Choh Yeung

The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis.

Metastatic cancers, once established, are the primary cause of mortality associated with cancer. Previously, we used a genomic approach to identify metastasis-associated genes in cancer. From this genomic data, we selected ezrin for further study based on its role in physically and functionally connecting the actin cytoskeleton to the cell membrane. In a mouse model of osteosarcoma, a highly metastatic pediatric cancer, we found ezrin to be necessary for metastasis. By imaging metastatic cells in the lungs of mice, we showed that ezrin expression provided an early survival advantage for cancer cells that reached the lung. AKT and MAPK phosphorylation and activity were reduced when ezrin protein was suppressed. Ezrin-mediated early metastatic survival was partially dependent on activation of MAPK, but not AKT. To define the relevance of ezrin in the biology of metastasis, beyond the founding mouse model, we examined ezrin expression in dogs that naturally developed osteosarcoma. High ezrin expression in dog tumors was associated with early development of metastases. Consistent with this data, we found a significant association between high ezrin expression and poor outcome in pediatric osteosarcoma patients.

Phosphoprotein Pathway Mapping: Akt/Mammalian Target of Rapamycin Activation Is Negatively Associated with Childhood Rhabdomyosarcoma Survival

Mapping of protein signaling networks within tumors can identify new targets for therapy and provide a means to stratify patients for individualized therapy. Despite advances in combination chemotherapy, the overall survival for childhood rhabdomyosarcoma remains approximately 60%. A critical goal is to identify functionally important protein signaling defects associated with treatment failure for the 40% nonresponder cohort. Here, we show, by phosphoproteomic network analysis of microdissected tumor cells, that interlinked components of the Akt/mammalian target of rapamycin (mTOR) pathway exhibited increased levels of phosphorylation for tumors of patients with short-term survival. Specimens (n = 59) were obtained from the Childrens Oncology Group Intergroup Rhabdomyosarcoma Study (IRS) IV, D9502 and D9803, with 12-year follow-up. High phosphorylation levels were associated with poor overall and poor disease-free survival: Akt Ser(473) (overall survival P < 0.001, recurrence-free survival P < 0.0009), 4EBP1 Thr(37/46) (overall survival P < 0.0110, recurrence-free survival P < 0.0106), eIF4G Ser(1108) (overall survival P < 0.0017, recurrence-free survival P < 0.0072), and p70S6 Thr(389) (overall survival P < 0.0085, recurrence-free survival P < 0.0296). Moreover, the findings support an altered interrelationship between the insulin receptor substrate (IRS-1) and Akt/mTOR pathway proteins (P < 0.0027) for tumors from patients with poor survival. The functional significance of this pathway was tested using CCI-779 in a mouse xenograft model. CCI-779 suppressed phosphorylation of mTOR downstream proteins and greatly reduced the growth of two different rhabdomyosarcoma (RD embryonal P = 0.00008; Rh30 alveolar P = 0.0002) cell lines compared with controls. These results suggest that phosphoprotein mapping of the Akt/mTOR pathway should be studied further as a means to select patients to receive mTOR/IRS pathway inhibitors before administration of chemotherapy.

Identification of FGFR4-activating mutations in human rhabdomyosarcomas that promote metastasis in xenotransplanted models

Rhabdomyosarcoma (RMS) is a childhood cancer originating from skeletal muscle, and patient survival is poor in the presence of metastatic disease. Few determinants that regulate metastasis development have been identified. The receptor tyrosine kinase FGFR4 is highly expressed in RMS tissue, suggesting a role in tumorigenesis, although its functional importance has not been defined. Here, we report the identification of mutations in FGFR4 in human RMS tumors that lead to its activation and present evidence that it functions as an oncogene in RMS. Higher FGFR4 expression in RMS tumors was associated with advanced-stage cancer and poor survival, while FGFR4 knockdown in a human RMS cell line reduced tumor growth and experimental lung metastases when the cells were transplanted into mice. Moreover, 6 FGFR4 tyrosine kinase domain mutations were found among 7 of 94 (7.5%) primary human RMS tumors. The mutants K535 and E550 increased autophosphorylation, Stat3 signaling, tumor proliferation, and metastatic potential when expressed in a murine RMS cell line. These mutants also transformed NIH 3T3 cells and led to an enhanced metastatic phenotype. Finally, murine RMS cell lines expressing the K535 and E550 FGFR4 mutants were substantially more susceptible to apoptosis in the presence of a pharmacologic FGFR inhibitor than the control cell lines expressing the empty vector or wild-type FGFR4. Together, our results demonstrate that mutationally activated FGFR4 acts as an oncogene, and these are what we believe to be the first known mutations in a receptor tyrosine kinase in RMS. These findings support the potential therapeutic targeting of FGFR4 in RMS.

An orthotopic model of murine osteosarcoma with clonally related variants differing in pulmonary metastatic potential.

