Role of Bach1/HMOX1 on the Metastatic Capability of B-Raf Active Melanoma

Metastatic melanoma has one of the worst prognoses of all malignancies with a median survival of only 6 months.  An estimated 70% of all melanomas harbor activating mutations in the B-Raf gene, the most common of which is a valine to glutamic acid substitution at amino acid 600 which renders the kinase constitutively active. Because of this, B-Raf inhibitors are an attractive targeted therapy.  While B-RAF^V600E-specific inhibitors initially showed clinical promise, most patients relapsed due to acquired resistance and adverse effects were observed including the development of squamous cell carcinoma in 20 to 30% of patients.  This highlights the importance of fully characterizing the role B-Raf plays in melanoma development, it’s cross talk with other signaling pathways and the molecular consequences of B-Raf inhibition. Oxidative stress is the accumulation of reactive oxygen species (ROS), which are synthesized as a byproduct of oxidative phosphorylation.  ROS are highly reactive and can cause cellular damage leading to diseases such as Alzheimer’s and cancer.  In response to oxidative stress, the transcription factor, Nrf2, is stabilized leading to the upregulation of a battery of cytoprotective genes. These proteins promote cell survival through a variety of mechanisms: removing harmful ROS, inhibiting apoptosis and promoting cell proliferation.  Nrf2 binds the antioxidant response element (ARE) promoting expression.  Bach1 is a repressor which competes for ARE binding. The primary target of Bach1 inhibition is HMOX1, which codes for Heme Oxygenase 1 (HMOX1), an enzyme that catalyzes the degradation of heme. While a vast body of literature supports the notion that these cytoprotective proteins are inhibitory of cancer, growing evidence has implicated many of these proteins in the initiation and progression of cancer.  HMOX1, for instance, has been implicated in promoting metastatic growth. The formation of spheres of melanoma cells, known as melanospheres, in non-adherent culture conditions is indicative of metastatic or tumor-initiating capabilities.  I have found that melanoma lines which harbor the B-Raf^V600E activating mutation (Sk-Mel-28, Sk-Mel-5, M14) form melanospheres while melanoma cell lines that have an activating mutation in N-Ras (HS936T, TXM13), an upstream kinase in the MAPK signaling cascade, do not form melanospheres.  By inhibiting B-Raf^V600E with the specific inhibitor, PLX-4032, I found that both frequency of melanosphere formation and melanosphere size decreased significantly in those cell lines that harbor the BRAF^V600E activating mutation.  By modulating the expression of HMOX1, B-Raf, and Bach1 through shRNA silencing and pharmacological inhibition, I have identified two potential roles for Bach1 in the formation of melanospheres in B-Raf active melanoma cell lines.  My findings can contribute to a more complete understanding of the roles Bach1 and HMOX1 play in the metastatic growth of melanoma.  Additionally, modulation of Bach1 may provide a novel therapeutic target.