Fouzi Boukhalfa

Mohamed Hadded

Paul Muhlethaler

Oyunchimeg Shagdar

Evaluation of a new Radio Technology and Visible Light Communication for a Platooning Application

Keywords: Autonomous vehicles, platooning, network simulation, OMNET, VEINS-VLC framework

The autonomous platoon is today one of the key tools for better road utilization. In fact, by optimizing the distance between vehicles, the air drag is reduced, and researchers have shown that 20% of energy can be saved by using this concept. From a network point of view, reducing the distance between vehicles will allow a new point-to-point communication link between the vehicles in front and behind by using Vehicular Visible Light Communication (V-VLC), thus providing an opportunity to have a hybrid communication. Our new radio design based on the AS-DTMAC protocol guarantees a high Quality of Service for real-time applications. However, with a very high density, we can reach the bandwidth dedicated to V2X radio. In the case of a platoon, this scenario can cause dangerous platoon instability. Assisting the radio with another communication vector such as V-VLC can help to maintain the high level of reliability that is necessary for the control of a platoon. In this paper, we first carry out an analytical analysis to investigate the capacity of our new radio technology to support the platoon control use case in terms of the quality of service (QoS) required for this type of application. Secondly, we show through extensive simulations the current level of V-VLC technology, compared to radio technology, in terms of packet loss and delay.

Introduction and motivation

Nowadays, our highways are becoming increasingly congested. For the best traveling experience, Intelligent Transportation Systems (ITS) aim to optimize road utilization and traffic management. To address congestion issues, conventional vehicle systems for traffic flow like the Adaptive Cruise Control (ACC) are not sufficient. This system automatically adjusts a vehicle's speed based on information from vehicles ahead. A more dynamic regulation strategy requires a cooperative system which will provide more information on the positions, speeds and accelerations of neighboring vehicles. Thanks to these cooperative systems, new vehicular applications can be established (an example of such a system being Cooperative Adaptive Cruise Control (CACC)). Platooning is one of the best-known applications that can enhance road capacity. A Platooning application involves a robust control with an efficient communication strategy. Due to high QoS requirements in terms of update frequency (at least 10Hz) with a recommended transmission latency of 20 ms and a reliability of 99.99%, researchers are looking to combine more than one communication technology. In the literature, many communication strategies have been proposed. To achieve low-latency and ultra-reliable communication, an additional vector of communication is required since relying only on the Radio Frequency (RF) link, as we developed in [START_REF] Boukhalfa | An Active Signaling Mechanism to Reduce Access Collisions in a Distributed TDMA Based MAC Protocol for Vehicular Networks[END_REF] [START_REF] Boukhalfa | An Analytical Model for Performance Analysis of An active Signaling-based MAC Protocol for Vehicular Networks[END_REF] [3] [4] [START_REF] Boukhalfa | Coexistence of IEEE 802.11p and the TDMA-based AS-DTMAC Protocol[END_REF], may not be sufficient. With a very high number of nodes, there is a risk of reaching the full capacity of the channel and, if that occurs, some nodes will be unable to communicate with the rest of the network. In such a case, Vehicular Visible Light Communication (V-VLC) is a potential technology that could assist RF communication. However, V-VLC in outdoor environments faces a number of challenges due to mobility, weather conditions and ambient light [START_REF] Memedi | Vehicular Visible Light Communications: A Survey[END_REF]. Nonetheless, it provides a large free spectrum at a relatively low cost as the Light Emitting Diodes (LEDs) are already embedded in modern vehicular light systems [START_REF] Memedi | Vehicular Visible Light Communications: A Survey[END_REF].

Our goal is twofold: first, to show that our new RF design is able to support platooning applications (to our best knowledge, we are the first to propose a radio technology based on a fully distributed TDMA which meets the QoS requirements necessary for platooning communication). Secondly, we set out to test the maturity of the V-VLC standard in a large-scale simulations for dynamic V-VLC scenarios such as platooning in the presence of interfering vehicles. For this, we have chosen to use a simulator based on a fairly realistic empirical model that takes into account the impact of the type of vehicle (the features of this framework are explained below). This study helps us to determine whether light links are sufficiently stable to be used in place of radio links, or whether we should use both communication links to improve the overall reliability.

The remainder of this paper is organized as follows. In the next section, we present related work. In Section 3, we discuss and prove analytically the capacity of our new radio design to support a platooning application. For this task, we select suitable metrics, namely average access time and path loss (under NLOS conditions). In Section 4, we show the performance of the V-VLC link in the presence of disturbing vehicles. We evaluate the PDR for various network conditions, different headlight modules (provided by Veins VLC [START_REF] Memedi | Impact of Realistic Light Radiation Pattern on Vehicular Visible Light Communication[END_REF] [8]) and speeds. We also compare the performance between RF and VLC in terms of PDR and delay. Finally, in Section 5, we conclude the paper outline future work..

Related work

The IEEE 802.15.7 [START_REF]IEEE Standard for Local and metropolitan area networks -Part 15.7: Short-Range Wireless Optical Communication Using Visible Light[END_REF], published in 2011, is the current Standard for Short-Range Optical Wireless Communications and includes outdoor communications. However, this standard is not specially designed for V-VLC and there is no effort being made in this direction: the standardisation efforts mainly focus on the system costs. Recent research has shown the need for a dedicated new standard for vehicular networks [START_REF] Cailean | Impact of IEEE 802.15.7 Standard on Visible Light Communications Usage in Automotive Applications[END_REF].

Light communications are classified into two categories depending on the environment: indoor (mainly using LiFi applications [10][11]), and outdoor (to establish communication between vehicles and the infrastructure [START_REF] Uysal | Visible Light Communication for Vehicular Networking: Performance Study of a V2V System Using a Measured Headlamp Beam Pattern Model[END_REF]). The VLC wavelength varies from 380nm to 780nm of the electromagnetic spectrum. VLC will benefit from a large available spectrum, which will result in high data rates. A VLC receiver can encompass either a Photodiode (PD) or a camera. The location of a vehicle's LED-based light makes the set-up of a full-duplex communication possible (see Figure 1). A vehicle has two tail-lights and two head-lights. Sunlight and outside light sources can cause, respectively, a shot noise and interference. V-VLC requires a line-ofsight (LOS) scenario but some research shows that it is possible to use ground reflections to have V-VLC NLOS communication [START_REF] Tebruegge | Empirical Characterization of the NLOS Component for Vehicular Visible Light Communication[END_REF]. However, the LOS scenario is not always available due to a variations in the vehicle's heading or to weather conditions. 2), which can not pass through objects, NLOS messages need to be forwarded in a multi-hop manner. VLC technology can provide an alternative for GPS-based positioning technology, which is not always available (e.g. in a tunnel or an indoor environment). The precision of such a system is about a few centimeters, whereas GPS gives an error of 10m [START_REF] Uysal | Visible Light Communication for Vehicular Networking: Performance Study of a V2V System Using a Measured Headlamp Beam Pattern Model[END_REF]. VLC uses multi-hop communication to reach vehicles that are not directly in front, resulting in high latency, whereas RF can reach all these vehicles immediately. On the other hand, in high density scenarios, V-VLC has good scalability compared to RF. If the RF band is restricted or unavailable (due to safety or military applications,) using VLC would be a suitable alternative. In Figure 3 (a) each platoon member is using V-VLC. Only the leader is using both V-VLC and RF to communicate with its followers. In Figure 3 (b) the platoon members are in redundant mode (both RF and V-VLC are being used). In Figure 4 (a) and (b) the vehicles are using only one vector at a time, either V-VLC or RF. Such an example can be found in [START_REF] Segata | Towards inter-vehicle communication strategies for platooning support[END_REF] where Segata et al. propose IEEE 802.11p/IEEE 1609.4 PHY/MAC to manage communication between the vehicle members of a platoon, They assume that each vehicle has the capacity to know its position in the platoon. The leader's beacon is used as a synchronisation signal to divide the time into slots. The number of slots depends on the size of the platoon, and the allocation of slots to vehicles will depend on their position with respect to the leader. The idea is to adapt the transmission power: the leader will use enough transmission power to reach each follower, whereas the followers will adapt their power to reach only those vehicles that require such data. This will reduce interference with other vehicles and increase spatial reuse.

Performance Analysis of AS-DTMAC for platooning control

Following the AS-DTMAC scheme described in previous work [START_REF] Boukhalfa | An Active Signaling Mechanism to Reduce Access Collisions in a Distributed TDMA Based MAC Protocol for Vehicular Networks[END_REF] [START_REF] Boukhalfa | Coexistence of IEEE 802.11p and the TDMA-based AS-DTMAC Protocol[END_REF], a vehicle loses its slot when it changes zones (see Figure 5: R equals the transmission range). Thanks to the active signaling mechanism, reserving another slot in the new area will take only a few milliseconds. Now, considering a platooning scenario, the members of the platoon are considered as a single entity. Therefore, in order to maintain communication between the leader and the followers, all platoon members must be allocated a slot in less than 100 ms (platoon update rate), otherwise, data are lost and this can affect the platoon's stability. We assume that we have n vehicles in the platoon and we wish to compute the average time needed for all the members of the platoon to obtain a slot in the new area, thus in a new DTMAC frame. We assume that the slots in the new areas are occupied with probability λ and free with probability 1 -λ 1 . We also assume that the free slots are 1. This is a simplified model for AS-DTMAC which assumes that the DTMAC frame is infinite obtained by AS-DTMAC in a greedy mode (the first slot available is reserved by a vehicle with AS-DTMAC without any collision since the collision rate is extremely small). We note by I 1 + 1,I 1 + I 2 + 2,. . . ,I 1 + I 2 + • • • + I n + n the slots successfully obtained by the vehicles in the platoon. The probability that

[2][3][4]
I 1 = i 1 ,I 2 = i 2 , . . . I n = i n is (1 -λ)λ i1 (1 -λ)λ i2 . . . (1 -λ)λ in (1)
and the total time needed for all the nodes to obtain a slot in the next frame is n +

I 1 + I 2 + • • • + I n .
The average time required for all the nodes to obtain a slot is thus

E((1 + I 1 ) + (1 + I 2 ) + • • • + (1 + I n-1 ) + (1 + I n )) (2)
where E means the expectation on all the possible events.

