CD44, γ-H2AX, and p-ATM Expressions in Short-Term Ex Vivo Culture of Tumour Slices Predict the Treatment Response in Patients with Oral Squamous Cell Carcinoma.

Squamous cell carcinoma is the most common type of head and neck cancer (HNSCC) with a disease-free survival at 3 years that does not exceed 30%. Biomarkers able to predict clinical outcomes are clearly needed. The purpose of this study was to investigate whether a short-term culture of tumour fragments irradiated ex vivo could anticipate patient responses to chemo- and/or radiotherapies. Biopsies were collected prior to treatment from a cohort of 28 patients with non-operable tumours of the oral cavity or oropharynx, and then cultured ex vivo. Short-term biopsy slice culture is a robust method that keeps cells viable for 7 days. Different biomarkers involved in the stemness status (CD44) or the DNA damage response (pATM and γ-H2AX) were investigated for their potential to predict the treatment response. A higher expression of all these markers was predictive of a poor response to treatment. This allowed the stratification of responder or non-responder patients to treatment. Moreover, the ratio for the expression of the three markers 24 h after 4 Gy irradiation versus 0 Gy was higher in responder than in non-responder patients. Finally, combining these biomarkers greatly improved their predictive potential, especially when the γ-H2AX ratio was associated with the CD44 ratio or the pATM ratio. These results encourage further evaluation of these biomarkers in a larger cohort of patients.

A pan-cancer analysis of the human tumor coagulome and its link to the tumor immune microenvironment.

OBJECTIVE: Solid tumors often establish a procoagulable state that can lead to venous thromboembolism (VTE). Although some of the key genes involved in this process are known, no previous study has compared the "coagulome", i.e., the expression of coagulation/fibrinolysis genes, across different primary tumor types. It is also unclear whether the coagulome is associated with specific characteristics of the tumor microenvironment (TME). We aimed to address this question. METHODS: We analyzed the expression of the genes F3, PLAU, PLAT, PLAUR, SERPINB2, and SERPINE1 in 32 cancer types using data from The Cancer Genome Atlas (TCGA) and other freely available resources. RESULTS: We identified specific expression patterns of procoagulant and fibrinolytic genes. The expression of the Tissue Factor (F3) was found to be tumor type dependent, with the highest expression in glioblastoma (GBM), a highly procoagulable tumor type. Conversely, high expression of the fibrinolysis gene cluster PLAU, PLAUR, SERPINE1 was consistently linked to the characteristics of the TME (monocytic infiltration) and high expression of important checkpoints of the immune response, such as PD-L2 and CD276/B7-H3. CONCLUSION: These tumor-specific patterns of expression might partially explain the differences in VTE risk among tumor types. We propose that biomarkers of coagulation fibrinolysis might provide valuable information about the TME in cancer patients.

Contribution of Genomics to the Surgical Management and Study of Oral Cancer.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most frequent type of tumor arising from the oral cavity. Surgery is the cornerstone of the treatment of these cancers. Tumor biology has long been overlooked as an important contributor to the outcome of surgical procedures, but recent studies are challenging this concept. Molecular analyses of tumor DNA or RNA provide a rich source of information about the biology of OSCC. METHODS: We searched for relevant articles using PubMed. We examined in particular the prospect of applying molecular methods for minimally invasive exploration of OSCC biology. RESULTS: We examined five potential applications of genomics to the surgical management and study of OSCC: i) assessing oral potentially malignant lesions; ii) tumor staging prior to surgery; iii) predicting postoperative risk in locally advanced tumors; iv) measuring minimal residual disease and optimizing the longitudinal monitoring of OSCC; and v) predicting the efficacy of medical treatment. CONCLUSIONS: Genomic information can be harnessed in order to identify new biomarkers that could improve the staging, choice of therapy and management of OSCC. The identification of new biomarkers is awaited for better personalization of the surgical treatment of OSCC.

SLC7A11 as a biomarker and therapeutic target in HPV-positive head and neck Squamous Cell Carcinoma.

