Activation of lysosomal iron triggers ferroptosis in cancer.

Iron catalyses the oxidation of lipids in biological membranes and promotes a form of cell death referred to as ferroptosis1-3. Identifying where this chemistry takes place in the cell can inform the design of drugs capable of inducing or inhibiting ferroptosis in various disease-relevant settings. Whereas genetic approaches have revealed underlying mechanisms of lipid peroxide detoxification1,4,5, small molecules can provide unparalleled spatiotemporal control of the chemistry at work6. Here, we show that the ferroptosis inhibitor liproxstatin-1 (Lip-1) exerts a protective activity by inactivating iron in lysosomes. Based on this, we designed the bifunctional compound fentomycin that targets phospholipids at the plasma membrane and activates iron in lysosomes upon endocytosis, promoting oxidative degradation of phospholipids and ferroptosis. Fentomycin effectively kills primary sarcoma and pancreatic ductal adenocarcinoma cells. It acts as a lipolysis-targeting chimera (LIPTAC), preferentially targeting iron-rich CD44high cell-subpopulations7,8 associated with the metastatic disease and drug resistance9,10. Furthermore, we demonstrate that fentomycin also depletes CD44high cells in vivo and reduces intranodal tumour growth in an immunocompetent murine model of breast cancer metastasis. These data demonstrate that lysosomal iron triggers ferroptosis and that lysosomal iron redox chemistry can be exploited for therapeutic benefits.

EHMT2 Inactivation in Pancreatic Epithelial Cells Shapes the Transcriptional Landscape and Inflammation Response of the Whole Pancreas.

The Euchromatic Histone Methyl Transferase Protein 2 (EHMT2), also known as G9a, deposits transcriptionally repressive chromatin marks that play pivotal roles in the maturation and homeostasis of multiple organs. Recently, we have shown that EHMT2 inactivation alters growth and immune gene expression networks, antagonizing KRAS-mediated pancreatic cancer initiation and promotion. Here, we elucidate the essential role of EHMT2 in maintaining a transcriptional landscape that protects organs from inflammation. Comparative RNA-seq studies between normal postnatal and young adult pancreatic tissue from EHMT2 conditional knockout animals ( EHMT2 fl/fl ) targeted to the exocrine pancreatic epithelial cells ( Pdx1-Cre and P48 Cre/+ ), reveal alterations in gene expression networks in the whole organ related to injury-inflammation-repair, suggesting an increased predisposition to damage. Thus, we induced an inflammation repair response in the EHMT2 fl/fl pancreas and used a data science-based approach to integrate RNA-seq-derived pathways and networks, deconvolution digital cytology, and spatial transcriptomics. We also analyzed the tissue response to damage at the morphological, biochemical, and molecular pathology levels. The EHMT2 fl/fl pancreas displays an enhanced injury-inflammation-repair response, offering insights into fundamental molecular and cellular mechanisms involved in this process. More importantly, these data show that conditional EHMT2 inactivation in exocrine cells reprograms the local environment to recruit mesenchymal and immunological cells needed to mount an increased inflammatory response. Mechanistically, this response is an enhanced injury-inflammation-repair reaction with a small contribution of specific EHMT2-regulated transcripts. Thus, this new knowledge extends the mechanisms underlying the role of the EHMT2-mediated pathway in suppressing pancreatic cancer initiation and modulating inflammatory pancreatic diseases.

Targeting NUPR1-dependent stress granules formation to induce synthetic lethality in KrasG12D-driven tumors.

We find that NUPR1, a stress-associated intrinsically disordered protein, induced droplet formation via liquid-liquid phase separation (LLPS). NUPR1-driven LLPS was crucial for the creation of NUPR1-dependent stress granules (SGs) in pancreatic cancer cells since genetic or pharmacological inhibition by ZZW-115 of NUPR1 activity impeded SGs formation. The KrasG12D mutation induced oncogenic stress, NUPR1 overexpression, and promoted SGs development. Notably, enforced NUPR1 expression induced SGs formation independently of mutated KrasG12D. Mechanistically, KrasG12D expression strengthened sensitivity to NUPR1 inactivation, inducing cell death, activating caspase 3 and releasing LDH. Remarkably, ZZW-115-mediated SG-formation inhibition hampered the development of pancreatic intraepithelial neoplasia (PanINs) in Pdx1-cre;LSL-KrasG12D (KC) mice. ZZW-115-treatment of KC mice triggered caspase 3 activation, DNA fragmentation, and formation of the apoptotic bodies, leading to cell death, specifically in KrasG12D-expressing cells. We further demonstrated that, in developed PanINs, short-term ZZW-115 treatment prevented NUPR1-associated SGs presence. Lastly, a four-week ZZW-115 treatment significantly reduced the number and size of PanINs in KC mice. This study proposes that targeting NUPR1-dependent SGs formation could be a therapeutic approach to induce cell death in KrasG12D-dependent tumors.

