S4.

2
Pathophysiology of Hormonal Resistance
Nama

: DR. Dr. Irza Wahid, SpPD-KHOM, FINASIM

Posisi

: Kepala Sub Bagian Hematologi-Onkologi Medik FK Unand/

RS Dr. M. Djamil Padang

Pendidikan

Dokter Umum FK Unand

Spesialis Penyakit Dalam FK Unand

Konsultan Hematologi-Onkologi Medik , Kolegium Ilmu Penyakit Dalam

Mechanisms of endocrine resistance in

breast cancer

Dr dr Irza Wahid SpPD KHOM

Division of Haematology Medical Oncology

Internal Medicine Departement Faculty of Medicine, Andalas University
General Hospital of Dr M Djamil Padang

Hormone Receptor-Positive (HR+)

Breast Cancer
Approximately 75% of invasive breast cancers
are classified as HR+1,2
ER signaling leads to

Staining of ER+ breast cancer nuclei

by immunohistochemistry (IHC)4

Cell proliferation
Time available for DNA repair
Risk of mutation

HR+ breast cancers are generally slower growing

and have a better prognosis than HR cancers1
ER expression correlates with improved
response to endocrine therapy1

A. Strong nuclear staining indicating widespread

expression of ER (Allred score = 8)

Effectiveness of endocrine therapy is limited by

denovo or acquired endocrine resistance
Multiple mechanisms within the ER pathway
allow development of resistance to endocrine
therapy3

B. Weak nuclear staining indicating low to

moderate expression of ER (Allred score = 4)

1. Cleator SJ, et al. Clin Breast Cancer. 2009;suppl 1:S6-S17; 2. Milani M, et al. Clin Med Ther. 2009;1:141-156;
3. Arpino G, et al. Endocr Rev. 2008;29(2):217-233; 4. www.breastpathology.info. Accessed September 14, 2011.
Images reprinted from NHS Trust, Edinburgh, UK. (www.breastpathology.info)

Estrogen Production
Normal tissue of subcutaneus fat, breast, muscle,
bone, ovarium. Conversion of androgen by
aromatase
ER is nuclear receptor encoded by the ESR1 gene
2 distinct transactivation domains : Activation
Function (AF 1 , AF 2)
Estrogen response element (E) Transcriptional
control proliferation, Time available for
DNA repair , Risk of mutation

Figure 1. Schematic representation of functional domains of human ER & ER.

* The A/B domain at the N-terminal contains AF-1 site.
* The C domain includes the DNA-binding domain (DBD) and a dimerization site.
* The D domain contains a nuclear localization signal.
* The E/F domain is located at the C-terminal and comprises the ligand binding, as
well as the AF-2 domain, a second nuclear localization signal, and another
dimerization site.

Berrera et al, Int. J. Mol. Sci. 2013

Figure 2: Diagram showing local estrogen production via the enzymatic onversion of
androgens to estrogen by aromatase and estrogen receptor signalling in breast cancer.
Estrogen (E), estrogen receptor alpha (ER), estrogen response element (ERE) and
heat-shock proteins (HSP).The targets of commonly used endocrine therapies in the
pathway are shown: aromatase inhibitors (AI), tamoxifen (T) and fulvestrant (F).
Dixon JM, New Journal of Science, 2014

A1. Genomic signalling (classic, direct binding)

Estrogen berikatan
dengan ER
sitoplasma/nukleus
Kompleks E-ER
menuju nukleus (NISSnuclear initiated steroid
sign)
Berikatan dengan
Estrogen Response
Element (ERE) di DNA
Kemudian akan
mengaktifkan faktor
transkripsi

A2. Genomic signal (Non Classic, tethering)

ER dan ER juga
dapat
memodulasi
ekspresi gen tanpa
ikatan langsung pada
DNA
Ada interaksi dengan
berbagai protein seperti
sp1, faktor transkripsi
fos/jun pada AP1, dan
melalui kompleks NFB

