Microbiome signatures in prostate cancer.

We have established a microbiome signature for prostate cancer using an array-based metagenomic and capture-sequencing approach. A diverse microbiome signature (viral, bacterial, fungal and parasitic) was observed in the prostate cancer samples compared with benign prostate hyperplasia controls. Hierarchical clustering analysis identified three distinct prostate cancer-specific microbiome signatures. The three signatures correlated with different grades, stages and scores of the cancer. Thus, microbiome signature analysis potentially provides clinical diagnosis and outcome predictions. The array data were validated by PCR and targeted next-generation sequencing (NGS). Specific NGS data suggested that certain viral genomic sequences were inserted into the host somatic chromosomes of the prostate cancer samples. A randomly selected group of these was validated by direct PCR and sequencing. In addition, PCR validation of Helicobacter showed that Helicobacter cagA sequences integrated within specific chromosomes of prostate tumor cells. The viral and Helicobacter integrations are predicted to affect the expression of several cellular genes associated with oncogenic processes.

Distinct Microbial Signatures Associated With Different Breast Cancer Types.

A dysbiotic microbiome can potentially contribute to the pathogenesis of many different diseases including cancer. Breast cancer is the second leading cause of cancer death in women. Thus, we investigated the diversity of the microbiome in the four major types of breast cancer: endocrine receptor (ER) positive, triple positive, Her2 positive and triple negative breast cancers. Using a whole genome and transcriptome amplification and a pan-pathogen microarray (PathoChip) strategy, we detected unique and common viral, bacterial, fungal and parasitic signatures for each of the breast cancer types. These were validated by PCR and Sanger sequencing. Hierarchical cluster analysis of the breast cancer samples, based on their detected microbial signatures, showed distinct patterns for the triple negative and triple positive samples, while the ER positive and Her2 positive samples shared similar microbial signatures. These signatures, unique or common to the different breast cancer types, provide a new line of investigation to gain further insights into prognosis, treatment strategies and clinical outcome, as well as better understanding of the role of the micro-organisms in the development and progression of breast cancer.

Acid Suspends the Circadian Clock in Hypoxia through Inhibition of mTOR.

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.

Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity.

Kidney cancer, one of the ten most prevalent malignancies in the world, has exhibited increased incidence over the last decade. The most common subtype is "clear cell" renal cell carcinoma (ccRCC), which features consistent metabolic abnormalities, such as highly elevated glycogen and lipid deposition. By integrating metabolomics, genomic, and transcriptomic data, we determined that enzymes in multiple metabolic pathways are universally depleted in human ccRCC tumors, which are otherwise genetically heterogeneous. Notably, the expression of key urea cycle enzymes, including arginase 2 (ARG2) and argininosuccinate synthase 1 (ASS1), is strongly repressed in ccRCC. Reduced ARG2 activity promotes ccRCC tumor growth through at least two distinct mechanisms: conserving the critical biosynthetic cofactor pyridoxal phosphate and avoiding toxic polyamine accumulation. Pharmacological approaches to restore urea cycle enzyme expression would greatly expand treatment strategies for ccRCC patients, where current therapies only benefit a subset of those afflicted with renal cancer.

The ovarian cancer oncobiome.

Humans and other mammals are colonized by microbial agents across the kingdom which can represent a unique microbiome pattern. Dysbiosis of the microbiome has been associated with pathology including cancer. We have identified a microbiome signature unique to ovarian cancers, one of the most lethal malignancies of the female reproductive system, primarily because of its asymptomatic nature during the early stages in development. We screened ovarian cancer samples along with matched, and non-matched control samples using our pan-pathogen array (PathoChip), combined with capture-next generation sequencing. The results show a distinct group of viral, bacterial, fungal and parasitic signatures of high significance in ovarian cases. Further analysis shows specific viral integration sites within the host genome of tumor samples, which may contribute to the carcinogenic process. The ovarian cancer microbiome signature provides insights for the development of targeted therapeutics against ovarian cancers.

ACSS2-mediated acetyl-CoA synthesis from acetate is necessary for human cytomegalovirus infection.