A guideline on the molecular ecosystem regulating ferroptosis.

Ferroptosis, an intricately regulated form of cell death characterized by uncontrolled lipid peroxidation, has garnered substantial interest since this term was first coined in 2012. Recent years have witnessed remarkable progress in elucidating the detailed molecular mechanisms that govern ferroptosis induction and defence, with particular emphasis on the roles of heterogeneity and plasticity. In this Review, we discuss the molecular ecosystem of ferroptosis, with implications that may inform and enable safe and effective therapeutic strategies across a broad spectrum of diseases.

Prediction of Adjuvant Gemcitabine Sensitivity in Resectable Pancreatic Adenocarcinoma Using the GemPred RNA Signature: An Ancillary Study of the PRODIGE-24/CCTG PA6 Clinical Trial.

PURPOSE: GemPred, a transcriptomic signature predictive of the efficacy of adjuvant gemcitabine (GEM), was developed from cell lines and organoids and validated retrospectively. The phase III PRODIGE-24/CCTG PA6 trial has demonstrated the superiority of modified folinic acid, fluorouracil, irinotecan, and oxaliplatin (mFOLFIRINOX) over GEM as adjuvant therapy in patients with resected pancreatic ductal adenocarcinoma at the expense of higher toxicity. We evaluated the potential predictive value of GemPred in this population. PATIENTS AND METHODS: Routine formalin-fixed paraffin-embedded surgical specimens of 350 patients were retrieved for RNA sequencing and GemPred prediction (167 in the GEM arm and 183 in the mFOLFIRINOX [mFFX] arm). Survival analyses were stratified by resection margins, lymph node status, and cancer antigen 19-9 level. RESULTS: Eighty-nine patients' tumors (25.5%) were GemPred+ and were thus predicted to be gemcitabine-sensitive. In the GEM arm, GemPred+ patients (n = 50, 30%) had a significantly longer disease-free survival (DFS) than GemPred- patients (n = 117, 70%; median 27.3 v 10.2 months, hazard ratio [HR], 0.43 [95% CI, 0.29 to 0.65]; P < .001) and cancer-specific survival (CSS; median 68.4 v 28.6 months, HR, 0.42 [95% CI, 0.27 to 0.66]; P < .001). GemPred had no prognostic value in the mFFX arm. DFS and CSS were similar in GemPred+ patients who received adjuvant GEM and mFFX (median 27.3 v 24.0 months, and 68.4 v 51.4 months, respectively). The statistical interaction between GEM and GemPred+ status was significant for DFS (P = .008) and CSS (P = .004). GemPred+ patients had significantly more adverse events of grade ≥3 in the mFFX arm (76%) compared with those in the GEM arm (40%; P = .001). CONCLUSION: This ancillary study of a phase III randomized trial demonstrates that among the quarter of patients with a GemPred-positive transcriptomic signature, survival was comparable with that of mFOLFIRINOX, whereas those receiving adjuvant gemcitabine had fewer adverse events.

Modular Self-Assembling Dendrimer Nanosystems for Magnetic Resonance and Multimodality Imaging of Tumors.

Bioimaging is a powerful tool for diagnosing tumors but remains limited in terms of sensitivity and specificity. Nanotechnology-based imaging probes able to accommodate abundant imaging units with different imaging modalities are particularly promising for overcoming these limitations. In addition, the nanosized imaging agents can specifically increase the contrast of tumors by exploiting the enhanced permeability and retention effect. A proof-of-concept study is performed on pancreatic cancer to demonstrate the use of modular amphiphilic dendrimer-based nanoprobes for magnetic resonance (MR) imaging (MRI) or MR/near-infrared fluorescence (NIRF) multimodality imaging. Specifically, the self-assembly of an amphiphilic dendrimer bearing multiple Gd3+ units at its terminals, generates a nanomicellar agent exhibiting favorable relaxivity for MRI with a good safety profile. MRI reveals an up to two-fold higher contrast enhancement in tumors than in normal muscle. Encapsulating the NIRF dye within the core of the nanoprobe yields an MR/NIRF bimodal imaging agent for tumor detection that is efficient both for MRI, at Gd3+ concentrations 1/10 the standard clinical dose, and for NIRF imaging, allowing over two-fold stronger fluorescence intensities. These self-assembling dendrimer nanosystems thus constitute effective probes for MRI and MR/NIRF multimodality imaging, offering a promising nanotechnology platform for elaborating multimodality imaging probes in biomedical applications.

