Metastatic Colorectal Cancer Cells Research Articles

Tu1668 Highly Metastatic Murine Colorectal Cancer Cells Share Transcriptome Identity Indicative of Cancer Cell Fusion With Macrophages

G A A b st ra ct s a*sqrt(X(i,j) > μ(i) and low if, X(i,j) + a*sqrt(X(i,j) < μ(i), where a=6. Genes were grouped into pathways using Ingenuity pathway analysis. RESULTS: Based on their relative expression levels at any one of 4 time points studied, all the genes from the raw RNAseq reads were grouped into 24 gene sets (table1). Four gene-sets figure (1 a-d). appear most interesting and coincide with change of morphology in the BEC-40W and loss of adherence to substrate (colony formation) observed in BEC-60W cells. FPKM data analysis supported upregulation of stem cell, immune response and epigenetic signatures, oxidative phosphorylation and stress response pathways. Pathway analysis revealed these genes are members of VEGF, RB, PTEN, ATF2, TP53 and other known oncogenic pathways. CONCLUSIONS: A statistical model identified four interesting gene sets from RNA sequencing data of the BEC model. These gene sets comprise oncogenes, tumor suppressors as well as regulators of signal transduction that correlated with and may be responsible for the transformed phenotype observed in BEC-40W and BEC-60W cells. Our observation from the BEC model corroborate that chronic exposure to B4 leads to genetic changes that can promote carcinogenesis in BE. Characterization of these potential candidate genes and pathways may lead to innovative biomarkers and therapeutic targets for potentially progressive BE. Table 1: The number of genes in each of the four interesting states (out of the 14 possible states). The expression state is denoted by binary digits 1=upregulation, 0= down regulation.

NOTCH3 Signaling Regulates MUSASHI-1 Expression in Metastatic Colorectal Cancer Cells

MUSASHI-1 (MSI-1) is a well-established stem cell marker in both normal and malignant colon cells and it acts by positively regulating the NOTCH pathway through inactivation of NUMB, a NOTCH signaling repressor. To date, the mechanisms of regulation of MSI-1 levels remain largely unknown. Here, we investigated the regulation of MSI-1 by NOTCH signaling in colorectal cancer cell lines and in primary cultures of colorectal cancer metastases. Stimulation by the NOTCH ligand DLL4 was associated with an increase of MSI-1 mRNA and protein levels, and this phenomenon was prevented by the addition of an antibody neutralizing NOTCH2/3 but not NOTCH1. Moreover, forced expression of activated NOTCH3 increased MSI-1 levels, whereas silencing of NOTCH3 by short hairpin RNA reduced MSI-1 levels in both colorectal cancer cells and CRC tumor xenografts. Consistent with these findings, enforced NOTCH3 expression or stimulation by DLL4 increased levels of activated NOTCH1 in colorectal cell lines. Finally, treatment of colorectal cancer cells with anti-NOTCH2/3 antibody increased NUMB protein while significantly reducing formation of tumor cell spheroids. This novel feed-forward circuit involving DLL4, NOTCH3, MSI-1, NUMB, and NOTCH1 may be relevant for regulation of NOTCH signaling in physiologic processes as well as in tumor development. With regard to therapeutic implications, NOTCH3-specific drugs could represent a valuable strategy to limit NOTCH signaling in the context of colorectal cancers overexpressing this receptor.

Modulation of Wnt Activity and Cell Physiology by Butyrate in LT97 Microadenoma Cells

