Osteosarcomas (OS) have highly chaotic genomes, yet their cancer drivers are poorly defined. Leveraging cross-species OS genomics we identify the frequent amplification of NSD3, a histone lysine methyltransferase and expose the NSD3-ARID3A axis as a core pathway in osteosarcomagenesis. Loss-and gain-of-function studies with CRISPR-Cas9 and lentivirus systems establish the causal role of NSD3 in OS tumor growth and spontaneous metastasis. NSD3 specifically enhances H3K27 di-methylation to initiate oncogenic reprogramming and inactivates the transcriptional repressor ARID3A. This culminates in altered expression of instructive genes RUNX2, MMP13, OCT4 and NANOG generating a shift in osteosarcoma cell differentiation to a primitive state. In human OS, NSD3 overexpression is seen in patient tumors and predicts a poor clinical outcome. Our study uncovers the crucial epigenetic dysregulation of histone lysine methyltransferase in osteosarcoma, opening new possibilities for therapy with epigenetic drugs.

Background Point-of-care testing (POCT) is increasingly being used in healthcare, including hospitals, and POCT-style tests are also used within some laboratories. The principles of biosafety, including risk assessment and containment of biohazardous agents, can be utilized as a foundation to establish policies and procedures guiding safe performance of POCT. However, specific biosafety guidelines for POCT are generally lacking, particularly for those performed outside laboratories by healthcare workers. This study aims to explore POCT biosafety program decision-making infrastructure and oversight in Ontario.Content The Institute of Quality Management in Healthcare distributed a survey to 249 laboratories in Ontario. There were 11 questions on POCT biosafety practices.Summary The survey had a high response rate of 88.7%. How POCT biosafety decisions were made was variable among respondents. For POCT-style tests conducted within laboratories, the biosafety officer (BSO) and/or the microbiologist were involved in biosafety decisions in 95% of microbiology labs or 55% of other labs. Only 27% of the respondents reported that biosafety decisions were made by BSOs and/or microbiologists when POCT was conducted outside the laboratory. When POCT is performed outside the laboratory, biosafety decisions were made largely by Infection Prevention and Control (IPAC) and POCT laboratory staff. Similarly, training and auditing of staff who perform POCT were mainly done by IPAC and POCT laboratory staff. The survey showed that a wide variety of POCT was being conducted for COVID-19 patients during the pandemic.

ABSTRACT Nasopharyngeal swabs (NPS) are considered the standard specimen for SARS-CoV-2 detection by PCR. We aimed to compare detection performance and patient experience of NPS with swish and gargle saliva (SGS) to determine its potential as an accurate, easy-to-collect, and comfortable alternative. We conducted a prospective study from March to May 2021 and recruited pediatric and adult outpatients seeking COVID-19 testing. Paired NPS and 5-mL SGS were collected from each participant during the same visit and tested in parallel using validated rRT-PCR assays. Variant of concern (VOC) analysis was performed for positive NPS specimens. The participant completed the surveys regarding their experience with the two collection methods. We included 238 participants in the SARS-CoV-2 detection performance analysis. Thirty-two participants tested positive for both NPS and SGS. In comparison to NPS, the sensitivity and specificity of SGS to detect SARS-CoV-2 were 94.1% (95%CI; 80.3%–99.3%) and 97.1% (95%CI; 93.7%–98.9%), respectively. Six participants tested positive only with SGS and only two with NPS. NPS is an imperfect gold standard. Thus, when compared to the COVID-19 case, that is, a patient with positive NPS and/or SGS, the sensitivity for NPS and SGS were 85.0% and 95.0%, respectively. VOC results (n = 26) revealed that 92% of the cases were alpha. From the 238 surveys, 89.9% of the participants described SGS collection as comfortable or very comfortable compared to 15.1% for NPS; 90.2% of the participants were likely or very likely to return for SGS collection compared to 59.3% for NPS. SGS is an alternative to NPS for SARS-CoV-2 detection in adult and pediatric outpatients as it is more patient-friendly while still maintaining comparable performance. IMPORTANCE Widespread and frequent testing for COVID-19 was an important strategy to identify infected patients to isolate and control the spread of the disease during the pandemic. The nasopharyngeal swab (NPS) global supply chain and access to trained healthcare professionals for standard NPS collection were often compromised. Patient discomfort and limited access challenged health systems to reach large numbers for testing in adult and pediatric populations. Our study revealed that swish and gargle saliva (SGS) was comparable to NPS in detecting SARS-CoV-2 and more patient-friendly than NPS. Patients were more likely to repeat the test with SGS. SGS was amenable to self-collection instead of relying on skilled professionals. This comprehensive evaluation highlights the challenges of comparing the accuracy of new methods to imperfect gold standards and identifies additional patient-centric factors that should be considered when defining such standards. Thus, SGS is an advantageous alternative specimen collection for outpatient en masse testing.

