Acid sphingomyelinase – a novel target for anti-cancer and degenerative diseases?

Dr Hong Sun, from the University of Nevada, Las Vegas, explores how acid sphingomyelinase (ASM) interacts with key components of the plasma membrane (PM) of the cell, thereby influencing cell signalling. Dr Sun and her colleagues have discovered that mutations or abnormal expression in the gene that encodes ASM can result in a wide range of impacts, from regulating lifespan to enhancing the risk of cancer and neuron degenerative disease.
The plasma membrane of the cell (PM) protects every cell in our body and maintains homeostasis via cell signalling. The PM is a highly dynamic and flexible structure consisting of a lipid bilayer, with embedded proteins. The structure and function of these proteins is regulated by membrane-associated lipid molecules, which influence cell communication and cellular processes. One of the critical regulators of this mechanism is the acid sphingomyelinase (ASM) enzyme, which hydrolyses sphingomyelin, an important lipid component of the PM, comprising around 10-20% of total PM lipids. The products of this reaction are ceramide and phosphocholine. Ceramide is a bioactive lipid molecule involved in a wide range of physiological processes, from cell proliferation, cell adhesion to cell death.

However, the exact mechanisms underpinning ASM function are not well understood. This inspired Dr Sun and her colleagues to investigate the specific mechanisms by which ASM influences protein components of the PM and the wider implications of these interactions. In particular, the team have studied the role of ASM in cancer, aging and neuron degeneration.
ASM reduces lifespan in Caenorhabditis elegans
The team used the nematode model organism Caenorhabditis elegans to study the effects of ASM on senescence. Model organisms are used to improve our understanding of biological phenomena. Evolution has resulted in the genetic conservation of metabolic pathways between different species, making it possible to reliably apply findings conducted on model organisms to other species, including humans. In this case, Dr Sun’s group discovered that the C.elegans genome harbours an ASM gene (asm-3) which encodes a protein that is 42% similar to the human ASM.

Aberrant function of ASM can cause a wide range of destructive diseases, including glioblastoma and Niemann Pick Disease, type A

In a previous study, Dr Sun’s group showed that the homolog of PTEN tumour suppressor regulates the conserved DAF-2/Insulin-like Growth Factor 1 Receptor signalling (IGF-1R) pathway (found in both mammals and C. elegans) and consequently regulates the animal’s lifespan. The team has since focused their studies to novel molecules that modulate DAF-2/IGF-1R signalling, regulating longevity. In order to discover whether ASM impacts this signalling pathway, the team performed RNA interference (RNAi) on asm-3. This is a technique used to silence a gene by effectively destroying the specific mRNA, meaning that the protein encoded by the gene cannot be synthesised. Effectively, the team reduced the effects of ASM-3 and the results showed that C.elegans then had a 19% lifespan increase, compared to controls. This suggests that asm-3 positively regulates the DAF2/IGF-1R pathway, limiting longevity.
To further explore this hypothesis, Dr Sun’s group studied the DAF-16/FOXO transcription factor, which is essential for regulating the DAF2/IGF-1R pathway. In fact, increased levels of this transcription factor results in lifespan extension. Reduced levels of asm-3 (by RNAi) resulted in a 100% increase in DAF-16/FOXO levels, which positively influences longevity. Therefore, these results indicate that ASM-3 naturally reduces DAF-16 protein expression, limiting potential lifespan.
The team also found that the two drugs desipramine and clomipramine significantly extend the lifespan of C. elegans, by inhibiting the asm gene, each reducing the enzyme activity by nearly 80%. This ground-breaking research could possibly be extended to mammals and be used to develop an anti-aging therapy.
Abnormal ASM and disease
Aberrant functions of the human ASM can cause a wide range of destructive diseases, including glioblastoma and Niemann Pick Disease, type A. Over-activation of ASM is found in glioblastoma, a very aggressive form of brain cancer, and patients usually die within a year of diagnosis. On the other hand, the loss-of-function of ASM is linked to Niemann Pick Disease, type A, an inherited neurodegenerative disease, characterised by destruction of neurons in the brain, and abnormal lipids build-up in the liver and spleen. Devastatingly, there is no cure and patients usually die within two or three years after prognosis. How is ASM linked to both cancer and a neurodegenerative disease? The research from Dr Sun’s group has revealed that the activities of ASM regulate the functions of the plasma membrane embedded proteins, in particular, the receptor tyrosine kinases (RTKs), which in turn may lead to cancer or neurogeneration, depending on how the activities of RTKs are affected.
In order to develop potential treatments for these life-debilitating diseases, Dr Sun and her colleagues have investigated the relationship between ASM and Met, a type of receptor tyrosine kinase (RTK), abundant in cancer cells.

