Notwithstanding the importance of laboratory mice in comprehension of carcinogenesis mechanisms, this cancer-prone model organism failed to provide satisfactory knowledge of cancer preventive mechanisms and treatment strategies in humans. (http://www.safermedicines.org/quotes/cancer.shtml). Therefore, elucidating mechanisms employed by a wild, non-inbred mammal that is naturally cancer-resistant raises promising opportunities.
In vivo studies of carcinogen-induced tumor
We report here that Spalax is resistant to two-stage DMBA/TPA, and 3-MCA carcinogen treatments. DMBA/TPA is commonly used to study malignant transformation, resembling formation of human squamous cell carcinoma . A single dose of DMBA induced substantial oxidative stress , and when followed by repetitive application of TPA led to persistent inflammation supporting tumorigenesis . In the present study, mice treated with DMBA/TPA initially developed benign papillomas, which subsequently transformed to squamous cell carcinomas. In contrast, treatment of Spalax led to necrotic wounds, which completely healed with no signs of malignancy. The carcinogen 3-MCA is known to produce fibrosarcomas through persistent inflammation and reactive metabolites causing severe oxidative damage . In our study, 100% of 3-MCA-injected mice and rats developed tumors at the injection site within two to three and four to six months, respectively. One year after 3MCA treatment no Spalax animals showed any pathological process. However, 2 out of 6 old individuals (from a total of 12 animals) developed benign fibrotic overgrowths after 14 and 16 months, respectively, and only one case of malignant transformation in a >10 year-old Spalax animal was recognized, 18 months after 3-MCA injection.
It is well established that oxidative stress drives tumor progression and metastasis . Thus, the mechanisms that Spalax evolved to survive hypoxia might be related to resistance to induced or spontaneous cancers. Spalax have recently been shown to have higher levels of reactive oxygen species (ROS) processing enzymes compared to hypoxia-intolerant rodents . Nrf2, a transcription factor critical for defense against oxidative stress, has a unique structure in Spalax. Whereas it is highly conserved among most mammals , Spalax Nrf2 carries 27 specific amino acid replacements, 6 within the Neh6-domain, which is critical for stabilizing the protein under ambient oxidative stress and for its transcriptional activity . Studies performed on Nrf2−/− mice have shown the essential role of Nrf2 for detoxification of DMBA metabolites and protection against DMBA-induced carcinogenesis . Unraveling the molecular mechanisms resulting in the healing of Spalax skin and inhibition of progression to tumor formation is the goal of our ongoing research. Hence, we have just initiated a comprehensive repetition of DMBA/TPA treatment where we will have a representative sample of animals from different stages following the application of this carcinogen in order to answer this question through quantification of apoptosis and senescence of Spalax skin and muscle tissue at the area of the carcinogen application. Furthermore, considering the high tolerance of Spalax to oxidative stress and the fact that DMBA is metabolized among others into ROS that cause oxidative DNA damage in the skin , the above experiment will allow us to compare the ROS levels upon DMBA application in Spalax and mice.
Another antioxidant enzyme, heme oxygenase-1 (HO-1), was shown to be elevated in Spalax tissues, and further increased under hypoxia [4, 11]. HO-1 is involved in the degradation and catabolism of heme and supports synthesis of ferritin, an iron storage protein, thus preventing oxidative damage caused by free heme and ROS . Indeed, most Spalax individuals, showing no external lesions following exposure to carcinogens, have probably resolved the initial inflammatory insult without excessive fibroplasias which can be attributed to more efficient anti-oxidation mechanisms. The benign fibroblastic proliferations observed in two 3-MCA-treated Spalax animals after 14 and 16 months suggest that Spalax is able to effectively arrest cancerous transformation. Nonetheless, whether Spalax tissues are able to prevent conversion of the 3-MCA pro-carcinogen into an active carcinogen, overcome its effect, or to inhibit previously transformed cells, remains to be clarified in future studies.
