Heme oxygenase 1 in BRAFV600 melanoma: Evidence for a role in chemoresistance, immuno-escape and angiogenesis.

Heme oxygenase 1 (HO-1) is widely recognized as a protective enzyme that counteracts cell damage through the antioxidant, antiapoptotic and anti-inflammatory activity of its metabolic products. By increasing cell resistance to stressors, HO-1 induction has been related with the gain of resistance and progression of different types of cancers and its involvement in favoring immune-escape and angiogenesis has been hypothesized. In this work, HO-1 expression in primary melanoma cells treated with the BRAF-mutated inhibitor Vemurafenib/PLX4032 has been investigated as a factor limiting cell death and recognition by Natural killer cells, that are the first line of defence of natural immune system against melanoma growth and spreading. The initial part of the work was carried out in collaboration with Professor Pietra’s research group at the Clinical Immunology Laboratory, Ospedale Policlinico San Martino in Genoa. Different cell lines, isolated in house from melanoma patients, were exposed to 1–10μM PLX4032. In three cell lines (MeOV-1, MeTA and MeMI), PLX4032 reduced melanoma cell viability up to 45%, while HO-1 expression was upregulated. HO-1 silencing/inhibition further significantly reduced melanoma cells viability following PLX4032 treatment. Furthermore, in PLX4032-treated melanoma cells, the expression of B7H6 and ULBP3, ligands of NKp30 and NKG2D activating receptors on NK cells, was downregulated and completely restored by HO-1 silencing/inhibition. This is the first report implicating HO-1 in limiting PLX4032 efficacy and melanoma recognition by NK cells, and these data contributed to a paper published in The International Journal of Cancer at the beginning of 2020. Further, the molecular mechanisms underlying HO-1 induction in melanoma cells exposed to PLX4032 have been studied. After treatment with 10μM PLX4032, MeOV-1 cells showed an early activation of the redox sensitive transcription factor Nrf2, and a long-lasting HO-1 induction. Notably, the upregulation of HO-1 was Nrf2-dependent, as it was prevented by Nrf2 silencing. Moreover, among the Nrf2-dependent genes, only HO-1 was induced in MeOV-1 exposed to PLX4032 since, under the same experimental conditions, other Nrf2-dependent genes such as GCLM, GCLC and NQO1 were not modulated. Moreover, a stable downregulation of Bach1, a negative regulator of HO-1 induction, was found. The present findings have identified a molecular mechanism, involving stable downregulation of Bach1 and early activation of Nrf2 that drives the induction of HO-1 in response to PLX4032 treatment. In an ERASMUS Plus collaboration with Professor Giovanni Mann at King's College London, UK, the effects of changes in the ambient oxygen levels on MeOV-1 cell responses to PLX4032 and HO-1 induction were investigated. PLX4032-treated MeOV-1 cells adapted long-term (5 days) to either 5kPa (physiological normoxia) and 1kPa (hypoxia) O2 showed pERK levels, cell viability, HO-1, Bach1 and Nrf2-dependent gene expression similar to cells cultured under standard conditions (18kPa O2). Moreover, HIF-1α expression was induced by cell adaption to 1kPa O2 but, interestingly, protein levels were already high in untreated cells cultured under 18kPa or 5kPa O2 due to the constitutive activation of pERK pathway. Furthermore, in MeOV-1 cells adapted to 18kPa and 5kPa O2, HIF-1α expression was almost completely abolished after 10μM PLX4032 treatment, while HIF-1α protein expression was only partially decreased in 1kPa O2-adapted MeOV-1 cells. Thus, no substantial differences in MeOV-1 cell response to PLX4032 were observed under altered O2 levels in cell culture, resulting most likely from the high basal expression of HIF-1α in this cell line. In order to evaluate the role of HO-1 in favouring melanoma angiogenesis, endothelial tube formation was examined in cultured bovine and human endothelial cells. Under standard culture conditions (18kPa O2), conditioned media (CM) derived from PLX4032-treated MeOV-1 cells increased the formation of tubes in bovine aortic endothelial cells (BAEC) seeded on Matrigel, while CM derived from HO-1 silenced MeOV-1 cells treated with PLX4032 significantly prevented tube formation. To verify the proangiogenic ability of CM derived from cells adapted to normoxia (5kPa O2) or hypoxia (1kPa O2), and the role played by HO-1 downregulation, additional experiments were conducted with human cerebral microvascular cells (hCMEC/D3) and human endothelial cells from vein (HECV) adapted to 5kPa O2. These endothelial cells were adapted at 5kPa O2, seeded on Matrigel with CM derived from PLX4032-treated MeOV-1 adapted to 5kPa and 1kPa O2. Some samples were co-treated with HO-1 inhibitor SnMP-IX. Unfortunately, due to the COVID-19 pandemic, these experiments have been performed only few times in the London laboratories, and thus only preliminary results are included in the thesis. HO-1 inhibition in MeOV-1 cells adapted at 5kPa and 1kPa O2 completely prevented the ability to form tubes. In these experimental conditions, no significant modulation of VEGF-A expression was observed suggesting that HO-1 dependent proangiogenic effects may not be related to the modulation of VEGF-A. In conclusion, the present results highlight the specific involvement of HO-1 in limiting the efficacy of Vemurafenib/PLX4032 on melanomas carrying on BRAFV600 mutation, by the combined modulation of Nrf2 and Bach1, and in favouring immune-escape by the downregulation of NK cell ligands B7H6 and ULBP3. Indeed, not only HO-1 silencing but also the use of HO-1 enzymatic inhibitor, already in clinical use, has been shown to increase PLX4032 efficacy and to improve NK recognition and killing. Furthermore, preliminary data highlight that HO-1 silencing/inhibition decreased the angiogenic potential of MeOV-1 cells in a VEGF-independent manner, even though the molecular mechanisms have yet to be investigated. Further studies of heme oxygenase 1, Nrf2 and Bach1 are warranted to confirm their potential as target co-treatment with Vemurafenib for the treatment of BRAF mutated melanoma.