Orthotopic Models Using New, Murine Lung Adenocarcinoma Cell Lines Simulate Human Non-Small Cell Lung Cancer Treated with Immunotherapy.

Understanding tumor-host immune interactions and the mechanisms of lung cancer response to immunotherapy is crucial. Current preclinical models used to study this often fall short of capturing the complexities of human lung cancer and lead to inconclusive results. To bridge the gap, we introduce two new murine monoclonal lung cancer cell lines for use in immunocompetent orthotopic models. We demonstrate how our cell lines exhibit immunohistochemical protein expression (TTF-1, NapA, PD-L1) and common driver mutations (KRAS, p53, and p110α) seen in human lung adenocarcinoma patients, and how our orthotopic models respond to combination immunotherapy in vivo in a way that closely mirrors current clinical outcomes. These new lung adenocarcinoma cell lines provide an invaluable, clinically relevant platform for investigating the intricate dynamics between tumor and the immune system, and thus potentially contributes to a deeper understanding of immunotherapeutic approaches to lung cancer treatment.

Alveolar epithelial cell growth hormone releasing hormone receptor in alveolar epithelial inflammation.

Purpose: Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that GHRH receptor (GHRH-R) in alveolar type 2 (AT2) cells could modulate pro-inflammatory and possibly subsequent pro-fibrotic effects of lipopolysaccharide (LPS) or cytokines, such that AT2 cells could participate in lung inflammation and fibrosis. Methods: We used human alveolar type 2 (iAT2) epithelial cells derived from induced pluripotent stem cells (iPSC) to investigate how GHRH-R modulates gene and protein expression. We tested iAT2 cells' gene expression in response to LPS or cytokines, seeking whether these mechanisms caused endogenous production of pro-inflammatory molecules or mesenchymal markers. Quantitative real-time PCR (RT-PCR) and Western blotting were used to investigate differential expression of epithelial and mesenchymal markers. Result: Incubation of iAT2 cells with LPS increased expression of IL1-ß and TNF-α in addition to mesenchymal genes, including ACTA2, FN1 and COL1A1. Alveolar epithelial cell gene expression due to LPS was significantly inhibited by GHRH-R peptide antagonist MIA-602. Incubation of iAT2 cells with cytokines like those in fibrotic lungs similarly increased expression of genes for IL1-ß, TNF-α, TGFß-1, Wnt5a, smooth muscle actin, fibronectin and collagen. Expression of mesenchymal proteins, such as N-cadherin and vimentin, were also elevated after prolonged exposure to cytokines, confirming epithelial production of pro-inflammatory molecules as an important mechanism that might lead to subsequent fibrosis. Conclusion: iAT2 cells clearly expressed the GHRH-R. Exposure to LPS or cytokines increased iAT2 cell production of pro-inflammatory factors. GHRH-R antagonist MIA-602 inhibited pro-inflammatory gene expression, implicating iAT2 cell GHRH-R signaling in lung inflammation and potentially in fibrosis.

Dual inhibition of IDO1/TDO2 enhances anti-tumor immunity in platinum-resistant non-small cell lung cancer.

