An Integrated View of Stressors as Causative Agents in OA Pathogenesis.

Cells in the body are exposed to dynamic external and internal environments, many of which cause cell damage. The cell's response to this damage, broadly called the stress response, is meant to promote survival and repair or remove damage. However, not all damage can be repaired, and sometimes, even worse, the stress response can overtax the system itself, further aggravating homeostasis and leading to its loss. Aging phenotypes are considered a manifestation of accumulated cellular damage and defective repair. This is particularly apparent in the primary cell type of the articular joint, the articular chondrocytes. Articular chondrocytes are constantly facing the challenge of stressors, including mechanical overloading, oxidation, DNA damage, proteostatic stress, and metabolic imbalance. The consequence of the accumulation of stress on articular chondrocytes is aberrant mitogenesis and differentiation, defective extracellular matrix production and turnover, cellular senescence, and cell death. The most severe form of stress-induced chondrocyte dysfunction in the joints is osteoarthritis (OA). Here, we summarize studies on the cellular effects of stressors on articular chondrocytes and demonstrate that the molecular effectors of the stress pathways connect to amplify articular joint dysfunction and OA development.

Wnt regulation of hematopoietic stem cell development and disease.

Hematopoietic stem cells (HSCs) are multipotent stem cells that give rise to all cells of the blood and most immune cells. Due to their capacity for unlimited self-renewal, long-term HSCs replenish the blood and immune cells of an organism throughout its life. HSC development, maintenance, and differentiation are all tightly regulated by cell signaling pathways, including the Wnt pathway. Wnt signaling is initiated extracellularly by secreted ligands which bind to cell surface receptors and give rise to several different downstream signaling cascades. These are classically categorized either ß-catenin dependent (BCD) or ß-catenin independent (BCI) signaling, depending on their reliance on the ß-catenin transcriptional activator. HSC development, homeostasis, and differentiation is influenced by both BCD and BCI, with a high degree of sensitivity to the timing and dosage of Wnt signaling. Importantly, dysregulated Wnt signals can result in hematological malignancies such as leukemia, lymphoma, and myeloma. Here, we review how Wnt signaling impacts HSCs during development and in disease.

Got WNTS? Insight into bone health from a WNT perspective.

WNT signaling, essential for many aspects of development, is among the most commonly altered pathways associated with human disease. While initially studied in cancer, dysregulation of WNT signaling has been determined to be essential for skeletal development and the maintenance of bone health throughout life. In this review, we discuss the role of Wnt signaling in bone development and disease with a particular focus on two areas. First, we discuss the roles of WNT signaling pathways in skeletal development, with an emphasis on congenital and idiopathic skeletal syndromes and diseases that are associated with genetic variations in WNT signaling components. Next, we cover a topic that has long been an interest of our laboratory, how high and low levels of WNT signaling affects the establishment and maintenance of healthy bone mass. We conclude with a discussion of the status of WNT-based therapeutics in the treatment of skeletal disease.

TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis.

Long bones are generated by mesoderm-derived skeletal progenitor/stem cells (SSCs) through endochondral ossification, a process of sequential chondrogenic and osteogenic differentiation tightly controlled by the synergy between intrinsic and microenvironment cues. Here, we report that loss of TRIM28, a transcriptional corepressor, in mesoderm-derived cells expands the SSC pool, weakens SSC osteochondrogenic potential, and endows SSCs with properties of ectoderm-derived neural crest cells (NCCs), leading to severe defects of skeletogenesis. TRIM28 preferentially enhances H3K9 trimethylation and DNA methylation on chromatin regions more accessible in NCCs; loss of this silencing upregulates neural gene expression and enhances neurogenic potential. Moreover, TRIM28 loss causes hyperexpression of GREM1, which is an extracellular signaling factor promoting SSC self-renewal and SSC neurogenic potential by activating AKT/mTORC1 signaling. Our results suggest that TRIM28-mediated chromatin silencing establishes a barrier for maintaining the SSC lineage trajectory and preventing a transition to ectodermal fate by regulating both intrinsic and microenvironment cues.

