Motility Of Colon Cancer Cells Research Articles

Modulation of intracellular calcium levels by calcium lactate affects colon cancer cell motility through calcium-dependent calpain.

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca2+) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca2+ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca2+ (iCa2+) levels for a prolonged period of time. Ca2+ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca2+ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca2+ supplementation to patient undergoing treatment for metastatic cancer.

CUB domain containing protein 1 (CDCP1) modulates adhesion and motility in colon cancer cells.

BackgroundDeregulated expression of the transmembrane glycoprotein CDCP1 (CUB domain-containing protein-1) has been detected in several cancers including colon, lung, gastric, breast, and pancreatic carcinomas. CDCP1 has been proposed to either positively or negatively regulate tumour metastasis. In this study we assessed the role of CDCP1 in properties of cells that are directly relevant to metastasis, namely adhesion and motility. In addition, association between CDCP1 and the tetraspanin protein CD9 was investigated.MethodsCDCP1 and CD9 protein expression was measured in a series of colon cancer cell lines by flow cytometry and Western blotting. Adhesion of Colo320 and SW480 cells was determined using a Matrigel adhesion assay. The chemotactic motility of SW480 cells in which CDCP1 expression had been reduced by RNA interference was analysed using the xCELLigence system Real-Time Cell Analyzer Dual Plates combined with 8 μm pore filters. Detergent-resistant membrane fractions were generated following density gradient centrifugation and the CDCP1 and CD9 protein composition of these fractions was determined by Western blotting. The potential association of the CDCP1 and CD9 proteins was assessed by co-immunoprecipitation.ResultsEngineered CDCP1 expression in Colo320 cells resulted in a reduction in cell adhesion to Matrigel. Treatment of SW480 cells with CDCP1 siRNA reduced serum-induced chemotaxis. CDCP1 and CD9 cell-surface protein and mRNA levels showed a positive correlation in colon cancer cell lines and the proteins formed a low-level, but detectable complex as judged by co-sedimentation of detergent lysates of HT-29 cells in sucrose gradients as well as by co-immunoprecipitation in SW480 cell lysates.ConclusionsA number of recent studies have assigned a potentially important role for the cell-surface protein CDCP1 in invasion and metastasis of a several types of human cancer cells. In this study, CDCP1 was shown to modulate cell-substratum adhesion and motility in colon cancer cell lines, with some variation depending on the colon cancer cell type. CDCP1 and CD9 were co-expressed at the mRNA and protein level and we obtained evidence for the presence of a molecular complex of these proteins in SW480 colon cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-754) contains supplementary material, which is available to authorized users.

PTyr421 Cortactin Is Overexpressed in Colon Cancer and Is Dephosphorylated by Curcumin: Involvement of Non-Receptor Type 1 Protein Tyrosine Phosphatase (PTPN1)

Cortactin (CTTN), first identified as a major substrate of the Src tyrosine kinase, actively participates in branching F-actin assembly and in cell motility and invasion. CTTN gene is amplified and its protein is overexpressed in several types of cancer. The phosphorylated form of cortactin (pTyr421) is required for cancer cell motility and invasion. In this study, we demonstrate that a majority of the tested primary colorectal tumor specimens show greatly enhanced expression of pTyr421-CTTN, but no change at the mRNA level as compared to healthy subjects, thus suggesting post-translational activation rather than gene amplification in these tumors. Curcumin (diferulolylmethane), a natural compound with promising chemopreventive and chemosensitizing effects, reduced the indirect association of cortactin with the plasma membrane protein fraction in colon adenocarcinoma cells as measured by surface biotinylation, mass spectrometry, and Western blotting. Curcumin significantly decreased the pTyr421-CTTN in HCT116 cells and SW480 cells, but was ineffective in HT-29 cells. Curcumin physically interacted with PTPN1 tyrosine phosphatases to increase its activity and lead to dephosphorylation of pTyr421-CTTN. PTPN1 inhibition eliminated the effects of curcumin on pTyr421-CTTN. Transduction with adenovirally-encoded CTTN increased migration of HCT116, SW480, and HT-29. Curcumin decreased migration of HCT116 and SW480 cells which highly express PTPN1, but not of HT-29 cells with significantly reduced endogenous expression of PTPN1. Curcumin significantly reduced the physical interaction of CTTN and pTyr421-CTTN with p120 catenin (CTNND1). Collectively, these data suggest that curcumin is an activator of PTPN1 and can reduce cell motility in colon cancer via dephosphorylation of pTyr421-CTTN which could be exploited for novel therapeutic approaches in colon cancer therapy based on tumor pTyr421-CTTN expression.

