Loss of Smad4 expression predicts liver metastasis in human colorectal cancer

Distant metastases represent the major cause of death after curative surgery of colorectal cancer. The aim of this study was to evaluate the role of Smad4 and KRAS genetic alterations in colorectal metastases taking into account both the site (hepatic versus extrahepatic) and the time (synchronous versus metachronous) of recurrence. We examined the immunohistochemical expression of Smad4 and frequency of KRAS mutation in primary colorectal tumors and in their corresponding metastatic tissues. Loss of Smad4 expression was noted in 37% (26/71) of the primary tumors and the corresponding metastases. Absence of Smad4 protein was more frequently observed in hepatic metastases, whether they were metachronous or synchronous, than in extrahepatic metastases (p<0.005). The frequency of KRAS mutations was high in the synchronous and extrahepatic metachronous metastases (68-80%), but was significantly lower in the hepatic metachronous metastases (11%). Our results indicate that absence of Smad4 expression correlated significantly with liver metastases regardless of the time of their occurrence and represents a promising new biomarker to predict liver metastasis in colorectal cancer patients. Therefore, this group of patients could benefit from a specific and appropriate pre- and/or post-operative therapy.     

Infrequent mutation of the tumour-suppressor gene Smad4 in early-stage colorectal cancer

Smad4 is a candidate tumour-suppressor gene identified recently on chromosome 18q21.1. Both alleles are inactivated in nearly one-half of pancreatic carcinomas, but its role in the tumorigenesis of other tumours is still unknown. The aim of this study was to investigate the potential involvement of the Smad4 locus in early-stage colorectal cancers (stages I-III) in tumour samples from a randomised multicentre trial. Of a large collection of DNA samples, 73 with a loss of one allele of the Smad4 gene were analysed for the presence of point mutations in the remaining gene. Patients, from whom biopsies were isolated, were part of a previous randomised multicentre study of the Swiss Group for Clinical Cancer Research on the benefit of adjuvant chemotherapy (SAKK study 40/81). Mutation analysis was restricted to the highly conserved C-terminal domain (exons 8, 9, 10 and 11) of Smad4, using PCR and single-strand conformational variant analysis. Two of the 73 patients (3%) with loss of one allele of Smad4 had a point mutation in the remaining allele. These results indicate that whereas Smad4 point mutations are prevalent in pancreatic carcinoma, they are infrequent in early stages (I-III) of colorectal cancer.     

Smad7 induces hepatic metastasis in colorectal cancer

Although Smad signalling is known to play a tumour suppressor role, it has been shown to play a prometastatic function also in breast cancer and melanoma metastasis to bone. In contrast, mutation or reduced level of Smad4 in colorectal cancer is directly correlated to poor survival and increased metastasis. However, the functional role of Smad signalling in metastasis of colorectal cancer has not been elucidated. We previously reported that overexpression of Smad7 in colon adenocarcinoma (FET) cells induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis. Here, we have observed that abrogation of Smad signalling by Smad7 induces liver metastasis in a splenic injection model. Polymerase chain reaction with genomic DNA from liver metastases indicates that cells expressing Smad7 migrated to the liver. Increased expression of TGF-beta type II receptor in liver metastases is associated with phosphorylation and nuclear accumulation of Smad2. Immunohistochemical analyses have suggested poorly differentiated spindle cell morphology and higher cell proliferation in Smad7-induced liver metastases. Interestingly, we have observed increased expression and junctional staining of Claudin-1, Claudin-4 and E-cadherin in liver metastases. Therefore, this report demonstrates, for the first time, that blockade of TGF-beta/Smad pathway in colon cancer cells induces metastasis, thus supporting an important role of Smad signalling in inhibiting colon cancer metastasis.     

Expression of Smad proteins in human colorectal cancer.

