Cancer stem cells (CSCs) within squamous cell carcinomas (SCCs) are hypothesized to contribute to chemotherapy and radiation resistance and represent potentially useful pharmacologic targets. Hallmarks of the stem cell phenotype that may contribute to therapy resistance of CSCs include quiescence, evasion of apoptosis, resistance to DNA damage, and expression of drug transporter pumps. A variety of CSC populations within SCCs of the head and neck and esophagus have been defined tentatively, based on diverse surface markers and functional assays. Stem-like self-renewal and differentiation capacities of these SCC subpopulations are supported by sphere formation and clonogenicity assays in vitro as well as limiting dilution studies in xenograft models. Early evidence supports a role for SCC CSCs in intrinsic therapy resistance, while detailed mechanisms by which these subpopulations evade treatment remain to be defined. Development of novel SCC therapies will be aided by pursuing such mechanisms as well as refining current definitions for CSCs and clarifying their relevance to hierarchical versus dynamic models of stemness.
cancer stem cells; drug resistance; squamous cell carcinoma
This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.
In photodynamic diagnosis, 5-aminolevulinic acid (5-ALA) is widely used for the fluorescence-guided resection of malignant brain tumors, where 5-ALA is converted to protoporphyrin IX, which exhibits strong fluorescence. Little is known, however, about the detailed molecular mechanisms underlying 5-ALA–induced fluorescence. To resolve this issue, we analyzed transcriptome profiles for the genes encoding enzymes, transporters, and a transcription factor involved in the porphyrin-biosynthesis pathway. By quantitative real-time (qRT)-PCR, we measured the mRNA levels of those genes in a total of 20 tumor samples that had been surgically resected from brain tumor patients at the Department of Neurosurgery of Osaka Medical College from 2008 to 2009. We selected 10 tumor samples with no 5-ALA–induced fluorescence, among which 2 were glioblastomas and 8 were metastatic brain tumors. Another 10 tumor samples were selected with strong fluorescence, among which 7 were glioblastomas and 3 were metastatic brain tumors. The qRT-PCR analysis study of these latter 10 samples revealed predominantly high levels of the mRNA of the coproporphyrinogen oxidase (CPOX) gene. The high mRNA level of CPOX expression was significantly well correlated with the phenotype of strong 5-ALA–induced fluorescence (P = .0003). These findings were further confirmed by immunohistochemical studies with a CPOX-specific antibody. It is concluded that induction of CPOX gene expression is one of the key molecular mechanisms underlying the 5-ALA–induced fluorescence of malignant brain tumors. The induction mechanism for the CPOX gene in brain tumors remains to be elucidated.
coproporphyrinogen oxidase; malignant glioma; metastatic brain tumor; photodynamic diagnosis
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.
Notch; EMT; squamous cell differentiation; ZEB1; miR-200
Squamous cell cancers account for more than half of all human cancers, and esophageal cancer is the sixth leading cause of cancer death worldwide. The majority of esophageal squamous cell carcinomas have identifiable p53 mutations, yet the same p53 mutations are found at comparable frequencies in pre-cancerous dysplasia, indicating that transformation requires additional somatic changes yet to be defined. Here we show that the zinc finger transcription factor KLF5 transactivates NOTCH1 in the context of p53 mutation or loss. KLF5 loss limited NOTCH1 activity and was sufficient on its own to transform primary human keratinocytes harboring mutant p53, leading to formation of invasive tumors. Restoration of NOTCH1 blocked transformation of KLF5-deficient and p53 mutant keratinocytes. While human dysplastic epithelia accumulated KLF5, KLF5 expression was lost concurrently with NOTCH1 in squamous cell cancers. Taken together, these results define KLF5 loss as a critical event in squamous cell transformation and invasion. Our findings suggest that KLF5 may be a useful diagnostic and therapeutic target in esophageal squamous carcinomas and possibly more generally in other cancers associated with p53 loss-of-function.
