Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

Background: Tumor-associated macrophages (TAMs) dominate the malignancy of cancers by perturbing the tumor microenvironment (TME). However, the clinical implications of heterogeneous subpopulations of TAMs in clear cell renal cell carcinoma (ccRCC) remain to be elucidated.Methods: We comprehensively evaluated the prognostic implications, biological behaviors, and immunogenomics features of the C-C Motif Chemokine Ligand 5 (CCL5) expression and CCL5⁺ TME in vitro and in 932 real-world ccRCC patients from testing and public validation cohorts. Flow cytometry was used to examine the functional patterns of CCL5⁺ TAMs with TME cell-infiltrating characterizations.Results: Our results identified distinct prognostic clusters with gradual changes in clinicopathological indicators based on CCL5 expression. Knockdown of CCL5 significantly restrained cell viability, migration capabilities of ccRCC cells, and the inhibits the proliferation and chemotaxis of THP1-derived TAMs. Mechanically, down-regulation of CCL5 arrested epithelial-mesenchymal transition by modulating the PI3K/AKT pathway in ccRCC cells. In ccRCC samples with CCL5 upregulation, the proportion of CCL5⁺ TAMs and PD-L1⁺ CD68⁺ TAMs were prominently increased, showing a typical suppressive tumor immune microenvironment (TIME). Besides, intra-tumoral CCL5⁺ TAMs showed distinct pro-tumorigenic TME features characterized by exhausted CD8⁺ T cells and increased expression of immune checkpoints. Furthermore, elevated CCL5⁺ TAMs infiltration was prominently associated with a dismal prognosis for patients with ccRCC.Conclusion: In conclusion, this study first revealed the predictive value of the chemokine CCL5 on the progression and TME of ccRCC. The intra-tumoral CCL5⁺ TAMs could be applied to comprehensively evaluate the prognostic patterns as well as unique TME characteristics among individuals, allowing for the identification of immunophenotypes and promotion of treatment efficiency for ccRCC.     

Prognostic and Predictive Value of Tumor-infiltrating Leukocytes and of Immune Checkpoint Molecules PD1 and PDL1 in Clear Cell Renal Cell Carcinoma

INTRODUCTION: Immune checkpoint inhibitors (ICI) have been approved for patients with clear cell renal cell carcinoma (ccRCC), but not all patients benefit from ICI. One reason is the tumor microenvironment (TME) that has substantial influence on patient's prognosis and therapy response. Thus, we comprehensively analyzed the TME of ccRCC regarding prognostic and predictive properties. METHODS: Tumor-infiltrating CD3-positive T-cells, CD8-positive cytotoxic T-lymphocytes (CTLs), regulatory T-cells, B-cells, plasma cells, macrophages, granulocytes, programmed cell death receptor 1 (PD-1), and its ligand PD-L1 were examined in a large hospital-based series of ccRCC with long-term follow-up information (n = 756) and in another patient collective with information on response to nivolumab therapy (n = 8). Tissue microarray technique and digital image analysis were used. Relationship between immune cell infiltration and tumor characteristics, cancer-specific survival (CSS), or response to ICI was examined. RESULTS: Univariate survival analysis revealed that increased tumor-infiltrating B-cells, T-cells, and PD-1-positive cells were significantly associated with favorable CSS and high levels of intratumoral granulocytes, macrophages, cytotoxic T-cells, and PD-L1 significantly with poor CSS. High CTL or B-cell infiltration and high PD-L1 expression of ccRCC tumor cells qualified as independent prognostic biomarkers for patients' CSS. Significantly higher densities of intratumoral T-cells, CTLs, and PD-1-positive immune cells were observed in ccRCC with response to ICI compared with patients with mixed or no response (CD3: p = 0.003; CD8: p = 0.006; PD-1: p = 0.01). DISCUSSION: This study shows that subsets of tumor-infiltrating leukocytes in the TME and also PD-1/PD-L1 provide prognostic and predictive information for patients with ccRCC.     

