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1.  Enhanced UV-Induced Skin Carcinogenesis in Transgenic Mice Overexpressing Proprotein Convertases1 
Neoplasia (New York, N.Y.)  2013;15(2):169-179.
The proprotein convertases (PCs) furin and PACE4 process numerous substrates involved in tumor growth, invasion, and metastasis. We have previously shown that PCs increase the susceptibility to chemical skin carcinogenesis. Because of the human relevancy of UV radiation in the etiopathogenesis of human skin cancer, we investigated whether or not transgenic mice overexpressing either furin alone or both furin and PACE4 show increased susceptibility to UV carcinogenesis. After backcrossing our previously described furin and PACE4 transgenic lines, targeted to the epidermis, into a SKH-1 background, we exposed both single and double transgenic mice to UV radiation for 34 weeks. The results showed an increase in squamous cell carcinoma (SCC) multiplicity of approximately 70% in the single furin transgenic mouse line SF47 (P < .002) and a 30% increase in the other single transgenic line SF49 when compared to wild-type (WT) SKH-1 mice. Interestingly, there was also an increase in the percentage of high histologic grade SCCs in the transgenic lines compared to the WT mice, i.e., WT = 9%, SF47 = 15%, and SF49 = 26% (P < .02). Targeting both furin and PACE4 to the epidermis in double transgenic mice did not have an additive effect on tumor incidence/multiplicity but did enhance the tumor histopathologic grade, i.e., a significant increase in higher grade SCCs was seen in the bigenic mouse line SPF47 (P < .02). Thus, we observed an increased susceptibility to UV in single furin transgenic mice that was not substantially enhanced in the double furin/PACE4 transgenic mice.
PMCID: PMC3579319  PMID: 23441131
2.  Optical Imaging of Periostin Enables Early Endoscopic Detection and Characterization of Esophageal Cancer in Mice 
Gastroenterology  2012;144(2):294-297.
Imaging strategies that detect early-stage esophageal squamous cell carcinoma (ESCC) could improve clinical outcomes, combined with endoscopic approaches. Periostin is an integrin-binding protein that is important in the tumor microenvironment. We created a fluorescent-labeled antibody that recognizes periostin and binds specifically to ESCC xenograft tumors in mice. In L2-cre;p120ctnLoxP/LoxP mice, which develop squamous cell cancers that resemble human ESCC, we visualized the probe in preneoplastic and neoplastic esophageal lesions using near-infrared fluorescent imaging with upper gastrointestinal endoscopy. Periostin might be a biomarker of the esophageal tumor microenvironment that can be used to detect preneoplastic lesions.
doi:10.1053/j.gastro.2012.10.030
PMCID: PMC3624041  PMID: 23085486
mouse model; neoplasm; extracellular matrix; POSTN
3.  Aurora Kinase A Mediates Epithelial Ovarian Cancer Cell Migration and Adhesion 
Oncogene  2013;33(5):539-549.
Aurora kinase A (AURKA) localizes to centrosomes and mitotic spindles where it mediates mitotic progression and chromosomal stability. Overexpression of AURKA is common in cancer, resulting in acquisition of alternate non-mitotic functions. In the current study, we identified a novel role for AURKA in regulating ovarian cancer cell dissemination and evaluated the efficacy of an AURKA-selective small molecule inhibitor, alisertib (MLN8237), as a single agent and combined with paclitaxel using an orthotopic xenograft model of epithelial ovarian cancer (EOC). Ovarian carcinoma cell lines were used to evaluate the effects of AURKA inhibition and overexpression on migration and adhesion. Pharmacologic or RNAi-mediated inhibition of AURKA significantly reduced ovarian carcinoma cell migration and adhesion and the activation-associated phosphorylation of the cytoskeletal regulatory protein SRC at tyrosine 416 (pSRCY416). Conversely, enforced expression of AURKA resulted in increased migration, adhesion and activation of SRC in cultured cells. In vivo tumor growth and dissemination were inhibited by alisertib treatment as a single agent. Moreover, combination of alisertib with paclitaxel, an agent commonly used in treatment of EOC, resulted in more potent inhibition of tumor growth and dissemination compared to either drug alone. Taken together, these findings support a role for AURKA in EOC dissemination by regulating migration and adhesion. They also point to the potential utility of combining AURKA inhibitors with taxanes as a therapeutic strategy for the treatment of EOC patients.