Klinge CM. Estrogen receptor interaction with estrogen response element. Nucleic
Acid Res 2001;29(14):2905-19

B. Ligand independent receptor activation

The ER signaling pathway is also regulated by membrane RTKs including EGFR, HER2,
and IGF1-R, FGFR. These membrane kinases activate signaling pathways that
eventually result in phosphorylation of ER as well as its coactivators and corepressors at
multiple sites to influence their specific functions
Crosstalk between the growth factor receptor and ER pathways has been established
through several other mechanisms . Estrogen can increase the expression of ligands
such as transforming growth factor- (TGF) and IGF1 which can then activate the
growth factor receptor pathway . On the other hand, estrogen signaling downregulates
the expression of EGFR and HER2 while increasing the expression of IGF1- R
Activation of the PI3K/AKT and the p42/44 mitogen-activated protein kinases (MAPK)
pathways by these receptors, in turn, downregulates the expression of ER and PR
Thus, while these RTKs can activate the transcriptional function of ER, they can also
reduce estrogen dependence by downregulating the expression of ER perhaps
contributing to the relative resistance to endocrine therapies in tumors amplified for
HER2 .

Osborne and Schiff. Annu Rev Med. 2011

C. Non-Genomic signal (MISS)

Membran Intiated Steroid Signaling(MISS), Non-transcriptional mechanisms

Low levels of ER have been found outside the nucleus in the membrane,
cytoplasm, or even in the mitochondria

Some of the non genomic action of estrogen appears to be too rapid for activate
GF receptor signaling, including the PI3K/AKT & Ras/p42,44, MAPK pathways .
Thus, ER can alter the expression of genes normally regulated by GF

The stress kinase pathway via p38 and JNK can also modulate ER function by
phosphorylation of ER & its coregulators . The microenvironment and its
associated integrin signaling may exert a similar activity .

Thus, ER activity and signaling is modulated by a variety of pathways which could

also contribute to resistance to ER-targeted therapies, especially when the
pathways display aberrant activity in a cancer cell.y regulated by growth factors

Osborne and Schiff. Annu Rev Med. 2011

Figure 1 Estrogen receptor action at molecular level. A: Ligand dependent activation: in classic estrogen
signaling, ligand-bound ER activates gene expression either through direct binding of dimeric ER to specific DNA
response elements in complexes including co-activators, or function as a coregulator through protein protein
interactions with other transcription factors to facilitate binding to serum response elements and activation of
transcription; B: Ligand independent activation: the ER can also be activated by ligand independent fashion, as a
consequence of signaling events downstream of membrane receptor tyrosine kinases (RTKs); C: Non genomic
mechanisms: signaling can be mediated through non-genomic mechanisms by ER that is localized at the cell
membrane or in the cytoplasm. mTOR: Mammalian target of rapamycin; FGFR: Fibroblast growth factor receptor;
IGF-1R: Insulin-like growth factor-1 receptor; EGFR: Epidermal growth factor receptor.

Zhao M et al (2014) . Advances in endocrine-resistant breast cancer

Endocrine theraphy agent

Anti estrogen
Selective estrogen
receptor modulator
(ESRM) AF1
Tamoxifen
Selective estrogen
receptor down regulator
(ESRD) AF1, AF2
Fulvestrant

Aromatase inhibitor
Non steroid inhibitor
letrozol,
anastrozol
Steroid inhibitor
exemestane

EGFR
HER2

Endocrine Therapy for HR+

Advanced Breast Cancer
Growth factor
receptor

Aromatase inhibitors
(AIs)
Nonsteroidal AIs
Anastrozole
Letrozole
Steroidal AIs
Exemestane

GRB2
SOS Shc

RAS
Cytoplasm

RAF

PI3K
MEK

Estrogen

Estrogen
receptor

Akt/
m-TOR

MAPK
Estrogen receptor
downregulator
(ERD)
Fulvestrant
P

Nucleus

Cofactor complex
LBD
LBD
P

AF1
AF1

DBD
DBD

Adapted from Yardley DA, et al. J Clin Oncol. 2011;29(suppl 27). Abstract 268.

