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1.  Assessing Computational Steps for CLIP-Seq Data Analysis 
BioMed Research International  2015;2015:196082.
RNA-binding protein (RBP) is a key player in regulating gene expression at the posttranscriptional level. CLIP-Seq, with the ability to provide a genome-wide map of protein-RNA interactions, has been increasingly used to decipher RBP-mediated posttranscriptional regulation. Generating highly reliable binding sites from CLIP-Seq requires not only stringent library preparation but also considerable computational efforts. Here we presented a first systematic evaluation of major computational steps for identifying RBP binding sites from CLIP-Seq data, including preprocessing, the choice of control samples, peak normalization, and motif discovery. We found that avoiding PCR amplification artifacts, normalizing to input RNA or mRNAseq, and defining the background model from control samples can reduce the bias introduced by RNA abundance and improve the quality of detected binding sites. Our findings can serve as a general guideline for CLIP experiments design and the comprehensive analysis of CLIP-Seq data.
doi:10.1155/2015/196082
PMCID: PMC4619761  PMID: 26539468
2.  Three-dimensional organotypic culture of stratified epithelia 
Cold Spring Harbor protocols  2015;2015(4):349-353.
One of the limitations of conventional tissue culture on flat 2D surfaces is the loss of complex interactions between the epithelium and stroma. We have devised a culture system that recreates the salient features of the stratified epithelium using primary cell cultures from mouse models. Stratified epithelial cells from various organs (e.g., skin and esophagus) can be grown. Once established, the system can be used to interrogate the effect of various pharmacologic and genetic manipulations on epithelial homeostasis and invasion. Below is applicable to the esophageal epithelium.
doi:10.1101/pdb.prot078311
PMCID: PMC5161091  PMID: 25834261
3.  Loss of Stromal IMP1 Promotes a Tumorigenic Microenvironment in the Colon 
Molecular cancer research : MCR  2015;13(11):1478-1486.
The colon tumor microenvironment (TME) is becoming increasingly recognized as a complex but central player in the development of many cancers. Previously, we identified an oncogenic role for the mRNA-binding protein IMP1 (IGF2BP1) in the epithelium during colon tumorigenesis. In the current study, we reveal the contribution of stromal IMP1 in the context of colitis-associated colon tumorigenesis. Interestingly, stromal deletion of Imp1 (Dermo1Cre;Imp1LoxP/LoxP, or Imp1ΔMes) in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis-associated cancer resulted in increased tumor numbers of larger size and more advanced histological grade than controls. In addition, Imp1ΔMes mice exhibited a global increase in pro-tumorigenic microenvironment factors, including enhanced inflammation and stromal components. Evaluation of purified mesenchyme from AOM/DSS-treated Imp1ΔMes mice demonstrated an increase in hepatocyte growth factor (HGF), which has not been associated with regulation via IMP1. Genetic knockdown of Imp1 in human primary fibroblasts confirmed an increase in HGF with Imp1 loss, demonstrating a specific, cell-autonomous role for Imp1 loss to increase HGF expression. Taken together, these data demonstrate a novel tumor-suppressive role for IMP1 in colon stromal cells and underscore an exquisite, context-specific function for mRNA-binding proteins, such as IMP1, in disease states.
doi:10.1158/1541-7786.MCR-15-0224
PMCID: PMC4644674  PMID: 26194191
Imp1; colon tumor microenvironment; colitis-associated colon cancer; HGF
4.  The Tumor Microenvironment in Esophageal Cancer 
Oncogene  2016;35(41):5337-5349.
