Valerie N. Barton

Multiple Molecular Subtypes of Triple-Negative Breast Cancer Critically Rely on Androgen Receptor and Respond to Enzalutamide In Vivo

Triple-negative breast cancer (TNBC) has the lowest 5-year survival rate of invasive breast carcinomas, and currently there are no approved targeted therapies for this aggressive form of the disease. The androgen receptor (AR) is expressed in up to one third of TNBC and we find that all AR+ TNBC primary tumors tested display nuclear localization of AR, indicative of transcriptionally active receptors. While AR is most abundant in the “luminal AR (LAR)” molecular subtype of TNBC, here, for the first time, we use both the new-generation anti-androgen enzalutamide and AR knockdown to demonstrate that the other non-LAR molecular subtypes of TNBC are critically dependent on AR protein. Indeed, AR inhibition significantly reduces baseline proliferation, anchorage-independent growth, migration, and invasion and increases apoptosis in four TNBC lines (SUM159PT, HCC1806, BT549, and MDA-MB-231), representing three non-LAR TNBC molecular subtypes (mesenchymal-like, mesenchymal stem–like, and basal-like 2). In vivo, enzalutamide significantly decreases viability of SUM159PT and HCC1806 xenografts. Furthermore, mechanistic analysis reveals that AR activation upregulates secretion of the EGFR ligand amphiregulin (AREG), an effect abrogated by enzalutamide in vitro and in vivo. Exogenous AREG partially rescues the effects of AR knockdown on proliferation, migration, and invasion, demonstrating that upregulation of AREG is one mechanism by which AR influences tumorigenicity. Together, our findings indicate that non-LAR subtypes of TNBC are AR dependent and, moreover, that enzalutamide is a promising targeted therapy for multiple molecular subtypes of AR+ TNBC. Mol Cancer Ther; 14(3); 769–78. ©2015 AACR.

Survey of MicroRNA expression in pediatric brain tumors

A better understanding of pediatric brain tumor biology is needed to assist in the development of less toxic therapies and to provide better markers for disease stratification. MicroRNAs (miRNA) may play a significant role in brain tumor biology. The present study provides an initial survey of miRNA expression in pediatric central nervous system (CNS) malignancies including atypical teratoid/rhabdoid tumor, ependymoma, glioblastoma, medulloblastoma, and pilocytic astrocytoma.

Claudin 6 Is a Positive Marker for Atypical Teratoid/Rhabdoid Tumors

Atypical teratoid/rhabdoid tumors (AT/RTs) are highly aggressive pediatric brain tumors characterized by the presence of rhabdoid cells and negative immunostaining for INI1 (BAF47). Histogenesis is unknown and diagnosis can be challenging because of their extreme morphological and immunophenotypic heterogeneity. Currently no signature markers other than INI1 loss have been identified. To search for possible candidate proteins of interest in AT/RTs, Affymetrix GeneChip® microarrays were utilized to investigate nine AT/RTs vs. 124 other tumor samples. The most distinctive gene identified was claudin 6 (CLDN6), a key component of tight junctions. CLDN6 showed moderate or higher mRNA expression in eight of nine AT/RTs, with little to no expression in 114 of 115 other tumors. Average expression was 38‐fold higher in AT/RTs vs. other samples. Immunohistochemical (IHC) staining of 33 tumor specimens found positive membrane staining in seven of seven AT/RTs, and was negative in 26 of 27 other brain tumor samples. Notably, none of the 16 medulloblastomas/primitive neuroectodermal tumors showed IHC staining for CLDN6. IHC staining results closely matched the level of mRNA expression detected by microarray. CLDN6 may be a useful positive marker to help further identify AT/RTs for diagnostic and treatment purposes.

