Samita Andreansky

Activation of the Unfolded Protein Response by 2-Deoxy-d-Glucose Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication and Gene Expression

ABSTRACT Lytic replication of the Kaposis sarcoma-associated herpesvirus (KSHV) is essential for the maintenance of both the infected state and characteristic angiogenic phenotype of Kaposis sarcoma and thus represents a desirable therapeutic target. During the peak of herpesvirus lytic replication, viral glycoproteins are mass produced in the endoplasmic reticulum (ER). Normally, this leads to ER stress which, through an unfolded protein response (UPR), triggers phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α), resulting in inhibition of protein synthesis to maintain ER and cellular homeostasis. However, in order to replicate, herpesviruses have acquired the ability to prevent eIF2α phosphorylation. Here we show that clinically achievable nontoxic doses of the glucose analog 2-deoxy-d-glucose (2-DG) stimulate ER stress, thereby shutting down eIF2α and inhibiting KSHV and murine herpesvirus 68 replication and KSHV reactivation from latency. Viral cascade genes that are involved in reactivation, including the master transactivator (RTA) gene, glycoprotein B, K8.1, and angiogenesis-regulating genes are markedly decreased with 2-DG treatment. Overall, our data suggest that activation of UPR by 2-DG elicits an early antiviral response via eIF2α inactivation, which impairs protein synthesis required to drive viral replication and oncogenesis. Thus, induction of ER stress by 2-DG provides a new antiherpesviral strategy that may be applicable to other viruses.

Chronic thoracic spinal cord injury impairs CD8+ T-cell function by up-regulating programmed cell death-1 expression

BackgroundChronic spinal cord injury (SCI) induces immune depression in patients, which contributes to their higher risk of developing infections. While defects in humoral immunity have been reported, complications in T-cell immunity during the chronic phase of SCI have not yet been explored.MethodsTo assess the impact of chronic SCI on peripheral T-cell number and function we used a mouse model of severe spinal cord contusion at thoracic level T9 and performed flow cytometry analysis on the spleen for T-cell markers along with intracellular cytokine staining. Furthermore we identified alterations in sympathetic activity in the spleen of chronic SCI mice by measuring splenic levels of tyrosine hydroxylase (TH) and norepinephrine (NE). To gain insight into the neurogenic mechanism leading to T-cell dysfunction we performed in vitro NE stimulation of T-cells followed by flow cytometry analysis for T-cell exhaustion marker.ResultsChronic SCI impaired both CD4+ and CD8+ T-cell cytokine production. The observed T-cell dysfunction correlated with increased expression of programmed cell death 1 (PD-1) exhaustion marker on these cells. Blocking PD-1 signaling in vitro restored the CD8+ T-cell functional defect. In addition, we showed that chronic SCI mice had higher levels of splenic NE, which contributed to the T-cell exhaustion phenotype, as PD-1 expression on both CD4+ and CD8+ T-cells was up-regulated following sustained exposure to NE in vitro.ConclusionsThese studies indicate that alteration of sympathetic activity following chronic SCI induces CD8+ T-cell exhaustion, which in turn impairs T-cell function and contributes to immune depression. Inhibition of the exhaustion pathway should be considered as a new therapeutic strategy for chronic SCI-induced immune depression.

Durable immune response to inactivated H1N1 vaccine is less likely in children with sickle cell anemia receiving chronic transfusions.

Defects in the immune system may affect vaccine responsiveness. Because of the splenic hypofunction and abnormal opsonic activity, it was unknown whether patients with sickle cell disease (SCD) would respond appropriately to H1N1 vaccination. The objective of this study was to assess seroprotective post‐vaccine H1N1 antibody response in children with SCD.

ERBB2 Overexpression Establishes ERBB3-Dependent Hypersensitivity of Breast Cancer Cells to Withaferin A

The catalytically deficient ERBB3 strongly synergizes with the receptor tyrosine kinase ERBB2, and elevated levels represent an overall risk factor for unfavorable disease outcomes in breast cancer. Although itself not a target of pan-ERBB kinase inhibitors, it contributes to resistance in ERBB2-targeted treatment regiments. The steroidal lactone Withaferin A (WA) has established broad anticancer properties through several modes of action and was shown to be effective against triple-negative breast cancers at elevated concentrations. We found that ERBB2 overexpression does render cells hypersensitive to WA. Although ERBB2 downregulation is one aspect of WA treatment at high concentrations, it is not causal for the elevated sensitivity at lower dosages. Instead, WA targets the ability of ERBB3 to amplify ERBB2 signaling. ERBB3 receptor levels, constitutive phosphorylation of both ERBB3 and ERBB2, as well as signaling through AKT are eliminated by WA treatment. By targeting ERBB2/ERBB3 as a functional unit, it is also effective in cases in which ERBB2-directed inhibitors, such as lapatinib, alone show reduced potency. Hence, WA or derivatives thereof may present a low toxicity addition to ERBB2-targeting therapeutics, especially in cases in which ERBB3 involvement is driving resistance or reduced overall sensitivity. Mol Cancer Ther; 15(11); 2750–7. ©2016 AACR.

