The LEAP2 Response to Cancer-Related Anorexia-Cachexia Syndrome in Male Mice and Patients.

The hormone ghrelin serves a protective role in cancer-related anorexia-cachexia syndrome (CACS)-a condition in which plasma levels of ghrelin rise, its administration lessens CACS severity, and experimentally reduced signaling by its receptor (GHSR) worsens fat loss and anorexia and accelerates death. Yet, actions for the related hormone liver-expressed antimicrobial peptide-2 (LEAP2), which is an endogenous GHSR antagonist, are unexplored in CACS. Here, we found that plasma LEAP2 and LEAP2/ghrelin ratio were lower in Lewis lung carcinoma (LLC) and RM-9 prostate cancer CACS mouse models. Ghrelin deletion exaggerated losses of tumor-free body weight and fat mass, reduced food intake, reduced soleus muscle weight, and/or lowered grip strength in LLC or RM-9 tumor-bearing mice. LEAP2 deletion lessened reductions in tumor-free body weight and fat mass and increased food intake in LLC or RM-9 tumor-bearing mice. In a 55-subject cohort of patients with CACS or weight-stable cancer, the plasma LEAP2/total ghrelin ratio was negatively correlated with 6-month weight change preceding blood collection. These data demonstrate that ghrelin deletion exacerbates CACS in the LLC and RM-9 tumor-bearing mouse models while contrastingly, LEAP2 deletion reduces measures of CACS in these tumor-bearing mouse models. Further, they suggest that lower plasma LEAP2/ghrelin ratio protects against worsened CACS.

ASMase is Essential for the Immune Response to Partial-Tumor Radiation Exposure.

BACKGROUND/AIMS: Tumor response to radiation is thought to depend on the direct killing of tumor cells. Our laboratory has called this into question. Firstly, we showed that the biology of the host, specifically the endothelial expression of acid sphingomyelinase (ASMase), was critical in determining tumor radiocurability. Secondly, we have shown that the immune system can enhance radiation response by allowing a complete tumor control in hemi-irradiated tumors. In this paper, we focus on the integration of these two findings. METHODS: We used Lewis Lung Carcinoma (LLC) cells, injected in the flank of either: (i) ASMase knockout or (ii) WT of matched background (sv129xBl/6) or (iii) C57Bl/6 mice. Radiation therapy (RT) was delivered to 50% or 100% of the LLC tumor volume. Tumor response, immune infiltration (CD8+ T cells), ICAM-1, and STING activation were measured. Radiotherapy was also combined with methyl-cyclodextrin, to inhibit the ASMase-mediated formation of ceramide-enriched lipid rafts. RESULTS: We recapitulated our previous finding, namely that tumor hemi-irradiation was sufficient for tumor control in the LLC/C57Bl/6 model. However, in ASMase KO mice hemi-irradiation was ineffective. Likewise, pharmacological inhibition of ASMase significantly reduced the tumor response to hemi-irradiation. Further, we demonstrated elevated ICAM-1 expression, increased levels of CD8+ T cells, ICAM-1, and STING activation in tumors growing in C57Bl/6 mice, as well as the ASMase WT strain. However, no such changes were seen in tumors growing in ASMase KO mice. CONCLUSION: ASMase and ceramide generation are necessary to mediate a radiation-induced anti-tumor immune response via STING activation.

Muscle-derived IL-1ß regulates EcSOD expression via the NBR1-p62-Nrf2 pathway in muscle during cancer cachexia.

Oxidative stress contributes to the loss of skeletal muscle mass and function in cancer cachexia. However, this outcome may be mitigated by an improved endogenous antioxidant defence system. Here, using the well-established oxidative stress-inducing muscle atrophy model of Lewis lung carcinoma (LLC) in 13-week-old male C57BL/6J mice, we demonstrate that extracellular superoxide dismutase (EcSOD) levels increase in the cachexia-prone extensor digitorum longus muscle. LLC transplantation significantly increased interleukin-1ß (IL-1ß) expression and release from extensor digitorum longus muscle fibres. Moreover, IL-1ß treatment of C2C12 myotubes increased NBR1, p62 phosphorylation at Ser351, Nrf2 nuclear translocation and EcSOD protein expression. Additional studies in vivo indicated that intramuscular IL-1ß injection is sufficient to stimulate EcSOD expression, which is prevented by muscle-specific knockout of p62 and Nrf2 (i.e. in p62 skmKO and Nrf2 skmKO mice, respectively). Finally, since an increase in circulating IL-1ß may lead to unwanted outcomes, we demonstrate that targeting this pathway at p62 is sufficient to drive muscle EcSOD expression in an Nrf2-dependent manner. In summary, cancer cachexia increases EcSOD expression in extensor digitorum longus muscle via muscle-derived IL-1ß-induced upregulation of p62 phosphorylation and Nrf2 activation. These findings provide further mechanistic evidence for the therapeutic potential of p62 and Nrf2 to mitigate cancer cachexia-induced muscle atrophy. KEY POINTS: Oxidative stress plays an important role in muscle atrophy during cancer cachexia. EcSOD, which mitigates muscle loss during oxidative stress, is upregulated in 13-week-old male C57BL/6J mice of extensor digitorum longus muscles during cancer cachexia. Using mouse and cellular models, we demonstrate that cancer cachexia promotes muscle EcSOD protein expression via muscle-derived IL-1ß-dependent stimulation of the NBR1-p62-Nrf2 signalling pathway. These results provide further evidence for the potential therapeutic targeting of the NBR1-p62-Nrf2 signalling pathway downstream of IL-1ß to mitigate cancer cachexia-induced muscle atrophy.

