Epithelial Cancer Progression Research Articles

Carcinoma‐associated fibroblasts: Non‐neoplastic tumour‐promoting mesenchymal cells

Cancerous stroma coevolves alongside tumour progression, thereby promoting the malignant conversion of epithelial carcinoma cells. To date, an abundance of data have supported crucial roles of the tumour microenvironment (TME) in providing cancer cells with proliferative, migratory, survival and invasive propensities favouring the processes of tumourigenesis. The cancerous reactive stroma is frequently populated by a large number of myofibroblasts (MFs), which are activated, non-transformed fibroblasts expressing α-smooth muscle actin (α-SMA). MFs together with non-MF cells present in the tumour-associated stroma are collectively referred to as carcinoma-associated fibroblasts (CAFs), one of the major stromal cell types recognised in various human carcinomas. Recruitment of fibroblasts and/or their progenitors to a tumour mass and their subsequent transdifferentiation into MFs, as well as ongoing maintenance of their activated state, are believed to be essential processes facilitating tumour progression. However, the complex networks of signalling pathways mediating the phenotypic conversion into CAFs, as well as those underlying their tumour-promoting interactions with other tumour-constituting cells, have yet to be fully explored. Histopathological confirmation of the presence of large numbers of CAF MFs within TME and their altered gene expression profiles are known to be associated with disease progression and to serve as independent negative prognostic factors for a wide range of tumour types. In this review, we examine the current evidence shedding light on the emerging roles of tumour-promoting CAFs, cells that are pivotal for epithelial cancer development and progression, and discuss the therapeutic potential of targeting these cells.

Metabolic changes during ovarian cancer progression as targets for sphingosine treatment

Tumor cells often exhibit an altered metabolic phenotype. However, it is unclear as to when this switch takes place in ovarian cancer, and the potential for these changes to serve as therapeutic targets in clinical prevention and intervention trials. We used our recently developed and characterized mouse ovarian surface epithelial (MOSE) cancer progression model to study metabolic changes in distinct disease stages. As ovarian cancer progresses, complete oxidation of glucose and fatty acids were significantly decreased, concurrent with increases in lactate excretion and 3H-deoxyglucose uptake by the late-stage cancer cells, shifting the cells towards a more glycolytic phenotype. These changes were accompanied by decreases in TCA flux but an increase in citrate synthase activity, providing substrates for de novo fatty acid and cholesterol synthesis. Also, uncoupled maximal respiration rates in mitochondria decreased as cancer progressed. Treatment of the MOSE cells with 1.5μM sphingosine, a bioactive sphingolipid metabolite, decreased citrate synthase activity, increased TCA flux, decreased cholesterol synthesis and glycolysis. Together, our data confirm metabolic changes during ovarian cancer progression, indicate a stage specificity of these changes, and suggest that multiple events in cellular metabolism are targeted by exogenous sphingosine which may be critical for future prevention trials.

The Ron Receptor Tyrosine Kinase: A Key Regulator of Inflammation and Cancer Progression

Numerous studies have documented abnormal expression and activation of the Ron receptor tyrosine kinase in a variety of human malignancies. Here we review the literature regarding the molecular mechanisms governing Ron regulation, the biological functions of Ron, the effect of Ron on cancer development, and potential therapeutic implications. In epithelial cells, activation of Ron by its ligand, macrophage stimulating protein, mediates a number of biological events including cell growth, motility, and epithelial to mesenchymal transition. Overexpression and/or activation of Ron has been implicated in the progression and metastasis of diverse epithelial cancers, where it plays a causal role in tumor development by promoting growth, survival, and motility of tumor cells. As a crucial regulator of inflammation, Ron inhibits classic macrophage activation and promotes alternative activation of macrophages, resulting in the resolution of inflammation and tissue repair. In addition, Ron alleviates antitumor immunity by promoting the alternative activation of tumor-associated macrophages, and Ron expression in the tumor microenvironment promotes the outgrowth of metastatic colonies. Hence, Ron is a promising therapeutic target for the treatment of epithelial cancers.

