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1.  Luteolin nanoparticle in chemoprevention – in vitro and in vivo anticancer activity 
Cancer prevention (chemoprevention) by using naturally occurring dietary agents has gained immense interest due to the broad safety window of these compounds. However, many of these compounds are hydrophobic and poorly soluble in water. They frequently display low bioavailability, poor systemic delivery, and low efficacy. To circumvent this problem, we explored a novel approach towards chemoprevention using nanotechnology to deliver luteolin, a natural compound present in green vegetables. We formulated water soluble polymer-encapsulated Nano-Luteolin from hydrophobic luteolin, and studied its anticancer activity against lung cancer and head and neck cancer. In vitro studies demonstrated that, like luteolin, Nano-Luteolin inhibited the growth of lung cancer cells (H292 cell line) and squamous cell carcinoma of head and neck (SCCHN) cells (Tu212 cell line). In Tu212 cells, the IC50 value of Nano-Luteolin was 4.13μM, and that of luteolin was 6.96μM. In H292 cells, the IC50 of luteolin was 15.56μM, and Nano-Luteolin was 14.96μM. In vivo studies using a tumor xenograft mouse model demonstrated that Nano-Luteolin has a significant inhibitory effect on the tumor growth of SCCHN in comparison to luteolin. Our results suggest that nanoparticle delivery of naturally occurring dietary agents like luteolin has many advantages and may have potential application in chemoprevention in clinical settings.
PMCID: PMC3888883  PMID: 24403290
Cancer; chemoprevention; luteolin; nanotechnology; nanomedicine
2.  P38 MAPK contributes to resistance and invasiveness of HER2-overexpressing breast cancer 
Current medicinal chemistry  2014;21(4):501-510.
Intrinsic or acquired resistance to the HER2-targeted therapy trastuzumab is a clinical concern in the treatment of patients with HER2-over-expressing metastatic breast cancers. We demonstrate here that multiple models of intrinsic and acquired resistance exhibit increased phosphorylation of p38 MAPK. Kinase inhibition of p38 rescued trastuzumab sensitivity in cells with acquired resistance. In addition, knockdown of p38 increased sensitivity to trastuzumab in an intrinsically resistant cell line. We previously reported that expression of growth differentiation factor 15 (GDF15) is increased in trastuzumab-resistant HER2-overexpressing breast cancer cells. In this study, we found that exogenous GDF15 or stable overexpression of GDF15 stimulated p38 phosphorylation in HER2-positive cells, suggesting a possible mechanism by which p38 is activated in resistant cells. GDF15 stable clones showed significantly increased invasiveness, which was rescued by p38 kinase inhibition, suggesting that p38 plays a role in the pro-invasive phenotype conferred by GDF15. Importantly, immunohistochemical analysis of a breast tumor tissue array indicated a significant (p=0.0053) correlation between HER2 and phosphorylated p38 specifically in GDF15-positive tissues. Our results suggest that p38 signaling drives trastuzumab resistance and invasiveness in HER2-overexpressing breast cancer. Upstream growth factor signals that have previously been implicated in trastuzumab resistance, such as GDF15, may contribute to the increased phosphorylation of p38 found in resistant cells.
PMCID: PMC4262834  PMID: 24251561
breast cancer; HER2; invasion; p38; trastuzumab
3.  Poly (ADP) ribose polymerase enzyme inhibitor, veliparib, potentiates chemotherapy and radiation in vitro and in vivo in small cell lung cancer 
Cancer Medicine  2014;3(6):1579-1594.
Poly (ADP) ribose polymerase (PARP) plays a key role in DNA repair and is highly expressed in small cell lung cancer (SCLC). We investigated the therapeutic impact of PARP inhibition in SCLC. In vitro cytotoxicity of veliparib, cisplatin, carboplatin, and etoposide singly and combined was determined by MTS in 9 SCLC cell lines (H69, H128, H146, H526, H187, H209, DMS53, DMS153, and DMS114). Subcutaneous xenografts in athymic nu/nu mice of H146 and H128 cells with relatively high and low platinum sensitivity, respectively, were employed for in vivo testing. Mechanisms of differential sensitivity of SCLC cell lines to PARP inhibition were investigated by comparing protein and gene expression profiles of the platinum sensitive and the less sensitive cell lines. Veliparib showed limited single-agent cytotoxicity but selectively potentiated (≥50% reduction in IC50) cisplatin, carboplatin, and etoposide in vitro in five of nine SCLC cell lines. Veliparib with cisplatin or etoposide or with both cisplatin and etoposide showed greater delay in tumor growth than chemotherapy alone in H146 but not H128 xenografts. The potentiating effect of veliparib was associated with in vitro cell line sensitivity to cisplatin (CC = 0.672; P = 0.048) and DNA-PKcs protein modulation. Gene expression profiling identified differential expression of a 5-gene panel (GLS, UBEC2, HACL1, MSI2, and LOC100129585) in cell lines with relatively greater sensitivity to platinum and veliparib combination. Veliparib potentiates standard cytotoxic agents against SCLC in a cell-specific manner. This potentiation correlates with platinum sensitivity, DNA-PKcs expression and a 5-gene expression profile.
PMCID: PMC4298385  PMID: 25124282
Carboplatin; cisplatin; etoposide; PARP; SCLC; veliparib (ABT-888)
4.  Targeted Iron-Oxide Nanoparticle for Photodynamic Therapy and Imaging of Head and Neck Cancer 
ACS nano  2014;8(7):6620-6632.
