Biology subjects

Przanowski, P.

Publications and source records attributed to Przanowski, P..

6 recordsLinked to original sources

bioRxiv2026

Critical role of cell competition in gliomagenesis

ABSTRACTMalignant glioma is incurable. Using a mouse genetic mosaic system to generate sporadic Trp53,Nf1-null OPCs, we previously identified oligodendrocyte precursor cell (OPC) as a cell-of-origin of glioma. Here, we report that pre-malignant Trp53,Nf1-null OPCs outcompete wildtype counterparts during their expansion. Blocking competition by mutating/strengthening wildtype OPCs impeded both pre-malignant progression and malignant expansion of glioma. "In-tissue" phosphoproteomic profiling revealed an enrichment of phosphopeptides related to RNA splicing and protein translation at the peak of cell competition, suggesting that competitiveness may stem from unique protein species. Among candidates was mTORC1, whose pharmacological inhibition or genetic disruption resulted in a loss of competitiveness in our mouse model. Finally, analysis of patient biopsies and interrogating the role of individual gliomagenic mutations in OPC competition supported its relevance in human gliomas. Together, these findings identified the driving role of competitive interactions among OPCs in gliomagenesis, and suggest unconventional therapeutic strategies to target this process.

cancer biology↗

bioRxiv2024

Endo-reduplication in mouse liver after conditional mutation of ORC2 and combined mutation of ORC1 and ORC2

The six subunit ORC is essential for initiation of DNA replication in eukaryotes. Cancer cell-lines in culture can survive and replicate DNA replication after genetic inactivation of individual ORC subunits, ORC1, ORC2 or ORC5. In primary cells, ORC1 was dispensable in the mouse liver for endo-reduplication, but this could be explained by the ORC1 homolog, CDC6, substituting for ORC1 to restore functional ORC. Here, we have created mice with a conditional deletion of ORC2, which does not have a homolog. Although mouse embryo fibroblasts require ORC2 for proliferation, mouse hepatocytes synthesize DNA in cell culture and endo-reduplicate in vivo without ORC2. Mouse livers endo-reduplicate after simultaneous deletion of ORC1 and ORC2 both during normal development and after partial hepatectomy. Since endo-reduplication initiates DNA synthesis like normal S phase replication these results unequivocally indicate that primary cells, like cancer cell lines, can load MCM2-7 and initiate replication without ORC.

genetics↗

bioRxiv2024

Patient-derived response estimates from zero-passage organoids of luminal breast cancer

BackgroundPrimary luminal breast cancer cells lose their identity rapidly in standard tissue culture, which is problematic for testing hormone interventions and molecular pathways specific to the luminal subtype. Breast cancer organoids are thought to retain tumor characteristics better, but long-term viability of luminal-subtype cases is a persistent challenge. Our goal was to adapt short-term organoids of luminal breast cancer for parallel testing of genetic and pharmacologic perturbations. MethodsWe freshly isolated patient-derived cells from luminal tumor scrapes, miniaturized the organoid format into 5 {micro}l replicates for increased throughput, and set an endpoint of 14 days to minimize drift. Therapeutic hormone targeting was mimicked in these "zero-passage" organoids by withdrawing {beta}-estradiol and adding 4-hydroxytamoxifen. We also examined sulforaphane as an electrophilic stress and commercial neutraceutical with reported anti-cancer properties. Downstream mechanisms were tested genetically by lentiviral transduction of two complementary sgRNAs and Cas9 stabilization for the first week of organoid culture. Transcriptional changes were measured by RT-qPCR or RNA sequencing, and organoid phenotypes were quantified by serial brightfield imaging, digital image segmentation, and regression modeling of cellular doubling times. ResultsWe achieved >50% success in initiating luminal breast cancer organoids from tumor scrapes and maintaining them to the 14-day zero-passage endpoint. Success was mostly independent of clinical parameters, supporting general applicability of the approach. Abundance of ESR1 and PGR in zero-passage organoids consistently remained within the range of patient variability at the endpoint. However, responsiveness to hormone withdrawal and blockade was highly variable among luminal breast cancer cases tested. Combining sulforaphane with knockout of NQO1 (a phase II antioxidant response gene and downstream effector of sulforaphane) also yielded a breadth of organoid growth phenotypes, including growth inhibition with sulforaphane, growth promotion with NQO1 knockout, and growth antagonism when combined. ConclusionsZero-passage organoids are a rapid and scalable way to interrogate properties of luminal breast cancer cells from patient-derived material. This includes testing drug mechanisms of action in different clinical cohorts. A future goal is to relate inter-patient variability of zero-passage organoids to long-term outcomes.