Regulated growth plate activity is essential for postnatal bone development and body stature, yet the systems regulating epiphyseal fusion are poorly understood. Here, we show that the tissue inhibitors of metalloprotease (TIMP) gene family is essential for normal bone growth after birth. Whole-body quadruple-knockout mice lacking all four TIMPs have growth plate closure in long bones, precipitating limb shortening, epiphyseal distortion, and widespread chondrodysplasia. We identify TIMP/FGF-2/IHH as a novel nexus underlying bone lengthening where TIMPs negatively regulate the release of FGF-2 from chondrocytes to allow IHH expression. Using a knock-in approach that combines MMP-resistant or ADAMTS-resistant aggrecans with TIMP deficiency, we uncouple growth plate activity in axial and appendicular bones. Thus, natural metalloprotease inhibitors are crucial regulators of chondrocyte maturation program, growth plate integrity, and skeletal proportionality. Furthermore, individual and combinatorial TIMP-deficient mice demonstrate the redundancy of metalloprotease inhibitor function in embryonic and postnatal development.

BACKGROUND:Multiple myeloma (MM) cells gain protection against drugs through interaction with bone marrow stromal cells (BMSCs). This form of resistance largely accounts for resistance to therapy in MM patients which warrants further exploration to identify more potential therapeutic targets.METHODS:We performed miRNA/mRNA qPCR arrays and western blotting to analyze transcriptional and translational changes in MM cells co-cultured with BMSCs. Drug cytotoxicity and apoptosis in MMGFP-BMSC co-cultures were measured using fluorescence plate reader and flowcytometry, respectively. miRNA was overexpressed in MM cell lines using Lentiviral transduction, miRNA-3'UTR binding was examined using luciferase assay.RESULTS:We found that BMSCs downregulated miR-101-3p and upregulated survivin (BIRC5) in MM cells. Survivin was downregulated by miR-101-3p overexpression and found to be a direct target of miR-101-3p using 3'UTR luciferase assay. Overexpression of survivin increased viability of MM cells in the presence of anti-myeloma drugs, and miR-101-3p inhibition by anti-miR against miR-101-3p upregulated survivin. Furthermore, overexpression of miR-101-3p or silencing of survivin triggered apoptosis in MM cells and sensitized them to anti-myeloma drugs in the presence of BMSCs overcoming the stroma-induced drug resistance.CONCLUSIONS:Our study demonstrates that BMSC-induced resistance to drugs is associated with survivin upregulation which is a direct target of miR-101-3p. This study also identifies miR-101-3p-survivin interaction as a druggable target involved in stroma-mediated drug resistance in MM and suggests it for developing more efficient therapeutic strategies.

