Rajani Rajbhandari

PERK-Dependent Activation of JAK1 and STAT3 Contributes to Endoplasmic Reticulum Stress-Induced Inflammation

ABSTRACT Neuroinflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. Here, we have examined the interaction between ER stress and JAK/STAT-dependent inflammation in glial cells. We show that ER stress is present in the central nervous system (CNS) concomitant with inflammation and astrogliosis in the multiple sclerosis (MS) mouse model of experimental autoimmune encephalomyelitis (EAE). Astrocytes do not easily succumb to ER stress but rather activate an inflammatory program involving activation of STAT3 in a JAK1-dependent fashion. ER stress-induced activation of the JAK1/STAT3 axis leads to expression of interleukin 6 (IL-6) and several chemokines. Moreover, the activation of STAT3 signaling is dependent on PERK, a central component of the ER stress response, which we show is phosphorylated by JAK1. Disruption of PERK abrogates ER stress-induced activation of STAT3 and subsequent gene expression. Additionally, ER-stressed astrocytes, via paracrine signaling, can stimulate activation of microglia, leading to production of IL-6 and oncostatin M (OSM). These IL-6 cytokines can then synergize with ER stress in astrocytes to drive inflammation. Together, this work describes a new PERK/JAK1/STAT3 signaling pathway that elicits a feed-forward inflammatory loop involving astrocytes and microglia to drive neuroinflammation, which may be relevant in diseases such as MS.

NF-κB-induced IL-6 ensures STAT3 activation and tumor aggressiveness in glioblastoma.

Glioblastoma (GBM) is the most aggressive, neurologically destructive and deadly tumor of the central nervous system (CNS). In GBM, the transcription factors NF-κB and STAT3 are aberrantly activated and associated with tumor cell proliferation, survival, invasion and chemoresistance. In addition, common activators of NF-κB and STAT3, including TNF-α and IL-6, respectively, are abundantly expressed in GBM tumors. Herein, we sought to elucidate the signaling crosstalk that occurs between the NF-κB and STAT3 pathways in GBM tumors. Using cultured GBM cell lines as well as primary human GBM xenografts, we elucidated the signaling crosstalk between the NF-κB and STAT3 pathways utilizing approaches that either a) reduce NF-κB p65 expression, b) inhibit NF-κB activation, c) interfere with IL-6 signaling, or d) inhibit STAT3 activation. Using the clinically relevant human GBM xenograft model, we assessed the efficacy of inhibiting NF-κB and/or STAT3 alone or in combination in mice bearing intracranial xenograft tumors in vivo. We demonstrate that TNF-α-induced activation of NF-κB is sufficient to induce IL-6 expression, activate STAT3, and elevate STAT3 target gene expression in GBM cell lines and human GBM xenografts in vitro. Moreover, the combined inhibition of NF-κB and STAT3 signaling significantly increases survival of mice bearing intracranial tumors. We propose that in GBM, the activation of NF-κB ensures subsequent STAT3 activation through the expression of IL-6. These data verify that pharmacological interventions to effectively inhibit the activity of both NF-κB and STAT3 transcription factors must be used in order to reduce glioma size and aggressiveness.

The Chinese Pueraria root extract (Pueraria lobata) ameliorates impaired glucose and lipid metabolism in obese mice.

The incidence of type 2 diabetes and metabolic disease is rapidly increasing, but effective therapies for their prevention and treatment have been poorly tolerated or minimally effective. In this study, chronic administration of kudzu root extract (8 months, 0.2%, w/w, in diet) decreased baseline fasting plasma glucose (183±14 vs. 148±11 mg/dl) and improved glucose and insulin tolerance in C57BL/6J ob/ob mice (1.67±0.17 ng/ml [kudzu treated] vs. 2.35±0.63 ng/ml [control]), but such treatment did not alter these parameters in lean control mice. Among the mice on the kudzu supplementation, plasma levels of isoflavone metabolites were significantly higher in ob/ob versus lean control mice, and unmetabolized puerarin (11.50±5.63 ng/g) was found in adipose tissue only in the treated mice. Together, these data demonstrate that a puerarin containing kudzu diet improves glucose and insulin responsiveness in ob/ob mice, suggesting that puerarin may be a beneficial adjuvant for treating metabolic disease.

