c-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting p53/Notch1 and breast cancer gene 1 expression.

Breast cancer is a heterogeneous disease with several subtypes carrying unique prognoses. Patients with differentiated luminal tumors experience better outcomes, while effective treatments are unavailable for poorly differentiated tumors, including the basal-like subtype. Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments. C-Jun N-terminal kinase 2 (JNK2) is important in mammary tumorigenesis and tumor progression. Using a variety of mouse models, human breast cancer cell lines and tumor expression data, studies herein support that JNK2 inhibits cell differentiation in normal and cancer-derived mammary cells. JNK2 prevents precocious pubertal mammary development and inhibits Notch-dependent expansion of luminal cell populations. Likewise, JNK2 suppresses luminal populations in a p53-competent Polyoma Middle T-antigen tumor model where jnk2 knockout causes p53-dependent upregulation of Notch1 transcription. In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition. JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression. These data suggest that therapies inhibiting JNK2 in breast cancer may promote tumor differentiation, improve endocrine therapy response, and inhibit metastasis.

c-Jun N-terminal Kinase 2 Regulates Multiple Receptor Tyrosine Kinase Pathways in Mouse Mammary Tumor Growth and Metastasis.

c-Jun N-terminal kinase 2 (JNK2) isoforms are transcribed from the jnk2 gene and are highly homologous with jnk1 and jnk3 transcriptional products. JNK proteins mediate cell proliferation, stress response, and migration when activated by a variety of stimuli, including receptor tyrosine kinases (RTKs), but their ability to influence tumor metastasis is ill defined. To evaluate JNK2 in this manner, we used the highly metastatic 4T1.2 mammary tumor cells. Short hairpin RNA expression directed toward JNK2 (shJNK2) decreases tumor cell invasion. In vivo, shJNK2 expression slows tumor growth and inhibits lung metastasis. Subsequent analysis of tumors showed that shJNK2 tumors express lower GRB2-associated binding protein 2 (GAB2). In vitro, knockdown of JNK2 or GAB2 inhibits Akt activation by hepatocyte growth factor (HGF), insulin, and heregulin-1, while phosphorylation of ERK is constitutive and Src dependent. Knockdown of GAB2 phenocopies knockdown of JNK2 in vivo by reducing tumor growth and metastasis, supporting that JNK2 mediates tumor progression by regulating GAB2. The influence of jnk2 in the host or microenvironment was also evaluated using syngeneic jnk2-/- and jnk2+/+ mice. Jnk2-/- mice experience longer survival and less bone and lung metastasis compared to jnk2+/+ mice after intracardiac injection of 4T1.2 cells. GAB2 has previously been shown to mediate osteoclast differentiation, and osteoclasts are critical mediators of tumor-related osteolysis. Thus, studies focusing on the role of JNK2 on osteoclast differentiation were undertaken. ShJNK2 expression impairs osteoclast differentiation, independently of GAB2. Further, shJNK2 4T1.2 cells express less RANKL, a stimulant of osteoclast differentiation. Together, our data support that JNK2 conveys Src/phosphotidylinositol 3-kinase (PI3K) signals important for tumor growth and metastasis by enhancing GAB2 expression. In osteoclast progenitor cells, JNK2 promotes differentiation, which may contribute to the progression of bone metastasis. These studies identify JNK2 as a tumor and host target to inhibit breast cancer growth and metastasis.

c-Jun N-terminal kinase 2 (JNK2) enhances cell migration through epidermal growth factor substrate 8 (EPS8).

Membrane-bound receptors induce biochemical signals to remodel the actin cytoskeleton and mediate cell motility. In association with receptor tyrosine kinases, several downstream mitogen-induced kinases facilitate cell migration. Here, we show a role for c-Jun N-terminal kinase 2 (JNK2) in promoting mammary cancer cell migration through inhibition of epidermal growth factor substrate 8 (EPS8) expression, a key regulator of EGF receptor (R) signaling and trafficking. Using jnk2(-/-) mice, we found that EPS8 expression is higher in polyoma middle T antigen (PyVMT)jnk2(-/-) mammary tumors and jnk2(-/-) mammary glands compared with the respective jnk2(+/+) controls. The inverse relationship between the jnk2 and eps8 expression was also associated with cancer progression in that patients with basal-type breast tumors expressing high jnk2 and low eps8 experienced poor disease-free survival. In mammary tumor cell lines, the absence of jnk2 greatly reduces cell migration that is rescued by EPS8 knockdown. Subsequent studies show that JNK2 enhances formation of the EPS8-Abi-1-Sos-1 complex to augment EGFR activation of Akt and ERK, whereas the absence of JNK2 promotes ESP8/RN-Tre association to inhibit endocytotic trafficking of the EGFR. Together, these studies unveil a critical role for JNK2 and EPS8 in receptor tyrosine kinase signaling and trafficking to convey distinctly different effects on cell migration.