Intervertebral disc degeneration (IDD) is induced by multiple factors including increased apoptosis, decreased survival, and reduced extracellular matrix (ECM) synthesis in the nucleus pulposus (NP) cells. The tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is the only known lipid phosphatase counteracting the PI3K/AKT pathway. Loss of PTEN leads to activated PI3K/AKT signaling, which plays a key role in a variety of cancers. However, the role of PTEN/PI3K/AKT signaling nexus in IDD remains unknown. Here, we report that PTEN is overexpressed in degenerative NP, which correlates with inactivated AKT. Using the PTEN knockdown approach by lentivirus-mediated short interfering RNA gene transfer technique, we report that PTEN decreases survival but induces apoptosis and senescence of NP cells. PTEN also inhibits expression and production of ECM components including collagen II, aggrecan, and proteoglycan. Furthermore, PTEN modulates the expression of ECM regulatory molecules SOX-9 and matrix metalloproteinase-3 (MMP-3). Using small-molecule AKT inhibitor GDC-0068, we confirm that PTEN regulates NP cell behaviors through its direct targeting of PI3K/AKT. These findings demonstrate for the first time that PTEN/PI3K/AKT signaling axis plays an important role in the pathogenesis of IDD. Targeting PTEN using gene therapy may represent a promising therapeutic approach against disc degenerative diseases.

Overexpression of myristoylated alanine-rich C kinase substrate (MARCKS) has been implicated in the progression of multiple cancer types including multiple myeloma (MM). Our previous study showed that overexpression of MARCKS in MM cells plays an important role in drug resistance. However; the mechanism(s) underlying MARCKS overexpression and its association with drug resistance in MM remain undefined. Using miRNA target scan algorithms, we identified miR-34a as a regulator of MARCKS, which is underexpressed in drug-resistant MM cells (8226-R5 and MM1R) in comparison to parental cells (8226 and MM1S, respectively). Using luciferase assays, we demonstrated direct targeting of MARCKS by miR-34a. Notably, targeting of MARCKS by miR-34a overexpression re-sensitized the resistant cells to anti-myeloma drugs. Moreover, since all known biological functions of MARCKS including its oncogenic effects are conducted by its phosphorylated form, we investigated whether inhibitor of MARCKS phosphorylation could antagonize drug resistance in MM cells. We showed that a MARCKS PSD peptide inhibitor (MPS) had a dose dependent cytotoxic effect on drug resistant MM cells in comparison to its mutated (inactive) form, but minimal cytotoxicity on normal peripheral blood mononuclear cells (PBMCs). Furthermore, MPS treatment sensitized drug-resistant MM cells to bortezomib (BTZ), a first line proteasome inhibitor that is widely used in the treatment of MM. We further investigated the effect of MPS on tumorigenesis in mouse xenograft models of MM resistant cells. Combination of BTZ plus MPS significantly suppressed tumor growth and prolonged overall survival as compared with MPS alone, BTZ alone or vehicle treated controls. IHC analysis of xenograft tumor sections revealed that combination treatment of MPS and BTZ resulted in decreased proliferation (Ki67) and increased apoptosis (TUNEL), compared to either BTZ or MPS alone. These results support that targeting of phospho-MARCKS by MPS contributes to drug sensitivity in MM resistant cells. Furthermore, we investigated the mechanisms by which MPS induces cell death and overcome bortezomib resistance. Following MPS treatment, PUMA was accumulated both in MM1R and 8226R5 MM cell lines, indicating that MPS treatment may lead to apoptosis. Interestingly, the level of LC3BII, a marker of autophagy, was also significantly upregulated, suggesting enhanced autophagic flux after MARCKS inhibition. The significant upregulation of LC3BII observed in both MM1R and 8226R5 MM cell lines after MARCKS silencing further confirmed the correlation between MARCKS inhibition and autophagy induction. Remarkably, MARCKS-silenced drug resistant MM cells were more vulnerable than negative control cells to the autophagy inhibitor chloroquine (CQ). More significant cytotoxicity of the BTZ plus CQ combo was also detected in MARCKS-inhibited MM cells than in negative control cells. Mechanistically, co-immuno-precipitation assays revealed increased interactions of PUMA with both Mcl-1 and Bcl-xL in MARCKS-silenced 8226R5 cells, while the interaction between Beclin-1 and Bcl-xL in MARCKS silenced 8226R5 cells was dampened. These results suggest that PUMA upregulation after MRCKS inhibition enhances the initiation of autophagic response by sequestering Bcl-2 family proteins like Bcl-xL from Beclin-1/Vps34 complexes. Taken together, our results demonstrate that the MPS peptide can sensitize drug resistant MM cell to anti-myeloma drugs by inhibiting MARCKS phosphorylation in vitro and in vivo. These findings underscore the importance of MARCKS suppression in antagonizing adaptive BTZ resistance and provide novel venues to treat MM.