STAT4 controls GM-CSF production by both Th1 and Th17 cells during EAE

BackgroundIn experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, mice genetically deficient in the transcription factor signal transducer and activator of transcription 4 (STAT4) are resistant to disease. In contrast, deletion or inhibition of the Th1-associated cytokines IL-12 or IFNγ which act upstream and downstream of STAT4, respectively, does not ameliorate disease. These discordant findings imply that STAT4 may act in a non-canonical role during EAE. Recently, STAT4 has been shown to regulate GM-CSF production by CD4 T cells and this cytokine is necessary for the induction of EAE. However, it is not known if STAT4 controls GM-CSF production by both Th1 and Th17 effector CD4 T cells.MethodsThis study utilized the MOG35–55 peptide immunization model of EAE. Intracellular cytokine staining and novel mixed bone marrow chimeric mice were used to study the CD4 T cell-intrinsic role of STAT4 during disease. STAT4 chromatin-immunoprecipitation (ChIP-PCR) experiments were performed to show STAT4 directly interacts with the Csf2 gene loci.ResultsHerein, we demonstrate that STAT4 controls CD4 T cell-intrinsic GM-CSF production by both Th1 and Th17 CD4 T cells during EAE as well as in vitro. Importantly, we show that STAT4 interacts with the Csf2 locus in MOG35–55-activated effector CD4 T cells demonstrating direct modulation of GM-CSF.ConclusionsOverall, these studies illustrate a previously unrecognized role of STAT4 to regulate GM-CSF production by not only Th1 cells, but also Th17 effector CD4 T cell subsets during EAE pathogenesis. Critically, these data highlight for the first time that STAT4 is able to modulate the effector profile of Th17 CD4 T cell subsets, which redefines our current understanding of STAT4 as a Th1-centric factor.

Attenuation of PKR-like ER Kinase (PERK) Signaling Selectively Controls Endoplasmic Reticulum Stress-induced Inflammation Without Compromising Immunological Responses

Inflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. ER stress is brought on by the accumulation of misfolded proteins in the ER, which leads to activation of the unfolded protein response (UPR), a conserved pathway that transmits signals to restore homeostasis or eliminate the irreparably damaged cell. We provide evidence that inhibition or genetic haploinsufficiency of protein kinase R-like endoplasmic reticulum kinase (PERK) can selectively control inflammation brought on by ER stress without impinging on UPR-dependent survival and adaptive responses or normal immune responses. Using astrocytes lacking one or both alleles of PERK or the PERK inhibitor GSK2606414, we demonstrate that PERK haploinsufficiency or partial inhibition led to reduced ER stress-induced inflammation (IL-6, CCL2, and CCL20 expression) without compromising prosurvival responses. In contrast, complete loss of PERK blocked canonical PERK-dependent UPR genes and promoted apoptosis. Reversal of eIF2α-mediated translational repression using ISRIB potently suppressed PERK-dependent inflammatory gene expression, indicating that the selective modulation of inflammatory gene expression by PERK inhibition may be linked to attenuation of eIF2α phosphorylation and reveals a previously unknown link between translational repression and transcription of inflammatory genes. Additionally, ER-stressed astrocytes can drive an inflammatory M1-like phenotype in microglia, and this can be attenuated with inhibition of PERK. Importantly, targeting PERK neither disrupted normal cytokine signaling in astrocytes or microglia nor impaired macrophage phagocytosis or T cell polarization. Collectively, this work suggests that targeting PERK may provide a means for selective immunoregulation in the context of ER stress without disrupting normal immune function.

