Endoplasmic reticulum stress regulators exhibit different prognostic, therapeutic and immune landscapes in pancreatic adenocarcinoma.

Endoplasmic reticulum stress (ERS) and unfolded protein response are the critical processes of tumour biology. However, the roles of ERS regulatory genes in pancreatic adenocarcinoma (PAAD) remain elusive. A novel ERS-related risk signature was constructed using the Lasso regression analysis. Its prognostic value, immune effect, metabolic influence, mutational feature and therapeutic correlation were comprehensively analysed through multiple bioinformatic approaches. The biofunctions of KDELR3 and YWHAZ in pancreatic cancer (PC) cells were also investigated through colony formation, Transwell assays, flow cytometric detection and a xenograft model. The upstream miRNA regulatory mechanism of KDELR3 was predicted and validated. ERS risk score was identified as an independent prognostic factor and could improve traditional prognostic model. Meanwhile, it was closely associated with metabolic reprogramming and tumour immune. High ERS risk enhanced glycolysis process and nucleotide metabolism, but was unfavourable for anti-tumour immune response. Moreover, ERS risk score could act as a potential biomarker for predicting the efficacy of ICBs. Overexpression of KDELR3 and YWHAZ stimulated the proliferation, migration and invasion of SW1990 and BxPC-3 cells. Silencing KDELR3 suppressed tumour growth in a xenograft model. miR-137 could weaken the malignant potentials of PC cells through inhibiting KDELR3 (5'-AGCAAUAA-3'). ERS risk score greatly contributed to PAAD clinical assessment. KDELR3 and YWHAZ possessed cancer-promoting capacities, showing promise as a novel treatment target.

N7-methylguanosine regulatory genes well represented by METTL1 define vastly different prognostic, immune and therapy landscapes in adrenocortical carcinoma.

Although N7-methylguanosine (m7G) is one of the most frequent RNA modifications, it has received little attention. Adrenocortical carcinoma (ACC) is a highly malignant and easily metastatic tumor, eagerly needing for novel therapeutic strategy. Herein, a novel m7G risk signature (METTL1, NCBP1, NUDT1 and NUDT5) was constructed using the Lasso regression analysis. It possessed highly prognostic value and could improve the predictive accuracy and clinical making-decision benefit of traditional prognostic model. Its prognostic value was also successfully validated in GSE19750 cohort. Through CIBERSORT, ESTIMATE, ssGSEA and GSEA analyzes, high-m7G risk score was found to be closely associated with increased enrichment of glycolysis and suppression of anti-cancer immune response. Therapeutic correlation of m7G risk signature was also investigated using tumor mutation burden, the expressions of immune checkpoints, TIDE score, IMvigor 210 cohort and TCGA cohort. m7G risk score was a potential biomarker for predicting the efficacy of ICBs and mitotane. Furthermore, we explored the biofunctions of METTL1 in ACC cells through a series of experimentations. Overexpression of METTL1 stimulated the proliferation, migration and invasion of H295R and SW13 cells. Immunofluorescence assays revealed that the infiltrating levels of CD8+ T cells was lower and that of macrophages was higher in clinical ACC samples with high METTL1 expression compared to that in low expression ones. Silencing METTL1 could significantly inhibited tumor growth in mouse xenograft model. Western blot assays showed that METTL1 positively regulated the expression of glycolysis rate-limiting enzyme HK1. Finally, miR-885-5p and CEBPB were predicted as the upstream regulators of METTL1 through data mining of the public databases. In conclusions, m7G regulatory genes well represented by METTL1 profoundly affected the prognosis, tumor immune, therapeutic outcomes, and malignant progression of ACC.

