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.
p53 mutation; c-Met; esophageal cancer; tumor invasion
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.
mouse model; neoplasm; extracellular matrix; POSTN
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.
Colon cancer; LIN28B; SNP; prognosis; molecular pathogenesis; genetics; genomics
Eosinophilic oesophagitis (EoE) is a chronic inflammatory condition of the oesophagus with limited treatment options. No previous transgenic model has specifically targeted the oesophageal mucosa to induce oesophageal eosinophilia.
We developed a mouse model that closely resembles EoE by utilising oxazolone haptenation in mice with transgenic overexpression of an eosinophil poietic and survival factor (interleukin (IL)-5) in resident squamous oesophageal epithelia.
Overexpression of IL-5 in the healthy oesophagus was achieved in transgenic mice (L2-IL5) using the squamous epithelial promoter Epstein–Barr virus ED-L2. Oxazolone-challenged L2-IL5 mice developed dose-dependent pan-oesophageal eosinophilia, including eosinophil microabscess formation and degranulation as well as basal cell hyperplasia. Moreover, oesophagi expressed increased IL-13 and the eosinophil agonist chemokine eotaxin-1. Treatment of these mice with corticosteroids significantly reduced eosinophilia and epithelial inflammation.
L2-IL5 mice provide a novel experimental model that can potentially be used in preclinical testing of EoE-related therapeutics and mechanistic studies identifying pathogenetic features associated with mucosal eosinophilia.
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.
Myeloid derived suppressor cells; docetaxol; RNA aptamer; CpG oligodeoxynucleotides (ODN); cyclophosphamide; gemcitabine; curcumin
Gastroesophageal reflux disease (GERD), Barrett's esophagus (BE), graft-versus-host disease (GVHD), and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease are common human gastrointestinal diseases that share inflammation as a key driver for their development. A general outcome resulting from these chronic inflammatory conditions is increased oxidative stress. Oxidative stress is caused by the generation of reactive oxygen and nitrogen species that are part of the normal inflammatory response, but are also capable of damaging cellular DNA, protein, and organelles. Damage to DNA can include DNA strand breaks, point mutations due to DNA adducts, as well as alterations in methylation patterns leading to activation of oncogenes or inactivation of tumor suppressors. There are a number of significant long-term consequences associated with chronic oxidative stress, most notably cancer. Infiltrating immune cells and stromal components of tissue including fibroblasts contribute to dynamic changes occurring in tissue related to disease development. Immune cells can potentiate oxidative stress, and fibroblasts have the capacity to contribute to advanced growth and proliferation of the epithelium and any resultant cancers. Disease models for GERD, BE, GVHD, and ulcerative colitis based on three-dimensional human cell and tissue culture systems that recapitulate in vivo growth and differentiation in inflammatory-associated microphysiological environments would enhance our understanding of disease progression and improve our ability to test for disease-prevention strategies. The development of physiologically relevant, human cell-based culture systems is therefore a major focus of our research. These novel models will be of enormous value, allowing us to test hypotheses and advance our understanding of these disorders, and will have a translational impact allowing us to more rapidly develop therapeutic and chemopreventive agents. In summary, this work to develop advanced human cell-based models of inflammatory conditions will greatly improve our ability to study, prevent, and treat GERD, BE, GVHD, and inflammatory bowel disease. The work will also foster the development of novel therapeutic and preventive strategies that will improve patient care for these important clinical conditions.
inflammation; oxidative stress; DNA damage; gastrointestinal disease; gastroesophageal reflux disease; Barrett's esophagus; graft-versus-host disease; inflammatory bowel disease; human three-dimensional organotypic model systems
Esophageal tumors provide unique challenges and opportunities for developing and testing surveillance imaging technology for different tumor microenvironment components, including assessment of immune cell modulation, with the ultimate goal of promoting early detection and response evaluation. In this context, accessibility through the lumen using a minimally invasive approach provides a means for repetitive evaluation longitudinally by combining fluorescent endoscopic imaging technology with novel fluorescent nanoparticles that are phagocytized by immune cells in the microenvironment. The agent we developed for imaging is synthesized from Feraheme (ferumoxytol), a Food and Drug Administration–approved monocrystaline dextran-coated iron oxide nanoparticle, which we conjugated to a near-infrared fluorochrome, CyAL5.5. We demonstrate a high level of uptake of the fluorescent nanoparticles by myeloid-derived suppressor cells (MDSCs) in the esophagus and spleen of L2Cre;p120ctnflox/flox mice. These mice develop esophageal dysplasia leading to squamous cell carcinoma; we have previously demonstrated that dysplastic and neoplastic esophageal lesions in these mice have an immune cell infiltration that is dominated by MDSCs. In the L2Cre;p120ctnflox/flox mice, evaluation of the spleen reveals that nearly 80% of CD45+ leukocytes that phagocytized the nanoparticle were CD11b+Gr1+ MDSCs. After dexamethasone treatment, we observed concordant decreased fluorescent signal from esophageal lesions during fluorescent endoscopy and decreased CyAL5.5-fluorescent–positive immune cell infiltration in esophageal dysplastic lesions by fluorescence-activated cell sorting analysis. Our observations suggest that this translatable technology may be used for the early detection of dysplastic changes and the serial assessment of immunomodulatory therapy and to visualize changes in MDSCs in the esophageal tumor microenvironment.
