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Andrea R. Genazzani , Mathias Sanchez, Marina Ines Flamini and Tommaso Simoncini
Division of Obstetrics and Gynecology Department of Clinical and Experimental Medicine -University of Pisa
Institute of Medicine and Experimental Biology of Cuyo (IMBECU), CCT-CONICET Mendoza, National University of Cuyo, Parque General San Martin s/n, Mendoza, CP:5500, Argentina
Breast cancer: Non gonadal effects of Gonadotropins
Prof. Andrea R. Genazzani , MD, PhD, HcD, FRCOG, FACOG
President of the International Society of Gynecological Endocrinology (ISGE)President of the European Society of Gynecology (ESG)General Secretary of the International Academy of Human Reproduction (IAHR)
University of Pisa, Italy
Ihave no financial relationships to disclose.
MENOPAUSE:LowE2 andHighFSH
0
100
200
300
400
500
600
700
800
900
1000
- 8 - 6 - 4 - 2 0 2 4 6 8 10
25
22.5
20
17.5
15
12.5
10
7.5
5
2.5
0
g/L
pMYears
FSH
LH
E2
Choietal.2007
LH/FSHINEPITHELIALOVARIANCELLSANDCANCER
EXTRA‐GONADALACTIONSOFGONADOTROPHINS
GnRH
LHFSH
EstrogensProgesteroneTestosterone
+
+
-
“ FSHR is expressed by the microvasculature of metastatic tumors.This fact strongly increases FSHR potential relevance as a clinicaltarget for cancer imaging and for therapy, especially for tumorsthat are highly resistant to currently available antiangiogenictreatments. “
FSH INCREASES THE RISK OF POSTMENOPAUSALOSTEOPOROSIS BY STIMULATING OSTEOCLASTDIFFERENTIATION
ModifiedbyE.D.Crawfordetal./UrologicOncology:SeminarsandOriginalInvestigations35(2017)183–191
SerumFSHwassignificantlyincreasedinwomenwithpostmenopausalosteoporosis
comparedwithwomenofthesameagewithnoosteoporosis
Wang etal.(1)demonstratedthatFSHincreasesthemRNAexpressionlevelsofRank,
Mmp‐9,TrapandCathepsin Kinosteoclasts.FSHmaydirectlyaffectthedifferentiationand
maturityofosteoclasts andmaypromoteboneabsorption
FOLLICLE‐STIMULATIONHORMONERECEPTORINHUMANUMBILICALVEINENDOTHELIALCELLS
Joanna Stelmaszewska et al.,Scientific Reports .2016,6:37095
Withregardtotumorvesselcells(primaryandmetastatic),FSHRmightserveasa
potentialcellularmarkerofdifferenttumors.
FSHRexpressionwasdemonstratedinhumanumbilicalveinendothelialcells
(HUVEC).
HUVECsrespondedtoFSHtreatmentinaseriesoffunctionaltestsliketube
formation,woundhealing,cellmigrationandproliferation,nitricoxideproductionand
cell survival. Morestudiesaredefinitelyneededtovalidatetherecentlydiscoveredextragonadal FSHRexpressionandfunction,especiallyintheendothelialcellsofdifferentvascularvesseltypesinnormalandtumortissues.
KeyandPike,Eur JCancerClin Oncol 1988
30–34 35–39 40–44 45–49 50–54 55–59 60–64 65–69 70–74
Menopause
Incidencerateper100,000women
Age(years)
10
20
50
100
200
MENOPAUSEANDBREASTCANCER
CollaborativeGrouponHormonalFactorsinBreastCancer,Lancet1997
0
10
20
30
40
50
60
70
80
90
50 55 60 65 70 75
Age (years)
Per 1000 women
77798389
Use for 15 yearsUse for 10 yearsUse for 5 yearsNever-use
ESTIMATEDCUMULATIVEINCIDENCEOFBREASTCANCER
BREASTCANCERANDHRT‐ CGHFBC
Chen,W.Y.Arch Intern Med (2006)
NHS:BREASTCANCER
0 0,5 1 1,5 2
Relative risk
0.96 (95% CI, 0.75-1.22)≤ 5 YEARS
0.90 (95% CI, 0.73-1.12)5 -10 YEARS
1.06 (95% CI, 0.87-1.30)10 - 15 YEARS
1.18 (95% CI, 0.95-1.48)15 - 20 YEARS
1.42 (95% CI, 1.13-1.77)≥ 20 YEARS
(P for trend <.001)
ESTROGENALONE
Stefanick, M. L. et al. JAMA 2006
0
0.01
0.02
0.03
0.04
0 1 2 3 4 5 6 7 8 9
HR 0.67(95% CI 0.47 – 0.97)
PLACEBOCEE ALONE (ADHERENT PATIENTS)
TIME (YEARS)
WHI: BREAST CANCER
RossiMet al,Eur JPharmacol.2009
INTERPLAYBETWEENESTROGENANDLHRECEPTOR
BREASTCANCER
ERα
E2
FSHR
FSH
LHR
LH
MOESINMOESIN
P
ACTIN
CELLMEMBRANE
ACTINFIBRES
MOESIN
ACTIN‐BINDINGPROTEINBELONGINGTOTHEEZRIN/RADIXIN/MOESIN(ERM)FAMILY.
