Untransformed Epithelial Cells Research Articles

Ovarian cancer cells exhibit diverse migration strategies on stiff collagenous substrata

In homoeostasis, the shape and sessility of untransformed epithelial cells are intricately linked together. Variations of this relationship in migrating cancer cells as they encounter different microenvironments are as yet ill understood. Here, we explore the interdependency of such traits in two morphologically distinct invasive ovarian cancer cell lines (OVCAR-3 and SK-OV-3) under mechanically variant contexts. We first established a metric toolkit that assessed traits associated with cell motion and shape, and rigorously measured their dynamical variation across trajectories of migration using a Shannon entropic distribution. Two stiffness conditions on polymerized collagen I with Young’s moduli of 0.5 kPa (soft) and 20 kPa (stiff) were chosen. Both the epithelioid OVCAR-3 and mesenchymal SK-OV-3 cells on soft substrata exhibited slow and undirected migration. On stiff substrata, SK-OV-3 showed faster persistent directed motion. Surprisingly, OVCAR-3 cells on stiffer substrata moved even faster than SK-OV-3 cells but showed a distinct angular motion. The polarity of SK-OV-3 cells on stiff substrata was well correlated with their movement, whereas, for OVCAR-3, we observed an unusual “slip” behavior, wherein the axes of cell shape and movement were poorly correlated. Whereas SK-OV-3 and OVCAR-3 showed greater mean deformation on stiffer substrata, the latter was anticorrelated with variation in angular motion or the mean deviation between shape and motility axis for SK-OV-3 but poorly correlated for OVCAR-3. Moreover, on softer substrata OVCAR-3 and SK-OV-3 were relatively rigid but showed greater shape variation (with OVCAR-3 showing a higher fold change) on stiffer substrata. Our findings suggest that greater deformability on stiffer milieu allow epithelioid cells to overcome constraints on the congruence in axis of shape and motion seen for mesenchymal cells and display distinct motile behaviors across this phenotypic spectrum.

Proteomic and yeast 2-hybrid screens to identify PTEN binding partners

PTEN is a phosphoinositide lipid phosphatase and an important tumour suppressor protein. PTEN function is reduced or lost in around a third of all human cancers through diverse mechanisms, from gene deletion to changes in the function of proteins which regulate PTEN through direct protein binding. Here we present data from SILAC (Stable Isotope Labelling by Amino acids in Cell culture) proteomic screens to identify proteins which bind to PTEN. These experiments using untransformed epithelial cells and glioma cells identified several novel candidate proteins in addition to many previously identified PTEN binding partners and many proteins which are recognised as common false positives using these methods. From subsequent co-expression pull-down experiments we provide further evidence supporting the physical interaction of PTEN with MMP1, Myosin 18A and SHROOM3. We also performed yeast two-hybrid screens which identify the previously recognised PTEN binding partner MSP58 in addition to the nuclear import export receptor TNPO3. These experiments identify several novel candidate binding partners of PTEN and provide further data addressing the set of proteins that interact with this important tumour suppressor.

Abstract 4038: Study of therapeutic effects of polyisoprenylated cysteinyl amide inhibitors on lung cancer cells of black patients using 3D-Printed alveolar model

