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1.  Aggressive estrogen-receptor-positive breast cancer arising in patients with elevated body mass index 
Background
Obese women with estrogen receptor (ER)-positive breast cancer may experience worse disease-free and overall survival. We hypothesize that this observation is due to intrinsically aggressive disease and that obesity will be associated with higher histologic grade and Ki67.
Methods
A sequential cohort of women with breast cancer diagnosed over 2 years was assembled from institutional tumor registries. Patient and tumor characteristics were abstracted from medical records; those with noninvasive tumors, or lacking body mass index (BMI), Ki67 or histologic grade data, were excluded. Univariate and multivariate analysis was performed to investigate the relationship between markers of aggressive disease (grade and Ki67) and multiple variables associated with obesity. A subgroup analysis was performed to investigate further whether ER and menopausal status influenced associations between BMI and aggressive phenotypes.
Results
Of the 1007 patients initially identified, 668 (68 %) met the eligibility criteria. In univariate analysis, histologic grade and Ki67 were strongly associated with increased BMI, younger age, and African-American race, but less so with diabetes, hypertension, and hyperlipidemia. Multivariate analysis confirmed that higher histologic grade was associated with increased BMI (p = 0.02), and that increased Ki67 was associated with younger age (p = 0.0003) and African-American race (p = 0.002). Additional analysis found that the association between increased BMI and higher-grade tumors was particularly significant in premenopausal women with ER-positive disease.
Conclusion
This study concludes that increased BMI is associated with aggressive-phenotype breast cancer and may be particularly relevant to ER-positive breast cancer developing in premenopausal African-American women.
doi:10.1007/s10147-014-0712-4
PMCID: PMC4362999  PMID: 24913910
Breast cancer; Obesity; Aggressive phenotype; Histologic grade; Ki67
2.  Prospective evaluation of plasma levels of ANGPT2, TuM2PK, and VEGF in patients with renal cell carcinoma 
BMC Urology  2015;15:24.
Background
To assess pathological correlations and temporal trends of Angiopoietin-2 (ANGPT2), vascular endothelial growth factor (VEGF) and M2 Pyruvate kinase (TuM2PK), markers of tumor vascular development and metabolism, in patients with renal cell carcinoma (RCC).
Methods
We prospectively collected plasma samples from 89 patients who underwent surgical/ablative therapy for RCC and 38 patients with benign disease (nephrolithiasis, hematuria without apparent neoplastic origin, or renal cysts). In RCC patients, marker levels were compared between at least 1 preoperative and 1 postoperative time point generally 3 weeks after surgery. Marker temporal trends were assessed using the Wilcoxon sign-rank test. Plasma VEGF, ANGPT2, and TuM2PK levels were determined by ELISA and tested for association with pathological variables.
Results
Median age was comparable between groups. 83/89 (93%) of the cohort underwent surgical extirpation. 82% of the tumors were organ confined (T ≤2, N0). Only ANGPT2 exhibited significantly elevated preoperative levels in patients with RCC compared to benign disease (p = 0.046). Elevated preoperative levels of ANGPT2 and TuM2PK significantly correlated with increased tumor size and advanced grade (p < 0.05). Chromophobe RCC exhibited higher levels of ANGPT2 compared to other histologies (p < 0.05). A decline in marker level after surgery was not observed, likely due to the timing of the analyses.
Conclusion
Our results suggest that ANGPT2 is a marker of RCC. Additionally, ANGPT2 and TuM2PK significantly correlated with several adverse pathological features. Further studies are needed to determine clinical applicability.
doi:10.1186/s12894-015-0019-4
PMCID: PMC4411704  PMID: 25885592
Biomarkers; Angiogenesis; Prospective; Renal cell carcinoma; Tumor metabolism
3.  Analysis of Internet Usage Among Cancer Patients in a County Hospital Setting: A Quality Improvement Initiative 
JMIR Research Protocols  2014;3(2):e26.
Background
Cancer is one of the most common diseases that patients research on the Internet. The Commission on Cancer (CoC) recommended that Parkland Memorial Hospital (PMH) improve the oncology services website. PMH is Dallas County’s public health care facility, serving a largely uninsured, minority population. Most research regarding patient Internet use has been conducted in insured, Caucasian populations, raising concerns that the needs of PMH patients may not be extrapolated from available data. The PMH Cancer Committee, therefore, adopted a quality improvement initiative to understand patients’ Internet usage.
Objective
The objective of the study was to obtain and analyze data regarding patients’ Internet usage in order to make targeted improvements to the oncology services section of the institutional website.
Methods
A task force developed an 11-question survey to ascertain what proportion of our patients have Internet access and use the Internet to obtain medical information as well as determine the specific information sought. Between April 2011 and August 2011, 300 surveys were administered to newly diagnosed cancer patients. Multivariate analyses were performed.
Results
Of 300 surveys, 291 were included. Minorities, primarily African-American and Hispanic, represented 78.0% (227/291) of patients. Only 37.1% (108/291) of patients had Internet access, most (256/291, 87.9%) having access at home. Younger patients more commonly had Internet access, with a mean age of 47 versus 58 years for those without (P<.001). Education beyond high school was associated with Internet access (P<.001). The most common reason for Internet research was to develop questions for discussion with one’s physician. Patients most frequently sought information regarding cancer treatment options, outcomes, and side effects.