Cuproptosis regulatory genes greatly contribute to clinical assessments of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common abdominal cancer with dissatisfactory therapeutic effects. The discovery of cuproptosis lights on new approach for cancer treatment and assessment. So far, there is extremely limited research investigating the roles of cuproptosis-related (CR) genes in cancers. METHODS: A novel CR risk signature was constructed using the Lasso regression analysis. Its prognostic value was assessed via a series of survival analyses and validated in three GEO cohorts. The effects of CR risk signature on tumor immune microenvironment (TIM) were explored through CIBERSORT, ESTIMATE, and ssGSEA algorithms. Using GESA, we investigated its impacts on various metabolism process. The somatic mutation features of CR signature genes were also explored via cBioPortal database. Using tumor mutation burden, expressions of immune checkpoints, TIDE score, IMvigor 210 cohort, and GSE109211 dataset, we explored the potential associations of CR risk score with the efficacy of immune checkpoint inhibitors (ICIs) and sorafenib. Finally, the biofunctions of DLAT in HCC cells were ascertained through qPCR, immunohistochemistry, colony formation, and Transwell assays. RESULTS: FDX1, DLAT, CDKN2A and GLS constituted the CR risk signature. CR risk signature possessed high prognostic value and was also applicable to three validation cohorts. Meanwhile, it could improve the accuracy and clinical making-decision benefit of traditional prognostic model. Moreover, high CR risk was indicative of unfavorable anti-tumor immune response and active metabolisms of glycolysis and nucleotide. As for therapeutic correlation, CR risk score was a potential biomarker for predicting the efficacy of ICIs and sorafenib. Through qPCR and immunohistochemistry detection in clinical samples, we reconfirmed DLAT was significantly upregulated in HCC samples. Overexpression of DLAT could promote the proliferation, migration, and invasion of HepG2 and HuH-7 cells. CONCLUSIONS: The novel CR risk signature greatly contributed to the clinical assessment of HCC. Cuproptosis regulatory gene DLAT possessed cancer-promoting capacities and was expected to be a promising therapeutic target for HCC.

Immature rat testis sustained long-term development using an integrative model.

BACKGROUND: Xenotransplantation has been primarily performed using fresh donor tissue to study testicular development for about 20 years, and whether the cultured tissue would be a suitable donor is unclear. In this study, we combined testicular culture and xenotransplantation into an integrative model and explored whether immature testicular tissue would survive and continue to develop in this model. METHODS: In the new integrative model group, the testes of neonatal rats on postnatal day 8 (PND 8) were cultured for 4 days ex vivo and then were transplanted under the dorsal skin of castrated nude mice. The xenografted testes were resected on the 57th day after xenotransplantation and the testes of rats in the control group were harvested on PND 69. The survival state of testicular tissue was evaluated from morphological and functional perspectives including H&E staining, immunohistochemical staining of 8-OH-dG, immunofluorescence staining, TUNEL assay, ultrastructural study, gene expression and protein analysis. RESULTS: (a) We found that complete spermatogenesis was established in the testes in the new integrative model group. Compared with the control in the same stage, the seminiferous epithelium in some tubules was a bit thinner and there were vacuoles in part of the tubules. Immunofluorescence staining revealed some ACROSIN-positive spermatids were present in seminiferous tubule of xenografted testes. TUNEL detection showed apoptotic cells and most of them were germ cells in the new integrative model group. 8-OH-dG immunohistochemistry showed strongly positive-stained in the seminiferous epithelium after xenotransplantation in comparison with the control group; (b) Compared with the control group, the expressions of FOXA3, DAZL, GFRα1, BOLL, SYCP3, CDC25A, LDHC, CREM and MKI67 in the new integrative model group were significantly elevated (P < 0.05), indicating that the testicular tissue was in an active differentiated and proliferative state; (c) Antioxidant gene detection showed that the expression of Nrf2, Keap1, NQO1 and SOD1 in the new integrative model group was significantly higher than those in the control group (P < 0.05), and DNA methyltransferase gene detection showed that the expression of DNMT3B was significantly elevated as well (P < 0.05). CONCLUSION: The new integrative model could maintain the viability of immature testicular tissue and sustain the long-term survival in vivo with complete spermatogenesis. However, testicular genes expression was altered, vacuolation and thin seminiferous epithelium were still apparent in this model, manifesting that oxidative damage may contribute to the testicular development lesion and it needs further study in order to optimize this model.

Corrigendum: A New Prognostic Risk Score: Based on the Analysis of Autophagy-Related Genes and Renal Cell Carcinoma.

[This corrects the article DOI: 10.3389/fgene.2021.820154.].

Tumor mutational burden presents limiting effects on predicting the efficacy of immune checkpoint inhibitors and prognostic assessment in adrenocortical carcinoma.