Escape from transforming growth factor-beta (TGF-beta)-induced inhibition of proliferation has been observed in many tumor cells and may contribute to loss of growth control. Smad proteins have been identified as major components in the intracellular signaling of TGF-beta family members. In this study, we examined the expression of receptor-activated, common-mediator and inhibitory Smads by immunohistochemistry in human colorectal cancers. We found increased expression of receptor-activated Smads in a fraction of the tumor cells, while no immunostaining for Smad2, Smad3 or Smad5 and only occasional staining for Smad1/8 was found in epithelial mucosa of normal colon. No or only weak staining for receptor-activated Smads, common-mediator Smad4 and inhibitory Smads was observed in the tumor stroma. Common-mediator Smad4 and inhibitory Smads were detected in cells of both tumor and normal tissues. We observed a distinct pattern of Smad4 immunostaining of epithelial cells along colon crypts, with high expression in zones of terminal differentiation. Our data show selective up-regulation of receptor-activated Smad proteins in human colorectal cancers and suggest involvement of Smad4 in differentiation and apoptosis of surface epithelial cells of normal crypts.     

Expression of angiopoietin-like 4 (ANGPTL4) in human colorectal cancer: ANGPTL4 promotes venous invasion and distant metastasis

There is strong evidence that the angiopoietin family is involved in the regulation of tumour progression. Recently, it has been reported that angiopoietin-like 4 (ANGPTL4) expression in cancer cells promotes the metastatic process by increasing vascular permeability. The present study was conducted to examine ANGPTL4 expression and its association with clinicopathological factors and prognosis in human colorectal cancers. We examined 144 cases of surgically-resected human colorectal adenocarcinomas by immunohistochemistry, RT-PCR and Western blot analysis. Also, overall survival was investigated. Among 144 cases of adenocarcinoma, 95 cases (66.0%) showed positive staining in the cytoplasm of the carcinoma cells for ANGPTL4. Histologically, well, moderately, poorly differentiated adenocarcinoma or mucinous carcinoma showed 55.2, 79.3, 61.5 or 44.4% expression of ANGPTL4, respectively. The expression of ANGPTL4 was correlated with the depth of tumour invasion (p<0.0005), Vienna classification (category 3-5)(p<0.00005), venous invasion (p<0.0005) and Duke's classification (p<0.005). However, ANGPTL4 expression was not correlated with overall survival. However, all patients (100%) with distant metastasis showed immunopositivity for ANGPTL4. The mRNA and the protein expression of ANGPTL4 were shown in four resected samples and cultured cell lines by RT-PCR or western blot analysis. These findings suggest that ANGPTL4 is one of the factors involved in the progression of human colorectal cancer, especially venous invasion and distant metastasis.     

KRAS 基因突变与结直肠癌转移模式相关性的研究简

目的：研究结直肠癌患者KRAS基因突变与根治后转移的相关性。方法：检测132例结直肠癌根治术患者石蜡组织中的KRAS基因,回顾性分析患者根治术后3年内转移、复发情况,采用单因素及多因素分析KRAS突变与转移的相关性。结果：各转移组KRAS基因突变率分别为：肺（63．2％）、脑（66．7％）、肝（30．4％）及无转移组（34．7％）。单因素分析提示肺、脑转移组KRAS基因突变率显著高于肝转移组及无转移组（P〈0．05）。纳入年龄和性别校正后采用Logistic回归分析提示KRAS突变显著增加肺转移及脑转移风险（P〈0．05）,而肝转移风险无显著升高（P〉0．05）。结论：结直肠癌转移模式与KRAS突变有关,检测KRAS突变状态有助于更加有效地制定复发监视策略。     

Targeting the ERK pathway reduces liver metastasis of Smad4-inactivated colorectal cancer

