Opposing roles for calcineurin and ATF3 in squamous skin cancer.

Calcineurin inhibitors such as cyclosporin A (CsA) are the mainstay of immunosuppressive treatment for organ transplant recipients. Squamous cell carcinoma (SCC) of the skin is a major complication of treatment with these drugs, with a 65 to 100-fold higher risk than in the normal population. By contrast, the incidence of basal cell carcinoma (BCC), the other major keratinocyte-derived tumour of the skin, of melanoma and of internal malignancies increases to a significantly lesser extent. Here we report that genetic and pharmacological suppression of calcineurin/nuclear factor of activated T cells (NFAT) function promotes tumour formation in mouse skin and in xenografts, in immune compromised mice, of H-ras(V12) (also known as Hras1)-expressing primary human keratinocytes or keratinocyte-derived SCC cells. Calcineurin/NFAT inhibition counteracts p53 (also known as TRP53)-dependent cancer cell senescence, thereby increasing tumorigenic potential. ATF3, a member of the 'enlarged' AP-1 family, is selectively induced by calcineurin/NFAT inhibition, both under experimental conditions and in clinically occurring tumours, and increased ATF3 expression accounts for suppression of p53-dependent senescence and enhanced tumorigenic potential. Thus, intact calcineurin/NFAT signalling is critically required for p53 and senescence-associated mechanisms that protect against skin squamous cancer development.

Molecular Genetics of FAM161A in North American Patients with Early-Onset Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a hereditary disease that leads to the progressive degeneration of retinal photoreceptor cells and to blindness. It is caused by mutations in several distinct genes, including the ciliary gene FAM161A, which is associated with a recessive form of this disorder. Recent investigations have revealed that defects in FAM161A represent a rather prevalent cause of hereditary blindness in Israel and the Palestinian territories, whereas they seem to be rarely present within patients from Germany. Genetic or clinical data are currently not available for other countries. In this work, we screened a cohort of patients with recessive RP from North America to determine the frequency of FAM161A mutations in this ethnically-mixed population and to assess the phenotype of positive cases. Out of 273 unrelated patients, only 3 subjects had defects in FAM161A. A fourth positive patient, the sister of one of these index cases, was also identified following pedigree analysis. They were all homozygous for the p.T452Sfx3 mutation, which was previously reported as a founder DNA variant in the Israeli and Palestinian populations. Analysis of cultured lymphoblasts from patients revealed that mutant FAM161A transcripts were actively degraded by nonsense-mediated mRNA decay. Electroretinographic testing showed 30 Hz cone flicker responses in the range of 0.10 to 0.60 microvolts in all cases at their first visit (age 12 to 23) (lower norm  =  50 μV) and of 0.06 to 0.32 microvolts at their most recent examination (age 27 to 43), revealing an early-onset of this progressive disease. Our data indicate that mutations in FAM161A are responsible for 1% of recessive RP cases in North America, similar to the prevalence detected in Germany and unlike the data from Israel and the Palestinian territories. We also show that, at the molecular level, the disease is likely caused by FAM161A protein deficiency.

Cross-presenting human gammadelta T cells induce robust CD8+ alphabeta T cell responses.

Gammadelta T cells are implicated in host defense against microbes and tumors but their mode of function remains largely unresolved. Here, we have investigated the ability of activated human Vgamma9Vdelta2(+) T cells (termed gammadelta T-APCs) to cross-present microbial and tumor antigens to CD8(+) alphabeta T cells. Although this process is thought to be mediated best by DCs, adoptive transfer of ex vivo antigen-loaded, human DCs during immunotherapy of cancer patients has shown limited success. We report that gammadelta T-APCs take up and process soluble proteins and induce proliferation, target cell killing and cytokine production responses in antigen-experienced and naïve CD8(+) alphabeta T cells. Induction of APC functions in Vgamma9Vdelta2(+) T cells was accompanied by the up-regulation of costimulatory and MHC class I molecules. In contrast, the functional predominance of the immunoproteasome was a characteristic of gammadelta T cells irrespective of their state of activation. Gammadelta T-APCs were more efficient in antigen cross-presentation than monocyte-derived DCs, which is in contrast to the strong induction of CD4(+) alphabeta T cell responses by both types of APCs. Our study reveals unexpected properties of human gammadelta T-APCs in the induction of CD8(+) alphabeta T effector cells, and justifies their further exploration in immunotherapy research.