BACKGROUND: Adrenocortical carcinoma (ACC) is a highly malignant urologic cancer and tends to metastasize. Although immune checkpoint inhibitors (ICIs) bring a glimmer of light to conquer ACC, only a fraction of patients have benefit from ICIs treatment. It is well known that tumor mutational burden (TMB) is closely associated with the efficacy and response rate of immunotherapy. However, its roles in ACC were not investigated. METHODS: Using somatic mutations data of 92 ACC samples in TCGA database, we calculated their TMB values by the 'maftools' package in R software (Ver 3.6.3). To explore the roles of TMB in ICIs therapy, we have addressed this issue from three perspectives. First, the effects of TMB levels on tumor immune microenvironment (TIM) were analyzed through CIBERSORT algorithm, ssGSEA method and TIMER web server. Second, we investigated the expressive correlations between TMB level and five pivotal immune checkpoints based on Pearson coefficient. Third, the difference in TIDE score between high- and low-TMB groups was compared. The prognostic value of TMB was also evaluated. Besides, GSEA was performed to determine the changes in the activities of signaling pathways caused by TMB. RESULTS: TMB values in ACC samples were not high. The average of total mutation counts in each sample was only 21.5. High TMB could lead metabolic reprogramming and poor survival outcomes. However, it was unable to affect the infiltration levels of lymphocytes, and failed to facilitate the activities of immune-related pathways. Regarding immune checkpoints (ICs), only PD-L1 upregulation could result in a good prognosis, and TMB level did not correlate with the expressions of other ICs except for LAG3. There was no significant difference in TIDE score between high- and low-TMB groups. Combining the present results and previous study, we speculated that inadequate stimulation for neoantigens formation, intrinsic immune-resistance and special genomic alterations were three possible reasons for TMB limiting functions in TIM and ICIs. Besides, TMB was toughly applied in clinical practice due to its high cost of determination and non-universal definition of high TMB. CONCLUSIONS: TMB presents limiting effects on prediction for ICIs efficacy and prognostic assessment for ACC patients.

Pyroptosis-Related Risk Signature Exhibits Distinct Prognostic, Immune, and Therapeutic Landscapes in Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is a common abdominal cancer. The existing therapeutic approaches often fail to achieve satisfactory results. Pyroptosis, an inflammatory form of programmed cell death, provides new ideas for anticancer treatment. However, the roles of pyroptosis-related (PR) genes (PRGs) in HCC remain elusive. Methods: Differentially expressed genes (DEGs) (n = 22) were screened out using TCGA and GTEx databases. A novel PR risk signature was constructed through Lasso regression analysis. Its prognostic value was evaluated through a series of survival analyses and was tested in ICGC and GSE14520 cohorts. CIBERSORT, ssGSEA, and ESTIMATE methods were employed to determine the effects of the PR risk score on the tumor immune microenvironment (TIM). The TIDE scoring system, IMvigor210 cohort, GSE109211 dataset, and GSDC database were applied to explore the associations of the PR risk score with therapeutic effects. The biofunctions of WNK1 in hepatocellular cancer (HC) cells were confirmed through qPCR, colony formation, and Transwell assays. Results: Overall, 22 of 45 PRGs (48.9%) were abnormally expressed in HCC samples. Then, a PR risk signature consisting of eight PRGs was constructed. A high PR risk score led to an unfavorable prognosis. The PR risk score was identified as an independent prognostic factor of HCC and could increase the decision-making benefit of the traditional TNM model. In addition, we established a nomogram containing the clinical stage and PR risk score to predict the survival rates of HCC patients. The prognostic value of the PR model was successfully validated in ICGC and GSE14520 cohorts. Moreover, high PR risk conferred the decreased infiltration level of CD8+ T cells and weakened the activities of "cytolytic activity" pathways. As for therapeutic correlation, a high PR risk score seemed to imply a poor efficacy of PD-1/L1 inhibitors and sorafenib. Finally, the overexpression of WNK1 could promote the proliferation, migration, and invasion of HC cells. Conclusions: The PR risk score was closely related to the prognosis, antitumor immune process, therapeutic outcomes, and malignant progression of HCC. WNK1, the core regulator of pyroptosis, possesses pro-oncogenic abilities, showing promise as a novel treatment target.

SLC1A5 Prefers to Play as an Accomplice Rather Than an Opponent in Pancreatic Adenocarcinoma.

