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1.  Ovarian cancer stem cells: Can targeted therapy lead to improved progression-free survival? 
World Journal of Stem Cells  2014;6(4):441-447.
Despite significant effort and research funds, epithelial ovarian cancer remains a very deadly disease. There are no effective screening methods that discover early stage disease; the majority of patients are diagnosed with advanced disease. Treatment modalities consist primarily of radical debulking surgery followed by taxane and platinum-based chemotherapy. Newer therapies including limited targeted agents and intraperitoneal delivery of chemotherapeutic drugs have improved disease-free intervals, but failed to yield long-lasting cures in most patients. Chemotherapeutic resistance, particularly in the recurrent setting, plagues the disease. Targeting the pathways and mechanisms behind the development of chemoresistance in ovarian cancer could lead to significant improvement in patient outcomes. In many malignancies, including blood and other solid tumors, there is a subgroup of tumor cells, separate from the bulk population, called cancer stem cells (CSCs). These CSCs are thought to be the cause of metastasis, recurrence and resistance. However, to date, ovarian CSCs have been difficult to identify, isolate, and target. It is felt by many investigators that finding a putative ovarian CSC and a chemotherapeutic agent to target it could be the key to a cure for this deadly disease. This review will focus on recent advances in this arena and discuss some of the controversies surrounding the concept.
doi:10.4252/wjsc.v6.i4.441
PMCID: PMC4172672  PMID: 25258665
Epithelial ovarian cancer; Cancer stem cells; Chemoresistance; Targeted therapy; Chemotherapy; Recurrent ovarian cancer
2.  S100A4 promotes pancreatic cancer progression through a dual signaling pathway mediated by Src and focal adhesion kinase 
Scientific Reports  2015;5:8453.
S100A4 expression is associated with poor clinical outcomes of patients with pancreatic cancer. The effects of loss or gain of S100A4 were examined in pancreatic cancer cell lines. S100A4 downregulation remarkably reduces cell migration and invasion, inhibits proliferation, and induces apoptosis in pancreatic tumor cells. S100A4 downregulation results in significant cell growth inhibition and apoptosis in response to TGF-β1, supporting a non-canonical role of S100A4 in pancreatic cancer. The role of S100A4 in tumor progression was studied by using an orthotopic human pancreatic cancer xenograft mouse model. Tumor mass is remarkably decreased in animals injected with S100A4-deficient pancreatic tumor cells. P27Kip1 expression and cleaved caspase-3 are increased, while cyclin E expression is decreased, in S100A4-deficient pancreatic tumors in vivo. S100A4-deficient tumors have lower expression of vascular endothelial growth factor, suggesting reduced angiogenesis. Biochemical assays revealed that S100A4 activates Src and focal adhesion kinase (FAK) signaling events, and inhibition of both kinases is required to maximally block the tumorigenic potential of pancreatic cancer cells. These findings support that S100A4 plays an important role in pancreatic cancer progression in vivo and S100A4 promotes tumorigenic phenotypes of pancreatic cancer cells through the Src-FAK mediated dual signaling pathway.
doi:10.1038/srep08453
PMCID: PMC4326725  PMID: 25677816
4.  Experimental virotherapy of chemoresistant pancreatic carcinoma using infectivity-enhanced fiber-mosaic oncolytic adenovirus 
Cancer gene therapy  2014;21(7):264-274.
Pancreatic cancer is a significant clinical problem and novel therapeutic approaches are desperately needed. Recent advances in conditionally replicative adenovirus-based (CRAd) oncolytic virus design allow the application of CRAd vectors as a therapeutic strategy to efficiently target and eradicate chemoresistant pancreatic cancer cells thereby improving the efficacy of pancreatic cancer treatment. The goal of this study was to construct and validate the efficacy of an infectivity-enhanced, liver-untargeted, tumor-specific CRAd vector. A panel of CRAds has been derived which embody the C-X-C chemokine receptor type 4 promoter for conditional replication, two fiber complex mosaicism for targeting expansion, and hexon hypervariable region 7 (HVR7) modification for liver untargeting. We evaluated CRAds for cancer virotherapy using a human pancreatic tumor xenograft model. Employment of the fiber mosaic approach improved CRAd replication in pancreatic tumor xenografts. Substitution of the HVR7 of the Ad5 hexon for Ad serotype 3 hexon resulted in decreased liver tropism of systemically administrated CRAd. Obtained data demonstrated that employment of complex mosaicism increased efficacy of the combination of oncolytic virotherapy with chemotherapy in a human pancreatic tumor xenograft model.
doi:10.1038/cgt.2014.26
PMCID: PMC4157623  PMID: 24903014
targeted oncolytic adenovirus; pancreatic cancer; gemcitabine; fiber; hexon
5.  Antagonistic Effects of Anti-EMMPRIN Antibody When Combined with Chemotherapy Against Hypovascular Pancreatic Cancers 
Purpose
To examine the antagonistic effects of anti-extracellular matrix metalloprotease inducer (anti-EMMPRIN) antibody when combined with chemotherapy using a hypovascular pancreatic tumor model.
