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Background. Because of its high metastatic potential, inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer. We previously demonstrated that Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein overexpression was associated with shorter metastasis-free survival (MFS) in IBC patients, but not in non-IBC (nIBC) patients. However, the mechanism of action of MARCKS and its particular association to poorer outcome in IBC compared to nIBC are poorly understood. Methods. We evaluated in vitro the inhibitory effect of MPS (MARCKS phosphorylation site domain), a peptide targeting MARCKS phosphorylation site domain (PSD) in single and in combination with paclitaxel treatment, on cell proliferation and cell motility in two cell lines of different phenotype (SUM149 for IBC and MDA-MB-231 for nIBC), as well as its distinct molecular mechanisms of action. We also assessed the clinical relevance of the protein expression of MARCKS and phosphatase and tensin homolog (PTEN) in a large series of IBC vs. nIBC patients. Results. In vitro, the treatment with MPS peptide impaired cell proliferation, migration, and invasion in SUM149 compared to MDA-MB-231 cells. More important, MARCKS inhibition increased paclitaxel sensitivity when using combination therapy in SUM149 cells compared to MDA-MB-231 cells. Interestingly, we could partially explain this specific inhibitory effect in IBC cells using western blot: MARCKS inhibition in single and in combination induced up and downregulation of the PTEN/AKT signaling pathway respectively in IBC compared to nIBC cells. Importantly, a negative correlation of MARCKS and PTEN was only found in the clinical IBC samples (180 patients) compared to nIBC samples (355 patients). More importantly, the group of patients with negative MARCKS and positive PTEN protein expression was associated to better 5-year MFS only in IBC patients. Conclusion. These results indicate two major findings: first, the important prognostic value of the negative correlation of MARCKS and PTEN expression in IBC patients, and second the specific role of MARCKS in regulating different downstream pathways and increasing the paclitaxel response in combination treatment in IBC compared to non-IBC. They suggest a potential IBC-specific targetable biomarker, the inhibition of which might impair disease aggressiveness and perhaps enhance patients’ survival. Citation Format: Maroua Manai, Ines Bini, Pascal Finetti, Haifa Bichiou, Carolina Reduzzi, Naziha Ben Hamida, Marc Lopez, Khaled Rahal, Karima Mrad, Mohamed Manai, Massimo Cristofanilli, Hamouda Boussen, Raoudha Doghri, Maher Kharrat, François Bertucci. Targeting MARCKS in inflammatory breast cancer increased paclitaxel sensitivity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-15.

Antibody–drug conjugates (ADC) represent a fast-growing drug class in oncology. However, ADCs are associated with resistance, and therapies able to overcome it are of utmost importance. Recently, enfortumab vedotin-ejfv (EV) was approved in nectin-4+ metastatic urothelial cancer. We previously described PVRL4/nectin-4 as a new therapeutic target in breast cancer and produced an efficient EV-like ADC comprising a human anti–nectin-4 mAb conjugated to monomethyl auristatin-E (MMAE) named N41mab-vcMMAE. To study the consequence of the long-term treatment with this ADC, we developed a preclinical breast cancer model in mice, and report a mechanism of resistance to N41mab-vcMMAE after 9-month treatment and a way to reverse it. RNA-sequencing pointed to an upregulation in resistant tumors of ABCB1 expression, encoding the multidrug resistance protein MDR-1/P-glycoprotein (P-gp), associated with focal gene amplification and high protein expression. Sensitivity to N41mab-vcMMAE of the resistant model was restored in vitro by P-gp pharmacologic inhibitors, like tariquidar. P-gp is expressed in a variety of normal tissues. By delivering the drug to the tumor more specifically than classical chemotherapy, we hypothesized that the combined use of ADC with P-gp inhibitors might reverse resistance in vivo without toxicity. Indeed, we showed that the tariquidar/N41mab-vcMMAE combination was well tolerated and induced a rapid regression of ADC-resistant tumors in mice. In contrast, the tariquidar/docetaxel combination was toxic and poorly efficient. These results show that ABC transporter inhibitors can be safely used with ADC to reverse ADC-induced resistance and open new opportunities in the fight against multidrug resistance.

Supplementary Figure from Overcoming Resistance to Anti–Nectin-4 Antibody-Drug Conjugate

Supplementary Table from Overcoming Resistance to Anti–Nectin-4 Antibody-Drug Conjugate

Antibody–drug conjugates (ADC) represent a fast-growing drug class in oncology. However, ADCs are associated with resistance, and therapies able to overcome it are of utmost importance. Recently, enfortumab vedotin-ejfv (EV) was approved in nectin-4+ metastatic urothelial cancer. We previously described PVRL4/nectin-4 as a new therapeutic target in breast cancer and produced an efficient EV-like ADC comprising a human anti–nectin-4 mAb conjugated to monomethyl auristatin-E (MMAE) named N41mab-vcMMAE. To study the consequence of the long-term treatment with this ADC, we developed a preclinical breast cancer model in mice, and report a mechanism of resistance to N41mab-vcMMAE after 9-month treatment and a way to reverse it. RNA-sequencing pointed to an upregulation in resistant tumors of ABCB1 expression, encoding the multidrug resistance protein MDR-1/P-glycoprotein (P-gp), associated with focal gene amplification and high protein expression. Sensitivity to N41mab-vcMMAE of the resistant model was restored in vitro by P-gp pharmacologic inhibitors, like tariquidar. P-gp is expressed in a variety of normal tissues. By delivering the drug to the tumor more specifically than classical chemotherapy, we hypothesized that the combined use of ADC with P-gp inhibitors might reverse resistance in vivo without toxicity. Indeed, we showed that the tariquidar/N41mab-vcMMAE combination was well tolerated and induced a rapid regression of ADC-resistant tumors in mice. In contrast, the tariquidar/docetaxel combination was toxic and poorly efficient. These results show that ABC transporter inhibitors can be safely used with ADC to reverse ADC-induced resistance and open new opportunities in the fight against multidrug resistance.