Recent studies have shown that human cytomegalovirus (HCMV) can induce a robust increase in lipid synthesis which is critical for the success of infection. In mammalian cells the central precursor for lipid biosynthesis, cytosolic acetyl CoA (Ac-CoA), is produced by ATP-citrate lyase (ACLY) from mitochondria-derived citrate or by acetyl-CoA synthetase short-chain family member 2 (ACSS2) from acetate. It has been reported that ACLY is the primary enzyme involved in making cytosolic Ac-CoA in cells with abundant nutrients. However, using CRISPR/Cas9 technology, we have shown that ACLY is not essential for HCMV growth and virally induced lipogenesis. Instead, we found that in HCMV-infected cells glucose carbon can be used for lipid synthesis by both ACLY and ACSS2 reactions. Further, the ACSS2 reaction can compensate for the loss of ACLY. However, in ACSS2-KO human fibroblasts both HCMV-induced lipogenesis from glucose and viral growth were sharply reduced. This reduction suggests that glucose-derived acetate is being used to synthesize cytosolic Ac-CoA by ACSS2. Previous studies have not established a mechanism for the production of acetate directly from glucose metabolism. Here we show that HCMV-infected cells produce more glucose-derived pyruvate, which can be converted to acetate through a nonenzymatic mechanism.

Identification of fungal pathogens in a patient with acute myelogenic leukemia using a pathogen detection array technology.

Invasive zygomycosis in immunocompromised patients results in a high mortality rate, and early identification is crucial to optimize therapy and to reduce morbidity. However, diagnosing specific species of zygomycetes fungi possess challenge in the clinical laboratories. A need for a rapid and sensitive diagnostic tool for early recognition of a zygomycetes fungus in clinical samples to the species level will lead to prompt and accurate therapy and the PathoChip provides one such platform. We utilized a pathogen array technology referred to as PathoChip, comprised of oligonucleotide probes that can detect all the sequenced viruses as well as known pathogenic bacteria, fungi and parasites and family-specific conserved probes, thus providing a means for detecting previously uncharacterized members of a family. We rapidly identified a zygomycetous fungus, Rhizomucor pusillus, an otherwise challenge for the clinical laboratories, predominantly in a patient with acute myelogenous leukemia. This report highlights the value of PathoChip as a diagnostic tool to identify micro-organisms to the species level, especially for those difficult to identify in most clinical laboratories. It will also help clinicians to obtain a critical snapshot of the infection profile of a patient to plan treatment strategies.

Distinct microbiological signatures associated with triple negative breast cancer.

Infectious agents are the third highest human cancer risk factor and may have a greater role in the origin and/or progression of cancers, and related pathogenesis. Thus, knowing the specific viruses and microbial agents associated with a cancer type may provide insights into cause, diagnosis and treatment. We utilized a pan-pathogen array technology to identify the microbial signatures associated with triple negative breast cancer (TNBC). This technology detects low copy number and fragmented genomes extracted from formalin-fixed paraffin embedded archival tissues. The results, validated by PCR and sequencing, define a microbial signature present in TNBC tissue which was underrepresented in normal tissue. Hierarchical clustering analysis displayed two broad microbial signatures, one prevalent in bacteria and parasites and one prevalent in viruses. These signatures demonstrate a new paradigm in our understanding of the link between microorganisms and cancer, as causative or commensal in the tumor microenvironment and provide new diagnostic potential.

Metagenomic assay for identification of microbial pathogens in tumor tissues.

