Erik R. Sampson

Synergistic response to oncogenic mutations defines gene class critical to cancer phenotype

Understanding the molecular underpinnings of cancer is of critical importance to the development of targeted intervention strategies. Identification of such targets, however, is notoriously difficult and unpredictable. Malignant cell transformation requires the cooperation of a few oncogenic mutations that cause substantial reorganization of many cell features and induce complex changes in gene expression patterns. Genes critical to this multifaceted cellular phenotype have therefore only been identified after signalling pathway analysis or on an ad hoc basis. Our observations that cell transformation by cooperating oncogenic lesions depends on synergistic modulation of downstream signalling circuitry suggest that malignant transformation is a highly cooperative process, involving synergy at multiple levels of regulation, including gene expression. Here we show that a large proportion of genes controlled synergistically by loss-of-function p53 and Ras activation are critical to the malignant state of murine and human colon cells. Notably, 14 out of 24 ‘cooperation response genes’ were found to contribute to tumour formation in gene perturbation experiments. In contrast, only 1 in 14 perturbations of the genes responding in a non-synergistic manner had a similar effect. Synergistic control of gene expression by oncogenic mutations thus emerges as an underlying key to malignancy, and provides an attractive rationale for identifying intervention targets in gene networks downstream of oncogenic gain- and loss-of-function mutations.

Teriparatide as a Chondroregenerative Therapy for Injury-Induced Osteoarthritis

Teriparatide is chondroprotective and chondroregenerative in a mouse model of injury-induced osteoarthritis of the knee. Extending the Service Life of Arthritic Joints Every year, millions of people with osteoarthritis are forced to scale back their physical activities hoping to alleviate pain and increase the longevity of their degenerating joints. The problem is serious: A decade from now, 25% or more of the U.S. population are predicted to suffer from osteoarthritis. The hallmark problem in osteoarthritis is the progressive and irreversible loss of cartilage. Ultimately, the only option is to surgically replace the lost cartilage with metal and plastic. But are there alternative strategies that could lead to cartilage replacement and reduce the cycle of pain and reduced quality of life? In this issue of Science Translational Medicine, Sampson and colleagues report that a naturally occurring hormone called parathyroid hormone (trade name Forteo), already approved by the Food and Drug Administration to build bone, can also boost the buildup of cartilage in a mouse model of injury-induced osteoarthritis. In this mouse model, injury to the meniscus and ligaments of the knee initiates a slow process of cartilage degeneration that is comparable to that seen in people suffering a similar injury. To approximate the clinical situation of treating someone with symptomatic osteoarthritis caused by a past trauma, the researchers administered parathyroid hormone to mice that were already osteoarthritic because of an injury to the medial meniscus and medial collateral ligament. Tissue and molecular analyses of the injured knee joints revealed that after 1 month of daily treatment with parathyroid hormone, the cartilage layer was 32% thicker than in injured mice that did not receive the hormone. In addition, the investigators noted an increase in production of matrix molecules by chondrocytes (the cells that produce cartilage), suppression of genes associated with inappropriate chondrocyte maturation, and a reduction in cartilage breakdown. The ability of parathyroid hormone to boost the addition of new cartilage matrix while blocking its degradation in osteoarthritic joints suggests that it may be a useful therapeutic for treating patients with osteoarthritis, a pervasive clinical condition with surgery as the only current solution. There is no disease-modifying therapy for osteoarthritis, a degenerative joint disease that is projected to afflict more than 67 million individuals in the United States alone by 2030. Because disease pathogenesis is associated with inappropriate articular chondrocyte maturation resembling that seen during normal endochondral ossification, pathways that govern the maturation of articular chondrocytes are candidate therapeutic targets. It is well established that parathyroid hormone (PTH) acting via the type 1 PTH receptor induces matrix synthesis and suppresses maturation of chondrocytes. We report that the PTH receptor is up-regulated in articular chondrocytes after meniscal injury and in osteoarthritis in humans and in a mouse model of injury-induced knee osteoarthritis. To test whether recombinant human PTH(1–34) (teriparatide) would inhibit aberrant chondrocyte maturation and associated articular cartilage degeneration, we administered systemic teriparatide (Forteo), a Food and Drug Administration–approved treatment for osteoporosis, either immediately after or 8 weeks after meniscal/ligamentous injury in mice. Knee joints were harvested at 4, 8, or 12 weeks after injury to examine the effects of teriparatide on cartilage degeneration and articular chondrocyte maturation. Microcomputed tomography revealed increased bone volume within joints from teriparatide-treated mice compared to saline-treated control animals. Immediate systemic administration of teriparatide increased proteoglycan content and inhibited articular cartilage degeneration, whereas delayed treatment beginning 8 weeks after injury induced a regenerative effect. The chondroprotective and chondroregenerative effects of teriparatide correlated with decreased expression of type X collagen, RUNX2 (runt-related transcription factor 2), matrix metalloproteinase 13, and the carboxyl-terminal aggrecan cleavage product NITEGE. These preclinical findings provide proof of concept that Forteo may be useful for decelerating cartilage degeneration and inducing matrix regeneration in patients with osteoarthritis.