[Antitumor Study of Neoantigen-reactive T Cells Co-expressing IL-7 and CCL19 
in Mouse Lung Cancer].

BACKGROUND: Neoantigen reactive T cell (NRT) has the ability to inhibit the growth of tumors expressing specific neoantigens. However, due to the difficult immune infiltration and the inhibition of tumor microenvironment, the therapeutic effect of NRT in solid tumors is limited. In this study, we designed NRT cells (7×19 NRT) that can express both interleukin-7 (IL-7) and chemokine C-C motif ligand 19 (CCL19) in mouse lung cancer cells, and evaluated the difference in anti-tumor effect between 7×19 NRT cells and conventional NRT cells. METHODS: We performed next-generation sequencing and neoantigen prediction for mouse Lewis lung carcinoma (LLC), prepared RNA vaccine, cultured NRT cells, constructed retroviral vectors encoding IL-7 and CCL19, transduced NRT cells and IL-7 and CCL19 were successfully expressed, and 7×19 NRT was successfully obtained. The anti-tumor effect was evaluated in vivo and in vitro in mice. RESULTS: The 7×19 NRT cells significantly enhanced the proliferation and invasion ability of T cells by secreting IL-7 and CCL19, achieved significant tumor inhibition in the mouse lung cancer and extended the survival period of mice. The T cell infiltration into tumor tissue and the necrosis of tumor tissue increased significantly after 7×19 NRT treatment. In addition, both 7×19 NRT treatment and conventional NRT treatment were safe. CONCLUSIONS: The anti-solid tumor ability of NRT cells is significantly enhanced by the arming of IL-7 and CCL19, which is a safe and effective genetic modification of NRT.

Tumour-induced alterations in single-nucleus transcriptome of atrophying muscles indicate enhanced protein degradation and reduced oxidative metabolism.

BACKGROUND: Tumour-induced skeletal muscle wasting in the context of cancer cachexia is a condition with profound implications for patient survival. The loss of muscle mass is a significant clinical obstacle and is linked to reduced tolerance to chemotherapy and increased frailty. Understanding the molecular mechanisms driving muscle atrophy is crucial for the design of new therapeutics. METHODS: Lewis lung carcinoma tumours were utilized to induce cachexia and muscle atrophy in mice. Single-nucleus libraries of the tibialis anterior (TA) muscle from tumour-bearing mice and their non-tumour-bearing controls were constructed using 10X Genomics applications following the manufacturer's guidelines. RNA sequencing results were analysed with Cell Ranger software and the Seurat R package. Oxygen consumption of mitochondria isolated from TA muscle was measured using an Oroboros O2k-FluoRespirometer. Mouse primary myotubes were treated with a recombinant ectodysplasin A2 (EDA-A2) protein to activate EDA-A2 receptor (EDA2R) signalling and study changes in gene expression and oxygen consumption. RESULTS: Tumour-bearing mice were sacrificed while exhibiting moderate cachexia. Average TA muscle weight was reduced by 11% (P = 0.0207) in these mice. A total of 12 335 nuclei, comprising 6422 nuclei from the control group and 5892 nuclei from atrophying muscles, were studied. The analysis of single-nucleus transcriptomes identified distinct myonuclear gene signatures and a shift towards type IIb myonuclei. Muscle atrophy-related genes, including Atrogin1, MuRF1 and Eda2r, were upregulated in these myonuclei, emphasizing their crucial roles in muscle wasting. Gene set enrichment analysis demonstrated that EDA2R activation and tumour inoculation led to similar expression patterns in muscle cells, including the stimulation of nuclear factor-kappa B, Janus kinase-signal transducer and activator of transcription and transforming growth factor-beta pathways and the suppression of myogenesis and oxidative phosphorylation. Muscle oxidative metabolism was suppressed by both tumours and EDA2R activation. CONCLUSIONS: This study identified tumour-induced transcriptional changes in muscle tissue at single-nucleus resolution and highlighted the negative impact of tumours on oxidative metabolism. These findings contribute to a deeper understanding of the molecular mechanisms underlying muscle wasting.

