The feedback loop of EFTUD2/c-MYC impedes chemotherapeutic efficacy by enhancing EFTUD2 transcription and stabilizing c-MYC protein in colorectal cancer.

BACKGROUND: Chemoresistance presents a significant obstacle in the treatment of colorectal cancer (CRC), yet the molecular basis underlying CRC chemoresistance remains poorly understood, impeding the development of new therapeutic interventions. Elongation factor Tu GTP binding domain containing 2 (EFTUD2) has emerged as a potential oncogenic factor implicated in various cancer types, where it fosters tumor growth and survival. However, its specific role in modulating the sensitivity of CRC cells to chemotherapy is still unclear. METHODS: Public dataset analysis and in-house sample validation were conducted to assess the expression of EFTUD2 in 5-fluorouracil (5-FU) chemotherapy-resistant CRC cells and the potential of EFTUD2 as a prognostic indicator for CRC. Experiments both in vitro, including MTT assay, EdU cell proliferation assay, TUNEL assay, and clone formation assay and in vivo, using cell-derived xenograft models, were performed to elucidate the function of EFTUD2 in sensitivity of CRC cells to 5-FU treatment. The molecular mechanism on the reciprocal regulation between EFTUD2 and the oncogenic transcription factor c-MYC was investigated through molecular docking, ubiquitination assay, chromatin immunoprecipitation (ChIP), dual luciferase reporter assay, and co-immunoprecipitation (Co-IP). RESULTS: We found that EFTUD2 expression was positively correlated with 5-FU resistance, higher pathological grade, and poor prognosis in CRC patients. We also demonstrated both in vitro and in vivo that knockdown of EFTUD2 sensitized CRC cells to 5-FU treatment, whereas overexpression of EFTUD2 impaired such sensitivity. Mechanistically, we uncovered that EFTUD2 physically interacted with and stabilized c-MYC protein by preventing its ubiquitin-mediated proteasomal degradation. Intriguingly, we found that c-MYC directly bound to the promoter region of EFTUD2 gene, activating its transcription. Leveraging rescue experiments, we further confirmed that the effect of EFTUD2 on 5-FU resistance was dependent on c-MYC stabilization. CONCLUSION: Our findings revealed a positive feedback loop involving an EFTUD2/c-MYC axis that hampers the efficacy of 5-FU chemotherapy in CRC cells by increasing EFTUD2 transcription and stabilizing c-MYC oncoprotein. This study highlights the potential of EFTUD2 as a promising therapeutic target to surmount chemotherapy resistance in CRC patients.

Expression mapping of GREM1 and functional contribution of its secreting cells in the brain using transgenic mouse models.

GREMLIN1 (GREM1) is a secreted protein that antagonizes bone morphogenetic proteins (BMPs). While abnormal GREM1 expression has been reported to cause behavioral defects in postpartum mice, the spatial and cellular distribution of GREM1 in the brain and the influence of the GREM1-secreting cells on brain function and behavior remain unclear. To address this, we designed a genetic cassette incorporating a 3×Flag-TeV-HA-T2A-tdTomato sequence, resulting in the creation of a novel Grem1Tag mouse model, expressing an epitope tag (3×Flag-TeV-HA-T2A) followed by a fluorescent reporter (tdTomato) under the control of the endogenous Grem1 promoter. This design facilitated precise tracking of the cell origin and distribution of GREM1 in the brain using tdTomato and Flag (or HA) markers, respectively. We confirmed that the Grem1Tag mouse exhibited normal motor, cognitive, and social behaviors at postnatal 60 days (P60), compared with C57BL/6J controls. Through immunofluorescence staining, we comprehensively mapped the distribution of GREM1-secreting cells across the central nervous system. Pervasive GREM1 expression was observed in the cerebral cortex (Cx), medulla, pons, and cerebellum, with the highest levels in the Cx region. Notably, within the Cx, GREM1 was predominantly secreted by excitatory neurons, particularly those expressing calcium/calmodulin-dependent protein kinase II alpha (Camk2a), while inhibitory neurons (parvalbumin-positive, PV+) and glial cells (oligodendrocytes, astrocytes, and microglia) showed little or no GREM1 expression. To delineate the functional significance of GREM1-secreting cells, a selective ablation at P42 using a diphtheria toxin A (DTA) system resulted in increased anxiety-like behavior and impaired memory in mice. Altogether, our study harnessing the Grem1Tag mouse model reveals the spatial and cellular localization of GREM1 in the mouse brain, shedding light on the involvement of GREM1-secreting cells in modulating brain function and behavior. Our Grem1Tag mouse serves as a valuable tool for further exploring the precise role of GREM1 in brain development and disease.

