transforming-growth-factor-alpha and Colorectal-Neoplasms

Colorectal cancer (CRC) is the second leading cause of cancer death in the western world. Even with the significant improvement in traditional chemotherapy, there remain limitations with this treatment. One of the most promising new targets in the treatment of CRC is the epithelial growth factor receptor (EGFR). Agents that inhibit the EGFR have demonstrated clinical activity as single agents and in combination with chemotherapy and the most promising of these agents is cetuximab, which blocks the binding of EGF and transforming growth factor-alpha (TGF-alpha) to EGFR. Thus, the finding that monoclonal antibodies against EGFR caused a response in patients, and reversed resistance to chemotherapy, was exciting news. However, expression of EGFR did not correlate with clinical benefit. Clearly, the search for markers of response to treatment against EGFR must go on.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Cetuximab; Colorectal Neoplasms; ErbB Receptors; Humans; Patient Selection; Transforming Growth Factor alpha; Treatment Failure; Treatment Outcome

Genetic changes related to cancer metastasis are overviewed. hst-1/int-2 co-amplification is closely related to the metastatic potential of esophageal carcinomas. Multiple autocrine and paracrine loops including EGF, TGF-alpha/EGF receptor system and HGF/c-met system are related to the biological malignancy of gastric carcinoma in general. On the other hand, K-sam and c-erbB2 amplification are frequently found in the metastatic foci of poorly and well-differentiated type gastric carcinoma. Various splice variants of cell adhesion molecule CD44 are the potent marker of human carcinomas themselves as well as metastases. Reduction in the expression of nm23 is a relatively common event in the metastasis of various human cancers, including stomach and colorectal carcinomas.

Topics: Animals; Carrier Proteins; Colorectal Neoplasms; ErbB Receptors; Gene Amplification; Genes, erbB-2; Humans; Hyaluronan Receptors; Neoplasm Metastasis; Oncogenes; Protein-Tyrosine Kinases; Receptors, Cell Surface; Receptors, Lymphocyte Homing; Stomach Neoplasms; Transforming Growth Factor alpha

Transforming growth factor alpha (TGFα) and TGFβ1 are growth-promoting and -inhibiting autocrine/paracrine growth factors, respectively, that may (1) affect risk for colorectal cancer and (2) be modifiable by anti-proliferative exposures. The effects of supplemental calcium and vitamin D3 on these two markers in the normal-appearing colorectal mucosa in humans are unknown. We conducted a pilot, randomized, double-blind, placebo-controlled, 2 × 2 factorial clinical trial (n = 92; 23/treatment group) of calcium 2 g and/or vitamin D3 800 IU/d versus placebo over 6 mo. TGFα and TGFβ1 expression was measured in biopsies of normal-appearing rectal mucosa using automated immunohistochemistry and quantitative image analysis at baseline and 6-mo follow-up. In the calcium, vitamin D3 , and calcium plus vitamin D3 groups relative to the placebo group (1) the mean overall expression of TGFβ1 increased by 14% (P= 0.25), 19% (P = 0.17), and 22% (P = 0.09); (2) the ratio of TGFα expression in the upper 40% (differentiation zone) to that in the lower 60 (proliferation zone) of the crypts decreased by 34% (P = 0.11), 31% (P = 0.22), and 26% (P = 0.33); and (3) the TGFα/TGFβ1 ratio in the upper 40% of the crypts decreased by 28% (P = 0.09), 14% (P = 0.41), and 22% (P = 0.24), respectively. These preliminary results, although not statistically significant, suggest that supplemental calcium and vitamin D3 may increase TGFβ1 expression and shift TGFα expression downward from the differentiation to the proliferation zone in the crypts in the normal-appearing colorectal mucosa of sporadic colorectal adenoma patients, and support further investigation in a larger clinical trial.

Topics: Adenoma; Aged; Anticarcinogenic Agents; Calcium, Dietary; Cholecalciferol; Colorectal Neoplasms; Dietary Supplements; Double-Blind Method; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Male; Middle Aged; Rectum; Transforming Growth Factor alpha; Transforming Growth Factor beta1; Transforming Growth Factors

Skin toxicity is a known clinical signature used to predict the prognosis of anti-epidermal growth factor receptor (EGFR) antibody treatment in metastatic colorectal cancer (mCRC). There are no biological markers to predict skin toxicity before anti-EGFR antibody treatment in mCRC patients. Between August 2008 and August 2011, pretreatment serum samples were obtained from KRAS wild-type (WT) patients who received anti-EGFR antibody treatment. Serum levels of ligands were measured by ELISA. A total of 103 KRAS WT patients were enrolled in the study. Progression-free survival and overall survival of patients with a high grade (grade 2-3) of skin toxicity were significantly longer than those with a low grade (grade 0-1) of skin toxicity (median progression-free survival, 6.4 months vs 2.4 months, P < 0.001; median overall survival, 14.6 months vs 7.1 months, P = 0.006). There were significant differences in distribution of serum levels of epiregulin (EREG), amphiregulin (AREG), and hepatocyte growth factor (HGF) between groups of low/high grade of skin toxicity (P < 0.048, P < 0.012, P < 0.012, respectively). In addition, serum levels of HGF, EREG, and AREG were inversely proportional to grades of skin toxicity as determined by the Cochran-Armitage test (P = 0.019, P = 0.047, P = 0.021, respectively). Our study indicated that serum levels such as HGF, EREG, and AREG may be significant markers to predict the grade of skin toxicity and the prognosis of anti-EGFR antibody treatment, which contribute to improvement of the management of skin toxicity and survival time in mCRC patients.

Topics: Adult; Aged; Aged, 80 and over; Amphiregulin; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Biomarkers, Pharmacological; Cetuximab; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; EGF Family of Proteins; Epiregulin; ErbB Receptors; Female; GTP Phosphohydrolases; Hepatocyte Growth Factor; Humans; Insulin-Like Growth Factor I; Male; Membrane Proteins; Middle Aged; Mutation; Panitumumab; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); ras Proteins; Skin; Transforming Growth Factor alpha; Treatment Outcome

This study evaluated the activity of 2 schedules of erlotinib in combination with chemotherapy, and the prognostic significance of serum amphiregulin (AREG) and transforming growth factor alpha (TGFa) in metastatic colorectal cancer.. A total of 60 untreated patients were randomized to a "continuous" (CON; erlotinib 100 mg daily) or an "intermittent" (INT; erlotinib 150 mg on alternate day on day 2 to 14, then 150 mg daily on days 15 to 21) schedule of erlotinib with a modified XELOX (capecitabine plus oxaliplatin) regimen. Serum levels of AREG and TGFa were determined serially.. Baseline characteristics were similar between the 2 arms. Of the 58 patients evaluated for response, there was a nonsignificant trend toward a slightly higher overall response rate in the INT arm (66.7%) versus the CON arm (56.7%). At a median follow-up of 2.8 years, the median overall survival was 18.8 months (95% confidence interval = 11.3-22.9 months) and 20.7 months (95% confidence interval = 12.5-31 months, P = .19) for the CON and INT arm, respectively. KRAS mutation did not predict drug response. The 2 arms did not differ significantly in toxicity. Baseline serum TGFa was an independent predictor of progression-free survival, whereas a drop in serum TGFa and AREG levels following 3 to 4 cycles of treatment were associated with shorter progression-free survival and overall survival, respectively.. The intermittent erlotinib schedule was associated with a higher response rate, although this is not statistically significant. Serum TGFa and AREG levels have prognostic significance in erlotinib-treated patients with colorectal cancer, and further studies are warranted.

Topics: Adult; Aged; Amphiregulin; Antineoplastic Combined Chemotherapy Protocols; Capecitabine; Colorectal Neoplasms; Deoxycytidine; Drug Administration Schedule; EGF Family of Proteins; Erlotinib Hydrochloride; Female; Fluorouracil; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Mutation; Neoplasm Metastasis; Oxaloacetates; Patient Compliance; Prognosis; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Quinazolines; ras Proteins; Transforming Growth Factor alpha

Data from rat experimental carcinogenesis studies indicate that supplemental dietary cellulose reduces the incidence of colon cancer. Epidemiology studies also indicate that high dietary fiber reduces the risk of colorectal cancer in humans. Patients diagnosed with sporadic adenomas were entered into a randomized clinical trial to determine if supplemental dietary cellulose would reduce the patients' risk for colon cancer. Immunohistochemical staining for transforming growth factor alpha (TGF-alpha) was done on biopsies of rectal mucosa taken from patients at the time of initial polypectomy and 1 year later. Results were evaluated for utility as a surrogate end point biomarker for reduction in colon cancer risk. There was a significant decrease in the fraction of the rectal crypt cells that stained for TGF-alpha in six of seven of the patients given the cellulose supplements but in only one of six of the patients not given cellulose. Thus, whether evaluated as a group or in individual patients, there was a significant decrease in TGF-alpha in rectal crypts due to cellulose intervention, which correlated with the expected ability of supplemental dietary cellulose to decrease the risk for colon cancer. Long-term testing of the ability of dietary cellulose to reduce adenoma recurrence is under way to validate the use of TGF-alpha as a surrogate end point biomarker.

