Expression of T-cadherin in tumor cells influences invasive potential of human hepatocellular carcinoma.

Overexpression of T-cadherin (T-cad) transcripts occurs in approximately 50% of human hepatocellular carcinomas (HCCs). To elucidate T-cad functions in HCC, we examined T-cad protein expression in normal and tumoral human livers and hepatoma cell lines and investigated its influence on invasive potential of HCC using RNA interference silencing of T-cad expression in Mahlavu cells. Whereas T-cad expression was restricted to endothelial cells (EC) from large blood vessels in normal livers, it was up-regulated in sinusoidal EC from 8/15 invasive HCCs. Importantly, in three of them (38%) T-cad was detected in tumor cells within regions in which E-cadherin expression was absent. Among six hepatoma cell lines, only Mahlavu expressed T-cad but not E-cadherin. T-cad exhibited a globally punctuate distribution in quiescent Mahlavu and additionally it concentrated at the leading edge of migrating cells. Matrigel invasion assay revealed that Mahlavu possess a high invasive potential that was significantly inhibited by T-cad silencing. Wound healing and random motility assays demonstrated that inhibition of T-cad expression in Mahlavu significantly reduced their motility. We propose that T-cad expression in tumor cells might occur by cadherin-switching during epithelial-mesenchymal transition and may represent an additional mechanism contributing to HCC metastasis.

Investigation in liver tissues and cell lines of the transcription of 13 genes mapping to the 16q24 region that are frequently deleted in hepatocellular carcinoma.

Many studies have associated chromosomal deletions in the 16q24 region with human cancers, including hepatocellular carcinoma. A more limited region around the microsatellite D16S402 has been shown implicated in the metastatic spread of hepatocellular carcinoma, prostate cancer, and Wilms' tumors. It is likely that one or more tumor suppressor genes are located in this 16q24 area. We used SYBR Green reagents to quantify, by real-time quantitative RT-PCR, the production of mRNA for 13 genes mapping to 16q24. The locations of these genes were determined from published human genome sequencing data. We studied mRNA levels in 10 liver tumor tissues, 10 nontumor liver tissues, five hepatoma cell lines, and in isolated hepatocytes. Results were compared with those for loss of heterozygosity observed in the D16S402 region and recurrence. No down-regulation was observed in tumor tissues. Two genes were consistently overexpressed: OKL38 and CDH13. CDH13, which functions in cell-cell adhesion, seems to be involved in liver carcinogenesis. However, no relationship was observed between the expression of this gene and changes in the D16S402 microsatellite or tumor recurrence. None of the other genes tested seemed to be a good candidate for a major tumor suppressor gene in liver carcinogenesis. Our results suggest that additional unknown genes involved in carcinogenesis are located in the 16q24 area.

Use of fusion proteins and procaryotic display systems for delivery of HIV-1 antigens: development of novel vaccines for HIV-1 infection.

Two non-pathogenic scaffolds (represented by the filamentous bacteriophage fd and the dihydrolipoyl acetyltransferase E2 protein of the Bacillus stearothermophilus pyruvate dehydrogenase (PDH) complex) able to deliver human immunodeficiency virus (HIV)-1 antigenic determinants, were designed in our laboratories and investigated in controlled assay conditions. Based on a modification of the phage display technology, we developed an innovative concept for a safe and inexpensive vaccine in which conserved antigenic determinants of HIV-1 reverse transcriptase (RTase) were inserted into the N-terminal region of the major pVIII coat protein of bacteriophagefd virions. Analogously, we developed another antigen delivery system based on the E2 component from the PDH complex and capable of displaying large intact proteins on the surface of an icosahedral lattice. Our data show that both of these systems can deliver B and T epitopes to their respective presentation compartments in target cells and trigger a humoral response as well as a potent helper and cytolytic response in vitro and in vivo.

Processing of filamentous bacteriophage virions in antigen-presenting cells targets both HLA class I and class II peptide loading compartments.

