Circulating tumour cells as a biomarker for diagnosis and staging in pancreatic cancer.

BACKGROUND: Current diagnosis and staging of pancreatic ductal adenocarcinoma (PDAC) has important limitations and better biomarkers are needed to guide initial therapy. We investigated the performance of circulating tumour cells (CTCs) as an adjunctive biomarker at the time of disease presentation. METHODS: Venous blood (VB) was collected prospectively from 100 consecutive, pre-treatment patients with PDAC. Utilising the microfluidic NanoVelcro CTC chip, samples were evaluated for the presence and number of CTCs. KRAS mutation analysis was used to compare the CTCs with primary tumour tissue. CTC enumeration data was then evaluated as a diagnostic and staging biomarker in the setting of PDAC. RESULTS: We found 100% concordance for KRAS mutation subtype between primary tumour and CTCs in all five patients tested. Evaluation of CTCs as a diagnostic revealed the presence of CTCs in 54/72 patients with confirmed PDAC (sensitivity=75.0%, specificity=96.4%, area under the curve (AUROC)=0.867, 95% CI=0.798-0.935, and P<0.001). Furthermore, a cut-off of ⩾3 CTCs in 4 ml VB was able to discriminate between local/regional and metastatic disease (AUROC=0.885; 95% CI=0.800-0.969; and P<0.001). CONCLUSION: CTCs appear to function well as a biomarker for diagnosis and staging in PDAC.

The proximal signaling network of the BCR-ABL1 oncogene shows a modular organization.

BCR-ABL1 is a fusion tyrosine kinase, which causes multiple types of leukemia. We used an integrated proteomic approach that includes label-free quantitative protein complex and phosphorylation profiling by mass spectrometry to systematically characterize the proximal signaling network of this oncogenic kinase. The proximal BCR-ABL1 signaling network shows a modular and layered organization with an inner core of three leukemia transformation-relevant adaptor protein complexes (Grb2/Gab2/Shc1 complex, CrkI complex and Dok1/Dok2 complex). We introduced an 'interaction directionality' analysis, which annotates static protein networks with information on the directionality of phosphorylation-dependent interactions. In this analysis, the observed network structure was consistent with a step-wise phosphorylation-dependent assembly of the Grb2/Gab2/Shc1 and the Dok1/Dok2 complexes on the BCR-ABL1 core. The CrkI complex demonstrated a different directionality, which supports a candidate assembly on the Nedd9 (Hef1, CasL) scaffold. As adaptor protein family members can compensate for each other in leukemic transformation, we compared members of the Dok and Crk protein families and found both overlapping and differential binding patterns. We identified an additional level of regulation for the CrkII protein via binding to 14-3-3 proteins, which was independent from its inhibitory phosphorylation. We also identified novel components of the inner core complexes, including the kinases Pragmin (Sgk223) and Lrrk1 (Lrrk2 paralog). Pragmin was found as a component of the CrkI complex and is a potential link between BCR-ABL1/CrkI and RhoA signaling. Lrrk1 is an unusual kinase with a GTPase domain. We detected Lrrk1 as a component of the Grb2/Gab2/Shc1 complex and found that it functionally interacts with the regulator of small GTPases Arap1 (Centd2) and possibly participates in the mitogen-activated protein kinase response to cellular stresses. This modular and phosphorylation-driven interaction network provides a framework for the integration of pleiotropic signaling effects of BCR-ABL1 toward leukemic transformation.

Maximal entropy inference of oncogenicity from phosphorylation signaling.

Point mutations in the phosphorylation domain of the Bcr-Abl fusion oncogene give rise to drug resistance in chronic myelogenous leukemia patients. These mutations alter kinase-mediated signaling function and phenotypic outcome. An information theoretic analysis of the correlation of phosphoproteomic profiling and transformation potency of the oncogene in different mutants is presented. The theory seeks to predict the leukemic transformation potency from the observed signaling by constructing a distribution of maximal entropy of site-specific phosphorylation events. The theory is developed with special reference to systems biology where high throughput measurements are typical. We seek sets of phosphorylation events most contributory to predicting the phenotype by determining the constraints on the signaling system. The relevance of a constraint is measured by how much it reduces the value of the entropy from its global maximum, where all events are equally likely. Application to experimental phospho-proteomics data for kinase inhibitor-resistant mutants shows that there is one dominant constraint and that other constraints are not relevant to a similar extent. This single constraint accounts for much of the correlation of phosphorylation events with the oncogenic potency and thereby usefully predicts the trends in the phenotypic output. An additional constraint possibly accounts for biological fine structure.

