Yanwen Yang

Regulation of NOTCH signaling by reciprocal inhibition of HES1 and Deltex 1 and its role in osteosarcoma invasiveness

The highly conserved NOTCH signaling pathway has many essential functions in the development of diverse cells, tissues and organs from Drosophila to humans, and dysregulated NOTCH signaling contributes to several disorders, including vascular and bone defects, as well as several cancers. Here we describe a novel mechanism of NOTCH regulation by reciprocal inhibition of two NOTCH downstream effectors: Deltex1 and HES1. This mechanism appears to regulate invasion of osteosarcoma cells, as Deltex1 blocks osteosarcoma invasiveness by downregulating NOTCH/HES1 signaling. The inhibitory effect of endogenous Deltex1 on NOTCH signaling is mediated through binding with the intracellular domain of NOTCH and ubiquitination and degradation of NOTCH receptors. Conversely, we show that the NOTCH target gene HES1 causes transcriptional inhibition of Deltex1 by directly binding to the promoter of Deltex1. An HES1 binding site is identified 400 bp upstream of the transcription start site of Deltex1. HES1-mediated repression of Deltex1 requires the C-terminal H3/H4 and WRPW domains of HES1, which associate with the TLE/Groucho corepressors. Taken together, we define a molecular mechanism regulating NOTCH signaling by reciprocal inhibition of the NOTCH target genes HES1 and Deltex1 in mammalian cells. This mechanism may have important clinical implications for targeting NOTCH signaling in osteosarcoma and other cancers.The highly conserved NOTCH signaling pathway has many essential functions in the development of diverse cells, tissues and organs from Drosophila to humans, and dysregulated NOTCH signaling contributes to several disorders, including vascular and bone defects, as well as several cancers. Here we describe a novel mechanism of NOTCH regulation by reciprocal inhibition of two NOTCH downstream effectors: Deltex1 and HES1. This mechanism appears to regulate invasion of osteosarcoma cells, as Deltex1 blocks osteosarcoma invasiveness by downregulating NOTCH/HES1 signaling. The inhibitory effect of endogenous Deltex1 on NOTCH signaling is mediated through binding with the intracellular domain of NOTCH and ubiquitination and degradation of NOTCH receptors. Conversely, we show that the NOTCH target gene HES1 causes transcriptional inhibition of Deltex1 by directly binding to the promoter of Deltex1. An HES1 binding site is identified 400 bp upstream of the transcription start site of Deltex1. HES1-mediated repression of Deltex1 requires the C-terminal H3/H4 and WRPW domains of HES1, which associate with the TLE/Groucho corepressors. Taken together, we define a molecular mechanism regulating NOTCH signaling by reciprocal inhibition of the NOTCH target genes HES1 and Deltex1 in mammalian cells. This mechanism may have important clinical implications for targeting NOTCH signaling in osteosarcoma and other cancers.

Slow Down to Stay Alive HER4 Protects Against Cellular Stress and Confers Chemoresistance in Neuroblastoma

Neuroblastoma (NBL) is a common pediatric solid tumor, and outcomes for patients with advanced neuroblastoma remain poor despite extremely aggressive treatment. Chemotherapy resistance at relapse contributes heavily to treatment failure. The poor survival of patients with high‐risk NBL prompted this investigation into novel treatment options with the objective of gaining a better understanding of resistance mechanisms. On the basis of previous work and on data from publicly available studies, the authors hypothesized that human epidermal growth factor receptor 4 (Her4) contributes to resistance.

Anti-tumor effects of the Notch pathway in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are driven by gain-of-function mutations of KIT or PDGFRa. The introduction of imatinib has significantly extended survival for patients. However, most patients develop resistances. Notch signaling is a conserved developmental pathway known to play a critical role in the development of several cancers, functioning as a tumor promoter or a tumor suppressor. Given that the normal progenitor cell for GIST, the interstitial cell of Cajal, has characteristics similar to those of cells of neuroendocrine origin, we hypothesized that Notch pathway impacts the biology of GIST cells. In this study, we retrovirally and pharmacologically manipulated the Notch pathway in human GIST cells. We also performed a retrospective analysis of a cohort on 15 primary tumors to determine the role of Hes1, a major target gene of Notch, as a prognostic marker for GIST. Constitutively, active intracellular domain of Notch1 (ICN1) expression potently induced growth arrest and downregulated KIT expression in vitro. Additionally, treatment with the histone deacetylase inhibitor suberoylanilide hydroxamic acid caused dose-dependent upregulation of Notch1 expression and a parallel decrease in viability in these cells. Retroviral silencing of downstream targets of Notch (dominant-negative Hes1) and pharmacological inhibition of Notch activation (γ-secretase inhibition) partially rescued GIST cells from suberoylanilide hydroxamic acid treatment. GIST patients with high Hes1 mRNA levels have a significantly longer relapse-free survival. These results identify a novel anti-tumor effect of Notch1 and cross talk between the Notch and KIT pathways. Thus, activation of this pathway by treatment with histone deacetylase inhibitors is an appealing potential therapeutic strategy for GISTs. Précis: This study is the first report of the tumor suppressor effects of Notch pathway in gastrointestinal stromal tumors via a negative feedback with the oncogene KIT and may lead the development of new therapeutic strategies for GISTs patients.

