Biology subjects

Horita, Y.

Publications and source records attributed to Horita, Y..

8 recordsLinked to original sources

bioRxiv2026

Metformin lowers HMGA1a and RBM3 protein in breast cancer cells, and its effect on ESR1 splicing depends on cell density and exposure time

The ESR1 gene produces ER46, an N-terminally truncated isoform generated by skipping of exon 1, which is reduced in tamoxifen-resistant breast cancer cells. We have reported that metformin moves the ratio of full-length ER to ER46 in opposite directions in MCF-7 cells and their tamoxifen-resistant subline TAMR1, and that metformin reduces TAMR1 xenograft growth (Harada et al., Am J Pharm Health Res 2022;10(11)). That report leaves two questions open: whether the effect is dosedependent and stable across culture conditions, and what splicing factor might carry it. Here we report the experiments that bear on both. Across a full dose range from 0.25 to 5 mM at high seeding density, metformin produced no consistent change in the full-length share of ESR1 transcript in either line: densitometry of the gel gave 42%, 43%, 42%, 43%, 45% and 48% in MCF-7 cells and 55%, 44%, 52%, 47%, 68% and 43% in TAMR1 cells. The reported shift therefore depends on the seeding density used, and the comparison that produced it is not reproduced at high density. At low density the MCF-7 response persisted to 48 h (52%, 62% and 68% at 0, 2.5 and 5 mM). Metformin lowered HMGA1a protein in MCF-7 cells and showed the same tendency in TAMR1 cells, at both seeding densities, and lowered RBM3 protein in TAMR1 cells at 24 and 48 h. HMGA1a is the factor we previously showed to generate ER46 by trapping U1 snRNP at an upstream pseudo-5' splice site, so a fall in HMGA1a predicts less ER46, the opposite of what was seen in TAMR1 cells at low density. The fall in RBM3, the protein through which metformin alters splicing in myotonic dystrophy, is the more plausible candidate. RT-PCR of the xenograft tumours from the earlier study detected full-length ESR1 transcript in the untreated and tamoxifen-plus-metformin groups but not in the singleagent groups, with amplification failing in several lanes; these panels do not separate the treatments. Taken together, the direction of the metformin effect on ESR1 splicing is conditional on culture density and does not survive a dose-response test at high density, and HMGA1a is unlikely to be the factor that carries it.

molecular biology↗

bioRxiv2026

Herboxidiene and EGFR expression in A172 glioblastoma cells

Glioblastoma carries a five-year survival of about 10%, and EGFR amplification is one of the molecular criteria that define the disease in the 2021 WHO classification of central nervous system tumours. SF3b inhibitors, which displace the U2 snRNP from the intron branch point, suppress EGFR expression in lung adenocarcinoma cells by causing retention of EGFR intron 2. We asked whether the same route operates in glioblastoma, using A172 cells, which express wild-type EGFR together with an in-frame tandem duplication of exons 18-26. Herboxidiene inhibited A172 proliferation: DMSO-treated cultures reached 2.32 times their day 0 reading by day 3 (SD 0.61), whereas every herboxidiene concentration from 0.125 to 1 M held the cultures at or below their starting value and differed from the vehicle on days 2 and 3 (Dunnett, p < 0.05). RT-PCR across EGFR exons 2-3 showed no intron 2-retained product at any concentration; instead the spliced product decreased relative to ACTB, to 0.43 of the vehicle value at 0.125 M, without deepening further up to 1 M. EGFR protein fell, but the two forms behaved differently: the 170 kDa wild-type band was no longer detectable from 0.13 M, whereas the 190 kDa duplicated form persisted at every concentration and declined only gradually, to 0.30 of the vehicle value at 1 M. RT-PCR of CDKN1B (p27) showed retention of introns 1 and 2 at 0.5 M in A172 and in A549 lung adenocarcinoma cells. In A172 cells, therefore, herboxidiene suppresses EGFR expression without the intron 2 retention seen in lung adenocarcinoma lines, and the growth inhibition is not accounted for by loss of EGFR alone. The proliferation data are the mean and SD of three series; the RT-PCR and western blot experiments were each performed once, without replicates or statistical testing, and should be read as preliminary.

molecular biology↗

bioRxiv2026

SF3b inhibitors suppress proliferation of nonsmall cell lung cancer cells irrespective of EGFR mutation status

