Biology subjects

Prochownik, E. V.

Publications and source records attributed to Prochownik, E. V..

12 recordsLinked to original sources

bioRxiv2026

MYC and RNA Polymerase II Binding Near Transcriptional End Sites Regulate the Expression of Functionally-Related Genes

MYC oncoprotein binding at promoters and enhancers influences RNA polymerase II (RNAPII)-driven gene expression. Numerous genes also bind MYC near their transcriptional end sites (TESs). This often allows direct promoter-TES contact via looping and further regulates total and "read-through" transcription that extends beyond standard termination sites. We aimed here to better clarify the rules governing TES-associated MYC and/or RNAPII binding cross-talk in human and murine cells. Using ChIPseq and RNAseq datasets from the ENCODE portal and elsewhere, MYC and RNAPII binding profiles were found to differ around TESs and transcriptional start sites (TSSs). Variations in E box flanking sequences likely accounted for the somewhat lower affinities of MYC for TES-associated sites. Motifs for numerous other transcription factors were also observed to cluster non-randomly and in close proximity to MYC and RNAPII binding site peak summits. On average, genes with TES-proximal MYC or RNAPII sites were more highly expressed than those without, although co-binding tended to be suppressive. Both normal and neoplastic proliferative stimuli altered the MYC and RNAPII binding patterns of many genes, indicating that "category switching" was common, subject to disparate external signals and often reversible. Functionally related gene sets with high levels of read-through transcription were uniformly marked by significant amounts of TES-associated MYC and/or RNAPII binding. These findings indicate that, both independently and together, MYC and RNAPII binding near TESs dynamically impact total and read-through transcription while also coordinating the expression of many common purpose gene sets. Significance StatementBinding of the MYC oncoprotein around transcriptional end sites (TESs) of genes can influence both total and "readthrough" transcription that extends beyond normal termination points. We have now studied how RNA polymerase II (RNAPII) cross-talks with MYC to regulate these processes and their consequences. We found that binding of these factors around TESs cooperates with their binding at transcriptional start sites (TSSs) and increases both absolute and read-through transcription. MYC and/or RNAPII binding around TSSs and TESs is often transient and reversible in response to both normal and neoplastic growth-promoting stimuli. TES-associated MYC and/or RNAPII binding thus not only influence the quantity and quality of gene expression but also mark gene subsets that participate in common pathways and functions.

bioinformatics↗

bioRxiv2025

Reversible Dissociation of Mitochondrial Complex V Balances Anabolic and Energy-Generating Needs in Cancer

Cancer cell metabolic re-programming provides the excess energy and anabolic precursors necessary to sustain uncontrolled growth. This is partly mediated by the Warburg effect, whereby glucose is converted into ATP and a subset of these anabolic substrates. Concurrently, mitochondrial mass and ATP production decline in most tumors. This raises the question of how increased supplies of glycolysis-derived anabolic substrates can be balanced with those generated by the TCA cycle. Using primary murine liver cancers and cell lines, we show that this can be explained by the dissociation of mitochondrial Complex V (CV or ATP synthase) into its component and functionally-independent Fo and F1 domains. This occurs as a result of marked reductions in MT-ATP6, a CV subunit that stabilizes the Fo-F1 association. Serving as a proton pore, Fo maintains a normal mitochondrial membrane potential without generating ATP, thus allowing the TCA cycle, electron transport chain and anaplerotic reactions to function at high levels. Concurrently, free F1 functions as an ATPase to prevent excessive ATP accumulation. The uncoupling of TCA cycle-derived anabolic substrate production from membrane hyperpolarization and ATP synthesis by a smaller population of more efficient mitochondria allows TCA cycle-generated anabolic precursors to match those generated via glycolysis.

cancer biology↗

bioRxiv2025

Derivation of Genetically-Defined Murine Hepatoblastoma Cell Lines with Angiogenic Potential