Esophageal cancer is a deadly disease, ranking sixth among all cancers in mortality. Despite incremental advances in diagnostics and therapeutics, esophageal cancer still carries a poor prognosis, and thus there remains a need to elucidate the molecular mechanisms underlying this disease. There is accumulating evidence that a comprehensive understanding of the molecular composition of esophageal cancer requires attention to not only tumor cells but also the tumor microenvironment, which contains diverse cell populations, signaling factors, and structural molecules that interact with tumor cells and support all stages of tumorigenesis. In esophageal cancer, environmental exposures can trigger chronic inflammation, which leads to constitutive activation of pro-inflammatory signaling pathways that promote survival and proliferation. Anti-tumor immunity is attenuated by cell populations such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), as well as immune checkpoints like programmed death-1 (PD-1). Other immune cells such as tumor-associated macrophages can have other pro-tumorigenic functions, including the induction of angiogenesis and tumor cell invasion. Cancer-associated fibroblasts secrete growth factors and alter the extracellular matrix (ECM) to create a tumor niche and enhance tumor cell migration and metastasis. Further study of how these TME components relate to the different stages of tumor progression in each esophageal cancer subtype will lead to development of novel and specific TME-targeting therapeutic strategies, which offer considerable potential especially in the setting of combination therapy.
doi:10.1038/onc.2016.34
PMCID: PMC5003768  PMID: 26923327
esophageal cancer; tumor microenvironment; cancer associated fibroblasts; immature myeloid cells
5.  CD38-Expressing Myeloid-Derived Suppressor Cells Promote Tumor Growth in a Murine Model of Esophageal Cancer 
Cancer research  2015;75(19):4074-4085.
Myeloid derived suppressor cells (MDSCs) are an immunosuppressive population of immature myeloid cells found in advanced stage cancer patients and mouse tumor models. Production of inducible nitric oxide synthase (iNOS) and arginase, as well as other suppressive mechanisms, allow MDSCs to suppress T cell-mediated tumor clearance and foster tumor progression. Using an unbiased global gene expression approach in conditional p120-catenin knockout mice (L2-cre;p120ctnf/f), a model of oral-esophageal cancer, we have identified CD38 as playing a vital role in MDSC biology, previously unknown. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation and neutrophil chemotaxis. We find that CD38 expression in MDSCs is evident in other mouse tumor models of esophageal carcinogenesis, and CD38high MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38high MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38low MDSCs, likely as a result of increased iNOS production. Additionally, we have identified novel tumor-derived factors, specifically IL-6, IGFBP-3 and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have detected an expansion of CD38-positive MDSCs in peripheral blood of advanced stage cancer patients and validated targeting CD38 in vivo as a novel approach to cancer therapy.
doi:10.1158/0008-5472.CAN-14-3639
PMCID: PMC4592477  PMID: 26294209
CD38; myeloid derived suppressor cells; immature myeloid cells; iNOS; NFκB
6.  Multiple gastrointestinal polyps in patients treated with BRAF inhibitors 
Purpose
BRAF inhibitors (BRAFi) extend survival in BRAF mutant melanoma but can promote the growth of Ras mutant neoplasms. This study determined if gastrointestinal polyps found in BRAFi-treated patients harbored Ras mutations.
Experimental Design
Colonic and gastric polyps were identified and resected from BRAFi-treated melanoma patients. Next generation sequencing (NGS) was performed on polyps. The ability of BRAFi to promote polyp formation was functionally characterized in Apc Min +/− mice. MAPK and beta catenin pathway activity was assessed by immunohistochemistry in mouse and human polyps.
Results
14 patients treated with BRAFi underwent endoscopy to assess for polyps. Seven out of 7 patients >40 years of age and treated for >2 years were found to have colonic tubular adenomas with 4 out of the 7 patients having 5 or more polyps. One patient presented with bleeding from hyperplastic gastric polyps that recurred 6 months after BRAFi rechallenge. NGS performed on polyps found no mutations in MAPK pathway genes, but found APC mutations in all tubular adenomas. A significant increase in the number of polyps was observed in BRAFi-treated compared to control-treated Apc Min +/− mice (20.8 ± 9.2 v. 12.8 ± 0.1; p=0.016). No polyps were observed in BRAFi-treated wild type mice.