Abstract 5500: Ormeloxifene modulates beta-catenin/TCF pathway in colon cancer.

Abstract BACKGROUND: Colorectal cancer is the leading cause of cancer-related death and the third most commonly diagnosed cancer in both males and females in the US. While the overall rates of colon cancer have been steadily falling in the past decade, the rates of colon cancer continue to increase in the under-50 age group. Therefore, developing newer strategies for the prevention and treatment of colon cancer is urgently needed. Aberrant β-catenin signaling has been detected in over 90% of colon cancer and is a central signaling pathway leading to the development of colon cancer. Mutations in components of this pathway lead to the accumulation of β-catenin in the nucleus where it functions as a co-transcription factor to transcribe proto-oncogenes. Therefore, strategies to regulate this specific signaling pathway can be developed as a novel therapeutic modality for the treatment of colon cancer. Ormeloxifene is a synthetic non-steroidal selective estrogen receptor modulator that has been widely used as an oral contraceptive and has demonstrated promising results in the inhibition of breast cancer. Herein, we have investigated the effect of ormeloxifene on colon cancer cell growth and its effect on β-catenin transcriptional activity. MATERIALS AND METHODS: SW480, SW620 colon cancer cells were used to investigate the effect of ormeloxifene (5-20 μM) on various cellular characteristics, including cell proliferation, clonogenic potential, motility and aggregation using standard procedures. β-catenin/T cell factor (TCF) transcription activity was measured by luciferase reporter assays using reporter constructs Topflash or Fopflash and pRL-TK (Renilla luciferase) and with Dual-Glo reagent. The expression and subcellular localization of proteins was determined by immunoblotting and confocal microscopy. RESULTS: Ormeloxifene demonstrated a concentration dependent (5-20 μM) inhibition of cell proliferation in the colon cells. In addition, ormeloxifene very effectively suppressed growth of colon cancer cells in both anchorage dependent and anchorage independent colony formation assays. Furthermore, ormeloxifene effectively inhibited cellular motility of colon cancer cells. Repression of these cellular phenotypes was correlated with attenuation of β-catenin/TCF4 transcriptional activity and the modulation of subcellular localization of β-catenin. Western blot analyses revealed reduction of TCF4 levels upon ormeloxifene treatment. CONCLUSION: Our findings suggest that ormeloxifene effectively inhibits tumorigenic characteristics of colon cancer cells via attenuating the β-catenin/TCF4 signaling pathway. It may therefore be a novel therapeutic modality for the prevention, treatment and management of colon cancer. Citation Format: Vasudha Sundram, Rishi Kumar Gara, Man Mohan Singh, Subhash C. Chauhan, Meena Jaggi. Ormeloxifene modulates beta-catenin/TCF pathway in colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5500. doi:10.1158/1538-7445.AM2013-5500

Abstract 1478: The role of ERBIN in suppressing migration in colon cancer.

