The TNF family member APRIL promotes colorectal tumorigenesis

Abstract

The tumor necrosis factor (TNF) family member APRIL (A proliferation inducing ligand) is a disease promoter in B-cell malignancies. APRIL has also been associated with a wide range of solid malignancies, including colorectal cancer (CRC). As evidence for a supportive role of APRIL in solid tumor formation was still lacking, we studied the involvement of APRIL in CRC. We observed that ectopic APRIL expression exacerbates the number and size of adenomas in Apc(Min) mice and in a mouse model for colitis-associated colon carcinogenesis. Furthermore, knockdown of APRIL in primary spheroid cultures of colon cancer cells and both mouse and human CRC cell lines reduced tumor clonogenicity and in vivo outgrowth. Taken together, our data therefore indicate that both tumor-derived APRIL and APRIL produced by non-tumor cells is supportive in colorectal tumorigenesis.

Figure 1

Ectopic expression of APRIL promotes tumor lesions in Apc^Min mice. Apc^Min mice were crossed with APRIL-Tg mice and offspring was analyzed for intestinal adenoma formation (Apc^Min n=7; APC^Min/APRIL-Tg n=12). Lesions were quantified after 13 weeks. (a) Apc^Min/APRIL-Tg mice display increased adenoma incidence in comparison with the Apc^Min littermates. Solid lines represent the mean value for each group. Mann–Whitney test, P-value is 0.0127. (b and c) Hematoxylin and eosin staining of adenomas in Apc^Min and Apc^Min/APRIL-Tg mice. Representative section of ‘Swiss rolled' ileum. (d–f) β-Catenin staining of representative lesions in the Apc^Min and Apc^Min/APRIL-Tg. Scale bar, 500 μm. Amplifications show nuclear and membrane-bound β-catenin. (e) Nuclear β-catenin quantification by colocalization with hematoxylin using spectral imaging. IHC photomicrograph, on the first step DAB is converted into fluorescent green, and hematoxylin into fluorescent blue by CRi Nuance v2.8 software, colocalization of both is represented in yellow in the second step. (f) Quantification of colocalization in the different scanned areas (T=tumor; N=normal). (g) PAS and (h) Alcian Blue staining of representative section. Lesions are surrounded by a black line. (i and j) Hematoxylin and eosin staining of representative adenomas in Apc^Min and Apc^Min/APRIL-Tg mice. Lesions are surrounded by a black line. (k) APC^Min/APRIL-Tg display larger lesion surface. Tumor size distribution in mm²

Figure 2

APRIL-Tg mice display increased tumor incidence and enlarged adenocarcinomas in a colitis-associated cancer model. Colorectal tumors were induced in 6- to 8-week-old littermate (n=15) and APRIL-Tg mice (n=18) treated with AOM/DSS. (a) After 60 days, tumor incidence per mouse was determined. Solid lines represent the mean value for each group. The statistical significance of the data was determined using Mann–Whitney test, P-value 0.0033. (b, c and e, f) Representative sections of ‘Swiss rolled' colons. Hematoxylin and eosin stainings of adenomas in littermates (b and e) or APRIL-Tg (c and f). Tumors are surrounded by a black line. (d) Tumor size distribution in littermate (n=15) and APRIL-Tg mice (n=18) treated with AOM/DSS. Data shown are mean±S.E.M. P-value 0.0221 for <5 mm² lesions, P-value 0.0047 for 5–10 mm² lesions. (g) APRIL-Tg mice induced with AOM/DSS display a reduced amount of low-grade dysplasia (LGD) and increased numbers of intramucosal carcinoma (IMC) as compared with their littermates; HGD, high-grade dysplasia are similar. (h–m) Representative images visualizing the three dysplastic categories of adenomas scored: (h and i) photomicrograph of an incipient low-grade lesion located on the tip of a mucosal fold. Inset shows basally oriented, small nuclei and diminished goblet cell differentiation (original magnification, × 64 (h); × 320 (i). (j and k) High-grade lesion displaying back-to-back glands with pseudostratified nuclei with open chromatin and frequent mitoses (original magnification, × 40 (j); × 200 (k). (l and m) Large lesion displaying irregular glandular arrangements with complete loss of apicobasal orientation, many apoptotic bodies and intraglandular necrotic debris, consistent with intramucosal carcinoma (original magnification, × 40 (l); × 200 (m)

Figure 3

APRIL is expressed in a panel of colorectal carcinoma cell lines and primary colon cancer spheroid cultures. (a) RT-PCR analysis for APRIL expression in CRC cell lines (left panel) and in primary colon cancer cells (right panel). The upper band at 750 represents the isoforms-α and -γ, whereas the lower band at 700 represents the β-form. GAPDH was used as loading control. (b) Western blot analysis for APRIL on lysates of CRC cell lines (left panel) and primary colon cancer cells (right panel). β-Actin is used as loading control

Figure 4

APRIL inducible knockdown. (a) Map of the inducible-lentiviral vector used to transduce CRC cell lines. (b) Representative images of LS180 cells transduced with an inducible shRNA vector treated with (right panel, I=induced) and without (left panel, NI=not induced) doxycycline. Pictures show overlay between red fluorescence (RFP) and brightfield microscopy. (c) Western blot analysis for APRIL expression in LS180 lines transduced with different shRNA targeting APRIL and cultured in the presence (I=induced) or absence (NI=not induced) of doxycycline. Constructs 17 and 18 show significant reduction of APRIL expression levels. β-actin is used as loading control

Figure 5

APRIL knockdown affects clonogenic growth of human and mice colorectal cancer cells and in vivo tumor outgrowth. (a) LS180, HT29, HCT-81, DLD-1 cells were sorted and plated at a density of 100 cells per well. After 10 days, cells in the colonies were counted and the plating efficiency was calculated. The data shown represent the mean±S.D. Samples are compared by one-way ANOVA analysis and Tukey's multiple comparison post-test. Right panel: CMT93 cell lines were transduced with shRNA targeting APRIL or control shRNA. 100 cells were sorted per well of a six-well plate. After 5 days, cells are fixed and stained with a crystal violet/glutaraldehyde solution. The amount of colonies was counted and the plating efficiency was calculated. P-value 0.011. (b) The shRNA-transduced primary colon cancer spheroid culture Co2 and Co1 were sorted based on RFP expression (shRNA expression), and limiting-dilution assay was performed as described previously. The clonal frequency and statistical significance are represented. The data shown represent the mean±S.D; **P<0.01. (c) Human LS180 cells (left panel) and mouse C26 (right panel) cells transduced with the different shRNA constructs (control is empty vector; APRIL-KD is APRIL-knockdown) were injected subcutaneously into nude mice. Tumor growth and survival was measured over time and used to generate a survival curve as depicted in the Kaplan–Meier curve. Log-rank test P-value 0.012 for LS180 and P-value 0.016 for C26