The role of AFAP1-AS1 in mitotic catastrophe and metastasis of triple-negative breast cancer cells by activating the PLK1 signaling pathway

Abstract

Triple-negative breast cancer (TNBC) is characterized by fast growth, high metastasis, high invasion, and a lack of therapeutic targets. Mitosis and metastasis of TNBC cells are two important biological behaviors in TNBC malignant progression. It is well known that the long noncoding RNA AFAP1-AS1 plays a crucial role in various tumors, but whether AFAP1-AS1 is involved in the mitosis of TNBC cells remains unknown. In this study, we investigated the functional mechanism of AFAP1-AS1 in targeting Polo-like Kinase 1 (PLK1) activation and participating in mitosis of TNBC cells. We detected the expression of AFAP1-AS1 in the TNBC patient cohort and primary cells by in situ hybridization (ISH), northern blot, fluorescent in situ hybridization (FISH) and cell nucleus/cytoplasm RNA fraction isolation. High AFAP1-AS1 expression was negatively correlated with overall survival (OS), disease-free survival (DFS), metastasis-free survival (MFS) and recurrence-free survival (RFS) in TNBC patients. We explored the function of AFAP1-AS1 by transwell, apoptosis, immunofluorescence (IF) and patient-derived xenograft (PDX) models in vitro and in vivo. We found that AFAP1-AS1 promoted TNBC primary cell survival by inhibiting mitotic catastrophe and increased TNBC primary cell growth, migration and invasion. Mechanistically, AFAP1-AS1 activated phosphorylation of the mitosis-associated kinase PLK1 protein. Elevated levels of AFAP1-AS1 in TNBC primary cells increased PLK1 pathway downstream gene expression, such as CDC25C, CDK1, BUB1 and TTK. More importantly, AFAP1-AS1 increased lung metastases in a mouse metastasis model. Taken together, AFAP1-AS1 functions as an oncogene that activates the PLK1 signaling pathway. AFAP1-AS1 could be used as a potential prognostic marker and therapeutic target for TNBC.

Keywords: AFAP1-AS1; Metastasis; Mitotic catastrophe; PLK1; TNBC.

Figure 1. AFAP1-AS1 is highly expressed and associated with TNBC patient prognosis. (A) Representative ISH images showed that AFAP1-AS1 was upregulated in TNBC cancer tissues compared with peritumour tissues (scale bar = 100 μm). (B) In 100 TNBC tissue RNA samples, AFAP1-AS1 was also upregulated in cancer tissues compared with peritumour tissues by qRT‒PCR (***p < 0.001). (C) Based on the AFAP1-AS1 ISH results of 1A in 368 TNBC tumor sample sections, the patients with high AFAP1-AS1 expression (ISH scores > 6, n = 228) had poorer OS than those with low AFAP1-AS1 expression (ISH scores ≤ 6, n = 140) according to Kaplan‒Meier curves (***p < 0.001 by paired Student’s t tests, mean ± SEM). (D) Kaplan‒Meier curves showed that the patients with high AFAP1-AS1 expression had poorer DFS than those with low AFAP1-AS1 expression (***p < 0.001 by paired Student’s t tests, mean ± SEM). (E) Kaplan‒Meier curves showed that the patients with high AFAP1-AS1 expression had poorer MFS than those with low AFAP1-AS1 expression (***p < 0.001 by paired Student’s t tests, mean ± SEM). (F) Kaplan‒Meier curves showed that the patients with high AFAP1-AS1 expression had poorer RFS than those with low AFAP1-AS1 expression (***p < 0.001 by paired Student’s t tests, mean ± SEM).

Figure 2. In TNBC primary cells, AFAP1-AS1 is highly expressed and mainly located in the cytoplasm. (A) The ratio of CD326 (epithelial marker) positive in extracted TNBC primary cells is up to 90%, and the ratio of FAP (stroma marker) positive in extracted TNBC primary cells is down to 5% by flow cytometry. (B) NB assays showed that AFAP1-AS1 is upregulated in 5 TNBC primary cell samples compared with 3 FA primary cell samples. (C) FISH assays showed that AFAP1-AS1 is upregulated in 5 TNBC primary cells compared with 3 FA primary cells and is mainly located in the cytoplasm. 18S and U6 were used as cytoplasmic and nuclear controls, respectively. (D) qRT‒PCR showed that AFAP1-AS1 was upregulated in 5 TNBC primary cell samples compared with 3 FA primary cell samples (***p < 0.001 by paired Student’s t tests, mean ± SEM). (E) Cell nuclear and cytoplasmic RNA fraction isolation assays showed that AFAP1-AS1 was mainly located in the cytoplasm, and 18S and U6 were used as the cytoplasmic and nuclear controls, respectively.

