Publications by authors named "Kurt Weiss"

55 Publications

Differential expression of angiogenesis markers HSP70, HSP90, VEGF and pERK1/2 in both components of dedifferentiated chondrosarcomas.

J Bone Oncol 2021 Aug 18;29:100370. Epub 2021 May 18.

Orthopedic Surgery, University of Pittsburgh Medical Center, United States.

Dedifferentiated chondrosarcomas (DDCS) are highly malignant bimorphic mesenchymal tumors with poor outcome and limited treatment options. Genes and proteins involved in angiogenesis play an important role in the development of invasion and metastasis. Immunohistochemical stains targeting HSP70, pERK1/2 and VEGFA were applied to a TMA containing 29 DDCS cases representing both tumor components. Higher expression of HSP70 and pERK1/2 was noted in the dedifferentiated component. RNA sequencing performed in 8 paired cases of DDCS comparing well differentiated and dedifferentiated components, showed higher expression of several family members and in the dedifferentiated component. Furthermore, high mobility group AT-hook 2 () and nuclear proto-oncogene demonstrated higher expression in the dedifferentiated component. Thus, the well differentiated and dedifferentiated components of DDCS are different, histologically and transcriptomically. The dedifferentiated component of DDCS shows higher expression of markers that are associated with malignant behavior. Some of these may represent future treatment targets.
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http://dx.doi.org/10.1016/j.jbo.2021.100370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167291PMC
August 2021

Prognostic factors and survival of patients undergoing surgical intervention for breast cancer bone metastases.

J Bone Oncol 2021 Aug 3;29:100363. Epub 2021 May 3.

Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

Introduction: Bone is the most common distant site of breast cancer metastasis. Skeletal lesions can cause significant morbidity due to pain, pathologic fracture, and electrolyte abnormalities. Current treatment for patients with bone metastases (BoM) from breast cancer is highly personalized and often involves a multidisciplinary approach with chemotherapy, hormone therapy, bone-targeted antiresorptive agents, radiation therapy, and surgery. We have retrospectively collected clinical data from a series of patients with bone metastases to evaluate the clinical characteristics, prognostic factors, and survival patterns of patients with breast cancer BoM receiving standard multimodal therapy.

Methods: A consecutive series of 167 patients with breast cancer BoM treated at a single institution between August 2013 and March 2020 were identified. Clinical information was obtained from the medical record and survival analyses were performed to evaluate patient outcomes and identify prognostic factors.

Results: Thirty-seven patients (22%) presented with BoM - bone metastases at the time of breast cancer diagnosis - and were 2.6 times more likely to die within the study period than those with asynchronous BoM (HR = 2.62, p = <0.0001). Patients who received bone-targeted medical therapy were 61% less likely to die after BoM diagnosis than those who did not (HR = 0.39, p = 0.001). Operative stabilization of BoM was more frequently employed in patients with lytic (p = 0.02) or mixed (p = 0.02) tumors than it was for those with blastic lesions. Patients treated with surgery had a lower overall bone metastasis survival than those treated without (p < 0.03).

Discussion: These findings reflect the current patterns in metastatic breast cancer treatment and associated outcomes. In a series of 167 consecutive patients, we demonstrate the natural history of breast cancer with BoM being treated with modern multimodal therapy. Understanding these treatment patterns and prognostic factors enhances the provider's ability to counsel patients and direct appropriate treatments.
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http://dx.doi.org/10.1016/j.jbo.2021.100363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8143999PMC
August 2021

Tutorial: practical considerations for tissue clearing and imaging.

Nat Protoc 2021 06 21;16(6):2732-2748. Epub 2021 May 21.

Morgridge Institute for Research, Madison, WI, USA.

Tissue clearing has become a powerful technique for studying anatomy and morphology at scales ranging from entire organisms to subcellular features. With the recent proliferation of tissue-clearing methods and imaging options, it can be challenging to determine the best clearing protocol for a particular tissue and experimental question. The fact that so many clearing protocols exist suggests there is no one-size-fits-all approach to tissue clearing and imaging. Even in cases where a basic level of clearing has been achieved, there are many factors to consider, including signal retention, staining (labeling), uniformity of transparency, image acquisition and analysis. Despite reviews citing features of clearing protocols, it is often unknown a priori whether a protocol will work for a given experiment, and thus some optimization is required by the end user. In addition, the capabilities of available imaging setups often dictate how the sample needs to be prepared. After imaging, careful evaluation of volumetric image data is required for each combination of clearing protocol, tissue type, biological marker, imaging modality and biological question. Rather than providing a direct comparison of the many clearing methods and applications available, in this tutorial we address common pitfalls and provide guidelines for designing, optimizing and imaging in a successful tissue-clearing experiment with a focus on light-sheet fluorescence microscopy (LSFM).
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http://dx.doi.org/10.1038/s41596-021-00502-8DOI Listing
June 2021

A Novel Mouse Model for SNP in Steroid Receptor Co-Activator-1 Reveals Role in Bone Density and Breast Cancer Metastasis.

Endocrinology 2021 Aug;162(8)

Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.

The steroid receptor coactivator-1 (SRC-1) is a nuclear receptor co-activator, known to play key roles in both estrogen response in bone and in breast cancer metastases. We previously demonstrated that the P1272S single nucleotide polymorphism (SNP; P1272S; rs1804645) in SRC-1 decreases the activity of estrogen receptor in the presence of selective estrogen receptor modulators (SERMs) and that it is associated with a decrease in bone mineral density (BMD) after tamoxifen therapy, suggesting it may disrupt the agonist action of tamoxifen. Given such dual roles of SRC-1 in the bone microenvironment and in tumor cell-intrinsic phenotypes, we hypothesized that SRC-1 and a naturally occurring genetic variant, P1272S, may promote breast cancer bone metastases. We developed a syngeneic, knock-in mouse model to study if the SRC-1 SNP is critical for normal bone homeostasis and bone metastasis. Our data surprisingly reveal that the homozygous SRC-1 SNP knock-in increases tamoxifen-induced bone protection after ovariectomy. The presence of the SRC-1 SNP in mammary glands resulted in decreased expression levels of SRC-1 and reduced tumor burden after orthotopic injection of breast cancer cells not bearing the SRC-1 SNP, but increased metastases to the lungs in our syngeneic mouse model. Interestingly, the P1272S SNP identified in a small, exploratory cohort of bone metastases from breast cancer patients was significantly associated with earlier development of bone metastasis. This study demonstrates the importance of the P1272S SNP in both the effect of SERMs on BMD and the development of tumor in the bone.
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http://dx.doi.org/10.1210/endocr/bqab094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8248588PMC
August 2021

Update on Osteosarcoma.

