Biology subjects

Padilla-Benavides, T.

Publications and source records attributed to Padilla-Benavides, T..

7 recordsLinked to original sources

bioRxiv2026

A DMT1-dependent iron-endoplasmic reticulum-extracellular matrix axis regulates cancer cell invasion

Intracellular iron homeostasis is increasingly recognized as a regulator of cancer cell behavior, but how iron distribution influences extracellular matrix (ECM) organization and invasion remains poorly understood. Here, we show that loss of divalent metal transporter 1 (DMT1/SLC11A2) disrupts intracellular iron homeostasis and promotes cancer cell invasion through an iron-ER-ECM axis. In MDA-MB-231 cells, DMT1 knockout (KO) reduced total iron content but increased the labile iron pool (LIP) in both 2D and 3D culture models, indicating altered intracellular iron distribution. Across transcriptomic and phenotypic readouts, DMT1-dependent effects were more evident in 3D than in 2D models, with DMT1 KO inducing endoplasmic reticulum (ER) stress and impaired collagen/ECM organization. Functionally, the DMT1-loss phenotype was marked by reduced 2D motility, whereas in 3D spheroid models DMT1 KO cells displayed enhanced invasive outgrowth in both Matrigel and collagen matrices. Iron chelation further modulated this phenotype in a DMT1-dependent manner. Pharmacologic induction of ER stress phenocopied the loose spheroid architecture and invasive behavior, supporting ER stress as a mechanistic link between altered iron handling and ECM destabilization. Together, these findings identify intracellular iron distribution, rather than total iron abundance alone, as a determinant of ECM integrity and context-dependent cancer cell invasion. One Sentence Summary: Iron misrouting, not iron excess, drives ER stress and collagen failure that unleash invasion in triple-negative breast cancer.

cancer biology↗

bioRxiv2024

Muscle-Specific Pyruvate Kinase Isoforms, Pkm1 and Pkm2, Regulate Mammalian SWI/SNF Proteins and Histone 3 Phosphorylation During Myoblast Differentiation

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, Pkm1 and Pkm2, function in glycolysis, but Pkm2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of Pkm1 and Pkm2 during myoblast differentiation. RNA-seq analysis revealed that Pkm2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. Dpf2 and Baf250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for activation of myogenic gene expression during differentiation. Pkm2 also mediated the incorporation of Dpf2 and Baf250a into the regulatory sequences controlling myogenic gene expression. Pkm1 did not affect expression but was required for nuclear localization of Dpf2. Additionally, Pkm2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters, but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for Pkm2 and a novel function for Pkm1 in gene expression and chromatin regulation during myoblast differentiation.

molecular biology↗

bioRxiv2023

Functional effect of indole-3 carbinol in the viability and invasive properties of cultured cancer cells.

Cancer treatment typically involves multiple strategies, such as surgery, radiotherapy, and chemotherapy, to remove tumors. However, chemotherapy often causes side effects, and there is a constant search for new drugs to alleviate them. Natural compounds are a promising alternative to this problem. Indole-3-carbinol (I3C) is a natural antioxidant agent that has been studied as a potential cancer treatment. I3C is an agonist of the aryl hydrocarbon receptor (AhR), a transcription factor that plays a role in the expression of genes related to development, immunity, circadian rhythm, and cancer. In this study, we investigated the effect of I3C on cell viability, migration, invasion properties, as well as mitochondrial integrity in hepatoma, breast, and cervical cancer cell lines. We found that all tested cell lines showed impaired carcinogenic properties and alterations in mitochondrial membrane potential after treatment with I3C. These results support the potential use of I3C as a supplementary treatment for various types of cancer.

cancer biology↗

bioRxiv2019

CK2-dependent phosphorylation of the Brg1 chromatin remodeling enzyme occurs during mitosis

Brg1 (Brahma related gene 1) is one of two mutually exclusive ATPases that can act as the catalytic subunit of mammalian SWI/SNF chromatin remodeling enzymes that facilitate utilization of the DNA in eukaryotic cells. Brg1 is a phospho-protein and its activity is regulated by specific kinases and phosphatases. Previously, we showed that Brg1 interacts with and is phosphorylated by casein kinase 2 (CK2) in a manner that regulates myoblast proliferation. Here we demonstrate that the Brg1-CK2 interaction occurred during mitosis in embryonic somites and in primary myoblasts derived from satellite cells isolated from muscle tissue. The interaction of CK2 activity with Brg1 and the incorporation of a number of other subunits into the mSWI/SNF enzyme complex were independent of CK2 enzymatic activity. CK2-mediated hyperphosphorylation of Brg1 was observed in mitotic cells derived from multiple cell types and organisms, suggesting functional conservation across tissues and species. The mitotically hyperphosphorylated form of Brg1 was localized with soluble chromatin, demonstrating that CK2-mediated phosphorylation of Brg1 is associated with specific partitioning of Brg1 within sub-cellular compartments. Thus CK2 acts a mitotic kinase that regulates Brg1 phosphorylation and sub-cellular localization.\n\nHIGHLIGHTSO_LIInteractions between CK2 and the Brg1 chromatin remodeling enzyme occur during mitosis\nC_LIO_LICK2-Brg1 interactions are independent of CK2 catalytic activity\nC_LIO_LICK2-mediated phosphorylation of Brg1 is a mitotic event\nC_LIO_LICK2-mediated phosphorylation of Brg1 is conserved across mammalian cell types\nC_LIO_LIThe mitotically hyperphosphorylated form of Brg1 is localized with soluble chromatin\nC_LI

cell biology↗

bioRxiv2019

Leptin promotes expression of EMT-related transcription factors and invasion in a Src and FAK-dependent pathway in MCF10A mammary epithelial cells

