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1.  Translational Phase I Trial of Vorinostat (Suberoylanilide Hydroxamic Acid) Combined with Cytarabine and Etoposide in Patients with Relapsed, Refractory, or High-Risk Acute Myeloid Leukemia 
To determine the maximum-tolerated dose (MTD) of the histone deacetylase inhibitor vorinostat combined with fixed doses of cytarabine (ara-C or cytosine arabinoside) and etoposide in patients with poor-risk or advanced acute leukemia, to obtain preliminary efficacy data, describe pharmacokinetics, and in vivo pharmacodynamic effects of vorinostat in leukemia blasts.
Experimental Design
In this open-label phase I study, vorinostat was given orally on days one to seven at three escalating dose levels: 200 mg twice a day, 200 mg three times a day, and 300 mg twice a day. On days 11 to 14, etoposide (100 mg/m2) and cytarabine (1 or 2 g/m2 twice a day if ≥65 or <65 years old, respectively) were given. The study used a standard 3+3 dose escalation design.
Eighteen of 21 patients with acute myelogenous leukemia (AML) treated on study completed planned therapy. Dose-limiting toxicities [hyperbilirubinemia/septic death (1) and anorexia/fatigue (1)] were encountered at the 200 mg three times a day level; thus, the MTD was established to be vorinostat 200 mg twice a day. Of 21 patients enrolled, seven attained a complete remission (CR) or CR with incomplete platelet recovery, including six of 13 patients treated at the MTD. The median remission duration was seven months. No differences in percentage S-phase cells or multidrug resistance transporter (MDR1 or BCRP) expression or function were observed in vivo in leukemia blasts upon vorinostat treatment.
Vorinostat 200 mg twice a day can be given safely for seven days before treatment with cytarabine and etoposide. The relatively high CR rate seen at the MTD in this poor-risk group of patients with AML warrants further studies to confirm these findings.
PMCID: PMC4332848  PMID: 23403629
2.  Blocking Immunosuppressive Checkpoints for Glioma Therapy: The More the Merrier! 
Immunosuppressive checkpoints mediated by IDO, CTLA4, and PD1/PDL1 play a critical role in glioma progression and the efficacy of immunotherapies. Combined blockade of these immunosuppressive checkpoints in a glioma model elicited long-term survival. This combined blockade adds to the armamentarium of anti-glioma therapies, which could be implemented in clinical trials.
PMCID: PMC4330468  PMID: 24879798
3.  A phase 1 study of the heat shock protein 90 inhibitor retaspimycin hydrochloride (IPI-504) in patients with gastrointestinal stromal tumors or soft tissue sarcomas 
Heat shock protein 90 (Hsp90) is required for the proper folding, function, and stability of various client proteins, two of which (KIT and PDGFRα) are critical in the pathogenesis and progression of gastrointestinal stromal tumors (GIST). This phase 1 study investigated the safety and maximum tolerated dose (MTD) of retaspimycin hydrochloride (IPI-504), a novel potent and selective Hsp90 inhibitor, in patients with metastatic and/or unresectable GIST or other soft-tissue sarcomas (STS).
Experimental Design
IPI-504 was administered intravenously at doses ranging from 90 to 500 mg/m2 twice weekly for 2 weeks on/1 week off. Safety, pharmacokinetic, and pharmacodynamic profiles were determined. Response was assessed by Response Evaluation Criteria for Solid Tumors (RECIST) 1.0 and optionally via 18-fluorodeoxyglucose positron emission tomography (18-FDG-PET) imaging.
Fifty-four patients received IPI-504; 37 with GIST and 17 with other STS. The MTD was 400 mg/m2 twice weekly for 2 weeks on/1 week off. Common related adverse events were fatigue (59%), headache (44%), and nausea (43%). Exposure to IPI-504, 17-AAG, and 17-AG increased with IPI-504 dose. Stable disease (SD) was observed in 70% (26/37) of patients with GIST and 59% (10/17) of patients with STS. There was one confirmed partial response (PR) in a patient with GIST and one PR in a patient with liposarcoma. Metabolic partial responses occurred in 11/29 (38%) of GIST patients.
In this study of advanced GIST or other STS, IPI-504 was generally well-tolerated with some evidence of anti-tumor activity, serving as a clinical proof-of-concept that HSP90 inhibition remains a promising strategy.