Osteosarcoma (OS) is the most common primary bone cancer, which occurs primarily in children and adolescents. Functional loss of the tumor suppressor PTEN has been demonstrated in bone malignancies including OS. We have recently reported that Pten expression inversely correlates with OS aggressiveness in mouse models. However, the mechanism whereby PTEN exerts its anti-tumor effect remains unknown. In this study, we first examined the expression of PTEN in human OS cell lines including U2OS, MG63 and Saos-2, and found that PTEN expression is reduced as compared to normal human osteoblasts. The downregulation of PTEN also associates with activation of AKT pathway. We then treated previously reported mouse OS tumor cells MOTO-RankΔ/ΔOC and human OS cell line U2OS with PTEN inhibitor VO-OHpic to investigate how PTEN impacts tumor cell behaviors. Our results showed that PTEN inhibits tumor cell proliferation, migration and invasion, but enhances tumor cell apoptosis. However, PTEN has no effects on tumor cell senescence and chemotaxis. PTEN also fails to induce tumor cells differentiation toward osteoblast lineage. On the other hand, PTEN inhibits tumor associated osteoclast differentiation. Moreover, overexpression of PTEN using gene transfer in U2OS cells inhibits proliferation but increases apoptosis. These findings indicate that PTEN not only targets tumor cells themselves by impacting cell behaviors, but also blocks osteoclast-mediated bone destruction, leading to interruption of the vicious cycle during osteosarcomagenesis. Loss of PTEN may consequently facilitate tumor growth and expansion in bone. Restoration of fully functional PTEN using gene therapy represents a potential approach against OS. J. Cell. Biochem. 118: 2684-2692, 2017. © 2017 Wiley Periodicals, Inc.

ABSTRACTBeing a tumor suppressor, PTEN functions as a dual‐specificity protein and phospholipid phosphatase and regulates a variety of cellular processes and signal transduction pathways. Loss of PTEN function has been detected frequently in different forms of cancers, such as breast, prostate and lung cancer, gastric and colon cancer, skin cancer, as well as endometrial carcinoma. In this review, we provide a summary of PTEN and its role in bone malignancies including bone metastases, multiple myeloma, and osteosarcoma, etc. We highlight the importance of PTEN loss leading to activation of the oncogenic PI3K/Akt/mTOR pathway in tumorigenesis and progression, which can be attributed to both genetic and non‐genetic alterations involving gene mutation, loss of heterozygosity, promoter hypermethylation, and microRNA mediated negative regulation. We also discuss the emerging therapeutic applications targeting PTEN loss for the treatment of these bone malignant diseases. J. Cell. Biochem. 116: 1837–1847, 2015. © 2015 Wiley Periodicals, Inc.

Osteosarcoma (OS) is the most common primary bone cancer, which occurs primarily in children and adolescents, severely affecting survivors' quality of life. Despite its chemosensitivity and treatment advances, long-term survival rates for OS patients have stagnated over the last 20 years. Thus, it is necessary to develop new molecularly targeted therapies for this metastatic bone cancer. Mutations in TP53 and RB are linked to OS predisposition and to the evolution of spontaneous OS. We established receptor activator of nuclear factor κB ligand (RANKL) as a therapeutic target for suppression and prevention of OS. Combined conditional osteoblast-specific deletions of Rb, p53, and the protein kinase A (PKA) regulatory subunit Prkar1α genes in genetically engineered mouse models (GEMMs) generate aggressive osteosarcomas, characterized by PKA, RANKL, and osteoclast hyperactivity. Whole-body Rankl deletion completely abrogates tumorigenesis. Although osteoblastic Rank deletion has little effect, osteoclastic Rank deletion delays tumorigenesis and prolongs life span. The latter is associated with inactivation of osteoclastogenesis and up-regulation of the tumor suppressor phosphatase and tensin homolog (PTEN). Further, we use these GEMMs as preclinical platforms to show that RANKL blockade with RANK-Fc arrests tumor progression and improves survival and also inhibits lung metastasis. Moreover, preemptive administration of RANK-Fc completely prevents tumorigenesis in mice highly predisposed to this aggressive cancer. Denosumab, a fully human monoclonal antibody against RANKL, is currently used to treat patients with osteoporosis or bone metastases. Our studies provide a strong rationale to consider RANKL blockade for the treatment and prevention of aggressive RANKL-overexpressing OS in humans.