Topics: Adult; Aged; Animals; Biomarkers, Tumor; Biopsy; Cell Transformation, Neoplastic; Cellulose; Colonic Polyps; Colorectal Neoplasms; Dietary Fiber; Female; Humans; Immunoenzyme Techniques; Intestinal Mucosa; Male; Middle Aged; Neoplasm Recurrence, Local; Prospective Studies; Rats; Risk Factors; Transforming Growth Factor alpha

Natural products are popular insights for researchers to investigate promising anti-cancer agents since some of these substances have lesser adverse effects restricting the treatment than traditional chemotherapeutic agents. A well-known monoterpene Carvacrol, widely consumed in Mediterranean cuisine and lower risks of cancer, has efficient anticancer effects. However, the mechanism of action is yet to be discovered.. The investigation aims to illuminate a new perceptive in the role of this substance on colorectal cancer treatment, by the means of differences in a well-defined range of soluble factors. Carvacrol effect on both HT-29 and HCT-116 cell lines was evaluated on proliferation and the IC50 values were calculated by the RTCA xCELLigence device. Then MAGPIX assay was performed to obtain the changes in soluble factors of the cell lines.. The Multiplexing assay suggests some of these factors were altered in favor of surviving and proliferation in aggressive cell line HCT-116 whereas they were altered against these characters in HT-29, were correlated with the increased IC50 concentration of HCT- 116 in carvacrol treatment.. The current study indicates that differences in the levels of these soluble factors could modulate the anticancer effect related to carvacrol.

Topics: Cell Proliferation; Colorectal Neoplasms; Cymenes; Granulocyte-Macrophage Colony-Stimulating Factor; HCT116 Cells; HT29 Cells; Humans; Leptin; Prolactin; Transforming Growth Factor alpha; Vascular Endothelial Growth Factor Receptor-2

Activating mutations in KRAS frequently occur in colorectal cancer (CRC) patients, leading to resistance to EGFR-targeted therapies.. To better understand the cellular reprogramming which occurs in mutant KRAS cells, we have undertaken a systems-level analysis of four CRC cell lines which express either wild type (wt) KRAS or the oncogenic KRAS. RNAseq revealed that genes involved in ribosome biogenesis, mRNA translation and metabolism were significantly upregulated in mtKRAS cells. Consistent with the transcriptional data, protein synthesis and cell proliferation were significantly higher in the mtKRAS cells. Targeted metabolomics analysis also confirmed the metabolic reprogramming in mtKRAS cells. Interestingly, mtKRAS cells were highly transcriptionally responsive to EGFR activation by TGFα stimulation, which was associated with an unexpected downregulation of genes involved in a range of anabolic processes. While TGFα treatment strongly activated protein synthesis in wtKRAS cells, protein synthesis was not activated above basal levels in the TGFα-treated mtKRAS cells. This was likely due to the defective activation of the mTORC1 and other pathways by TGFα in mtKRAS cells, which was associated with impaired activation of PKB signalling and a transient induction of AMPK signalling.. We have found that mtKRAS cells are substantially rewired at the transcriptional, translational and metabolic levels and that this rewiring may reveal new vulnerabilities in oncogenic KRAS CRC cells that could be exploited in future.

Topics: AMP-Activated Protein Kinases; Cell Line, Tumor; Colorectal Neoplasms; ErbB Receptors; Humans; Mechanistic Target of Rapamycin Complex 1; Metabolomics; Mutation; Proto-Oncogene Proteins p21(ras); Ribosomes; Signal Transduction; Transcription, Genetic; Transforming Growth Factor alpha

Colorectal cancer (CRC) is the 3rd most common cancer worldwide. Recently, long non-coding RNAs (lncRNAs) were found to be critical modulators in the CRC progression. The aim of this study is to investigate the potential roles of lncRNA P73 antisense RNA 1T (TP73-AS1) in CRC development and progression.. Quantitative real-time PCR (qRT-PCR) was performed to determine relevant gene expression levels; western blot was performed to determine protein expression levels; CCK-8, colony formation, wound healing and Transwell invasion assays were used to determined CRC cell proliferation, migration and invasion; in vivo tumor growth was assessed in xenograft mice model.. TP73-AS1 was up-regulated in both CRC tissues and CRC cell lines. Overexpression of TP73-AS1 was associated with metastasis and advanced clinical stages in CRC patients. Overexpression of TP73-AS1 promoted CRC cell growth, proliferation, migration and invasion in vitro; and knockdown of TP73-AS1 significantly inhibited CRC cell growth, proliferation, migration and invasion in vitro as well as tumor growth in vivo. Bioinformatics analysis and luciferase reporter assay indicated that TP73-AS1 could bind directly with miR-194, and TP73-AS1 negatively regulated the expression of miR-194 in CRC cells. Further study indicated that miR-194 negatively regulated the downstream target of transforming growth factor alpha (TGFα) via targeting its 3' untranslated region, and TP73-AS1 positively regulated the expression of TGFα in CRC cells. Moreover, overexpression of miR-194 suppressed CRC cell proliferation and invasion, and attenuated the effects of TP73-AS1 overexpression on CRC cell proliferation and invasion. Silence of TGFα inhibited CRC cell proliferation and invasion, and also reversed the effects of TP73-AS1 overexpression on CRC cell proliferation and invasion.. this study demonstrated that TP73-AS1 regulated CRC progression by acting as a competitive endogenous RNA to sponge miR-194 to modulate the expression of TGFα.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; MicroRNAs; Neoplasm Invasiveness; Neoplasm Staging; RNA, Antisense; RNA, Long Noncoding; Transforming Growth Factor alpha; Xenograft Model Antitumor Assays

Anti-EGFR mAb is reported to induce EGFR internalization in colorectal cancer cells. However, the biological relevance of EGFR internalization with anti-EGFR mAb is unknown. Therefore, the relevance of EGFR downregulation with anti-EGFR mAb to antitumor activity in colorectal cancer cells was investigated. Quantification of EGFR on the cell surface before cetuximab treatment was assessed by flow cytometry, and its growth-inhibitory effects were measured by Trypan blue exclusion, in 10

Topics: Adult; Aged; Antineoplastic Agents, Immunological; Caco-2 Cells; Cell Line, Tumor; Cell Survival; Cetuximab; Colorectal Neoplasms; Down-Regulation; Endosomes; ErbB Receptors; Female; HCT116 Cells; HT29 Cells; Humans; Male; Middle Aged; Proto-Oncogene Proteins B-raf; ras Proteins; Transforming Growth Factor alpha

In some biomedical studies, biomarkers are measured repeatedly along some spatial structure or over time and are subject to measurement error. In these studies, it is often of interest to evaluate associations between a clinical endpoint and these biomarkers (also known as functional biomarkers). There are potentially two levels of correlation in such data, namely, between repeated measurements of a biomarker from the same subject and between multiple biomarkers from the same subject; none of the existing methods accounts for correlation between multiple functional biomarkers. We propose a Bayesian approach to model a clinical outcome of interest (e.g. risk for colorectal cancer) in the presence of multiple functional biomarkers while accounting for potential correlation. Our simulations show that the proposed approach achieves good performance in finite samples under various settings. In the presence of substantial or moderate correlation, the proposed approach outperforms an existing approach that does not account for correlation. The proposed approach is applied to a study of biomarkers of risk for colorectal neoplasms and our results show that the risk for colorectal cancer is associated with two functional biomarkers, APC and TGF-α, in particular, with their values in the region between the proliferating and differentiating zones of colorectal crypts.