Dietary fiber intake is linked to a reduced risk of colon cancer. This effect may in part be due to butyrate, the fermentation product of fiber in the colon. Butyrate is a short-chain fatty acid that acts as a histone deacetylase inhibitor (HDACi). Butyrate induces apoptosis and represses clonal growth of colorectal cancer (CRC) cells, in a manner dependent upon the hyperactivation of Wnt /beta-catenin signaling. While fiber has been linked to CRC prevention, in vitro studies on the action of butyrate have used CRC cell lines, instead of cells representative of earlier stages of colonic neoplasia, which are the likely target of butyrate-mediated preventive activity. The LT97 cell line is derived from a microadenoma, the earliest stage of colonic neoplasia from which cells can be isolated. We characterized LT97 cells with respect to effects of butyrate on Wnt signaling and apoptosis, and we determined whether modulation of CREB binding protein (CBP)/p300 activity influences the ability of butyrate to induce Wnt activity and apoptosis. We report that in LT97 cells, butyrate induces apoptosis, strongly upregulates Wnt signaling, and the upregulation of Wnt signaling is dependent upon CBP/p300 activity. In addition, findings from overexpression experiments suggest differences between CBP and p300 in their ability to influence Wnt signaling in LT97 cells; p300, but not CBP, stimulates basal Wnt activity. We also evaluated differences in gene expression between early stage LT97 cells and late stage metastatic SW620 CRC cells that exhibit markedly different cellular phenotypes. The comparative gene expression analyses revealed differences that may impact neoplastic progression and the sensitivity to the effects of butyrate. The findings have implications for the prevention of CRC by fiber/butyrate.

MicroRNA-221 promotes colorectal cancer cell invasion and metastasis by targeting RECK

MicroRNAs (miRNAs) have recently emerged as regulators of metastasis. We provide insight into the behavior of miR-221 in colorectal cancer (CRC) metastasis by showing that miR-221 is significantly upregulated in metastatic CRC cell lines and tissues. miR-221 overexpression enhances, whereas miR-221 depletion reduces CRC cell migration and invasion in vitro and metastasis in vivo. We identify RECK as a direct target of miR-221, reveal its expression to be inversely correlated with miR-221 in CRC samples and show that its re-introduction reverses miR-221-induced CRC invasiveness. Collectively, miR-221 is an oncogenic miRNA which may regulate CRC migration and invasion through targeting RECK.

Abstract A65: Characterization of CBP/p300-mediated butyrate-induced Wnt activity in colonic microadenoma cells.

Abstract Objectives of the study: We wished to (a) compare gene expression profiles between a colonic microadenoma cell line (LT97) and a metastatic colorectal cancer (CRC) cell line (SW620) in the presence and absence of butyrate, (b) determine whether the sensitivity of LT97 cells to the growth suppressive effects of butyrate correlates to a high-fold upregulation of Wnt signaling in these cells, and (c) evaluate how modulation of CBP/p300-mediated Wnt signaling influences the ability of butyrate to upregulate Wnt activity in LT97 cells. Methods: RNA was isolated from mock treated or butyrate treated LT97 microadenoma cells or SW620 metastatic CRC cells, followed by full human genome microarray analysis (Genus Biosystems). Colonic cells were transfected with Wnt reporter plasmids and treated with ICG-001 (which inhibits CBP-mediated Wnt activity) or co-transfected with a p300 expression vector or with an expression vector for KLF4, which inhibits CBP/p300 association with beta-catenin. Reporter assays, western blotting, and caspase assays were performed. Results: We demonstrate that LT97 colonic microadenoma cells differ substantially in gene expression profiles compared to a metastatic CRC cell line (SW620), both in the presence and absence of butyrate. Further, we demonstrate that the high degree of sensitivity to the growth repressive effects of butyrate of LT97 cells correlates to a marked butyrate-induced hyperactivation of Wnt activity in that cell line. Further, the induction of Wnt activity by butyrate in LT97 cells is sensitive to treatment with ICG-001, overexpression of CBP or p300, or blockade of CBP/p300-mediated Wnt activity by the transcription factor KLF4. Conclusions: Microadenoma cells, reflecting very early stage colonic neoplasia, are highly sensitive to the effects of butyrate on Wnt signaling, in a manner dependent upon CBP/p300. The findings generated from this study may lead to approaches to modulate the action of CBP/p300-mediated Wnt signaling transcriptional programs for therapeutic or chemopreventive effect. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A65. Citation Format: Darina L. Lazarova, Christopher Chiaro, Terrence Wong, Michael Bordonaro. Characterization of CBP/p300-mediated butyrate-induced Wnt activity in colonic microadenoma cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A65.

