Poly- and perfluoroalkylated substances (PFAS) are chemicals that persist and bioaccumulate in the environment and are found in nearly all human populations through several routes of exposure. Human occupational and community exposure to PFAS has been associated with several cancers, including cancers of the kidney, testis, prostate, and liver. While evidence suggests that PFAS are not directly mutagenic, many diverse mechanisms of carcinogenicity have been proposed. In this mini-review, we organize these mechanisms into three major proposed pathways of PFAS action—metabolism, endocrine disruption, and epigenetic perturbation—and discuss how these distinct but interdependent pathways may explain many of the proposed pro-carcinogenic effects of the PFAS class of environmental contaminants. Notably, each of the pathways is predicted to be highly sensitive to the dose and window of exposure which may, in part, explain the variable epidemiologic and experimental evidence linking PFAS and cancer. We highlight testicular and prostate cancer as models to validate this concept.

The potential effects of poly- and perfluoroalkyl substances (PFAS) are a recently emergent human and environmental health concern. There is a consistent link between PFAS exposure and cancer, but the mechanisms are poorly understood. Although epidemiological evidence supporting PFAS exposure and cancer in general is conflicting, there is relatively strong evidence linking PFAS and testicular germ cell tumors (TGCTs). However, no mechanistic studies have been performed to date concerning PFAS and TGCTs. In this report, the effects of the legacy PFAS perfluorooctanesulfonic acid (PFOS) and the newer “clean energy” PFAS lithium bis(trifluoromethylsulfonyl)imide (LiTFSi, called HQ-115), on the tumorigenicity of TGCTs in mice, TGCT cell survival, and metabolite production, as well as gene regulation were investigated. In vitro, the proliferation and survival of both chemo-sensitive and -resistant TGCT cells were minimally affected by a wide range of PFOS and HQ-115 concentrations. However, both chemicals promoted the growth of TGCT cells in mouse xenografts at doses consistent with human exposure but induced minimal acute toxicity, as assessed by total body, kidney, and testis weight. PFOS, but not HQ-115, increased liver weight. Transcriptomic alterations of PFOS-exposed normal mouse testes were dominated by cancer-related pathways and gene expression alterations associated with the H3K27me3 polycomb pathway and DNA methylation, epigenetic pathways that were previously showed to be critical for the survival of TGCT cells after cisplatin-based chemotherapy. Similar patterns of PFOS-mediated gene expression occurred in PFOS-exposed cells in vitro. Metabolomic studies revealed that PFOS also altered metabolites associated with steroid biosynthesis and fatty acid metabolism in TGCT cells, consistent with the proposed ability of PFAS to mimic fatty acid-based ligands controlling lipid metabolism and the proposed role of PFAS as endocrine disrupters. Our data, is the first cell and animal based study on PFAS in TGCTs, support a pro-tumorigenic effect of PFAS on TGCT biology and suggests epigenetic, metabolic, and endocrine disruption as potential mechanisms of action that are consistent with the non-mutagenic nature of the PFAS class.

