Transcriptomic analysis of pancreatic adenocarcinoma specimens obtained from Black and White patients.

In pancreatic cancer clinical trials, Black patients are under-represented while having higher morbidity and mortality rates as compared to other racial groups. Multiple factors, including socioeconomic and lifestyle factors may contribute to this disparity, but genomic contributions remain unclear. In an exploratory project to identify genes that may contribute to differences in survival between Black (n = 8) and White (n = 20) patients with pancreatic cancer, transcriptomic sequencing of over 24,900 genes was performed in human pancreatic tumor and non-tumor tissue obtained from Black and White patients. Over 4,400 genes were differentially expressed in tumor and non-tumor tissue, irrespective of race. To validate these results, the expression of four genes (AGR2, POSTN, TFF1, and CP) reported to be up-regulated in pancreatic tumor tissue as compared to non-tumor tissue were confirmed using quantitative PCR. Transcriptomic analysis that compared pancreatic tumor tissue from Black and White patients revealed differential expression in 1,200 genes, while a comparison of the non-tumor and tumor gene expression differences within each race revealed over 1,500 tumor-specific differentially expressed genes in pancreatic tumor and non-tumor tissue from Black patients. We identified TSPAN8 as a potential tumor-specific gene significantly overexpressed in pancreatic tumor tissue in Black patients as compared to White patients. Using Ingenuity Pathway Analysis software to compare the race-associated gene expression profiles, over 40 canonical pathways were identified to be potentially impacted by the gene expression differences between the races. Heightened expression of TSPAN8 was associated with poor overall survival, suggesting TSPAN8 as one potential genetic factor contributing to the differential outcomes in Black patients with pancreatic cancer, supporting the potential utility of larger genomic studies to further explore the role of TSPAN8 in pancreatic cancer.

SARS-CoV-2 spike protein variant binding affinity to an angiotensin-converting enzyme 2 fusion glycoproteins.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (Covid-19) pandemic, continues to evolve and circulate globally. Current prophylactic and therapeutic countermeasures against Covid-19 infection include vaccines, small molecule drugs, and neutralizing monoclonal antibodies. SARS-CoV-2 infection is mainly mediated by the viral spike glycoprotein binding to angiotensin converting enzyme 2 (ACE2) on host cells for viral entry. As emerging mutations in the spike protein evade efficacy of spike-targeted countermeasures, a potential strategy to counter SARS-CoV-2 infection is to competitively block the spike protein from binding to the host ACE2 using a soluble recombinant fusion protein that contains a human ACE2 and an IgG1-Fc domain (ACE2-Fc). Here, we have established Chinese Hamster Ovary (CHO) cell lines that stably express ACE2-Fc proteins in which the ACE2 domain either has or has no catalytic activity. The fusion proteins were produced and purified to partially characterize physicochemical properties and spike protein binding. Our results demonstrate the ACE2-Fc fusion proteins are heavily N-glycosylated, sensitive to thermal stress, and actively bind to five spike protein variants (parental, alpha, beta, delta, and omicron) with different affinity. Our data demonstrates a proof-of-concept production strategy for ACE2-Fc fusion glycoproteins that can bind to different spike protein variants to support the manufacture of potential alternative countermeasures for emerging SARS-CoV-2 variants.

An etanercept O-glycovariant with enhanced potency.

Most therapeutic proteins are glycosylated with N-glycans and/or O-glycans. N-glycans on therapeutic proteins have been extensively studied for their control strategy and impact on drug product quality. However, knowledge of O-glycosylation in therapeutic protein production and its impact on product quality remains elusive. To address this gap, we generated an O-glycoengineered Chinese Hamster Ovary (CHO) cell line platform to modulate O-glycosylation of therapeutic proteins and investigated the impact of O-glycans on the physicochemical and biological properties of etanercept. Our results demonstrate that this CHO cell line platform produces controlled O-glycosylation profiles containing either truncated O-glycans (sialylTn and/or Tn), or sialylCore 3 alone, or sialylCore 1 with sialylTn or sialylCore 3 O-glycans on endogenous and recombinant proteins. Moreover, the platform demonstrated exclusive modulation of O-glycosylation without affecting N-glycosylation. Importantly, certain O-glycans on etanercept enhanced tumor necrosis factor-α binding affinity and consequent potency. This is the first report that describes the systematic establishment of an O-glycoengineered CHO cell line platform with direct evidence that supports the applicability of the platform in the production of engineered proteins with desired O-glycans. This platform is valuable for identifying O-glycosylation as a critical quality attribute of biotherapeutics using the quality by design principle.

