This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.clon.2015.10.00

Baird, RD

Carroll, JS

Apollo (Cambridge)

Clinical	Oncology	-	Science	in	focus	-	Editorial	TITLE:	Understanding	oestrogen	receptor	function	in	breast	cancer,	and	its	interaction	with	the	progesterone	receptor.	New	preclinical	findings	and	their	clinical	implications.		AUTHORS:	Richard	D	Baird1,	Jason	S	Carroll2		1	Department	of	Oncology,	University	of	Cambridge	2	Cancer	Research	UK	Cambridge	Institute		Correspondence	to:	richard.baird@medschl.cam.ac.uk	or	jason.carroll@cruk.cam.ac.uk	.		Background	Oestrogen	 antagonists	 have	 been	 used	 for	 decades	 for	 the	 treatment	 of	 patients	 with	oestrogen	receptor-	(ER-)	positive	breast	cancers,	to	the	benefit	of	many	millions	of	women	worldwide.	 	However,	the	clinical	outcomes	of	these	women	vary	considerably,	something	that	 has	 been	 an	 important	 focus	 for	 research,	 but	 an	 unresolved	 issue.	 	 One	 key	observation	 has	 been	 that	 patients	 with	 ER-positive	 and	 progesterone	 receptor-	 (PR-)	positive	breast	cancers	 tend	 to	have	better	clinical	outcomes	 than	 those	with	ER-positive,	PR-negative	tumours.			PR	is	an	oestrogen	regulated	target	gene	and	for	many	years,	the	accepted	explanation	was	that	PR-positivity	was	a	passive	marker	of	a	functional	oestrogen	receptor	(Figure	1	A).	ER-positive,	 PR-positive	 tumours	 were	 more	 likely	 to	 be	 sensitive	 to	 treatment	 with	 ER	antagonists,	 and	 therefore	 have	 a	 better	 outcome.	 Conversely,	 ER-positive,	 PR-negative	tumours	were	 thought	 to	 have	 a	 "non-functional"	 oestrogen	 receptor,	 and	 therefore	 are	less	likely	to	respond	to	anti-oestrogens.		However,	 recently	 published	 preclinical	 data1	 suggest	 an	 alternative	 explanation,	 namely	that	progesterone	receptor	activity	can	change	where	ER	binds	to	DNA,	directly	modulating	ER	 function	 and	 thereby	 potentially	 improving	 the	 tumour	 response	 to	 ER	 antagonists	(Figure	 1	B).	 This	 is	 important,	 since	 such	 combination	 endocrine	 therapies	might	 lead	 to	improved	clinical	outcomes.	This	new	preclinical	data	sheds	new	light	on	where	ER	makes	contact	 with	 DNA	 and	 the	 importance	 of	 parallel	 pathways	 that	 can	 influence	 the	 DNA	binding	profile	and	ultimately,	activity	of	the	ER	complex	in	breast	cancer.		Where	does	ER	make	contact	with	DNA?	It	might	be	thought	that	after	stimulation	by	oestrogen,	ER	binds	to	DNA	and	regulates	the	transcription	 of	 genes	 encoded	 immediately	 adjacent	 to	 the	 ER	 binding	 site.	 In	 fact,	transcription	 factor	 mapping	 techniques	 (eg.	 Chromatin	 Immunoprecipitation-sequencing	(ChIP-seq))	have	shown	that	in	most	cases,	ER	regulates	its	target	genes	from	considerable	distances.	 ER	 and	 its	 associated	proteins	 (termed	 co-factors)	 bind	 to	 ER	binding	 sites	 and	subsequently,	DNA	loops	form	to	bring	this	ER	complex	adjacent	to	its	distant	target	genes.	Interestingly,	 when	 ER+	 PR+	 (good	 outcome)	 tumours	 are	 compared	 with	 ER+	 PR-	 (poor	outcome)	tumours,	the	ER	DNA	binding	sites	are	distinct,	with	different	genes	being	switch	on	and	off	as	a	result2.				How	does	ER	make	contact	with	DNA?	The	role	of	ER-interacting	proteins.	ER	 function	 is	 heavily	 influenced	 by	 ER-binding	 proteins,	 which	 can	make	 the	 DNA	more	accessible	 for	 the	 ER	 complex,	 or	 assist	 in	 stabilisation	 of	 the	 ER-DNA	 interaction.	 