tRF-27 competitively Binds to G3BPs and Activates MTORC1 to Enhance HER2 Positive Breast Cancer Trastuzumab Tolerance.

About 20% of breast cancer patients are positive for HER2. The efficacy of current treatments is limited by primary and secondary resistance to trastuzumab. tRNA-derived fragments (tRFs) have shown crucial regulatory roles in various cancers. This study aimed to evaluate the role of tRF-27 in regulating the resistance of HER2-positive breast cancer against trastuzumab. tRF-27 was highly expressed in trastuzumab-resistant cells, and its expression level could predict the resistance to trastuzumab. High expression of tRF-27 promoted the growth and proliferation of trastuzumab-exposed cells. RNA-pulldown assay and mass spectrometry were performed to identify Ras GTPase-activating protein-binding proteins 1 and 2 (G3BPs) (two proteins targeted by tRF-27); RNA-immunoprecipitation (RIP) to confirm their bindings; co-immunoprecipitation (co-IP) and RNA-pulldown assay to determine the binding domains between G3BPs and tRF-27.tRF-27 bound to the nuclear transport factor 2 like domain(NTF2 domain) of G3BPs through a specific sequence. tRF-27 relied on G3BPs and NTF2 domain to increase trastuzumab tolerance. tRF-27 competed with lysosomal associated membrane protein 1(LAMP1) for NTF2 domain, thereby inhibiting lysosomal localization of G3BPs and tuberous sclerosis complex (TSC). Overexpression of tRF-27 inhibited phosphorylation of TSCs and promoted the activation of mechanistic target of rapamycin complex 1(MTORC1) to enhance cell proliferation and entice the resistance of HER2-positive breast cancer against trastuzumab.

Unveiling Alterations of Epigenetic Modifications and Chromatin Architecture Leading to Lipid Metabolic Reprogramming during the Evolutionary Trastuzumab Adaptation of HER2-Positive Breast Cancer.

Secondary trastuzumab resistance represents an evolutionary adaptation of HER2-positive breast cancer during anti-HER2 treatment. Most current studies have tended to prioritize HER2 and its associated signaling pathways, often overlooking broader but seemingly less relevant cellular processes, along with their associated genetic and epigenetic mechanisms. Here, transcriptome data is not only characterized but also examined epigenomic and 3D genome architecture information in both trastuzumab-sensitive and secondary-resistant breast cancer cells. The findings reveal that the global metabolic reprogramming associated with trastuzumab resistance may stem from genome-wide alterations in both histone modifications and chromatin structure. Specifically, the transcriptional activities of key genes involved in lipid metabolism appear to be regulated by variant promoter H3K27me3 and H3K4me3 modifications, as well as promoter-enhancer interactions. These discoveries offer valuable insights into how cancer cells adapt to anti-tumor drugs and have the potential to impact future diagnostic and treatment strategies.

An Imbalance in Histone Modifiers Induces tRNA-Cys-GCA Overexpression and tRF-27 Accumulation by Attenuating Promoter H3K27me3 in Primary Trastuzumab-Resistant Breast Cancer.

tRNA-derived fragments (tRFs) play crucial roles in cancer progression. Among them, tRF-27 has been identified as a key factor in promoting naïve trastuzumab resistance in HER2-positive breast cancer. However, the origin of tRF-27 remains uncertain. In this study, we propose that the upregulated expression of specific cysteine tRNAs may lead to the increased accumulation of tRF-27 in trastuzumab-resistant JIMT1 cells. Mechanistically, the reduced inhibitory H3K27me3 modification at the promoter regions of tRF-27-related tRNA genes in JIMT1 cells, potentially resulting from decreased EZH2 and increased KDM6A activity, may be a critical factor stimulating the transcriptional activity of these tRNA genes. Our research offers fresh insights into the mechanisms underlying elevated tRF-27 levels in trastuzumab-resistant breast cancer cells and suggests potential strategies to mitigate trastuzumab resistance in clinical treatments.

Exosome-Transmitted tRF-16-K8J7K1B Promotes Tamoxifen Resistance by Reducing Drug-Induced Cell Apoptosis in Breast Cancer.

