Search

Prone whole breast irradiation results in lower dose to organs at risk compared with supine position, especially lung dose. However, the adoption of prone position for whole breast irradiation + lymph node irradiation remains limited and data on lymph node irradiation in 5 fractions are lacking. Although the study was ended prematurely for the primary endpoint (breast retraction at 2 years), we decided to report acute toxicity for prone and supine positions and 5 and 15 fractions. Additionally, dosimetry and set-up accuracy between prone and supine positions were evaluated.A randomized open-label factorial 2 × 2 design was used for an acute toxicity comparison between prone and supine positions and 5 and 15 fractions. The primary endpoint of the trial was breast retraction 2 years after treatment. In total, 57 patients were evaluated. Dosimetry and set-up errors were compared between prone and supine positions. All patients were positioned on either our in -house developed prone crawl breast couch or a Posirest-2 (Civco).No difference in acute toxicity between prone and supine positions was found, but 5 fractions did result in a lower risk of desquamation (15% vs 41%; P = .04). Prone positioning resulted in lower mean ipsilateral lung dose (2.89 vs 4.89 Gy; P.001), mean thyroid dose (3.42 vs 6.61 Gy; P = .004), and mean contralateral breast dose (0.41 vs 0.54 Gy; P = .007). No significant difference in mean heart dose (0.90 vs 1.07 Gy; P = .22) was found. Set-up accuracy was similar between both positions.Unfortunately, the primary endpoint of the trial was not met due to premature closure of the trial. Acceleration in 5 fractions resulted in a lower risk of desquamation. Prone positioning did not influence acute toxicity or set-up accuracy, but did result in lower ipsilateral mean lung dose, thyroid dose, and contralateral breast dose.

Introduction A simultaneous integrated boost (SIB) leads to less acute toxicity. Less is known for late toxicity due to SIB. In this first and only randomized trial, two-years toxicity is analysed. Materials and methods Physician-assessed toxicity, using the LENT SOMA scale, and photographs, analysed with the BCCT.core software, was examined for 150 patients, randomized between SIB and sequential boost (SEB). Results Differences in physician-assessed two-years toxicity and photographic analysis between SIB and SEB are very small and not significant. Conclusion There is no indication that a SIB leads to an excess in toxicity or worse cosmetic outcome at 2 years.

Introduction Acceleration of radiotherapy in 5 fractions for breast cancer can reduce the burden of treatment. We report on acute toxicity after whole-breast irradiation with a simultaneous integrated boost in 5 fractions over 10–12 days. Material and methods Acute toxicity and health-related quality of life (HRQoL) of 200 patients, randomized between a 15- or 5-fractions schedule, were collected, using the CTCAE toxicity scoring system, the Multidimensional Fatigue Inventory, EORTC QLQ-C30 and BR23 and the BREAST-Q questionnaire. The prescribed dose to the breast was either 15∗2.67 Gy (40.05 Gy) or 5∗5.7 Gy (28.5 Gy). 90% of patients received a SIB to a cumulative dose of 46.8 Gy (15∗3.12 Gy) or 31 Gy (5∗6.2 Gy). Results Physician-assessed toxicity was lower for the 5-fractions group. A significant difference was observed for breast pain (p = 0.002), fatigue (p, Highlights • Patients treated in 5 fractions show less physician-assessed and less patient-relatedcacute toxicity. • Patients treated in 5 fractions show less physician-assessed and patient-related pain and fatigue. • Patients treated in 5 fractions show less deterioration for physical and social functioning.

After breast conserving surgery (BCS) for breast cancer, radiotherapy (RT) reduces the risk of locoregional recurrences and improves overall survival [1,2]. Despite the benefits, late toxicity may ...

Background Most trials in head and neck cancer emphasize locoregional control, as this is the main pattern of therapy failure. However, up to 15% of patients develop distant metastases. The purpose of this study was to present the investigated factors associated with distant metastasis in a single-center patient cohort. Methods A retrospective analysis of a single-center patient cohort over an 18-year period has been performed. We report on prevalence and incidence of distant metastasis, timing in relation to locoregional failure, Kaplan–Meier analysis for actuarial distant control rates, and univariate analysis taking into account histological, etiologic, surgical, site-dependent, stage-dependent characteristics, modality of primary therapy, and locoregional control. Results Of 1022 patients, 141 (13.8%) were diagnosed with distant metastases involving 283 sites. Actuarial rates of distant control were 88%, 84%, 80%, and 79% at 1, 2, 5, and 10 years, respectively. Factors associated with distant metastasis are stage grouping and regional node positivity, extranodal extension, locoregional residual disease, and human papillomavirus (HPV) negative status in oropharyngeal squamous cell carcinoma. Conclusion Distant metastases in head and neck cancer led to dismal prognosis. Factors associated with distant metastasis are related to characteristics of the primary tumor. © 2017 Wiley Periodicals, Inc. Head Neck 39: 1733–1743, 2017

PURPOSE: REQUITE aimed to establish a resource for multi-national validation of models and biomarkers that predict risk of late toxicity following radiotherapy. The purpose of this article is to provide summary descriptive data. METHODS: An international, prospective cohort study recruited cancer patients in 26 hospitals in eight countries between April 2014 and March 2017. Target recruitment was 5300 patients. Eligible patients had breast, prostate or lung cancer and planned potentially curable radiotherapy. Radiotherapy was prescribed according to local regimens, but centres used standardised data collection forms. Pre-treatment blood samples were collected. Patients were followed for a minimum of 12 (lung) or 24 (breast/prostate) months and summary descriptive statistics were generated. RESULTS: The study recruited 2069 breast (99% of target), 1808 prostate (86%) and 561 lung (51%) cancer patients. The centralised, accessible database includes: physician- (47,025 forms) and patient- (54,901) reported outcomes; 11,563 breast photos; 17,107 DICOMs and 12,684 DVHs. Imputed genotype data are available for 4223 patients with European ancestry (1948 breast, 1728 prostate, 547 lung). Radiation-induced lymphocyte apoptosis (RILA) assay data are available for 1319 patients. DNA (n = 4409) and PAXgene tubes (n = 3039) are stored in the centralised biobank. Example prevalences of 2-year (1-year for lung) grade ≥2 CTCAE toxicities are 13% atrophy (breast), 3% rectal bleeding (prostate) and 27% dyspnoea (lung). CONCLUSION: The comprehensive centralised database and linked biobank is a valuable resource for the radiotherapy community for validating predictive models and biomarkers. PATIENT SUMMARY: Up to half of cancer patients undergo radiation therapy and irradiation of surrounding healthy tissue is unavoidable. Damage to healthy tissue can affect short- and long-term quality-of-life. Not all patients are equally sensitive to radiation "damage" but it is not possible at the moment to identify those who are. REQUITE was established with the aim of trying to understand more about how we could predict radiation sensitivity. The purpose of this paper is to provide an overview and summary of the data and material available. In the REQUITE study 4400 breast, prostate and lung cancer patients filled out questionnaires and donated blood. A large amount of data was collected in the same way. With all these data and samples a database and biobank were created that showed it is possible to collect this kind of information in a standardised way across countries. In the future, our database and linked biobank will be a resource for research and validation of clinical predictors and models of radiation sensitivity. REQUITE will also enable a better understanding of how many people suffer with radiotherapy toxicity. ispartof: RADIOTHERAPY AND ONCOLOGY vol:138 pages:59-67 ispartof: location:Ireland status: published

Radiotherapy remains one of the corner stones in the treatment of various malignancies and often leads to an improvement in overall survival. Nonetheless, pre-clinical evidence indicates that radiation can entail pro-metastatic effects via multiple pathways. Via direct actions on cancer cells and indirect actions on the tumor microenvironment, radiation has the potential to enhance epithelial-to-mesenchymal transition, invasion, migration, angiogenesis and metastasis. However, the data remains ambiguous and clinical observations that unequivocally prove these findings are lacking. In this review we discuss the pre-clinical and clinical data on the local and systemic effect of irradiation on the metastatic process with an emphasis on the molecular pathways involved.

