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
The MAPK pathway has emerged as a central target for melanoma therapy due to its persistent activation in the majority of tumors. Several BRAF inhibitors aimed at curbing MAPK pathway activity are currently in advanced stages of clinical investigation. However, their therapeutic success is limited by the emergence of drug resistance, as responses are transient and tumors eventually recur. Understanding the mechanisms underlying resistance to BRAF inhibitors is essential to develop effective and long-lasting therapies for melanoma patients. Here, we briefly review recent pre-clinical studies providing insight into the molecular mechanisms of resistance to BRAF inhibitors and discuss potential strategies to treat drug resistant melanomas.
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.
This proof-of-concept study was designed to determine if functional, pharmacodynamic profiles relevant to targeted therapy could be derived from live human melanoma samples using a novel automated platform.
A series of 13 melanoma cell lines was briefly exposed to a BRAF inhibitor (PLX-4720) on a platform employing automated fluidics for sample processing. Levels of the phosphoprotein p-ERK in the mitogen-activated protein kinase (MAPK) pathway from treated and untreated sample aliquots were determined using a bead-based immunoassay. Comparison of these levels provided a determination of the pharmacodynamic effect of the drug on the MAPK pathway. A similar ex vivo analysis was performed on fine needle aspiration (FNA) biopsy samples from four murine xenograft models of metastatic melanoma, as well as 12 FNA samples from patients with metastatic melanoma.
Melanoma cell lines with known sensitivity to BRAF inhibitors displayed marked suppression of the MAPK pathway in this system, while most BRAF inhibitor-resistant cell lines showed intact MAPK pathway activity despite exposure to a BRAF inhibitor (PLX-4720). FNA samples from melanoma xenografts showed comparable ex vivo MAPK activity as their respective cell lines in this system. FNA samples from patients with metastatic melanoma successfully yielded three categories of functional profiles including: MAPK pathway suppression; MAPK pathway reactivation; MAPK pathway stimulation. These profiles correlated with the anticipated MAPK activity, based on the known BRAF mutation status, as well as observed clinical responses to BRAF inhibitor therapy.
Pharmacodynamic information regarding the ex vivo effect of BRAF inhibitors on the MAPK pathway in live human melanoma samples can be reproducibly determined using a novel automated platform. Such information may be useful in preclinical and clinical drug development, as well as predicting response to targeted therapy in individual patients.
The role of tumour-stromal interactions in progression is generally well accepted but their role in initiation or treatment is less well understood. It is now generally agreed that rather than consisting solely of malignant cells, tumours consist of a complex dynamic mixture of cancer cells, host fibroblasts, endothelial cells, and immune cells that interact with each other and micro-environmental factors to drive tumour progression. We are particularly interested in stromal cells (for example fibroblasts) and stromal factors (for example fibronectin) as important players in tumour progression since they have also been implicated in drug resistance. Here we develop an integrated approach to understand the role of such stromal cells and factors in the growth and maintenance of tumours as well as their potential impact on treatment resistance, specifically in application to melanoma. Using a suite of experimental assays we show that melanoma cells can stimulate the recruitment of fibroblasts and activate them, resulting in melanoma cell growth by providing both structural (extra-cellular matrix proteins) and chemical support (growth factors). Motivated by these experimental results we construct a compartment model and use it to investigate the roles of both stromal activation and tumour aggressiveness in melanoma growth and progression. We utilise this model to investigate the role fibroblasts might play in melanoma treatment resistance and the clinically observed flare phenomena that is seen when a patient, who appears resistant to a targeted drug, is removed from that treatment. Our model makes the unexpected prediction that targeted therapies may actually hasten tumour progression once resistance has occurred. If confirmed experimentally, this provocative prediction may bring important new insights into how drug resistance could be managed clinically.
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
Several melanoma cell subpopulations with tumor-initiating and/or tumor-maintaining properties exist that may contribute to chemoresistance and tumor recurrence after standard therapies. One of these subpopulations expresses a B-cell marker, CD20. In a small pilot trial, we showed that a subset of Stage IV melanoma patients may potentially benefit from an adjuvant treatment using the anti-CD20 antibody rituximab.
CD20; adjuvant immunotherapy; melanoma; rituximab; tumor-infiltrating B cells; tumor-initiating subpopulations
The utility of different models to identify cancer stem cells continues to be a subject of intense debate. Here, we summarize recent efforts to characterize intra-tumoral heterogeneity of melanoma and delineate key questions for future studies. Within a developing or already established tumor microenvironment, we propose that continuous tumor maintenance is assured by specific subpopulations whose phenotype is not static but instead is dynamically regulated. These small and temporarily distinct subpopulations likely play critical roles in tumor progression. They are important therapeutic targets but only in the context of combination therapies that also eliminate the bulk of the tumor.
