Increased Cancer Cell Invasion Research Articles

Significance of PI3K/AKT signaling pathway in metastasis of esophageal squamous cell carcinoma and its potential as a target for anti-metastasis therapy.

Metastasis is the most lethal hallmark of esophageal squamous cell carcinoma (ESCC). The aim of the study is to identify key signaling pathways that control metastasis in ESCC. Highly invasive ESCC sublines (designated I3 cells) were established through three rounds of selection of cancer cells invading through matrigel-coated chambers. Gene expression profile of one of the I3 sublines was compared with that of its parental cell line using cDNA microarray analysis. Gene ontology and pathway analyses of the differentially expressed genes (both upregulated and downregulated) indicated that genes associated with cellular movement and the AKT pathway were associated with increased cancer cell invasiveness. Western blot analysis confirmed increased phosphorylated AKT (p-AKT), N-cadherin and decreased E-cadherin expression in the I3 cells. Immunohistochemistry was used to evaluate the clinical significance of p-AKT expression in ESCC, and the results showed higher p-AKT nuclear expression in lymph node metastases when compared with primary carcinoma. Inactivation of the PI3K/AKT pathway with specific inhibitors, or with PTEN overexpression, resulted in reversed cadherin switching and inhibited cancer cell motility. Inhibition of the pathway by treatment with wortmannin markedly suppressed experimental metastasis in nude mice. Our data demonstrated the importance of the PI3K/AKT signaling pathway in ESCC metastasis and support PI3K/AKT as a valid therapeutic target in treatment of metastatic ESCC.

Abstract IA01: Recent advances in studying EGFR and its ligands in colorectal cancer

Abstract At Vanderbilt, we have fashioned a three-pronged approach to study colorectal cancer (CRC): from in vitro studies to those both in mouse and humans. In vitro, we have analyzed trafficking and cell-surface delivery of EGFR and its ligands in a battery of CRC cell lines that retain the ability to form uniform polarizing monolayers on Transwell filters. In the mouse, we have discovered that the pan-ERBB inhibitor, LRIG1, marks a largely quiescent population of colonic stem cells distinct from LGR5 and that administration of tamoxifen to Lrig1-CreET2;Apcfl/+ mice eliminates one allele of Apc and results in highly penetrant, multiple, histologically advanced adenomas in the distal colon 50-100 days later; these adenomas show the expected loss of the 2nd Apc allele. Later this month, we will submit the 4th renewal of our GI Specialized Programs of Research Excellence (SPORE) that focuses on CRC. An advance in each of these areas will be discussed. Based on our trafficking studies, we have discovered that EGFR ligands can be released in endosome-derived small vesicles called exosomes. The ligands are signaling competent and bind to EGFR on recipient cells. We have termed this new mode of signaling, Exosomal Targeted Receptor Activation (ExTRAcrine). We have recently advanced this work to studying EGFR in exosomes isolated from mouse and human plasma using a method we have developed, fluorescence-based vesicle sorting (FAVS). Second, a limitation to studying EGFR in the mouse is the lack of robust reagents to monitor EGFR by immunohistochemistry. To circumvent this limitation, we have used CRISPR gene editing to target the endogenous mouse locus to create a chimeric EGFR-Emerald (green) reporter mouse. We will present data showing the value of this mouse in monitoring EGFR in the normal colon and in colonic tumors generated in our Lrig1-CreERT2;Apcfl/+ mouse model. Finally, we have developed a new 3D system to study CRC. Using this system, we have discovered a novel mode of cetuximab resistance and will show its relevance to human CRC. 1. Singh B, Coffey RJ. Trafficking of epidermal growth factor receptor ligands in polarized epithelial cells. Annu Rev Physiol 76: 275-300, 2014. PMC4180094. 2. Higginbotham JN, Demory Beckler M, Gephart JD, Franklin JL, Bogatcheva G, Kremers GJ, Piston DW, Ayers GD, McConnell RE, Tyska MJ, Coffey RJ. Amphiregulin exosomes increase cancer cell invasion. Curr Biol 21: 779-86, 2011. PMC3417320. 3. Higginbotham JN, Zhang Q, Jeppesen DK, Scott AM, Manning HC, Ochieng J, Franklin JL, Coffey RJ. Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting. J Extracell Vesicles 5: 29254, 2016. PMC4921784. 4. Powell AE, Wang Y, Li Y, Poulin EJ, Means AL, Washington MK, Higginbotham JN, Juchheim A, Prasad N, Levy SE, Guo Y, Shyr Y, Aronow BJ, Haigis KM, Franklin JL, Coffey RJ. The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor. Cell 149: 146-58, 2012. PMCPMC3563328. 5. Powell AE, Vlacich G, Zhao ZY, McKinley ET, Washington MK, Manning HC, Coffey RJ. Inducible loss of one Apc allele in Lrig1-expressing progenitor cells results in multiple distal colonic tumors with features of familial adenomatous polyposis. Am J Physiol Gastrointest Liver Physiol 307: G16-23, 2014. PMC4080164. 6. Wang Y, Shi C, Lu Y, Poulin EJ, Franklin JL, Coffey RJ. Loss of Lrig1 leads to expansion of Brunner glands followed by duodenal adenomas with gastric metaplasia. Am J Pathol 185: 1123-34, 2015. PMC4380843. 7. Poulin EJ, Powell AE, Wang Y, Li Y, Franklin JL, Coffey RJ. Using a new Lrig1 reporter mouse to assess differences between two Lrig1 antibodies in the intestine. Stem Cell Res 13: 422-430, 2014. PMC638408. 8. Kondo J, Powell AE, Wang Y, Musser MA, Southard-Smith EM, Franklin JL, Coffey RJ. LRIG1 Regulates Ontogeny of Smooth Muscle-Derived Subsets of Interstitial Cells of Cajal in Mice. Gastroenterology 149: 407-19 e8, 2015. PMC4527342. Citation Format: Robert J. Coffey. Recent advances in studying EGFR and its ligands in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr IA01.