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are standard care for EGFR-mutant non-small cell lung cancer (NSCLC), but acquired resistance limits their durability, and alternatives that do not depend on EGFR kinase activity are needed. We asked whether inhibitors of SF3b, a core component of the U2 small nuclear ribonucleoprotein particle, act on NSCLC cells regardless of EGFR genotype. Three lung adenocarcinoma lines were treated with pladienolide B or herboxidiene at 0.01, 0.1 and 1 uM: PC9 and HCC827, both carrying an EGFR exon 19 deletion and both inhibited by gefitinib at nanomolar concentrations, and A549, which is EGFR wild type and gefitinibin sensitive. Proliferation was followed for three days by crystal violet staining, EGFR intron 2 retention was assayed by RTPCR, and EGFR protein was measured by western blotting. Both compounds produced a concentration-dependent increase in the intron 2 retained product in all three lines, and both held proliferation at or near the day 0 cell number in all three, at concentrations down to 0.01 uM. EGFR protein was lost in PC9, reduced in A549, and only partly reduced in HCC827, where a residual band persisted in lanes whose growth was fully suppressed. Since A549 is EGFR wild-type, and published knockdown data show that removing EGFR does not suppress proliferation of wild-type lines, the growth inhibition cannot be attributed to loss of EGFR alone. We read EGFR intron 2 retention as a marker of target engagement rather than as the mechanism of the growth effect. These are the results of single experiments without replicates or statistical testing and should be read as preliminary.

molecular biology↗

bioRxiv2026

Sodium butyrate inhibits the proliferation of temozolomide-resistant glioblastoma (A172) and lung adenocarcinoma (A549) cells and biphasically regulates EGFR expression

Glioblastoma carries one of the poorest prognoses of any solid tumour, and once resistance to temozolomide (TMZ) emerges there is little left to offer. Because the TMZ response is governed largely by epigenetic silencing of MGMT, histone deacetylase (HDAC) inhibitors are a rational partner for TMZ. We asked whether sodium butyrate (NaB), a short-chain fatty acid produced by the gut microbiota and a class I/IIa HDAC inhibitor, retains activity against TMZ-resistant cells. In A172 glioblastoma and A549 lung adenocarcinoma cells, NaB changed EGFR expression biphasically: EGFR exon 2-5 mRNA was induced at 0.15 mM in A172 and at 0.30 mM in A549, and was suppressed at higher concentrations, with EGFR protein following the same pattern in A172. NaB reduced proliferation of both lines from 0.31 mM in a concentration-dependent manner, more markedly in A172 than in A549. At concentrations that were inactive alone (NaB 0.30 mM, TMZ 3 {micro}M), the combination inhibited proliferation of A172 on day 3 and of A549 on days 2 and 3. TMZ-resistant sublines (A172 rTMZ, A549 rTMZ) established by stepwise exposure showed no response to TMZ 10 {micro}M alone, but NaB at 0.5 and 2.5 mM inhibited their proliferation concentration-dependently. The ratio of combination to TMZ alone was not lower in the resistant sublines than in the parental lines (A172 0.70 vs 0.86; A549 0.49 vs 0.58), indicating that the effect in resistant cells is attributable to NaB itself rather than to restored TMZ sensitivity. Millimolar exposure is the main barrier to translation.

molecular biology↗

bioRxiv2026

Decoy and antisense RNA oligonucleotides targeting the EGFR exon 3 splice site suppress proliferation of A172 glioblastoma cells

Short RNA oligonucleotides directed at the 3' splice site region of EGFR exon 3 reduced the proliferation of A172 glioblastoma cells, while the same oligonucleotides had no detectable effect on A549 lung adenocarcinoma cells. Glioblastoma carries EGFR amplification in roughly a third of IDH-wildtype cases, and the A172 line harbours a tandem duplication in the EGFR tyrosine kinase domain. Because exon definition in human genes depends on RNA-binding proteins that engage exonic sequence near splice sites, we used the SpliceAid database to identify candidate RNA-binding proteins at EGFR exons 2 and 3. Y-box binding protein 1 (YB-1, encoded by YBX1), reported to drive glioblastoma growth through its RNA-binding function, scored highly immediately adjacent to the exon 3 3' splice site. We designed four 2'-modified RNA oligonucleotides carrying a nuclear localisation signal: two antisense oligonucleotides against the exon 3 region (asRNA1, spanning the splice site; asRNA2, downstream of it), a decoy complementary to the asRNA2 target (dRNA2), and a decoy carrying the YB-1 consensus binding sequence (dYB-1). In A172 cells, all four reduced EGFR protein in a single Western blot experiment, and asRNA1, asRNA2 and dRNA2 reduced proliferation relative to scrambled RNA at days 1 to 3; dYB-1 produced a comparable reduction at day 3. None of these effects was seen in A549 cells. YB-1 protein level and subcellular distribution were unchanged by the oligonucleotides, arguing against an indirect route through altered YB-1 abundance. These observations are preliminary. Every experiment reported here was performed once: the proliferation data come from single plates with three wells per group, analysed by Dunnett's test against the scrambled-RNA control, and the Western blot, RT-PCR and immunocytochemistry panels are single runs presented as representative observations. Direct binding of YB-1 to the targeted EGFR sequence has not been demonstrated. We post this work as a preprint so that the design strategy and the initial observations are available while these questions remain open.

