A treatise on endothelial biology and exosomes: homage to Theresa Maria Listowska Whiteside.

Exosomes are the current primary research focus of Dr. Theresa L. Whiteside. They are key mediators of intercellular communication in the head and neck, as well as other sites. Their effects in the tumor microenvironment are manifold and include suppression of immunity, promotion of angiogenesis, enabling of metastasis, as well as reprogramming of fibroblasts and mesenchymal stromal cells. The aim of this communication is to summarize Dr. Whiteside's contribution to the field of exosome research and details the interactions of exosomes with endothelial cells leading to recent findings on how to target endothelial cells using exosomes as a therapeutic approach.

Efficacy of adoptive therapy with tumor-infiltrating lymphocytes and recombinant interleukin-2 in advanced cutaneous melanoma: a systematic review and meta-analysis.

Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) has been tested in advanced melanoma patients at various centers. We conducted a systematic review and meta-analysis to assess its efficacy on previously treated advanced metastatic cutaneous melanoma. The PubMed electronic database was searched from inception to 17 December 2018 to identify studies administering TIL-ACT and recombinant interleukin-2 (IL-2) following non-myeloablative chemotherapy in previously treated metastatic melanoma patients. Objective response rate (ORR) was the primary end point. Secondary end points were complete response rate (CRR), overall survival (OS), duration of response (DOR) and toxicity. Pooled estimates were derived from fixed or random effect models, depending on the amount of heterogeneity detected. Analysis was carried out separately for high dose (HD) and low dose (LD) IL-2. Sensitivity analyses were carried out. Among 1211 records screened, 13 studies (published 1988 - 2016) were eligible for meta-analysis. Among 410 heavily pretreated patients (some with brain metastasis), 332 received HD-IL-2 and 78 LD-IL-2. The pooled overall ORR estimate was 41% [95% confidence interval (CI) 35% to 48%], and the overall CRR was 12% (95% CI 7% to 16%). For the HD-IL-2 group, the ORR was 43% (95% CI 36% to 50%), while for the LD-IL-2 it was 35% (95% CI 25% to 45%). Corresponding pooled estimates for CRR were 14% (95% CI 7% to 20%) and 7% (95% CI 1% to 12%). The majority of HD-IL-2 complete responders (27/28) remained in remission during the extent of follow-up after CR (median 40 months). Sensitivity analyses yielded similar results. Higher number of infused cells was associated with a favorable response. The ORR for HD-IL-2 compared favorably with the nivolumab/ipilimumab combination following anti-PD-1 failure. TIL-ACT therapy, especially when combined with HD-IL-2, achieves durable clinical benefit and warrants further investigation. We discuss the current position of TIL-ACT in the therapy of advanced melanoma, particularly in the era of immune checkpoint blockade therapy, and review future opportunities for improvement of this approach.

First-in-human phase I study of copanlisib (BAY 80-6946), an intravenous pan-class I phosphatidylinositol 3-kinase inhibitor, in patients with advanced solid tumors and non-Hodgkin's lymphomas.