Abstract Lung cancer is one of the top five deadliest cancers, and black Americans have a higher mortality rate when compared to white Americans even though they smoke relatively fewer cigarettes. Lung adenocarcinoma (LUAD) is the most common histological lung cancer subtype in all population groups including blacks. There are no in vitro model systems to study lung cancer in black subjects, and there is only one lung adenocarcinoma cell line from a black patient (NCI-H23). Here we aim to address the lack of in vitro models to study black LUAD. Using three-dimensional (3D) biofabrication technologies, we’ve developed a thick vascularized tissue model to recapitulate physiologically relevant heterogeneous human alveoli. Specifically, the blood-air barrier of the alveolar model is simulated with two perfusable channels embedded in a thick vascularized tissue construct: a helical blood channel and a straight air channel. Spatially, the air channel is in the center and is surrounded by the blood channel. Human endothelial cells are seeded onto the inner surface of the blood channel and NCI-H23 cells (which carry a KRAS mutation) or untransformed alveolar epithelial cells derived from black subjects, are seeded on the inner surface of the air channel. The blood and air channels are perfused using cell medium and oxygenated air, respectively. The model is used for the study of potential LUAD therapeutic agents such as polyisoprenylated cysteinyl amide inhibitors (PCAIs), which target and disrupt the RAS-mediated signaling pathway. KRAS is the most commonly mutated oncogene in LUAD in blacks, and our experiments are aimed at ensuring that PCAIs are effective in black LUAD and may offer better treatment outcomes for ethnically/racially diverse patients in whom the RAS signaling pathway is frequently altered and for whom pharmaceutical therapy is necessitated by the late diagnoses. Citation Format: Nadine Belony, Bing Ren, Phuc Pham, Matthew Gregory, Pablo E. Puente, Nazarius S. Lamango, Ite A. Offringa, Yong Huang. Study of therapeutic effects of polyisoprenylated cysteinyl amide inhibitors on lung cancer cells of black patients using 3D-Printed alveolar model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4038.

Serum amyloid A1: Innocent bystander or active participant in cell migration in triple-negative breast cancer?

The Serum Amyloid A (SAA) family of proteins is associated with various pathological conditions, including cancer. However, their role in cancer is incompletely understood. Here, we investigated the role of SAA1 in cell cycle regulation, apoptosis, survival signaling, metabolism, and metastasis in models of triple-negative breast cancer (TNBC), using RNAi. Our data show that in untransformed epithelial cells (MCF12A), the knockdown of SAA1 induces the expression of cell cycle regulators (MCM2, p53), the activation of DNA repair (PARP synthesis), and survival signaling (NFκB). In contrast, knockdown of SAA1 in the TNBC cell line (MDA-MB-231) induced the expression p16 and shifted cells in the cell cycle from the S to G2/M phase, without the activation of DNA repair. Moreover, in SAA1-deficient MDA-MB-231 and HCC70 cells, metabolism (NADH oxidation) continually increased while cell migration (% wound closure and the rate of wound closure) decreased. However, silencing of SAA1 altered epithelial and mesenchymal markers in MCF12A (E-cadherin, Laminin 1β, Vimentin) and MDA-MB-231 (α-Smooth muscle actin) cells, associated with the metastatic program of epithelial-mesenchymal transition. Nonetheless, our data provide evidence that SAA1 could potentially serve as a therapeutic target in TNBC.

Nippon Shokubai succeeded in improving the performance of the electrolyte for all solid polymer batteries

The Serum Amyloid A (SAA) family of proteins is associated with various pathological conditions, including cancer. However, their role in cancer is incompletely understood. Here, we investigated the role of SAA1 in cell cycle regulation, apoptosis, survival signaling, metabolism, and metastasis in models of triple-negative breast cancer (TNBC), using RNAi. Our data show that in untransformed epithelial cells (MCF12A), the knockdown of SAA1 induces the expression of cell cycle regulators (MCM2, p53), the activation of DNA repair (PARP synthesis), and survival signaling (NFκB). In contrast, knockdown of SAA1 in the TNBC cell line (MDA-MB-231) induced the expression p16 and shifted cells in the cell cycle from the S to G2/M phase, without the activation of DNA repair. Moreover, in SAA1-deficient MDA-MB-231 and HCC70 cells, metabolism (NADH oxidation) continually increased while cell migration (% wound closure and the rate of wound closure) decreased. However, silencing of SAA1 altered epithelial and mesenchymal markers in MCF12A (E-cadherin, Laminin 1β, Vimentin) and MDA-MB-231 (α-Smooth muscle actin) cells, associated with the metastatic program of epithelial-mesenchymal transition. Nonetheless, our data provide evidence that SAA1 could potentially serve as a therapeutic target in TNBC.

Mechanical mismatch between Ras transformed and untransformed epithelial cells.