doi:10.1038/onc.2012.632
PMCID: PMC3640671  PMID: 23334327
Aurora kinase A; SRC; ovarian cancer; migration; alisertib; adhesion
4.  IGFBP3 promotes esophageal cancer growth by suppressing oxidative stress in hypoxic tumor microenvironment 
Insulin-like growth factor binding protein 3 (IGFBP3), a hypoxia-inducible gene, regulates a variety of cellular processes including cell proliferation, senescence, apoptosis and epithelial-mesenchymal transition (EMT). IGFBP3 has been linked to the pathogenesis of cancers. Most previous studies focus upon proapoptotic tumor suppressor activities of IGFBP3. Nevertheless, IGFBP3 is overexpressed in certain cancers including esophageal squamous cell carcinoma (ESCC), one of the most aggressive forms of squamous cell carcinomas (SCCs). The tumor-promoting activities of IGFBP3 remain poorly understood in part due to a lack of understanding as to how the tumor microenvironment may influence IGFBP3 expression and how IGFBP3 may in turn influence heterogeneous intratumoral cell populations. Here, we show that IGFBP3 overexpression is associated with poor postsurgical prognosis in ESCC patients. In xenograft transplantation models with genetically engineered ESCC cells, IGFBP3 contributes to tumor progression with a concurrent induction of a subset of tumor cells showing high expression of CD44 (CD44H), a major cell surface receptor for hyaluronic acid, implicated in invasion, metastasis and drug resistance. Our gain-of-function and loss-of-function experiments reveal that IGFBP3 mediates the induction of intratumoral CD44H cells. IGFBP3 cooperates with hypoxia to mediate the induction of CD44H cells by suppressing reactive oxygen species (ROS) in an insulin-like growth factor-independent fashion. Thus, our study sheds light on the growth stimulatory functions of IGFPB3 in cancer, gaining a novel mechanistic insight into the functional interplay between the tumor microenvironment and IGFBP3.
PMCID: PMC3902230  PMID: 24482736
CD44; esophageal; squamous cell carcinoma; hypoxia; IGFBP3 and reactive oxygen species
5.  Correction: The putative Tumor Suppressor VILIP-1 Counteracts Epidermal Growth Factor-Induced Epidermal-Mesenchymal Transition in Squamous Carcinoma Cells 
PLoS ONE  2013;8(12):10.1371/annotation/9453b87a-098a-44ee-9e76-97eebb50b641.
doi:10.1371/annotation/9453b87a-098a-44ee-9e76-97eebb50b641
PMCID: PMC3876944
7.  A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT competent cells that express the ZEB transcription factors 
Cancer research  2011;71(21):6836-6847.
Zinc finger E-box binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated senescence, epithelial to mesenchymal transition (EMT) and cancer stem cell function. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1 enriched EMT competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage independent growth and tumor formation in nude mice. RNA interference (RNAi) experiments suggested the involvement of ZEBs in anchorage independent colony formation, invasion and TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation was recapitulated upon Notch inhibition by DNMAML1 in organotypic 3D culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of squamous esophageal cancers.
doi:10.1158/0008-5472.CAN-11-0846
PMCID: PMC3206139  PMID: 21890822
Notch; EMT; squamous cell differentiation; ZEB1; miR-200
8.  Klf4 Overexpression Activates Epithelial Cytokines and Inflammation-Mediated Esophageal Squamous Cell Cancer in Mice 
Gastroenterology  2010;139(6):2124-2134.e9.
BACKGROUND & AIMS
Esophageal squamous cell cancer accounts for more than 90% of cases of esophageal cancers. Its pathogenesis involves chronic epithelial irritation, although the factors involved in the inflammatory process and the mechanisms of carcinogenesis are unknown. We sought to develop a mouse model of this cancer.
METHODS
We used the ED-L2 promoter of Epstein-Barr virus to overexpress the transcriptional regulator Krüppel-like factor 4 (Klf4) in esophageal epithelia of mice; we used mouse primary esophageal keratinocytes to examine the mechanisms by which KLF4 induces cytokine production.
RESULTS
KLF4 was an epithelial-specific mediator of inflammation; we developed a new mouse model of esophageal squamous dysplasia and inflammation-mediated squamous cell cancer. KLF4 activated a number of proinflammatory cytokines, including TNF-α, CXCL5, G-CSF and IL-1α, within keratinocytes in an NF-κB– dependent manner. KLF4 was not detected in proliferating or cancer cells, indicating a non-cell autonomous effect of KLF4 on proliferation and carcinogenesis.
CONCLUSIONS
KLF4 has distinct functions in carcinogenesis; upregulation of Klf4 specifically in esophageal epithelial cells induces inflammation. This mouse model might be used to determine the molecular mechanisms of esophageal squamous cell cancer and inflammation-mediated carcinogenesis.
doi:10.1053/j.gastro.2010.08.048
PMCID: PMC3457785  PMID: 20816834
Keratinocyte; Esophagus; Tumor Formation; Transformation
9.  Thymine DNA Glycosylase Is Essential for Active DNA Demethylation by Linked Deamination-Base Excision Repair 
Cell  2011;146(1):67-79.
Summary
DNA methylation is a major epigenetic mechanism for gene silencing. While methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here we show that either knockout or catalytic inactivation of the DNA repair enzyme Thymine DNA Glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific, developmentally- and hormonally-regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage-response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.
doi:10.1016/j.cell.2011.06.020
PMCID: PMC3230223  PMID: 21722948
embryonic lethality; base excision repair; CpG dinucleotides; DNA demethylation; promoter methylation profiles
10.  Biomechanical remodeling of the microenvironment by stromal Caveolin-1 favors tumor invasion and metastasis 
Cell  2011;146(1):148-163.
