Cancer metastasis is a dynamic process that begins with dissemination of cells from a primary tumor and culminates in metastases at one or more discontinuous secondary sites. The process of invasion has been thoroughly studied but the final steps in metastasis, metastatic colonization, remain largely unknown [5]. The PI identified a novel role in metastatic colonization for JNKK1, a dual-specificity kinase that activates the JNK and/or p38 MAPKs in response to extracellular stimuli. In the AT6.1 prostate cancer (CaP) model and the SKOV3ip.1 human ovarian cancer model, activated JNKK1 reduced the number of metastases by ~ 90% and significantly prolonged survival [1,2]. In vivo mechanistic studies showed that in AT6.1 cells JNKK1 signals through the JNK MAPK to suppress colonization, while in SKOv3ip.1 cells it signals through the p38 MAPK. These findings prompt important questions. What is the mechanism(s) responsible for JNKK1-mediated suppression of metastatic colonization? What functional gene expression changes are caused by activated JNKK1? Are these gene expression changes consistent or distinct when JNKK1 signals through JNK or p38 to inhibit colonization? Do cells become resistant to JNKK1's effects? What are the cellular consequences? I hypothesize that in vivo activation of JNKK1 by different MAP3Ks results in signaling through the p38 and JNK MAPKs in SKOV3ip.1 and AT6.1 cells, respectively. JNKK1 activation then causes altered transcription of a discrete subset of target genes, thereby impairing metastatic colonization. I further hypothesize that JNKK1 regulation of metastatic colonization involves reversible controls on cellular proliferation at the metastatic site.
Three Specific Aims will test these assertions using tools developed in my laboratory.
AIM I will test the hypothesis that distinct MAP3Ks activate JNKK1 in AT6.1 and SKOV3ip.1 cells and that JNKK1's metastasis suppressor function will be conserved in human CaP models. Studies will functionally test candidate upstream MAP3Ks as activators of JNKK1-mediated metastasis suppression in AT6.1 and SKOV3ip.1 cells using gene depletion approaches. The metastasis suppressor function of JNKK1 in human CaP cells will be evaluated by ectopic expression of JNKK1 in PC3 (metastatic) cells or its depletion in LNCaP (low metastatic) cells and by testing their metastatic ability in an orthotopic model.
AIM II will test the hypothesis that JNKK1 activation causes altered transcription of a discrete set of target genes when signaling is mediated by JNK or p38 MAPKs. Transcriptional profiling and bioinformatic analyses will identify a set of genes for validation and subsequent in vivo functional assays.
AIM III will test the novel hypothesis that activated JNKK1 induces a reversible cell cycle arrest in disseminated AT6.1 and SKOV3ip.1 cells. Studies will examine the mechanism by which JNKK1 suppresses proliferation and how cells bypass suppression and go onto form overt metastases. These studies will provide mechanistic data critical to development of approaches to control disseminated cell growth, and to confirm JNNK1 as a molecular target for the control of metastatic colonization.

Public Health Relevance

To devise methods to reduce mortality from metastatic disease, we must identify cellular pathways controlling the growth of cancer cells at metastatic sites. Studies in this proposal focus on mechanisms controlling such growth. This work will generate much needed information about the processes regulating metastasis formation and will identify new targets for development of antimetastatic therapies.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Tumor Cell Biology Study Section (TCB)
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Sathyamoorthy, Neeraja
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University of Chicago
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Krishnan, Venkatesh; Clark, Robert; Chekmareva, Marina et al. (2015) In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose. J Vis Exp :e52721
Clark, Robert; Krishnan, Venkatesh; Schoof, Michael et al. (2013) Milky spots promote ovarian cancer metastatic colonization of peritoneal adipose in experimental models. Am J Pathol 183:576-91
Bainer, Russell O; Veneris, Jennifer Taylor; Yamada, S Diane et al. (2012) Time-dependent transcriptional profiling links gene expression to mitogen-activated protein kinase kinase 4 (MKK4)-mediated suppression of omental metastatic colonization. Clin Exp Metastasis 29:397-408
Krishnan, Venkatesh; Stadick, Nathan; Clark, Robert et al. (2012) Using MKK4's metastasis suppressor function to identify and dissect cancer cell-microenvironment interactions during metastatic colonization. Cancer Metastasis Rev 31:605-13
Szmulewitz, Russell Z; Clark, Robert; Lotan, Tamara et al. (2012) MKK4 suppresses metastatic colonization by multiple highly metastatic prostate cancer cell lines through a transient impairment in cell cycle progression. Int J Cancer 130:509-20
Knopeke, Matthew T; Ritschdorff, Eric T; Clark, Robert et al. (2011) Building on the foundation of daring hypotheses: using the MKK4 metastasis suppressor to develop models of dormancy and metastatic colonization. FEBS Lett 585:3159-65
Shoushtari, Alexander N; Szmulewitz, Russell Z; Rinker-Schaeffer, Carrie W (2011) Metastasis-suppressor genes in clinical practice: lost in translation? Nat Rev Clin Oncol 8:333-42
Thobe, Megan N; Clark, Robert J; Bainer, Russell O et al. (2011) From prostate to bone: key players in prostate cancer bone metastasis. Cancers (Basel) 3:478-93
Thiolloy, Sophie; Rinker-Schaeffer, Carrie W (2011) Thinking outside the box: using metastasis suppressors as molecular tools. Semin Cancer Biol 21:89-98
Khan, Shaheena M; Funk, Holly M; Thiolloy, Sophie et al. (2010) In vitro metastatic colonization of human ovarian cancer cells to the omentum. Clin Exp Metastasis 27:185-96

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