Experimental evidence for cancer resistance in a bat species

Bats are exceptionally long-lived relative to their body size and have been hypothesized to possess intrinsic cancer resistance mechanisms, similar to other long-lived mammals like naked and blind mole-rats. Prior molecular studies suggested anticancer adaptations in bats (e.g., altered microRNAs, adaptive changes in growth hormone receptor and p53), but direct experimental evidence remained scarce. This study systematically tests cancer resistance across seven bat species by inducing malignant transformation using oncogenic HRAS(G12V) and SV40 large T antigen in primary fibroblasts, and compares outcomes to mouse cells. The goal is to identify whether any bat species demonstrates resistance to malignant transformation and to uncover underlying molecular mechanisms, with implications for understanding natural cancer resistance and longevity.

Long-lived mammals often evolve cancer resistance mechanisms linked to lifespan, including repressed telomerase activity, reduced proliferation, and enhanced genome stability (e.g., naked mole rats, blind mole rats). In bats, prior evidence includes downregulation of oncogenic microRNAs and upregulation of cancer-resistant microRNAs in Myotis myotis, and adaptive changes in growth hormone receptor and p53 associated with cancer resistance. However, experimental validation of cancer resistance in bats was limited. Comparative oncogenic transformation studies in rodents showed that HRAS(G12V) and SV40 LT readily transform mouse fibroblasts but fail in naked and blind mole-rat fibroblasts, motivating similar assays in bats.

Samples and cell culture: Primary skin fibroblasts were obtained for seven bat species (Myotis pilosus, Myotis altarium, Rhinolophus pusillus, Rhinolophus ferrumequinum, Rhinolophus sinicus, Hipposideros armiger, Rousettus leschenaultii) and laboratory mouse Mus musculus. Cells were maintained in DMEM +10% FBS at 5% CO2, 3% O2, 37°C. Oncogenic transformation: Lentiviral vectors were constructed to stably express HRAS(G12V) and SV40 Large T antigen in each species’ fibroblasts; selection with puromycin generated stable lines. Protein expression of HRAS(G12V) and SV40 LT was verified by immunoblotting to ensure comparable levels across species. Anchorage-independent growth: Soft agar assays (base 0.8% agarose, top 0.4% agar with 2×10^5 cells/well) were incubated 4 weeks and colony areas quantified. In vivo xenografts: Stable lines were infected with luciferase lentivirus, selected with blasticidin, and 1×10^6 cells in saline +30% Matrigel were injected subcutaneously into immunodeficient B-NDG mice. Bioluminescence imaging was performed weekly after D-luciferin administration; tumors were harvested and weighed at 3 weeks or when volume approached ethical limits. Transcriptomics and co-expression: RNA-seq (MGISEQ 2000, PE150) with three biological replicates per species; reads trimmed and assembled (Trinity). FPKM quantified; 6,314 one-to-one orthologs (FPKM>0.5 in >50% samples) were used for WGCNA (signed network, power=12) to identify modules. Differential expression and module eigengenes were compared across species. Network and prognosis analyses: PPI network (STRING) for genes in upregulated module M1 and downregulated module M2; node degree and betweenness centrality computed in Cytoscape. For each gene in M1/M2, KMplot data across 21 tumor types were analyzed for associations between gene expression and patient survival. Functional validation: CRISPR/Cas9 (lentiCRISPRv2-hygro) knockouts of HIF1A, COPS5, RPS3, EP300, EIF5B in mouse HRAS(G12V)+SV40LT fibroblasts; knockout efficiency verified by immunoblotting. Overexpression of bat (MPI) HIF1A, COPS5, RPS3 in MPI fibroblasts using pLenti CMV vectors; effects on proliferation measured by MTT assays; soft agar and xenograft assays conducted for combined overexpression. Genome sequencing and comparative genomics: High-quality genome assembly for MPI generated using Nanopore long reads (206.4 Gb; N50 28.1 kb; polished with NextDenovo/NextPolish; BUSCO complete ~96.3%). Repeats annotated with RepeatModeler/RepeatMasker (~41.04% repetitive). Gene annotation using RNA-seq, homology, and ab initio pipelines, resulting in 20,742 protein-coding genes. Whole-genome alignments across mouse, human, and bat genomes with lastz, chain/net, hal tools. Identification of conserved non-coding elements (CNEs): Neutral model (fourfold degenerate sites) estimated with PHAST tools; conserved elements detected with PhastCons; accelerated evolution on the MPI branch detected with phyloP-ACC (FDR≤0.05), yielding 437,414 CNEs overall and 20,231 accelerated in MPI. Chromatin accessibility and regulatory element mapping: ATAC-seq performed on mouse and MPI fibroblasts; peaks called by MACS2; peaks compared between species using liftOver. Overlap with ENCODE candidate cis-regulatory elements (cCREs) to prioritize putative regulatory regions near HIF1A, COPS5, RPS3. Motif analysis and reporter assays: Predicted TF binding motifs in CNEs using HOMER/FIMO; dual-luciferase assays in HEK293T and NIH3T3 to test enhancer activity of candidate elements (e.g., CNE143336 near HIF1A; a potential regulatory sequence upstream of COPS5, RSC). dCas9-KRAB with sgRNAs used to inhibit enhancer activity of CNE143336; impact on endogenous HIF1A measured by immunoblotting. ChIP-qPCR: Anti-HIF1A ChIP performed in mouse and MPI fibroblasts; qPCR with primers spanning the 17 bp motif region in RSC assessed HIF1A binding enrichment. Additional analyses: Tested proliferation impact of COPS5 knockdown in human epithelial and cancer cell lines (HK-2, MCF-7, PANC-1). Assessed potential relationship between accelerated CNEs and transposable elements.