But all the I j with j ∈ 1, 2, . . . , n are independent with the same law and we have:

E((1 + I 1 ) + • • • + (1 + I n )) = nE(1 + I 1 ) = n 1 -λ . (3)
We also have

P ((n + I 1 + • • • + I n = n + p) = n + p -1 n -1 λ p (1 -λ) n .
(4) We have to notice that the last slot must be free, this explains the presence of the -1 in the formula. We can easily compute the distribution of n + I 1 + • • • + I n . In Figure 6, we plot the probability that the last slot successfully reserved by a platoon of 10 vehicles is at most slot number N . We observe that unless there is a high load, the reservation stops before the end of the AS-DTMAC frame whose duration is 33 slots.

In order to further investigate the capacity of our new radio technology design to support the platoon application, we analyze the connectivity between platoon members in fading channel conditions, and under our active signaling mechanism.

V2X applications operate on the 5.9GHz bandwidth, and the platooning application requires a safety distance (d) between platoon members of between 10 m and 30 m. We can consider that d >> λ (λ here refers to the radio wave length) and thus we use statistical models to take into account the fading effect (including the random channel gain and random phase shift). The following formula gives the path loss with channel fading:

P L(d) = 10 * γ * log(d/d 0 ) + P L 0 (d 0 ) + X (5)
X is a zero mean complex normal distribution variable with standard deviation σ. The γ is the path loss exponent. The values of σ and γ were taken from [START_REF] Fernandez | Path Loss Characterization for Vehicular Communications at 700 MHz and 5.9 GHz Under LOS and NLOS Conditions[END_REF]. AS-DTMAC uses the physical layer of the IEEE 802.11p thus the RX sensitivity is -85dbm (64-QAM with 3/4 coding) [START_REF] Yacheur | Analysis and Comparison of IEEE[END_REF]. In a previous paper [START_REF] Boukhalfa | Physical and MAC Layer Design for Active Signaling Schemes in Vehicular Networks[END_REF] The following results were obtained by using the Monte-Carlo simulation. Figure 7 shows the probability of correctly detecting and decoding (P d ) a packet under fading conditions versus the distance between the transmitter and the receiver. From this curve we can see that up to 150 m, 100% of packets are received correctly. Beyond this value, the P d gradually decreases by increasing the distance. In Figure 8, we vary the variance (σ) of the fading (the sweep values were taken from the reference [START_REF] Chandrasekharamenon | Connectivity analysis of one-dimensional vehicular ad hoc networks in fading channels[END_REF]) and show their impact on P d . we plot four curves, each one corresponding to a particular distance (150 m, 170 m, 190 m, 210 m). The results show that there is an impact of this parameter on the detection: by raising σ, the probability decreases; however, the probability decreases more rapidly when the distance grows. Finally, in Figure 9 we vary the path loss exponent (γ) from 2.2 to 3 [START_REF] Chandrasekharamenon | Connectivity analysis of one-dimensional vehicular ad hoc networks in fading channels[END_REF]. As we can observe, this last parameter also has an impact on P d . Furthermore, for each curve plotted, we can see that the threshold to have 100% of packets moves with the distance (i.e. the greater the distance is, the more the threshold moves to the left).

From this connectivity analysis, we can conclude that safe connectivity can be achieved at a distance of 150 m. However, the above study (i.e. the probability of a platoon having a completely reserved slot) shows that regarding the AS-DTMAC functionality we limit the platoon size to 10 vehicles with an assumed inter-vehicle distance of 10 m and a mean vehicle length of 4-5 m.

Simulation results

The simulation tool for V-VLC used in this section is based on VEINS-VLC. This simulator uses an empirical model (i.e. it uses photometric data and thus takes into account the NLOS component coming from the ground). As the data are collected from a real headlight module, this radiation pattern includes the effect of a high and low beam 2 in the headlight coming from real vehicular headlight modules. These modules are from HELLA GmbH & Co. KGaA [START_REF] Memedi | Impact of Realistic Light Radiation Pattern on Vehicular Visible Light Communication[END_REF] [START_REF] Memedi | Impact of Vehicle Type and Headlight Characteristics on Vehicular VLC Performance[END_REF]. Figure 10 summarizes the integration of the empirical model into the VEINS framework (for more details see [START_REF] Memedi | Impact of Vehicle Type and Headlight Characteristics on Vehicular VLC Performance[END_REF]). 2. The low beam in the headlight is to implement an asymmetric light distribution.

Figure 10. Summary of the integration of the empirical model in VEINS-VLC (the images were taken from [START_REF] Memedi | Impact of Vehicle Type and Headlight Characteristics on Vehicular VLC Performance[END_REF]): the measurements were done at the HELLA's Lichtkanal company in a light channel room of dimension 145m * 11m. For the emissions, a real pair of headlights module was used, whereas the receiver side uses two Photodiodes (PDs) of type PDA100A placed at the same level as the license plate and the tail lights.

V-VLC performance with presence of interference

In this section, we show the impact of interference on the performance of visible light communication in a vehicular platoon. Our study focuses on the vehicles that are arriving in parallel to the platoon's lane. When there is packet in the channel transmitted between the platoon vehicles, the channel is disturbed by the light of these arriving vehicles. This will continue to be be the case until all the disturbing vehicles have overtaken the platoon (see Figure 11). Figure 12 shows the Packet Delivery Ratio (PDR) versus the number of disturbing vehicles. We plot different curves, each one using a particular model available on the market (LbSedan1, LbSedan2, Lbsuv1, HbSedan2, HbSedan1). All the curves decrease as a function of the number of disturbing vehicles. As we can observe, the interference caused by the vehicles' lights has an important impact, particularly for high numbers of disturbing vehicles (for 100 disturbing vehicles: 40% of the packets are lost). We also see that the types of vehicles in platoon impact the PDR and the difference can reach 10% for high densities. Figure 13 shows the impact of the speed of disturbing vehicles on the PDR. We vary the speed between 50 km/h to 120 km/h, and we plot three curves for 40, 70, and 100 disturbing vehicles respectively. The results show that the PDR is better when the speed increases. In fact, the greater the speed of the disturbing vehicles, the shorter the interference time is, which results in a better PDR.

Figure 14 shows the impact of platoon vehicle interdistance on the PDR. We vary the distance between 10 m to 40 m, and we plot three curves for 40, 70, and 100 disturbing vehicles respectively. The results show that the PDR is better when the communication is under 10 m. In fact, beyond 10 m, the PDR decreases rapidly until reaching a communication distance of 25 m, then the PDR becomes more stable for all curves.

Performance comparision between V-VLC and RF

In this section, we discuss and compare the performance of RF and V-VLC in terms of PDR and delays. We consider the same scenario as in Figure 11 extended by a parallel highway for vehicles moving on the opposite side. As concerns VLC propagation, only the two lanes near the platoon lane will have an impact on the performance, the rest will have no impact, whatever the density is. As described before, AS-DTMAC divides the road into different zones, and each zone has a set of slots (about 33 slots per zone), thus, the performance of the RF can be impacted when the number of vehicles rises since more vehicles are reserving a slot and there is a risk of reaching the total resource capacity.

Figure 15 shows the delay for V-VLC versus platoon beacon packet length. Obviously, the delay increases as the packet length increases. The delay varies from 1 ms to 9 ms for packets with lengths ranging from 100 to 1200 bytes. Moreover, in a platoon, if, for example, the last member attempts to communicate with the leader via V-VLC (see Figure 11), messages need to be forwarded via multihop communication, which increases the latency. Thus, to respect the QoS requirements of this application, the platoon size must be taken into consideration.

In Figure 16 we plot the PDR versus the number of disturbing vehicle for both RF and VLC. As we can see in this scenario, RF is largely reliable compared to V-VLC. In fact, even for high numbers of disturbing vehicles, RF is not greatly affected (less then 3% of packets are lost) whereas

Conclusion

In this paper, we showed the ability of our radio technology, based on AS-DTMAC, to respond to the QoS requirements of the platooning application. We conducted largescale platoon simulations based on the Veins-vlc framework, which uses a realistic V-VLC model. We also presented the state-of-the-art of V-VLC and showed the lack of maturity of this technology compared to RF. However, V-VLC is still an excellent assistant technology in the platoon use case. By highlighting the limitations of this technology, we will challenge them in the future in order to achieve the full capacity of this technology in terms of data rate, latency and inter vehicular distance. Finally, we compared the performance of RF and V-VLC in the platooning scenario in terms of PDR and delay.

In future work, we will focus on integrating RF and VLC communication in a heterogeneous network in order to keep the reliability as high as possible. We plan to propose a smart switching protocol at the handover level to choose the best communication technology depending on the mobility scenario. This protocol will use a dynamic threshold and make decisions based on a vehicular realtime system such as the Channel Busy Ratio (CBR), which gives information about the radio channel quality, but also on V2X by exploiting the information obtained from CAMs to estimate the network load. Thus, the protocol will be able to propose a redundant mode based on the use of RF and V-VLC together in the case when the network load is low.

