formation tour amsterdam

A Bit Of Pop Music

Concert Review: Beyoncé Formation World Tour at Amsterdam Arena

Beyoncé on top of her game in Amsterdam Arena Beyoncé delivered her without a doubt most controversial album so far,  Lemonade , earlier this year. The record is full of raw lyrics about a cheating husband and at the same time touches upon political issues. Both her personal life and the position of Afro Americans are discussed. She became the talk of the town at the Super Bowl with a powerful rendition of ‘Formation’ and has recently become more and more vocal for the BlackLivesMatter movement. The pops star also became an activist. On Saturday Night she treated her Dutch fans in the Amsterdam Arena to the Formation World Tour!

Miss Carter started the tour in April in the United States and moved to Europe in June for the second part. The setlist contains as many as 35 songs, although the tracks which are not on her last album, will be performed only partially. She leaves out some big hits and fan favourites like ‘If I Were A Boy’, ‘Listen’, ‘Single Ladies’ and ‘XO’, while she does perform ‘Me, Myself & I’, a moderate hit from her debut. The sometimes unexpected choices don’t seem to bother the crowd who cheer for her loudly from the moment she comes on stage.

That entry is quite a big one to be fair. Her dancers come up in black dresses with hats and Beyoncé follows suit from underneath the stage in a similar outfit with some added bling. The excitement in the room is bursting when Queen B’s face becomes visible when she nods her head to the beat of ‘ Formation ‘. Full of attitude she immediately gets the crowd going when she screams ‘I Slay!’. What follows is an impressive choreography of her dancers with Beyoncé as the ever glowing centre of attention. This is something that continues throughout the whole show and it is definitely outstanding how the star of the night never misses a note or even a dance move.

Visually, the Formation World Tour is definitely a spectacle. The stage is filled with a huge decor, but Beyoncé does not need anything to hide behind and forms the point of attention from start to finish. The wind machines are working hard to blow her stunning hair out of her sweaty face. The style of her dance goes from slow and sensual to fast and powerful. The latter is most evident on a literally splashing rendition of ‘Freedom’, performed on the second smaller stage that is filled up with water. She continues with her feet in the water while singing Destiny’s Child classic ‘Survivor’, which is a nice reference to the famous music video of the track. Although the water sequence is definitely one of the highlights of the show, Beyoncé also impresses with just her vocals when she sings songs like ‘Irreplaceable’ and ‘Love On Top’ a capella. These are the moments when the crowd forms a huge choir that sings along every word.

Although Beyoncé gave it her all, she couldn’t stop her voice from getting lost into the awful echoes that the Amsterdam Arena seems to create. This recently badly affected Rihanna’s concert here too . The bass sounds overrule badly and even Beyoncé’s powerful vocals during the choruses can’t help the sound quality from declining towards the end. She closes the show with the emotional hit ‘Halo’, which is dedicated to the victims of the recent tragic events in the world. During fan favourite ‘All Night’ a lot of balloons appear in the room. Beyoncé shows herself thankful for this spontaneous action by the Dutch fan club and the loyal 19(!) year long support of her Dutch followers. Other than that, Beyoncé doesn’t talk much on stage, which gives the show a serious vibe, but never too dark. She touches upon the problems of the American police and black citizens with some footage on the screen and during songs like ‘Formation’ and ‘Freedom’, but the main goal still is entertainment. She twists, jumps, stamps her feet, conducts with her arms and shows she is completely in a league of her own in the current music industry.

Written by: Joana Chung

——————————————— Concert Recensie: Beyoncé in Amsterdam Arena.

Beyoncé toont zich een onevenaarbare superster in Amsterdam Arena Met het eerder dit jaar verschenen Lemonade heeft Beyoncé haar meest spraakmakende album tot nu toe de wereld in geslingerd. De plaat bevat rauwe teksten over ontrouw en is tegelijkertijd politiek beladen. Ze bespreekt zowel gebeurtenissen uit haar privéleven als ontwikkelingen rondom de Afro-Amerikaanse bevolking. Ze wist alle ogen op zich te richten met haar krachtige ‘Formation’ optreden bij de Super Bowl en steekt haar sympathie voor de BlackLivesMatter beweging niet meer onder stoelen of banken. De popster is ook activiste geworden. Zaterdagavond stond ze in de Amsterdam Arena om het Nederlandse publiek te trakteren op de Formation World Tour.

Miss Carter begon de tour in april in de Verenigde Staten en reisde in juni naar Europa af voor het vervolg. De setlist telt maar liefst 35 tracks, hoewel de meeste liedjes die niet op Lemonade staan, niet volledig worden vertolkt. Opvallend is wel dat hits als ‘If I Were A Boy’, ‘Listen’, ‘Single Ladies’ en ‘XO’ de grote afwezigen vormen, terwijl ‘Me, Myself & I’, een bescheiden hit van haar eerste album, wel op het programma staat. Deze soms onverwachte keuzes lijken het publiek echter geen moment te deren, want vanaf het eerste moment wordt er enthousiast voor haar gegild.

De opening is dan ook zeker een groots moment! Wanneer de eerste figuren met hoge hoeden en zwarte jurken aan komen lopen, barst het gejuich los en Beyoncé zelf in dito kostuum met bling komt vanuit het podium langzaam omhoog. De hoge hoeden verbergen nog de gezichten, maar wanneer ze op de beat van ‘ Formation ‘ knikken, zien we eindelijk Beyoncé en haar dansers. Met veel swag zweept ze het publiek op en de “I slay” kreten kunnen op veel respons rekenen. Een strakke choreografie volgt, waarbij een dozijn vrouwelijke dansers krachtige moves maakt met Beyoncé in het middelpunt. Vergelijkbare routines worden ons gedurende de hele show voorgeschoteld en het is indrukwekkend om te zien hoe Beyoncé vocaal altijd overeind blijft en geen danspasje mist.

Visueel is de gehele show een spektakel. Het podium wordt gevuld door een immens decor met verschillende verdiepingen. Ze heeft het echter niet nodig om zich achter te verschuilen, want met haar dansers achter zich vormt ze veelal de punt van een driehoekige formatie, waarbij de windmachines overuren maken om haar weelderige lokken uit haar bezwete gezicht te blazen. Haar stijl varieert van sensueel verleidend tot een krachtige powerhouse zoals het optreden van ‘Freedom’. Het b-podium is dan gevuld met een laag water waar Bey en haar dansers een letterlijk spetterende choreografie neerzetten. Ze vervolgt met haar voeten in het water tijdens de Destiny’s child klassieker ‘Survivor’, wat een leuke knipoog naar de video van het nummer is. Hoewel dit waterballet absoluut één van de hoogtepunten van de avond is, maakt Beyoncé vooral ook veel indruk met de liedjes die ze a capella ten gehore brengt, zoals ‘Irreplaceable’ en ‘Love On Top’. Juist dan wordt ook het publiek het meest aangespoord om uit volle borst mee te zingen.

Hoewel Beyoncé haar alles geeft, kan ze niet voorkomen dat haar stem soms verdwaalt in de beruchte galm van de Arena, waardoor onlangs nog het concert van Rihanna behoorlijk in het water viel . De basgeluiden resoneren langer door en ondanks Beyoncé’s krachtige zang, wordt de galm naarmate de show vordert, steeds erger. Het slotnummer ‘Halo’ draagt ze op aan de slachtoffers van de recente drama’s in de wereld en tijdens het meeslepende ‘All Night’ wordt ze door haar fanclub verrast met ballonnen door de hele zaal. Ze bedankt hen voor al 19(!) jaar lang loyale steun. Beyoncé is op het podium niet een vrouw van veel woorden, wat de show serieus maakt, maar niet te somber. Er is zeker aandacht voor de problematiek rondom de Amerikaanse politie en Afro-Amerikaanse inwoners, vooral op beelden en tijdens liedjes als ‘Freedom’ en ‘Formation’, maar de show blijft toch vooral entertainment. Ze draait, springt, stampt en dirigeert anderen met haar armen en steekt qua choreografie, maar ook vocaal, nog steeds met kop en schouders boven haar collega’s uit.

Geschreven door: Joana Chung

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The Formation World Tour

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The Formation World Tour is the seventh concert tour by Beyoncé . It is in support of her sixth studio album, Lemonade . The tour began on April 27, 2016, at Marlins Park in Miami, Florida, and concluded at Metlife Stadium in East Rutherford, New Jersey on October 7, 2016.

• 1 Background
• 2 Production and development
• 4 Tour dates
• 6.1 Clothing
• 6.2 Accessories

Background [ ]

On February 6, 2016, Beyoncé released " Formation " for free on the music streaming service TIDAL and its accompanying music video on her official YouTube. On February 7, 2016, Beyoncé performed "Formation" during her guest appearance at the Super Bowl 50 halftime show. Immediately after the performance, a commercial aired announcing The Formation World Tour, which would kick off in Miami on April 27, with the first pre-sales going on sale just two days after the announcement on February 9, 2016.

Production and development [ ]

The focal point of the tour's main-stage was a 60 feet tall rotating cuboid, nicknamed the 'Monolith' by designers. The stage also consists of a runway, which also acts as a treadmill leading onto a B-stage that fills with a pool of water. The treadmill on the catwalk was designed to be waterproof in order to withstand unpredictable weather found in outdoor stadiums. The B-stage stores 2,000 gallons of water inside of it and takes approximately 10 minutes to fill up, which occurs without the audience even realizing.

Setlist [ ]

The setlist varies from date to date, but in total it has been composed of 45 songs: 9 from Lemonade , 7 from Beyoncé , 7 from 4 , 4 from Dangerously In Love , 2 from B'Day , 2 from I Am... Sasha Fierce , 2 collaborations, 1 soundtrack song, 1 Destiny's Child song, and 6 other songs that are used as intros/interludes that Beyoncé does not perform.

• " No Angel " (video intro) (contains elements of "Formation)
• " Formation "
• " Sorry " (contains elements of "I'm Sorry")
• " Kitty Kat " (a capella version) *
• " Irreplaceable " (a capella version) *
• " Irremplazable " *
• " Bow Down " (contains elements of "Tom Ford")
• " Run The World (Girls) "
• " Superpower " (video interlude) (containing elements of "Made It")
• " Mine " (contains elements of "Standing on the Sun Remix")
• " Baby Boy " (contains elements of "Freaks")
• " Hold Up " (contains elements of "Bam Bam")
• " Countdown " (contains elements of "Pop My Trunk")
• " Me, Myself and I "
• " Runnin' (Lose It All) "
• " All Night "
• " 6 Inch " *
• " I Care " (video interlude) (contains elements of " Ghost ", "New Slaves", "The Hills" and "6 Inch" )
• " Don't Hurt Yourself "
• " Ring the Alarm " (contains elements of "Five to One", "Ring the Alarm (Tenor Saw song)", "Independent Woman Part I", "Lost Yo Mind", "Naughty Girl" and " I Been On ")
• " Diva " (contains elements of "U Mad", "Still Tippin'", "Panda" "Cut It", "Man", "I Got The Keys" and "Dey Know")
• " Flawless (Remix) "
• "Feeling Myself"
• " Drunk in Love " (contains elements of "Swimming Pools (Drank)")
• " Partition " *
• "Hip Hop Star" / " Freakum Dress" (video interlude)
• "Daddy Lessons"
• " Single Ladies (Put A Ring On It) " *
• " Love On Top "
• "The Beautiful Ones" *
• "Purple Rain" (video interlude with the original recording)
• " Crazy in Love " (contains elements of the 2014 Remix and excerpts from "Bootylicious")
• " Naughty Girl "
• " Party " (contains elements of "La Di Da Di")
• " Blow " (contains elements of "Give It to Me Baby" and "Nasty Girl") *
• " Sweet Dreams " (contains elements of "Sweet Dreams (Are Made of This)") *
• "Die With You" (video interlude) (contains elements of "Blue")
• " Freedom "
• " End of Time " (contains elements of "Grown Woman")

Songs marked with an asterisk (*) were not performed at every date.

Tour dates [ ]

a. The show on June 14, 2016 in Detroit, United States was originally scheduled to take place on May 29, 2016, but was postponed due to scheduling changes.

b. The show on October 2, 2016 in Nashville, United States was originally scheduled to take place on May 5, 2016, but was rescheduled for unknown reasons.

c. The show on October 7, 2016 in East Rutherford, United States was originally scheduled to take place on September 7, 2016, but was rescheduled due to doctor's order for vocal rest.

