The Role of JAK2 in ILEI-Induced Cancer Stem Cell Formation

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 DESCRIPTION (provided by applicant) Metastatic disease causes the mortality of most breast cancer patients, but the molecular mechanism of metastasis remains unclear. One hypothesis is that a specific subset of primary breast cancer cells known as cancer stem cells (CSCs) contributes to metastatic outgrowths (1). It has been shown that cells that have undergone a TGF-β-induced epithelial-to-mesenchymal transition (EMT) possess CSC properties (2). Because traditional chemotherapeutics cannot eradicate CSCs, clarifying the molecular mechanism of TGF-β-induced CSC formation could allow for targeted therapies against CSCs and provide a paradigm shift for cancer care. One of the ways TGF-β can induce EMT is the tumor suppressor hnRNP-E1 (3-5). Under normal conditions hnRNP-E1 binds and inhibits the translation of EMT-related mRNAs, but TGF-β treatment releases hnRNP-E1 from the mRNAs, allows for translation, and induces EMT. As stated previously, cells that have undergone a TGF-β-induced EMT possess CSC properties so we wanted to know the downstream effectors of TGF- β/hnRNP-E1 that confer CSC properties. We began our search by focusing on genes that were regulated by both TGF-β and hnRNP-E1, and chose the cytokine Interleukin-like EMT Inducer (ILEI). Preliminary results have shown that ILEI is necessary but not sufficient to activate STAT3 signaling and initiate CSC formation in vitro. Our previous work suggested that JAK2, a canonical activator of STAT3 signaling, was regulated by the same TGF-β/hnRNP-E1 mechanism as ILEI. These findings lead us to hypothesize that ILEI requires an additional TGF-β/hnRNP-E1-regulated gene such as JAK2 to phosphorylate STAT3 and initiate CSC formation. This hypothesis will be addressed through the following Specific Aims. Aim 1 will establish the role of TGF-β and hnRNP-E1 in JAK2 expression. Polysome profiling and RNA-Immunoprecipitations will confirm whether JAK2 is translationally regulated by hnRNP-E1. Aim 2 will determine the role of JAK2 in ILEI signaling. JAK2 will be modulated in several cell lines, and we will measure its effect on STAT3 activation as well as mammosphere formation. Aim 3 will validate the in vivo significance of ILEI signaling in CSC formation. We are interested in CSCs because of their putative role in metastatic outgrowths. Thus, in our final aim we will use a murine breast cancer metastasis model and apply the JAK inhibitor ruxolitinib to block ILEI signaling. These Aims will support the targeting of ILEI signaling as a novel therapy against metastatic breast cancer.