Immune Gene and Cell Enrichment Is Associated with a Good Prognosis in Ependymoma

Approximately 50% of children with ependymoma will suffer from tumor recurrences that will ultimately lead to death. Development of more effective therapies and patient stratification in ependymoma mandates better prognostication. In this study, tumor gene expression microarray profiles from pediatric ependymoma clinical samples were subject to ontological analyses to identify outcome-associated biological factors. Histology was subsequently used to evaluate the results of ontological analyses. Ontology analyses revealed that genes associated with nonrecurrent ependymoma were predominantly immune function-related. Additionally, increased expression of immune-related genes was correlated with longer time to progression in recurrent ependymoma. Of those genes associated with both the nonrecurrent phenotype and that positively correlated with time to progression, 95% were associated with immune function. Histological analysis of a subset of these immune function genes revealed that their expression was restricted to a subpopulation of tumor-infiltrating cells. Analysis of tumor-infiltrating immune cells showed increased infiltration of CD4+ T cells in the nonrecurrent ependymomas. No genomic sequences for SV40, BK, JC, or Merkel polyomaviruses were found in nonrecurrent ependymoma. This study reveals that up-regulation of immune function genes is the predominant ontology associated with a good prognosis in ependymoma and it provides preliminary evidence of a beneficial host proinflammatory and/or Ag-specific immune response.

Aurora kinase A as a rational target for therapy in glioblastoma

OBJECT Despite advances in the knowledge of tumor biology, the outcome of glioblastoma tumors remains poor. The design of many molecularly targeted therapies in glioblastoma has focused on inhibiting molecular abnormalities present in tumor cells compared with normal tissue rather than patient outcome-associated factors. As an alternative approach, the present study identified genes associated with shorter survival as potential therapeutic targets. It was hypothesized that inhibition of a molecular target associated with poor outcome would impact glioblastoma cell proliferation. METHODS The present study correlated patient survival data with tumor gene expression profiling and gene ontology analysis. Genes associated with shorter survival were identified and one of these was selected for therapeutic targeting in an in vitro system. Glioblastoma cell growth suppression was measured by H(3)-thymidine uptake, colony formation, and flow cytometry. RESULTS The gene expression microarray and ontology analysis revealed that genes involved in mitotic processes, including AURKA, were associated with poor prognosis in glioblastoma. Inhibition of AURKA suppressed glioblastoma cell growth. Moreover, inhibition of AURKA was synergistic with radiation in glioblastoma cells at high radiation doses. CONCLUSIONS Relative expression of AURKA may be of prognostic value and warrants further investigation with larger, prospective studies. Pharmacological inhibition of AURKA is a potentially promising therapy for glioblastoma.

PARP1 expression in pediatric central nervous system tumors

Despite advances in therapy, outcome in many high‐grade pediatric central nervous system (CNS) tumors remains poor. The focus of neuro‐oncology research has thus turned towards identifying novel therapeutic targets. Poly(ADP‐ribose) polymerase‐1 (PARP1) is a DNA repair protein that has been studied in a variety of malignancies and may interfere with therapy‐induced DNA damage, however expression in pediatric CNS tumors is unknown.

Cooperative Dynamics of AR and ER Activity in Breast Cancer

Androgen receptor (AR) is expressed in 90% of estrogen receptor alpha–positive (ER+) breast tumors, but its role in tumor growth and progression remains controversial. Use of two anti-androgens that inhibit AR nuclear localization, enzalutamide and MJC13, revealed that AR is required for maximum ER genomic binding. Here, a novel global examination of AR chromatin binding found that estradiol induced AR binding at unique sites compared with dihydrotestosterone (DHT). Estradiol-induced AR-binding sites were enriched for estrogen response elements and had significant overlap with ER-binding sites. Furthermore, AR inhibition reduced baseline and estradiol-mediated proliferation in multiple ER+/AR+ breast cancer cell lines, and synergized with tamoxifen and fulvestrant. In vivo, enzalutamide significantly reduced viability of tamoxifen-resistant MCF7 xenograft tumors and an ER+/AR+ patient-derived model. Enzalutamide also reduced metastatic burden following cardiac injection. Finally, in a comparison of ER+/AR+ primary tumors versus patient-matched local recurrences or distant metastases, AR expression was often maintained even when ER was reduced or absent. These data provide preclinical evidence that anti-androgens that inhibit AR nuclear localization affect both AR and ER, and are effective in combination with current breast cancer therapies. In addition, single-agent efficacy may be possible in tumors resistant to traditional endocrine therapy, as clinical specimens of recurrent disease demonstrate AR expression in tumors with absent or refractory ER. Implications: This study suggests that AR plays a previously unrecognized role in supporting E2-mediated ER activity in ER+/AR+ breast cancer cells, and that enzalutamide may be an effective therapeutic in ER+/AR+ breast cancers. Mol Cancer Res; 14(11); 1054–67. ©2016 AACR.