PMCID: PMC4330562  PMID: 24045182
IPI-504; Hsp90; GIST; STS; phase 1; retaspimycin hydrochloride
4.  Inhibition of Established Micrometastases by Targeted Drug Delivery via Cell Surface Associated GRP78 
The major cause of morbidity in breast cancer is development of metastatic disease, for which few effective therapies exist. Since tumor cell dissemination is often an early event in breast cancer progression and can occur prior to diagnosis, new therapies need to focus on targeting established metastatic disease in secondary organs. We report an effective therapy based on targeting cell surface-localized glucose regulated protein 78 (GRP78). GRP78 is expressed normally in the endoplasmic reticulum, but many tumors and disseminated tumor cells are subjected to environmental stresses and exhibit elevated levels of GRP78, some of which is localized at the plasma membrane.
Experimental Design and Results
Here we show that matched primary tumors and metastases from patients who died from advanced breast cancer also express high levels of GRP78. We utilized a peptidomimetic targeting strategy that employs a known GRP78-binding peptide fused to a pro-apoptotic moiety (designated BMTP78) and show that it can selectively kill breast cancer cells that express surface-localized GRP78. Further, in preclinical metastasis models, we demonstrate that administration of BMTP78 can inhibit primary tumor growth as well as prolong overall survival by reducing the extent of outgrowth of established lung and bone micrometastases.
The data presented here provide strong evidence that it is possible to induce cell death in established micrometastases by peptide mediated targeting of cell surface localized GRP in advanced breast cancers. The significance to patients with advanced breast cancer of a therapy that can reduce established metastatic disease should not be underestimated.
PMCID: PMC4331071  PMID: 23470966
Breast cancer; metastasis; GRP78; targeted therapy; cell stress
5.  Therapeutic Potential of HSP90 Inhibition for Neurofibromatosis Type 2 
The growth and survival of neurofibromatosis type 2 (NF2)–deficient cells are enhanced by the activation of multiple signaling pathways including ErbBs/IGF-1R/Met, PI3K/Akt, and Ras/Raf/Mek/Erk1/2. The chaperone protein HSP90 is essential for the stabilization of these signaling molecules. The aim of the study was to characterize the effect of HSP90 inhibition in various NF2-deficient models.
Experimental Design
We tested efficacy of the small-molecule NXD30001, which has been shown to be a potent HSP90 inhibitor. The antiproliferative activity of NXD30001 was tested in NF2-deficient cell lines and in human primary schwannoma and meningioma cultures in vitro. The antitumor efficacy of HSP90 inhibition in vivo was verified in two allograft models and in one NF2 transgenic model. The underlying molecular alteration was further characterized by a global transcriptome approach.
NXD30001 induced degradation of client proteins in and suppressed proliferation of NF2-deficient cells. Differential expression analysis identified subsets of genes implicated in cell proliferation, cell survival, vascularization, and Schwann cell differentiation whose expression was altered by NXD30001 treatment. The results showed that NXD30001 in NF2-deficient schwannoma suppressed multiple pathways necessary for tumorigenesis.
HSP90 inhibition showing significant antitumor activity against NF2-related tumor cells in vitro and in vivo represents a promising option for novel NF2 therapies.
PMCID: PMC4331126  PMID: 23714726
6.  Rb Loss is Characteristic of Prostatic Small Cell Neuroendocrine Carcinoma 
Small cell neuroendocrine carcinoma of the prostate is likely to become increasingly common with recent advances in pharmacologic androgen suppression. Thus, developing molecular markers of small cell differentiation in prostate cancer will be important to guide diagnosis and therapy of this aggressive tumor.
Experimental Design
We examined the status of RB1, TP53 and PTEN in prostatic small cell and acinar carcinomas via immunohistochemistry (IHC), copy number alteration analysis and sequencing of formalin fixed paraffin-embedded specimens.
We found Rb protein loss in 90% (26/29) of small cell carcinoma cases with RB1 allelic loss in 85% (11/13) of cases. Of acinar tumors occurring concurrently with prostatic small cell carcinoma, 43% (3/7) showed Rb protein loss. In contrast, only 7% (10/150) of primary high grade acinar carcinomas, 11% (4/35) of primary acinar carcinomas with neuroendocrine differentiation, and 15% (2/13) of metastatic castrate resistant acinar carcinomas showed Rb protein loss. Loss of PTEN protein was seen in 63% (17/27) of small cell carcinomas, with 38% (5/13) showing allelic loss. By IHC, accumulation of p53 was observed in 56% (14/25) of small cell carcinomas, with 60% (6/10) of cases showing TP53 mutation.
Loss of RB1 by deletion is a common event in prostatic small cell carcinoma and can be detected by validated IHC assay. As Rb protein loss rarely occurs in high grade acinar tumors, these data suggest that Rb loss is a critical event in the development of small cell carcinomas and may be a useful diagnostic and potential therapeutic target.
