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, 9 (22), 6468-6484
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BCAP31 Drives TNBC Development by Modulating Ligand-Independent EGFR Trafficking and Spontaneous EGFR Phosphorylation

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BCAP31 Drives TNBC Development by Modulating Ligand-Independent EGFR Trafficking and Spontaneous EGFR Phosphorylation

Wenyan Fu et al. Theranostics.

Abstract

Identification of novel targets for triple-negative breast cancer (TNBC) is an urgent task as targeted therapies have increased the lifespans of Oestrogen Receptor +/ Progesterone Receptor + and HER2+ cancer patients. Methods: genes involved in protein processing in the endoplasmic reticulum, which have been reported to be key players in cancer, were used in loss-of-function screening to evaluate the oncogenic roles of these genes to identify candidate target genes in TNBC. In vitro and in vivo function assays as well as clinical prognostic analysis were used to study the oncogenic role of the gene. Molecular and cell based assays were further employed to investigate the mechanisms. Results: B Cell Receptor Associated Protein 31 (BCAP31), the expression of which is correlated with early recurrence and poor survival among patients, was identified an oncogene in our assay. In vitro studies further suggested that BCAP31 acts as a key oncogene by promoting TNBC development. We also showed that BCAP31 interacts with epidermal growth factor receptor (EGFR) and serves as an inhibitor of ligand-independent EGFR recycling, sustaining EGFR autophosphorylation and activation of downstream signalling. Conclusion: These findings reveal the functional role of BCAP31, an ER-related protein, in EGFR dysregulation and TNBC development.

