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1.  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
2.  Tumor Suppressor Alterations Cooperate to Drive Aggressive Mesotheliomas with Enriched Cancer Stem Cells via a p53-miR34a-c-Met Axis 
Cancer research  2013;74(4):1261-1271.
Malignant mesothelioma (MM) is a highly aggressive, asbestos-related cancer frequently marked by mutations of both NF2 and CDKN2A. We demonstrate that germline knockout of one allele of each of these genes causes accelerated onset and progression of asbestos-induced MM compared to asbestos-exposed Nf2+/− or wild-type (WT) mice. Ascites from some Nf2+/−;Cdkn2a+/− mice exhibited large tumor spheroids, and tail vein injections of MM cells established from these mice, but not from Nf2+/− or WT mice, produced numerous tumors in the lung, suggesting increased metastatic potential of tumor cells from Nf2+/−;Cdkn2a+/− mice. Intraperitoneal injections of MM cells derived from Nf2+/−;Cdkn2a+/− mice into SCID mice produced tumors that penetrated the diaphragm and pleural cavity and harbored an increased cancer stem cells (CSCs). MM cells from Nf2+/−;Cdkn2a+/− mice stained positively for CSC markers and formed CSC spheroids in vitro more efficiently than counterparts from WT mice. Moreover, tumor cells from Nf2+/−;Cdkn2a+/− mice showed elevated c-Met expression/activation, which was partly dependent on p53-mediated regulation of miR34a and required for tumor migration/invasiveness and maintenance of the CSC population. Collectively, these studies demonstrate in vivo that inactivation of Nf2 and Cdkn2a cooperate to drive the development of highly aggressive MMs characterized by enhanced tumor spreading capability and the presence of a CSC population associated with p53/miR34a-dependent activation of c-Met. These findings suggest that cooperativity between losses of Nf2 and Cdkn2a plays a fundamental role in driving the highly aggressive tumorigenic phenotype considered to be a hallmark of MM.
doi:10.1158/0008-5472.CAN-13-2062
PMCID: PMC3945416  PMID: 24371224
malignant mesothelioma; tumor suppressor genes; cancer stem cells; metastasis; mouse models
3.  IMP1 promotes tumor growth, dissemination and a tumor-initiating cell phenotype in colorectal cancer cell xenografts 
Carcinogenesis  2013;34(11):2647-2654.
Igf2 mRNA binding protein 1 (IMP1, CRD-BP, ZBP-1) is a messenger RNA binding protein that we have shown previously to regulate colorectal cancer (CRC) cell growth in vitro. Furthermore, increased IMP1 expression correlates with enhanced metastasis and poor prognosis in CRC patients. In the current study, we sought to elucidate IMP1-mediated functions in CRC pathogenesis in vivo. Using CRC cell xenografts, we demonstrate that IMP1 overexpression promotes xenograft tumor growth and dissemination into the blood. Furthermore, intestine-specific knockdown of Imp1 dramatically reduces tumor number in the Apc Min/+ mouse model of intestinal tumorigenesis. In addition, IMP1 knockdown xenografts exhibit a reduced number of tumor cells entering the circulation, suggesting that IMP1 may directly modulate this early metastatic event. We further demonstrate that IMP1 overexpression decreases E-cadherin expression, promotes survival of single tumor cell-derived colonospheres and promotes enrichment and maintenance of a population of CD24+CD44+ cells, signifying that IMP1 overexpressing cells display evidence of loss of epithelial identity and enhancement of a tumor-initiating cell phenotype. Taken together, these findings implicate IMP1 as a modulator of tumor growth and provide evidence for a novel role of IMP1 in early events in CRC metastasis.
doi:10.1093/carcin/bgt217
PMCID: PMC3810836  PMID: 23764754
4.  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
5.  Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3 
Cancer research  2004;64(21):7711-7723.
Epidermal growth factor receptor (EGFR) is frequently overexpressed in esophageal carcinoma and its precursor lesions. To gain insights into how EGFR overexpression affects cellular functions in primary human esophageal cells, we performed gene expression profiling and identified insulin-like growth factor-binding protein (IGFBP)-3 as the most up-regulated gene. IGFBP-3 regulates cell proliferation through both insulin-like growth factor-dependent and independent mechanisms. We found that IGFBP-3 mRNA and protein expression was increased in EGFR-overexpressing primary and immortalized human esophageal cells. IGFBP-3 was also up-regulated in EGFR-overexpressing cells in organotypic culture and in EGFR transgenic mice. Furthermore, IGFBP-3 mRNA was overexpressed in 80% of primary esophageal squamous cell carcinomas and 60% of primary esophageal adenocarcinomas. Concomitant up-regulation of EGFR and IGFBP-3 was observed in 60% of primary esophageal squamous cell carcinomas. Immunohistochemistry revealed cytoplasmic localization of IGFBP-3 in the preponderance of preneoplastic and neoplastic esophageal lesions. IGFBP-3 was also overexpressed in esophageal cancer cell lines at both mRNA (60%) and protein (40%) levels. IGFBP-3 secreted by cancer cells was capable of binding to insulin-like growth factor I. Functionally, epidermal growth factor appeared to regulate IGFBP-3 expression in esophageal cancer cell lines. Finally, suppression of IGFBP-3 by small interfering RNA augmented cell proliferation, suggesting that IGFBP-3 may inhibit tumor cell proliferation as a negative feedback mechanism. In aggregate, we have identified for the first time that IGFBP-3 is an aberrantly regulated gene through the EGFR signaling pathway and it may modulate EGFR effects during carcinogenesis.
doi:10.1158/0008-5472.CAN-04-0715
PMCID: PMC4140096  PMID: 15520175
6.  A requirement for Nedd9 in luminal progenitor cells prior to mammary tumorigenesis in MMTV-HER2/ErbB2 mice 
Oncogene  2013;33(4):411-420.
