Protease‐activated receptor 1 drives and maintains ductal cell fates in the premalignant pancreas and ductal adenocarcinoma

Pancreatic acinar cells have high plasticity and can transdifferentiate into ductal‐like cells. This acinar‐to‐ductal metaplasia (ADM) contributes to tissue maintenance but may also contribute to the premalignant transformation that can eventually progress to pancreatic ductal adenocarcinoma (PDAC). Macrophages are key players in ADM, and macrophage‐secreted matrix metalloproteinase (MMP)‐9 induces ADM through yet unknown mechanisms. As we previously identified MMP9 as a novel agonist of protease‐activated receptor 1 (PAR1), a receptor that is known to orchestrate the cross‐talk between macrophages and tumor cells in PDAC, we here assessed the contribution of PAR1 to pancreatic cell fates. We found that genetic deficiency for PAR1 increases acinar gene expression programs in the healthy pancreas and that PAR1 deficiency limits ductal transdifferentiation in experimental systems for ADM. Moreover, PAR1 silencing in PDAC cells increases acinar marker expression. Changes in PDAC cell lines were associated with a downregulation of known Myc‐target genes, and Myc inhibition mimics PAR1 deficiency in enhancing acinar programs in healthy organoids and PDAC cells. Overall, we identify the PAR1‐Myc axis as a driver of ductal cell fates in premalignant pancreas and PDAC. Moreover, we show that cellular plasticity is not unique to acinar cells and that ductal regeneration into acinar‐like cells is possible even in the context of oncogenic KRAS activation.     

Aberrant expression of claudin‐6 contributes to malignant potentials and drug resistance of cervical adenocarcinoma

Recent studies have revealed that aberrant expression of tight junction (TJ) proteins is a hallmark of various solid tumors and it is recognized as a useful therapeutic target. Claudin‐6 (CLDN6), a member of the family of TJ transmembrane proteins, is an ideal therapeutic target because it is not expressed in human adult normal tissues. In this study, we found that CLDN6 is highly expressed in uterine cervical adenocarcinoma (ADC) and that high CLDN6 expression was correlated with lymph node metastasis and lymphovascular infiltration and was an independent prognostic factor. Shotgun proteome analysis revealed that cell‐cell adhesion‐related proteins and drug metabolism‐associated proteins (aldo‐keto reductase [AKR] family proteins) were significantly increased in CLDN6‐overexpressing cells. Furthermore, overexpression of CLDN6 enhanced cell‐cell adhesion properties and attenuated sensitivity to anticancer drugs including doxorubicin, daunorubicin, and cisplatin. Taken together, the results indicate that aberrant expression of CLDN6 enhances malignant potentials and drug resistance of cervical ADC, possibly due to increased cell‐cell adhesion properties and drug metabolism. Our findings provide an insight into a new therapeutic strategy, a CLDN6‐targeting therapy, against cervical ADC.     

Metabolic enzyme ACSL3 is a prognostic biomarker and correlates with anticancer effectiveness of statins in non‐small cell lung cancer

Lung cancer is one of the most common cancers, still characterized by high mortality rates. As lipid metabolism contributes to cancer metabolic reprogramming, several lipid metabolism genes are considered prognostic biomarkers of cancer. Statins are a class of lipid‐lowering compounds used in treatment of cardiovascular disease that are currently studied for their antitumor effects. However, their exact mechanism of action and specific conditions in which they should be administered remains unclear. Here, we found that simvastatin treatment effectively promoted antiproliferative effects and modulated lipid metabolism‐related pathways in non‐small cell lung cancer (NSCLC) cells and that the antiproliferative effects of statins were potentiated by overexpression of acyl‐CoA synthetase long‐chain family member 3 (ACSL3). Moreover, ACSL3 overexpression was associated with worse clinical outcome in patients with high‐grade NSCLC. Finally, we found that patients with high expression levels of ACSL3 displayed a clinical benefit of statins treatment. Therefore, our study highlights ACSL3 as a prognostic biomarker for NSCLC, useful to select patients who would obtain a clinical benefit from statin administration.