Mitochondrial dysfunction generates aggregates that resist lysosomal degradation in human breast cancer cells.

Disrupting functional protein homeostasis is an established therapeutic strategy for certain tumors. Ongoing studies are evaluating autophagy inhibition for overcoming chemotherapeutic resistance to such therapies by neutralizing lysosomal pH. New and sensitive methods to monitor autophagy in patients are needed to improve trial design and interpretation. We report that mitochondrial-damaged breast cancer cells and rat breast tumors accumulate p53-positive protein aggregates that resist lysosomal degradation. These aggregates were localized to enzymatically-active autolysosomes that were degrading autophagosomes and the autophagic receptor proteins TAX1BP1 and NDP52. NDP52 was identified to associate with aggregated proteins and knocking down NDP52 led to the accumulation of protein aggregates. TAX1BP1 was identified to partly localize with aggregates, and knocking down TAX1BP1 enhanced aggregate formation, suppressed autophagy, impaired NDP52 autophagic degradation and induced cell death. We propose that quantifying aggregates and autophagic receptors are two potential methods to evaluate autophagy and lysosomal degradation, as confirmed using primary human tumor samples. Collectively, this report establishes protein aggregates and autophagy receptors, TAX1BP1 and NDP52, as potential endpoints for monitoring autophagy during drug development and clinical studies.

Doxorubicin-induced cardiotoxicity is suppressed by estrous-staged treatment and exogenous 17ß-estradiol in female tumor-bearing spontaneously hypertensive rats.

BACKGROUND: Doxorubicin (DOX), an anthracycline therapeutic, is widely used to treat a variety of cancer types and known to induce cardiomyopathy in a time and dose-dependent manner. Postmenopausal and hypertensive females are two high-risk groups for developing adverse effects following DOX treatment. This may suggest that endogenous reproductive hormones can in part suppress DOX-induced cardiotoxicity. Here, we investigated if the endogenous fluctuations in 17ß-estradiol (E2) and progesterone (P4) can in part suppress DOX-induced cardiomyopathy in SST-2 tumor-bearing spontaneously hypersensitive rats (SHRs) and evaluate if exogenous administration of E2 and P4 can suppress DOX-induced cardiotoxicity in tumor-bearing ovariectomized SHRs (ovaSHRs). METHODS: Vaginal cytology was performed on all animals to identify the stage of the estrous cycle. Estrous-staged SHRs received a single injection of saline, DOX, dexrazoxane (DRZ), or DOX combined with DRZ. OvaSHRs were implanted with time-releasing pellets that contained a carrier matrix (control), E2, P4, Tamoxifen (Tam), and combinations of E2 with P4 and Tam. Hormone pellet-implanted ovaSHRs received a single injection of saline or DOX. Cardiac troponin I (cTnI), E2, and P4 serum concentrations were measured before and after treatment in all animals. Cardiac damage and function were further assessed by echocardiography and histopathology. Weight, tumor size, and uterine width were measured for all animals. RESULTS: In SHRs, estrous-staged DOX treatment altered acute estrous cycling that ultimately resulted in prolonged diestrus. Twelve days after DOX administration, all SHRs had comparable endogenous circulating E2. Thirteen days after DOX treatment, SHRs treated during proestrus had decreased cardiac output and increased cTnI as compared to animals treated during estrus and diestrus. DOX-induced tumor reduction was not affected by estrous-staged treatments. In ovaSHRs, exogenous administration of E2 suppressed DOX-induced cardiotoxicity, while P4-implanted ovaSHRs were partly resistant. However, ovaSHRs treated with E2 and P4 did not have cardioprotection against DOX-induced damage. CONCLUSIONS: This study demonstrates that estrous-staged treatments can alter the extent of cardiac damage caused by DOX in female SHRs. The study also supports that exogenous E2 can suppress DOX-induced myocardial damage in ovaSHRs.

Loss of sirtuin 1 and mitofusin 2 contributes to enhanced ischemia/reperfusion injury in aged livers.

