Conserved features of cancer cells define their sensitivity to HAMLET-induced death; c-Myc and glycolysis

Abstract

HAMLET is the first member of a new family of tumoricidal protein-lipid complexes that kill cancer cells broadly, while sparing healthy, differentiated cells. Many and diverse tumor cell types are sensitive to the lethal effect, suggesting that HAMLET identifies and activates conserved death pathways in cancer cells. Here we investigated the molecular basis for the difference in sensitivity between cancer cells and healthy cells. Using a combination of small hairpin RNA inhibition, proteomic and metabolomic technology we identified the c-Myc oncogene as one essential determinant of HAMLET sensitivity. Increased c-Myc expression levels promoted the sensitivity to HAMLET and shRNA knockdown of c-Myc suppressed the lethal response, suggesting that oncogenic transformation with c-Myc creates a HAMLET-sensitive phenotype. Furthermore, the HAMLET sensitivity was modified by the glycolytic state of the tumor cells. Glucose deprivation sensitized tumor cells to HAMLET-induced cell death and in the shRNA screen Hexokinase 1, PFKFB1 and HIF1α modified HAMLET sensitivity. Hexokinase 1 was shown to bind HAMLET in a protein array containing approximately 8000 targets and Hexokinase activity decreased within 15 minutes of HAMLET treatment, prior to morphological signs of tumor cell death. In parallel, HAMLET triggered rapid metabolic paralysis in carcinoma cells. The glycolytic machinery was modified and glycolysis was shifted towards the pentose phosphate pathway. Tumor cells were also shown to contain large amounts of oleic acid and its derivatives already after 15 minutes. The results identify HAMLET as a novel anti-cancer agent that kills tumor cells by exploiting unifying features of cancer cells such as oncogene-addiction or the Warburg effect.

The Alpha-Lactalbumin/Oleic Acid Complex and Its Cytotoxic Activity

“…Furthermore, a reduction in the extracellular glucose levels or degree of glycolysis, predominantly the aerobic glycolysis of tumor cells known as the Warburg Effect, of A549 lung carcinoma cells resulted in an enhanced HAMLET-sensitivity [83]. HAMLET was shown to bind and reduce the activity of the glycolytic enzyme hexokinase 1 in the A549 carcinoma cells, which could partly explain the inhibitory effect of HAMLET on glycolysis [83].…”

“…An elevated expression level of c-Myc, an oncogene that has the ability to bind a significant fraction of all known gene promoters, has been shown to increase HAMLET-sensitivity of various tumor cells, suggesting that the level of c-Myc expression could be a direct determinant of HAMLET susceptibility [83]. Furthermore, a reduction in the extracellular glucose levels or degree of glycolysis, predominantly the aerobic glycolysis of tumor cells known as the Warburg Effect, of A549 lung carcinoma cells resulted in an enhanced HAMLET-sensitivity [83].…”

“…Anti-adhesion [1,82,102] c-Myc oncogene status [83] Inhibition of 20S proteasome activity [69] Inhibition of glycolysis [83] Lysosomal pathway [61] p38 pathway [82] Ribosome interactions [2] MAL co-localizes with mitochondria and causes release of cytochrome c from the IMM to the cytosol MAL causes loss of mitochondrial membrane potential For elaboration of the specific tumor cells prone to the mentioned effect, the reader is encouraged to see the references. HK1, hexokinase 1; IMM, inner mitochondrial membrane; MAL, multimeric -lactalbumin; MPT, mitochondrial permeability transition.…”

Lipids as Tumoricidal Components of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET)

“…Studies in patients and animal models have confirmed this selectivity and demonstrated therapeutic efficacy against several tumor types ( 16 -20). HAMLET has broad tumoricidal activity unrelated to well defined cellular responses such as apoptosis (21)(22)(23). Essential aspects of the conserved death response to HAMLET have been defined, including the dependence on oncogenic transformation (22), proteasome inhibition (24), and nucleosome-histone binding (25).…”

“…HAMLET has broad tumoricidal activity unrelated to well defined cellular responses such as apoptosis (21)(22)(23). Essential aspects of the conserved death response to HAMLET have been defined, including the dependence on oncogenic transformation (22), proteasome inhibition (24), and nucleosome-histone binding (25). We have recently identified ion channel activation as a new, unifying mechanism of HAMLET tumoricidal effect (23).…”