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FRENCH MUSICAL BROADCASTING FROM 1965 TO 1974: A MULTIPLICATION OF THE PRODUCTIONS BUT A REDUCED NUMBER OF THE STATIONS by Christophe BENNET

In 1965, Henry Barraud retired. One can suggest, like historian Karine Lebail, that "this was the end of an age for broadcasting music. 1 " Although no longer in charge of music, he was still exerting an administrative supervision on its broadcasting, but the director of the Chaîne nationale's manager fiercely opposed the project of a specialization of the various possible stations of the ORTF. In fact, he was afraid of a ghettoisation of classical music, already criticized as being elitist : culture is indivisible. He however wouldn't be able to slow down this inexorable movement of specialization of the stations. Henceforth, non specialized stations object to broadcasting classical music, since it now has its own station and listeners. The years 1960 were the time of the blossoming of the microgroove and the authorities draw the conclusions, painful as they are for the bands linked with the Radio. The steady disappearance of the links between the diverse provincial orchestras and the Radio leave only three remaining regional orchestras in 1970: in Lyon, Nice and Toulouse. The splitting up of the ORTF, in 1974, will be the occasion of a radical reorganisation: the last provincial orchestras will be part of the regionalization plan implemented by the new "Directeur de la Musique", Marcel Landowski, as decided by a"Ministry of Cultural Affairs" led by André Malraux2 .

-FRANCE CULTURE KEEPS A STRONG MUSICAL BROADCASTING POLICY

A sampling of about thirty days over the 1965-74 decade gives an idea of what the musical broadcasting of France Culture was. The station which actually continues Henry Barraud's station keeps a wide space dedicated to classical music, even though it tends to decrease. Fed by 75 programs (that is to say one third, anyway, of the programs of France Musique on the same days when we picked up the samplings 3 !), this panel is dominated by the 15 slots during which the regional orchestras are performing. Among them, the most frequently broadcasted one is the Strasbourg orchestra, often conducted by Charles Bruck or Georges Tzipine 4 . Like all the others, the Nice orchestra is often scheduled at mid-day. On Saturday November 12 th 1966, André Jouve is conducting it, and he notably proposes Fingal's Cave overture, by Mendelssohn 5 . As early as 1970, these bands are even sometimes broadcasted on late hours, like the concert of the Nice orchestra, conducted by Michel Plasson, on Sunday November 21 st 1971, from midnight to 1 am.

Considering the number of non classifiable programs like the "didactic cycles" (9), we can hail the longevity of the program hosted by Henry Barraud, on Sundays from 11 am to noon from 1967 on. His analyses and explanations of works like Saint-Sébastian's martyrdom (Debussy), Orfeo (Monteverdi), Missa Solemnis (Beethoven) enables France Culture to broadcast the recordings of prestigious bands 6 . We could also mention a program entitled "A Musical Encyclopedia through records". The episode of Monday November 20 th 1967 is honored with the presence of Jacques Chailley, Myriam Soumagnac, Rémy Stricker 7 , Claude Roland-Manuel, Jacques Loey and Jean-Paul Aron.

The station also relays music festivals (7 in our sampling) such as those of Bregenz (1962), Saint-Paul-de-Vence (1967), "Festival estival de Paris" (1972), Besançon and Bayreuth (1973). On Saturday September 29 th 1973, in a 5-hour program, lyrical art amateurs can hear Wagner's Mastersingers from Nuremberg, by the Norbert Balatsch Choir and the Orchestra of the Festival, conducted by Silvio Varviso.

One can also notice some "recorded and isolated pieces" (7) which are curiously very precisely described in the programs 8 . We could mention for example the four short minutes in which the Minuetto of Haydn's Divertimento in D major is performed under the masterly baton of Jean-François Paillard 9 . On the other hand, we can find programs that almost last a full hour, like Charles Ives's Symphony #4, performed by the American Symphony Orchestra, conducted by Léopold Stokowski with the Schola Cantorum of New York 10 .

The sequences of chamber music (6) are as numerous as those that could be called "original productions around the classical music" (owing to their theme and the tone of their announcement). In the program "Prestige de la Musique" realized by Jean Fontaine in the evening of Monday November 13 th 1972, the latter proposes nothing less than a concert at the salle Pleyel, by the Orchestre national of the ORTF, conducted by Lorin Maazel with Christa Ludwig. The program: Schubert's Symphony #8, Mahler's "Lieder eines junges Wanderer" and Chaikovsky's Symphony #5 11 . 4 As early as 1965, this band became known as "Orchestre radio-symphonique de Strasbourg", and then "Orchestre symphonique d'Alsace" from 1974. 5 At that time, he was "more or less the official conductor of the orchestra" (interview of André Jouve on Ferbruary 15 th 2007). André Jouve was the "Directeur des services et programmes musicaux de Radio France". 6 With, for the mentioned examples, nothing less than the "Choir, Maitrise and the National Orchestra of the ORTF, cond. Inghelbrecht"; the "Ensemble Vocal et Instrumental de Lauzanne",cond. Michel Corboz," and the "Berlin Philharmonia,cond. Herbert von Karajan." 7 Those are musicologists who also produce programs on France Music. 8 We could here tell the story of a certain Madame Launay, from France Inter, who was an expert in the art of dividing these into short pieces of "stop-gap" music between two actual programs in relation with the timing required. The presence of these stop-gaps pieces on the program scripts can be explained by the fact that these documents were used as guideline of the programs. These were anyway prepared by the department in charge of the regulation of the programs (Pierre Guitton, first, then Jacques Guinchard). 9 On Saturday November 12 th 1966, from 7.36 to 7.40 am ! 10 On Monday November 20 th 1967, from 6 to 6.40 pm. 11 For the genesis of this program, one could read the statement by Agathe Mella in the previous article.

We should mention that this program is simultaneously broadcasted in stereo on France Musique. It's also the case for the relays of the great concerts (6), like the one released in 1966 at the "Théatre des Champs-Elysées" by the Montreal Symphony Orchestra12 . We should also add some "focuses on the composers", like the program "Musiciens français contemporains13 " (4), which anyway existed before 1965. It's also the case of the "cult programs" (3). The very popular productions "Les grands musiciens" by Jean Witold and "Plaisir de la musique" by Alexis Roland-Manuel however hardly lasted two years after Henry Barraud's dismissal from the "Chaîne nationale14 ". The latter is anyway not entirely disconnected from the life of the station, since his Sunday program will soon replace the one of Alexis Roland-Manuel. By assuring a transition, he is the one who hosts one of the last editions of "Plaisir de la musique", on Sunday November 13 th 1966: a "tribute to Alexis Roland-Manuel, by Henry Barraud, with the Orchestre Philharmonique de l'ORTF, cond. Daniel Chabrun."

With an average of 2.5 programs of about one hour everyday15 , France Culture still has a genuine policy of musical program. Along the years, one can see that the part dedicated to music is shrinking. But music still has its own station.

-FRANCE MUSIQUE: THE STATION OF MUSIC LOVERS AND MUSICIANS A COHERENT PROGRAM OF QUALITY TARGETING CLASSICAL AND MODERN MUSIC

After Marius Constant's departure, Charles Chaynes remains alone at the helm of the musical station "almost abandoned, as we could say 16 ". He expects to assure a balance between symphonic music, the great soloists, vocal music, chamber music, religious music, entertaining music, romantic and modern music. Jean-Louis Alibert has well shown that symphonic music constantly gets the biggest share. It absorbs 17.7% of broadcasting time. Then comes instrumental music (especially concertos) with 17.5%, chamber music absorbing 15.7 and vocal music 9.1% of broadcasting time 17 . These genres are those most in demand with the audience, but other genres are also broadcasted, like lyrical music (5.1%), light music (6%) or modern music. Concerning the latter, even if this musical genre does not usually get a wide success, it must be present in the program schedule. France Musique, as a station of public service, makes efforts and has an obligation of promoting musical creation in France. Furthermore, Charles Chanes is himself a composer, and particularly sensitive to these new forms of "sophisticated music".

Concerning the broadcasting of these genres, we should notice, on the one hand, Charles Chayne's devotion to multiplying "live music" to the detriment of records 18 . On the other hand, the broadcasting of this station on the MF band guarantees a much better quality of listening than on the long waves. The number of programs broadcasted in stereophony on France Musique keeps growing: 17 hours a week in 1966-1967; 21 hours in 1967-1968; 31 hours in 1968-1969; 35 hours in 1969-1970; 40 hours in 1970-1971 19 . In connection with the Public Relations Service of the ORTF, some days are coordinated by Jacques Vaudoux, a producer at France Musique 20 . "Every month, a city of France [is] chosen, either for its importance, or because the local station [has] just been equipped in stereophony 21 ." The purpose actually consists in inviting the retailers of radio sets to a studio of the local radio or in a local venue in order to show them the quality of the sound in stereophony / MF, by making them listen to a program of classical music. The sellers are targeted by these campaigns, yet the operations aimed at the audience are the ones that most mobilize the ORTF's attention. By studying the activity reports and the presentation of the program schedules of every new season during Charles Chaynes's period, one can realize how important for France Musique the contact with the audience is. The station mobilizes all its energy in order to offer its audience programs of an ever increased quality and to increase its audience.