DDX39 as a predictor of clinical prognosis and immune checkpoint therapy efficacy in patients with clear cell renal cell carcinoma

DEAD-box protein 39 (DDX39) has been demonstrated to be a tumorigenic gene in multiple tumor types, but its role in the progression and immune microenvironment of clear cell renal cell cancer (ccRCC) remains unclear. The aim of the present study was to investigate the role of DDX39 in the ccRCC tumor progression, immune microenvironment and efficacy of immune checkpoint therapy.The DDX39 expression level was first detected in tumors in the public data and then verified in ccRCC samples from Changzheng Hospital. The prognostic value of DDX39 expression was assessed in the Cancer Genome Atlas (TCGA) and ccRCC patients from Changhai Hospital. The role of DDX39 in promoting ccRCC was analyzed by bioinformatic analysis and in vitro experiments. The association between DDX39 expression and immune cell infiltration and immune inhibitory markers was analyzed, and its value in predicting the immune checkpoint therapy efficacy in ccRCC were evaluated in the public database. DDX39 expression was elevated in Oncomine, GEO and TCGA ccRCC databases, as well as in Changzheng ccRCC samples. In TCGA ccRCC patients, increased DDX39 expression predicted worse overall survival (OS) (p<0.0001) and progression-free interval (PFI) (p<0.0001), and was shown as an independent predictive factor for OS (p=0.002). These findings were consistent with those from Changhai ccRCC patients. In addition, GO and GSEA analysis identified DDX39 as a pro-ccRCC gene. In vitro experiments confirmed the role of DDX39 in promoting ccRCC cell. Finally, DDX39 was found to be positively correlated with a variety of immune inhibitory markers, and could predict the adverse efficacy of immune checkpoint therapy in TIDE analysis. In conclusion, Increased DDX39 in ccRCC patients predicted worse clinical prognosis, promoted ccRCC cell proliferation, migration and invasion, and also predicted adverse efficacy of immune checkpoint therapy.     

Identification of a four immune-related genes signature based on an immunogenomic landscape analysis of clear cell renal cell carcinoma

Renal clear cell carcinoma (ccRCC) is the most common type of renal cell carcinoma, which has strong immunogenicity. A comprehensive study of the role of immune-related genes (IRGs) in ccRCC is of great significance in finding ccRCC treatment targets and improving patient prognosis. In this study, we comprehensively analyzed the expression of IRGs in ccRCC based on The Cancer Genome Atlas datasets. The mechanism of differentially expressed IRGs in ccRCC was analyzed by bioinformatics. In addition, Cox regression analysis was used to screen prognostic related IRGs from differentially expressed IRGs. We also identified a four IRGs signature consisting of four IRGs (CXCL2, SEMA3G, PDGFD, and UCN) through lasso regression and multivariate Cox regression analysis. Further analysis results showed that the four IRGs signature could effectively predict the prognosis of patients with ccRCC, and its predictive power is independent of other clinical factors. In addition, the correlation analysis of immune cell infiltration showed that this four IRGs signature could effectively reflect the level of immune cell infiltration of ccRCC. We also found that the expression of immune checkpoint genes CTLA-4, LAG3, and PD-1 in the high-risk group was higher than that in the low-risk group. Our research revealed the role of IRGs in ccRCC, and developed a four IRGs signature that could be used to evaluate the prognosis of patients with ccRCC, which will help to develop personalized treatment strategies for patients with ccRCC and improve their prognosis. In addition, these four IRGs may be effective therapeutic targets for ccRCC.     

Traditional Chinese medicine Bu-Shen-Jian-Pi-Fang attenuates glycolysis and immune escape in clear cell renal cell carcinoma: results based on network pharmacology

Clear cell renal cell carcinoma (ccRCC) is the most common malignant type of kidney cancer. The present study aims to explore the underlying mechanism and potential targets of the traditional Chinese medicine Bu-Shen-Jian-Pi-Fang (BSJPF) in the treatment of ccRCC based on network pharmacology. After obtaining the complete composition information for BSJPF from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, we analyzed its chemical composition and molecular targets and then established a pharmacological interaction network. Twenty-four significantly differentially expressed genes and nine pathways mainly related to tumor proliferation were identified and screened. Functional enrichment analysis indicated that the potential targets might be significantly involved in glycolysis and the HIF-1 signaling pathway. To further confirm the effect of BSJPF on ccRCC cell proliferation, a BALB/c xenograft mouse model was constructed. Potential targets involved in regulating glycolysis and the tumor immune microenvironment were evaluated using RT-qPCR. VEGF-A expression levels were markedly decreased, and heparin binding-EGF expression was increased in the BSJPF group. BSJPF also inhibited tumor proliferation by enhancing GLUT1- and LDHA-related glycolysis and the expression of the immune checkpoint molecules PD-L1 and CTLA-4, thereby altering the immune-rejection status of the tumor microenvironment. In summary, the present study demonstrated that the mechanism of BSJPF involves multiple targets and signaling pathways related to tumorigenesis and glycolysis metabolism in ccRCC. Our research provides a novel theoretical basis for the treatment of tumors with traditional Chinese medicine and new strategies for immunotherapy in ccRCC patients.     

Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions

The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34⁺ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.Subject terms: Cell biology, Cancer     

Phosphoinositide-dependent kinase 1–associated glycolysis is regulated by miR-409-3p in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most popular kidney cancer in adults. Metabolic shift toward aerobic glycolysis is a fundamental factor for ccRCC therapy. MicroRNAs (miRNAs) are thought to be important regulators in ccRCC development and progression. Phosphoinositide-dependent kinase 1 (PDK1) is required for metabolic activation; however, the role of PDK1-induced glycolytic metabolism regulated by miRNAs is unclear in ccRCC. So, the purpose of the current study is to elucidate the underlying mechanism in ccRCC cell metabolism mediated by PDK1. Our results revealed that miR-409-3p inhibited glycolysis by regulating PDK1 expression in ccRCC cells. We also found that miR-409-3p was regulated by hypoxia. Our results indicated that PDK1 facilitated ccRCC cell glycolysis, regulated by miR-409-3p in hypoxia.     

Effect of lentivirus-mediated shRNA inactivation of HK1, HK2, and HK3 genes in colorectal cancer and melanoma cells

Background

The switch from oxidative phosphorylation to glycolysis in proliferating cancer cells, even under aerobic conditions, has been shown first in 1926 by Otto Warburg. Today this phenomenon is known as the “Warburg effect” and recognized as a hallmark of cancer. The metabolic shift to glycolysis is associated with the alterations in signaling pathways involved in energy metabolism, including glucose uptake and fermentation, and regulation of mitochondrial functions. Hexokinases (HKs), which catalyze the first step of glycolysis, have been identified to play a role in tumorigenesis of human colorectal cancer (CRC) and melanoma. However, the mechanism of action of HKs in the promotion of tumor growth remains unclear.

Results

The purpose of the present study was to investigate the effect of silencing of hexokinase genes (HK1, HK2, and HK3) in colorectal cancer (HT-29, SW 480, HCT-15, RKO, and HCT 116) and melanoma (MDA-MB-435S and SK-MEL-28) cell lines using short hairpin RNA (shRNA) lentiviral vectors. shRNA lentiviral plasmid vectors pLSLP-HK1, pLSLP-HK2, and pLSLP-HK3 were constructed and then transfected separately or co-transfected into the cells. HK2 inactivation was associated with increased expression of HK1 in colorectal cancer cell lines pointing to the compensation effect. Simultaneous attenuation of HK1 and HK2 levels led to decreased cell viability. Co-transfection with shRNA vectors against HK1, HK2, and HK3 mRNAs resulted in a rapid cell death via apoptosis.

Conclusions

We have demonstrated that simultaneous inactivation of HK1 and HK2 was sufficient to decrease proliferation and viability of melanoma and colorectal cancer cells. Our results suggest that HK1 and HK2 could be the key therapeutic targets for reducing aerobic glycolysis in examined cancers.

