Erratum: Hypoxia induced exosomal circRNA promotes metastasis of Colorectal Cancer via targeting GEF-H1/RhoA axis: Erratum.

[This corrects the article DOI: 10.7150/thno.44419.].

The microprotein encoded by exosomal lncAKR1C2 promotes gastric cancer lymph node metastasis by regulating fatty acid metabolism.

Lymph node metastasis (LNM) is the prominent route of gastric cancer dissemination, and usually leads to tumor progression and a dismal prognosis of gastric cancer. Although exosomal lncRNAs have been reported to be involved in tumor development, whether secreted lncRNAs can encode peptides in recipient cells remains unknown. Here, we identified an exosomal lncRNA (lncAKR1C2) that was clinically correlated with lymph node metastasis in gastric cancer in a VEGFC-independent manner. Exo-lncAKR1C2 secreted from gastric cancer cells was demonstrated to enhance tube formation and migration of lymphatic endothelial cells, and facilitate lymphangiogenesis and lymphatic metastasis in vivo. By comparing the metabolic characteristics of LN metastases and primary focuses, we found that LN metastases of gastric cancer displayed higher lipid metabolic activity. Moreover, exo-lncAKR1C2 encodes a microprotein (pep-AKR1C2) in lymphatic endothelial cells and promotes CPT1A expression by regulating YAP phosphorylation, leading to enhanced fatty acid oxidation (FAO) and ATP production. These findings highlight a novel mechanism of LNM and suggest that the microprotein encoded by exosomal lncAKR1C2 serves as a therapeutic target for advanced gastric cancer.

Screening of ferroptosis-related genes with prognostic effect in colorectal cancer by bioinformatic analysis.

Colorectal cancer (CRC) remains a common malignant tumor of digestive tract with high incidence rate and high mortality in the worldwide. The current clinical treatments of CRC often fail to achieve satisfactory results. Searching for more effective prediction or prognosis biomarkers, or developing more targeted therapeutic schedule may help to improve the outcomes of CRC patients. Here, we tried to study the effect of ferroptosis-related genes on CRC prognosis and make it clearer that ferroptosis has connection with immune environment. First, we obtained gene expression data of CRC and normal tissues, as well as corresponding clinical data from the Gene Expression Omnibus (GEO) database and the Cancer Genome Atlas (TCGA) database. The differentially expressed genes (DEGs) were intersected with ferroptosis-related gene set downloaded from FerrDb database, and 93 abnormally expressed ferroptosis-related genes were obtained. Then, these genes were analyzed for functional enrichment. Univariate Cox regression and multivariate Cox regression analyses were performed to establish prognostic model based on ferroptosis-related genes. In the process of exploring the correlation between prognostic genes and immune infiltration, we found that these genes were closely related to B cells, CD8+ T cells, CD4+ T cells, macrophages and other cells in CRC. In addition, we found a large proportion of plasma cells and macrophages in TCGA-COADREAD. Finally, a prognostic nomogram of ferroptosis-related genes was established, including age, sex, grade and other predicted values. To summary, we established a prognostic model of colorectal cancer (CRC) based on ferroptosis-related genes and further explored the relationship between these genes with immune microenvironment.

Comprehensive Characterization of Transforming Growth Factor Beta Receptor 1 in Stomach Adenocarcinoma Identifies a Prognostic Signature for Predicting Clinical Outcomes and Immune Infiltrates.