Writers and readers of H3K9me2 form distinct protein networks during the cell cycle that include candidates for H3K9 mimicry.

Histone H3 lysine 9 methylation (H3K9me), which is written by the Euchromatic Histone Lysine Methyltransferases EHMT1 and EHMT2 and read by the heterochromatin protein 1 (HP1) chromobox (CBX) protein family, is dysregulated in many types of cancers. Approaches to inhibit regulators of this pathway are currently being evaluated for therapeutic purposes. Thus, knowledge of the complexes supporting the function of these writers and readers during the process of cell proliferation is critical for our understanding of their role in carcinogenesis. Here, we immunopurified each of these proteins and used mass spectrometry to define their associated non-histone proteins, individually and at two different phases of the cell cycle, namely G1/S and G2/M. Our findings identify novel binding proteins for these writers and readers, as well as corroborate known interactors, to show the formation of distinct protein complex networks in a cell cycle phase-specific manner. Furthermore, there is an organizational switch between cell cycle phases for interactions among specific writer-reader pairs. Through a multi-tiered bioinformatics-based approach, we reveal that many interacting proteins exhibit histone mimicry, based on an H3K9-like linear motif. Gene ontology analyses, pathway enrichment, and network reconstruction inferred that these comprehensive EHMT and CBX-associated interacting protein networks participate in various functions, including transcription, DNA repair, splicing, and membrane disassembly. Combined, our data reveals novel complexes that provide insight into key functions of cell cycle-associated epigenomic processes that are highly relevant for better understanding these chromatin-modifying proteins during cell cycle and carcinogenesis.

Combining the antianginal drug perhexiline with chemotherapy induces complete pancreatic cancer regression in vivo.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the human cancers with the poorest prognosis. Interestingly, we found that mitochondrial respiration in primary human PDAC cells depends mainly on the fatty acid oxidation (FAO) to meet basic energy requirements. Therefore, we treated PDAC cells with perhexiline, a well-recognized FAO inhibitor used in cardiac diseases. Some PDAC cells respond efficiently to perhexiline, which acts synergistically with chemotherapy (gemcitabine) in vitro and in two xenografts in vivo. Importantly, perhexiline in combination with gemcitabine induces complete tumor regression in one PDAC xenograft. Mechanistically, this co-treatment causes energy and oxidative stress promoting apoptosis but does not exert inhibition of FAO. Yet, our molecular analysis indicates that the carnitine palmitoyltransferase 1C (CPT1C) isoform is a key player in the response to perhexiline and that patients with high CPT1C expression have better prognosis. Our study reveals that repurposing perhexiline in combination with chemotherapy is a promising approach to treat PDAC.

Pacpaint: a histology-based deep learning model uncovers the extensive intratumor molecular heterogeneity of pancreatic adenocarcinoma.

Two tumor (Classical/Basal) and stroma (Inactive/active) subtypes of Pancreatic adenocarcinoma (PDAC) with prognostic and theragnostic implications have been described. These molecular subtypes were defined by RNAseq, a costly technique sensitive to sample quality and cellularity, not used in routine practice. To allow rapid PDAC molecular subtyping and study PDAC heterogeneity, we develop PACpAInt, a multi-step deep learning model. PACpAInt is trained on a multicentric cohort (n = 202) and validated on 4 independent cohorts including biopsies (surgical cohorts n = 148; 97; 126 / biopsy cohort n = 25), all with transcriptomic data (n = 598) to predict tumor tissue, tumor cells from stroma, and their transcriptomic molecular subtypes, either at the whole slide or tile level (112 µm squares). PACpAInt correctly predicts tumor subtypes at the whole slide level on surgical and biopsies specimens and independently predicts survival. PACpAInt highlights the presence of a minor aggressive Basal contingent that negatively impacts survival in 39% of RNA-defined classical cases. Tile-level analysis ( > 6 millions) redefines PDAC microheterogeneity showing codependencies in the distribution of tumor and stroma subtypes, and demonstrates that, in addition to the Classical and Basal tumors, there are Hybrid tumors that combine the latter subtypes, and Intermediate tumors that may represent a transition state during PDAC evolution.