Supplementary Figures. Fig.S1: Additional confocal images showing that expression of EFA6Bvsvg in MCF7 cells restores normal apico-basal polarity and promotes lumen formation Fig.S2: Analysis of the specificity of EFA6B repression using siRNAs Fig.S3: Additional confocal images showing that repression of EFA6B in MCF7 cells abrogates the assembly of TJ and leads to unorganized aggregates Fig.S4: Exogenous expression of EFA6Bvsvg abrogates the TGFβ-induced EMT in MDCK cells Fig.S5: Analysis of EFA6B/PSD4 mutations and copy number alterations from the cBioPortal website

The adenosine A2A receptor (A2AR), dopamine D2 receptor (D2R) and metabotropic glutamate receptor type 5 (mGluR5) form A2AR-D2R-mGluR5 heteroreceptor complexes in living cells and in rat striatal neurons. In the current study, we present experimental data supporting the view that the A2AR protomer plays a major role in the inhibitory modulation of the density and the allosteric receptor-receptor interaction within the D2R-mGluR5 heteromeric component of the A2AR-D2R-mGluR5 complex in vitro and in vivo. The A2AR and mGluR5 protomers interact and modulate D2R protomer recognition and signalling upon forming a trimeric complex from these receptors. Expression of A2AR in HEK293T cells co-expressing D2R and mGluR5 resulted in a significant and marked increase in the formation of the D2R-mGluR5 heteromeric component in both bioluminescence resonance energy transfer and proximity ligation assays. A highly significant increase of the the high-affinity component of D2R (D2RKi High) values was found upon cotreatment with the mGluR5 and A2AR agonists in the cells expressing A2AR, D2R and mGluR5 with a significant effect observed also with the mGluR5 agonist alone compared to cells expressing only D2R and mGluR5. In cells co-expressing A2AR, D2R and mGluR5, stimulation of the cells with an mGluR5 agonist like or D2R antagonist fully counteracted the D2R agonist-induced inhibition of the cAMP levels which was not true in cells only expressing mGluR5 and D2R. In agreement, the mGluR5-negative allosteric modulator raseglurant significantly reduced the haloperidol-induced catalepsy in mice, and in A2AR knockout mice, the haloperidol action had almost disappeared, supporting a functional role for mGluR5 and A2AR in enhancing D2R blockade resulting in catalepsy. The results represent a relevant example of integrative activity within higher-order heteroreceptor complexes.

The adenosine A

Early diagnosis and prompt management of acute mesenteric ischaemia (AMI) are key to survival but remain extremely difficult, due to vague and non-specific symptoms. Serum lactate (SL) is commonly presented as a useful biomarker for the diagnosis or prognosis of AMI. The aim of our study was test SL (1) as a diagnostic marker and (2) as a prognostic marker for AMI.This was an ancillary multicentre case-control study. Patients with AMI at intensive care unit (ICU) admission were included (AMI group) and matched to ICU patients without AMI (control group). SL was measured and compared on day 0 (D0) and day 1 (D1). Diagnosis and prognosis accuracy were assessed by receiver operating characteristic (ROC) and their area under the curve (AUC).Each group consisted of 137 matched ICU patients. There was no significant difference of SL between the two groups at D0 or at D1 (p = 0.26 and p = 0.29 respectively). SL was a poor marker of AMI: at D0 and D1, AUC were respectively 0.57 [0.51; 0.63] and 0.60 [0.53; 0.67]. SL at D0 and D1 correctly predicted ICU mortality, independently of AMI (AUC D0: 0.69 [0.59; 0.79] vs. 0.74 [0.65; 0.82]; p = 0.51 and D1: 0.74 [0.64; 0.84] vs. 0.76 [0.66; 0.87]; p = 0.77, respectively, for control and AMI groups].SL has no specific link with AMI, both for diagnosis and prognosis. SL should not be used for the diagnosis of AMI but, despite its lack of specificity, it may help to assess severity.