Conclusions
Less than one-half of PMH oncology patients have Internet access. This is influenced by age, educational level, and ethnicity. Those with access use it to obtain information related to their cancer diagnosis. The most effective way of addressing our patients’ needs using the institutional website is to provide links to reputable disease-specific sites.
doi:10.2196/resprot.2806
PMCID: PMC4034116  PMID: 24824330
Internet; cancer; quality; quality improvement; patient education
4.  Axillary lymph node dissection for breast cancer utilizing Harmonic Focus® 
Background
For patients with axillary lymph node metastases from breast cancer, performance of a complete axillary lymph node dissection (ALND) is the standard approach. Due to the rich lymphatic network in the axilla, it is necessary to carefully dissect and identify all lymphatic channels. Traditionally, these lymphatics are sealed with titanium clips or individually sutured. Recently, the Harmonic Focus®, a hand-held ultrasonic dissector, allows lymphatics to be sealed without the utilization of clips or ties. We hypothesize that ALND performed with the Harmonic Focus® will decrease operative time and reduce post-operative complications.
Methods
Retrospective review identified all patients who underwent ALND at a teaching hospital between January of 2005 and December of 2009. Patient demographics, presenting pathology, treatment course, operative time, days to drain removal, and surgical complications were recorded. Comparisons were made to a selected control group of patients who underwent similar surgical procedures along with an ALND performed utilizing hemostatic clips and electrocautery. A total of 41 patients were included in this study.
Results
Operative time was not improved with the use of ultrasonic dissection, however, there was a decrease in the total number of days that closed suction drainage was required, although this was not statistically significant. Complication rates were similar between the two groups.
Conclusion
In this case-matched retrospective review, there were fewer required days of closed suction drainage when ALND was performed with ultrasonic dissection versus clips and electrocautery.
doi:10.1186/1477-7819-9-90
PMCID: PMC3170616  PMID: 21843361
5.  Translating Research into Practice Intervention Improves Management of Acute Pain in Older Hip Fracture Patients 
Health Services Research  2009;44(1):264-287.
Objective
To test an interdisciplinary, multifaceted, translating research into practice (TRIP) intervention to (a) promote adoption, by physicians and nurses, of evidence-based (EB) acute pain management practices in hospitalized older adults, (b) decrease barriers to use of EB acute pain management practices, and (c) decrease pain intensity of older hospitalized adults.
Study Design
Experimental design with the hospital as the unit of randomization.
Study Setting
Twelve acute care hospitals in the Midwest.
Data Sources
(a) Medical records (MRs) of patients ≥65 years or older with a hip fracture admitted before and following implementation of the TRIP intervention and (b) physicians and nurses who care for those patients.
Data Collection
Data were abstracted from MRs and questions distributed to nurses and physicians.
Principal Findings
The Summative Index for Quality of Acute Pain Care (0–18 scale) was significantly higher for the experimental (10.1) than comparison group (8.4) at the end of the TRIP implementation phase. At the end of the TRIP implementation phase, patients in the experimental group had a lower mean pain intensity rating than those in the comparison group (p<.0001).
Conclusion
The TRIP intervention improved quality of acute pain management of older adults hospitalized with a hip fracture.
doi:10.1111/j.1475-6773.2008.00913.x
PMCID: PMC2669630  PMID: 19146568
Translation; implementation; intervention; pain; elderly; hip fracture
6.  Cost of Hospital Care for Older Adults with Heart Failure: Medical, Pharmaceutical, and Nursing Costs 
Health Services Research  2008;43(2):635-655.
Objective
To determine the impact of patient characteristics, clinical conditions, hospital unit characteristics, and health care interventions on hospital cost of patients with heart failure.
Data Sources/Study Setting
Data for this study were part of a larger study that used electronic clinical data repositories from an 843-bed, academic medical center in the Midwest.
Study Design
This retrospective, exploratory study used existing administrative and clinical data from 1,435 hospitalizations of 1,075 patients 60 years of age or older. A cost model was tested using generalized estimating equations (GEE) analysis.
Data Collection/Extraction Methods
Electronic databases used in this study were the medical record abstract, the financial data repository, the pharmacy repository; and the Nursing Information System repository. Data repositories were merged at the patient level into a relational database and housed on an SQL server.
Principal Findings
The model accounted for 88 percent of the variability in hospital costs for heart failure patients 60 years of age and older. The majority of variables that were associated with hospital cost were provider interventions. Each medical procedure increased cost by $623, each unique medication increased cost by $179, and the addition of each nursing intervention increased cost by $289. One medication and several nursing interventions were associated with lower cost. Nurse staffing below the average and residing on 2–4 units increased hospital cost.