• MPI fibroblasts resist malignant transformation: Despite comparable expression of HRAS(G12V) and SV40 LT across species, MPI fibroblasts formed markedly smaller soft-agar colonies than mouse and six other bat species (P<0.001; also ****P<0.0001 in figure). MPI fibroblasts from multiple tissues (skin, intestine, tail) consistently failed to form large colonies.
• In vivo resistance: Mouse HRAS(G12V)+SV40LT fibroblasts formed rapidly growing tumors, whereas MPI HRAS(G12V)+SV40LT fibroblasts produced significantly smaller tumors. Bioluminescence was lower for MPI xenografts at weeks 2 and 3 (e.g., P=0.017 and P=0.006), and tumor weights at week 3 were significantly reduced for MPI-derived tumors vs mouse (P=2.21E-05).
• Transcriptomics and co-expression modules: WGCNA identified 34 modules; module M1 upregulated and M2 downregulated in MPI. Hub genes from PPI analysis were HIF1A, EP300, EIF5B, COPS5, RPS3 (all in M2, downregulated in MPI). Lower expression of these, especially HIF1A and EIF5B, associated with improved patient survival across multiple cancer types in KMplot analysis.
• Functional validation of candidate genes: CRISPR suppression of HIF1A, COPS5, and RPS3 significantly inhibited proliferation in mouse HRAS(G12V)+SV40LT fibroblasts (P<0.05), while EP300 and EIF5B knockdown showed no remarkable effect. Overexpression of HIF1A and RPS3 in MPI HRASV40LT fibroblasts increased proliferation (P<0.05); combined overexpression of HIF1A, COPS5, and RPS3 further enhanced proliferation and significantly increased soft agar colony size and xenograft tumor size (P<0.001).
• Regulatory mechanisms: • HIF1A: An accelerated conserved non-coding element, CNE143336 (~190 kb downstream of HIF1A), showed enhancer activity in reporter assays; the MPI ortholog exhibited significantly weaker enhancer activity than mouse/RSI in HEK293T and NIH3T3 (P<0.05; overall vs control P<0.001). Blocking CNE143336 with dCas9-KRAB and sgRNAs reduced HIF1A expression (e.g., P=0.002), indicating its role in regulating HIF1A. • COPS5: A potential enhancer ~40 kb upstream of COPS5 (RSC) present in mouse but lacking an intact 17 bp HIF1A motif in MPI. RSC displayed enhancer activity; in NIH3T3, MPI RSC activity was significantly weaker than mouse/RSI (P<0.05). Deleting the 17 bp motif in mouse RSC reduced activity (P=1.04E-06); inserting the 17 bp motif from mouse/RSI into MPI RSC increased activity (P<0.05). ChIP-qPCR showed stronger HIF1A binding at mouse RSC than MPI RSC (P<0.001). Protein analysis under transformation showed COPS5 increased in mouse HRAS(G12V)+SV40LT (P<0.01) but not in MPI; HIF1A increased in both; RPS3 unchanged.
• In vivo impact of COPS5 downregulation: CRISPR depletion of COPS5 in mouse HRAS(G12V)+SV40LT fibroblasts reduced xenograft bioluminescence (weeks 2–3, P<0.05) and tumor weight (P=0.006). Overexpressing COPS5 in MPI HRAS(SV40LT) fibroblasts increased xenograft mass (P<0.05).
• Senescence propensity and longevity context: MPI fibroblasts exhibited higher β-galactosidase activity and elevated p21/p53 after etoposide-induced senescence vs other bats and mouse (P<0.01), consistent with a pro-senescence, anti-cancer phenotype. Literature links depletion of COPS5/HIF1A with premature senescence.
• Transposon analysis: Among accelerated CNEs in MPI, overlap with TEs was less frequent than in non-accelerated CNEs (P<0.001), suggesting accelerated CNE evolution is not broadly driven by TE activity, though TEs are near the lost COPS5 enhancer region.