cancer biology↗

bioRxiv2021

ANKLE1 cleaves mitochondrial DNA and contributes to cancer risk by driving the Warburg effect and apoptosis resistance

Alleles within the chr19p13.1 locus are associated with increased risk of both ovarian and breast cancer and increased expression of the ANKLE1 gene. ANKLE1 is molecularly characterized as an endonuclease that efficiently cuts branched DNA and shuttles between the nucleus and cytoplasm. However, the role of ANKLE1 in mammalian development and homeostasis remains unknown. In normal development ANKLE1 expression is limited to the erythroblast lineage and we found that ANKLE1s role is to cleave the mitochondrial genome during erythropoiesis. We show that ectopic expression of ANKLE1 in breast epithelial-derived cells leads to genome instability and mitochondrial DNA (mtDNA) cleavage. mtDNA degradation then leads to mitophagy and causes a shift from oxidative phosphorylation to glycolysis (Warburg effect). Moreover, mtDNA degradation activates STAT1 and expression of epithelial-mesenchymal transition (EMT) genes. Reduction in mitochondrial content contributes to apoptosis resistance, which may allow precancerous cells to avoid apoptotic checkpoints and proliferate. These findings provide evidence that ANKLE1 is the causal cancer susceptibility gene in the chr19p13.1 locus and describe mechanisms by which higher ANKLE1 expression promotes cancer risk.

cancer biology↗

bioRxiv2021

Distinct MUNC lncRNA structural domains regulate transcription of different promyogenic factors

Many lncRNAs have been discovered using transcriptomic data, however, it is broadly unclear what fraction of lncRNAs are functional and what structural properties affect their phenotype. MUNC lncRNA, also known as DRReRNA, stimulates skeletal muscle differentiation. The prevailing hypothesis is that MUNC stimulates the Myod1 gene in cis as an enhancer RNA and stimulates expression of several other promyogenic genes in trans by recruiting the cohesin complex to their promoters. Experimental probing of the RNA structure revealed that MUNC contains multiple structural domains not detected by RNA structure prediction algorithms in the absence of experimental information. We discovered that these specific and structurally distinct domains are required for induction of different promyogenic genes, for binding at different genomic sites to regulate the expression of adjacent genes, and for binding the cohesin complex. Moreover, we found that induction of Myod1 or interaction with cohesin comprise only a subset of the broad regulatory impact of this lncRNA. Our study thus reveals unexpectedly complex, structure-driven functions for the MUNC lncRNA and emphasizes the importance of experimentally determined structures for understanding structure-function relationships in lncRNAs.

molecular biology↗

bioRxiv2019

miR-206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro

miR-206, miR-1a-1 and miR-1a-2 are induced during differentiation of skeletal myoblasts and promote myogenesis in vitro. miR-206 is required for skeletal muscle regeneration in vivo. Although this microRNA family is hypothesized to play an essential role in differentiation, a triple knockout of the three genes has not been done to test this hypothesis. We report that triple KO C2C12 myoblasts generated using CRISPR/Cas9 method differentiate despite the expected de-repression of the microRNA targets. Surprisingly, their mitochondrial function is diminished. Triple KO mice demonstrate partial embryonic lethality, most likely due to the role of miR-1a in cardiac muscle differentiation. Two triple KO mice survive and grow normally to adulthood with smaller myofiber diameter and diminished physical performance. Thus, unlike other microRNAs important in other differentiation pathways, the miR-206 family is not absolutely essential for myogenesis and is instead a modulator of optimal differentiation of skeletal myoblasts.

molecular biology↗