Ashley M. Holder

Genetic Factors in Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) is a relatively common birth defect associated with high mortality and morbidity. Although the exact etiology of most cases of CDH remains unknown, there is a growing body of evidence that genetic factors play an important role in the development of CDH. In this review, we examine key findings that are likely to form the basis for future research in this field. Specific topics include a short overview of normal and abnormal diaphragm development, a discussion of syndromic forms of CDH, a detailed review of chromosomal regions recurrently altered in CDH, a description of the retinoid hypothesis of CDH, and evidence of the roles of specific genes in the development of CDH.

Chromosome 8p23.1 deletions as a cause of complex congenital heart defects and diaphragmatic hernia.

Recurrent interstitial deletion of a region of 8p23.1 flanked by the low copy repeats 8p‐OR‐REPD and 8p‐OR‐REPP is associated with a spectrum of anomalies that can include congenital heart malformations and congenital diaphragmatic hernia (CDH). Haploinsufficiency of GATA4 is thought to play a critical role in the development of these birth defects. We describe two individuals and a monozygotic twin pair discordant for anterior CDH all of whom have complex congenital heart defects caused by this recurrent interstitial deletion as demonstrated by array comparative genomic hybridization. To better define the genotype/phenotype relationships associated with alterations of genes on 8p23.1, we review the spectrum of congenital heart and diaphragmatic defects that have been reported in individuals with isolated GATA4 mutations and interstitial, terminal, and complex chromosomal rearrangements involving the 8p23.1 region. Our findings allow us to clearly define the CDH minimal deleted region on chromosome 8p23.1 and suggest that haploinsufficiency of other genes, in addition to GATA4, may play a role in the severe cardiac and diaphragmatic defects associated with 8p23.1 deletions. These findings also underscore the importance of conducting a careful cytogenetic/molecular analysis of the 8p23.1 region in all prenatal and postnatal cases involving congenital defects of the heart and/or diaphragm.

Genomic alterations that contribute to the development of isolated and non-isolated congenital diaphragmatic hernia

Background Congenital diaphragmatic hernia (CDH) is a life threatening birth defect. Most of the genetic factors that contribute to the development of CDH remain unidentified. Objective To identify genomic alterations that contribute to the development of diaphragmatic defects. Methods A cohort of 45 unrelated patients with CDH or diaphragmatic eventrations was screened for genomic alterations by array comparative genomic hybridisation or single nucleotide polymorphism based copy number analysis. Results Genomic alterations that were likely to have contributed to the development of CDH were identified in 8 patients. Inherited deletions of ZFPM2 were identified in 2 patients with isolated diaphragmatic defects and a large de novo 8q deletion overlapping the same gene was found in a patient with non-isolated CDH. A de novo microdeletion of chromosome 1q41q42 and two de novo microdeletions on chromosome 16p11.2 were identified in patients with non-isolated CDH. Duplications of distal 11q and proximal 13q were found in a patient with non-isolated CDH and a de novo single gene deletion of FZD2 was identified in a patient with a partial pentalogy of Cantrell phenotype. Conclusions Haploinsufficiency of ZFPM2 can cause dominantly inherited isolated diaphragmatic defects with incomplete penetrance. These data define a new minimal deleted region for CDH on 1q41q42, provide evidence for the existence of CDH related genes on chromosomes 16p11.2, 11q23-24 and 13q12, and suggest a possible role for FZD2 and Wnt signalling in pentalogy of Cantrell phenotypes. These results demonstrate the clinical utility of screening for genomic alterations in individuals with both isolated and non-isolated diaphragmatic defects.

Targeting the PI3-Kinase/Akt/mTOR Signaling Pathway

This article presents an overview of the PI3K/Akt/mTOR signaling pathway. As a central regulator of cell growth, protein translation, survival, and metabolism, activation of this signaling pathway contributes to the pathogenesis of many tumor types. Biochemical and genetic aberrations of this pathway observed in various cancer types are explored. Last, pathway inhibitors both in development and already approved by the Food and Drug Administration are discussed.