SDF-1/CXCR4 axis promotes directional migration of colorectal cancer cells through upregulation of integrin αvβ6

Colorectal cancer (CRC) displays a predilection for metastasis to liver. Although stromal cell-derived factor-1 (SDF-1)/CXCR4 plays an important role in the liver metastasis, the molecular mechanism still remains obscure. We previously reported that integrin αvβ6 was implicated in the progression of CRC. However, no data are currently available on the cross talk between CXCR4 and αvβ6. In the present study, we first demonstrated the cross talk between CXCR4 and αvβ6 and their role in liver metastasis of CRC. We analyzed 159 human CRC samples and found that expression of CXCR4 and αvβ6 was significantly associated with liver metastasis, and interestingly expression of αvβ6 significantly correlated with expression of CXCR4. Both CXCR4 and αvβ6 were highly expressed in metastatic CRC cell lines HT-29 and WiDr, whereas both of them were exiguous in non-metastatic cell line Caco-2. Furthermore, inhibition of αvβ6 significantly decreased SDF-1α-induced cell migration in vitro. SDF-1/CXCR4 could upregulate αvβ6 expression through phosphorylation of ERK and activation of Ets-1 transcription factor. In conclusion, we demonstrate that SDF-1/CXCR4 induces directional migration and liver metastasis of CRC cells by upregulating αvβ6 through ERK/Ets-1 pathway. These data support combined inhibition of CXCR4 and αvβ6 to prevent development of liver metastasis of CRC.

Dietary restriction-resistant human tumors harboring the PIK3CA-activating mutation H1047R are sensitive to metformin

Cancer cells expressing constitutively active phosphatidylinositol-3 kinase (PI3K) are proliferative regardless of the absence of insulin, and they form dietary restriction (DR)-resistant tumors in vivo. Because the binding of insulin to its receptors activates the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling cascade, activating mutations in the PIK3CA oncogene may determine tumor response to DR-like pharmacological strategies targeting the insulin and mTOR pathways. The anti-diabetic drug metformin is a stereotypical DR mimetic that exerts its anti-cancer activity through a dual mechanism involving insulin-related (systemic) and mTOR-related (cell-autonomous) effects. However, it remains unclear whether PIK3CA-activating mutations might preclude the anti-cancer activity of metformin in vivo. To model the oncogenic PIK3CA-driven early stages of cancer, we used the clonal breast cancer cell line MCF10DCIS.com, which harbors the gain-of-function H1047R hot-spot mutation in the catalytic domain of the PI3KCA gene and has been shown to form DR-refractory xenotumors. To model PIK3CA-activating mutations in late stages of cancer, we took advantage of the isogenic conversion of a PIK3CA-wild-type tumor into a PIK3CA H1047R-mutated tumor using the highly metastatic colorectal cancer cell line SW48. MCF10DCIS.com xenotumors, although only modestly affected by treatment with oral metformin (approximately 40% tumor growth inhibition), were highly sensitive to the intraperitoneal (i.p.) administration of metformin, the anti-cancer activity of which increased in a time-dependent manner and reached >80% tumor growth inhibition by the end of the treatment. Metformin treatment via the i.p. route significantly reduced the proliferation factor mitotic activity index (MAI) and decreased tumor cellularity in MCF10DCIS.com cancer tissues. Whereas SW48-wild-type (PIK3CA+/+) cells rapidly formed metformin-refractory xenotumors in mice, ad libitum access to water containing metformin significantly reduced the growth of SW48-mutated (PIK3CAH1047R/+) xenotumors by approximately 50%. Thus, metformin can no longer be considered as a bona fide DR mimetic, at least in terms of anti-cancer activity, because tumors harboring the insulin-unresponsive, DR-resistant, PIK3CA-activating mutation H1047R remain sensitive to the anti-tumoral effects of the drug. Given the high prevalence of PIK3CA mutations in human carcinomas and the emerging role of PIK3CA mutation status in the treatment selection process, these findings might have a significant impact on the design of future trials evaluating the potential of combining metformin with targeted therapy.