PMCID: PMC3931005  PMID: 24323898
Prostatic adenocarcinoma; small cell carcinoma; tumor suppressor; RB1; TP53; PTEN
7.  Cellular uptake of imatinib into leukemic cells is independent of human organic cation transporter 1 (OCT1) 
In addition to mutated BCR-ABL1 kinase, the organic cation transporter 1 (OCT1, encoded by SLC22A1) has been considered to contribute to imatinib resistance in patients with chronic myeloid leukemia (CML). Since data are conflicting as to whether OCT1 transports imatinib and may serve as clinical biomarker we used a combination of different approaches including animal experiments to elucidate comprehensively the impact of OCT1 on cellular imatinib uptake.
Experimental Design
Transport of imatinib was studied using OCT1-expressing Xenopus oocytes, mammalian cell lines (HEK293, MDCK, V79) stably expressing OCT1, human leukemic cells, and Oct1-knockout mice. OCT1 mRNA and protein expression were analyzed in leukemic cells from imatinib naïve CML patients as well as in cell lines.
Transport and inhibition studies showed that overexpression of functional OCT1 protein in Xenopus oocytes or mammalian cell lines did not lead to an increased cellular accumulation of imatinib. The CML cell lines (K562, Meg-01, LAMA84) and leukemic cells from patients expressed neither OCT1 mRNA nor protein as demonstrated by immunoblotting and immunofluorescence microscopy, yet they showed a considerable imatinib uptake. Oct1 deficiency in mice had no influence on plasma and hepatic imatinib concentrations.
These data clearly demonstrate that cellular uptake of imatinib is independent of OCT1 and therefore OCT1 is apparently not a valid biomarker for imatinib resistance.
PMCID: PMC3932302  PMID: 24352644
imatinib resistance; organic cation transporter 1; CML; drug transport; SLC22A1
8.  EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebral glioma and generates host immunity against tumor-antigen loss 
Chimeric antigen receptor (CAR) transduced T cells represent a promising immune therapy that has been shown to successfully treat cancers in mice and humans. However, CARs targeting antigens expressed in both tumors and normal tissues have led to significant toxicity. Preclinical studies have been limited by the use of xenograft models that do not adequately recapitulate the immune system of a clinically relevant host. EGFRvIII is a constitutively activated mutant of the naturally occurring epidermal growth factor receptor and is antigenically identical in both human and mouse glioma, but is also completely absent from any normal tissues.
Experimental Design
We developed a third-generation, EGFRvIII-specific murine CAR (mCAR), and performed tests to determine its efficacy in a fully immune-competent mouse model of malignant glioma.
At elevated doses, infusion with EGFRvIII mCAR T cells led to cures in all mice with brain tumors. Additionally, antitumor efficacy was found to be dependent on lymphodepletive host conditioning. Selective blockade with EGFRvIII soluble peptide significantly abrogated the activity of EGFRvIII mCAR T cells in vitro and in vivo, and may offer a novel strategy to enhance the safety profile for CAR-based therapy. Lastly, mCAR-treated, cured mice were resistant to rechallenge with EGFRvIIINEG tumors, suggesting generation of host immunity against additional tumor antigens.
All together, these data support that third-generation, EGFRvIII specific mCARs are effective against gliomas in the brain and highlight the importance of syngeneic, immune-competent models in the preclinical evaluation of tumor immunotherapies.
PMCID: PMC3943170  PMID: 24352643
central nervous system neoplasms; epidermal growth factor receptor; immunotherapy; glioma; glioblastoma; GBM; chimeric antigen receptor; CAR; murine model
9.  Are Short Telomeres Hallmarks of Cancer Recurrence? 
Exposure to radiation and some chemotherapeutic agents is associated with an increased risk of developing second cancers. Short telomeres are almost universally associated with malignant cancer progression. An unanswered question is whether inherited short telomeres or therapy related telomere shortening is a biomarker of development of second malignant neoplasms.
PMCID: PMC3944321  PMID: 24366690
10.  Complement Activation and Intraventricular Rituximab Distribution in Recurrent Central Nervous System Lymphoma 
To elucidate the mechanistic basis for efficacy of intrathecal rituximab. We evaluated complement activation as well as the pharmacokinetics of intraventricular rituximab in patients who participated in two phase 1 multicenter studies.
Experimental Design
We evaluated complement activation as a candidate mediator of rituximab within the CNS. Complement C3 and C5b-9 were quantified by ELISA in serial CSF specimens after intraventricular rituximab administration. We determined rituximab concentration profiles in CSF and serum. A population three- compartment pharmacokinetic model was built to describe the disposition of rituximab following intraventricular administration. The model was derived from results of the first trial and validated with results of the second trial.
