Sara M Steinmann

Anabel Sánchez-Martín

Elisabeth Tanzer

Antonio Cigliano

Giovanni M Pes

Maria M Simile

Laurent Désaubry

Jose J G Marin

Matthias Evert

Diego F Calvisi
email: calvisid@uniss.it

Anabel Sánchmartín

Laurent Desaubry

eIF4A1 Is a Prognostic Marker and Actionable Target in Human Hepatocellular Carcinoma

Keywords: hepatocellular carcinoma, eIF4A1, translation inhibitors, targeted therapies

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Liver cancer is one of the most frequent malignancies and has poor prognosis, ranked as the third leading cause of cancer-related deaths worldwide and thus remains a global health issue [START_REF] Sung | GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[END_REF]. The incidence of liver cancer is increasing, and the World Health Organization has estimated that the number of deaths from this cancer will reach one million annually in 2030 [START_REF] Sleeman | The escalating global burden of serious health-related suffering: Projections to 2060 by world regions, age groups, and health conditions[END_REF]. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, accounting for ~90% of cases. HCC commonly develops in the setting of underlying liver diseases such as cirrhosis or chronic liver inflammation. Although hepatitis B or C virus infection and alcohol abuse remain major HCC risk factors, the number of HCC cases related to non-alcoholic fatty liver disease (NAFLD) or associated with metabolic syndrome or diabetes mellitus is rapidly growing, and this condition might become the leading cause of HCC in Western countries in the near future [START_REF] Akinyemiju | The Burden of Primary Liver Cancer and Underlying Etiologies From 1990 to 2015 at the Global, Regional, and National Level: Results from the Global Burden of Disease Study 2015[END_REF][START_REF] Younossi | Nonalcoholic Steatohepatitis Is the Fastest Growing Cause of Hepatocellular Carcinoma in Liver Transplant Candidates[END_REF]. Surgical resection, radiofrequency ablation, transarterial chemoembolization, and liver transplantation are potentially curative treatments for early-stage HCC lesions [START_REF] Grigorie | Curative Intent Treatment of Hepatocellular Carcinoma-844 Cases Treated in a General Surgery and Liver Transplantation Center[END_REF][START_REF] Vogel | Updated treatment recommendations for hepatocellular carcinoma (HCC) from the ESMO Clinical Practice Guidelines[END_REF]. However, most patients with HCC are diagnosed in an advanced stage of the disease when they are no longer good candidates for curative strategies. Palliative systemic therapy is the only therapeutic option for these patients. Several tyrosine multikinase inhibitors, including sorafenib, lenvatinib, regorafenib, and cabozantinib, are approved as the first-or second-line treatments for unresectable advanced HCC. However, only a slight increase in overall survival has been reached in these patients [START_REF] Marin | Mechanisms of Pharmacoresistance in Hepatocellular Carcinoma: New Drugs but Old Problems[END_REF][START_REF] Llovet | Sorafenib in advanced hepatocellular carcinoma[END_REF][START_REF] Bruix | Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): A randomised, double-blind, placebo-controlled, phase 3 trial[END_REF][START_REF] Abou-Alfa | Cabozantinib in Patients with Advanced and Progressing Hepatocellular Carcinoma[END_REF]. Immune-based therapies are also emerging for the treatment of advanced HCC. The combination of azetolizumab (anti-PD-L1) with bevacizumab (anti-VEGF) has shown superior efficacy compared to sorafenib, and it is now recommended as the standard first-line treatment for advanced HCC [START_REF] Vogel | Updated treatment recommendations for hepatocellular carcinoma (HCC) from the ESMO Clinical Practice Guidelines[END_REF][START_REF] Finn | Atezolizumab plus Bevacizumab in Unresectable Hepatocellular Carcinoma[END_REF]. Nevertheless, the benefits of immunotherapy are still modest, and many patients remain non-responders. Therefore, developing novel therapeutic strategies is imperative to improving the outcome of HCC patients.

Targeted inhibition of translation machinery is emerging as a promising cancer treatment approach. Protein synthesis is a highly controlled process that plays a major role in gene expression regulation. However, this mechanism is frequently dysregulated in human malignancies [START_REF] Bhat | Targeting the translation machinery in cancer[END_REF]. Studies have shown that altered mRNA translation leads to tumorigenesis and cancer progression by selectively enhancing the synthesis of proteins involved in cell proliferation, activation of invasion and metastasis, and other neoplastic characteristics-related processes [START_REF] Silvera | Translational control in cancer[END_REF]. Translation initiation is the rate-limiting step; it is regulated by multiple eukaryotic initiation factors (eIFs) [START_REF] Sonenberg | Regulation of translation initiation in eukaryotes: Mechanisms and biological targets[END_REF][START_REF] Jackson | The mechanism of eukaryotic translation initiation and principles of its regulation[END_REF]. The eIF4F complex, which includes eIF4E, eIF4G, and eIF4A proteins, is essential for cap-dependent ribosome recruitment and translation initiation. Thus, eIF4E binds to the m 7 G-cap structure at the mRNA '5' end, whereas eIF4G acts as a scaffold protein recruiting eIF4E and eIF4A. The latter, the only component of the eIF4F complex with enzymatic activity, is an ATP-dependent DEAD-box RNA helicase that unwinds the mRNA secondary structures in the 5 untranslated regions (5 -UTRs) to enable ribosome scanning. The helicase eIF4A is required to efficiently translate mRNAs with long and highly structured 5 -UTRs, including those with G-quadruplex structures [START_REF] Wolfe | RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer[END_REF]. These complex structures characterize the 5-UTRs of many oncogenes considered eIF4A-dependent genes, such as KRAS, BLC2, NOTCH1, CDK6, and CCND1 [START_REF] Singh | Targeting eIF4A-Dependent Translation of KRAS Signaling Molecules[END_REF][START_REF] Rubio | Transcriptome-wide characterization of the eIF4A signature highlights plasticity in translation regulation[END_REF].

There are two eIF4A isoforms involved in mRNA translation, eIF4A1 and eIF4A2, which share ~90% homology in their amino acid sequence [START_REF] Li | Eukaryotic translation initiation factor 4AIII (eIF4AIII) is functionally distinct from eIF4AI and eIF4AII[END_REF]. Despite their similarity, eIF4A1, but not eIF4A2, is essential to cell viability. In addition, eIF4A2 cannot compensate for eIF4A1 dysfunction, suggesting that both isoforms have distinct biological properties [START_REF] Lu | The diverse roles of the eIF4A family: You are the company you keep[END_REF][START_REF] Galicia-Vázquez | A cellular response linking eIF4AI activity to eIF4AII transcription[END_REF]. Their expression also varies between different types of tissues, with eIF4A1 being the most abundant paralog [START_REF] Nielsen | The mouse protein synthesis initiation factor 4A gene family includes two related functional genes which are differentially expressed[END_REF]. The overexpression of the eIF4A1 isoform occurs in a wide variety of malignancies, where it correlates with metastasis and poor prognoses [START_REF] Gao | High intratumoral expression of eIF4A1 promotes epithelial-to-mesenchymal transition and predicts unfavorable prognosis in gastric cancer[END_REF][START_REF] Liang | Decreased expression of EIF4A1 after preoperative brachytherapy predicts better tumor-specific survival in cervical cancer[END_REF][START_REF] Modelska | The malignant phenotype in breast cancer is driven by eIF4A1-mediated changes in the translational landscape[END_REF]. The c-Myc protooncogene upregulates EIF4A1 transcription [START_REF] Lin | c-Myc and eIF4F are components of a feedforward loop that links transcription and translation[END_REF]; in addition, the levels of free functional eIF4A1 protein are increased by the degradation of the tumor suppressor programmed cell death 4 (PDCD4), which is itself regulated by mammalian target of rapamycin (mTOR) signaling [START_REF] Dorrello | S6K1-and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth[END_REF][START_REF] Roux | Signaling Pathways Involved in the Regulation of mRNA Translation[END_REF]. Although little is known about eIF4A2 in cancer, recent studies have revealed the upregulation of this isoform in colorectal cancer [START_REF] Chen | Eukaryotic initiation factor 4A2 promotes experimental metastasis and oxaliplatin resistance in colorectal cancer[END_REF] and paclitaxel-resistant breast cancer [START_REF] Liu | MicroRNA-5195-3p enhances the chemosensitivity of triple-negative breast cancer to paclitaxel by downregulating EIF4A2[END_REF]. High eIF4A2 levels are also associated with poor prognosis in colorectal and esophageal squamous cell carcinomas [START_REF] Chen | Eukaryotic initiation factor 4A2 promotes experimental metastasis and oxaliplatin resistance in colorectal cancer[END_REF][START_REF] Lyu | High expression of eIF4A2 is associated with a poor prognosis in esophageal squamous cell carcinoma[END_REF].

