mRNA and miRNA Profiles of Exosomes from Cultured Tumor Cells Reveal Biomarkers Specific for HPV16-Positive and HPV16-Negative Head and Neck Cancer.

Human papillomavirus (HPV)(+) and HPV(-) head and neck cancer (HNC) cells' interactions with the host immune system are poorly understood. Recently, we identified molecular and functional differences in exosomes produced by HPV(+) vs. HPV(-) cells, suggesting that genetic cargos of exosomes might identify novel biomarkers in HPV-related HNCs.

Downregulation of estrogen receptor and modulation of growth of breast cancer cell lines mediated by paracrine stromal cell signals.

Purpose: Breast cancers have a poorer prognosis if estrogen receptor expression was lost during recurrence. It is unclear whether this conversion is cell autonomous or whether it can be promoted by the microenvironment during cancer dormancy. We explored the ability of marrow-derived stromal cell lines to arrest co-cultured breast cancer cells and suppress estrogen receptor alpha (ER) expression during arrest, facilitating the emergence of estrogen-independent breast cancer clones.

The Shu complex is a conserved regulator of homologous recombination.

Homologous recombination (HR) is an error-free DNA repair mechanism that maintains genome integrity by repairing double-strand breaks (DSBs). Defects in HR lead to genomic instability and are associated with cancer predisposition. A key step in HR is the formation of Rad51 nucleoprotein filaments which are responsible for the homology search and strand invasion steps that define HR.

Insights into APC/C: from cellular function to diseases and therapeutics.

Anaphase-promoting complex/cyclosome (APC/C) is a multifunctional ubiquitin-protein ligase that targets different substrates for ubiquitylation and therefore regulates a variety of cellular processes such as cell division, differentiation, genome stability, energy metabolism, cell death, autophagy as well as carcinogenesis. Activity of APC/C is principally governed by two WD-40 domain proteins, Cdc20 and Cdh1, in and beyond cell cycle. In the past decade, the results based on numerous biochemical, 3D structural, mouse genetic and small molecule inhibitor studies have largely attracted our attention into the emerging role of APC/C and its regulation in biological function, human diseases and potential therapeutics.

Phenotypic and functional characteristics of CD39 human regulatory B cells (Breg).

CD39 and CD73 are key enzymes in the adenosine (ADO) pathway. ADO modulates pathophysiological responses of immune cells, including B cells. It has recently emerged that a subpopulation of ADO-producing CD39CD73 B cells has regulatory properties.

New insights into posttranslational modifications of Hippo pathway in carcinogenesis and therapeutics.

PTMs (posttranslational modifications) such as ubiquitylation, sumoylation, acetylation and protein methylation are pivotal modifiers that determine the activation, deactivation or subcellular localization of signaling proteins, facilitating the initiation, amplification and transduction of signaling. Accumulating evidence suggest that several key signaling molecules in Hippo signaling pathway are tightly regulated by various types of PTMs. Malfunction of these critical signaling modules such as YAP/TAZ, MAT1/2 and LATS1/2 due to deregulated PTMs has been linked to a variety of human diseases such as cancer.

Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer.

Objective: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable.

Methods: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC).

Small-molecule inhibitors targeting INK4 protein p18(INK4C) enhance ex vivo expansion of haematopoietic stem cells.

Among cyclin-dependent kinase inhibitors that control the G1 phase in cell cycle, only p18 and p27 can negatively regulate haematopoietic stem cell (HSC) self-renewal. In this manuscript, we demonstrate that p18 protein is a more potent inhibitor of HSC self-renewal than p27 in mouse models and its deficiency promoted HSC expansion in long-term culture. Single-cell analysis indicated that deleting p18 gene favoured self-renewing division of HSC in vitro.

Kinetic gating mechanism of DNA damage recognition by Rad4/XPC.

The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA.

Measurements of natural killer (NK) cells.

In this article, we describe currently available methods for measuring NK cell functions: cytotoxicity and cytokine expression using flow cytometry-based assays and cytokine production using the Luminex-based technology. Quality control measures necessary for assay accuracy and reliability are also addressed.