A	comprehensive	 list	of	these	proteins	can	be	obtained	using	unbiased	techniques	 like	RIME	(Rapid	 Immunoprecipitation	 Mass	 Spectrometry	 of	 Endogenous	 protein),	 in	 which	 the	protein	of	interest	is	pulled	out	of	cell	lysates	with	an	antibody,	and	mass	spectrometry	used	to	discover	which	other	proteins	it	physically	associates	with.	Using	this	technique,	over	100	ER-binding	 proteins	 have	 been	 identified,	 including	 FoxA1	 and	 GATA3,	 as	 would	 be	expected3.	Out	 of	 these,	 the	 pioneer	 factor	 FoxA1	 has	 been	 found	 to	 be	 essential	 for	 ER	function,	 by	 stabilising	 ER-DNA	 interactions.	 In	 the	 absence	 of	 FoxA1,	 ER-positive	 breast	cancer	 cells	 stop	 growing4,	 revealing	 a	 dependence	 on	 FoxA1	 for	 ER	 functioning.	 These	findings	make	FoxA1	itself	a	major	focus	of	research,	including	understanding	the	structural	interactions	between	ER	and	FoxA1,	the	role	of	potential	chemical	FoxA1	post-translational	modifications	 and	 the	 discovery	 of	 chemical	 inhibitors	 that	 block	 FoxA1	 function.	Interestingly,	 in	 addition	 to	 the	 known	 ER	 interacting	 protein,	 PR	 was	 purified	 as	 an	 ER	associated	protein,	suggesting	a	putative	functional	role	for	PR	in	the	ER	complex.		What	happens	when	a	progesterone	agonist	is	added?	When	breast	cancer	cells	are	treated	 in	vitro	with	a	progesterone	agonist,	 the	same	RIME	technique	can	be	used	to	examine	changes	in	protein	binding	under	these	new	conditions.	Firstly	focusing	on	changes	 in	PR	 interaction,	there	 is	an	 increase	 in	a	number	of	proteins,	including	ER	and	the	ER-binding	proteins	FoxA1	and	GATA3.	However,	when	the	focus	is	on	ER	interactors,	the	only	progesterone-induced	change	observed	is	an	increase	in	PR	itself1.	Furthermore,	the	addition	of	progesterone	causes	a	rapid	redistribution	of	ER	binding	sites,	with	ER	binding	reprogrammed	to	thousands	of	new	DNA	sites	within	3	hours	of	treatment.	This	 new	 list	 of	 binding	 sites	 is	 highly	 reproducible,	 and	 the	 same	 effect	 is	 seen	whether	endogenous	or	synthetic	progestogens	are	used.	The	progesterone	induced	ER-DNA	binding	sites	are	mediated	by	PR,	such	that	ER	becomes	sequestered	by	PR	to	new	locations	in	the	genome.	The	net	result	of	 this	 is	 inhibition	of	ER	gene	expression	activity	and	consequent	decreases	 in	 cellular	 growth.	 In	 support	 of	 this	 observation,	 primary	 tumour	 samples	cultivated	 ex	 vivo	 for	 up	 to	 two	 weeks	 showed	 a	 reduction	 in	 tumour	 cell	 growth	 in	response	 to	 progesterone	 treatment	 and	 a	 reversal	 of	 the	 oestrogen-induced	 growth.	 In	vivo	 experiments	 have	 also	 now	 shown	 that	 the	 addition	 of	 progesterone	 to	 tamoxifen	therapy	of	breast	cancer	cell	line	xenografts	both	inhibits	proliferation	as	measured	by	Ki67,	but	also	prevents	tumour	growth	more	effectively	than	tamoxifen	alone1.			An	alternative	explanation	to	the	"non-functional"	ER	theory	For	 many	 years,	 the	 "non-functional"	 ER	 theory	 has	 been	 used	 to	 explain	 why	 ER+	 PR-	breast	cancers	have	a	worse	clinical	outcome,	however	there	are	now	multiple	reasons	to	think	that	this	theory	might	be	too	simplistic.	