Potential therapeutic effect of epigenetic therapy on treatment-induced neuroendocrine prostate cancer.

Although adenocarcinomas of the prostate are relatively indolent, some patients with advanced adenocarcinomas show recurrence of treatment-induced neuroendocrine prostate cancer, which is highly aggressive and lethal. Detailed biological features of treatment-induced neuroendocrine prostate cancer have not been characterized owing to limited biopsies/resections and the lack of a cellular model. In this study, we used a unique cellular model (LNCaP/NE1.8) to investigate the potential role of cancer stem cells in treatment-induced neuroendocrine prostate cancer with acquired resistance to hormonal therapy and chemotherapy. We also studied the role of cancer stem cells in enhancing invasion in treatment-induced neuroendocrine prostate cancer cells that recurred after long-term androgen-ablation treatment. Using an in vitro system mimicking clinical androgen-ablation, our results showed that the neuroendocrine-like subclone NE1.8 cells were enriched with cancer stem cells. Compared to parental prostate adenocarcinoma LNCaP cells, NE1.8 cells are more resistant to androgen deprivation therapy and chemotherapeutic agents and show increased cancer cell invasiveness. Results from this study also suggest a potential epigenetic therapeutic strategy using suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, as a chemotherapeutic agent for therapy-resistant treatment-induced neuroendocrine prostate cancer cells to minimize the risk of prostate cancer recurrence and metastasis.

MicroRNA-21 induces breast cancer cell invasion and migration by suppressing smad7 via EGF and TGF-β pathways.

MicroRNA-21 (miR-21) upregulation, smad family member 7 (smad7) downregulation, epidermal growth factor (EGF) and transforming growth factor-β (TGF-β) actions contribute to breast cancer cell aggressiveness. However, their correlation and the relevant molecular mechanisms involved remain to be elucidated. The present study was undertaken to determine the association of miR-21, smad7, EGF and TGF-β with breast cancer cell invasion and migration and to identify the molecular mechanisms involved using immunohistochemistry and western blot analysis. In the present study, the plasma miR-21 levels were significantly increased in patients with breast cancer, as compared to the controls. Smad7 was confirmed to be a direct target of miR-21, by luciferase reporter and western blot assays. The downregulation of smad7 by miR-21 or sismad7 enhanced EGF-dependent invasion and migration, as well as TGF-β-dependent invasion and migration. The actions of miR-21 were abrogated by expressing a modified smad7 cDNA resistant to miR-21. Moreover, miR-21, EGF and TGF-β combined to markedly increase cancer cell invasion and migration, and this effect was blocked by the combination of erlotinib (an EGF receptor kinase inhibitor) and SB505124 (a type I TGF-β receptor inhibitor). A lower smad7 expression was identified in poorly differentiated breast cancers, as compared to well- to moderately differentiated breast cancers. Notably, antagonism of miR-21 decreased breast cancer cell proliferation and tumor growth in mouse models. In conclusion, our results demonstrated that plasma miR-21 levels may serve as a diagnostic marker in breast cancers, whereas miR-21 promotes breast cancer cell proliferation and invasion by suppressing smad7, which enhances EGF and TGF-β pathways.

Ubiquitin C-terminal hydrolase-L1 increases cancer cell invasion by modulating hydrogen peroxide generated via NADPH oxidase 4.