Transforming growth factor β (TGF-β)/Smad signaling is involved in colorectal carcinoma (CRC) development and progression. The frequent loss of SMAD4 is associated with liver metastasis and poor prognosis of CRC, but the underlying mechanism remains elusive. This study aimed to elucidate the role of Smad-independent TGF-β signaling in CRC metastasis. Immunohistochemistry showed that Smad4 level was negatively correlated with TNM stage and phospho-ERK level in human CRCs and liver metastasis samples. Knockdown of Smad4 in CT26 and HCT116 cells activated ERK pathway, altered the expression of MMP2 and COX-2, promoted cell motility, migration, and invasion in vitro, enhanced metastasis, and shortened the survival of metastatic tumor-bearing mice. MEK inhibitor U0126 and GSK1120212 inhibited the motility, migration, and invasion of Smad4 knockdown cells, inhibited metastasis, and prolonged the survival of metastatic tumor-bearing mice. Furthermore, MEK inhibitor could reverse the changes of phospho-ERK, MMP2, and COX-2 levels. In conclusion, our results indicate that ERK pathway plays a key oncogenic role in CRC with SMAD4 inactivation mutations, and implicate ERK as a potential therapeutic target for CRC liver metastasis.     

Resection of isolated distant nodal metastasis in metastatic colorectal cancer

BackgroundLittle is known regarding the role of resection in patients with colorectal cancer (CRC) who present with isolated non-regional lymph node metastasis (NRLNM).MethodsUsing the Surveillance, Epidemiology and End Results database, we identified patients diagnosed with CRC and NRLNM from 2004 to 2013.ResultsA total of 849 patients presented with CRC and isolated NRLNM. Of these, 90 (10.6%) underwent resection of NRLNM. Median overall survival (OS) did not differ for patients who underwent resection of NRLNM compared to those who did not (33 versus 29 months, p = 0.68). Subgroup analysis by primary tumor site, also did not demonstrate a difference in median OS. Cox proportional hazard model revealed older age (Hazard ratio [HR] 1.34, 95% Confidence Interval [CI] 1.17–1.53, p < 0.0001), higher tumor grade (HR 1.81, 95% CI 1.52–2.16, p < 0.0001), and earlier year of diagnosis (HR 1.34, 95% CI 1.17–1.53, p < 0.0001) were associated with decreased OS. There was no survival difference between those who underwent resection of NRLNM compared to those who had not (HR 0.997, p = 0.28).ConclusionResection of NRLNM in patients with CRC is not associated with an OS benefit. Further studies are needed to determine if there is a subset of patients who could potentially benefit from this resection strategy.     

C-reactive protein is associated with distant metastasis of T3 colorectal cancer

Few studies have examined the relationship between systemic inflammatory response (SIR) and distant metastasis in patients with T3 colorectal cancer (T3 CRC). Uni- and multivariate analyses were performed in order to evaluate the influence of SIR on distant metastasis in patients with T3 CRC using collected clinical data. Between January 2000 and August 2009, 335 patients with pathologically diagnosed T3CRC were enrolled. Univariate analysis revealed that tumor differentiation, lymphatic invasion, venous invasion, lymph node metastasis, serum carcinoembryonic antigen (CEA) level, carbohydrate antigen 19-9 (CA 19-9) level, C-reactive protein (CRP) level and the Glasgow Prognostic Score (GPS) were associated with distant metastasis. Multivariate analysis using these selected characteristics disclosed that the CRP level was associated with distant metastasis of T3 CRC, as well as with lymph node metastasis, and CEA and CA19-9 levels. The level of CRP is one of the important clinical characteristics associated with distant metastasis of T3 CRC.     

Background mutation frequency in microsatellite-unstable colorectal cancer

Microsatellite instability (MSI) is observed in approximately 12% of colorectal cancers. Genes containing a mononucleotide microsatellite in the coding sequence are particularly prone to inactivation in MSI tumorigenesis, and much work has been conducted to identify genes with high repetitive tract mutation rates in these tumors. Much less attention has been paid to background mutation frequencies, and no work has focused on nontranscribed regions. Here, we studied 114 nontranscribed intergenic A/T and C/G repeats 6 to 10 bp in length, located distant from known genes, to examine background mutation frequencies in MSI colorectal cancers. A strong correlation with tract length was observed, and mutation frequencies of up to 87% were observed in 8 to 10 bp tracts. Subsequently, to compare the background mutation rate in transcribed and nontranscribed noncoding repeats, we screened nine randomly selected intronic C/G8 repeats. In addition, the coding repeats of seven suggested MSI target genes, and nine previously published intronic A8 and G8 repeats were analyzed. Intronic repeats seemed to mutate less frequently than nontranscribed intergenic repeats. Our results show that strand slippage mutations in mismatch repair-deficient cells are as abundant in short intergenic repeats as in many proposed MSI target genes. However, under mismatch repair deficiency, strand slippage mutations in transcribed sequences seem to be repaired more efficiently than in intergenic nontranscribed sequences. The mechanisms causing these differences are not yet understood and should be a subject for further studies. For MSI target gene identification, repeats in transcribed sequences seem to be the most appropriate reference group for coding region repeat mutations.     

