Radiotheranostics in oncology: Making precision medicine possible.

A quintessential setting for precision medicine, theranostics refers to a rapidly evolving field of medicine in which disease is diagnosed followed by treatment of disease-positive patients using tools for the therapy identical or similar to those used for the diagnosis. Against the backdrop of only-treat-when-visualized, the goal is a high therapeutic index with efficacy markedly surpassing toxicity. Oncology leads the way in theranostics innovation, where the approach has become possible with the identification of unique proteins and other factors selectively expressed in cancer versus healthy tissue, advances in imaging technology able to report these tissue factors, and major understanding of targeting chemicals and nanodevices together with methods to attach labels or warheads for imaging and therapy. Radiotheranostics-using radiopharmaceuticals-is becoming routine in patients with prostate cancer and neuroendocrine tumors who express the proteins PSMA (prostate-specific membrane antigen) and SSTR2 (somatostatin receptor 2), respectively, on their cancer. The palpable excitement in the field stems from the finding that a proportion of patients with large metastatic burden show complete and partial responses, and this outcome is catalyzing the search for more radiotheranostics approaches. Not every patient will benefit from radiotheranostics; but, for those who cross the target-detected line, the likelihood of response is very high.

AMPK, a Regulator of Metabolism and Autophagy, Is Activated by Lysosomal Damage via a Novel Galectin-Directed Ubiquitin Signal Transduction System.

AMPK is a central regulator of metabolism and autophagy. Here we show how lysosomal damage activates AMPK. This occurs via a hitherto unrecognized signal transduction system whereby cytoplasmic sentinel lectins detect membrane damage leading to ubiquitination responses. Absence of Galectin 9 (Gal9) or loss of its capacity to recognize lumenal glycans exposed during lysosomal membrane damage abrogate such ubiquitination responses. Proteomic analyses with APEX2-Gal9 have revealed global changes within the Gal9 interactome during lysosomal damage. Gal9 association with lysosomal glycoproteins increases whereas interactions with a newly identified Gal9 partner, deubiquitinase USP9X, diminishes upon lysosomal injury. In response to damage, Gal9 displaces USP9X from complexes with TAK1 and promotes K63 ubiquitination of TAK1 thus activating AMPK on damaged lysosomes. This triggers autophagy and contributes to autophagic control of membrane-damaging microbe Mycobacterium tuberculosis. Thus, galectin and ubiquitin systems converge to activate AMPK and autophagy during endomembrane homeostasis.

TSC2 regulates tumor susceptibility to TRAIL-mediated T-cell killing by orchestrating mTOR signaling.

Resistance to cancer immunotherapy continues to impair common clinical benefit. Here, we use whole-genome CRISPR-Cas9 knockout data to uncover an important role for Tuberous Sclerosis Complex 2 (TSC2) in determining tumor susceptibility to cytotoxic T lymphocyte (CTL) killing in human melanoma cells. TSC2-depleted tumor cells had disrupted mTOR regulation following CTL attack, which was associated with enhanced cell death. Wild-type tumor cells adapted to CTL attack by shifting their mTOR signaling balance toward increased mTORC2 activity, circumventing apoptosis, and necroptosis. TSC2 ablation strongly augmented tumor cell sensitivity to CTL attack in vitro and in vivo, suggesting one of its functions is to critically protect tumor cells. Mechanistically, TSC2 inactivation caused elevation of TRAIL receptor expression, cooperating with mTORC1-S6 signaling to induce tumor cell death. Clinically, we found a negative correlation between TSC2 expression and TRAIL signaling in TCGA patient cohorts. Moreover, a lower TSC2 immune response signature was observed in melanomas from patients responding to immune checkpoint blockade. Our study uncovers a pivotal role for TSC2 in the cancer immune response by governing crosstalk between TSC2-mTOR and TRAIL signaling, aiding future therapeutic exploration of this pathway in immuno-oncology.

HCMV-secreted glycoprotein gpUL4 inhibits TRAIL-mediated apoptosis and NK cell activation.

Human cytomegalovirus (HCMV) is a paradigm of pathogen immune evasion and sustains lifelong persistent infection in the face of exceptionally powerful host immune responses through the concerted action of multiple immune-evasins. These reduce NK cell activation by inhibiting ligands for activating receptors, expressing ligands for inhibitory receptors, or inhibiting synapse formation. However, these functions only inhibit direct interactions with the infected cell. To determine whether the virus also expresses soluble factors that could modulate NK function at a distance, we systematically screened all 170 HCMV canonical protein-coding genes. This revealed that UL4 encodes a secreted and heavily glycosylated protein (gpUL4) that is expressed with late-phase kinetics and is capable of inhibiting NK cell degranulation. Analyses of gpUL4 binding partners by mass spectrometry identified an interaction with TRAIL. gpUL4 bound TRAIL with picomolar affinity and prevented TRAIL from binding its receptor, thus acting as a TRAIL decoy receptor. TRAIL is found in both soluble and membrane-bound forms, with expression of the membrane-bound form strongly up-regulated on NK cells in response to interferon. gpUL4 inhibited apoptosis induced by soluble TRAIL, while also binding to the NK cell surface in a TRAIL-dependent manner, where it blocked NK cell degranulation and cytokine secretion. gpUL4 therefore acts as an immune-evasin by inhibiting both soluble and membrane-bound TRAIL and is a viral-encoded TRAIL decoy receptor. Interestingly, gpUL4 could also suppress NK responses to heterologous viruses, suggesting that it may act as a systemic virally encoded immunosuppressive agent.