Renal-cell carcinoma is considered to be a radioresistant tumour, but this notion might be wrong. If given in a few (even single) fractions, but at a high fraction dose, stereotactic body radiotherapy becomes increasingly important in the management of renal-cell carcinoma, both in primary settings and in treatment of oligometastatic disease. There is an established biological rationale for the radiosensitivity of renal-cell carcinoma to stereotactic body radiotherapy based on the ceramide pathway, which is activated only when a high dose per fraction is given. Apart from the direct effect of stereotactic body radiotherapy on renal-cell carcinoma, stereotactic body radiotherapy can also induce an abscopal effect. This effect, caused by immunological processes, might be enhanced when targeted drugs and stereotactic body radiotherapy are combined. Therefore, rigorous, prospective randomised trials involving a multidisciplinary scientific panel are needed urgently. © 2014 Elsevier Ltd.

In colon cancer, the biological significance of lymphatic tumour spread remains a matter of debate, which impacts on related questions such as the ideal extent of lymphadenectomy and the prognostic significance of lymph node counts. Several lines of evidence suggest that metastasis to locoregional nodes occurs early and is a stochastic, rather than a stepwise phenomenon, and in essence reflects the tumour-host-metastasis relationship. Not surprisingly, therefore, several clinical trials failed to identify a survival benefit from extensive lymphadenectomy compared to standard resection. The recently described complete mesocolic excision technique, which aims to improve survival by maximizing nodal clearance, should be subjected to a prospective randomized trial. There has been a fairly consistent and intriguing relation between nodal counts and survival in colon cancer. Therapeutic effects of more extensive removal of invaded nodes seem an unlikely explanation for the observed association. Similarly, several findings argue against stage migration as the only or even the most important explanation. The available literature shows an extensive array of factors confounding the nodal count-survival relationship, which are correlated to the patient's clinical characteristics, pathology variables, and factors relating to the individual (treating surgeon and pathologist) and institutional healthcare levels. More research into the biology of nodal spread and the nodal count-survival relationship is indicated and may have important implications for therapy such as the further introduction of minimally invasive surgery and the identification of novel and potentially modifiable factors impacting on both nodal counts and survival.

Background:Four-and-a-half LIM domains protein 2 (FHL2) is a component of the focal adhesion structures and has been suggested to have a role in cancer progression. It has been shown to be overexpressed in the colorectal cancer (CRC).Methods:Here, we examined a possible prognostic value of FHL2 in CRC. Immunohistochemistry for FHL2 was performed on 296 CRCs without distant metastases at the time of surgery. Staining in the epithelial compartment was quantitatively evaluated using image analysis, and results were related to clinical variables. Antibody specificity was tested using small-interfering RNA transfection in hTERT-immortalised myofibroblasts.Results:Varying degrees of cytoplasmic FHL2 expression by neoplastic epithelial cells were detectable in all cases. Higher FHL2 expression in the epithelial compartment was an independent adverse prognostic factor. Multivariate Cox analysis shows that expression in the tumour invasion front (P, SCOPUS: ar.j, info:eu-repo/semantics/published

The slow aggregation assay is generally used to study the functionality of cell-cell adhesion complexes. Single cells are seeded on a semisolid agar substrate in a 96-well plate and the cells spontaneously aggregate. We used HEK FLAG-MOP cells that stably overexpress the mu opioid receptor and the mu-opioid-receptor-selective agonists DAMGO and morphine to study whether other factors than functionality of cell-cell adhesions complexes can contribute to changes in the pattern of slow aggregation on agar. HEK FLAG-MOP cells formed small compact aggregates. In the presence of DAMGO and morphine, larger and fewer aggregates were formed in comparison to the vehicle control. These aggregates were localized in the center of the agar surface, whereas in the vehicle control they were dispersed over the substrate. However, in suspension culture on a Gyrotory shaker, no stimulation of aggregation was observed by DAMGO and morphine, showing that opioids do not affect affinity. A dissociation experiment revealed that HEK FLAG-MOP aggregates formed in the absence or presence of opioids are resistant to de-adhesion. We demonstrated that the larger aggregates are neither the result of cell growth stimulation by DAMGO and morphine. Since manipulations of the substrate such as increasing the agar concentration or mixing agar with agarose induced the same changes in the pattern of slow aggregation as treatment with opioids, we suggest that cell-substrate adhesion may be involved in opioid-stimulated aggregation.

Background and purpose Cancer-associated fibroblasts (CAFs) are increasingly recognised as promoters of tumour progression. It is poorly investigated whether cancer management protocols, such as neoadjuvant radio(chemo)therapy, have an impact on CAFs and, by consequence, on tumour progression. This prompted us to study the impact of neoadjuvant radio(chemo)therapy on the α-SMA/epithelial area ratio in rectal cancer, and the impact of this ratio on recurrence-free survival. Material and methods Immunohistochemistry for the CAF marker α-SMA and the proliferation marker Ki67 was performed on sections from 98 rectal cancers of which 62 had undergone neoadjuvant radio(chemo)therapy. Results Computer-assisted quantitative analysis showed that the α-SMA/neoplastic epithelial area ratio was higher after neoadjuvant therapy, and that rectal cancers with high α-SMA/epithelial area ratio had low proliferation rates. Interestingly, the α-SMA/epithelial area ratio was an adverse prognostic factor with regard to recurrence-free survival in univariate analysis. In addition, multivariate analysis showed that an α-SMA/epithelial area ratio above 1 provides an independent prognostic value associated with a poor recurrence-free survival. Conclusion These results suggest that neoadjuvant treatment has an impact on CAFs in rectal cancer. The correlation of CAFs with decreased recurrence-free survival and abundant experimental data in the literature suggest that under certain circumstances, not yet very well understood, CAFs may favour tumour progression.

Invasiveness, the ability of cancer cells to migrate beyond the normal tissue boundaries, often leads to metastasis and thereby usually turns cancer into a fatal disease. At the molecular level, the E-cadherin/catenin complex is an example of a powerful invasion suppressor in epithelial cells. Since the absence of melanocytes has been associated with disturbances in epithelial organization, we decided to investigate the influence of molecules secreted by melanocytes on the function of the E-cadherin/catenin complex. We used the Bowes melanoma cell line as a source of such molecules. The conditioned medium of Bowes melanoma stimulated aggregation of human MCF-7/6 mammary adenocarcinoma cells at short (30 min) and long (24 –72 hr) notice. This effect could be inhibited by MB2, an antibody against human E-cadherin. Conditioned medium of Bowes melanoma also inhibited invasion of MCF-7/6 cells into precultured chick heart fragments. Candidate molecules such as insulin, insulin-like growth factor I, follistatin and interleukins were ruled out to be responsible for the effects, but heregulin mimicked some of the effects of the conditioned medium. Our data indicate that heregulin stimulates aggregation and inhibits invasion of MCF-7/6 cells via activation of the E-cadherin/catenin complex. © 2004 Wiley-Liss, Inc.