Multiple myeloma (MM) is incurable in virtually all patients due to the presence of innate and emergent drug-resistance. To identify potential drug resistance mechanisms in MM we used iTRAQ (isobaric tags for relative and absolute quantitation) mass spectrometry to compare protein expression profiles of drug-resistant (RPMI 8226-R5) and sensitive (RPMI 8226-S) isogenic cell lines. We identified selective overexpression of myristoylated alanine-rich C-kinase substrate (MARCKS) in drug-resistant R5 cells. MARCKS overexpression was also observed in several drug-resistant human myeloma cell lines (HMCLs) and in drug-resistant primary MM samples. Functionally, inhibition of MARCKS phosphorylation by enzastaurin or knockdown of the gene by RNAi significantly enhanced the sensitivity of resistant HMCLs and primary MM samples to bortezomib and to other anti-myeloma drugs, providing evidence that MARCKS can modulate drug response. Mechanistically, pMARCKS (phosphorylated form of MARCKS) was found to function as an E2F-1 cofactor to regulate SKP2 transcription. pMARCKS promoted cell-cycle progression by facilitating SKP2 expression, suppressing p27(Kip1) and potentially counteracting drug-induced cell-cycle arrest by promoting Cyclin E/CDK2 activity. Importantly, MARCKS knockdown in combination with bortezomib treatment overcame bortezomib resistance, significantly inhibited tumor growth and prolonged host survival in a MM xenograft model. These data provide a rationale for therapeutic targeting of pMARCKS to improve the outcome of patients with refractory/relapsed MM.

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Although pathologic characteristics of this disease are clear and well established, much remains to be understood about this tumor, particularly at the molecular signaling level. Secreted signaling molecules of the Wnt family have been widely investigated and found to play a central role in controlling embryonic bone development, bone mass, and postnatal bone regeneration. A variety of studies also suggest that Wnt signaling pathway is closely associated with bone malignancies, including breast or prostate cancer induced bone metastasis, multiple myeloma, as well as osteosarcoma. Here, we provide an overview of the role of Wnt signaling pathway in osteosarcoma development and progression, highlighting the aberrant activation of Wnt pathway in this bone malignancy. We also discuss the potential therapeutic applications for the treatment of osteosarcoma targeting Wnt pathway. J. Cell. Biochem. 115: 625-631, 2014. (c) 2013 Wiley Periodicals, Inc.

Lung cancer remains a major worldwide health problem and patients have high rate of metastasis including bone. Although pathologic characteristics of this disease are clear and well established, much remains to be understood about this tumor, particularly at the molecular signaling level. Secreted signaling molecules of the Wnt family have been widely investigated and found to play a prominent role to induce human malignant diseases, such as breast and prostate cancer. A variety of studies have also demonstrated that the Wnt signaling pathway is closely associated with bone malignancies including osteosarcoma, multiple myeloma, and breast or prostate cancer induced bone metastasis. The aim of this review is to provide a summary regarding the role of the Wnt signaling pathway in lung cancer and bone metastasis, highlighting the aberrant activation of Wnt in this malignancy. We also discuss the potential therapeutic applications for the treatment of lung cancer and cancer induced bone metastasis targeting the Wnt pathway.