BACKGROUND: To evaluate the safety, tolerability, pharmacokinetics, and maximum tolerated dose (MTD) of copanlisib, a phosphatidylinositol 3-kinase inhibitor, in patients with advanced solid tumors or non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: Phase I dose-escalation study including patients with advanced solid tumors or NHL, and a cohort of patients with type 2 diabetes mellitus. Patients received three weekly intravenous infusions of copanlisib per 28-day cycle over the dose range 0.1-1.2 mg/kg. Plasma copanlisib levels were analyzed for pharmacokinetics. Biomarker analysis included PIK3CA, KRAS, BRAF, and PTEN mutational status and PTEN immunohistochemistry. Whole-body [(18)F]-fluorodeoxyglucose positron emission tomography ((18)FDG-PET) was carried out at baseline and following the first dose to assess early pharmacodynamic effects. Plasma glucose and insulin levels were evaluated serially. RESULTS: Fifty-seven patients received treatment. The MTD was 0.8 mg/kg copanlisib. The most frequent treatment-related adverse events were nausea and transient hyperglycemia. Copanlisib exposure was dose-proportional with no accumulation; peak exposure positively correlated with transient hyperglycemia post-infusion. Sixteen of 20 patients treated at the MTD had reduced (18)FDG-PET uptake; 7 (33%) had a reduction >25%. One patient achieved a complete response (CR; endometrial carcinoma exhibiting both PIK3CA and PTEN mutations and complete PTEN loss) and two had a partial response (PR; both metastatic breast cancer). Among the nine NHL patients, all six with follicular lymphoma (FL) responded (one CR and five PRs) and one patient with diffuse large B-cell lymphoma had a PR by investigator assessment; two patients with FL who achieved CR (per post hoc independent radiologic review) were on treatment >3 years. CONCLUSION: Copanlisib, dosed intermittently on days 1, 8, and 15 of a 28-day cycle, was well tolerated and the MTD was determined to be 0.8 mg/kg. Copanlisib exhibited dose-proportional pharmacokinetics and promising anti-tumor activity, particularly in patients with NHL. CLINICALTRIALSGOV: NCT00962611; https://clinicaltrials.gov/ct2/show/NCT00962611.

The receptor for advanced glycation end products (RAGE) enhances autophagy and neutrophil extracellular traps in pancreatic cancer.

Neutrophil extracellular traps (NETs) are formed when neutrophils expel their DNA, histones and intracellular proteins into the extracellular space or circulation. NET formation is dependent on autophagy and is mediated by citrullination of histones to allow for the unwinding and subsequent expulsion of DNA. NETs have an important role in the pathogenesis of several sterile inflammatory diseases, including malignancy, therefore we investigated the role of NETs in the setting of pancreatic ductal adenocarcinoma (PDA). Neutrophils isolated from two distinct animal models of PDA had an increased propensity to form NETs following stimulation with platelet activating factor (PAF). Serum DNA, a marker of circulating NET formation, was elevated in tumor bearing animals as well as in patients with PDA. Citrullinated histone H3 expression, a marker of NET formation, was observed in pancreatic tumors obtained from murine models and patients with PDA. Inhibition of autophagy with chloroquine or genetic ablation of receptor for advanced glycation end products (RAGE) resulted in decreased propensity for NET formation, decreased serum DNA and decreased citrullinated histone H3 expression in the pancreatic tumor microenvironment. We conclude that NETs are upregulated in pancreatic cancer through RAGE-dependent/autophagy mediated pathways.

You eat what you are: autophagy inhibition as a therapeutic strategy in leukemia.

A deeper understanding of the role of autophagy, literally 'self-eating', in normal and cancer cell biology has emerged over the last few years. Autophagy serves as a vehicle for cells to respond to various stressors including genomic, hypoxic and nutrient stress, and to oppose mechanisms of 'programmed' cell death. Here, we review not only mechanisms of cell death and cell survival but also the early successes in applying autophagy inhibition strategies in solid tumors using the only currently available clinical inhibitor, oral hydroxychloroquine. In acute leukemia, currently available chemotherapy drugs promote cell death and demonstrate clinical benefit, but relapse and subsequent chemotherapy resistance is common. Increasing preclinical data suggest that autophagy is active in leukemia as a means of promoting cell survival in response to chemotherapy. We propose coupling autophagy inhibition strategies with current cytotoxic chemotherapy and discuss synergistic combinations of available anti-leukemic therapies with autophagy inhibition. Furthermore, novel autophagy inhibitors are in development and promise to provide new therapeutic opportunities for patients with leukemia.

Plasma biosignature and brain pathology related to persistent cognitive impairment in late-life depression.

