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Gene trapping identifies chloride channel 4 as a novel inducer of colon cancer cell migration, invasion and metastases.

Ishiguro T, Avila H, Lin SY, Nakamura T, Yamamoto M, Boyd DD - Br. J. Cancer (2010)

Bottom Line: Rapid amplification of cDNA ends identified the trapped gene as the chloride channel 4 (CLCN4) exchanger.Stable expression of a CLCN4 cDNA enhanced motility, whereas cells knocked down or for this transcript showed reduced migration/invasion.Intra-splenic delivery of RKO-CLCN4 transfectants, but not controls, yielded liver metastases, and transcript levels were higher in colon cancer metastases to the liver when compared with primary tumours.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Department, MD Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT

Background: To date, there are few reports on gene products contributing to colon cancer progression.

Methods: We used a gene trap comprised of an enhanced retroviral mutagen (ERM) cassette that includes a tetracycline-responsive promoter upstream of a haemagglutinin (HA) tag and a splice donor site. Integration of the ERM within an endogenous gene yields a tetracycline-regulated HA-tagged transcript. We transduced RKO colon cancer cells expressing a tetracycline trans-activator-off with the ERM-encoding retrovirus and screened for enhanced migration.

Results: One clone showed fivefold enhanced migration with tetracycline withdrawal. Rapid amplification of cDNA ends identified the trapped gene as the chloride channel 4 (CLCN4) exchanger. Stable expression of a CLCN4 cDNA enhanced motility, whereas cells knocked down or for this transcript showed reduced migration/invasion. CLCN4-overexpressing RKO colon cancer cells were more resistant than controls to proton load-induced cytotoxicity, consistent with the H(+)-extruding function of this antiporter. Intra-splenic delivery of RKO-CLCN4 transfectants, but not controls, yielded liver metastases, and transcript levels were higher in colon cancer metastases to the liver when compared with primary tumours.

Conclusions: CLCN4 is a novel driver of colon cancer progression.

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Related in: MedlinePlus

Schematic of gene-trapping experimental strategy. LTR, long terminal repeat; SA, splice acceptor; tTA, tetracycline-responsive trans-activator.
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fig1: Schematic of gene-trapping experimental strategy. LTR, long terminal repeat; SA, splice acceptor; tTA, tetracycline-responsive trans-activator.

Mentions: Although the aforementioned observations have provided invaluable insight into the molecular mechanisms responsible for colorectal cancer development, there have been relatively few studies that identify gene products contributing to colon cancer cell migration, invasion and metastases (Cajot et al, 1997; Guan et al, 2000; Minard et al, 2005; Samuels et al, 2005). As one example, Drg-1, identified by differential display, is downregulated in its expression in metastatic colon cancer and its forced overexpression reduces in vitro invasion of colon cancer, these findings leading the authors to propose Drg-1 as a metastatic suppressor (Guan et al, 2000). Similarly, expression of motility-related protein (MRP1/CD9) is highly repressed in metastases compared with primary colorectal tumours, and anti-MRP1/CD9 antibodies abrogate tumour cell migration (Cajot et al, 1997). Conversely, the PIK3CA gene is mutated in ∼30% of colorectal cancers (Samuels et al, 2004) and these alterations generate a protein that facilitates tumour invasion (Samuels et al, 2005). Considering the paucity of studies to identify genes regulatory for colon cancer cell migration/invasion, we undertook an unbiased, genome-wide approach to discover novel genes stimulatory for colon cancer cell migration. Towards this end, we used a gene-trap method (Liu et al, 2000; Lin and Elledge, 2003) (see Figure 1 for strategy) using a retroviral enhanced retroviral mutagen (ERM) cassette. The ERM includes a haemagglutinin (HA) tag and a splice donor site under the control of a tetracycline-responsive promoter. On retroviral transduction of tetracycline trans-activator-expressing cells, if the ERM is integrated intra-genically, the endogenous gene is brought under the control of the tetracycline-responsive promoter and the spliced ‘gene-trapped' transcript can be identified by the HA tag sequence using rapid amplification of cDNA ends (3′-RACE). Using this gene-trap system, we report herein the identification of chloride channel 4 (CLCN4) as a novel gene product that enhances colon cancer migration and metastases.