The candidate tumor suppressor gene Ecrg4 as a wound terminating factor in cutaneous injury

The Esophageal cancer-related gene-4 (Ecrg4) is a candidate tumor suppressor gene whose secreted protein product has been implicated in the development and progression of epithelial cancers, neuroprogenitor cell activation after central nervous system injury, cell senescence in neurodegeneration, and the survival of hematopoietic stem cells. Here, we investigated the temporal and spatial localization of Ecrg4 expression in healthy and injured mouse skin, and evaluated the biological activity of Ecrg4 using viral-mediated gene delivery in cutaneous wound healing models. Using in situ hybridization and immunohistochemistry, we found both Ecrg4 mRNA and its protein product localized to the epidermis, dermis, and hair follicles of healthy mouse skin. Upon cutaneous injury, Ecrg4 redistributed to the wound margins where gene microarray and quantitative RT-PCR showed an increased gene expression5-10days post-injury as a late phase injury response gene. Ecrg4 over-expression inhibited the directional migration of fibroblasts in modified Boyden chambers in vitro, but had no effect on rates of fibroblast proliferation. Ecrg4 over-expression in vivo at the wound margins delayed the rate of wound closure at 1 and 2days after full-thickness punch injury. These findings point to the candidate tumor suppressor gene Ecrg4 as a novel, biologically active, constituent of skin and skin injury. The possibility that Ecrg4 serves as a wound termination factor during wound resolution is discussed.

MiRNA expression as a predictor of survival in patients with metastatic colorectal cancer treated with EGFR inhibitors.

e14059 Background: The epidermal growth factor receptor (EGFR) pathway is crucial in the development and progression of human epithelial cancers. EGFR inhibitors have been tested for treatment of a variety of cancers but they are generally plagued by low response rates, leading to ineffective or non-optimal chemotherapy, unnecessary drug toxicity and expense. In colorectal cancer, KRAS mutations are clearly associated with resistance to anti-EGFR antibodies. A major challenge is to identify, in non-mutated KRAS patients, markers that can predict response to this therapy. This study aimed to develop a miRNA expression-based model for evaluating the responsiveness of patients with KRAS wild-type metastatic colorectal cancer to one or more EGFR inhibitors prior to treatment. Methods: miRNA profiling of KRAS wild-type tumors was performed on a discovery set of 44 patients with metastatic colorectal cancer treated with anti-EGFR inhibitors. A miRNA expression-based predictor of survival risk group was calculated by combining a Cox proportional hazards model and a supervised principal component method. Results: miRNA profiling of the discovery set identified hsa-miRNA-31* whose expression in tumor samples was highly correlated with survival and displayed prediction prospective for disease-free and overall survivals. Parameters of the hsa-miRNA-31* expression-based prediction model were determined on a subgroup of 23 patients. Its discriminative ability was evaluated on the complementary 21 samples that included 19 events. In this validation set, 4 of the 6 patients classified as “low risk” by the model survived for more than 25 weeks as disease-free whereas 11 of the 13 patients classified as “high risk” displayed disease progression within 25 weeks, suggesting a specificity of 85% [95% CI: 54%-98%] and a sensitivity of 67% [95% CI: 22%-96%] for the prediction model. These values were associated with a PPV of 67%, [95% CI: 22%-95%] and a NPV of 85% [95% CI: 54%-98%]. Conclusions: Our findings demonstrated that a prediction model based on the level of expression of hsa-miRNA-31* could evaluate whether a patient with a KRAS wild-type metastatic colorectal cancer is likely to respond to an EGFR inhibitor.

Abstract 4218: Calcitonin receptor increases invasion of prostate cancer cells by activating cAMP-dependent protein kinase (PKA) and promoting phosphorylation of tight junction proteins

Abstract The role of calcitonin (CT) and its receptor (CTR) in epithelial cancer progression is an emerging concept with great clinical potential. Expression of CT and CTR is frequently elevated in prostate cancers (PCs) and activation of CT-CTR axis in non-invasive PC cells induces an invasive phenotype. Invasive phenotype is acquired by the EMT, a process involving the loss of cell-cell adhesion and cytoskeletal rearrangement. Tight (TJs) and adherens junctions (AJs) are shown to play an important role in this process. Present studies focused on the actions of CTR-mediated intracellular events associated with TJ functionality and the induction of EMT. We have recently identified a type 1 PDZ-binding motif in CTR-C tail, and showed that CTR-C PDZ-binding motif interacts with the PDZ3 domain of ZO-1, a critical scaffolding protein of TJs. CTR-ZO-1 interaction is critical for proinvasive actions CTR on PC cells, and deletion of either CTR-C PDZ-binding motif abolishes the ability of PC cells to form distant metastases in orthotopic xenograft model. Therefore, we first examined the kinetics of CTR-ZO-1 interaction and the effect of CTR on TJ assembly. PC3 cells expressing either CTRwt or CTRαPDZ were acutely treated with CT, and subcellular distribution of TJ proteins was examined by immunocytochemistry and immunoblotting. Since CTR is coupled to stimulatory G protein, we examined effect of PKA inhibitors on CTR actions. Next, we tested whether CTR phosphorylates TJ proteins. Finally, we generated novel peptides and delivered them intracellularly to attenuate CTR-ZO-1 interaction. The results have shown that CTR-ZO-1 interaction occurs rapidly and peaks within 30 seconds of CT stimulus; this is rapidly followed by TJ disassembly and endocytosis of TJ proteins. Endocytosis of ZO-1 occurs first, which is followed with the endocytosis of occludin and claudin 3. We then identified that CTR-mediated TJ disassembly requires PKA-mediated phosphorylation of claudin 3 and ZO-1, and this phosphorylation may cause conformational changes and lead to TJ disassembly. Next, we demonstrated that CTR-mediated PKA phosphorylation must occur within TJ complex, and this spatio-temporal specificity of PKA activation is provided by the CTR-ZO-1 interaction. Finally, we have shown that CTR-ZO-1 interaction as well as CTR-stimulated invasion of PC cells can be attenuated by novel synthetic peptides that we have generated. For the first time, we report here the chain of PKA-mediated events that lead to the disassembly of tight junctions, loss of cell-cell adhesion, induction of EMT, invasion and metastasis of PC cells. We also provide a novel translational approach to generate synthetic peptides to attenuate these events. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4218. doi:1538-7445.AM2012-4218