Driven to death: Inhibition of farnesylation increases Ras activity in osteosarcoma and promotes growth arrest and cell death

To improve cancer outcomes, investigators are turning increasingly to small molecule medicines that disrupt vital signaling cascades, inhibit malignant growth, or induce apoptosis. One vital signaling molecule is Ras, and a key step in Ras activation is membrane anchoring of Ras through prenylation, the C-terminal addition of a lipid anchor. Small molecule inhibitors of farnesyltransferase (FTI), the enzyme most often responsible for prenylating Ras, showed clinical promise, but development of FTIs such as tipifarnib has been stalled by uncertainty about their mechanism of action, because Ras seemed unimpeded in tipifarnib-treated samples. Interpretation was further complicated by the numerous proteins that may be farnesylated, as well as availability of an alternate prenylation pathway, geranylgeranylation. Our initial observations of varied response by cancer cell lines to tipifarnib led us to evaluate the role of FTI in Ras signal alteration using various tumor models. We describe our novel counterintuitive finding that endogenous Ras activity increases in cancer cell lines with low endogenous Ras activity when farnesyltransferase is inhibited by either tipifarnib or short hairpin RNA. In response to tipifarnib, variable growth arrest and/or cell death correlated with levels of activated extracellular signal–regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Sensitivity to tipifarnib treatment was shown by growth inhibition and by an increase in subdiploid cell numbers; cells with such sensitivity had increased activation of ERK and p38 MAPK. Because Ras must be prenylated to be active, our findings suggest that geranylgeranylated N-Ras or K-Ras B interacts differently with downstream effector proteins in sensitive cancer cells responding to tipifarnib, switching the balance from cell proliferation to growth inhibition. Mol Cancer Ther; 9(5); 1111–9. ©2010 AACR.

Driven to Death: Inhibition of Farnesylation Increases Ras Activity and Promotes Growth Arrest and Cell Death

To improve cancer outcomes, investigators are turning increasingly to small molecule medicines that disrupt vital signaling cascades, inhibit malignant growth, or induce apoptosis. One vital signaling molecule is Ras, and a key step in Ras activation is membrane anchoring of Ras through prenylation, the C-terminal addition of a lipid anchor. Small molecule inhibitors of farnesyltransferase (FTI), the enzyme most often responsible for prenylating Ras, showed clinical promise, but development of FTIs such as tipifarnib has been stalled by uncertainty about their mechanism of action, because Ras seemed unimpeded in tipifarnib-treated samples. Interpretation was further complicated by the numerous proteins that may be farnesylated, as well as availability of an alternate prenylation pathway, geranylgeranylation. Our initial observations of varied response by cancer cell lines to tipifarnib led us to evaluate the role of FTI in Ras signal alteration using various tumor models. We describe our novel counterintuitive finding that endogenous Ras activity increases in cancer cell lines with low endogenous Ras activity when farnesyltransferase is inhibited by either tipifarnib or short hairpin RNA. In response to tipifarnib, variable growth arrest and/or cell death correlated with levels of activated extracellular signal–regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Sensitivity to tipifarnib treatment was shown by growth inhibition and by an increase in subdiploid cell numbers; cells with such sensitivity had increased activation of ERK and p38 MAPK. Because Ras must be prenylated to be active, our findings suggest that geranylgeranylated N-Ras or K-Ras B interacts differently with downstream effector proteins in sensitive cancer cells responding to tipifarnib, switching the balance from cell proliferation to growth inhibition. Mol Cancer Ther; 9(5); 1111–9. ©2010 AACR.

