Analysis of the 2017 American Society for Radiation Oncology (ASTRO) Research Portfolio.

PURPOSE: Research in radiation oncology (RO) is imperative to support the discovery of new uses of radiation and improvement of current approaches to radiation delivery and to foster the continued evolution of our field. Therefore, in 2016, the American Society of Radiation Oncology performed an evaluation of research grant funding for RO. METHODS AND MATERIALS: Members of the Society of Chairs of Academic Radiation Oncology Programs (SCAROP) were asked about funded and unfunded grants that were submitted by their departments between the fiscal years 2014 and 2016. Grants were grouped according to broad categories defined by the 2017 American Society of Radiation Oncology Research Agenda. Additionally, active grants in the National Institutes of Health (NIH) Research Portfolio Online Reporting Tools database were collated using RO faculty names. RESULTS: Overall, there were 816 funded (44%) and 1031 unfunded (56%) SCAROP-reported grants. Total grant funding was over $196 million. The US government funded the plurality (42.2%; 345 of 816) of grants compared with nonprofit and industry funders. Investigators from 10 institutions accounted for >75% of funded grants. Of the funded grants, 43.5% were categorized as "genomic influences and targeted therapies." The proportion of funded to unfunded grants was highest within the category of "tumor microenvironment, normal tissue effects, and reducing toxicity" (53.4% funded). "New clinical trial design and big data" had the smallest share of SCAROP grant applications and the lowest percent funded (38.3% of grants). NIH grants to RO researchers in 2014 to 2016 accounted for $85 million in funding. From the 31 responding SCAROP institutions, there was a 28% average success rate for RO proposals submitted to the NIH during this period. CONCLUSIONS: Though RO researchers from responding institutions were relatively successful in obtaining funding, the overall amount awarded remains small. Continued advocacy on behalf of RO is needed, as well as investment to make research careers more attractive areas for emerging faculty.

Assessing the Training and Research Environment for Genomics, Bioinformatics, and Immunology in Radiation Oncology.

PURPOSE: To assess radiation oncologists' perceptions of training and research opportunities in the fields of genomics, bioinformatics, and immunology. MATERIALS AND METHODS: A 13-item electronic survey was sent to 101 radiation oncology department chairs and administrators. A separate 30-item electronic survey was sent to 132 members of the American Society for Radiation Oncology Science Council as well as to 565 members of the Association of Residents in Radiation Oncology. Survey responses were collected, and results were analyzed using descriptive statistics. RESULTS: Twenty-six department chairs and 91 general respondents submitted responses. Among general respondents, 69% were current trainees and 31% had completed training. The majority of respondents (92%) were affiliated with an academic/university main campus. Approximately half of respondents (43% to 53%) reported no prior formal training in bioinformatics, genomics, or immunology. More than half of department chairs (54% to 58%) and general respondents (57% to 63%) thought that current training opportunities in these areas were absolutely or moderately insufficient. A majority of respondents (53% to 65%) thought that additional training in these areas would provide opportunity for career advancement, and 80% could identify a current or future research project that additional training in these fields would allow them to pursue. More than half of respondents expressed interest in attending a formal training course, and the majority of department chairs (22 of 26 [85%]) reported that they would probably or definitely send trainees or faculty members to a formal training course. CONCLUSION: Among radiation oncologists surveyed, there is a perceived lack of current training opportunities in bioinformatics, genomics, and immunology. A majority of respondents reported an interest in obtaining additional training in these areas and believed that training would provide opportunity for career advancement.

pp32 (ANP32A) expression inhibits pancreatic cancer cell growth and induces gemcitabine resistance by disrupting HuR binding to mRNAs.