A STATION OPEN TO ITS LISTENERS

Charles Chaynes recalls that the levels of the audience figures were constantly being monitored:

"First there was the 'cumulated audience figure over one week' ('I'm listening at least one day a week'): we reached 2.7 million listeners, that is to say 7.5% of the listeners; it was not so bad compared to today (with the competition of Radio Classique). Anyway, you can see that by 1973, we had reached strong figures and very deeply monitored: so many men, women, the little company owners, and so on. 22 " 17 Jean-Louis Alibert, "Dix neuf heures de musique par jour : les fonctions de France Musique", third cycle thesis in information and communication, Grenoble III, November 1982, p. 87. 18 Cf. in the frame our interview of May 3 rd 2007. 19 According to the "Archives de la Direction de la Musique". 20 In 1965 and 1966, together with Charles Chanes he presents on Saturday mornings a program which will last the whole decade: "Pour l'amateur de stéréophonie". The argument of the program is technical, but its content is artistic, like the genesis of the station. 21 Jacques Chardonnier, "La promotion de France Musique et de la stéréophonie", in "Les Cahiers d'Histoire de la Radiodiffusion", #55, janvier-mars 1998, p. 63. 22 According to the 1973 report provided by Charles Chaynes.

PRODUCERS AND FAMOUS PRODUCTIONS AT FRANCE CULTURE by Charles CHAYNES

Christophe Bennet: -"How did you begin the job as manager of France Musique, after Marius Constant had left?"

Charles Chaynes: -"At the beginning, I continued in the same way, yet I added some things. For example, we had several public programs with highly qualified producers, pioneers like Myriam Soumagnac (a well-known musicologist); Claude Lehmann; Rémi Stricker (who had been professor of musicology at the Conservatoire for a long time); François Serette (a composer who was very interested in the radio).

I had attributed themes to each of them: François Serette produced a program entitled "Les jeunes filles sont musiciens" (young ladies are musicians). Every week, he focused his interest on a junior high school or a high school. He chose guinea-pigs, let them hear some music, and it was the occasion to broadcast music with young people who hardly knew it.

Just before Marius's leaving (in 1964), there was a great program called: "France Musique reçoit". In 1963, the main studios were open to public programs, with invited artists. This was really the star program. Great celebrities used to come, because we were linked to the record labels, which sent their stars as guests. For example, we made the first program with Elisabeth Schwarzkopf. We also had many famous people, through the launching of their records. The records' producers were glad that the station should be placed under the auspices of their favorites, and it enabled us to provide great moments. This took place at the "Maison de la Radio": we opened three studios at the same time: studios 105, 106 and sometimes104. The idea of this program was a free broadcast: the spectators could go from one program to another. From 8 to 11 pm, these actions took place simultaneously in the three studios where the producer of the program chose to make people hear such or such a thing.

Gilles Cantagrel, a famous musicologist and fecund writer, was a producer on France Musique. After my leaving, he was the manager of the program for a while. There was also a very famous producer, Philippe Caloni, whom I had appointed. A friend had told me: "there is a good guy, he knows the microphone as well as music"; I then gave him a program: he was remarkable at the microphone. He was still there in 1975.

I have mentioned four producers who made public programs, but there were many others. I had a team of almost 40 producers! Apart from their programs, which were most often weekly ones, there was a star daily program, in the morning from 10 am to noon. It lasted for ages (it's still going on 40 years later under other names). At the beginning we called it : "Que savons-nous de…?" (What do we know about…?). Actually, for the whole week, there was a top producer with a musical background (a composer, an artist), who took up an issue and developed it throughout the week: "What do we know about such performer, such school, such music?" This program lasted until it became, a few years ago (I don't know if it still exists): "Le matin des musiciens" (The morning of the musicians). Yet, it was my idea in 1965! CB: -"So, in 1965 you remained alone at the head of France Musique and for some years?" CC: -"Yes, for 10 years, from 1965 to 1975. During those ten years, I was responsible for the whole program. Then I struggled to bring more and more "live music", because we only used records at the beginning. We multiplied the concerts of chamber music, the thematic concerts. For example, among the producers that I mentioned, there was Rémi Stricker (who has recently retired from the Conservatoire). He used to produce a program called "Lever de Rideau" (Curtain up !). He used to broadcast young, not very well-known artists. This was successful and interesting. "What do you think about this or that one?" he would ask me. We chose together, but I gave him a free rein because I knew his capabilities. It led to live music in the studio and in public, in studios 105 or 104 (depending on the importance of the band). As early as 1964, there were more public programs; my purpose was to bring in constant live music (with or without spectators) in order to break the idea of records." CB: -"As far as the concerts are concerned, I suppose that you didn't stay in France?" CC: -"Indeed not! We had many relays from abroad. We were in touch with the service of international relays. Every week, I was sent a choice of foreign programs, and we sometimes broadcasted in the evening, as concerts, programs coming from Germany, Austria, a little from everywhere. They were proposed by the EUR (European Union of Radio), which still exists. Gilles Cantagrel has anyway dealt with that for years." CB: -"In what circumstances did you leave France Musique?" CC: -"The pivotal years 1974/1975 were a tormented period. On January 1 st 1975, there was what was called the "explosion of the ORTF". This reshaping of the French audiovisual organization was a drama for many people: in the last months of 1974, no one knew what was going to happen; which company we were going to join. I even didn't know where I was about to land! Pierre Vozlinsky, who became the manager of the music department in 1973, had made me a proposal: "Listen, it would be a good idea if you dealt with the international relations of France Musique; you would invite foreign concerts, via the records companies, via the foreign radios. Are you interested?" -"Yes, why not?" I answered. This was the situation until the day when Tolia Nikiprovetzki, who was in charge of the "Illustations musicales" department (he succeeded Henri Dutilleux after his leaving in 1962) came and talked to Pierre Vozlinsky. He told him about his plan to retire. Immediately, Pierre called me and say: "Niki is about to leave: I'm ready to make a big effort on musical creation; if you want this department, I double the budget and you'll be able to lead a policy of creation. This time, I didn't say no. With further financial means, I was going to develop a great sector. This is how, after leading France Musique for 10 years, I was appointed head of Radio France's department of musical orders, and remained in charge for 15 years."

(Abstract of an interview realized on May 3 rd 2007)

In a survey carried out over 11 000 people during the second trimester 1971, a question was asked to the listeners of France Musique about their musical tastes. In this rating, a person was considered as a listener of France Musique if "they listened to France Musique at least once a week." Defined as such, the population almost reached 2 million people. One question was: "here is a list of the different musical genres, what are the two genres you are most interested in?" Here is the outcome of the survey 23 : The hierarchy is not the same, according to the frequency of the listening. The attraction of certain genres is growing with the recurrence of the listening: symphonic music, chamber music, opera, and soloists' recitals.

One can clearly see that the result of the survey is entirely consistent with the level of the broadcasting of the genres pointed out by Jean-Louis Alibert. Yet, as Gilbert Amy stated when he was appointed musical adviser of the radio in 1973: "Music is not only an object of consumption and digestion." In a long interview given to the daily newspaper "Le Monde", the man who will soon create the "Nouvel Orchestre Philharmonique" explains that it is urgent to reform the music played on the radio, to struggle against a kind of disaffection of the audience, and to properly use an incomparable potential. To this purpose, "The commented programs must obey to the same wish of a sophisticated culture, targeting a wide audience. 24 " These commented programs are in fact constituting the major activity of the station. 23 Table presented "in order of importance" and realized upon the survey :"Tastes and preferences of the listeners of France Musique" JPA / CD / 17.11;71, Fonds Jacques Durand, consultable at INA. 24 "Le Monde" of June 10th 1973 : "La musique n'est pas seulement un objet de consommation", an interview of Gilbert Amy, by Jacques Lonchampt.

CONSTANCY AND STABILITY OF THE PROGRAM SCHEDULES

Though the broadcasting essentially consists of continuous recorded music, framed by the "announcement -disannouncement" of the works, the station offers different kinds of programs realized by the producers, such as the magazines dealing with artistic news, including the famous "Quotidien de la Musique" by Philippe Caloni or the information about the new records, with the "Tribune des critiques de disques" by Armand Panigel. This program had been created at the Club d'Essai in 1947 and knew an exceptional longevity and success 25 on France Musique.

