Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic-helix-loop-helix-PAS transcription factor consisting of HIF-1 alpha and HIF-1 beta subunits. HIF-1 alpha expression and HIF-1 transcriptional activity increase exponentially as cellular O2 concentration is decreased. Several dozen target genes that are transactivated by HIF-1 have been identified, including those encoding erythropoietin, glucose transporters, glycolytic enzymes, and vascular endothelial growth factor. The products of these genes either increase O2 delivery or allow metabolic adaptation to reduced O2 availability. HIF-1 is required for cardiac and vascular development and embryonic survival. In fetal and postnatal life, HIF-1 is required for a variety of physiological responses to chronic hypoxia. HIF-1 expression is increased in tumor cells by multiple mechanisms and may mediate adaptation to hypoxia that is critical for tumor progression. HIF-1 thus appears to function as a master regulator of O2 homeostasis that plays essential roles in cellular and systemic physiology, development, and pathophysiology.

Regulation of mammalian o2 homeostasis by hypoxia-inducible factor 1

The placenta is the first organ to form during mammalian embryogenesis. Problems in its formation and function underlie many aspects of early pregnancy loss and pregnancy complications in humans. Because the placenta is critical for survival, it is very sensitive to genetic disruption, as reflected by the ever-increasing list of targeted mouse mutations that cause placental defects. Recent studies of mouse mutants with disrupted placental development indicate that signalling interactions between the placental trophoblast and embryonic cells have a key role in placental morphogenesis. Furthering our understanding of mouse trophoblast development should provide novel insights into human placental function.

Placental development: Lessons from mouse mutants

Hypoxia-inducible factor 1: master regulator of O2 homeostasis

Over the past decade, PAS domains have been identified in dozens of signal transduction molecules and various forms have been found in animals, plants, and prokaryotes. In this review, we summarize this rapidly expanding research area by providing a detailed description of three signal transduction pathways that utilize PAS protein heterodimers to drive their transcriptional output. It is hoped that these model pathways can provide a framework for use in understanding the biology of the less well-understood members of this emerging superfamily, as well as of those to be characterized in the days to come. We use this review to develop the idea that most eukaryotic PAS proteins can be classified by functional similarities, as well as by predicted phylogenetic relationships. We focus on the a-class proteins, which often act as sensors of environmental signals, and the b-class proteins, which typically act as broad-spectrum partners that target these heterodimers to their genomic targets.

The PAS Superfamily: Sensors of Environmental and Developmental Signals

Early detection remains the most promising approach to improve long-term survival of patients with ovarian cancer. In a five-center case-control study, serum proteomic expressions were analyzed on 153 patients with invasive epithelial ovarian cancer, 42 with other ovarian cancers, 166 with benign pelvic masses, and 142 healthy women. Data from patients with early stage ovarian cancer and healthy women at two centers were analyzed independently and the results cross-validated to discover potential biomarkers. The results were validated using the samples from two of the remaining centers. After protein identification, biomarkers for which an immunoassay was available were tested on samples from the fifth center, which included 41 healthy women, 41 patients with ovarian cancer, and 20 each with breast, colon, and prostate cancers. Three biomarkers were identified as follows: (a) apolipoprotein A1 (down-regulated in cancer); (b) a truncated form of transthyretin (down-regulated); and (c) a cleavage fragment of inter-alpha-trypsin inhibitor heavy chain H4 (up-regulated). In independent validation to detect early stage invasive epithelial ovarian cancer from healthy controls, the sensitivity of a multivariate model combining the three biomarkers and CA125 [74% (95% CI, 52-90%)] was higher than that of CA125 alone [65% (95% CI, 43-84%)] at a matched specificity of 97% (95% CI, 89-100%). When compared at a fixed sensitivity of 83% (95% CI, 61-95%), the specificity of the model [94% (95% CI, 85-98%)] was significantly better than that of CA125 alone [52% (95% CI, 39-65%)]. These biomarkers demonstrated the potential to improve the detection of early stage ovarian cancer.

https://cancerres.aacrjournals.org/content/canres/64/16/5882.full.pdf

Three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer

ARNT-deficient mice and placental differentiation.

We have previously reported the identification of three ovarian cancer biomarker panels comprised of SELDI-TOF-MS peaks representing 14 differentially expressed serum proteins for the diagnosis of ovarian cancer. Using micro-LC-MS/MS, we identified five m/z peaks as transthyretin (TTR 13.9 kDa, TTR fragment 12.9 kDa), beta-hemoglobin (Hb, 15.9 kDa), apolipoprotein AI (ApoAI, 28 kDa) and transferrin (TF, 79 kDa). Western and/or ELISA methods confirmed the differential expression of TTR, Hb, and TF, and multivariate analyses resulted in improving the detection of early stage ovarian tumors (low malignant potential and malignant; receiver operating characteristic, ROC 0.933) as compared to cancer antigen CA125 alone (ROC 0.833). Interestingly, when CA125 was included with our markers in the multivariate analysis, the ROC increased to 0.959. Furthermore, multivariate analysis with only the mucinous subtype of early stage ovarian tumors, showed our markers to greatly improve the detection of disease (ROC 0.959) as compared to CA125 alone (ROC 0.613). Interestingly, the combination of CA125 with our markers did not seem to further improve the detection of mucinous tumors (ROC 0.955). We conclude that TTR, Hb, ApoAI and TF, when combined with CA125 should significantly improve the detection of early stage ovarian cancer.