SUMOylation in Skeletal Development, Homeostasis, and Disease.

The modification of proteins by small ubiquitin-related modifier (SUMO) molecules, SUMOylation, is a key post-translational modification involved in a variety of biological processes, such as chromosome organization, DNA replication and repair, transcription, nuclear transport, and cell signaling transduction. In recent years, emerging evidence has shown that SUMOylation regulates the development and homeostasis of the skeletal system, with its dysregulation causing skeletal diseases, suggesting that SUMOylation pathways may serve as a promising therapeutic target. In this review, we summarize the current understanding of the molecular mechanisms by which SUMOylation pathways regulate skeletal cells in physiological and disease contexts.

Physically-cross-linked poly(vinyl alcohol) cell culture plate coatings facilitate preservation of cell-cell interactions, spheroid formation, and stemness.

We employed aqueous solutions of highly-hydrolyzed (>99+%) poly(vinyl alcohol), PVA, to coat plastic dishes as a method to efficiently induce three-dimensional (3D) culturing of cells. The coatings were prepared by simple evaporation of 3 wt/vol% solutions of PVA in water and require no additional processing steps after air drying under sterile conditions. The coating allows spheroids to form in solution. Spheroid formation is usually preferable to two-dimensional (2D) culturing as it creates a more realistic ex vivo model of some human tissues and tumors. Using PVA-coated cell culture plates, we demonstrated that we can grow reproducibly sized spheroids using several human glioma cell lines, including LN229, U87 MG, and Gli36, and the embryonic kidney cell line, 293T. Spheroids formed on PVA-coated plates grow as well as on other commercially-available, low-attachment plates, and have excellent optical imaging properties. As spheroids, LN229 cells express markers of cancer stem cells. Finally, we confirmed that spheroids generated on PVA-coated plates are sensitive to molecular perturbations, as increased expression of the cell adhesion molecule PTPµ significantly increased the size of spheroids. The PVA hydrogel layer is an effective tool for creating a more realistic ex vivo culture system than traditional 2D culture and can be used to generate cell spheroids for potential application in drug screening and personalized medicine for diseases such as cancer.

Detection of Tumor-Specific PTPmu in Gynecological Cancer and Patient Derived Xenografts.

BACKGROUND: We developed a fluorophore-conjugated peptide agent, SBK4, that detects a tumor-specific proteolyzed form of the cell adhesion molecule, PTPmu, found in the tumor microenvironment. We previously demonstrated its tissue specific distribution in high-grade brain tumors. To extend those studies to other aggressive solid tumor types, we assessed the tissue distribution of PTPmu/SBK4 in a set of matched gynecologic cancer patient derived xenografts (PDXs) and primary patient tumors, as well as a limited cohort of tumors from gynecological cancer patients. PDXs isolated from the tissues of cancer patients have been shown to yield experimentally manipulatable models that replicate the clinical characteristics of individual patients' tumors. In this study, gynecological cancer PDXs and patient biopsies were examined to determine if tumor-specific proteolyzed PTPmu was present. METHODS: We used the peptide agent SBK4 conjugated to the fluorophore Texas Red (TR) to label tumor tissue microarrays (TMAs) containing patient and/or PDX samples from several high-grade gynecologic cancer types, and quantified the level of staining with Image J. In one TMA, we were able to directly compare the patient and the matched PDX tissue on the same slide. RESULTS: While normal tissue had very little SBK4-TR staining, both primary tumor tissue and PDXs have higher labeling with SBK4-TR. Matched PDXs and patient samples from high-grade endometrial and ovarian cancers demonstrated higher levels of PTPmu by staining with SBK4 than normal tissue. CONCLUSION: In this sample set, all PDXs and high-grade ovarian cancer samples had increased labeling by SBK4-TR compared with the normal controls. Our results indicate that proteolyzed PTPmu and its novel peptide detection agent, SBK4, allow for the visualization of tumor-specific changes in cell adhesion molecules by tissue-based staining, providing a rationale for further development as an imaging agent in aggressive solid tumors, including gynecological cancers.

PTPmu-targeted nanoparticles label invasive pediatric and adult glioblastoma.