Conclusions
The model and data analysis techniques used here provide an innovative and useful methodology to describe and quantify significant health care processes and their impact on cost per hospitalization. The findings indicate the importance of conducting research using existing clinical data in health care.
doi:10.1111/j.1475-6773.2007.00789.x
PMCID: PMC2442365  PMID: 18370971
Heart failure; hospital cost; interventions; RN staffing
7.  Prediction of post-operative necrosis after mastectomy: A pilot study utilizing optical diffusion imaging spectroscopy 
Introduction
Flap necrosis and epidermolysis occurs in 18-30% of all mastectomies. Complications may be prevented by intra-operative detection of ischemia. Currently, no technique enables quantitative valuation of mastectomy skin perfusion. Optical Diffusion Imaging Spectroscopy (ViOptix T.Ox Tissue Oximeter) measures the ratio of oxyhemoglobin to deoxyhemoglobin over a 1 × 1 cm area to obtain a non-invasive measurement of perfusion (StO2).
Methods
This study evaluates the ability of ViOptix T.Ox Tissue Oximeter to predict mastectomy flap necrosis. StO2 measurements were taken at five points before and at completion of dissection in 10 patients. Data collected included: demographics, tumor size, flap length/thickness, co-morbidities, procedure length, and wound complications.
Results
One patient experienced mastectomy skin flap necrosis. Five patients underwent immediate reconstruction, including the patient with necrosis. Statistically significant factors contributing to necrosis included reduction in medial flap StO2 (p = 0.0189), reduction in inferior flap StO2 (p = 0.003), and flap length (p = 0.009).
Conclusion
StO2 reductions may be utilized to identify impaired perfusion in mastectomy skin flaps.
doi:10.1186/1477-7819-7-91
PMCID: PMC2788547  PMID: 19939277
8.  Bayesian optimal discovery procedure for simultaneous significance testing 
BMC Bioinformatics  2009;10:5.
Background
In high throughput screening, such as differential gene expression screening, drug sensitivity screening, and genome-wide RNAi screening, tens of thousands of tests need to be conducted simultaneously. However, the number of replicate measurements per test is extremely small, rarely exceeding 3. Several current approaches demonstrate that test statistics with shrinking variance estimates have more power over the traditional t statistic.
Results
We propose a Bayesian hierarchical model to incorporate the shrinkage concept by introducing a mixture structure on variance components. The estimates from the Bayesian model are utilized in the optimal discovery procedure (ODP) proposed by Storey in 2007, which was shown to have optimal performance in multiple significance tests. We compared the performance of the Bayesian ODP with several competing test statistics.
Conclusion
We have conducted simulation studies with 2 to 6 replicates per gene. We have also included test results from two real datasets. The Bayesian ODP outperforms the other methods in our study, including the original ODP. The advantage of the Bayesian ODP becomes more significant when there are few replicates per test. The improvement over the original ODP is based on the fact that Bayesian model borrows strength across genes in estimating unknown parameters. The proposed approach is efficient in computation due to the conjugate structure of the Bayesian model. The R code (see Additional file 1) to calculate the Bayesian ODP is provided.
doi:10.1186/1471-2105-10-5
PMCID: PMC2628883  PMID: 19126217
9.  DAB2IP Status in High-Risk Prostate Cancer Correlates with Outcome for Patients Treated with Radiation Therapy 
Purpose
This pilot study investigates the role of DAB2IP and EZH2 as prognostic biomarkers in high-risk prostate cancer patients receiving definitive radiation therapy.
Methods and Materials
Immunohistochemistry was performed and scored by an expert genitourinary pathologist. Clinical endpoints evaluated were freedom from biochemical failure (FFBF), castration resistance-free survival (CRFS), and distant metastasis-free survival (DMFS). Log-rank test and Cox regression were used to determine significance of biomarker levels with clinical outcome.
Results
Fifty-four patients with high-risk prostate cancer (stage ≥T3a, or Gleason score ≥8, or PSA ≥20) treated with radiation therapy from 2005-2012 at our institution were evaluated. Nearly all patients expressed EZH2 (98%), whereas 28% of patients revealed DAB2IP-reduction and 72% retained DAB2IP. Median follow up was 34.0 months for DAB2IP-reduced patients, 29.9 months for DAB2IP-retained patients, and 32.6 months in the EZH2 study. DAB2IP reduction portended worse outcome compared to DAB2IP-retained patients, including FFBF (4-year: 37% vs. 89%, p = 0.04), CRFS (4-year: 50% vs. 90%, p = 0.02), and DMFS (4-year: 36% vs. 97%, p = 0.05). Stratified EZH2 expression trended toward significance for worse FFBF and CRFS (p = 0.07). Patients with reduced DAB2IP or highest intensity EZH2 expression exhibited worse FFBF (4-year: 32% vs. 95%, p = 0.02), CRFS (4-year: 28% vs. 100%, p < 0.01), and DMFS (4-year: 39% vs. 100%, p = 0.04) compared to the control group.
Conclusion
DAB2IP loss is a potent biomarker that portends worse outcome despite definitive radiotherapy for patients with high-risk prostate cancer. EZH2 is expressed in most high-risk tumors and is a less potent discriminator of outcome in this study. DAB2IP status in combination with degree of EZH2 expression may be useful for determining patients with worse outcome within the high-risk prostate cancer population.
doi:10.1016/j.ijrobp.2014.03.035
PMCID: PMC4464555  PMID: 24867541
Biochemical failure; Castration resistance; DAB2IP; EZH2; Prostate cancer
10.  Orexin Regulates Bone Remodeling via a Dominant Positive Central Action and a Subordinate Negative Peripheral Action 
Cell metabolism  2014;19(6):927-940.
