Hui-Wen Lo, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Epidermal growth factor receptor (EGFR) is critically involved in the genesis and progression of human cancers and is considered as an attractive target for anti-cancer therapy. However, clinical success with anti-EGFR therapy remains limited in part due to our incomplete knowledge of the EGFR pathway. Accumulating evidences revealed a novel mode of EGFR signaling in which EGF ligand shuttles EGFR into the nucleus, leading to cyclin D1 gene activation. Patients with breast tumors that contain high nuclear EGFR survived poorly compared to those with no/low levels. However, the nature and pathological significance of this novel EGFR network remain largely unknown. We will test the hypothesis that nuclear EGFR functions as both a transcriptional regulator and a tyrosine kinase and that de-regulated nuclear EGFR pathway contributes to a more aggressive biology of human tumors. Preminary data indicate a novel nuclear interaction between EGFR and the oncogenic transcription factor, signal transducer and activator of transcription-3, STATS, leading to increased expression of inducible nitric oxide synthase (iNOS). Aim 1 will characterize nuclear EGFR/STAT3 interaction and determine its role in iNOS gene regulation. Moreover, whether nuclear EGFR also functions as a tyrosine kinase remains unknown. Interestingly, preliminary results suggest that nuclear EGFR phosphorylates c-jun. In addition, we found that EGF activates expression of TWIST, a mediator for epithelial-mesenchymal transition (EMT)/metastasis and that TWIST gene promoter can be regulated by EGFR, c-jun and STATS. Aim 2 will thus determine the effect of EGFR/ c-jun/STAT3 interplay on TWIST gene activation and TWIST-mediated EMT/tumor progression. A role of nuclear EGFR in Taxol/5-Fu resistance is suggested by our preliminary data. Aim 3 will determine the significance and mechanisms for nuclear EGFR-mediated chemoresistance. This proposal is highly relevant to public health and its outcome will shed light into the nuclear EGFR signaling network in human cancers. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Alternative splicing is observed in ~40-60% of human genes; however, its full impact on human cancers is still not well understood. In this proposal, we will functionally characterize a novel, alternatively spliced transcription factor discovered in the PI's laboratory, namely, tGLI1 (truncated glioma-associated oncogene homolog 1). Belonging to the GLI1 family of zinc finger transcription factors, tGLI1 has an in-frame deletion of entire exon 3 and part of exon 4 of the GLI1 gene. Like GLI1, tGLI1 responds to sonic hedgehog ligand, undergoes nuclear transport, and regulates known GLI1 target genes. However, our preliminary results showed that tGLI1 may differ from GLI1 in regulating tumor phenotypes, transcriptional targets and expression pattern. Glioblastoma multiforme (GBM) xenografts engineered to express tGLI1 appeared to be more aggressive in growth, invasiveness, and angiogenesis than those with GLI1. tGLI1 has gained the ability to enhance expression of several invasion- and angiogenesis-promoting genes. tGLI1 was highly expressed in nearly 50% of GBM specimens we had examined, but undetectable in normal brain or other normal tissues. These observations have led us to hypothesize that the novel tGLI1 transcription factor behaves as a gain-of-function GLI1 that regulates expression of a unique set of genes not targeted by GLI1, and that because of its overexpression in GBM and its ability to enhance genes important for tumor growth, invasion and angiogenesis, tGLI1 supports some of the predominant features of GBM, i.e. high degrees of proliferation, infiltration and vascularity. Three Specific Aims are proposed to test this hypothesis. (1) Determine whether tGLI1 mediates malignant phenotypes of GBM. There has been no systematic study that investigated the role of tGLI1 in distinctive GBM malignant phenotypes. Here, we will create intracranial GBM xenografts with differential levels of tGLI1 via knock-in and knock-down approaches, examine the xenografts for growth, invasiveness and angiogenesis, and monitor the xenograft-carrying mice for survival. (2) Elucidate the mechanisms by which tGLI1 regulates gene expression in GBM. tGLI1 has gained the ability to enhance expression of several genes; however, the underlying mechanisms for this unique ability is currently unknown. Here, we will investigate two potential mechanisms: a) tGLI1 binds to unique DNA elements, not recognized by GLI1, within its target gene promoters and thereby regulates genes not targeted by GLI1; and b) tGLI1 has gained the ability to interact with transcription regulators that do not interact with GLI1, thereb regulating genes not targeted by GLI1. (3) Investigate alternative splicing process that leads to tGLI1 mRNA synthesis in GBM. Alternative splicing process for tGLI1 mRNA synthesis is presently unknown. We will identify the cis-acting elements within the pre-mRNA and the RNA-binding splicing factors that together mediate alternative splicing for tGLI1 mRNA synthesis. Our study will be the first step towards defining tGLI1 as a novel mediator of GBM malignancy, and understanding the full spectrum of tGLI1 functionality and the molecular basis for tGLI1 mRNA synthesis.