The organization of the actin cytoskeleton plays a key role in regulating cell mechanics. It is fundamentally altered during transformation, affecting how cells interact with their environment. We investigated mechanical properties of cells expressing constitutively active, oncogenic Ras (RasV12) in adherent and suspended states. To do this, we utilized atomic force microscopy and a microfluidic optical stretcher. We found that adherent cells stiffen and suspended cells soften with the expression of constitutively active Ras. The effect on adherent cells was reversed when contractility was inhibited with the ROCK inhibitor Y-27632, resulting in softer RasV12 cells. Our findings suggest that increased ROCK activity as a result of Ras has opposite effects on suspended and adhered cells. Our results also establish the importance of the activation of ROCK by Ras and its effect on cell mechanics.

The Chromatin Assembly Factor Complex 1 (CAF1) and 5-Azacytidine (5-AzaC) Affect Cell Motility in Src-transformed Human Epithelial Cells

Tumor invasion into surrounding stromal tissue is a hallmark of high grade, metastatic cancers. Oncogenic transformation of human epithelial cells in culture can be triggered by activation of v-Src kinase, resulting in increased cell motility, invasiveness, and tumorigenicity and provides a valuable model for studying how changes in gene expression cause cancer phenotypes. Here, we show that epithelial cells transformed by activated Src show increased levels of DNA methylation and that the methylation inhibitor 5-azacytidine (5-AzaC) potently blocks the increased cell motility and invasiveness induced by Src activation. A proteomic screen for chromatin regulators acting downstream of activated Src identified the replication-dependent histone chaperone CAF1 as an important factor for Src-mediated increased cell motility and invasion. We show that Src causes a 5-AzaC-sensitive decrease in both mRNA and protein levels of the p150 (CHAF1A) and p60 (CHAF1B), subunits of CAF1. Depletion of CAF1 in untransformed epithelial cells using siRNA was sufficient to recapitulate the increased motility and invasive phenotypes characteristic of transformed cells without activation of Src. Maintaining high levels of CAF1 by exogenous expression suppressed the increased cell motility and invasiveness phenotypes when Src was activated. These data identify a critical role of CAF1 in the dysregulation of cell invasion and motility phenotypes seen in transformed cells and also highlight an important role for epigenetic remodeling through DNA methylation for Src-mediated induction of cancer phenotypes.

Sulforaphane Regulates NFE2L2/Nrf2-Dependent Xenobiotic Metabolism Phase II and Phase III Enzymes Differently in Human Colorectal Cancer and Untransformed Epithelial Colon Cells

ABSTRACTSulforaphane (SFN), a naturally occurring chemopreventive and anticancer agent, is a nuclear factor, erythroid 2-like 2 (NFE2L2/Nrf2) inducer. Nrf2 plays a critical role in coordinating the cell defense system by initiating the transcription of cytoprotective genes, including detoxification enzymes such as NAD(P)H quinone dehydrogenase 1 (NQO1) and transport proteins such as ATP-binding cassette, subfamily C (CFTR/MRP). Recently, the essential role of Nrf2 in tumor development and progression and in the development of multidrug resistance in cancer cells has been highlighted. The aim of this study was to compare the effect of SFN on the Nrf2 system and the Nrf2-target enzymes NQO1 and MRP in human untransformed epithelial colon CRL-1790 cells and in HT-29 and Caco-2 colorectal cancer cells to elucidate the role of SFN in cancer prevention and treatment. We have demonstrated that SFN has excellent cytoprotective properties in CRL-1790 cells, as it induced Nrf2-dependent expression of MRP1 and NQO1. SFN induced Nrf2 target enzyme activity in HT-29 and Caco-2 cancer cells but regulated the Nrf2/ARE signaling pathway differently in cancer and untransformed cells.

Abstract PR02: Cyclin E1 deregulation occurs early during fallopian tube tumorigenesis and promotes secretory cell transformation

