Abstract
There are no reports of cancer in sponges, despite them having somatic cell turnover, long lifespans and no specialized adaptive immune cells. In order to investigate whether sponges are cancer resistant, we exposed a species of sponge, Tethya wilhelma, to X-rays. We found that T. wilhelma can withstand 600 Gy of X-ray radiation. That is approximately 100 times the lethal dose for humans. A single high dose of X-rays did not induce cancer in sponges, providing the first experimental evidence of cancer resistance in the phylum, Porifera. Following X-ray exposure, we found an overexpression of genes involved in DNA repair, signaling transduction pathways and epithelial to mesenchymal transition. Sponges have the highest level of radiation resistance that has yet been observed in animals that have sustained somatic cell turnover. This may make them an excellent model system for studying cancer resistance and developing new approaches for cancer prevention and treatment.
Introduction
To date there have been no reports of cancer in sponges1. Sponges are part of the phylum Porifera, and they have a long lifespan and somatic cell turnover2, which should make them susceptible to cancer because over the course of their lifespans they would be expected to accumulate carcinogenic mutations. Here we set out to investigate whether sponges are particularly cancer resistant, and, if so, what the mechanisms underlying this cancer resistance are. Through a combination of microscopy and transcriptomics, we were able to observe changes in Tethya wilhelma (Demosponges)3,4 after X-ray exposure and assess the organism-level, cell-level and gene expression changes over time.
Basal invertebrates like sponges that lack5 immune specialized cells6 or with primitive elements of an adaptive immune system7,8, may lack the ability to detect and eliminate mutant cells. They should be particularly susceptible to cancer. However, the fact that no cancer has been reported in sponges1 suggests that they might have a physiology that is resilient to mutations or possess effective mechanisms for DNA damage prevention, DNA repair, and tissue homeostasis.
Sponges are an effective model system for studying cancer resistance
In order to investigate cancer resistance in sponges and evaluate this hypothesis, we studied the sponge T. wilhelma, which is a sessile, filter-feeding demosponge that originally came from the Indo-Pacific oceans9 (Fig.1). Demosponges possess a canal system which is characterized by a highly complex network of chambers lined with choanocytes, which are flagellated cells that are specialized in creating a flow of water and capturing food particles10. Water enters from the pores located mainly on the lateral walls of sponges and is discarded through a large medial excurrent canal opening on the apex of sponges, the osculum10. T. wilhelma have a globular shape and the largest specimens can reach the size of 15-20 mm in diameter (Fig.1). T. wilhelma reproduce asexually by budding in the laboratory11. These sponges are capable of contractile and slow locomotory behavior 3,4 and adapt well to being cultured in aquaria3,4.
The genome sequence of T. wilhelma is available12 and genomic analyses have shown that many molecular pathways (e.g. signal transduction mechanisms) are well conserved on sponges12. T. wilhelma has a remarkably long lifespan but sufficiently short generation time3 to make it possible to rapidly obtain experimental results.
This is the first study of radiation resistance and cancer resistance in porifera, the phylum at the very base of animal life on earth.
Materials and Methods
Lab cultures
We built a sponge culture system that consists of a main saltwater aquarium (340 liters) that develops an ecosystem (coral reef) capable of supporting the growth of the sponges. The sponges are grown in 3 smaller (30 liter) culture aquariums connected to the main one, with controlled temperature (24°C) and water flow. This setting allows easy access to the sponges, including the observation of the sponges under a microscope without having to remove them from the aquarium.
We fed the sponges with artificial plankton (Aquakultur Genzel GmbH, Germany) twice daily. In order to obtain fine (<25μm) food particles assimilable by sponges, we homogenized the artificial plankton with an IKA T10 Basic Ultra Turrax homogenizer.
The culturing of the sponges started 4 years prior to this study, with 20 animals. The original specimens are still alive, thus the lifespan of the sponges in our laboratory setting is > 4 years.
