The coordination of nutrient and energy availability with cell growth and division is essential for proper immune cell development and function. By using a chemical mutagenesis strategy in mice, we ...identified a pedigree that has a complete block in B cell development at the pre-B cell stage resulting from a deletion in the Fnip1 gene. Enforced expression of an immunoglobulin transgene failed to rescue B cell development. Whereas essential pre-B cell signaling molecules were activated normally in Fnip1-null pre-B cells, the metabolic regulators AMPK and mTOR were dysregulated, resulting in excessive cell growth and enhanced sensitivity to apoptosis in response to metabolic stress (pre-B cell receptor crosslinking, oncogene activation). These results indicate that Folliculin-interacting protein 1 (Fnip1) is vital for B cell development and metabolic homeostasis and reveal a metabolic checkpoint that may ensure that pre-B cells have sufficient metabolic capacity to support division, while limiting lymphomagenesis caused by deregulated growth.
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► A deletion in the Fnip1 gene blocks B cell development at the pre-B cell stage ► Fnip1 controls metabolic homeostasis and survival during pre-B cell activation ► Fnip1 is required for AMPK to inhibit mTOR-mediated pre-B cell growth ► Fnip1−/− pre-B cells are resistant to lymphoma formation induced by c-Myc
Several recent insights into the roles of mitochondria in cancer have renewed efforts to develop nongenotoxic therapies targeting mitochondrial proteins and functions. Mitochondria are central hubs ...for intrinsic apoptotic pathways that are activated by cellular stress and injury, and as a consequence, cancers often have defects in these pathways. Bcl-2, the first identified regulator of apoptotic cell deaths, was discovered as an oncogene in human cancers. BCL-2 inhibits mitochondrial pathways of apoptosis through local effects at mitochondrial and endoplasmic reticulum membranes. Increased expression of BCL-2 and the related antiapoptotic proteins BCL-XL, MCL-1, and BCL-W occurs in significant subsets of common cancer types (Table I) and is generally correlated with poor response. Although incomplete, the emerging understanding of BCL-2 functions through structural, biochemical, and organelle physiology studies has provided paths for targeting BCL-2 with small molecules. Cancer cells also exhibit metabolic differences with their normal cell counterparts, including aerobic glycolysis, known as the Warburg effect, mitochondrial membrane hyperpolarization, and unusual dependence on nutrient substrates such as glucose and glutamine. This knowledge has prompted reexamination of the potential cancer selectivity of previously identified mitochondriotoxic compounds, including approved drugs for other indications, and screening programs to identify new compounds with mitochondrial activities. Environ. Mol. Mutagen. 2010.
Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog ...of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.
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•A specific inhibitor of an EBV prosurvival Bcl-2 protein was computationally designed•The likelihood of designed proteins correctly folding correlates with activity•The designed inhibitor can initiate apoptosis in EBV-infected cancer cells•Intracellular delivery with a micelle carrier suppresses xenograft tumor growth
A novel computationally designed protein, called BINDI, binds to Epstein-Barr virus Bcl-2 protein with picomolar affinity. BINDI initiates apoptosis in EBV-infected cancer lines, can suppress tumor growth, and extends survival in a xenograft disease model of EBV-positive human lymphoma.
Kaposi's sarcoma (KS) is the most commonly reported tumor in parts of Africa and is the most common tumor of AIDS patients worldwide. KS-associated herpesvirus (KSHV) is the etiologic agent of KS. ...Although KS tumors contain many cell types, the predominant cell is the spindle cell, a cell of endothelial origin that maintains KSHV latency. KSHV activates many cell-signaling pathways but little is known about how KSHV alters cellular metabolism during latency. The Warburg effect, a common metabolic alteration of most tumor cells, is defined by an increase in aerobic glycolysis and a decrease in oxidative phosphorylation as an energy source. The Warburg effect adapts cells to tumor environments and is necessary for the survival of tumor cells. During latent infection of endothelial cells, KSHV induces aerobic glycolysis and lactic acid production while decreasing oxygen consumption, thereby inducing the Warburg effect. Inhibitors of glycolysis selectively induce apoptosis in KSHV-infected endothelial cells but not their uninfected counterparts. Therefore, similar to cancer cells, the Warburg effect is necessary for maintaining KSHV latently infected cells. We propose that KSHV induction of the Warburg effect adapts infected cells to tumor microenvironments, aiding the seeding of KS tumors. Additionally, inhibitors of glycolysis may provide a unique treatment strategy for latent KSHV infection and ultimately KS tumors.
