Epithelial Cell Tumorigenesis Research Articles

Abstract 5635: TPM4 overexpression promotes colon epithelial cell tumorigenesis in vitro and correlates with human colon tumor progression in vivo

Abstract Because colon cancer still exerts a heavy burden on human health, we are interested in identifying very early events in colon epithelial cell tumorigenesis in order to develop strategies to treat and possibly prevent colon carcinogenesis. We hypothesize that aberrant expression of genes involved in crypt-luminal colon epithelial cell migration, needed for proliferative, undifferentiated stem cells at the crypt bottom to mature into growth arrested, mature effector cells near the lumen, may disrupt normal differentiation and initiate events that can trigger the transition from a normal to premalignant state in colon epithelial cells. Forced upregulation of tropomyosin 4 (TPM4), an actin-binding protein that exerts many effects on contractility and migration, prevents certain aspects of Caco2 maturation induced by contact inhibition of growth, including increased expression of the digestive enzyme sucrose isomaltase and glutathione S-transferase alpha 1. Furthermore, TPM4.2-overexpressing cells exhibit numerous morphological abnormalities. Compared to untransfected Caco2 cells and empty-vector stable transfectant controls, TPM4.2-overexpressing Caco2 cell clones elaborate aberrant thin membrane protrusions and expanded membrane ruffles. Particularly striking are defective maintenance of dome structures at middle (day 14) and late (day 21) time points of induced differentiation in TPM4.2-overeexpressing clones. We further hypothesize that TPM4 is overexpressed in human colon tumors, particularly at early stages of tumorigenesis. In order to test this hypothesis, we examined TPM4 RNA expression in datasets obtained from human colon tissue at premalignant as well as tumorigenic stages. TPM4 trends toward overexpression in premalignant, relative to normal, human colon tissue (uninvolved mucosa from patients with familial adenomatous polyposis, or FAP) and is significantly overexpressed in adenocarcinoma tissue relative to normal controls. Interestingly, TPM4 overexpression may be more pronounced at earlier, rather than later, stages of adenocarcinoma. Our results suggest that aberrant TPM4 overexpression in colon epithelial cells can promote adoption of a tumorigenic state. Certain aspects of Caco2 cell maturation are disrupted by forced TPM4 overexpression during induced differentiation, and TPM4 overexpression correlates with early stages of colon tumorigenesis. Misregulation of TPM4 gene expression may therefore represent a critical molecular event that can block normal differentiation and induce early events in colon epithelial cell tumorigenesis. TPM4 expression level may therefore prove to be a useful biomarker that can, along with other diagnostics, help to determine tumorigenic progression in human colon tissue and to develop novel strategies to prevent colon cancer and treat it at early stages. Citation Format: Giulio Ferrero, Barbara Pardini, Alessio Naccarati, Michael Papetti. TPM4 overexpression promotes colon epithelial cell tumorigenesis in vitro and correlates with human colon tumor progression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5635.