Firstly,	in	patients	with	metastatic	ER+	breast	cancer,	 resistance	 to	 one	 endocrine	 therapy	 does	 not	 necessarily	 mean	 resistance	 to	another	endocrine	therapy;	 indeed,	 it	 is	standard	of	care	for	patients	to	be	managed	with	sequential	endocrine	therapy	in	the	absence	of	rapidly	progressive	visceral	disease.	Second,	we	know	from	preclinical	oestrogen-responsive	element	(ERE)	reporter	experiments	that	in	endocrine	therapy-resistant	tumours,	there	is	still	evidence	of	ER-associated	transcriptional	activity.	 Furthermore,	we	 know	 from	 such	models	 that	 continued	 tumour	 proliferation	 is	dependent	 on	 the	 ER-binding	 protein	 FoxA1,	 implying	 maintenance	 of	 a	 functional	 ER	complex.	In	support	of	this,	recent	discoveries	have	revealed	that	ER	is	frequently	mutated	in	metastases	that	arise	from	ER+	breast	cancers5–7	and	the	mutations	occur	in	a	predictably	part	of	ER	that	renders	ER	independent	of	oestrogen.	These	new	findings	support	a	role	for	a	functional,	albeit	constitutive,	ER	complex	in	endocrine	resistant	patients.	In	fact	the	non-functional	ER	theory	has	been	challenged	before8,9,	but	perhaps	it	 is	only	now	that	we	are	able	to	propose	a	plausible	alternative;	namely	that	progesterone	receptor	expression	is	not	just	 a	 passive	 consequence	 of	 an	 active	 oestrogen	 receptor;	 but	 that	 PR	 can	 actively	reprogram	ER	binding	to	alternative	sites	and	PR	negativity	might	actually	contribute	to	an	altered,	 but	 still	 functional,	 ER	 complex.	 The	 loss	 of	 PR	 can	 be	 the	 cause,	 not	 the	consequence	of	altered	ER	activity	and	tumour	progression.	An	alternative	explanation	for	low	PR	 levels	has	been	 identified,	namely	a	 frequent	deletion	 in	the	genomic	regions	that	encodes	 the	 PR	 gene	 (PGR),	 essentially	 removing	 the	 PR	 ‘molecular	 handbrake’	 that	 can	sequester	and	inactivate	ER.	This	raises	the	possibility	that	we	can	activate	this	 ‘molecular	handbrake’	by	modulating	PR	with	existing	compounds	with	potential	clinical	benefit.		Potential	clinical	significance	The	 potential	 clinical	 significance	 of	 these	 findings	 is	 clear.	 First,	 the	 addition	 of	 a	progesterone	 agonist	 might	 enhance	 the	 anti-proliferative	 activity	 of	 anti-oestrogen	therapies,	and	therefore	prove	a	more	effective	combination	therapy.	Clinical	data	already	exist	 which	 suggest	 that	 the	 PR	 agonist	 megestrol	 acetate	 ("Megace")	 may	 help	 control	tumour	 growth	 in	 patients	 with	 ER-positive	 metastatic	 breast	 cancer	 after	 aromatase	inhibitor	 treatment	 failure.	 In	 a	 single-arm	 phase	 II	 study,	 48	 postmenopausal	 "hormone	sensitive"	patients	were	 treated	with	megestrol	 acetate	 at	 the	dose	of	 160	mg	daily.	 The	treatment	was	reasonably	well	tolerated,	and	yielded	a	clinical	benefit	rate	of	40%,	with	a	median	duration	for	this	benefit	of	10	months10.			It	should	also	be	noted	that	there	is	a	body	of	evidence	supporting	the	use	of	low-dose	PR	agonists	 as	 supportive	 therapies	 to	 help	 ameliorate	 the	 hot	 flashes	 associated	with	 anti-oestrogen	therapy11.	A	second	important	motive	for	such	combination	therapy	is	therefore	to	improve	the	quality	of	life	for	women	taking	anti-oestrogens.	This	is	even	more	important	at	a	 time	when	 the	 intensity12	 and	 /	or	duration13	of	adjuvant	endocrine	 therapy	 is	being	increased	for	many	patients.		As	 the	 preclinical	 findings	 described	 here	 are	 translated	 into	 the	 clinic,	 a	 number	 of	 key	questions	 remain.	 