Published OnlineFirst August 8, 2019; DOI: 10.1158/0008-5472.
CAN-19-0708
Molecular Cell Biology
CDK4 Regulates Lysosomal Function and mTORC1
Activation to Promote Cancer Cell Survival
Laia Martínez-Carreres1, Julien Puyal2, Lucía C. Leal-Esteban1, Meritxell Orpinell3,
Judit Castillo-Armengol1, Albert Giralt1, Oleksandr Dergai1, Catherine Moret1,
Valentin Barquissau1, Anita Nasrallah1, Ange  lique Pabois4,5, Lianjun Zhang4,
Pedro Romero , Isabel C. Lopez-Mejia , and Lluis Fajas1
4 1
Abstract
Cyclin-dependent kinase 4 (CDK4) is well-known for its
role in regulating the cell cycle, however, its role in cancer
metabolism, especially mTOR signaling, is undefined. In this
study, we established a connection between CDK4 and lyso-
somes, an emerging metabolic organelle crucial for mTORC1
activation. On the one hand, CDK4 phosphorylated the tumor
suppressor folliculin (FLCN), regulating mTORC1 recruit-
ment to the lysosomal surface in response to amino acids.
On the other hand, CDK4 directly regulated lysosomal func-
tion and was essential for lysosomal degradation, ultimately
regulating mTORC1 activity. Pharmacologic inhibition or
genetic inactivation of CDK4, other than retaining FLCN at
the lysosomal surface, led to the accumulation of undigested
material inside lysosomes, which impaired the autophagic flux
and induced cancer cell senescence in vitro and in xenograft
Introduction
Cyclin-dependent kinase 4 (CDK4) has a well-established role
in cell-cycle control (1) and CDK4–cyclin complexes are com-
monly deregulated in tumorigenesis (2). These complexes are of
great interest as therapeutic targets, and the FDA has approved the
specific CDK4/6 kinase inhibitors PD0332991 (palbociclib),
LEE011 (ribociclib), and LY2835219 (abemaciclib) for treating
advanced or metastatic hormone receptor (HR)-positive and
1
Center for Integrative Genomics, University of Lausanne, Lausanne,
Switzerland. 2Department of Fundamental Neurosciences, University of
Lausanne, Lausanne, Switzerland. 3Department of Physiology, University of
Lausanne, Lausanne, Switzerland. 4Department of Fundamental Oncology,
University of Lausanne, Epalinges, Switzerland. 5Ludwig Institute for Cancer
Research, University of Lausanne, Epalinges, Switzerland.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Current addresses for L. Zhang: Center for Systems Medicine, Institute of Basic
Medical Sciences, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing, China; or Suzhou Institute of Systems Medicine, Suzhou,
Jiangsu, China.
Corresponding Author: Lluis Fajas, Center for Integrative Genomics, University
of Lausanne, Lausanne 1015, Switzerland. Phone: 41766111675; E-mail:
lluis.fajas@unil.ch
Cancer Res 2019;79:5245–59
doi: 10.1158/0008-5472.CAN-19-0708