Genomic Instability, Inflammation, and Cancer

Accumulating evidence supports that the inflammatory milieu plays a key role in the initiation and progression of epithelial cancer, the most characteristic example being that of ulcerative colitis which is associated with increased risk of colonic adenocarcinoma. Other less frequent and studied inflammatory lesions include asbestos-induced cellular damages, oral lichen planus (premalignant lesion), and cardiac myxoma. The mechanistic basis of this association is just beginning to be clarified. In the center of this picture lies the cytokine network which seems to link genomic instability, an evolving hallmark of cancer, with cancer development. In this special issue which deals with genomic instability, inflammation, and cancer, several research groups present evidence supporting this connection. H. Matsuzaki et al. have demonstrated a relationship between chronic inflammation and carcinogenesis through asbestos-induced cellular and molecular alteration of immunocompetent cells, resulting in a decline in tumor immunity. Induction of chronic inflammation in the areas of the lung, regional lymph nodes and the pleural cavity has been shown to be due to the production of reactive oxygen/nitrogen species. Cellular and molecular features of immunocompetent cells may be altered by asbestos fibers which, combined with the surrounding inflammation, eventually lead to decreased tumor immunity. M. Murata et al, have investigated the role of nitrative and oxidative DNA damage in inflammation-related carcinogenesis. In their work they revealed that infectious and noninfectious agents induce iNOS-dependent formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in cancer tissues and precancerous regions. Their results suggest that DNA base damage may lead to double-stranded breaks. They also demonstrated IL-6-modulated iNOS expression via STAT3 and EGFR in Epstein-Barr virus-associated nasopharyngeal carcinoma and found promoter hypermethylation in several tumor suppressor genes, while proposing 8-nitroguanine as potentially useful biomarkers for predicting the risk of inflammation-related cancers. I. S. Pateras et al. have investigated the presence of herpes simplex virus (HSV) DNA in a cohort of cardiac myxomas assessing the possibility that HSV infection might be involved in the development of these lesions with potential therapeutic applications. E. Georgakopoulou et al. have examined whether oral lichen planus exhibits malignant potential, with relation to the development of oral squamous cell carcinoma, and may therefore represent a model of preneoplastic inflammation. I. Aivaliotis et al. have reviewed the effects of cytokines as crucial components of inflammation, participating in the interaction between the cells of the tumor microenvironment, and have focused on their potential role in the development of genomic instability. This special issue of the Journal of Biomedicine and Biotechnology is devoted to the presentation of such inflammatory conditions with predisposition to genomic instability and cancer development. I hope that it will help stimulate research that is aimed at understanding and therapeutic targeting of these lesions. Vassilis G. Gorgoulis

Efficacy and Safety of Gefitinib or Platinum plus Taxane in Egfr-Mutant Advanced Non-Small Cell Lung Cancers: A Meta-Analysis of First-Line Randomized Controlled Trials