Consequently, the characteristics of eIF4A isoforms make them attractive drug targets for anti-tumor therapy. Rocaglates, also known as flavaglines, are a group of small molecules harboring a common cyclopean[b]benzofuran core that suppresses cap-dependent translation by inhibiting eIF4A activity. More than one hundred natural rocaglates have been isolated from Aglaia species, including silvestrol and rocaglamide. In addition, numerous synthetic derivatives have recently been developed to improve their potency and bioavailability [START_REF] Kim | Potential of cyclopentabbenzofurans from Aglaia species in cancer chemotherapy[END_REF][START_REF] Pan | silvestrol and structurally related bioactive compounds from Aglaia species[END_REF][START_REF] Ebada | Chemistry and biology of rocaglamides (= flavaglines) and related derivatives from aglaia species (meliaceae)[END_REF]. The rocaglates stabilize the non-specific binding of eIF4A to RNA, which prevents its incorporation into the eIF4F complex [START_REF] Iwasaki | Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor[END_REF][START_REF] Cencic | Antitumor Activity and Mechanism of Action of the Cyclopentabbenzofuran, Silvestrol[END_REF], and they target both eIF4A isoforms [START_REF] Chu | Amidino-Rocaglates: A Potent Class of eIF4A Inhibitors[END_REF]. Silvestrol, one of the best-studied drugs of this family, has demonstrated potent anti-tumor activity both in vitro and in vivo [START_REF] Bordeleau | Therapeutic suppression of translation initiation modulates chemosensitivity in a mouse lymphoma model[END_REF][START_REF] Chen | Silvestrol induces early autophagy and apoptosis in human melanoma cells[END_REF], and the antiproliferative potency of other active rocaglates derivatives, such as the synthetic compound CR-1-31-B, has also been reported in several tumors [START_REF] Zhang | Targeting translation initiation by synthetic rocaglates for treating MYC-driven lymphomas[END_REF][START_REF] Cao | Targeting eIF4A using rocaglate CR-1-31B sensitizes gallbladder cancer cells to TRAIL-mediated apoptosis through the translational downregulation of c-FLIP[END_REF][START_REF] Manier | Inhibiting the oncogenic translation program is an effective therapeutic strategy in multiple myeloma[END_REF][START_REF] Chan | eIF4A supports an oncogenic translation program in pancreatic ductal adenocarcinoma[END_REF]. Similarly, the synthetic flavagline FL3 exhibited anticancer properties in various experimental cancer models [START_REF] Wang | Prohibitin ligands: A growing armamentarium to tackle cancers, osteoporosis, inflammatory, cardiac and neurological diseases[END_REF]. Furthermore, the synthetic eIF4A inhibitor, Zotatifin, is currently being evaluated in a phase I/II clinical trial for solid tumors [START_REF]A Phase 1-2 Dose-Escalation and Cohort-Expansion Study of Intravenous Zotatifin (eFT226) in Subjects with Selected Advanced Solid Tumor Malignancies[END_REF].

In this study, we investigated the pathogenetic role of eIF4A1 in human HCC and its potential usefulness as a novel molecular target for the development of novel pharmacological strategies.

Results

2.1. eIF4A1 Is Significantly Overexpressed in Human Hepatocellular Carcinoma and Correlates with a Worse Outcome First, eIF4A1 mRNA levels in human cancers were investigated using data obtained from The Cancer Genome Atlas (TCGA) and the UALCAN (http://ualcan.path.uab. edu/; accessed on 24 November 2022) analysis tool. eIF4A1 mRNA expression was elevated in various cancer types, including colon adenocarcinoma (COAD), glioblastoma multiforme (GBM), esophageal carcinoma (ESCA), and kidney renal clear cell carcinoma (KIRC), among others (Figure 1A). In HCC (normal tissue: n = 50; tumor tissue: n = 371), eIF4A1 was significantly overexpressed (Figure 1A; LIHC: liver hepatocellular carcinoma; Figure 1B; P = 8.871× 10 -10 ) with comparable incidences for women (P = 2.566 × 10 -10 ) and men (P = 2.155 × 10 -10 ; Figure 1C) and elevated expression for patients aged between 21 and 80 years (Figure 1D; 21-40 years: P = 1.463 × 10 -06 ; 41-60 years: P = 2.841 × 10 -08 ; 61-80 years: P = 7.070 × 10 -09 ) compared to that in normal tissue. Maximal eIF4A1 expression was detected in tumors of young patients between 21 and 40 years of age (Figure 1D; 21-40 vs. 61-80 years: P = 2.678 × 10 -02 ). Examination of multiple clinicopathological features revealed that HCC patients with high eIF4A1 expression had poorer prognoses. The survival time of patients with high eIF4A1 expression was significantly shorter than that of patients with low or medium eIF4A1 levels (Figure 1E; P = 0.027). Furthermore, the TCGA data showed that in comparison to normal tissue, eIF4A1 expression was increased in all tumor grades, with grades 3 and 4 tumors revealing even higher levels than grades 1 and 2 tumors (Figure 1F; Normal vs. Grade 1: P = 2.359 × 10 -03 ; Normal vs. Grade 2: P = 3.212 × 10 -09 ; Normal vs. Grade 3: P = 4.581 × 10 -09 ; Normal vs. Grade 4: P = 6.219 × 10 -03 ; Grade 1 vs. Grade 3: P = 5.378 × 10 -04 ; Grade 1 vs. Grade 4: P = 3.380 × 10 -02 ; Grade 2 vs. Grade 3: P = 2.050 × 10 -02 ; Grade 2 vs. Grade 4: P = 2.020 × 10 -02 ). The analysis of HCC cancer stages (displayed significantly increased eIF4A1 expression in stage 1 to 3 tumors (Figure 1G; Normal vs. Stage 1: P = 1.878 × 10 -08 ; Normal vs. Stage 2: P = 3.364 × 10 -07 ; Normal vs. Stage 3: P = 1.058 × 10 -06 ; Stage 1 vs. Stage 3: P = 2.366 × 10 -02 ). Importantly, high eIF4A1 expression was found in N0 and N1 nodal metastasis status (Figure 1H; Normal vs. N0: P = 4.421 × 10 -12 ; Normal vs. N1: P = 1.023 × 10 -04 ), with a clear tendency toward enhanced eIF4A1 expression in N1 specimens.

To confirm the findings in the TCGA dataset analysis, the eIF4A1 levels in 47 human HCC samples and paired normal tissues collected at the University Regensburg were evaluated. In compliance with the TCGA data set, western blot analysis revealed that eIF4A1 protein levels were significantly increased in HCC specimens compared to those in paired non-tumorous tissues (Figure 2; *** p = 0.0002; Wilcoxon test; Supplementary Figure S1). In addition, immunoblotting showed augmented protein expression of the eukaryotic initiation factor (eIF) family member eIF4A2 (* p = 0.0249; Wilcoxon test) and the translation initiator and rocaglate target DEAD-box protein 3 DDX3 [START_REF] Chen | Dual targeting of DDX3 and eIF4A by the translation inhibitor rocaglamide A[END_REF] (** p = 0.0088; Wilcoxon test) in HCC lesions (Figure 2A,B; Supplementary Figure S1). Remarkably, when assessing the prognostic relevance of the three proteins, eIF4A1 levels were inversely associated with patient survival time (p < 0.0001; Figure 3A; Supplementary Materials). In contrast, levels of eIF4A2 did not separate HCC patients based on survival length (P = 0.741; Figure 3B; Supplementary Materials). Moreover, DDX3 displayed a trend toward more prolonged survival without reaching significance (P = 0.142; Figure 3C; Supplementary Materials). Subsequently, we evaluated the levels of eIF4A1 in a large collection of formalinfixed, paraffin-embedded HCC samples (n = 356) by immunohistochemistry (Figure 4). In agreement with western blot data, increased immunoreactivity for eIF4A1 was detected ubiquitously in HCC lesions compared to that in the matching non-neoplastic liver tissues (Figure 4, first lower panel). In contrast, non-tumorous epithelial cells exhibited faint or absent eIF4A1 staining (Figure 4, second lower panel). HCC lesions displayed either homogeneous or scattered upregulation of eIF4A1 (Figure 4, third to fifth lower panels). Overall, the present data indicated that eIF4A1 is highly expressed in primary HCC lesions and associated with a poor outcome. These findings suggest the usefulness of eIF4A1 as a prognostic biomarker in human HCC.

Targeting eIF4A1 with Rocaglates Inhibits Tumor Cell Growth in HCC Cell Lines

Next, to evaluate the therapeutic potential of targeting eIF4A1 activity in HCC, we challenged human HCC cell lines in vitro with five distinct rocaglate derivatives, namely CR-1-31-B, FL3, Rocaglamide, Silvestrol, and Zotatifin. These drugs promote the binding affinity of eIF4A1 to mRNA species containing polypurine sequence motifs, sequentially impeding the translation initiation during protein synthesis by blocking the formation of the heterotrimeric eIF4F cap-binding complex. Prior to pharmacological treatments, we assessed the endogenous eIF4A1, eIF4A2, and DDX3 protein levels using western blot analysis in various human HCC cell lines. eIF4A1, eIF4A2, and DDX3 proteins were expressed in all HCC cell lines tested (Figure 5A,B; Supplementary Figure S2).