Background: SLC1A5, a ferroptosis regulator gene, plays a dual role in cancer regulation. However, the roles of SLC1A5 in pancreatic adenocarcinoma (PAAD) remain elusive. Methods: SLC1A5's expression and somatic mutation information were determined by TCGA, GEO, Oncomine, and cBioPortal databases. Its prognostic value was assessed in TCGA cohort and was validated in three independent cohorts. The effects of SLC1A5 on the tumor immune microenvironment were analyzed by the CIBERSORT algorithm, ssGSEA method, and TISIDB and TIMER databases. The "oncoPredict" R package, TIDE algorithm, ImmuCellAI online tool, and GSE35141 and GSE59357 datasets were used to ascertain its therapeutic correlations. GSEA and Western blot were applied to reveal the effects of SLC1A5 on the mTORC1 signaling pathway and ferroptosis process. The biofunctions of SLC1A5 were assessed by MTT, wound-healing, Transwell, and xenograft assays. Results: SLC1A5 was significantly upregulated in the PAAD samples but was not commonly accompanied with somatic mutation (2.3%). Overexpression of SLC1A5 led to a poor prognosis and was identified as an independent prognostic factor. Moreover, high SLC1A5 expression suppressed the antitumor immune process by changing the infiltrating levels of immune cells. As for therapeutic correlations, SLC1A5 was related to the efficacy of dasatinib, sunitinib, sorafenib, and imatinib but may not predict that of radiotherapy, chemotherapeutic drugs, and immune checkpoints inhibitors (ICIs). Notably, the overexpression of SLC1A5 could activate the mTORC1 signaling pathway and may increase the cellular sensitivity to ferroptosis. Finally, the overexpression of SLC1A5 markedly promoted proliferation, migration, and invasion of pancreatic cancer cells. At the in vivo level, SLC1A5 deletion inhibited tumor growth in a mice xenograft model. Conclusions: SLC1A5 prefers to play as an accomplice rather than an opponent in PAAD. Our findings provide novel insights into PAAD treatment.

Abnormal expression and the significant prognostic value of aquaporins in clear cell renal cell carcinoma.

Aquaporins (AQPs) are a kind of transmembrane proteins that exist in various organs of the human body. AQPs play an important role in regulating water transport, lipid metabolism and glycolysis of cells. Clear cell renal cell carcinoma (ccRCC) is a common malignant tumor of the kidney, and the prognosis is worse than other types of renal cell cancer (RCC). The impact of AQPs on the prognosis of ccRCC and the potential relationship between AQPs and the occurrence and development of ccRCC are demanded to be investigated. In this study, we first explored the expression pattern of AQPs by using Oncomine, UALCAN, and HPA databases. Secondly, we constructed protein-protein interaction (PPI) network and performed function enrichment analysis through STRING, GeneMANIA, and Metascape. Then a comprehensive analysis of the genetic mutant frequency of AQPs in ccRCC was carried out using the cBioPortal database. In addition, we also analyzed the main enriched biological functions of AQPs and the correlation with seven main immune cells. Finally, we confirmed the prognostic value of AQPs throughGEPIA and Cox regression analysis. We found that the mRNA expression levels of AQP0/8/9/10 were up-regulated in patients with ccRCC, while those of AQP1/2/3/4/5/6/7/11 showed the opposite. Among them, the expression differences of AQP1/2/3/4/5/6/7/8/9/11 were statistically significant. The differences in protein expression levels of AQP1/2/3/4/5/6 in ccRCC and normal renal tissues were consistent with the change trends of mRNA. The biological functions of AQPs were mainly concentrated in water transport, homeostasis maintenance, glycerol transport, and intracellular movement of sugar transporters. The high mRNA expression levels of AQP0/8/9 were significantly correlated with worse overall survival (OS), while those of AQP1/4/7 were correlated with better OS. AQP0/1/4/9 were prognostic-related factors, and AQP1/9 were independent prognostic factors. In general, this research has investigated the values of AQPs in ccRCC, which could become new survival markers for ccRCC targeted therapy.

Prognostic value of circulating tumor cells and immune-inflammatory cells in patients with renal cell carcinoma.