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.
LIN28B; LIN28; let-7; colon cancer; differentiation; metastasis
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.
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.
Lin28b is an RNA-binding protein that inhibits biogenesis of let-7 microRNAs. LIN28B is overexpressed in diverse cancers, yet a specific role in the molecular pathogenesis of colon cancer has yet to be elucidated. We have determined that human colon tumors exhibit decreased levels of mature let-7 isoforms and increased expression of LIN28B. In order to determine LIN28B's mechanistic role in colon cancer, we expressed LIN28B in immortalized colonic epithelial cells and human colon cancer cell lines. We found that LIN28B promotes cell migration, invasion, and transforms immortalized colonic epithelial cells. In addition, constitutive LIN28B expression increases expression of intestinal stem cell markers LGR5 and PROM1 in the presence of let-7 restoration. This may occur as a result of Lin28b protein binding LGR5 and PROM1 mRNA, suggesting that a subset of LIN28B functions are independent of its ability to repress let-7. Our findings establish a new role for LIN28B in human colon cancer pathogenesis, and suggest LIN28B post-transcriptionally regulates LGR5 and PROM1 through a let-7 independent mechanism.
LIN28B; LIN28; Let-7; colon cancer; LGR5; PROM1
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.
tumor microenvironment; periostin; EGFR; p53
The pancreas is a complex organ comprised of three critical cell lineages: islet (endocrine), acinar, and ductal. This review will focus upon recent insights and advances in the biology of pancreatic ductal cells. In particular, emphasis will be placed upon the regulation of ductal cells by specific transcriptional factors during development as well as the underpinnings of acinar-ductal metaplasia as an important adaptive response during injury and regeneration. We also address the potential contributions of ductal cells to neoplastic transformation, specifically in pancreatic ductal adenocarcinoma.
Squamous cell carcinomas (SCCs) with an infiltrative invasion pattern carry a higher risk of treatment failure. Such infiltrative invasion may be mediated by a mesenchymal-like subpopulation of malignant cells that we have previously shown to arise from epithelial to mesenchymal transition (EMT) and resist epidermal growth factor receptor (EGFR) targeting. Here we demonstrate that SCCs with infiltrative, high risk invasion patterns contain abundant mesenchymal-like cells, which are rare in tumors with low risk patterns. This cellular heterogeneity was modeled accurately in three dimensional culture using collagen-embedded SCC spheroids, which revealed distinct invasive fronts created by collective migration of E-cadherin-positive cells versus infiltrative migration of individual mesenchymal-like cells. Because EGFR expression by mesenchymal-like cells was diminished in the spheroid model and in human SCCs, we hypothesized that SCCs shift toward infiltrative invasion mediated by this subpopulation during anti-EGFR therapy. Anti-EGFR treatment of spheroids using erlotinib or cetuximab enhanced infiltrative invasion by targeting collective migration by E-cadherin-positive cells while sparing mesenchymal-like cells; by contrast, spheroid invasion in absence of mesenchymal-like cells was abrogated by erlotinib. Similarly, cetuximab treatment of xenografts containing mesenchymal-like cells created an infiltrative invasive front comprised of this subpopulation, whereas no such shift was observed upon treating xenografts lacking these cells. These results implicate mesenchymal-like SCC cells as key mediators of the infiltrative invasion seen in tumors with locally aggressive behavior. They further demonstrate that EGFR inhibition can promote an infiltrative invasion front comprised of mesenchymal-like cells preferentially in tumors where they are abundant prior to therapy.
