Journal of Experimental & Clinical Cancer Research最新文献

Background: Glioblastoma multiforme (GBM) exhibits a cellular hierarchy with a subpopulation of stem-like cells known as glioblastoma stem cells (GSCs) that drive tumor growth and contribute to treatment resistance. NAD(H) emerges as a crucial factor influencing GSC maintenance through its involvement in diverse biological processes, including mitochondrial fitness and DNA damage repair. However, how GSCs leverage metabolic adaptation to obtain survival advantage remains elusive.

Methods: A multi-step process of machine learning algorithms was implemented to construct the glioma stemness-related score (GScore). Further in silico and patient tissue analyses validated the predictive ability of the GScore and identified a potential target, CYP3A5. Loss-of-function or gain-of-function genetic experiments were performed to assess the impact of CYP3A5 on the self-renewal and chemoresistance of GSCs both in vitro and in vivo. Mechanistic studies were conducted using nontargeted metabolomics, RNA-seq, seahorse, transmission electron microscopy, immunofluorescence, flow cytometry, ChIP‒qPCR, RT‒qPCR, western blotting, etc. The efficacy of pharmacological inhibitors of CYP3A5 was assessed in vivo.

Results: Based on the proposed GScore, we identify a GSC target CYP3A5, which is highly expressed in GSCs and temozolomide (TMZ)-resistant GBM patients. This elevated expression of CYP3A5 is attributed to transcription factor STAT3 activated by EGFR signaling or TMZ treatment. Depletion of CYP3A5 impairs self-renewal and TMZ resistance of GSCs. Mechanistically, CYP3A5 maintains mitochondrial fitness to promote GSC metabolic adaption through the NAD⁺/NADH-SIRT1-PGC1α axis. Additionally, CYP3A5 enhances the activity of NAD-dependent enzyme PARP to augment DNA damage repair. Treatment with CYP3A5 inhibitor alone or together with TMZ effectively suppresses tumor growth in vivo.

Conclusion: Together, this study suggests that GSCs activate STAT3 to upregulate CYP3A5 to fine-tune NAD⁺/NADH for the enhancement of mitochondrial functions and DNA damage repair, thereby fueling tumor stemness and conferring TMZ resistance, respectively. Thus, CYP3A5 represents a promising target for GBM treatment.

Background: Tumor-associated macrophages (TAMs), particularly M2-polarized TAMs, are significant contributors to tumor progression, immune evasion, and therapy resistance in gastric cancer (GC). Despite efforts to target TAM recruitment or depletion, clinical efficacy remains limited. Consequently, the identification of targets that specifically inhibit or reprogram M2-polarized TAMs presents a promising therapeutic strategy.

Objective: This study aims to identify a dual-function target in GC cells that drives both malignant phenotypes and M2 macrophage polarization, revealing its molecular mechanisms to provide novel therapeutic targets for selectivly targeting M2-polarized TAMs in GC.

Methods: Transcriptomic and clinical data from GC and adjacent tissues were utilized to identify mRNAs associated with high M2 macrophage infiltration and poor prognosis. Single-cell sequencing elucidated cell types expressing the target gene. Transwell co-culture and exosome intervention experiments demonstrated its role in M2 polarization. Small RNA sequencing of exosomes, western blotting, and CoIP assays revealed the molecular mechanisms underlying exosome-mediated M2 polarization. Protein array, ChIP and dual-luciferase reporter assays clarified the molecular mechanisms by which the target gene regulated exosomal miRNA. In vivo validation was performed using xenograft tumor models.

Results: SERPINE1 was identified as a highly expressed mRNA in GC tissues and cells, significantly associated with advanced clinical stages, worse prognosis, and higher M2 macrophage infiltration in patients with GC. SERPINE1 overexpression in GC cells promoted tumor growth and M2 macrophage polarization. SERPINE1 facilitated the transfer of let-7 g-5p to macrophages via cancer-derived exosomes, inducing M2 polarization. Exosomal let-7 g-5p internalized by macrophages downregulated SOCS7 protein levels, disrupting its interaction with STAT3 and relieving the inhibition of STAT3 phosphorylation, thereby leading to STAT3 hyperactivation, which consequently drove M2 polarization. Additionally, in GC cells, elevated SERPINE1 expression activated JAK2, enhancing STAT3 binding to the let-7 g-5p promoter and promoting its transcription, thereby increasing let-7 g-5p levels in exosomes.