Abstract The LAP (leucine-rich repeat (LRR) and PDZ domain) family protein, ERBIN, is basolaterally localized protein that has previously been identified as a regulator of epithelial cell polarity and an ERBB2/HER2 binding protein. In addition, ERBIN inhibits RAF activation by disrupting by the RAS/RAF interaction. However, the functional importance of ERBIN in cancer remains elusive. In this study, we examined the role of ERBIN in regulating colon cancer progression and metastasis. Stable colon cancer cell lines, including SW480, LIM2405, and Caco2, were generated using lentiviral shRNA to deplete endogenous ERBIN expression. Our results showed that knockdown of ERBIN induced epithelial-mesenchymal transition (EMT)-like phenotype as marked by spindle-like morphology, a decrease in E-cadherin and an increase in vimentin expression. Moreover, the overall rate of cell migration was significantly increased in ERBIN knockdown cells as determined using Transwell assays. When tracking the movement of individual cells using live cell imaging, we found that ERBIN knockdown cells were considerably more motile with faster migrating speed when compared to control cells. Mechanistically, knockdown of ERBIN expression resulted in an increase in both Akt and ERK signaling activation. Taken together, we have identified a novel role of ERBIN in suppressing EMT and cell motility in colon cancer cells. Citation Format: Payton D. Stevens, Mike Yang, Tianyan Gao. The role of ERBIN in suppressing migration in colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1478. doi:10.1158/1538-7445.AM2013-1478

Abstract 4295: PHLPP suppresses epithelial-mesenchymal transition and cancer cell invasion by negatively regulating RAF1 activity.

Abstract PHLPP belongs to a family of protein phosphatases that have previously been shown to inhibit Akt-mediated cell proliferation and survival signaling in cancers. Here, we demonstrate that PHLPP exerts its tumor suppressor function by negatively regulating the RAF/MEK/ERK pathway to inhibit cell migration and invasion. Specifically, we identify the oncogenic protein kinase RAF1 as an interacting protein and a novel substrate of PHLPP. PHLPP dephosphorylates the S338 site of RAF1 and inactivates the kinase both in vitro and in cells. Loss of PHLPP expression largely increases the amplitude and duration of RAF/MEK/ERK signaling in cells, whereas overexpression of PHLPP inhibits the pathway. Functionally, knockdown of PHLPP induces epithelial-mesenchymal transition (EMT)-like phenotype, which coincides with a significant increase in migration and invasion in colon cancer cells. Furthermore, we show that upregulation of transcription factors ZEB1 and ZEB2 is responsible for increased invasiveness observed in PHLPP knockdown cells. Taken together, we have identified a novel role of PHLPP in suppressing EMT and cell motility in colon cancer cells by directly antagonizing RAF1 function. Citation Format: Xin Li, Payton D. Stevens, Tianyan Gao. PHLPP suppresses epithelial-mesenchymal transition and cancer cell invasion by negatively regulating RAF1 activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4295. doi:10.1158/1538-7445.AM2013-4295

레스베라트롤의 HT-29 대장암 세포증식 및 이동성 억제효과

Resveratrol, natural polyphenol in grapes and red wine, is known to have the anti-proliferatory and anti-angiogenic effects in various cancer cells. In this study, we have investigated the effects of resveratrol in HT-29 colon cancer cells. Treatment of resveratrol in different concentrations and time inhibited proliferation of HT-29 colon cancer cells. We explored the effects of resveratrol on HT-29 colon cancer cell motility using a wound healing assay. In the absence of the resveratrol, the HT-29 cells are migrated along the edges of the wound and showed a large-scale migration, whereas dose- and time-dependent inhibition of cell flattening and spreading was observed in the presence of resveratrol. Resveratrol inhibited MMP-9 in a dose- and time-dependent on HT-29 colon cancer cells by Western blotting. In addition, resveratrol increased AMPK activity and decreased COX-2, VASP and VEGF expression. Treatment of compound C inhibited AMPK activity, however, the expression of VASP and COX-2 increased thus, COX-2 and VASP are modulated by AMPK. However treatment of celecoxib could not control AMPK activity but decreased VEGF expression. We suggest that resveratrol inhibits cell proliferation and migration through activation of AMPK and decreased COX-2, VASP and VEGF expression in HT-29 colon cancer cells.