pattern of invasion; EGFR inhibition; squamous cell carcinoma; EMT; tumor heterogeneity
MicroRNAs (miRNAs) are single-stranded, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Genes encoding miRNAs are located in regions of the genome that are commonly amplified, deleted or rearranged. They are commonly dysregulated in human cancers and known to act as oncogenes or tumor suppressors. Members of the miR-200 miRNA family are downregulated in human cancer cells and tumors due to aberrant epigenetic gene silencing and play a critical role in the suppression of epithelial-to-mesenchymal transition (EMT), tumor cell adhesion, migration, invasion and metastasis, by targeting and repressing the expression of key mRNAs that are involved in EMT (ZEB1 and ZEB2), β-catenin/Wnt signaling (β-catenin), EGFR inhibitor resistance (ERRFI-1) and chemoresistance to therapeutic agents (TUBB3). Since the miR-200 family functions as putative tumor suppressors and represent biomarkers for poorly differentiated and aggressive cancers, restoration of miR-200 expression may have therapeutic implications for the treatment of metastatic and drug-resistant tumors.
miRNAs; mir-200; epithelial-mesenchymal transition; β-catenin/Wnt signaling; microenvironment; metastasis; RNA
In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and loss of E-cadherin is a hallmark of tumor progression fostering cancer cell invasion and metastasis. To examine E-cadherin loss in squamous cell cancers, we used primary human esophageal epithelial cells (keratinocytes) as a platform and retrovirally transduced wild-type and dominant-negative forms of E-cadherin into these cells. We found decreased cell adhesion in the cells expressing dominant-negative E-cadherin, thereby resulting in enhanced migration and invasion. To analyze which molecular pathway(s) may modulate these changes, we conducted microarray analysis and found up-regulation of transforming growth factor β receptor II (TβRII) in the wild-type E-cadherin-overexpressing cells, which was confirmed by real-time PCR and Western blot analyses. To investigate the in vivo relevance of this finding, we analyzed tissue microarrays of paired esophageal squamous cell carcinomas and adjacent normal esophagus, and we could show a coordinated loss of E-cadherin and TβRII in ~80% of tumors. To determine if there may be an E-cadherin-dependent regulation of TβRII, we show the physical interaction of E-cadherin with TβRII and that this is mediated through the extracellular domains of E-cadherin and TβRII, respectively. In addition, TβRI is recruited to this complex. When placed in the context of three-dimensional cell culture, which reflects the physiologic microenvironment, TβRII-mediated cell signaling is dependent upon intact E-cadherin function. Our results, which suggest that E-cadherin regulates TβRII function, have important implications for epithelial carcinogenesis characterized through the frequent occurrence of E-cadherin and TβRII loss.
Lung squamous cell carcinoma (SCC) is a deadly disease for which current treatments are inadequate. We demonstrate that biallelic inactivation of Lkb1 and Pten in the mouse lung leads to SCC that recapitulates the histology, gene expression, and microenvironment found in human disease. Lkb1;Pten null (LP) tumors expressed the squamous markers KRT5, p63 and SOX2, and transcriptionally resembled the basal subtype of human SCC. In contrast to mouse adenocarcinomas, the LP tumors contained immune populations enriched for tumor-associated neutrophils. SCA1+NGFR+ fractions were enriched for tumor-propagating cells (TPCs) that could serially transplant the disease in orthotopic assays. TPCs in the LP model and NGFR+ cells in human SCCs highly expressed Pd-ligand-1 (PD-L1), suggesting a mechanism of immune escape for TPCs.
Embryonic development of the pancreas is marked by an early phase of dramatic morphogenesis, in which pluripotent progenitor cells of the developing pancreatic epithelium give rise to the full array of mature exocrine and endocrine cell types. The genetic determinants of acinar and islet cell lineages are somewhat well defined; however, the molecular mechanisms directing ductal formation and differentiation remain to be elucidated. The complex ductal architecture of the pancreas is established by a reiterative program of progenitor cell expansion and migration known as branching morphogenesis, or tubulogenesis, which proceeds in mouse development concomitantly with peak Pdx1 transcription factor expression. We therefore evaluated Pdx1 expression with respect to lineage-specific markers in embryonic sections of the pancreas spanning this critical period of duct formation and discovered an unexpected population of nonislet Pdx1-positive cells displaying physical traits of branching. We then established a 3D cell culture model of branching morphogenesis using primary pancreatic duct cells and identified a transient surge of Pdx1 expression exclusive to branching cells. From these observations we propose that Pdx1 might be involved temporally in a program of gene expression sufficient to facilitate the biochemical and morphological changes necessary for branching morphogenesis.