Abstract The fallopian tube has been implicated as the dominant site of origin for high-grade serous ovarian carcinoma. However, the molecular pathogenesis of fallopian tube-derived serous cancer is poorly understood and there are few experimental studies examining the transformation of human fallopian tube cells. Recent genomic analyses have identified CCNE1 (Cyclin E1) gene amplification as a potential oncogenic driver of high-grade serous ovarian carcinoma development. Here, we examine the oncogenic role of Cyclin E1 in serous carcinogenesis, first by characterizing its expression in both late-stage and early-stage human tumor samples, and secondly, by generating an experimental model of Cyclin E1-mediated transformation using primary human fallopian tube secretory epithelial cells. Using fluorescent in situ hybridization and immunohistochemical analyses, we show that Cyclin E1 is strongly expressed in a subset of late-stage high-grade serous carcinomas harboring CCNE1 amplification and that aberrant Cyclin E1 expression can occur very early during serous tumorigenesis, evident in non-invasive tubal carcinoma and putative precursor lesions. To examine the effects of constitutive Cyclin E1 expression in untransformed cells, we immortalized human fallopian tube secretory epithelial cells with TERT (telomerase reverse transcriptase) and mutant TP53R175H (representing early genetic alterations in serous tumorigenesis), followed by overexpression of CCNE1 or a vector control. Using a series of in vitro assays, we show that inappropriate Cyclin E1 expression imparts malignant characteristics to immortal fallopian tube secretory epithelial cells, including accelerated proliferation, loss of contact inhibition, and clonal growth ability. In addition, we use a combination of DNA damage assays and PCR array profiling to demonstrate that Cyclin E1 induces DNA double-strand and single-strand breaks, accompanied by up-regulation of DNA damage response genes involved in managing DNA replication stress. In sum, our results suggest that constitutive Cyclin E1 expression drives over-proliferation of untransformed but p53-compromised fallopian tube secretory epithelial cells, accompanied by an accumulation of DNA damage and altered expression of DNA damage response genes. Our data supports the hypothesis that Cyclin E1 promotes fallopian tube transformation by inducing replication stress-associated DNA damage, thus leading to chromosomal instability, and that CCNE1 amplification is a major driver of Cyclin E1 overexpression in high-grade serous carcinoma. This abstract is also presented as Poster A1. Citation Format: Alison M. Karst, Paul M. Jones, Natalie Vena, Azra H. Ligon, Joyce F. Liu, Michelle S. Hirsch, Dariush Etemadmoghadam, David D. Bowtell, Ronny Drapkin. Cyclin E1 deregulation occurs early during fallopian tube tumorigenesis and promotes secretory cell transformation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR02.

Trask Loss Enhances Tumorigenic Growth by Liberating Integrin Signaling and Growth Factor Receptor Cross-Talk in Unanchored Cells

The cell surface glycoprotein Trask/CDCP1 is phosphorylated during anchorage loss in epithelial cells in which it inhibits integrin clustering, outside-in signaling, and cell adhesion. Its role in cancer has been difficult to understand, because of the lack of a discernible pattern in its various alterations in cancer cells. To address this issue, we generated mice lacking Trask function. Mammary tumors driven by the PyMT oncogene and skin tumors driven by the SmoM2 oncogene arose with accelerated kinetics in Trask-deficient mice, establishing a tumor suppressing function for this gene. Mechanistic investigations in mammary tumor cell lines derived from wild-type or Trask-deficient mice revealed a derepression of integrin signaling and an enhancement of integrin-growth factor receptor cross-talk, specifically in unanchored cell states. A similar restrictive link between anchorage and growth in untransformed epithelial cells was observed and disrupted by elimination of Trask. Together our results establish a tumor-suppressing function in Trask that restricts epithelial cell growth to the anchored state.

Abstract 267: Large-scale RNAi screening of human kinome identifies putative breast cancer related molecular targets