Topics: Adenomatous Polyposis Coli Protein; Bayes Theorem; Biomarkers; Colorectal Neoplasms; Humans; Research Design; Risk; Transforming Growth Factor alpha

Increased colorectal epithelial cell proliferation is an early, common event in colorectal carcinogenesis. We conducted a pilot, colonoscopy-based case-control study (n = 49 cases, 154 controls) of incident, sporadic colorectal adenoma to investigate endogenous cell growth factors and receptor, as well as the balance of growth factors, as potential modifiable pre-neoplastic biomarkers of risk for colorectal neoplasms. We measured transforming growth factor alpha (TGFα), TGFβ(1), and TGFβ receptor II (TGFβRII) expression in normal-appearing mucosa from the rectum, sigmoid colon, and ascending colon using automated immunohistochemistry and quantitative image analysis. Diet and lifestyle were assessed via questionnaires. The mean ratio of rectal TGFα to TGFβ(1) expression and mean rectal TGFα expression were, respectively, 110% (P = 0.02) and 49% (P = 0.04) higher in cases than in controls, and associated with a more than two-fold (OR 2.42, 95% CI 0.85-6.87) and a 62% (OR 1.62, 95% CI 0.63-4.19) higher risk of colorectal adenoma. TGFβ(1) and TGFβRII expression were 6.7% (P = 0.75) and 7.2% (P = 0.49), respectively, lower in cases than in controls. The TGFα/TGFβ(1) expression ratio was 105% higher among smokers than among non-smokers (P = 0.03). These preliminary data suggest that the balance of TGFα and TGFβ(1) expression, and to a lesser extend TGFα alone, in the normal-appearing rectal mucosa may be directly associated with risk for incident, sporadic colorectal neoplasms, as well as with modifiable risk factors for colorectal neoplasms.

Topics: Adult; Aged; Biomarkers, Tumor; Case-Control Studies; Colon; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; Receptors, Transforming Growth Factor beta; Rectum; Risk Factors; Transforming Growth Factor alpha; Transforming Growth Factor beta1

The epidermal growth factor family (EGF) has been suggested to influence the sensitivity to anti-epidermal growth factor receptor therapy. We examined the correlation between circulating levels of the epidermal growth factors amphiregulin and transforming growth factor-α (TGF-α) and the MET ligand hepatocyte growth factor and sensitivity to the anti-epidermal growth factor receptor antibody in colorectal cancer (CRC) patients.. Plasma levels of each ligand were measured by enzyme-linked immunosorbent assay in 51 patients with wild-type KRAS CRC.. Patients with high hepatocyte growth factor (HGF) levels had a significantly lower disease control rate (DCR) and shorter median progression-free survival (PFS) and overall survival (OS) than those with low expression levels. Amphiregulin was correlated with objective response rate (ORR) but not with PFS or OS. Cetuximab response and survival were not associated with TGF-α.. Circulating HGF may help identify CRC patients most likely to benefit from anti-epidermal growth factor receptor antibody therapy.

Topics: Adult; Aged; Aged, 80 and over; Amphiregulin; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cetuximab; Colorectal Neoplasms; Drug Resistance, Neoplasm; Female; Hepatocyte Growth Factor; Humans; Male; Middle Aged; Proto-Oncogene Proteins c-met; Transforming Growth Factor alpha

Colon cancer is a cancer of the epithelial cells lining the colon. It is mainly divided into different stages according to the invasiveness and metastatic ability of the tumor. Many mutations are acquired which leads to the development of this malignancy. These occur in entities that greatly affect the cell cycle, cell signaling pathways and cell motility, which all involve the action of Rho GTPases. The protein of interest in the present study was DLC2, also known as StarD13 or START-GAP2, a GTPase-activating protein (GAP) for Rho and Cdc42. Literature data indicate that this protein is considered a tumor-suppressor in hepatocellular carcinoma. Previous research in our laboratory confirmed StarD13 as a tumor suppressor in astrocytoma and in breast cancer. In the present study, we investigated the role of StarD13 in colon cancer. When overexpressed, StarD13 was found to lead to a decrease in cell proliferation in colon cancer cells. Consistently, knockdown of StarD13 led to an increase in cell proliferation. This showed that, similarly to its role in astrocytoma and breast cancer, StarD13 appears to be a tumor suppressor in colon cancer as well. We also examined the role of StarD13 in cell motility. StarD13 knockdown resulted in the inhibition of 2D cell motility. This was due to the inhibition of Rho; consequently Rac-dependent focal complexes were not formed nor detached for the cells to move forward. However, StarD13 knockdown led to an increase in 3D cell motility. Although StarD13 was indeed a tumor suppressor in our colon cancer cells, as evidenced by its effect on cell proliferation, it was required for cancer cell invasion. The present study further describes the role of StarD13 as a tumor suppressor as well as a Rho GAP.

Topics: bcl-2-Associated X Protein; Caco-2 Cells; cdc42 GTP-Binding Protein; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Genes, Tumor Suppressor; GTPase-Activating Proteins; HT29 Cells; Humans; Neoplasm Invasiveness; Proto-Oncogene Proteins c-bcl-2; rac1 GTP-Binding Protein; rhoA GTP-Binding Protein; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor alpha; Transforming Growth Factor beta1; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Targeted inhibition of EGFR with the mAbs cetuximab or panitumumab is a valuable treatment for RAS wild-type colorectal cancers. The efficacy of EGFR blockade is limited by the emergence of acquired resistance often attributed to secondary KRAS mutations. Remarkably, tumor biopsies from resistant patients show that only a fraction of the resilient cells carry KRAS mutations. We hypothesized that a paracrine cross-talk driven by the resistant subpopulation may provide in trans protection of surrounding sensitive cells.. Conditioned medium assays and three-dimensional cocultures were used to assess paracrine networks between cetuximab-sensitive and -resistant cells. Production of EGFR ligands by cells sensitive to cetuximab and panitumumab was measured. The ability of recombinant EGFR ligands to protect sensitive cells from cetuximab was assessed. Biochemical activation of the EGFR signaling pathway was measured by Western blotting.. Colorectal cancer cells sensitive to EGFR blockade can successfully grow despite cetuximab treatment when in the company of their resistant derivatives. Media conditioned by resistant cells protect sensitive parental cells from cetuximab. EGFR blockade triggers increased secretion of TGFα and amphiregulin. Increased secretion of ligands by resistant cells can sustain EGFR/ERK signaling in sensitive cells.. Colorectal cancer cells that develop resistance to cetuximab and panitumumab secrete TGFα and amphiregulin, which protect the surrounding cells from EGFR blockade. This paracrine protective mechanism might be therapeutically exploitable.

Topics: Amphiregulin; Antibodies, Monoclonal, Humanized; Cell Line, Tumor; Cell Proliferation; Cetuximab; Colorectal Neoplasms; Culture Media, Conditioned; Drug Resistance, Neoplasm; ErbB Receptors; Genes, ras; Humans; MAP Kinase Signaling System; Mutation; Paracrine Communication; Protein Kinase Inhibitors; Transforming Growth Factor alpha

Increased secretion of EGFR ligands amphiregulin and TGFα by limited KRAS-mutant clones is suggested as a paracrine resistance mechanism to anti-EGFR antibodies in colorectal cancer models. These findings are biologically sound but need to be replicated, including in the clinical setting, to foresee whether they are clinically relevant and therapeutically exploitable.

Topics: Amphiregulin; Colorectal Neoplasms; Drug Resistance, Neoplasm; ErbB Receptors; Humans; Paracrine Communication; Transforming Growth Factor alpha

Extracellular S468R mutation of the epidermal growth factor receptor (EGFR) was recently identified as the cause of resistance to cetuximab, a widely used drug in colorectal cancer treatment. Here, we have determined the binding free energies of cetuximab's Fab V(H)-V(L) domains and endogenous EGF ligand to wild type and S468R EGFR by high-throughput molecular dynamics. This work provides a possible mechanism of resistance in terms of increased competition, an hypothesis that can be further validated experimentally.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cetuximab; Colorectal Neoplasms; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Molecular Dynamics Simulation; Mutation; Neuregulin-1; Single-Domain Antibodies; Thermodynamics; Transforming Growth Factor alpha

Meprinα, an astacin-type metalloprotease is overexpressed in colorectal cancer cells and is secreted in a non-polarized fashion, leading to the accumulation of meprinα in the tumor stroma. The transition from normal colonocytes to colorectal cancer correlates with increased meprinα activity at primary tumor sites. A role for meprinα in invasion and metastatic dissemination is supported by its pro-angiogenic and pro-migratory activity. In the present study, we provide evidence for a meprinα-mediated transactivation of the EGFR signaling pathway and suggest that this mechanism is involved in colorectal cancer progression. Using alkaline phosphatase-tagged EGFR ligands and an ELISA assay, we demonstrate that meprinα is capable of shedding epidermal growth factor (EGF) and transforming growth factor-α (TGFα) from the plasma membrane. Shedding was abrogated using actinonin, an inhibitor for meprinα. The physiological effects of meprinα-mediated shedding of EGF and TGFα were investigated with human colorectal adenocarcinoma cells (Caco-2). Proteolytically active meprinα leads to an increase in EGFR and ERK1/2 phosphorylation and subsequently enhances cell proliferation and migration. In conclusion, the implication of meprinα in the EGFR/MAPK signaling pathway indicates a role of meprinα in colorectal cancer progression.