Subsequently, to assess the cytotoxic potential of eIF4A inhibitors, the HLE, HLF, and PLC/PRF/5 tumor cell lines were randomly selected and treated with a range of concentrations between 0 and 200 nM per eIF4A inhibitor, and colorimetric MTT assays were performed 48 h after starting compound exposure. Principally, cellular metabolic activity was measured to indicate cell viability, proliferation, and cytotoxicity. Overall, all five rocaglates efficiently reduced tumor cell viability in the three cell lines in a dosedependent manner (Figure 6A-C). Notably, in HCC cell lines, the calculated half-maximal inhibitory concentration (IC50) values for the five drugs resided in the low nanomolar range (Figure 6D). To further elucidate the mechanisms of action of eIF4A inhibitors in HCC cell lines, we selected the two most effective drugs, i.e., CR-1-31-B and Zotatifin, for the following experiments. Zotatifin was also selected because, to date, it is the only eIF4A inhibitor in clinical trials. When evaluating cell proliferation, we found that incubation with CR-1-31-B and Zotatifin induced a marked reduction in BrdU incorporation in both HLE and HLF cell lines, with a slightly more pronounced anti-growth effect in the case of CR-1-31-B (Figure 7A,B). Next, we assessed the effects of CR-1-31-B and Zotatifin on some of the most relevant oncogenic pathways in hepatocarcinogenesis by western blot analysis (Figure 9; Supplementary Figures S3 andS4). Both drugs did not affect eIF4A1 and DDX3 protein levels, supporting the hypothesis that they act on eIF4A1 and DDX3 activity without af-fecting their expression. In contrast, similarly to what has been described in other tumor types, the treatment of HCC cells with these drugs led to eIF4A2 upregulation, which is considered a compensatory response to marked eIF4A1 suppression. Nonetheless, it has been shown that eIF4A2 induction cannot functionally compensate for eIF4A1 loss or inactivation [START_REF] Lu | The diverse roles of the eIF4A family: You are the company you keep[END_REF][START_REF] Galicia-Vázquez | A cellular response linking eIF4AI activity to eIF4AII transcription[END_REF]. Regarding oncogenic pathways, both CR-1-31-B and Zotatifin administration resulted in the downregulation of activated/phosphorylated (p-)STAT3, AKT, ERK1/2, and total SKP2 proteins in a dose-dependent manner. In addition, levels of the Hippo pathway effector protein TAZ were decreased, whereas the effects on the ortholog YAP were inconsistent. Furthermore, the levels of prohibitin 1 and 2 (PHB1 and 2), which are supposed to be critical targets of rocaglates [START_REF] Nebigil | Flavaglines as natural products targeting eIF4A and prohibitins: From traditional Chinese medicine to antiviral activity against coronaviruses[END_REF], were decreased in CR-1-31-B-and Zotatifin-treated cells. In contrast, levels of mTOR effectors, such as activated/phosphorylated (p-)RPS6 and phosphorylated/inactivated (p-)4EBP1, were either increased or unchanged following CR-1-B-31 and Zotatifin administration.

Targeting the mTOR Pathway Synergizes with eIF4A Inhibitors to Restrain HCC Cell Growth In Vitro

Inspired by the findings that eIF4A inhibitors did not reduce the activation of mTOR effectors, we tested whether mTOR suppression could synergize with eIF4A inhibitors to hamper HCC cell growth. For this purpose, the HLE and HLF cell lines were treated with the pan mTOR inhibitor, Rapalink-1 [START_REF] Rodrik-Outmezguine | Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor[END_REF], either alone or in combination with CR-1-31-B or Zotatifin. Rapalink-1 was chosen over common mTORC1 inhibitors, such as Rapamycin or Rapamycin homologs, because the latter drugs effectively inhibit RPS6 but not 4EBP1 [START_REF] Lu | Role of the Mammalian Target of Rapamycin Pathway in Liver Cancer: From Molecular Genetics to Targeted Therapies[END_REF].

When assessing HLE and HLF metabolic activity with the MTT assay, we discovered that Rapalink-1 inhibited HCC cell growth at nanomolar concentrations (IC50: 20 nM). Significantly, the combination of Rapalink-1 with CR-1-31-B or Zotatifin triggered an even more potent, synergistic reduction of HLE and HLF metabolic activity than that induced by treatments alone (Figure 10). Subsequently, we assessed proliferation and apoptosis in the same cell lines using Rapalink-1 alone and in combination with CR-1-B-R1 or Zotatifin. Again, the combined treatments resulted in a significant reduction in proliferation (Figure 11A) and a robust increase in apoptosis (Figure 11B) in the two cell lines. Finally, western blot analysis confirmed that the mTOR inhibitor, Rapalink-1, alone and in combination with the eIF4A inhibitors, CR-1-31-B or Zotatifin, fully inhibited p-RPS6 and p-4EBP1 levels. In addition, Rapalink-1 alone resulted in the downregulation of eIF4A1, eIF4A2, and DDX3, whereas inconsistent results were obtained for these proteins when Rapalink-1 was combined with CR-1-31-B or Zotatifin (Figure 12; Supplementary Figure S5). These data indicated that targeting the eIF4A family and mTOR oncogenic pathway might be a potent anti-tumor treatment strategy in HCC cell lines (Figure 12; Supplementary Figure S5).

Discussion

HCC, the most common liver primary cancer, is characterized by its late diagnosis, increasing incidence worldwide, and limited treatment options [START_REF] Llovet | Hepatocellular carcinoma[END_REF]. Despite the most recent and effective therapeutic strategies [START_REF] Finn | Atezolizumab plus Bevacizumab in Unresectable Hepatocellular Carcinoma[END_REF][START_REF] D'alessio | The evolving treatment paradigm of advanced hepatocellular carcinoma: Putting all the pieces back together[END_REF][START_REF] Garuti | The changing scenario of hepatocellular carcinoma in Italy: An update[END_REF][START_REF] Facciorusso | Efficacy of Regorafenib in Hepatocellular Carcinoma Patients: A Systematic Review and Meta-Analysis[END_REF], the prognosis for advanced HCC remains poor [START_REF] Finn | Evolution of Systemic Therapy for Hepatocellular Carcinoma[END_REF]. Therefore, the identification of novel molecular targets and development of more efficacious therapies are imperative to improving the life expectancy of HCC patients.

Cumulating evidence indicates that translation dysregulation is critical to tumor development and progression [START_REF] Xue | Expression and Functional Roles of Eukaryotic Initiation Factor 4A Family Proteins in Human Cancers[END_REF]. Therefore, targeting translation initiation components might be a potential strategy for therapeutic interventions against cancer [START_REF] Jiang | Targeting translation regulators improves cancer therapy[END_REF]. In the present study, we investigated in silico and in vitro the pathogenetic and therapeutic relevance of eIF4A1, a pivotal player in initiating cap-dependent protein translation, in HCC [START_REF] Li | Eukaryotic translation initiation factor 4AIII (eIF4AIII) is functionally distinct from eIF4AI and eIF4AII[END_REF][START_REF] Jiang | Targeting translation regulators improves cancer therapy[END_REF]. Our results strongly suggest a pro-tumorigenic function of eIF4A1. Specifically, we show that HCC patients with high levels of eIF4A1 mRNA and protein carry poor outcomes. Thus, eIF4A1 might represent a novel prognostic marker in this disease. Accordingly, a negative prognostic role of eIF4A1 has been previously detected in other tumor types, such as lung adenocarcinoma [START_REF] Wu | Characterization of the Oncogenic Potential of Eukaryotic Initiation Factor 4A1 in Lung Adenocarcinoma via Cell Cycle Regulation and Immune Microenvironment Reprogramming[END_REF], clear cell renal carcinoma [START_REF] Zhang | High expression of eIF4A1 predicts unfavorable prognosis in clear cell renal cell carcinoma[END_REF], and gastric cancer [START_REF] Gao | High intratumoral expression of eIF4A1 promotes epithelial-to-mesenchymal transition and predicts unfavorable prognosis in gastric cancer[END_REF], among many others.

Furthermore, we revealed that targeting eIF4A1 using rocaglates induced strong growth suppression of human HCC cells by inhibiting proliferation and triggering apoptosis. Notably, the growth inhibitory activity of these compounds on HCC cells occurred at low nanomolar concentrations, implying their potency, at least in vitro. These data, confirming previous findings obtained in different tumor types, support the targeting of eIF4A1 as a promising therapeutic strategy against HCC [START_REF] Jiang | Targeting translation regulators improves cancer therapy[END_REF].

At the molecular level, we discovered that various oncoproteins belonging to the JAK/STAT, ERK/MAPK, AKT, and Hippo/TAZ pathways are markedly downregulated by eIF4A inhibitors. On the other hand, we revealed that treatment with these compounds did not affect the activity of members of the mTOR signaling cascade. Strikingly, exposure of HCC cells to combinations of eIF4A inhibitors and the mTOR inhibitor, Rapalink-1, a thirdgeneration mTOR inhibitor that links rapamycin and MLN0128 [START_REF] Rodrik-Outmezguine | Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor[END_REF], resulted in synergistic cytostatic effects. Indeed, this treatment led to higher suppression of proliferation and induction of massive apoptosis compared to that induced by incubation with one of the compounds alone. To the best of our knowledge, this is the first report showing the synergistic effects of eIF4A and mTOR inhibitors against the growth of HCC cells. Furthermore, preliminary data from our laboratory indicate that this drug combination is also synergistic in other tumor entities in vitro, suggesting that cancer cells might be highly vulnerable to eIF4A and mTOR pathway inhibition.

Moreover, this is the first study to demonstrate in the setting of HCC that Rapalink-1-induced inhibition of mTOR signaling can be achieved through suppression of two key downstream substrates, p-RPS6 Ser235/236 and p-4EBP1 Thr37/46 , even in the presence of eIF4A inhibitors. Hence, we suggest that a combinatory therapy with eIF4A inhibitors and Rapalink-1, a drug that was originally developed to overcome mTOR resistance mutations, might serve as a refinement strategy for systemic management of HCC by simultaneously and effectively targeting both mTOR signaling and eIF4A-dependent translation. Overall, the present data indicate that eIF4A is a potential prognostic biomarker in HCC patients and a promising therapeutic target in this deadly tumor type. In particular, eIF4A inhibitors displayed formidable cytostatic activity against HCC cells in vitro, further augmented in combination with an mTOR inhibitor. Although the antineoplastic effectiveness of these drugs, alone and in combination, requires validation in vivo, the present data suggest that targeting translation initiation can provide an innovative and valuable strategy in the development of novel treatments against human HCC.