Tamoxifen resistance remains a challenge in hormone receptor-positive (HR+) breast cancer. Recent evidence suggests that transfer ribonucleic acid (tRNA)-derived fragments play pivotal roles in the occurrence and development of various tumors. However, the relationship between tRNA-derived fragments and tamoxifen resistance remains unclear. In this study, we found that the expression of tRF-16-K8J7K1B was upregulated in tamoxifen-resistant cells in comparison with tamoxifen-sensitive cells. Higher levels of tRF-16-K8J7K1B were associated with shorter disease-free survival in HR+ breast cancer. Overexpression of tRF-16-K8J7K1B promotes tamoxifen resistance. Moreover, extracellular tRF-16-K8J7K1B could be packaged into exosomes and could disseminate tamoxifen resistance to recipient cells. Mechanistically, exosomal tRF-16-K8J7K1B downregulates the expression of apoptosis-related proteins, such as caspase 3 and poly (ADP-ribose) polymerase, by targeting tumor necrosis factor-related apoptosis-inducing ligand in receptor cells, thereby reducing drug-induced cell apoptosis. Therapeutically, the inhibition of exosomal tRF-16-K8J7K1B increases the sensitivity of breast cancer cells to tamoxifen in vivo. These data demonstrate that exosomal tRF-16-K8J7K1B may be a novel therapeutic target to overcome tamoxifen resistance in HR+ breast cancer.

CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma.

Background: Circular ribonucleic acids (circRNAs) play a key role in the development of different types of cancer. Ferroptosis is a type of programmed cell death that contributes to cancer progression. However, the role of circRNAs in lung adenocarcinoma (LUAD) ferroptosis remains unclear. Methods: The gene expression levels of circRNA P4HB (circP4HB), microRNA-1184 (miR-1184) and Solute carrier family 7 member 11 (Slc7a11), also known as Xct were detected using quantitative real-time polymerase chain reaction (qRT-PCR). Ferroptosis of established LUAD cells was induced by erastin. Cell viability was examined via Cell Counting Kit 8 assays. Ferroptosis was evaluated by malondialdehyde (MDA), Prostaglandin-endoperoxide Synthase 2 (Ptgs2), lipid reactive oxygen species (lipid ROS), and JC-1 detection. The mechanism of circP4HB/miR-1184/SLC7A11 was investigated by luciferase reporter assays, RNA immunoprecipitation, RNA pull-down, and western blot assays. A functional for circP4HB in vivo was determined using xenograft nude mice models. Results: CircP4HB expression levels were increased in LUAD. It triggered glutathione (GSH) synthesis and, therefore protected LUAD cells from ferroptosis induced by erastin. CircP4HB may function as a competing endogenous RNA by modulating miR-1184 to regulate SLC7A11. CircP4HB inhibited ferroptosis by regulating miR-1184/ SLC7A11-mediated GSH synthesis. In vivo, overexpression of circP4HB promoted tumor growth and inhibited ferroptosis. Conclusions: The circRNA, circP4HB acts as a novel ferroptosis suppressor in LUAD. Furthermore, circP4HB protects LUAD from ferroptosis via modulation of the miR-1184/SLC7A11 axis. Our findings identified circP4HB as a novel biomarker in LUAD and warrants further investigation in the early diagnosis and treatment of LUAD.

A positive feedback loop: RAD18-YAP-TGF-ß between triple-negative breast cancer and macrophages regulates cancer stemness and progression.

As a key regulator of the DNA translesion synthesis (TLS) pathway, RAD18 is error-prone and contributes to the accumulation of DNA mutations. Our previous study showed that it plays an essential role in the progression of multiple tumors. However, the mechanism through which RAD18 influences triple-negative breast cancer (TNBC), especially the interaction between tumor cells and the tumor microenvironment, remains elusive. In this study, we showed that RAD18 expression is markedly higher in patients with high T stage TNBC and inversely correlated with prognosis. High expression of RAD18 facilitated a highly stem-cell phenotype through the Hippo/YAP pathway, which supports the proliferation of TNBC. In addition, the cytokine byproduct TGF-ß activates macrophages to have an M2-like tumor-associated macrophage (TAM) phenotype. Reciprocally, TGF-ß from TAMs activated RAD18 in TNBC to enhance tumor stemness, forming a positive feedback loop. Inhibition of YAP or TGF-ß breaks this loop and suppresses cancer stemness and proliferation In nude mice, RAD18 promoted subcutaneous transplanted tumor growth and M2-type TAM recruitment. Collectively, the RAD18-YAP-TGF-ß loop is essential for the promotion of the stemness phenotype by TNBC and could be a potential therapeutic target for TNBC.

Tumor-derived exosomal miR-3157-3p promotes angiogenesis, vascular permeability and metastasis by targeting TIMP/KLF2 in non-small cell lung cancer.

