A human breast cancer-derived xenograft and organoid platform for drug discovery and precision oncology.

Models that recapitulate the complexity of human tumors are urgently needed to develop more effective cancer therapies. We report a bank of human patient-derived xenografts (PDXs) and matched organoid cultures from tumors that represent the greatest unmet need: endocrine-resistant, treatment-refractory and metastatic breast cancers. We leverage matched PDXs and PDX-derived organoids (PDxO) for drug screening that is feasible and cost-effective with in vivo validation. Moreover, we demonstrate the feasibility of using these models for precision oncology in real time with clinical care in a case of triple-negative breast cancer (TNBC) with early metastatic recurrence. Our results uncovered a Food and Drug Administration (FDA)-approved drug with high efficacy against the models. Treatment with this therapy resulted in a complete response for the individual and a progression-free survival (PFS) period more than three times longer than their previous therapies. This work provides valuable methods and resources for functional precision medicine and drug development for human breast cancer.

A Cancer-Selective Zinc Ionophore Inspired by the Natural Product Naamidine A.

We present data demonstrating the natural product mimic, zinaamidole A (ZNA), is a modulator of metal ion homeostasis causing cancer-selective cell death by specifically inducing cellular Zn2+-uptake in transformed cells. ZNA's cancer selectivity was evaluated using metastatic, patient-derived breast cancer cells, established human breast cancer cell lines, and three-dimensional organoid models derived from normal and transformed mouse mammary glands. Structural analysis of ZNA demonstrated that the compound interacts with zinc through the N2-acyl-2-aminoimidazole core. Combination treatment with ZnSO4 strongly potentiated ZNA's cancer-specific cell death mechanism, an effect that was not observed with other transition metals. We show that Zn2+-dyshomeostasis induced by ZNA is unique and markedly more selective than other known Zn2+-interacting compounds such as clioquinol. The in vivo bioactivity of ZNA was also assessed and revealed that tumor-bearing mice treated with ZNA had improved survival outcomes. Collectively, these data demonstrate that the N2-acyl-2-aminoimidazole core of ZNA represents a powerful chemotype to induce cell death in cancer cells concurrently with a disruption in zinc homeostasis.

miR-34a Regulates Expression of the Stathmin-1 Oncoprotein and Prostate Cancer Progression.

In aggressive prostate cancers, the oncoprotein STMN1 (also known as stathmin 1 and oncoprotein 18) is often overexpressed. STMN1 is involved in various cellular processes, including cell proliferation, motility, and tumor metastasis. Here, it was found that the expression of STMN1 RNA and protein is elevated in metastatic prostate cancers. Knockdown of STMN1 resulted in reduced proliferation and invasion of cells and tumor growth and metastasis in vivo Furthermore, miR-34a downregulated STMN1 by directly binding to its 3'-UTR. Overexpression of miR-34a in prostate cancer cells reduced proliferation and colony formation, suggesting that it is a tumor suppressor. The transcriptional corepressor C-terminal binding protein 1 (CtBP1) negatively regulated expression of miR-34a. Furthermore, gene expression profiling of STMN1-modulated prostate cancer cells revealed molecular alterations, including elevated expression of growth differentiation factor 15 (GDF15), which is involved in cancer progression and potentially in STMN1-mediated oncogenesis. Thus, in prostate cancer, CtBP1-regulated miR-34a modulates STMN1 expression and is involved in cancer progression through the CtBP1\miR-34a\STMN1\GDF15 axis.Implications: The CtBP1\miR-34a\STMN1\GDF15 axis is a potential therapeutic target for treatment of aggressive prostate cancer. Mol Cancer Res; 16(7); 1125-37. ©2017 AACR.

Expression and Role of PAICS, a De Novo Purine Biosynthetic Gene in Prostate Cancer.

BACKGROUND: Our goal was to investigate de novo purine biosynthetic gene PAICS expression and evaluate its role in prostate cancer progression. METHODS: Next-generation sequencing, qRTPCR and immunoblot analysis revealed an elevated expression of a de novo purine biosynthetic gene, Phosphoribosylaminoimidazole Carboxylase, Phosphoribosylaminoimidazole Succinocarboxamide Synthetase (PAICS) in a progressive manner in prostate cancer. Functional analyses were performed using prostate cancer cell lines- DU145, PC3, LnCaP, and VCaP. The oncogenic properties of PAICS were studied both by transient and stable knockdown strategies, in vivo chicken chorioallantoic membrane (CAM) and murine xenograft models. Effect of BET bromodomain inhibitor JQ1 on the expression level of PAICS was also studied. RESULTS: Molecular staging of prostate cancer is important factor in effective diagnosis, prognosis and therapy. In this study, we identified a de novo purine biosynthetic gene; PAICS is overexpressed in PCa and its expression correlated with disease aggressiveness. Through several in vitro and in vivo functional studies, we show that PAICS is necessary for proliferation and invasion in prostate cancer cells. We identified JQ1, a BET bromodomain inhibitor previously implicated in regulating MYC expression and demonstrated role in prostate cancer, abrogates PAICS expression in several prostate cancer cells. Furthermore, we observe loss of MYC occupancy on PAICS promoter in presence of JQ1. CONCLUSIONS: Here, we report that evaluation of PAICS in prostate cancer progression and its role in prostate cancer cell proliferation and invasion and suggest it as a valid therapeutic target. We suggest JQ1, a BET-domain inhibitor, as possible therapeutic option in targeting PAICS in prostate cancer. Prostate 77:10-21, 2017. © 2016 Wiley Periodicals, Inc.

Sulindac sulfide inhibits colon cancer cell growth and downregulates specificity protein transcription factors.

BACKGROUND: Specificity protein (Sp) transcription factors play pivotal roles in maintaining the phenotypes of many cancers. We hypothesized that the antineoplastic effects of sulindac and its metabolites were due, in part, to targeting downregulation of Sp transcription factors. METHODS: The functional effects of sulindac, sulindac sulfone and sulindac sulfide on colon cancer cell proliferation were determined by cell counting. Effects of these compounds on expression of Sp1, Sp3, Sp4 and pro-oncogenic Sp-regulated genes were determined by western blot analysis of whole cell lysates and in transient transfection assays using GC-rich constructs. RESULTS: Sulindac and its metabolites inhibited RKO and SW480 colon cancer cell growth and the order of growth inhibitory potency was sulindac sulfide>>sulindac sulfone>sulindac. Treatment of SW480 and RKO cells with sulindac sulfide downregulated expression of Sp1, Sp3 and Sp4 proteins. Sulindac sulfide also decreased expression of several Sp-regulated genes that are critical for cancer cell survival, proliferation and angiogenesis and these include survivin, bcl-2, epidermal growth factor receptor (EGFR), cyclin D1, p65 subunit of NFκB and vascular endothelial growth factor (VEGF). Sulindac sulfide also induced reactive oxygen species (ROS) and decreased the level of microRNA-27a in colon cancer cells, which resulted in the upregulation of the Sp-repressor ZBTB10 and this resulted in downregulation of Sp proteins. CONCLUSIONS: The results suggest that the cancer chemotherapeutic effects of sulindac in colon cancer cells are due, in part, to its metabolite sulindac sulfide which downregulates Sp transcription factors and Sp-regulated pro-oncogenic gene products.

Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer.

Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention.

zRICH, a protein induced during optic nerve regeneration in zebrafish, promotes neuritogenesis and interacts with tubulin.

Mammals do not regenerate axons in their central nervous system (CNS) spontaneously. This phenomenon is the cause of numerous medical conditions after damage to nerve fibers in the CNS of humans. The study of the mechanisms of nerve regeneration in other vertebrate animals able to spontaneously regenerate axons in their CNS is essential for understanding nerve regeneration from a scientific point of view, and for developing therapeutic approaches to enhance nerve regeneration in the CNS of humans. RICH proteins are a novel group of proteins implicated in nerve regeneration in the CNS of teleost fish, yet their mechanisms of action are not well understood. A number of mutant versions of the zebrafish RICH (zRICH) protein were generated and characterized at biochemical and cellular levels in our laboratory. With the aim of understanding the effects of RICH proteins in neuronal axon outgrowth, stable transfectants derived from the neuronal model PC12 cell line expressing zRICH Wild-Type or mutant versions of zRICH were studied. Results from differentiation experiments suggest that RICH proteins enhance neuronal plasticity by facilitating neurite branching. Biochemical co-purification results have demonstrated that zRICH binds to the cytoskeletal protein tubulin. The central domain of the protein is sufficient for tubulin binding, but a mutant version of the protein lacking the terminal domains, which cannot bind to the plasma membrane, was not able to enhance neurite branching. RICH proteins may facilitate axon regeneration by regulating the axonal cytoskeleton and facilitating the formation of new neurite branches.

Induction of the transcriptional repressor ZBTB4 in prostate cancer cells by drug-induced targeting of microRNA-17-92/106b-25 clusters.

Androgen-insensitive DU145 and PC3 human prostate cancer cells express high levels of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and treatment of cells with methyl 2-cyano-3,11-dioxo-18ß-olean-1,12-dien-30-oate (CDODA-Me) inhibited cell growth and downregulated Sp1, Sp3, and Sp4 expression. CDODA-Me (15 mg/kg/d) was a potent inhibitor of tumor growth in a mouse xenograft model (PC3 cells) and also decreased expression of Sp transcription factors in tumors. CDODA-Me-mediated downregulation of Sp1, Sp3, and Sp4 was due to induction of the transcriptional repressor ZBTB4, which competitively binds and displaces Sp transcription factors from GC-rich sites in Sp1-, Sp3-, Sp4-, and Sp-regulated gene promoters. ZBTB4 levels are relatively low in DU145 and PC3 cells due to suppression by miR paralogs that are members of the miR-17-92 (miR-20a/17-5p) and miR-106b-25 (miR-106b/93) clusters. Examination of publically available prostate cancer patient array data showed an inverse relationship between ZBTB4 and miRs-20a/17-5p/106b/93 expression, and increased ZBTB4 in patients with prostate cancer was a prognostic factor for increased survival. CDODA-Me induces ZBTB4 in prostate cancer cells through disruption of miR-ZBTB4 interactions, and this results in downregulation of pro-oncogenic Sp transcription factors and Sp-regulated genes.

Pharmacologic doses of ascorbic acid repress specificity protein (Sp) transcription factors and Sp-regulated genes in colon cancer cells.

Ascorbic acid (vitamin C) inhibits cancer cell growth, and there is a controversy regarding the cancer chemoprotective effects of pharmacologic doses of this compound that exhibits prooxidant activity. We hypothesized that the anticancer activity of pharmacologic doses of ascorbic acid (<5 mM) is due, in part, to reactive oxygen species-dependent downregulation of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 and Sp-regulated genes. In this study, ascorbic acid (1-3 mM) decreased RKO and SW480 colon cancer cell proliferation and induced apoptosis and necrosis, and this was accompanied by downregulation of Sp1, Sp3, and Sp4 proteins. In addition, ascorbic acid decreased expression of several Sp-regulated genes that are involved in cancer cell proliferation [hepatocyte growth factor receptor (c-Met), epidermal growth factor receptor and cyclin D1], survival (survivin and bcl-2), and angiogenesis [vascular endothelial growth factor (VEGF) and its receptors (VEGFR1 and VEGFR2)]. Other prooxidants such as hydrogen peroxide exhibited similar activities in colon cancer cells, and cotreatment with glutathione inhibited these responses. This study demonstrates for the first time that the anticancer activities of ascorbic acid are due, in part, to ROS-dependent repression of Sp transcription factors.

GT-094, a NO-NSAID, inhibits colon cancer cell growth by activation of a reactive oxygen species-microRNA-27a: ZBTB10-specificity protein pathway.

Ethyl 2-((2,3-bis(nitrooxy)propyl)disulfanyl)benzoate (GT-094) is a novel nitric oxide (NO) chimera containing an nonsteroidal anti-inflammatory drug (NSAID) and NO moieties and also a disulfide pharmacophore that in itself exhibits cancer chemopreventive activity. In this study, the effects and mechanism of action of GT-094 were investigated in RKO and SW480 colon cancer cells. GT-094 inhibited cell proliferation and induced apoptosis in both cell lines and this was accompanied by decreased mitochondrial membrane potential (MMP) and induction of reactive oxygen species (ROS), and these responses were reversed after cotreatment with the antioxidant glutathione. GT-094 also downregulated genes associated with cell growth [cyclin D1, hepatocyte growth factor receptor (c-Met), epidermal growth factor receptor (EGFR)], survival (bcl-2, survivin), and angiogenesis [VEGF and its receptors (VEGFR1 and VEGFR2)]. Results of previous RNA interference studies in this laboratory has shown that these genes are regulated, in part, by specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 that are overexpressed in colon and other cancer cell lines and not surprisingly, GT-094 also decreased Sp1, Sp3, and Sp4 in colon cancer cells. GT-094-mediated repression of Sp and Sp-regulated gene products was due to downregulation of microRNA-27a (miR-27a) and induction of ZBTB10, an Sp repressor that is regulated by miR-27a in colon cancer cells. Moreover, the effects of GT-094 on Sp1, Sp3, Sp4, miR-27a, and ZBTB10 were also inhibited by glutathione suggesting that the anticancer activity of GT-094 in colon cancer cells is due, in part, to activation of an ROS-miR-27a:ZBTB10-Sp transcription factor pathway.