Abbreviations

3‐HMGCR

3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase

95% CI

95% confidence intervals

ACSL3

acyl‐CoA synthetase long‐chain family member 3

ACSLs

long‐chain acyl‐CoA synthetases

ALP

alkaline phosphatase

APOA1

apolipoprotein A1

ATCC

American Type Culture Collection

CASP9

caspase 9

ECAR

extracellular acidification rate

ECOG

Eastern Cooperative Oncology Group

EMT

epithelial‐to‐mesenchymal transition

ER

endoplasmic reticulum

FAs

fatty acids

FFPE

formalin‐fixed, paraffin‐embedded

GTEx

genotype‐tissue expression

HR

Hazard ratio

IC50

half‐maximal inhibitory concentration

LDH

lactate dehydrogenase

MTT

3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium

NID1

nidogen 1

No ORF

no open reading frame

NSCLC

non‐small cell lung cancer

OCR

oxygen consumption rate

OS

overall survival

PGE2

prostaglandins E2

RETN

resistin

TCGA

The Cancer Genome Atlas

TMA

tumor tissue microarray

     

IL‐7 is expressed in malignant mesothelioma and has a prognostic value

Malignant pleural mesothelioma (MPM) is an aggressive cancer mainly related to asbestos exposure. Despite recent therapeutic advances, notably immunotherapies, the benefit remains limited and restricted to a small percentage of patients. Thus, a better understanding of the disease is needed to identify new therapeutic strategies. Recently, interleukin 7 receptor (IL‐7R) has been described as being expressed by MPM cells and associated with poorer patient survival. Thus, the aim of this work was to study the IL‐7R/IL‐7 pathway in MPM using patient samples. We found that, although more than 40% of MPM cells expressed IL‐7R, IL‐7 had no effect on their intracellular signaling. Accordingly, the addition of IL‐7 to the culture medium did not affect MPM cell growth. Using The Cancer Genome Atlas (TCGA) database, we showed that high IL7 gene expression in MPM tumors was associated with a higher overall patient survival and an induction of genes involved in the immune response. In pleural effusions (PEs), we found that IL‐7 concentration was not a good diagnostic biomarker. However, we observed that high IL‐7 levels in PEs were associated with shorter survival of MPM patients, but not of lung cancer patients. The prognostic value of IL‐7 was also conserved when only patients with epithelioid mesothelioma, the most common histological type of MPM, were analyzed. Taken together, our study suggests that, although the IL‐7R/IL‐7 signaling pathway is not functional in MPM cells, IL‐7 expression in PEs may have prognostic value in MPM patients.     

The lncRNA ‘UCA1’ modulates the response to chemotherapy of ovarian cancer through direct binding to miR‐27a‐5p and control of UBE2N levels

Ovarian cancer (OC) is the leading cause of death in patients with gynecologic cancers. Due to late diagnosis and resistance to chemotherapy, the 5‐year survival rate in patients with OC is below 40%. We observed that UCA1, a lncRNA previously reported to play an oncogenic role in several malignancies, is overexpressed in the chemoresistant OC cell line OAW42‐R compared to their chemotherapy‐sensitive counterpart OAW42. Additionally, UCA1 overexpression was related to poor prognosis in two independent patient cohorts. Currently, the molecular mechanisms through which UCA1 acts in OC are poorly understood. We demonstrated that downregulation of the short isoform of UCA1 sensitized OC cells to cisplatin and that UCA1 acted as competing endogenous RNA to miR‐27a‐5p. Upon UCA1 downregulation, miR‐27a‐5p downregulated its direct target UBE2N leading to the upregulation of BIM, a proapoptotic protein of the Bcl2 family. The upregulation of BIM is the event responsible for the sensitization of OC cells to cisplatin. In order to model response to therapy in patients with OC, we used several patient‐derived organoid cultures, a model faithfully mimicking patient’s response to therapy. Inhibition of UBE2N sensitized patient‐derived organoids to platinum salts. In conclusion, response to treatment in patients with OC is regulated by the UCA1/miR‐27a‐5p/UBE2N axis, where UBE2N inhibition could potentially represent a novel therapeutic strategy to counter chemoresistance in OC.     