Metastasis is the main cause of death in patients with advanced lung cancer. The exosomes released by cancer cells create tumor microenvironment, and then accelerate tumor metastasis. Cancer-derived exosomes are considered to be the main driving force for metastasis niche formation at foreign sites, but the mechanism in Non-small cell lung carcinoma (NSCLC) is unclear. In metastatic NSCLC patients, the expression level of miR-3157-3p in circulating exosomes was significantly higher than that of non-metastatic NSCLC patients. Here, we found that miR-3157-3p can be transferred from NSCLC cells to vascular endothelial cells through exosomes. Our work indicates that exosome miR-3157-3p is involved in the formation of pre-metastatic niche formation before tumor metastasis and may be used as a blood-based biomarker for NSCLC metastasis. Exosome miR-3157-3p has regulated the expression of VEGF/MMP2/MMP9 and occludin in endothelial cells by targeting TIMP/KLF2, thereby promoted angiogenesis and increased vascular permeability. In addition, exosome miR-3157-3p promoted the metastasis of NSCLC in vivo.

hsa_circ_0008234 inhibits the progression of lung adenocarcinoma by sponging miR-574-5p.

circRNAs are a novel type of noncoding RNA (ncRNA) that have been identified as an important regulator of gene expression and play a part in the progression of various diseases. However, the function of circ_0008234 in lung adenocarcinoma (LUAC) remains unknown. Through the GEO (Gene Expression Omnibus) database, circ_0008234 was first found to be downregulated in LUAC tissues. It could inhibit cell growth and accelerate apoptosis in vitro and in vivo. In terms of its possible mechanism, circ_0008234 mainly was present in the cytoplasm and competed with miR-574-5p to regulate RND3 (Rho family GTPase 3). Our results revealed that circ_0008234 inhibited the progression of LUAC through a competing endogenous RNA (ceRNA)-based mechanism and provided potential biomarkers and therapeutic targets for LUAC treatment.

miR-550a-5p Functions as a Tumor Promoter by Targeting LIMD1 in Lung Adenocarcinoma.

Lung adenocarcinoma accounts for half of all lung cancer cases in most countries. Mounting evidence has demonstrated that microRNAs play important roles in cancer progression, and some of them can be identified as potential biomarkers. This study aimed to explore the role of miR-550a-5p, a lung adenocarcinoma-associated mature microRNA screened out from the TCGA database via R-studio and Perl, with abundant expression in samples and with 5-year survival prognosis difference, as well as having not been studied in lung cancer yet. Potential target genes were predicted by the online database. Gene ontology enrichment, pathway enrichment, protein-protein interaction network, and hub genes-microRNA network were constructed by FunRich, STRING database, and Cytoscape. Then, LIMD1, a known tumor suppressor gene reported by multiple articles, was found to have a negative correlation with miR-550a-5p. The expression of miR-550a-5p was up-regulated in tumor samples and tumor-associated cell lines. Its high expression was also correlated with tumor size. Cell line A549 treated with miR-550a-5p overexpression promoted tumor proliferation, while H1299 treated with miR-550a-5p knockdown showed the opposite result. Mechanically, miR-550a-5p negatively regulated LIMD1 by directly binding to its 3'-UTR validated by dual luciferase assay. In summary, a new potential prognostic and therapeutic biomarker, miR-550a-5p, has been identified by bioinformatics analysis and experimental validation in vitro and in vivo, which promotes lung adenocarcinoma by silencing a known suppressor oncogene LIMD1.

Circular RNA LPAR3 sponges microRNA-198 to facilitate esophageal cancer migration, invasion, and metastasis.

In this study, we explored expression and functions of circular RNA LPAR3 (circLPAR3) in esophageal squamous cell carcinoma (ESCC). The differential expression of circular RNAs (circRNAs) in 10 ESCC and corresponding paracarcinoma tissues was analyzed through circRNA microarray, then the candidate circRNAs were detected and verified through quantitative RT-PCR, and a novel circRNA was screened, which was circLPAR3. Circular RNA LPAR3 showed apparently high expression in ESCC tissues and cells, which was closely correlated with the clinical stage and lymph node metastasis of ESCC patients. Circular RNA LPAR3 was mainly located in the cytoplasm of ESCC cells, which was more stable than the baseline gene. Circular RNA LPAR3 upregulated MET gene expression through sponge adsorption of microRNA (miR)-198, activated the RAS/MAPK and the PI3K/Akt pathways, and promoted ESCC cell migration, invasion, and metastasis in vivo and in vitro. However, it had no effect on ESCC cell proliferation. Circular RNA LPAR3 can regulate the miR-198-MET signal axis to promote the migration, invasion, and metastasis of esophageal cancer cells, which can thereby serve as a potential diagnostic and therapeutic target of esophageal cancer.