Procedures
Severely compromised immunodeficient mice bearing orthotopic MIA PaCa-2 tumors were used (five to six animals per group). Dynamic contrast-enhanced magnetic resonance imaging was used to examine the relationship between tumor vascularity and size. Therapy was initiated when tumors were hypovascular. Treatments included: (1) gemcitabine alone, (2) anti-EMMPRIN antibody alone, and (3) combination, each for 2 weeks. Additionally, another treatment arm included β-lapachone, an NAD(P)H/quinone 1 (NQO1) bioactivated agent. 18F-fluoro-D-glucose-positron emission tomography/computed tomography imaging was used weekly to monitor therapeutic effects.
Results
Gemcitabine or anti-EMMPRIN monotherapy significantly delayed tumor growth, but the combination therapy showed an antagonistic effect. Similarly, tumor growth was significantly suppressed by β-lapachone alone, and additive effects were noted when combined with gemcitabine, but the therapeutic efficacy was reduced when anti-EMMPRIN antibody was added.
Conclusions
Anti-EMMPRIN antibody with chemotherapy in hypovascular tumors results in antagonistic effects.
doi:10.1007/s11307-013-0665-4
PMCID: PMC4062967  PMID: 23836505
Pancreatic cancer; EMMPRIN; β-Lapachone; Gemcitabine; DCE-MRI; FDG-PET/CT
6.  Loss of N-Myc interactor promotes epithelial-mesenchymal-transition by activation of TGF-β/SMAD signaling 
Oncogene  2013;33(20):2620-2628.
Epithelial-Mesenchymal-Transition (EMT) is one of the critical cellular programs that facilitate the progression of breast cancer to an invasive disease. We have observed that the expression of N-myc interactor (NMI) decreases significantly during progression of breast cancer, specifically in invasive and metastatic stages. Recapitulation of this loss in breast cell lines with epithelial morphology [MCF10A (non-tumorigenic) and T47D (tumorigenic)] by silencing NMI expression causes mesenchymal-like morphological changes in 3-D growth, accompanied by up-regulation of SLUG and ZEB2 and increased invasive properties. Conversely, we found that restoring NMI expression attenuated mesenchymal attributes of metastatic breast cancer cells accompanied by distinctly circumscribed 3-D growth with basement membrane deposition and decreased invasion. Further investigations into the downstream signaling modulated by NMI revealed that NMI expression negatively regulates SMAD signaling, which is a key regulator of cellular plasticity. We demonstrate that NMI blocks TGF-β/SMAD signaling via up-regulation of SMAD7, a negative feedback regulator of the pathway. We also provide evidence that NMI activates STAT signaling which negatively modulates TGF-β/SMAD signaling. Taken together, our findings suggest that loss of NMI during breast cancer progression could be one of the driving factors that enhance invasive ability of breast cancer by aberrant activation of TGF-β/SMAD signaling.
doi:10.1038/onc.2013.215
PMCID: PMC4267223  PMID: 23770854
N-Myc interactor; EMT; SMAD; Breast cancer
7.  ST6Gal-I protein expression is upregulated in human epithelial tumors and correlates with stem cell markers in normal tissues and colon cancer cell lines 
Cancer research  2013;73(7):2368-2378.
The ST6Gal-I sialyltransferase adds an α2–6-linked sialic acid to the N-glycans of certain receptors. ST6Gal-I mRNA has been reported to be upregulated in human cancer, but a prior lack of antibodies has limited immunochemical analysis of the ST6Gal-I protein. Here we show upregulated ST6Gal-I protein in several epithelial cancers, including many colon carcinomas. In normal colon, ST6Gal-I localized selectively to the base of crypts, where stem/progenitor cells are found, and the tissue staining patterns were similar to the established stem cell marker ALDH1. Similarly, ST6Gal-I expression was restricted to basal epidermal layers in skin, another stem/progenitor cell compartment. ST6Gal-I was highly expressed in induced pluripotent stem (iPS) cells, with no detectable expression in the fibroblasts from which iPS cells were derived. On the basis of these observations we investigated further an association of ST6Gal-I with cancer stem cells (CSCs). Selection of irinotecan resistance in colon carcinoma cells led to a greater proportion of CSCs compared with parental cells, as measured by the CSC markers CD133 and ALDH1 activity (Aldefluor). These chemoresistant cells exhibited a corresponding upregulation of ST6Gal-I expression. Conversely, shRNA-mediated attenuation of ST6Gal-I in colon carcinoma cells with elevated endogenous expression decreased the number of CD133/ALDH1-positive cells present in the cell population. Collectively, our results suggest that ST6Gal-I promotes tumorigenesis and may serve as a regulator of the stem cell phenotype in both normal and cancer cell populations.
doi:10.1158/0008-5472.CAN-12-3424
PMCID: PMC4038408  PMID: 23358684
glycosylation; ST6Gal-I; colon carcinoma; stem cells; cancer stem cells
8.  Catalase abrogates β-lapachone-induced PARP1 hyperactivation-directed programmed necrosis in NQO1-positive breast cancers 
Molecular cancer therapeutics  2013;12(10):10.1158/1535-7163.MCT-12-0962.