Characterization of serum biomarkers for detection of early stage ovarian cancer.

One hundred eighty-four serum samples from patients with ovarian cancer (n = 109), patients with benign tumors (n = 19), and healthy donors (n = 56) were analyzed on strong anion-exchange surfaces using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry technology. Univariate and multivariate statistical analyses applied to protein-profiling data obtained from 140 training serum samples identified three biomarker protein panels. The first panel of five candidate protein biomarkers, termed the screening biomarker panel, effectively diagnosed benign and malignant ovarian neoplasia [95.7% sensitivity, 82.6% specificity, 89.2% accuracy, and receiver operating characteristic (ROC) area under the curve of 0.94]. The other two panels, consisting of five and four candidate protein biomarkers each, effectively distinguished between benign and malignant ovarian neoplasia and were therefore referred to as validation biomarker panel I (81.5% sensitivity, 94.9% specificity, 88.2% accuracy, and ROC = 0.94) and validation biomarker panel II (72.8% sensitivity, 94.9% specificity, 83.9% accuracy, and ROC = 0.90). The three ovarian cancer biomarker protein panels correctly diagnosed 41 of the 44 blinded test samples: 21 of 22 malignant ovarian neoplasias [10 of 11 early-stage ovarian cancer (I/II) and 11 of 11 advanced-stage ovarian cancer (III/IV)], 6 of 6 low malignant potential, 5 of the 6 benign tumors, and 9 of 10 normal patient samples. In conclusion, we have discovered three ovarian cancer biomarker protein panels that, when used together, effectively distinguished serum samples from healthy controls and patients with either benign or malignant ovarian neoplasia.

Identification of biomarkers for ovarian cancer using strong anion-exchange ProteinChips: Potential use in diagnosis and prognosis

Endometrial adenocarcinoma is the most common gynecologic malignancy in the United States. However, reliable diagnostic or prognostic tumor markers have not been identified for endometrial cancer. In this study, we examined whether urokinase plasminogen activator receptor (UPAR), a glycosyl-phosphatidylinositol-linked membrane protein, is a candidate diagnostic or prognostic marker for patients with cancer of the endometrium. Sixty-five surgically excised, formalin-fixed endometrial tissue specimens were accessioned through the Department of Pathology Registry at the University of California, Los Angeles, and analyzed for UPAR expression by using immunohistochemical techniques. A retrospective review was also performed to determine stage and histopathologic grade of disease, recurrence, and mortality. No expression of UPAR protein was present in seven patients with benign neoplasia of the endometrium. UPAR protein expression highly correlated with stage of disease (ungrouped Spearman correlation = 0.625, P < 0.0001): 40% of patients with stage I, 66% of patients with stage II, 100% of patients with stage III, and 85% with stage IV demonstrated the highest level of UPAR expression. Moreover, high UPAR expression positively correlated with grade of disease (ungrouped Spearman correlation = 0.71, P < 0.0001): 29% of grade 1 specimens, 57% of grade 2, and over 90% of specimens with grade 3, the majority representing uterine papillary serous carcinoma and mixed malignant mesodermal tumor. Finally, UPAR protein expression also positively correlated with rate of recurrence and mortality in patients with adenocarcinoma of the endometrium (ungrouped P = 0.034). Our data suggest that UPAR is a useful prognostic marker for biologically aggressive forms of endometrial cancer.

https://www.pnas.org/content/pnas/99/16/10647.full.pdf

Urokinase plasminogen activator receptor: Prognostic biomarker for endometrial cancer

Rat PC12 pheochromocytoma cells treated with nerve growth factor (NGF) extend "neurites" and initiate a neuronal differentiation pathway. Although neurotrophins, growth factors [e.g., epidermal growth factor (EGF)], and other ligands induce many common primary response genes (PRGs) in PC12 cells, a unique PRG subset is induced preferentially by NGF. Expression of one NGF preferentially induced gene, urokinase plasminogen activator receptor (UPAR), is required for NGF-induced neurite extension and neuronal differentiation. A 2.1-kb fragment of the rat UPAR 5' regulatory region confers differential expression by NGF versus EGF, following transfection of a luciferase reporter construct into PC12 cells. Deletion studies identified a region between -100 and -50 nucleotides from the transcription start site as the region conferring preferential NGF induction. Sequence comparisons among rat, human, and murine UPAR promoters identified two common potential regulatory regions. Site-directed mutation identified an activator protein-1 (AP-1) region between -66 and -72 bp, required for luciferase reporter activation by NGF. Electrophoretic mobility shift and antibody supershift assays demonstrated that specific Fos and Jun family members preferentially bind to this site following NGF treatment. We conclude that preferential activation of transcription factor binding at this AP-1 site mediates preferential NGF activation of the UPAR gene.

Katherine R. Kozak

Papers

ARNT-deficient mice and placental differentiation.

Characterization of serum biomarkers for detection of early stage ovarian cancer.

Identification of biomarkers for ovarian cancer using strong anion-exchange ProteinChips: Potential use in diagnosis and prognosis

Urokinase plasminogen activator receptor: Prognostic biomarker for endometrial cancer

Characterization of the rat urokinase plasminogen activator receptor promoter in PC12 cells.