Summary
Mechanotransduction, a key determinant of tissue homeostasis and tumor progression, is driven by intercellular adhesions, cell contractility and forces generated with the microenvironment, dependent on extracellular matrix composition, organization and compliance. Caveolin-1 (Cav1) favors cell elongation in 3D cultures and promotes Rho-and force-dependent contraction, matrix alignment and microenvironment stiffening through regulation of p190RhoGAP. In turn, microenvironment remodeling by Cav1-fibroblasts forces cell elongation. Cav1-deficient mice have disorganized stromal tissue architecture. Stroma associated with human carcinomas and melanoma metastases is enriched in Cav1-expressing carcinoma-associated fibroblasts (CAFs). Cav1 expression in breast CAFs correlates with low survival, and Cav1 depletion in CAFs decreases CAF contractility. Consistently, fibroblast expression of Cav1, through p190RhoGAP regulation, favors directional migration and invasiveness of carcinoma cells in vitro. In vivo, stromal Cav1 remodels peri- and intratumoral microenvironments to facilitate tumor invasion, correlating with increased metastatic potency. Thus, Cav1 modulates tissue responses through force-dependent architectural regulation of the microenvironment.
doi:10.1016/j.cell.2011.05.040
PMCID: PMC3244213  PMID: 21729786
Caveolin-1; stiffness; mechanotransduction; cell-derived matrices; cell motility; microenvironment; tumor stroma
11.  The mTOR pathway affects proliferation and chemosensitivity of urothelial carcinoma cells and is upregulated in a subset of human bladder cancers 
BJU international  2010;108(2B):E84-E90.
SUMMARY
OBJECTIVE
To investigate whether mammalian target of rapamycin (mTOR) inhibition by rapamycin is therapeutically efficacious in combination with cisplatin for bladder cancer.
MATERIALS AND METHODS
Using a panel of human urothelial carcinoma cell lines, we determined the effect of rapamycin on cell viability, cell-cycle progression, signalling and apoptosis.
The effect of mTOR inhibition on chemosensitivity was investigated by treating cells with rapamycin, alone, or with cisplatin.
The effect of rapamycin or cisplatin treatment was assessed in xenograft mice inoculated with urothelial carcinoma cells.
Expression of p-mTOR in human bladder cancer specimens was assessed using a tissue microarray.
RESULTS
Treatment with rapamycin significantly decreased cell viability in UMUC3 (P=0.004) and 253J (P<0.001) cells. It induced arrest in the G0–G1 phase and decreased activation of p-mTOR and its downstream effector, p-S6K, in both cell lines.
Treatment with rapamycin increased the ability of cisplatin to inhibit cell viability in UMUC3 (P=0.002) and 253J (P=0.03) cells. No evidence for apoptosis induction was noted after treatment with rapamycin alone.
Mouse xenografts of UMUC3 cells revealed that rapamycin significantly prolonged survival and enhanced the therapeutic efficacy of cisplatin.
In patient urothelial carcinoma specimens, p-mTOR expression was increased in cancer vs. non-tumour bladder tissue in 65/203 (32.0%) tumours.
CONCLUSION
mTOR blockade inhibits urothelial carcinoma cell proliferation and enhances the effectiveness of cisplatin.
Suppression of the mTOR pathway has the potential to be a therapeutic target in bladder cancer for selected patients.
doi:10.1111/j.1464-410X.2010.09844.x
PMCID: PMC3116980  PMID: 21050361
bladder cancer; mTOR; chemotherapy; rapamycin; radical cystectomy
12.  Notch receptor inhibition reveals the importance of cyclin D1 and Wnt signaling in invasive esophageal squamous cell carcinoma 
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of squamous cell carcinomas. Common genetic lesions in ESCC include p53 mutations and EGFR overexpression, both of which have been implicated in negative regulation of Notch signaling. In addition, cyclin D1 is overexpressed in ESCC and can be activated via EGFR, Notch and Wnt signaling. To elucidate how these genetic lesions may interact during the development and progression of ESCC, we tested a panel of genetically engineered human esophageal cells (keratinocytes) in organotypic 3D culture (OTC), a form of human tissue engineering. Notch signaling was suppressed in culture and mice by dominant negative Mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor. DNMAML1 mice were subjected to 4-Nitroquinoline 1-oxide-induced oral-esophageal carcinogenesis. Highly invasive characteristics of primary human ESCC were recapitulated in OTC as well as DNMAML1 mice. In OTC, cyclin D1 overexpression induced squamous hyperplasia. Concurrent EGFR overexpression and mutant p53 resulted in transformation and invasive growth. Interestingly, cell proliferation appeared to be regulated differentially between those committed to squamous-cell differentiation and those invading into the stroma. Invasive cells exhibited Notch-independent activation of cyclin D1 and Wnt signaling. Within the oral-esophageal squamous epithelia, Notch signaling regulated squamous-cell differentiation to maintain epithelial integrity, and thus may act as a tumor suppressor by preventing the development of a tumor-promoting inflammatory microenvironment.
PMCID: PMC3410579  PMID: 22860235
Esophageal squamous cell carcinoma; organotypic 3D culture; EGFR; P53; cyclin D1; Wnt; Notch; squamous-cell differentiation; invasion; 4-Nitroquinoline 1-oxide
13.  Contribution of Abcc10 (Mrp7) to in vivo paclitaxel resistance as assessed in Abcc10−/− mice 
Cancer research  2011;71(10):3649-3657.