This study explored the role of ubiquitin C-terminal hydrolase-L1 (UCH-L1) in the production of ROS and tumor invasion. UCH-L1 was found to increase cellular ROS levels and promote cell invasion. Silencing UCH-L1, as well as inhibition of H2O2 generation by catalase or by DPI, a NOX inhibitor, suppressed the migration potential of B16F10 cells, indicating that UCH-L1 promotes cell migration by up-regulating H2O2 generation. Silencing NOX4, which generates H2O2, with siRNA eliminated the effect of UCH-L1 on cell migration. On the other hand, NOX4 overexpressed in HeLa cells happens to be ubiquitinated, and NOX4 following deubiquitination by UCH-L1, restored H2O2-generating activity. These in vitro findings are consistent with the results obtained in vivo with catalase (-/-) C57BL/6J mice. When H2O2 and UCH-L1 levels were independently varied in these animals, the former by infecting with H2O2-scavenging adenovirus-catalase, and the latter by overexpressing or silencing UCH-L1, pulmonary metastasis of B16F10 cells overexpressing UCH-L1 increased significantly in catalase (-/-) mice. In contrast, invasion did not increase when UCH-L1 was silenced in the B16F10 cells. These findings indicate that H2O2 levels regulated by UCH-L1 are necessary for cell invasion to occur and demonstrate that UCH-L1 promotes cell invasion by up-regulating H2O2 via deubiquitination of NOX4.

Methylation-mediated silencing of Dlg5 facilitates bladder cancer metastasis

Dlg5 (Discs large homolog 5), a member of the membrane-associated guanylate kinase adaptor family of scaffolding proteins, has been shown to participate in cancer progression. However, little is known about whether abnormal expression of Dlg5 facilitates bladder cancer metastasis. In the current study we initiated a study analyzing Dlg5 expression and its roles in human bladder cancer metastasis. The expression of Dlg5 is decreased in most bladder cancer tissues compared with adjacent normal tissues, and Dlg5 expression is further downregulated in patients with muscle-invasive tumors. DNA methylation analysis showed a methylation of Dlg5 gene in bladder cancer cell lines and in bladder cancer tumors, especially in muscle-invasive tumors. Hypermethylation of Dlg5 in bladder tumors is tightly correlated with silencing of Dlg5 expression, which is further functionally validated by demethylation analysis in bladder cancer cell lines. Knockdown of Dlg5 increases cancer cell invasion in vitro and promotes cancer metastasis in vivo. Of clinical significance, Kaplan–Meier analysis showed that downregulation of Dlg5 is significantly associated with reduced overall survival in patients with bladder cancer. ConclusionThese data suggest that inhibition of Dlg5 by DNA hypermethylation contributes to provoke invasive phenotypes in bladder tumor.

Abstract 973: Tumor-associated fibroblasts induce long-term changes in breast cancer invasion, cancer stem cell populations, and metastasis through modulation of the Tiam1-ostepontin pathway

Abstract We have previously shown that silencing of the Rac exchange factor Tiam1 in tumor-associated fibroblasts induces increased invasion and metastasis in epithelial and cancer cells using 3D-tissue culture models and a mouse model of human breast cancer. Here we investigate the underlying mechanism using a novel method for isolating human breast cancer cells out of 3D spheroid co-cultures containing mammary fibroblasts. Co-culture of human breast cancer cells with Tiam1-deficient fibroblasts induces increased cancer cell invasion into matrix, while co-culture with Tiam1-overexpressing fibroblasts induces decreased invasion into matrix. Strikingly, breast cancer cells isolated from 3D culture with Tiam1-deficient fibroblasts exhibit persistent increases in migration, epithelial-mesenchymal transition (EMT), and cancer stem cell characteristics as assayed by tumorsphere assay, flow cytometry, and limiting dilution mouse xenograft implantation experiments. In contrast, breast cancer cells exposed to Tiam1-overexpressing fibroblasts and then isolated from 3D co-cultures persistently display the opposite phenotypes. The observed changes in the breast cancer cells are dependent on fibroblast-produced osteopontin (OPN), as OPN silencing or antibody-mediated inhibition abrogates the increased invasion, migration, and cancer stem cell populations induced by Tiam1-deficient fibroblasts. Treatment of co-cultures by chemotherapeutic agents does not prevent effects induced by Tiam1-deficient fibroblasts. In contrast, treatment of co-cultures by the OPN inhibitor Agelastatin A reverses all effects induced by Tiam1-deficient fibroblasts, and blocks subsequent development of lung metastases in the mouse xenograft model. Finally, in human breast cancer samples Tiam1 expression is significantly decreased in fibroblasts associated with invasive human breast cancers compared to fibroblasts associated with ductal carcinoma in situ (DCIS). Conversely, OPN expression is significantly increased in fibroblasts associated with invasive breast cancers compared to fibroblasts associated with DCIS. These results suggest that regulated Tiam1 expression in breast cancer-associated fibroblasts modulates breast cancer invasion, EMT, cancer stem cell populations, and metastasis through regulation of fibroblast OPN secretion. At least some breast cancers demonstrate commensurate changes in fibroblast Tiam1 and OPN expression correlating with invasiveness, suggesting a mechanism for therapeutic targeting. Citation Format: Kun Xu, Xuejun Tian, Stephen P. Naber, Sun Oh, Charlotte Kuperwasser, Rachel J. Buchsbaum. Tumor-associated fibroblasts induce long-term changes in breast cancer invasion, cancer stem cell populations, and metastasis through modulation of the Tiam1-ostepontin pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 973. doi:10.1158/1538-7445.AM2014-973

