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Characteriza5on of Wnt/β-‐catenin signaling and its eﬀect on Schwann cell tumorigenesis using a tamoxifen-‐inducible Plp-‐Cre to drive the expression of ac5vated β-‐catenin in Schwann cells. Jus5n Knight1, 2, Dr. Adrienne L. Watson1, 2, 3, 4, Rory Williams1, 2, Leah Anderson1, 2, Dr. David A. Largaespada1, 2, 3, 4 1Masonic Cancer Center, 2Department of Gene5cs, Cell Biology and Development, 3Center for Genome Engineering, 4Brain Tumor Program, University of Minnesota, Minneapolis, MN 55455, USA. Abstract Introduc5on Neuroﬁbromatosis Type 1 (NF1) •  NF1 is a heritable gene5c disorder where indviduals are born with a loss of one copy of the Neuroﬁbromin (NF1) gene, and subsequent loss of the second copy in Schwann cells is characterized by the forma5on of benign tumors throughout the peripheral nervous system(1) •  NF1 is known to be a tumor suppressor gene that confers its eﬀect via nega5ve regula5on of Ras through Ras-‐GAP ac5vity(2) Malignant Peripheral Nerve Sheath Tumors (MPNSTs): •  MPNSTs are derived from Schwann cells and grow on the peripheral nerves in the body •  Currently the only treatments available are broad, non-‐speciﬁc chemotherapy and surgical resec5on(3,4, 5) •  The tumors oVen form via secondary gene5c changes following biallelic loss of NF1 •  5 year survival rate of less than 40%(3,5) •  MPNSTs can develop both sporadically and in the context of NF1 where 10% of pa5ents with NF1 will develop MPNSTs(3,6,7,8) Wnt/β-‐catenin signaling in Schwann cell tumorigenesis •  MPNST analysis from human and mice has shown ac5va5on of this pathway(9) •  Wnt/β-‐catenin signaling has been shown to induce oncogenic phenotypes in immortalized human Schwann cells and its down regula5on leads to a decrease of these phenotypes in MPNST cells(9) •  Wnt/β-‐catenin signaling is a known cancer pathway for lung, breast, ovarian and prostate cancer (10) Plp-‐Cre recombina5on of β-catenin
Plp promoter B-‐catenin exon 3 (Phosphoryla5on site) Image adapted from: hip://www.scq.ubc.ca/targe5ng-‐your-‐dna-‐with-‐the-‐crelox-‐system/ F i g u r e 1 : S c h w a n n c e l l – s p e c i ﬁ c recombinaGon of β-‐catenin exon 3 will occur with the use of PlpCre In this scheme, a myelin proteolipid protein (Plp) that is tamoixfen inducible (Plp-‐Cre-‐ERT2) is used to control expression of the Cre recombinase. Cre recombinase recognizes 34 bp loxP sites and removes any DNA sequence ﬂanked by loxP sites (ﬂoxed) as circular DNA. When tamoxifen is introduced to the organism the Cre is ac5vated and the ﬂoxed exon 3 of β-‐catenin (β-‐cat) is removed in the peripheral nervous system. β-‐cat then becomes cons5tu5vely ac5ve due to the absence of exon 3 which is an important inhibitory phosphoryla5on site . PlpCre will only be ac5ve in Schwann cells and thus recombina5on will only occur in those cells or their precursors. Ac5vated β-‐cat shortens lifespan and causes developmental heart problems and embryonic lethality A. 450 100 400 A. 350 B. Percent Survival 300 250 n=13 200 n=20 150 100 50 n=4 0 n=3 n=10 Dhh; Nf1 ﬂ/+ Dhh; Nf1 ﬂ/ﬂ Dhh; β-‐cat Dhh; Nf1 ﬂ/+ ; ﬂ β-‐cat ﬂ C. Dhh; Nf1 ﬂ/ﬂ ; β-‐cat ﬂ Brain Bottom Dorsal Root Ganglia
70 ﬂ/ﬂ Dhh; Nf1
60 50 30 0 200 400 600 Dhh; Nf1 ﬂ/ﬂ ; β-‐cat ﬂ Time (Days) Enlarged Heart
Loss of Nf1 using the tamoxifen inducible PlpCre results in the forma5on of grade 1 neuroﬁbromas (12) GEM Grade 1 Neurofibromas
20:20 (100%)
Thoracic
17:20 (85%)
Lumbar/Sacral
16:20 (80%)
GEM Grade III PNSTs
Back/Abdomen
1:20 (5%)
Base of Tail
2:20 (10%)
Hind Limb
1:20 (5%)
Figure 3: Loss of Nf1 in adult mice using tamoxifen induced PlpCre results in formaGon of grade 1 neuroﬁbromas and some grade 3 Peripheral Nerve Sheath Tumors (PNSTs)(11). Data table adapted from Mayes et al. 2011 where Plp-‐Cre; Nf1 ﬂ/ﬂ mice were injected with tamoxifen aVer 60 days of life. The cohort experienced high percentages (66-‐100%) of mice experiencing grade 1 Cervical, Thoracic and Lumbar/Sacral neuroﬁbromas. The adult tamoxifen cohort also experienced Grade 3 PNSTs (5-‐10%) in the Back, Base of Tail and Hindlimb. In this experiment we will be comparing our results to those ajained by Mayes et al. for their adult tamoxifen cohort to see if Plp-‐Cre ac5vated β-‐catenin expression will worsen this phenotype. It is important to note that this study only used Plp-‐Cre; Nf1 ﬂ/ﬂ mice. A.
