Adele G. Woolley

Mutations in the tumour suppressor, TP53 , are prevalent in human cancers yet their functional implications remain unclear. Through comprehensive pan-cancer database analysis, we identified H179 mutations in TP53 as significantly associated with poor disease-free survival across multiple cancer types. Functional studies in lung, ovarian, and prostate cancer cells over-expressing the p53 ^H179R/Y mutants revealed that these mutants are not only defective in tumour-suppressive functions but also actively promote tumour progression. These mutants drive metabolic reprogramming by increasing neutral lipid levels through de novo fatty acid synthesis (p53 ^H179R/Y ) and fatty acid uptake (p53 ^H179Y ), accompanied by increased lipid droplets and APOE expression, collectively promoting enhanced invasiveness relative to p53-null cells. Our findings demonstrate that TP53 missense mutations are not functionally equivalent to TP53 loss. Notably, H179 mutations act as oncogenic drivers, and our data highlight APOE and lipid metabolism as potential therapeutic targets, underscoring the clinical relevance of precise TP53 mutation characterisation.

Mutations in the tumor suppressor, TP53, are prevalent in human cancers; yet their functional implications remain unclear. Through comprehensive pan-cancer database analysis, we identified H179 mutations in TP53 as significantly associated with poor disease-free survival across multiple cancer types. Functional studies in lung, ovarian, and prostate cancer cells overexpressing the p53^H179R/Y mutants revealed that these mutants are not only defective in tumor-suppressive functions but also actively promote tumor progression. These mutants induce metabolic reprogramming by increasing neutral lipid levels, lipid droplet formation, and the expression of apolipoprotein E (APOE), leading to enhanced invasiveness in 3D collagen models compared to p53 null cells. Moreover, APOE levels were also elevated in patients whose tumor had mutations at codon 179 of TP53. These findings underscore the critical role of TP53 codon 179 mutations in driving cancer progression and highlight potential therapeutic targets for mitigating their impact.

When a tooth is diseased or damaged through caries, bioactive molecules are liberated from the pulp and dentin as part of the natural response to injury and these are key molecules for stimulating stem cell responses for tissue repair. Incorporation of these extracellular‐matrix (ECM)‐derived molecules into a hydrogel model can mimic in vivo conditions to enable dentin–pulp complex regeneration. Here, a chitosan/alginate (C/A) hydrogel is developed to sequester bovine ECM extracts. Human dental pulp cells (hDPCs) are cultured with these constructs and proliferation and cytotoxicity assays confirm that these C/A hydrogels are bioactive. Sequential z‐axis fluorescent imaging visualizes hDPCs protruding into the hydrogel as it degraded. Alizarin red S staining shows that hDPCs cultured with the hydrogels display increased calcium‐ion deposition, with dentin ECM stimulating the highest levels. Alkaline phosphatase activity is increased, as is expression of transforming growth factor‐beta as demonstrated using immunocytochemistry. Directional analysis following phase contrast kinetic image capture demonstrates that both dentin and pulp ECM molecules act as chemoattractants for hDPCs. Data from this study demonstrate that purified ECM from dental pulp and dentin when delivered in a C/A hydrogel stimulates dental tissue repair processes in vitro.

The initiation of CD8⁺ T cell responses against dead cell–associated Ags is tightly regulated, facilitating adaptive immunity against pathogens and tumors while preventing autoimmunity. It is now well established that dying cells actively regulate the generation of CD8⁺ T cell responses via the release or exposure of damage-associated molecular patterns. However, it is unclear whether nonproteasomal proteases (activated in stressed and dying cells) can influence the availability of Ags for cross-presentation. Using a mouse model of immunogenic necrosis, we investigated the role of tumor-derived proteases in the priming of CD8⁺ T cells. We demonstrate that proteases released from necrotic tumor cells can degrade whole-protein Ag, generating proteolytic intermediates that are efficiently cross-presented by dendritic cells and enhance CD8⁺ T cell cross-priming. We identify a dominant role for calpain proteases, which are activated during necrotic cell death induced by severe heat shock. Mechanistically, proteolytic intermediates generated by tumor-derived proteases associate with necrotic tumor cell debris, which acts as a vehicle for Ag transfer that facilitates highly efficient cross-presentation in dendritic cells. Our results suggest that proteolytic systems activated in Ag donor cells during cell death may influence the availability of antigenic substrates for cross-presentation, thereby regulating the antigenicity of cell death. Visual Abstract Key Points Severe heat shock enhances the immunogenicity of necrotic tumor cells. Severe heat shock promotes cross-presentation, but not increased adjuvanticity or phagocytosis. Enhanced cross-presentation is mediated by tumor-derived calpains.

