Lu-Yuan Li

Human rhomboid family-1 gene silencing causes apoptosis or autophagy to epithelial cancer cells and inhibits xenograft tumor growth.

The rhomboid family of genes carry out a wide range of important functions in a variety of organisms. Little is known, however, about the function of the human rhomboid family-1 gene (RHBDF1). We show here that RHBDF1 function is essential to epithelial cancer cell growth. RHBDF1 mRNA level is significantly elevated in clinical specimens of invasive ductal carcinoma of the breast, and the protein is readily detectable in human breast cancer or head and neck cancer cell lines. Silencing the RHBDF1 gene with short interfering RNA (siRNA) results in apoptosis in breast cancer MDA-MB-435 cells and autophagy in head and neck squamous cell cancer 1483 cells. The treatment also leads to significant down-modulation of activated AKT and extracellular signal-regulated kinase in the cells, suggesting that critically diminished strength of these growth signals may be the key attributes of the induction of cell death. Furthermore, silencing the RHBDF1 gene in MDA-MB-435 or 1483 xenograft tumors on athymic nude mice by using i.v. administered histidine-lysine polymer nanoparticle-encapsulated siRNA results in marked inhibition of tumor growth. Our findings indicate that RHBDF1 has a pivotal role in sustaining growth signals in epithelial cancer cells and thus may serve as a therapeutic target for treating epithelial cancers. [Mol Cancer Ther 2008;7(6):1355–64]

Human rhomboid family-1 gene RHBDF1 participates in GPCR-mediated transactivation of EGFR growth signals in head and neck squamous cancer cells

Epidermal growth factor receptor (EGFR) is an activated oncogene in many cancers. It can be transactivated by ligands of G protein‐coupled receptors (GPCRs). We show here that a novel gene, human rhomboid family‐1 (RHBDF1), which was recently reported to have a pivotal role in epithelial cancer cell growth in culture and in xenograft tumors, participates in the modulation of GPCR‐mediated EGFR transactivation. The RHBDF1 protein localizes mainly in the endoplasmic reticulum. Silencing the RHBDF1 gene in head and neck squamous cancer cell line 1483 cells with siRNA causes an inhibition of gastrin‐releasing peptide (GRP) ‐induced phosphorylation of EGFR and EGFR‐dependent signaling proteins p44/42 MAPK and AKT, accompanied by an inhibition of GRP‐induced survival, proliferation, and invasion of the cells. The EGFR signaling pathway itself remains intact, however, as the cells remain responsive to exogenous EGF. In addition, RHBDF1 gene silencing disrupts GRP‐stimulated secretion of EGFR ligand TGF‐α, but not the production of latent TGF‐α, whereas engineered overexpression of RHBDF1 markedly accelerates the secretion of TGF‐α. These findings are consistent with the view that RHBDF1 is critically involved in a GPCR ligand‐stimulated process leading to the activation of latent EGFR ligands.—Zou, H., Thomas, S. M., Yan, Z.‐W., Grandis, J. R, Vogt, A., Li, L.‐Y. Human rhomboid family‐1 gene RHBDF1 participates in GPCR‐mediated transactivation of EGFR growth signals in head and neck squamous cancer cells. FASEB J. 23, 425–432 (2009)

Inhibition of endothelial progenitor cell differentiation by VEGI

Endothelial progenitor cells (EPCs) play a critical role in postnatal and tumor vasculogenesis. Vascular endothelial growth inhibitor (VEGI; TNFSF15) has been shown to inhibit endothelial cell proliferation by inducing apoptosis. We report here that VEGI inhibits the differentiation of EPCs from mouse bone marrow-derived Sca1(+) mononuclear cells. Analysis of EPC markers indicates a significant decline of the expression of endothelial cell markers, but not stem cell markers, on VEGI-treated cells. Consistently, the VEGI-treated cells exhibit a decreased capability to adhere, migrate, and form capillary-like structures on Matrigel. In addition, VEGI induces apoptosis of differentiated EPCs but not early-stage EPCs. When treated with VEGI, an increase of phospho-Erk and a decrease of phospho-Akt are detected in early-stage EPCs, whereas activation of nuclear factor-kappaB, jun N-terminal kinase, and caspase-3 is seen in differentiated EPCs. Furthermore, VEGI-induced apoptosis of differentiated EPC is, at least partly, mediated by death receptor-3 (DR3), which is detected on differentiated EPC only. VEGI-induced apoptosis signals can be inhibited by neutralizing antibodies against DR3 or recombinant extracellular domain of DR3. These findings indicate that VEGI may participate in the modulation of postnatal vasculogenesis by inhibiting EPC differentiation.

