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. 2015 Sep 8;6(26):22467-79.
doi: 10.18632/oncotarget.4164.

DMXL2 drives epithelial to mesenchymal transition in hormonal therapy resistant breast cancer through Notch hyper-activation

Monica Faronato  1 , Van T M Nguyen  1 , Darren K Patten  1 , Ylenia Lombardo  1 , Jennifer H Steel  1 , Naina Patel  1 , Laura Woodley  1 , Sami Shousha  1 , Giancarlo Pruneri  2 , R Charles Coombes  1 , Luca Magnani  1
Affiliations

DMXL2 drives epithelial to mesenchymal transition in hormonal therapy resistant breast cancer through Notch hyper-activation

Monica Faronato et al. Oncotarget. .

Abstract

The acquisition of endocrine therapy resistance in estrogen receptor α (ERα) breast cancer patients represents a major clinical problem. Notch signalling has been extensively linked to breast cancer especially in patients who fail to respond to endocrine therapy. Following activation, Notch intracellular domain is released and enters the nucleus where activates transcription of target genes. The numerous steps that cascade after activation of the receptor complicate using Notch as biomarker. Hence, this warrants the development of reliable indicators of Notch activity. DMXL2 is a novel regulator of Notch signalling not yet investigated in breast cancer. Here, we demonstrate that DMXL2 is overexpressed in a subset of endocrine therapy resistant breast cancer cell lines where it promotes epithelial to mesenchymal transition through hyper-activation of Notch signalling via V-ATPase dependent acidification. Following DMXL2 depletion or treatment with Bafilomycin A1, both EMT targets and Notch signalling pathway significantly decrease. We show for the first time that DMXL2 protein levels are significantly increased in ERα positive breast cancer patients that progress after endocrine therapy. Finally, we demonstrate that DMXL2 is a transmembrane protein with a potential extra-cellular domain. These findings identify DMXL2 as a novel, functional biomarker for ERα positive breast cancer.

Keywords: DMXL2; EMT; Notch; breast cancer; endocrine therapy.

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Conflict of interest statement

CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. Notch pathway is upregulated in LTED and is dependent on DMXL2 overexpression
A–D. Representative western blot showing (reported) Notch transmembrane domain (TM) and Notch target Hey2 in MCF7 and LTED cells (clone 1 and 2 respectively) Membranes were blotted with commercially available antibodies against Notch total level B–E. q(RT) PCR mRNA normalised to 28S of Notch receptors and Notch targets C–F. Cells were fractionated and the chromatin fraction was loaded. Membranes were blotted with in house-developed antibodies against the intracellular domain (Notch ICD).
Figure 2
Figure 2. DMXL2 is overexpressed in metastatic tumors resistant to endocrine therapy
A. Primary and matched Metastatic samples were processed using DMXL2 IHC B. H scores were plotted. Pair-wise T-test between the average score (3 independent scorers, duplicate sections) was used to establish significance. DMXL2 is overexpressed in ETR cells and it is epigenetically activated. C. Representative western blot showing DMXL2 is overexpressed in LTED resistant cells. Graph shows protein normalised to loading control (Actin). q(RT) PCR mRNA levels normalised to 28S. In (C) cells were cultured as described in material and methods. In D. cells were cultured in MCF7 media (3 days). (*) P < 0.05 (**) P < 0.01 (***) P < 0.001 E. Different clone of MCF7 and LTED showing DMXL2 upregulation. F. Snapshots from DNaseI hypersensitivity site (DHS-seq) and Chip-seq for K27ac histone mark near DMXL2 promoter across all 6 breast cancer cell models. Chip-seq tracks display the active regulatory elements near DMXL2 promoter are uniquely found in estrogen deprived cell lines (LTED cells), indicating the activation of DMXL2 gene in LTED cells, but not MCF7 cells.
Figure 3
Figure 3. DMXL2 regulates Notch pathway
A. q(RT) PCR mRNA levels of Notch targets following DMXL2 depletion B. Representative immunofluorescence images of Hey2 following siDMXL2 showing Hey2 nuclear signal was impaired. Bars represent 400 uM C. Representative western blot showing reported TM Notch accumulates following DMXL2 depletion. Antibodies were used as in 1A D. Representative blot showing Notch3 and 4ICD reduced chromatin accumulation following siDMXL2. Antibodies were used as in 2C. Knock down was performed for 48 or 72 hrs (for mRNA or protein extraction respectively). (*) P < 0.05 (**) P < 0.01 (***) P < 0.001. Every experiment is an average of three independent experiments. Bars represent standard deviation.
Figure 4
Figure 4. DMXL2 depletion is mirrored by V-ATPase inhibition
A. Representative immunofluorescence showing reduced Lysotracker staining following 72 hours DMXL2 depletion. Bars represent 400 uM B. Lysotracker staining of acidic intracellular compartments in LTED cells following Bafilomycin A1 treatment (6 hours). Bars represent 400 uM. C. Representative western blot showing Notch ICDs stop accumulating in the nucleus and the chromatin fraction following Bafilomycin A1 (BafA1) treatment in a time dependent manner D. q(RT) PCR mRNA of Notch targets levels following BafA1 treatment. In B, C, and D cells were treated with 125 pM of Bafilomycin A1. E. Notch targets are rescued by ectopic expression of Notch ICD. Cells were pre-treated for 6 hrs with 500 pM of BafA1 followed by 24 hrs Notch ICD over-expression. Every experiment is an average of three independent experiments. Bars represent standard deviation (*) P < 0.05 (**) P < 0.01 (***) P < 0.001.
Figure 5
Figure 5. DMXL2 regulates mesenchymal switch in LTED cells
A. Representative western blot showing EMT targets genes in LTED cells compared to MCF7 B. q(RT) PCR mRNA levels of EMT targets normalised to 28S C. Representative western blot showing regulation of EMT genes following DMXL2 knock down D. q(RT) PCR mRNA of EMT genes after siDMXL2 E, F. Knock down was performed on 6 well plate for 24/48 hours. Cells were harvested and counted. 40,000 were seeded on 16 wells ECM plates with/without matrigel (for invasion and migration respectively) and left migrating or invading for 48/24 hours respectively. The graphs represent the difference in cell number compared to siControl G. 3D Invasion assay organoids. Representative pictures of organoids embedded in matrigel after 5 days knock down (performed during the hanging time) at day 0 and 2. The graph represents the difference in the area between day 0 (cells just embedded) and day 2 (cells left invading. Bars represent 400 uM. H. Bafilomycin A1 impairs cell invasion. Cells were treated as in 4C. qRT-PCR mRNA for EMT genes is shown. I. Representative pictures of organoids. Medium containing 500 pM BafA1 was added to the medium for 48 hours after embedding the organoids in matrigel. Day 0 and day 2 images are shown. L. EMT targets are rescued by ectopic expression of Notch ICD. Cells were pre-treated for 6 hrs with 500 pM of BafA1 followed by 24 hours Notch ICD over-expression. Every experiment is an average of three independent experiments. Bars represent standard deviation (*) P < 0.05 (**) P < 0.01 (***) P < 0.001.
Figure 6
Figure 6. DMXL2 has a potential extracellular domain
A. Western blot showing cells labeled with biotin and processed as described in material and methods. EGFR and Actin are used as controls B. Graphs showing DMXL2 putative transmembrane domain C. Representative live immunofluorescence images showing DMXL2 is detected only with the antibody interacting with the potential extracellular domain (AbCAm antibody). Bars represent 400 uM.
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