Progress in the research of p53 tumour suppressor activity controlled by Numb in triple‐negative breast cancer

Abstract Numb is known as a cell fate determinant as it identifies the direction of cell differentiation via asymmetrical partitioning during mitosis. It is considered as a tumour suppressor, and a frequent loss of Numb expression in breast cancer is noted. Numb forms a tri‐complex with p53 and E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing the ubiquitination and degradation of p53. In this study, we examined Numb expression in 125 patients with triple‐negative breast cancer (TNBC). The results showed that 61 (48.8%) patients presented with a deficient or decreased Numb expression. The percentage of Ki67 > 14% in the retained Numb group was significantly lower than that in the decreased and deficient Numb groups (86.00% vs. 98.40%, P = .0171). This study aimed to detect the expression and migration of Numb, HDM2 and p53 in the membrane, cytoplasmic and nuclear fractions of normal mammary epithelial cell line MCF‐10A and basal‐like TNBC cell line MDA‐MB‐231. We obtained the cell fractions to identify changes in these three protein levels after the re‐expression of NUMB in the MDA‐MB‐231 cells and the knocking down of NUMB in the MCF‐10A cells. Results showed that Numb regulates p53 levels in the nucleus where the protein levels of Numb are positively correlated with p53 levels, regardless if it is re‐expressed in the MDA‐MB‐231 cells or knocked down in the MCF‐10A cells. Moreover, HDM2 was remarkably decreased only in the membrane fraction of NUMB knock‐down cells; however, its mRNA levels were increased significantly. Our results reveal a previously unknown molecular mechanism that Numb can migrate into the nucleus and interact with HDM2 and p53.

cells and promote epithelial-to-mesenchymal transition leading to an unsuccessful lactation. 7,8 Furthermore, the loss of Numb expression was associated with the occurrence of breast cancer. Decreased Numb expression also affects cell cycle proteins, thus accelerating the transformation of G1/S phase and promoting the proliferation of tumour cells. 9 Breast cancer is a common malignant tumour in women, and its incidence rate has been increasing annually. Triplenegative breast cancer (TNBC) is a type of malignant breast cancer that represents a sub-group of breast cancers that tests negative for oestrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2). 10,11 In breast cancers, a significant positive correlation was noted between Numb expression (deficient and decreased vs. retained) and ER and PR status. 12 The immunohistochemical staining of the pathological sections from 241 patients with breast cancer revealed that approximately 44% of deficient or decreased Numb expression was noted in 25 patients with basal-like TNBC, and its loss or decrease was associated with the degree of tumour malignancy, prognosis and 5-year survival rate. 12 We performed immunohistochemical staining on the paraffin sec- in the retained Numb group was significantly lower than that in the decreased and deficient Numb groups (P = .0171). However, no significant difference was noted in terms of age, tumour size, lymph node status and histological type between the retained Numb and decreased and deficient Numb groups.
The mutation and decrease of the tumour suppressor p53 in breast cancer is common. HDM2 oncoprotein can degrade P53, resulting in shorter half-life and decreased p53 activity. [13][14][15][16] Numb interacts with HDM2 and prevents the ubiquitination and p53 degradation, thus maintaining the activity and stability of p53.
However, the specific mechanism of underlying their interactions remains unclear. 17 The present study aimed to further explore the migration and expression of Numb, HDM2 and p53 in the cell membrane, cytoplasm and nucleus of MCF-10A and MDA-MB-231 cells.
Our results demonstrated that Numb and HDM2 were detected in the cell membrane, cytoplasm and nucleus and that p53 was pri- level of p53 and Numb in the nucleus. Meanwhile, no significant changes were observed in the HDM2 levels. These results demonstrated that Numb can migrate into the nucleus to inhibit the degradation of p53 by HDM2 in the NUMB-EGFP-transfected cells. In the nucleus of NUMB knock-down cells, the expression of Numb and p53 significantly increased, and no changes were observed in the HDM2 and p53 mRNA levels. Importantly, these results validated that Numb regulates p53 levels in the nucleus and that it is positively correlated with p53 levels.

| Clinical samples
We collected archival formalin-fixed paraffin-embedded mammary tissue specimens from 125 patients with TNBC who were diagnosed at the Clinical pathology diagnosis centre of Medical University of Chongqing between 2012 and 2017. Numb status was attributed to the tumours by measuring the levels of Numb expression by immunohistochemical staining (IHC). Normal mammary tissues showed a strong and homogeneous NUMB expression. Tumours were classified based on an IHC scale from 0 to 2 (score 2, <10% positive; score 1, 10%-50% positive and score 0, >50% positive tumour cell expression of Numb).

| Ethics statement
The study was approved by an ethics committee and follows the tenants of the Declaration of Helsinki. This study was a retrospective clinical study. All patients had signed an informed consent before surgery and agreed to use pathological specimens for scientific research.