Background/ObjectivesHepatoblastoma (HB) is the most common form of pediatric liver cancer, with the vast majority of these tumors evidence of mutation and/or deregulation of the oncogenic transcription factors {beta}-catenin (B), YAP (Y) and NRF2 (N). HB research has been hampered by a paucity of established cell lines, particularly those bearing these molecular drivers. All combinations of B, Y and N (i.e. BY, BN, YN and BYN) are tumorigenic when over-expressed in murine livers but it has not been possible to establish cell lines from primary tumors. Recently, we found that concurrent Crispr-mediate targeting of the Cdkn2a tumor suppressor locus allows for such immor-talized cell lines to be generated with high fidelity. MethodsWe generated 5 immortalized cell lines from primary Cdkn2a-targeted BN and YN HBs and characterized their properties. Notably, 4 of the 5 retain their ability to grow as subcutaneous or pulmonary tumors in the immune-competent mice from which they originated. Most notably, when maintained under hypoxia conditions for as little as 2 days, BN cells reversibly up-regulated the expression of numerous endothelial cell (EC)-specific genes and ac-quired EC-like properties that benefited tumor growth. ConclusionsThe above approach is currently the only means by which HB cell lines with pre-selected, clinically relevant oncogenic drivers can be generated and the only ones that can be studied in immune-competent mice. Its generic nature should allow HB cell lines with other oncogenic drivers to be derived. A collection of such cell lines will be useful for studying tumor cell-EC trans-differentiation, interactions with the immune environment and drug sensitivities. Simple SummaryMost hepatoblastomas (HB) are associated with aberrant expression of {beta}-catenin (B), YAP (Y) and/or NRF2 (N) transcription factors and can be modeled in mice by over-expressing pairwise of triple combination of these. Virtually no human or murine HB cell lines exist that bear these mutations. We describe here an efficient way to generate cell lines from primary BN and YN tumors. Moreover, one of the BN lines shows a remarkable ability to trans-differentiate into endothelial cells under hypoxic conditions that may facilitate angiogenesis. These cell lines along with previousl-derived BN and BYN lines showed similar sensitivities to drugs commonly used to treat HB. Because the approach for cell line derivation we describe is quite general, it should allow for the generation of additional lines driven by less common factors. A collection of such permanent and well-characterized cell lines will facilitate studies that are difficult or impractical to perform in vivo.

cancer biology↗

bioRxiv2024

Gas1-Mediated Suppression of Hepatoblastoma Tumorigenesis

Background and AimsHepatoblastoma (HB), the most common pediatric liver cancer, often dysregulates the Wnt/{beta}-catenin, Hippo and NFE2L2/NRF2 pathways. Pairwise combinations of oncogenically active forms of the terminal transcription factor effectors of these pathways, namely {beta}-catenin (B), YAP (Y) and NRF2 (N) generate HBs in mice, with the triple combination (B+Y+N) being particularly potent. Each tumor group alters the expression of thousands of B-,Y- and N-driven unique and common target genes. Identifying those most responsible for transformation is thus an important question as it might reveal new mechanistic insights and therapeutic opportunities. Approach and ResultsTranscriptional profiling of >60 murine HBs driven by the above oncogenic combinations and different B mutants and in genetic backgrounds that impair tumor growth rates but not initiation has revealed a common set of 22 "BYN genes" that are similarly deregulated in all cases. Many are associated with multiple "Cancer Hallmarks" and their expression levels often correlate with survival in human HBs, hepatocellular carcinomas and other cancers. Among the most down-regulated of these is Gas1, which encodes a Glycosylphosphatidylinositol (GPI)-linked outer membrane protein. We show here that restoring Gas1 expression impairs B+Y+N-driven HB tumor growth in vivo and in HB-derived immortalized cell lines in vitro in a manner than requires membrane anchoring of the protein via its GPI moiety. ConclusionsOur findings implicate Gas1 as a proximal mediator of HB pathogenesis and validate the BYN gene set as one deserving of closer additional scrutiny in future studies.

cancer biology↗

bioRxiv2024

Efficient Derivation of Immortalized, Isogenic Cell Lines from Genetically Defined Murine Hepatoblastomas

