Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins.

Pancreatic ductal adenocarcinomas are characterised by a dense connective tissue reaction. To test the hypothesis that stroma components are synthesised and produced by the tumour cells themselves, eight cell lines as well as six xenografted tumours from human ductal adenocarcinomas of the pancreas were examined for the expression of extracellular matrix proteins (ECM), using cDNA probes and antibodies to collagen types I, III and IV, vitronectin, fibronectin, undulin and laminin. All tumour cell lines (CAPAN-1, CAPAN-2, AsPC-1, BxPC-3, PANC-1, PaCa-2, PaCa-3, PaCa-44) and xenografted human pancreatic tumours expressed at least one of the examined ECM at the RNA (collagen type IV > laminin = fibronectin = vitronectin > collagen type III > undulin > collagen type I) or protein level (collagen type IV = collagen type III > vitronectin > laminin > collagen type I = fibronectin > undulin). In nude mouse tumours expression of laminin and collagen I was most pronounced in well-differentiated carcinomas. In a few tumours, collagen type III, vitronectin and undulin were expressed on the luminal side of the neoplastic glands, suggesting loss of normal polar differentiation. Incubation with fetal calf serum modulated ECM RNA levels to a varying extent in all but one cell line (AsPC-1). The results suggest that human pancreatic ductal adenocarcinomas cells are capable of synthesising and producing extracellular matrix proteins in vitro and in vivo, but that the extent and pattern of ECM expression differs between the various tumours and conditions tested.     

Predictive value of Smac, VEGF and Ki-67 in rectal cancer treated with neoadjuvant therapy

The present study aimed to identify whether second mitochondria-derived activator of caspase (Smac), vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (Ki-67) expression in pre-treatment tumor biopsies are useful predictive markers of tumor response in patients with rectal cancer undergoing pre-operative chemoradiotherapy (CRT). Paraffin-embedded tissues obtained before and after therapy were evaluated by immunohistochemical staining for Smac, VEGF and Ki-67. The study evaluated the correlation of Smac, VEGF and Ki-67 immunoreactivity in tumor biopsies before treatment of tumor response to pre-operative CRT. Regarding Smac, patients with a favorable response to neoadjuvant CRT had higher pre-therapy levels (p=0.011). The level of Smac expression decreased after neoadjuvant therapy (p=0.044). However, VEGF expression was found to be negatively and significantly correlated with a favorable tumor response to neoadjuvant CRT (p=0.010). A transient increase in VEGF expression was detected in the resected specimens following neoadjuvant therapy (p=0.030). In addition, tumors with a low Ki-67 labeling index (Ki-67-LI) expression were found to be more sensitive to neoadjuvant therapy than those with a high expression of Ki-67-LI (p=0.034). In contrast to VEGF, the Ki-67 expression level decreased after neoadjuvant therapy. Smac, VEGF and Ki-67 expression levels, assessed immunohistochemically from pre-treatment tumor biopsies, may be useful predictive markers of rectal tumor response to pre-operative CRT.     

Synthetic peptides inhibit adhesion of human tumor cells to extracellular matrix proteins