Virions of filamentous bacteriophage fd are capable of displaying multiple copies of peptide epitopes and generating powerful immune responses to them. To investigate the antigen processing mechanisms in human B cell lines used as antigen presenting cells, the major coat protein (pVIII) in intact virions was fluorescently labeled, and its localization in various intracellular compartments was followed using confocal microscopy. We show that the virions were taken up and processed to yield peptides that reach both the major histocompatibility complex (MHC) class II compartment and the endoplasmic reticulum. Moreover, when exposed to bacteriophages displaying a cytotoxic T lymphocyte (CTL) epitope from the reverse transcriptase of human immunodeficiency virus type-1 (HIV-1), B cells were lysed by specific cytotoxic lymphocytes. This confirms that filamentous bacteriophage virions are capable of being taken up and processed efficiently by MHC class I and class II pathways, even in nonprofessional antigen presenting cells. These remarkable features explain, at least in part, the unexpected ability of virions displaying foreign T-cell epitopes to prime strong T-helper-dependent CTL responses. These findings have important implications for the development of peptide-based vaccines, using filamentous bacteriophage virions as scaffolds.

A functional and regulatory network associated with PIP expression in human breast cancer.

BACKGROUND: The PIP (prolactin-inducible protein) gene has been shown to be expressed in breast cancers, with contradictory results concerning its implication. As both the physiological role and the molecular pathways in which PIP is involved are poorly understood, we conducted combined gene expression profiling and network analysis studies on selected breast cancer cell lines presenting distinct PIP expression levels and hormonal receptor status, to explore the functional and regulatory network of PIP co-modulated genes. PRINCIPAL FINDINGS: Microarray analysis allowed identification of genes co-modulated with PIP independently of modulations resulting from hormonal treatment or cell line heterogeneity. Relevant clusters of genes that can discriminate between [PIP+] and [PIP-] cells were identified. Functional and regulatory network analyses based on a knowledge database revealed a master network of PIP co-modulated genes, including many interconnecting oncogenes and tumor suppressor genes, half of which were detected as differentially expressed through high-precision measurements. The network identified appears associated with an inhibition of proliferation coupled with an increase of apoptosis and an enhancement of cell adhesion in breast cancer cell lines, and contains many genes with a STAT5 regulatory motif in their promoters. CONCLUSIONS: Our global exploratory approach identified biological pathways modulated along with PIP expression, providing further support for its good prognostic value of disease-free survival in breast cancer. Moreover, our data pointed to the importance of a regulatory subnetwork associated with PIP expression in which STAT5 appears as a potential transcriptional regulator.

Deciphering cellular states of innate tumor drug responses.

BACKGROUND: The molecular mechanisms underlying innate tumor drug resistance, a major obstacle to successful cancer therapy, remain poorly understood. In colorectal cancer (CRC), molecular studies have focused on drug-selected tumor cell lines or individual candidate genes using samples derived from patients already treated with drugs, so that very little data are available prior to drug treatment. RESULTS: Transcriptional profiles of clinical samples collected from CRC patients prior to their exposure to a combined chemotherapy of folinic acid, 5-fluorouracil and irinotecan were established using microarrays. Vigilant experimental design, power simulations and robust statistics were used to restrain the rates of false negative and false positive hybridizations, allowing successful discrimination between drug resistance and sensitivity states with restricted sampling. A list of 679 genes was established that intrinsically differentiates, for the first time prior to drug exposure, subsequently diagnosed chemo-sensitive and resistant patients. Independent biological validation performed through quantitative PCR confirmed the expression pattern on two additional patients. Careful annotation of interconnected functional networks provided a unique representation of the cellular states underlying drug responses. CONCLUSION: Molecular interaction networks are described that provide a solid foundation on which to anchor working hypotheses about mechanisms underlying in vivo innate tumor drug responses. These broad-spectrum cellular signatures represent a starting point from which by-pass chemotherapy schemes, targeting simultaneously several of the molecular mechanisms involved, may be developed for critical therapeutic intervention in CRC patients. The demonstrated power of this research strategy makes it generally applicable to other physiological and pathological situations.