Purpose: The discovery of EGFR mutations renewed interest in lung cancer translational research since EGFR-dependent pathway plays an important role in the development and progression of human epithelial cancers, including non-small cell lung cancer (NSCLC). The present meta-analysis was performed to review the recent advances with the selective oral EGFR-TK inhibitor gefitinib among EGFR mutation positive patients with NSCLC. Methods: Using the keywords “gefitinib” and “lung cancer” MEDLINE was searched. The primary reports of interest were the randomized controlled trials in NSCL published in peer-reviewed journals. Three recent studies concerning the effect of gefitinib in NSCLC were identified to be relevant for the meta-analysis based on their similarity in terms of study design. PFS and objective response rate (ORR) in gefitinib and platinum/taxane combination were compared by the Hazard ratio (HR) or Odds Ratio (OR) of meta-analysis. Results: The HR (95%CI) of the meta-analysis of 0.41 (0.34 - 0.49) demonstrated that in patients EFGR mutation positive patients, PFS was significantly longer among those who received gefitinib compared to platin derivative/taxane combination. Furthermore, OR of the meta-analysis for ORR was 3.83 (2.72 - 5.40). While hematological toxicity was observed in the majority of the taxane/platinum group, major adverse events in gefitinib patients were skin rashes/acnea, dry skin, elevated liver enzymes and diarrhea. Conclusions: This meta-analysis strongly confirms the efficacy and better tolerability of gefitinib in EGFR positive NSCLC, and the importance of EGFR mutation testing in order to plan first-line treatment in routine clinical practice in NSCLC.

Structural Basis of Cooperativity in Human UDP-Glucose Dehydrogenase

BackgroundUDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics.MethodologyPreviously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle.ConclusionIn all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD+ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD+ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.

Polymorphonuclear granulocytes in human head and neck cancer: Enhanced inflammatory activity, modulation by cancer cells and expansion in advanced disease

The progression of epithelial cancer is associated with an intense immunological interaction between the tumor cells and immune cells of the host. However, little is known about the interaction between tumor cells and polymorphonuclear granulocytes (PMNs) in patients with head and neck squamous cell carcinoma (HNSCC). In our study, we investigated systemic PMN-related alterations in HNSCC, the role of tumor-infiltrating PMNs and their modulation by the tumor microenvironment. We assessed the infiltration of HNSCC tissue by PMNs (retrospectively) and systemic PMN-related alterations in blood values (prospectively) in HNSCC patients (n = 99 and 114, respectively) and control subjects (n = 41). PMN recruitment, apoptosis and inflammatory activity were investigated in an in vitro system of peripheral blood PMNs and a human HNSCC cell line (FaDu). HNSCC tissue exhibited considerable infiltration by PMNs, and strong infiltration was associated with poorer survival in advanced disease. PMN count, neutrophil-to-lymphocyte ratio and serum concentrations of CXCL8 (interleukin-8), CCL4 (MIP-1β) and CCL5 (RANTES) were significantly higher in the peripheral blood of HNSCC patients than in that of controls. In vitro, HNSCC-conditioned medium inhibited apoptosis of PMNs, increased chemokinesis and chemotaxis of PMNs, induced release of lactoferrin and matrix metalloproteinase 9 by PMNs and enhanced the secretion of CCL4 by PMN. Our findings demonstrate alterations in PMN biology in HNSCC patients. In vitro, tumor-derived factors modulate cellular functions of PMNs and increase their inflammatory activity. Thus, the interaction between HNSCC and PMNs may contribute to host-mediated changes in the tumor microenvironment.

PRSS14/Epithin is induced in macrophages by the IFN-γ/JAK/STAT pathway and mediates transendothelial migration

PRSS14/Epithin (also known as matriptase and ST14), a member of the type II transmembrane serine proteases, is primarily found in a subpopulation of normal epithelial cells and in epithelial cancers. Its known functions include maintaining the epithelial barrier, thymic development, and cancer progression. In this study, we show that several macrophage cell lines and activated bone marrow-derived macrophages also express PRSS14/Epithin. Surface expression, as well as cytoplasmic expression, was detectable upon activation by IFN-γ, but not TNF-α or TGF-β. Induction of the protein appeared to be restricted to macrophages. IFN-γ showed a biphasic regulation in RAW264.7 cells, and upregulated expression was sustained for several days. This induction by IFN-γ was partially through the increase of PRSS14/Epithin mRNA production, which is downstream of the JAK pathway, shown by the inhibition by tyrphostin AG490. Using chromatin immunoprecipitation, we verified that two sites among six putative STAT1 binding sites in the PRSS14/Epithin promoter were occupied by STAT1 upon activation. Treatment with IFN-γ enhanced the serum-triggered transendothelial migration of RAW264.7 cells, but not that of PRSS14/Epithin knock-down RAW264.7 cells, although they express multiple markers such as ICAM1, CD80, and CD40 at normal levels. These data strongly suggest that PRSS14/Epithin plays an important role in the transendothelial migration of activated macrophages in the inflammatory microenvironment, and the mode of action is similar to the events in cancer metastasis.