Complement C3 and C5b-9 were reproducibly activated in CSF after intraventricular rituximab. Ectopic expression of C3 mRNA and protein within CNS lymphoma lesions was localized to myeloid cells. Constitutive high C3 activation at baseline was associated with adverse prognosis. A PK model was built which contains three distinct compartments to describe the distribution of rituximab within the neuroaxis after intraventricular administration.
We provide the first evidence of C3 activation within the neuroaxis with intraventricular immunotherapy and suggest that complement may contribute to immunotherapeutic responses of rituximab in CNS lymphoma. Penetration of rituximab into neural tissue is supported by this pharmacokinetic model and may contribute to efficacy. These findings have general implications for intraventricular immunotherapy. Our data highlight potential innovations to improve efficacy of intraventricular immunotherapy both via modulation of the innate immune response as well as innovations in drug delivery.
PMCID: PMC3944388  PMID: 24190981
Complement; Immunotherapy; Non-Hodgkin’s Lymphoma; Rituximab; Pharmacokinetics
11.  Molecular Pathways: Targeting resistance in the androgen receptor for therapeutic benefit 
Androgen receptor (AR) signaling is critical in the development and progression of prostate cancer, leading to intensive efforts to elucidate all potential points of inflection for therapeutic intervention. These efforts have revealed new mechanisms of resistance and raise the possibility that known mechanisms may become even more relevant in the context of effective AR suppression. These mechanisms include tumoral appropriation of alternative androgen sources, alterations in AR expression, AR mutations, truncated AR variants, alterations and cross-talk in recruitment of co-factors to AR binding sites in the genome, and AR-driven oncogenic gene fusions. New agents such as enzalutamide, EPI-001, AR-specific peptidomimetics, novel HSP90 inhibitors and PARP inhibitors, as well as new approaches to co-targeting the AR pathway point to the potential for more complete and durable control of AR mediated growth.
PMCID: PMC3944407  PMID: 24305618
prostate cancer; androgens; androgen receptor
12.  T lymphocytes redirected against the chondroitin sulfate proteoglycan-4 control the growth of multiple solid tumors both in vitro and in vivo 
Because of its high expression on various types of tumors and its restricted distribution in normal tissues, chondroitin sulfate proteoglycan-4 (CSPG4) represents an attractive target for the antibody-based therapy of several solid tumors. We tested whether T cells transduced with a CSPG4-specific chimeric antigen receptor (CAR) inhibited the growth of CSPG4-expressing tumor cells both in vitro and in vivo.
Experimental Design
We first independently validated by immunohistochemistry (IHC) the expression of CSPG4 in an extensive panel of tumor arrays and normal tissues as well as queried public gene expression profiling datasets of human tumors. We constructed a second generation CSPG4-specific CAR also encoding the CD28 costimulatory endodomain (CAR.CSPG4). We then evaluated human T lymphocytes expressing this CAR for their ex vivo and in vivo anti-tumor activity against a broad panel of solid tumors.
IHC showed that CSPG4 is highly expressed in melanoma, breast cancer, head and neck squamous cell carcinoma (HNSCC) and mesothelioma. In addition, in silico analysis of microarray expression data identified other important potential tumors expressing this target, including glioblastoma, clear cell renal carcinoma and sarcomas. T lymphocytes genetically modified with a CSPG4-CAR controlled tumor growth in vitro and in vivo in NSG mice engrafted with human melanoma, HNSCC and breast carcinoma cell lines.
CAR.CSPG4-redirected T cells should provide an effective treatment modality for a variety of solid tumors.
PMCID: PMC3944408  PMID: 24334762
13.  Molecular Pathways: Gene-environment interactions regulating dietary fiber induction of proliferation and apoptosis via butyrate for cancer prevention 
Gene-environment interactions are so numerous and biologically complicated that it can be challenging to understand their role in cancer. However, dietary fiber and colorectal cancer prevention may represent a tractable model system. Fiber is fermented by colonic bacteria into short-chain fatty acids such as butyrate. One molecular pathway that has emerged involves butyrate having differential effects depending on its concentration and the metabolic state of the cell. Low-moderate concentrations, which are present near the base of colonic crypts, are readily metabolized in the mitochondria to stimulate cell proliferation via energetics. Higher concentrations, which are present near the lumen, exceed the metabolic capacity of the colonocyte. Unmetabolized butyrate enters the nucleus and functions as a histone deacetylase (HDAC) inhibitor that epigenetically regulates gene expression to inhibit cell proliferation and induce apoptosis as the colonocytes exfoliate into the lumen. Butyrate may therefore play a role in normal homeostasis by promoting turnover of the colonic epithelium. Because cancerous colonocytes undergo the Warburg effect, their preferred energy source is glucose instead of butyrate. Consequently, even moderate concentrations of butyrate accumulate in cancerous colonocytes and function as HDAC inhibitors to inhibit cell proliferation and induce apoptosis. These findings implicate a bacterial metabolite with metaboloepigenetic properties in tumor suppression.