Merchandise [1] [ ]

Clothing [ ].

Accessories [ ]

• ↑ https://www.teenvogue.com/gallery/beyonce-boycott-formation-tour-merch
• 1 Filmography
• 2 Cowboy Carter

Beyonce Announces ‘Formation’ World Tour Following Super Bowl Show

By Jerry Portwood

Jerry Portwood

One day after surprise-releasing a new song and video ,  Beyoncé  followed her Super Bowl 50 halftime show performance with news of the Formation World Tour.

The tour is the singer’s first solo trek since her ambitious Mrs. Carter Show World Tour in 2013. It kicks off April 27th in Miami, Florida at Marlins Park, with an additional 21 stadium dates that include New York City, Los Angeles, Chicago, Toronto, Philadelphia, Dallas. The European leg of the tour starts June 28th in Sunderland, U.K. at Stadium of Light and includes dates in London, Manchester, Zurich, Amsterdam, Paris, Milan, Stockholm, Frankfurt and more.

Bey debuted “Formation” live Sunday at Santa Clara, California’s Levi’s Stadium dressed in a tight black jumpsuit with gold military “ammunition” that seemed to reference Michael Jackson’s signature look from the Eighties. Bruno Mars, another previous Super Bowl halftime show headliner, also appeared during Coldplay’s set.

“Formation” is available to stream and download for free exclusively through Tidal .

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Beyoncé Formation World Tour Dates

April 27 – Miami @ Marlins Park April 29 – Tampa @ Raymond James Stadium May 1 – Atlanta @ Georgia Dome May 3 – Raleigh @ Carter-Finley Stadium May 5 – Nashville @ Nissan Stadium May 7 – Houston @ NRG Stadium May 9 – Dallas @ AT&T Stadium May 12 – San Diego @ Qualcomm Stadium May 14 – Los Angeles @ Rose Bowl May 16 – Santa Clara @ Levi’s Stadium May 18 – Seattle @ CenturyLink Field May 20 – Edmonton, Alberta @ Commonwealth Stadium May 23 – Minneapolis @ TCF Bank Stadium May 25 – Toronto @ Rogers Centre May 27 – Chicago @ Soldier Field May 29 – Detroit @ Ford Field May 31 – Pittsburgh @ Heinz Field June 3 – Boston @ Gillette Stadium June 5 – Philadelphia @ Lincoln Financial Field June 7 – New York City @ Citi Field June 10 – Baltimore @ M&T Bank Stadium June 12 – Hershey @ Hersheypark Stadium June 28 – Sunderland, UK @ Stadium of Light June 30 – Cardiff, UK @ Millennium Stadium July 2 – London, UK @ Wembley Stadium July 5 – Manchester, UK @ Emirates Old Trafford July 7 – Glasgow, UK @ Hampden Park July 9 – Dublin, Ireland @ Croke Park July 12 – Dusseldorf, Germany @ Esprit Arena July 14 – Zurich, Switzerland @ Letzigrund July 16 – Amsterdam @ Arena July 18 – Milan, Italy @ Stadio San Siro July 21 – Paris @ Stade de France July 24 – Copenhagen @ Parken July 26 – Stockholm, Sweden @ Friends Arena July 29 – Frankfurt, Germany @ Commerzbank Arena July 31 – Brussels, Belgium @ Roi Boudoin

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Beyonce Adds Dates to Formation Tour

First there was a surprise tour announcement following Beyonce's Super Bowl halftime performance. Now there's the surprise announcement that more dates have been added to the Formation Tour.

By Katie Atkinson

Katie Atkinson

Executive Digital Director, West Coast

First there was a surprise tour announcement following Beyonce ‘s Super Bowl halftime performance . Now there’s the surprise announcement that more dates have been added to the Formation Tour.

Beyonce to Embark on Formation Stadium Tour

Beyonce revealed Wednesday (Feb. 17) that new shows have been added in Chicago and London following sold-out dates over the past week. The new Chicago show (on sale Feb. 25) will be May 28 at Soldier Field, while the London show at Wembley Stadium (on sale Feb. 23) is set for July 3.

How Shaboozey Leveraged a Beyoncé Collaboration Into a Solo Chart Smash

Below, see the full Formation Tour itinerary in North America and Europe — plus which shows have already sold out and when the next tickets go on sale.

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Beyonce Formation Tour 2016 stomps in beautiful rhythm all over Citi Field, NY

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The Beyonce Formation tour took over Citi Field in Queens, N.Y., for two nights starting Tuesday, June 7, 2016. Since April 27, 2016, Beyonce has been touring the nations, performing her hit album “Lemonade.” She first released her album as a visual album on HBO. Upon experiencing the album, her fans became confused as to whether or not she was accusing her husband Jay-Z of cheating, and they wanted to know who exactly was this “Becky.” But some believe the album was not directed at her own relationship but rather at her parents’ relationship.

Regardless of who the album is about, Beyonce keeps us guessing. Critics believe her visual album was pure art. Her album has wowed music and social critics alike. With her poetry, current cultural and political references and the African tribal paint, it sent a message to women, especially Black women, that as women we will go through things that men will never understand, but at the end of the day it’s okay to be independent and strong. She said as much during the brilliant execution of hit after hit on Tuesday.

Her true fans, Bey Hive diehards, and her casual admirers seemed to know every lyric to every song, old or new. Fans wore their Beyonce attire and dressed to impress. Some fans, including myself, bought her overpriced merchandise, to be worn later. Before she came out on stage, DJ Khalid hosted the preshow. He brought out several rappers who were born and raised in New York City and performed with them. Rappers like Remy Ma, Fat Joe and Fabulous performed their latest hit songs. He also brought out other performers like T.I., Travis Scott, Busta Rhymes and French Montana. After DJ Khalid woke everyone up, Beyonce appeared on stage about two hours later and performed. The fans, including myself, went crazy and sang every word to every song she performed. It was also Prince’s birthday, so she dedicated some of her onstage time to performing his song “Purple Rain” as the audience soaked in the moment, bathed in a purple hue.

Overall, the chatter as folks made their way home on packed trains was that her show was brilliant. Everyone was excited by her selection, the grand set, her fabulous costume changes and tight dance routines. Beyonce and her Formation squad of approximately 20 dancers were excellent. Everyone left the field beaming, agreeing loudly that Beyonce is a great entertainer as they gently pushed to get on the after-midnight trains home.

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How To Get Beyoncé Tickets In 2024

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Waiting for an announcement that Beyoncé will be touring to promote her Cowboy Carter album while you play it on repeat? If so, you’re not alone; the album has surpassed one billion streams on Spotify. Even though a tour has not yet been announced, there are rumors and speculation aplenty which are keeping us hopeful. Well be updating this article often, so check back for the latest information—we can all “Countdown” together.

Beyoncé onstage last year during her “Renaissance” tour in Amsterdam. She's not touring right now, ... [+] but here's the scoop on when Beyoncé might perform live again.

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The 8 best running hats that deliver breathable, sweat-wicking shade, where to buy tickets to beyoncé’s “cowboy carter” tour.

We won’t get any specific details about ticketing until a Cowboy Carter tour is officially announced. But here’s a certainty: Beyoncé’s Ticketmaster page is a good place to start. And when tickets are available, they will probably sell out quickly, so you might need to turn to a reseller such as StubHub , VividSeats or TickPick instead. Some outlets—like TickPick—let you sign up for updates so you can be notified as soon as Beyoncé tickets are available.

When Do Beyoncé Tickets Go On Sale?

It’s been less than a year since the Renaissance tour ended, so it might seem premature to be wondering about how to get Beyoncé tickets. Is it, though? Her own mother, Tina Knowles, didn’t say no when she was asked if she knew anything about an upcoming Cowboy Carter tour. She just smiled and said that the interviewer would have to ask Beyoncé herself. That’s not a no, right?

Additionally, The Dream, one of Bey’s friends who also worked on the Cowboy Carter album, posted on X in March, “So proud! BB / Let’s Go / This tour bout to B’ Incredible!” with a photo of Beyoncé in Western gear. It was deleted swiftly, but not before fans grabbed screenshots.

That’s not all. Amari Marshall, Beyoncé’s co-dance captain, was asked about an upcoming Cowboy Carter tour in a recent interview. She also didn’t say no, but emphasized that she can’t talk about it, answering, “[Beyoncé] knows I don’t play about her. I don’t know nothing.” The Queen herself has fueled anticipation and rumors by posting videos on her Instagram account of what could be construed as tour vehicles, emblazoned with the words “Cowboy Carter.”

Even with no tour announcement, Queen Bey can’t stop making news, as her name was officially included in a French dictionary, Petit Larousse Illustré, in early May. Her entry reads, “American singer of R&B and pop.” The book might need to amend that by including “country” in her definition, after the incredible success of Cowboy Carter .

If you type “CowboyCarterWorldTour.com” into a web browser, you’ll find that it automatically redirects to Beyoncé’s Ticketmaster page . The page features a header photo showing the singer on a horse, wearing a silver cowboy hat. But there’s a twist: The owner of the X (formerly Twitter) account @CowboyCarterWT —an unofficial fan account— has posted that she is the owner of that site, too, so it isn’t evidence that Beyoncé plans to tour soon.

But Bey has debuted a real, authorized website: BeenCountry . It displays three photos, one of a ticket stub to her last tour stamped with “sold out.” Blink and you miss it, but in teeny tiny letters in the lower left hand corner, it says Join . Click there, and you’re on a page that says act ii and Cowboy Carter , with a field to submit an email address and phone number to sign up for latest info and updates.

It seems reasonable that the first announcement of a tour will arrive there, along with info about how to get Beyoncé tickets. But you can always check back here as well, since we’ll be updating this article as soon as we know when Beyoncé tickets go on sale.

How Much Are Beyoncé Tickets?

Until they are on sale, it’s really hard to say how much Beyoncé tickets will cost. But if the Renaissance tour is any indication—and it probably is—prices will likely start at about $150 for the least expensive seats, while more premium seats could sell for $400 to $500 each. As with any tour, prices will vary based on geography and where the seat is in relation to the stage.

Ironically, some members of the Beyhive are hoping she holds off , at least for a little while. There have been a multitude of posts on every social media platform from fans complaining that they can’t handle another big expense right now. Some have even been requesting that Beyoncé hold off and not tour until next year, since they can’t afford pricey concert tickets right now. Some say that they haven’t yet recovered from the expense of the Renaissance tour. We suspect they’re crying “Alligator Tears,” though, and they’re as anxious as we are to find out how to get Beyoncé tickets.

Beyoncé’s “Cowboy Carter” 2024 Tour Dates

We will “Ring the Alarm” here as soon as we know the Cowboy Carter tour dates and how to get Beyoncé tickets. Check back soon.

What Is The Best Way To Get Beyoncé Tickets?

When Beyoncé took her Renaissance tour on the road, Ticketmaster employed a lottery for the release of tickets. Verified fans were able to sign up and were given access codes, until all tickets released had been sold. After that, fans could sign up on waitlists to be notified if more tickets were released.

We expect something similar to happen for the Cowboy Carter tour, so get in “Formation” and keep your eye on Beyoncé’s Ticketmaster page for the opportunity to sign up.

How Much Do Beyoncé Tickets Usually Cost?

Most relatively decent seats to Beyoncé’s Renaissance tour initially cost around $500. Once tickets became available on broker sites, prices varied wildly. Expect to see ticket prices reach into the thousands for the best seats in big cities, as well as great deals on cheaper tickets, especially at the last minute before shows.

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Nicki Minaj is released after Amsterdam arrest for allegedly 'carrying drugs': Reports

Nicki Minaj has apparently been released after being arrested in Amsterdam on Saturday, according to reports by multiple outlets. Footage from an Instagram Live by Minaj showed her exchange with an officer. It was then recorded and posted on X by various fan accounts.

Multiple reports state that Minaj's release was seemingly confirmed by Amsterdam police in a post on the agency's X account in Dutch although her name was not mentioned. In English it reads: "We have just released a 41-year-old American woman who we arrested this afternoon at Schiphol on suspicion of exporting soft drugs. After consultation with the Public Prosecution Service, the suspect was fined and can continue her journey."

The 12-time Grammy nominee appeared to be detained after a video, lasting over three minutes, shows the rapper discussing her apparent arrest with an Amsterdam police officer, who said the star is being detained "because you're carrying drugs" after she asked why she was being arrested.