Desmoid tumours are locally invasive soft tissue tumours in which beta-catenin mediated TCF-dependent transcription is activated. The role of soluble factors secreted by the myofibroblastic desmoid tumour, which could stimulate tumour invasiveness, was investigated. Using collagen gel invasion assays, the presence of factors stimulating invasion in desmoid conditioned media (CM) could be established. Since matrix metalloproteinases (MMPs) have been implicated in the process of tumoral invasion, the expression levels of the MMP family members were evaluated. Quantitative reverse transcription-PCR was used to determine the expression levels of MMP1, MMP2, MMP3, MMP7, MMP11, MMP12, MMP13, MMP14 and the inhibitors TIMP1, TIMP2 and TIMP3. Besides overexpression of MMP7, a known TCF-dependent target gene, a striking upregulation of the expression levels of MMP1, MMP3, MMP11, MMP12 and MMP13 in desmoid tumours, compared to unaffected fibroblasts from the same patients, was found. Treating the CM of desmoids with a synthetic and a physiologic MMP inhibitor reduced the invasion-stimulating capacity of the desmoid CM by approximately 50%. These results suggest the involvement of soluble factors, released by the desmoid cells, in stimulating invasion and implicate the MMPs as facilitators of invasion.

Maintenance of epithelial tissues needs the stroma. When the epithelium changes, the stroma inevitably follows. In cancer, changes in the stroma drive invasion and metastasis, the hallmarks of malignancy. Stromal changes at the invasion front include the appearance of myofibroblasts, cells sharing characteristics with fibroblasts and smooth muscle cells. The main precursors of myofibroblasts are fibroblasts. The transdifferentiation of fibroblasts into myofibroblasts is modulated by cancer cell-derived cytokines, such as transforming growth factor-beta (TGF-beta). TGF-beta causes cancer progression through paracrine and autocrine effects. Paracrine effects of TGF-beta implicate stimulation of angiogenesis, escape from immunosurveillance and recruitment of myofibroblasts. Autocrine effects of TGF-beta in cancer cells with a functional TGF-beta receptor complex may be caused by a convergence between TGF-beta signalling and beta-catenin or activating Ras mutations. Experimental and clinical observations indicate that myofibroblasts produce pro-invasive signals. Such signals may also be implicated in cancer pain. N-Cadherin and its soluble form act as invasion-promoters. N-Cadherin is expressed in invasive cancer cells and in host cells such as myofibroblasts, neurons, smooth muscle cells, and endothelial cells. N-Cadherin-dependent heterotypic contacts may promote matrix invasion, perineural invasion, muscular invasion, and transendothelial migration; the extracellular, the juxtamembrane and the beta-catenin binding domain of N-cadherin are implicated in positive invasion signalling pathways. A better understanding of stromal contributions to cancer progression will likely increase our awareness of the importance of the combinatorial signals that support and promote growth, dedifferentiation, invasion, and ectopic survival and eventually result in the identification of new therapeutics targeting the stroma.

Invasion causes cancer malignancy. We review recent data about cellular and molecular mechanisms of invasion, focusing on cross-talk between the invaders and the host. Cancer disturbs these cellular activities that maintain multicellular organisms, namely, growth, differentiation, apoptosis, and tissue integrity. Multiple alterations in the genome of cancer cells underlie tumor development. These genetic alterations occur in varying orders; many of them concomitantly influence invasion as well as the other cancer-related cellular activities. Examples discussed are genes encoding elements of the cadherin/catenin complex, the nonreceptor tyrosine kinase Src, the receptor tyrosine kinases c-Met and FGFR, the small GTPase Ras, and the dual phosphatase PTEN. In microorganisms, invasion genes belong to the class of virulence genes. There are numerous clinical and experimental observations showing that invasion results from the cross-talk between cancer cells and host cells, comprising myofibroblasts, endothelial cells, and leukocytes, all of which are themselves invasive. In bone metastases, host osteoclasts serve as targets for therapy. The molecular analysis of invasion-associated cellular activities, namely, homotypic and heterotypic cell-cell adhesion, cell-matrix interactions and ectopic survival, migration, and proteolysis, reveal branching signal transduction pathways with extensive networks between individual pathways. Cellular responses to invasion-stimulatory molecules such as scatter factor, chemokines, leptin, trefoil factors, and bile acids or inhibitory factors such as platelet activating factor and thrombin depend on activation of trimeric G proteins, phosphoinositide 3-kinase, and the Rac and Rho family of small GTPases. The role of proteolysis in invasion is not limited to breakdown of extracellular matrix but also causes cleavage of proinvasive fragments from cell surface glycoproteins.

Gelsolin is a widely distributed actin binding protein involved in controlling cell morphology, motility, signaling and apoptosis. The role of gelsolin in tumor progression, however, remains poorly understood. Here we show that expression of green fluorescent protein (GFP)-tagged gelsolin in MDCK-AZ, MDCKtsSrc or HEK293T cells promotes invasion into collagen type I. In organ culture assays, MDCK cells expressing gelsolin-GFP invaded pre-cultured chick heart fragments. Gelsolin expression inhibited E-cadherin-mediated cell aggregation but did not disrupt the E-cadherin-catenin complex. Co-expression of dominant-negative Rac1N17, but not RhoAN19 or Cdc42N17, counteracted gelsolin-induced invasion, suggesting a requirement for Rac1 activity. Increased ARF6, PLD or PIP5K 1alpha activity canceled out gelsolin-induced invasion. Furthermore, we found that invasion induced by gelsolin is dependent on Ras activity, acting through the PI3K-Rac pathway via the Ras guanine nucleotide exchange factor Sos-1. These findings establish a connection between gelsolin and the Ras oncogenic signaling pathway.

Bile acids are implicated in colorectal carcinogenesis as evidenced by epidemiological and experimental studies. We examined whether bile acids stimulate cellular invasion of human colorectal and dog kidney epithelial cells at different stages of tumor progression. Colon PC/AA/C1, PCmsrc, and HCT-8/E11 cells and kidney MDCKT23 cells were seeded on top of collagen type I gels and invasive cells were counted after 24 h incubation. Activation of the Rac1 and RhoA small GTPases was investigated by pull-down assays. Haptotaxis was analysed with modified Boyden chambers. Lithocholic acid, chenodeoxycholic acid, cholic acid and deoxycholic acid stimulated cellular invasion of SRC- and RhoA-transformed PCmsrc and MDCKT23-RhoAV14 cells, and of HCT-8/E11 cells originating from a sporadic tumor, but were ineffective in premalignant PC/AA/C1 and MDCKT23 cells. Bile acid-stimulated invasion occurred through stimulation of haptotaxis and was dependent on the RhoA/Rho-kinase pathway and signaling cascades using protein kinase C, mitogen-activated protein kinase, and cyclooxygenase-2. Accordingly, BA-induced invasion was associated with activation of the Rac1 and RhoA GTPases and expression of the farnesoid X receptor. We conclude that bile acids stimulate invasion and haptotaxis in colorectal cancer cells via several cancer invasion signaling pathways.