UNLABELLED: Screening for thousands of viruses and other pathogenic microorganisms, including bacteria, fungi, and parasites, in human tumor tissues will provide a better understanding of the contributory role of the microbiome in the predisposition for, causes of, and therapeutic responses to the associated cancer. Metagenomic assays designed to perform these tasks will have to include rapid and economical processing of large numbers of samples, supported by straightforward data analysis pipeline and flexible sample preparation options for multiple input tissue types from individual patients, mammals, or environmental samples. To meet these requirements, the PathoChip platform was developed by targeting viral, prokaryotic, and eukaryotic genomes with multiple DNA probes in a microarray format that can be combined with a variety of upstream sample preparation protocols and downstream data analysis. PathoChip screening of DNA plus RNA from formalin-fixed, paraffin-embedded tumor tissues demonstrated the utility of this platform, and the detection of oncogenic viruses was validated using independent PCR and deep sequencing methods. These studies demonstrate the use of the PathoChip technology combined with PCR and deep sequencing as a valuable strategy for detecting the presence of pathogens in human cancers and other diseases. IMPORTANCE: This work describes the design and testing of a PathoChip array containing probes with the ability to detect all known publicly available virus sequences as well as hundreds of pathogenic bacteria, fungi, parasites, and helminths. PathoChip provides wide coverage of microbial pathogens in an economical format. PathoChip screening of DNA plus RNA from formalin-fixed, paraffin-embedded tumor tissues demonstrated the utility of this platform, and the detection of oncogenic viruses was validated using independent PCR and sequencing methods. These studies demonstrate that the PathoChip technology is a valuable strategy for detecting the presence of pathogens in human cancers and other diseases.

ChREBP, a glucose-responsive transcriptional factor, enhances glucose metabolism to support biosynthesis in human cytomegalovirus-infected cells.

Carbohydrate-response element binding protein (ChREBP) plays a key role in regulating glucose metabolism and de novo lipogenesis in metabolic tissues and cancer cells. Here we report that ChREBP is also a critical regulator of the metabolic alterations induced during human cytomegalovirus (HCMV) infection. The expression of both ChREBP-α and ChREBP-ß is robustly induced in HCMV-infected human fibroblasts; this induction is required for efficient HCMV infection. Depletion of ChREBP in HCMV-infected cells results in reduction of HCMV-induced glucose transporter 4 and glucose transporter 2 expression, leading to inhibition of glucose uptake, lactate production, nucleotide biosynthesis, and NADPH generation. We previously reported that HCMV infection induces lipogenesis through the activation of sterol regulatory element binding protein 1, which is mediated by the induction of PKR-like endoplasmic reticulum kinase. Data from the present study show that HCMV-induced lipogenesis is also controlled by the induction of ChREBP, in a second mechanism involved in the regulation of HCMV-induced de novo lipogenesis. These results suggest that ChREBP plays a key role in reprogramming glucose and lipid metabolism in HCMV infection.

Dynein mediates the localization and activation of mTOR in normal and human cytomegalovirus-infected cells.

Activation of stress signaling pathways normally leads to inhibition of the mammalian target of rapamycin complex 1 (mTORC1); however, human cytomegalovirus (HCMV) infection maintains mTORC1 activity in the presence of numerous types of stress. We previously demonstrated that HCMV infection maintains mTORC1 activity during amino acid deprivation through a Ras-related GTP-binding (Rag) protein-independent mechanism. This depends on the colocalization of mTOR and its activator, Rheb (Ras homology enriched in brain)-GTP, to a perinuclear position that corresponds to the viral cytoplasmic assembly compartment (AC). The data presented here show that the HCMV-induced, amino acid depletion-resistant perinuclear localization and activation of mTORC1 occurs as early as 8 h post-infection, prior to AC formation. We show that the molecular motor dynein is required for perinuclear localization of mTORC1 in both uninfected and HCMV-infected cells. Association between dynein and mTOR is shown by coimmunoprecipitation, and inhibition of dynein function using RNAi or the small molecule inhibitor ciliobrevin A inhibits mTORC1 activity in both uninfected and HCMV-infected cells. The data suggest that mTORC1 activation requires dynein-dependent transport to a position in the cell where it can be activated. Thus, the HCMV commandeers a cellular dynein-dependent mTORC1 activation mechanism to maintain stress-resistant mTORC1 activity during infection and to form the AC.

Human cytomegalovirus induces multiple means to combat reactive oxygen species.

