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Inducible Knockout of Twist1 in Young and Adult Mice Prolongs Hair Growth Cycle and Has Mild Effects on General Health, Supporting Twist1 as a Preferential Cancer Target

2013; Elsevier BV; Volume: 183; Issue: 4 Linguagem: Inglês

10.1016/j.ajpath.2013.06.021

1525-2191

Yan Xu, Yixiang Xu, Lan Liao, Niya Zhou, Sarah M. Theissen, Xin-Hua Liao, Hoang Nguyen, Thomas Ludwig, Li Qin, Jarrod D. Martinez, Jun Jiang, Jianming Xu,

Epigenetics and DNA Methylation

Twist1 promotes epithelial–mesenchymal transition, invasion, metastasis, stemness, and chemotherapy resistance in cancer cells and thus is a potential target for cancer therapy. However, Twist1-null mice are embryonic lethal, and people with one Twist1 germline mutant allele develop Saethre–Chotzen syndrome; it is questionable whether Twist1 can be targeted in patients without severe adverse effects. We found that Twist1 is expressed in several tissues, including fibroblasts of the mammary glands and dermal papilla cells of the hair follicles. We developed a tamoxifen-inducible Twist1 knockout mouse model; Twist1 knockout in 6-week-old female mice did not affect mammary gland morphogenesis and function during pregnancy and lactation. In both males and females, the knockout did not influence body weight gain, heart rate, or total lean and fat components. The knockout also did not alter blood pressure in males, although it slightly reduced blood pressure in females. Although Twist1 is not cyclically expressed in dermal papilla cells, knockout of Twist1 at postnatal day 13 (when hair follicles have developed) drastically extended the anagen phase and accelerated hair growth. These results indicate that Twist1 is not essential for maintaining an overall healthy condition in young and adult mice and that loss of function facilitates hair growth in adulthood, supporting Twist1 as a preferential target for cancer therapy. Twist1 promotes epithelial–mesenchymal transition, invasion, metastasis, stemness, and chemotherapy resistance in cancer cells and thus is a potential target for cancer therapy. However, Twist1-null mice are embryonic lethal, and people with one Twist1 germline mutant allele develop Saethre–Chotzen syndrome; it is questionable whether Twist1 can be targeted in patients without severe adverse effects. We found that Twist1 is expressed in several tissues, including fibroblasts of the mammary glands and dermal papilla cells of the hair follicles. We developed a tamoxifen-inducible Twist1 knockout mouse model; Twist1 knockout in 6-week-old female mice did not affect mammary gland morphogenesis and function during pregnancy and lactation. In both males and females, the knockout did not influence body weight gain, heart rate, or total lean and fat components. The knockout also did not alter blood pressure in males, although it slightly reduced blood pressure in females. Although Twist1 is not cyclically expressed in dermal papilla cells, knockout of Twist1 at postnatal day 13 (when hair follicles have developed) drastically extended the anagen phase and accelerated hair growth. These results indicate that Twist1 is not essential for maintaining an overall healthy condition in young and adult mice and that loss of function facilitates hair growth in adulthood, supporting Twist1 as a preferential target for cancer therapy. Twist1 is a class B member of the basic helix-loop-helix (bHLH) transcription factor superfamily. Twist1 forms heterodimers with class A members of the same superfamily, such as E12 and E47, to bind NdeI E-box DNA elements to regulate the expression of genes essential for mesodermal induction and organogenesis.1Thisse B. el Messal M. Perrin-Schmitt F. The twist gene: isolation of a Drosophila zygotic gene necessary for the establishment of dorsoventral pattern.Nucleic Acids Res. 1987; 15: 3439-3453Crossref PubMed Scopus (248) Google Scholar, 2Qin Q. Xu Y. He T. Qin C. Xu J. 