Photodynamic therapy (PDT) is a highly specific anticancer treatment modality for various cancers, particularly for recurrent cancers that no longer respond to conventional anticancer therapies. PDT has been under development for decades, but light-associated toxicity limits its clinical applications. To reduce the toxicity of PDT, we recently developed a targeted nanoparticle (NP) platform that combines a second-generation PDT drug, Pc 4, with a cancer targeting ligand, and iron oxide (IO) NPs. Carboxyl functionalized IO NPs were first conjugated with a fibronectin-mimetic peptide (Fmp), which binds integrin β1. Then the PDT drug Pc 4 was successfully encapsulated into the ligand-conjugated IO NPs to generate Fmp-IO-Pc 4. Our study indicated that both nontargeted IO-Pc 4 and targeted Fmp-IO-Pc 4 NPs accumulated in xenograft tumors with higher concentrations than nonformulated Pc 4. As expected, both IO-Pc 4 and Fmp-IO-Pc 4 reduced the size of HNSCC xenograft tumors more effectively than free Pc 4. Using a 10-fold lower dose of Pc 4 than that reported in the literature, the targeted Fmp-IO-Pc 4 NPs demonstrated significantly greater inhibition of tumor growth than nontargeted IO-Pc 4 NPs. These results suggest that the delivery of a PDT agent Pc 4 by IO NPs can enhance treatment efficacy and reduce PDT drug dose. The targeted IO-Pc 4 NPs have great potential to serve as both a magnetic resonance imaging (MRI) agent and PDT drug in the clinic.
PMCID: PMC4155749  PMID: 24923902
iron-oxide nanoparticle; Fmp-IO-Pc 4; photodynamic therapy; head and neck cancer; magnetic resonance imaging; integrin β1
5.  High-Risk Medulloblastoma: A Pediatric Oncology Group Randomized Trial of Chemotherapy Before or After Radiation Therapy (POG 9031) 
Journal of Clinical Oncology  2013;31(23):2936-2941.
To compare event-free survival (EFS) in children with high-risk medulloblastoma randomly assigned to receive either chemotherapy before radiation or chemotherapy after radiation.
Patients and Methods
One hundred twelve patients were randomly assigned to each arm. Criteria used to categorize patients as high risk included M1-4 disease by modified Chang staging classification, T3b/T4 disease, or greater than 1.5 cm3 of residual tumor after surgery. Postoperatively, children with high-risk medulloblastoma were randomly assigned to two arms, either chemotherapy entailing three cycles of cisplatin and etoposide before radiation (chemotherapy first [CT1]) or the same chemotherapy regimen after radiation (radiation therapy first [RT1]). Both groups received consolidation chemotherapy consisting of vincristine and cyclophosphamide.
The median follow-up time was 6.4 years. Five-year EFS was 66.0% in the CT1 arm and 70.0% in the RT1 arm (P = .54), and 5-year overall survival in the two groups was 73.1% and 76.1%, respectively (P = .47). In the CT1 arm, 40 of the 62 patients with residual disease achieved either complete or partial remission.
Five-year EFS did not differ significantly whether, after surgery, patients received chemotherapy before or after radiotherapy.
PMCID: PMC3732312  PMID: 23857975
6.  Phase II Study of Docetaxel in Combination with Everolimus for Second- or Third-Line Therapy of Advanced Non-Small-Cell Lung Cancer 
We conducted a phase II study of docetaxel in combination with everolimus, a mammalian target of rapamycin (mTOR) inhibitor, for salvage therapy of advanced non–small-cell lung cancer (NSCLC) based on promising preclinical and early-phase clinical data. Patients with advanced-stage NSCLC treated with one or two previous systemic therapy regimens were given docetaxel (60 mg/m2) and everolimus (5 mg orally once daily on days 1–19) every 3 weeks. Archived tumor specimens were evaluated for markers of mTOR pathway activation (total and phosphorylated mTOR, Akt, S6, eIF4e, and 4EBP1). Twenty-eight patients were enrolled (median age: 62 years; male: 13; Caucasians: 19; adenocarcinoma: 20; performance status 0, 3; performance status 1, 23; 1 previous regimen, 16). A median of 3.5 cycles of therapy was administered. Two patients experienced partial response and 15 had stable disease (clinical benefit rate, 70%). The 6-month progression-free survival rate was 5%, and the median overall survival was 9.6 months. Low pAkt expression correlated with clinical benefit rate (p = 0.01) but not with progression-free survival or overall survival. The combination of everolimus and docetaxel was tolerated well, but the efficacy was relatively modest in an unselected population of patients with NSCLC.
PMCID: PMC4126230  PMID: 23407561
Non–small-cell lung cancer; Docetaxel; Everolimus
7.  Progesterone and vitamin D: improvement after traumatic brain injury in middle-aged rats 
Hormones and behavior  2013;64(3):10.1016/j.yhbeh.2013.06.009.
Progesterone (PROG) and vitamin D hormone (VDH) have both shown promise in treating traumatic brain injury (TBI). Both modulate apoptosis, inflammation, oxidative stress, and excitotoxicity. We investigated whether 21 days of VDH deficiency would alter cognitive behavior after TBI and whether combined PROG and VDH would improve behavioral and morphological outcomes more than either hormone alone in VDH-deficient middle-aged rats given bilateral contusions of the medial frontal cortex. PROG (16 mg/kg) and VDH (5 µg/kg) were injected intraperitoneally 1 hour post-injury. Eight additional doses of PROG were injected subcutaneously over 7 days post-injury. VDH deficiency itself did not significantly reduce baseline behavioral functions or aggravate impaired cognitive outcomes. Combination therapy showed moderate improvement in preserving spatial and reference memory but was not significantly better than PROG monotherapy. However, combination therapy significantly reduced neuronal loss and the proliferation of reactive astrocytes, and showed better efficacy compared to VDH or PROG alone in preventing MAP-2 degradation. VDH+PROG combination therapy may attenuate some of the potential long-term, subtle, pathophysiological consequences of brain injury in older subjects.