Reactive oxygen species (ROS) are generated as by-products of many cellular processes and can modulate cellular signaling pathways. However, high ROS levels are toxic; thus, intracellular ROS need to be tightly controlled. Therefore, cells use a group of antioxidant molecules and detoxifying enzymes that remove or detoxify reactive species. We found that the level of the antioxidant glutathione is greatly increased in human cytomegalovirus (HCMV)-infected cells due to activation of glutathione synthetic enzymes. In addition, our data suggest that virus-specific mechanisms are used to induce the expression of target antioxidant and detoxifying enzymes critical for the success of the infection. As a result of this virus-induced anti-ROS environment, key signaling kinases, such as the mammalian target of rapamycin (mTOR) kinase in mTOR complex 1 (mTORC1), are protected from inhibition by exogenous hydrogen peroxide (H(2)O(2)). In this regard, we found that phosphorylation of mTOR kinase at serine 2448 (suggested to be activating) was maintained during infection even under ROS stress conditions that inhibited it in uninfected cells. We also show that AMP-dependent kinase (AMPK)-mediated phosphorylation of serine 792 of raptor, the specificity subunit of mTORC1, increases in infected cells after H(2)O(2) treatment. This phosphorylation is normally inhibitory for mTORC1. However, in infected cells this did not result in inhibition of mTORC1 activity, suggesting that inhibitory effects of raptor phosphorylation are circumvented. Overall, our data suggest that HCMV utilizes virus-specific mechanisms to activate a variety of means to protect the cell and mTORC1 from the effects of ROS.

Viruses and metabolism: alterations of glucose and glutamine metabolism mediated by human cytomegalovirus.

Recent studies of human cytomegalovirus (HCMV) infection have demonstrated that the virus significantly alters cellular metabolism, especially the utilization of glucose and glutamine. Glucose is not broken down by the tricarboxylic acid (TCA) cycle in infected cells; instead, it is used biosynthetically for fatty acid synthesis for membranes needed during the infection. In this chapter, we discuss the possibility that HCMV integrates its mechanisms for manipulating cellular signaling and stress responses to induce novel adipocyte-like differentiation in order to alter metabolism so that glucose can be used synthetically, that is, for fatty acids and lipids. This process diverts glucose from the TCA cycle and requires induction of enzymes that can convert glutamine to α-ketoglutarate to maintain the TCA cycle (anaplerosis). We discuss data proposing that the anaplerotic utilization of glutamine may be mediated, in part, by c-Myc activation, and the induction of adipocyte-like differentiation may result from the activation of the endoplasmic reticulum resident kinase PKR-like ER kinase. These alterations in metabolism during HCMV infection are comparable to those seen in many tumor cells. Indeed, the alterations in cellular signaling, stress responses, and metabolism that have been characterized could result in unexpected pathogenesis, potentially implicating HCMV as an agent or subtle cofactor in many maladies. Better understanding of HCMV's effects on cell signaling and metabolism will show how HCMV-mediated modifications of cellular processes relate to pathogenesis and will suggest novel avenues for antiviral therapy.

Viral effects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection.

Human cytomegalovirus (HCMV) infection causes dramatic alterations of intermediary metabolism, similar to those found in tumor cells. In infected cells, glucose carbon is not completely broken down by the tricarboxylic acid (TCA) cycle for energy; instead, it is used biosynthetically. This process requires increased glucose uptake, increased glycolysis and the diversion of glucose carbon, in the form of citrate, from the TCA cycle for use in HCMV-induced fatty acid biosynthesis. The diversion of citrate from the TCA cycle (cataplerosis) requires induction of enzymes to promote glutaminolysis, the conversion of glutamine to α-ketoglutarate to maintain the TCA cycle (anaplerosis) and ATP production. Such changes could result in heretofore uncharacterized pathogenesis, potentially implicating HCMV as a subtle cofactor in many maladies, including oncogenesis. Recognition of the effects of HCMV, and other viruses, on host cell metabolism will provide new understanding of viral pathogenesis and novel avenues for antiviral therapy.

Role of the endoplasmic reticulum chaperone BiP, SUN domain proteins, and dynein in altering nuclear morphology during human cytomegalovirus infection.