Background: Stomach adenocarcinoma (STAD) ranks as the third leading cause of cancer death worldwide. TGF­ß receptor 1 (TGFBR1), serving important roles in the TGF­ß family, the mechanisms whereby TGFBR1 governs tumor progression, immune cell infiltration in STAD remains unintelligible. Methods: We used the TCGA, GEPIA, and HPA databases to explore TGFBR1 expression in STAD, the correlation between TGFBR1 expression and the clinical features. A receiver operating characteristic (ROC) curve and nomogram were constructed, and LASSO (the Least Absolute Shrinkage and Selection Operator)-selected features were used to build the TGFBR1 prognostic signature. GSEA is used to find the potential mechanism of TGFBR1 to promote the malignant process of STAD. We explored the influence of the TGFBR1 on the immune microenvironment of STAD through the TIMER2.0 and GEPIA database. Results: In our study, TGFBR1 expression was significantly elevated in STAD and positively co-expression with pathologic stage, lymph node metastases (LNM) stage and histopathological grade. Nine factors with non-zero coefficients were identified by LASSO-selected features. Survival analysis revealed that patients with high TGFBR1 had shorter OS, FP, and PPS. Multivariate Cox analysis revealed that TGFBR1 was an independent prognostic factor for OS in STAD. The ROC analysis suggested that high diagnostic value with the AUC of TGFBR1 was 0.739. GSEA revealed that high TGFBR1 expression was correlated with pathway in cancer, MAPK signaling pathway, NOTCH signaling pathway, and VEGF-C production. ssGSEA showed that TGFBR1 is correlated with NK cells, Tem and Th17 cells. Furthermore, elevated TGFBR1 expression was found to be significantly correlated with several immune checkpoint and immune markers associated with immune cell subsets. Conclusion: In summary, TGFBR1 could be a prognostic biomarker and an important regulator of immune cell infiltration in STAD. The present study revealed the probable underlying molecular mechanisms of TGFBR1 in STAD and provided a potential target for improving the prognosis.

Erratum: Hypoxia induced exosomal circRNA promotes metastasis of Colorectal Cancer via targeting GEF-H1/RhoA axis: Erratum.

[This corrects the article DOI: 10.7150/thno.44419.].

LncRNA PPM1A-AS Regulate Tumor Development Through Multiple Signal Pathways in T-Cell Acute Lymphoblastic Leukemia.

ALL (Acute lymphoblastic leukemia) is the most common pediatric malignancy and T-ALL (T-cell acute lymphoblastic leukemia) comprises about 15% cases. Compared with B-ALL (B-cell acute lymphoblastic leukemia), the prognosis of T-ALL is poorer, the chemotherapy is easier to fail and the relapse rate is higher. Previous studies mainly focused in Notch1-related long non-coding RNAs (lncRNAs) in T-ALL. Here, we intend to investigate lncRNAs involved in T-ALL covering different subtypes. The lncRNA PPM1A-AS was screened out for its significant up-regulation in 10 T-ALL samples of different subtypes than healthy human thymus extracts. Besides, the PPM1A-AS expression levels in 3 T-ALL cell lines are markedly higher than that in CD45+ T cells of healthy human. We further demonstrate that PPM1A-AS can promote cell proliferation and inhibit cell apoptosis in vitro and can influence T-ALL growth in vivo. Finally, we verified that PPM1A-AS can regulate core proteins, Notch4, STAT3 and Akt, of 3 important signaling pathways related to T-ALL. These results confirm that lncRNA PPM1A-AS can act as an oncogene in T-ALL and maybe a potential clinical target of patients resistant to current chemotherapy or relapsed cases.

Chemotoxicity-induced exosomal lncFERO regulates ferroptosis and stemness in gastric cancer stem cells.