Age 60 days Phenotypes 171 151 143 Plp-‐Cre; B-‐cat ﬂ/ﬂ Plp-‐Cre; Nf1 ﬂ/+; B-‐ M2588 (0) cat ﬂ/+ M2649 (1) M2745 (1) 163 161 152 M2746 (3) M2812 (0) Plp-‐Cre; Nf1 ﬂ/ﬂ; B-‐ M2592 (30) cat ﬂ/+ F2773 (0) 152 143 164 70 (deceased) F2775 (3) M2813 (1) M2814 (3) Tamoxifen injected 143 143 143 Tumor like buldge Shakey, bloated abdomen, darkened and enlarged spleen (0.3g) Plp-‐Cre; Nf1 ﬂ/+; B-‐
cat ﬂ/ﬂ Plp-‐Cre; Nf1 ﬂ/ﬂ; B-‐
Discussion Is acGvated β-‐catenin only expressed in peripheral nerve Gssue using Plp-‐Cre? From Figure 4A it is shown that only in peripheral nerve 5ssue does recombina5on happen and cons5tu5ve β-‐cat ac5va5on is achieved. In all other 5ssue loca5ons, Plp-‐
Cre does not seem to be eﬀec5ng β-cat ac5va5on through recombina5on. What are the expected results? We believe the results of the experiment will show that when Nf1 is lost and β-cat is ac5vated, the oncogenic phenotype will be enhanced in comparison to Mayes et al. where just Nf1 was lost. This could be in the form of higher tumor grades and/or tumor burden, or faster development of tumors. How will Gssues be examined? AVer subjects experience severe phenotypes such as paralysis, they will be sacriﬁced and 5ssues will be harvested. These 5ssues will be examined by: •  Observa5on (color, enlargement, tumor number and size) •  Independent pathological tumor grading after parafilm embedding •  Histologic Staining •  H&E-‐ Iden5ﬁca5on of cellularity and mito5c ﬁgures •  S100-‐ Iden5ﬁca5on of Schwann cell origin and diﬀeren5a5on status •  Ki67-‐ Iden5ﬁca5on of prolifera5ng cells Acknowledgements • 
University of Minnesota Undergraduate Research OpportuniGes Program (UROP) Dr. Adrienne Watson (post-‐doc), Rory Williams (Undergraduate Research Assistant), Leah Anderson (Undergraduate Research Assistant), and Largaespada Lab members Dr. David Largaespada (Principal InvesGgator) Funding B. Β-cat Recombined Allele
Nf1ﬂ/ﬂ; β-‐cat ﬂ/ﬂ Plp-‐Cre-‐ErT2 a
WT allele Recombined allele Plp-‐Cre-‐ErT2; Nf1ﬂ/+; β-‐cat ﬂ/+ Plp-‐Cre-‐ErT2; Nf1ﬂ/+; β-‐cat ﬂ/+ a Figure 4: β-‐cat RecombinaGon occurs only in Peripheral Nervous Tissue and Breeding Scheme A) DNA Recombina5on PCR taken from M2548 Plp-‐Cre-‐ErT2; Nf1ﬂ/+; β-‐cat ﬂ/+ , a tamoxifen injected mouse. Tissues were harvested at 90 days of age, 30 days aVer tamoxifen injec5ons. M2548 was injected twice a day for 3 days with tamoxifen (1.0mg intraperitoneal) at 60 days of life to circumvent lethality and cardiac defects. Recombina5on of β-‐cat is seen in Scia5c Nerve (Scia5c), Brachial Plexus (BP) and Dorsal Root Ganglia (DRG) as characterized by the shorter fragment represen5ng the deleted exon 3 of the now ac5vated β-‐cat . Recombina5on occurs only in these 5ssues due to the Schwann cell speciﬁcity of the PlpCre driver. Other 5ssues including the heart, lungs, brain, kidney, and gut lack a second fragment sugges5ng no β-‐cat recombina5on and thus no Wnt signaling ac5va5on is occurring in those areas. B) General breeding scheme to generate desired cohorts. M2543 (0) M2650 (3) M2815 (10) Age Tissues harvested cat ﬂ/ﬂ for