Curr Oncol Rep 2021 Apr 21;23(6):71. Epub 2021 Apr 21.

Division of Orthopaedic Oncology, Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

Purpose Of Review: Osteosarcoma (OSA) is the most common primary tumor of bone, mainly affecting children and adolescents. Here we discuss recent advances in surgical and systemic therapies, and highlight potentially new modalities in preclinical evaluation and prognostication.

Recent Findings: The advent of neoadjuvant and adjuvant chemotherapy has markedly improved the disease-free recurrence and overall survival of OSA. However, treatment efficacy has been stagnant since the 1980s. This plateau has prompted preclinical and clinical research into in precision surgery, inhaled chemotherapy to increase pulmonary drug concentration without systemic side effects, and novel immunomodulators intended to block molecular pathways associated with OSA proliferation and metastasis. With the advent of novel surgical techniques and new forms and vectors for chemotherapy, it is hoped that OSA treatment outcomes will exceed their currently sustained plateau in the near future.
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http://dx.doi.org/10.1007/s11912-021-01053-7DOI Listing
April 2021

The Role of ALDH in the Metastatic Potential of Osteosarcoma Cells and Potential ALDH Targets.

Adv Exp Med Biol 2020 ;1258:157-166

Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

Aldehyde dehydrogenases are a family of enzymes that oxidize aldehydes to carboxylic acids. These enzymes are important in cellular homeostasis during oxidative stress by the elimination of toxic aldehyde by-products from various cellular processes. In osteosarcoma, aldehyde dehydrogenase 1A1has been described as a cancer stem cell marker. Its activity has been found to correlate with metastatic potential and the metastatic phenotype. As such, a more complete understanding of aldehyde dehydrogenase in osteosarcoma will give us a deeper knowledge of its impact on osteosarcoma metastatic potential. Our hope is that this knowledge can be translated into novel antimetastatic therapeutic strategies and thus improve osteosarcoma prognoses.
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http://dx.doi.org/10.1007/978-3-030-43085-6_10DOI Listing
December 2020

Do Patient-derived Spheroid Culture Models Have Relevance in Chondrosarcoma Research?

Clin Orthop Relat Res 2021 03;479(3):477-490

R. Ma, J. Mandell, F. Lu, T. Heim, R. Watters, K. R. Weiss, Musculoskeletal Oncology Laboratory, University of Pittsburgh School of Medicine Department of Orthopaedic Surgery, Pittsburgh, PA, USA.

Background: In high-grade chondrosarcoma, 5-year survival is lower than 50%. Therefore, it is important that preclinical models that mimic the disease with the greatest possible fidelity are used to potentially develop new treatments. Accumulating evidence suggests that two-dimensional (2-D) cell culture may not accurately represent the tumor's biology. It has been demonstrated in other cancers that three-dimensional (3-D) cancer cell spheroids may recapitulate tumor biology and response to treatment with greater fidelity than traditional 2-D techniques. To our knowledge, the formation of patient-derived chondrosarcoma spheroids has not been described.

Questions/purposes: (1) Can patient-derived chondrosarcoma spheroids be produced? (2) Do spheroids recapitulate human chondrosarcoma better than 2-D cultures, both morphologically and molecularly? (3) Can chondrosarcoma spheroids provide an accurate model to test novel treatments?

Methods: Experiments to test the feasibility of spheroid formation of chondrosarcoma cells were performed using HT-1080, an established chondrosarcoma cell line, and two patient-derived populations, TP19-S26 and TP19-S115. Cells were cultured in flasks, trypsinized, and seeded into 96-well ultra-low attachment plates with culture media. After spheroids formed, they were monitored daily by bright-field microscopy. Spheroids were fixed using paraformaldehyde and embedded in agarose. After dehydration with isopropanol, paraffin-embedded spheroids were sectioned, and slides were stained with hematoxylin and eosin. To compare differences and similarities in gene expression between 2-D and 3-D chondrosarcoma cultures and primary tumors, and to determine whether these spheroids recapitulated the biology of chondrosarcoma, RNA was extracted from 2-D cultures, spheroids, and tumors. Quantitative polymerase chain reaction was performed to detect chondrosarcoma markers of interest, including vascular endothelial growth factor alpha, hypoxia-inducible factor 1α, COL2A1, and COL10A1. To determine whether 2-D and 3-D cultures responded differently to novel chondrosarcoma treatments, we compared their sensitivities to disulfiram and copper chloride treatment. To test their sensitivity to disulfiram and copper chloride treatment, 10,000 cells were seeded into 96-well plates for 2-D culturing and 3000 cells in each well for 3-D culturing. After treating the cells with disulfiram and copper for 48 hours, we detected cell viability using quantitative presto-blue staining and measured via plate reader.

Results: Cell-line and patient-derived spheroids were cultured and monitored over 12 days. Qualitatively, we observed that HT-1080 demonstrated unlimited growth, while TP19-S26 and TP19-S115 contracted during culturing relative to their initial size. Hematoxylin and eosin staining of HT-1080 spheroids revealed that cell-cell attachments were more pronounced at the periphery of the spheroid structure than at the core, while the core was less dense. Spheroids derived from the intermediate-grade chondrosarcoma TP19-S26 were abundant in extracellular matrix, and spheroids derived from the dedifferentiated chondrosarcoma TP19-S115 had a higher cellularity and heterogeneity with spindle cells at the periphery. In the HT-1080 cells, differences in gene expression were appreciated with spheroids demonstrating greater expressions of VEGF-α (1.01 ± 0.16 versus 6.48 ± 0.55; p = 0.003), COL2A1 (1.00 ± 0.10 versus 7.46 ± 2.52; p < 0.001), and COL10A1 (1.01 ± 0.19 versus 22.53 ± 4.91; p < 0.001). Differences in gene expressions were also noted between primary tumors, spheroids, and 2-D cultures in the patient-derived samples TP19-S26 and TP19-S115. TP19-S26 is an intermediate-grade chondrosarcoma. With the numbers we had, we could not detect a difference in VEGF-α and HIF1α gene expression compared with the primary tumor. COL2A1 (1.00 ± 0.14 versus 1.76 ± 0.10 versus 335.66 ± 31.13) and COL10A1 (1.06 ± 0.378 versus 5.98 ± 0.45 versus 138.82 ± 23.4) expressions were both greater in the tumor (p (COL2A1) < 0.001; p (COL10A1) < 0.0001) and 3-D cultures (p (COL2A1) = 0.004; p (COL10A1) < 0.0001) compared with 2-D cultures. We could not demonstrate a difference in VEGF-α and HIF1α expressions in TP19-S115, a dedifferentiated chondrosarcoma, in the tumor compared with 2-D and 3-D cultures. COL2A1 (1.00 ± 0.02 versus 1.86 ± 0.18 versus 2.95 ± 0.56) and COL10A1 (1.00 ± 0.03 versus 5.52 ± 0.66 versus 3.79 ± 0.36) expressions were both greater in spheroids (p (COL2A1) = 0.003; p (COL10A1) < 0.0001) and tumors (p (COL2A1) < 0.001; p (COL10A1) < 0.0001) compared with 2-D cultures. Disulfiram-copper chloride treatment demonstrated high cytotoxicity in HT-1080 and SW-1353 chondrosarcoma cells grown in the 2-D monolayer, but 3-D spheroids were highly resistant to this treatment.