PMCID: PMC3944646  PMID: 24270685
14.  Mesothelin Overexpression Is a Marker of Tumor Aggressiveness and Is Associated with Reduced Recurrence-free and Overall Survival in Early-Stage Lung Adenocarcinoma 
In an effort to identify molecular markers of tumor aggressiveness and therapeutic targets in lung adenocarcinoma (ADC), we investigated the expression of mesothelin (MSLN) in lung ADC, as well as its biological and clinical relevance.
Experimental Design
In a training and validation set of patients with early-stage (I–III) lung ADC (n=1209), a tissue microarray consisting of tumors and normal lung tissue was used to examine the association between MSLN expression and recurrence-free survival (RFS) and overall survival (OS). The influence of MSLN overexpression on lung ADC was investigated in vitro and in vivo by use of clinically relevant orthotopic and metastatic xenogeneic and syngeneic mouse models.
MSLN was expressed in 69% of lung ADC tumors, with one in five patients strongly expressing MSLN and no expression in normal lung tissue. Increased MSLN expression was associated with reduced OS (HR, 1.78 [95% CI, 1.26–2.50]; P<0.01) and RFS (HR, 1.67 [95% CI, 1.21–2.27]; P<0.01) in multivariate analyses, even after adjustment for currently known markers of tumor aggressiveness in lung ADC: male sex, smoking history, increasing stage, morphologic pattern, visceral pleural invasion, lymphatic or vascular invasion, and mutation status. In vitro, lung ADC cells overexpressing MSLN demonstrated increased cell proliferation, migration, and invasion; in vivo, mice with MSLN(+) tumors demonstrated decreased survival (P=0.001).
MSLN expression in patients with early-stage lung ADC is associated with increased risk of recurrence and reduced OS, indicating that MSLN expression is a molecular marker of tumor aggressiveness and a potential target for therapy.
PMCID: PMC3944669  PMID: 24334761
Mesothelin; lung adenocarcinoma; prognosis; targeted therapy; non-small cell lung cancer
15.  Telomere Content and Risk of Second Malignant Neoplasm in Survivors of Childhood Cancer: A Report from the Childhood Cancer Survivor Study 
Shorter constitutional telomere length has been associated with increased cancer incidence. Furthermore, telomere shortening is observed in response to intensive chemotherapy and/or ionizing radiation exposure. We aimed to determine if less telomere content was associated with treatment-related second malignant neoplasms (SMNs) in childhood cancer survivors.
Using a nested case-control design, 147 cancer survivors with breast cancer, thyroid cancer, or sarcoma developing after treatment for childhood cancer (cases) were matched (1:1) with childhood cancer survivors without a SMN (controls). Cases and controls were matched by primary cancer diagnosis, years since diagnosis, age at time of sample collection, years of follow up from childhood cancer diagnosis, exposure to specific chemotherapy agents, and to specific radiation fields. We performed conditional logistic regression using telomere content (TC) as a continuous variable to estimate odds ratios (ORs) with corresponding 95% confidence intervals (CI) for development of SMN. ORs were also estimated for specific SMN types, i.e., breast cancer, thyroid cancer, and sarcoma.
There was an inverse relationship between TC and SMN, with an adjusted OR of 0.3 per unit change in telomere length to single copy gene ratio (95% CI, 0.09–1.02, p=0.05). Patients with thyroid cancer SMN were less likely to have more telomere content (OR 0.04, 95% CI, 0.00–0.55, p=0.01), but statistically significant associations could not be demonstrated for breast cancer or sarcoma.
A relation between less telomere content and treatment-related thyroid cancer was observed, suggesting that shorter telomeres may contribute to certain SMNs in childhood cancer survivors.
PMCID: PMC3944671  PMID: 24277454
telomere; second malignant neoplasm; risk; childhood cancer
16.  PID1 (NYGGF4), a new growth-inhibitory gene in embryonal brain tumors and gliomas 
We present here the first report of PID1 (Phosphotyrosine Interaction Domain containing 1; NYGGF4) in cancer. PID1 was identified in 2006 as a gene that modulates insulin signaling and mitochondrial function in adipocytes and muscle cells.
