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BCL6 and the Notch pathway: a signaling axis leading to a novel druggable biotarget in triple negative breast cancer

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Abstract

Background

The transcriptional repressor B-cell lymphoma 6 (BCL6) is dysregulated in several neoplasms, but its role in triple negative breast cancer (TNBC), a highly aggressive subtype which lacks effective treatment, is unclear. The presence of intratumoral cancer stem cells (CSCs) is a main cause of tumor relapse. The Notch signaling pathway is crucial for regulating CSC self-renewal and promoting breast cancer (BC) development and resistance to anticancer therapies. Here, we investigated signaling cascades of BCL6 in the CSC compartment of TNBCs, and the mechanisms that govern its activity, mainly through Notch signaling.

Methods

Gene expression, somatic copy number alterations and clinical data from the Cancer Genome Atlas and METABRIC were accessed through the Xena and cbioportal browsers. Public transcriptome profiles from TNBC datasets were retrieved from the Gene Expression Omnibus. Mammosphere formation efficiency was calculated after BCL6 knockdown via transient siRNA transfection, stable silencing or pharmacological inhibition. The effects exhibited via BCL6 inhibition in putative TNBC stem-like cells were evaluated by immunofluorescence and qRT-PCR analyses. Chromatin immunoprecipitation experiments were performed to validate a putative BCL6 responsive element located in the first intron of the Numb gene and to define the circuit of corepressors engaged by BCL6 following its inhibition. Immunoprecipitation assays were carried out to investigate a novel interaction at the basis of BCL6 control of CSC activity in TNBC.

Results

In silico analyses of benchmarked public datasets revealed a significant enrichment of BCL6 in cancer stemness related pathways, particularly of Notch signaling in TNBC. In vitro stable inhibition of BCL6 significantly reduced tumor cell growth and, accordingly, we found that the mammosphere formation efficiency of BCL6 silenced cells was significantly impaired by pharmacological inhibition of Notch signaling. BCL6 was found to be expressed at significantly higher levels in TNBC mammospheres than in their adherent counterparts, and loss of BCL6 function significantly decreased mammosphere formation with preferential targeting of CD44-positive versus ALDH-positive stem-like cells. Functional interplay between BCL6 and the chromatin remodeling factor EZH2 triggered the BCL6/Notch stemness signaling axis via inhibition of Numb transcription.

Conclusions

Our results may be instrumental for the prospective design of combination treatment strategies that selectively target novel TNBC-associated biomarker(s) whose activity is implicated in the regulation of cancer stemness (such as BCL6) and molecules in developmentally conserved signaling pathways (such as Notch) to achieve long-lasting tumor control and improve patient outcomes.

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Availability of data and materials

All data supporting the conclusions of this article are included within the article and its additional files. TCGA data from RNA-seq (HiSeq counts) and somatic CN alterations were accessed using the Xena browser. METABRIC-processed data were retrieved from BioPortal. Normalized data and clinical information including TNBC datasets were retrieved from GEO. The ChIP seq data relative to the HER2-positive SKBR3 BC cell line analyzed in the present study are available upon request from the corresponding author of [6].

Abbreviations

BCL6:

B-cell lymphoma protein 6

TNBC:

Triple negative breast cancer

CSCs:

Cancer stem cells

BC:

Breast cancer

2nd gen:

Second generation of mammospheres

SMRT:

Silencing mediator for retinoid and thyroid receptors

BCoR:

BCL6 corepressor

HDACs:

Histone deacetylases

EZH2:

Enhancer of zeste homolog 2

HIF-1α:

Hypoxia-inducible factor 1-alpha

EMT:

Epithelial–mesenchymal transition

ZEB1:

Zinc finger e-box binding homeobox 1

Wnt:

Wingless-related integration site

RI-BPI:

Retro-inverso BCL6 peptide inhibitor

BTB:

Broad-complex, tramtrack, and bric-à-brac

TCGA:

The cancer genome atlas

CN:

Copy number

GEO:

Gene expression omnibus datasets

IQR:

Interquartile range

ER:

Estrogen receptor

PR:

Progesterone receptor

HER2:

