Breast cancer is a heterogeneous disease with vastly different tumor progression kinetics and survival outcomes depending upon the differentiation state and gene expression patterns of the tumor. Effective treatments exist for patients with endocrine therapy sensitive or HER2 overexpressing tumors, but targeted treatments are not available for other tumor types. The mechanisms governing mammary tumor phenotype generation could prove critical to finding treatments. The c-Jun N-terminal kinase (JNK) pathway has recently been implicated in the inhibition of breast tumor luminal differentiation (1, 2) and JNK2, in particular, is important in mammary tumorigenesis and tumor progression (3-8). Therefore, the involvement of JNK2 in inhibition of mammary luminal cell differentiation was investigated in normal glands and tumors. Studies found that JNK2 inhibits luminal cell populations in normal mammary ducts. Additionally, JNK2 suppresses Notch activity in stem cell niche of the developing mammary gland. In vitro assays show that control over differentiation by JNK2 is due to suppression of p53-dependent Notch1 expression. Inhibition of luminal cell populations by JNK2 is also apparent in tumor cell models regardless of p53 expression. In the p53-competent Polyoma Middle T-antigen model, Notch1 expression is suppressed by JNK2. In the absence of p53, JNK2 suppresses luminal populations independent of Notch1. In this model, decreased luminal marker expression is accompanied by increased epithelial to mesenchymal transition. It was also found that JNK2-dependent epithelial to mesenchymal transition inhibits luminal populations and is driven by JNK2-dependent suppression of Brca1. JNK2 also confers resistance to estrogen signaling inhibition, and increases the metastatic ability of tumor cells in vivo. These data establish the importance of JNK2 in mammary epithelial cell differentiation in normal glands and tumors. They also suggest that JNK2 may be an effective prognostic marker or treatment target. / text

Receptor tyrosine kinase (RTK) inhibitors are emerging as an effective therapeutic option for treatment of breast cancer patients overexpressing particular RTKs. However, more patients may benefit from an inhibitor targeting a common effector protein downstream several RTKs. The presented studies herein identify c-Jun N-Terminal Kinase 2 (JNK2), a kinase downstream multiple RTKs, as a novel target to effectively inhibit Phosphatidylinositol 3-kinase/AKT activation and metastasis. Knockdown of JNK2 in the highly metastatic 4T1.2 mammary cancer cells significantly decreased growth factor induced invasion in Boyden chambers, orthotopic tumor growth, and metastatic lesions in lungs and bone. Intra-cardiac introduction of cancer cells is utilized to specifically study the later steps in the metastatic cascade including travel of disseminated cancer cells to a secondary location. Thus, earlier steps such as the process of acquiring a malignant phenotype leading to escape from the primary tumor are bypassed. Survival was prolonged in mice receiving intra-cardiac injection of cells deficient of JNK2 either in the host or in the tumor cells, suggesting a potential role for JNK2 as a therapeutic target for advanced stage breast cancer patients. Using siRNA and inhibitors against Src and PI3K, we determined that JNK2 activity is dependent on Src and PI3K, positioning JNK2 downstream of two critical factors involved in tumor progression. Microarray analysis of JNK2 deficient tumors revealed that JNK2 positively regulates the adaptor protein Grb2 associated binding protein 2 (Gab2). Knockdown of Gab2 in 4T1.2 cells resulted in decreased tumor growth and a trend for decreased lung metastasis. In vitro, stimulation of 4T1.2 shJNK2 or 4T1.2 shGab2 cells with HGF, heregulin, or insulin resulted in impaired AKT activation, suggesting involvement of Gab2 and JNK2 in multiple RTK signaling pathways. Understanding of the intricate mechanisms involved in RTK signal transduction can contribute to drug design geared towards more effective targets, namely JNK2. The secondary goal of this research was to decipher the individual roles of JNK2 and JNK1 in metastatic breast cancer. Survival was significantly shortened in mice injected intra-cardiac with 4T1.2 shJNK1 cells. In congruence, serum Cathepsin K levels were increased and bone lesions observed were higher in mice injected with shJNK1 expressing tumor cells compared to mice injected with control cells. In sharp contrast, jnk1-/- mice displayed dramatically increased survival and fewer bone lesions upon intra-cardiac injections of 4T1.2 cells. Collectively, these data suggest cell and isoform specific roles for JNKs. / text