Systematic cancer-testis gene expression analysis identified CDCA5 as a potential therapeutic target in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies with poor prognosis. Cancer-testis genes (CTGs) have been vigorously pursued as targets for cancer immunotherapy, but the expressive patterns and functional roles of CTGs remain unclear in ESCC. METHODS: A systematic screening strategy was adopted to screen CTGs in ESCC by integrating multiple public databases and RNA expression microarray data from 119 ESCC subjects. For the newly identified ESCC prognosis-associated CTGs, an independent cohort of 118 patients with ESCC was recruited to validate the relationship via immunohistochemistry. Furthermore, functional assays were performed to determine the underlying mechanisms. FINDINGS: 21 genes were recognized as CTGs, in particular, CDCA5 was aberrantly upregulated in ESCC tissues and significantly associated with poor prognosis (HR = 1.85, 95%CI: 1.14-3.01, P = .013). Immunohistochemical staining confirmed that positive CDCA5 expression was associated with advanced TNM staging and a shorter overall survival rate (45.59% vs 28.00% for CDCA5-/+ subjects, P = 1.86 × 10-3). H3K27 acetylation in CDCA5 promoter might lead to the activation of CDCA5 during ESCC tumorigenesis. Functionally, in vitro assay of gain- and loss-of-function of CDCA5 suggested that CDCA5 could promote ESCC cells proliferation, invasion, migration, apoptosis resistance and reduce chemosensitivity to cisplatin. Moreover, in vivo assay showed that silenced CDCA5 could inhibit tumor growth. Mechanistically, CDCA5 knockdown led to an arrest in G2/M phase and changes in the expression of factors that played fundamental roles in the cell cycle pathway. INTERPRETATION: CDCA5 contributed to ESCC progression and might serve as an attractive target for ESCC immunotherapy. FUND: This work was supported by the Natural Science Foundation of Jiangsu Province (No. BK20181083 and BK20181496), Jiangsu Top Expert Program in Six Professions (No. WSW-003 and WSW-007), Major Program of Science and Technology Foundation of Jiangsu Province (No. BE2016790 and BE2018746), Jiangsu Medical Young Talent Project (No. QNRC2016566), the Program of Jiangsu Medical Innovation Team (No. CXTDA2017006), Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX18_1487) and Jiangsu Province 333 Talents Project (No. BRA2017545).

hsa_circ_0006168 sponges miR-100 and regulates mTOR to promote the proliferation, migration and invasion of esophageal squamous cell carcinoma.

Circle RNAs (circRNAs) are the novel noncoding RNAs with the covalent closed-loop structure, which play a crucial role in a variety of pathological processes, including cancer. Nevertheless, the expression profiles and functions of circRNAs in esophageal squamous cell cancer (ESCC) remain largely unknown. In this paper, 10 pairs of ESCC tissues were utilized to screen the circRNA expression profiles by means of microarray assay; further, a novel circular RNA named hsa_circ_0006168 was investigated. Meanwhile, the expression of hsa_circ_0006168 was measured in 52 ESCC tissues and in cell lines. Our results suggested that, hsa_circ_0006168 was remarkably increased not only in ESCC tissues but also in cell lines compared with those in normal cases. Besides, high hsa_circ_0006168 expression was positively connected with lymph node metastasis and TNM stage of ESCC patients. In vitro, the proliferation, invasion and migration capacities of ESCC cells were suppressed through down-regulating hsa_circ_0006168 expression. Besides, RNase R digestion assay confirmed that hsa_circ_0006168 was more stable than its linear CNOT6L mRNA form. Moreover, nuclear and cytoplasmic fraction assay indicated that hsa_circ_0006168 was mainly distributed in the cytoplasm of Kyse450 and TE13 cells. Mechanically, it was discovered in this study that hsa_circ_0006168 might regulate the expression of Mammalian Target of Rapamycin (mTOR) by sponging microRNA-100 (miR-100). Taken together, hsa_circ_0006168 can promote ESCC proliferation, migration and invasion through the competing endogenous RNA (ceRNA) mechanism, which has been first confirmed in our results. In ESCC, hsa_circ_0006168 can serve as a potential diagnostic biomarker and therapeutic target.

Genome-wide copy number variation analysis identified ANO1 as a novel oncogene and prognostic biomarker in esophageal squamous cell cancer.