Sphingosine-1-Phosphate Mediates a Reciprocal Signaling Pathway between Stellate Cells and Cancer Cells that Promotes Pancreatic Cancer Growth

Sphingosine-1-phosphate (S1P) is produced by sphingosine kinase 1 and is implicated in tumor growth, although the mechanisms remain incompletely understood. Pancreatic stellate cells (PSCs) reside within the tumor microenvironment and may regulate tumor progression. We hypothesized that S1P activates PSCs to release paracrine factors, which, in turn, increase cancer cell invasion and growth. We used a combination of human tissue, invitro, and invivo studies to mechanistically evaluate this concept. Sphingosine kinase 1 was overexpressed in human pancreatic tissue, especially within tumor cells. S1P activated PSCs invitro and conditioned medium from S1P-stimulated PSCs, increased pancreatic cancer cell migration, and invasion, which was dependent on S1P2, ABL1 (alias c-Abl) kinase, and matrix metalloproteinase-9. Invivo studies showed that pancreatic cancer cells co-implanted with S1P2 receptor knockdown PSCs led to less cancer growth and metastasis in s.c. and orthotopic pancreatic cancer models compared with control PSCs. Pancreatic cancer cell-derived S1P activates PSCs to release paracrine factors, including matrix metalloproteinase-9, which reciprocally promotes tumor cell migration and invasion invitro and cancer growth invivo.

Extracellular Tissue Transglutaminase Activates Noncanonical NF-κB Signaling and Promotes Metastasis in Ovarian Cancer

Tissue transglutaminase (TG2) is a multifunctional protein that binds to fibronectin and exerts protein transamidating activity in the presence of Ca2+. We previously reported that TG2 is upregulated in ovarian tumors and enhances intraperitoneal (i.p.) metastasis. TG2 is secreted abundantly in ovarian cancer (OC) ascites as an active enzyme, yet its function in the extracellular compartment remains unknown. To study the distinct functions of secreted TG2, we used recombinant His6-tagged TG2 and catalytically inactive enzyme in vitro and in vivo. By using i.p. and orthotopic ovarian xenografts, we show that extracellular transglutaminase promoted OC peritoneal metastasis. The main pathway activated by extracellular TG2 was noncanonical nuclear factor-kappa B (NF-κB) signaling, and the enzymatic function of the protein was required to induce phosphorylation of IκBkinaseá and processing of the precursor protein p100 into the active p52 subunit. A specific target of TG2-activated p52/RelB complex is the hyaluronan receptor, CD44. Noncanonical NF-κB activation by extracellular TG2 induced CD44 up-regulation and epithelial-to-mesenchymal transition, contributing to increased cancer cell invasiveness and OC peritoneal dissemination. Taken together, our data support that noncanonical NF-κB activation is the pathway through which extracellular TG2 promotes OC metastasis.

Decreased carbonyl reductase 1 expression promotes malignant behaviours by induction of epithelial mesenchymal transition and its clinical significance

Human carbonyl reductase 1 (CBR1) is an enzyme that catalyse the reduction of many compounds by using NADPH-dependent oxydoreductase activity. Although CBR1 is known to regulate the tumour progression, the molecular mechanisms of CBR1 in cancer progression and the clinical significance of CBR1 status remain unclear. Here, we investigated the molecular mechanisms by which CBR1 affects cancer cell behaviour in vitro and the clinical significance of CBR1 using immunohistochemical analyses in endometrial cancer. Here, the role of CBR1 in cancer cell invasion and metastasis, and its molecular mechanisms were investigated by transfection of sense and antisense CBR1 cDNAs into a human endometrial adenocarcinoma cell line. The relationship between CBR1 expression analysed by immunohistochemistry and prognosis such as progression free survival (PFS) and overall survival (OS) was examined in endometrial cancer tissues from FIGO stage I–IV (n=109). Suppression of CBR1 by antisense CBR1 cDNA increased cancer cell invasion, and suppressed E-cadherin expression and capacity for cellular aggregation. In contrast, over-expression of CBR1 by sense CBR1 cDNA increased E-cadherin expression and capacity for cellular aggregation, and suppressed cancer cell invasion. The expression of transcriptional suppressors of E-cadherin, Snail and ZEB1, were increased by CBR1 suppression, but suppressed by CBR1 over-expression. Immunohistochemical analyses showed that decreased CBR1 expression is significantly related with poor PFS and OS compared with strong CBR1 expression. In multivariate analyses, decreased CBR1 expression was an independent prognostic factor for PFS and OS. CBR1 regulates cancer cell invasion in endometrial adenocarcinomas by regulating the epithelial mesenchymal transition. A decreased CBR1 expression can be a useful marker of an unfavourable clinical outcome in patients with endometrial cancer.