Vascular endothelial growth inhibitor (VEGI; TNFSF15) inhibits bone marrow-derived endothelial progenitor cell incorporation into Lewis lung carcinoma tumors

Bone marrow (BM)-derived endothelial progenitor cells (EPC) have a critical role in tumor neovascularization. Vascular endothelial growth inhibitor (VEGI) is a member of the TNF superfamily (TNFSF15). We have shown that recombinant VEGI suppresses tumor angiogenesis by specifically eliminating proliferating endothelial cells (EC). We report here that treatment of tumor bearing mice with recombinant VEGI leads to a significantly decreased population of BM-derived EPC in the tumors. We transplanted whole bone marrow from green fluorescent protein (GFP) transgenic mice into C57BL/6 recipient mice, which were then inoculated with Lewis lung carcinoma (LLC) cells. Intraperitoneal injection of recombinant VEGI led to significant inhibition of tumor growth and decrease of vasculature density compared to vehicle-treated mice. Tumor implantation yielded a decrease of BM-derived EPC in the peripheral blood, while VEGI-treatment resulted in an initial delay of such decrease. Analysis of the whole bone marrow showed a decrease of Lin−-c-Kit+-Sca-1+ hematopoietic stem cell (HSC) population in tumor bearing mice; however, VEGI-treatment caused a significant increase of this cell population. In addition, the number of BM-derived EPC in VEGI-treated tumors was notably less than that in the vehicle-treated group, and most of the apoptotic cells in the VEGI-treated tumors were of bone marrow origin. These findings indicate that VEGI inhibits BM-derived EPC mobilization and prevents their incorporation into LLC tumors by inducing apoptosis specifically of BM-derived cells, resulting in the inhibition of EPC-supported tumor vasculogenesis and tumor growth.

Down-modulation of TNFSF15 in ovarian cancer by VEGF and MCP-1 is a pre-requisite for tumor neovascularization

Persistent inflammation and neovascularization are critical to cancer development. In addition to upregulation of positive control mechanisms such as overexpression of angiogenic and inflammatory factors in the cancer microenvironment, loss of otherwise normally functioning negative control mechanisms is likely to be an important attribute. Insights into the down-modulation of such negative control mechanisms remain largely unclear, however. We show here that tumor necrosis factor superfamily-15 (TNFSF15), an endogenous inhibitor of neovascularization, is a critical component of the negative control mechanism that operates in normal ovary but is missing in ovarian cancer. We show in clinical settings that TNFSF15 is present prominently in the vasculature of normal ovary but diminishes in ovarian cancer as the disease progresses. Vascular endothelial growth factor (VEGF) produced by cancer cells and monocyte chemotactic protein-1 (MCP-1) produced mainly by tumor-infiltrating macrophages and regulatory T cells effectively inhibits TNFSF15 production by endothelial cells in vitro. Using a mouse syngeneic tumor model, we demonstrate that silencing TNFSF15 by topical shRNA treatments prior to and following mouse ovarian cancer ID8 cell inoculation greatly facilitates angiogenesis and tumor growth, whereas systemic application of recombinant TNFSF15 inhibits angiogenesis and tumor growth. Our findings indicate that downregulation of TNFSF15 by cancer cells and tumor infiltrating macrophages and lymphocytes is a pre-requisite for tumor neovascularization.