NOX4-dependent ROS production by stromal mammary cells modulates epithelial MCF-7 cell migration

Background:The influence of the stromal microenvironment on the progression of epithelial cancers has been demonstrated. Unravelling the mechanisms by which stromal cells affect epithelial behaviour will contribute in understanding cellular malignancy. It has been proposed that redox environment has a role in the acquisition of malignancy. In this work, we studied the influence of epithelial cells on the stromal redox status and the consequence of this phenomenon on MCF-7 cell motility.Methods:We analysed in a co-culture system, the effect of RMF-EG mammary stromal cells on the migratory capacity of MCF-7 cell line. To test whether the NOX-dependent stromal redox environment influences the epithelial migratory behaviour, we knocked down the expression of NOX4 using siRNA strategy. The effect of TGF-β1 on NOX4 expression and activity was analysed by qPCR, and intracellular ROS production was measured by a fluorescent method.Results:Migration of MCF-7 breast epithelial cells was stimulated when co-cultured with RMF-EG cells. This effect depends on stromal NOX4 expression that, in turn, is enhanced by epithelial soluble factors. Pre-treatment of stromal cells with TGF-β1 enhanced this migratory stimulus by elevating NOX4 expression and intracellular ROS production. TGF-β1 seems to be a major component of the epithelial soluble factors that stimulate NOX4 expression.Conclusions:Our results have identified that an increased stromal oxidative status, mainly provided by an elevated NOX4 expression, is a permissive element in the acquisition of epithelial migratory properties. The capacity of stromal cells to modify their intracellular ROS production, and accordingly, to increase epithelial motility, seems to depend on epithelial soluble factors among which TGF-β1 have a decisive role.

Abstract B18: Expression of CTIP2/Bcl11b is linked to disease progression in head and neck cancer and is a critical mediator of epithelial cell migration and motility during wound healing processes

Abstract We have previously shown that transcriptional regulator Ctip2 controls epidermal homeostasis during skin morphogenesis and plays a crucial role in epidermal permeability barrier (EPB) formation (1,2). In a recent study we have shown that CTIP2 expression is upregulated in human head and neck tumors and in atopic dermatitis (3,4). It is known that “cancers are wounds that do not heal” and genes implicated in wound healing are known to influence cancer progression. Interestingly, Ctip2 expression was also induced in mouse skin following tape stripping (mild injury by abrasion) and in cutaneous full thickness wounds. We therefore investigated function of Ctip2 during cutaneous wound healing using both in vitro scratch assay and in vivo full thickness wound healing assay. In the present study we looked at the Ctip2 expression levels by immunohistochemical (IHC) and western blot on wild-type mice skin after inducing mild abrasion injury by tape stripping and in full thickness wounds by punch biopsy. We have also analyzed the effect of Ctip2 deletion in the epidermis of mice skin using Ctip2ep-/- mice in these two processes using histology, immunohistochemistry (IHC), and Western blotting. Our data on in vitro scratch migration assay demonstrated a delay in keratinocyte migration and wound closure in mutant keratinocytes. Similarly, healing of cutaneous wounds was significantly delayed by 2-3 days in Ctip2ep-/- mice (selectively lacking the gene for Ctip2 in epidermal keratinocytes) in comparison to wild-type control (Ctip2L2/L2) mice where wounds closed much faster. Histological analyses of skin wounds revealed that the distance between the two migratory tongues of epithelial cells was larger in the mutant mice [1.2 fold (p<0.001)] compared to the wild-type controls (Ctip2L2/L2) suggesting a possible role of Ctip2 in re-epithelialization process probably effecting the keratinocyte migration similar to what was seen in our in vitro data on scratch migration assay. Immunohistochemistry (IHC) and western blotting confirmed a persistant expression of PCNA and the delayed induction of both K16 and K6 (markers of activated keratinocytes) in the epidermal keratinocytes from the mutant wounds compared to control mice wounds suggesting a possible delay in the onset of restoration of differentiation in the newly formed epidermis of Ctip2ep-/- mutant mice. Altogether, we showed for the first time that keratinocytic Ctip2 modulated cutaneous wound healing by affecting the re- epithelialization and differentiation processes. Identification of genes regulated by Ctip2 during wound healing which are also involved in epithelial cancer progression is expected to improve diagnosis and treatment of human epithelial cancers.

Soluble epithin/PRSS14 secreted from cancer cells contains active angiogenic potential.

