KDF1, a Novel Tumor Suppressor in Clear Cell Renal Cell Carcinoma

Abstract

KDF1 has been identified as a key regulator of epidermal proliferation and differentiation, but it is unknown whether KDF1 is involved in the pathogenesis of malignancy. No study has reported the expression and function of KDF1 in renal cancer. To explore the pathologic significance of KDF1 in clear cell renal cell carcinoma (ccRCC), the expression level of KDF1 protein in the tumor tissue of ccRCC patients was examined by immunohistochemistry and Western blot while the expression level of KDF1 mRNA was analyzed by using the data from TCGA database. In vitro cell experiments and allogeneic tumor transplantation tests were performed to determine the effects of altered KDF1 expression on the phenotype of ccRCC cells. Both the KDF1 mRNA and protein were found to be decreasingly expressed in the tumor tissue of ccRCC patients when compared with the adjacent non-tumor control tissue. The expression level of KDF1 in the tumor tissue was found to correlate negatively with the tumor grade. Patients with higher KDF1 in the tumor tissue were found to have longer overall survival and disease-specific survival time. KDF1 was shown to be an independent factor influencing the disease-specific survival of the ccRCC patients. Overexpression of KDF1 was found to inhibit the proliferation, migration and invasion of ccRCC cells, which could be reversed by decreasing the expression of KDF1 again. ccRCC cells with KDF1 overexpression were found to produce smaller transgrafted tumors. These results support the idea that KDF1 is involved in ccRCC and may function as a tumor suppressor.

Keywords: KDF1; clear cell renal cell carcinoma; prognosis; tumor grade; tumor suppressor.

Figure 1

Expression of KDF1 mRNA in the tumor tissue of patients with clear cell renal cell carcinoma (ccRCC) and its association with overall survival. The expression level of KDF1 mRNA in the tumor tissue of 530 ccRCC patients was compared with that in the 72 normal renal samples (A). Patients were divided into higher KDF1 mRNA subgroup (with the KDF1 mRNA level >1.415 Fragments Per Kilobase per Million (FPKM)) and lower KDF1 mRNA subgroup (with the KDF1 mRNA level ≤1.415 FPKM) according to the level of KDF1 mRNA in the tumor tissue and overall survival were compared between the two subgroups by using Kaplan–Meier method (B). In the analysis of overall survival, 23 patients who died within a month after operation were excluded and a total of 507 patients were included. **p < 0.01. FPKM, Fragments per kilobase Million.

Figure 2

Expression of KDF1 in the tumor tissue of patients with clear cell renal cell carcinoma (ccRCC). Immunohistochemical staining was performed on the tumor tissue of 241 ccRCC patients while 39 non-tumor renal tissue samples were used as controls. The expression of KDF1 in the tumor tissue of 10 ccRCC patients was confirmed by Western blot while 10 non-tumor renal tissues were used as controls. (A) Representative pictures of immunohistochemistry. a: A representative picture from non-tumor renal tissue; b: A representative picture from ccRCC patients showing negative immunostaining for KDF1 (the KDF1 level was scored as 0); c: A representative picture from ccRCC patients showing weak immunostaining for KDF1 (the KDF1 level was scored as 1); d: A representative picture from ccRCC patients showing medium immunostaining for KDF1 (the KDF1 level was scored as 2); e: A representative picture from ccRCC patients showing strong immunostaining for KDF1 (the KDF1 level was scored as 3). (B) Results of Western blot analysis for KDF1 in the tumor tissue of 10 ccRCC patients and the matched non-tumor tissue. (C) Comparison of the KDF1 protein level between the tumor tissue of ccRCC patients and the non-tumor renal tissue according to the results of immunohistochemistry. (D) Quantitative analysis of the KDF1 protein level in the tumor tissue of 10 ccRCC patients compared with the non-tumor renal tissue according to the results of Western blot. a1-e1 is the local amplification of a-e respectively. N1-N10: Non-tumor tissue; T1-T10: ccRCC tumor tissue. **P < 0.01. Scale bar: 50 μm.