DNA damage by X-ray irradiation
We exposed young adult sponges (diameter>5mm) to X-rays utilizing a RS-2000 Biological System X-ray irradiator. We exposed the sponges to a single dose of 600 Gy for the experiments. Considering X-ray absorbance of the 10 mm column of water above the animals during the X-ray irradiation, we estimated the actual X-ray exposure of specimens to be 13.7% lower, 518 Gy.
Experimental settings
Based on preliminary morphological observations, we selected three time points: 24 hours, 7 days, and 21 days after X-ray exposure for both histological and transcriptomic analyses (RNA-seq). We randomly selected 3 sponges for each time point plus 3 specimens of control for both histological and transcriptome analyses, for a total of 36 sponges. We treated 4 additional independent groups of sponges (n=6, n=5, n=5, n=5, total 21 sponges) and relative controls (5 sponges for each group, total 20 sponges), for long-term morphological observations. Each group was exposed to X-rays at different times.
Morphological analysis
We observed the morphological changes in the animals in vivo using ImageJ software13. Sponges are partially translucent, but only superficial structures can be observed in vivo. However, their shape is regular, and any morphological changes are easily observable.
For histological examination we fixed the specimens with Pampl’s fluid14 for 24 hours at 4°C. Then, we dissolved the siliceous spicules that make up its skeleton by submerging the specimens in 4% hydrofluoric acid (MilliporeSigma, cat. n. 1.00338) for an additional 24 hours at 4°C, then we followed standard histological protocols15,16.
For transmission electron microscopy, we fixed specimens in 2.5% glutaraldehyde (Electron Microscopy Sciences, cat.n. 16020) in 0.2 M Na-cacodylate sucrose buffer (pH 7.2; Electron Microscopy Sciences, cat.n. 12300) for 2.5 hours at 4°C. Then, we rinsed the specimens 3 times with a 0.2 cacodylate sucrose buffer for 45-60 min total, post fixed them for 2 hours in 1% osmium tetroxide (Electron Microscopy Sciences, cat n. 19150) 0.2 cacodylate sucrose buffer and washed them 1 time with buffer, then 3 times with deionized water for 45-60 min total. We stained them en bloc with 1% aqueous uranyl acetate (Electron Microscopy Sciences, cat n. 22400) for 16 hours at 4°C. After washing the specimens 4 times with water for 45-60 min total. We dehydrated them with an ascending ethanol series up to 70% ethanol. Then, we disilicate the specimens with 4% hydrofluoric acid for 1 hour at 4°C. Afterwards, we washed specimens in 70% ethanol, and we completed the dehydration with an ethanol series up to 100%. Then we transferred the specimens to anhydrous propylene oxide (cat. n. 14300) for 30 minutes (replacing the anhydrous propylene oxide with fresh one after 15 minutes). We infiltrated samples with 5% Spurr’s epoxy resin (in anhydrous propylene oxide 3 hours with rotation; 50% resin in anhydrous propylene oxide overnight with rotation (18 hr); 75% resin in anhydrous propylene oxide with rotation (6 hr); 100% pure resin 3x for 24 hr total (6 hr, 12 hr, 6 hr). Finally, we flat-embedded the specimens and polymerized them at 60°C for 27 hrs. We used a diamond knife to cut ultrathin sections. We observed the sections under a Philips CM12 transmission electron microscope.
Quantification of DNA damage
We quantified the DNA damage caused by X-ray exposure by the silver-stained Comet alkaline assay (Travigen®, Cat#4251-050-K)17,18 according to the manufacturer’s specifications and we used ImageJ software13 to quantify the DNA fragmentation.