Cancer stem cells (CSCs) are an important cause of tumor recurrence and drug resistance. As a new type of cell death that relies on iron ions and is strictly regulated by intracellular and extracellular signals, the role of ferroptosis in tumor stem cells deserves extensive attention. Mass spectrum was applied to screen for ferroptosis-related proteins in gastric cancer (GC). Sphere-formation assay was used to estimate the stemness of gastric cancer stem cells (GCSCs). Exosomal lnc-ENDOG-1:1 (lncFERO) was isolated by ultracentrifugation. Ferroptosis was induced by erastin and was assessed by detecting lipid ROS, mitochondrial membrane potential, and cell death. Furthermore, a series of functional in vitro and in vivo experiments were conducted to evaluate the effects of lncFERO on regulating ferroptosis and chemosensitivity in GCSCs. Here, we showed that stearoyl-CoA-desaturase (SCD1) played a key role in regulating lipid metabolism and ferroptosis in GCSCs. Importantly, exosomal lncFERO (exo-lncFERO) derived from GC cells was demonstrated to promote SCD1 expression by directly interacting with SCD1 mRNA and recruiting heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which resulted in the dysregulation of PUFA levels and the suppression of ferroptosis in GCSCs. Moreover, we found that hnRNPA1 was also involved in lncFERO packing into exosomes in GC cells, and both in vitro and in vivo data suggested that chemotoxicity induced lncFERO secretion from GC cells by upregulating hnRNPA1 expression, leading to enhanced stemness and acquired chemo-resistance. All these data suggest that GC cells derived exo-lncFERO controls GCSC tumorigenic properties through suppressing ferroptosis, and targeting exo-lncFERO/hnRNPA1/SCD1 axis combined with chemotherapy could be a promising CSC-based strategy for the treatment of GC.

iRGD-modified exosomes effectively deliver CPT1A siRNA to colon cancer cells, reversing oxaliplatin resistance by regulating fatty acid oxidation.

Fatty acid oxidation (FAO) plays a vital role in drug resistance in cancer cells. Carnitine palmitoyltransferase 1A (CPT1A), a key enzyme of FAO, is widely recognized as an emerging therapeutic target. Here, we confirmed that CPT1A was heterogeneously expressed in colon cancer cells, with a high expression in oxaliplatin-resistant cells but low expression in oxaliplatin-sensitive cells, and expression could be increased by oxaliplatin stimulation. In addition, we verified that CPT1A was more highly expressed in colon cancer tissues than in noncancerous tissues. Silencing CPT1A by siRNA or etomoxir, a specific small-molecule inhibitor of CPT1A, could reverse the sensitivity of drug-resistant colon cancer cells to oxaliplatin. Subsequently, the combination of oxaliplatin with CPT1A inhibition promoted apoptosis and inhibited proliferation. In addition, exosomes were generated with the iRGD peptide on the surface, which showed highly efficient targeting compared with control exosomes in vivo. Furthermore, we loaded and therapeutically applied iRGD-modified exosomes with siCPT1A to specifically deliver siCPT1A into tumours to suppress FAO. As a consequence, iRGD-modified exosomes showed the significant inhibition of CPT1A in tumour tissues and exhibited the ability to reverse oxaliplatin resistance and inhibit tumour growth by inhibiting FAO with high safety in vivo.

Exosome-delivered miR-221/222 exacerbates tumor liver metastasis by targeting SPINT1 in colorectal cancer.

MicroRNAs (miRNAs) are involved in the progression of many cancers through largely unelucidated mechanisms. The results of our present study identified a gene cluster, miR-221/222, that is constitutively upregulated in serum exosome samples of patients with colorectal carcinoma (CRC) with liver metastasis (LM); this upregulation predicts a poor overall survival rate. Using an in vitro cell coculture model, we demonstrated that CRC exosomes harboring miR-221/222 activate liver hepatocyte growth factor (HGF) by suppressing SPINT1 expression. Importantly, miR-221/222 plays a key role in forming a favorable premetastatic niche (PMN) that leads to the aggressive nature of CRC, which was further shown through in vivo studies. Overall, our results show that exosomal miR-221/222 promotes CRC progression and may serve as a novel prognostic marker and therapeutic target for CRC with LM.

Exosomal miR-208b related with oxaliplatin resistance promotes Treg expansion in colorectal cancer.