Recently we reported that the ATP-binding cassette transporter Abcc10, also known as multidrug resistance protein 7 (Mrp7), is able to confer resistance to a variety of anticancer agents including taxanes. However, the in vivo functions of the pump have not been determined to any extent. Here we generated and analyzed Abcc10−/− mice in order to investigate the ability of Abcc10 to function as an endogenous resistance factor. Mouse embryo fibroblasts derived from Abcc10 −/− mice were hypersensitive to docetaxel, paclitaxel, vincristine and Ara-C and exhibited increased cellular drug accumulation, relative to wild type controls. Abcc10 null mice treated with paclitaxel exhibited increased lethality associated with neutropenia and marked bone marrow toxicity. Toxicity in spleen and thymus was also evident. These findings indicate that Abcc10 is dispensable for health and viability, and that it is an endogenous resistance factor for taxanes, other natural product agents and nucleoside analogs. This is the first demonstration that an ATP-binding cassette transporter other than P-glycoprotein can affect in vivo tissue sensitivity towards taxanes.
doi:10.1158/0008-5472.CAN-10-3623
PMCID: PMC3096848  PMID: 21576088
Drug transport; drug resistance; Mrp7; Abcc10; taxanes; paclitaxel
14.  The Fbx4 Tumor Suppressor Regulates Cyclin D1 Accumulation and Prevents Neoplastic Transformation ▿  
Molecular and Cellular Biology  2011;31(22):4513-4523.
Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complexes modulate the accumulation of key cell cycle regulatory proteins. Following the G1/S transition, SCFFbx4 targets cyclin D1 for proteasomal degradation, a critical event necessary for DNA replication fidelity. Deregulated cyclin D1 drives tumorigenesis, and inactivating mutations in Fbx4 have been identified in human cancer, suggesting that Fbx4 may function as a tumor suppressor. Fbx4+/− and Fbx4−/− mice succumb to multiple tumor phenotypes, including lymphomas, histiocytic sarcomas and, less frequently, mammary and hepatocellular carcinomas. Tumors and premalignant tissue from Fbx4+/− and Fbx4−/− mice exhibit elevated cyclin D1, an observation consistent with cyclin D1 as a target of Fbx4. Molecular dissection of the Fbx4 regulatory network in murine embryonic fibroblasts (MEFs) revealed that loss of Fbx4 results in cyclin D1 stabilization and nuclear accumulation throughout cell division. Increased proliferation in early passage primary MEFs is antagonized by DNA damage checkpoint activation, consistent with nuclear cyclin D1-driven genomic instability. Furthermore, Fbx4−/− MEFs exhibited increased susceptibility to Ras-dependent transformation in vitro, analogous to tumorigenesis observed in mice. Collectively, these data reveal a requisite role for the SCFFbx4 E3 ubiquitin ligase in regulating cyclin D1 accumulation, consistent with tumor suppressive function in vivo.
doi:10.1128/MCB.05733-11
PMCID: PMC3209253  PMID: 21911473
15.  The putative Tumor Suppressor VILIP-1 Counteracts Epidermal Growth Factor-Induced Epidermal-Mesenchymal Transition in Squamous Carcinoma Cells 
PLoS ONE  2012;7(3):e33116.
Epithelial-mesenchymal transition (EMT) is a crucial step for the acquisition of invasive properties of carcinoma cells during tumor progression. Epidermal growth factor (EGF)-treatment of squamous cell carcinoma (SCC) cells provokes changes in the expression of lineage markers, morphological changes, and a higher invasive and metastatic potential. Here we show that chronic stimulation with EGF induces EMT in skin-derived SCC cell lines along with the down-regulation of the epithelial marker E-cadherin, and of the putative tumor suppressor VILIP-1 (visinin-like protein 1). In esophageal squamous cell carcinoma and non-small cell lung carcinoma the loss of VILIP-1 correlates with clinicopathological features related to enhanced invasiveness. VILIP-1 has previously been shown to suppress tumor cell invasion via enhancing cAMP-signaling in a murine SCC model. In mouse skin SCC cell lines the VILIP-1-negative tumor cells have low cAMP levels, whereas VILIP-1-positive SCCs possess high cAMP levels, but low invasive properties. We show that in VILIP-1-negative SCCs, Snail1, a transcriptional repressor involved in EMT, is up-regulated. Snail1 expression is reduced by ectopic VILIP-1-expression in VILIP-1-negative SCC cells, and application of the general adenylyl cyclase inhibitor 2′,3′-dideoxyadenosine attenuated this effect. Conversely, EGF-stimulation of VILIP-1-positive SCC cells leads to the down-regulation of VILIP-1 and the induction of Snail1 expression. The induction of Snail is inhibited by elevated cAMP levels. The role of cAMP in EMT was further highlighted by its suppressive effect on the EGF-induced enhancement of migration in VILIP-1-positive SCC cells. These findings indicate that VILIP-1 is involved in EMT of SCC by regulating the transcription factor Snail1 in a cAMP-dependent manner.
doi:10.1371/journal.pone.0033116
PMCID: PMC3316558  PMID: 22479362
16.  Involvement of VILIP-1 (visinin-like protein) and opposite roles of cyclic AMP and GMP signaling in in vitro cell migration of murine skin squamous cell carcinoma 
Molecular Carcinogenesis  2010;50(5):319-333.