Poor prognosis for glioblastoma (GBM) is a consequence of the aggressive and infiltrative nature of gliomas where individual cells migrate away from the main tumor to distant sites, making complete surgical resection and treatment difficult. In this manuscript, we characterize an invasive pediatric glioma model and determine if nanoparticles linked to a peptide recognizing the GBM tumor biomarker PTPmu can specifically target both the main tumor and invasive cancer cells in adult and pediatric glioma models. Using both iron and lipid-based nanoparticles, we demonstrate by magnetic resonance imaging, optical imaging, histology, and iron quantification that PTPmu-targeted nanoparticles effectively label adult gliomas. Using PTPmu-targeted nanoparticles in a newly characterized orthotopic pediatric SJ-GBM2 model, we demonstrate individual tumor cell labeling both within the solid tumor margins and at invasive and dispersive sites.

A PTPmu Biomarker is Associated with Increased Survival in Gliomas.

An integrated approach has been adopted by the World Health Organization (WHO) for diagnosing brain tumors. This approach relies on the molecular characterization of biopsied tissue in conjunction with standard histology. Diffuse gliomas (grade II to grade IV malignant brain tumors) have a wide range in overall survival, from months for the worst cases of glioblastoma (GBM) to years for lower grade astrocytic and oligodendroglial tumors. We previously identified a change in the cell adhesion molecule PTPmu in brain tumors that results in the generation of proteolytic fragments. We developed agents to detect this cell surface-associated biomarker of the tumor microenvironment. In the current study, we evaluated the PTPmu biomarker in tissue microarrays and individual tumor samples of adolescent and young adult (n = 25) and adult (n = 69) glioma populations using a fluorescent histochemical reagent, SBK4-TR, that recognizes the PTPmu biomarker. We correlated staining with clinical data and found that high levels of the PTPmu biomarker correlate with increased survival of glioma patients, including those with GBM. Patients with high PTPmu live for 48 months on average, whereas PTPmu low patients live only 22 months. PTPmu high staining indicates a doubling of patient survival. Use of the agent to detect the PTPmu biomarker would allow differentiation of glioma patients with distinct survival outcomes and would complement current molecular approaches used in glioma prognosis.

Quantitative Molecular Imaging with a Single Gd-Based Contrast Agent Reveals Specific Tumor Binding and Retention in Vivo.

Magnetic resonance imaging (MRI) has become an indispensable tool in the diagnosis and treatment of many diseases, especially cancer. However, the poor sensitivity of MRI relative to other imaging modalities, such as PET, has hindered the development and clinical use of molecular MRI contrast agents that could provide vital diagnostic information by specifically locating a molecular target altered in the disease process. This work describes the specific and sustained in vivo binding and retention of a protein tyrosine phosphatase mu (PTPµ)-targeted, molecular magnetic resonance (MR) contrast agent with a single gadolinium (Gd) chelate using a quantitative MRI T1 mapping technique in glioma xenografts. Quantitative T1 mapping is an imaging method used to measure the longitudinal relaxation time, the T1 relaxation time, of protons in a magnetic field after excitation by a radiofrequency pulse. T1 relaxation times can in turn be used to calculate the concentration of a gadolinium-containing contrast agent in a region of interest, thereby allowing the retention or clearance of an agent to be quantified. In this context, retention is a measure of molecular contrast agent binding. Using conventional peptide chemistry, a PTPµ-targeted peptide was linked to a chelator that had been conjugated to a lysine residue. Following complexation with Gd, this PTPµ-targeted molecular contrast agent containing a single Gd ion showed significant tumor enhancement and a sustained increase in Gd concentration in both heterotopic and orthotopic tumors using dynamic quantitative MRI. This single Gd-containing PTPµ agent was more effective than our previous version with three Gd ions. Differences between nonspecific and specific agents, due to specific tumor binding, can be determined within the first 30 min after agent administration by examining clearance rates. This more facile chemistry, when combined with quantitative MR techniques, allows for widespread adoption by academic and commercial entities in the field of molecular MRI ultimately leading to improved detection of disease.