Gankyrin Activates IL-8 to Promote Hepatic Metastasis of Colorectal Cancer

Hepatic metastasis is responsible for the majority of colorectal cancer (CRC)-related mortalities. Although Gankyrin (PSMD10) has been implicated in cancer metastasis, its exact role and underlying mechanisms of CRC hepatic metastasis remain largely unknown. Herein, we showed that the expression of Gankyrin was higher in primary CRC with hepatic metastasis compared with CRC without metastasis. RNAi-mediated silencing of Gankyrin expression in highly metastatic human CRC cells impaired their migratory and metastatic capacity in vivo. Genome-wide transcriptome profiling revealed activation of the interleukin (IL)-8 signaling pathway by Gankyrin. Protein levels of IL-8 and cyclin D1 (CCND1), the two important molecules in the IL-8 pathway, were positively correlated with Gankyrin expression in human CRC specimens. Furthermore, genetic deletion of cyclin D1 showed its requirement in Gankyrin-mediated cell migration. Finally, administration of recombinant IL-8 rescued the migratory defect of CRC cells where Gankyrin expression was silenced. Together, our findings highlight the importance of Gankyrin in hepatic metastasis of CRC and point out its candidature as a potential prognostic marker and therapeutic target to improve the clinical management of metastatic CRC.

Decreased Levels of miR-224 and the Passenger Strand of miR-221 Increase MBD2, Suppressing Maspin and Promoting Colorectal Tumor Growth and Metastasis in Mice

Little is known about functions of microRNA (miR) passenger strands (miR*) or their roles in tumor development or progression. We screened for miRs and miR* with levels that were altered in metastatic colorectal cancer (CRC) cells and human tumor samples and investigated their targets and effects on cell function and tumor progression in mice. We performed array-based profile analysis to identify miRs with levels that were increased more than 2-fold in metastatic (SW620) CRC cells compared with nonmetastatic (SW480) cells. Quantitative polymerase chain reaction and in situ hybridization analyses were used to measure miRNA levels in CRC cell lines and human tumor samples. We used miRNA duplex mimics or inhibitors to increase and decrease levels of miRNA in CRC cells and assessed their activities and ability to form metastatic xenograft tumors in nude mice. Levels of miR-221* and miR-224 were reduced in metastatic compared with nonmetastatic CRC cells; levels in human tumor samples correlated inversely with tumor stage and metastasis to lymph nodes as well as patient survival times. SW480 cells transfected with miR-221* or miR-224 inhibitors had increased motility in vitro compared with SW480 control cells and formed larger, more metastatic tumors when injected into mice. SW620 cells transfected with miR-221* or miR-224 mimics had reduced migration and motility in vitro and formed smaller tumors with fewer metastases in mice compared with control SW620 cells. We identified the 3' untranslated region of MBD2 messenger RNA as a target of miR-221* and miR-224. MBD2 silences the gene encoding maspin, a suppressor of metastasis. In CRC cells, we found that miR-221* and miR-224 increase the expression of maspin through MBD2 down-regulation. In metastatic CRC cells, reduced levels of miR-221* and miR-224 increase levels of MBD2, thereby decreasing expression of the metastasis suppressor maspin. Increased activities of miR-221* and miR-224 reduce growth and metastasis of CRC xenograft tumors in mice; these miRs might be developed as therapeutic reagents or biomarkers of CRC progression.

In-depth Characterization of the Secretome of Colorectal Cancer Metastatic Cells Identifies Key Proteins in Cell Adhesion, Migration, and Invasion