Cells treated with hsp90 inhibitors exhibit pleiotropic changes, including an expansion of the mitochondrial compartment, accompanied by mitochondrial fragmentation and condensed mitochondrial ...morphology, with ultimate compromise of mitochondrial integrity and apoptosis.
We identified several mitochondrial oxidative phosphorylation complex subunits, including several encoded by mtDNA, that are upregulated by hsp90 inhibitors, without corresponding changes in mRNA abundance. Post-transcriptional accumulation of mitochondrial proteins observed with hsp90 inhibitors is also seen in cells treated with proteasome inhibitors. Detailed studies of the OSCP subunit of mitochondrial F1F0-ATPase revealed the presence of mono- and polyubiquitinated OSCP in mitochondrial fractions. We demonstrate that processed OSCP undergoes retrotranslocation to a trypsin-sensitive form associated with the outer mitochondrial membrane. Inhibition of proteasome or hsp90 function results in accumulation of both correctly targeted and retrotranslocated mitochondrial OSCP.
Cytosolic turnover of mitochondrial proteins demonstrates a novel connection between mitochondrial and cytosolic compartments through the ubiquitin-proteasome system. Analogous to defective protein folding in the endoplasmic reticulum, a mitochondrial unfolded protein response may play a role in the apoptotic effects of hsp90 and proteasome inhibitors.
Severe intestinal graft-vs-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT) causes mucosal ulceration and induces innate and adaptive immune responses that amplify and ...perpetuate GVHD and the associated barrier dysfunction. Pharmacological agents to target mucosal barrier dysfunction in GVHD are needed. We hypothesized that induction of Wnt signaling by lithium, an inhibitor of glycogen synthase kinase (GSK3), would potentiate intestinal crypt proliferation and mucosal repair and that inhibition of GSK3 in inflammatory cells would attenuate the deregulated inflammatory response to mucosal injury. We conducted an observational pilot study to provide data for the potential design of a randomized study of lithium. Twenty patients with steroid refractory intestinal GVHD meeting enrollment criteria were given oral lithium carbonate. GVHD was otherwise treated per current practice, including 2 mg/kg per day of prednisone equivalent. Seventeen patients had extensive mucosal denudation (extreme endoscopic grade 3) in the duodenum or colon. We observed that 8 of 12 patients (67%) had a complete remission (CR) of GVHD and survived more than 1 year (median 5 years) when lithium administration was started promptly within 3 days of endoscopic diagnosis of denuded mucosa. When lithium was started promptly and less than 7 days from salvage therapy for refractory GVHD, 8 of 10 patients (80%) had a CR and survived more than 1 year. In perspective, a review of 1447 consecutive adult HCT patients in the preceding 6 years at our cancer center showed 0% one-year survival in 27 patients with stage 3-4 intestinal GVHD and grade 3 endoscopic appearance in the duodenum or colon. Toxicities included fatigue, somnolence, confusion or blunted affect in 50% of the patients. The favorable outcomes in patients who received prompt lithium therapy appear to support the future conduct of a randomized study of lithium for management of severe GVHD with extensive mucosal injury.
ClinicalTrials.gov NCT00408681.
Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that coordinates nutrient and growth factor availability with cellular growth, division, and differentiation. Studies examining the ...roles of mTOR signaling in immune function revealed critical roles for mTOR in regulating T cell differentiation and function. However, few studies have investigated the roles of mTOR in early B cell development. In this study, we found that mTOR is highly activated during the pro- and pre-B stages of mouse B cell development. Conditional disruption of the mTOR coactivating protein Raptor in developing mouse B cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B cells and loss of Ag-specific Ab production. Pre-B cell survival and proliferation were significantly reduced in Raptor-deficient mice. Forced expression of a transgenic BCR or a BclxL transgene on Raptor-deficient B cells failed to rescue B cell development, suggesting that pre-BCR signaling and B cell survival are impaired in a BclxL-independent manner. Raptor-deficient pre-B cells exhibited significant decreases in oxidative phosphorylation and glycolysis, indicating that loss of mTOR signaling in B cells significantly impairs cellular metabolic capacity. Treatment of mice with rapamycin, an allosteric inhibitor of mTOR, recapitulated the early B cell developmental block. Collectively, our data reveal a previously uncharacterized role for mTOR signaling in early B cell development, survival, and metabolism.