PRPS2-mediated modulation of the antitumor immune response in lung cancer through CCL2-mediated tumor-associated macrophages and myeloid-derived suppressor cells.

BACKGROUND: Phosphoribosyl pyrophosphate synthetase 2 (PRPS2) is known as an oncogene in many types of cancers, including lung cancer. However, its role in regulating tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) remains unclear. Our study aimed to explore the involvement of PRPS2 in TAM and MDSC regulation. METHODS: Stable Lewis lung cancer (LLC) cell lines were established using a lentivirus system. These LLC lines were then used to establish tumor model in mice. The levels of target genes were determined using qPCR, western blotting, and ELISA assays. The percentage of different immune cell types was analyzed using fluorescence-activated cell sorting. The chemotaxis ability of TAM and MDSC was evaluated using an in vitro transwell chemotaxis assay. RESULTS: Notably, PRPS2 was found to regulate the chemotaxis of TAM and MDSC in tumor cells, as evidenced by the positive correlation of PRPS2 expression levels and abundance of TAM and MDSC populations. In addition, the expression of CCL2, mediated by PRPS2, was identified as a key factor in the chemotaxis of TAM and MDSC, as evidenced by a significant reduction in macrophages and MDSC numbers in the presence of the CCL2 antibody. Furthermore, in vivo experiments confirmed the involvement of PRPS2 in mediating CCL2 expression. PRPS2 was also found to regulate immune cell infiltration into tumors, whereas knockdown of CCL2 reversed the phenotype induced by PRPS2 overexpression. In tumor tissues from mice implanted with LLC-PRPS2-shCCL2 cells, a notable increase in CD4+ and CD8+ T cell percentages, alongside a marked decrease in TAMs, M-MDSC, and PMN-MDSC, was observed. CONCLUSION: Taken together, PRPS2 plays a crucial role in modulating the antitumor immune response by reprogramming CCL2-mediated TAM and MDSC.

Tumor promoting effect of PDLIM2 downregulation involves mitochondrial ROS, oncometabolite accumulations and HIF-1α activation.

BACKGROUND: Cancer is characterized by dysregulated cellular metabolism. Thus, understanding the mechanisms underlying these metabolic alterations is important for developing targeted therapies. In this study, we investigated the pro-tumoral effect of PDZ and LIM domain 2 (PDLIM2) downregulation in lung cancer growth and its association with the accumulation of mitochondrial ROS, oncometabolites and the activation of hypoxia-inducible factor-1 (HIF-1) α in the process. METHODS: Databases and human cancer tissue samples were analyzed to investigate the roles of PDLIM2 and HIF-1α in cancer growth. DNA microarray and gene ontology enrichment analyses were performed to determine the cellular functions of PDLIM2. Seahorse assay, flow cytometric analysis, and confocal microscopic analysis were employed to study mitochondrial functions. Oncometabolites were analyzed using liquid chromatography-mass spectrometry (LC-MS). A Lewis lung carcinoma (LLC) mouse model was established to assess the in vivo function of PDLIM2 and HIF-1α. RESULTS: The expression of PDLIM2 was downregulated in lung cancer, and this downregulation correlated with poor prognosis in patients. PDLIM2 highly regulated genes associated with mitochondrial functions. Mechanistically, PDLIM2 downregulation resulted in NF-κB activation, impaired expression of tricarboxylic acid (TCA) cycle genes particularly the succinate dehydrogenase (SDH) genes, and mitochondrial dysfunction. This disturbance contributed to the accumulation of succinate and other oncometabolites, as well as the buildup of mitochondrial reactive oxygen species (mtROS), leading to the activation of hypoxia-inducible factor 1α (HIF-1α). Furthermore, the expression of HIF-1α was increased in all stages of lung cancer. The expression of PDLIM2 and HIF-1α was reversely correlated in lung cancer patients. In the animal study, the orally administered HIF-1α inhibitor, PX-478, significantly reduces PDLIM2 knockdown-promoted tumor growth. CONCLUSION: These findings shed light on the complex action of PDLIM2 on mitochondria and HIF-1α activities in lung cancer, emphasizing the role of HIF-1α in the tumor-promoting effect of PDLIM2 downregulation. Additionally, they provide new insights into a strategy for precise targeted treatment by suggesting that HIF-1α inhibitors may serve as therapy for lung cancer patients with PDLIM2 downregulation.