As we saw, there were also programs with invited audiences ("France Musique reçoit", "Invitation au concert") and musicology programs. The latter were devoted to the explanation of the works or the history of music, and concerned diverse periods and styles (like "Que savons-nous de… ?"). As a whole, the 17 hours of daily programs during the working week are spread over the morning, from 7 am to 9 or 10 am, with uncommented musical programs. From 10 to 11.30 am or 12.30 pm, there is a magazine slot, or more elaborated and musical topics. In a second part of the day, from lunchtime on to 2.30 pm, one can again hear musical and information programs. From early afternoon until 6 pm the programs are more pedagogical, focusing on a topic. From 6 to 8 pm, the uncommented programs are coming back until the evening concert, from 8.30 to 11 pm. The end of the evening is usually dedicated to connoisseurs, with more elaborate programs, targetting more original styles.

In the making of the program schedules, Saturdays and Sundays are always different from working days, in which there are regular rendezvous from one day to the other26 . On the weekend, the program slots are longer, for the listeners are liable to devoting a more important time to listening to France Musique. Besides, in summer, the program schedules are also different from the rest of the year, especially to leave a broad space to the relays from festivals.

THE TYPOLOGY OF THE PROGRAMS SHOWS THEIR DIVERSITY

Among the 230 programs or so that constitute this survey corpus, one can make out 26 different categories, which well illustrate the diversity of the productions. The following table doesn't claim to be exhaustive, owing to the narrow sampling. It however gives a rather realistic idea of the proportion and longevity of the types of programs. For each of the sequences, one can read a significant title and observe a frequency. 25 Cf the framed interviews of Charles Chaynes and Paulette Vernerey. Compared to the previous period, the rhythm of the listeners' occupation is carefully respected (with the "dinner-concert" and "supper-concert"), with the desire to match their musical tastes. Let's notice the significant proportion of record programs chosen by the listeners, a concept born during the thirties 28 : "Disques classiques demandés par les auditeurs, réunis par Denise Chanal" (in 1965); "Carte blanche aux auditeurs, prés. par Pierre Massé" (in 1966); "Nos disques sont les vôtres, microsillons demandés par les auditeurs et réunis par Berthe Bailly (ou par Denise Chanal 29 )" (from 1967 to 1974).

Typology of the France Musique programs of the 1965-1974 period

On the issue of the bonds with the listeners, we also can notice some quizzes or games punctuating the program schedules, whose origins are not less old than those of records on request. From 1969 to 1972, the program "Comment, pourquoi, de qui?" by Claude Roland-Manuel is recurrent. Broadcasted on Saturdays, this program is split into two short sequences of 5 minutes each 30 , and systematically mentioned by the newspapers (a proof that the listeners were interested in them), followed by a wide slot, from 11 pm to midnight: "L'heure des connaisseurs, comment, pourquoi, de qui?: Claude Roland-Manuel répond"'.

Charles Chaynes perfectly recalls the genesis and the form of this program:

"There was also a program a little more sophisticated that I used to produce with Claude Roland-Manuel, a program of musical riddles. Marius [Constant] had told me: 'If you agree to team with Roland-Manuel, he's quite good at the microphone, but he needs someone to find out the ideas of riddles', like going and searching Schubert in Mendelssohn's style, Monterverdi in Vittoria's, difficult things, the relations between the composers. We would propose 5 or 6 riddles per program broadcasted by the station and the listeners would send their answer. The following week, Claude released the report of the previous week. And so it went on from one week to the other! Sometimes, we quizzed them on the performers; this was quite an arduous program, which was eventually addressed the great music lovers, and there was a loyal audience. I used to say: 'they are impressive, they are unbeatable!"I sought the most twisted questions, the most complicated; but they always answered them! 31 " By watching the table above, one can understand that the classification is not easy. In fact, the "original productions 32 ", which are actually commented programs, sometimes overlap several categories of the typology proposed. We could for example mention the programs dedicated to "modern music", by means of a "focus on a composer" (precisely a modern one), or through a "symphonic concert". A program that could be classified in the category of "baroque music", but coming from a "festival" recording can also be considered as a "recital" or a "focus on a performer". As for the program with the delightful and promising title "Beethoven en stéréophonie", the promotion of "stereophony" could similarly be associated with : "focus on a composer"… Charles Chaynes' term is therefore homogeneous as a whole, even if modifications were brought in and little arrangement occurred from one year to the next. For example, "the return from the 1968 summer holidays will be dedicated to a renovation of France Musique's program.", and "the programs of France Musique were shaken in 1973 by the reform starting on January I st , whose key lines were reaffirmed at the start of the school year in October 33 ". In 1973, an attempt at harmonization was in fact made, since the producers were entrusted with programs at the same hours daily throughout the week. These little arrangements, however, were little in comparison with the huge movement of reform which hit the ORTF and France Musique in 1974-1975 and ended Charles Chaynes' managerial career 34 . In 1975 there was indeed a clear and distinct decision to really reform the station. It differed from the previous tendency that had been torn between the desire of conquering new audiences and the fear of disappointing the former ones, the basis of its listeners.

As early as July 1974, Pierre Vozlinsky, the deputy chairman of the ORTF for music, in charge of the orchestras and the choirs publicly stated the terms in which the problems of music were settled at the "Office":

"The difficulties that we face in the organization of the musical programs, which are held by a genuine coherence and allow long term projections, come from the scattering of the forces. The example of France Musique is particularly meaningful. This station is dedicated to 31 Abstract from the interview of May 3 rd 2007. 32 We use inverted commas for the quotation of some of the items of the typology of programs. 33 Archives of the Direction de la Musique, 1960-1976, V. 92 W.C. "Origine du programme France Musique". 34 One can read the frame section on this topic.

music, but it is practically separated from the performing areas, that is to say the orchestras and even the services of production of the radio's music. […] The Government must therefore have an interventionist position; a national radio and television must have a combined cultural attitude. There is no other answer to the visual and auditive terrorism of industrialized hammering. 35 " One can immediately see that the reshaping of the station will be significant and in proportion to the upheaval in the audiovisual landscape. At the beginning, it will curiously be embodied by Louis Dandrel, the journalist who interviews Vozlinsky for the newspaper "Le Monde".

By comparison with the new program schedules sparked by the latter (whose team will remain only two years in charge of France Musique 36 ), the long years which preceded may appear conventional. Charles Chaynes however says that he has "always fought" to bring substance to his station, so that France Musique should not be a 'music tap', but a lively station, close to its listeners. The myriad of new productions and the level of the audience figures, never equaled since then, respectively prove this energy display and the good reception of the musical programs by the audience.

The frequencies of France Musique in 1970

Many thanks to Gérard Hocmard for his help with the language issues.

35 "Radio-Télévision : un entretien avec Pierre Vozlinsky", "Le Monde" of June 30 th -July 1st 1974, p. 1;11. 36 Concerning the Dandrel period, one could read : Christophe Bennet, "France-Musique en 1975 : une réforme qui décoiffe !", in "Les Cahiers d'Histoire de la Radiodiffusion", #86, octobre-décembre 2005, p. 120-149 ; and id,. "France-Musique : la fin de l'épisode Dandrel", Ibid., # 94, octobre-décembre 2007, p. 147-166. In the backstage of the program La Tribune des critiques de disques by Paulette VERNEREY Christophe Bennet: -"Before your occupation as 'chargée de réalisation' in the Orchestre National, didn't you do anything else?" Paulette Vernerey: -"Of course, I had been working for six years for the program "La tribune des critiques de disques" by Armand Panigel, a two-hour program which was broadcasted every Sunday afternoon. The recordings took place every Monday from 5 to 8 pm. Then a 4 of 5-hour-long editing session was following in the week, because the duration often exceeded the expected length… The choice of the program was inspired by a recent publication that had drawn the interest of Armand Panigel. I had to select the chosen records from our rich record library. In a listening room, just before the recording, the producer chose five or six versions that were to be compared with the new publication.

When he went for an opera, A. Panigel listed for me the sequences and the tunes he was interested in. In the listening room, I marked the spots thanks to a previously prepared graduated sheet. This had to be very accurate. At the time when I was assigned this mission (coming after Gérard Auzanneau, who afterward became the manager of the principal record library of the ORTF), Armand Panigel, Jacques Bourgeois, José Bruyr, Antoine Goléa, Michel Hofmann (and sometimes a guest) used to gather around the microphone.

I recall rather sharp arguments between Jacques Bourgeois and Antoine Goléa about the performance of an opera role by some performers. Public programs were broadcasted liveor recordedespecially during the Aix en Provence Festival scheduled during the summer of 1966: Mozart's Idoménéo and Ariane à Naxos by Richard Strauss. In 1969: Berlioz's La Damnation de Faust.

