Charles G. Bailey

Molecular sequelae of proteasome inhibition in human multiple myeloma cells

The proteasome inhibitor PS-341 inhibits IκB degradation, prevents NF-κB activation, and induces apoptosis in several types of cancer cells, including chemoresistant multiple myeloma (MM) cells. PS-341 has marked clinical activity even in the setting of relapsed refractory MM. However, PS-341-induced apoptotic cascade(s) are not yet fully defined. By using gene expression profiling, we characterized the molecular sequelae of PS-341 treatment in MM cells and further focused on molecular pathways responsible for the anticancer actions of this promising agent. The transcriptional profile of PS-341-treated cells involved down-regulation of growth/survival signaling pathways, and up-regulation of molecules implicated in proapoptotic cascades (which are both consistent with the proapoptotic effect of proteasome inhibition), as well as up-regulation of heat-shock proteins and ubiquitin/proteasome pathway members (which can correspond to stress responses against proteasome inhibition). Further studies on these pathways showed that PS-341 decreases the levels of several antiapoptotic proteins and triggers a dual apoptotic pathway of mitochondrial cytochrome c release and caspase-9 activation, as well as activation of Jun kinase and a Fas/caspase-8-dependent apoptotic pathway [which is inhibited by a dominant negative (decoy) Fas construct]. Stimulation with IGF-1, as well as overexpression of Bcl-2 or constitutively active Akt in MM cells also modestly attenuates PS-341-induced cell death, whereas inhibitors of the BH3 domain of Bcl-2 family members or the heat-shock protein 90 enhance tumor cell sensitivity to proteasome inhibition. These data provide both insight into the molecular mechanisms of antitumor activity of PS-341 and the rationale for future clinical trials of PS-341, in combination with conventional and novel therapies, to improve patient outcome in MM.

Orchestrated intron retention regulates normal granulocyte differentiation.

Intron retention (IR) is widely recognized as a consequence of mis-splicing that leads to failed excision of intronic sequences from pre-messenger RNAs. Our bioinformatic analyses of transcriptomic and proteomic data of normal white blood cell differentiation reveal IR as a physiological mechanism of gene expression control. IR regulates the expression of 86 functionally related genes, including those that determine the nuclear shape that is unique to granulocytes. Retention of introns in specific genes is associated with downregulation of splicing factors and higher GC content. IR, conserved between human and mouse, led to reduced mRNA and protein levels by triggering the nonsense-mediated decay (NMD) pathway. In contrast to the prevalent view that NMD is limited to mRNAs encoding aberrant proteins, our data establish that IR coupled with NMD is a conserved mechanism in normal granulopoiesis. Physiological IR may provide an energetically favorable level of dynamic gene expression control prior to sustained gene translation.

Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19

Hartnup disorder (OMIM 234500) is an autosomal recessive abnormality of renal and gastrointestinal neutral amino acid transport noted for its clinical variability. We localized a gene causing Hartnup disorder to chromosome 5p15.33 and cloned a new gene, SLC6A19, in this region. SLC6A19 is a sodium-dependent and chloride-independent neutral amino acid transporter, expressed predominately in kidney and intestine, with properties of system B0. We identified six mutations in SLC6A19 that cosegregated with disease in the predicted recessive manner, with most affected individuals being compound heterozygotes. The disease-causing mutations that we tested reduced neutral amino acid transport function in vitro. Population frequencies for the most common mutated SLC6A19 alleles are 0.007 for 517G → A and 0.001 for 718C → T. Our findings indicate that SLC6A19 is the long-sought gene that is mutated in Hartnup disorder; its identification provides the opportunity to examine the inconsistent multisystemic features of this disorder.