Malignant astrocytic tumors are characterized by the pronounced and diffuse migration of tumor astrocytes into the brain parenchyma. The present study shows that gastrin is a brain neuropeptide that is able to significantly modulate astrocytic tumor migration at both invasion and motility levels. In the matter of invasion, gastrin severely reduces the in vitro invasive abilities of C6 rat glioma, 9L rat gliosarcoma, and U373 human glioma cells in a collagen matrix. In vitro, gastrin also markedly modifies the motility features in both C6 and U373 cells, at least partly through a decrease in the expression of the RhoA small GTPase, and so brings about some dramatic modifications to the organization in the actin cytoskeleton. The in vitro preincubation of C6 tumor cells with gastrin significantly increases the life spans of rats stereotactically implanted with these cells as compared with the survival periods of rats implanted with gastrin-untreated C6 cells. As suggested by our in vitro experiments, these effects, observed in vivo cannot relate to only the gastrin-induced decrease in tumor astrocyte migratory abilities. Indeed, gastrin also induces immunomodulatory effects, because we observed a marked gastrin-induced recruitment of lymphocytes into C6 gliomas and 9L gliosarcomas. These data all suggest that gastrin can act as an endogenous modulator of glioma progression.

Matrix metalloproteases from the cell surface cleave an 80 kDa Ecadherin fragment (sECAD) that induces invasion of cancer cells into collagen type I and inhibits cellular aggregation. Conditioned media from MDCKts.srcCl2 cells at 40 C and 35 C, PCm.src5 and COLO-16 cells at 37 C contained spontaneously released sECAD; these 48 h old conditioned media were capable of inhibiting Ecadherin functions in a paracrine way. Here we show direct cleavage of the extracellular domain of Ecadherin by the serine protease plasmin. sECAD released by plasmin inhibits Ecadherin functions as evidenced by induction of invasion into collagen type I and inhibition of cellular aggregation. This functional inhibition by sECAD was reversed by aprotinin or by immunoadsorption on protein Sepharose 4 fast flow beads with antibodies against the extracellular part of Ecadherin. Our results demonstrate that plasmin produces extracellular Ecadherin fragments which regulate Ecadherin function in cells containing an intact Ecadherin/ catenin complex.

Cancer is caused by a series of genomic changes leading directly or indirectly to disturbance of growth, differentiation and tissue integrity. Genomic, transcriptional or posttranscriptional alterations of E-cadherin/catenin complexes that are implicated in various steps of cancer development comprise mutational inactivation, transcriptional downregulation of E-cadherin sometimes accompanied by upregulation of N-cadherin, proteolysis of E-cadherin and posttranslational stabilisation of beta-catenin and plakoglobin. The E-cadherin/catenin complex serves not only cell-cell adhesion but also transduces signals to the nucleus and to the cytoskeleton, either directly or through its connections with multiple other complexes. We review here the expression of E-cadherin/catenin in human cancers, emphasising methods of observation and prognostic interpretation of results. This is illustrated in thyroid lesions from the benign follicular adenoma to the extremely malignant anaplastic carcinoma. The eye is an organ largely neglected by students of cadherins and catenins. The implication of a variety of members of these molecular families in the embryonic development of the eye strongly suggests that disturbances of cadherin/catenin complexes are crucial also in the development of ocular tumours.

OBJECTIVE: Previously an upregulation of E-cadherin and its associated molecules alpha-catenin, beta-catenin and plakoglobin has been demonstrated in clinically overt inflammatory bowel disease (IBD). The aim of this study was to investigate the expression of the E-cadherin/catenin complex in subclinically inflamed bowel mucosa from spondyloarthropathy (SpA) patients. METHODS: Ileal and colonic biopsy specimens from 19 SpA patients with subclinical inflammatory gut lesions and from seven controls were stained with monoclonal antibodies against E-cadherin, beta-catenin and plakoglobin and a polyclonal antibody against alpha-catenin. E-cadherin mRNA was detected using a riboprobe. Inflammation was histologically classified into acute, chronic active and chronic quiescent forms. RESULTS: In acute and chronic active bowel inflammation of SpA patients, upregulation of the E-cadherin/catenin glycoprotein complex could be observed. Chronic lesions in a quiescent state did not show such an upregulation. Furthermore, chronic inflammation was associated with an increase in E-cadherin mRNA. CONCLUSIONS: As some of the SpA patients with subclinical gut inflammation develop IBD, upregulation of the E-cadherin/catenin complex in inflamed bowel mucosa from SpA patients may point to early cellular changes in the development of IBD. However, at present it cannot be excluded that increased E-cadherin/catenin complex expression is a bystander phenomenon of active inflammation

We have investigated the role of sialylation on cell-cell adhesion mediated by E-cadherin. Two MCF-7 human breast cancer cell variants were studied: MCF-7/AZ cells showed a spontaneous cell-cell adhesion in the fast and slow aggregation assay, whereas the adhesion deficient MCF-7/6 cell variant failed to form larger aggregates, suggesting that E-cadherin was not functional under the conditions of both assays. We measured the sialyltransferase activities using Galβ1-3GalNAcα-O-benzyl and Galβ1- 4GlcNAcα-O-benzyl as acceptor substrates as well as mRNA levels of four sialyltransferases, ST3Gal I, ST3Gal III, ST3Gal IV, ST6Gal I, using multiplex RT-PCR in MCF-7 cell variants. The α2-6 and α2-3 sialylation of E-cadherin was investigated by immuno-blot using Sambucus nigra agglutinin and Maackia amurensis agglutinin. Compared to the adhesion-proficient MCF-7/AZ cells, the adhesion-deficient MCF-7/6 cell line apparently lacks ST6Gal I mRNA, has a lower ST3Gal I mRNA, a lower ST3Gal I sialyltransferase activity, and no α2-3 linked sialic acid moieties on E-cadherin. The potential anti-cancer drug 1-O-octadecyl-2-O-methylglycero-3-phosphocholine (ET-18-OMe, 48 h, 25 μg/ml) belonging to the class of alkyllysophospholipids restored the E-cadherin function in the adhesion-deficient MCF-7/6 cells as evidenced by an increased aggregation. ET-18-OMe caused loss of ST6Gal I mRNA in MCF-7/AZ cells but no changes of sialyltransferase activities or sialic acid moieties on E-cadherin could be observed. We conclude that Ca2+-dependent, E-cadherin-specific homotypic adhesion of MCF-7/AZ or MCF-7/6 cells treated with ET-18-OMe was not affected by sialylation of E-cadherin.

