Qingxia Wei

Side Population Cells Isolated from Mesenchymal Neoplasms Have Tumor Initiating Potential

Although many cancers are maintained by tumor-initiating cells, this has not been shown for mesenchymal tumors, in part due to the lack of unique surface markers that identify mesenchymal progenitors. An alternative technique to isolate stem-like cells is to isolate side population (SP) cells based on efflux of Hoechst 33342 dye. We examined 29 mesenchymal tumors ranging from benign to high-grade sarcomas and identified SP cells in all but six samples. There was a positive correlation between the percentage of SP cells and the grade of the tumor. SP cells preferentially formed tumors when grafted into immunodeficient mice, and only cells from tumors that developed from the SP cells had the ability to initiate tumor formation upon serial transplantation. Although SP cells are able to efflux rhodamine dye in addition to Hoechst 33342, we found that the ability to efflux rhodamine dye did not identify a population of cells enriched for tumor-initiating capacity. Here, we identify a subpopulation of cells within a broad range of benign and malignant mesenchymal tumors with tumor-initiating capacity. In addition, our data suggest that the proportion of SP cells could be used as a prognostic factor and that therapeutically targeting this subpopulation of cells could be used to improve patient outcome.

Beta-catenin regulates wound size and mediates the effect of TGF-beta in cutaneous healing

After cutaneous injury, a variety of cell types are activated to reconstitute the epithelial and dermal components of the skin. β‐Catenin plays disparate roles in keratinocytes and fibroblasts, inhibiting keratinocyte migration and activating fibroblast proliferation, suggesting that β‐catenin could either inhibit or enhance the healing process. How β‐catenin functions in concert with other signaling pathways important in the healing process is unknown. Wound size was examined in mice expressing conditional null or conditional stabilized alleles of β‐catenin, regulated by an adenovirus expressing cre‐recombinase. The size of the wounds in the mice correlated with the protein level of β‐catenin. Using mice expressing these conditional alleles, we found that the wound phenotype imparted by Smad3 deficiency and by the injection of TGF+ before wounding is mediated in part by β‐catenin. TGF+ was not able to regulate proliferation in β‐catenin null fibroblasts, whereas keratinocyte proliferation rate was independent of β‐catenin. When mice are treated with lithium, β‐catenin‐mediated signaling was activated in cutaneous wounds, which healed with a larger size. These results demonstrate a crucial role for β‐catenin in regulating cutaneous wound size. Furthermore, these data implicate mesenchymal cells as playing a critical role regulating wound size.‐Cheon, S. S., Wei, Q., Gurung, A., Youn, A., Bright, T., Poon, R., Whetstone, H., Guha, A., Alman, B. A. Beta‐catenin regulates wound size and mediates the effect of TGF‐beta in cutaneous healing. FASEB J. 20, 692–701 (2006)

High-Grade Glioma Formation Results from Postnatal Pten Loss or Mutant Epidermal Growth Factor Receptor Expression in a Transgenic Mouse Glioma Model

High-grade gliomas are devastating brain tumors associated with a mean survival of <50 weeks. Two of the most common genetic changes observed in these tumors are overexpression/mutation of the epidermal growth factor receptor (EGFR) vIII and loss of PTEN/MMAC1 expression. To determine whether somatically acquired EGFRvIII expression or Pten loss accelerates high-grade glioma development, we used a previously characterized RasB8 glioma-prone mouse strain, in which these specific genetic changes were focally introduced at 4 weeks of age. We show that both postnatal EGFRvIII expression and Pten inactivation in RasB8 mice potentiate high-grade glioma development. Moreover, we observe a concordant loss of Pten and EGFR overexpression in nearly all high-grade gliomas induced by either EGFRvIII introduction or Pten inactivation. This novel preclinical model of high-grade glioma will be useful in evaluating brain tumor therapies targeted to the pathways specifically dysregulated by EGFR expression or Pten loss.

Macrophages Promote Osteoblastic Differentiation In Vivo: Implications in Fracture Repair and Bone Homeostasis

Macrophages are activated in inflammation and during early phases of repair processes. Interestingly, they are also present in bone during development, but their function during this process is unclear. Here, we explore the function of macrophages in bone development, growth, and repair using transgenic mice to constitutively or conditionally deplete macrophages. Depletion of macrophages led to early skeletal growth retardation and progressive osteoporosis. By 3 months of age, macrophage‐deficient mice displayed a 25% reduction in bone mineral density and a 70% reduction in the number of trabecular bone compared to control littermates. Despite depletion of macrophages, functional osteoclasts were still present in bones, lining trabecular bone and the endosteal surface of the cortical bone. Furthermore, ablation of macrophages led to a 60% reduction in the number of bone marrow mesenchymal progenitor cells and a decrease in the ability of these cells to differentiate to osteoblasts. When macrophages were depleted during fracture repair, bone union was impaired. Calluses from macrophage‐deficient animals were smaller, and contained less bone and more fibrotic tissue deposition. Taken together, this shows that macrophages are crucial for maintaining bone homeostasis and promoting fracture repair by enhancing the differentiation of mesenchymal progenitors.