SUMMARY
Orexin neuropeptides promote arousal, appetite, reward, and energy expenditure. However, whether orexin affects bone mass accrual is unknown. Here we show that orexin functions centrally through orexin receptor 2 (OX2R) in the brain to enhance bone formation. OX2R-null mice exhibit low-bone-mass owing to elevated circulating leptin; whereas central administration of an OX2R-selective agonist augments bone mass. Conversely, orexin also functions peripherally through orexin receptor 1 (OX1R) in the bone to suppress bone formation. OX1R-null mice exhibit high-bone-mass owing to a mesenchymal stem cell differentiation shift from adipocyte to osteoblast that results from higher osseous ghrelin expression. The central action is dominant over the peripheral action because bone mass is reduced in orexin-null and OX1R2R-double-null mice but enhanced in orexin over-expressing transgenic mice. These findings reveal orexin as a critical rheostat of skeletal homeostasis that exerts a yin-yang dual regulation, and highlight orexin as a therapeutic target for osteoporosis.
doi:10.1016/j.cmet.2014.03.016
PMCID: PMC4047150  PMID: 24794976
11.  Aspirin improves outcome in high risk prostate cancer patients treated with radiation therapy 
Cancer Biology & Therapy  2014;15(6):699-706.
Purpose
High-risk prostate cancer (PC) has poor outcomes due to therapeutic resistance to conventional treatments, which include prostatectomy, radiation, and hormone therapy. Previous studies suggest that anticoagulant (AC) use may improve treatment outcomes in PC patients. We hypothesized that AC therapy confers a freedom from biochemical failure (FFBF) and overall survival (OS) benefit when administered with radiotherapy in patients with high-risk PC.
Materials and Methods
Analysis was performed on 74 high-risk PC patients who were treated with radiotherapy from 2005 to 2008 at UT Southwestern. Of these patients, 43 were on AC including aspirin (95.6%), clopidogrel (17.8%), warfarin (20%), and multiple ACs (31.1%). Associations between AC use and FFBF, OS, distant metastasis, and toxicity were analyzed.
Results
Median follow-up was 56.6 mo for all patients. For patients taking any AC compared with no AC, there was improved FFBF at 5 years of 80% vs. 62% (P = 0.003), and for aspirin the FFBF was 84% vs. 65% (P = 0.008). Aspirin use was also associated with reduced rates of distant metastases at 5 years (12.2% vs. 26.7%, P = 0.039). On subset analysis of patients with Gleason score (GS) 9–10 histology, aspirin resulted in improved 5-year OS (88% vs. 37%, P = 0.032), which remained significant on multivariable analysis (P < 0.05).
Conclusions
AC use was associated with a FFBF benefit in high-risk PC which translated into an OS benefit in the highest risk PC patients with GS 9–10, who are most likely to experience mortality from PC. This hypothesis-generating result suggests AC use may represent an opportunity to augment current therapy.
doi:10.4161/cbt.28554
PMCID: PMC4049786  PMID: 24658086
aspirin; anticoagulant; prostate cancer; radiotherapy
12.  Radiation-enhanced Lung Cancer Progression in a Transgenic Mouse Model of Lung Cancer is Predictive of Outcomes in Human Lung and Breast Cancer 
Purpose
Carcinogenesis is an adaptive process between nascent tumor cells and their microenvironment including the modification of inflammatory responses from anti-tumorigenic to pro-tumorigenic. Radiation exposure can stimulate inflammatory responses that inhibit or promote carcinogenesis. The purpose of this study is to determine the impact of radiation exposure on lung cancer progression in vivo and assess the relevance of this knowledge to human carcinogenesis.
Experimental Design
K-rasLA1 mice were irradiated with various doses and dose regimens and then monitored till death. Microarray analyses were performed using Illumina® BeadChips on whole lung tissue 70 days post-irradiation with a fractionated or acute dose of radiation and compared to age-matched unirradiated controls. Unique group classifiers were derived by comparative genomic analysis of three experimental cohorts. Survival analyses were performed using principal component analysis and k-means clustering on three lung adenocarcinoma, three breast adenocarcinoma, and two lung squamous carcinoma annotated microarray datasets.
Results
Radiation exposure accelerates lung cancer progression in the K-rasLA1 lung cancer mouse model with dose fractionation being more permissive for cancer progression. A non-random inflammatory signature associated with this progression was elicited from whole lung tissue containing only benign lesions and predicts human lung and breast cancer patient survival across multiple datasets. Immunohistochemical analyses suggest that tumor cells drive predictive signature.