BACKGROUND: The impact of non-small cell lung cancer (NSCLC) metabolism on the immune microenvironment is not well understood within platinum resistance. We have identified crucial metabolic differences between cisplatin-resistant (CR) and cisplatin-sensitive (CS) NSCLC cells with elevated indoleamine 2,3-dioxygenase-1 (IDO1) activity in CR, recognized by increased kynurenine (KYN) production. METHODS: Co-culture, syngeneic, and humanize mice models were utilized. C57BL/6 mice were inoculated with either Lewis lung carcinoma mouse cells (LLC) or their platinum-resistant counterpart (LLC-CR) cells. Humanized mice were inoculated with either A (human CS cells) or ALC (human CR cells). Mice were treated with either IDO1 inhibitor or TDO2 (tryptophan 2,3-dioxygenase-2) inhibitor at 200 mg/kg P.O. once a day for 15 days; or with a new-in-class, IDO1/TDO2 dual inhibitor AT-0174 at 170 mg/kg P.O. once a day for 15 days with and without anti-PD1 antibody (10 mg/kg, every 3 days). Immune profiles and KYN and tryptophan (TRP) production were evaluated. RESULTS: CR tumors exhibited a more highly immunosuppressive environment that debilitated robust anti-tumor immune responses. IDO1-mediated KYN production from CR cells suppressed NKG2D on immune effector natural killer (NK) and CD8+ T cells and enhanced immunosuppressive populations of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Importantly, while selective IDO1 inhibition attenuated CR tumor growth, it concomitantly upregulated the TDO2 enzyme. To overcome the compensatory induction of TDO2 activity, we employed the IDO1/TDO2 dual inhibitor, AT-0174. Dual inhibition of IDO1/TDO2 in CR mice suppressed tumor growth to a greater degree than IDO1 inhibition alone. Significant enhancement in NKG2D frequency on NK and CD8+ T cells and a reduction in Tregs and MDSCs were observed following AT-1074 treatment. PD-L1 (programmed death-ligand-1) expression was increased in CR cells; therefore, we assessed dual inhibition + PD1 (programmed cell death protein-1) blocking and report profound anti-tumor growth and improved immunity in CR tumors which in turn extended overall survival in mice. CONCLUSION: Our study reports the presence of platinum-resistant lung tumors that utilize both IDO1/TDO2 enzymes for survival, and to escape immune surveillance as a consequence of KYN metabolites. We also report early in vivo data in support of the potential therapeutic efficacy of the dual IDO1/TDO2 inhibitor AT-0174 as a part of immuno-therapeutic treatment that disrupts tumor metabolism and enhances anti-tumor immunity.

Exploiting the Innate Plasticity of the Programmed Cell Death-1 (PD1) Receptor to Design Pembrolizumab H3 Loop Mimics.

Checkpoint blockade of the immunoreceptor programmed cell death-1 (PD1) with its ligand-1 (PDL1) by monoclonal antibodies such as pembrolizumab provided compelling clinical results in various cancer types, yet the molecular mechanism by which this drug blocks the PD1/PDL1 interface remains unclear. To address this question, we examined the conformational motion of PD1 associated with the binding of pembrolizumab. Our results revealed that the innate plasticity of both C'D and FG loops is crucial to form a deep binding groove (371 Å3 ) across several distant epitopes of PD1. This analysis ultimately provided a rational-design to create pembrolizumab H3 loop mimics [RDYRFDMGFD] into ß-hairpin scaffolds. As a result, a 20-residue long ß-hairpin peptide 1 e was identified as a first-in-class potent PD1-inhibitor (EC50 of 0.29 µM; Ki of 41 nM).

Enhancing the Effect of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Signaling and Arginine Deprivation in Melanoma.

Melanoma as a very aggressive type of cancer is still in urgent need of improved treatment. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and arginine deiminase (ADI-PEG20) are two of many suggested drugs for treating melanoma. Both have shown anti-tumor activities without harming normal cells. However, resistance to both drugs has also been noted. Studies on the mechanism of action of and resistance to these drugs provide multiple targets that can be utilized to increase the efficacy and overcome the resistance. As a result, combination strategies have been proposed for these drug candidates with various other agents, and achieved enhanced or synergistic anti-tumor effect. The combination of TRAIL and ADI-PEG20 as one example can greatly enhance the cytotoxicity to melanoma cells including those resistant to the single component of this combination. It is found that combination treatment generally can alter the expression of the components of cell signaling in melanoma cells to favor cell death. In this paper, the signaling of TRAIL and ADI-PEG20-induced arginine deprivation including the main mechanism of resistance to these drugs and exemplary combination strategies is discussed. Finally, factors hampering the clinical application of both drugs, current and future development to overcome these hurdles are briefly discussed.

Cisplatin Resistance and Redox-Metabolic Vulnerability: A Second Alteration.