Conclusion
BRAF inhibitors may increase the risk of developing hyperplastic gastric polyps and colonic adenomatous polyps. Due to the risk of gastrointestinal bleeding and the possibility of malignant transformation, further studies are needed to determine whether or not endoscopic surveillance should be recommended for patients treated with BRAF inhibitors.
doi:10.1158/1078-0432.CCR-15-0469
PMCID: PMC4668213  PMID: 26202952
7.  A LIN28B-RAN-AURKA Signaling Network Promotes Neuroblastoma Tumorigenesis 
Cancer cell  2015;28(5):599-609.
SUMMARY
A more complete understanding of aberrant oncogenic signaling in neuroblastoma, a malignancy of the developing sympathetic nervous system, is paramount to improving patient outcomes. Recently, we identified LIN28B as an oncogenic driver in high-risk neuroblastoma. Here, we identify the oncogene RAN as a LIN28B target and show regional gain of chromosome 12q24 as an additional somatic alteration resulting in increased RAN expression. We show that LIN28B influences RAN expression by promoting RAN Binding Protein 2 expression and by directly binding RAN mRNA. Further, we demonstrate a convergence of LIN28B and RAN signaling on Aurora kinase A activity. Collectively, these findings demonstrate that LIN28B-RAN-AURKA signaling drives neuroblastoma oncogenesis, suggesting that this pathway may be amenable to therapeutic targeting.
doi:10.1016/j.ccell.2015.09.012
PMCID: PMC4643330  PMID: 26481147
8.  SOX15 governs transcription in human stratified epithelia and a subset of esophageal adenocarcinomas 
Background & Aims
Intestinal metaplasia (Barrett's esophagus, BE) is the principal risk factor for esophageal adenocarcinoma (EAC). Study of the basis for BE has centered on intestinal factors, but loss of esophageal identity likely also reflects absence of key squamous-cell factors. As few determinants of stratified epithelial cell-specific gene expression are characterized, it is important to identify the necessary transcription factors.
Methods
We tested regional expression of mRNAs for all putative DNA-binding proteins in the mouse digestive tract and verified esophagus-specific factors in human tissues and cell lines. Integration of diverse data defined a human squamous esophagus-specific transcriptome. We used chromatin immunoprecipitation (ChIP-seq) to locate transcription factor binding sites, computational approaches to profile transcripts in cancer datasets, and immunohistochemistry to reveal protein expression.
Results
The transcription factor SOX15 is restricted to esophageal and other murine and human stratified epithelia. SOX15 mRNA levels are attenuated in BE and its depletion in human esophageal cells reduced esophageal transcripts significantly and specifically. SOX15 binding is highly enriched near esophagus-expressed genes, indicating direct transcriptional control. SOX15 and hundreds of genes co-expressed in squamous cells are reactivated in up to 30% of EAC specimens. Genes normally confined to the esophagus or intestine appear in different cells within the same malignant glands.
Conclusions
These data identify a novel transcriptional regulator of stratified epithelial cells and a subtype of EAC with bi-lineage gene expression. Broad activation of squamous-cell genes may shed light on whether EACs arise in the native stratified epithelium or in ectopic columnar cells.
doi:10.1016/j.jcmgh.2015.07.009
PMCID: PMC4620585  PMID: 26516633
Esophageal gene regulation; Barrett's esophagus; esophageal transcriptome; SOX15 cistrome
9.  Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4 
Cell Cycle  2015;13(24):3857-3866.
Barrett's esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.
doi:10.4161/15384101.2014.972875
PMCID: PMC4614677  PMID: 25558829
Barrett's esophagus; KLF4; metaplasia; Notch signaling; transdifferentiation
10.  Multiparametric profiling of non–small-cell lung cancers reveals distinct immunophenotypes 
JCI Insight  null;1(14):e89014.
BACKGROUND. Immune checkpoint blockade improves survival in a subset of patients with non–small-cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking. Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited.
METHODS. We performed comprehensive flow cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next-generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC).
