Acquisition of Relative Interstrand Crosslinker Resistance and PARP Inhibitor Sensitivity in Fanconi Anemia Head and Neck Cancers

PURPOSE: Fanconi anemia is an inherited disorder associated with a constitutional defect in the Fanconi anemia DNA repair machinery that is essential for resolution of DNA interstrand crosslinks. Individuals with Fanconi anemia are predisposed to formation of head and neck squamous cell carcinomas (HNSCC) at a young age. Prognosis is poor, partly due to patient intolerance of chemotherapy and radiation requiring dose reduction, which may lead to early recurrence of disease. EXPERIMENTAL DESIGN: Using HNSCC cell lines derived from the tumors of patients with Fanconi anemia, and murine HNSCC cell lines derived from the tumors of wild-type and Fancc(-/-) mice, we sought to define Fanconi anemia-dependent chemosensitivity and DNA repair characteristics. We utilized DNA repair reporter assays to explore the preference of Fanconi anemia HNSCC cells for non-homologous end joining (NHEJ). RESULTS: Surprisingly, interstrand crosslinker (ICL) sensitivity was not necessarily Fanconi anemia-dependent in human or murine cell systems. Our results suggest that the increased Ku-dependent NHEJ that is expected in Fanconi anemia cells did not mediate relative ICL resistance. ICL exposure resulted in increased DNA damage sensing and repair by PARP in Fanconi anemia-deficient cells. Moreover, human and murine Fanconi anemia HNSCC cells were sensitive to PARP inhibition, and sensitivity of human cells was attenuated by Fanconi anemia gene complementation. CONCLUSIONS: The observed reliance upon PARP-mediated mechanisms reveals a means by which Fanconi anemia HNSCCs can acquire relative resistance to the ICL-based chemotherapy that is a foundation of HNSCC treatment, as well as a potential target for overcoming chemoresistance in the chemosensitive individual

The Pediatric Cell Atlas:Defining the Growth Phase of Human Development at Single-Cell Resolution

Single-cell gene expression analyses of mammalian tissues have uncovered profound stage-specific molecular regulatory phenomena that have changed the understanding of unique cell types and signaling pathways critical for lineage determination, morphogenesis, and growth. We discuss here the case for a Pediatric Cell Atlas as part of the Human Cell Atlas consortium to provide single-cell profiles and spatial characterization of gene expression across human tissues and organs. Such data will complement adult and developmentally focused HCA projects to provide a rich cytogenomic framework for understanding not only pediatric health and disease but also environmental and genetic impacts across the human lifespan

Pleuropulmonary Blastoma: Evolution of an Entity as an Entry into a Familial Tumor Predisposition Syndrome.

Pleuropulmonary blastomas (PPB) is the most common primary malignant neoplasm of the lung in children. Like the other solid dysontogenic neoplasms, this tumor typically presents before the age of seven years. The earliest manifestation is the presence of a lung cyst(s) which is usually recognized in the first year of life and is difficult on the basis of imaging studies to differentiate it from non-neoplastic cysts of early childhood. From a multilocular cyst, PPB has the potential to progress to a high grade multipatterned primitive sarcoma. Over 65% of all affected children have a heterozygous germline mutation in DICER1. The DICER1-PPB familial tumor predisposition syndrome is initially recognized in most cases on the basis of PPB alone, but also several other unique and characteristic extrapulmonary tumors including pediatric cystic nephroma, nasal chondromesenchymal hamartoma, nodular lesions of the thyroid, embryonal rhabdomyosarcoma of the cervix and ciliary body medulloepithelioma

The DEK Oncogene Is a Target of Steroid Hormone Receptor Signaling in Breast Cancer

<div><p>Expression of estrogen and progesterone hormone receptors indicates a favorable prognosis due to the successful use of hormonal therapies such as tamoxifen and aromatase inhibitors. Unfortunately, 15–20% of patients will experience breast cancer recurrence despite continued use of tamoxifen. Drug resistance to hormonal therapies is of great clinical concern so it is imperative to identify novel molecular factors that contribute to tumorigenesis in hormone receptor positive cancers and/or mediate drug sensitivity. The hope is that targeted therapies, in combination with hormonal therapies, will improve survival and prevent recurrence. We have previously shown that the DEK oncogene, which is a chromatin remodeling protein, supports breast cancer cell proliferation, invasion and the maintenance of the breast cancer stem cell population. In this report, we demonstrate that DEK expression is associated with positive hormone receptor status in primary breast cancers and is up-regulated <em>in vitro</em> following exposure to the hormones estrogen, progesterone, and androgen. Chromatin immunoprecipitation experiments identify <em>DEK</em> as a novel estrogen receptor α (ERα) target gene whose expression promotes estrogen-induced proliferation. Finally, we report for the first time that DEK depletion enhances tamoxifen-induced cell death in ER+ breast cancer cell lines. Together, our data suggest that DEK promotes the pathogenesis of ER+ breast cancer and that the targeted inhibition of DEK may enhance the efficacy of conventional hormone therapies.</p> </div

Association of DEK expression with clinical and pathological variables in invasive adenocarcinomas of the breast.