ACTIVATEDTHROUGHPHOSPHORYLATION.
WHENACTIVEBINDSACTINANDINDUCESITSREMODELINGTOWARDTHECELLMEMBRANE,WHEREBRIDGESWITHANCHORAGEPROTEINS,SUCHASINTEGRINS,AREFORMED.
ACTINREMODELINGANDCELLMOVEMENT
17β‐ESTRADIOL0’ 5’ 10’ 15’ 20’ 30’ 45’ 60’
MEANMEMBRANETHICKNESS(pixel ± SD)
MEANMEMBRANEINTENSITY(mean graylevel ± SD)
MEANCYTOSOLINTENSITY(mean graylevel ± SD)
MEMBRANE/CYTOSOLINTENSITYRATIO
0’ 6,3 ± 2,1 62,3 ± 5,6 62,0 ± 7,8 1
5’ 7,0 ± 2,6 75,4 ± 7,8 71,9 ± 7,8 1,1
10’ 36,7 ± 14.5 * 120,5 ± 9,4 * 66,4 ± 2,8 1,8 *
15’ 48,3 ± 4.7 * 114,7 ± 11,8 * 45,9 ± 9,0 2,5 *
20’ 49,3 ± 4.7 * 112,4 ± 13,5 * 66,7 ± 2 1,7 *
30’ 22,3 ± 4,2 96,4 ± 11,4 * 72,3 ± 4,9 1,3
45’ 7,6 ± 2,8 71,1 ± 14,2 61,9 ± 2,5 1,1
60’ 5,0 ± 1 61,5 ± 1,9 62,4 ± 2,2 0,99
Mem
bran
e Th
ickn
ess
(Pix
els,
mea
n±
SD)
17β-ESTRADIOL
ESTROGENANDBCACTINREMODELING
M.S.GirettiPLoS One 2008
P‐MOESIN
MOESIN
20X
DETAIL
20X
DETAIL
NORMALBREAST FAD BREASTCANCER
MEMBRANE MEMBRANE FOCAL MEMBRANEMEMBRANE‐
CYTOPLASMATICCYTOPLASMIC
M.S.GirettiPLoS One 2008
CON(1)
E2+TAM(1.2)E2+Y(0.3)
E2+PTX (0.26)E2(2.2)
TAM(0.8)
E2‐BSA (2.4) E2+ICI(0.71)
AS(0.4) S(2.53)
E2
E2ANDER+BCCELLINVASION
M.S.GirettiPLoS One 2008
FAK
Src
P
ACTIN
CELL MEMBRANE
ACTIN FIBRES
FOCALADHESIONCOMPLEXES
POINTSOFINTERACTIONBETWEENTHECELLANDTHEECM.
DURINGMIGRATION,ADHESIONSASSEMBLEATTHE
LEADINGEDGEANDDISASSEMBLEATTHETRAILINGEDGE.
CONTROLLEDBYADHESION‐RELATEDMULTIPROTEIN
COMPLEXESSUCHASFAKANDSRC
VINCULIN
FOCALADHESIONANDCELLMOVEMENT
E2ACTIVATESFAK
A.M.Sanchezet al,MolecularEndocrinology,2010
CCON 2’ 5’ 10’ 30’15’ 60’
17β-ESTRADIOL (10 nM)
pFA
K39
7M
ERG
EA
CTI
N
20’
DCON 2’ 5’ 10’ 30’15’ 60’20’
17β-ESTRADIOL (10 nM)
VIN
CU
LIN
/DA
PI
CON E2 ICI PTX PP2
WM PD siRNA FAK siRNA cdc42 siRNA WASP
E2 +
E2 +
ERSIGNALINGTOFAK,CDC42 ANDWASP:BCINVASION
BREASTCANCER
ERα
E2
FSHR
FSH
LHR
LH
5 mUI/mL
5 mUI/mL
50 mUI/mL
50 mUI/mL
FSH/LHANDBREASTCANCER
FSH/LH
FSH/LH
FSH/LH
40 h 8 h
A.M.Sanchezet al./Molecular andCellular Endocrinology 437(2016)22e34
p‐MOESIN
FAK
FSHR
MOESIN
p‐FAK397
LHR
CON 5 5+50 50
LH (UI/ml) FSH (UI/ml)
505+505CON
LH+FSH (UI/ml)
505+505CON
T47‐DER (+)
FSH/LHANDBREASTCANCER
A.M.Sanchezet al./Molecular andCellular Endocrinology 437(2016)22e34
Vinculin
FAK
FOCAL ADHESION ASSEMBLY
Gαi Gβ
PI3K c‐Src
LH/FSH
LHR/FSHR
Gα13Gβ
ROCK2
Moesin
ACTIN CYTOSKELETON REMODELING
Signalingcascadesofgonadotrophins tomoesin andFAKinbreast cancer cells.