Contractile Forces Contribute to Increased Glycosylphosphatidylinositol-anchored Receptor CD24-facilitated Cancer Cell Invasion

The malignancy of a tumor depends on the capability of cancer cells to metastasize. The process of metastasis involves cell invasion through connective tissue and transmigration through endothelial monolayers. The expression of the glycosylphosphatidylinositol-anchored receptor CD24 is increased in several tumor types and is consistently associated with increased metastasis formation in patients. Furthermore, the localization of β1-integrins in lipid rafts depends on CD24. Cell invasion is a fundamental biomechanical process and usually requires cell adhesion to the extracellular matrix (ECM) mainly through β1 heterodimeric integrin receptors. Here, we studied the invasion of A125 human lung cancer cells with different CD24 expression levels in three-dimensional ECMs. We hypothesized that CD24 expression increases cancer cell invasion through increased contractile forces. To analyze this, A125 cells (CD24 negative) were stably transfected with CD24 and sorted for high and low CD24 expression. The invasiveness of the CD24(high) and CD24(low) transfectants was determined in three-dimensional ECMs. The percentage of invasive cells and their invasion depth was increased in CD24(high) cells compared with CD24(low) cells. Knockdown of CD24 and of the β1-integrin subunit in CD24(high) cells decreased their invasiveness, indicating that the increased invasiveness is CD24- and β1-integrin subunit-dependent. Fourier transform traction microscopy revealed that the CD24(high) cells generated 5-fold higher contractile forces compared with CD24(low) cells. To analyze whether contractile forces are essential for CD24-facilitated cell invasion, we performed invasion assays in the presence of myosin light chain kinase inhibitor ML-7 as well as Rho kinase inhibitor Y27632. Cell invasiveness was reduced after addition of ML-7 and Y27632 in CD24(high) cells but not in CD24(neg) cells. Moreover, after addition of lysophosphatidic acid or calyculin A, an increase in pre-stress in CD24(neg) cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase or STAT3 strongly reduced the invasiveness of CD24(high) cells, slightly reduced that of CD24(low) cells, and did not alter the invasiveness of CD24(neg) cells. Taken together, these results suggest that CD24 enhances cell invasion through increased generation or transmission of contractile forces.

Radiation-enhancement of MDA-MB-231 breast cancer cell invasion prevented by a cyclooxygenase-2 inhibitor.

Background:Recent evidences support that radiation can promote the invasion of cancer cells. As interactions between cancer cells and surrounding stromal cells can have an important role in tumour progression, we determined whether an irradiation to fibroblasts can enhance the invasiveness of breast cancer cells. The role of cyclooxygenase-2 (COX-2), an inflammatory enzyme frequently induced by radiotherapy, was investigated.Methods:Irradiated 3T3 fibroblasts were plated in the lower compartment of invasion chambers and used as chemoattractant for non-irradiated human breast cancer cell MDA-MB-231, which are oestrogen receptor negative (ER(−)) and the oestrogen receptor positive (ER(+)) MCF-7 cells. Stimulation of COX-2 expression in irradiated 3T3 cells was measured by a semi-quantitative qPCR and western blot. Capacity of the major product of COX-2, the prostaglandin E2 (PGE2), to stimulate the production of the matrix metalloproteinase-2 (MMP-2) and cancer cell invasion were assessed with a zymography gel and invasion chambers.Results:Irradiation (5 Gy) of 3T3 fibroblasts increased COX-2 expression and enhanced by 5.8-fold the invasiveness of non-irradiated MDA-MB-231 cells, while their migration was not modified. Addition of the COX-2 inhibitor NS-398 completely prevented radiation-enhancement of cancer cell invasion. Further supporting the potential role of COX-2, addition of PGE2 has increased cancer cell invasion and release of MMP-2 from the MDA-MB-231 cells. This effect of radiation was dependant on the expression of membrane type 1 (MT1)–MMP, which is required to activate the MMP-2, but was not associated with the ER status. Although irradiated fibroblasts stimulated the invasiveness of MDA-MB-231 ER(−) cells, no enhancement was measured with the ER(+) cell line MCF-7.Conclusions:Radiation-enhancement of breast cancer cell invasion induced by irradiated 3T3 fibroblasts is not dependant on the ER status, but rather the expression of MT1–MMP. This adverse effect of radiation can be prevented by a specific COX-2 inhibitor.

