Craniofacial anomalies (CFA) are the most frequent human congenital disease and a major cause of infant mortality and childhood morbidity. Although CFA appear to arise from a combination of genetic factors and environmental influences, the underlying gene defects and pathomechanisms for the majority of CFA are currently unknown. Here we reveal an unknown role for the E3 ubiquitin ligase Wwp2 in regulating craniofacial patterning. Mice deficient for Wwp2 develop malformations of the craniofacial region. Wwp2 is present in cartilage where its expression is controlled by Sox9. Our studies demonstrate that Wwp2 influences craniofacial patterning through its interactions with Goosecoid (Gsc), a paired-like homeobox transcription factor that plays an important role in craniofacial development. We show that Wwp2 associated Gsc is a transcriptional activator of the key cartilage regulatory protein Sox6. Wwp2 interacts with Gsc to facilitate its mono-ubiquitination, a post-translational modification required for optimal transcriptional activation of Gsc. Our results identify the first physiological pathway regulated by Wwp2 in vivo as well as identify a unique non-proteolytic mechanism through which the Wwp2 controls craniofacial development.

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Aberrant activation of fibroblast growth factor receptors (FGFRs) contributes to breast cancer growth, progression and therapeutic resistance. Due to the complex nature of the FGF/FGFR axis, and the numerous effects of FGFR activation on tumor cells and the surrounding microenvironment, the specific mechanisms through which aberrant FGFR activity contributes to breast cancer are not completely understood. We show here that FGFR activation induces accumulation of hyaluronan (HA) within the extracellular matrix (ECM) and that blocking HA synthesis decreases proliferation, migration and therapeutic resistance. Furthermore, FGFR-mediated HA accumulation requires activation of the signal transducer and activator of transcription 3 (STAT3) pathway, which regulates expression of hyaluronan synthase 2 (HAS2) and subsequent HA synthesis. Using a novel in vivo model of FGFR-dependent tumor growth, we demonstrate that STAT3 inhibition decreases both FGFR-driven tumor growth and HA levels within the tumor. Finally, our results suggest that combinatorial therapies inhibiting both FGFR activity and HA synthesis is more effective than targeting either pathway alone and may be a relevant therapeutic approach for breast cancers associated with high levels of FGFR activity. In conclusion, these studies indicate a novel targetable mechanism through which FGFR activation in breast cancer cells induces a pro-tumorigenic microenvironment.

Macrophages are critical mediators of inflammation and important regulators of developmental processes. As a key phagocytic cell type, macrophages evolved as part of the innate immune system to engulf and process cell debris and pathogens. Macrophages produce factors that act directly on their microenvironment and also bridge innate immune responses to the adaptive immune system. Resident macrophages are important for acting as sensors for tissue damage and maintaining tissue homeostasis. It is now well-established that macrophages are an integral component of the breast tumor microenvironment, where they contribute to tumor growth and progression, likely through many of the mechanisms that are utilized during normal wound healing responses. Because macrophages contribute to normal mammary gland development and breast cancer growth and progression, this review will discuss both resident mammary gland macrophages and tumor-associated macrophages with an emphasis on describing how macrophages interact with their surrounding environment during normal development and in the context of cancer.