I left Armand Panigel's Tribune in order to collaborate to a great series commanded by Charles Chaynes, who then was the manager of France Musique, and entitled Que savons-nous de… ?: every week, from Monday to Friday (10 to 10.30 am), a different release was screened by a musicologist or a musician. For example, "Que savons-nous de…" : Heinrich Schütz, by Paul Mefano; of Spanish music, by Martine Cadieu and Maurice Ohana; of the Pléiade poets by Myriam Soumagnac; of Monteverdi, by Maurice Le Roux; of Charles Ives, by Harry Halbreich, etc.

For 180 programs or so, I stuck the famous red label "READY TO BE BROADCASTED"! on the recording lists.

Abstracts from an interview realized on November 24 th 2006.

  Charles CHAYNES and Paul RICOUD, on November 15 th 1960 28 Christophe Bennet, "Musique et radio pendant les années trente. La création d'un genre radiophonique", PhD thesis in History of Music and Musicology, Paris-Sorbonne, 2007, p. 269-298: "L'amateur au micro". 29 This title already existed in 1964, when Denise Chanal was accompanied by Philippe Moreuil. 30 At noon for the first and in the middle of the afternoon for the second.

The French version of this article can be read in : Christophe Bennet, 1925-1975 : Cinquante ans de musique classique à la radio (direction), Les Cahiers d'Histoire de la Radiodiffusion, Cahier thématique n°95, janviermars 2008, 250 p.: http://cohira.fr/cahier_n_95_de_1925_a_1975_janvier_a_mars/

3 For each year of the decade, one following Saturday, Sunday and Monday were monitored under the angle of the classical music programs (at least when the contents were significant).

On Sunday November

th 1966, from 5.48 to 7.30 pm. Zubin Mehta especially conducts Richard Strauss's Don Giovanni.13 On Sunday November 17 th 1968, from 8.45 to 9.45 pm, the program is dedicated to André Jolivet. The ORTF band "Ars Nova", conducted by Daniel Chabrun, notably performs his "Suite en concert pour flute et quatre percussions."

Concerning the program "Les grands musiciens", Jean Witold actually disappeared in 1966. His wife Joëlle took up the program for a while in 1967.

Over the thirty days monitored, the 75 programs represent a little more than 78 hours of programs. For France Musique, the sampling for the same days shows, a figure of 233 programs, for a cumulated duration of 252 hours.

Interview of Charles Chanes on May 3 rd 2007 : "In 1962, Marius tells me he's going to found a band of modern music, Ars Nova: 'I can no longer deal with France Musique's programs, and I agree with the general direction that France Music has two heads.' Eventually, he completely abandoned his post in 1964. I then became the manager of France Music, since he had left."

This is why the samplings that we have collected for this article are focusing on the Saturdays, Sundays and Mondays.


Manuel Garcin

M Vendé-Leclerc

B Robineau

Pierre Maurizot

Le Gonéri

Alexandre Cozannet

Nicolae-Lerma

Islets of New-Caledonia lagoons in the perspective of Climate change and sea level rise

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Method Around twenty islets from the southwestern and eastern lagoons of Grande-Terre (New Caledonia main island) and one from Nokanhui atoll (Ile des Pins) have been studied. Such studies integrate field work and observations (erosion scars, accretion area, sedimentology, etc.) and analysis of historical photographs and satellites images. Geomorphological and sedimentological data have been collected during 2013 and 2014 field surveys. Past extensions of each islet have been interpreted using available aerial views and satellite sensing. Old aerial photographs are rather rare because islets being sometimes quite away from the shore, they were not covered by aerial survey of the main island. All available data have been integrated in a GIS. Thus, islets's reconstitution extends from one decade to 70 years. Time evolutions of shape and surface of each islet have been mapped in order to compute surface changes and the present percentage of coast's lengths in erosion, in accretion or stable. Moreover, the forcing factors such as winds, wave, tropical storms or cyclone, and the ENSO have been analyzed, in order to understand potential links with the islet behavior.

Results

In terms of size and shape, a high diversity and a variety of evolution trends of islets during the past decades can be noticed. Actually, processes affecting the coast of each islet (erosion, accretion, stability) are highly variable. All islets have at least 50% of their coasts affected by erosion and for four this rate is close to 100%. Islets showing increasing surface during the past years are very rare. Analysis and observations show that five main stages constitute the life-cycle of Caledonian islets, namely: nucleation, growing, maturity, decay, relic or endangered. Changes of environmental parameters and forcing factors as well as the inherited geomorphology lead the islets from one stage to another. The becoming of each islet is linked to its past evolution, its present state and future evolution of environmental parameters. Parameters are linked to the climate variability like ENSO or IPO which control the intensity and direction of trade winds and the average sea level in the SW Pacific. Forcing factors include also extreme events like cyclones, storms and austral swells which can trigger at very short term powerful erosion or accretion with high impact on the islet. Sea level rise induced by the anthropogenic climate change has also to be taken into account. Using our data and informations and postulating that the current situation remains identical, we consider that : 19% of the islets are in a critical state with a very likely disappearance in the next future (few years); 10% of the islets are in a critical situation with a likely disappearance in the next future and very likely disappearance in the middle term (next decades); 19% of the islets show a rapid evolution which can lead to their disappearance in the middle term but not in the next future; 10% of the islets are not endangered at short and middle time scale and 43% of the islets are not endangered at all (stable or accreting, large surface, relatively high altitude). Our results show that situations are contrasted from one islet to another. We have also to emphasize that uncertainties are higher for middle and long term future due to uncertainties about the sea level. Uncertainties are also linked to the potential reaching of a thresholds value (value and rates) which would lead to a modification of the resilience capacity of each islet. Thus, a coastal observatory will be of first importance in monitoring the impact of climate change and sea level rise on such systems.

Islets of New-Caledonia lagoons in the perspective of Climate change and sea level rise

  . Garcin 1 , M. Vendé-Leclerc 2 , B. Robineau 2 , P. Maurizot 2 , G. Le Cozannet 1 , A. Nicolae-Lerma 1 On July 7th 2008, UNESCO included parts of Lagoons of New Caledonia in the list of the Reef Diversity and Associated Ecosystems. Sandy islets from New Caledonian lagoons lie on lagoonal reef platforms or on the reef barrier.They have a major role in these specific and rich ecosystems, being the seat of nesting for turtles, seabirds, sea kraits etc. In addition, islets have a high importance in the Caledonian culture and way of life; economy of the islands has been developed around tourism and services with specific activities such as sports and relaxation. One of the recurrent issues for people and governmental agencies concerns the islets's future in the perspective of Climate Change and Sea Level Rise. In this context, the Coastal Observatory of New Caledonia (OBLIC) has initiated research about the recent and present evolution of islets in order to predict their "behavior".

	1 -BRGM/ Coastal Risks and Climate Change Unit; FRANCE; 2 -DIMENC/ Geological Survey of New-Caledonia, NEW-
	CALEDONIA
	Context and problematic:

M


Angélique Gougelet
email: angelique.gougelet@inserm.fr

Chiara Sartor

Laura Bachelot

Cécile Godard

Carmen Marchiol

Gilles Renault

Frédéric Tores

Patrick Nitschke

Catherine Cavard

Benoit Terris

Christine Perret

Sabine Colnot

Antitumor activity of an inhibitor of miR-34a in liver cancer with β-cateninmutations miR-34a oncogenicity in β-catenin-mutated liver cancer

Keywords: hepatocellular carcinoma, β-catenin, miR-34a, HNF-4α, miRNA-based therapies

come

Antitumor activity of an inhibitor of miR-34a in liver cancer with β-catenin-mutations miR-34a oncogenicity in β-catenin-mutated liver cancer

INTRODUCTION

Liver cancer is the third leading cause of cancer-related death worldwide.

Hepatocellular carcinoma (HCC) is the most frequent form of primary liver cancer, which mostly affects men with cirrhosis. HCC frequently appears in a context of chronic liver disease caused by infection with the hepatitis B and C viruses (HBV and HCV, respectively), alcohol consumption, obesity or genotoxic exposure ( [START_REF] Knudsen | The changing landscape of hepatocellular carcinoma: etiology, genetics, and therapy[END_REF] for a review). We, and others, have shown that mutations of the CTNNB1 gene, encoding β-catenin, are observed in 15 to 40% of HCCs [START_REF] De | Somatic mutations of the betacatenin gene are frequent in mouse and human hepatocellular carcinomas[END_REF][START_REF] Miyoshi | Activation of the beta-catenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3[END_REF]. HCCs with β-catenin mutation constitute one of the two subclasses of HCC [START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF][START_REF] Lee | Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling[END_REF], and are thought to follow a particular pattern of pathogenesis, as most are well differentiated, cholestatic and with a better prognosis [START_REF] Audard | Cholestasis is a marker for hepatocellular carcinomas displaying beta-catenin mutations[END_REF]. Several years ago, we engineered a mouse model in which β-catenin signaling was overactivated exclusively in the liver, by the tamoxifeninducible and Cre-Lox-mediated inactivation of the Apc (Apc KO ) gene, encoding a tumor suppressor, which is an inhibitor of the Wnt/β-catenin pathway, andthe liver "zonation-keeper" [START_REF] Benhamouche | Apc tumor suppressor gene is the "zonation-keeper" of mouse liver[END_REF][START_REF] Colnot | Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas[END_REF]. In this model, the absence of Apc in single hepatocytes results in the development of β-catenin-activated liver tumors with properties similar to those observed in human patients [START_REF] Colnot | Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas[END_REF]. IfApcis deleted in all hepatocytes, the βcatenin signaling in hepatocytes leads to the induction of a transcriptional program very similar to that of human HCC with β-catenin mutation [START_REF] Colnot | Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas[END_REF][START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF].