Conclusions
These results demonstrate that radiation exposure can cooperate with benign lesions in a transgenic model of cancer by impacting inflammatory pathways, and that clinically relevant similarities exist between human lung and breast carcinogenesis.
doi:10.1158/1078-0432.CCR-13-2589
PMCID: PMC3961755  PMID: 24486591
Radiation; Lung Cancer; Progression; Mouse models; Genomics
13.  Inhibition of DNA Double-Strand Break Repair by the Dual PI3K/mTOR Inhibitor NVP-BEZ235 as a Strategy for Radiosensitization of Glioblastoma 
Purpose
Inhibitors of the DNA damage response (DDR) have great potential for radiosensitization of numerous cancers including glioblastomas (GBM), which are extremely radio- and chemo-resistant brain tumors. Currently, there are no DNA double-strand break (DSB) repair inhibitors that have been successful in treating GBM. Our lab has previously demonstrated that the dual PI3K/mTOR inhibitor NVP-BEZ235 can potently inhibit the two central DDR kinases, DNA-PKcs and ATM, in vitro. Here, we tested whether NVP-BEZ235 could also inhibit ATM and DNA-PKcs in tumors in vivo and assessed its potential as a radio- and chemo-sensitizer in pre-clinical mouse GBM models.
Experimental design
The radiosensitizing effect of NVP-BEZ235 was tested by following tumor growth in subcutaneous and orthotopic GBM models. Tumors were generated using the radioresistant U87-vIII glioma cell line and GBM9 neurospheres in nude mice. These tumors were then treated with ionizing radiation (IR) and/or NVP-BEZ235 and analyzed for DNA-PKcs and ATM activation, DSB repair inhibition, and attenuation of growth.
Results
NVP-BEZ235 potently inhibited both DNA-PKcs and ATM kinases and attenuated the repair of IR-induced DNA damage in tumors. This resulted in striking tumor radiosensitization, which extended the survival of brain tumor-bearing mice. Notably, tumors displayed a higher DSB-load when compared to normal brain tissue. NVP-BEZ235 also sensitized a subset of subcutaneous tumors to temozolomide, a drug routinely used concurrently with IR for the treatment of GBM.
Conclusions
These results demonstrate that it may be possible to significantly improve GBM therapy by combining IR with potent and bioavailable DNA repair inhibitors like NVP-BEZ235.
doi:10.1158/1078-0432.CCR-13-1607
PMCID: PMC3947495  PMID: 24366691
Glioblastoma; DNA double-strand break; Radiosensitization; NVP-BEZ235; Temozolomide
14.  miR-34a Blocks Osteoporosis and Bone Metastasis by Inhibiting Osteoclastogenesis and Tgif2 
Nature  2014;512(7515):431-435.
The bone resorbing osteoclasts significantly contribute to osteoporosis and cancer bone metastases1-3. MicroRNAs (miRNAs) play important roles in physiology and disease4,5, and present tremendous therapeutic potential6. Nonetheless, how miRNAs regulate skeletal biology is underexplored. Here we identify miR-34a as a novel and critical suppressor of osteoclastogenesis, bone resorption and the bone metastatic niche. miR-34a is down-regulated during osteoclast differentiation. Osteoclastic miR-34a over-expressing transgenic mice exhibit lower bone resorption and higher bone mass. Conversely, miR-34a knockout and heterozygous mice exhibit elevated bone resorption and reduced bone mass. Consequently, ovariectomy-induced osteoporosis, as well as bone metastasis of breast and skin cancers, are diminished in osteoclastic miR-34a transgenic mice, and can be effectively attenuated by miR-34a nanoparticle treatment. Mechanistically, we identify Tgif2 (transforming growth factor-beta-induced factor 2) as an essential direct miR-34a target that is pro-osteoclastogenic. Tgif2 deletion reduces bone resorption and abolishes miR-34a regulation. Together, using mouse genetic, pharmacological and disease models, we reveal miR-34a as a key osteoclast suppressor and a potential therapeutic strategy to confer skeletal protection and ameliorate bone metastasis of cancers.
doi:10.1038/nature13375
PMCID: PMC4149606  PMID: 25043055
15.  A Novel Germline Mutation in BAP1 Predisposes to Familial Clear-Cell Renal Cell Carcinoma 
Molecular cancer research : MCR  2013;11(9):1061-1071.
Renal cell carcinoma (RCC) clusters in some families. Familial RCC arises from mutations in several genes, including VHL, which is also mutated in sporadic RCC. However, a significant percentage of familial RCC remains unexplained. Recently, we discovered that the BAP1 gene is mutated in sporadic RCC. BAP1, which encodes a nuclear deubiquitinase, is a two-hit tumor suppressor gene. Somatic BAP1 mutations are associated with high-grade ccRCC and poor patient outcomes. To determine whether BAP1 predisposes to familial RCC, we sequenced the BAP1 gene in 83 unrelated probands with unexplained familial RCC. We identified a novel variant (c.41T>A; p.L14H), which cosegregated with the RCC phenotype. The p.L14H variant disrupts a highly conserved residue in the catalytic domain, a domain frequently targeted by missense mutations. The family with the BAP1 variant was characterized by early-onset clear cell RCC, occasionally of high Fuhrman grade, and lacked other features that characterize von Hippel-Lindau syndrome. These findings suggest that BAP1 is a familial RCC predisposing gene.
doi:10.1158/1541-7786.MCR-13-0111
PMCID: PMC4211292  PMID: 23709298
renal cell carcinoma; BAP1; cancer; inherited RCC; predisposition; tumor suppressor
16.  Cooperation and antagonism among cancer genes: the renal cancer paradigm 
Cancer research  2013;73(14):4173-4179.
