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.

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.

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.

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.

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.

An orthotopic model of human osteosarcoma growth and spontaneous pulmonary metastasis.

Osteosarcoma is the most common primary malignancy of bone and patients often develop pulmonary metastases. In order to investigate the pathogenesis of human osteosarcoma, there is a great need to develop a clinically relevant animal model. Here we report the development of an osteosarcoma animal model using three related human osteosarcoma lines, the parental TE-85 and two derivative lines MNNG/HOS and 143B. In vitro characterization demonstrated that the 143B line had the greatest cell migration and the least cell adhesion activities among the three lines. The 143B line also exhibited the greatest ability for anchorage independent growth. When GFP-tagged osteosarcoma cells were injected into the proximal tibia of athymic mice, we found that 143B cells were highly tumorigenic and metastatic, and MNNG/HOS cells were tumorigenic but significantly less metastatic. TE85 cells were neither tumorigenic nor metastatic. The number of pulmonary metastases was found 50-fold higher in 143B injected animals than that in MNNG/HOS injected mice. No pulmonary metastases were detected in TE85 injected animals for up to 8 weeks. Primary tumors formed by MNNG/HOS and 143B cells could be visualized by whole body fluorescence imaging, while the pulmonary metastases were visualized on the necropsied samples. The GFP tagged 143B cells (and to a lesser extent, MNNG/HOS cells) were readily recovered from lung metastases. This clinically relevant model of human osteosarcoma provides varying degrees of tumor growth at the primary site and metastatic potential. Thus, this orthotopic model should be a valuable tool to investigate factors that promote or inhibit osteosarcoma growth and/or metastasis.

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

In the past decade, findings from various disciplines of research have stimulated a reevaluation 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.

Adjunctive therapy for men with high risk localized and locally advanced prostate cancer: targeting disseminated tumor cells.

PURPOSE: Our research into the pathophysiology of micrometastatic dissemination and cancer recurrence has resulted in the initiation of a clinical trial for men with clinically localized and locally advanced disease. MATERIALS AND METHODS: We describe the development of this trial, which exploits anti-angiogenesis therapy, and delineate how our understanding of prostate cancer metastasis influenced its design. RESULTS: Prostate cancer is a heterogeneous disease. Although many men can be cured with local therapy, a large majority with clinically localized disease will experience a relapse usually at a distant site. This result is most likely due to micrometastatic dissemination early in the disease process. Therefore, successful contemporary treatment of many men with prostate cancer should include a combination of local and systemic therapies. Fortunately, cellular, molecular and genetic features that may predict which men are most in need of this therapeutic approach are being identified and characterized. This insight not only supports the rationale for a combination therapeutic approach to prostate cancer management, but will help identify the pathways and agents that provide the most promising targets for intervention. CONCLUSIONS: Despite advances in prevention and early detection, refinements in surgical technique, and improvements in radiation and systemic therapies, the ability to cure all men with prostate cancer remains unattainable. The continuing challenge is the successful eradication of recurrent and metastatic disease.