The molecular pathogenesis of HCC has been elucidated [START_REF] Guichard | Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma[END_REF], but the incidence and mortality of HCC remain high, despite the use of the tyrosine kinase inhibitor sorafenib. Different therapeutic strategies could therefore be used to target tumors in accordance with theirpathogenic pathways. MicroRNAs (miRNAs) have recently emerged as potent diagnostic and prognostic biomarkers, and numbers of reportsdevoted to miRNAs have shown that these molecules act as oncogenes or tumor suppressors [START_REF] Babashah | The oncogenic and tumour suppressive roles of microRNAs in cancer and apoptosis[END_REF]. The global analysis of the miRNAs expressed in liver tumors highlighted a miRNA signature common to all types of HCC [START_REF] Gougelet | microRNA: new diagnostic and therapeutic tools in liver disease?[END_REF][START_REF] Ladeiro | MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations[END_REF][START_REF] Murakami | Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues[END_REF]. A loss of miR-122 [START_REF] Ladeiro | MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations[END_REF], miR-29 [START_REF] Xiong | Effects of microRNA-29 on apoptosis, tumorigenicity, and prognosis of hepatocellular carcinoma[END_REF], and miR-124 [START_REF] Furuta | miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma[END_REF] has been described. Inversely, some oncogenic miRNAs have been identified: miR-21 [START_REF] Meng | MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer[END_REF], miR-221/miR-222 [START_REF] Pineau | miR-221 overexpression contributes to liver tumorigenesis[END_REF], and miR-224 [START_REF] Wang | Profiling microRNA expression in hepatocellular carcinoma reveals microRNA-224 up-regulation and apoptosis inhibitor-5 as a microRNA-224-specific target[END_REF]. Despite the number of global analyses, the role of miR-34a in liver tumorigenesis remains unclear. Even if this miRNA is currently described as a tumor suppressor in various cancers [START_REF] Lodygin | Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer[END_REF], two studies suggested that miR-34a levels decrease in HCCs, mainly HBV+ [START_REF] Li | miR-34a inhibits migration and invasion by downregulation of c-Met expression in human hepatocellular carcinoma cells[END_REF][START_REF] Lou | Oncolytic adenovirus co-expressing miRNA-34a and IL-24 induces superior antitumor activity in experimental tumor model[END_REF], while other studies reported an induction of miR-34a, particularly in HCC with β-cateninmutations [START_REF] Meng | MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer[END_REF][START_REF] Pineau | miR-221 overexpression contributes to liver tumorigenesis[END_REF].

We aimed to identify candidate microRNAs, regulated by β-catenin, potentially involved in liver tumorigenesis. We observed that miR-34a was overexpressed in response to β-catenin overactivation; the levels of miR-34a being inversely correlated with those of HNF-4α (hepatocyte nuclear receptor α), another key factor for hepatocyte differentiation, which we have shown to antagonize β-catenin function [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF]. MiR-34a induction was observed specifically in mouse and human liver tumors with β-catenin mutations, and miR-34a was found to play an oncogenic role in the liver.

These findings call into question the systematic use of miR-34a mimics to combat liver tumor growth (http://www.mirnarx.com/pipeline/mirna-MRX34.html). Instead, we provide here proof-of-concept that a locked nucleic acid (LNA)-based inhibitor of miR-34a may be useful for treatment, exclusively in HCCs with β-catenin mutation.

METHODS

Animals and reagents

Transgenic mice, liver sampling and immunostaining have been described elsewhere [START_REF] Benhamouche | Apc tumor suppressor gene is the "zonation-keeper" of mouse liver[END_REF][START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF][START_REF] Torre | The transforming growth factoralpha and cyclin D1 genes are direct targets of beta-catenin signaling in hepatocyte proliferation[END_REF], and detailed in the supporting materials.

Hepatocyte isolation

To directly lyse hepatocytes after collagenase perfusion, i.e. for RNA or protein extraction, the transgenic livers were perfused six days after tamoxifen injection (1.5 mg)as previously described [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF][START_REF] Torre | The transforming growth factoralpha and cyclin D1 genes are direct targets of beta-catenin signaling in hepatocyte proliferation[END_REF]. Non-parenchymal cells were excluded in the supernatant, and hepatocytes preparations were used for subsequent experiments.

Periportal and pericentral subpopulations of wt hepatocytes were isolated by perfusion with digitonin and collagenase, as previously reported [START_REF] Braeuning | Differential gene expression in periportal and perivenous mouse hepatocytes[END_REF].

Cell culture

For primary culture of hepatocytes, livers were perfused three days after tamoxifen injection to Apc lox/lox mice, the cells were dispersed in William's medium (supplemented with 10% fetal bovine serum, penicillin-streptomycin, fungizone, 25nM dexamethasone, insulin 4 μg/mL and 1% BSA), and plated in wells coated with rattail collagen I (GIBCO).

Transfection assays

The cells were transfected 4h after plating, as described in the supporting materials.

xCELLigence assay

Hepatocyte adhesion and cell cycle progression were measured with the xCELLigence system (Roche). Cells were transfected with 100 nMLNA or mimic 4 h after plating, as described above (see supporting Materials). The impedance was then measured for 48 h.

Western-Blotting

The membrane with 50μg proteins was probed overnight with a primary antibody against the protein of interest (see Supporting Materials), and then incubated with a HRP-conjugated secondary antibody (Cell Signaling) for detection with the ECL system (Biorad).

In situ hybridization

Sections were deparaffinized and treated with 0.1 mg/ml proteinase K, then hybridized for one hour at 55°C with 100 nmol digoxigenin-labeled-LNA-scramble or LNA-34a (Exiqon). Signals were detected with anti-digoxigenin (Roche) and NBT/BCIP (Roche).

Samples processing for miRNA-seq:

Total RNA was extracted from purified hepatocytes six days after tamoxifen injection with Trizol® reagent (Life Technologies). For each set of conditions, we analyzed four distinct hepatocyte preparations by deep sequencing on an Illumina platform (Fasteris, Switzerland). The reads generated by the HiSeq were analyzed with miRDeep2 and miRAnalyzer software (See Supporting Materials).

Chromatin sonication and immunoprecipitation

The ChIP protocol was adapted from that of Nelson [START_REF] Nelson | Protocol for the fast chromatin immunoprecipitation (ChIP) method[END_REF], as previously described [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF](See Supporting Materials).

Human samples

We included 44 patients treated for liver cancer at Cochin hospital in this study (Table S3). All tumor samples were frozen after surgery.

RNA extraction and RT-qPCR

Total RNA was extracted from purified hepatocytes with Trizol® reagent (Life Technologies). Levels of miRNAwere determinedon 2 ng total mRNA, with a specific Taqman miRNA assay (Applied Biosystems). Levelof cyclin D1 and HNF-4α was determined on 100 ng total mRNA relative to 18S RNA (see Supporting Materials).

Statistical analysis

We assessed the significance of differences between two groups of samples in Wilcoxon tests if n<30 and Student tests if n>30. ANOVA was used to compare three groups of samples. P<0.05 was considered statistically significant.