Conclusion: We provide preliminary findings that it is possible to generate 3-D spheroids from chondrosarcoma cell lines and two human chondrosarcomas (one dedifferentiated chondrosarcoma and one intermediate-grade chondrosarcoma). Chondrosarcoma spheroids derived from human tumors demonstrated morphology more reminiscent of primary tumors than cells grown in 2-D culture. Spheroids displayed similar expressions of cartilage markers as the primary tumor, and we observed a higher expression of collagen markers in the spheroids compared with cells grown in monolayer. Spheroids also demonstrated greater chemotherapy resistance than monolayer cells, but more patient-derived spheroids are needed to further conclude that 3-D cultures may mimic the chemoresistance that chondrosarcomas demonstrate clinically. Additional studies on patient-derived chondrosarcoma spheroids are warranted.

Clinical Relevance: Chondrosarcomas demonstrate resistance to chemotherapy and radiation, and we believe that if they can be replicated, models such as 3-D spheroids may provide a method to test novel treatments for human chondrosarcoma. Additional comprehensive genomic studies are required to compare 2-D and 3-D models with the primary tumor to determine the most effective way to study this disease in vitro.
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http://dx.doi.org/10.1097/CORR.0000000000001317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899730PMC
March 2021

Evaluating the reoperation rate and hardware durability of three stabilizing implants for 105 malignant pathologic humerus fractures.

Injury 2020 Apr 27;51(4):947-954. Epub 2020 Feb 27.

Department of Orthopaedic Surgery, University of Pittsburgh Medical Center Shadyside, 5200 Centre Ave, Suite 415 Pittsburgh, PA 15232, USA.

Introduction: Many patients sustaining a malignant pathologic humerus fracture (MPHF) elect for surgical stabilization. Complications prompting reoperation can occur, leading to additional quality of life and financial cost. One common event preceding reoperation is a broken implant (BI). The purpose of this study was to identify the rate of reoperation following surgical stabilization of MPHF with three techniques - photodynamic bone stabilization (PBS), intramedullary nail (IMN), and cemented plate fixation (CPF) - and estimate to what extent improved implant durability might prevent reoperation.

Materials And Methods: Retrospective data collection was performed, identifying 105 procedures (100 patients) who underwent non-articular MPHF surgery from 2010-2016: 19 PBS, 65 IMN, 21 CPF. All patients were followed for at least two years or until death.

Results: Reoperation rates were similar at one year (10.5%,6.2%,4.8%, p = 737), two years (15.8%,6.2%,9.5%, p = 375), and final evaluation (15.8%,7.7%,14.3%, p = 248). The rate of BI for PBS, IMN, and CPF was 10.5%,0%, and 4.8% (p = 049 PBS/IMN) at one year, 15.8%,0%, and 9.5% (p = 010 PBS/IMN) at two years, and 15.8%,0%, and 14.3% (p = 010 IMN/PBS, p = 013 IMN/CPF) at final evaluation.

Conclusions: Reoperation rate was not significantly different at any time point. However, IMN surgery resulted in the lowest rate of broken implants (zero), statistically significant versus PBS at all time periods and versus CPF at final follow-up. PBS may eventually offer selected advantages for MPHF management, but current data suggests fragility must be thoughtfully considered.
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http://dx.doi.org/10.1016/j.injury.2020.02.124DOI Listing
April 2020

A Novel Sulforaphane-Regulated Gene Network in Suppression of Breast Cancer-Induced Osteolytic Bone Resorption.

Mol Cancer Ther 2020 02 29;19(2):420-431. Epub 2019 Nov 29.

Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania.

Bone is the most preferred site for colonization of metastatic breast cancer cells for each subtype of the disease. The standard of therapeutic care for breast cancer patients with bone metastasis includes bisphosphonates (e.g., zoledronic acid), which have poor oral bioavailability, and a humanized antibody (denosumab). However, these therapies are palliative, and a subset of patients still develop new bone lesions and/or experience serious adverse effects. Therefore, a safe and orally bioavailable intervention for therapy of osteolytic bone resorption is still a clinically unmet need. This study demonstrates suppression of breast cancer-induced bone resorption by a small molecule (sulforaphane, SFN) that is safe clinically and orally bioavailable. osteoclast differentiation was inhibited in a dose-dependent manner upon addition of conditioned media from SFN-treated breast cancer cells representative of different subtypes. Targeted microarrays coupled with interrogation of The Cancer Genome Atlas data set revealed a novel SFN-regulated gene signature involving cross-regulation of and and their downstream effectors. Both RUNX2 and p65/p50 expression were higher in human breast cancer tissues compared with normal mammary tissues. RUNX2 was recruited at the promotor of Inhibition of osteoclast differentiation by SFN was augmented by doxycycline-inducible stable knockdown of RUNX2. Oral SFN administration significantly increased the percentage of bone volume/total volume of affected bones in the intracardiac MDA-MB-231-Luc model indicating suppression of osteolytic bone resorption by SFN. These results indicate that SFN is a novel inhibitor of breast cancer-induced osteolytic bone resorption and .
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007818PMC
February 2020

Comparison of cachectic and non-cachectic sarcoma patients reveals an important role of Notch signaling in metastasis and myogenesis.

Am J Cancer Res 2019 1;9(8):1746-1756. Epub 2019 Aug 1.

