Omental macrophages secrete chemokine ligands that promote ovarian cancer colonization of the omentum via CCR1.

The omentum is the most common site of ovarian cancer metastasis. Immune cell clusters called milky spots are found throughout the omentum. It is however unknown if these immune cells contribute to ovarian cancer metastasis. Here we report that omental macrophages promote the migration and colonization of ovarian cancer cells to the omentum through the secretion of chemokine ligands that interact with chemokine receptor 1 (CCR1). We found that depletion of macrophages reduces ovarian cancer colonization of the omentum. RNA-sequencing of macrophages isolated from mouse omentum and mesenteric adipose tissue revealed a specific enrichment of chemokine ligand CCL6 in omental macrophages. CCL6 and the human homolog CCL23 were both necessary and sufficient to promote ovarian cancer migration by activating ERK1/2 and PI3K pathways. Importantly, inhibition of CCR1 reduced ovarian cancer colonization. These findings demonstrate a critical mechanism of omental macrophage induced colonization by ovarian cancer cells via CCR1 signaling.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

SPOP promotes tumorigenesis by acting as a key regulatory hub in kidney cancer.

Hypoxic stress and hypoxia-inducible factors (HIFs) play important roles in a wide range of tumors. We demonstrate that SPOP, which encodes an E3 ubiquitin ligase component, is a direct transcriptional target of HIFs in clear cell renal cell carcinoma (ccRCC). Furthermore, hypoxia results in cytoplasmic accumulation of SPOP, which is sufficient to induce tumorigenesis. This tumorigenic activity occurs through the ubiquitination and degradation of multiple regulators of cellular proliferation and apoptosis, including the tumor suppressor PTEN, ERK phosphatases, the proapoptotic molecule Daxx, and the Hedgehog pathway transcription factor Gli2. Knockdown of SPOP specifically kills ccRCC cells, indicating that it may be a promising therapeutic target. Collectively, our results indicate that SPOP serves as a regulatory hub to promote ccRCC tumorigenesis.

Milky spots promote ovarian cancer metastatic colonization of peritoneal adipose in experimental models.

The goal of controlling ovarian cancer metastasis formation has elicited considerable interest in identifying the tissue microenvironments involved in cancer cell colonization of the omentum. Omental adipose is a site of prodigious metastasis in both ovarian cancer models and clinical disease. This tissue is unusual for its milky spots, comprised of immune cells, stromal cells, and structural elements surrounding glomerulus-like capillary beds. The present study shows the novel finding that milky spots and adipocytes play distinct and complementary roles in omental metastatic colonization. In vivo assays showed that ID8, CaOV3, HeyA8, and SKOV3ip.1 cancer cells preferentially lodge and grow within omental and splenoportal fat, which contain milky spots, rather than in peritoneal fat depots. Similarly, medium conditioned by milky spot-containing adipose tissue caused 75% more cell migration than did medium conditioned by milky spot-deficient adipose. Studies with immunodeficient mice showed that the mouse genetic background does not alter omental milky spot number and size, nor does it affect ovarian cancer colonization. Finally, consistent with the role of lipids as an energy source for cancer cell growth, in vivo time-course studies revealed an inverse relationship between metastatic burden and omental adipocyte content. Our findings support a two-step model in which both milky spots and adipose have specific roles in colonization of the omentum by ovarian cancer cells.

Flexible peritoneal windows for quantitative fluorescence and bioluminescence preclinical imaging.