Breast cancer is a heterogeneous disease for which prognosis and treatment strategies are largely governed by the receptor status (estrogen, progesterone and Her2) of the tumor cells. Gene expression profiling of whole breast tumors further stratifies breast cancer into several molecular subtypes which also co-segregate with the receptor status of the tumor cells. We postulated that cancer associated fibroblasts (CAFs) within the tumor stroma may exhibit subtype specific gene expression profiles and thus contribute to the biology of the disease in a subtype specific manner. Several studies have reported gene expression profile differences between CAFs and normal breast fibroblasts but in none of these studies were the results stratified based on tumor subtypes.
To address whether gene expression in breast cancer associated fibroblasts varies between breast cancer subtypes, we compared the gene expression profiles of early passage primary CAFs isolated from twenty human breast cancer samples representing three main subtypes; seven ER+, seven triple negative (TNBC) and six Her2+.
We observed significant expression differences between CAFs derived from Her2+ breast cancer and CAFs from TNBC and ER + cancers, particularly in pathways associated with cytoskeleton and integrin signaling. In the case of Her2+ breast cancer, the signaling pathways found to be selectively up regulated in CAFs likely contribute to the enhanced migration of breast cancer cells in transwell assays and may contribute to the unfavorable prognosis of Her2+ breast cancer.
These data demonstrate that in addition to the distinct molecular profiles that characterize the neoplastic cells, CAF gene expression is also differentially regulated in distinct subtypes of breast cancer.
A new metal-containing scaffold for the design of protein kinase inhibitors is introduced. Key feature is a 3-(2-pyridyl)-1,8-naphthalimide “pharmacophore chelate ligand” which is designed to form two hydrogen bonds with the hinge region of the ATP-binding site and is at the same time capable of serving as a stable bidentate ligand through C-H-activation at the 4-position of the electron-deficient naphthalene moiety. This C-H-activation leads to a reduced demand for coordinating heteroatoms and thus sets the basis for a very efficient three-step synthesis starting from 1,8-naphthalic anhydride. The versatility of this ligand is demonstrated with the discovery of a ruthenium complex that functions as a nanomolar inhibitor for myosin light-chain kinase (MYLK or MLCK).
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
Curative eradication of all cells within carcinomas is seldom achievable with chemotherapy alone. This limitation may be partially attributable to tumor cell subpopulations with intrinsic resistance to current drugs. Within squamous cell carcinoma (SCC) cell lines, we previously characterized a subpopulation of mesenchymal-like cells displaying phenotypic plasticity and increased resistance to both cytotoxic and targeted agents. These mesenchymal-like (Ecad-lo) cells are separable from epithelial-like (Ecad-hi) cells based on loss of surface E-cadherin and expression of vimentin. Despite their long-term plasticity, both Ecad-lo and Ecad-hi subsets in short-term culture maintained nearly uniform phenotypes after purification. This stability allowed testing of segregated subpopulations for relative sensitivity to the cytotoxic agent cisplatin in comparison to salinomycin, a compound with reported activity against CD44+CD24− stem-like cells in breast carcinomas. Salinomycin showed comparable efficacy against both Ecad-hi and Ecad-lo cells in contrast to cisplatin, which selectively depleted Ecad-hi cells. An in vivo correlate of these mesenchymal-like Ecad-lo cells was identified by immunohistochemical detection of vimentin-positive malignant subsets across a part of direct tumor xenografts (DTXs) of advanced stage SCC patient samples. Cisplatin treatment of mice with established DTXs caused enrichment of vimentin-positive malignant cells in residual tumors, but salinomycin depleted the same subpopulation. These results demonstrate that mesenchymal-like SCC cells, which resist current chemotherapies, respond to a treatment strategy developed against a stem-like subset in breast carcinoma. Further, they provide evidence of mesenchymal-like subsets being well-represented across advanced stage SCCs, suggesting that intrinsic drug resistance in this subpopulation has high clinical relevance.
