Perfluorooctanesulfonic Acid Alters Pro-Cancer Phenotypes and Metabolic and Transcriptional Signatures in Testicular Germ Cell Tumors.

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.

G0S2 promotes antiestrogenic and pro-migratory responses in ER+ and ER- breast cancer cells.

G0/G1 switch gene 2 (G0S2) is known to inhibit lipolysis by inhibiting adipose triglyceride lipase (ATGL). In this report, we dissect the role of G0S2 in ER+ versus ER- breast cancer. Overexpression of G0S2 in ER- cells increased cell proliferation, while G0S2 overexpression in ER+ cells decreased cell proliferation. Transcriptome analysis revealed that G0S2 mediated distinct but overlapping transcriptional responses in ER- and ER+ cells. G0S2 reduced genes associated with an epithelial phenotype, especially in ER- cells, including CDH1, ELF3, STEAP4 and TACSTD2, suggesting promotion of the epithelial-mesenchymal transition (EMT). G0S2 also repressed estrogen signaling and estrogen receptor target gene signatures, especially in ER+ cells, including TFF1 and TFF3. In addition, G0S2 overexpression increased cell migration in ER- cells and increased estrogen deprivation sensitivity in ER+ cells. Interestingly, two genes downstream of ATGL in fat utilization and very important in steroid hormone biosynthesis, HMGCS1 and HMGCS2, were downregulated in G0S2 overexpressing ER+ cells. In addition, HSD17B11, a gene that converts estradiol to its less estrogenic derivative, estrone, was highly upregulated in G0S2 overexpressing ER+ cells, suggesting G0S2 overexpression has a negative effect on estradiol production and maintenance. High expression of G0S2 and HSD17B11 was associated with improved relapse-free survival in breast cancer patients while high expression of HMGSC1 was associated with poor survival. Finally, we deleted G0S2 in breast cancer-prone MMTV-PyMT mice. Our data indicates a complex role for G0S2 in breast cancer, dependent on ER status, that may be partially mediated by suppression of the estrogen signaling pathway.

Toward a Mechanistic Understanding of Poly- and Perfluoroalkylated Substances and Cancer.

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.

Nephrotoxicity of perfluorooctane sulfonate (PFOS)-effect on transcription and epigenetic factors.

Perfluorooctane sulfonate (PFOS) is a widespread persistent environmental pollutant implicated in nephrotoxicity with altered metabolism, carcinogenesis, and fibrosis potential. We studied the underlying epigenetic mechanism involving transcription factors of PFOS-induced kidney injury. A 14-day orally dosed mouse model was chosen to study acute influences in vivo. Messenger RNA expression analysis and gene set enrichment analysis were performed to elucidate the relationship between epigenetic regulators, transcription factors, kidney disease, and metabolism homeostasis. PFOS was found to accumulate in mouse kidney in a dose-dependent manner. Kidney injury markers Acta2 and Bcl2l1 increased in expression significantly. Transcription factors, including Nef2l2, Hes1, Ppara, and Ppard, were upregulated, while Smarca2 and Pparg were downregulated. Furthermore, global DNA methylation levels decreased and the gene expression of histone demethylases Kdm1a and Kdm4c were upregulated. Our work implicates PFOS-induced gene expression alterations in epigenetics, transcription factors, and kidney biomarkers with potential implications for kidney fibrosis and kidney carcinogenesis. Future experiments can focus on epigenetic mechanisms to establish a panel of PFOS-induced biomarkers for nephrotoxicity evaluation.

Reciprocal epigenetic remodeling controls testicular cancer hypersensitivity to hypomethylating agents and chemotherapy.

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.

Between a Rock and a Hard Place: An Epigenetic-Centric View of Testicular Germ Cell Tumors.