2019 American Association for Cancer Research.
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Cancer
Research
models. Importantly, the use of CDK4 inhibitors in therapy is
known to cause senescence but not cell death. To overcome
this phenomenon and based on our findings, we increased the
autophagic flux in cancer cells by using an AMPK activator in
combination with a CDK4 inhibitor. The cotreatment induced
autophagy (AMPK activation) and impaired lysosomal func-
tion (CDK4 inhibition), resulting in cell death and tumor
regression. Altogether, we uncovered a previously unknown
role for CDK4 in lysosomal biology and propose a novel
therapeutic strategy to target cancer cells.
Significance: These findings uncover a novel function of
CDK4 in lysosomal biology, which promotes cancer progres-
sion by activating mTORC1; targeting this function offers a
new therapeutic strategy for cancer treatment.
HER2-negative breast cancer. Clinical studies using CDK4/6 inhi-
bitors to treat other malignancies are being conducted (3).
Research from our group and others has shown that the role of
CDK4 is not limited to the control of the cell cycle. Indeed, CDK4
is also a major regulator of energy homeostasis (4–6) through
E2F1–RB complex (7), AMPK (8), and IRS2 (9). Importantly, the
CDK4 pathway has been shown to cross-talk with the mTOR
pathway, which is a major regulator of cell growth and metab-
olism (10, 11). CDK4/6 inhibition attenuates mTOR Complex 1
(mTORC1) activity in some cancer models (12, 13), yet the effects
of CDK4/6 inhibitors on mTORC1 seem to be cell-type specific
because opposite results were observed in other cancer types (14).
The exact mechanism underlying the CDK4–mTOR cross-talk in
mammals is unknown, although in Drosophila it occurs via
the phosphorylation of TSC2 (15). Given that mTOR activity is
increased in numerous cancers and participates in the transla-
tional regulation of several oncogenic proteins, mTOR inactiva-
tion constitutes an attractive strategy for cancer treatment (16).
Lysosomes, considered for years as only the digestive system
of the cell, have emerged as key effectors in cell metabolism, due
to their role as platforms in the activation of mTOR pathway
(17–19). mTORC1 is recruited to the surface of lysosomes in a
complex amino acid (AA)-dependent manner (17). Among the
multiple regulators of this process, we focused on folliculin
(FLCN), a tumor suppressor that functions as a complex with
FNIP. The FLCN–FNIP complex interacts with Rag GTPases in the
absence of AAs repressing their activity. When AAs are sensed,
FLCN–FNIP complexes dissociate from Rag GTPases eliciting
their activation. The activation of Rag GTPases is crucial for
5245
 Published OnlineFirst August 8, 2019; DOI: 10.1158/0008-5472.CAN-19-0708
Martínez-Carreres et al.
mTORC1 recruitment to lysosomes (20). Importantly, mTORC1
activation is also triggered by the accumulation of AAs in the
lysosomal lumen (21). Therefore, alterations in the lysosomal
function directly impact mTORC1 activity (22, 23). Additionally,
these organelles play roles in cell survival and cell proliferation,
thus becoming emerging targets for cancer therapy (24–26).
In this study, we demonstrate that CDK4 is capable of mod-
ulating mTORC1 activity in a direct manner, through the phos-
phorylation of FLCN, and indirectly, by promoting lysosomal
function. When CDK4 inhibitors are used, the lack of lysosomal
function induces senescence in triple-negative breast cancer
(TNBC) cells and impairs tumor growth in a mouse xenograft
model. Moreover, a combination of AMPK activation and CDK4
inhibition was used in an attempt to trigger autophagy in con-
ditions when lysosomes are dysfunctional and resulted in cell
death and tumor regression. This finding is of high relevance in
TNBC, a highly invasive and aggressive cancer type that does not
have a clear therapeutic strategy yet (27).
Materials and Methods
Materials
LY2835219, PD0332991, and LEE011 were purchased from
MedChem Express and used at 0.5 mmol/L, unless otherwise
indicated. R3 human IGF-1 (I11146; Sigma) was used at 30 ng/mL.
Minimal essential media (MEM) amino acids solution (50X;
Gibco) was used at the indicated concentrations. Rapamycin
(LC Laboratories) was kindly provided by Pedro Romero
(University of Lausanne) and used at the indicated concentra-
tions. Bafilomycin A1 (BafA1) was purchased from Enzo Life
Sciences (ALX-380-030-M001) and used at 0.3 mmol/L. Cell perme-
able aKG analog DMKG (349631; Sigma) was used at 5 mmol/L.
Cell culture and transfection
MDA-MB-231, CCRF-CEM, HTC116, IB115, HT29, SKOV,
MCF7, and PC3 cell lines, all obtained from ATCC, were cultured
in RPMI1640 þ GlutaMAX containing, 10 mmol/L HEPES, 1
mmol/L sodium pyruvate (All from Gibco), and 10% FBS (PAA
Laboratories). Thawed cells were allowed one passage to
reach exponential growth phase before being used. Cells were
used during maximum of 10 passages in the experiments per-
formed in this study. PCR-based mycoplasma tests were done
routinely using specific primers: 50 -TGCACCATCTGTCACTCTGT-
TAACCTC-30 and 3-GGGAGCAAACAGGATTAGATACCCT-50 , the
last test was performed in June 2019.
MDA-MB-231 CDK4 knockout (KO), E2F1 KO, and FLCN KO
stable cell lines were generated with CRISPR/Cas9 technology.
The lentiCRISPR v2 plasmid was a gift from Feng Zhang (MIT;
Addgene, plasmid #52961; ref. 28). The pMD2.G plasmid was a
gift from Didier Trono (EPFL; Addgene, #12259). The pCMV-
dR8.91 plasmid and the guide RNA for FLCN were gifts from
Christian Widmann (University of Lausanne). The target sequences
for the guide RNAs are shown in Supplementary Table S1.
Synthetic oligonucleotides were purchased and cloned into the
digested LentiCrispR vector as described in Shalem and collea-
gues (28). Lentiviral production was based on the standard
protocol established by Salmon and Trono (29). The resulting
lentivirus (2 mL) was then used to infect MDA-MB-231 cells for
72 hours. Infected cells were selected with 5 mg/mL puromycin for
5 consecutive days. Western blotting was performed to ensure that
the protein of interest was no longer expressed.
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Plasmid transfection was performed using X-tremeGENE HP.
pRK5-FLAG-FLCN was obtained from Addgene (#72290; ref. 30).
MIG-human RagC WT and MIG-human RagC S75N plasmids
were kindly provided by Alejo Efeyan (CNIO). pCMV-R24C
plasmid was a gift from Mariano Barbacid (CNIO).
For insulin pathway stimulation, cells were first cultured in FBS-
free media for 15 hours and subsequently treated with IGF-1 for
20 minutes before lysis or fixation.
For AA stimulation, after a 15 hours treatment with FBS-free
media with or without CDK4/6 inhibitor, the cells were incubated
for 2 hours in KRBB media containing 111 mmol/L NaCl,
25 mmol/L NaHCO3, 5 mmol/L KCl, 2.5 mmol/L CaCl2, 1
mmol/L MgCl2, 25 mmol/L HEPES, 25 mmol/L glucose,
and dialyzed FBS with or without CDK4/6 inhibitor. For the last
20 minutes, 2X MEM amino acids solution and 2 mmol/L
glutamine were added to the cells.
For acute CDK4/6 inhibition (3 hours), LY2835219 was dilut-
ed in RPMI complete media at the indicated concentrations.
Western blotting
Western blotting was performed as described previously (7).
The band intensities were quantified using the Fiji image-
processing package (31). The following antibodies were
used: anti-CDK4 (H22), anti-CDK6 (C21), and anti-RB
(C15) from Santa Cruz Biotechnology; anti-phospho Rb-
S780 (D59B7), anti-phospho P70S6K-T389 (108D2), anti-
4E-BP1 (53H11), anti-phospho AKT-T308 (244F9), anti-
phospho AKT- S472/3 (D9E), anti-p70S6 kinase (49D7),
anti-LC3B (polyclonal), anti-SQSTM1 (polyclonal), anti-
AcetylCoA Carboxylase (ACC; polyclonal), anti-phospho Acet-
ylCoA Carboxylase (p-ACC), anti-ULK1 (D8H5), and anti-
phospho ULK-S757 from Cell Signaling Technology; and
anti-a-tubulin (DM1A) from Sigma.
Site-specific rabbit polyclonal antibody against phospho-FLCN
was a gift from Kei Sakamoto (Nestle Institute of Health Sciences,
Lausanne, Switzerland). It was generated by YenZym Antibodies
by immunization and affinity purification with a phosphorylated
peptide of the human sequence (CQMNSRMRAH- S-PAEG-amide
for pS62 FLCN, the prefix  denotes the phosphorylated residue).
Mass spectrometry
Protein samples were separated on SDS-PAGE gels. Bands
corresponding to FLCN-GST or FLCN-FLAG were excised and
digested with trypsin. Peptide mixtures were analyzed by
HPLC/MS-MS. MS data were analyzed using Mascot software
2.6 (Matrix Science). Scaffold software (version 4.8.4; Prote-
ome Software Inc.) was used to validate MS/MS-based peptide
and protein identifications, and to perform dataset alignment.
MsViz software (32) was used for comparison of sequence
coverage and phosphorylation of FLCN protein. A full descrip-
tion of the methods can be found in Supplementary Materials
and Methods.
Flow cytometry
Cells were trypsinized and stained with Annexin V-PE (Bio-
Legend, 640907) according to the manufacturer's protocol. The
cells were analyzed on either a Gallios (Beckman Coulter) or
LSR II (BD Biosciences) flow cytometer. For intracellular Ki67
staining, cells were fixed and permeabilized according to the
manufacturer's protocol (BioLegend). Cells were then incubat-
ed with anti-Ki67-FITC (BioLegend) for 30 minutes on ice.
Cancer Research
 Published OnlineFirst August 8, 2019; DOI: 10.1158/0008-5472.CAN-19-0708
After washing twice with permeabilization buffer, cells were
resuspended in PBS prior to analysis. For each sample, at least
10,000 events were acquired.
For LysoTracker experiments, fluorescence was analyzed with
an ImageStream III Flow Cytometer. After treating the cells, 100
nmol/L Lysotracker Green DND-26 (Life Technologies) was
added to live cells for 1 hour. The cells were then trypsinized
and fixed with 4% PFA for 15 minutes at room temperature,
washed twice with PBS, and resuspended with 2% FBS. Nuclear
staining was performed with DAPI. Ten thousand events were
acquired per condition at a magnification power equivalent to
60.
Lysosomal Intracellular Activity Assay Kit (Cell-Based; Biovi-
sion) was used according to the manufacturer's instructions. Ten
thousand events were acquired with an ACCURI C6 flow
cytometer.
All flow cytometry analyses were performed with FlowJo soft-
ware (Version 7.6.5).
Cathepsin B Activity Assay Kit (fluorometric)
Cathepsin B activity was assessed with a Fluorometric Kit
(ab65300; Abcam). Fluorescence was measured with a Tecan
plate reader (Ex/Em ¼ 400/505 nm).
Colorimetric detection of senescence-associated
b-galactosidase
For senescence-associated b-galactosidase (SA-b-Gal) analy-
sis, cells in culture were fixed with 2% PFA and 0.2% glutar-
aldehyde for 5 minutes at room temperature, washed with
PBS, and stained with a solution containing 40 mmol/L citric
acid Na phosphate buffer, 5 mmol/L K4 [Fe(CN)6] 3H2O,
5 mmol/L K3 [Fe(CN)6], 150 mmol/L NaCl, 2 mmol/L MgCl2,
and 1 mg/mL X-gal in distilled water for 15 hours at 37 C. After
staining, the cells were washed twice with PBS and once with
methanol, and the plates were allowed to air dry. Bright-field
images were obtained with an upright light microscope (Zeiss)
with a 20 objective. Positive cells and the total number of
cells per field were counted manually.
For SA-b-Gal analysis of mouse tumors, fresh tissues were
frozen embedded in OCT, and stored at 80 C. Tumors were
cut into 8-mm-thick sections and mounted onto glass slides.
After air-drying for 30 minutes, the sections were fixed with 2%
PFA and 0.2% glutaraldehyde and stained as previously. The
sections were counterstained with 0.1% Nuclear Fast Red
(Sigma).
Electron microscopy
For transmission electron microscopy (TEM), a standard fixa-
tion, embedding, and staining protocol of mice tumors and cells
was established. Ultrathin sections were prepared and imaged
using a Philips CM100 at 80 kV acceleration voltage equipped
with a TVIPS TemCam-F416 digital camera. For further details see
the Supplementary Materials and Methods.
Animal studies
MDA-MB-231 cells were injected into the fourth mammary
gland of 8-week-old female NSG mice (NOD.Cg-Prkdcscid
Il2rgtm1Wjl/Sz strain; The Jackson Laboratory). Tumor growth
and body weight were measured twice per week until the tumor
size reached 50 mm3 per mouse. Mice were randomized into
2 groups for the 8-days treatment. LY2835219 (75 mg/kg),
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CDK4 and Lysosomes
formulated in 1% HEC in distilled water, was administrated
orally. A-769662 (20 mg/kg) was administrated by intraperi-
toneal injection in NaCl 0.9%. Body weight and tumor growth
were monitored every other day. After 8-days treatment,
the animals were anesthetized with isoflurane (3%) and sacri-
ficed by cervical dislocation. Tumors from 10 mice per
group were dissected and cut into two pieces; one piece was
fixed in PFA for histological staining, and the other piece
was snap-frozen in liquid N2 for protein and RNA analysis.
Tumors from 5 mice per group were processed for TEM analysis
and SA-b-Gal staining. All animal care and treatment proce-
dures were performed in accordance with Swiss guidelines
and were approved by the Canton of Vaud, Service de la
Consommation et des Affaires Veterinaires (SCAV; authoriza-
tion VD 2797.1).
Quantification and statistical analyses
The results are expressed as the means  SEM. Comparisons
between 2 groups were performed with unpaired 2-tailed Student
t tests, and multiple group comparisons were performed by
unpaired 1-way ANOVA and 2-way ANOVA, both followed
by Tukey test or otherwise indicated. All P values below 0.05
were considered significant. Statistically significant values are re-
presented by asterisks corresponding to  , P < 0.05;  , P < 0.01;