Materials and Methods

Human Tissue Specimens

Forty-seven human HCC tumor tissue samples and paired surrounding non-tumorous tissues were collected at the Institute of Pathology at the University of Regensburg (Regensburg, Germany). Patient features are summarized in Supplementary Table S1. HCC tumors were divided between shorter survival/poorer outcome (HCCP; n = 25) and longer survival/better outcome (HCCB; n = 22), characterized by <3 and >3 years' survival following partial liver resection, respectively. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Clinical Research Ethics Committee of the Medical University of Regensburg (protocol code 17-1015-101; 4 July 2018).

Cell Lines and Reagents

The human HCC cell lines HLE (JCRB0404, Xenotech, Kansas City, MO, USA), HLF (JCRB0405, Tebu-bio GmbH, Offenbach, Germany), PLC/PRF/5 (300315, CLS Cell lines Service GmbH, Eppelheim, Germany), SNU182 (CRL-2235, LGC Standards GmbH), and SNU449 (CRL-2234, LGC Standards GmbH) were cultured at 37 • C in a 5% CO 2 humidified atmosphere. HLE, HLF, and PLC/PRF/5 cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM, high glucose, Anprotec, Bruckberg, Germany), and SNU182 and SNU449 cell lines were maintained in RPMI 1640 (Anprotec) supplemented with 10% (v/v) FCS (Anprotec, Bruckberg, Germany), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 1% penicillin/streptomycin solution (all from Anprotec). Mycoplasma-free status for all cell lines was monitored regularly using the PCR Mycoplasma Test Kit I (PK-CA91-1096, PromoCell, Heidelberg, Germany), and cell line authentication was performed by Cell Lines Service (Eppelheim, Germany). CR-1-31-B (HY-136453, MedChemExpress, Cas. No. 1352914-52-3), FL3 [START_REF] Thuaud | Synthetic analogue of rocaglaol displays a potent and selective cytotoxicity in cancer cells: Involvement of apoptosis inducing factor and caspase-12[END_REF]

MTT Viability, Proliferation, and Apoptosis Assays

Cells were seeded in flat-bottom 96 well plates at a density of 10 x 10 4 cells per well. After an overnight attachment period, cells were exposed to various concentrations of CR-1-31-B, FL3, Rocaglamide, Silvestrol, Zotatifin, and Rapalink-1 alone or CR-1-31-B-Rapalink-1 and Zotatifin-Rapalink-1 combinations for 48 h. Cells treated with DMSO and wells containing only culture medium served as negative and background controls, respectively.

For the MTT assay, following treatment, 10 µL of 5 mg/mL methyl-thiazolyl-diphenyltetrazolium bromide (MTT) solution was added per 96 well and incubated at 37 • C and under 5% CO 2 for 2 h. The medium was fully aspirated, and 100 µL of 100% (v/v) DMSO was added per well to dissolve the formazan crystals. After 15 min of shaking at room temperature, the absorbances were measured at 570 and 630 nm using the FLUORstar Omega multiplate reader and MARS data analysis software (both from BMG Labtech, Ortenberg, Germany). All experiments were performed in triplicate or quadruplicate and repeated at least twice. The average absorbance of the DMSO-treated cells was defined as 100% cell viability. IC50 values and standard deviations were calculated using GraphPad Prism software. Average cell viability values of three independent MTT assay experiments were employed to determine dose-effect curves (Fa = fraction affected) and the combination indices of two drugs using CompuSyn PC software version 1 (ComboSyn Inc., Paramus, NJ, USA).

Cell proliferation was evaluated in the cell lines at the 48-h time point using the BrdU Cell Proliferation Assay Kit (Cell Signaling Technology, Danvers, MA, USA). Briefly, following drug treatment, cells were incubated with 1× bromodeoxyuridine (BrdU) for 2 h and fixed for 30 min at room temperature. The fixing solution was discarded, and cells were incubated with the BrdU mouse detection antibody for 1 h at room temperature. After washing, cells were stained with HRP-conjugated anti-mouse secondary antibody for 30 min at room temperature and washed again. After incubation with TMB substrate solution for a further 30 min at room temperature, stop solution was added, and the absorbance was measured at 450 nm. The results are expressed as fold-change over DMSO control.

Apoptosis was determined in the HCC cell lines using the Cell Death Detection Elisa plus Kit (Roche Molecular Biochemicals, Indianapolis, IN, USA), following the manufacturer's instructions. All cell line experiments were repeated at least three times in triplicate.

Protein Extraction, Western Blot Analysis, and Smart Protein Layers (SPL) Approach

Human HCC tumor and non-tumorous liver tissues were homogenized in 100-250 µL of T-PER TM lysis buffer (78510, Thermo Fisher Scientific) containing 1x Halt TM Protease and Phosphatase Inhibitor Cocktail (78443, Thermo Scientific) in the Bullet Blender Storm 24 (Next Advance, Troy, NY, USA) for 3 min at speed 8, placed in the rotator mixer for 30-60 min at +4 • C and sonicated. Lysis of pellets from the cell lines was performed in the same lysis buffer for 1 h on ice. Protein concentrations were determined using the colorimetric BioRad Protein Assay Dye Reagent Concentrate (500-0006, Bio-Rad, Hercules, CA, USA) with bovine serum albumin (BSA) as the standard.

For western blot analysis, protein lysates were denatured in 1× LDS Bolt TM sample buffer (B0007) and 1× Bolt TM reducing agent (B00009, both from Invitrogen) by boiling for 5 min at 95 • C. Samples with 10 µL of protein per lane were separated by SDS-PAGE on Bolt™ 4-12% Bis-Tris Mini Protein Gels (NW04125BOX) in 1× Bolt™ MES SDS Running Buffer (B000202, both from Invitrogen) and transferred onto nitrocellulose membranes (iBlot TM 2 Mini/Regular Transfer Stacks, IB23001/2) by electroblotting running program P0 in the iBlot™ 2 Gel Transfer Device (IB21001, both from Thermo Fisher Scientific). Membranes were blocked in EveryBlot Blocking Buffer (12010020, Bio-Rad) and probed at +4 • C overnight with specific antibodies: eIF4A1 (1:1000; ab31217, Abcam, Cambridge, UK); eIF4A2 (1:1000; ab31218, Abcam); DDX3 (1:1000; A300-474A, Bethyl Laboratories, Montgomery, USA); p-RPS6 Ser235//236 (4856), p-4EBP1 Thr3747 (2885), p-STAT3 Tyr705 (9145), AKT (4691),), ERK1/2 (4695), p-ERK1/2 Thr202/204 (4370), SKP2 (2652), YAP/TAZ (8418), p-YAP/TAZ (13008), PHB1 (2426), PHB2 (14085) (all 1:1000; Cell Signaling Technology); p-AKT Ser473 (1:1000; 66444-1-IG, Proteintech (Rosemont, IL, USA); and β-actin (1:5000; ab20272, Abcam). Horseradish peroxidase-conjugated secondary antibody (1:20,000) was incubated for 1 h at room temperature, and blots were imaged with Clarity Max ECL Western Blotting Substrate (1705062S, Bio-Rad) using ChemiDoc MP (17001402, Bio-Rad). Band volumes were quantified with Image Lab TM software version 6.0.1 (Bio-Rad) and normalized to GAPDH or β-actin.

High patient-to-patient variation of reference protein expression, such as GAPDH and β-actin, impedes proper quantification of western blot data. Hence, for accurate target protein quantification of human HCC samples, western blotting was performed using the Smart Protein Layers (SPL) Kit Red from NH DyeAgnostics (PR913, Halle, Germany) according to the manufacturer's instructions. The SPL technology is based on three components: (i) red-fluorescent Smart Label (SMA label) reagent for labeling and visualization of total protein on gels and blots, (ii) bi-fluorescent sample-specific standard Smartalyzer L (SMA basic L) for normalization, standardization, and quantification of total protein per sample, and (iii) a calibrator (CAL) for comparison of fluorescence and target protein signals derived from different gels. Briefly, before gel electrophoresis, every protein sample was labeled with 2 µL of SMA basic L and 1 µL of Smart label working solution. Samples with of 10 µg of protein per sample, 12 µL of calibrator (CAL; 12.5, 25, and 80 kDa), and 5 µL of Biotinylated Protein Ladder (SeeBlue TM Plus 2, Thermo Fisher Scientific) were applied to the gels. After conventional gel electrophoresis, the gel fluorescence of SMA basic L (signal: band at 80 kDa; channel: Alexa Fluor 488; gel loading control = GLO) and SMA label (signal: whole lane; channel: Cy5; gel total protein = GTO) were imaged. Following blotting, the blot fluorescence of the SMA label (signal: whole lane; channel: Cy5; blot total protein = BTO) and the blot chemiluminescent signal of the target protein (signal: band; chemiluminescence; band target protein = BTA) were detected. Finally, the normalization of gel load (based on SMA basic; GLO), normalization of Smart Label (based on SMA label), and normalization of target protein signal (based on total protein) were conducted using the SPL-normalization template following the manufacturer's protocol. Results were expressed as SPL normalized target volume. Visualization and quantification were performed using the Chemidoc MP Imaging System and Image Lab TM software version 6.0.1 (both from Bio-Rad).