Molecular genetic analysis
We treated the T. wilhelma specimens with 600 Gy (actually 518 Gy due to water absorbance). After 24 hours, 7 days, and 21 days following X-ray exposure we extracted the total RNA (RNeasy® mini kit, Qiagen, cat. n. 74104) from 3 sponges for each treatment and control. After verifying the purity and integrity of the RNA using an Agilent 2200 TapeStation, part of the extracted RNA (11.06 ng per sample on average) was utilized for RNA-seq analysis. We sequenced the samples using an Illumina NextSeq 500 instrument. We checked the quality of the RNA-seq reads for each sample using FastQC v0.10.1 and we aligned the reads with the reference genome (NCBI, SRA, SRR2163223) using STAR v2.5.1b (22.68 million reads uniquely mapped on average per sample). Cufflinks v2.2.1 was used to report FPKM values (Fragments Per Kilobase of transcripts per Million mapped reads) and read counts. We uniquely mapped 17.05 million reads to the reference genome on average, per sample. We performed a differential expression analysis using the EdgeR package from Bioconductor v3.2 in R 3.2.3. For each pairwise comparison, genes with false discovery rate (FDR) <0.05 were considered significant and 2 log2-fold changes of expression between conditions were reported after Bonferroni correction. We analyzed the differentially expressed genes using BLAST19, Ensembl20 and their functional annotations, including fold enrichment (FE), using DAVID21,22 and PANTHER23 software, and protein domains24. We focused on the overexpressed genes because the decrease of gene expression can be an nonspecific effect of X-ray exposure due to cellular damage.
Results
We initially conducted a dose finding experiment to quantify the maximum tolerance to increasing doses (range:160-800 Gy) of radiation on sponges. After the treatment we observed the sponges daily. An 800 Gy dose is lethal for sponges (n=5, 80% lethality). In contrast, all sponges (n=7) exposed to 600 Gy suffered transitory morphological changes but survived. We then conducted the subsequent experiments using a single dose of 600 Gy.
Morphological observations
We observed a general pattern in the morphological changes over time in sponges after X-ray exposure (Fig. 2, 3 and Fig. 1S).
Initially (2-7 days) the sponges begin to shrink, producing short and thin body extensions, and the pores and the osculum are no longer visible (Fig. 2, 3 and Fig. 1S). Sponges reach the minimum size after 21-25 days (Fig. 2, 3; paired t-test, df=16, t=6.341, p<0.0001), their surface became smooth and they produced large and long body projections, causing sponges to acquire an irregular star shape (Fig. 2, 3 and Fig. 1S).
Then, sponges reverse the shrinking process, but their morphologic features appear to be still altered or progress to further dissolvement (Fig. 2): body projections increased their surface and additional body projections were generated (Fig. 2). Body extensions were either re-absorbed or broke off, generating new satellite sponges, observed in 33.3% of sponges (Fig. 2-4 and Fig. 1S).
Sponges gradually reacquired their original anatomical organization and appeared normal after ∼180 days. Four out of 21 treated sponges died after an average of 162 (±30.9 S.D.) days. At the time of this writing, it has been over 1 year since treatment. 17 of 21 sponges treated with 600 Gy are alive, do not show any morphological changes, and are indistinguishable from untreated sponges (Fig. 2, 3 and Fig. 1S).
Histological analysis
After X-ray exposure, sponges lose their typical anatomical organization16 (Fig. 5). The filtering structures of sponges (choanoderm) and the specialized water flowing and feeding cells (choanocytes) are lost (Fig. 5), and the choanoderm appear to be filled with undifferentiated cells25. The histological analyses did not show necrotic areas at any time after X-ray exposure (Fig. 5).
Electron microscopy analysis
We further investigated the morphological changes of the choanoderm after 7 days from X-ray exposure. Indeed, the choanoderm of treated sponges is disorganized and the choanocyte chambers are deeply altered or absent. The choanocytes are not anymore recognizable (Fig. 6). The mutualistic bacteria, presumably Cyanobacteria26, are able to survive the X-ray treatment as well.
DNA damage analysis
DNA fragmentation analysis (Comet assay) showed limited DNA degradation immediately after a submaximal (600 Gy) X-ray exposure (DNA fragmentation, treated: 8.23% ± 16.32 S.D., controls 1.34% ± 6.99 S.D. (Fig. 7).