At present, there is considerable interest in the use of in vivo fluorescence and bioluminescence imaging to track the onset and progression of pathologic processes in preclinical models of human disease. Optical quantitation of such phenomena, however, is often problematic, frequently complicated by the overlying tissue's scattering and absorption of light, as well as the presence of endogenous cutaneous and subcutaneous fluorophores. To partially circumvent this information loss, we report here the development of flexible, surgically implanted, transparent windows that enhance quantitative in vivo fluorescence and bioluminescence imaging of optical reporters. These windows are metal and glass free and thus compatible with computed tomography, magnetic resonance imaging, positron emission tomography, and single-photon emission computed tomography; they also permit visualization of much larger areas with fewer impediments to animal locomotion and grooming than those previously described. To evaluate their utility in preclinical imaging, we surgically implanted these windows in the abdominal walls of female athymic nude mice and subsequently inoculated each animal with 1 × 10(4) to 1 × 10(6) bioluminescent human ovarian cancer cells (SKOV3ip.1-luc). Longitudinal imaging studies of fenestrated animals revealed up to 48-fold gains in imaging sensitivity relative to nonfenestrated animals, with relatively few complications, allowing wide-field in vivo visualization of nascent metastatic ovarian cancer colonization.

Using MKK4's metastasis suppressor function to identify and dissect cancer cell-microenvironment interactions during metastatic colonization.

Host tissue microenvironment plays key roles in cancer progression and colonization of secondary organs. One example is ovarian cancer, which colonizes the peritoneal cavity and especially the omentum. Our research indicates that the interaction of ovarian cancer cells with the omental microenvironment can activate a stress-kinase pathway involving the mitogen-activated protein kinase kinase 4 (MKK4). A combination of clinical correlative and functional data suggests that MKK4 activation suppresses growth of ovarian cancer cells lodged in omentum. These findings prompted us to turn our focus to the cellular composition of the omental microenvironment and its role in regulating cancer growth. In this review, in addition to providing an overview of MKK4 function, we highlight a use for metastasis suppressors as a molecular tool to study cancer cell interaction with its microenvironment. We review features of the omentum that makes it a favorable microenvironment for metastatic colonization. In conclusion, a broader, evolutionary biology perspective is presented which we believe needs to be considered when studying the evolution of cancer cells within a defined microenvironment. Taken together, this approach can direct new multi-dimensional lines of research aimed at a mechanistic understanding of host tissue microenvironment, which could be used to realize novel targets for future research.

Time-dependent transcriptional profiling links gene expression to mitogen-activated protein kinase kinase 4 (MKK4)-mediated suppression of omental metastatic colonization.

Although metastasis is the most lethal attribute of cancer, critical gaps in our knowledge of how cancer cells effectively colonize distant sites remain. For example, little is known about the cellular and molecular events that occur during the timecourse of metastatic colonization. To address this we are using the mitogen-activated protein kinase kinase 4 (MKK4) metastasis suppressor as a tool to identify these events. Specifically, we report a microarray expression-based strategy to identify genes whose transcription is altered in SKOV3ip.1 human ovarian cancer cells that express ectopic MKK4 throughout the course of in vivo metastatic colonization. The majority of genes identified fell into the categories of cytokinesis, cytoskeleton remodeling, and cell adhesion, and their expression was repressed in MKK4-expressing cells relative to vector controls. The greatest transcriptional divergence was concomitant with impaired proliferation at 14 days post injection (dpi). Specifically, 763 genes were differentially expressed (FDR < 0.05) between lesions that expressed ectopic MKK4 and paired controls. In contrast, only seven genes were differentially expressed at the experimental endpoint, when MKK4-expressing and control cells had formed macroscopic metastases. Application of our cohort of differentially expressed genes to three independent clinical datasets demonstrated a strong correlation between our findings and metastatic phenotypes in patient samples. Our results highlight the dynamic nature of metastatic colonization and reinforce the importance of examining both molecular and cellular phenotypes over time when studying metastasis formation.

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.

Building on the foundation of daring hypotheses: using the MKK4 metastasis suppressor to develop models of dormancy and metastatic colonization.

The identification of a novel metastasis suppressor function for the MAP Kinase Kinase 4 protein established a role for the stress-activated kinases in regulating the growth of disseminated cancer cells. In this review, we describe MKK4's biological mechanism of action and how this information is being used to guide the development of new models to study cancer cell dormancy and metastatic colonization. Specifically, we describe the novel application of microvolume structures, which can be modified to represent characteristics similar to those that cancer cells experience at metastatic sites. Although MKK4 is currently one of many known metastasis suppressors, this field of research started with a single daring hypothesis, which revolutionized our understanding of metastasis, and opened up new areas of exploration for basic research. The combination of our increasing knowledge of metastasis suppressors and such novel technologies provide hope for possible clinical interventions to prevent suffering from the burden of metastatic disease.