Immunohistochemistry

Human liver specimens were harvested and fixed in 10% formalin overnight at 4 • C and embedded in paraffin. Hematoxylin and eosin (Thermo Fisher Scientific, Waltham, MA, USA) staining was conducted using a standard protocol on liver sections. Specifically, antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) by heating in a microwave on high for 10 min, followed by a 20-min cool down at room temperature. After blocking with 5% goat serum and the Avidin-Biotin blocking kit (Vector Laboratories, Burlingame, CA, USA), the slides were incubated with the anti-eIF4A1 primary antibody (1:100; ab31217, Abcam) overnight at 4 • C. Slides were then subjected to 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity and, subsequently, the biotin-conjugated secondary antibody was applied at a 1:500 dilution for 30 min at room temperature. Reaction detection was achieved using the Vectastain ABC-Elite Peroxidase Kit (Vector Laboratories, # PK-6100) with ImmPACT DAB (Vector Laboratories, SK-4105) as the chromogen. Slides were counterstained with Mayer's hematoxylin.

Statistical Analysis

GraphPad Prism version 9.3.1 software (GraphPad Software Inc., La Jolla, CA, USA) and IBM SPSS version 26 software (IBM Corp., Armonk, NY, USA) were employed to evaluate statistical significance. The p values for TCGA data were obtained from the UALCAN analysis tool. The non-parametric Wilcoxon signed-rank test was used for paired sample comparison. Kaplan-Meier curves were evaluated using the log-rank test. For calculation of IC50s, data were transformed to log2, normalized, and non-linear regression (log)inhibitor vs. response-variable slope (four parameters) was performed. For the transformation of SEM to SD, SEM was multiplied by the square root of the sample size n. Tukey's multiple comparison test was applied for multiple comparisons. Two-tailed values of * p < 0.5, ** p < 0.1, *** p < 0.01, and **** p < 0.001 were considered significant. Lowercase letters denoted statistical significance, as stated in the associated figure legends. All data are expressed as the mean ± SD or SEM.

Conclusions

Our results, obtained in silico and in vitro, constitute the initial step in an innovative direction to develop pharmacological strategies to treat liver cancer. This is particularly valuable owing to the lack of available tools for efficaciously treating advanced nonsurgically resectable HCC. The fact that eIF4A1 was consistently upregulated in HCC in comparison with adjacent non-tumor liver tissue and the magnitude of this change was negatively correlated with the prognosis of HCC patients supports a role of eIF4A1 in HCC malignancy. This led us to evaluate the impact of eIF4A1 inhibitors on HCC cell viability. Interestingly, even at low nanomolar concentrations, these inhibitors triggered a remarkable reduction in proliferation and augmented apoptosis, paralleled by the inhibition of several oncogenic pathways. Moreover, the cytostatic effect of eIF4A1 inhibitors was further increased by the pan mTOR inhibitor, Rapalink-1.

During the last decade, considerable efforts have been made to characterize the molecular basis for the high refractoriness of HCC to currently available first-and second-line drugs [START_REF] Marin | Molecular bases of the poor response of liver cancer to chemotherapy[END_REF]. This, together with the identification of new sensitizing targets, will permit us to overcome the current marked limitations in treating these patients, which in clinical practice means that they are subjected to treatments of low efficacy but with harmful side effects. In addition, unsatisfactory management delays the selection of alternative therapeutic options while allowing a Darwinian selection of the most resistant clones, which complicates the situation due to the development of cross-resistance.

In summary, the results of the present study highlight the pathogenic relevance of eIF4A1 in HCC and constitute an encouraging advance in developing new sensitizing strategies. Thus, the usefulness of combining eIF4A1 and mTOR inhibitors with currently used chemotherapeutic agents deserves further investigation.

Supplementary Materials:

The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms24032055/s1. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Figure 1 .

 1 Figure 1. Biostatistical analysis from TGCA data of the eIF4A1 gene in various tumor types and human hepatocellular carcinoma (HCC). Please refer to the main text for detailed explanations. (A) mRNA levels of eIF4A1 in various tumor types, including liver hepatocellular carcinoma (LIHC). (B) mRNA levels of eIF4A1 in non-tumorous (normal) and HCC (primary tumor) specimens. (C) mRNA levels of eIF4A1 in HCC specimens based on gender. (D) mRNA levels of eIF4A1 in HCC specimens based on age (Yrs: Years). (E) Kaplan-Meier curves in HCC patients based on eIF4A1 mRNA levels. (F) mRNA levels of eIF4A1 in HCC specimens based on tumor grade (grade 1: well-differentiated; grade 2: moderately differentiated; grade 3: poorly differentiated; grade 4: undifferentiated). (G) mRNA levels of eIF4A1 in HCC specimens based on the individual cancer stage (stage 1: cancers localized to one part of the body; stage 2 and 3: locally advanced cancers; stage 4: spread to other organs and often metastasized cancers). (H) mRNA levels of eIF4A1 in HCC specimens based on nodal metastasis status (N0: no evidence of cancer in regional lymph nodes; N1: cancer has spread to a single lymph node near the liver).

Figure 2 .

 2 Figure 2. eIF4A1 is upregulated in human hepatocellular carcinoma (HCC; n = 47). (A) Levels of eIF4A1, eIF4A2, and DDX3 proteins in human HCC samples, as detected by western blotting using the SPL method. (B) Quantification of western blot band intensities. Dots representing the individual protein expression of one patient and their medians are shown (* p < 0.5; ** p < 0.1; *** p < 0.01; Wilcoxon matched-pairs signed rank test). Abbreviations: P, patient; T, tumor; N, non-tumor; GLO: Gel loading control (green); GTO: Total protein (red).

Figure 3 .

 3 Figure 3. Levels of eIF4A1 protein negatively correlate with the survival of patients with hepatocellular carcinoma (HCC) (n = 47). Kaplan-Meyer curves in HCC patients for eIF4A1 (A), eIF4A2 (B), and DDX3 (C) proteins.

Figure 4 .

 4 Figure 4. Representative immunohistochemistry patterns of eIF4A1 protein in human hepatocellular carcinoma (HCC; n = 356). The first left panels show an example of human HCC (denominated HCC1) at low magnification. Note the enhanced immunoreactivity for eIF4A1 in the tumorous part (T) compared with the neighboring non-tumorous surrounding tissue (ST), which exhibits faint/absent eIF4A1 staining. The second and third left panels depict higher magnifications for ST and HCC1. A second tumor (HCC2), characterized by intense, homogeneous eIF4A1 immunoreactivity, is shown in the fourth panel from the left. Finally, a third tumor (HCC3) in the right panels displays heterogeneous eIF4A1 immunolabeling. Abbreviation: H&E, hematoxylin and eosin staining. Original magnifications: 40× in the first left panels, 200× in all the other panels. Scale bar: 500 µm in the first left panels, 100 µm in all the other panels.

Figure 5 .

 5 Figure 5. Human hepatocellular carcinoma cell lines express eIF4A1, eIF4A2, and DDX3 proteins. (A) The levels of the three proteins were assessed in HLE, HLF, PLF/PRF/5, SNU182, and SNU449 cell lines by western blot analysis. GAPDH was used as a loading control, and protein band intensities of eIF4A1, eIF4A2, and DDX3 were normalized to GAPDH levels (B).

Figure 6 .

 6 Figure 6. Cell viability of human hepatocellular carcinoma cell lines exposed to eIF4A inhibitors, as assessed by MTT assay. The eIF4A1 inhibitors, FL3, Zotatifin, CR-1-31-B, Rocaglamide, and Silvestrol, were administered to HLE (A), HLF (B), and PLC/PRF/5 (C) cell lines. Similar data were obtained in the SNU449 cell line (not shown). Data of two independent experiments with n = 4 technical replicates are represented as the percentage of DMSO-treated cells ± SD. A summary of the IC50 ± SD of the five drugs in the three cell lines is depicted in (D).

Figure 7 .

 7 Figure 7. Effects of the eIF4A inhibitors, CR-1-31-B and Zotatifin, on the proliferation of hepatocellular carcinoma cell line monolayer cultures indicated by BrdU incorporation. BrdU incorporation assay was conducted on (A) HLE and (B) HLF cells treated for 48 h with 10 nM CR-1-31-B and 20 nM Zotatifin. Untreated cells and cells treated with DMSO served as controls. The fold-changes over DMSO of optical densities (OD) at 450 nm are presented. All results are expressed as mean ± SD of three independent experiments in triplicate. For statistical analysis, Tukey's multiple comparisons test was performed (** p < 0.1; **** p < 0.001).Subsequently, apoptosis was determined in the same cell lines treated with the two eIF4A inhibitors. Both CR-1-31-B and Zotatifin induced higher apoptotic cell death in the cell lines than was observed in untreated and DMSO-treated cells (Figure8A,B).

Figure 8 .

 8 Figure 8. Effects of the eIF4A inhibitors, CR-1-31-B and Zotatifin, on the apoptosis of hepatocellular carcinoma cell line monolayer cultures. Cell death assay was conducted on (A) HLE and (B) HLF cells treated for 48 h with 10 nM CR-1-31-B and 20 nM Zotatifin. Untreated cells and cells treated with DMSO served as controls. The fold-changes over DMSO of optical densities (OD) at 405 nm are presented. All results are expressed as mean ± SD of three independent experiments in triplicate. For statistical analysis, Tukey's multiple comparisons test was performed (* p < 0.05; **** p < 0.001).

Figure 9 .

 9 Figure 9. Effects of the eIF4A inhibitors, CR-1-31-B and Zotatifin, in HCC cell lines on the levels of elements of oncogenic pathways involved in hepatocarcinogenesis. Western blot analysis was used to assess the levels of several effectors of oncogenic cascades in HLE (A) and HLF (B) cell lines exposed to increasing concentrations of CR-1-31-B and Zotatifin inhibitors (0, 10, 20, 50, and 100 nM). Cells were treated for 48 h, and western blot analysis was conducted at this time point.