Experimental Design and Results
Using four independent medulloblastoma datasets, we show that mean PID1 mRNA levels were lower in unfavorable medulloblastomas (Groups 3 and 4, and anaplastic histology) compared with favorable medulloblastomas (SHH and WNT groups, and desmoplastic/nodular histology) and with fetal cerebellum. In two large independent glioma datasets PID1 mRNA was lower in glioblastomas (GBMs), the most malignant gliomas, compared to other astrocytomas, oligodendrogliomas and non-tumor brains. Neural and proneural GBM subtypes had higher PID1 mRNA compared to classical and mesenchymal GBM. Importantly, overall survival and radiation-free progression-free survival were longer in medulloblastoma patients with higher PID1 mRNA (univariate and multivariate analyses). Higher PID1 mRNA also correlated with longer overall survival in glioma and GBM patients. In cell culture, overexpression of PID1 inhibited colony formation in medulloblastoma, atypical teratoid rhabdoid tumor (ATRT) and GBM cell lines. Increasing PID1 also increased cell death and apoptosis, inhibited proliferation, induced mitochondrial depolarization, and decreased serum-mediated phosphorylation of AKT and ERK in medulloblastoma, ATRT and/or GBM cell lines, whereas siRNA to PID1 diminished mitochondrial depolarization.
These data are the first to link PID1 to cancer and suggest that PID1 may have a tumor inhibitory function in these pediatric and adult brain tumors.
PMCID: PMC3962776  PMID: 24300787
ATRT; apoptosis; brain cancer; glioma; glioblastoma; medulloblastoma; PID1 (NYGGF4); proliferation
Triple-negative breast cancer (TNBC) is a heterogeneous disease; gene expression (GE) analyses recently identified six distinct TNBC subtypes, each displaying a unique biology. Exploring novel approaches to treatment of these subtypes is critical, since less than 30% of women with metastatic breast cancer survive five years and virtually all women with metastatic TNBC will ultimately die of their disease despite systemic therapy. To date, not a single targeted therapy has been approved for the treatment of TNBC and cytotoxic chemotherapy remains the standard treatment. We will discuss the current and upcoming therapeutic strategies being explored in an attempt to “target” TNBC.
PMCID: PMC3962777  PMID: 24536073
18.  Clusterin is a Gene Specific Target of MicroRNA-21 in Head and Neck Squamous Cell Carcinoma 
MicroRNA-21 (miRNA-21) has proto-oncogenic properties, though no miRNA-21 specific targets have been found in head and neck squamous cell carcinoma (HNSCC). Further study of miRNA-21 and its specific targets is essential to understanding HNSCC biology.
Experimental Design:
miRNA expression profiles of 10 HNSCC and 10 normal mucosa samples were investigated using a custom miRNA microarray. 13 HNSCC and 5 normal mucosa primary tissue specimens underwent mRNA expression microarray analysis. To identify miRNA-21 downstream targets, oral keratinocyte cells were subjected to microarray analysis after miRNA-21 transient transfection. miRNA and mRNA expression were validated by RT-qPCR in a separate cohort of 16 HNSCC and 15 normal mucosal samples. Microarray and bioinformatics analyses were integrated to identify potential gene targets. In vitro assays looked at the function and interaction of miRNA-21 and its specific gene targets.
miRNA-21 was upregulated in HNSCC and stimulated cell growth. Integrated analyses identified Clusterin (CLU) as a potential miRNA-21 gene target. CLU was downregulated after forced expression of miRNA-21 in normal and HNSCC cell lines. The activity of a luciferase construct containing the 3’UTR of CLU was repressed by the ectopic expression of miRNA-21. CLU was also downregulated in primary HNSCC and correlated with miRNA-21 over-expression. CLU variant 1 (CLU-1) was the predominant splice variant in HNSCC, and showed growth suppression function that was reversed by miRNA-21 over-expression.
CLU is a specific, functional target of oncogenic miRNA-21 in HNSCC. CLU-1 isoform is the predominant growth suppressive variant targeted by miRNA-21.
PMCID: PMC3970211  PMID: 24327270
Clusterin; microRNA-21; gene target; tumor-suppressor gene; head and neck cancer
19.  PBX1 is a Favorable Prognostic Biomarker as it Modulates 13-cis Retinoic Acid-mediated Differentiation in Neuroblastoma 
Neuroblastoma is an embryonic childhood cancer with high mortality. 13-cis retinoic acid (13-cisRA) improves survival for some patients, but many recur, suggesting clinical resistance. The mechanism of resistance, and the normal differentiation pathway, are poorly understood. Three-Amino-acid Loop Extension (TALE) family genes are master regulators of differentiation. Since retinoids promote differentiation in neuroblastoma, we evaluated TALE family gene expression in neuroblastoma.
Experimental Design
We evaluated expression of TALE family genes in RA-sensitive and -resistant neuroblastoma cell lines, with and without 13cis-RA treatment, identifying genes whose expression correlate with retinoid sensitivity. We evaluated the roles of one gene, PBX1, in neuroblastoma cell lines, including proliferation and differentiation. We evaluated PBX1 expression in primary human neuroblastoma samples by RT-qPCR, and three independent clinical cohort microarray datasets.
