Leon_Machado_Jorge_Alfonso_MSc_2017.pdf (3.345Mb)

INTERACTION BETWEEN ESTROGEN AND INTERFERON- SIGNALING
PATHWAYS IN THE REGULATION OF MAJOR HISTOCOMPATIBILITY
COMPLEX CLASS II EXPRESSION IN BREAST CANCER CELLS
By
Jorge Alfonso Leon Machado
Thesis presented to the Département de Biologie as condition for
the achievement of a Master’s degree in science (M. Sc.)
FACULTÉ DES SCIENCES
UNIVERSITÉ DE SHERBROOKE
Sherbrooke, Québec, Canada, January 2017

January 20 2017
Members of the jury
Professor Viktor Steimle
Directeur de recherche
Université de Sherbrooke, Département de Biologie
Professor Nicolas Gévry
Président rapporteur
Université de Sherbrooke, Département de Biologie
Professor Richard Blouin
Membre interne
Université de Sherbrooke, Département de Biologie

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SUMMARY
Activation of the antigen presentation mechanisms by cancer cells is one of the main
pathways used by the immune system for tumor detection and suppression. Induction
of the expression of molecules of the Major Histocompatibility complex class II (MHC-
II) by the interferon- (IFN) is important for the efficient presentation of tumor antigens.
Nevertheless, it has been observed that expression of these molecules is supressed in
tissular contexts where the concentration of estradiol (E2) is high.
In this work we attempted to explain if the down-regulation exerted by estradiol on the
expression of the MHC-II molecules in breast cancer cells was mediated by a silencing
effect of the estrogen receptor- (ER) through a possible estrogen receptor binding
site (ERBS) in the locus of promoter IV (pIV) of the master regulator of MHC-II
expression, the class II transactivator (CIITA). The breast cancer cell line MDA-MB-231
(ER-/ERβ-) and its stable transfectants MC2 (ER+) and VC5 (empty vector) were
used as model cell lines. Expression of the MCH-II molecules is controlled by CIITA,
and stimulation with IFN activates the transcription of the pIV of CIITA. Stimulation of
these cell lines with IFN induced expression of the MCH-II molecules and addition of
E2 repressed such expression only in the MC2 cell line, as observed by flow cytometry
analysis. Six other breast cancer cell lines were tested, with only the MCF7 cell line
showing a significant inhibition. Then we analyzed if the inhibition of the MHC-II
expression was due to a down-regulation of CIITA. Protein analysis performed by
western blot and mRNA quantification by RT-qPCR both revealed down-regulation of
CIITA in the cells exposed to IFN+E2 compared to those treated only with IFN.
However, reporter gene analysis did not demonstrate any influence of our candidate
ERBS in the inhibition of the activation of CIITA-pIV. ChIP-seq analysis of the VC5 and
MC2 cell lines for ER also failed to demonstrate any binding of the receptor anywhere
in the vicinity of the CIITA locus. However gene ontology and disease ontology analysis

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of the sequencing data showed a higher activation of tumorigenic cellular pathways in
the cells treated with IFN + E2 than in the cells treated only with E2. These results
suggest that activation of the inflammatory pathways by IFN could exert a detrimental
effect on the cancer development.
Key words: breast cancer, antigenic presentation, CIITA, MHC-II, estrogen, interferon
gamma, estrogen receptor binding site.