Anti-hormonal agents in breast cancer (DONE)

Question 1

Adenocarcinomas

Answer 1

Most breast cancers are adenocarcinomas (invasive cells derived from breast ducts or lobular glandular tissue)
Recent gene expression studies have found multiple molecular subtypes of breast cancer exist

Question 2

Sporadic breast cancer

Answer 2

Most breast cancers are sporadic (somatic gene mutation), rather than familial (7%)
80% of familial have inherited faulty BRCA genes

Question 3

Operable (early, primary) disease

Answer 3

Within breast and axillary lymph node only
Remove with surgery
Followed by adjuvant systemic therapy to block re-growth of any cancer cells that may have spread from the tumour prior to surgery
Adjuvant systemic therapy aims to cure and prevent relapse
Useful to initially treat with systemic therapy to treat large tumours, allowing more conservative surgery- neoadjuvant treatment

Question 4

Advanced (inoperable, stage 4, metastatic) disease

Answer 4

Breast cancer has already spread out to distant, potentially life threatening metastatic sites
Systemic therapy aims to limit further tumour growth, improve QoL and modestly improve survival for this poorer outlook group
Key systemic agents: anti-hormones that block actions of oestrogen, a female steroid hormone which commonly drives breast cancer

Question 5

Natural oestrogens in women

Answer 5

17B-estradiol (most potent and predominant), also estriol and estrone
Tetracyclic structure based around estrane nucleus: 4 cycloalkane rings- planar 18 carbons, aromatic A ring, methyl group at position 1

Question 6

Functions of oestrogen

Answer 6

Oestrogens regulate normal breast growth, reproductive tract and menstrual cycle, fertility, circulating blood lipids, bone density (beneficial), also impact on brain and cognitive function
Circulating 17B-estradiol levels in women: 400pmols/L premenopausal, 40pmols/L postmenopausal

Question 7

Evidence that oestrogen is important in driving growth of many human breast cancers

Answer 7

13% breast cancer incidence in women vs 1% in men
Age: increased lifetime exposure of breast to oestrogens can increase breast cancer risk
70% of breast cancer occurs in women >50 years
Earlier menstruation, later menopause, increased circulating oestrogen levels after menopause
Protective effect of removing ovaries on breast cancer development, some breast cancers regress after surgical ovariectomy

Question 8

How does oestrogen drive breast tumour growth?

Answer 8

Via the oestrogen receptor (ER), a nuclear protein overexpressed in >70% breast cancers
Oestrogen binds to ER
Complex molecular events increase ER-regulated gene expression to promote proliferation and cell survival
Causes breast tumour growth

Question 9

Classical mechanism of oestrogen/ER signalling involving nuclear ER (i)

Answer 9

Oestrogen diffuses into cell to nucleus, binds to hydrophobic pocket in nuclear ER
Conformational change so helix 12 region of ER protein fully seals pocket containing E ligand
Hsp90 chaperones are lost and two E-bound receptors dimerize
ERdimer binds to a target sequence in DNA promoter of oestrogen sensitive genes to drive their expression: the oestrogen response element (ELE)
Interaction and transcription is helped by a pioneer factor called FOXA1

Question 10

Classical mechanism of oestrogen/ER signalling involving nuclear ER (ii)

Answer 10

On binding of ER dimer to ERE, gene expression regulated by synergistic activity of two transcriptional activation function regions within ER protein: AF2 and AF1
When oestrogen is bound to the receptor (with associated conformational changes), AF2 recruits co-activator proteins to help bridge ER to transcriptional machinery, triggering transcription
AF1 also recruits co-activators, after it is phosphorylated by kinase enzymes
Both AF2 and AF1 activity is required for maximum gene expression, which promotes proliferation, cell survival and growth

Question 11

ER protein has five domains relating to its complex function

Answer 11

A/B domain: amino terminal, contains AF1 for transcriptional activation with several phosphorylation sites
C domain: binds ERE sequences in DNA
D domain: hinge for dimerization, binds Hsp90, contributes to DNA binding, has a nuclear localisation signal
E domain: ligand binding domain, contains hydrophobic pocket that binds oestrogen, also contains AF2 for transcriptional activation
F domain: carboxy terminal, modulates transcriptional activation and dimerisation

Question 12

Anti-hormonal agents have been developed as treatments to interfere with oestrogen/ER signalling

Answer 12

The first targeted cancer treatments:
Anti-hormonal agents block oestrogenic signals in cells
Hinder expression of genes central to proliferation and cell survival
ER+ breast tumour growth inhibited