Musculoskeletal Oncology Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine Pittsburgh, PA, USA.

Cancer-associated cachexia is a wasting syndrome that affects up to 50% of cancer patients. It is defined as unintentional weight loss ≥5% over 6 months and characterized by muscle atrophy, fatigue, and anorexia that are refractory to nutritional support. Sarcoma describes a diverse group of malignancies arising from the connective tissues. Sarcoma patients are uniquely susceptible to cancer-associated cachexia given its origins in the musculoskeletal system. Our previous research suggests that sarcoma cells may contribute to sarcoma-associated cachexia (SAC) via establishment of TNF-α-mediated inflammation and dysregulation of muscle homeostasis by abnormal Notch signaling. Here, we examine the role of the Notch pathway and pro-inflammatory cytokines in cells derived from cachectic and non-cachectic human sarcoma patients. We observed increased expression of Notch pathway genes in the cachexia group while no differences in pro-inflammatory cytokines were observed. Co-culture of muscle-derived stem cells (MDSCs) and sarcoma cells demonstrated the inhibition of MDSC maturation with both cachectic and non-cachectic patient cells, corresponding to elevated and Notch pathway expression in MDSCs. Our findings suggest that there is no difference in inflammatory profile between cachexia and non-cachexia sarcoma samples. However, Cachectic sarcoma samples express increased Notch that mediates muscle wasting possibly through inhibition of myogenesis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726982PMC
August 2019

Combination Therapy with Disulfiram, Copper, and Doxorubicin for Osteosarcoma: Support for a Novel Drug Repurposing Strategy.

Sarcoma 2019 11;2019:1320201. Epub 2019 Jul 11.

Musculoskeletal Oncology Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.

Although many cancer cells have significantly higher copper concentrations compared with normal cells and tissues, the role of copper in cancer biology and metastatic disease remains poorly understood. Here, we study the importance of copper in osteosarcoma, which frequently metastasizes to the lungs and is often chemoresistant. K12 and K7M2 are murine OS cells with differing metastatic phenotypes: K7M2 is highly metastatic, whereas K12 is much less so. Intracellular copper levels were determined using atomic absorption. Copper transporters were quantified by qPCR. Cytotoxicity of doxorubicin, disulfiram, and copper(II) chloride was assessed with a cell viability fluorescence stain. Additionally, K7M2 viable cell counts were determined by trypan blue exclusion staining after 72 hours of treatment. Copper levels were found to be significantly higher in K12 OS cells than in K7M2 cells. qPCR showed that K12 cells upregulate the copper influx pump CTR1 and downregulate the copper efflux pump ATP7A compared to K7M2 OS cells. Combination treatment of copper chloride (50 nM) with disulfiram (80 nM) was only cytotoxic to K12 cells. Triple treatment with doxorubicin, disulfiram, and copper displayed potent and durable cytotoxicity of highly metastatic K7M2 cells. We demonstrate here that murine OS cell lines differing in metastatic potential also vary in endogenous copper levels and regulation. Additionally, these differences in copper regulation may contribute to selective cytotoxicity of K12 cells by extremely low doses of copper-potentiated disulfiram. The combination of doxorubicin, disulfiram, and copper should be explored as a therapeutic strategy against OS metastases.
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http://dx.doi.org/10.1155/2019/1320201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657614PMC
July 2019

FGFR4 overexpression and hotspot mutations in metastatic ER+ breast cancer are enriched in the lobular subtype.

NPJ Breast Cancer 2019 27;5:19. Epub 2019 Jun 27.

1Women's Cancer Research Center, UPMC Hillman Cancer Center, Pittsburgh, PA USA.

Invasive lobular carcinoma (ILC) is an understudied subtype of breast cancer that requires novel therapies in the advanced setting. To study acquired resistance to endocrine therapy in ILC, we have recently performed RNA-Sequencing on long-term estrogen deprived cell lines and identified FGFR4 overexpression as a top druggable target. Here, we show that FGFR4 expression also increases dramatically in endocrine-treated distant metastases, with an average fold change of 4.8 relative to the paired primary breast tumor for ILC, and 2.4-fold for invasive ductal carcinoma (IDC). In addition, we now report that FGFR4 hotspot mutations are enriched in metastatic breast cancer, with an additional enrichment for ILC, suggesting a multimodal selection of FGFR4 activation. These data collectively support the notion that FGFR4 is an important mediator of endocrine resistance in ILC, warranting future mechanistic studies on downstream signaling of overexpressed wild-type and mutant FGFR4.
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http://dx.doi.org/10.1038/s41523-019-0114-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597581PMC
June 2019

ASO Author Reflections: Tumor Painting with Indocyanine Green Dye: Why not Sarcoma, too?

Authors:
Kurt R Weiss

Ann Surg Oncol 2019 12 11;26(Suppl 3):628-629. Epub 2019 Jun 11.

Musculoskeletal Oncology Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.

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http://dx.doi.org/10.1245/s10434-019-07397-6DOI Listing
December 2019

A germline BARD1 mutation in a patient with Ewing Sarcoma: Implications for familial testing and counseling.

Pediatr Blood Cancer 2019 09 3;66(9):e27824. Epub 2019 Jun 3.

Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.

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http://dx.doi.org/10.1002/pbc.27824DOI Listing
September 2019

Overall survival and tumor recurrence after surgical resection for primary malignant chest wall tumors: a single-center, single-surgeon experience.

J Orthop Surg (Hong Kong) 2019 May-Aug;27(2):2309499019838296

6 Department of Orthopedic Surgery and Musculoskeletal Oncology, University of Pittsburgh Medical Center and Hillman Cancer Institute, Pittsburgh, PA, USA.

Background And Objectives: Malignant primary chest wall tumors (PCWTs) comprise a rare group of thoracic tumors with unique anatomical considerations, and experience with wide surgical resection is limited to specialty referral centers and specific diagnoses. We investigated the tumor recurrence and overall survival (OS) for patients with a variety of PCWTs diagnoses at our institution.

Methods: From 1991 to 2010, patients with malignant PCWT undergoing wide surgical resection for curative intent under a single surgeon were reviewed. Diagnosis and grade (if applicable) of surgical pathology, along with patient demographics, neoadjuvant chemotherapy or radiation therapy, and outcomes (complications, recurrence, and OS) at follow-up were analyzed.