RESULTS

MiR-34a is induced following β-catenin activation

We searched for candidate microRNAs regulated by β-catenin in hepatocytes, by deep-sequencing the miRNAs expressed in the pre-tumoral Apc deletion (Apc KO ) model, in which there is pan-lobular β-catenin overactivation, and comparing the results with those for wild-type (wt) hepatocytes. A statistical analysis showed that 123 miRNAs discriminated between Apc KO and wt hepatocytes (Tables S1 andS2), and that these miRNAs mostly targeted the Wnt/β-catenin pathway (Figure S1). We confirmed that miR-375 levels were significantly decreased by β-catenin activation (Table S2), as reported in HCCs with β-catenin mutation [START_REF] Ladeiro | MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations[END_REF]. We also identified 54 miRNAs generated from the imprinted Dlk1/Gtl2 locus as positive targets of β-catenin (Table S1). We discarded this locus for further studies, as this is not a relevant target of β-catenin in human HCCs (see the supplemental materials, Tables S1,S3 and Figure S2). We thus focused on miR-34a, which was induced by a factor of about three after β-catenin activation, with respect to wild-type hepatocytes, in the miRNAseq analysis (Table S1), and in qPCR (Figure 1A). This miRNA displayed the most significant induction following β-catenin activation after the miRNAs from the Dlk1/Gtl2 locus, and its overexpression in HCC with β-catenin mutation has already been reported [START_REF] Pineau | miR-221 overexpression contributes to liver tumorigenesis[END_REF]. We collected 24 human tumors with a mutation of CTNNB1 leading to β-catenin activation, and 20 tumors without CTNNB1 mutation. This cohort covered the major etiologies observed for HCC (HBV, HCV, alcohol abuse) (Table S3). In our cohort of patients, miR-34a levels were higher in tumors than in normal tissue (by a factor of 2 in the absence of CTNNB1 mutation, and 5 in presence of CTNNB1 mutation, Figure 1B); this induction was significantly stronger in tumors with β-catenin mutations (p<0.005). The overexpression of miR-34a in tumors with mutations of β-catenin was correlated with a context of HCV infection or no particular risk factors (Figure 1C). The sample size was too small to obtain a significant difference in miR-34a overexpression as a function of alcohol abuse status (Figure 1C). The overexpression of miR-34a was confirmed by in situ hybridization in another panel of tumors with β-catenin mutation (Figure 1D). Thus, miR-34a is overexpressed in human HCCs with CTNNB1 mutations, and could constitute a major mediator of the oncogenic signal initiated by β-catenin activation in hepatocytes.

β-catenin directly regulates miR-34a expression

For confirmation of miR-34a regulation by β-catenin, we transfected isolated wt and Apc KO hepatocytes with a siRNA against β-catenin. β-catenin silencing decreased miR-34a levels by about 47% for wt hepatocytes and 33% for Apc KO hepatocytes (Figure 2A). We thus investigated whether β-catenin could directly control miR-34a expression, by ChIP experiments with an antibody against TCF-4 (Tcell factor 4), the main LEF/TCF factor associated with β-catenin in liver [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF], and an antibody against β-catenin. We measured the binding of TCF-4 and β-catenin tosites upstream from miR-34a identified as a TCF-4 binding site in our ChIP-seq data [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF]. βcatenin activation led to a significant increase in the binding of TCF-4 (Figure 2B) and β-catenin (Figure 2C) to a site 21kb upstream from miR-34a. β-catenin binding was also significantly increased following Apc depletion on a more proximal TCF-4 site (Figure S3). Overall, our data suggest that an excess of β-catenin could significantly increase miR-34a transcription in hepatocytes.

MiR-34a zonal redistribution after β-catenin overactivation

We then assessed the hepatic distribution of miR-34a by in situ hybridization.

Exceptions, such as cyclin D1 [START_REF] Torre | The transforming growth factoralpha and cyclin D1 genes are direct targets of beta-catenin signaling in hepatocyte proliferation[END_REF], are known, but the direct targets of β-catenin are generally expressed in pericentral (PC) hepatocytes, in which Wnt/β-catenin signaling is activated [START_REF] Benhamouche | Apc tumor suppressor gene is the "zonation-keeper" of mouse liver[END_REF]. Counterintuitively, in normal livers, miR-34a was found to be preferentially expressed in hepatocytes around the portal vein and, more particularly, in bile duct cells (Figure 3A). For confirmation of this prevalence of miR-34a in the periportal (PP)subpopulation, we isolated PP hepatocytes and PC hepatocytes separately, by injecting digitonin into the central vein and the portal vein, respectively. This differential perfusion also showed that PP samples were enriched in miR-34a (Figure 3B). The results were non-significant, since using this technique, biliary cells are mainly lost during washing steps. In addition, β-catenin overactivation led to a loss of miR-34a zonation, and to miR-34a expression of in all hepatocytes (Figure 3A). In conclusion, miR-34a appears to be expressed in hepatocytes throughout the liver only when β-catenin is overactivated.

We then searched the DIANA database (http://diana.imis.athenainnovation.gr/DianaTools/index.php?r=microT_CDS/index), a database with experimentally validated targets for miRNAs, for potential targets of miR-34a.

Number of potent targets have been identified (Table S4), but we focused on three interesting proteins: LEF-1, a transcription factor associated with β-catenin, caspase 2, recently identified as a direct target of miR-34a in the liver [START_REF] Meng | Epigenetic regulation of miR-34a expression in alcoholic liver injury[END_REF], and HNF-4α, which we recently showed to be an antagonist of β-catenin in the liver [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF]. These opposing activities result in a reciprocal physiological distribution in the liver, with miR-34a found in biliary cells (Figure 3A) and HNF-4α in hepatocytes, consistent with the hypothesis that miR-34a could repress HNF-4α expression in biliary cells [START_REF] Nagy | Expression of hepatic transcription factors during liver development and oval cell differentiation[END_REF].

MiR-34a inhibition improves HNF-4α transactivation capacity in hepatocytes displaying β-catenin-activation

We confirmed by western blotting that the levels of HNF-4α, caspase 2, and LEF-1 proteins (Figure 3C) were inversely correlated with miR-34a levels in Apc KO mice.

Similarly, the overexpression of miR-34a in human HCCs with CTNNB1 mutations was also associated with weaker immunostaining for HNF-4α in these tumors (Figure S4). In a previous study, we showed that β-catenin impaired the transcriptional activity of HNF-4α on a luciferase reporter gene under the control of an HNF-4αresponse element (FOP) [START_REF] Gougelet | T-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice[END_REF]. Having shown that β-catenin induces miR-34a, which could in turn regulate HNF-4α levels, we investigated the involvement of miR-34a in the antagonism between HNF-4α and β-catenin in Apc KO hepatocytes. An inhibitor of miR-34a based on locked nucleic acid technology (LNA-34a), which consists of the complementary sequence for miR-34a with a methylene group in the ribose, significantly activated the FOP reporter (Figure 3D). A mimic of miR-34a had no effect, probably due to the maximal miR-34a overexpression in Apc KO hepatocytes (Figure 3C). In conclusion, miR-34a represses HNF-4α transcriptional activity, and appears to be involved in the β-catenin/HNF-4α antagonism in our model of β-catenin overactivation.

MiR-34a is induced in tumors displayingβ-catenin activation

We then assessed the level of miR-34a in liver tumours mutated for β-catenin in mice. We compared 35 tumors with seven normal tissues obtained from wt mice that had received tamoxifen injections. MiR-34a levels in these tumors were twice those in normal tissues (Figure 4A). HNF-4α mRNA levels in these tumors were also half those in normal tissues (Figure 4B), confirming its tumor suppressor role in the liver [START_REF] Lazarevich | Deregulation of hepatocyte nuclear factor 4 (HNF4)as a marker of epithelial tumors progression[END_REF][START_REF] Ning | Hepatocyte nuclear factor 4 alpha suppresses the development of hepatocellular carcinoma[END_REF]. Interestingly, the tumors with the highest miR-34a levels also had the lowest HNF-4α levels (Figures 4C and4D). The deregulation of miR-34a in these tumors was independent of p53 mRNA levels (Figure S5), despite miR-34a being a target of p53 [START_REF] He | A microRNA component of the p53 tumour suppressor network[END_REF]. Finally, we confirmed that overexpression of miR-34a in tumors with β-catenin overactivation, by in situ hybridization, was associated to a loss of HNF-4α (Figure 4E).

MiR-34ainhibitionby a locked nucleic acid

These data highlight the unique oncogenic role of miR-34a in a context of βcatenin overactivation. This led us to try to block miR-34a activity with a LNA, which has proved promising in human liver disease [START_REF] Lanford | Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection[END_REF]. We assessed the efficacy of this approach in cultures of hepatocytes isolated from wt and Apc KO mice. No change in the morphology of hepatocytes transfected with LNAs was observed, on microscopy, 72 hours after transfection (data not shown). The efficacy of LNA-34a was assessed by cotransfection with a luciferase reporter construct with a miR-34a binding site in its 3'UTR. As expected, following cotransfection with LNA-34a, miR-34a activity was impaired, resulting in an increase in luciferase activity (about three times higher than that obtained after cotransfection with LNA-scramble, Figure 5A). The LNA efficacy was also confirmed by western blotting for HNF-4α: HNF-4α levels were higher in the presence of LNA-34a (Figure 5B).