Erratum: Exposure to a youthful circulation rejuvenates bone repair through modulation of β-catenin.

The capacity for tissues to repair and regenerate diminishes with age. We sought to determine the age-dependent contribution of native mesenchymal cells and circulating factors on in vivo bone repair. Here we show that exposure to youthful circulation by heterochronic parabiosis reverses the aged fracture repair phenotype and the diminished osteoblastic differentiation capacity of old animals. This rejuvenation effect is recapitulated by engraftment of young haematopoietic cells into old animals. During rejuvenation, β-catenin signalling, a pathway important in osteoblast differentiation, is modulated in the early repair process and required for rejuvenation of the aged phenotype. Temporal reduction of β-catenin signalling during early fracture repair improves bone healing in old mice. Our data indicate that young haematopoietic cells have the capacity to rejuvenate bone repair and this is mediated at least in part through β-catenin, raising the possibility that agents that modulate β-catenin can improve the pace or quality of fracture repair in the ageing population.

Mutant IDH is sufficient to initiate enchondromatosis in mice.

Significance Current genomic and biochemical analysis revealed mutations in isocitrate dehydrogenase (IDH) genes associated with several neoplasms and a novel enzymatic activity of IDH mutations to catalyze α-ketoglutarate to d-2-hydroxyglutarate, contributing to tumorigenesis. We identified a broad range of IDH1 mutations, including a previously unidentified IDH1-R132Q mutation, in cartilage tumors. Cartilage-specific Col2a1-Cre/ERT2;Idh1-R132 mutant knock-in mice developed multiple enchondroma-like lesions. These data show that mutant Idh in growth-plate cells causes persistence of chondrocytes, giving rise to enchondromas adjacent to the growth cartilage in bone. Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas.

Hedgehog and Notch Signaling Regulate Self-Renewal of Undifferentiated Pleomorphic Sarcomas

Like many solid tumors, sarcomas are heterogeneous and include a small fraction of the so-called side population (SP) cells with stem-like tumor-initiating potential. Here, we report that SP cells from a soft tissue tumor of enigmatic origin termed undifferentiated pleomorphic sarcoma (also known as malignant fibrous histiocytoma or MFH sarcoma) display activation of both the Hedgehog and Notch pathways. Blockade to these pathways in murine xenograft models, this human cancer decreased the proportion of SP cells present and suppressed tumor self-renewal, as illustrated by the striking inability of xenograft tumors subjected to pathway blockade to be serially transplanted to new hosts. In contrast, conventional chemotherapies increased the proportion of SP cells present in tumor xenografts and did not affect their ability to be serially transplanted. SP cells from these tumors displayed an unexpectedly high proliferation rate which was selectively inhibited by Hedgehog and Notch blockade compared with conventional chemotherapies. Together, our findings deepen the concept that Hedgehog and Notch signaling are fundamental drivers of tumor self-renewal, acting in a small population of tumor-initiating cells present in tumors. Furthermore, our results suggest not only novel treatment strategies for deadly recurrent unresectable forms of this soft tumor subtype, but also potential insights into its etiology which has been historically controversial.

Hedgehog Pathway Inhibition in Chondrosarcoma Using the Smoothened Inhibitor IPI-926 Directly Inhibits Sarcoma Cell Growth

Hedgehog (Hh) pathway inhibition in cancer has been evaluated in both the ligand-independent and ligand-dependent settings, where Hh signaling occurs either directly within the cancer cells or within the nonmalignant cells of the tumor microenvironment. Chondrosarcoma is a malignant tumor of cartilage in which there is ligand-dependent activation of Hh signaling. IPI-926 is a potent, orally delivered small molecule that inhibits Hh pathway signaling by binding to Smoothened (SMO). Here, the impact of Hh pathway inhibition on primary chondrosarcoma xenografts was assessed. Mice bearing primary human chondrosarcoma xenografts were treated with IPI-926. The expression levels of known Hh pathway genes, in both the tumor and stroma, and endpoint tumor volumes were measured. Gene expression profiling of tumors from IPI-926–treated mice was conducted to identify potential novel Hh target genes. Hh target genes were studied to determine their contribution to the chondrosarcoma neoplastic phenotype. IPI-926 administration results in downmodulation of the Hh pathway in primary chondrosarcoma xenografts, as demonstrated by evaluation of the Hh target genes GLI1 and PTCH1, as well as inhibition of tumor growth. Chondrosarcomas exhibited autocrine and paracrine Hh signaling, and both were affected by IPI-926. Decreased tumor growth is accompanied by histopathologic changes, including calcification and loss of tumor cells. Gene profiling studies identified genes differentially expressed in chondrosarcomas following IPI-926 treatment, one of which, ADAMTSL1, regulates chondrosarcoma cell proliferation. These studies provide further insight into the role of the Hh pathway in chondrosarcoma and provide a scientific rationale for targeting the Hh pathway in chondrosarcoma. Mol Cancer Ther; 13(5); 1259–69. ©2014 AACR.