Figure 10 .

 10 Figure 10. Highly synergistic cytotoxicity of combining the pan mTOR inhibitor, Rapalink-1, with CR-1-31-B and Zotatifin in hepatocarcinoma cell lines. (A) HLE and (D) HLF cells were exposed to various concentrations of compounds, alone or in combination, for 48 h. Cell proliferation based on the metabolic activity was assessed using the MTT assay and expressed as fold-change over DMSO control. The mean ± SEM of three independent experiments performed in technical triplicates are shown. (B,E) Dose effect curves of obtained fractions of dead cells (Fa) were used to determine the combination index (CI) (C,F) and synergism of drug combinations using CompuSyn software (https://www.combosyn.com, accessed on 1 November 2022) based on the Chou and Talalay method [50]. CI < 1 suggests synergism.

Figure 11 .

 11 Figure 11. Combining the pan mTOR inhibitor, Rapalink-1, with CR-1-31-B and Zotatifin results in synergistic anti-growth activity in hepatocarcinoma cell lines. (A) HLE and HLF cells were exposed to the three drugs, alone or in combination, for 48 h and proliferation was assessed. (B) HLE and HLF cells were exposed to the three drugs, alone or in combination, for 48 h and apoptosis was determined. For the experiments in (A,B), the drugs were administered at their IC50 concentrations. All results are expressed as mean ± SD of three independent experiments in triplicate. For statistical analysis, Tukey's multiple comparisons test was performed. Lowercase letters are used to denote statistical significance (a-f: p > 0.0001; a, vs. Control; b, vs. DMSO; c, vs. CR-1-31-B, d, vs. Zotatifin; e, vs. Rapalink-1; f, CR-1-31-B + Rapalink-1).

Figure 12 .

 12 Figure 12. Complete inactivation of mTOR effectors after synergistic eIF4A and mTOR inhibition using CR-1-31-B and Zotatifin in combination with Rapalink-1 in HCC cell lines. Western blot analysis in HLE and HLF cell lines challenged with CR-1-31-B (10 nM) and Zotatifin (10 nM) and Rapalink-1 (10 nM) inhibitors, alone or in combination (1:1). Cells were treated for 48 h, and western blot analysis was conducted at this time point.

  , Rapalink-1 (HY-111373, MedChemExpress, Cas. No. 1887095-82-0), Rocaglamide (HY-19356, MedChemExpress, Cas. No. 84573-16-0), Silvestrol (HY-13251, MCE, Cas. No. 697235-38-4), and Zotatifin (HY-112163, MedChemExpress, Cas. No. 2098191-53-6) were used for in vitro experiments. Stock solutions (1 and 10 mM) were prepared in dimethylsulfoxide (DMSO) and aliquots were stored at -20 • C.

Author Contributions:

  Individual contributions for this research article: Conceptualization, D.F.C.; methodology, A.S.-M., S.M.S., M.M.S., A.C., L.D. and E.T.; validation, D.F.C.; formal analysis, A.S.-M. and S.M.S.; investigation, A.S.-M., D.F.C. and S.M.S.; resources, D.F.C.; data curation, D.F.C.; writing-original draft preparation, A.S.-M., D.F.C. and S.M.S.; writing-review and editing, D.F.C., S.M.S., A.S.-M. and J.J.M.; visualization, A.S.-M., D.F.C. and S.M.S.; supervision, D.F.C., J.J.M. and G.M.P.; project administration, D.F.C.; funding acquisition, M.E. and D.F.C. All authors have read and agreed to the published version of the manuscript. Funding: DFC was supported by a grant from the Italian Association Against Cancer (AIRC) (grant number IG19175) and the Fondo di Ateneo per la Ricerca 2019 from the University of Sassari (Sassari, Italy). The funders had no role in the study design, data collection and analysis, publication decision, or manuscript preparation. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Clinical Research Ethics Committee of the Medical University of Regensburg (protocol code 17-1015-101; 4 July 2018).

Acknowledgments:

We thank Manfred Meyer and Ingrid Winkel (Institute of Pathology, University Regensburg, Regensburg, Germany) for their excellent technical support.

Data Availability Statement: Not applicable.

Conflicts of Interest:

The authors declare no conflict of interest.


Adèle Mennerat
email: adele.mennerat@cefe.cnrs.fr

Philippe Perret

Samuel P Caro

Philipp Heeb

Marcel M Lambrechts

Aromatic plants in blue tit Cyanistes caeruleus nests: no negative effect on bloodsucking Protocalliphora blow fly larvae

Keywords: blue tit, Cyanistes caeruleus, blow fly, Protocalliphora sp., ectoparasite, aromatic plants, nest protection hypothesis, anti-parasite defence

Nesting birds use several behavioural or physiological defence mechanisms against parasites. On Corsica, female blue tits (Cyanistes caeruleus) incorporate fresh fragments of a limited number of aromatic plants in the nest cup, from the end of nest construction until fledging. Some of these plants negatively affect bacterial growth and host location by blood-sucking mosquitoes in laboratory conditions. In natural populations, Corsican blue tit chicks are exposed to the highest levels of blood-sucking ectoparasitic blow flies (Protocalliphora sp.) reported in Europe. These ectoparasites can have severe negative effects on chick development and survival probabilities, especially when food constraints are elevated. Here we investigated in several natural Corsican blue tit populations the hypothesis that aromatic plants brought to the nest have anti-blow fly effects during the chick-rearing stage. We predicted that: 1) the amount of aromatic plants should be negatively related to blow fly infestation intensity across nests, 2) experimental addition of aromatic plants in nests should reduce blow fly infestation intensity, and 3) nestlings should be in better physical condition in nests where aromatic plants were experimentally added. No significant relation was found between the amount of aromatic plants in nests and blow fly infestation intensity. Experimental addition of aromatic plants did not reduce blow fly infestation intensity and did not affect the chick phenotypic parameters we measured. We conclude that aromatic plants in blue tit nests are not used as a defence against ectoparasitic Protocalliphora blow flies in our study population.

Introduction

Some bird species bring to their nests green plants which are rich in volatile secondary compounds (Wimberger 1984, Clark and[START_REF] Clark | Use of nest material as insecticidal and anti-pathogenic agents by the European Starling[END_REF]. The selected plant species often represent a small, non-random fraction of the available species in the habitat [START_REF] Gwinner | The function of green plants in nests of European starlings (Sturnus vulgaris)[END_REF][START_REF] Gwinner | Green plants in starling nests: effects on nestlings[END_REF][START_REF] Lambrechts | Aromatic herbs in Corsican blue tit nests: The 'potpourri' hypothesis[END_REF]. Three, non-mutually exclusive, main hypotheses on the functional significance of the use of greenery by birds have been proposed in the literature. First, breeding birds may exploit the anti-parasite properties of plant secondary compounds to repel, kill or impair the development of nest ectoparasites, and therefore affect host-parasite interactions (the nest protection hypothesis, e.g. Wimberger 1984, Clark and[START_REF] Clark | Use of nest material as insecticidal and anti-pathogenic agents by the European Starling[END_REF]. Second, the chemical compounds of fresh plant fragments may stimulate the host immune system independently from potential detrimental effects on ectoparasites, and perhaps reduce endoparasite loads or pathogen infection probabilities (the drug hypothesis, [START_REF] Gwinner | Green plants in starling nests: effects on nestlings[END_REF]. Finally, the use of green plants by males during nest building may play a role in mate attraction, for instance when the ability to find particular plants reflects aspects related to territory and/or individual quality beneficial to chicks (the courtship hypothesis, [START_REF] Fauth | Ectoparasitism and the role of green nesting material in the European starling[END_REF][START_REF] Gwinner | The function of green plants in nests of European starlings (Sturnus vulgaris)[END_REF][START_REF] Brouwer | Green nesting material has a function in mate attraction in the European starling[END_REF][START_REF] Polo | Female starlings adjust primary sex ratio in response to aromatic plants in the nests[END_REF][START_REF] Veiga | Nest green plants as a male status signal and courtship display in the spotless starling[END_REF].

Field investigations experimentally demonstrating influences of fresh plant material on nest ectoparasite loads or offspring characteristics are rare. [START_REF] Clark | Effect of biologically active plants used as nest material and the derived benefit to starling nestlings[END_REF] first demonstrated a positive effect of green plant addition in nests of the European starling Sturnus vulgaris. Experimental addition of green plant fragments from day 10 before-hatching until day 13 post-hatching reduced the number of hematophagous mites in nests relative to control nests. Adding green material to the nest did not influence chick mass or feather development, but resulted in higher levels of blood haemoglobin in nestlings. Clark and Mason's experimental design did not simulate starling natural behaviour because males usually stop bringing greenery to the nest before the start of incubation (Gwinner 1997, Brouwer and[START_REF] Brouwer | Green nesting material has a function in mate attraction in the European starling[END_REF]. In another starling study, [START_REF] Fauth | Ectoparasitism and the role of green nesting material in the European starling[END_REF] experimentally removed green nest material after the end of clutch completion, whereas control nests still contained greenery added by males during courtship and nest building. The two treatments did not differ in ectoparasite loads, breeding success, chick condition or post-fledging survival. In a third experiment comparing herb and grass nests, herbs selected by starlings did not reduce ectoparasite loads, but affected both chick body mass and blood parameters (hematocrit, lymphocyte and basophil counts; [START_REF] Gwinner | Green plants in starling nests: effects on nestlings[END_REF][START_REF] Gwinner | European starlings: nestling condition, parasites and green nest material during the breeding season[END_REF]. Most investigations in bird species other than starlings also failed to demonstrate a significant negative influence of fresh plants on nest-dwelling ectoparasites. In the wood stork Mycteria americana, green plant material in nests had no repelling effect on dermestid beetle larvae Dermestid nidum [START_REF] Rodgers | The use and function of green nest material by wood storks[END_REF]. [START_REF] Dawson | Does fresh vegetation protect avian nests from ectoparaites? An experiment with tree swallows[END_REF] found that experimentally-added yarrow Achillea millefolium in nests of the non greenery-using tree swallows Tachycineta bicolor had no negative effects on blow fly or flea infestation levels. An exception is [START_REF] Shutler | Experimental addition of greenery reduces flea loads in nests of a non-greenery using species, the tree swallow Tachycineta bicolor[END_REF] recent study in tree swallow nests showing negative effects of yarrow on flea number, although not on blow fly pupae numbers.