Induction of an Osteoarthritis-like Phenotype and Degradation of Phosphorylated Smad3 by Smurf2 in Transgenic Mice

OBJECTIVE To determine whether Smurf2, an E3 ubiquitin ligase known to inhibit transforming growth factor beta (TGFbeta) signaling, is expressed in human osteoarthritic (OA) cartilage and can initiate OA in mice. METHODS Human OA cartilage was obtained from patients undergoing knee arthroplasty. Samples were graded histologically using the Mankin scale and were examined immunohistochemically for Smurf2 expression. A transgene driven by the collagen 2alpha1 promoter was used to overexpress Smurf2 in mice. Smurf2 overexpression in mouse sternal chondrocytes was confirmed by reverse transcription-polymerase chain reaction and Western blotting. Changes in articular cartilage area, chondrocyte number, and chondrocyte diameter were assessed histomorphometrically using OsteoMeasure software. Alterations in type X collagen and matrix metalloproteinase 13 (MMP-13) in articular chondrocytes were examined by in situ hybridization and immunohistochemistry, respectively. Joint bone phenotypes were evaluated by microfocal computed tomography. The effects of Smurf2 overexpression on TGFbeta signaling were examined using a luciferase-based reporter and immunoprecipitation/Western blotting. RESULTS Human OA cartilage strongly expressed Smurf2 as compared with nonarthritic human cartilage. By 8 months of age, Smurf2-transgenic mice exhibited decreased articular cartilage area, fibrillation, clefting, eburnation, subchondral sclerosis, and osteophytes. Increased expression of type X collagen and MMP-13 were also detected in articular cartilage from transgenic mice. Transgenic sternal chondrocytes showed reduced TGFbeta signaling as well as decreased expression and increased ubiquitination of pSmad3. CONCLUSION Smurf2 is up-regulated during OA in humans, and Smurf2-transgenic mice spontaneously develop an OA-like phenotype that correlates with decreased TGFbeta signaling and increased pSmad3 degradation. Overall, these results suggest a role of Smurf2 in the pathogenesis of OA.

High-fat diet accelerates progression of osteoarthritis after meniscal/ligamentous injury

IntroductionIncreasing obesity and type 2 diabetes, in part due to the high-fat (HF) Western diet, parallels an increased incidence of osteoarthritis (OA). This study was undertaken to establish a causal relation between the HF diet and accelerated OA progression in a mouse model and to determine the relative roles of weight gain and metabolic dysregulation in this progression.MethodsFive-week-old C57BL/6 mice were placed on HF (60% kcal) or low-fat (lean, 10% kcal) diets for 8 or 12 weeks before transecting the medial collateral ligament and excising a segment of the medial meniscus of the knee to initiate OA. One group was switched from lean to HF diet at the time of surgery.ResultsBody weight of mice on the HF diet peaked at 45.9 ± 2.1 g compared with 29.9 ± 1.8 g for lean diets, with only those on the HF becoming diabetic. Severity of OA was greater in HF mice, evidenced by the Osteoarthritis Research Society International (OARSI) histopathology initiative scoring method for mice and articular cartilage thickness and area. To assess the importance of weight gain, short- and long-term HF diets were compared with the lean diet. Short- and long-term HF groups outweighed lean controls by 6.2 g and 20.5 g, respectively. Both HF groups became diabetic, and OA progression, evidenced by increased OARSI score, decreased cartilage thickness, and increased osteophyte diameter, was comparably accelerated relative to those of lean controls.ConclusionsThese results demonstrate that the HF diet accelerates progression of OA in a type 2 diabetic mouse model without correlation to weight gain, suggesting that metabolic dysregulation is a comorbid factor in OA-related cartilage degeneration.