The development of drug resistance in tumors is a major obstacle to effective cancer chemotherapy and represents one of the most significant complications to improving long-term patient outcomes. Despite early positive responsiveness to platinum-based chemotherapy, the majority of lung cancer patients develop resistance. The development of a new combination therapy targeting cisplatin-resistant (CR) tumors may mark a major improvement as salvage therapy in these patients. The recent resurgence in research into cellular metabolism has again confirmed that cancer cells utilize aerobic glycolysis ("the Warburg effect") to produce energy. Hence, this observation still remains a characteristic hallmark of altered metabolism in certain cancer cells. However, recent evidence promotes another concept wherein some tumors that acquire resistance to cisplatin undergo further metabolic alterations that increase tumor reliance on oxidative metabolism (OXMET) instead of glycolysis. Our review focuses on molecular changes that occur in tumors due to the relationship between metabolic demands and the importance of NAD+ in redox (ROS) metabolism and the crosstalk between PARP-1 (Poly (ADP ribose) polymerase-1) and SIRTs (sirtuins) in CR tumors. Finally, we discuss a role for the tumor metabolites of the kynurenine pathway (tryptophan catabolism) as effectors of immune cells in the tumor microenvironment during acquisition of resistance in CR cells. Understanding these concepts will form the basis for future targeting of CR cells by exploiting redox-metabolic changes and their consequences on immune cells in the tumor microenvironment as a new approach to improve overall therapeutic outcomes and survival in patients who fail cisplatin.

Cardiomyopathic mutations in essential light chain reveal mechanisms regulating the super relaxed state of myosin.

In this study, we assessed the super relaxed (SRX) state of myosin and sarcomeric protein phosphorylation in two pathological models of cardiomyopathy and in a near-physiological model of cardiac hypertrophy. The cardiomyopathy models differ in disease progression and severity and express the hypertrophic (HCM-A57G) or restrictive (RCM-E143K) mutations in the human ventricular myosin essential light chain (ELC), which is encoded by the MYL3 gene. Their effects were compared with near-physiological heart remodeling, represented by the N-terminally truncated ELC (Δ43 ELC mice), and with nonmutated human ventricular WT-ELC mice. The HCM-A57G and RCM-E143K mutations had antagonistic effects on the ATP-dependent myosin energetic states, with HCM-A57G cross-bridges fostering the disordered relaxed (DRX) state and the RCM-E143K model favoring the energy-conserving SRX state. The HCM-A57G model promoted the switch from the SRX to DRX state and showed an ∼40% increase in myosin regulatory light chain (RLC) phosphorylation compared with the RLC of normal WT-ELC myocardium. On the contrary, the RCM-E143K-associated stabilization of the SRX state was accompanied by an approximately twofold lower level of myosin RLC phosphorylation compared with the RLC of WT-ELC. Upregulation of RLC phosphorylation was also observed in Δ43 versus WT-ELC hearts, and the Δ43 myosin favored the energy-saving SRX conformation. The two disease variants also differently affected the duration of force transients, with shorter (HCM-A57G) or longer (RCM-E143K) transients measured in electrically stimulated papillary muscles from these pathological models, while no changes were displayed by Δ43 fibers. We propose that the N terminus of ELC (N-ELC), which is missing in the hearts of Δ43 mice, works as an energetic switch promoting the SRX-to-DRX transition and contributing to the regulation of myosin RLC phosphorylation in full-length ELC mice by facilitating or sterically blocking RLC phosphorylation in HCM-A57G and RCM-E143K hearts, respectively.

Perspective on the treatment of non-small cell lung cancer in the context of potential SARS-CoV-2 infection during the pandemic.