Recombinant GnRH-p53 protein sensitizes breast cancer cells to 5-fluorouracil-induced apoptosis in vitro and in vivo

An ideal approach to treat cancers with dysfunctional p53 tumor suppressor gene is to reinstate p53 functionality by directly using p53 protein as a therapeutic agent. However, this has not been possible because the cells cannot readily internalize the protein. We constructed a fusion protein consisting of gonadotropin-releasing hormone (GnRH-p53) and p53 moieties. The recombinant protein was directly used to treat human breast cancer cells and athymic nude mice bearing breast cancer xenografts, with or without DNA synthesis-arresting agent 5-fluorouracil (5-FU). Treatments of cells from breast cancer cell-lines MDA-MB-231, T47D, or SKBR-3 with GnRH-p53 in combination with 5-FU significantly enhanced p53-activated apoptosis signals, including PUMA expression, BAX translocation to mitochondria, and activated caspase-3. Intratumoral injection of the GnRH-p53 protein inhibited MDA-MB-231 xenograft growth and induced p53-mediated apoptosis in the tumors. Systemic treatment of the tumor-bearing mice via tail vein injection of GnRH-p53 markedly augmented the anticancer efficacy of 5-FU. Substitution of GnRH-p53 with wild type p53 protein had no effect. Recombinant GnRH-p53 is able to function as a surrogate of p53 with regard to its apoptosis-inducing activity. Combination of GnRH-p53 with DNA-damaging drugs may be of important therapeutic value for cancer treatment.

Abstract 468: Down-regulation of TNFSF15 (VEGI) in tumor vasculature under inflammatory conditions is a pre-requisite of tumor neovascularization

Tumor necrosis factor superfamily-15 (TNFSF15; VEGI) is an endogenous inhibitor of neovascularization. TNFSF15 is largely produced by quiescent vascular endothelial cells in an established vasculature but is markedly down-regulated in endothelial cells undergoing proliferation such as those in tumors or at a wound site. Recombinant TNFSF15 is a potent inhibitor of endothelial cell proliferation and tumor growth. We hypothesize that TNFSF15 as a negative regulator of angiogenesis must be down-regulated prior to the initiation of angiogenesis, and that the inflammatory conditions created by cancer cells and infiltrating lymphocytes lead to TNFSF15 down-modulation in the microenvironment, permitting new blood vessel growth. We analyzed normal ovary tissues (n=12) and clinical specimens of ovarian cancer (n=86; including stages I, II, III and IV) for the expression of TNFSF15, the endothelial cell marker PECAM (CD31), and tumor-infiltrating T-cell markers CD4 and CD25. We found that the degree of T-cell infiltration increases markedly while TNFSF15 expression diminishes as the disease progresses. We then determined the influence of human ovarian cancer cell line OVCAR-3 cell conditioned media on cytokine production by human CD4+ T-cells and CD4+CD25+ Treg cells, as well as the influence of the conditioned media of the latter two cells on TNFSF15 expression in human umbilical vein endothelial cells (HUVEC). We found that the conditioned media of Th1 or Treg cells can inhibit HUVEC TNFSF15 expression only after the lymphocytes being treated with OVCAR-3 conditioned media. Interestingly, we further found that mouse ovarian cancer ID8 cells implanted subcutaneously on mice treated with TNFSF15 shRNA at the injection sites exhibit significantly greater tumor growth rates compared with control shRNA treated ones. Our findings indicate that TNFSF15 plays a critical role in the maintenance of vascular stability as its down-modulation is a pre-requisite for the initiation of tumor neovascularization, and that TNFSF15 down-regulation is carried out by tumor-infiltrating T-cells under the influence of the cancer cells in the microenvironment. 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 468. doi:10.1158/1538-7445.AM2011-468