Improving patient outcome by personalized therapy involves a thorough understanding of an agent’s mechanism of action. β-Lapachone (clinical forms, Arq501/Arq761) has been developed to exploit dramatic cancer-specific elevations in the phase II detoxifying enzyme, NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is dramatically elevated in solid cancers, including primary and metastatic (e.g., triple-negative (ER-, PR-, Her2/Neu-)) breast cancers. To define cellular factors that influence the efficacy of β-lapachone using knowledge of its mechanism of action, we confirmed that NQO1 was required for lethality and mediated a futile redox cycle where ~120 moles of superoxide were formed per mole of β-lapachone in 5 min. β-Lapachone induced reactive oxygen species (ROS), stimulated DNA single strand break-dependent PARP1 hyperactivation, caused dramatic loss of essential nucleotides (NAD+/ATP) and elicited programmed necrosis in breast cancer cells. While PARP1 hyperactivation and NQO1 expression were major determinants of β-lapachone-induced lethality, alterations in catalase expression, including treatment with exogenous enzyme, caused marked cytoprotection. Thus, catalase is an important resistance factor, and highlights H2O2 as an obligate ROS for cell death from this agent. Exogenous superoxide dismutase (SOD) enhanced catalase-induced cytoprotection. β-Lapachone-induced cell death included AIF translocation from mitochondria to nuclei, TUNEL+ staining, atypical PARP1 cleavage, and GAPDH S-nitrosylation, which were abrogated by catalase. We predict that the ratio of NQO1:catalase activities in breast cancer versus associated normal tissue are likely to be the major determinants affecting the therapeutic window of β-lapachone and other NQO1 bioactivatable drugs.
doi:10.1158/1535-7163.MCT-12-0962
PMCID: PMC3807805  PMID: 23883585
Breast cancer; Catalase; Super Oxide Dismutase, β-Lapachone; NADPH quinone oxidoreductase-1; Poly (ADP-ribose) polymerase-1; Programmed necrosis
9.  DCE-MRI Detects Early Vascular Response in Breast Tumor Xenografts Following Anti-DR5 Therapy 
Purpose
Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) measured the early vascular changes after administration of TRA-8, bevacizumab, or TRA-8 combined with bevacizumab in breast tumor xenografts.
Procedures
Groups 1–4 of nude mice bearing human breast carcinoma were injected with phosphate-buffered saline, TRA-8, bevacizumab, and TRA-8 + bevacizumab on day0, respectively. DCE-MRI was performed on days0, 1, 2, and 3, and thereafter tumors were collected for terminal deoxynucleotidyl transferase-mediated dUT nick end labeling and CD31 staining.
Results
DCE-MRI measured a significant Ktrans change within 3 days after TRA-8 therapy that correlated with tumor growth arrest, whichwas not shown with statistical significance by histopathology at these early time points posttreatment. The Ktrans changes followed quadratic polynomial curves.
Conclusion
DCE-MRI detected significantly lower Ktrans levels in breast tumor xenografts following TRA-8 monotherapy or combined therapy with bevacizumab.
doi:10.1007/s11307-010-0320-2
PMCID: PMC4138021  PMID: 20383593
DCE-MRI; Reference region model; DR5; TRAIL; TRA-8; Bevacizumab; Breast cancer; Novel biomarker
10.  Recurrent read-through fusion transcripts in breast cancer 
Read-through fusion transcripts that result from the splicing of two adjacent genes in the same coding orientation are a recently discovered type of chimeric RNA. We sought to determine if read-through fusion transcripts exist in breast cancer. We performed paired-end RNA-seq of 168 breast samples, including 28 breast cancer cell lines, 42 triple negative breast cancer primary tumors, 42 estrogen receptor positive (ER+) breast cancer primary tumors, and 56 non-malignant breast tissue samples. We analyzed the sequencing data to identify breast cancer associated read-through fusion transcripts. We discovered two recurrent read-through fusion transcripts that were identified in breast cancer cell lines, confirmed across breast cancer primary tumors, and were not detected in normal tissues (SCNN1A-TNFRSF1A and CTSD-IFITM10). Both fusion transcripts use canonical splice sites to join the last splice donor of the 5′ gene to the first splice acceptor of the 3′ gene, creating an in-frame fusion transcript. Western blots indicated that the fusion transcripts are translated into fusion proteins in breast cancer cells. Custom small interfering RNAs targeting the CTSD-IFITM10 fusion junction reduced expression of the fusion transcript and reduced breast cancer cell proliferation. Read-through fusion transcripts between adjacent genes with different biochemical functions represent a new type of recurrent molecular defect in breast cancer that warrant further investigation as potential biomarkers and therapeutic targets. Both breast cancer associated fusion transcripts identified in this study involve membrane proteins (SCNN1A-TNFRSF1A and CTSD-IFITM10), which raises the possibility that they could be breast cancer-specific cell surface markers.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-014-3019-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s10549-014-3019-2
PMCID: PMC4085473  PMID: 24929677
11.  The impact of novel retinoids in combination with platinum chemotherapy on ovarian cancer stem cells 
Gynecologic oncology  2011;125(1):226-230.