Inhibition of ASM in cancer cells leads to a strong reduction of the cell surface levels of Met, a RTK important for cancer cell growth

RTK proteins are embedded in the PM and bind to specific ligands, triggering an intracellular cascade, which results in cell growth and differentiation. In particular, Met is found in epithelial cells of a wide range of organs. Met is activated by the ligand, hepatocyte growth factor (HGF), triggering mitogenesis and morphogenesis. However, enhanced Met RTK activity can have severe oncogenic affects, and is significantly associated with glioblastoma.

The team performed an in-depth study which indicated that ASM is a key regulator of Met RTK. The ASM gene was down-regulated in glioblastoma cells using two different methods: i) RNAi and ii) inhibition of the ASM enzyme by desipramine. Overall, Met protein levels at the PM decreased by approximately 40%. Interestingly, Dr Sun and her colleagues found that this reduced level of PM-associated Met is due to its accumulation in the Golgi Apparatus, an organelle that regulates the PM-destined proteins and lipids. This suggests that the ASM protein is required for the transportation of Met from the Golgi Apparatus to the PM. Therefore, over-expression of the ASM gene results in an increased quantity of ASM protein and consequently enhances Met RTK activity in cancerous cells. Interestingly, the team also discovered that inactivation of ASM resulted in an increased trafficking of Met to lysozymes, where the protein is degraded. The latter may offer an approach to down-regulates Met for anti-cancer therapy.
Future research and potential therapies
The innovative research of Dr Sun and her team has greatly enhanced our understanding of the multiple roles of ASM and the adverse implications of its mutated form including increased disease risk and limited longevity. Dr Sun is now focusing her research efforts on further determining how the enhancement of ASM/RTK activity causes cancer while the loss of ASM/RTK activity causes neuron degeneration, resulting in Niemann Pick Disease, type A. By furthering our knowledge, the team aims to develop novel therapies to then target ASM and potentially save many lives.

