Genes regulated by JNK signaling in MDA231-LM2 breast cancer cells

In advanced malignancies, cancer cells have acquired capabilities to resist a variety of stress-inducing insults. We show that c-Jun N-terminal kinase (JNK) stress signaling is highly active in cancer cells from patients with late stage breast cancer and promotes tumor growth and metastasis in mouse models. Transcriptomic analysis revealed that JNK activity induces genes associated with extracellular matrix (ECM), wound healing and mammary stem cells. The ECM proteins and niche components osteopontin (SPP1) and tenascin C (TNC) are induced by JNK signaling and promote metastatic colonization of the lungs. Notably, treatment with chemotherapeutic drugs induces JNK activity in breast cancer cells, reinforcing the production of SPP1 and TNC. Inhibition of JNK or reduction of SPP1 or TNC expression sensitizes primary tumors and metastases in mice to chemotherapy. We used Affymetrix microarrays to analyze the transcriptomic output modulated by JNK activity in a lung metastatic derivative of the MDA-MB-231 breast cancer cell line, MDA231-LM2. Modulation of JNK activity in metastatic breast cancer cells MDA231-LM2 was achieved either by overexpression of constitutively active JNK or by exposing the cells to a selective JNK inhibitor. Constitutively active JNK was overexpressed in MDA231-LM2 cells via lentiviral transduction using a pLVX-Puro vector, into which we subcloned the Flag-MKK7B2Jnk1a1 (MKK7-JNK) or the mutated Flag-MKK7B2Jnk1a1(APF) (MKK7-JNK(mut)) fusion genes (Addgene plasmids #19726 and #19730, provided by Roger Davis). Cells transduced with an empty pLVX-Puro vector (Control) or overexpressing MKK7-JNK(mut) were used as controls. Inhibition of JNK activity in MDA231-LM2 breast cancer cells was attained by treating the cells with the second generation ATP-competitive anthrapyrazolone JNK inhibitor CC-401 (JNKi). MDA231-LM2 cells exposed to DMSO (vehicle) were used as controls. For each condition, three biological replicates were analyzed.