PMCID: PMC3833454  PMID: 23896206
Aging; Combination treatments; Functional repair; Progesterone; Traumatic brain injury; Vitamin D deficiency; Vitamin D3 hormone
8.  Acetylated Tubulin (AT) as a Prognostic Marker in Squamous Cell Carcinoma of the Head and Neck 
Head and Neck Pathology  2013;8(1):66-72.
Acetylated tubulin (AT) expression has been proposed as a marker for sensitivity to taxane chemotherapy. We wanted to explore AT as a prognostic marker in squamous cell carcinoma of the head and neck (SCCHN). We assessed AT expression in archival tissue from our institutional tissue bank of primary SCCHN specimens. We also examined AT expression on pre-therapy tissues of patients with SCCHN receiving induction chemotherapy with docetaxel, cisplatin and 5FU (TPF IC). AT expression was assessed on archival cases of SCCHN with (N = 63) and without (N = 82) locoregional lymph node metastases (LNM). The predominant tumor site was oral cavity (52 %). Immunohistochemistry staining was based on staining intensity and percentage of tumor cells stained to create a weighted index (WI). A total of nine patients who received TPF IC were evaluable for response by RECIST and also had pre-therapy tissues available. A significant independent correlation between AT and tumor grade (p = 0.001) and primary location (p = 0.008) was noted. There was a trend of higher AT in patients with presence of LNM (p = 0.052) and a trend in improved OS for patients with an AT WI below the median compared to those above the median for patients with no LNM (p = 0.054). For patients treated with induction TPF, we observed an inverse correlation between AT expression and response to TPF IC (p = 0.0071). AT expression is correlated with tumor grade and primary site. There was an observed trend correlating AT with presence nodal metastases. The observed inverse correlation with response to taxane based chemotherapy needs validation in a larger sample size.
PMCID: PMC3950380  PMID: 23881549
Taxane sensitivity; Acetylated tubulin; Head and neck cancer; Induction therapy; Nodal metastases
9.  RRM2 Regulates Bcl-2 in Head and Neck and Lung Cancers: A Potential Target for Cancer Therapy 
Ribonucleotide reductase subunit M2 (RRM2) plays an active role in tumor progression. Recently, we reported that depletion of RRM2 by systemic delivery of a nanoparticle carrying RRM2-specific siRNA suppresses head and neck tumor growth. The aim of this study is to clarify the underlying mechanism by which RRM2 depletion inhibits tumor growth.
siRNA-mediated gene silencing was performed to downregulate RRM2. Immunoblotting, RT-PCR, confocal microscopy, tissue fractionation, gene overexpression and knockdown were employed to analyze critical apoptosis signaling. Conventional immunohistochemistry (IHC) and quantum dot-based IHF were applied to detect RRM2 and Bcl2 expression and localization in tissue samples from patients and mice.
Knockdown of RRM2 led to apoptosis through the intrinsic pathway in head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) cell lines. We demonstrated that Bcl-2 is a key determinant controlling apoptosis, both in vitro and in vivo and that RRM2 depletion significantly reduces Bcl-2 protein expression. We observed that RRM2 regulates Bcl-2 protein stability, with RRM2 suppression leading to increased Bcl-2 degradation, and identified their co-localization in HNSCC and NSCLC cells. In a total of 50 specimens each from HNSCC and NSCLC patients, we identified the co-localization of Bcl-2 and RRM2 and found a significant positive correlation between their expression in HNSCC (R=0.98, p<0.0001) and NSCLC (R=0.92, p<0.0001) tumor tissues.
Our novel findings add to the knowledge of RRM2 in regulating expression of the anti-apoptotic protein Bcl-2 and reveal a critical link between RRM2 and Bcl-2 in apoptosis signaling.
PMCID: PMC3747783  PMID: 23719266
RRM2; Bcl-2; RNAi; Apoptosis
10.  Dose Escalation with Overdose Control using a Quasi-Continuous Toxicity Score in Cancer Phase I Clinical Trials 
Contemporary clinical trials  2012;33(5):949-958.
Escalation with overdose control (EWOC) is a Bayesian adaptive design for selecting dose levels in cancer Phase I clinical trials while controlling the posterior probability of exceeding the maximum tolerated dose (MTD). EWOC has been used by clinicians to design many cancer Phase I clinical trials, see e.g [1-4]. However, this design treats the toxicity response as a binary indicator of dose limiting toxicity (DLT) and does not account for the number and specific grades of toxicities experienced by patients during the trial. Chen et al. (2010) proposed a novel toxicity score system to fully utilize all toxicity information using a normalized equivalent toxicity score (NETS). In this paper, we propose to incorporate NETS into EWOC using a quasi-Bernoulli likelihood approach to design cancer Phase I clinical trials. We call the design escalation with overdose control using normalized equivalent toxicity score (EWOC-NETS). Simulation results show that this design has good operating characteristics and improves the accuracy of MTD, trial efficiency, therapeutic effect, and overdose control relative to EWOC which is used as a representative of designs treating toxicity response as a binary indicator of DLT. We illustrate the performance of this design using real trial data in identifying the Phase II dose.