Let-7 Status Is Crucial for PARP1 Expression in HER2-Overexpressing Breast Tumors

HER2+ breast tumors have been shown to express elevated levels of PARP1 protein. Yet, the mechanism by which PARP1 is upregulated in HER2+ breast cancer is unknown. Here, knockdown of HER2 (ERBB2) in HER2+ breast cancer cells resulted in a reduction in PARP1 protein. Conversely, ectopic overexpression of HER2 in a non-HER2–overexpressing cell line resulted in increased PARP1 protein levels. Alterations in HER2 expression had no significant effect on PARP1 transcript levels. Instead, HER2 mRNA status was inversely correlated with let-7a miRNA levels in breast cancer cells. Ectopic expression of let-7a miRNA resulted in downregulation of PARP1 protein, whereas expression of the let-7a anti-miRNA increased PARP1 protein. Furthermore, luciferase assays demonstrate that let-7a regulates PARP1 via its 3′UTR. Importantly, let-7a was significantly lower in human HER2+ breast tumors compared with HER2− breast tumors and inversely correlated with PARP1 protein levels. Finally, HER2+ breast cancer cells exhibited similar cytotoxicity to ectopic let-7a expression as the PARP inhibitor veliparib (ABT-888). Collectively, these results reveal that increased PARP1 expression in HER2+ breast cancers is regulated by the let-7a miRNA, and that let-7a is a potential strategy to suppress PARP1 activity. Implications: This study reports the novel findings that HER2 increases PARP1 protein via suppression of the let-7a miRNA, which regulates the PARP1 3′-UTR. Moreover, HER2 status correlates with high PARP1 and low let-7a in breast cancer clinical specimens. Mol Cancer Res; 15(3); 340–7. ©2016 AACR.

Protein kinase CK2 is important for the function of glioblastoma brain tumor initiating cells

Protein kinase CK2 is a ubiquitously expressed serine/threonine kinase composed of two catalytic subunits (α) and/or (α′) and two regulatory (β) subunits. The expression and kinase activity of CK2 is elevated in many different cancers, including glioblastoma (GBM). Brain tumor initiating cells (BTICs) are a subset of cells that are highly tumorigenic and promote the resistance of GBM to current therapies. We previously reported that CK2 activity promotes prosurvival signaling in GBM. In this study, the role of CK2 signaling in BTIC function was examined. We found that expression of CK2α was increased in CD133+ BTICs compared to CD133− cells within the same GBM xenolines. Treatment with CX-4945, an ATP-competitive inhibitor of CK2, led to reduced expression of Sox2 and Nestin, transcription factors important for the maintenance of stem cells. Similarly, inhibition of CK2 also reduced the frequency of CD133+ BTICs over the course of 7 days, indicating a role for CK2 in BTIC persistence and survival. Importantly, using an in vitro limiting dilution assay, we found that inhibition of CK2 kinase activity with CX-4945 or siRNA knockdown of the CK2 catalytic subunits reduced neurosphere formation in GBM xenolines of different molecular subtypes. Lastly, we found that inhibition of CK2 led to decreased EGFR levels in some xenolines, and combination treatment with CX-4945 and Gefitinib to inhibit CK2 and EGFR, respectively, provided optimal inhibition of viability of cells. Therefore, due to the integration of CK2 in multiple signaling pathways important for BTIC survival, CK2 is a promising target in GBM.

MicroRNA-31 is required for astrocyte specification

Previously, we determined microRNA‐31 (miR‐31) is a noncoding tumor suppressive gene frequently deleted in glioblastoma (GBM); miR‐31 suppresses tumor growth, in part, by limiting the activity of NF‐κB. Herein, we expand our previous studies by characterizing the role of miR‐31 during neural precursor cell (NPC) to astrocyte differentiation. We demonstrate that miR‐31 expression and activity is suppressed in NPCs by stem cell factors such as Lin28, c‐Myc, SOX2 and Oct4. However, during astrocytogenesis, miR‐31 is induced by STAT3 and SMAD1/5/8, which mediate astrocyte differentiation. We determined miR‐31 is required for terminal astrocyte differentiation, and that the loss of miR‐31 impairs this process and/or prevents astrocyte maturation. We demonstrate that miR‐31 promotes astrocyte development, in part, by reducing the levels of Lin28, a stem cell factor implicated in NPC renewal. These data suggest that miR‐31 deletions may disrupt astrocyte development and/or homeostasis.