Matrix Metalloproteinase-2 Cleavage of the β1 Integrin Ectodomain Facilitates Colon Cancer Cell Motility

Cancer cell invasion is a key element in metastasis that requires integrins for adhesion/de-adhesion, as well as matrix metalloproteinases (MMPs) for focalized proteolysis. Herein we show that MMP-2 is up-regulated in resected colorectal tumors and degrades β1 integrins with the release of fragments containing the β1 I-domain. The β1 cleavage pattern is similar to that produced by digestion of α5β1 and α2β1 with MMP-2. Two such fragments, at 25 and 75 kDa, were identified after immunoprecipitation, with monoclonal antibody BD610468 reacting with the NH(2)-terminal I-like ectodomain followed by SDS-PAGE and microsequencing using electrospray (ISI-Q-TOF-Micromass) spectrometry. Cleavage of the β1 integrin can be abolished by inhibition of MMP-2 activity; it can be induced by up-regulation of MMP-2 expression, as exemplified by HT29 colon cancer cells transfected with pCMV6-XL5-MMP-2. Co-immunoprecipitation studies of colon cancer cells showed that the β1 integrin subunit is associated with MMP-2. The MMP-2-mediated shedding of the I-like domain from β1 integrins resulted in decreased adhesion of colon cancer cells to collagen and fibronectin, thus abolishing their receptivity. Furthermore, such cells showed enhanced motility as evaluated by a "wound healing-like" assay and time-lapse microscopy, indicating their increased invasiveness. Altogether, our data demonstrate that MMP-2 amplifies the motility of colon cancer cells, not only by digesting the extracellular matrix components in the vicinity of cancer cells but also by inactivating their major β1 integrin receptors.

An RNA Interference Phenotypic Screen Identifies a Role for FGF Signals in Colon Cancer Progression

In tumor cells, stepwise oncogenic deregulation of signaling cascades induces alterations of cellular morphology and promotes the acquisition of malignant traits. Here, we identified a set of 21 genes, including FGF9, as determinants of tumor cell morphology by an RNA interference phenotypic screen in SW480 colon cancer cells. Using a panel of small molecular inhibitors, we subsequently established phenotypic effects, downstream signaling cascades, and associated gene expression signatures of FGF receptor signals. We found that inhibition of FGF signals induces epithelial cell adhesion and loss of motility in colon cancer cells. These effects are mediated via the mitogen-activated protein kinase (MAPK) and Rho GTPase cascades. In agreement with these findings, inhibition of the MEK1/2 or JNK cascades, but not of the PI3K-AKT signaling axis also induced epithelial cell morphology. Finally, we found that expression of FGF9 was strong in a subset of advanced colon cancers, and overexpression negatively correlated with patients' survival. Our functional and expression analyses suggest that FGF receptor signals can contribute to colon cancer progression.

S100A4-induced cell motility and metastasis is restricted by the Wnt/β-catenin pathway inhibitor calcimycin in colon cancer cells

The calcium-binding protein S100A4 is a central mediator of metastasis formation in colon cancer. S100A4 is a target gene of the Wnt/β-catenin pathway, which is constitutively active in the majority of colon cancers. In this study a high-throughput screen was performed to identify small-molecule compounds targeting the S100A4-promoter activity. In this screen calcimycin was identified as a transcriptional inhibitor of S100A4. In colon cancer cells calcimycin treatment reduced S100A4 mRNA and protein expression in a dose- and time-dependent manner. S100A4-induced cellular processes associated with metastasis formation, such as cell migration and invasion, were inhibited by calcimycin in an S100A4-specific manner. Calcimycin reduced β-catenin mRNA and protein levels despite the expression of Δ45-mutated β-catenin. Consequently, calcimycin inhibited Wnt/β-catenin pathway activity and the expression of prominent β-catenin target genes such as S100A4, cyclin D1, c-myc, and dickkopf-1. Finally, calcimycin treatment of human colon cancer cells inhibited metastasis formation in xenografted immunodeficient mice. Our results demonstrate that targeting the expression of S100A4 with calcimycin provides a functional strategy to restrict cell motility in colon cancer cells. Therefore calcimycin may be useful for studying S100A4 biology, and these studies may serve as a lead for the development of treatments for colon cancer metastasis.

