TGF Beta Signaling Pathways and Microrna Function in the Female Reproductive Tract

TGF Beta signaling pathways and microRNA function in the female reproductive tract

Prof. Martin Matzuk Baylor College of Medicine Intellectual and Developmental Disabilities Research Center Houston, TX, USA TGFb signaling pathways and microRNA function in the female reproductive tract Topic 1

TGFb Superfamily

N Pro RRRR Mature C

• Largest family of growth factor ligands in mammals • Function as secreted homodimers or heterodimers in multiple developmental and physiologic processes • We have been studying 12 family members including growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), activins, inhibins, and GDF3 GDF9 functions in folliculogenesis & cumulus expansion Pre-Ovulatory Follicle

Cumulus GC Early Antral Follicle

Ovulating Follicle Mural GC

Fertilization Secondary & Embryo Follicle Development GDF9 KO

Primary Primordial Follicle Follicles Dong et. al. Nature 1996 Elvin et al. Mol Endo, 1999a, b TGFb Superfamily “Canonical” Signaling Pathways – GDF9 and BMP15 appear to signal in different pathways

GDF9/BMP b b Extracellular Cytoplasm Type II Type I (Kinase) (Kinase) “BMP” SMAD1/5/8 SMAD2/3 “Activin/TGFb” Pathway SMAD4 SMAD4 Pathway (BMP15) (GDF9)

Nucleus BMP-Specific Activin-Specific Genes Genes What is the ovarian GDF9 type 1 receptor? ALK1 Activin receptor like-1 ALK2 Activin receptor 1 ALK3 BMP receptor 1A ALK4 Activin receptor 1B ALK5 TGFb receptor 1 ALK6 BMP receptor 1B ALK7 Activin receptor 1C b b Extracellular Cytoplasm Type II Type I (Kinase) (Kinase) “BMP” ALK2, ALK5, “GDF9” Pathway ALK3, ALK6 AMHR2-Cre Pathway

Nucleus Strategy to define the function of ALK5 in the female reproductive tract and determine if it is the type 1 receptor for GDF9

ALK5 floxed allele

Exon3 AMHR2-Cre Recombinase

AMHR2-Cre recombines floxed alleles in granulosa cells

ALK5 null allele Control Mice

Exon3 *Lacz-Neo Exon3 ALK5flox/-

PKKKRKV * ALK5 cKO Mice ALK5flox/- ; AMHR2cre/+ ALK5 cKO mice are sterile

n=10 n=7 n=10 n=7 Expression of ALK5 in the mouse ovary at 8 wks: ALK5-Lacz staining in the granulosa cells is not consistent with ALK5 as a GDF9 receptor

TC CL

GC CL Ovary

PMSG48h200 M hCG7h

ALK5 expression Present in theca and corpora lutea COC Absent in preantral granulosa cells 2F Absent in cumulus granulosa cells BMP15 and GDF9 suppress ALK5 in granulosa cells

1.2 a 1

0.8

0.6 b 0.4 b 0.2

Con

GDF9 BMP15 In vivo cumulus expansion occurs in ALK5 cKO mice

5 x 20 x

Control CC (#7371) Oo

cKO Oo (#7373) CC

TC What is the cause of the sterility in the ALK5 cKO? AMHR2-Cre conditional knockout in somatic cells

Ovarian follicle Müllerian duct (E11.5)

Mesenchyme

Smooth muscle (+ Stroma)

Ovary Oviduct

Uterus Granulosa cells

Cervix AMHR2-Cre Anti-Müllerian hormone receptor type 2 ALK5 cKO mice develop oviductal diverticuli (outpouchings) Control ALK5 cKO

Oviduct Diverticuli

3 wks

Diverticuli

12 wks Oviduct Pol II MiRNA gene Pri-miRNA

Drosha

Pre-miRNA

Pre-miRNA Dicer

Mature 22nt miRNA Cytoplasm

Regulate gene expression (degrade mRNA and block translation)

Dicer cKO  Oviductal diverticuli  Sterile  Diverticuli prevent embryos from reaching the uterus The oviductal diverticuli trap oocytes and embryos

Diverticulum

Zona pellucida remnants Morula stage embryos Dying embryo

Hatched blastocyst ALK5 is expressed in smooth muscle of the oviduct and uterus In the absence of ALK5, smooth muscle is abnormal

