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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

Knockdown of gene-trapped CLCN4 counters tumour cell migration. RKO-ERM no. 28 cells, induced for CLCN4 expression by tetracycline withdrawal, were transiently transfected with a CLCN4-targeting (siRNA no. 1, 2, 3) or a non-targeting siRNA using lipofectamine 2000. After 48 h, cells were either RNA extracted for semi-quantitation of CLCN4 transcript levels by RT–PCR (35 cycles) using primers located in exons 11 and 13 (A) or 5 × 104 cells were analysed for cell migration (B) as per Figure 2. Data in B represent mean±s.e. values for triplicate determinations. asterisk indicates, P<0.05 compared with that of the non-targeting siRNA.
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fig3: Knockdown of gene-trapped CLCN4 counters tumour cell migration. RKO-ERM no. 28 cells, induced for CLCN4 expression by tetracycline withdrawal, were transiently transfected with a CLCN4-targeting (siRNA no. 1, 2, 3) or a non-targeting siRNA using lipofectamine 2000. After 48 h, cells were either RNA extracted for semi-quantitation of CLCN4 transcript levels by RT–PCR (35 cycles) using primers located in exons 11 and 13 (A) or 5 × 104 cells were analysed for cell migration (B) as per Figure 2. Data in B represent mean±s.e. values for triplicate determinations. asterisk indicates, P<0.05 compared with that of the non-targeting siRNA.

Mentions: It remained a formal possibility that the tetracycline-responsive promoter was modulating a gene(s) neighbouring CLCN4 that was causal for the altered migration. Further, as the endogenous CLCN4 gene was trapped at exon 7 (determined by 3′ RACE), the expressed truncated protein (Supplementary Data 1), while retaining much of the chloride channel structure, might invoke a biological effect unrelated to its physiological role. Thus, to validate the gene-trapped CLCN4 as an inducer of tumour cell migration, the following experiments were performed. First, we used a siRNA strategy to determine whether silencing of the endogenous gene (induced by tetracycline withdrawal) in RKO-ERM no. 28 cells would counter cell migration. Of three independent siRNAs tested, two (siRNA no. 1 and no. 2) were effective in countering CLCN4 expression in RKO-ERM no. 28 cells (Figure 3A) induced by tetracycline withdrawal. Moreover, transient transfection of CLCN4-induced RKO-ERM no. 28 cells with siRNA no. 1 or no. 2 reduced (P<0.05) cell migration (Figure 3B) when compared with the non-targeting siRNA. Second, we determined whether the expression of a full-length CLCN4 cDNA would reiterate the effects on migration of the endogenously trapped CLCN4 gene. Towards this end, an HA-tagged full-length CLCN4 cDNA was subcloned into the pIRES-EGFP2 bicistronic expression construct and stably transfected into RKO cells. Cells were then selected with G418, and the GFP-positive pooled population was analysed by western blotting using an anti-HA antibody (Figure 4A). As expected, we detected the expression of the full-length HA-tagged CLCN4 protein at the predicted size of 95 kDa, but not non-HA-tagged CLCN4. More importantly, RKO cells expressing full-length CLCN4 cDNA showed enhanced migration (P<0.05) in two independent assays (Figure 4B, Supplementary Data 2) and in vitro invasion (Figure 4C), compared with transfectants harbouring the empty vector. Monolayer proliferation was unaffected by CLCN4 expression (data not shown). Moreover, transient transfection of the CLCN4 siRNA no. 2 countered (P<0.05) this increased migration/invasion, concomitant (Figure 4B, C) with silencing of the exogenous construct (Figure 4D).


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)

Knockdown of gene-trapped CLCN4 counters tumour cell migration. RKO-ERM no. 28 cells, induced for CLCN4 expression by tetracycline withdrawal, were transiently transfected with a CLCN4-targeting (siRNA no. 1, 2, 3) or a non-targeting siRNA using lipofectamine 2000. After 48 h, cells were either RNA extracted for semi-quantitation of CLCN4 transcript levels by RT–PCR (35 cycles) using primers located in exons 11 and 13 (A) or 5 × 104 cells were analysed for cell migration (B) as per Figure 2. Data in B represent mean±s.e. values for triplicate determinations. asterisk indicates, P<0.05 compared with that of the non-targeting siRNA.
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fig3: Knockdown of gene-trapped CLCN4 counters tumour cell migration. RKO-ERM no. 28 cells, induced for CLCN4 expression by tetracycline withdrawal, were transiently transfected with a CLCN4-targeting (siRNA no. 1, 2, 3) or a non-targeting siRNA using lipofectamine 2000. After 48 h, cells were either RNA extracted for semi-quantitation of CLCN4 transcript levels by RT–PCR (35 cycles) using primers located in exons 11 and 13 (A) or 5 × 104 cells were analysed for cell migration (B) as per Figure 2. Data in B represent mean±s.e. values for triplicate determinations. asterisk indicates, P<0.05 compared with that of the non-targeting siRNA.
Mentions: It remained a formal possibility that the tetracycline-responsive promoter was modulating a gene(s) neighbouring CLCN4 that was causal for the altered migration. Further, as the endogenous CLCN4 gene was trapped at exon 7 (determined by 3′ RACE), the expressed truncated protein (Supplementary Data 1), while retaining much of the chloride channel structure, might invoke a biological effect unrelated to its physiological role. Thus, to validate the gene-trapped CLCN4 as an inducer of tumour cell migration, the following experiments were performed. First, we used a siRNA strategy to determine whether silencing of the endogenous gene (induced by tetracycline withdrawal) in RKO-ERM no. 28 cells would counter cell migration. Of three independent siRNAs tested, two (siRNA no. 1 and no. 2) were effective in countering CLCN4 expression in RKO-ERM no. 28 cells (Figure 3A) induced by tetracycline withdrawal. Moreover, transient transfection of CLCN4-induced RKO-ERM no. 28 cells with siRNA no. 1 or no. 2 reduced (P<0.05) cell migration (Figure 3B) when compared with the non-targeting siRNA. Second, we determined whether the expression of a full-length CLCN4 cDNA would reiterate the effects on migration of the endogenously trapped CLCN4 gene. Towards this end, an HA-tagged full-length CLCN4 cDNA was subcloned into the pIRES-EGFP2 bicistronic expression construct and stably transfected into RKO cells. Cells were then selected with G418, and the GFP-positive pooled population was analysed by western blotting using an anti-HA antibody (Figure 4A). As expected, we detected the expression of the full-length HA-tagged CLCN4 protein at the predicted size of 95 kDa, but not non-HA-tagged CLCN4. More importantly, RKO cells expressing full-length CLCN4 cDNA showed enhanced migration (P<0.05) in two independent assays (Figure 4B, Supplementary Data 2) and in vitro invasion (Figure 4C), compared with transfectants harbouring the empty vector. Monolayer proliferation was unaffected by CLCN4 expression (data not shown). Moreover, transient transfection of the CLCN4 siRNA no. 2 countered (P<0.05) this increased migration/invasion, concomitant (Figure 4B, C) with silencing of the exogenous construct (Figure 4D).

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