Cell
growth

Selective estrogen
receptor modulators
(SERMs)
Tamoxifen
Toremifene
13

Endocrine Resistance in HR+ Advanced Breast Cancer

Definition of endocrine resistance is evolving

Primary resistance: PD within 6 months
of starting treatment1
Secondary resistance: Initial response with relapse
6 months or later1
Approximately 50% of patients with HR+ advanced
breast cancer do not respond to initial endocrine
therapy2
The majority of patients with HR+ advanced breast
cancer will ultimately progress despite endocrine therapy
Abbreviations: HR, hormone receptor; PD, progressive disease.
1. Bachelot T, et al. Breast Cancer Res Treat. 2010;100(suppl 1):Abstract S1-S6; 2. Bedard PL, et al. Breast Cancer Res Treat.
2008;108(3):307-317.

Endocrine Resistance in HR+ Advanced Breast Cancer

Clinical
Histopatology
Marker
- High expression of cycle machinery genes
Cyclin-D1, Cyclin-E1, CDK4, CDK6
- High expression of antiapoptotic proteins
- Abberant expression of other regulation cycle cell
C-MYC, RB1, p21, P27KIPI
Abbreviations: HR, hormone receptor; PD, progressive disease.
1. Bachelot T, et al. Breast Cancer Res Treat. 2010;100(suppl 1):Abstract S1-S6; 2. Bedard PL, et al. Breast Cancer Res Treat.
2008;108(3):307-317.

Mechanisms of Resistance to Endocrine Therapy

De novo
resistance in
breast cancer is
characterized by
loss of ER (the
ER isoform)
expression and
ER gene
mutations such
as deletion and
point mutation

Patients carrying
inactive alleles of
cytochrome
P4502D6 (CYP2D6)
deficiency cannot
convert tamoxifen to
its active metabolite,
endoxifen, therefore
are resistant to
tamoxifen.

By contrast,
multiple
mechanisms
have been
detected to
account for the
acquired
resistance to
endocrine
therapies

Dixon JM, New Journal of Science, 2014

1 A. Level of estrogen receptors

Studies have suggested that innate

resistance may be linked to lower
levels of ER, which might suggest that the
drive to proliferation of these cancers is
not as dependent on estrogen as those
expressing higher levels of ER.

1 B. ER Post-translational Modifications.
A number of post-translational modifications of ER have been reported,
including phosphorylation, methylation and sumoylation which influence its
interaction with other members of the ER signalling pathway.

It has been suggested that aberrations in the posttranslational modification of

ER could be linked to endocrine therapy resistance . ER can be phosphorylated
at a serine-118, serine-167 and threonine-311 within the AF1 binding domain as
well as in other domains.
Phosphorylation and activation of ER at key positions can result from a
number of pathways including: the MAPK/ERK pathway in response to
growth factors such as epidermal growth factor (EGF), the PI3K-AKT
pathway in response to insulin-like growth factors and the p38-MAPK
pathway in response to stress or various cytokines .
It has been suggested that aberrations in the posttranslational modification of
ER could be linked to endocrine therapy resistance

1 C. Differential ER Binding
A recent study looked at genome-wide ER binding events in primary breast tumours
of patients sensitive and resistant to tamoxifen and revealed that in tamoxifenresistant cancers ER is still recruited to the chromatin and binds regulatory regions in
a pattern that is unique to resistant tumours
The resistant phenotype may be due to selection and expansion of a resistant
subpopulation of cells, or alternatively could involve the rapid reprogramming of ER
binding by FOXA1, which has a known role in ER-chromatin interactions in response
to growth stimuli
Forkhead motifs and EREs were found to be enriched within the DNA regions which
showed increased ER binding in tamoxifen-resistant cell lines and in primary tumour
specimens of patients with a poor clinical outcome, providing further evidence for the
FOXA1- mediated reprogramming model of ER binding .
These findings suggest that ER may have an important role to play in tamoxifen
resistance by binding to a distinct set of regulatory elements giving rise to a unique
gene expression profile which promotes tumour progression and confers resistance
to therapy.

