Gene-expression analyses identify altered transcription factors and supports the antitumor activity of novel bromodomain inhibitors in triple negative breast cancer Ocana A¹, Perez-Peña J¹, Serrano-Heras G¹, Corrales-Sánchez V¹, Montero JC², Martin M³ and Pandiella A². 1 Translational
Research Unit, Albacete University Hospital, Albacete, Spain. 2 Cancer Research Unit, CSIC-University of Salamanca, Spain. 3 Health Research Institute Gregorio Marañón, Complutense University, Madrid, Spain.
INTRODUCTION
RESULTS Expression of TFs in human basal-like tumors is reduced by treatment with JQ1
CANCER
426 deregulated genes
30 transcription factors
• Gene transcription profiling and Gene-set enrichment analyses. qRT-PCR of identified genes
Angiogenesis 1 00
2
6 12 44 88 10 12 % of genes included % of genes included
Annexin V+
MDA-MB-231 Cyclin D1 Cyclin D3 Cyclin A Cyclin B
14
Cyclin E
JQ1 (µM) - 0.2 0.5 0.2 0.5 - 0.2 0.5 0.2 0.5 12h 24h 12h 24h
0.2
JQ1 (µM) Vinorelbine (nM) Docetaxel (nM) Cisplatin (µM) Carboplatin (µM)
p27
0.2 0.2 0.2 0.2 1 1 0.5 0.5 2.5 2.5 25 25
pRb S807/811
Annexin V+
1.6 1.4 1.2 1 0.8
Top modified genes
0.6 0.4 0.2
Annexin V-
pRb S780
HS-578T
Annexin V+
HS578T
Wee 1 pcdc2 Y15
9 8 7 6 5
BUBR1 CDK2 CDK4
4
GAPDH
3 2
JQ1 (µM) - 0.2 0.5 0.2 0.5 - 0.2 0.5 0.2 0.5
1
0 0
JQ1 (µM) Vinorelbine (nM) Docetaxel (nM) Cisplatin (µM) Carboplatin (µM)
24h
55
10 10
15 15
20 20
48h
24h
48h
0.2
0.2 0.2 0.2 0.2 1 1 0.5 0.5 2.5 2.5 25 25
25
% of genes included
0
In vivo effect of JQ1
Effect of BET inhibitors on proliferation and synergistic interaction with chemotherapy Concentration JQ1 (µM) 72 hours
MTT metabolization (A 562) (% of control)
MDA-MB-231 HS578T BT549 HCC3153
100 100
8080
90 90 80 80 Mda-Mb-231 70 Hs-578-T 70 BT549 60 60 HCC3153 50 50 40 40 30 30 20 20 10 10 00
*
60
60
4040 2020
** **
MDA-MB-231
0
0,1
0
0.1
0,2
0.2
0,4
0.4
0,6
0.6
0,8
0.8
1
CONTROL JQ1 0.1 µM
HS-578-T
BT549
HCC3153
5
1
5
Concentration JQ1 (µM) 72 hours
Concentration JQ1 (µM)
2
Antagonism Additivity Synergy
1
Antagonism Additivity Synergy
JQ1 (nM) Carboplatin (µM) Cisplatin (µM) Docetaxel (nM) Vinorelbine (nM)
40 80 120 160 200 40 80 120 160 200 20 40 60 80 100 20 40 60 80 100 5 10 15
20 25 0.5 1 1.5
2
2.5 0.2 0.4 0.6 0.8 1 0.4 0.8 1.2 1.6 2
For the evaluation of JQ1, we used a panel of four triple negative cell lines including MDA-MB-231, HS578T, BT549 and HCC3153. Treatment reduced proliferation measured by MTT uptake in a dose and time dependent. Studies using clonogenic cell survival assays also showed the effect of the drugs on the number of colonies formed at ten days in MDA-MB-231, HS578T, BT549 and HCC3153 using doses below the IC50. As chemotherapy is the standard of care for the treatment of TNBC, we decided to combine BET inhibitors with chemotherapies that are routinely used in the clinical setting. For this purpose we used the two more sensitive cell lines MDA-MB-231 and HS578T.
1200 1200
CONTROL JQ1 50 mg/kg (i.p daily)
1000 1000
*
MDA-MB-231 JQ1, 50 mg/kg
800 800 600 600 400 400 p27
48 h
MDA-MB-231 as a model and the non-transformed cell line MCF-10A as a control, we evaluated if treatment with the BET inhibitor JQ1 was able to inhibit some of the identified upregulated genes. After treatment with JQ1 at different time points the expression of some of these genes were reduced.
