Potential Mechanisms for Cancer Resistance in Elephants

Potential Mechanisms for Cancer Resistance in Elephants and Comparative Cellular Response to DNA Damage in Humans
Lisa M. Abegglen, PhD, Aleah F. Caulin, PhD, [...], and Joshua D. Schiffman, MD

Additional article information

Abstract
IMPORTANCE
Evolutionary medicine may provide insights into human physiology and pathophysiology, including tumor biology.

OBJECTIVE
To identify mechanisms for cancer resistance in elephants and compare cellular response to DNA damage among elephants, healthy human controls, and cancer-prone patients with Li-Fraumeni syndrome (LFS).

DESIGN, SETTING, AND PARTICIPANTS
A comprehensive survey of necropsy data was performed across 36 mammalian species to validate cancer resistance in large and long-lived organisms, including elephants (n = 644). The African and Asian elephant genomes were analyzed for potential mechanisms of cancer resistance. Peripheral blood lymphocytes from elephants, healthy human controls, and patients with LFS were tested in vitro in the laboratory for DNA damage response. The study included African and Asian elephants (n = 8), patients with LFS (n = 10), and age-matched human controls (n = 11). Human samples were collected at the University of Utah between June 2014 and July 2015.

EXPOSURES
Ionizing radiation and doxorubicin.

MAIN OUTCOMES AND MEASURES
Cancer mortality across species was calculated and compared by body size and life span. The elephant genome was investigated for alterations in cancer-related genes. DNA repair and apoptosis were compared in elephant vs human peripheral blood lymphocytes.

RESULTS
Across mammals, cancer mortality did not increase with body size and/or maximum life span (eg, for rock hyrax, 1% [95%CI, 0%–5%]; African wild dog, 8%[95%CI, 0%–16%]; lion, 2%[95%CI, 0% –7%]). Despite their large body size and long life span, elephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95%CI, 3.14%–6.49%), compared with humans, who have 11% to 25%cancer mortality. While humans have 1 copy (2 alleles) of TP53, African elephants have at least 20 copies (40 alleles), including 19 retrogenes (38 alleles) with evidence of transcriptional activity measured by reverse transcription polymerase chain reaction. In response to DNA damage, elephant lymphocytes underwent p53-mediated apoptosis at higher rates than human lymphocytes proportional to TP53 status (ionizing radiation exposure: patients with LFS, 2.71% [95%CI, 1.93%–3.48%] vs human controls, 7.17%[95%CI, 5.91%–8.44%] vs elephants, 14.64%[95%CI, 10.91%–18.37%]; P < .001; doxorubicin exposure: human controls, 8.10% [95%CI, 6.55%–9.66%] vs elephants, 24.77%[95%CI, 23.0%–26.53%]; P < .001).

CONCLUSIONS AND RELEVANCE
Compared with other mammalian species, elephants appeared to have a lower-than-expected rate of cancer, potentially related to multiple copies of TP53. Compared with human cells, elephant cells demonstrated increased apoptotic response following DNA damage. These findings, if replicated, could represent an evolutionary-based approach for understanding mechanisms related to cancer suppression.

The mechanisms that prevent accumulation of genetic damage and subsequent uncontrolled proliferation of somatic cells in multicellular organisms remain poorly understood. A greater number of cells and cell divisions increases the chance of accumulating mutations resulting in malignant transformation.1 If all mammalian cells are equally susceptible to oncogenic mutations, then cancer risk should increase with body size (number of cells) and species life span (number of cell divisions). The Peto paradox describes the observation that cancer incidence across animals does not appear to increase as theoretically expected for larger body size and life span.2,3 To our knowledge, the cellular mechanism for this phenomenon of cancer resistance has never been demonstrated experimentally in organisms other than rodents.4–6

TP53 (encoding the protein p53 [RefSeq NM_000546]) is a crucial tumor suppressor gene, mutated in the majority of human cancers.7 Referred to as the “guardian of the genome,” inactivation of p53 leads to 3 cancer cell characteristics including suppression of apoptosis, increased proliferation, and genomic instability.8,9 Humans contain 1 copy (2 alleles) of TP53, and both functioning alleles are crucial to prevent cancer development. Absence of 1 functional allele leads to Li-Fraumeni syndrome (LFS), a cancer predisposition with more than a 90% lifetime risk for cancer, multiple primary tumors, and early childhood cancers.10,11 Understanding the cellular mechanism of cancer suppression in animals could benefit humans at high risk of cancer, such as patients with LFS, and even the healthy, aging population.

This study investigated the cancer rate in different mammals (including elephants), identified potential molecular mechanisms of cancer resistance, and compared response to DNA damage in elephants with that in healthy human controls and individuals with LFS.

Methods
Ethical and scientific institutional review board approval was obtained from each participating research organization for all elephant and human participation, including written informed consent from human participants. Experiments were performed on peripheral blood lymphocytes (PBLs) from African and Asian elephants, from a representative clinical cohort of patients with LFS enrolled in a separate study (the Cancer Genetics Study, University of Utah), and from age-matched human controls without a significant family history of cancer also enrolled in the Cancer Genetics Study. Patients with LFS were selected for inclusion as a representative sample based on TP53 mutation status, varied cancer history, and availability for blood draw. Human subject materials were collected at the University of Utah from June 2014 to July 2015. Laboratory experiments were also performed on African elephant fibroblasts, human fibroblasts, and HEK293 cells to confirm these findings.