Background & AimsMolecularly, hepatoblastoma (HB), the most common childhood liver cancer, is the simplest of all human neoplasms, with the vast majority deregulating the Wnt/{beta}-catenin, Hippo and/or NFE2/NRF2 signaling pathways. Murine HBs can be generated by over-expressing any pairwise or triple combination of mutant forms of these pathways terminal effectors, namely {beta}-catenin (B), YAP (Y) and NFE2L2/NRF (N). Each molecular subtypes displays distinct features resembling those of human HBs. However, research has been hampered by a paucity of established cell lines of any species. MethodsWe show here that immortalized cell lines can be routinely established from murine HBs that over-express B+Y and B+Y+N. This is facilitated by the concurrent in vivo, Crispr-mediated inactivation of the Cdkn2a tumor suppressor locus. ResultsEight BY and 3 BYN cell lines have been generated and characterized and are available to the HB research community. Ten of these lines can be regrown as subcutaneous and metastatic lung tumors in the immuno-competent mice from which they originated while retaining their original histologic features. During maintenance as spheroids in vitro, or during in vivo propagation, tumor cells express endothelial cell markers, particularly in regions that are hypoxic and/or in proximity to incipient blood vessels. ConclusionsThe ability to generate isogenic HB cell lines with defined oncogenic drivers should facilitate studies that are best performed in vitro. The approach may also be useful for deriving HB cell lines associated with less common molecular drivers and from human tumors. SynopsisThe derivation of multiple immortalized murine hepatoblastoma cell lines driven by defined oncogenes is described. These lines are isogenic, retain their tumorigenicity in immuno-competent mice, readily form spheroids and express endothelial markers in response to hypoxia. They will allow studies that have heretofore been difficult or impossible to perform in vivo.

cancer biology↗

bioRxiv2024

MYC Binding Near Transcriptional End Sites Regulates Basal Gene Expression, Read-Through Transcription and Intragenic Contacts

The MYC oncoprotein regulates numerous genes involved in cellular processes such as cell cycle and mitochondrial and ribosomal structure and function. This requires heterodimerization with its partner, MAX, and binding to specific promoter and enhancer elements. Here, we show that MYC and MAX also bind near transcriptional end sites (TESs) of over one-sixth of all annotated genes. These interactions are dose-dependent, evolutionarily conserved, stabilize the normally short-lived MYC protein and regulate expression both in concert with and independent of MYCs binding elsewhere. MYCs TES binding occurs in association with other transcription factors, alters the chromatin landscape, increases nuclease susceptibility and can alter transcriptional read-through, particularly in response to certain stresses. MYC-bound TESs can directly contact promoters and may fine-tune gene expression in response to both physiologic and pathologic stimuli. Collectively, these findings support a previously unrecognized role for MYC in regulating transcription and its read-through via direct intragenic contacts between TESs and promoters.

molecular biology↗

bioRxiv2023

The Myc-Like Mlx Network Impacts Aging and Metabolism

The "Mlx" and "Myc" Networks share many common gene targets. Just as Mycs activity depends upon its heterodimerization with Max, the Mlx Network requires that the Max-like factor Mlx associate with the Myc-like factors MondoA or ChREBP. We show here that body-wide Mlx inactivation, like that of Myc, accelerates numerous aging-related phenotypes pertaining to body habitus and metabolism. The deregulation of numerous aging-related Myc target gene sets is also accelerated. Among other functions, these gene sets often regulate ribosomal and mitochondrial structure and function, genomic stability and aging. Whereas "MycKO" mice have an extended lifespan because of a lower cancer incidence, "MlxKO" mice have normal lifespans and a somewhat higher cancer incidence. Like Myc, Mlx, MondoA and ChREBP expression and that of their target genes, deteriorate with age in both mice and humans, underscoring the importance of life-long and balanced cross-talk between the two Networks to maintain normal aging. TeaserInactivation of the Myc-like "Mlx Network" in mice leads to phenotypic and molecular signs of premature aging and a cancer predisposition.

cancer biology↗

bioRxiv2023

Premature Aging and Reduced Cancer Incidence Associated with Body-Wide Loss of Myc