From prostate to bone: key players in prostate cancer bone metastasis.

Bone is the most common site for metastasis in human prostate cancer patients. Skeletal metastases are a significant cause of morbidity and mortality and overall greatly affect the quality of life of prostate cancer patients. Despite advances in our understanding of the biology of primary prostate tumors, our knowledge of how and why secondary tumors derived from prostate cancer cells preferentially localize bone remains limited. The physiochemical properties of bone, and signaling molecules including specific chemokines and their receptors, are distinct in nature and function, yet play intricate and significant roles in prostate cancer bone metastasis. Examining the impact of these facets of bone metastasis in vivo remains a significant challenge, as animal models that mimic the natural history and malignant progression clinical prostate cancer are rare. The goals of this article are to discuss (1) characteristics of bone that most likely render it a favorable environment for prostate tumor cell growth, (2) chemokine signaling that is critical in the recruitment and migration of prostate cancer cells to the bone, and (3) current animal models utilized in studying prostate cancer bone metastasis. Further research is necessary to elucidate the mechanisms underlying the extravasation of disseminated prostate cancer cells into the bone and to provide a better understanding of the basis of cancer cell survival within the bone microenvironment. The development of animal models that recapitulate more closely the human clinical scenario of prostate cancer will greatly benefit the generation of better therapies.

Metastasis-suppressor genes in clinical practice: lost in translation?

Over the past 25 years, an expanding set of metastasis-suppressor genes (MSGs) has been identified that specifically regulate metastasis formation without affecting primary growth. MSGs are involved in diverse molecular processes in multiple tumor types. Given the wealth of metastasis biology that underlies their functions, treatment strategies based on MSGs have an unparalleled potential to improve patient care. Using NM23 as a prime example, we discuss how specific MSGs have been used as prognostic markers, tools for predicting response to treatment, and targets for the development of novel therapies. Barriers specific to the translation of MSG biology into clinical practice are reviewed and future research directions necessary for clinical advances are delineated. Although to date the impact of MSGs on patient care is limited, it is an expanding field with vast potential to help develop new treatments and identify patients who will most benefit from them.

Thinking outside the box: using metastasis suppressors as molecular tools.

Metastasis, the process in which tumor cells move from a primary tumor through the circulation, lodge, and grow in distant locations, is a significant contributor to cancer patient morbidity and mortality, yet remains poorly understood. The molecular processes regulating tumorigenicity and metastasis are distinguishable, suggesting that it is possible to design therapeutic interventions to specifically control metastasis formation. Metastasis suppressors, which specifically regulate metastasis, are being used in "reverse genetics" approaches to discover the phenotypic alterations caused by modulating their levels and/or activity. This strategy is allowing the identification of tumor-host interactions that are crucial for efficient colonization and their disruption can be targeted to suppress metastases formation. In this review we discuss studies addressing invasion and migration, key functions for both early and late in the metastatic process. Metastasis suppressor functions, which modulate lodging and subsequent colonization of the secondary site, are also described. In sum this review focuses on metastasis suppressors that have yielded insight into mechanisms controlling metastasis formation. These serve as platform for out of the box thinking which will enable the discovery of new paradigms in metastasis research.

Disrupting ovarian cancer metastatic colonization: insights from metastasis suppressor studies.