Results: One hundred fifteen patients were included in the study. The most common tumor diagnoses included pleomorphic sarcoma and liposarcoma. Negative margins were achieved in 70 (74%) of cases. Postoperative complications were reported in 21 (20%) cases. The 5-year survival rate was 54%, while the 10-year survival rate was 29%. The local and distant recurrence rates were 50% and 38%, respectively. OS was significantly less in patients with any recurrence ( p < 0.001) but not significantly different between pathology grades ( p = 0.28).

Conclusions: Wide resection for malignant PCWT is feasible when undertaken for a heterogenous group of diagnoses.
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http://dx.doi.org/10.1177/2309499019838296DOI Listing
April 2020

Atypical Lipomatous Tumors: Does Our Inconsistent Terminology Have Patient Repercussions? Results of a Meta-Analysis.

Am J Clin Oncol 2019 05;42(5):487-492

Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA.

Objectives: Misnaming low-grade lipomatous tumors poses a clinical and medicolegal challenge, potentially subjecting patients to expensive and unnecessary surgeries. The terms atypical lipomatous tumor (ALT) and "well-differentiated" liposarcoma (WDL) have been used interchangeably in pathology reports, scholarly works and consensus recommendations, creating vagaries between low-virulence extremity tumors and retroperitoneal disease with metastatic potential.

Methods: A systematic review was performed on all studies that reported on the local recurrence rate and metastasis of ALTs and WDLs in living human subjects. Local recurrence and metastases were compared using Fisher's Exact Test.

Results: In total, 20 studies evaluated ALTs (n=936), whereas 13 studied WDLs (n=626). Mean follow-up was 6.6±2.0 years (median, 7.0 y). No metastatic disease was observed among ALTs, whereas 15 patients with WDLs (2.7%, P<0.0001) had metastases. The local recurrence rate of ALTs was significantly lower than WDLs after both marginal (15.1%, 141/936 vs. 46.0%, 288/626, P<0.0001) and wide excisions (3.3%, 2/59 in ALT vs. 17.4%, 19/109, P=0.007).

Conclusions: ALT should be reserved for extremity lesions meeting appropriate histopathologic criteria that represent nonmetastatic disease, reducing over-diagnosis, over-treatment, and patient risk.
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http://dx.doi.org/10.1097/COC.0000000000000540DOI Listing
May 2019

Tumor Resection Guided by Intraoperative Indocyanine Green Dye Fluorescence Angiography Results in Negative Surgical Margins and Decreased Local Recurrence in an Orthotopic Mouse Model of Osteosarcoma.

Ann Surg Oncol 2019 Mar 27;26(3):894-898. Epub 2018 Dec 27.

Musculoskeletal Oncology Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.

Background: Surgical resection with negative margins is the foundation of extremity sarcoma management. Failure to achieve negative surgical margins can result in local recurrence (LR), a potentially devastating complication. Indocyanine green (ICG) is a US FDA-approved fluorophore previously used to guide carcinoma resections. We investigated the potential of ICG as an intraoperative guide during experimental sarcoma resection.

Methods: Fifty 6-week-old immunocompetent Balb/c female mice received left proximal tibia paraphyseal injections of 5 × 10 K7M2 murine osteosarcoma cells. Animals were separated into two groups (n = 25 each): (1) ICG-assisted surgical resection; and (2) no ICG-assisted resection. Resections were performed 4 weeks after primary tumor engraftment. All animals received 7.5 ug ICG via retro-orbital injection 12 h prior to surgery. ICG fluorescence measurements and clinical evaluations were performed 4 weeks after resection to detect LR.

Results: Eleven of 25 animals from each group developed gross tumors. Four weeks after resection, group 1 had 0/11 tumor recurrences, while group 2 had recurrences in 9/11 (81.8%) experimental mice (p < 0.0002) (Fig. 2). There was a 100% NPV in group 1, and no tumor recurrence with fluorescence-free margins after the primary surgery. Group 2 had a 100% positive predictive value for the development of an LR if any fluorescent signal was present at the surgical margin after resection.

Conclusion: Intraoperative ICG guidance led to reliably negative surgical margins and a diminished LR rate. Given the benign safety profile of ICG and its prior clinical success, these results could be immediately translatable to the clinical realm.
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http://dx.doi.org/10.1245/s10434-018-07114-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373227PMC
March 2019

Frequent ESR1 and CDK Pathway Copy-Number Alterations in Metastatic Breast Cancer.

Mol Cancer Res 2019 02 24;17(2):457-468. Epub 2018 Oct 24.

Women's Cancer Research Center, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, Pennsylvania.

DNA sequencing has identified a limited number of driver mutations in metastatic breast cancer beyond single base-pair mutations in the estrogen receptor (). However, our previous studies and others have observed that structural variants, such as ESR1 fusions, may also play a role. Therefore, we expanded upon these observations by performing a comprehensive and highly sensitive characterization of copy-number (CN) alterations in a large clinical cohort of metastatic specimens. NanoString DNA hybridization was utilized to measure CN gains, amplifications, and deletions of 67 genes in 108 breast cancer metastases, and in 26 cases, the patient-matched primary tumor. For ESR1, a copyshift algorithm was applied to identify CN imbalances at exon-specific resolution and queried large data sets (>15,000 tumors) that had previously undergone next-generation sequencing (NGS). Interestingly, a subset of ER tumors showed increased ESR1 CN (11/82, 13%); three had CN amplifications (4%) and eight had gains (10%). Increased CN was enriched in metastatic specimens versus primary tumors, and this was orthogonally confirmed in a large NGS data set. -amplified tumors showed a site-specific enrichment for bone metastases and worse outcomes than nonamplified tumors. No CN amplifications and only one gain was identified in ER tumors. copyshift was present in 5 of the 11 ESR1-amplified tumors. Other frequent amplifications included , and cell-cycle pathway members and , which showed mutually exclusivity with deletions of , and . IMPLICATIONS: Copy-number alterations of and key CDK pathway genes are frequent in metastatic breast cancers, and their clinical relevance should be tested further.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359977PMC
February 2019

Disulfiram reduces metastatic osteosarcoma tumor burden in an immunocompetent or-thotopic mouse model.

Oncotarget 2018 Jul 10;9(53):30163-30172. Epub 2018 Jul 10.

Musculoskeletal Oncology Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.

Introduction: The overall survival rate of patients with osteosarcoma (OS) and pulmonary metastases has remained stagnant at 15-30% for several decades. Disulfiram (DSF) is an FDA-approved aldehyde dehydrogenase inhibitor that reduces the metastatic phenotype of OS cells . Here we evaluate its efficacy, as compared to doxorubicin chemotherapy, in a previously-validated orthotopic model of metastatic OS.

