SMAD4 Expression in Renal Cell Carcinomas Correlates With a Stem-Cell Phenotype and Poor Clinical Outcomes

Abstract

Renal cell carcinoma (RCC) is the most lethal neoplasm of common urologic cancers with poor prognoses. SMAD4 has a principal role in TGF-β (Transformis growth factorβ)-induced epithelial to mesenchymal transition (EMT) as a key factor in gaining cancer stem cell (CSC) features and tumor aggressiveness. This study aimed to evaluate the expression patterns and clinical significance of SMAD4 in RCC and the impact of its targeting on stem cell/mesenchymal cells and EMT characteristics in renal spheroid derived cells (SDCs) compared to parental cells (PCs) in RCC. The expression pattern and clinical significance of SMAD4 was evaluated in RCC. SDCs were enriched using a sphere culture system. Then SDCs and their PCs were compared with respect to their sphere and colony formation, expression of putative CSC markers, invasiveness as well as expression of genes, including stemness/mesenchymal, SMAD4 and TGFβ1genes. Finally, the effect of SMAD4 knockdown on SDCs was analyzed. We demonstrated that SMAD4 is positively correlated with decreased disease specific survival (DSS) in RCC patients and clear cell RCC (ccRCC) subtype and associates with poor DSS in patients with RCC, especially in ccRCC as the most metastatic RCC subtype. SDCs exhibited higher stem cell/mesenchymal properties. Inhibition of SMAD4 in PCs accelerated the dissociation of SDCs and decreased their clonogenicity, invasiveness, expression of mesenchymal markers and expression of SMAD4 and TGFβ1 genes compared to SDCs before transfection. We suggest that targeting SMAD4 may be useful against renal CSCs and may improve RCC prognosis.

Keywords: TGFβ signaling pathway; cancer stem cells; epithelial to mesenchymal transition; renal cancer; targeting.

Figure 1

Immunohistochemical (IHC) Analysis of Nuclear SMAD4 Expression in Different 707 Renal Cell Carcinoma (RCC) Samples. RCC samples expressed SMAD4 at various levels. SMAD4 expression in clear cell RCC at various levels: weak (A), moderate (B), and strong (C). Moderate expression of SMAD4 in chromophobe RCC (D) and strong expression of SMAD4 in papillary RCC (E). IHC staining of breast cancer tissue as positive (F) and negative (G) controls. (A–C) are presented with a magnification of 400×, (D–G) are presented with a magnification of 200×).

Figure 2

Association Between Expression of SMAD4 and Survival Rates in Patients With Renal Cell Carcinoma (RCC). Disease-specific survival (DSS) with nuclear SMAD4 expression grouped into high versus low expression levels in (A) RCC and (B) ccRCC patients. Number of events and at risk patients on the Kaplan Meier estimates are presented underneath the graphs (A, B).

Figure 3

Sphere formation by ACHN cells. Sphere Formation by ACHN Cells. (A) Spheroid derived cells (SDCs) from parental ACHN cells (PCs) cultured anchorage-independent conditions formed typical spheroids in the presence of growth factors at the second passage. (B) ACHN adherent monolayer cells. Figures are shown with a magnification of 100 X.

Figure 4

Clonogenicity and Sphere Formation Potential of Parental Cells (PCs) and Spheroid-Derived Cells (SDCs). (A) Sphere formation capacity of SDCs was significantly higher compared to PCs and was decreased in SDCs after transfection with SiSMAD4 (P < 0.01). The colony and sphere numbers were counted under a dissection microscope. (B) Three types of colonies termed holo, mero, and paraclones were identified during the colony formation assay in 3 cell populations, including PCs and SDCs before and after transfection with SiSMAD4. (C, D) The potential of clonogenicity was significantly higher in SDCs than PCs and was significantly decreased in SDCs after transfection with SiSMAD4. (E) The final results of cell invasion assay showed that SDCs were significantly more invasive (2.27-fold) than PCs, and invasive potential was significantly decreased (1.96-fold) in SDCs after transfection of PCs with SiSMAD4. Data are represented as mean ± SD (n = 3 each). **P < 0.01. (n = 3 each) and *P < 0.05.

Figure 5

Expression Levels of Stemness, EMT, and Angiogenesis-Related Genes and TGF-β1 using qRT-PCR in Parental Cells (PCs) and Spheroid-Derived Cells (SDCs) From Before and After Transfection of PCs with SiSMAD4. (A) Stem cell marker, (B) Mesenchymal marker, (C) ABC transporters, Angiogenesis-related and TGF-β1 genes expression of PCs and SDCs in SFDM and SDCs established by SMAD4 inhibition were quantified by qRT-PCR. Data are represented as mean ± SD (n = 3 each). *P < 0.05, and **P < 0.01.

Figure 6

Expression Levels of Putative CSC Markers in Parental Cells (PCs) and Pre transfected and Post Transfected Spheroid-Derived Cells (SDCs) Using Flow Cytometry. (A) Expression of CSC markers in PCs. (B) Expression of CSC markers in SDCs before transfection with SiSMAD4. (C) Expression of CSC markers in SDCs after transfection with SiSMAD4. Data are represented as mean ± SD (n = 3 each).

Figure 7

Transfection of Parental Cells (PCs) With SiSMAD4 and its Effect on Expression Level of SMAD4. (A) Relative SMAD4 mRNA level in PCs was measured 24,48 and 72 hours after SMAD4 knockdown compared to PCS. (B) SMAD4 gene expression level of PCs and SDCs before and after transfection with SiSMAD4. (C) Measurement of protein SMAD4 expression by Western blotting in PCs and SDCs before and after SMAD4 knockdown. Data are represented as mean ± SD (n = 3 each). **P < 0.05, and ***P < 0.0001.

Figure 8

Spheroid Derived Cells (SDCs) With Stem Cell-Related Features Before and After SMAD4 Transfection. Sphere formation assay in serum-free defined medium (SFDM) compared to the self-renewal capacity 3D sphere derivatives of the PCs after SiSMAD4 inhibition during 6 days (A:1st to G:7th day). (H) SDCs before transfection with SiSMAD4 showed significantly higher sphere-forming ability after 10 days. Figures are shown with a magnification of 100×.

Figure 9

Expression Levels of Stemness, EMT, and Angiogenesis-Related Genes, TGF-β1 and miR204 using qRT-PCR in Spheroid-Derived Cells (SDCs) From Before and After Transfection of PCs with SiSMAD4, cytoplasmic Twist1 expression and survival rates in patients with clear cell Renal Cell Carcinoma. (A) Stem cell markers, (B2) Mesenchymal markers, (C) TGF-β1 gene and (D) (miR204) expression of SDCs in SFDM and SDCs before and after Transfection by SMAD4 inhibition were quantified by qRT-PCR. Data are represented as mean ± SD (n = 3 each). *P < 0.05, **P < 0.01 and ***P < 0.0001. (B1) Correlation between cytoplasmic Twist1 expression and survival rates in patients with clear cell Renal Cell Carcinoma (ccRCC). Progression-free survival (PFS) with cytoplasmic Twist1 expression grouped into low- versus moderate- versus high expression levels.