Conclusion: GC cell-derived SERPINE1, functioning as a primary driver of GC growth and TAM M2 polarization, promotes M2 polarization through the regulation of exosomal let-7 g-5p transfer via autocrine activation of the JAK2/STAT3 signaling pathway. These findings elucidate a novel mechanism of SERPINE1-induced M2 polarization and highlight SERPINE1 as a promising target for advancing immunotherapy and targeted treatments in GC.

Background: Targeting the TGF-β pathway in tumor therapy has proven challenging due to the highly context-dependent functions of TGF-β. Integrin αvβ8, a pivotal activator of TGF-β, has been implicated in TGF-β signaling within tumors, as demonstrated by the significant anti-tumor effects of anti-αvβ8 antibodies. Nevertheless, the expression profile of αvβ8 remains a subject of debate, and the precise mechanisms underlying the anti-tumor effects of anti-αvβ8 antibodies are not yet fully elucidated.

Methods: We utilized single-cell RNA sequencing to assess αvβ8 expression across various human tumors. An anti-αvβ8 antibody was developed and characterized for its binding and blocking properties in vitro. Cryo-EM single-particle analysis was employed to study the detailed interaction between αvβ8 and the antibody Fab fragment. The anti-tumor efficacy of the antibody was evaluated in syngeneic mouse models with varying levels of αvβ8 expression, both as a monotherapy and in combination with PD-1 antibodies. Human PBMCs were isolated to investigate αvβ8 expression in myeloid cells, and macrophages were exposed to the antibody to study its impact on macrophage polarization. Pharmacokinetic studies of the αvβ8 antibody were conducted in cynomolgus monkeys.

Results: Integrin αvβ8 is notably expressed in certain tumor types and tumor-infiltrating macrophages. The specific αvβ8 antibody 130H2 demonstrated high affinity, specificity, and blocking potency in vitro. Cryo-EM analysis further revealed that 130H2 interacts exclusively with the β8 subunit, without binding to the αv subunit. In vivo studies showed that this antibody significantly inhibited tumor growth and alleviated immunosuppression by promoting immune cell infiltration. Furthermore, combining the antibody with PD-1 inhibition produced a synergistic anti-tumor effect. In human PBMCs, monocytes exhibited high αvβ8 expression, and the antibody directly modulated macrophage polarization. Tumors with elevated αvβ8 expression were particularly responsive to 130H2 treatment. Additionally, favorable pharmacokinetic properties were observed in cynomolgus monkeys.

Conclusions: In summary, integrin αvβ8 is highly expressed in certain tumors and tumor-infiltrating macrophages. Targeting αvβ8 with a blocking antibody significantly inhibits tumor growth by modulating macrophage polarization and enhancing immune cell infiltration. Combining αvβ8 targeting with PD-1 treatment markedly increases the sensitivity of immune-excluded tumors. These results support further clinical evaluation of αvβ8 antibodies.

Background: Multiple Myeloma (MM) is the second most common hematological malignancy, characterized by the accumulation of monoclonal plasmocytes in the bone marrow. Despite advancements with proteasome inhibitors, immunomodulatory agents, and CD38-targeting antibodies, MM remains largely incurable due to resistant clones and frequent relapses. The success of the proteasome inhibitor bortezomib (BTZ) in MM treatment highlights the critical role of the ubiquitin-proteasome system (UPS) in this disease. Deubiquitinases (DUBs), which regulate protein stability, interactions, and localization by removing ubiquitin modifications, have emerged as promising therapeutic targets in various cancers, including MM.

Methods: Through a comprehensive loss-of-function screen, we identified USP39 as a critical survival factor for MM cells. Gene Set Enrichment Analysis (GSEA) was employed to correlate USP39 mRNA levels with clinical outcomes in MM patients. USP39 protein expression was evaluated via immunohistochemistry (IHC) on bone marrow samples from MM patients and healthy controls. The impact of USP39 knockdown via SiRNA was assessed through in vitro assays measuring cellular metabolism, clonogenic capacity, cell cycle progression, apoptosis, and sensitivity to BTZ. Co-immunoprecipitation and deubiquitination assays were conducted to elucidate the interaction and regulation of ZEB1 by USP39. Finally, in vitro and in vivo zebrafish experiments were used to characterize the biological consequences of ZEB1 regulation by USP39.