In vitro studies of Spalax cancer resistance
Tumor growth and invasion are dependent on growth factors and cytokines produced by stromal cells . Normal stroma contains a relatively small number of fibroblasts associated with ECM. However, during wound healing, fibrosis or malignant transformations, stromal fibroblasts proliferate, intensively produce growth factors and cytokines, express α-smooth muscle actin and become cancer-associated fibroblasts (CAFs) [12, 34, 35]. CAFs are removed from the granulation tissue rapidly after healing, though in cancer stroma they persist, contributing to epithelial mesenchymal transition of cancer cells. The later phenomenon is important for cancer progression and is mediated, at least in part, by metalloproteinases secretion and ROS generation [36, 37]. Although the reports published to date have been mainly addressed to a cancer-promoting role of stromal fibroblasts, evidence suggests that normal stroma and normal fibroblasts could impede tumorigenesis [14–16, 38, 39]. Early studies  demonstrated that normal dermal fibroblasts suppressed development of malignant phenotypes of RAS-transformed keratinocytes when grafted into animals. Similarly, normal fibroblasts were able to retard melanomagenesis in its early stages . Inhibition of growth and induction of differentiation were found in breast cancer pre-neoplastic MCF10-AT1-EIII8 cells when co-cultivated with normal fibroblasts, even in the presence of estrogen . It is still unclear what events in the stroma, along with its interaction with precancerous cells, lead to a transition of the stromal function from cancer-protective to cancer-promoting, or, as in the present case of Spalax, what are the molecular mechanisms that Spalax evolved to escape cancerous transformation and to develop anti-cancer ability.
In a recent study , cancer resistance in Spalax was discussed. It was suggested that pro-growth signals originating from the fetal bovine serum, routinely added to culture medium, are conceived as cancerous transformation-like stimuli, driving Spalax fibroblast necrotic death, triggered through release of interferon-β (IFN-β). Nonetheless, in the same study, higher and earlier death rates were also shown in serum-reduced or serum-free media. Furthermore, the possibility that CM from “dying” cells may lack beneficial nutrients, or contain toxic metabolites, or other factors beyond IFN-β, was not addressed. Additionally, measurements of IFN-β in Spalax CM were performed indirectly using human cell lines . The first, VSV (Vesicular Stomatitis Virus)-GFP (encoding a Green Fluorescent Protein) gene assay, measures IFN-β expression levels by VSV-GFP reporter assay. In this assay, HT1080 cell line (human fibrosarcoma cells) had been incubated with Spalax CM, and then infected with a GFP-encoding VSV. The level of IFN-β in the media corresponds to the reduction in the number of GFP positive human HT1080 cells. In the second assay, IFN-β release by “dying” Spalax cells is determined by HEK (Human Embryo Kidney cells)-Blue cells assay. In this assay the induction of β-gal reporter in human EK cells under IFN-β-inducible promoter is measured. Both assays use human cells for indirectly measuring Spalax IFN-β, which is inconsistent with the authors’ declaration that human cells are nonresponsive to Spalax CM stimuli possibly due to species divergence of IFN-β . Likewise, no proof was given that the ability to kill “dying” fibroblasts is unique to Spalax’s CM, for example, by trying to compare the fate of the cells when grown with CM of the other species tested in the study, namely, mice or human. Additionally, the method used in this study for declaring necrotic death is based on the Annexin V/propidium iodide assay . Briefly, floating and adherent cells were harvested, stained with Annexin-V and propidium iodide, and analyzed by flow cytometery. The known disadvantage of this method is that it cannot conclusively prove that cell death is solely the result of necrosis, nor eliminate the possibility of apoptotic mechanisms. Also, the authors have not provided evidence for interrelations between their three declared observations (transformation-like stimuli, necrotic death and release of IFN-β). Overall, it is our impression that the above mentioned study  does not provide direct evidence to Spalax cancer resistance, certainly not its anti-tumor properties. Alternatively, we show here that viable, proliferating Spalax fibroblasts, from adult and newborn animals, inhibit growth of cancer cells derived from different tissues and species, most importantly human, but do not affect non-cancerous cells, including those of Spalax (Figure 7F), thereby highlighting a strategy used by Spalax to identify and target malignancies. This unique interaction is further strengthened by the observation that the growth of cancer cells is regained once the immediate interaction with Spalax cells is terminated (Figure 7D). Importantly, no inhibitory effect on cancer cell growth was found when fibroblasts from above-ground species (rat, mice and Acomys) were tested.