Figure 1 .

 1 Figure 1. Vehicle LED-based light

Figure 2 .

 2 Figure 2. A LOS & NLOS V-VLC scenario

Figure 3 .Figure 4 .

 34 Figure 3. Communication strategy based on RF-VLC

Figure 5 .

 5 Figure 5. Platoon scenario with RF based on AS-DTMAC

Figure 6 .

 6 Figure 6. Probability of a platoon of 10 vehicles completely reserved before slot N+1

Figure 7 .

 7 Figure 7. Probability of detecting & decoding versus RX-TX vehicle interdistance

Figure 8 .Figure 9 .

 89 Figure 8. Probability of detecting & decoding versus Rayleigh-fading variance σ

Figure 11 .

 11 Figure 11. A highway scenario with a platoon in the lower lane, the other lanes are for connecting vehicles.

Figure 12 .

 12 Figure 12. V-VLC PDR versus number of disturbing vehicles: Simulation time = 116s; platoon size = 8; platoon inter-vehicle distance = 10 m; Disturbing vehicles' speed = 100 km/h ; Platoon vehicles' speed = 15 km/h.

Figure 13 .

 13 Figure 13. V-VLC PDR versus disturbing vehicle speed: Simulation time = 208s; platoon size = 8; platoon inter-vehicle distance = 10 m; Disturbing vehicles' speed = 100 km/h; Platoon vehicles' speed = 15 km/h; Headlight module = "HbSedan2".

Figure 14 .

 14 Figure 14. V-VLC PDR versus platooning vehicle inter-distance: Simulation time = 116 s; platoon size = 8; Platoon vehicles' speed = 15 km/h; Disturbing vehicles' speed = 100 km/h; Headlight module = "HbSedan2".

Figure 15 .

 15 Figure 15. V-VLC delay versus platoon beacon packet length

Figure 16 .

 16 Figure 16. V-VLC and RF PDR versus number of disturbing vehicles

TABLE 1 .

 1 Simulation parameters

	Parameter	Value
	PacketBytelength	1024byte
	BeaconingFrequency	10Hz
	Channel model	veins-vlc
	Car-Following-Model	CACC
	Bitrate	1Mbps
	Disturbing vehicles speed	50-120km/h


Karim Mrouj

Nuria Andrés-Sánchez

Geronimo Dubra

Priyanka Singh

Michal Sobecki

Dhanvantri Chahar

Emile Al Ghoul

Ana Bella Aznar

Susana Prieto

Nelly Pirot

Florence Bernex

Benoit Bordignon

Cedric Hassen-Khodja

Martin Villalba

Liliana Krasinska

Daniel Fisher
email: daniel.fisher@igmm.cnrs.fr

Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Ki-67 is a nuclear protein expressed only in proliferating vertebrate cells, a property underlying its widespread use in oncology as a biomarker [START_REF] Endl | The Ki-67 protein: fascinating forms and an unknown function[END_REF]. Its expression is routinely assessed in histopathology to grade tumours; there are also indications for its use as a prognostic marker [START_REF] Cuzick | Prognostic value of a combined estrogen receptor, progesterone receptor, Ki-67, and human epidermal growth factor receptor 2 immunohistochemical score and comparison with the Genomic Health recurrence score in early breast cancer[END_REF], although uncertainty over the relationship between Ki-67 index and prognosis remains. The cellular functions of Ki-67 are not well understood, and whether it is involved in tumourigenesis is unclear.

For a long time, Ki-67 was thought to be required for cell proliferation [START_REF] Kausch | Antisense treatment against Ki-67 mRNA inhibits proliferation and tumor growth in vitro and in vivo[END_REF][START_REF] Rahmanzadeh | Chromophore-assisted light inactivation of pKi-67 leads to inhibition of ribosomal RNA synthesis[END_REF][START_REF] Schluter | The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins[END_REF][START_REF] Starborg | The murine Ki-67 cell proliferation antigen accumulates in the nucleolar and heterochromatic regions of interphase cells and at the periphery of the mitotic chromosomes in a process essential for cell cycle progression[END_REF][START_REF] Zheng | Inhibition of renal cancer cell growth in vitro and in vivo with oncolytic adenovirus armed short hairpin RNA targeting Ki-67 encoding mRNA[END_REF][START_REF] Zheng | Knockdown of Ki-67 by small interfering RNA leads to inhibition of proliferation and induction of apoptosis in human renal carcinoma cells[END_REF] and early work suggested that it promotes ribosomal RNA transcription [START_REF] Rahmanzadeh | Chromophore-assisted light inactivation of pKi-67 leads to inhibition of ribosomal RNA synthesis[END_REF][START_REF] Booth | Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery[END_REF]. However, recent genetic studies have shown that despite promoting formation of the perichromosomal layer of mitotic chromosomes [START_REF] Booth | Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery[END_REF][START_REF] Cuylen | Ki-67 acts as a biological surfactant to disperse mitotic chromosomes[END_REF][START_REF] Sobecki | The cell proliferation antigen Ki-67 organises heterochromatin[END_REF][START_REF] Takagi | Perichromosomal protein Ki67 supports mitotic chromosome architecture[END_REF], it is not required for cell proliferation [START_REF] Cuylen | Ki-67 acts as a biological surfactant to disperse mitotic chromosomes[END_REF][START_REF] Sobecki | The cell proliferation antigen Ki-67 organises heterochromatin[END_REF][START_REF] Takagi | Perichromosomal protein Ki67 supports mitotic chromosome architecture[END_REF][START_REF] Cidado | Ki-67 is required for maintenance of cancer stem cells but not cell proliferation[END_REF]. It is also dispensable for ribosomal RNA synthesis and processing, and mice lacking Ki-67 develop and age normally [START_REF] Sobecki | The cell proliferation antigen Ki-67 organises heterochromatin[END_REF]. In addition, Ki-67 is not overexpressed in cancers; rather, Ki-67 expression is controlled by cell cycle regulators, including CDKs, the activating subunit of the ubiquitin ligase APC/C-CDH1, which is required for destruction of mitotic cyclins, and the cell cycle transcription factor B-Myb [START_REF] Sobecki | The cell proliferation antigen Ki-67 organises heterochromatin[END_REF][START_REF] Sobecki | Cell-Cycle Regulation Accounts for Variability in Ki-67 Expression Levels[END_REF][START_REF] Miller | Ki67 is a Graded Rather than a Binary Marker of Proliferation versus Quiescence[END_REF]. These recent studies raise the question of whether Ki-67 plays any role in tumourigenesis, which has not been addressed genetically.

Despite not being essential for cell proliferation, Ki-67 might be important in carcinogenesis for other reasons. We previously identified over 50 Ki-67-interacting proteins that are involved in transcription and chromatin regulation. We also found that Ki-67 organises heterochromatin: in NIH3T3 cells with TALEN-mediated biallelic disruption of the Ki-67 gene, or human cancer cells with stable knockdown of Ki-67, repressive histone marks H3K9me3 and H4K20me3 are dispersed, the heterochromatin is less compact, and association between centromeres and nucleoli is disrupted. Conversely, overexpression of Ki-67 caused ectopic heterochromatin formation [START_REF] Sobecki | The cell proliferation antigen Ki-67 organises heterochromatin[END_REF].

Involvement of Ki-67 in chromatin organisation was corroborated by a study showing that Ki-67 is required to maintain heterochromatin marks at inactive X-chromosomes in non-transformed cells [START_REF] Sun | Ki-67 Contributes to Normal Cell Cycle Progression and Inactive X Heterochromatin in p21 Checkpoint-Proficient Human Cells[END_REF]. Heterochromatin is a phenotypic marker of multiple cancers [START_REF] Norton | Perinucleolar compartment prevalence is a phenotypic pancancer marker of malignancy[END_REF], suggesting that it might be involved in carcinogenesis. We thus tested whether and how Ki-67 is required for different steps of carcinogenesis.

RESULTS

Ki-67 is dispensable in cancer cell lines yet is rarely mutated in human cancers.

Ki-67 expression is widely used as a marker for cell proliferation in cancer, but whether it is important for carcinogenesis is unclear. We previously found no adverse effects on cell proliferation of either shRNA-mediated knockdown of Ki-67 in U2OS or HeLa cells, or TALENmediated disruption of the gene encoding Ki-67 in NIH/3T3 cells [START_REF] Sobecki | The cell proliferation antigen Ki-67 organises heterochromatin[END_REF]. To see whether this is true across different cancer types, we interrogated the Cancer Dependency Map project DepMap (https://depmap.org/portal/) [START_REF] Tsherniak | Defining a Cancer Dependency Map[END_REF] using the latest CRISPR (Avana 20Q1) and RNAi (Broad Institute) datasets. As controls, we compared Ki-67 with two proto-oncogenes that have roles in cell proliferation, MYC and CCND1, encoding c-Myc and cyclin D1, respectively, and with PCNA, encoding Proliferating Cell Nuclear Antigen, whose expression profile is similar to that of Ki-67. Ki-67, PCNA and c-Myc were universally expressed, while cyclin D1 was expressed in most cell lines, though not all (Fig. 1A), possibly due to compensation by a different D-type cyclin. In CRISPR-Cas9 screens, PCNA was essential for proliferation of 775 of 777 cell lines tested, while 719 out of 726 lines required c-Myc, and 579 out of 726 required cyclin D1 (Fig. 1B). In contrast, Ki-67 knockout did not affect cell proliferation in 725 out of 739 cell lines (Fig. 1B), confirming that it is generally dispensable for cell proliferation in human cancer cells. Nevertheless, like PCNA, MKI67

showed almost no copy number variations among the different cell lines, in contrast to the two cancer driver oncogenes MYC and CCND1, which were frequently amplified (Fig. 1C). The same is true in data from clinical samples of cancer patients in the cBioPortal database, which further showed that only 5% of cancers presented mutations in MKI67 (Fig. 1D). Almost all of these were mis-sense mutations, which might be passenger mutations.