RESULTS. Cytometric profiling identified an immunologically “hot” cluster with abundant CD8+ T cells expressing high levels of PD-1 and TIM-3 and an immunologically “cold” cluster with lower relative abundance of CD8+ T cells and expression of inhibitory markers. The “hot” cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function and high PD-L1 expression by IHC. There was no correlation between immunophenotype and KRAS or EGFR mutation, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the “hot” cluster. Additionally, approximately 20% of cases had high B cell infiltrates with a subset producing IL-10.
CONCLUSIONS. Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer.
FUNDING. The Robert A. and Renée E. Belfer Family Foundation, Expect Miracles Foundation, Starr Cancer Consortium, Stand Up to Cancer Foundation, Conquer Cancer Foundation, International Association for the Study of Lung Cancer, National Cancer Institute (R01 CA205150), and the Damon Runyon Cancer Research Foundation.
NSCLCs stratify into clusters based on the prevalence of CD8+ T cells and their expression of the immune checkpoint molecules PD-1 and TIM-3.
doi:10.1172/jci.insight.89014
PMCID: PMC5033841  PMID: 27699239
11.  Radiofrequency Ablation is Associated with Decreased Neoplastic Progression in Patients with Barrett’s Esophagus and Confirmed Low-Grade Dysplasia 
Gastroenterology  2015;149(3):567-576.e3.
Background & Aims
Barrett’s esophagus (BE) with low-grade dysplasia (LGD) can progress to high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC). Radiofrequency ablation (RFA) has been shown to be an effective treatment for LGD in clinical trials but its effectiveness in clinical practice is unclear. We compared the rate of progression of LGD following RFA to that with endoscopic surveillance alone in routine clinical practice.
Methods
We performed a retrospective study of patients who either underwent RFA (n=45) or surveillance endoscopy (n=125) for LGD, confirmed by at least 1 expert pathologist, from October 1992 through December 2013 at 3 medical centers in the US. Cox regression analysis was used to assess the association between progression and RFA.
Results
Data were collected over median follow-up periods of 889 days (inter-quartile range, 264–1623 days) after RFA and 848 days (inter-quartile range, 322–2355 days) after surveillance endoscopy (P=.32). The annual rates of progression to HGD or EAC was 6.6% in the surveillance group and 0.77% in the RFA group. The risk of progression to HGD or EAC was significantly lower among patients who underwent RFA than those who underwent surveillance (adjusted hazard ratio, 0.06; 95% confidence interval, 0.008–0.48).
Conclusions
Among patients with BE and confirmed LGD, rates of progression to a combined endpoint of HGD and EAC were lower among those treated with RFA than among untreated patients. Although selection bias cannot be excluded, these findings provide additional evidence for the use of endoscopic ablation therapy for LGD.
doi:10.1053/j.gastro.2015.04.013
PMCID: PMC4550488  PMID: 25917785
eradication; clinical setting; prevention; esophageal cancer
12.  KrasG12D induces EGFR-MYC cross signaling in murine primary pancreatic ductal epithelial cells 
Oncogene  2015;35(29):3880-3886.
Epidermal growth factor receptor (EGFR) signaling has a critical role in oncogenic Kras-driven pancreatic carcinogenesis. However, the downstream targets of this signaling network are largely unknown. We developed a novel model system utilizing murine primary pancreatic ductal epithelial cells (PDECs), genetically engineered to allow time-specific expression of oncogenic KrasG12D from the endogenous promoter. We show that primary PDECs are susceptible to KrasG12D-driven transformation and form pancreatic ductal adenocarcinomas (PDAC) in vivo after Cdkn2a inactivation. In addition, we demonstrate that activation of KrasG12D induces an EGFR signaling loop to drive proliferation. Interestingly, pharmacological inhibition of EGFR fails to decrease KrasG12D-activated ERK or PI3K signaling. Instead our data provide novel evidence that EGFR signaling is needed to activate the oncogenic and pro-proliferative transcription factor c-MYC. EGFR and c-MYC have been shown to be essential for pancreatic carcinogenesis. Importantly, our data link both pathways and thereby, explain the crucial role of EGFR for KrasG12D-driven carcinogenesis in the pancreas.
doi:10.1038/onc.2015.437
PMCID: PMC4877299  PMID: 26592448
pancreatic cancer; EGFR; Kras; MYC
13.  Whole genome sequencing defines the genetic heterogeneity of familial pancreatic cancer 
Cancer discovery  2015;6(2):166-175.