Ovarian cancer affects approximately 25,000 women in the United States each year and remains one of the most lethal female malignancies. A standard approach to therapy is surgical cytoreduction, after which the remaining microscopic residual disease is treated with chemotherapy. The vast majority of patients have disease recurrence, underscoring the crucial need for approaches to control the regrowth, or colonization, of tissues after local treatment. Improved therapies require mechanistic information about the process of metastatic colonization, the final step in metastasis, in which cancer cells undergo progressive growth at secondary sites. Studies of metastasis suppressors are providing insights into events controlling metastatic colonization. This paper reviews our laboratory's approach to the identification, characterization, and functional testing of the JNKK1/MKK4 metastasis suppressor in ovarian cancer metastatic colonization. Specifically, we demonstrate that interaction of ovarian caner cells with the omental microenvironment activates JNKK1/MKK4 resulting in decreased proliferation without affecting apoptosis. The potential role of the omental microenvironment, specifically milky spot structures, is also described. It is our goal to provide this work as a usable paradigm that will enable others to study metastasis suppressors in clinical and experimental ovarian cancer metastases.

In vitro metastatic colonization of human ovarian cancer cells to the omentum.

Despite the potentially crucial contributions of the omentum in the regulation of ovarian cancer metastatic growth, it remains a poorly understood organ. Due to its anatomic location and structural fragility, the omentum presents inherent challenges to mechanism-based in vivo studies. Thus, the availability of an ex vivo omental model would, in part, address some of these difficulties posed. Here we describe a technique for identifying, isolating and maintaining ex vivo cultures of omenta from immune-compromised and -competent mice. Ex vivo culture conditions were developed that maintain tissue viability, architecture, and function for up to 10 days. Further experiments demonstrate that the ex vivo culture conditions allow for the proliferation of ovarian cancer cells in vitro and support a similar pattern of microscopic lesions after either intraperitoneal injection of ovarian cancer cells or co-culture of ovarian cancer cells with the omentum. In agreement with previous studies from our laboratory, histologic evaluation of these specimens found that ovarian cancer cells, as well as other peritoneal cancer cells, preferentially accumulate in, and colonize, omental areas rich in immune cells. We now recognize that these are specific, functional structures referred to as milky spots. In sum, these are foundational studies of a readily accessible model, which is easily manipulated and can be immediately used to study the dynamic process of omental colonization. It is hoped that investigators will use the data herein as a starting point for refinements and modifications which will enable them to tailor the model to the specific needs of the experimental question(s) they wish to pursue.

Increased MKK4 abundance with replicative senescence is linked to the joint reduction of multiple microRNAs.

MKK4 (mitogen-activated protein kinase kinase 4) is a pivotal upstream activator of c-Jun N-terminal kinase and p38. Here, we report that the abundance of MKK4 increases in senescent human diploid fibroblasts through enhanced translation. We identified four microRNAs (miR-15b, miR-24, miR-25, and miR-141) that target the MKK4 messenger RNA (mRNA); the abundance of these microRNAs decreased during replicative senescence. Individually modulating the amount of each microRNA did not modify MKK4 abundance, but their concomitant overexpression decreased and their joint reduction increased MKK4 abundance. Reporter analyses indicated that these microRNAs acted through the MKK4 5' and 3' untranslated regions. Elevated MKK4 abundance inhibited cell proliferation and increased the phosphorylation and activity of p38 and PRAK (p38-regulated/activated protein kinase). Thus, multiple microRNAs acting on a single target, the MKK4 mRNA, collectively influence MKK4 abundance during replicative senescence.

FYN is overexpressed in human prostate cancer.

