Ilaria Stefania Pagani

OTX1 expression in breast cancer is regulated by p53

The p53 transcription factor has a critical role in cell stress response and in tumor suppression. Wild-type p53 protein is a growth modulator and its inactivation is a critical event in malignant transformation. It has been recently demonstrated that wild-type p53 has developmental and differentiation functions. Indeed an over-expression of p53 in tumor cells induces asymmetrical division avoiding self-renewal of cancer stem cells (CSCs) and instead promoting their differentiation. In this study, 28 human breast carcinomas have been analyzed for expression of wild-type p53 and of a pool of non-clustered homeobox genes. We demonstrated that orthodenticle homolog 1 gene (OTX1) is transcribed in breast cancer. We established that the p53 protein directly induces OTX1 expression by acting on its promoter. OTX1 has been described as a critical molecule for axon refinement in the developing cerebral cortex of mice, and its activity in breast cancer suggests a synergistic function with p53 in CSC differentiation. Wild-type p53 may regulate cellular differentiation by an alternative pathway controlling OTX1 signaling only in breast cancer cells and not in physiological conditions.

Expression of VEGF-A, Otx homeobox and p53 family genes in proliferative vitreoretinopathy.

Introduction. Proliferative vitreoretinopathy (PVR) is a severe inflammatory complication of retinal detachment. Pathological epiretinal membranes grow on the retina surface leading to contraction, and surgery fails in 5% to 10% of the cases. We evaluated the expression of VEGF-A, Otx1, Otx2, Otx3, and p53 family members from PVR specimens to correlate their role in inducing or preventing the pathology. Methods. Twelve retinal samples were taken from patients affected by PVR during therapeutic retinectomies in vitreoretinal surgery. Gene expression was evaluated using quantitative real-time reverse transcriptase PCR analysis and immunohistochemistry, using four healthy human retinae as control. Result. Controls showed basal expression of all genes. PVR samples showed little or no expression of Otx1 and variable expression of VEGF-A, Otx2, Otx3, p53, and p63 genes. Significant correlation was found among VEGF-A, Otx2, p53, and p63 and between Otx1 and Otx3. Conclusions. Otx homeobox, p53 family, and VEGF-A genes are expressed in PVR human retina. We individuated two possible pathways (VEGF-A, Otx2, p53, p63 and Otx1 and Otx3) involved in PVR progression that could influence in different manners the course of the pathology. Individuating the genetic pathways of PVR represents a novel approach to PVR therapies.

The mammary gland and the homeobox gene Otx1

Abstract:  The mammary gland, the unique organ that primarily form at puberty, is an ideal model to study the functions of homeobox (HB) genes in both development and tumorigenesis. HB genes comprise a large family of developmental regulators that have a critical role in cell growth and differentiation. In the normal mammary gland, homeobox genes are involved in ductal formation, epithelial branching, and lobulo‐alveolar development by regulating epithelial proliferation and differentiation. The HB genes are controlled in a spatial and temporal manner in both stromal and epithelial cells. They are coordinately regulated by hormones and extracellular matrix, suggesting that many signaling pathways are involved in homeobox gene functions. When homeobox genes are misexpressed in animal models, different defects are displayed in mammary gland development. Aberrant expression of homeobox genes, overexpressed or downregulated, is found in primary carcinomas and in breast cancer. The Otx1 HB gene is a classic regulatory of nervous system development during embryogenesis. Postnatally Otx1 is transcribed in the anterior pituitary gland, where activates transcription of the pituitary hormones, and plays a role in hematopoiesis, enhancing pluripotent cells, and erythroid differentiation. Otx1 can still be detected in mature cells of the erythroid and megacaryocytic lineage. During cyclical development of mammary gland, the Otx1 gene is overexpressed in lactation, confirming a role of this transcription factor in cell differentiation. Recent studies report that Otx1 is overexpressed in breast cancer. Otx1 is expressed during embryogenesis, and it is expressed again during carcinogenesis, implying its possible function in differentiation of neoplastic cells.

gDNA Q-PCR for clinical monitoring of CML

Therapy with tyrosine kinase inhibitors (TKIs) is sufficient to prevent the progression to advanced chronic myeloid leukemia (CML) and inhibit recurrence. This has revolutionized the management of CML; but to maintain such “operational cure,” the Imatinib Mesylate (IM) is required indefinitely, incurring considerable cost and side effects due to the therapy.1

BCR-ABL1 genomic DNA PCR response kinetics during first-line imatinib treatment of chronic myeloid leukemia.