Results: Our study found that elevated USP39 mRNA levels are directly associated with shorter survival in MM patients. USP39 protein expression is significantly higher in MM patient plasmocytes compared to healthy individuals. USP39 knockdown inhibits clonogenic capacity, induces cell cycle arrest, triggers apoptosis, and overcomes BTZ resistance. Gain-of-function assays revealed that USP39 stabilizes the transcription factor ZEB1, enhancing the proliferation and the trans-migratory potential of MM cells.

Conclusions: Our findings highlight the critical role of the deubiquitinase USP39, suggesting that the USP39/ZEB1 axis could serve as a potential diagnostic marker and therapeutic target in MM.

On September 23-24 (2024) the 6th Workshop IRE on Translational Oncology, titled "Cancer Organoids as Reliable Disease Models to Drive Clinical Development of Novel Therapies," took place at the IRCCS Regina Elena Cancer Institute in Rome. This prominent international conference focused on tumor organoids, bringing together leading experts from around the world.A central challenge in precision oncology is modeling the dynamic tumor ecosystem, which encompasses numerous elements that evolve spatially and temporally. Patient-derived 3D culture models, including organoids, explants, and engineered or bioprinted systems, have recently emerged as sophisticated tools capable of capturing the complexity and diversity of cancer cells interacting within their microenvironments. These models address critical unmet needs in precision medicine, particularly in aiding clinical decision-making. The rapid development of these human tissue avatars has enabled advanced modeling of cellular alterations in disease states and the screening of compounds to uncover novel therapeutic pathways.Throughout the event, distinguished speakers shared their expertise and research findings, illustrating how organoids are transforming our understanding of treatment resistance, metastatic dynamics, and the interaction between tumors and the surrounding microenvironment.This conference served as a pivotal opportunity to strengthen international collaborations and spark innovative translational approaches. Its goal was to accelerate the shift from preclinical research to clinical application, paving the way for increasingly personalized and effective cancer therapies.

Background: Liquid biopsies offer less burdensome sensitive disease monitoring. Bone marrow (BM) metastases, common in various cancers including neuroblastoma, is associated with poor outcomes. In pediatric high-risk neuroblastoma most patients initially respond to treatment, but in the majority the disease recurs with only 40% long-term survivors, stressing the need for more sensitive detection of disseminated disease during therapy.

Methods: To validate sensitive neuroblastoma mRNA RT-qPCR BM testing, we prospectively assessed serial BM samples from 345 international high-risk neuroblastoma patients, treated in trials NB2004 (GPOH) or NBL2009 (DCOG), using PHOX2B, TH, DDC, CHRNA3, and GAP43 RT-qPCR mRNA markers and BM GD2-immunocytology. Association between BM-infiltration levels and event-free survival (EFS) and overall survival (OS) was estimated by using Cox regression models and Kaplan-Meier's methodology.

Results: BM infiltration >10% by RT-qPCR at diagnosis was prognostic for survival (adjusted hazard ratio (HR) 1.82 [95%CI 1.25-2.63] and 2.04 [1.33-3.14] for EFS and OS, respectively). Any post-induction RT-qPCR positivity correlated with poor EFS and OS, with a HR of 2.10 [1.27-3.49] and 1.76 [1.01-3.08] and 5-years EFS of 26.6% [standard error 5.2%] versus 60.4% [6.7] and OS of 43.8% [5.9] versus 65.7% [6.6] for RT-qPCR-positive patients versus RT-qPCR-negative patients. In contrast, post-induction immunocytology positivity was not associated with EFS or OS (HR 1.22 [0.68-2.19] and 1.26 [0.54-2.42]).

Conclusion: This study validates the association of not clearing of BM metastases by sensitive RT-qPCR detection with very poor outcome. We therefore propose implementation of RT-qPCR for minimal residual disease testing in neuroblastoma to guide therapy.