Objective
Retinoids are important modulators of cell growth, differentiation, and proliferation. 9cUAB30, 9cUAB124, and 9cUAB130 are three novel retinoid compounds that show cytotoxic effects in other malignancies. We evaluated these novel retinoids in combination with chemotherapy against ovarian cancer stem cells (CSCs) in vitro and in an ex vivo model.
Methods
A2780 cells were plated in 96-well plates and treated with retinoid, carboplatin, or combination therapy. Cell viability was evaluated using ATPLite assay. The A2780 cell line was also analyzed for CSCs by evaluating ALDH activity using flow cytometry. A2780 cells treated ex vivo with retinoids and chemotherapy were injected into the flank of athymic nude mice in order to evaluate subsequent tumor initiating capacity.
Results
A2780 cells were sensitive to treatment with retinoids and carboplatin. The best treatment resulted from the combination of retinoid 9cUAB130 and carboplatin. Untreated A2780 cells demonstrated ALDH activity in 3.3% of the cell population. Carboplatin treatment enriched ALDH activity to 27.3%, while 9cUAB130±carboplatin maintained the ALDH positive levels similar to untreated controls (2.3% and 6.7%, respectively). Similar results were found in tumorsphere-forming conditions. Flank injections of ex vivo treated A2780 cells resulted in 4/4 mice developing tumors at 40 days in the untreated group, while 0/4 tumors developed in the 9cUAB130 and carboplatin treated group.
Conclusion
Combination treatment with carboplatin and retinoids reduced cell-viability, reduced CSC marker expression, and inhibited tumorigenicity, making it a more effective treatment when compared with carboplatin alone.
doi:10.1016/j.ygyno.2011.12.425
PMCID: PMC4049085  PMID: 22155260
Retinoid; Ovarian cancer; Stem cell
12.  Early Therapy Evaluation of Combined Cetuximab and Irinotecan in Orthotopic Pancreatic Tumor Xenografts by Dynamic Contrast-Enhanced Magnetic Resonance Imaging 
Molecular imaging  2011;10(3):153-167.
Early pancreatic cancer response following cetuximab and/or irinotecan therapies was measured by serial dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) before and during therapy. Groups 1 to 4 (n = 6/group) of SCID mice bearing orthotopic pancreatic adenocarcinoma xenografts expressing luciferase were treated with phosphate-buffered saline, cetuximab, irinotecan, or cetuximab combined with irinotecan, respectively, twice weekly for 3 weeks. DCE-MRI was performed on days 0, 1, 2, and 3 after therapy initiation, whereas anatomic magnetic resonance imaging was performed on days 0, 1, 2, 3, 6, and 13. Bioluminescence imaging was performed on days 0 and 21. At day 21, all tumors were collected for further histologic analyses (Ki-67 and CD31 staining), whereas tumor dimensions were measured by calipers. The Ktrans values in the 0.5 mm–thick peripheral tumor region were calculated, and the changes in Ktrans during the 3 days posttherapy were compared to tumor volume changes, bioluminescent signal changes, and histologic findings. The Ktrans changes in the peripheral tumor region after 3 days of therapy were linearly correlated with 21-day decreases in tumor volume (p < .001), bioluminescent signal (p = .050), microvessel densities (p = .002), and proliferating cell densities (p = .001). This study supports the clinical use of DCE-MRI for pancreatic cancer patients for early assessment of an anti–epidermal growth factor receptor therapy combined with chemotherapy.
PMCID: PMC3957340  PMID: 21496446
13.  Mechanisms of drug sensitization to TRA-8, an agonistic death receptor 5 antibody, involve modulation of the intrinsic apoptotic pathway in human breast cancer cells 
Molecular cancer research : MCR  2011;9(4):403-417.
TRA-8, a monoclonal antibody to death receptor 5 induces apoptosis in various cancer cells; however the degree of sensitivity varies from highly sensitive to resistant. We have previously shown resistance to TRA-8 can be reversed using chemotherapeutic agents, but the mechanism underlying this sensitization was not fully understood. Here, we examined the combination of TRA-8 with doxorubicin or bortezomib in breast cancer cells. In TRA-8 resistant BT-474 and T47D cells, both chemotherapy agents synergistically sensitized cells to TRA-8 cytotoxicity with enhanced activation of apoptosis demonstrated by cleavage of caspases and PARP, reduced Bid, increased pro-apoptotic Bcl-2 proteins, and increased mitochondrial membrane depolarization. Doxorubicin or bortezomib combined with TRA-8 also reduced Bcl-XL and XIAP in treated cells. Furthermore, targeting these proteins with pharmacological modulators, AT-101, BH3I-2′ and AT-406, produced sensitization to TRA-8. TRA-8 combined with AT-101 or BH3I-2′, inhibitors of anti-apoptotic Bcl-2 proteins, produced synergistic cytotoxicity against ZR-75-1, BT-474, and T47D cells. The IAP targeting compound, AT-406, was synergistic with TRA-8 in BT-474 cells and to a lesser extent T47D cells. Activation of the intrinsic apoptotic pathway was a common mechanism associated with sensitization of TRA-8 resistant breast cancer cell lines. Collectively, these studies show that the Bcl-2 and IAP families of proteins are involved in TRA-8 and chemotherapy resistance via their modulation of the intrinsic apoptotic pathway. Targeting these proteins with novel agents sensitized TRA-8 resistant breast cancer cells, suggesting this approach may represent a potent therapeutic strategy in the treatment of breast cancer.