Abstract To identify vulnerable genes in breast cancer cells that may be therapeutic candidates for treatment of breast cancer, we conducted an unbiased, large-scale, synthetic siRNA-mediated RNAi screen. Using cell viability as the read-out, we first screened a whole human kinome library plus 350 additional genes (four siRNAs per gene) in a cell line representative of triple negative (ER -ve, PR -ve, Her2/Neu -ve) breast cancer, MDA-MB-468. We identified approximately 40 kinases whose loss-of-function reduced the viability of MDA-MB-468 cells based on the criteria of at least two of four siRNAs per gene generating a Z score of ≤ −1.5. To validate the top candidates from our screen, PLK1, AURKB and PCTK3/CDK18), we tested additional siRNAs (from multiple vendors) in different breast cancer cell lines including ER positive cell lines, CAMA-1 and MCF-7, HER-2 amplified cell lines, HCC-1954 and AU565, triple-Negative (Basal A) cell lines HCC-1937 and MDA-MB-468, and triple-Negative (Basal B) cell lines BT549 and MDA-MB-231. We found silencing of PLK1, AURKB or PCTK3/CDK18 reduced the cell viability in majority of these cell lines to a certain degree and also induced cell cycle arrest and apoptosis. PLK1 and AURKB are being actively pursued as molecular targets in cancer but little is known of the function of the PCTK3/CDK18 protein, with most of the limited functional studies of PCTK3/CDK18 having focused on a role in neuronal cell signaling. One study has though reported an increase in the expression of PCTK3/CDK18 in breast cancer (Valladares et al., Cancer Genet Cytogenet. 2006 170:147). We found silencing of PCTK3/CDK18 is lethal to most breast cancer cells, but it is well tolerated in the untransformed breast epithelial cell line MCF10A. The PCTK3/CDK18 gene encodes a member of the PCTAIRE protein kinase subfamily of CDC2-related serine/threonine-specific protein kinases. We observed that with multiple siRNAs, we induced an 80% decrease in the viability of MDA-MB-468 cells 96 hours following siRNA transfection. To ensure that the observed inhibitory effect is indeed due to silencing of PCTK3/CDK18 we performed an RNAi rescue experiment, by creating a silent third-codon point mutation within the region targeted by one of the PCTK3 siRNAs. We found the RNAi-induced inhibition and cell cycle arrest is countered by expression of a functional version of the target gene that is resistant to the silencing siRNA. No specific inhibitor of PCTK3/CDK18 is available, but a proteome-wide CDK/CRK-specific kinase inhibitor RGB-286147 does decrease cell viability, and induce cell cycle arrest and apoptosis of MDA-MB-468 cells. We are currently further investigating the normal and cancer-related functional roles of PCTK3/CDK18 and are pursuing strategies for the identification of PCTK3/CDK18 specific inhibitors. 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 267. doi:1538-7445.AM2012-267

Abstract 3358: Acquired epithelial-mesenchymal transition synergizes with normal epithelial cells to induce hepatic metastases

Abstract Identification of the required hallmarks for the tumor cells to disseminate to the liver represents an important milestone to delay cancer progression, and consequently to avoid the harmful consequences for patients. Largely unknown, a myriad of cell properties seem to cooperate in a successful metastatic process and in the chemo-/radio- therapeutic resistance. Our aim is to define the intrinsic characteristics of tumor cells that may contribute to epithelial-mesenchymal transition (EMT) in early and late metastatic events. We previously observed that: i) metastatic cells reaching the liver microenvironment display cancer stem features associated with EMT and increased TGFβ1-signaling (Zubeldia et al. Proceedings AACR, 2010; 51, #2304); ii) hepatic secondary tumors are enriched with a subpopulation of cells with a distinctive Sca1+ phenotype and a concurrent increased expression of other cancer stem markers (Zubeldia et al. Proceedings MRS-AACR, 2010; #A105). First, we demonstrated the high tumorigenicity of these metastatic liver-derived cell lines after subcutaneous injection in nude mice, without obvious correlation with their origin regarding TGFβ1-signaling. However, as shown by bioluminescent imaging, some of the novel metastatic liver-derived cell sublines were unable to achieve metastases following intrasplenic injection in C57BL/6J mice. We also noted a differential efficiency of each clone in primary tumor formation in this experimental model. Importantly, we found that co-injection of these cells (isolated from liver metastases) with non transformed hepatocytes (AML12) resulted in a significant increase of metastasis formation. We show an important role of normal cells for metastatic spread in early steps of tumor formation, and suggest the protective activity that the untransformed epithelial cells may have throughout the metastatic progression. We will present the therapeutic implications of our findings for the clinical treatment of carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3358. doi:10.1158/1538-7445.AM2011-3358

A gene expression signature associated with structural differentiation of prostate acini predicts postoperative recurrence of prostate cancer

