Biology subjects

Hrstka, R.

Publications and source records attributed to Hrstka, R..

6 recordsLinked to original sources

bioRxiv2026

Hypoxia-conditioned HNSCC cell line secretomes drive phenotypic, functional, and transcriptional reprogramming of human neutrophils

Neutrophils display marked functional plasticity in cancer; however, it remains poorly understood how soluble factors derived from hypoxic and irradiated head and neck squamous cell carcinoma (HNSCC) cells reprogram neutrophil phenotype and function. Here, we employed a well characterized and controlled in vitro model to examine how tumor-conditioned media (TCM) from HNSCC cell lines cultured under ambient (21% O2) or hypoxic (1% O2) conditions, with or without 6 Gy gamma irradiation, modulate human neutrophil phenotype, and functional and transcriptional responses. Initial analyses were performed using TCM from three different HNSCC cell lines, whereas subsequent mechanistic characterization focused on FaDu-derived TCM. We show that TCM prolongs neutrophil survival in a cell line-dependent manner. Among the tested cell lines, hypoxia-conditioned FaDu-derived TCM promoted immunomodulatory neutrophil state characterized by enhanced survival, selective priming of ROS production, and elevated TRAIL-R3/TRAIL-R2 ratio. Induction of classical activation markers (CD11b, CD62L) was not evident. Transcriptomic analysis revealed minimal effects of normoxic TCM. Hypoxia-conditioned TCM induced a pronounced transcriptional program enriched in hypoxia- and stress-associated pathways. In contrast, irradiation of tumor cells had a limited additional impact on neutrophil reprogramming. Together, these findings indicate hypoxia-conditioned tumor secretomes as important drivers of neutrophil functional adaptation in vitro, supporting a model in which soluble factors alone are sufficient to induce a persistent, immunomodulatory neutrophil phenotype. This work provides mechanistic insight into tumor-neutrophil crosstalk, highlighting hypoxia-driven signaling as a potential therapeutic target in radioresistant HNSCC and supporting a role for neutrophil reprogramming in this context.

cancer biology↗

bioRxiv2025

HPV status and oxygen tension shape transcriptomic, inflammatory, and cell cycle responses in HNSCC treated with ionizing radiation

Head and neck squamous cell carcinoma (HNSCC) comprises biologically distinct subtypes defined by human papillomavirus (HPV) status, which may influence treatment outcomes. Hypoxia is a common feature of solid tumors, including HNSCC, and can reduce radiotherapy efficacy by modulating DNA repair, cell-cycle progression, and inflammatory signaling. However, the combined impact of hypoxia and HPV status on cellular radiosensitivity remains poorly defined. We investigated the influence of hypoxia (1% O2) and gamma-irradiation on proliferation, cell cycle, apoptosis, transcriptomic profiles, and cytokine/chemokine secretion in three HNSCC cell lines: HPV-negative FaDu and Detroit-562 (metastatic origin), and HPV-positive 2A3. Cells were cultured under chronic hypoxic or ambient conditions, with or without irradiation. Independent of HPV status or metastatic phenotype, hypoxia prolonged the cell doubling time, while only irradiated cells (under both oxygen conditions) exhibited features of an intermediate epithelial-mesenchymal transition phenotype. Treatment of HPV-positive cells significantly decreased the number of upregulated genes, reduced cytokine secretion (IL-8, SERPINE1), and affected all phases of the cell cycle compared with HPV-negative cells. Furthermore, only the metastatic cell line showed no change in cleaved caspase-3 levels after irradiation in hypoxia and produced higher levels of cytokines (IL-8, SERPINE1). Our findings highlight how tumor oxygenation and HPV status intersect to modulate the radiation response in HNSCC. These insights may guide the development of personalized, biomarker-driven radiotherapy strategies that account for both the tumor microenvironment and viral etiology.

