Wnt/IL-1ß/IL-8 autocrine circuitries control chemoresistance in mesothelioma initiating cells by inducing ABCB5.

Malignant pleural mesothelioma (MPM) is a tumor with high chemoresistance and poor prognosis. MPM-initiating cells (ICs) are known to be drug resistant, but it is unknown if and how stemness-related pathways determine chemoresistance. Moreover, there are no predictive markers of IC-associated chemoresistance. Aim of this work is to clarify if and by which mechanisms the chemoresistant phenotype of MPM IC was due to specific stemness-related pathways. We generated MPM IC from primary MPM samples and compared the gene expression and chemo-sensitivity profile of IC and differentiated/adherent cells (AC) of the same patient. Compared to AC, IC had upregulated the drug efflux transporter ABCB5 that determined resistance to cisplatin and pemetrexed. ABCB5-knocked-out (KO) IC clones were resensitized to the drugs in vitro and in patient-derived xenografts. ABCB5 was transcriptionally activated by the Wnt/GSK3ß/ß-catenin/c-myc axis that also increased IL-8 and IL-1ß production. IL-8 and IL-1ß-KO IC clones reduced the c-myc-driven transcription of ABCB5 and reacquired chemosensitivity. ABCB5-KO clones had lower IL-8 and IL-1ß secretion, and c-myc transcriptional activity, suggesting that either Wnt/GSK3ß/ß-catenin and IL-8/IL-1ß signaling drive c-myc-mediated transcription of ABCB5. ABCB5 correlated with lower time-to-progression and overall survival in MPM patients treated with cisplatin and pemetrexed. Our work identified multiple autocrine loops linking stemness pathways and resistance to cisplatin and pemetrexed in MPM IC. ABCB5 may represent a new target to chemosensitize MPM IC and a potential biomarker to predict the response to the first-line chemotherapy in MPM patients.

Small Nucleolar RNAs Determine Resistance to Doxorubicin in Human Osteosarcoma.

Doxorubicin (Dox) is one of the most important first-line drugs used in osteosarcoma therapy. Multiple and not fully clarified mechanisms, however, determine resistance to Dox. With the aim of identifying new markers associated with Dox-resistance, we found a global up-regulation of small nucleolar RNAs (snoRNAs) in human Dox-resistant osteosarcoma cells. We investigated if and how snoRNAs are linked to resistance. After RT-PCR validation of snoRNAs up-regulated in osteosarcoma cells with different degrees of resistance to Dox, we overexpressed them in Dox-sensitive cells. We then evaluated Dox cytotoxicity and changes in genes relevant for osteosarcoma pathogenesis by PCR arrays. SNORD3A, SNORA13 and SNORA28 reduced Dox-cytotoxicity when over-expressed in Dox-sensitive cells. In these cells, GADD45A and MYC were up-regulated, TOP2A was down-regulated. The same profile was detected in cells with acquired resistance to Dox. GADD45A/MYC-silencing and TOP2A-over-expression counteracted the resistance to Dox induced by snoRNAs. We reported for the first time that snoRNAs induce resistance to Dox in human osteosarcoma, by modulating the expression of genes involved in DNA damaging sensing, DNA repair, ribosome biogenesis, and proliferation. Targeting snoRNAs or down-stream genes may open new treatment perspectives in chemoresistant osteosarcomas.

Enhanced effect of geldanamycin nanocomposite against breast cancer cells growing in vitro and as xenograft with vanquished normal cell toxicity.

Despite enormous advances in remedies developed for breast cancer, an effective therapeutic strategy by targeting malignant cells with the least normal tissue toxicity is yet to be developed. Hsp90 is considered to be an important therapeutic target to inhibit cell proliferation. Geldanamycin (GDM), a potent inhibitor of Hsp90 was withdrawn from clinical trials due to its undesirable hepatotoxicity. We report a superparamagnetic iron oxide (SPION) based polymeric nanocomposite of GDM augmenting anticancer competence with decreased hepatic toxicity. The particle size of nanocomposite was ascertained to be 76±10nm with acceptable stability. A comparative dose dependent in vitro validation of cytotoxicity showed an enhanced cellular damage and necrosis in breast cancer (MCF-7) cell line at a low dose of 5.49nM (in GDM nanocomposite) in contrast to 20nM of pure GDM, while normal breast epithelial cells (MCF-10A) were least affected. Besides, in vivo study (in breast cancer xenografts) substantiated 2.7 fold delay in tumor progression mediated by redundancy in the downstream functions of p-Akt and MAPK-Erk leading to apoptosis with negligible hepatotoxicity. Pure GDM disrupted the function and morphology of liver with lesser therapeutic efficacy than the GDM nanocomposite. These findings deduce that GDM based polymeric magnetite nanocomposite play a vital role in efficacious therapy while vanquishing normal cells and hepatic toxicity and thereby promising it to be reinstated in clinics.

