Sankar Addya

Loss of stromal caveolin-1 leads to oxidative stress, mimics hypoxia and drives inflammation in the tumor microenvironment, conferring the "reverse Warburg effect": a transcriptional informatics analysis with validation.

Cav-1 (-/-) deficient stromal cells are a new genetic model for myofibroblasts and cancer-associated fibroblasts. Using an unbiased informatics analysis of the transcriptional profile of Cav-1 (-/-) deficient mesenchymal stromal cells, we have now identified many of the major signaling pathways that are activated by a loss of Cav-1, under conditions of metabolic restriction (with low glucose media). Our informatics analysis suggests that a loss of Cav-1 induces oxidative stress, which mimics a constitutive pseudo-hypoxic state, leading to 1) aerobic glycolysis and 2) inflammation in the tumor stromal microenvironment. This occurs via the activation of 2 major transcription factors, namely HIF (aerobic glycolysis) and NF-kB (inflammation) in Cav-1 (-/-) stromal fibroblastic cells. Experimentally, we show that Cav-1 deficient stromal cells may possess defective mitochondria, due to the over-production of nitric oxide (NO), resulting in the tyrosine nitration of the mitochondrial respiratory chain components (such as complex I). Elevated levels of nitro-tyrosine were observed both in Cav-1 (-/-) stromal cells, and via acute knock-down with siRNA targeting Cav-1. Finally, metabolic restriction with mitochondrial (complex I) and glycolysis inhibitors was synthetically lethal with a Cav-1 (-/-) deficiency in mice. As such, Cav-1 deficient mice show a dramatically reduced mitochondrial reserve capacity. Thus, a mitochondrial defect in Cav-1 deficient stromal cells could drive oxidative stress, leading to aerobic glycolysis, and inflammation, in the tumor microenvironment. These stromal alterations may underlie the molecular basis of the “Reverse Warburg Effect”, and could provide the key to targeted anti-cancer therapies using metabolic inhibitors. In direct support of these findings, the transcriptional profile of Cav-1 (-/-) stromal cells overlaps significantly with Alzheimer’s disease, which is characterized by oxidative stress, NO over-production (peroxynitrite formation), inflammation, hypoxia, and mitochondrial dysfunction. We conclude that Cav-1 (-/-) deficient mice are a new whole-body animal model for an activated lethal tumor micro-environment, i.e., “tumor stroma” without the tumor. Since Cav-1 (-/-) mice are also an established animal model for pro-fibrotic disease, our current results may have implications for understanding the pathogenesis of scleroderma (systemic sclerosis) and pulmonary fibrosis, which are also related to abnormal mesenchymal stem cell function.

Roles of β -catenin signaling in phenotypic expression and proliferation of articular cartilage superficial zone cells

The superficial zone (SFZ) of articular cartilage has unique structural and biomechanical features, is thought to promote self-renewal of articular cartilage, and is thus important for joint long-term function, but the mechanisms regulating its properties remain unclear. Previous studies revealed that Wnt/β-catenin signaling is continuously active in SFZ, indicating that it may be essential for SFZ function. Thus, we examined whether Wnt/β-catenin signaling regulates proliferation and phenotypic expression in SFZ cells. Using transgenic mice, we found that acute activation of Wnt/β-catenin signaling increases SFZ thickness, Proteoglycan 4 (Prg4, also called lubricin) expression and the number of slow-cell cycle cells, whereas conditional ablation of β-catenin causes the opposite. We developed a novel method to isolate SFZ cell-rich populations from the epiphyseal articular cartilage of neonatal mice, and found that the SFZ cells in culture exhibit a fibroblastic cytoarchitecture and higher Prg4 and Ets-related gene (Erg) expression and lower aggrecan expression compared with chondrocyte cultures. Gene array analyses indicated that SFZ cells have distinct gene expression profiles compared with underlying articular chondrocytes. Treatment of Wnt3a strongly stimulated SFZ cell proliferation and maintained strong expression of Prg4 and Erg, whereas ablation of β-catenin strongly impaired proliferation and phenotypic expression. When the cells were transplanted into athymic mice, they formed Prg4- and aggrecan-expressing cartilaginous masses attesting to their autonomous phenotypic capacity. Ablation of β-catenin caused a rapid loss of Prg4 gene expression and strong increases in expression of aggrecan and collagen 10, the latter being a trait of hypertrophic chondrocytes. Together, the data reveal that Wnt/β-catenin signaling is a key regulator of SFZ cell phenotype and proliferation, and may be as important for articular cartilage long-term function.