Liver metastasis in colorectal cancer is the major cause of cancer-related deaths. To identify and characterize proteins associated with colon cancer metastasis, we have compared the conditioned serum-free medium of highly metastatic KM12SM colorectal cancer cells with the parental, poorly metastatic KM12C cells using quantitative stable isotope labeling by amino acids in cell culture (SILAC) analyses on a linear ion trap-Orbitrap Velos mass spectrometer. In total, 1337 proteins were simultaneously identified in SILAC forward and reverse experiments. For quantification, 1098 proteins were selected in both experiments, with 155 proteins showing >1.5-fold change. About 52% of these proteins were secreted directly or using alternative secretion pathways. GDF15, S100A8/A9, and SERPINI1 showed capacity to discriminate cancer serum samples from healthy controls using ELISAs. In silico analyses of deregulated proteins in the secretome of metastatic cells showed a major abundance of proteins involved in cell adhesion, migration, and invasion. To characterize the tumorigenic and metastatic properties of some top up- and down-regulated proteins, we used siRNA silencing and antibody blocking. Knockdown expression of NEO1, SERPINI1, and PODXL showed a significant effect on cellular adhesion. Silencing or blocking experiments with SOSTDC1, CTSS, EFNA3, CD137L/TNFSF9, ZG16B, and Midkine caused a significant decrease in migration and invasion of highly metastatic cells. In addition, silencing of SOSTDC1, EFNA3, and CD137L/TNFSF9 reduced liver colonization capacity of KM12SM cells. Finally, the panel of six proteins involved in invasion showed association with poor prognosis and overall survival after dataset analysis of gene alterations. In summary, we have defined a collection of proteins that are relevant for understanding the mechanisms underlying adhesion, migration, invasion, and metastasis in colorectal cancer.

Syndecan-2 is upregulated in colorectal cancer cells through interactions with extracellular matrix produced by stromal fibroblasts

BackgroundThe extracellular matrix (ECM) influences the structure, viability and functions of cells and tissues. Recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ECM components and the secretion of growth factors. Syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. Syndecan-2 has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. We have examined the effect of stromal fibroblast-produced ECM on the production of proteoglycans by colorectal cancer cell lines.ResultsOur results showed that in a highly metastatic colorectal cancer cell line, HCT-116, syndecan-2 expression is enhanced by fibroblast ECM, while the expression of other syndecans decreased. Of the various components of the stromal ECM, fibronectin was the most important in stimulating the increase in syndecan-2 expression. The co-localization of syndecan-2 and fibronectin suggests that these two molecules are involved in the adhesion of HCT-116 cells to the ECM. Additionally, we demonstrated an increase in the expression of integrins alpha-2 and beta-1, in addition to an increase in the expression of phospho-FAK in the presence of fibroblast ECM. Furthermore, blocking syndecan-2 with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments.ConclusionsOverall, these results show that interactions between cancer cells and stromal ECM proteins induce significant changes in the behavior of cancer cells. In particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan-2 may have implications in the migratory behavior of highly metastatic tumor cells.

Proteome profiling of exosomes derived from human primary and metastatic colorectal cancer cells reveal differential expression of key metastatic factors and signal transduction components

Exosomes are small extracellular 40-100 nm diameter membrane vesicles of late endosomal origin that can mediate intercellular transfer of RNAs and proteins to assist premetastatic niche formation. Using primary (SW480) and metastatic (SW620) human isogenic colorectal cancer cell lines we compared exosome protein profiles to yield valuable insights into metastatic factors and signaling molecules fundamental to tumor progression. Exosomes purified using OptiPrep™ density gradient fractionation were 40-100 nm in diameter, were of a buoyant density ~1.09 g/mL, and displayed stereotypic exosomal markers TSG101, Alix, and CD63. A major finding was the selective enrichment of metastatic factors (MET, S100A8, S100A9, TNC), signal transduction molecules (EFNB2, JAG1, SRC, TNIK), and lipid raft and lipid raft-associated components (CAV1, FLOT1, FLOT2, PROM1) in exosomes derived from metastatic SW620 cells. Additionally, using cryo-electron microscopy, ultrastructural components in exosomes were identified. A key finding of this study was the detection and colocalization of protein complexes EPCAM-CLDN7 and TNIK-RAP2A in colorectal cancer cell exosomes. The selective enrichment of metastatic factors and signaling pathway components in metastatic colon cancer cell-derived exosomes contributes to our understanding of the cross-talk between tumor and stromal cells in the tumor microenvironment.

