Mitogen-activated protein kinase kinase 4 (MKK4) acts as a metastasis suppressor gene in human ovarian carcinoma

Despite improvements in chemotherapy and the recognition that aggressive surgical cytoreduction is beneficial, the majority of patients diagnosed with ovarian cancer will die as a result of metastatic disease. The molecular changes associated with acquisition of metastatic ability in ovarian cancer are poorly understood. We hypothesize that metastasis suppressor gene inactivation or down-regulation plays a role in ovarian cancer progression. Mitogen-activated protein kinase kinase 4 (MKK4), a member of the stress-activated protein kinase signaling cascade, has been identified recently as a metastasis-suppressor gene. An immunohistochemical approach was taken to test the possibility that MKK4 dysregulation occurs during the development of clinical ovarian cancer metastases. MKK4 expression was evaluated in normal and metastatic ovarian tissues. Normal ovarian epithelial cells showed high intensity staining for MKK4, whereas metastatic tissues showed a statistically significant decrease in expression. These results support a role for MKK4 dysregulation in the development of clinical disease. A functional approach was taken to test the ability of MKK4 to suppress metastatic colonization, the process whereby disseminated cancer cells lodge and grow at a secondary site in vivo. The SKOV3ip.1 human ovarian cancer cell line was chosen for these studies because it lacks endogenous MKK4 expression but retains both upstream and downstream components of the signaling cascade of MKK4. Ectopic expression of MKK4 in these cells, when injected into female SCID mice, suppressed the number of overt metastatic implants by nearly 90%. Furthermore, MKK4 expression increased the life span of the animals by 70%. Taken together, these data support a role for MKK4 in the suppression of metastatic colonization in ovarian cancer.     

E-cadherin and DAP kinase in pancreatic adenocarcinoma and corresponding lymph node metastases

E-cadherin and DAP kinase have been implicated as 'invasion suppressor' genes in human cancer. The aim of this study was to analyze the methylation status of E-cadherin and DAP kinase and the expression of the protein in the metastatic lesions and to compare it with the expression in the primary tumor. Methylation-specific PCR of the DAP kinase and E-cadherin promoter was performed in 28 primary adenocarcinomas of the pancreas and in 13 corresponding regional lymph node metastases. The presence of E-cadherin and DAP kinase protein was assessed by immunohistochemistry. Metastatic lymph nodes showed a significant different expression profile from the primary tumor. E-cadherin methylation was observed in 8/28 (29%) and loss of protein expression was observed in 16/28 (57%) of pancreatic carcinomas. E-cadherin methylation was observed in 7/13 (54%) and loss of protein expression was observed in 11/13 (85%) lymph node metastases (p=0.047). DAP kinase methylation occurred in 11/28 (39%) pancreatic carcinomas and loss of protein expression was observed in 13/28 (46%). DAP kinase was methylated in 6/13 (46%) lymph node meta-stases and loss of protein expression was observed in 10/13 (77%) (p=0.039). Comparing primary tumor and corresponding lymph node metastases in 13 cases, the status of E-cadherin methylation was discordant in 2 cases. The protein expression pattern of E-cadherin and DAP kinase was discordant in 4 and 3 cases respectively. Unmethylated tumor samples did not express E-cadherin in 12 and DAP kinase protein in 6 cases. Our results demonstrate that reduction of E-cadherin and DAP kinase expression is more frequent in lymph node metastases than in the primary tumor and methylation of the promoter region contributes to this reduction; however, an alternative mechanism of inactivation seems to exist.     

Suppression of metastatic colonization by the context-dependent activation of the c-Jun NH2-terminal kinase kinases JNKK1/MKK4 and MKK7