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Donaher J.L. Murphy D.A. Chau S. Yang J. Spatiotemporal regulation of epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis.Cancer Cell. 2012; 22: 725-736Abstract Full Text Full Text PDF PubMed Scopus (801) Google Scholar These studies suggest that Twist1 may be an important and useful target for controlling cancer metastasis and enhancing the efficacy of cancer therapy. However, given the lethal phenotype of Twist1-null mice, it remains to be evaluated whether inhibition of Twist1 in cancer patients causes any severe health problems. The process of carcinogenesis from normal epithelial cells can essentially be considered as a dedifferentiation process that is commonly associated with the expression of many genes that are only or predominantly expressed during early embryonic development. We hypothesize that, although Twist1 is required for embryonic development and for cancer cell EMT and metastasis, Twist1 is not essential for maintaining a generally healthy physiological condition in the adult organism. To test this hypothesis, we first examined Twist1 protein distribution in adult mice, then developed an inducible knockout model to delete Twist1 in adult mice at different stages as needed, and finally defined the effect of Twist1 knockout on physiological functions after embryonic morphogenesis was fully accomplished. Mouse tissues were dissected and fixed in a 4% paraformaldehyde solution at 4°C overnight. After a PBS wash, the fixed tissues were dehydrated and embedded in paraffin blocks as described previously.55Kuang S.Q. Liao L. Zhang H. Lee A.V. O'Malley B.W. Xu J. AIB1/SRC-3 deficiency affects insulin-like growth factor I signaling pathway and suppresses v-Ha-ras-induced breast cancer initiation and progression in mice.Cancer Res. 2004; 64: 1875-1885Crossref PubMed Scopus (159) Google Scholar Tissue sections (5 μm thick) were prepared, deparaffinized in xylene, and hydrated using an ethanol gradient. H&E staining, antigen retrieval, IHC, and immunohistofluorescence (IHF) were performed as described previously.55Kuang S.Q. Liao L. Zhang H. Lee A.V. O'Malley B.W. Xu J. AIB1/SRC-3 deficiency affects insulin-like growth factor I signaling pathway and suppresses v-Ha-ras-induced breast cancer initiation and progression in mice.Cancer Res. 2004; 64: 1875-1885Crossref PubMed Scopus (159) Google Scholar For immunostaining, sections were blocked with either 10% normal serum or a M.O.M mouse-on-mouse immunodetection kit (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature and incubated with primary antibodies overnight at 4°C. The primary antibodies were against Twist1 (ab50887; Abcam, Cambridge, MA), α-smooth muscle actin (α-SMA) (Dako M0851; Agilent Technologies, Santa Clara, CA), lymphoid enhancer-binding factor 1 (Lef-1) (C12A5; Cell Signaling Technology, Danvers, MA), vimentin (ab8978; Abcam), and Ki-67 (550609; BD Biosciences, San Jose, CA). Secondary antibodies (Vector Laboratories) were diluted 1:400. The IHC signal was enhanced using a Vectastain ABC system and was visualized with a peroxidase substrate kit containing 3,3′-diaminobenzidine (Vector Laboratories). Slides were counterstained with Harris modified hematoxylin and mounted with Permount mounting medium (Thermo Fisher Scientific, Waltham, MA) for microscopy. For IHF, a tyramide signal amplification kit (Life Technologies, Carlsbad, CA) was used according to the manufacturer's instructions. TUNEL assay was performed on hydrated mouse skin sections using an apoptosis detection kit (Upstate; EMD Millipore, Billerica, MA) according to the manufacturer's instructions. The mouse line with floxed Twist1 alleles (Twist1f/f) for conditional knockout has been described previously56Chen Y.T. Akinwunmi P.O. Deng J.M. Tam O.H. Behringer R.R. Generation of a Twist1 conditional null allele in the mouse.Genesis. 2007; 45: 588-592Crossref PubMed Scopus (43) Google Scholar and was obtained from Mutant Mouse Regional Resource Centers (016842-UNC). In this line, two loxP sites were placed flanking the entire coding region of the Twist1 gene.56Chen Y.T. Akinwunmi P.O. Deng J.M. Tam O.H. Behringer R.R. Generation of a Twist1 conditional null allele in the mouse.Genesis. 2007; 45: 588-592Crossref PubMed Scopus (43) Google Scholar The Rosa26-CreERT2 mouse line has been described previously57Guo K. McMinn J.E. Ludwig T. Yu Y.H. Yang G. Chen L. Loh D. Li C. Chua Jr., S. Zhang Y. Disruption of peripheral leptin signaling in mice results in hyperleptinemia without associated metabolic abnormalities.Endocrinology. 