Keywords: BCAP31; Cancer Development; EGFR; TNBC.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
RNAi screening and KMplot strategy to identify regulators of basal-like breast cancer. (A) Schematic highlighting the criteria for gene selection and the experimental setup of the RNAi screen. (B) Heat map representing the expression of selected transcripts in different cells as determined by qPCR analysis. (C) Column showing the average Z-score of triple-negative cell lines minus the average Z-score of luminal cell lines (ΔZ-score) for all the genes included in the RNAi screen. A negative value indicates that the siRNA decreased cell proliferation more in the triple-negative cell lines. * P < 0.05 comparing triple-negative and luminal Z-scores. (D) The average triple-negative and luminal Z-scores for all cell lines for BCAP31. The P value indicates a statistically significant difference between triple-negative and luminal lines. Error bars represent the SD. (E) Kaplan-Meier survival curves showed poorer RFS and OS with high BCAP31 expression than those with low BCAP31 expression in breast cancer, left two panels, and in basal-like breast cancer, right panel.
Figure 2
Figure 2
Upregulation of BCAP31 correlates with a poor prognosis for human breast cancer. (A) The scores indicate BCAP31 levels in representative tumour tissues. The scores were calculated by the intensity and percentage of stained cells as described in the Methods. (B) Patients with high BCAP31 expression (score of 2-3) have poorer overall survival and a higher probability of recurrence than patients with low BCAP31 expression (score of 0-1).
Figure 3
Figure 3
The roles of BCAP31 in promoting breast cancer development. (A) Confirmation of BCAP31 knockdown (KD, shBCAP31) and re-expression (shRES) in TNBC cell lines. (B-E) The effects of BCAP31 knockdown or re-expression on the in vitro proliferation (B), (C and D) migration, invasion and (E) colony formation of HCC cells. Error bars indicate the means ± SEM. (F) In vivo tumour growth in MDA-MB231 shBCAP31 mice was significantly inhibited compared with control mice. Representative images of tumour-bearing mice are shown. (G) The dynamic change in tumour volume in subcutaneous models is shown. Error bars indicate the means ± SEM. Knockdown of BCAP31 significantly decreased tumour growth (H) and spontaneous liver metastasis (I) in MDA-MB-231 xenograft nude mice models. Representative images of H&E-stained liver tissues from the xenograft groups. Arrowheads indicate metastatic nodules in the liver.
Figure 4
Figure 4
BCAP31 interacts with EGFR and facilitates ligand-independent EGFR/AKT pathway activation. (A) Mass spectrometry (MS) analysis of BCAP31-associated proteins. The purified protein complex was resolved on SDS-PAGE, followed by silver staining; then, the bands were retrieved and analysed by MS. (B) Analyses of the identified BCAP31 interactors. The diagram depicts BCAP31 interactors as detected by MS. The network was built based on the interaction network of EGFR-associated signalling and trafficking processes in the KEGG database overlaid with MS data. (C and D) The interaction between exogenous and endogenous BCAP31 and EGFR. Co-IP assay and immunoblot analyses evaluating the BCAP31-EGFR interaction in COS-7 cells and TNBC cells. (E) Downregulation of BCAP31 significantly attenuates ligand-independent signalling. TNBC cells stably expressed shBCAP31, control shRNA, or shRES when treated with EGF or EGF combined with gefitinib. IB examinations of phosphorylated EGFR and downstream signalling are shown. (F) Re-expression of the K721A kinase-dead (EGFRKD) EGFR rescues the ligand-independent phosphorylation of EGFR at Tyr 845 and downstream AKT signalling in EGFR-knockdown MDA-MB-231 cells expressing control shRNA but not shBCAP31. SiRNA-resistant EGFR-KD was expressed in control or shBCAP31-MDA-MB-231 cells by lentivirus-mediated infection. Endogenous EGFR was knocked down by siRNA, and whole-cell lysate was harvested for western blot analysis. (G) Tumours of MDA-MB-231 tumour xenografts were harvested, homogenized, and analysed for pEGFR (Tyr1068, Tyr845), pAKT (Ser473), total EGFR and total AKT by ELISA. Statistical significance was determined using Dunnett's test.
Figure 5
Figure 5
BCAP31 inhibits EGFR auto-recycling. (A) MDA-MB-231 cells were fixed for costaining of endogenous BCAP31 (green) and Calnexin (red). (B) MDA-MB-231 cells were fixed for costaining of endogenous EGFR (green) and BCAP31 (red). (C) Transfected COS-7 cells were fixed for costaining of exogenous EGFR (green) and BCAP31 (red). (D) (A) EGFR mRNA expression was determined by qPCR analysis. (E) Cycloheximide (CHX) did not suppress EGFR degradation in BCAP31 KD cells. An EGFR degradation assay was performed in the absence or presence of CHX (50 μg/ml). (F) TNBC cells were surface biotinylated, and surface and total EGFR levels were analysed by western blotting. (G) Representative flow cytometric histograms of TNBC cells examined for the expression of cell surface EGFR with or without BCAP31 knockdown. The experiment was repeated three times independently with similar results. (H) The ligand-induced recycling of EGFR was determined in TNBC cells stably expressing shNT, shBCAP31, and shRES vectors as indicated. Error bars indicate the means ± SEM. (I) The ligand-independent recycling of EGFR was determined in TNBC cells. MDA-MB-231 cells were fixed for costaining of endogenous EGFR (green) and RAB11 (J), EEA (K), and LAMP1 (L) (red). Bar, 10 μm
Figure 6
Figure 6
RAB11 is required for BCAP31-mediated EGFR recycling inhibition and cancer development. (A) BCAP31 interacts with RAB11. The indicated constructs (BCAP31-FLAG and RAB11-HA) were transiently expressed in COS-7 cells, and the whole-cell lysates were immunoprecipitated (IP) with the indicated antibody. (B) In vitro interaction between BCAP31 and RAB11. Whole-cell lysates from MDA-MB-231 cells were prepared, and IP and IB were performed with antibodies as indicated. (C) MDA-MB-231 cells were fixed for costaining of endogenous BCAP31 (green) and RAB11 (red). (D) Confirmation of RAB11 knockdown using western blotting. (E-H) The effects of KD of BCAP31 and RAB11 on the ligand-independent recycling (E), ligand-independent signalling (F), cell proliferation (G), and colony formation (H) of TNBC cells stably expressing the indicated shRNA vectors. The fold differences represent the mean of the experimental group compared with that of the controls. Error bars indicate the means ± SEM. * p < 0.05 compared with the CTRL. (I) Immunoprecipitation of endogenous EGFR from control or BCAP31-knockdown MDA-MB-231 cells. Bar, 10 μm.

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