Mouse model reveals the role of SOX7 in the development of congenital diaphragmatic hernia associated with recurrent deletions of 8p23.1

Recurrent microdeletions of 8p23.1 that include GATA4 and SOX7 confer a high risk of both congenital diaphragmatic hernia (CDH) and cardiac defects. Although GATA4-deficient mice have both CDH and cardiac defects, no humans with cardiac defects attributed to GATA4 mutations have been reported to have CDH. We were also unable to identify deleterious GATA4 sequence changes in a CDH cohort. This suggested that haploinsufficiency of another 8p23.1 gene may contribute, along with GATA4, to the development of CDH. To determine if haploinsufficiency of SOX7-another transcription factor encoding gene-contributes to the development of CDH, we generated mice with a deletion of the second exon of Sox7. A portion of these Sox7(Δex2/+) mice developed retrosternal diaphragmatic hernias located in the anterior muscular portion of the diaphragm. Anterior CDH is also seen in Gata4(+/-) mice and has been described in association with 8p23.1 deletions in humans. Immunohistochemistry revealed that SOX7 is expressed in the vascular endothelial cells of the developing diaphragm and may be weakly expressed in some diaphragmatic muscle cells. Sox7(Δex2/Δex2) embryos die prior to diaphragm development with dilated pericardial sacs and failure of yolk sac remodeling suggestive of cardiovascular failure. Similar to our experience screening GATA4, no clearly deleterious SOX7 sequence changes were identified in our CDH cohort. We conclude that haploinsufficiency of Sox7 or Gata4 is sufficient to produce anterior CDH in mice and that haploinsufficiency of SOX7 and GATA4 may each contribute to the development of CDH in individuals with 8p23.1 deletions.

Ability to Generate Patient-Derived Breast Cancer Xenografts Is Enhanced in Chemoresistant Disease and Predicts Poor Patient Outcomes.

Background Breast cancer patients who are resistant to neoadjuvant chemotherapy (NeoCT) have a poor prognosis. There is a pressing need to develop in vivo models of chemo resistant tumors to test novel therapeutics. We hypothesized that patient-derived breast cancer xenografts (BCXs) from chemo- naïve and chemotherapy-exposed tumors can provide high fidelity in vivo models for chemoresistant breast cancers. Methods Patient tumors and BCXs were characterized with short tandem repeat DNA fingerprinting, reverse phase protein arrays, molecular inversion probe arrays, and next generation sequencing. Results Forty-eight breast cancers (24 post-chemotherapy, 24 chemo-naïve) were implanted and 13 BCXs were established (27%). BCX engraftment was higher in TNBC compared to hormone-receptor positive cancer (53.8% vs. 15.6%, p = 0.02), in tumors from patients who received NeoCT (41.7% vs. 8.3%, p = 0.02), and in patients who had progressive disease on NeoCT (85.7% vs. 29.4%, p = 0.02). Twelve patients developed metastases after surgery; in five, BCXs developed before distant relapse. Patients whose tumors developed BCXs had a lower recurrence-free survival (p = 0.015) and overall survival (p<0.001). Genomic losses and gains could be detected in the BCX, and three models demonstrated a transformation to induce mouse tumors. However, overall, somatic mutation profiles including potential drivers were maintained upon implantation and serial passaging. One BCX model was cultured in vitro and re-implanted, maintaining its genomic profile. Conclusions BCXs can be established from clinically aggressive breast cancers, especially in TNBC patients with poor response to NeoCT. Future studies will determine the potential of in vivo models for identification of genotype-phenotype correlations and individualization of treatment.