The expression of protein phosphatase 32 (PP32, ANP32A) is low in poorly differentiated pancreatic cancers and is linked to the levels of HuR (ELAV1), a predictive marker for gemcitabine response. In pancreatic cancer cells, exogenous overexpression of pp32 inhibited cell growth, supporting its long-recognized role as a tumor suppressor in pancreatic cancer. In chemotherapeutic sensitivity screening assays, cells overexpressing pp32 were selectively resistant to the nucleoside analogs gemcitabine and cytarabine (ARA-C), but were sensitized to 5-fluorouracil; conversely, silencing pp32 in pancreatic cancer cells enhanced gemcitabine sensitivity. The cytoplasmic levels of pp32 increased after cancer cells are treated with certain stressors, including gemcitabine. pp32 overexpression reduced the association of HuR with the mRNA encoding the gemcitabine-metabolizing enzyme deoxycytidine kinase (dCK), causing a significant reduction in dCK protein levels. Similarly, ectopic pp32 expression caused a reduction in HuR binding of mRNAs encoding tumor-promoting proteins (e.g., VEGF and HuR), while silencing pp32 dramatically enhanced the binding of these mRNA targets. Low pp32 nuclear expression correlated with high-grade tumors and the presence of lymph node metastasis, as compared to patients' tumors with high nuclear pp32 expression. Although pp32 expression levels did not enhance the predictive power of cytoplasmic HuR status, nuclear pp32 levels and cytoplasmic HuR levels associated significantly in patient samples. Thus, we provide novel evidence that the tumor suppressor function of pp32 can be attributed to its ability to disrupt HuR binding to target mRNAs encoding key proteins for cancer cell survival and drug efficacy.

The role of HuR in gemcitabine efficacy in pancreatic cancer: HuR Up-regulates the expression of the gemcitabine metabolizing enzyme deoxycytidine kinase.

RNA-binding protein HuR binds U- or AU-rich sequences in the 3'-untranslated regions of target mRNAs, stabilizing them and/or modulating their translation. Given the links of HuR with cancer, we studied the consequences of modulating HuR levels in pancreatic cancer cells. HuR-overexpressing cancer cells, in some instances, are roughly up to 30-fold more sensitive to treatment with gemcitabine, the main chemotherapeutic component of treatment regimens for pancreatic ductal adenocarcinoma (PDA), compared with control cells. In pancreatic cancer cells, HuR associates with deoxycytidine kinase (dCK) mRNA, which encodes the enzyme that metabolizes and thereby activates gemcitabine. Gemcitabine exposure to pancreatic cancer cells enriches the association between HuR and dCK mRNA and increases cytoplasmic HuR levels. Accordingly, HuR overexpression elevates, whereas HuR silencing reduces, dCK protein expression in pancreatic cancer cells. In a clinical correlate study of gemcitabine treatment, we found a 7-fold increase in risk of mortality in PDA patients with low cytoplasmic HuR levels compared with patients with high HuR levels, after adjusting for other treatments and demographic variables. These data support the notion that HuR is a key mediator of gemcitabine efficacy in cancer cells, at least in part through its ability to regulate dCK levels posttranscriptionally. We propose that HuR levels in PDA modulate the therapeutic efficacy of gemcitabine, thus serving as a marker of the clinical utility of this common chemotherapeutic agent and a potential target for intervention in pancreatic cancer.

Genome-wide SNP assay reveals structural genomic variation, extended homozygosity and cell-line induced alterations in normal individuals.

The recent hapmap effort has placed focus on the application of genome-wide SNP analysis to assess the contribution of genetic variability, particularly SNPs, to traits such as disease. Here, we describe the utility of genome-wide SNP analysis in the direct detection of extended homozygosity and structural genomic variation. We use this approach to assess the frequency of genomic alterations resulting from the lymphoblast immortalization and culture processes commonly used in cell repositories. We have assayed 408 804 SNPs in 276 DNA samples extracted from Epstein-Barr virus immortalized cell lines, which were derived from lymphocytes of elderly neurologically normal subjects. These data reveal extended homozygosity (contiguous tracts >5 Mb) in 9.5% (26/272) and 340 structural genomic alterations in 182 (66.9%) DNA samples assessed, 66% of which did not overlap with previously described structural variations. Examination of DNA extracted directly from the blood of 30 of these subjects confirmed all examined instances of extended homozygosity (6/6), 75% of structural genomic alteration <5 Mb in size (12/16) and 13% (1/8) of structural genomic alteration >5 Mb in size. These data suggest that structural genomic variation is a common phenomenon in the general population. While a proportion of this variability may be caused or its relative abundance altered by the immortalization and clonal process this will have only a minor effect on genotype and allele frequencies in a large cohort. It is likely that this powerful methodology will augment existing techniques in the identification of chromosomal abnormalities.