SARS-CoV-2 infections are rising at an alarming rate and various aspects of this pandemic must be quickly and adequately addressed in order to enhance effective healthcare delivery and protect at risk populations such as cancer patients. Preventing Covid-19 infection must be a top system wide priority to avoid mortality, and considerable financial and disease burden. Most cancer patients, and in particular those with tumors resistant to chemotherapy are particularly vulnerable to infection. In this review, we connect potential viral infection of patients with lung tumors that have somewhat quiescence the immune response in the tumor microenvironment and categorize target molecules in metabolism that may be used to identify at risk patients leading to more effective treatment regimens; keeping continuity of therapy and disease prevention during a very tumultuous period of time surrounding the pandemic.

Growth Hormone-Releasing Hormone in Lung Physiology and Pulmonary Disease.

Growth hormone-releasing hormone (GHRH) is secreted primarily from the hypothalamus, but other tissues, including the lungs, produce it locally. GHRH stimulates the release and secretion of growth hormone (GH) by the pituitary and regulates the production of GH and hepatic insulin-like growth factor-1 (IGF-1). Pituitary-type GHRH-receptors (GHRH-R) are expressed in human lungs, indicating that GHRH or GH could participate in lung development, growth, and repair. GHRH-R antagonists (i.e., synthetic peptides), which we have tested in various models, exert growth-inhibitory effects in lung cancer cells in vitro and in vivo in addition to having anti-inflammatory, anti-oxidative, and pro-apoptotic effects. One antagonist of the GHRH-R used in recent studies reviewed here, MIA-602, lessens both inflammation and fibrosis in a mouse model of bleomycin lung injury. GHRH and its peptide agonists regulate the proliferation of fibroblasts through the modulation of extracellular signal-regulated kinase (ERK) and Akt pathways. In addition to downregulating GH and IGF-1, GHRH-R antagonist MIA-602 inhibits signaling pathways relevant to inflammation, including p21-activated kinase 1-signal transducer and activator of transcription 3/nuclear factor-kappa B (PAK1-STAT3/NF-κB and ERK). MIA-602 induces fibroblast apoptosis in a dose-dependent manner, which is an effect that is likely important in antifibrotic actions. Taken together, the novel data reviewed here show that GHRH is an important peptide that participates in lung homeostasis, inflammation, wound healing, and cancer; and GHRH-R antagonists may have therapeutic potential in lung diseases.

Targeting the Kynurenine Pathway for the Treatment of Cisplatin-Resistant Lung Cancer.

Cisplatin resistance is a major barrier in the effective treatment of lung cancer. Cisplatin-resistant (CR) lung cancer cells do not primarily use glucose but rather consume amino acids such as glutamine and tryptophan (Trp) for survival. CR cells activate the kynurenine (KYN) pathway (KP) to cope with excessive reactive oxygen species (ROS) and maintain homeostasis for growth and proliferation. Consequently, indoleamine 2,3-dioxygenase-1 (IDO1) becomes an essential enzyme for CR cells' survival because it initiates and regulates the first step in the KP. Increased IDO1 activities and ROS levels are found in CR cells versus cisplatin-sensitive lung cancer. Importantly, significantly greater KYN/Trp ratio (P = 0.005) is detected in serum of patients who fail cisplatin when compared with naïve treatment. Knocking down IDO1 using shRNA or IDO1 inhibitors heightens ROS levels and results in a significant growth inhibitory effect only on CR cells and not on cisplatin-sensitive cells. Exposing CR cells to antioxidant (TIRON) results in suppression of IDO1 activity and confers resistance to IDO1 inhibition, indicating an interrelationship between ROS and IDO1. Because KYN plays a critical role in reprogramming naïve T cells to the immune-suppressive regulatory T-cell (T-reg) phenotype, we observed higher expression of TGFß, FoxP3, and CD4+CD25+ in mice bearing CR tumors compared with tumors from cisplatin-sensitive counterparts. IMPLICATIONS: Findings suggest that the enzyme-inhibitory activity and antitumor efficacy of IDO1 inhibitors rely in part on ROS levels, arguing that IDO1 expression alone may be insufficient to determine the clinical benefits for this class of experimental cancer drugs. Importantly, IDO1 inhibitors may be more suitable to treat patients with lung cancer who failed cisplatin therapy than naïve treatment patients.