histology, pathology (tumor Status. A) The procedure that will be followed in this experiment. Mice grade), as well as Figure 5: Experimental SchemaGc and ﬂ/+Current ﬂ/+ with desired genotypes, PLP-‐Cre; Nf1 ; B-‐cat , PLP-‐Cre; Nf1 ﬂ/ﬂ; B-‐catﬂ/+ , PLP-‐Cre; Nf1 ﬂ/+; B-‐catﬂ/ﬂ, PLP-‐Cre; Nf1 ﬂ/ﬂ; Wnt signaling. B-‐catﬂ/ﬂ , are aged out to 60 days where they are then injected with tamoxifen to ac5vate Wnt signaling. We will then observe them un5l they experience peripheral nervous system phenotypes such as paralysis. At that point the mouse will be sacriﬁced and its 5ssues will be harvested for analysis of tumor grade, burden, ac5va5on of Wnt pathway, histology and pathology. B) Current numbers of desired genotypes that have been injected and are aging out. • 
• 
Schwann cell speciﬁc Wnt/β-cat signaling with PlpCre a Observe for phenotype (sickness, paralysis, observable tumors). 2-‐4 months. Dhh; Nf1 ﬂ/+
; β-‐cat ﬂ 20 0 Proportion of Mice Experiencing
Phenotype (n=20)
Plp-‐-‐Cre; Nf1 ﬂ/ﬂ Plp-‐Cre; B-‐cat ﬂ/+ Dhh; β-‐cat ﬂ 40 10 C. Cervical
Plp-‐Cre; Nf1 ﬂ/+ ; Bcat ﬂ/+ PLP mice injected Breed for desired B. Genotype Mice: B. genotypes Plp-‐-‐Cre; Nf1 ﬂ/+ CO2 euthanizaGon when severe phenotype is present 80 Figure 2: Expression of acGvated β-‐catenin in Schwann cells using Dhh-‐Cre dramaGcally reduces lifespan and confers abnormal phenotypes. A) Average
length of life among the experimental cohorts. Dhh; Nf1fl/+ and Dhh; Nf1fl/fl
have significantly longer average lifespans than Dhh; β-cat fl, Dhh; Nf1 fl/+; βcat fl, and Dhh; Nf1fl/fl; β-cat fl as determined by a two-tailed t test. Average
lifespans among β-cat fl cohorts were not significantly different as determined
by a two-tailed t test. Sample size and SEM bars included, no asterisk means
no significance between cohorts (p>0.05), (**) means high significance
between cohorts (p<<0.01). B) Kaplan Meier graph showing the percent
survival of experimental cohorts at specific times (days) after birth. Mice were
studied everyday for phenotypes and were euthanized when they displayed
symptoms such as paralysis, failure to groom, and severe weight loss. Note: βcat fl refers to mice having both 1 or both β-cat alleles floxed. C) Pictures of
phenotypes of Dhh-Cre; Nf1fl/+; β-cat fl mice. Pictures were taken during
necropsy which immediately followed CO2 euthanization. Dhh-Cre; Nf1 fl/+; βcat fl mice did not experience enlarged nerves, and instead had nerves closely
resembling wild type nerves. However, Dhh-Cre; Nf1 fl/+; β-cat fl and DhhCre; Nf1fl/fl; β-cat fl often displayed enlarged hearts. Enlarged nerve pictures
are given for comparison and were taken from a Dhh-Cre; Pten fl/fl mouse in
Keng et al.(11)
Phenotypes and Location of PlpCre-ERT+; Nf1 Adult Mice
Injected with Tamoxifen
A. ﬂ/+ Dhh; Nf1