Fine–Lubinsky syndrome: Sibling pair suggests possible autosomal recessive inheritance†

In 1983, Fine and Lubinsky reported a single patient with craniofacial anomalies, hearing loss, cataracts, microstomia, CNS anomalies, and developmental delay. Since that time, three additional patients with similar findings have been described. In each case the patients was the only affected child in his or her family. Here we describe the first brother and sister sibling pair with features suggestive of Fine–Lubinsky syndrome. Features present in one or both of our patients, and the majority of previously described individuals with Fine–Lubinsky syndrome, include: craniosynostosis/abnormal calvaria, prominent frontal bones, flat facial profiles, small noses, microstomia, hearing loss, developmental delay/mental retardation, and abnormal digits. Unusual anomalies present in our patients include marked brachydactyly of fingers and toes, camptodactyly most severely affecting the second fingers, and permanence of decidual teeth. The identification of a sibling pair with Fine–Lubinsky suggests a possible autosomal recessive inheritance pattern. It is important that parents of children with Fine–Lubinsky be informed of the increased recurrence risk associated with that type of inheritance.

Abstract 1186: Patient-derived breast cancer xenografts demonstrate molecular evolution in the phosphatidylinositol 3-kinase pathway upon engraftment

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Patients with breast cancer who do not respond to standard systemic therapy have a poor prognosis. There is a pressing need to develop in vivo models of breast cancer to test novel therapeutics. Directly implanting tumors into immunodeficient mice may more accurately recapitulate human tumor characteristics compared to cell line xenografts. We tested the feasibility of generating breast cancer xenografts (BCXs) and the effect of serial passage on tumor characteristics. Methods: Tumors from 48 patients with breast cancer were implanted. Patient tumors and BCXs were characterized with short tandem repeat DNA fingerprinting, and a series of BCXs were characterized with next generation sequencing, molecular inversion probe arrays and reverse phase protein arrays. Results: BCXs were established in 15 (31%) of 48 patients and 13 have been serially passaged. BCX engraftment was higher in patients with triple negative compared to ER or PR+ breast cancer (11 of 18, 61% vs. 4 of 30, 13%, p=0.001), and in patients who received neoadjuvant chemotherapy (13 of 25, 52% vs. 2 of 23, 9%, p=0.002). 7 patients developed metastases after surgery; in 5, BCXs developed before distant relapse. Compared to patient tumors, BCXs demonstrated genomic instability. Although mutation status, copy numbers and proteomic profiles were often maintained, an activating PIK3CA mutation was acquired in one BCX lineage and loss of PTEN in another. Proteomics demonstrated activation in PI3K/mTOR signaling in BCXs compared with patient tumors. Notably, 2 of 48 models demonstrated a transformation to induce mouse tumors. Conclusions: BCXs can be established prior to relapse from breast cancer, especially in patients with poor response to neoadjuvant chemotherapy. Although molecular profiles of BCX are mostly similar to the patient tumor that was implanted, differences such as aberrations in PI3K signaling can occur. Future studies will determine whether molecular evolution in BCXs reflect that seen upon progression/relapse and the potential of in vivo models for individualization of treatment. Citation Format: Priscilla F. McAuliffe, Argun Akcakanat, Kurt Evans, Agda Karina Eterovic, Hao Zhao, Ken Chen, Takafumi Sangai, Huiqin Chen, Kim-Anh Do, Ashley M. Holder, Chandeshwar Sharma, William Fraser Symmans, Mihai Gagea, Katherine A. Naff, Aysegul Sahin, Asha S. Multani, Gordon B. Mills, Ana Maria Gonzalez-Angulo, Funda Meric-Bernstam. Patient-derived breast cancer xenografts demonstrate molecular evolution in the phosphatidylinositol 3-kinase pathway upon engraftment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1186. doi:10.1158/1538-7445.AM2014-1186