Advances in clinical, translational, and basic studies of metastasis have identified molecular changes associated with specific facets of the metastatic process. Studies of metastasis suppressor gene function are providing a critical mechanistic link between signaling cascades and biological outcomes. We have previously identified c-Jun NH2-terminal kinase (JNK) kinase 1/mitogen-activated protein kinase (MAPK) kinase 4 (JNKK1/MKK4) as a prostate cancer metastasis suppressor gene. The JNKK1/MKK4 protein is a dual-specificity kinase that has been shown to phosphorylate and activate the JNK and p38 MAPKs in response to a variety of extracellular stimuli. In this current study, we show that the kinase activity of JNKK1/MKK4 is required for suppression of overt metastases and is sufficient to prolong animal survival in the AT6.1 model of spontaneous metastasis. Ectopic expression of the JNK-specific kinase MKK7 suppresses the formation of overt metastases, whereas the p38-specific kinase MKK6 has no effect. In vivo studies show that both JNKK1/MKK4 and MKK7 suppress the formation of overt metastases by inhibiting the ability of disseminated cells to colonize the lung (secondary site). Finally, we show that JNKK1/MKK4 and MKK7 from disseminated tumor cells are active in the lung but not in the primary tumor, providing a biochemical explanation for why their expression specifically suppressed metastasis while exerting no effect on the primary tumor. Taken together, these studies contribute to a mechanistic understanding of the context-dependent function of metastasis regulatory proteins.     

Mitogen-activated protein kinase kinase 4/stress-activated protein/Erk kinase 1 (MKK4/SEK1), a prostate cancer metastasis suppressor gene encoded by human chromosome 17.

The introduction of a discontinuous approximately 70-cM portion of human chromosome 17 significantly suppresses the metastatic ability of AT6.1 rat prostate cancer cells without affecting tumorigenicity (M. A. Chekmareva et al., Prostate, 33: 271-280, 1997). We have recently demonstrated that AT6.1 cells containing the approximately 70-cM region (AT6.1-17-4 cells) escape from the primary tumor and arrest in the lung but are growth-inhibited unless the metastasis suppressor region is lost (M. A. Chekmareva et al., Cancer Res., 58: 4963-4969, 1998). A series of in vivo studies indicated that the observed growth inhibition was due to the effect of a gene(s) at the metastatic site (M. A. Chekmareva et al., Cancer Res., 58: 4963-4969, 1998). We have now identified the mitogen-activated protein kinase kinase 4/stress-activated protein/Erk kinase 1 (MKK4/SEK1) gene as a candidate metastasis suppressor gene encoded by the approximately 70-cM region. AT6.1 cells were transfected with a MKK4/SEK1 expression construct, and the cells were tested in standard spontaneous metastasis assays. Whereas the metastatic ability of the AT6.1-MKK4/SEK1 cells was significantly reduced as compared with that of transfection controls, the growth rate of the primary tumors was not affected; the average tumor volume at day 29 after injection was approximately 2 cm. Furthermore, histological examination of the lungs of AT6.1-MKK4/SEK1 tumor-bearing animals revealed that the suppression by MKK4/SEK1 is due to an effect at the metastatic site, consistent with the phenotype conferred by the original approximately 70-cM chromosomal region. These studies implicate MKK4/SEK1 as a metastasis suppressor gene encoded by human chromosome 17.     

Role of mitogen-activated protein kinase kinase 4 in cancer

Mitogen-activated protein (MAP) kinase kinase 4 (MKK4) is a component of stress activated MAP kinase signaling modules. It directly phosphorylates and activates the c-Jun N-terminal kinase (JNK) and p38 families of MAP kinases in response to environmental stress, pro-inflammatory cytokines and developmental cues. MKK4 is ubiquitously expressed and the targeted deletion of the Mkk4 gene in mice results in early embryonic lethality. Further studies in mice have indicated a role for MKK4 in liver formation, the immune system and cardiac hypertrophy. In humans, it is reported that loss of function mutations in the MKK4 gene are found in approximately 5% of tumors from a variety of tissues, suggesting it may have a tumor suppression function. Furthermore, MKK4 has been identified as a suppressor of metastasis of prostate and ovarian cancers. However, the role of MKK4 in cancer development appears complex as other studies support a pro-oncogenic role for MKK4 and JNK. Here we review the biochemical and functional properties of MKK4 and discuss the likely mechanisms by which it may regulate the steps leading to the formation of cancers.     

c-Jun NH2-terminal kinase activating kinase 1/mitogen-activated protein kinase kinase 4-mediated inhibition of SKOV3ip.1 ovarian cancer metastasis involves growth arrest and p21 up-regulation