Predicting the mechanism of action of YQYYJD prescription in the treatment of non-small cell lung cancer using transcriptomics analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: The efficacy of the herbal formula Yiqi Yangyin Jiedu (YQYYJD) in the treatment of advanced lung cancer has been reported in clinical trials. However, the key anti-lung cancer herbs and molecular mechanisms underlying its inhibition of lung cancer are not well-understood. AIM OF THE STUDY: To identify the key anti-lung cancer herbs in the YQYYJD formula and investigate their therapeutic effect and potential mechanism of action in non-small cell lung cancer (NSCLC) using transcriptomics and bioinformatics techniques. MATERIALS AND METHODS: A mouse Lewis lung carcinoma (LLC) subcutaneous inhibitory tumor model was established with 6 mice in each group. Mice were treated with the YQYYJD split formula: Yiqi Formula (YQ), Yangyin Formula (YY), and Ruanjian Jiedu Formula (RJJD) for 14 days. The tumor volume and mouse weight were recorded, and the status of tumor occurrence was further observed by taking photos. The tumor was stained with hematoxylin-eosin to observe its histopathological changes. Immunohistochemistry was used to detect the expression of the proliferation marker Ki67 and the apoptotic marker Caspase-3 in tumor tissues. Flow cytometry was used to detect the number of CD4+ and CD8+ T cells and cytokines interleukin-2 (IL-2) and interferon-gamma (IFN-γ) in the spleen and tumor tissues. The differential genes of key drugs against tumors were obtained by transcriptome sequencing of tumors. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) enrichment analyses were performed on differential genes to obtain pathways and biological processes where targets were aggregated. TIMER2.0 and TISIDB databases were used to evaluate the impact of drugs on immune cell infiltration and immune-related genes. The binding activity of the key targets and compounds was verified by molecular docking. RESULTS: YQ, YY, and RJJD inhibited the growth of subcutaneous transplanted tumors in LLC mice to varying degrees and achieved antitumor effects by inhibiting the expression of tumor cell proliferation, apoptosis, and metastasis-related proteins. Among the three disassembled prescriptions, YQ better inhibited the growth of subcutaneous transplanted tumors in LLC mice, significantly promoted tumor necrosis, significantly increased the expression of Caspase-3 protein in tumor tissue, and significantly decreased the expression of Ki-67 (P < 0.05), thereby increasing the infiltration of CD8+ T cells. YQ significantly increased the expression of CD4+ and CD8+ T cells in tumor and splenic tissues of tumor-bearing mice and up-regulated the expression of IL-2 and IFN-γ. Transcriptome sequencing and bioinformatics results showed that after YQ intervention, differentially expressed genes were enriched in more than one tumor-related pathway and multiple immune regulation-related biological functions. There were 12 key immune-related target genes. CONCLUSION: YQ was the key disassembled prescription of YQYYJD, exerting significant antitumor effects and immune regulation effects on NSCLC. It may have relieved T cell exhaustion and regulated the immune microenvironment to exert antitumor effects by changing lung cancer-related targets, pathways, and biological processes.

Diet-induced obesity does not exacerbate cachexia in male mice bearing Lewis-lung carcinoma tumors.

Cachexia is a muscle-wasting syndrome commonly observed in patients with cancer, which can significantly worsen clinical outcomes. Because of a global rise in obesity, the coexistence of cachexia in obese individuals poses unique challenges, with the impact of excessive adiposity on cachexia severity and underlying pathophysiology not well defined. Understanding the interplay between cachexia and obesity is crucial for improving diagnosis and treatment strategies for these patients; therefore, the present study examined differences in cachexia between lean and obese mice bearing Lewis lung carcinoma (LLC) tumors. Nine-week-old, male C57Bl6J mice were placed on either a chow or a high-fat diet (HFD) for 9 wk. After the diet intervention, mice were inoculated with LLC or vehicle. Markers of cachexia, such as body and muscle loss, were noted in both chow and HFD groups with tumors. Tumor weight of HFD animals was greater than that of chow. LLC tumors reduced gastrocnemius, plantaris, and soleus mass, regardless of diet. The tibialis anterior and plantaris mass and cross-sectional area of type IIb/x fibers in the gastrocnemius were not different between HFD-chow, HFD-tumor, and chow-tumor. Using RNA sequencing (RNA-seq) of the plantaris muscle from chow-tumor and HFD-tumor groups, we identified ∼400 differentially expressed genes. Bioinformatic analysis identified changes in lipid metabolism, mitochondria, bioenergetics, and proteasome degradation. Atrophy was not greater despite larger tumor burden in animals fed an HFD, and RNA-seq data suggests that partial protection is mediated through differences in mitochondrial function and protein degradation, which may serve as future mechanistic targets.NEW & NOTEWORTHY This study provides timely information on the interaction between obesity and cancer cachexia. Lean and obese animals show signs of cachexia with reduced body weight, adipose tissue, and gastrocnemius muscle mass. There was not significant wasting in the tibialis anterior, plantaris, or fast twitch fibers in the gastrocnemius muscle of obese animals with tumors. RNA-seq analysis reveals that obese tumor bearing animals had differential expression of mitochondria- and degradation-related genes, which may direct future studies in mechanistic research.