OBJECTIVE: To test the hypothesis that FYN, a member of the SRC family of kinases (SFKs), is up-regulated in prostate cancer, as FYN is functionally distinct from other SFKs, and interacts with FAK and paxillin (PXN), regulators of cell morphology and motility. MATERIALS AND METHODS: Through data-mining in Oncomine (http://www.oncomine.org), cell-line profiling with immunoblotting, quantitative reverse transcription and polymerase chain reaction (RT-PCR) and immunohistochemical analysis, we described FYN expression in prostate cancer. The analysis included 32 cases of prostate cancer, nine of prostatic intraepithelial neoplasia (PIN) and 19 normal prostates. Samples were scored for the percentage of stained glands and intensity of staining (from 0 to 3). Each sample was assigned a composite score generated by multiplying percentage and intensity. RESULTS: Data-mining showed an eight times greater FYN expression in prostate cancer than in normal tissue; this was specific to FYN and not present for other SFKs. Expression of FYN in prostate cancer cell lines (LNCaP, 22Rv1, PC3, DuPro) was detected using quantitative RT-PCR and immunoblotting. Expression of FYN and its signalling partners FAK and PXN was detected in human tissue. Comparing normal with cancer samples, there was a 2.1-fold increase in median composite score for FYN (P < 0.001) 1.7-fold increase in FAK (P < 0.001), and a doubling in PXN (P < 0.05). There was a 1.7-fold increase in FYN (P < 0.05) and a 1.6-fold increase in FAK (P < 0.01) in cancer compared with PIN. CONCLUSIONS: These studies support the hypothesis that FYN and its related signalling partners are up-regulated in prostate cancer, and support further investigation into the role of the FYN as a therapeutic target.

Societal interactions in ovarian cancer metastasis: a quorum-sensing hypothesis.

The biochemical and biological mechanisms metastatic cancer cells use to function as communities and thwart internal and external growth control mechanisms remain undefined. In this work, we present the hypothesis that cancer cells may use a Quorum-Sensing mechanism to regulate multicellular functions and control steps in metastatic colonization. Quorum sensing is a bacterial cell-cell communication process used to track increasing cell-population density and, in response to changes in cell number, coordinate gene expression and behavior on a community-wide scale. Important parallels between the behavior of societies of bacterial cells and societies of malignant cancer cells exist in the bacterial literature. Of relevance to metastasis is the finding that pathogenic bacteria use quorum sensing to determine when their population numbers are high enough to collectively form biofilms in or on host organisms. Biofilms are complex, heterogeneous communities of bacterial cells encased within an extracellular matrix attached to a solid surface. Biofilms exacerbate disease and are refractory to a battery of therapies. We suggest that the quorum-sensing-controlled bacterial biofilm formation process closely parallels the steps in metastatic colonization. Cells migrate toward/on target surfaces (organ-specific homing), show cell-cell and cell-matrix interactions (tumor cell-stromal cell crosstalk), remain subclinical until they can mount an effective attack (dormancy), form complex structures with channels for nutrient flow (vascularized lesions), and contain resistant cells which can cause disease recurrence (persistors). Using ovarian cancer as an example, we present data supporting the connection between metastatic colonization and quorum sensing and discuss the implications for understanding and controlling metastasis formation.

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.

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/c-Jun NH2-terminal kinase kinase 1 protein expression is subject to translational regulation in prostate cancer cell lines.

Mitogen-activated protein kinase kinase 4/c-Jun NH(2)-terminal kinase kinase 1 (MKK4/JNKK1; hereafter referred to as MKK4) is a dual-specificity kinase with a critical role in regulating the activity of c-Jun NH(2)-terminal kinase and p38 kinases. We identified a novel biological function for MKK4 in the regulation of growth of ovarian and prostate cancer metastases. Clinical correlative studies showed that MKK4 protein levels were reduced in high-grade prostate cancer and prostate and ovarian cancer metastases compared with normal tissue, which prompted investigation into the mechanism(s) responsible for down-regulation of MKK4 in a panel of cancer cell lines. Initial studies found that low levels of MKK4 protein did not correlate with either exon deletion or decreased levels of MKK4 mRNA, suggesting that MKK4 protein levels were regulated posttranscriptionally by either reduced translation or reduced protein stability. Endogenous MKK4 was highly stable and not subject to altered proteolysis. Instead, MKK4 biosynthesis seemed to be regulated by altered translation. In support of this assertion, we found that cytosolic MKK4 mRNA was shifted toward active polysomes in cells with higher levels of MKK4 protein, suggesting that MKK4 mRNA was translated more efficiently in these cells. This study supports a novel mechanism for the regulation of MKK4 protein levels. Further, these findings have potential therapeutic implications for modulating the expression of a signaling kinase involved in the regulation of metastatic growth.