Abstract Glandular malignancies such as prostate carcinoma display a considerable heterogeneity in the degree of structural organization while the prognostic role of the associated molecular changes awaits further exploration. Using a three-dimensional (3-D) tissue organization culture model that permits the structural differentiation of prostate glandular epithelium we profiled the transcriptomes of multicellular organoids formed by untransformed prostate epithelial cells (PECs) or prostate carcinoma cells as well as those cultured as cell monolayers. Interrogation into the multiple (n = 6) sets of differentially expressed genes (DEGs) identified that those altered significantly during the structural differentiation process of polarized PEC acini strongly associated with the probability of recurrence in a cohort of 50 prostate cancer patients receiving radical prostatectomy (RP). By contract, the DEGs related to prostate tumor spheroids did not correlate significantly with prognosis. To further explore this association, we prioritized the genes and applied supervised classification algorithms whereby identified a 39-gene structure-associated prognosis signature (SAPS) that shows a remarkable predictivity for the likelihood of post-RP recurrence (overall accuracy 100%, P < 0.001). The SAPS outperformed standard clinicopathological variables, including Gleason scores, as well as existing multigene prognostic predictors of prostate cancer. Our results suggest a distinct contribution by the glandular structural differentiation program to prostate cancer pathology and the usefulness of a robust and biologically relevant genomic predictor of the prognosis of prostate cancer (Supported by NHRI CA-099-PP-19 and Department of Health of Taiwan H99-TD-C-111-004 to KKC).

Migfilin contributes to cell‐matrix adhesion‐mediated survival signaling through direct interaction with Src

Integrin‐mediated cell‐extracellular matrix (ECM) adhesion is essential for protection of epithelial cells against apoptosis, but the underlying mechanism is incompletely understood. Here we show that migfilin, an integrin‐proximal adaptor protein, interacts with Src and contributes to cell‐ECM‐mediated survival signaling. Loss of cell‐ECM adhesion markedly reduces the migfilin level in untransformed epithelial cells and concomitantly induces apoptosis(anoikis). Overexpression of migfilin substantially desensitizes cell detachment‐induced apoptosis. Conversely, depletion of migfilin promotes apoptosis despite the presence of cell‐ECM adhesion. At the molecular level, migfilin directly interacts with Src and the migfilin binding surface overlaps with the inhibitory intramolecular interaction sites in Src. Consequently, the binding of migfilin activates Src, resulting in suppression of apoptosis. Our results reveal a novel mechanism by which cell‐ECM adhesion regulates Src activation and survival signaling. This migfilin‐mediated signaling pathway is dysfunctional in multiple types of carcinoma cells, which likely contributes to aberrant Src activation and anoikis resistance in the cancerous cells.

Migfilin Interacts with Src and Contributes to Cell-Matrix Adhesion-mediated Survival Signaling

Integrin-mediated cell-extracellular matrix (ECM) adhesion is essential for protection of epithelial cells against apoptosis, but the underlying mechanism is incompletely understood. Here we show that migfilin, an integrin-proximal adaptor protein, interacts with Src and contributes to cell-ECM-mediated survival signaling. Loss of cell-ECM adhesion markedly reduces the migfilin level in untransformed epithelial cells and concomitantly induces apoptosis. Overexpression of migfilin substantially desensitizes cell detachment-induced apoptosis. Conversely, depletion of migfilin promotes apoptosis despite the presence of cell-ECM adhesion. At the molecular level migfilin directly interacts with Src, and the migfilin binding surface overlaps with the inhibitory intramolecular interaction sites in Src. Consequently, the binding of migfilin activates Src, resulting in suppression of apoptosis. Our results reveal a novel mechanism by which cell-ECM adhesion regulates Src activation and survival signaling. This migfilin-mediated signaling pathway is dysfunctional in multiple types of carcinoma cells, which likely contributes to aberrant Src activation and anoikis resistance in the cancerous cells.