doi:10.1158/1541-7786.MCR-10-0133
PMCID: PMC3086592  PMID: 21357440
breast neoplasms; TRAIL; death receptor antibody
14.  Combined modality therapy with TRAIL or agonistic death receptor antibodies 
Cancer Biology & Therapy  2011;11(5):431-449.
Molecularly targeted therapies, such as antibodies and small molecule inhibitors have emerged as an important breakthrough in the treatment of many human cancers. One targeted therapy under development is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) due to its ability to induce apoptosis in a variety of human cancer cell lines and xenografts, while lacking toxicity in most normal cells. TRAIL and apoptosis-inducing agonistic antibodies to the TRAIL death receptors have been the subject of many preclinical and clinical studies in the past decade. However, the sensitivity of individual cancer cell lines of a particular tumor type to these agents varies from highly sensitive to resistant. Various chemotherapy agents have been shown to enhance the apoptosis-inducing capacity of TRAIL receptor-targeted therapies and induce sensitization of TRAIL-resistant cells. This review provides an overview of the mechanisms associated with chemotherapy enhancement of TRAIL receptor-targeted therapies including modulation of the apoptotic (death receptor expression, FLIP and Bcl-2 or inhibitors of apoptosis [IAP] families) as well as cell signaling (NFκB, Akt, p53) pathways. These mechanisms will be important in establishing effective combinations to pursue clinically and in determining relevant targets for future cancer therapies.
doi:10.4161/cbt.11.5.14671
PMCID: PMC3087899  PMID: 21263219
TRAIL; death receptor antibodies; death receptor 4; death receptor 5; combination chemotherapy; apoptosis; cancer therapy
15.  Combination Treatment with TRA-8 Anti – Death Receptor 5 Antibody and CPT-11 Induces Tumor Regression in an Orthotopic Model of Pancreatic Cancer 
Purpose
Evaluate the response of human pancreatic cancer cell lines and orthotopic tumors to TRA-8, an agonistic antibody to death receptor 5, in combination with irinotecan (CPT-11).
Experimental Design
MIA PaCa-2 and S2VP10 cells were treated with TRA-8 and/or CPT 11. Cell viability was determined by ATP assay. JC-1 mitochondrial depolarization and Annexin V assays confirmed cell death by apoptosis. Immunoblotting was used to evaluate protein changes. MIA PaCa-2 cells were injected into the pancreas of severe combined immunodeficient mice. Mice underwent abdominal ultrasound to quantitate tumor size before and after treatment with twice weekly injections of 200 µg TRA-8 and/or 25 mg/kg CPT-11 for one or two treatment cycles, each lasting 2 weeks.
Results
MIA PaCa-2 cells were more sensitive to TRA-8 and showed additive cytotoxicity, whereas S2VP10 cells showed synergistic cytotoxicity when treated with TRA-8 and CPT-11. Cell death occurred via apoptosis with increased cleavage of caspase-3, caspase-8, and caspase-9 and proapoptotic proteins Bid and poly(ADP)ribose polymerase after combination treatment compared with either agent alone. XIAP and Bcl-XL inhibitors of apoptosis were down-regulated. After a single cycle of in vivo combination therapy, tumor sizes had diminished significantly (P < 0.001) at 8 days posttreatment compared with no treatment, CPT-11, and TRA-8; and there was a 50-day increase in survival with combination treatment over untreated controls (P = 0.0002), 30 days over TRA-8, and a 36-day increase over CPT-11 monotherapy (P = 0.0003). With two cycles of TRA-8/CPT-11 treatment, mean survival time increased significantly (P < 0.001) to 169 days versus untreated controls, TRA-8 or CPT-11 (76, 121, or 108 days, respectively).
Conclusions
Combination TRA-8 and CPT-11 therapy produced enhanced cytotoxicity and survival in the MIA PaCa-2 orthotopic model of pancreatic cancer.