, P < 0.001; and  , P < 0.0001.
Supplementary methods
Additional details about the methods used in this paper can be
found in the Supplementary Materials and Methods and in
Supplementary Tables S2 to S5.
Results
CDK4 activity is required for mTORC1 localization at
lysosomes
CDK4/6 inhibition was previously shown to decrease mTORC1
activity in some models of human cancer (12–14). To determine
the cell-type specificity of this cross-talk, we activated mTORC1 in
8 human cancer cell lines with IGF-1 in the presence of CDK4/6
inhibitor LY2835219. The efficiency of CDK4/6 inhibition was
measured by RB phosphorylation. Treatment with LY2835219
caused a decrease in p70S6K and 4E-BP1 phosphorylation (2
well-known targets of mTORC1), both in the unstimulated and in
the IGF-1 stimulated conditions (Supplementary Fig. S1A).
Despite showing considerable mTORC1 inhibition, some cell
lines showed only a mild decrease in AKT phosphorylation in
the presence of LY2835219 (HCT116, IB115, MDA-MB-231,
SKOV3, PC3), suggesting that the effects observed in mTORC1
activity were at least partially independent of decreased AKT
signaling.
We focused our experiments on the TNBC cell line MDA-MB-
231 because it was one of the most responsive to CDK4/6
inhibition. The treatment with PD0332991 and LEE011, 2 other
CDK4/6 inhibitors, also resulted in decreased mTORC1 activity in
this cell line (Supplementary Fig. S1B and S1C).
We next found that MDA-MB-231 wild-type (WT) cells treated
with LY2835219, or CDK4 KO MDA-MB-231 cells showed
impaired translocation of mTORC1 to the lysosomes in response
to AAs, a key step for mTORC1 activation (Fig. 1A and B), which
correlated with decreased phosphorylation of 4E-BP1, p70S6K,
and ULK (Fig. 1C and D). Inhibition of glutaminolysis has been
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Martínez-Carreres et al.

Figure 1.
CDK4 inhibition or depletion prevents the recruitment of mTOR to the lysosomal surface in response to amino acid stimulation. A, Confocal immunofluorescence
analysis showing mTOR colocalization with lysosomes (LAMP1 and mTOR staining) in WT and CDK4 KO MDA-MB-231 cells, with or without AA stimulation and in
the presence of 0.5 mmol/L of the CDK4/6 inhibitor LY2835219 or DMSO. B, Quantification of mTOR–LAMP1 colocalization from A using Pearson coefficient. At
least 40 fields per treatment condition from three independent experiments were analyzed. C, Western blot analysis showing levels of phospho-RB and total RB,
CDK4, and mTORC1 target genes (phospho-p70S6K and total p70S6K, phospho-ULK and total ULK, and 4E-BP1) in WT and CDK4 KO MDA-MB-231 cells with or
without AA stimulation and with or without 0.5 mmol/L LY2835219. a-Tubulin was used as a loading control. D, Quantification of phospho-p70S6K normalized
to a-tubulin from three independent experiments as in C.  , P < 0.05;    , P < 0.001;     , P < 0.0001.

