Mechanisms of HAMLET-induced cancer cell death

• HAMLET, a complex of α-lactalbumin and oleic acid, preferentially kills cancer cells and is also effective in vivo. HAMLET causes apoptosis but cells die even if this pathway is inhibited. Thus, the role of autophagy, an alternative cell death pathway, was examined. During autophagy double membrane-enclosed autophagosomes form around cellular material and deliver it to the lysosomes for degradation. After HAMLET treatment, such vesicles were observed and HAMLET also increased granular LC3-GFP staining confirming autophagosome accumulation. In addition, the autophagic form of LC3 increased if lysosomal degradation was inhibited, indicating a true increase in autophagic flux. Several factors that may activate autophagy were identified, including mitochondrial damage, inhibition of the autophagy inhibitor mTOR and increased the expression of autophagy components. Inhibition of autophagy by Atg5 and Beclin-1 siRNAs reduced cell death suggesting that autophagy is an important part of the cell death programme. As autophagy can be initiated by metabolic stress and as metabolism is altered in cancer cells we investigated if HAMLET-induced cell death involves an effect on metabolism. Healthy cells, which were less sensitive to HAMLET, had lower levels of c-Myc, an important regulator of glucose metabolism, and knockdown of c-Myc in cancer cells reduced their sensitivity. In contrast, cancer cell death was enhanced by glycolysis inhibition. HAMLET was also shown to bind the glycolytic enzyme hexokinase 1 (HK1) and to reduce HK activity, ATP levels and lactate release. Also, knockdown of HK1 and the glycolysis-enhancing HIF1A sensitized cancer cells to HAMLET whereas knockdown of the glycolysis-regulating protein PFKFB1 reduced sensitivity. Finally, mass spectrometry-based analysis indicated that HAMLET has a broad impact on metabolism possibly due to binding of HK1. In an additional screen for HAMLET-binding proteins prohibitin-2, a multifunctional protein found at the same cellular sites as HAMLET, was identified. The binding was confirmed in vitro and HAMLET was shown to also bind to the related prohibitin. Co-staining for HAMLET and prohibitins revealed colocalization in the cytoplasm. In addition, nuclear staining for both prohibitins was reduced, as described in camptothecin-induced cancer cell death. Furthermore, prohibitin-2 siRNA reduced cell death after HAMLET treatment suggesting that prohibitins may play a role in HAMLET-induced cell death. Besides cell death, HAMLET also causes cancer cell detachment and α-actinin-4, which crosslinks the actin cytoskeleton and focal adhesions, was identified in a screen as a potential HAMLET target. The binding between HAMLET and α-actinins was confirmed by co-immunoprecipitation and the actin and integrin binding sites on α-actinin were identified as possible sites of HAMLET binding in a peptide binding assay. HAMLET was also shown to colocalize with α-actinin-4 in the cell periphery and to reduce its granular surface staining and intracellular trabecular staining. In addition, HAMLET altered the staining of other focal adhesion components and reduced the level of active focal adhesion kinase and ERK, which can both be regulated via focal adhesions. Healthy cells did not detach after HAMLET treatment but the detachment of cancer cells was even further enhanced by knockdown of α-actinins. In contrast, overexpression of α-actinin-4-GFP delayed blebbing and rounding up further suggesting that α-actinin is involved in HAMLET-induced detachment. In summary, this study indicates that HAMLET activates autophagy and disrupts metabolism at least partly by binding to HK1 and that these events contribute to cell death. The results also suggest that c-Myc and glycolysis are important determinants of HAMLET sensitivity. Furthermore, the study identifies prohibitins as novel HAMLET targets with a potential role in cell death and suggests that HAMLET binding to α-actinins disrupts focal adhesions leading to cell detachment.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Immunologi inom det medicinska området (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Immunology in the medical area (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Mikrobiologi inom det medicinska området (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Microbiology in the medical area (hsv//eng)

Nyckelord

alpha-actinin

prohibitin

alpha-lactalbumin

HAMLET

cancer

cell death

autophagy

focal adhesions

c-Myc

hexokinase

glycolysis

Publikations- och innehållstyp

dok (ämneskategori)

vet (ämneskategori)