Inositol polyphosphate 4-phosphatase II regulates PI3K/Akt signaling and is lost in human basal-like breast cancers

Inositol polyphosphate 4-phosphatase-II (INPP4B) is a regulator of the phosphoinositide 3-kinase (PI3K) signaling pathway and is implicated as a tumor suppressor in epithelial carcinomas. INPP4B loss of heterozygosity (LOH) is detected in some human breast cancers; however, the expression of INPP4B protein in breast cancer subtypes and the normal breast is unknown. We report here that INPP4B is expressed in nonproliferative estrogen receptor (ER)-positive cells in the normal breast, and in ER-positive, but not negative, breast cancer cell lines. INPP4B knockdown in ER-positive breast cancer cells increased Akt activation, cell proliferation, and xenograft tumor growth. Conversely, reconstitution of INPP4B expression in ER-negative, INPP4B-null human breast cancer cells reduced Akt activation and anchorage-independent growth. INPP4B protein expression was frequently lost in primary human breast carcinomas, associated with high clinical grade and tumor size and loss of hormone receptors and was lost most commonly in aggressive basal-like breast carcinomas. INPP4B protein loss was also frequently observed in phosphatase and tensin homolog (PTEN)-null tumors. These studies provide evidence that INPP4B functions as a tumor suppressor by negatively regulating normal and malignant mammary epithelial cell proliferation through regulation of the PI3K/Akt signaling pathway, and that loss of INPP4B protein is a marker of aggressive basal-like breast carcinomas.

Lethal toxin of Bacillus anthracis causes apoptosis of macrophages

Lethal toxin is a major anthrax virulence factor, causing the rapid death of experimental animals. Lethal toxin can enter most cell types, but only certain macrophages and cell lines are susceptible to toxin-mediated cytolysis. We have shown that in murine RAW 264.7 cells, sublytic amounts of lethal toxin trigger intracellular signaling events typical for apoptosis, including changes in membrane permeability, loss of mitochondrial membrane potential, and DNA fragmentation. The cells were protected from the toxin by specific inhibitors of caspase-1, -2, -3, -4, -6, and -8. Phagocytic activity of macrophages was inhibited by sublytic concentrations of lethal toxin. Infection of cells with anthrax (Sterne) spores impaired their bactericidal capacity, which could be reversed by a lethal toxin inhibitor, bestatin. We suggest that apoptosis rather than direct lysis is biologically relevant to lethal toxin intracellular activity.

Effect of Bacillus anthracis lethal toxin on human peripheral blood mononuclear cells

Lethal toxin (LeTx) plays a central role in anthrax pathogenesis, however a cytotoxicity of LeTx has been difficult to demonstrate in vitro. No cytolytic effect has been reported for human cells, in contrast to murine cell lines, indicating that cell lysis can not be considered as a marker of LeTx activity. We have recently shown that murine macrophage‐like RAW 264.7 cells treated with LeTx or infected with anthrax spores underwent changes typical of apoptotic death. Here we demonstrate that cells from human peripheral blood display a proapoptotic behavior similar to murine cells. TUNEL assay detected a nucleosomal degradation typical of apoptosis in peripheral blood mononuclear cells (PBMC) treated with LeTx. Membrane staining with apoptotic dyes was detected in macrophages derived from monocytes in presence of LeTx. The toxin inhibited production of proinflammatory cytokines in PBMC stimulated with a preparation of Bacillus anthracis cell wall. Infection of PBMC with anthrax spores led to the appearance of a large population of cells stained positively for apoptosis, with a reduced capacity to eliminate spores and vegetative bacteria. The aminopeptidase inhibitor, bestatin, capable of protecting cells from LeTx, restored a bactericidal activity of infected cells. These findings may be explained by LeTx expression within phagocytes and support an important role of LeTx as an early intracellular virulence factor contributing to bacterial dissemination and disease progression.

ASCT2/SLC1A5 controls glutamine uptake and tumour growth in triple-negative basal-like breast cancer

Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) mediates uptake of glutamine, a conditionally essential amino acid in rapidly proliferating tumour cells. Uptake of glutamine and subsequent glutaminolysis is critical for activation of the mTORC1 nutrient-sensing pathway, which regulates cell growth and protein translation in cancer cells. This is of particular interest in breast cancer, as glutamine dependence is increased in high-risk breast cancer subtypes. Pharmacological inhibitors of ASCT2-mediated transport significantly reduced glutamine uptake in human breast cancer cell lines, leading to the suppression of mTORC1 signalling, cell growth and cell cycle progression. Notably, these effects were subtype-dependent, with ASCT2 transport critical only for triple-negative (TN) basal-like breast cancer cell growth compared with minimal effects in luminal breast cancer cells. Both stable and inducible shRNA-mediated ASCT2 knockdown confirmed that inhibiting ASCT2 function was sufficient to prevent cellular proliferation and induce rapid cell death in TN basal-like breast cancer cells, but not in luminal cells. Using a bioluminescent orthotopic xenograft mouse model, ASCT2 expression was then shown to be necessary for both successful engraftment and growth of HCC1806 TN breast cancer cells in vivo. Lower tumoral expression of ASCT2 conferred a significant survival advantage in xenografted mice. These responses remained intact in primary breast cancers, where gene expression analysis showed high expression of ASCT2 and glutamine metabolism-related genes, including GLUL and GLS, in a cohort of 90 TN breast cancer patients, as well as correlations with the transcriptional regulators, MYC and ATF4. This study provides preclinical evidence for the feasibility of novel therapies exploiting ASCT2 transporter activity in breast cancer, particularly in the high-risk basal-like subgroup of TN breast cancer where there is not only high expression of ASCT2, but also a marked reliance on its activity for sustained cellular proliferation.

Secreted Neutral Metalloproteases of Bacillus anthracis as Candidate Pathogenic Factors

To evaluate the pathogenic potential of Bacillus anthracis-secreted proteases distinct from lethal toxin, two neutral zinc metalloproteases were purified to apparent homogeneity from the culture supernatant of a non-virulent delta Ames strain (pXO1–, pXO2–). The first (designated Npr599) is a thermolysin-like enzyme highly homologous to bacillolysins from other Bacillus species. The second (designated InhA) is a homolog of the Bacillus thuringiensis immune inhibitor A. These proteases belong to the M4 and M6 families, respectively. Both enzymes digested various substrates, including extracellular matrix proteins, endogenous inhibitors, and coagulation proteins, with some differences in specificity. In addition, InhA accelerated urokinase-mediated plasminogen activation, suggesting that InhA acts as a modulator of plasmin in the host inflammatory system. Relevant to epithelial barrier function, Npr599 and InhA significantly enhanced syndecan-1 shedding from cultured normal murine mammary gland cells without affecting their viability through stimulation of the host cell ectodomain shedding mechanism. In addition, Npr599 and InhA directly cleaved recombinant syndecan-1 fused to glutathione S-transferase. Mass spectrometric analysis suggested that the cleavage sites of Npr599 and InhA are the Asp39–Asp40 and Gly48–Thr49 bonds, respectively. We propose that Npr599 and InhA from B. anthracis are multifunctional pathogenic factors that may contribute to anthrax pathology through direct degradation of host tissues, increases in barrier permeability, and/or modulation of host defenses.

Targeting ASCT2-mediated glutamine uptake blocks prostate cancer growth and tumour development

Glutamine is conditionally essential in cancer cells, being utilized as a carbon and nitrogen source for macromolecule production, as well as for anaplerotic reactions fuelling the tricarboxylic acid (TCA) cycle. In this study, we demonstrated that the glutamine transporter ASCT2 (SLC1A5) is highly expressed in prostate cancer patient samples. Using LNCaP and PC‐3 prostate cancer cell lines, we showed that chemical or shRNA‐mediated inhibition of ASCT2 function in vitro decreases glutamine uptake, cell cycle progression through E2F transcription factors, mTORC1 pathway activation and cell growth. Chemical inhibition also reduces basal oxygen consumption and fatty acid synthesis, showing that downstream metabolic function is reliant on ASCT2‐mediated glutamine uptake. Furthermore, shRNA knockdown of ASCT2 in PC‐3 cell xenografts significantly inhibits tumour growth and metastasis in vivo, associated with the down‐regulation of E2F cell cycle pathway proteins. In conclusion, ASCT2‐mediated glutamine uptake is essential for multiple pathways regulating the cell cycle and cell growth, and is therefore a putative therapeutic target in prostate cancer.

Targeting glutamine transport to suppress melanoma cell growth

Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAFWT (C8161 and WM852) and BRAFV600E mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2‐mediated glutamine transport is a potential therapeutic target for both BRAFWT and BRAFV600E melanoma.