Birt-Hogg-Dube' Syndrome (BHDS) is a rare genetic disorder in humans characterized by skin hamartomas, lung cysts, pneumothorax, and increased risk of renal tumors. BHDS is caused by mutations in the ...BHD gene, which encodes for Folliculin, a cytoplasmic adapter protein that binds to Folliculin interacting proteins-1 and -2 (Fnip1, Fnip2) as well as the master energy sensor AMP kinase (AMPK). Whereas kidney-specific deletion of the Bhd gene in mice is known to result in polycystic kidney disease (PKD) and renal cell carcinoma, the roles of Fnip1 in renal cell development and function are unclear. In this study, we utilized mice with constitutive deletion of the Fnip1 gene to show that the loss of Fnip1 is sufficient to result in renal cyst formation, which was characterized by decreased AMPK activation, increased mTOR activation, and metabolic hyperactivation. Using RNAseq, we found that Fnip1 disruption resulted in many cellular and molecular changes previously implicated in the development of PKD in humans, including alterations in the expression of ion and amino acid transporters, increased cell adhesion, and increased inflammation. Loss of Fnip1 synergized with Tsc1 loss to hyperactivate mTOR, increase Erk activation, and greatly accelerate the development of PKD. Our results collectively define roles for Fnip1 in regulating kidney development and function, and provide a model for how loss of Fnip1 contributes to PKD and perhaps renal cell carcinoma.
To investigate combined MRI and
F-FDG PET for assessing breast tumor metabolism/perfusion mismatch and predicting pathological response and recurrence-free survival (RFS) in women treated for breast ...cancer.
Patients undergoing neoadjuvant chemotherapy (NAC) for locally-advanced breast cancer were imaged at three timepoints (pre, mid, and post-NAC), prior to surgery. Imaging included diffusion-weighted and dynamic contrast-enhanced (DCE-) MRI and quantitative
F-FDG PET. Tumor imaging measures included apparent diffusion coefficient, peak percent enhancement (PE), peak signal enhancement ratio (SER), functional tumor volume, and washout volume on MRI and standardized uptake value (SUVmax), glucose delivery (K
) and FDG metabolic rate (MRFDG) on PET, with percentage changes from baseline calculated at mid- and post-NAC. Associations of imaging measures with pathological response (residual cancer burden RCB 0/I vs. II/III) and RFS were evaluated.
Thirty-five patients with stage II/III invasive breast cancer were enrolled in the prospective study (median age: 43, range: 31-66 years, RCB 0/I: N = 11/35, 31%). Baseline imaging metrics were not significantly associated with pathologic response or RFS (p > 0.05). Greater mid-treatment decreases in peak PE, along with greater post-treatment decreases in several DCE-MRI and
F-FDG PET measures were associated with RCB 0/I after NAC (p < 0.05). Additionally, greater mid- and post-treatment decreases in DCE-MRI (peak SER, washout volume) and
F-FDG PET (K
) were predictive of prolonged RFS. Mid-treatment decreases in metabolism/perfusion ratios (MRFDG/peak PE, MRFDG/peak SER) were associated with improved RFS.
Mid-treatment changes in both PET and MRI measures were predictive of RCB status and RFS following NAC. Specifically, our results indicate a complementary relationship between DCE-MRI and
F-FDG PET metrics and potential value of metabolism/perfusion mismatch as a marker of patient outcome.
Cell proliferation requires the coordinated activity of cytosolic and mitochondrial metabolic pathways to provide ATP and building blocks for DNA, RNA and protein synthesis. Many metabolic pathway ...genes are targets of the c-myc oncogene and cell-cycle regulator. However, the contribution of c-Myc to the activation of cytosolic and mitochondrial metabolic networks during cell-cycle entry is unknown. Here, we report the metabolic fates of U-(13)C glucose in serum-stimulated myc(-/-) and myc(+/+) fibroblasts by (13)C isotopomer NMR analysis. We demonstrate that endogenous c-myc increased (13)C labeling of ribose sugars, purines and amino acids, indicating partitioning of glucose carbons into C1/folate and pentose phosphate pathways, and increased tricarboxylic acid cycle turnover at the expense of anaplerotic flux. Myc expression also increased global O-linked N-acetylglucosamine protein modification, and inhibition of hexosamine biosynthesis selectively reduced growth of Myc-expressing cells, suggesting its importance in Myc-induced proliferation. These data reveal a central organizing function for the Myc oncogene in the metabolism of cycling cells. The pervasive deregulation of this oncogene in human cancers may be explained by its function in directing metabolic networks required for cell proliferation.
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