Gene trapping identifies chloride channel 4 as a novel inducer of colon cancer cell migration, invasion and metastases.

Ishiguro T, Avila H, Lin SY, Nakamura T, Yamamoto M, Boyd DD - Br. J. Cancer (2010)

Schematic of gene-trapping experimental strategy. LTR, long terminal repeat; SA, splice acceptor; tTA, tetracycline-responsive trans-activator.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2837579&req=5

fig1: Schematic of gene-trapping experimental strategy. LTR, long terminal repeat; SA, splice acceptor; tTA, tetracycline-responsive trans-activator.
Mentions: Although the aforementioned observations have provided invaluable insight into the molecular mechanisms responsible for colorectal cancer development, there have been relatively few studies that identify gene products contributing to colon cancer cell migration, invasion and metastases (Cajot et al, 1997; Guan et al, 2000; Minard et al, 2005; Samuels et al, 2005). As one example, Drg-1, identified by differential display, is downregulated in its expression in metastatic colon cancer and its forced overexpression reduces in vitro invasion of colon cancer, these findings leading the authors to propose Drg-1 as a metastatic suppressor (Guan et al, 2000). Similarly, expression of motility-related protein (MRP1/CD9) is highly repressed in metastases compared with primary colorectal tumours, and anti-MRP1/CD9 antibodies abrogate tumour cell migration (Cajot et al, 1997). Conversely, the PIK3CA gene is mutated in ∼30% of colorectal cancers (Samuels et al, 2004) and these alterations generate a protein that facilitates tumour invasion (Samuels et al, 2005). Considering the paucity of studies to identify genes regulatory for colon cancer cell migration/invasion, we undertook an unbiased, genome-wide approach to discover novel genes stimulatory for colon cancer cell migration. Towards this end, we used a gene-trap method (Liu et al, 2000; Lin and Elledge, 2003) (see Figure 1 for strategy) using a retroviral enhanced retroviral mutagen (ERM) cassette. The ERM includes a haemagglutinin (HA) tag and a splice donor site under the control of a tetracycline-responsive promoter. On retroviral transduction of tetracycline trans-activator-expressing cells, if the ERM is integrated intra-genically, the endogenous gene is brought under the control of the tetracycline-responsive promoter and the spliced ‘gene-trapped' transcript can be identified by the HA tag sequence using rapid amplification of cDNA ends (3′-RACE). Using this gene-trap system, we report herein the identification of chloride channel 4 (CLCN4) as a novel gene product that enhances colon cancer migration and metastases.

Bottom Line: Rapid amplification of cDNA ends identified the trapped gene as the chloride channel 4 (CLCN4) exchanger.Stable expression of a CLCN4 cDNA enhanced motility, whereas cells knocked down or for this transcript showed reduced migration/invasion.Intra-splenic delivery of RKO-CLCN4 transfectants, but not controls, yielded liver metastases, and transcript levels were higher in colon cancer metastases to the liver when compared with primary tumours.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Department, MD Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT

Background: To date, there are few reports on gene products contributing to colon cancer progression.

Methods: We used a gene trap comprised of an enhanced retroviral mutagen (ERM) cassette that includes a tetracycline-responsive promoter upstream of a haemagglutinin (HA) tag and a splice donor site. Integration of the ERM within an endogenous gene yields a tetracycline-regulated HA-tagged transcript. We transduced RKO colon cancer cells expressing a tetracycline trans-activator-off with the ERM-encoding retrovirus and screened for enhanced migration.

Results: One clone showed fivefold enhanced migration with tetracycline withdrawal. Rapid amplification of cDNA ends identified the trapped gene as the chloride channel 4 (CLCN4) exchanger. Stable expression of a CLCN4 cDNA enhanced motility, whereas cells knocked down or for this transcript showed reduced migration/invasion. CLCN4-overexpressing RKO colon cancer cells were more resistant than controls to proton load-induced cytotoxicity, consistent with the H(+)-extruding function of this antiporter. Intra-splenic delivery of RKO-CLCN4 transfectants, but not controls, yielded liver metastases, and transcript levels were higher in colon cancer metastases to the liver when compared with primary tumours.

Conclusions: CLCN4 is a novel driver of colon cancer progression.

Show MeSH
Related in: MedlinePlus