VILIP-1 (visinin-like protein 1) is downregulated in various human squamous cell carcinoma. In a mouse skin SCC model VILIP-1 expression is reduced in aggressive tumor cells, accompanied by reduced cAMP levels. Overexpression of VILIP-1 in aggressive SCC cells led to enhanced cAMP production, in turn causing a reduction in invasive properties. Moreover, in primary neurons and neuronal tumor lines VILIP-1 enhanced cGMP-signaling. Here, we set out to determine whether and how cAMP and cGMP-signaling contribute to the VILIP-1 effect on enhanced SCC model cell migration, and thus most likely invasiveness in vivo. We found stronger increase in cGMP levels in aggressive, VILIP-1-negative SCC cells following stimulation of guanylyl cyclases NPR-A and -B with the natriuretic peptides ANP and CNP, respectively. Incubation with ANP or 8Br-cGMP to increase cGMP levels further enhanced the migration capacity of aggressive cells, whereas cell adhesion was unaffected. Increased cGMP was caused by elevated expression levels of NPR-A and NPR-B. However, the expression level of VILIP-1 did not affect cGMP signaling and guanylyl cyclase expression in SCC. In contrast, VILIP-1 led to reduced migration of aggressive SCC cells depending on cAMP levels as shown by use of adenylyl cyclase inhibitor 2′,3′-dideoxyadenosine. Involvement of cAMP-effectors PKA and EPAC play a role downstream of adenylyl cyclase activation. VILIP-1-positive and -negative cells did not differ in mRNA expression of adenylyl cyclases, but an effect on enhanced protein expression and membrane localization of ACs was shown to underlie enhancement of cAMP production and, thus, reduction in cell migration by VILIP-1.
doi:10.1002/mc.20707
PMCID: PMC3202085  PMID: 21480386
squamous cell carcinoma; cAMP-; cGMP-signaling; cell migration; calcium-binding protein; tumor invasion suppressor
17.  Influence of Affinity and Antigen Internalization on the Uptake and Penetration of Anti-HER2 Antibodies in Solid Tumors 
Cancer research  2011;71(6):2250-2259.
Antibody drugs are widely used in cancer therapy, but conditions to maximize tumor penetration and efficacy have yet to be fully elucidated. In this study, we investigated the impact of antibody binding affinity on tumor targeting and penetration with affinity variants that recognize the same epitope. Specifically, we compared four derivatives of the C6.5 monoclonal antibody (MAb) which recognizes the same HER2 epitope (monovalent KDs ranging from 270nM to 0.56nM). Moderate affinity was associated with the highest tumor accumulation at 24hr and 120hr post i.v. injection, whereas high affinity was found to produce the lowest tumor accumulation. Highest affinity MAb were confined to the perivascular space of tumors with an average penetration of 20.4 +/− 7.5 microns from tumor blood vessels. Conversely, lowest affinity MAb exhibited a broader distribution pattern with an average penetration of 84.8 +/− 12.8 microns. In vitro internalization assays revealed that antibody internalization and catabolism generally increased with affinity, plateauing once the rate of HER2 internalization exceeded the rate of antibody dissociation. Effects of internalization and catabolism on tumor targeting were further examined using antibodies of moderate (C6.5) or high affinity (trastuzumab) labeled with residualizing (111In-labeled) or non-residualizing (125I-labeled) radioisotopes. Significant amounts of antibody of both affinities were degraded by tumors in vivo. Further, moderate to high affinity MAbs targeting the same HER2 epitope with monovalent affinity above 23nM had equal tumor accumulation of residualizing radiolabel over 120hrs. Results indicated equal tumor exposure, suggesting that MAb penetration and retention in tumors reflected affinity-based differences in tumor catabolism. Together, these results suggest that high-density, rapidly internalizing antigens subject high-affinity antibodies to greater internalization and degradation, thereby limiting their penetration of tumors. In contrast, lower affinity antibodies penetrate tumors more effectively when rates of antibody-antigen dissociation are higher than rates of antigen internalization. Together, our findings offer insights into how to optimize the ability of therapeutic antibodies to penetrate tumors.
doi:10.1158/0008-5472.CAN-10-2277
PMCID: PMC3077882  PMID: 21406401
18.  Periostin, a cell adhesion molecule, facilitates invasion in the tumor microenvironment and annotates a novel tumor invasive signature in esophageal cancer 
Cancer Research  2010;70(13):5281-5292.