Abstract 1487: TPM4 overexpression promotes colon epithelial cell tumorigenesis

Abstract Although much progress has been made in colon cancer treatment and prevention, high morbidity and mortality still associated with this disease are tremendous burdens to human health. Therefore, novel strategies are needed to provide insight into how colon cancer can be prevented and treated, particularly before malignancy. One approach is to identify genes whose misregulation disrupts crypt-luminal colon epithelial cell migration, needed for stem cell progeny at the bottom of the crypt to transition from a highly proliferative, immature state to a growth arrested, mature phenotype near the lumen. Aberrant expression of these genes may contribute to early stages of tumorigenesis, before adoption of a malignant state. Tropomyosins, including tropomyosin 4 (TPM4), are actin-binding proteins that regulate contractility and migration in many cell types. We have shown that TPM4 RNA is overexpressed in premalignant (flat mucosa) of FAP (Familial Adenomatous Polyposis) patients 2.59 fold over the level in normal controls. Furthermore, TPM4 RNA and protein is highly overexpressed in proliferating, undifferentiated Caco2 (human adenocarcinoma) cells relative to growth-arrested, differentiated cells. Therefore, we hypothesize that TPM4 overexpression can block colon epithelial cell differentiation and induce early, premalignant events in colon epithelial cell tumorigenesis. To test this hypothesis, we determined whether Caco2 cell clones stably overexpressing TPM4.2, the TPM4 isoform expressed in Caco2 cells, are defective in the ability to adopt a mature phenotype measured by RNA expression of differentiation-specific genes, including intestinal alkaline phosphatase (ALPi), upon induction of spontaneous differentiation using quantitative real-time polymerase chain reaction (qRT-PCR). Stable TPM4.2 transfectants exhibited a 2.61-fold decreased expression of intestinal alkaline phosphatase (ALPi) relative to control transfectants after 20-21 days of spontaneous induction. Lgr5 RNA was quickly suppressed in both TPM4.2 and control transfectants upon induction of differentiation but was re-expressed in TPM4.2 transfectants 12.3 fold above control levels at day 14 of spontaneous induction. Our results suggest that proper regulation of TPM4 expression in colon epithelial cells is critical to preventing adoption of a tumorigenic state. TPM4 RNA is overexpressed in premalignant human colon tissue, and forced overexpression of TPM4 suppresses maturation and promotes proliferation in an in vitro model of colon epithelial cell differentiation. Misregulation of TPM4 gene expression may therefore block normal differentiation and induce early events in colon epithelial cell tumorigenesis. Based on these studies, we suggest that TPM4 expression levels may be used as diagnostic tools for determining tumorigenic potential and may offer therapeutic strategies for early intervention in colon cancer treatment. Citation Format: Rajsumeet Macwan, Piotr B. Kozlowski, Michael Papetti. TPM4 overexpression promotes colon epithelial cell tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1487.

Co-Expression of Coxsackievirus/Adenovirus Receptors and Desmoglein 2 in Lung Adenocarcinoma: A Comprehensive Analysis of Bioinformatics and Tissue Microarrays.

Introduction: Coxsackievirus/adenovirus receptors (CARs) and desmoglein-2 (DSG2) are similar molecules to adenovirus-based vectors in the cell membrane. They have been found to be associated with lung epithelial cell tumorigenesis and can be useful markers in predicting survival outcome in lung adenocarcinoma (LUAD). Methods: A gene ontology enrichment analysis disclosed that DSG2 was highly correlated with CAR. Survival analysis was then performed on 262 samples from the Cancer Genome Atlas, forming “Stage 1A” or “Stage 1B”. We therefore analyzed a tissue microarray (TMA) comprised of 108 lung samples and an immunohistochemical assay. Computer counting software was used to calculate the H-score of the immune intensity. Cox regression and Kaplan–Meier analyses were used to determine the prognostic value. Results: CAR and DSG2 genes are highly co-expressed in early stage LUAD and associated with significantly poorer survival (p = 0.0046). TMA also showed that CAR/DSG2 expressions were altered in lung cancer tissue. CAR in the TMA was correlated with proliferation, apoptosis, and epithelial–mesenchymal transition (EMT), while DSG2 was associated with proliferation only. The Kaplan–Meier survival analysis revealed that CAR, DSG2, or a co-expression of CAR/DSG2 was associated with poorer overall survival. Conclusions: The co-expression of CAR/DSG2 predicted a worse overall survival in LUAD. CAR combined with DSG2 expression can predict prognosis.