Stat5 promotes metastatic behavior of human prostate cancer cells in vitro and in vivo.

There are no effective therapies for disseminated prostate cancer. Constitutive activation of Stat5 in prostate cancer is associated with cancer lesions of high histological grade. We have shown that Stat5 is activated in 61% of distant metastases of clinical prostate cancer. Active Stat5 increased metastases formation of prostate cancer cells in nude mice by 11-fold in an experimental metastases assay. Active Stat5 promoted migration and invasion of prostate cancer cells, and induced rearrangement of the microtubule network. Active Stat5 expression was associated with decreased cell surface E-cadherin levels, while heterotypic adhesion of prostate cancer cells to endothelial cells was stimulated by active Stat5. Activation of Stat5 and Stat5-induced binding of prostate cancer cells to endothelial cells were decreased by inhibition of Src but not of Jak2. Gene expression profiling indicated that 21% of Stat5-regulated genes in prostate cancer cells were related to metastases, while 7.9% were related to proliferation and 3.9% to apoptosis. The work presented here provides the first evidence of Stat5 involvement in the induction of metastatic behavior of human prostate cancer cells in vitro and in vivo. Stat5 may provide a therapeutic target protein for disseminated prostate cancer.

ChIP sequencing of cyclin D1 reveals a transcriptional role in chromosomal instability in mice

Chromosomal instability (CIN) in tumors is characterized by chromosomal abnormalities and an altered gene expression signature; however, the mechanism of CIN is poorly understood. CCND1 (which encodes cyclin D1) is overexpressed in human malignancies and has been shown to play a direct role in transcriptional regulation. Here, we used genome-wide ChIP sequencing and found that the DNA-bound form of cyclin D1 occupied the regulatory region of genes governing chromosomal integrity and mitochondrial biogenesis. Adding cyclin D1 back to Ccnd1(-/-) mouse embryonic fibroblasts resulted in CIN gene regulatory region occupancy by the DNA-bound form of cyclin D1 and induction of CIN gene expression. Furthermore, increased chromosomal aberrations, aneuploidy, and centrosome abnormalities were observed in the cyclin D1-rescued cells by spectral karyotyping and immunofluorescence. To assess cyclin D1 effects in vivo, we generated transgenic mice with acute and continuous mammary gland-targeted cyclin D1 expression. These transgenic mice presented with increased tumor prevalence and signature CIN gene profiles. Additionally, interrogation of gene expression from 2,254 human breast tumors revealed that cyclin D1 expression correlated with CIN in luminal B breast cancer. These data suggest that cyclin D1 contributes to CIN and tumorigenesis by directly regulating a transcriptional program that governs chromosomal stability.

Transcription Factor Stat3 Stimulates Metastatic Behavior of Human Prostate Cancer Cells in Vivo, whereas Stat5b Has a Preferential Role in the Promotion of Prostate Cancer Cell Viability and Tumor Growth

Identification of the molecular changes that promote viability and metastatic behavior of prostate cancer is critical for the development of improved therapeutic interventions. Stat5a/b and Stat3 are both constitutively active in locally-confined and advanced prostate cancer, and both transcription factors have been reported to be critical for the viability of prostate cancer cells. We recently showed that Stat3 promotes metastatic behavior of human prostate cancer cells not only in vitro but also in an in vivo experimental metastases model. In this work, we compare side-by-side Stat5a/b versus Stat3 in the promotion of prostate cancer cell viability, tumor growth, and induction of metastatic colonization in vivo. Inhibition of Stat5a/b induced massive death of prostate cancer cells in culture and reduced both subcutaneous and orthotopic prostate tumor growth, whereas Stat3 had a predominant role over Stat5a/b in promoting metastases formation of prostate cancer cells in vivo in nude mice. The molecular mechanisms underlying the differential biological effects induced by these two transcription factors involve largely different sets of genes regulated by Stat5a/b versus Stat3 in human prostate cancer model systems. Of the two Stat5 homologs, Stat5b was more important for supporting growth of prostate cancer cells than Stat5a. This work provides the first mechanistic comparison of the biological effects induced by transcription factors Stat5a/b versus Stat3 in prostate cancer.