Thus, even though Ki-67 is dispensable for cell proliferation in virtually all cancer cells, it is ubiquitously expressed at similar levels in all cancers, corroborating our previous findings that variability in Ki-67 expression is accounted for by its regulation through the cell cycle [START_REF] Sobecki | Cell-Cycle Regulation Accounts for Variability in Ki-67 Expression Levels[END_REF].

Together, these observations suggest that Ki-67 may provide benefit to cancer cells. In contrast, its overexpression may be counter-selected in cancers, which would fit with our finding that increased levels of Ki-67 arrest cell proliferation [START_REF] Sobecki | The cell proliferation antigen Ki-67 organises heterochromatin[END_REF].

The lack of overexpression or deletion of Ki-67 in cancers implies that correlating Ki-67 expression with patient survival is likely to simply reflect the impact of the fraction of proliferating cells. If so, then PCNA expression should give similar results. To address this question, we queried survival correlations in different cancer types with MKI67 and PCNA mRNA levels in TCGA expression data using OncoLnc [START_REF] Anaya | OncoLnc: linking TCGA survival data to mRNAs, miRNAs, and lncRNAs[END_REF]. For both genes, there was either no correlation (breast and colorectal cancer), a modest positive correlation (lung cancer), or a strong negative correlation (liver cancer and renal cancer) of expression levels with survival (SI Appendix Fig. S1). Thus, in several different cancers, despite the contrasting requirements for PCNA and Ki-67 for cell proliferation, the correlation of expression of each gene with survival is very similar.

Mice lacking Ki-67 are resistant to intestinal tumourigenesis.

The fact that Ki-67 is ubiquitously expressed in cancers but is not required for cell proliferation raises the question of whether it has functional roles in carcinogenesis. To see whether Ki-67 knockout affects initiation of tumourigenesis in vivo, we used a germline TALEN-disruption of Ki-67 (Mki67 2nt∆/2nt∆ ) that we generated [START_REF] Sobecki | The cell proliferation antigen Ki-67 organises heterochromatin[END_REF]. We first employed chemical induction of colon carcinogenesis by azoxymethane / dextran sodium sulphate (AOM-DSS) treatment [START_REF] Robertis | The AOM/DSS murine model for the study of colon carcinogenesis: From pathways to diagnosis and therapy studies[END_REF] in WT and Mki67 2nt∆/2nt∆ mice. We observed that DSS alone induced a similar decrease in body weight in control and mutated mice compared to the controls (SI Appendix Fig. S2A). Histopathological examination of colonic sections revealed a typical DSS-induced colitis in both genotypes (SI Appendix Fig. S2B), with increased numbers of immune cells in the lamina propria, moderate crypt cells damage and hyperplasia. As expected, AOM-DSS efficiently induced colon tumours within 16 weeks in both wild-type and Mki67 2nt∆/+ mice (indeed, heterozygous mice had bigger lesions; future studies will address effects of Ki-67 gene dosage on tumour growth). However, no macroscopic lesions were observed in Mki67 2nt∆/2nt∆ mice (Fig. 2A,B). This suggests that Ki-67 is specifically required for initiation of tumourigenesis. To confirm these findings, we used a genetic model of intestinal tumourigenesis. We crossed Mki67 2nt∆/2nt∆ mice with Apc ∆14/+ mice, which rapidly develop tumours in the intestine due to loss of the second allele of the Apc tumour suppressor gene [START_REF] Colnot | Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers[END_REF]. While, as expected, Apc ∆14/+ Mki67 2nt∆/+ mice formed multiple colon tumours, tumour burden was strongly reduced in Apc ∆14/+ Mki67 2nt∆/2nt∆ mice (Fig. 2C,D). Thus, Ki-67 is required for efficient initiation of tumourigenesis induced by chemical mutagenesis or loss of a tumour suppressor in vivo, suggesting that it might be required for cell transformation. To test this, we transduced our previously generated Mki67 +/+ or TALEN-mutated Mki67 -/-NIH/3T3 fibroblasts [START_REF] Sobecki | The cell proliferation antigen Ki-67 organises heterochromatin[END_REF] with oncogenic mutant H-Ras (G12V), and evaluated colony formation as an indicator of transformation. While H-Ras G12V -transduced control 3T3 cells efficiently formed colonies, Mki67 -/-3T3 cells did not, despite having similar rates of cell proliferation (SI Appendix Fig. S3). This indicates that Ki-67 expression facilitates oncogeneinduced transformation in these cells.

Loss of Ki-67 causes global transcriptome changes and deregulates pathways involved in cancer.

Since we previously found that knockdown of Ki-67 in cancer cells altered their chromatin organisation and affected gene expression [START_REF] Sobecki | The cell proliferation antigen Ki-67 organises heterochromatin[END_REF], we hypothesised that the resistance of Ki-67 knockout cells to transformation might also result from gene expression changes. We first performed RNA-sequencing analysis of Ki-67 wild-type and knockout NIH/3T3 cells. This revealed surprisingly wide-ranging effects of Ki-67 loss on the transcriptome, with 2558 genes significantly deregulated in independent clones of Mki67 -/-cells (q < 0.05) (Fig. 3A; Dataset S1). This level of transcriptome alteration suggested a global effect on chromatin rather than a direct involvement of Ki-67 in controlling specific pathways or transcription factors, which is consistent with our previous finding that Ki-67 interacts with many general chromatin regulators in the U2OS cancer cell line [START_REF] Sobecki | The cell proliferation antigen Ki-67 organises heterochromatin[END_REF]. We therefore expected that Ki-67 knockout would also extensively affect the transcriptome of established cancer cells, with possible consequences for tumourigenicity. To investigate this, we used the syngeneic 4T1 mouse mammary carcinoma model, which is derived from BALB/c mice. This cell line mimics human triple-negative breast cancer, is highly invasive and spontaneously metastasises to distant organs [START_REF] Dexter | Heterogeneity of tumor cells from a single mouse mammary tumor[END_REF][START_REF] Heppner | Nontransgenic models of breast cancer[END_REF]. As expected, 4T1 cell proliferation rates were unaffected by CRISPR-Cas9-mediated Mki67 gene knockout (SI Appendix Fig. S4A-D).

In 4T1 cells, Mki67 knockout caused even more extensive gene expression alterations than in NIH/3T3 cells: 4979 genes were deregulated, of which 1239 and 585 genes were >2-fold downregulated and up-regulated, respectively (Fig. 3B,C; Dataset S2). There was little overlap in the deregulated genes between Mki67 -/-4T1 (epithelial) and NIH/3T3 (mesenchymal) cells (Fig. 3C, Tables S1,S2) in accordance with our hypothesis that, by organising chromatin, Ki-67 enables global gene regulation in different cell types rather than directly controlling specific genes.

To see whether the global effect of Ki-67 knockout on gene expression is conserved across cancer cell types and species, we next disrupted the MKI67 gene by CRISPR-Cas9 in human MDA-MB-231 triple negative breast cancer cells (SI Appendix Fig. S5A,B), which is a highly mesenchymal-like cell line due to an extensive epithelial-mesenchymal transition (EMT). As expected, MKI67 -/-MDA-MB-231 cells proliferated normally in vitro (SI Appendix Fig. S5C-D).

Transcriptome analysis by RNA-seq showed that Ki-67 knockout in this cell line also caused genome-scale alterations in gene expression (Fig. 3D), with 9127 genes deregulated, 914 of which were up-or downregulated by a factor of >2 (Dataset S3).

We investigated whether the extensive transcriptome changes seen in cancer cells upon Ki-67 knockout affect pathways involved in tumourigenesis. In 4T1 cells, bioinformatic analysis of the most up-and downregulated genes revealed deregulation of various components of inflammation, apoptosis, p53, the EMT, estrogen response, K-Ras signalling and hypoxia (Fig. 3E).

We also manually analysed transcriptome data and noticed upregulation of the Notch pathway, downregulation of the EMT, the Wnt pathway, antigen presentation, and aldehyde metabolism, which we validated by qRT-PCR (Fig. 3F). Downregulated genes were enriched in targets of nuclear factor erythroid 2-related factor 2 (NFE2L2), one of the major orchestrators of responses to oxidative stress; polycomb-repression complex 2 (PRC2), which mediates Histone H3 lysine-27 trimethylation (H3K27me3) and is a well-characterised regulator of the EMT [START_REF] Tiwari | Sox4 is a master regulator of epithelial-mesenchymal transition by controlling Ezh2 expression and epigenetic reprogramming[END_REF][START_REF] Chase | Aberrations of EZH2 in Cancer[END_REF]; the pluripotency factors Nanog and Sox2; and interferon regulatory factor 8 (IRF8) (Fig. 3G). All of these pathways have previously been implicated in tumourigenesis. In MDA-MB-231 cells, like 4T1 cells, pathway analysis also revealed genes involved in the EMT, inflammatory response, early estrogen response, K-RAS signalling and hypoxia, while a significant portion of the deregulated genes were under the control of PRC2 and ESR1 (estrogen receptor 1; Fig. 3H,I and Dataset S3). In summary, similar pathways involved in cancer are affected upon Ki-67 knockout in different cancer cell lines.