Galectin-3 binding protein promotes cell motility in colon cancer by stimulating the shedding of protein tyrosine phosphatase kappa by proprotein convertase 5

It has previously been reported that shedding of the PTPκ ectodomain drives enhanced motility of colon cancer cells. Herein, we provide mechanism underlying the regulation of PTPκ shedding by galectin-3 binding protein. PTPκ was inarguably scissored by the processed form of proprotein convertase 5 (subtilisin/kexin type 5), and galectin-3 binding protein which is over-produced in colon cancer cells and tissues contributed to increased cancer cell motility by acting as a negative regulator of galectin-3 at the cell surface. The high expression ratio of galectin-3 binding protein to galectin-3 was clinically correlated to lymphatic invasion. These results suggest that galectin-3 binding protein may be a potential therapeutic target for treatment of, at least, colon cancer patients with high expression of galectin-3 binding protein.

The induction of S100p expression by the Prostaglandin E2 (PGE2)/EP4 receptor signaling pathway in colon cancer cells

Background: Prostaglandin E2 (PGE2) levels are frequently elevated in colorectal carcinomas. PGE2 is perceived via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is most relevant. PGE2/EP4-receptor interaction activates CREB via the ERK/MEK pathway. However, the downstream target genes activated by this pathway remained to be investigated.Methodology/Prinicipal Findings: Here, we have identified S100P (an EF-hand calcium binding protein) as a novel downstream target. We show by realtime RT-PCR that S100P mRNA levels are elevated in 14/17 (82%) colon tumor tissues as compared to paired adjacent normal colonic tissues. S100P expression is stimulated in the presence of PGE2 in a time dependent manner at mRNA and protein levels in colon, breast and pancreatic cancer cells. Pharmacological and RNAi-mediated inhibition of the EP4 receptor attenuates PGE2-dependent S100P mRNA induction. RNAi-mediated knockdown of CREB inhibits endogenous S100P expression. Furthermore, using luciferase reporter analysis and EMSA we show that mutation and/or deletion of the CRE sequence within the S100P promoter abolished PGE2-mediated transcriptional induction. Finally, we demonstrate that RNAi-mediated knockdown of S100P compromised invadopodia formation, colony growth and motility of colon cancer cells. Interestingly, endogenous knock down of S100P decreases ERK expression levels, suggesting a role for ERK in regulating S100P mediated cell growth and motility.Conclusions/Significance: Together, our findings show for the first time that S100P expression is regulated by PGE2/EP4-receptor signaling and may participate in a feedback signaling that perpetuates tumor cell growth and migration. Therefore, our data suggest that dysregulated S100P expression resulting from aberrant PGE2/EP4 receptor signaling may have important consequences relevant to colon cancer pathogenesis.

Inhibition of COX‐2 with NS‐398 decreases colon cancer cell motility through blocking epidermal growth factor receptor transactivation: possibilities for combination therapy