Little is known about the the signalling pathways driving the adenoma-to-carcinoma sequence in human colonic epithelial cells. Accumulation and activation of the src tyrosine kinase in colon cancer suggest a potential role of this oncogene in this early progression. Therefore, we introduced either activated src (m-src), polyoma-MT alone or combined with normal c-src in the adenoma PC/AA/C1 cell line (PC) to define the function and phenotypic transformations induced by these oncogenes in familial adenomatous polyposis (FAP) colonic epithelial cells. Functional expression of these oncoproteins induced the adenoma-to-carcinoma conversion, overexpression of the hepatocyte growth factor (HGF) receptor Met, but failed to confer invasiveness in vivo and in vitro, or to produce alterations in cell proliferation and differentiation. In contrast, PC-msrc cells became susceptible to the HGF-induced invasion of collagen gels and exhibited sustained activation of the pp60src tyrosine kinase and Tyr phosphorylation of the 120-kDa E-cadherin, which was further increased by HGF Transcripts of HGF were clearly identified by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot in the parental and transformed PC cells, suggesting an autocrine mechanism. Taken together, the data indicate that: (1) experimental activation of src and PyMT pathways directly induces tumorigenicity and Met upregulation in a colon adenoma cell line; (2) HGF-activated Met and src cooperate in inducing invasion; (3) in view of the molecular associations between catenins and cadherin or the tumour-suppressor gene product APC, the cell adhesion molecule E-cadherin may constitute a downstream effector of src and Met. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6

Malignant cancer cells do not act as lone wolves to achieve metastasis, as they exist within a complex ecosystem consisting of an extracellular matrix scaffold populated by carcinoma-associated fibroblasts (CAFs), endothelial cells and immune cells. We recognize local (primary tumor) and distant ecosystems (metastasis). CAFs, also termed myofibroblasts, may have other functions in the primary tumor versus the metastasis. Cellular origin and tumor heterogeneity lead to the expression of specific markers. The molecular characteristics of a CAF remain in evolution since CAFs show operational flexibility. CAFs respond dynamically to a cancer cell's fluctuating demands by shifting profitable signals necessary in metastasis. Local, tissue-resident fibroblasts and mesenchymal stem cells (MSCs) coming from reservoir sites such as bone marrow and adipose tissue are the main progenitor cells of CAFs. CAFs may induce awakening from metastatic dormancy, a major cause of cancer-specific death. Cancer management protocols influence CAF precursor recruitment and CAF activation. Since CAF signatures represent early changes in metastasis, including formation of pre-metastatic niches, we discuss whether liquid biopsies, including exosomes, may detect and monitor CAF reactions allowing optimized prognosis of cancer patients.

Background Surgery-induced acceleration of tumour growth has been observed since several centuries. Methods We reviewed recent insights from in vitro data, animal experimentation, and clinical studies on how surgery-induced wound healing or resection of a primary cancer influences the tumour–host ecosystem in patients harbouring minimal residual or metastatic disease. Results Most of the growth factors, chemokines, and cytokines orchestrating surgical wound healing promote tumour growth, invasion, or angiogenesis. In addition, resection of a primary tumour may accelerate synchronous metastatic growth. In the clinical setting, indirect evidence supports the relevance of the above findings. Randomized clinical trials are underway comparing resection versus observation in metastatic breast and colon cancer with asymptomatic primary tumours. Conclusions In depth knowledge of how surgical intervention alters the tumour–host-metastasis communicating ecosystems could have important implications for clinical decision making in patients with synchronous metastatic disease and for the design and timing of multimodality treatment strategies.

Invasion is a major challenge for cancer therapy. Invasion or noninvasion results from the cross talk between cancer cells and host cells, building molecular invasion-promoter and invasion-suppressor complexes. The E-cadherin/catenin invasion-suppressor complex is attractive as a target for a putative antiinvasive therapy because of its multifactorial regulation at multiple levels and sometimes in a reversible way. Mutations in the E-cadherin gene combined with loss of the wild type allele causes irreversible downregulation in some human cancers. Posttranslational and reversible downregulation may occur by tyrosine phosphorylation of β-catenin. Phosphorylation is implicated also in transmembrane receptor signal transduction through the E-cadherin/catenin complex. Homophilic interaction with E-cadherin on another cell through a dimeric adhesion zipper, involving the HAV sequence of the first extracellular domains, is the major extracellular link of the E-cadherin/catenin complex. Intracellularly, the list of proteins that bind to or signal through the complex or one or more of its elements is growing. In vitro, insulin-like growth factor-I, and tamoxifen may upregulate the functions of the E-cadherin/catenin complex and inhibit invasion, demonstrating that this complex may serve as a target for antiinvasive therapy. © 1996 Wiley-Liss, Inc.

Inappropriate activation of the Wnt/APC/beta-catenin signaling pathways plays a critical role at early stages in a variety of human cancers. However, their respective implication in tumor cell invasion is still hypothetical. Here, we show that two activators of the canonical Wnt/beta-catenin transcription pathway, namely Dvl-2, the Axin 501-560 fragment binding glycogen synthase kinase -3beta (GSK-3beta), and the negative Wnt regulator wt-Axin did not alter cell invasion into type I collagen. In addition, both Dvl-2 and Axin 501-560 exerted a permissive action on the proinvasive activity of HGF and intestinal trefoil factor. Upstream activation of Wnt signaling by the Wnt-2 and Wnt-3a ligands, stable overexpression of Wnt-2, as well as GSK-3beta inhibition by lithium, SB216763, and GSK-3beta dominant negative forms (K85R and R96E) conferred the invasive phenotype through several proinvasive pathways. Induction of the matrix metalloprotease MMP-7 (matrilysin) gene and protein by Wnt-2 was abolished by inactivation of the AP-1 binding site in the promoter. Accordingly, invasion induced by Wnt-2 was prevented by soluble FRP-3 and FRP-1, sequestration of Gbetagamma subunits, depletion of the GSK-3beta protein by RNA interference, the c-Jun dominant negative mutant TAM67 and was not reversed by wt-Axin. Thus, the proinvasive activity of Wnt-2 is mediated by a noncanonical Wnt pathway using GSK-3beta and the AP-1 oncogene. Our data provide a potential clue for our understanding of the action and crosstalk between Wnt activators and other proinvasive pathways, in relation with matrix substrates and proteases in human cancers.

The gas-chromatographic determination of hexadecylphosphocholine (HePC), an experimental antitumor agent of the alkyllysophospholipid group, in Caco-2T cell culture and cell culture media is described. The Caco-2T cells were treated with HePC at a concentration of 40 micrograms/ml (9.8 microM) and the uptake of the drug into the cells (calculated per milligram protein) was measured after 48 h culture (37 degrees C, 10% CO2). Also, a reversibility test for another 48 h was carried out in which the retention of the drug was measured. The toxicity of HePC on Caco-2T cells in viability assays was determined. Before the capillary gas-chromatographic determination, sample cleanup was performed by solid-phase extraction (SPE) on a weak cation-exchange column of the CBA (carboxylic acid) type. For quantitation, racemic 1-O-octadecyl-2-O-methylglycero-3-phosphorylcholine (ET-18-OMe) was added as internal standard, followed by derivatization with trimethylsilylbromide. The results showed that HePC taken up by the cells during 48 h of treatment was still detectable 48 h after removal of the drug from the medium.