Ferroptosis, a regulated form of cell necrosis was previously reported to be induced upon pharmacological targeting of the cystine transporter SLC7A11 in Head and neck Squamous Cell Carcinoma (HNSCC). Whether tumors arising in a context of chronic infection with Human Papillomavirus (HPV) are sensitive to ferroptosis is unknown. Using RNAseq data (both whole-tumor and single-cell sequencing) we report that HPV positive (HPV+ve) tumors have lower expression levels of SLC7A11 compared to HPV negative (HPV-ve) HNSCC. We examined in vitro the effect of erastin, a specific blocker of SLC7A11, applied on two HNSCC cell lines with stable expression of HPV16 E6 and E7 oncoproteins. We report a decrease in total GSH levels and an increased sensitivity to erastin-induced ferroptosis in E6-E7 cells. Cell sensitivity to ferroptosis was specificaly related to a defect in cystine transport since we found no difference in ferroptosis induced by the direct inhibition of GPX4, and N-Acetyl Cystein abolished the difference between WT and E6-E7-expressing cells. Our findings point to SLC7A11 as an HPV-related biomarker of potential therapeutic relevance in HNSCC. Targeting cystine import to promote ferroptosis might be a promising strategy against HPV+ve HNSCC. (188 words).

Vasoplegia After Cardiac Surgery Is Associated With Endothelial Glycocalyx Alterations.

OBJECTIVE: To assess endothelial glycocalyx (EG) alteration in vasoplegic syndrome after cardiac surgery with cardiopulmonary bypass. DESIGN: Prospective analysis SETTING: Single university hospitals. PARTICIPANTS: Patients undergoing elective cardiac surgery under cardiopulmonary bypass. INTERVENTIONS: Observational study METHODS: Heparan sulfate and syndecan-1 levels were assessed in plasma before surgery, on intensive care unit admission, and on the first postoperative day. The primary outcome was comparisons of heparan sulfate and syndecan-1 levels in patients with and without vasoplegic syndrome. RESULTS: A total of 46 patients were analyzed. Only syndecan-1 was modified by cardiac surgery (p < 0.05). Plasma syndecan-1 levels were lower in patients with vasoplegic syndrome at the 3 time-points and were associated with the cumulative norepinephrine dose. Baseline plasma syndecan-1 predicted the development of vasoplegic syndrome with an area under the curve of 0.7 (confidence interval 95%: 0.51-0.85, p = 0.045). Heparan sulfate levels were not modified by cardiac surgery. CONCLUSION: Patients with vasoplegic syndrome after cardiac surgery present a different pattern of EG components. Lower syndecan-1 levels were associated with vasoplegic syndrome. These preliminary results suggest a specific phenotype that may reflect endothelial activation leading to alteration of the EG.

Impact and Relevance of the Unfolded Protein Response in HNSCC.

Head and neck squamous cell carcinomas (HNSCC) encompass a heterogeneous group of solid tumors that arise from the upper aerodigestive tract. The tumor cells face multiple challenges including an acute demand of protein synthesis often driven by oncogene activation, limited nutrient and oxygen supply and exposure to chemo/radiotherapy, which forces them to develop adaptive mechanisms such as the Unfolded Protein Response (UPR). It is now well documented that the UPR, a homeostatic mechanism, is induced at different stages of cancer progression in response to intrinsic (oncogenic activation) or extrinsic (microenvironment) perturbations. This review will discuss the role of the UPR in HNSCC as well as in the key processes that characterize the physiology of HNSCC. The role of the UPR in the clinical context of HNSCC will also be addressed.

Combining genomic analyses with tumour-derived slice cultures for the characterization of an EGFR-activating kinase mutation in a case of glioblastoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) gene alterations and amplification are frequently reported in cases of glioblastoma (GBM). However, EGFR-activating mutations that confer proven sensitivity to tyrosine kinase inhibitors (TKIs) in lung cancer have not yet been reported in GBM. CASE PRESENTATION: Using next-generation sequencing, array comparative genomic hybridization and droplet digital PCR, we identified the p.L861Q EGFR mutation in a case of GBM for the first time. The mutation was associated with gene amplification. L861Q may be a clinically valuable mutation because it is known to sensitize non-small-cell lung cancers to treatment with the second-generation EGFR TKI afatinib in particular. Furthermore, we used slice culture of the patient's GBM explant to evaluate the tumour's sensitivity to various EGFR-targeting drugs. Our results suggested that the tumour was not intrinsically sensitive to these drugs. CONCLUSIONS: Our results highlight (i) the value of comprehensive genomic analyses for identifying patient-specific, targetable alterations, and (ii) the need to combine genomic analyses with functional assays, such as tumour-derived slice cultures.