doi:10.1158/1078-0432.CCR-07-1075
PMCID: PMC3045836  PMID: 17875786
16.  KISS1 over-expression suppresses metastasis of pancreatic adenocarcinoma in a xenograft mouse model 
Identifying molecular targets for treatment of pancreatic cancer metastasis is critical due to the high frequency of dissemination prior to diagnosis of this lethal disease. Because the KISS1 metastasis suppressor is expressed at reduced levels in advanced pancreatic cancer, we hypothesized that re-expression of KISS1 would reduce metastases. Highly metastatic S2VP10 cells expressing luciferase (S2VP10L) were transfected with a FLAG-tagged version of KISS1 (KFM), KFMΔSS (with deleted secretion signal sequence), or pcDNA3 control plasmid (CP) and expression was confirmed by RTQ-PCR. SCID mice were implanted orthotopically with S2VP10L cells or transfectants and tumor growth and metastases were monitored using bioluminescence imaging. Mice with S2VP10L-KISS1 tumors developed fewer liver (98%) and lung (99%) metastases than S2VP10L. Unexpectedly, mice with S2VP10L-KFMΔSS tumors also had reduced liver and lung metastases, but had more metastases than mice with S2VP10L-KISS. KISS1 protein was found in the cytoplasm of both KFMΔSS and KISS1-expressing orthotopic tumors by immunohistochemistry. Metastases were not found in lungs of mice with S2VP10L-KISS1 tumors; whereas, KFMΔSS lung sections had regions of concentrated KISS1 staining, suggesting that secretion of KISS1 is needed to reduce metastasis significantly. These data suggest induction of KISS1 expression has potential as an adjuvant treatment for pancreatic cancer.
doi:10.1007/s10585-010-9349-5
PMCID: PMC2975384  PMID: 20844932
KISS1; metastasis suppressor; pancreatic adenocarcinoma
17.  A deimmunized bispecific ligand directed toxin that shows an impressive anti-pancreatic cancer effect in a systemic nude mouse orthotopic model 
Pancreas  2012;41(5):789-796.
Objective
The objective was to test a bispecific ligand directed toxin (BLT), with reduced immunogenicity for enhanced efficacy in targeting orthotopic pancreatic cancer in vivo.
Method
A new BLT was created in which both human EGF and IL-4 cytokines were cloned onto the same single chain molecule with deimmunized pseudomonas exotoxin (dEGF4KDEL). Key amino acids dictating B cell generation of neutralizing anti-toxin antibodies were mutated. Bioassays were used to determine whether mutation reduced potency, and ELISA studies were performed to determine whether anti-toxin antibodies were reduced. A genetically altered luciferase MIA PaCa-2 xenograft model was used to image in real time and determine affects on systemic malignant human cancer. BLTs targeting B cells were used as specificity controls.
Results
dEGF4KDEL was significantly effective following systemic injection against established orthotopic MIA PaCa-2 pancreatic cancer and selectively prevented metastasis. Mutagenesis significantly reduced anti-toxin levels in vivo with no apparent activity loss in vitro. The drug was effective against three human pancreatic cancer lines in vitro, MIA PaCa-2, SW1990, and S2VP10.
Conclusions
Despite the metastatic nature of the MIA PaCa-2 orthotopic tumor xenografted in nude mice, high percentages of tumors responded to extended dEGFKDEL treatment resulting in significant anti-cancer effects and disease-free survivors.
doi:10.1097/MPA.0b013e31823b5f2e
PMCID: PMC3336038  PMID: 22258068
immunotoxin; pancreatic cancer; xenograft model; EGF; pseudomonas exotoxin; immunoconjugate therapy
18.  Cancer Treatment with Gene Therapy and Radiation Therapy 
Advances in cancer research  2012;115:221-263.
Radiation therapy methods have evolved remarkably in recent years which have resulted in more effective local tumor control with negligible toxicity of surrounding normal tissues. However, local recurrence and distant metastasis often occur following radiation therapy mostly due to the development of radioresistance through the deregulation of the cell cycle, apoptosis, and inhibition of DNA damage repair mechanisms. Over the last decade, extensive progress in radiotherapy and gene therapy combinatorial approaches has been achieved to overcome resistance of tumor cells to radiation. In this review, we summarize the results from experimental cancer therapy studies on the combination of radiation therapy and gene therapy.
doi:10.1016/B978-0-12-398342-8.00007-0
PMCID: PMC3664947  PMID: 23021246
19.  Therapy of Head and Neck Squamous Cell Carcinoma with Replicative Adenovirus Expressing Tissue Inhibitor of Metalloproteinase-2 and Chemoradiation 
Cancer gene therapy  2008;16(3):246-255.
Objective
Recent studies have demonstrated efficacy of targeted therapy combined with radiotherapy in HNSCC. We hypothesized that a combination treatment including a replicating adenovirus armed with tissue inhibitor metalloproteinase-2 (TIMP-2), radiation, and Cisplatin will augment treatment response and reduce tumor growth in vivo of HNSCC xenografts.
Design
Both single-agent (TIMP-2 virus, radiation, and Cisplatin) and the combination therapies were evaluated in vitro and in vivo. The efficacy of both single agent and combination therapies in vivo was determined by monitoring of tumor growth and immunohistochemistry.
Results
Treatment with replicative Ad-TIMP-2 virus and radiation decreased cell viability in vitro and resulted in an additional anti-angiogenic response in vivo. Tumor response rates to treatment with replicative Ad-TIMP-2, radiation, Cisplatin, or combination therapies ranged from limited inhibition of tumor growth of the single-agent therapy to a statistically significant additive anti-tumor response with the combination therapies. Replicative Ad-TIMP-2 + radiation + Cisplatin in the SCC1 nude mice demonstrated the greatest response rates in tumor growth and angiogenesis.