Gene expression analysis
We performed the transcriptome analysis (RNA-seq) of 3 T. wilhelma specimens collected at each of 3 different time points (24 hours, 7 and 21 days) after X-ray treatment. We found a total of 639 overexpressed transcripts in the three experimental time points compared to untreated sponges at the same time points (Fig. 8). Each group had different gene expression levels and differs from the controls (Fig. 2S). Many of the expressed genes have a human homolog (given in parentheses). There are genes overexpressed only at a specific time point (24 hours, 7 or 21 days) and genes overexpressed at 2 or 3 time points (Tab. 3S, fig. 8 and fig. 3S).
Genes overexpressed 24 hours after X-ray exposure
We found 195 transcripts overexpressed 24 hours after X-ray exposure, of which 97 have a human homolog (Fig. 8, tab. 1S and fig. 3S). Sixty-four of those 195 transcripts were only overexpressed after 24 hours but not at later time points. Fourty-four of those 64 overexpressed transcripts specific to the 24-hour time point have a human homolog. We detected an enrichment of human homolog genes involved in DNA repair (FE=10.3, FDR= 0.037, DAVID) such as twi_ss.22376.1 (LIG3), DNA ligase and twi_ss.21448.1 (MRE11), involved in the double strand break repair mechanisms, twi_ss.11375.1 (PCNA), proliferating cell nuclear antigen, has a key role in DNA damage response, twi_ss.28211.1 (RPA1), DNA repair pathway and the replication protein A1, is a cofactor of DNA polymerase delta involved in the RAD6-dependent DNA repair pathway, Twi_ss.25822.1-3 (GINS4), GINS subunit, domain A, is involved in double-strand break repair via break-induced replication.
We found that twi_ss.17326.7 gene (CUBN), cubilin, is the most differentially expressed gene (logFC=8.55) specific to the 24-hour time point after X-ray exposure. CUBN is an endocytic receptor expressed in the epithelium of intestines and kidneys27, and down-regulated in renal cell carcinoma28.
In addition, we identified overexpression of genes involved in stress response such as twi_ss.28284.1 (HSPA1A), the heat shock protein family A (Hsp70) member 1A.
Genes overexpressed 7 days after X-ray exposure
We found 408 transcripts overexpressed, of which 141 have a human homolog (Fig. 8, tab. 1S and fig. 3S). Among them, 125 transcripts were specifically overexpressed after 7 days, of which 53 have a human homolog. We detected an enrichment of ankyrin containing domain genes (FE=19, FDR< 0.001), signal proteins (FE=2.4, FDR= 0.005) and extracellular matrix remodeling genes (FE=68.1, FDR= 0.03).
We identified genes specifically overexpressed after 7 days involved in DNA repair such as twi_ss.10792.1 (FAN1), twi_ss.27976.3 (KAT5) and twi_ss.19635.1 (TRIP12) and, importantly, we found a correspondence with the morphological changes observed 7 days after X-ray exposure and the function of genes overexpressed at the same time. For instance, we found the overexpression of genes involved in development, adult tissue homeostasis and mesenchymal transition such as twi_ss.2267.1 (NOTCH1), twi_ss.5628.6 (MET) MET proto-oncogene, receptor tyrosine kinase, twi_ss.6204.1 (POSTN), periostin. We also found genes involved in embryonic stem cell regulation such as twi_ss.7082.1 (ETV4), twi_ss.4355.1 (TRIM71), twi_ss.329.1 (ZFP36L1) together with a variety of genes such as twi_ss.26378.1 (COL6A3) collagen type VI alpha 3 chain, twi_ss.31853a.1 (COL6A6) collagen type VI alpha 6 chain, twi_ss.25970.2 (LTBP1) latent transforming growth factor beta binding protein 1, twi_ss.20516a.5 (SORL1) sortilin related receptor 1, twi_ss.2619.7 (VWF) von Willebrand factor, twi_ss.19885.1 (NRXN3) neurexin 3, twi_ss.1244.5 (ADGRE5) adhesion G protein-coupled receptor E5 involved with the epithelial-mesenchymal transition.