The goal of this proposal is to determine the role of tGLI1 (truncated glioma-associated oncogene homolog 1) in breast cancer brain metastasis (BCBM). Our laboratory discovered tGLI1 as an alternatively spliced GLI1 that lacks entire exon 3 and part of exon 4, but retains the ability to undergo nuclear transport and respond to sonic hedgehog-smoothened signaling. In addition to activating known GLI1 target genes, tGLI1 gains the ability to activate genes not regulated by GLI1, leading to increased migration, invasion and angiogenesis. Whether tGLI1 plays any role in metastasis of any tumor type is unknown. Our mouse studies indicated that tGLI1 promotes breast cancer preferential metastasis to the brain and conversely, tGLI1 knockdown selectively suppressed BCBM. tGLI1 protein is overexpressed in lymph node metastases and BCBM samples. In elucidating how tGLI1 promotes BCBM, we found that exosomes secreted from tGLI1-high cancer cells strongly activated astrocytes, the most abundant brain cells known to promote tumor growth when activated, and that tGLI1-high cancer cells had an increased ability to interact with and activate astrocytes in vitro and in vivo. Since microRNAs can be loaded into exosomes and circulating tumor exosomal miRNAs can prime distant organs for organ-specific metastasis, we conducted an exosomal miRNA microarray followed by validations and found that tGLI1-high cancer cells secreted high levels of exosomal miR-1290 and miR-1246 in vitro and in vivo. Whether miR-1290 and miR-1246 play any role in brain metastasis of any cancer or astrocytes is unknown. We observed that miR-1290/1246 inhibited expression of two transcription repressors (FOXA2 and SOX9), which leads to secretion of ciliary neurotropic factor (CNTF) to activate astrocytes and stimulate cancer cells. We hypothesize that tumoral tGLI1 promotes brain metastasis by priming astrocytes in the metastatic niche, and activated astrocytes in turn facilitate BCBM through secreting CNTF. We further hypothesize that tGLI1-high breast cancer cells prime astrocytes via the novel signaling axis: tumoral tGLI1?exosomal miR-1290/1246?astrocyte FOXA2/ SOX9?astrocyte CNTF?astrocyte activation. In Aim 1, we will determine the extent to which tGLI1 promotes BCBM using three mouse models (cell line-derived xenograft, PDX, and transgenic mice), overexpression and knockdown approaches, two inoculation routes (intracardiac and mammary fat pad injections), and human patient samples. In Aim 2, we will examine whether and how tumoral tGLI1 upregulates exosomal miR- 1290/1246 to activate astrocytes in the metastatic niche, and clarify how miR-1290/1246 suppress FOXA2 and SOX9 expression within astrocytes, how FOXA2 and SOX9 repress CNTF expression, and the role of CNTF in astrocyte activation and BCBM progression. In Aim 3, we will examine if miR-1290 and miR-1246 play essential roles in tGLI1-mediated BCBM, identify their downstream targets, and elucidate the roles of intracellular miR- 1290/1246 in BCBM. The project could define tGLI1, miR-1290/1246, and CNTF as novel mediators of BCBM and regulators of astrocytes in the metastatic niche, thus providing novel mechanistic insights into BCBM.