Results: All treatment groups displayed a significantly reduced quantitative OS metastatic burden compared with controls. The metastatic burden of Lo DSF-treated animals was equivalent to the DXR group. Ninety-five percent of control animals displayed evidence of metastatic disease, which was significantly greater than all treatment groups.

Discussion: Disulfiram treatment resulted in a reduced burden of OS metastatic disease compared with controls. This was statistically-equivalent to doxorubicin. No additive effect was observed between these two therapies.

Materials And Methods: One-hundred twenty immunocompetent Balb/c mice received proximal tibia paraphyseal injections of 5 × 10 K7M2 murine OS cells. Therapy began three weeks after injection: saline (control), low-dose disulfiram (Lo DSF), high-dose disulfiram (Hi DSF), doxorubicin (DXR), Lo DSF + DXR, and Hi DSF + DXR. Transfemoral amputations were performed at 4 weeks. Quantitative metastatic tumor burden was measured using near-infrared indocyanine green (ICG) angiography.
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http://dx.doi.org/10.18632/oncotarget.25733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6059028PMC
July 2018

Nrf2 prevents Notch-induced insulin resistance and tumorigenesis in mice.

JCI Insight 2018 03 8;3(5). Epub 2018 Mar 8.

Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Insulin resistance is associated with increased incidence and enhanced progression of cancers. However, little is known about strategies that can effectively ameliorate insulin resistance and consequently halt cancer progression. Herein, we propose that the transcription factor Nrf2 (also known as Nfe2l2) may be such a target, given its central role in disease prevention. To this end, we developed a mouse that overexpresses the Notch intracellular domain in adipocytes (AdNICD), leading to lipodystrophy-induced severe insulin resistance and subsequent development of sarcomas, as a model reflecting that Notch signaling is deregulated in cancers and shows positive associations with insulin resistance and fatty liver disease in humans. Nrf2 pathway activation was achieved by knocking down Keap1, a repressor of Nrf2, in the AdNICD background. Constitutively enhanced Nrf2 signaling in this setting led to prevention of hepatic steatosis, dyslipidemia, and insulin resistance by repressing hepatic lipogenic pathways and restoration of the hepatic fatty acid profile to control levels. This protective effect of Nrf2 against diabetes extended to significant reduction and delay in sarcoma incidence and latency. Our study highlights that the Nrf2 pathway, which has been induced by small molecules in clinical trials, is a potential therapeutic target against insulin resistance and subsequent risk of cancer.
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http://dx.doi.org/10.1172/jci.insight.97735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5922294PMC
March 2018

Considerations for the Long Term Treatment of Pediatric Sarcoma Survivors.

Indian J Orthop 2018 Jan-Feb;52(1):77-80

Karmanos Cancer Center, Detroit, Michigan, USA.

Sarcomas are primary malignancies of the connective tissues. They are exceedingly rare in adults, but much more common in children. The historically recent advent of cytotoxic chemotherapy for pediatric sarcomas has revolutionized the treatment of these diseases and dramatically improved their prognoses. There is thus a population of pediatric sarcoma survivors that are "coming of age" as adults. However, this progress is not without consequences. Due to aggressive treatment protocols that include various combinations of surgery, chemotherapy, and radiation therapy, pediatric sarcoma survivors are at risk of myriad physical, medical, and psychological difficulties as they enter adulthood. These include but are not limited to physical disabilities, chemotherapy-induced cardiac issues, second malignancies, and anxiety. These patients pose unique challenges to their adult primary care physicians. One possible solution to these challenges is multidisciplinary sarcoma survivorship clinics. By paying greater attention to the unique issues of pediatric sarcoma survivors, involved physicians can maximize the physical and emotional health of pediatric sarcoma survivors.
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http://dx.doi.org/10.4103/ortho.IJOrtho_248_17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5791236PMC
February 2018

Quantitative Primary Tumor Indocyanine Green Measurements Predict Osteosarcoma Metastatic Lung Burden in a Mouse Model.

Clin Orthop Relat Res 2018 03;476(3):479-487

M. S. Fourman, J. B. Mandell, S. Yu, J. C. Tebbets, J. A. Crasto, K. R. Weiss Cancer Stem Cell Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA A. Mahjoub School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA; and the Cancer Stem Cell Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA P. E. Alexander Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.

Background: Current preclinical osteosarcoma (OS) models largely focus on quantifying primary tumor burden. However, most fatalities from OS are caused by metastatic disease. The quantification of metastatic OS currently relies on CT, which is limited by motion artifact, requires intravenous contrast, and can be technically demanding in the preclinical setting. We describe the ability for indocyanine green (ICG) fluorescence angiography to quantify primary and metastatic OS in a previously validated orthotopic, immunocompetent mouse model.

Questions/purposes: (1) Can near-infrared ICG fluorescence be used to attach a comparable, quantitative value to the primary OS tumor in our experimental mouse model? (2) Will primary tumor fluorescence differ in mice that go on to develop metastatic lung disease? (3) Does primary tumor fluorescence correlate with tumor volume measured with CT?

Methods: Six groups of 4- to 6-week-old immunocompetent Balb/c mice (n = 6 per group) received paraphyseal injections into their left hindlimb proximal tibia consisting of variable numbers of K7M2 mouse OS cells. A hindlimb transfemoral amputation was performed 4 weeks after injection with euthanasia and lung extraction performed 10 weeks after injection. Histologic examination of lung and primary tumor specimens confirmed ICG localization only within the tumor bed.

Results: Mice with visible or palpable tumor growth had greater hindlimb fluorescence (3.5 ± 2.3 arbitrary perfusion units [APU], defined as the fluorescence pixel return normalized by the detector) compared with those with a negative examination (0.71 ± 0.38 APU, -2.7 ± 0.5 mean difference, 95% confidence interval -3.7 to -1.8, p < 0.001). A strong linear trend (r = 0.81, p < 0.01) was observed between primary tumor and lung fluorescence, suggesting that quantitative ICG tumor fluorescence is directly related to eventual metastatic burden. We did not find a correlation (r = 0.04, p = 0.45) between normalized primary tumor fluorescence and CT volumetric measurements.