Cognitive impairment is highly prevalent among individuals with late-life depression (LLD) and tends to persist even after successful treatment. The biological mechanisms underlying cognitive impairment in LLD are complex and likely involve abnormalities in multiple pathways, or 'cascades,' reflected in specific biomarkers. Our aim was to evaluate peripheral (blood-based) evidence for biological pathways associated with cognitive impairment in older adults with LLD. To this end, we used a data-driven comprehensive proteomic analysis (multiplex immunoassay including 242 proteins), along with measures of structural brain abnormalities (gray matter atrophy and white matter hyperintensity volume via magnetic resonance imaging), and brain amyloid-ß (Aß) deposition (PiB-positron emission tomography). We analyzed data from 80 older adults with remitted major depression (36 with mild cognitive impairment (LLD+MCI) and 44 with normal cognitive (LLD+NC)) function. LLD+MCI was associated with differential expression of 24 proteins (P<0.05 and q-value <0.30) related mainly to the regulation of immune-inflammatory activity, intracellular signaling, cell survival and protein and lipid homeostasis. Individuals with LLD+MCI also showed greater white matter hyperintensity burden compared with LLD+NC (P=0.015). We observed no differences in gray matter volume or brain Aß deposition between groups. Machine learning analysis showed that a group of three proteins (Apo AI, IL-12 and stem cell factor) yielded accuracy of 81.3%, sensitivity of 75% and specificity of 86.4% in discriminating participants with MCI from those with NC function (with an averaged cross-validation accuracy of 76.3%, sensitivity of 69.4% and specificity of 81.8% with nested cross-validation considering the model selection bias). Cognitive impairment in LLD seems to be related to greater cerebrovascular disease along with abnormalities in immune-inflammatory control, cell survival, intracellular signaling, protein and lipid homeostasis, and clotting processes. These results suggest that individuals with LLD and cognitive impairment may be more vulnerable to accelerated brain aging and shed light on possible mediators of their elevated risk for progression to dementia.

RAGE is essential for oncogenic KRAS-mediated hypoxic signaling in pancreatic cancer.

A hypoxic tumor microenvironment is characteristic of many cancer types, including one of the most lethal, pancreatic cancer. We recently demonstrated that the receptor for advanced glycation end products (RAGE) has an important role in promoting the development of pancreatic cancer and attenuating the response to chemotherapy. We now demonstrate that binding of RAGE to oncogenic KRAS facilitates hypoxia-inducible factor 1 (HIF1)α activation and promotes pancreatic tumor growth under hypoxic conditions. Hypoxia induces NF-κB-dependent and HIF1α-independent RAGE expression in pancreatic tumor cells. Moreover, the interaction between RAGE and mutant KRAS increases under hypoxia, which in turn sustains KRAS signaling pathways (RAF-MEK-ERK and PI3K-AKT), facilitating stabilization and transcriptional activity of HIF1α. Knock down of RAGE in vitro inhibits KRAS signaling, promotes HIF1α degradation, and increases hypoxia-induced pancreatic tumor cell death. RAGE-deficient mice have impaired oncogenic KRAS-driven pancreatic tumor growth with significant downregulation of the HIF1α signaling pathway. Our results provide a novel mechanistic link between NF-κB, KRAS, and HIF1α, three potent molecular pathways in the cellular response to hypoxia during pancreatic tumor development and suggest alternatives for preventive and therapeutic strategies.

The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics.

Tumor cells require increased adenosine triphosphate (ATP) to support anabolism and proliferation. The precise mechanisms regulating this process in tumor cells are unknown. Here, we show that the receptor for advanced glycation endproducts (RAGE) and one of its primary ligands, high-mobility group box 1 (HMGB1), are required for optimal mitochondrial function within tumors. We found that RAGE is present in the mitochondria of cultured tumor cells as well as primary tumors. RAGE and HMGB1 coordinately enhanced tumor cell mitochondrial complex I activity, ATP production, tumor cell proliferation and migration. Lack of RAGE or inhibition of HMGB1 release diminished ATP production and slowed tumor growth in vitro and in vivo. These findings link, for the first time, the HMGB1-RAGE pathway with changes in bioenergetics. Moreover, our observations provide a novel mechanism within the tumor microenvironment by which necrosis and inflammation promote tumor progression.

Strange attractors: DAMPs and autophagy link tumor cell death and immunity.