The "Anaconda" rapper responded by saying "I'm not carrying drugs." Minaj continued the conversation by telling the officer "I need a lawyer present" and "no, I need a lawyer present now" when the officer told her to get in a vehicle. The officer then appeared to say, "we'll get (you to) next show." Minaj responded and said, "you're talking about my show? What about it?" according to an apparent Instagram Live posted from the rapper's own page.

USA TODAY has reached out to reps for Minaj. In a emailed statement to USA TODAY, Amsterdam Police said "we cannot provide information on persons older than 18 years. This is because of the data privacy law. Therefore, we cannot confirm anything."

Need a break? Play the USA TODAY Daily Crossword Puzzle.

Minaj was slated to perform in Manchester, England on Saturday, according to her official tour website. But that has now been postponed due to the incident, according to reports.

Arrest follows X posts claiming Amsterdam airport security was trying to 'sabotage' her world tour

The star first made noise earlier Saturday before her alleged arrest after claiming apparent Amsterdam Airport Schiphol officials were trying to "sabotage" her tour.

In a lengthy series of X posts, she took aim at the airport saying, "they've been trying everything they can to TRY to stop this tour" and adding, "they took my luggage & when I asked where it is they said it's on the plane. It couldn't have been, I just pulled up." Minaj added that "this is how they plant things on your luggage."

The rap superstar is currently on her Pink Friday 2 World Tour and was en route to Manchester, her next tour stop, when the incident took place.

She posted a nearly one-minute-long video of herself discussing the luggage issue with an airport official, who appeared to be an apparent airport security guard that said they want to "open" her bags. Later, Minaj posted that "they said they found weed." As she noted in a previous X post, marijuana is legal in Amsterdam .

Minaj explained that the alleged luggage issue was happening because "they" try to make her book a new plane "every time" and added that she "fired mngmnt who I found out for years were adding on 30-50K on my jet & pocketing it." Minaj also said she fired a tour manager recently for the same thing and continued, saying "their goal is to make me late, & to pocket 40K."

"They’re being paid big money to try to sabotage my tour b/c soooooo many ppl are mad that it’s this successful & they can’t eat off me. They got caught stealing money from my travel/jets. Got fired. Got mad. Etc," Minaj wrote on X.

Minaj also reposted a post on X by a fan suggesting that the bag checks are being done to further sabotage the star.

"The odds that this happens before an international flight right after Nickis announcement for the second leg of her US tour is suspect. The opps are really out here. There is not one single person that can convince me otherwise. The proof is in the second bag check," the user said.

Nicki Minaj airline, airport posts on X follow Megan Thee Stallion rant from earlier this year

In addition to this spat with airport officials, Minaj is known for her public feuds with other female rappers including Megan Thee Stallion and Cardi B. Earlier this year, she lashed out at Megan Thee Stallion in a series of tweets and a diss track, "Big Foot."

"Swearing on your dead mother when you lie," Minaj  said in one line . "Cause she was lying on your dead momma."

Megan Thee Stallion's Hiss hits No. 1 on Billboard Hot 100 amid Nicki Minaj feud

She also told Megan Thee Stallion she "fell off, I said 'get up on your good foot," referencing her Houston-born rival's 2020 shooting by Canadian rapper Tory Lanez. Later on the track, Minaj raps "like a bodybuilder I keep raisin' the bar" before rhyming and questioning "you get shot with no scar?"

Nicki Minaj's diss track and on X came after her rap peer returned to music with hit single "Hiss" (which followed the similarly reptilian track "Cobra"), rapping that people "don't be mad at Megan," but they're "mad at Megan's Law." Minaj's husband, Kenneth Petty, and her brother, Jelani Maraj, are registered sex offenders.

A development path for teachers in higher education

That is the question that underpinned the VU-CTL development path; a visual representation of the professionalisation pathways teachers can follow at VU-CTL. The VU-CTL development path enables lecturers to choose professionalisation paths that fit their situation and learning needs, and to plan and oversee these paths in the broad perspective of their continuous development. How does the development path work? Each honeycomb represents a path that can be followed independently. The path starts with a Start-to-teach day for all beginner teachers. After this introduction, usually the University Teaching Qualification (UTQ/BKO) follows, unless teachers are hired for a limited teaching task - as mentor or supervisor of thesis or internship. After obtaining the UTQ/BKO, teachers can deepen their knowledge and choose to specialise, for example in the field of assessment, or to deepen their knowledge in a broader sense. Teachers decide for themselves which honeycombs they want to pursue. This allows them to choose offerings that match their current learning needs. On the website ctl-vu.nl , teachers can consult a current, interactive version of the development path and select examples of possible development paths. White paper ‘A development path for lecturers in higher education’ Curious about how the VU-CTL development path came about? And on which didactic principles it is based? In the white paper ‘ A development path for teachers in higher education'(.pdf) you can read more about the didactics within the development path, the underlying vision on teaching and our plans for the future. Are you involved in a CTL/TLC in formation, the development of a CTL or do you have ideas from another field? Contact us and we are happy to talk with you: [email protected] .

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Do you want to ask a question? Please contact Carin Weitering, Higher Education Student Affairs, at [email protected] . You can also call us on +31 (0)20 598 4263 .

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• Media Centre Emirates

Emirates, IATA et Airbus lancent un programme inédit de formation des pilotes pour la qualification de type A350

Dubaï et Paris, 6 juin 2024 : Emirates, l'Association du transport aérien international (IATA) et Airbus ont uni leurs forces pour créer un programme amélioré de formation et d'évaluation basé sur les compétences ( Competency-Based Training and Assessment - CBTA) pour la qualification de type A350, alors que la compagnie aérienne se prépare à la livraison de 65 A350 à partir de septembre 2024. Un premier groupe de 256 pilotes sera formé dans le cadre de ce nouveau cursus au Training college d'Emirates, à Dubaï, à partir de juillet 2024.

Le programme de formation avancée des pilotes utilise la méthodologie CBTA qui associe les directives de l'IATA à l'expertise et aux ressources opérationnelles d'Emirates, ainsi qu'à l'expérience de formation CBTA d'Airbus sur l'A350, qui s'étend sur plus de 10 ans.

Axé sur les compétences et le comportement des pilotes, le programme CBTA A350 utilise la philosophie de formation CBTA éprouvée qui permet aux instructeurs d'évaluer la formation des pilotes et de rechercher des améliorations immédiates de leurs performances. Le programme se déroulera en deux phases et comprendra 20 jours de formation sur simulateur et d'évaluation, répartis en 15 sessions distinctes.

“ Avec ce programme, les pilotes d'Emirates vont recevoir une formation interne basée sur les compétences avec des instructeurs hautement qualifiés, les dotant ainsi de très fortes capacités pour soutenir les opérations mondiales de la compagnie aérienne. Le programme CBTA adapté à l'A350 soutient l'intégration du nouvel appareil dans notre flotte, 1 000 pilotes devant suivre le cours de qualification de type A350. Cette dernière initiative s'inscrit dans le cadre de l'engagement d'Emirates à offrir les normes de service et de confort les plus élevées, tout en soutenant notre croissance opérationnelle et notre expansion ”, a déclaré le capitaine Bader Al Marzooqi, vice-président principal de la formation au pilotage d'Emirates.

“ Combiner l'expertise d'Emirates, d'Airbus et de l'IATA pour concevoir et dispenser la formation à la qualification de type A350 est une opportunité unique. Notre objectif commun est d'exploiter pleinement les avantages de la CBTA pour qualifier les pilotes de l'A350 de la manière la plus efficace possible. En le faisant conjointement, les trois organisations acquerront également une expérience précieuse qui pourra renforcer leurs autres activités de formation ”, a déclaré Nick Careen, vice-président principal de l'IATA pour les opérations, la sécurité et la sûreté.

“ L'A350 est un avion de pointe qui nécessite des solutions de formation tout aussi avancées. Notre partenariat avec l'IATA et Emirates garantit que les pilotes d'Emirates reçoivent la formation la plus complète et la plus efficace, ce qui contribuera à une mise en service de l'A350 dans le monde entier, dans des conditions d’excellence ”, a déclaré le capitaine Stéphan Labrucherie, responsable mondial de la formation aéronautique chez Airbus.

Le programme de qualification de type de l'A350 est l'un des moyens utilisés par Emirates pour préparer l'entrée en service de son nouvel appareil dans sa flotte. Outre la formation des pilotes, d'autres services de la compagnie travaillent également à la préparation de l'entrée en service de l'A350, notamment les équipes chargées de la prestation de services et de l'ingénierie. 

CBTA et IATA

Bénéficier des avantages et de l'efficacité de la formation CBTA est un objectif de longue date de l'industrie. Pour le personnel naviguant, l'IATA soutient cet objectif avec son guide CBTA pour la formation des équipages de bord, qui est entièrement aligné sur les normes CBTA de l'OACI (Organisation de l'aviation civile internationale) et comprend des bibliothèques spécifiques pour les compagnies aériennes/opérateurs et les organismes de formation.

L'IATA met également l'accent sur la formation CBTA dans le cadre de la réglementation de l'IATA sur les marchandises dangereuses . Le Centre CBTA de l'IATA soutient les organisations de l'industrie aéronautique – y compris les opérateurs, les autorités de l'aviation civile et les organismes de formation – dans le développement des capacités et des ressources pour les programmes de formation sur les marchandises dangereuses. Il propose également des formations CBTA spécialisées pour diverses fonctions des compagnies aériennes, depuis les agents d'escale jusqu'aux agents de service aux passagers, en passant par les équipes de planification des chargements, le personnel de bord, etc.

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L'histoire d'Emirates débute en 1985 lorsque nous avons lancé nos opérations avec seulement deux avions. Aujourd'hui, nous opérons la plus grande flotte au monde d'Airbus A380 et Boeing 777, offrant à nos clients le confort des gros porteurs les plus récents et les plus efficaces du ciel.

A travers le monde, nous inspirons les voyageurs avec notre réseau croissant de destinations internationales, nos divertissements à bord de premier ordre, notre cuisine d'inspiration régionale, et notre service de classe mondiale.

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• Open access
• Published: 04 June 2024

Alpha-6 integrin deletion delays the formation of Brca1/p53-deficient basal-like breast tumors by restricting luminal progenitor cell expansion

• Marisa M. Faraldo 1 ,
• Mathilde Romagnoli 2 , 3 ,
• Loane Wallon 1 , 4 ,
• Pierre Dubus 5 , 6 ,
• Marie-Ange Deugnier 2 &
• Silvia Fre 1  

Breast Cancer Research volume  26 , Article number:  91 ( 2024 ) Cite this article

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The aberrant amplification of mammary luminal progenitors is at the origin of basal-like breast cancers associated with BRCA1 mutations. Integrins mediate cell–matrix adhesion and transmit mechanical and chemical signals that drive epithelial stem cell functions and regulate tumor progression, metastatic reactivation, and resistance to targeted therapies. Consistently, we have recently shown that laminin-binding integrins are essential for the expansion and differentiation of mammary luminal progenitors in physiological conditions. As over-expression of the laminin-binding α6 integrin (Itgα6) is associated with poor prognosis and reduced survival in breast cancer, we here investigate the role of Itgα6 in mammary tumorigenesis.

We used Blg-Cre; Brca1 F/F ; Trp53 F/F mice, a model that phenocopies human basal-like breast cancer with BRCA1 mutations. We generated mutant mice proficient or deficient in Itgα6 expression and followed tumor formation. Mammary tumors and pretumoral tissues were characterized by immunohistochemistry, flow cytometry, RT-qPCR, Western blotting and organoid cultures. Clonogenicity of luminal progenitors from preneoplastic glands was studied in 3D Matrigel cultures.

We show that Itga6 deletion favors activation of p16 cell cycle inhibitor in the preneoplastic tissue. Subsequently, the amplification of luminal progenitors, the cell of origin of Brca1-deficient tumors, is restrained in Itgα6-deficient gland. In addition, the partial EMT program operating in Brca1/p53-deficient epithelium is attenuated in the absence of Itgα6. As a consequence of these events, mammary tumor formation is delayed in Itgα6-deficient mice. After tumor formation, the lack of Itgα6 does not affect tumor growth but rather alters their differentiation, resulting in reduced expression of basal cell markers.

Conclusions

Our data indicate that Itgα6 has a pro-tumorigenic role in Blg-Cre; Brca1 F/F ; Trp53 F/F mice developing basal-like mammary tumors. In particular, we reveal that Itgα6 is required for the luminal progenitor expansion and the aberrant partial EMT program that precedes the formation of BRCA1 deficient tumors.