Artificial neural network for prediction of distant metastasis in colorectal cancer

Background and Objectives: Artificial neural networks (ANNs) are flexible and nonlinear models which can be used by clinical oncologists in medical research as decision making tools. This study aimed to predict distant metastasis (DM) of colorectal cancer (CRC) patients using an ANN model. Methods: The data of this study were gathered from 1219 registered CRC patients at the Research Center for Gastroenterology and Liver Disease of Shahid Beheshti University of Medical Sciences, Tehran, Iran (January 2002 and October 2007). For prediction of DM in CRC patients, neural network (NN) and logistic regression (LR) models were used. Then, the concordance index (C index) and the area under receiver operating characteristic curve (AUROC) were used for comparison of neural network and logistic regression models. Data analysis was performed with R 2.14.1 software. Results: The C indices of ANN and LR models for colon cancer data were calculated to be 0.812 and 0.779, respectively. Based on testing dataset, the AUROC for ANN and LR models were 0.82 and 0.77, respectively. This means that the accuracy of ANN prediction was better than for LR prediction. Conclusion: The ANN model is a suitable method for predicting DM and in that case is suggested as a good classifier that usefulness to treatment goals.     

Higher frequency of diploidy in young-onset microsatellite-stable colorectal cancer.

PURPOSE: Colorectal carcinoma (CRC) can be divided into two nonoverlapping groups: those that are chromosomally unstable but microsatellite stable (MSS CIN+) and those that are chromosomally stable but microsatellite unstable (MSI CIN-). However, a third group with neither chromosome nor microsatellite instability (MSS CIN-) makes a substantial contribution to the total CRC burden. The clinicopathologic features of MSS CIN- CRC are not well delineated. We assessed the relationship between age and chromosomal instability (CIN) status as measured by ploidy and allelic imbalance in a series of MSS tumors. EXPERIMENTAL DESIGN: We studied a prospectively collected series of CRC patients at Mayo Clinic Rochester. A total of 84 samples of MSS CRC in patients or=65 years old served as a comparison group. CIN status was assessed using two techniques: ploidy analysis by flow cytometry and small chromosome changes as measured by genomewide fractional allelic imbalance. RESULTS: CRC in the young-onset group was more likely to involve the rectum and to be high stage. MSS tumors in the young-onset group were more often diploid (46%) than those in older patients (26%; P = 0.006). This difference was maintained in the subset of MSS CRC that were high stage (42% versus 18%; P = 0.02) and in rectal cancers (50% versus 23%; P = 0.04). CONCLUSION: A greater proportion of young patients with MSS CRC has diploid tumors than patients who develop MSS CRC over age 65.     

结直肠癌中K-ras基因突变研究

目的研究K-ras基因突变表达与结直肠癌的相关性。方法采用实时荧光定量PCR方法检测89例患者结直肠癌组织中K-ras基因2号外显子12与13编码子突变情况,并结合病理资料进行分析。结果89例患者结直肠癌组织中K-ras基因突变者为37例,突变率为41．6％。其中12编码子突变为29例,突变率为32．6％。13编码子突变为8例,突变率为9．O％。K-ras基因突变与肿瘤位置、分化程度无明显相关性（P〉0．05）,与浸润深度、淋巴结转移、肝转移有相关性（P〈0．05）。淋巴结转移越多K-ras基因突变率越高,有肝转移者K-ras基因突变率高。结论K-ras基因突变在结直肠癌的发生、发展中起重要作用,与浸润深度、淋巴结转移和肝转移密切相关。     

Higher frequency of DPC4/Smad4 alterations in pancreatic cancer cell lines than in primary pancreatic adenocarcinomas.