Pancreatic cancer is projected to become the second leading cause of cancer-related death in the United States by 2020. A familial aggregation of pancreatic cancer has been established, but the cause of this aggregation in most families is unknown. To determine the genetic basis of susceptibility in these families, we sequenced the germline genome of 638 familial pancreatic cancer patients. We also sequenced the exomes of 39 familial pancreatic adenocarcinomas. Our analyses support the role of previously identified familial pancreatic cancer susceptibility genes such as BRCA2, CDKN2A and ATM, and identify novel candidate genes harboring rare, deleterious germline variants for further characterization. We also show how somatic point mutations that occur during hematopoiesis can affect the interpretation of genome-wide studies of hereditary traits. Our observations have important implications for the etiology of pancreatic cancer and for the identification of susceptibility genes in other common cancer types.
doi:10.1158/2159-8290.CD-15-0402
PMCID: PMC4744563  PMID: 26658419
14.  Imaging of Secreted Extracellular Periostin, an Important Marker of Invasion in the Tumor Microenvironment in Esophageal Cancer 
Periostin, an extracellular matrix protein, plays key role in cell adhesion and motility within the tumor microenvironment and is correlated with tumor invasion. We developed and characterized a PET tracer that specifically targets periostin and evaluated the probe in preclinical models of esophageal squamous cell carcinoma (ESCC).
Methods
The Institutional Animal Care and Use Committee approved all animal studies. Antiperiostin-F(ab′)2 was generated from a monoclonal antibody by enzymatic digestion, conjugated to DOTA, and labeled with 64Cu. Human ESCC cell lines, TE-11 with high and TT with minimal periostin expression, were implanted in nu/nu mice to generate the positive and control tumor models, respectively. PET/CT imaging was performed at 6, 12, and 24 h and organ-specific biodistribution at 24 h after probe injection. Additionally the probe was tested in a genetically engineered mouse model of periostin-expressing distal esophageal/forestomach ESCC. Tissue microarrays of esophageal neoplasms and ESCC as well as extracted tumor samples were stained for periostin.
Results
We generated a 64Cu-DOTA-antiperiostin-F(ab′)2 with a dissociation constant of 29.2 ± 3.0 nM. PET/CT images and biodistribution studies showed significantly higher tracer uptake in TE-11 than TT tumors (maximum standardized uptake value, 24 h: 0.67± 0.09 vs. 0.36 ± 0.03, P < 0.0005; percentage injected dose per gram, 24 h: 3.24 ± 0.65 vs. 1.63 ± 0.49, P < 0.0001). In genetically engineered mouse models, ESCC high periostin tracer uptake anatomically correlated with the 18F-FDG uptake at the gastroesophageal junction. All of the ESCC cores and 96.2% of adenocarcinoma stained positive for periostin, with most stained strongly (67.3% and 69.3%, respectively).
Conclusion
We demonstrated that specific imaging of extracellular matrix periostin in ESCC is feasible using a targeted PET tracer. Detection of periostin in the tumor microenvironment may help with early detection, postsurgical follow-up, and in situ characterization of primary and metastatic lesions.
doi:10.2967/jnumed.115.156216
PMCID: PMC4826779  PMID: 26069303
periostin; immuno-PET imaging; extracellular matrix; tumor microenvironment; esophageal cancer
15.  A common p53 mutation (R175H) activates c-Met receptor tyrosine kinase to enhance tumor cell invasion 
Cancer Biology & Therapy  2013;14(9):853-859.