Priming therapy by targeting enhancer-initiated pathways in patient-derived pancreatic cancer cells.

BACKGROUND: Systems biology leveraging multi-OMICs technologies, is rapidly advancing development of precision therapies and matching patients to targeted therapies, leading to improved responses. A new pillar of precision oncology lies in the power of chemogenomics to discover drugs that sensitizes malignant cells to other therapies. Here, we test a chemogenomic approach using epigenomic inhibitors (epidrugs) to reset patterns of gene expression driving the malignant behavior of pancreatic tumors. METHODS: We tested a targeted library of ten epidrugs targeting regulators of enhancers and super-enhancers on reprogramming gene expression networks in seventeen patient-derived primary pancreatic cancer cell cultures (PDPCCs), of both basal and classical subtypes. We subsequently evaluated the ability of these epidrugs to sensitize pancreatic cancer cells to five chemotherapeutic drugs that are clinically used for this malignancy. FINDINGS: To comprehend the impact of epidrug priming at the molecular level, we evaluated the effect of each epidrugs at the transcriptomic level of PDPCCs. The activating epidrugs showed a higher number of upregulated genes than the repressive epidrugs (χ2 test p-value <0.01). Furthermore, we developed a classifier using the baseline transcriptome of epidrug-primed-chemosensitized PDPCCs to predict the best epidrug-priming regime to a given chemotherapy. Six signatures with a significant association with the chemosensitization centroid (R ≤ -0.80; p-value < 0.01) were identified and validated in a subset of PDPCCs. INTERPRETATION: We conclude that targeting enhancer-initiated pathways in patient-derived primary cells, represents a promising approach for developing new therapies for human pancreatic cancer. FUNDING: This work was supported by INCa (Grants number 2018-078 to ND and 2018- 079 to JI), Canceropole PACA (ND), Amidex Foundation (ND), and INSERM (JI).

Cargo-selective and adaptive delivery of nucleic acid therapeutics by bola-amphiphilic dendrimers.

Nucleic acid therapeutics are becoming an important drug modality, offering the unique opportunity to address "undruggable" targets, respond rapidly to evolving pathogens, and treat diseases at the gene level for precision medicine. However, nucleic acid therapeutics have poor bioavailability and are chemolabile and enzymolabile, imposing the need for delivery vectors. Dendrimers, by virtue of their well-defined structure and cooperative multivalence, represent precision delivery systems. We synthesized and studied bola-amphiphilic dendrimers for cargo-selective and on-demand delivery of DNA and small interfering RNA (siRNA), both important nucleic acid therapeutics. Remarkably, superior performances were achieved for siRNA delivery with the second-generation dendrimer, yet for DNA delivery with the third generation. We systematically studied these dendrimers with regard to cargo binding, cellular uptake, endosomal release, and in vivo delivery. Differences in size both of the dendrimers and their nucleic acid cargos impacted the cooperative multivalent interactions for cargo binding and release, leading to cargo-adaptive and selective delivery. Moreover, both dendrimers harnessed the advantages of lipid and polymer vectors, while offering nanotechnology-based tumor targeting and redox-responsive cargo release. Notably, they allowed tumor- and cancer cell-specific delivery of siRNA and DNA therapeutics for effective treatment in different cancer models, including aggressive and metastatic malignancies, outperforming the currently available vectors. This study provides avenues to engineer tailor-made vectors for nucleic acid delivery and precision medicine.