shown to prevent lysosomal recruitment and subsequent activa-
tion of mTORC1 (33). The effects of LY2835219 could not be
rescued, however, with a-ketoglutarate (aKG) stimulation, indi-
cating that CDK4 inhibition or depletion does not affect mTORC1
translocation to the lysosomal surface by impairing glutamine
metabolism (Supplementary Fig. S2A and S2B). Interestingly,
MDA-MB-231 cells lacking E2F1 showed normal mTORC1 trans-
location to the lysosomal surface and increased mTORC1 activa-
tion (Supplementary Fig. S2C–S2F) but were still sensitive to
CDK4 inhibition. These suggest that the effects of CDK4 on
mTORC1 activity might be independent of E2F1 transcriptional
activity.
CDK4 regulates mTORC1 activity through phosphorylating
FLCN
We hypothesized that CDK4 potentially regulates mTORC1
pathway through phosphorylation of one of its regulators. A
bioinformatics search of the proteins of mTORC1 pathway iden-
tified proteins containing the putative phosphorylation site for
CDK4: (S/T)PX(K/R/P) using phosphosite.org (Supplementary

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CDK4 and Lysosomes

Figure 2.
CDK4 regulates mTORC1 activity through phosphorylating FLCN. A, Representation of the phosphorylation sites detected by mass spectrometry analysis in the
in vitro kinase assay with FLCN-GST and CDK4/CyclinD3. Green rectangles represent protein coverage. Blue dots represent phosphorylation sites, and their size
correlates with their abundance. B, Confocal immunofluorescence analysis showing the colocalization of overexpressed FLCN with lysosomes (LAMP1 and FLCN
staining) in WT and CDK4 KO MDA-MB-231 cells, with or without AA stimulation and in the presence of 0.5 mmol/L of LY2835219 or DMSO. C, Quantification of
FLCN-LAMP1 colocalization from B using Pearson coefficient. At least 40 fields per treatment condition from three independent experiments were analyzed.
D, Western blot analysis of WT and FLCN KO MDA-MB-231 cells, treated with DMSO or LY2835219 for 3 hours. Phospho-FLCN and total FLCN, phospho-p70S6K
and total p70S6K, phospho-RB and total RB, and a-tubulin as a loading control. E, Quantification of phospho-FLCN levels normalized to a-tubulin from D. F,
Immunofluorescence of phospho-S6 from WT and FLCN KO MDA-MB-231 after 20 minutes of AA stimulation, in the presence or absence of LY2935219.
G, Quantification of phospho-S6 intensity from F.  , P < 0.05;   , P < 0.01;     , P < 0.0001.

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Martínez-Carreres et al.
Fig. S3A). The corresponding peptides were used for a kinase
competition assay versus RB consensus peptide. The peptides
belonging to 4E-BP1, LARP1, and FLCN, triggered a decrease of
RB phosphorylation, indicating a competition with RB for CDK4
phosphorylation (Supplementary Fig. S3B). In vitro kinase assays
using CDK4/CycD3 recombinant protein and glutathione S-
transferase (GST) fusion of full-length FLCN followed by mass
spectrometry revealed that FLCN could be phosphorylated by
recombinant CDK4/CycD3 at 4 different sites (S62, S73, T227,
and S571; Fig. 2A). Importantly, S62, S73, and S571 phos-
phorylation sites were found when FLCN was immunopreci-
pitated from MDA-MB-231 cells upon AA and IGF-1 stimula-
tion (Supplementary Fig. S3C). Radioactive in vitro kinase
assays confirmed that CDK4/CycD3 can phosphorylate the
full-length FLCN in vitro (Supplementary Fig. S3D). When
FLCN S62, S73 and S571 were mutated to alanine (nonphos-
phorylatable forms), the intensity of the autoradiography
bands was reduced, indicating a decrease in FLCN phosphor-
ylation. This result was even more striking in the triple mutant
(Supplementary Fig. S3D).
Importantly, FLCN is retained at the lysosomes in the presence
of AAs upon CDK4 inhibition or depletion (Fig. 2B and C), which
is consistent with the absence of mTOR. To further study the
effects of CDK4 on FLCN, we generated an MDA-MB-231 FLCN
KO stable cell line. We used a phospho-specific antibody directed
against phospho-S62 FLCN, one of the phosphorylation sites that
we found in CDK4-FLCN in vitro kinase assays. Phosphorylation
of FLCN decreases significantly in WT cells treated with
LY2835219 for only 3 hours (Fig. 2D and E), indicating an acute
control of CDK4 over FLCN, potentially resulting in mTORC1
regulation. FLCN KO cells were still sensitive to mTORC1 inac-
tivation by CDK4/6 inhibition, as shown by decreased p70S6K
phosphorylation, but to a lesser extent than WT cells (Fig. 2D).
Moreover, WT cells showed a 2-fold decrease in the phosphory-
lation of S6 when treated with LY2835219, whereas FLCN
KO cells showed a milder, yet significant, reduction of levels of
S6 ribosomal protein phosphorylation. S6 phosphorylation
remained significantly higher in LY2835219-treated FLCN KO
cells, compared with LY2835219-treated WT cells (Fig. 2F and G).
Our results suggest that complementary mechanisms to FLCN
phosphorylation account for the effects of CDK4 on mTORC1
activation.
Interestingly, the overexpression of the R24C CDK4 mutant,
which abolishes the ability p16(INK4a) to inhibit CDK4 (34),
highly increased phospho-p70S6K levels under FBS starvation
conditions as compared with untransfected cells (Supplemen-
tary Fig. S4A and S4B). Increased pS62-FLCN/FLCN ratio was
also observed with R24C overexpression (Supplementary
Fig. S4A and S4C), indicating that the R24C mutation bypasses
the effect of starvation on the activation of the mTORC1
pathway.
Additionally, the overexpression of RAGC S75N mutant
confers insensitivity to mTORC1 activity upon acute treatment
with LY2835219 (Supplementary Fig. S4D and S4E). This
reinforces the link between CDK4 and mTORC1, not only
through FLCN, but also through other mechanisms of nutrient
sensing.
CDK4 inhibition or depletion increases lysosomal mass
Lysosomal biogenesis is a biological process coordinated by the
transcription factor TFEB, which is repressed by mTORC1. Under
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nutrient-rich conditions, TFEB is phosphorylated by mTORC1,
causing its retention in the cytoplasm. By contrast, when
mTORC1 is inactivated, unphosphorylated TFEB translocates to
the nucleus and promotes the transcription of numerous genes
encoding lysosomal and autophagic proteins (19). To test wheth-
er CDK4 inhibition or depletion affected TFEB target genes, we
treated WT and CDK4 KO MDA-MB-231 cells with complete or
serum-starvation media, with or without LY2835219. As
expected, under starvation conditions, WT MDA-MB-231 cells
showed increased expression of genes regulated by TFEB (Fig. 3A).
Moreover, we found that CDK4/6 inhibition synergized with
starvation to further increase the expression of those genes. CDK4
KO cells also presented increased expression of those genes under
basal conditions, but no further increase was found upon CDK4/6
inhibition (Fig. 3A).
We next used LysoTracker staining and flow cytometry to
look at the percentage of lysosome-positive cells upon CDK4
inhibition or depletion. The percentage of LysoTracker positive
cells and the size of the LysoTracker positive particles were
consistently and markedly increased in the absence of
CDK4 activity (Fig. 3B–D). Similarly, when we quantified the
size of lysosomal-associated membrane protein 1 (LAMP1)-
positive particles using immunofluorescence, we found a sig-
nificant increase in lysosomal density in CDK4 KO cells com-
pared to WT MDA-MB-231 cells (Fig. 3E and F). Consistently,
PD0332991 and LEE011 treatments also increased LysoTracker
intensity in MDA-MB-231 cells (Supplementary Fig. S5A).
Immortalized MEFs treated with LY2835219 also showed
increased LysoTracker intensity (Supplementary Fig. S5B).
These results suggest a new function of CDK4 in the control
of lysosomal biology.
CDK4 is required for lysosomal function
It is well known that the inhibition of mTORC1 results in the
induction of autophagy, a conserved catabolic process that
triggers the degradation of intracellular constituents and orga-
nelles in the lysosome (35). Serum-starvation conditions, as
well as mTOR inhibition by rapamycin, increased the amounts
of the autophagosome marker LC3-II (Fig. 4A and B, long
exposure). CDK4/6 inhibition similarly increased LC3-II levels.
Moreover, CDK4 KO cells showed increased levels of LC3-II in
basal conditions and were more sensitive to starvation-
mediated autophagic stimuli (Fig. 4A and B). These indicate
either an increase of autophagosome biogenesis or an
impairment of the autophagic flux in the absence of CDK4.
Indeed, these effects could be secondary to mTOR inactivation,
because the decreased mTORC1 activity caused by CDK4 inhi-
bition or depletion shown in Fig. 1 could ultimately induce
lysosomal biogenesis.
In WT cells, under starvation conditions and in the presence of
rapamycin treatment, BafA1, which is a potent V-ATPase inhibitor
that blocks autophagosome-lysosome fusion, further increased
LC3-II levels, indicating that rapamycin induces autophagosome
biogenesis. In contrast, in CDK4 KO cells or cells treated with
CDK4 inhibitor, BafA1 failed to cause any additional increase in
LC3-II levels (Fig. 4A–C). These results suggest that CDK4 does
not directly participate in autophagosome biogenesis. On the
other hand, no abnormal SQSTM1 accumulation was observed
after CDK4 inhibition or depletion, despite observing a consistent
SQSTM1 increase with BafA1 (Fig. 4A). SQSTM1 protein levels are
often negatively correlated with autophagic degradation.
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CDK4 and Lysosomes