In many patients without clinical metastases, cancer cells have already escaped from the primary tumor and entered a distant organ. A long-standing question in metastasis research is why some disseminated cancer cells fail to complete steps of metastatic colonization for extended periods of time. Our laboratory identified c-Jun NH(2)-terminal kinase activating kinase 1/mitogen-activated protein kinase kinase 4 (JNKK1/MKK4) as a metastasis suppressor protein in a mouse xenograft model of experimental i.p. ovarian cancer metastasis. In this model, expression of JNKK1/MKK4 via activation of p38 delays formation of >or=1-mm implants and prolongs animal survival. Here, we elucidate the time course of this delay as well as the biological mechanisms underpinning it. Using the Gompertz function to model the net accumulation of experimental omental metastases, we show that MKK4-expressing implants arise, on average, 30 days later than controls. Quantitative real-time PCR shows that MKK4 expression does not have a substantial effect on the number of cancer cells initially adhering to the omentum, and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling analysis shows that there is no increase in apoptosis in these cells. Instead, immunohistochemical quantitation of cell cycle proteins reveals that MKK4-expressing cells fail to proliferate once they reach the omentum and up-regulate p21, a cell cycle inhibitor. Consistent with the time course data, in vitro kinase assays and in vivo passaging of cell lines derived from macroscopic metastases show that the eventual outgrowth of MKK4-expressing cells is not due to a discrete selection event. Rather, the population of MKK4-expressing cells eventually uniformly adapts to the consequences of up-regulated MKK4 signaling.     

Mitogen-activated protein kinase kinase 4 metastasis suppressor gene expression is inversely related to histological pattern in advancing human prostatic cancers

We have shown recently (B. A. Yoshida et al., Cancer Res., 59: 5483-5487) that mitogen-activated protein kinase kinase 4 (MKK4) can suppress AT6.1 rat prostate cancer metastases in vivo. Evaluation of the expression of components of the MKK4 signaling cascade showed a loss or down-regulation of expression of MKK4 or c-Jun, a downstream mediator of MKK4, in six of eight human prostate cancer cell lines. Given these findings, we next assessed whether MKK4 dysregulation occurs during the development of clinical prostate cancer. Immunohistochemical studies showed high levels of MKK4 expression in the epithelial but not the stromal compartment of normal prostatic tissues. In neoplastic tissues, a statistically significant, direct, inverse relationship between Gleason pattern and MKK4 was established. These results demonstrate that MKK4 protein is consistently down-regulated during prostate cancer progression and support a role for dysregulation of its signaling cascade in clinical disease. To test the possibility that down-regulation of MKK4 protein is the result of allelic loss, metastatic prostate cancer lesions were examined for loss of heterozygosity (LOH) within the MKK4 locus (D17S969). These studies showed a 31% (5 of 16) LOH of MKK4 that is not associated with coding region mutations, which suggests that the nucleotide sequence of the gene in the remaining allele is infrequently mutated.     

A case of multiple liver metastases from nonfunctioning neuroendocrine pancreatic tumor curatively resected after successful intra-arterial chemotherapy

We report a case of 33-year-old woman for nonfunctioning neuroendocrine pancreatic tumor with synchronous multiple liver metastases. For the primary pancreatic legion, we performed pylorus-preserving pancreatoduodenectomy at first. Three weeks after the surgery, the patient started to undergo intra-arterial chemotherapy with a weekly administration of high-dose 5-FU (1,000 mg/body) for residual liver metastases. Any severe adverse events were not observed. After 20 courses, a partial response was achieved and she underwent curative operation with partial resection for 4 metastatic legions and RFA for others. Histopathological findings of resected specimens revealed no viable neoplastic tissues. She is alive with no sign of recurrence 3 months after the surgery. Although a surgical resection is accepted as the standard therapy for liver metastases of nonfunctioning neuroendocrine pancreatic tumor, intra-arterial chemotherapy might be an alternative therapeutic option for unresectable case.     