Background: Osteosarcoma (OS), the most prevalent primary malignant bone tumor in children and adolescents, arises from bone-forming mesenchymal cells. Despite advancements in surgical resection and neoadjuvant chemotherapy (cisplatin, doxorubicin, and methotrexate), chemotherapy resistance remains a significant challenge, leading to poor survival rates in patients with metastatic or recurrent OS.

Methods: In this study, we focused on the role of OTULIN, a key linear deubiquitinating enzyme, in OS chemoresistance. In addition, mechanistic investigations were carried out to identify potential downstream targets of OTULIN involved in cisplatin resistance.

Results: Our results demonstrated that OTULIN expression was significantly upregulated in OS tissues and cell lines following cisplatin treatment but not in response to doxorubicin or methotrexate. High OTULIN expression was associated with reduced survival in sarcoma patients. Furthermore, immunohistochemical analysis of prechemotherapy and postchemotherapy OS tissues revealed increased OTULIN expression in postchemotherapy samples. In vitro results demonstrated that OTULIN plays a critical role in mediating cisplatin resistance in OS. Mechanistically, GPX4 could be a downstream target of OTULIN, conferring cisplatin resistance to OS by blocking the mitochondrial apoptotic pathway but not ferroptosis. Specifically, OTULIN prevents the proteasomal degradation of GPX4 by reducing its ubiquitin level, thereby conferring resistance to cisplatin in OS cells.

Conclusion: This study highlights the importance of OTULIN in OS chemoresistance and provides a promising approach for targeting the OTULIN-GPX4 axis to improve the prognosis of OS patients. Our findings offer new insights into the molecular mechanisms underlying OS chemoresistance and suggest potential therapeutic targets for future clinical interventions.

Background: Head and neck squamous cell carcinoma (HNSCC) is a very aggressive disease characterized by a heterogeneous tumor immune microenvironment (TIME). Tumor-associated macrophages (TAMs) constitute the major innate immune population in the TIME where they facilitate crucial regulatory processes that participate in malignant tumor progression. SPP1 + macrophages (SPP1 + Macs) are found in many cancers, but their effects on HNSCC remain unknown. This study aimed to identify and validate the role and function of SPP1 + Macs in the malignant progression of HNSCC.

Methods: In this study, we applied single-cell RNA sequencing (scRNA-seq) analyses of paired tumor and normal tissues from 5 HNSCC patients to identify tumor-specific SPP1 + Macs. RT-qPCR and multiplex immunohistochemical and multiplex immunofluorescence staining were used to verify the presence of SPP1 + Macs in the clinical samples. Gene set variation analysis suggested that SPP1 + Macs were actively involved in cytokine production. Thus, we constructed SPP1-OE macrophages and SPP1-KD macrophages (both differentiated from THP-1 cells), performed a Luminex liquid suspension chip detection assay to detect differential cytokines, and further assessed their biological functions and mechanisms in several HNSCC cell lines and adjacent macrophages. An in vivo experiment was used to verify the function of SPP1 + Macs in HNSCC progression.

Results: The scRNA-seq results revealed that myeloid cells were heterogeneous and strongly correlated with tumor cells in the TIME in HNSCC and identified tumor-specific SPP1 + Macs, which were positively correlated with poor prognosis of HNSCC patients. Gene set variation analysis (GSVA) suggested that SPP1 + Macs were actively involved in cytokine production. Luminex liquid suspension chip detection assay indicated that SPP1 + Mac-derived TNF-α and IL-1β played important roles. Both in vitro and in vivo experiments and the use of VGX-1027, an inhibitor of macrophage-derived TNF-α and IL-1β, confirmed that SPP1 + Mac-derived TNF-α and IL-1β promoted HNSCC progression by supporting tumor cell proliferation and migration. Mechanistically, we found that TNF-α and IL-1β were upregulated due to NF-kappa B signaling pathway activation in SPP1 + Macs. Moreover, SPP1 + Mac-derived TNF-α and IL-1β promoted the expression of OPN in both tumor cells and other adjacent macrophages through different signaling pathways.

Conclusions: SPP1 + Macs increase the secretion of TNF-α and IL-1β via the NF-kappa B pathway to promote HNSCC cell proliferation, and TNF-α and IL-1β in turn upregulate the expression of OPN in tumor cells and macrophages; thus, SPP1 + Macs may be a candidate target through which antitumor efficacy can be enhanced.