The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). pDac resembles duct cells morphologically and, to some extent, at a molecular level. recently, genetic-lineage labeling has become popular in the field of tumor biology in order to study cell-fate decisions or to trace cancer cells in the mouse. However, certain biological questions require a nongenetic labeling approach to purify a distinct cell population in the pancreas. Here we describe a protocol for isolating mouse pancreatic ductal epithelial cells and duct-like cells directly in vivo using ductal-specific Dolichos biflorus agglutinin (DBA) lectin labeling followed by magnetic bead separation. Isolated cells can be cultured (in two or three dimensions), manipulated by lentiviral transduction to modulate gene expression and directly used for molecular studies. this approach is fast (∼4 h), affordable, results in cells with high viability, can be performed on the bench and is applicable to virtually all genetic and nongenetic disease models of the pancreas.
In eukaryotic cells, MCM, the minichromosome maintenance proteins, form a heterohexamer during G1 phase in a cell cycle and constitute a DNA helicase activity at the onset of replication. MCM proteins are downregulated and dissociated from chromatin when cells exit the cell cycle. MCM proteins are upregulated frequently in a variety of dysplastic and cancer cells. To delineate the role of MCM in esophageal epithelial biology, we determined the MCM family gene expression during the cellular senescence, immortalization, differentiation and apoptosis. All of the MCM2-7 proteins appeared to be downregulated in primary human esophageal keratinocytes upon replicative senescence. Their expression was restored by ectopic expression of a catalytic subunit of human telomerase, resulting in immortalization. Interestingly, we found a reciprocal induction of a novel MCM2-related protein fragment upon cell growth inhibition associated with senescence, contact inhibition or terminal differentiation, but not apoptosis. Epitope mapping of this MCM2-related fragment suggested the lack of amino- and carboxyl-terminal regions including one of the putative nuclear localization signals and the ATPase domain, the MCM box. The absence of multiple MCM2 transcripts implied a possible posttranslational molecular cleavage in generation of the MCM2-related fragment, and a potential functional role in regulation of the activity of the MCM protein complex.
MCM2; senescence; differentiation; esophageal keratinocytes
The esophageal epithelium is a prototypical stratified squamous epithelium that exhibits an exquisite equilibrium between proliferation and differentiation. After basal cells proliferate, they migrate outward toward the luminal surface, undergo differentiation, and eventually slough due to apoptosis. The identification and characterization of stem cells responsible for the maintenance of the esophageal epithelium remains elusive. Here, we employed Hoechst dye extrusion and BrdU label–retaining assays to identify in mice a potential esophageal stem cell population that localizes to the basal cell compartment. The self-renewing capacity of this population was characterized using a clonogenic assay and a 3D organotypic culture model. The putative esophageal stem cells were also capable of epithelial reconstitution in vivo in direct esophageal epithelial injury models. In both the 3D organotypic culture and direct mucosal injury models, the putative stem cells gave rise to undifferentiated and differentiated cells. These studies therefore provide a basis for understanding the regenerative capacity and biology of the esophageal epithelium when it is faced with injurious insults.
BACKGROUND & AIMS
Radiofrequency ablation (RFA) is an established treatment for dysplastic Barrett’s esophagus (BE). Although short-term endpoints of ablation have been ascertained, there have been concerns about recurrence of intestinal metaplasia (IM) after ablation. We aimed to estimate the incidence and identify factors that predicted the recurrence of IM after successful RFA.
We analyzed data from 592 patients with BE treated with RFA from 2003 through 2011 at 3 tertiary referral centers. Complete remission of intestinal metaplasia (CRIM) was defined as eradication of IM (in esophageal and gastro esophageal junction biopsies), documented by 2 consecutive endoscopies. Recurrence was defined as presence of IM or dysplasia after CRIM in surveillance biopsies. Two experienced gastrointestinal pathologists confirmed pathology findings.
Based on histology analysis, before RFA, 71% of patients had high-grade dysplasia or esophageal adenocarcinoma, 15% had low-grade dysplasia, and 14% had non-dysplastic BE. Of patients treated, 448 (76%) were assessed following RFA. 55% of patients underwent endoscopic mucosal resection before RFA. The median time to CRIM was 22 months, with 56% of patients in CRIM by 24 months. Increasing age and length of BE segment were associated with a longer times to CRIM. Twenty-four months after CRIM, the incidence of recurrence was 33%; 22% of all recurrences observed were dysplastic BE. There were no demographic or endoscopic factors associated with recurrence. Complications developed in 6.5% of subjects treated with RFA; strictures were the most common complication.
Of patients with BE treated by RFA, 56% are in complete remission after 24 months. However, 33% of these patients have disease recurrence within the next 2 years. Most recurrences were non-dysplastic and endoscopically manageable, but continued surveillance after RFA is essential.
esophageal cancer; prevention; endoscopic therapy; EAC