Clonal origin of lymph node metastases in bladder carcinoma

BACKGROUND: Evidence of genetic heterogeneity within urothelial carcinomas of the bladder has raised questions about the clonal origin of urothelial carcinoma and its metastases. High-grade urothelial carcinoma of the bladder frequently metastasizes to multiple regional lymph nodes in the pelvis. Whether or not these multiple lymph node metastases originate from the same tumor clone is uncertain. Molecular analysis of microsatellite alterations and X-chromosome inactivation status of distinct tumor cell populations from the same patient may further our understanding of the genetic basis of carcinoma progression and metastasis. METHODS: The authors examined 24 patients who underwent radical cystectomy for urothelial carcinoma. All patients had multiple (from two to four) lymph node metastases. Genomic DNA samples were prepared from formalin fixed, paraffin embedded tissue sections using laser-assisted microdissection. Loss of heterozygosity (LOH) assays for 3 microsatellite polymorphic markers on chromosome 9p21 (D9S171, region of putative tumor suppressor gene p16), 9q32 (D9S177, putative tumor suppressor gene involved in urothelial carcinoma tumorigenesis), and 17p13 (TP53, the p53 locus) were performed. In addition, X-chromosome inactivation analysis was performed in primary tumors and metastases from 10 female patients. RESULTS: In total, 79 tumors were analyzed. The overall frequency of allelic loss was 67% (16 of 24 tumors) in the primary urothelial carcinomas and 79% (19 of 24 tumors) in the metastatic carcinomas. The primary urothelial carcinoma showed LOH at the D9S171, D9S177, and TP53 loci in 39% (9 of 23 tumors), 30% (6 of 20 tumors), and 30% (7 of 23 tumors) of informative samples, respectively. LOH in > or = 1 lymph node metastases was seen at the D9S171, D9S177, and TP53 loci in 35% (8 of 23 tumors), 45% (9 of 20 tumors), and 48% (11 of 23 tumors) of informative samples, respectively. Eleven tumors demonstrated identical allelic loss patterns at all DNA loci both in the primary carcinoma and in all corresponding lymph node metastases. Three tumors showed allelic loss in the metastatic carcinoma but not in its matched primary carcinoma. Six tumors demonstrated a different LOH pattern in each of its lymph node metastases. Clonality analysis showed the same pattern of nonrandom X-chromosome inactivation both in the primary urothelial carcinoma and in all of the lymph node metastases in five of nine informative tumors studied. Four tumors showed a random pattern of X-chromosome inactivation in both the primary carcinoma and in the metastases. CONCLUSIONS: LOH and X-chromosome inactivation assays showed that multiple lymph node metastases and matched primary urothelial carcinomas of the bladder had the same clonal origin, suggesting that the capability for metastasis often arises in only a single clonal population in the primary tumor. The variable LOH patterns observed in some of the tumors likely reflect genetic divergence during the clonal evolution of urothelial carcinoma.     

The p38 kinases MKK4 and MKK6 suppress metastatic colonization in human ovarian carcinoma

Despite considerable efforts to improve early detection of ovarian cancer, the majority of women at time of diagnosis will have metastatic disease. Understanding and targeting the molecular underpinnings of metastasis continues to be the principal challenge in the clinical management of ovarian cancer. Whereas the multistep process of metastasis development has been well established in both clinical and experimental models, the molecular factors and signaling pathways involved in successful colonization of a secondary site by disseminated cancer cells are not well defined. We have previously identified mitogen-activated protein kinase (MAPK) kinase 4/c-Jun NH2-terminal kinase (JNK)-activating kinase (MKK4/JNKK1/SEK1, hereafter referred to as MKK4) as a metastasis suppressor protein in ovarian carcinoma. In this study, we elucidate key mechanisms of MKK4-mediated metastasis suppression. Through the use of a kinase-inactive mutant, we show that MKK4 kinase activity is essential for metastasis suppression and prolongation of animal survival. Because MKK4 can activate either of two MAPKs, p38 or JNK, we expressed MKK6 or MKK7, specific activators of these MAPKs, respectively, to delineate which MAPK signaling module was involved in MKK4-mediated metastasis suppression. We observed that MKK6 expression suppressed metastatic colonization whereas MKK7 had no effect. Our finding that MKK4 and MKK6 both suppress metastasis points to the p38 pathway as an important regulatory pathway for metastatic colonization in ovarian cancer.     

Relationship of 4F2 antigen with local growth and metastatic potential of squamous cell carcinoma of the larynx

The 4F2 antigen is a cell-membrane glycoprotein which arises early in the G0-G1 phases of the cell cycle. This molecule is present in all established human cell lines and most malignant human cells. The authors used an indirect immunophosphatase method to study 50 squamous cell carcinomas of the larynx and ten lymph-node metastases, corresponding to six primary tumors, for 4F2 expression. The tumors showed several patterns of 4F2 staining which were correlated with different behaviors and prognoses of the neoplasms. Three different patterns (no staining, peripheral staining, and diffuse 4F2 expression) are described as are their relationships with metastatic behavior of the carcinomas. Tumors with metastases were found only in the third group (P = 0.0001). These results led to the following conclusions: (1) the 4F2 antigen is present in squamous cell carcinomas; (2) its distribution reflects the tumor-spreading pattern; and (3) it correlates with differentiation and metastatic behavior.     

