Relevance of biopsy-derived pancreatic organoids in the development of efficient transcriptomic signatures to predict adjuvant chemosensitivity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) patients are frequently treated by chemotherapy. Even if personalized therapy based on molecular analysis can be performed for some tumors, PDAC regimens selection is still mainly based on patients' performance status and expected efficacy. Therefore, the establishment of molecular predictors of chemotherapeutic efficacy could potentially improve prognosis by tailoring treatments. We have recently developed an RNA-based signature that predicts the efficacy of adjuvant gemcitabine using 38 PDAC primary cell cultures. While demonstrated its efficiency, a significant association with the classical/basal-like PDAC spectrum was observed. We hypothesized that this flaw was due to the basal-like biased phenotype of cellular models used in our strategy. To overcome this limitation, we generated a prospective cohort of 27 consecutive biopsied derived pancreatic organoids (BDPO) and include them in the signature identification strategy. As BDPO's do not have the same biased phenotype as primary cell cultures we expect they can compensate one with each other and cover a broader range of molecular phenotypes. We then obtained an improved signature predicting gemcitabine sensibility that was validated in a cohort of 300 resected PDAC patients that have or have not received adjuvant gemcitabine. We demonstrated a significant association between the improved signature and the overall and disease-free survival in patients predicted as sensitive and treated with adjuvant gemcitabine. We propose then that including BDPO along primary cell cultures represent a powerful strategy that helps to overcome primary cell cultures limitations producing unbiased RNA-based signatures predictive of adjuvant treatments in PDAC.

A transcriptomic signature to predict adjuvant gemcitabine sensitivity in pancreatic adenocarcinoma.

BACKGROUND: Chemotherapy is the only systemic treatment approved for pancreatic ductal adenocarcinoma (PDAC), with a selection of regimens based on patients' performance status and expected efficacy. The establishment of a potent stratification associated with chemotherapeutic efficacy could potentially improve prognosis by tailoring treatments. PATIENTS AND METHODS: Concomitant chemosensitivity and genome-wide RNA profiles were carried out on preclinical models (primary cell cultures and patient-derived xenografts) derived from patients with PDAC included in the PaCaOmics program (NCT01692873). The RNA-based stratification was tested in a monocentric cohort and validated in a multicentric cohort, both retrospectively collected from resected PDAC samples (67 and 368 patients, respectively). Forty-three (65%) and 203 (55%) patients received adjuvant gemcitabine in the monocentric and the multicentric cohorts, respectively. The relationships between predicted gemcitabine sensitivity and patients' overall survival (OS) and disease-free survival were investigated. RESULTS: The GemPred RNA signature was derived from preclinical models, defining gemcitabine sensitive PDAC as GemPred+. Among the patients who received gemcitabine in the test and validation cohorts, the GemPred+ patients had a higher OS than GemPred- (P = 0.046 and P = 0.00216). In both cohorts, the GemPred stratification was not associated with OS among patients who did not receive gemcitabine. Among gemcitabine-treated patients, GemPred+ patients had significantly higher OS than the GemPred-: 91.3 months [95% confidence interval (CI): 61.2-not reached] versus 33 months (95% CI: 24-35.2); hazard ratio 0.403 (95% CI: 0.221-0.735, P = 0.00216). The interaction test for gemcitabine and GemPred+ stratification was significant (P = 0.0245). Multivariate analysis in the gemcitabine-treated population retained an independent predictive value. CONCLUSION: The RNA-based GemPred stratification predicts the benefit of adjuvant gemcitabine in PDAC patients.

TP53INP1, a tumor suppressor, interacts with LC3 and ATG8-family proteins through the LC3-interacting region (LIR) and promotes autophagy-dependent cell death.