The tumor suppressor gene DPC4/Smad4 at 18q21.1 is inactive in about 50% of pancreatic carcinoma xenografts and cell lines. However, the role of DPC4 in the multistep carcinogenesis of primary pancreatic adenocarcinomas remains uncertain. Therefore, we examined 45 primary human pancreatic adenocarcinomas and 12 pancreatic cancer cell lines for DPC4 alterations by single-strand conformational variant (SSCV) analysis and a PCR-based deletion assay. DPC4 was inactivated by either homozygous deletion or point mutation in 6 of 12 cell lines (50%). None of the primary pancreatic carcinomas carried a DPC4 mutation, although 66% revealed LOH of 18q21 sequences. These findings suggest that inactivation of DPC4 occurs more frequently in tumor-derived cell lines than in primary pancreatic adenocarcinomas. In addition, another, yet unidentified, tumor suppressor gene(s) may be linked with the frequent LOH of 18q21 in primary pancreatic adenocarcinomas.     

Genistein suppresses FLT4 and inhibits human colorectal cancer metastasis

Dietary consumption of genistein, found in soy, has been associated with a potentially protective role in colorectal cancer (CRC) development and progression. Herein we demonstrate that genistein will inhibit human CRC cell invasion and migration, that it does so at non-cytotoxic concentrations and we demonstrate this in multiple human CRC cell lines. After orthotopic implantation of human CRC tumors into mice, oral genistein did not inhibit tumor growth, but did inhibit distant metastasis formation, and was non-toxic to mice. Using a qPCR array, we screened for genistein-induced changes in gene expression, followed by Western blot confirmation, demonstrating that genistein downregulated matrix metalloproteinase 2 and Fms-Related Tyrosine Kinase 4 (FLT4; vascular endothelial growth factor receptor 3). After demonstrating that genistein suppressed neo-angiogenesis in mouse tumors, we examined FLT4 expression in primary CRC and adjacent normal colonic tissue from 60 human subjects, demonstrating that increased FLT4 significantly correlates with increased stage and decreased survival. In summary, we demonstrate for the first time that genistein inhibits human CRC metastasis at dietary, non-toxic, doses. FLT4 is identified as a marker of metastatic disease, and as a response marker for small molecule therapeutics that inhibit CRC metastasis.     

Alteration of the PTEN/MMAC1 gene locus in primary lung cancer with distant metastasis.

The PTEN/MMAC1 gene located at 10q23, has been proposed to be a tumor suppressor gene. To determine the involvement of alteration of the PTEN/MMAC1 gene in carcinogenesis and the progression of primary lung cancers, we analyzed tumor samples of primary and distant metastatic sites and normal lung tissue samples of 30 patients with advanced lung cancer with distant metastasis. The tissues were analyzed for allelic deletion and mutational inactivation of PTEN/MMAC1 by loss of heterozygosity (LOH) analysis, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), and direct sequence analysis. LOH of the PTEN/MMAC1 locus was common in each histologic type of primary lung cancer. In this study, the overall allelic deletion rate was 33.3% (7/21). Allelic loss at the primary site and that at the metastatic site of each patient, were identical; in most cases, it seemed that the allelic loss had occurred before metastasis. Sequence analysis of the PTEN/MMAC1 gene revealed a G to C substitution located 8 bp upstream of the coding region of exon 1 and which seems to be a polymorphism, in 4 of the 30 cases. Somatic mutations of the PTEN/MMAC1 gene were not identified in any of the tumors at the primary and metastatic sites. These data indicate that point mutations in the PTEN/MMAC1 gene are probably not an important factor in tumorigenesis and the progression of a major subset of lung cancers. Due to frequent allelic loss at the PTEN/MMAC1 locus occurring at a stage earlier than the metastatic process, alternative mechanisms in which the remaining allele is inactivated such as methylation or homozygous deletion of a small region of the gene that can not be detected by the usual analysis, or alteration of other important tumor suppressor genes lying close to the PTEN/MMAC1 gene on 10q23, may be involved in the tumorigenesis of lung cancers of all histologic subtypes.     