The mammary gland undergoes two major morphogenetic events postnatally: the elongation and branching of a ductal tree during puberty and the expansion of the mammary epithelium at pregnancy, accompanied by the generation of secretory alveoli, the milk-secreting units [ 1 ]. The regulation of the different steps of postnatal mammary morphogenesis and differentiation is a complex process involving the action of systemic hormones and growth factors [ 2 , 3 ]. In addition, interaction of epithelial cells with the extracellular matrix (ECM) is essential for mammary gland development and function by modulating cellular responses to soluble factors [ 4 , 5 , 6 ].

The mammary epithelium is organized as a bilayer, with a layer of luminal cells lining the ductal or alveolar lumen, and a surrounding layer of basal cells. From mid-pregnancy and during lactation, the luminal cells produce and secrete milk in response to hormone stimulation. The basal (also called myoepithelial) cells express the basal-specific pair of cytokeratins 5/14 (K5 and K14), the transcription factors p63 and Slug, and smooth muscle (SM) contractile proteins like α-SM-actin and SM-myosin. The luminal cells specifically express the cytokeratins 8/18 (K8 and K18). They include a subset of cells expressing the receptors for estrogen (ER) and progesterone (PR) that act as hormone sensors through the production of paracrine signals regulating basal and luminal cell function, and a population devoid of hormone receptor expression (ER/PR-), comprising the luminal progenitors at the origin of the expansion of the mammary epithelium during pregnancy [ 2 ]. In recent years, using lineage tracing techniques, numerous studies have revealed that the different mammary lineages originate from embryonic multipotent stem cells. In the postnatal gland, at homeostasis, the three mammary lineages, basal, luminal ER/PR + and luminal ER/PR-, are essentially maintained by their own lineage-restricted unipotent stem/progenitor cells (for review see [ 1 , 7 ]).

Human breast cancer can be classified into six main molecular subtypes based on transcriptional signatures: luminal A, luminal B, HER2-enriched, normal-like, claudin-low and basal-like [ 8 , 9 , 10 ]. The basal-like tumors represent 15–20% of all breast cancers and are characterized by the expression of basal markers such as K5/K14 and p63. They belong to the so-called triple-negative tumors that lack ER and PR and display low levels of HER2 receptor, making them resistant to hormonal or HER2 targeted therapies [ 11 ]. Most basal-like tumors display a loss of p53 function [ 12 ]. Several studies in the last decade suggest that the distinct breast cancer subtypes originate from different populations of stem and progenitor cells in the mammary hierarchy [ 7 ]. Notably, deregulation of luminal ER/PR- progenitors is believed to be at the origin of basal-like breast tumors, particularly those associated with Brca1 mutations [ 13 , 14 ]. Given the absence of lineage inter-conversion during normal tissue homeostasis, this would imply a switch or a loss of cell identity during tumorigenesis. Accordingly, Brca1 germline mutation has been shown to alter the fate of mammary luminal cells and cause luminal-to-basal transformation [ 15 , 16 ]. Furthermore, Brca1 loss induces the expression of EMT (epithelial-to-mesenchymal transition) related genes, which are associated with the expansion of cancer stem cells and the formation of basal-like tumors in mice [ 17 ].

Among the adhesion systems contributing to the maintenance of the mammary epithelial bilayer, integrins constitute the main cell surface receptors to ECM. They connect the matrix network to the cytoskeleton and trigger biochemical and mechanical signals that, in coordination to soluble factors, control important cell functions [ 18 ]. Integrins are heterodimers, composed of an α and a β subunit, and 24 integrin dimers with distinct substrate specificities have been described [ 19 ]. Integrins are present in both basal and luminal cells, at all stages of mammary development, and numerous studies have reported their essential roles in controlling mammary cell growth and differentiation [ 4 , 20 , 21 ]. Furthermore, integrin signaling is often deregulated in cancer, affecting the ability of tumor cells to proliferate without control, to become invasive or to survive in adverse conditions [ 18 ].

The whole mammary epithelium is surrounded by a specialized ECM, the basement membrane (BM). Laminins are major components of the BM, and the laminin-binding integrin dimers (α3β1, α6β1 and α6β4) are highly expressed in mammary epithelial cells [ 22 ]. We have recently reported that laminin-binding integrins have essential roles in the regulation of stem/progenitor cell function during normal mammary gland development [ 22 , 23 ]. Importantly, α6-integrins (that is, the dimers containing α6-subunit) are involved in the progression of some cancers and in the regulation of normal and cancer stem cells [ 24 ]. Here, we explore the role of the α6-integrin in mammary tumorigenesis, using an established mouse model of basal-like cancer, the Blg-Cre;Brca1 F/F ;Trp53 F/F mice, that develop invasive tumors phenocopying BRCA1-deficient human breast tumors at the molecular and morphological level [ 14 ]. Our results reveal that lack of α6-integrin (Itgα6) in luminal progenitors delays the induction of the EMT program that takes place in the early steps of tumorigenesis caused by Brca1 deficiency [ 15 , 17 ]. Concomitantly, the absence of Itgα6 favors the activation of the p16 cell cycle inhibitor, thereby limiting luminal progenitor expansion in the preneoplastic epithelium. As a result of these alterations, tumor initiation is delayed in Itgα6-deficient Brca1/p53 mutant mice. These results indicate that α6-integrins have a pro-tumorigenic role in basal-like breast cancer and suggest its potential use to dissect the initial steps involved in the establishment of this highly malignant type of breast cancer.

The generation of Brca1 F/F , Trp53 F/F and Itga6 F/F mice has been previously described [ 25 , 26 , 27 ]. BlgCre transgenic mice were purchased from The Jackson Laboratory. Mice were bred in a mixed 129SV/C57BL6 genetic background. In all experiments, unless specified, BlgCre -negative littermates were used as controls. Adult females were assessed by palpation twice a week to monitor tumor appearance. For growth curves, after the first palpation, tumor diameters were measured every 2–3 days and used to calculate tumor volume [ 28 ]. Animals were analyzed before the overall tumor burden reached the maximal permitted by the ethics committee (1500 mm 3 ). For necrosis analysis, only tumors larger than 1200 mm 3 were used. Husbandry, supply of animals, as well as maintenance and care in the Animal Facility of Institut Curie (facility license #C75-05-18) before and during experiments fully satisfied the animal’s needs and welfare. All mice were housed and bred in a specific-pathogen-free (SPF) barrier facility with a 12:12 h light–dark cycle and food and water available ad libitum. Mice were sacrificed by cervical dislocation.

Whole-mount analyses, histology and immunolabeling

For whole-mount Carmine-Alum staining, mammary fat pads were spread onto glass slides, fixed overnight in Methacarn (methanol:chloroform:acetic acid in 6:3:1 proportion) and stained with carmine as previously described [ 29 ]. Whole-mount images were acquired with a Leica DFZ420C color video camera in a Leica MZ8 binocular using the LAS (Leica Applications Suite) software. For histological analysis, mammary tissues (tumors or glands) were fixed in 4% paraformaldehyde (PFA) overnight at 4 °C and embedded in paraffin. Sections of 5–7 µm were cut and de-waxed for hematoxylin/eosin staining or immunolabeling. Antigen retrieval was performed by incubating sections in 10 mM citrate buffer pH 5 at 98 °C for 10 min. For cryosections, after fixation in 4% PFA as previously, tumors were incubated in 30% sucrose at 4 °C for 48 h, then frozen on Tissu-Tek (Sakura) and 5–7 µm sections were obtained using a Leica LM1950 cryostat. Sections were incubated with primary antibodies overnight at 4 °C in a humidified chamber then with secondary Alexa-fluor conjugated antibodies and 1 μM DAPI (Sigma) for one hour at room temperature. Finally, sections were mounted in Poly-mount medium (Polysciences), and pictures were obtained in an Upright Spinning Disk Confocal microscope (Roper/Zeiss) with a CoolSnap HQ2 camera. Details of the antibodies used are provided in Additional File 5 : Table S1 .

FACS analysis and primary mammary cell preparation

Thoracic and inguinal mammary glands from single virgin females were pooled, dissociated and processed for single-cell suspension and flow cytometry as described elsewhere [ 30 , 31 ]. Tumors of 1000–1200 mm 3 were used for dissociation, following the same procedure. For cell sorting, cells were incubated at 4 °C for 20 min with the following antibodies: anti-CD45-APC (clone 30-F11), anti-CD31-APC (clone MEC13.3), anti-mouse Ter119-APC, anti-CD24-PE (clone M1/69), anti-CD49f-APC/Cy7 (clone GoH3), anti-ICAM1-PE/Cy7 (clone YN1/1.7.4), anti-CD29-PE/Cy7 (clone HMb1-1) and anti-CD104-PE/Cy7 (clone 346-11A) all from Biolegend. Labelled cells were analyzed and sorted out using a MoFlo Astrios (Beckman Coulter) or a FACSAria Fusion (BD Biosciences) cell sorters. Sorted luminal progenitor cells (CD31/CD45/Ter119 − ) CD24 high CD49f low ICAM + population) were used for single-cell organoid assays and gene and protein expression analysis. Data were analyzed using FlowJo software (v10.10.0).

Organoid culture and immunostaining

Mammary organoids were derived from fourth-fifth mammary fat pads or tumors as previously described [ 32 ]. Briefly, mammary fat pads were minced with scalpels to approximately 1 mm 3 pieces and digested by incubation for 1 h at 37 °C with 3 mg/ml of collagenase A (Roche) and 1.5 mg/ml of pancreatic trypsin (Sigma) in serum-free Leibowitz L15 medium (Gibco). To eliminate residual single cells such as fibroblast or other stromal cells, four consecutive differential centrifugations at low speed (450 g for 10 s) were performed. The final organoid pellet was mixed with growth-factor reduced Matrigel (BD Biosciences) and seeded in 8-well coverslip bottom chambers (Ibidi) for immunostaining or in 24-well plates for passaging. A suspension containing 50–100 organoids in 30 µl of Matrigel was added to each well. Organoids were cultured for 10–15 days in DMEM/F12 medium containing penicillin/streptomycin, Glutamax, 10 mM HEPES, N2, B27, 100 ng/ml Nrg1 (R&D) 100 ng/ml Noggin (Peprotech/Thermo Fisher Scientific) and 100 ng/ml R-spondin 1 (R&D). The medium was replaced twice a week. To replate organoids, Matrigel was mechanically disrupted by pipetting up and down in ice-cold PBS with a P1000 pipette. After centrifugation at 450 g for 5 min, organoid pellets were resuspended in fresh Matrigel and plated as described above. For single cell organoids, 15,000 sorted cells were resuspended in a 20 µl drop of Matrigel and cultured for 12–14 days in 24-well plates as described above.

For immunostaining, organoids were fixed in 4% PFA at RT for 1 h and permeabilized with 1% Triton-X-100. Non-specific epitopes were blocked by incubating organoids in 2% BSA, 5% FBS and 0.25% Triton-X-100 for 1 h. Antibodies were diluted in blocking solution and incubated overnight (primary antibodies) and 5 h (secondary antibodies) at RT. For EdU incorporation assays, 10 µM EdU was added to the organoids 2 h before fixation. EdU was revealed after organoid immunostaining using a Click-iT EdU Imaging kit (Invitrogen) following manufacturer’s instructions. After staining, organoids were kept in 50% glycerol in PBS at 4 °C until imaging using an Inverted Spinning Disk Wide Confocal Microscope CSU-W1 (Roper/Nikon/Gattaca) and a sCMOS BSI camera (Nikon).

RNA extraction and RT-qPCR

Total RNA was isolated from sorted cells using RNeasy Microkit (Qiagen). To avoid eventual DNA contamination, purified RNA was treated with RNAse-free DNAse (Qiagen). RNAs were reverse-transcribed using SuperScript IV Vilo (Thermo Fisher Scientific). Quantitative PCR was performed using the QuantiNova SYBR Green PCR Kit (Qiagen) on a LightCycler 480 real-time PCR system (Roche). The values obtained were normalized to Gapdh levels. The primers used for RT-qPCR analysis were purchased from SABiosciences/Qiagen or designed using Oligo 6.8 software (Molecular biology Insights) and synthesized by Eurogentec. Primers used in this study are listed in Additional File 6 : Table S2 .

