Tumor-suppressive function of UNC5D in papillary thyroid cancer

Abstract

Background: Studies have shown an association of the UNC5D gene with kidney and bladder cancer and neuroblastoma. We investigated whether UNC5D acts as a tumor suppressor in papillary thyroid carcinoma (PTC).

Methods: Primary PTC tumors and matched normal thyroid tissues were obtained from 112 patients to detect UNC5D mRNA by real-time PCR. Genomic DNA sequencing was performed to detect BRAF mutation in PTC tumors. The association between UNC5D expression and clinicopathological data from PTC patients was reviewed retrospectively. PTC-derived cancer cell lines TPC-1 and K1 with stable transfection of UNC5D were used to investigate the functions of UNC5D. Flow cytometry, CCK-8, Transwell assay and scratch tests were used to examine cell cycle distribution, proliferation and migration.

Results: The expression of UNC5D was significantly decreased in PTC compared with adjacent normal thyroid tissues. Lower UNC5D expression was significantly associated with aggressive tumor behaviors, such as lymph node metastasis and BRAF mutation. Overexpression of UNC5D significantly suppressed malignant cell behaviors, including cell proliferation and migration, as well as tumor growth in vivo.

Conclusions: These findings suggest a potential tumor suppressor role of UNC5D in PTC progression; and provide insight into potential clinical relevance for the prognosis of PTC.

Keywords: BRAF mutation; UNC5D; lymph node metastasis; papillary thyroid carcinoma; tumor-suppressive function.

Figure 1. UNC5D is attenuated or silenced in PTC tissues

(A) comparison of the relative expression levels of UNC5D in 112 paired PTC and adjacent noncancerous tissues as measured by real-time PCR. “^***” indicates P< 0.01. (B) UNC5D mRNA expression in 4 representative pairs of PTC and adjacent noncancerous tissue as assessed by RT-PCR. (C) Western blot analysis of UNC5D protein in PTC and adjacent normal tissues. N, matched normal thyroid tissue; C, cancer tissue.

Figure 2. Aggressive tumor behaviors affected UNC5D mRNA expression levels in PTC

(A) the representative electropherogram of the hotspot mutation in the BRAF gene. A case of PTC with the wild-type BRAF and a case with the BRAF T1799A mutation are shown. The red rectangle indicates the nucleotide where the mutation occurs. (B-D) contrast of UNC5D mRNA expression levels between subgroups divided by with or without the BRAF^V600E hotspot mutation (B), LNM or non-LNM(C), multifocality (D), and tumor size (E). “^*” indicates P< 0.05.

Figure 3. UNC5D inhibits PTC cell growth and cell cycle status

K1 and TPC-1 cells were infected with control/UNC5D-expressing lentiviruses and monitored for growth using various assays. Each assay was repeated at least 3 times. (A) real time-PCR analysis of UNC5D in 2 PTC-derived cell lines (TPC-1 and K1) and 3 normal thyroid gland tissues (mean value of expression was set to 1). Means ± SEM of triplicate samples compared to each normal control are shown. NT = three normal thyroid tissues used as a control. (B) UNC5D mRNA expression in three normal thyroid tissues and 2 thyroid cancer cell lines as assessed by RT-PCR. (C) Western blot results show the protein expression of FLAG and GAPDH. (D) cell proliferation was analyzed using CCK-8. The results are presented as absorbance (OD) at 450 nm for triplicate wells. “^*” indicates P< 0.05. (E) colony formation of K1 and TPC-1 cells in monolayer culture. Overexpression of UNC5D inhibited cell proliferation and the colony formation of K1 and TPC-1 cells in a monolayer culture, P <0.05. (F) the flow cytometric analysis of the cell cycle showed that UNC5D induced G2-M arrest in K1 and TPC-1 cells. The results quantified in the cell cycle analysis are shown as a percentage of the total number of cells. Data are expressed as the means±SEM of three independent experiments. “^*” indicates P< 0.05.

Figure 4. Effect of UNC5D on the migration of K1 and TPC-1 cells

Transwell assay was performed to determine the migration ability of K1 (A) and TPC-1 cells (B). Representative images showing cell migration in Transwell assay. The number of migration cells was counted in 6 randomly chosen fields and averaged for each of the triplicate wells. Data are expressed as the means±SEM of three independent experiments. “^*” indicates P < 0.05, “^***” indicates P < 0.01. Representative images showing the migration of K1 (C) and TPC-1 cells (D) in the scratch wound-healing assay at 2 points.

Figure 5. UNC5D inhibits PTC growth in vivo

(A) twelve 4-5-week old male BALB/c nude mice were separated into 2 groups and injected with K1-Control cells or K1-UNC5D cells. (B) solid tumors were peeled from mouse subcutaneous tissue at 35 days post-injection. (C) tumor growth curves showed the volume of xenograft tumors changed in a time-dependent manner. (D) tumor weights were measured when mice were sacrificed. The means±SEM are reported. “^*”, P < 0.05.