Ras‐mediated intestinal epithelial cell transformation requires cyclooxygenase‐2‐induced prostaglandin E2 signaling

Ras-mediated transformation is associated with upregulation of cyclooxygenase-2 (COX-2), which in turn promotes prostaglandin E2 (PGE2) synthesis and secretion. Although recent studies have identified molecular mechanisms by which Ras mediates upregulation of COX-2, conflicting observations have been made. Furthermore, while COX-2 upregulation has been shown to be important for Ras transformation, the signaling pathways initiated by PGE2-stimulation of EP family of heterotrimeric G protein-coupled receptors (GPCR) and contribution of PGE2 signaling to Ras-mediated transformation are issues that remain unresolved. In this study, we first determined that Raf effector pathway activation of the extracellular-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) cascade alone was sufficient and necessary for COX-2 and PGE2 upregulation. However, Raf-independent regulation of the c-jun N-terminal kinase (JNK) and p38 MAPK cascades is also involved in COX-2 and PGE2 upregulation, with the JNK and p38 pathways exhibiting opposing roles in COX-2 and PGE2 upregulation. Furthermore, in contrast to previous studies, we found that an epidermal growth factor (EGF) receptor autocrine growth mechanism, another Raf-independent signaling mechanism, was necessary for COX-2 and PGE2 upregulation. Second, we determined that inhibition of EP1/2 receptor function blocked growth transformation by Ras, demonstrating that PGE2 upregulation is a key transforming function of COX-2. Finally, we found that PGE2 stimulated the activation of Ras and ERK, but not Akt, and reduced matrix deprivation-induced apoptosis, in untransformed epithelial cells. In summary, our studies define additional, multiple signaling mechanisms that promote COX-2 and PGE2 expression and show that COX-2-stimulated PGE2-EP receptor signaling is required for growth and survival transformation by Ras.

Nuclear Factor-κB and Manganese Superoxide Dismutase Mediate Adaptive Radioresistance in Low-Dose Irradiated Mouse Skin Epithelial Cells

Mechanisms governing inducible resistance to ionizing radiation in untransformed epithelial cells pre-exposed to low-dose ionizing radiation (LDIR; </=10 cGy) are not well understood. The present study provides evidence that pre-exposure to 10 cGy X-rays increases clonogenic survival of mouse skin JB6P+ epithelial cells subsequently exposed to 2 Gy doses of gamma-rays. To elucidate the molecular pathways of LDIR-induced adaptive radioresistance, the transcription factor nuclear factor-kappaB (NF-kappaB) and a group of NF-kappaB-related proteins [i.e., p65, manganese superoxide dismutase (MnSOD), phosphorylated extracellular signal-regulated kinase, cyclin B1, and 14-3-3zeta] were identified to be activated as early as 15 min after LDIR. Further analysis revealed that a substantial amount of both 14-3-3zeta and cyclin B1 accumulated in the cytoplasm at 4 to 8 h when cell survival was enhanced. The nuclear 14-3-3zeta and cyclin B1 were reduced and increased at 4 and 24 h, respectively, after LDIR. Using YFP-fusion gene expression vectors, interaction between 14-3-3zeta and cyclin B1 was visualized in living cells, and LDIR enhanced the nuclear translocation of the 14-3-3zeta/cyclin B1 complex. Treatment of JB6P+ cells with the NF-kappaB inhibitor IMD-0354 suppressed LDIR-induced expression of MnSOD, 14-3-3zeta, and cyclin B1 and diminished the adaptive radioresistance. In addition, treatment with small interfering RNA against mouse MnSOD was shown to inhibit the development of LDIR-induced radioresistance. Together, these results show that NF-kappaB, MnSOD, 14-3-3zeta, and cyclin B1 contribute to LDIR-induced adaptive radioresistance in mouse skin epithelial cells.

C‐Fos is required for TGFβ1 production and the associated paracrine migratory effects of human colon carcinoma cells