We confirmed induction of PBX1 expression, and no other TALE family genes, was associated with 13-cisRA responsiveness in NB cell lines. Exogenous PBX1 expression in neuroblastoma cell lines, mimicking induced PBX1 expression, significantly impaired proliferation and anchorage-independent growth, and promoted RA-dependent and -independent differentiation. Reduced PBX1 protein levels produced an aggressive growth phenotype and RA resistance. PBX1 expression correlated with histological neuroblastoma subtypes, with highest expression in benign ganglioneuromas and lowest in high-risk neuroblastomas. High PBX1 expression is prognostic of survival, including in multivariate analysis, in the three clinical cohorts.
PBX1 is an essential regulator of differentiation in neuroblastoma and potentiates retinoid-induced differentiation. Neuroblastoma cells and tumors with low PBX1 expression have an immature phenotype with poorer prognosis, independent of other risk factors.
PMCID: PMC4134768  PMID: 24947929
Neuroblastoma; Differentiation; 13-cis retinoic acid; PBX1
20.  BET-bromodomain inhibition of MYC-amplified medulloblastoma 
MYC-amplified medulloblastomas are highly lethal tumors. BET bromodomain inhibition has recently been shown to suppress MYC-associated transcriptional activity in other cancers. The compound JQ1 inhibits BET bromodomain-containing proteins, including BRD4. Here we investigate BET bromodomain targeting for the treatment of MYC-amplified medulloblastoma.
Experimental Design
We evaluated the effects of genetic and pharmacological inhibition of BET bromodomains on proliferation, cell cycle, and apoptosis in established and newly generated patient- and GEMM-derived medulloblastoma cell lines and xenografts that harbored amplifications of MYC or MYCN. We also assessed the effect of JQ1 on MYC expression and global MYC-associated transcriptional activity. We assessed in vivo efficacy of JQ1 in orthotopic xenografts established in immunocompromised mice.
Treatment of MYC-amplified medulloblastoma cells with JQ1 decreased cell viability associated with arrest at G1 and apoptosis. We observed down-regulation of MYC expression and confirmed inhibition of MYC-associated transcriptional targets. Exogenous expression of MYC from a retroviral promoter reduced the effect of JQ1 on cell viability, suggesting that attenuated levels of MYC contribute to the functional effects of JQ1. JQ1 significantly prolonged survival of orthotopic xenograft models of MYC-amplified medulloblastoma (p<0.001). Xenografts harvested from mice after five doses of JQ1 had reduced expression of MYC mRNA and a reduced proliferative index.
JQ1 suppresses MYC expression and MYC-associated transcriptional activity in medulloblastomas, resulting in an overall decrease in medulloblastoma cell viability. These preclinical findings highlight the promise of BET bromodomain inhibitors as novel agents for MYC-amplified medulloblastoma.
PMCID: PMC4198154  PMID: 24297863
BET-bromodomain; JQ1; MYC; MYCN; MYCL1; medulloblastoma
21.  Power in Numbers: Meta-analysis to Identify Inhibitor-Sensitive Tumor Genotypes 
Widespread tumor genotyping has increased the complexity of lung cancer care, often identifying mutations of uncertain clinical significance. In the accompanying article, the authors carried out a meta-analysis of the published literature on EGF receptor (EGFR) genotype and erlotinib/gefitinib sensitivity to develop a publicly accessible database to inform patient care.
PMCID: PMC4327821  PMID: 23403632
22.  New Strategies in Lung Cancer: Epigenetic Therapy for Non–Small Cell Lung Cancer 
Recent discoveries that non–small cell lung cancer (NSCLC) can be divided into molecular subtypes based on the presence or absence of driver mutations have revolutionized the treatment of many patients with advanced disease. However, despite these advances, a majority of patients are still dependent on modestly effective cytotoxic chemotherapy to provide disease control and prolonged survival. In this article, we review the current status of attempts to target the epigenome, heritable modifications of DNA, histones, and chromatin that may act to modulate gene expression independently of DNA coding alterations, in NSCLC and the potential for combinatorial and sequential treatment strategies.
PMCID: PMC4325981  PMID: 24644000
23.  Proteomic characterization of breast cancer xenografts identifies early and late bevacizumab-induced responses and predicts effective drug combinations 
Neoangiogenesis is an important feature in tumor growth and progression, and combining chemotherapy and antiangiogenic drugs have demonstrated clinical efficacy. However, as treatment induced resistance often develops our goal was to identify pathways indicating response and/or evolving resistance to treatment, and inhibit these pathways to optimize the treatment strategies.