Wild-type cKO

EP Oviduct

Uterus SM

(Cross section) Control cKO Uterus

Uterus

Circular Longitudinal (Smooth Muscle) Disruption of smooth muscle layers of the uterus in ALK5 cKO (Anti-smooth muscle actin immunohistochemistry) Control ALK5 cKO 12 wks - #7411 8 wks - #7114 12 wks - #7412

2.5 x 5 x 5 x Uterus (Cross section)

CM CM LM

LM CM Circular Longitudinal (Smooth Muscle) 20 x 20 x 20 x Why do the ALK5 cKO and Dicer cKO mice have similar oviductal phenotypes and sterility?  MicroRNAs including miR-143 and miR-145 have been shown to regulate vascular smooth muscle cell proliferation and differentiation

 TGFb signaling through SMAD2 and SMAD3 also function in the maturation of microRNAs by DROSHA in smooth muscle differentiation

One Possible Model

DROSHA DICER Smooth pri-miRNA pre-miRNA mature miRNA Muscle Differentiation ALK5 TGFb SMAD2/3 TGFBR2 BMP Signaling Pathways

BMPs b b Extracellular Cytoplasm Type II Type I (Kinase) (Kinase) “BMP” SMAD1/5/8 SMAD2/3 “Activin/TGFb” Pathway SMAD4 SMAD4 Pathway (GDF9)

Nucleus BMP-Specific Activin-Specific Genes Genes Analysis of BMP type 1 receptor function ALK1 Activin receptor like-1 ALK2 Activin receptor 1 ALK3 BMP receptor 1A ALK4 Activin receptor 1B ALK5 TGFb receptor 1 ALK6 BMP receptor 1B ALK7 Activin receptor 1C b b Extracellular Cytoplasm Type II Type I (Kinase) (Kinase) “BMP” ALK2, ALK5, “GDF9” Pathway ALK3, ALK6 AMHR2-Cre Pathway

Nucleus Anti-Mullerian Hormone Receptor 2 (AMHR2)-Cre and Progesterone Receptor (PR)-Cre to define BMP type 1 receptor signaling in the uterus

UTERUS PR-Cre Amhr2-Cre

Epithelium and (strong) Stroma Smooth muscle and (weak) Stroma John Lydon Richard Behringer Franco DeMayo ALK3 AMHR2-Cre cKO in the ovary results in subfertility

60 Control 50 Bmpr1a cKO

% Time in Each Stage 0

Estrus Diestrus Proestrus Metestrus ALK3/6 DKO mice

100

% Females %Tumors with 0 8-10 11-13 14-16 Age (Months)  ALK3/6 DKO mice develop ovarian granulosa cell tumors

 Findings phenocopy SMAD1/5 DKO mice The implantation process in the mouse uterus ALK6 null and ALK3 AMHR2-Cre models – Normal decidualization is observed

Control ALK3 cKO ALK6 KO (AMHR2-Cre) PR-Cre models to identify BMP type 1 receptors in decidualization and peri-implantation  PR-Cre deletion of either ALK3 or ALK2 results in sterility ALK3 Decidualization ALK3 (Post-implantation) Control cKO Control cKO Control ALK3 cKO

No Implantation

E7.5 E7.5 Decidualization Control ALK2 cKO ALK2 (Post-implantation) Control ALK2 cKO

Embryo Resorptions

E8.5 E8.5  Both ALK2 and ALK3 are required for normal uterine function Summary of BMP signaling in the uterus (Model based on studies by DeMayo, Tsai, and others)

Peri- Implantation Implantation PR LIF PR IHH

COUP-TFII HOXA10

BMP2 ?

PTGS2 ALK3 PRL ALK2

? IGFBP1 WNT4/6 FKBP3/4/5 ? Fallopian tube Topic 2 (Oviduct)

MicroRNAs in ovarian cancer Uterus Follicles Ovary

Endometrium

Stroma/Muscle

Cervix

Overall Hypothesis MicroRNAs play key roles in ovarian cancer Ovarian Cancer Introduction

 The fifth leading cause of cancer death in women, and the leading cause of death from gynecological malignancy  ~22,430 U.S. women diagnosed yearly with ovarian cancer  5 year survival <50% and ~15,280 women die yearly  The four major histologic types are: serous (70%), endometrioid (10%), clear cell (10%), and mucinous (5%) MicroRNAs function as tumor suppressors and oncogenes