EMT; squamous cell carcinoma; head and neck cancer; esophageal cancer; chemotherapy resistance; salinomycin; tumor heterogeneity
Metastatic melanoma is the most aggressive skin cancer. Recently, phenotypically distinct subpopulations of tumor cells were identified. Among them, ABCB5-expressing cells were proposed to display an enhanced tumorigenicity with stem cell-like properties. In addition, ABCB5+ cells are thought to participate to chemoresistance through a potential efflux function of ABCB5. Nevertheless, the fate of these cells upon drugs that are used in melanoma chemotherapy remains to be clarified. Here we explored the effect of anti-melanoma treatments on the ABCB5-expressing cells. Using a melanoma xenograft model (WM266-4), we observed in vivo that ABCB5-expressing cells are enriched after a temozolomide treatment that induces a significant tumor regression. These results were further confirmed in a preliminary study conducted on clinical samples from patients that received dacarbazine. In vitro, we showed that ABCB5-expressing cells selectively survive when exposed to dacarbazine, the reference treatment of metastatic melanoma, but also to vemurafenib, a new inhibitor of the mutated kinase V600E BRAF and other various chemotherapeutic drugs. Our results show that anti-melanoma chemotherapy might participate to the chemoresistance acquisition by selecting tumor cell subpopulations expressing ABCB5. This is of particular importance in understanding the relapses observed after anti-melanoma treatments and reinforces the interest of ABCB5 and ABCB5-expressing cells as potential therapeutic targets in melanoma.
Cutaneous melanoma originates from pigment producing melanocytes or their precursors and is considered the deadliest form of skin cancer. For the last 40 years, few treatment options were available for patients with late-stage melanoma. However, remarkable advances in the therapy field were made recently, leading to the approval of two new drugs, the mutant BRAF inhibitor vemurafenib and the immunostimulant ipilimumab. Although these drugs prolong patients' lives, neither drug cures the disease completely, emphasizing the need for improvements of current therapies. Our knowledge about the complex genetic and biological mechanisms leading to melanoma development has increased, but there are still gaps in our understanding of the early events of melanocyte transformation and disease progression. In this review, we present a summary of the main contributing factors leading to melanocyte transformation and discuss recent novel findings and technologies that will help answer some of the key biological melanoma questions and lay the groundwork for novel therapies.
Molecular biology; oncogenes; tumor biology
Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare disorder characterized by segmental accelerated aging and early death from coronary artery disease or stroke. Nearly 90% of HGPS sufferers carry a G608G mutation within exon 11 of LMNA, producing a truncated form of prelamin A, referred to as “progerin”. Here, we report the isolation of naïve multipotent skin-derived precursor (SKP) cells from dermal fibroblast cultures from HGPS donors. These cells form spheres and express the neural crest marker, nestin, in addition to the multipotent markers, OCT4, Sox2, Nanog and TG30; these cells can self-renew and differentiate into smooth muscle cells (SMCs) and fibroblasts. The SMCs derived from the HGPS-SKPs accumulate nuclear progerin with increasing passages. A subset of the HGPS-naïve SKPs express progerin in vitro and in situ in HGPS skin sections. This is the first in vivo evidence that progerin is produced in adult stem cells, and implies that this protein could induce stem cells exhaustion as a mechanism contributing to aging. Our study provides a basis on which to explore therapeutic applications for HGPS stem cells and opens avenues for investigating the pathogenesis of other genetic diseases.
Lamin A; Progerin; HGPS; Progeria; Adult stem cells
Melanoma is the deadliest form of skin cancer and its incidence has been increasing. The disease manifests itself as clinically and genetically distinct subgroups, indicating the need for patient-specific diagnostic and treatment tools. The discovery of activating mutations (V600E) in the BRAF kinase in approximately 50% of patients spurred the development of compounds to inhibit aberrant BRAF activity, and the first drug candidate to show promising clinical activity is PLX4032 (also known as RG7204). Most recent clinical data from a phase II trial indicate that PLX4032 causes tumor regression and stabilized disease in >50% of advanced melanoma patients harboring BRAF V600E tumors. These data validate the effectiveness of oncogene-targeted therapy against advanced melanoma and offer hope that the disease can be overcome. However, as melanoma is dynamic and heterogeneous, careful treatment strategies and combination therapies are warranted to obtain long-term clinical effects.