Genetics of normal and pathological sleep in humans.

The complexity of sleep-wake regulation, in addition to the many environmental influences, includes genetic predisposing factors, which begin to be discovered. Most of the current progress in the study of sleep genetics comes from animal models (dogs, mice, and drosophila). Multiple approaches using both animal models and different genetic techniques are needed to follow the segregation and ultimately to identify 'sleep genes' and molecular bases of sleep disorders. Recent progress in molecular genetics and the development of detailed human genome map have already led to the identification of genetic factors in several complex disorders. Only a few genes are known for which a mutation causes a sleep disorder. However, single gene disorders are rare and most common disorders are complex in terms of their genetic susceptibility, environmental factors, gene-gene, and gene-environment interactions. We review here the current progress in the genetics of normal and pathological sleep and suggest a few future perspectives.

Immunogenicity of the carcinoembryonic antigen derived peptide 694 in HLA-A2 healthy donors and colorectal carcinoma patients.

Carcinoembryonic antigen (CEACAM5) is commonly overexpressed in human colon cancer. Several antigenic peptides recognized by cytolytic CD8+ T-cells have been identified and used in colon cancer phase-I vaccination clinical trials. The HLA-A*0201-binding CEA(694-702) peptide was recently isolated from acid eluted MHC-I associated peptides from a human colon tumor cell line. However, the immunogenicity of this peptide in humans remains unknown. We found that the peptide CEA(694-702) binds weakly to HLA-A*0201 molecules and is ineffective at inducing specific CD8+ T-cell responses in healthy donors. Immunogenic-altered peptide ligands with increased affinity for HLA-A*0201 were identified. Importantly, the elicited cytolytic T lymphocyte (CTL) lines and clones cross-reacted with the wild-type CEA(694-702) peptide. Tumor cells expressing CEA were recognized in a peptide and HLA-A*0201 restricted fashion, but high-CEA expression levels appear to be required for CTL recognition. Finally, CEA-specific T-cell precursors could be readily expanded by in vitro stimulation of peripheral blood mononuclear cell (PBMC) from colon cancer patients with altered CEA peptide. However, the CEA-specific CD8+ T-cell clones derived from cancer patients revealed low-functional avidity and impaired tumor-cell recognition. Together, using T-cells to demonstrate the processing and presentation of the peptide CEA694-702, we were able to corroborate its presentation by tumor cells. However, the low avidity of the specific CTLs generated from cancer patients as well as the high-antigen expression levels required for CTL recognition pose serious concerns for the use of CEA694-702 in cancer immunotherapy.

