Faculty Bibliography

INTRODUCTION: The mouse mammary gland provides a powerful model system for studying processes involved in epithelial tissue development. Although markers that enrich for mammary stem cells and progenitors have been identified, our understanding of the mammary developmental hierarchy remains incomplete. METHODS: We used the MMTV promoter linked to the reverse tetracycline transactivator to induce H2BGFP expression in the mouse mammary gland. Mammary epithelial cells (MECs) from virgin mice were sorted by flow cytometry for expression of the mammary stem cell/progenitor markers CD24 and CD29, and H2BGFP. Sorted populations were analyzed for in vivo repopulation ability, expression of mammary lineage markers, and differential gene expression. RESULTS: The reconstituting activity of CD24(+)/CD29(+) cells in cleared fat pad transplantation assays was not distinguished in GFP(+) compared to GFP(-) subpopulations. However, within the CD24(+)/CD29(lo) luminal progenitor-enriched population, H2BGFP(+), but not H2BGFP(-), MECs formed mammary structures in transplantation assays; moreover, this activity was dramatically enhanced in pregnant recipients. These outgrowths contained luminal and myoepithelial mammary lineages and produced milk, but lacked the capacity for serial transplantation. Transcriptional microarray analysis revealed that H2BGFP(+)/CD24(+)/CD29(lo) MECs are distinct from H2BGFP(-)/CD24(+)/CD29(lo) MECs and enriched for gene expression signatures with both the stem cell (CD24(+)/CD29(+)) and luminal progenitor (CD24(+)/CD29(lo)/CD61(+)) compartments. CONCLUSIONS: We have identified a population of MECs containing pregnancy-activated multipotent progenitors that are present in the virgin mammary gland and contribute to the expansion of the mammary gland during pregnancy.

OBJECTIVE: The receptor-like protein-tyrosine phosphatase (PTP) CD148 and the nontransmembrane PTP1-B have been shown to be net positive regulators of Src family kinases in platelets. In the present study, we compared the relative contributions of these PTPs in platelet activation by the major glycoprotein, glycoprotein VI, alpha(IIb)beta(3), and C-type lectin-like receptor 2 (CLEC-2). METHODS AND RESULTS: PTP-1B-deficient mouse platelets responded normally to the glycoprotein VI-specific agonist collagen-related peptide and antibody-mediated CLEC-2 activation. However, they exhibited a marginal reduction in alpha(IIb)beta(3)-mediated Src family kinase activation and tyrosine phosphorylation. In contrast, CD148-deficient platelets exhibited a dramatic reduction in activation by glycoprotein VI and alpha(IIb)beta(3) and a marginal reduction in response to activation by CLEC-2, which was further enhanced in the absence of PTP-1B. These defects were associated with reduced activation of Src family kinase and spleen tyrosine kinase, suggesting a causal relationship. Under arteriolar flow conditions, there was defective aggregate formation in the absence of PTP-1B and, to a greater extent, CD148 and a severe abrogation of both adhesion and aggregation in the absence of both PTPs. CONCLUSIONS: Findings from this study demonstrate that CD148 plays a dominant role in activating Src family kinases in platelets relative to PTP-1B. Both PTPs are required for optimal platelet activation and aggregate formation under high arterial shear rates.

Macrophages migrate to sites of insult during normal inflammatory responses. Integrins guide such migration, but the transmission of signals from integrins into the requisite cytoskeletal changes is poorly understood. We have discovered that the hematopoietic adaptor protein Skap2 is necessary for macrophage migration, chemotaxis, global actin reorganization and local actin reorganization upon integrin engagement. Binding of phosphatidylinositol [3,4,5]-triphosphate to the Skap2 pleckstrin-homology (PH) domain, which relieves its conformational auto-inhibition, is critical for this integrin-driven cytoskeletal response. Skap2 enables integrin-induced tyrosyl phosphorylation of Src-family kinases (SFKs), Adap, and Sirpalpha, establishing their roles as signaling partners in this process. Furthermore, macrophages lacking functional Sirpalpha unexpectedly have impaired local integrin-induced responses identical to those of Skap2(-/-) macrophages, and Skap2 requires Sirpalpha for its recruitment to engaged integrins and for coordinating downstream actin rearrangement. By revealing the positive-regulatory role of Sirpalpha in a Skap2-mediated mechanism connecting integrin engagement with cytoskeletal rearrangement, these data demonstrate that Sirpalpha is not exclusively immunoinhibitory, and illuminate previously unexplained observations implicating Skap2 and Sirpalpha in mouse models of inflammatory disease.