Leptin is one of the main adipokines secreted in breast tissue, and has been associated with epithelial-mesenchymal transition (EMT) and tumor progression in breast cancer. Leptin promotes EMT, cell migration and invasion in epithelial breast cells, leading to tumor progression. However, the molecular mechanism that underlies these events is not fully understood; however, the activation of different signaling pathways appears to be essential. In this sense, the effect of leptin on the activation of kinases like Src and FAK, which regulate signaling pathways that activate the EMT program, has not been completely described. Therefore, we investigated the involvement of these kinases using an in vitro model for leptin-induced EMT process in the non-tumorigenic MCF10A cell line. To this end, MCF10A cells were stimulated with leptin, and Src and FAK activation was assessed. Specific events occurring during EMT were also evaluated in the presence or absence of the kinasess chemical inhibitors PP2 and PF-573228. For instance, we tested the expression and subcellular localization of the EMT-related transcription factors Twist and {square}-catenin, by western blot and immunofluorescence. We also evaluated the secretion and activation of matrix metalloproteases (MMP-2 and MMP-9) by gelatin zymography. Invasiveness properties of leptin-stimulated cells were determined by invadopodia formation assays, and by the transwell chamber method. Our results showed that leptin promotes EMT through Src and FAK activation, which leads to the secretion and activation of MMP-2 and MMP-9, invadopodia formation and cell invasion in MCF10A cells. In conclusion, our data suggest that leptin promotes an increase in the expression levels of Twist and {square}-catenin, the secretion of MMP-2, MMP-9, the invadopodia formation and invasion in MCF10A cells in a Src and FAK-dependent manner.

cell biology↗

bioRxiv2019

Calcineurin broadly regulates the initiation of skeletal muscle-specific gene expression by binding target promoters and facilitating the interaction of the SWI/SNF chromatin remodeling enzyme

Calcineurin (Cn) is a calcium-activated serine/threonine protein phosphatase that is broadly implicated in diverse cellular processes, including the regulation of gene expression. During skeletal muscle differentiation, Cn activates the NFAT transcription factor but also promotes differentiation by counteracting the negative influences of protein kinase C beta (PKC{beta}) via dephosphorylation and activation of BRG1, an enzymatic subunit of the mammalian SWI/SNF ATP-dependent chromatin remodeling enzyme. Here we identified four major temporal patterns of Cn-dependent gene expression in differentiating myoblasts and determined that Cn is broadly required for the activation of the myogenic gene expression program. Mechanistically, Cn promotes gene expression through direct binding to myogenic promoter sequences and facilitating the binding of BRG1, other SWI/SNF subunit proteins, and MyoD, a critical lineage determinant for skeletal muscle differentiation. We conclude that the Cn phosphatase directly impacts the expression of myogenic genes by promoting ATP-dependent chromatin remodeling and formation of transcription-competent promoters.

molecular biology↗

bioRxiv2019

Ouabain enhances cell-cell adhesion mediated by β1-subunits of the Na+,K+-ATPase in CHO fibroblasts.

Adhesion is an important characteristic of epithelial cells to provide a crucial barrier to pathogens and substances. In polarized epithelial cells, cell-adhesion depends on tight junctions, adherent junctions and the Na+,K+-ATPase. All these are located in the basolateral membrane of the cells. The hormone ouabain, a cardiotonic steroid, binds to the subunit of the Na+,K+-ATPase, and inhibits the pump activity when used at above M concentrations. At physiological nM concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through activation of signaling pathways associated to the subunit. Our group showed that non-adherent CHO cells transfected with the canine {beta}1 subunit become adhesive, and that homotypic interactions between {beta}1 subunits of the Na+,K+-ATPase occur between neighboring epithelial cells. Therefore, in this study we investigated whether the adhesion between {beta}1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the {beta}1 subunit of the Na+,K+-ATPase (CHO-{beta}1) and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO-{beta}1 cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the {beta}1 subunit of the Na+,K+-ATPase at the cell membrane. We also screened the effect of ouabain on activation of signaling pathways in CHO-{beta}1 cells, and their effect on cell adhesion. We found that c-Src, is activated by ouabain and is therefore likely to regulate the adhesive properties of CHO-{beta}1 cells. Collectively, our findings suggest that the {beta}1 subunits adhesion is modulated by the levels of expression and activation of the Na+,K+-ATPase at the plasma membrane, which is regulated by ouabain.

cell biology↗