This work provides direct experimental evidence that Myotis pilosus exhibits resistance to malignant transformation in vitro and reduced tumorigenicity in vivo compared to mouse and six other bat species under identical oncogenic drivers. Systems-level transcriptomics highlighted a downregulated module in MPI enriched for hub genes HIF1A, COPS5, and RPS3, and functional perturbation experiments established that reduced HIF1A and COPS5, with a contributory role for RPS3, underlie decreased proliferation, colony formation, and tumor growth. Mechanistically, the study links MPI-specific regulatory changes to decreased oncogene expression: an accelerated enhancer element (CNE143336) modulates HIF1A expression, and a 17 bp deletion in a COPS5 upstream enhancer disrupts an HIF1A binding motif, weakening enhancer activity and HIF1A recruitment. Given the reciprocal regulatory interplay between HIF1A and COPS5, coordinated downregulation likely attenuates hypoxia signaling and oncogenic pathways, contributing to cancer resistance. The observed pro-senescence tendencies in MPI fibroblasts align with known anti-cancer mechanisms in long-lived mammals, suggesting that cancer resistance and longevity co-evolved in this species. While COPS5 downregulation reduces tumor burden, it does not fully recapitulate MPI’s resistance, indicating additional mechanisms remain to be discovered. These findings advance our understanding of naturally evolved mammalian anticancer strategies and point to regulatory genome evolution as a driver of cancer resistance.

The study identifies Myotis pilosus as a bat species with experimental evidence of cancer resistance in primary fibroblasts and xenografts. It implicates downregulation of HIF1A and COPS5, with supporting effects from RPS3, as key contributors, mechanistically linked to MPI-specific regulatory sequence changes, including an accelerated enhancer near HIF1A and loss of an HIF1A motif in a COPS5 enhancer. The work highlights regulatory evolution as a mechanism for anti-cancer phenotypes and reinforces the connection between longevity and cancer resistance. Future research should: (1) extend analyses to additional bat species and cell types, especially epithelial cells; (2) dissect additional regulatory elements and pathways contributing to resistance; (3) explore the systemic physiology of MPI (e.g., immune, metabolic adaptations) in cancer suppression; and (4) translate insights into therapeutic strategies targeting HIF1A/COPS5 regulatory axes.

Primary assays used fibroblasts, whereas most human cancers arise from epithelial cells; although some epithelial and cancer lines were tested for COPS5 knockdown effects, broader validation is needed. The transformation protocol relied on HRAS(G12V)+SV40 LT; species- or cell type-specific oncogenic requirements could differ, so resistance in other bat species may have been missed. The lifespan of MPI is undocumented, limiting direct correlation with longevity. COPS5 downregulation only partially accounts for the resistance compared to MPI cells, indicating additional mechanisms. The enhancer near RPS3 (CNE563305) could not be functionally tested due to amplification challenges. While TE proximity to the lost COPS5 enhancer was noted, causality was not established. Cross-species transcriptomic comparisons and module inferences, while controlled, may still carry biases.