Polyamine analogues down-regulate estrogen receptor alpha expression in human breast cancer cells.

The critical role of polyamines in cell growth has led to the development of a number of agents that interfere with polyamine metabolism including a novel class of polyamine analogues, oligoamines. Here we demonstrate that oligoamines specifically suppress the mRNA and protein expression of estrogen receptor alpha (ERalpha) and ERalpha target genes in ER-positive human breast cancer cell lines, whereas neither ERbeta nor other steroid hormonal receptors are affected by oligoamines. The constitutive expression of a cytomegalovirus promoter-driven exogenous ERalpha in ER-negative MDA-MB-231 human breast cancer cells was not altered by oligoamines, suggesting that oligoamines specifically suppress ERalpha transcription rather than affect mRNA or protein stability. Further analysis demonstrated that oligoamines disrupted the DNA binding activity of Sp1 transcription factor family members to an ERalpha minimal promoter element containing GC/CA-rich boxes. Treatment of MDA-MB-231 cells with the JNK-specific inhibitor SP600125 or expression of the c-Jun dominant negative inhibitor TAM67 blocked the oligoamine-activated JNK/c-Jun pathway and enhanced oligoamine-inhibited ERalpha expression, suggesting that AP-1 is a positive regulator of ERalpha expression and that oligoamine-activated JNK/AP-1 activity may antagonize the down-regulation of ERalpha induced by oligoamines. Taken together, these results suggest a novel antiestrogenic mechanism for specific polyamine analogues in human breast cancer cells.

Characterization of a novel PMA-inducible pathway of interleukin-13 gene expression in T cells.

Although interleukin 13 (IL-13) is an important mediator of asthma and allergic diseases, the molecular mechanisms regulating IL-13 gene expression are not well understood. This study was designed to define the molecular mechanisms governing IL-13 gene expression in T cells. IL-13 expression was examined in human peripheral blood T cells and in the EL-4 T-cell line by enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction. An IL-13 promoter deletion analysis was performed using luciferase-based reporter plasmids transiently transfected into EL-4 cells by electroporation. DNA binding factors were investigated using electrophoretic mobility shift assays. In contrast to IL-4 expression, which required concomitant activation of calcium- and protein kinase C- (PKC-) dependent signalling pathways, PKC activation alone was sufficient for IL-13 protein secretion in mitogen-primed (but not resting) peripheral blood T cells, and for IL-13 mRNA expression and promoter activity in EL-4 T cells. Promoter deletion analysis localized a phorbol 12-myristate 13-acetate (PMA)-sensitive element to a proximal promoter region between -109 and -79 base pairs upstream from the IL-13 transcription start site. This promoter region supported the binding of both constitutive and PMA-inducible nuclear factors in gel shift assays.

Protein phosphatase 2A regulates estrogen receptor alpha (ER) expression through modulation of ER mRNA stability.