Inhibiting NR5A2 targets stemness in pancreatic cancer by disrupting SOX2/MYC signaling and restoring chemosensitivity.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a profoundly aggressive and fatal cancer. One of the key factors defining its aggressiveness and resilience against chemotherapy is the existence of cancer stem cells (CSCs). The important task of discovering upstream regulators of stemness that are amenable for targeting in PDAC is essential for the advancement of more potent therapeutic approaches. In this study, we sought to elucidate the function of the nuclear receptor subfamily 5, group A, member 2 (NR5A2) in the context of pancreatic CSCs. METHODS: We modeled human PDAC using primary PDAC cells and CSC-enriched sphere cultures. NR5A2 was genetically silenced or inhibited with Cpd3. Assays included RNA-seq, sphere/colony formation, cell viability/toxicity, real-time PCR, western blot, immunofluorescence, ChIP, CUT&Tag, XF Analysis, lactate production, and in vivo tumorigenicity assays. PDAC models from 18 patients were treated with Cpd3-loaded nanocarriers. RESULTS: Our findings demonstrate that NR5A2 plays a dual role in PDAC. In differentiated cancer cells, NR5A2 promotes cell proliferation by inhibiting CDKN1A. On the other hand, in the CSC population, NR5A2 enhances stemness by upregulating SOX2 through direct binding to its promotor/enhancer region. Additionally, NR5A2 suppresses MYC, leading to the activation of the mitochondrial biogenesis factor PPARGC1A and a shift in metabolism towards oxidative phosphorylation, which is a crucial feature of stemness in PDAC. Importantly, our study shows that the specific NR5A2 inhibitor, Cpd3, sensitizes a significant fraction of PDAC models derived from 18 patients to standard chemotherapy. This treatment approach results in durable remissions and long-term survival. Furthermore, we demonstrate that the expression levels of NR5A2/SOX2 can predict the response to treatment. CONCLUSIONS: The findings of our study highlight the cell context-dependent effects of NR5A2 in PDAC. We have identified a novel pharmacological strategy to modulate SOX2 and MYC levels, which disrupts stemness and prevents relapse in this deadly disease. These insights provide valuable information for the development of targeted therapies for PDAC, offering new hope for improved patient outcomes. A Schematic illustration of the role of NR5A2 in cancer stem cells versus differentiated cancer cells, along with the action of the NR5A2 inhibitor Cpd3. B Overall survival of tumor-bearing mice following allocated treatment. A total of 18 PDX models were treated using a 2 x 1 x 1 approach (two animals per model per treatment); n=36 per group (illustration created with biorender.com ).

Sound-based assembly of a microcapillary network in a saturn-like tumor model for drug testing.

The tumor microenvironment (TME), consisting of extracellular matrix, proteins, stromal cells, and a vascular system, is reported to have a key role in cancer progression and prognosis. Thereby, the interaction between the vascular network and tumor mass is an important feature of the TME since the anticancer agents which are delivered to the TME can trigger the vascular response and influence the therapeutic outcome of the treatment. To identify and develop new therapeutic strategies, 3D in vitro models that recapitulate the complexity of the TME are urgently needed. Among them, vascularized tumor models are a promising approach, allowing to target tumor angiogenesis and reduce tumor growth. By using sound patterning, cells can be condensed locally into highly reproducible patterns through the action of mild hydrodynamic forces. Here, we use a soundwave-driven cell assembly approach to create a ring-shaped microcapillary network in fibrin hydrogel. Then, we generate a 3D vascularized tumor model by combining a tumor heterotypic spheroid, consisting of fibroblasts and Malignant Pleural Mesothelioma (MPM) cells, with the surrounding vascular ring. Based on its shape, we name it Saturn-like vascularized Tumor Model (STM). The growth of the microcapillary network is monitored over time by fluorescence imaging. The area covered by the microcapillary network, and its continuous increase in presence of the heterotypic tumor spheroid was monitored. Interestingly, this effect is enhanced when treating the STM with the anticancer agent Cisplatin. Overall, we show the use of sound patterning as a fast and cell-friendly approach to spatially organize and condense cells, to generate a 3D in vitro platform from which simple readouts of drug tests can be extracted by image analysis, with the potential to provide a model system for tailored tumor therapy.

SKP2 drives the sensitivity to neddylation inhibitors and cisplatin in malignant pleural mesothelioma.