Role of the unfolded protein response in tumor cell characteristics and cancer outcome.

PURPOSE OF REVIEW: In the present review, we discuss the possible role of the unfolded protein response (UPR) in the acquisition of tumor cell characteristics and in the prognosis of cancer outcome, which could assist and contribute to the development of more promising therapeutic strategies. RECENT FINDINGS: Accumulating evidence supports the idea that alteration of endoplasmic reticulum proteostasis is a key player in cancer development and aggressiveness. Some UPR components were reported as independent prognostic biomarker. Recent evidence supports a relationship between the UPR activation status and prognosis of tumors. This may represent an interesting avenue for better characterization of carcinogenesis and tumor type. SUMMARY: The contribution of the UPR to the characteristics of malignant tumors is complex and dependent on both intrinsic (e.g. oncogene addiction) and extrinsic (e.g. hypoxia) contexts. Through adaptation to severe microenvironmental conditions, UPR branches are generally a survival strategy for cancer cells, which are able to cope with this challenging context. We address the question of whether the activation status of the UPR is related to tumor properties and discuss the role of the UPR in the clinical context.

A potential role of the unfolded protein response in post-transplant cancer.

Cancer is one of the major causes of mortality in organ transplant patients receiving immunosuppressive regimen based on Cyclosporin A (CsA). Organ transplantation and chronic immunosuppression are typically associated with skin cancers (both squamous cell carcinoma and melanoma) and renal cell carcinoma (RCC). Recent studies have shown that in addition to its immunosuppressive effects, accounted for by the inhibition of calcineurin and the modulation of the transcriptional programme of lymphocytes, CsA also directly stimulates the growth and aggressive behaviour of various cancer cells. Using renal carcinogenesis as an example, we discuss the current evidence for a role of cellular proteostasis, i.e. the regulation of the production, maturation and turnover of proteins in eukaryotic cells, in tumorigenesis arising under conditions of chronic immunosuppression. We present the recent studies showing that CsA induces the unfolded protein response (UPR) in normal and transformed kidney cells. We examine how the UPR might be important, considering in particular the genomic analyses showing the existence of a correlation between the levels of expression of the actors of the UPR, the chaperones of the endoplasmic reticulum (ER) and the aggressiveness of renal carcinoma. The UPR may offer a possible explanation for how immunosuppressive regimens based on CsA promote renal carcinogenesis. We discuss the opportunities offered by this biological knowledge in terms of screening, diagnosis and treatment of post-transplant cancers, and propose possible future translational studies examining the role of tumour proteostasis and the UPR in this context.

Metallothionein-1 as a biomarker of altered redox metabolism in hepatocellular carcinoma cells exposed to sorafenib.

BACKGROUND: Sorafenib, a kinase inhibitor active against various solid tumours, induces oxidative stress and ferroptosis, a new form of oxidative necrosis, in some cancer cells. Clinically-applicable biomarkers that reflect the impact of sorafenib on the redox metabolism of cancer cells are lacking. METHODS: We used gene expression microarrays, real-time PCR, immunoblot, protein-specific ELISA, and gene reporter constructs encoding the enzyme luciferase to study the response of a panel of cancer cells to sorafenib. Tumour explants prepared from surgical hepatocellular carcinoma (HCC) samples and serum samples obtained from HCC patients receiving sorafenib were also used. RESULTS: We observed that genes of the metallothionein-1 (MT1) family are induced in the HCC cell line Huh7 exposed to sorafenib. Sorafenib increased the expression of MT1G mRNA in a panel of human cancer cells, an effect that was not observed with eight other clinically-approved kinase inhibitors. We identified the minimal region of the MT1G promoter that confers inducibility by sorafenib to a 133 base pair region containing an Anti-oxidant Response Element (ARE) and showed the essential role of the transcription factor NRF2 (Nuclear factor erythroid 2-Related Factor 2). We examined the clinical relevance of our findings by analysing the regulation of MT1G in five tumour explants prepared from surgical HCC samples. Finally, we showed that the protein levels of MT1 increase in the serum of some HCC patients receiving sorafenib, and found an association with reduced overall survival. CONCLUSION: These findings indicate that MT1 constitute a biomarker adapted for exploring the impact of sorafenib on the redox metabolism of cancer cells.

