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Class 3 semaphorins are axon guidance factors implicated in tumor and vascular biology, including invasive activity. Recent studies indicate that semaphorin 3F (SEMA3F) is a potent inhibitor of metastasis; however, its functional role in breast cancer is not fully understood. We found that exogenous SEMA3F inhibited phosphorylation of Akt and mTOR downstream kinase S6K in MDA-MB-231 and MCF7 cells via neuropilin-2 (NRP2) receptor. We also examined the effect of SEMA3F on breast cancer progression in vivo allograft model. The mouse 4T1 breast cancer cells or 4T1 cells overexpressing SEMA3F (4T1-SEMA3F) were implanted into mammary fat pads of Balb/c mice. We found that tumor growth was significantly inhibited in 4T1-SEMA3F injected mice compared to controls. Immunostaining revealed a remarkable reduction in the expression of vimentin, a mesenchymal cell marker, in 4T1-SEMA3F tumors. We also observed that mice injected with 4T1-SEMA3F cells had minimal metastasis to the liver and lungs, compared to controls. As a novel feature, SEMA3F suppressed TGFβ-induced Smad2 phosphorylation, resulting in the inhibition of cell invasiveness and epithelial-to-mesenchymal transition (EMT) in breast cancer. Consistently, a significant correlation between reduced expression of SEMA3F and poor outcome in patients with breast cancer. We conclude that SEMA3F acts as a dual inhibitor of the Akt-mTOR and TGFβ signaling pathways; thus, it has the potential to treat metastatic breast cancer., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Androgen is widely acknowledged to regulate skeletal muscle mass. However, the specific mechanism driving muscle atrophy resulting from androgen deficiency remains elusive. Systemic androgen receptor knockout (ARKO) mice exhibit reduction in both muscle strength and muscle mass while skeletal muscle fiber specific ARKO mice have decreased muscle strength without affecting skeletal muscle mass in the limbs. Therefore, androgens may indirectly regulate skeletal muscle mass through effects on non-myofibers. Considering this, our investigation focused on blood fluid factors that might play a role in the regulation of skeletal muscle mass under the influence of androgens. Using a male mouse model of sham, orchidectomy and DHT replacement, mass spectrometry for serum samples of each group identified epidermal growth factor receptor (EGFR) as a candidate protein involving the regulation of skeletal muscle mass affected by androgens. Egfr expression in both liver and epididymal white adipose tissue correlated with androgen levels. Furthermore, Egfr expression in these tissues was predominantly elevated in male compared to female mice. Interestingly, male mice exhibited significantly elevated serum EGFR concentrations compared to their female counterparts, suggesting a connection with androgen levels. Treatment of EGFR to C2C12 cells promoted phosphorylation of AKT and its downstream S6K, and enhanced the protein synthesis in vitro. Furthermore, the administration of EGFR to female mice revealed a potential role in promoting an increase in skeletal muscle mass. These findings collectively enhance our understanding of the complex interplay among androgens, EGFR, and the regulation of skeletal muscle mass.