Gremlin-1 Promotes Colorectal Cancer Cell Metastasis by Activating ATF6 and Inhibiting ATF4 Pathways.

Cancer cell survival, function and fate strongly depend on endoplasmic reticulum (ER) proteostasis. Although previous studies have implicated the ER stress signaling network in all stages of cancer development, its role in cancer metastasis remains to be elucidated. In this study, we investigated the role of Gremlin-1 (GREM1), a secreted protein, in the invasion and metastasis of colorectal cancer (CRC) cells in vitro and in vivo. Firstly, public datasets showed a positive correlation between high expression of GREM1 and a poor prognosis for CRC. Secondly, GREM1 enhanced motility and invasion of CRC cells by epithelial-mesenchymal transition (EMT). Thirdly, GREM1 upregulated expression of activating transcription factor 6 (ATF6) and downregulated that of ATF4, and modulation of the two key players of the unfolded protein response (UPR) was possibly through activation of PI3K/AKT/mTOR and antagonization of BMP2 signaling pathways, respectively. Taken together, our results demonstrate that GREM1 is an invasion-promoting factor via regulation of ATF6 and ATF4 expression in CRC cells, suggesting GREM1 may be a potential pharmacological target for colorectal cancer treatment.

Lnc-DC promotes estrogen independent growth and tamoxifen resistance in breast cancer.

Selective estrogen receptor modulators (SERMs) such as tamoxifen have proven to be effective in the treatment of estrogen receptor (ER) positive breast cancer. However, a major obstacle for such endocrine therapy is estrogen independent growth, leading to resistance, and the underlying mechanism is not fully understood. The purpose of this study was to determine whether long non-coding RNAs (lncRNAs) are involved in regulation of estrogen independent growth and tamoxifen resistance in ER positive breast cancer. Using a CRISPR/Cas9-based SAM (synergistic activation mediator) library against a focus group of lncRNAs, we identify Lnc-DC as a candidate lncRNA. Further analysis suggests that Lnc-DC is able to reduce tamoxifen-induced apoptosis by upregulation of anti-apoptotic genes such as Bcl2 and Bcl-xL. Furthermore, Lnc-DC activates STAT3 by phosphorylation (pSTAT3Y705), and the activated STAT3 subsequently induces expression of cytokines which in turn activate STAT3, forming an autocrine loop. Clinically, upregulation of Lnc-DC is associated with poor prognosis. In particular, analysis of a tamoxifen-treated patient cohort indicates that Lnc-DC expression can predict the response to tamoxifen. Together, this study demonstrates a previously uncharacterized function of Lnc-DC/STAT3/cytokine axis in estrogen independent growth and tamoxifen resistance, and Lnc-DC may serve as a potential predictor for tamoxifen response.

Loganetin and 5-fluorouracil synergistically inhibit the carcinogenesis of gastric cancer cells via down-regulation of the Wnt/ß-catenin pathway.