*<p>Chi-Squared statistic.</p

Model for DEK transcriptional up-regulation following 17β-estradiol exposure.

<p>Upon 17β-estradiol exposure, ERα is activated and binds to the <i>DEK</i> promoter at least at two locations – an ERE half site at −716 bp and at ERα/Sp1 binding sites more proximal to the transcriptional start site. A second potential mechanism of up-regulation is the ERα-mediated up-regulation of E2F proteins (particularly E2F3) that also increase <i>DEK</i> transcription. Increased levels of DEK then promote proliferation. DEK expression can be targeted with the anti-estrogen tamoxifen to inhibit cell proliferation. The knockdown of DEK by RNAi can increase tamoxifen sensitivity of ER+ cell lines by synergistically inducing an apoptotic response.</p

DEK is necessary for 17β-estradiol stimulated cell proliferation and modulates sensitivity to tamoxifen.

<p>(A) DEK expression is required for 17β-estradiol stimulated cellular proliferation. Hormone starved MCF7 cells transduced with non-targeting shRNA (NTsh) or DEK shRNA (DEKsh2) were untreated (CS-FBS) or exposed to 10 nM 17β-estradiol, then cultured in BrdU. The percentage of BrdU positive cells was determined by flow cytometry. Asterisk (*) denotes p<0.05 using Student’s t-test. (B and C) DEK depletion by shRNA (DEKsh2) works synergistically with tamoxifen to induce apoptosis in breast cancer cell lines. (B) Bright field images (100× magnification) of MCF7 cells expressing either NTsh or DEKsh2 were cultured in low serum media and either untreated or treated with tamoxifen for 18 hours. (C) DEK depletion by shRNA (DEKsh2) enhances the cytotoxic effect of tamoxifen. DEK proficient and deficient MCF7 (left) and T47D (right) cells were grown in low serum media then treated with 3 µg/ml tamoxifen for 22 hours. Cells were labeled with 7AAD then analyzed for sub-G1 content by flow cytometry as a measure of apoptosis. Results shown are the average of triplicate experiments. Two asterisks (**) indicate p<0.01 as determined using a 2-way ANOVA test for significance. For MCF7 cells, p = 0.08. (A and B insets) DEK shRNA knockdown is shown by western blot analysis for normally cultured cells that were transduced with lentivirus carrying either non-targeting shRNA (NTsh) or DEK specific shRNA (DEKsh2).</p

DEK expression is associated with positive hormone receptor status in human primary and cultured breast cancers.

<p>(A) Estrogen receptor (ER) negative tumors were often negative for DEK staining (right) while ER positive tumors were often positive for DEK staining (left). Immunohistochemical staining for DEK in two invasive ductal carcincomas showing positive DEK staining (DEK+) in a hormone receptor positive (ER+/PR+) tumor and lack of DEK expression (DEK-) in a hormone receptor negative tumor (ER−/PR−). Low power images are at 200× and all high power images are at 1000× magnification. (B) Western blotting for DEK showed increased expression following exposure to 17β-estradiol for 48 hours in ER+ MCF7 and T47D cells but not in ER- BT20 cells. (C) Cell morphology of T47D cells in CS-FBS (left), 48 hours of 17β-estradiol treatment (middle), and under normal culture conditions (right). Bright field images of cultured cells were obtained at 100× total magnification. (D) <i>DEK</i> expression increases in 17β-estradiol and R1881 treated cells. Quantitative RT-PCR was performed to detect <i>DEK</i> expression in hormone starved MCF7 cells treated with 10 nM 17β-estradiol or 1 µM methyltrienolone (R1881) for six hours. <i>GAPDH</i> was used as a control and values are normalized to the untreated sample. (E) Western blotting for DEK shows increased protein levels after treatment of hormone starved MCF7 cells with 10 nM 17β-estradiol, 10 nM progesterone, or 1 µM methyltrienolone (R1881) over the course of 48 hours.</p