DOI: 10.5281/zenodo.16947455
title: GYLATL1 promotes endocrine therapy resistance in luminal breast cancer and is regulated by estrogen-independent ERα signaling

description: Supplementary Information for study:

GYLATL1 promotes endocrine therapy resistance in luminal breast cancer and is regulated by estrogen-independent ERα signaling

Janina Müller1,2, Emre Sofyali1,2, Luisa Schwarzmüller1,2, Yael Aylon3, Eviatar Weizman4, Lisa Schlicker5, Katherine Kelly2,6, Simone Borgoni1,2, Simin Oz6, Birgitta E Michels1, Sara Burmester1, Angelika Wörner1, Sabine Karolus1, Daniela Heiss7, Rainer Will7, Veronica Rodrigues de Melo Costa1, Cole Stocker1,2, Pavlo Lutsik6, Dieter Weichenhan6, Ilse Hofmann8, Nishanth Belugali Nataraj9, Yosef Yarden9, Luca Magnani10, Christoph Plass6, Almut Schulze5, Cindy Körner1, Moshe Oren3, Stefan Wiemann1

Affiliations

1 Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany

2 Faculty of Biosciences, University of Heidelberg, Im Neuenheimer Feld 234, 69120 Heidelberg, Germany

3 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel

4 G-INCPM, Weizmann Institute of Science, Rehovot 76100, Israel

5 Division of Tumor Metabolism and Microenvironment, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg, Germany

6 Division of Cancer Epigenomics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany

7 Cellular Tools Core Facility, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany

8 Antibody Core Facility, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

9 Department of Immunology and Regenerative Biology, Weizmann Institute of Science, Rehovot 76100, Israel

10 Department of Oncology and Hematology-Oncology, Università degli Studi di Milano, Via Festa del Perdono 7, 20122 Milan, Italy

 

Experimental data

Epigenetic-focused cytometry time-of-flight (EpiTOF) [1, 2]

Experiment carried out by Janina Müller, in the lab of Yael Aylon and Moshe Oren. Data analyzed by Eviatar Weizmann and Luisa Schwarzmüller.

MCF7 (RRID:CVCL_0031) single-cell suspensions were generated and washed with Maxpar PBS (#201058, Fluidigm, San Francisco, CA. USA) and subsequently stained with 1.25 µM Cisplatin for one minute to identify dead cells. The staining reaction was quenched using DMEM supplemented with 10% FBS. Cells were washed with Maxpar Cell Staining Buffer (#201068, Fluidigm, San Francisco, CA, USA), and about 3 × 106 cells per sample were fixed, permeabilized using the Maxpar Nuclear Antigen Staining Buffer Set (#201063, Fluidigm, San Francisco, CA, USA), and barcoded with the Cell-ID 20-Plex Pd Barcoding Kit (#201060, Fluidigm, San Francisco, CA, USA) according to the manufacturer's instructions. Following two washing steps with Maxpar Nuclear Antigen Staining Buffer Set permeabilization buffer, equal amounts of barcoded samples of the different MCF7 cell lines (i.e., parental, LTED, KO1, and KO2) were combined and incubated with an antibody cocktail (Supplementary Table 11) for 30 minutes at room temperature. Following antibody incubation, the cells were washed twice with Maxpar Cell Staining Buffer and fixed at 4 °C with fresh 4% formaldehyde (#28908, Thermo Fisher Scientific, Waltham, MA, USA), ensuring gentle rocking to prevent clumping. Following overnight incubation, 125 nM Cell-ID Intercalator-Ir (#201192A, Fluidigm, San Francisco, CA, USA) was added and incubated for 45 minutes at room temperature to label DNA. Afterward, cells were washed twice with Maxpar Cell Staining Buffer and once with Maxpar Water (#201069, Fluidigm, San Francisco, CA, USA). Cells were resuspended in a dilution of EQ Four Element Calibration Beads (#28908, Fluidigm, San Francisco, CA, USA) in Maxpar Water, achieving a concentration of approximately 250K cells/ml, and subsequently filtered through a 35 μm mesh. Data was acquired on a CyTOF Helios platform (Fluidigm, San Francisco, CA, USA). Normalization and data cleanup to isolate live single cells were conducted as described by Bagwell et al. [3]. Metal-conjugated antibodies were either procured from Fluidigm or conjugated utilizing appropriate Maxpar X8 Antibody Labeling Kits (Fluidigm, San Francisco, CA, USA). The mass cytometry antibody panel was designed to minimize signal spillover utilizing the Maxpar Panel Designer (Fluidigm, San Francisco, CA, USA). Analysis of EpiTOF data was executed through an R-based pipeline [4]. Data were imported into R (version 4.0.2) and transformed using arcsine transformation with a cofactor of 5 and regressed to H3 and H3.3 expression. For statistical comparison, the median arcsine-transformed intensities were used (n = 2). Using a two-sided, unpaired T-test, LTED was compared against the parental cells and cells from both GLYATL1 KO clones simultaneously.

The tables contain raw data (signal intensities for every identified cell and for every tested mark) from two replicate experiments/batches. WT: parental MCF7 cell line (ATCC); LTED: long-time estrogen deprived MCF7 cell line (proxy for resistance to aromatase inhibition) [5]; KO1 and KO2: LTED cell line with CRISPR/Cas9 knockout of GLYATL1 gene, clones LTED GLYATL1 KO1 and LTED GLYATL1 KO2 [this study].