Evaluation of 18F-FDG and 18F-FLT for monitoring therapeutic responses of colorectal cancer cells to radiotherapy

In order to compare the efficacy of 18F-fluorothymidine (FLT) and 18F-fluorodeoxyglucose (FDG) for monitoring early responses to irradiation, two human colorectal cancer (CRC) cell lines SW480 and SW620, which were derived from the primary lesions and the metastatic lymph node, underwent X-ray irradiation of 0, 10, or 20Gy and were examined at 0, 24 and 72h After irradiation, reduced proliferation of both SW480 and SW620 cells was observed in a dose-dependent manner (P<0.001), G0-G1 arrest was also noted in both cell types after 72h in the 20Gy group (P<0.001). Although increased apoptosis was observed in both cell lines after irradiation (P<0.001), a greater percentage of SW480 cells underwent apoptosis in response to irradiation than SW620 cells. Increased Hsp27 and decreased integrin β3, Ki67 and VEGFR2 expression was observed over time via immunocytochemistry and Western blot analysis (P<0.001), however, no significant changes were noted in response to irradiation. Finally, reduced uptake of 18F-FLT by SW480 or SW620 cells was observed at 24-h post-irradiation, however, reduced 18F-FDG uptake was only observed after 72h. Therefore, we conclude that 18F-FLT is a more suitable positron emission tomography (PET) tracer for monitoring early responses to irradiation in primary and metastatic lymph node CRC cells.

Abstract 1484: Effects of UCN2 and its receptor CRHR2 in colorectal cancer growth and metastasis.

Abstract Background and aims: Colorectal cancer (CRC) represents the third most common form of cancer worldwide and is associated with chronic colonic inflammation. Inflammation is partially regulated by the Corticotropin Releasing Hormone (CRH) family of neuropeptides and their receptors CRHR1 and CRHR2 which are expressed in CNS and periphery including the gastrointestinal tract. However, the effect of CRH system on tumorigenesis is poorly studied and controversial. Up-to-date there are no studies implicating the CRH family of peptides in CRC development and progression. Here, we aim to identify the role the CRH family members play in CRC growth and metastasis in vitro and in vivo and explore the underlying molecular mechanism. Methods: mRNA levels of CRH family members were monitored by qRT-PCR in 46 and 56 normal human colon and CCA biopsies, respectively, as well as in 7 CCA cell lines. CCA cell lines were lentivirally transduced for CRHR2 expression (CRHR2+). The parental and CRHR2+ cells were tested for expression of UCN2, IL-6 and IL-6R using qRT-PCR. Tumor progression was assessed in vitro by proliferation, invasion and migration assays after treatment with IL-6, astressin 2B or Ucn2. Single cell colony formation was assessed by soft agar assay. Expression of pSTAT3 and EMT markers was determined by western blot analysis. An orthotopic transplantation mouse model was used for validation of metastasis in vivo. Results: CCA biopsies and cell lines had almost lost expression of CRHR2 and elevated levels of its selective agonist UCN2, compared to their normal counterparts. CRHR2+ clones had reduced endogenous UCN2 and pro-inflammatory cytokine mRNA levels including IL-6 and its receptor IL-6R. These effects were more pronounced in highly metastatic CRC cell lines and after exogenous administration of UCN2. CRHR2+ clones with downregulated IL-6R levels showed a significantly reduced pSTAT3 levels compared to parental cells, followed by decreased cell proliferation, colony formation, invasion and migration. The above events were able to be reversed by the selective CRHR2 antagonist, astressin 2B, indicating that they were CRHR2 specific. EMT markers were also significantly dysregulated in CRHR2+ clones, suggesting EMT as a downstream target of CRHR2 signaling in CRC. The decreased metastatic potential of CRHR2+ clones, observed in vitro is currently validated in vivo. Conclusions: This is the first demonstration of the protective role of CRHR2 in preventing CRC growth rates and aggressiveness and a novel molecular evidence linking chronic inflammation and CRC through modulation of the Stat3 signaling pathway. Furthermore, monitoring CRHR2 and Ucn2 levels in CRC tissues may benefit early diagnosis of metastasis, and might serve as a novel drug target for prevention and treatment of advanced stages of CRC. Citation Format: Stavroula Baritaki, Jorge Rodriguez, Dimitrios Iliopoulos, Charalabos Pothoulakis. Effects of UCN2 and its receptor CRHR2 in colorectal cancer growth and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1484. doi:10.1158/1538-7445.AM2013-1484