Ischemia/reperfusion (I/R) injury is a causative factor contributing to morbidity and mortality during liver resection and transplantation. Livers from elderly patients have a poorer recovery from these surgeries, indicating reduced reparative capacity with aging. Mechanisms underlying this age-mediated hypersensitivity to I/R injury remain poorly understood. Here, we investigated how sirtuin 1 (SIRT1) and mitofusin 2 (MFN2) are affected by I/R in aged livers. Young (3 months) and old (23-26 months) male C57/BL6 mice were subjected to hepatic I/R in vivo. Primary hepatocytes isolated from each age group were also exposed to simulated in vitro I/R. Biochemical, genetic, and imaging analyses were performed to assess cell death, autophagy flux, mitophagy, and mitochondrial function. Compared to young mice, old livers showed accelerated liver injury following mild I/R. Reperfusion of old hepatocytes also showed necrosis, accompanied with defective autophagy, onset of the mitochondrial permeability transition, and mitochondrial dysfunction. Biochemical analysis indicated a near-complete loss of both SIRT1 and MFN2 after I/R in old hepatocytes, which did not occur in young cells. Overexpression of either SIRT1 or MFN2 alone in old hepatocytes failed to mitigate I/R injury, while co-overexpression of both proteins promoted autophagy and prevented mitochondrial dysfunction and cell death after reperfusion. Genetic approaches with deletion and point mutants revealed that SIRT1 deacetylated K655 and K662 residues in the C-terminus of MFN2, leading to autophagy activation. The SIRT1-MFN2 axis is pivotal during I/R recovery and may be a novel therapeutic target to reduce I/R injury in aged livers.

Mitochondrial dysfunction activates lysosomal-dependent mitophagy selectively in cancer cells.

Molecules designed to target and accumulate in the mitochondria are an emerging therapeutic approach for cancer and other indications. Mitochondria-targeted redox agents (MTAs) induce mitochondrial damage and autophagy in cancer cells. However, the mechanisms for these molecules to induce mitophagy, the clearance of damaged mitochondria, are largely unknown. Using breast derived cell lines and a series of targeted molecules, mitochondrial dysfunction and autophagy was established to be selective for MDA-MB-231 cancer cells as compared to the non-cancerous MCF-12A cells. Kinetic analyses revealed that mitochondrial dysfunction precedes the activation of autophagy in these cancer cells. To determine the onset of mitophagy, stably expressing mitochondrial mKeima, a mitochondrial pH sensor, cell lines were generated and revealed that these drugs activate lysosomal dependent mitochondrial degradation in MDA-MB-231 cells. Mitophagy was confirmed by identifying the accumulation of a PINK1, mitochondria located in autophagosomes, and the formation of an autophagosome-mitochondria protein (MFN2-LC3-II) complex. These results are the first to demonstrate that mitochondrial redox agents selectively induce mitophagy in a breast cancer cell line and their potential application both as tools for investigating mitochondrial biomechanics and as therapeutic strategies that target mitochondrial metabolism.

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity.

Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis.

Carbamazepine suppresses calpain-mediated autophagy impairment after ischemia/reperfusion in mouse livers.

Onset of the mitochondrial permeability transition (MPT) plays a causative role in ischemia/reperfusion (I/R) injury. Current therapeutic strategies for reducing reperfusion injury remain disappointing. Autophagy is a lysosome-mediated, catabolic process that timely eliminates abnormal or damaged cellular constituents and organelles such as dysfunctional mitochondria. I/R induces calcium overloading and calpain activation, leading to degradation of key autophagy-related proteins (Atg). Carbamazepine (CBZ), an FDA-approved anticonvulsant drug, has recently been reported to increase autophagy. We investigated the effects of CBZ on hepatic I/R injury. Hepatocytes and livers from male C57BL/6 mice were subjected to simulated in vitro, as well as in vivo I/R, respectively. Cell death, intracellular calcium, calpain activity, changes in autophagy-related proteins (Atg), autophagic flux, MPT and mitochondrial membrane potential after I/R were analyzed in the presence and absence of 20 µM CBZ. CBZ significantly increased hepatocyte viability after reperfusion. Confocal microscopy revealed that CBZ prevented calcium overloading, the onset of the MPT and mitochondrial depolarization. Immunoblotting and fluorometric analysis showed that CBZ blocked calpain activation, depletion of Atg7 and Beclin-1 and loss of autophagic flux after reperfusion. Intravital multiphoton imaging of anesthetized mice demonstrated that CBZ substantially reversed autophagic defects and mitochondrial dysfunction after I/R in vivo. In conclusion, CBZ prevents calcium overloading and calpain activation, which, in turn, suppresses Atg7 and Beclin-1 depletion, defective autophagy, onset of the MPT and cell death after I/R.