A Common Role for Various Human Truncated Adenomatous Polyposis Coli Isoforms in the Control of Beta-Catenin Activity and Cell Proliferation

The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancer cases, leading to the synthesis of truncated APC products and the stabilization of β-catenin. Truncated APC is almost always retained in tumour cells, suggesting that it serves an essential function. Here, RNA interference has been used to down-regulate truncated APC in several colorectal cancer cell lines expressing truncated APCs of different lengths, thereby performing an analysis covering most of the mutation cluster region (MCR). The consequences on proliferation in vitro, tumour formation in vivo and the level and transcriptional activity of β-catenin have been investigated. Down-regulation of truncated APC results in an inhibition of tumour cell population expansion in vitro in 6 cell lines out of 6 and inhibition of tumour outgrowth in vivo as analysed in one of these cell lines, HT29. This provides a general rule explaining the retention of truncated APC in colorectal tumours and defines it as a suitable target for therapeutic intervention. Actually, we also show that it is possible to design a shRNA that targets a specific truncated isoform of APC without altering the expression of wild-type APC. Down-regulation of truncated APC is accompanied by an up-regulation of the transcriptional activity of β-catenin in 5 out of 6 cell lines. Surprisingly, the increased signalling is associated in most cases (4 out of 5) with an up-regulation of β-catenin levels, indicating that truncated APC can still modulate wnt signalling through controlling the level of β-catenin. This control can happen even when truncated APC lacks the β-catenin inhibiting domain (CiD) involved in targeting β-catenin for proteasomal degradation. Thus, truncated APC is an essential component of colorectal cancer cells, required for cell proliferation, possibly by adjusting β-catenin signalling to the “just right” level

Knockdown of truncated APC inhibits tumour formation by HT29 cells in nude mice.

<p>Lentivirus transduced HT29 cells expressing either shVEC, shN-APC or shVACO4A were injected subcutaneously into both flanks of nude mice (n, number of injections). Tumours were measured once a week over a period of 6 weeks post injection. (<b>A</b>) Tumour volumes upon termination shown as a dot plot. Bar, mean tumour size. (<b>B</b>) Increase of the mean tumour volume over time, +/−standard deviation.</p

Knockdown of truncated APC reduces proliferation of SW948 cells in vitro and increases wnt signalling.

<p>SW948 cells were transduced with lentiviruses for the expression of either shVEC or shN-APC and the transduced cells were sorted by FACS based on GFP co-expressed by the lentivirus. Data are representative of at least two independent experiments. (<b>A</b>) Efficiency of APC knockdown and β-catenin level. (<b>B</b>) Effect of APC knockdown on cellular proliferation, as described in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034479#pone-0034479-g002" target="_blank">figure 2B</a>. (<b>C</b>) β-catenin-dependent reporter assay, as described in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034479#pone-0034479-g003" target="_blank">figure 3C</a>. To visualize FOP activity, values have been multiplied by 10.</p

Consequences of APC down-regulation.

<p>LOH, loss of heterozygocity.</p>1<p>, reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034479#pone.0034479-Schneikert3" target="_blank">[25]</a>.</p>2<p>, as measured by using the TOP/FOP assay.</p>3<p>, as measured by RT-PCR of the axin2 and Lgr5 target genes.</p>4<p>, reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034479#pone.0034479-Schneikert2" target="_blank">[15]</a>.</p>5<p>, this study.</p

Transient knockdown of APC reduces colorectal cancer cell proliferation in vitro and increases wnt signalling.

<p>GP2D and CaCo2 cells were transiently transfected with either siGFP or siAPC and further processed two days after transfection. Data are representative of at least two independent experiments. (<b>A</b>) Efficiency of APC knockdown and β-catenin level. (<b>B</b>) Effect of APC knockdown on cellular proliferation, as described in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034479#pone-0034479-g002" target="_blank">figure 2B</a>. (<b>C</b>) β-catenin-dependent reporter assay. Cells were transiently transfected with either the TOPglow or control FOPglow reporters, together with a β-galactosidase expression vector to correct for variations in the transfection efficiency. Luciferase activity was measured 48 h post-transfection and normalized to β-galactosidase values. Data are represented as the mean value of triplicates+/−standard deviation. To visualize FOP activity, values have been multiplied by 4 and 100 for GP2D and CaCo2 cells, respectively.</p

Truncated APC products from SW480, DLD1, HT29, GP2D, CaCo2, LoVo, SW948 and VACO4A cells.

<p>Functional domains of APC are indicated and include the Armadillo repeat (Arm) domain, the 15 (15RA–15RD) and 20 (20R1–20R3) amino acid repeats that are β-catenin binding sites, the β-catenin inhibitory domain (CID) which is necessary to target β-catenin for degradation and the first axin/conductin binding site (SAMP). The mutation cluster region (MCR) extends from the end of the 15RA to the middle of the 20R3 and corresponds to the region where most mutations associated with colorectal cancer have been found. The indicated amino acid positions refer to the length of the various truncated APCs expressed in the different colorectal cancer cell lines. SW480, DLD1, GP2D, CaCo2 and VACO4A cells have a truncating mutation at one allele and underwent loss of heterozygocity at the second allele, whereas SW948, HT29 and LoVo cells carry each two different truncating mutations.</p

Stable knockdown of APC reduces colorectal cancer cell proliferation in vitro and increases wnt signalling.