Clinical perspective of human colorectal cancer metastasis

Summary Death from colorectal cancer frequently results from manifestations of recurrent local or metastatic disease following initial curative therapy. Presently, the attempted curative treatment of recurrent colorectal cancer lays in the hands of the surgeon. This paper reviews the natural history of surgically treated large bowel cancer and summarizes published and National Cancer Institute experience with the surgical therapy of recurrent disease. A schema with rationale is offerred for follow-up of the patient following curative bowel resection. The treatment of recurrent disease incorporating the use of CEA initiated second-look laparotomy is advocated. Hepatic and pulmonary resections for metastatic disease are accepted as important therapeutic endeavours, and are discussed in some detail.It is concluded that of all patients diagnosed as having large bowel cancer, roughly 70% are resectable for cure at time of presentation, and 45% will indeed be cured by primary resection. Of the 25% who fail primary therapy, approximately 20% (5% of all colorectal cancer patients) can be cured by local re-resection or hepatic or pulmonary resection.     

Increased MUTYH mutation frequency among Dutch families with breast cancer and colorectal cancer

Homozygous and compound heterozygous MUTYH mutations predispose for MUTYH-associated polyposis (MAP). The clinical phenotype of MAP is characterised by the multiple colorectal adenomas and colorectal carcinoma. We previously found that female MAP patients may also have an increased risk for breast cancer. Yet, the involvement of MUTYH mutations in families with both breast cancer and colorectal cancer is unclear. Here, we have genotyped the MUTYH p.Tyr179Cys, p.Gly396Asp and p.Pro405Leu founder mutations in 153 Dutch families with breast cancer patients and colorectal cancer patients. Families were classified as polyposis, revised Amsterdam criteria positive (FCRC-AMS positive), revised Amsterdam criteria negative (FCRC-AMS negative), hereditary breast and colorectal cancer (HBCC) and non-HBCC breast cancer families. As anticipated, biallelic MUTYH mutations were identified among 13% of 15 polyposis families, which was significantly increased compared to the absence of biallelic MUTYH mutations in the population (P = 0.0001). Importantly, six heterozygous MUTYH mutations were identified among non-polyposis families with breast and colorectal cancer. These mutations were identified specifically in FCRC-AMS negative and in HBCC breast cancer families (11% of 28 families and 4% of 74 families, respectively; P = 0.02 for both groups combined vs. controls). Importantly, the 11% MUTYH frequency among FCRC-AMS negative families was almost fivefold higher than the reported frequencies for FCRC-AMS negative families unselected for the presence of breast cancer patients (P = 0.03). Together, our results indicate that heterozygous MUTYH mutations are associated with families that include both breast cancer patients and colorectal cancer patients, independent of which tumour type is more prevalent in the family.     

Higher frequency of point mutations in the c-K-ras 2 gene in human colorectal adenomas with severe atypia than in carcinomas.

Human colorectal carcinomas may be induced from adenomas or they may occur de novo. To examine which is the main pathway, we analyzed point mutations at codon 12 in the c-K-ras 2 gene in 73 colorectal carcinomas, 13 metastatic tumors, 72 adenomas and 30 normal tissues. The c-K-ras 2 codon 12 mutation frequency was 0/30 in normal tissues, 0/17 in adenomas with mild atypia, 3/37 (8.1%) in adenomas with moderate atypia, 15/18 (83.3%) in adenomas with severe atypia, 19/73 (26.0%) in primary carcinomas and 3/13 (23.1%) in metastatic tumors. The mutation frequency in adenomas with severe atypia was much higher than that in carcinomas. These results indicate that many colorectal carcinomas may not be induced through adenomas with severe atypia.