Topics: Anti-Bacterial Agents; Caco-2 Cells; Cell Movement; Cell Proliferation; Colorectal Neoplasms; ErbB Receptors; Humans; Hydroxamic Acids; Ligands; MAP Kinase Signaling System; Metalloendopeptidases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Proteins; Phosphorylation; Transforming Growth Factor alpha

Oncogenic mutations in Kras occur in 40% to 45% of patients with advanced colorectal cancer (CRC). We have previously shown that chemotherapy acutely activates ADAM17, resulting in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. In this study, we examined the role of mutant Kras in regulating growth factor shedding and ADAM17 activity, using isogenic Kras mutant (MT) and wild-type (WT) HCT116 CRC cells. Significantly higher levels of TGF-α and VEGF were shed from KrasMT HCT116 cells, both basally and following chemotherapy treatment, and this correlated with increased pErk (phosphorylated extracellular signal regulated kinase)1/2 levels and ADAM17 activity. Inhibition of Kras, MEK (MAP/ERK kinase)1/2, or Erk1/2 inhibition abrogated chemotherapy-induced ADAM17 activity and TGF-α shedding. Moreover, we found that these effects were not drug or cell line specific. In addition, MEK1/2 inhibition in KrasMT xenografts resulted in significant decreases in ADAM17 activity and growth factor shedding in vivo, which correlated with dramatically attenuated tumor growth. Furthermore, we found that MEK1/2 inhibition significantly induced apoptosis both alone and when combined with chemotherapy in KrasMT cells. Importantly, we found that sensitivity to MEK1/2 inhibition was ADAM17 dependent in vitro and in vivo. Collectively, our findings indicate that oncogenic Kras regulates ADAM17 activity and thereby growth factor ligand shedding in a MEK1/2/Erk1/2-dependent manner and that KrasMT CRC tumors are vulnerable to MEK1/2 inhibitors, at least in part, due to their dependency on ADAM17 activity.

Topics: ADAM Proteins; ADAM17 Protein; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cell Line, Tumor; Cetuximab; Colorectal Neoplasms; Gefitinib; Genes, ras; HCT116 Cells; HT29 Cells; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mice; Mice, Inbred BALB C; Mice, SCID; Point Mutation; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Quinazolines; ras Proteins; Transforming Growth Factor alpha

Transforming growth factor-alpha (TGF-alpha), a stimulatory growth factor and member of the epidermal growth factor family, is a mediator of oncogenesis and malignant progression in colorectal carcinogenesis. Limited evidence suggests its utility as a growth-related biomarker of risk for colorectal cancer.. We measured expression of TGF-alpha in biopsies of normal-appearing colorectal mucosa using automated immunohistochemistry and quantitative image analysis in a subsample of 29 cases and 31 controls from a colonoscopy-based case-control study (n = 203) of biomarkers of risk for incident sporadic colorectal adenoma. Diet, lifestyle, and medical history were assessed with validated questionnaires.. TGF-alpha expression in the rectum was 51% higher in cases compared with controls (P = 0.05) and statistically significantly associated with accepted risk factors for colorectal neoplasms (36% lower among nonsteroidal anti-inflammatory drug users, 49% lower among women using hormone replacement therapy, 79% higher among persons with a family history of colorectal cancer).. TGF-alpha expression in the normal-appearing rectal mucosa shows promise as an early, potentially modifiable biomarker of risk for colorectal cancer.

Topics: Adenoma; Adult; Aged; Biomarkers, Tumor; Case-Control Studies; Chi-Square Distribution; Colonoscopy; Colorectal Neoplasms; Diet; Female; Humans; Immunohistochemistry; Incidence; Intestinal Mucosa; Male; Middle Aged; Phenotype; Regression Analysis; Risk Factors; Surveys and Questionnaires; Transforming Growth Factor alpha

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) has emerged as a promising anticancer agent. However, resistance to TRAIL is likely to be a major problem, and sensitization of cancer cells to TRAIL may therefore be an important anticancer strategy. In this study, we examined the effect of the epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) gefitinib and a human epidermal receptor 2 (HER2)-TKI (M578440) on the sensitivity of human colorectal cancer (CRC) cell lines to recombinant human TRAIL (rhTRAIL). A synergistic interaction between rhTRAIL and gefitinib and rhTRAIL and M578440 was observed in both rhTRAIL-sensitive and resistant CRC cells. This synergy correlated with an increase in EGFR and HER2 activation after rhTRAIL treatment. Furthermore, treatment of CRC cells with rhTRAIL resulted in activation of the Src family kinases (SFK). Importantly, we found that rhTRAIL treatment induced shedding of transforming growth factor-alpha (TGF-alpha) that was dependent on SFK activity and the protease ADAM-17. Moreover, this shedding of TGF-alpha was critical for rhTRAIL-induced activation of EGFR. In support of this, SFK inhibitors and small interfering RNAs targeting ADAM-17 and TGF-alpha also sensitized CRC cells to rhTRAIL-mediated apoptosis. Taken together, our findings indicate that both rhTRAIL-sensitive and resistant CRC cells respond to rhTRAIL treatment by activating an EGFR/HER2-mediated survival response and that these cells can be sensitized to rhTRAIL using EGFR/HER2-targeted therapies. Furthermore, this acute response to rhTRAIL is regulated by SFK-mediated and ADAM-17-mediated shedding of TGF-alpha, such that targeting SFKs or inhibiting ADAM-17, in combination with rhTRAIL, may enhance the response of CRC tumors to rhTRAIL.

Topics: ADAM Proteins; ADAM17 Protein; Apoptosis; Colorectal Neoplasms; Drug Resistance, Neoplasm; ErbB Receptors; Gefitinib; HCT116 Cells; HT29 Cells; Humans; Metalloproteases; Quinazolines; Receptor, ErbB-2; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Recombinant Proteins; src-Family Kinases; TNF-Related Apoptosis-Inducing Ligand; Transforming Growth Factor alpha

Activation of the epidermal growth factor receptor (EGFR) requires cell surface cleavage of EGFR ligands, uptake of soluble ligand by the receptor, and initiation of EGFR tyrosine kinase activity. We define these collective events as the EGFR axis. Transforming growth factor-alpha (TGF-alpha) and amphiregulin are two EGFR ligands that are delivered preferentially to the basolateral surface of polarized epithelial cells where the EGFR resides. TACE/ADAM-17 (tumor necrosis factor-alpha converting enzyme/a disintegrin and metalloprotease) has been implicated in ectodomain cleavage of TGF-alpha and amphiregulin.. Using a human polarizing colorectal cancer (CRC) cell line, HCA-7, and a tissue array of normal colonic mucosa and primary and metastatic CRC, we determined the intracellular localization of TACE and the effects of EGFR axis inhibition in CRC.. Herein, we show that TACE is localized to the basolateral plasma membrane of polarized HCA-7 cells. TACE is overexpressed in primary and metastatic CRC tumors compared with normal colonic mucosa; the intensity of its immunoreactivity is inversely correlated with that of TGF-alpha and amphiregulin. Pharmacologic blockade of HCA-7 cells with an EGFR monoclonal antibody, a selective EGFR tyrosine kinase inhibitor, and a selective TACE inhibitor results in concentration-dependent decreases in cell proliferation and active, phosphorylated mitogen-activated protein kinase. Combining suboptimal concentrations of these agents results in cooperative growth inhibition, increased apoptosis, and reduced mitogen-activated protein kinase pathway activation. Furthermore, an EGFR tyrosine kinase-resistant clone of HCA-7 cells is growth-inhibited by combined monoclonal antibody and TACE inhibition.. These results implicate TACE as a promising target of EGFR axis inhibition in CRC.