Western blot analysis

Sorted cell pellets were resuspended in hot 1.5 × Laemli buffer, vortexed and boiled for 5 min. Samples were run on NuPAGE Novex 4–12% Bis Tris gels (Life Technologies/Invitrogen) and transferred onto nitrocellulose. Membranes were incubated with 5% BSA in TBS containing 0.1% Tween 20 (TBST) for 1 h at room temperature and with primary antibodies overnight at 4 °C. The primary antibodies used are indicated in Additional File 5 : Table S1 . Secondary antibodies coupled to horseradish peroxidase were from Cell Signalling Technology. Detection was performed by chemiluminiscence (Super signal West Pico+, Pierce) using a ChemiDoc MP imaging system (Bio-Rad).

In silico gene expression analysis

Illumina RNAseq gene expression data of 1247 breast cancer cases in TCGA cohorts classified according to PAM50 signature were downloaded from Xena Browser ( https://xenabrowser.net ). mRNA expression levels of ITGA6 and ITGA3 were imported as normalized log 2 values and visualized as box and whisker plots using Prism (GraphPad) v10.2.2.

Statistical analysis of the data

At least n = 3 animals were used for each experiment. Statistical tests and further graphs were prepared in Prism (GraphPad) v10.2.2. For survival curves, a long-rank (Mantel-Cox) test was used. All graphs show mean ± standard error of the mean (SEM). Except when specifically indicated, differences between groups were assessed with Student’s t test with two-tailed distribution and unequal variance (Welch’s correction). The significance threshold was p  < 0.05. * indicates p  < 0.05, ** indicates p  < 0.01, *** indicates p  < 0.001, and **** indicates p  < 0.0001.

Tumor formation is delayed in basal-like Brca1/p53-deficient tumors lacking Itga6

We have recently shown that laminin-binding integrins (containing the α3- and α6- integrins subunits, encoded by the Itga3 and Itga6 genes, respectively) are essential for the regulation of mammary stem/progenitor cell function and for mammary development during pregnancy and lactation [ 22 , 23 ]. To study their role in tumorigenesis, we first interrogated the TCGA database comprising the main molecular subtypes of breast cancer, for the expression of ITGA3 and ITGA6 transcripts. Interestingly, ITGA6 levels were significantly higher in basal-like tumors when compared with the other breast cancer subtypes, while ITGA3 levels were lower in this group (Fig.  1 A, Additional File 1 : Fig. S1 A). This prompted us to investigate whether Itgα6 plays a role in the formation of basal-like breast cancer. To this purpose, we used Blg-Cre; Brca1 F/F ;Trp53 F/F mice (hereafter referred to as Brca1p53-KO), an established mouse model of basal-like cancer closely resembling BRCA1-deficient human tumors [ 14 ]. In these mice, the Blg (beta-lactoglobulin) promoter targets Cre recombinase expression to the ER/PR- luminal progenitor population, inducing the specific Brca1 and Trp53 gene deletion in these cells [ 14 , 23 ]. Brca1p53-KO mice were crossed with Itga6 F/F mice, to obtain Blg-cre;Brca1 F/F ; Trp53 F/F ;Itga6 F/WT and Blg-cre;Brca1 F/F ;Trp53 F/F ;Itga6 F/F mice (hereafter referred to as α6 ± ;Brca1p53-KO and α6KO/Brca1p53-KO, respectively) and cohorts of mice of the three genotypes were monitored for tumor formation (Fig.  1 B). All mice developed mammary tumors within a period of 13.5 months. Brca1p53-KO females developed palpable mammary tumors with a mean latency of 7.2 months. A slight, non-statistically significant delay in tumor onset was observed in α6 ± ; Brca1p53-KO females (mean latency of 8.3 months). Strikingly, homozygous Itga6 deficiency induced a significant delay in tumor formation, with a mean latency of 9 months in α6KO/Brca1p53-KO mice (Fig.  1 B). Furthermore, the number of tumors per animal was not affected in a6 ± ; Brca1p53-KO but was significantly reduced in α6KO/Brca1p53-KO females, when compared to Brca1p53-KO females (Fig.  1 C). The α6KO/Brca1p53-KO (α6-deficient) females were then used for further analysis and compared to female mice expressing normal levels of Itgα6 throughout the study. Small (< 5 mm diameter) α6KO/Brca1p53-KO mammary tumors had histological characteristics comparable to Brca1p53-KO small tumors and were mainly classified as invasive ductal carcinoma of non-special type (IDC-NST) (Fig.  1 D). In larger tumors (≥ 1200 mm 3 ), extended or multifocal necrotic areas were evidenced in half of Brca1p53-KO tumors but not in α6KO/Brca1p53-KO tumors (Additional File 1 : Fig. S1 B). In both cohorts, the tumors were mostly positive for the luminal marker Keratin 8 (K8) but devoid of progesterone receptor (PR) as previously described for Brca1p53-KO tumors (Fig.  1 E; [ 14 , 25 ]). PR was, however, detected in normal-looking ducts adjacent to the tumors, as well as in tumor-free mammary tissues (Fig.  1 E, Additional File 1 : Fig. S1 C). The absence of the Itgα6 subunit in the majority of tumor cells of α6KO/Brca1p53-KO mice was confirmed by immunostaining (Additional File 1 : Fig. S1 D). As we previously reported in normal mammary glands of Blg-cre; Itga6 F/F mice, most α6-deficient tumor cells also lack β4 integrin at the membrane (Additional File 1 : Fig. S1 D; [ 23 ]).

Tumor formation is delayed in basal-like Brca1/p53-deficient tumors lacking Itga6 . A In silico analysis of ITGA6 mRNA expression in human breast cancer subtypes classified by the PAM50 signature: Normal-like (n = 8), Luminal A (n = 231), Luminal B (n = 127), HER2-enriched (n = 58), and basal-like (n = 98). B Kaplan–Meier tumor-free survival curve of Brca1p53-KO (n = 56) α6 ± ;Brca1p53-KO (n = 19) and α6KO/Brca1p53-KO (n = 44) females. P  = 0.13 (Brca1p53-KO vs. α6 ± ;Brca1p53-KO); P  = 0.0001 (Brca1p53-KO vs. α6KO/Brca1p53-KO); P  = 0.001 (α6 ± ;Brca1p53-KO vs. α6KO/Brca1p53-KO). C Number of mammary tumors per mouse in Brca1p53-KO (n = 55) α6 ± ;Brca1p53-KO (n = 11) and α6KO/Brca1p53-KO (n = 43) females. D, E Histological analysis of Brca1p53-KO and α6KO/Brca1p53-KO tumors. D Hematoxylin/eosin staining. E Immunofluorescent staining with anti-PR (red), anti-K8 (white) and anti α-SMA (green) antibodies. Nuclear DAPI staining is shown in blue. In the tumors, α-SMA staining is mostly restricted to tumor fibroblast. White arrows indicate the presence of normal ductal luminal cells positively stained for PR. Scale bar: 100 µm ( D ) and 20 µm ( E ). In A , C : * P  < 0.05, **** P  < 0.0001, n.s., non-significant

These results indicate that the lack of Itgα6 delays the formation of basal-like mammary tumors in Brca1/p53 deficient mice without inducing major changes in their global histopathological phenotype.

Itga6 deletion alters the differentiation of Brca1/p53-deficient tumor cells

To investigate if tumor growth was affected by the lack of Itgα6, we first analyzed cell proliferation by immunostaining with an anti-Ki67 antibody. Similar proliferation rates were found in α6-deficient and α6-proficient tumors, independently of the tumor size (Fig.  2 A). Likewise, tumor organoid cultures presented analogous proliferation rates in both groups, even after several organoid passages, as shown in EdU-incorporation assays (Additional File 2 : Fig. S2 A, B). Integrin activation has been involved in cell survival in different tissues [ 33 ]. We then assessed apoptosis by cleaved-caspase-3 (CC3) staining and found that apoptosis rates were low and only slightly increased in α6KO/Brca1p53-KO tumors (around 2–3% of CC3 + tumor cells; Fig.  2 B). In agreement with these data, systematic measurement of tumor size after initial palpation showed that tumor growth was not significantly affected by the absence of Itga6 (Fig.  2 C).

Effect of Itga6 deletion on growth and differentiation of Brca1/p53-deficient tumors. A Immunofluorescent staining with anti-Itgα6 (white), and anti-Ki67 (red) antibodies. The graph shows the percentage of Ki67 + cells (mean ± SEM) in small (< 750 mm 3 ; n = 8 animals per genotype) and large (> 750 mm 3 ; n = 7 animals per genotype) tumors. B Immunofluorescent staining with anti-K8 (white), and anti-cleaved caspase 3 (CC3, red) antibodies. The graph shows the percentage of CC3 + cells (mean ± SEM) in small (< 750 mm 3 ; n = 4 animals per genotype) and large (≥ 750 mm 3 ; n = 4 animals per genotype) tumors. A and B : Nuclear DAPI staining is shown in blue. Scale bar: 20 µm. C Tumor growth curves of Brca1p53-KO (n = 7) and α6KO/Brca1p53-KO (n = 7) tumors. The difference between groups is not significant (Kruskal–Wallis test). D Upper panels: Representative FACS analysis of a Brca1p53-KO and a α6KO/Brca1p53-KO tumor with CD24 and Lin (CD31/CD45/Ter-119) antibodies. The red rectangle shows the CD24 + Lin − population sorted for gene expression analysis. Lower panels: Representative FACS analysis of tumors with CD24 and CD49f (α6-integrin) antibodies showing depletion of surface Itgα6 in most epithelial cells of the α6KO/Brca1p53-KO tumor. E RT-qPCR analysis of the CD24 + Lin − tumor cell population from Brca1p53-KO (n = 5) and α6KO/Brca1p53-KO (n = 5) animals (mean ± SEM). F Representative immunofluorescent staining with anti-K5 (red) and anti-K8 (white) antibodies. Magnification of the white rectangle is shown in the right panels of Brca1p53-KO and α6KO/Brca1p53-KO tumors. Nuclear DAPI staining is shown in blue. Scale bar: 75 µm (left panels for each tumor) and 20 µm (magnification in the right panels). In A , B , E * P  < 0.05, n.s., non-significant

We subsequently isolated tumor cells (CD24 + Lin − ) by flow cytometry and performed gene expression analyses for deeper characterization (Fig.  2 D, upper panels; Additional File 2 : Fig. S2 C). As previously found by immunostaining of tumor sections (Additional File 1 : Fig. S1 D), flow cytometry and RT-qPCR analysis further confirmed the reduction of Itga6 expression in α6KO/Brca1p53-KO tumor cells (Fig.  2 D, lower panels; Additional File 2 : Fig. S2 D). Trp53 and Brca1 genes, although similarly expressed in the tumor cells of both groups, were significantly down-regulated when compared to wild-type luminal cells (Additional File 2 : Fig. S2 D). Expression of the luminal genes Krt18 , Esr1 and Cdh1 (encoding respectively for cytokeratin 18, estrogen receptor α and E-cadherin) was not significantly changed in α6KO/Brca1p53-KO tumors (Fig.  2 E, Additional File 2: Fig. S2E). Similarly, the level of Tnfsf11 and Tnfrsf11a encoding respectively for the secreted factor RANKL and its receptor, RANK, involved in the oncogenesis of Brca1 mutation-driven breast cancer, was not altered in the absence of Itgα6 (Additional File 2 : Fig. S2 E; [ 34 ]). In contrast, the basal genes Krt5 (encoding for cytokeratin 5) and Snai2 (encoding for the transcription factor Slug) were significantly down-regulated in α6-deficient tumors (Fig.  2 E). Diminished expression of Krt5 was confirmed by immunostaining (Fig.  2 F).

Collectively, these results indicate that lack of Itgα6 does not affect tumor growth but alters the differentiation of Brca1/p53-deficient mammary tumor cells.

Itga6 deletion impairs luminal progenitor cell activity in Brca1/p53-deficient preneoplastic glands

To further characterize the early events leading to the observed delay in tumorigenesis in α6KO/Brca1p53-KO mice, we analyzed the mammary tissue of 4–5 month-old females, before the appearance of tumors (here after referred to as preneoplastic glands). In contrast to wild-type glands, comprising essentially mammary ducts with limited branching, the glands of Brca1p53-KO females were hyper-branched and contained a high number of alveolar-like structures, as assessed by Carmine staining and histological analyses (Fig.  3 A). This phenotype was previously reported in similar Brca1-deficient mouse models [ 35 , 36 ]. However, in α6KO/Brca1p53-KO mice this aberrant mammary phenotype was less pronounced (Fig.  3 A). Consistently, proliferation rates were increased in Brca1p53-KO mammary epithelium but were close to normal in α6-deficient glands (Fig.  3 B).