In tumor cells that have lost responsiveness to the growth inhibitory effects of transforming growth factor beta (TGFbeta), increased TGFbeta production by the tumor cells often contributes to cancer progression, primarily through paracrine mechanisms. Here we investigated the major components of the activator protein-1 (AP-1) complex in the TGFbeta1 promoter of human colon carcinoma cells (HCCCs). In contrast to untransformed epithelial cells (UECs), HCCCs displayed constitutive activation of AP-1 at the proximal AP-1 site in the human TGFbeta1 promoter. Further, in contrast to the JunD and Fra-2 components present in the AP-1 complex at this AP-1 site in UECs, c-Fos was the major detectable AP-1 component in HCCCs. Thus, transcriptional factor switching had occurred in HCCCs relative to the UECs, with regard to the proximal AP-1 site of the human TGFbeta1 promoter. Small interfering RNAs (siRNAs) against c-Fos significantly suppressed AP-1 activity at the relevant AP-1 site, and led to a decrease in TGFbeta1 secretion by the HCCCs. Our results indicate for the first time that c-Fos binding at the TGFbeta1 promoter proximal AP-1 site in HCCCs is required for TGFbeta1 production by the tumor cells. Further, we demonstrated that blockade of TGFbeta1 secretion by c-Fos siRNA led to a suppression of the cellular migration and mitogenesis of NIH 3T3 fibroblasts in a paracrine fashion. Thus, c-Fos may have utility as a target for blocking tumor cell-secreted TGFbeta1, thereby suppressing the migratory behavior associated with the malignant phenotype of HCCCs.

Ubiquitin–proteasome degradation of KLF5 transcription factor in cancer and untransformed epithelial cells

Ubiquitin-mediated proteolysis plays a central role in controlling intracellular levels of essential regulatory molecules such as p53, cyclins, myc, BRCA1, HIF-1alpha, etc. The Kruppel-like factor 5 (KLF5) transcription factor regulates biological processes involved in carcinogenesis, angiogenesis, and smooth muscle cell differentiation. In carcinogenesis, KLF5's role has been indicated by frequent genetic deletion as well as functional studies. Here we show that KLF5 is an unstable protein with a short half-life. Destruction of KLF5 was prevented by each of the proteasome-specific inhibitors tested but not by an inhibitor for trypsin-like proteases and cysteine proteases or by a lysosome inhibitor in epithelial cells. Furthermore, KLF5 underwent ubiquitination, and deletion of a 56-amino-acid sequence adjacent to a known transactivation domain of KLF5 significantly reduced its ubiquitination and degradation. Interestingly, cancer cells appeared to be more active in KLF5 degradation than untransformed epithelial cells, yet their proteasome activity was not higher. These results suggest that KLF5 protein is degraded at least in part through ubiquitination-proteasome pathway, which may have become hyperactive for KLF5 in cancer cells.

KLF11 mediates a critical mechanism in TGF-β signaling that is inactivated by Erk-MAPK in pancreatic cancer cells

Background & Aims: Smad-regulated transcription plays a central role in transforming growth factor (TGF)-β-induced cell growth inhibition and tumor suppression. Like the Smads, KLF11 is an early response transcription factor that mediates TGF-β-induced growth inhibition in untransformed epithelial cells. Here, we investigated the functional implications of KLF11 in TGF-β signaling and transcription in normal epithelial as well as pancreatic cancer cells. Methods: The effects of KLF11 on TGF-β signaling and transcription were examined on the levels of reporter transactivation, Smad2 phosphorylation, and expression of endogenous TGF-β-regulated genes. Promoter analysis, real-time polymerase chain reaction, and coimmunoprecipitation studies were performed to study KLF11-induced and mSin3A corepressor-mediated repression of Smad7. Erk-induced KLF11 phosphorylation was examined in vitro and in vivo, and its impact on KLF11-mSin3A-mediated Smad7 repression was verified in pancreatic cancer cells using site-directed mutagenesis. Results: KLF11 potentiates TGF-β signaling by terminating the inhibitory Smad7 loop. Mechanistically, KLF11 represses TGF-β-induced transcription from the Smad7 promoter by recruiting mSin3a via GC-rich sites. This function is inhibited in pancreatic cancer cells with oncogenic Ras mutations, in which Erk/mitogen-activated protein kinase phosphorylates KLF11, leading to disruption of KLF11-mSin3a interaction. Expression of an Erk-insensitive KLF11 mutant restores both mSin3a binding and Smad7 repression and results in enhanced TGF-β signaling in pancreatic cancer cells. Conclusions: These results define a novel mechanism in TGF-β-regulated gene expression. KLF11 potentiates Smad-signaling activity in normal epithelial cells through termination of the negative feedback loop imposed by Smad7. The fact that this function of KLF11 is inhibited by oncogenic Erk/mitogen-activated protein kinase in pancreatic cancer cells emphasizes the importance of this mechanism for oncogenesis.