Mammary and extramammary Paget’s Diseases (PD) are a malignant skin cancer characterized by the appearance of Paget cells. Although easily diagnosed, its pathogenesis remains unknown. Here, single-cell RNA-sequencing identified distinct cellular states, novel biomarkers, and signaling pathways — including mTOR, associated with extramammary PD. Interestingly, we identified MSI1 ectopic overexpression in basal epithelial cells of human PD skin, and show that Msi1 overexpression in the epidermal basal layer of mice phenocopies human PD at histopathological, single-cell and molecular levels. Using this mouse model, we identified novel biomarkers of Paget-like cells that translated to human Paget cells. Furthermore, single-cell trajectory, RNA velocity and lineage-tracing analyses revealed a putative keratinocyte-to-Paget-like cell conversion, supporting the in situ transformation theory of disease pathogenesis. Mechanistically, the Msi1-mTOR pathway drives keratinocyte-Paget-like cell conversion, and suppression of mTOR signaling with Rapamycin significantly rescued the Paget-like phenotype in Msi1-overexpressing transgenic mice. Topical Rapamycin treatment improved extramammary PD-associated symptoms in humans, suggesting mTOR inhibition as a novel therapeutic treatment in PD.
Subscribe to Journal
Get full journal access for 1 year
We are sorry, but there is no personal subscription option available for your country.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Kanitakis, J. Mammary and extramammary Paget’s disease. J. Eur. Acad. Dermatol. Venereol. 21, 581–590 (2007).
Lopes Filho, L. L. et al. Mammary and extramammary Paget’s disease. An. Bras. Dermatol. 90, 225–231 (2015).
Sandoval-Leon, A. C., Drews-Elger, K., Gomez-Fernandez, C. R., Yepes, M. M. & Lippman, M. E. Paget’s disease of the nipple. Breast Cancer Res. Treat. 141, 1–12 (2013).
Lam, C. & Funaro, D. Extramammary Paget’s disease: summary of current knowledge. Dermatol. Clin. 28, 807–826 (2010).
Smith, K. J., Tuur, S., Corvette, D., Lupton, G. P. & Skelton, H. G. Cytokeratin 7 staining in mammary and extramammary Paget’s disease. Mod. Pathol. 10, 1069–1074 (1997).
Liegl, B. et al. Mammary and extramammary Paget’s disease: an immunohistochemical study of 83 cases. Histopathology 50, 439–447 (2007).
Hikita, T., Ohtsuki, Y., Maeda, T. & Furihata, M. Immunohistochemical and fluorescence in situ hybridization studies on noninvasive and invasive extramammary Paget’s disease. Int. J. Surg. Pathol. 20, 441–448 (2012).
Keatings, L. et al. c-erbB-2 oncoprotein expression in mammary and extramammary Paget’s disease: an immunohistochemical study. Histopathology 17, 243–247 (1990).
Fu, W., Lobocki, C. A., Silberberg, B. K., Chelladurai, M. & Young, S. C. Molecular markers in Paget disease of the breast. J. Surg. Oncol. 77, 171–178 (2001).
Mori, O., Hachisuka, H., Nakano, S., Sasai, Y. & Shiku, H. Expression of ras p21 in mammary and extramammary Paget’s disease. Arch. Pathol. Lab. Med. 114, 858–861 (1990).
Liegl, B., Horn, L. C. & Moinfar, F. Androgen receptors are frequently expressed in mammary and extramammary Paget’s disease. Mod. Pathol. 18, 1283–1288 (2005).
Morbeck, D. et al. GATA3 expression in primary vulvar Paget disease: a potential pitfall leading to misdiagnosis of pagetoid urothelial intraepithelial neoplasia. Histopathology 70, 435–441 (2017).
Zhang, G., Zhao, Y., Abdul-Karim, F. W. & Yang, B. P16 expression in primary vulvar extramammary Paget Disease. Int. J. Gynecol. Pathol. https://doi.org/10.1097/PGP.0000000000000602 (2019).
Ito, T., Kaku-Ito, Y. & Furue, M. The diagnosis and management of extramammary Paget’s disease. Exp. Rev. Anticancer Ther. 18, 543–553 (2018).