Tumor suppressor Oncogene

oncogene tumor suppressor AAAAA AAAAA

miRNA miRNA

Decreased miRNA activity Increased miRNA activity increases levels of target decreases levels of target oncogene tumor suppressor Use Illumina sequencing to profile miRNAs in ovarian surface epithelium (versus ovarian cancer)

miR-21

miR-31

miR-29a

miR-103

miR-140

miR-320a

miR-221

% sequences total of % miR-191 miR-31 was universally downregulated >30-fold in our human serous ovarian cancers and cell lines

Increased

Decreased

miR-31 Hypothesis: miR-31 is an ovarian tumor suppressor miR-31 overexpression in OVCAR8 serous ovarian cancer cell line halts proliferation mainly by inducing apoptosis The region encoding miR-31 (9p21.3) is frequently deleted in serous ovarian cancer Model: miR-31 is a novel tumor suppressor that functions in conjunction with mutations in the CDKN2A/p53 pathway to cause ovarian cancer

9p21.3 deletion miR-31 CDKN2A p14ARF p16INK4A

E2F2 TFDP2 MDM2

p53 Other Targets (e.g., CEBPA) E2F pathway cyclinD CDK4/6

CANCER MicroRNAs, the PI3K Pathway, and Cancer  Our data suggests that specific microRNAs including miR-31 are downregulated in ovarian cancer

 Anil Sood’s group found that low DICER levels are correlated with reduced survival in ovarian cancer patients

 Tyler Jacks’ lab showed that suppression of miRNAs promotes tumorigenesis in a lung cancer model

 In about 75% of ovarian cancers, the PI3K/AKT pathway is activated

 Conditional knockout of either DICER or PTEN using AMHR2-Cre fails to produce cancer

Thus, we decided to make a double mutant mouse lacking DICER and the tumor suppressor, PTEN AMHR2-Cre DICER single KO – First mouse model with diverticuli in the oviduct

Diverticulum

(But No Cancer)

Ovary Uterus Cancers were seen in the ovaries of DICER/PTEN DKO The cancers have papillary structures and are cytokeratin 8 and WT1 positive consistent with papillary serous ovarian cancer DICER/PTEN DKO mice quickly die between 6 & 13 months

100

90 Control (n=20)

80 DKODKO (n=23)(n=23)

Survival (%) (%) Survival Survival

0 20 24 28 32 36 40 44 48 52 56 Age (weeks) Ovarian cancer mice quickly develop ascites that contains tumor cells that can be propagated in vivo in SCID mice and in cell culture

Control DKO Metastases are observed throughout the peritoneum

DKO with Peritoneal Metastasis Ovarian cancer histotypes resemble cells at various locations in the reproductive tract

HISTOTYPES

High grade serous

Low-grade serous

Endometrioid

Mucinous

VAGINA Clear cell DICER/PTEN DKO mice initially develop tumors in the fallopian tube

4.5 month old mouse Summary of progression of high-grade serous carcinomas in DICER/PTEN DKO Revised Model: PI3K, miRNA, p53, CDKN2A pathways are interrelated to regulate high-grade serous carcinoma formation and progression Topic 2 Conclusions & Future Directions

 miR-31 is a novel tumor suppressor miRNA that functions in cancer cell lines that have mutations in the p53/CDKN2A pathways -> we will test the effects of gene therapy delivery of miR-31 to cancers in vivo

 Activation of the PI3K pathway through cKO of PTEN in the absence of DICER results in fallopian tube primary serous carcinomas that metastasize to the ovary and peritoneum and eventually kill the mice -> we will determine which additional pathways are altered in these tumor cells, define particular miRNAs and PI3K pathway inhibitors that reverse the phenotype, and identify the cancer stem cells that allow propagation of the ovarian cancers Matzuk Lab Members ACKNOWLEDGMENTS Julio Agno Denise Archambeault Ruihong Chen Caterina Clementi Mike Fountain Naoki Iwamori Tokuko Iwamori Jaeyeon Kim Qinglei Li Lang Ma Takashi Nagashima Derek O’Neil Adithya Rangarajan Zhifeng Yu

Baylor Collaborators Past Lab Members Matt Anderson Greg Buchold Chad Creighton Mark Edson Franco DeMayo Ankur Nagaraja Preethi Gunaratne Roopa Nalam Shannon Hawkins Angshumoy Roy Jaewook Jeong Other Collaborators Dorrie Lamb Richard Behringer John Lydon John Eppig Stephanie Pangas Vesa Kaartinen Alex Yatsenko Tom Thompson Karen Lyons Funding: NIH, DLDCC, OCRF, YTAC, Mary Kay