To provide an investigative tool for the study of osteosarcoma (OSA) biology we have developed a syngeneic (balb/c) murine model of OSA, using cell lines derived from a spontaneously occurring murine OSA (Schmidt et al. Differentiation 1988; 39: 151-60). This model is characterized by orthotopic primary tumor growth, a period of minimal residual disease, spontaneous pulmonary metastasis, and clonally related variants (K7M2 and K12) that differ in pulmonary metastatic potential. Primary tumor and pulmonary metastasis histology was consistent with OSA in human patients. Expression of bone sialoprotein, biglyan, decorrin, and osteopontin was suggestive of bone lineage cells. The development and use of a more aggressive OSA cell line (K7M2) resulted in spontaneous metastasis to the lungs in over 90% of mice, whereas metastases were seen in only 33% of mice when a less aggressive OSA cell line (K12; Schmidt et al. Differentiation 1988; 39: 151-60) was used. Death from metastasis occurred at a median of 76 days using K7M2 whereas no median was achieved after 140 days using K12. Angiogenic potential, characterized by CD31 and factor VIII staining of primary tumors and pulmonary metastases, was greater in the K7M2 model compared to the K12 model. No significant differences in the in vitro or in vivo expression of angiogenesis associated genes (flt1, flt4, TIE1, TIE2, and VEGF) was found between K7M2 and K12. This well characterized and relevant model of OSA will be a valuable resource to improve our understanding of the biology and treatment of metastasis in OSA.

Beta4 integrin promotes osteosarcoma metastasis and interacts with ezrin

The development of pulmonary metastasis is the major cause of death in osteosarcoma, and its molecular basis is poorly understood. In this study, we show that β4 integrin is highly expressed in human osteosarcoma cell lines and tumor samples. Furthermore, highly metastatic MNNG-HOS cells have increased levels of β4 integrin. Suppression of β4 integrin expression by shRNA and disruption of β4 integrin function by transfection of dominant-negative β4 integrin was sufficient to revert this highly metastatic phenotype in the MNNG-HOS model without significantly affecting primary tumor growth. These findings suggest a role for β4 integrin expression in the metastatic phenotype in human osteosarcoma cells. In addition, we identified a previously uncharacterized interaction between β4 integrin and ezrin, a membrane-cytoskeletal linker protein that is implicated in the metastatic behavior of osteosarcoma. The β4 integrin–ezrin interaction appears to be critical for maintenance of β4 integrin expression. These data begin to integrate ezrin and β4 integrin expression into a model of action for the mechanism of osteosarcoma metastases.

Dual targeting of EWS-FLI1 activity and the associated DNA damage response with Trabectedin and SN38 synergistically inhibits Ewing sarcoma cell growth

Purpose: The goal of this study is to optimize the activity of trabectedin for Ewing sarcoma by developing a molecularly targeted combination therapy. Experimental Design: We have recently shown that trabectedin interferes with the activity of EWS-FLI1 in Ewing sarcoma cells. In this report, we build on this work to develop a trabectedin-based combination therapy with improved EWS-FLI1 suppression that also targets the drug-associated DNA damage to Ewing sarcoma cells. Results: We demonstrate by siRNA experiments that EWS-FLI1 drives the expression of the Werner syndrome protein (WRN) in Ewing sarcoma cells. Because WRN-deficient cells are known to be hypersensitive to camptothecins, we utilize trabectedin to block EWS-FLI1 activity, suppress WRN expression, and selectively sensitize Ewing sarcoma cells to the DNA-damaging effects of SN38. We show that trabectedin and SN38 are synergistic, demonstrate an increase in DNA double-strand breaks, an accumulation of cells in S-phase and a low picomolar IC50. In addition, SN38 cooperates with trabectedin to augment the suppression of EWS-FLI1 downstream targets, leading to an improved therapeutic index in vivo. These effects translate into the marked regression of two Ewing sarcoma xenografts at a fraction of the dose of camptothecin used in other xenograft studies. Conclusions: These results provide the basis and rationale for translating this drug combination to the clinic. In addition, the study highlights an approach that utilizes a targeted agent to interfere with an oncogenic transcription factor and then exploits the resulting changes in gene expression to develop a molecularly targeted combination therapy. Clin Cancer Res; 20(5); 1190–203. ©2013 AACR.

Diminished G1 checkpoint after gamma-irradiation and altered cell cycle regulation by insulin-like growth factor II overexpression.