TP53INP1 (tumor protein 53-induced nuclear protein 1) is a tumor suppressor, whose expression is downregulated in cancers from different organs. It was described as a p53 target gene involved in cell death, cell-cycle arrest and cellular migration. In this work, we show that TP53INP1 is also able to interact with ATG8-family proteins and to induce autophagy-dependent cell death. In agreement with this finding, we observe that TP53INP1, which is mainly nuclear, relocalizes in autophagosomes during autophagy where it is eventually degraded. TP53INP1-LC3 interaction occurs via a functional LC3-interacting region (LIR). Inactivating mutations of this sequence abolish TP53INP1-LC3 interaction, relocalize TP53INP1 in autophagosomes and decrease TP53INP1 ability to trigger cell death. Interestingly, TP53INP1 binds to ATG8-family proteins with higher affinity than p62, suggesting that it could partially displace p62 from autophagosomes, modifying thereby their composition. Moreover, silencing the expression of autophagy related genes (ATG5 or Beclin-1) or inhibiting caspase activity significantly decreases cell death induced by TP53INP1. These data indicate that cell death observed after TP53INP1-LC3 interaction depends on both autophagy and caspase activity. We conclude that TP53INP1 could act as a tumor suppressor by inducing cell death by caspase-dependent autophagy.

Expression of Deltex1 during mouse embryogenesis: comparison with Notch1, 2 and 3 expression.

The Notch signalling pathway defines a phylogenetically conserved cell-cell communication process that enables cell-fate specification in multicellular organisms. Deltex is a component of the Notch signalling network that physically interacts with the ankyrin repeats of Notch. Here, we report on the expression pattern of the Deltex1 gene during mouse embryonic development and, furthermore, we compare its expression with that of the Notch1, 2 and 3 genes. Complementary and combinatorial expression patterns between Deltex1 and the three Notch genes were observed throughout embryogenesis since Deltex1 expression was related either to cytodifferentiation (i.e. neuronal tissues) or to cell proliferation events (i.e. eye, vascular structures, hematopoiesis).

A new alternative transcript encodes a 60 kDa truncated form of integrin beta 3.

A cDNA for integrin beta 3 isolated from a human erythroleukaemia (HEL) cell library contained a 340 bp insert at position 1281. This mRNA, termed beta 3c, results from the use of a cryptic AG donor splice site in intron 8 of the beta 3 gene, and is different from a previously described alternative beta 3 mRNA. The predicted open reading frame of beta 3C stops at a TAG stop codon 69 bp downstream from position 1281. It starts with the signal peptide and the 404 N-terminal extracellular residues of beta 3, encompassing the ligand binding sites, followed by 23 C-terminal intron-derived residues, corresponding to a truncated form of beta 3 lacking the cysteine-rich, transmembrane and cytoplasmic domains. Expression of beta 3C mRNA was demonstrated in human platelets, megakaryocytes, endothelial cells and HEL cells by reverse transcriptase/PCR. The beta 3C transcript was also demonstrated in the mouse, suggesting its conservation through evolution. Finally, a 60 kDa polypeptide corresponding to the beta 3C alternative transcript was demonstrated in platelets by Western blotting using a polyclonal antibody raised against a synthetic peptide designed from the beta 3C intronic sequence. Taken together, these results suggest a biological role for beta 3C, the first alternative transcript showing an altered extracellular domain of a beta integrin.

Characterization of HIV1-PAR, a macrophage-tropic strain: cell tropism, virus/cell entry and nucleotide sequence of the envelope glycoprotein.

The HIV1-PAR strain, isolated from the cerebrospinal fluid of an HIV1-seropositive man suffering from encephalopathy, replicated well in cord blood lymphocytes, poorly in peripheral blood mononuclear cells, and to different levels in blood-derived macrophage (BDM) cultures prepared from different blood donors. In marked contrast to its replication in primocultures, it did not grow in CEM and U937 cell lines. HIV1-PAR production in BDM was inhibited by more than 90% after treatment with OKT4A or 13B8.2 monoclonal antibodies (mAb) binding to adjacent epitopes of the D1 domain of the CD4 molecules. A lower but significant inhibitory effect was observed after BDM treatment with BL4 and OKT4 mAb, directed to the D2 and D3 domain of the CD4 molecule, respectively. The entire HIV1-PAR envelope glycoprotein gene was amplified by polymerase chain reaction and sequenced. The deduced amino acid sequence of HIV1-PAR gp160 revealed the presence of 847 amino acids and 86% homology with the HIV1 LAV virus prototype. An alignment of the amino acid sequence of the envelope glycoprotein of HIV1-PAR and HIV1-LAV showed that the differences were mostly clustered within the five variable regions. Five CD4-binding domains, the gp120/gp41 cleavage site, the putative gp41 fusion domain and 21 out of the 22 cysteine residues were conserved in both isolates. The results further confirm the macrophage-tropic character of the HIV1-PAR virus.