cancer biology↗

bioRxiv2025

ALG13 loss-of-function alters glycosylation, impairs neuronal maturation, and drives network hypoactivity in a cortical organoid model of CDG

BackgroundCongenital disorders of glycosylation (CDGs) are a group of rare metabolic diseases recognized for their neurological presentations, including developmental delay and seizures. However, the link between glycosylation defects and cortical brain network pathology remains elusive. MethodsTo address this unmet need, we generated iPSC derived human cortical organoids (hCOs) for ALG13-CDG, which is the second most common CDG that is also X-linked. To comprehensively understand the impact of glycosylation defects on cortical pathology in CDG, we combined electrophysiological recordings using multi-electrode arrays (MEA) with comprehensive molecular profiling via multiomics, including scRNA-seq, proteomics, glycoproteomics, N-glycan imaging, lipidomics, and metabolomics. X-inactivation status was also evaluated in both iPSCs and organoids. ResultsALG13-CDG hCOs revealed reduced glycosylation of proteins critical for extracellular matrix (ECM), neuronal migration, lipid metabolism, calcium ion homeostasis, and neuronal excitability. Dysregulation in related pathways was corroborated by proteomics and scRNA-seq, which also showed altered communication patterns in these pathways. Trajectory analysis revealed an inversion in neuronal development, with early inhibitory and delayed excitatory development, indicating an excitatory and inhibitory (E/I) imbalance. MEA recordings demonstrated early network hypoactivity with reduced firing rates, immature burst dynamics, and shorter axonal extensions. Despite this, transcriptomic and proteomic data revealed upregulation of excitatory receptors suggesting latent hyperexcitability. Altered lipid and sugar (GlcNAc) metabolism and skewed X-inactivation were also observed. ConclusionsOur study provides the first evidence of glycosylation defects in an ALG13-CDG human cortical organoid (hCO) model and links these defects to disrupted neuronal developmental trajectories and dysregulation of key pathways essential for brain function. We identify mistimed neuronal maturation and an excitatory/inhibitory (E/I) imbalance as early drivers of network hypoactivity and immature burst dynamics, with downstream compensatory hyperexcitability that may contribute to seizure susceptibility. While specific to ALG13-CDG, these mechanisms likely extend to other glycosylation disorders with overlapping neurological features. This work offers new mechanistic insight into cortical dysfunction associated with impaired protein glycosylation and highlights potential targets for therapeutic intervention.

neuroscience↗

bioRxiv2025

PGM1 deficiency disrupts sarcomere and mitochondrial function in a stem-cell cardiomyocyte model

BackgroundPhosphoglucomutase-1 (PGM1) plays a pivotal role in glycolysis, glycogen metabolism, and glycosylation. Pathogenic variants in PGM1 cause PGM1-congenital disorder of glycosylation (PGM1-CDG), a multisystem disorder with cardiac involvement. While glycosylation abnormalities in PGM1-CDG are treatable with galactose, cardiomyopathy does not improve suggesting a glycosylation-independent pathomechanism. Recently, mitochondrial abnormalities have been shown in a heart of a PGM1-deficicient patient and PGM1-mouse model. In addition, PGM1 has been associated with LDB3 (ZASP/Cypher), a sarcomeric Z-disk protein also associated with cardiomyopathy. However, the cardiac-specific role of PGM1 remains poorly understood, and targeted therapies for PGM1-related cardiomyopathy are currently lacking. MethodsInduced pluripotent stem cell-derived cardiomyocytes (iCMs) were generated from PGM1-deficient patient fibroblasts. Multielectrode array (MEA) recordings, untargeted (glyco)proteomics, and pathway analysis were performed to assess functional and molecular changes. Key findings were validated using tracer metabolomics and mitochondrial respiration assays. ResultsPGM1-deficient iCMs exhibited reduced beating frequency, impaired contractility, and prolonged contraction kinetics. Proteomic analyses revealed depletion of Z-disk components, including LDB3. AlphaFold3 structural modeling predicted a direct interaction between PGM1 and LDB3, implicating PGM1 in Z-disk integrity, which was confirmed in vitro. In addition, mitochondrial proteins were severely depleted, prompting us to investigate mitochondrial function. Functional validation confirmed extensive metabolic rewiring, energy depletion, and severely impaired mitochondrial respiration. Finally, the in silico drug repurposing identified possible therapeutic options that could target PGM1-deficient cardiomyopathy. ConclusionPGM1 is a key regulator of cardiomyocyte function, linking sarcomeric Z-disk integrity with mitochondrial metabolism. These mechanistic insights offer a foundation for developing targeted therapies for PGM1-CDG and potentially other cardiomyopathies involving Z-disk dysfunction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=175 HEIGHT=200 SRC="FIGDIR/small/662580v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@17da449org.highwire.dtl.DTLVardef@1acb669org.highwire.dtl.DTLVardef@1fbbeecorg.highwire.dtl.DTLVardef@b38b6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Internal Translation of p53 Oncoproteins During Integrated Stress Response Confers Survival Advantage on Cancer Cells

