Role of the pro-survival molecule Bfl-1 in melanoma

Abstract

Melanoma, the cancer derived from the melanocytes of the skin, is becoming an ever more common cancer in the ageing population of the developed world and displays an intrinsic resistance to current therapies. This chemo resistance makes metastatic melanoma an extremely difficult cancer to treat leading to a 5 year survival rate of just 20%. As such, it is important to unearth new potential drug targets to increase the prospects for melanoma patients.Bfl-1 is a pro-survival protein of the Bcl-2 family of apoptosis regulating proteins. It has been found to be over-expressed in a small subset of chemo resistant tumours, including melanoma. Accordingly, I characterised the molecule in melanoma cells and determined its role in protecting these cells from chemotherapy-induced apoptosis. I elucidated the expression profile and half-lives of the protein and mRNA across a panel of melanoma cell-lines and healthy melanocytes. I also confirmed, using pathway specific inhibitors, that Bfl-1 was transcriptionally regulated by the NFB pathway and concluded through pulsechase experiments that Bfl-1 protein was degraded, at least in part, by the proteasome. Subcellular fractionation and indirect immunofluorescence techniques elucidated that Bfl-1 was located mostly at the mitochondria in both resting and apoptotic cells, with a diffuse level present throughout the cytoplasm. As well as characterising the protein in both melanoma cells and healthy primary melanocytes, I determined its role in the resistance of these cells to apoptosis. Over-expressed Bfl-1 was found to protect melanoma cells from apoptosis caused by a range of chemotherapeutic agents in A375 melanoma cells, which naturally expressed very low levels of Bfl-1. Further, knock down of Bfl-1 using siRNA technology in melanoma cells revealed the dependence of these cells on Bfl-1 for their resistance to certain chemotherapeutic agents currently used in melanoma treatment, including the MEK inhibitor PD901. All together, this research contributes to our understanding of Bfl-1 and highlights it as a potentially important therapeutic target in metastatic melanoma.

Selective Covalent Targeting of Anti-Apoptotic BFL-1 by Cysteine-Reactive Stapled Peptide Inhibitors

“…Importantly, we observed comparative enhancement in cytotoxicity and caspase 3/7 activation for BIM SAHB A -3 in two additional BFL-1 expressing melanoma cell lines (SK-MEL-2, SK-MEL-28)(Haq et al, 2013; Hind et al, 2015) (Figure S4), but no evidence of this phenomenon in non-melanoma lines that either lack or maintain BFL-1 expression, but are driven by alternate oncogenic mechanisms (e.g. A549, MCF7, H929)(Acquaviva et al, 2012; Haq et al, 2013; Leverson et al, 2015) (Figure S5).…”

“…Whereas BIM SAHB A and BIM SAHB A -3 demonstrated equivalent binding to anti-apoptotic MCL-1, as previously observed in the context of competitive interaction with recombinant anti-apoptotic proteins (Figure 3B), the warhead-bearing construct again showed markedly increased engagement of BFL-1 (Figure 6A). To verify that BIM SAHB A -3 can indeed label native mitochondrial BFL-1(Hind et al, 2015), we incubated mitochondria purified from A375P cells with biotinylated BIM SAHBs and observed BIM SAHB A -3-selective biotinylation of mitochondrial protein at the identical molecular weight as immunoreactive BFL-1 (Figure 6B). Live confocal microscopy imaging of A375P cells treated with FITC-BIM SAHB A -3 further revealed the stapled peptide’s striking intracellular localization at the mitochondria, the physiologic site of BFL-1 activity, in both morphologically normal A375P cells (Figure 6C) and those undergoing apoptosis induction, as reflected by cell shrinkage, nuclear condensation, and membrane blebbing (Figure 6D).…”

“…In melanoma, for example, BFL-1 overexpression correlates with chemoresistance and metastasis(Hind et al, 2015; Riker et al, 2008), and is directly regulated by the microphthalmia-associated transcription factor (MITF), which has proven essential to melanomagenesis(Haq et al, 2013). In lymphoma, upregulation of BFL-1 underlies the resistance to selective inhibition of BCL-2 and BCL-X L , underscoring the importance of developing BFL-1 inhibitors in the era of ABT-199(Yecies et al, 2010).…”

Targeting PI3K/mTOR Signaling Displays Potent Antitumor Efficacy against Nonfunctioning Pituitary Adenomas

“…In a physiologic context, Bfl-1 is mainly expressed in the hematopoietic system, where it facilitates the survival of selected leukocytes subsets. Bfl-1 has been found overexpressed in a subset of chemoresistant tumors, where it protects tumor cells from chemotherapy-induced apoptosis (52). Recently, peptide aptamers specifically targeting Bfl-1 have been generated and shown to sensitize B-cell lymphoma cell lines to chemotherapeutic drugs through induction of apoptosis (53,54).…”

BH3 mimetics induce apoptosis independent of DRP-1 in melanoma

“…High BFL-1 expression occurs in at least a subset of melanoma and can be an important pro-survival player 49,50 . Therefore, we examined the effects of knocking down BFL-1 on the combination-induced killing, in two cell lines with high endogenous BFL-1 expression (Supplementary Figure 6).…”