     

Targeting HLA-F suppresses the proliferation of glioma cells via a reduction in hexokinase 2-dependent glycolysis

HLA-F, a nonclassical HLA class I molecule, is required for regulating immune tolerance. In recent years, HLA-F has been found to play a role in a variety of cancers, including glioma (GM). Additionally, high expression of HLA-F predicts the poor overall survival of individuals with GM. However, the functions of HLA-F in GM remain to be further elucidated. In this study, we found that HLA-F expression was elevated in GM tissues. High levels of HLA-F resulted in a high cell proliferation index and predicted GM recurrence. Forced expression of HLA-F promoted the growth of murine C8-D1A cells transplanted in immunodeficient Rag2^-/- mice. In contrast, silencing HLA-F inhibited cell growth in vitro. Furthermore, targeting HLA-F with an anti-HLA-F antibody suppressed the growth of C8-D1A cells stably expressing HLA-F transplanted in immunodeficient Rag2^-/- mice. In further experiments, we found that forced expression of HLA-F contributed to the aerobic glycolysis phenotype in C8-D1A cells along with an increase in HK2 protein stabilization. Conversely, silencing HK2 by shRNA reduced HLA-F-mediated glycolysis and cell proliferation. Our data indicated that HLA-F promoted cell proliferation via HK2-dependent glycolysis. HLA-F could be a potential therapeutic target for the treatment of GM.     

Sodium butyrate inhibits aerobic glycolysis of hepatocellular carcinoma cells via the c‐myc/hexokinase 2 pathway

Aerobic glycolysis is a well‐known hallmark of hepatocellular carcinoma (HCC). Hence, targeting the key enzymes of this pathway is considered a novel approach to HCC treatment. The effects of sodium butyrate (NaBu), a sodium salt of the short‐chain fatty acid butyrate, on aerobic glycolysis in HCC cells and the underlying mechanism are unknown. In the present study, data obtained from cell lines with mouse xenograft model revealed that NaBu inhibited aerobic glycolysis in the HCC cells in vivo and in vitro. NaBu induced apoptosis while inhibiting the proliferation of the HCC cells in vivo and in vitro. Furthermore, the compound inhibited the release of lactate and glucose consumption in the HCC cells in vitro and inhibited the production of lactate in vivo. The modulatory effects of NaBu on glycolysis, proliferation and apoptosis were related to its modulation of hexokinase 2 (HK2). NaBu downregulated HK2 expression via c‐myc signalling. The upregulation of glycolysis in the HCC cells induced by sorafenib was impeded by NaBu, thereby enhancing the anti‐HCC effect of sorafenib in vitro and in vivo. Thus, NaBu inhibits the expression of HK2 to downregulate aerobic glycolysis and the proliferation of HCC cells and induces their apoptosis via the c‐myc pathway.     

Circular RNA circDVL1 inhibits clear cell renal cell carcinoma progression through the miR-412-3p/PCDH7 axis

Clear cell renal cell carcinoma (ccRCC) is a primary kidney cancer with high aggressive phenotype and extremely poor prognosis. Accumulating evidence suggests that circular RNAs (circRNAs) play pivotal roles in the occurrence and development of various human cancers. However, the expression, clinical significance and regulatory role of circRNAs in ccRCC remain largely unclear. Here we report that circDVL1 to be reduced in the serums and tissues from ccRCC patients, and to negatively correlate with ccRCC malignant features. Overexpression of circDVL1 inhibits proliferation, induces G1/S arrest, triggers apoptosis, and reduces migration and invasion in different ccRCC cells in vitro. Correspondingly, circDVL1 overexpression suppresses ccRCC tumorigenicity in a mouse xenograft model. Mechanistically, circDVL1 serves as a sponge for oncogenic miR-412-3p, thereby preventing miR-412-3p-mediated repression of its target protocadherin 7 (PCDH7) in ccRCC cells. Collectively, our results demonstrate that circDVL1 exerts tumor-suppressive function during ccRCC progression through circDVL1/miR-412-3p/PCDH7 axis, and suggest that circDVL1 could be a novel diagnostic and prognositc marker and therapeutic target for ccRCC.     