ASB3 promotes hepatocellular carcinoma progression by mediating DR5 ubiquitination in TRAIL resistance.

Ankyrin-repeat proteins with a suppressor of cytokine signaling box (ASB) proteins belong to the E3 ubiquitin ligase family. 18 ASB members have been identified whose biological functions are mostly unexplored. Here, we discovered that ASB3 was essential for hepatocellular carcinoma (HCC) development and high ASB3 expression predicted poor clinical outcomes. ASB3 silencing induced HCC cell growth arrest and apoptosis in vitro and in vivo. Liver-specific deletion of Asb3 gene suppressed diethylnitrosamine (DEN)-induced liver cancer development. Mechanistically, ASB3 interacted with death receptor 5 (DR5), which promoted ubiquitination and degradation of DR5. We further showed that ASB3 knockdown stabilized DR5 and increased the sensitivity of liver cancer cells to the treatment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a DR5-dependent manner in cellular and in animal models. In summary, we demonstrated that ASB3 promoted ubiquitination and degradation of DR5 in HCC, suggesting the potential of targeting ASB3 to HCC treatment and overcome TRAIL resistance.

The TRAIL-Induced Cancer Secretome Promotes a Tumor-Supportive Immune Microenvironment via CCR2.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known for specifically killing cancer cells, whereas in resistant cancers, TRAIL/TRAIL-R can promote metastasis via Rac1 and PI3K. It remains unknown, however, whether and to what extent TRAIL/TRAIL-R signaling in cancer cells can affect the immune microenvironment. Here we show that TRAIL-triggered cytokine secretion from TRAIL-resistant cancer cells is FADD dependent and identify the TRAIL-induced secretome to drive monocyte polarization to myeloid-derived suppressor cells (MDSCs) and M2-like macrophages. TRAIL-R suppression in tumor cells impaired CCL2 production and diminished both lung MDSC presence and tumor growth. In accordance, the receptor of CCL2, CCR2, is required to facilitate increased MDSC presence and tumor growth. Finally, TRAIL and CCL2 are co-regulated with MDSC/M2 markers in lung adenocarcinoma patients. Collectively, endogenous TRAIL/TRAIL-R-mediated CCL2 secretion promotes accumulation of tumor-supportive immune cells in the cancer microenvironment, thereby revealing a tumor-supportive immune-modulatory role of the TRAIL/TRAIL-R system in cancer biology.

Caspase-8 Acts in a Non-enzymatic Role as a Scaffold for Assembly of a Pro-inflammatory "FADDosome" Complex upon TRAIL Stimulation.

TRAIL is a potent inducer of apoptosis and has been studied almost exclusively in this context. However, TRAIL can also induce NFκB-dependent expression of multiple pro-inflammatory cytokines and chemokines. Surprisingly, whereas inhibition of caspase activity blocked TRAIL-induced apoptosis, but not cytokine production, knock down or deletion of caspase-8 suppressed both outcomes, suggesting that caspase-8 participates in TRAIL-induced inflammatory signaling in a scaffold role. Consistent with this, introduction of a catalytically inactive caspase-8 mutant into CASP-8 null cells restored TRAIL-induced cytokine production, but not cell death. Furthermore, affinity precipitation of the native TRAIL receptor complex revealed that pro-caspase-8 was required for recruitment of RIPK1, via FADD, to promote NFκB activation and pro-inflammatory cytokine production downstream. Thus, caspase-8 can serve in two distinct roles in response to TRAIL receptor engagement, as a scaffold for assembly of a Caspase-8-FADD-RIPK1 "FADDosome" complex, leading to NFκB-dependent inflammation, or as a protease that promotes apoptosis.

TRAIL-driven targeting and reversing cervical cancer radioresistance by seleno-nanotherapeutics through regulating cell metabolism.