Oxaliplatin resistance is a challenge in the treatment of colorectal cancer (CRC) patients. Regulatory T cells (Tregs) are well known for their immunosuppressive roles, and targeting Tregs is an effective way to improve chemosensitivity. Exosome-delivered microRNA (miRNA) might be used as a potential biomarker for predicting chemosensitivity. However, the relationship between Tregs and exosomal miRNAs remains largely unknown. TaqMan low-density array was performed to screen the differentially expressed serum miRNAs from pooled serum of patients who had FOLFOX treatment. Differential expression was validated using qRT-PCR in individual samples. Exosomes were isolated by sequential differential centrifugation, and they were verified by transmission electron microscopy. The RNA and protein levels were determined by quantitative real-time PCR and western blotting. A mouse xenograft model was adopted to evaluate the correlation between exosome-derived miR-208b and Tregs in vivo. We demonstrated that circulating miR-208b is a non-invasive marker for predicting FOLFOX sensitivity in CRC. miR-208b in colon cancer was secreted by tumor cells in the pattern of exosomes, and oxaliplatin-resistant cells showed the most obvious phenomenon of miR-208b increase. Colon cancer cell-secreted miR-208b was sufficiently delivered into recipient T cells to promote Treg expansion by targeting programmed cell death factor 4 (PDCD4). Furthermore, in vivo studies indicated that Treg expansion mediated by cancer cell-secreted miR-208b resulted in tumor growth and oxaliplatin resistance. Our results demonstrate that tumor-secreted miR-208b promotes Treg expansion by targeting PDCD4, and it may be related to a decrease of oxaliplatin-based chemosensitivity in CRC. These findings highlight a potential role of exosomal miR-208b as a predictive biomarker for oxaliplatin-based therapy response, and they provide a novel target for immunotherapy.

Serum microRNAs as Biomarkers for the Noninvasive Early Diagnosis of Biliary Tract Cancer.

BACKGROUND: Biliary tract cancers (BTCs) are aggressive malignancies with difficult early diagnosis and poor prognosis. Studies have shown that microRNAs (miRNAs) are expected to be biomarkers of the disease, which indicates that we can diagnose cancers according to the miRNAs that have significant changes. The aim of this study was to explore miRNA biomarkers of BTCs. METHODS: A total of 163 samples were collected and divided into the control group, the benign group and the malignant group. High-throughput low-density chips were used to screen miRNAs with significant changes. Then, the preliminary screening test and the verification test were performed by quantitative real time PCR (qRT-PCR). Finally, the level of miRNAs in serum exosomes was measured. RESULTS: MiR-10a, miR-21, miR-135b, miR-221, and miR-214 were upregulated in the BTCs group compared to the control group. The change in the miR-221 level was statistically significant when the malignant group was compared with the benign group (P<0.01). Meanwhile, miR-135b and miR-214 were enriched in serum exosomes. CONCLUSION: Five miRNAs in the serum were found to be significantly upregulated in patients with BTCs. Among them, miR-221 can serve as an early diagnostic marker for BTCs patients. MiR-10a, miR-21, miR-135b and miR-214 can be used as biomarkers for the diagnosis of biliary diseases.

Hypoxia induced exosomal circRNA promotes metastasis of Colorectal Cancer via targeting GEF-H1/RhoA axis.

Hypoxia is one of the important properties of solid tumor. However, oxygen supply within tumors is generally heterogeneous according to the distance from the nearest blood vessel. The discrepancy of metastatic potential exists between hypoxic cancer cells and relatively normoxic cancer cells. But the molecular mechanism remains poorly understood. Methods: Differential expression of circRNAs in plasma exosomes of CRC patients and normal subjects was performed by screening. Exosomes were isolated by ultra-centrifugation and RNA expressions were determined by RT-qPCR. The migratory capacity of cells was performed by high intension imaging, wound healing assay and transwell chamber migration assay. Results: Circ-133 is enriched in the plasma exosomes of CRC patients and increased with the disease progression. Exosomal circ-133 derived from hypoxic cells delivered into normoxic cells and promoted cancer metastasis by acting on miR-133a/GEF-H1/RhoA axis. Meanwhile, animal experiments revealed that knockdown of circ-133 can inhibit tumor metastasis. Circ-133 is expected to be a new biomarker for monitoring tumor progression and might be a novel therapeutic target. Conclusions: Hypoxia-derived exosomal circ-133 transported into normaxic cancer cells and promoted cell migration via miR-133a/GEF-H1/RhoA axis. This study reveals a potential mechanism for that the intra-tumor heterogeneity of oxygen promote cancer progression.