Human tumor cell progression and metastasis are partially dependent on the ability of a tumor cell to adhere to the proteins of the extracellular matrix (ECM) and survive at the distant location. Six novel D-amino acid-containing peptides were analyzed for their ability to adhere to human prostate tumor cells, support tumor cell adhesion, and inhibit tumor cell adhesion to ECM proteins or human dermal fibroblasts. Of these, two peptides called RZ-3 (kmviywkag) and HYD-1 (kikmviswkg) bound to tumor cell surfaces and compared favorably with the previously reported AG-73 (RKRLQVQLSIRT) L-amino acid peptide, as determined by fluorescence-activated cell sorting analysis. A scrambled peptide derivative of HYD-1, called HYDS-1 (wiksmkivkg), was not active. The RZ-3, HYD-1, and AG-73 peptides supported maximal cancer cell adhesion at 5 microg, 10 microg, and 50 microg/well, respectively. The ECM proteins fibronectin, laminin 1, and collagen IV supported maximal cell adhesion at 1 microg, >10 microg, and 50 microg/well, respectively. Prostate tumor cell adhesion to immobilized RZ-3 and HYD-1 peptides was inhibited by alpha2-6- and beta1-integrin-blocking antibodies. Conversely, tumor cell adhesion to a beta1-integrin-specific antibody was blocked by both RZ-3 and HYD-1. Epithelial cell adhesion to dermal fibroblasts was inhibited by HYD-1 and unaffected by the scrambled peptide, HYDS-1. Cell adhesion to immobilized peptides was unaffected by EDTA. The soluble RZ-3 and HYD-1 peptides inhibited tumor cell adhesion to each of the immobilized four ECM proteins (1.0 microg/well) in a time- and concentration-dependent manner. The IC(50) of the RZ-3 peptide for blocking adhesion to fibronectin, laminin 1, laminin 5, and collagen IV was 2.4 microg, 1.8 microg, 4.6 microg, and 2.8 microg/well, respectively. The IC(50) of the HYD-1 peptide for blocking adhesion to fibronectin, laminin 1, laminin 5, and collagen IV was 6.9 microg, 5.7 microg, >10 microg, and 6.2 microg/well, respectively. Taken together, these results indicate that RZ-3 and HYD-1 are biologically active D-amino acid-containing peptides that can themselves support tumor cell adhesion and can inhibit tumor cell adhesion to immobilized ECM proteins or dermal fibroblasts.     

Products of cells from gliomas: IX. Evidence that two fundamentally different mechanisms change extracellular matrix expression by gliomas

Summary Four human astrocytic gliomas of high grade of malignancy were each evaluated in tissue andin vitro for percentages of cells expressing glial fibrillary acidic protein (GFAP), collagen type IV, laminin and fibronectin assessed by immunofluorescence with counterstaining of nuclear DNA. Percentages of cells with reticulin and cells binding fluorescein-labeledUlex europaeus agglutinin were also assessed. In tissue, each extracellular matrix (ECM) component was associated with cells in the walls of abnormal proliferations of glioma vessels, and all four tumors had the same staining pattern. Two strikingly different patterns of conversion of gene product expression emerged duringin vitro cultivation. (1). In the most common pattern, percentages of all six markers consistently shifted toward the exact phenotype of mesenchymal cells in abnormal vascular proliferations: increased reticulin, collagen type IV, laminin and fibronectin; markedly decreased glial marker GFAP and absent endothelial markerUlex europaeus agglutinin. The simplest explanation of this constellation of changes coordinated toward expression of vascular ECM markers is that primary glioma cell cultures are overgrown by mesenchymal cells from the abnormal vascular proliferations of the original glioma. These cell cultures were tested forin situ hybridization (ISH) signals of chromosomes 7 and 10. Cells from one glioma had diploid signals. Cells from the other glioma had aneuploid signals indicating they were neoplastic; however, their signals reflected different numerical chromosomal aberrations than those common to neoplastic glia. (2). The second pattern was different. Cells with ISH chromosomal signals of neoplastic glia retained GFAP, and gained collagen type IV. Their laminin and fibronectin diminished, but persisted among a lower percentage of cells. Cloning and double immunofluorescence confirmed the presence of individual cells with glial and mesenchymal markers. A cell expressing GFAP in addition to either fibronectin, reticulin or collagen type IV is not a known constituent of glioblastoma tissue. This provides evidence of a second mechanism of conversion of gene expression in gliomas.     