The use of non-steroidal anti-inflammatory drugs has proved of great interest in the prevention and treatment of colorectal cancer, although their precise mechanisms of action remain unclear. Overexpression of cyclooxygenase-2 (COX-2) and subsequent prostaglandin production promote metastasis and have been shown to increase cell motility in vitro. We have aimed to elucidate whether specific inhibition of COX-2 with NS-398 (NS-398 is a selective inhibitor of COX-2) would be able to inhibit motility of colorectal cancer cells and whether this was modulated through epidermal growth factor receptor (EGFR) transactivation. A transwell filter assay was used to study cell motility. Expression of COX-2, EGFR phosphorylation and prostaglandin E(2) (PGE(2)) receptors were assessed by Western blot analysis and reverse transcriptase-polymerase chain reaction. PGE(2) concentrations after NS-398 treatment were estimated by enzyme immunoassay. Treatment with NS-398 significantly reduced PGE(2) levels and reduced cell migration in the HT29 and HCA7 colorectal carcinoma cell lines and this effect was rescued by addition of PGE(2). Furthermore, specific inhibition of COX-2 with NS-398 reduced EGFR phosphorylation in colorectal cancer cells. Direct inhibition of EGFR activity with AG1478 reduced PGE(2)-stimulated motility, clearly demonstrating that PGE(2 )acts via the EGFR-signalling pathway. The novel combination of NS-398 and AG1478 dramatically reduced migration of colorectal cancer cells. The data presented indicate that the use of NS-398 in chemoprevention and adjuvant therapy for colorectal cancer may work in part, through the inhibition of cell motility. Furthermore, our data suggest that the combined use of non-steroidal anti-inflammatory drugs with EGFR antagonists could be explored further for future use in the clinic.

EPS8 Facilitates Cellular Growth and Motility of Colon Cancer Cells by Increasing the Expression and Activity of Focal Adhesion Kinase

In an attempt to study the role of Eps8 in human carcinogenesis, we observe that ectopic overexpression of Eps8 in SW480 cells (low Eps8 expression) increases cell proliferation. By contrast, expressing eps8 small interference RNA in SW620 and WiDr cells (high Eps8 expression) reduces their proliferation rate. Interestingly, attenuation of Eps8 decreases Src Pi-Tyr-416, Shc Pi-Tyr-317, and serum-induced FAK Pi-Tyr-397 and Pi-Tyr-861. Remarkably, by virtue of mammalian target of rapamycin/STAT3 Pi-Ser-727, Eps8 modulates FAK expression required for cell proliferation. Within 62% of colorectal tumor specimens examined, >2-fold enhancement of Eps8 as compared with their normal counterparts is observed, especially for those from the advanced stage. In agreement with the modulation of FAK by Eps8, the concomitant expression of these two proteins in tumor specimens is observed. Notably, Eps8 attenuation also impedes the motility of SW620 and WiDr cells, which can be rescued by ectopically expressed FAK. This finding discloses the indispensability of Eps8 and FAK in cell locomotion. These results provide a novel mechanism for Eps8-mediated FAK expression and activation in colon cancer cells.

The MIG-2/Integrin Interaction Strengthens Cell-Matrix Adhesion and Modulates Cell Motility

Integrin-mediated cell-matrix adhesion plays an important role in control of cell behavior. We report here that MIG-2, a widely expressed focal adhesion protein, interacts with beta1 and beta3 integrin cytoplasmic domains. Integrin binding is mediated by a single site within the MIG-2 FERM domain. Functionally, the MIG-2/integrin interaction recruits MIG-2 to focal adhesions. Furthermore, using alphaIIbbeta3 integrin-expressing Chinese hamster ovary cells, a well described model system for integrin activation, we show that MIG-2 promotes integrin activation and enhances cell-extracellular matrix adhesion. Although MIG-2 is expressed in many cell types, it is deficient in certain colon cancer cells. Expression of MIG-2, but not of an integrin binding-defective MIG-2 mutant, in MIG-2-null colon cancer cells strengthened cell-matrix adhesion, promoted focal adhesion formation, and reduced cell motility. These results suggest that the MIG-2/integrin interaction is an important element in the cellular control of integrin-mediated cell-matrix adhesion and that loss of this interaction likely contributes to high motility of colon cancer cells.