Fluorescent-Guided Surgical Resection of Glioma with Targeted Molecular Imaging Agents: A Literature Review.

The median life expectancy after a diagnosis of glioblastoma is 15 months. Although chemotherapeutics may someday cure glioblastoma by killing the highly dispersive malignant cells, the most important contribution that clinicians can currently offer to improve survival is by maximizing the extent of resection and providing concurrent chemo-radiation, which has become standard. Strides have been made in this area with the advent and implementation of methods of improved intraoperative tumor visualization. One of these techniques, optical fluorescent imaging with targeted molecular imaging agents, allows the surgeon to view fluorescently labeled tumor tissue during surgery with the use of special microscopy, thereby highlighting where to resect and indicating when tumor-free margins have been obtained. This advantage is especially important at the difficult-to-observe margins where tumor cells infiltrate normal tissue. Targeted fluorescent agents also may be valuable for identifying tumor versus nontumor tissue. In this review, we briefly summarize nontargeted fluorescent tumor imaging agents before discussing several novel targeted fluorescent agents being developed for glioma imaging in the context of fluorescent-guided surgery or live molecular navigation. Many of these agents are currently undergoing preclinical testing. As the agents become available, however, it is necessary to understand the strengths and weaknesses of each.

Regulation of development and cancer by the R2B subfamily of RPTPs and the implications of proteolysis.

The initial cloning of receptor protein tyrosine phosphatases (RPTPs) was met with excitement because of their hypothesized function in counterbalancing receptor tyrosine kinase signaling. In recent years, members of a subfamily of RPTPs with homophilic cell-cell adhesion capabilities, known as the R2B subfamily, have been shown to have functions beyond that of counteracting tyrosine kinase activity, by independently influencing cell signaling in their own right and by regulating cell adhesion. The R2B subfamily is composed of four members: PTPmu (PTPRM), PTPrho (PTPRT), PTPkappa (PTPRK), and PCP-2 (PTPRU). The effects of this small subfamily of RPTPs is far reaching, influencing several developmental processes and cancer. In fact, R2B RPTPs are predicted to be tumor suppressors and are among the most frequently mutated protein tyrosine phosphatases (PTPs) in cancer. Confounding these conclusions are more recent studies suggesting that proteolysis of the full-length R2B RPTPs result in oncogenic extracellular and intracellular protein fragments. This review discusses the current knowledge of the role of R2B RPTPs in development and cancer, with special detail given to the mechanisms and implications that proteolysis has on R2B RPTP function. We also touch upon the concept of exploiting R2B proteolysis to develop cancer imaging tools, and consider the effects of R2B proteolysis on axon guidance, perineural invasion and collective cell migration.

A protease storm cleaves a cell-cell adhesion molecule in cancer: multiple proteases converge to regulate PTPmu in glioma cells.

Cleavage of the cell-cell adhesion molecule, PTPµ, occurs in human glioblastoma multiforme brain tumor tissue and glioma cell lines. PTPµ cleavage is linked to increased cell motility and growth factor independent survival of glioma cells in vitro. Previously, PTPµ was shown to be cleaved by furin in the endoplasmic reticulum to generate membrane associated E- (extracellular) and P- (phosphatase) subunits, and by ADAMs and the gamma secretase complex at the plasma membrane. We also identified the presence of additional extracellular and intracellular PTPµ fragments in brain tumors. We set out to biochemically analyze PTPµ cleavage in cancer cells. We determined that, in addition to the furin-processed form of PTPµ, a pool of 200 kDa full-length PTPµ exists at the plasma membrane that is cleaved directly by ADAM to generate a larger shed form of the PTPµ extracellular segment. Notably, in glioma cells, full-length PTPµ is also subject to calpain cleavage, which generates novel PTPµ fragments not found in other immortalized cells. We also observed glycosylation and phosphorylation differences in the cancer cells. Our data suggest that an additional serine protease also contributes to PTPµ shedding in glioma cells. We hypothesize that a "protease storm" occurs in cancer cells whereby multiple proteases converge to reduce the presence of cell-cell adhesion molecules at the plasma membrane and to generate protein fragments with unique biological functions. As a consequence, the "protease storm" could promote the migration and invasion of tumor cells.