Tumor-Infiltrating PD-1+ Immune Cell Density is Associated with Response to Neoadjuvant Chemoradiotherapy in Rectal Cancer

BackgroundElevated tumor-infiltrating T-cell density is associated with favorable outcomes in patients with rectal cancer treated with neoadjuvant chemoradiotherapy (CRT). Here, we evaluated the significance of programmed cell death 1 (PD-1)-positive cells, regulatory T cells, and macrophages in response to CRT and prognosis.Patients and MethodsWe assessed CD8+, PD-1+, FOXP3+, CD68+, and CD163+ intratumoral and stromal cell densities by immunohistochemistry using pre-treatment biopsies from 275 patients with rectal cancer treated with neoadjuvant CRT. We determined the impact of these measurements on response to CRT and survival. Response to CRT was determined by tumor regression grade (TRG) of surgical specimens, with good responders defined as TRG3-4.ResultsIntratumoral CD8+ and PD-1+ cell densities were significantly higher in good responders than in poor responders, whereas stromal CD68+ cell density was significantly lower in good responders as compared with poor responders. The multivariable analysis revealed high intratumoral CD8+ and PD-1+ cell densities to be independently associated with good responders (CD8: odds ratio [OR], 2.27; 95% confidence interval [CI], 1.21 – 4.34, P = .010; PD-1: OR, 1.97; 95%CI, 1.03 – 3.84, P = .039), and improved recurrence-free survival (CD8: hazard ratio [HR], 0.56; 95%CI, 0.32 – 0.98, P = .044; PD-1: HR, 0.37; 95%CI, 0.19 – 0.71, P = .002). Only high intratumoral CD8+ cell density was associated with improved overall survival (P = .022).ConclusionPre-treatment high intratumoral PD-1+ and CD8+ cell densities were independently associated with good response to CRT and improved recurrence-free survival, with high intratumoral CD8+ cell density additionally associated with improved overall survival. These values may serve as predictive and prognostic biomarkers in rectal cancer.     

Relationship of a Mr 140 fibronectin receptor and other adhesion-related glycoproteins to tumor cell-cell interaction

Primary melanocytes attach poorly to collagen type IV and laminin, in contrast to their firm attachment to collagen type I/III and fibronectin [Gilchrest et al., In vitro (Rockville), 21: 114-120, 1985]. We have now found that metastatic B16 melanoma cells attach well to collagen type IV, laminin, vitronectin, and fibronectin but show a selective defect in attachment and cell aggregation on native collagen type I. Both flattened and aggregated melanoma cells revealed the presence of a Mr 120,000 surface-iodinated species with affinity for a matrix containing the hexapeptide (glycylarginylglycylaspartylserylproline) which includes the fibronectin cell attachment sequence, but only flattened cells showed significant exposure of a Mr 140,000 iodinated component with affinity for a large cell attachment-promoting fibronectin polypeptide. Decrease of the Mr 140,000 fibronectin-binding external protein in the collagen-cultured melanoma cells was also associated with an inability to respond to the cell attachment activity of fibronectin, laminin, or vitronectin added to the collagen gels. Metabolic labeling with [3H]glucosamine and electrophoretic analysis showed that lack of attachment and cell aggregation was associated with an increase in high molecular weight wheat germ agglutinin-binding glycoconjugates and an increase in Mr 55,000 concanavalin A-binding glycoprotein species. Our data suggest that: (a) melanoma cell attachment requires the expression of the Mr 140,000 fibronectin receptor which appears to be down regulated in cells exposed to poorly adhesive substrates; (b) expression of the Mr 120,000 iodinated species with affinity for the fibronectin attachment sequence (arginylglycylaspartic acid) may be necessary but not sufficient for firm cell-substratum interactions; (c) increased tumor cell-cell interaction may involve a decreased attachment to substrate and the expression of different glycoproteins which may modulate cell-cell association.     

X-rays modulate extracellular matrix in vivo.