Resistance to 'apoptotic' cell death is one of the major hallmarks of cancer, contributing to tumor development and therapeutic resistance. Damage-associated molecular patterns (DAMPs) are molecules released or exposed by dead, dying, injured, or stressed non-apoptotic cells, with multiple roles in inflammation and immunity. Release of DAMPs not only contributes to tumor growth and progression but also mediates skewing of antitumor immunity during so-called immunogenic tumor cell death (ICD). Autophagy is a lysosome-mediated homeostatic degradation process in which cells digest their own effete organelles and macromolecules to meet bioenergetic needs and enable protein synthesis. For tumor cells, autophagy is a double-edged sword. Autophagy, in balance with apoptosis, can function as a tumor suppressor; autophagy deficiency, associated with alterations in apoptosis, initiates tumorigenesis in many settings. In contrast, autophagy-related stress tolerance generally promotes cell survival, which enables tumor growth and promotes therapeutic resistance. Most anticancer therapies promote DAMP release and enhance autophagy. Autophagy not only regulates DAMP release and degradation, but also is triggered and regulated by DAMPs. This interplay between autophagy and DAMPs, serving as 'strange attractors' in the dynamic system that emerges in cancer, regulates the effectiveness of antitumor treatment. This interplay also shapes the immune response to dying cells upon ICD, culling the least fit tumor cells and promoting survival of others. Thus, DAMPs and autophagy are suitable emergent targets for cancer therapy, considering their more nuanced role in tumor progression.

The Beclin 1 network regulates autophagy and apoptosis.

Beclin 1, the mammalian orthologue of yeast Atg6, has a central role in autophagy, a process of programmed cell survival, which is increased during periods of cell stress and extinguished during the cell cycle. It interacts with several cofactors (Atg14L, UVRAG, Bif-1, Rubicon, Ambra1, HMGB1, nPIST, VMP1, SLAM, IP(3)R, PINK and survivin) to regulate the lipid kinase Vps-34 protein and promote formation of Beclin 1-Vps34-Vps15 core complexes, thereby inducing autophagy. In contrast, the BH3 domain of Beclin 1 is bound to, and inhibited by Bcl-2 or Bcl-XL. This interaction can be disrupted by phosphorylation of Bcl-2 and Beclin 1, or ubiquitination of Beclin 1. Interestingly, caspase-mediated cleavage of Beclin 1 promotes crosstalk between apoptosis and autophagy. Beclin 1 dysfunction has been implicated in many disorders, including cancer and neurodegeneration. Here, we summarize new findings regarding the organization and function of the Beclin 1 network in cellular homeostasis, focusing on the cross-regulation between apoptosis and autophagy.

Apoptosis promotes early tumorigenesis.

One hallmark of cancer is intrinsic or acquired resistance to apoptosis. Surprisingly, recent studies demonstrate that CD95/Fas/Apo1 and p53 upregulated mediator of apoptosis/PUMA (potent inducers of the death receptor and the mitochondrial apoptotic pathways, respectively) promote tumorigenesis. These findings provide important insights into the multifaceted roles of apoptosis in tumorigenesis.

HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells.

Autophagy, a tightly regulated lysosome-dependent catabolic pathway, is important in the regulation of cancer development and progression and in determining the response of tumor cells to anticancer therapy. However, the role of autophagy in leukemia still remains largely unknown. Here we show that high-mobility group box 1 (HMGB1), the best characterized damage-associated molecular pattern, was released from leukemia cell lines after chemotherapy-induced cytotoxicity and activated autophagy to protect against injury. Treatment with HMGB1-neutralizing antibodies increased the sensitivity of leukemia cells to chemotherapy; whereas, exogenous HMGB1 rendered these cells more resistant to drug-induced cytotoxicity. Moreover, exogenous HMGB1 increased autophagy as evaluated by increased expression of the autophagic marker microtubule-associated protein light chain 3-II, degradation of sequestosome 1 (p62) and autophagosome formation. Furthermore, knockdown or pharmacological inhibition of either phosphoinositide 3-kinase-III or extracellular signal-regulated kinase kinase mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase inhibited HMGB1-induced autophagy. Taken together, these results suggest that HMGB1 release after chemotherapy is a critical regulator of autophagy and a potential drug target for therapeutic interventions in leukemia.