Runx2-mediated activation of the Bax gene increases osteosarcoma cell sensitivity to apoptosis

The Runx family of transcription factors regulate cell growth and differentiation, and control the expression of target genes involved in cell fate decisions. We examined the role of the bone-related member of this family, Runx2, in regulating apoptosis via modulation of the Bcl2 family of genes in the osteosarcoma cell line Saos2. Our data demonstrate that Runx2 directly binds to two Runx-specific regulatory elements on the human bax promoter thereby inducing Bax expression. Furthermore, bone morphogenetic protein-induced or vector-mediated expression of Runx2 resulted in upregulation of Bax expression, and subsequent increased sensitivity of Saos2 cells to apoptosis. Finally, the observed upregulation of Bax expression and increased apoptosis were Runx2 dependent as Runx2 loss of function abrogated these effects. Our study provides the first evidence for Bax as a direct target of Runx2, suggesting that Runx2 may act as a proapoptotic factor in osteosarcoma cells.

Conditional activation of β‐catenin signaling in mice leads to severe defects in intervertebral disc tissue

OBJECTIVE The incidence of low back pain is extremely high and is often linked to intervertebral disc (IVD) degeneration. The mechanism of this disease is currently unknown. This study was undertaken to investigate the role of β-catenin signaling in IVD tissue function. METHODS β-catenin protein levels were measured by immunohistochemical analysis of disc samples obtained from patients with disc degeneration and from normal subjects. To generate β-catenin conditional activation (cAct) mice, Col2a1-CreER(T2) -transgenic mice were bred with β-catenin(fx(Ex3)/fx(Ex3)) mice. Changes in disc tissue morphology and function were examined by micro-computed tomography, histologic analysis, and real-time polymerase chain reaction assays. RESULTS β-catenin protein was up-regulated in disc tissue samples from patients with disc degeneration. To assess the effects of increased β-catenin levels on disc tissue, we generated β-catenin cAct mice. Overexpression of β-catenin in disc cells led to extensive osteophyte formation in 3- and 6-month-old β-catenin cAct mice, which were associated with significant changes in the cells and extracellular matrix of disc tissue and growth plate. Gene expression analysis demonstrated that activation of β-catenin enhanced runt-related transcription factor 2-dependent Mmp13 and Adamts5 expression. Moreover, genetic ablation of Mmp13 or Adamts5 on the β-catenin cAct background, or treatment of β-catenin cAct mice with a specific matrix metalloproteinase 13 inhibitor, ameliorated the mutant phenotype. CONCLUSION Our findings indicate that the β-catenin signaling pathway plays a critical role in disc tissue function.

Smurf2 Induces Degradation of GSK-3β and Upregulates β-Catenin in Chondrocytes: A Potential Mechanism for Smurf2-Induced Degeneration of Articular Cartilage

We have previously demonstrated that Smurf2 is highly expressed in human osteoarthritis (OA) tissue, and overexpression of Smurf2 under the control of the type II collagen promoter (Col2a1) induces an OA-like phenotype in aged Col2a1-Smurf2 transgenic mice, suggesting that Smurf2 is located upstream of a signal cascade which initiates OA development. However, the factors downstream of Smurf2 in this signal cascade and how Smurf2-induced OA is initiated are largely unknown. In this study, we further characterized the phenotypic changes in Col2a1-Smurf2 transgenic and WT articular cartilage from the postnatal stage to adulthood. We found that the articular cartilage degeneration occurring at the cartilage surface in 6 month-old Col2a1-Smurf2 transgenic mice progressed from an expanded hypertrophic domain in the basal layer of the deep articular cartilage at 2.5 weeks of age, which may lead to an accelerated calcification and ectopic ossification of this region at 1 month of age, and aggregation and maturation of articular chondrocytes in the middle and deep zones at 2 months and 4.5 months of age, respectively. Furthermore, we discovered that ectopically expressed Smurf2 interacted with GSK-3beta and induced its ubiquitination and subsequent proteasomal degradation, and hence upregulated beta-catenin in Col2a1-Smurf2 transgenic chondrocytes ex vivo. It is therefore likely that Smurf2-mediated upregulation of beta-catenin through induction of proteasomal degradation of GSK-beta in chondrocytes may activate articular chondrocyte maturation and associated alteration of gene expression, the early events of OA.

Aberrant Hypertrophy in Smad3-Deficient Murine Chondrocytes Is Rescued by Restoring Transforming Growth Factor β-Activated Kinase 1/ Activating Transcription Factor 2 Signaling: A Potential Clinical Implication for Osteoarthritis