Enhanced expression of cholecystokinin‐2 receptor promotes the progression of colon cancer through activation of focal adhesion kinase

Focal adhesion kinase (FAK) is suggested to be intimately involved in the progression of malignancies. Our previous research has demonstrated that activation of cholecystokinin-2 receptor (CCK2R) by gastrin stimulates a rapid activation of FAK pathway in human colon cancer cells. The purpose of this study is to determine the role of CCK2R and FAK in the progression of colon cancer. In this study, matched tissue samples of primary colon cancer and adjacent normal colon mucosa from the same patient were collected from 45 patients with colon cancer undergoing surgical resection. The gastrin expression was detected using reverse transcription polymerase chain reaction (RT-PCR). The CCK2R expression was examined by in situ hybridization and RT-PCR. The expression of FAK and phosphorylated FAK at tyrosine 397 (phospho-FAK) were detected using immunohistochemistry and immunoblotting. Colo320 and SW787, 2 colon cancer cell lines with or without CCK2R expression, were recruited in this study. Antisense oligonucleotide of FAK was used to block the expression of FAK. Invasiveness and motility of colon cancer cells were detected by Boyden chamber. In this series, enhanced expression of gastrin, CCK2R, FAK and phospho-FAK were observed in colon cancer tissues. CCK2R expression correlated with expression of phospho-FAK. Coexpression of CCK2R and phospho-FAK associated with invasion and lymph node metastasis. Increased invasion and motility was induced by gastrin in Colo320 cells. Overexpression of CCK2R by stable transfection of CCK2R plasmid amplified this increase and incubation with 1 microM L-365,260, a specific CCK2R antagonist, completely inhibited the effect of gastrin. FAK antisense largely blocked the increase of invasion and motility in Colo320 cells. Our data represent the evidence for the CCK2R regulating invasion and motility of colon cancer cells, and support a role of CCK2R in the progression of colon cancer. FAK play a critical role in this CCK2R-mediated effect.

Transient upregulation of GRP and its receptor critically regulate colon cancer cell motility during remodeling

Gastrin-releasing peptide (GRP) is typically viewed as a growth factor in cancer. However, we have suggested that in colon cancer, GRP acts primarily as a morphogen when it and its receptor (GRP-R) are aberrantly upregulated. As such, GRP/GRP-R act(s) primarily to modulate processes contributing to the assumption or maintenance of tumor differentiation. One of the most important such processes is the ability of tumor cells to achieve directed motility in the context of tissue remodeling. Yet the cellular conditions affecting GRP/GRP-R expression, and the biochemical pathways involved in mediating its morphogenic properties, remain to be established. To study this, we evaluated the human colon cancer cell lines Caco-2 and HT-29 cells. We found that confluent cells do not express GRP/GRP-R. In contrast, disaggreation and plating at subconfluent densities results in rapid GRP/GRP-R upregulation followed by their progressive decrease as confluence is achieved. GRP/GRP-R coexpression correlated with that of focal adhesion kinase (FAK) phosphorylation of Tyr(397), Tyr(407), Tyr(861), and Tyr(925) but not Tyr(576) or Tyr(577). To more specifically evaluate the kinetics of GRP/GRP-R upregulation, we wounded confluent cell monolayers. At t = 0 h GRP/GRP-R were not expressed, yet cells immediately began migrating into the gap created by the wound. GRP/GRP-R were first detected at approximately 2 h, and maximal levels were observed at approximately 6 h postwounding. The GRP-specific antagonist [d-Phe(6)]-labeled bombesin methyl ester had no effect on cell motility before GRP-R expression. In contrast, this agent increasingly attenuated cell motility with increasing GRP-R expression such that from t = 6 h onward no further cell migration into the gap was observed. Overall, these findings indicate the existence of GRP-independent and -dependent phases of tumor cell remodeling with the latter mediating colon cancer cell motility during remodeling via FAK.