Identification of mutations in the PYRIN-containing NLR genes (NLRP) in Head and Neck Squamous Cell Carcinoma.

Head and Neck Squamous Cell Carcinoma (HNSCC) encompasses malignancies that arise in the mucosa of the upper aerodigestive tract. Recent high throughput DNA sequencing revealed HNSCC genes mutations that contribute to several cancer cell characteristics, including dysregulation of cell proliferation and death, intracellular proinflammatory signaling, and autophagy. The PYRIN-domain containing NLR (Nucleotide-binding domain, Leucine rich Repeats - containing) proteins have recently emerged as pivotal modulators of cell death, autophagy, inflammation, and metabolism.

A Complex between Atg7 and Caspase-9: A NOVEL MECHANISM OF CROSS-REGULATION BETWEEN AUTOPHAGY AND APOPTOSIS.

Several cross-talk mechanisms between autophagy and apoptosis have been identified, in which certain co-regulators are shared, allowing the same protein to participate in these opposing processes. Our studies suggest that caspase-9 is a novel co-regulator of apoptosis and autophagy and that its caspase catalytic activity is dispensable for its autophagic role. We provide evidence that caspase-9 facilitates the early events leading to autophagosome formation; that it forms a complex with Atg7; that Atg7 is not a direct substrate for caspase-9 proteolytic activity; and that, depending on the cellular context, Atg7 represses the apoptotic capability of caspase-9, whereas the latter enhances the Atg7-mediated formation of light chain 3-II.

A phase I trial of isolated hepatic perfusion (IHP) using 5-FU and oxaliplatin in patients with unresectable isolated liver metastases from colorectal cancer.

Background: Isolated hepatic perfusion (IHP) with melphalan is an established approach for patients with unresectable metastatic liver lesions. This study determined the safety and maximum tolerated dose (MTD) of 5-FU with oxaliplatin via IHP.

Methods: Standard 3 × 3 Phase I design.

Targeting Stat3 abrogates EGFR inhibitor resistance in cancer.

Purpose: EGF receptor (EGFR) is upregulated in most epithelial cancers where signaling through EGFR contributes to cancer cell proliferation and survival. The limited clinical efficacy of EGFR inhibitors suggests that identification of resistance mechanisms may identify new pathways for therapeutic targeting. STAT3 is upregulated in many cancers and activated via both EGFR-dependent and -independent pathways.

For breast cancer prognosis, immunoglobulin kappa chain surfaces to the top.

The stromal immunoglobulin kappa chain (IGKC) has been validated as an immunologic biomarker of prognosis and response to therapy in human breast cancer and other cancers. This validation emphasizes the key role of humoral immunity in control of cancer progression and has major implications for determining prognosis of patients with cancer.

Regulation of KLF4 turnover reveals an unexpected tissue-specific role of pVHL in tumorigenesis.

The transcription factor Krüppel-like factor 4 (KLF4) is an important regulator of cell-fate decision, including cell-cycle regulation, apoptosis, and stem cell renewal, and plays an ambivalent role in tumorigenesis as a tissue-specific tumor suppressor or oncogene. Here, we report that the Von Hippel-Lindau gene product, pVHL, physically interacts with KLF4 and regulates its rapid turnover observed in both differentiated and stem cells. We provide mechanistic insights into KLF4 degradation and show that pVHL depletion in colorectal cancer cells leads to cell-cycle arrest concomitant with increased transcription of the KLF4-dependent p21 gene.

Disarming suppressor cells to improve immunotherapy.

Human tumors can use many different mechanisms to induce dysfunction in the host immune system. Accumulations of inducible regulatory T cells (iTreg, Tr1) are commonly seen in the tumor microenvironment. These Treg express CD39 and up-regulate CD73 ectonucleotidases, hydrolyze exogenous adenosine triphosphate (ATP) to AMP and adenosine and produce prostaglandin E(2) (PGE(2)).

Posttraumatic growth in women one year after diagnosis for gynecologic cancer or benign conditions.