KLF5; p53; NOTCH1; squamous cell cancer; esophageal cancer
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.
Esophageal squamous cell carcinoma; organotypic 3D culture; EGFR; P53; cyclin D1; Wnt; Notch; squamous-cell differentiation; invasion; 4-Nitroquinoline 1-oxide
The authors present a novel method of the recapping hemilaminoplasty in a retrospective study of patients with spinal surgical disorders. This report describes the surgical technique and the results of hemilaminoplasty using an ultrasonic bone curette. The aim of this study was to examine the safety and effectiveness of the hemilaminoplasty technique with ultrasonic bone curette.
Between April 2003 and July 2011, 33 patients with various spinal diseases (17 spinal tumors, 5 dural arteriovenous fistulas, 3 syringomyelia, 2 sacral perineural cysts, and 2 arachnoid cysts) were treated microsurgically by using an ultrasonic bone curette with scalpel blade and lightweight handpiece. The ultrasonic bone curette was used for division of lamina. After resection of the lesion, the excised lamina was replaced exactly in situ to its original anatomic position with a titanium plate and screw. Additional fusion technique was not required and the device was easy to handle. All patients were observed both neurologically and radiologically by dynamic plain radiographs and computed tomography (CT) scan.
The operation was performed successfully and there were no instrument-related complications such as dural laceration, nerve root injury, and vessels injury. The mean number of resected and restored lamina was 1.7. CT confirmed primary bone fusion in all patients by 12 months after surgery.
The ultrasonic bone curette is a useful instrument for recapping hemilaminoplasty in various spinal surgeries. This method allows anatomical reconstruction of the excised bone to preserve the posterior surrounding tissues.
Recapping hemilaminoplasty; spinal surgery; ultrasonic bone curette
The procedure of catheter ablation for the treatment of drug resistant atrial fibrillation (AF) has evolved but still relies on lesion sets intended to isolate areas of focal firing, mainly the myocardial sleeves of the pulmonary veins (PVs), from the rest of the atria. However the success rates for this procedure have varied inversely with the type of AF. At best success rates have been 20 to 30% below that of other catheter ablation procedures for Wolff-Parkinson-White syndrome, atrioventricular junctional re-entrant tachycardia and atrial flutter. Basic and clinical evidence has emerged suggesting a critical role of the ganglionated plexi (GP) at the PV-atrial junctions in the initiation and maintenance of the focal form of AF. At present the highest success rates have been obtained with the combination of PV isolation and GP ablation both as catheter ablation or minimally invasive surgical procedures. Various lines of evidence from earlier and more recent reports provide that both neurally based and myocardially based forms of AF can separately dominate or coexist within the context of atrial remodeling. Future studies are focusing on non-pharmacological, non-ablative approaches for the prevention and treatment of AF in order to avoid the substantive complications of both these regimens.
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
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.
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.
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.
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.
NOTCH1; NOTCH3; esophageal epithelium; squamous differentiation
Cyclin D1 elicits transcriptional effects through inactivation of the retinoblastoma protein and direct association with transcriptional regulators. The current work reveals a molecular relationship between cyclin D1/CDK4 kinase and protein arginine methyltransferase 5 (PRMT5), an enzyme associated with histone methylation and transcriptional repression. Primary tumors of a mouse lymphoma model exhibit increased PRMT5 methyltransferase activity and histone arginine methylation. Analyses demonstrate that MEP50, a PRMT5 co-regulatory factor, is a CDK4 substrate, and phosphorylation increases PRMT5/MEP50 activity. Increased PRMT5 activity mediates key events associated with cyclin D1-dependent neoplastic growth including CUL4 repression, CDT1 overexpression, and DNA re-replication. Importantly, human cancers harboring mutations in Fbx4, the cyclin D1 E3 ligase, exhibit nuclear cyclin D1 accumulation and increased PRMT5 activity.