Patient-derived organoids represent a novel platform to recapitulate the cancer cells in the patient tissue. While cancer heterogeneity has been extensively studied by a number of omics approaches, little is known about the spatiotemporal kinase activity dynamics. Here we applied a live imaging approach to organoids derived from 10 pancreatic ductal adenocarcinoma (PDAC) patients to comprehensively understand their heterogeneous growth potential and drug responses. By automated wide-area image acquisitions and analyses, the PDAC cells were non-selectively observed to evaluate their heterogeneous growth patterns. We monitored single-cell ERK and AMPK activities to relate cellular dynamics to molecular dynamics. Furthermore, we evaluated two anti-cancer drugs, a MEK inhibitor, PD0325901, and an autophagy inhibitor, hydroxychloroquine (HCQ), by our analysis platform. Our analyses revealed a phase-dependent regulation of PDAC organoid growth, where ERK activity is necessary for the early phase and AMPK activity is necessary for the late stage of organoid growth. Consistently, we found PD0325901 and HCQ target distinct organoid populations, revealing their combination is widely effective to the heterogeneous cancer cell population in a range of PDAC patient-derived organoid lines. Together, our live imaging quantitatively characterized the growth and drug sensitivity of human PDAC organoids at multiple levels: in single cells, single organoids, and individual patients. This study will pave the way for understanding the cancer heterogeneity and promote the development of new drugs that eradicate intractable cancer., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Extracellular signal-regulated kinase (ERK) regulates multiple cellular functions through distinct activation patterns. Genetically encoded fluorescent probes are instrumental in dissecting the ERK activity dynamics in living cells. Here we modified a previously reported Förster resonance energy transfer (FRET) probe for ERK, EKAREN5 by replacing its mTurquoise2 and YPet sequences with mTurquoise-GL and a synonymous codon variant of YPet, respectively. The modified biosensor, EKAREN5-gl, showed an increased sensitivity to EGF-induced ERK activation responding to a very low dose (20 pg/ml) of EGF stimulation. We quantitatively characterized two FRET-based ERK probes, EKAREN5 and EKAREN5-gl, and a subcellular kinase translocation-based probe, ERK-KTR. We found the three biosensors differently respond to EGF stimulations with different intensity, duration, and latency. Furthermore, we investigated how the minimal EGF-induced ERK activation affects the downstream transcription in HeLa cells by comprehensive transcriptional analysis. We found the minimal ERK activation leads to a distinct transcriptional pattern from those induced by higher ERK activations. Our study highlights the significance of sensitive fluorescent probes to understand cellular signal dynamics and the role of minimal ERK activation in regulating transcription.Key words: fluorescent probe, ERK, FRET, KTR.

Impairment of ribosome biogenesis (RiBi) triggered by inhibition of ribosomal RNA (rRNA) synthesis and processing leads to various biological effects. We report that Schlafen 11 (SLFN11) induces TP53-independent apoptosis through RiBi impairment. Upon replication stress, SLFN11 inhibits rRNA synthesis with RNA polymerase I accumulation and increased chromatin accessibility in the ribosomal DNA (rDNA) genes. SLFN11-dependent RiBi impairment preferentially depletes short-lived proteins, particularly MCL1, leading to apoptosis in response to replication stress. SLFN11's Walker B motif (E669), DNA-binding site (K652), dephosphorylation site for single-strand DNA binding (S753), and RNase sites (E209/E214) are all required for the SLFN11-mediated RiBi impairment. Comparable effects were obtained with direct RNA polymerase I inhibitors and other RiBi inhibitory conditions regardless of SLFN11. These findings were extended across 34 diverse human cancer cell lines. Thus, we demonstrate that RiBi impairment is a robust inactivator of MCL1 and an additional proapoptotic mechanism by which SLFN11 sensitizes cancer cells to chemotherapeutic agents., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.)

Prostate-specific membrane antigen (PSMA) protein expression is induced during prostate cancer progression and metastasis. Recently, we reported that PSMA-positive vesicles released by prostate cancer cell lines enhanced vascular endothelial cell angiogenesis and that PSMA may be involved in tumor angiogenesis. Similarly, it is known that PSMA is upregulated in peritumoral vessels in renal cell carcinoma (RCC). In this study, we investigated the significance and molecular function of PSMA in RCC. PSMA immunohistochemical staining confirmed PSMA presence only in perinephric tumor vessels, and PSMA intensity was strongly correlated with recurrence rate and venous invasion. Spatial gene expression analysis revealed that FOLH1 expression, which codes PSMA, was upregulated in tumor blood vessels around renal cancer, and that angiogenesis-related pathways were enhanced. The 10,000 g pellet fraction of the renal cancer cell lines Caki1- and ACHN-conditioned medium (CM) induced PSMA positivity in human umbilical vein endothelial cells (HUVECs) and enhanced tube formation. Mass spectrometry indicated that the 10,000 g pellet fraction contained various kinds of growth factors, like GDF15 and MYDGF. RNA sequencing showed that supplementing HUVECs with RCC cell CM-enhanced angiogenesis-related signaling pathways. Conclusively, microvesicle components secreted by RCC cells transform vascular endothelial cells into PSMA-positive cells, enhancing angiogenesis.