p53 is the most known and studied tumour suppressor gene. Yet we have recently shown that p53 is also a proto-oncogene, as it encodes the {Delta}160p53 oncoprotein. Integrated stress response (ISR) is a survival pathway frequently activated in cancers, marked by the phosphorylation of eukaryotic initiation factor 2alpha (eIF2) and a defined reprogramming in mRNA translation. Here we identified ISR as a powerful trigger of p53 oncogene, leading to the induction of not only {Delta}160p53 but also {Delta}133p53, another protein variant of the p53 gene. Upon ISR the two isoforms were translated internally from p53 full-length (FL) transcript through an internal regulator of expression site (IRES) located in the vicinity of codon 160. Frameshift mutations upstream of codons 133 and 160 demonstrated that FLp53 protein synthesis is not required for making {Delta}133p53 and {Delta}160p53. Instead, targeting IRES(160) with an antisense oligo was sufficient to efficiently and specifically impair the expression of these isoforms without affecting FLp53 levels. This in turn averted ISRs protective program culminating in cancer cell cycle arrest and death. Mechanistically, FLp53 showed 3 times more affinity to {Delta}160p53 than to other isoforms, {Delta}40p53 or {Delta}133p53. During ISR {Delta}160p53 localized to the nucleus and strongly inhibited FLp53-mediated activation of pro-apoptotic gene p53 upregulated modulator of apoptosis (PUMA). Our results uncover a new branch of the ISR network essential for cancer cell survival and growth and establish the proof of concept for a new strategy to target cancer.

cancer biology↗

bioRxiv2019

Breast cancer classification based on proteotypes obtained by SWATH mass spectrometry

Accurate breast cancer classification is vital for patient management decisions, and better tumour classification is expected to enable more precise and eventually personalized treatment to improve patient outcomes. Here, we present a novel quantitative proteotyping approach based on SWATH mass spectrometry and establish key proteins for breast tumour classification derived from proteotype data. The study was based on 96 tissue samples representing five breast cancer subtypes according to conventional classification. Correlation of SWATH proteotype patterns indicated groups that largely recapitulate these subtypes. However, the proteotype-based classification also revealed varying degrees of heterogeneity within the conventional subtypes, with triple negative tumours being the most heterogeneous. Proteins that contributed most strongly to the proteotype-based classification include INPP4B, CDK1, and ERBB2, which are associated with oestrogen receptor status, tumour grade, and HER2 status, respectively. While these three key proteins exhibited high levels of correlation between protein and transcript levels (R>0.67), general correlation did not exceed R=0.29, indicating the value of protein-level measurements of biomarkers and disease-regulated genes. Overall, our data shows how large-scale protein-level measurements by next-generation proteomics can lead to improved patient stratification for precision medicine.

cancer biology↗