Biology subjects

Pujana, M. A.

Publications and source records attributed to Pujana, M. A..

8 recordsLinked to original sources

bioRxiv2026

Aurora kinase A enables collective invasion and metastasis by endowing a leader cell phenotype and stabilizing Eplin-mediated cohesion with follower cells

The metastatic process initiates with collective cell invasion into surrounding tissues and axillary nodes, and subsequent colonization at a distant site. Previously, we found collective invasion is augmented during the G2 cell cycle phase, facilitated through Aurora kinase A (AURKA)-mediated centrosome polarization in the leader cell. Here, we identify cell cycle-associated gene signatures as overrepresented in axilla and liver metastatic sites, with AURKA expression strongly correlated with breast cancer metastasis signatures, and pan-cancer patient survival. Then, we show GFP-AURKA expression endows breast epithelia cells with the ability to form metastatic outgrowths within immune-incompetent chicken embryos. Multi-parametric imaging of wound closure assays reveals phenotypes enabled by, and dependent upon AURKA expression. We discover leader cells express AURKA and acquire front-polarized centrosomes, which differentiates them from other cells in the migrating group. Ectopic expression of GFP-AURKA induces a leader cell phenotype. Conversely, inhibition of AURKA activity alters actin dynamics, promotes turnover of cell contacts, and reduces coordination within migrating groups. Specifically, AURKA interacts with the actin regulator EPLIN, and AURKA inhibition localizes EPLIN to lamellipodia and away from E-cadherin-positive contacts. Inhibiting these necessary roles for AURKA may provide a critical barrier against the metastatic spread of human breast carcinoma cells.

cancer biology↗

bioRxiv2023

A rise in double-strand breaks sensitizes tumours to oxidative metabolism inhibitors

ABSTRACTDouble strand brakes (DSB) accumulate in cellular DNA as a result of deficiencies in homologous recombination repair systems, such as mutations in BRCA genes, or upon antitumoral treatments. In the present study we show that the accumulation of DSB, regardless of its origins, leads to a shift towards oxidative metabolism. We have identified that DSB-induced reactive oxygen species (ROS) promote the activation of NRF2 which downregulates the glycolytic transcription factor HIF-1. HIF-1 inhibition is a key step in this metabolic shift, because leads to the reduction of PDHK1 levels and the consequential activation of pyruvate dehydrogenase, a mitochondrial gatekeeper of cellular metabolism, promoting this metabolic shift. Remarkably, after the induction of DSBs, the tumour is more sensitive to the inhibition of oxidative metabolism since both treatments synergize in vivo, resulting in reduced tumour growth. Therefore, we demonstrate a significant feedback between DSBs induction and cancer cell metabolism that ultimately limits the cells potential for metabolic plasticity, hence sensitizing it to the action of counteracting drugs.

cancer biology↗

bioRxiv2023

DNA double-strand break-capturing nuclear envelope tubules drive DNA repair

The nuclear envelope is a membrane separating nuclear from cytoplasmic processes. Existing models suggest that damaged DNA moves to the envelope at the edge of the nucleus for repair. Yet, most damaged human DNA does not reposition to the nuclear periphery during repair. Here we show that human cells relocate the nuclear envelope to non-peripheral damaged DNA, providing solid support promoting the reconnection of DNA break ends. Upon DNA double-strand break (DSB) induction, cytoplasmic microtubules poke the nuclear envelope inwards, inducing an extensive network of DSB-capturing nuclear envelope tubules (dsbNETs). The formation of dsbNETs, which encompass the nuclear lamina and the inner and outer nuclear membranes, depends on DNA damage response kinases, dynamic microtubules, the linker of the nucleoskeleton and cytoskeleton (LINC) proteins SUN1 and SUN2, nuclear pore protein NUP153, and kinesin KIF5B. Repressing dsbNETs compromises the reassociation of DSB ends. The timely reversal of dsbNETs by the kinesin KIFC3 also promotes repair. DSB ends reconnection is restored in dsbNETs-deficient cells by enlarging the 53BP1 DNA repair center. The lamina-binding domain of SUN1 mediates its entry into the tubules and DSB capture by the envelope. Fusing truncated SUN1 to the NHEJ repair protein KU70 fails to localize SUN1 to the tubules but rescues DSB targeting only to the boundary envelope. Although dsbNETs typically promote accurate DSB repair and cell survival, they are co-opted by the PARP inhibitor olaparib to induce aberrant chromosomes restraining BRCA1-deficient breast cancer cells. We uncover dsbNETs, which bring the nuclear envelope to DSBs for repair and potentiate the efficacy of anti-cancer agents. Our findings revise theories of the structure-function relationship of the nuclear envelope and identify dsbNETs as a critical factor in DNA repair and nuclear organization, with implications for health and disease.