PURPOSE: The relationships among circulating tumor cells (CTCs), inflammatory cells, and platelets in patients with renal cell carcinoma (RCC) are not transparent. We evaluated the correlations among CTCs, blood inflammatory cells, and platelets in patients with RCC and their prognostic value for metastasis-free survival. METHODS: CTC and typical tumor cell chip data were collected and analyzed by the GEO database. The baseline data, survival data, CTCs data, and blood test results were statistically analyzed. RESULTS: Bioinformatics analysis showed that the function of the differentially expressed genes between CTCs and normal tumor cells mainly involved platelets and immune inflammation. A total of 82 patients whose follow-up time was 3 to 68 months were included in the analysis. Clinical data of the patients confirmed that there is a correlation between platelets and mesenchymal CTCs. Simultaneously, there was a correlation between immune inflammatory cells and platelets. The univariate Cox proportional hazards model indicated that staging, mesenchymal CTCs, and the monocyte-to-neutrophil ratio (MNR) had prognostic value. The multivariate Cox proportional hazards model indicated that staging and the MNR had prognostic value and high accuracy. CONCLUSIONS: Bioinformatics analysis showed that CTCs were related to platelets and immune-inflammatory cells. Furthermore, the clinical data confirmed that platelets were correlated with mesenchymal CTCs and immune-inflammatory cells in the blood. By using mesenchymal CTCs, the MNR, or staging respectively, it is possible to predict the risk of postoperative metastasis in RCC patients. As a compound prognostic factor, staging, and the MNR can provide more convenient and accurate condition monitoring.

Pyroptosis regulators exert crucial functions in prognosis, progression and immune microenvironment of pancreatic adenocarcinoma: a bioinformatic and in vitro research.

Pyroptosis is an inflammatory programmed cell death, showing potentials to be a novel anti-cancer approach. However, the roles of pyroptosis-related (PR) genes (PRGs) in pancreatic adenocarcinoma (PAAD) remain elusive. In the present study, we constructed a novel PR risk signature through the lasso regression analysis. The risk signature was greatly conducive to PAAD prognostic assessment. PR risk score was identified as an independent prognostic factor and could distinguish the prognostic differences of most clinical subgroups. Meanwhile, it could improve the traditional prognostic models based on TNM-staging. Next, its prognostic value was also tested in five validation cohorts. Using CIBERSORT, ESTIMATE, and ssGSEA algorithms, the effects of PR risk signature on tumor immune microenvironment (TIM) were explored. High PR risk suppressed antitumor immune through decreasing the infiltrating levels of CD8 T and NK cells. The genomic information and histological expression of risk PRGs were uncovered by USCA and HPA databases. Somatic mutation, methylation alteration, and homozygous CNV of eight PRGs barely occurred in PAAD samples. As for therapeutic correlation, PR risk score may not predict the efficacy of PD-1/L1 inhibitors and was weakly associated with multiple drug susceptibilities. Finally, the biofunctions of toll like receptor 3 (TLR3) in pancreatic cancer (PC) cells were investigated through qPCR, MTT, colony formation, and Transwell assays. Overexpression of TLR3 could promote the proliferation, migration, and invasion of PC cells. In conclusion, PRGs play crucial roles in prognosis, progression, and immune microenvironment of PAAD. TLR3 is expected to be a promising therapeutic target.

Immature rat testis sustained long-term development using an integrative model

BACKGROUND: Xenotransplantation has been primarily performed using fresh donor tissue to study testicular development for about 20 years, and whether the cultured tissue would be a suitable donor is unclear. In this study, we combined testicular culture and xenotransplantation into an integrative model and explored whether immature testicular tissue would survive and continue to develop in this model. METHODS: In the new integrative model group, the testes of neonatal rats on postnatal day 8 (PND 8) were cultured for 4 days ex vivo and then were transplanted under the dorsal skin of castrated nude mice. The xenografted testes were resected on the 57th day after xenotransplantation and the testes of rats in the control group were harvested on PND 69. The survival state of testicular tissue was evaluated from morphological and functional perspectives including H&E staining, immunohistochemical staining of 8-OH-dG, immunofluorescence staining, TUNEL assay, ultrastructural study, gene expression and protein analysis. RESULTS: (a) We found that complete spermatogenesis was established in the testes in the new integrative model group. Compared with the control in the same stage, the seminiferous epithelium in some tubules was a bit thinner and there were vacuoles in part of the tubules. Immunofluorescence staining revealed some ACROSIN-positive spermatids were present in seminiferous tubule of xenografted testes. TUNEL detection showed apoptotic cells and most of them were germ cells in the new integrative model group. 8-OH-dG immunohistochemistry showed strongly positive-stained in the seminiferous epithelium after xenotransplantation in comparison with the control group; (b) Compared with the control group, the expressions of FOXA3, DAZL, GFRα1, BOLL, SYCP3, CDC25A, LDHC, CREM and MKI67 in the new integrative model group were significantly elevated (P < 0.05), indicating that the testicular tissue was in an active differentiated and proliferative state; (c) Antioxidant gene detection showed that the expression of Nrf2, Keap1, NQO1 and SOD1 in the new integrative model group was significantly higher than those in the control group (P < 0.05), and DNA methyltransferase gene detection showed that the expression of DNMT3B was significantly elevated as well (P < 0.05). CONCLUSION: The new integrative model could maintain the viability of immature testicular tissue and sustain the long-term survival in vivo with complete spermatogenesis. However, testicular genes expression was altered, vacuolation and thin seminiferous epithelium were still apparent in this model, manifesting that oxidative damage may contribute to the testicular development lesion and it needs further study in order to optimize this model.