X-rays have an antiangiogenic effect in the chicken embryo chorioallantoic membrane (CAM) model of in vivo angiogenesis. Our study demonstrates that X-rays induce an early apoptosis of CAM cells, modulate the synthesis and deposition of extracellular matrix (ECM) proteins involved in regulating angiogenesis and affect angiogenesis induced by tumour cells implanted onto the CAM. Apoptosis was evident within 1-2 hr, but not later than 6 hr after irradiation. Fibronectin, laminin, collagen type I, integrin alpha(v)beta3 and MMP-2 protein amounts were all decreased 6 hr after irradiation. In contrast, collagen type IV, which is restricted to basement membrane, was not affected by irradiation of the CAM. There was a similar decrease of gene expression for fibronectin, laminin, collagen type I and MMP-2, 6 hr after irradiation. The levels of mRNA for integrin alpha(v)beta3 and collagen type IV were unaffected up to 24 hr after irradiation. The decrease in both protein and mRNA levels was reversed at later time points and 48 hr after irradiation, there was a significant increase in the expression of all the genes studied. When C6 glioma tumour cells were implanted on irradiated CAMs, there was a significant increase in the angiogenesis induced by tumour cells, compared to that in non-irradiated CAMs. Therefore, although X-rays have an initial inhibitory effect on angiogenesis, their action on the ECM enhances new vessel formation induced by glioma cells implanted on the tissue.     

Cytoskeletal-induced alterations in the adhesion of HT-1080 fibrosarcoma cells to extracellular matrix

We have investigated the adhesion of the human fibrosarcoma cell line, HT-1080, transfected with glial fibrillary acidic protein (GFAP) to a variety of extracellular matrix macromolecules (ECM) including collagen type IV, laminin, and fibronectin. The GFAP-transfectants demonstrated altered adhesiveness to extracellular matrix substrates when compared to controls. GFAP-positive, heavy metal-induced fibrosarcoma cells were more adherent to plastic and collagen type IV than were the parental or uninduced cells. In contrast, GFAP-positive fibrosarcoma cells were less adherent to laminin- or fibronectin-coated dishes than controls. Time course adhesion studies over 9 days showed that the heavy metal-induced fibrosarcoma cells progressively became more adherent to collagen type IV and less adherent to laminin- or fibronectin-coated dishes than did uninduced cells. However, with the removal of heavy metal from the medium, the HT-1080 fibrosarcoma cells were restored to their original adhesive potential. By phase microscopy, uninduced and induced HT-1080 cells demonstrated different morphological features and remained viable in an anchorage-dependent fashion on collagen type IV as a substrate. By way of contrast, GFAP-induced HT-1080 cells were not particularly viable in monolayer culture and readily detached from laminin as a substrate. The expression of beta1 integrin in GFAP-positive fibrosarcoma cells was decreased following heavy metal induction by Western blot analyses. In contrast, the expression of alpha2 integrin was increased whereas alpha5 integrin was unchanged in HT-1080 cells following the induction of GFAP. Gelatin zymography showed that 72 kDa collagenase was less expressed in GFAP-induced clones than in controls. Our data suggest that the forced expression of the intermediate filament, GFAP, in HT-1080 cells may modulate cell adhesion to different ECM substrates through alterations in expression of integrins.     

Altered cell-matrix contact: a prerequisite for breast cancer metastasis?

The integrins are receptors that regulate interaction between epithelial cells and the extracellular matrix. Previous studies have shown that a reduction in the expression of the alpha2beta1, alpha3beta1, alpha6beta1, alpha(v)beta1 and alpha(v)beta5 integrins in primary breast cancer is associated with positive nodal status. In order to assess the functional significance of altered integrin expression, primary breast cancer cells were derived from individual patients with known tumour characteristics using immunomagnetic separation. Purified human fibronectin, vitronectin, laminin and type IV collagen were used to represent the principal extracellular matrix proteins in an in vitro adhesion assay. Primary breast cancer cells from lymph node-positive patients were significantly less adhesive to each of the matrix proteins studied (P<0.001, Mann-Whitney U-test). Matrix adhesion of primary breast cancer cells from node-negative patients was inhibited by appropriate integrin monoclonal antibodies (P<0.001, paired Wilcoxon test). Adhesion to fibronectin, vitronectin and laminin, but not type IV collagen, was influenced by the inhibitor arginine-glycine-aspartate, suggesting that breast cancer cell recognition of collagen IV is mediated through alternative epitopes. Weak matrix adhesion correlated with loss of integrin expression in tissue sections from corresponding patients assessed using immunohistochemistry. This study demonstrates a link between altered integrin expression and function in primary breast cancers predisposed to metastasize.     

