Jason S Pierce’s research while affiliated with Medical University of South Carolina and other places

The metazoan lifespan is determined in part by a complex signaling network that regulates energy metabolism and stress responses. Key signaling hubs in this network include insulin/IGF‐1, AMPK, mTOR, and sirtuins. The Hippo/Mammalian Ste20‐like Kinase1 (MST1) pathway has been reported to maintain lifespan in Caenorhabditis elegans, but its role has not been studied in higher metazoans. In this study, we report that overexpression of Hpo, the MST1 homolog in Drosophila melanogaster, decreased lifespan with concomitant changes in lipid metabolism and aging‐associated gene expression, while RNAi Hpo depletion increased lifespan. These effects were mediated primarily by Hpo‐induced transcriptional activation of the RNA‐binding protein maternal expression at 31B (Me31b)/RCK, resulting in stabilization of mRNA‐encoding a lipolytic hormone, Akh. In mouse adipocytes, Hpo/Mst1 mediated adipocyte differentiation, phosphorylation of RNA‐binding proteins such as Rck, decapping MRNA 2 (Dcp2), enhancer Of MRNA decapping 3 (Edc3), nucleolin (NCL), and glucagon mRNA stability by interacting with Rck. Decreased lifespan in Hpo‐overexpressing Drosophila lines required expression of Me31b, but not DCP2, which was potentially mediated by recovering expression of lipid metabolic genes and formation of lipid droplets. Taken together, our findings suggest that Hpo/Mst1 plays a conserved role in longevity by regulating adipogenesis and fatty acid metabolism.

Oncogenic multidrug resistance (MDR) is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin-rich domains. As ceramides are the sole source for sphingomyelins, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-seq databases, we found patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1⁺ clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (∼2-fold) in chemoresistant ABCB1high (A498, Caki-1) compared to chemosensitive ABCB1low (ACHN, HPCT) cells. In addition, lipidomics analyses by HPLC-MS/MS showed bias towards CerS2-associated C20:0/C20:1-ceramides compared to CerS5/6-associated C14:0/C16:0-ceramides (2:1). Sphingomyelins were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1high cells was partially reversed by inhibitors of de novo ceramide synthesis (L-cycloserine) and CerS (fumonisin B1) in cell viability assays. Additionally, downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123⁺ efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically-inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and sphingomyelin composition to ultra long-chain species (C22-C26). Inhibitors of ER-associated degradation (ERAD) (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/sphingomyelin species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse MDR in renal cancers.

COVID-19 pandemic exerts a health care emergency around the world. The illness severity is heterogeneous. It is mostly unknown why some individuals who are positive for SARS-CoV-2 antibodies stay asymptomatic while others show moderate to severe disease symptoms. Reliable biomarkers for early detection of the disease are urgently needed to attenuate the virus’s spread and help make early treatment decisions. Bioactive sphingolipids play a crucial role in the regulation of viral infections and pro-inflammatory responses involved in the severity of COVID-19. However, any roles of sphingolipids in COVID-19 development or detection remain unknown. In this study, lipidomics measurement of serum sphingolipids demonstrated that reduced sphingosine levels are highly associated with the development of symptomatic COVID-19 in the majority (99.24%) SARS-CoV-2-infected patients compared to asymptomatic counterparts. The majority of asymptomatic individuals (73%) exhibited increased acid ceramidase (AC) in their serum, measured by Western blotting, consistent with elevated sphingosine levels compared to SARS-CoV-2 antibody negative controls. AC protein was also reduced in almost all of the symptomatic patients’ serum, linked to reduced sphingosine levels, measured in longitudinal acute or convalescent COVID-19 samples. Thus, reduced sphingosine levels provide a sensitive and selective serologic biomarker for the early identification of asymptomatic versus symptomatic COVID-19 patients.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950 Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each lab using a different lipidomics workflow. A total of 1527 unique lipids were measured across all laboratories, and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and inter-laboratory quality control and method validation. These analyses were performed using non-standardized laboratory-independent workflows. The consensus locations were also compared to a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Plasma deoxy-sphingoid bases are elevated in type 2 diabetes patients and correlate with the stage of diabetic distal sensorimotor polyneuropathy; however, associations between deoxy-sphingolipids (DSL) and neuropathy in type 1 diabetes have not been examined. The primary aim of this exploratory pilot study was to assess the associations between multiple sphingolipid species including DSL and free amino acids and the presence of symptomatic neuropathy in a DCCT/EDIC type 1 diabetes subcohort. Using mass spectroscopy, plasma levels of DSL and free amino acids in DCCT/EDIC type 1 diabetes participants (n = 80), with and without symptoms of neuropathy, were investigated. Patient-determined neuropathy was based on 15-item self-administered questionnaire (Michigan Neuropathy Screening Instrument) developed to assess distal symmetrical peripheral neuropathy in diabetes. Patients who scored ≥4, or reported inability to sense their feet during walking or to distinguish hot from cold water while bathing were considered neuropathic. Plasma levels of ceramide, sphingomyelin, hexosyl- and lactosylceramide species, and amino acids were measured and analyzed relative to neuropathy status in the patient. Deoxy-C24-ceramide, C24- and C26-ceramide were higher in patients with neuropathy than those without neuropathy. Cysteine was higher in patients with neuropathy. No differences in other sphingolipids or amino acids were detected. The covariate-adjusted Odds Ratios of positive patient-reported neuropathy was associated with increased levels of deoxy-C24-, and deoxy-C24:1-ceramide; C22-, C24-, and C26-ceramide; and cysteine. Plasma deoxy-ceramide and ceramide species may have potential diagnostic and prognostic significance in diabetic neuropathy.