Computational design of antagonist peptides based on the structure of secreted frizzled-related protein-1 (SFRP1) aiming to inhibit Wnt signaling pathway

Aberrant activation of Wnt/β-catenin signaling pathway, due to the genetic or epigenetic changes, is responsible for tumorigenesis in epithelial cells of different types of cancer such as colorectal cancer. Secreted Frizzled-Related Protein-1 (SFRP1), as one of the antagonist proteins of this pathway, is hyper-methylated in colorectal cancer leading to the formation of Wnt-Fz-LRP and activation of Wnt/β-catenin signaling pathway. We aimed to design antagonist peptides based on SFRP1 structure against wingless-type 2 (Wnt2), a highly expressed ligand in different cancers like colorectal cancer, to inhibit the formation of the initial triple complex of Wnt-Fz-LRP. After homology modeling of SFRP1, molecular docking showed that Wnt2 and SFRP1 interact in the same mode of xWnt8-mFz8 and hWnt3-mFz8 through the thumb and finger binding sites. These binding sites were selected for designing peptides using either substitution or deep learning-based approaches. The efficiency of each designed peptide in interacting with Wnt2 was evaluated by molecular docking. Stability assessment of Wnt2-peptide complexes via molecular dynamic (MD) revealed that the designed peptides could effectively interact with Wnt2 binding sites during the simulation. However, the designed peptides against the thumb site had higher binding affinity and hydrogen bonds compared to the initial sequence. The secondary structure of the designed peptides indicated an alpha-helix structure which is a favorable structure for peptide drugs. Computing the physicochemical properties of peptides predicted a fairly acceptable structure which made them promising candidates in the treatment of cancers like CRC.

Identification of pathways and genes in the process of E6/E7-induced carcinogenesis of esophageal epithelial cells.

Human papillomavirus (HPV) infection was associated with some carcinomas, especially malignant tumors in upper digestive tract, upper respiratory tract, and genitourinary system. The mechanism of the viral transformation of normal cells is still not very clear. To investigate the tumorigenesis of epithelial cells, E6/E7-induced malignant transformation model cells were used for expression profiling analysis by performing RNA expression microarray detection. Bioinformatics analysis was applied to investigate the cellular process changes along with the E6/E7 expression in SHEE cells. The differentially expressed genes were further grouped and uploaded for Search Tool for the Retrieval of Interacting Genes analysis. The protein-protein interaction results were visualized. The hub genes and their first-neighbors genes were selected, followed by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The obtained results demonstrated that tumor-related biological processes began to emerge during the carcinogenesis process from 48th passage to 76th passage of SHEE cells after E6/E7 expression. Ten hub genes were identified and analyzed during the E6/E7-induced tumorigenesis. This study explores the gene expression network in the progressive transformation of immortalized esophageal epithelial cells induced by E6/E7 expression. Understanding the biological processes and hub genes that first appear during the transformation will provide some clues to the mechanism of E6/E7-induced carcinogenesis of esophageal epithelial cells.

Role of tropomyosin 4 gene misregulation in colon epithelial cell tumorigenesis.

e15184 Background: Ordered migration of colon epithelial cells from the bottom to the top of the crypt is required for their differentiation. Because aberrant colon cell migration may contribute to early stages of colon tumorigenesis, elucidation of how this migration is regulated provides insight into how colon cancer can be prevented and treated before malignancy. Certain actin-binding tropomyosins, including tropomyosin 4 (TPM4), regulate cytoskeletal contractility and migration in many different cell types. Methods: To determine whether aberrant TPM4 regulation may be an early event in colon cell tumorigenesis, we measured TPM4 RNA expression in uninvolved, grossly normal colon tissue from 10 human familial adenomatous polyposis (FAP) patients genetically predisposed to colon cancer versus 7 patients not genetically predisposed (normal). We also examined TPM4 expression in a colon adenocarcinoma cell line (Caco2). These are highly proliferative, undifferentiated cells but can be induced to growth arrest and differentiate, thus acquiring many characteristics of normal absorptive colon epithelial cells. Reporter assays were used to identify sequences that regulate TPM4 gene expression in Caco2 cells. Results: Average TPM4 RNA expression is 2.59 fold higher in the FAP versus normal tissue. TPM4 RNA and protein are highly expressed in proliferating, undifferentiated Caco2 cells but are severely reduced in growth arrested, differentiated cells. Reporter assays have identified genetic regions, near the TPM4 transcriptional start site, that may regulate TPM4 gene expression in proliferating Caco2 cells. Conclusions: This data suggests that aberrantly upregulated TPM4 may be an early event in colon tumorigenesis and that normal TPM4 expression, by fostering ordered colon epithelial cell migration, may promote normal cell differentiation and mitigate adoption of a premalignant state.