Growth Hormone-Releasing Hormone Receptor Antagonist Modulates Lung Inflammation and Fibrosis due to Bleomycin.

PURPOSE: Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that a GHRH receptor (GHRH-R) antagonist, MIA-602, would inhibit bleomycin-induced lung inflammation and/or fibrosis in C57Bl/6J mice. METHODS: We tested whether MIA-602 (5 µg or vehicle given subcutaneously [SC] on days 1-21) would decrease lung inflammation (at day 14) and/or fibrosis (at day 28) in mice treated with intraperitoneal (IP) bleomycin (0.8 units on days 1, 3, 7, 10, 14, and 21). Bleomycin resulted in inflammation and fibrosis around airways and vessels evident histologically at days 14 and 28. RESULTS: Inflammation (histopathologic scores assessed blindly) was visibly less evident in mice treated with MIA-602 for 14 days. After 28 days, lung hydroxyproline (HP) content increased significantly in mice treated with vehicle; in contrast, lung HP did not increase significantly compared to naïve controls in mice treated with GHRH-R antagonist. GHRH-R antagonist increased basal and maximal oxygen consumption of cultured lung fibroblasts. Multiple genes related to chemotaxis, IL-1, chemokines, regulation of inflammation, and extracellular signal-regulated kinases (ERK) were upregulated in lungs of mice treated with bleomycin and MIA-602. MIA-602 also prominently suppressed multiple genes related to the cellular immune response including those for T-cell differentiation, receptor signaling, activation, and cytokine production. CONCLUSIONS: MIA-602 reduced lung inflammation and fibrosis due to bleomycin. Multiple genes related to immune response and T-cell functions were downregulated, supporting the view that MIA-602 can modulate the cellular immune response to bleomycin lung injury.

Phase 2 study of pembrolizumab and circulating biomarkers to predict anticancer response in advanced, unresectable hepatocellular carcinoma.

BACKGROUND: Checkpoint inhibitors have shown modest activity in patients with advanced hepatocellular carcinoma (HCC). Herein, the authors report a prospective single-institution clinical/translational phase 2 study of pembrolizumab in patients with advanced HCC and circulating biomarkers closely related to response. METHODS: Pembrolizumab was administered at a dose of 200 mg intravenously every 3 weeks among patients who may have developed disease progression while receiving, were intolerant of, or refused sorafenib. The circulating levels of cytokines, chemokines, programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and PD-L2 were correlated with response, tumor PD-L1 expression, and other clinicopathological features. RESULTS: A total of 29 patients were treated and 28 patients were evaluable for response. The most common laboratory grade 3/4 adverse events were increases in aspartate aminotransferase and/or alanine aminotransferase and serum bilirubin, which for the most part were reversible. In terms of efficacy, one patient achieved a complete response and 8 patients achieved partial responses for an overall response rate of 32%. Four other patients had stable disease. The median progression-free survival was 4.5 months and the median overall survival was 13 months. Response did not correlate with prior sorafenib therapy, PD-L1 tumor staining, or a prior history of hepatitis. Correlative studies revealed that high baseline plasma TGF-ß levels (≥200 pg/mL) significantly correlated with poor treatment outcomes after pembrolizumab. Tumor PD-L1 and plasma PD-L1/PD-1 levels were associated with plasma IFN-γ or IL-10. CONCLUSIONS: Pembrolizumab was found to demonstrate activity in patients with advanced HCC. Toxicity generally was tolerable and reversible. A set of immunological markers in blood plasma as well as PD-L1 staining indicated that baseline TGF-ß could be a predictive biomarker for response to pembrolizumab.

Phase II trial of SOM230 (pasireotide LAR) in patients with unresectable hepatocellular carcinoma.