Question 13

Treatment with an anti-hormone

Answer 13

5 years of an anti-hormone is beneficial in 75% of ER+ early disease. Substantially delays or prevents relapse, and extends survival or cure for many patients. 5 year survival rate is now 80%, due to increased awareness, screening, and improved treatments including anti-hormones
Can be supplemented by chemotherapy regime of ER+ tumour is aggressive
Anti-hormones also beneficial in 60% ER+ advanced disease patients

Question 14

Anti-hormones in tumours lacking ER

Answer 14

No benefit of anti-hormones in tumours lacking ER, so use chemotherapy in ER negative disease e.g. anthracycline or taxane-based treatments

Question 15

Key classes of anti-hormones for ER+ breast cancer

Answer 15

Oestrogen binding to ER in the breast cancer: non-steroidal anti-oestrogens or SERMs e.g. tamoxifen
Oestrogen binding to ER in the breast cancer, with additional catastrophic effects on ER protein level and ER activity: steroidal anti-oestrogens or SERDs e.g. Faslodex
Oestrogen production throughout the body: aromatase inhibitors for postmenopausal women, non-steroidal and steroidal; LH-RH agonist for premenopausal women e.g. Zoladex

Question 16

Inhibitors of oestrogen binding to ER: non-steroidal anti-oestrogens

Answer 16

From 1950s, non-steroidal agents developed. Some areas of their structure resemble 17B oestradiol, so they can compete with oestrogen for binding to ER (anti-oestrogenic) to prevent oestrogen stimulation
Tamoxifen- a trans isomer of substituted triphenylethylene with a bulky side chain, potent metabolites include 4-OH-Tam

Question 17

Tamoxifen

Answer 17

Inhibitory (anti-oestrogenic) on normal breast with low toxicity
Reduces relapse by 50% to improve survival and so has contributed to >20% improval in breast cancer patient survival over last 10 years
Can reduce risk of breast cancer developing by 50%- prevention in high risk women
Administered to ER+ breast cancer patients orally, 20mg per day for 5 years after surgery

Question 18

Tamoxifen binding in other tissues

Answer 18

Breast and CNS: beneficial effects- reduces ER+ breast cancer growth, unwanted effects- menopausal like hot flushes
Bone, CVS, uterus and liver: beneficial effects- anti-osteoporotic, lowers blood lipids to reduce cardiac problems; unwanted effects- stimulates uterus, can induce thromboembolic events/strokes
Tamoxifen is a Selective Estrogen Receptor Moldulator = SERM = a partial antioestrogen because of its tissue-specific activity

Question 19

Mechanism of anti-oestrogenic activity of tamoxifen in some tissues (i)

Answer 19

Tamoxifen has some mimicry with oestrogen, so competes for ER binding in ER pocket. ER binding to ERE sequences can then occur with tamoxifen, all as seen for oestrogen
Bulky basic side chain alters ER conformation, so helix 12 is displaced and cannot properly seal the pocket

Question 20

Mechanism of anti-oestrogenic activity of tamoxifen in some tissues (ii)

Answer 20

This tamoxifen-altered ER conformation does not allow recruitment of co-activators to AF2 (which actually recruits co-repressors)
Result of this AF2 blockade: transcription of proliferation/cell survival genes is decreased so growth is inhibited: antioestrogenic

Question 21

Mechanism of oestrogenic activity of tamoxifen in other tissues

Answer 21

While AF2 is blocked by conformation change, AF1 remains unaffected: can still be phosphorylated by kinases and recruits coactivators
Result of retained AF1 activity: some transcription of proliferation and cell survival genes, so growth is stimulated: oestrogenic
SERM behaviour: depends on tissue context e.g. balance of co-activators, co-repressors and kinase regulators of AF1 phosphorylation

Question 22

Drug features required to improve on the SERM tamoxifen

Answer 22

No anti-oestrogenic hot flushes
Improve anti-oestrogenic effect in breast cancer
No oestrogenic thromboembolic effects
No oestrogenic effect on uterus
Maintain oestrogenic effect on blood lipids
Maintain oestrogenic effect on bone

Question 23

Other novel SERMs (1990s)

Answer 23

Further triphenylethylenes based on tamoxifen (or its potent 4-OH metabolite) developed, all retain anti-oestrogenic side chain
Like tamoxifen, competitively block oestrogen binding to ER
Such SERMs e.g. toremifene are generally no better than tamoxifen