Short-term culture of tumour slices reveals the heterogeneous sensitivity of human head and neck squamous cell carcinoma to targeted therapies.

BACKGROUND: Despite recent progress, investigating the impact of targeted therapies on Head and Neck Squamous Cell Carcinoma (HNSCC) remains a challenge. We investigated whether short-term culture of tumour fragments would permit the evaluation of tumour sensitivity to targeted therapies at the individual level. METHODS: We cultivated tumour slices prepared from 18 HNSCC tumour samples obtained during surgical resection. The samples were treated for 48 h with a panel of 8 targeted therapies directed against selected oncogenic transduction pathways. We analysed the cell proliferation index (CPI) of tumour cells using Ki67 labelling and the activation status of the RAF-MEK-ERK cascade through ERK phosphorylation analysis. RESULTS: Fourteen tumours were successfully analysed after short-term culture of tumour samples, revealing a striking individual heterogeneity of HNSCC in terms of tumour cell sensitivity to targeted therapies. Using 50% inhibition of CPI as threshold, sorafenib was shown to be active in 5/14 tumours. Cetuximab, the only approved targeted drug against HNSCC, was active in only 2/14 tumours. A more than 50% inhibition was observed with at least one drug out of the eight tested in 10/14 tumours. Cluster analysis was carried out in order to examine the effect of the drugs on cell proliferation and the RAF-MEK-ERK cascade. CONCLUSIONS: In vitro culture of tumour fragments allows for the evaluation of the effects of targeted therapies on freshly resected human tumours, and might be of value as a possible guide for the design of clinical trials and for the personalization of the medical treatment of HNSCC.

[Ferroptosis, a new form of cell death relevant to the medical treatment of cancer]. / La ferroptose, une nouvelle forme de mort cellulaire applicable au traitement médical des cancers.

Ferroptosis is a form of cell death that has recently been reported during exposure to erastin, a chemical compound identified in a screen for molecules able to kill cancer cells carrying an active Ras oncogene. In cells exposed to inducers of ferroptosis, a catastrophic alteration of the cellular redox metabolism occurs, resulting in massive lipid peroxidation in the plasma membrane and loss of cell viability. We present our recent observations suggesting that sorafenib, the only medical treatment with proven efficacy against hepatocellular carcinoma, induces ferroptosis, a new anti-oncogenic mode of action of this drug. The discovery of ferroptosis sheds light on the critical adaptations of the redox metabolism in cancer cells. It might also foster the discovery of new biomarkers and innovative approaches for the treatment of cancer.

Transcriptional Induction of SERPINE1 and Fibrinolysis Inhibition as Predominant Effects of Glucocorticoids on the Cancer Coagulome.

BACKGROUND/AIM: How tumors regulate the genes of the coagulome is crucial for cancer-associated thrombosis and the occurrence of venous thromboembolic complications in patients with cancer. We have previously reported potent yet complex effects of glucocorticoids (GC) on the expression of three genes that play a key role in the regulation of thrombin/plasmin activation (F3, PLAU, and SERPINE1). This study aimed to extend the investigation of GC effects to the whole tumor coagulome and assess the resulting impact on the ability of cancer cells to activate thrombin and plasmin. MATERIALS AND METHODS: Cancer RNA expression data were retrieved from various sources. Additionally, oral squamous cell carcinoma (OSCC) cells exposed to GC in vitro were analyzed using QPCR, enzymatic assays measuring thrombin and urokinase-type Plasminogen Activator (uPA) activity, and D-dimer concentrations. RESULTS: Our findings highlight the potent and specific stimulatory effect of GC on SERPINE1 expression across different types of cancer. Consistently, GC were found to inhibit uPA proteolytic activity and reduce the concentrations of D-dimers in OSCC in vitro. CONCLUSION: Fibrinolysis inhibition is a key consequence of cancer cell exposure to GC, possibly leading to the stabilization of the fibrin clot in cancer.