LNA-34a impairshepatocyte proliferationafterβ-catenin overactivation

We then investigated the effect of LNA-34a on the proliferation of hepatocytes. We used the xCELLigence system, which measures cell impedance in real-time, through the use of gold microelectrodes placed under each well. WT hepatocytes underwent one round of cell division, due to the proproliferative effect of tamoxifen and collagenase perfusion (Figure S6A). WT hepatocytes had a lower cell proliferation index than Apc KO hepatocytes (Figure S6A), confirming that an absence of Apc leads to hepatocyte hyperproliferation [START_REF] Colnot | Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas[END_REF]. Transfection with LNA-34a significantly impaired Apc KO hepatocyte proliferation (35% inhibition), but had no effect on wt hepatocytes (Figure 5C). Similar results were obtained with the Cell Titer assay, measuring the reduction of tetrazolium in formazan, a process dependent on NADPH (data not shown). The activity of lactate deshydrogenase in cell supernatant was not altered following LNA-34a transfection, suggesting an absence of LNA-34a toxicity (Figure S6B). This suggests that LNA-34a has a specific antiproliferative effect on hepatocytes displaying β-catenin overactivation.In addition, the LNA-34a transfected three days successively increased the amount of caspase-2 propeptide, as well as caspase-2L, its proapototic isoform, and also decreasedthe level offull-length PARP-1, suggesting its increased cleavage (Figure 5D). This was not observed for wt hepatocytes (Figure S6C). In vivo, the LNA-34a injected to mice only increased caspase-2L level in Apc KO mice (Figure S6D).In conclusion, LNA-34a promotes a pro-apoptotic signal in the Apc KO hepatocytes.

Cyclin D1 has been identified as a target of miR-34a [START_REF] Sun | Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest[END_REF]. We therefore assessed cyclin D1 levels after transfection with LNA-34a. Contrary to expectations, cyclin D1 protein and mRNA levels, at a lower extent,were decreased in the presence of LNA-34a (Figure 5E). However, this is consistent with the antiproliferative effects of LNA-34a on Apc KO hepatocytes (Figure 5C). The loss of cyclin D1 may be a consequence of the increase in HNF-4α amount in the presence of LNA-34a (Figure 5B), as HNF-4α has been shown to repress cyclin D1 [START_REF] Walesky | Hepatocyte-specific deletion of hepatocyte nuclear factor-4alpha in adult mice results in increased hepatocyte proliferation[END_REF] (Figure 6E). In agreement with this hypothesis, no change in cyclin D1 protein and mRNA levels was observed when Apc KO hepatocytes also lacking hnf-4α, were transfected with LNA-34a (Figure 6A).

LNA-34a had also a weaker antiproliferative effect on these hepatocytes isolated from a transgenic mouse model, in which both Apc and hnf4a had been deleted by Cre-Lox strategy(Figure 6B). The higher proliferation rate observed for hnf4/Apc KO hepatocytes in the presence of LNA-34a was associated to a minor induction of caspase-2L level, even if caspase-2 propeptide was efficiently increased (Figure 6C); PARP amount was also not altered in this case (Figure 6C). Finally, we realized ChIP experiments with an antibody against HNF-4α in Apc KO hepatocytes,and analyzed the binding of HNF-4α in the presence of LNA-34a on two binding sites identified in published ChIP-seq experiments done in HepG2 cells and in murine livers (Figure S7). We showed that the binding of HNF-4α to cyclin D1 promoter tended to increasefollowing exposure to LNA-34a, in particular for peak 1 (Figure 6D). This finding corroborates that HNF-4α mediates the decrease in cyclin D1 levels in response to LNA-34a. In conclusion, following its overactivation, β-catenin binds to the miR-34a promoter, thereby increasing its expression. MiR-34a in turn decreases the amount of HNF-4α, leading to increases in cyclin D1 transcription, and hepatocyte proliferation (Figure 6E).

MiR-34a inhibition reduces tumor progression in Apc KO mice

We thus developed a tumor suppressive strategy based on miR-34a inhibition, which could constitute the first targeted therapy for liver tumors displaying β-catenin activation. We assessed the impact of LNA-34a on liver tumor progression in an Apc KO context. Once the tumors became detectable on ultrasound scans, we injected LNA-34a (10 mg/kg) once weekly, and followed tumor development twice monthly, by 2D-ultrasound (Figure S8). We found that LNA-34a slowed the progression of tumors displaying β-catenin activation, to rates about half those for untreated tumors (Figure 7A andS8). This treatment also inhibited the development of new tumors, with only one new tumor on average occurring on LNA-34a treatment, versus four on LNAscramble treatment (Figure 7B). As expected, HNF-4α levels increased and cyclin D1 levels decreased in tumors treated with LNA-34a (Figure S9A). Even if cyclin D1 level was impaired, Ki-67 labeling was similar in tumors treated with LNA-34a, and in those treated with the scramble LNA (Figure S9B). Similar results were obtained with western-blots for phospho-Histone H3 and cyclin A labeling (data not shown).However, LNA-34a promoted caspase 2 and 3 activation in tumors, in both western-blot and immunohistochemistry experiments, confirming its pro-apoptotic effect (Figures 7C and7E), with minor effects on non-tumoral liver (Figure 7D).Thus, treatment based on the use of LNA-34a could reduce tumor development through an increase in HNF-4α levels, leading to a loss of cyclin D1, and through a pro-apoptotic program associated with the induction of caspase 2 and caspase 3 cleavage.

DISCUSSION

The microRNA miR-34a has been widely described as a tumor suppressor.

Indeed, its expression is frequently impairedin various cancers following an hypermethylation of its promoter [START_REF] Lodygin | Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer[END_REF], including breast and prostate cancers [START_REF] Liu | The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44[END_REF][START_REF] Yang | MicroRNA-34 suppresses breast cancer invasion and metastasis by directly targeting Fra-1[END_REF]. A preclinical trial is currently underway with a mimic of miR-34a encapsulated into liposomes in various murine models of solid tumors (miRNATherapeutics, http://www.mirnarx.com/pipeline/mirna-MRX34.html). However, the role of miR-34a in liver tumorigenesis remains unclear. One study suggested that miR-34a level decrease in HCC [START_REF] Lou | Oncolytic adenovirus co-expressing miRNA-34a and IL-24 induces superior antitumor activity in experimental tumor model[END_REF], andis associated to migration and invasion [START_REF] Li | miR-34a inhibits migration and invasion by downregulation of c-Met expression in human hepatocellular carcinoma cells[END_REF], but another two studies reported the induction of miR-34a in HCC samples [START_REF] Meng | MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer[END_REF][START_REF] Pineau | miR-221 overexpression contributes to liver tumorigenesis[END_REF], in HCV+ sera [START_REF] Cermelli | Circulating microRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver disease[END_REF], and in HCC cells in response to alcohol [START_REF] Meng | Epigenetic regulation of miR-34a expression in alcoholic liver injury[END_REF]. Our findings suggest that miR-34a is an oncogenic mediator in tumors displaying β-catenin activation, both in mice and humans, confirming the data reported by Pineau et al. [START_REF] Pineau | miR-221 overexpression contributes to liver tumorigenesis[END_REF]. Our results could reconcile the data of Lou and Li, which suggested that miR-34a was lost in two thirds of the HCCs studied [START_REF] Li | miR-34a inhibits migration and invasion by downregulation of c-Met expression in human hepatocellular carcinoma cells[END_REF][START_REF] Lou | Oncolytic adenovirus co-expressing miRNA-34a and IL-24 induces superior antitumor activity in experimental tumor model[END_REF]. These samples corresponded mostly to HBV + tumors, a subgroup with a lower frequency of CTNNB1 mutations [START_REF] Guichard | Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma[END_REF]. The other third of patients in their cohort, with HCV infection, displayed an increase in miR-34a expression, and this group is likely to be enriched in CTNNB1 mutations, according to transcriptomic classifications [START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF][START_REF] Lee | Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling[END_REF]. In our cohort, tumors without β-catenin mutations displayed heterogeneity in terms of miR-34a amount. A greater understanding of this variability would be very useful,

In our study, we used a LNA approach to block cell cycle progression (Figure 5C), to decrease cyclin D1 protein levels (Figure 5E), and to induce caspase-2 activation (Figure 5D), in isolated Apc KO hepatocytes. The decrease in cyclin D1 protein levels was actually due to HNF-4α, because the invalidation of hnf4a in the Apc KO model resulted in an absence of change in cyclin D1 levels (Figure 6A), and a smaller decrease in cell proliferation (Figure 6B), in the presence of LNA-34a. In addition, LNA-34a increases HNF-4α protein level (Figure 5B), but also its transcriptional activity on a luciferase construct under the control of a HNF-4 responsive element (Figure 3D), and on cyclin D1 promoter (Figure 6D).Thus, HNF-4α, a tumor suppressor in the liver [START_REF] Lazarevich | Deregulation of hepatocyte nuclear factor 4 (HNF4)as a marker of epithelial tumors progression[END_REF], appears to be a key mediator of the effects of miR-34a in the Apc KO model (Figure 6E). More importantly, miR-34a emerges as a crucial component of the oncogenic signal induced by β-catenin in hepatocytes.