The FABP12/PPARγ pathway promotes metastatic transformation by inducing epithelial‐to‐mesenchymal transition and lipid‐derived energy production in prostate cancer cells

Early stage localized prostate cancer (PCa) has an excellent prognosis; however, patient survival drops dramatically when PCa metastasizes. The molecular mechanisms underlying PCa metastasis are complex and remain unclear. Here, we examine the role of a new member of the fatty acid‐binding protein (FABP) family, FABP12, in PCa progression. FABP12 is preferentially amplified and/or overexpressed in metastatic compared to primary tumors from both PCa patients and xenograft animal models. We show that FABP12 concurrently triggers metastatic phenotypes (induced epithelial‐to‐mesenchymal transition (EMT) leading to increased cell motility and invasion) and lipid bioenergetics (increased fatty acid uptake and accumulation, increased ATP production from fatty acid β‐oxidation) in PCa cells, supporting increased reliance on fatty acids for energy production. Mechanistically, we show that FABP12 is a driver of PPARγ activation which, in turn, regulates FABP12''s role in lipid metabolism and PCa progression. Our results point to a novel role for a FABP‐PPAR pathway in promoting PCa metastasis through induction of EMT and lipid bioenergetics.

Abbreviations

AR

androgen receptor

ATP

adenosine triphosphate

CN

copy number

CPT1

carnitine palmitoyltransferase I

CS

citrate synthase

EMT

epithelial–mesenchymal transition

ET

electron transfer‐state

FABP

fatty acid‐binding protein

LD

lipid droplet

OA

oleic acid

PCa

prostate cancer

PPAR

peroxisome proliferator‐activated receptor

PPRE

peroxisome proliferator‐activated receptor response element

TZD

thiazolidinediones

     

A novel anti‐c‐Kit antibody–drug conjugate to treat wild‐type and activating‐mutant c‐Kit‐positive tumors

c‐Kit overexpression and activating mutations, which are reported in various cancers, including gastrointestinal stromal tumor (GIST), small‐cell lung cancer (SCLC), acute myeloid leukemia, acral melanoma, and systemic mastocytosis (SM), confer resistance to tyrosine kinase inhibitors (TKIs). To overcome TKI resistance, an anti‐c‐Kit antibody–drug conjugate was developed in this study to treat wild‐type and mutant c‐Kit‐positive cancers. NN2101, a fully human IgG1, was conjugated to DM1, a microtubule inhibitor, through N‐succinimidyl‐4‐(N‐maleimidomethyl) cyclohexane‐1‐carboxylate (SMCC) (to give NN2101‐DM1). The antitumor activity of NN2101‐DM1 was evaluated in vitro and in vivo using various cancer cell lines. NN2101‐DM1 exhibited potent growth‐inhibitory activities against c‐Kit‐positive cancer cell lines. In a mouse xenograft model, NN2101‐DM1 exhibited potent growth‐inhibitory activities against imatinib‐resistant GIST and SM cells. In addition, NN2101‐DM1 exhibited a significantly higher anti‐cancer effect than carboplatin/etoposide against SCLC cells where c‐Kit does not mediate cancer pathogenesis. Furthermore, the combination of NN2101‐DM1 with imatinib in imatinib‐sensitive GIST cells induced complete remission compared with treatment with NN2101‐DM1 or imatinib alone in mouse xenograft models. These results suggest that NN2101‐DM1 is a potential therapeutic agent for wild‐type and mutant c‐Kit‐positive cancers.     

Pancreatic ductal adenocarcinoma cells regulated the gemcitabine‐resistance function of CAFs by LINC00460

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal malignancy with extremely poor prognosis. Gemcitabine resistance is a major challenge in the treatment of PDAC. Here, we showed that LINC00460 was associated with the response to gemcitabine both in PDAC patients and PDAC‐PDX. After knocking down LINC00460 in PDAC tumor cells, results of RNA sequencing followed by gene ontology analysis indicated that LINC00460 influenced the activity of growth factors and modified the extracellular matrix. FISH showed that LINC00460 is mostly located in the cytoplasm. Results of RNA pull‐down, LC–MS/MS, RIP, and immunoblotting confirmed that LINC00460 could directly bind to PDAP1. Furthermore, we demonstrated that LINC00460 mediated the cellular communication of PDAC tumor cells and CAFs by PDAP1/PDGFA/PDGFR signaling pathway and regulated the gemcitabine‐resistance function of CAFs, which could be reversed by treatment with a PDGFR inhibitor (crenolanib). PDAC‐PDX tumors with lower expression of LINC00460 showed a better response to gemcitabine plus crenolanib treatment. Our finding supported the application of LINC00460 in precision medicine that uses gemcitabine plus crenolanib to treat PDAC with low expression of LINC00460.     