BACKGROUND: A phase II trial of pasireotide was performed to assess its efficacy and safety in advanced or metastatic hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Patients with advanced HCC and Child-Pugh score ≤7 received pasireotide LAR 60 mg intramuscularly every 28 days. Primary endpoint was disease control rate. Secondary endpoints were time to tumor progression, response rate, treatment-related adverse events, and overall survival. Serum insulin growth factor-1 was measured before and after pasireotide. RESULTS: Twenty patients were treated and evaluable. Eighteen patients (90%) had prior therapy; 16 patients (80%) had multiple therapies. Median age was 65, 75% had Barcelona Clinic Liver Cancer stage C, and 55% had metastatic disease. The main toxicity was hyperglycemia. Rare adverse effects included reversible grade 4 elevation in alanina transaminase/aspartate transaminase in one patient. The best response was stable disease in 9 patients (45%). Median time to tumor progression for the 20 patients was 3 months, and median survival was 9 months. CONCLUSION: Pasireotide had limited clinical benefit as second-line or third-line treatment in patients with advanced or metastatic HCC. Low baseline insulin growth factor-1 level may be indicative when SOM230 treatment may be ineffective, and decreasing levels after treatment may be indicative of disease control.

A Kinetic Dearomatization Strategy for an Expedient Biomimetic Route to the Bielschowskysin Skeleton.

Bielschowskysin (1), the flagship of the furanocembranoid diterpene family, has attracted attention from chemists owing to its intriguing and daunting polycyclic architecture and medicinal potential against lung cancer. The high level of functionalization of 1 poses a considerable challenge to synthesis. Herein, a stereoselective furan dearomatization strategy of furanocembranoids was achieved via the intermediacy of chlorohydrins. The stereochemical course of the kinetic dearomatization was established, and the C3 configuration of the resulting exo-enol ether intermediates proved to be essential to complete the late-stage transannular [2+2] photocycloaddition. Overall, this biomimetic strategy starting from the natural product acerosolide (9) featured an unprecedented regio- and highly stereoselective furan dearomatization, which provided rapid access to the pivotal exo-enol ethers en route to the intricate bielschowskyane skeleton.

Degradation of AMPK-α1 sensitizes BRAF inhibitor-resistant melanoma cells to arginine deprivation.

Melanomas harboring BRAF mutation (V600E) are known to recur frequently following treatment with BRAF inhibitors (BRAFi) despite a high initial response rate. Our previous study has uncovered that BRAFi-resistant melanoma (BR) cells are vulnerable to arginine deprivation. It has been reported that naïve melanoma cells undergo autophagy and re-express argininosuccinate synthetase 1 (ASS1) to enable them to synthesize arginine for survival when encountering arginine deprivation. Abolishing these two factors in BR cells confers sensitivity to arginine deprivation. In this report, we further demonstrated that downregulation of AMPK-α1 in BR cells is a major factor contributing to impairment of autophagy as evidenced by decreased autophagosome formation. These BR cells also showed a metabolic shift from glucose to arginine dependence, which was supported by decreased expressions of GLUT1 (glucose transporter) and hexokinase II (HKII) coupled with less glucose uptake but high levels of arginine transporter CAT-2 expression. Furthermore, silencing CAT-2 expression also distinctly attenuated BR cell proliferation. Notably, when naïve melanoma cells became BR cells by long-term exposure to BRAFi, a stepwise degradation of AMPK-α1 was initiated via ubiquitin-proteasome system (UPS). We discovered that a novel E3 ligase, RING finger 44 (RNF44), is responsible for promoting AMPK-α1 degradation in BR cells. RNF44 expression in BR cells was upregulated by transcription factor CREB triggered by hyperactivation of ERK/AKT. High levels of RNF44 corresponding to low levels of AMPK-α1 appeared in BR xenografts and melanoma tumor samples from BR and BRAFi/MEK inhibitor (MEKi)-resistant (BMR) melanoma patients. Similar to BR cells, BMR cells were also sensitive to arginine deprivation. Our study provides a novel insight into the mechanism whereby BRAFi or BRAFi/MEKi resistance drives proteasomal degradation of AMPK-α1 and consequently regulates autophagy and metabolic reprogramming in melanoma cells.