Human squamous cell cancers are the most common epithelially derived malignancies. One example is esophageal squamous cell carcinoma (ESCC), which is associated with a high mortality rate (1) that is related to a propensity for invasion and metastasis (2). Here we report that periostin, a highly expressed cell adhesion molecule, is a key component of a novel tumor invasive signature obtained from an organotypic culture model of engineered ESCC. This tumor invasive signature classifies with human ESCC microarrays, underscoring its utility in human cancer. Genetic modulation of periostin promotes tumor cell migration and invasion as revealed in gain of and loss of function experiments. Inhibition of EGFR signaling and restoration of wild-type p53 function were each found to attenuate periostin, suggesting interdependence of two common genetic alterations with periostin function. Collectively, our studies reveal periostin as an important mediator of ESCC tumor invasion and they indicate that organotypic (3D) culture can offer an important tool to discover novel biologic effectors in cancer.
doi:10.1158/0008-5472.CAN-10-0704
PMCID: PMC3274349  PMID: 20516120
tumor microenvironment; periostin; EGFR; p53
19.  Estrogen and Cytochrome P450 1B1 Contribute to Both Early- and Late-Stage Head and Neck Carcinogenesis 
Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common type of cancer in the U.S. The goal of this study was to evaluate the contribution of estrogens to the development of HNSCCs. Various cell lines derived from early- and late-stage head and neck lesions were used to: characterize the expression of estrogen synthesis and metabolism genes, including cytochrome P450 (CYP)1B1, examine the effect of estrogen on gene expression and evaluate the role of CYP1B1 and/or estrogen in cell motility, proliferation and apoptosis. Estrogen metabolism genes (CYP1B1, CYP1A1, catechol-o-methyltransferase, UDP-glucuronosyltransferase 1A1, and glutathione-S-transferase P1) and estrogen receptor (ER)β were expressed in cell lines derived from both premalignant (MSK-Leuk1) and malignant (HNSCC) lesions. Exposure to estrogen induced CYP1B1 2.3 to 3.6 fold relative to vehicle-treated controls (P=0.0004) in MSK-Leuk1 cells but not in HNSCC cells. CYP1B1 knockdown by shRNA reduced the migration and proliferation of MSK-Leuk1 cells by 57% and 45%, respectively. Exposure of MSK-Leuk1 cells to estrogen inhibited apoptosis by 26%, while supplementation with the antiestrogen fulvestrant restored estrogen-dependent apoptosis. Representation of the estrogen pathway in human head and neck tissues from 128 patients was examined using tissue microarrays. The majority of the samples exhibited immunohistochemical staining for ERβ (91.9%), CYP1B1 (99.4%) and 17β-estradiol (88.4%). CYP1B1 and ERβ were elevated in HNSCCs relative to normal epithelium (P=0.024 and 0.008, respectively). These data provide novel insight into the mechanisms underlying head and neck carcinogenesis and facilitate the identification new targets for chemopreventive intervention.
doi:10.1158/1940-6207.CAPR-10-0133
PMCID: PMC3043603  PMID: 21205741
estrogen; CYP1B1; leukoplakia; HNSCC; cancer progression
20.  NOTCH1 and NOTCH3 coordinate esophageal squamous differentiation through a CSL-dependent transcriptional network 
Gastroenterology  2010;139(6):2113-2123.
Background & Aims
The Notch receptor family regulates cell fate through cell-cell communication. CSL (CBF-1/RBP-jκ, Su(H), Lag-1) drives canonical Notch-mediated gene transcription during cell lineage specification, differentiation and proliferation in the hematopoietic system, the intestine, the pancreas and the skin. However, the functional roles of Notch in esophageal squamous epithelial biology remain unknown.
Methods
Normal esophageal keratinocytes were stimulated with calcium chloride to induce terminal differentiation. The squamous epithelia were reconstituted in organotypic three-dimensional culture, a form of human tissue engineering. Notch was inhibited in culture with a γ-secretase inhibitor or dominant negative mastermind-like1 (DNMAML1). The roles of Notch receptors were evaluated by in vitro gain-of-function and loss-of-function experiments. Additionally, DNMAML1 was targeted to the mouse esophagus by cytokeratin K14 promoter-driven Cre (K14Cre) recombination of Lox-STOP-Lox-DNMAML1. Notch-regulated gene expression was determined by reporter transfection, chromatin immunoprecipitation (ChIP) assays, quantitative reverse-transcription polymerase chain reactions (RT-PCR), Western blotting, immunofluorescence and immunohistochemistry.
Results
NOTCH1 (N1) was activated at the onset of squamous differentiation in the esophagus. Intracellular domain of N1 (ICN1) directly activated NOTCH3 (N3) transcription, inducing HES5 and early differentiation markers such as involucrin (IVL) and cytokeratin CK13 in a CSL-dependent fashion. N3 enhanced ICN1 activity and was required for squamous differentiation. Loss of Notch signaling in K14Cre;DNMAML1 mice perturbed esophageal squamous differentiation and resulted in N3 loss and basal cell hyperplasia.
Conclusions
Notch signaling is important for esophageal epithelial homeostasis. In particular, the crosstalk of N3 with N1 during differentiation provides novel, mechanistic insights into Notch signaling and squamous epithelial biology.
doi:10.1053/j.gastro.2010.08.040
PMCID: PMC2997138  PMID: 20801121
NOTCH1; NOTCH3; esophageal epithelium; squamous differentiation
21.  Study of FoxA Pioneer Factor at Silent Genes Reveals Rfx-Repressed Enhancer at Cdx2 and a Potential Indicator of Esophageal Adenocarcinoma Development 
PLoS Genetics  2011;7(9):e1002277.