Although most gastrointestinal tumours are sensitive to 5-fluorouracil (5FU), drug resistance is commonly occurred after 5FU therapy in gastric cancer (GC). Loganetin is the primary active compound in Cornus officinali. However, the synergetic effects of loganetin and 5FU on GC remain unknown. Here, we investigated the synergetic effects and the underlying mechanism of loganetin and 5FU on proliferation, stem-like properties, migration, and invasion of GC both in vitro and in vivo. We found that loganetin alone inhibited the proliferation, stem-like properties, migration and invasion of GC cells in vitro. Importantly, the loganetin remarkably enhanced the anti-cancer effect of 5FU on GC cells and the Wnt/ß-catenin pathway might be involved in this process. Animal experiments further confirmed the synergistic effects of 5FU and loganetin on inhibiting cell growth and metastasis of GC. These results suggested that loganetin could synergistically increase the effect of 5FU against GC, which sheds light on effective combinational drug strategies for GC treatment.

Identification and Investigation of miRNAs From Gastrodia elata Blume and Their Potential Function.

Gastrodia elata Blume (G. elata) is a valuable traditional Chinese medicine with neuroprotection, anti-inflammatory, and immune regulatory functions. MicroRNAs (miRNA) is a kind of endogenous noncoding small RNAs that plays distinctly important roles for gene regulation of organisms. So far, the research on G. elata is mainly focused on the pharmacological functions of the natural chemical ingredients, and the function of G. elata miRNA remains unknown. In this study, 5,718 known miRNAs and 38 novel miRNAs were identified by high-throughput sequencing from G. elata. Based on GO and KEGG analysis, we found that the human genes possibly regulated by G. elata miRNAs were related to the cell cycle, immune regulation, intercellular communication, etc. Furthermore, two novel miRNAs as Gas-miR01 and Gas-miR02 have stable and high expression in the medicinal tissues of G. elata. Further bioinformatics prediction showed that both Gas-miR01 and Gas-miR02 could target Homo sapiens A20 gene, furthermore, the dual-luciferase reporter gene assay and Western Blotting verified the interaction of Gas-miR01 or Gas-miR02 with A20. These evidences suggested that G. elata-unique miRNAs might be involved in certain physiological processes. The animal experiment showed that Gas-miR01 and Gas-miR02 could be detected in some tissues of mice by intragastric administration; meanwhile, the A20 expression in some tissues of mice was downregulated. These results supported for the functional study of G. elata miRNAs.

KLF4 p.A472D Mutation Contributes to Acquired Resistance to Cetuximab in Colorectal Cancer.

With the increase of treatment course, resistance to EGFR blockade is inevitable in patients with metastatic colorectal cancer (mCRC). KRAS mutations have been considered to be primary drivers of this resistance; however, the potential function of other genes has not been extensively investigated. This study collected 17 plasma samples from patients with mCRC with cetuximab resistance, and target-capture deep sequencing was used to identify mutations in circulating tumor DNA (ctDNA). Analysis of mutational prevalence in ctDNA was performed from three colorectal cancer tissue-based datasets and one ctDNA dataset. The prevalence of mutations identified in ctDNA was consistent with both colorectal cancer tissue-based and ctDNA datasets. Clonal analysis revealed that 41.2% of patients were positive for at least one subclone. Multiple mechanisms of cetuximab resistance were coexisted in individual patients, and one of the patients even harbored nine distinct mutations. In particular, functional study of Krüppel-like factor 4 (KLF4) p.A472D revealed increased cetuximab resistance in colorectal cancer cells, which was associated with the increased phosphorylation of downstream EGFR signaling proteins. These results suggest that KLF4 p.A472D may contribute to cetuximab resistance in patients with mCRC and thus may serve as a new biomarker in clinical application. Monitoring somatic mutations related to cetuximab resistance in patients with mCRC through ctDNA may provide real-time insights for clinical reference and treatment planning.

IGF2BP2 regulates DANCR by serving as an N6-methyladenosine reader.