BackgroundEstrogen and progesterone are potent breast mitogens. In addition to steroid hormones, multiple signaling pathways input to estrogen receptor (ER) and progesterone receptor (PR) actions via posttranslational events. Protein kinases commonly activated in breast cancers phosphorylate steroid hormone receptors (SRs) and profoundly impact their activities.MethodsTo better understand the role of modified PRs in breast cancer, we measured total and phospho-Ser294 PRs in 209 human breast tumors represented on 2754 individual tissue spots within a tissue microarray and assayed the regulation of this site in human tumor explants cultured ex vivo. To complement this analysis, we assayed PR target gene regulation in T47D luminal breast cancer models following treatment with progestin (promegestone; R5020) and antiprogestins (mifepristone, onapristone, or aglepristone) in conditions under which the receptor is regulated by Lys388 SUMOylation (K388 intact) or is SUMO-deficient (via K388R mutation to mimic persistent Ser294 phosphorylation). Selected phospho-PR-driven target genes were validated by qRT-PCR and following RUNX2 shRNA knockdown in breast cancer cell lines. Primary and secondary mammosphere assays were performed to implicate phospho-Ser294 PRs, epidermal growth factor signaling, and RUNX2 in breast cancer stem cell biology.ResultsPhospho-Ser294 PR species were abundant in a majority (54%) of luminal breast tumors, and PR promoter selectivity was exquisitely sensitive to posttranslational modifications. Phospho-PR expression and target gene programs were significantly associated with invasive lobular carcinoma (ILC). Consistent with our finding that activated phospho-PRs undergo rapid ligand-dependent turnover, unique phospho-PR gene signatures were most prevalent in breast tumors clinically designated as PR-low to PR-null (luminal B) and included gene sets associated with cancer stem cell biology (HER2, PAX2, AHR, AR, RUNX). Validation studies demonstrated a requirement for RUNX2 in the regulation of selected phospho-PR target genes (SLC37A2). In vitro mammosphere formation assays support a role for phospho-Ser294-PRs via growth factor (EGF) signaling as well as RUNX2 as potent drivers of breast cancer stem cell fate.ConclusionsWe conclude that PR Ser294 phosphorylation is a common event in breast cancer progression that is required to maintain breast cancer stem cell fate, in part via cooperation with growth factor-initiated signaling pathways and key phospho-PR target genes including SLC37A2 and RUNX2. Clinical measurement of phosphorylated PRs should be considered a useful marker of breast tumor stem cell potential. Alternatively, unique phospho-PR target gene sets may provide useful tools with which to identify patients likely to respond to selective PR modulators that block PR Ser294 phosphorylation as part of rational combination (i.e., with antiestrogens) endocrine therapies designed to durably block breast cancer recurrence.Electronic supplementary materialThe online version of this article (doi:...

Coordination between osteoblasts and osteoclasts is required for bone health and homeostasis. Here we show that mice deficient in SMURF2 have severe osteoporosis in vivo. This low bone mass phenotype is accompanied by a pronounced increase in osteoclast numbers, although Smurf2-deficient osteoclasts have no intrinsic alterations in activity. Smurf2-deficient osteoblasts display increased expression of RANKL, the central osteoclastogenic cytokine. Mechanistically, SMURF2 regulates RANKL expression by disrupting the interaction between SMAD3 and vitamin D receptor by altering SMAD3 ubiquitination. Selective deletion of Smurf2 in the osteoblast lineage recapitulates the phenotype of germline Smurf2-deficient mice, indicating that SMURF2 regulates osteoblast-dependent osteoclast activity rather than directly affecting the osteoclast. Our results reveal SMURF2 as an important regulator of the critical communication between osteoblasts and osteoclasts. Furthermore, the bone mass phenotype in Smurf2- and Smurf1-deficient mice is opposite, indicating that SMURF2 has a non-overlapping and, in some respects, opposite function to SMURF1.