Sek, P., Zawrocki, A., Biernat, W. & Piekarski, J. H. HER2 molecular subtype is a dominant subtype of mammary Paget’s cells. An immunohistochemical study. Histopathology 57, 564–571 (2010).
Caliskan, M. et al. Paget’s disease of the breast: the experience of the European Institute of Oncology and review of the literature. Breast Cancer Res. Treat. 112, 513–521 (2008).
Marucci, G. et al. Toker cells are probably precursors of Paget cell carcinoma: a morphological and ultrastructural description. Virchows Arch. 441, 117–123 (2002).
Mehta, N. J., Torno, R. & Sorra, T. Extramammary Paget’s disease. South Med. J. 93, 713–715 (2000).
Nakamura, M., Okano, H., Blendy, J. A. & Montell, C. Musashi, a neural RNA-binding protein required for Drosophila adult external sensory organ development. Neuron 13, 67–81 (1994).
Sakakibara, S. et al. Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell. Dev. Biol. 176, 230–242 (1996).
Sakakibara, S., Nakamura, Y., Satoh, H. & Okano, H. Rna-binding protein Musashi2: developmentally regulated expression in neural precursor cells and subpopulations of neurons in mammalian CNS. J. Neurosci. 21, 8091–8107 (2001).
Kayahara, T. et al. Candidate markers for stem and early progenitor cells, Musashi-1 and Hes1, are expressed in crypt base columnar cells of mouse small intestine. FEBS Lett. 535, 131–135 (2003).
Li, N. et al. The Msi family of RNA-binding proteins function redundantly as intestinal oncoproteins. Cell Rep. 13, 2440–2455 (2015).
Yousefi, M. et al. Msi RNA-binding proteins control reserve intestinal stem cell quiescence. J. Cell Biol. 215, 401–413 (2016).
Macosko, E. Z. et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 (2015).
Satija, R., Farrell, J. A., Gennert, D., Schier, A. F. & Regev, A. Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol. 33, 495–502 (2015).
Stuart, T. et al. Comprehensive Integration of single-cell data. Cell 177, 1888–1902 (2019).
Hu, H. B., Yang, X. P., Zhou, P. X., Yang, X. A. & Yin, B. High expression of keratin 6C is associated with poor prognosis and accelerates cancer proliferation and migration by modulating epithelial-mesenchymal transition in lung adenocarcinoma. Genes Genom. 42, 179–188 (2019).
Wang, S. et al. Single cell transcriptomics of human epidermis reveals basal stem cell transition states. bioRxiv https://doi.org/10.1101/784579 (2019).
Kowalczyk, M. S. et al. Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells. Genome Res. 25, 1860–1872 (2015).
Mori, O., Karashima, T., Matsuo, K. & Hashimoto, T. Epidermal cell cultures from involved skin of patients with mammary and extramammary Paget’s disease. Kurume Med. J. 44, 165–169 (1997).
Thomas, P. D. et al. PANTHER: a library of protein families and subfamilies indexed by function. Genome Res. 13, 2129–2141 (2003).
Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30 (2000).
Tirosh, I. et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science 352, 189–196 (2016).
Iga, N. et al. Accumulation of exhausted CD8+ T cells in extramammary Paget’s disease. PLoS One 14, e0211135 (2019).
Sugiyama-Nakagiri, Y., Akiyama, M., Shibata, S., Okano, H. & Shimizu, H. Expression of RNA-binding protein Musashi in hair follicle development and hair cycle progression. Am. J. Pathol. 168, 80–92 (2006).
Benoit, S. et al. Elevated serum levels of calcium-binding S100 proteins A8 and A9 reflect disease activity and abnormal differentiation of keratinocytes in psoriasis. Br. J. Dermatol. 155, 62–66 (2006).
Marionnet, C. et al. Modulation of gene expression induced in human epidermis by environmental stress in vivo. J. Invest. Dermatol. 121, 1447–1458 (2003).
Leyva-Castillo, J. M., Hener, P., Jiang, H. & Li, M. TSLP produced by keratinocytes promotes allergen sensitization through skin and thereby triggers atopic march in mice. J. Invest. Dermatol. 133, 154–163 (2013).
Hildebrand, J. D. Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network. J. Cell Sci. 118, 5191–5203 (2005).