Conclusions
Combination Ad-TIMP-2 gene therapy with radiation and the triple treatment group resulted in an augmented therapeutic response. This is the first report of the potential benefits of combining radiation and MMP inhibitor treatment.
doi:10.1038/cgt.2008.76
PMCID: PMC2713186  PMID: 18846112
TIMP-2; chemoradiation; gene therapy; HNSCC
20.  Effect of anti-DR5 and chemotherapy on basal-like breast cancer 
The purpose is to evaluate sensitivity of basal-like breast cancer to treatment with anti-DR5 alone and in combination with chemotherapy. Cytotoxicity of TRA-8 anti-DR5 alone and in combination with doxorubicin or paclitaxel was examined. The role of a DR5-associated molecule (DDX3) in the regulation of apoptosis by recruitment of cIAP1 to the DR5/DDX3 complex was studied. SUM159 and 2LMP orthotopic xenografts were treated with TRA-8 alone and in combination with Abraxane or doxorubicin, and tumor growth inhibition determined. Diffusion-weighted magnetic resonance imaging was used to monitor early tumor response. The majority (12/15) of basal-like cell lines were very sensitive to TRA-8-induced cytotoxicity (IC50 values of 1.0–49 ng/ml). In contrast, 8/11 luminal or HER2-positive cell lines were resistant (IC50 > 1,000 ng/ml). Enhanced killing of basal-like cell lines was produced by combination treatment with TRA-8 and doxorubicin. Majority of basal cell lines expressed lower levels of DR5-associated DDX3 and cIAP1 than luminal and HER2-positive cell lines. TRA-8 inhibited growth of basal xenografts and produced 20% complete 2LMP tumor regressions. TRA-8 and chemotherapy produced greater 2LMP growth inhibition than either alone. An increase in apparent diffusion coefficient in 2LMP tumors was measured in a week of therapy with TRA-8 and Abraxane. Basal-like cell lines were more sensitive to TRA-8-mediated cytotoxicity than HER2-over-expressing and luminal cell lines, and chemotherapy enhanced cytotoxicity. High sensitivity of basal cells to TRA-8 correlated with low expression of DR5/DDX3/cIAP1 complex. Treatment with TRA-8 and chemotherapy may be an effective therapy for basal-like breast cancer.
doi:10.1007/s10549-011-1755-0
PMCID: PMC3613128  PMID: 21901385
Basal-like breast cancer; Anti-DR5 antibody; Chemotherapy
21.  Basal-like breast cancer stem cells are sensitive to anti-DR5 mediated cytotoxicity 
Breast cancer stem cells (BrCSC) are resistant to common therapeutic modalities including chemotherapy, radiation, and hormonal agents. They are thought to contribute to treatment resistance, relapse, and metastases. This study examines the effect of a monoclonal anti-DR5 antibody (TRA-8) and chemotherapy (adriamycin, taxol) on BrCSC populations from basal-like breast cancer cell lines. Doubly enriched BrCSC (CD44+, CD24−, ALDH+) cells were exposed to TRA-8 and control reagents and examined for cytotoxicity, caspase activation, tumorsphere formation and tumorigenicity. Doubly enriched BrCSC populations expressed cell surface DR5 and were sensitive to TRA-8 mediated cytotoxicity with induction of caspase 8 and 3 activation. TRA-8 at sub-nanomolar concentrations inhibited 2LMP and SUM159 BrCSC tumorsphere formation and was more than 50-fold more inhibitory than TRAIL or anti-DR4 at equimolar concentrations. Chemotherapy treatment of 2LMP and SUM159 cell lines resulted in a relative increase of BrCSC, whereas TRA-8 produced a decrease in the percentage of BrCSC. TRA-8 exposure to 2LMP and SUM159 BrCSC preparations produced significant inhibition of tumorigenicity. DR5 maybe a therapeutic target on the surface of basal-like BrCSC which is amenable to agonistic monoclonal anti-DR5 therapy.
doi:10.1007/s10549-011-1763-0
PMCID: PMC3609658  PMID: 21915634
Anti-DR5; Tigatuzumab; Basal-like breast cancer; Breast cancer stem cells; Tumor initiating cells; Tumorspheres; Death receptor 5
22.  The C-Terminal Region Mesd Peptide Mimics Full-Length Mesd and Acts as an Inhibitor of Wnt/β-Catenin Signaling in Cancer Cells 
PLoS ONE  2013;8(2):e58102.
While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/β-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/β-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer.
doi:10.1371/journal.pone.0058102
PMCID: PMC3585277  PMID: 23469146
23.  Cellular Model of Warburg Effect Identifies Tumor Promoting Function of UCP2 in Breast Cancer and Its Suppression by Genipin 
PLoS ONE  2011;6(9):e24792.
