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ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA for protein acetylation. ACLY has two major splice isoforms: the full-length canonical "long" isoform and an uncharacterized "short" isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within an intrinsically disordered region and includes at least one dynamically phosphorylated residue. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the percent spliced in (PSI) of exon 14 is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types. This prompted us to explore potential biochemical and functional differences between ACLY isoforms. Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology nor did it attenuate tumor burden in a genetic model of intestinal cancer. Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive.

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The replicative senescence of human amniotic epithelial stem cells (hAECs) is a major concern towards its clinical application. This study found that a 300-kDa hyaluronic acid (HA) could effectively delay the replicative senescence of hAECs, as indicated by the downregulation of cellular senescence markers and alteration of the cell cycle, and substantially improve the differentiation capacities of hAECs. HA was confirmed to regulate the CD44 isoform switch by upregulating the CD44s and downregulating the CD44v, thus exerting an anti-aging effect. We further found that HA induced the upregulation of hyaluronan synthase (HAS) 2, resulting in the activation of epithelial splicing regulatory protein 1 (ESRP1) and alternative splicing of CD44 mRNA, thereby promoting CD44s expression and inhibiting CD44v expression. Knockdown of HAS2 blocked ESRP1 expression and attenuated the anti-aging effects of HA. Hermes-1, a specific blocker of CD44, caused partial loss of the anti-aging effect of HA, upregulated senescence markers, and downregulated stemness markers. Furthermore, CD44s receptor activation was shown to initiate the AKT/mTOR downstream signaling. Conclusively, the study suggested that HA delayed hAEC senescence by regulating CD44 isoform switch to activate the AKT/mTOR signaling pathway, and there is potential for the clinical application of hAECs in combination with HA.

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As aberrant alternative splicing by either dysregulation or mutations of splicing factors contributes to cancer initiation and progression, splicing factors are emerging as potential therapeutic targets for cancer therapy. Therefore, pharmacological modulators targeting splicing factors have been under development. Epithelial splicing regulatory protein 1 (ESRP1) is an epithelial cell-specific splicing factor, whose downregulation is associated with epithelial-mesenchymal transition (EMT) by regulating alternative splicing of multiple genes, such as CD44, CTNND1, ENAH, and FGFR2. Consistent with the downregulation of ESRP1 during EMT, it has been initially revealed that high ESRP1 expression is associated with favorable prognosis and ESRP1 plays a tumor-suppressive role in cancer progression. However, ESRP1 has been found to promote cancer progression in some cancers, such as breast and ovarian cancers, indicating that it plays a dual role in cancer progression depending on the type of cancer. Furthermore, recent studies have reported that ESRP1 affects tumor growth by regulating the metabolism of tumor cells or immune cell infiltration in the tumor microenvironment, suggesting the novel roles of ESRP1 in addition to EMT. ESRP1 expression was also associated with response to anticancer drugs. This review describes current understanding of the roles and mechanisms of ESRP1 in cancer progression, and further discusses the emerging novel roles of ESRP1 in cancer and recent attempts to target splicing factors for cancer therapy.

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During intravasation, cancer cells cross the endothelial barrier and enter the circulation. Extracellular matrix stiffening has been correlated with tumor metastatic potential; however, little is known about the effects of matrix stiffness on intravasation. Here, we utilize in vitro systems, a mouse model, specimens from patients with breast cancer, and RNA expression profiles from The Cancer Genome Atlas Program (TCGA) to investigate the molecular mechanism by which matrix stiffening promotes tumor cell intravasation. Our data show that heightened matrix stiffness increases MENA expression, which promotes contractility and intravasation through focal adhesion kinase activity. Further, matrix stiffening decreases epithelial splicing regulatory protein 1 (ESRP1) expression, which triggers alternative splicing of MENA, decreases the expression of MENA11a, and enhances contractility and intravasation. Altogether, our data indicate that matrix stiffness regulates tumor cell intravasation through enhanced expression and ESRP1-mediated alternative splicing of MENA, providing a mechanism by which matrix stiffness regulates tumor cell intravasation.

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Intratumoral heterogeneity is tightly associated with the failure of anticancer treatment modalities including conventional chemotherapy, radiation therapy, and molecularly targeted therapy. Such heterogeneity is generated in an evolutionary manner not only as a result of genetic alterations but also by the presence of cancer stem cells (CSCs). CSCs are proposed to exist at the top of a tumor cell hierarchy and are undifferentiated tumor cells that manifest enhanced tumorigenic and metastatic potential, self-renewal capacity, and therapeutic resistance. Properties that contribute to the robustness of CSCs include the abilities to withstand redox stress, to rapidly repair damaged DNA, to adapt to a hyperinflammatory or hyponutritious tumor microenvironment, and to expel anticancer drugs by the action of ATP-binding cassette transporters as well as plasticity with regard to the transition between dormant CSC and transit-amplifying progenitor cell phenotypes. In addition, CSCs manifest the phenomenon of metabolic reprogramming, which is essential for maintenance of their self-renewal potential and their ability to adapt to changes in the tumor microenvironment. Elucidation of the molecular underpinnings of these biological features of CSCs is key to the development of novel anticancer therapies. In this review, we highlight the pathological relevance of CSCs in terms of their hallmarks and identification, the properties of their niche-both in primary tumors and at potential sites of metastasis-and their resistance to oxidative stress dependent on system xc (-).