Background/aim: Because patients with cancer of apparently equivalent stage often have different outcomes, it is necessary to gather additional information to complement cancer staging. Dysregulated sphingolipid metabolism contributes to carcinogenesis. In this retrospective pilot study, we tested the hypothesis that changes in serum levels of sphingolipids are associated with stage IV colorectal cancer (CRC). Patients and methods: We used commercially available serum samples from healthy males and patients with CRC (adenocarcinoma of the large intestine, stage IV with metastases). Blood samples were obtained immediately prior to anesthesia/surgery. We measured sphingolipid levels in sera using mass spectrometry. Results: In serum of patients with CRC, the levels of C16-, C18-, C18:1-, and C24:1-ceramide, as well as those of sphingosine, were significantly higher than those of controls. In contrast, the levels of C24-sphingomyelin were significantly lower than those of controls. A global test of association showed that ceramides and sphingomyelins but not hexosylceramides were significantly associated with stage IV CRC. Conclusion: Sphingolipids have a potential of serving as novel, non-invasive, inexpensive, and effective blood-based biomarkers to complement CRC staging for better prognosis and more personalized medicine.

Background/aim: Combining an anticancer agent fenretinide (HPR) or C6-pyridinium ceramide (LCL29) with Foscan-mediated photodynamic therapy (FoscanPDT) is expected to augment anticancer benefits of each substance. We showed that treatment with FoscanPDT+HPR enhanced accumulation of C16-dihydroceramide, and that fumonisin B1 (FB), an inhibitor of ceramide synthase, counteracted caspase-3 activation and colony-forming ability of head and neck squamous cell carcinoma (HNSCC) cells. Because cancer cells appear to be more susceptible to increased levels of the endoplasmic reticulum (ER) stress than normal cells, herein we tested the hypothesis that FoscanPDT combined with HPR or LCL29 induces FB-sensitive ER stress-associated apoptosis that affects cell survival. Materials and methods: Using an HNSCC cell line, we determined: cell survival by clonogenic assay, caspase-3 activity by spectrofluorometry, the expression of the ER markers BiP and CHOP by quantitative real-time polymerase chain reaction and western immunoblotting, and sphingolipid levels by mass spectrometry. Results: Similar to HPR+FoscanPDT, LCL29+FoscanPDT induced enhanced loss of clonogenicity and caspase-3 activation, that were both inhibited by FB. Our additional pharmacological evidence showed that the enhanced loss of clonogenicity after the combined treatments was singlet oxygen-, ER stress- and apoptosis-dependent. The combined treatments induced enhanced, FB-sensitive, up-regulation of BiP and CHOP, as well as enhanced accumulation of sphingolipids. Conclusion: Our data suggest that enhanced clonogenic cell killing after the combined treatments is dependent on oxidative- and ER-stress, apoptosis, and FB-sensitive sphingolipid production, and should help develop more effective mechanism-based therapeutic strategies.

Because photodynamic therapy (PDT) alone is not always effective as an anticancer treatment, PDT is combined with other anticancer agents for improved efficacy. The clinically-relevant fenretinide [N-(4-hydroxyphenyl) retinamide; 4HPR], was combined with the silicon phthalocyanine photosensitizer Pc4-mediated PDT to test for their potential to enhance killing of SCC17B cells, a clinically-relevant model of human head and neck squamous cell carcinoma. Because each of these treatments induces apoptosis and regulates the de novo sphingolipid (SL) biosynthesis pathway, the role of ceramide synthase, the pathway-associated enzyme, in PDT+4HPR-induced apoptotic cell death was determined using the ceramide synthase inhibitor fumonisin B1 (FB). PDT+4HPR enhanced loss of clonogenicity. zVAD-fmk, a pan-caspase inhibitor, and FB, protected cells from death post-PDT+4HPR. In contrast, the anti-apoptotic protein Bcl2 inhibitor ABT199 enhanced cell killing after PDT+4HPR. Combining PDT with 4HPR led to FB-sensitive, enhanced Bax associated with mitochondria and cytochrome c redistribution. Mass spectrometry data showed that the accumulation of C16-dihydroceramide, a precursor of ceramide in the de novo SL biosynthesis pathway, was enhanced after PDT+4HPR. Using quantitative confocal microscopy, we found that PDT+4HPR enhanced dihydroceramide/ceramide accumulation in the ER, which was inhibited by FB. The results suggest that SCC17B cells are sensitized to PDT by 4HPR via the de novo SL biosynthesis pathway and apoptosis, and imply potential clinical relevance of the combination for cancer treatment.

Combining photodynamic therapy (PDT) with another anticancer treatment modality is an important strategy for improved efficacy. PDT with Pc4, a silicon phthalocyanine photosensitizer, was combined with C6-pyridinium ceramide (LCL29) to determine their potential to promote death of SCC17B human head and neck squamous cell carcinoma cells. PDT+LCL29-induced enhanced cell death was inhibited by zVAD-fmk, a pan-caspase inhibitor, and fumonisin B1 (FB), a ceramide synthase inhibitor. Quantitative confocal microscopy showed that combining PDT with LCL29 enhanced FB-sensitive ceramide accumulation in the mitochondria. Furthermore, PDT+LCL29 induced enhanced FB-sensitive redistribution of cytochrome c and caspase-3 activation. Overall, the data indicate that PDT+LCL29 enhanced cell death via FB-sensitive, mitochondrial ceramide accumulation and apoptosis.