Trastuzumab-Resistant HER2+ Breast Cancer Cells Retain Sensitivity to Poly (ADP-Ribose) Polymerase (PARP) Inhibition

HER2-targeted therapies, such as trastuzumab, have increased the survival rates of HER2+ breast cancer patients. However, despite these therapies, many tumors eventually develop resistance to these therapies. Our lab previously reported an unexpected sensitivity of HER2+ breast cancer cells to poly (ADP-ribose) polymerase inhibitors (PARPi), agents that target homologous recombination (HR)–deficient tumors, independent of a DNA repair deficiency. In this study, we investigated whether HER2+ trastuzumab-resistant (TR) breast cancer cells were susceptible to PARPi and the mechanism behind PARPi induced cytotoxicity. We demonstrate that the PARPi ABT-888 (veliparib) decreased cell survival in vitro and tumor growth in vivo of HER2+ TR breast cancer cells. PARP-1 siRNA confirmed that cytotoxicity was due, in part, to PARP-1 inhibition. Furthermore, PARP-1 silencing had variable effects on the expression of several NF-κB–regulated genes. In particular, silencing PARP-1 inhibited NF-κB activity and reduced p65 binding at the IL8 promoter, which resulted in a decrease in IL8 mRNA and protein expression. Our results provide insight in the potential mechanism by which PARPi induces cytotoxicity in HER2+ breast cancer cells and support the testing of PARPi in patients with HER2+ breast cancer resistant to trastuzumab. Mol Cancer Ther; 17(5); 921–30. ©2018 AACR.

Withaferin A and sulforaphane regulate breast cancer cell cycle progression through epigenetic mechanisms

ABSTRACT Little is known about the effects of combinatorial dietary compounds on the regulation of epigenetic mechanisms involved in breast cancer prevention. The human diet consists of a multitude of components, and there is a need to elucidate how certain compounds interact in collaboration. Withaferin A (WA), found in the Indian winter cherry and documented as a DNA methyltransferase (DNMT) inhibitor, and sulforaphane (SFN), a well‐known histone deacetylase (HDAC) inhibitor found in cruciferous vegetables, are two epigenetic modifying compounds that have only recently been studied in conjunction. The use of DNMT and HDAC inhibitors to reverse the malignant expression of certain genes in breast cancer has shown considerable promise. Previously, we found that SFN + WA synergistically promote breast cancer cell death. Herein, we determined that these compounds inhibit cell cycle progression from S to G2 phase in MDA‐MB‐231 and MCF‐7 breast cancer. Furthermore, we demonstrate that this unique combination of epigenetic modifying compounds down‐regulates the levels of Cyclin D1 and CDK4, and pRB; conversely, the levels of E2F mRNA and tumor suppressor p21 are increased independently of p53. We find these events coincide with an increase in unrestricted histone methylation. We propose SFN + WA‐induced breast cancer cell death is attributed, in part, to epigenetic modifications that result in the modulated expression of key genes responsible for the regulation of cancer cell senescence. Graphical abstract Figure. No caption available. HighlightsTumor suppressor p21protein is reactivated via withaferin A (WA) and sulforaphane (SFN).Combinatorial nutritive compounds have the ability to impede cell cycle progression.Mutant p53 is down‐regulated via the studied compounds where wild type p53 is not.Epigenetic mechanisms are regulated by WA and SFN.The H3K4Me3 marker is increased at the p21 promoter after WA and SFN treatment.