The prevalence of downregulation of gene expression in Ki-67 knockout cells, the enrichment in PRC2 targets among these genes, and our previous observations that Ki-67 associates with the essential PRC2 component SUZ12 [START_REF] Sobecki | The cell proliferation antigen Ki-67 organises heterochromatin[END_REF], prompted us to ask whether loss of Ki-67 affects genome-wide distribution of H3K27me3. To answer this question, we performed chromatin immunoprecipitation with high-throughput sequencing (ChIP-Seq) on WT and Ki-67 knockout 4T1 cells. While there were no genome-wide changes in H3K27me3 distribution, a substantial subfraction of genes showed an increase in this mark (SI Appendix Fig. 6A). This was particularly evident for genes strongly repressed in Ki-67 knockout cells (Fig. 3J, left). To investigate correlations between changes in the levels of histone modifications and of gene expression, we assigned an average value of the repressive H3K9me3, as well as activatory H3K4me3 and H3K27ac reads across 10kb surrounding the transcription start site, and plotted the differences in these values between wild-type and Ki-67 knockout cells with gene expression changes. This showed a strong correlation between the change in gene expression and H3K27me3 and H3K4me3 (Figure 3K), while H3K27ac levels only correlated well with the most highly downregulated genes (SI Appendix Fig. S6B). Of 9 genes whose downregulated expression in Ki-67 knockout cells we confirmed by qRT-PCR, we only found an obvious increase of H3K27me3 on the EMT-promoting transcription factor Twist1. This correlated with downregulation of active promoter-associated H3K4me3 and H3K27ac (Fig. 3L). There was also a slight increase of H3K27me3 and reduction in H3K27ac at the Vimentin promoter (SI Appendix Fig. S6C). Taken together, these results suggest that Ki-67 loss generally increases PRC2-mediated repressive histone marks at downregulated genes, but most of the expression alterations resulting from Ki-67 knockout may not be directly due to modulation by PRC2. Instead, they are likely knock-on effects of altered expression of other transcriptional regulators that result in changes in active promoter-associated histone marks.

Ki-67 promotes stem cell characteristics and controls the EMT in mammary carcinoma.

We next investigated the biological consequences resulting from these global transcriptome alterations in cancer cells lacking Ki-67. Importantly, although the Notch pathway is oncogenic in T-cell acute lymphoid leukaemia, it can act as a tumour suppressor in specific cellular contexts [START_REF] Nowell | Notch as a tumour suppressor[END_REF], can block Wnt signalling [START_REF] Nicolas | Notch1 functions as a tumor suppressor in mouse skin[END_REF][START_REF] Devgan | p21WAF1/Cip1 is a negative transcriptional regulator of Wnt4 expression downstream of Notch1 activation[END_REF] (a driver of tumourigenesis, cell stemness and the EMT) and induce drug resistance [START_REF] Meng | γ-Secretase Inhibitors Abrogate Oxaliplatin-Induced Activation of the Notch-1 Signaling Pathway in Colon Cancer Cells Resulting in Enhanced Chemosensitivity[END_REF]. We confirmed Notch pathway upregulation at the protein level (Fig. 4A). While the EMT is closely associated with a stem-like state [START_REF] Mani | The epithelial-mesenchymal transition generates cells with properties of stem cells[END_REF][START_REF] Morel | Generation of breast cancer stem cells through epithelial-mesenchymal transition[END_REF][START_REF] Wellner | The EMT-activator ZEB1 promotes tumorigenicity by repressing stemnessinhibiting microRNAs[END_REF], the most stem-like states appear to show a hybrid expression of both epithelial and mesenchymal characteristics, with the most epithelial and mesenchymal cells losing stemness [START_REF] Kröger | Acquisition of a hybrid E/M state is essential for tumorigenicity of basal breast cancer cells[END_REF][START_REF] Pastushenko | Identification of the tumour transition states occurring during EMT[END_REF][START_REF] Ye | Distinct EMT programs control normal mammary stem cells and tumourinitiating cells[END_REF]. We found that 4T1 cells express both E-cadherin and vimentin, suggesting a highly stem-like state (Fig. 4B), but Ki-67 knockout 4T1 cells had reduced expression of the mesenchymal marker vimentin and upregulated E-cadherin (Fig. 4B,C). To see whether this translates to a loss of stemlike character, we analysed aldehyde dehydrogenase (ALDH) activity, which is a bona fide marker of stem and progenitor cells [START_REF] Ginestier | ALDH1 Is a Marker of Normal and Malignant Human Mammary Stem Cells and a Predictor of Poor Clinical Outcome[END_REF][START_REF] Huang | Aldehyde Dehydrogenase 1 Is a Marker for Normal and Malignant Human Colonic Stem Cells (SC) and Tracks SC Overpopulation during Colon Tumorigenesis[END_REF]. ALDH activity was strongly reduced in 4T1 Ki-67 knockout cells (Fig. 4D). Furthermore, the ability to form spheroids in the absence of adhesion to a surface, another characteristic of stem cells [START_REF] Mani | The epithelial-mesenchymal transition generates cells with properties of stem cells[END_REF][START_REF] Ishiguro | Tumor-derived spheroids: Relevance to cancer stem cells and clinical applications[END_REF] was also largely decreased (Fig. 4E).

To test whether repression of the EMT in Ki-67 knockouts depends on PRC2, we additionally disrupted PRC2 components Suz12 or Ezh2 in WT and Mki67 -/-4T1 cells using CRISPR-Cas9 (SI Appendix Fig. S7A). This partially rescued vimentin expression, indicative of an increased ability to undergo an EMT (SI Appendix Fig. S7B), but did not restore it to levels observed in WT cells. This corroborates the results of the H3K27me3 ChIP-seq analysis described above, indicating that the influence of PRC2 activity on gene expression changes observed in Ki-67 knockout cells is limited.

Analysis of EMT markers in MDA-MB-231 cells showed that Ki-67 knockout cells retained a mesenchymal character, with vimentin but no E-cadherin staining (Fig. 4B,C), suggesting that they are far from a hybrid-like EMT state. Importantly, Ki-67 knockout cells had even higher vimentin expression, implying a further distance from the putative stem-like hybrid state. In agreement with this hypothesis, neither WT nor Ki-67 knockout MDA-MB-231 cells showed significant ALDH activity (SI Appendix Fig. S8A). Importantly, Ki-67 knockout cells were unable to form mammospheres, indicating a conserved requirement for Ki-67 in maintaining ability to seed formation of new cell colonies (SI Appendix Fig. S8B). As such, loss of Ki-67 reverses cells to a more epithelial or a more mesenchymal state, depending on the cell type of origin. This perturbation of the EMT correlates with a reduction of stem-like character in different cell lines.

Ki-67 expression promotes tumour growth.

To determine whether these phenotypic alterations affect the tumourigenicity of cancer cells, we first engrafted WT and Ki-67 knockout 4T1 cells orthotopically into mouse mammary fat pads.

Since Ki-67 knockout caused alteration of inflammatory response genes (Fig. 3E), we initially used athymic-nude and NOD/SCID mice, allowing us to assess roles of Ki-67 in tumour growth and metastasis while minimising possible confounding effects of an altered immune response. RNAseq of early-stage tumours from WT and Ki-67 mutant 4T1 cells grafted into nude mice showed that Ki-67-dependent transcriptome changes were well preserved in vivo (Fig. 5A, Dataset S4), including down-regulation of mesenchymal genes, upregulation of epithelial genes and of the Notch pathway, which was validated by increased HES1 staining in tumours (SI Appendix Fig. S9A). Reduced vimentin staining of Ki-67-mutant tumours confirmed the loss of EMT in vivo (Fig. 5B). As assessed by PCNA and phospho-histone H3S10 staining, cell proliferation in vivo was unaffected by loss of Ki-67 (Fig. 5C,D). However, tumours from Mki67 -/-4T1 cells grew significantly slower than from control cells in both types of immunodeficient mice (Fig. 5E,F).

There were no apparent differences in apoptosis upon Ki-67 knockout in 4T1 cells in vitro (SI Appendix Fig. S9B), and, while there was significantly increased apoptosis in one of the two 4T1 Ki-67 knockout clones in vivo (SI Appendix Fig. S9C,D), both clones had similarly slowed tumour progression. Analysis of necrosis revealed variability between tumours and clones (SI Appendix Fig. S9E,F). As assessed by -H2A.X staining, we did not find evidence for increased DNA damage in Ki-67 knockout cells nor tumours (SI Appendix Fig. S9G,H). Thus, in vivo differences in tumour growth between WT and Ki-67 knockout tumours cannot be explained by either reduced cell proliferation or by increased DNA damage or cell death. It is theoretically possible that the slower tumour growth of Ki-67 knockouts might be explained by a marginally lower intrinsic cell proliferation rate that we did not detect when culturing WT and knockout cell lines individually, but might become visible over longer time-scales and when co-cultured with wild type cells. To test this, we performed competition experiments in vitro between WT and knockout 4T1 cells over 12 days. We found that the initial 50/50 ratio of wild-type and Ki-67 knockout 4T1 cells was maintained after two weeks of co-culture (SI Appendix Fig. S10A-C), ruling out this possible explanation.