Figure 3

Results of survival analysis of patients with clear cell renal cell carcinoma (ccRCC) based on KDF1 protein level in the tumor tissue. A total of 241 ccRCC patients were included. The patients were divided into lower KDF1 protein subgroup (with immunostaining score for KDF1 in the tumor tissue being 0 or 1, n=136) and higher KDF1 protein subgroup (with immunostaining score for KDF1 in the tumor tissue being 2 or 3, n=105) according to the level of KDF1 protein in the tumor tissue of the patients. The overall survival (A) and disease-specific survival (B) were compared between the two subgroups by using Kaplan-Meier method.

Figure 4

Effect of KDF1 overexpression on the proliferation, migration and invasion of ccRCC cells. Two ccRCC cell lines, 786-O and ACHN, were used in the experiments. The KDF1 overexpression ccRCC cells, 786-O-KDF1 and ACHN-KDF1, were constructed via stably infecting 786-O and ACHN cells with a recombinant KDF1 expression lentivirus. The overexpression of KDF1 in ccRCC cells were confirmed by quantitative RT-PCR and Western blot (A) and the influence of KDF1 overexpression in the proliferation (B), migration (C) and invasion (D) of the ccRCC cells were evaluated by using CCK-8, wound healing and Matrigel invasion chamber methods. All the experiments were repeated at least three times. A, untransduced ACHN cells; A-VC, control virus transduced ACHN cells; A-K, KDF1 overexpression ACHN cells; O, untransduced 786-O cells; O-VC, control virus transduced 786-O cells; O-K, KDF1 overexpression 786-O cells. *p < 0.05; **p < 0.01. Scale bar, 100µm.

Figure 5

Overexpression of KDF1 significantly decreased the growth of transgrafted tumors and the ratio of ki-67 positive cells in the tumor. Four-week old male nude mice were randomly divided into non-transduced cell group, control virus transduced cell group and KDF1 overexpression cell group. Six mice were used in each group. For each mouse, 2×10⁶ cells (untransduced ACHN cells for the untransduced cell group, control virus transduced ACHN cells for the control virus transduced cell group, and KDF1 overexpression ACHN cells for the KDF1 overexpression cell group) were transplanted subcutaneously on the side of the body. Six weeks later, the mice were euthanized and the tumors were removed, measured and weighed. Paraffin sections of the transgrafted tumors were used in Hematoxylin-Eosin (HE) staining and immunohistochemical staining for ki-67. Figure part (A) Results of the tumor transplant trial showing that overexpression of KDF1 decreased the growth of transgrafted tumors. Figure part (B) Results of immunohistochemical staining for ki-67 showing that overexpression of KDF1 decreased the ratio of ki-67 positive cells in the transgrafted tumors. Figure part (C) Results of HE staining showing no structural difference among the tumor tissues. A, the untransduced cell group; A-VC, the control virus transduced cell group; A-K, the KDF1 overexpression cell group. a1-f1 is a partial magnification of a-f, respectively. **P < 0.01.

Figure 6

Knockdown of KDF1 reversed the effect of KDF1 overexpression on the ccRCC cell’s proliferation, migration and invasion. A recombinant KDF1 shRNA expression lentivirus was used to knock down the expression of KDF1 in the KDF1 overexpression ccRCC cells. The knockdown of KDF1 expression was confirmed by quantitative RT-PCR and Western blot analysis (A) and the influence of KDF1 knockdown in the proliferation (B), migration (C) and invasion (D) of the KDF1 overexpression ccRCC cells were evaluated by using CCK-8, wound healing and Matrigel invasion chamber methods. All the experiments were repeated at least three times. A, untransduced ACHN cells; A-K, KDF1 overexpression ACHN cells; A-K-sh, the KDF1 knockdown A-K; O, untransduced 786-O cells; O-K, KDF1 overexpression 786-O cells; O-K-sh, the KDF1 knockdown O-K; *p < 0.05; **p < 0.01. Scale bar, 100µm.