MicroRNA-mediated GABAAα-1 receptor subunit down-regulation in adult spinal cord following neonatal cystitis-induced chronic visceral pain in rats

Summary In an experimental model of neonatal cystitis, microRNA‐mediated post‐transcriptional deregulation of spinal GABAergic system is involved in long‐lasting visceral hyperalgesia. Abstract The nociceptive transmission under pathological chronic pain conditions involves transcriptional and/or translational alteration in spinal neurotransmitters, receptor expressions, and modification of neuronal functions. Studies indicate the involvement of microRNA (miRNA) – mediated transcriptional deregulation in the pathophysiology of acute and chronic pain. In the present study, we tested the hypothesis that long‐term cross‐organ colonic hypersensitivity in neonatal zymosan‐induced cystitis is due to miRNA‐mediated posttranscriptional suppression of the developing spinal GABAergic system. Cystitis was produced by intravesicular injection of zymosan (1% in saline) into the bladder during postnatal (P) days P14 through P16 and spinal dorsal horns (L6–S1) were collected either on P60 (unchallenged groups) or on P30 after a zymosan re‐challenge on P29 (re‐challenged groups). miRNA arrays and real‐time reverse transcription–polymerase chain reaction (RT‐PCR) revealed significant, but differential, up‐regulation of mature miR‐181a in the L6–S1 spinal dorsal horns from zymosan‐treated rats compared with saline‐treated controls in both the unchallenged and re‐challenged groups. The target gene analysis demonstrated multiple complementary binding sites in miR‐181a for GABAA receptor subunit GABAAα‐1 gene with a miRSVR score of −1.83. An increase in miR‐181a concomitantly resulted in significant down‐regulation of GABAAα‐1 receptor subunit gene and protein expression in adult spinal cords from rats with neonatal cystitis. Intrathecal administration of the GABAA receptor agonist muscimol failed to attenuate the viscero‐motor response (VMR) to colon distension in rats with neonatal cystitis, whereas in adult zymosan‐treated rats the drug produced significant decrease in VMR. These results support an integral role for miRNA‐mediated transcriptional deregulation of the GABAergic system in neonatal cystitis‐induced chronic pelvic pain.

Inflammatory breast cancer (IBC): clues for targeted therapies

Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer characterized by rapid proliferation, early metastatic development and poor prognosis. Since there are few preclinical models of IBC, there is a general lack of understanding of the complexity of the disease. Recently, we have developed a new model of IBC derived from the pleural effusion of a woman with metastatic secondary IBC. FC-IBC02 cells are triple negative and form clusters (mammospheres) in suspension that are strongly positive for E-cadherin, β-catenin and TSPAN24, all adhesion molecules that play an important role in cell migration and invasion. FC-IBC02 cells expressed stem cell markers and some, but not all of the characteristics of cells undergoing epithelial mesenchymal transition (EMT). Breast tumor FC-IBC02 xenografts developed quickly in SCID mice with the presence of tumor emboli and the development of lymph node and lung metastases. Remarkably, FC-IBC02 cells were able to produce brain metastasis in mice on intracardiac or intraperitoneal injections. Genomic studies of FC-IBC02 and other IBC cell lines showed that IBC cells had important amplification of 8q24 where MYC, ATAD2 and the focal adhesion kinase FAK1 are located. MYC and ATAD2 showed between 2.5 and 7 copies in IBC cells. FAK1, which plays important roles in anoikis resistance and tumor metastasis, showed 6–4 copies in IBC cells. Also, CD44 was amplified in triple-negative IBC cells (10–3 copies). Additionally, FC-IBC02 showed amplification of ALK and NOTCH3. These results indicate that MYC, ATAD2, CD44, NOTCH3, ALK and/or FAK1 may be used as potential targeted therapies against IBC.

Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

The CCND1 gene, which is frequently overexpressed in cancers, encodes the regulatory subunit of a holoenzyme that phosphorylates the retinoblastoma protein. Although it is known that cyclin D1 regulates estrogen receptor (ER)α transactivation using heterologous reporter systems, the in vivo biological significance of cyclin D1 to estrogen-dependent signaling, and the molecular mechanisms by which cyclin D1 is involved, are yet to be elucidated. Herein, genome-wide expression profiling conducted of 17β-estradiol-treated castrated virgin mice deleted of the Ccnd1 gene demonstrated that cyclin D1 determines estrogen-dependent gene expression for 88% of estrogen-responsive genes in vivo. In addition, expression profiling of 17β-estradiol-stimulated cyclin D1 small interfering RNA treated MCF7 cells shows cyclin D1 is required for estrogen-mediated gene expression in vitro. Genome-wide chromatin immunoprecipitation-Seq analysis revealed a cyclin D1-DNA bound form associated with genes that were regulated by estrogen in a cyclin D1-dependent manner. The cyclin D1-dependent estrogen signaling pathways identified in vivo were highly enriched for extracellular membrane-associated growth factor receptors (epidermal growth factor receptor, ErbB3, and EphB3) and their ligands (amphiregulin, encoded by AREG gene), and matrix metalloproteinase. The AREG protein, a pivotal ligand for epidermal growth factor receptors to promote cellular proliferation, was induced by cyclin D1 via the AREG promoter. Chromatin immunoprecipitation analysis demonstrated the recruitment of cyclin D1 to the breast cancer 1 (Brca1)/ERα binding site of the Areg gene. Cyclin D1 genetic deletion demonstrated the in vivo requirement for cyclin D1 in assembling the estrogen-dependent amplified in breast cancer 1-associated multiprotein complex. The current studies define a requirement for cyclin D1 in estrogen-dependent signaling modules governing growth factor receptor and ligand expression in vivo and reveal a noncanonical function of cyclin D1 at ERα target gene promoters. Cyclin D1 mediates the convergence of ERα and growth factor signaling at a common cis-element of growth factor genes.

Dachshund Binds p53 to Block the Growth of Lung Adenocarcinoma Cells

Hyperactive EGF receptor (EGFR) and mutant p53 are common genetic abnormalities driving the progression of non-small cell lung cancer (NSCLC), the leading cause of cancer deaths in the world. The Drosophila gene Dachshund (Dac) was originally cloned as an inhibitor of hyperactive EGFR alleles. Given the importance of EGFR signaling in lung cancer etiology, we examined the role of DACH1 expression in lung cancer development. DACH1 protein and mRNA expression was reduced in human NSCLC. Reexpression of DACH1 reduced NSCLC colony formation and tumor growth in vivo via p53. Endogenous DACH1 colocalized with p53 in a nuclear, extranucleolar location, and shared occupancy of -15% of p53-bound genes in ChIP sequencing. The C-terminus of DACH1 was necessary for direct p53 binding, contributing to the inhibition of colony formation and cell-cycle arrest. Expression of the stem cell factor SOX2 was repressed by DACH1, and SOX2 expression was inversely correlated with DACH1 in NSCLC. We conclude that DACH1 binds p53 to inhibit NSCLC cellular growth.

Immune clearance of attenuated rabies virus results in neuronal survival with altered gene expression.

Rabies virus (RABV) is a highly neurotropic pathogen that typically leads to mortality of infected animals and humans. The precise etiology of rabies neuropathogenesis is unknown, though it is hypothesized to be due either to neuronal death or dysfunction. Analysis of human brains post-mortem reveals surprisingly little tissue damage and neuropathology considering the dramatic clinical symptomology, supporting the neuronal dysfunction model. However, whether or not neurons survive infection and clearance and, provided they do, whether they are functionally restored to their pre-infection phenotype has not been determined in vivo for RABV, or any neurotropic virus. This is due, in part, to the absence of a permanent “mark” on once-infected cells that allow their identification long after viral clearance. Our approach to study the survival and integrity of RABV-infected neurons was to infect Cre reporter mice with recombinant RABV expressing Cre-recombinase (RABV-Cre) to switch neurons constitutively expressing tdTomato (red) to expression of a Cre-inducible EGFP (green), permanently marking neurons that had been infected in vivo. We used fluorescence microscopy and quantitative real-time PCR to measure the survival of neurons after viral clearance; we found that the vast majority of RABV-infected neurons survive both infection and immunological clearance. We were able to isolate these previously infected neurons by flow cytometry and assay their gene expression profiles compared to uninfected cells. We observed transcriptional changes in these “cured” neurons, predictive of decreased neurite growth and dysregulated microtubule dynamics. This suggests that viral clearance, though allowing for survival of neurons, may not restore them to their pre-infection functionality. Our data provide a proof-of-principle foundation to re-evaluate the etiology of human central nervous system diseases of unknown etiology: viruses may trigger permanent neuronal damage that can persist or progress in the absence of sustained viral antigen.