Jonathan A. Hickson

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.

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.

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.

Metastasis suppressor genes: from gene identification to protein function and regulation.

In the past decade, findings from various disciplines of research have stimulated a re-evaluation of fundamental concepts of the biology of metastasis. The convergence of two avenues of research has largely been responsible for this shift. First, clinical and experimental studies of specific steps of the metastatic cascade have shown that cancer cells often disseminate early in the natural history of disease and can persist at secondary sites for extended periods of time. These findings suggest that disseminated cells remain subject to growth regulation at distant sites as “dormant” single cells or microscopic metastases consisting of small numbers of cells. Second, complementary functional, biochemical, and signal transduction studies have identified a specific class of proteins that suppress the formation of overt metastases. These proteins are encoded by metastasis suppressor genes which are operationally defined as genes that suppress in vivo metastasis without inhibiting primary tumor growth when expressed ectopically in metastatic cell lines. While metastasis suppressor proteins may affect many steps in metastatic development, recent evidence specifically implicates several of these proteins in the regulation of growth of disseminated cells at secondary sites. This review describes the evolving understanding of rate-limiting steps of metastatic growth, and the role of metastasis suppressor proteins in the regulation of these processes. We will give an overview of the studies of metastasis suppressor protein function which have shifted our attention toward mechanisms of growth control at the secondary site (i.e. “metastatic colonization”). Emphasis is placed upon the complimentary research in the fields of metastasis and signal transduction that has identified signaling pathways controlling metastatic colonization. We also discuss the regulation of metastasis suppressor proteins and the potential biological and biochemical mechanisms responsible for their organ-type specificity. Finally, the implication of these emerging concepts on the development of therapeutic strategies will be presented.

MKK4 and metastasis suppression: A marriage of signal transduction and metastasis research

MAP kinase kinase 4 (MKK4) is a member of the stress-activated protein kinase (SAPK) signaling cascade and is involved in the regulation of many cellular processes. We have recently demonstrated a functional role for MKK4 in the suppression of metastases. In this review, we discuss the established cellular and biochemical functions of MKK4, as well as a new function for MKK4 as a metastasis suppressor gene. Because of the importance of signaling studies to this translational work, a detailed example of the strategy and tools that can be employed to define the biochemical mechanism of MKK4-mediated metastasis suppression is presented. Finally, the potential therapeutic utility of these findings is discussed.

Using metastasis suppressor proteins to dissect interactions among cancer cells and their microenvironment

Cancer metastasis is a complex, dynamic process that begins with dissemination of cells from the primary tumor and culminates in the formation of clinically detectable, overt metastases at one or more discontinuous secondary sites. Evidence from in vivo video microscopy as well as PCR and immunohistochemical studies suggest that cancer cell dissemination is an early event in tumor progression and that cells may persist in a potentially dormant state for a prolonged period. Similarly, the mechanisms by which these disseminated cells initiate growth and complete the process of metastatic colonization remain largely unknown. Understanding signal transduction pathways regulating this final step of metastasis is therefore critical for successful clinical management. While genetic mutations or epigenetic changes may be required for a cell or group of cells to separate and survive distant from the primary tumor, the microenvironment within secondary tissues plays a substantial role in influencing whether disseminated cells survive and proliferate. Our work is focused on using metastasis suppressor proteins to gain insight into why the majority of disseminated cells, which should be fully malignant, do not proliferate immediately at secondary sites. The translational goal of this work is to identify targets for inhibiting metastatic growth and prolonging disease-free survival.

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.

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.

Stopping cancer before it colonizes.

Controlling the growth of cancer cells at metastatic sites is one goal of cancer drug development. Studies of metastasis suppressor function bring this long-sought goal closer (pages 933–938).

Inhibition of prostate cancer metastatic colonization by ∼4.2 Mb of human chromosome 12

Our previous studies demonstrate that introduction of a ∼70 cM region (now estimated at 63.75 Mb by the Human Genome Project) of human chromosome 12 into the highly metastatic Dunning rat prostate cancer cell line AT6.1 results in >30‐fold (≥90%) reduction in the number of overt metastases in spontaneous metastasis assays. We report the further localization and biological characterization of the metastasis‐suppressor activity encoded by a reduced region of chromosome 12. To localize this metastasis‐suppressor activity, a panel of AT6.1 microcell hybrids that retain varying portions of human chromosome 12 was constructed and subjected to sequence‐tagged site (STS)‐based PCR analysis and assessment of in vivo metastatic ability. Data from these complementary approaches localized the metastasis‐suppressor activity to a ∼4.2 Mb portion of human chromosome 12q24.3 comprised of 3 separate regions. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) and immunoblotting were used for differential expression analyses to identify which characterized genes, predicted gene sequences and expressed sequence tags (ESTs) within this region could be responsible for the observed metastasis suppression. Comprehensive in vivo studies showed that suppressed AT6.1‐12 hybrids that retain the metastasis‐suppressor region on 12q24.3 are capable of arriving at the secondary site, but are not able to persist there. Thus, unlike other metastasis‐suppressor genes characterized to date, the metastasis‐suppressor gene encoded by this region appears to utilize a different biologic mechanism to suppress the growth of overt metastases at the secondary site.