Yihui Shi

Metallothionein Induction by Hypoxia Involves Cooperative Interactions between Metal-Responsive Transcription Factor-1 and Hypoxia-Inducible Transcription Factor-1α

Mammalian metallothionein (MT) genes are transcriptionally activated by the essential metal zinc as well as by environmental stresses, including toxic metal overload and redox fluctuations. In addition to playing a key role in zinc homeostasis, MT proteins can protect against metal- and oxidant-induced cellular damage, and may participate in other fundamental physiologic and pathologic processes such as cell survival, proliferation, and neoplasia. Previously, our group reported a requirement for metal-responsive transcription factor-1 (MTF-1) in hypoxia-induced transcription of mouse MT-I and human MT-IIA genes. Here, we provide evidence that the protumorigenic hypoxia-inducible transcription factor-1α (HIF-1α) is essential for induction of MT-1 by hypoxia, but not zinc. Chromatin immunoprecipitation assays revealed that MTF-1 and HIF-1α are both recruited to the mouse MT-I promoter in response to hypoxia, but not zinc. In the absence of HIF-1α, MTF-1 is recruited to the MT-I promoter but fails to activate MT-I gene expression in response to hypoxia. Thus, HIF-1α seems to function as a coactivator of MT-I gene transcription by interacting with MTF-1 during hypoxia. Coimmunoprecipitation studies suggest interaction between MTF-1 and HIF-1α, either directly or as mediated by other factors. It is proposed that association of these important transcription factors in a multiprotein complex represents a common strategy to control unique sets of hypoxia-inducible genes in both normal and diseased tissue. (Mol Cancer Res 2008;6(3):483–90)

The metal-responsive transcription factor-1 protein is elevated in human tumors

We previously identified metal-responsive transcription factor-1 (MTF-1) as a positive contributor to mouse fibrosarcoma growth through effects on cell survival, proliferation, tumor angiogenesis, and extracellular matrix remodeling. In the present study, we investigated MTF-1 protein expression in human tissues by specific immunostaining of both normal and tumor tissue samples. Immunohistochemical (IHC) staining of a human tissue microarray (TMA), using a unique anti-human MTF-1 antibody, indicated constitutive MTF-1 expression in most normal tissues, with liver and testis displaying comparatively high levels of expression. Nevertheless, MTF-1 protein levels were found to be significantly elevated in diverse human tumor types, including breast, lung, and cervical carcinomas. IHC analysis of a separate panel of full-size tissue sections of human breast cancers, including tumor and normal adjacent, surrounding tissue, confirmed and extended the results of the TMA analysis. Taken with our previous findings, this new study suggests a role for MTF-1 in human tumor development, growth or spread. Moreover, the study suggests that MTF-1 could be a novel therapeutic target that offers the opportunity to manipulate metal or redox homeostasis in tumor cells.

Pharmacodynamic assays to facilitate preclinical and clinical development of pre‐mRNA splicing modulatory drug candidates

The spliceosome has recently emerged as a new target for cancer chemotherapy and novel antitumor spliceosome targeted agents are under development. Here, we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small‐molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel‐based assay for the measurement of drug exposure in human leukocytes. The first assay utilizes a highly specific bioluminescent splicing reporter, based on the skipping of exons 4–11 of a Luc‐MDM2 construct, which specifically yields active luciferase when treated with small‐molecule spliceosome modulators. We demonstrate that this reporter can be used to monitor alternative splicing in whole cells in vitro. We describe here that cell lines carrying the reporter can be used in vivo for the efficient pharmacodynamic analysis of agents during drug optimization and development. We also demonstrate dose‐ and time‐dependent on‐target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression. The second assay relies on the treatment of freshly drawn human blood with SD6 ex vivo treatment. Changes in alternative splicing are determined by RT‐PCR using genes previously identified in in vitro experiments. The Luc‐MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

A Novel Tumor-Activated Prodrug Strategy Targeting Ferrous Iron Is Effective in Multiple Preclinical Cancer Models

Here we describe a new approach for tumor targeting in which augmented concentrations of Fe(II) in cancer cells and/or the tumor microenvironment triggers drug release from an Fe(II)-reactive prodrug conjugate. The 1,2,4-trioxolane scaffold developed to enable this approach can in principle be applied to a broad range of cancer therapeutics and is illustrated here with Fe(II)-targeted forms of a microtubule toxin and a duocarmycin-class DNA-alkylating agent. We show that the intrinsic reactivity/toxicity of the duocarmycin analog is masked in the conjugated form and this greatly reduced toxicity in mice. This in turn permitted elevated dosing levels, leading to higher systemic exposure and a significantly improved response in tumor xenograft models. Overall our results suggest that Fe(II)-dependent drug delivery via trioxolane conjugates could have significant utility in expanding the therapeutic index of a range of clinical and preclinical stage cancer chemotherapeutics.

Abstract 3532: Antitumor activity of SR16388 on multiple myeloma targets factors needed for adaptation to bone marrow microenvironment

Multiple myeloma (MM) is a B-cell malignancy that is characterized by accumulation of clonal plasma cells in the bone marrow. The bone marrow microenvironment plays a critical role in MM cell pathogenesis and progression. In the bone marrow milieu, MM cells adhere to the bone marrow stromal cells and trigger the secretion of growth factors such as vascular endothelial growth factor (VEGF). VEGF in turn upregulates the secretion of interleukin-6 (IL-6), the major cytokine that mediates MM cell growth and survival in part through the activation of Janus kinase (Jak)/signal transducers and activators of transcription 3 (STAT3). VEGF also stimulates the proliferation of endothelial and stromal cells and induces angiogenesis via its receptors. Thus, targeting both myeloma cells and bone marrow microenvironment is a valid approach for the development of therapeutic agents for MM. At SRI International, we have developed an antiestrogen, SR16388, that binds to ER-α, ER-β and ERR-α with potent antitumor and antiangiogenic properties. We have previously reported that SR16388 inhibited MM cell proliferation in vitro and MM tumors in vivo. In the current study, we are presenting data to show that the inhibitory effect of SR16388 on MM cell proliferation was achieved by inducing apoptosis, and arresting cells at the G2 phase. Furthermore, at nanomolar concentrations SR16388 inhibited IL-6-induced cell proliferation and blocked the activation of STAT3 induced by IL-6 in RPMI-8226 human MM cells. In human endothelial cells, SR16388 inhibited VEGF-induced cell proliferation and VEGF-induced phosphorylation of STAT3. In a RPIM-8226 tumor xenograft model, the microvessel density in the tumor tissue was markedly reduced by treatment with SR16388. We are currently investigating the effect of SR16388 in combination with known therapeutic agents on the growth of MM tumors in vivo. 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 3532. doi:10.1158/1538-7445.AM2011-3532

Near-infrared fluorescent labeling of tissue transglutaminase substrates for imaging tumor boundaries

A novel strategy is developed to optically image tumor boundaries by cross-linking near-infrared fluorescent-labeled tissue trans glutaminase substrates into tumor boundary tissue.

Abstract 4193: A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor scaffold

The splicing of pre-mRNA is a critical process in normal cells and is deregulated in cancer. Compounds that modulate this process have recently been shown to target a specific vulnerability in tumors. We have developed a novel cell-based assay that specifically activates luciferase in cells exposed to SF3B1 targeted compounds, such as sudemycin D6. This assay was used to screen a combined collection of approved drugs and bioactive compounds. This screening approach identified several active hits, the most potent of which were CGP-74514A and aminopurvalanol A, both have been reported to be cyclin-dependent kinases (CDKs) inhibitors. We found that these compounds, and their analogs, show significant cdc2-like kinase (CLK) inhibition and clear structure-activity relationships (SAR) at CLKs. We prepared a set of analogs and were able to ‘dial out’ the CDK activity and simultaneously developed CLK inhibitors with low nanomolar activity. Thus, we have demonstrated the utility of our exon-skipping assay and identified new molecules that exhibit potency and selectivity for CLK, as well as some structurally related dual CLK/CDK inhibitors. Citation Format: Yihui Shi, Jaehyeon Park, Chandraiah Lagisetti, Wei Zhou, Lidia C. Sambucetti, Thomas R. Webb. A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor scaffold [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4193. doi:10.1158/1538-7445.AM2017-4193

Abstract 4365: Targeting the SF3B1 spliceosome protein: Development of a reporter for HTS screen and pharmacodynamic profiling of small molecule drug leads

The spliceosome is an important emerging target for cancer therapy that has only recently been uncovered that represents an exciting new opportunity for therapeutic intervention in cancer. Two naturally occurring bacterial products show striking selective toxicity to tumors and have recently been found to act by targeting the SF3b subunit of the spliceosome, thus modulating pre-mRNA splicing in tumor cells. However, these natural products, and analogs derived from them, are synthetically quite complex. We have designed and optimized a class of novel synthetic small molecules, called sudemycins, which also modulate alternative splicing and have substantial antitumor activity. Previous work in our lab has shown that our lead compound, sudemycin D6 has significant in vivo antitumor activity. We now report the development of a high-throughput cell-based luciferase reporter system that has multiple applications in facilitating splicing modulatory drug discovery and development. Citation Format: Yihui Shi, Chandraiah Lagisetti Lagisetti, Amanda S. Joyner, Lidia C. Sambucetti, Thomas R. Webb. Targeting the SF3B1 spliceosome protein: Development of a reporter for HTS screen and pharmacodynamic profiling of small molecule drug leads. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4365. doi:10.1158/1538-7445.AM2015-4365

Abstract 2146: Development of functional assays for p97/VCP inhibition.

p97 (also called VCP in metazoans and CDC48 in yeast) is a highly conserved, ubiquitously expressed, and essential AAA ATPase. p97 plays a key role in endoplasmic reticulum-associated degradation of misfolded secretory and membrane proteins as well as ubiquitin-dependent turnover of a subset of cytoplasmic substrates of the ubiquitin-proteasome system.

Abstract 1569: Antitumor activity of SR16388 against human multiple myeloma in vitro and in vivo

Multiple myeloma (MM) is the cancer of plasma cells that derive from a clonal disorder of terminally differentiated B cells in the bone marrow. MM is generally thought to be refractory against chemotherapeutics and is incurable. Development of novel agents for the treatment of this disease is urgently needed. Previous studies have indicated that MM cells express estrogen receptors (ERs), and anti-estrogens such as ICI 182,780 and Tamoxifen have demonstrated inhibitory effects on proliferation of MM cell lines as well as on MM cells isolated from patients. MM cells also over-express proangiogenic factors such as VEGF to induce angiogenesis and support their growth and survival. At SRI International, we have developed a steroidal anti-angiogenic molecule (SR16388) that potently inhibits angiogenesis in lung and prostate cancers, and it also binds to ER-α and ER-s with high affinities in the nanomolar range. In this study, we examined the in vitro and in vivo effects of SR16388 on growth of MM cells. In RPMI-8226 multiple myeloma cells, SR16388 inhibited cell proliferation with an IC50 value of 0.9 µM. Cell cycle analysis revealed that SR16388 arrested the RPMI-8226 cells in G2 phase. In an in vivo murine xenograft model, SR16388 significantly inhibited the growth of RPMI-8226 tumors at the dose levels of 50 and 100 mg/kg with P 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 1569.