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human cancer with poor prognosis due to late diagnosis and metastasis. Common genomic alterations in ESCC include p53 mutation, p120ctn inactivation, and overexpression of oncogenes such as cyclin D1, EGFR, and c-Met. Using esophageal epithelial cells transformed by the overexpression of EGFR and p53R175H, we find novel evidence of a functional link between p53R175H and the c-Met receptor tyrosine kinase to mediate tumor cell invasion. Increased c-Met receptor activation was observed upon p53R175H expression and enhanced further upon subsequent EGFR overexpression. We inhibited c-Met phosphorylation, resulting in diminished invasion of the genetically transformed primary esophageal epithelial cells (EPC-hTERT-EGFR-p53R175H), suggesting that the mechanism of increased invasiveness upon EGFR and p53R175H expression may be the result of increased c-Met activation. These results suggest that the use of therapeutics directed at c-Met in ESCC and other squamous cell cancers.
doi:10.4161/cbt.25406
PMCID: PMC3909554  PMID: 23792586
p53 mutation; c-Met; esophageal cancer; tumor invasion
16.  Krt19(+)/Lgr5(−) cells are radioresistant cancer initiating stem cells in the colon and intestine 
Cell stem cell  2015;16(6):627-638.
The colonic and intestinal epithelium are renewed every 3 days. In the intestine there are at least two principal stem cell pools. The first contains rapid cycling crypt based columnar (CBC) Lgr5+ cells, while the second is comprised of slower cycling Bmi1-expressing cells at the +4 position above the crypt base. In the colon, however, the identification of Lgr5-negative stem cell pools has proven more challenging. Here, we demonstrate that the intermediate filament, keratin-19 (Krt19), marks long-lived, radiation resistant cells above the crypt base that generate Lgr5+ CBCs in the colon and intestine. In colorectal cancer models, Krt19+ cancer initiating cells are also radioresistant while Lgr5+ stem cells are radiosensitive. Moreover, Lgr5+ stem cells are dispensable in both the normal and neoplastic colonic epithelium, as ablation of Lgr5+ stem cells results in their regeneration from Krt19 expressing cells. Thus, Krt19+ stem cells are a discrete target relevant for cancer therapy.
Graphical Abstract
doi:10.1016/j.stem.2015.04.013
PMCID: PMC4457942  PMID: 26046762
Krt19; intestine; colon; stem cells; cancer
18.  Detection of Tumor Suppressor Genes in Cancer Development by a Novel shRNA-based method 
Molecular cancer research : MCR  2015;13(5):863-869.
Pancreatic cancer is one of the deadliest cancers with poor survival rates and limited therapeutic options. To improve the understanding of this disease's biology, a prerequisite for the generation of novel therapeutics, new platforms for rapid and efficient genetic and therapeutic screening are needed. Therefore, a combined in vitro/in vivo hybrid shRNA-assay was developed using isolated murine primary pancreatic ductal cells (PDCs), in which oncogenic KrasG12D could be activated in vitro by genomic recombination through 4OH-tamoxifen-induced nuclear translocation of Cre-ERT2 expressed under control of the ROSA26 promoter. Further genetic manipulation was achieved through selective and stable RNA interference (RNAi) against the tumor suppressors p16Ink4a (CDKN2A) or p53 (TP53) using lentiviral gene delivery. Treatment of PDCs with 4OH-tamoxifen increased phosphorylation of extracelluar signal-regulated kinase (ERK) downstream of KRAS, and subsequent lentiviral transduction resulted in sustained target gene repression. Double-mutant PDCs were then re-introduced into the pancreata of NOD-SCID-gamma (NSG) mice and monitored for tumor growth. Orthotopic implantation of PDCs carrying the activated KrasG12D-allele and shRNA against p16Ink4a or p53 resulted in tumor growth, metastasis and reduced survival of NSG mice. In contrast, KrasG12D alone was not sufficient to induce tumor growth. Implications: The combinatory in vitro/in vivo approach described in this study allows for rapid and efficient identification of genes involved in carcinogenesis and opens new avenues for the development of therapeutic strategies to improve cancer treatment.
doi:10.1158/1541-7786.MCR-14-0709
PMCID: PMC4433420  PMID: 25724428
Pancreatic cancer; shRNA; tumor suppressor; carcinogenesis; genetic screen
19.  WNT10A promotes an invasive and self-renewing phenotype in esophageal squamous cell carcinoma 
Carcinogenesis  2015;36(5):598-606.