Figure 3.
CDK4 inhibition or depletion increases lysosomal mass. A, qPCR analysis showing the expression of transcription factor EB (TFEB)–regulated genes that are
involved in lysosomal or autophagic processes (cathepsins B and D and SQSTM1) in WT or CDK4 KO MDA-MB-231 cells in complete (þFBS) or serum-starvation
media (FBS), treated with or without 0.5 mmol/L LY2835219. Each condition was assessed in duplicates for three independent experiments. B, Representative
flow cytometry images of WT or CDK4 KO MDA-MB-231 cells treated with or without LY2835219 and stained with LysoTracker Green DND-26 under the indicated
conditions. C, Proportions of cells containing lysosomes, estimated by quantification of fluorescence from LysoTracker Green DND-26–stained cells from two
independent experiments. At least 10,000 events were acquired. Data are subdivided into categories of the number of lysosomes per cell. Significant differences
between WT and KO MDA-MB-231 cells are indicated by:  , total lysosomal particles (P < 0.05); $, lysosomal particles per cell (3–5 particles; P < 0.05); &,
lysosomal particles per cell (>5 particles; P < 0.05); 2-way ANOVA followed by Tukey multiple comparisons test. D, As in C, but subdivided into categories of the
sizes of lysosomes per cell. Significant differences between WT and KO MDA-MB-231 cells are indicated by:  , lysosomal particles <0.5 mm2 (P < 0.05); $,
lysosomal particles per cell (<0.5 mm2; P < 0.05). E, Immunofluorescence analysis showing LAMP1 and CDK4 expression in WT and CDK4 KO MDA-MB-231 cells in
complete media. F, Quantification of the total volume of LAMP1-positive particles in z-stack images. At least 30 cells in total from three independent experiments
were analyzed.  , P < 0.05;   , P < 0.01;    , P < 0.001.

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Martínez-Carreres et al.

Figure 4.
Absence of CDK4 alters lysosomal function. A, Western blot analysis of autophagosomal markers LC3-I, LC3-II, and SQSTM1 in WT and CDK4 KO MDA-MB-231
cells treated 24 hours with complete media (þFBS) or serum-starvation media (FBS), presence/absence of 0.5 mmol/L rapamycin and of 0.5 mmol/L
LY2835219 and presence/absence of 0.3 mmol/L BafA1, 6 hours before the treatment. B, Quantification of protein expression of LC3-II protein levels normalized
to a-tubulin from three independent experiments as in A (only the conditions without BafA1). C, Quantification of LC3-II protein levels normalized to a-tubulin
from three independent experiments as in A (only the conditions with BafA1). Statistical analysis was performed using a paired t test. D, Representative electron
micrographs of WT and CDK4 KO MDA-MB-321 cells showing that cells lacking CDK4 have increased autolysosome (arrows) density and size. In CDK4 KO cells,
autolysosomes accumulated nondegraded material, such as undegraded autophagosomes (arrowheads), in both complete (þFBS) and serum-starvation media
(FBS). In cell cultures without FBS, an increase in autophagosomes (arrowheads) was also observed in CDK4 KO cells as compared with WT cells. N, nucleus.
E–G, Quantification of the autophagosome (E) and autolysosome (F) area per cell, and of the percentage of autolysosomes containing degraded material (G), for
the conditions shown in D. n ¼ 20 cells per condition. H, Quantification of intracellular lysosomal activity of WT and CDK4 KO MDA-MB-231 cells in complete
media. I, Quantification of cathepsin B activity in WT and CDK4 KO MDA-MB-231 cells in complete media.  , P < 0.05;   , P < 0.01;    , P < 0.001.

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CDK4 and Lysosomes

However, it has been already observed that the expression of
SQSTM1 does not always inversely correlate with autophagic
activity, given that they can be restored during prolonged
starvation (36).
We next used TEM to analyze the ultrastructure of
lysosomes in WT and CDK4 KO MDA-MB-231 cells incubated
with serum-starvation media to trigger autophagy. Serum-
starvation induced autophagosome and lysosome formation
in MDA-MB-231 WT cells. CDK4 KO MDA-MB-231 cells dis-
played higher densities of autophagosomes and lysosomes.
Moreover, TEM analysis revealed that the lysosomes in CDK4
KO cells were filled with electron-dense undigested material
(Fig. 4D–G; Supplementary Fig. S5C). LY2835219-treated WT
cells mimicked CDK4 KO cells (Supplementary Fig. S5D–S5H).