Driven to death

To improve cancer outcomes, investigators are turning increasingly to small molecule medicines that disrupt vital signaling cascades, inhibit malignant growth, or induce apoptosis. One vital signaling molecule is Ras, and a key step in Ras activation is membrane anchoring of Ras through prenylation, the C-terminal addition of a lipid anchor. Small molecule inhibitors of farnesyltransferase (FTI), the enzyme most often responsible for prenylating Ras, showed clinical promise, but development of FTIs such as tipifarnib has been stalled by uncertainty about their mechanism of action, because Ras seemed unimpeded in tipifarnib-treated samples. Interpretation was further complicated by the numerous proteins that may be farnesylated, as well as availability of an alternate prenylation pathway, geranylgeranylation. Our initial observations of varied response by cancer cell lines to tipifarnib led us to evaluate the role of FTI in Ras signal alteration using various tumor models. We describe our novel counterintuitive finding that endogenous Ras activity increases in cancer cell lines with low endogenous Ras activity when farnesyltransferase is inhibited by either tipifarnib or short hairpin RNA. In response to tipifarnib, variable growth arrest and/or cell death correlated with levels of activated extracellular signal–regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Sensitivity to tipifarnib treatment was shown by growth inhibition and by an increase in subdiploid cell numbers; cells with such sensitivity had increased activation of ERK and p38 MAPK. Because Ras must be prenylated to be active, our findings suggest that geranylgeranylated N-Ras or K-Ras B interacts differently with downstream effector proteins in sensitive cancer cells responding to tipifarnib, switching the balance from cell proliferation to growth inhibition. Mol Cancer Ther; 9(5); 1111–9. ©2010 AACR.

Hes4: A potential prognostic biomarker for newly diagnosed patients with high-grade osteosarcoma

Prognostic biomarkers for osteosarcoma (OS) at the time of diagnosis are lacking. Necrotic response of OS to preoperative chemotherapy correlates with survival and is determined 3–4 months after diagnosis. The purpose of this study is to identify biomarkers that will stratify patients into good or poor responders to chemotherapy at diagnosis and determine the role of potential biomarkers in OS pathogenesis.

Abstract 197: Multi-cellular tumor spheroids in vitro model reveals a critical role of ERBB4 in survival of osteosarcoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Multi-cellular tumor spheroids (MCTS) model is an in vitro model holding an intermediate complexity between monolayer culture and solid tumor. It is cell-cell adhesion dependent and closely resembles avascular tumor regions and micrometastasis in pro-angiogenesis stage. Osteosarcoma (OS) is the most common malignant bone tumor with high incidence of early lung metastasis; therefore, multi-cellular tumor spheroid can be used to model its in vivo behaviors. Our group previously reported the expression profile of ERBB family in osteosarcoma tissue samples and cell lines. To further study ERBB pathways in osteosarcoma tumor spheroids, we generated OS tumor spheroids by plating cells on non-adherent poly-HEMA pre-coated dishes. Under anchorage-independent conditions, OS cells spontaneously generate multi-cellular spheroids to acquired anoikis resistant by cell-cell adhesion; blocking the formation of spheroids will result rapid cell death and increased PARP cleavage. MCTSs showed lower proliferation rate than monolayer culture, and they held a chemo-resistant phenotype, as shown by proliferation assay and subdiploid DNA content. Surprisingly, ERBB4 expression was up-regulated in non-adherent tumor spheroids compared with monolayer culture (1.2, 2.9, 6.3 fold respectively in 3 cell lines) while EGFR and ERBB2 did not. With knockdown of ERBB4 expression by shRNA, OS cells became less tolerant to anoikis and serum starvation, as shown by PARP cleavage and subdiploid DNA content. Moreover, knockdown of ERBB4 increased the chemo-sensitivity of OS cells against multiple chemotherapy including cisplatin, doxorubicin, and methotrexate (71%, 71%, 60% in survival compared to scramble control). In conclusion, our data showed that ERBB4 contributes to the tumorigenicity of osteosarcoma and plays a protective role against multiple exogenous apoptotic stimuli including matrix detachment, nutrition insufficiency and cytotoxic reagents. Although wildly expressed in osteosarcoma, the role of ERBB4 in tumor biology is largely unknown. These findings indicate that ERBB4 is a key factor regulating survival pathway of OS under various cellular stress condition. Thus, in primary osteosarcoma and metastatic lesion, ERBB4 might be a potential therapeutic target to facilitate multi-modal therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 197. doi:10.1158/1538-7445.AM2011-197

Driven to death: Inhibition of farnesylation increases Ras activity and promotes growth arrest and cell death [corrected].