Melanoma cell adhesion to basement membrane mediated by integrin-related complexes

During invasion and metastasis, tumor cells use a variety of surface adhesion receptors to attach to and invade basement membranes and interstitial stroma. We examined the role of the cell surface integrin-like complex in the attachment of the invasive murine B16-BL6 melanoma cell line to basement membrane. Polyclonal antibodies prepared against integrin-related complexes isolated from hamster BHK cells (anti-ECMR) or mouse erythroleukemia cells (anti-mouse FnR) inhibited the attachment of B16 cells to complex basement membrane matrices and to substrates coated with purified extracellular matrix components (fibronectin, laminin, and type IV collagen). The expression of integrin-like receptors on the surface of B16 cells was confirmed by selective immunoprecipitation of radiolabeled and solubilized membrane proteins with the antibodies. Both antibodies also reacted with an integrin-related fibronectin-binding receptor complex purified by ligand affinity chromatography on fibronectin-Sepharose columns. The anti-integrin antibodies failed to react with the Mr 68,000 laminin-binding protein, suggesting that their inhibition of cell attachment to laminin and complex basement membrane was not due to contaminating antibodies against the Mr 68,000 laminin receptor. The results indicate that the integrin receptor complexes on B16-BL6 cells either interact directly with a diverse set of extracellular-matrix-associated components or somehow modulate the activity and function of other receptors. Thus integrins may have an important role in tumor cell invasion of tissue barriers.     

Neural cell adhesion molecule-deficient beta-cell tumorigenesis results in diminished extracellular matrix molecule expression and tumour cell-matrix adhesion.

To understand by which mechanism neural cell adhesion molecule (N-CAM) limits beta tumour cell disaggregation and dissemination, we searched for potential downstream genes of N-CAM during beta tumour cell progression by gene expression profiling. Here, we show that N-CAM-deficient beta-cell tumorigenesis is associated with changes in the expression of genes involved in cell-matrix adhesion and cytoskeletal dynamics, biological processes known to affect the invasive and metastatic behaviour of tumour cells. The extracellular matrix (ECM) molecules emerged as the primary target, i.e. N-CAM deficiency resulted in down-regulated mRNA expression of a broad range of ECM molecules. Consistent with this result, deficient deposition of major ECM stromal components, such as fibronectin, laminin 1 and collagen IV, was observed. Moreover, N-CAM-deficient tumour cells displayed defective matrix adhesion. These results offer a potential mechanism for tumour cell disaggregation during N-CAM-deficient beta tumour cell progression. Prospective consequences of these findings for the role of N-CAM in beta tumour cell dissemination are discussed.     

Regulation of human colon-carcinoma cell adhesion to extracellular matrix by transforming growth factor beta 1.