Induction of specific T-helper and cytolytic responses to epitopes displayed on a virus-like protein scaffold derived from the pyruvate dehydrogenase multienzyme complex.

The icosahedral protein scaffold (1.5MDa) generated by self-assembly of the catalytic domains of the dihydrolipoyl acetyltransferase core of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus has been engineered to display 60 copies of one or more peptide epitopes on a single molecule (E2DISP). An E2DISP scaffold displaying pep23, a 15-residue B- and T-helper epitope from the reverse transcriptase of HIV-1, was able to induce a pep23-specific T-helper response in cell lines in vitro. The same scaffold displaying both pep23 and peptide RT2, a nine-residue CTL epitope from HIV-1 reverse transcriptase, was able to prime an RT2-specific CD8(+) T-cell response in human cell lines in vitro and in HLA-A2 transgenic mice in vivo. This was accompanied by a humoral antibody response specific for E2DISP-presented epitopes. Thus, the icosahedral acetyltransferase core constitutes a simple and flexible scaffold for multiple epitope display with access to both cellular and humoral immune response pathways.

Analysis of chromosomal instability in pulmonary or liver metastases and matched primary hepatocellular carcinoma after orthotopic liver transplantation.

To investigate the genetic mechanism of metastatic spread in hepatocellular carcinoma (HCC), we analyzed genomic changes in lung or liver metastases and the corresponding primary tumors (83 tumor samples) in 18 patients who underwent orthotopic liver transplantation. We studied the incidence of microsatellite instability (MSI) and loss of heterozygosity (LOH) involving 8 highly polymorphic microsatellite markers and the polyA tract, Bat26. We also sought alterations of p53 and beta-catenin gene mutations. High MSI (>30-40% of the loci analyzed) was found only in primary tumors (11%), whereas LOH was observed in 50% of primary and in 39% of recurrent tumors. p53 mutations were found in 2 cases of primary HCC but not in the corresponding metastases. P53 was overexpressed in 4 primary HCC (22%) and 7 metastases (39%). The percentage of beta-catenin gene mutations was low (6%). Lung metastases retained the D16S402 microsatellite abnormalities observed in the primary tumors, whereas recurrent liver tumor did not (p = 0.02). In conclusion, LOH and P53 protein overexpression, rather than mutations in the p53 or beta-catenin genes or MSI, seem to be involved in the spreading of HCC, suggesting the presence of metastasis suppressor genes in the vicinity of the chromosomal loci in question.

Expression and molecular characterization of alternative transcripts of the ARHGEF5/TIM oncogene specific for human breast cancer.

The ARHGEF5/TIM oncogene belongs to the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. It is well established that Rho-GEFs play an important role in tumorigenesis and metastasis through the activation of their substrates, the Rho GTPases. Little is known about ARHGEF5/TIM oncogene expression and cellular functions. Because of its localization close to the common fragile site FRA7I, which has been shown to be responsible for an inverted duplication of the 7q34-q35 region in breast carcinoma cells, we examined the expression of the ARHGEF5/TIM oncogene in normal and tumoral breast tissue. We report here the identification of five novel ARHGEF5/TIM alternative transcripts specifically expressed in breast tumors. These variant transcripts were characterized by the absence of one or several exons, all coding for the catalytic Dbl-homology domain and generating modified or truncated predicted variant proteins. The variant transcripts were predominantly expressed in breast carcinoma cell lines and in the most aggressive primary breast carcinomas, suggesting they may play a role in breast tumor progression. Moreover, we demonstrate that the expression of recombinant ARHGEF5/TIM protein in transfected COS-7 and NIH-3T3 cells generated a loss of actin stress fibers and the formation of membrane ruffles and filopodia. This pattern suggests that ARHGEF5/TIM activates Rac1, Cdc42 or RhoG rather than RhoA, as previously demonstrated in in vitro guanine nucleotide exchange assays. We anticipate that the activation of the ARHGEF5/TIM oncogene, possibly by the variant isoforms detected here, may play an important role in proliferative breast disease.