Cadherin‐3 is a novel oncogenic biomarker with prognostic value in glioblastoma

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. The prognosis of patients is very poor, with a median overall survival of ~ 15 months after diagnosis. Cadherin‐3 (also known as P‐cadherin), a cell–cell adhesion molecule encoded by the CDH3 gene, is deregulated in several cancer types, but its relevance in GBM is unknown. In this study, we investigated the functional roles, the associated molecular signatures, and the prognostic value of CDH3/P‐cadherin in this highly malignant brain tumor. CDH3/P‐cadherin mRNA and protein levels were evaluated in human glioma samples. Knockdown and overexpression models of P‐cadherin in GBM were used to evaluate its functional role in vitro and in vivo. CDH3‐associated gene signatures were identified by enrichment analyses and correlations. The impact of CDH3 in the survival of GBM patients was assessed in independent cohorts using both univariable and multivariable models. We found that P‐cadherin protein is expressed in a subset of gliomas, with an increased percentage of positive samples in grade IV tumors. Concordantly, CDH3 mRNA levels in glioma samples from The Cancer Genome Atlas (TCGA) database are increased in high‐grade gliomas. P‐cadherin displays oncogenic functions in multiple knockdown and overexpression GBM cell models by affecting cell viability, cell cycle, cell invasion, migration, and neurosphere formation capacity. Genes that were positively correlated with CDH3 are enriched for oncogenic pathways commonly activated in GBM. In vivo, GBM cells expressing high levels of P‐cadherin generate larger subcutaneous tumors and cause shorter survival of mice in an orthotopic intracranial model. Concomitantly, high CDH3 expression is predictive of shorter overall survival of GBM patients in independent cohorts. Together, our results show that CDH3/P‐cadherin expression is associated with aggressiveness features of GBM and poor patient prognosis, suggesting that it may be a novel therapeutic target for this deadly brain tumor.     

AMP‐activated protein kinase activation primes cytoplasmic translocation and autophagic degradation of the BCR‐ABL protein in CML cells

Chronic myeloid leukemia is driven by the BCR‐ABL oncoprotein, a constitutively active protein tyrosine kinase. Although tyrosine kinase inhibitors (TKIs) have greatly improved the prognosis of CML patients, the emergence of TKI resistance is an important clinical problem, which deserves additional treatment options based on unique biological properties to CML cells. In this study, we show that metabolic homeostasis is critical for survival of CML cells, especially when the disease is in advanced stages. The BCR‐ABL protein activates AMP‐activated protein kinase (AMPK) for ATP production and the mTOR pathway to suppress autophagy. BCR‐ABL is detected in the nuclei of advanced‐stage CML cells, in which ATP is sufficiently supplied by enhanced glucose metabolism. AMP‐activated protein kinase is further activated under energy‐deprived conditions and triggers autophagy through ULK1 phosphorylation and mTOR inhibition. In addition, AMPK phosphorylates 14‐3‐3 and Beclin 1 to facilitate cytoplasmic translocation of nuclear BCR‐ABL in a BCR‐ABL/14‐3‐3τ/Beclin1/XPO1 complex. Cytoplasmic BCR‐ABL protein undergoes autophagic degradation when intracellular ATP is exhausted by disruption of the energy balance or forced autophagy flux with AMP mimetics, mTOR inhibitors, or arsenic trioxide, leading to apoptotic cell death. This pathway represents a novel therapeutic vulnerability that could be useful for treating TKI‐resistant CML.     