Conclusions: We demonstrate a novel methodology for quantifying primary and metastatic OS in a previously validated, immunocompetent, orthotopic mouse model. Quantitative fluorescence of the primary tumor with ICG angiography is linearly related to metastatic burden, a relationship that does not exist with respect to clinical tumor size. This highlights the potential utility of ICG near-infrared fluorescence imaging as a valuable preclinical proof-of-concept modality. Future experimental work will use this model to evaluate the efficacy of novel OS small molecule inhibitors.

Clinical Relevance: Given the histologic localization of ICG to only the tumor bed, we envision the clinical use of ICG angiography as an intraoperative margin and tumor detector. Such a tool may be used as an alternative to intraoperative histology to confirm negative primary tumor margins or as a valuable tool for debulking surgeries in vulnerable anatomic locations. Because we have demonstrated the successful preservation of ICG in frozen tumor samples, future work will focus on blinded validation of this modality in observational human trials, comparing the ICG fluorescence of harvested tissue samples with fresh frozen pathology.
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http://dx.doi.org/10.1007/s11999.0000000000000003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260021PMC
March 2018

Exome-capture RNA sequencing of decade-old breast cancers and matched decalcified bone metastases.

JCI Insight 2017 09 7;2(17). Epub 2017 Sep 7.

Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Bone metastases (BoM) are a significant cause of morbidity in patients with estrogen receptor-positive (ER-positive) breast cancer; yet, characterizations of human specimens are limited. In this study, exome-capture RNA sequencing (ecRNA-seq) on aged (8-12 years), formalin-fixed, paraffin-embedded (FFPE), and decalcified cancer specimens was evaluated. Gene expression values and ecRNA-seq quality metrics from FFPE or decalcified tumor RNA showed minimal differences when compared with matched flash-frozen or nondecalcified tumors. ecRNA-seq was then applied on a longitudinal collection of 11 primary breast cancers and patient-matched synchronous or recurrent BoMs. Overtime, BoMs exhibited gene expression shifts to more Her2 and LumB PAM50 subtype profiles, temporally influenced expression evolution, recurrently dysregulated prognostic gene sets, and longitudinal expression alterations of clinically actionable genes, particularly in the CDK/Rb/E2F and FGFR signaling pathways. Taken together, this study demonstrates the use of ecRNA-seq on decade-old and decalcified specimens and defines recurrent longitudinal transcriptional remodeling events in estrogen-deprived breast cancers.
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http://dx.doi.org/10.1172/jci.insight.95703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621874PMC
September 2017

RhoA/ROCK inhibition improves the beneficial effects of glucocorticoid treatment in dystrophic muscle: implications for stem cell depletion.

Hum Mol Genet 2017 08;26(15):2813-2824

Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, Houston, TX 77054, USA.

Glucocorticoid treatment represents a standard palliative treatment for Duchenne muscular dystrophy (DMD) patients, but various adverse effects have limited this treatment. In an effort to understand the mechanism(s) by which glucocorticoids impart their effects on the dystrophic muscle, and potentially reduce the adverse effects, we have studied the effect of prednisolone treatment in dystrophin/utrophin double knockout (dKO) mice, which exhibit a severe dystrophic phenotype due to rapid muscle stem cell depletion. Our results indicate that muscle stem cell depletion in dKO muscle is related to upregulation of mTOR, and that prednisolone treatment reduces the expression of mTOR and other pro-inflammatory mediators, consequently slowing down muscle stem cell depletion. However, prednisolone treatment was unable to improve the myogenesis of stem cells and reduce fibrosis in dKO muscle. We then studied whether glucocorticoid treatment can be improved by co-administration of an inhibitor of RhoA/ROCK signaling, which can be activated by glucocorticoids and was found in our previous work to be over-activated in dystrophic muscle. Our results indicate that the combination of RhoA/ROCK inhibition and glucocorticoid treatment in dystrophic muscle have a synergistic effect in alleviating the dystrophic phenotype. Taken together, our study not only shed light on the mechanism by which glucocorticoid imparts its beneficial effect on dystrophic muscle, but also revealed the synergistic effect of RhoA/ROCK inhibition and glucocorticoid treatment, which could lead to the development of more efficient therapeutic approaches for treating DMD patients.
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http://dx.doi.org/10.1093/hmg/ddx117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886088PMC
August 2017

Lung cells support osteosarcoma cell migration and survival.

BMC Cancer 2017 01 25;17(1):78. Epub 2017 Jan 25.

Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.

Background: Osteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined.

Methods: Three human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment.

Results: Lung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p <0.05). Lung cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline phosphatase staining.

Conclusions: Lung endothelial HULEC-5a cells are attractants for OS cell migration, proliferation, and survival. The SJSA-1 osteosarcoma cell line demonstrated greater metastatic potential than Saos-2 and U-2 cells. ALDH appears to be involved in the interaction between lung and OS cells, and ALP may be a valuable biomarker for monitoring functional OS changes during metastasis.
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http://dx.doi.org/10.1186/s12885-017-3047-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5267399PMC
January 2017

EZH2 or HDAC1 Inhibition Reverses Multiple Myeloma-Induced Epigenetic Suppression of Osteoblast Differentiation.

Mol Cancer Res 2017 04 23;15(4):405-417. Epub 2017 Jan 23.

Department of Medicine, Division of Hematology-Oncology, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.

In multiple myeloma, osteolytic lesions rarely heal because of persistent suppressed osteoblast differentiation resulting in a high fracture risk. Herein, chromatin immunoprecipitation analyses reveal that multiple myeloma cells induce repressive epigenetic histone changes at the locus that prevent osteoblast differentiation. The most pronounced multiple myeloma-induced changes were at the promoter, converting it from a poised bivalent state to a repressed state. Previously, it was observed that multiple myeloma induces the transcription repressor GFI1 in osteoblast precursors, which correlates with decreased expression, thus prompting detailed characterization of the multiple myeloma and TNFα-dependent GFI1 response element within the promoter. Further analyses reveal that multiple myeloma-induced GFI1 binding to in osteoblast precursors and recruitment of the histone modifiers HDAC1, LSD1, and EZH2 is required to establish and maintain repression in osteogenic conditions. These GFI1-mediated repressive chromatin changes persist even after removal of multiple myeloma. Ectopic GFI1 is sufficient to bind to , recruit HDAC1 and EZH2, increase H3K27me3 on the gene, and prevent osteogenic induction of endogenous expression. knockdown in MC4 cells blocked multiple myeloma-induced recruitment of HDAC1 and EZH2 to , acquisition of repressive chromatin architecture, and suppression of osteoblast differentiation. Importantly, inhibition of EZH2 or HDAC1 activity in pre-osteoblasts after multiple myeloma exposure or in osteoblast precursors from patients with multiple myeloma reversed the repressive chromatin architecture at and rescued osteoblast differentiation. This study suggests that therapeutically targeting EZH2 or HDAC1 activity may reverse the profound multiple myeloma-induced osteoblast suppression and allow repair of the lytic lesions. .
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0242-TDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524543PMC
April 2017

Notch Signaling Mediates Skeletal Muscle Atrophy in Cancer Cachexia Caused by Osteosarcoma.