Copy number variations (CNVs) represent one of the most common genomic alterations. This study aimed to evaluate the roles of genes within highly aberrant genome regions in the prognosis of esophageal squamous cell cancer (ESCC). Exome sequencing data from 81 paired ESCC tissues were used to screen aberrant genomic regions. The associations between CNVs and gene expression were evaluated using gene expression data from the same individuals. Then, an RNA expression array profile from 119 ESCC samples was adopted for differential gene expression and prognostic analyses. Two independent ESCC cohorts with 315 subjects were further recruited to validate the prognostic value using immunohistochemistry tests. Finally, we explored the potential mechanism of our identified novel oncogene in ESCC. In total, 2003 genes with CNVs were observed, of which 76 genes showed recurrent CNVs in more than three samples. Among them, 32 genes were aberrantly expressed in ESCC tumor tissues and statistically correlated with CNVs. Strikingly, 4 (CTTN, SHANK2, INPPL1 and ANO1) of the 32 genes were significantly associated with the prognosis of ESCC patients. Patients with a positive expression of ANO1 had a poorer prognosis than ANO1 negative patients (overall survival rate: 42.91% versus 26.22% for ANO1-/+ samples, P < 0.001). Functionally, ANO1 promoted ESCC cell proliferation, migration and invasion by activating transforming growth factor-ß pathway. Knockdown of ANO1 significantly inhibited tumor progression in vitro and in vivo. In conclusion, ANO1 is a novel oncogene in ESCC and may serve as a prognostic biomarker for ESCC.

MicroRNA-1204 promotes cell proliferation by regulating PITX1 in non-small-cell lung cancer.

MicroRNA-1204 (miR-1204), a member of the PVT1 region, may improve B cell differentiation and metastasis in breast cancer. However, the role of miR-1204 in non-small-cell lung cancer (NSCLC) and its mechanism remain unclear. The GEO public database was first employed to find differentially expressed genes. The expression level of miR-1204 in patient tissues and NSCLC cell lines was determined using qRT-PCR. Cell proliferation assays were performed to investigate the impact of miR-1204 on cell growth. Bioinformatics analysis and dual-luciferase reporter assays were conducted to find potential target genes. Finally, we performed in vivo experiments to identify the effect of miR-1204 on tumor formation in nude mice. It was first found that miR-1204 was overexpressed in NSCLC tissues and cells. miR-1204 increased the proliferation of NSCLC cells and reduced cell cycle arrest in vitro. PITX1 (paired like homeodomain 1) was found as a potential target gene. In addition, PITX1 was also found to be low in expression in NSCLC tissues and cells. To show that PITX1 reversed the function of miR-1204 in promoting proliferation, confirmatory experiments were performed. Moreover, high miR-1204 and low PITX1 expression was highly correlated with tumor size, lymph node metastasis, and the TNM stage in patients diagnosed with NSCLC. Our results suggested that upregulated miR-1204 in NSCLC is associated with NSCLC progression and promotes NSCLC cell proliferation by downregulating PITX1. miR-1204 may act as a poor prognostic factor and a potential therapeutic target for NSCLC.

MicroRNA-1258 suppresses tumour progression via GRB2/Ras/Erk pathway in non-small-cell lung cancer.

OBJECTIVES: Lung cancer is still a disease with high morbidity and mortality in the world. MicroRNAs have been proven to act as an indispensable role in the reuse of multiple solid tumours. Although miR-1258 plays a vital role in suppressing metastasis in breast cancer and gastric cancer, the specific biological function of miR-1258 in non-small-cell lung cancer remains unclear. METHODS: The differential expression of miR-1258 in NSCLC tissues and corresponding paracancerous tissues was detected by qRT-PCR and ISH. Flow cytometry and CCK-8, EdU, tubule formation, and senescence assays were performed, and xenograft models were studied to explore the function of miR-1258. Potential targets of miR-1258 were verified by dual luciferase reporter assay, qRT-PCR, IHC and Western blotting. RESULTS: In vitro and in vivo gain- and loss-of-function assays suggested that miR-1258 inhibits NSCLC cell proliferation and induces senescence and apoptosis. The luciferase reporter assay, IHC and Western blotting analysis showed that GRB2 is one of the direct targets of miR-1258. The GRB2 overexpression plasmid can reverse the functional changes after overexpression of miR-1258. In contrast, miR-1258 inhibitor significantly reversed si-GRB2-induced GRB2 down-regulation. Mechanistically, overexpression of miR-1258 inhibits GRB2 expression and then leads to inactivation of the Ras/Erk oncogenic pathway. CONCLUSIONS: Our results indicate that miR-1258 can suppress NSCLC progression by targeting the GRB2/Ras/Erk pathway, which may lead to different insights into potential biomarkers and novel therapeutic strategies for NSCLC patients.