Exosomal IL-8 derived from Lung Cancer and Colon Cancer cells induced adipocyte atrophy via NF-κB signaling pathway.

BACKGROUND: Cytokines secreted in the tumor microenvironment function in cancer cachexia (CC), a common clinicopathological syndrome associated with adipocyte wasting and skeletal muscle atrophy. Extracellular vesicles (EVs) secreted by cancer cells actively engage in inter-tissue communication; EVs and enclosed cytokines are largely undefined in CC adipocytes wasting. METHODS: EVs derived from Lewis lung carcinoma (LLC) and colorectal cancer C26 cells were extracted and characterized. Conditioned medium and EVs from cancer cells were applied to 3 T3-L1 adipocytes. Recombinant IL-8, IL-8 neutralizing antibody, CXCR2 and NF-κB inhibitor were examined in functional assays. Lipolysis of adipocytes was monitored by Western blots, Oil red O staining and glycerol assays. Furthermore, LLC and C26 cell lines were established as cachexia model to explore the relevance of IL-8 and NF-κB signaling in CC adipose wasting. Adipose tissues were collected for histology analyses. RESULTS: LLC and C26 cell-derived EVs induced lipolysis of 3 T3-L1 adipocytes. Specially, Dil-labeled EVs were effectively taken up by 3 T3-L1 adipocytes, which were motivated by the delivered IL-8 to elicit the NF-κB pathway. In comparison, special IL-8 neutralizing antibody relieved that lipolysis of 3 T3-L1 adipocytes induced by EVs together with conditioned medium of LLC and C26 cells, respectively. Consistently, both CXCR2 and NF-κB inhibitors would lessen the phenotype of lipolysis in 3 T3-L1 adipocytes. In the in vivo settings, both LLC and C26-tumor bearing mice had higher serum IL-8 levels as compared to the control groups. Two typical lipolysis markers, PGC1α and UCP1, were also up-regulated in the adipose tissues of LLC and C26-tumor mice groups, respectively. CONCLUSIONS: EVs secreted by LLC and C26 tumor cells would induce adipocyte wasting via extracellular IL-8-mediated NF-κB signaling. Our study pointed out the physiological and therapeutic values of exosomal IL-8 in CC lipolysis.

As a prognostic biomarker of clear cell renal cell carcinoma RUFY4 predicts immunotherapy responsiveness in a PDL1-related manner.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is one of the most lethal malignancies in the urinary system and the existing immunotherapy has not achieved satisfactory outcomes. Therefore, this study aims at establishing a novel gene signature for immune infiltration and clinical outcome (overall survival and immunotherapy responsiveness) in ccRCC patients. METHODS: Based on RNA sequencing data and clinical information in The Cancer Genome Atlas (TCGA) database, we calculated proportions of immune cells in 611 samples using an online tool CIBERSORTx. Multivariate survival analysis was conducted to determine crucial survival-associated immune cells and immune-infiltration-related genes (IIRGs). Next, the clinical specimens and common renal cancer cell lines were applied to confirm IIRGs expression at protein and RNA levels. Finally, functional enrichment analyses and siRNA technology targeted to RUFY4 were implemented to verify its function of predicting immunotherapy response. RESULTS: Follicular helper T cells (TFHs) and Regulatory T cells (Tregs) were highly infiltrated in the tumor microenvironment (TME) and their relative proportions were independent prognostic factors for patients. Among IIRGs of TFHs and TREGs, RUFY4 was found to be highly activated in tumor microenvironment and its co-expression network was enriched in PDL1/PD1 checkpoint pathway in cancer. Additionally, knockdown of RUFY4 led to the decline of PDL1 and proliferation ability in ccRCC cell lines. CONCLUSION: TFHs and Tregs were considered as prognostic biomarkers and RUFY4 was an immunotherapeutic predictor of ccRCC patients in a PDL1-Related manner.

Downregulation of miR-29c promotes muscle wasting by modulating the activity of leukemia inhibitory factor in lung cancer cachexia.

BACKGROUND: Cancer cachexia is a wasting disorder characterized by significant weight loss, and is attributed to skeletal muscle weakness. In the process of cancer development, microRNAs act as oncogenes or tumor suppressors. Moreover, they are implicated in muscle development and wasting. This study sought to explore the mechanisms and correlation between miR-29c and muscle wasting in lung cancer cachexia. METHODS: Data for expression analysis were retrieved from the Cancer Genome Atlas (TCGA) database. qRT-PCR analyses were performed to explore the expression levels of miR-29c and Leukemia Inhibitory Factor (LIF). Lewis lung carcinoma (LLC) cell line was used to establish a cachexia model to explore the functions of miR-29c and LIF in lung cancer cachexia. Furthermore, in vitro (in C2C12 myotubes) and in vivo (in LLC tumor-bearing mice) experiments were performed to explore the mechanisms of miR-29c and LIF in lung cachexia. RESULTS: Analysis of the lung cancer cachexia model showed that miR-29c was down-regulated, and its expression was negatively correlated with muscle catabolic activity. Overexpression of miR-29c mitigated the cachectic phenotype. Mechanistic studies showed that LIF was a direct target gene of miR-29c, and LIF was upregulated in vitro and in vivo. Analysis showed that LIF promoted muscle wasting through the JAK/STAT and MAP-kinase pathways. CONCLUSIONS: The findings indicated that miR-29c was negatively correlated with the cachectic phenotype, and the miR-29c-LIF axis is a potential therapeutic target for cancer cachexia.