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AIM:To investigate the expression of miR-23a and epithelial splicing regulatory protein 1(ESRP1) in rectal cancer tissues and cell lines as well as their effects on rectal cancer cell viability and apoptosis.METHODS:The relative levels of miR-23a in the rectal cancer tissues and cultured cells were assessed by RT-qPCR.The positive expression of ESRP1 in the rectal cancer tissues and non-cancer tissues was detected by immunohistochemical staining.The sequences of miR-23a inhibitor and inhibitor negative control (NC) were synthesized, and transfected into the SW480 cells.The cell viability was measured by CCK-8 assay.The apoptotic rate was analyzed by flow cytometry.The cell invasion was evaluated by Matrigel counting assay.The expression of ESRP1 was determined by Western blot.The wild-type pGL3-ESRP1-3'UTR (wt-pGL3-ESRP1-3'UTR) or mutant pGL3-ESRP1-3'UTR (mut-pGL3-ESRP1-3'UTR) plasmid and miR-23a inhibitor or inhibitor NC were co-transfected into the HEK293 and SW480 cells.The dual luciferase activity was detected according to Promega dual luciferase reporter gene assay kit instructions.The cell viability and apoptosis were evaluated by CCK-8 assay and flow cytometry analysis, respectively, after the SW480 cells were transfected with ESRP1 mimic or mimic NC.The expression of ESRP1, caspase-3, Smac and X-linked inhibitor of apoptosis protein (XIAP) in the SW480 cells was detected by Western blot.RESULTS:The expression of miR-23a was significantly up-re-gulated in the rectal cancer tissues and cell lines, while the positive expression of ESRP1 was significantly decreased in the rectal cancer specimens.The miR-23a expression was also closely related to lymphnode metastasis and TNM stages of rectal cancer patients.ESRP1 was inversely correlated with miR-23a in the rectal cancer tissues.After transfection with miR-23a inhibitor in human rectal cancer SW480 cells, the down-regulation of miR-23a induced significant inhibition of cell viability as compared with the cells transfected with inhibitor NC (P<0.01).Furthermore, the apoptotic rate induced by the miR-23a inhibitor transfection was markedly higher than that of control (P<0.01).Luciferase assay showed that ESRP1 was a direct target gene of miR-23a.The cell viability and apoptosis were inhibited and promoted, respectively, after transfection with ESRP1 mimic in the SW480 cells.Promoted expression of ESRP1 significantly up-regulated the levels of caspase-3 and Smac as well as down-regulated the expression of XIAP in the SW480 cells.CONCLUSION:The expression of miR-23a is significantly associated with the growth and apoptosis of human rectal cancer cells by targeting ESRP1.miR-23a may be a potential therapeutic target for the treatment of rectal cancer in the future.

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Despite the development of various therapeutic approaches, recurrence and metastasis remain major problems for patients with advanced cancer. Recent studies have shown that cancer stem cells (CSCs) play an important role in cancer aggressiveness. In cancer tissues, a small number of CSCs are able to self-renew and differentiate into heterogeneous cancer cells. CSCs usually remain in the resting phase of the cell cycle and possess efficient drug efflux pathways. Thus, they are resistant to chemoradiotherapy and surviving CSCs contribute to recurrence. During cancer metastasis, CSCs undergo epithelial-mesenchymal transition (EMT), thereby acquiring mesenchymal features, migrating to adjacent stromal tissues, and invading blood or lymph vessels. Recent studies showed that EMT-inducible factors also enhance or induce CSC-like features in cancer cells. These findings suggest that EMT is closely correlated with cancer recurrence and metastasis. Inhibition of nestin, a CSC marker, reduces the aggressiveness of several types of cancer. Suppression of the mesenchymal variant of fibroblast growth factor (FGFR)-2, FGFR-2 IIIc, and regulation of the EMT using epithelial splicing regulatory protein 1 (ESRP1) are effective in the treatment of immunodeficient mice with pancreatic cancer. The roles of CSCs and EMT in cancer and possible therapies are discussed in this review.

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Objective: To investigate the effects of silencing epithelial splicing regulatory protein 1 (ESRP 1) gene expression on isoform switch of fibroblast growth factor receptor 2 (FGFR2) in ovarian cancer OVCAR5 cells. Methods: The two lentivirus expression vectors carrying ESRP1- shRNA1/2 targeting ESRP 1 gene (named as pLVTHM/ESRP1-shRNA1 and pLVTHM/ESRP1-shRNA2, respectively) were constructed, while the vector pLVTHM/ NC-shRNA was used as the negative control. The three plasmids were separately transfected into 293T cells to prepare the recombinant lentivirus, and the recombinant lentivirus carrying ESRP1- shRNA1/2 and NC-shRNA were infected into OVCAR5 cells, respectively. Then the expression levels of ESRP1 mRNA and protein in OVCAR5 cells were determined by real-Time fluorescent quantitative PCR and Western blotting, respectively. The effect of ESRP 1 gene silencing on FGFR2 isoform switch was detected by restrictive endonucleases Ava I and Hin C II The effect of ESPR 1 gene silencing on the proliferation of OVCAR5 cells was detected using MTT method. The changes of E-cadherin, N-cadherin and β-catenin expressions were detected by Western blotting. Results: The lentivirus expression vectors containing ESRP1-shRNA1/2 were constructed successfully, and their recombinant lentivirus were obtained. Compared with the negative control group, the expressions of ESRP1 mRNA and protein in OVCAR5 cells were significantly inhibited by ESRP1 shRNA (both P < 0.05). ESPR 1 gene silencing could induce the FGFR2 isoform switch from epithelial subtype (FGFR2b) to mesenchymal subtype (FGFR2c) in OVCAR5 cells, and could promote cell proliferation, down-regulate the expression of E-cadherin, up-regulate the expression of N-cadherin, and activate β-catenin in OVCAR5 cells (all P < 0.05). Conclusion: ESPR 1 gene silencing could induce the isoform switch of FGFR 2 gene switch, promote cell proliferation and induce epithelialmesenchymal transition (EMT) in OVCAR5 cells. The processes may involve the regulation of Wnt/β-catenin signaling pathway.