Figure 5.
Dysfunctional lysosomes, but not mTORC1 inhibition, induce senescence. A, Proliferation of WT MDA-MB-231 cells cultured in either complete (þFBS) or serum-
starvation (FBS) media, with or without LY2835219, shown as the percentage of Ki67-positive cells. B, Apoptosis under the same conditions, shown as the
percentage of Annexin V-positive cells. C, Quantification of the percentage of SA-b-Gal-positive WT and CDK4 KO MDA-MB-231 cells from triplicates, at least
5 fields per replicate. D, Quantification of the fold induction of SA-b-Gal induced by LY2835219 or rapamycin treatment from triplicates, at least 5 fields per
replicate. E, mTOR-independent induction of senescence by LY2835219, visualized by colorimetric senescence-associated SA-b-Gal staining of WT and CDK4 KO
MDA-MB-231 cells cultured in /þ FBS media for the last 16 hours after 8 days of DMSO, 0.5 mmol/L LY2835219, or 0.5 mmol/L rapamycin treatment. F, qPCR
analysis showing expression of genes usually upregulated in senescence in WT MDA-MB-231 cells treated for 8 days with DMSO, LY2835219, or rapamycin in
complete media. Triplicates were analyzed for each condition.  , P < 0.05;   , P < 0.01;    , P < 0.001;     , P < 0.0001. ns, nonsignificant.

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Martínez-Carreres et al.
Consistently, the intracellular lysosomal activity and cathepsin
B activity were decreased in the CDK4 KO cells (Fig. 4H and I).
Overall, these results suggest that CDK4 is fundamental for the
activity of lysosomes and that its absence impairs autophagic
flux at the lysosomal degradation step.
Dysfunctional lysosomes, but not mTORC1 inhibition, induce
senescence
Lysosomal processes are associated with aging and longev-
ity (37), and the increase of lysosomal content is a characteristic
of senescence progression (38). This led us to investigate the
fate of cells lacking CDK4 activity. Analysis of Ki-67 levels
showed that CDK4/6 inhibition significantly decreased the
proliferation rate of cells in complete medium (Fig. 5A). Yet,
when the cells were serum-starved, no differences were
observed in response to LY2835219 (Fig. 5A). Apoptosis,
measured by Annexin V staining, was not significantly induced
by LY2835219 in complete medium (Fig. 5B). A slight increase
in apoptosis was seen when CDK4/6 inhibition was combined
with serum starvation, but the overall percentage of apoptotic
cells was extremely low (Fig. 5B). Interestingly, 8 days of
treatment with LY2835219 resulted in higher senescence, as
measured by senescence-associated beta-galactosidase (SA-
bGal) staining. CDK4 KO cells also showed increased senes-
cence (Fig. 5C–E). No differences were observed under serum
starvation conditions or upon rapamycin treatment, indicating
that the induction of senescence in the absence of CDK4 was
not due to mTORC1 inhibition (Fig. 5C–E).
Consistent with the SA-bGal data, LY2835219 treatment
induced the expression of most of the senescence-related genes
evaluated (Fig. 5F). However, CDKN1A and RBL2, which are
p53-regulated genes, did not respond to CDK4/6 inhibition in
the CDKN2A and p53 mutant MDA-MB-231 cell line. This
reinforces the idea that the induction of senescence by CDK4
inhibition or depletion is more likely related to the lysosome than
to any effect on p53. With the exception of RKHD3 and IGFBP5,
mTOR inhibition by rapamycin also failed to induce the expres-
sion of genes related to senescence (Fig. 5F).
These results suggest that the lysosomal dysfunction induced
by CDK4 inhibition or depletion is the direct cause of the
senescent phenotype in these cells, independent of mTORC1
inhibition.
The CDK4 inhibitor LY2835219 alters lysosomal function,
attenuates mTORC1 activity, and decreases tumor growth in a
breast cancer xenograft mouse model
To investigate the effects of CDK4/6 inhibition on lyso-
somal function in vivo, we used a breast cancer xenograft
model by injecting MDA-MB-231 cells into the mammary
glands of NSG mice. Intratumoral decrease in RB phosphor-
ylation in response to LY2835219 confirmed that CDK4/6 was
indeed inhibited in the tumors (Fig. 6A and B). Consistent
with previous reports (39), LY2835219-treated mice showed
halted tumor growth with reduced cell proliferation (Fig. 6C–
E), compared with mice treated with vehicle. The lack of
immune cell infiltration in the tumors suggested that the
effects were independent of the immune system (Supplemen-
tary Fig. S6A).
The tumors of LY2835219-treated mice had increased expres-
sion of the lysosomal marker LAMP1 (Fig. 6F and G) and of TFEB
target genes (Supplementary Fig. S6B). In addition, LY2835219
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treatment also decreased the activity of mTORC1 (Fig. 6H and I).
TEM analysis proved that tumors from LY2835219 treated mice
had higher densities of autophagosomes and lysosomes, and that
those lysosomes accumulated nondigested material (Fig. 6J–M;
Supplementary Fig. S6C). Furthermore, we showed that the
tumors of these mice had typical features of senescence
(Fig. 6N; Supplementary Fig. S6D). Together, these results further
demonstrate that CDK4 plays an essential role in the regulation of
lysosomal function in vivo and that alterations in lysosomal
function lead to tumor cell senescence in a mouse model of breast
cancer.
The CDK4 inhibitor LY2835219 in combination with the AMPK
activator A769662 induces cell death in breast cancer cells and
tumors
Lysosomes are essential for autophagy. We speculated that
triggering autophagy in LY2835219-treated mice, which have
impaired lysosomal function, would trigger the collapse of the
system and result in cell death. In vivo, the allosteric AMPK
activator A769662, which is a potent inducer of autophagy, in
combination with LY2835219, resulted in tumor regression, in
contrast to the treatment with LY2835219 or A769662 alone
(Fig. 7A–D). Indeed, almost 50% of the tumors decreased their
size upon cotreatment, although no major differences were
found in proliferation (Fig. 7E and F). In contrast, cleaved-
caspase-3 staining revealed a 6-fold induction of apoptosis in
mice cotreated with A769662 and LY2835219 (Fig. 7E–G),
suggesting that increased apoptosis was underlying the
decrease in tumor size in these mice. CDK4 inhibition resulted
in decreased RB phosphorylation, but no further decrease was
observed in cotreated tumors (Fig. 7H and I). As for mTORC1
activity, LY2835219 treatment, as well as AMPK activation,
decreased p70S6K phosphorylation (Fig. 7H and J). Interest-
ingly, CDK4 inhibition triggered AMPK activation, as measured
by the phosphorylation of ACC, a known target of AMPK
(Fig. 7H and K).
In vitro, CDK4 inhibition and AMPK activation as single treat-
ments failed to induce cell death after 1-week treatment in MDA-
MB-231 cells as shown by the low levels of Annexin V-positive
cells (Supplementary Fig. S7A). Only the combination of
LY2835219 and A769662 increased notably the percentage of
Annexin V-positive cells (Supplementary Fig. S7A).
TEM analysis of the tumors revealed that the percentage of
the autophagosome and lysosome area per cell was increased in
the single treatments, as well as in the A769662 and
LY2835219 cotreated group (Supplementary Fig. S7B–S7D).
Mice cotreated with both A769662 and LY2835219 still dis-
played a significant decrease in lysosomes with digested mate-
rial (Supplementary Fig. S7B and S7E). The underlying mech-
anism of this apparent paradox could be that A769662 induced
cell death only in cells in which LY2835219 treatment impaired
lysosomal function. Indeed, some of the cotreated cells dis-
played mixed morphological features of apoptotic cell death
(highly condensed chromatin, shrinkage of the cytoplasm) and
autophagic cell death (numerous autophagosomes and auto-
lysosomes, focal swelling of the perinuclear membrane
[Supplementary Fig. S7B (indicated by  )].
Taken together, these results show that a combination treat-
ment using A769662 and LY2835219 provides a better outcome
than LY2835219 alone, inducing cell death and tumor regression
in the MDA-MB-231 breast cancer xenograft model.
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CDK4 and Lysosomes