Protein phosphatase 2A (PP2A) is a ubiquitously expressed member of the serine-threonine phosphatase family that is involved in regulation of many cellular processes including transcription, translation, cellular metabolism, and apoptosis. Because of a correlation between PP2A and estrogen receptor alpha (ER) expression in several human breast cancer cell lines, the effect of PP2A on regulation of ER expression in the human breast cancer cell line MCF-7 was studied. Inhibition of PP2A using the pharmacologic inhibitor okadaic acid at 250 nm for 16 h resulted in a 60% reduction in PP2A activity in MCF-7 cells concurrent with a 75% reduction in ER mRNA and protein expression. Similar results were obtained with a small interfering RNA probe that specifically inhibited PP2A expression. ER promoter studies showed that regulation of ER through the PP2A pathway did not occur through transcriptional activation. Rather, PP2A mediated ER expression through modulation of ER mRNA stability through degradation of ER mRNA, reversible with concomitant treatment with the proteasomal inhibitor MG 132. These data suggest a novel pathway controlling ER expression resulting from the activation of PP2A, potentially providing a novel therapeutic target.

Regulation of polyamine analogue cytotoxicity by c-Jun in human MDA-MB-435 cancer cells.

Several polyamine analogues have efficacy against a variety of epithelial tumor models including breast cancer. Recently, a novel class of polyamine analogues designated as oligoamines has been developed. Here, we demonstrate that several representative oligoamine compounds inhibit in vitro growth of human breast cancer MDA-MB-435 cells. The activator protein-1 (AP-1) transcriptional factor family members, c-Jun and c-Fos, are up-regulated by oligoamines in MDA-MB-435 cells, suggesting a possible AP-1-dependent induction of apoptosis. However, the use of a novel c-Jun NH(2)-terminal kinase (JNK) inhibitor, SP600125, suggests that inhibition of c-Jun activity sensitized tumor cells to oligoamine-induced cell death. To directly test this hypothesis, cells were stably transfected with the dominant-negative mutant c-Jun (TAM67), which lacks the NH(2)-terminal transactivation domain. Cells overexpressing TAM67 exhibit normal growth kinetics but demonstrate a significantly increased sensitivity to oligoamine cytotoxicity and attenuated colony formation after oligoamine treatment. Furthermore, oligoamine treatment leads to more profound caspase-3 activation and poly(ADP-ribose) polymerase cleavage in TAM67 transfectants, suggesting that c-Jun acts as an antiapoptosis factor in MDA-MB-435 cells in response to oligoamine treatment. These findings indicate that oligoamine-inducible AP-1 plays a prosurvival role in oligoamine-treated MDA-MB-435 cells and that JNK/AP-1 might be a potential target for enhancing the therapeutic efficacy of polyamine analogues in human breast cancer.

Epigenetic regulation of protein phosphatase 2A (PP2A), lymphotactin (XCL1) and estrogen receptor alpha (ER) expression in human breast cancer cells.

Absence of the estrogen receptor alpha (ER) in human breast cancer cells is an indicator of poor prognosis, and predictive of lack of response to hormonal therapy. Previous studies in our laboratory and others have shown that epigenetic regulation, including DNA methylation and histone deacetylation, are common mechanisms leading to ER gene silencing. Through the use of pharmacologic inhibitors, 5-aza 2'deoxycytidine (AZA) and Trichostatin A (TSA), we have shown that alterations in both of these mechanisms results in synergistic reexpression of ER mRNA and functional protein. These alterations may play a larger role in stimulation of cell signaling pathways leading to ER expression. We have utilized newly developed genome wide screening microarray techniques to identify gene(s) contributing to the hormone independent phenotype and AZA/TSA mediated ER expression. From this screen, we identified and confirmed expression of 4 candidate genes (PP2A, XCL1, THY1 and NBC4) as potential regulators of the hormone independent phenotype. Expression of two genes, XCL1 and PP2A, appeared to be correlated with ER expression. PP2A expression was not changed with ER degradation using ICI 182,780 whereas XCL1 expression decreased in the presence of AZA/TSA and ICI 182,780. This suggests that PP2A may be a determinant of ER expression while XCL1 appears to be ER responsive and downstream of ER expression. These gene products may be novel targets to be further explored in the development of new therapeutics for ER negative breast cancer.