BACKGROUND: The combination of pemetrexed and cisplatin remains the reference first-line systemic therapy for malignant pleural mesothelioma (MPM). Its activity is moderate because of tumor aggressiveness, immune-suppressive environment and resistance to chemotherapy-induced immunogenic cell death (ICD). Preliminary and limited findings suggest that MPM cells have deregulated ubiquitination and proteasome activities, although proteasome inhibitors achieved disappointing clinical results. METHODS: Here, we investigated the role of the E3-ubiquitin ligase SKP/Cullin/F-box (SCF) complex in cell cycle progression, endoplasmic reticulum (ER)/proteostatic stress and ICD in MPM, and the therapeutic potential of the neddylation/SCF complex inhibitor MLN4924/Pevonedistat. RESULTS: In patient-derived MPM cultures and syngenic murine models, MLN4924 and cisplatin showed anti-tumor effects, regardless of MPM histotype and BAP1 mutational status, increasing DNA damage, inducing S- and G2/M-cell cycle arrest, and apoptosis. Mechanistically, by interfering with the neddylation of cullin-1 and ubiquitin-conjugating enzyme UBE2M, MLN4924 blocks the SCF complex activity and triggers an ER stress-dependent ICD, which activated anti-MPM CD8+T-lymphocytes. The SKP2 component of SCF complex was identified as the main driver of sensitivity to MLN4924 and resistance to cisplatin. These findings were confirmed in a retrospective MPM patient series, where SKP2 high levels were associated with a worse response to platinum-based therapy and inferior survival. CONCLUSIONS: We suggest that the combination of neddylation inhibitors and cisplatin could be worth of further investigation in the clinical setting for MPM unresponsive to cisplatin. We also propose SKP2 as a new stratification marker to determine the sensitivity to cisplatin and drugs interfering with ubiquitination/proteasome systems in MPM.

Potential Diagnostic and Prognostic Role of Microenvironment in Malignant Pleural Mesothelioma.

INTRODUCTION: A comprehensive analysis of the immune cell infiltrate collected from pleural fluid and from biopsy specimens of malignant pleural mesothelioma (MPM) may contribute to understanding the immune-evasion mechanisms related to tumor progression, aiding in differential diagnosis and potential prognostic stratification. Until now such approach has not routinely been verified. METHODS: We enrolled 275 patients with an initial clinical diagnosis of pleural effusion. Specimens of pleural fluids and pleural biopsy samples used for the pathologic diagnosis and the immune phenotype analyses were blindly investigated by multiparametric flow cytometry. The results were analyzed using the Kruskal-Wallis test. The Kaplan-Meier and log-rank tests were used to correlate immune phenotype data with patients' outcome. RESULTS: The cutoffs of intratumor T-regulatory (>1.1%) cells, M2-macrophages (>36%), granulocytic and monocytic myeloid-derived suppressor cells (MDSC; >5.1% and 4.2%, respectively), CD4 molecule-positive (CD4+) programmed death 1-positive (PD-1+) (>5.2%) and CD8+PD-1+ (6.4%) cells, CD4+ lymphocyte activating 3-positive (LAG-3+) (>2.8% ) and CD8+LAG-3+ (>2.8%) cells, CD4+ T cell immunoglobulin and mucin domain 3-positive (TIM-3+) (>2.5%), and CD8+TIM-3+ (>2.6%) cells discriminated MPM from pleuritis with 100% sensitivity and 89% specificity. The presence of intratumor MDSC contributed to the anergy of tumor-infiltrating lymphocytes. The immune phenotype of pleural fluid cells had no prognostic significance. By contrast, the intratumor T-regulatory and MDSC levels significantly correlated with progression-free and overall survival, the PD-1+/LAG-3+/TIM-3+ CD4+ tumor-infiltrating lymphocytes correlated with overall survival. CONCLUSIONS: A clear immune signature of pleural fluids and tissues of MPM patients may contribute to better predict patients' outcome.

Loss of C/EBP-ß LIP drives cisplatin resistance in malignant pleural mesothelioma.

OBJECTIVES: Cisplatin-based chemotherapy is moderately active in malignant pleural mesothelioma (MPM) due to intrinsic drug resistance and to low immunogenicity of MPM cells. CAAT/enhancer binding protein (C/EBP)-ß LIP is a pro-apoptotic and chemosensitizing transcription factor activated in response to endoplasmic reticulum (ER) stress. MATERIALS AND METHODS: We investigated if LIP levels can predict the clinical response to cisplatin and survival of MPM patients receiving cisplatin-based chemotherapy. We studied the LIP-dependent mechanisms determining cisplatin-resistance and we identified pharmacological approaches targeting LIP, able to restore cisplatin sensitiveness, in patient-derived MPM cells and animal models. Results were analyzed by a one-way analysis of variance test. RESULTS: We found that LIP was degraded by constitutive ubiquitination in primary MPM cells derived from patients poorly responsive to cisplatin. LIP ubiquitination was directly correlated with cisplatin chemosensitivity and was associated with patients' survival after chemotherapy. Overexpression of LIP restored cisplatin's pro-apoptotic effect by activating CHOP/TRB3/caspase 3 axis and up-regulating calreticulin, that triggered MPM cell phagocytosis by dendritic cells and expanded autologous anti-tumor CD8+CD107+T-cytotoxic lymphocytes. Proteasome inhibitor carfilzomib and lysosome inhibitor chloroquine prevented LIP degradation. The triple combination of carfilzomib, chloroquine and cisplatin increased ER stress-triggered apoptosis and immunogenic cell death in patients' samples, and reduced tumor growth in cisplatin-resistant MPM preclinical models. CONCLUSION: The loss of LIP mediates cisplatin resistance, rendering LIP a possible predictor of cisplatin response in MPM patients. The association of proteasome and lysosome inhibitors reverses cisplatin resistance by restoring LIP levels and may represent a new adjuvant strategy in MPM treatment.