PMCID: PMC4046335  PMID: 22561391
Escalation with Overdose Control; Maximum Tolerated Dose; Multiple Toxicities; Quasi-continuous; Normalized Equivalent Toxicity Score; Toxicity Score System
11.  Clinical Efficacy of Targeted Biologic Agents as Second-Line Therapy of Advanced Thyroid Cancer 
The Oncologist  2013;18(12):1262-1269.
The authors report an experience using biologic agents as second-line treatment for advanced thyroid cancer and show that patients derived a modest additional benefit in comparison to the front-line treatment. The findings are relevant for the clinical management of patients and for future studies of second-line targeted therapy of thyroid cancer.
Learning Objectives
List the therapeutic opportunities with tyrosine kinase inhibitors for advanced thyroid cancer, especially in the frontline setting.Describe and discuss the current knowledge and experience with salvage therapy with biologic agents for thyroid cancer.
Targeted biologic agents showed clinically meaningful efficacy as front-line therapy for advanced radioiodine-refractory and medullary thyroid cancer. The clinical benefit of these agents beyond the front line has yet to be established.
We assessed the clinical benefit of targeted agents in patients with advanced differentiated and medullary thyroid cancer treated at a single academic cancer center. We determined efficacy and compared front-line and second-line benefit using biochemical and anatomic response, time to treatment failure, and progression-free survival (PFS). Statistical differences were assessed by t test and chi-square test. Survival curves were generated by the Kaplan-Meier method. Differences in survival were assessed using the log-rank test, and a p value <.05 was considered significant.
We identified 39 patients with advanced differentiated and medullary thyroid cancer treated with targeted biologic agents. Median age was 56.3 years. Overall, 25 men and 14 women participated. Histology showed 23% medullary and 77% differentiated cancer. Nineteen patients progressed on front-line therapy and subsequently received second-line therapy. Targeted agents conferred clinically meaningful benefit in the second-line setting in terms of biochemical response (13.3%), clinical benefit (83.3%), median time to treatment failure (4.0 months; 95% confidence interval: 2.6–8.2), and median PFS (4.6 months; 95% confidence interval: 3.2–8.2). Second-line benefit (median PFS) was more modest in comparison to the front-line setting in both genders (women: 3 months vs. 12.2 months; men: 6 months vs. 19.7 months), in differentiated cancers (4.1 months vs. 15.7 months), and with vascular targeting agents (4.4 months vs. 20.1 months).
Patients with advanced thyroid cancer derived meaningful clinical benefit from additional therapy with a biologic agent following disease progression on front-line targeted therapy.
PMCID: PMC3868420  PMID: 24153240
Thyroid cancer; Biologic agents; Second-line
12.  Real-World Effectiveness of Systemic Agents Approved for Advanced Non-Small Cell Lung Cancer: A SEER–Medicare Analysis 
The Oncologist  2013;18(5):600-610.
Disparity exists between patients with lung cancer enrolled in clinical trials and patients treated in the community setting. This study assessed the real-world effectiveness of cytotoxic agents that became available for the treatment of non-small cell lung cancer (NSCLC) in the last 2 decades using the Surveillance, Epidemiology, and End Results–Medicare database. Study findings support the effectiveness of currently approved drugs for the treatment of advanced NSCLC in the real-world oncology practice.
Disparity exists between patients with lung cancer enrolled in clinical trials and patients treated in the community setting. This study assessed the real-world effectiveness of cytotoxic agents that became available for the treatment of non-small cell lung cancer (NSCLC) in the last 2 decades.
We employed the linked Surveillance, Epidemiology, and End Results (SEER)–Medicare database for patients diagnosed with stage IIIB/IV NSCLC between 1988 and 2005 to assess the effectiveness of newly approved agents. Effectiveness of specific agents was assessed at time periods immediately following the approval of the agent for NSCLC: baseline, 1988–1994; platinum, 1995–1999; docetaxel, 1999–2003; pemetrexed and bevacizumab, 2004–2005. Significant associations between specific drug treatment and survival improvement were determined using the Kaplan-Meier method, Cox proportional hazard model, and propensity score analyses. Significant differences were established by log-rank test.
This analysis employed data from 143,548 patients by sex (58% male, 42% female), cancer stage (35% stage IIIB, 65% stage IV), and age (12% 20–64 years, 22% 65–69 years, 45% 70–79 years, 22% 80 years and older). There was temporal improvement in survival for patients treated with newly approved chemotherapy (1-year survival rates: 32.41% in 1988–1994, 32.95% in 1995–1998, 37.40% in 1999–2003, and 39.55% in 2004–2005). Patients treated with a newly approved drug during the relevant treatment era had a significant reduction in the risk of death when compared with patients treated with chemotherapy other than the newly approved agent (hazard ratios [95% confidence interval] were 0.76 [0.71–0.81] for platinum, 0.73 [0.70–0.75] for docetaxel, 0.40 [0.37–0.44] for pemetrexed, and 0.33 [0.27–0.40] for bevacizumab; p < .001). Propensity score adjustment did not significantly alter these results.
Currently approved drugs for the treatment of advanced NSCLC are associated with improved survival in the U.S. Medicare patient population. Our findings support the effectiveness of these agents in the real-world oncology practice.
PMCID: PMC3662852  PMID: 23635558
SEER–Medicare; Lung cancer; Chemotherapy; Effectiveness; Survival; Real world
13.  Soluble FAS ligand as a biomarker of disease recurrence in differentiated thyroid cancer 
Cancer  2013;119(8):1503-1511.