Perspective: Contribution of Epstein-Barr virus (EBV) Reactivation to the Carcinogenicity of Nasopharyngeal Cancer Cells.

Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma derived from the epithelium of the post-nasal cavity, with a unique geographic and ethnic distribution. Epstein–Barr virus (EBV) is an etiological agent of NPC, but how it contributes to carcinogenesis is not completely clear. Although it is thought that EBV latency participates in the development of NPC, increasing evidence reveals that the lytic cycle also plays an important role in the carcinogenic process. In this review, we summarize our recent studies on how EBV reactivation causes genomic instability and accelerates tumorigenesis in epithelial cells. The roles of three lytic genes, namely, BRLF1, BGLF5 and BALF3, in this process are also introduced. Moreover, blocking EBV reactivation using natural compounds may help delay the progression of NPC tumorigenesis. These studies provide a new insight into NPC carcinogenesis and raise the possibility that inhibition of EBV reactivation may be a novel approach to prevent the relapse of NPC.

Tumor-associated myeloid cells promote tumorigenesis of non-tumorigenic human and murine prostatic epithelial cell lines

The etiology of prostate cancer is poorly understood, but it is a multi-step process that has been linked to environmental factors that induce inflammation within the gland. Glands of prostate cancer patients frequently contain multiple zones of disease at various stages of progression. The factors that drive disease progression from an indolent benign stage to aggressive disease are not well-defined. Prostate inflammation and carcinoma are associated with high levels of myeloid cell infiltration; these cells are linked to disease progression in other cancers, but their role in prostate cancer is unclear. To determine whether myeloid cells contribute to prostate cancer progression, the ability of prostate tumor-associated CD11b+ cells (TAMC) to drive prostate epithelial cell tumorigenesis was tested. Co-culture of CD11b+ TAMC with non-tumorigenic genetically primed prostate epithelial cells resulted in stable transformation and induction of tumorigenesis. RNA sequencing identified the IL-1α pathway as a potential molecular mechanism responsible for tumor promotion by TAMC. Inhibition of IL-1α delayed growth of TAMC-induced tumors. Further analysis showed that IL-1α inhibition led to decreased angiogenesis within tumors, suggesting that IL-1α promotes prostate tumor progression, potentially through augmentation of angiogenesis.

The B-cell tumor promoter Bcl-3 suppresses inflammation-associated colon tumorigenesis in epithelial cells.

Bcl-3 is an atypical member of the IκB family. It associates with p50/NF-κB1 and p52/NF-κB2 homodimers in nuclei where it modulates transcription in a context-dependent manner. A subset of B cell tumors exhibits recurrent translocations of Bcl-3, resulting in overexpression. Elevated expression without translocations is also observed in various B cell lymphomas and even some solid tumors. Here we investigated the role of Bcl-3 in AOM/DSS-induced colon tumors, a mouse model for colitis-associated colorectal cancers in humans. Contrary to expectations, Bcl-3 suppressed colorectal tumor formation: Bcl-3-deficient mice were relatively protected from DSS-induced epithelial damage and developed more polyps after AOM/DSS treatment, though polyp size was unaffected. DSS-challenged mutant mice exhibited increased recruitment of myeloid-derived suppressor cells (MDSCs), consistent with protection of the epithelium. Loss of Bcl-3 in intestinal epithelial cells was sufficient to increase tumorigenesis. The added tumor burden in mutant mice was dependent on TNFα, a tumorigenic, NF-κB-mediated signaling pathway that was dampened by Bcl-3. These findings reveal a tumor-suppressive role for Bcl-3 in this inflammation-associated cancer model. Bcl-3 thus functions as a tumor promoter or suppressor, depending on the cellular and environmental context.