HMGB1 release and redox regulates autophagy and apoptosis in cancer cells.

The functional relationship and cross-regulation between autophagy and apoptosis is complex. In this study we show that the high-mobility group box 1 protein (HMGB1) is a redox-sensitive regulator of the balance between autophagy and apoptosis. In cancer cells, anticancer agents enhanced autophagy and apoptosis, as well as HMGB1 release. HMGB1 release may be a prosurvival signal for residual cells after various cytotoxic cancer treatments. Diminished HMGB1 by short hairpin RNA transfection or inhibition of HMGB1 release by ethyl pyruvate or other small molecules led predominantly to apoptosis and decreased autophagy in stressed cancer cells. In this setting, reducible HMGB1 binds to the receptor for advanced glycation end products (RAGEs), but not to Toll-like receptor 4, induces Beclin1-dependent autophagy and promotes tumor resistance to alkylators (melphalan), tubulin disrupting agents (paclitaxel), DNA crosslinkers (ultraviolet light) and DNA intercalators (oxaliplatin or adriamycin). On the contrary, oxidized HMGB1 increases the cytotoxicity of these agents and induces apoptosis mediated by the caspase-9/-3 intrinsic pathway. HMGB1 release, as well as its redox state, thus links autophagy and apoptosis, representing a suitable target when coupled with conventional tumor treatments.

The receptor for advanced glycation end products (RAGE) sustains autophagy and limits apoptosis, promoting pancreatic tumor cell survival.

Activation of the induced receptor for advanced glycation end products (RAGE) leads to initiation of NF-kappaB and MAP kinase signaling pathways, resulting in propagation and perpetuation of inflammation. RAGE-knockout animals are less susceptible to acute inflammation and carcinogen-induced tumor development. We have reported that most forms of tumor cell death result in release of the RAGE ligand, high-mobility group protein 1 (HMGB1). We now report a novel role for RAGE in the tumor cell response to stress. Targeted knockdown of RAGE in the tumor cell, leads to increased apoptosis, diminished autophagy and decreased tumor cell survival . In contrast, overexpression of RAGE is associated with enhanced autophagy, diminished apoptosis and greater tumor cell viability. RAGE limits apoptosis through a p53-dependent mitochondrial pathway. Moreover, RAGE-sustained autophagy is associated with decreased phosphorylation of mammalian target of rapamycin (mTOR) and increased Beclin-1/VPS34 autophagosome formation. These findings show that the inflammatory receptor, RAGE, has a heretofore unrecognized role in the tumor cell response to stress. Furthermore, these studies establish a direct link between inflammatory mediators in the tumor microenvironment and resistance to programmed cell death. Our data suggest that targeted inhibition of RAGE or its ligands may serve as novel targets to enhance current cancer therapies.

Coordinate NF-kappaB and STAT1 activation promotes development of myeloid type 1 dendritic cells.

NF-kappaB and STAT1 are critically involved in the initiation of the inflammatory cascade. Using semi-automated imaging cytometry and fluorescent antibodies, we screened several factors for their ability to induce nuclear translocation of RelA/NF-kappaB and STAT1 in subsets of monocyte-derived dendritic cells (DC). Detailed kinetics and dose-response studies are presented for IL-1-, LPS-, CD40L-, IFN-gamma- and IFN-alpha-stimulated responses. The results are consistent with the notion that simultaneous activation of both STAT1 and NF-kappaB pathways at the initiation of differentiation culture is required for efficient priming of IL-12 production by DC. Maturation of DC led to characteristic NF-kappaB and STAT1 distribution and response patterns. During the resting stage, DC differentiated under the presence of IFN-gamma showed sustained STAT activation and remained responsive to LPS. By contrast, PGE2-supplemented DC could be characterized by negligible responses to LPS and IFN-gamma and a remarkable NF-kappaB response to CD40L. STAT1 pathway was suppressed in PGE2-supplemented cells. We conclude that the magnitude and temporal kinetics of the nuclear shift of STAT1 and NF-kappaB in myeloid DC are associated with IL-12p70 production and are dependent on the nature of the stimulating factors and the polarization state of cells.

