NPEPPS Is a Druggable Driver of Platinum Resistance.

There is an unmet need to improve the efficacy of platinum-based cancer chemotherapy, which is used in primary and metastatic settings in many cancer types. In bladder cancer, platinum-based chemotherapy leads to better outcomes in a subset of patients when used in the neoadjuvant setting or in combination with immunotherapy for advanced disease. Despite such promising results, extending the benefits of platinum drugs to a greater number of patients is highly desirable. Using the multiomic assessment of cisplatin-responsive and -resistant human bladder cancer cell lines and whole-genome CRISPR screens, we identified puromycin-sensitive aminopeptidase (NPEPPS) as a driver of cisplatin resistance. NPEPPS depletion sensitized resistant bladder cancer cells to cisplatin in vitro and in vivo. Conversely, overexpression of NPEPPS in sensitive cells increased cisplatin resistance. NPEPPS affected treatment response by regulating intracellular cisplatin concentrations. Patient-derived organoids (PDO) generated from bladder cancer samples before and after cisplatin-based treatment, and from patients who did not receive cisplatin, were evaluated for sensitivity to cisplatin, which was concordant with clinical response. In the PDOs, depletion or pharmacologic inhibition of NPEPPS increased cisplatin sensitivity, while NPEPPS overexpression conferred resistance. Our data present NPEPPS as a druggable driver of cisplatin resistance by regulating intracellular cisplatin concentrations. SIGNIFICANCE: Targeting NPEPPS, which induces cisplatin resistance by controlling intracellular drug concentrations, is a potential strategy to improve patient responses to platinum-based therapies and lower treatment-associated toxicities.

Effects of Metformin and its Metabolite Guanylurea on Fathead Minnow (Pimephales promelas) Reproduction.

Metformin, along with its biotransformation product guanylurea, is commonly observed in municipal wastewaters and subsequent surface waters. Previous studies in fish have identified metformin as a potential endocrine-active compound, but there are inconsistencies with regard to its effects. To further investigate the potential reproductive toxicity of metformin and guanylurea to fish, a series of experiments was performed with adult fathead minnows (Pimephales promelas). First, explants of fathead minnow ovary tissue were exposed to 0.001-100 µM metformin or guanylurea to investigate whether the compounds could directly perturb steroidogenesis. Second, spawning pairs of fathead minnows were exposed to metformin (0.41, 4.1, and 41 µg/L) or guanylurea (1.0, 10, and 100 µg/L) for 23 days to assess impacts on reproduction. Lastly, male fathead minnows were exposed to 41 µg/L metformin, 100 µg/L guanylurea, or a mixture of both compounds, with samples collected over a 96-h time course to investigate potential impacts to the hepatic transcriptome or metabolome. Neither metformin nor guanylurea affected steroid production by ovary tissue exposed ex vivo. In the 23 days of exposure, neither compound significantly impacted transcription of endocrine-related genes in male liver or gonad, circulating steroid concentrations in either sex, or fecundity of spawning pairs. In the 96-h time course, 100 µg guanylurea/L elicited more differentially expressed genes than 41 µg metformin/L and showed the greatest impacts at 96 h. Hepatic transcriptome and metabolome changes were chemical- and time-dependent, with the largest impact on the metabolome observed at 23 days of exposure to 100 µg guanylurea/L. Overall, metformin and guanylurea did not elicit effects consistent with reproductive toxicity in adult fathead minnows at environmentally relevant concentrations. Environ Toxicol Chem 2022;41:2708-2720. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Assessing effects of aromatase inhibition on fishes with group-synchronous oocyte development using western mosquitofish (Gambusia affinis) as a model.

Exposure to certain anthropogenic chemicals can inhibit the activity to cytochrome P450 aromatase (CYP19) in fishes leading to decreased plasma 17ß-estradiol (E2), plasma vitellogenin (VTG), and egg production. Reproductive dysfunction resulting from exposure to aromatase inhibitors has been extensively investigated in several laboratory model species of fish. These model species have ovaries that undergo asynchronous oocyte development, but many fishes have ovaries with group-synchronous oocyte development. Fishes with group-synchronous oocyte development have dynamic reproductive cycles which typically occur annually and are often triggered by complex environmental cues. This has resulted in a lack of test data and uncertainty regarding sensitivities to and adverse effects of aromatase inhibition. The present study used the western mosquitofish (Gambusia affinis) as a laboratory model to investigate adverse effects of chemical aromatase inhibition on group-synchronous oocyte development. Adult female western mosquitofish were exposed to either 0, 2, or 30 µg/L of the model nonsteroidal aromatase inhibiting chemical, fadrozole, for a complete reproductive cycle. Fish were sampled at four time-points representing pre-vitellogenic resting, early vitellogenesis, late vitellogenesis/early ovarian recrudescence, and late ovarian recrudescence. Temporal changes in numerous reproductive parameters were measured, including gonadosomatic index (GSI), plasma sex steroids, and expression of selected genes in the brain, liver, and gonad that are important for reproduction. In contrast to fish from the control treatment, fish exposed to 2 and 30 µg/L of fadrozole had persistent elevated expression of cyp19 in the ovary, depressed expression of vtg in the liver, and a low GSI. These responses suggest that completion of a group-synchronous reproductive cycle was unsuccessful during the assay in fish from either fadrozole treatment. These adverse effects data show that exposure to aromatase inhibitors has the potential to cause reproductive dysfunction in a wide range of fishes with both asynchronous and group-synchronous reproductive strategies.

Quantitative Response-Response Relationships Linking Aromatase Inhibition to Decreased Fecundity are Conserved Across Three Fishes with Asynchronous Oocyte Development.

Quantitative adverse outcome pathways (qAOPs) describe quantitative response-response relationships that can predict the probability or severity of an adverse outcome for a given magnitude of chemical interaction with a molecular initiating event. However, the taxonomic domain of applicability for these predictions is largely untested. The present study began defining this applicability for a previously described qAOP for aromatase inhibition leading to decreased fecundity developed using data from fathead minnow (Pimephales promelas). This qAOP includes quantitative response-response relationships describing plasma 17ß-estradiol (E2) as a function of plasma fadrozole, plasma vitellogenin (VTG) as a function of plasma E2, and fecundity as a function of plasma VTG. These quantitative response-response relationships simulated plasma E2, plasma VTG, and fecundity measured in female zebrafish (Danio rerio) exposed to fadrozole for 21 days but not these responses measured in female Japanese medaka (Oryzias latipes). However, Japanese medaka had different basal levels of plasma E2, plasma VTG, and fecundity. Normalizing basal levels of each measurement to equal those of female fathead minnow enabled the relationships to accurately simulate plasma E2, plasma VTG, and fecundity measured in female Japanese medaka. This suggests that these quantitative response-response relationships are conserved across these three fishes when considering relative change rather than absolute measurements. The present study represents an early step toward defining the appropriate taxonomic domain of applicability and extending the regulatory applications of this qAOP.