Next, since Ki-67 4T1 knockout cells had downregulated expression of several angiogenic factors, including angiopoietin-1, both in cell culture and in tumours (Dataset S2, S4), we analysed angiogenesis in WT and Ki-67 knockout tumours by CD31-staining of endothelial cells. Indeed, we found that the mean blood vessel density was significantly reduced in tumours from both Ki-67 knockout clones in NOD/SCID mice (Fig. 5G,H). Tumours lacking Ki-67 also appeared more fibrotic than controls, as indicated by Sirius-red staining of collagen (Fig. 5I). However, blood vessel density was comparable between wild-type and Ki-67 knockout tumours in nude mice (Fig. 5J). Moreover, the distribution of vessels of different sizes was similar between all genotypes and mouse backgrounds (SI Appendix Fig. S9I). Therefore, while we cannot rule out a contribution of either reduced or altered angiogenesis to the slow growth of knockout tumours, this cannot explain the differences seen in all situations.

We also analysed tumours from xenografts of wild-type and Ki-67 mutant MDA-MB-231 cells in nude mice. We similarly observed reduced tumour growth rate despite normal cell proliferation (SI Appendix Fig. S11A-C). As with 4T1 cells, there was no detectable increase in apoptosis in MDA-MB-231 cells lacking Ki-67 in vitro (SI Appendix Fig. S9B). However, apoptosis, but not DNA damage (SI Appendix Fig. S11D), was significantly increased in Ki-67 knockout MDA-MB-231 tumours (SI Appendix Fig. S11E), necrosis was reduced (SI Appendix Fig. S11F), fibrosis was more apparent (SI Appendix Fig. S11G), while mean blood vessel density and distribution of vessels of different sizes was comparable with control tumours (SI Appendix Fig. S11H,I).

To assess the generality of these observations in a different tumour type, we stably knocked down Ki-67 by shRNA in a commonly used aggressive human cervical cancer cell line, HeLa S3, and grafted the resulting cells and an shRNA control line into opposing flanks of athymic nude mice. Ki-67 knockdown was maintained in vivo (SI Appendix Fig. S12A,B). Again, tumour growth was severely impaired (SI Appendix Fig. S12C), despite unaffected cell proliferation (as shown by unchanged PCNA and mitotic indices; SI Appendix Fig. S12D,E), while there was increased necrosis and apoptosis (SI Appendix Fig. S12F,G). It is interesting to note that shRNA of Ki-67 was sufficient to induce strong phenotypes.

In conclusion, Ki-67 knockout or knockdown in several different established cancer cell lines consistently results in slower tumour growth upon grafts in mice despite unchanged cell proliferation, and often leads to non-cell autonomous increases in apoptosis or fibrosis, with necrosis and angiogenesis more variably affected. However, none of these plausible explanations for the reduced tumour growth is true in all experimental situations. These results show that effects of Ki-67 loss are wide-ranging and multifactorial.

Lastly, since many of the genes repressed in the absence of Ki-67 are under the control of PRC2 and concurrent knock-out of PRC2 genes partly restores the EMT to Ki-67 KO cells (SI Appendix Fig. S7), we tested whether the inactivation of PRC2 in cells lacking Ki-67 could restore tumour growth. We injected Suz12 -/-, Mki67 -/-and Suz12 -/-Mki67 -/-4T1 cells orthotopically into nude mice, and found that Suz12 knockout did not restore Mki67 -/-4T1 tumour growth rates, nor affect tumourigenicity of control cells (SI Appendix Fig. S13). As such, PRC2 contributes to suppressing the EMT in the absence of Ki-67, but it does not in itself have either pro-or anti-tumour activity in this context, in contrast to other systems [START_REF] Chase | Aberrations of EZH2 in Cancer[END_REF][START_REF] Bracken | Polycomb group proteins: navigators of lineage pathways led astray in cancer[END_REF]. This result also suggests that Ki-67 roles in tumourigenesis depend on several chromatin regulatory pathways, consistent with it being a hub for interactions with multiple chromatin regulators.

Ki-67 promotes metastasis but enables efficient anti-tumour immune responses.

We observed that in orthotopic grafts, control 4T1 cells metastasised to the lungs in 4 weeks in nude mice, but metastases were largely absent at this point in mice bearing Mki67 -/-tumours (Fig. 6A). We tested whether this was due to differences in detachment from the primary tumour or ability to seed metastases. To do this, we quantified rates of metastasis formation of control and Ki-67 knockout 4T1 cells by injecting cells directly into the tail vein, then dissociating lung tissue after 3 weeks and growing cells in the presence of 6-thioguanine, to which 4T1 cells are resistant.

The number of metastatic cells that formed colonies was reduced nearly 100-fold in Ki-67 knockouts (Fig. 6B). This points to an essential requirement for Ki-67 in seeding metastasis, in accordance with its apparent role in conferring stem-like characteristics. We could not test whether this reduced metastatic capacity was conserved in MDA-MB-231 cells since, in our experiments, neither Ki-67 knockout nor control MDA-MB-231 xenografts generated any visible lung metastases, consistent with their lack of stem-like character.

We next investigated how Ki-67 expression affects tumourigenesis and metastasis in the context of an intact immune system, by engrafting WT or Mki67 -/-4T1 cells into immune-proficient BALB/c mice. As expected [START_REF] Tao | Imagable 4T1 model for the study of late stage breast cancer[END_REF], control 4T1 tumours established quickly and initially regressed before regrowing (Fig. 6C). This initial regression has been attributed to a strong anti-tumour immune response [START_REF] Tao | Imagable 4T1 model for the study of late stage breast cancer[END_REF]. Surprisingly, no such initial regression occurred when Mki67 -/-4T1 cells were engrafted (Fig. 6C), suggesting that Ki-67 knockout cells fail to trigger an efficient immune response. Nevertheless, despite the consequently higher tumour burden, there were either similar or less metastases than from control 4T1 cells (Fig. 6D), although differences failed to reach statistical significance (p = 0.13). We surmised that injection of 4T1 cells directly into the circulation via the tail vein should circumvent the immune-targeting of tumour cells at the primary-site, allowing direct assessment of the capacity of cells to establish a metastatic niche. Importantly, WT 4T1 cells again more efficiently colonised lungs in this setting than Mki67 -/-4T1 cells (Fig. 6E), despite the higher overall tumour burden in the latter. Together, the results of these experiments underline the requirement for Ki-67 in seeding metastasis. They also suggest a defective immune response to Ki-67 knockout tumours. In agreement, immunohistological analysis revealed increased infiltration of Myeloid-Derived Suppressor Cells [START_REF] Yang | Myeloid-Derived Suppressor Cells in Tumors: From Mechanisms to Antigen Specificity and Microenvironmental Regulation[END_REF] in knockout tumours (using the MDSC marker, GR1; Fig. 6F), possibly indicating an immunosuppressive environment that protects the tumours against immune-mediated cytotoxicity. To investigate why Ki-67 knockout cells fail to induce an efficient anti-tumour immune response, we analysed the expression of the mouse major histocompatibility complex (MHC) class I, responsible for antigen presentation. Transcriptome analysis showed downregulated expression of factors implicated in antigen processing and loading on MHC I: Tapasin, Tap1, Tap2, Psmb8 and Psmb9 in Mki67 -/-4T1 cells, while tumours showed a further downregulation of Erap1 and B2M (Dataset S2, S4 and Fig. 3B,F). Flow cytometry revealed lower expression of MHC class I molecules H2D and H2K (Fig. 6G). MHC class I expression was also downregulated in MKI67 -/-MDA-MB-231 cells (SI Appendix Fig. S14), suggesting that roles of Ki-67 in maintaining MHC expression are conserved across species.

Cells lacking Ki-67 are sensitised to drugs.

The above results show that Ki-67 enables cell transformation, tumour growth and metastasis, yet also confers efficient targeting of tumours by the immune system. Finally, we asked whether Ki-67 would also influence drug responses, as, in addition to stem-like characteristics, the EMT has also been associated with resistance to chemotherapeutic drugs [START_REF] Zheng | Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer[END_REF]. We noticed that 26 genes involved in drug metabolism were downregulated in Ki-67 knockout 4T1 cells, while only one was upregulated, suggesting that Ki-67 expression might affect sensitivity to chemotherapeutic drugs (Fig. 7A, SI Appendix Fig. S15A). To test this, we performed an automated gene-drug screen using the Prestwick chemical library, composed of 1283 FDAapproved small molecules. We also included salinomycin, a positive control found to target cancer stem cells [START_REF] Gupta | Identification of selective inhibitors of cancer stem cells by highthroughput screening[END_REF], and 6-thioguanine, which was originally used to isolate 4T1 cells [START_REF] Dexter | Heterogeneity of tumor cells from a single mouse mammary tumor[END_REF]. Control 4T1 cells were sensitive to 102 drugs at 10µM concentration, while the two Mki67 -/-clones were sensitive to 99 and 98 respectively, with 82 hits common to the three cell lines (SI Appendix Fig. S15B; Dataset S5). This suggests that Ki-67 loss does not qualitatively alter the drugsensitivity profiles. We next determined the IC 50 of 10 hits commonly used in cancer therapy.

Importantly, Mki67 -/-cells were markedly more sensitive to all the molecules tested (Fig. 7B). As such, by supporting expression of xenobiotic metabolism genes, Ki-67 provides cancer cells with a degree of protection against therapeutic drugs.

DISCUSSION

The above results show that Ki-67 is not required for cancer cell proliferation in vitro or in vivo in any cell type tested, but its expression critically influences all steps of tumourigenesis, including initiation, progression and metastasis, as well as immune responses and drug sensitivity. In both human and mouse mammary carcinoma cells, we present evidence that this is because Ki-67 sustains transcriptional programmes needed for tumour cells to adapt to their environment. This is indicated by the failure to generate intestinal tumours in Ki-67-mutant mice, the reduced ability of some Ki-67 knockout cancer cells to induce angiogenesis, the presence of both epithelial and mesenchymal characteristics in Ki-67-positive epithelial cancer cells, which are altered in cells lacking Ki-67, the inability of the latter to colonise other tissues and give rise to metastases, their increased sensitivity to drugs, and their reduced interactions with the immune system. These phenotypes correlate well with alterations of expression of genes involved in the EMT, antigen presentation, drug metabolism, and other cancer-associated hallmarks.