Topics: ADAM Proteins; ADAM17 Protein; Amphiregulin; Apoptosis; Blotting, Western; Cell Line, Tumor; Colorectal Neoplasms; EGF Family of Proteins; Enzyme Inhibitors; ErbB Receptors; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Tissue Array Analysis; Transforming Growth Factor alpha

Autocrine tumour growth factor alpha (TGFalpha)/epidermal growth factor receptor (EGFR) stimulation in colorectal carcinoma (CRC) cells regulates cell adhesion and invasiveness via ribosomal protein S6 kinase (S6K) phosphorylation in pre-clinical studies. The aim of this study was to evaluate whether TGFalpha and EGFR expression might be correlated with a higher metastatic behaviour in human tumours. Paraffin-embedded material was retrospectively collected from 101 primitive CRCs including all stage IV patients at diagnosis treated at our Institution from 1999 to 2004 (50 cases, Group B) and 51 stage II-III control cases (Group A). EGFR and TGFalpha expression, together with signalling molecules (including signal transducer and activator of transcription [STAT3], serine-treonine kinase [Akt], mitogen-activated protein kinase [MAPK], mammalian target of rapamycin [mTOR] and S6K) in selected samples, was evaluated by immunohistochemistry using the EGFR Dako antibody. A total of 68/101 (67.3%) cases were EGFR positive and 79/101 (78.2%) cases were TGFalpha positive. EGFR/TGFalpha co-expression differed significantly (p = 0.02) between Group A and Group B tumours (23/51, 45.1% vs 34/50, 68.0%, respectively), whereas no differences in STAT, Akt, mTOR expression was evident between the two groups. Conversely, there was a significantly higher expression of phosphorylated S6K in stage IV cases (Group B) than in the controls (Group A; 70.4% vs 38.7%; p = 0.02). In agreement with in vitro data, EGFR, TGFalpha and S6K co-expression in human CRC was significantly higher in patients with advanced stage at diagnosis.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Colorectal Neoplasms; ErbB Receptors; Female; Humans; Immunoenzyme Techniques; Male; Mitogen-Activated Protein Kinase Kinases; Neoplasm Metastasis; Neoplasm Staging; Protein Kinases; Proto-Oncogene Proteins c-akt; Retrospective Studies; Ribosomal Protein S6 Kinases; STAT3 Transcription Factor; TOR Serine-Threonine Kinases; Transforming Growth Factor alpha

Evaluation of the relationship between clinicopathological and immunohistochemical risk factors for liver metastasis, including CD10 expression, is meaningful in colorectal carcinoma (CRC). The purpose of the present study was to clarify what kind of risk factors are significant and independent factors for the development of liver metastasis in CRC. Sixty cases of advanced CRC with synchronous liver metastasis and sixty cases of advanced CRC without liver metastasis at least 5 years after resection of the primary CRC were randomly selected. We analysed the clinicopathological factors and the expression of four biological factors, CD44, transforming growth factor alpha (TGF-alpha), vascular endothelial growth factor (VEGF) and CD10, by immunohistochemistry. Univariate analysis revealed that the incidence of vascular invasion, the incidence of lymph node metastasis and the expression of CD44, TGF-alpha, VEGF and CD10 were all significantly higher in the cases of CRC with liver metastasis than in cases of non-metastatic CRC. Logistic regression analysis showed that lymph node metastasis, the expression of CD10 and the expression of VEGF were significant factors and independent of the other variables. If all three markers are positive, the probability of liver metastasis becomes 92.7%. In this study, lymph node metastasis, CD10 and VEGF were all found to be significant risk factors for the development of liver metastasis in the cases of CRC. These risk factors according to multivariate analysis are candidate markers for predicting the development of liver metastasis.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Colorectal Neoplasms; Female; Humans; Hyaluronan Receptors; Immunohistochemistry; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neprilysin; Risk Factors; Transforming Growth Factor alpha; Vascular Endothelial Growth Factor A

Incapacitating symptom burden in cancer patients contributes to poor quality of life (QOL) and can influence treatment outcomes because of poor tolerance to therapy. In this study, the role of circulating cytokines in the production symptoms in cancer patients is evaluated.. Eighty patients with metastatic colorectal cancer with either normal (group I, n = 40) or dampened (group II, n = 40) 24-hour rest/activity patterns measured by actigraphy were identified. Actigraphy patterns were correlated with QOL indices, serum cortisol obtained at 8:00 a.m. and 4:00 p.m. and with serum levels of transforming growth factor-alpha, tumor necrosis factor-alpha, and interleukin 6 (IL-6) obtained at 8:00 a.m. and analyzed in duplicate by ELISA. Cytokine levels and survival were also correlated.. Group II patients had significantly higher pre treatment levels of all three cytokines, displayed significantly poorer emotional and social functioning, had higher fatigue, more appetite loss, and poorer performance status compared with group I patients. Transforming growth factor-alpha (TGF-alpha) and IL-6 were significantly increased in the patients with WHO performance status >1 and in those with appetite loss. Fatigue was significantly associated with elevated TGF-alpha only. IL-6 was increased in those patients with extensive liver involvement and multiple organ replacement, and it was significantly correlated with dampened cortisol rhythm. In a multivariate analysis, IL-6 was correlated with poor treatment outcome.. Significant correlations were found between serum levels of TGF-alpha and IL-6, circadian patterns in wrist activity and serum cortisol and tumor-related symptoms in patients with metastatic colorectal cancer. These data support the hypothesis that some cancer patient's symptoms of fatigue, poor QOL, and treatment outcome are related to tumor or host generated cytokines and could reflect cytokine effects on the circadian timing system. This interplay between cytokine signaling pathways, the hypothalamic-pituitary-adrenal axis, the autonomic nervous system, and efferent pathways of the suprachiasmatic nucleus that control circadian physiology, opens the way to new rational interventions for symptom management in cancer patients.

Topics: Activities of Daily Living; Adult; Aged; Appetite; Circadian Rhythm; Colorectal Neoplasms; Fatigue; Female; Humans; Hydrocortisone; Interleukin-6; Male; Middle Aged; Neoplasm Metastasis; Prognosis; Prospective Studies; Quality of Life; Sleep; Social Behavior; Survival Analysis; Transforming Growth Factor alpha; Tumor Necrosis Factor-alpha

Matrix metalloproteinase-7 (MMP-7) plays an important role in carcinoma invasion and metastasis of cancer. Recent studies focus on diverse roles of MMP-7, other than as a protease, during cancer progression. MMP-7 activates the epidermal growth factor (EGF) receptor by releasing an EGF ligand, tumor growth factor (TGF)-alpha.. We examined expression of MMP-7 and EGF receptor in an immunohistochemical study of 40 colorectal cancer (CRC) cases. To determine the relationship between the EGF receptor and MMP-7, with a potential curative application, we compared the antitumor activity of the EGF receptor tyrosine kinase inhibitor (gefitinib) between MMP-7 transfectant, KYSE150 and HT29, and control cells.. We found a statistically significant correlation (P = 0.04) between MMP-7 and activated (phosphorylated) EGF receptor expression, both being positive in six (15%) cases. Gefitinib reduced the cell number ratio more for MMP-7 transfectant than mock cells, and the proportion of apoptotic cells was 1.5 times higher in MMP-7 transfectant than mock cells by annexin/propidium iodide staining. This was mediated by activation of a TGF-beta signal as confirmed by the abundant expression of TGF-beta protein, the cytoplasmic to nuclear translocation of Smad4 protein by the administration of gefitinib, and the quantitative assay of the plasminogen activator inhibitor-1 promoter/luciferase construction.. We propose that there are some cancers with up-regulated MMP-7 expression that leads to the activation of apoptotic activity of TGF-beta, which is susceptible to treatment with EGF receptor tyrosine kinase inhibitor.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Apoptosis; Cell Nucleus; Colorectal Neoplasms; Cytoplasm; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Humans; Immunoenzyme Techniques; Male; Matrix Metalloproteinase 7; Middle Aged; Phosphorylation; Protein Transport; Quinazolines; Smad4 Protein; Trans-Activators; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Colon and rectal mucosal crypt epithelium is a rapidly renewing cell population, where cell proliferation is normally balanced by cell loss. This report concerns the putative paracrine action of transforming growth factor alpha(TGF-alpha) in this homeostatic process. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and TGF-alpha was performed on biopsy specimens of rectal mucosa taken from consenting patients. The height of the proliferative compartment in mid-axially sectioned crypts in each individual was determined from the distribution of PCNA stained cells. The number of TGF-alpha stained cells that exhibited intense positive staining in a continuous column from the mouth down the side of the crypt was also scored in each individual patient. There was a significant positive correlation (P=0.05,n =22 patients) between the height of the proliferative compartment and the number of cells staining for TGF-alpha. Non-cellular TGF-alpha reactivity was also observed in the lamina propria adjacent to the TGF-alpha reactive epithelial cells, indicating secretion of TGF-alpha by these epithelial cells. These findings suggest that TGF-alpha is released from epithelial cells in the upper compartment of the crypt into the adjacent lamina propria and then diffuses to the epithelial cells in the lower part of the crypt, resulting in expansion of the proliferative compartment.