Cyclin D1; CDK4; CUL4; CDT1; PRMT5; MEP50; Arginine Methylation
Insulin-like growth factor-binding protein (IGFBP)-3 is overexpressed frequently in esophageal squamous cell carcinoma. Yet, the role of IGFBP3 in esophageal tumor biology remains to be elucidated. We find that IGFBP3 facilitates transforming growth factor (TGF)-β1-mediated epithelial-to-mesenchymal transition (EMT) in transformed human esophageal epithelial cells, EPC2–hTERT–EGFR–p53R175H. In organotypic 3D culture, a form of human tissue engineering, laser-capture microdissection revealed concurrent upregulation of TGF-β target genes, IGFBP3 and EMT-related genes in the cells invading into the stromal compartment. IGFBP3 enhanced TGF-β1-mediated EMT as well as transcription factors essential in EMT by allowing persistent SMAD2 and SMAD3 phosphorylation. TGF-β1-mediated EMT and cell invasion were enhanced by ectopically expressed IGFBP3 and suppressed by RNA interference directed against IGFBP3. The IGFBP3 knockdown effect was rescued by IGFBP3I56G/L80G/L81G, a mutant IGFBP3 lacking an insulin-like growth factor (IGF)-binding capacity. Thus, IGFBP3 can regulate TGF-β1-mediated EMT and cell invasion in an IGF or insulin-like growth factor 1 receptor-independent manner. IGFBP3I56G/L80G/L81G also promoted EMT in vivo in a Ras-transformed human esophageal cell line T-TeRas upon xenograft transplantation in nude mice. In aggregate, IGFBP3 may have a novel IGF-binding independent biological function in regulation of TGF-β1-mediated EMT and cell invasion.
Transforming growth factor (TGF)-β is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive as to which molecular mechanisms determine the cellular capacity to undergo EMT in response to TGF-β. We have found that both epidermal growth factor receptor (EGFR) overexpression and mutant p53 tumor suppressor genes contribute to enrichment of an EMT-competent cellular subpopulation amongst telomerase-immortalized human esophageal epithelial cells during malignant transformation. EGFR overexpression triggers oncogene-induced senescence, accompanied by induction of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and p21. Interestingly, a subpopulation of cells emerges by negating senescence without loss of EGFR overexpression. Such cell populations express increased levels of zinc finger E-box binding (ZEB) transcription factors ZEB1 and ZEB2, and undergo EMT upon TGF-β stimulation. Enrichment of EMT-competent cells was more evident in the presence of p53 mutation, which diminished EGFR-induced senescence. RNA interference directed against ZEB resulted in induction of p15INK4B and p16INK4A, reactivating the EGFR-dependent senescence program. Importantly, TGF-β-mediated EMT did not take place when cellular senescence programs were activated by either ZEB knockdown or activation of wild-type p53 function. Thus, senescence checkpoint functions activated by EGFR and p53 may be evaded through the induction of ZEB, thereby allowing expansion of an EMT-competent unique cellular subpopulation, providing novel mechanistic insights into the role of ZEB in esophageal carcinogenesis.