Overexpression of the NEDD9/HEF1/Cas-L scaffolding protein is frequent, and drives invasion and metastasis in breast, head and neck, colorectal, melanoma, lung, and other types of cancer. We have examined the consequences of genetic ablation of Nedd9 in the MMTV-HER2/ERBB2/neu mouse mammary tumor model. Unexpectedly, we found that only a limited effect on metastasis in MMTV-neu;Nedd9−/− mice compared to MMTV-neu;Nedd9+/+ mice, but instead a dramatic reduction in tumor incidence (18% versus 80%), and a significantly increased latency until tumor appearance. Orthotopic reinjection and tail vein injection of cells arising from tumors, coupled with in vivo analysis, indicated tumors arising in MMTV-neu;Nedd9−/− mice had undergone mutational selection that overcame the initial requirement for Nedd9. To better understand the defects in early tumor growth, we compared mammary progenitor cell pools from MMTV-neu;Nedd9−/− versus MMTV-neu;Nedd9+/+ mice. The MMTV-neu;Nedd9−/− genotype selectively reduced both the number and colony-forming potential of mammary luminal epithelial progenitor cells, while not affecting basal epithelial progenitors. MMTV-neu;Nedd9−/−mammospheres had striking defects in morphology and cell polarity. All of these defects were seen predominantly in the context of the HER2/neu oncogene, and were not associated with randomization of the plane of mitotic division, but rather with depressed expression the cell attachment protein FAK, accompanied by increased sensitivity to small molecule inhibitors of FAK and SRC. Surprisingly, in spite of these significant differences, only minimal changes were observed in the gene expression profile of Nedd9−/− mice, indicating critical Nedd9-dependent differences in cell growth properties were mediated via post-transcriptional regulation of cell signaling. Coupled with emerging data indicating a role for NEDD9 in progenitor cell populations during the morphogenesis of other tissues, these results indicate a functional requirement for NEDD9 in the growth of mammary cancer progenitor cells.
doi:10.1038/onc.2012.607
PMCID: PMC3628996  PMID: 23318423
breast cancer; HER2; mammary precursor cells; drug resistance
7.  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
8.  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
9.  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
10.  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
11.  Correction: VILIP-1 Downregulation in Non-Small Cell Lung Carcinomas: Mechanisms and Prediction of Survival 
PLoS ONE  2013;8(10):10.1371/annotation/ad0e4775-c4d5-454c-96c0-755ba9fcc806.
doi:10.1371/annotation/ad0e4775-c4d5-454c-96c0-755ba9fcc806
PMCID: PMC4046811
12.  Inflammatory breast cancer (IBC): clues for targeted therapies 
Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer characterized by rapid proliferation, early metastatic development and poor prognosis. Since there are few preclinical models of IBC, there is a general lack of understanding of the complexity of the disease. Recently, we have developed a new model of IBC derived from the pleural effusion of a woman with metastatic secondary IBC. FC-IBC02 cells are triple negative and form clusters (mammospheres) in suspension that are strongly positive for E-cadherin, β-catenin and TSPAN24, all adhesion molecules that play an important role in cell migration and invasion. FC-IBC02 cells expressed stem cell markers and some, but not all of the characteristics of cells undergoing epithelial mesenchymal transition (EMT). Breast tumor FC-IBC02 xenografts developed quickly in SCID mice with the presence of tumor emboli and the development of lymph node and lung metastases. Remarkably, FC-IBC02 cells were able to produce brain metastasis in mice on intracardiac or intraperitoneal injections. Genomic studies of FC-IBC02 and other IBC cell lines showed that IBC cells had important amplification of 8q24 where MYC, ATAD2 and the focal adhesion kinase FAK1 are located. MYC and ATAD2 showed between 2.5 and 7 copies in IBC cells. FAK1, which plays important roles in anoikis resistance and tumor metastasis, showed 6–4 copies in IBC cells. Also, CD44 was amplified in triple-negative IBC cells (10–3 copies). Additionally, FC-IBC02 showed amplification of ALK and NOTCH3. These results indicate that MYC, ATAD2, CD44, NOTCH3, ALK and/or FAK1 may be used as potential targeted therapies against IBC.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-013-2600-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s10549-013-2600-4
PMCID: PMC4273486  PMID: 23784380
CD44; NOTCH3; MYC; ATAD2; ALK; FAK1/PTK2
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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