MYC proto-oncogene dysregulation alters metabolism, translation and other functions in ways that support tumor induction and maintenance. Although Myc+/- mice are healthier and longer-lived than control mice, the long-term ramifications of more complete Myc loss remain unknown. We now describe the chronic consequences of body-wide Myc inactivation initiated postnatally. "MycKO" mice acquire numerous features of premature aging including altered body composition and habitus, metabolic dysfunction, hepatic steatosis and the dysregulation of numerous gene sets involved in functions that normally deteriorate with aging. Yet, MycKO mice have extended life spans that correlate with a 3-4-fold lower lifetime cancer incidence. Aging tissues from normal mice and humans also down-regulate Myc and gradually deregulate many of the same Myc target gene sets that are dysregulated in MycKO mice. Normal aging and its associated cancer predisposition are thus highly linked via Myc and its target genes and can be genetically separated.

cancer biology↗

bioRxiv2021

Coordinated Cross-Talk Between the Myc and Mlx Networks in Liver Regeneration and Neoplasia

Background & AimsThe c-Myc (Myc) bHLH-ZIP transcription factor is deregulated in most cancers. In association with Max, Myc controls target genes that supervise metabolism, ribosome biogenesis, translation and proliferation. This "Myc Network" cross-talks with the "Mlx Network", which consists of the Myc-like proteins MondoA and ChREBP and Max-like Mlx. Together, this "Extended Myc Network" regulates both common and distinct genes targets. Here we studied the consequence of Myc and/or Mlx ablation in the liver, particularly those pertaining to hepatocyte proliferation, metabolism and spontaneous tumorigenesis. MethodsWe examined the ability of hepatocytes lacking Mlx (MlxKO) or Myc+Mlx (double KO or DKO) to repopulate the liver over an extended period of time in a murine model of Type I tyrosinemia. We also compared this and other relevant behaviors, phenotypes and transcriptomes of the livers to those from previously characterized MycKO, ChrebpKO and MycKO x ChrebpKO mice. ResultsHepatocyte regenerative potential deteriorated as the Extended Myc Network was progressively dismantled. Genes and pathways dysregulated in MlxKO and DKO hepatocytes included those pertaining to translation, mitochondrial function and non-alcoholic fatty liver disease (NAFLD). The Myc and Mlx Networks were shown to cross-talk, with the latter playing a disproportionate role in target gene regulation. All cohorts also developed NAFLD and molecular evidence of early steatohepatitis. Finally, MlxKO and DKO mice displayed extensive hepatic adenomatosis. ConclusionsIn addition to demonstrating cooperation between the Myc and Mlx Networks, this study revealed the latter to be more important in maintaining proliferative, metabolic and translational homeostasis, while concurrently serving as a suppressor of benign tumorigenesis. SynopsisThe Myc and Mlx Networks exhibit extensive cross-talk and regulate distinct but overlapping sets of transcriptional targets. The current work demonstrates the cooperation between these two Networks in supporting the regenerative capabilities of normal hepatocytes while also revealing that the Mlx Network serves as a suppressor of spontaneous hepatic adenomatosis

cancer biology↗

bioRxiv2020

Patient-Derived Mutant Forms of NFE2L2/NRF2 Drive Aggressive Murine Hepatoblastomas