HIF2α promotes tumour growth in clear cell renal cell carcinoma by increasing the expression of NUDT1 to reduce oxidative stress.

BACKGROUND: The key role of hypoxia-inducible factor 2alpha (HIF2α) in the process of renal cancer has been confirmed. In the field of tumour research, oxidative stress is also considered to be an important influencing factor. However, the relationship and biological benefits of oxidative stress and HIF2α in ccRCC remain unclear. This research attempts to explore the effect of oxidative stress on the cancer-promoting effect of HIF2α in ccRCC and reveal its mechanism of action. METHODS: The bioinformatics analysis for ccRCC is based on whole transcriptome sequencing and TCGA database. The detection of the expression level of related molecules is realised by western blot and PCR. The expression of Nucleoside diphosphate-linked moiety X-type motif 1 (NUDT1) was knocked down by lentiviral infection technology. The functional role of NUDT1 were further investigated by CCK8 assays, transwell assays and cell oxidative stress indicator detection. The exploration of related molecular mechanisms is realised by Luciferase assays and Chromatin immunoprecipitation (ChIP) assays. RESULTS: Molecular screening based on knockdown HIF2α sequencing data and oxidative stress related data sets showed that NUDT1 is considered to be an important molecule for the interaction of HIF2α with oxidative stress. Subsequent experimental results showed that NUDT1 can cooperate with HIF2α to promote the progression of ccRCC. And this biological effect was found to be caused by the oxidative stress regulated by NUDT1. Mechanistically, HIF2α transcription activates the expression of NUDT1, thereby inhibiting oxidative stress and promoting the progression of ccRCC. CONCLUSIONS: This research clarified a novel mechanism by which HIF2α stabilises sirtuin 3 (SIRT3) through direct transcriptional activation of NUDT1, thereby inhibiting oxidative stress to promote the development of ccRCC. It provided the possibility for the selection of new therapeutic targets for ccRCC and the study of combination medication regimens.

Extracellular vesicles-released parathyroid hormone-related protein from Lewis lung carcinoma induces lipolysis and adipose tissue browning in cancer cachexia.

Cancer cachexia is a metabolic disorder characterized by skeletal muscle wasting and white adipose tissue browning. Specific functions of several hormones, growth factors, and cytokines derived from tumors can trigger cachexia. Moreover, adipose tissue lipolysis might explain weight loss that occurs owing to cachexia. Extracellular vesicles (EVs) are involved in intercellular communication. However, whether EVs participate in lipolysis induced by cancer cachexia has not been thoroughly investigated. Using Lewis lung carcinoma (LLC) cell culture, we tested whether LLC cell-derived EVs can induce lipolysis in 3T3-L1 adipocytes. EVs derived from LLC cells were isolated and characterized biochemically and biophysically. Western blotting and glycerol assay were used to study lipolysis. LLC cell-derived EVs induced lipolysis in vivo and vitro. EVs fused directly with target 3T3-L1 adipocytes and transferred parathyroid hormone-related protein (PTHrP), activating the PKA signaling pathway in 3T3-L1 adipocytes. Blocking PTHrP activity in LLC-EVs using a neutralizing antibody and by knocking down PTHR expression prevented lipolysis in adipocytes. Inhibiting the PKA signaling pathway also prevents the lipolytic effects of EVs. In vivo, suppression of LLC-EVs release by knocking down Rab27A alleviated white adipose tissue browning and lipolysis. Our data showed that LLC cell-derived EVs induced adipocyte lipolysis via the extracellular PTHrP-mediated PKA pathway. Our data demonstrate that LLC-EVs induce lipolysis in vitro and vivo by delivering PTHrP, which interacts with PTHR. The lipolytic effect of LLC-EVs was abrogated by PTHR knockdown and treatment with a neutralizing anti-PTHrP antibody. Together, these data show that LLC-EV-induced lipolysis is mediated by extracellular PTHrP. These findings suggest a novel mechanism of lipid droplet loss and identify a potential therapeutic strategy for cancer cachexia.

Folic acid-modified Exosome-PH20 enhances the efficiency of therapy via modulation of the tumor microenvironment and directly inhibits tumor cell metastasis.