Androgen Receptor Biology in Triple Negative Breast Cancer: a Case for Classification as AR+ or Quadruple Negative Disease

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) amplification. Due to the absence of these receptors, TNBC does not respond to traditional endocrine or HER2-targeted therapies that improve patient prognosis in other breast cancer subtypes. TNBC has a poor prognosis, and currently, there are no effective targeted therapies. Some TNBC tumors express androgen receptor (AR) and may benefit from AR-targeted therapies. Here, we review the literature on AR in TNBC and propose that TNBC be further sub-classified as either AR+ TNBC or quadruple negative breast cancer since targeting AR may represent a viable therapeutic option for a subset of TNBC.

Unique Molecular Characteristics of Pediatric Myxopapillary Ependymoma

Myxopapillary ependymoma (MEPN) generally can be cured by gross total surgical resection and usually manifest a favorable prognosis. However, surgery is less curative in tumors that are large, multifocal or extend outside the thecal sac. Late recurrences may occur, particularly in pediatric patients. The role of adjuvant therapy is unclear in the clinical management of recurrent tumors. Clinical trial design requires a better understanding of tumor biology. Unique molecular features of MEPN were investigated by using microarray technology to compare the gene expression of five pediatric MEPN to 24 pediatric intracranial ependymoma (EPN). The upregulation of three genes of interest, homeobox B13 (HOXB13), neurofilament, light polypeptide (NEFL) and PDGFRα, was further studied by immunohistochemistry in a larger cohort that included adult MEPN and EPN specimens. Protein expression in MEPN was compared to subependymoma, spinal EPN, intracranial EPN and normal fetal and adult ependyma. Immunoreactivity for HOXB13, NEFL and PDGFRα was strongest in MEPN and virtually absent in subependymoma. Spinal and intracranial EPN generally expressed weak or focal staining. MEPN manifests unique gene and protein expression patterns compared to other EPNs. Aberrant expression of HOXB13 suggests possible recapitulation of developmental pathways in MEPN tumorigenesis. PDGFRα may be a potential therapeutic target in recurrent MEPN.

Clinical and molecular characteristics of congenital glioblastoma

Congenital glioblastoma (cGBM) is an uncommon tumor of infancy with a reported variable but often poor cure rate, even with intensive therapy. Five patients with cGBMs, arising de novo and not in familial tumor predisposition kindreds, were studied for histological and biological features, using Affymetrix microarray. Tumors were large, often associated with hemorrhage, extended into the thalamus, and often bulged into the ventricles. One patient died acutely from bleeding at the time of operation. The 4 surviving patients underwent surgery (1 gross total resection, 3 subtotal resections or biopsies) and moderate intensity chemotherapy without radiation, and remain progression-free at a median time of 36 months (range, 30-110 months). Affymetrix microarrays measured gene expression on the 3 cGBMs from which frozen tissue was available. Unsupervised hierarchical clustering of cGBMs versus 168 other central nervous system tumors demonstrated that cGBMs clustered most closely with other high-grade gliomas. Gene expression profiles of cGBMs were compared with non-congenital pediatric and adult GBMs. cGBMs demonstrated marked similarity to both pediatric and adult GBMs, with only 31 differentially expressed genes identified (false discovery rate, <0.05). Unique molecular features of cGBMs included over-expression of multiple genes involved in glucose metabolism and tissue hypoxia. cGBMs show histological and biological overlap with pediatric and adult GBMs but appear to have a more favorable outcome, with good response to moderate intensity chemotherapy with only subtotal resection or biopsy. Further study may determine whether identified gene expression differences contribute to the improved survival seen in these tumors.