Aerobic exercise training mitigates tumor growth and cancer-induced splenomegaly through modulation of non-platelet platelet factor 4 expression.

Exercise training reduces the incidence of several cancers, but the mechanisms underlying these effects are not fully understood. Exercise training can affect the spleen function, which controls the hematopoiesis and immune response. Analyzing different cancer models, we identified that 4T1, LLC, and CT26 tumor-bearing mice displayed enlarged spleen (splenomegaly), and exercise training reduced spleen mass toward control levels in two of these models (LLC and CT26). Exercise training also slowed tumor growth in melanoma B16F10, colon tumor 26 (CT26), and Lewis lung carcinoma (LLC) tumor-bearing mice, with minor effects in mammary carcinoma 4T1, MDA-MB-231, and MMTV-PyMT mice. In silico analyses using transcriptome profiles derived from these models revealed that platelet factor 4 (Pf4) is one of the main upregulated genes associated with splenomegaly during cancer progression. To understand whether exercise training would modulate the expression of these genes in the tumor and spleen, we investigated particularly the CT26 model, which displayed splenomegaly and had a clear response to the exercise training effects. RT-qPCR analysis confirmed that trained CT26 tumor-bearing mice had decreased Pf4 mRNA levels in both the tumor and spleen when compared to untrained CT26 tumor-bearing mice. Furthermore, exercise training specifically decreased Pf4 mRNA levels in the CT26 tumor cells. Aspirin treatment did not change tumor growth, splenomegaly, and tumor Pf4 mRNA levels, confirming that exercise decreased non-platelet Pf4 mRNA levels. Finally, tumor Pf4 mRNA levels are deregulated in The Cancer Genome Atlas Program (TCGA) samples and predict survival in multiple cancer types. This highlights the potential therapeutic value of exercise as a complementary approach to cancer treatment and underscores the importance of understanding the exercise-induced transcriptional changes in the spleen for the development of novel cancer therapies.

Exercise training induces alteration of clock genes and myokines expression in tumor-bearing mice.

To investigate the impact of different exercise training schedules (following a fixed schedule or at random times of the day) on clock genes and myokine expression patterns in the skeletal muscle of tumor-bearing mice. Mice were divided into three groups: tumor (LLC), tumor + exercise training (LLC + T) always performed at the same time of the day (ZT2) and exercise training at random times of the day (ZTAlt). Mice were inoculated subcutaneously with Lewis lung carcinoma cells. The gastrocnemius muscle was dissected and the clock gene expression (Clock/Per1/Per2/Per3/Rev-Erbα/GAPDH) was investigated by quantitative reverse transcription polymerase chain reaction with SYBR® Green. Myokine content in muscle (tumour necrosis factor alpha/IL-10/IL-4) was assessed by enzyme-linked immunosorbent assay. At the end of the protocol, the trained groups showed a reduction in total weight, when compared to Lewis lung carcinoma. Tumor weight was lower in the LLC + T (ZTAlt), when compared to LLC. Clock gene mRNA expression showed a significant increase for ZT20 in the groups that performed physical exercise at LLC + T (ZTAlt), when compared with LLC. The Per family showed increased mRNA expression in ZT4 in both trained mice groups, when compared with LLC. LLC + T (ZTAlt) presented reduction of the expression of anti-inflammatory myokines (Il-10/IL-4) during the night, compared with LLC + T(ZT2). Exercise training is able to induce marked modification of clock gene expression and of the production of myokines, in a way that is dependent on schedule exercise training strategy. Taken together, the results show that exercise is a potent Zeitgeber and may thus contribute to change clock genes expression and myokines that are able to reduce the tumor weight.

Dendritic cells infected with recombinant adenoviral vector encoding mouse fibroblast activation protein-α and human livin α exert an antitumor effect against Lewis lung carcinoma in mice.