Suppression of carbonyl reductase expression enhances malignant behaviour in uterine cervical squamous cell carcinoma: Carbonyl reductase predicts prognosis and lymph node metastasis

Carbonyl reductase (CR) is an NADPH-dependent, mostly monomeric, cytosolic enzyme with broad substrate specificity for carbonyl compounds. CR appears to be involved in the regulation of tumour progression. However, molecular mechanisms of CR in tumour progression and clinical significance of CR status remain unclear in human uterine squamous cell carcinoma (SCC). Here, we investigated the clinical significance of CR using immunohistochemical analyses of human uterine cervical SCC tissues and how CR affects cancer cell behaviour in vitro. Paraffin sections from uterine cervical SCC tissues, FIGO stage Ib1-IIb ( n = 67) were immunostained with anti-CR antibodies. Overall survival (OS) and progression-free survival (PFS) were analyzed by the Kaplan–Meier method. Sense and antisense CR cDNAs were transfected into a human uterine SCC cell line (SiHa) to investigate the role of CR in cancer cell invasion and metastasis. Immunohistochemical analyses showed that reduced CR expression patterns in primary cancer lesions were closely associated with a high incidence of pelvic lymph node metastasis, poor OS, and poor PFS. In an in vitro experiment, suppression of CR increased cancer cell invasion, secretion of MMP-2, -9 and cyclooxygenase-2 (COX-2) expression and decreased E-cadherin expression. On the other hand, over-expression of CR increased E-cadherin expression and decreased MMP-2, -9 secretion and COX-2 expression. The reduced CR expression pattern, as measured by immunohistochemistry, can be a useful predictor of lymph node metastasis and poor prognosis in patients with uterine SCC. This clinical result is supported by the in vitro data which show that suppression of CR expression promotes cancer cell invasion with decreased E-cadherin expression and increased MMP-2, -9 secretion.

MiR-200c at the nexus of epithelial-mesenchymal transition, resistance to apoptosis, and the breast cancer stem cell phenotype

Decreased expression of miRNAs of the miR-200 family has been implicated in the growth and metastasis of breast cancer cells. Of this family, miR-200c has garnered particular attention as a consequence of its ability to target ZEB1 and ZEB2, mediators of epithelial- mesenchymal transition. An article in the previous issue of Breast Cancer Research identifies additional targets of miR-200c that link increased cancer cell invasiveness, resistance to apoptosis, and induction of breast cancer stem cell characteristics.

Abstract 387: Prostate cancer cell invasion is stimulated by cross communication with human monocyte-lineage cells

Abstract Tumor-associated macrophages (TAMs) produce factors that may stimulate tumor growth, angiogenesis and progression. In this study, co-cultures with monocyte-derived THP-1, U-937 cells or normal peripheral blood monocytes stimulated PC-3, DU145 and LNCaP prostate cancer cell basement membrane invasion. This study investigated the interactions between monocytes and prostate cancer cells leading to increased cancer cell invasion. The PC-3 prostate cancer cells expressed high levels of IL-6 and IL-8, at similar levels to co-cultures with human U-937 monocyte-derived cells. However, the co-culture supernatants showed increased MCP-1 levels, up to 5 ng/mL, compared with prostate cancer cells cultured alone with MCP-1 less than 10 pg/mL. During the co-cultures, MCP-1 was first detected at 8 hours and increased over 3 days. Co-culture of U-937 or THP-1 monocytes with either DU145 or LNCaP cells also increased MCP-1 expression significantly above cancer cell or monocyte cultures alone. When cultured separately, the monocyte-lineage cells produced more MCP-1 than the cancer cells; however, MCP-1 expression increased more than 10-fold in the co-cultured prostate cancer cells. Addition of purified MCP-1 and IL-8 stimulated prostate cancer cell invasion to levels similar to the co-cultures. Further, addition of anti-MCP-1 neutralizing antibodies inhibited prostate cancer cell invasion when compared with control antibodies. Co-culture with U-937 cells also stimulated NF-κB DNA binding activity in the prostate cancer cells. Transfection of the prostate cancer cells with dominant negative IκBα or treatment of the co-cultures with Bay11-7082 or Lacticystin inhibited NF-κB DNA binding and prostate cancer cell invasion. These experiments demonstrate that human monocyte-derived cells can cross-communicate with prostate cancer cells leading to increased cancer cell invasion. These studies have potential to identify treatments to target MCP-1 and NF-κB activity to inhibit monocyte-induced cancer cell invasion and progression. Support by Northwestern University Prostate S.P.O.R.E. P50CA90386 and CDMRP DAMD-17-02-1-0162 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 387. doi:10.1158/1538-7445.AM2011-387