Topics: Aged; Colorectal Neoplasms; Epithelial Cells; Female; Humans; Intestinal Mucosa; Linear Models; Male; Middle Aged; Paracrine Communication; Rectum; Transforming Growth Factor alpha

Although pathologic stage is currently the main prognostic indicator for patients with colorectal carcinoma (CRC), mounting evidence suggests that, in its current form, it is insufficient to predict clinical outcome. To assess biologic markers of primary CRC that may improve clinical staging and provide useful information for the application of novel therapeutic strategies, the authors investigated a panel of markers that included transforming growth factor alpha (TGF alpha), epithelial growth factor receptor (EGF-R; the protein product of the c-erb B2/HER-2 oncogene), matrix metalloproteinase 2 (MMP-2), insulin-like growth factor II (IGF-II), vascular endothelial growth factor (VEGF), and angiogenesis, as evaluated by microvessel density (MVD).. Two groups of CRC were studied: 1) surgical samples from patients who achieved a disease free survival of at least 6 years (CRC-M0) and 2) surgical specimens of both primary tumors and synchronous or metachronous liver metastases (CRC-M1).. Chi-square analysis revealed that expression levels of TGF-alpha, c-erb B2/HER-2, MMP-2, IGF-II, VEGF, and MVD (but not EGF-R) were significantly higher in CRC-M1 samples compared with CRC-M0 samples (P < 0.001, P < 0.05, P < 0.001, P < 0.001, P < 0.01, and P < 0.001, respectively). Logistic regression analysis showed that TGF-alpha, IGF-II, and MMP-2 had significantly greater expression in CRC-M1 samples independent of the other variables (including tumor classification, histologic grade, and patient age). If all three markers had > or = 25% expression, then the probability of developing liver metastasis was 99.5%.. Based on the evidence of this study, TGF-alpha MMP-2, and IGF-II seem suitable candidates for a selective panel of markers designed to provide significant additional information with respect to the current pathologic staging system for patients with colorectal carcinoma.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma; Colorectal Neoplasms; Disease-Free Survival; DNA, Complementary; Female; Humans; Immunohistochemistry; In Situ Hybridization; Insulin-Like Growth Factor II; Liver Neoplasms; Male; Matrix Metalloproteinase 2; Middle Aged; Neoplasm Staging; Predictive Value of Tests; Prognosis; Retrospective Studies; Transforming Growth Factor alpha

Expression of TGFalpha and the EGF receptor was studied in relation to apoptosis in human colorectal mucosa and premalignant and malignant tumors. In normal mucosa the proteins colocalized both in the proliferation compartment and at the luminal pole of the crypts in cells committed to undergo apoptosis. While staining for the EGF receptor was increased in premalignant and malignant lesions, TGFalpha was undetectable in aberrant crypt foci as well as large areas of adenomas. Incidence of apoptosis (AI) was high in these areas ranging from 8.83-24.59. Adenomas did, however, contain islands of high TGFalpha expression where AI was decreased to a range of 0.76-4.00 (decreased at P=0.0027). In carcinomas TGFalpha expression was increased above both normal and adenoma levels corresponding to the decrease in apoptosis in the malignant tumors. Tissue localization of TGFalpha and AI were still inversely related ( P=0.022), but interpatient variability was much larger than for adenomas. The data indicate that TGFalpha is the main survival factor in premalignant tumor cells of the colon, while additional factors moderate its effect in carcinomas. This suggests the possibility of targeting the EGF receptor pathway not only for treatment but also for the reversal of adenoma growth and the prevention of malignant colorectal tumors.

Topics: Apoptosis; Colorectal Neoplasms; Humans; Immunohistochemistry; Intestinal Mucosa; Precancerous Conditions; Transforming Growth Factor alpha

Our hypothesis is that rotation increases apoptosis in standard tissue culture medium at shear stresses of greater than approximately 0.3 dyn/cm2. Human MIP-101 poorly differentiated colorectal carcinoma cells were cultured for 6 d in complete medium in monolayers, on Teflon-coated nonadherent surfaces (static three-dimensional [3D]) or in rotating 3D cultures either in microgravity in low-earth orbit (3D microg) or in unit gravity on the ground (3D 1g). Apoptosis (determined morphologically), proliferation (by MIB1 staining), and the expression of epidermal growth-factor receptor (EGF-R), TGF-alpha, or TGF-beta were assessed by immunohistochemistry, while the expression of the differentiation marker carcinoembryonic antigen (CEA) was assessed on Western blots. Over the course of 6 d, static 3D cultures displayed the highest rates of proliferation and lowest apoptosis. This was associated with high EGF-R, TGF-alpha, and TGF-beta expression which was greater than that of a monolayer culture. Both rotated 3D lg and 3D microg cultures displayed lower expression of EGF-R, TGF-alpha, or TGF-beta and proliferation than that of monolayer or static 3D cultures. However, rotated 3D microg displayed significantly less apoptosis and greater CEA expression than rotated 3D 1g cultures. When rotated cultures of MIP-101 cells were grown uncler static conditions for another 3 d, proliferation increased and apoptosis decreased. Thus, rotation appears to increase apoptosis and decrease proliferation, whereas static 3D cultures in either unit or microgravity have less apoptosis, and reduced rotation in microgravity increases CEA expression.

Topics: Apoptosis; Bioreactors; Carcinoembryonic Antigen; Cell Adhesion; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Size; Colorectal Neoplasms; Culture Media; ErbB Receptors; Humans; Rotation; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Weightlessness

This study was conducted to investigate the serum levels of transforming growth factor-alpha in patients with colorectal cancer and to investigate the clinical significance of these levels in association with tumor stage and histologic differentiation. Also, serum levels of transforming growth factor-alpha were measured after curative surgical resection.. Serum levels of transforming growth factor-alpha were measured in 42 consecutive patients with colorectal cancer before surgery, in 21 patients after surgical resection (part of the 42 preoperative patients), and in 20 healthy volunteers. We used TGF-alpha Assay.. Serum levels of transforming growth factor-alpha in patients with colorectal cancer were significantly higher than in the healthy control group (P = 0.001). Significant elevations in serum levels of transforming growth factor-alpha were found in 50 percent (21/42) of patients with colorectal cancer when the mean + 2 standard deviations (80.4 pg/ml) of the control group were used as the upper limit of the normal range. Serum levels of transforming growth factor-alpha tended to decrease with increasing tumor size (n = 31; r = -0.52; P = 0.002). Serum levels of transforming growth factor-alpha before surgery (89.7 +/- 44.4 pg/ml; n = 21) significantly decreased to 60.3 +/- 19.8 pg/ml after surgical resections of tumors (P = 0.017). Serum levels of transforming growth factor-alpha completely decreased to the same serum levels of the control group after surgical resections in all patients who had serum levels of transforming growth factor-alpha greater than mean + 2 standard deviations (80.4 pg/ml) of the control group preoperatively (n = 11; P = 0.002).. Levels of preoperative transforming growth factor-alpha in patients with colorectal cancer appeared to be higher than levels measured in control subjects. Serum levels of transforming growth factor-alpha before surgery significantly decreased after surgical resections of tumors. Additional studies are warranted to determine if serum levels of transforming growth factor-alpha may be useful as a potential biomarker in the management of patients with colorectal cancer.

Topics: Adenocarcinoma; Adult; Biomarkers, Tumor; Colorectal Neoplasms; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Middle Aged; Transforming Growth Factor alpha

To evaluate the association among known angiogenic growth factors or factors related to the plasminogen activation system and clinicopathological factors in patients with colorectal cancer, we examined the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-alpha (TGF-alpha), urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PA-R) and plasminogen activator inhibitor-1 (PAI-1) in clinical specimens of colorectal cancers by Northern blot analysis. In comparison with the expression of these angiogenesis-related genes in 7 paired samples of colorectal cancers and the adjacent normal mucosa, VEGF mRNA level was significantly higher in the cancer tissues than in the adjacent normal mucosa (p < 0.05). We analyzed expression of these genes in 44 cases of primary colorectal cancers. Among the 3 angiogenic growth factors we examined, VEGF mRNA expression was significantly higher in the cancer tissues with blood vessel invasion or with lymphatic vessel invasion than in those without, respectively (p < 0.05). On the other hand, u-PA-R mRNA expression was significantly higher in the cancers with blood vessel invasion than in those without (p < 0.05). In addition, there was a correlation between the expression levels of VEGF and u-PA-R mRNA in the cancer tissues we have examined. Using immunohistochemistry, strong staining of VEGF or u-PA-R was observed in the cancer cells invading the microvessels. Our findings suggest that malignant transformation might accompany the upregulation of VEGF expression in colorectal cancers and that VEGF and u-PA-R might contribute cooperatively to increase angiogenesis around the tumor as well as the metastasis via microvessels.