Treatment with the BET inhibitor JQ1 induced arrest at the G1 phase in MDA-MB-231 and HS578T cells. Biochemical evaluation of the mechanism of action confirmed that JQ1 increased the expression of proteins that negatively regulate cell cycle progression in this phase like p21 and p27. Elevated levels of cyclin D1 and D3 were also observed. We also identified a reduction of proteins involved in progression along G2 or M phases such as cyclin B3 or BUBR1. Effect on apoptosis mediated by JQ1 alone or in combination. An increase in apoptosis was observed when administered with platinum agents.
Control
X-fold mRNa Expression
1.8
1
and
2
Endocrine process Chromatin organization Stress response Cell differentiation Apoptosis and cell death Other functions Immune response Biosynthetic process Transcription
MCF10A MDA-MB-231 (untreated) MDA-MB-231 (24h treatment, JQ1) MDA-MB-231 (48h treatment, JQ1)
0
animals
Endocrine process
Annexin V-
HS578T
12 24 48 C 12 24 48
Cell percentage
2
• MTTs and biochemical studies using western-blots
• In vivo effect using xenografted pharmacodynamic assessment
3
C
JQ1 500 nM 24h treatment
2
• Analyses of synergism with the CalcuSyn v.2.0 software program
Cell differentiation
MDA-MB-231 Sub G0 G0/G1 S G2/M
MDA-MB-231
0
• Evaluation of apoptosis and cell cycle arrest by flow cytometry using Annexin V and propidium iodide, respectively.
Other functions 4
HS-578T
Annexin V-
00
• Triple negative cell lines and xenografted nude mice
MDA-MB-231
p21
120 120
MATERIAL AND METHODS
Apoptosis and cell death 6 Biosynthetic process / 5 Nitrogen metabolism
Cell percentage
BREAST
BREAST
• To identify genes that encoded for transcription factors (TFs) that were upregulated in basal-like tumors. • To evaluate the antitumor effect of BET inhibitors: JQ1 alone and JQ1 combined with chemotherapy in vitro and in vivo
SIPB1. Spi-B transcription factor (Spi-1/PU.1 related) SOX11. SRY (sex determining region Y) -box 11 FOXC1. Forkhead box C1 ZNF 367. Zinc finger protein 367 EN1. Engrailed homolog 1 PITX1. Paired-like homeodomain transcription factor 1 UHRF1. Ubiquitin-like, containing PHD and RING finger domains 1 ZIC1. Zic family member 1 (odd-paired homolog, Drosophila) NFE2L3. Nuclear factor (erythroid-derived 2)- like 3
Cell percentage
NORMAL
Control 1
2
24 h 1
2
48 h 1
2 WB: α-p27
200 200 00
GAPDH 1
0
55
1010
WB: α-GAPDH
1414
Days post-treatment
Weight tumor (g)
OBJECTIVES
BASAL LIKE
Tumor volume (mm³)
In this work using gene-expression analyses we identify transcription factors overexpressed in TNBC that are druggable by BET inhibitors.
GOTERM_BP_5
Transcription factors with increased expression
Epigenetic regulators have been considered key druggable players in some cancers and inhibition of their action produced clinical benefit. A novel family of epigenomic mediators includes agents that act on bromodomains. Proteins with bromodomains play a central role regulating the expression of several genes involved in malignant transformation. In the last years, studies have explored the antitumor effect of agents that inhibit the bromodomain and extraterminal (BET) family of bromodomain containing proteins. In silico evaluation of human tumors by gene expression analyses can identify functions that could be pharmacologically inhibited helping to select druggable targets.
Cell cycle arrest and apoptotic effect by BET inhibitors alone and in combination with chemoterapy
JQ1 500 nM 12h treatment
Number of colonies
Triple negative breast cancer (TNBC) is an incurable disease with poor prognosis and limited therapeutic options beyond treatment with chemotherapy. It is characterized by its rapid proliferation and metastatic potential. In this context, the identification of novel anticancer agents with potential clinical activity is an important objective.
1 0.8 0.6 0.4 0.2 0 CONTROL JQ1 50 mg/kg (i.p daily)
In vivo activity of BET inhibitors on MDA-MB-231 xenografted tumors. Nude mice were treated with JQ1 at 50mg/kg i.p. daily for two weeks. Treatment with this compound produced an inhibition of the tumor growth and a clear reduction of tumor weight. No toxicities were observed. To evaluate the mechanism of action in vivo, we treated animal at the mentioned dose and extracted tumors at 24 and 48 hours. The biochemical evaluation of the treated tumors demonstrated an increased expression of p27, as observed in cell lines, demonstrating its effect on G1.
CONCLUSION Gene-expression profiling identified upregulated transcription factors in triple negative tumors compared with normal breast human samples. BET bromodomain inhibitors reduced the expression of some of these genes in cellular models. JQ1 shows clear antitumor activity in vitro and in vivo. Corresponding author email address: Dr. Alberto Ocaña,
[email protected]
