Dorothy Hu

XBP1 promotes triple-negative breast cancer by controlling the HIF1α pathway

Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE1 (ref. 2) and its substrate XBP1 (ref. 3). Previous studies report UPR activation in various human tumours, but the role of XBP1 in cancer progression in mammary epithelial cells is largely unknown. Triple-negative breast cancer (TNBC)—a form of breast cancer in which tumour cells do not express the genes for oestrogen receptor, progesterone receptor and HER2 (also called ERBB2 or NEU)—is a highly aggressive malignancy with limited treatment options. Here we report that XBP1 is activated in TNBC and has a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumour growth and tumour relapse and reduced the CD44highCD24low population. Hypoxia-inducing factor 1α (HIF1α) is known to be hyperactivated in TNBCs. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1α that regulates the expression of HIF1α targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1α and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and indicate that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.

The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice

Nearly every extracellular ligand that has been found to play a role in regulating bone biology acts, at least in part, through MAPK pathways. Nevertheless, much remains to be learned about the contribution of MAPKs to osteoblast biology in vivo. Here we report that the p38 MAPK pathway is required for normal skeletogenesis in mice, as mice with deletion of any of the MAPK pathway member-encoding genes MAPK kinase 3 (Mkk3), Mkk6, p38a, or p38b displayed profoundly reduced bone mass secondary to defective osteoblast differentiation. Among the MAPK kinase kinase (MAP3K) family, we identified TGF-beta-activated kinase 1 (TAK1; also known as MAP3K7) as the critical activator upstream of p38 in osteoblasts. Osteoblast-specific deletion of Tak1 resulted in clavicular hypoplasia and delayed fontanelle fusion, a phenotype similar to the cleidocranial dysplasia observed in humans haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2). Mechanistic analysis revealed that the TAK1-MKK3/6-p38 MAPK axis phosphorylated Runx2, promoting its association with the coactivator CREB-binding protein (CBP), which was required to regulate osteoblast genetic programs. These findings reveal an in vivo function for p38beta and establish that MAPK signaling is essential for bone formation in vivo. These results also suggest that selective p38beta agonists may represent attractive therapeutic agents to prevent bone loss associated with osteoporosis and aging.

The E3 ubiquitin ligase Wwp2 regulates craniofacial development through mono-ubiquitylation of Goosecoid

Craniofacial anomalies (CFAs) are the most frequently occurring human congenital disease, and a major cause of infant mortality and childhood morbidity. Although CFAs seems to arise from a combination of genetic factors and environmental influences, the underlying gene defects and pathophysiological mechanisms for most CFAs are currently unknown. Here we reveal a role for the E3 ubiquitin ligase Wwp2 in regulating craniofacial patterning. Mice deficient in 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 has 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-ubiquitylation, a post-translational modification required for optimal transcriptional activation of Gsc. Our results identify for the first time a physiological pathway regulated by Wwp2 in vivo, and also a unique non-proteolytic mechanism through which Wwp2 controls craniofacial development.

Cdh1 Regulates Osteoblast Function through an APC/C-Independent Modulation of Smurf1

The APC/Cdh1 E3 ubiquitin ligase plays an essential role in both mitotic exit and G1/S transition by targeting key cell-cycle regulators for destruction. There is mounting evidence indicating that Cdh1 has other functions in addition to cell-cycle regulation. However, it remains unclear whether these additional functions depend on its E3 ligase activity. Here, we report that Cdh1, but not Cdc20, promotes the E3 ligase activity of Smurf1. This is mediated by disruption of an autoinhibitory Smurf1 homodimer and is independent of APC/Cdh1 E3 ligase activity. As a result, depletion of Cdh1 leads to reduced Smurf1 activity and subsequent activation of multiple downstream targets, including the MEKK2 signaling pathway, inducing osteoblast differentiation. Our studies uncover a cell-cycle-independent function of Cdh1, establishing Cdh1 as an upstream component that governs Smurf1 activity. They further suggest that modulation of Cdh1 is a potential therapeutic option for treatment of osteoporosis.

MLK3 regulates bone development downstream of the faciogenital dysplasia protein FGD1 in mice

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.

Regulation of Sclerostin Expression in Multiple Myeloma by Dkk‐1; A Potential Therapeutic Strategy for Myeloma Bone Disease

Sclerostin is a potent inhibitor of osteoblastogenesis. Interestingly, newly diagnosed multiple myeloma (MM) patients have high levels of circulating sclerostin that correlate with disease stage and fractures. However, the source and impact of sclerostin in MM remains to be defined. Our goal was to determine the role of sclerostin in the biology of MM and its bone microenvironment as well as investigate the effect of targeting sclerostin with a neutralizing antibody (scl‐Ab) in MM bone disease. Here we confirm increased sclerostin levels in MM compared with precursor disease states like monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM. Furthermore, we found that a humanized MM xenograft mouse model bearing human MM cells (NOD‐SCID.CB17 male mice injected intravenously with 2.5 million of MM1.S‐Luc‐GFP cells) demonstrated significantly higher concentrations of mouse‐derived sclerostin, suggesting a microenvironmental source of sclerostin. Associated with the increased sclerostin levels, activated β‐catenin expression levels were lower than normal in MM mouse bone marrow. Importantly, a high‐affinity grade scl‐Ab reversed osteolytic bone disease in this animal model. Because scl‐Ab did not demonstrate significant in vitro anti‐MM activity, we combined it with the proteasome inhibitor carfilzomib. Our data demonstrated that this combination therapy significantly inhibited tumor burden and improved bone disease in our in vivo MM mouse model. In agreement with our in vivo data, sclerostin expression was noted in marrow stromal cells and osteoblasts of MM patient bone marrow samples. Moreover, MM cells stimulated sclerostin expression in immature osteoblasts while inhibiting osteoblast differentiation in vitro. This was in part regulated by Dkk‐1 secreted by MM cells and is a potential mechanism contributing to the osteoblast dysfunction noted in MM. Our data confirm the role of sclerostin as a potential therapeutic target in MM bone disease and provides the rationale for studying scl‐Ab combined with proteasome inhibitors in MM.

