Regular articleMitogen-activated protein kinases (MAPK) as predictors of clinical outcome in serous ovarian carcinoma in effusions☆
Introduction
The evolution and progression of cancer is a multistep process, during which malignant cells develop aberrations affecting major aspects of cellular function, such as adhesion, proliferation, differentiation, and apoptosis [1]. Many of the characteristics of malignant cells, including the synthesis of metastasis-associated molecules (e.g., proteolytic enzymes, angiogenic factors), refractoriness to apoptotic signals, and immortality, originate from a variety of extracellular signals, including stress, growth factors, cytokines, and mitogens, and are mediated via membrane receptors, such as tyrosine kinase receptors [2], [3]. These in turn relay their messages to the nucleus using a complex network of intracellular signaling pathways.
The mitogen-activated protein kinase (MAPK) intracellular signaling mode is a four-kinase component cassette, in which each kinase activates the following kinase substrate through a complex network, enabling the cell to maintain diversity and specificity while responding to various extracellular stimuli. The first kinase in the cascade, MAPK kinase kinase kinases (MAPKKKK), includes molecules such as PAK (p21-activated kinase) that are phosphorylated through interaction with small GTP-binding proteins (e.g., Ras, CDC42, and Rac-1) [2], [4]. The 14 MAPK kinase kinases (MAPKKK) in the second level of the cascade are a heterogenous group that includes Raf-1, A-Raf, and B-Raf, MAPK/ERK kinase kinases 1–4 (MEKK1–4), apoptosis-stimulating kinase-1 (ASK-1), and mixed lineage kinase-3 (MLK-3) [3], [4].
The third level of the cascade includes the MKKK-activated 7 MAPK kinases, MEK1,2 and MKK3–7. These are activated through double phosphorylation of serine and threonine residues, an interaction that owes its specificity largely to scaffolding of the former by other proteins and their subcellular localization [3], [4], [5]. Autocrine positive and negative loops and specific docking regions in proteins targeted for phosphorylation confer additional specificity [5]. The final level consists of 12 MAPK, including extracellular-regulated kinase (ERK1–5), c-jun amino-terminal kinase (JNK1–3), and the high-osmolarity glycerol response kinase in its different isoforms (p38 α,β,γ,δ). Tyrosine and threonine phosphorylation of MAPK occurs in a specific manner. Thus, MEK1,2 activate ERK, MKK5 activates ERK5, MKK4,7 activate JNK, and MKK3,6 activate p38 subfamily members [3], [4]. Activation of MAPK is followed by phosphorylation of a variety of cytosolic substrates, as well as their translocation to the nucleus, where they activate a large number of transcription factors, such as AP-1, p53, Elk-1, Ets-1, c-Myc, and STATs [3]. This results in a variety of biological effects, some of which are induced by several members belonging to the three groups of MAPK.
The JNK and p38 subfamily of kinases is activated by a large spectrum of stress-related stimuli. These include osmotic shock, inhibition of protein synthesis, and formation of oxygen radical species [6]. Signaling by p38, for example, affects gene expression, signaling via the adrenergic, arachidonate and nitric oxide pathways, apoptosis, and proliferation and differentiation, and is involved in the pathology of ischemic injury, infection, and wound healing [7]. The ERK subfamily of kinases is largely activated by growth factor signals, as those mediated by receptor tyrosine kinases [3]. The net result is growth, differentiation, and proliferation [3]. Thus, p38 and JNK are thought to largely mediate apoptotic signals, while ERK promotes the opposing effect [8]. However, overlaps are now known to exist in these functions. Pathological modulation of MAPK has been increasingly recognized as a critical component of stress-induced cellular responses and diseases [9], [10], [11].
The expression and role of several MAPK members in ovarian carcinoma have been previously investigated in vitro. Activation of MAPK members was detected following stimulation of cells with transforming growth factor α (TNFα)[12], as well as through epidermal growth factor receptor activation by EGF [13], [14] or endothelin-1 [15]. Activation of ERK signaling through c-Ha-Ras stimulation has been shown to be involved in promoter activation of the urokinase-type plasminogen activator (u-PA) [16], while JNK1 has been found to have a role in up-regulation of u-PA receptor [17]. Treatment of ovarian carcinoma cell lines in vitro with paclitaxel or cisplatin resulted in measurable changes in MAPK levels, with resulting effects toward proliferation or apoptosis [18], [19], [20], [21], [22]. Despite this expanding research effort in vitro, little or none is known about MAPK expression and activation in clinical specimens of ovarian carcinoma. The possible effect of MAPK on tumor progression or disease outcome in this malignancy is similarly unknown.
To initiate this study we took advantage of a series of phosphorylation state-specific antibodies ideally suited for studying complex patterns of phosphoregulation [23]. The present study evaluated MAPK expression in 64 serous effusions from ovarian carcinoma patients. Specimens consisted of both primary diagnosis and postchemotherapy samples, enabling correlation of treatment effect to kinase activity, as well as the specific association between kinase expression and activity and the use of platinum and paclitaxel in the therapeutic protocol. MAPK expression was studied for possible correlation with established proliferation and apoptosis markers. The comparison of peritoneal and pleural effusions facilitated the evaluation of in vivo tumor progression. Finally, full clinico-pathologic data for all patients enabled us to relate findings to established prognostic parameters and survival.
Section snippets
Effusion specimens
The material consisted of 64 fresh nonfixed peritoneal and pleural effusions submitted to the Division of Cytology, Department of Pathology, The Norwegian Radium Hospital, during the period January 1998–September 2000. Thirty-seven specimens were obtained preoperatively or intraoperatively, while an additional 27 were obtained at disease recurrence, from 59 patients diagnosed with serous ovarian carcinoma and 2 patients diagnosed with primary peritoneal carcinoma (PPC). Effusion specimens
MAPK level and activity
Pan- and p-ERK were detected in 63/64 (98%) and 59/64 (92%) specimens, respectively (Fig. 1). Values for pan-ERK ranged from 0 to 393% of the level detected in A375SM cells. p-ERK activity showed a range of 0–98%. pan- and p-JNK were detected in 62/64 (97%) and 44/64 (69%) specimens, respectively (Fig. 1). Values for pan-JNK ranged from 0 to 319% of the level detected in A375SM cells. p-JNK activity showed a similar range of 0–253%. pan-p38 was detected in 63/64 (98%) specimens, and its
Discussion
Epithelial ovarian carcinoma, especially of the serous type, shows unique predilection for the serosal surface of the peritoneal cavity and its lining abdominal organs. In the majority of cases, solid metastases are found concomitantly to the presence of malignant cells in a peritoneal effusion. The pleural space may be involved as well, either at diagnosis or, more commonly, at a later stage. The biological characteristics of ovarian carcinoma cells in effusions have been poorly characterized
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This study was supported by Grant D-02019 from the Norwegian Cancer Society.
- 1
Supported by the Yeshaya Horowitz fellowship grant.
- 2
Contributed equally to this study.
- 3
Affiliated with the David R. Bloom Center for Pharmacy at the Hebrew University.