1 D. Activating Mutations in ER
A recent clinical sequencing study in patients with advanced ER-positive breast
cancer identified a D538G mutation within ER in endocrine therapy resistant patients
causing a change from aspartic acid to glycine at position 538 within the ligand
binding domain.
Importantly, the mutation was found in distant metastatic sites but not in the primary
tumour. The D538G mutant ER was found to confer constitutive ligand-independent
transcriptional activity which mimicked that of estrogen bound wild-type ER with
reduced tamoxifen binding affinity.
Overexpression of mutant ER was found to enhance proliferation
and confer resistance to tamoxifen

Similar studies have also identified additional ER mutations in the ligandbinding

domain which also result in constitutive activity and may represent potential
mechanisms for acquired endocrine therapy resistance

1 E. ER-Independent Signalling
It should be noted that the estrogen receptor exists as two distinct isoforms: ER
(ER) and ER. The exact role of ER is not clear, however studies have shown
that tamoxifen can bind ER and that tamoxifen-bound ER can activate AP1
regulated genes, possibly by altering the balance of associated coactivators and
corepressors at the promoter site.
Increased ER expression has been reported in tamoxifen resistant breast cancers
and data from a recent study suggested that the ratio of ER to ER may be
important in predicting response to tamoxifen and anastrozole in the neoadjuvant
setting
Alternative mechanisms involve expression of truncated isoforms of ER such as ER
36 or other estrogen-related receptors such as estrogen-related receptor
gamma (ERR), associated with reduced response to tamoxifen
Resistance related to ERR overexpression might suggest an important role for
this molecule in an alternative estrogen signalling pathway

2. Progesterone receptors
Estrogen signalling via the ER has been shown to upregulate
the expression of the PR and thus the majority of ER +
patients are also PR +.
Tumours which are ER + and PR - display a poorer response
to endocrine therapy and a more aggressive phenotype than
ER +/ PR + tumours,

Some reports have shown that the ER +/PR + population has

a significantly better prognosis compared with the ER + / PR -

3. EPIGENETICS AND ENDOCRINE RESISTANCE

Epigenetics is defined as reversible changes in gene expression
without change in the DNA sequence.
There is increasing evidence that epigenetic modification plays a
potential role in the development of endocrine resistance in breast
cancer.
The epigenetic regulation of ER is mediated though the recruitment
of multimolecular complexes containing HDAC1, DNMT1 and other
co-repressors to the promoter region. Methylation of the gene
encoding ER- is one of the mechanisms of loss of ER expression
in ER negative breast cancer cell.
The epigenetic silencing of ER target genes is crucial to the
development of ER independent growth and endocrine treatment
resistance.

4. Crosstalk with Growth Factor Signalling Pathways

Receptor tyrosine kinases (RTKs) HER2

Receptors for epidermal growth factor (EGFR)
Mitogen actifated protein kinase (MAPK)

Insulin-like growth factor 1 (IGF1)

PI3K-AKT-mTOR Pathway

Figure 2. Pathways involved in endocrine resistance. (a) While tamoxifen (T), aromatase
inhibitors (AIs), or fulvestrant (F) inhibit estrogen (E) signalization, GFR pathways promote ER phosphorylation,
transcription factors (TFs), and their coactivators (CoA) in a ligand-independent manner. E-ER complex outside
the nucleus can interact with GFRs, Src, CoA and matrix metalloproteinases that release heparin-binding-EGF; (b)
Stress may trigger signalization leading to ER and its coregulators phosphorylation; (c) Notch regulates the
migration and invasion of breast cancer cells. E inhibits this pathway while T activates it; (d) High levels of CoA,
low levels of corepressors (CoR) and altered expression of miRNAs (e) have been implicated in endocrine
resistance development.