The roles of ferroptosis regulatory gene SLC7A11 in renal cell carcinoma: A multi-omics study.

BACKGROUND: The ferroptosis inhibitory gene Solute carrier family 7 member 11 (SLC7A11) provides a new strategy for anticancer treatment. However, its function in renal cell carcinoma (RCC) remains elusive. METHODS: The expression and somatic mutation information of SLC7A11 in RCC samples were determined using The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), Gene Expression Omnibus (GEO), Oncomine, and cBioPortal databases. The prognostic value of SLC7A11 was assessed through survival analysis, Receiver operating characteristic curve (ROC) analysis, independent prognostic analysis, clinical subgroup analysis, and nomogram. Its prognostic value was also validated in the ICGC and GSE29607 cohorts. Gene set enrichment analysis (GSEA) was employed to investigate the effects of SLC7A11 on multiple metabolic pathways. The CIBERSORT algorithm and single-sample gene set enrichment analysis (ssGSEA) method were applied to evaluate the effects of SLC7A11 on the tumor immune microenvironment (TIM). SLC7A11's therapeutic correlations were analyzed using the GSE87121, GSE67501, and GSDC datasets. Finally, the biofunctions of SLC7A11 in renal cancer cells and ferroptosis were ascertained by MTT, wound healing, transwell, and western blot assays. RESULTS: Through multiple datasets, SLC7A11 was found to be markedly upregulated in RCC. In terms of prognosis, SLC7A11 overexpression conferred a worse prognosis and was identified as an independent prognostic factor. Its prognostic value was validated in ICGC cohort. Moreover, high SL7CA11 expression could stimulate nucleotides, fatty acids, and amino acid metabolism to meet the proliferative consumption of tumor cells. As for the immune effect, SLC7A11 suppressed antitumor immunity by reducing the abundances of CD8+ T and NK cells. Regarding the therapeutic response, SLC7A11 expression was not correlated with the sensitivities of most chemotherapy and targeted drugs. Finally, SLC7A11 promoted the proliferation, migration, and invasion of renal cancer cells by enhancing GPX4 output, which in turn inhibits ferroptosis. CONCLUSIONS: SLC7A11 not only deeply influences RCC prognosis and TIM, but also promotes RCC progression by inhibiting ferroptosis and inducing metabolic reprogramming. In addition, SLC7A11 weakly affects the therapeutic effect and sensitivities of multiple chemotherapy and targeted drugs.

Ferroptosis regulators, especially SQLE, play an important role in prognosis, progression and immune environment of breast cancer.