It is poorly understood how driver mutations in cancer genes work together to promote tumor development. Renal cell carcinoma (RCC) offers a unique opportunity to study complex relationships among cancer genes. The four most commonly mutated genes in RCC of clear-cell type (the most common type) are two-hit tumor suppressor genes and they cluster in a 43 Mb region on chromosome 3p that is deleted in ~90% of tumors: VHL (mutated in ~80%), PBRM1 (~50%), BAP1 (~15%) and SETD2 (~15%). Meta-analyses that we conducted show that mutations in PBRM1 and SETD2 co-occur in tumors at a frequency higher than expected by chance alone, indicating that these mutations may cooperate in tumorigenesis. In contrast, consistent with our previous results, mutations in PBRM1 and BAP1 tend to be mutually exclusive. Mutation exclusivity analyses (often confounded by lack of statistical power) raise the possibility of functional redundancy. However, mutation exclusivity may indicate negative genetic interactions, as proposed herein for PBRM1 and BAP1, and mutations in these genes define RCC with different pathologic features, gene expression profiles, and outcomes. Negative genetic interactions among cancer genes point toward broader context-dependencies of cancer gene action beyond tissue dependencies. Understanding cancer genes dependencies may unravel vulnerabilities that can be exploited therapeutically.
doi:10.1158/0008-5472.CAN-13-0360
PMCID: PMC4051157  PMID: 23832661
17.  Differential Radiosensitivity Phenotypes of DNA-PKcs Mutations Affecting NHEJ and HRR Systems following Irradiation with Gamma-Rays or Very Low Fluences of Alpha Particles 
PLoS ONE  2014;9(4):e93579.
We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.
doi:10.1371/journal.pone.0093579
PMCID: PMC3979685  PMID: 24714417
18.  Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer 
Scientific Reports  2014;4:4480.
Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071–40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.
doi:10.1038/srep04480
PMCID: PMC3967199  PMID: 24670678
19.  BAP1 loss defines a new class of renal cell carcinoma 
Nature genetics  2012;44(7):751-759.
The molecular pathogenesis of renal cell carcinoma (RCC) is poorly understood. Whole-genome and exome sequencing followed by innovative tumorgraft analyses (to accurately determine mutant allele ratios) identified several putative two-hit tumor suppressor genes including BAP1. BAP1, a nuclear deubiquitinase, is inactivated in 15% of clear-cell RCCs. BAP1 cofractionates with and binds to HCF-1 in tumorgrafts. Mutations disrupting the HCF-1 binding motif impair BAP1-mediated suppression of cell proliferation, but not H2AK119ub1 deubiquitination. BAP1 loss sensitizes RCC cells in vitro to genotoxic stress. Interestingly, BAP1 and PBRM1 mutations anticorrelate in tumors (P=3×10−5), and combined loss of BAP1 and PBRM1 in a few RCCs was associated with rhabdoid features (q=0.0007). BAP1 and PBRM1 regulate seemingly different gene expression programs, and BAP1 loss was associated with high tumor grade (q=0.0005). Our results establish the foundation for an integrated pathological and molecular genetic classification of RCC, paving the way for subtype-specific treatments exploiting genetic vulnerabilities.
doi:10.1038/ng.2323
PMCID: PMC3788680  PMID: 22683710
20.  A high-throughput screen identifies miRNA inhibitors regulating lung cancer cell survival and response to paclitaxel 
RNA Biology  2013;10(11):1700-1713.
microRNAs (miRNAs) are small RNAs endogenously expressed in multiple organisms that regulate gene expression largely by decreasing levels of target messenger RNAs (mRNAs). Over the past few years, numerous studies have demonstrated critical roles for miRNAs in the pathogenesis of many cancers, including lung cancer. Cellular miRNA levels can be easily manipulated, showing the promise of developing miRNA-targeted oligos as next-generation therapeutic agents. In a comprehensive effort to identify novel miRNA-based therapeutic agents for lung cancer treatment, we combined a high-throughput screening platform with a library of chemically synthesized miRNA inhibitors to systematically identify miRNA inhibitors that reduce lung cancer cell survival and those that sensitize cells to paclitaxel. By screening three lung cancer cell lines with different genetic backgrounds, we identified miRNA inhibitors that potentially have a universal cytotoxic effect on lung cancer cells and miRNA inhibitors that sensitize cells to paclitaxel treatment, suggesting the potential of developing these miRNA inhibitors as therapeutic agents for lung cancer. We then focused on characterizing the inhibitors of three miRNAs (miR-133a/b, miR-361-3p, and miR-346) that have the most potent effect on cell survival. We demonstrated that two of the miRNA inhibitors (miR-133a/b and miR-361-3p) decrease cell survival by activating caspase-3/7-dependent apoptotic pathways and inducing cell cycle arrest in S phase. Future studies are certainly needed to define the mechanisms by which the identified miRNA inhibitors regulate cell survival and drug response, and to explore the potential of translating the current findings into clinical applications.
doi:10.4161/rna.26541
PMCID: PMC3907480  PMID: 24157646
cell viability; drug response; lung cancer; miRNA; paclitaxel
21.  A Validated Tumorgraft Model Reveals Activity of Dovitinib Against Renal Cell Carcinoma 
Science translational medicine  2012;4(137):137ra75.
