Endoplasmic reticulum protein BI-1 regulates Ca²âº-mediated bioenergetics to promote autophagy.

Autophagy is a lysosomal degradation pathway that converts macromolecules into substrates for energy production during nutrient-scarce conditions such as those encountered in tumor microenvironments. Constitutive mitochondrial uptake of endoplasmic reticulum (ER) Ca²âº mediated by inositol triphosphate receptors (IP3Rs) maintains cellular bioenergetics, thus suppressing autophagy. We show that the ER membrane protein Bax inhibitor-1 (BI-1) promotes autophagy in an IP3R-dependent manner. By reducing steady-state levels of ER Ca²âº via IP3Rs, BI-1 influences mitochondrial bioenergetics, reducing oxygen consumption, impacting cellular ATP levels, and stimulating autophagy. Furthermore, BI-1-deficient mice show reduced basal autophagy, and experimentally reducing BI-1 expression impairs tumor xenograft growth in vivo. BI-1's ability to promote autophagy could be dissociated from its known function as a modulator of IRE1 signaling in the context of ER stress. The results reveal BI-1 as a novel autophagy regulator that bridges Ca²âº signaling between ER and mitochondria, reducing cellular oxygen consumption and contributing to cellular resilience in the face of metabolic stress.

Vaccinia virus F1L protein promotes virulence by inhibiting inflammasome activation.

Host innate immune responses to DNA viruses involve members of the nucleotide-binding domain, leucine-rich repeat and pyrin domain containing protein (NLRP) family, which form "inflammasomes" that activate caspase-1, resulting in proteolytic activation of cytokines interleukin (IL)-1ß and IL-18. We hypothesized that DNA viruses would target inflammasomes to overcome host defense. A Vaccinia virus (VACV) B-cell CLL/lymphoma 2 (Bcl-2) homolog, F1L, was demonstrated to bind and inhibit the NLR family member NLRP1 in vitro. Moreover, infection of macrophages in culture with virus lacking F1L (ΔF1L) caused increased caspase-1 activation and IL-1ß secretion compared with wild-type virus. Virulence of ΔF1L virus was attenuated in vivo, causing altered febrile responses, increased proteolytic processing of caspase-1, and more rapid inflammation in lungs of infected mice without affecting cell death or virus replication. Furthermore, we found that a hexapeptide from F1L is necessary and sufficient for inhibiting the NLRP1 inflammasome in vitro, thus identifying a peptidyl motif required for binding and inhibiting NLRP1. The functional importance of this NLRP1-binding motif was further confirmed by studies of recombinant ΔF1L viruses reconstituted either with the wild-type F1L or a F1L mutant that fails to bind NLRP1. Cellular infection with wild-type F1L reconstituted virus-suppressed IL-1ß production, whereas mutant F1L did not. In contrast, both wild-type and mutant versions of F1L equally suppressed apoptosis. In vivo, the NLR nonbinding F1L mutant virus exhibited an attenuated phenotype similar to ΔF1L virus, thus confirming the importance of F1L interactions with NLRP1 for viral pathogenicity in mice. Altogether, these findings reveal a unique viral mechanism for evading host innate immune responses.

Tumour cell survival mechanisms in lethal metastatic prostate cancer differ between bone and soft tissue metastases.