Gene overexpressed 21 days after X-ray exposure
we found genes 410 overexpressed transcripts of which 150 of these have a human homolog (Fig. 8, tab. 1S and fig. 3S). We identified 157 transcripts specifically overexpressed after 21 days of which 72 have a human homolog. There is not a functional signature specific to the genes expressed specifically after 21 days. We found genes involved in DNA double-strand break repair: twi_ss.10068.1 (PARP3) poly (ADP-ribose) polymerase family member 3, twi_ss.28718.1 (PARPF19) PHD finger protein 19 and, DNA repair: twi_ss.7757.3 (UBR5) ubiquitin protein ligase E3 component n-recognin 5.
Gene overexpressed 24 hours, 7 and 21 days after X-ray exposure
We found 81 transcripts overexpressed at all 3 time points of which 34 have a human homolog (Fig. 8, tab. 1S and fig. 3S). For instance, Twi_ss.16656.2 (PHF8), PHD Finger Protein 8 one of the most differentially expressed gene (9.3±1.8 S.D. logFC), the C. elegans homolog promotes DNA repair via homologous recombination29. Twi_ss.4977.9 (8.9±0.4 S.D. logFC) is a homolog of the human gene Spatacsin (SPG11). The function of SPG11 is not well understood. It has a role in a form of spastic paraplegia, a neurodegenerative disorder and it appears to be also involved in DNA repair30.
Gene overexpressed 24 hours and 7 days after X-ray exposure
we found 40 transcripts overexpressed at both time points of which 19 of these genes have a human homolog (Fig. 7-9, tab. 1S). The most differential expressed gene (8±1.8 S.D. logFC) is twi_ss.12458.1 (TTN), a gene unknown to be activated after X-ray exposure.
Gene overexpressed 24 hours and 21 days after X-ray exposure
we found only 10 overexpressed transcripts of which 5 of these genes have a human homolog gene (Fig. 8, tab. 1S and fig. 3S). With only 5 genes with known homolog functions, there were not statistically significantly enriched pathways. However, twi_ss.19378.2 (ERCC1) is involved in DNA repair.
Gene overexpressed 7 days and 21 days after X-ray exposure
we found genes 162 overexpressed transcripts of which 45 of these have a human homolog gene (Fig. 8, tab. 1S and fig. 3S). Overall, there is an enrichment of genes involved in extracellular matrix organization such as fibronectin (FE=15.7, FDR< 0.0001), with an EGF-like domain (FE=19.7, FDR<0.0001) and signal peptides (FE=3.3, FDR<0.0001). One of the most overexpressed genes (10.1±0.7 S.D. logFC) is twi_ss.21105.9 (SCUBE1) signal peptide, CUB and EGF-like domain-containing protein 1, which may function as an adhesive molecule31.
Discussion
T. wilhelma sponges can withstand 600 Gy (actual 517.6 Gy) of X-ray radiation. That is approximately 60 times the lethal dose for mice32,33 and 100 times the lethal dose for humans34. According to conventional wisdom, this amount of radiation should shatter the sponge’s DNA, however the Comet assay suggests this does not happen in T. wilhelma.
Early organisms evolved in an environment with higher levels of background radiation35. Despite the fact that water partially shields aquatic organisms from direct radiation exposure, radionuclides can accumulate in the sea. As filter-feeding animals, sponges could be particularly exposed to the accumulation of radionuclides and other toxic agents36. Moreover, sponges are sessile organisms without a nervous system thus not capable of rapidly escaping or quickly reducing the water flow through their bodies if the concentration of radioactive agents increases. For these reasons sponges are considered biological indicators of environmental pollution such as radionucleotides37,38.