<p>HT29, GP2D, CaCo2, LoVo and VACO4A cells were transduced with lentiviruses for the expression of either shVEC, shN-APC or shVACO4A and the transduced cells were sorted by FACS based on GFP co-expressed by the lentivirus. Data are representative of at least two independent experiments. (<b>A</b>) Efficiency of APC knockdown and β-catenin level as determined by western blotting in Triton-X100 (Tx) or hypotonic (H) cell lysates. α-tubulin and β-actin were used as controls for equal sample loading. Molecular weights of APC isoforms (kD) are indicated. (<b>B</b>) Effect of APC knockdown on cellular proliferation, analyzed using the colorimetric MTT assay over the indicated time course. Data are the mean of triplicates+/−standard deviation. (<b>C</b>) Semi-quantitative RT-PCR of APC, axin2, Lgr5 and GAPDH (21 and 26 cycles).</p

Functional Comparison of Human Adenomatous Polyposis Coli (APC) and APC-Like in Targeting Beta-Catenin for Degradation

<div><p>Truncating mutations affect the adenomatous polyposis coli (APC) gene in most cases of colon cancer, resulting in the stabilization of β-catenin and uncontrolled cell proliferation. We show here that colon cancer cell lines express also the paralog APC-like (APCL or APC2). RNA interference revealed that it controls the level and/or the activity of β-catenin, but it is less efficient and binds less well to β-catenin than APC, thereby providing one explanation as to why the gene is not mutated in colon cancer. A further comparison indicates that APCL down-regulates the β-catenin level despite the lack of the 15R region known to be important in APC. To understand this discrepancy, we performed immunoprecipitation experiments that revealed that phosphorylated β-catenin displays a preference for binding to the 15 amino acid repeats (15R) rather than the first 20 amino acid repeat of APC. This suggests that the 15R region constitutes a gate connecting the steps of β-catenin phosphorylation and subsequent ubiquitination/degradation. Using RNA interference and domain swapping experiments, we show that APCL benefits from the 15R of truncated APC to target β-catenin for degradation, in a process likely involving heterodimerization of the two partners. Our data suggest that the functional complementation of APCL by APC constitutes a substantial facet of tumour development, because the truncating mutations of APC in colorectal tumours from familial adenomatous polyposis (FAP) patients are almost always selected for the retention of at least one 15R.</p></div

The inhibition of the transcriptional activity of β-catenin mediated by APCL depends on truncated APC.

<p><b>A</b>, CaCo2 cells were transiently transfected on day 1 with the indicated siRNAs, on day 2 with reporter plasmids together with 30 ng of either an empty plasmid or an expression vector for the APCL construct yAPCL1257 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068072#pone-0068072-g001" target="_blank">figure 1</a>) and subjected on day 3 to the TOP/FOP reporter assay to measure the transcriptional activity of β-catenin (see Methods). Shown is the mean of three independent values +/− standard deviation from a representative experiment. <b>B</b>, CaCo2 cells were transiently transfected on day 1 with the indicated siRNAs and on day 2 with either 2 µg of an empty plasmid (flag) or expression vectors for either truncated APC (yAPCL1289, 2 µg) or APCL (yAPCL1257, 0,5 µg) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068072#pone-0068072-g001" target="_blank">figure 1</a>). Whole cell extracts were prepared on day 3 and subjected to western blotting analysis, using the indicated antibodies. Molecular weights are shown in kD. Note that yAPCL1257 is running slower than yAPCL1289.</p

Expression of APCL in colon cancer cell lines and functional consequences after down-regulation by RNA interference.

<p><b>A,</b> APCL is expressed in CaCo2, DLD1, HT29, LoVo, SW480 and SW837 colorectal cancer cell lines. Hypotonic cell extracts from cells grown to approximately the same confluence were subjected in duplicate to western blotting analysis, using the indicated antibodies. See material and methods to retrieve a description of the different truncating APC mutations in the cell lines. <b>B,</b> APCL down-regulation increases the level of β-catenin in CaCo2 cells and stimulates its transcriptional activity in CaCo2 and DLD1 cells. Cells were transiently transfected with siRNAs to GFP, APC and APCL on day 1 and with reporter plasmids on day 2. TOP/FOP reporter assays were performed on day 4 to measure the transcriptional activity of β-catenin (see Material and Methods). Shown is the mean of three independent values +/− standard deviation from a representative experiment. Hypotonic cell extracts were prepared on day 4 from cells not transfected with plasmids and subjected to western blotting analysis, using the indicated antibodies. Molecular weights are shown in kD. The dotted lines indicate the removal of intervening lanes.</p