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