Epithin (PRSS14/matriptase/ST14), a type II membrane protein, is involved in progression of epithelial cancers and metastasis as well as in the normal epidermal barrier function. When activated, it translocates into the cell-cell contacts and sheds into media. In order to understand the specific mechanism during tumor progression, we tested the angiogenic potential of secreted form of epithin. Epithin produced from the cancer cells shed more in hypoxia and induced motility of endothelial cells. Epithin enhanced the migration and invasion of mouse and bovine endothelial cells without cell proliferation. Furthermore, soluble epithin induced endothelial differentiation in the assay of the human endothelial microvessel-like tube formation and in that of the chicken chorioallantoic membrane. The knock-down of epithin in the 427 thymoma cell line abolished the protease activity of secreted epithin fraction, reduced the invasion of endothelial cells through matrigel, and tube formation activity. Only specific antibodies abolished the migration of endothelial cell and the vessel morphogenesis, suggesting that epithin specifically functions in these systems. Therefore, we propose that the secreted epithin in the hypoxic cancer microenvironment plays a role as a proangiogenic factor, and can be modulated with specific antibodies.

Abstract 2302: Knockdown of ZEB1, a master epithelial-to-mesenchymal transition (EMT) inducing gene, suppresses growth of pleural mesothelioma cell lines

Abstract Background and Objective: Malignant pleural mesothelioma (MPM) is a highly aggressive disease, usually caused by asbestos exposure. The underlying molecular mechanism contributing to aggressive phenotype of MPM remains to be fully elucidated. Epithelial-to-mesenchymal transition (EMT), an early developmental program, causes a wide variety of human epithelial cancers to have the ability to invade surrounding tissues and metastasize to distant sites. MPM cells usually exhibit mesenchymal characteristics probably because of their mesodermal origin. Such characteristics include fibroblast-like morphology and expression of master EMT-inducing genes. Thus, EMT may contribute to aggressive phenotype of MPM. Among EMT-inducing genes, ZEB1, a repressor of E-cadherin, has emerged as a key player in the progression of several epithelial cancers. To explore the potential of ZEB1 as a therapeutic target for MPM, we examined the effect of ZEB1 knockdown on growth of MPM cell lines. In addition, we evaluated the effect of ZEB1 knockdown on the levels of IL-6, which is known to induce growth of mesothelioma cells. Materials and Methods: 18 human MPM cell lines and one non-tumorigenic mesothelial cell line were used. Transient and stable knockdown of ZEB1 were done in two MPM cell lines. Quantitative real-time RT-PCR and western blot of ZEB1, E-cadherin, and Vimentin were done. Soluble IL-6 in cellular supernatants was measured by enzyme-linked immunosorbent assay. Cell proliferation was measured by WST-1 and clonogenic growth was measured by liquid and soft agar colony formation assays. FACS with PI staining was done to examine apoptosis and cell cycle. Apoptosis was also evaluated by western blot of cleaved caspase-3. Results: The majority of MPM cells expressed higher levels of ZEB1 than a non-tumorigenic mesothelial cell. We performed ZEB1 knockdown experiments in two MPM cells, ACC-MESO-1(MESO-1) and H2052, which express high and moderate levels of ZEB1. Transient knockdown of ZEB1 caused MESO-1 but not H2052 cells to reexpress E-cadherin. Transient ZEB1 knockdown suppressed cell proliferation and liquid colony formation in the two lines. Most importantly, the ZEB1 knockdown dramatically suppressed soft agar colony formation in the two lines. We did not see apoptosis or cell cycle arrest in either of the two lines. Stable knockdown of ZEB1 induced morphologic changes suggestive of mesenchymal-to-epithelial transition (MET) in MESO-1 cells but not in H2052 cells. ZEB1 stable knockdown resulted in a decreased IL-6 production in MESO-1 cells, suggesting that growth inhibition by ZEB1 knockdown may in part due to decreased IL-6 production. Conclusion: These results suggest that ZEB1 serves as an attractive therapeutic target for MPM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2302.

Abstract 1428: Necessary contribution of wild-type stroma to prostate tumorigenesis