EGFR; EMT; senescence; ZEB1; ZEB2
Hypoxia-inducible factors (HIFs), in particular HIF-1α, have been implicated in tumor biology. However, HIF target genes in the esophageal tumor microenvironment remain elusive. Gene expression profiling was performed upon hypoxia-exposed non-transformed immortalized human esophageal epithelial cells, EPC2-hTERT, and comparing with a gene signature of esophageal squamous cell carcinoma (ESCC). In addition to known HIF-1α target genes such as carbonic anhydrase 9, insulin-like growth factor binding protein-3 (IGFBP3) and cyclooxygenase (COX)-2, prostaglandin E synthase (PTGES) was identified as a novel target gene among the commonly upregulated genes in ESCC as well as the cells exposed to hypoxia. The PTGES induction was augmented upon stabilization of HIF-1α by hypoxia or cobalt chloride under normoxic conditions and suppressed by dominant-negative HIF-1α. Whereas PTGES messenger RNA (mRNA) was negatively regulated by normoxia, PTGES protein remained stable upon reoxygenation. Prostaglandin E2 (PGE2) biosynthesis was documented in transformed human esophageal cells by ectopic expression of PTGES as well as RNA interference directed against PTGES. Moreover, hypoxia stimulated PGE2 production in a HIF-1α-dependent manner. In ESCC, PTGES was overexpressed frequently at the mRNA and protein levels. Finally, COX-2 and PTGES were colocalized in primary tumors along with HIF-1α and IGFBP3. Activation of the COX-2–PTGES axis in primary tumors was further corroborated by concomitant upregulation of interleukin-1β and downregulation of hydroxylprostaglandin dehydrogenase. Thus, PTGES is a novel HIF-1α target gene, involved in prostaglandin E biosynthesis in the esophageal tumor hypoxic microenvironment, and this has implications in diverse tumors types, especially of squamous origin.
IGF and EGF regulate various physiological and pathological processes. IGF binding protein (IGFBP)-3 regulates cell proliferation in IGF-dependent and -independent fashions. Recently, we identified IGFBP-3 as a novel EGF receptor (EGFR) downstream target molecule in primary and immortalized human esophageal epithelial cells, suggesting an interplay between the EGF and IGF signaling pathways. However, the regulatory mechanisms for IGFBP-3 expression and its functional role in esophageal cell proliferation remain to be elucidated. Herein, we report that IGFBP-3 mRNA and protein were induced upon growth factor deprivation in primary and immortalized human esophageal cells through mechanisms requiring p53-independent de novo mRNA transcription and protein synthesis. This occurred in the face of the activated phosphatidylinositol 3-OH-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. Secreted IGFBP-3 neutralized IGFs and prevented IGF-I receptor (IGF-IR) activation. In contrast, EGF suppressed IGFBP-3 mRNA and protein expression through activation of MAPK in an EGFR-tyrosine kinase-dependent manner to restore the cellular response to IGF-I. When stably overexpressed, wild-type IGFBP-3 but not I56G/L80G/L81G (GGG) mutant IGFBP-3, which has a reduced affinity to IGFs, prevented IGF-I from activating IGF-IR and Akt as well as stimulating cell proliferation. However, unlike other cell types where IGFBP-3 exerts antiproliferative effects, neither wild-type nor GGG mutant IGFBP-3 alone affected cell proliferation or EGFR activity. These results indicate that IGF signaling is subject to negative regulation through IGFBP-3 and positive regulation by EGF, the latter of which suppresses IGFBP-3. This provides a platform for understanding the novel cross talk between EGF- and IGF-mediated pathways.
insulin-like growth factor binding protein-3; epidermal growth factor receptor; mammalian target of rapamycin; esophageal epithelial cells
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.
Insulin-like growth factor binding protein (IGFBP)-3 exerts either proapoptotic or growth stimulatory effects depending upon the cellular context. IGFBP-3 is overexpressed frequently in esophageal cancer. Yet, the role of IGFBP-3 in esophageal tumor biology remains elusive. To delineate the functional consequences of IGFBP-3 overexpression, we stably transduced Ha-RasV12-transformed human esophageal cells with either wild-type or mutant IGFBP-3, the latter incapable of binding Insulin-like growth factor (IGFs) as a result of substitution of amino-terminal Ile56, Leu80, and Leu81 residues with Glycine residues. Wild-type, but not mutant, IGFBP-3 prevented IGF-I from activating the IGF-1 receptor and AKT, and suppressed anchorage-independent cell growth. When xenografted in nude mice, in vivo bioluminescence imaging demonstrated that wild-type, but not mutant IGFBP-3, abrogated tumor formation by the Ras-transformed cells with concurrent induction of apoptosis, implying a prosurvival effect of IGF in cancer cell adaptation to the microenvironment. Moreover, there was more aggressive tumor growth by mutant IGFBP-3 overexpressing cells than control cell tumors, without detectable caspase-3 cleavage in tumor tissues, indicating an IGF-independent growth stimulatory effect of mutant IGFBP-3. In aggregate, these data suggest that IGFBP-3 contributes to esophageal tumor development and progression through IGF-dependent and independent mechanisms.