The Combination of Arginine Deprivation and 5-Fluorouracil Improves Therapeutic Efficacy in Argininosuccinate Synthetase Negative Hepatocellular Carcinoma.

Argininosuccinate synthetase (ASS), a key enzyme to synthesize arginine is down regulated in many tumors including hepatocellular carcinoma (HCC). Similar to previous reports, we have found the decrease in ASS expression in poorly differentiated HCC. These ASS(-) tumors are auxotrophic for arginine. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine, has shown activity in these tumors, but the antitumor effect is not robust and hence combination treatment is needed. Herein, we have elucidated the effectiveness of ADI-PEG20 combined with 5-Fluorouracil (5-FU) in ASS(-)HCC by targeting urea cycle and pyrimidine metabolism using four HCC cell lines as model. SNU398 and SNU387 express very low levels of ASS or ASS(-) while Huh-1, and HepG2 express high ASS similar to normal cells. Our results showed that the augmented cytotoxic effect of combination treatment only occurs in SNU398 and SNU387, and not in HepG2 and Huh-1 (ASS(+)) cells, and is partly due to reduced anti-apoptotic proteins X-linked inhibitor of apoptosis protein (XIAP), myeloid leukemia cell differentiation protein (Mcl-1) and B-cell lymphoma-2 (Bcl-2). Importantly, lack of ASS also influences essential enzymes in pyrimidine synthesis (carbamoyl-phosphate synthetase2, aspartate transcarbamylase and dihydrooratase (CAD) and thymidylate synthase (TS)) and malate dehydrogenase-1 (MDH-1) in TCA cycle. ADI-PEG20 treatment decreased these enzymes and made them more vulnerable to 5-FU. Transfection of ASS restored these enzymes and abolished the sensitivity to ADI-PEG20 and combination treatment. Overall, our data suggest that ASS influences multiple enzymes involved in 5-FU sensitivity. Combining ADI-PEG20 and 5-FU may be effective to treat ASS(-)hepatoma and warrants further clinical investigation.

Autophagic Mechanism in Anti-Cancer Immunity: Its Pros and Cons for Cancer Therapy.

Autophagy, a self-eating machinery, has been reported as an adaptive response to maintain metabolic homeostasis when cancer cells encounter stress. It has been appreciated that autophagy acts as a double-edge sword to decide the fate of cancer cells upon stress factors, molecular subtypes, and microenvironmental conditions. Currently, the majority of evidence support that autophagy in cancer cells is a vital mechanism bringing on resistance to current and prospective treatments, yet whether autophagy affects the anticancer immune response remains unclear and controversial. Accumulated studies have demonstrated that triggering autophagy is able to facilitate anticancer immunity due to an increase in immunogenicity, whereas other studies suggested that autophagy is likely to disarm anticancer immunity mediated by cytotoxic T cells and nature killer (NK) cells. Hence, this contradiction needs to be elucidated. In this review, we discuss the role of autophagy in cancer cells per se and in cancer microenvironment as well as its dual regulatory roles in immune surveillance through modulating presentation of tumor antigens, development of immune cells, and expression of immune checkpoints. We further focus on emerging roles of autophagy induced by current treatments and its impact on anticancer immune response, and illustrate the pros and cons of utilizing autophagy in cancer immunotherapy based on preclinical references.

Exploiting ROS and metabolic differences to kill cisplatin resistant lung cancer.