Association of lipocalin‐2 in colorectal cancer metastasis to the liver

Metastasis of colorectal cancer (CRC) to the liver reduces the five‐year survival rate to less than 12% and is the major cause of death from CRC. Metastasis occurs due to productive collaborations between tumor cells and host‐derived cells in the tumor microenvironment. A pre‐metastatic niche is created to prime for cancer cell invasion into the target organ. In Balb/c mice bearing cecal tumors derived from a highly metastatic CRC cell line, microarray analysis revealed that lipocalin‐2 (LCN2) is the most highly expressed protein in the liver prior to metastasis. Western blot analysis and examination of blood serum by ELISA illustrated increased levels of LCN2 as tumors progressed into metastasis. RT‐PCR analysis showed that greater levels of LCN2 mRNA were found in highly metastatic cells as compared to less metastatic CRC cells. Our results show that LCN2 is highly associated with the development of colorectal cancer metastasis to the liver, with increased levels indicating advanced stages of metastatic progression.Center for Colon Cancer Research, University of South Carolina, Columbia, South Carolina

Toxicity of Bevacizumab in Combination with Chemotherapy in Older Patients

Bevacizumab leads to improved survival for patients with metastatic colorectal cancer (CRC) or non-small cell lung cancer (NSCLC) when added to chemotherapy. Little is known about factors associated with receipt of bevacizumab, or whether bevacizamab is associated with increased toxicity when added to chemotherapy. We conducted a prospective study of patients aged ≥65 years, which evaluated the association between geriatric assessment (GA) metrics and chemotherapy toxicity. We examined differences in characteristics and outcomes of patients with CRC and NSCLC cancers who received bevacizumab with chemotherapy versus chemotherapy alone. From a total of 207 patients, 27 (13%) received bevacizumab plus chemotherapy and 180 (87%) received chemotherapy alone. Groups were similar in sociodemographic and cancer characteristics. There were no baseline differences in GA domains except that patients with heart disease were less likely to receive bevacizumab (4% vs. 26%, p = .01). Seventy-eight percent of patients who had bevacizumab had grade 3-5 toxicity compared to only 57% who received chemotherapy alone (p = .06). Patients receiving bevacizumab were more likely to develop grade 3 hypertension than those who received chemotherapy alone (15% vs. 2%, p < .01). In multivariable analysis, factors associated with grade 3 or more toxicity included: bevacizumab (OR: 2.86, p = .04), CRC (OR: 2.54, p < .01), and baseline anemia (OR: 2.58, p = .03). Heart disease was more common in those who did not receive bevacizumab. Older patients who receive bevacizumab with chemotherapy have a higher odds of developing a grade 3-5 toxicity compared with those who receive chemotherapy alone.

Comprehensive fluorogenic derivatization–liquid chromatography/tandem mass spectrometry proteomic analysis of colorectal cancer cell to identify biomarker candidate

Existing colorectal cancer biomarkers are insufficient for providing a quick and accurate diagnosis, which is critical for a good prognosis. More appropriate biomarkers are thus needed. To identify new colorectal cancer biomarker candidates, we conducted a comprehensive differential proteomic analysis of six cancer cell lines and a normal cell line, utilizing a fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) approach. Two sets of intracellular biomarker candidates were identified: one for colorectal cancer, and the other for metastatic colorectal cancer. Our results suggest that cooperative expression of FABP5 and cyclophilin A might be linked to Her2 signaling. Upregulation of LDHB and downregulation of GAPDH suggest the existence of a specific nonglycolytic energy production pathway in metastatic colorectal cancer cells. Downregulation of 14-3-3ζ/δ, cystatin-B, Ran and thioredoxin could be a result of their secretion, which then stimulates metastasis via activity in the sera and ascitic fluids. We propose a possible flow scheme to describe the dynamics of protein expression in colorectal cancer cells leading to tumor progression and metastasis via cell proliferation, angiogenesis, disorganization of actin filaments and epithelial-mesenchymal transition. Our results suggest that colorectal tumor progression may be regulated by signaling mediated by Her2, hypoxia, and TGFβ.