BACKGROUND: Ferroptosis, a new form of programmed cell death, has great potential for cancer treatment. However, the roles of ferroptosis-related (FR) genes in breast cancer (BC) remain elusive. MATERIALS AND METHODS: Using TCGA database, a novel FR risk signature was constructed through the Lasso regression analysis. Meanwhile, its prognostic value was assessed by a series of survival analyses. Besides, a nomogram was constructed to predict the overall survival rate (OSR) of individual at 1,3,5 year. Four validation cohorts (n = 2248), including METABRIC, GSE58812, GSE20685 and ICGC-KR datasets, were employed to test the prognostic value of FR risk signature. The effects of FR risk signature on BC immune microenvironment were explored by CIBERSORT algorithm and ssGSEA method. The histological expressions of FR risk genes were presented by HPA database. The biofunctions of SQLE were determined by qPCR, MTT, wound-healing and Transwell assays. RESULTS: We constructed a novel FR risk signature consisting of eight genes. High FR risk led a poor prognosis and was identified as an independent prognostic factor. Besides, A higher proportion of patients with luminal A type was observed in low-risk group (53%), while a higher proportion of patients with basal type in high-risk group (24%). FR risk score could discriminate the prognostic difference of most clinical subgroups, except for M1 stage, HER2 and basal types. Moreover, its prognostic value was successfully validated in other four cohorts. Through immune analyses, we found that the reduced infiltration levels of CD8+ and NK cells, whereas the enhanced activity of antigen presentation process appeared in high FR risk. Then, FR risk score was found to weakly correlate with the expressions of six immune checkpoints. Through the experiments in vitro, we confirmed that overexpression of SQLE could promote, whereas blocking SQLE could inhibit the proliferative, migrative and invasive abilities of BC cells. CONCLUSIONS: FR risk signature was conducive to BC prognostic assessment. High FR risk level was closely associated with BC immunosuppression, but may not predict ICIs efficacy. Moreover, SQLE was identified as a crucial cancer-promoting gene in BC. Our findings provide new insights into prognostic assessment and molecular mechanism of BC.

The prognostic value of circulating tumour cells (CTCs) and CTC white blood cell clusters in patients with renal cell carcinoma.

PURPOSE: Circulating tumour cell (CTC) and CTC-white blood cell (CTC-WBC) clusters are related to the prognosis of tumour patients. However, the relationship between CTC-WBC clusters and prognosis in renal cell carcinoma (RCC) patients is not clear. We evaluated the prognostic value of CTC-WBC clusters using metastasis-free survival (MFS) and overall survival (OS) in patients with RCC. MATERIALS AND METHODS: The baseline, survival, and CTC data of patients with RCC were statistically analysed by R. RESULTS: The Cox risk proportional regression model suggests that the total CTCs, pathology type, and CTC-WBC clusters can be used as prognostic indicators for the MFS of RCC patients. Total CTCs and solid tumour diameter can be used as prognostic indicators for the OS of RCC patients. Using Kaplan-Meier survival analysis, we found that patients with total CTCs, pathology, and CTC-WBC clusters greater than the cut-off value had a worse MFS, and patients with total CTCs greater than the cut-off value had a worse OS. CONCLUSION: The analysis of the clinical sample data in patients with RCC shows that CTC-WBC clusters play an important role in monitoring the prognosis of RCC. Among them, total CTCs, pathology, and CTC-WBC clusters were combined as prognostic factors for the MFS of RCC patients. Total CTCs and solid tumour diameter can be combined as prognostic factors for the OS of RCC patients. These prognostic factors provide more convenient and accurate condition monitoring for renal cancer patients and can be used to actively improve the prognosis of patients.

Bioinformatic mining and validation of the effects of ferroptosis regulators on the prognosis and progression of pancreatic adenocarcinoma.

Ferroptosis, a new form of programmed cell death, provides a new option for anti-tumor treatment. However, the roles of ferroptosis-related (FR) genes in pancreatic adenocarcinoma (PAAD) were not fully elaborated. In the present study, 185 TCGA samples and 81 ICGC samples were used as training and validation cohorts, respectively. A novel FR risk signature (ALOX5, ALOX12, PTGS2, SAT1, STEAP3 and SQLE) was constructed via the Lasso regression analysis. In TCGA cohort, the risk signature was identified as an independent prognostic factor. Decision curve analysis (DCA) indicated that FR risk score could increase the net benefit when making clinical-decision. In addition, we constructed a nomogram to predict the overall survival rate (OSR) of individual at 1,2,3 year. Meanwhile, the prognostic value was partly validated in ICGC cohort. Through immune analyses, we found that high FR risk could affect the immune abundances of five lymphocytes but not effectively affect the activities of immune-related pathways. The expressions of most FR risk genes did not correlate with that of PD-L1(CD274) and CTLA4. Further, through RT-qPCR tests, the expressions of PTGS2 and SQLE were proven to be significantly upregulated in normal pancreatic duct epithelia cell (HPDE6-C7) compared to pancreatic cancer cells (SW1990 and BxPC-3). MTT, wound-healing and transwell assays revealed that silencing PTGS2 and SQLE could inhibit the proliferation, migration and invasion of pancreatic cancer cells. Besides, western-blot assays showed that blocking PTGS2 and SQLE expressions could suppress the protein expressions of cyclin D1 and N-cadherin, but facilitate that of E-cadherin, which suggested that they were involved in the epithelial to mesenchymal transition (EMT). Collectively, FR risk signature provides an important complement for PAAD prognostic analysis. High FR risk level can adversely affect anti-tumor immune process, but may not serve as a predictive marker of ICIs efficacy. PTGS2 and SQLE are proven to possess cancer-promoting abilities in PAAD.