OBJECTIVE To investigate the biologic significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and activating transcription factor 2 (ATF-2) in the transforming growth factor beta (TGFbeta) signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation. METHODS Joint disease in Smad3-knockout (Smad3(-/-)) mice was examined by microfocal computed tomography and histologic analysis. Numerous in vitro methods including immunostaining, real-time polymerase chain reaction, Western blotting, an ATF-2 DNA-binding assay, and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3(-/-) mice. RESULTS In Smad3(-/-) mice, an end-stage OA phenotype gradually developed. TGFbeta-activated kinase 1 (TAK1)/ATF-2 signaling was disrupted in Smad3(-/-) mouse chondrocytes at the level of p38 MAP kinase (MAPK) activation, resulting in reduced ATF-2 phosphorylation and transcriptional activity. Reintroduction of Smad3 into Smad3(-/-) cells restored the normal p38 response to TGFbeta. Phosphorylated p38 formed a complex with Smad3 by binding to a portion of Smad3 containing both the MAD homology 1 and linker domains. Additionally, Smad3 inhibited the dephosphorylation of p38 by MAPK phosphatase 1 (MKP-1). Both ATF-2 overexpression and p38 activation repressed type X collagen expression in wild-type and Smad3(-/-) chondrocytes. P38 was detected in articular cartilage and perichondrium; articular and sternal chondrocytes expressed p38 isoforms alpha, beta, and gamma, but not delta. CONCLUSION Smad3 is involved in both the onset and progression of OA. Loss of Smad3 abrogates TAK1/ATF-2 signaling, most likely by disrupting the Smad3-phosphorylated p38 complex, thereby promoting p38 dephosphorylation and inactivation by MKP-1. ATF-2 and p38 activation inhibit chondrocyte hypertrophy. Modulation of p38 isoform activity may provide a new therapeutic approach for OA.

The orally bioavailable met inhibitor PF-2341066 inhibits osteosarcoma growth and osteolysis/matrix production in a xenograft model.

Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Ninety percent of patients who present with metastatic and 30% to 40% of patients with nonmetastatic disease experience relapse, creating an urgent need for novel therapeutic strategies. The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are important for mitosis, motility, and cell survival. Upregulation of Met/HGF signaling via receptor overexpression, amplification, or mutation drives the proliferation, invasiveness, and metastasis of a variety of cancer cells, including OS, prompting the development of Met/HGF inhibitors. OS cells depend on Met overexpression because introduction of dominant‐negative Met inhibits in vivo tumorigenicity. Despite the importance of Met/HGF signaling in the development and maintenance of OS, the potential efficacy of pharmacologic Met inhibition in OS has been addressed only in in vitro studies. PF‐2341066 is an orally bioavailable, selective ATP‐competitive Met inhibitor that showed promising results recently in a phase I clinical trial in non–small cell lung cancer (NSCLC) patients. We tested the ability of PF‐2341066 to inhibit malignant properties of osteosarcoma cells in vitro and orthotopic xenograft growth in vivo. In vitro, PF‐2341066 inhibited osteosarcoma behavior associated with primary tumor growth (eg, proliferation and survival) as well as metastasis (eg, invasion and clonogenicity). In nude mice treated with PF‐2341066 via oral gavage, the growth and associated osteolysis and extracortical bone matrix formation of osteosarcoma xenografts were inhibited by PF‐2341066. PF‐2341066 may represent an effective new systemic therapy for localized and potentially disseminated osteosarcoma.

Establishment of an index with increased sensitivity for assessing murine arthritis

The goals of our study were to establish quantitative outcomes for assessing murine knee arthritis and develop an Arthritis Index that incorporates multiple outcomes into a single calculation that provides enhanced sensitivity. Using an accepted model of meniscal/ligamentous injury (MLI)‐induced osteoarthritis (OA), we assessed mouse knee arthritis using several approaches. Histology‐based methods were performed to visualize joint tissues including articular cartilage and subchondral bone. Accepted histologic scoring methods and histomorphometry were performed to grade cartilage degeneration and determine articular cartilage area, respectively. MicroCT was used to visualize and quantify the bony structures of the joint including osteophytes and joint bone volume. A statistical algorithm was then developed that combined histologic scores and cartilage areas into a single Arthritis Index. MLI induced progressive, OA‐like articular cartilage degeneration characterized by increasing (worsening) histologic score and decreasing cartilage area. MicroCT revealed osteophytes and increased joint bone volume between the femoral and tibial physes following MLI. Lastly, an Arthritis Index calculation was established, which incorporated histologic scoring and cartilage area. The Arthritis Index provided enhanced quantitative sensitivity in assessing the level of joint degeneration compared to either histologic scoring or cartilage area determination alone; when using the Index, between 29% and 43% fewer samples are needed to establish statistical significance in studies of murine arthritis. Arthritis in the mouse knee can be quantitatively assessed by histologic scoring, measuring cartilage area, and determining joint bone volume. Enhanced sensitivity can be achieved by performing the Arthritis Index calculation, a novel method for quantitatively assessing mouse knee arthritis.