Intragenic amplification and formation of extrachromosomal small circular DNA molecules from the PIP gene on chromosome 7 in primary breast carcinomas.

The PIP gene is expressed in exocrine glands and, in pathologic conditions, in breast cysts and breast cancers exhibiting apocrine features. It is localized on the long arm of chromosome 7, a region frequently alterated in mammary tumors. We previously described an abnormal restriction pattern of the PIP gene in 33% of prostate carcinomas analyzed. Here, we analyze the structure of the PIP gene in primary breast carcinomas. We report that part of the 3' end, including exon 3, intron C, two-thirds of exon 4 and a small portion of intron B, is amplified and involved in the formation of extrachromosomal spcDNA molecules in 3/14 (21.4%) breast cancers analyzed. The involvement of a well-defined intragenic region of a gene in the formation of spcDNA appears to be unprecedented. Since spcDNA has been suggested to serve as an enhancer of genetic instability, the PIP gene may be the target of genomic variability processes in breast cancer.

Study of disabling T-cell activation and inhibiting T-cell-mediated immunopathology reveals a possible inverse agonist activity of CD4 peptidomimetics.

We designed a new class of aromatically modified exocyclic peptides based on the structure of CD4 by engineering one of the cysteine residues in a peptidomimetic derived from the CDR3 region of the CD4 molecule. All three species mediate inhibition of T-cell proliferation at concentrations ranging from 10 to 100 microM. The mimetics CD4-Cys and CD4-Met bind to sCD4 with affinities ranging from 1 to 2 microM, while CD4-Ser shows poor binding in radioisotope assay. Though these mimetics have similar structures, they exhibit different biochemical and biological functions. Activation of T-cells as measured by thymidine incorporation or IL-2 production revealed that CD4-Cys and CD4-Ser mimetics behave as classical antagonists. On the other hand, the CD4-Met species inhibited T-cell proliferation with an IC(50) of 30 microM but unexpectedly increased IL-2 secretion modestly at a less than 3 microM concentration. In experimental autoimmune encephalitis (EAE), CD4-Ser and CD4-Cys mimetics reduced the severity of EAE symptoms while the CD4-Met mimetic exacerbated the conditions. We propose that CD4-Cys and CD4-Ser are classical antagonists, but CD4-Met may possess properties of an inverse agonist. The structure-activity relationship of mimetics reveals that a minor change in the net hydropathic value is enough to alter the dynamic nature of the receptor-ligand complex.

Initiation of the breakage-fusion-bridge mechanism through common fragile site activation in human breast cancer cells: the model of PIP gene duplication from a break at FRA7I.

Gene amplification plays a critical role in tumor progression. Hence, understanding the factors triggering this process in human cancers is an important concern. Unfortunately, the structures formed at early stages are usually unavailable for study, hampering the identification of the initiating events in tumors. Here, we show that the region containing the PIP gene, which is overexpressed in 80% of primary and metastatic breast cancers, is duplicated in the breast carcinoma cell line T47D. The two copies are organized as a large palindrome, lying 'in loco' on one chromosome 7. Such features constitute the landmark of the breakage-fusion-bridge (BFB) cycle mechanism. In hamster cells selected in vitro to resist cytotoxic drugs, common fragile site (CFS) activation has been shown to trigger this mechanism. Here, we characterize FRA7I at the molecular level and demonstrate that it lies 2 Mb telomeric to the PIP gene and sets the distal end of the repeated sequence. Moreover, our results suggest that the BFB process was frozen within the first cycle by healing of the broken chromosome. T47D cells thus offer a unique opportunity to observe the earliest products of the BFB cycle mechanism. Our findings constitute the first evidence that this amplification mechanism can be initiated in vivo by fragile site activation.