Up-Regulation of RFC3 Promotes Triple Negative Breast Cancer Metastasis and is Associated With Poor Prognosis Via EMT

Abstract

Triple-negative breast cancer (TNBC) was regarded as the most aggressive and mortal subtype of breast cancer (BC) since the molecular subtype system has been established. Abundant studies have revealed that epithelial-mesenchymal transition (EMT) played a pivotal role during breast cancer metastasis and progression, especially in TNBC. Herein, we showed that inhibition the expression of replication factor C subunit 3 (RFC3) significantly attenuated TNBC metastasis and progression, which was associated with EMT signal pathway. In TNBC cells, knockdown of RFC3 can down-regulate mesenchymal markers and up-regulate epithelial markers, significantly attenuated cell proliferation, migration and invasion. Additionally, silencing RFC3 expression can decrease nude mice tumor volume, weight and relieve lung metastasis in vivo. Furthermore, we also demonstrated that overexpression of RFC3 in TNBC showed increased metastasis, progression and poor prognosis. We confirmed all of these results by immunohistochemistry analysis in 127 human TNBC tissues and found that RFC3 expression was significantly associated with poor prognosis in TNBC. Taken all these findings into consideration, we can conclude that up-regulation of RFC3 promotes TNBC progression through EMT signal pathway. Therefore, RFC3 could be an independent prognostic factor and therapeutic target for TNBC.

Figure 1

RFC3 is up-regulated in primary breast cancer. (A) Quantitative real time RT-PCR analysis of RFC3 mRNA from the same four pairs of breast cancer and adjacent non-tumor breast tissues. Error bars represent SDs calculated from triplicate experiments. (B) RT-PCR analysis of RFC3 protein in four pairs of human primary breast tumor tissues (T) and paired adjacent non-tumor breast tissue (N). (C) Western blotting analysis of RFC3 protein in four human primary breast cancer (T) and paired adjacent non-tumor breast tissues (N), with each pair taken from a same patient.

Figure 2

Up-regulation of RFC3 is associated with poor prognostic phenotype in breast cancer. (A) RFC3 expression level in different differentiation stage (benign tissue, carcinoma in situ, invasive carcinoma) analyzed by immunohistochemistry. (B) Expression levels of RFC3 in 30 paired breast cancer and adjacent non-tumor breast tissues. Alteration of expression is shown as box plot presentations and the mean level of RFC3 expression in breast cancer were significantly higher than that in non-tumor tissues. (P < .0001, independent t test). Expression levels of RFC3 between 127 BCs with and without metastasis. The mean level of RFC3 expression in BCs with metastasis was significantly higher than that in BCs without metastasis (P < .0001, independent t test). (C) Overall survival, disease-free survival and metastasis-free survival rate for cases with high RFC3 expression versus that of cases with low RFC3 expression.

Figure 3

RFC3 expression is up-regulated in breast cancer cells lines (A) Quantitative real time PCR analysis was performed in 6 breast cancer cell lines and a non-tumor immortalized human breast epithelial cell line MCF-10A. (B) RFC3 protein level in 6 breast cancer cell lines and a non-tumor immortalized human breast epithelial cell line MCF-10A were analyzed by western blotting.

Figure 4

Inhibition of RFC3 attenuated BC cell proliferation, motility and invasion. (A) The knockdown efficiency of shRNA against RFC3 was examined by Western blotting in MDA-MB-231 and MDA-MB-468 cells. (B) The proliferation ability was measured in MDA-MB-231 and MDA-MB-468 cells using colony formation assay. (C) The migration and (D) invasiveness abilities were analyzed in MDA-MB-231 and MDA-MB-468 cells by Boyden chamber assay (*P < .05, **P < .01).

Figure 5

Knockdown of RFC3 can relieve xenograph tumor progression in null mice. Xenograft model in nude mice. MDA-MB-231, shVector and shRFC3 cells were inoculated in the fat pat of nude mice (n = 5/group). (A) Images of the tumors from all mice in each group. (B) The lung metastasis staining in H&E, (C) tumor volume and (D) mean tumor weight was analyzed.

Figure 6

Knockdown of RFC3 can reversed epithelial-mesenchymal transition (EMT) The expression of EMT markers of E-Cadherin, ZO-1, Vimentin and N-Cadherin were analyzed by western blotting both in MDA-MB-231 and MDA-MB-468 breast cancer cells. GAPDH was probed as the loading control.