90 ** Future direc5ons Plp-‐Cre; Nf1 ﬂ/ﬂ ; Bcat ﬂ/+ Plp-‐Cre; Nf1 ﬂ/+ ; Bcat ﬂ/+ Plp-‐Cre; Nf1 ﬂ/+ ; Bcat ﬂ/ﬂ B. 500 Average Lifespan (Days) Malignant peripheral nerve sheath tumors (MPNSTs) are soV 5ssue sarcomas which form in about 10% of Neuroﬁbromatosis Type 1 (NF1) pa5ents but can also form sporadically in the general popula5on. NF1 is an autosomal dominant disease in which pa5ents with a hereditary mutant copy of NF1 develop benign neuroﬁbromas throughout the peripheral nervous system aVer loss of the second copy of NF1 in Schwann cells. Upon addi5onal gene5c modiﬁca5ons, these benign neuroﬁbromas can undergo malignant transforma5on into MPNSTs. There are few treatment op5ons for MPNSTs other than surgical resec5on and broad, high-‐dose chemotherapy. Such treatments are oVen ineﬀec5ve, resul5ng in a ﬁve-‐year survival rate of only 40%. Recently, ac5va5on of the Wnt/β-‐
catenin signaling pathway, a pathway that has been implicated in many cancer types, has been shown to induce oncogenic phenotypes in vitro in immortalized Schwann cell lines. Down regula5on of Wnt/β-‐catenin signaling has also been shown to reduce oncogenic phenotypes in MPNST cell lines. While promising, this characteriza5on of Wnt/β-‐catenin signaling in Schwann cell tumorigenesis has yet to be tested in the context of an in vivo mouse model. We sought to induce Wnt/β-‐catenin signaling in the Schwann cells of mice via a Cre-‐Lox system. In this system, Cre Recombinase was expressed by the Desert Hedgehog Promoter (Dhh-‐Cre), ac5ve in Schwann cells and their precursors, to drive expression of a cons5tu5vely ac5ve form of β-‐catenin. The genera5on of mice expressing the ac5vated β-‐catenin was signiﬁcantly less than expected from Mendelian inheritance pajerns, sugges5ng embryonic lethality. Mice that did survive past birth had short lifespans and enlarged hearts, but did not display nerve hyperplasia or tumorigenesis. We concluded that Wnt/β-‐signaling is important for cardiac development and that our Dhh promoter was likely expressed in the heart. From this experiment we were not able to make any conclusions about the role of Wnt/β-‐catenin signaling in Schwann cells. To circumvent this, we are using a myelin proteolipid protein Cre recombinase (PlpCre). This PlpCre driver, which is speciﬁc to Schwann cells, is tamoxifen-‐ inducible, and will allow ac5va5on of Wnt/β-‐catenin signaling aVer birth and cardiac development. PlpCre has previously been shown to be eﬀec5ve at inducing recombina5on and inac5va5on of target genes in the Schwann cells of adult mice. The loss of Nf1 func5on in these adult mice led to tumor forma5on and morbidity beginning four months aVer injec5on. We hypothesized the ac5va5on of Wnt/β-‐catenin signaling in Schwann cells would also induce oncogenic phenotypes. We further hypothesized that, combined with inac5va5on of Nf1, ac5va5on of Wnt/ β-‐catenin signaling will lead to greater Schwann cell tumorigenesis and progression in mice than would be observed with either single gene5c change. Currently, transgenic mice containing PlpCre, ﬂoxed β-‐cat, and ﬂoxed Nf1 are being injected with tamoxifen at 60 days of age, well aVer development has completed, in order to avoid the semi-‐