Understanding how silent genes can be competent for activation provides insight into development as well as cellular reprogramming and pathogenesis. We performed genomic location analysis of the pioneer transcription factor FoxA in the adult mouse liver and found that about one-third of the FoxA bound sites are near silent genes, including genes without detectable RNA polymerase II. Virtually all of the FoxA-bound silent sites are within conserved sequences, suggesting possible function. Such sites are enriched in motifs for transcriptional repressors, including for Rfx1 and type II nuclear hormone receptors. We found one such target site at a cryptic “shadow” enhancer 7 kilobases (kb) downstream of the Cdx2 gene, where Rfx1 restricts transcriptional activation by FoxA. The Cdx2 shadow enhancer exhibits a subset of regulatory properties of the upstream Cdx2 promoter region. While Cdx2 is ectopically induced in the early metaplastic condition of Barrett's esophagus, its expression is not necessarily present in progressive Barrett's with dysplasia or adenocarcinoma. By contrast, we find that Rfx1 expression in the esophageal epithelium becomes gradually extinguished during progression to cancer, i.e, expression of Rfx1 decreased markedly in dysplasia and adenocarcinoma. We propose that this decreased expression of Rfx1 could be an indicator of progression from Barrett's esophagus to adenocarcinoma and that similar analyses of other transcription factors bound to silent genes can reveal unanticipated regulatory insights into oncogenic progression and cellular reprogramming.
Author Summary
FoxA transcriptional regulatory proteins are “pioneer factors” that engage silent genes, helping to endow the competence for activation. About a third of the DNA sites we found to be occupied by FoxA in the adult liver are at genes that are silent. Analysis of transcription factor binding motifs near the FoxA sites at silent genes revealed a co-occurrence of motifs for the transcriptional repressors Rfx1 and type II nuclear hormone receptors (NHR-II). Further analysis of one such region downstream of the Cdx2 gene shows that it is a cryptic enhancer, in that it functions poorly unless Rfx1 or NHR-II binding is prevented, in which case FoxA1 promotes enhancer activity. Cdx2 encodes a transcription factor that promotes intestinal differentiation; ectopic expression of Cdx2 in the esophagus can help promote metaplasia and cancer. By screening numerous staged samples of human tissues, we show that Rfx1 expression is extinguished during the progression to esophageal adenocarcinoma and thus may serve as a marker of cancer progression. These studies exemplify how the analysis of pioneer factors bound to silent genes can reveal a basis for the competence of cells to deregulate gene expression and undergo transitions to cancer.
doi:10.1371/journal.pgen.1002277
PMCID: PMC3174211  PMID: 21935353
22.  Insulin-like growth factor-binding protein-3 promotes transforming growth factor-β1-mediated epithelial-to-mesenchymal transition and motility in transformed human esophageal cells 
Carcinogenesis  2010;31(8):1344-1353.
Insulin-like growth factor-binding protein (IGFBP)-3 is overexpressed frequently in esophageal squamous cell carcinoma. Yet, the role of IGFBP3 in esophageal tumor biology remains to be elucidated. We find that IGFBP3 facilitates transforming growth factor (TGF)-β1-mediated epithelial-to-mesenchymal transition (EMT) in transformed human esophageal epithelial cells, EPC2–hTERT–EGFR–p53R175H. In organotypic 3D culture, a form of human tissue engineering, laser-capture microdissection revealed concurrent upregulation of TGF-β target genes, IGFBP3 and EMT-related genes in the cells invading into the stromal compartment. IGFBP3 enhanced TGF-β1-mediated EMT as well as transcription factors essential in EMT by allowing persistent SMAD2 and SMAD3 phosphorylation. TGF-β1-mediated EMT and cell invasion were enhanced by ectopically expressed IGFBP3 and suppressed by RNA interference directed against IGFBP3. The IGFBP3 knockdown effect was rescued by IGFBP3I56G/L80G/L81G, a mutant IGFBP3 lacking an insulin-like growth factor (IGF)-binding capacity. Thus, IGFBP3 can regulate TGF-β1-mediated EMT and cell invasion in an IGF or insulin-like growth factor 1 receptor-independent manner. IGFBP3I56G/L80G/L81G also promoted EMT in vivo in a Ras-transformed human esophageal cell line T-TeRas upon xenograft transplantation in nude mice. In aggregate, IGFBP3 may have a novel IGF-binding independent biological function in regulation of TGF-β1-mediated EMT and cell invasion.
doi:10.1093/carcin/bgq108
PMCID: PMC2915630  PMID: 20513670
23.  VEGF is a Promising Therapeutic Target for the Treatment of Clear Cell Carcinoma of the Ovary 
Molecular cancer therapeutics  2010;9(8):2411-2422.
