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 0 Con GDF9 BMP15 In vivo cumulus expansion occurs in ALK5 cKO mice 5 x 20 x GC Control CC (#7371) Oo TC GC 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 SM 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 40 30 20 10 % Time in Each Stage 0 Estrus Diestrus Proestrus Metestrus ALK3/6 DKO mice 100 80 60 40 20 % Females% with Tumors 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 of total % 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) 70 60 50 40 30 Survival (%) (%) Survival Survival 20 10 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