The PAC-1 dual specificity phosphatase predicts poor outcome in serous ovarian carcinoma
Introduction
Intracellular signal transduction pathways have a central role in conveying messages that originate from a variety of extracellular stimuli, including stress, growth factors, cytokines, and mitogens. These in turn lead to critical events in the evolution and progression of cancer, such as the synthesis of metastasis-associated molecules (e.g., proteolytic enzymes, angiogenic factors), refractoriness to apoptotic signals and immortality.
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 [1], [2], [3]. 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). The JNK and p38 subfamilies of kinases are activated by stress-related stimuli, including osmotic shock, inhibition of protein synthesis, and formation of oxygen radical species [4], while the ERK subfamily is largely activated by growth factor signals, as those mediated by receptor tyrosine kinases [3]. Tyrosine and threonine phosphorylation of MAPK leads to their activation, which 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. 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 [5]. JNK and p38 were thought to largely mediate apoptotic signals, with ERK promoting the opposing effect [6]. However, overlaps are now known to exist in these functions.
Signaling via MAPK has been shown to affect central pathways, such as those activated by transforming growth factor α (TNFα) [7] and epidermal growth factor receptor (EGFR) [8], [9] in cell line studies of ovarian cancer. Treatment of ovarian carcinoma cell lines in vitro with paclitaxel or cisplatin resulted in measurable changes in MAPK levels, with resulting effects towards proliferation or apoptosis [10], [11], [12], [13].
Since uncontrolled activation of MAPK may have deleterious effects on cellular function, these enzymes are tightly regulated. MAPK deactivation is achieved through the action of the dual specificity phosphatases (DUSP), a family of nine enzymes with considerable sequence homology, including the presence of a common active site sequence motif and two N-terminal CH2 domains [14], [15]. Despite their common role, DUSP are expressed at variable levels in benign tissues and differ in their ability to inhibit members of the three MAPK sub-families. PAC-1 is a 32-kDa nuclear DUSP, which is primarily expressed in hematopoietic cells. PAC-1 inhibits ERK and p38, but not JNK, and is induced by ERK [15]. MKP-1 (CL100), the human homologue of the first cloned DUSP, is a nuclear protein of 39.5 kDa with wide tissue distribution that is induced in response to a variety of stimuli, including osmotic shock, oxidative stress, growth factors, and UV radiation [14], [15]. MKP-1 is induced through MAPK activation and is in turn able to dephosphorylate threonine and tyrosine residues of p38, JNK, and ERK, thereby forming a regulatory loop [15]. MKP-4 is a 41.8-kDa DUSP localized mainly to the cytoplasm with moderate preferential specificity for ERK over p38 and JNK [15]. MKP-5 is a 52.6-kDa DUSP localized to both the nucleus and cytoplasm with specific inhibitory activity against p38 and JNK [15].
Several studies have evaluated MKP-1 expression in malignant epithelial cells. MKP-1 induction was seen in the SiHa cervical carcinoma line following hypoxia and JNK activation, with subsequent inactivation of JNK [16]. Inhibition of JNK-mediated apoptosis has been reported in the DU145 prostate cancer line [17], as well as in clinical specimens of this tumor [18]. Higher MKP-1 expression and MAPK phosphorylation were seen in gastric carcinoma specimens compared to corresponding benign tissues [19]. Reduced expression of MKP-1 in high-grade tumors and metastases of prostate, colon, and bladder carcinoma compared to primary tumors of lower grade and normal tissue was reported [20]. These differences were not found in breast carcinoma [20]. MKP-1 expression did not affect ERK expression or activity in the latter study [20]. Finally, MKP-1 expression was reduced in solid ovarian carcinoma compared to normal tissue and adenomas, but cancer cell expression of MKP-1 predicted worse outcome [21]. PAC-1, MKP-4, and MKP-5 expression has not been studied to date in carcinomas.
The present study evaluated the expression of MKP-1, MKP-4, MKP-5, and PAC-1 mRNA and its correlation with clinicopathologic parameters in 39 pleural and peritoneal malignant effusions from ovarian carcinoma patients. Only specimens with predominant cancer cell population (80–100% of cells) were studied. Specimens consisted of both primary diagnosis and post-chemotherapy samples, enabling correlation of treatment effect to DUSP expression. The comparison of peritoneal and pleural effusions enabled us to evaluate in vivo tumor progression. Finally, the possible relationship between DUSP expression and clinicopathologic parameters, including survival, was analyzed.
Section snippets
Effusion specimens
The material consisted of 39 fresh non-fixed peritoneal and pleural effusions submitted to the Section of Cytology, Department of Pathology, The Norwegian Radium Hospital, during the period of January 1998–September 2002. Specimens were obtained pre-operatively or intra-operatively (15 specimens) or at disease recurrence (24 specimens) from 32 patients diagnosed with serous ovarian carcinoma (35 specimens) and three patients diagnosed with primary peritoneal carcinoma (PPC) (four specimens).
MKP-1 and PAC-1 are the DUSP expressed by metastatic ovarian carcinoma cells in effusions
Our objective in the present study was to investigate which members of the DUSP family are expressed in ovarian carcinoma cells in effusions. For this purpose, we analyzed 39 effusions containing a high percentage of tumor cells. MKP-1 and PAC-1 mRNA was found in 36 and 37 effusions, respectively, with expression levels showing more than 50-fold variation in the studied effusions (Fig. 1). MKP-4 and MKP-5 were not expressed in any of the studied specimens (Fig. 2). These results suggest
PAC-1 is a novel predictor of poor survival in ovarian carcinoma
In view of our recent report, in which MAPK activation was shown to correlate with clinicopathologic parameters of improved outcome and longer survival, we wished to analyze the relationship between DUSP mRNA expression and these parameters. Neither MKP-1 nor PAC-1 levels showed association with FIGO stage, the extent of residual disease or tumor histological grade (data not shown). Follow-up period ranged from 1 to 81 months (mean = 34 months, median = 27 months). At the time of last
Discussion
The spreading of ovarian carcinoma cells to the peritoneal and pleural cavities constitute major events along tumor progression of this disease and are, together with solid metastases, by far a more frequent cause of patient mortality than the primary tumor. Despite this fact, molecular studies of ovarian cancer center most often on primary tumors. Consequently, the biological characteristics of ovarian carcinoma cells in effusions are considerably less characterized on both the phenotypic and
Acknowledgements
The work of Vered Givant-Horwitz is supported by the Yeshaya Horowitz fellowship grant.
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Affiliated with the David R. Bloom Center for Pharmacy at the Hebrew University.