BACKGROUND: Fibroblast activation protein-α (FAP) and livin α are considered as cancer-associated fibroblasts (CAFs) and tumor-specific targets, respectively, for immunogenic tumor vaccines. This study is designed to decipher the antitumor effect of double-gene modified dendritic cells (DCs) on Lewis lung carcinoma (LLC). METHODS: By encoding mouse FAP cDNA and human livin α (i.e., hlivin α) cDNA into recombinant adenoviral vector (rAd), rAd-FAP, rAd-hlivin α, and rAd-FAP/hlivin α were constructed, which were then transduced into mouse DCs. LLC-bearinig mice were immunized with the infected DCs (5 × 105 cells/mouse), followed by calculation of tumor volume and survival rate. The identification of CAFs from mouse LLC as well as the determination on expressions of FAP and livin α, was accomplished by western blot. Cytotoxic T lymphocyte assay was harnessed to assess the effect of the infected DCs on inducing splenic lymphocytes to lyse CAFs. RESULTS: DCs were successfully transduced with rAd-FAP/hlivin α in vitro. FAP was highly expressed in CAFs. CAFs were positive for α-SMA and negative for CD45 and CD31. Livin α level was upregulated in mouse LLC. Immunization with rAd-FAP/hlivin α-transduced DCs suppressed LLC volume and improved the survival of tumor-bearing mice. Immunization with rAd-FAP/hlivin α-transduced DCs enhanced the cytotoxic effect of splenic lymphocytes on LLC tumor-derived CAFs. CONCLUSION: Injection with rAd-FAP/hlivin α-transduced DCs promotes immune-enhanced tumor microenvironment by decreasing CAFs and suppresses tumor growth in LLC mouse models.

LP07 and LLC preclinical models of lung cancer induce divergent anabolic deficits and expression of pro-inflammatory effectors of muscle wasting.

Preclinical models have been instrumental to elucidate the mechanisms underlying muscle wasting in lung cancer (LC). We investigated anabolic deficits and the expression of proinflammatory effectors of muscle wasting in the LP07 and Lewis lung carcinoma (LLC) tumor models. Tumor growth resulted in significant weakness in LP07 but not in LLC mice despite similar reductions in gastrocnemius muscle mass in both models. The LP07 tumors caused a reduction in ribosomal (r)RNA and a decrease in rRNA gene (rDNA) transcription elongation, whereas no changes in ribosomal capacity were evident in LLC tumor-bearing mice. Expression of RNA Polymerase I (Pol I) elongation-associated subunits Polr2f, PAF53, and Znrd1 mRNAs was significantly elevated in the LP07 model, whereas Pol I elongation-related factors FACT and Spt4/5 mRNAs were elevated in the LLC mice. Reductions in RPS6 and 4E-BP1 phosphorylation were similar in both models but were independent of mTOR phosphorylation in LP07 mice. Muscle inflammation was also tumor-specific, IL-6 and TNF-α mRNA increased with LLC tumors, and upregulation of NLRP3 mRNA was independent of tumor type. In summary, although both models caused muscle wasting, only the LP07 model displayed muscle weakness with reductions in ribosomal capacity. Intracellular signaling diverged at the mTOR level with similar reductions in RPS6 and 4E-BP1 phosphorylation regardless of tumor type. The increase in proinflammatory factors was more pronounced in the LLC model. Our results demonstrate novel divergent anabolic deficits and expression of proinflammatory effectors of muscle wasting in the LP07 and LLC preclinical models of lung cancer.NEW & NOTEWORTHY We provide novel data demonstrating significant divergence in anabolic deficits and the expression of proinflammatory effectors of muscle wasting consequent to different lung-derived tumors.

PGC-1α overexpression is not sufficient to mitigate cancer cachexia in either male or female mice.

Cancer cachexia (CC) accounts for 20%-40% of cancer-related deaths. Mitochondrial aberrations have been shown to precede muscle atrophy in different atrophy models, including cancer. Therefore, this study investigated potential protection from the cachectic phenotype through overexpression of peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α). First, to establish potential of mitochondria-based approaches we showed that the mitochondrial antioxidant MitoTEMPO (MitoT) attenuates myotube atrophy induced by Lewis lung carcinoma (LLC) cell conditioned media. Next, cachexia was induced in muscle-specific PGC-1α overexpressing (MCK-PCG1α) or wildtype (WT) littermate mice by LLC implantation. MCK-PCG1α did not protect LLC-induced muscle mass loss. In plantaris, Atrogin mRNA content was 6.2-fold and ∼11-fold greater in WT-LLC vs WT-phosphate-buffered saline (PBS) for males and females, respectively (p < 0.05). MitoTimer red:green ratio for male PGC was ∼65% higher than WT groups (p < 0.05), with ∼3-fold more red puncta in LLC than PBS (p < 0.05). Red:green ratio was ∼56% lower in females WT-LLC vs PGC-LLC (p < 0.05). In females, no change in red puncta was noted across conditions. Lc3 mRNA content was ∼73% and 2-fold higher in male and female LLC mice, respectively, vs PBS (p < 0.05). While MitoT could mitigate cancer-induced atrophy in vitro, PGC-1α overexpression was insufficient to protect muscle mass and mitochondrial health in vivo despite mitigation of cachexia-associated signaling pathways.