High accumulation of hyaluronan (HA) in the tumor microenvironment leads to an increase in the interstitial pressure and reduction perfusion of drugs. Furthermore, high molecular-weight (HMW)-HA suppresses M1 macrophage polarization, enhances M2 polarization, and induces immunosuppression. Hyaluronidase treatment have attempted to decrease the quantity of HA in tumors. However, hyaluronidase-driven HA degradation driven accelerates tumor cell metastasis, which is a major cause of mortality in cancer patients. Thus, we designed a novel exosome-based drug delivery system (DDS), named Exos-PH20-FA, using genetic engineering to express human hyaluronidase (PH20) and self-assembly techniques to modify the exosomes with folic acid (FA). Our results show that Exos-PH20-FA degraded HMW-HA to low-molecular-weight (LMW)-HA. Moreover, LMW-HA polarized macrophages to the M1 phenotype and reduced the number of relevant immunosuppressive immunocytes which changed the immune microenvironment from an immunosuppressive to immunosupportive phenotype. Furthermore, we demonstrated Exos-PH20-FA directly reduced hyaluronidase-induced metastasis of tumor cells. This tumor treatment also allowed an enhanced delivery of chemotherapy by tumor-targeting effect with FA modification. Our findings indicate that Exos-PH20-FA improves tumor treatment efficiency and reduces the side effects of hyaluronidase treatment, namely tumor cell metastasis. This all-in-one exosome-based HA targeting DDS maybe a promising treatment that yields more efficient and safer results.

Impact of inflammation and immunotherapy in renal cell carcinoma.

Substantial research attention has been directed at exploring the mechanisms and treatment of renal cell carcinoma (RCC). Indeed, the association between inflammation and tumor phenotypes has been at the center of cancer research. Concomitant with research on the inflammation response and inflammatory molecules involved in RCC, new breakthroughs have emerged. A large body of knowledge now shows that treatments targeting inflammation and immunity in RCC provide substantial clinical benefits. Adequate analysis and a better understanding of the mechanisms of inflammatory factors in the occurrence and progression of RCC are highly desirable. Currently, numerous RCC treatments targeted at inflammation and immunotherapy are available. The current review describes in detail the link between inflammation and RCC.

Downregulation of ubiquitin-specific protease 2 possesses prognostic and diagnostic value and promotes the clear cell renal cell carcinoma progression.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC), characterized by high mortality, invasion, metastasis, recurrence and drug resistance, is the most common malignant tumor of the urinary system. A clear understanding of the underlying molecular mechanisms and its role during tumorigenesis of RCC can contribute to development of prognostic and targeted therapies. METHODS: We analyzed datasets from the public database, TCGA, Oncomine, for differential expression of ubiquitin-specific protease 2 (USP2), and further investigated its relationship with the clinical stage, pathological grade and prognosis of renal cancer. We used real-time quantitative PCR and western blot analysis to validate USP2 expression in clinical samples and renal cancer cell lines. Finally, we used CCK-8 and transwell assays to determine its effects on biological functions in cells. RESULTS: We observed significantly lower levels of USP2 mRNA in renal cancer, relative to normal, tissues across the four datasets from the Oncomine database (P<0.001), 533 cases from TCGA database (P<0.0001) and 30 pairs of clinical samples (P<0.0001). Similarly, a decreased USP2 protein expression in ccRCC was detected following immunohistochemical (IHC) and western blot analyses. Furthermore, the aberrant expression of USP2 resulted in significant relationship with clinical stage, pathological grade and lower USP2 mRNA expression was interrelated to poor prognosis of renal cell carcinoma. USP2 acted as an independent factor for ccRCC diagnosis, with an AUC of 0.8888 (95% CI: 0.8529 to 0.9246; P<0.0001). Exogenous restoration of USP2 in ccRCC cells resulted in repression of cell proliferation, migration, and invasion. CONCLUSIONS: Overall, these results show that USP2 acts as an anti-oncogene and an independent factor for ccRCC prognosis. Positive modulation of USP2 might lead to development of a novel strategy for ccRCC treatment.

Overexpression of PPT2 Represses the Clear Cell Renal Cell Carcinoma Progression by Reducing Epithelial-to-mesenchymal Transition.

Clear cell renal cell carcinoma (ccRCC) is one of the most common malignant tumors of the urinary system and has a poor response to radiotherapy and chemotherapy. To date, it is urgent to find effective biomarkers for the prevention and treatment of ccRCC. The occurrence and development of ccRCC is closely related to metabolic disturbances. Palmitoyl protein thioesterase 2 (PPT2) is a lysosomal thioesterase which is highly associated with metabolism, and it has never been studied in ccRCC. In this study, we first revealed PPT2 is significantly downregulated in ccRCC, and its expression level is highly correlated with clinicopathological parameters of ccRCC patients. Our ROC curve analyses evaluated the potential of PPT2 as a novel diagnostic marker and prognostic factor. Functional experiment results showed overexpression of PPT2 represses the proliferation, migration and invasion of ccRCC cells in vitro. Mechanistic investigations demonstrated that overexpression of PPT2 represses the ccRCC progression by reducing epithelial-to-mesenchymal transition (EMT). In conclusion, PPT2 is downregulated in ccRCC. Decreased PPT2 expression may be considered as a novel diagnostic marker and prognostic factor and serve as a therapeutic target for ccRCC.