The SMAD4 protein and prognosis of pancreatic ductal adenocarcinoma.

PURPOSE: SMAD4 (also called Dpc4) is a tumor suppressor in the TGF-beta signaling pathway that is genetically inactivated in approximately 55% of all pancreatic adenocarcinomas. We investigated whether prognosis after surgical resection for invasive pancreatic adenocarcinoma is influenced by SMAD4 status. EXPERIMENTAL DESIGN: Using immunohistochemistry, we characterized the SMAD4 protein status of 249 pancreatic adenocarcinomas resected from patients who underwent pancreaticoduodenectomy (Whipple resection) at The Johns Hopkins Hospital, Baltimore, MD, between 1990 and 1997. The SMAD4 gene status of 56 of 249 (22%) pancreatic carcinomas was also determined. A multivariate Cox proportional hazards model assessed the relative risk of mortality associated with SMAD4 status, adjusting for known prognostic variables. RESULTS: Patients with pancreatic adenocarcinomas with SMAD4 protein expression had significantly longer survival (unadjusted median survival was 19.2 months as compared with 14.7 months in patients with pancreatic cancers lacking SMAD4 protein expression; P = 0.03). This SMAD4 survival benefit persisted after adjustment for prognostic factors including tumor size, margins, lymph node status, pathological stage, blood loss, and use of adjuvant chemoradiotherapy. The relative hazard of mortality for cancers lacking SMAD4 after adjusting for other prognostic factors was 1.36 (95% confidence interval, 1.01-1.83; P = 0.04). CONCLUSION: Patients undergoing Whipple resection for pancreatic adenocarcinoma survive longer if their cancers express SMAD4.     

MKK4 suppresses metastatic colonization by multiple highly metastatic prostate cancer cell lines through a transient impairment in cell cycle progression

Metastatic dissemination in prostate cancer is often early, but not all cancer cells form clinical metastases. Map kinase kinase 4 (MKK4) suppresses metastasis in a preclinical prostate cancer model. We hypothesize that MKK4 will specifically inhibit metastatic colonization through impaired proliferation. Three highly metastatic rat prostate cancer cell lines (AT6.1, Mat-Lu and AT3.1) were employed. Stably over-expressing HA-MKK4 or vector control lines were injected into immunocompromised mice. These experiments validated that HA-MKK4 specifically affects metastatic colonization and increases survival. Median survival (days) with HA-MKK4 vs. vector was 42 vs. 28 (p < 0.0001) for AT6.1, 25 vs. 19 (p < 0.0001) for Mat-Lu and 27 vs. 20 (p < 0.0001) for AT3.1. HA-MKK4 suppresses colonization within 14 days post dissemination, after which exponential proliferation resumes. Although overt metastases retain HA-MKK4, it is inactive within these lesions. Nonetheless, metastasis-derived cell lines were shown to retain functional HA-MKK4 and like their parental HA-MKK4 line are suppressed for experimental metastasis formation in vivo. Disseminated AT6.1-HA-MKK4 cells were analyzed and were found to have an alteration in cell cycle. Specifically, there was an accumulation of cells in G1-phase (p = 0.024) and decrease in S-phase (p = 0.037) compared with vector. In multiple prostate cancer lines, HA-MKK4 suppresses an early step in metastatic colonization. These data support a model in which MKK4 activation at the metastatic site causes a cell-cycle arrest, which is eventually overcome despite presence of functional HA-MKK4. Further studies will specifically interrogate the regulation of MKK4 activation within the metastatic microenvironment and the down-stream molecular events critical for metastasis suppression.     

Successful treatment with cetuximab combination chemotherapy in a case of FOLFOX-refractory rectal cancer with previously unresectable multiple liver metastases leading to complete resection

Most colorectal cancer patients with liver metastases are not resectable upon initial diagnosis. Recently, chemotherapy improves overall survival of initially unresectable patients by allowing tumor downstaging and complete resection. We report a FOLFOX-refractory rectal cancer patient with unresectable multiple liver metastases, whose tumors could be downstaged and completely resected after initiation of FOLFIRI with cetuximab. Case: A 41-year-old male demonstrated rectal cancer with unresectable multiple liver metastases. He was treated by FOLFOX4 therapy as first-line chemotherapy. After initiating 14 courses, he was treated by FOLFIRI with cetuximab because of disease progression. After initiation of chemotherapy, radiographic examination demonstrated remarkable reduction of primary rectal tumor and metastatic liver tumors. He underwent complete rectal tumor resection after 13 courses of chemotherapy, and metastatic liver tumor resection after 18 courses of chemotherapy.     