Human epidermal growth factor receptor 2

GSEA:

Gene set enrichment analysis

MSigDB:

Molecular signature database

ssGSEA:

Single-sample GSEA

IC50 :

The concentration that inhibits 50% of cell growth

DMSO:

Dimethyl sulfoxide

EED:

Embryonic ectoderm development

PRC2:

Polycomb repressive complex 2

H3K27me3:

Tri-methylated lysine 27 of histone H3

MFE:

Mammosphere-forming efficiency

qRT-PCR:

Quantitative real-time PCR

siRNA:

Small RNA interference

siBCL6:

SiRNAs specific for BCL6

siSCR:

Scramble control siRNA

shRNA:

Short hairpin RNA interference

shBCL6-SUM149 and shBCL6-SUM159:

Stable BCL6-silenced SUM149 and SUM159 cells

shSCR-SUM149 and shSCR-SUM159:

Stable scramble control-silenced SUM149 and SUM159 cells

GFP:

Green fluorescent protein

FACS:

Fluorescence activated cell sorting

MFI:

Mean fluorescence intensity

ALDH:

Aldehyde dehydrogenases

BAAA:

BODIPY-aminoacetaldehyde

DEAB:

Diethylaminobenzaldehyde

ALDH+ :

ALDH-positive cells

ALDH-:

ALDH-negative cells

FITC:

Fluorescein isothiocyanate

WB:

Western blot

IP:

Immunoprecipitation

ChIP:

Chromatin immunoprecipitation

FDR:

False discovery rate

BL1:

Basal-like 1 BC subtype

BL2:

Basal-like 2 BC subtype

IM:

Immunomodulatory BC subtype

LAR:

Luminal androgen receptor BC subtype

M:

Mesenchymal BC subtype

UNS:

Unstable BC subtype

mRNA:

Messenger RNA

3D:

3-Dimensional mammosphere-forming cell culture conditions

2D:

2-Dimension adherent cell culture conditions

CD44+ :

CD44-positive cells

CTRL:

Parental control

ChIP seq:

Chromatin immunoprecipitation by deep sequencing

NT:

Untreated cells

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Acknowledgements

We are indebted to Dr. Leandro Cerchietti, Department of Medicine, Division of Hematology & Medical Oncology, Weill Cornell Medicine, New York, USA, for providing FX1 and for critical discussions. We are grateful to Dr. P. Longoni for his excellent technical assistance and support in the cytofluorimetric analyses, and L. Mameli for her excellent assistance at the Fondazione IRCCS Istituto Nazionale dei Tumori di Milano.

Funding

This work has been supported by: -BANDO RICERCA STRATEGICA ISTITUZIONALE-2016 “Molecular and phenotypic landascape of high-grade breast cancer (HGBC) tumor microenvironment: characterization and reconversion of the immunosuppressive tumor-surrounding stroma and cell compartment” to MDN; -MICROTHER– “Improving immunotherapy of solid tumors by targeting the immunosuppressive tumor microenvironment: from preclinical "proof-of-concept" to the development of phase I studies” to MDN; - Associazione Italiana Ricerca Cancro (AIRC) to S.M. Pupa.

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Author notes

  1. Serenella M. Pupa and Massimo Di Nicola contributed equally to this work.

Authors and Affiliations

  1. Unit of Immunotherapy and Anticancer Innovative Therapeutics, Department of Medical Oncology and Hematology Fondazione, IRCCS Istituto Nazionale Dei Tumori, Milan, Italy

    Francesca De Santis, Giovanni Fucà & Massimo Di Nicola

  2. Department of Genomic Medicine and Toxicology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico

    Sandra L. Romero-Cordoba

  3. Biochemistry Department, Instituto Nacional de Ciencias Médicas Y Nutrición Salvador Zubirán, Mexico City, Mexico

    Sandra L. Romero-Cordoba

  4. Molecular Targeting Unit, Department of Research, Fondazione IRCCS Istituto Nazionale Dei Tumori, Milan, Italy

    Lorenzo Castagnoli, Simona Faraci, Elda Tagliabue & Serenella M. Pupa

  5. The New York Stem Cell Foundation Research Institute, New York, NY, USA

    Tatiana Volpari

  6. Department of Medical Oncology and Hematology, Fondazione IRCCS Istituto Nazionale Dei Tumori, Milan, Italy

    Filippo De Braud

  7. Department of Oncology and Oncohematology, University of Milan, Milan, Italy

    Filippo De Braud

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  1. Francesca De Santis
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Contributions