There are examples of extreme radioresistance in bacteria39 and multicellular organisms capable of anhydrobiosis (desiccation) such as rotifers40 and tardigrades41–43, which is unlikely to apply to T. wilhelma. Tolerance for both desiccation and radiation may originate from similar DNA protective or repair mechanisms40,44. Indeed, desiccation causes DNA breakage40,42, similar to damage induced by radiation, that may be repaired upon rehydration40. Tardigrades possess molecular mechanisms to prevent DNA damage. Dsup is a tardigrade-specific nucleosome-binding protein that protects chromatin from hydroxyl radicals and contributes to the organism’s radio-tolerance45. The combination of DNA protective and DNA repair mechanisms determines the level of radioresistance of these organisms. Importantly, rotifers and tardigrades have no or highly restricted somatic cell turnover41,46,47, and the species tested for radioresistance have a short (∼60 days) lifespan41,48 which prevents mutant clones and accumulating further mutations, leading to cancer. In these conditions, the DNA damage that occurs does not propagate and so might not be apparent49. Thus, there is little chance for cancer to develop in tardigrades.
In order to investigate the long-term effect of DNA damage and cancer in invertebrates, somatic cell turnover and long lifespan should be an essential feature of any experimental organism. However, the main invertebrate model organisms currently in use (e.g. Caenorhabditis elegans and Drosophila melanogaster) have limited or no somatic cell turnover50 and their lifespans51,52 are too short to study the long term effects of radiation. In contrast, sponges have somatic cell turnover2 and a remarkably long lifespan: the largest Xestospongia muta specimen described on Caribbean reefs is estimated to be more than 2,300 years old53, a specimen of the sponge Monorhaphis chuni is thought to be 11,000 years old54 and radiocarbon dating of the sponge Rossella racovitzae racovitzae determined that was around 440 years old55. Selection for this long lifespan may have also selected for cancer suppression mechanisms.
Considering all these factors: somatic cell turnover, long life span and a primitive immune system56, we would expect the development of tumors in sponges, but there have been no reports of cancer in the entire Porifera phylum1 (with 8500 described living species57).
In our experimental setting, (single high dose) radiation did not induce cancer development in T. wilhelma during 1 year following X-ray exposure. Malignant cancer risk in humans is estimated to be 8% per Gy58. A small fraction of the dose to which the sponges have been subjected would generally have given rise to cancer in mice59 and humans58.
The molecular data and morphological observations suggest that sponges protect their DNA from damage in the first place and then activate mechanisms of DNA repair and cell death as they go through a complex phase of tissue reorganization. Finally, they rebuild their tissues’ original features. Our findings suggest that the cancer resistance in sponges might be linked to their radioresistance.
Genes involved in DNA repair are activated at different times and for different lengths of time, providing insight into the temporal activation of these genes during the DNA repair process (Fig. 8, tab. 1S and fig. 3S). We found the overexpression of genes known to have a role in DNA double strand break repair (Tab. 1S), confirming that our experimental setting is capable of induced the typical DNA damage produced by X-ray exposure and that sponges respond to radiation by upregulating DNA repair. As expected, we observed a higher number of genes involved DNA repair 24 hours and 7 days after X-ray exposure, but we also identified genes involved in this process specifically expressed 21 days after X-ray exposure. This observation suggests that after 21 days the sponges are still actively repairing the DNA damage induced by radiation. Treated sponges overexpressed only 81 transcripts (12.7% of all overexpressed transcripts) in all 3 time points, but overexpressed 64 transcripts only at 24 hours, 125 transcripts only at 7 days, and 157 transcripts only at 21 days, suggesting waves of sequential transcriptional events.
We found overexpressed genes previously not linked to X-ray induced damage, or with unknown function in humans (Table 3S). For example, twi_ss.17326.7 (CUBN), cubilin in humans. CUBN is an endocytic receptor expressed in the epithelium of intestines and kidneys27. Interestingly, low expression of CUBN in renal cell carcinoma is significantly associated with early disease progression and poor patient outcome28. We hypothesize that the CUBN gene has a protective function against DNA damage or is involved in DNA repair and thus its downregulation in kidney cancer increases the chance of an aggressive evolution of the disease.
Epigenetic changes such as methylation are induced by X-ray exposure and contribute to regulate the cell response to stress60. Though we did not directly measure methylation, we detected the overexpression of twi_ss.21704.3 (NSUN7), a gene involved in methylation. Methylation induced by radiation can be a persistent epigenetic change after radiation 60 regulating gene activity long after expression normalization.