Exosome-Delivered c-Met siRNA Could Reverse Chemoresistance to Cisplatin in Gastric Cancer.

BACKGROUND: Drug resistance often occurs in the treatment of gastric cancer, which is the main cause of poor prognosis of chemotherapy. c-Met is overexpressed in a variety of tumors including gastric cancer, often leads to poor prognosis of gastric cancer, therefore regarded as a key target for the treatment of gastric cancer. This study aims to determine whether exosomes with si-c-Met could inhibit the resistance to cisplatin in gastric cancer (GC). METHODS: The protein expression levels of c-Met in tumor tissues and normal tissues of patients were evaluated by Western blot (WB) and immunohistochemistry (IHC), HEK293T cells were transfected with si-c-Met, exosomes were isolated, then co-cultured with gastric cancer cell lines and confirmed that it was incorporated into the cells by transmitted electron microscopy. Functional experiments were performed to examine the inhibitory effect of exo-si-c-Met on gastric cancer cell resistance in vitro, and xenograft models were used to reveal that exo-si-c-Met can enhance the sensitivity of tumors to cisplatin in vivo. RESULTS: High expression of c-Met is associated with poor prognosis of GC patients. si-c-Met significantly inhibited migration, invasion and promoted apoptosis in vitro, which indicated that si-c-Met sensitizes the response of gastric cancer cells to cisplatin. Exo-si-c-Met sharply reduced c-Met expression in gastric cancer cells and reverse the resistance to cisplatin in vitro and in vivo. CONCLUSION: Our results indicate that exo-si-c-Met can inhibit the invasion and migration of gastric cancer cells and promote apoptosis in vitro and inhibit tumor growth in vivo, reversing the resistance to cisplatin in gastric cancer.

Exosome-delivered circRNA promotes glycolysis to induce chemoresistance through the miR-122-PKM2 axis in colorectal cancer.

Malignant tumors, including colorectal cancer (CRC), usually rely on ATP generation through aerobic glycolysis for both rapid growth and chemotherapy resistance. The M2 isoform of pyruvate kinase (PKM2) has a key role in catalyzing glycolysis, and PKM2 expression varies even within a single tumor. In this study, we confirmed that expression of PKM2 is heterogeneous in CRC cells, namely high in oxaliplatin-resistant cells but relatively low in sensitive cells, and found that chemoresistant cells had enhanced glycolysis and ATP production. In addition, we report a PKM2-dependent mechanism through which chemosensitive cells may gradually transform into chemoresistant cells. The circular RNA hsa_circ_0005963 (termed ciRS-122 in this study), which was determined to be a sponge for the PKM2-targeting miR-122, was positively correlated with chemoresistance. In vitro and in vivo studies showed that exosomes from oxaliplatin-resistant cells delivered ciRS-122 to sensitive cells, thereby promoting glycolysis and drug resistance through miR-122 sponging and PKM2 upregulation. Moreover, si-ciRS-122 transported by exosomes could suppress glycolysis and reverse resistance to oxaliplatin by regulating the ciRS-122-miR-122-PKM2 pathway in vivo. Exosomes derived from chemoresistant CRC cells could transfer ciRS-122 across cells and promote glycolysis to reduce drug susceptibility in chemosensitive cells. This intercellular signal delivery suggests a potential novel therapeutic target and establishes a foundation for future clinical applications in drug-resistant CRC.

Exosomal circRNA derived from gastric tumor promotes white adipose browning by targeting the miR-133/PRDM16 pathway.