Summary
WNT10A is upregulated in invasive esophageal tumor cells versus non-invasive cells in 3D-organotypic cultures. Functionally, WNT10A promotes proliferation, migration, invasion and self-renewal. WNT10A is expressed during development, downregulated postnatally and re-expressed during carcinogenesis, where it correlates with poor prognosis.
Esophageal cells overexpressing epidermal growth factor receptor (EGFR) and TP53 mutation can invade into the extracellular matrix when grown in 3D-organotypic cultures (OTC) and mimic early invasion in esophageal squamous cell carcinoma (ESCC). We have performed laser capture microdissection with RNA microarray analysis on the invasive and non-invasive tumor cells of p53R175H-overexpressing OTC samples to determine candidate genes facilitating tumor invasion. WNT10A was found to be >4-fold upregulated in the invasive front. Since WNT10A is also prominently upregulated during placode promotion in hair follicle development, a process that requires epithelial cells to thicken and elongate, in order to allow downward growth, we hypothesized that WNT10A may be important in mediating a similar mechanism of tumor cell invasion in ESCC. We have found that WNT10A expression is significantly upregulated in human ESCC, when compared with normal adjacent tissue. Furthermore, high WNT10A expression levels correlate with poor survival. Interestingly, we observe that WNT10A is expressed early in embryogenesis, but is reduced dramatically postnatally. We demonstrate that overexpression of WNT10a promotes migration and invasion, and proliferation of transformed esophageal cells. Lastly, we show that WNT10A overexpression induces a greater CD44High/CD24Low population, which are putative markers of cancer stem cells, and increases self-renewal capability. Taken together, we propose that WNT10A acts as an oncofetal factor that is highly expressed and may promote proper development of the esophagus. During tumorigenesis, it is aberrantly overexpressed in order to promote ESCC migration and invasion, and may be linked to self-renewal of a subset of ESCC cells.
doi:10.1093/carcin/bgv025
PMCID: PMC4498148  PMID: 25795715
20.  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
21.  A LIN28B polymorphism predicts for colon cancer survival 
Cancer Biology & Therapy  2012;13(14):1390-1395.
The pathogenesis of sporadic colorectal cancer involves distinct pathways, with characteristic genomic alterations. The first pathway, chromosome instability (CIN), is driven by APC mutations and is typified by Kras mutations, p53 mutation/loss of heterozygosity, and deletions at chromosome 18q. The second pathway is referred to as microsatellite instability (MSI), a genetic hallmark of the accumulated mutations that occur as a consequence of derangements in the mismatch repair genes. Finally, proximal colon cancers may involve methylation of a number of genes, which is frequently referred to as the CpG island methylator phenotype (CIMP), and are associated with B-raf mutations. The ability to stratify colorectal cancers by risk would be facilitated by the identification of polymorphisms that might be utilized as biomarkers. LIN28B is an RNA binding protein that is overexpressed in colon cancers. We find that LIN28B rs314277 is associated with significant recurrence of colorectal cancer in Stage II disease, which may have translational therapeutic implications.
doi:10.4161/cbt.22336
PMCID: PMC3542229  PMID: 23052130
Colon cancer; LIN28B; SNP; prognosis; molecular pathogenesis; genetics; genomics
22.  FBXO4 loss facilitates carcinogen induced papilloma development in mice 
Cancer Biology & Therapy  2015;16(5):750-755.