Su1863 CD177+ Neutrophils Preventing Epithelial Cell Tumorigenesis through Promotion of Migration and Cytotoxicity

Su1863 CD177+ Neutrophils Preventing Epithelial Cell Tumorigenesis through Promotion of Migration and Cytotoxicity

The oncogenicity of Epstein-Barr virus: being “more or less” is to be concerned

Epstein-Barr virus (EBV) ubiquitously infects human beings and is associated with several malignancies. However, the mechanism of the viral oncogenicity remains largely to be understood. In our investigations, we have reported a tumor model in which that EBV facilitated the tumorigenesis in epithelial cells latently infected by EBV genome. Using this model, we have found several clues and further confirmed them for the viral pathogenesis. We have recently reported that the EBV copy number is related with the potential malignancy of the infected cells, corresponding to the activation level of LMP1 and NF-κB signaling. Though the DNA load in patient blood has been well-documented as an potential indicator of EBV-related diseases, this was the first experimental model to verify that the EBV copy number in cancer cells directly correlates with the tumorigenesis. We emphasize that not only “with or without” but also “more or less” should be concerned in EBV-related approaches at gene expression level or in genome context.

Abstract A29: YAP interacts with the ERBB signaling pathway to control ovarian surface epithelial cell tumorigenesis.

Abstract Introduction: The Hippo signaling pathway controls organ size and inhibits tumorigenesis through a kinase cascade that leads to the phosphorylation of the transcriptional co-activator Yes-associated protein 1 (YAP1, more commonly referred to as YAP). Recent studies have shown that YAP may play a role in the progression of ovarian cancer. However, current reports considering the relationships between YAP expression and clinicopathological outcomes in ovarian cancer are inconsistent. Additionally, the role and functional mechanism of YAP on ovarian tumorigenesis and ovarian cancer progression are not fully understood. Aims: The aims of the present study are to examine the role of YAP in the tumorigenesis of ovarian surface epithelial cells, and to uncover the molecular mechanisms underlying YAP regulation of OSE malignant transformation. Methods: We used a large cohort of ovarian cancer patient samples, an ovarian tissue-specific YAP-overexpression mouse model, and a series of unique cellular models to address above questions. Results: Immunohistochemical analysis using a large cohort of patient samples indicated that YAP expression is associated with poor clinical outcomes in ovarian cancer patients. Overexpression of wild-type YAP or constitutively active YAP in immortalized human ovarian surface epithelial cells (HOSE) induced anchorage-independent growth of these cells in a soft agar assay and tumorigenesis in a xenograft mouse model, suggesting that YAP is sufficient to induce transformation of immortalized human OSEs. To confirm the pathological relevance of our findings, we created an ovarian tissue-specific YAP overexpression mouse model by breeding AMHR2-cre mice to ROSA26-rtTA mice and tetO-YAPS127A mice. The created AMHR2-Cre-rtTA-tetO-YAPS127A mice specifically express YAPS127A (constitutively active YAP) in ovarian tissues once these mice are treated with doxycycline (Dox). We found that AMHR2-induced ovarian tissue specific expression of YAPS127A, which led to drastic neoplasia of ovarian surface epithelial (OSE) cells in AMHR2-Cre-rtTA-tetO-YAPS127A mice after induction with Dox for 3 weeks. In addition, our mechanistic studies indicated that activation of YAP in human OSE cells induced expression of EGFR, ERBB3, HBEGF, NRG1 and NRG2 both in vitro and in vivo. Intriguingly, the activated ERBB signaling in turn suppressed the Hippo pathway and activated YAP protein. Conclusion: Our results demonstrate the existence of a NRGs & HBEGF/ERBBs/YAP/NRGs&HBEGF autocrine loop that may play a critical role in regulating the development and progression of ovarian cancer. Our study clearly indicates that combined targeting of ERBB and the Hippo/YAP pathways represents a novel strategy for treatment of ovarian cancer. Citation Format: Chunbo He, Xiangmin LV, John S. Davis, Wang Cheng. YAP interacts with the ERBB signaling pathway to control ovarian surface epithelial cell tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A29.