Most anticancer drugs entering clinical trials fail to achieve approval from the US FDA. Drug development is hampered by the lack of preclinical models with therapeutic predictive value. Herein, we report the development and validation of a tumorgraft model of renal cell carcinoma (RCC) and its application to the evaluation of an experimental drug. Tumor samples from 94 patients were implanted in the kidney of mice without additives or disaggregation. Tumors from 35 patients formed tumorgrafts, and 16 stable lines were established. Samples from metastatic sites engrafted at high frequency, and stable engraftment of primary tumors in mice correlated with decreased patient survival suggesting that tumor growth in mice may reveal the acquisition by the tumor of an ability to thrive at distant sites and metastasize. Tumorgrafts retained the histology, gene expression, DNA copy number alterations, and over 90% of the protein-coding gene mutations of the corresponding tumors. As determined by the induction of hypercalcemia in tumorgraft-bearing mice, tumorgrafts were able to act on the host causing paraneoplastic syndromes. In studies simulating drug exposures in patients, RCC tumorgraft growth was inhibited by sunitinib and sirolimus (into which temsirolimus is converted in humans), but not by erlotinib, which was used as a control. Dovitinib, a drug in clinical development, showed greater activity than sunitinib and sirolimus. The routine incorporation of models recapitulating the molecular genetics and drug sensitivities of human tumors into preclinical programs has the potential to improve oncology drug development.
doi:10.1126/scitranslmed.3003643
PMCID: PMC3570965  PMID: 22674553
xenograft; tumor graft; orthotopic; PK; NOD/SCID; kidney cancer; clear-cell renal cell carcinoma
22.  Tamoxifen Downregulates Ets-oncogene Family Members ETV4 and ETV5 in Benign Breast Tissue: Implications for Durable Risk Reduction 
Background
Five years of tamoxifen reduces breast cancer risk by nearly 50% but is associated with significant side-effects and toxicities. A better understanding of the direct and indirect effects of tamoxifen in benign breast tissue could elucidate new mechanisms of breast carcinogenesis, suggest novel chemoprevention targets, and provide relevant early response biomarkers for Phase II prevention trials.
Methods
Seventy-three women at increased risk for breast cancer were randomized to tamoxifen (20 mg daily) or placebo for three months. Blood and breast tissue samples were collected at baseline and post-treatment. Sixty-nine women completed all study activities (37 tamoxifen and 32 placebo). The selected biomarkers focused on estradiol and IGFs in the blood, DNA methylation and cytology in random periareolar fine needle aspirates, and tissue morphometry, proliferation, apoptosis, and gene expression (microarray and RT-PCR) in the tissue core samples.
Results
Tamoxifen downregulated ets-oncogene transcription factor family members ETV4 and ETV5 and reduced breast epithelial cell proliferation independent of CYP2D6 genotypes or effects on estradiol, ESR1 or IGFs. Reduction in proliferation was correlated with downregulation of ETV4 and DNAJC12. Tamoxifen reduced the expression of ETV4- and ETV5-regulated genes implicated in epithelial-stromal interaction and tissue remodeling. Three months of tamoxifen did not affect breast tissue composition, cytological atypia, preneoplasia or apoptosis.
Conclusions
A plausible mechanism for the chemopreventive effects of tamoxifen is restriction of lobular expansion into stroma through downregulation of ETV4 and ETV5. Multipotential progenitor cap cells of terminal end buds may be the primary target.
doi:10.1158/1940-6207.CAPR-11-0186
PMCID: PMC3208724  PMID: 21778330
Tamoxifen; Biomarkers; Gene Expression; Proliferation; DNA Methylation
23.  Interplay between pVHL and mTORC1 pathways in clear-cell renal cell carcinoma 
Molecular cancer research : MCR  2011;9(9):1255-1265.
Mammalian target of rapamycin complex 1 (mTORC1) is implicated in cell growth control and is extensively regulated. We previously reported that in response to hypoxia, mTORC1 is inhibited by the protein regulated in development and DNA damage response 1 (REDD1). REDD1 is upregulated by HIF-1, and forced REDD1 expression is sufficient to inhibit mTORC1. REDD1-induced mTORC1 inhibition is dependent on a protein complex formed by the tuberous sclerosis complex (TSC)1 and 2 (TSC2) proteins. In clear-cell renal cell carcinoma (ccRCC), the von Hippel-Lindau (VHL) gene is frequently inactivated leading to constitutive activation of HIF-2 and/or HIF-1, which may be expected to upregulate REDD1 and inhibit mTORC1. However, mTORC1 is frequently activated in ccRCC and mTORC1 inhibitors are effective against this tumor type; a paradox herein examined. REDD1 was upregulated in VHL-deficient ccRCC by in silico microarray analyses, as well as by quantitative real-time PCR, Western blot, and immunohistochemistry. Vhl disruption in a mouse model was sufficient to induce Redd1. Using ccRCC-derived cell lines, we show that REDD1 upregulation in tumors is VHL-dependent, and that both HIF-1 and HIF-2 are, in a cell-type dependent manner, recruited to, and essential for, REDD1 induction. Interestingly, whereas mTORC1 is responsive to REDD1 in some tumors, strategies have evolved in others, such as mutations disrupting TSC1, to subvert mTORC1 inhibition by REDD1. Sequencing analyses of 77 ccRCCs for mutations in TSC1, TSC2 and REDD1, using PTEN as a reference, implicate the TSC1 gene, and possibly REDD1, as tumor suppressors in sporadic ccRCC. Understanding how ccRCCs become refractory to REDD1-induced mTORC1 inhibition should shed light into the development of ccRCC and may aid in patient selection for molecular targeted therapies.