Berrera et al, Int. J. Mol. Sci. 2013

Historic Timeline of Therapies for HR+

Advanced Breast Cancer
Others1
Anthracyclines1 Taxanes1
Chemotherapy

Endocrine
Therapy

1896

Doxorubicin
Epirubicin

Paclitaxel
Docetaxel

1980s

1990s

1977

Oopho- SERMS4
rectomy2,3 Tamoxifen

Toremifene

Capecitabine
Gemcitabine
Ixabepilone
Eribulin
Nabpaclitaxel

2000s

2002

2010

AIs4

ERDs5

ERDs5

Anastrozole
Letrozole
Exemestane

Fulvestrant High-dose
Fulvestrant*

1990s

2012
Targeting
mechanisms
of endocrine
resistance

Abbreviations: AI, aromatase inhibitor; ERDs, estrogen receptor downregulator; HR+; hormone receptor positive; SERMS, selective estrogen receptor
modulators.
* Marginal improvement over lower dose fulvestrant.
27
1. http://www.advancedbreastcancercommunity.org/treatment/drugs.htm; 2. Beatson CT. Lancet. 1896;2:104-107; 3. Beatson CT. Lancet. 1896;2:162-165;
4. Cohen MH, et al. Oncologist. 2001;6:4-11; 5. Faslodex [package insert]. Wilmington, DE: AstraZeneca Pharmaceuticals LP; 2011.

Figure 3: Summary of resistance in breast cancer showing the clinical manifestations of

resistance in the neoadjuvant and adjuvant settings, the clinical need to accurately identify high
risk patients, an overview of some of the best described resistance mechanisms and potential
treatments and therapeutic strategies currently under investigation to combat resistance.

Dixon JM, New Journal of Science, 2014

Endocrine Therapy Everolimus: Positive Results

Dual Inhibition of E2 and MTOR

Reprinted from Prat A and Baselga J. Nat Clin Pract Oncol.

2008;5(9):531-542.

BOLERO-2: AI +/ mTOR Inhibitor

(Progression-free Survival)

Reprinted from Baselga J, et al. N Engl J Med. 2012;366(2):109-119.

Copyright 2011 Massachusetts Medical Society. All rights reserved.
Update presented by Hortobagyi GN. SABCS 2011. Abstract S3-7.

29

TAMRAD Time to Progression and Overall Survival

Time to
Progression

Overall Survival
(Secondary Endpoint)

(Secondary Endpoint)

Probability of Survival

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

1.0

Probability of Survival

HR = 0.53 (95% CI = 0.35,

0.81)
Exploratory log-rank P =
.0026
TAM + Everolimus: 8.6 months
TAM: 4.5 months

1.0

0.9
0.8
0.7

HR = 0.32 (95% CI = 0.15,

0.68)
Exploratory log-rank P =
.0019

0.6
0.5
0.4
0.3

TAM + Everolimus: not reached

TAM: 32.9 mo

0.2
0.1

2 4

8 10 12 14 16 18 20 22 24 26 28

Time, mo

Abbreviations: CI , confidence interval; HR, hazard ratio; TAM, tamoxifen.

Adapted from Bachelot T, et al. SABCS 2010. Abstract S1-6.

0.0

12 15

18 21

Time, mo

24 27

30

Conclusions

Breast cancer is a heterogeneous disease

Gene profiling and sequencing would help to
assess better the prognosis and to discover targets
(evolving field)
Multiple mechanisms within the ER pathway
allow development of resistance to endocrine thy .
Endocrine agents (Tamoxifen, AI) + Everolimus
improve the outcome of HR+ patients

31

THANK

YOU