Melanoma is an excellent model to study molecular mechanisms of tumor progression because melanoma usually develops through a series of architecturally and phenotypically distinct stages that are progressively more aggressive, culminating in highly metastatic cells. In this study, we used an in-depth, three-dimensional (3-D) protein level, comparative proteome analysis of two genetically, very-closely-related, melanoma cell lines with low- and high-metastatic potentials to identify proteins and key pathways involved in tumor progression. This proteome comparison utilized fluorescent tagging of cell lysates followed by microscale solution isoelectric focusing (MicroSol-IEF) prefractionation and subsequent analysis of each fraction on narrow-range 2-D gels. LC-MS/MS analysis of gel spots exhibiting significant abundance changes identified 110 unique proteins. The majority of observed abundance changes closely correlate with biological processes central to cancer progression, such as cell death and growth and tumorigenesis. In addition, the vast majority of protein changes mapped to six cellular networks, which included known oncogenes (JNK, c-myc, and N-myc) and tumor suppressor genes (p53 and TGF-β) as critical components. These six networks showed substantial connectivity, and most of the major biological functions associated with these pathways are involved in tumor progression. These results provide novel insights into cellular pathways implicated in melanoma metastasis.
2-D difference in gel electrophoresis (2-D DIGE); Metastatic melanoma; Microscale solution isoelectric focusing (MicroSol-IEF); Protein networks; Systems biology
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
BRAF is an attractive target for melanoma drug development. However, resistance to BRAF inhibitors is a significant clinical challenge. We describe a model of resistance to BRAF inhibitors developed by chronic treatment of BRAFV600E melanoma cells with the BRAF inhibitor SB-590885; these cells are cross resistant to other BRAF-selective inhibitors. Resistance involves flexible switching among the three RAF isoforms, underscoring the ability of melanoma cells to adapt to pharmacological challenges. IGF-1R/PI3K signaling was enhanced in resistant melanomas, and combined treatment with IGF-1R/PI3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells. Increased IGFR-1R and pAKT levels in a post-relapse human tumor sample are consistent with a role for IGF-1R/PI3K-dependent survival in the development of resistance to BRAF inhibitors.
melanoma; BRAF; MEK; IGF-1R; targeted therapy; drug resistance
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
Expression of the α2β1 integrin, a receptor for collagens and laminin, is altered during tumor progression. Recent studies have linked polymorphisms in the α2 integrin gene with oral, squamous cell carcinoma (SCC). To determine the α2β1 integrin's role in SCC progression, we crossed α2-null mice with K14-HPV16 transgenic animals. Pathological progression to invasive carcinoma was evaluated in HPV-positive, α2-null (HPV/KO) and HPV-positive, wild-type (HPV/WT) animals. α2β1 integrin expression stimulated progression from hyperplasia and papillomatosis to dysplasia with concomitant dermal mast cell infiltration. Moreover, lymph node metastasis was decreased by 31.3% in HPV/KO, compared to HPV/WT, animals. To evaluate the integrin-specific impact on the malignant epithelium versus the microenvironment, we developed primary tumor cell lines. Although transition from dysplasia to carcinoma was unaltered during spontaneous tumor development, isolated primary HPV/KO SCC cell lines demonstrated decreased migration and invasion, compared to HPV/WT cells. When HPV/WT and HPV/KO SCC cells were orthotopically injected into WT or KO hosts, tumor α2β1 integrin expression resulted in decreased tumor latency, regardless of host integrin status. HPV/WT SCC lines failed to demonstrate a proliferative advantage in vitro, however, the HPV/WT tumors demonstrated increased growth compared to HPV/KO SCC lines in vivo. Although contributions of the integrin to the microenvironment cannot be excluded, our studies indicate that α2β1 integrin expression by HPV-transformed keratinocytes modulates SCC growth and progression.
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
Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.
Bioengineering; Issue 54; 3D model; melanocyte; melanoma; skin
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.
Tenascin-C (TNC) is highly expressed in melanoma; however, little is known about its functions. Recent studies indicate that TNC plays a role within the stem cell niche. We hypothesized that TNC creates a specific environment for melanoma cells to exhibit a stem cell-like phenotype, driving tumor growth and evading conventional therapies. TNC expression was strongly up-regulated in melanoma cells grown as 3D spheres (enriched for stem-like cells) when compared to adherent cells. Down-modulation of TNC by shRNA-lentiviruses significantly decreased the growth of melanoma spheres. The incidence of pulmonary metastases after intravenous injection of TNC knockdown cells was significantly lower in NOD/SCID IL2Rγnull mice compared to control cells. Melanoma spheres contain and increased number of side population (SP) cells, which exhibited stem cell characteristics and the potential for drug resistance due to their high efflux capacity. Knockdown of TNC dramatically decreased the SP fraction in melanoma spheres and lowered their resistance to doxorubicin treatment, likely due to the down-regulation of multiple ABC transporters, including ABCB5. These data suggest that TNC plays a critical role in melanoma progression by mediating protective signals in the therapy-resistant population of melanoma.