doi:10.1158/1541-7786.MCR-11-0302
PMCID: PMC3234675  PMID: 21798997
REDD1; DDIT4; mTORC1; VHL; RCC; HIF; nucleolus
24.  Upregulation of TRAG3 gene in urothelial carcinoma of the bladder 
Conventional chemotherapy is commonly used for advanced stages of bladder cancer with modest success and high morbidity. Identifying markers of resistance will allow clinicians to tailor treatment to a specific patient population. T24-tumorigenic cell line was grown orthotopically in nude mice and monitored using bioluminescence imaging and microcomputed tomography until they developed metastases. Stable sublines were then developed from primary bladder (T24-P), lung (T24-L) and bone (T24-B) tissues. Chromosomal analysis and DNA microarray were used to characterize these sublines. qRT-PCR and immunohistochemistry (IHC) were used for validation. Epigenetic modifiers were used to study gene regulation. The cell viability was quantified with MTT assay. Chromosomal analysis revealed multiple alterations in metastatic cell lines compared to T24-P. DNA microarray analysis showed that Taxol-Resistance-Associated-Gene-3 (TRAG3) gene was the most upregulated gene. From qRT-PCR and IHC, TRAG3 was significantly higher in T24-L and T24-B than T24-P. TRAG3 gene expression is likely controlled by DNA methylation, but not histone acetylation. Interestingly, T24-B and T24-L cells were more resistant than T24-P to treatment with anti-microtubule agents such as docetaxel, paclitaxel and vinblastine. TRAG3 mRNA expression was higher in 20% of patients with ≤pT2 (n=10) and 60% of patients with ≥pT3 (n=20) compared to normal adjacent tissue (p=0.05). In addition, the median TRAG3 expression was 6.7-fold higher in ≥pT3 tumors compared to ≤pT2 tumors. Knowing the status of TRAG3 expression could help clinicians tailor treatment to a particular patient population that could benefit from treatment, while allocating patients with resistant tumors to new experimental therapies.
doi:10.1002/ijc.25631
PMCID: PMC3082622  PMID: 20734393
urothelial carcinoma; bladder; TRAG3; resistance
25.  Phase I Dose-Escalation Study of Stereotactic Body Radiation Therapy for Low- and Intermediate-Risk Prostate Cancer 
Journal of Clinical Oncology  2011;29(15):2020-2026.
Purpose
To evaluate the tolerability of escalating doses of stereotactic body radiation therapy in the treatment of localized prostate cancer.
Patients and Methods
Eligible patients included those with Gleason score 2 to 6 with prostate-specific antigen (PSA) ≤ 20, Gleason score 7 with PSA ≤ 15, ≤ T2b, prostate size ≤ 60 cm3, and American Urological Association (AUA) score ≤ 15. Pretreatment preparation required an enema and placement of a rectal balloon. Dose-limiting toxicity (DLT) was defined as grade 3 or worse GI/genitourinary (GU) toxicity by Common Terminology Criteria of Adverse Events (version 3). Patients completed quality-of-life questionnaires at defined intervals.
Results
Groups of 15 patients received 45 Gy, 47.5 Gy, and 50 Gy in five fractions (45 total patients). The median follow-up is 30 months (range, 3 to 36 months), 18 months (range, 0 to 30 months), and 12 months (range, 3 to 18 months) for the 45 Gy, 47.5 Gy, and 50 Gy groups, respectively. For all patients, GI grade ≥ 2 and grade ≥ 3 toxicity occurred in 18% and 2%, respectively, and GU grade ≥ 2 and grade ≥ 3 toxicity occurred in 31% and 4%, respectively. Mean AUA scores increased significantly from baseline in the 47.5-Gy dose level (P = .002) as compared with the other dose levels, where mean values returned to baseline. Rectal quality-of-life scores (Expanded Prostate Cancer Index Composite) fell from baseline up to 12 months but trended back at 18 months. In all patients, PSA control is 100% by the nadir + 2 ng/mL failure definition.
Conclusion
Dose escalation to 50 Gy has been completed without DLT. A multicenter phase II trial is underway treating patients to 50 Gy in five fractions to further evaluate this experimental therapy.
doi:10.1200/JCO.2010.31.4377
PMCID: PMC3138546  PMID: 21464418

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