Neoplastic transformation of porcine mammary epithelial cells in vitro and tumor formation in vivo.

BackgroundThe mammary glands of pigs share many functional and morphological similarities with the breasts of humans, raising the potential of their utility for research into the mechanisms underlying normal mammary function and breast carcinogenesis. Here we sought to establish a model for the efficient manipulation and transformation of porcine mammary epithelial cells (pMEC) in vitro and tumor growth in vivo.MethodsWe utilized a vector encoding the red florescent protein tdTomato to transduce populations of pMEC from Yorkshire –Hampshire crossbred female pigs in vitro and in vivo. Populations of primary pMEC were then separated by FACS using markers to distinguish epithelial cells (CD140a-) from stromal cells (CD140a+), with or without further enrichment for basal and luminal progenitor cells (CD49f+). These separated pMEC populations were transduced by lentivirus encoding murine polyomavirus T antigens (Tag) and tdTomato and engrafted to orthotopic or ectopic sites in immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice.ResultsWe demonstrated that lentivirus effectively transduces pMEC in vitro and in vivo. We further established that lentivirus can be used for oncogenic-transformation of pMEC ex vivo for generating mammary tumors in vivo. Oncogenic transformation was confirmed in vitro by anchorage-independent growth, increased cell proliferation, and expression of CDKN2A, cyclin A2 and p53 alongside decreased phosphorylation of Rb. Moreover, Tag-transformed CD140a- and CD140a-CD49f + pMECs developed site-specific tumors of differing histopathologies in vivo.ConclusionsHerein we establish a model for the transduction and oncogenic transformation of pMEC. This is the first report describing a porcine model of mammary epithelial cell tumorigenesis that can be applied to the study of human breast cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1572-7) contains supplementary material, which is available to authorized users.

Real-Time Methylomic Aberrations During Initiation and Progression of Induced Human Mammary Epithelial Cell Tumorigenesis

Neoplastic transformation provides one of the few existing opportunities to analyze molecular changes in real time during the initiation and progression of breast cancer. Human mammary epithelial cells underwent neoplastic reprogramming, generating one line of semitransformed, premalignant cells and two separate, temporal lines of fully transformed human mammary epithelial cells (THMECs). An Illumina Infinium HumanMethylation27 BeadChip was used to analyze DNA methylation alterations in 27,578 CpG loci at three consecutive time points over an 80-day (d) transformation period. The mean β value for semitransformed human mammary epithelial cells CpG loci (0.245) was much greater than for either THMEC-40d (0.055) or THMEC-80d (0.066), indicating a large loss of methylation after neoplastic induction. In addition, 54% of CpG loci were hypermethylated during the THMEC-40d to THMEC-80d transition. We observed that the CpG loci exhibiting DNA methylation changes during early oncogenesis were enriched for biological functions like cellular movement; this was distinctly different than in the later, more progressive stages of the transformation process enriched for processes involving differentiation. The timing of major methylomic changes may be important in directing the cell toward a more cancerous phenotype. In addition, gene-specific hypermethylation appears to silence developmentally related genes, leading to dedifferentiation.

Differential Tumorigenic Potential and Matriptase Activation between PDGF B versus PDGF D in Prostate Cancer