Topics: Adult; Aged; Aged, 80 and over; Cell Line; Colon; Colonic Neoplasms; Colorectal Neoplasms; DNA Probes; Endothelial Growth Factors; Female; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Intestinal Mucosa; Lymphokines; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Urokinase-Type Plasminogen Activator; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The over-expression of epidermal growth factor receptor (EGF-R), EGF and transforming growth factor alpha (TGFalpha) could be a mechanism for colorectal tumor cells to escape normal growth controls. Our aims were: (i) to evaluate EGF, TGFalpha, and EGF-R concentrations in neoplastic tissue and surrounding mucosa from 40 patients with colorectal adenocarcinoma, and (ii) to assess the expression of these growth factors and their receptor in relation to the tumor site. EGF, TGFalpha, and EGF-R were detected in either colorectal neoplastic tissue or surrounding mucosa. Significantly increased levels of EGF and EGF-R were present in neoplastic samples compared to surrounding mucosa. Furthermore, a significant increase in TGFalpha and in EGF levels was observed in the left-sided surrounding mucosa and left-sided neoplastic tissue, respectively. EGF-R stimulation by its ligands may play an important role in colorectal neoplastic tissue. Moreover, the higher content of growth factors in the left-side than the right-side colon suggests different growth properties in the proximal and distal colon.

Topics: Adenocarcinoma; Aged; Colorectal Neoplasms; ErbB Receptors; Female; Functional Laterality; Humans; Intestinal Mucosa; Male; Middle Aged; Transforming Growth Factor alpha

To determine whether the expression of transforming growth factor alpha (TGF-alpha), its receptor (epidermal growth factor receptor [EGFr]), p53 nuclear protein, and proliferation influences prognosis of patients with liver metastases, a study was performed in 45 liver metastases and 33 corresponding primary colorectal carcinomas in patients referred for liver surgery. The expression of TGF-alpha, EGFr, p53 nuclear protein, and proliferation rate was correlated with clinicopathological characteristics and survival after partial liver resection. In liver metastases, TGF-alpha expression was low in 42%, intermediate in 35%, and high in 23%. TGF-alpha expression was higher in liver metastases derived from lymph node-positive primary carcinomas, in synchronous and in irresectable liver metastases compared with those derived from lymph node-negative primary carcinomas, metachronous, and resectable liver metastases. Nuclear p53 expression was found in 83% of primary tumors and 71% of liver metastases. p53 expression did not correlate with the various clinicopathological characteristics. Ki67 expression was not associated with clinicopathological characteristics in primary and metastatic tumors. In the 38 patients in whom a partial liver resection was performed, median survival was 25 months in patients with a higher TGF-alpha expression in the metastasis than in the primary tumor and 60 months in patients with comparable or lower TGF-alpha expression in the metastasis than in the primary tumor (P = .036). Median survival after liver resection was 21 months in patients with p53-negative liver metastases and 58 months in patients with p53-positive metastases (P = .043). By multivariate analysis, p53 and EGFr expression on liver metastases were the best predictors of disease-free survival after partial liver resection, with relative risks of 2.38 and 3.33, respectively. In patients with colorectal liver metastases, referred for liver surgery, a higher TGF-alpha expression is associated with unfavorable tumor characteristics, whereas p53 and absence of EGFr expression is associated with a better survival after partial liver resection.

Topics: Adenocarcinoma; Adult; Aged; Cell Division; Cell Nucleus; Colonic Neoplasms; Colorectal Neoplasms; Disease-Free Survival; ErbB Receptors; Female; Follow-Up Studies; Humans; Ki-67 Antigen; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prognosis; Survival Analysis; Time Factors; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

8-Chloro-cyclic AMP (8-Cl-cAMP) is a cAMP analogue that specifically down-regulates type I protein kinase A, a signaling protein directly involved in cell proliferation and neoplastic transformation, and that causes growth inhibition in a variety of human cancer cell types. In this report, we have investigated the effects of 8-Cl-cAMP on the expression of several growth factors in human colon (GEO and LS174T) and breast (MDA-MB468) cancer cell lines. 8-Cl-cAMP treatment caused in the three cancer cell lines a significant dose- and time-dependent inhibition in the expression of various endogenous autocrine growth factors, such as transforming growth factor alpha, amphiregulin, and CRIPTO, and of two angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor, at both the mRNA and protein levels. Furthermore, 8-Cl-cAMP treatment markedly inhibited the ability of all three cell lines to invade a basement membrane matrix in a chemoinvasion assay. Finally, 8-Cl-cAMP-induced inhibition of GEO tumor growth in nude mice was accompanied by a significant suppression of transforming growth factor alpha, amphiregulin, CRIPTO, basic fibroblast growth factor, and vascular endothelial growth factor production by the tumor cells, and of neoangiogenesis, as detected by factor VIII staining of host blood cells. These results demonstrate that 8-Cl-cAMP is a novel anticancer drug that inhibits the production of various autocrine and paracrine tumor growth factors that are important in sustaining autonomous local growth and facilitate invasion and metastasis.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Amphiregulin; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Division; Colonic Neoplasms; Colorectal Neoplasms; EGF Family of Proteins; Endothelial Growth Factors; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; Membrane Glycoproteins; Mice; Mice, Nude; Neoplasm Proteins; Neovascularization, Pathologic; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Results of recent studies indicate that some cultured human carcinoma cell lines are capable of proliferating autonomously in serum-free medium as a result of the synthesis and secretion of transforming growth factor alpha (TGF alpha). TGF alpha interacts with epidermal growth factor receptor (EGFR) and induces its activation. In an attempt to extend these observations, we evaluated TGF alpha-mediated autonomous growth and constitutive EGFR activation in the human adenocarcinoma cell line SW403. The cell line shows synthesis of EGF receptors and TGF alpha but not EGF, and exhibits constitutive phosphorylation of the 170-kDa EGFR. Use of blocking anti-EGFR monoclonal antibodies (mAb) inhibits autonomous growth of SW403 cells and leads to a significant reduction of receptor phosphorylation. The inhibitory effect of the blocking anti-EGFR mAb is reversible upon addition of TGF alpha. In contrast, autonomous proliferation of SW403 cells is not inhibited by addition of neutralizing anti-EGF mAb. Our findings suggest that the proliferation of cells of the human SW403 adenocarcinoma cell line is regulated by an autocrine TGF alpha loop and that this regulatory pathway can be interrupted by using anti-EGFR mAb.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Antibodies, Monoclonal; Cell Division; Colorectal Neoplasms; ErbB Receptors; Female; Humans; Male; Middle Aged; Phosphorylation; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

The frequency of expression and localization of cripto-1 (CR-1), amphiregulin (AR), transforming growth factor alpha (TGF alpha), epidermal growth factor receptor (EGFR) and erbB-2 were examined by immunohistochemistry in 45 carcinomas and adjacent non-involved normal colon mucosa. Thirty (66.7%), 24 (53.3%), 23 (51.1%), 23 (51.1%) and 13 (28.9%) of the 45 carcinomas showed positive staining for CR-1, AR, TGF alpha, EGFR and erbB-2, respectively, whereas 7 (15.5%), 17 (37.7%), 15 (33.3%), 20 (44.4%) and 0 (0%) of the corresponding non-involved normal mucosa specimens were reactive. Among 13 carcinomas with lymph node involvement, 10 (76.9%), 8 (61.5%), 10 (76.9%), 8 (61.5%) and 7 (53.8%) exhibited positive staining for CR-1, AR, TGF-alpha, EGFR and erbB-2, respectively. There was a statistically significant association between the frequency of either TGF alpha (P < 0.05) or erbB-2 (P < 0.05) expression and lymph node metastasis. In addition, a significantly higher frequency of positive staining for TGF alpha was observed in Dukes' grade C carcinomas (P < 0.05). Finally, significant trends for coexpression of EGFR and either TGF alpha (P < 0.01) or AR (P < 0.05) were detected in carcinomas. These data suggest that AR and TGF alpha may play an important role in the development of colorectal carcinomas through an autocrine mechanism involving EGFR, and demonstrate that TGF alpha and erbB-2 may be more reliable indicators of metastasis or prognosis than CR-1, AR or EGFR in human colon cancers.

