Rania Georges

The insulin-like growth factor binding proteins 3 and 7 are associated with colorectal cancer and liver metastasis.

Over the last few decades, a great deal of attention has been directed to the IGF system for its vital role in regulating cell and tissue survival, growth and differentiation. The insulin-like growth factor binding proteins (IGFBPs), a main constituent of this system, have been implicated in the tumorigenesis of colorectal cancer (CRC). In this study, we intended to shed more light on two essential members; IGFBP3 as representative for the six main IGFBPs and IGFBP7 to represent their related proteins (IGFBP-rps). Our experiments on silencing IGFBP3 or IGFBP7 in the two human CRC cell lines SW480, Caco2, and in the rat CRC cell line CC531 show reduced proliferation, colony formation, and for IGFBP3, also reduced migration. The expression of both genes in 68 human CRC samples was higher in UICC stages II and III than in stages I and IV. Additionally, IGFBP3 was negatively correlated with age (p = 0.05) and positively related to IGFBP7 expression (p = 0.0001). Further, in a liver metastasis experiment, the expression of both genes was drastically increased in response to early metastatic growth in vivo. Since these high levels returned gradually to normal thereafter, it could be assumed that the up-regulation of IGFBPs is vital during the process of homing into the liver and early metastatic dissemination. Our results indicate that IGFBP3 and 7 cannot be simply considered as tumor suppressors but have additional properties, which become evident only during cancer progression and metastasis formation.

Sequential biphasic changes in claudin1 and claudin4 expression are correlated to colorectal cancer progression and liver metastasis

Terminal progression of colorectal cancer (CRC) culminates in liver metastasis. To identify genes that are involved in the metastatic phenotype, cDNA microarrays were used to analyse mRNA expression profiles of colorectal carcinoma (CC)531 rat colon adenocarcinoma cells for changes related to their homing into the liver. Briefly, CC531 cells were intraportally implanted into the liver of Wag‐Rij rats and re‐isolated after 3, 6, 9, 14 and 21 days. Compared to control CC531 cells, claudin1 and claudin4 were among the ≥8‐fold initially down‐regulated genes. The co‐culture of tumour cells with isolated rat hepatocytes and Kupffer cells did not induce down‐regulation of either claudin1 or 4. When the environment effective on circulating tumour cells was simulated by cell culture conditions favouring their adhesion, only claudin4 showed augmented expression. Knockdown of claudin1 and claudin4 mediated by small interfering RNA caused significantly increased migration and decreased clonogenic growth of tumour cells (P < 0.05), but had no effect on their proliferation. These experimental results were paralleled by increased claudin1 and claudin4 expression in human CRC samples in Union for International Cancer Control (UICC) stages I–III, as evaluated by real‐time PCR. Increased claudin4 levels were correlated with significantly reduced overall survival (log‐rank test, P= 0.018). Further, significantly (P < 0.05) reduced expression of claudin1 and claudin4 was observed in stage IV and liver metastasis by immunohistochemistry. In conclusion, sequential biphasic changes in claudin1 and claudin4 expression occur during the homing of rat CC531 CRC cells to the liver. This modulation is reflected by significant changes in claudin expression in human primary and metastatic CRC.