Molecular genetics of ocular diseases

The eye is a complex organ, which provides one of our most important senses, sight. The retina is the neuronal component of the eye and represents the connection with the central nervous system for the transmission of the information that leads to image processing. Retinitis pigmentosa (RP) is one of the most common forms of inherited retinal degeneration, in which the primary death of rods, resulting in night blindness, is always followed by the loss of cones, which leads to legal blindness. Clinical and genetic heterogeneity in retinitis pigmentosa is not only due to different mutations in different genes, but also to different effects of the same mutation in different individuals, sometimes even within the same family. My thesis work has been mainly focused on an autosomal dominant form of RP linked to mutations in the PRPF31 gene, which often shows reduced penetrance. Our study has led to the identification of the major regulator of the penetrance of PRPF31 mutations, the CNOT3 protein, and to the characterization of its mechanism of action. Following the same rationale of investigating molecular mechanisms that are responsible for clinical and genetic heterogeneity of retinitis pigmentosa, we studied a recessive form of the disease associated with mutations in the recently-identified gene FAMI61 A, where mutations in the same gene give rise to variable clinical manifestations. Our data have increased the knowledge of the relationship between genotype and phenotype in this form of the disease. Whole genome sequencing technique was also tested as a strategy for disease gene identification in unrelated patients with recessive retinitis pigmentosa and proved to be effective in identifying disease-causing variants that might have otherwise failed to be detected with other screening methods. Finally, for the first time we reported a choroidal tumor among the clinical manifestations of PTEN hamartoma tumor syndrome, a genetic disorder caused by germline mutations of the tumor suppressor gene PTEN. Our study has highlighted the heterogeneity of this choroidal tumor, showing that genetic and/or epigenetic alterations in different genes may contribute to the tumor development and growth. - L'oeil est un organe complexe, à l'origine d'un de nos sens les plus importants, la vue. La rétine est la composante neuronale de l'oeil qui constitue la connexion avec le système nerveux central pour la transmission de l'information et qui conduit à la formation des images. La rétinite pigmentaire (RP) est une des formes les plus courantes de dégénérescence rétinienne héréditaire, dans laquelle la mort primaire de bâtonnets, entraînant la cécité nocturne, est toujours suivie par la perte de cônes qui conduit à la cécité complète. L'hétérogénéité clinique et génétique dans la rétinite pigmentaire n'est pas seulement due aux différentes mutations dans des gènes différents, mais aussi à des effets différents de la même mutation chez des individus différents, parfois même dans la même famille. Mon travail de thèse s'est principalement axé sur une forme autosomique dominante de RP liée à des mutations dans le gène PRPF31, associées souvent à une pénétrance réduite, me conduisant à l'identification et à la caractérisation du mécanisme d'action du régulateur principal de la pénétrance des mutations: la protéine CNOT3. Dans la même logique d'étude des mécanismes moléculaires responsables de l'hétérogénéité clinique et génétique de la RP, nous avons étudié une forme récessive de la maladie associée à des mutations dans le gène récemment identifié FAMI61 A, dont les mutations dans le même gène donnent lieu à des manifestations cliniques différentes. Nos données ont ainsi accru la connaissance de la relation entre le génotype et le phénotype dans cette forme de maladie. La technique de séquençage du génome entier a été ensuite testée en tant que stratégie pour l'identification du gène de la maladie chez les patients atteints de RP récessive. Cette approche a montré son efficacité dans l'identification de variantes pathologiques qui n'auraient pu être détectées avec d'autres méthodes de dépistage. Enfin, pour la première fois, nous avons identifié une tumeur choroïdienne parmi les manifestations cliniques du PTEN hamartoma tumor syndrome, une maladie génétique causée par des mutations germinales du gène suppresseur de tumeur PTEN. Notre étude a mis en évidence l'hétérogénéité de cette tumeur choroïdienne, montrant que les altérations génétiques et/ou épigénétiques dans les différents gènes peuvent contribuer au développement et à la croissance tumorale.

Vaccination with a recombinant protein encoding the tumor-specific antigen NY-ESO-1 elicits an A2/157-165-specific CTL repertoire structurally distinct and of reduced tumor reactivity than that elicited by spontaneous immune responses to NY-ESO-1-expressing Tumors.

In a recent vaccination trial assessing the immunogenicity of an NY-ESO-1 (ESO) recombinant protein administered with Montanide and CpG, we have obtained evidence that this vaccine induces specific cytolytic T lymphocytes (CTL) in half of the patients. Most vaccine-induced CTLs were directed against epitopes located in the central part of the protein, between amino acids 81 and 110. This immunodominant region, however, is distinct from another ESO CTL region, 157-165, that is a frequent target of spontaneous CTL responses in A2+ patients bearing ESO tumors. In this study, we have investigated the CTL responses to ESO 157-165 in A2+ patients vaccinated with the recombinant protein. Our data indicate that after vaccination with the protein, CTL responses to ESO 157-165 are induced in some, but not all, A2+ patients. ESO 157-165-specific CTLs induced by vaccination with the ESO protein were functionally heterogeneous in terms of tumor recognition and often displayed decreased tumor reactivity as compared with ESO 157-165-specific CTLs isolated from patients with spontaneous immune responses to ESO. Remarkably, protein-induced CTLs used T-cell receptors similar to those previously isolated from patients vaccinated with synthetic ESO peptides (Vbeta4.1) and distinct from those used by highly tumor-reactive CTLs isolated from patients with spontaneous immune responses (Vbeta1.1, Vbeta8.1, and Vbeta13.1). Together, these results demonstrate that vaccination with the ESO protein elicits a repertoire of ESO 157-165-specific CTLs bearing T-cell receptors that are structurally distinct from those of CTLs found in spontaneous immune responses to the antigen and that are heterogeneous in terms of tumor reactivity, being often poorly tumor reactive.

Essential role of MALT1 protease activity in activated B cell-like diffuse large B-cell lymphoma.