Abstract 4314: A mouse model of cerebral cavernous malformations

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Cerebral cavernous malformations (CCM) are prevalent cerebral vascular lesions involving aberrant angiogenesis that can lead to hemorrhage, epilepsy, and other neurological manifestations. CCM consists of brittle, proliferating caverns filled with blood and lined with endothelial cells. However, the underlying mechanism of CCM development is poorly understood. The cytokine vascular endothelial cell growth inhibitor (VEGI; TNFSF15; TL1A) is an endogenous negative regulator of angiogenesis produced largely by endothelial cells and exerts a specific inhibitory activity on the proliferation of endothelial cells. We found that the production of VEGI by the endothelium is nearly completely diminished in the CCM tissues. In sharp contrast, the expression of VEGI is readily detectable on the vasculature in nearby brain tissues. On the other hand, angiogenic factors such as vascular endothelial cell growth factor (VEGF) is known to be highly expressed in CCM lesions. We show here that an imbalance of angiogenesis promoters and inhibitors, namely, the down-regulation of VEGI and up-regulation of VEGF, are attributable to the development of CCM. We have constructed a mouse CCM model by implanting mouse endothelial cell line bEnd3 cells in the brain of a female BALB/C mouse. The cavernous blood vessels formed in the model closely resemble those observed in CCM and thus may be used to study the pathology and treatment options of CCM. 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 4314. doi:10.1158/1538-7445.AM2011-4314

Abstract 1112: Human rhomboid family-1 gene RHBDF1 assists trafficking of TGFa for GPCR-mediated transactivation of EGFR

Epidermal growth factor receptor (EGFR) can be transactivated by ligands of G protein-coupled receptors (GPCR). GPCR signaling pathways often involve the formation of clathrin-coated endosomes, which interact with endoplasmic reticulum/Golgi complex to form recycling endosome and secretion vesicles containing EGFR pro-ligands such as pro-TGFα, which is proteolytically processed and released on the cell surface to activate EGFR in an autocrine manner. We showed previously that human rhomboid family-1 gene RHBDF1 is essential for GPCR-EGFR transactivation by assisting the secretion of TGFα, a ligand of EGFR. We show here silencing the RHBDF1 with shRNA leads to inhibition of EGFR ligand-dependent transactivation induced by GPCR ligands such as gastrin release peptide (GRP) and sphingosine-1-phosphate. However, RHBDF1 is not involved in EGFR ligand-independent GPCR-EGFR transactivation induced by isoproterenol. Inhibitors of endosome formation and membrane trafficking such as chlorpromazine, nocodazole, and cytochalasin-D also inhibit GRP-induced EGFR phosphorylation. Inhibitors of endosome acidification such as chloroquine and BFA lessen the inhibitory effect of RHBDF1 shRNA on GRP-induced EGFR phosphorylation. The RHBDF1 protein localizes mainly to the endoplasmic reticulum with pro-TGFα and GRASP55 in starved, quiescent cancer cells, but is phosphorylated and translocated to the cell surface upon GPCR ligands stimulation. GPCR ligands also activate the intracellular signaling molecule Src which then co-translocates with RHBDF1 and clathrin to the plasma membrane. Src-clathrin interaction and co-localization to the cell surface are RHBDF1-dependent. These findings are consistent with the view that, in response to GPCR activation, RHBDF1 assists pro-TGFα trafficking to the cell surface through clathrin coated trafficking. 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 1112. doi:10.1158/1538-7445.AM2011-1112

Abstract 1307: Downregulation of vascular endothelial cell growth inhibitor (VEGI; TNFSF15) expression in endothelial cells by interferon-γ

Vascular endothelial growth inhibitor (VEGI) is a member of the tumor necrosis factor superfamily (TNFSF15) produced largely by endothelial cells. It functions as an endogenous inhibitor of neovascularization. We showed previously that VEGI inhibits angiogenesis by enforcing a growth arrest on quiescent endothelial cells while inducing apoptosis in proliferating endothelial cells. We also found that VEGI gene expression is down-regulated in growing endothelial cells in vitro as well as in tumor vasculatures. Here we show that, among a variety of inflammation-modulating cytokines we evaluated, interferon-γ (IFNγ) can down-regulate VEGI expression in human umbilical vein endothelial cells in a dose- and time-dependent manner. Neutralizing antibodies of IFNγ receptor 1 restores VEGI expression. IFNγ modulation of VEGI gene expression is mediated by unphosphorylated STAT1 rather than phosphorylated STAT1. These findings are consistent with the view that down-regulation of VEGI gene expression in endothelial cells by IFNγ is a prerequisite for the initiation and continuation of angiogenesis during inflammation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1307.