Cyclin D1 is frequently overexpressed in esophageal squamous cell carcinoma (ESCC) and is considered a key driver of this disease. Mutations in FBXO4, F-box specificity factor that directs SCF-mediated ubiquitylation of cyclin D1, occur in ESCC with concurrent overexpression of cyclin D1 suggesting a potential tumor suppressor role for FBXO4. To evaluate the contribution of FBXO4-dependent regulation cyclin D1 in esophageal squamous cell homeostasis, we exposed FBXO4 knockout mice to N-nitrosomethylbenzylamine (NMBA), an esophageal carcinogen. Our results revealed that loss of FBXO4 function facilitates NMBA induced papillomas in FBXO4 het (+/−) and null (−/−) mice both by numbers and sizes 11 months after single dose NMBA treatment at 2mg/kg by gavage when compared to that in wt (+/+) mice (P < 0.01). No significant difference was noted between heterozygous or nullizygous mice consistent with previous work. To assess cyclin D1/CDK4 dependence, mice were treated with the CDK4/6 specific inhibitor, PD0332991, for 4 weeks. PD0332991 treatment (150mg/kg daily), reduced tumor size and tumor number. Collectively, our data support a role for FBXO4 as a suppressor of esophageal tumorigenesis.
doi:10.1080/15384047.2015.1026512
PMCID: PMC4622573  PMID: 25801820
cyclin D1; CDK4; ESCC; FBXO4; PD0332991
23.  Myeloid derived suppressor cells 
Oncoimmunology  2013;2(4):e24117.
The goal of achieving measurable response with cancer immunotherapy requires counteracting the immunosuppressive characteristics of tumors. One of the mechanisms that tumors utilize to escape immunosurveillance is the activation of myeloid derived suppressor cells (MDSCs). Upon activation by tumor-derived signals, MDSCs inhibit the ability of the host to mount an anti-tumor immune response via their capacity to suppress both the innate and adaptive immune systems. Despite their relatively recent discovery and characterization, anti-MDSC agents have been identified, which may improve immunotherapy efficacy.
doi:10.4161/onci.24117
PMCID: PMC3654606  PMID: 23734336
Myeloid derived suppressor cells; docetaxol; RNA aptamer; CpG oligodeoxynucleotides (ODN); cyclophosphamide; gemcitabine; curcumin
24.  Lin28b promotes colon cancer progression and metastasis 
Cancer research  2011;71(12):4260-4268.
LIN28B is a homolog of LIN28 which induces pluripotency when expressed in conjunction with OCT4, SOX2, and KLF4 in somatic fibroblasts. LIN28B represses biogenesis of let-7 microRNAs and is implicated in both development and tumorigenesis. Recently, we have determined that LIN28B overexpression occurs in colon tumors. We conducted a comprehensive analysis of Lin28b protein expression in human colon adenocarcinomas. We found that LIN28B overexpression correlates with reduced patient survival and increased probability of tumor recurrence. In order to elucidate tumorigenic functions of LIN28B, we constitutively expressed LIN28B in colon cancer cells and evaluated tumor formation in vivo. Tumors with constitutive Lin28b expression exhibit increased expression of colonic stem cell markers LGR5 and PROM1, mucinous differentiation, and metastasis. Together, our findings point to a function for LIN28B in promoting colon tumor pathogenesis, especially metastasis.
doi:10.1158/0008-5472.CAN-10-4637
PMCID: PMC3117110  PMID: 21512136
LIN28B; LIN28; let-7; colon cancer; differentiation; metastasis
25.  Deletion of p120-catenin results in a tumor microenvironment with inflammation and cancer that establishes it as a tumor suppressor gene 
Cancer cell  2011;19(4):470-483.
Summary
P120ctn interacts with E-cadherin, but no formal proof that p120ctn functions as a bone fide tumor suppressor gene has emerged. We report herein that p120ctn loss leads to tumor development in mice. We have generated a conditional knockout model of p120ctn whereby mice develop pre-neoplastic and neoplastic lesions in the oral cavity, esophagus and squamous forestomach. Tumor derived cells secrete granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α (TNFα). The tumors contain significant desmoplasia and immune cell infiltration. Immature myeloid cells comprise a significant percentage of the immune cells present, and likely participate in fostering a favorable tumor microenvironment, including the activation of fibroblasts.
doi:10.1016/j.ccr.2011.02.007
PMCID: PMC3077713  PMID: 21481789

Results 1-25 (96)