To improve cancer outcomes, investigators are turning increasingly to small molecule medicines that disrupt vital signaling cascades, inhibit malignant growth, or induce apoptosis. One vital signaling molecule is Ras, and a key step in Ras activation is membrane anchoring of Ras through prenylation, the C-terminal addition of a lipid anchor. Small molecule inhibitors of farnesyltransferase (FTI), the enzyme most often responsible for prenylating Ras, showed clinical promise, but development of FTIs such as tipifarnib has been stalled by uncertainty about their mechanism of action, because Ras seemed unimpeded in tipifarnib-treated samples. Interpretation was further complicated by the numerous proteins that may be farnesylated, as well as availability of an alternate prenylation pathway, geranylgeranylation. Our initial observations of varied response by cancer cell lines to tipifarnib led us to evaluate the role of FTI in Ras signal alteration using various tumor models. We describe our novel counterintuitive finding that endogenous Ras activity increases in cancer cell lines with low endogenous Ras activity when farnesyltransferase is inhibited by either tipifarnib or short hairpin RNA. In response to tipifarnib, variable growth arrest and/or cell death correlated with levels of activated extracellular signal–regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Sensitivity to tipifarnib treatment was shown by growth inhibition and by an increase in subdiploid cell numbers; cells with such sensitivity had increased activation of ERK and p38 MAPK. Because Ras must be prenylated to be active, our findings suggest that geranylgeranylated N-Ras or K-Ras B interacts differently with downstream effector proteins in sensitive cancer cells responding to tipifarnib, switching the balance from cell proliferation to growth inhibition. Mol Cancer Ther; 9(5); 1111–9. ©2010 AACR.

Abstract 3970: EGF has stimulatory and survival effects in osteosarcoma in vitro and pan-ERBB kinase inhibition causes cell growth inhibition in vitro and reduces lung metastases in vivo

Osteosarcoma is the most common primary bone tumor in children and adolescents, with recurring disease or lung metastases being the primary cause of death in patients. Expression of EGFR, Her-2, and Her-4 (ERBB1, 2 and 4) have been reported in human primary osteosarcoma, suggesting that they may be contributing to osteosarcoma pathogenesis, but the correlation of expression levels with patient outcomes has been controversial. We have shown that a pan-ERBB small molecule kinase inhibitor causes growth inhibition and apoptosis of osteosarcoma cell in vitro, suggesting a potential therapeutic benefit of using this drug in vivo. In the present study, we show that protein expression levels of EGFR, Her-2 and Her-4 are higher in osteosarcoma lung metastases than in primary tumors by TMA, and that these levels are significantly higher for Her-2 and Her-4. We created eight new osteosarcoma cell lines from primary patient tissues and found that all eight, in addition to six commonly used osteosarcoma cell lines, express detectable levels of EGFR and Her-2, and several express detectable Her-4. EGF ligand stimulation in vitro produces robust phosphorylation of EGFR, Her-2 and Her-4 in most cell lines and moderate activation in others. EGF stimulation also produces phosphorylation of Akt, MAPK and p70S6K in most cell lines and of STAT5 in six cell lines, suggesting that EGF has growth and survival stimulation effects. When adding a pan-ERBB kinase inhibitor, Pf-299804, to cells pre-stimulated with EGF, phosphorylation of EGFR, Her-2, Her-4, Akt, etc. are inhibited in a dose-dependent manner. Reverse phase protein array also shows an increase in pro-apoptotic proteins Bim and cleaved PARP with Pf-299804 treatment, confirmed by Western blots. Mouse xenograft experiments, where cells injected in the tibia spontaneously form lung metastases, showed a significant decrease in three categories of Her-4-positive lung metastases in drug-treated mice based on size: 1-4 cell oligomets (p=0.0055); 5 cell-200 micron micromets (p=0.0276); and greater than 200 micron macromets (p=0.0055). These data suggest that EGF produces mitogenic and survival stimulation in osteosarcoma cells and that pan ERBB-family kinase inhibition causes growth inhibition and apoptosis in vitro and reduction of lung metastases in a mouse model in vivo. This provides evidence that pan-ERBB kinase inhibition has therapeutic potential for osteosarcoma lung metastases. Citation Format: Laura D. Nelson, Tiffany N. Lynch, Yanwen Yang, Wei-Lien Wang, Diane Liu, Dafydd G. Thomas, Dennis P. M. Hughes. EGF has stimulatory and survival effects in osteosarcoma in vitro and pan-ERBB kinase inhibition causes cell growth inhibition in vitro and reduces lung metastases in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3970. doi:10.1158/1538-7445.AM2014-3970