Reliable predictive biomarkers are required to address the challenge of disease recurrence following thyroid cancer surgery. We assessed the association of cellular- and serum-based immunologic mediators with thyroid cancer recurrence.
Leukocyte subset counts and immune regulatory cytokines were determined in peripheral blood samples using multiparameter flow cytometry and 51-panel multiplex ELISA (Luminex) assays, respectively. Functional activity of circulating B-, T- and NK lymphocytes was assessed ex vivo. Differences in mean biomarker levels between defined subject groups and correlations between biomarkers and cancer recurrence were assessed by t-test or Wilcoxon test and by univariate and multivariate analyses with Cox model. Optimal cut-off values of significantly correlated biomarkers that best predict recurrence post surgery were established by receiver operating characteristics and validated by the optimal cut-point determination algorithm.
We enrolled 35 patients (median age - 49.4 years; 24 females; 15 patients with disease recurrence) and 21 controls. Patients without recurrence had higher levels of soluble FAS ligand (sFASL), TGF-b, T-regs and PD-1/PD-L1 expressing leukocytes. sFASL (HR:0.60; 95%CI:0.38–0.95; p=0.031) and IFN-α (HR:1.55; 95%CI:1.03–2.34; p=0.038) showed significant association with cancer recurrence. There was a significant difference in PFS between patient groups stratified by sFASL optimal cut-point of 15 pg/ml (Logrank p=0.0009).
sFASL and IFN-α showed significant correlation with thyroid cancer recurrenceand may be useful for risk-adapted surveillance strategies for thyroid cancer.
PMCID: PMC3799834  PMID: 23340930
Thyroid; cancer; biomarker; recurrence; FASL
14.  Interruption of nuclear localization of ATBF1 during the histopathologic progression of head and neck squamous cell carcinoma 
Head & neck  2012;35(7):1007-1014.
The AT-motif Binding Factor 1 (ATBF1) gene is frequently altered at the genetic level in several types of cancer, but its protein expression and subcellular localization have not been well studied in human cancers including head and neck squamous cell carcinomas (HNSCCs).
ATBF1 expression and localization were examined in five cell lines and 197 clinical specimens of HNSCC, and correlated with pathological and clinical characteristics.
ATBF1 was predominantly localized in the nucleus of hyperplastic squamous epithelium. Whereas nuclear ATBF1 dramatically decreased in invasive tumors (p=0.0012), cytoplasmic ATBF1 levels progressively increased from dysplasia to invasive tumors (p<0.0001), and the increase correlated with poor survival. Reduced nuclear ATBF1 level was also detected in HNSCC cell lines.
Nuclear localization of ATBF1 is frequently interrupted in HNSCC, and the interruption is significantly associated with the progression of HNSCC. The cytoplasmic ATBF1 level could be useful for predicting patient survival.
PMCID: PMC3978689  PMID: 22791392
HNSCC; ATBF1; mislocalization; survival; tumor suppressor
16.  Chemoprevention of Head and Neck Cancer by Simultaneous Blocking of Epidermal Growth Factor Receptor and Cyclooxygenase-2 Signaling Pathways: Preclinical and Clinical Studies 
We investigated the efficacy and underlying molecular mechanism of a novel chemopreventive strategy combining epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with cyclooxygenase-2 inhibitor (COX-2I).
Experimental Design
We examined the inhibition of tumor cell growth by combined EGFR-TKI (erlotinib) and COX-2I (celecoxib) treatment using head and neck cancer (HNC) cell lines and a preventive xenograft model. We studied the antiangiogenic activity of these agents and examined the affected signaling pathways by immunoblotting analysis in tumor cell lysates and immunohistochemistry (IHC) and enzyme immunoassay (EIA) analyses on the mouse xenograft tissues and blood, respectively. Biomarkers in these signaling pathways were studied by IHC, EIA, and an antibody array analysis in samples collected from participants in a phase I chemoprevention trial of erlotinib and celecoxib.
The combined treatment inhibited HNC cell growth significantly more potently than either single agent alone in cell line and xenograft models, and resulted in greater inhibition of cell cycle progression at G1 phase than either single drug. The combined treatment modulated the EGFR and mTOR signaling pathways. A phase I chemoprevention trial of combined erlotinib and celecoxib revealed an overall pathologic response rate of 71% at time of data analysis. Analysis of tissue samples from participants consistently showed downregulation of EGFR, pERK and pS6 levels after treatment, which correlated with clinical response.
Treatment with erlotinib combined with celecoxib offers an effective chemopreventive approach through inhibition of EGFR and mTOR pathways, which may serve as potential biomarkers to monitor the intervention of this combination in the clinic.
PMCID: PMC3693760  PMID: 23422093
17.  Arylsulfonamide KCN1 inhibits in vivo glioma growth and interferes with HIF signaling by disrupting HIF-1α interaction with co-factors p300/CBP 
The hypoxia inducible factor-1 (HIF-1) plays a critical role in tumor adaptation to hypoxia, and its elevated expression correlates with poor prognosis and treatment failure in cancer patients. In this study, we determined whether 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, KCN1, the lead inhibitor in a novel class of arylsulfonamide inhibitors of the HIF-1 pathway, had anti-tumorigenic properties in vivo and further defined its mechanism of action.
Experimental Design
We studied the inhibitory effect of systemic KCN1 delivery on the growth of human brain tumors in mice. To define mechanisms of KCN1 anti-HIF activities, we examined its influence on the assembly of a functional HIF1α/HIF1β/p300 transcription complex.