Accurate quantification of minimal residual disease (MRD) during treatment of chronic myeloid leukemia (CML) guides clinical decisions. The conventional MRD method, RQ-PCR for BCR-ABL1 mRNA, reflects a composite of the number of circulating leukemic cells and the BCR-ABL1 transcripts per cell. BCR-ABL1 genomic DNA only reflects leukemic cell number. We used both methods in parallel to determine the relative contribution of the leukemic cell number to molecular response. BCR-ABL1 DNA PCR and RQ-PCR were monitored up to 24 months in 516 paired samples from 59 newly-diagnosed patients treated with first-line imatinib in the TIDEL-II study. In the first three months of treatment, BCR-ABL1 mRNA values declined more rapidly than DNA. By six months, the two measures aligned closely. The expression of BCR-ABL1 mRNA was normalized to cell number to generate an expression ratio. The expression of e13a2 BCR-ABL1 was lower than that of e14a2 transcripts at multiple time points during treatment. BCR-ABL1 DNA was quantifiable in 48% of samples with undetectable BCR-ABL1 mRNA, resulting in MRD being quantifiable for an additional 5-18 months (median 12 months). These parallel studies show for the first time that the rapid decline in BCR-ABL1 mRNA over the first three months of treatment is due to a reduction in both cell number and transcript level per cell, whereas beyond three months, falling levels of BCR-ABL1 mRNA are proportional to the depletion of leukemic cells.

A Method for Next-Generation Sequencing of Paired Diagnostic and Remission Samples to Detect Mitochondrial DNA Mutations Associated with Leukemia

Somatic mitochondrial DNA (mtDNA) mutations have been identified in many human cancers, including leukemia. To identify somatic mutations, it is necessary to have a control tissue from the same individual for comparison. When patients with leukemia achieve remission, the remission peripheral blood may be a suitable and easily accessible control tissue, but this approach has not previously been applied to the study of mtDNA mutations. We have developed and validated a next-generation sequencing approach for the identification of leukemia-associated mtDNA mutations in 26 chronic myeloid leukemia patients at diagnosis using either nonhematopoietic or remission blood samples as the control. The entire mt genome was amplified by long-range PCR and sequenced using Illumina technology. Variant caller software was used to detect mtDNA somatic mutations, and an empirically determined threshold of 2% was applied to minimize false-positive results because of sequencing errors. Mutations were called against both nonhematopoietic and remission controls: the overall concordance between the two approaches was 81% (73/90 mutations). Some discordant results were because of the presence of somatic mutations in remission samples, because of either minimal residual disease or nonleukemic hematopoietic clones. This method could be applied to study somatic mtDNA mutations in leukemia patients who achieve minimal residual disease, and in patients with nonhematopoietic cancers who have a matched uninvolved tissue available.

Erratum: OTX1 expression in breast cancer is regulated by p53

Correction to: Oncogene (2011) 30, 3096-3103; doi:10.1038/onc.2011.31; published online 11 April 2011 Since the publication of the above manuscript, the authors have identified an error in the author list; the name of the fourteenth author was incorrectly presented. The corrected author list is shown above.

Erratum: The mammary gland and the homeobox gene otx1. (Breast Journal (2010) 16:1 (S53-S56))

Pagani IS, Terrinoni A, et al. The mammary gland and the homeobox gene otx1. Breast J 2010;16(suppl 1): S53–S56. Two authors’ names were misspelled in the above article. The correct authors’ names are: Ilaria S. Pagani, PhD, Alessandro Terrinoni, PhD, Laura Marenghi, PhD, Ileana Zucchi, PhD, Anna M. Chiaravalli, PhD, Valeria Serra, PhD, Francesca Rovera, MD, Silvia Sirchia, PhD, Gianlorenzo Dionigi, MD, Monica Miozzo, MD, Annalisa Frattini, PhD, Alberta Ferrari, MD, Carlo Capella, MD, Francesco Pasquali, MD, Francesco Lo Curto, PhD, Alberto Albertini, MD, Gerry Melino, MD, and Giovanni Porta, MD