In this study we investigated the tumorigenicity, growth pattern and spontaneous metastatic ability of a series of nine human colorectal carcinoma cell lines after subcutaneous and intracaecal xenografting in nude mice. CaCo2 cells were found to be poorly tumorigenic to non-tumorigenic in either site; the other cell lines were tumorigenic in both sites. SW1116, SW480 and SW620 did not show local invasive in the NCI-H716 and LS174T cells were both invasive in the caecum, but only NCI-H716 was invasive in the subcutis. HT29 and 5583 (S and E) cells were invasive in the caecum and from that site metastatic to the lungs and/or the liver. HT29 and 5583S cells were both invasive in the subcutis, but 5583E cells were not. Of each category of in vivo behaviour in the caecum, one cell line was further investigated with regard to invasion in vitro (into embryonic chick heart fragments), E-cadherin expression in vivo and in vitro and in vitro production of u-PA and t-PA. These parameters were chosen in view of their purported role in extracellular matrix degradation and intercellular adhesion, which are all involved in the invasive and metastatic cascade. Invasion in vitro was not predictive for invasion or metastasis in vivo. In the cell line which showed invasion in embryonic chick heart tissue, heterogeneous E-cadherin expression was observed in vitro together with a relatively high production of u-PA. The non-invasive cell lines showed in vitro homogeneous expression of E-cadherin with a relatively low production of u-PA. In vivo expression of E-cadherin was either absent or heterogeneous. We conclude that: (1) colorectal carcinoma xenografts show site-specific modification of in vivo invasive and metastatic behaviour; (2) invasion in vitro does not correlate with invasion and metastasis in vivo; (3) in vitro non-invasion might be associated with homogeneous E-cadherin expression and low production of u-PA; (4) E-cadherin expression in vitro differs from E-cadherin expression in vivo. The results support the notion that the microenvironment in which cancer cells grow is one of the factors involved in the regulation of invasive and metastatic behaviour. Images Figure 1 Figure 2 Figure 3 Figure 4

All-trans-retinoic acid (RA), like insulin-like growth factor I (IGF-I) and tamoxifen, inhibit invasion of human MCF-7/6 mammary cancer cells in vitro. For tamoxifen and for IGF-I, activation of the invasion-suppressor function of the E-cadherin/catenin complex was shown to be the most probable mechanism of the anti-invasive action. We did a series of experiments to determine whether the anti-invasive effect of RA also implicated the invasion-suppressor E-cadherin/catenin complex. Human MCF-7/6 mammary and HCT-8/R1 colon cancer cells, both with a dysfunctional E-cadherin/catenin complex, were treated with RA and the function of the complex was evaluated through Ca(2+)-dependent fast aggregation. Fast aggregation of both MCF-7/6 and HCT-8/R1 cells was induced by 1 microM RA. This effect was abolished by antibodies against E-cadherin. RA-induced fast aggregation was not sensitive to cycloheximide, tyrosine kinase inhibitors or antibodies against IGF-I or against the IGF-I receptor. RA did not stimulate IGF-I receptor phosphorylation or alter the E-cadherin/catenin complex, as evidenced by immunoprecipitation. RA up-regulates the function of the invasion-suppressor complex E-cadherin/catenin. Its action mechanism is different from that of IGF-I. RA may act as an anti-invasive agent with unique mechanisms of action. Images Figure 7 Figure 8

Invasive growth of a tumor occurs within an ecosystem where a continuous communication exists between cancer cells and a wide network of tumor-associated host cells. Secretory factors from the cancer cells activate the recruitment of host cells, both near to and far from the primary tumor site, as well as promote the departure of cancer- and host cells to distant tissues. The present review focuses on the prominent role of myofibroblasts in local and distant ecosystems namely the primary tumor, the bone marrow, the circulation, the sites of lymph node and of distant metastases and the nervous system. We believe that there exist distinct types of myofibroblasts with distinct reaction patterns that affect invasive tumor growth in different ways. Mathematical models predict that the specific conditions in local ecosystems determine the invasive phenotype of a tumor. Experimental cell culture models incorporating cancer cells, primary tumor-derived myofibroblasts and matrix proteins in a three-dimensional context confirm the pro-invasive activity of myofibroblasts. New methodologies will facilitate the direct observation of invasive cells including their interaction with myofibroblasts in clinically relevant ecosystems. A better understanding of the local and distant tumor ecosystems may help us to design personalized strategies in the treatment of cancer.

Loss of histotypic organization of epithelial cells is a common feature in normal development as well as in the invasion of carcinomas. Here we show that the v-src oncogene is a potent effector of epithelial differentiation and invasiveness. MDCK epithelial cells transformed with a temperature-sensitive mutant of v-src exhibit a strictly epithelial phenotype at the nonpermissive temperature for pp60v-src activity (40.5 degrees C) but rapidly loose cell-to-cell contacts and acquire a fibroblast-like morphology after culture at the permissive temperature (35 degrees C). Furthermore, the invasiveness of the cells into collagen gels or into chick heart fragments was increased at the permissive temperature. The profound effects of v-src on intercellular adhesion were not linked to changes in the levels of expression of the epithelial cell adhesion molecule E-cadherin. Rather, we observed an increase in tyrosine phosphorylation of E-cadherin and, in particular, of the associated protein beta-catenin. These results suggest a mechanism by which v-src counteracts junctional assembly and thereby promotes invasiveness and dedifferentiation of epithelial cells through phosphorylation of the E-cadherin/catenin complex.

Three clonal subpopulations (A, B, C) isolated from the same rhabdomyosarcoma of the rat and differing in their degree of spontaneous differentiation were tested for their invasive potential before and after differentiation induction with retinoic acid (RA), N-monomethylformamide (NMF) and sodium butyrate (NaBut). Invasive potential was analysed in an in vitro assay using embryonic chick heart fragments in organotypic culture. In standard culture medium, all three subpopulations were shown to be invasive, progressively replacing the chick heart fragments within 7-11 days after confrontation. After exposure to RA, NMF or NaBut, marked differences in the invasive potential of these subpopulations were, however, observed. Subpopulation C exhibited a pronounced decline in invasive potential, as evidenced by a significant decrease (P = 0.005) in the proportion of chick heart fragments with advanced stages of invasion. This response, however, was confined to the differentiation-inducing agents RA and NaBut, which had also produced a marked increase in morphological and/or biochemical differentiation (P = 0.0001). In contrast, NMF, which had only minor effects on differentiation, failed to affect the invasive potential of subpopulation C. In subpopulation B, a transient inhibition of single cell invasion became evident after exposure to RA, whereas NMF and NaBut failed to affect the invasive potential in spite of minor effects on differentiation. In the least differentiated subpopulation A, which was shown to be refractory to the differentiation-inducing effects of RA, NMF and NaBut, there was also no observation of any reduction of invasive potential. The results of our study demonstrate that differentiation-inducing agents can significantly reduce the invasive potential of malignant tumors, although marked differences of response are to be expected between the different subpopulations of a tumor.

Head and neck squamous cell carcinoma (HNSCC) is a major healthcare problem worldwide affecting more than half a million patients each year. Despite considerable advances in the treatment of HNSCC, a high rate of recurrences aggravates the clinical situation and disease outcomes have only modestly improved. Recent insights show that cancer is not only a disease of the transformed epithelium but is also influenced and dependent on its stromal environment. In this review we suggest that resident and bone marrow (BM)-derived mesenchymal stem cells (MSCs) are precursors of the stroma associated with HNSCC and contribute to blood- and lymph angiogenesis, modulate the immune system and produce tumor-associated myofibroblasts. In addition, the impact of radiation therapy on the stromal reaction in HNSCC is discussed. Understanding the mechanisms of how MSCs promote invasive growth and metastasis in HNSCC and respond to cancer management strategies is of profound medical importance and will help us to design improved therapeutic protocols.