In addition to mechanisms of DNA repair, we detected the overexpression of genes involved in apoptosis and cell death. For example, Twi_ss.31154.2 (NOX5), NADPH oxidase–generated ROS, a gene involved in heavy ion irradiation–induced cell death60, suggesting the activation mechanisms of cell death, presumably to remove cells too compromised by radiation.
Notably, we observed a distinct pattern in the morphological changes in the treated sponges. Seven days after X-ray exposure, sponges lose their typical anatomical organization and most of the cells seem to acquire undifferentiated features. Totipotent cells (archeocytes) are part of the mesohyl and they can replace damaged cells61. Choanocytes, one of the most specialized types of sponge cells, can also transdifferentiate into archaeocytes (stem cells) and serve the same function61,62. In transcriptomic analysis 7 days following X-ray exposure, we found overexpression of genes known to be involved in cell adhesion, signaling, embryonic cell regulation and EMT such as NOTCH1 and MET, consistent with our observations of changes in sponge morphology.
Studying radiation resistance in sponges might help improve radiation therapy
Ionizing radiation is part of the natural environment and evolution of organisms. Thus, the study of radiation responses in sponges could contribute to understanding the molecular evolution of cell radioresistance. This could help us understand the evolution of radioresistance in cancer cells as well, and potentially lead to methods for protecting human cells from radiation damage.
Over the course of a human lifetime, cells can evolve mechanisms that allow them to endure the effects of X-ray exposure63,64 through somatic evolution. In human cancers, radiotherapy often induces EMT, and leads to increased radioresistance accompanied by increased cell migration and invasion65–68.
The finding that sponges react to X-ray exposure by increasing the number of undifferentiated cells suggests that EMT is not a cancer cell specific response to radiotherapy, but rather a natural cellular response to radiation both in organisms and human cells69. Sponges are particularly suitable to study this process because their simple tissue organization facilitates identification and analysis of tissue alterations. Sponges give us the unique opportunity to study mesenchymal proliferation and EMT in response to radiation, removing the confounding effect of thousands of mutations or deregulated genes described in cancers which may be unrelated to EMT. It is important to note that cancer cells do not utilize de novo cancer specific mechanisms to survive radiation exposure, but rather employ a biological process that is shared with sponges and has been evolutionarily conserved in our genomes for 100’s of millions of years. Understanding this biological process in response to radiotherapy in sponges could provide the basis for new treatment and cancer prevention strategies.
The study of the evolution of anti-cancer mechanisms such as radiation resistance can contribute to the understanding of multicellular organisms more generally, as the evolution of multicellularity itself depended on the multicellular organisms’ ability to prevent somatic cells proliferating out of control70. Our work suggests that sponges may be particularly resistant to cancer because of their radiation resistance, and shows that sponges are an viable model system for studying anti-cancer mechanisms and radiation resistance.
Author contributions
A.F, A.A. and C.C.M. designed the study. A.F. designed and performed the experiments, collected and analyzed the data. J.T. was an undergraduate student, he contributed to perform the experiments and collecting data. J.S. is an undergraduate student, he contributed to perform the bioinformatic analysis. A. Fortunato, A.A. and C.C.M. wrote the manuscript.
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
We would like to thank Erik Southard for helping to maintain the sponges and assistance during the experiments; Joy Blain and Shanshan Yang (ASU Genomics Facility) for RNA sequencing. David Lowry (Life Science Electron Microscopy Lab) for TEM imaging; Debra Baluch (W.M. Keck Bioimaging Laboratory) for histological samples preparation for light microscopy. We are also grateful to Tim Chan for his advice and suggestions. This work was supported in part by NIH grants U54 CA217376, U2C CA233254, P01 CA91955, R01 CA170595, R01 CA185138 and R01 CA140657 as well as CDMRP Breast Cancer Research Program Award BC132057 and the Arizona Biomedical Research Commission grant ADHS18-198847. The findings, opinions and recommendations expressed here are those of the authors and not necessarily those of the universities where the research was performed or the National Institutes of Health.
Footnotes
↵* co-senior authors