The regulatory effect of transforming growth factor beta 1 (TGF-beta 1) on the adhesion of human colon-carcinoma cells to the extracellular matrix (ECM) was investigated. ECMs used in this study included tissue-culture wells coated with fibronectin, laminin, collagen and BSA, as well as plastic wells. Three phenotypically different human colon-carcinoma cell lines (Moser, HCT116, and KM12SM) were used. The Moser cell line is moderately differentiated and, in terms of the diversity of responses elicited by TGF-beta 1, is the human colon-carcinoma cell line most responsive to TGF-beta 1 as reported to date. By comparison, the undifferentiated HCT116 and the highly metastatic KM12SM cells are unresponsive to this growth factor. We showed that TGF-beta 1 regulated the adhesion responses of all 3 cell lines. However, the response profiles as well as the endogenous adhesive properties of each cell line were quite different from those of the others. Endogenous Arg-Gly-Asp(RGD)-related receptors were present on the HCT116 but not on the other cells. The observed regulatory effect of TGF-beta 1 was contingent on the cell line, the type of ECM, and the growth-factor treatment protocol used. When cells were treated with TGF-beta 1 for 16 hr prior to exposure to ECM in a 4-hr adhesion assay, a significant increase in adhesion to fibronectin and collagen was observed for the Moser cells. For the identical experimental protocol, the KM12SM cells responded by increasing adhesion to fibronectin, while the HCT116 cells responded by decreasing adhesion to collagen. Kinetic analyses of TGF-beta 1 treatment showed that increased adhesion response to laminin was induced in the Moser cells after 2 hr of growth-factor treatment. This response declined rapidly upon further exposure of the cells to TGF-beta 1. Simultaneous exposure of cells to both TGF-beta 1 and ECM negated the adhesion responses described above. The up-modulation of adhesion to fibronectin, laminin and collagen by TGF-beta 1 was mediated through RGD-related integrin receptors. RGD-containing peptides effectively blocked the enhanced adhesion responses induced by TGF-beta 1.     

Serum CEA as a predictor for the response to preoperative chemoradiation in rectal cancer

BACKGROUND AND OBJECTIVES: Recent data suggest that good responders to preoperative chemoradiation (CRT) have a favorable prognosis in rectal cancer patients. The aim of this study was to investigate the predictive value of serum carcinoembryonic antigen (CEA) levels for the tumor response to preoperative CRT in rectal cancer patients. METHODS: The study comprised 141 rectal adenocarcinoma patients who underwent preoperative radiotherapy with 5-fluorouracil (FU) based chemotherapy, followed by radical surgery. The staging workup was consisted of endorectal ultrasound, abdominopelvic computed tomography scan, or magnetic resonance imaging. The outcome parameters were cancer-specific survival and disease-free survival. Pre-CRT clinicopathologic features, including age, gender, location of the tumor, clinical tumor (cT) classification, clinical nodal (cN) classification, and serum CEA levels were investigated as possible predictors for the response to preoperative CRT. RESULTS: Pathologic complete or near complete responses (good responders, GR) occurred in 26 (19%) patients, while partial or no response (poor responders, PR) occurred in the remaining 115 (81%) patients. GR showed better cancer-specific survival (P = 0.028) and disease-free survival rates (P = 0.011) than PR. Univariate analysis revealed that positive cN and elevated (>5 ng/ml) pre-CRT serum CEA levels are associated with poor tumor response to preoperative CRT. Using logistic regression analysis, elevated pre-CRT serum CEA levels were the only significant predictor for the poor response to CRT (Odd ratio = 2.876, 95% confidence interval = 1.04-7.46, P = 0.041). CONCLUSIONS: Our data suggest that elevated pre-CRT serum CEA levels are associated with poor tumor response to CRT. Therefore, pre-CRT serum CEA levels provide useful information about tumor response to preoperative CRT in rectal cancer patients.     