The authors sought to examine levels and predictors of posttraumatic growth over one year after surgery in women diagnosed with gynecologic cancer or benign conditions necessitating surgical intervention. Women with advanced-stage cancer (n = 16), early-stage cancer (n = 18), benign gynecologic disease (n = 21), and no disease (n = 14; postannual pelvic exam) completed questionnaires (Perceived Threat, PTSD Checklist [PCL]) 1 week prior to surgery and completed the Posttraumatic Growth Inventory (PTGI) 16 months postsurgery. The four groups' scores varied significantly on the PTGI.

XPF expression correlates with clinical outcome in squamous cell carcinoma of the head and neck.

Purpose: Tumor-specific biomarkers that predict resistance to DNA damaging agents may improve therapeutic outcomes by guiding the selection of effective therapies and limiting morbidity related to ineffective approaches. XPF (ERCC4) is an essential component of several DNA repair pathways and XPF-deficient cells are exquisitely sensitive to DNA damaging agents. The purpose of this study was to determine whether XPF expression levels predict clinical response to DNA damaging agents in head and neck squamous cell carcinoma (HNSCC).

Turnover of BRCA1 involves in radiation-induced apoptosis.

Background: Germ-line mutations of the breast cancer susceptibility gene-1 (BRCA1) increase the susceptibility to tumorigenesis. The function of BRCA1 is to regulate critical cellular processes, including cell cycle progression, genomic integrity, and apoptosis. Studies on the regulation of BRCA1 have focused intensely on transcription and phosphorylation mechanisms.

Phosphorylation of the anaphase-promoting complex/Cdc27 is involved in TGF-beta signaling.

Loss of TGF-β-induced growth inhibition is a hallmark of many human tumors. Previous studies implied that activation of the anaphase-promoting complex (APC/cyclosome) is involved in the TGF-β signaling pathway, which facilitates the destruction of SnoN, a transcriptional co-suppressor, which leads in turn to the transactivation of TGF-β-responsive genes for cell cycle arrest. The function of APC was demonstrated in TGF-β signal transduction, but the mechanism by which it is activated in response to TGF-β signaling remains unclear.

Rare event detection and analysis in flow cytometry: bone marrow mesenchymal stem cells, breast cancer stem/progenitor cells in malignant effusions, and pericytes in disaggregated adipose tissue.

One of the major strengths of Flow Cytometry is its ability to perform multiple measurements on single cells within a heterogeneous mixture. When the populations of interest are relatively rare, analytical methodology that is adequate for more prevalent populations is often overcome by sources of artifacts that become apparent only when large numbers of cells are acquired. This chapter presents three practical examples of rare event problems and gives detailed instructions for preparation of single cell suspensions from bone marrow, malignant effusions, and solid tissue.

APC/C-Cdh1: from cell cycle to cellular differentiation and genomic integrity.

Anaphase-promoting complex/cyclosome (APC/C) is a multifunctional ubiquitin-protein ligase that targets various substrates for proteolysis inside and outside of the cell cycle. The activation of APC/C is dependent on two WD-40 domain proteins, Cdc20 and Cdh1. While APC/Cdc20 principally regulates mitotic progression, APC/Cdh1 shows a broad spectrum of substrates in and beyond cell cycle.

Proteolysis of Rad17 by Cdh1/APC regulates checkpoint termination and recovery from genotoxic stress.

Recent studies have shown a critical function for the ubiquitin-proteasome system (UPS) in regulating the signalling network for DNA damage responses and DNA repair. To search for new UPS targets in the DNA damage signalling pathway, we have carried out a non-biased assay to identify fast-turnover proteins induced by various types of genotoxic stress. This endeavour led to the identification of Rad17 as a protein exhibiting a distinctive pattern of upregulation followed by subsequent degradation after exposure to UV radiation in human primary cells.

Reciprocal granzyme/perforin-mediated death of human regulatory and responder T cells is regulated by interleukin-2 (IL-2).

Human CD4(+)CD25(high)FOXP3(+) T regulatory cells (Treg) can suppress responder T cell (RC) functions by various mechanisms. In co-cultures of Treg and autologous activated RC, both cell subsets up-regulate the expression of granzymes and perforin, which might contribute to Treg-mediated suppression. Here, we investigate the sensitivity and resistance of Treg and RC to granzyme/perforin-mediated death.