Sarcoma 2016 9;2016:3758162. Epub 2016 Jun 9.

Cancer Stem Cell Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA.

Skeletal muscle atrophy in cancer cachexia is mediated by the interaction between muscle stem cells and various tumor factors. Although Notch signaling has been known as a key regulator of both cancer development and muscle stem cell activity, the potential involvement of Notch signaling in cancer cachexia and concomitant muscle atrophy has yet to be elucidated. The murine K7M2 osteosarcoma cell line was used to generate an orthotopic model of sarcoma-associated cachexia, and the role of Notch signaling was evaluated. Skeletal muscle atrophy was observed in the sarcoma-bearing mice, and Notch signaling was highly active in both tumor tissues and the atrophic skeletal muscles. Systemic inhibition of Notch signaling reduced muscle atrophy. In vitro coculture of osteosarcoma cells with muscle-derived stem cells (MDSCs) isolated from normal mice resulted in decreased myogenic potential of MDSCs, while the application of Notch inhibitor was able to rescue this repressed myogenic potential. We further observed that Notch-activating factors reside in the exosomes of osteosarcoma cells, which activate Notch signaling in MDSCs and subsequently repress myogenesis. Our results revealed that signaling between tumor and muscle via the Notch pathway may play an important role in mediating the skeletal muscle atrophy seen in cancer cachexia.
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http://dx.doi.org/10.1155/2016/3758162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917717PMC
July 2016

Characterization of axonal transport defects in Drosophila Huntingtin mutants.

J Neurogenet 2016 Sep - Dec;30(3-4):212-221. Epub 2016 Jul 22.

a Department of Biology , The Picower Institute for Learning and Memory, Massachusetts Institute of Technology , Cambridge , MA , USA.

Polyglutamine (polyQ) expansion within Huntingtin (Htt) causes the fatal neurodegenerative disorder Huntington's Disease (HD). Although Htt is ubiquitously expressed and conserved from Drosophila to humans, its normal biological function is still being elucidated. Here we characterize a role for the Drosophila Htt homolog (dHtt) in fast axonal transport (FAT). Generation and expression of transgenic dHtt-mRFP and human Htt-mRFP fusion proteins in Drosophila revealed co-localization with mitochondria and synaptic vesicles undergoing FAT. However, Htt was not ubiquitously associated with the transport machinery, as it was excluded from dense-core vesicles and APLIP1 containing vesicles. Quantification of cargo movement in dHtt deficient axons revealed that mitochondria and synaptic vesicles show a decrease in the distance and duration of transport, and an increase in the number of pauses. In addition, the ratio of retrograde to anterograde flux was increased in mutant animals. Dense-core vesicles did not display similar defects in processivity, but did show altered retrograde to anterograde flux along axons. Given the co-localization with mitochondria and synaptic vesicles, but not dense-core vesicles, the data suggest dHtt likely acts locally at cargo interaction sites to regulate processivity. An increase in dynein heavy chain expression was also observed in dHtt mutants, suggesting that the altered flux observed for all cargo may represent secondary transport changes occurring independent of dHtt's primary function. Expression of dHtt in a milton (HAP1) mutant background revealed that the protein does not require mitochondria or HAP1 to localize along axons, suggesting Htt has an independent mechanism for coupling with motors to regulate their processivity during axonal transport.
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http://dx.doi.org/10.1080/01677063.2016.1202950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5525331PMC
December 2017

Retinal Targets ALDH Positive Cancer Stem Cell and Alters the Phenotype of Highly Metastatic Osteosarcoma Cells.

Sarcoma 2015 24;2015:784954. Epub 2015 Dec 24.

Cancer Stem Cell Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA 15219, Kazakhstan.

Aldehyde dehydrogenase (ALDH) is a cancer stem cell marker. Retinoic acid has antitumor properties, including the induction of apoptosis and inhibition of proliferation. Retinal, the precursor of retinoic acid, can be oxidized to retinoic acid by dehydrogenases, including ALDH. We hypothesized that retinal could potentially be transformed to retinoic acid with higher efficiency by cancer stem cells, due to the higher ALDH activity. We previously observed that ALDH activity is greater in highly metastatic K7M2 osteosarcoma (OS) cells than in nonmetastatic K12 OS cells. We also demonstrated that ALDH activity correlates with clinical metastases in bone sarcoma patients, suggesting that ALDH may be a therapeutic target specific to cells with high metastatic potential. Our current results demonstrated that retinal preferentially affected the phenotypes of ALDH-high K7M2 cells in contrast to ALDH-low K12 cells, which could be mediated by the more efficient transformation of retinal to retinoic acid by ALDH in K7M2 cells. Retinal treatment of highly metastatic K7M2 cells decreased their proliferation, invasion capacity, and resistance to oxidative stress. Retinal altered the expression of metastasis-related genes. These observations indicate that retinal may be used to specifically target metastatic cancer stem cells in OS.
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http://dx.doi.org/10.1155/2015/784954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706975PMC
January 2016

Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary Tumors, Metastatic Lesions, and Cell-Free DNA of Breast Cancer Patients.

Clin Cancer Res 2016 Mar 23;22(5):1130-7. Epub 2015 Oct 23.

Womens Cancer Research Center, Magee-Women Research Institute, Pittsburgh, Pennsylvania. Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania.

Purpose: Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell-free DNA (cfDNA).

Experimental Design: Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n = 43), bone (n = 12) and brain metastases (n = 38), and cfDNA (n = 29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed.

Results: ESR1 mutations were detected for D538G (n = 13), Y537S (n = 3), and Y537C (n = 1), and not for K303R, S463P, or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3% to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n = 5), mutations were detected in both (n = 3) or in cfDNA only (n = 2). Treatment was associated with changes in ESR1 mutation detection and allele frequency.

Conclusions: ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1-mutant clones are enriched by endocrine therapy. Further studies should address whether sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease. See related commentary by Gu and Fuqua, p. 1034.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-1534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775406PMC
March 2016
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