Transcriptional profiling identifies genes induced by hepatocyte-derived extracellular matrix in metastatic human colorectal cancer cell lines

The milieu of the liver, and in particular hepatocyte-derived extracellular matrix (hECM), is a critical factor regulating development of liver metastases of colorectal cancer (CRC) cells. The present study has investigated genes altered by hECM in CRC cells and particularly by heparan sulfate chains of hepatocyte proteoglycans. Gene profiling analysis shows that after 2days on hECM, 226 genes are up-regulated more than 2-fold in strongly metastatic SM cells, including genes involved in growth arrest and apoptosis, signal transduction, cell migration, proliferation, communication and angiogenesis, with activation of the erbB signaling network and p53 effectors. Genes down-regulated by hECM include genes involved in lipogenesis and the S phase of the cell cycle. Further studies exploring the kinetics of gene expression after 4 and 7days culture on hECM show induction of EGF family members and of stem cell markers. In particular, hECM, but not collagen, increases mRNA expression of HB-EGF and colon stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5). Expression of these genes is not induced by hECM depleted of the heparan sulfate chains of proteoglycans. Lastly, a specific cell population positive for cancer stem cell (CSC) markers LGR5, epCAM and CD133, but negative for CD44, appears after 7days culture on hECM, a population which is reduced by 50% in cells grown on heparan sulfated-depleted hECM. Collectively, the data suggest that hECM induces growth factors and receptors regulating proliferation of metastatic CRC in the liver and offers a growth advantage for specific populations expressing CSC markers.

Colorectal Cancer Migration and Invasion Initiated by microRNA-106a

MicroRNAs have been implicated in the regulation of several cellular signaling pathways of colorectal cancer (CRC) cells. Although emerging evidence proves that microRNA (miR)-106a is expressed highly in primary tumor and stool samples of CRC patients; whether or not miR-106a mediates cancer metastasis is unknown. We show here that miR-106a is highly expressed in metastatic CRC cells, and regulates cancer cell migration and invasion positively in vitro and in vivo. These phenotypes do not involve confounding influences on cancer cell proliferation. MiR-106a inhibits the expression of transforming growth factor-β receptor 2 (TGFBR2), leading to increased CRC cell migration and invasion. Importantly, miR-106a expression levels in primary CRCs are correlated with clinical cancer progression. These observations indicate that miR-106a inhibits the anti-metastatic target directly and results in CRC cell migration and invasion.

High Expression of IL-13 Receptor α2 in Colorectal Cancer Is Associated with Invasion, Liver Metastasis, and Poor Prognosis

Autocrine secretion of cytokines by metastatic colorectal cancer cells and their role during invasion and liver homing has been poorly characterized. In this study, we used cytokine arrays to analyze the secretomes of poorly and highly metastatic colorectal cancer cells. Compared with poorly metastatic cancer cells, highly metastatic cells expressed increased levels of the immunosuppressive cytokines interleukin (IL)-4 and IL-13 in addition to increased surface expression of the high affinity IL-13 receptor IL-13Rα2, suggesting that IL-13Rα2 mediates IL-13 effects in colorectal cancer cells. Silencing of IL-13Rα2 in highly metastatic cells led to a decrease in adhesion capacity in vitro and a reduction in liver homing and increased survival in vivo, revealing a role for this receptor in cell adhesion, migration, invasion, and metastatic colonization. In support of this, IL-13 signaling activated the oncogenic signaling molecules phosphoinositide 3-kinase, AKT, and SRC in highly metastatic cells. Clinically, high expression of IL-13Rα2 was associated with later stages of disease progression and poor outcome in patients with colorectal cancer. Our findings therefore support a critical role for IL-13Rα2 expression in colon cancer invasion and metastasis.