Aging impairs peritoneal but not bone marrow‐derived macrophage phagocytosis

Aging results in deterioration of the immune system, which is associated with increased susceptibility to infection and impaired wound healing in the elderly. Phagocytosis is an essential process in both wound healing and immune defence. As such, age‐related impairments in phagocytosis impact on the health of the elderly population. Phagocytic efficiency in peritoneal macrophages, bone marrow‐derived macrophages and bone marrow monocytes from young and old mice was investigated. Aging significantly impaired phagocytosis by peritoneal macrophages, both in vitro and in vivo. However, bone marrow‐derived macrophages and bone marrow monocytes did not exhibit age‐related impairments in phagocytosis, suggesting no intrinsic defect in these cells. We sought to investigate underlying mechanisms in age‐related impairments in phagocytosis by peritoneal macrophages. We hypothesized that microenvironmental factors in the peritoneum of old mice impaired macrophage phagocytosis. Indeed, macrophages from young mice injected into the peritoneum of old mice exhibited impaired phagocytosis. Proportions of peritoneal immune cells were characterized, and striking increases in numbers of T cells, B1 and B2 cells were observed in the peritoneum of old mice compared with young mice. In addition, B cell‐derived IL‐10 was increased in resting and LPS‐activated peritoneal cell cultures from old mice. These data demonstrate that aging impairs phagocytosis by tissue‐resident peritoneal macrophages, but not by bone marrow‐derived macrophages/monocytes, and suggest that age‐related defects in macrophage phagocytosis may be due to extrinsic factors in the tissue microenvironment. As such, defects may be reversible and macrophages could be targeted therapeutically in order to boost immune function in the elderly.     

VNN3 is a potential novel biomarker for predicting prognosis in clear cell renal cell carcinoma

Although pathological observations provide approximate prognoses, it is difficult to achieve prognosis in patients with existing prognostic factors. Therefore, it is very important to find appropriate biomarkers to achieve accurate cancer prognosis. Renal cell carcinoma (RCC) has several subtypes, the discrimination of which is crucial for proper treatment. Here, we present a novel biomarker, VNN3, which is used to prognose clear cell renal cell carcinoma (ccRCC), the most common and aggressive subtype of kidney cancer. Patient information analyzed in our study was extracted from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) cohorts. VNN3 expression was considerably higher in stages III and IV than in stages I and II. Moreover, Kaplan–Meier curves associated high VNN3 expression with poor prognoses (TCGA, p?p?=?.00076), confirming that ccRCC prognosis can be predicted via VNN3 expression patterns. Consistent with all patient results, the prognosis of patients with higher VNN3 expression was worse in both low stage (I and II) and high stage (III and IV) (TCGA, p < 0.0001 in stage I and II; ICGC, p = 0.028 in stage I and II; TCGA, p = 0.005 in stage III and IV). Area under the curve and receiver operating characteristic curves supported our results that highlighted VNN3 expression as a suitable ccRCC biomarker. Multivariate analysis also verified the prognostic performance of VNN3 expression (TCGA, p?p?=?.017). Altogether, we suggest that VNN3 is applicable as a new biomarker to establish prognosis in patients with ccRCC.     

Construction and validation of a novel ten miRNA-pair based signature for the prognosis of clear cell renal cell carcinoma

BackgroundClear cell renal cell carcinoma (ccRCC) is the most predominate pathological subtype of renal cell carcinoma, causing a recurrence or metastasis rate as high as 20% to 40% after operation, for which effective prognostic signature is urgently needed.MethodsThe mRNA and miRNA profiles of ccRCC specimens were collected from the Cancer Genome Atlas. MiRNA-pair risk score (miPRS) for each miRNA pair was generated as a signature and validated by univariate and multivariate Cox proportional hazards regression analysis. Functional enrichment was performed, and immune cells infiltration, as well as tumor mutation burden (TMB), and immunophenoscore (IPS) were evaluated between high and low miPRS groups. Target gene-prediction and differentially expressed gene-analysis were performed based on databases of miRDB, miRTarBase, and TargetScan. Multivariate Cox proportional hazards regression analysis was adopted to establish the prognostic model and Kaplan-Meier survival analysis was performed.FindingsA novel 10 miRNA-pair based signature was established. Area under the time-dependent receiver operating curve proved the performance of the signature in the training, validation, and testing cohorts. Higher TMB, as well as the higher CTLA4-negative PD1-negative IPS, were discovered in high miPRS patients. A prognostic model was built based on miPRS (1 year-, 5 year-, 10 year- ROC-AUC=0.92, 0.84, 0.82, respectively).InterpretationThe model based on miPRS is a novel and valid tool for predicting the prognosis of ccRCC.FundingThis study was supported by research grants from the China National Natural Scientific Foundation (81903972, 82002018, and 82170752) and Shanghai Sailing Program (19YF1406700 and 20YF1406000).