is still low and/or possibly as prevention of tumor metastasis.

Results of ERK activation
With such a large body of evidence supporting a role for ERK MAP kinase signal transduction pathway in promoting tumorigenesis in prostate cancer, we recognize that p42 / p44 MAP kinase signal transduction pathway may serve as a novel target for the treatment of prostate cancer.

C-Jun N-terminal kinase (JNK) and the prostate Expression and activation of JNK in tissue
The presence of active JNK in normal tissues from prostatectomy specimens is somewhat controversial, even in the same research groups [1,2]. Most studies show that JNK expression or activation is increased in neoplastic cells [3]. Additionally, JNK activity appears to be inversely related to MKP-1 expression. An interesting sidebar to the use of transgenic adenocarcinoma of mouse prostate (TRAMP) mice by Uzgare et al showed that JNK expression can increase in poorly differentiated tumors without an apparent increase in activation. JNK is generally activated by cellular stress, however, numerous molecules are able to phosphorylate the enzyme [  Regarding gross cellular function in the cell, authors have shown that activation of JNK regulates the cytoskeleton; prevention of nucleosome formation and mitochondrial dysfunction appear to also be major events following JNK activation [59,66,69]. JNK appears to be overwhelmingly involved in apoptotic pathways shown by multiple studies using a variety of molecules, protein and hormones to activate JNK. JNK activation has been shown to be a crucial step in the apoptosis induced by nonsteroidal antiinflammatory drugs in human colon cancer cells [74]. Hypoxia appears to be another condition in which JNK is phosphorylated. This was shown in male rats that were castrated and subsequently had the environment of the ventral prostate gland examined [75].

Results of JNK activation
JNK activation is not a necessity for apoptosis as demonstrated in multiple studies using inhibitors or dominant negative cell lines. Despite overwhelming studies suggesting JNK activation and apoptosis, several well-performed studies suggest that JNK inactivation is beneficial. Different roles for JNK1 and JNK2 are of interest and poorly understood. As mentioned previously, JNK2 inactivation prevented the up regulation of genes involved in DNA repair, mRNA turnover and drug resistance [72]. Other studies using anti-sense forms of JNK have revealed some interesting findings. One study has suggested that JNK is more active in growth and proliferation [76]. These authors exposed human prostate cancer lines to anti-sense JNK1 and JNK2 and found that anti-sense JNK1 inhibited growth and anti-sense JNK2 inhibited proliferation. This study suggests that JNK is a potential target for prostate cancer growth. Another study reviewed the methods for sensitizing prostate cancer cells to cisplatin, by expression of p53 and anti-sense JNK [77].

p38 MAP kinase MAP kinase (p38) and the prostate
In non-cancerous human prostate tissue p38 MAP kinase protein is present in the basal cells and epithelial cells of the prostate gland, but one study has shown it to be absent in the epithelial layer [2]. While likely present it is not normally activated in epithelial tissue samples that have been studied, but has stronger activity in the prostatic stroma similar to ERK. However, epithelial p38 MAP kinase can become active in situations of neoplasia and benign hypertrophy of the prostate gland [3]. One study using TRAMP mice suggested that the strong epithelial p38 MAP kinase activation present in intraepithelial neoplasia and well-differentiated tumors but might be lost in poorly differentiated tumors [78]. Similar to JNK, p38 MAP kinase is a kinase primarily activated by external stresses.

Conclusions and future directions
MAPK signal transduction pathways seem to play diverse role in prostate physiology. Significant differences have been observed in the activation pattern of all three major MAPK families (ERK, JNK and p38 MAPK) in prostate epithelial and stromal cells and under normal and pathophysiological conditions. Modulation of these MAP   1]. Identification of the signaling cascades that are selectively activated in normal prostate and in hormone responsive and hormone refractory prostate cancer cells is critical in identification of selective targets and development of new and rational therapies for treatment of prostate cancer.