Single cell molecular recognition of migrating and invading tumor cells using a targeted fluorescent probe to receptor PTPmu.

Detection of an extracellular cleaved fragment of a cell-cell adhesion molecule represents a new paradigm in molecular recognition and imaging of tumors. We previously demonstrated that probes that recognize the cleaved extracellular domain of receptor protein tyrosine phosphatase mu (PTPmu) label human glioblastoma brain tumor sections and the main tumor mass of intracranial xenograft gliomas. In this article, we examine whether one of these probes, SBK2, can label dispersed glioma cells that are no longer connected to the main tumor mass. Live mice with highly dispersive glioma tumors were injected intravenously with the fluorescent PTPmu probe to test the ability of the probe to label the dispersive glioma cells in vivo. Analysis was performed using a unique three-dimensional (3D) cryo-imaging technique to reveal highly migratory and invasive glioma cell dispersal within the brain and the extent of colabeling by the PTPmu probe. The PTPmu probe labeled the main tumor site and dispersed cells up to 3.5 mm away. The cryo-images of tumors labeled with the PTPmu probe provide a novel, high-resolution view of molecular tumor recognition, with excellent 3D detail regarding the pathways of tumor cell migration. Our data demonstrate that the PTPmu probe recognizes distant tumor cells even in parts of the brain where the blood-brain barrier is likely intact. The PTPmu probe has potential translational significance for recognizing tumor cells to facilitate molecular imaging, a more complete tumor resection and to serve as a molecular targeting agent to deliver chemotherapeutics to the main tumor mass and distant dispersive tumor cells.

Novel cryo-imaging of the glioma tumor microenvironment reveals migration and dispersal pathways in vivid three-dimensional detail.

Traditional methods of imaging cell migration in the tumor microenvironment include serial sections of xenografts and standard histologic stains. Current molecular imaging techniques suffer from low resolution and difficulty in imaging through the skull. Here we show how computer algorithms can be used to reconstruct images from tissue sections obtained from mouse xenograft models of human glioma and can be rendered into three-dimensional images offering exquisite anatomic detail of tumor cell dispersal. Our findings identify human LN-229 and rodent CNS-1 glioma cells as valid systems to study the highly dispersive nature of glioma tumor cells along blood vessels and white matter tracts in vivo. This novel cryo-imaging technique provides a valuable tool to evaluate therapeutic interventions targeted at limiting tumor cell invasion and dispersal.

Should I stay or should I go? Shedding of RPTPs in cancer cells switches signals from stabilizing cell-cell adhesion to driving cell migration.

Dissolution of cell-cell adhesive contacts and increased cell-extracellular matrix adhesion are hallmarks of the migratory and invasive phenotype of cancer cells. These changes are facilitated by growth factor binding to receptor protein tyrosine kinases (RTKs). In normal cells, cell-cell adhesion molecules (CAMs), including some receptor protein tyrosine phosphatases (RPTPs), antagonize RTK signaling by promoting adhesion over migration. In cancer, RTK signaling is constitutive due to mutated or amplified RTKs, which leads to growth factor independence, or autonomy. An alternative route for a tumor cell to achieve autonomy is to inactivate cell-cell CAMs such as RPTPs. RPTPs directly mediate cell adhesion and regulate both cadherin-dependent adhesion and signaling. In addition, RPTPs antagonize RTK signaling by dephosphorylating molecules activated following ligand binding. Both RPTPs and cadherins are downregulated in tumor cells by cleavage at the cell surface. This results in shedding of the extracellular, adhesive segment and displacement of the intracellular segment, altering its subcellular localization and access to substrates or binding partners. In this commentary we discuss the signals that are altered following RPTP and cadherin cleavage to promote cell migration. Tumor cells both step on the gas (RTKs) and disconnect the brakes (RPTPs and cadherins) during their invasive and metastatic journey.

Tumor-derived extracellular fragments of receptor protein tyrosine phosphatases (RPTPs) as cancer molecular diagnostic tools.