Question 24

Inhibitors of oestrogen/ER binding, with additional catastrophic effects on ER protein

Answer 24

7a derivatives of oestradiol-17B with long, unbranched 7a-alkysulfinyl side chain
1987: Faslodex
100 fold improved ER binding affinity vs. tamoxifen
Potent competitive inhibitor of oestrogen binding to ER; also gives catastrophic decreases in ER level and ER activity
Behaves as pure antioestrogen (no oestrogenic activity; entirely antioestrogenic)

Question 25

Faslodex mechanism of pure antioestrogenicity

Answer 25

Steroidal faslodex competitively inhibits oestrogen binding to ER, but long side chain also very severely disrupts ER conformation
Impairs ER dimerization and decreases nuclear localisation of ER. ER collects in cytoplasm and is degraded: profound ER protein loss
Also prevents helix 12 closure of ER pocket, blocking all co-activator recruitment but favouring co-repressor recruitment, silencing both AF2 and AF1 activity of ER
No ER regulated proliferation/cell survival gene expression is possible

Question 26

Faslodex clinical pharmacology

Answer 26

Superior anti-oestrogenicity in ER+ breast cancer cell in lab vs tamoxifen
While potent anti-oestrogen, fewer hot flushes (does not cross BBB)
No unwanted oestrogenic effects on uterus or thromboembolic events
While equally no beneficial oestrogenicity on blood lipids or bone, no adverse events reported: well tolerated
Since IM delivery, oral pure antioestrogens with enhanced bioavailability are now under evaluation

Question 27

Inhibiting oestrogen production in the body

Answer 27

Aromatases are the enzymes central in producing oestrogens required for breast tumour growth
Premenopausal women: oestrogens made by aromatisation during menstrual cycle by aromatase enzymes in ovarian granulosa cells
Postmenopausal women: no ovarian function, but some oestrogens derived by peripheral aromatization. Adrenal glands make androgens from cholesterol, that are then aromatized to make oestrogens by aromatases predominantly in peripheral fat/muscle
Breast cancers and surrounding adipose fibroblasts also contain aromatases that make oestrogens

Question 28

Aromatization reaction: androgens to oestrogens

Answer 28

Aromatase enzyme binds androgen substrate into its active site
Aromatase catalyses aromatization of A ring in androgen (C19) to oestrogen (C18) = introduction of aromatic ring and loss of C19 methyl e.g. testosterone to estradiol)

Question 29

Aromatase enzyme CYP450 comprises two functional units

Answer 29

CYP450 hemoprotein: haem (Fe) group binds androgen in active site and catalyses its aromatization to oestrogen
Flavoprotein NADPH-cytochrome P450 reductase:L transfers e- from NADPH to cytochrome P450 arom to support this catalysis

Question 30

Aromatase inhibitors can be used to block oestrogen synthesis in postmenopausal ER+ breast cancer patients

Answer 30

Able to block oestrogen production by all aromatases, causing severe oestrogen deprivation, so breast cancer growth is inhibited
Only currently approved in ER+ postmenopausal women
Non-steroidal and steroidal AIs
Third generation AIs are the current gold standard for ER+ postmenopausal breast cancer treatment

Question 31

Steroidal aromatase inhibitors

Answer 31

Early prototype 4-hydroxyandrostenedione (formestane), now more selective, potent and well tolerated AIs are used e.g. exemestane
Androgen like structure so can compete with substrate for binding to aromatase active site
Aromatase then metabolizes bound AI to reactive intermediates, these covalently and irreversibly bind active site, which irreversibly inactivates the enzyme
98% aromatase inhibition and >90% oestrogen deprivation

Question 32

Non-steroidal aromatase inhibitors

Answer 32

Early prototype aminoglutethimide; now more selective, potent and well tolerated non-steroidal triazole AIs are used e.g. letrozole, anastrozole
Compete with substrate for binding to aromatase active site, but binding is reversible
AI then reversibly binds haem group in aromatase enzyme via azole nitrogens (which have free e- pair), blocking enzyme activity
Again, 98% aromatase inhibition and >90% oestrogen deprivation

Question 33

Superior clinical effectiveness of AIs

Answer 33

Superior to tamoxifen in ER+ postmenopausal advance breast cancer, valuable after tamoxifen fails
Superior in ER+ postmenopausal early breast cancer to improve survival in 5 year AI treatment
Able to prevent breast cancer in high risk postmenopausal women
No oestrogenicity- no uterus or thromboembolism problems
AI is now gold standard for ER+ postmenopausal breast cancer