Our data suggest that Ki-67 does not work by directly controlling expression of specific genes, since in Ki-67 knockout mouse fibroblast or epithelial cancer cells there are relatively few common deregulated genes. Instead, we find evidence that Ki-67 regulates general chromatin states that affect expression of genes in fundamental biological processes. This is supported by the fact that gene expression alterations are global, i.e. well within an order of magnitude of the number of genes in the genome, with around 2500 genes altered in non-transformed fibroblasts, nearly 5000 genes deregulated in Ki-67 knockout mouse mammary carcinoma cells, and over 9000 (i.e. the majority of expressed genes) in human mammary cancer cells. We compared changes in expression of genes involved in biological processes that are significantly enriched in Ki-67 knockout 3T3, 4T1, 4T1 tumour grafts and human MDA-MB-231 cells (SI Appendix Fig. S16).

This clearly shows the similarity of affected pathways, and even genes, among these different knockouts, and highlights the fact that within common pathways different individual genes may be affected among different cell types. The similarity between the changes in different mammary carcinoma cells is apparent both in vitro and in tumours.

Our interpretation is also consistent with the extensive alterations in chromatin histone marks. With such wide-ranging transcriptome changes, it is almost impossible to attribute the loss of tumourigenicity to changes in expression of candidate genes, or even single regulatory mechanisms. Indeed, inactivation of the PRC2 complex did not restore tumourigenicity to Ki-67 knockout 4T1 cells. However, similar cancer-associated pathways are deregulated in 4T1 and

MDA-MB-231 cells, suggesting general conservation of regulatory mechanisms.

A role in cancer-promoting transcriptional programmes provides a plausible explanation for the ubiquitous expression of Ki-67 across essentially all cancer types. Indeed, we show that Ki-67 is required for efficient tumour growth, independently of cell proliferation, in different human and mouse cancer cell types. Although to some observers it might appear paradoxical that tumour progression is reduced in Ki-67 knockouts without changes in cell proliferation rates, our data suggest several possible mechanisms. We observed non-cell autonomous effects in vivo, including increases in apoptosis or fibrosis, to which altered angiogenesis could be a contributor.

Other forms of cell death cannot be ruled out, but we did not find a consistent increase in necrosis. Additionally, it is possible that tumours might arise from a smaller number of initiating cells, due to a loss of stem-like character. In support of this, spheroid formation is decreased in both highly epithelial (4T1) and highly mesenchymal (MDA-MB-231) cancer cells. The fact that Ki-67 knockout reduces ability to seed metastases is consistent with this idea.

Whether or not Ki-67 expression maintains cancer stem cells (CSC) as such, is debatable. A recent study in the human colorectal cancer cell line DLD-1 showed that Ki-67 knockout reduces the number of cells expressing the antigen CD133, commonly assumed to be a marker of cancer stem cells [START_REF] Cidado | Ki-67 is required for maintenance of cancer stem cells but not cell proliferation[END_REF]. However, in intestinal crypts, CD133 expression is not specific to stem cells, and CD133-negative cancer cells were equally capable of sustaining tumourigenesis in a long-term serial transplantation model [START_REF] Shmelkov | CD133 expression is not restricted to stem cells, and both CD133+ and CD133-metastatic colon cancer cells initiate tumors[END_REF]. Furthermore, the concept that CSCs are rare pre-existing populations of cells with hard-wired CSC properties, a model which emerged from xenograft experiments using sorted populations of hematopoietic stem cells, no longer appears valid, at least for solid tumours [START_REF] Batlle | Cancer stem cells revisited[END_REF]. Lineage tracing experiments in the intestine showed that tumours arise from stem cells which divide rapidly, make up around 10% of the cell in intestinal crypts, and can be regenerated from non-stem cells [START_REF] Batlle | Cancer stem cells revisited[END_REF]. A more recent concept is that epithelial cells are phenotypically plastic, in a manner dependent on EMT-inducing transcription factors [START_REF] Chaffer | Poised chromatin at the ZEB1 promoter enables breast cancer cell plasticity and enhances tumorigenicity[END_REF], with no such fixed entity as CSC or non-CSC. Emerging evidence suggests that cells can reside in various phenotypic stages along the EMT-spectrum with concurrent expression of epithelial and mesenchymal traits, and that such hybrid states are important for carcinogenesis [START_REF] Kröger | Acquisition of a hybrid E/M state is essential for tumorigenicity of basal breast cancer cells[END_REF][START_REF] Pastushenko | Identification of the tumour transition states occurring during EMT[END_REF][START_REF] Ye | Distinct EMT programs control normal mammary stem cells and tumourinitiating cells[END_REF]. Our data showing that ALDH activity, which is a bona fide marker for intestinal stem cells and breast and colon CSC [START_REF] Ginestier | ALDH1 Is a Marker of Normal and Malignant Human Mammary Stem Cells and a Predictor of Poor Clinical Outcome[END_REF][START_REF] Huang | Aldehyde Dehydrogenase 1 Is a Marker for Normal and Malignant Human Colonic Stem Cells (SC) and Tracks SC Overpopulation during Colon Tumorigenesis[END_REF], is expressed by a significant fraction (≈10%) of WT epithelial cancer cells but far fewer (<3%) Ki-67 knockout cells, are consistent with this model.

The EMT and stemness have previously been correlated with drug metabolism [START_REF] Zheng | Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer[END_REF][START_REF] Shibue | CSCs, and drug resistance: the mechanistic link and clinical implications[END_REF].

We find that, in cancer cells with a reversion of the EMT, Ki-67 loss leads to a reduced expression of genes encoding xenobiotic metabolism factors, which translates into increased sensitivity to all drugs tested in 4T1 cells. This is likely a conserved phenotype of Ki-67 loss, since Ki-67 knockout HeLa, DLD1 and MCF10A cells were also found to be more sensitive to all drugs tested [START_REF] Cuylen | Ki-67 acts as a biological surfactant to disperse mitotic chromosomes[END_REF][START_REF] Cidado | Ki-67 is required for maintenance of cancer stem cells but not cell proliferation[END_REF].

Previous studies of links between stem cell characteristics and carcinogenesis have focused on transcriptional states, converging on Myc-regulated targets as the strongest link between stem cell and cancer cell transcriptional signatures [START_REF] Kim | A Myc network accounts for similarities between embryonic stem and cancer cell transcription programs[END_REF]. However, while in Ki-67 knockout NIH/3T3 cells Myc was slightly downregulated (Dataset S1), it was significantly upregulated in 4T1 cells (Dataset S2) ruling out the possibility that the reduced oncogenicity of these cells is simply due to loss of Myc, and again favouring a global effect of Ki-67 loss on the chromatin state.

Our hypothesis is consistent with our previous identification of many general chromatin regulators interacting with Ki-67, including components of PRC1 and PRC2, REST, NuRD, NIF1, NuA4, MLL, SET1, NoRC and NCOA6 complexes [START_REF] Sobecki | The cell proliferation antigen Ki-67 organises heterochromatin[END_REF]. Disrupting one such interactor, the PRC2 complex, partially, but not fully, rescued mesenchymal traits in Ki-67 mutant epithelial cancer cells, consistent with the idea that Ki-67 acts through multiple chromatin regulatory complexes.

In accordance, loss of PRC2 did not restore tumourigenicity to Mki67 -/-4T1 cells. Identifying the exact biochemical mechanisms by which Ki-67 affects the chromatin state and gene expression will require further studies. Ki-67 has no enzymatic activity and appears to be a largely intrinsically disordered protein, potentially providing hub-like properties for protein-protein interactions.

How this confers the ability to adapt to the environment is not clear, but one can speculate that its binding of a large number of general transcription regulators is involved in maintenance of meta-stable states between different transcriptional programmes -in other words, transcriptional plasticity.

In conclusion, Ki-67, which is universally expressed in proliferating cells, enables multiple steps in carcinogenesis in different cancer types that require drastic changes in transcriptional programmes. Therapeutic targeting of Ki-67 itself will likely be challenging, since it is an intrinsically-disordered protein with no inherent enzyme activity. However, it could be of therapeutic benefit to inhibit its effectors in control of cellular adaptation to the environment. Alternatively, our results also suggest that Ki-67 confers an Achilles' heel to cancer cells, namely, their recognition by the immune-system. It will be interesting to see if non-proliferating cells, which do not express Ki-67, are consequently more resistant to immune-mediated killing. If this is the case, then, paradoxically, it might be advantageous to promote, rather than hinder, cell proliferation in order for immunotherapy to be optimally effective.