molecular biology↗

bioRxiv2021

Loss of epigenetic regulation disrupts lineage integrity, reactivatesmultipotency and promotes breast cancer

Systematically investigating the scores of genes mutated in cancer and discerning real drivers from inconsequential bystanders is a prerequisite for Precision Medicine, but remains challenging. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 recurrent long-tail breast cancer genes, which revealed epigenetic regulation as major tumor suppressive mechanism. We report that core or accessory components of the COMPASS histone methylase complex including KMT2C, KDM6A, BAP1 and ASXL2 ("EpiDrivers") cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, we find that Cre-mediated activation of PIK3CAH1047R elicited an aberrant alveolar lactation program in luminal cells, which was exacerbated upon loss of EpiDrivers. Remarkably, EpiDriver loss in basal cells also triggered an alveolar-like lineage conversion and accelerated formation of luminal-like tumors, suggesting a basal origin for luminal tumors. As EpiDrivers are mutated in 39% of human breast cancers, lineage infidelity and lactation mimicry may significantly contribute to early steps of breast cancer progression. Statement of significanceInfrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In-vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of lactation genes as potential early events in tumorigenesis.

cancer biology↗

bioRxiv2021

Mammary lineage dictates homologous recombination repair and PARP inhibitor vulnerability

It has long been assumed that all normal cells have the same capacity to engage homologous recombination (HR) and non-homologous end joining (NHEJ) to repair DNA double-strand breaks (DSBs), a concept exploited for DNA-damaging chemotherapeutics. We show that mammary epithelial lineage dictates the DSB repair pathway choice. Primary mammary proteomes and DSB repair enumeration by {gamma}-H2AX, Rad51 and DNA-PKc foci reveal that NHEJ operates in all epithelial cells, but high-fidelity HR is restricted to the luminal lineage. This translates to divergent poly (ADP-ribose) polymerase inhibitor (PARPi) vulnerability of mammary epithelial progenitor activity in both mouse and human, irrespective of the BRCA1/2 status. Proteome-defined lineage-specific signatures correlate to breast cancer subtypes and predict PARPi response of triple-negative human breast cancer xenografts. These intrinsically divergent HR characteristics of mammary cell types underpin a new strategy for identifying PARPi responders.

cancer biology↗

bioRxiv2019

Mammary epithelial cells have lineage-restricted metabolic identities

Cancer metabolism adapts the metabolic network of its tissue-of-origin. However, breast cancer is not a disease of a singular origin. Multiple epithelial populations serve as the culprit cell-of-origin for specific breast cancer subtypes, yet knowledge surrounding the metabolic network of normal mammary epithelial cells is limited. Here, we show that mammary populations have cell type-specific metabolic programs. Primary human breast cell proteomes of basal, luminal progenitor, and mature luminal populations revealed their unique enrichment of metabolic proteins. Luminal progenitors had higher abundance of electron transport chain subunits and capacity for oxidative phosphorylation, whereas basal cells were more glycolytic. Targeting oxidative phosphorylation and glycolysis with inhibitors exposed distinct metabolic vulnerabilities of the mammary lineages. Computational analysis indicated that breast cancer subtypes retain metabolic features of their putative cell-of-origin. Lineage-restricted metabolic identities of normal mammary cells partly explain breast cancer metabolic heterogeneity and rationalize targeting subtype-specific metabolic vulnerabilities to advance breast cancer therapy.