Recently, several studies investigated the unique cancer-resistance properties of the naked mole rat (Heterocephalus glaber), another subterranean, long-lived, rodent species. The most recent study suggested a connection between a high viscosity of media conditioned by Heterocephalus fibroblast cells due to exceptional secretion of high-molecular mass hyaluronan (HMM-HA) , which was suggested to mediate what was named by the authors “early contact inhibition”, previously described by the same group as an anticancer mechanism in Heterocephalus cells, and was initially ascribed to p16(Ink4a) and p27(Kip1) activity . In the same paper , it is reported that HMM-HA was detected also in Spalax fibroblasts even in higher levels compared to Heterocephalus fibroblasts, though no experiments were carried out to clarify its role in Spalax fibroblasts. Nevertheless, this may explain the prevalent high viscosity of the medium of cultured Spalax fibroblasts we noticed, though we find that it does not prevent Spalax cells from reaching confluence or influences their anti-cancer properties. Furthermore, CM from Spalax with apparent normal viscosity was also able to inhibit cancer cells proliferation (ongoing study). In light of the fact that hyaluronan-cancer cell interactions were shown to promote, and not inhibit, cancer invasion , the correlation between HMM-HA, the potential of cells to reach confluence and the resistance to oncogenic transformation or anti-cancer activity, requires further direct experimental support, especially in the case of our model organism, the Spalax. Another study endorsed Heterocephalus cells’ cancer-resistance to rapid cell crisis following oncogenic transformation, which is characterized by abnormal chromatin material and nuclei, leading to a failure to successfully complete cell division, hence the inability of the cells to progress into malignancy . These observations are somewhat similar to our findings of fragmented and deformed nuclei and chromatin condensation (Figure 8), disturbed cell division and proliferation (Figure 9E) of human cancer cells, as well as the 3MCA-induced Spalax and mice fibrosarcoma cell line, upon their interaction with Spalax fibroblasts. In view of the similar ability of Heterocephalus fibroblasts to kill cancer cells (Figure 6), and as the efficiency of experimental oncogenic transduction of cells is never 100%, it is possible that the Heterocephalus cells that escaped malignant transformation killed the oncogenic-transduced ones.
Our findings demonstrated that Spalax fibroblasts or their CM target human cancer cells growth machinery, triggering programmed cancer cell death (Figures 4, 5, 8 and 9). Following co-culture with Spalax fibroblasts or their CM, cancer cells (Hep3B, HepG2 and MCF7) undergo morphological changes typical of apoptosis : swelling, rounding, detachment, shrinkage and floating. Moreover, nuclear condensation and abnormal mitochondrial fission as well as accumulation of cells in sub-G1 (Figures 8 and 9) also suggest apoptotic modes of cancer cell death. BrdU incorporation, reflecting cell proliferation, confirmed that Spalax CM contains anti-proliferative factors, inhibiting cell division in a time-dependent pattern. We further showed that the effect of Spalax CM on cancer cells is transient and reversible. That is, replacing the CM with regular fresh medium leads to recovery of those cancer cells that had not been affected by the CM. Last but not least, Spalax fibroblasts presumably impair the aggressive behavior of tumor cells: the invasive phenotype of highly metastatic MDA-MB-231 breast carcinoma cells was markedly reduced (Figure 10). Noteworthy, the ability to form colonies in soft agar by 3MCA-induced, Spalax-derived fibrosarcoma was significantly suppressed by homologous fibroblasts, whereas heterologous fibroblasts (rat and mouse) increased tumor formation (Figure 11). Spalax fibroblasts also inhibited colony formation in soft agar by 3MCA-induced, mouse-derived fibrosarcoma.
