High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat

Abstract

The naked mole rat (Heterocephalus glaber) displays exceptional longevity, with a maximum lifespan exceeding 30 years. This is the longest reported lifespan for a rodent species and is especially striking considering the small body mass of the naked mole rat. In comparison, a similarly sized house mouse has a maximum lifespan of 4 years. In addition to their longevity, naked mole rats show an unusual resistance to cancer. Multi-year observations of large naked mole-rat colonies did not detect a single incidence of cancer. Here we identify a mechanism responsible for the naked mole rat's cancer resistance. We found that naked mole-rat fibroblasts secrete extremely high-molecular-mass hyaluronan (HA), which is over five times larger than human or mouse HA. This high-molecular-mass HA accumulates abundantly in naked mole-rat tissues owing to the decreased activity of HA-degrading enzymes and a unique sequence of hyaluronan synthase 2 (HAS2). Furthermore, the naked mole-rat cells are more sensitive to HA signalling, as they have a higher affinity to HA compared with mouse or human cells. Perturbation of the signalling pathways sufficient for malignant transformation of mouse fibroblasts fails to transform naked mole-rat cells. However, once high-molecular-mass HA is removed by either knocking down HAS2 or overexpressing the HA-degrading enzyme, HYAL2, naked mole-rat cells become susceptible to malignant transformation and readily form tumours in mice. We speculate that naked mole rats have evolved a higher concentration of HA in the skin to provide skin elasticity needed for life in underground tunnels. This trait may have then been co-opted to provide cancer resistance and longevity to this species.

Figure 1. Naked mole-rat cells secrete HA of exceptionally high molecular weight

(a) Naked mole-rat cells make the culture media viscous. The viscosity of water, media or media conditioned with human skin (HSF), guinea pig skin (GP SF), mouse skin (MSF) or naked mole-rat skin (NMR SF) fibroblasts for 20 days. The NMR SF + HAase bar shows naked mole-rat conditioned media digested with HAase to specifically digest HA. The NMR SF Mut are naked mole-rat skin fibroblasts that spontaneously lost the ECI phenotype. The NMR EF are naked mole-rat embryonic fibroblasts that do not show ECI. The experiment was repeated three times; error bars show s.d. (b) Purified HA separated on pulse-field gel. Each sample was either run intact or pre-digested with HAase. The experiment was repeated five times, using both skin and lung fibroblasts (Supplementary Figure 9), and a representative gel is shown. (c) Western blot showing the levels of HA synthases in naked mole-rat adult skin fibroblasts, naked mole-rat embryonic fibroblasts, human skin fibroblasts or mouse skin fibroblasts. (d) Conserved catalytic domain of mammalian HAS2 proteins. The top sequence is the naked mole-rat HAS2. Dots indicate amino acids identical to the naked mole-rat sequence. The two amino acid changes unique to the naked mole-rat are indicated by red boxes.

Figure 2. Naked mole-rat tissues contain high levels of HA

(a) Naked mole-rat HAS2 overexpressed in human HEK293 cells secretes HMW-HA. Small panel on the right shows immunoblot with anti HAS2 antibodies on whole cell extracts from the control and HAS2-transfected cells. (b) Tissues from the naked mole-rat, mouse, and guinea pig stained with Alcian Blue. The control samples treated with HAase do not show blue staining, demonstrating that the staining is specific to HA. Staining was performed on three different animals and representative skin and heart images are shown. Brain and kidney are shown in Supplementary Figure 2. (c) Naked mole-rat fibroblasts have low HAase activity. Naked mole-rat skin fibroblasts (NMR SF), guinea pig skin fibroblasts (GP SF), human skin fibroblasts (HSF), mouse skin fibroblasts (MSF) or HeLa cells were incubated with the media containing HMW-HA for four days. The levels of HA were then analyzed by pulse-field gel. Control samples were incubated in the absence of cells. The experiments were repeated four times (all samples except GP), and three times for GF, error bars show s.d.; asterisk indicates P<0.01 by t-test. (d) Naked mole-rat tissues have low HAase activity. Media containing HMW-HA was incubated with corresponding tissue fragments from naked mole-rats (NMR) or mice for six hours and HA levels analyzed by pulse-field gel. The experiments were repeated three times and error bars show s.d.; asterisk indicates P<0.05 by t-test.

Figure 3. HMW-HA is required for ECI

(a) Naked mole rat cells (NMR SF) grown in the presence of HAase do not display ECI and proliferate to high cell density. (b) Quantification of cell growth, showing the maximum cell number per plate achieved under indicated growth conditions. The last bar shows naked mole-rat cells grown in the presence of CD44-blocking antibody. The experiments were repeated four times (except the last bar, which was repeated three times) and error bars show s.d.; asterisk indicates P<0.001 by t-test. (c) Naked mole-rat cells were grown in the presence of HAase for 12 days, then HAase was removed. (d) Naked mole-rat cells have higher affinity to HA. Cells were incubated with fluorescein-labeled HA, and the average fluorescence was plotted. Experiment was repeated four times; error bars are s.e.m.; asterisk indicates P<0.001 by t-test.

Figure 4. Removal of HMW-HA makes naked mole-rat cells susceptible to malignant transformation

(a) Soft agar assays of anchorage-independent growth. Mouse (MSF) or naked mole-rat (NMR SF) cells were transfected with vectors encoding SV40 LT (LT) or its mutant derivatives K1 or Δ434 and H-Ras V12 (Ras), and plated in soft agar. Cells were cultured with or without HAase. The image shows colonies after 3 weeks of growth at 200× magnification. The experiment was repeated three times. (b) Mouse xenograft experiment with naked mole-rat cells in which HMW-HA was abolished. NIH-III immunodeficient mice were injected with mouse (MSF) cells expressing SV40LT and H-Ras V12 as a positive control, or naked mole rat (NMR SF) cells expressing SV40LT and H-Ras V12 and either control shRNA, Hyal2 cDNA, or shRNA to HAS2. All xenografts with mouse cells formed large tumors. Xenografts with naked mole-rat cells expressing control shRNA did not form tumors, while naked mole-rat cells overexpressing Hyal2 or HAS2 shRNA formed tumors in mice. The images show xenograft sites (blue arrow) and representative tumors. The number of xenografts resulting in tumor formation per the total number of xenografts with each cell type is shown on the right.