The subterranean mole rat Spalax is an excellent model for studying adaptation of a mammal toward chronic environmental hypoxia. Neuroglobin (Ngb) and cytoglobin (Cygb) are O ₂ -binding respiratory proteins and thus candidates for being involved in molecular hypoxia adaptations of Spalax . Ngb is expressed primarily in vertebrate nerves, whereas Cygb is found in extracellular matrix-producing cells and in some neurons. The physiological functions of both proteins are not fully understood but discussed with regard to O ₂ supply, the detoxification of reactive oxygen or nitrogen species, and apoptosis protection. Spalax Ngb and Cygb coding sequences are strongly conserved. However, mRNA and protein levels of Ngb in Spalax brain are 3-fold higher than in Rattus norvegicus under normoxia. Importantly, Spalax expresses Ngb in neurons and additionally in glia, whereas in hypoxia-sensitive rodents Ngb expression is limited to neurons. Hypoxia causes an approximately 2-fold down-regulation of Ngb mRNA in brain of rat and mole rat. A parallel regulatory response was found for myoglobin (Mb) in Spalax and rat muscle, suggesting similar functions of Mb and Ngb. Cygb also revealed an augmented normoxic expression in Spalax vs. rat brain, but not in heart or liver, indicating distinct tissue-specific functions. Hypoxia induced Cygb transcription in heart and liver of both mammals, with the most prominent mRNA up-regulation (12-fold) in Spalax heart. Our data suggest that tissue globins contribute to the remarkable tolerance of Spalax toward environmental hypoxia. This is consistent with the proposed cytoprotective effect of Ngb and Cygb under pathological hypoxic/ischemic conditions in mammals.

Background: Subterranean blind mole rats(Spalax)are hypoxia tolerant (down to 3% O₂), long lived (>20 years) rodents showing no clear signs of aging or aging related disorders. In 50 years ofSpalaxresearch, spontaneous tumors have never been recorded among thousands of individuals.Here we addressed the questions of (1) whetherSpalaxis resistant to chemically-induced tumorigenesis, and (2) whether normal fibroblasts isolated fromSpalaxpossess tumor-suppressive activity.Results: Treating animals with 3-Methylcholantrene (3MCA) and 7,12-Dimethylbenz(a) anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA), two potent carcinogens, confirmedSpalaxhigh resistance to chemically induced cancers. While all mice and rats developed the expected tumors following treatment with both carcinogens, amongSpalaxno tumors were observed after DMBA/TPA treatment, while 3MCA induced benign fibroblastic proliferation in 2Spalaxindividuals out of12, and only a single animal from the advanced age group developed malignancy 18 months post-treatment. The remaining animals are still healthy 30 months post-treatment.In vitroexperiments showed an extraordinary ability of normalSpalaxcultured fibroblasts to restrict malignant behavior in a broad spectrum of human-derived and in newly isolatedSpalax3MCA-induced cancer cell lines. Growth of cancer cells was inhibited by either direct interaction withSpalaxfibroblasts or with soluble factors released into culture media and soft agar. This was accompanied by decreased cancer cell viability, reduced colony formation in soft agar, disturbed cell cycle progression, chromatin condensation and mitochondrial fragmentation. Cells from another cancer resistant subterranean mammal, the naked mole rat, were also tested for direct effect on cancer cells and, similar toSpalax, demonstrated anti-cancer activity. No effect on cancer cells was observed using fibroblasts from mouse, rat orAcomys.Spalaxfibroblast conditioned media had no effect on proliferation of noncancerous cells.Conclusions: This report provides pioneering evidence thatSpalaxis not only resistant to spontaneous cancer but also to experimentally induced cancer, and shows the unique ability ofSpalaxnormal fibroblasts to inhibit growth and kill cancer cells, but not normal cells, either through direct fibroblast-cancer cell interaction or via soluble factors. Obviously, along with adaptation to hypoxia,Spalaxhas evolved efficient anti-cancer mechanisms yet to be elucidated. Exploring the molecular mechanisms allowingSpalaxto survive in extreme environments and to escape cancer as well as to kill homologous and heterologous cancer cells may hold the key for understanding the molecular nature of host resistance to cancer and identify new anti-cancer strategies for treating humans.