Background: Advanced cutaneous T-cell lymphomas (CTCLs) remain an unmet medical need. Brentuximab vedotin (BV), an anti-CD30 antibody-drug conjugate (ADC) linked to monomethyl auristatin E (MMAE), do not deliver significant long-term improvements in patient outcomes. More recently, mogamulizumab and anti-KIR3DL2 provided encouraging results but new targeted therapies are needed. Inducible Co-Stimulator (ICOS), a T-cell costimulatory receptor involved in the development of CTCLs, arouses interest. Methods: We used immunohistochemistry to study ICOS expression in skin biopsies of 23 patients with early-stage mycosis fungoides (MF), 12 with transformed MF (TMF) and 17 with Sézary Syndrome (SS), at diagnosis or in relapse. Skin samples from 12 patients with B-cell lymphomas, 14 with CD30 + lymphoproliferative disease (LPD), 12 with primary cutaneous CD4+ small/medium T-cell lympho-proliferative disorder and 13 with angioimmunoblastic T-cell lymphoma (AITL) were used as controls. ICOS expression by circulating Sézary cells and regulatory T cells (Tregs) in patients with SS was evaluated using flow cytometry and compared to healthy donors (HD) lymphocytes. In 5 patients with SS, we also analyzed concomitant biopsies from involved nodes. Then, we investigated the efficacy of anti-ICOS ADCs generated by coupling murine anti-ICOS 314.8 monoclonal antibodies with MMAE and pyrrolobenzodiazepine (PBD), in comparison to BV. We used ICOS + CTCL cell lines (MyLa, MJ and HUT78), murine xenograft models with MyLa and ICOS + Patient Derived Xenografts (PDXs) from patients with SS and AITL. In order to identify the best anti-ICOS clone that we should develop for a clinical trial, we evaluated the affinity of the antibody on the receptor, the internalization capacity of the antibody using pHAb Reactive Dyes kit (Promega®), and the ability of the antibody to act as an ADC using a secondary conjugate (Mab-ZAP kit, Advanced Targeting Systems®). Results: ICOS was highly expressed by the cutaneous atypical lymphocytic infiltrates in respectively 61%, 75% and 88% of patients with early-stage MF, TMF and SS, such as in all the involved nodes. Double staining experiments which were performed in both skin and lymph node revealed that ICOS expression appears mainly restricted to neoplastic CD4 + T-cells, with rare ICOS +CD8 + T-cells in the tumor micro-environment. ICOS expression by circulating Sézary cells was strong: 69 ± 7.3% versus 38.8 ± 7.1% of non-tumoral CD4 + cells (p In CTCL cell lines, we observed a significant dose-dependent decrease in cell viability in the presence of anti-ICOS-MMAE (IC50 = 8.2ng/mL) and anti-ICOS-PBD (IC50 = 1.2ng/mL) ADCs. In a mouse xenograft model (MyLa), anti-ICOS-MMAE ADCs provided a longer overall survival (OS) than BV (HR=15.2; CI95%: 3.2-71.1; p Among 8 different anti-ICOS clones, clone 314.8 had the best affinity on MyLa and MJ cell lines. Clones 53.3, 293.1, 92.17, 88.2 and 279.1 also had good affinity to receptor, whereas clones 145.1 and 121.4 had poor affinity. Using the internalization pHAb reactive dyes kit, we found that clones 314.8, 53.3, 92.17, 88.2 internalized significantly better and faster than the other ones. In order to verify if there is a correlation between internalization capacity and ADC effect, clones 53.3, 92.17 and 145.1 were coupled to MMAE. While anti-ICOS-53.3 and anti-ICOS-92.17 ADCs had similar efficacy to anti-ICOS-314.8 ADCs on MyLa, anti-ICOS 145.1 ADCs resulted in significantly lower cell death. Finally, all clones showed good ability to act as ADCs with Mab-ZAP, excluding clones 279.1, 145.1 and 121.4. Discussion: ICOS is a promising therapeutic target because it is expressed both by tumor T-cells and regulatory T-cells. We report for the first time the strong and frequent expression of ICOS in CTCLs, as well as the preclinical efficacy of anti-ICOS ADCs on CTCL cell line and PDXs. These results could be extended to the spectrum of follicular variant peripheral T-cell lymphomas. Conclusion: Collectively, our findings provide the preliminary basis for a therapeutic trial Figure 1 Figure 1. Disclosures Lopez: Emergence Therapeutics: Current holder of individual stocks in a privately-held company. Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees. Olive: ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Cancer is initiated by somatic mutations in oncogenes or tumor suppressor genes. However, additional alterations provide selective advantages to the tumor cells to resist treatment and develop metastases. Their identification is of paramount importance. Reduced expression of EFA6B (Exchange Factor for ARF6, B) is associated with breast cancer of poor prognosis. Here, we report that loss of EFA6B triggers a transcriptional reprogramming of the cell-to-ECM interaction machinery and unleashes CDC42-dependent collective invasion in collagen. In xenograft experiments, MCF10 DCIS.com cells, a DCIS-to-IDC transition model, invades faster when knocked-out for EFA6B. In addition, invasive and metastatic tumors isolated from patients have lower expression of EFA6B and display gene ontology signatures identical to those of EFA6B knock-out cells. Thus, we reveal an EFA6B-regulated molecular mechanism that controls the invasive potential of mammary cells; this finding opens up avenues for the treatment of invasive breast cancer., Reduced expression of EFA6B is found in highly metastatic breast cancer subtypes. Here, the authors report that loss of EFA6B promotes collective invasion through activation of the epithelial-to-mesenchymal transition program and CDC42-dependent signalling pathways in breast cancer.

The treatment of advanced-stage cutaneous T-cell lymphoma (CTCL) remains an unmet medical need. Mogamulizumab, anti-KIR3DL2, and brentuximab vedotin (BV), an anti-CD30 antibody–drug conjugate (ADC) coupled with monomethyl-auristatin-E (MMAE), provided encouraging results, but new targeted therapies are needed. Inducible T-cell costimulator (ICOS), a T-cell costimulatory receptor, is a promising therapeutic target, not only because it is expressed by malignant T cells in CTCL but also because of its connection with the suppressive activity of regulatory T (Treg) cells. Immunohistochemical analysis revealed that ICOS was widely expressed by malignant cells in skin biopsy specimens from 52 patients with mycosis fungoides and Sézary syndrome (SS), as well as in involved node biopsy specimens from patients with SS. Furthermore, flow cytometry demonstrated its strong expression by circulating tumor cells in all our patients with SS. Percentages of ICOS+ Treg cells were significantly higher in patients with SS than in healthy donors. We then investigated the preclinical efficacy of anti-ICOS ADCs generated by coupling murine anti-ICOS monoclonal antibodies with MMAE and pyrrolobenzodiazepine. In 3 CTCL cell lines (Myla, MJ, and HUT78), we observed a significant dose-dependent decrease in cell viability in the presence of anti-ICOS ADCs. In addition, anti-ICOS-MMAE ADCs had an in vitro and in vivo efficacy superior to BV in a mouse xenograft model (MyLa). Finally, we assessed the efficacy of anti-ICOS ADCs in ICOS+ patient-derived xenografts from patients with SS and angioimmunoblastic T-cell lymphoma. Collectively, our findings provide the preliminary basis for a therapeutic trial.

[Resumen] Este trabajo presenta el diseño de un desacoplador dinámico en un rango de frecuencia determinado para la detección de ataques de reproducción. Inicialmente se estudia el comportamiento del desacoplador al variar su orden. Después, se realiza una comparación cuantitativa para la cual se propone una función de coste, que permitirá comparar el valor que toma la función en una frecuencia dada respecto al valor deseado para el funcionamiento correcto del desacoplador. Finalmente, se ilustra este enfoque a través de un ejemplo numérico en el cual los resultados del desacople son satisfactorios y a su vez la efectividad del mismo en la detección de ataques de reproducción. [Abstract] This paper presents the design of a dynamical decoupler in a predetermined range of frequencies, for the detection of replay attacks. At first, the behavior of the decoupler is studied by changing its order. Later, a quantitative comparison is performed by using a cost function, which allows comparing the decoupling performance at a given frequency with respect to the desired value needed for a satisfactory performance of the decoupler. Finally, this technique is illustrated by means of a numerical example in which the decoupling performance and effectiveness in detecting replay attacks are deemed to be satisfactory. Ministerio de Economía y Empresa; DPI2014-58104-R Ministerio de Economía y Empresa; DPI2017-88403-R Agencia Estatal de Investigación; MDM-2016-0656 Agencia Estatal de Investigación; FJCI-2016-29019