The platelet-derived growth factors (PDGF A, B, C, and D) and their receptors (α-PDGFR and β-PDGFR) play an indispensible role in physiologic and pathologic conditions, including tumorigenesis. The transformative β-PDGFR is overexpressed and activated during prostate cancer progression, but the identification and functional significance of its complementary ligand have not been elucidated. This study examined potential oncogenic functions of β-PDGFR ligands PDGF B and PDGF D, using nonmalignant prostate epithelial cells engineered to overexpress these ligands. In our models, PDGF D induced cell migration and invasion more effectively than PDGF B in vitro. Importantly, PDGF D supported prostate epithelial cell tumorigenesis in vivo and showed increased tumor angiogenesis compared with PDGF B. Autocrine signaling analysis of the mitogen-activated protein kinase and phosphoinositide 3-kinase pathways found PDGF D-specific activation of the c-jun-NH2-kinase (JNK) signaling cascade. Using short hairpin RNA and pharmacologic inhibitors, we showed that PDGFD-mediated phenotypic transformation is β-PDGFR and JNK dependent. Importantly, we made a novel finding of PDGF D-specific increase in the shedding and activation of the serine protease matriptase in prostate epithelial cells. Our study, for the first time to our knowledge, showed ligand-specific β-PDGFR signaling as well as PDGF D-specific regulation of matriptase activity and its spatial distribution through shedding. Taken together with our previous finding that matriptase is a proteolytic activator of PDGF D, this study provides a molecular insight into signal amplification of the proteolytic network and PDGF signaling loop during cancer progression.

Involvement of WRN helicase in immortalization and tumorigenesis by the telomeric crisis pathway (Review)

The repeated replication of cells shortens telomeres, culminating in their instability, after which most cells cease to replicate and die. However, a small fraction of the cells become immortalized by maintaining telomeres with activated telomerase activity. It has been proposed that WRN helicase encoded by the WRN gene, the causative gene of Werner syndrome (WS), is required for immortalization by the telomeric crisis pathway (TCP) in a system that uses lymphoblastoid cell lines transformed by the Epstein-Barr virus. Taken together, these characteristics indicate that WRN helicase is also required for the immortalization of epithelial cells by TCP and consequent carcinogenesis, suggesting that the tumorigenesis of epithelial cells by TCP is suppressed in WS lacking the WRN helicase function. Notably, in WS the pathway of alternative lengthening of telomeres without activation of telomerase activity has been suggested to be involved in immortalization and tumorigenesis. This factor is consistent with the abundance of non-epithelial cancers in WS in that the ratio of epithelial to non-epithelial cancers is approximately 1:1 in WS patients compared to 10:1 in the general population. A hypothetical scheme showing the role of WRN helicase in immortalization by means of the supposed 'breakage-fusion-bridge cycle' of chromosomes at telomeric crisis is described.

Proteinase‐activated receptors induce interleukin‐8 expression by intestinal epithelial cells through ERK/RSK90 activation and histone acetylation

Proteinase-activated receptors (PARs) are involved in both inflammation and tumorigenesis in epithelial cells. Interleukin (IL)-8 is a potent chemoattractant and is also involved in angiogenesis. The molecular mechanism whereby PARs induce epithelial IL-8 expression is not known. In HT-29 colonic epithelial cells, PAR(1) or PAR(2) agonists stimulated the expression of IL-8 through a NF-kappaB-dependent pathway without inducing IkappaB degradation and disassociation of IkappaB from NF-kappaB. Further studies revealed that PAR activation induced the phosphorylation of p65 at Ser-276 in the nucleus, which increased the recruitment of histone acetyltransferase (HAT) p300 to p50. Inhibition of ERK activation completely blocked PAR-induced IL-8 expression, phosphorylation of p65 and HAT activity. We also demonstrated that RSK p90 was the downstream kinase that mediated ERK-induced nuclear p65 phosphorylation. In conclusion, activation of either PAR(1) or PAR(2) stimulated the transcriptional up-regulation of IL-8 in HT-29 colonic epithelial cells through a pathway that involved ERK/RSK p90, NF-kappaB phosphorylation, and HAT activity. These studies provide evidence of a new role for serine proteinases and PARs in the regulation of gene expression in colonic inflammation and tumorigenesis.