Figure 6.
The CDK4/6 inhibitor LY2835219 alters lysosome morphology in a breast cancer xenograft mouse model. A, Western blot analysis for RB and phospho-RB in
xenograft protein extracts from both groups. B, Quantification of A as arbitrary levels of phospho-RB normalized to total RB. C, Volume of MDA-MB-231 tumors
from mice (n ¼ 8–9 mice per condition). D, IHC with Ki67 antibody and DAPI for tumor sections from DMSO- or LY2835219-gavaged mice. E, Percentage of Ki67-
positive cells from D. Three images per section were analyzed for 8 or 9 mice. F, IHC with LAMP1 antibody and DAPI of tumor sections from mouse xenograft
models treated with either LY2835219 or DMSO. G, Quantification in arbitrary units of the area of LAMP1 staining normalized to the number of nuclei per field.
Three images per section were analyzed for 8 or 9 mice. H, Western blot analysis for the mTOR target protein p70S6K and phospho-p70S6K from tumor
lysates, following gavage with either DMSO or LY2835219. I, Quantification of H showing arbitrary levels of phospho-p70S6K normalized to total p70S6K.
J, Representative electron micrographs of xenografts showing that LY2835219 treatment notably increases the density of autolysosomes (arrows) and that these
autolysosomes accumulate nondegraded materials, such as undegraded autophagosomes (arrowheads). N, nucleus. K–M, Quantification of conditions shown in
J as the percentage of autophagosome area per cell (K), percentage of autolysosome area per cell (L), and the percentage of autolysosomes containing
degraded material per cell (M). n ¼ 30 cells per condition. N, SA-b-Gal staining of tumor cell senescence in tumor xenograft sections taken from mice gavaged
with LY2835219 or DMSO.  , P < 0.05;    , P < 0.001;     , P < 0.0001.

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Martínez-Carreres et al.

Figure 7.
The CDK4 inhibitor LY2835219 in combination with the AMPK activator A769661 induces cell death in breast cancer cells and tumors. A, Tumor
volume of breast cancer xenografts in NSG mice was monitored throughout the whole experiment. Treatment started on day 21 and lasted for 8 days
(n ¼ 9–10). B, Representative images from tumors at the day of sacrifice, after the corresponding treatment. C, Increment of tumor volume per
mouse: Vol day 29/Vol day 21. D, Representation of percentage of tumors, which increases more than 1.5-fold, from 1 to 1.5-fold, or less than 1-fold.
E, Ki67 and cleaved caspase-3 immunostaining in tumor sections after the corresponding treatment. F, Quantification of E, percentage of Ki67
positive cells. G, Quantification of E, cleaved caspase-3 area per field, comparing LY2835219 and A769662þLY2935219. H, Western blot analysis of
phosphorylation of RB, p70S6K, and ACC proteins in tumor samples. I, Quantification of H, phospho-RB normalized to total RB. J, Quantification
of H, phospho-p70S6K normalized to total p70S6K. K, Quantification of H, phospho-ACC normalized to total ACC.  , P < 0.05;   , P < 0.01;

, P < 0.0001.