Gene expression profiles of tumor regression grade in locally advanced rectal cancer after neoadjuvant chemoradiotherapy

Tumor regression grading (TRG) reportedly has prognostic value in rectal cancer patients after pre-operative chemoradiotherapy (CRT). The aim of this retrospective study was to differentiate gene expression profiles based on TRG in residual cancer cells after CRT. We evaluated pathological response using the criteria of four TRG systems: the Japanese Society for the Cancer of Colon and Rectum (JSCCR), Mandard, Dworak and R?del. Total RNA was obtained using microdissection from 52 locally advanced rectal cancer specimens from patients who underwent pre-operative CRT to examine the expression levels of 20 genes [PCNA, MKI67, CDKN1A (p21Cip1), CDK2, CHEK1, PDRG1, LGR5, PROM1 (CD133), CD44, SOX2, POU5F1 (OCT4), LKB1, VEGF, EGFR, HGF, MET, HIF1, GLUT1, BAX and BCL2] using real-time quantitative RT-PCR. Gene expression was compared across the four TRG systems. LGR5 gene expression levels in CRT non-responders were significantly higher than in responders in all four grading systems. Patients with elevated PDRG1 and GLUT1 gene expression had poor pathological response in three TRG systems (JSCCR, Dworak and R?del). MKI67 gene expression in non-responders was significantly higher than in responders in two grading systems (JSCCR and R?del). While, BAX gene expression in responders was significantly higher than in non-responders in the Mandard TRG system. The results of this study suggest that TRG may reflect characteristics, such as proliferative activity, stemness potency and resistance to hypoxia, of residual cancer cells following pre-operative CRT.     

Switching in discoid domain receptor expressions in SLUG-induced epithelial-mesenchymal transition

BACKGROUND: Acquired features of cells under epithelial-mesenchymal transition (EMT) have not yet been fully identified. The current study was conducted to assess alterations in both the proliferative potential and the responsiveness to extracellular matrices (ECMs) in EMT. METHODS: MDCK cells and SLUG-transfected MDCK clones (SLUG-MDCK) were used in this study. The cell cycle was analyzed by using flow cytometry and Western blotting. ECM-stimulated cell proliferation was examined by using the following ECMs, type I collagen, type IV collagen, fibronectin, and laminin. Protein phosphorylation was detected by immunoprecipitation-Western by using the 4G10 antibody. RESULTS: Both G1 and G2/M arrest were found in the SLUG-MDCK cells, and the responsible molecules for the cell-cycle arrests were, at least in part, p21WAF1/Cip1 and Wee1. Once in contact with type I collagen, the SLUG-MDCK cells, showing the Wee1 degradation, dramatically started to proliferate up to 6-fold in cell number at Day 5, in contrast to only a 2-fold increase in the control. The analysis of the collagen receptors in the SLUG-MDCK cells disclosed a striking increase in the discoid domain receptor (DDR) 2 expression and a clear decrease in the DDR1 expression. The immunoprecipitated DDR2 protein extracted from SLUG-MDCK cells, which were cultured on collagen for 30 minutes, was tyrosine-phosphorylated, indicating valid functionality of the up-regulated receptor. The altered expression from DDR1 to DDR2 was also found in the naturally dedifferentiated sister cell lines of human liver cancer. CONCLUSIONS: Collectively, SLUG-induced EMT may alter the expression profile of receptor tyrosine kinases, including DDRs.     

MicroRNA in rectal cancer

In rectal cancer, one of the most common cancers worldwide, the proper staging of the disease determines the subsequent therapy. For those with locally advanced rectal cancer, a neoadjuvant chemoradiotherapy (CRT) is recommended before any surgery. However, response to CRT ranges from complete response (responders) to complete resistance (non-responders). To date we are not able to separate in advance the first group from the second, due to the absence of a valid biomarker. Therefore all patients receive the same therapy regardless of whether they reap benefits. On the other hand almost all patients receive a surgical resection after the CRT, although a watch-and-wait procedure or an endoscopic resection might be sufficient for those who responded well to the CRT. Being highly conserved regulators of gene expression, microRNAs (miRNAs) seem to be promising candidates for biomarkers. Many studies have been analyzing the miRNAs expressed in rectal cancer tissue to determine a specific miRNA profile for the ailment. Unfortunately, there is only a small overlap of identified miRNAs between different studies, posing the question as to whether different methods or differences in tissue storage may contribute to that fact or if the results simply are not reproducible, due to unknown factors with undetected influences on miRNA expression. Other studies sought to find miRNAs which correlate to clinical parameters (tumor grade, nodal stage, metastasis, survival) and therapy response. Although several miRNAs seem to have an impact on the response to CRT or might predict nodal stage, there is still only little overlap between different studies. We here aimed to summarize the current literature on rectal cancer and miRNA expression with respect to the different relevant clinical parameters.     