LKB1/PEA3/ΔNp63 Pathway Regulates PTGS‐2 (COX‐2) Transcription in Lung Cancer Cells Upon Cigarette Smoke Exposure

This is the first study to show that cigarette smoking induced the LKB1/PEA 3/ΔNp63-dependent transcriptional regulation of inflammatory molecules, such as COX-2/PTGS-2. Using mainstream smoke extract (MSE) and sidestream smoke extract (SSE) as modeling tools for primary and secondhand smoking, we found that both MSE and SSE downregulated protein levels for LKB1, while upregulated protein levels for PEA 3 and COX-2 in a dose-dependent manner. Using the endogenous ChIP analysis, we further found that the C/EBPβ, NFκB, NF-Y (CHOP), PEA 3 (ETS) and ΔNp63 proteins bound to the specific area (-550 to -130) of the COX-2 promoter, while forming multiple protein complexes in lung cancer cells exposed to MSE and SSE. Our results define a novel link between various transcription factors occupying the COX-2 promoter and cellular response to cigarette smoke exposure bringing a new component, ΔNp63α, showing a critical role for cooperation between various chromatin components in regulation of COX-2 expression and, therefore strengthening the central role of inflammatory process in tumorigenesis of epithelial cells, especially after cigarette smoke exposure (both primary and secondhand).

TGF-ß signaling, tumor microenvironment and tumor progression: the butterfly effect

Transforming growth factor-beta (TGF-beta) signals through receptor serine/threonine kinases and intracellular Smad effectors, regulating numerous epithelial cell processes. TGF-beta plays a crucial role in the cancer initiation and progression through tumor cell autonomous signaling and interactions with tumor microenvironment, but is featured with a butterfly effect upon the stages of tumorigenesis. TGF-beta signaling acts as a suppressor of epithelial cell tumorigenesis at early stages, but promotes tumor progression by enhancing migration, invasion, and survival of the tumor cells during the later stages. TGF-beta signaling also cross-talks with other cell survival signaling pathways. Tumor microenvironment contains many distinct cell types, which substantially influences the tumor cell growth and survival, and the invasion and metastasis. TGF-beta in the microenvironment, produced by cancer and/or stromal cells, is high and negatively correlates with disease progression and patient prognosis. Therefore, TGF-beta may affect tumor progression by multiple mechanisms in addition to its direct action on tumor cells, and the diversities of TGF-beta signaling in tumors imply a need for caution to TGF-beta-targeted strategies of tumor prevention and/or therapeutics.

Abstract B92: P38 MAPK activation is critical for FGFR3-induced cell transformation in human bladder carcinoma

Abstract Bladder cancer is estimated to be the fifth most prevalent type of cancer in industrialized countries. In most of the cases (more than 90 %), tumors are urothelial cell carcinoma (UCC). Activating mutations of the fibroblast growth factor receptor 3 (FGFR3) are the most frequent genetic alteration in UCC (up to 80% in low grade Ta tumors). We have previously described that the mutated receptor displays oncogenic properties (Bernard-Pierrot et al., 2006). In this study, we aimed to better understand the signaling properties of the oncogenic mutated FGFR3. We performed a systematic analysis of phosphoproteins involved in classical signaling pathways activated by FGFRs coupled to a pharmacological study in NIH-3T3 cells expressing the mutated receptor or not. We identified p38 MAPK as a key down-stream signaling molecule of FGFR3-induced NIH-3T3 cell transformation. We further showed that activation of p38 is also critical for FGFR3-induced tumorigenesis of epithelial cells. Indeed, in vitro, p38- siRNAs and SB203580, a specific p38 MAPK inhibitor, inhibited the transforming properties of MGH-U3 cells derived from a human bladder tumor and expressing mutated FGFR3. Moreover, in vivo, treatment with SB203580, inhibited mutated FGFR3-induced proliferation both in MGH-U3 xenografted tumors and in transgenic mice expressing mutated FGFR3 in the skin (Logié et al.,2005). Finally, we identified p38 activation in all FGFR3 mutated human bladder tumors studied and more generally in all human bladder tumors presenting identified FGFR3 signaling pathway alterations. Taken together, those results suggest that p38 is a key signaling molecule in a high proportion of human bladder tumors and could represent an interesting therapeutic target both in muscle non-invasive and invasive tumors mutated for FGFR3 or more generally in tumors with FGFR3 pathway activation. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B92.