First Locally Acquired Congenital Zika Syndrome Case in the United States: Neonatal Clinical Manifestations

In the spring of 2017, a full-term infant with microcephaly was delivered in South Florida. During first trimester, the mother presented with fever, nausea, and vomiting. She reported no foreign travel for herself or her partner. The infants neurologic, ophthalmologic, neuroradiologic, and audiologic findings were highly suggestive of congenital Zika syndrome (CZS), confirmed by IgM antibodies and plaque reduction neutralization test. New observations, including peripheral temporal retinal avascularity and peripapillary retinal nerve fiber layer thinning, are presented from this first known case of non-travel-associated CZS in the United States. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:e93-e98.].

Abstract A115: Withaferin A downregulates her-2/neu protein expression in human breast cancer cells.

HER-2/neu protein is over-expressed in approximately 20-30% of breast cancers. Overexpression of this oncoprotein results in a more aggressive tumor phenotype and poor prognosis for patients. Current treatment strategies include passive administration of anti-HER-2/neu monoclonal antibody Trastuzumab as well as the small molecule inhibitor lapatinib. Both of these molecules function at the receptor level to block HER-2/neu signaling resulting in inhibition of cell growth and tumor regression in the majority of patients. However, a significant number of patients relapse or develop progressive disease due to acquired mechanisms of resistance to current therapy. Alternative approaches, that utilize multiple modes of action, are therefore necessary to combat this severe form of breast cancer. Withaferin A (WA) is a natural compound isolated from the Indian ayurvedic medicinal plant Withania Somnifera. Natural compounds are notorious for their multiple mechanisms of action, which make them ideal therapeutics for complex diseases such as cancer. Recent research has demonstrated that WA is effective in inducing cell death in several cancer cell lines by inhibiting molecular pathways such as Akt and MAPK proliferative cascades which are downstream of her-2/neu activation. Additionally, WA has also been shown to inhibit function of the molecular chaperone heat shock protein 90 (HSP90) in pancreatic carcinoma cells. Since HER-2/neu is a client protein of HSP90 and the chaperone functions to stabilize HER-2/neu protein expression on the cell surface, our goal was to determine whether WA would directly affect HER-2/neu expression. We initially established the cytotoxic activity of WA on HER-2/neu expressing human breast cancer cell lines such as SKBr3, MCF-7her-2neu and BT474 by MTT assay. We report that WA exhibits potent cytotoxic activity in these cells with IC50 concentrations of 1.6, 2.1 and 0.6 μM respectively after 48hr treatment. Comparison of WA sensitivity in a low HER-2/neu expressing cell line MCF-7 generated an IC50 of 5.1μM, indicating that cell lines that overexpress HER-2/neu may be more susceptible to the compound. Analysis of HER-2/neu expression both at the cell surface and intracellular localization were next performed in response to WA. WA treatment completely downregulated the expression of HER-2/neu protein from cell surface by 24hr and was both time and concentration dependent. Abrogation of intracellular protein expression also correlated to the downregulation from the cell surface. We determined that WA does not alter transcription of HER-2/neu, as there was no change in the gene expression with WA treatment compared to untreated cells. Further experiments are ongoing to address whether WA specifically targets signaling pathways that alter HER-2/neu signaling. Understanding the mechanisms by which WA induces down-regulation of HER-2/neu would serve as a means of developing WA as an alternative therapeutic strategy for HER-2/neu cancers not amenable to standard treatments. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A115. Citation Format: Ananlise Smith, Sarah Poliquin, Darlah Lopez-Rodriguez, Samita Andreansky. Withaferin A downregulates her-2/neu protein expression in human breast cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A115.

Abstract B57: A natural plant-derived compound, Withaferin A, exploits the stress pathway to induce antitumor immunity.

Recent research involving chemotherapeutic drugs such as anthracyclines have aided in redefining the definition of apoptosis from a form of cell death that is immunologically silent to the one that can activate an anti-cancer immune response. This form of apoptosis has been characterized as immunogenic cell death (ICD) and possess certain hallmarks such as the cell surface expression of heat shock proteins (HSPs) including HSP70 and HSP90 as well as ER-stress mediated pre-apoptotic expression of the ER chaperone calreticulin (CRT). One of the mechanisms by which ICD occurs is linked to the induction of an unfolded protein response (UPR) due to prolonged chemotherapeutic stress on cancer cells. Our research focuses on the development of Withaferin A (WA), a steroidal lactone derived from the Indian ayurvedic medicinal plant Withania somnifera as a chemoimmunotherapeutic agent. The benefit of using natural plant derived products is their low toxicity, multimodal action and potential ability to enhance the immune system. We initially screened the effect of Withaferin A by microarray analysis on breast cancer cells. WA induced significant alteration of 1160 genes (p-value 0.005). A pathway analysis determined that WA induces a robust heat shock response through up-regulation of several genes related to this pathway including response to unfolded proteins. The gene expression data was confirmed by the induction of intracellular HSP70, HSP40 as well as the UPR marker Bip78 by western blot. Furthermore we are able to demonstrate that WA treatment results in the translocation of known immunogenic HSPs HSP70, CRT and gp96 to the cell surface of several cancer cell lines. Additionally, WA treatment results in the induction of apoptosis and these WA mediated apoptotic cells can confer immunological protection against re-challenge with live tumor cells in vivo. Our long-term goal is to elucidate the anti-tumor immunomodulatory mechanisms of WA. Funded by: American Cancer Society (S.A.) Citation Format: Annalise R. Smith, Darlah Lopez, Samita Andreansky. A natural plant-derived compound, Withaferin A, exploits the stress pathway to induce antitumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B57.