The complexity of survival mechanisms in cancer cells from patients remains poorly understood. To obtain a comprehensive picture of tumour cell survival in lethal prostate cancer metastases, we examined five survival proteins that operate within three survival pathways in a cohort of 185 lethal metastatic prostate metastases obtained from 44 patients. The expression levels of BCL-2, BCL-XL, MCL-1, cytoplasmic survivin, nuclear survivin, and stathmin were measured by immunohistochemistry in a tissue microarray. Simultaneous expression of three or more proteins occurred in 81% of lethal prostate cancer metastases and BCL-2, cytoplasmic survivin and MCL-1 were co-expressed in 71% of metastatic sites. An unsupervised cluster analysis separated bone and soft tissue metastases according to patterns of survival protein expression. BCL-2, cytoplasmic survivin and MCL-1 had significantly higher expression in bone metastases (p < 10(-5)), while nuclear survivin was significantly higher in soft tissue metastases (p = 3 × 10(-14)). BCL-XL overexpression in soft tissue metastases almost reached significance (p = 0.09), while stathmin expression did not (p = 0.28). In addition, the expression of MCL-1 was significantly higher in AR-positive tumours. Neuroendocrine differentiation was not associated with specific survival pathways. These studies show that bone and soft tissue metastases from the same patient differ significantly in expression of a panel of survival proteins and that with regard to survival protein expression, expression is associated with the metastatic site and not the patient. Altogether, this suggests that optimal therapeutic inhibition may require combinations of drugs that target both bone and soft tissue-specific survival pathways.

A role for ATF2 in regulating MITF and melanoma development.

The transcription factor ATF2 has been shown to attenuate melanoma susceptibility to apoptosis and to promote its ability to form tumors in xenograft models. To directly assess ATF2's role in melanoma development, we crossed a mouse melanoma model (Nras(Q61K)::Ink4a⁻/⁻) with mice expressing a transcriptionally inactive form of ATF2 in melanocytes. In contrast to 7/21 of the Nras(Q61K)::Ink4a⁻/⁻ mice, only 1/21 mice expressing mutant ATF2 in melanocytes developed melanoma. Gene expression profiling identified higher MITF expression in primary melanocytes expressing transcriptionally inactive ATF2. MITF downregulation by ATF2 was confirmed in the skin of Atf2⁻/⁻ mice, in primary human melanocytes, and in 50% of human melanoma cell lines. Inhibition of MITF transcription by MITF was shown to be mediated by ATF2-JunB-dependent suppression of SOX10 transcription. Remarkably, oncogenic BRAF (V600E)-dependent focus formation of melanocytes on soft agar was inhibited by ATF2 knockdown and partially rescued upon shMITF co-expression. On melanoma tissue microarrays, a high nuclear ATF2 to MITF ratio in primary specimens was associated with metastatic disease and poor prognosis. Our findings establish the importance of transcriptionally active ATF2 in melanoma development through fine-tuning of MITF expression.

A Unique Multiplex ELISA to Profile Growth Factors and Cytokines in Cerebrospinal Fluid.

Multiplex arrays designed for enzyme-linked immunosorbent assays (ELISAs) are robust and cost-effective for profiling biomarkers. Identification of relevant biomarkers in biological matrices or fluids helps in the understanding of disease pathogenesis. Here, we describe a sandwich ELISA-based multiplex assay to assess growth factor and cytokine levels in cerebrospinal fluid (CSF) samples derived from multiple sclerosis patients, amyotrophic lateral sclerosis patients, and control subjects without any neurological disorder. Results indicate that multiplex assay designed for the sandwich ELISA method is a unique, robust, and cost-effective method for profiling growth factors and cytokines present in CSF samples.

Small-molecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity.

Apoptosis resistance commonly occurs in cancers, preventing activation of Caspase family cell death proteases. XIAP is an endogenous inhibitor of Caspases overexpressed in many cancers. We developed an enzyme derepression assay, based on overcoming XIAP-mediated suppression of Caspase-3, and screened mixture-based combinatorial chemical libraries for compounds that reversed XIAP-mediated inhibition of Caspase-3, identifying a class of polyphenylureas with XIAP-inhibitory activity. These compounds, but not inactive structural analogs, stimulated increases in Caspase activity, directly induced apoptosis of many types of tumor cell lines in culture, and sensitized cancer cells to chemotherapeutic drugs. Active compounds also suppressed growth of established tumors in xenograft models in mice, while displaying little toxicity to normal tissues. These findings validate IAPs as targets for cancer drug discovery.