Background: Small molecule MIRA-1 induced mutant p53-dependent apoptosis in several types of solid tumours. However, anti-tumour activity of MIRA-1 in haematological malignancies including multiple myeloma (MM) is unknown. In this study, we evaluated the effect of MIRA-1 in MM. Methods: We examined the anti-tumour activity of MIRA-1 alone or in combination with current anti-myeloma agents in a panel of MM cell lines, primary MM samples, and in a mouse xenograft model of MM. Results: MIRA-1 treatment resulted in the inhibition of viability, colony formation, and migration and increase in apoptosis of MM cells irrespective of p53 status accompanied by upregulation of Puma and Bax and downregulation of Mcl-1 and c-Myc. Genetic knockdown of p53 did not abrogate apoptotic response of MIRA-1. MIRA-1 triggered activation of PERK and IRE- α leading to splicing of XBP1 indicating an association of endoplasmic reticulum stress response. Furthermore, combined treatment of MIRA-1 with dexamethasone, doxorubicin or velcade displayed synergistic response in MM cells. Importantly, MIRA-1 alone or in combination with dexamethasone retarded tumour growth and prolonged survival without showing any untoward toxicity in the mice bearing MM tumour. Conclusions: Our data provide the preclinical framework for clinical evaluation of MIRA-1 as a novel therapeutic agent to improve patient outcome in MM.

In adults with precursor-B lymphoblastic leukemia (BCP-ALL) there remain a majority of patients who fall in an intermediate cytogenetics risk category with a heterogenous outcome. We analyzed immunophenotypic and cytogenetic factors retrospectively in 126 consecutive adults with BCR-ABL negative BCP-ALL who were treated with a pediatric-based protocol at a single institution over a 10 year period. In addition to age, WBC and cytogenetic findings, CD13 positivity was an independent poor prognostic indicator for overall survival (OS, p=0.049), event-free survival (EFS, p=0.013), and relapse-free survival (RFS, p<0.001). The prognostic value of CD13 was primarily seen in patients with normal or intermediate risk cytogenetics. A risk model that includes age>60 years, WBC>30×109/L, SWOG high/very high risk cytogenetics and CD13 positivity, performs better than a risk model of cytogenetics alone for stratifying patients by OS (p=0.001), EFS (p=7×10−4) and RFS (p=8×10−4). Incorporating CD13 into a scoring system provides high discrimination for relapse risk and survival.

The following article from Proteomics, “Isocratic method for affinity enrichment of covalently‐linked peptides in cyanogen bromide cleavage of proteins” by T. Shi, J. Liu, C. Yan and X. Wang, published online as “Accepted Article” on 4 July 2011 (DOI: 10.1002/pmic.201100126) in the Wiley Online Library (http://www.onlinelibrary.wiley.com), has been retracted by agreement between the authors, the Editor‐in‐Chief and Wiley‐VCH Verlag GmbH & Co. KGaA. The retraction has been agreed due to partial overlap between this article and the following article published in Analytical Chemistry, “Method for the affinity purification of covalently linked peptides following cyanogens bromide cleavage of proteins” by T. Shi et al. (Anal. Chemistry 2009, 81, 9885–9895). In addition, the author T. Shi was wrongly affiliated with the Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.

The low resolution structure of a protein can sometimes be inferred from information about existing disulfide bridges or experimentally introduced chemical crosslinks. Frequently, this task involves enzymatic digestion of a protein followed by mass spectrometry-based identification of covalently linked peptides. To facilitate this task, we developed a method for the enrichment of covalently linked peptides following the chemical cleavage of a protein. The method capitalizes on the availability of homoserine lactone moieties at the C-termini of cyanogen bromide cleavage products which support selective conjugation of affinity tags. The availability of two C-termini within covalently linked peptides allows for the conjugation of two distinct affinity tags and thereby enables subsequent removal of unmodified peptides by tandem affinity chromatography. Here, we demonstrate the stepwise implementation of this method using a polyhistidine tag and a biotin tag for the selective two-step purification of covalently linked cyanogen bromide fragments from increasingly complex protein samples. The method is independent of the nature of the covalent bond, is adaptable to fully denaturing conditions, and requires only low picomole quantities of starting material.