The chemokines CCR1 and CCRL2 have a role in colorectal cancer liver metastasis

C-C chemokine receptor type 1 (CCR1) and chemokine C-C motif receptor-like 2 (CCRL2) have not yet been sufficiently investigated for their role in colorectal cancer (CRC). Here, we investigated their expression in rat and human CRC samples, their modulation of expression in a rat liver metastasis model, as well as the effects on cellular properties resulting from their knockdown. One rat and five human colorectal cancer cell lines were used. CC531 rat colorectal cells were injected via the portal vein into rats and re-isolated from rat livers after defined periods. Following mRNA isolation, the gene expression was investigated by microarray. In addition, all cell lines were screened for mRNA expression of CCR1 and CCRL2 by reverse transcription polymerase chain reaction (RT-PCR). Cell lines with detectable expression were used for knockdown experiments; and the respective influence was determined on the cells’ proliferation, scratch closure, and colony formation. Finally, specimens from the primaries of 50 patients with CRC were monitored by quantitative RT-PCR for CCR1 and CCRL2 expression levels. The microarray studies showed peak increases of CCR1 and CCRL2 in the early phase of liver colonization. Knockdown was sufficient at mRNA but only moderate at protein levels and resulted in modest but significant inhibition of proliferation (p < 0.05), scratch closure, and colony formation (p < 0.05). All human CRC samples were positive for CCR1 and CCRL2 and showed a significant pairwise correlation (p < 0.0004), but there was no correlation with tumor stage or age of patients. In summary, the data point to an important role of CCR1 and CCRL2 under conditions of organ colonization and both chemokine receptors qualify as targets of treatment during early colorectal cancer liver metastasis.

Investigation of Metastasis-Related Genes: A Rat Model Mimicking Liver Metastasis of Colorectal Carcinoma

Liver is the main target of colorectal cancer (CRC) metastasis. Currently, the number of reports is small, which describe changes in gene expression supporting liver metastasis. Here, a rat model was used for analyzing mRNA modulations during liver colonization and compared with available literature. In the model, CC531 rat CRC cells were injected via a mesenteric vein into isogenic WAG/Rij rats and re-isolated at early, intermediate, advanced, and terminal stages of liver colonization. These cells were used for RNA isolation. Microarrays were used for analyzing mRNA profiles of expression. The number of deregulated genes is comparatively large and only part of it has been studied so far. As reported to date, claudins and insulin-like growth factor-binding proteins (IGFBPs) were found to be deregulated. The fact that the chosen method is efficient is confirmed by the study of claudins and IGFBPs, which show altered expression in the initial stages of liver colonization and then return to normalcy. In addition, cadherin was described to be downregulated in epithelial–mesenchymal transition models. It can, therefore, be concluded that the models used are helpful in finding genes, which are instrumental for metastatic liver colonization.

Experimental Colorectal Cancer Liver Metastasis

With estimated 1 080 000 diagnosed cases each year, which account for 1.1% of all deaths, colorectal carcinoma (CRC) ranks fourth in cancer-related deaths in both sexes worldwide (WHOSIS, 2008). In Europe, CRC is the third most lethal malignancy after lung and stomach cancers in men, and it ranks second after breast cancer in women (WHOSIS, 2008). CRC progression is characterized by increased growth of the primary carcinoma as well as lymphatic and haematogenic spread. The liver is often the first vascular bed in which disseminating colorectal cancer cells are trapped and therefore is affected in up to 10-20% of CRC patients at the time of presentation (Berney, et al., 1998). Another 40-50% of patients will eventually develop liver metastasis during the course of their illness, which is commonly the cause of death (Bentrem, et al., 2005, Stangl, et al., 1994, Sugarbaker, 1990). At present, liver resection is considered the treatment of choice for suited patients with colorectal liver metastases, offering a five-year survival rate of 25-44% (Choti, et al., 2002, Garden, et al., 2006, Zacharias, et al., 2004) to those 20-25% of patients with isolated liver metastasis (Adson, et al., 1984, Bismuth, et al., 1996, Fong, et al., 1999). Unfortunately, this procedure is feasible only in patients with no signs of irresectable extra-hepatic disease, whereas the median survival is only 9–19 months for patients with unresectable disease who receive systemic chemotherapy (de Gramont, et al., 2000, Giacchetti, et al., 2000, Meyerhardt and Mayer, 2005, Saltz, et al., 2000). However, the fact that CRC malignancy develops over a long period and can only be efficiently controlled if detected early provokes many efforts to better understand the neoplastic progression of this cancer. It is well known, that there is a continuous shedding of tumor cells from a primary CRC (Chambers, et al., 2002), but not all disseminated CRC cells develop into macrometastases. It was hypothesized that sub-populations of malignant cells evolve a genetic advantage to become “highly metastatic”. These clones are skilled to dissociate from the primary cancer, to intravasate into nearby blood and lymphatic vessels, to travel through the lymphatic and hematogenous systems, to survive the immune surveillance, to extravasate into distant tissues forming micrometastases, and to eventually colonize the target organ. In this cascade, the epithelial-mesenchymal transition (EMT), characterized by the loss of cell-to-cell adhesion and cell polarity (Thiery, 2003), plays a crucial role in different stages;