This study examined the role of VEGF as a therapeutic target in clear cell carcinoma (CCC) of the ovary, which has been regarded as a chemoresistant histological subtype. Immunohistochemical analysis using tissue microarrays of 98 primary ovarian cancers revealed that VEGF was strongly expressed both in early stage and advanced stage CCC of the ovary. In early stage CCCs, patients who had tumors with high levels of VEGF had significantly shorter survival than those with low levels of VEGF. In vitro experiments revealed that VEGF expression was significantly higher in cisplatin-refractory human clear cell carcinoma cells (RMG1-CR and KOC7C-CR), compared to the respective parental cells (RMG1 and KOC7C) in the presence of cisplatin. In vivo treatment with bevacizumab markedly inhibited the growth of both parental CCC cells-derived (RMG1 and KOC7C) and cisplatin-refractory CCC cells-derived (RMG1-CR and KOC7C-CR) tumors as a result of inhibition of tumor angiogenesis. The results of the current study indicate that VEGF is frequently expressed and can be a promising therapeutic target in the management of CCC. Bevacizumab may be efficacious not only as a first-line treatment but also as a second-line treatment of recurrent disease in patients previously treated with cisplatin.
doi:10.1158/1535-7163.MCT-10-0169
PMCID: PMC2941981  PMID: 20663925
VEGF; survival; bevacizumab; cisplatin; clear cell carcinoma
24.  Experimental Treatment of Estrogen Receptor (ER) Positive Breast Cancer with Tamoxifen and Brivanib Alaninate, a VEGFR-2/FGFR-1 Kinase Inhibitor: a potential clinical application of angiogenesis inhibitors 
Purpose
Tamoxifen, a selective estrogen receptor modulator (SERM), and brivanib alaninate, a VEGFR-2 inhibitor, are two target specific agents that result in a substantial decrease in tumor growth when given alone. Tamoxifen activates SERM stimulated breast and endometrial tumor growth. Tamoxifen and brivanib alaninate have side effects that can affect therapeutic outcomes. The primary goal of the current study was to evaluate the therapeutic effects of lower doses of both agents when given in combination to mice with SERM sensitive, estrogen stimulated tumor xenografts (MCF-7 E2 tumors). Experiments were conducted to evaluate the response of SERM stimulated breast (MCF-7 Tam, MCF-7 Ral) and endometrial tumors (EnCa 101) to demonstrate the activity of brivanib alaninate in SERM resistant models.
Experimental Design
In the current study, tumor xenografts were minced and bi-transplanted into the mammary fat pads of athymic, ovariectomized mice. Preliminary experiments were conducted to determine an effective oral dose of tamoxifen and brivanib alaninate that had minimal effect on tumor growth. Doses of 125 µg of tamoxifen and 0.05 mg/g of brivanib alaninate were evaluated. An experiment was designed to evaluate the effect of the two agents together when started at the time of tumor implantation. An additional experiment was done in which tumors were already established and then treated, to obtain enough tumor tissue for molecular analysis.
Results
Brivanib alaninate was effective at inhibiting tumor growth in SERM sensitive (MCF-7 E2) and SERM stimulated (EnCa 101, MCF-7 Ral, MCF-7 Tam) models. The effect of the low dose drug combination as an antitumor strategy for SERM sensitive (MCF-7 E2) in early treatment was as effective as higher doses of either drug used alone. In established tumors, the combination is successful at decreasing tumor growth, while neither agent alone is effective. Molecular analysis revealed a decreased phosphorylation of VEGFR-2 in tumors that were treated with brivanib alaninate and an increase in VEGFA transcription to compensate for the blockade of VEGFR-2 by increasing the transcription of VEGFA. Tamoxifen increases the phosphorylation of VEGFR-2 and this effect is abrogated by brivanib alaninate. There was also increased necrosis in tumors treated with brivanib alaninate.
Conclusion
Historically, tamoxifen has a role in blocking angiogenesis as well as the blockade of the ER. Tamoxifen and a low dose of an angiogenesis inhibitor, brivanib alaninate, can potentially be combined not only to maximize therapeutic efficacy but also to retard SERM resistant tumor growth.
doi:10.1016/j.ejca.2010.02.018
PMCID: PMC2927957  PMID: 20303261
25.  EGFR and mutant p53 expand esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors 
Cancer research  2010;70(10):4174-4184.
Transforming growth factor (TGF)-β is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive as to which molecular mechanisms determine the cellular capacity to undergo EMT in response to TGF-β. We have found that both epidermal growth factor receptor (EGFR) overexpression and mutant p53 tumor suppressor genes contribute to enrichment of an EMT-competent cellular subpopulation amongst telomerase-immortalized human esophageal epithelial cells during malignant transformation. EGFR overexpression triggers oncogene-induced senescence, accompanied by induction of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and p21. Interestingly, a subpopulation of cells emerges by negating senescence without loss of EGFR overexpression. Such cell populations express increased levels of zinc finger E-box binding (ZEB) transcription factors ZEB1 and ZEB2, and undergo EMT upon TGF-β stimulation. Enrichment of EMT-competent cells was more evident in the presence of p53 mutation, which diminished EGFR-induced senescence. RNA interference directed against ZEB resulted in induction of p15INK4B and p16INK4A, reactivating the EGFR-dependent senescence program. Importantly, TGF-β-mediated EMT did not take place when cellular senescence programs were activated by either ZEB knockdown or activation of wild-type p53 function. Thus, senescence checkpoint functions activated by EGFR and p53 may be evaded through the induction of ZEB, thereby allowing expansion of an EMT-competent unique cellular subpopulation, providing novel mechanistic insights into the role of ZEB in esophageal carcinogenesis.
doi:10.1158/0008-5472.CAN-09-4614
PMCID: PMC3007622  PMID: 20424117
EGFR; EMT; senescence; ZEB1; ZEB2

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