Abstract Introduction: A previously described mouse model, Tgfbr2-fspKO, with a conditional stromal knockout of the TGF-beta type II receptor (Tgfbr2), developed prostatic intra-epithelial neoplasia (PIN) lesions and subsequently progressed to prostate adenocarcinoma. Tissue recombination of wild-type (WT) prostatic organoids with 100% Tgfbr2-floxE2/floxE2 prostatic stroma resulted in grafts with normal prostatic glandular architecture, whereas recombination with 100% Tgfbr2-fspKO stroma resulted in grafts with PIN lesions. Interestingly, only grafts made with a mixture of WT and KO stroma resulted in prostatic adenocarcinoma. Furthermore, loss of stromal Tgfbr2 expression occurs in a proportion of human prostate cancer tissues. The objective of the study was to elucidate the mechanism(s) contributing to the cancer progression in the context of a mixed heterogeneous stroma. Method: Prostate stromal-epithelial interactions were described by a hybrid computational model involving a hypothesized 2-step mechanism (initial transformation followed by invasion), where these steps are controlled by stromal-derived paracrine morphogenic factors. Biological parameters for the candidate morphogens (Wnt3a and SDF-1/CXCL12) were assessed by qPCR and ELISA from stromal mixing co-culture experiments. Results: Modeling indicated that the expression of the initiating morphogen increases as a function of the % of Tgfbr2-KO stromal cells. The incidence of invasion promoted by the pro-invasive morphogen was biphasic with a 50/50 mixture of WT and Tgfbr2-KO stromal cells resulting in maximal cancer progression. Wnt3a is produced at 4-fold higher levels in Tgfbr2-KO vs WT stroma and antibody mediated depletion of Wnt3a from stromal conditioned media reduced the growth rate of LNCaP cells. SDF-1 is produced at nearly 4-fold higher levels in WT vs. Tgfbr2-KO stroma and SDF-1 mRNA expression is synergistically increased when WT and Tgfbr2-KO stroma are co-cultured. Additionally, CD90 expression is heterogeneously expressed by stromal tissues associated with primary human prostate cancer as compared to benign tissue as demonstrated by immunofluorescence staining. Conclusions: The model and data support the importance of stromal heterogeneity in the development of prostatic adenocarcinoma in two independent tumorigenic steps. Each step is facilitated by a different stromal component, so that stromal heterogeneity results in the maximal tumorigenesis. Tgfbr2-KO stromal cells produce an altered cytokine profile that drives the proliferation of the epithelial compartment and likely primes the WT stromal cells to produce additional cytokines, like SDF-1, that promote epithelial invasion and cancer progression. While most studies tend to focus on the altered stroma, this study suggests that the WT stroma cells play a necessary and important role required as a rate-limiting step for prostate cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1428.

Novel Therapies Against Aggressive and Recurrent Epithelial Cancers by Molecular Targeting Tumor- and Metastasis-Initiating Cells and Their Progenies

A growing body of experimental evidence has revealed that the highly tumorigenic cancer stem/progenitor cells endowed with stem cell-like properties might be responsible for initiation and progression of numerous aggressive epithelial cancers into locally invasive, metastatic and incurable disease states. The malignant transformation of tissue-resident adult stem/progenitor cells or their progenies into tumorigenic and migrating cancer stem/progenitor cells and their resistance to current cancer therapies have been associated with their high expression levels of specific oncogenic products and drug resistance-associated molecules. In this regard, we describe the tumorigenic cascades that are frequently activated in cancer stem/progenitor cells versus their differentiated progenies during the early and late stages of the epithelial cancer progression. The emphasis is on the growth factor signaling pathways involved in the malignant behavior of prostate and pancreatic cancer stem/progenitor cells and their progenies. Of clinical interest, the potential molecular therapeutic targets to eradicate the tumor- and metastasis-initiating cells and their progenies and develop new effective combination therapies against locally advanced and metastatic epithelial cancers are also described.

Human steroidogenic factor-1 (hSF-1) regulates progesterone biosynthesis and growth of ovarian surface epithelial cancer cells

The majority of cancers derived from ovarian surface epithelial (OSE) cells are lethal. Estrogens promote proliferation of OSE cells, whereas progesterone inhibits proliferation and promotes apoptosis of OSE cells. Human steroidogenic factor-1 (hSF-1) induction of the steroidogenic acute regulatory protein ( StAR) gene, and the steroidogenic enzymes CYP11A1 and HSD3B2 is central to progesterone biosynthesis. Whereas hSF-1 and StAR are expressed in human ovarian surface epithelial (HOSE) cells, hSF-1 and StAR protein were not expressed in a panel of malignant ovarian cancer cell lines (SKOV-3, BG-1, and Caov-3), and in human OSE cells immortalized by SV40 large T antigen (IOSE-121). Transient expression of hSF-1 in SKOV-3 cells activated the expression of StAR, p450scc and 3βHSD-II mRNAs, and induced progesterone biosynthesis. Additionally, hSF-1 suppressed proliferation and promoted apoptosis of SKOV-3 cells and suppressed SKOV-3 cell growth induced by ERα and estradiol. These findings suggest that hSF-1 is central to progesterone biosynthesis in OSE cells. Human SF-1 may decrease OSE cancer cell numbers directly by apoptosis, and indirectly by opposing estradiol-induced proliferation. These findings are consistent with the hypothesis, that down-regulation of hSF-1 contributes to progression of ovarian epithelial cancers.