On Corsica, hole-nesting female blue tits (Cyanistes caeruleus) actively incorporate fresh fragments of aromatic plants in the nest cup (e.g. Lavandula stoechas, Achillea ligustica, Helichrysum italicum, Mentha suaveolens). They daily add fresh aromatic plant fragments in the nest between the end of nest construction until fledging, and replenish the nest with fresh fragments of the same plant species quickly after experimental removal (Lambrechts andDos Santos 2000, Petit et al. 2002, Mennerat pers. obs.). The plant species found in blue tit nests represent only a small fraction of the plants available in the habitat [START_REF] Petit | Blue tits use selected plants and olfaction to maintain an aromatic environment for nestlings[END_REF]. A noticeable difference between starling and blue tit plant-adding behaviours is that blue tits keep adding aromatic plants during the whole nestling period, which suggests that these plants have a little role in mate attraction. Some of the selected plants possess antiseptic, fungicidal or insecticidal properties [START_REF] Petit | Blue tits use selected plants and olfaction to maintain an aromatic environment for nestlings[END_REF][START_REF] Rossi | Antibacterial action of essential oils from Corsica[END_REF], and repel blood-sucking mosquitoes in laboratory conditions [START_REF] Lafuma | Aromatic plants in bird nests as a protection against blood-sucking flying insects?[END_REF]. In free-ranging blue tit populations on Corsica, nests are infested with very high numbers of Protocalliphora blood-sucking blow fly larvae attacking nestlings [START_REF] Hurtrez-Boussès | Variations in prevalence and intensity of blow fly infestations in an insular Mediterranean population of blue tits. -Can[END_REF]. Adult female blow flies visit bird nests and lay eggs in the nest material just after the chicks hatch, and keep visiting bird nests throughout the whole nestling period [START_REF] Bennett | Studies on the life history of some species of Protocalliphora (Diptera: Calliphoridae). -Can[END_REF]Whitworth 1991, Hurtrez-Boussès et al. 1999). Blow fly larvae grow into three successive, blood-sucking larval stages, and remain in the nest material until they emerge as adults after their pupal phase [START_REF] Bennett | Studies on the life history of some species of Protocalliphora (Diptera: Calliphoridae). -Can[END_REF]Whitworth 1991, Heeb et al. 2000). In Corsican blue tit populations, blow fly larvae have pronounced detrimental effects on nestling development, fledgling mass, hematocrit and post-fledging survival; they also influence aspects related to chick behaviour and parental care, including investment in nest sanitation (Hurtrez-Boussès et al. 1997, 1998, 2000[START_REF] Banbura | Effects of Protocalliphora parasites on nestling food composition in Corsican Blue 1 Tits Parus caeruleus: consequences for nestling performance[END_REF][START_REF] Simon | Physiological Ecology of Mediterranean Blue Tits (Parus caeruleus L): effects of ectoparasites (Protocalliphora spp.) and food abundance on metabolic capacity of nestlings[END_REF], 2005, Charmantier et al. 2004). Other nest ectoparasites, such as fleas, ticks, lice or mites, are rarely observed in these populations (prevalences <3%, Hurtrez-Boussès et al. 1997, A. Mennerat pers.

obs.).

Here we investigate whether fresh aromatic plant fragments incorporated in Corsican blue tit nests have significant negative effects on blow fly infestation, as predicted by the nest protection hypothesis. We used both an observational approach to examine the relationship between aromatic plants and blow fly infestation in nonmanipulated nests, and an experimental test of the effects of aromatic plants on blow fly infestation intensity and nestling body condition.

Material and Methods a) Study sites and field protocols

The study was carried out in 2005 and 2006 in three Corsican blue tit populations accepting nestboxes for breeding ("Muro-deciduous", 42°33' N, 08°55' E, broad-leaved deciduous oakwood, Quercus humilis; "Muro-evergreen", 42°36' N, 08°58' E, evergreen oakwood, Q. ilex; "Pirio", 42°31' N, 08°46' E, evergreen oakwood, Q. ilex; see [START_REF] Blondel | Breeding strategies of the Blue Tit and Coal Tit (Parus) in mainland and island Mediterranean habitats: a comparison[END_REF][START_REF] Lambrechts | 1 Adaptive inter-population differences in blue tit life-history traits on Corsica[END_REF][START_REF] Dawson | Does fresh vegetation protect avian nests from ectoparaites? An experiment with tree swallows[END_REF] for a detailed description of the sites). All nestboxes were monitored throughout the breeding season to determine the onset of egg laying (March 1 st = day 1), clutch size, hatching date, the number of hatchlings, and the number of chicks fledged (for a description of the field protocols, see e.g. [START_REF] Blondel | Breeding strategies of the Blue Tit and Coal Tit (Parus) in mainland and island Mediterranean habitats: a comparison[END_REF][START_REF] Lambrechts | 1 Adaptive inter-population differences in blue tit life-history traits on Corsica[END_REF]. Nestlings were weighed at day 9-11 post-hatching when chick demands are considered to be the highest, and measured and weighted again when chicks had reached their asymptotic mass at day 14-15 post-hatching. Body mass was measured to the nearest 0.1g with a Pesola spring balance and tarsus length to the nearest 0.1mm with a digital calliper. Chick body mass and tarsus length are often associated with the probability to be recruited in the local breeding population [START_REF] Blondel | Is the small clutch size of a Corsican Blue tit population optimal ?[END_REF][START_REF] Heeb | Ectoparasite infestation and sex-biased local recruitment of hosts[END_REF]). Together with body mass, hematocrit determines chick aerobic capacity, which is related to post-fledging survival in Corsican blue tit populations [START_REF] Thomas | Common paths link food abundance and ectoparasite loads to physiological performance and recruitment in nestling blue tits[END_REF]). We therefore used hematocrit as a measure of physiological condition in nestlings from Pirio. For each chick, around 20 μL of blood was collected from the brachial vein into a heparinised microcapillary tube, and centrifugated for 3 minutes at 13000 rpm.

The hematocrit value was defined as the percentage volume of erythrocytes in the total blood sample.

b) Blow fly quantification procedures

To avoid loss of blow fly larvae during nest removal, we placed each monitored nest in a tissue bag, following the method applied by [START_REF] Hurtrez-Boussès | Interactions hôte-parasite: le système Mésange bleue-Protocalliphora en région méditerranéenne[END_REF]. A bag was inserted under the nest 2 to 4 days before hatching. When chicks were 2-3 days old, the edge of the bag was pulled up to reach the same level as the top of the nest cup. At day 14-16 post-hatching, after measuring the chicks, nests were collected, enclosed in hermetic plastic bags and replaced by the same amount of fresh moss. In the laboratory, blow fly larvae and pupae were sorted out of the nest material and counted. Blow fly larvae develop into three successive larval stages before pupating. First-stage larvae are particularly difficult to detect. Therefore, our estimate of blow fly infestation intensity only included the total number of second-stage larvae, third-stage larvae and pupae, following the protocols applied by [START_REF] Hurtrez-Boussès | Interactions hôte-parasite: le système Mésange bleue-Protocalliphora en région méditerranéenne[END_REF] and [START_REF] Heeb | Bird-ectoparasite interactions, nest humidity and ectoparasite communities[END_REF]. Aromatic plant fragments were easily identified using morphological characteristics that clearly differed from moss and twigs. A herbarium of local plants identified by a botanical specialist was also used as reference.

d) Experimental protocol

In Pirio 2005, two experimental groups of 21 nests were monitored. In nests randomly assigned to a "treated" group, we added 1g of fresh leaves of two locally abundant aromatic plants often found in nests (0.5g of Lavandula stoechas and 0.5g of Helichrysum italicum), first 3 days before hatching, then daily from day 2-3 posthatching until day 14-15 post-hatching. Before adding fresh aromatic plants, we carefully removed all detectable aromatic plant fragments added by female blue tits or by the experimenter during previous days. Therefore the quantity of fresh plant fragments in nests remained constant through time and across nests. Nests assigned to the "control" group received the same treatment as treated nests, but fresh moss (1g) was added instead of aromatic plants. After drying, 1g of the aromatic plants used in this experiment weighed approximately 0.3g, which is within the natural range of fresh aromatic plant fragments daily added in nests by Corsican blue tits (0.03 -0.31g dry mass per nest per day, A. Mennerat unpub. data.). Nests were collected at day 14-15 post-hatching, when chicks were measured and blow fly infestation intensity was quantified as described before. The two groups did not differ in egg laying date (mean ± SD, treated nests: 65.95±2.31, control nests: 65.19±2.58; t-test, d.f.=40, t=-1.01, P=0.32) and clutch size (mean ± SD, treated nests: 7.19±0.81, control nests: 7.43±1.60; t-test, d.f.=40, t=0.61 , P=0.55 ), so we assume we controlled for aspects related to the quality of the parents or the territory.