Background: In a previous study, we reported the strong expression of Inducible T-cell costimulatory (ICOS) by Sézary cells, and presented the excellent preclinical efficacy results of anti-ICOS antibody drug conjugates (ADCs) in both Sézary syndrome (SS) and angioimmunoblastic T-cell lymphoma. Although exerting a potent direct action on the tumor cell, ADCs have the disadvantage of being associated with a cumulative toxicity, related to the conjugated drug. The development of antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/ADCP)-inducing anti-ICOS monoclonal antibodies (mAbs) is therefore of great importance to ensure long-term maintenance therapy. Methods: We first determined which anti-ICOS clone was the best candidate to induce an ADCC effect on ICOS + cell lines (MyLa, MJ and HUT78), using Mouse FcγRIII ADCC Bioassay (Promega®). The selected mAb was then chimerized and afucosylated (GlymaxX® technology, Evitria®). Secondly, we evaluated in vitro ADCC and ADCP effect of the chimeric anti-ICOS mAb against ICOS + CTCL cell lines, compared to both positive controls (mogamulizumab (moga) and alemtuzumab) and negative controls (IgG1 isotype control, and rituximab). To perform ADCC experiments, we co-incubated target cells with mAbs and allogenic NK lymphocytes from healthy volunteers (HV). Cellular apoptosis was measured by flow cytometry using the Caspase 3/7 assay (Promega®). For ADCP, monocytes were sorted from HV blood samples and treated with M-CSF. Target cells were labeled with PKH67 (Sigma-Aldrich®) and after co-incubation, CD14 +CD11b +PKH67 + monocytes were analyzed by flow cytometry. Finally, we verified the ADCC/ADCP potency of anti-ICOS mAbs on primary Sézary cells isolated from 16 moga-naïve SS patients, and 6 patients who had developed resistance to moga. We also questioned whether anti-ICOS mAbs were able to promote the autologous apoptosis of Sézary cells and T regulatory cells (Tregs) when directly incubated with peripheral blood mononuclear cells (PBMCs) from patients with SS. Results: Among 7 different anti-ICOS clones, 314.8, 92.17 and 293.1 clones had the higher ADCC activity against MyLa, MJ and HUT78. Of these 3 clones, 314.8 had the best affinity to the receptor, and the best ability to inhibit binding between ICOS receptor and a recombinant ICOS ligand protein. Anti-ICOS 314.8 mAb was then chosen to be chimerized and glyco-engineered. ICOS and CCR4 were strong and similarly expressed on MyLa and MJ. HUT78 had only mild expression of ICOS and CD52. Anti-ICOS mediated potent ADCC of cell lines (respectively 39.1±5% and 52.6±2.4% for MyLa and MJ cells), without significant difference when compared to mogamulizumab. In HUT78 cells, anti-ICOS induced a specific apoptosis of 35.7±5% versus 15.6±5.6% with alemtuzumab (p=0.02; CI95%: 4.1-36.1). Moreover, phagocytosis induced by anti-ICOS was significantly increased than that induced by negative controls. On MyLa cells, anti-ICOS had a greater phagocytosis activity than moga (59.4±5.2% vs 39.4±5.1%, p=0.031). Expression of ICOS by circulating tumor cells was found in all the 16 moga-naïve patients. The expression was strong, as 61±6% of tumor cells expressed ICOS vs 20±8% of non-tumoral CD4 + cells. CCR4 was more expressed than ICOS on both Sézary cells and non-tumoral CD4 + cells (91±6%, and 44±9% respectively). Anti-ICOS induced the apoptosis of 57.1±4.7% Sézary cells, compared to 16.9±2.2% with rituximab (p Ex vivo, anti-ICOS allowed 39.4±19.9% and 70.1±20.1% lysis of Sézary cells and Tregs respectively, with no difference with moga and alemtuzumab. However, the depletion of non-tumoral CD4 + and total PBMCs was significantly lower with anti-ICOS mAbs than with moga and alemtuzumab. Discussion: In a recent study, we showed that Treg cells of patients with SS have a high expression of ICOS. Here, we demonstrate that anti-ICOS mAbs induce Tregs depletion, which may improve immune profiles and emphasize Sézary cells killing. Our data suggest robust anti-tumor activity of anti-ICOS mAbs in SS, and xenograft experiments are underway to confirm these findings. Disclosures Lopez: Emergence Therapeutics: Current holder of individual stocks in a privately-held company. Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees. Olive: Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

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// Maroua Manai 1, 5, 6, 7 , Jeanne Thomassin-Piana 2, * , Amor Gamoudi 6, * , Pascal Finetti 1, * , Marc Lopez 1 , Radhia Eghozzi 6 , Sinda Ayadi 6 , Olfa Ben Lamine 6 , Mohamed Manai 5 , Khaled Rahal 6 , Emmanuelle Charafe-Jauffret 2, 3 , Jocelyne Jacquemier 2 , Patrice Viens 3, 4 , Daniel Birnbaum 1 , Hamouda Boussen 5, 7 , Max Chaffanet 1 , Francois Bertucci 1, 3, 4 1 Departement d’Oncologie Moleculaire, Centre de Recherche en Cancerologie de Marseille, Aix Marseille Universite, Marseille, France 2 Departement de Bio-Pathologie, Institut Paoli-Calmettes, Marseille, France 3 UFR de Medecine, Aix Marseille Universite, Marseille, France 4 Departement d’Oncologie Medicale, Institut Paoli-Calmettes, Marseille, France 5 Departement de Biologie, Unite de Biochimie et Biologie Moleculaire, Faculte des Sciences de Tunis, Universite de Tunis El Manar, Tunisie 6 Departement d’Oncologie Medicale, Institut Salah Azaiez, Tunis, Tunisie 7 Service d’Oncologie Medicale, Hopital l’Ariana, Tunis, Tunisie * These authors contributed equally to second authors Correspondence to: Francois Bertucci, email: bertuccif@ipc.unicancer.fr Keywords: expression, immunohistochemistry, inflammatory breast cancer, MARCKS, survival Received: July 29, 2016 Accepted: December 14, 2016 Published: December 21, 2016 ABSTRACT Background: Inflammatory breast cancer (IBC) is the most aggressive form of locally-advanced breast cancer. Identification of new therapeutic targets is crucial. We previously reported MARCKS mRNA overexpression in IBC in the largest transcriptomics study reported to date. Here, we compared MARCKS protein expression in IBC and non-IBC samples, and searched for correlations between protein expression and clinicopathological features. Results: Tumor samples showed heterogeneity with respect to MARCKS staining: 18% were scored as MARCKS-positive (stained cells ≥ 1%) and 82% as MARCKS-negative. MARCKS expression was more frequent in IBC (36%) than in non-IBC (11%; p = 1.4E−09), independently from molecular subtypes and other clinicopathological variables. We found a positive correlation between protein and mRNA expression in the 148/502 samples previously analyzed for MARCKS mRNA expression. MARCKS protein expression was associated with other poor-prognosis features in the whole series of samples such as clinical axillary lymph node or metastatic extension, high pathological grade, ER-negativity, PR-negativity, HER2-positivity, and triple-negative and HER2+ statutes. In IBC, MARCKS expression was the sole tested variable associated with poor MFS. Materials and Methods: We retrospectively analyzed MARCKS protein expression by immunohistochemistry in 502 tumors, including 133 IBC and 369 non-IBC, from Tunisian and French patients. All samples were pre-therapeutic clinical samples. We searched for correlations between MARCKS expression and clinicopathological features including the IBC versus non-IBC phenotype and metastasis-free survival (MFS). Conclusions: MARCKS overexpression might in part explain the poor prognosis of IBC. As an oncogene associated with poor MFS, MARCKS might represent a new potential therapeutic target in IBC.