cell biology↗

bioRxiv2018

Differential metabolic activity and discovery of therapeutic targets using summarized metabolic pathway models

Backgroundin spite of the increasing availability of genomic and transcriptomic data, there is still a gap between the detection of perturbations in gene expression and the understanding of their contribution to the molecular mechanisms that ultimately account for the phenotype studied. Alterations in the metabolism are behind the initiation and progression of many diseases, including cancer. The wealth of available knowledge on metabolic processes can therefore be used to derive mechanistic models that link gene expression perturbations to changes in metabolic activity that provide relevant clues on molecular mechanisms of disease and drug modes of action (MoA). In particular, pathway modules, which recapitulate the main aspects of metabolism, are especially suitable for this type of modeling.\n\nResultswe present Metabolizer, a web-based application that offers an intuitive, easy-to-use interactive interface to analyze differences in pathway module metabolic activities that can also be used for class prediction and in silico prediction of Knock-Out (KO) effects. Moreover, Metabolizer can automatically predict the optimal KO intervention for restoring a diseased phenotype. We provide different types of validations of some of the predictions made by Metabolizer.\n\nConclusionsMetabolizer is a web tool that allows understanding molecular mechanisms of disease or the MoA of drugs within the context of the metabolism by using gene expression measurements. In addition, this tool automatically suggests potential therapeutic targets for individualized therapeutic interventions.\n\nMetabolizer can be found at: http://metabolizer.babelomics.org.

bioinformatics↗

bioRxiv2018

Subjugation of TGFβ Signaling by Human Papilloma Virus in Head and Neck Squamous Cell Carcinoma Shifts DNA Repair from Homologous Recombination to Alternative End-Joining

Purpose: Following cytotoxic therapy, 70% of patients with human papillomavirus (HPV) positive oropharyngeal head and neck squamous cell carcinoma (HNSCC) are alive at 5 years compared to 30% of those with similar HPV-negative cancer, which is thought to be due to dysregulation of DNA repair. Loss of transforming growth factor {beta} (TGF{beta}) signaling is a poorly studied consequence of HPV that could contribute to this phenotype.\n\nExperimental Design: Human HNSCC cell lines (n=9), patient-derived xenografts (n=9), tissue microarray (n=194), TCGA expression data and primary tumor specimens (n=10) were used to define the relationship between TGF{beta} competency, response to DNA damage, and type of DNA repair.\n\nResults: Analysis of HNSCC specimens in situ and in vitro showed that HPV associates with loss of TGF{beta} signaling that increases the response to radiation or cisplatin. TGF{beta} suppressed miR-182 that inhibited both BRCA1, necessary for homologous recombination repair, and FOXO3, which is required for ATM kinase activity. TGF{beta} signaling blockade by either HPV or inhibitors released this control, compromised HRR and increased response to PARP inhibition. Antagonizing miR-182 rescued the homologous recombination deficit in HPV+ cells. Loss of TGF{beta} signaling unexpectedly increased error-prone, alternative end-joining repair.\n\nConclusions: HPV-positive HNSCC cells are unresponsive to TGF{beta}. Abrogated TGF{beta} signaling compromises homologous recombination and shifts reliance on alt-EJ repair that provides a mechanistic basis for sensitivity to PARP inhibitors. The effect of HPV in HNSCC provides critical validation of TGF{beta}s role in DNA repair proficiency and further raises the translational potential of TGF{beta} inhibitors in cancer therapy.

cancer biology↗