A computational approach to resolve cell level contributions to early glandular epithelial cancer progression

BackgroundThree-dimensional (3D) embedded cell cultures provide an appropriate physiological environment to reconstruct features of early glandular epithelial cancer. Although these are orders of magnitude simpler than tissues, they too are complex systems that have proven challenging to understand. We used agent-based, discrete event simulation modeling methods to build working hypotheses of mechanisms of epithelial 3D culture phenotype and early cancer progression. Starting with an earlier software analogue, we validated an improved in silico epithelial analogue (ISEA) for cardinal features of a normally developed MDCK cyst. A set of axiomatic operating principles defined simulated cell actions. We explored selective disruption of individual simulated cell actions. New framework features enabled recording detailed measures of ISEA cell activities and morphology.ResultsEnabled by a small set of cell operating principles, ISEA cells multiplied and self-organized into cyst-like structures that mimicked those of MDCK cells in a 3D embedded cell culture. Selective disruption of "anoikis" or directional cell division caused the ISEA to develop phenotypic features resembling those of in vitro tumor reconstruction models and cancerous tissues in vivo. Disrupting either process, or both, altered cell activity patterns that resulted in morphologically similar outcomes. Increased disruption led to a prolonged presence of intraluminal cells.ConclusionsISEA mechanisms, behaviors, and morphological properties may have biological counterparts. To the extent that in silico-to-in vitro mappings are valid, the results suggest plausible, additional mechanisms of in vitro cancer reconstruction or reversion, and raise potentially significant implications for early cancer diagnosis based on histology. Further ISEA development and use are expected to provide a viable platform to complement in vitro methods for unraveling the mechanistic basis of epithelial morphogenesis and cancer progression.

161 Combining the adhesion pathway inhibition with radiotherapy – from the bench to the clinic

The mechanisms involved in the epithelial to mesenchymal transition (EMT) are integrated in concert with master developmental and oncogenic pathways regulating in tumor growth, angiogenesis, metastasis, as well as the reprogrammation of specific gene repertoires ascribed to both epithelial and mesenchymal cells. Consequently, it is not unexpected that EMT has profound impacts on the neoplastic progression, patient survival, as well as the resistance of cancers to therapeutics (taxol, vincristine, oxaliplatin, EGF-R targeted therapy and radiotherapy), independent of the “classical” resistance mechanisms linked to genotoxic drugs. New therapeutic combinations using genotoxic agents and/or EMT signaling inhibitors are therefore expected to circumvent the chemotherapeutic resistance of cancers characterized by transient or sustained EMT signatures. Thus, targeting critical orchestrators at the convergence of several EMT pathways, such as the transcription pathways NF-κB, AKT/mTOR axis, MAPK, β-catenin, PKC and the AP-1/SMAD factors provide a realistic strategy to control EMT and the progression of human epithelial cancers. Several inhibitors targeting these signaling platforms are already tested in preclinical and clinical oncology. In addition, upstream EMT signaling pathways induced by receptor and nonreceptor tyrosine kinases (e.g. EGF-R, IGF-R, VEGF-R, integrins/FAK, Src) and G-protein-coupled receptors (GPCR) constitute practical options under preclinical research, clinical trials or are currently used in the clinic for cancer treatment: e.g. small molecule inhibitors (Iressa: targeting selectively the EGF-R; CP-751,871, AMG479, NVP-AEW541, BMS-536924, PQIP, AG1024: IGF-R; AZD2171, ZD6474: VEGF-R; AZD0530, BMS-354825, SKI606: Src; BIM-46174: GPCR; rapamycin, CCI-779, RAD-001: mTOR) and humanized function blocking antibodies (Herceptin: ErbB2; Avastin: VEGF-A; Erbitux: EGF-R; Abegrin: αvβ3 integrins). We can assume that silencing RNA and adenovirus-based gene transfer of therapeutic miR and dominant interferring expression vectors targeting EMT pathways and signaling elements will bring additional ways for the treatment of epithelial cancers. Identification of the factors that initiate, modulate and effectuate EMT signatures and their underlying upstream oncogenic pathways should provide the basis of more efficient strategies to fight cancer progression as well as genetic and epigenetic forms of drug resistance. This goal can be accomplished using global screening of human clinical tumors by EMT-associated cDNA, proteome, miRome, and tissue arrays.