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Glioblastoma is the most aggressive primary brain tumor in adults. Androgens are reported to influence the development of glioblastoma. Dihydrotestosterone (DHT) is formed from testosterone by action of the enzyme steroid 5α-reductase and is the most potent growth-inducing androgen metabolite. 24-Hydroxycholesterol, another steroid in the brain, is pivotal for brain cholesterol homeostasis and has been suggested to influence glioblastoma cells. However, a connection between 24-hydroxycholesterol and androgen metabolism related to glioblastoma has not previously been reported. The present study reports that human T98G glioblastoma cells metabolize testosterone into DHT, 3αandrostanediol and androstenedione. The 5α-reductase pathway converted testosterone to DHT and further to 3α-androstanediol. The 17β-hydroxysteroid dehydrogenase pathway metabolized testosterone to androstenedione. Results indicated that the 5α-reductase pathway is the major pathway for testosterone metabolism in this cell line. 24-Hydroxycholesterol significantly suppressed the conversion of testosterone to DHT and 3α-androstanediol, to a similar degree as the synthetic 5α-reductase inhibitor finasteride. Suppression of DHT formation resulted in increased metabolism to androstenedione. Similar effects on DHT formation were observed with the LXR agonist T0901317 as with 24-hydroxycho-lesterol. In addition, 24-hydroxycholesterol suppressed DHT formation in patient-derived primary GB cell lines U3009 and U3013, indicating that the observed connection between 24-hydroxycholesterol and androgen metabolism is not unique for T98G cells. Furthermore, 24-hydroxycholesterol-mediated suppression of DHT formation was also observed in human neuroblastoma SH-SY5Y cells. To summarize, the present data provide information on androgen metabolism in glioblastoma cells and indicate a previously unknown link between cholesterol homeostasis and growth-inducing androgens in glioblastoma and potentially other cell types.

Background: Increased stromal Platelet-derived growth factor receptor beta (PDGFRβ) expression is a hallmark of the desmoplastic tissue reaction in cancer and marks subsets of cancer-associated fibroblasts, pericytes, and smooth muscle cells. However, its functional status in situ has been anticipated from static expression measures, which cannot determine whether high receptor abundance reflects active signaling.

Methods: We established two second-generation proximity ligation assays (PLAs) to quantify PDGFRβ activation in the in situ environment of human lung cancer by detecting either phosphorylated PDGFRβ or its interaction with the adaptor protein Grb2. The immunofluorescence-based assays were applied to tissue-microarrays including diagnostic samples from over 600 non-small cell lung cancer (NSCLC) patients.

Results: In lung cancer tissue, activation scores correlated with PDGFRβ expression but revealed a more nuanced receptor status, indicating variable activation despite similar expression levels. Higher PDGFRβ activation was associated with increased recurrence risk exclusively in squamous cell carcinoma, a finding not captured by conventional immunohistochemistry. This activation was accompanied by a specific stromal profile enriched for LRRC15- and FAP-positive cells, a pattern absent in adenocarcinomas.

Conclusion: PDGFRβ activation status provides functional information beyond receptor expression, uncovering clinically relevant, otherwise overlooked, stromal phenotypes. The approach illustrates the diagnostic potential of functional protein assays in the era of precision medicine.

The active form of vitamin D, 1,25-dihydroxyvitamin D₃, is known to act via VDR (vitamin D receptor), affecting several physiological processes. In addition, PDIA3 (protein disulphide-isomerase A3) has been associated with some of the functions of 1,25-dihydroxyvitamin D₃. In the present study we used siRNA-mediated silencing of PDIA3 in osteosarcoma and prostate carcinoma cell lines to examine the role(s) of PDIA3 for 1,25-dihydroxyvitamin D₃-dependent responses. PDIA3 silencing affected VDR target genes and significantly altered the 1,25-dihydroxyvitamin D₃-dependent induction of CYP24A1, essential for elimination of excess 1,25-dihydroxyvitamin D₃. Also, PDIA3 silencing significantly altered migration and proliferation in prostate PC3 cells, independently of 1,25-dihydroxyvitamin D₃. 1,25-Dihydroxyvitamin D₃ increased thermostability of PDIA3 in cellular thermal shift assay, supporting functional interaction between PDIA3 and 1,25-dihydroxyvitamin D₃-dependent pathways. In summary, our data link PDIA3 to 1,25-dihydroxyvitamin D₃-mediated signalling, underline and extend its role in proliferation and reveal a novel function in maintenance of 1,25-dihydroxyvitamin D₃ levels.

This study explored the signaling interplay between the vitamin D receptor (VDR) and receptor tyrosine kinases (RTKs). Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-BB promotes cell proliferation in normal and cancer cells. At the same time, the active form of vitamin D (1,25(OH)₂-vitamin D₃) inhibits proliferation in some cells. Although EGF receptors (EGFR) and PDGF receptors (PDGFR) activate similar downstream pathways, we found that they interact with VDR signaling in distinct ways. We confirmed that 1,25(OH)₂-vitamin D₃ induces CYP24A1 gene expression in U2OS, T98G, and U251 cells. We found this to be potentiated when combined with EGF. In contrast, PDGF-BB did not impact 1,25(OH)₂-vitamin D₃-induced CYP24A1 expression in U2OS cells. The increase in CYP24A1 expression due to the combined action of EGF and 1,25(OH)₂-vitamin D₃ was dependent on AKT and ERK1/2 activation. Another VDR-responsive gene, CYP27B1, was unaffected by the addition of EGF, suggesting that EGF may have gene-specific effects on VDR signaling. While PDGF-BB did not influence CYP24A1 expression, 1,25(OH)₂-vitamin D₃ significantly influenced PDGF-BB-induced receptor phosphorylation and cell proliferation. In summary, we found that EGF, but not PDGF-BB, influenced the expression of the VDR-dependent gene CYP24A1, while 1,25(OH)₂-vitamin D₃ had an inhibitory effect on PDGFR signaling and proliferation. These findings highlight unique crosstalk between 1,25(OH)₂-vitamin D₃ signaling and EGF or PDGF-BB.