Cancer-related cachexia is a metabolic syndrome characterized by a wasting disorder of adipose and skeletal muscle and is accompanied by body weight loss and systemic inflammation. The treatment options for cancer cachexia are limited, and the molecular mechanism remains poorly understood. Circular RNAs (circRNAs) are a novel family of endogenous noncoding RNAs that have been proposed to regulate gene expression in mammals. Exosomes are small vesicles derived from cells, and recent studies have shown that circRNAs are stable in exosomes. However, little is known about the biological role of circRNAs in exosomes. In our study, we showed that circRNAs in plasma exosomes have specific expression features in gastric cancer (GC), and ciRS-133 is linked with the browning of white adipose tissue (WAT) in GC patients. Exosomes derived from GC cells deliver ciRS-133 into preadipocytes, promoting the differentiation of preadipocytes into brown-like cells by activating PRDM16 and suppressing miR-133. Moreover, knockdown of ciRS-133 reduced cancer cachexia in tumor-implanted mice, decreasing oxygen consumption and heat production. Thus, exosome-delivered circRNAs are involved in WAT browning and play a key role in cancer-associated cachexia.

Exosome circRNA secreted from adipocytes promotes the growth of hepatocellular carcinoma by targeting deubiquitination-related USP7.

Hepatocellular carcinoma (HCC), the major form of liver cancer, has shown increasing incidence and poor prognosis. Adipose tissue is known to function in energy storage and metabolism regulation by the secretion of adipokines. Circular RNAs (circRNAs), a novel type of noncoding RNA, have recently been recognized as key factors in tumor development, but the role of exosome circRNAs derived from adipose tissues has not been defined yet. Here, adipose-secreted circRNAs were found to regulate deubiquitination in HCC, thus facilitating cell growth. It was observed that exosome circ-deubiquitination (circ-DB) is upregulated in HCC patients with higher body fat ratios. Moreover, in vitro and in vivo studies showed that exo-circ-DB promotes HCC growth and reduces DNA damage via the suppression of miR-34a and the activation of deubiquitination-related USP7. Finally, the results showed that the effects of adipose exosomes on HCC cells can be reversed by knockdown of circ-DB. These results indicate that exosome circRNAs secreted from adipocytes promote tumor growth and reduce DNA damage by suppressing miR-34a and activating the USP7/Cyclin A2 signaling pathway.

Plasma Exosomal Long Non-Coding RNAs Serve as Biomarkers for Early Detection of Colorectal Cancer.

BACKGROUND/AIMS: Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the second leading cause of cancer-related deaths worldwide. Thus, methods for early diagnosis of CRC are urgently needed. We aimed to identify potential long non-coding RNAs (lncRNAs) in circulatory exosomes that may serve as biomarkers for the detection of early-stage CRC. METHODS: Exosomes from the plasma of CRC patients (n = 50) and healthy individuals (n = 50) were isolated by ultracentrifugation, followed by extraction of total exosomal RNAs using TRIzol reagent. Microarray analysis was used for exosomal lncRNA profiling in the two groups, and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine the expression level of lncRNAs in all patients and healthy subjects. RESULTS: The expression of six lncRNAs (LNCV6_116109, LNCV6_98390, LNCV6_38772, LNCV_108266, LNCV6_84003, and LNCV6_98602) was found to be significantly up-regulated in CRC patients compared with that in healthy individuals by qRT-PCR. The receiver operating characteristic curve was used to verify their diagnostic accuracy. The values of the area under the curve for these lncRNAs were 0.770 (LNCV6_116109), 0.7500 (LNCV6_98390), 0.6500 (LNCV6_38772), 0.6900 (LNCV_108266), 0.7500 (LNCV6_84003), and 0.7200 (LNCV6_98602). CONCLUSION: Our study suggested that the expression of these six exosomal lncRNAs (LNCV6_116109, LNCV6_98390, LNCV6_38772, LNCV_108266, LNCV6_84003, and LNCV6_98602) was significantly up-regulated in the plasma of CRC patients, and that they may serve as potential non-invasive biomarkers for early diagnosis of CRC.