Neoadjuvant treatment of Dermatofibrosarcoma Protuberans of pancreas with Imatinib: case report and systematic review of literature

Dermatofibrosarcoma Protuberans (DFSP) is a rare skin tumor, characterized by frequent local recurrence but is seldom metastatic. It is histologically characterized by storiform arrangement of spindle cells. Cytogenetically, most tumors are characterized by translocation 17:22 leading to overexpression of tyrosine kinase PDGFB which can be targeted with tyrosine kinase inhibitor, Imatinib. We describe the first case of unresectable pancreatic metastases from DFSP treated with neoadjuvant Imatinib and subsequently R0 metastectomy. Additionally, a comprehensive systematic review of DFSP pancreatic metastases and the current published data on the use of Imatinib in DFSP is summarized.     

Loss of expression of Dpc4 in pancreatic intraepithelial neoplasia: evidence that DPC4 inactivation occurs late in neoplastic progression

Infiltrating adenocarcinomas of the pancreas are believed to arise from histologically identifiable intraductal precursors [pancreatic intraepithelial neoplasias (PanINs)] that undergo a series of architectural, cytological, and genetic changes. The role of DPC4 tumor suppressor gene inactivation in this progression has not been defined. Immunohistochemistry for the Dpc4 protein in formalin-fixed, paraffin-embedded tissue is a sensitive and specific marker for DPC4 gene status, providing a tool to examine DPC4 status in these putative precursor lesions. A total of 188 PanINs were identified in 40 pancreata, 38 (95%) of which also contained an infiltrating adenocarcinoma. Sections containing these 188 duct lesions were labeled with a monoclonal antibody to Dpc4. All 82 flat (PanIN-1A), all 54 papillary (PanIN-1B), and all 23 atypical papillary (PanIN-2) intraductal lesions expressed Dpc4. In contrast, 9 of 29 (31%) severely atypical lesions (PanIN-3 lesions, carcinomas in situ) did not. The difference in Dpc4 expression between histologically low-grade (PanIN-1 and -2) and histologically high-grade (PanIN-3) duct lesions was statistically significant (P < 0.0001). In three cases, the pattern of Dpc4 expression in the PanIN-3 lesions did not match the pattern of expression in the associated infiltrating carcinomas, indicating that these high-grade lesions did not simply represent infiltrating carcinoma growing along benign ducts. Loss of Dpc4 expression occurs biologically late in the neoplastic progression that leads to the development of infiltrating pancreatic cancer, at the stage of histologically recognizable carcinoma.     

New paradigms for the function of JNKK1/MKK4 in controlling growth of disseminated cancer cells

Much work has been done in the 20 years since the discovery of the first metastasis suppressor gene to investigate the diverse biochemical functions of the proteins these genes encode. The function of metastasis suppressors cannot be solely predicted from correlative clinical data or in vitro studies. Instead, careful design of in vivo experiments to test broader hypotheses is necessary to pinpoint the mechanism of action of these novel proteins. Our laboratory identified c-Jun NH2-terminal kinase activating kinase 1 (JNKK1)/Mitogen-activated protein kinase (MAPK) kinase 4 (JNKK1/MKK4) as a metastasis suppressor in prostate and ovarian cancer. JNKK1/MKK4 is a stress activated protein kinase (SAPK) involved in a variety of signaling events, ranging from the regulation of hepatoblast survival during mammalian development to metastasis suppression in adult ovarian and prostate cancers. JNKK1/MKK4 function has typically been associated with the c-Jun NH2-terminal kinase (JNK) signaling pathway, particularly in the immune system where JNK plays a role in inflammatory signaling and apoptosis. However, evidence continues to accumulate that JNKK1/MKK4 is also a physiologic activator of p38 under certain conditions, and that activation of p38 arrests cell cycle progression. This review will provide a historical perspective on the role of JNKK1/MKK4 in SAPK signaling, including some recent findings from our own laboratory that shed light on the complicated role for JNKK1/MKK4 in metastatic colonization.