Analysis of the preneoplastic gland of Brca1/p53-deficient mice. A Representative microphotographs of mammary glands from 5-month-old virgin control, Brca1p53-KO and α6KO/Brca1p53-KO mouse. Upper panels: fragments of glands stained with Carmine in whole-mount. Lower panels: Hematoxylin and Eosin staining of gland sections. Scale bar: 1 mm (upper panels) and 100 µm (lower panels). B Immunofluorescent staining with anti-K8 (white), and anti-Ki67 (red) antibodies. Nuclear DAPI staining is shown in blue. Scale bar: 20 µm. The graph shows the percentage of Ki67 + cells (mean ± SEM) obtained from five animals per group. C, D Representative FACS analysis of mammary glands from 5-months-old virgin mouse. C separation of basal (green) and luminal (orange) populations in control and Brca1/p53-deficient mammary glands. Note that in the α6KO/Brca1p53-KO gland, a fraction of luminal cells are depleted of Itgα6 expression. Right: graphs showing the percentage of luminal and basal cells in the Lin- population, in 7 independent cell sorting experiments (mean ± SEM). D Analysis of ICAM1 expression gated in the luminal cells. Right: graphs showing the percentage of luminal ICAM1 + cells in the luminal gate and the ICAM1 + and ICAM1- in the total MEC population in 7 independent cell sorting experiments (mean ± SEM). E Representative microphotographs of organoids formed by sorted luminal ICAM1 + cells after 12 days of culture. Scale bar: 250 µm. Right: graph showing the number of organoids per well obtained in 4 independent cell sorting experiments. In B - E *P  < 0.05, * *P  < 0.01, n.s., non-significant

Flow cytometry analysis was performed with preneoplastic glands, to separate basal and luminal mammary cells as previously reported [ 30 , 31 ]. Compared to control glands, luminal cells were amplified in Brca1/p53 mutants, and this increase was clearly more pronounced in α6-expressing mice, at the expense of the basal population which was decreased in both mutant mice relative to control glands (Fig.  3 C). In addition, we could confirm that Itgα6 was depleted in a substantial fraction of luminal cells in the preneoplastic tissue of α6KO/Brca1p53-KO mice (Fig.  3 C). Itgα6 chain can bind to β1 or β4 chains to form α6β1 and α6β4 laminin-receptor dimers. In the luminal cells of α6KO/Brca1p53-KO glands, Itgβ4 surface levels were decreased when compared to Brca1p53-KO mice, while basal cells displayed similar Itgβ4 levels in both groups (Additional File 3 : Fig. S3 A). No differences were detected in Itgβ1 expression between both groups in luminal and in basal cells (Additional File 3 : Fig. S3 A).

We have previously reported that in wild-type glands, ICAM1 expression discriminates two luminal cell populations, consisting of highly clonogenic progenitors (ICAM1 + , mostly ER/PR-) and poorly clonogenic, mature (ICAM1-, ER/PR +) luminal cells [ 37 , 38 ]. Similarly, two populations were separated by ICAM1 in Brca1/p53 mutant tissues: ICAM1 + (hereafter referred to as luminal progenitors or LP) and ICAM1- (hereafter referred to as luminal mature or LM) which is enriched in cells expressing the hormone receptor ERα (Fig.  3 D, Additional File 3 : Fig. S3 B). The Blg promoter has been reported to specifically target luminal progenitor cells [ 14 , 23 ] and we confirmed these results in our mice, showing that Cre expression was found predominantly in ICAM1 + LP (Additional File 3 : Fig. S3 C). Accordingly, FACS analysis revealed that, in α6KO/Brca1p53-KO glands, integrin depletion is mostly detected in the ICAM1 + luminal progenitor population and this was confirmed by RT-qPCR analysis (Additional File 3 : Fig. S3 D, E). Furthermore, immuno-staining of α6KO/Brca1p53-KO mammary gland sections showed that the PR + cells retain expression of Itgα6 at their surface, indicating that mature luminal cells are not targeted for integrin deletion by BlgCre (Additional File 3 : Fig. S3 F).

Within the luminal cell population, the proportion of ICAM1 + LP was not significantly changed in Brca1/p53 mutants when compared to control animals, confirming previously reported results in a similar mouse model (Fig.  3 D; [ 14 ]). However, when quantified over the proportion of total mammary epithelial cells (MECs), the percentage of ICAM1 + LP was increased in Brca1p53-KO glands and, of interest, it was rescued to control numbers in α6-deficient glands (Fig.  3 D). Thus, in α6KO/Brca1p53-KO mice, the observed increase in luminal cells (Fig.  3 C), appears to derive from an over-representation of ML cells, that indeed show a trend to increase, although without reaching statistical significance. Gene expression analysis of sorted mutant luminal progenitors confirmed a decrease in Brca1 and Trp53 transcripts compared to control cells, while the expression of known markers of these cells (i.e. Krt18 , Elf5, Csn2 ) was similar to control (Additional File 3 : Fig. S3 E). Furthermore, we cultured sorted luminal progenitors in Matrigel as 3D organoids [ 32 ] and found that their ability to form organoids, was severely impaired upon Itgα6 deletion (Fig.  3 E).

Altogether these results indicate that deletion of Itga6 inhibits the expansion of luminal progenitors induced by Brca1/p53 loss and impairs their clonogenicity during the preneoplastic steps taking place before tumor development.

Itga6 deletion inhibits the ectopic expression of basal/EMT-like genes in Brca1/p53-deficient luminal progenitors

We then explored tumor progression, by analyzing the non-tumoral glands from mice harboring palpable tumors (hereafter referred to as juxta-tumoral glands). At this stage, mammary gland morphology was often perturbed, with hyperplastic areas developing in both Itgα6-deficient and -proficient glands (Fig.  4 A). Consistently, proliferation rates were increased in the glands of both Brca1 mutant groups when compared with the normal epithelium; however, the increase in proliferation was attenuated in α6KO/Brca1p53-KO glands (Fig.  4 B). Enhanced proliferation was also detected in organoids derived from tissue fragments of mutant mice, indicating an epithelium cell-autonomous phenotype (Additional File 4 : Fig. S4 A). Decreased expression of Itgα6 was confirmed in the luminal cells of α6KO/Brca1p53-KO glands by flow cytometry and RT-qPCR (Fig.  4 C; Additional File 4 : Fig. S4 B). Of note, the fraction of luminal cells depleted of Itgα6 was significantly higher in juxta-tumoral glands (8–10-month-old females) than in preneoplastic tissue (4–5-months-old females) in α6KO/Brca1p53-KO mice, probably due to the increased expression of the Blg promoter with age, as previously reported (Additional File 4 : Fig. S4 C; [ 14 ]). The proportion of luminal and basal cells was not significantly different in Brca1p53-KO and α6KO/Brca1p53-KO juxta-tumoral glands (Fig.  4 C). However, within the luminal cell population, the proportion of ICAM1 + LP was still decreased in Itgα6-deficient glands, similarly to what observed in the preneoplastic gland (Fig.  4 D).

The aberrant expression of basal/EMT-like genes in luminal progenitors of Brca1/p53-deficient mice is reduced by Itga6 deletion. A Hematoxylin and Eosin staining of juxta-tumoral mammary gland sections from Brca1p53-KO and α6KO/Brca1p53-KO mice (bearing a tumor in another gland) and a control littermate. Scale bar: 100 µm. B Immunofluorescent staining with anti-K8 (white), and anti-Ki67 (red) antibodies. Nuclear DAPI staining is shown in blue. Scale bar: 20 µm. The graph shows the percentage of Ki67 + cells (mean ± SEM) obtained from 4 controls, 6 Brca1p53-KO and 6 α6KO/Brca1p53-KO mice. C , D Representative FACS analysis of juxta-tumoral glands from Brca1p53-KO and α6KO/Brca1p53-KO mice. C separation of basal (green) and luminal (orange) populations. Right: graph showing the percentage of basal and luminal cells in the Lin- population in 5 independent cell sorting experiments. D Analysis of ICAM1 expression gated in the luminal cells. Right: graph showing the percentage of luminal ICAM1 + and ICAM1- cells relative to the total luminal population in five independent cell sorting experiments. E RT-qPCR analysis of the ICAM1 + LP from Brca1p53-KO and α6KO/Brca1p53-KO juxta-tumoral glands and control glands (5 animals per group) The graphs present mean ± SEM. F Immunofluorescent staining with anti-K8 (green), and anti-K14 (red) antibodies of control normal gland and normal-looking ducts (upper panels), or of mammary hyperplasic lesions (lower panels) developed in Brca1p53-KO and α6KO/Brca1p53-KO mice. In normal ducts, dashed rectangles indicate magnifications shown in the right panels. Note the presence of numerous cells co-expressing K8 and K14, indicated by white arrowheads in normal ducts, or marked in yellow in lesions (lower panels). Nuclear DAPI staining is shown in blue. Scale bar: 20 µm (12 µm in magnifications). G Western blot analysis of ICAM1 + LP isolated from Brca1p53-KO and α6KO/Brca1p53-KO juxta-tumoral glands. Control cells are shown for comparison. In B – E , * P  < 0.05, ** P  < 0.01, *** P  < 0.001, n.s., non-significant

Before tumor formation, Brca1/p53-deficient luminal cells have been reported to acquire some basal-like traits and to express EMT-related genes [ 17 , 39 ]. Consistently, in Brca1p53-KO luminal progenitors, we found a significant increase in several basal transcripts (such as Trp63 , encoding for the transcription factor p63) and EMT-related genes, such a Fn1 (encoding for the ECM protein fibronectin), Vim (encoding for the cytoskeletal protein vimentin) as well as the transcription factor Twist1 , compared to control cells (Fig.  4 E). Notably, in the α6KO/Brca1p53-KO luminal progenitors, expression of these genes was reduced when compared to the Brca1p53-KO cells (Fig.  4 E). In addition, the basal marker cytokeratin 14 (K14) was readily detected in luminal (K8 +) cells of Brca1p53-KO epithelium, both in normal-looking ducts detected at this stage and in the pretumoral lesions (Fig.  4 F). Interestingly, such cells co-expressing luminal and basal markers (K8 + /K14 +) were rarely found in α6KO/Brca1p53-KO epithelium, just like in nascent mammary lesions of juxta-tumoral glands (Fig.  4 F). Western blot analysis performed with extracts of sorted luminal progenitor cells confirmed K14 differential expression in Brca1p53 mutant cells and the partial rescue of its expression upon α6 knock-out (Fig.  4 G).

Taken together, these results indicate that Itgα6 contributes to the acquisition of the expression of basal/EMT-associated genes in Brca1/p53-deficient luminal progenitors prior to tumor formation. It is tempting to speculate that cells with mixed luminal/basal and EMT features might represent tumor cells of origin or the cells responsible for tumor growth and progression. If that was the case, the decrease of such cells in α6 KO mice might reflect the delay in tumor formation observed in these compound mice.

Lack of Itga6 favors the induction of p16 caused by Brca1 deficiency in preneoplastic glands

Among invasive breast cancer, basal-like tumors are characterized by the high expression of the cell cycle inhibitor p16 at the transcriptional level [ 40 ]. Moreover, the presence of a p16-expressing luminal cell population in preneoplastic Blg-Cre;Brca1 F/F ;Trp53 F/F mammary tissue has been recently reported [ 41 ]. In agreement with these recent findings, we detected p16 expression in luminal cells (K8 + /α-SMA) from 5-month-old Brca1 mutant mice, but not in control mice (Fig.  5 A). In Brca1p53-KO mice, expression of the Cdkn2a gene (encoding for the p16 protein) in luminal progenitors progressively increased from preneoplastic (predominantly normal-looking epithelium) to juxta-tumoral glands (presenting some hyperplastic lesions), but not in fully grown tumors (Fig.  5 B). Interestingly, in α6KO/Brca1p53-KO mutants, luminal progenitors displayed higher levels of Cdkn2a transcripts than Itgα6-proficient cells, while no difference was detected between both groups at the tumor stage (Fig.  5 B). We then investigated the spatial distribution of p16 expression in the mammary epithelium. Due to the heterogeneity in morphology of the juxta-tumoral glands, we estimated p16 levels in normal-looking structures and lesion areas separately. Consistent with our gene expression analysis, we found that the percentage of p16 + cells was higher in normal-looking epithelium of Itgα6-deficient females, whereas this difference disappeared in the areas with lesions (Fig.  5 C). Additionally, Western blot analysis with protein extracts from sorted LP of preneoplastic glands showed higher p16 levels in α6-deficient cells (Fig.  5 D).