// Anthony Goncalves 1, 2, * , Francois Bertucci 1, 2, 3, * , Arnaud Guille 2, 3 , Severine Garnier 2, 3 , Jose Adelaide 2, 3 , Nadine Carbuccia 2, 3 , Oliver Cabaud 2, 3 , Pascal Finetti 2, 3 , Serge Brunelle 4 , Gilles Piana 4 , Jeanne Tomassin-Piana 5 , Maria Paciencia 5 , Eric Lambaudie 6 , Cornel Popovici 2, 7 , Renaud Sabatier 1, 2, 3 , Carole Tarpin 1 , Magali Provansal 1 , Jean-Marc Extra 1 , Francois Eisinger 2, 7 , Hagay Sobol 2, 7 , Patrice Viens 1, 2 , Marc Lopez 2, 3 , Christophe Ginestier 2, 3 , Emmanuelle Charafe-Jauffret 2, 3, 5 , Max Chaffanet 2, 3, # , Daniel Birnbaum 2, 3, # 1 Department of Medical Oncology, Institut Paoli-Calmettes, Marseille, France 2 Aix Marseille Univ, CNRS U7258, INSERM U1068, Institut Paoli-Calmettes, CRCM, Marseille, France 3 Department of Molecular Oncology, Institut Paoli-Calmettes, Marseille, France 4 Department of Imaging, Institut Paoli-Calmettes, Marseille, France 5 Department of Biopathology, Institut Paoli-Calmettes, Marseille, France 6 Department of Surgical Oncology, Institut Paoli-Calmettes, Marseille, France 7 Department of Oncogenetics, Institut Paoli-Calmettes, Marseille, France * both authors equally contributed # both authors equally supervised Correspondence to: Anthony Goncalves, email: goncalvesa@ipc.unicancer.fr Keywords: precision medicine, advanced breast cancer, NGS, CGH, patient-derived xenograft Received: July 10, 2016 Accepted: September 29, 2016 Published: October 18, 2016 ABSTRACT Background: Routine feasibility and clinical impact of genomics-based tumor profiling in advanced breast cancer (aBC) remains to be determined. We conducted a pilot study to evaluate whether precision medicine could be prospectively implemented for aBC patients in a single center and to examine whether patient-derived tumor xenografts (PDX) could be obtained in this population. Results: Thirty-four aBC patients were included. Actionable targets were found in 28 patients (82%). A targeted therapy could be proposed to 22 patients (64%), either through a clinical trial (n=15) and/or using already registered drugs (n=21). Ten patients (29%) eventually received targeted treatment, 2 of them deriving clinical benefit. Of 22 patients subjected to mouse implantation, 10 had successful xenografting (45%), mostly in triple-negative aBC. Methods: aBC patients accessible to tumor biopsy were prospectively enrolled at the Institut Paoli-Calmettes in the BC-BIO study ( ClinicalTrials.gov , NCT01521676). Genomic profiling was established by whole-genome array comparative genomic hybridization (aCGH) and targeted next-generation sequencing (NGS) of 365 candidate cancer genes. For a subset of patients, a sample of fresh tumor was orthotopically implanted in humanized cleared fat pads of NSG mice for establishing PDX. Conclusions: Precision medicine can be implemented in a single center in the context of clinical practice and may allow genomic-driven treatment in approximately 30% of aBC patients. PDX may be obtained in a significant fraction of cases.

Nectin-4 is a tumor antigen present on the surface of breast, ovarian and lung carcinoma cells. It is rarely present in normal adult tissues and is therefore a candidate target for cancer immunotherapy. Here, we identified a Nectin-4 antigenic peptide that is naturally presented to T cells by HLA-A2 molecules. We first screened the 502 nonamer peptides of Nectin-4 (510 amino acids) for binding to and off-rate from eight different HLA class I molecules. We then combined biochemical, cellular and algorithmic assays to select 5 Nectin-4 peptides that bound to HLA-A*02:01 molecules. Cytolytic T lymphocytes were obtained from healthy donors, that specifically lyzed HLA-A2(+) cells pulsed with 2 out of the 5 peptides, indicating the presence of anti-Nectin-4 CD8(+) T lymphocytes in the human T cell repertoire. Finally, an HLA-A2-restricted cytolytic T cell clone derived from a breast cancer patient recognized peptide Nectin-4145-153 (VLVPPLPSL) and lyzed HLA-A2(+) Nectin-4(+) breast carcinoma cells. These results indicate that peptide Nectin-4145-153 is naturally processed for recognition by T cells on HLA-A2 molecules. It could be used to monitor antitumor T cell responses or to immunize breast cancer patients.