MATERIALS AND METHODS

Details of all materials and methods can be found in the SI Appendix. Materials include all animals, cell lines, antibodies, plasmids, PCR primers and reagents. Methods include CRISPR/Cas9-mediated genome editing, AOM-DSS-mediated colon carcinogenesis, DNA replication assay, Mammosphere assay, Aldehyde Dehydrogenase (ALDH) activity assay, Xenografts, Visualisation of lung metastases, Cell extracts and Western-blotting, Histology and immunostaining, qRT-PCR, Colony formation assay, RNA sequencing library preparation and sequencing, Chromatin Immunoprecipitation and sequencing (ChIP-Seq), Sequence data processing, Gene set enrichment analysis, Automated drug library screen and Statistical analysis. Fig. 3. Ki-67 ablation deregulates global gene expression programs in 4T1 cells. Dot plot analysis of differentially expressed genes (DEGs) in NIH/3T3 Mki67 -/-cells (A), 4T1 Mki67 -/-cells (B), and MDA-MB-231 MKI67 -/-cells (D). Red dots: DEGs with p-value < 0.05; purple dots: log 2 fold change, LFC >1 or <-1, p-value < 0.05; grey dots: not significant, NS. (C) Venn diagrams of DEGs in NIH/3T3 and 4T1 Mki67 -/-cells under condition of p-value < 0.05 (top), and p-value (LFC >1 or <-1) < 0.05 (bottom). Gene set enrichment analysis of highly deregulated genes in 4T1 Mki67 -/-cells (E) and MDA-MB-231 MKI67 -/-cells (H). (F) Quantitative RT-PCR analysis of differentially expressed genes in 4T1 Mki67 -/-cells; fold change in expression + SD is shown. Gene set enrichment analysis of ENCODE and ChEA Consensus transcription factors from ChIP-X gene sets in Mki67 -/-4T1 cells (G) and MDA-MB-231 MKI67 -/-cells (I). FDR, false discovery rate adjusted p-values. (J) Left, heatmaps of ChIP-seq analysis of H3K27me3 in most down-and upregulated genes. Right, gene set enrichment analysis associated with these genes (K) The average values of the H3K27me3 (in green) and H3K4me3 (in apricot) ChIP-seq reads over the 10kb region surrounding the gene in 4T1 Mki67 -/-cells were subtracted from the values of wild-type cells, and the plotted against the log of the fold-change for each gene in RNA-seq. (L) ChIP-seq profiles of histone marks at the Twist1 locus.

Fig. 1 .

 1 Fig. 1. Ki-67 is dispensable for human cancer cells but is rarely mutated. (A) Expression levels of Ki-67 (MKI67), PCNA, cyclin D1 (CCND1) and c-Myc (MYC) in cell lines from the Cancer Cell Line Encyclopaedia. Values are log2(Transcripts Per Kilobase Million [TPM]+1) of TPM-nomalised RNA-seq data. (B) Relative copy number values for each gene from Sanger and Broad Institute whole exome sequencing data of cancer cell lines. (C) Dependency Mapper (DepMap) analysis of dependency of cancer cell lines on each gene, from genome-wide CRISPR (blue) and RNAi (violet) screens. Lower values of Gene Effect indicate higher likelihood of dependency in a given cell line. 0 corresponds to a non-essential gene, -1 is the median of all common essential genes. (D) cBioPortal mutation analysis of each gene in Pan-cancer TCGA data.

Fig. 2 .

 2 Fig. 2. Germline disruption of Ki-67 protects mice against intestinal tumourigenesis. (A, C) IHC staining of β-catenin in whole intestines from 6-7-month-old Apc +/Δ14 Mki67 +/2ntΔ (A) and Apc +/Δ14 Mki67 2ntΔ/2ntΔ (C) mice. Scale bar, 5mm. Insets show accumulation of β-catenin in nuclei. Scale bar, 500µm. (B, D) Quantification of the number (top) and total area (bottom) of neoplastic lesions. Error bars, SEM (n= 7 Apc +/Δ14 Mki67 +/2ntΔ mice; n= 6 Apc +/Δ14 Mki67 2ntΔ/2ntΔ mice).

Fig. 4 .

 4 Fig. 4. Ki-67 promotes stem cell characteristics and controls the EMT in mammary carcinoma.(A) Immunoblot analysis of Hes1 expression in CTRL and two different clones (#1 and #2) of Mki67 -/-4T1 cells. Immunoblot (B) and immunofluorescence (C; scale bar, 30µm) analysis of vimentin and E-cadherin expression in CTRL and Mki67 -/-4T1 and MDA-MB-231 cells. (D) Aldehyde dehydrogenase activity measured using a flow-cytometry assay in 4T1 CTRL and Mki67 - /-cells. DAEB, inhibitor of ALDH was used as a negative control. Top, flow cytometry profiles; bottom, quantification of ALDH+ cells. Error bars, SEM (n=2 independent analyses). (E) Mammosphere formation assay of 4T1 CTRL or Mki67 -/-cells. Representative images (left; scale bar, 400 µm) and quantification (right; error bars, SEM, n=10) after 7 days.

Fig. 5 .

 5 Fig.5. Ki-67 promotes tumour growth. (A) Dot plot analysis of differentially expressed genes (DEGs) in 4T1 Mki67 -/-cells versus tumours derived from grafting 4T1 Mki67 -/-cells into nude mice, showing a highly significant correlation. (B) Fluorescent IHC analysis of vimentin in 4T1 CTRL and Mki67 -/-tumours in NOD/SCID mice (scale bar, 30 µm). (C) Fluorescent IHC analysis of H3S10ph in 4T1 CTRL and Mki67 -/-tumours in NOD/SCID mice (red; pan cytokeratin (PCK) in green, DAPI in blue; scale bar 30 µm), top, and quantification, bottom. (D) IHC staining for Ki-67 and PCNA in 4T1 CTRL or Mki67 -/-tumours. Scale bar, 100 µm. 4T1 CTRL or Mki67 -/-orthotopic xenografts in NOD/SCID (E) and nude (F) mice. Tumour growth was monitored for 3 weeks. Error bars, SEM (n=6 mice). CD-31 staining of endothelial cells in CTRL and Mki67 -/-tumours in NOD-SCID mice: quantification of mean vessel density (MVD; G) and representative IHC images (H; scale bar 500 µm). (I) Sirius-red staining of collagen in CTRL and Mki67 -/-tumours in NOD-SCID mice; insets indicate tumours areas presented at higher magnification (scale bar, 250 µm). (J) Quantification of MVD in CTRL and Mki67 -/-tumours in nude mice.

Fig. 6 .

 6 Fig. 6. Ki-67 promotes metastasis and anti-tumour immune responses. (A) Quantification of lung metastases in nude mice injected orthotopically with 4T1 CTRL or Mki67 -/-cells. Error bars, SEM. Top: representative images of lungs stained to visualise metastases (white nodules); scale bar, 10mm. (B) Lung tissue from nude mice injected via tail vein with 4T1 CTRL or Mki67 -/-cells (2 per condition) was dissociated after 2 weeks and resulting cells were maintained in the presence of 6-thioguanine, to select for 4T1 cells. Left, crystal violet staining of resulting colonies. Right, quantification. (C) Tumour growth over 6 weeks of 4T1 CTRL or Mki67 -/-orthotopic xenografts in immunocompetent BALB/c mice. Error bars, SEM (n=8 mice), and quantification of lung metastases in each group (D). Error bars, SEM. (E) 4T1 CTRL or Mki67 -/-#2 cells were injected via tail vein into immune-competent BALB/c mice. Representative images of stained lungs (day 21 post-injection), metastases are white. Scale bar, 10mm. (F) IHC analysis of 4T1 CTRL or Mki67 -/- tumours (week-4 post-transplantation) stained for Gr-1, a myeloid-derived suppressor cell (MDSC) marker. Scale bar, 100 µm. (G) 4T1 CTRL and Mki67 -/-cells were stained with anti-H2D d -FITC and anti-H2K d -PE, or control isotypes. Left, flow cytometry profiles. Right, quantification (n=1 of each clone).

Fig. 7 .

 7 Fig. 7. Ki-67 promotes cancer cell drug resistance. (A) Xenobiotic metabolism is a hallmark of genes downregulated in Mki67 -/-cells. (B) IC 50 of 4T1 CTRL or Mki67 -/-cells to indicated compounds, derived from dose-response experiments.

ACKNOWLEDGEMENTS

We thank D. Grimanelli for initial guidance on analysing transcriptomes, A. Covinhes and Y. Buscail, for IHC development, Montpellier Resources in Imaging (MRI) for imaging and flow cytometry services, MGX for sequencing services, and P. Jay, D. Santamaria, M.E. Hochberg and M. Serrano for comments on the manuscript. K.M and M.S. were funded by the Ligue Nationale Contre le Cancer (LNCC); N.A. was funded by the University of Montpellier; P.S. and K.M. received funding from Worldwide Cancer Research (WWCR); D.C. and G.D. were funded by the French National Cancer Institute (INCa); D.F. and L.K. are Inserm employees. This work was initiated and finalised with support from the LNCC (EL2013.LNCC/DF and EL2018.LNCC/DF) and continued with support from WWCR (16-0006) and INCa (PLBIO18-094). The chemical library screen relied on support provided by the Programme Opérationnel FEDER-FSE 2014-2020 Languedoc Roussillon. RHEM histology facility was supported by SIRIC Montpellier Cancer Grant INCa_Inserm_DGOS_12553, the European regional development foundation and the Occitanie region (FEDER-FSE 2014-2020 Languedoc Roussillon).

Data availability

All RNA-seq and ChIP-seq data have been deposited in GEO as a reference series, with the accession number GSE163114. This data in GEO will be made publicly available prior to publication of the manuscript.

SUPPORTING INFORMATION

Supporting information includes 16 figures and 5 Datasets.

AUTHOR CONTRIBUTIONS

D.F. conceived and supervised the project. K.M., L.K., and D.F. designed experiments and interpreted the data. K.M., N.A., D.C., M.S., P.S., E.A., A.A., S.P., N.P., F.B., B.B., and C.H-K. performed experiments and interpreted the data. G.D. performed data analysis and interpretation. L.K. and D.F. wrote the manuscript, with contributions from all authors.

COMPETING FINANCIAL INTERESTS

The authors declare that they have no competing interests.