molecular biology↗

bioRxiv2026

Androgen receptor determines skeletal muscle sexual dimorphism

The molecular and functional bases of sexual dimorphism in skeletal muscle remain poorly understood. The androgen receptor (AR) is a major regulator of sex-biased gene expression in muscle, but its genomic targets and associated coregulators in vivo are incompletely defined. Using ChIL-seq and an AirID-AR knock-in mouse, we mapped AR-bound genes and AR-associated proteins in skeletal muscle and identified histone deacetylase-linked corepressors. We further identified myosin binding protein H (Mybph) as a female-biased AR-repressed gene conserved in mouse and human muscle. Mybph loss disrupted sarcomeric organization and selectively delayed postinjury force recovery in female mice. These findings define an in vivo AR regulatory network and identify AR-dependent Mybph repression as a potential mechanism contributing to skeletal muscle sexual dimorphism.

molecular biology↗

bioRxiv2023

Ikzf1 association with Foxp3 for Foxp3-dependent gene repression in Treg cells: induction of autoimmunity and tumor immunity by disrupting the association

The transcription factor Foxp3 specifically expressed in regulatory T (Treg) cells controls Treg function by repressing some genes and activating others. We have shown here that the transcription factor Ikzf1 associates with Foxp3 via its exon 5 (called IkE5) and that conditional deletion of IkE5 up-regulated the genes, including Ifng, normally repressed by Foxp3 upon TCR stimulation. IkE5-deletion in Treg cells indeed incurred IFN-{gamma} overproduction, which destabilized Foxp3 expression and impaired suppressive function, consequently producing fatal systemic autoimmune diseases and evoking strong anti-tumor immunity. In addition, pomalidomide, which degrades IKZF1 and IKZF3, induced IFN-{gamma} overproduction in human Treg cells. Mechanistically, the Foxp3/Ikzf1/Ikzf3 complex exerted gene-repressing function by competing with epigenetic co-activators, such as p300 and NFAT1, for binding to the target gene loci via chromatin remodeling. Collectively, the association of Ikzf1 with Foxp3 is essential for repressive function of Foxp3, and can be targeted to control autoimmunity and tumor immunity.

immunology↗

bioRxiv2022

PTEN-induced kinase 1 gene single-nucleotide variants as biomarkers in adjuvant chemotherapy for colorectal cancer

BackgroundFluoropyrimidine-based adjuvant chemotherapy is globally recommended for postoperative stage III colon cancer and high-risk stage II patients. However, adjuvant chemotherapy is often associated with severe adverse events and is not highly effective in preventing recurrence. Therefore, a recurrence-prevention biomarker of adjuvant chemotherapy for colorectal cancer is necessary for providing such treatments to appropriate patients. Autophagy (including mitophagy) is activated under chemotherapy-induced stress and contributes to chemotherapy resistance. Expression of autophagy-related genes and their single-nucleotide polymorphisms are reported to be effective predictors of chemotherapy response in some cancers. Our goal was to evaluate the relationship between the single-nucleotide variants of autophagy-related genes and recurrence rates to identify the recurrence-prevention biomarkers of adjuvant chemotherapy in colorectal cancer. MethodsWe analyzed surgical or biopsy specimens from 84 patients who underwent radical surgery followed by fluoropyrimidine-based adjuvant chemotherapy at Saitama Medical University International Medical Center between January and December 2016. Using targeted enrichment sequencing, we identified single-nucleotide variants and insertions/deletions in 50 genes, including autophagy-related genes, and examined their association with colorectal cancer patient relapse rates. ResultsWe detected 560 single-nucleotide variants or insertions/deletions in the target region. The results of Fishers exact test indicated that the recurrence rate of colorectal cancer after adjuvant chemotherapy was significantly lower in patients with the single- nucleotide variants (c.1018G>A [p < 0.005] or c.1562A>C [p < 0.01]) of the mitophagy-related gene PTEN-induced kinase 1. ConclusionsThe two single-nucleotide variants of this mitophagy-related gene may be biomarkers of non-recurrence in colorectal cancer patients who received postoperative adjuvant chemotherapy.

cancer biology↗