lethal phenotype and heart defects. Tissues and tumors will be analyzed for pathology, histology, and tumor grade, and tumors will also be examined for the presence of Wnt signaling ac5va5on in vivo. With the results from this study, we hope to further elucidate the role of Wnt/β-‐catenin signaling in Schwann cell tumorigenesis in the context of spontaneous MPNSTs and those that develop in NF1 pa5ents. 45 day old male
Enlarged Nerves
fl/+
Dhh-Cre; Pten fl/fl Dhh-Cre; Nf1 ;
β-cat fl
Plp-‐Cre-‐ErT2; Nf1ﬂ/ﬂ; β-‐cat ﬂ/fL Plp-‐Cre-‐ErT2; Nf1ﬂ/+; β-‐cat ﬂ/ﬂ Plp-‐Cre-‐ErT2; Nf1ﬂ/ﬂ; β-‐cat ﬂ/+ Plp-‐Cre-‐ErT2; Nf1ﬂ/+; β-‐cat ﬂ/+ Plp-‐Cre-‐ErT2; Nf1ﬂ/+ Plp-‐Cre-‐ErT2; Nf1ﬂ/ﬂ Plp-‐Cre-‐ErT2; β-‐cat ﬂ/+ Plp-‐Cre-‐ErT2; β-‐cat ﬂ/ﬂ • 
• 
• 
• 
NaGonal InsGtutes of Health (NINDS, NCI) Children's Cancer Research Fund The Jacqueline Dunlap NF Research Fund The Zachary NF Research Fund References 1. Side, L., Taylor, B., Cayoueie, M., Conner, E., Thompson, P., Luce, M., and Shannon, K. (1997). Homozygous TrovóMarqui, A., and Tajara, E. (2006). Neuroﬁbromin: a general outlook. Clinical Gene5cs 70, 1–13. 2. Widemann, B.C. (2009). Current status of sporadic and neuroﬁbromatosis type 1-‐associated malignant peripheral nerve sheath tumors. Curr Oncol Rep 11, 322– 328. 3. Gregorian, C., Nakashima, J., Dry, S.M., Nghiemphu, P.L., Smith, K.B., Ao, Y., Dang, J., Lawson, G., Mellinghoﬀ, I.K., Mischel, P.S., et al. (2009). PTEN dosage is essen5al for neuroﬁbroma development and malignant transforma5on. Proc. Natl. Acad. Sci. U.S.A. 106, 19479–19484. 4.Katz, D., Lazar, A., and Lev, D. (2009). Malignant peripheral nerve sheath tumor (MPNST): the clinical implica5ons of cellular signaling pathways. Expert Reviews in Molecular Medicine 11, null–null. 5. Uhlmann, E.J., and Plotkin, S.R. (2012). Neuroﬁbromatoses. Adv. Exp. Med. Biol. 724, 266–277. 6. Carroll, S.L., and Ratner, N. (2008). How Does the Schwann Cell Lineage Form Tumors in NF1? Glia 56, 1590–1605. 7. Carroll, S.L. (2012). Molecular mechanisms promo5ng the pathogenesis of Schwann cell neoplasms. Acta Neuropathol 123, 321–348. 8.Watson, A.L., Rahrmann, E.P., Moriarity, B.S., Choi, K., Conboy, C.B., Greeley, A.D., Halfond, A.L., Anderson, L.K., Wahl, B.R., Keng, V.W., et al. (2013). Canonical Wnt/β-‐catenin Signaling Drives Human Schwann Cell Transforma5on, Progression, and Tumor Maintenance. Cancer Discovery 3, 674–689. 10. MacDonald BT , Tamai K , He X . Wnt/beta-‐catenin signaling: components,mechanisms, and diseases . Dev Cell 2009 ; 17 : 9 – 26 . 11. Keng, V.W., Watson, A.L., Rahrmann, E.P., Li, H., Tschida, B.R., Moriarity, B.S., Choi, K., Rizvi, T.A., Collins, M.H., Wallace, M.R., et al. (2012). Condi5onal Inac5va5on of Pten with EGFR Overexpression in Schwann Cells Models Sporadic MPNST. Sarcoma 2012, 620834. 12. Mayes, D.A., Rizvi, T.A., Cancelas, J.A., Kolasinski, N.T., Ciraolo, G.M., Stemmer-‐Rachamimov, A.O., and Ratner, N. (2011). Perinatal or adult Nf1 inac5va5on using tamoxifen-‐inducible PlpCre each cause neuroﬁbroma forma5on. Cancer Res 71, 4675–4685. Inac5va5on of the NF1 Gene in Bone Marrow Cells from Children with Neuroﬁbromatosis Type 1 and Malignant Myeloid Disorders. New England Journal of Medicine 336, 1713–1720.