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Functional Genetic Characterization of Mammary and Cancer Stem Cells

Benjamin Spike

Appointment Period: 2009-2012, Grant Years: [25,26,27]

Benjamin SpikeMy work has aimed to identify stem cell signaling networks co-opted in breast cancer. The work identified a strong correlation between stem cell-like expression patterns and loss of p53 tumor suppressor function in the most aggressive forms of breast and lung cancers. This finding supports the idea that p53 loss may facilitate the reprogramming of cells to primitive cell states in vivo during progression. Such tumor associated reprogramming provides a potential explanation for the long recognized parallels between embryogenesis and tumorigenesis. Alternatively, as first suggested in the mid-1800s by Conheim and Virchow, embryonic features could derive from cells that persist and retain embryonic characteristics into adulthood but which remain hidden amongst the more plentiful adult somatic cells until they emerge as a cancerous outgrowth. To address these possibilities, we recently isolated embryonic mouse mammary stem cells and characterized gene expression programs found in these cells and their neighbors. We discovered that the embryonic programs are also evident in various subtypes of breast cancers in humans. In our subsequent efforts to leverage this information toward elucidation of specific functional embryonic mammary stem cell markers that could serve as biomarkers and stem cell directed therapeutic targets, we have uncovered a role for soluble Cripto and its surface receptor GRP78 in normal mammary stem cell maintenance.  We have subsequently determined that GRP78 is present on the surface of both fetal and adult mammary stem cells and that both are sensitive to Cripto pathway activation and antagonism.  Importantly, both proteins have been previously implicated in cancer and may be induced by stresses such as hypoxia or nutrient deprivation that often prevail in the tumor microenvironment. This provides yet another potential mechanism contributing to the resurrection of stem-like cellular states in cancer.

Because of their paracrine signaling potential and stress sensitivity, these mammary stem cell factors and others implicated in our studies above, also point to cellular heterogeneity, and consequently, a complex set of microenvironment cues and responses as defining features of the mammary stem cell state. Without deconvolution, however, this same heterogeneity represents a confounding factor in stem cell analyses.  To address this problem and better understand the role played by cellular heterogeneity both within the stem cell population and in the stem cell microenvironment, we and our collaborators have begun to develop the tools and obtain the data needed for a high resolution, systems level view of mammary stem cell identity using single cell RNA-Sequencing.  These studies promise to provide the first complete RNA sequence analysis of pure embryonic mammary stem cells, and are also critical for obtaining refined signatures to identify and molecularly tailor therapies for breast cancers that share targetable features of these cells.  The support of the Growth Regulation and Oncogenesis Training Grant has allowed me to develop the complex set of laboratory and computational skills needed to carry out these challenging studies and to effectively communicate with a diverse network of essential collaborators.

PUBLICATIONS (resulting from this training)

Spike BT, Kelber JA, Booker E, Kalathur M, Rodewald LW,  Lipianskaya J, La J, He M, Wright  T Klemke R, Wahl GM, Gray PC.  A developmentally conserved role for Cripto/GRP78 self-renewal signaling in fetal and adult mammary stem cells. Genes and Development. Submitted.

Spike BT*, Engle D*, Lin J*, Cheung S, Wahl GM. (*Lin, Engle, Spike are co-equal first authors) A mammary stem cell population identified and characterized in late embryogenesis reveals similarities to human breast cancer. (2012) Cell Stem Cell.  10:183-197. PMID: 22305568. PMC3277444

Mizuno H*, Spike BT*, Wahl GM, Levine AJ. Inactivation of p53 in breast cancers correlates with stem cell transcriptional signatures. Proc Natl Acad Sci USA. (2010) 107:22745-50. PMID: 21149740; PMCID: PMC3012457. *co-equal contribution

Spike BT, Wahl GM. p53, Stem Cells, and Reprogramming: Tumor Suppression beyond Guarding the Genome. Genes Cancer. (2011) 2:404-19. PMID: 21779509; PMCID: PMC3135646.

Spike BT, Engle D, Lin J, Cheung S, Wahl GM. A mammary stem cell population identified and characterized in late embryogenesis reveals similarities to human breast cancer. (2012) Cell Stem Cell.  10:183-197. PMID: 22305568.

Jennifer Lin*, Dannielle Engle*, Benjamin Spike*, Samantha Cheung, and Geoffrey M. Wahl (*Lin, Engle, Spike are co-equal first authors) Mouse mammary stem cells identified late in embryogenesis reveal gene signatures in human breast cancer. In revision to Nature

Other publications (prior to Training Grant appointment):

Liu H, Knabb JR, Spike BT, Macleod KF. Elevated poly-(ADP-ribose)-polymerase activity sensitizes retinoblastoma-deficient cells to DNA damage-induced necrosis. Mol Cancer Res. 2009 Jul;7(7):1099-109. Epub 2009 Jul 7. PMID: 19584263.

Dirlam A, Spike BT, Macleod KF. Deregulated E2f-2 underlies cell cycle and maturation defects in retinoblastoma null erythroblasts. Mol Cell Biol. 2007 Dec;27(24):8713-28. Epub 2007 Oct 8. PMID: 17923680; PMC2169398.

Spike BT, Macleod KF. Effects of hypoxia on heterotypic macrophage interactions. Cell Cycle. 2007 Nov 1;6(21):2620-4. Epub 2007 Aug 13. Review. PMID: 17873523.

Tracy K, Dibling BC, Spike BT, Knabb JR, Schumacker P, Macleod KF. BNIP3 is an RB/E2F target gene required for hypoxia-induced autophagy. Mol Cell Biol. 2007 Sep;27(17):6229-42. Epub 2007 Jun 18. PMID: 17576813; PMC1952167.

Spike BT, Dibling BC, Macleod KF. Hypoxic stress underlies defects in erythroblast islands in the Rb-null mouse. Blood. 2007 Sep 15;110(6):2173-81. Epub 2007 Jun 8. PMID: 17557897; PMC1976369.

Diwan A, Koesters AG, Odley AM, Pushkaran S, Baines CP, Spike BT, Daria D, Jegga AG, Geiger H, Aronow BJ, Molkentin JD, Macleod KF, Kalfa TA, Dorn GW 2nd. Unrestrained erythroblast development in Nix-/- mice reveals a mechanism for apoptotic modulation of erythropoiesis. Proc Natl Acad Sci U S A. 2007 Apr 17;104(16):6794-9. Epub 2007 Apr 9. PMID: 17420462; PMC1849960.

Spike BT, Macleod KF. The Rb tumor suppressor in stress responses and hematopoietic homeostasis. Cell Cycle. 2005 Jan;4(1):42-5. Epub 2005 Jan 29. Review. PMID: 15611658.

Spike BT, Dirlam A, Dibling BC, Marvin J, Williams BO, Jacks T, Macleod KF. The Rb tumor suppressor is required for stress erythropoiesis. EMBO J. 2004 Oct 27;23(21):4319-29. Epub 2004 Sep 30. PMID: 15457215; PMC524396.

Rowley AH, Shulman ST, Spike BT, Mask CA, Baker SC. Oligoclonal IgA response in the vascular wall in acute Kawasaki disease. J Immunol. 2001 Jan 15;166(2):1334-43. PMID: 11145718.

Aladjem MI, Spike BT, Rodewald LW, Hope TJ, Klemm M, Jaenisch R, Wahl GM. ES cells do not activate p53-dependent stress responses and undergo p53-independent apoptosis in response to DNA damage. Curr Biol. 1998 Jan 29;8(3):145-55. PMID: 9443911.