Pancreatic ductal adenocarcinoma ubiquitination profiling reveals specific prognostic and theranostic markers.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has been widely studied at multiomics level. However, little is known about its specific ubiquitination, a major post-translational modification (PTM). As PTMs regulate the final function of any gene, we decided to establish the ubiquitination profiles of 60 PDAC. METHODS: We used specific proteomic tools to establish the ubiquitin dependent proteome (ubiquitinome) of frozen PDXs (Patients' derived xenographs). Then, we performed bioinformatics analysis to identify the possible associations of these ubiquitination profiles with tumour phenotype, patient survival and resistance to chemotherapies. Finally, we used proximity ligation assays (PLA) to detect and quantify the ubiquitination level of one identified marker. FINDINGS: We identified 38 ubiquitination site profiles correlating with the transcriptomic phenotype of tumours and four had notable prognostic capabilities. Seventeen ubiquitination profiles displayed potential theranostic marker for gemcitabine, seven for 5-FU, six for oxaliplatin and thirteen for irinotecan. Using PLA, we confirmed the use of one ubiquitination profile as a drug-response marker, directly on paraffin embedded tissues, supporting the possible application of these biomarkers in the clinical setting. INTERPRETATION: These findings bring new and important insights on the relationship between ubiquitination levels of proteins and different molecular and clinical features of PDAC patients. Markers identified in this study could have a potential application in clinical settings to help to predict response to chemotherapies thereby allowing the personalization of treatments. FUNDING: Fondation ARC (PJA 20181208270 and PGA 12021010002840_3562); INCa; Canceropôle PACA; DGOS; Amidex Foundation; Fondation de France; and INSERM.

Low plasma PD-L1 levels, early tumor onset and absence of peritoneal carcinomatosis improve prognosis of women with advanced high-grade serous ovarian cancer.

BACKGROUND: The most common subtype of ovarian cancer (OC) showing immunogenic potential is represented by the high-grade serous ovarian cancer (HGSOC), which is characterized by the presence of tumor-infiltrating immune cells able to modulate immune response. Because several studies showed a close correlation between OC patient's clinical outcome and expression of programmed cell death protein-1 or its ligand (PD-1/PD-L1), the aim of our study was to investigate if plasma levels of immunomodulatory proteins may predict prognosis of advanced HGSOC women. PATIENTS AND METHODS: Through specific ELISA tests, we analyzed plasma concentrations of PD-L1, PD-1, butyrophilin sub-family 3A/CD277 receptor (BTN3A1), pan-BTN3As, butyrophilin sub-family 2 member A1 (BTN2A1), and B- and T-lymphocyte attenuator (BTLA) in one hundred patients affected by advanced HGSOC, before surgery and therapy. The Kaplan-Meier method was used to generate the survival curves, while univariate and multivariate analysis were performed using Cox proportional hazard regression models. RESULTS: For each analyzed circulating biomarker, advanced HGSOC women were discriminated based on long (≥ 30 months) versus short progression-free survival (PFS < 30 months). The concentration cut-offs, obtained by receiver operating characteristic (ROC) analysis, allowed to observe that poor clinical outcome and median PFS ranging between 6 and 16 months were associated with higher baseline levels of PD-L1 (> 0.42 ng/mL), PD-1 (> 2.48 ng/mL), BTN3A1 (> 4.75 ng/mL), pan-BTN3As (> 13.06 ng/mL), BTN2A1 (> 5.59 ng/mL) and BTLA (> 2.78 ng/mL). Furthermore, a lower median PFS was associated with peritoneal carcinomatosis, age at diagnosis > 60 years or Body Mass Index (BMI) > 25. A multivariate analysis also suggested that plasma concentrations of PD-L1 ≤ 0.42 ng/mL (HR: 2.23; 95% CI: 1.34 to 3.73; p = 0.002), age at diagnosis ≤ 60 years (HR: 1.70; 95% CI: 1.07 to 2.70; p = 0.024) and absence of peritoneal carcinomatosis (HR: 1.87; 95% CI: 1.23 to 2.85; p = 0.003) were significant prognostic marker for a longer PFS in advanced HGSOC patients. CONCLUSIONS: The identification of high-risk HGSOC women could be improved through determination of the plasma PD-L1, PD-1, BTN3A1, pan-BTN3As, BTN2A1 and BTLA levels.

Intrinsically Disordered Chromatin Protein NUPR1 Binds to the Enzyme PADI4.