High PTX3 expression is associated with a poor prognosis in diffuse large B‐cell lymphoma

Tumor‐associated macrophages (TAMs) are associated with a poor prognosis of diffuse large B‐cell lymphoma (DLBCL). As macrophages are heterogeneous, the immune polarization and their pathological role warrant further study. We characterized the microenvironment of DLBCL by immunohistochemistry in a training set of 132 cases, which included 10 Epstein–Barr virus‐encoded small RNA (EBER)‐positive and five high‐grade B‐cell lymphomas, with gene expression profiling in a representative subset of 37 cases. Diffuse large B‐cell lymphoma had a differential infiltration of TAMs. The high infiltration of CD68 (pan‐macrophages), CD16 (M1‐like), CD163, pentraxin 3 (PTX3), and interleukin (IL)‐10‐positive macrophages (M2c‐like) and low infiltration of FOXP3‐positive regulatory T lymphocytes (Tregs) correlated with poor survival. Activated B cell‐like DLBCL was associated with high CD16, CD163, PTX3, and IL‐10, and EBER‐positive DLBCL with high CD163 and PTX3. Programmed cell death‐ligand 1 positively correlated with CD16, CD163, IL‐10, and RGS1. In a multivariate analysis of overall survival, PTX3 and International Prognostic Index were identified as the most relevant variables. The gene expression analysis showed upregulation of genes involved in innate and adaptive immune responses and macrophage and Toll‐like receptor pathways in high PTX3 cases. The prognostic relevance of PTX3 was confirmed in a validation set of 159 cases. Finally, in a series from Europe and North America ({"type":"entrez-geo","attrs":{"text":"GSE10846","term_id":"10846"}}GSE10846, R‐CHOP‐like treatment, n = 233) high gene expression of PTX3 correlated with poor survival, and moderately with CSF1R, CD16, MITF, CD163, MYC, and RGS1. Therefore, the high infiltration of M2c‐like immune regulatory macrophages and low infiltration of FOXP3‐positive Tregs is associated with a poor prognosis in DLBCL, for which PTX3 is a new prognostic biomarker.     

Orthogonal MET analysis in a population‐representative stage II–III colon cancer cohort: prognostic and potential therapeutic implications

Clinical trials for MET inhibitors have demonstrated limited success for their use in colon cancer (CC). However, clinical efficacy may be obscured by a lack of standardisation in MET assessment for patient stratification. In this study, we aimed to determine the molecular context in which MET is deregulated in CC using a series of genomic and proteomic tests to define MET expression and identify patient subgroups that should be considered in future studies with MET‐targeted agents. To this aim, orthogonal expression analysis of MET was conducted in a population‐representative cohort of stage II/III CC patients (n = 240) diagnosed in Northern Ireland from 2004 to 2008. Targeted sequencing was used to determine the relative incidence of MET R970C and MET T992I mutations within the cohort. MET amplification was assessed using dual‐colour dual‐hapten brightfield in situ hybridisation (DDISH). Expression of transcribed MET and c‐MET protein within the cohort was assessed using digital image analysis on MET RNA in situ hybridisation (ISH) and c‐MET immunohistochemistry (IHC) stained slides. We found that less than 2% of the stage II/III CC patient population assessed demonstrated a genetic MET aberration. Determination of a high MET RNA‐ISH/low c‐MET IHC protein subgroup was found to be associated with poor 5‐year cancer‐specific outcomes compared to patients with concordant MET RNA‐ISH and c‐MET IHC protein expression (HR 2.12 [95%CI: 1.27–3.68]). The MET RNA‐ISH/c‐MET IHC protein biomarker paradigm identified in this study demonstrates that subtyping of MET expression may be required to identify MET‐addicted malignancies in CC patients who will truly benefit from MET inhibition.     

Multiplex immune protein profiling of fine‐needle aspirates from patients with non‐small‐cell lung cancer reveals signatures associated with PD‐L1 expression and tumor stage

Biomarker signatures identified through minimally invasive procedures already at diagnosis of non‐small‐cell lung cancer (NSCLC) could help to guide treatment with immune checkpoint inhibitors (ICI). Here, we performed multiplex profiling of immune‐related proteins in fine‐needle aspirate (FNA) samples of thoracic lesions from patients with NSCLC to assess PD‐L1 expression and identify related protein signatures. Transthoracic FNA samples from 14 patients were subjected to multiplex antibody‐based profiling by proximity extension assay (PEA). PEA profiling employed protein panels relevant to immune and tumor signaling and was followed by Qlucore^® Omics Explorer analysis. All lesions analyzed were NSCLC adenocarcinomas, and PEA profiles could be used to monitor 163 proteins in all but one sample. Multiple key immune signaling components (including CD73, granzyme A, and chemokines CCL3 and CCL23) were identified and expression of several of these proteins (e.g., CCL3 and CCL23) correlated to PD‐L1 expression. We also found EphA2, a marker previously linked to inferior NSCLC prognosis, to correlate to PD‐L1 expression. Our identified protein signatures related to stage included, among others, CXCL10 and IL12RB1. We conclude that transthoracic FNA allows for extensive immune and tumor protein profiling with assessment of putative biomarkers of important for ICI treatment selection in NSCLC.     