IGFBP-3; IGF; Ras; esophageal cancer; in vivo bioluminescence
The cyclin D1 oncogene is frequently amplified/overexpressed in oral squamous cell carcinomas. Mice with overexpression of cyclin D1 targeted to the stratified squamous epithelia of the tongue, esophagus and forestomach develop a phenotype of epithelial dysplasia at these sites. In this study, we examined the effect cyclin D1 overexpression on susceptibility of mice to carcinogen-induced tumorigenesis, using 4-nitroquinoline-1-oxide (4NQO), an established potent oral carcinogen in mice. Cyclin D1 overexpressing mice and non-transgenic littermates were administered 4NQO (20 ppm or 50 ppm in the drinking water) for 8 weeks and monitored for an additional 16 weeks. Histopathological analyses of the tongue revealed significantly higher severity of dysplasia in the cyclin D1 overexpression mice, compared with non-transgenic controls and with untreated controls. Moreover, only the cyclin D1 overexpression mice developed neoplastic lesions in the oro-esophageal epithelia. Examination of the dysplastic and neoplastic lesions reveled abnormal proliferation. Our findings suggest that cyclin D1 overexpression enhances susceptibility to carcinogen-induced oral tumorigenesis. These results underscore the importance of cyclin D1 in the process of oral neoplastic development. Further, they emphasize the value of this transgenic model to study the pathogenesis of oral precancer and cancer and establish it as a model system to test candidate agents for chemoprevention of upper aero-digestive cancer.
Mouth neoplasms/genetics; Cyclin D1/genetics; Disease Models; Animal Mice; Transgenic
Accumulating evidence indicates that ATP-binding cassette (ABC) transporter ABCG2 plays a key role in regulating the cellular accumulation of porphyrin derivatives in cancer cells and thereby affects the efficacy of photodynamic therapy and photodynamic diagnosis. The activity of porphyrin efflux can be affected by genetic polymorphisms in the ABCG2 gene. On the other hand, Nrf2, an NF-E2-related transcription factor, has been shown to be involved in oxidative stress-mediated induction of the ABCG2 gene. Since patients have demonstrated individual differences in their response to photodynamic therapy, transcriptional activation and/or genetic polymorphisms of the ABCG2 gene in cancer cells may affect patients' responses to photodynamic therapy. Protein kinase inhibitors, including imatinib mesylate and gefitinib, are suggested to potentially enhance the efficacy of photodynamic therapy by blocking ABCG2-mediated porphyrin efflux from cancer cells. This review article provides an overview on the role of human ABC transporter ABCG2 in photodynamic therapy and photodynamic diagnosis.
Lineage survival oncogenes are activated by somatic DNA alterations in cancers arising from the cell lineages in which these genes play a role in normal development.1,2 Here we show that a peak of genomic amplification on chromosome 3q26.33, found in squamous cell carcinomas (SCCs) of the lung and esophagus, contains the transcription factor gene SOX2—which is mutated in hereditary human esophageal malformations3 and necessary for normal esophageal squamous development4, promotes differentiation and proliferation of basal tracheal cells5 and co-operates in induction of pluripotent stem cells.6,7,8
SOX2 expression is required for proliferation and anchorage-independent growth of lung and esophageal cell lines, as shown by RNA interference experiments. Furthermore, ectopic expression of SOX2 cooperated with FOXE1 or FGFR2 to transform immortalized tracheobronchial epithelial cells. SOX2-driven tumors show expression of markers of both squamous differentiation and pluripotency. These observations identify SOX2 as a novel lineage survival oncogene in lung and esophageal SCC.