FDS, TV, SF and LC performed most experiments; FDS, SLR-C and LC performed the statistical analyses; FDS, SLR-C, SMP and MDN designed the study; FDS, SLR-C and SMP integrated the results and drafted the manuscript; FDS, SLR-C, LC, GF, SMP and MDN assisted with data analyses and interpretation; ET and FDB revised the manuscript; SMP and MDN were responsible for the study supervision. All authors have read and approved the final submitted manuscript.

Corresponding author

Correspondence to Massimo Di Nicola.

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Supplementary file1 (PDF 449 KB)

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Supplementary file2: Correlation plot between protein and mRNA expression levels in BC from Krug et. al, Cell 2020 (upper side) and TCGA (lower side) samples for Bcl6, Numb and Hes1 genes. In all cases correlations were computed by Pearson metric (PDF 112 KB)

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Supplementary file3: (a-d) WB analysis of protein extracts from CTRL, siSCR- or siBCL6-transduced SUM149 (a) and SUM159 (c) TNBC cell lines to evaluate BCL6 expression. β-Actin was used to normalize protein loading. (b, d) Densitometric analyses of the plots shown in a and c. (e, f) Cell counts of CTRL, siBCL6-SUM149 (e) and siBcl6-SUM159 (f) cells evaluated 48 h after transient BCL6 silencing. Data were normalized to proper siSCR samples. Column bars indicate the means ± SD (n = 3). Significance was calculated by Student’s t-test. (g, h) Cell counts of CTRL, shBCL6-SUM149 (g) and shBCL6-SUM159 (h) cells evaluated at different time points. Data were normalized to proper shSCR samples. Column bars indicate the means ± SD (n = 3). Significance was calculated by Student’s t-test (PDF 50 KB)

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Supplementary file4: CSC-related biomarker evaluation in siBCL6- or matched siSCR--knockdown SUM149 or SUM159 cells. (a, b) FACS analysis of CD44+ (a) or ALDH+ (b) cell subsets compared to matched isotype-stained cells and DEAB control samples (PDF 250 KB)

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Supplementary file5: FX1 IC50 titration. (a-d) IC50 values of the FX1 inhibitor in SUM149-derived (a, c) or SUM159-derived (b, d) mammospheres (a, b) or their 2D cell counterparts (c, d) (PDF 32 KB)

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Supplementary file6: (a, b) Percentage of the MFE of SUM159 (a) or SUM149 (b) cells treated with FX1 or DMSO. Column bars indicate the means ± SD (n = 3). Significance was calculated by the Student’s t-test. (c, d) Microphotographs showing SUM159-derived (c) or SUM149-derived (d) mammospheres cultured for seven days in presence of FX1 or DMSO (magnification 20x) (PDF 24 KB)

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Supplementary file7: BCL6 binding to positive and negative control genomic regions. (a, b) ChIP analysis of NT or FX1- or DMSO-treated SUM149 (a) or SUM159 (b) cell lines for 12 h or 24 h at the genomic loci identified as region B and the CTRL region. Column bars indicate the means ± SD (n = 3). The black column represents the IgG background. ChIP results are given as 2.5% ChIP compared to the input material. Screen shots were taken from the UCSC Genome Browser on Human Dec. 2013 (GRCh38/hg38) Assembly and represent the region B genomic locus (c) or the CTRL region (d) (PDF 110 KB)

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De Santis, F., Romero-Cordoba, S.L., Castagnoli, L. et al. BCL6 and the Notch pathway: a signaling axis leading to a novel druggable biotarget in triple negative breast cancer. Cell Oncol. 45, 257–274 (2022). https://doi.org/10.1007/s13402-022-00663-y

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  • DOI: https://doi.org/10.1007/s13402-022-00663-y

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