SIX2 and CITED1, markers of nephronic progenitor self-renewal, remain active in primitive elements of Wilms' tumor

Abstract

Purpose: SIX2 and CITED1 are transcriptional regulators that specify self-renewing nephronic progenitor cells of the embryonic kidney. We hypothesized that SIX2, which promotes and maintains this stem cell population, and CITED1 remain active in Wilms' tumor (WT).

Methods: To evaluate expression domains and the pathogenic significance of SIX2 and CITED1 across WT, the Children's Oncology Group provided 40 WT specimens of stages I to IV (n = 10 per stage), which were enriched for unfavorable histology (n = 20) and treatment failure (relapse or death, n = 20). SIX2 and CITED1 protein expression was evaluated qualitatively (immunohistochemistry) and quantitatively (Western blot, or WB). Gene transcription was estimated using quantitative real-time polymerase chain reaction (qRT-PCR).

Results: SIX2 was visualized by immunohistochemistry in 36 (94.7%) of 38 specimens. Protein and messenger RNA expression of SIX2 were quantitatively similar across all stages of disease (P = .48 WB; P = 0.38 qPCR), in favorable or unfavorable histology (P = 0.51 WB; P = 0.58 qPCR), and in treatment failure or success (P = 0.86 WB; P = 0.49 qPCR). Although CITED1 expression paralleled SIX2 qualitatively, no quantitative correlation between SIX2 and CITED1 expression was observed (Spearman correlation coefficient, 0.28; P = 0.08). As in the fetal kidney, overlapping, but also distinct, WT cellular expression domains were observed between SIX2 and CITED1.

Conclusion: SIX2 and CITED1 remain active across all disease characteristics of WT. Activity of these genes in WT potentially identifies a population of self-renewing cancer cells that exhibit an embryonic, stemlike phenotype. Taken together, these transcriptional regulators may be fundamental to WT cellular self-renewal and may represent targets for novel therapies that promote terminal differentiation.

Figure 1

Serial sections from a favorable histology WT show blastemal immunopositivity for SIX2 (A) and CITED1 (B). Epithelia and stroma are immunonegative, similar to the embryonic kidney staining pattern. Magnification = 20X. Serial sections from unfavorable histology WT with similar overlap in blastemal expression of SIX2 (C) and CITED1 (D). Areas surrounding epithelial differentiation are weakly CITED1 positive, but SIX2 negative, demonstrating divergent staining patterns unique to a proportion of tumors examined. Magnification = 40X. Normal kidney controls show immunonegativity for SIX2 (E) and CITED1 (F). Magnification = 20X.

Figure 2

Serial sections from e18.5 mouse fetal kidney show a population of cells in the ventral cap mesenchyme and in pre-tubular aggregates (arrowheads) that are SIX2 positive (A), but CITED1 negative (B). Magnification = 20X. Double immunofluorescence for SIX2 and CITED1 on the same section (C) identifies this SIX2 positive, CITED1 negative population in the ventral cap mesenchyme (arrowheads). Magnification = 20X.

Figure 3

Serial sections from a 24 wk human fetal kidney show SIX2 (A) and CITED1 (B) immunopositivity corresponding to the cap mesenchyme. Magnification = 20X. Double immunofluorescence on a 16 wk human fetal kidney demonstrates SIX2 (C) and CITED1 (D) positivity in the cap mesenchyme (E, merged image). Magnification = 20X.

Figure 4

Double immunofluorescence co-localizes SIX2 (A, E) and CITED1 (B, F) within the blastema of favorable histology WT. Individual cells within the tumor blastema are heterogeneous with respect to SIX2 and CITED1 intensity (D, H). An area of aggregated blastema is negative for SIX2 and CITED1 (G, asterix; Bl = blastema, St= stroma). Co-localization of SIX2 (I, M) and CITED1 (J, N) in unfavorable histology WT (L, P merged images). Areas of epithelial differentiation (K, asterix) may be either positive for SIX2 or CITED1 (L), or negative (O, Ep) depending on the tumor or region within a given tumor. A–D magnification = 20X; E–P magnification = 40X.

Figure 5

Boxplot representation of SIX2 and CITED1 demonstrates ubiquitous expression across samples grouped by tumor stage (A), histology (B), and treatment outcome (C; failure = relapse or death). (D) Western blots for SIX2 and CITED1 in favorable histology (FH) and unfavorable histology (UH) WT specimens confirm ubiquitous expression, yet variability among samples. The CITED1 band at 27 kDa was utilized for densitometry and ran at same molecular weight as MCF7 positive control. Lower panels depict positive and negative controls (MCF7 = MCF7 cell lysate; COS = COS cell lysate; MFK = e18.5 mouse fetal kidney; CMN = congenital mesoblastic nephroma; VUWT = Wilms tumor).