Targeting endothelin receptor signalling overcomes heterogeneity driven therapy failure

Approaches to prolong responses to BRAF targeting drugs in melanoma patients are challenged by phenotype heterogeneity. Melanomas of a “MITF‐high” phenotype usually respond well to BRAF inhibitor therapy, but these melanomas also contain subpopulations of the de novo resistance “AXL‐high” phenotype. > 50% of melanomas progress with enriched “AXL‐high” populations, and because AXL is linked to de‐differentiation and invasiveness avoiding an “AXL‐high relapse” is desirable. We discovered that phenotype heterogeneity is supported during the response phase of BRAF inhibitor therapy due to MITF‐induced expression of endothelin 1 (EDN1). EDN1 expression is enhanced in tumours of patients on treatment and confers drug resistance through ERK re‐activation in a paracrine manner. Most importantly, EDN1 not only supports MITF‐high populations through the endothelin receptor B (EDNRB), but also AXL‐high populations through EDNRA, making it a master regulator of phenotype heterogeneity. Endothelin receptor antagonists suppress AXL‐high‐expressing cells and sensitize to BRAF inhibition, suggesting that targeting EDN1 signalling could improve BRAF inhibitor responses without selecting for AXL‐high cells

Investigating the functional role of the amyloid precursor protein’s copper binding domain

© 2011 Dr. Loredana SpoerriAlzheimer’s disease is a progressive and eventually fatal neurodegenerative disorder characterized by specific proteins deposition in the brain: amyloid plaques and tau tangles. While age represents the major risk factor for AD, the mechanisms triggering the pathology remain unclear. According to the amyloid cascade hypothesis, accumulation of the main component of the amyloid plaques, the β-amyloid (Aβ) peptide, is crucial for the onset of the disease. This phenomenon is mainly accounted for by mis-metabolism of the Amyloid Precursor Protein (APP) from which Aβ is derived and by low Aβ clearance. Copper dyshomeostasis, which has been reported in AD, contributes to Aβ accumulation by influencing APP metabolism. Conversely, APP regulates copper homeostasis, a process in which imbalances can lead to oxidative stress and inflammatory response. This study investigated the relationship between APP and copper in terms of APP-mediated copper homeostasis and toxicity, as well as copper-mediated APP metabolism. The role of the APP copper binding domain (CuBD), and in particular the copper binding site histidine residues, was examined with the results revealing that the domain mediates APP metabolism and structure stability. This study has significantly contributed to the understanding of APP CuBD in modulating APP metabolism and stability, and highlights the potential of this domain as a novel therapeutic target in AD

Role of the amyloid precursor protein and copper in Alzheimer's disease

The Amyloid Precursor Protein is a highly studied protein due to its assumed central role in the pathogenic pathway that leads to Alzheimer's disease. It is expressed into a number of isoforms and undergoes complex proteolytic processing. Structural, biochemical and cellular data identify the Amyloid Precursor Protein as copper binding protein. This interaction modulates Amyloid Precursor Protein metabolism as well as copper homeostasis. This interaction could also have a direct impact upon the pathogenic pathway that causes Alzheimer's diseases

Real-time cell cycle imaging in a 3D cell culture model of melanoma

Aberrant cell cycle progression is a hallmark of solid tumors; therefore, cell cycle analysis is an invaluable technique to study cancer cell biology. However, cell cycle progression has been most commonly assessed by methods that are limited to temporal snapshots or that lack spatial information. Here, we describe a technique that allows spatiotemporal real-time tracking of cell cycle progression of individual cells in a multicellular context. The power of this system lies in the use of 3D melanoma spheroids generated from melanoma cells engineered with the fluorescent ubiquitination-based cell cycle indicator (FUCCI). This technique allows us to gain further and more detailed insight into several relevant aspects of solid cancer cell biology, such as tumor growth, proliferation, invasion, and drug sensitivity

Reciprocal regulation of BRN2 and NOTCH1/2 signaling synergistically drives melanoma cell migration and invasion

Phenotypic plasticity drives cancer progression, impacts on treatment response and is a major driver of therapeutic resistance. In melanoma, a regulatory axis between the MITF and BRN2 transcription factors has been reported to promote tumor heterogeneity by mediating switching between proliferative and invasive phenotypes respectively. Despite strong evidence that subpopulations of cells that exhibit a BRN2high/MITFlow expression profile switch to a predominantly invasive phenotype, the mechanisms by which this switch is propagated and promotes invasion remain poorly defined. We have found that a reciprocal relationship between BRN2 and NOTCH1/2 signaling exists in melanoma cells in vitro, within patient datasets and in vivo primary and metastatic human tumors that bolsters acquisition of invasiveness. Working through the epigenetic modulator EZH2, the BRN2- NOTCH1/2 axis is potentially a key mechanism by which the invasive phenotype is maintained. Given the emergence of agents targeting both EZH2 and NOTCH, understanding the mechanism through which BRN2 promotes heterogeneity may provide crucial biomarkers to predict treatment response to prevent metastasis

The amyloid precursor protein copper binding domain histidine residues 149 and 151 mediate APP stability and metabolism

One of the key pathological hallmarks of Alzheimer disease (AD) is the accumulation of the APP-derived amyloid β peptide (Aβ) in the brain. Altered copper homeostasis has also been reported in AD patients and is thought to increase oxidative stress and to contribute to toxic Aβ accumulation and regulate APP metabolism. The potential involvement of the N-terminal APP copper binding domain (CuBD) in these events has not been investigated. Based on the tertiary structure of the APP CuBD, we examined the histidine residues of the copper binding site (His, His, and His ). We report that histidines 149 and 151 are crucial for CuBD stability and APP metabolism. Co-mutation of the APP CuBD His and His to asparagine decreased APP proteolytic processing, impaired APP endoplasmic reticulum-to-Golgi trafficking, and promoted aberrant APP oligomerization in HEK293 cells. Expression of the triple H147N/H149N/H151N-APP mutant led to up-regulation of the unfolded protein response. Using recombinant protein encompassing the APP CuBD, we found that insertion of asparagines at positions 149 and 151 altered the secondary structure of the domain. This study identifies two APP CuBD residues that are crucial for APP metabolism and suggests an additional role of this domain in APP folding and stability besides its previously identified copper binding activity. These findings are of major significance for the design of novel AD therapeutic drugs targeting this APP domain

Decatenation checkpoint defective melanomas are dependent on PI3K for survival

Melanoma cell lines are commonly defective for the G2-phase cell cycle checkpoint that responds to incomplete catenation of the replicated chromosomes. Here, we demonstrate that melanomas defective for this checkpoint response are less sensitive to genotoxic stress, suggesting that the defective cell lines compensated for the checkpoint loss by increasing their ability to cope with DNA damage. We performed an siRNA kinome screen to identify kinases responsible and identified PI3K pathway components. Checkpoint-defective cell lines were three-fold more sensitive to small molecule inhibitors of PI3K. The PI3K inhibitor PF-05212384 promoted apoptosis in the checkpoint-defective lines, and the increased sensitivity to PI3K inhibition correlated with increased levels of activated Akt. This work demonstrates that increased PI3K pathway activation is a necessary adaption for the continued viability of melanomas with a defective decatenation checkpoint

A novel ATM-dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma

Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM-dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM-dependent checkpoint arrest, and over-expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM-dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over-express PLK1, and a significant proportion of melanomas have high levels of PLK1 over-expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM-dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected