Peter T. White

Regulation of pathogenic IL-17 responses in collagen-induced arthritis: roles of endogenous interferon-gamma and IL-4

IntroductionInterleukin (IL)-17 plays an important role in the pathogenesis of rheumatoid arthritis and the mouse model collagen-induced arthritis (CIA). Interferon(IFN)-γ and IL-4 have been shown to suppress Th17 development in vitro, but their potential immunoregulatory roles in vivo are uncertain. The goals of this study were to determine the relationship between Th17 responses and disease severity in CIA and to assess regulation of IL-17 by endogenous IFN-γ and IL-4.MethodsDBA1/LacJ mice were immunized with type II collagen in complete Freunds adjuvant (CFA) to induce arthritis, and treated with neutralizing antibody to IFN-γ and/or IL-4. Systemic IL-17, IFN-γ, and IL-4 were measured in serum. At the peak of disease, cytokine production was measured by ELISA of supernatants from spleen, lymph node and paw cultures. Paws were also scored for histologic severity of arthritis.ResultsJoint inflammation was associated with a higher ratio of systemic IL-17/IFN-γ. Neutralization of IFN-γ accelerated the course of CIA and was associated with increased IL-17 levels in the serum and joints. The IFN-γ/IL-4/IL-17 responses in the lymphoid organ were distinct from such responses in the joints. Neutralization of IL-4 led to increased arthritis only in the absence of IFN-γ and was associated with increased bone and cartilage damage without an increase in the levels of IL-17.ConclusionsIL-4 and IFN-γ both play protective roles in CIA, but through different mechanisms. Our data suggests that the absolute level of IL-17 is not the only determinant of joint inflammation. Instead, the balance of Th1, Th2 and Th17 cytokines control the immune events leading to joint inflammation.

Interactions of T Cells with Fibroblast-Like Synoviocytes: Role of the B7 Family Costimulatory Ligand B7-H3

Fibroblast-like synoviocytes (FLS) and T cells can activate each other in vitro, and in vivo interactions between these cells may be important in rheumatoid arthritis (RA), yet FLS lack significant expression of CD28 ligands. We sought to identify molecules homologous to CD28 ligands that are strongly expressed by FLS, and documented strong B7-H3 expression on FLS and by fibroblasts of other tissues, which was unaffected by a variety of cytokines. Western blot analysis of FLS lysates showed predominant expression of the larger, four Ig-like domain isoform of B7-H3. Immunohistological sections of RA synovial tissue showed strong staining for B7-H3 on FLS. Cells expressing B7-H3 were distinct from but in close proximity to cells that expressed CD45, CD20, and CD3. Confocal microscopy of FLS and T cell cocultures showed localization of B7-H3 in the region of the T cell-FLS contact point, but distinct from the localization of T cell CD11a/CD18 (LFA-1) and FLS CD54 (ICAM-1). Reduction of B7-H3 expression on FLS by RNA interference affected interactions of FLS with resting T cells or cytokine-activated T cells. Resting T cells showed increased production of TNF-α, IFN-γ, and IL-2, whereas cytokine-activated T cells showed reduced cytokine production relative to control. However, cytokine production by T cells activated through their TCR was not notably altered by knock down of B7-H3. These observations suggest that B7-H3 may be important for the interactions between FLS and T cells in RA, as well as other diseases, and the outcome of such interactions depends on the activation state of the T cell.

Novel HSP90 inhibitors effectively target functions of thyroid cancer stem cell preventing migration and invasion

BACKGROUND Thyroid cancer stem cells (CSCs) with ALDH and CD44 markers contribute to tumor growth and aggressiveness. We hypothesized that novel HSP90 inhibitors (KU711, WGA-TA) and 17-AAG can effectively target the function of thyroid CSCs in vitro and prevent migration and invasion. METHODS Validated papillary (TPC1), follicular (FTC238,WRO), and anaplastic (ACT1) human thyroid cancer cell lines were treated with 3 HSP90 inhibitors. CSCs were quantified for aldehyde dehydrogenase by flow cytometry, CD44 expression by Western blot, and thyrosphere formation assay. Cellular pathway proteins were analyzed by Western blot and migration/invasion by Boyden-chambers. RESULTS WGA-TA and 17-AAG induced HSP70 compensation (not observed with KU711) on Western blot in all cell lines (>1,000 fold vs controls). Only WGA-TA degraded HSP90-Cdc37 complexing by 60-70% versus controls. Expression of HSP90 clients β-catenin, BRAF, Akt, and phospho-Akt were significantly inhibited by WGA-TA treatment (50-80%, 50-90%, >80%, and >90%) compared with controls, KU711, and 17-AAG treatment. KU711 and WGA-TA decreased CD44 expression in all cell lines (25-60% vs controls/17-AAG), decreased ALDEFLOR activity by 69-98% (P < .005), and decreased sphere formation by 64-99% (P < .05 each). Finally, cell migration was decreased by 31-98%, 100%, and 30-38%, and invasion by 75-100%, 100%, and 47% by KU711,WGA-TA, and 17-AAG treatment (P < .05) each, respectively. CONCLUSION KU711 and WGA-TA are novel HSP90 inhibitors targeting CSC function and inhibiting cell migration/invasion in differentiated and anaplastic thyroid cancers, warranting further translational evaluation in vivo.

The discovery and development of sorafenib for the treatment of thyroid cancer

Introduction: Although the prognosis for most differentiated thyroid cancers (DTCs) remains excellent, recurrence and insensitivity to radioactive iodine (RAI) lead to therapeutic challenges and poorer outcomes. In defining the pathogenesis of DTC, multiple genetic alterations have been identified in key pathways focused around receptor tyrosine kinases (RTKs) and the MAPK cascade. Sorafenib was specifically developed to target rapidly accelerated fibrosarcoma (RAF) kinase in the MAPK pathway. It has been shown, however, to have potent inhibition of several key RTKs, RAF kinase and the V600E BRAF mutation, gaining FDA approval in November 2013 for advanced RAI-refractory DTC. Areas covered: The authors provide a review of the targeted RAF kinase discovery strategy as well as the preclinical and clinical development of sorafenib, leading to FDA approval of DTC. The authors also provide some insight into the clinical use of sorafenib and look at important considerations for treatment. Expert opinion: Sorafenib significantly improves progression-free survival in metastatic DTC patients who are RAI-refractory. However, the overall survival benefit is still unproven and requires additional follow up. Despite its cost and significant side-effect profile, which results in dose reductions in the majority of DTC patients, sorafenib should be considered for the treatment of RAI-refractory advanced DTC patients following evaluation of their individual risk–benefit stratification.

Synthetic high-density lipoprotein nanoparticles: A novel therapeutic strategy for adrenocortical carcinomas

BACKGROUND Chemotherapeutic strategies for adrenocortical carcinoma (ACC) carry substantial toxicities. Cholesterol is critical for ACC cell growth and steroidogenesis, and ACC cells overexpress scavenger receptor BI, which uptakes cholesterol from circulating high-density lipoprotein (HDL) cholesterol. We hypothesize that cholesterol-free synthetic-HDL nanoparticles (sHDL) will deplete cholesterol and synergize with chemotherapeutics to achieve enhanced anticancer effects at lesser (less toxic) drug levels. METHODS The antiproliferative efficacy of ACC cells for the combinations of sHDL with chemotherapeutics was tested by Cell-Titer Glo. Cortisol levels were measured from the culture media. Effects on steroidogenesis was measured by real-time polymerase chain reaction (RT-PCR). Induction of apoptosis was evaluated by flow cytometry. RESULTS Combination Index (CI) for sHDL and either etoposide (E), cisplatin (P), or mitotane (M) demonstrated synergy (CI < 1) for antiproliferation. Alone or in combination with the chemotherapy drugs, sHDL was able to decrease cortisol production by 70-90% compared with P alone or controls (P < .01). RT-PCR indicated inhibition of steroidogenic enzymes for sHDL (P < .01 vs no sHDL). Combination therapy with sHDL increased apoptosis by 30-50% compared with drug or sHDL alone (P < .03), confirmed by a decrease in the mitochondrial potential. CONCLUSION sHDL can act synergistically and lessen the amount of M/E/P needed for anticancer efficacy in ACC in part owing to cholesterol starvation. This novel treatment strategy warrants further investigation translationally.

Synthetic high-density lipoprotein nanodisks for targeted withalongolide delivery to adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy and has a 5-year survival rate of <35%. ACC cells require cholesterol for steroid hormone production, and this requirement is met via expression on the cell surface of a high level of SRB1, responsible for the uptake of high-density lipoproteins (HDLs), which carry and transport cholesterol in vivo. Here, we describe how this natural lipid carrier function of SRB1 can be utilized to improve the tumor-targeted delivery of a novel natural product derivative – withalongolide A 4,19,27-triacetate (WGA-TA) – which has shown potent antitumor efficacy, but poor aqueous solubility. Our strategy was to use synthetic HDL (sHDL) nanodisks, which are effective in tumor-targeted delivery due to their smallness, long circulation half-life, documented safety, and ability to bind to SRB1. In this study, we prepared sHDL nanodisks using an optimized phospholipid composition combined with ApoA1 mimetic peptide (22A), which has previously been tested in clinical trials, to load WGA-TA. Following optimization, WGA-TA nanodisks showed drug encapsulation efficiency of 78%, a narrow particle size distribution (9.81±0.41 nm), discoid shape, and sustained drug release in phosphate buffered saline. WGA-TA-sHDL nanodisks exhibited higher cytotoxicity in the ACC cell line H295R half maximal inhibitory concentration ([IC50] 0.26±0.045 μM) than free WGA-TA (IC50 0.492±0.115 μM, P<0.05). Fluorescent dye-loaded sHDL nanodisks efficiently accumulated in H295R adrenal carcinoma xenografts 24 hours following dosing. Moreover, daily intraperitoneal administration of 7 mg/kg WGA-TA-loaded sHDL nanodisks significantly inhibited tumor growth during 21-day administration to H295R xenograft-bearing mice compared to placebo (P<0.01). Collectively, these results suggest that WGA-TA-loaded nanodisks may represent a novel and beneficial therapeutic strategy for the treatment of ACC.

Natural withanolides in the treatment of chronic diseases

Withanolides, and in particular extracts from Withania somnifera, have been used for over 3,000 years in traditional Ayurvedic and Unani Indian medical systems as well as within several other Asian countries. Traditionally, the extracts were ascribed a wide range of pharmacologic properties with corresponding medical uses, including adaptogenic, diuretic, anti-inflammatory, sedative/anxiolytic, cytotoxic, antitussive, and immunomodulatory. Since the discovery of the archetype withaferin A in 1965, approximately 900 of these naturally occurring, polyoxygenated steroidal lactones with 28-carbon ergostane skeletons have been discovered across 24 diverse structural types. Subsequently, extensive pharmacologic research has identified multiple mechanisms of action across key inflammatory pathways. In this chapter we identify and describe the major withanolides with anti-inflammatory properties, illustrate their role within essential and supportive inflammatory pathways (including NF-κB, JAK/STAT, AP-1, PPARγ, Hsp90 Nrf2, and HIF-1), and then discuss the clinical application of these withanolides in inflammation-mediated chronic diseases (including arthritis, autoimmune, cancer, neurodegenerative, and neurobehavioral). These naturally derived compounds exhibit remarkable biologic activity across these complex disease processes, while showing minimal adverse effects. As novel compounds and analogs continue to be discovered, characterized, and clinically evaluated, the interest in withanolides as a novel therapeutic only continues to grow.

Synthetic high-density lipoprotein nanoconjugate targets neuroblastoma stem cells, blocking migration and self-renewal

Background: Pathways critical for neuroblastoma cancer stem cell function are targeted by 4,19,27‐triacetyl withalongolide A (WGA‐TA). Because neuroblastoma cells and their cancer stem cells highly overexpress the scavenger receptor class B type 1 receptor that binds to synthetic high‐density lipoprotein, we hypothesized that a novel mimetic synthetic high‐density lipoprotein nanoparticle would be an ideal carrier for the delivery of 4,19,27‐triacetyl withalongolide to neuroblastoma and neuroblastoma cancer stem cells. Methods: Expression of scavenger receptor class B type 1 in validated human neuroblastoma cells was evaluated by quantitative polymerase chain reaction (qPCR) and Western blot. In vitro cellular uptake of synthetic high‐density lipoprotein nanoparticles was observed with a fluorescence microscope. In vivo biodistribution of synthetic high‐density lipoprotein nanoparticles was investigated with IVIS imaging. Self‐renewal and migration/invasion were assessed by sphere formation and Boyden chamber assays, respectively. Viability was analyzed by CellTiter‐Glo assay. Cancer stem cell markers were evaluated by flow cytometry. Results: qPCR and Western blot analysis revealed a higher level of scavenger receptor class B type 1 expression and drug uptake in N‐myc amplified neuroblastoma cells. In vitro uptake of synthetic high‐density lipoprotein was almost completely blocked by excess synthetic high‐density lipoprotein. The synthetic high‐density lipoprotein nanoparticles mainly accumulated in the tumor and liver, but not in other organs. Synthetic HDL‐4,19,27‐triacetyl withalongolide showed a 1,000‐fold higher potency than the carrier (synthetic high‐density lipoprotein) alone (P < .01) to kill neuroblastoma cells. Additionally, a dose‐dependent decrease in sphere formation, invasion, migration, and cancer stem cell markers was observed after treatment of neuroblastoma cells with synthetic high‐density lipoprotein–4,19,27‐triacetyl withalongolide A. Conclusion: Synthetic high‐density lipoprotein is a promising platform to improve the delivery of anticancer drug 4,19,27‐triacetyl withalongolide A to neuroblastomas and neuroblastoma cancer stem cells through SR‐B1 targeting in vitro and in vivo.

Abstract B24: Natural withanolides: A new group of anticancer drugs that selectively target the PI3K-mTOR pathway as novel potent therapeutics against colon cancers in vitro and in vivo