Laminin, a major basement membrane component, arrested the migration of MCF-7/AZ human breast adenocarcinoma cells that were not invasive in vitro. Migration of invasive MCF-7/6 cells was not affected by laminin. Both cell types expressed the 67 kD laminin receptor, at both mRNA and protein level, but did not express the alpha 6 subunit of the VLA-6 integrin-type laminin receptor. The presence of YIGSR peptides (100 micrograms/ml), reported to block the interaction between laminin and its 67 kD receptor, did not change the migratory response of MCF-7/AZ or MCF-7/6 cells when meeting laminin lanes. In addition, the migration of these cell types was not affected by the presence of 17-beta-estradiol (10(-6) M) or all-trans retinoic acid (10(-6) M), which were both reported to increase the number of 67 kD receptors. We could therefore not assign an involvement of the 67 kD receptors in migration of MCF-7 cells on laminin, nor did we find evidence that conditioned medium of MCF-7/6 cells contains factors that are able to initiate migration of MCF-7/AZ cells on laminin.

The invasiveness of MCF-7 human mammary carcinoma cells was tested in vitro via confronting cultures with embryonic chick heart fragments. Invasive (e.g. MCF-7/6) and non-invasive (e.g. MCF-7/AZ) variants were detected. Automated image analysis of time-lapse video-microscopy recordings showed that the plasma membrane ruffling activity of the invasive MCF-7/6 variant was higher than the ruffling activity of the non-invasive MCF-7/AZ variant. Addition of all-trans-retinoic acid to the culture medium (10(-6) M) inhibited both invasion and ruffling of MCF-7/6 cells, while MCF-7/AZ cells became invasive and acquired an increased ruffling by the same type of treatment. A similar opposite effect on MCF-7 cells was not found after treatment with other ligands of the nuclear steroid/thyroid receptor superfamily. Triiodo-l-thyronine (up to 10(-5) M) and beta-oestradiol (up to 10(-6) M) did not alter the invasiveness of the cells, while dexamethasone (10(-6) M) and the pure anti-oestrogen ICI 164,384 inhibited both invasion and ruffling. Our data show that retinoic acid can modulate invasiveness in opposite directions. Images Figure 1

The involvement of phosphoinositide 3-kinases class IA (PI3K-alpha and -beta) in cancer cell proliferation, survival, motility, and invasiveness is now well established. However, the possible contribution of the class IB PI3Kgamma in cancer cell transformation remains to be explored. In this study, we have stably transfected the PI3Kgamma-deficient human colon cancer cell line HCT8/S11 with expression vectors encoding either wild-type PI3Kgamma, its plasma membrane targeted form CAAX-PI3Kgamma, or the PI3Kgamma lipid and protein kinase-dead mutant (CAAX-K832R). We provide evidence that the constitutively active CAAX-PI3Kgamma variant induced collagen type I invasion in HCT8/S11 cells through disruption of cell-cell adhesion, with no apparent impact on cell proliferation and motility. The proinvasive activity of CAAX-PI3K-gamma was abolished by pharmacological inhibitors targeting PI3-K activities (wortmannin), Rho-GTPases, and the Rho-Rho kinase axis (C3T exoenzyme and Y27632, respectively). Conversely, the wild-type PI3Kgamma and its double mutant CAAX-K832R were ineffective on cancer cell invasion measured under control or stimulated conditions operated with the proinvasive agents leptin and intestinal trefoil factor. Taken together, our data indicate that PI3Kgamma exerts transforming functions via several mechanisms in human colon epithelial cancer cells, including alterations of homotypic cell-cell adhesion and induction of collagen type I invasion through canonical proinvasive pathways.

Tissue integrity is maintained by the stroma in physiology. In cancer, however, tissue invasion is driven by the stroma. Myofibroblasts and cancer-associated fibroblasts are important components of the tumor stroma. The origin of myofibroblasts remains controversial, although fibroblasts and bone marrow-derived precursors are considered to be the main progenitor cells. Myofibroblast reactions also occur in fibrosis. Therefore, we wonder whether nontumorous myofibroblasts have different characteristics and different origins as compared to tumor-associated myofibroblasts. The mutual interaction between cancer cells and myofibroblasts is dependent on multiple invasive growth-promoting factors, through direct cell-cell contacts and paracrine signals. Since fibrosis is a major side effect of radiotherapy, we address the question how the main methods of cancer management, including chemotherapy, hormonotherapy and surgery affect myofibroblasts and by inference the surrogate endpoints invasion and metastasis.

Since its introduction more than a decade ago, intensity-modulated radiotherapy (IMRT) has spread to most radiotherapy departments worldwide for a wide range of indications. The technique has been rapidly implemented, despite an incomplete understanding of its advantages and weaknesses, the challenges of IMRT planning, delivery, and quality assurance, and the substantially increased cost compared with non-IMRT. Many publications discuss the theoretical advantages of IMRT dose distributions. However, the key question is whether the use of IMRT can be exploited to obtain a clinically relevant advantage over non-modulated external-beam radiation techniques. To investigate which level of evidence supports the routine use of IMRT for various disease sites, we did a review of clinical studies that reported on overall survival, disease-specific survival, quality of life, treatment-induced toxicity, or surrogate endpoints. This review shows evidence of reduced toxicity for various tumour sites by use of IMRT. The findings regarding local control and overall survival are generally inconclusive.

Human mammary carcinoma cell lines (MCF-7) were analysed for their hormone sensitivity before and after transfection with a v-Ha-ras oncogene or with a neomycin-resistance gene followed by selection in vitro or in vivo. Our aim was to test how the expression of the ras oncogene would influence the estradiol sensitivity of MCF-7 cells. In culture, MCF-7 cells expressing the viral p21 oncogene product, as compared to parental MCF-7 cells and their control derivatives, showed lower levels of a 67-kDa estrogen receptor. Progesterone receptors, however, remained sensitive to up-regulation by estrogens. The oncogene-expressing cells were less sensitive than all controls to stimulation of proliferation by 10(-8)M estradiol or to inhibition of proliferation by 2-CH3-4-OH tamoxifen, and this was not dependent upon the type of culture medium used. After s.c. or i.p. injection into female athymic nude mice, ovariectomized or left intact, the growth of MCF-7 cells expressing the ras oncogene product and of all control cells was sensitive to stimulation by estrogen supplementation. Conversely, cell lines derived from tumors generated with long latency in untreated athymic nude mice by v-ras-expressing MCF-7 cells showed efficient formation of quickly growing tumors in the absence of estrogen supplementation. No differences were observed in invasion and metastasis of the different MCF-7 cell types injected into athymic nude mice that were supplemented with estrogens or not.

The changes in glycosylation of an immortalized epithelial cell line (MDCK) before and after progression towards a more malignant phenotype have been studied. The parental MDCK-3 cells were immortalized after long-term passage in vitro and have shown no tendency for spontaneous acquisition of malignancy-related phenotypes such as tumorigenicity, invasion and metastasis. They conserved morphological and functional characteristics of the epithelial tissue of origin. The ras-MDCK cells acquired the fully malignant phenotype after transformation with a Harvey murine sarcoma virus; they were immortalized, invasive in vitro and produced invasive and also metastatic tumors after subcutaneous injection into nude mice. Using immobilized lectins and gel chromatography, before and after liberation of O-linked glycans from the peptide moieties and also after removal of terminal sialic acid, we have found differences in the glycosylpeptides of both whole cells and cell surface trypsinates from ras-MDCK cultures as compared to the parental MDCK-3 cultures: (i) more sialic acid in the N-linked tri- and tetra-antennary structures; (ii) more fucosylation in the N-glycosylpeptides; (iii) more bi-antennary N-glycosylpeptides and less O-linked glycans; and (iv) a lower molecular weight of the O-linked glycans probably due to a decreased sialylation. It is concluded that alterations in sialylation and fucosylation of the cell surface exposed glycans accompanied progression of MDCK-3 cells towards a more malignant phenotype.