Bioinformatic analyses and experimental validation of the role of m6A RNA methylation regulators in progression and prognosis of adrenocortical carcinoma.

M6A-related genes have been proven to play an important role in many cancers. However, the role of that in adrenocortical carcinoma (ACC) has not been fully elucidated. In the present study, 77 ACC samples from TCGA database were divided into localized (n = 46) and metastatic (n = 31) groups. Three differential expression genes (DEGs) and five prognostic m6A genes were screened out. M6A-related risk signature (RBM15 and HNRNPC) was constructed by the Lasso regression analysis. In TCGA cohort (training cohort), the risk signature was identified as an ACC-independent prognostic factor and can distinguish the prognostic difference of ACC patients with clinical stage I-II, T3-4 and N0 stages. A nomogram combining T stage and m6A risk score was constructed to predict the overall survival rate (OSR) of individual at 1,2,3 year. Meanwhile, its prognostic value was also confirmed in the validation cohort (GSE33371 dataset). The potential associations between m6A risk level and immune checkpoint inhibitors (ICIs) therapy were also investigated via the TISIDB online tool. High m6A risk not only can suppress immunotherapy-related biological processes, but also repress the expressions of immune-checkpoint markers. Moreover, five pairs of clinical specimens were collected to confirm the overexpression of HNRNPC and non-ectopic expression of RBM15 in tumor tissues. HNRNPC was proven to promote the proliferation, migration and invasion of H295R and SW13 cells through MTT and Transwell assays. In conclusion, the m6A-related risk signature was beneficial for prognostic analysis and can affect immune microenvironment in ACC. HNRNPC played a pro-cancer role in ACC progression.

A New Prognostic Risk Score: Based on the Analysis of Autophagy-Related Genes and Renal Cell Carcinoma.

Introduction: Clear cell renal cell carcinoma (ccRCC) patients suffer from its high recurrence and metastasis rate, and a new prognostic risk score to predict individuals with high possibility of recurrence or metastasis is in urgent need. Autophagy has been found to have a dual influence on tumorigenesis. In this study we aim to analyze autophagy related genes (ATGs) and ccRCC patients and find a new prognostic risk score. Method: Analyzing differential expression genes (DEGs) in TCGA-KIRC dataset, and took intersection with ATGs. Through lasso, univariate, and multivariate cox regression, DEGs were chosen, and the coefficients and expression levels of them were components constructing the formula of risk score. We analyzed mRNA expression of DEGs in tumor and normal tissue in ONCOMINE database and TCGA-KIRC dataset. The Human Protein Atlas (HPA) was used to analyze protein levels of DEGs. The protein-protein interaction (PPI) network was examined in STRING and visualized in cytoscape. Functional enrichment analysis was performed in RStudio. To prove the ability and practicibility of risk score, we analyzed univariate and multivariate cox regression, Kaplan-Meier curve (K-M curve), risk factor association diagram, receiver operating characteristic curve (ROC curve) of survival and nomogram, and the performance of nomogram was evaluated by calibration curve. Then we further explored functional enrichment related to risk groups through Gene Set Enrichment Analysis (GSEA), weighted gene co-expression network analysis (WGCNA), and Metascape database. At last, we investigated immune cell infiltration of DEGs and two risk groups through TIMER database and "Cibersort" algorithm. Result: We identified 7 DEGs (BIRC5, CAPS, CLDN7, CLVS1, GMIP, IFI16, and TCIRG1) as components of construction of risk score. All 7 DEGs were differently expressed in ccRCC and normal tissue according to ONCOMINE database and TCGA-KIRC dataset. Functional enrichment analysis indicated DEGs, and their most associated genes were shown to be abundant in autophagy-related pathways and played roles in tumorigenesis and progression processes. A serious analysis proved that this risk score is independent from the risk signature of ccRCC patients. Conclusion: The risk score constructed by 7 DEGs had the ability of predicting prognosis of ccRCC patients and was conducive to the identification of novel prognostic molecular markers. However, further experiment is still needed to verify its ability and practicability.