e20040 Background: Advanced stage cutaneous T-cell lymphomas (CTCLs) remain an unmet medical need. Immunomodulatory agents such as mogamulizumab, anti-KIR3DL2 and brentuximab vedotin (BV), an anti-CD30 antibody–drug conjugate (ADC) coupled to monomethyl-auristatin-E (MMAE), provided encouraging results but new targeted therapies are needed. Inducible Co-Stimulator (ICOS), a T-cell costimulatory receptor involved in the development of CTCLs, arouses interest. Methods: We used immunohistochemistry to study ICOS expression in skin biopsies of 23 patients with early-stage mycosis fungoides (MF), 12 with transformed MF (TMF) and 17 with Sézary Syndrome (SS), at diagnosis or in relapse. ICOS expression by circulating Sézary cells and regulatory T cells (Tregs) in patients with SS was evaluated using flow cytometry, and compared to healthy donors (HD) lymphocytes. In 5 patients with SS, we also analyzed concomitant biopsies from involved nodes. Then, we investigated the efficacy of anti-ICOS ADCs generated by coupling murine anti-ICOS monoclonal antibodies with MMAE, in comparison to BV. We used ICOS+ CTCL cell lines (MyLa and MJ), murine xenograft models with MyLa and ICOS+ Patient Derived Xenografts (PDXs) from patients with SS and angioimmunoblastic T-cell lymphoma (AITL). Results: ICOS was highly expressed by the cutaneous atypical lymphocytic infiltrates in respectively 61%, 75% and 88% of patients with early-stage MF, TMF and SS, such as in the 5 patients with node involvement. ICOS expression by circulating Sézary cells was strong: 69±7.3% versus 38.8±7.1% of non-tumoral CD4+ cells ( p< 0.009; CI95%: 8.7-51.6); and 31±3.2% of CD4+ cells in HD ( p< 0.0001; CI95%:20.3-46.3). Percentages of ICOS+ Tregs were significantly higher in patients with SS than in HD. In CTCL cell lines, we observed a significant dose-dependent decrease in cell viability in the presence of anti-ICOS ADCs. In a mouse xenograft model (MyLa), anti-ICOS ADCs provided a longer overall survival (OS) than BV (HR = 15.2;CI95%:3.2-71.1; p< 0.0006). Finally, in ICOS+ PDXs anti-ICOS ADCs significantly improved OS, and reduced the number of tumor cells in the blood, bone marrow and spleen. No evidence of ADC toxicity was observed in treated mice. Conclusions: All together our results show that ICOS is a therapeutic target of interest in CTCLs and provide the preliminary basis for a therapeutic trial

Trabajo presentado en el 8th International Conference on Systems and Control (ICSC), celebrado en Marrakech (Marruecos), del 23 al 25 de octubre de 2019, Unmanned aerial vehicles (UAVs) are reported to be highly exposed as possible targets of cyber attacks, due to their strong strategic and economic value, and their increasing use in a wide range of operations. Among the most critical cyber attacks, replay attacks are performed by replacing the real data coming from the sensors with previously recorded data. In this way, the attacker may provoke performance degradation, cause instability, and allow to perform undetectable physical attacks. The main contribution of this paper is to investigate the applicability of a frequency-based detection method, which uses a sine wave with time-varying frequency as authentication signal, to a UAV affected by replay attacks. The effectiveness of the method is illustrated through simulation scenarios.

The emergence of SARS-CoV and MERS-CoV, triggered the discovery of a high diversity of coronaviruses in bats. Studies from Europe have shown that coronaviruses circulate in bats in France but this reflects only a fraction of the whole diversity. In the current study the diversity of coronaviruses circulating in western Europe was extensively explored. Ten alphacoronaviruses in eleven bat species belonging to the Miniopteridae, Vespertilionidae and Rhinolophidae families and, a SARS-CoV-related Betacoronavirus in Rhinolophus ferrumequinum were identified. The diversity and prevalence of bat coronaviruses presently reported from western Europe is much higher than previously described and includes a SARS-CoV sister group. This diversity demonstrates the dynamic evolution and circulation of coronaviruses in this species. That said, the identified coronaviruses were consistently associated with a particular bat species or genus, and these relationships were maintained no matter the geographic location. The observed phylogenetic grouping of coronaviruses from the same species in Europe and Asia, emphasizes the role of host/pathogen coevolution in this group., Graphical abstract fx1, Highlights • A SARS-CoV sister clade member, Betacoronavirus EPI1, found in Western Europe. • Betacoronavirus EPI1 circulates in Rhinolophus ferrumequinum bat in Western Europe. • 9 alphacoronaviruses species found in Vespertillionidae and Miniopteridae bats. • Rhinolophus ferrumequinum hosts Betacov EPI1 and Alphacov EPI4, EPI6 and EPI7. • Alphacoronavirus EPI6 is strictly associated with Rhinolophus ferrumequinum.