In normal livers, miR-34a was particularly expressed in bile duct cells (Figure 3A and 3B). This specific pattern of expression may hinder the accumulation of HNF-4α protein in the biliary cells, which is required for bile cell lineage patterning [START_REF] Nagy | Expression of hepatic transcription factors during liver development and oval cell differentiation[END_REF],probably independently of β-catenin, which is not activated in normal bile duct cells [START_REF] Hu | Wnt/beta-catenin signaling in murine hepatic transit amplifying progenitor cells[END_REF]. Thepattern of miR-34a expression is not consistent with the usual model, in which β-catenin targets are located near the central vein. Cyclin D1 is one exception to this model;its pattern of expression is not restricted to the PC zone [START_REF] Torre | The transforming growth factoralpha and cyclin D1 genes are direct targets of beta-catenin signaling in hepatocyte proliferation[END_REF].

Interestingly, the region bound to TCF-4/β-catenin located in miR-34a promoter contains no classical Wnt-responsive element, suggesting that the control on miR-34a is complex (Figure 2B). This complexityis lost when β-catenin is overactivated, as in HCCs.

The key finding of this study was the decrease in liver tumor growth afforded by the delivery of LNA-34a (Figures 7A andS8). Only a few studies to date have demonstrated the potency of miRNA-based approaches to cancer treatment.

However, the great advantage of this approach for HCC is that these molecules are preferentially delivered to the liver [START_REF] Roberts | Efficient and persistent splice switching by systemically delivered LNA oligonucleotides in mice[END_REF]. The first authorization for clinical trials with miRNA-based therapies, concernsthe LNA against miR-122 (miravirsen) for

HCVtreatment [START_REF] Lanford | Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection[END_REF]. The use of these modulators poses a number of challenges, particularly for their specificity. Our therapeutic approach, involving the inhibition of miR-34a, displays no toxicity (Figure S6B), and has the great advantage of restoring the tumor suppressor HNF-4α, targeting cyclin D1 and the metabolic pathways regulated by HNF-4α (Figure S9). In addition, this approach may also alter the antiinflammatory loop orchestrated by HNF-4α, controlling interleukin-6 production by hepatocytes [START_REF] Gougelet | MicroRNA-feedback loop as a key modulator of liver tumorigenesis and inflammation[END_REF][START_REF] Hatziapostolou | An HNF4alpha-miRNA inflammatory feedback circuit regulates hepatocellular oncogenesis[END_REF]. More importantly, the LNA-34a promotes a pro-apoptotic signal orchestrated by caspase 2 and 3 (Figure 7C-E). In conclusion, our data reveal that miR-34a inhibition mainly induces a pro-apototic program centered onto caspase-2, and could affect cell proliferation through cyclin D1 inhibition. However, the fact that Ki-67 and cyclin A labeling were not affected by LNA-34a treatment, suggeststhat miR-34a effects on cell proliferation are more complex.

In conclusion, despite the current preclinical trials being run by miRNAtherapeutic, our findings suggest that the precise differential diagnosis of HCC, including an assessment of the mutational status of β-catenin, for which miR-34a inhibitors would be more appropriate than miR-34a mimics, is vital. Using this approach, we hope to affect not only the tumor itself, but also its environment, through the first effective targeted therapy for one third of HCCs worldwide.

Figure 1 :

 1 Figure 1: miR-34a is induced following β-catenin activation in mouse

Figure 2 :

 2 Figure 2: miR-34a is induced following β-catenin activation, through the direct

Figure 3A -

 3A Figure 3A-B: miR-34a induction following β-catenin activation is associated

Figure 4 :

 4 Figure 4: miR-34a is induced in tumors from Apc KO mice.

Figure 5 :

 5 Figure 5: LNA-34a inhibits Apc KO hepatocyte proliferation, decreases cyclin D1 levels, and promotes apoptosis. A: Isolated Apc KO hepatocytes were transfected

Figure 6 :

 6 Figure 6: LNA-34a impact depends on HNF4α expression A: Cyclin D1 mRNA and protein level in isolated hnf-4α KO /Apc KO hepatocytes transfected with LNA-scramble or LNA-34a for 48 h. B: Cell proliferation of Apc KO or Apc/hnf-4α KO cells, transfected with LNA-scramble or LNA-34a for 48 h. Results are

Figure 7 :

 7 Figure 7: LNA-34a slows the development of tumors from APC KO mice, by

ACKNOWLEDGMENTS

We thank Dr F. Gonzalez for the hnf4 -/-mice. We are grateful to Dr P. Bossard and M. Caüzac for LDH protocol and to Dr. A-F. Burnol for the gift of PARP-1 antibody.

SUMMARY BOX

What is already known on this subject ?

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and the third leading cause of death by cancer worldwide, due to the lack of effective treatments. We, and others, have shown that mutations of the CTNNB1 gene, encoding β-catenin, are observed in 15 to 40% of HCCs. HCCs with β-catenin mutation constitute one of the two subclasses of HCC, and are thought to follow a particular pattern of pathogenesis.

What are the new findings?

Using a transgenic mouse model developing tumors with β-catenin activation, we provide the first evidence of an oncogenic role of miR-34a in liver tumors with βcatenin mutations. An inhibitor against miR-34a slows tumor progression, and inhibits the development of new tumors in this model.

How might it impact on clinical practice in the foreseeable future?

These findings call into question the preclinical trial designed by miRNAtherapeutics, using miR-34a mimics to combat liver tumor growth. Instead, we provide here proof-of-concept that a locked nucleic acid-based inhibitor of miR-34a may be useful for treatment, exclusively in HCCs with β-catenin mutation.

STUDY APPROVAL

All procedures were carried out according to the French guidelines for the care and use of experimental animals. All animal studies were approved by the regional


Sea-level rise impact on European shelf tide dynamics D. Idier (1) ; F. Paris (1) ; G. Le Cozannet (2) ; F. Boulahya (1)

(1) BRGM, Orléans, France; (2) BRGM / C NRS, BRGM-DRP-R3C / C NRS-LGP (UMR-8591), Orleans, France

Abstract content Sea-level rise (SLR) affects not only the total water level, but can also modify the tidal dynamics. Several studies investigated the SLR effect on the tide of the Western European continental shelf, analysing the tidal dynamics (mainly the M2 component) for various SLR scenarios or coastal defence schemes. The present study aims at completing existing knowledge, using a modelling based approach, investigating 11 uniform SLR scenarios from -0.25 to +10m, and analysing the effect on high/low tide water level as well as the amplitude and phase tidal components. Sea-level rise scenarios include the present sea-level, as well as several idealized scenarios that correspond to plausible sea-levels for more or less distant past or future. On the one hand, the -0.25m scenario can be considered as a low bound for the preindustrial sea-level. On the other hand, positive SLR scenarios correspond to more or less plausible sea-levels in the future: according to IPCC, a rise of sea-level of +25cm is likely by 2046-2065 whatever the climate change scenario, and the likely range for high emissions scenarios reaches 0.5 to 1m by 2100. While higher SLR scenarios are not excluded by 2100, sea-level will continue to rise beyond 2100, and it is likely to reach values of several meters by 2200 and beyond. Finally, the +10m SLR scenario corresponds to a situation where most Greenland and west-Antarctic ice-sheets have melted. Assuming that coastal defences along the present day shoreline are maintained and upgraded, the patterns of increase / decrease of high tide level (annual maximum water level) are almost stationary in most of the area (70%), with an increase in most of the domain and a decrease mainly in the Western English Channel. These changes are globally varying linearly with the SLR, till the +2m SLR scenario, with rate ranging between -15% and +15% of SLR. The main patterns are: (1) a decrease in the Western English Channel, (2) an increase in the Irish Sea, Southern North Sea and German Bight. The analysis of tidal component contributions shows that high tide level changes patterns are not exactly similar to M2 pattern changes (e.g. along the French Atlantic coast, high tide level increases whereas M2 amplitude decreases). This highlights the need to take into account all the components when analysing SLR effect on the tide. The main changes in the maximum water level result from the changes of the M2, S2, N2, M4 and MS4 components. Sea-level rise pushes several areas (e.g. Atlantic coast, Irish Sea) closer to resonance, leading to the increase of the resonant tidal components (especially semi-diurnal or quart-diurnal, depending on the area). The linear behaviour of tide dynamics with the SLR is highly sensitive to the coastal defence strategy (i.e. let flood or not), the high tide level varying much less linearly with SLR when flooding is allowed, like for instance in the German Bight. However, several areas appear not sensitive to this choice, such that the estimated trends there are highly probable: an increase of ~6% SLR (resp. decrease of ~15% SLR) in the North of Irish Sea (resp. in the Western English Channel). Finally, preliminary investigations show that, at least, for the non-uniform SLR scenario we computed, the high water level is very weakly sensitive to the (non)-uniformity, local rates of increase/decrease (relative to local SLR) being similar to the ones obtained from uniform SLR scenarios.