The pentaspan membrane glycoprotein CD133 marks lineage-specific cancer progenitor cells and is associated with poor prognosis in a number of tumor types. Despite its utility as a cancer progenitor cell marker, CD133 protein regulation and molecular function remain poorly understood. We find that the deacetylase HDAC6 physically associates with CD133 to negatively regulate CD133 trafficking down the endosomal-lysosomal pathway for degradation. We further demonstrate that CD133, HDAC6, and the central molecule of the canonical Wnt signaling pathway, beta-catenin, can physically associate as a ternary complex. This association stabilizes beta-catenin via HDAC6 deacetylase activity, which leads to activation of beta-catenin signaling targets. Downregulation of either CD133 or HDAC6 results in increased beta-catenin acetylation and degradation, which correlates with decreased proliferation in vitro and tumor xenograft growth in vivo. Given that CD133 marks progenitor cells in a wide range of cancers, targeting CD133 may be a means to treat multiple cancer types.

Noonan syndrome (NS) is a relatively common autosomal dominant disorder characterized by congenital heart defects, short stature, and facial dysmorphia. NS is caused by germ line mutations in several components of the RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway, including both kinase-activating and kinase-impaired alleles of RAF1 ( approximately 3 to 5%), which encodes a serine-threonine kinase for MEK1/2. To investigate how kinase-impaired RAF1 mutants cause NS, we generated knock-in mice expressing Raf1(D486N). Raf1(D486N/+) (here D486N/+) female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1(D486N)-expressing cells compared with controls. RAF1(D486N), as well as other kinase-impaired RAF1 mutants, showed increased heterodimerization with BRAF, which was necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also required heterodimerization to enhance MEK/ERK activation. Our results suggest that an increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations.

Cell polarity plays a key role in development and is disrupted in tumors, yet the molecules and mechanisms that regulate polarity remain poorly defined. We found that the scaffolding adaptor GAB1 interacts with two polarity proteins, PAR1 and PAR3. GAB1 binds PAR1 and enhances its kinase activity. GAB1 brings PAR1 and PAR3 into a transient complex, stimulating PAR3 phosphorylation by PAR1. GAB1 and PAR6 bind the PAR3 PDZ1 domain and thereby compete for PAR3 binding. Consequently, GAB1 depletion causes PAR3 hypophosphorylation and increases PAR3/PAR6 complex formation, resulting in accelerated and enhanced tight junction formation, increased transepithelial resistance, and lateral domain shortening. Conversely, GAB1 overexpression, in a PAR1/PAR3-dependent manner, disrupts epithelial apical-basal polarity, promotes multilumen cyst formation, and enhances growth factor-induced epithelial cell scattering. Our results identify GAB1 as a negative regulator of epithelial cell polarity that functions as a scaffold for modulating PAR protein complexes on the lateral membrane.