Inhibition of epidermal growth factor receptor suppresses parathyroid hormone-related protein expression in tumours and ameliorates cancer-associated cachexia.

BACKGROUND: Lung cancer is the primary cause of cancer deaths worldwide. Activation of epidermal growth factor receptor (EGFR) leads to lung cancer progression and poor prognosis while involuntary weight loss remains a major problem. Tumour-derived parathyroid hormone-related protein (PTHrP) emerged as a potential mediator of cachexia. Here, we investigated the modulatory role of EGFR signalling in PTHrP (encoded by Pthlh) gene expression and the impact of this relationship on cancer cachexia. METHODS: Global gene expression profiles of Lewis lung carcinoma (LLC) cells were analysed. Pthlh mRNA levels were measured by qRT-PCR in LLC cells treated with EGFR ligands and tyrosine kinase inhibitors (TKIs). LLC tumour-bearing mice received EGFR TKI erlotinib for 7 days via intraperitoneal injection or oral gavage. Tumour Pthlh mRNA, weight of fat/muscle tissue, and grip strength were assessed. RNA-seq data from The Cancer Genome Atlas and gene expression analysis tools were used to characterize expression profiles of PTHLH and EGFR along with correlation analysis of PTHLH with EGFR and transforming growth factor alpha (TGFA) in human lung cancer and head and neck squamous carcinoma (HNSC). Survival of lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) patients with EGFR gene alterations was analysed in regard to PTHLH expression. RESULTS: Expression of EGFR ligands, EGFR itself, and PTHrP co-clusters in LLC cells. Activation of EGFR signalling with its ligands significantly increases (3.8-fold, P < 0.0005) while EGFR TKIs significantly decrease (90%, P < 0.0005) Pthlh mRNA levels in LLC cells. Pthlh mRNA drops 65-75% (P < 0.0005) in tumours upon treatment of LLC tumour-bearing mice with erlotinib while their muscle mass and grip strength increase (9.2% P < 0.05, 23% P < 0.005, respectively) compared with tumour-bearing control mice. PTHLH is overexpressed in tumours of LUSC (45.8-fold, P < 0.05) and HNSC (17.5-fold, P < 0.05) compared with normal tissue. PTHLH expression correlates with EGFR and its ligand TGFA in both cancers (LUSC: n = 745, R = 0.32, P < 0.0001 and R = 0.51, P < 0.0001; HNSC: n = 545, R = 0.34, P < 0.001 and R = 0.50, P < 0.001, respectively). High PTHLH mRNA associates with poor overall survival in LUAD patients with activating EGFR mutations (n = 40, log-rank test, P = 0.0451). CONCLUSIONS: Epidermal growth factor receptor signalling regulates expression of cachexia mediator PTHrP. EGFR inhibition reduces PTHrP expression in LLC tumours and ameliorates cachexia in LLC tumour-bearing mice.

Hyperglycemia-induced oxidative stress promotes tumor metastasis by upregulating vWF expression in endothelial cells through the transcription factor GATA1.

Diabetes mellitus (DM) characterized by hyperglycemia is a chronic metabolic disorder that leads to many symptoms and vascular complications. Despite the close association between DM and cancer progression, the response and role of endothelial cells (ECs) under diabetic conditions in tumor metastasis remain to be elucidated. In this study, we sought to determine whether and how ECs under diabetic conditions contribute to tumor metastasis. We have taken advantage of syngeneic mouse tumor models of Lewis lung carcinoma (LLC) and melanoma (B16F10) cells and a streptozotocin (STZ)-induced hyperglycemia model. We demonstrated that hyperglycemia increased the metastasis of LLC and B16F10 cells in an experimental metastasis model with an intravenous injection of the tumor cells. We also found that hyperglycemia promoted lung metastasis of tumor cells by increasing the adhesiveness of ECs to facilitate the adhesion of tumor cells to ECs rather than affecting the metastatic behavior of tumor cells themselves. From the analysis of gene expression in primary lung ECs from STZ-treated mice, we identified that vWF promoted the adhesion of tumor cells to ECs and the transendothelial migration of tumor cells. Mechanistically, hyperglycemia-induced oxidative stress in ECs, and increased oxidative stress enhanced vWF expression in ECs through an increase in the transcription factor GATA1. These results provide evidence for the role of vWF in ECs in promoting hyperglycemia-induced tumor metastasis and potential therapeutic targets for the regulation of vWF expression in ECs and hyperglycemia-induced tumor metastasis.

Macrophages are metabolically heterogeneous within the tumor microenvironment.