The catalytic α-subunit of the enzyme phosphatidylinositol-3-OH kinase (PI(3)Kα) relays signals from G- protein-coupled receptors at the cell membrane and mediates leukocyte responses to chemokines and chemoattractants1, 2, ³. Sasaki et al.⁴ report that mice that cannot produce PI(3)Kα have a high incidence of colorectal carcinomas, causing weight loss and premature death. However, PI(3)Kα-null mouse strains have been independently generated in three other laboratories; none of these mice developed tumours and their weight and lifespan were normal. This casts coubt on the idea that loss of functional PI(3)Kα leads directly to transformation of colon epithelial cells and tumour progression.

Down-regulation of the epithelial cell-cell adhesion molecule E-cadherin is frequently associated with tumor formation and progression. Besides its role in physical cell-cell adhesion, E-cadherin is also thought to be involved in intracellular signaling in normal epithelial cells. In these cells, the Armadillo catenin p120ctn binds to the cytoplasmic domain of E-cadherin and stabilizes the adhesion complexes. On loss of E-cadherin, cytoplasmic p120ctn might accumulate and contribute to tumor malignancy. We used suppression subtractive hybridization to search for genes regulated by E-cadherin expression. We isolated human Nanos1 as a transcript of which levels decrease on E-cadherin reexpression in a human breast cancer cell line. The hNanos1 protein bears a COOH-terminal (CCHC)2 zinc finger domain and belongs to an evolutionarily conserved protein family sharing functions in germ cell development in both vertebrates and invertebrates. We found an inverse correlation between E-cadherin and hNanos1 expression in various cell lines and under diverse conditions. Conditional expression of hNanos1 in human colorectal DLD1 cancer cells functionally abolished cell-cell adhesion. It induced cytoplasmic translocation of p120ctn, as well as strong migratory and invasive properties. We also found that the NH2-terminal domain of hNanos1, which is conserved only among mammals, interacts with p120ctn. hNanos1 counteracted the stimulatory effect of p120ctn on cell protrusion formation. Together, these findings describe a new function for hNanos1 as a downstream effector of E-cadherin loss contributing to tumor progression. Targeting hNanos1 might be a promising strategy in the treatment of E-cadherin–negative tumors in particular. (Cancer Res 2006; 12(20): 10007-15)

Colon cancer results from erroneous renewal of the enteric epithelium. Mutations in stem cells, or their proliferative progenitors, cause accumulation of cells that invade into the stroma and continue to divide rather than migrating on top of the basement membrane prior to entering into apoptosis. Many of these changes in invasive activity appear to be related to the invasion-suppressor role of E-cadherin. We have also investigated Listeria monocytogenes and other enteric bacteria, since these bacteria stimulate invasion through the production of a beta-casein-derived 13-amino acid peptide which is produced by enzymes present in the colon cancer ecosystem. The pro-invasive 13-amino acid peptide signals via small guanosine triphosphatases, which modulate the actin cytoskeleton, and via phosphorylation of the delta opioid receptor. The pro-invasive activity of this peptide is neutralized by the delta opioid receptor antagonist, naloxone. Since the delta opioid receptor belongs to the family of G protein-coupled receptors, implicated in colon cancer cell invasion signalling pathways, it is tempting to speculate that opioids could play a role in mediating this trait of malignant tumours.

P-cadherin expression in breast carcinomas has been associated with tumors of high histologic grade and lacking estrogen receptor-α, suggesting a link between these proteins. In the MCF-7/AZ breast cancer cell line, blocking estrogen receptor-α signaling with the antiestrogen ICI 182,780 induced an increase of P-cadherin, which coincided with induction of in vitro invasion. Retroviral transduction of MCF-7/AZ cells, as well as HEK 293T cells, showed the proinvasive activity of P-cadherin, which requires the juxtamembrane domain of its cytoplasmic tail. This study establishes a direct link between P-cadherin expression and the lack of estrogen receptor-α signaling in breast cancer cells and suggests a role for P-cadherin in invasion, through its interaction with proteins bound to the juxtamembrane domain.

CapG (gCap39) is a ubiquitous gelsolin-family actin modulating protein involved in cell signalling, receptor-mediated membrane ruffling, phagocytosis and motility. CapG is the only gelsolin-related actin binding protein that localizes constitutively to both nucleus and cytoplasm. Structurally related proteins like severin and fragmin are cytoplasmic because they contain a nuclear export sequence that is absent in CapG. Increased CapG expression has been reported in some cancers but a causal role for CapG in tumour development, including invasion and metastasis, has not been explored. We show that moderate expression of green fluorescent protein-tagged CapG (CapG-EGFP) in epithelial cells induces invasion into collagen type I and precultured chick heart fragments. Nuclear export sequence-tagged CapG-EGFP fails to induce invasion, whereas point mutations in the nuclear export sequence permitting nuclear re-entry restore cellular invasion. Nuclear import of CapG is energy-dependent and requires the cytosolic receptor importin β but not importin α. Nuclear CapG does not possess intrinsic transactivation activity but suppresses VP16 transactivation of a luciferase reporter gene in a dose-dependent manner. Furthermore, invasion requires signalling through the Ras-phosphoinositide 3-kinase pathway and Cdc42 or RhoA, but not Rac1. We show for the first time active nuclear import of an actin binding protein, and our findings point to a role for nuclear CapG in eliciting invasion, possibly through interfering with the cellular transcription machinery.

Tight junctions and microvilli constitute an anti-invasive barrier at the luminal side of enteric cell layers. Both subcellular structures are disrupted following adhesion of Entamoeba histolytica trophozoites to enteric cell layers in vitro. It was our aim to analyse the molecular mechanism underlying this disruption. Therefore, we cocultured enteric T84 cell layers established on filter inserts with E. histolytica trophozoites and tested various modulators of enteric molecules, involved in the functional regulation of tight junctions, as well as inhibitors of trophozoite virulence factors on their capacity to maintain the transepithelial electrical resistance. Pretreatment of trophozoites with the proteinase inhibitor N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone prevented the decrease in transepithelial electrical resistance whereas none of the modulators used to pretreat enterocytes were successful. Moreover, zymography and Western blot analysis revealed that both N-Tosyl-Phenylalanine chloromethyl ketone and N-Tosyl-l-Lysine chloromethyl ketone inhibited E. histolytica cysteine proteinases and prevented proteolysis of tight junction molecules ZO-1 and ZO-2 and of villin, the major actin bundling molecule in microvilli. Immunocytochemistry with an antibody against ezrin, an actin-binding molecule in microvilli, and phase contrast microscopy demonstrated that pretreatment of trophozoites with N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone also prevented disturbance of microvilli and destruction of Caco-2 enteric cell layers in cocultures. Taken together, our results indicate that trophozoites use their proteinases to overcome microvilli and tight junction barriers during the invasion of enteric cell layers, that these phenomena could be prevented by pretreatment of trophozoites with N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone, and that such pretreatment disabled trophozoites to destroy enteric cell layers in vitro.