Downregulation of miR‐26 promotes invasion and metastasis via targeting interleukin‐22 in cutaneous T‐cell lymphoma

It has been reported that certain microRNAs (miRNA) are associated with the pathogenesis of lymphoma. We have previously demonstrated that histone deacetylase inhibitors restore tumor‐suppressive miRNAs, such as miR‐16, miR‐29, miR‐150, and miR‐26, in advanced cutaneous T‐cell lymphoma (CTCL). Among these, the function of miR‐26 remains unclear. In this study, we aimed to reveal the function of miR‐26 in CTCL oncogenesis. First, we confirmed that the miR‐26 family was markedly dysregulated in CTCL cell lines and primary samples. In vivo analysis using miR‐26a‐transduced CTCL cells injected into immunodeficient NOG mice demonstrated the significant prolonged survival of the mice, suggesting that the miRNA had a tumor‐suppressive function. We performed gene expression assays and identified 12 candidate miR‐26 targets, namely RGS13, FAM71F1, OAF, SNX21, CDH2, PTPLB, IL22, DNAJB5, CASZ1, CACNA1C, MYH10, and CNR1. Among these, IL22 was the most likely candidate target because the IL‐22–STAT3–CCL20–CCR6 cascade is associated with tumor invasion and metastasis of advanced CTCL. In vitro analysis of IL22 and IL22RA knockdown and miR‐26 transduction demonstrated inhibited CTCL cell migration. In particular, IL22 knockdown induced cell apoptosis. Finally, we conducted in vivo inoculation analysis of mice injected with shIL22‐transfected CTCL cells, and found no tumor invasion or metastasis in the inoculated mice, although the control mice showed multiple tumor invasions and metastases. These results, along with our previous data, demonstrated that miR‐26 is a tumor suppressor that is associated with tumor invasion and the metastasis of advanced CTCL by regulating the IL‐22–STAT3–CCL20 cascade. Therefore, a IL‐22‐targeting therapy could be a novel therapeutic strategy for advanced CTCL.     

Pseudogene KRT17P3 drives cisplatin resistance of human NSCLC cells by modulating miR‐497‐5p/mTOR

Chemoresistance is a major obstacle in non–small cell lung cancer (NSCLC) treatment. The pseudogene keratin 17 pseudogene 3 (KRT17P3) has been previously shown to be upregulated in lung cancer tissues of patients with cisplatin resistance. In the present study, RT‐qPCR was performed to evaluate KRT17P3 levels in plasma samples collected from 30 cisplatin‐resistant and 32 cisplatin‐sensitive patients. We found that the plasma level of KRT17P3 is upregulated in cisplatin‐resistant patients, and the increased expression of plasma KRT17P3 is associated with poor chemotherapy response. Functional studies demonstrated that KRT17P3 overexpression in cultured NSCLC cells increases cell viability and decreases apoptosis upon cisplatin treatment in vitro and in vivo, while KRT17P3 knockdown has the opposite effect. Mechanistically, bioinformatics analysis, RNA immunoprecipitation, and dual luciferase reporter assay indicated that KRT17P3 acts as a molecular sponge for miR‐497‐5p and relieves the binding of miR‐497‐5p to its target gene mTOR. Rescue experiments validated the functional interaction between KRT17P3, miR‐497‐5p, and mTOR. Taken together, our findings indicate that KRT17P3/miR‐497‐5p/mTOR regulates the chemosensitivity of NSCLC, suggesting a potential therapeutic target for cisplatin‐resistant NSCLC patients. KRT17P3 may be a potential peripheral blood marker of NSCLC patients resistant to cisplatin.