The process of assembly and egress of human cytomegalovirus (HCMV) virions requires significant morphological alterations of the nuclear and cytoplasmic architecture. In the studies presented we show that the nuclear periphery is dramatically altered, especially near the cytoplasmic assembly compartment, where the nuclear lamina is specifically rearranged, the outer nuclear membrane is altered, and the nucleus becomes permeable to large molecules. In addition, the tethering of the inner and outer nuclear membranes is lost during infection due to a decrease in levels of the SUN domain proteins. We previously demonstrated that the endoplasmic reticulum protein BiP functions as a component of the assembly compartment and disruption of BiP causes the loss of assembly compartment integrity. In this study we show that the depletion of BiP, and the loss of assembly compartment integrity, results in the loss of virally induced lamina rearrangement and morphology of the nucleus that is characteristic of HCMV infection. BiP functions in lamina rearrangement through its ability to affect lamin phosphorylation. Depletion of BiP and disruption of the assembly compartment result in the loss of lamin phosphorylation. The dependency of lamin phosphorylation on BiP correlates with an interaction between BiP and UL50. Finally, we confirm previous data (S. V. Indran, M. E. Ballestas, and W. J. Britt, J. Virol. 84:3162-3177, 2010) suggesting an involvement of dynein in assembly compartment formation and extend this observation by showing that when dynein is inhibited, the nuclear morphology characteristic of an HCMV infection is lost. Our data suggest a highly integrated assembly-egress continuum.

Glutamine metabolism is essential for human cytomegalovirus infection.

Human fibroblasts infected with human cytomegalovirus (HCMV) were more viable than uninfected cells during glucose starvation, suggesting that an alternate carbon source was used. We have determined that infected cells require glutamine for ATP production, whereas uninfected cells do not. This suggested that during infection, glutamine is used to fill the tricarboxylic acid (TCA) cycle (anaplerosis). In agreement with this, levels of glutamine uptake and ammonia production increased in infected cells, as did the activities of glutaminase and glutamate dehydrogenase, the enzymes needed to convert glutamine to alpha-ketoglutarate to enter the TCA cycle. Infected cells starved for glutamine beginning 24 h postinfection failed to produce infectious virions. Both ATP and viral production could be rescued in glutamine-starved cells by the TCA intermediates alpha-ketoglutarate, oxaloacetate, and pyruvate, confirming that in infected cells, a program allowing glutamine to be used anaplerotically is induced. Thus, HCMV infection activates the mechanisms needed to switch the anaplerotic substrate from glucose to glutamine to accommodate the biosynthetic and energetic needs of the viral infection and to allow glucose to be used biosynthetically.

The endoplasmic reticulum chaperone BiP/GRP78 is important in the structure and function of the human cytomegalovirus assembly compartment.

We previously demonstrated that the endoplasmic reticulum (ER) chaperone BiP functions in human cytomegalovirus (HCMV) assembly and egress. Here, we show that BiP localizes in two cytoplasmic structures in infected cells. Antibodies to the extreme C terminus, which includes BiP's KDEL ER localization sequence, detect BiP in regions of condensed ER near the periphery of the cell. Antibodies to the full length, N terminus, or larger portion of the C terminus detect BiP in the assembly compartment. This inability of C-terminal antibodies to detect BiP in the assembly compartment suggests that BiP's KDEL sequence is occluded in the assembly compartment. Depletion of BiP causes the condensed ER and assembly compartments to dissociate, indicating that BiP is important for their integrity. BiP and pp28 are in association in the assembly compartment, since antibodies that detect BiP in the assembly compartment coimmunoprecipitate pp28 and vice versa. In addition, BiP and pp28 copurify with other assembly compartment components on sucrose gradients. BiP also coimmunoprecipitates TRS1. Previous data show that cells infected with a TRS1-deficient virus have cytoplasmic and assembly compartment defects like those seen when BiP is depleted. We show that a fraction of TRS1 purifies with the assembly compartment. These findings suggest that BiP and TRS1 share a function in assembly compartment maintenance. In summary, BiP is diverted from the ER to associate with pp28 and TRS1, contributing to the integrity and function of the assembly compartment.

The TORrid affairs of viruses: effects of mammalian DNA viruses on the PI3K-Akt-mTOR signalling pathway.