Necropsy data were examined from zoo animals to determine if empirical evidence supports that cancer incidence does not increase with body size or life span. Fourteen years of necropsy data collected by the San Diego Zoo12 was compiled and tumor incidence was calculated for 36 mammalian species, spanning up to 6 orders of magnitude in size and life span.13 Data from the Elephant Encyclopedia14 were analyzed on the cause of death in captive African (Loxodonta africana) and Asian (Elephas maximus) elephants to estimate age incidence and overall lifetime cancer risk. Using the cancer transformation model from Calabrese and Shibata,15 the percentage decrease in cellular mutation rate was calculated to account for a 100× increase in cell mass (the difference between elephants and humans) without cancer development.

Genomic sequence analysis was next performed on the publicly available scaffolds of the African elephant genome in the Ensembl database (release 72; http://www.enssemble.org/) and the NCBI Gene database (http://www.ncbi.nlm.nih.gov/gene), with examination of cancer-related genes including oncogenes and tumor suppressors. TP53 sequence alignments were explored in related species, and African and Asian elephant TP53 retrogenes were cloned and resequenced. Capillary sequencing was performed on single elephants to avoid issues of single-nucleotide polymorphisms between elephants. Whole genome sequencing (Illumina HiSeq 2500) was performed on freshly extracted DNA from an African elephant at 40× average sequence coverage, with more than 100× coverage within areas of TP53.

Functional molecular analysis of TP53 and its retrogenes was performed on peripheral blood mononuclear cells from African and Asian elephants and fibroblasts from an African elephant. To determine if TP53 retrogenes are expressed in the elephant, reverse transcription–polymerase chain reaction was performed on RNA collected from African elephant peripheral blood mononuclear cells and African elephant fibroblasts. Polymerase chain reaction primers were designed to distinguish the TP53 retrogenes from the ancestral sequence and splice variants. Human vs elephant DNA repair efficiency (measured by double-strand breaks indicated by number of phospho-histone H2AX [pH2AX] foci), apoptosis (annexin V [AV] and propidium iodide [PI] by flow cytometry and Apotox-Glo, Promega), and cell cycle arrest (Apotox-Glo, Promega) were compared at different time points (1, 5, 10, 18, 24, and 72 hours) after DNA damage (doxorubicin, 0.005–30 µM; and ionizing irradiation, 0.5, 2, 5, 6, 10, and 20 Gy). Late apoptosis was defined as AV+PI+ and early apoptosis was defined as AV+PI−. Experiments were performed in either triplicate or quadruplicate. p53 plays a critical role in p21 and mouse double minute 2 homolog (Mdm2 or E3 ubiquitin-protein ligase Mdm2) protein induction following DNA damage,16,17 so p21 immunoblots were performed to validate a p53-dependent DNA damage response in elephant cells. p53 retrogene 9 (GenBank KF715863) was cloned into an expression vector to produce a protein fused to an epitope from the Myc protein. HEK293 cells were transfected with this Myc-tagged p53 retrogene 9 expression vector and p53 retrogene protein expression was measured by immunoblot using an antibody to the Myc tag. Retrogene protein product was co-immunoprecipitated from HEK293 cell lysates with Myc antibody, followed by immunoblots for phospho-p53 (serine-15) and Mdm2. The HEK293 cell line was chosen for these experiments because it is a human cell line (human embryonic kidney) that is easy to transfect and measure protein expression.

Cross-species lifetime cancer incidence was estimated by the number of animals in each species that reportedly died of cancer. A logistic regression model was fit to determine if body mass and maximum life span are variables associated with cancer incidence (R software, version 3.2.1). Additionally, all combinations of mass, life span, and mass-specific basal metabolic rate were examined for evidence of cancer association. An inverse cancer association was specifically tested in the largest existing terrestrial mammal, the elephant. For the DNA damage analysis, a χ2 test was used to compare pH2AX foci, and an unpaired 2-sided t test with α = .05 was used for apoptosis and cell cycle arrest (R software, version 3.2.1, and Graph-Pad Prism, version 6.0e). Both a linear regression and a Jonckheere-Terpstra test were used to assess if apoptotic response decreased with age.

Details of the experimental methods are further described in the eAppendix in the Supplement.

Results
Zoo Necropsies and Cancer Mortality
The 36 mammalian species analyzed spanned from the striped grass mouse (weight, 51 g, with a maximum life span of 4.5 years) to the elephant (weight, 4800 kg, with a maximum life span of 65 years). Cancer risk did not increase with mammalian body size and maximum life span among 36 species analyzed (eg, for rock hyrax, 1% [95% CI, 0%–5%]; African wild dog, 8% [95% CI, 0%–16%]; lion, 2% [95% CI, 0%–7%]) (Figure 1). No significant relationship was found with any combinations of mass, life span, and basal metabolic rate and cancer incidence (eFigure 1 and eTable 1 in the Supplement). Among 644 annotated elephant deaths from the Elephant Encyclopedia database, the lifetime cancer incidence was 3.11% (95% CI, 1.74%–4.47%) (Table 1). To obtain a more conservative estimate, an inferred cancer incidence was calculated for cases that lacked adequate details for the cause of death, leading to an estimated elephant cancer mortality rate of 4.81% (95% CI, 3.14%–6.49%). Based on an algebraic model of carcinogenesis,15 a 2.17-fold decrease in mutation rate was calculated as sufficient to protect elephants from cancer development given their 100× increased cellular mass compared with humans.