Mechanisms for breast cancer metastasis remain unclear. Whether truncated glioma-associated oncogene homolog 1 (TGLI1), a transcription factor known to promote angiogenesis, migration and invasion, plays any role in metastasis of any tumor type has never been investigated. In this study, results of two mouse models of breast cancer metastasis showed that ectopic expression of TGLI1, but not GLI1, promoted preferential metastasis to the brain. Conversely, selective TGLI1 knockdown using antisense oligonucleotides led to decreased breast cancer brain metastasis (BCBM) in vivo. Immunohistochemical staining showed that TGLI1, but not GLI1, was increased in lymph node metastases compared to matched primary tumors, and that TGLI1 was expressed at higher levels in BCBM specimens compared to primary tumors. TGLI1 activation is associated with a shortened time to develop BCBM and enriched in HER2-enriched and triple-negative breast cancers. Radioresistant BCBM cell lines and specimens expressed higher levels of TGLI1, but not GLI1, than radiosensitive counterparts. Since cancer stem cells (CSCs) are radioresistant and Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Triple-negative breast cancer (TNBC) and HER2-positive breast cancer are particularly aggressive and associated with unfavorable prognosis. TNBC lacks effective treatments. HER2-positive tumors have treatment options but often acquire resistance to HER2-targeted therapy after initial response. To address these challenges, we determined whether novel combinations of JAK2-STAT3 and SMO-GLI1/tGLI1 inhibitors synergistically target TNBC and HER2 breast cancer since these two pathways are concurrently activated in both tumor types and enriched in metastatic tumors. Herein, we show that novel combinations of JAK2 inhibitors (ruxolitinib and pacritinib) with SMO inhibitors (vismodegib and sonidegib) synergistically inhibited in vitro growth of TNBC and HER2-positive trastuzumab-resistant BT474-TtzmR cells. Synergy was also observed against breast cancer stem cells. To determine if the combination is efficacious in inhibiting metastasis, we treated mice with intracardially inoculated TNBC cells and found the combination to inhibit lung and liver metastases, and prolong host survival without toxicity. The combination inhibited orthotopic growth, VEGF-A expression, and tumor vasculature of both TNBC and HER2-positive trastuzumab-refractory breast cancer. Lung metastasis of orthotopic BT474-TtzmR xenografts was suppressed by the combination. Together, our results indicated that dual targeting of JAK2 and SMO resulted in synergistic suppression of breast cancer growth and metastasis, thereby supporting future clinical testing.

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The goal of this study is to identify pharmacological inhibitors that target a recently identified novel mediator of breast cancer brain metastasis (BCBM), truncated glioma-associated oncogene homolog 1 (tGLI1). Inhibitors of tGLI1 are not yet available. To identify compounds that selectively kill tGLI1-expressing breast cancer, we screened 1527 compounds using two sets of isogenic breast cancer and brain-tropic breast cancer cell lines engineered to stably express the control, GLI1, or tGLI1 vector, and identified the FDA-approved antifungal ketoconazole (KCZ) to selectively target tGLI1-positive breast cancer cells and breast cancer stem cells, but not tGLI1-negative breast cancer and normal cells. KCZ’s effects are dependent on tGLI1. Two experimental mouse metastasis studies have demonstrated that systemic KCZ administration prevented the preferential brain metastasis of tGLI1-positive breast cancer and suppressed the progression of established tGLI1-positive BCBM without liver toxicities. We further developed six KCZ derivatives, two of which (KCZ-5 and KCZ-7) retained tGLI1-selectivity in vitro. KCZ-7 exhibited higher blood–brain barrier penetration than KCZ/KCZ-5 and more effectively reduced the BCBM frequency. In contrast, itraconazole, another FDA-approved antifungal, failed to suppress BCBM. The mechanistic studies suggest that KCZ and KCZ-7 inhibit tGLI1’s ability to bind to DNA, activate its target stemness genes Nanog and OCT4, and promote tumor proliferation and angiogenesis. Our study establishes the rationale for using KCZ and KCZ-7 for treating and preventing BCBM and identifies their mechanism of action.

Tumor suppressor candidate 2 (TUSC2, also known as FUS1) was identified as a candidate tumor suppressor gene located in a region on chromosome 3p21.3 that undergoes allelic loss in lung and breast cancers. Loss of TUSC2 expression has been reported in various cancers and is associated with poor survival. Evidence to date indicates that TUSC2 behaves as a tumor suppressor in lung cancer; however, its role as a tumor suppressor in other tumor types has not been fully established. Since the mechanism for gliomagenesis is still unclear, we investigated the role of TUSC2 in the development and progression of glioblastoma (GBM), the most common and deadliest brain cancer in adults. Here, we found that forced TUSC2 expression suppressed neurosphere-forming capability of glioma stem cells, regardless of molecular subtypes. Forced expression of TUSC2 in GBM cell lines inhibited their ability to form colonies and neurospheres. To further determine whether TUSC2 plays a tumor suppressive role in GBM, we knocked down TUSC2 expression in TUSC2-expressing GBM cells using siRNA and CRISPR/Cas9, and found TUSC2 knockdown to significantly enhance neurosphere formation of GBM cells. Using an orthotopic GBM xenograft mouse model, we further observed that CRISPR/Cas9-mediated TUSC2 knockout significantly promoted the intracranial growth of GBM tumors. To gain inisghts into the mechanisms underlying TUSC2’s tumor suppressive function in GBM, we conducted RNA-Seq using control and TUSC2-knockout GBM cell lines, and identified a number of genes whose expression was altered in response to TUSC2 loss. Ongoing studies are being conducted to validate idenified genes, and elucidate their involvement in gliomagenesis and GBM progression. Furthermore, we speculated that TUSC2 may interact with cellular proteins leading to tumor suppression. To test this hypothesis, we conducted protein interactome analysis using immunoprecipitaton followed by mass spectrometry in which cell lysates from GBM cells and human astrocytes (a cell-of-origin for GBM) were used. This study has identified approximately 40 proteins that differentially interact with TUSC2 in GBM cells versus human astrocytes. Roles of these TUSC2-interacting proteins in GBM suppression and gliomagenesis are being examined in onging studies. Finally, we have generated conditional TUSC2-knockout mice to further address the role that TUSC2 plays in gliomagenesis. Collectively, these findings support a novel role that TUSC2 plays in GBM progression and gliomagenesis, thereby advancing our understanding of GBM pathobiology. Citation Format: Tadas K. Rimkus, Dongqin Zhu, Richard L. Carpenter, Ivy Paw, Austin Arrigo, Sherona Sirkisoon, Daniel Doheny, Noah Aguayo, Jingyun Lee, Guangxu Jin, Eric Spooner, Boris Pasche, Waldemar Debinski, Hui-Wen Lo. Roles of tumor suppressor candidate 2 (TUSC2) in glioblastoma progression and gliomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3481.