Multianalyte profiling of serum cytokines for detection of pancreatic cancer.

Early detection of pancreatic cancer might improve clinical outcome. Significant alterations in the levels of individual serum cytokines have been reported in pancreatic cancer. We hypothesized that a multicytokine panel could serve as biomarkers for pancreatic cancer. To evaluate the diagnostic utility of such a panel, we have utilized a novel multianalyte LabMAP profiling technology that allows simultaneous measurement of multiple markers. In this study, a panel of 31 serological markers including cytokines, chemokines, growth and angiogenic factors in combination with CA 19-9 was analyzed in sera of pancreatic cancer patients, patients with chronic pancreatitis, and matched control healthy subjects. Statistical analysis identified a multicytokine panel that was able to distinguish pancreatic cancer from healthy controls with a sensitivity of 85.7% and specificity of 92.3%, which was superior to performance of CA 19-9 alone. Importantly, a multicytokine panel allowed the discrimination of pancreatic cancer from chronic pancreatitis with high sensitivity of 98% and specificity of 96.4%. In conclusion, we demonstrated that analysis of multiple serum cytokines using a novel LabMAP technology is a promising approach for development of a diagnostic assay for pancreatic cancer.

Therapeutic and specific antitumor immunity induced by co-administration of immature dendritic cells and adenoviral vector expressing biologically active IL-18.

Interleukin-18 is a potent cytokine expressed early in the immune response following cleavage in activated composes. We have investigated the in vivo antitumor effects of intratumoral (i.t.) administration of an adenoviral vector expressing biologically active murine interleukin (IL)-18 (Ad.PTH.IL-18). Substantial antitumor effects were observed when established MCA205 fibrosarcoma was treated in syngeneic immunocompetent mice with intratumoral injection of Ad.PTH.IL-18 (P = 0.0025 versus control vector treatment), generating potent cytotoxic T lymphocytes (CTLs) in culture. In contrast, the antitumor effect was absent, and cytotoxic activity was significantly less (P = 0.021) in gld mice (Fas ligand deficient). To enhance the in vivo antitumor activity of the treatment using Ad.PTH.IL-18, we co-injected immature DC and Ad.PTH.IL-18 i.t. into established, day 7 MCA205 fibrosarcoma and MC38 adenocarcinoma. Co-injection of both Ad.PTH.IL-18 and DC was associated with complete abrogation of injected tumors. Furthermore, the antitumor effects were also observed on distant tumors inoculated i.d. in the contralateral flank of the animal. The induced cytolytic activity was tumor-specific and MHC class I-restricted. As we have previously demonstrated in vitro (Tanaka F et al, Cancer Res 2000; 60: 4838-4844) and consistent with these findings in vivo, NK, T and dendritic cells coactivately mediate the IL-18 enhanced antitumor effect. This study suggests that the coactivate strategy could be used in the clinical setting to treat patients with cancer. do

Mitochondrial lipid alterations during Fas- and radiation-induced apoptosis.

In the present study, we investigated the dynamic alterations in mitochondrial lipids occurring during Fas- and radiation-induced cell death. Cross-linking of CD-95 on Fas-sensitive Jurkat cells produced rapid increases in two species of mitochondrial phosphatidylglycerol. By 2.5 h, phosphatidylglycerol decreases below basal levels, concomitant with an increase in mitochondrial ceramide. In addition, between 1.5 and 3.0 h after anti-Fas crosslinking, there is a continued loss of mitochondrial cardiolipin. When gamma irradiation was used to induce apoptosis, similar lipid changes occurred, although with somewhat slower kinetics. Fas-resistant Jurkat cells exhibited phosphatidylglycerol as the dominant lipid species in their mitochondria. Following Fas ligation, there is a transient decrease in phosphatidylglycerol, but cardiolipin and ceramide remained unchanged. The high basal levels of PG in Fas-resistant cells and the increase in PG levels in Fas-sensitive cells undergoing apoptosis was determined to be due to increased PGP synthase activity. Thus, critical mitochondrial lipids could potentially serve as novel targets in regulating the apoptotic process.