The protein-tyrosine phosphatase Shp1 negatively regulates insulin action on glucose homeostasis in liver and muscle, but its potential role in obesity-linked insulin resistance has not been examined. To investigate the role of Shp1 in hepatic insulin resistance, we generated hepatocyte-specific Shp1 knockout mice (Ptpn6(H-KO)), which were subjected to extensive metabolic monitoring throughout an 8-week standard chow diet (SD) or high-fat diet (HFD) feeding. We report for the first time that Shp1 expression is upregulated in metabolic tissues of HFD-fed obese mice. When compared with their Shp1-expressing Ptpn6(f/f) littermates, Ptpn6(H-KO) mice exhibited significantly lowered fasting glycemia and heightened hepatic insulin sensitivity. After HFD feeding, Ptpn6(H-KO) mice developed comparable levels of obesity as Ptpn6(f/f) mice, but they were remarkably protected from liver insulin resistance, as revealed by euglycemic clamps and hepatic insulin signaling determinations. Although Ptpn6(H-KO) mice still acquired diet-induced peripheral insulin resistance, they were less hyperinsulinemic during a glucose tolerance test because of reduced insulin secretion. Ptpn6(H-KO) mice also exhibited increased insulin clearance in line with enhanced CC1 tyrosine phosphorylation in liver. These results show that hepatocyte Shp1 plays a critical role in the development of hepatic insulin resistance and represents a novel therapeutic target for obesity-linked diabetes.

The Runx1 transcription factor is post-translationally modified by seryl/threonyl phosphorylation, acetylation, and methylation that control its interactions with transcription factor partners and epigenetic coregulators. In this issue of Genes & Development, Huang and colleagues (pp. 1587-1601) describe how the regulation of Runx1 tyrosyl phosphorylation by Src family kinases and the Shp2 phosphatase toggle Runx1's interactions between different coregulatory molecules.

Noonan syndrome (NS), a genetic disease caused in half of cases by activating mutations of the tyrosine phosphatase SHP2 (PTPN11), is characterized by congenital cardiopathies, facial dysmorphic features, and short stature. How mutated SHP2 induces growth retardation remains poorly understood. We report here that early postnatal growth delay is associated with low levels of insulin-like growth factor 1 (IGF-1) in a mouse model of NS expressing the D61G mutant of SHP2. Conversely, inhibition of SHP2 expression in growth hormone (GH)-responsive cell lines results in increased IGF-1 release upon GH stimulation. SHP2-deficient cells display decreased ERK1/2 phosphorylation and rat sarcoma (RAS) activation in response to GH, whereas expression of NS-associated SHP2 mutants results in ERK1/2 hyperactivation in vitro and in vivo. RAS/ERK1/2 inhibition in SHP2-deficient cells correlates with impaired dephosphorylation of the adaptor Grb2-associated binder-1 (GAB1) on its RAS GTPase-activating protein (RASGAP) binding sites and is rescued by interfering with RASGAP recruitment or function. We demonstrate that inhibition of ERK1/2 activation results in an increase of IGF-1 levels in vitro and in vivo, which is associated with significant growth improvement in NS mice. In conclusion, NS-causing SHP2 mutants inhibit GH-induced IGF-1 release through RAS/ERK1/2 hyperactivation, a mechanism that could contribute to growth retardation. This finding suggests that, in addition to its previously shown beneficial effect on NS-linked cardiac and craniofacial defects, RAS/ERK1/2 modulation could also alleviate the short stature phenotype in NS caused by PTPN11 mutations.

In recent years, the increasing awareness that somatic mutations and other genetic aberrations drive human malignancies has led us within reach of personalized cancer medicine (PCM). The implementation of PCM is based on the following premises: genetic aberrations exist in human malignancies; a subset of these aberrations drive oncogenesis and tumor biology; these aberrations are actionable (defined as having the potential to affect management recommendations based on diagnostic, prognostic, and/or predictive implications); and there are highly specific anticancer agents available that effectively modulate these targets. This article highlights the technology underlying cancer genomics and examines the early results of genome sequencing and the challenges met in the discovery of new genetic aberrations. Finally, drawing from experiences gained in a feasibility study of somatic mutation genotyping and targeted exome sequencing led by Princess Margaret Hospital-University Health Network and the Ontario Institute for Cancer Research, the processes, challenges, and issues involved in the translation of cancer genomics to the clinic are discussed.