Recently, radioresistance has become a major obstacle in the radiotherapy of cervical cancer. To demonstrate enhanced radiosensitization against radioresistant cervical cancer, radioresistant cervical cancer cell line was developed and the mechanism of radioresistance was explored. Due to the overexpression of (death receptor 5, DR5) in cervical cancer, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-overexpressed cervical cancer cell membrane-camouflaged Cu2-xSe nanomedicine (CCMT) was designed. Since the CCMT was encapsulated with TRAIL-modified cell membrane, it represented high target to cervical cancer cell and immune evasion. Furthermore, Cu2-xSe had the ability to scavenge glutathione (GSH) and produce ·OH with excess H2O2 in the tumor microenvironment. The presence of CCMT combined with radiation therapy could effectively increase the 1O2 produced by X-rays. In vitro and in vivo studies elaborated that CCMT exhibited excellent radiosensitization properties to reverse radiotolerance by scavenging GSH and promoting DNA damage, apoptosis, mitochondrial membrane potential damage and metabolic disruption. Collectively, this study suggested that the development of TRAIL-overexpressed cell membrane-camouflaged Cu2-xSe nanomedicine could advance future cervical cancer treatment and minimize the disadvantages associated with radiation treatment.

The Identification of New c-FLIP Inhibitors for Restoring Apoptosis in TRAIL-Resistant Cancer Cells.

The catalytically inactive caspase-8-homologous protein, c-FLIP, is a potent antiapoptotic protein highly expressed in various types of cancers. c-FLIP competes with caspase-8 for binding to the adaptor protein FADD (Fas-Associated Death Domain) following death receptors' (DRs) activation via the ligands of the TNF-R family. As a consequence, the extrinsic apoptotic signaling pathway involving DRs is inhibited. The inhibition of c-FLIP activity in tumor cells might enhance DR-mediated apoptosis and overcome immune and anticancer drug resistance. Based on an in silico approach, the aim of this work was to identify new small inhibitory molecules able to bind selectively to c-FLIP and block its anti-apoptotic activity. Using a homology 3D model of c-FLIP, an in silico screening of 1880 compounds from the NCI database (National Cancer Institute) was performed. Nine molecules were selected for in vitro assays, based on their binding affinity to c-FLIP and their high selectivity compared to caspase-8. These molecules selectively bind to the Death Effector Domain 2 (DED2) of c-FLIP. We have tested in vitro the inhibitory effect of these nine molecules using the human lung cancer cell line H1703, overexpressing c-FLIP. Our results showed that six of these newly identified compounds efficiently prevent FADD/c-FLIP interactions in a molecular pull-down assay, as well as in a DISC immunoprecipitation assay. The overexpression of c-FLIP in H1703 prevents TRAIL-mediated apoptosis; however, a combination of TRAIL with these selected molecules significantly restored TRAIL-induced cell death by rescuing caspase cleavage and activation. Altogether, our findings indicate that new inhibitory chemical molecules efficiently prevent c-FLIP recruitment into the DISC complex, thus restoring the caspase-8-dependent apoptotic cascade. These results pave the way to design new c-FLIP inhibitory molecules that may serve as anticancer agents in tumors overexpressing c-FLIP.

Apoptotic Receptors and CD107a Expression by NK Cells in an Interaction Model with Trophoblast Cells.

Natural killer cells (NK cells) exert cytotoxicity towards target cells in several ways, including the expression of apoptosis-mediating ligands (TRAIL, FasL). In addition, NK cells themselves may be susceptible to apoptosis due to the expression of TRAIL receptors. These receptors include TRAIL-R1 (DR4), TRAIL-R2 (DR5), capable of inducing apoptosis, and TRAIL-R3 (DcR1), TRAIL-R4 (DcR2), the so-called "decoy receptors", which lack an intracellular domain initiating activation of caspases. Of particular interest is the interaction of uterine NK cells with cells of fetal origin, trophoblasts, which are potential targets for natural killer cells to carry out cytotoxicity. The aim of this work was to evaluate the expression of proapoptotic receptors and their ligands as well as CD107a expression by NK cells in a model of interaction with trophoblast cells. To evaluate NK cells, we used cells of the NK-92 line; cells of the JEG-3 line were used as target cells. The cytokines IL-1ß, IL-15, IL-18, TNFα, IL-10, TGFß and conditioned media (CM) of the first and third trimester chorionic villi explants were used as inducers. We established that cytokines changed the expression of apoptotic receptors by NK cells: in the presence of TNFα, the amount and intensity of Fas expression increased, while in the presence of TGFß, the amount and intensity of expression of the DR5 receptor decreased. Soluble chorionic villi factors alter the expression of TRAIL and FasL by NK-92 cells, which can reflect the suppression of the TRAIL-dependent mechanism of apoptosis in the first trimester and stimulating the Fas-dependent mechanism in the third trimester. In the presence of trophoblast cells, the expression of TRAIL and DcR1 by NK cells was reduced compared to intact cells, indicating an inhibitory effect of trophoblast cells on NK cell cytotoxicity. In the presence of chorionic villi CM and trophoblast cells, a reduced number of NK-92 cells expressing DR4 and DR5 was found. Therefore, soluble factors secreted by chorionic villi cells regulate the resistance of NK cells to death by binding TRAIL, likely maintaining their activity at a certain level in case of contact with trophoblast cells.