NFATc1 and NFATc2 repress spontaneous osteoarthritis

Significance Currently, little is understood about how the transcriptional regulation of cartilage breakdown contributes to pathogenesis of osteoarthritis (OA). Here, we report that, within cartilage, the transcription factor Nuclear factor of activated T cells c1 (NFATc1) displays selective expression in superficial articular chondrocytes. Accordingly, mice lacking both NFATc1 and NFATc2 in cartilage were generated and found to develop a severe, spontaneous and early-onset OA. These findings establish NFATc1 as a key transcriptional repressor of cartilage breakdown and OA. Additionally, these findings provide a unique model of OA that is an attractive platform for the preclinical development of treatments to alter the course of OA. Osteoarthritis (OA) was once viewed originally as a mechanical disease of “wear and tear,” but advances made during the past two decades suggest that abnormal biomechanics contribute to active dysregulation of chondrocyte biology, leading to catabolism of the cartilage matrix. A number of signaling and transcriptional mechanisms have been studied in relation to the regulation of this catabolic program, but how they specifically regulate the initiation or progression of the disease is poorly understood. Here, we demonstrate that cartilage-specific ablation of Nuclear factor of activated T cells c1 (Nfatc1) in Nfatc2−/− mice leads to early onset, aggressive OA affecting multiple joints. This model recapitulates features of human OA, including loss of proteoglycans, collagen and aggrecan degradation, osteophyte formation, changes to subchondral bone architecture, and eventual progression to cartilage effacement and joint instability. Consistent with the notion that NFATC1 is an OA-suppressor gene, NFATC1 expression was significantly down-regulated in paired lesional vs. macroscopically normal cartilage samples from OA patients. The highly penetrant, early onset, and severe nature of this model make it an attractive platform for the preclinical development of treatments to alter the course of OA. Furthermore, these findings indicate that NFATs are key suppressors of OA, and regulating NFATs or their transcriptional targets in chondrocytes may lead to novel disease-modifying OA therapies.

Administration of BMP2/7 in utero partially reverses Rubinstein-Taybi syndrome–like skeletal defects induced by Pdk1 or Cbp mutations in mice

Mutations in the coactivator CREB-binding protein (CBP) are a major cause of the human skeletal dysplasia Rubinstein-Taybi syndrome (RTS); however, the mechanism by which these mutations affect skeletal mineralization and patterning is unknown. Here, we report the identification of 3-phosphoinositide-dependent kinase 1 (PDK1) as a key regulator of CBP activity and demonstrate that its functions map to both osteoprogenitor cells and mature osteoblasts. In osteoblasts, PDK1 activated the CREB/CBP complex, which in turn controlled runt-related transcription factor 2 (RUNX2) activation and expression of bone morphogenetic protein 2 (BMP2). These pathways also operated in vivo, as evidenced by recapitulation of RTS spectrum phenotypes with osteoblast-specific Pdk1 deletion in mice (Pdk1osx mice) and by the genetic interactions observed in mice heterozygous for both osteoblast-specific Pdk1 deletion and either Runx2 or Creb deletion. Finally, treatment of Pdk1osx and Cbp+/- embryos with BMPs in utero partially reversed their skeletal anomalies at birth. These findings illustrate the in vivo function of the PDK1-AKT-CREB/CBP pathway in bone formation and provide proof of principle for in utero growth factor supplementation as a potential therapy for skeletal dysplasias.

Schnurri-3 regulates ERK downstream of WNT signaling in osteoblasts

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.

Klotho expression in osteocytes regulates bone metabolism and controls bone formation

Osteocytes within the mineralized bone matrix control bone remodeling by regulating osteoblast and osteoclast activity. Osteocytes express the aging suppressor Klotho, but the functional role of this protein in skeletal homeostasis is unknown. Here we identify Klotho expression in osteocytes as a potent regulator of bone formation and bone mass. Targeted deletion of Klotho from osteocytes led to a striking increase in bone formation and bone volume coupled with enhanced osteoblast activity, in sharp contrast to what is observed in Klotho hypomorphic (kl/kl) mice. Conversely, overexpression of Klotho in cultured osteoblastic cells inhibited mineralization and osteogenic activity during osteocyte differentiation. Further, the induction of chronic kidney disease with high-turnover renal osteodystrophy led to downregulation of Klotho in bone cells. This appeared to offset the skeletal impact of osteocyte-targeted Klotho deletion. Thus, our findings establish a key role of osteocyte-expressed Klotho in regulating bone metabolism and indicate a new mechanism by which osteocytes control bone formation.