This study investigates the role of Rho GTPases, specifically Cdc42, Rac1, and RhoA, in platelet-derived growth factor receptors (PDGFRα and PDGFRβ) signaling. Signal transducer and activator of transcription (STAT) proteins, essential for cellular processes such as proliferation and immune response, are activated downstream of PDGFRs. Dysregulation of these pathways is linked to various diseases, including cancer. The current study examines the effects of Rho GTPase depletion on PDGFR phosphorylation, STAT protein stability, and downstream signaling. Results indicate that depletion of Cdc42, Rac1, or RhoA impairs PDGFR phosphorylation and reduces STAT1 and STAT3 signaling, without significantly affecting AKT and ERK1/2 pathways. The findings highlight the critical regulatory roles of Rho GTPases in PDGFR-mediated STAT signaling.

The interplay between membrane subregions and receptor tyrosine kinases (RTK) will influence signaling in both normal and pathological RTK conditions. In this study, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor β (PDGFR-β) internalizations were investigated by immunofluorescent microscopy following simultaneous treatment with EGF and PDGF-BB. We found that the two receptors utilize separate routes of internalization, which merges in a common perinuclear endosomal compartment after 45 min of stimulation. This is further strengthened when contrasting the recruitment of either EGFR or PDGFR-β to either clathrin or caveolin-1: PDGFR-β dissociates from caveolin-1 upon stimulation, and engages clathrin, whilst an increased recruitment of EGFR, to both clathrin and caveolin-1, was observed upon EGF stimulation. The association between EGFR and caveolin-1 is supported by the observation that EGFR was localized in lipid raft associated fractions, whereas PDGFR-β was not. We also found that disruption of lipid rafts using MβCD led to an increased EGFR dimerization and phosphorylation in response to ligand, as well as a dramatic decrease in AKT- and a smaller but robust decrease in ERK1/2 phosphorylation. This suggest that lipid rafts may be important to effectively connect the EGFR with downstream proteins to facilitate signaling. Our data implies that cholesterol depletion of the plasma membrane affect the signaling of EGFR and PDGFRβ differently.

Ras is a key switch controlling cell behavior. In the GTP-bound form, Ras interacts with numerous effectors in a mutually ex-clusive manner, where individual Ras-effectors are likely part of larger cellular (sub)complexes. The molecular details of these (sub)complexes and their alteration in specific contexts are not understood. Focusing on KRAS, we performed affinity puri-fication (AP)-mass spectrometry (MS) experiments of exoge-nously expressed FLAG-KRAS WT and three oncogenic mutants ("genetic contexts") in the human Caco-2 cell line, each exposed to 11 different culture media ("culture contexts") that mimic conditions relevant in the colon and colorectal cancer. We identified four effectors present in complex with KRAS in all genetic and growth contexts ("context-general effectors"). Seven effectors are found in KRAS complexes in only some contexts ("context-specific effectors"). Analyzing all interactors in complex with KRAS per condition, we find that the culture contexts had a larger impact on interaction rewiring than genetic contexts. We investigated how changes in the interactome impact functional outcomes and created a Shiny app for interactive visualization. We validated some of the functional differences in metabolism and proliferation. Finally, we used networks to evaluate how KRAS-effectors are involved in the modulation of functions by random walk analyses of effector-mediated (sub)complexes. Altogether, our work shows the impact of environmental contexts on network rewiring, which provides insights into tissue-specific signaling mechanisms. This may also explain why KRAS oncogenic mutants may be causing cancer only in specific tissues despite KRAS being expressed in most cells and tissues.

It has become clear that lipid rafts functions as signaling hotspots connecting cell surface receptors to intracellular signaling pathways. However, the exact involvement of lipid rafts in receptor tyrosine kinase signaling is still poorly understood. In this study, we have analyzed platelet-derived growth factor (PDGF) receptor β (PDGFR-β) signaling in two different cell lines depleted of cholesterol, and as a consequence, disruption of lipid rafts. Cholesterol depletion of BJ-hTERT fibroblasts using methyl-β-cyclodextrin (MβCD) did not affect PDGFR-β activation as measured by its tyrosine phosphorylation. However, we did observe a small reduction in AKT phosphorylation and a more robust decrease of ERK1/2 activation. In contrast, in the osteosarcoma cell line U2OS, we noticed a deficient receptor activation. Interestingly, in U2OS cells, the ERK1/2 pathway was unaffected, but instead AKT and SRC signaling was reduced. These results suggest that cell type specific wiring of signaling pathways can lead to differential sensitivity to cholesterol depletion. Furthermore, MβCD treatment had a much more pronounced morphological effect on U2OS compared to BJ-hTERT cells. This is consistent with a previous report claiming that cancer cells are more sensitive to cholesterol depletion than normal cells. Our data supports the possibility that cholesterol lowering drugs may impede tumor growth.