An MRI Radiomics Approach to Predict the Hypercoagulable Status of Gliomas.

Venous thromboembolic events are frequent complications of Glioblastoma Multiforme (GBM) and low-grade gliomas (LGGs). The overexpression of tissue factor (TF) plays an essential role in the local hypercoagulable phenotype that underlies these complications. Our aim was to build an MRI radiomics model for the non-invasive exploration of the hypercoagulable status of LGG/GBM. Radiogenomics data from The Cancer Genome Atlas (TCGA) and REMBRANDT (Repository for molecular BRAin Neoplasia DaTa) cohorts were used. A logistic regression model (Radscore) was built in order to identify the top 20% TF-expressing tumors, considered to be at high thromboembolic risk. The most contributive MRI radiomics features from LGG/GBM linked to high TF were identified in TCGA using Least Absolute Shrinkage and Selection Operator (LASSO) regression. A logistic regression model was built, whose performance was analyzed with ROC in the TCGA/training and REMBRANDT/validation cohorts: AUC = 0.87 [CI95: 0.81-0.94, p < 0.0001] and AUC = 0.78 [CI95: 0.56-1.00, p = 0.02], respectively. In agreement with the key role of the coagulation cascade in gliomas, LGG patients with a high Radscore had lower overall and disease-free survival. The Radscore was linked to the presence of specific genomic alterations, the composition of the tumor coagulome and the tumor immune infiltrate. Our findings suggest that a non-invasive assessment of the hypercoagulable status of LGG/GBM is possible with MRI radiomics.

Iron-dependent cell death of hepatocellular carcinoma cells exposed to sorafenib.

The multikinase inhibitor sorafenib is currently the treatment of reference for advanced hepatocellular carcinoma (HCC). In our report, we examined the cytotoxic effects of sorafenib on HCC cells. We report that the depletion of the intracellular iron stores achieved by using the iron chelator deferoxamine (DFX) strikingly protects HCC cells from the cytotoxic effects of sorafenib. The protective effect of the depletion of intracellular iron stores could not be explained by an interference with conventional forms of programmed cell death, such as apoptosis or autophagic cell death. We also found that DFX did not prevent sorafenib from reaching its intracellular target kinases. Instead, the depletion of intracellular iron stores prevented sorafenib from inducing oxidative stress in HCC cells. We examined the possibility that sorafenib might exert a cytotoxic effect that resembles ferroptosis, a form of cell death in which iron-dependent oxidative mechanisms play a pivotal role. In agreement with this possibility, we found that pharmacological inhibitors (ferrostatin-1) and genetic procedures (RNA interference against IREB-2) previously reported to modulate ferroptosis, readily block the cytotoxic effects of sorafenib in HCC cells. Collectively, our findings identify ferroptosis as an effective mechanism for the induction of cell death in HCC. Ferroptosis could potentially become a goal for the medical treatment of HCC, thus opening new avenues for the optimization of the use of sorafenib in these tumors.

The coagulome of oral squamous cell carcinoma: examining the role and regulation of coagulation in oral cancers using a systems approach.

PURPOSE OF REVIEW: Solid tumors often establish a locally hypercoagulant state that promotes vascular complications, such as venous thromboembolism (VTE). Oral squamous cell carcinoma (OSCC) is associated with a broad range of hemostatic complications. Although VTE rarely occurs in ambulatory patients with OSCC, the coagulation cascade is typically activated by surgical resection and local hemorrhage. We present the recent progress in the understanding of the role and regulation of coagulation in OSCC. RECENT FINDINGS: Application of systems biology, using bulk tumor and single cell genomic analyses, unveiled the landscape of the tumor coagulome. Of all tumor types, OSCC express the highest mRNA levels of F3 and PLAU, the genes that encode the tissue factor (TF) and urokinase-type plasminogen activator (uPA), the key regulators of coagulation and fibrinolysis, respectively. It also brought to light the intimate and reciprocal regulation between coagulation/fibrinolysis and the tumor microenvironment (TME). SUMMARY: OSCC have a specific coagulome, with consequences that likely extend beyond the vascular risk. We discuss the attractive possibility that biomarkers of the coagulation cascade might reflect some important characteristics of the TME, offering new opportunities to better understand the impact of surgical procedures, better predict their oncological outcome and improve current therapeutic approaches.