Compared to many common solid tumors, the main genetic drivers of most testicular germ cell tumors (TGCTs) are unknown. Decades of focus on genomic alterations in TGCTs including awareness of a near universal increase in copies of chromosome 12p have failed to uncover exceptional driver genes, especially in genes that can be targeted therapeutically. Thus far, TGCT patients have missed out on the benefits of targeted therapies available to treat most other malignancies. In the past decade there has been a greater appreciation that epigenetics may play an especially prominent role in TGCT etiology, progression, and hypersensitivity to conventional chemotherapy. While genetics undoubtedly plays a role in TGCT biology, this mini-review will focus on the epigenetic "states" or features of testicular cancer, with an emphasis on DNA methylation, histone modifications, and miRNAs associated with TGCT susceptibility, initiation, progression, and response to chemotherapy. In addition, we comment on the current status of epigenetic-based therapy and epigenetic biomarker development for TGCTs. Finally, we suggest a unifying "rock and a hard place" or "differentiate or die" model where the tumorigenicity and curability of TGCTs are both dependent on common but still ill-defined epigenetic states.

Hypermethylation and global remodelling of DNA methylation is associated with acquired cisplatin resistance in testicular germ cell tumours.

Testicular germ cell tumours (TGCTs) respond well to cisplatin-based therapy. However, cisplatin resistance and poor outcomes do occur. It has been suggested that a shift towards DNA hypermethylation mediates cisplatin resistance in TGCT cells, although there is little direct evidence to support this claim. Here we utilized a series of isogenic cisplatin-resistant cell models and observed a strong association between cisplatin resistance in TGCT cells and a net increase in global CpG and non-CpG DNA methylation spanning regulatory, intergenic, genic and repeat elements. Hypermethylated loci were significantly enriched for repressive DNA segments, CTCF and RAD21 sites and lamina associated domains, suggesting that global nuclear reorganization of chromatin structure occurred in resistant cells. Hypomethylated CpG loci were significantly enriched for EZH2 and SUZ12 binding and H3K27me3 sites. Integrative transcriptome and methylome analyses showed a strong negative correlation between gene promoter and CpG island methylation and gene expression in resistant cells and a weaker positive correlation between gene body methylation and gene expression. A bidirectional shift between gene promoter and gene body DNA methylation occurred within multiple genes that was associated with upregulation of polycomb targets and downregulation of tumour suppressor genes. These data support the hypothesis that global remodelling of DNA methylation is a key factor in mediating cisplatin hypersensitivity and chemoresistance of TGCTs and furthers the rationale for hypomethylation therapy for refractory TGCT patients.

A phase 1 study of combined guadecitabine and cisplatin in platinum refractory germ cell cancer.

PURPOSE: Germ cell tumors (GCTs) are cured with therapy based on cisplatin, although a clinically significant number of patients are refractory and die of progressive disease. Based on preclinical studies indicating that refractory testicular GCTs are hypersensitive to hypomethylating agents (HMAs), we conducted a phase I trial combining the next-generation HMA guadecitabine (SGI-110) with cisplatin in recurrent, cisplatin-resistant GCT patients. METHODS: Patients with metastatic GCTs were treated for five consecutive days with guadecitabine followed by cisplatin on day 8, for a 28-day cycle for up to six cycles. The primary endpoint was safety and toxicity including dose-limiting toxicity (DLT) and maximum tolerated dose (MTD). RESULTS: The number of patients enrolled was 14. The majority of patients were heavily pretreated. MTD was determined to be 30 mg/m2 guadecitabine followed by 100 mg/m2 cisplatin. The major DLTs were neutropenia and thrombocytopenia. Three patients had partial responses by RECIST criteria, two of these patients, including one with primary mediastinal disease, completed the study and qualified as complete responses by serum tumor marker criteria with sustained remissions of 5 and 13 months and survival of 16 and 26 months, respectively. The overall response rate was 23%. Three patients also had stable disease indicating a clinical benefit rate of 46%. CONCLUSIONS: The combination of guadecitabine and cisplatin was tolerable and demonstrated activity in patients with platinum refractory germ cell cancer.

Mechanisms of cisplatin sensitivity and resistance in testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) are a cancer pharmacology success story with a majority of patients cured even in the highly advanced and metastatic setting. Successful treatment of TGCTs is primarily due to the exquisite responsiveness of this solid tumor to cisplatin-based therapy. However, a significant percentage of patients are, or become, refractory to cisplatin and die from progressive disease. Mechanisms for both clinical hypersensitivity and resistance have largely remained a mystery despite the promise of applying lessons to the majority of solid tumors that are not curable in the metastatic setting. Recently, this promise has been heightened by the realization that distinct (and perhaps pharmacologically replicable) epigenetic states, rather than fixed genetic alterations, may play dominant roles in not only TGCT etiology and progression but also their curability with conventional chemotherapies. In this review, it discusses potential mechanisms of TGCT cisplatin sensitivity and resistance to conventional chemotherapeutics.