Experimental Design
To identify markers of response and/or resistance Reverse Phase Protein Array (RPPA) was utilized to characterize treatment-induced changes in a bevacizumab responsive and a nonresponsive human breast cancer xenograft. Results were combined with bioinformatic modeling to predict druggable targets for optimization of the treatment.
RPPA analysis showed that both tumor models responded to bevacizumab with an early (day 3) upregulation of growth factor receptors and downstream signaling pathways, with persistent mTOR signaling until the end of the in vivo experiment. Adding doxorubicin to bevacizumab showed significant and superior growth inhibition of basal-like tumors, whereas no additive effect was seen in the luminal-like model. The combination treatment corresponded to a continuous late attenuation of mTOR signaling in the basal-like model, while the inhibition was temporary in the luminal-like model. Integrating the bevacizumab-induced dynamic changes in protein levels with bioinformatic modeling predicted inhibition of PI3K-pathway to increase the efficacy of bevacizumab monotherapy. In vivo experiments combining bevacizumab and the PI3K/mTOR inhibitor BEZ235 confirmed their significant and additive growth inhibitory effect in the basal-like model.
Treatment with bevacizumab caused compensatory upregulation of several signaling pathways. Targeting such pathways increased the efficacy of antiangiogenic therapy.
PMCID: PMC4326070  PMID: 24192926
24.  Integrated Next-Generation Sequencing and Avatar Mouse Models for Personalized Cancer Treatment 
Current technology permits an unbiased massive analysis of somatic genetic alterations from tumor DNA as well as the generation of individualized mouse xenografts (Avatar models). This work aimed to evaluate our experience integrating these two strategies to personalize the treatment of patients with cancer.
We performed whole-exome sequencing analysis of 25 patients with advanced solid tumors to identify putatively actionable tumor-specific genomic alterations. Avatar models were used as an in vivo platform to test proposed treatment strategies.
Successful exome sequencing analyses have been obtained for 23 patients. Tumor-specific mutations and copy-number variations were identified. All samples profiled contained relevant genomic alterations. Tumor was implanted to create an Avatar model from 14 patients and 10 succeeded. Occasionally, actionable alterations such as mutations in NF1, PI3KA, and DDR2 failed to provide any benefit when a targeted drug was tested in the Avatar and, accordingly, treatment of the patients with these drugs was not effective. To date, 13 patients have received a personalized treatment and 6 achieved durable partial remissions. Prior testing of candidate treatments in Avatar models correlated with clinical response and helped to select empirical treatments in some patients with no actionable mutations.
The use of full genomic analysis for cancer care is encouraging but presents important challenges that will need to be solved for broad clinical application. Avatar models are a promising investigational platform for therapeutic decision making. While limitations still exist, this strategy should be further tested.
PMCID: PMC4322867  PMID: 24634382
25.  Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma 
Myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) play a key role in the progression of head and neck squamous cell carcinoma (HNSCC). On the basis of our preclinical data demonstrating that phosphodiesterase-5 (PDE5) inhibition can modulate these cell populations, we evaluated whether the PDE5 inhibitor tadalafil can revert tumor-induced immunosuppression and promote tumor immunity in patients with HNSCC.
Experimental Design
First, we functionally and phenotypically characterized MDSCs in HNSCCs and determined, retrospectively, whether their presence at the tumor site correlates with recurrence. Then, we performed a prospective single-center, double-blinded, randomized, three-arm study in which patients with HNSCC undergoing definitive surgical resection of oral and oropharyngeal tumors were treated with tadalafil 10 μg/day, 20 μg/day, or placebo for at least 20 days preoperatively. Blood and tumor MDSC and Treg presence and CD8+ T-cell reactivity to tumor antigens were evaluated before and after treatment.
MDSCs were characterized in HNSCC and their intratumoral presence significantly correlates with recurrence. Tadalafil treatment was well tolerated and significantly reduced both MDSCs and Treg concentrations in the blood and in the tumor (P < 0.05). In addition, the concentration of blood CD8+ T cells reactive to autologous tumor antigens significantly increased after treatment (P < 0.05). Tadalafil immunomodulatory activity was maximized at an intermediate dose but not at higher doses. Mechanistic analysis suggests a possible off-target effect on PDE11 at high dosages that, by increasing intracellular cAMP, may negatively affect antitumor immunity.
Tadalafil seems to beneficially modulate the tumor micro- and macro-environment in patients with HNSCC by lowering MDSCs and Tregs and increasing tumor-specific CD8+ T cells in a dose-dependent fashion.
PMCID: PMC4322895  PMID: 25320361

Results 1-25 (2078)