Figure 3. Knockdown of AFAP1-AS1 inhibited TNBC primary cell migration, invasion, and proliferation and induced apoptosis. (A) Representative images of AFAP1-AS1 knockdown-inhibited migration and invasion of TNBC primary cells (scale bar = 100 μm). (B) The statistical results of 3 A (***p < 0.001 by paired Student’s t tests, mean ± SD). (C) AFAP1-AS1 knockdown inhibited the proliferation of TNBC primary cells, as determined by CCK8 assay (***p < 0.001 by paired Student’s t tests, mean ± SD). (D) Representative images of AFAP1-AS1 knockdown-induced TNBC primary cell apoptosis by flow cytometry assay. (E) The statistical results of 3 D (***p < 0.001 by paired Student’s t tests, mean ± SD). (F) Efficiency of AFAP1-AS1 knockdown in TNBC primary cells by NB assays.

Figure 4. Knockdown of AFAP1-AS1 induced mitotic catastrophe in TNBC primary cells. (A) IF staining with α-tubulin and γ-tubulin in TNBC primary cells showed that AFAP1-AS1 knockdown induced mitotic catastrophe in the mitosis cycle. (B) The statistics of mitotic catastrophe cells in A (***p < 0.001 by paired Student’s t tests, mean ± SD). (C) Representative images of abnormal nuclei (micronuclei, bin-nuclei and multinuclei) stained with DAPI. (D) The statistics of abnormal nuclei (including micronuclei, bin nuclei and multinuclei) in C (***p < 0.001 by paired Student’s t tests, mean ± SD).

Figure 5. Knockdown of AFAP1-AS1 inhibited PLK1 phosphorylation and downstream gene expression. (A) AFAP1-AS1 knockdown significantly inhibited PLK1 phosphorylation rather than the phosphorylation of ERK, AKT, C-jun, IKBα, P38, JNK, and STAT3 in 2 TNBC primary cell lines by WB assay. (B) AFAP1-AS1 knockdown significantly inhibited the expression of PLK1 pathway downstream genes, including CCDC25C, CDK1, BUB1 and TTK. AFAP1-AS1 knockdown did not affect other mitosis-associated proteins, such as HSET and Eg5, in TNBC primary cells (case 4 and case 5) by WB assay. (C) AFAP1-AS1 was overexpressed in TNBC primary cells (case 1: low expression AFAP1-AS1), the expression of PLK1 pathway downstream genes, including CCDC25C, CDK1, BUB1 and TTK, was increased, and the expression of other mitosis-associated proteins, such as HSET and Eg5, was not changed.

Figure 6. AFAP1-AS1 mediates the malignant biological function of TNBC primary cells through activation of the PLK1 pathway. (A) Representative images of IHC in serial tissue sections showed that PLK1 and p-PLK1 were upregulated in TNBC cancer tissues compared with peritumour tissues (scale bar = 400 μm). (B) The expression of AFAP1-AS1 was positively correlated with p-PLK1 in 368 TNBC tissues, R² = 0.3769, p < 0.0001. (C) WB assays showed that PLK1 and p-PLK1 were upregulated in 5 TNBC primary cell samples compared with 3 FA primary cell samples. (D) Transwell assays showed that AFAP1-AS1 knockdown significantly inhibited migration and invasion abilities, and PLK1 overexpression rescued the inhibition of migration and invasion abilities by AFAP1-AS1 knockdown in TNBC primary cells (scale bar = 100 μm). (E) The statistics of positive cells about D (***p < 0.001 by paired Student’s t tests, mean ± SD). (F) The statistical analyses showed that AFAP1-AS1 knockdown significantly increased the abnormal mitotic cells, and PLK1 overexpression inhibited the augmentation of abnormal mitotic cells by AFAP1-AS1 knockdown in TNBC primary cells (***p < 0.001 by paired Student’s t tests, mean ± SD).

Figure 7. Knockdown of AFAP1-AS1 inhibited the proliferation and metastasis of TNBC in vivo. (A) The summarized procedure of the PDX model in vivo in this study. (B) Tumor size in different groups was calculated every 7 days over 2 months. (***p < 0.001 between the NC group and LNA groups by paired Student’s t tests, mean ± SD). (C) Representative images of PDXs in the different groups. AFAP1-AS1 knockdown by LNA inhibited tumor growth (scale bar: 1 cm). (D) The tumor weight of the representative images of 4C with AFAP1-AS1 knockdown by LNA was reduced compared to the NC group. (E) The expression of AFAP1-AS1 was decreased in the knockdown groups by LNA compared to the NC group. (F) PDX tissue sections were subjected to HE and IHC staining against Ki-67 and p-PLK1. Ki-67 and p-PLK1 were decreased in the knockdown groups by LNA compared to the NC group. (G) To explore the influence of AFAP1-AS1 on TNBC metastasis, live imaging in vivo was conducted by intravenous injection of MDA-MB-231 cells with luciferase, and the metastases in the lung were significantly fewer in the sh-AFAP1-AS1 groups than in the scramble group. (H) Representative lung metastasis nodes in the scramble group by HE staining (scale bar: 200 μm). (I) The efficiency of AFAP1-AS1 stable knockdown was reduced compared to that of the scramble group.