Epigenetic Remodeling through Downregulation of Polycomb Repressive Complex 2 Mediates Chemotherapy Resistance in Testicular Germ Cell Tumors.

A greater understanding of the hypersensitivity and curability of testicular germ cell tumors (TGCTs) has the potential to inform strategies to sensitize other solid tumors to conventional chemotherapies. The mechanisms of cisplatin hypersensitivity and resistance in embryonal carcinoma (EC), the stem cells of TGCTs, remain largely undefined. To study the mechanisms of cisplatin resistance we generated a large panel of independently derived, acquired resistant clones from three distinct parental EC models employing a protocol designed to match standard of care regimens of TGCT patients. Transcriptomics revealed highly significant expression changes shared between resistant cells regardless of their parental origin. This was dominated by a highly significant enrichment of genes normally repressed by H3K27 methylation and the polycomb repressive complex 2 (PRC2) which correlated with a substantial decrease in global H3K27me3, H2AK119 ubiquitination, and expression of BMI1. Importantly, repression of H3K27 methylation with the EZH2 inhibitor GSK-126 conferred cisplatin resistance to parental cells while induction of H3K27 methylation with the histone lysine demethylase inhibitor GSK-J4 resulted in increased cisplatin sensitivity to resistant cells. A gene signature based on H3K27me gene enrichment was associated with an increased rate of recurrent/progressive disease in testicular cancer patients. Our data indicates that repression of H3K27 methylation is a mechanism of cisplatin acquired resistance in TGCTs and that restoration of PRC2 complex function is a viable approach to overcome treatment failure.

Towards a Unified Understanding of Lithium Action in Basic Biology and its Significance for Applied Biology.

Lithium has literally been everywhere forever, since it is one of the three elements created in the Big Bang. Lithium concentration in rocks, soil, and fresh water is highly variable from place to place, and has varied widely in specific regions over evolutionary and geologic time. The biological effects of lithium are many and varied. Based on experiments in which animals are deprived of lithium, lithium is an essential nutrient. At the other extreme, at lithium ingestion sufficient to raise blood concentration significantly over 1 mM/, lithium is acutely toxic. There is no consensus regarding optimum levels of lithium intake for populations or individuals-with the single exception that lithium is a generally accepted first-line therapy for bipolar disorder, and specific dosage guidelines for sufferers of that condition are generally agreed on. Epidemiological evidence correlating various markers of social dysfunction and disease vs. lithium level in drinking water suggest benefits of moderately elevated lithium compared to average levels of lithium intake. In contrast to other biologically significant ions, lithium is unusual in not having its concentration in fluids of multicellular animals closely regulated. For hydrogen ions, sodium ions, potassium ions, calcium ions, chloride ions, and magnesium ions, blood and extracellular fluid concentrations are closely and necessarily regulated by systems of highly selective channels, and primary and secondary active transporters. Lithium, while having strong biological activity, is tolerated over body fluid concentrations ranging over many orders of magnitude. The lack of biological regulation of lithium appears due to lack of lithium-specific binding sites and selectivity filters. Rather lithium exerts its myriad physiological and biochemical effects by competing for macromolecular sites that are relatively specific for other cations, most especially for sodium and magnesium. This review will consider what is known about the nature of this competition and suggest using and extending this knowledge towards the goal of a unified understanding of lithium in biology and the application of that understanding in medicine and nutrition.

Three-Dimensional Encapsulation of Saccharomyces cerevisiae in Silicate Matrices Creates Distinct Metabolic States as Revealed by Gene Chip Analysis.