In order to strengthen our findings of Spalax cells’ anti-cancer activity, compared to cells from laboratory, in-bred, aboveground mice and rats, we decided to follow the cancer activity pattern of two other, wild, out-bred, species. Hence, fibroblast cells were propagated from the aboveground, wild, short-lived rodent Acomys, and the subterranean, wild, long-lived Heterocephalus. We have shown here that, similar to Spalax cells, Heterocephalus fibroblasts restrict growth and effectively kill cancer cells, while Acomys cells behave similarly to rat and mice, that is, have no anti-cancer activity (Figure 6). We may assume that this anti-cancer ability might be shared by species living under extreme conditions and adapted to stress, such as hypoxia, which is directly related to cancer initiation and progression. It would be interesting to investigate this phenomenon in other hypoxia-tolerant species, such as other subterranean, high altitude and diving mammals.
Previous studies showed that key hypoxia-regulatory genes in stromal fibroblasts, such as HIF1-α and VEGF, negatively influence tumorigenesis . HIF1-α is a known tumor-promoting transcription factor in most malignancies ; however, its expression in tumor stromal fibroblasts could suppress cancer cell growth . We have previously shown that HIF1-α as well as ROS-scavenging enzymes  are constitutively highly expressed in Spalax. Similar to our explanations of the failure to induce cancer in vivo in live Spalax animals, in vitro studies, using fibroblast cells, demonstrated a significant role in adaptive response to oxidative stress, at least in part, via expression of HO-1 . High levels of mitochondrial ROS produced by cancer cells were shown to drive tumor development via remodeling of the stromal environment and enhancing invasion. Recently, the roles of ROS produced by fibroblasts in their trans-differentiation to myofibroblasts and in cancer cell invasiveness were reported . ROS-generating CM of mutated fibroblasts promoted metastasis of A375 melanoma through the increasing of ROS and HIF1-α stabilization in melanoma cells. However, when N-acetyl cysteine, a ROS scavenger, was added to the system, HIF1-α accumulation and melanoma cell invasion were inhibited .
Adaptive tolerance to hypoxia stress in Spalax, both in vivo and in vitro, may grant the unique resistance to cancer through strong antioxidant mechanisms, among others (for example, as mentioned here, the unique activity of its p53  and heparanse ), that quench ROS before they spread and damage DNA and other macromolecules, thus providing cellular homeostasis and cancer protection. As such, they are a milestone in our efforts in understanding the mechanisms by which the long-lived, hypoxia-tolerant Spalax hinders cancer initiation and progression.
Collectively, we have shown here an outstanding cancer resistance of the whole, live Spalax, and not just in cultured cells, and anticancer activity of Spalax cells on human cancer cells, and not just resisting transformation of its own healthy cells. This phenomenon extensively described here using different methodologies on cells from different ages of Spalax, together with our initial observation of a similar ability of cells originated from another subterranean, long-lived, hypoxia- and cancer-resistant animal, the Heterocephalus, highlight the importance to adopt such animal models with exceptional genetic-embedded tolerance to environmental stress, in cancer research.
Our ongoing research is focused on identifying the factors secreted by Spalax cells, and their selective interaction with cancer cells to suppress tumorigenesis. Our first step to exploring the nature of the secreted factors was the heat-inactivation preliminary experiment presented here (Figure 7G). The heat treatment of Spalax CM caused only partial loss of the anti-cancer activity of Spalax-generated CM. Although not conclusive, this may indicate the involvement of protein factors in the observed phenomenon. We are also studying the signaling mechanisms and death receptors whose activation triggers cancer cell death. These studies will hopefully contribute to the identification of new anti-cancer mechanisms and future tumor preventive or therapeutic strategies. To our knowledge, the present study demonstrates, for the first time, Spalax tolerance to chemically induced carcinogenesis along with direct anticancer effect of Spalax fibroblasts on human cancer cells.