Lymphocyte-specific TRAF3 transgenic mice have enhanced humoral responses and develop plasmacytosis, autoimmunity, inflammation, and cancer.

Tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) regulates both innate and adaptive immunity by modulating signaling by Toll-like receptors (TLR) and TNF receptors. TRAF3 was recently identified as a tumor suppressor in human multiple myeloma, suggesting a prominent role in plasma cell homeostasis. We have generated transgenic mice expressing human TRAF3 in lymphocytes. These mice are normal at birth, but they develop over time plasmacytosis and hypergammaglobulinemia, as well as systemic inflammation and tertiary lymphoid organ formation. The analysis of the humoral responses of the TRAF3 mice demonstrated increased responses to T-dependent and T-independent antigens with increased production of antigen-specific immunoglobulin Gs (IgGs) compared with wild-type mice. Furthermore, TLR-mediated IgG production is also increased in TRAF3 B cells. In addition, TRAF3 mice develop autoimmunity and are predisposed to cancer, particularly squamous cell carcinomas of the tongue ( approximately 50% incidence) and salivary gland tumors. In summary, TRAF3 renders B cells hyperreactive to antigens and TLR agonists, promoting autoimmunity, inflammation, and cancer, hereby providing a new model for studying de novo carcinogenesis promoted by B cell-initiated chronic inflammation.

Salmonella typhimurium engineered to produce CCL21 inhibit tumor growth.

Intravenously-applied bacteria tend to accumulate in tumors and can sporadically lead to tumor regression. Systemic administration of attenuated Salmonella typhimurium is safe and has shown no significant adverse effects in humans. The purpose of this study was to test the hypothesis that engineering S. typhimurium to express a chemokine, CCL21, would increase anti-tumor activity. We engineered an attenuated strain of S. typhimurium to produce the chemokine CCL21. Attenuated S. typhimurium expressing CCL21 significantly inhibited the growth of primary tumors and pulmonary metastases in preclinical models of multi-drug-resistant murine carcinomas, while control bacteria did not. Histological analysis of tumors showed marked inflammatory cell infiltrates in mice treated with CCL21-expressing but not control bacteria. Levels of cytokines and chemokines known to be induced by CCL21 [e.g., interferon-gamma (INFgamma), CXCL9, and CXCL10] were significantly elevated in tumors of mice treated with CCL21-expressing but not control S. typhimurium. The anti-tumor activity was found to be dependent on CD4- and CD8-expressing cells, based on antibody-mediated in vivo immuno-depletion experiments. Anti-tumor activity was achieved without evidence of toxicity. In summary, chemokine-expressing, attenuated bacteria may provide a novel approach to cancer immunotherapy for effective and well-tolerated in vivo delivery of immunomodulatory proteins.

Image analysis algorithms for immunohistochemical assessment of cell death events and fibrosis in tissue sections.

Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson's trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections.

Development of diabesity in mice with neuronal deletion of Shp2 tyrosine phosphatase.

Obesity and diabetes, termed "diabesity," are serious health problems that are increasing in frequency. However, the molecular mechanisms and neuronal regulation of these metabolic disorders are not fully understood. We show here that Shp2, a widely expressed Src homology 2-containing Tyr phosphatase, plays a critical role in the adult brain to control food intake, energy balance, and metabolism. Mice with a neuron-specific, conditional Shp2 deletion were generated by crossing a pan-neuronal Cre-line (CRE3) with Shp2(flox/flox) mice. These congenic mice, CRE3/Shp2-KO, developed obesity and diabetes and the associated pathophysiological complications that resemble those encountered in humans, including hyperglycemia, hyperinsulinemia, hyperleptinemia, insulin and leptin resistance, vasculitis, diabetic nephropathy, urinary bladder infections, prostatitis, gastric paresis, and impaired spermatogenesis. This mouse model may help to elucidate the molecular mechanisms that lead to the development of diabesity in humans and provide a tool to study the in vivo complications of uncontrolled diabetes.