Single nucleotide polymorphisms (SNPs) of the ATP-binding cassette (ABC) transporter ABCG2 gene have been suggested to be a significant factor in patients’ responses to medication and/or the risk of diseases. We aimed to evaluate the impact of the major non-synonymous SNP Q141K on lysosomal and proteasomal degradations.
ABCG2 WT and the Q141K variant were expressed in Flp-In-293 cells by using the Flp recombinase system. Their expression levels and cellular localization was measured by immunoblotting and immunofluorescence microscopy, respectively.
The protein level of the Q141K variant expressed in Flp-In-293 cells was about half that of ABCG2 WT, while their mRNA levels were equal. The protein expression level of the Q141K variant increased about two-fold when Flp-In-293 cells were treated with MG132. In contrast, the protein level of ABCG2 WT was little affected by the same treatment. After treatment with bafilomycin A1, the protein levels of ABCG2 WT and Q141K increased 5- and 2-fold in Flp-In-293 cells, respectively.
The results strongly suggest that the major non-synonymous SNP Q141K affects the stability of the ABCG2 protein in the endoplasmic reticulum and enhances its susceptibility to ubiquitin-mediated proteasomal degradation.
ABCG2; SNP; endoplasmic reticulum associated degradation (ERAD); ubiquitin; proteasome
Barrett's esophagus (BE) is the replacement of normal squamous esophageal mucosa with an intestinalized columnar epithelium. The molecular mechanisms underlying its development are not understood. Cdx2 is an intestine-specific transcription factor that is ectopically expressed in BE, but its role in this process is unclear. Herein, we describe a novel cell culture model for BE. Retroviral-mediated Cdx2 expression in immortalized human esophageal keratinocytes [EPC-human telomerase reverse transcriptase (hTERT)] could transiently be established but not maintained and was associated with a reduction in cell proliferation. Coexpression of cyclin D1, but not a dominant-negative p53, rescued proliferation in the Cdx2-expressing cells. Cdx2 expression in the EPC-hTERT.D1 cells decreased cell proliferation but did not induce intestinalization. We investigated for other treatments to enhance intestinalization and found that acidic culture conditions uniformly killed EPC-hTERT.D1.Cdx2 cells. However, treatment with 5-aza-2-deoxycytidine (5-AzaC) to demethylate epigenetically silenced genes did appear to be tolerated. Multiple Cdx2 target genes, markers of intestinal differentiation and markers of BE, were induced by this 5-AzaC treatment. More interestingly, the expression level of several of these genes was enhanced only in the EPC-hTERT.D1-Cdx2 cells treated with 5-AzaC. Two of these, SLC26a3/DRA (downregulated in adenoma) and Na+/H+ exchanger 2 (NHE2), were not previously known to be elevated in BE; however, we confirmed their elevation in BE tissue samples. 5-AzaC treatment also induced cell senescence, even at low doses. We conclude that ectopic proliferation signals, alterations in epigenetic gene regulation and the inhibition of tumor suppressor mechanisms are required for Cdx2-mediated intestinalization of human esophageal keratinocytes in BE.
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
SCFFbx4 was recently identified as the E3 ligase for cyclin D1. We now describe cell cycle-dependent phosphorylation and dimerization of Fbx4 that is regulated by GSK3β and defective in human cancer. We present data demonstrating that a pathway involving Ras-Akt-GSK3β controls the temporal phosphorylation and dimerization of the SCFFbx4 E3 ligase. Inhibition of Fbx4 activity results in accumulation of nuclear cyclin D1 and oncogenic transformation. The importance of this regulatory pathway for normal cell growth is emphasized by the prevalence of mutations in Fbx4 in human cancer that impair dimerization. Collectively, this data reveals that inactivation of the cyclin D1 E3 ligase will likely contribute to cyclin D1 overexpression in a significant fraction of human cancer.
Cyclin D1; Fbx4; esophageal cancer; GSK3β
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.