In order to design hybrid cellular/synthetic devices such as sensors and vaccines, it is important to understand how the metabolic state of living cells changes upon physical confinement within three-dimensional (3D) matrices. We analyze the gene expression patterns of stationary phase Saccharomyces cerevisiae (S. cerevisiae) cells encapsulated within three distinct nanostructured silica matrices and relate those patterns to known naturally occurring metabolic states. Silica encapsulation methods employed were lipid-templated mesophase silica thin films formed by cell-directed assembly (CDA), lipid-templated mesophase silica particles formed by spray drying (SD), and glycerol-doped silica gel monoliths prepared from an aqueous silicate (AqS+g) precursor solution. It was found that the cells for all three-encapsulated methods enter quiescent states characteristic of response to stress, albeit to different degrees and with differences in detail. By the measure of enrichment of stress-related gene ontology categories, we find that the AqS+g encapsulation is more amenable to the cells than CDA and SD encapsulation. We hypothesize that this differential response in the AqS+g encapsulation is related to four properties of the encapsulating gel: (1) oxygen permeability, (2) relative softness of the material, (3) development of a protective sheath around individual cells (visible in TEM micrographs vide infra), and (4) the presence of glycerol in the gel, which has been previously noted to serve as a protectant for encapsulated cells and can serve as the sole carbon source for S. cerevisiae under aerobic conditions. This work represents a combination of experiment and analysis aimed at the design and development of 3D encapsulation procedures to induce, and perhaps control, well-defined physiological behaviors.

Multifactorial Understanding of Ion Abundance in Tandem Mass Spectrometry Experiments.

In a bottom-up shotgun approach, the proteins of a mixture are enzymatically digested, separated, and analyzed via tandem mass spectrometry. The mass spectra relating fragment ion intensities (abundance) to the mass-to-charge are used to deduce the amino acid sequence and identify the peptides and proteins. The variables that influence intensity were characterized using a multi-factorial mixed-effects model, a ten-fold cross-validation, and stepwise feature selection on 6,352,528 fragment ions from 61,543 peptide ions. Intensity was higher in fragment ions that did not have neutral mass loss relative to any mass loss or that had a +1 charge state. Peptide ions classified for proton mobility as non-mobile had lowest intensity of all mobility levels. Higher basic residue (arginine, lysine or histidine) counts in the peptide ion and low counts in the fragment ion were associated with lower fragment ion intensities. Higher counts of proline in peptide and fragment ions were associated with lower intensities. These results are consistent with the mobile proton theory. Opposite trends between peptide and fragment ion counts and intensity may be due to the different impact of factor under consideration at different stages of the MS/MS experiment or to the different distribution of observations across peptide and fragment ion levels. Presence of basic residues at all three positions next to the fragmentation site was associated with lower fragment ion intensity. The presence of proline proximal to the fragmentation site enhanced fragmentation and had the opposite trend when located distant from the site. A positive association between fragment ion intensity and presence of sulfur residues (cysteine and methionine) on the vicinity of the fragmentation site was identified. These results highlight the multi-factorial nature of fragment ion intensity and could improve the algorithms for peptide identification and the simulation in tandem mass spectrometry experiments.

POLYAR, a new computer program for prediction of poly(A) sites in human sequences.

BACKGROUND: mRNA polyadenylation is an essential step of pre-mRNA processing in eukaryotes. Accurate prediction of the pre-mRNA 3'-end cleavage/polyadenylation sites is important for defining the gene boundaries and understanding gene expression mechanisms. RESULTS: 28761 human mapped poly(A) sites have been classified into three classes containing different known forms of polyadenylation signal (PAS) or none of them (PAS-strong, PAS-weak and PAS-less, respectively) and a new computer program POLYAR for the prediction of poly(A) sites of each class was developed. In comparison with polya_svm (till date the most accurate computer program for prediction of poly(A) sites) while searching for PAS-strong poly(A) sites in human sequences, POLYAR had a significantly higher prediction sensitivity (80.8% versus 65.7%) and specificity (66.4% versus 51.7%) However, when a similar sort of search was conducted for PAS-weak and PAS-less poly(A) sites, both programs had a very low prediction accuracy, which indicates that our knowledge about factors involved in the determination of the poly(A) sites is not sufficient to identify such polyadenylation regions. CONCLUSIONS: We present a new classification of polyadenylation sites into three classes and a novel computer program POLYAR for prediction of poly(A) sites/regions of each of the class. In tests, POLYAR shows high accuracy of prediction of the PAS-strong poly(A) sites, though this program's efficiency in searching for PAS-weak and PAS-less poly(A) sites is not very high but is comparable to other available programs. These findings suggest that additional characteristics of such poly(A) sites remain to be elucidated. POLYAR program with a stand-alone version for downloading is available at http://cub.comsats.edu.pk/polyapredict.htm.