Background Genomic-based approaches in advanced breast cancer (ABC) were recently demonstrated as feasible in the clinical practice, but only a limited number of patients were actually treated with targeted therapies matching genomic alterations, with low antitumor activity. We conducted a pilot study to evaluate whether precision medicine using NGS and aCGH could be implemented prospectively at a single center in ABC patients. In addition, we examined whether PDX could be derived from ABC and thus could help inform therapeutic decision. Methods ABC patients accessible to tumor biopsy were prospectively enrolled at the Institut Paoli-Calmettes in the BC-BIO study (ClinicalTrials.gov, NCT01521676). Tumor tissue from locally recurrent or metastatic disease was immediately frozen after dedicated biopsy. Genomic profiling included high-resolution 4x180K aCGH (Agilent Technologies, Massy, France) and DNA sequencing, using a library of 365 cancer candidate genes (HaloPlex target enrichment kit, Agilent technologies, Santa Clara, CA, USA) and MiSeq analyzer (Illumina, San Diego, CA, USA) with 2x150-bp, paired-end at about 300x coverage. In a subset of patients, fresh tumor was implanted orthotopically in humanized cleared fat pads of NSG mice for establishing xenotransplants. Results A total of 34 ABC patients were included, with the following characteristics: median age 54 years (35-77); molecular subtypes: 11 triple-negative (32%), 12 luminal non-HER2 (35%), 4 luminal HER2 (12%), 3 HER2 non-luminal (9%), and 4 unknown (12%); 33 with previous chemotherapy (97%); 22 with previous endocrine treatment (35%); 7 with previous anti-HER2 (21%). Tumor biopsies were obtained from liver (15), skin (6), peritoneum (4), breast (3), node (3), lung (1), pleura (1), and ascitis (1), with a median tumor cellularity of 70% (range 10-90%). aCGH and NGS were available from 34 and 33 patients, respectively. An actionable target was found in 28 patients (82%), corresponding to 66 targets, including 37 mutations (8 in PIK3CA, 7 TP53, 4 ESR1, 2 AKT1, 2 BRCA2, 2 HER2), 22 amplifications (7 for CCND1, 2 CCNE1, 2 FGFR1, 2 IGF1R) and 7 homozygous deletions (3 for PTEN, 2 CDKN2A/B,1 BRCA2, 1 STK11). A targeted therapeutic proposal was possible, either in a clinical trial (N=18, 52%) or using already registered drugs (N=17, 50%). Ten patients actually received a targeted treatment, 1 of them experienced objective response and 1 showed stable disease for more than 6 months. Of 26 patients subjected to mouse implantation, 10 had successful xenografting (6 triple-negative, 2 HER2, 1 luminal non-HER2, 1 subtype non-attributed), with a median time to reach 10 mm of 148 days. These PDX will be used as models to understand the patient's therapeutic response. Conclusion Precision medicine using high-throughput DNA sequencing and aCGH can be implemented at a single center in the context of clinical practice and may allow direct therapeutic proposal in 1/3 of patients, but antitumor activity was minimal. PDX may be obtained in a significant fraction of patients, especially in triple-negative and HER2 subtypes, and could phenotypically complement genomic data. Citation Format: Gonçalves A, Bertucci F, Chaffanet M, Guille A, Garnier S, Adelaide J, Carbuccia N, Brunelle S, Piana G, Cabaud O, Thomassin-Piana J, Paciencia-Gros M, Chereau-Ewald E, Lambaudie E, Sabatier R, Tarpin C, Provansal M, Jalaguier-Coudray A, Extra J-M, Sarran A, Pakradouni J, Viens P, Lopez M, Ginestier C, Charafe-Jauffret E, Birnbaum D. Next-generation sequencing (NGS), array comparative genomic hybridization (aCGH) and patient-derived tumor xenograft (PDX) for precision medicine in advanced breast cancer: A single-center prospective study. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-13-23.

International audience; Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Identification of new therapeutic targets is crucial. MARCKS, myristoylated alanine-rich C-kinase substrate, has been implicated in aggressiveness of several cancers and MARCKS inhibitors are in development. Using immunohistochemistry (IHC), we retrospectively assessed MARCKS expression in epithelial and stromal cells of 118 pre-chemotherapy EOC samples and 40 normal ovarian samples from patients treated at Salah Azaiez Institute. We compared MARCKS expression in normal versus cancer samples, and searched for correlations with clinicopathological features, including overall survival (OS). Seventy-five percent of normal samples showed positive epithelial MARCKS staining versus 50% of tumor samples (p = 6.02 x 10(-3)). By contrast, stromal MARCKS expression was more frequent in tumor samples (77%) than in normal samples (22%; p = 1.41 x 10(-9)). There was no correlation between epithelial and stromal IHC MARCKS statutes and prognostic clinicopathological features. Stromal MARCKS expression was correlated with shorter poor OS in uni- and multivariate analyses. Stromal MARCKS overexpression in tumors might contribute to cancer-associated fibroblasts activation and to the poor prognosis of EOC, suggesting a potential therapeutic interest of MARCKS inhibition for targeting the cooperative tumor stroma.

International audience; Background: Triple-negative breast cancers (TNBCs) are associated with a poor prognosis. In contrast to other molecular sub-types, they have no identified specific target and chemotherapy remains the only available systemic treatment. The adhesion molecule nectin-4 represents a new potential therapeutic target in different cancer models. Here, we have tested the prognos-tic value of nectin-4 expression and assessed the therapeutic efficiency of an anti-nectin 4 antibody drug conjugate (ADC) on localised and metastatic TNBC in vitro and in vivo. Materials and methods: We analysed nectin-4/PVRL4 mRNA expression in 5673 invasive breast cancers and searched for correlations with clinicopathological features including metastasis-free survival (MFS). Immunohistochemistry was carried out in 61 TNBCs and in samples of primary TNBC Patient-Derived Xenografts (PDXs). An anti-nectin-4 antibody eligible for ADC was produced and tested in vitro and in vivo in localised and metastatic TNBC PDXs. Results: High nectin-4/PVRL4 mRNA expression was associated with poor-prognosis features including the TN and basal sub-types. High PVRL4 mRNA expression showed independent negative prognostic value for MFS in multivariate analysis in TNBCs. Nectin-4 protein expression was not detected in adult healthy tissues including mammary tissue. Membranous protein expression was found in 62% of TNBCs, with strong correlation with mRNA expression. We developed an ADC (N41mab-vcMMAE) comprising a human anti-nectin-4 monoclonal antibody conjugated to monomethyl auristatin-E (MMAE). In vitro, this ADC bound to nectin-4 with high affinity and specificity and induced its internalisation as well as dose-dependent cytotoxicity on nectin-4-expressing breast cancer cell lines. In vivo, this ADC induced rapid, complete and durable responses on nectin-4-positive xenograft TNBC samples including primary tumours, metastatic lesions, and local relapses; efficiency was dependent on both the dose and the nectin-4 tumour expression level. Conclusion: Nectin-4 is both a new promising prognostic biomarker and specific therapeutic target for ADC in the very limited armamentarium against TNBC.