Background: Current targeted inhibitors for colon cancer can reduce tumor burdens or stabilize disease, but are limited by their toxicity and lack of durable efficacy. Efforts continue to identify novel agents for this disease and newer multi-kinase inhibitors are currently being evaluated in clinical trials. Novel natural withanolides have been shown to selectively inhibit the PI3K-Akt-mTOR pathway in vitro and in vivo in adrenal cancer and medullary thyroid cancers. We hypothesize that this new class of drug compounds will be potent, selectively targeted inhibitors of this pathway network in colon cancers in vitro as well as in vivo as a promising novel therapeutics for this disease. Methods: In vitro: Withaferin A (WA), withalongolide A (WGA) and acetylated derivatives of each were first tested for anticancer effect on tumor cell viability on 5 different colon cancer lines (SW48, SW480, HT-29, SW-620, HCT-116) by MTS assay, then for apoptosis by flow cytometry and confirmed by western analysis for caspase 3 activation and PARP cleavage. PI3K-mTOR inhibition was confirmed by western blot analysis for total and phosphorylated levels of: mTOR, Akt, 4EBP1, p70-S6K; as well as for specific effects on colon cancer regulatory pathways including: GSK3β, β-catenin, Notch1 cleavage, and cyclin D1. In vivo: Athymic nude mice were injected with 10^7 HT-29 human colon cancer-cells in the left posterior flank and treated x 21days once tumors were >100mm3(N=10/drug) and followed for endpoints of toxicity (decreased body weight or body score),tumor growth>1500mm3 and survival. Results: IC50 levels of the parent compound WA averaged 640nM whereas the acetylated derivatives were as low as 30-100nM in the 5 cell lines compared to 4500nM in normal fibroblast cells. The withanolides potently induced apoptosis (starting at 250-500nM drug) on flow cytometry and this was confirmed by caspase 3 activation and PARP cleavage on Western analysis. These drugs also selectively inhibited phosphorylation of mTOR, Akt, 4EBP1, and p70S6K in a dose-dependent manner in the colon cancer lines starting at only 500nM of drug. Specific to colon cancers, the withanolides modulated GSK3β, β-catenin, Notch1 cleavage, and cyclin D1 levels in a dose-dependent manner all more potently than targeted tyrosine kinase inhibitors like ventadenib and cabozantenib which did not inhibit the PI3K-mTOR pathway. In vivo, all control animals progressed to metastatic disease to the liver and a terminal study endpoint within 6 weeks. All treatment arms inhibited HT-29 colon cancer growth at varying rates during the treatment period with only the WA treatment arm at 8mg/kg/day demonstrating a complete pathologic response to treatment in 80% of mice (p Conclusions: Natural withanolides are a novel class of chemotherapy drugs that selectively target PI3K-mTOR in colon cancers and demonstrate potent efficacy in vitro and in vivo compared to TKIs being evaluated in clinical trials with improved tolerability and less toxicity in vivo. These compounds warrant further evaluation to determine their clinical potential in this disease. Citation Format: Chitra Subramanian, Peter White, Alicia Gingrich, Barbara N. Timmermann, Mark Steven Cohen. Natural withanolides: A new group of anticancer drugs that selectively target the PI3K-mTOR pathway as novel potent therapeutics against colon cancers in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B24.

Abstract 1739: A novel HSP90 inhibitor induces metabolic oxidative stress in head and neck squamous cancers and prevents migration and stem cell EMT even under hypoxic conditions

INTRODUCTION: Advanced head and neck squamous cell carcinoma (HNSCC) remains a challenge to treat. In HNSCC, hypoxia is a negative prognostic marker with altered metabolism, increased invasiveness, and resistance to therapy. Natural withanolides exhibit potent, highly selective anticancer effects in HNSCCs in vitro and in vivo through a novel anticancer mechanism. These drugs [withaferin A and the novel compound, withalongolide A-triacetate(WGA-TA)] selectively induce metabolic oxidative stress in cancer cells while inhibiting HSP90 chaperone function via disruption of CDC37 (co-chaperone) binding. We hypothesize that WGA-TA is a potent, novel therapeutic drug for HNSCC that will decrease tumor migration and cancer stem cell endothelial-mesenchymal transition (EMT) even in the setting of hypoxia. METHODS: Human HNSCC cell lines MDA-1986, UMSCC11A, 11B and 12 were grown either under normoxic or hypoxic conditions. CM-H2DCFDA level defined accumulation of intracellular reactive oxygen species. Effects on proliferation and apoptosis were evaluated by flow cytometry (FC), Western blotting (WB), and RT PCR. Viability was evaluated by MTS and clonigenic assay. Scratch wound-healing and Boyden chamber assays were used to follow cell migration. RESULTS: WGA-TA treatment of both hypoxic and normoxic cells showed dose dependent induction of oxidation (p CONCLUSION: WGA-TA potently induces glycolytic oxidative stress in HNSCC leading to decrease in CSC EMT as well as,induction of apoptosis and loss of tumor cell migration even in hypoxic conditions. These exciting findings warrant further evaluation in translational in vivo models. Citation Format: Chitra Subramanian, Qing Zhu, Eileen Brandes, Peter T. White, Barbara N. Timmermann, Mark S. Cohen. A novel HSP90 inhibitor induces metabolic oxidative stress in head and neck squamous cancers and prevents migration and stem cell EMT even under hypoxic conditions. [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 1739. doi:10.1158/1538-7445.AM2015-1739