2007; 148: 3987-3997Crossref PubMed Scopus (95) Google Scholar and was provided by T.L. In this mouse line, the Rosa26 locus drives the expression of the Cre-ERT2 fusion protein consisting of the Cre recombinase and the mutant ligand-binding domain of the estrogen receptor α (ER-α). This mutant ligand-binding domain binds only 4-hydroxytamoxifen (the active metabolite of tamoxifen), but not endogenous estrogen. On tamoxifen binding, the Cre-ERT2 fusion protein translocates into the nucleus from the cytoplasm, allowing its Cre recombinase to excise any floxed DNA fragment in the genome.57Guo K. McMinn J.E. Ludwig T. Yu Y.H. Yang G. Chen L. Loh D. Li C. Chua Jr., S. Zhang Y. Disruption of peripheral leptin signaling in mice results in hyperleptinemia without associated metabolic abnormalities.Endocrinology. 2007; 148: 3987-3997Crossref PubMed Scopus (95) Google Scholar Rosa-CreERT2 and Twist1f/f mice were crossbred for two generations to generate Rosa-CreERT2+/−;Twist1f/f (hereafter referred to as CreERT2;Twist1f/f or, after tamoxifen treatment, as Twist1Δ/Δ) and Rosa-CreERT2−/−;Twist1f/f (hereafter referred to as Twist1f/f or control) mice with the same genetic background for experiments. For analyzing the genotypes of these mice, genomic DNA samples were extracted from a small piece of mouse ear tissue to serve as PCR template as described previously.58Liu Z. Zhou S. Liao L. Chen X. Meistrich M. Xu J. Jmjd1a demethylase-regulated histone modification is essential for cAMP-response element modulator-regulated gene expression and spermatogenesis.J Biol Chem. 2010; 285: 2758-2770Crossref PubMed Scopus (116) Google Scholar Allele-specific PCR primers were synthesized and allele-specific PCR reactions were performed as described previously.57Guo K. McMinn J.E. Ludwig T. Yu Y.H. Yang G. Chen L. Loh D. Li C. Chua Jr., S. Zhang Y. Disruption of peripheral leptin signaling in mice results in hyperleptinemia without associated metabolic abnormalities.Endocrinology. 2007; 148: 3987-3997Crossref PubMed Scopus (95) Google Scholar Animal protocols were approved by the Animal Care and Use Committee of Baylor College of Medicine. Tamoxifen (Sigma-Aldrich, St. Louis, MO) was dissolved in corn oil (Sigma-Aldrich) at a concentration of 20 mg/mL. For inducible knockout of the floxed Twist1 alleles in adult mice, 6-week-old CreERT2;Twist1f/f and Twist1f/f mice were injected with 1 mg tamoxifen/day intraperitoneally for five consecutive days. At 7 days after the last injection, various mouse organs were collected for examining histology, for assessing Twist1 knockout efficiency by PCR-based genotype analysis after organ-specific genomic DNA samples were prepared, and for analyzing Twist1 protein by IHC. The maintenance of tamoxifen-induced Twist1 knockout efficiency was also examined by IHC at different time points after tamoxifen treatment. The body weights of the tamoxifen-treated mice were measured once a week at different time points. For analysis of the hair follicle cycle, 13-day-old CreERT2;Twist1f/f and Twist1f/f mice were injected with 0.4 mg tamoxifen/day intraperitoneally for three consecutive days. Dorsal skin specimens were collected at various time points after tamoxifen treatment for histological examination and immunostaining. The staining of whole-mounted mammary glands was performed as described previously.59Xu J. Liao L. Ning G. Yoshida-Komiya H. Deng C. O'Malley B.W. The steroid receptor coactivator SRC-3 (p/CIP/RAC3/AIB1/ACTR/TRAM-1) is required for normal growth, puberty, female reproductive function, and mammary gland development.Proc Natl Acad Sci USA. 2000; 97: 6379-6384Crossref PubMed Scopus (458) Google Scholar In brief, whole mammary glands were dissected out from mice and fixed on glass slides in Carnoy's fixative (1:3:7 glacial acetic acid–CHCl3–ethanol). They were then hydrated in 70% ethanol, rinsed in water, and stained overnight in carmine alum, followed by dehydration in increasing series of alcohol and clearing in xylene. The glands were mounted under coverslips with Permount. Body composition was measured using a PIXImus body composition and densitometry system (Piximus, Fitchburg, WI). Mice were weighed on a compact scale (cs200; Ohaus, Pine Brook, NJ), and body lengths were measured from the nose to the base of the tail. Mice were anesthetized with 2% isoflurane during the procedure. Data analysis was performed at the Mouse Phenotyping Core Facili