Mice deficient in Schnurri-3 (SHN3; also known as HIVEP3) display increased bone formation, but harnessing this observation for therapeutic benefit requires an improved understanding of how SHN3 functions in osteoblasts. Here we identified SHN3 as a dampener of ERK activity that functions in part downstream of WNT signaling in osteoblasts. A D-domain motif within SHN3 mediated the interaction with and inhibition of ERK activity and osteoblast differentiation, and knockin of a mutation in Shn3 that abolishes this interaction resulted in aberrant ERK activation and consequent osteoblast hyperactivity in vivo. Additionally, in vivo genetic interaction studies demonstrated that crossing to Lrp5 -/-mice partially rescued the osteosclerotic phenotype of Shn3 -/-mice; mechanistically, this corresponded to the ability of SHN3 to inhibit ERK-mediated suppression of GSK3β. Inducible knockdown of Shn3 in adult mice resulted in a high-bone mass phenotype, providing evidence that transient blockade of these pathways in adults holds promise as a therapy for osteoporosis. IntroductionAdult bone mass reflects the balance between production of bone by osteoblasts and resorption of bone by osteoclasts, and disturbance of this balance results in bone pathology such as osteoporosis. As osteoblasts have a limited ability to directly perceive the thickness of the bone they overlay, they rely on the ability to integrate extracellular cues to appropriately adjust the rate of bone formation. One such extracellular cue, the WNT/ β-catenin pathway, is well established as a positive regulator of osteoblast differentiation, appearing to divert early progenitors away from a chondrocyte fate into becoming osteoblasts (1, 2). Deletion or inhibition of individual WNT signaling components results in dysregulation of osteoblast activity and differentiation and, by extension, altered anabolic bone formation in mice (3). Under basal conditions, cytosolic β-catenin undergoes constitutive ubiquitination and proteasome-dependent degradation via the destruction complex that consists of adenomatous polyposis coli (APS), Axin, CK1α, and glycogen synthase kinase 3-β (GSK3β) (4). A subset of ligands of the WNT family binds to the 7-pass transmembrane receptor Frizzled and the single-pass low-density lipoprotein receptor-related protein 5 (LRP5) or LRP6. This inhibits the constitutive phosphorylation of β-catenin by GSK3β, which in turn prevents β-catenin ubiquitination and proteasome-dependent degradation. Subsequently, β-catenin translocates into the nucleus to activate its transcrip-

Mutations in human FYVE, RhoGEF, and PH domain-containing 1 (FGD1) cause faciogenital dysplasia (FGDY; also known as Aarskog syndrome), an X-linked disorder that affects multiple skeletal structures. FGD1 encodes a guanine nucleotide exchange factor (GEF) that specifically activates the Rho GTPase CDC42. However, the mechanisms by which mutations in FGD1 affect skeletal development are unknown. Here, we describe what we believe to be a novel signaling pathway in osteoblasts initiated by FGD1 that involves the MAP3K mixed-lineage kinase 3 (MLK3). We observed that MLK3 functions downstream of FGD1 to regulate ERK and p38 MAPK, which in turn phosphorylate and activate the master regulator of osteoblast differentiation, Runx2. Mutations in FGD1 found in individuals with FGDY ablated its ability to activate MLK3. Consistent with our description of this pathway and the phenotype of patients with FGD1 mutations, mice with a targeted deletion of Mlk3 displayed multiple skeletal defects, including dental abnormalities, deficient calvarial mineralization, and reduced bone mass. Furthermore, mice with knockin of a mutant Mlk3 allele that is resistant to activation by FGD1/CDC42 displayed similar skeletal defects, demonstrating that activation of MLK3 specifically by FGD1/CDC42 is important for skeletal mineralization. Thus, our results provide a putative biochemical mechanism for the skeletal defects in human FGDY and suggest that modulating MAPK signaling may benefit these patients. IntroductionIn 1970, Aarskog described an X-linked recessive syndrome characterized by an upturned nose, short stature, multiple dental defects, delayed skeletal age, and multiple bone malformations (1, 2). Later work confirmed these observations, naming the disorder faciogenital dysplasia (FGDY) or Aarskog-Scott syndrome and identified the gene mutated as FYVE, RhoGEF, and PH domain-containing 1 (FGD1) (3). FGD1 encodes a member of the guanine nucleotide exchange factor (GEF) family, which catalyses the exchange of GDP for GTP and promotes the activity of Rho family GTPases (3-6). More than 16 distinct FGD1 mutations have been reported to cosegregate with FGDY. These mutations include deletions and premature truncations, implying that a loss of FGD1 function underlies FGDY (6, 7). Despite these insights into the genetic basis of FGDY, it remains unclear how FGD1 affects bone development.Expression analysis demonstrates that FGD1 is highly expressed in osteoblasts, suggesting that FGD1 signaling plays a critical role in osteoblast differentiation and function (8). Microinjection studies show that FGD1 specifically activates CDC42, a member of the Rho (Ras homology) family of GTPase proteins (4, 5). A