The nuclear protein 1 (NUPR1) is an intrinsically disordered protein involved in stress-mediated cellular conditions. Its paralogue nuclear protein 1-like (NUPR1L) is p53-regulated, and its expression down-regulates that of the NUPR1 gene. Peptidyl-arginine deiminase 4 (PADI4) is an isoform of a family of enzymes catalyzing arginine to citrulline conversion; it is also involved in stress-mediated cellular conditions. We characterized the interaction between NUPR1 and PADI4 in vitro, in silico, and in cellulo. The interaction of NUPR1 and PADI4 occurred with a dissociation constant of 18 ± 6 µM. The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch surrounding the key residue Ala33, as pinpointed by: (i) computational results; and, (ii) site-directed mutagenesis of residues of NUPR1. The association between PADI4 and wild-type NUPR1 was also assessed in cellulo by using proximity ligation assays (PLAs) and immunofluorescence (IF), and it occurred mainly in the nucleus. Moreover, binding between NUPR1L and PADI4 also occurred in vitro with an affinity similar to that of NUPR1. Molecular modelling provided information on the binding hot spot for PADI4. This is an example of a disordered partner of PADI4, whereas its other known interacting proteins are well-folded. Altogether, our results suggest that the NUPR1/PADI4 complex could have crucial functions in modulating DNA-repair, favoring metastasis, or facilitating citrullination of other proteins.

Dendrimer nanosystems for adaptive tumor-assisted drug delivery via extracellular vesicle hijacking.

Drug delivery systems (DDSs) that can overcome tumor heterogeneity and achieve deep tumor penetration are challenging to develop yet in high demand for cancer treatment. We report here a DDS based on self-assembling dendrimer nanomicelles for effective and deep tumor penetration via in situ tumor-secreted extracellular vesicles (EVs), an endogenous transport system that evolves with tumor microenvironment. Upon arrival at a tumor, these dendrimer nanomicelles had their payload repackaged by the cells into EVs, which were further transported and internalized by other cells for delivery "in relay." Using pancreatic and colorectal cancer-derived 2D, 3D, and xenograft models, we demonstrated that the in situ-generated EVs mediated intercellular delivery, propagating cargo from cell to cell and deep within the tumor. Our study provides a new perspective on exploiting the intrinsic features of tumors alongside dendrimer supramolecular chemistry to develop smart and effective DDSs to overcome tumor heterogeneity and their evolutive nature thereby improving cancer therapy.

GLI1, a novel target of the ER stress regulator p97/VCP, promotes ATF6f-mediated activation of XBP1.

Upon accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER), the Unfolded Protein Response (UPR) is triggered to restore ER homeostasis. The induction of stress genes is a sine qua non condition for effective adaptive UPR. Although this requirement has been extensively described, the mechanisms underlying this process remain in part uncharacterized. Here, we show that p97/VCP, an AAA+ ATPase known to contribute to ER stress-induced gene expression, regulates the transcription factor GLI1, a primary effector of Hedgehog (Hh) signaling. Under basal (non-ER stress) conditions, GLI1 is repressed by a p97/VCP-HDAC1 complex while upon ER stress GLI1 is induced through a mechanism requiring both USF2 binding and increase histone acetylation at its promoter. Interestingly, the induction of GLI1 was independent of ligand-regulated Hh signaling. Further analysis showed that GLI1 cooperates with ATF6f to induce promoter activity and expression of XBP1, a key transcription factor driving UPR. Overall, our work demonstrates a novel role for GLI1 in the regulation of ER stress gene expression and defines the interplay between p97/VCP, HDAC1 and USF2 as essential players in this process.

Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling, and suppresses age-related bone loss in male mice.

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-ß signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6-19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6-19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.

Stroma-targeting strategies in pancreatic cancer: a double-edged sword.

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer with limited treatment options and terrible long-term survival, and it is expected to become the second leading cause of cancer-related death by 2030. One reason why this cancer is so aggressive and resistant is the formation of dense stroma that surrounds the neoplastic epithelium, which promotes tumor progression, invasion, metastasis, and resistance. The three major components of PDAC stroma are extracellular matrix (ECM), cancer-associated fibroblasts (CAFs), and vasculature. The dense ECM acts as a natural physical barrier, impeding drug penetration to PDAC tumor cells. Consequently, the method that combines stroma-targeting with anticancer therapy may be a viable alternative for increasing drug penetration. Additionally, blood vessels are key entities of the tumor stroma, serving as a pathway for nutrition as well as the only way for chemical medicines and immune cells to act. Finally, PDAC CAFs and tumor cells have crosstalk effects in the tumor microenvironment, where they are responsible for enhanced matrix deposition. In this review, we aim to provide an overview of our current comprehension of the three key components of PDAC stroma and the new promising therapeutic targets for PDAC.