High levels of insulin-like growth factor II (IGFII) mRNA expression are detected in many human tumors of different origins including rhabdomyosarcoma, a tumor of skeletal muscle origin. To investigate the role of IGFII in tumorigenesis, we have compared the mouse myoblast cell line C2C12-2.7, which was stably transfected with human IGFII cDNA and expressed high and constant amounts of IGFII, to a control cell line C2C12-1.1. A rhabdomyosarcoma cell line, RH30, which expresses high levels of IGFII and contains mutated p53, was also used in these studies. IGFII overexpression in mouse myoblast C2C12 cells causes a reduced cycling time and higher growth rate. After γ-irradiation treatment, C2C12-1.1 cells were arrested mainly in G0/G1 phase. However, C2C12-2.7 and RH30 cells went through a very short G1 phase and then were arrested in an extended G2/M phase. To verify further the effect of IGFII on the cell cycle, we developed a Chinese hamster ovary (CHO) cell line with tetracycline-controlled IGFII expression. We found that CHO cells with high expression of IGFII have a shortened cycling time and a diminished G1 checkpoint after treatment with methylmethane sulfonate (MMS), a DNA base-damaging agent, when compared with CHO cells with very low IGFII expression. It was also found that IGFII overexpression in C2C12 cells was associated with increases in cyclin D1, p21, and p53 protein levels, as well as mitogen-activated protein kinase activity. These studies suggest that IGFII overexpression shortens cell cycling time and diminishes the G1 checkpoint after DNA damage despite an intact p53/p21 induction. In addition, IGFII overexpression is also associated with multiple changes in the levels and activities of cell cycle regulatory components following γ-irradiation. Taken together, these changes may contribute to the high growth rate and genetic alterations that occur during tumorigenesis.

Identification of FoxM1/Bub1b Signaling Pathway as a Required Component for Growth and Survival of Rhabdomyosarcoma

We identified Bub1b as an essential element for the growth and survival of rhabdomyosarcoma (RMS) cells using a bar-coded, tetracycline-inducible short hairpin RNA (shRNA) library screen. Knockdown of Bub1b resulted in suppression of tumor growth in vivo, including the regression of established tumors. The mechanism by which this occurs is via postmitotic endoreduplication checkpoint and mitotic catastrophe. Furthermore, using a chromatin immunoprecipitation assay, we found that Bub1b is a direct transcriptional target of Forkhead Box M1 (FoxM1). Suppression of FoxM1 either by shRNA or the inhibitor siomycin A resulted in reduction of Bub1b expression and inhibition of cell growth and survival. These results show the important role of the Bub1b/FoxM1 pathway in RMS and provide potential therapeutic targets.

IGF-1R Inhibition Activates a YES/SFK Bypass Resistance Pathway: Rational Basis for Co-Targeting IGF-1R and Yes/SFK Kinase in Rhabdomyosarcoma

The insulin-like growth factor 1 receptor (IGF-1R) has surfaced as a significant target in multiple solid cancers due to its fundamental roles in pro-survival and anti-apoptotic signaling. However, development of resistance to IGF-1R blockade represents a significant hindrance and limits treatment efficacy in the clinic. In this study, we identified acquired resistance to IGF-1R blockade with R1507, an antibody against IGF-1R, and with BMS-754807, a small molecular inhibitor of IGF-1R/insulin receptor (IR). We showed that treatment with an IGF-IR antibody, R1507, or an IR/IGF-IR kinase inhibitor, BMS-754807, was associated with increased activation of YES/SRC family tyrosine kinase (SFK) in rhabdomyosarcoma (RMS). Combining anti–IGF-1R agents with SFK inhibitors resulted in blockade of IGF-1R inhibition–induced activation of YES/SFK and displayed advantageous antitumor activity in vitro and in vivo. Our data provide evidence that IGF-1R blockade results in activation of the YES/SRC family kinase bypass resistance pathway in vitro and in vivo. This may be of particular clinical relevance since both Yes and IGF components are overexpressed in RMS. Increased YES/SFK activation might serve as a clinical biomarker for predicting tumor resistance to IGF-1R inhibition. Dual inhibition of IGF-1R and SFK may have a broader and enhanced clinical benefit for patients with RMS.

Autocrine transforming growth factor-β growth pathway in murine osteosarcoma cell lines associated with inability to affect phosphorylation of retinoblastoma protein

Purpose. Production of active transforming growth factor-β (TGF-β ) by human osteosarcoma may contribute to malignant progression through mechanisms that include induction of angiogenesis, immune suppression and autocrine growth stimulation of tumor cell growth.To study events associated with induction of cell proliferation by TGF-β , we have evaluated the TGF-β pathway in two murine osteosarcoma cell lines, K7 and K12. Results. Northern and immunohistochemical analyses show that each cell line expressesTGF-β1 and TGF-β3 mRNA and protein. Both cell lines secrete activeTGF-β 1 and display a 30–50% reduction in growth when cultured in the presence of a TGF-β blocking antibody. Expression of TGF-β receptors TβRI, TβRII and TβRIII is demonstrated by affinity labeling with 125 -TGF-β 1, and the intermediates, Smads 2, 3 and 4, are uniformly expressed. Smads 2 and 3 are phosphorylated in response toTGF-β , while pRb phosphorylation in each osteosarcoma cell line is not affected by either exogenousTGF-β or TGF-β antibody. Conclusions. The data implicate events downstream of Smad activation, including impaired regulation of pRb, in the lack of a growth inhibitory response toTGF-β , and indicate that this murine model of osteosarcoma is valid for investigating the roles of autocrineTGF-β in vivo.