The GPI-Anchored Receptor CD24 Increases Cancer Cell Invasion through Enhanced Contractile Forces

The malignancy of a tumor depends on the capability of cancer cells to metastasize. The process of metastasis involves cell invasion through connective tissue and transmigration through endothelial monolayers. The GPI-anchored receptor CD24 expression is increased in several tumor types and is consistently associated with increased metastasis formation in patients. Furthermore, the localization of β1 integrins in lipid rafts depends on CD24. Cell invasion is a fundamental biomechanical process and usually requires cell adhesion to the extracellular matrix (ECM) through mainly β1 heterodimeric integrin receptors. Here, we studied the invasion of human lung A125 cancer cells with different CD24 expression levels in 3D ECMs. We hypothesized that CD24 expression increases the invasiveness of cancer cells by enhanced contractile force transmission or generation.To analyze this, CD24neg cells (CD24-negative) were stably transfected with CD24 and sorted for high and low CD24 expression. The invasiveness of the CD24high and CD24low transfectants were determined in 3D-ECMs. The percentage of invasive cells and their invasion depth was increased in CD24high cells compared to CD24low cells.Knock-down of CD24 and of the β1 integrin subunit in CD24high cells decreased their invasiveness, indicating that the increased invasiveness is CD24 and β1 integrin subunit dependent.Fourier transform traction microscopy revealed that the CD24high cells generated 5-fold higher contractile forces compared to CD24low cells. To test whether contractile forces were functional related to cell invasion. Cell invasiveness was reduced after addition of myosin light chain kinase inhibitor ML-7 as well as Rho kinase inhibitor Y27632 in CD24high cells, but not in CD24neg cells, whereas an increase in prestress in CD24neg cells after addition of LPA increased cell invasiveness. Taken together, these results suggest that CD24 enhances cell invasion through increased transmission or generation of contractile forces.

PGF2α-F-prostanoid receptor signalling via ADAMTS1 modulates epithelial cell invasion and endothelial cell function in endometrial cancer

BackgroundAn increase in cancer cell invasion and microvascular density is associated with a poorer prognosis for patients with endometrial cancer. In endometrial adenocarcinoma F-prostanoid (FP) receptor expression is elevated, along with its ligand prostaglandin (PG)F2α, where it regulates expression and secretion of a host of growth factors and chemokines involved in tumorigenesis. This study investigates the expression, regulation and role of a disintegrin and metalloproteinase with thrombospondin repeat 1 (ADAMTS1) in endometrial adenocarcinoma cells by PGF2α via the FP receptor.MethodsHuman endometrium and adenocarcinoma tissues were obtained in accordance with Lothian Research Ethics Committee guidance with informed patient consent. Expression of ADAMTS1 mRNA and protein in tissues was determined by quantitative RT-PCR analysis and immunohistochemistry. Signal transduction pathways regulating ADAMTS1 expression in Ishikawa cells stably expressing the FP receptor to levels seen in endometrial cancer (FPS cells) were determined by quantitative RT-PCR analysis. In vitro invasion and proliferation assays were performed with FPS cells and human umbilical vein endothelial cells (HUVECs) using conditioned medium (CM) from PGF2α-treated FPS cells from which ADAMTS1 was immunoneutralised and/or recombinant ADAMTS1. The role of endothelial ADAMTS1 in endothelial cell proliferation was confirmed with RNA interference. The data in this study were analysed by T-test or ANOVA.ResultsADAMTS1 mRNA and protein expression is elevated in endometrial adenocarcinoma tissues compared with normal proliferative phase endometrium and is localised to the glandular and vascular cells. Using FPS cells, we show that PGF2α-FP signalling upregulates ADAMTS1 expression via a calmodulin-NFAT-dependent pathway and this promotes epithelial cell invasion through ECM and inhibits endothelial cell proliferation. Furthermore, we show that CM from FPS cells regulates endothelial cell ADAMTS1 expression in a rapid biphasic manner. Using RNA interference we show that endothelial cell ADAMTS1 also negatively regulates cellular proliferation.ConclusionsThese data demonstrate elevated ADAMTS1 expression in endometrial adenocarcinoma. Furthermore we have highlighted a mechanism whereby FP receptor signalling regulates epithelial cell invasion and endothelial cell function via the PGF2α-FP receptor mediated induction of ADAMTS1.