Heparanase is an endoglycosidase that degrades heparan sulfate (HS) at the cell surface and in the extracellular matrix. Heparanase is expressed mainly by cancer cells, and its expression is correlated with increased tumor aggressiveness, metastasis, and angiogenesis. Here, we report the cloning of a unique splice variant (splice 36) of heparanase from the subterranean blind mole rat (Spalax). This splice variant results from skipping part of exon 3, exons 4 and 5, and part of exon 6 and functions as a dominant negative to the wild-type enzyme. It inhibits HS degradation, suppresses glioma tumor growth, and decreases experimental B16–BL6 lung colonization in a mouse model. Intriguingly,Spalaxsplice variant 7 of heparanase (which results from skipping of exon 7) is devoid of enzymatic activity, but unlike splice 36 it enhances tumor growth. Our results demonstrate that alternative splicing of heparanase regulates its enzymatic activity and might adapt the heparanase function to the fluctuating normoxic–hypoxic subterranean environment thatSpalaxexperiences. Development of anticancer drugs designed to suppress tumor growth, angiogenesis, and metastasis is a major challenge, of which heparanase inhibition is a promising approach. We anticipate that the heparanase splicing model, evolved during 40 million years of Spalacid adaptation to underground life, would pave the way for the development of heparanase-based therapeutic modalities directed against angiogenesis, tumor growth, and metastasis.

The blind subterranean mole rat (Spalax ehrenbergi superspecies) is a model animal for survival under extreme environments due to its ability to live in underground habitats under severe hypoxic stress and darkness. Here we report the transcriptome sequencing of Spalax galili, a chromosomal type of S. ehrenbergi. cDNA pools from muscle and brain tissues isolated from animals exposed to hypoxic and normoxic conditions were sequenced using Sanger, GS FLX, and GS FLX Titanium technologies. Assembly of the sequences yielded over 51,000 isotigs with homology to ∼12,000 mouse, rat or human genes. Based on these results, it was possible to detect large numbers of splice variants, SNPs, and novel transcribed regions. In addition, multiple differential expression patterns were detected between tissues and treatments. The results presented here will serve as a valuable resource for future studies aimed at identifying genes and gene regions evolved during the adaptive radiation associated with underground life of the blind mole rat.

Background: The development of complex responses to hypoxia has played a key role in the evolution of mammals, as inadequate response to this condition is frequently associated with cardiovascular diseases, developmental disorders, and cancers. Though numerous studies have used mice and rats in order to explore mechanisms that contribute to hypoxia tolerance, these studies are limited due to the high sensitivity of most rodents to severe hypoxia. The blind subterranean mole ratSpalaxis a hypoxia tolerant rodent, which exhibits unique longevity and therefore has invaluable potential in hypoxia and cancer research.Results: Using microarrays, transcript abundance was measured in brain and muscle tissues fromSpalaxand rat individuals exposed to acute and chronic hypoxia for varying durations. We found thatSpalaxglobal gene expression response to hypoxia differs from that of rat and is characterized by the activation of functional groups of genes that have not been strongly associated with the response to hypoxia in hypoxia sensitive mammals. Using functional enrichment analysis ofSpalaxhypoxia induced genes we found highly significant overrepresentation of groups of genes involved in anti apoptosis, cancer, embryonic/sexual development, epidermal growth factor receptor binding, coordinated suppression and activation of distinct groups of transcription factors and membrane receptors, in addition to angiogenic related processes. We also detected hypoxia induced increases of different criticalSpalaxhub gene transcripts, including antiangiogenic genes associated with cancer tolerance in Down syndrome human individuals.Conclusions: This is the most comprehensive study ofSpalaxlarge scale gene expression response to hypoxia to date, and the first to use customSpalaxmicroarrays. Our work presents novel patterns that may underlie mechanisms with critical importance to the evolution of hypoxia tolerance, with special relevance to medical research.