Experimental evidence for cancer resistance in a bat species

Bats represent a substantial portion of mammalian diversity (~20%, >1400 species) and demonstrate remarkable adaptations including flight, echolocation, virus tolerance, and exceptional longevity. Many bat species live three times longer than similarly sized mammals, with 18 out of 19 mammalian species exhibiting lifespans longer than humans (relative to body size) belonging to the bat order. Long-lived mammals like naked mole-rats and blind mole-rats showcase notable cancer resistance due to evolved longevity mechanisms, such as repressed telomerase activity, reduced cell proliferation, and enhanced genome stability. This suggests bats might also have evolved cancer resistance, especially in long-lived species. Previous molecular and genomic studies support this: oncogenic microRNAs are downregulated while cancer-resistant microRNAs are upregulated in the long-lived bat *Myotis myotis*. Adaptive changes in the growth hormone receptor and p53 have also been observed in long-lived bats. However, direct experimental evidence for cancer resistance in bats has been lacking, necessitating systematic investigation. Understanding the anticancer properties of mammals could provide crucial insights into natural cancer resistance mechanisms. This study aims to address this gap by conducting in vitro and in vivo experiments on seven bat species to investigate their cancer resistance, focusing on the potential for malignant transformation.

The relationship between longevity and cancer resistance has been a subject of extensive research. Studies on long-lived species such as naked mole-rats and blind mole-rats have highlighted various mechanisms contributing to their remarkable resistance to cancer. These mechanisms include alterations in telomere maintenance, changes in cell cycle regulation, and improved genome stability. Research on bats, known for their exceptional longevity, has shown evidence of potentially cancer-resistant mechanisms. These include changes in gene expression related to the regulation of cell growth, apoptosis, and DNA repair. However, the available evidence was primarily observational or correlative, lacking in direct experimental verification of cancer resistance. This study aims to provide direct experimental evidence through in vitro and in vivo experiments focusing on malignant transformation.

The study utilized primary skin fibroblasts from seven bat species (*Myotis pilosus*, *Myotis altarium*, *Rhinolophus pusillus*, *Rhinolophus ferrumequinum*, *Rhinolophus sinicus*, *Hipposideros armiger*, and *Rousettus leschenaultii*) and laboratory mice (*Mus musculus*) as a control. Stable cell lines expressing oncogenic HRAS(G12V) and SV40 LT were generated using lentiviral transduction. Anchorage-independent growth assays in soft agar were performed to assess malignant transformation potential. In vivo experiments involved subcutaneous injection of luciferase-expressing fibroblasts into immunodeficient mice to monitor tumor growth. Transcriptome sequencing (RNA-seq) was conducted on fibroblasts from all eight species to identify differentially expressed genes. Weighted gene co-expression network analysis (WGCNA) was employed to identify gene modules associated with cancer resistance. Protein-protein interaction (PPI) networks were constructed using STRING database. Survival data from the KMplot database were analyzed to assess the association between gene expression and cancer patient survival. CRISPR-Cas9 gene editing was used to manipulate the expression of candidate genes. The MPI genome was sequenced using PacBio technology to identify evolutionarily conserved non-coding elements (CNEs) and assess their regulatory activity using ATAC-seq and luciferase reporter assays. ChIP-qPCR was used to investigate transcription factor binding.

Fibroblasts from *Myotis pilosus* (MPI) demonstrated significantly higher resistance to malignant transformation compared to other bat species and mice in both in vitro (soft agar assay) and in vivo (xenograft) experiments. Transcriptome sequencing and WGCNA identified three genes (*HIF1A*, *COPSS*, and *RPS3*) whose downregulation in MPI correlated with cancer resistance. CRISPR-Cas9 mediated knockdown of these genes in mouse fibroblasts inhibited cell proliferation, while overexpression in MPI cells promoted proliferation and tumor growth in vivo. Analysis of the MPI genome revealed the loss of a potential enhancer containing a *HIF1A* binding site upstream of *COPSS*, likely causing *COPSS* downregulation. Further experiments confirmed that blocking the regulatory activity of this enhancer reduced *HIF1A* expression. The downregulation of *COPSS* alone was shown to significantly reduce tumor size in vivo. The study also found evidence that MPI fibroblasts exhibit higher senescence than other species, suggesting a potential link to longevity and cancer resistance. Analysis of transposable elements did not reveal a strong association with the loss of the potential enhancer near *COPSS*. Inhibition of *COPSS* in human epithelial and cancer cell lines (HK-2, MCF-7, PANC-1) also decreased proliferation, suggesting broader relevance of the finding. Differences in COPS5 protein size between MPI and other species were noted; however, further investigation indicated that this was not likely due to antibody non-specificity.

This study provides the first direct experimental evidence of cancer resistance in a bat species, supporting the hypothesis of a link between longevity and cancer resistance. The downregulation of *HIF1A*, *COPSS*, and *RPS3* in *Myotis pilosus* appears to be a crucial mechanism for this resistance. The identified loss of a *HIF1A* binding site in a potential enhancer upstream of *COPSS* offers a molecular explanation for the downregulation of *COPSS*. The results highlight the potential of exploring long-lived species for discovering novel anticancer mechanisms. The findings suggest that the downregulation of these genes, particularly *COPSS*, could be a promising target for future anticancer therapies. While the study focuses on fibroblasts, the impact of *COPSS* downregulation on epithelial cells suggests broader implications. However, the complex interplay between *HIF1A* and *COPSS*, and the potential role of *RPS3*, warrant further investigation.

This research provides compelling experimental evidence for cancer resistance in the big-footed bat (*Myotis pilosus*), linking this resistance to the downregulation of *HIF1A*, *COPSS*, and *RPS3*. The loss of a functional enhancer sequence upstream of *COPSS* is identified as a potential mechanism for this downregulation. These findings highlight the potential of studying long-lived mammals for identifying novel cancer-resistant mechanisms and suggest further research into the specific roles of *HIF1A*, *COPSS*, and *RPS3* in cancer suppression and the potential for therapeutic interventions.

The study primarily focused on fibroblasts, which represent a part of the tumor microenvironment but not the primary tumor cells. While the results suggest a broader impact on epithelial cells, further research is needed to confirm the role of *COPSS* downregulation in various human cancers. The study didn't explicitly measure the lifespan of *Myotis pilosus*, relying on inferences from related species. The mechanistic details underlying the loss of the *COPSS* enhancer require further investigation.