Abstract 1496: Epithelial to mesenchymal transition and rapamycin sensitivity in cancer cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Activation of the mTOR signaling pathway is common in multiple cancers. Rapamycin has inhibitory effects on solid tumors through inhibition of the mTOR kinase and alteration of downstream signaling. Though rapamycin and its analogs have yielded promising results from in vitro studies in sensitive cell lines, they have generated only a modest effect in clinical application. Epithelial to mesenchymal transition (EMT) in cancer cells involves not only the loss of cell-cell adhesion and acquisition of migratory and invasive properties but also resistance to cell death, senescence, and chemotherapy. We hypothesized that the EMT status of cancer cells regulates their sensitivity to rapamycin. We classified a panel of immortalized cancer cell lines as resistant or sensitive to rapamycin based on their IC50 as determined by Sulforhodamine B (SRB) assay. Using reverse phase proteomic array, we found that EMT markers were differentially expressed in sensitive compared to resistant immortalized cell lines. Rapamycin sensitive (RS) cell lines had increased expression of E-cadherin and N-cadherin while rapamycin resistant (RR) cell lines had increased expression of Smad3 (p<0.02). To validate these findings, we performed western blotting for EMT markers in selected RR and RS cell lines. E-cadherin expression was higher in RS cell lines than RR cell lines; in turn, RR cell lines expressed increased levels of the mesenchymal marker Vimentin. To determine if inducing EMT decreases rapamycin sensitivity, we assessed E-cadherin and Vimentin expression by western blotting in the epithelial, immortalized breast cancer cell line, MCF7, which was transfected with a constitutively-active mutant of Snail, a known EMT driver. Constitutively-active Snail mutant MCF7 demonstrated loss of E-cadherin and gain of Vimentin expression compared to cells transfected with nonfunctional mutant Snail or Wild-type Snail. Furthermore, we found decreased growth inhibition with rapamycin by SRB assay in constitutively-active Snail mutant MCF7 compared to controls (p<0.05). To determine if modulating EMT increases rapamycin sensitivity, we induced MET using ZEB1 siRNA knockdown and miR200b/c transfection in two mesenchymal cancer cell lines_ACHN and MDA-MB-231. Although miR200b/c transfection induced E-cadherin expression by immunoblotting in both cell lines, there was no significant increase in growth inhibition by SRB assay. In contrast, ZEB1 siRNA knockdown partially reversed EMT in these cells, which exhibited increased sensitivity to rapamycin by SRB assay. Most but not all mechanisms of EMT modulation altered rapamycin sensitivity in immortalized cancer cell lines. Future studies to determine the mechanism of EMT modulation that results in increased sensitivity to rapamycin could have important clinical applications for combination therapies utilizing rapamycin and its analogs. Citation Format: Ashley M. Holder, Farrell Adkins, Argun Akcakanat, Huiqin Chen, Kim-Anh Do, Mien-Chie Hung, Funda Meric-Bernstam. Epithelial to mesenchymal transition and rapamycin sensitivity in cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1496. doi:10.1158/1538-7445.AM2013-1496

Abstract 682: Increased stearoyl-CoA desaturase 1 expression is associated with shorter survival in breast cancer patients

Stearoyl-CoA desaturase 1 (SCD1) is an essential regulator of fatty acid synthesis that affects cell membrane composition and may play a role in cancer cell metabolism. We have previously shown that SCD1 expression is increased by mTOR signaling and that SCD1 increases breast cancer cell growth in vitro. The purpose of this study was to determine protein levels of SCD1 in breast cancer and to determine whether there is an association between SCD1 levels and survival. Fine needle aspirates were collected from the primary breast cancers of 253 patients with Stage I-III breast cancer and protein lysates were prepared. SCD1 expression was analyzed using reverse phase proteomic array. Patient demographic and clinical characteristics including age, ethnicity, menopausal status, clinical stage, tumor grade, and tumor subtype were reviewed. Samples were divided into high or low SCD1 levels based on a cut-off determined from martingale residual plots and regression tree analysis. SCD1 levels were significantly higher in older patients and lower in triple negative (estrogen, progesterone, and HER2 negative) cancers, After adjusting for tumor subtype, tumor grade, age, and clinical stage, patients who had high SCD1 levels had significantly shorter relapse-free survival (RFS) (p=0.013) and overall survival (OS) (p=0.038). SCD1 level varies by breast cancer subtype and predicts significantly shorter RFS and OS. Future studies are needed to define the role of SCD1 in breast cancer and evaluate its potential as a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 682. doi:1538-7445.AM2012-682