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, β-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division.

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and cancer progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cell lines resulted in cytokinesis failure, multinucleation and an increase in G ₁ transit time. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging of cells undergoing cytokinesis both in vitro and in zebrafish embryos, we found that YB-1 was critical for microtubule organization during cytokinesis. Using mass spectrometry we identified multiple novel phosphorylation sites on YB-1. We show that phosphorylation of YB-1 at multiple serine residues was essential for its function during cytokinesis. Using atomistic modelling we show how multiple phosphorylations alter YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining for the first time precisely how YB-1 regulates cell division. SUMMARY: Y-box-binding protein-1, YB-1, is essential for cell division, but it is not clear how it functions. Using live imaging and confocal microscopy we show that YB-1 functions only in the last step of division, specifically being required to initiate cytokinesis.

Elevated levels of nuclear Y-box binding protein 1 (YB-1) are linked to poor prognosis in cancer. It has been proposed that entry into the nucleus requires specific proteasomal cleavage. However, evidence for cleavage is contradictory and high YB-1 levels are prognostic regardless of cellular location. Here, using confocal microscopy and mass spectrometry, we find no evidence of specific proteolytic cleavage. Doxorubicin treatment, and the resultant G₂ arrest, leads to a significant increase in the number of cells where YB-1 is not found in the cytoplasm, suggesting that its cellular localisation is variable during the cell cycle. Live cell imaging reveals that the location of YB-1 is linked to progression through the cell cycle. Primarily perinuclear during G₁ and S phases, YB-1 enters the nucleus as cells transition through late G₂/M and exits at the completion of mitosis. Atomistic modelling and molecular dynamics simulations show that dephosphorylation of YB-1 at serine residues 102, 165 and 176 increases the accessibility of the nuclear localisation signal (NLS). We propose that this conformational change facilitates nuclear entry during late G₂/M. Thus, the phosphorylation status of YB-1 determines its cellular location.

GLI pathogenesis-related 1 (GLIPR1) was previously identified as an epigenetically regulated tumor suppressor in prostate cancer and, conversely, an oncoprotein in glioma. More recently, GLIPR1 was shown to be differentially expressed in other cancers including ovarian, acute myeloid leukemia, and Wilms’ tumor. Here we investigated GLIPR1 expression in metastatic melanoma cell lines and tissue. GLIPR1 was variably expressed in metastatic melanoma cells, and transcript levels correlated with degree of GLIPR1 promoter methylation in vitro. Elevated GLIPR1 levels were correlated with increased invasive potential, and siRNA-mediated knockdown of GLIPR1 expression resulted in reduced cell migration and proliferation in vitro. Immunohistochemical studies of melanoma tissue microarrays showed moderate to high staining for GLIPR1 in 50% of specimens analyzed. GLIPR1 staining was observed in normal skin in merocrine sweat glands, sebaceous glands, and hair follicles within the dermis.

The literature concerning the subcellular location of Y-box binding protein 1 (YB-1), its abundance in normal and cancer tissues, and its prognostic significance is replete with inconsistencies. An explanation for this could be due in part to the use of different antibodies in immunohistochemical and immunofluorescent labeling of cells and tissues. The inconsistencies could also be due to poor resolution of immunohistochemical data. We analyzed two cohorts of breast tumours for both abundance and subcellular location of YB-1 using three different antibodies; two targeting N-terminal epitopes (AB-a and AB-b) and another (AB-c) targeting a C-terminal epitope. We also investigated stress-induced nuclear translocation of YB-1 in cell culture. We report that both AB-a and AB-c detected increased YB-1 in the cytoplasm of high-grade breast cancers, and in those lacking estrogen and progesterone receptors; however the amount of YB-1 detected by AB-a in these cancers is significantly greater than that detected by AB-c. We confirm our previously published findings that AB-b is also detecting hnRNP A1, and cannot therefore be used to reliably detect YB-1 by immunohistochemistry. We also report that AB-a detected nuclear YB-1 in some tumour tissues and stress treated cells, whereas AB-c did not. To understand this, cancer cell lines were analyzed using native gel electrophoresis, which revealed that the antibodies detect different complexes in which YB-1 is a component. Our data suggest that different YB-1 antibodies show different staining patterns that are determined by the accessibility of epitopes, and this depends on the nature of the YB-1 complexes. It is important therefore to standardize the protocols if YB-1 is to be used reproducibly as a prognostic guide for different cancers.