Wolber, R. A., Dupuis, B. A. & Wick, M. R. Expression of c-erbB-2 oncoprotein in mammary and extramammary Paget’s disease. Am. J. Clin. Pathol. 96, 243–247 (1991).
Xu, T. et al. [CMTM5 inhibits the tumor cell behavior of prostate cancer by downregulation of HER2]. Beijing Da Xue Xue Bao Yi Xue Ban 42, 386–390 (2010).
Fu, Y. F., Gui, R. & Liu, J. HER-2-induced PI3K signaling pathway was involved in the pathogenesis of gastric cancer. Cancer Gene. Ther. 22, 145–153 (2015).
Zhong, H. et al. Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res. 60, 1541–1545 (2000).
Hudson, C. C. et al. Regulation of hypoxia-inducible factor 1alpha expression and function by the mammalian target of rapamycin. Mol. Cell Biol. 22, 7004–7014 (2002).
Norrenberg, S. et al. Retrospective study: Rapamycin or rapalog 0.1% cream for facial angiofibromas in tuberous sclerosis complex: evaluation of treatment effectiveness and cost. Br. J. Dermatol. 179, 208–209 (2018).
Lin, N. et al. Expression of the p38 MAPK, NF-kappaB and cyclin D1 in extramammary Paget’s disease. J. Dermatol. Sci. 45, 187–192 (2007).
Chen, S. Y. et al. Concordant overexpression of phosphorylated ATF2 and STAT3 in extramammary Paget’s disease. J. Cutan. Pathol. 36, 402–408 (2009).
Cosgarea, I., Zaremba, A. & Hillen, U. Extramammary Paget’s disease. Hautarzt 70, 670–676 (2019).
Ghazizadeh, Z. et al. Prospective isolation of ISL1(+) cardiac progenitors from human ESCs for myocardial infarction therapy. Stem Cell Rep. 10, 848–859 (2018).
Smith, N. R. et al. Cell adhesion molecule CD166/ALCAM functions within the crypt to orchestrate murine intestinal stem cell homeostasis. Cell. Mol. Gastroenterol. Hepatol. 3, 389–409 (2017).
Du, X. et al. Extramammary Paget’s disease mimicking acantholytic squamous cell carcinoma in situ: a case report. J. Cutan. Pathol. 37, 683–686 (2010).
Tanskanen, M., Jahkola, T., Asko-Seljavaara, S., Jalkanen, J. & Isola, J. HER2 oncogene amplification in extramammary Paget’s disease. Histopathology 42, 575–579 (2003).
Cho, Z. et al. Podoplanin expression in peritumoral keratinocytes predicts aggressive behavior in extramammary Paget’s disease. J. Dermatol. Sci. 87, 29–35 (2017).
Kudinov, A. E. et al. Musashi-2 (MSI2) supports TGF-beta signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis. Proc. Natl. Acad. Sci. USA 113, 6955–6960 (2016).
Chen, S. et al. Immunohistochemical analysis of the mammalian target of rapamycin signalling pathway in extramammary Paget’s disease. Br. J. Dermatol. 161, 357–363 (2009).
Hata, H. et al. mTOR expression correlates with invasiveness and progression of extramammary Paget’s disease. J. Eur. Acad. Dermatol. Venereol. 30, 1238–1239 (2016).
Janku, F., Yap, T. A. & Meric-Bernstam, F. Targeting the PI3K pathway in cancer: are we making headway? Nat. Rev. Clin. Oncol. 15, 273–291 (2018).
Kato, R. et al. Efficacy of everolimus in patients with advanced renal cell carcinoma refractory or intolerant to VEGFR-TKIs and safety compared with prior VEGFR-TKI treatment. Jpn. J. Clin. Oncol. 44, 479–485 (2014).
Yao, J. C. et al. Everolimus for advanced pancreatic neuroendocrine tumors. N. Engl. J. Med. 364, 514–523 (2011).
Sasaki, M. et al. Anorectal mucinous adenocarcinoma associated with latent perianal Paget’s disease. Am. J. Gastroenterol. 85, 199–202 (1990).
Yim, J. H., Wick, M. R., Philpott, G. W., Norton, J. A. & Doherty, G. M. Underlying pathology in mammary Paget’s disease. Ann. Surg. Oncol. 4, 287–292 (1997).