The successful replication of mammalian DNA viruses requires that they gain control of key cellular signalling pathways that affect broad aspects of cellular macromolecular synthesis, metabolism, growth and survival. The phosphatidylinositol 3'-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) pathway is one such pathway. Mammalian DNA viruses have evolved various mechanisms to activate this pathway to obtain the benefits of Akt activation, including the maintenance of translation through the activation of mTOR. In addition, viruses must overcome the inhibition of this pathway that results from the activation of cellular stress responses during viral infection. This Review will discuss the range of mechanisms that mammalian DNA viruses use to activate this pathway, as well as the multiple mechanisms these viruses have evolved to circumvent inhibitory stress signalling.

Interaction between simian virus 40 large T antigen and insulin receptor substrate 1 is disrupted by the K1 mutation, resulting in the loss of large T antigen-mediated phosphorylation of Akt.

The cellular kinase Akt is a key controller of cellular metabolism, growth, and proliferation. Many viruses activate Akt due to its beneficial effects on viral replication. We previously showed that wild-type (WT) simian virus 40 (SV40) large T antigen (TAg) inhibits apoptosis via the activation of PI3K/Akt signaling. Here we show that WT TAg expressed from recombinant adenoviruses in U2OS cells induced the phosphorylation of Akt at both T308 and S473. In contrast, Akt phosphorylation was eliminated by the K1 mutation (E107K) within the retinoblastoma protein (Rb) binding motif of TAg. This suggested that Akt phosphorylation may depend on TAg binding to Rb or one of its family members. However, in Rb-negative SAOS2 cells depleted of p107 and p130 by using small hairpin RNAs (shRNAs), WT TAg still mediated Akt phosphorylation. These results suggested that the K1 mutation affects another TAg function. WT-TAg-mediated phosphorylation of Akt was inhibited by a PI3K inhibitor, suggesting that the effects of TAg originated upstream of PI3K; thus, we examined the requirement for insulin receptor substrate 1 (IRS1), which binds and activates PI3K. Depletion of IRS1 by shRNAs abolished the WT-TAg-mediated phosphorylation of Akt. Immunoprecipitation studies showed that the known interaction between TAg and IRS1 is significantly weakened by the K1 mutation. These data indicate that the K1 mutation disrupts not only Rb binding but also IRS1 binding, contributing to the loss of activation of PI3K/Akt signaling.

Human cytomegalovirus infection alters the substrate specificities and rapamycin sensitivities of raptor- and rictor-containing complexes.

Signaling mediated by the mammalian target of rapamycin kinase (mTOR) is activated during human cytomegalovirus (HCMV) infection. mTOR is found in two complexes differing by the binding partner, rictor or raptor. Activated mTOR-raptor promotes cap-dependent translation through the hyperphosphorylation of the eIF4E-binding protein (4E-BP). This activity of the raptor complex is normally inhibited by cell stress responses or the drug rapamycin. However, we previously showed that this inhibition of mTOR signaling can be circumvented during HCMV infection such that hyperphosphorylation of 4E-BP is maintained. Here we show that HCMV infection also activates the rictor complex, as indicated by increased phosphorylation of Akt S473; this phosphorylation is insensitive to rapamycin but sensitive to caffeine in both uninfected and infected cells. By using short-hairpin RNAs to deplete rictor and raptor, we find that rictor is more significant than raptor for the viral infection. Surprisingly, the inhibitory effects of rapamycin on viral growth are primarily due to the presence of rictor, not raptor. Raptor and rictor depletion experiments show that in HCMV-infected cells, both raptor- and rictor-containing complexes can mediate the hyperphosphorylation of 4E-BP and the phosphorylation of p70S6 kinase. Under these conditions, the rictor complex is rapamycin-sensitive for the hyperphosphorylation of 4E-BP, but the raptor complex is not. These data suggest that, during HCMV infection, the rictor- and raptor-containing complexes are modified such that their substrate specificities and rapamycin sensitivities are altered. Our data also suggest that the present understanding of rapamycin's inhibitory effects is incomplete.