Human platelets express the SERCA2-b isoform of Ca(2+)-transport ATPase.

Previous biochemical studies suggested that the human platelet Ca2+ATPase system may be cell-specific. To test this hypothesis, we first undertook the molecular cloning of Ca2+ATPase from human erythroleukaemia (HEL) cells, because this human cell line exhibits megakaryocytic features and expresses a Ca2+ATPase that cross-reacts with platelet Ca(2+)-ATPase. For this cloning, an HEL-cell cDNA library was screened with a rat cardiac Ca2+ATPase cDNA probe. The insert of the longest clone isolated was 3.9 kb and its sequence displayed a 100% identity with that of the non-muscle human Ca2+ATPase 2-b isoform, termed SERCA2-b (sarco-endoplasmic-reticulum Ca2+ATPase). The 3.9 kb cDNA covered a subtotal coding region and part of the 3' non-coding end of the SERCA2-b mRNA. It cross-hybridized with the 4 kb transcript species of cardiac SERCA2-a and with non-muscle SERCA2-b mRNAs, but not with fast-skeletal-muscle SERCA1 mRNA. We next confirmed that SERCA2-b was a component of the platelet Ca2+ATPase system because (1) the platelet clones isolated from a platelet cDNA library exhibited a 100% homology with HEL-cell cDNA; (2) SERCA2-b mRNA was amplified by PCR on total platelet RNA and (3) platelet Ca2+ATPase cross-reacted with a polyclonal SERCA2-b-specific antiserum. Platelets therefore contain a Ca2+ATPase definitely identified as the SERCA2-b isoform of Ca2+ATPase, thus eliminating the possibility that they only contain a single specific Ca2+ATPase.

Expression of chimeric human transferrin genes in transfected human tumor cell lines.

The iron-binding plasma protein transferrin (TF) is essential for supplying iron to cells and the prevention of iron toxicity. Our laboratory has cloned and characterized the human TF gene. Comparison of promoter regions of TF genes from human, chicken, and mouse reveals a strong nucleotide sequence conservation. This study demonstrates that 5' flanking regions of the TF gene are sufficient for directing expression of a heterologous gene in transgenic mice and transfected cells. For cell-specific expression, more than 150 base pairs appear to be required.

Cloning of glycoprotein IIIa cDNA from human erythroleukemia cells and localization of the gene to chromosome 17.

Platelet aggregation requires the binding of adhesive proteins such as fibrinogen to the heterodimer of membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Human erythroleukemia (HEL) cells synthesize both GPIIb and GPIIIa. Using poly(A+) RNA purified from HEL cells, we constructed a cDNA library in the lambda gt10 phage vector. This library was screened with a 38mer oligonucleotide derived from a platelet GPIIIa peptide, and three overlapping cDNAs were isolated. The three inserts encompassed 3.5 kilobases (kb), including the entire coding region of mature GPIIIa (2,286 basepairs, bp) and 1.3 kb of 3' untranslated sequence. All 222 residues determined directly from platelet GPIIIa tryptic peptides exactly matched the HEL cell-deduced amino acid sequence. The HEL cell sequence matched a previously reported endothelial cell cDNA sequence except for eight nucleotides. Five of these nucleotide differences were silent changes consistent with genetic polymorphisms. The other three differences resulted in changes in the deduced amino acid sequence of GPIIIa; reexamination of the endothelial cell cDNA sequence in these three areas revealed that it is actually identical to the HEL cell sequence. The virtual identity of the endothelial and HEL cell cDNA sequences provides direct evidence that GPIIIa is a subunit common to cell-adhesion receptors present in more than one cell type. We localized the gene for GPIIIa to chromosome 17, the same chromosome to which we had previously mapped the gene for GPIIb.