KCN1 specifically inhibited HIF reporter gene activity in several glioma cell lines at the nanomolar level. KCN1 also downregulated transcription of endogenous HIF-1 target genes, such as VEGF, Glut-1 and carbonic anhydrase 9, in an HRE-dependent manner. KCN1 potently inhibited the growth of subcutaneous malignant glioma tumor xenografts with minimal adverse effects on the host. It also induced a temporary survival benefit in an intracranial model of glioma but had no effect in a model of melanoma metastasis to the brain. Mechanistically, KCN1 did not down-regulate levels of HIF-1α or other components of the HIF transcriptional complex; rather, it antagonized hypoxia-inducible transcription by disrupting the interaction of HIF-1α with transcriptional co-activators p300/CBP.
Our results suggest that the new HIF pathway inhibitor KCN1 has antitumor activity in mouse models, supporting its further translation for the treatment of human tumors displaying hypoxia or HIF overexpression.
PMCID: PMC3518680  PMID: 22923450
18.  Wnt signaling in triple negative breast cancer is associated with metastasis 
BMC Cancer  2013;13:537.
Triple Negative subset of (TN) Breast Cancers (BC), a close associate of the basal-like subtype (with limited discordance) is an aggressive form of the disease which convey unpredictable, and poor prognosis due to limited treatment options and lack of proven effective targeted therapies.
We conducted an expression study of 240 formalin-fixed, paraffin-embedded (FFPE) primary biopsies from two cohorts, including 130 TN tumors, to identify molecular mechanisms of TN disease.
The annotation of differentially expressed genes in TN tumors contained an overrepresentation of canonical Wnt signaling components in our cohort and others. These observations were supported by upregulation of experimentally induced oncogenic Wnt/β-catenin genes in TN tumors, recapitulated using targets induced by Wnt3A. A functional blockade of Wnt/β-catenin pathway by either a pharmacological Wnt-antagonist, WntC59, sulidac sulfide, or β-catenin (functional read out of Wnt/β-catenin pathway) SiRNA mediated genetic manipulation demonstrated that a functional perturbation of the pathway is causal to the metastasis- associated phenotypes including fibronectin-directed migration, F-actin organization, and invasion in TNBC cells. A classifier, trained on microarray data from β-catenin transfected mammary cells, identified a disproportionate number of TNBC breast tumors as compared to other breast cancer subtypes in a meta-analysis of 11 studies and 1,878 breast cancer patients, including the two cohorts published here. Patients identified by the Wnt/β-catenin classifier had a greater risk of lung and brain, but not bone metastases.
These data implicate transcriptional Wnt signaling as a hallmark of TNBC disease associated with specific metastatic pathways.
PMCID: PMC4226307  PMID: 24209998
Breast cancer; Triple negative; Wnt; FFPE; Microarray
19.  Heterozygous deletion of Atbf1 by the Cre-loxP system in mice causes preweaning mortality 
Genesis (New York, N.Y. : 2000)  2012;50(11):819-827.
ATBF1 is a large nuclear protein that contains multiple zinc-finger motifs and four homeodomains. In mammals, ATBF1 regulates differentiation, and its mutation and/or downregulation is involved in tumorigenesis in several organs. To gain more insight into the physiological functions of ATBF1, we generated and validated a conditional allele of mouse Atbf1 in which exons 7 and 8 were flanked by loxP sites (Atbf1flox). Germline deletion of a single Atbf1 allele was achieved by breeding to EIIa-cre transgenic mice, and Atbf1 heterozygous mice displayed reduced body weight, preweaning mortality, increased cell proliferation, and attenuated cytokeratin 18 (CK18) expression, indicating haploinsufficiency of Atbf1. Floxed Atbf1 mice will help us understand such biological processes as neuronal differentiation and tumorigenesis.
PMCID: PMC3443507  PMID: 22644989
Atbf1; Cre-loxP; conditional knockout; heterozygous; preweaning mortality
20.  The Pivotal Role of Integrin β1 in Metastasis of Head and Neck Squamous Cell Carcinoma 
This study aimed to understand the prognostic value of integrin β1 expression in head and neck squamous cell carcinoma (HNSCC) and the mechanism underlying its association with metastatic HNSCC.
Experimental Design
Archival HNSCC tissues including 99 non-metastatic primary tumors and 101 metastatic primary tumors were examined for the association of integrin β1 expression with metastasis and disease prognosis by appropriate statistical methods. Fluorescence activated cell sorting was used to separate the integrin β1high/+ cell population from the integrin β1low/− population in HNSCC cell lines. These two populations and integrin β1 shRNA knock-down HNSCC cells were examined for the effect of integrin β1 on invasion in vitro and on lymph node and lung metastases in a xenograft mouse model. Expression and activation of matrix metalloproteinases (MMPs) were examined by zymography.
Statistical analysis showed that integrin β1 expression was significantly higher in the metastatic primary tumors than in the non-metastatic tumors (42.6% vs 24.8%, p<0.0001 and p<0.0001 by univariate and multivariate analyses, respectively). In patients with lymph node metastasis, integrin β1 expression was inversely correlated with overall survival (p=0.035). The integrin β1 knock-down or integrin β1low/− HNSCC cells showed a significant reduction in lymph node and lung metastases in vivo (p<0.001 and p<0.05, respectively). Significantly reduced matrigel invasion capability was also found in integrin β1 knock-down or integrin β1low/− HNSCC cells (p< 0.01). Finally, zymography results showed integrin β1 affected HNSCC invasion by regulating MMP-2 activation.