Abstract 5252: Sequential biphasic changes in claudin1 and claudin4 expression correlate to colorectal cancer progression and liver metastasis

Terminal progression of colorectal cancer culminates in liver metastasis. To identify genes that are involved in the metastatic phenotype, cDNA microarrays were used to analyze mRNA expression profiles of CC531 rat colon adenocarcinoma cells for changes related to their homing into the liver. Briefly, CC531 cells were intraportally implanted into the liver of Wag-Rij rats and re-isolated after 3, 6, 9, 14 and 21 days. Compared to control CC531 cells, claudin1 and claudin4 were among the ≥8 fold initially down-regulated genes. The co-culture of tumor cells with isolated rat hepatocytes and Kupffer cells did not induce down-regulation of either claudin1 or 4. When the environment effective on circulating tumor cells was simulated by cell culture conditions favoring their adhesion, only claudin4 showed augmented expression. Knockdown of claudin1 and claudin4 mediated by siRNA caused significantly increased migration and decreased clonogenic growth of tumor cells (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5252. doi:10.1158/1538-7445.AM2011-5252

Abstract 1741: Quantitative Real-Time PCR analysis of SPARCL1 and SPARC expression in colorectal cancer tissues

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The SPARC family of proteins comprises 10 members, which share structural similarities in one or more protein domains. SPARC-like protein 1 (SPARCL1; also known as hevin) and SPARC are two important family members, which have been found to be involved in various tumors. The relationship between these two molecules and colorectal cancer and its liver metastasis has not yet been fully studied and understood. cDNA microarray was used to analyze the expression profiles of 22523 genes in CC531 rat colon adenocarcinoma cells. Briefly, 2 × 106 CC531 were injected into the portal vein of male Wag/Rij rats to grow in the liver. The tumor cells were then re-isolated from rat livers at different time points (after 3, 6, 9, 14 and 21 days). SPARCL 1 and SPARC were among the genes, which were up-regulated in the re-isolated metastatic cells as compared to control CC531 cells grown in vitro, especially on days 3 (two- and nine-fold for SPARCL1 and SPARC) and 6 (four- and eleven-fold for SPARCL1 and SPARC) following tumor implantation. These results motivated us to analyze the expression of these two genes in 65 colorectal cancer tissues taken from patients in different UICC tumor stages (I, n=11; II, n=23; III, n=17; IV, n=14). A mixture of 10 mucosa samples was taken for comparison as normal control. Quantitative real time-PCR results showed that SPARCL1 mRNA was significantly down-regulated in 44 samples (68%) with these levels being on average fourfold lower (0.24 ± 0.21) than those in normal mucosa. Normal levels were found in 18 samples (28%), and up-regulated levels were detected in only 3 samples (4%) with these levels being on average threefold higher than those in normal mucosa (3.0 ± 1.1). No correlation between this down- or up-regulation and tumor stage could be found. In contrast to the results for SPARCL1, SPARC showed an almost consistent up-regulation in 60 of the tumor samples (92%). The average level of SPARC up-regulation was higher than that of SPARCL1 (6.5 fold higher than in normal mucosa). It is worth noting here that in those 21 samples, in which SPARCL1 mRNA was not down-regulated, SPARC expression was on average twofold higher (13fold higher than in normal mucosa) than the average expression level of all samples. These results suggest a relationship between the two family members, but it is still not clear, whether they have opposing or complementary functions in the context of tumor initiation and progression. More studies should be performed to uncover the possible role of these proteins in the course of colorectal cancer and its liver metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1741.