Receptor protein tyrosine phosphatase (RPTPs) are involved in many cellular processes, including the regulation of adhesion, migration and cellular signaling. Many RPTPs are putative tumor suppressors because of the transcriptional and translational changes observed in their expression during tumorigenesis. Recently, RPTPs were shown to be post-translationally regulated during tumorigenesis by proteolysis in a manner similar to proteolysis of the Notch receptor. There is accumulating evidence that proteolysis of RPTPs influence their cellular function and that RPTP fragments may function as oncogenes. By exploiting what is known about RPTP ligand binding domains and crystal structures of ligand-RPTP interfaces, we describe novel molecular diagnostics that have been or can be developed to identify tumor margins and target tumor tissues.

Identification of phospholipase C gamma1 as a protein tyrosine phosphatase mu substrate that regulates cell migration.

The receptor protein tyrosine phosphatase PTPµ has a cell-adhesion molecule-like extracellular segment and a catalytically active intracellular segment. This structure gives PTPµ the ability to transduce signals in response to cell-cell adhesion. Full-length PTPµ is down-regulated in glioma cells by proteolysis which is linked to increased migration of these cells in the brain. To gain insight into the substrates PTPµ may be dephosphorylating to suppress glioma cell migration, we used a substrate trapping method to identify PTPµ substrates in tumor cell lines. We identified both PKCδ and PLCγ1 as PTPµ substrates. As PLCγ1 activation is linked to increased invasion of cancer cells, we set out to determine whether PTPµ may be upstream of PLCγ1 in regulating glioma cell migration. We conducted brain slice assays using U87-MG human glioma cells in which PTPµ expression was reduced by shRNA to induce migration. Treatment of the same cells with PTPµ shRNA and a PLCγ1 inhibitor prevented migration of the cells within the brain slice. These data suggest that PLCγ1 is downstream of PTPµ and that dephosphorylation of PLCγ1 is likely to be a major pathway through which PTPµ suppresses glioma cell migration.

Cancer cells cut homophilic cell adhesion molecules and run.

The term contact inhibition (CI) encompasses the cellular changes that result in cessation of cell migration and of proliferation due to signals transduced when one cell comes into physical contact with another cell. Cancer cells, however, do not contact inhibit. A molecular understanding of the loss of CI in cancer cells is important for understanding tumor progression. In this Perspective, we propose that the loss of CI observed in cancer cells is the result of extracellular proteolysis of transmembrane cell-cell cell adhesion molecules (CAM) in the tumor microenvironment. Proteolysis of homophilic cell-cell CAMs results in a shed extracellular fragment and released cytoplasmic fragment(s) that disrupts adhesion and induces signals that promote proliferation and/or migration. The importance of this observation in tumor progression is supported by the presence of the shed extracellular fragments of homophilic cell-cell CAMs in serum and tumor tissue of cancer patients suggesting that instead of acting as tumor suppressors, the shed CAM extracellular and cytoplasmic fragments actually function as oncogenes. The study of cell-cell CAM cleavage will provide important and novel means of diagnosing, imaging, and treating tumor progression.

Characterization of the adhesive properties of the type IIb subfamily receptor protein tyrosine phosphatases.

Receptor protein tyrosine phosphatases (RPTPs) have cell adhesion molecule-like extracellular domains coupled to cytoplasmic tyrosine phosphatase domains. PTPmu is the prototypical member of the type IIb subfamily of RPTPs, which includes PTPrho, PTPkappa, and PCP-2. The authors performed the first comprehensive analysis of the subfamily in one system, examining adhesion and antibody recognition. The authors evaluated if antibodies that they developed to detect PTPmu also recognized other subfamily members. Notably, each antibody recognizes distinct subsets of type IIb RPTPs. PTPmu, PTPrho, and PTPkappa have all been shown to mediate cell-cell aggregation, and prior work with PCP-2 indicated that it can mediate bead aggregation in vitro. This study reveals that PCP-2 is unique among the type IIb RPTPs in that it does not mediate cell-cell aggregation via homophilic binding. The authors conclude from these experiments that PCP-2 is likely to have a distinct biological function other than cell-cell aggregation.