Background and AimsHepatoblastoma (HB), the most common pediatric liver cancer, often bears {beta}-catenin mutations and deregulates the Hippo tumor suppressor pathway. Murine HBs can be generated by co-expressing {beta}-catenin mutants and the constitutively active Hippo effector YAPS127A. Some HBs and other cancers also express mutants of NFE2L2/NRF2 (NFE2L2), a transcription factor that tempers oxidative and electrophilic stress. In doing so, NFE2L2 either suppresses or facilitates tumorigenesis. MethodsWe evaluated NFE2L2s role in HB pathogenesis by co-expressing all combinations of mutant {beta}-catenin, YAPS127A and the patient-derived NFE2L2 mutants L30P and R34P in murine livers. We evaluated growth, biochemical and metabolic profiles and transcriptomes of the ensuing tumors. ResultsIn association with {beta}-catenin+YAPS127A, L30P and R34P markedly accelerated HB growth and generated widespread cyst formation and necrosis, which are otherwise uncommon features. Surprisingly, any two members of the mutant {beta}-catenin-YAPS127A-L30P/R34P triad were tumorigenic, thus directly establishing NFE2L2s oncogenicity. Each tumor group displayed distinct features but shared 22 similarly deregulated transcripts, 10 of which perfectly correlated with survival in human HBs and 17 of which correlated with survival in multiple adult cancers. One highly up-regulated transcript encoded serpin E1, a serine protease inhibitor that regulates fibrinolysis, growth and extracellular matrix. The combination of mutant {beta}-catenin, YAPS127A and Serpin E1, while not accelerating cystogenic tumor growth, did promote the wide-spread necrosis associated with mutant {beta}-catenin-YAPS127A-L30P/R34P tumors. ConclusionsOur findings establish the direct oncogenicity of NFE2L2 mutants and key transcripts, including serpin E1, that drive specific HB features.

cancer biology↗

bioRxiv2020

Acquired deficiency of the peroxisomal enzyme enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase is a metabolic vulnerability in hepatoblastoma

Metabolic reprogramming provides transformed cells with proliferative and/or survival advantages. However, capitalizing on this therapeutically has been only moderately successful due to the relatively small magnitude of these differences and because cancers may re-program their metabolism to evade metabolic pathway inhibition. Mice lacking the peroxisomal bi-functional enzyme enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) and supplemented with the 12-carbon fatty acid lauric acid (C12) accumulate dodecanedioic acid (DDDA), a toxic C12 metabolite that causes hepatocyte necrosis and acute liver failure. In a murine model of pediatric hepatoblastoma (HB), down-regulation of Ehhadh also occurs in combination with a more general suppression of mitochondrial {beta}- and peroxisomal {omega}-fatty acid oxidation (FAO) pathways. HB-bearing mice provided with C12 and/or DDDA-supplemented diets survived significantly longer than those on standard diets. The tumors also developed massive necrosis in response to short-term DDDA supplementation. Reduced Ehhadh was noted in murine hepatocellular carcinomas (HCCs) and in substantial subsets of human cancers, including HCCs. Acquired DDDA resistance was not associated with Ehhadh re-expression but was associated with 129 transcript differences ~90% of which were down-regulated in DDDA-resistant tumors and ~two-thirds of which correlated with survival in several human cancers. These transcripts often encoded components of the extracellular matrix suggesting that DDDA resistance arises from its reduced intracellular transport. Our results demonstrate the feasibility of a metabolic intervention that is non-toxic, inexpensive and likely compatible with traditional therapies. C12 and/or DDDA-containing diets could potentially be used to supplement other treatments or as alternative therapeutic choices.

cancer biology↗

bioRxiv2017

Diagnostic and Prognostic Implications of Ribosomal Protein Transcript Coordination in Human Cancers

Ribosomes, the organelles responsible for the translation of mRNA, are comprised of rRNA and ~80 ribosomal proteins (RPs). Although canonically assumed to be maintained in equivalent proportions, some RPs have been shown to possess differential expression across tissue types. Dysregulation of RP expression occurs in a variety of human diseases, notably in many cancers, and altered expression of some RPs correlates with different tumor phenotypes and patient survival. To investigate the impact of global RP transcript (RPT) expression patterns on tumor phenotypes, we analyzed RPT expression of ~10,000 human tumors and 700 normal tissues with t-distributed stochastic neighbor embedding (t-SNE). We show here that normal tissues and cancers possess readily discernible RPT expression patterns. In tumors, this patterning is distinct from normal tissues, distinguishes tumor subtypes from one another, and in many cases correlates with molecular, pathological, and clinical features, including survival. Collectively, RPT expression can be used as a powerful and novel method of tumor classification, offering a potential clinical tool for prognosis and therapeutic stratification.

cancer biology↗