HAMLET: Functional Properties and Therapeutic Potential

James Ho CS; Anna Rydström; Maria Trulsson; Johannes Bålfors; Petter Storm; Manoj Puthia; Aftab Nadeem; Catharina Svanborg


Future Oncol. 2012;8(10):1301-1313. 

In This Article

Cell Death in Response to HAMLET

Susceptibility of Tumor Cells to HAMLET

HAMLET appears to identify and exploit conserved features of cancer cells for its tumoricidal activity. To date, more than 40 different tumor cell lines have been exposed to HAMLET in vitro and shown to be sensitive, regardless of tumor type, species and tissue origin.[9] Arguably, such conserved features may either represent general features of tumor cells that also render them susceptible to HAMLET or may reflect the presence of specific, conserved targets critical for the cell death response. To identify properties that render tumor cells susceptible to HAMLET, a combination of shRNA inhibition, proteomic and metabolomic technology were used. Elevated c-MYC expression was shown to create a HAMLET-sensitive phenotype and the c-MYC and RAS oncogenes were identified as essential determinants of HAMLET sensitivity.[12] Furthermore, the shRNA screen identified Hexokinase 1, PFKFB1 and HIF1α as determinants of HAMLET sensitivity. Hexokinase 1 was also shown to bind HAMLET in a protein array containing approximately 8000 targets.[12] Importantly, glucose deprivation sensitized tumor cells to HAMLET-induced cell death and HAMLET triggered rapid metabolic paralysis in carcinoma cells. By mass spectrometry, the glycolytic machinery was shown to be modified by HAMLET and glycolysis was shifted towards the pentose phosphate pathway. These findings link the HAMLET sensitivity of tumor cells to conserved features defined by oncogenic transformation and the metabolic state (the Warburg effect).

The Plasma Membrane: The First Barrier

HAMLET discriminates tumor cells from normal differentiated cells, and interactions at the plasma membrane level determine the difference in sensitivity. The key molecular interactions are not fully understood, but in early studies, HAMLET was shown to trigger ion fluxes across tumor cell membranes.[9] Recently, in lipid membrane models, HAMLET has been shown to interact with phospholipid bilayers in the absence of specific tumor cell membrane constituents.[45] HAMLET, bound to egg yolk and soybean membranes at physiological pH, perturbed membrane integrity and caused leakage of vesicular contents to the exterior. Vesicles composed of natural lipid mixtures showed drastically altered morphology in response to HAMLET, although the natively folded protein or oleic acid did not have these effects.[45,46] Interestingly, the membrane elongation and a change in fluidity further emphasize the potential for ion channel activation by HAMLET through mechanosensing or, alternatively, the formation of HAMLET-specific ion channels.[47] The results suggest that the membrane is perturbed in concert by the partially unfolded protein and the fatty acid by a mechanism requiring both the protein and the fatty acid.

Relevance of these membrane perturbations and resulting ion fluxes for tumor cell death has also been documented.[47] HAMLET was shown to trigger rapid cation fluxes and the inhibition of these fluxes prevented many aspects of the cell death response to HAMLET. Furthermore, tumor specificity was suggested, as HAMLET was shown to alter membrane properties in plasma membrane vesicles from tumor cells while plasma membrane vesicles from normal, differentiated cells were not affected.[45] This may reflect differences in membrane composition, structural organization and functional properties between tumor cells and normal, differentiated cells, including membrane fluidity and the fatty acid composition of the phospholipids. Membranes of tumor cells have altered lipid composition and fluidity, which may alter the properties of membrane-bound receptors, enzymes and endocytic pathways and thereby the activation of cell death.[48] Thus, tumor cell membranes may favor HAMLET binding and facilitate HAMLET-induced membrane perturbations activating the cell death response.

An Unfolded Protein Response: Endoplasmic Reticulum Stress & Proteasome Fragmentation in HAMLET Response

After perturbing the membranes of tumor cells, HAMLET enters the cytoplasm and translocates to the nuclei.[10,13,49,50] The authors have hypothesized that the internalization of HAMLET creates an unfolded protein-overload scenario that triggers endoplasmic reticulum stress and targets HAMLET to the proteasomes for degradation. Proteasomes normally control the level of endogenous unfolded proteins by degrading them in the proteolytic core, proteins that resist degradation or proteasome inhibition may cause cell death. Endogenous, unfolded proteins are degraded by 26S and 20S proteasomes but unfolded α-lactalbumin interacts mainly with the 20S proteasomes in vitro.[51] The authors have shown that HAMLET is targeted to 20S proteasomes in tumor cells and triggers a change in proteasomes structure, with modifications of catalytic (β1 and β5) and structural subunits.[52] Evidence for a direct interaction of HAMLET with intact proteasomes and proteasome subunits was obtained in vitro.[52] Interestingly, HAMLET resisted degradation by proteasomal enzymes and inhibited proteasome activity. Thus, targeting of internalized HAMLET to the proteasomes and perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.

Nuclear Receptors & Chromatin Interactions of HAMLET

The nuclear translocation of HAMLET is rapid, with 75% of the complex reaching the nuclei within 1 h at 35 µM concentrations. Healthy cells, by contrast, only take up small amounts of HAMLET and there is no evidence that HAMLET reaches the nuclei of healthy cells.[13,49] The nuclear accumulation of HAMLET has been proposed to reflect the role of histones as nuclear receptors for HAMLET, and the affinity binding, mainly to histones H3 and H4, creates virtually insoluble complexes.[49] In nuclear extracts, HAMLET, histones and DNA form virtually insoluble complexes. As chromatin accessibility is controlled by the acetylation state of the histone tail, histone deacetylase inhibitors are used to modify the chromatin accessibility of tumoricidal agents. Histone deacetylase inhibitors enhance the tumoricidal effects of HAMLET, in part by enhancing the hyperacetylation response.[53] Future studies in tumor models will be of interest to investigate whether the combination of HAMLET and histone deacetylase inhibitors may be used to increase the therapeutic efficiency in vivo.