Oncogenic K-RAS Is Required to Maintain Changes in Cytoskeletal Organization, Adhesion, and Motility in Colon Cancer Cells

RAS oncogenes are thought to play a role at multiple stages of tumorigenesis. The role and mechanisms by which RAS oncogenes maintain the transformed state of human cancer cells are poorly understood. Here, we have studied the role of oncogenic K-RAS in maintaining cytoskeletal disruption, cell adhesion and motility in metastatic colon carcinoma cells. Targeted deletion of K-RAS(G13D) from HCT116 colon carcinoma cells restored their ability to assemble stress fibers and focal adhesions/complexes, accompanied by increased cell-matrix adhesion and reduced motility. We further show that oncogenic K-Ras induces high Rho activity, but uncouples Rho from stress fiber formation. This uncoupling required the maintenance of high levels of the activator protein-1 family member, Fra-1, via a mitogen-activated protein/extracellular signal-regulated kinase-dependent pathway. We also show that PI3-kinase signaling is required for the motility of HCT116 cells downstream of oncogenic K-Ras. Our findings suggest that mutated K-RAS oncogenes are essential for maintenance of the transformed and invasive phenotype of human colon cancer cells.

Motility of colon cancer cells: modulation by CD44 isoform expression

The invasion of cancer cells at primary tumor sites and their migration during metastatic spread require the expression of cell adhesion and motility proteins. Whether accelerated cell motility is necessary in these two processes is not universally accepted. In this study we took advantage that CD44, a cell adhesion protein, has different metastatic potentials depending on its splicing isoforms to examine how they affect cell motility. We established stable transfectants of standard and variant isoforms of CD44 in SW620 cells, a human colon carcinoma cell line that does not express CD44. The morphology of the cells varied according to the CD44 isoform expressed, but actin filament distribution remained unchanged. Using the wound assay in a two-dimensional in vitro cell motility system, we found that the expression of standard CD44 increases cell motility, whereas CD44 isoforms containing an exon sequence associated with metastatic dissemination has a slowing effect. Cell proliferation was also decreased by the expression of variant CD44 isoforms. Overall, colon cancer cells expressing variant CD44 isoforms had slower cell motility, possibly due to alterations in their cell adhesion properties. In conclusion, this study suggests that, contrary to common models, the metastatic phenotype is associated with a slow rate of cell migration when tested in vitro.

Laminin-10 Mediates Basal and EGF-Stimulated Motility of Human Colon Carcinoma Cells via α3β1 and α6β4 Integrins

Signals from the epidermal growth factor (EGF) receptor and integrin-dependent adhesion to laminin contribute to the progression and metastasis of colonic tumors. However, little is know about the mechanisms by which these signals cooperate. Recently, we have reported that the colon cancer cell line LIM1215 secretes and adhere to autocrine laminin-10 via multiple integrin receptors and that EGF stimulates spreading of these cells on the same substrate. In this report, we investigate the effect of EGF and laminin-10 on colon cancer cell migration in vitro. EGF stimulates migration of LIM1215 cells in a wound healing assay. The response to EGF is inhibited by anti-EGF receptor antibody 528, the EGF receptor kinase inhibitor AG-1478, or the MAP kinase kinase inhibitor PD98059 but not the PI3-K inhibitor wortmannin. Using Transwell migration chambers, we demonstrate that laminin-10 but not collagen-I, collagen-IV, or a commercial preparation of human placental laminin is a potent motility factor for LIM1215 cells. The migration response to laminin-10 is increased upon stimulation of the cells with EGF and correlates with the up-regulation of α6β4 integrin expression as measured by analysis of Triton X-100-soluble cellular extracts. The results from integrin inhibition experiments indicate that basal migration on laminin-10 is mediated by α3β1 but not α2β1 nor α6β4 integrins. Alpha3 blocking antibodies also inhibited EGF-stimulated chemokinetic migration of LIM1215 cells on laminin-10. However, in contrast to unstimulated cells, α6 or β4 integrin-blocking antibodies inhibited the migration of EGF-stimulated cells by up to 50%. Taken together, these results support the cooperative role of EGF receptor and laminin-10 on colon cancer cell motility and suggest a critical role for both the α3β1 and the α6β4 integrins in this process.