Bioinformatic identification of potential autocrine signaling loops in cancers from gene expression profiles.

Many biological signaling pathways involve autocrine ligand-receptor loops; misregulation of these signaling loops can contribute to cancer phenotypes. Here we present an algorithm for detecting such loops from gene expression profiles. Our method is based on the hypothesis that for some autocrine pathways, the ligand and receptor are regulated by coupled mechanisms at the level of transcription, and thus ligand-receptor pairs comprising such a loop should have correlated mRNA expression. Using our database of experimentally known ligand-receptor signaling partners, we found examples of ligand-receptor pairs with significantly correlated expression in five cancer-based gene expression datasets. The correlated ligand-receptor pairs we identified are consistent with known autocrine signaling events in cancer cells. In addition, our algorithm predicts new autocrine signaling loops that can be verified experimentally. Chemokines were commonly members of these potential autocrine pathways. Our analysis also revealed ligand-receptor pairs with expression patterns that may indicate cellular mechanisms for preventing autocrine signaling.

Rapid gene repression triggered by interleukin-6 at the onset of monocyte differentiation.

To date, the majority of characterized extracellular ligand-induced rapid changes in gene expression involve upregulation. Hence, rapid gene repression is either less common or less well studied. To study rapid gene repression during cytokine-initiated differentiation programs, we used the mRNA subtractive hybridization technique of representational difference analysis to isolate repressed genes. Cultures of the myeloid leukemia cell line M1 were induced to terminally differentiate by treatment with interleukin-6 (IL-6). The repressed genes identified in our subtraction products include the genes encoding the growth factor receptor Flt3/Flk2/STK-1 (CD135) and the costimulatory protein CD24 [heat-stable antigen] and the c-myb oncogene. Following 4 h of IL-6 treatment, mRNA levels of these genes are decreased by 45-65% relative to controls and after 8 h by 65-80%. Lipopolysaccharide also triggers the repression of these genes. Protein synthesis inhibitors do not block the IL-6-stimulated repression of c-myb, or c-myc, mRNA, yet they do block the repression of flt3 and CD24 mRNA, demonstrating the existence of both protein synthesis-independent and -dependent mechanisms of cytokine-triggered rapid gene repression during differentiation.

Selection of human cervical epithelial cells that possess reduced apoptotic potential to low-oxygen conditions.

Since human papillomavirus (HPV) infection is strongly associated with cervical neoplasia and tumor hypoxia has prognostic significance in human cervical carcinomas, we examined the relationship between hypoxia and apoptosis in human cervical epithelial cells expressing high-risk HPV type 16 oncoproteins. In vitro, hypoxia stimulated both p53 induction and apoptosis in primary cervical epithelial cells infected with the HPV E6 and E7 genes but not in cervical fibroblasts infected with E6 and E7. Furthermore, cell lines derived from HPV-associated human cervical squamous cell carcinomas were substantially less sensitive to apoptosis induced by hypoxia, indicating that these cell lines have acquired additional genetic alterations that reduced their apoptotic sensitivity. Although the process of long-term cell culturing resulted in selection for subpopulations of HPV oncoprotein-expressing cervical epithelial cells with diminished apoptotic potential, the exposure of cells to hypoxia greatly accelerated the selection process. These results provide evidence for the role of hypoxia-mediated selection of cells with diminished apoptotic potential in the progression of human tumors and can in part explain why cervical tumors that possess low pO2 values are more aggressive.

Modulation of c-Myc activity and apoptosis in vivo.

We have developed an animal tumor model system to study the effects of c-Myc activation on apoptosis induction in vivo. Tumors were generated in SCID mice from Rat-1 fibroblasts that constitutively express an inactive c-Myc-estrogen receptor fusion protein (T.D. Littlewood et al, Nucleic Acids Res., 23: 1686 -1690, 1995), which is activated in vivo by the administration of 4-hydroxytamoxifen in time release pellets. We demonstrate that activation of c-Myc results in a substantial increase in the number of apoptotic tumor cells and that this apoptosis is predominant in regions of tumor hypoxia. c-Myc-induced apoptosis of hypoxic cells is inhibited in tumors that overexpress the human Bcl-2 protein. Bcl-2, however, does not prevent p53 protein accumulation or the down-regulation of the cyclin-cdk inhibitor p27 protein following c-Myc activation by 4-hydroxytamoxifen. This result suggests that Bcl-2 does not affect c-Myc function directly but acts downstream of c-Myc to inhibit apoptosis. We propose that the ability of activated c-Myc to enhance cellular proliferation might contribute to the genesis of early neoplasms that are held in check by the alternate ability of c-Myc to induce apoptosis of cells that have outgrown their supply of oxygen or other factors associated with hypoxic regions of solid tumors. Secondary genetic lesions downstream of c-Myc that suppress the apoptotic potential of tumor cells, such as Bcl-2 overexpression, might play an important role in the malignant progression of these tumors because they would disrupt the balance between apoptosis and proliferation initiated by c-Myc deregulation.

Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours.

Apoptosis is a genetically encoded programme of cell death that can be activated under physiological conditions and may be an important safeguard against tumour development. Regions of low oxygen (hypoxia) and necrosis are common features of solid tumours. Here we report that hypoxia induces apoptosis in oncogenically transformed cells and that further genetic alterations, such as loss of the p53 tumour-suppressor gene or overexpression of the apoptosis-inhibitor protein Bcl-2, substantially reduce hypoxia-induced cell death. Hypoxia also selects for cells with defects in apoptosis, because small numbers of transformed cells lacking p53 overtake similar cells expressing wild-type p53 when treated with hypoxia. Furthermore, highly apoptotic regions strongly correlate with hypoxic regions in transplanted tumours expressing wild-type p53, whereas little apoptosis occurs in hypoxic regions of p53-deficient tumours. We propose that hypoxia provides a physiological selective pressure in tumours for the expansion of variants that have lost their apoptotic potential, and in particular for cells acquiring p53 mutations.

Attenuated response of p53 and p21 in primary cultures of human prostatic epithelial cells exposed to DNA-damaging agents.

The multifocal origin of prostate cancer suggests a pan-organ defect in a tumor suppressor pathway. Although structural mutations in the p53 gene have been implicated in late-stage prostate cancer, little is known about the p53 response to genotoxic stress in normal human prostatic epithelial cells from which adenocarcinomas originate. We found that the majority (10 of 12) of epithelial cell cultures derived from histologically normal tissues of radical prostatectomy specimens failed to exhibit p53 accumulation in response to ionizing radiation. Epithelial cell cultures derived from benign prostatic hyperplasia and a primary prostatic adenocarcinoma also failed to accumulate p53 in response to ionizing radiation. In contrast, cultures of prostatic stromal cells derived from normal, benign prostatic hyperplasia, or adenocarcinoma tissues exhibited a 3-9-fold induction of p53 within 1-3 h after irradiation. Since p53 regulates a cell cycle checkpoint through the induction of the cyclin-cdk inhibitor p21, we examined p21 accumulation and cell cycle arrest following exposure to ionizing radiation. With one exception, epithelial cells that did not display increased p53 or p21 induction did not demonstrate a significant G1-S arrest in response to ionizing radiation, whereas stromal cells that accumulated p53 and p21 exhibited a large cell cycle arrest. These results indicate a functional difference between the DNA damage response of epithelial and stromal prostatic cells and suggest a possible mechanism for the increased susceptibility of prostatic epithelial cells to accumulate genetic alterations.

Hypoxia induces accumulation of p53 protein, but activation of a G1-phase checkpoint by low-oxygen conditions is independent of p53 status.

It has been convincingly demonstrated that genotoxic stresses cause the accumulation of the tumor suppressor gene p53. One important consequence of increased p53 protein levels in response to DNA damage is the activation of a G1-phase cell cycle checkpoint. It has also been shown that G1-phase cell cycle checkpoints are activated in response to other stresses, such as lack of oxygen. Here we show that hypoxia and heat, agents that induce cellular stress primarily by inhibiting oxygen-dependent metabolism and denaturing proteins, respectively, also cause an increase in p53 protein levels. The p53 protein induced by heat is localized in the cytoplasm and forms a complex with the heat shock protein hsc70. The increase in nuclear p53 protein levels and DNA-binding activity and the induction of reporter gene constructs containing p53 binding sites following hypoxia occur in cells that are wild type for p53 but not in cells that possess mutant p53. However, unlike ionizing radiation, the accumulation of cells in G1 phase by hypoxia is not strictly dependent on wild-type p53 function. In addition, cells expressing the human papillomavirus E6 gene, which show increased degradation of p53 by ubiquitination and fail to accumulate p53 in response to DNA-damaging agents, do increase their p53 levels following heat and hypoxia. These results suggest that hypoxia is an example of a "nongenotoxic" stress which induces p53 activity by a different pathway than DNA-damaging agents.