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Discussion
We show here for the first time that CDK4 regulates lysosomal
function and mTORC1 activity in cancer cells. We demonstrate
that CDK4, through phosphorylation of FLCN, facilitates the
migration of mTOR to the lysosomes in order to be activated.
The abrogation of mTOR activation by depletion or chemical
inhibition of CDK4 consequently resulted in a substantial
increase in the numbers of lysosomes and autophagosomes.
These prove that CDK4 is necessary for the dissociation of
FLCN from the lysosomes, and for the subsequent recruitment
and activation of mTORC1. However, this increase was not
correlated with greater lysosomal activity, which suggested that
CDK4 has an mTOR-independent role in this process. mTORC1
activation is initiated at the lysosome and requires the presence
of AAs in the cytosol and in the lysosomal lumen (21). Impor-
tantly, we prove that CDK4 inhibition or depletion leads to the
accumulation of intra-lysosomal undigested material, which
probably results in impaired AA recycling and blunted
mTORC1 activation. We show here an additional mechanism
in which CDK4 favors mTORC1 activation by promoting the
digestion of proteins in the lysosome, in turn providing met-
abolic intermediates that sustain cell growth and survival via
downstream effectors. This pathway would be particularly
crucial during prolonged starvation conditions, such as the
typical environment of some tumors.
Cells initially respond to nutrient deprivation by inactivat-
ing their energy-consuming processes, such as protein or lipid
biosynthesis, and by activating catabolism. At the same time,
other mechanisms are activated to recycle molecules to provide
the cell with enough substrates and metabolic intermediates
to survive—an important function of autophagy. In the long
term, autophagy reactivates the mTORC1 pathway by replen-
ishing the lysosomes with digested proteins and AAs (40).
CDK4 inhibition or depletion could therefore mimic a star-
vation signal. This hypothesis is in agreement with previous
studies showing that CDK4/6 inhibitors induce autop-
hagy (41, 42). We also observed an increase in the number
of autophagosomes upon CDK4 inhibition. However, when
analyzing the ultimate fate of the autophagosomes, we found
that they accumulate due to the impairment of lysosomal
degradation upon CDK4 inhibition or depletion. Indeed,
others have demonstrated that the inhibition of lysosomal
activity causes decreased fusion with autophagosomes and
vice-versa (43–45). In this study, we show that inhibiting or
depleting CDK4, in addition to inducing autophagy, likely
through mTORC1 inactivation, impairs the autophagic flux at
the lysosomal degradation step. This observation may explain
the increased susceptibility of CDK4 KO cells to autophagic
stimuli. We also found that CDK4 inhibition or depletion
increased the expression of many lysosomal genes. It is well
known that mTORC1 negatively regulates lysosomal biogen-
esis (46). This could be secondary to mTORC1 inhibition, or
due to a compensatory mechanism to create new lysosomes, as
the existing ones are dysfunctional.
CDK4/6 inhibitors have been shown to accumulate into lyso-
somes, a phenomenon called lysosomal trapping (47). However,
the use of CDK4 KO cells in our work demonstrates that CDK4
rather stimulates lysosomal function and that the lysosomal
trapping of the drug is secondary to the lysosomal impairment
induced by CDK4 inhibition. In addition, CDK4/6 inhibition has
been found to result in proteasomal activation (48). A negative-
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CDK4 and Lysosomes
feedback exists between proteasomal activity and autophagic
flux (49). Thus, the impairment of autophagic flux at the lyso-
somal degradation step can result in proteasomal activation and
vice versa.
Despite the known requirement for lysosomes in cell-cycle
progression (50, 51), little is known about the relationship
between lysosomes and senescence. Importantly, in eukaryotes,
autophagy impairment via lysosomal dysfunction has been
described to be an important characteristic of oxidative
stress-induced senescence (52). Our findings suggest that the
ultimate fate of cells that lack CDK4 is the activation of
senescence due to lysosomal dysfunction. The absence of
senescence when cells are treated with rapamycin further
demonstrates that mTORC1 inactivation is a consequence of
lysosomal impairment due to CDK4 inhibition or depletion.
However, CDK4 inhibitor-induced lysosomal dysfunction was
not sufficient to prompt cell death in the tumors of the treated
mice. Instead, cancer cells were arrested but were still alive,
which explained that the tumor burden was not decreased but
only stabilized (Fig. 6). We reasoned that further forcing the
autophagic flux would create additional stress that could kill
these tumor cells. Autophagic induction in conditions where
the lysosomal function is impaired abrogates the restoration of
metabolic intermediates in the cytoplasm and the synthesis of
macromolecules. Altogether, this might create a situation in
which cells collapse and enter into apoptosis. With this aim,
we used the AMPK activator A769662, which is known to
increase autophagy, in combination with the CDK4 inhibitor.
Indeed, cotreatment resulted in increased cancer cell death and
therefore tumor regression (Fig. 7). This is a major finding that
represents a paradigm switch regarding the use of CDK4 inhi-
bitors for the treatment of cancer. Strikingly, and consistent
with our findings, CDK4 inhibitors are not efficient as a single
drug in the clinical practice and are often used in combination
with other drugs (53–55).
We show here that the effects of CDK4 in MDA-MB-231 cells are
likely to be independent of E2F1, the transcription factor mod-
ulated by CDK4 during the cell cycle. It has been reported,
however, that E2F1 regulates lysosomal positioning and activates
mTORC1 by promoting its recruitment to the lysosomal sur-
face (56, 57). These suggest a dual and complementary role for
CDK4 in the regulation of the mTORC1 pathway: first, through
regulation of the lysosomal function; and second, through FLCN
phosphorylation.
It was previously described that CDK4/6 inhibitors display
antitumor activity only in RB-positive cells (58). In addition, it
was unclear whether CDK4/6 inhibition had an effect on
TNBCs. Our findings are consistent with other studies showing
that CDK4/6 inhibitors can still have some effects on RB-
negative cells (59) and that CDK4/6 inhibitors do have anti-
tumor effects in TNBCs. Indeed, depending on the cell type,
CDK4/6 inhibition triggers either a quiescence or senescence
response, not necessarily via the canonical RB-E2F pathway
(reviewed in refs. 53, 60).
Overall, this study demonstrates a new role for CDK4 in the
regulation of lysosomal function, which ultimately leads to
senescence in cancer cells and mTORC1 inactivation. In addition,
we highlight the importance of lysosomes in cancer and we
propose that CDK4/6 inhibitors could be used in combination
with other drugs to target lysosomal function as a novel anticancer
strategy.
Cancer Res; 79(20) October 15, 2019 5257
 Published OnlineFirst August 8, 2019; DOI: 10.1158/0008-5472.CAN-19-0708
Martínez-Carreres et al.
Disclosure of Potential Conflicts of Interest
P. Romero is an Editor-in-Chief at the Journal for Immunotherapy of Cancer,
reports receiving a commercial research grant from Roche pRED – Zurich, has
received speakers bureau honoraria from Roche, and has an unpaid consultant/
advisory board relationship with Immatics Biotechnologies. No potential
conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: L. Martínez-Carreres, J. Puyal, M. Orpinell, A. Giralt,
P. Romero, I.C. Lopez-Mejia, L. Fajas
Development of methodology: L. Martínez-Carreres, M. Orpinell, J. Castillo-
Armengol, A. Pabois
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): L. Martínez-Carreres, J. Puyal, L.C. Leal-Esteban,
M. Orpinell, J. Castillo-Armengol, A. Giralt, O. Dergai, V. Barquissau,
A. Nasrallah, A. Pabois, L. Zhang, P. Romero, I.C. Lopez-Mejia
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): L. Martínez-Carreres, J. Puyal, L.C. Leal-Esteban,
M. Orpinell, J. Castillo-Armengol, A. Giralt, L. Zhang, I.C. Lopez-Mejia, L. Fajas
Writing, review, and/or revision of the manuscript: L. Martínez-Carreres,
J. Puyal, M. Orpinell, A. Giralt, V. Barquissau, A. Nasrallah, L. Zhang,
P. Romero, I.C. Lopez-Mejia, L. Fajas
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): L. Martínez-Carreres, M. Orpinell
Study supervision: M. Orpinell, L. Fajas
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Acknowledgments
The authors acknowledge all the members of the Fajas laboratory for
support and discussions. The authors thank Jean Daraspe (University of
Lausanne, Switzerland) for technical assistance and the Electron Microscopy
Facility at the University of Lausanne for the use of electron microscopes. The
authors thank Patrice Waridel and Manfredo Quadroni (from the Protein
Analysis Facility, Center for Integrative Genomics, Faculty of Biology and
Medicine, University of Lausanne, Switzerland) for their help with mass
spectrometry analysis. This work from Prof. Fajas lab is supported by the
Swiss National Foundation (31003A-159586). The work on autophagy of
Julien Puyal is supported by the Swiss National Foundation (310030-
163064 and 310030_182332). The work of I.C. Lopez-Mejia is supported
by the Swiss National Science Foundation (Ambizione PZ00P3_168077).
The authors thank Prof. Kei Sakamoto, Prof. Christian Widmann, Prof. Alejo
Efeyan, and Prof. Mariano Barbacid for kindly providing some material used
in this work.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received February 28, 2019; revised June 28, 2019; accepted August 1, 2019;
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 Published OnlineFirst August 8, 2019; DOI: 10.1158/0008-5472.CAN-19-0708

CDK4 Regulates Lysosomal Function and mTORC1 Activation to

Promote Cancer Cell Survival
Laia Martínez-Carreres, Julien Puyal, Lucía C. Leal-Esteban, et al.

Cancer Res 2019;79:5245-5259. Published OnlineFirst August 8, 2019.

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