P120ctn and P-Cadherin but Not E-Cadherin Regulate Cell Motility and Invasion of DU145 Prostate Cancer Cells

BackgroundAdherens junctions consist of transmembrane cadherins, which interact intracellularly with p120ctn, ß-catenin and α-catenin. p120ctn is known to regulate cell-cell adhesion by increasing cadherin stability, but the effects of other adherens junction components on cell-cell adhesion have not been compared with that of p120ctn.Methodology/Principal FindingsWe show that depletion of p120ctn by small interfering RNA (siRNA) in DU145 prostate cancer and MCF10A breast epithelial cells reduces the expression levels of the adherens junction proteins, E-cadherin, P-cadherin, ß-catenin and α-catenin, and induces loss of cell-cell adhesion. p120ctn-depleted cells also have increased migration speed and invasion, which correlates with increased Rap1 but not Rac1 or RhoA activity. Downregulation of P-cadherin, β-catenin and α-catenin but not E-cadherin induces a loss of cell-cell adhesion, increased migration and enhanced invasion similar to p120ctn depletion. However, only p120ctn depletion leads to a decrease in the levels of other adherens junction proteins.Conclusions/SignificanceOur data indicate that P-cadherin but not E-cadherin is important for maintaining adherens junctions in DU145 and MCF10A cells, and that depletion of any of the cadherin-associated proteins, p120ctn, ß-catenin or α-catenin, is sufficient to disrupt adherens junctions in DU145 cells and increase migration and cancer cell invasion.

Chloramphenicol Causes Mitochondrial Stress, Decreases ATP Biosynthesis, Induces Matrix Metalloproteinase-13 Expression, and Solid-Tumor Cell Invasion

Overuse and abuse of antibiotics can increase the risk of cancer. Chloramphenicol can inhibit both bacterial and mitochondrial protein synthesis, causing mitochondrial stress and decreased ATP biosynthesis. Chloramphenicol can accelerate cancer progression; however, the underlying mechanisms of chloramphenicol in carcinogenesis and cancer progression are still unclear. We found that chloramphenicol can induce matrix metalloproteinase (MMP)-13 expression and increase MMP-13 protein in conditioned medium, resulting in an increase in cancer cell invasion. Chloramphenicol also activated c-Jun N-terminal kinases (JNK) and phosphatidylinositol 3-kinase (PI-3K)/Akt signaling, leading to c-Jun protein phosphorylation. The activated c-Jun protein has been proven to activate binding to the MMP-13 promoter and also upregulate the amount of MMP-13. Both the SP 600125 (JNK inhibitor) and LY 294002 (PI-3K/Akt inhibitor) can inhibit chloramphenicol-induced c-Jun phosphorylation, MMP-13 expression, and cell invasion. Overexpression of the dominant-negative JNK and PI-3K p85 subunit also negate chloramphenicol-induced responses. Other antibiotics that cause mitochondrial stress and a decrease in ATP biosynthesis also induce MMP-13 expression. These findings suggest that chloramphenicol-induced PI-3K/Akt, JNK phosphorylation, and activator protein 1 activation might function as a novel mitochondrial stress signal that result in an increase of MMP-13 expression and MMP-13-associated cancer cell invasion. The findings of this study confirms that chloramphenicol, and other 70S ribosomal inhibitors, should be administered with caution, especially during cancer therapy.

Induction of invasion in an organotypic oral cancer model by CoCl2, a hypoxia mimetic

Invasion is a hallmark of malignancy. The aim of this study was to develop an in vitro model that can be used for experimental studies of cancer cell invasion. The organotypic oral cancer model was constructed by growing oral squamous cell carcinoma (OSCC) cells on a collagen matrix in which normal human fibroblasts were incorporated. Immunohistochemical staining of the model showed that the expression of invasion-related molecules such as phosphorylated extracellular signal-regulated kinases 1 and 2 (p-ERK1/2), cyclooxygenase-2 (COX-2), p75(NTR), and hepatocyte growth factor receptor (Met) was similar to that seen in OSCC. Treatment of the model with cobalt chloride (CoCl(2)) to mimic hypoxic conditions increased cancer cell invasion, defined as the appearance of cancer cell islands protruding into the matrix. Models treated with CoCl(2) showed increased expression of p75(NTR) and laminin-5 in the cancer cells, and a more pronounced fragmentation of collagen IV in the basal membrane area, in contrast to models that were left untreated. The results indicate that the present model is well suited for studies on cancer cell invasion in the matrix and that the addition of CoCl(2) on day 3 of the experiment is indicated because it markedly increases the invasion and improves the model.