The Tumor Coagulome as a Transcriptional Target and a Potential Effector of Glucocorticoids in Human Cancers.

BACKGROUND: The coagulome, defined as the repertoire of genes that locally regulate coagulation and fibrinolysis, is a key determinant of vascular thromboembolic complications of cancer. In addition to vascular complications, the coagulome may also regulate the tumor microenvironment (TME). Glucocorticoids are key hormones that mediate cellular responses to various stresses and exert anti-inflammatory effects. We addressed the effects of glucocorticoids on the coagulome of human tumors by investigating interactions with Oral Squamous Cell Carcinoma, Lung Adenocarcinoma, and Pancreatic Adenocarcinoma tumor types. METHODS: We analyzed the regulation of three essential coagulome components, i.e., the tissue factor (TF), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1) in cancer cell lines exposed to specific agonists of the glucocorticoid receptor (GR) (dexamethasone and hydrocortisone). We used QPCR, immunoblots, small-interfering RNA, Chromatin immunoprecipitation sequencing (ChIPseq) and genomic data from whole tumor and single-cell analyses. RESULTS: Glucocorticoids modulate the coagulome of cancer cells through a combination of indirect and direct transcriptional effects. Dexamethasone directly increased PAI-1 expression in a GR-dependent manner. We confirmed the relevance of these findings in human tumors, where high GR activity/high SERPINE1 expression corresponded to a TME enriched in active fibroblasts and with a high TGF-ß response. CONCLUSION: The transcriptional regulation of the coagulome by glucocorticoids that we report may have vascular consequences and account for some of the effects of glucocorticoids on the TME.

EGFR activation is a potential determinant of primary resistance of hepatocellular carcinoma cells to sorafenib.

Sorafenib is currently the medical treatment of reference for hepatocellular carcinoma (HCC), but it is not known whether sorafenib is equally active in all HCC. Here, our aim was to explore intrinsic differences in the response of HCC cells to sorafenib, to identify potential mechanisms leading to primary resistance to this treatment. We analyzed a panel of six human HCC cell lines and compared the activity of the main oncogenic kinase cascades, their clonogenic potential, proliferation and apoptosis upon exposure to sorafenib. We report that HCC cells present important differences in their response to sorafenib, and that some cell lines are more resistant to the actions of sorafenib than others. We identify the activated epidermal growth factor receptor (EGFR) as a parameter that promotes the resistance of HCC cells to sorafenib. In resistant cells, the efficacy of sorafenib was increased when EGFR was inhibited, as was demonstrated using two chemical inhibitors (erlotinib or gefitinib), a monoclonal antibody directed against EGFR (cetuximab), and RNA interference directed against EGFR. A combination of EGFR inhibitors and sorafenib affords a better control over HCC proliferation, most likely through an improved blockade of the RAF kinases. Our findings therefore confirm the importance of RAF kinases as therapeutic targets in HCC, and identify EGFR as a determinant of the sensitivity of HCC cells to sorafenib. Our findings bear possible implications for the improvement of the efficacy of sorafenib in HCC, and might be useful for the identification of predictive biomarkers in this context.

Upregulation of BAD, a pro-apoptotic protein of the BCL2 family, in vascular smooth muscle cells exposed to uremic conditions.

Chronic kidney disease (CKD) has recently emerged as a major risk factor for cardiovascular pathology. CKD patients display accelerated atherosclerotic process, leading to circulatory complications. However, it is currently not clear how uremic conditions accelerate atherosclerosis. Apoptosis is an important homeostatic regulator of vascular smooth cells under pathological conditions. In the present study, we explored the regulation of apoptosis in cells of the vascular wall in the uremic context. We analysed the expression and regulation of the proteins of the BCL2 family that play an essential role in apoptosis. Our results, obtained in mice and primary human smooth muscle cells exposed to two uremic toxins, point to the existence of an alteration in expression and function of one pro-apoptotic member of this family, the protein BAD. We explore the regulation of BAD by uremic toxins and report the sensitization of vascular smooth muscle cells to apoptosis upon BAD induction.

Helicobacter pylori VacA toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane.

The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% beta-strands, similar to pore-forming beta-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.