Firstly,	 can	 we	 confirm	 in	 the	 clinic	 that	 the	 addition	 of	 a	 PR	 agonist	enhances	 the	 anti-proliferative	 activity	 of	 anti-oestrogen	 therapy?	 Second,	 what	 is	 the	lowest	dose	of	progesterone	therapy	which	can	achieve	a	significant	biological	effect	with	an	acceptable	side	effect	profile?	Third,	and	most	 importantly,	can	the	combination	of	PR	agonist	with	 ER	 antagonist	 improve	 clinical	 outcomes	 for	 both	metastatic	 and	early	 stage	breast	 cancer	 patients.	 Clinical	 trials	 are	 currently	 being	 set	 up	 to	 test	 these	 and	 other	questions,	in	an	attempt	to	improve	the	outcome	for	millions	of	patients	who	are	diagnosed	with	ER-positive	breast	cancer	every	year.		References		1.	 Mohammed,	H.	et	al.	Progesterone	receptor	modulates	ERα	action	in	breast	cancer.	Nature	523,	313–317	(2015).		2.	 Ross-Innes,	C.	S.	et	al.	Differential	oestrogen	receptor	binding	is	associated	with	clinical	outcome	in	breast	cancer.	Nature	(2012).	doi:10.1038/nature10730		3.	 Mohammed,	H.	et	al.	Endogenous	purification	reveals	GREB1	as	a	key	estrogen	receptor	regulatory	factor.	Cell	Rep.	3,	342–9	(2013).		4.	 Hurtado,	A.,	Holmes,	K.	A.,	Ross-Innes,	C.	S.,	Schmidt,	D.	&	Carroll,	J.	S.	FOXA1	is	a	key	determinant	of	estrogen	receptor	function	and	endocrine	response.	Nat.	Genet.	43,	27–33	(2011).		5.	 Toy,	W.	et	al.	ESR1	ligand-binding	domain	mutations	in	hormone-resistant	breast	cancer.	Nat.	Genet.	45,	1439–1445	(2013).		6.	 Robinson,	D.	R.	et	al.	Activating	ESR1	mutations	in	hormone-resistant	metastatic	breast	cancer.	Nat.	Genet.	45,	1446–1451	(2013).		7.	 Merenbakh-Lamin,	K.	et	al.	D538G	Mutation	in	Estrogen	Receptor- :	A	Novel	Mechanism	for	Acquired	Endocrine	Resistance	in	Breast	Cancer.	Cancer	Res.	73,	6856–6864	(2013).		8.	 Arpino,	G.	et	al.	Estrogen	receptor-positive,	progesterone	receptor-negative	breast	cancer:	association	with	growth	factor	receptor	expression	and	tamoxifen	resistance.	J.	Natl.	Cancer	Inst.	97,	1254–61	(2005).		9.	 Cui,	X.,	Schiff,	R.,	Arpino,	G.,	Osborne,	C.	K.	&	Lee,	A.	V.	Biology	of	progesterone	receptor	loss	in	breast	cancer	and	its	implications	for	endocrine	therapy.	J.	Clin.	Oncol.	23,	7721–7735	(2005).		10.	 Bines,	J.	et	al.	Activity	of	megestrol	acetate	in	postmenopausal	women	with	advanced	breast	cancer	after	nonsteroidal	aromatase	inhibitor	failure:	a	phase	II	trial.	Ann.	Oncol.	25,	831–6	(2014).		11.	 Loprinzi,	C.	L.	et	al.	Megestrol	acetate	for	the	prevention	of	hot	flashes.	N.	Engl.	J.	Med.	331,	347–52	(1994).		12.	 Francis,	P.	A.	et	al.	Adjuvant	Ovarian	Suppression	in	Premenopausal	Breast	Cancer.	N.	Engl.	J.	Med.	372,	436–446	(2015).		13.	 Davies,	C.	et	al.	Long-term	effects	of	continuing	adjuvant	tamoxifen	to	10	years	versus	stopping	at	5	years	after	diagnosis	of	oestrogen	receptor-positive	breast	cancer:	ATLAS,	a	randomised	trial.	Lancet	6736,	1–12	(2012).			 		Figure	1.	Our	changing	understanding	of	the	interaction	between	oestrogen	receptor	(ER)	and	progesterone	receptor	(PR)	function.				

English

Understanding Oestrogen Receptor Function in Breast Cancer and its Interaction with the Progesterone Receptor. New Preclinical Findings and their Clinical Implications.

Understanding Oestrogen Receptor Function in Breast Cancer and its Interaction with the Progesterone Receptor. New Preclinical Findings and their Clinical Implications.

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