The Warburg Effect is characterized by an irreversible injury to mitochondrial oxidative phosphorylation (OXPHOS) and an increased rate of aerobic glycolysis. In this study, we utilized a breast epithelial cell line lacking mitochondrial DNA (rho0) that exhibits the Warburg Effect associated with breast cancer. We developed a MitoExpress array for rapid analysis of all known nuclear genes encoding the mitochondrial proteome. The gene-expression pattern was compared among a normal breast epithelial cell line, its rho0 derivative, breast cancer cell lines and primary breast tumors. Among several genes, our study revealed that over-expression of mitochondrial uncoupling protein UCP2 in rho0 breast epithelial cells reflects gene expression changes in breast cancer cell lines and in primary breast tumors. Furthermore, over-expression of UCP2 was also found in leukemia, ovarian, bladder, esophagus, testicular, colorectal, kidney, pancreatic, lung and prostate tumors. Ectopic expression of UCP2 in MCF7 breast cancer cells led to a decreased mitochondrial membrane potential and increased tumorigenic properties as measured by cell migration, in vitro invasion and anchorage independent growth. Consistent with in vitro studies, we demonstrate that UCP2 over-expression leads to development of tumors in vivo in an orthotopic model of breast cancer. Genipin, a plant derived small molecule, suppressed the UCP2 led tumorigenic properties, which were mediated by decreased reactive oxygen species and down-regulation of UCP2. However, UCP1, 3, 4 and 5 gene expression was unaffected. UCP2 transcription was controlled by SMAD4. Together, these studies suggest a tumor-promoting function of UCP2 in breast cancer. In summary, our studies demonstrate that i) the Warburg Effect is mediated by UCP2; ii) UCP2 is over-expressed in breast and many other cancers; iii) UCP2 promotes tumorigenic properties in vitro and in vivo and iv) genipin suppresses the tumor promoting function of UCP2.
doi:10.1371/journal.pone.0024792
PMCID: PMC3174207  PMID: 21935467
24.  A new drug delivery method of bispecific ligand-directed toxins that reduces toxicity and promotes efficacy in a model of orthotopic pancreatic cancer 
Pancreas  2010;39(6):913-922.
Objective
Biologicals targeting EGFR and IL-13R react with over-expressed markers on cancer cells, but also react with receptor on normal cells. Since we developed novel bispecific ligand directed toxins (BLT) synthesized by cloning EGF and IL-13 on the same molecule with toxin, our objective was to determine whether we could block normal receptors while still targeting receptors over-expressed on cancer cells thereby decreasing toxicity while maintaining efficacy.
Methods
A method, ToxBloc, was developed in which a bolus IP dose of recombinant EGF13 (without toxin) was given to mice about 15-20 minutes prior to DTEGF13. Experiments were then performed to determine if the MTD was reduced and whether we still were able to eliminate progression of aggressive human, metastatic, systemic pancreatic cancer induced by orthotopic injection in nude mice.
Results
ToxBloc permitted us to safely exceed the DTEGF13 MTD by 15-fold. This approach permitted repetitive high dosing with the BLT resulting in tumor regression (p<0.01). Tumor affects were documented using a tumor imaging model in which tumor growth was monitored noninvasively in real time. ToxBloc was selective since other bispecific peptides did not block.
Conclusions
ToxBloc represents a new method of drug delivery and a potential solution to the toxicity problem.
doi:10.1097/MPA.0b013e3181cbd908
PMCID: PMC2907476  PMID: 20182395
immunotoxin; pancreatic cancer; nude mice; EGF; IL-13; toxicity
25.  Anti-EMMPRIN Monoclonal Antibody as a Novel Agent for Therapy of Head and Neck Cancer 
Purpose
Extracellular matrix metalloprotease inducer (EMMPRIN) is a tumor surface protein that promotes growth and is overexpressed in head and neck cancer. These features make it a potential therapeutic target for monoclonal antibody (mAb) – based therapy. Because molecular therapy is considered more effective when delivered with conventional cytotoxic agents, anti-EMMPRIN therapy was assessed alone and in combination with external beam radiation.
Experimental Design
Using a murine flank model, loss of EMMPRIN function was achieved by transfection with a small interfering RNA against EMMPRIN or treatment with a chimeric anti-EMMPRIN blocking mAb. Cytokine expression was assessed for xenografts, tumor cells, fibroblasts, and endothelial cells.
Results
Animals treated with anti-EMMPRIN mAb had delayed tumor growth compared with untreated controls, whereas treatment with combination radiation and anti-EMMPRIN mAb showed the greatest reduction in tumor growth (P = 0.001). Radiation-treated EMMPRIN knockdown xenografts showed a reduction in tumor growth compared with untreated knockdown controls (P = 0.01), whereas radiation-treated EMMPRIN – expressing xenografts did not show a delay in tumor growth. Immunohistochemical evaluation for Ki67 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) resulted in a reduction in proliferation (P = 0.007) and increased apoptosis in anti-EMMPRIN mAb – treated xenografts compared with untreated controls (P = 0.087). In addition, we provide evidence that EMMPRIN suppression results in decreased interleukin 1β (IL-1β), IL-6, and IL-8 cytokine production, in vitro and in vivo.
Conclusions
These data suggest that anti-EMMPRIN antibody inhibits tumor cell proliferation in vivo and may represent a novel targeted treatment option in head and neck squamous cell carcinoma.
doi:10.1158/1078-0432.CCR-09-0212
PMCID: PMC2796106  PMID: 19509148

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