These findings indicate that integrin β1 has a major impact on HNSCC prognosis through its regulation of metastasis.
PMCID: PMC3462074  PMID: 22829201
21.  Single agent maintenance therapy for advanced stage non-small cell lung cancer: A meta-analysis 
Maintenance therapy is a new treatment paradigm for advanced non-small cell lung cancer (NSCLC). We conducted a meta-analysis of randomized studies with single agent maintenance therapy.
An electronic literature search of public databases (MEDLINE, EMBASE, Cochrane library) and manual search of relevant conference proceedings was performed. A formal meta-analysis was conducted using Comprehensive Meta Analysis software (Version 2.0). Outcome data were pooled and reported as hazard ratio (HR). The primary outcome of interest was overall survival (OS) and secondary outcome was progression free survival (PFS).
Twelve studies were included (5 meeting abstracts, 7 full manuscripts) with a total of 4286 patients (maintenance arm/control arm- 2449/1837, median age 61 years, males -69 %). The OS (HR 0.86, 95% confidence intervals [CI] 0.80-0.92; P=0.0003) and PFS (HR 0.80, 95% CI 0.77-0.84; P<0.0001) were superior with maintenance therapy. ‘Switch’ maintenance was associated with significant OS and PFS improvement (OS HR 0.84, 95% CI 0.77-0.91; P=0.00026; PFS HR 0.62, 95% CI 0.57-0.67; P<0.0001). Despite a modest improvement in PFS (HR 0.90, 95%CI 0.85-0.95; P=0.007), “continuation” maintenance was not associated with survival benefit (HR 0.927, 95%CI 0.78-1.09; P=0.33). Improvements in OS and PFS were observed with both EGFR-targeted agents (HR 0.83, 95% CI 0.74-0.92; P=0.004; HR 0.64, 95% CI 0.58-0.71 P<0.0001) and cytotoxic agents (HR 0.89, 95% CI 0.80-0.98; P=0.018; HR 0.85, 95% CI 0.80-0.89; P < 0.0001).
Single agent maintenance therapy improves overall survival, though statistical significance was only noted with ‘switch’ maintenance.
PMCID: PMC3396772  PMID: 22546678
Maintenance therapy; non-small cell lung cancer; NSCLC; EGFR; pemetrexed; erlotinib; gefitinib
22.  Comparison of Quantum Dot Technology with Conventional Immunohistochemistry in Examining Aldehyde Dehydrogenase 1A1 as a Potential Biomarker for Lymph Node Metastasis of Head and Neck Cancer 
European Journal of Cancer  2012;48(11):1682-1691.
This study explored whether expression of aldehyde dehydrogenase 1 (ALDH1A1) in the primary tumor correlated with lymph node metastasis (LNM) of squamous cell carcinoma of the head and neck (HNSCC). We used both quantum dot (QD)-based immunohistofluorescence (IHF) and conventional immunohistochemistry (IHC) to quantify ALDH1A1 expression in primary tumor samples taken from 96 HNSCC patients, 50 with disease in the lymph nodes and 46 without. The correlation between the quantified level of ALDH1A1 expression and LNM in HNSCC patients was evaluated with univariate and multivariate analysis. The prognostic value of ALDH1A1 was examined by Kaplan-Meier analysis and Wald test. ALDH1A1 was highly correlated with LNM in HNSCC patients (p < 0.0001 by QD-based IHF and 0.039 by IHC). The two methods (QD-based IHF and conventional IHC) for quantification of ALDH1A1 were found to be comparable (R = 0.75, p < 0.0001), but QD-IHF was more sensitive and objective than IHC. The HNSCC patients with low ALDH1A1 expression had a higher 5-year survival rate than those with high ALDH1A1 level (p = 0.025). Our study suggests that ALDH1A1 is a potential biomarker for predicting LNM in HNSCC patients, though it is not an independent prognostic factor for survival of HNSCC patients. Furthermore, QD-IHF has advantages over IHC in quantification of ALDH1A1 expression in HNSCC tissues.
PMCID: PMC3381072  PMID: 22341992
Aldehyde dehydrogenase 1; biomarker; metastasis; immunohistochemistry; head and neck cancer; quantum dot; nanotechnology
23.  A systematic analysis of efficacy of second line chemotherapy in sensitive and refractory small cell lung cancer 
Journal of Thoracic Oncology  2012;7(5):866-872.
SCLC patients unresponsive or relapsing within 90 days following frontline chemotherapy have poor prognosis and are treated with regimens different than the first-line regimen. Potential differences in the efficacy of second line therapy for refractory and sensitive SCLC have not been well studied.
Studies that enrolled sensitive and refractory (relapse more than or less than 90 days) SCLC patients for second-line therapy were identified using electronic databases (MEDLINE, EMBASE, and Cochrane library) and meeting abstracts databases. A systematic analysis was conducted using Comprehensive Meta Analysis (Version 2.2.048) software to calculate the Odds ratio of response and 95% confidence limits. Median overall survival time for sensitive and resistant SCLC patients was compared by 2-sided Student’s T-Test. We tested for significant heterogeneity by Cochran’s chi-square test and I square index.
Twenty one studies published between 1984 and 2011 were eligible for this analysis with a total of 1692 patients enrolled; 912 with sensitive and 780 with refractory SCLC. The overall RR was 17.9% with a higher RR of 27.7% (range: 0 – 77%) for sensitive SCLC versus 14.8% (range: 0 – 70%) for refractory patients; p=0.0001. Pooled overall Odds ratio of response was 2.235 (95% CI: 1.518 – 3.291; p=0.001) favoring patients with sensitive disease. Median overall survival time was 6.7 months with a weighted survival of 7.7 and 5.4 months for sensitive and refractory SCLC respectively (p=0.0035).