Induction of the cell cycle inhibitor p16 in the Brca1/p53-deficient preneoplastic glands. A Immunofluorescent staining with anti-p16 (red), anti-K8 (white) and anti α-SMA (green) antibodies. Nuclear DAPI staining is shown in blue. Scale bar: 20 µm. B RT-qPCR analysis of Cdkn2a expression (coding for the p16 protein) in ICAM1 + LP cells (n = 4) and CD24 + tumor cells (n = 3) from Brca1p53-KO and α6KO/Brca1p53-KO mice at different stages. Control ICAM1 + LP values are shown for comparison (n = 4). C Immunofluorescent staining with anti-p16 (red) and anti-K8 (white) antibodies. Nuclear DAPI staining is shown in blue. Scale bar: 20 µm. The graph shows the percentage of p16 + cells (mean ± SEM) obtained from 3–4 animals per group. D Western blot analysis of luminal progenitor cells isolated from Brca1p53-KO and α6KO/Brca1p53-KO juxta-tumoral glands. Cells isolated from control littermate females are shown for comparison. E Immunofluorescent staining with anti-p16 (red), and anti-Ki67 (green) antibodies. Nuclear DAPI staining is shown in blue. Scale bar: 20 µm. The graph shows the fraction of p16 + that are also Ki67 + (mean ± SEM) obtained from 3–4 animals per group. F Immunofluorescent staining with anti-phospho-Rb (red), and anti-K8 (white) antibodies. Nuclear DAPI staining is shown in blue. Scale bar: 20 µm. The graph shows the percentage of phospho-Rb + luminal (K8 +) cells (mean ± SEM) obtained from 4 animals per group. In B , C , E , F , * P  < 0.05, ** P  < 0.01, *** P  < 0.001, n.s., non-significant

Furthermore, in agreement with previous studies, we found that, in Brca1/p53-deficient epithelium, a non-negligible fraction of p16 + cells was abnormally cycling, as shown by their Ki67 status (Fig.  5 E; [ 40 , 41 ]. Of interest, we found that the amount of cycling p16 + cells was reduced in Itgα6-deficient glands, predominantly in normal-looking epithelial structures, suggesting cell cycle misregulation in cells expressing p16 in preneoplastic glands (Fig.  5 E).

The mode of action of the cell cycle inhibitor p16 involves the inhibition of the CDK4/6 kinase, resulting in hypo-phosphorylation of the retinoblastoma (Rb) protein, locking cells in a cell cycle arrest [ 42 ]. Consistent with a higher amount of non-cycling p16 + cells, we also detected a reduction in the proportion of cells expressing phosho-Rb in α6KO/Brca1p53-KO mutant mammary epithelium, particularly in the normal-looking ducts (Fig.  5 F). Accordingly, the levels of phospho-Rb protein were reduced in luminal cells depleted of Itgα6 (Fig.  5 D). Taken together, these results indicate that lack of Itgα6 favors the cell cycle inhibitor function of p16, leading to reduced proliferation in Brca1/p53 mutant preneoplastic glands.

Dysregulated integrin expression and function can impact tumor development at several levels: from the initial transformation of epithelial cells at tumor onset, to cancer cell migration and invasion through tissue boundaries and colonization of distant tissues. In breast cancer, Itgα6 has been associated with tumor cell invasion and metastasis [ 24 ]. However, its role in the early steps of tumorigenesis has not been investigated. We describe here the important contribution of Itgα6 to the formation of basal-like mammary tumors. Using a mouse model of aggressive breast cancer due to the induced deficiency of Brca1 and p53 in the mammary luminal epithelium, we show that deletion of Itga6 in the tumor cells of origin, the luminal progenitors, impairs tumor formation through a mechanism involving: (i) inhibition of cell cycle progression associated to over-expression of p16 in pre-tumoral glands and (ii) interference with an EMT-like program normally occurring during the first stages of tumorigenesis triggered by Brca1/p53 loss.

We have chosen to examine the phenotype of induced loss of Itga6 in Blg-Cre;Brca1 F/F ;Trp53 F/F mice, a well-established model of basal-like tumors, mimicking features characteristic of human BRCA1-deficient breast cancer [ 14 ]. In these mice, the Blg promoter targets gene deletion in luminal progenitors, which are considered the cells of origin of Brca1-mutant basal-like tumors. Consistently with our previous work in homeostatic glands, we detected Blg-driven Cre expression and targeted gene deletion predominantly in ICAM1 + LP [ 23 ]. However, some contribution of the ICAM1- mature luminal cells to tumorigenesis i.e. providing mitogenic signals, cannot be excluded. The role of mature luminal cell transformation in the genesis of Brca1-deficient tumors would require further investigation and the use of promoters specifically targeting this cell population, such as ER or Prominin-1 promoters.

Although in many types of cancers, including breast, Itgα6 has been reported to promote tumorigenesis, its expression is anti-correlated with tumor progression and invasion in some types of leukemia and prostate cancer [ 43 , 44 ]. Furthermore, Itgα6 has been categorized as a tumor suppressor in the gut, where its ablation, by disrupting hemidesmosomes, leads to intestinal epithelium detachment and inflammatory lesions that progress to colorectal carcinoma [ 27 ]. Therefore, the role of this integrin in tumorigenesis is pleiotropic and appears to be context dependent.

The integrin α6 subunit can associate with β1 and β4 to form α6β1 and α6β4 laminin-receptor dimers. While the β1-subunit is highly promiscuous, α6 is the only known partner of the β4 subunit. Accordingly, we found that Itgβ4 levels are reduced in luminal cells as well as in tumors in α6KO/Brca1p53-KO mice. In agreement with our results, deletion of the Itgβ4 signaling domain in a mouse model of Erbb2-induced mammary carcinoma resulted in impaired tumor formation [ 45 ]. Moreover, Itgβ1 is dispensable for tumor induction in the same mouse model, although its deletion leads to a delayed tumor onset and reduced tumor volume [ 46 ]. We have not detected significant changes in the cell surface levels of Itgβ1 in our α6KO/Brca1p53-KO mice. Therefore, our results suggest that the decreased levels of α6β4 (rather than those of α6β1) in the α6KO/Brca1p53-KO epithelium predominantly contribute to the observed phenotypes.

High expression of Itgα6 serves as a marker for isolating stem cells in different tissues [ 47 ]. We have previously shown that the simultaneous depletion of α3 and α6 integrins results in a significant perturbation of mammary gland development and function [ 23 ]. Depletion of Itgα6 alone led to mild mammary phenotype during lactation, while depletion of Itgα3 did not have significant effects. In particular, during pregnancy-associated alveologenesis, Itgα6 seems dispensable for the amplification of luminal progenitors [ 23 ]. On the contrary, here we found that, in the absence of Itgα6, expansion of the functional luminal progenitor population that precedes tumorigenesis in Brca1/p53-deficient mice is impaired, indicating that Itgα6 has different roles in normal physiology and tumor formation.

Itgα6 has also been shown to enrich for tumor initiating activity in breast and other tumor types [ 24 ]. In ER- breast tumors, cells with high Itgα6 expression display heightened tumorigenicity and self-renewal in vivo [ 48 ]. In the same vein, a subpopulation of CD24 + Itgα6 + Itgβ1 + cells with increased proliferation and enhanced tumor-forming ability has been found in mouse cell lines derived from p53 ± ;Brca1-deficient tumors [ 49 ]. Itgα6 exists as two different cytoplasmic variants, α6A and α6B, generated by alternative splicing [ 50 ]. The Mercurio’s lab reported that the α6B isoform promotes tumorigenesis in human breast cancer cell lines, unlike the α6A isoform, which is dispensable for tumor formation [ 51 , 52 ]. The loxP cassette used for Itga6 deletion in our study includes the transmembrane and the cytoplasmic exons specific of each of the two splicing variants, leading to the lack of expression of both isoforms in the α6KO/Brca1p53-KO mice [ 27 ]. Identification of the isoform involved in tumor induction in our model warrants further investigation.

Previous studies using similar Brca1-deficient mouse models described aberrant mammary alveolar development and single cell RNAseq analysis revealed the existence, at the premalignant stage, of a molecular cell cluster resembling luminal alveolar cells typical of gestation in the homeostatic gland [ 35 , 36 ]. Alveologenesis mimicry and lineage infidelity may significantly contribute to establishment of breast cancer [ 53 ]. We indeed found aberrant alveolar-like structures in the mutant preneoplastic glands associated to increased epithelial proliferation. However, these phenotypes were attenuated in the absence of Itgα6 and this was accompanied by the aforementioned reduction in luminal progenitor expansion. Notably, the clonogenic potential of luminal progenitors was reduced by Itga6 deletion, potentially contributing to the delayed tumor onset observed in α6KO/Brca1p53-KO mice.

Partial epithelial-to-mesenchymal transition (pEMT) has been associated to Brca1-induced tumorigenesis [ 16 , 17 ]. This process implies the existence of intermediate cellular states displaying both epithelial and mesenchymal (E/M) traits, that have been correlated to increased tumorigenicity in different types of cancer (reviewed in [ 54 , 55 ]). Furthermore, a high frequency of cells co-expressing basal and luminal specific markers (K8 + /K14 + cells) has been associated with malignancy in human Brca1-mutation carriers and in tumor mouse models [ 39 , 56 ]. In line with these data, we have detected an increased expression of basal and EMT-related genes in the luminal progenitors from juxta-tumoral tissue in Brca1p53-KO mice. This phenotype appears mitigated in the cells lacking Itgα6 expression suggesting that Itgα6 is required for the luminal-to basal switch and the concomitant triggering of a pEMT program in the Brca1/p53-deficient epithelium. Consistently, using a model of TNBC cells, Bierie et al. have shown the existence of Itgβ4 + cells residing in an intermediate E/M phenotypic state and displaying high tumorigenicity [ 57 ].

Of interest, in α6KO/Brca1p53-KO mice we have detected the overexpression of the cell cycle inhibitor p16, associated with hypo-phosphorylated Rb and a reduced proportion of cycling cells. Compared to other breast cancer types, basal-like invasive tumors display increased activation of the p16/Rb pathway [ 40 ]. Furthermore, single cell RNAseq analysis has recently revealed the presence of a p16-expressing luminal population in the pre-malignant tissue of the same Brca1/p53-deficient mice used in our study [ 41 ]. Suppression of Itgα6 has been shown to induce the expression of the cell cycle inhibitor p27 resulting in compromised cell cycle progression in a triple-negative breast cancer cell line [ 58 ]. In addition, a recent study reported that in the absence of Rb, Brca1p53-KO mice develop luminal ER + (instead of basal-like) tumors, indicating that Rb signaling is involved in the luminal-to-basal switch in Brca1/p53-deficient epithelium [ 59 ]. Therefore, the perturbation of the p16/Rb pathway at early stages of malignancy could provide a mechanistic explanation for the observed phenotypes (reduced proliferation, inhibition of luminal-to-basal switch) resulting in delayed tumorigenesis in α6KO/Brca1p53-KO mice (Fig.  6 ).

Model of the impact of Itgα6 loss in the formation of Brca1p53-deficient tumors. Loss of Brca1 and p53 in luminal progenitor cells induces epithelial proliferation that is, in first instance, counterbalanced by the concomitant activation of the p16/Rb pathway. With time and the plausible accumulation of DNA damage, a cell cycle arrest bypass occurs that, accompanied by the luminal-to-basal switch and pEMT, leads to tumor formation. In the Itgα6-deficient animals, overactivation of p16/Rb pathway in preneoplastic tissue restrains epithelial proliferation and limits luminal-to basal switch and pEMT, resulting in delayed tumor initiation

High Itgα6 levels are associated with poor survival in breast cancer and ITGA6 expression is an independent prognosis factor in ER-negative breast cancer [ 60 , 61 , 62 , 63 ]. Furthermore, this integrin subunit contributes to breast cancer cell dissemination and metastasis [ 64 , 65 ]. The Brca1p53-KO model used in this study is poorly metastatic, precluding the analysis of the involvement of Itgα6 in metastatic stages. However, we found that in advanced stages of tumorigenesis, Itgα6 appears dispensable for tumor growth, but its deletion affects tumor cell differentiation, presenting lower expression of basal markers, probably reflecting the inefficient induction of luminal/basal double-positive at the early steps of tumor formation. Importantly, our results have uncovered a critical role for Itgα6 in the first stages of breast cancer initiation, suggesting that its expression in luminal cells could be causally linked to tumor progression and malignant phenotype and as such represent a promising tool to predict the evolution of early breast lesions.

