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

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 housemouse has amaximum lifespan of 4 years. In addition to their longevity, nakedmole 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 amechanism responsible for the nakedmole rat’s cancer resistance.We found thatnakedmole-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 ofHA-degrading enzymes and aunique 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 comparedwithmouse or human cells. Perturbation of the signalling pathways sufficient for malignant transformation of mouse fibroblasts fails to transform naked mole-rat cells. However, once highmolecular-mass HA is removed by either knocking down HAS2 or overexpressing theHA-degrading enzyme, HYAL2, nakedmole-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. Mice and rats are standard animalmodels for cancer research due in part to their short lifespan and high incidence of cancer. However, these traits indicate that mice and rats have fewer anticancer mechanisms, and novel tumour resistance mechanisms are less likely to be discovered using these models. Here we focused our research on a small rodent, the naked mole rat, which in contrast to mice and rats is long-lived and cancer resistant. Our previous studies identified a novel anticancermechanism in the nakedmole rat, termed early contact inhibition (ECI). Contact inhibition is a process of arresting cell growth when cells come in to contact with each other or the extracellularmatrix. Contact inhibition is a powerful anticancer mechanism that is lost in cancer cells. Naked mole-rat cells arrest at amuch lower density thanmouse cells, and the loss of ECI makes cells more susceptible to malignant transformation. However, the signals triggering ECI in naked mole rats remained unknown. While culturing multiple lines of naked mole-rat fibroblasts we noticed that the culture media became very viscous after a few days. Viscosity measurements confirmed that the media conditioned by the naked mole-rat cells was more viscous than the media conditioned by human, guinea-pig or mouse cells (Fig. 1a). We included the guineapig because it is phylogenetically closer to the naked mole rat than the mouse. We identified the viscous ‘substance’ secreted by the naked mole-rat fibroblasts as high-molecular-mass HA (HMM-HA). Treatment with hyaluronidase (HAase), which specifically digests HA, reduced the media viscosity to background levels (Fig. 1a). Naked molerat embryonic fibroblasts, which do not display ECI, did not increase viscosity of the culture media (Fig. 1a and Supplementary Fig. 1). HA is anunbrancheddisaccharide glucuronic acid/N-acetylglucosamine polymer and is one of themain components of the extracellularmatrix. Biological responses triggered byHAdependon theHApolymer length. HMM-HA represses mitogenic signalling and has anti-inflammatory properties, whereas low-molecular-mass HA promotes proliferation and inflammation. Analysis of HA from tissue culture media using pulse-field electrophoresis showed that the HA secreted by naked mole-rat cells has a molecular mass of 6–12MDa, whereas mouse and guinea-pig HA range from 0.5 to 3MDa (Fig. 1b); human HA has a molecular mass of 0.5–2MDa. Naked mole-rat embryonic fibroblasts did not secrete HMM-HA (Fig. 1b). Notably, a mutated clone NMR SF Mut (mutated naked mole-rat skin fibroblasts), which spontaneously lost the ECI phenotype and p16 expression, still produced HMM-HA (Fig. 1a, b), indicating that the physical presence of HMM-HA is not sufficient for the ECI phenotype; rather, the intact signalling pathway leading from HMMHA to induction of p16 INK4a is required. These experiments establish HMM-HA as the extracellular signal that triggers ECI. In vertebrate cells,HA is producedbyHAsynthasesHAS1,HAS2 and HAS3 that differ in tissue distribution and the size of HA produced. Nakedmole-rat skin fibroblasts overexpressHAS2, the enzyme responsible for the synthesis of HMM-HA, in comparison with mouse and human fibroblasts (Fig. 1c). Nakedmole-rat embryonic fibroblasts, which do not secrete HMM-HA, did not show increased levels of HAS2. The levels of HAS1 and HAS3 were similar between mouse, human and nakedmole-rat cells (Fig. 1c). Collectively, these results show thatnaked mole-rat cells, which display ECI, secrete HA of exceptionally high molecular mass. Hyaluronan synthases are highly conserved in vertebrates. The HAS2 protein has 98.7% identity and 100% similarity between human and mouse. We cloned and sequenced HAS2 complementary DNA from the naked mole rat and compared it to other mammalian Has2 genes (Fig. 1d). Two asparagines that are 100% conserved among mammals were replaced with serines in the naked mole-rat HAS2. This change occurs in no other mammalian Has2 genes deposited in GenBank, including the close relative of the nakedmole rat, the guineapig. HAS2 contains seven putative transmembrane domains and a cytoplasmic loop. The conserved regions carrying asparagine to serine substitutions correspond to the cytoplasmic loop containing the enzyme’s active site. These unique amino acid changesmaybe responsible for the high processivity of the nakedmole-ratHAS2. Indeed, when the cDNA for the naked mole-rat HAS2 was overexpressed in human HEK293 cells, they began secreting HMM-HA (Fig. 2a).