Many viruses utilize cell adhesion molecules of the immunoglobulin superfamily as receptors. In particular, viruses of different classes exploit nectins. The large DNA viruses, herpes simplex and pseudorabies viruses, use ubiquitous nectins 1 and 2. The negative-strand RNA virus measles virus (MeV) uses tissue-specific nectin-4, and the positive-strand RNA virus poliovirus uses nectin-like 5 (necl-5), also known as poliovirus receptor. These viruses contact the BC, C′C″, and FG loops on the upper tip of their receptor's most membrane-distal domain. This location corresponds to the newly defined canonical adhesive interface of nectins, but how viruses utilize this interface has remained unclear. Here we show that the same key residues in the BC and FG loops of nectin-4 govern binding to the MeV attachment protein hemagglutinin (H) and cell entry, nectin-4 homodimerization, and heterodimerization with nectin-1. On the other hand, residues in the C′C″ loop necessary for homo- and heterotypic interactions are dispensable for MeV-induced fusion and cell entry. Remarkably, the C′C″ loop governs dissociation of the nectin-4 and H ectodomains. We provide formal proof that H can interfere with the formation of stable nectin-1/nectin-4 heterodimers. Finally, while developing an alternative model to study MeV spread, we observed that polarized primary pig airway epithelial sheets cannot be infected. We show that a single amino acid variant in the BC loop of pig nectin-4 fully accounts for restricted MeV entry. Thus, the three loops forming the adhesive interface of nectin-4 have different roles in supporting MeV H association and dissociation and MeV-induced fusion. IMPORTANCE Different viruses utilize nectins as receptors. Nectins are immunoglobulin superfamily glycoproteins that mediate cell-cell adhesion in vertebrate tissues. They interact through an adhesive interface located at the top of their membrane-distal domain. How viruses utilize the three loops forming this interface has remained unclear. We demonstrate that while nectin-nectin interactions require residues in all three loops, the association of nectin-4 with the measles virus hemagglutinin requires only the BC and FG loops. However, we discovered that residues in the C′C″ loop modulate the dissociation of nectin-4 from the viral hemagglutinin. Analogous mechanisms may support cell entry of other viruses that utilize nectins or other cell adhesion molecules of the immunoglobulin superfamily as receptors.

In the developing area of personalized medicine, targeted therapies are mainly based on genomic characterization of each tumor, and is currently proposed as promising strategies for advanced breast cancer (ABC). Despite the promises of advanced genome sequencing, many patients still fail therapy, resulting in disease progression, recurrence, and metastases. Cancer stem cells (CSCs) concept illustrates the non-genetic intrinsic resistance, recapitulates tumor heterogeneity that creates hierarchically organized tumor tissues where a subpopulation of self-renewing cancer stem cells (CSCs) sustains the long- term clonal maintenance of the neoplasm. Evidences indicate that CSCs survive many commonly employed cancer therapeutics. Patient-derived tumor xenograft (PDXs) models recapitulate tumor complexity and heterogeneity at cellular and molecular level. We aimed to specifically address the therapeutic sensitivity in ABC, by using an ex vivo assay based on PDX prospective collection, fully characterized for genomic alterations. In this work, we aim at defining for each tumor the best therapy to target breast cancer intratumor heterogeneity, the CSC component. For that, we defined a panel of 44 FDA-approved compounds used for cancer treatment, including breast and other types of cancer, cancer stem cell drugs, chemo or targeted therapies. For each drug, we screened the differential sensitivity of the bulk tumor cells and the CSC components for 12 PDX models using an ex vivo screening approach on short term culture. To assess intra tumor heterogeneity, we set up an original dual strategy: for the bulk cells, an ex vivo assay based on IC50, and for breast CSC component a miniaturized Aldefluor assay. First, we demonstrate that bulk cells and CSCs sensitivity may be dissociated for the same drug in the same PDX models. Then, we observed that whereas bulk cell sensitivity may be correlated to tumor genomic abnormalities, CSC drug sensitivity seems not to follow the rule.CSC are selectively sensitive to specific compounds. We are exploring the pathways that sustain this selective sensitivity in the CSCs components. We are currently identifying targets using mass spectrometry in CSCs and bulk cells.Then, we validated the hits predicted from ex vivo screening assays by in vivo treatment of using PDX models for the selected drugs, and in a patient with ABC. In that work, we demonstrated that CSCs display different sensitivity profiles than bulk cells to the same agents, irrespective to their genomic background and are identifying the CSC specific targets. Here, we propose a new model of precision medicine based on ex vivo CSC assays for personalized testing of drug susceptibility in advanced breast cancer. Citation Format: Charafe-Jauffret E, Wicinski J, Cabaud O, Lopez M, Audebert S, Adelaide J, Chaffanet M, Guille A, Goncalves A, Bertucci F, Birnbaum D, Ginestier C. Ex vivo CSC assays for personalized testing of drug susceptibility in advanced breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-06-02.

International audience; Measles virus is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths. Although it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to infect initially macrophages and dendritic cells of the airways using signalling lymphocytic activation molecule family member 1 (SLAMF1; also called CD150) as a receptor. These cells then cross the respiratory epithelium and transport the infection to lymphatic organs where measles virus replicates vigorously. How and where the virus crosses back into the airways has remained unknown. On the basis of functional analyses of surface proteins preferentially expressed on virus-permissive human epithelial cell lines, here we identify nectin-4 (ref. 8; also called poliovirus-receptor-like-4 (PVRL4)) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains measles virus entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is downregulated in infected epithelial cells, including those of macaque tracheae. Although other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that measles virus targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer, which has implications for ongoing measles-virus-based clinical trials of oncolysis.

The mechanisms of seasonal acclimatization in eurythermal fish such as common carp are not fully understood. Here, we concentrate on the regulation of pituitary factors, as this organ was shown to be highly affected by seasonal changes. We cloned and sequenced two different cDNAs for each of the transcription factors Pitx2 and Rpx, known to play a role in pituitary development. We show that these genes are conserved throughout evolution, to different degrees depending on the specific domain considered. Finally, we show that the cDNAs for both factors are clearly up-regulated during the winter season, in sharp contrast to other regulators such as Pit1 or pituitary hormone genes such as prolactin (prl) and growth hormone (gh). Our results suggest that increased expression of Pitx2 and Rpx contributes to seasonal adaptation of common carp to winter conditions.

Nectin and nectin-like (necl) proteins form a family of 9 adhesion molecules that belong to the immunoglobulin superfamily. They play a key role in different biological processes such as cell polarity, proliferation, differentiation and migration in epithelial, endothelial, immune and nervous systems. Besides their role in physiology, they have been involved in different pathological processes in humans. They serve as virus receptors (poliovirus and herpes simplex virus), they are involved in orofacial malformation (CLPED1) and recently they have been described as markers, actors and potential therapeutics targets in cancer. Among them, necl-5, nectin-2 and nectin-4 are overexpressed in tumors, and are associated with a poor prognosis. On the opposite, necl-1, necl-2 and necl-4 act as tumor suppressors and are repressed in cancer. The involvement of nectins and necls molecules in cancer and their potential used in therapy is discussed in this review.