The major function of Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is to regulate cell metabolism. However, emerging evidence indicates that IGF2BP2 plays a role in cancer, but the underlying mechanism is largely unknown. Here we showed that upregulation of IGF2BP2 is associated with poor outcomes of pancreatic cancer patients and suppression of IGF2BP2 inhibits cell proliferation. We further showed that IGF2BP2 regulates lncRNA DANCR. Ectopic expression IGF2BP2 enhances, whereas knockdown (KD) or knockout (KO) of IGF2BP2 suppresses DANCR expression. Moreover, in vivo RNA precipitation and reciprocal RNA immunoprecipitation revealed that IGF2BP2 interacts with DANCR. DANCR promotes cell proliferation and stemness-like properties. Experiments with xenograft models revealed that while ectopic expression of DANCR promotes, DANCR KO suppresses tumor growth. Mechanistically, DANCR is modified at N6-methyladenosine (m6A) and mutagenesis assay identified that adenosine at 664 of DANCR is critical to the interaction between IGF2BP2 and DANCR where IGF2BP2 serves a reader for m6A modified DANCR and stabilizes DANCR RNA. Together, these results suggest that DANCR is a novel target for IGF2BP2 through m6A modification, and IGF2BP2 and DANCR work together to promote cancer stemness-like properties and pancreatic cancer pathogenesis.

Immunotherapy in pancreatic cancer: New hope or mission impossible?

Pancreatic cancer (PC), one of the most lethal diseases, remains a challenging problem. Novel cancer therapy targeting the immune system has been explored. Although immunotherapy has yielded a favorable response in pre-clinical models, no significant improvement has been confirmed in clinical trials for PC. This may be partly attributable to the unique immunosuppressive tumor microenvironment of PC. Studies focusing on combination therapy showed the ability to break the immunosuppressive tumor microenvironment and enhance the immune response, which can translate to clinical benefits. Moreover, the application of sequencing techniques and neo-antigen vaccines has achieved promising results in clinical trials, which promote the development of personalized immunotherapy. However, lack of effective biomarkers is another challenge for the realization of personalized immune medicine. Biomarkers are urgently needed to identify subgroup of patients who would benefit from immunotherapy. In this review, we discuss advances in immunotherapy for pancreatic cancer, as well as the challenges and prospects for personalized immune medicine.

Prospects of Noncoding RNAs in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a global health problem and one of the most common malignant tumors. Recent studies have shown that noncoding RNAs (ncRNAs) contribute to the pathogenesis of hepatocellular carcinoma (HCC). These RNAs may be involved in a variety of pathological processes such as cell proliferation, apoptosis, angiogenesis, invasion, and metastasis. In addition, abnormal expression of ncRNAs in HCC may provide potential prognostic or diagnostic biomarkers. This review provides an overview of the role and potential applications of ncRNAs, miRNAs, lncRNAs, circRNAs, and snoRNAs in liver cancer.

Cloning, expression profiling, and acetylation identification of alpha-tubulin N-acetyltransferase 1 from Bombyx mori.

Alpha-tubulin N-acetyltransferase 1 (ATAT1) is an acetyltransferase specific to α-tubulin and performs important functions in many cellular processes. Bombyx mori is an economic insect and also known as a model lepidoptera insect. In this study, we cloned a B. mori ATAT1 gene (BmATAT1) (Gen Bank accession number: XP_004932777.1). BmATAT1 contained an open reading frame (ORF) of 1,065 bp encoding 355 amino acids (aa). Expression profiling of BmATAT1 protein showed that the expression levels of BmATAT1 at different developmental stages and different tissues in fifth-instar larvae differ. BmATAT1 was highly expressed at the egg stage and in the head of the fifth-instar larvae. Subcellular localization showed that BmATAT1 was distributed in the cytoplasm and nucleus. Furthermore, BmATAT1 may lead to time-dependent induction of cell cycle arrest in the G2/M phase by flow cytometry analysis. Interestingly, using site-specific mutation, immunoprecipitation, and Western blotting, we further found a BmATAT1 acetylated site at K156, suggesting that this acetyltransferase could be regulated by acetylation itself.