Macrophages are often prominently present in the tumor microenvironment, where distinct macrophage populations can differentially affect tumor progression. Although metabolism influences macrophage function, studies on the metabolic characteristics of ex vivo tumor-associated macrophage (TAM) subsets are rather limited. Using transcriptomic and metabolic analyses, we now reveal that pro-inflammatory major histocompatibility complex (MHC)-IIhi TAMs display a hampered tricarboxylic acid (TCA) cycle, while reparative MHC-IIlo TAMs show higher oxidative and glycolytic metabolism. Although both TAM subsets rapidly exchange lactate in high-lactate conditions, only MHC-IIlo TAMs use lactate as an additional carbon source. Accordingly, lactate supports the oxidative metabolism in MHC-IIlo TAMs, while it decreases the metabolic activity of MHC-IIhi TAMs. Lactate subtly affects the transcriptome of MHC-IIlo TAMs, increases L-arginine metabolism, and enhances the T cell suppressive capacity of these TAMs. Overall, our data uncover the metabolic intricacies of distinct TAM subsets and identify lactate as a carbon source and metabolic and functional regulator of TAMs.

LncRNA Snhg6 regulates the differentiation of MDSCs by regulating the ubiquitination of EZH2.

Myeloid-derived suppressor cells (MDSCs) are derived from bone marrow progenitor cells commonly, which is a heterogeneous cell group composed of immature granulocytes, dendritic cells, macrophages and early undifferentiated bone marrow precursor cells. Its differentiation and immunosuppressive function are regulated by complex network signals, but the specific regulation mechanisms are not yet fully understood. In this study, we found that in mouse of Lewis lung cancer xenograft, long non-coding RNA Snhg6 (lncRNA Snhg6) was highly expressed in tumor-derived MDSCs compared with spleen-derived MDSCs. LncRNA Snhg6 facilitated the differentiation of CD11b+ Ly6G- Ly6Chigh monocytic MDSCs (Mo-MDSCs) rather than CD11b+ Ly6G+ Ly6Clow polymorphonuclear MDSCs (PMN-MDSCs), but did not affect the immunosuppressive function of MDSCs. Notably, lncRNA Snhg6 could inhibit the expression of EZH2 by ubiquitination pathway at protein level rather than mRNA level during the differentiation of mouse bone marrow cells into MDSCs in vitro. EZH2 may be an important factor in the regulation of lncRNA Snhg6 to promote the differentiation of Mo-MDSCs. So what we found may provide new ideas and targets for anti-tumor immunotherapy targeting MDSCs.

Tet2 deficiency in immune cells exacerbates tumor progression by increasing angiogenesis in a lung cancer model.

Immune cells harboring somatic mutations reportedly infiltrate cancer tissues in patients with solid cancers and accompanying clonal hematopoiesis. Loss-of-function TET2 mutations are frequently observed in clonal hematopoiesis in solid cancers. Here, using a mouse lung cancer model, we evaluated the activity of Tet2-deficient immune cells in tumor tissues. Myeloid-specific Tet2 deficiency enhanced tumor growth in mice relative to that seen in controls. Single-cell sequencing analysis of immune cells infiltrating tumors showed relatively high expression of S100a8/S100a9 in Tet2-deficient myeloid subclusters. In turn, treatment with S100a8/S100a9 promoted Vegfa production by cancer cells, leading to a marked increase in the tumor vasculature in Tet2-deficient mice relative to controls. Finally, treatment of Tet2-deficient mice with an antibody against Emmprin, a known S100a8/S100a9 receptor, suppressed tumor growth. These data suggest that immune cells derived from TET2-mutated clonal hematopoiesis exacerbate lung cancer progression by promoting tumor angiogenesis and may provide a novel therapeutic target for lung cancer patients with TET2-mutated clonal hematopoiesis.

Ganoderma formosanum Exopolysaccharides Inhibit Tumor Growth via Immunomodulation.

Ganoderma formosanum (GF) is a medicinal mushroom endemic to Taiwan. Previous research established the optimal culture conditions to produce exopolysaccharide rich in ß-glucan (GF-EPS) from submerged fermentation of GF. The present study investigated the antitumor effects of GF-EPS in a Lewis lung carcinoma cell (LLC1) tumor-bearing mice model. In the preventive model, GF-EPS was orally administered to mice before LLC1 injection. In the therapeutic model, GF-EPS oral administration was initiated five days after tumor cell injection. The tumor size and body weight of the mice were recorded. After sacrifice, the lymphocyte subpopulation was analyzed using flow cytometry. Spleen tissues were used to analyze cytokine mRNA expression. The results showed that GF-EPS (80 mg/kg) effectively suppressed LLC1 tumor growth in both the preventive and therapeutic models. GF-EPS administration increased the proportion of natural killer cells in the spleen and activated gene expression of several cytokines. Our results provide evidence that GF-EPS promotes tumor inhibition through immunomodulation in tumor-bearing mice.