Cisplatin resistance remains a major problem in the treatment of lung cancer. We have discovered that cisplatin resistant (CR) lung cancer cells, regardless of the signaling pathway status, share the common parameter which is an increase in reactive oxygen species (ROS) and undergo metabolic reprogramming. CR cells were no longer addicted to the glycolytic pathway, but rather relied on oxidative metabolism. They took up twice as much glutamine and were highly sensitive to glutamine deprivation. Glutamine is hydrolyzed to glutamate for glutathione synthesis, an essential factor to abrogate high ROS via xCT antiporter. Thus, blocking glutamate flux using riluzole (an amyotropic lateral sclerosis approved drug) can selectively kill CR cells in vitro and in vivo. However, we discovered here that glutathione suppression is not the primary pathway in eradicating the CR cells. Riluzole can lead to further decrease in NAD+ (nicotinamide adenine dinucleotide) and lactate dehydrogenase-A (LDHA) expressions which in turn further heightened oxidative stress in CR cells. LDHA knocked-down cells became hypersensitive to riluzole treatments and possessed increased levels of ROS. Addition of NAD+ re-stabilized LDHA and reversed riluzole induced cell death. Thus far, no drugs are available which could overcome cisplatin resistance or kill cisplatin resistant cells. CR cells possess high levels of ROS and undergo metabolic reprogramming. These metabolic adaptations can be exploited and targeted by riluzole. Riluzole may serve as a dual-targeting agent by suppression LDHA and blocking xCT antiporter. Repurposing of riluzole should be considered for future treatment of cisplatin resistant lung cancer patients.

Cisplatin-induced synthetic lethality to arginine-starvation therapy by transcriptional suppression of ASS1 is regulated by DEC1, HIF-1α, and c-Myc transcription network and is independent of ASS1 promoter DNA methylation.

Many human tumors require extracellular arginine (Arg) for growth because the key enzyme for de novo biosynthesis of Arg, argininosuccinate synthetase 1 (ASS1), is silenced. These tumors are sensitive to Arg-starvation therapy using pegylated arginine deiminase (ADI-PEG20) which digests extracellular Arg. Many previous studies reported that ASS1 silencing is due to epigenetic inactivation of ASS1 expression by DNA methylation, and that the demethylation agent 5-aza-deoxycytidine (Aza-dC) can induce ASS1 expression. Moreover, it was reported that cisplatin suppresses ASS1 expression through ASS1 promoter methylation, leading to synthetic lethality to ADI-PEG20 treatment. We report here that cisplatin supppresses ASS1 expression is due to upregulation of HIF-1α and downregulation of c-Myc, which function as negative and positive regulators of ASS1 expression, respectively, by reciprocal bindings to the ASS1 promoter. In contrast, we found that Aza-dC induces ASS1 expression by downregulation of HIF-1α but upregulation of c-Myc. We further demonstrated that the clock protein DEC1 is the master regulator of HIF-1α and c-Myc that regulate ASS1. cDDP upregulates DEC1, whereas Aza-dC suppresses its expression. Using two proteasomal inhibitors bortezomib and carfilzomib which induce HIF-1α accumulation, we further demonstrated that HIF-1α is involved in ASS1 silencing for the maintenance of Arg auxotrophy for targeted Arg-starvation therapy.

Radiofrequency ablation in primary non-small cell lung cancer: What a radiologist needs to know.

Lung cancer continues to be one of the leading causes of death worldwide. In advanced cases of lung cancer, a multimodality approach is often applied, however with poor local control rates. In early non-small cell lung cancer (NSCLC), surgery is the standard of care. Only 15-30% of patients are eligible for surgical resection. Improvements in imaging and treatment delivery systems have provided new tools to better target these tumors. Stereotactic body radiation therapy (SBRT) has evolved as the next best option. The role of radiofrequency ablation (RFA) is also growing. Currently, it is a third-line option in stage 1 NSCLC, when SBRT cannot be performed. More recent studies have demonstrated usefulness in recurrent tumors and some authors have also suggested combination of RFA with other modalities in larger tumors. Following the National Lung Screening Trial (NLST), screening by low-dose computed tomography (CT) has demonstrated high rates of early-stage lung cancer detection in high-risk populations. Hence, even considering the current role of RFA as a third-line option, in view of increasing numbers of occurrences detected, the number of potential RFA candidates may see a steep uptrend. In view of all this, it is imperative that interventional radiologists be familiar with the techniques of lung ablation. The aim of this article is to discuss the procedural technique of RFA in the lung and review the current evidence regarding RFA for NSCLC.