A key element for the development of suitable anti-cancer drugs is the identification of cancer-specific enzymatic activities that can be therapeutically targeted. Mucosa-associated lymphoid tissue transformation protein 1 (MALT1) is a proto-oncogene that contributes to tumorigenesis in diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype, the least curable subtype of DLBCL. Recent data suggest that MALT1 has proteolytic activity, but it is unknown whether this activity is relevant for tumor growth. Here we report that MALT1 is constitutively active in DLBCL lines of the ABC but not the GCB subtype. Inhibition of the MALT1 proteolytic activity led to reduced expression of growth factors and apoptosis inhibitors, and specifically affected the growth and survival of ABC DLBCL lines. These results demonstrate a key role for the proteolytic activity of MALT1 in DLBCL of the ABC subtype, and provide a rationale for the development of pharmacological inhibitors of MALT1 in DLBCL therapy.

Different behaviour of mouse-human chimeric antibody F(ab')2 fragments of IgG1, IgG2 and IgG4 sub-class in vivo.

Mouse-human chimeric monoclonal antibodies (MAbs) of 3 different human IgG sub-classes directed against carcinoembryonic antigen (CEA) have been produced in SP-0 cells transfected with genomic chimeric DNA. F(ab')2 fragments were obtained by pepsin digestion of the purified chimeric MAbs of human IgG1, IgG2 and IgG4 sub-class and of parental mouse MAb IgG1. The 4 F(ab')2 fragments exhibit similar molecular weight by SDS-PAGE. They were labelled with 125I or 131I and high binding (80 to 87%) to purified unsolubilized CEA was observed. In vivo, double labelling experiments indicate that the longest biological half-life and the highest tumour-localization capacity is obtained with F(ab')2 from chimeric MAb of human IgG2 sub-class, whereas F(ab')2 from chimeric MAb IgG4 give very low values for these 2 parameters. F(ab')2 from chimeric MAb IgG1 and from parental mouse MAb yield intermediate results in vivo. Our findings should help to select the appropriate human IgG sub-class to produce chimeric or reshaped MAb F(ab')2 to be used for tumour detection by immunoscintigraphy and for radioimmunotherapy.

A soluble form of B cell maturation antigen, a receptor for the tumor necrosis factor family member APRIL, inhibits tumor cell growth.

A proliferation-inducing ligand (APRIL) is a ligand of the tumor necrosis factor (TNF) family that stimulates tumor cell growth in vitro and in vivo. Expression of APRIL is highly upregulated in many tumors including colon and prostate carcinomas. Here we identify B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI), two predicted members of the TNF receptor family, as receptors for APRIL. APRIL binds BCMA with higher affinity than TACI. A soluble form of BCMA, which inhibits the proliferative activity of APRIL in vitro, decreases tumor cell proliferation in nude mice. Growth of HT29 colon carcinoma cells is blocked when mice are treated once per week with the soluble receptor. These results suggest an important role for APRIL in tumorigenesis and point towards a novel anticancer strategy.

Increased Wnt signaling triggers oncogenic conversion of human breast epithelial cells by a Notch-dependent mechanism.

Wnt and Notch signaling have long been established as strongly oncogenic in the mouse mammary gland. Aberrant expression of several Wnts and other components of this pathway in human breast carcinomas has been reported, but evidence for a causative role in the human disease has been missing. Here we report that increased Wnt signaling, as achieved by ectopic expression of Wnt-1, triggers the DNA damage response (DDR) and an ensuing cascade of events resulting in tumorigenic conversion of primary human mammary epithelial cells. Wnt-1-transformed cells have high telomerase activity and compromised p53 and Rb function, grow as spheres in suspension, and in mice form tumors that closely resemble medullary carcinomas of the breast. Notch signaling is up-regulated through a mechanism involving increased expression of the Notch ligands Dll1, Dll3, and Dll4 and is required for expression of the tumorigenic phenotype. Increased Notch signaling in primary human mammary epithelial cells is sufficient to reproduce some aspects of Wnt-induced transformation. The relevance of these findings for human breast cancer is supported by the fact that expression of Wnt-1 and Wnt-4 and of established Wnt target genes, such as Axin-2 and Lef-1, as well as the Notch ligands, such as Dll3 and Dll4, is up-regulated in human breast carcinomas.