Role of extracellular matrix in regulation of staurosporine-induced apoptosis in breast cancer cells

Autocrine and paracrine mechanisms modulate the synthesis and secretion of extracellular matrix (ECM); moreover, each component of the ECM is capable of modulating the synthesis and release of other ECM molecules. Therefore, the synthesis of ECM glycoprotein fibronectin and laminin was studied in the human breast cancer cell lines MCF7 and MDA MB 23, plated on different ECM. Our results showed that the cells plated on a fibronectin substrate increased laminin synthesis: this event correlated with an increase in alpha2 and alpha3 integrin subunits. Staurosporine-induced apoptosis was then analyzed in the cell lines plated on different ECM. Staurosporine treatment determined the apoptosis of 35 and 33% respectively of MDA MB 231 and MCF7; these values increased to 60 and 64% in cells plated on laminin, to 48 and 63% in cells plated on fibronectin and to 64 and 69% in cells plated on matrigel. Moreover, staurosporine treatment decreased bcl-2 expression in the cells plated on fibronectin and laminin. Yet, staurosporine treatment determined PARP cleavage and PARP partial disappearance when the cells were plated on matrigel. Finally, a partial loss of function mutant Ras protein that activated only Raf pathway, was expressed in MCF7, in order to identify whether the increase of apoptosis induced by extracellular matrix involved the Raf/MAP kinase pathway. The increase of apoptosis of the cells plated on matrigel suggested that the activation of the Raf pathway is probably involved in the decrease of survival on matrigel. These data demonstrate that the modification of ECM modulates the apoptotic process of breast cancer cells and suggest that it is worthwhile to dissect the role of ECM in the control of apoptotic process.     

Global DNA methylation is altered by neoadjuvant chemoradiotherapy in rectal cancer and may predict response to treatment – A pilot study

Aim

In rectal cancer, not all tumours display a response to neoadjuvant treatment. An accurate predictor of response does not exist to guide patient-specific treatment. DNA methylation is a distinctive molecular pathway in colorectal carcinogenesis. Whether DNA methylation is altered by neoadjuvant treatment and a potential response predictor is unknown. We aimed to determine whether DNA methylation is altered by neoadjuvant chemoradiotherapy (CRT) and to determine its role in predicting response to treatment.

Patients and methods

Fifty-three (n = 53) patients with locally advanced rectal cancers treated with neoadjuvant CRT followed by surgery were identified from the pathology databases of 2 tertiary referral centres over a 4-year period. Immunohistochemical staining of treatment specimens was carried out using the 5-Methylcytidine (Eurogentec, Seraing, Belgium) antibody. Quantitative analysis of staining was performed using an automated image analysis platform. The modified tumour regression grading system was used to assess tumour response to neoadjuvant therapy.

Results

Seven (13%) patients showed complete pathological response while 46 (87%) patients were partial responders to neoadjuvant treatment. In 38 (72%) patients, significant reduction in methylation was observed in post-treatment resection specimens compared to pre-treatment specimens (171.5 vs 152.7, p = 0.01); in 15 (28%) patients, methylation was increased. Pre-treatment methylation correlated significantly with tumour regression (p < 0.001), T-stage (p = 0.005), and was able to predict complete and partial pathological responders (p = 0.01).

Conclusion

Neoadjuvant CRT appears to alter the rectal cancer epigenome. The significant correlation between pre-treatment DNA methylation with tumour response suggests a potential role for methylation as a biomarker of response.