CELL MOTILITY AND INVASION
FSH/LH AND BREAST CANCER
A.M.Sanchezet al./Molecular andCellular Endocrinology 437(2016)22e34
LH 50LH 5+50CON LH5
LH 50LH 5+50CON LH5
shRNA LHR +
C 68,8±4,92 134,3±3,33 100,3±6,96 88,6±3,21
68,5±2,65 65,5±6,50 67,5±6,66 66,67±6,81
FSH 50FSH 5+50CON FSH5
FSH 50FSH 5+50CON FSH5
shRNA FSHR +
G76,6±4,219 100±5,831 120,6±7,345 97,7±7,76
79±7,937 79,8±3,347 74±2,01 74,5±1,291
F
= NS vs. CON = P≤ 0,05 vs CON
I
= P≤ 0,05 vs. CON = P≤ 0,05 vs CON
A.M.Sanchezet al./Molecular andCellular Endocrinology 437(2016)22e34
Planeix etal.JournalofExperimental&ClinicalCancerResearch(2015)34:12
ENDOTHELIALFSHREXPRESSIONININVASIVEBREASTCANCERANDVASCULARREMODELINGATTUMORPERIPHERYThebloodvesselsthatexpressedFSHRwerelocatedinalayerthatextended
2mmintoand5mmoutsideofthetumor.
100%ofbloodvesselsexpressingFSHRatthedemarcationlinebetween
thetumorandthenormaltissue.
EndothelialFSHRplayanimportantroleinvascularremodelingand
generationofshortlow‐resistance,high‐flowpathwaysofbloodattumor
periphery.
FSH/LHANDBREASTCANCER– RATBCPROGRESSION
15 WEEKS 30 DAYS
NMU
30 DAYS 30 DAYS
OVX
BC DEVELOPMENT
E2
GnRH ANALOGUEEND
TUMOR SIZE MONITORED/3 DAYS
EXPERIMENTAL GROUPS:
1) SHAM2) NMU3) NMU + OVX4) NMU + OVX + E25) NMU + OVX + GnRH ANALOGUE6) NMU + OVX + GnRH ANALOGUE + E2
NMU NMU+OVX
NMU+OVX+LH ANALOGUE NMU+OVX+E2 NMU+OVX+LH ANALOGUE+E2
POSITIVE CONTROL (KIDNEY)
LHRANDBREASTCANCER– RATBCPROGRESSION
B NMU NMU+OVX
NMU+OVX+LH ANALOGUE NMU+OVX+E2 NMU+OVX+LH ANALOGUE+E2
POSITIVE CONTROL (OVARY)
FSHR AND BREAST CANCER – RAT BC PROGRESSION
FSH/LH AND BREAST CANCER – RAT BC PROGRESSION
A.M. Sanchez et al, unpublished
WORKINGHYPOTHESIS
IfFSHandLHactonnormalorbreastcancercells,
modulationoftheirreceptorsmaybeclinicallyuseful.
GnRH analogues/antagonists may be useful, since they
decrease FSH and LH independently from estrogen.
FSHRorLHRmodulatorsmaybeengineered.
Todate,GnRH antagonistsareonlyusedinfertilewomento
decreaseestrogens.
CONCLUSIONS
Steroidreceptors(ER,PR)extranuclear signalingdrivesaseriesofcytoskeletal changesinbreastcancercells.
ThesechangesarerelatedtotheabilityofBCcellstomove,migrateandinvadematrices.
LHandFSHseemtoactonsimilarpathways,throughdirect,extragonadal actionsonbreastcancercells.
Identification of the mechanisms of recruitment of thesepathways might help to design new strategies to interferewith BC progression in postmenopausal women.
MATIAS SANCHEZ
T. SIMONCINI
MARINA INES FLAMINI