References:

1.       Aylon Y, Furth N, Mallel G, Friedlander G, Nataraj NB, Dong M, Hassin O, Zoabi R, Cohen B, Drendel V, Salame TM, Mukherjee S, Harpaz N, Johnson R, Aulitzky WE, Yarden Y, Shema E & Oren M (2022) Breast cancer plasticity is restricted by a LATS1-NCOR1 repressive axis. Nat Commun 13, 7199, doi: 10.1038/s41467-022-34863-9.

2.       Harpaz N, Mittelman T, Beresh O, Griess O, Furth N, Salame TM, Oren R, Fellus-Alyagor L, Harmelin A, Alexandrescu S, Marques JG, Filbin MG, Ron G & Shema E (2022) Single-cell epigenetic analysis reveals principles of chromatin states in H3.3-K27M gliomas. Mol Cell 82, 2696-2713 e2699, doi: 10.1016/j.molcel.2022.05.023.

3.       Bagwell CB, Inokuma M, Hunsberger B, Herbert D, Bray C, Hill B, Stelzer G, Li S, Kollipara A, Ornatsky O & Baranov V (2020) Automated Data Cleanup for Mass Cytometry. Cytometry A 97, 184-198, doi: 10.1002/cyto.a.23926.

4.       Nowicka M, Krieg C, Crowell HL, Weber LM, Hartmann FJ, Guglietta S, Becher B, Levesque MP & Robinson MD (2017) CyTOF workflow: differential discovery in high-throughput high-dimensional cytometry datasets. F1000Res 6, 748, doi: 10.12688/f1000research.11622.3.

5.       Nguyen VT, Barozzi I, Faronato M, Lombardo Y, Steel JH, Patel N, Darbre P, Castellano L, Gyorffy B, Woodley L, Meira A, Patten DK, Vircillo V, Periyasamy M, Ali S, Frige G, Minucci S, Coombes RC & Magnani L (2015) Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion. Nat Commun 6, 10044, doi: 10.1038/ncomms10044.

List of antibodies used in the experiment:

     


Name
target
metal
isotope
provider
cat#


Histone H3 Antibody (D1H2) - 115In
H3
In
115
IonPath
711501


Anti-Histone H3.3 antibody [EPR17899] - ChIP Grade
H3.3
Gd
155
Abcam
ab208690


Histone H4 (D2X4V) Rabbit mAb #13919 
H4
Tb
159
Cell Signaling
13919


Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb
H3K4me3
Nd
145
Cell Signaling
9751


Di-Methyl-Histone H3 (Lys36) (C75H12) Rabbit mAb
H3K36me2
Sm
149
Cell Signaling
2901


Acetyl-Histone H4 (Lys16) (E2B8W) Rabbit mAb #13534
H4K16AC
Sm
152
Cell Signaling
13534


Mono-Methyl-Histone H3 (Lys4) (D1A9) XP® Rabbit mAb (BSA and Azide Free)
H3K4me1
Sm
154
Cell Signaling
53138


Anti-Histone H3 (acetyl K64) antibody [EPR20713] - BSA and Azide free
H3K64ac
Gd
156
Abcam
ab251549


Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb
H3K27ac
Gd
160
Cell Signaling
8173P


Histone H3K27me3 antibody (mAb) - 168Er
H3K27me3
Er
168
Active Motif 
61017


Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb #13969
H3K9me3
Er
170
Cell Signaling
13969


Anti-Histone H3 (di methyl K27) antibody - ChIP Grade
H3K27me2
ND
142
Abcam
ab24684


Anti-Histone H3 (di methyl K9) antibody [Y49] - BSA and Azide free (ab173325)
H3K9me2
Eu
151
Abcam
ab173325


Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (BSA and Azide Free) #45772
H2Aub
Eu
153
Cell Signaling
45772


Anti-Histone H4 (tri methyl K20) antibody [EPR17001(2)] - BSA and Azide free
H4K20me3
Dy
161
Abcam
ab239410


Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAb #4909
H3K36me3
Ho
165
Cell Signaling
4909


Histone H3K9ac antibody (mAb)
H3K9ac
Tm
169
Active Motif
61663


Anti-Human CD326/EpCAM (9C4)-141Pr
EpCAM
Pr
141
FLUIDIGM
3141006B


Anti-Human CD24 (ML5)-166Er
CD24
Er
166
FLUIDIGM
3166007B


Anti-Human/Mouse CD49F (GoH3)-164Dy
CD49f
Dy
164
FLUIDIGM
3164006B


Anti-Human/Mouse CD44 (IM7)-171Yb
CD44
Yb
171
FLUIDIGM
3171003


Anti-Cytokeratin 5 antibody [SP27] - BSA and Azide free
K5
Ce
140
Abcam
ab236216 


Anti-Estrogen Receptor alpha antibody [E115] - Low endotoxin, Azide free
ERa
Dy
163
Abcam
ab167611


(3174014A) Anti-Human Keratin 8/18 (C51)-174Yb
K8/18
Yb
174
FLUIDIGM
3174014A


Anti-pHistone H3 [S28] (HTA28)-175Lu
pH3
Lu
175
FLUIDIGM
3175012A


(3162012B) Anti-Ki-67 (B56)-162Dy
Ki-67
Yb
172
FLUIDIGM
3162012B