Testicular germ cell tumors (TGCTs) are aggressive but sensitive to cisplatin‐based chemotherapy. Alternative therapies are needed for tumors refractory to cisplatin with hypomethylating agents providing one possibility. The mechanisms of cisplatin hypersensitivity and resistance in TGCTs remain poorly understood. Recently, it has been shown that TGCTs, even those resistant to cisplatin, are hypersensitive to very low doses of hypomethylating agents including 5‐aza deoxy‐cytosine (5‐aza) and guadecitabine. We undertook a pharmacogenomic approach in order to better understand mechanisms of TGCT hypomethylating agent hypersensitivity by generating a panel of acquired 5‐aza‐resistant TGCT cells and contrasting these to previously generated acquired isogenic cisplatin‐resistant cells from the same parent. Interestingly, there was a reciprocal relationship between cisplatin and 5‐aza sensitivity, with cisplatin resistance associated with increased sensitivity to 5‐aza and 5‐aza resistance associated with increased sensitivity to cisplatin. Unbiased transcriptome analysis revealed 5‐aza‐resistant cells strongly downregulated polycomb target gene expression, the exact opposite of the finding for cisplatin‐resistant cells, which upregulated polycomb target genes. This was associated with a dramatic increase in H3K27me3 and decrease in DNMT3B levels in 5‐aza‐resistant cells, the exact opposite changes seen in cisplatin‐resistant cells. Evidence is presented that reciprocal regulation of polycomb and DNMT3B may be initiated by changes in DNMT3B levels as DNMT3B knockdown alone in parental cells resulted in increased expression of H3K27me3, EZH2, and BMI1, conferred 5‐aza resistance and cisplatin sensitization, and mediated genome‐wide repression of polycomb target gene expression. Finally, genome‐wide analysis revealed that 5‐aza‐resistant, cisplatin‐resistant, and DNMT3B‐knockdown cells alter the expression of a common set of polycomb target genes. This study highlights that reciprocal epigenetic changes mediated by DNMT3B and polycomb may be a key driver of the unique cisplatin and 5‐aza hypersensitivity of TGCTs and suggests that distinct epigenetic vulnerabilities may exist for pharmacological targeting of TGCTs.

Background/Objectives: Testicular germ cell tumors (TGCTs) are uniquely curable with cisplatin-based therapies even when widely metastatic; however, cisplatin resistance does occur, resulting in very poor prognosis. The mechanisms to explain TGCT hypersensitivity to cisplatin and mechanisms of resistance are not well-understood. Methods: The global transcriptional response to acute cisplatin treatment (24 h after a 6 h pulse of cisplatin) was assessed in three parental embryonal carcinoma TGCT cells lines compared to multiple isogenic, stable, cisplatin-resistant clonal lines from these parental cells. Results: Cisplatin treatment of parental cells consistently showed a more robust overall transcriptional response to cisplatin compared to their cisplatin-resistant cellular counterparts for a common set of genes and pathways including the upregulation of genes associated with histone modifications and p53, EMT, and KRAS signaling and the downregulation of genes normally upregulated by MYC. Focusing on genes exclusively altered in parental cells revealed upregulated genes known to be induced by p53 and downregulated by MYC and the transferrin receptor, TFRC1. Several of these p53/MYC/TFRC1 targets were associated with a higher instance of disease-free survival in a cohort of TGCT patients. Conclusions: Cisplatin resistance in TGCT cells is associated with a diminished alteration in cisplatin-responsive genes, especially genes known to be regulated by p53, MYC and TFRC1, that may be linked to cisplatin hypersensitivity and survival in TGCTs.

Studying the ecology of ancient organisms requires a tremendous amount of data, as in the case of prehistoric sharks where our knowledge is based mostly on teeth. Shark Tooth Forensics is a participatory science project tackling the problem of deciphering the ecology of ancient sharks with assistance from public school students. Traditionally, paleontological data collection protocols use research staff or a cadre of trained volunteers to collect paleontological data, whereas participatory science provides a means to increase the number of people involved in paleontological research. Students contributing to our project search for fossil shark teeth in sediments from various geologic time periods, and measure and record the teeth they discover for accessioning in natural history museums. Statistical analyses reveal that middle school students provide measurements of shark teeth that are indistinguishable from those same measurements taken by research staff. However, it is necessary to collect data from large student populations, ~10–30 students, before obtaining a statistically reliable sample. In addition, we find students are able to recognize shark teeth from a variety of species, not just stereotypical triangular teeth of species like the Great White (Carcharodon carcharias). Teachers can utilize Shark Tooth Forensics curricular activities that use student data in curriculum as a way to gain more personal investment. We estimate over 2,000 students have already participated, and with the potential for this project to last for decades, many thousands more will have the opportunity to discover fossil sharks and reconstruct the lives of these ancient fish.