Topics: Adenocarcinoma; Amphiregulin; Colorectal Neoplasms; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Lymphatic Metastasis; Membrane Glycoproteins; Neoplasm Proteins; Peptides; Receptor, ErbB-2; Transforming Growth Factor alpha

The DiFi colorectal carcinoma cell line, derived from a patient with familial adenomatous polyposis, was examined for gene expression and production of the autocrine growth factor transforming growth factor alpha (TGF-alpha) and for epidermal growth factor receptor (EGFR) gene expression and gene copy number. DiFi cells expressed TGF-alpha transcripts as identified on Northern (RNA) blots. Addition of TGF-alpha (10 ng/ml) or EGF (10 ng/ml) to DiFi cell cultures (lacking EGF or serum) up-regulated DiFi cell basal TGF-alpha mRNA levels, suggesting that autoinduction of TGF-alpha occurs in these cells. DiFi cell cultures in log phase growth secreted measurable amounts of TGF-alpha (347 pg/10(6) cells/24 h) into their culture medium, as determined by radioimmunoassay. DiFi cells showed strong overexpression of the EGFR gene on Northern blots relative to three other colon cancer cell lines examined. Immunoperoxidase staining showed enhanced EGFR expression in a cell subpopulation among the original (uncultured) ascitic fluid cells from which the DiFi cell line was established. This cell subpopulation was observed to expand dramatically between passages 1 and 25. Immune complex kinase assay of DiFi cells showed that EGFR were functional as determined by their ability to autophosphorylate. The EGFR gene in these cells was not found to be rearranged or genetically altered using Southern blot analysis. Dot blot analysis of DiFi cell DNA revealed EGFR gene amplification in the range of 60-80 copies/cell, which is approximately twice the copy number seen in A-431 epidermoid carcinoma cells. To our knowledge DiFi cells represent the first example of EGFR gene amplification in a colorectal adenocarcinoma. Because DiFi colorectal cancer cells uniquely show production and auto-induction of TGF-alpha in addition to amplification and overexpression of the EGFR gene, these cells represent a valuable tool for studying the role(s) of the EGFR in the regulation of tumor cell growth.

Topics: Blotting, Northern; Blotting, Southern; Colorectal Neoplasms; DNA, Neoplasm; ErbB Receptors; Gene Amplification; Humans; Phosphorylation; Transforming Growth Factor alpha; Tumor Cells, Cultured; Up-Regulation

A total of 117 colorectal tissue specimens were examined immunohistochemically for the production of immunoreactive (IR-) transforming growth factor (TGF)-alpha and IR-epidermal growth factor (EGF). IR-TGF-alpha was detected in 26/32 (81.3%) invasive cancers, 14/27 (51.9%) carcinomas in situ, and 14/58 (24.1%) adenomas. IR-EGF was detected in 14/32 (43.8%) invasive cancers, 12/27 (44.4%) carcinomas in situ, and 12/58 (20.7%) adenomas. The staining intensity of IR-TGF-alpha was related to the histologic grade of malignancy, but that of IR-EGF was not. These suggest that IR-TGF-alpha plays a more important role than IR-EGF in the growth of colorectal neoplasms, and that further study of these growth factors would be helpful in understanding the biology of colorectal carcinoma.

Topics: Adenoma; Carcinoma; Carcinoma in Situ; Colorectal Neoplasms; Epidermal Growth Factor; Humans; Immunohistochemistry; Transforming Growth Factor alpha

We have tested growth factor responsiveness of a panel of eight human colorectal carcinoma cell lines. Insulin-like growth factors I and II (IGF-I and IGF-II) stimulated growth of five lines (HT-29, LS411N, LS513, SW480, WiDr). At 30 ng ml-1 both factors enhanced growth up to 3-fold. They induced half-maximal stimulation at 1.9-6.51 ng ml-1. Even after delayed addition IGF-I and II significantly enhanced growth in a short-term proliferation assay. They exerted maximal effects under limiting serum conditions (0.5% FCS) and at low cell density (1.25-5 x 10(4) ml-1). Using these conditions transforming growth factor alpha (TGF alpha) enhanced proliferation of all IGF-responsive cell lines, except SW480. 1.11-3.31 ng ml-1 were required to obtain a half-maximal response. With 10-20 ng ml-1 maximal stimulation occurred at plateau values different from those for IGF-I/II. Proliferation of all cell lines responsive to both IGF-I and TGF alpha was further enhanced by combining both factors, resulting a synergistic response of LS513, while the effects on HT-29, LS411N and WiDr were additive. With HT-29 and LS411N a 24 h exposure to TGF alpha was sufficient to obtain a full response in the co-stimulatory assay. Our results illustrate the importance of IGF-I/II and TGF alpha as stimulators of growth of colorectal carcinomas.

Topics: Cell Division; Cell Line; Colorectal Neoplasms; Dose-Response Relationship, Drug; Drug Synergism; Humans; In Vitro Techniques; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Serum Albumin; Transforming Growth Factor alpha

Surgical specimens from 19 patients with invasive colorectal cancers and 12 specimens of normal mucosa from the same patients were examined immunohistochemically for the production of the immunoreactive (IR-) transforming growth factor (TGF)-alpha and IR-epidermal growth factor (EGF) with an anti-TGF-alpha monoclonal antibody (MAb) OAL-MTG01 and anti-EGF MAb KEM-10. Immunoreactive TGF-alpha was detected in 16 (84.2%) of 19 colorectal cancers. In contrast, there was no IR-TGF-alpha in the gland cells of normal mucosa. Immunoreactive EGF was detected in 7 (36.8%) of 19 colorectal cancers and 1 (8.3%) of 12 cases of normal mucosa. The production of both IR-TGF-alpha and IR-EGF in colorectal cancer did not differ by histologic type and Dukes' stage. Immunoreactive TGF-alpha was detected at significantly higher incidence than IR-EGF in colorectal cancer. These results indicate that IR-TGF-alpha should prove valuable as a possible tumor marker in colorectal cancers, and it may be very useful in understanding the biology of colorectal cancer.

Topics: Antibodies; Antibody Specificity; Colorectal Neoplasms; Cross Reactions; Humans; Immunohistochemistry; Intestinal Mucosa; Reference Values; Transforming Growth Factor alpha

We have examined the expression of mRNAs for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), EGF receptor (EGFR), PDGF-A chain (PDGFA), PDGF-B chain (PDGFB) and PDGF receptor (PDGFR) genes in seven human colorectal carcinoma cell lines and 18 human colorectal carcinomas. In surgically resected specimens of the 18 colorectal tumors, TGF-alpha, EGFR, PDGFA, PDGFB and PDGFR mRNAs were detected at various levels in 15 (83%), 9 (50%), 18 (100%), 8 (44%) and 12 (67%), respectively. They were also detected in normal tissues. Interestingly, EGF mRNA was detected in only five (28%) of the tumors, but not in normal mucosa. Expression of EGF was also confirmed immunohistochemically in tumor cells. Of the five tumors expressing EGF, four expressed EGFR mRNA and showed a tendency to invade veins and lymphatics. All the colorectal carcinoma cell lines expressed TGF-alpha mRNA, and five cell lines expressed EGFR mRNA simultaneously. Production of TGF-alpha protein by DLD-1 and CoLo320DM cells was confirmed by TGF-alpha specific monoclonal antibody binding assay. The spontaneous 3H-thymidine uptake by DLD-1 was suppressed by an anti-TGF-alpha monoclonal antibody. PDGFA and PDGFB mRNA were also expressed in four cell lines, but PDGFR and EGF mRNA was not detected. These results suggest that human colorectal carcinomas express multi-loops of growth factors and that TGF-alpha produced by tumor cells functions as an autocrine growth factor in human colonic carcinoma.

Topics: Antibodies, Monoclonal; Blotting, Northern; Colorectal Neoplasms; DNA Probes; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Immunohistochemistry; Iodine Radioisotopes; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; RNA, Neoplasm; Thymidine; Transforming Growth Factor alpha; Tritium; Tumor Cells, Cultured