Why is the hydrolysis of sphingomyelins by acid sphingomyelinase (ASM) important?
This question goes back to the basic properties of PM lipid bilayer. Sphingomyelins, together with cholesterol, can form tightly packed, ordered lipid microdomains, the ‘lipid rafts’, according to the pioneering studies from Dr Kai Simons’ group. However, sphingomyelins are asymmetrically localised in the outer layer of the PM. On the other hand, ceramides, the hydrolysed products of sphingomyelins, are present in both outer and inner layers of PM. This is because ceramide is much more hydrophobic than sphingomyelin, as ceramide lacks the phosphate and polar head group of sphingomyelin, and can therefore rapidly flip-flop in the lipid bilayer. Ceramides also have a remarkable biophysical property of self-association, allowing them to form unique lipid microdomains. When ceramides are produced by ASM at the outer layer of the PM, as suggested by both ours and others’ studies, the inner layer of the PM is able to rapidly acquire ceramides from the outer layer. The presence of ceramides in both layers of the PM provides a favourable lipid microenvironment to facilitate transmembrane receptors interaction and signalling.
Mutant ASMs play a key role in senescence and disease, can it affect any other processes?
We’ve identified C. elegans ASM homolog as a novel regulator for the conserved IGF-1R signalling pathway involved in regulation of animal aging. Interestingly, in both mouse and human, IGF-1R signalling pathway is intimately associated with stem cells maintenance and survival. So ASM may be potentially involved in regulation of stem cell senescence. The second, and also possibly related, process is the response to oxidative stress. In C. elegans, knocking down the homologs of ASM or IGF-1R each confers a resistance towards oxidative stress and increases animal’s lifespan. Accordingly, the similar process may be affected in the mammalian systems.
How did you use RNA interference (RNAi) to study the effects of ASM?
RNAi is a magic bullet that researchers use to silence (to inhibit the expression) of a particular gene, and this phenomenon was first discovered in C. elegans. In C. elegans, one can also perform an RNAi-based genomic screen, i.e. using RNAi to silence all genes in the genome and then look for a particular phenotype. This approach is facilitated by the fact that C. elegans eat bacteria as food, so the RNAi can be achieved by feeding the worms with bacteria expressing the RNAi clones. This is in fact how we have identified the worm ASM homolog at the first place, by conducting a genomic RNAi screen and looking for genes, when silenced by RNAi, giving to a similar phenotype as that due to loss of DAF-2, the IGF-1R homolog.
Can novel anti-cancer therapies be developed which target ASM?
Yes, we believe that ASM (and the ‘ceramide lipid island’) is a vulnerable target for cancer cells. Since ASM is highly expressed in cancer cells, if we block ASM, we shall be able to destroy the ceramide-enriched lipid microenvironment that harbours RTKs (such as Met and IGF-1R), critical for cancer cell proliferation and survival. ASM is also an attractive molecular target for anti-cancer therapy because the enzyme is localised in the outer layer of PM and therefore is readily accessible to both macromolecule (e.g., antibodies) and small molecule inhibitors.
What are your future research goals?
Our future research goals are several-fold. First, we plan to conduct more mechanistic studies to understand how ASM/ceramides regulate RTK signalling. Secondly, we’d like to employ preclinical models to validate that ASM is a critical target for anti-cancer therapy. Third, we will use mouse knockout models to determine if and how ASM is involved in regulation of neuron survival or stem cell maintenance, processes related to neuron-degeneration and aging.

Research Objectives
Dr Hong Sun’s lab explores mechanisms of cell signalling and was one of the original groups to clone the PTEN tumour suppressor gene. Her current research aims to develop novel anti-cancer therapy that target lipid enzymes.
Funding

• National Institutes of Health (NIH)
• Department of Defence (DOD)
• University of Nevada, Las Vegas, College of Sciences Cancer Research Gift Fund

Collaborators

• Members of Dr Sun’s Laboratory at the University of Nevada, Las Vegas: Dr Yongsoon Kim, Postdoctoral Fellow;
• Dr Xiahui Xiong, Postdoctoral Fellow;
• Ms Linyu Zhu, Graduate Student; Ms Wenjing Li, Graduate Student
• Dr Hui Zhang, Dept of Chemistry and Biochemistry, University of Nevada, Las Vegas, USA
• Dr Tao Ye, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China

Bio
Dr Sun received her PhD degree in 1991 from Harvard University, under the mentorship of Nobel Laureate Dr Jack Szostak. She was appointed as an Assistant Professor and then Associate Professor at Yale University School of Medicine between 1995-2007. In 2007, she became a Professor and the Director of Cancer Genomics at Nevada Cancer Institute. She joined the faculty of University of Nevada, Las Vegas in 2012.
Contact
Dr Hong Sun
Associate Professor
Department of Chemistry and Biochemistry
University of Nevada, Las Vegas
4505 S. Maryland Pkwy
Las Vegas, NV 89154
USA
E: [email protected]
T: +1 702 774 1485
W: www.unlv.edu/people/hong-sun