Morandi, L. et al. Intraepidermal cells of Paget’s carcinoma of the breast can be genetically different from those of the underlying carcinoma. Hum. Pathol. 34, 1321–1330 (2003).
Katz, Y. et al. Musashi proteins are post-transcriptional regulators of the epithelial-luminal cell state. Elife 3, e03915 (2014).
Espersen, M. L., Olsen, J., Linnemann, D., Hogdall, E. & Troelsen, J. T. Clinical implications of intestinal stem cell markers in colorectal cancer. Clin. Colorectal Cancer 14, 63–71 (2015).
Fan, J. et al. Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis. Nat. Methods 13, 241–244 (2016).
Guerrero-Juarez, C. F. et al. Single-cell analysis reveals fibroblast heterogeneity and myeloid-derived adipocyte progenitors in murine skin wounds. Nat. Commun. 10, 650 (2019).
Cheng, J. B. et al. Transcriptional programming of normal and inflamed human epidermis at single-cell resolution. Cell Rep. 25, 871–883 (2018).
Qiu, X. et al. Single-cell mRNA quantification and differential analysis with Census. Nat. Methods 14, 309–315 (2017).
Trapnell, C. et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat. Biotechnol. 32, 381–386 (2014).
Qiu, X. et al. Reversed graph embedding resolves complex single-cell trajectories. Nat. Methods 14, 979–982 (2017).
Jin, S., MacLean, A. L., Peng, T. & Nie, Q. scEpath: energy landscape-based inference of transition probabilities and cellular trajectories from single-cell transcriptomic data. Bioinformatics 34, 2077–2086 (2018).
Zhang, H. M. et al. AnimalTFDB 2.0: a resource for expression, prediction and functional study of animal transcription factors. Nucleic Acids Res. 43, D76–D81 (2015).
La Manno, G. et al. RNA velocity of single cells. Nature 560, 494–498 (2018).
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).
Chen, E. Y. et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinform. 14, 128 (2013).
Wang, S. et al. Transformation of the intestinal epithelium by the MSI2 RNA-binding protein. Nat. Commun. 6, 6517 (2015).
Ma, X. et al. Msi2 maintains quiescent state of hair follicle stem cells by directly repressing the Hh signaling pathway. J. Invest. Dermatol. 137, 1015–1024 (2017).
Z. Yu is supported by the National Natural Science Foundation of China (81772984, 81572614); Beijing Nature Foundation Grant (5162018); the Major Project for Cultivation Technology (2016ZX08008001, 2014ZX08008001); Basic Research Program (2019TC227, 2019TC088); and SKLB Open Grant (2020SKLAB6–18). B.A. is supported by a NIH Grant R01AR42288. M.V.P. is supported by a Pew Charitable Trust Grant, NIH Grants R01-AR067273 and R01-AR069653, NSF Grant DMS1763272, and Simons Foundation Grant (594598, Q.N.). This work is also supported by NIH grants U01-AR073159 (to Q.N., M.V.P., and X.D.) and U54-CA217378 (to Arthur Lander, John Lowengrub, and Marian Waterman). Q.N. is also supported by NSF grants DMS176372 and DMS1562176, the Jayne Koskinas Ted Giovanis Foundation for Health and Policy jointly with the Breast Cancer Research Foundation, and NSF-Simons Foundation (594598). C.F.G.-J. is supported by UC Irvine Chancellor’s ADVANCE Postdoctoral Fellowship Program, NSF-Simons Postdoctoral Fellowship, NSF Grant DMS1763272, and Simons Foundation Grant (594598, Q.N.) and a kind gift from the Howard Hughes Medical Institute Hanna H. Gray Postdoctoral Fellowship Program. G.Z. is supported by the Key Project of the National Natural Science Foundation of China (81830093) and the CAMS Innovation Fund for Medical Sciences (CIFMS; No. 2019-I2M-1-003).
The authors declare no competing interests.
About this article
Cite this article
Song, Y., Guerrero-Juarez, C.F., Chen, Z. et al. The Msi1-mTOR pathway drives the pathogenesis of mammary and extramammary Paget’s disease. Cell Res 30, 854–872 (2020). https://doi.org/10.1038/s41422-020-0334-5
Molecular Therapy - Oncolytics (2021)
EMBO Molecular Medicine (2021)