Refractory SCLC patients derive modest clinical benefit from second line chemotherapy. However, response and survival outcomes are superior with chemosensitive disease.
PMCID: PMC3381878  PMID: 22722788
small cell lung cancer; chemotherapy; sensitive; resistant; refractory
24.  Reliability of quantitative ultrasonic assessment of normal-tissue toxicity in breast cancer radiotherapy 
We have recently reported that ultrasound imaging, together with ultrasound tissue characterization (UTC), can provide quantitative assessment of radiation-induced normal-tissue toxicity. This study’s purpose is to evaluate the reliability of our quantitative ultrasound technology in assessing acute and late normal-tissue toxicity in breast cancer radiotherapy.
Method and Materials
Our ultrasound technique analyzes radio-frequency echo signals and provides quantitative measures of dermal, hypodermal, and glandular-tissue toxicities. To facilitate easy clinical implementation, we further refined this technique by developing a semi-automatic ultrasound-based toxicity assessment tool (UBTAT). Seventy-two ultrasound studies of 26 patients (720 images) were analyzed. Images of 8 patients were evaluated for acute toxicity (<6 months post radiotherapy) and those of 18 patients were evaluated for late toxicity (≥6 months post radiotherapy). All patients were treated according to a standard radiotherapy protocol. To assess intra-observer reliability, one observer analyzed 720 images in UBTAT and then repeated the analysis 3 months later. To assess inter-observer reliability, three observers (two radiation oncologists and one ultrasound expert) each analyzed 720 images in UBTAT. An intraclass correlation coefficient (ICC) was used to evaluate intra- and inter-observer reliability. Ultrasound assessment and clinical evaluation were also compared.
Intra-observer ICC was 0.89 for dermal toxicity, 0.74 for hypodermal toxicity, and 0.96 for glandular-tissue toxicity. Inter-observer ICC was 0.78 for dermal toxicity, 0.74 for hypodermal toxicity, and 0.94 for glandular-tissue toxicity. Statistical analysis found significant changes in dermal (p < 0.0001), hypodermal (p=0.0027), and glandular-tissue (p < 0.0001) assessments in the acute toxicity group. Ultrasound measurements correlated with clinical RTOG toxicity scores of patients in the late toxicity group. Patients with RTOG grade 1 or 2 had greater ultrasound-assessed toxicity percentage changes than patients with RTOG grade 0.
Early and late radiation-induced effects on normal tissue can be reliably assessed using quantitative ultrasound.
PMCID: PMC3156354  PMID: 21435799
breast cancer; radiation toxicity; ultrasound; tissue characterization
25.  Characterization of primary prostate carcinoma by anti-1-amino-2-[18F] -fluorocyclobutane-1-carboxylic acid (anti-3-[18F] FACBC) uptake 
Anti-1-amino-3-[18F] fluorocyclobutane-1-carboxylic acid (anti-3-[18F] FACBC) is a synthetic amino acid positron emission tomography (PET) radiotracer with utility in the detection of recurrent prostate carcinoma. The aim of this study is to correlate uptake of anti-3-[18F] FACBC with histology of prostatectomy specimens in patients undergoing radical prostatectomy and to determine if uptake correlates to markers of tumor aggressiveness such as Gleason score. Ten patients with prostate carcinoma pre-radical prostatectomy underwent 45 minute dynamic PET-CT of the pelvis after IV injection of 347.8 ± 81.4 MBq anti-3-[18F] FACBC. Each prostate was co-registered to a separately acquired MR, divided into 12 sextants, and analyzed visually for abnormal focal uptake at 4, 16, 28, and 40 min post-injection by a single reader blinded to histology. SUVmax per sextant and total sextant activity (TSA) was also calculated. Histology and Gleason scores were similarly recorded by a urologic pathologist blinded to imaging. Imaging and histologic analysis were then compared. In addition, 3 representative sextants from each prostate were chosen based on highest, lowest and median SUVmax for immunohistochemical (IHC) analysis of Ki67, synaptophysin, P504s, chromogranin A, P53, androgen receptor, and prostein. 79 sextants had malignancy and 41 were benign. Highest combined sensitivity and specificity was at 28 min by visual analysis; 81.3% and 50.0% respectively. SUVmax was significantly higher (p<0.05) for malignant sextants (5.1±2.6 at 4 min; 4.5±1.6 at 16 min; 4.0±1.3 at 28 min; 3.8±1.0 at 40 min) compared to non-malignant sextants (4.0±1.9 at 4 min; 3.5±0.8 at 16 min; 3.4±0.9 at 28 min; 3.3±0.9 at 40 min), though there was overlap of activity between malignant and non-malignant sextants. SUVmax also significantly correlated (p<0.05) with Gleason score at all time points (r=0.28 at 4 min; r=0.42 at 16 min; r=0.46 at 28 min; r=0.48 at 40 min). There was no significant correlation of anti-3-[18F] FACBC SUVmax with Ki-67 or other IHC markers. Since there was no distinct separation between malignant and non-malignant sextants or between Gleason score levels, we believe that anti-3-[18F] FACBC PET should not be used alone for radiation therapy planning but may be useful to guide biopsy to the most aggressive lesion.
PMCID: PMC3545368  PMID: 23342303
Positron emission tomography (PET); prostate carcinoma; anti-3-[18F] FACBC

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