Availability of data and materials

Data sharing is not applicable to this article as no datasets were generated during the current study.

Abbreviations

Basement membrane

Cleaved-caspase-3

Cyclin-dependent kinase

4′6-Diamidino-2-phenylindole

Extracellular matrix

5-Ethynyl-2′-deoxyuridine

Estrogen receptor

Fluorescence-activated cell sorting

Intercellular adhesion molecule-1

Cytokeratin 14, 5, 8

Luminal mature

Luminal progenitor

Mammary epithelial cell

Partial epithelial-mesenchymal transition

Progesterone receptor

Receptor activator of nuclear factor kappa-B ligand

Retinoblastoma

Reverse-transcription-quantitative polymerase chain reaction

Standard error of the mean

α-Smooth-muscle actin

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Acknowledgements

The authors thank Dr. J. Jonkers (Netherlands Cancer Institut, Amsterdam, NL) for generously sharing the Brca1-flox and the Trp53-Flox mice with us and Dr. Georges-Labouesse and Dr. de Arcangelis (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Ilkirch, France) for kindly sharing the Itga6-Flox mice. We are particularly grateful to M. Glukhova for the initiation of the study and to Céline Vallot for helpful discussions. We acknowledge A. di Cicco for excellent technical assistance. We thank S. Jannet, M. Garcia, C. Pauchard, and the personnel of the Animal facility at Institut Curie for taking care of the mice, and to O. Renaud and the Cell and Tissue Imaging Platform of the Genetics and Developmental Biology Department (UMR3215/U934) for their expertise. We also thank C. Guerin, A. Viguier, S. Grondin and A. Chipont and the personnel of the Flow Cytometry Core facility for excellent assistance with FACS analyses . We acknowledge all the members of the Fre laboratory for support and constructive discussions.

The work was supported by grants from La Ligue Nationale Contre le Cancer Ile de France (RS16/75-70) and Labex CelTisPhybio (ANR-10-LABX-0038), part of the Idex PSL to MMF and from the Medical Research Foundation FRM “FRM Equipes” EQU201903007821, the FSER (Fondation Schlumberger pour l’éducation et la recherche) FSER20200211117, the Association for Research against Cancer (ARC) label ARCPGA2021120004232_4874 and by Labex DEEP ANR-Number 11-LBX-0044 to S.F. MR received funding from Marie Curie Fellowship Program; SF is Directeur de Recherche , MMF and MAD are Chargé de Recherche at the Institut National de la Santé et de la Recherche Médicale (INSERM) .

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Marisa M. Faraldo, Loane Wallon & Silvia Fre

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MMF, MAD and SF conceived and designed the experiments. MMF, MR and LW performed all the experiments. MMF and PD analyzed the data. MMF and SF provided funding of the study. MMF, MAD and SF wrote the manuscript. All authors read and approved the final manuscript.

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Faraldo, M.M., Romagnoli, M., Wallon, L. et al. Alpha-6 integrin deletion delays the formation of Brca1/p53-deficient basal-like breast tumors by restricting luminal progenitor cell expansion. Breast Cancer Res 26 , 91 (2024). https://doi.org/10.1186/s13058-024-01851-4

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Ukraine : Emmanuel Macron annonce l’envoi d’avions Mirage 2000-5

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Le chef de l’État, qui reçoit le président ukrainien vendredi à l’Élysée, a aussi annoncé la formation d’une brigade de soldats ukrainiens.

Après avoir dit, depuis plus d'un an, non systématiquement, Emmanuel Macron a fini par céder . Le chef de l'État a annoncé jeudi soir lors d'une interview sur France 2 et Tf1 que la France allait livrer des avions de chasse Mirage 2000-5 à l'Ukraine d’ici à la fin de l’année.

• Suivez les informations sur la guerre en Ukraine avec l'application du Figaro

« Nous allons bâtir une coalition avec d'autres partenaires » , a-t-il expliqué, sans préciser le nombre d'appareils qui seront fournis. Paris cherche à convaincre d’autres pays possédant aussi des Mirage d’en céder des exemplaires. La Grèce, l'Inde, le Qatar, les Émirats arabes unis ou le Brésil utilisent cet avion sorti des usines de Dassault Aviation (*). «Dès cet été» , des pilotes ukrainiens seront formés en France sur ces appareils. Pour l'instant, l'armée de l'air n'accueille qu'une dizaine de pilotes ukrainiens pour des formations initiales sur Alpha jet. Il faudra plusieurs mois pour qu’ils soient opérationnels.

Jusqu'à présent, la France avait écarté l'hypothèse de livrer des Mirage 2000 pour ne pas affaiblir la flotte de l'armée de l'air, taillée au plus juste, et par souci de cohérence. Pour l'Ukraine, équipée de Mig21 de conception soviétique, et qui s'apprête à recevoir dans les prochains mois des avions F16  américains, ajouter un nouveau type d'appareil pourrait se transformer en casse-tête logistique. L'armée de l'air n'a pas non plus les effectifs pour former beaucoup de pilotes.

Mais toutes ces réserves n'ont pas tenu face à l'urgence. Le chef de l'État semble décidé à prouver à Volodymyr Zelensky, qu'il reçoit vendredi à l'Élysée après l'avoir accueilli jeudi aux cérémonies du Débarquement, que la France veut rehausser son engagement vis-à-vis de l'Ukraine. «Nous ne voulons pas d'escalade de la guerre» , a assuré Emmanuel Macron, tout en souhaitant donner à Kiev les moyens de se défendre. Grâce aux avions occidentaux, même en nombre limité, l’Ukraine sera en mesure de renforcer ses défenses aériennes.

Une brigade de 4500 hommes

En difficulté sur le champ de bataille, l'Ukraine a des besoins urgents… Emmanuel Macron a annoncé la prochaine formation d'une brigade de 4500 soldats, équipée et armée . L’annonce est plus symbolique qu’autre chose, au moment où l’Ukraine cherche à mobiliser plusieurs centaines de milliers d’hommes. La formation de la future brigade ne se déroulera pas en Ukraine. Le chef de l'État n'a pas encore réussi à entraîner derrière lui d'autres pays européens prêts à envoyer des instructeurs en Ukraine, comme il l’aurait souhaité. Le ministre de la Défense ukrainien, Roustem Oumerov , «a adressé une demande officielle il y a 48 heures» à ses partenaires pour des formations dans son pays, a rapporté Emmanuel Macron. Les réticences sont fortes entre Occidentaux autour des risques d'engrenage.

«Pourquoi est-ce que cela serait un facteur d'escalade ?» , a interrogé le chef de l'État pour balayer l'accusation. Il a rappelé que des citoyens français qui se sont engagés en Ukraine ont déjà péri là-bas. «Est-ce que la mort d'un civil est moins grave que celle d'un soldat ?» Le président de la République veut dédramatiser les risques pour permettre aux Occidentaux de franchir un cap, malgré les menaces de la Russie. En envoyant des soldats sur le terrain, non pas dans les zones de combat mais en arrière, Emmanuel Macron veut ancrer le soutien occidental dans la durée. Ce serait une condition pour concrétiser les garanties de sécurité signées entre l’Ukraine et ses partenaires.

Le Kremlin menace

Alors que de nouvelles lignes rouges s'effacent, le Kremlin brandit le spectre de l'escalade . La Russie a averti qu'elle pourrait fournir des armes aux adversaires des pays qui livrent des armes à l'Ukraine, sans dire qui ou quoi. Le porte-parole du Kremlin a aussi désigné de possibles formateurs militaires français envoyés en Ukraine comme des cibles légitimes. «Les limites (du conflit) sont fixées par ce que font les Russes. Ce n'est pas nous qui attaquons» , a répété le chef de l'État. Mais l’Occident doit participer à la riposte pour être crédible.

Même critiquée, l’initiative française est observée de près en Europe. «Les Russes ne font pas qu’écouter ce que nous pouvons dire, ils regardent ce que nous faisons » , explique un responsable balte à propos de la proposition d’envoyer des soldats en Ukraine. «La Russie doit être battue» , poursuit-on en s’inquiétant qu’un manque de clarté en Europe ne laisse croire au Kremlin que les Occidentaux n’ont pas envie de se défendre. «C’est l’inaction qui provoque Poutine» , estime cette source. Emmanuel Macron semble décidé à contrer cette logique, quitte à prendre des risques.

(*) Le groupe Dassault est propriétaire du Figaro.

• Troupes françaises en Ukraine : Macron réfléchit, la Russie menace
• Les Occidentaux évoluent sur l’opportunité de frappes ukrainiennes contre la Russie
• Avions de combat à l’Ukraine, Gaza, européennes... Ce qu’il faut retenir de l’interview d’Emmanuel Macron
• guerre en Ukraine
• Emmanuel Macron

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gauloisrefractaires

le 07/06/2024 à 23:02

Feu d'artifice garanti ! La Russie possède avec les S400 et S500, le système défense aérienne le plus performant au monde ! Quant aux chasseurs SU35, le Rafale ne fait pas le poids, à plus forte raison des vieux Mirage dépassés, quelle que soit le modèle. Pour donner une idée des forces en présence, les missiles antiaériens Russes dépassent la vitesse opérationnelle de mach 10, quand les Mirages plafonnent à Mach 2,2. 5m plus long que le Mirage, le SU 35 vole à Mach 2,7 contre 2.3 pour le Mirage et possède une capacité d'emport deux fois plus importante. Aussi bien que les Russes ont brûlé les "redoutables" Abrams, Leopard et AMX, ils feront des confettis des Mirage et F16. L'inutile de l'Élysée ne fait que s'humilier un peu plus à la face du monde.

Minet du Phare

le 07/06/2024 à 19:11

Là, EM commence vraiment à jouer à la roulette russe avec la France.

Patin-couffin

le 07/06/2024 à 18:20

Macron a annoncé que la France proposerait de céder ses derniers Mirage 2000-5 à l'Ukraine. Cependant, cette décision soulève de nombreuses questions quant à sa faisabilité et son impact sur les capacités militaires françaises. En effet, si la France devait se séparer de ses Mirage 2000-5, cela représenterait environ un tiers de sa flotte, composée de seulement trois escadrons, soit environ 45 avions réellement opérationnels. Une telle réduction compromettrait la défense aérienne du pays dans le role attribué aux F5. Quant aux Mirage 2000-5 des autres pays, la situation n'est guère plus favorable, les nations arabes, le Chili ou l'inde equipés de 2000-5 sont peu enclines à les fournir à l'Ukraine. La Grèce, seule autre détentrice potentielle, utilise des modèles datant de 1985, en voie de déclassement depuis deux ans. Bien que ces avions soient performants, ils nécessitent un entretien minutieux par du personnel hautement qualifié. Face aux avions et missiles russes, ces appareils sont dépassés. De plus, l'Inde, en quête de pièces détachées, négocie leur achat depuis 2013. En définitive, cette initiative de Macron semble irréaliste et peu susceptible d'influencer le cours de la guerre en Ukraine. Elle n'apportera qu'une solution temporaire et inefficace, tandis que les pertes humaines continueront de s'accumuler, servant principalement les intérêts américains.

Conflit Israël-Hamas : Netanyahou «surpris et déçu» que Biden ne soutienne pas de sanctions contre la CPI

La Maison-Blanche a estimé que sanctionner cette organisation ne serait pas «la bonne approche » , même si Joe Biden avait jugé «scandaleuse» la demande de mandats d'arrêt contre des dirigeants israéliens.

Guerre en Ukraine : la France est prête à participer «directement au conflit militaire», prétend le Kremlin

Le chef de l’État français a déclaré jeudi que la France prévoyait de fournir des avions de chasse Mirage 2000-5 à l'Ukraine d'ici à la fin de l'année.

80 ans du Débarquement : Rishi Sunak fait polémique après avoir quitté prématurément les commémorations

Le premier ministre britannique a écourté son séjour en France pour une interview télévisée au Royaume-Uni. Rishi Sunak a été contraint de présenter ses excuses ce vendredi.

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