e) Statistical analyses

Blow fly infestation intensity was square-root transformed prior to analyses. The relation between blow fly infestation intensity and amount of aromatic plants in nonmanipulated nests was investigated using a generalised linear model (type 3 'GLM' procedure, SAS 9.1, SAS Institute Inc.). We included year and habitat type (deciduous vs evergreen) as fixed factors and brood size as covariate in the model because they may influence blow fly infestation intensity [START_REF] Hurtrez-Boussès | Variations in prevalence and intensity of blow fly infestations in an insular Mediterranean population of blue tits. -Can[END_REF]. We also tested for the interaction between year and amount of aromatic plants because the relation between aromatic plants and blow fly infestation may vary according to yearly environmental fluctuations. A mixed-effects model was also performed with year as a random factor, 1 habitat type (deciduous vs evergreen) as a fixed factor and brood size as a covariate ('MIXED' procedure, SAS 9.1, SAS Institute Inc.). Since the results of both MIXED and GLM procedure were approximately the same, we will only present the results from the GLM analysis, which allows testing for the interaction between year and aromatic plants.

To investigate whether adding aromatic plants had an effect on blow fly infestation and nestling survival, blow fly infestation intensity and brood size at days 14-15 post-hatching in the two experimental groups were compared with t-tests. As nestling mass at days 9-11 post-hatching and nestling wing length at days 14-15 post-hatching were positively correlated with chick age (d.f.=1, t=19.6, P<10 -4 and d.f.=1, t=20.89, P<10 -4 respectively), we considered residuals of the regression of both variables against chick age as corrected variables. We used the first axis of a Principal Component Analysis (PCA) from the correlation matrix of the three measurements of nestling mass, tarsus length and wing length (corrected for age) as an estimate of nestling body condition at day 14-15 post-hatching. The first axis ('PC1') accounted for 53% of the total variance. We investigated the effects of treatment, brood size and blow fly infestation intensity on nestling corrected mass at days 9-11, nestling size (PC1) and nestling hematocrit with mixed-effects models considering chick as statistical unit and nest as random factor ('MIXED' procedure, SAS 9.1, SAS Institute Inc.). The interaction between treatment and blow fly infestation intensity was also included because effects of treatment may be more pronounced under high infestation intensities.

Results

Blow fly infestation intensity was not significantly related to the total dry mass of 1 aromatic plant fragments found in non-manipulated nests (d.f.=1, F=1.90, P=0.41, Figure1). In addition, it was not significantly related to the interaction between year and the total dry mass of aromatic plant fragments (d.f.=1, F=0.99, P=0.32), nor to habitat type (d.f.=1, F=0.16, P=0.69). However, nests with larger broods contained more blow fly larvae (d.f.=1, F=13.85, P<0.001) and blow fly infestation intensity tended to be higher in 2006 than in 2005(mean ± SD, 2005: 35.9±26.34;: 35.9±26.34;2006: 44.39±27.84;[START_REF] Shutler | Experimental addition of greenery reduces flea loads in nests of a non-greenery using species, the tree swallow Tachycineta bicolor[END_REF]f.=1, F=3.55, P=0.06, Table 1).

Experimentally adding aromatic plants in nests did not affect blow fly infestation intensity: treated and control groups did not significantly differ in blow fly infestation intensity (mean ± SD number of blow fly larvae, treated nests: 64.00±36.29, control nests: 55.67±31.11; t-test on square-root transformed numbers: d.f.=40, t=-0.64, P=0.53; Figure2). Adding aromatic plants did not either affect nestling survival: brood size did not differ between treated and control nests (mean ± SD, treated nests: 6.51±1.20, control nests: 6.24±1.23; t-test: d.f.=40, t=-0.74, P=0.46).

Adding aromatic plants in nests did not affect nestling mass at days 9-11 posthatching (corrected for chick age), nestling body condition or nestling hematocrit at days 14-15 post-hatching. None of the three response variables was significantly related to experimental treatment, nor to its interaction with blow fly infestation intensity (Table2).

Nestlings at days 9-11 post-hatching were heavier in smaller broods (d.f.=1, F=6.99, P<0.01), whereas nestling hematocrit was lower in nests with more blow fly larvae (d.f.=1, F=10.24, P<0.01) and higher in larger broods (d.f.=1, F=10.24, P<0.01, see Table2).

1

Discussion

We initially hypothesized that aromatic plant fragments in blue tits nests could alter nestling detection capacities in adult blow flies, repel adults or larvae, or contain compounds toxic for adults and/or larvae (e.g. Lambrechts andDos Santos 2000, Lafuma et al. 2001). Because blow flies were exposed to aromatic plants at the time when they lay eggs in blue tit nests, we predicted lower numbers of blow fly larvae in our aromatictreated nests, which was not the case. In addition, our two-year study involving one hundred sampled nests did not reveal any significant correlation between the total dry mass of aromatic plant fragments and blow fly infestation intensities across blue tit nests in two oak habitat types. We therefore conclude that the aromatic plant fragments sampled and used in our experiment have no anti-blow fly effects and therefore could not protect chicks against blow fly attack. This is also supported by our analyses that chick traits influenced by blow fly attack (e.g. nestling hematocrit, see Introduction), were not related to the amount of aromatic plant fragments added in the nest.

Although we are confident that the aromatic plants sampled do not protect blue tit chicks against blow fly attack, significant correlations between the aromatic plants and offspring characteristics or breeding success may have been masked by unidentified confounding variables, the particular environmental conditions during the study, or the offspring traits investigated. For instance, inconsistent with former investigations in the same study population, we did not find a significant relationship between blow fly infestation intensity and chick size or mass, perhaps because our study was carried out under highly favourable environmental conditions (own observations, see also [START_REF] Thomas | Common paths link food abundance and ectoparasite loads to physiological performance and recruitment in nestling blue tits[END_REF]). [START_REF] Gwinner | Green plants in starling nests: effects on nestlings[END_REF] found that the positive effect of green nesting material 1 on nestling body mass was greater in highly mite-infested nests. [START_REF] Gwinner | European starlings: nestling condition, parasites and green nest material during the breeding season[END_REF] also found that the positive effect of herbs on nestling body mass was particularly revealed under unfavourable conditions (high mite load, low ambient temperature, prolonged rainfall). Also, aromatic plants may have positive effects on post-fledging survival that we were not able to estimate with our measures of nestling condition, e.g. via repellent effects on blood-sucking flying insects that can be vectors of avian endoparasites [START_REF] Lafuma | Aromatic plants in bird nests as a protection against blood-sucking flying insects?[END_REF], or anti-bacterial effects reducing the risk of pathogen infection of nestlings [START_REF] Rossi | Antibacterial action of essential oils from Corsica[END_REF]). More work on potential ectoparasites other than Protocalliphora blow flies is therefore required to better understand ectoparasitearomatic plant interactions in blue tit nests, their underlying mechanisms and their potential consequences on chick survival in wild populations. Table 1. Relation between (square-root transformed) blow fly infestation intensity and total dry mass of aromatic plants, brood size, habitat type, year and the interaction between year and total dry mass of aromatic plants, as tested by a type 3 GLM (see Methods).

(

  c) Quantification of aromatic plants in non-manipulated nestsA hundred nests were sampled to examine the relationship between the total amount of aromatic plants and blow fly infestation intensity at day 14-15 post-hatching(Murodeciduous: 2005, 35 nests and 2006: 25 nests; Muro-evergreen: 2005, 25 nests and 2006,15 nests). To avoid damages to plant fragments caused by e.g. mites or micro-organisms before sampling of aromatic plant fragments, all nests were microwave-disinfected after blow fly quantification. During sampling, aromatic plant fragments were separated from the rest of the nest material, stored in paper bags and allowed to dry at ambient temperature for several weeks. Dried samples were weighed with a precision balance (Acculab Pocket Pro C/50) to the nearest 0.002g to obtain the total dry mass of aromatic plants per nest.

414 415Figure 1 .Figure 2 .

 41412 Figure 1. Number of blow fly larvae plotted against dry mass of aromatic plants in nests

Table 1

 1

	424	)
			Blow fly infestation intensity
			d.f.	F	P
		Aromatic plants	1	1.90	0.41
		Brood size	1	13.85	<0.001
		Habitat type	1	0.16	0.69
		Year	1	3.55	0.06
		Year*Aromatic plants 1	0.99	0.32
	425

Table 2 .

 2 Effects of experimental treatment (aromatic vs control nests), blow fly infestation intensity, brood size and the treatment*infestation intensity interaction on nestling mass at days 9-11 post-hatching ('Mass1', corrected for age), nestling body condition ('PC1') and nestling hematocrit, as tested by mixed-effects models with nest as random factor (see Methods).

Acknowledgments

This project was partly funded by a research grant from the ANR to P. Heeb and M.M.

Lambrechts. Special thanks to P. Bourgault for significant help in the field and discussions. We thank D.W. Thomas for helpful discussion and J. Blondel for advice and comments on the manuscript. We are also grateful to D. Beaune, P.A. Bernier, D.

Monroe and C. Babin for field assistance and to three anonymous referees for helpful comments on earlier versions of the manuscript.