| Immunohistochemistry
Serial sections were cut from paraffin blocks, deparaffinized with xylene and then rehydrated in a graded ethanol series. For antigen retrieval, the samples were microwaved for 14 minutes in a citrate buffer (pH = 6). Subsequently, the sections were treated with 3% hydrogen peroxide for 15 minutes. All sections were incubated with Anti-NUMB monoclonal antibody (1:100, ab14140; Abcam) overnight at 4°C. These antibodies were identified using a biotinylated secondary antibody (PV-9001, Beijing Sequoia Jinqiao) labelled with streptavidin-horseradish peroxidase and a DAB staining kit (KIT-5020, Maixin Biotechnology. As for the negative control, the primary antibody was substituted with phosphate-buffered saline (PBS).
Human adrenal gland tissue is the positive control recommended by the antibody, and it is the reliable basis to verify the quality of the antibody.

| Immunofluorescence
Cells were seeded onto glass coverslips in 24-well plates, washed with PBS, fixed in 4% paraformaldehyde for 20 minutes at room temperature and permeabilized with 0.1% Triton X-100. They were incubated in blocking buffer (PBS with 5% bovine serum albumin

| Cell fractionation experiments and immunoblotting
The cytoplasmic, membrane and organelle and cytoskeletal and nuclear fractions of cells were obtained by lysing using the Cell

| Statistical analysis
Student's t test (two-tailed) was used to determine the significance of the differences between the groups. A P value of <.05 was considered statistically significant. NUMB expression intensities in human breast cancer samples were analysed using chi-square and Fisher's exact tests. The SAS version 9.4 software (Copyright © 2016 SAS Institute Inc Cary) was used for analysis. A significant difference was determined at α = 0.05.

| Numb expression in normal mammary tissue and triple-negative breast cancers
We performed immunohistochemical staining on the paraffin sections from 125 patients with TNBC to detect the Numb ex-   and MCF-10A cells. In addition, p53 was not expressed in the cell membrane ( Figure 2E).

| Numb, HDM2 and p53 levels in the different cell fractions of the NUMB-EGFP-transfected MDA-MB-231 cells
The above-mentioned experimental data suggested that Numb  Figure 3A). However, in the fraction of the membrane and cytoplasm, Numb, HDM2 and p53 have not been affected ( Figure 3A).

Meanwhile, no significant change was observed in HDM2 and P53
in the mRNA level after the re-expression of NUMB in MDA-MB-231 ( Figure 3E). Importantly, the expression of Numb in the membrane fraction did not significantly change, and in the cytoplasmic fraction, no expression was observed. Moreover, a significant increase was ob-  Figure 3D).

| Effects of NUMB knock-down on Numb, HDM2 and p53 expression in the different cell fractions of MCF-10A cells
Transfection with NUMB siRNA decreased Numb expression in the MCF-10A cell line, and the protein level of HDM2 and p53 decreased accordingly ( Figure 4A). q-PCR indicated that the mRNA level of HDM2 was significantly increased, whereas p53 did not change significantly ( Figure 4E). Furthermore, after separating the cell fractions for Western blot experiments, the protein level of Numb and p53 both decreased in the nuclear fraction, whereas no changes were noted in HDM2 ( Figure 4A). In the membrane fraction of MCF-10A, Numb expression did not change significantly, whereas the HDM2 decreased remarkably. Moreover, p53 was not detected ( Figure 4A).
The Numb, HDM2 and p53 levels were not significantly different in the cytoplasmic fraction ( Figure 4A). Importantly, the Numb levels were significantly decreased in the nuclear fraction after tampering with NUMB in the MCF-10A cell line, and it did not significantly change in the membrane and cytoplasmic fractions ( Figure 4B). The expression of HDM2 significantly decreased in the membrane fraction of MCF-10A, and that in the cytoplasmic and nuclear fractions did not change ( Figure 4C). The p53 levels in the nucleus of the MCF-10A cells significantly increased and that in the cytoplasm remained unchanged. No expression was noted in the cell membrane ( Figure 4D).

| D ISCUSS I ON
A higher percentage of tumours from the triple-negative (SR-/ HER2-) sub-group displayed decreased or deficient Numb expression compared with those in the other sub-groups (44% [11/25] of the basal-like triple-negative cell line). 18 We performed immunohistochemical staining on the paraffin sections from 125 patients with TNBC to detect Numb expression. 19,20 The results showed  Note: The categorical variables were reported as numbers (n) and percentages of the total (%), and chi-square test and Fisher's exact test were used to test the difference. The data analysis for this study was generated using SAS 9.4 software (Copyright © 2016 SAS Institute Inc Cary). Significant difference was determined at the α level of 0.05. All P values were calculated using chi-square test, except when calculating correlation between histological type, Ki67 and Numb expression when Fisher's exact was used. There is no significant difference in age, tumour size, lymph node status, histological type between the retained Numb group and reduced and deficient Numb group. The per cent of Ki67 > 14% in retained Numb group was significantly lower than that in the reduced and deficient Numb group (86.00% vs. 98.40%, P = .0171).
*The P value is significant.

ACK N OWLED G EM ENTS
We thank all the breast cancer patients who donated their samples for research. This work was supported by grants from the Chongqing science and technology commission to Youde.C and JX.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
JX and XQ performed experimental work and analysed data. YC, YQL and JX performed data analysis and wrote the manuscript.
Youde.C planned and supervised the project, provided samples and supervised the histopathological analysis.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article and its additional information files.