Identification of CD146 as a marker enriched for tumor-propagating capacity reveals targetable pathways in primary human sarcoma

Tumor-propagating cells (TPCs) are believed to drive cancer initiation, progression and recurrence. These cells are characterized by enhanced tumorigenicity and self-renewal. The ability to identify such cells in primary human sarcomas relies on the dye exclusion ability of tumor side population (SP) cells. Here, we performed a high-throughput cell surface antigen screen and found that CD146 is enriched in the SP population. In vivo serial transplantation assays showed that CD146+ cells are highly tumorigenic, capable of self-renewal and thus enriches for the TPC population. In addition, depletion of SP cells from the CD146+ population show that CD146+ cells and SP cells are a distinct and overlapping TPC populations. Gene expression profiling of CD146+ and SP cells revealed multiple pathways commonly upregulated in both of these populations. Inhibition of one of these upregulated pathways, Notch signaling, significantly reduced tumor growth and self-renewal. Our data demonstrate that CD146 is an effective cell surface marker for enriching TPCs in primary human sarcomas. Targeting differentially activated pathways in TPCs may provide new therapeutic strategies for treating sarcoma.

Abstract A04: Molecular characterization of undifferentiated pleomorphic sarcomas

Objective: Undifferentiated pleomorphic sarcomas (UPS) are high-grade soft-tissue sarcomas (STS) with no apparent line of differentiation. Despite overall gains in the management of STS, progress in therapies based on molecular targets and understanding the unknown genetic diversity is needed, especially in UPS where response to therapy is limited. Using integrated multi-omic analysis, we aim to characterize UPS for recurrent genetic mutations, altered pathways, and the mutational spectrum. These insights into UPS may enable robust classification, identify prognostic markers, and lead to novel treatment approaches. Methods: Clinically well-annotated cases of UPS with matched blood specimens were retrieved from the Sarcoma Tumor Bank at Mount Sinai Hospital, Toronto. Using current World Health Organization histopathologic classification, reevaluated cases were subjected to whole-genome (WGS) or whole-exome sequencing (WES) and RNA sequencing (RNA-seq). Cell lines and/or mouse xenografts were established from some of the UPS tumors. The goal of this study was to integrate annotated somatic mutations, expressed transcripts, mRNA fusions, structural rearrangements, and copy number aberrations to detect recurrent molecular features in UPS. Results: At present, 43 UPS tumor, 8 matched metastatic samples, 6 mouse xenografts, and 4 cell lines have been sequenced by WGS or WES and RNA-seq. Among the 34 cases analyzed by WES, known cancer genes TP53, LRP1B, ATRX, and RB1 are found to recurrently contain somatic variants. Among these genes, nonsilent somatic variants were found in 18/34 tumors. The somatic variant burden among the WES tumors is approximately 5 muts/Mb. Two UPS cases were hypermutated with a somatic variant burden >10 muts/Mb. In examining the base changes in the hypermutated cases, the two display a C>A bias, in which the 59 and 39 bases flanking the C>A alterations in the hypermutated cases have a distinct pattern (TpCpN, CpCpA) that differ from the other samples. These somatic C>A biases in their trinucleotide context were not detected in >10K tumor cases across 33 cancer types sequenced by TCGA. An RB1 and ATXN10 fusion transcript that lacks the RB1 tumor-suppressing domain was confirmed in one case, while the derived cell line also expressed the RB1-ATXN10 fusion. The cell lines and xenograft models retained 8-71% of their matched tumors9 somatic variants. The metastatic samples contained between 13-63% of the somatic variants also identified in their matched primary tumors, among the 4 WES metastatic samples analyzed thus far. Conclusion: Initial analyzes of UPS tumors has identified recurrently mutated cancer-related genes and a unique pattern of C>A bias not previously seen. Ongoing multi-omic analysis, integrating the RNA-seq with the somatic alterations, of UPS tumors may reveal further genomic events that may aid in stratification of UPS, understand the drivers of the disease, identify actionable targets, and realize novel therapeutics. Citation Format: Andrew Seto, Nalan Gokgoz, Qingxia Wei, Yael Babichev, Brendan C. Dickson, Benjamin Alman, Rebecca A. Gladdy, Adam Shlien, Jay Wunder, Irene L. Andrulis. Molecular characterization of undifferentiated pleomorphic sarcomas [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A04.